key: cord- -nrw zztp authors: egan, timothy j title: drug-resistant plasmodium falciparum: are recent advances a cause for optimism? date: - - journal: future microbiol doi: . /fmb. . sha: doc_id: cord_uid: nrw zztp nan the period since has seen dramatic advances in the fight against malaria. the disease has been eliminated in countries and is in the elimination or pre-elimination phase in others. in addition, by there had been a % decrease in malaria cases, a % decrease in prevalence of childhood malaria and a % decrease in malaria deaths worldwide since . these advances can be attributed to several strategies, notably the use of insecticide treated bed nets, indoor residual spraying of insecticides, intermittent preventative treatment and improved diagnosis and treatment of malarial infections. in the case of treatment, the use of artemisinin combination therapy has been shown to reduce childhood mortality by an average of to %. this remarkable progress can be attributed to the commitment of resources. in the period - funding for malaria control and elimination increased almost threefold, with the three largest sources being the global fund to fight aids, tb and malaria, the united states president's malaria initiative together with usaid and the governments of malaria endemic countries themselves [ ] . the tools that have been developed to control and eliminate malaria and the funds devoted to this cause have in many ways rivaled the achievements of the s and s, which were brought about by the use of ddt and chloroquine (cq). ominously, however, in much the same way as cq-resistance began in southeast asia near the thai-cambodia border approximately years ago, so artemisinin-resistance has become apparent in the same region in the last years [ , ] . this was later shown to have spread to the thai-myanmar border area [ ] . while artemisinin combination therapy largely remains effective, evidence soon emerged of increased failure rates of artesunate mefloquine combination therapy linked to artemisinin-resistance near the thailand-cambodia border [ ] . thus the problem of artemisinin resistance represents a serious risk to the future success of the malaria control and elimination program. it is now widely accepted that mutations in the gene encoding pfcrt, a digestive vacuole (dv) transporter located in the dv membrane is the primary factor in cq resistance in plasmodium falciparum, albeit that other proteins probably also contribute to the level of resistance [ ] . discovery of the pfcrt gene and its role in cq resistance only occurred years after the first appearance of resistance [ ] , by which time it was almost universal. even today, the natural physiological role of pfcrt is not known. in the case of artemisinin, clinical evidence for resistance was first published in [ ] . molecular markers for resistance in the cambodian strains were identified within years [ ] . using whole-genome sequencing of an artemisinin-sensitive parasite line from tanzania cultured under escalating drug pressure for years and clinical artemisinin-resistant isolates from cambodia, ariey et al. were able to identify mutations in the pf d _ kelch propeller domain, or pfkelch , as markers of artemisinin-resistance. three pfkelch polymorphisms (c y, r t and y h) were found to be correlated with significant increases in parasite clearance time in patients, with the c y mutation showing the largest increase. these changes, especially the c y polymorphism, were found at high frequency in provinces of western cambodia where clinical artemisinin resistance is prevalent and were largely absent in provinces where artemisinin resistance is not encountered. these molecular markers have already proved extraordinarily valuable in mapping the spread of artemisinin resistance across myanmar, which is now perilously close to the border of india [ ] . indeed, they will likely revolutionize surveillance of artemisinin resistance and may provide more time and better information for devising strategies to mitigate the effects of resistance. in the last few months two studies have investigated the physiological role of pfcrt and mechanism of pfkelch -based artemisinin resistance, respectively. in the first of these studies, moriyama and co-workers expressed recombinant pfcrt fused at the n-and c-terminal ends with a soluble escherichia coli protein [ ]. this was incorporated into liposomes. by varying the solution in which the liposomes were prepared as well as the solution in which they were dispersed together with the use of the potassiumselective ionophore valinomycin, the authors could explore the effects of ph and membrane potential on the transport of various substrates as well as antimalarials into the liposomes. using radiolabeled tetraethyl ammonium ions (tea), they demonstrated that substrate uptake into the liposomes required a ph gradient and membrane potential with lower ph and positive polarization on the outside of the liposome membrane. in the case of cq-sensitive pfcrt d , a wide range of naturally occurring cationic species, including among others amino acids, peptides derived from hemoglobin degradation and glutathione, exerted a cis-inhibition of tea transport into the liposomes. cq, quinidine and the cq-chemosensitizer verapamil had a similar effect. furthermore, amino acids and cq were shown to be taken up into the liposomes, demonstrating that pfcrt is capable of transporting these cationic substances across the membrane. neutral and anionic amino acids and other biomolecules had no effect on tea uptake and were not transported into the liposomes. this work suggests that the natural role of pfcrt is as a polyspecific organic cation transporter. two surprises were that cq was transported even by cq-sensitive pfcrt and that much less cisinhibition was exerted by this drug in the presence of the crucial k t mutation in pfcrt that is required for resistance. indeed, with pfcrt exhibiting the additional mutations present in cq-resistant strains, namely pfcrt g and pfcrt dd , cq did not inhibit tea transport. more detailed investigation indicated that cq has lower affinity for mutant forms of pfcrt, but a higher maximal rate of transport (v max ). this results in substantially faster cq transport by cq-resistant mutants of pfcrt. finally, the results also hint at an additional role of cq in inhibiting export of peptides derived from hemoglobin degradation from the dv. in the second study, haldar and co-workers used a fluorescent early endosome antigen construct (ss-eea -mcherry) as a phosphatidylinositol- -phosphate (pi p) reporter [ ] . in the absence of pi p, wild-type ss-eea -mcherry is secreted from the endoplasmic reticulum to the parasitophorous vacuole of the parasite. this system had been used by the authors to screen for inhibitors of plasmodium falciparum phosphatidylinositol- -kinase (pfpi k). they went on to show that dihydroartemisinin, the active metabolite of the artemisinins, inhibits pfpi k in a reversible fashion in early ring stage parasites as visualized by the appearance of ss-eea -mcherry in the parasitophorous vacuole. the inactive analog, deoxyartemisinin caused no such effect so that pi p formation occurred uninhibited and ss-eea -mcherry remained "the problem of artemisinin resistance represents a serious risk to the future success of the malaria control and elimination program." drug-resistant plasmodium falciparum: are recent advances a cause for optimism? editorial future science group www.futuremedicine.com bound to pi p in the endoplasmic reticulum. artemisinins did not inhibit mammalian pi k. furthermore, homology modeling of the active site of pfpi k indicated that dihydroartemisinin can make specific interactions with amino acids in this site. parasites with mutant pfkelch associated with clinical resistance contained raised levels of pfpi k. wild-type pfkelch was shown to bind to pfpi k and bring about ubiquitination of the pfpi k, which results in protein degradation. the mutant forms of pfkelch were found not to bind pfpi k, resulting in decreased ubiquitination and thus increased levels of the enzyme. ring stage survival was found to correlate with raised pi p levels. while these studies are of undoubted scientific importance and will likely engender much new research and debate, it is also interesting to consider whether the implications of these findings are cause for optimism. given that artemisinin resistance during the early ring stage is not related to any structural changes in the proposed pfpi k target, but rather to increased concentration of this target arising from mutations in the pfkelch protein, prospects for avoiding resistance to this class of drug seem bleaker than might have been hoped. only extraordinarily potent analogs or a substantial increase in dose would be likely to be able to overcome this form of resistance. regarding drugs with cq-like mechanisms of action, it would seem that these likely have a natural propensity to be transported by pfcrt and hence a predisposition for development of resistance. however, in this case it may at least in principle be possible to discover compounds that inhibit transport of cationic substrates without themselves being subject to such transport. if the findings with proteoliposomes are an accurate reflection of what happens in the dv, new thinking will thus be required in the design of any new drugs in this class. the author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. no writing assistance was utilized in the production of this manuscript. evidence of artemisininresistant malaria in western cambodia artemisinin resistance in plasmodium falciparum malaria emergence of artemisinin-resistant malaria on the western border of thailand: a longitudinal study artemisinin-resistant malaria in asia know your enemy: understanding the role of pfcrt in drug resistance could lead to new antimalarial tactics mutations in the p. falciparum digestive vacuole transmembrane protein pfcrt and evidence for their role in chloroquine resistance a molecular marker of artemisinin-resistant plasmodium falciparum malaria spread of artemisinin-resistant plasmodium falciparum in myanmar: a cross-sectional survey of the k molecular marker key: cord- - d esp authors: walker, peter j.; firth, cadhla; widen, steven g.; blasdell, kim r.; guzman, hilda; wood, thomas g.; paradkar, prasad n.; holmes, edward c.; tesh, robert b.; vasilakis, nikos title: evolution of genome size and complexity in the rhabdoviridae date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d esp rna viruses exhibit substantial structural, ecological and genomic diversity. however, genome size in rna viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. here we conduct a large-scale analysis of the genome sequences of animal rhabdoviruses, including genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. all but seven of the rhabdoviruses clustered into well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. we show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive orfs within the major structural protein genes, and the insertion and loss of additional orfs in each gene junction in a clade-specific manner. changes in the lengths of gene junctions accounted for as much as . % of the variation in genome size from the smallest to the largest genome, and the frequency with which new orfs were observed increased in the ’ to ’ direction along the genome. we also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving turbs-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. we conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded rna genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the rhabdoviridae. rna viruses are among the most structurally and ecologically diverse of all life forms [ ] . their genomes may consist of positive (+) sense, negative (-) sense or ambi-sense singlestranded (ss) rna, or double-stranded (ds) rna, and may take the form of a single or multiple segments that are packaged in single or multiple particles. rna viruses also employ a plethora of strategies for replication and gene expression, and encode a vast array of structural and nonstructural proteins, many of which are unique and have multiple, highly specialized functions [ ] . despite their diversity, rna virus genomes are ubiquitously small, averaging only kb, and with a maximum size of~ kb for some members of the order nidovirales [ , ] . this size limitation has been linked to high mutation rates (a mean rate of~ mutation /genome /replication) due to replication with an error-prone rna-dependent rna polymerase that lacks proofreading capability [ , ] . high error rates are thought to limit genome sizes because, as size increases, the number of deleterious mutations also increases to levels beyond which reproduction of the fittest variant cannot be guaranteed [ , ] . due to this fundamental evolutionary constraint, rna viruses have employed various mechanisms of genome compression, such as the use of alternative or overlapping open reading frames (orfs) and the evolution of multiple functions for individual proteins [ , , ] . for some rna viruses, increases in genome size have been associated with increases in the size of replicative proteins [ ] and the presence of helicase and proof-reading exonuclease domains [ , [ ] [ ] [ ] . however, the mechanisms and evolutionary context that would favour increased genome size and complexity, given constraints on replication efficiency, are currently unknown [ , ] . the rhabdoviridae is one of the most ecologically diverse families of rna viruses. rhabdoviruses have been identified in a very wide range of plants and animals, including mammals, birds, reptiles, and fish with many transmitted by arthropod vectors [ , ] . the family includes rabies virus (rabv), which causes over , human deaths annually [ ] , vesicular stomatitis indiana virus (vsiv), which has served as an important model for the study of many aspects of mammalian virus replication and virus-host interactions, and many other important pathogens of humans, livestock, farmed aquatic animals and food crops. the nonsegmented [-] ssrna rhabdovirus genome is packaged within a characteristic bullet-or rodshaped particle comprising five structural proteins-the nucleoprotein (n), polymeraseassociated phosphoprotein (p), matrix protein (m), glycoprotein (g) and rna-dependent rna polymerase (l) [ ] . the genome features partially complementary, untranslated leader (l) and trailer (t) sequences and five orfs arranged in the order '-n-p-m-g-l- '. each orf is flanked by relatively conserved transcription initiation (ti) and transcription termination/ polyadenylation (ttp) sequences which orchestrate expression of the five corresponding capped and polyadenylated mrnas [ ] . rhabdovirus genomes may also contain additional orfs encoding putative proteins, which are mostly of unknown function. these may occur as alternative or overlapping orfs within the major structural protein genes or as independent orfs flanked by ti or ttp sequences in the regions between the structural protein genes [ ] , some of which appear to have arisen by gene duplication [ , [ ] [ ] [ ] [ ] [ ] . here we undertake the first large-scale analysis of the evolution of genome size and complexity in a family of [-] ssrna viruses. we demonstrate that remarkable changes in genome size and complexity have occurred in rhabdoviruses in a clade-specific manner, primarily by extension and insertion of additional transcriptional units in the structural protein gene junctions, followed by occasional losses. we also show that rhabdoviruses have evolved a large number of accessory proteins and that the use of non-canonical gene expression strategies appears to be common, particularly amongst vector-borne rhabdoviruses. our data set comprised the complete or near-complete genome sequences of animal rhabdoviruses, including viruses isolated from various vertebrates and arthropods for which we determined the sequences de novo (s table) . incomplete genomes lacked only the extreme terminal sequences. all rhabdovirus genomes contained the five canonical structural protein genes (n, p, m, g and l); however, there was remarkable diversity in the number and location of other long orfs. across the data set, we identified additional orfs nt in length of which shared no detectable protein sequence similarity with any other protein in our data set or with those in public databases (s table) . these additional orfs were located either within the structural protein genes or in additional transcriptional units located in regions between these genes (fig. ) . the additional transcriptional units were annotated by using relatively conserved ti and ttp motifs. the core ti sequence (uugu) was conserved with some minor variations (cugu, uugc, uuga, ucgu, ugau) employed in some viruses. the ttp motif g[u] was also conserved, with the variation a[u] occurring only in several genes of one virus (chov). due to the large number and diversity of additional orfs, we adopted a standard nomenclature that does not necessarily reflect structural homology. unless previously assigned a distinctive name (e.g., befv g ns , α , α , β and γ proteins), all orfs nt were assigned names according to the following rules: i) each additional transcriptional unit was designated u (unknown) followed by a number as they appeared in order in the genome presented in positive polarity (i.e., u , u , u , etc); ii) the first orf within each transcriptional unit was assigned the same designation as the transcriptional unit; and iii) each subsequent orf within any transcriptional unit (alternative, overlapping or consecutive) was designated by letter (i.e., u x, u y, u z) (s table) . alternative orfs are defined here as those which occur in a different frame within another longer orf; overlapping orfs are alternative orfs which extend beyond the end of the primary orf; and consecutive orfs are those which do not overlap but follow consecutively within the same transcriptional unit. the arbitrary cut-off of nt ( aa) was selected on the basis that two small basic proteins of and amino acids (c and c') have been shown to be expressed from an alternative orf within the vsiv p gene [ , ] . these are the smallest known rhabdovirus proteins. to determine the evolutionary history of the rhabdoviruses studied here, we inferred a phylogenetic tree using conserved regions of the l protein of all viruses in our data set as well as the recently described north creek virus (norcv) [ , ] (fig. ) . all but two of these rhabdoviruses (norcv and mouv) clustered into well-supported monophyletic groups (bootstrap proportion [bsp] ); however, many of the deeper nodes were unresolved throughout the phylogeny. eight of the well-supported clades corresponded to the eight established genera (lyssavirus, vesiculovirus, perhabdovirus, sigmavirus, ephemerovirus, tibrovirus, tupavirus and sprivivirus) and we assigned a further seven clades as proposed new genera (almendravirus, bahiavirus, curiovirus, hapavirus, ledantevirus, sawgravirus and sripuvirus). the taxonomic assignment of the two remaining clades was considered to be ambiguous (s table) . for simplicity of expression we refer here to all as 'genera', whether existing or proposed, but we recognise that taxonomic proposals require consideration and ratification by the international committee on taxonomy of viruses (ictv). although the analysis was limited by the availability of single isolates of most viruses, apparent structure by geographic location or reservoir host was not observed in the phylogeny. however, multiple genera appeared to be primarily associated with bats (i.e., ledanteviruses, lyssaviruses), fish (i.e., perhabdoviruses, spriviviruses) or ungulates (i.e., ephemeroviruses, tibroviruses, vesiculoviruses). vector-borne rhabdoviruses were present in of the groups, dominating the dimarhabdovirus supergroup, but were largely absent from clades associated with bats (lyssavirus), flies (sigmavirus) and fish (perhabdovirus, sprivivirus) (fig. ) . the exception to this trend was the tupavirus clade, which comprised viruses that have not yet been associated with a vector species, and for which little is known about their ecology or distribution. each of the seven newly proposed rhabdovirus genera formed an independent, well-supported monophyletic group in the l protein phylogeny (bsp ), and comprised viruses with similar genome organization ( fig. ; fig. ). in several instances, viruses clustered closely with other members of a genus, yet we considered them to be unassigned species due to major differences in genomic architecture (see below). for example, the newly proposed genus curiovirus comprises a monophyletic group of four viruses isolated from biting midges (culicoides sp.), sandflies (lutzomyia spp.) and mosquitoes (coqillettidia and trichoprosopon spp.) from the forests of south america and the caribbean (s table) . the genomes of curv, irirv, rbuv and itav all have one or more orfs located between the m and g genes, and the g and l genes. in contrast, the closely related aruv and inhv lack additional genes between the m and g and for this reason we have excluded them from the genus curiovirus at this time. we also recognize the previous suggestion that curv and itav should be assigned to a new genus for which the name bracorhabdovirus (brazilian amazonian culicoides rhabdoviruses) was proposed [ ] . however, our analysis clearly indicates that this monophyletic group has a broader host range and geographic distribution than this regionally-derived name suggests. five of the novel viruses (comprising four putative new species) identified in this study were assigned to established genera. two of these, koolv and yatv, clustered within the existing ephemerovirus clade, (bsp ) and possessed the characteristic genome organization of ephemeroviruses, including a non-structural glycoprotein gene (g ns ) followed by a viroporin newly proposed genera are indicated by a † symbol. cytorhabdovirus, novirhabdovirus and nucleorhabdovirus outgroup sequences were excluded from the tree as they were too divergent to establish a reliable rooting. the tree is therefore rooted arbitrarily on one of two basal clades (genera almendravirus and bahiavirus) that comprise viruses isolated from mosquitoes. (α ) and several other small proteins ( fig. ; fig. ). similarly, two novel viruses isolated from biting midges (culicoides insignis), swbv and bav, clustered within the genus tibrovirus (bsp ) and exhibited the conserved n-p-m-u -u -g-u -l genome organisation ( fig. ; fig. ; s table) . swbv was assigned as a new species (sweetwater branch virus), but bav is closely related to tibv and may be regarded as the same species (tibrogargan virus). finally, a novel tupavirus (klav) identified from two species of vole (microtus and clethrionomys spp.), clustered with the tupv and durv clade in the l protein phylogeny ( fig. ; s table) . a more detailed rationale for the assignment of viruses to existing and proposed new genera is provided as supplementary text. we identified a . % variation in genome size from the smallest genome (fukv, ledantevirus; , nt) to the largest in our data set (koolv, ephemerovirus; , nt). all genomes, including those for which extreme terminal sequences were unresolved, appeared to fall within this range. variations in genome size were associated with: i) variation in the length of intergenic regions (igrs) between transcriptional units; ii) variation in the length of ' and ' untranslated regions (utrs) within individual transcriptional units; iii) the presence of additional transcriptional units containing long orfs; and iv) the presence of overlapping or consecutive long orfs within individual transcriptional units. an examination of genome size across the phylogeny revealed a general trend towards larger genomes in the lower third of the tree, which is comprised of the hapaviruses, curioviruses, tibroviruses and ephemeroviruses, as well as several unassigned viruses (s fig.) . although this may indicate that an enhanced capacity for genome expansion is a property specific to this group, variation in genome size can also be observed between viruses in the majority of genera in the data set. several clade-specific patterns were evident when the lengths of the transcriptional units and igrs were compared within and between rhabdovirus genera (table ) . ledantevirus genomes were smallest on average ( . × the length of the l) whereas ephemeroviruses genomes were the largest ( . × the length of the l, table ). interestingly, although substantial variation in the length of gene junctions was observed in several genera (including ephemeroviruses and lyssaviruses), most variation in genome size occurred as the result of the presence of new, non-canonical orfs in the regions between the structural protein genes (table ) . although new orfs were observed in each igr across the phylogeny (n-p, p-m, m-g and g-l) their location was primarily restricted to a single igr within each genus. for example, while hapavirus genome expansion occurred primarily in the p-m junction, genome expansion in the ephemeroviruses occurred at the g-l junction and tibrovirus and curiovirus genomes contained additional orfs primarily in the m-g junction (table ). this suggests that once a new orf arises at a particular gene junction within a lineage, further expansion is more likely to continue at the same gene junction, rather than begin anew elsewhere in the genome. whilst the genome architecture in some viruses was highly compact, others featured long stretches of sequence with non-ascribed function that occurred primarily as 'utrs and 'utrs within transcriptional units (fig. ) . the proportion of untranslated sequences within or between transcriptional units ranged from . % (fukv; nt) to . % (wcbv; nt) and did not correlate with genome size. furthermore, although all lyssaviruses (such as wcbv) featured a high proportion of untranslated sequences (primarily evident as a very long 'utr in the g gene), there was no consistent association between the proportion of untranslated sequences and genus assignment (fig. ). for example, in the genus hapavirus, the proportion of untranslated sequences in the two largest genomes varied from . % (ngav) to . % (ljv). similarly, in the genus ephemerovirus the proportion of untranslated sequences varied from . % in the smallest genome (yatv) to . % in the largest genome (koolv). the presence of long stretches of untranslated sequence, which occurred primarily within transcriptional units, suggests these regions may be functional. however, it is unclear at this time why they are present in some rhabdoviruses and not in others. gene duplication. previous studies have provided evidence of gene duplication in the rhabdoviridae, involving the g and g ns genes [ , ] and the β and γ genes [ ] in the ephemeroviruses, and the u , u and u genes in the hapaviruses flav and wonv [ , , ] . to identify further examples of gene duplication, we conducted a blast analysis of all proteins in our database (e-value < e- ) and used clustalx alignments to confirm sequence similarity. by this analysis, orfs located between the p and m genes of most hapaviruses encode proteins which share detectable sequence similarity. this family of homologous p-m intergenic region proteins (pmips) includes the u , u and u proteins of ljv, wonv, pcv, orv, ljav, manv, mqov, flav, hpv, kamv and mosv (s fig. and s fig.) , as well as the u x proteins of manv and glov which are encoded in orfs overlapping their respective u orfs (s fig.) . although pairwise alignments provide clear evidence for homology, the hapavirus pmips share generally low levels of sequence identity and no universally conserved motifs, indicating considerable structural and functional divergence from their ancestral homolog. proteins encoded in the p-m region in other hapaviruses (i.e., joiv u , ngav u , u x and ngav u ) failed to display significant similarity with the pmips or evidence of gene duplication but this may be due to further structural divergence. additional evidence of gene duplication included the u and u proteins of joiv (encoded in orfs located between the g and l genes), and the n-terminal regions of the p proteins and the upstream u accessory proteins of the sripuviruses chov and smv, each of which share significant sequence similarity (s fig.) . these data suggest that the u protein of the sripuviruses originated from a duplication of the p gene, with the downstream copy of the gene retaining the parental function. similarly, in the curioviruses there is extensive amino acid sequence similarity between the u proteins of curv and irirv and the n-terminal region of the g proteins, suggesting evolution of u through partial duplication of the g gene, which lies immediately downstream. putative accessory genes were found to be abundant and varied greatly in number and location in each genome (fig. ) . a complete list of orfs > nt is annotated in s table. in most cases, homology searches detected no significant amino acid sequence identity with entries in genbank. however, various rhabdovirus accessory gene families were identified based on amino acid sequence identity in our custom blast searches, or common structural characteristics. viroporins. viroporins are small hydrophobic proteins that oligomerize in host cell membranes to form hydrophilic pores, disrupting various cellular processes and promoting virus replication [ ] . orfs encoding viroporin-like proteins were found in more than one-third of the rhabdoviruses in the data set, either as overlapping or consecutive orfs within the g gene, or in additional transcriptional units following the g (or g ns ) gene (fig. ) . orfs encoding putative viroporins were evident in the genomes of all ephemeroviruses, tibroviruses, hapaviruses, bahiaviruses, almendraviruses and curioviruses, as well as the unassigned species aruv and inhv (fig. ) . several of these proteins have been identified previously [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . like the befv α protein for which viroporin activity has been confirmed experimentally, these proteins have the structure characteristics of class ia viroporins, including a central transmembrane and a highly basic c-terminal domain. however, although located in similar positions in the genomes, they are generally too divergent in sequence to establish orthology [ , ] . other small transmembrane proteins. small proteins with a predicted central transmembrane domain but lacking other characteristics of class a viroporins were identified in several other rhabdoviruses (s fig.; s table) . transmembrane proteins with an n-terminal ectodomain are encoded in the gx orf of sripuviruses and the u orf of one curiovirus (rbuv). however, in other curioviruses (curv and irirv), transmembrane proteins are encoded in the u orf and are predicted to have the reverse membrane topology to the rbuv u protein. sequence alignments further suggest these proteins are not orthologous. there is also a small double-membrane spanning protein with a predicted short ectodomain loop encoded in an alternative orf in the fukv m gene that is not present in other ledanteviruses. other small hydrophobic (sh) proteins. small highly hydrophobic proteins ( . - . kd) lacking predicted transmembrane domains are encoded in all tupaviruses (as independent transcriptional units following the m gene) and sripuviruses (as overlapping orfs within the m gene) (s fig.; s table) . all have similar hydropathy profiles with a highly hydrophilic n-terminal domain extending to the centre of the sequence, but sequence identity indicative of orthology is restricted to closely-related viruses. several of these sh proteins have been identified previously but their function remains unknown [ ] [ ] [ ] [ ] . large class i transmembrane glycoproteins. all ephemeroviruses encode a class i transmembrane glycoprotein (g ns ) in the orf following the g gene [ , , , ] . ngav (assigned to the proposed new genus hapavirus) also encodes a g ns protein with similar structural characteristics [ ] . however, as we found no evidence to support recombination between ngav and any ephemerovirus, the ngav g ns gene is likely to have arisen by an independent duplication event of the upstream g gene with which it shares amino acid sequence identity. orf u immediately following the mcov g gene (genus hapavirus) also encodes a large class i transmembrane glycoprotein but lacks the set of conserved cysteine residues that are characteristic of g and g ns proteins, and our homology searches failed to identify similarity with any known protein (s fig.) . other genus-specific accessory gene families. orthologous sets of accessory genes occur in genus-specific patterns in each of the structural protein gene junctions ( fig. ; s table) . in addition to the hapavirus pmip genes, these include genes in the n-p junction of sripuviruses chov and smv (u proteins), the m-g junction of curioviruses (u and u x proteins) and tibroviruses (u and u proteins), and the g-l junction of curioviruses (u x proteins) and ephemeroviruses (α , β, γ and δ proteins) (s fig. to s fig.) . some of these orthologous gene sets have been described previously [ ] . most encode proteins without remarkable structural characteristics and of unknown function (s table) . several general architectural patterns in the arrangement of orfs were evident, implicating several mechanisms of non-canonical gene expression. non-cannonical expression mechanisms are used commonly in other families of rna viruses to increase genome complexity without significantly increasing genome size [ ] . the patterns we observed in this data set were associated with consecutive, overlapping of alternative orfs within individual transcriptional units. consecutive orfs and turbs motifs. consecutive long orfs with termination and initiation codons that are either overlapping (e.g., uaaug) or separated by a short stretch of nucleotides were common in several groups of rhabdoviruses (fig. ) . as previously observed for flav, this 'stop-start' arrangement is commonly preceded by a 'termination upstream ribosome-binding site' (turbs), which contains a short sequence motif that is complementary to the loop region of helix of s ribosomal rna [ , ] . the turbs may also contain flanking anti-complementary sequence motifs that are predicted to form a stem-loop structure. this arrangement was found in the m transcriptional unit in the sripuviruses, the g transcriptional unit of several hapaviruses (flav, hpv, manv, mqov, kamv, mosv and glov) and the transcriptional unit between the p and m genes of glov. the 'stop-start' arrangement also occurs in the transcriptional unit between the g and l genes of aruv, allowing expression of the u orf, but in this case the turbs appears to be further upstream of the stopstart site. finally, the α gene transcriptional unit in most ephemeroviruses contains consecutive orfs encoding a viroporin (α ) and a second protein of unknown function (α ). in kotv, a tubrs is evident upstream of the stop-start site but in other ephemeroviruses the turbs appears to be more cryptic. overlapping orfs and ribosomal-frame shift (rfs) sites. overlapping orfs are common in rhabdovirus genomes and represent a second common architectural arrangement requiring non-canonical gene expression. overlapping orfs occur within the n transcriptional unit (wonv, orv, pcv, mcov, manv), the g transcriptional unit (wonv, orv, pcv, bgv, harv) or within additional transcriptional units between the p and m genes (manv, ngav) or the m and g genes (curv, irirv, rbuv). expression of the second orfs in these arrangements would require either internal initiation in an alternative reading frame or another mechanism such as rna editing or a ribosomal frame-shift (rfs) to extend the first orf. use of alternative initiation codons has been reported in the m and p genes of vsv and the p gene of rabv, and rna editing has been described in the p gene of paramyxoviruses [ , [ ] [ ] [ ] [ ] . although not described previously in mononegaviruses, potential rfs sites were identified in some of these rhabdovirus gene overlap regions, featuring the 'slippery' sequence motifs uaruuuuuuca (bgv, harv, msv) or ccnuuuuuuga (wonv, orv, pcv) followed by a predicted stem-loop structure (s fig.) . these sequence motifs and associated stem-loop structures most closely resemble the- rfs that allows expression of gag-pol in hiv- and other lentiviruses [ , ] . alternative orfs and leaky ribosomal scanning. the third architectural arrangement involves the use of alternative orfs within a longer orf. this arrangement was described previously in vsiv, in which two small basic proteins of and amino acids (c and c') are expressed from an alternative orf within the p gene [ , ] . on this basis, we scanned the rhabdovirus genome data set for alternative orfs of various size ranges and observed that the frequency varied from~ . /genome for orfs in the range of - nt ( - amino acids) tõ . /genome for range - nt ( - amino acids) (fig. ). alternative orfs amino acids occurred in each of the structural protein genes (n, p, m, g and l) and in the additional transcriptional units between the p and m genes. they were most common in the p and least common in the m genes. as observed in other viruses, expression of these alternative orfs could occur by leaky ribosomal scanning, allowing initiation of transcription by a proportion of ribosomes on the alternative start codon [ ] . although, it is not known which (if any) of these alternative orfs are expressed, several factors are likely to be important in determining the probability and level of expression: i) the kozak contexts of the first and alternative initiation codons; ii) the length of the alternative orf (longer orfs are less likely to occur by chance); iii) the location of the alternative orf (distally located orfs are less likely to be expressed in long transcripts); and iv) the expression level of the transcript (l gene transcripts are likely to be the least abundant). for example, short orfs with initiation codons in poor kozak context at the distal end of the l gene are not likely to be expressed at significant levels, if at all. however, in some cases, closely related viruses were found to contain alternative orfs at the same genome location, with initiation codons in good context and encoding predicted polypeptides with high levels of sequence identity (s table) . such arrangements occurred in the n table. doi: . /journal.ppat. .g genes of hpv and flav, the p genes of manv and mqov, the u and m genes of kamv and mosv, and near the start of the g genes of the sripuviruses (niav, sriv, chov and smv); these proteins are considered very likely to be both expressed and functional. we have conducted a detailed analysis of the structural organisation and genome evolution of a family of negative-sense rna viruses-the rhabdoviridae. previous studies have surveyed known rhabdoviruses for biological and genomic diversity, revealed phylogenetic relationships, and considered factors that may have determined their rates of evolution [ , , , ] . in this study, we greatly expanded the repertoire of rhabdovirus genome sequences, which demonstrate extensive variation in genome size and complexity, allowing the assignment of seven proposed new genera. we also identified patterns of accessory gene evolution and expression, and showed that changes in rhabdovirus genome length and composition have occurred throughout the evolutionary history of the family, primarily through the generation and loss of new transcriptional units. this observation is especially striking given the obvious constraints on viral genome size [ ] . the most remarkable aspect of this analysis is the number and variety of additional orfs identified in rhabdovirus genomes, which provides a very different perspective of the family and its evolution than had been obtained from studies of the traditional prototype members (vsiv and rabv). as many of these orfs occur as additional transcriptional units complete with conserved transcriptional control sequences, there is a high likelihood that they would be expressed in infected cells. expression of orfs located in additional transcriptional units has been demonstrated previously for several ephemeroviruses and for the hapavirus wonv [ , , , , , ] . others occur as either alternative or overlapping orfs. further studies are required to determine which of these orfs may be expressed, but we suggest that expression is likely when both the encoded amino acid sequence and the translational context are conserved in related species. notably, very few of the additional orfs detected in this analysis encode proteins with identifiable sequence similarity to other known proteins. sequence similarity, when detected, occurred only between closely related viruses assigned to a genus and, although some accessory protein families were identified, these were more commonly related by shared structural characteristics, such as charged or transmembrane domains, than by sequence. this has been observed previously for so-called orphan ('orfan') proteins in other viruses and bacteria. it has been suggested that the uniqueness of orphan proteins, or their restriction to a single species or genus, is the result of creation de novo, rather than by recombination or lateral gene transfer, and that they play an 'accessory' role in viral pathogenicity or transmission instead of having functions in virion structure or replication [ ] [ ] [ ] . it has also been observed that many orphan proteins are predicted to be highly disordered in structure or, when ordered, structural resolution has revealed unique folds [ ] . as such, future determination of the biological activities of the plethora of novel proteins identified here will require functional studies that may well provide important insights into aspects of infection and immunity as well as fundamental cellular processes and pathways. substantial variation in genome size and complexity was also observed in many rhabdovirus genera, suggesting that the length of the genome is not heavily constrained in all members of the family. indeed, the presence of new orfs and/or very long stretches of non-coding sequence within or between transcriptional units was noted frequently. previous observations have demonstrated that foreign genes of up to~ kb can be inserted into the vsiv genome without significant disruption to viral replication in vitro [ , ] . expanded vsiv genomes were morphologically similar but proportionally longer than wild-type viruses, suggesting that the unique morphology of the rhabdovirus particle may more readily accommodate genome expansion than other virion structures. a significant body of evidence suggests that genome size in rna viruses is likely to be constrained by low replication fidelity [ , ] , and a relationship between genome size and error rate has been observed in a diverse array of organisms [ ] . however, if the genome sizes of rhabdoviruses are constrained by selective pressures other than (or in addition to) those imposed by the background mutation rate, genome expansion may not require a concomitant reduction in polymerase error rates. as the mutation rate of rhabdoviruses has only been determined experimentally for vsiv thus far (~ × - subs/ nucleotide/replication), it is impossible to assess whether the increases in genome size observed here have been associated with concomitant reductions in mutation rate [ ] . it is also striking that while some rhabdovirus genomes appear to have undergone major changes in length and complexity, others contain only the ' and ' promoter regions and five canonical transcriptional units with minimal ' and 'utrs. this suggests that the acquisition and loss of new genes and intergenic regions may be a regular feature of rhabdovirus evolution. previous studies of rna viruses have concluded that constraints on genome size imposed by polymerase error have led to various strategies to minimize genome size while increasing functional complexity, such as gene overlaps and protein multi-functionality [ , ] . given these size constraints, it is unclear why long non-coding regions would arise both within and between transcriptional units and be maintained throughout the evolution of some rhabdovirus genera. it has been known for many years that a long '-utr of unknown function (ψ region) in the g gene of rabv is unnecessary for efficient replication in cell culture or in mice, but may play a role in neuroinvasion [ ] [ ] [ ] . indeed, the retention of similar ψ regions in all lyssaviruses and the existence of long utrs and igrs in other rhabdoviruses suggests that they must provide some fitness advantage in vivo, such as stabilising rna secondary structure, serving as a source of, or targets for, micro rnas, or attenuating transcription of downstream genes to achieve the most effective balance of gene expression. indeed, an analysis of patterns and rates of sequence evolution in the rhabdoviridae and other families in the mononegavirales revealed that, although non-coding regions are less conserved than those that encode proteins, their evolutionary rates are associated with relative genomic position, suggesting that they impact on gene expression [ ] . additional orfs and non-coding sequences occurred at all junctions of the canonical structural protein genes (i.e., n-p, p-m, m-g, and g-l), although there was variation in both the frequency of insertion and the extent of expansion. notably, insertions at the n-p junction are rare, with a single additional orf present in the closely related sripuviruses chov and smv, and short overlapping orfs present within the n gene transcriptional unit in some hapaviruses. it has been reported previously in a study of vsiv recombinants that only the n-p gene junction was refractory to the stable expression of an inserted transcriptional unit, and resulted in a virus with significantly reduced replication efficiency [ ] . in contrast, transcriptional units inserted at other gene junctions were stably expressed, maintained through repeated passages and had no effect on replication efficiency. as the insertion of additional transcriptional units attenuates expression levels of all downstream genes, this may be associated with the importance of maintaining precise control of n and p protein ratios in infected cells to ensure efficient switching between the transcription and replication modes of the ribonucleoprotein complex [ , ] . the relationships, locations and contexts of additional orfs in various viruses lead us to propose a general model for rhabdovirus genome plasticity, which can account for both gains and losses in genome size and complexity (fig. ) . in each of these viruses, small orfs of various lengths occur within most transcriptional units; and although only those nt have genomic evolution in rhabdoviruses been catalogued here, there are numerous other smaller orfs throughout most genomes. it is reasonable to assume that, although the polypeptides encoded in many of these orfs may not be expressed at all during infection, some may be expressed through leaky ribosomal scanning. these are likely to represent a rich genetic resource for the evolution of new functional genes in rna viruses [ ] , triggering the rapid evolution of highly specialised functions. contemporarily, the evolution of a suitable kozak context, turbs motifs and ribosomal frame-shift sites would allow optimal expression within the parental transcriptional unit. ultimately, these new orfs may become uncoupled from the parental gene through gene (sequence) duplication [ ] . as observed previously, this process would allow unconstrained evolution of the new orf and loss of the redundant copy of the parental orf [ , ] . alternatively, new genes may also evolve independently of existing orfs. in some rhabdoviruses in our data set, very long non-coding regions (up to nt) were present either within or between transcriptional units that could serve as a resource to spawn genes de novo in the absence of the evolutionary constraints imposed on alternative or overlapping orfs. this is most likely to occur when orfs are present in transcribed non-coding regions (utrs) such as the ψ region of wcbv in which, uniquely amongst lyssaviruses, an orf of nt has been identified [ ] . the creation of new genes de novo in non-transcribed igrs, such as those present in the g-l gene junctions of ljv, kotv and koolv, almost certainly would require prior or simultaneous evolution of new or modified transcriptional control sequences to allow their expression. we recognise that other mechanisms of genome expansion are also possible. in central american isolates of vsiv, for example, imprecise reiterative insertions of up to nt in the '-utr of the g-gene (variations of '-uuuuuaa- ') have been attributed to non-templated extension by polymerase stutter at the ttp sequence [ , ] . although homologous recombination appears to be very rare in mononegaviruses [ ] , and we found no evidence of lateral gene transfer, we cannot exclude their involvement in rhabdovirus genome expansion. it is also evident that although there is an overall trend toward an expansion of genome size and complexity in the rhabdoviruses, gene loss is also likely to have occurred periodically throughout the evolution of the family. for example, the ephemerovirus γ proteins appear to have been lost in arv and obov, and the hapavirus pmips are entirely absent only from mcov (fig. ) . although our data suggests that gene gain is a more frequent process than gene loss, we acknowledge that, if loss is very frequent, we might not be able to observe it given the available data. this may be resolved in the future with the acquisition of significantly more genomes sampled more closely in time. indeed, as defective-interfering particles are known to occur commonly in rhabdoviruses, a mechanism for purging redundant sequences appears to be readily available [ ] [ ] [ ] . nevertheless, it is evident that a remarkable capacity for genomic plasticity through the gain and loss of accessory functions has been a central theme of rhabdovirus evolution. although our analysis was limited to the rhabdoviridae, similar mechanisms of genome expansion appear to occur in other families of non-segmented (-) ssrna viruses (mononegavirales). for example, amongst the paramyxoviridae genome length varies by . % from human metapneumovirus ( , nt) to beilong virus ( , nt) , and paramyxoviruses also contain novel accessory genes in transcriptional units inserted at various gene junctions [ ] . the apparent propensity for genome expansion in mononegaviruses may be due to their discontinuous transcription strategy which generates multiple viral mrnas. sequence insertions within and between the individual transcriptional units of mononegaviruses are less likely to disrupt gene expression than in (+) ssrna viruses in which the genome commonly encodes a single polyprotein which is processed post-translationally. finally, this study has also provided an important advance in rhabdovirus taxonomy, allowing the assignment of six new species to existing genera and the assignment of species to seven proposed new genera as well as the identification of six new unassigned species. there are currently no formal criteria for genus demarcation in rhabdoviruses. a system of genetic classification (demarc) that allows demarcation of viral taxa based on pairwise evolutionary distances has been proposed and, for picornaviruses, was shown to be comparable to expertbased taxonomic classification [ , ] . however, the application of this approach to the rhabdoviridae would likely require a larger set of sequenced genomes at lower taxonomic levels [ ] , and would be compromised by extensive rate variation among lineages (as this leads to biases in genetic distance measurements). in the taxonomy of higher organisms, to be descriptively useful, a genus should be monophyletic, reasonably compact, and ecologically, morphologically, or biogeographically distinct [ ] . our assignment of new genera in the rhabdoviridae has been based primarily on the identification of well-supported monophyletic groups using unambiguously aligned regions of the l gene, together with a consideration of common features of genome organisation and known aspects of viral ecology. genome organisation has proven here to be a useful taxonomic marker as similar arrangements of accessory genes and other conserved elements of genome architecture appear to be the result of significant evolutionary events that provide resolution between the family and species levels. for some of the new genera, host and/or vector associations have also been relatively informative but in many cases, only single isolates of a species are available and else little is known of their ecology. it is likely that the proposed assignments of viruses to genera and the placement of the proposed unassigned species will evolve into a more complete taxonomic description as more viruses are discovered and as ecological data accumulates. details of the viruses included in this study, including taxonomic status, sources and dates of isolation, and genbank accession numbers of genome sequences are given in s table. all but three viruses sequenced in this study were obtained from the world reference center for emerging viruses and arboviruses (wrceva), located at the university of texas medical branch, galveston. of the remaining viruses, fukv and koolv were obtained from the collection held at the csiro australian animal health laboratory, geelong, and joiv was obtained from the qimr collection held at the queensland university of technology, brisbane, and kindly provided by dr john aaskov. viruses sequenced in this study were prepared as described previously [ ] . with the exception of hpv, itav, curv, glov, inhv, nmv, mebv, yatv, ldv, garv, cntv, irirv, rbuv, barv, ljav, keuv, mcov, smv, chov, pcv and bav, which were sequenced directly from infected suckling mouse brain, viruses were sequenced from viral preparations grown in bhk-bsr, c / or vero cells monolayers. sequencing was performed using either the illumina hiseq or miseq platforms. viral rna was fragmented by incubation at °c for min in . l of fragmentation buffer (illumina ). a sequencing library was prepared from the sample rna using an illumina truseq rna v kit following the manufacturer's protocol. samples were sequenced using the × paired-end protocol. reads in fastq format were quality-filtered and any adapter sequences were removed using trimmomatic software [ ] . the de novo assembly program abyss [ ] was used to assemble the reads into contigs using several different sets of reads and k values from to . the longest contigs were selected and reads were mapped back to the contigs using bowtie [ ] and visualized with the integrated genomics viewer [ ] to verify that the assembled contigs were correct. total reads ranged from . to million and the percentage of reads mapping to the virus genome in each sample ranged from . % to %. details are available upon request. assembly of full genome sequences was performed as previously described [ ] and predicted orfs > amino acids in length were identified across each genome using geneious . . (biomatters ltd). for each non-canonical orf > amino acids in length, we sought to identify putative homologues by first comparing the protein sequence to the complete non-redundant protein sequence database available on genbank using the blastp and psi-blast search algorithms, as well as to the uniprot database using the hidden markov model alignment-based algorithm hhblits [ ] . for these searches, we investigated all matches with an evalue < . we then created a custom protein database containing all orfs > amino acids in length from our data set ( proteins) and performed a custom blast search to identify homologues within this data set. here, an e-value of < e- was considered a significant match. amino acid sequence alignments containing all putative matches to each orf were then created using clustal x and evidence of structural and sequence similarity was investigated by visual inspection. structural predictions for proteins were conducted using compute pi/mw, sig-nalp, tmhmm, tmpred, netnes and netnglyc available through the expasy bioinformatics resource portal (http://www.expasy.org/). to quantify the location and extent of variation in genome size in our data set, we compared the average length of each genomic region within and between rhabdovirus genera. for all viruses, we normalized the length of each gene region (from the ti to ttp sequences, inclusively) and intergenic region by dividing by the length of the corresponding l gene, which varied least across the data set (coefficients of variation: n = . , p = . , m = . , g = . , l = . ). as there was substantial variability in the proportion of the ' and ' utrs that were included in the sequence data set, we considered each genome to begin at the first ti sequence and end at the final ttp sequence for this analysis. to infer evolutionary relationships among animal rhabdoviruses, we compiled sequences of the l (rna-dependent rna polymerase) protein, as this was the most highly conserved protein across the data set. we initially attempted to root the tree using a standard outgroup method. members of the rhabdovirus genera that infect plants (i.e., cytorhabdovirus and nucleorhabdovirus) were excluded as their sequences were highly divergent. we therefore utilized four members of the genus novirhabdovirus (infectious haematopoietic necrosis virus adb ; viral hemorrhagic septicaemia virus bah ; hirame rhabdovirus aco ; and snakehead rhabdovirus np ) as outgroups. unfortunately, these novirhabdovirus sequences were also far too divergent (>> amino acid change per site under multiple amino acid substitution models; results available on request) to establish a reliable rooting for our data set, as three different basal groups were identified using different models of amino acid substitution, although overall tree topologies were similar among substitution models (results available on request). in addition, the use of the novirhabdoviruses as outgroups resulted in excessive numbers of residues being removed following gblocks pruning (see below). based on the observation that most known rhabdoviruses are either insect viruses or replicate in insect vectors, it has been reasonably argued that plant and animal rhabdoviruses may have origins in insects [ ] . we therefore selected the rooting scheme that best fit this theory. to this end, we choose one of the two basal clades from the novirhabdovirus-rooted tree, comprising viruses isolated from mosquitoes (i.e., the almendraviruses), as the most divergent group. we then repeated the phylogenetic analysis (procedure described below) excluding the novirhabdoviruses and rooting it on the almendraviruses. importantly, the choice of outgroup did not influence relationships either between or within the major clades demonstrating strong bootstrap support (bsp ). the alignment used for the final tree inference (i.e., excluding the novirhabdoviruses) was comprised of amino acid sequences aligned using the muscle program [ ] , with ambiguously aligned regions removed using the gblocks program with default parameters [ ] . this resulted in a final sequence alignment of taxa, amino acid residues in length. the phylogenetic relationships among these sequences were determined using the maximum likelihood (ml) method available in phyml . [ ] employing the wag+g model of amino acid substitution and subtree pruning and regrafting (spr) branch-swapping. the phylogenetic robustness of each node was determined using , bootstrap replicates and nearest-neighbour branch-swapping. fig. (a-d) . amino acid sequence alignments of small accessory proteins encoded in the genomes of ephemeroviruses. (pdf) s fig. (a-d) . amino acid sequence alignments of the u , u x proteins, u x and u x proteins of the curioviruses, and of the rbuv u protein with the itav u protein. (pdf) s fig. (a, b) . amino acid sequence alignments of the u and u proteins of tibroviruses. (pdf) s fig. (a-e) . analysis of the potential ribosomal frame-shift sites in the sequence overlap regions of curioviruses and some hapaviruses. 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divergent and ambiguously aligned blocks from protein sequence alignments new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . key: cord- -ndz oarf authors: ayithan, natarajan; bradfute, steven b.; anthony, scott m.; stuthman, kelly s.; bavari, sina; bray, mike; ozato, keiko title: virus-like particles activate type i interferon pathways to facilitate post-exposure protection against ebola virus infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ndz oarf ebola virus (ebov) causes a severe hemorrhagic disease with high fatality. virus-like particles (vlps) are a promising vaccine candidate against ebov. we recently showed that vlps protect mice from lethal ebov infection when given before or after viral infection. to elucidate pathways through which vlps confer post-exposure protection, we investigated the role of type i interferon (ifn) signaling. we found that vlps lead to accelerated induction of ifn stimulated genes (isgs) in liver and spleen of wild type mice, but not in ifnar(-/-) mice. accordingly, ebov infected ifnar(-/-) mice, unlike wild type mice succumbed to death even after vlp treatment. the isgs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. importantly, proinflammatory cytokine/chemokine expression was much higher in wt mice without vlps than mice treated with vlps. in ebov infected ifnar(-/-) mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of vlp treatment, supporting the view that type i ifn signaling helps to limit viral replication and attenuate inflammatory responses. further analyses showed that vlp protection requires the transcription factor, irf known to amplify type i ifn signaling in dendritic cells and macrophages, the probable sites of initial ebov infection. together, this study indicates that vlps afford post-exposure protection by promoting expeditious initiation of type i ifn signaling in the host. ebola viruses (ebovs) are enveloped, negative-sense rna filoviruses that can cause a severe hemorrhagic fever in humans and non-human primates (nhps) [ , ] . mouse-adapted ebov causes similar acute disease in mice, offering a useful animal model to study ebov infection [ , ] . ebov infection is characterized by rapid viral replication and dysregulated innate and adaptive immune responses. the disease follows profound suppression of type i ifn signaling and a contrasting excess inflammation that leads to mucosal hemorrhages and multi-organ failure resembling septic shock syndrome [ , ] . virally encoded anti-ifn proteins, vp and vp play major roles in ebov virulence [ , ] . vp blocks type i ifn induction in dendritic cells (dcs) and macrophages, and acts as a virulence factor necessary for a recombinant virus to attain infectivity in the host [ ] [ ] [ ] [ ] . vp , on the other hand, blocks ifn signaling by interfering with ifn activated jak/stat pathways [ ] . lines of evidence support the critical importance of type i ifn signaling in providing resistance against ebov infection; mice deficient in stat , a transcription factor required for ifn induction, or ifnar , encoding the membrane receptor for type i ifns, are susceptible to wild type zaire ebov, against which wild type mice are resistant [ ] [ ] [ ] . a study of sudan ebov infection in humans showed that ifnα levels are significantly higher in surviving patients than those with fatal ebov infection, who had higher levels of proinflammatory cytokines/chemokines such as il- , and mip- β [ , ] . high ifnα production is reported to correlate with increased resistance against ebov in mice as well [ ] . administration of recombinant ifnα or ifnβ confers delayed time-to-death in nhps [ , ] . furthermore, ifnα, used as an adjunct therapy for monoclonal antibody treatment, is shown to enhance protection in nhps [ ] . ebov infection remains a potential threat to public health, which is compounded with the lack of effective prevention or treatment. to overcome this problem, various vaccine candidates have been developed, including various dna constructs, recombinant viruses, vlps, as well as treatment with anti-sense sirna [ ] [ ] [ ] . vlps are subunit-based vaccines, extensively studied for a variety of infectious pathogens [ , ] . vlps prepared from ebov and other filoviruses are composed of the matrix protein (vp ), glycoprotein (gp), and at times nucleoprotein (np) and represent a potentially promising candidate for ebov vaccine. ebov vlps have been shown to confer protection upon rodents and nhps when given prior to infection [ ] [ ] [ ] . in the accompanying paper, we show that post-exposure administration of trivalent vlps protects mice from lethal ebov infection, further crediting the potential of vlps as a possible vaccine [ ] . in that study, we show that vlp protection requires macrophages, dendritic cells (dcs) as well as b and either cd or cd t lymphocytes, indicating that both innate and adaptive immunity are involved in conferring protection. the aim of this study was to further investigate molecular bases of postexposure protection by vlps. based on our previous report that vlps stimulate type i ifn expression in dcs and macrophages, in vitro, we focused on the role of type i ifn signaling, and found that post-exposure vlp treatment leads to accelerated activation of ifn signaling, resulting in early induction of isgs. significantly, vlp stimulated isg induction coincided with the attenuation of proinflammatory cytokine surge in ebov infected mice. the reduced inflammatory responses was attributed to activation of type i ifn signaling, since vlp treated ifnar -/mice were unable to inhibit not only viral replication but proinflammatory responses, and succumbed to death. our results indicate that early type i ifn response is a major mechanism that contributes to vlp mediated protection against ebov infection. ifnar -/-, ifnar +/+ mice of balb/c background and irf -/and irf +/+ mice of c bl/ background were bred in the nichd animal facility and transferred to the facility of the united states army medical research institute of infectious diseases (usamriid) for ebov infection studies. research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, . the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. the iacuc committee approving this protocol is the united states army medical research institute of infectious diseases (usamriid) iacuc. animals were monitored at least once daily and their status was evaluated according to an intervention score sheet approved by usamriid iacuc. monitoring increased to three times daily if the animals were given a score of three or four. euthanization was by co inhalation followed by confirmatory cervical dislocation. analgesics and anesthetics were not used in this study and animals were euthanized for humane purposes if they reached a score of five or more, which would be indicated if the animals exhibited ruffled fur, weakness, unresponsiveness, and/or difficulty walking. otherwise, animals were euthanized at the end of the study. vlps were composed of ebov gp, np and vp and were generated in mammalian t cells as reported previously [ ] . vlp preparations used in this study contained < . endotoxin u/mg. mice were infected with~ pfu (~ , ld ) of mouse-adapted ebov via intraperitoneal (i.p.) route [ ] . mice were injected with vlps ( μg) diluted in pbs through i.p. h after ebov infection. morbidity and mortality of ebov infected mice were monitored twice daily for up to days. total rna from liver and spleen of ebov infected mice were extracted by trizol method (invitrogen) and cdna was synthesized from μg total rna by superscript ii reverse transcriptase (invitrogen). qpcr amplification was done with ng cdna in μl sybr green pcr master mix (applied biosystems) with μm of both reverse and forward primers used in the abi prism sequence detection system (applied biosystems). mrna of expression of indicated genes were analyzed as described in detail elsewhere [ ] . the primer pairs were used for ebov gp, '-tgggctgaaaactgctacaatc- ' and '-ctttgtgcacataccggcac- '; nlrp , '-tgctcttcactgctatcaagccct- ' and '-acaagcctttgctccagaccctat- '. all other gene primer sequences were followed from the previous publications [ , ] . transcript levels were normalized with hprt, and expressed as relative expression. statistical analysis was carried out by excel software using two-tail paired student's t test. data represent the mean of at least three independent assay ± sem. a p value < . was considered significant. to assess the role of type i ifn signaling in vlp-mediated protection against ebov infection, we tested ifnar -/mice for protection by vlps. in fig. a , wild type (wt) and ifnar -/mice (both balb/c background, n = ) were injected with μg of vlps h after infection by the mouse adapted (ma) ebov, and the morbidity and mortality were checked daily for the subsequent days. wt mice without vlp injection all died between day and day , whereas % of mice that received vlps survived after ebov infection, confirming that vlps protect mice even when they were given post-infection. in contrast, ifnar -/mice that received vlps all died before or at day as those without vlp injection (fig. a) . these results are in agreement with previous report on early death of ma-ebov infected ifnar -/mice [ ] . ebovs are thought to initially infect dcs and macrophages in liver and spleen, making these tissues the major sites of ebov replication in the mouse, although the virus infects many other organs later [ , ] . to ascertain whether vlps inhibit viral replication, we measured ebov glycoprotein (gp) mrna expression in liver and spleen from wt and ifnar -/mice with or without vlp administration. qrt-pcr analysis in fig. b and c, found that levels of gp mrna rose sharply in ifnar -/mice on day of infection when gp mrna was still at background in wt mice. ifnar -/mice that received vlps also expressed considerable amounts of gp mrna, although levels varied between liver and spleen in the vlp treated group. thus, ebov appeared to replicate faster and to a greater extent in ifnar -/mice than wt mice. it should be noted here that in wt mice, gp mrna levels began to increase rapidly after day , peaking on day to day , and that vlp injection inhibited gp mrna expression by more than half (s fig.) . pfu ma ebov, followed h later by injection i.p. with μg of ebov vlps. one group of ifnar +/+ (wt) mice infected with ebov without vlp served as control. mortality is expressed as percent survival of each group on indicated days. results are a representative of three independent experiments, which gave very similar outcomes. qrt-pcr detection of ebov gp mrna level on day post-ebov infection with or without vlps from liver (b) and spleen (c) of wt or ifnar -/mice. gp transcripts were normalized by hprt and values represent the mean ± sem of duplicate samples from three independent experiments. asterisk denotes significant differences compared to wt controls (*p . , **p . ). these results are in line with the results that ifnar -/and stat -/mice are more susceptible to ebov infection, suggesting the possibility that vlp mediated protection is linked to the activation of type i ifn signaling [ ] [ ] [ ] . however, vlp injection may not have prevented ebov pathogenesis in ifnar -/mice, possibly because the disease manifests more severely in these mice than in wt mice. on the other hand, it has been recently shown that adenovirus based vaccine can protect ifnar -/mice from lethal evob infection presumably through antibody responses, which indicates that ifnar -/mice are not universally vulnerable, and anti-ebov resistance can be attained in some cases [ ] . we recently reported that ebov vlps activate type i ifn transcription in dcs and macrophages in vitro, leading to induction of many isgs in these cells [ ] . here we asked whether vlps stimulate isg induction in vivo. wt mice were infected with ma ebov and received vlps h later, and induction of isg mrnas was tested on days . and . isgs encoding anti-viral proteins were first examined, as they may provide early protection against ebov infection. upper panels in fig. a and b compare induction of anti-viral isgs, ifit , mx , oas a and stat with or without vlp injection in liver and spleen. in this early stage, levels of these isgs were consistently higher in the vlp-injected groups than those without vlps. at later stages of infection, however, the situation reversed, in that mice without vlps had higher levels of isgs, as seen on day (s fig. for complete kinetics) . these results indicate that vlp administration accelerated type i ifn and isg induction, which presumably provide early anti-viral activity, not afforded without vlps. we next tested whether vlps induce other isgs, particularly those with negative regulatory activities. this question was of interest to us, since mice that did not receive vlps expressed higher levels of proinflammatory cytokines and chemokines, which raised the possibility that ifn signaling exerts negative regulatory activity towards proinflammatory responses, perhaps by controlling nf-κb activation [ ] . shown in the lower panels in fig. a and b is induction of irgm , usp , trim and trim . irgm is an ifn inducible gtpase that inhibits lps induced endotoxin shock in mice [ ] . usp is an isg deconjugating factor that negatively regulates tlr signaling and resultant cytokine induction [ ] . trim and trim are members of the tripartite motif family that downregulate tlr induced inflammatory responses [ ] [ ] [ ] [ ] . expression of these isgs was also higher in the vlp injected group than that without vlps both in liver and spleen. similar to anti-viral isgs, expression of these negative regulatory factors changed at the later stage (s fig). these data indicate that vlps accelerate induction of anti-viral and negative regulatory isgs, which may help suppress ebov's anti-ifn antagonism (see discussion). to confirm that vlp induction of isg is dependent on type i ifn signaling, we next tested isg induction in ifnar -/mice. as expected, none of the isgs tested in fig. were induced in ifnar -/mice after vlp treatment or ebov infection (s fig). vlps lower expression of proinflammatory cytokines in ebov infected mice ebov pathophysiology such as severe hemorrhagic symptoms and tissue damage is thought to be associated with dysregulated inflammatory cytokine production [ , ] . given that vlps accelerated induction of negative regulatory isgs, we next evaluated whether vlps modulate expression of proinflammatory genes. in fig. , expression of tnfα, il- and il- β, chemokines such as mcp- (ccl ), mip- α (ccl ), mip- β (ccl ), kc (cxcl ) and inflammasome gene nlrp was measured in ebov infected mice with or without vlps. these genes were all strongly induced upon ebov infection and peaked on day with a gradual decline on days in all cases, their expression was significantly attenuated in the vlp-treated group as compared to the group without vlps. the difference was most dramatic in the early stage on day , where the expression was reduced at least by %. in agreement with these results, we noted that serum levels of some of these proinflammatory cytokines were higher in ebov infected mice that were treated with vlps as compared those without vlps [ ] . these results support the view that limiting superfluous inflammatory responses contribute to vlp mediated protection. ifnar -/mice increasing evidence indicates that type i ifns antagonize inflammatory responses in a variety of settings [ ] [ ] [ ] . in light of the results that vlps stimulate those isgs known to suppress proinflammatory responses, it was of importance to determine whether type i ifn signaling by and of itself affects ebov induction of proinflammatory cytokines and chemokines. results in fig. and s fig compare expression of the above proinflammatory factors in ifnar +/+ and ifnar -/mice infected with ebov. all cytokines and chemokines tested were induced after ebov infection in both strains. importantly, their levels were much higher in ifnar -/mice than ifnar +/+ mice. these results indicate that type i ifn signaling downregulates ebov stimulated induction of proinflammatory factors, possibly through isgs with negative regulatory activities. the above results indicated that type i ifns attenuate proinflammatory responses during ebov infection. to explore whether type i ifns have a similar activity in settings other than ebov infection, we next tested lps and ifnβ induced inflammatory responses in macrophages in vitro. lps activates nf-κb mediated proinflammatory cytokine induction, which can result in endotoxin shock [ ] . as shown in fig. , combined treatment with lps and ifnβ led to hyper induction of tnfα, il- , il- β and a chemokine kc in ifnar -/macrophages as compared to wt cells. lps and ifnβ also induced negative feedback factors, trim and trim , with much lower expression in ifnar -/cells than ifnar +/+ cells. these results support a model in which type i ifns negatively regulate proinflammatory cytokine/chemokine responses at least in some situations. we found that mip- α, mip- β and mcp- were not hyperinduced in ifnar -/cells, suggesting that some proinflammatory genes are regulated not only by type i ifns but other factors (data not shown). alternatively, these differences may reflect variances between in vivo and in vitro conditions. to further define pathways downstream of ifnar activity, important for vlp protection, we directed our attention on irf , a transcription factor expressed in macrophages and dcs [ ] [ ] [ ] . irf is induced by ifns and tlr ligands in a stat dependent manner, and plays a pivotal role in facilitating innate immune responses. although irf is not involved in initial triggering of type i ifn induction, it amplifies ifn transcription in dcs and macrophages [ ] . irf promotes induction of multiple anti-microbial factors and is required for innate resistance against a variety of pathogens [ ] [ ] [ ] . irf stimulates expression of mhc and costimulatory molecules to boost antigen presentation [ , ] . we thus tested whether irf disruption affects vlp-mediated protection against ebov. survival data in fig. a show that approximately % of irf -/mice that received vlps died between day and , which is nearly identical to the mortality curve of wt mice without vlps. as expected, the majority of wt mice that received vlps survived against ebov infection. it is of note that ifnar -/mice died to days earlier than irf -/mice, which may be attributed to the difference in the mouse background. correlating with the lack of protection, ebola gp mrna levels were much higher role of type i ifn in vlp-mediated protection against ebov infection in ebov infected irf -/mice than irf +/+ mice with or without vlps (fig. b) . we next examined whether induction of anti-viral and negative feedback isgs is dependent on irf . data in fig. c illustrate that induction of these isgs was very meager in irf -/mice, in contrast to robust induction in irf +/+ mice. importantly, vlps did not rescue isg induction in irf -/mice. results were similar in liver and spleens ( fig. c and s fig). these results indicate that vlps, upon initial activation of type i ifn cascade, rely subsequently on the activation of downstream pathways represented by irf to confer protection against ebov. to gain insight into the pathways through which vlps confer resistance against ebov infection, we investigated the role of type i ifn signaling in vivo and found that it significantly contributes to vlp-mediated protection. this conclusion is supported by the observation that post-exposure vlp treatment accelerated isg induction in ebov infected mice, leading to reduced viral replication and inflammatory gene expression. further supporting the critical role of type i ifn signaling in the protection, vlps did not induce isgs in ifnar -/mice, and did not protect the mice from lethal ebov infection. these results are consistent with the report that post-exposure ifnβ or ifnα treatment increases protection against ebov infection in nhps [ , , , ] . it is likely that vlps initially stimulated type i ifn genes, which in turn led to early induction of isgs. in line with this notion, we recently showed that exogenous vlps stimulate transcription of ifnα and ifnβ in dcs and macrophages in vitro, an event coupled with immediate and robust isg induction [ ] . it may be reasonable to assume that ifnar -/mice were not protected by vlps primarily because isg induction was absent. however, ifnar -/mice may be susceptible to infection due to additional defects in innate immunity that are a secondary consequence of defective ifn signaling, which obliterates vlps protection. contouring this notion however, it is of note that ifnar -/mice can be protected against ebov by an adenovirus-based vaccine, indicating that ifnar -/mice are not totally without defense [ ] . rather, it is possible that ifnar -/mice are not protected by vlps that rely on isg induction for protection, whereas they are protected by the adenovirus vaccine that depends on antibody response. vlp-induced isgs included anti-viral proteins known to inhibit replication of rna viruses such as ifit , mx and oas a, as well as negative feedback factors that curb excess inflammatory responses, such as irgm , usp , trim and trim . although the question of which antiviral isgs are effective in inhibiting ebov replication awaits further research, it is anticipated that some of anti-viral isgs induced by vlps may interfere with ebov life cycle [ ] . what is the significance of accelerated ifn response in vlp mediated protection? available evidence suggests that vlps may overcome ebov's anti-ifn antagonism. the virally encoded vp and vp disable the entire ifn system in the host; while vp blocks the jak/stat pathway of ifn signaling, vp , an ebov virulence factor, inhibits type i ifn induction in many cell types [ , , , ] . we previously showed that vp inhibits type i ifn induction in murine dcs by premature sumoylation and inactivation of irf [ ] . it is thought that vp and vp have a decisive effect on the subsequent host resistance, since abated ifn signaling would impair proper innate immune responses, leading to deficiency in dc maturation, defective antigen presentation and aberrant inflammation. compromised innate immunity would consequently undermine development of adaptive immunity [ ] (see a model in fig. ) . it is remarkable that in the vlp treated mice, isg induction began early within . to days after ebov infection (which was only . to days after vlp treatment), when little to no isg induction was seen in mice without vlps. the delayed isg induction in ebov infected mice is reminiscent of the reports showing that influenza virus delays isg induction in lung epithelial cells through ns , an influenza anti-ifn protein that is linked to disease pathology [ , ] . an influenza virus strain deficient in ns is shown to induce isgs earlier than wild type virus, although the wild type strain does stimulate isgs later on [ , ] . supporting the view that viral anti-ifn factors stall isg induction, rather than completely abrogate the induction, we also observed isg induction on day and later in mice without vlps. it may be envisaged that vlps trigger ifn activation early on, thereby eluding the activity of the ebov anti-ifn proteins (a model in fig. ) . the most striking observation made in this study is the vlp-dependent suppression of proinflammatory responses. this suppression was a result of type i ifn signaling, as ifnar -/mice expressed higher levels of proinflammatory cytokines and chemokines, observed not only after ebov infection but also by ifnβ and lps stimulation. these results are in accordance with the growing recognition that type i ifns are linked to attenuation of inflammatory responses [ , ] . for example, pinto et al., reported, in the west nile virus infection model that ifnar -/mice express excess proinflammatory cytokines, including those found in this study, as compared to wt mice, which correlated with increased disease pathology. in this system, the overt inflammatory responses were attributed to ifn signaling in macrophages and dcs [ ] . induction of proinflammatory cytokines and chemokines may be negatively regulated by ifn signaling through a series of negative feedback factors [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . irgm , induced by ifn signaling restricts lps induced endotoxin shock without limiting ifnβ expression [ ] . trim and trim inhibit proinflammatory cytokine induction, at least in part by interfering with the nf-κb dependent arm of transcription [ ] [ ] [ ] . in addition, these factors may act by post-transcriptional mechanisms, affecting inflammasome activation [ ] . in this regard, guarda et al. [ ] reported that type i ifns inhibit production of il- by inhibiting activity of the nlrp and nlrp inflammasomes and by il- induction. thus, isgs with negative regulatory activity may preferentially attenuate proinflammatory pathways, while sparing ifn induction pathways. given our earlier observations that vp does not grossly affect nf-κb activation, while strongly inhibiting type i ifn activation, ebov may promote proinflammatory pathways at least in part through vp [ ] . lastly, we show that the transcription factor irf is required for vlp mediated post-exposure protection. our results offer an added mechanistic insight into the pathways through which vlps provide protection. irf is expressed predominantly in macrophages and dcs, and helps to amplify type i ifn gene induction and boosts ifns biological activities [ ] . given that macrophages and dcs are the putative early sites of ebov infection, vlps may exert a major impact on these cells to facilitate early innate immunity, in an irf dependent manner. in conclusion, vlps confer post-exposure protection upon ebov infected mice by rapidly inducing isgs, thereby permitting timely establishment of anti-viral and anti-inflammatory states in the host. vlps may act primarily by relieving ebov's antagonism against type i ifns, resulting in reduced systemic inflammation and subsequent enhancement in acquired immune responses (a model in fig. ). pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells a compendium of years of epidemiological, clinical, and laboratory studies mouse models for filovirus infections a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete mip- alpha and tnf-alpha and inhibit poly-ic-induced ifn-alpha in vitro how ebola and marburg viruses battle the immune 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signalling and immune regulation the content of this publication does not necessarily reflect the views or policies of the us department of defense or the united states army medical institute of infectious diseases. we thank members of pgd for in depth discussions and critical reading of the manuscript. we also thank ms. monica gupta for breeding ifnar -/mice for this study. key: cord- -ror z h authors: liu, xiaoli; zhang, hua; su, lijie; yang, peng; xin, zhiqiang; zou, junwei; ren, shuangyi; zuo, yunfei title: low expression of dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-related protein in lung cancer and significant correlations with brain metastasis and natural killer cells date: - - journal: mol cell biochem doi: . /s - - - sha: doc_id: cord_uid: ror z h dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin-related protein (dc-signr) is a type ii transmembrane protein which has been reported to bind a variety of pathogens as well as participate in immunoregulation. but the association between the level of dc-signr and lung cancer is unknown. to investigate the clinical diagnostic significance of dc-signr in lung cancer, we investigated serum dc-signr levels in lung cancer patients and healthy individuals using enzyme-linked immunosorbent assay (elisa). results showed that serum dc-signr levels in lung cancer patients were lower than that in healthy controls (p = . ). a cut-off value of . ng/l for dc-signr predicted the presence of lung cancer with . % sensitivity and . % specificity (area under the curve = . , p = . ). strikingly, serum dc-signr levels were significantly higher in lung cancer patients with brain metastasis compared to those without metastasis (p = . ). moreover, the serum concentrations of dc-signr in lung cancer patients also correlated significantly with serum natural killer cells percentage (p = . ). in addition, immunohistochemistry assay demonstrated that the expression of dc-signr in lung tissues of lung cancer patients and tuberculosis patients was significantly lower than that in normal lung tissues (p = . , . ), and there is no significant difference between tuberculosis tissues and lung cancer tissues (p = . ). these results suggest that dc-signr maybe a promising biological molecule that has the potential for clinical research of lung cancer, whereas its underlying roles are needed to be investigated in further studies. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. lung cancer is the leading cause of cancer death worldwide among men and the second leading cancer site in women [ ] . the high mortality rate of this disease is primarily due to the difficulty of early diagnosis, the high metastatic potential, and the poor responses to chemical therapy and radiotherapy [ ] . unfortunately, the unclear etiology of lung cancer presents a huge challenge for the treatment of lung cancer. many groups' results showed that smoking is the most important modifiable risk factor for lung cancer [ ] [ ] [ ] . otherwise, lung cancer susceptibility is also determined by host genetic factors [ , ] . infection may play a role in lung cancer, including human papilloma virus (ppv), epstein-barr virus, and human immunodeficiency virus (hiv) [ ] [ ] [ ] . in addition, there are some biological molecules correlated with the risk of lung cancer such as p-selectin [ ] and chondrolectin (chodl) [ ] . other known risk factors for lung cancer include exposure to electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. several occupational and environmental carcinogens such as asbestos, arsenic, and polycyclic aromatic hydrocarbons [ ] . recent studies from several groups suggest that lectins may play an important role in a variety of physiological and pathological processes, typically with tumor progression and metastasis. for example, läubli and borsig [ ] reported that selectins were upregulated the pulmonary metastatic microenvironment is facilitated by p or l-selectin mediated interactions between tumor cells and blood components, which can significantly reduced metastasis. other groups results also showed that high mannose-binding lectin (mbl) levels were associated with poorer lung cancer survival [ ] , and low mbl levels increased gastric cancer risk [ ] . additionally, liver sinusoidal endothelial cell lectin (lsectin) can adhere to colorectal cells and plays an important role in colorectal carcinoma liver metastasis [ ] . recently, our laboratory has been reported that dc-sign and dc-signr are low expressions in patients with non-hodgkin's lymphoma (nhl) and could be potentially useful in nhl clinical settings [ , ] . dc-sign and dc-signr are novel blood-based molecular markers which can potentially be used for the diagnosis of early stage of colon cancer patients [ ] . therefore, we are interested in the expression of dc-signr in lung cancer patients. dc-signr, a closest homologues to the c-type lectin superfamily members dc-sign, lsectin, and cd , which consists of an n-terminal cytoplasmic tail, a transmembrane domain, an extracellular c-terminal neck region, and a c-type carbohydrate recognition domain (crd) [ , ] . the crd domain of dc-signr can interact with high mannose carbohydrates, and the neck domains that support the crd may influence the ligand-binding properties of these lectins, and the intra-cellular domain located in the cytoplasmic tail containing recycling and internalization motifs [ ] . dc-signr is highly expressed on liver sinusoidal endothelial cells, lymph node endothelial cells, and a significant proportion in terms of placenta capillary endothelial cells but was not expressed in peripheral bloodderived dcs [ ] . furthermore, dc-signr is also expressed in the lung in both cytokeratin positive alveolar epithelia, as well as a subset of cells that co-expressed angiotensin converting enzyme- (ace ) but were negative for cytokeratin [ ] . like dc-sign, dc-signr can recognize and bind several pathogens, including viruses, bacteria, fungi, and parasites [ ] . dc-signr also recognizes endogenous ligands, such as icam molecules [ ] , although their glycan epitopes have not been fully characterized. however, the role of dc-signr in lung cancer was not been elucidated. in our research, we compared the serum levels of dc-signr between lung cancer patients and healthy volunteers using an elisa. we found that lung cancer patients showed lower levels of dc-signr compared with healthy controls and that there were correlations between serum levels of dc-signr and clinical parameters of lung cancer patients. in immunohistochemistry (ihc) study, the dc-signr is lower expression in lung tissues from lung cancer patients compared to those in normal lung tissues. serum samples of lung cancer patients were obtained from the first and second affiliated hospital of dalian medical university during march -june and were stored at - °c until they were analyzed. the mean age of the patients was years (range, - years), were male and were female. according to the world health organization (who) classification, the lung cancer cases consisted of small cell lung cancer (sclc) and non-small cell lung cancer (nsclc), the nsclc is further divided into three major pathologic subtypes, including adenocarcinoma (adc), squamous cell carcinoma (scc), and large cell carcinoma [ ] . clinical staging of lung cancer patients was performed according to the iaslc lung cancer staging [ ] . clinical data including gender, age, cancer stage, histological subtype, concentrations of tumor markers, and the percentage of t cell subsets were acquired from hospitalization records. the basic characteristics of these subjects were shown in table and supplementary table . the control group was composed of healthy blood donor volunteers. among the healthy subjects, the mean age was years (range, - years). each healthy control performed routine physical examinations, and the results were within the normal range. lung cancer patients and healthy individuals with various pathogen infections and poor performance status were excluded in present study. in addition, formalin-fixed and paraffin-embedded tissues of lung cancer patients, lung tissues from tuberculosis patients, normal lung tissues, and lymph nodes from to were obtained from the second affiliated hospital of dalian medical university. lung tissues from tuberculosis patients and normal lung tissues were used as control groups, and normal lymph nodes were used as either positive or negative controls. the clinical data for these subjects are summarized in table and supplementary table . we used standard sandwich elisa to detect the levels of dc-signr in human serum. -well microplates were coated with ll anti-dc-signr goat polyclonal antibody (pab, santa cruz biotechnology, inc., usa, catalog number: sc- ) at a final concentration of . lg/ml and incubated overnight at °c. washed the plates times with phosphate buffered saline (pbs) containing . % tween- (pbst) (ph . ), and the wells were blocked with blocking buffer ( % bovine serum albumin) at °c for h. the plates were washed three times with pbst. and then, ll serum from lung cancer patients and healthy controls was added to the wells. as a negative control, ll pbs was used. each plate was incubated at °c for h. subsequently, pbst was used to wash the plates times, and ll anti-dc-signr mouse monoclonal antibody (mab, r&d systems, inc., usa, catalog number: mab ) diluted to a concentration of . lg/ml was added to each wells. the plates were incubated at °c for . h. after washing, ll peroxidase-conjugated goat anti-mouse (zsgb-bio) was added and the plates were incubated at °c for h. finally, the plates were washed times and incubated with , , , -tetramethylbenzidine (tmb, tiangen bio-tech co, ltd.) at °c for . h, and then mol/l h so was added to stop the reaction. the optical density (od) was measured at the nm wavelength on a microplate reader. the quantitative dc-signr concentrations were determined by comparing the optical density values with the standard curve. the s b levels in lung cancer patients with brain metastasis were detected by elisa kit (westang bio-tech, ltd). the patients who have other diseases which can affect s b levels were excluded in this study. the concentrations of s b were shown in supplementarytable . before being deparaffinized in xylene and rehydrated in a graded ethanol series, sections from paraffin-embedded blocks were incubated at °c for min. sections were washed with pbs, and then endogenous peroxidase activity was blocked with % hydrogen peroxide diluted in double-distilled water for min at room temperature. antigen retrieval was performed by heating slides in citrate buffer ( . mol/l citric acid, ph . ) for min in a microwave oven. after being washed with pbs, the sections were incubated with goat serum for the purpose of blocking. sections were incubated with anti-dc-signr rabbit pab (abcam, inc.) overnight at °c ( : ). the next day, after being washed with pbs, the sections were incubated with horseradish peroxidase-labeled anti-rabbit immunoglobulin ( : ) (zsgb-bio) at °c for h and were then washed again. finally, all the sections were developed with , -diaminobenzidine tetrahydrochloride (dab,zsgb-bio) within the cells and then counterstained with hematoxylin. photographs were recorded on an olympus bx microscope. the mean density of dc-signr expression was assessed using the image-pro plus version . (media cybernetics, inc. silver spring, md usa). integrated optical density (iod) sum represents the protein content of dc-signr in the area of interest (aoi), while area equals the area of aoi. (iod sum)/area stands for ''mean density''. briefly, images were captured at magnification from aois/case, which were selected based on areas with maximal dc-signr staining. the mean density values are shown in supplementary table . this quantitation was positively correlated with dc-signr expression in tissue. non-parametric mann-whitney u test was used to determine the statistical significance among two groups. the statistical significance among more than two groups was determined with the kruskal-wallis non-parametric test. correlation of dc-signr values with clinical parameters was tested by the non-parametric spearman's rank correlation coefficient test. in all tests, -sided p values below . were considered significant. all statistical analyses were performed using graphpad prism (graphpad software, inc., san diego, ca, usa). we detected serum dc-signr levels in participants, composing of with lung cancer and healthy controls by elisa. the dc-signr level in the serum of patients with lung cancer ( . ± . mg/ml) was significantly lower than that in healthy controls ( . ± . mg/ml) (p = . ; fig. a) . a roc curve is often used to assess the power of a novel serum marker for tumor prediction. in the present study, a cut-off value of . ng/l for dc-signr predicted the presence of lung cancer with . % sensitivity and . % specificity, and the area under curve (auc) was . (p = . ) (fig. b) . therefore, serum dc-signr levels demonstrated low accuracy for the detection of lung cancer. serum concentrations of dc-signr correlated significantly with lung cancer patients who have brain metastasis we assessed the correlations between serum levels of dc-signr and clinical data, including gender, age, cancer stage, histologic subtype, and metastasis to different organs. strikingly, serum concentrations of dc-signr of lung cancer patients with brain metastasis were higher than that without metastasis (p = . ; fig. b ). however, the levels of dc-signr in patients with lung cancer showed no significant difference between metastasis and non-metastasis (p = . ; fig. a) . furthermore, the serum dc-signr levels were not significantly different between bone metastasis and non-metastasis (p = . ; fig. b ), lung metastasis and non-metastasis (p = . ; fig. b ), lymph node metastasis and non-metastasis (p = . ; fig. b ). otherwise, we found that the serum levels of dc-signr correlated significantly with serum nk cells percentage with the spearman's correlation coefficient of - . (p = . ; fig. a ). however, there was no significant correlation between serum dc-signr and cd , with a spearman's correlation coefficient of . (p = . ; fig. b ). moreover, serum levels of dc-signr showed no association with cd with a spearman's correlation coefficient of . (p = . ; fig. c ). in addition, serum levels of dc-signr displayed no correlation with cd with a spearman's correlation coefficient of . (p = . ; fig. d ). no significant correlations were observed between serum levels of dc-signr and clinical parameters, such as age, gender, cancer stage, and histologic subtype contrary to above results, serum levels of dc-signr in patients with lung cancer showed no significant difference between male and female (p = . ; fig. a ). serum levels of dc-signr also showed no significant difference between patients of b years old and patients [ years old (p = . ; fig. b ). serum levels of dc-signr expressed no association with age with a spearman's correlation coefficient of . (p = . ; fig. c ). furthermore, the serum dc-signr levels were not significantly different among the stage i-ii, and stage iii-iv (p = . ; fig. d ). additionally, for different histologic subtype, no significant difference was observed between lung cancer patients with sclc and those with nsclc (p = . ; fig. e) . similarly, the difference of serum dc-signr levels was not apparent between sclc, adc and scc (p = . ; fig. f ). serum dc-signr levels showed no significant correlation with s b, carcinoembryonic antigen (cea), neuron-specific enolase (nse), and cyfra - in our study, the levels of dc-signr in lung cancer patients with brain metastasis is higher than that without metastasis. s b as a marker of brain metastasis, so we compare the correlation between the two molecular. however, there is no significant correlation between dc-signr and s b, with a spearman's correlation coefficient of . (p = . ; fig. a ). in present study, serum levels of dc-signr expressed no association with cea, with a spearman's correlation coefficient of . (p = . ; fig. b) . similarly, serum levels of dc-signr were not significantly correlated with nse, with a spearman's correlation coefficient of - . (p = . ; fig. c ). furthermore, the data also showed no correlation between serum dc-signr and cyfra - levels, with a spearman's correlation coefficient of . (p = . ; fig. d ). the expression of dc-signr in lung cancer patients and tuberculosis patients was significantly lower than that in normal lung tissues we determined the expression level of dc-signr in serum. moreover, it has been reported that dc-signr is fig. serum dc-signr levels were significantly correlated with brain metastasis. the serum levels of dc-signr were not significantly different between metastasis and non-metastasis (p = . ) (a). serum concentrations of dc-signr were higher in brain metastasis than in non-metastasis (p = . ) (b). however, the serum dc-signr levels were not significantly different between bone metastasis and non-metastasis (p = . ), lung metastasis and non-metastasis (p = . ), lymph node metastasis and nonmetastasis (p = . ) (b) mol cell biochem ( ) : - expressed in the lung in both cytokeratin positive alveolar epithelia, as well as a subset of cells that co-expressed ace [ ] . therefore we want to know whether the dc-signr expression in lung cancer tissues. we performed ihc analysis, using anti-dc-signr pab in lung cancer tissues ( sclc, adc, and scc) and lung tissues of tuberculosis patients, and normal lung tissues were used as controls. strikingly, the mean density of dc-signr expression in the lung cancer patients and tuberculosis patients was significantly lower than that in normal (figs. , ) . the normal lymph nodes of tumor patients were used as positive controls and negative controls. the expression of dc-signr mainly occurs in infective diseases, such as acquired immune deficiency syndrome (aids) with hiv infection, hepatitis c infection disease, and ebola virus infection disease [ ] . recently, our laboratory reported that dc-signr expression is lower in patients with nhl than in healthy individuals and higher expression in colon cancer patient serum [ , ] . to extend the knowledge of the possible clinical applications of dc-signr, we studied the serum levels of dc-signr in patients with lung cancer, and we also studied the expression and location levels of dc-signr in lung cancer tissues. our study demonstrated that serum levels of dc-signr in lung cancer patients were significantly lower than those in healthy individuals. furthermore, we found that the serum dc-signr levels correlated significantly with brain metastasis and serum nk cells percentage in lung cancer patients. brain metastasis as one of the most dreaded complications of lung cancer has a poor prognosis [ ] . up to % of lung cancer patients develop brain metastasis during their course of disease [ ] . the occurrence of brain metastasis is associated with poor prognosis and high morbidity in patients with advanced lung cancer, even after intensive multimodal therapy [ ] . furthermore, little is known about the pathobiology and the molecular mechanisms involved in the brain metastatic cascade which limits the possibilities for focused drug development and clinical trial [ ] . s b and proapolipoprotein a as a serum marker of brain metastasis in lung cancer have been investigated in a recent study [ ] [ ] [ ] . in our study, the dc-signr levels in lung cancer patients with brain metastasis are significantly higher than those without metastasis. so, we compared the correlation between serum dc-signr and s b levels. however, there is no significant correlation between them. s b which is a protein found in astrocytes and only released into serum when the blood-brain barrier is breached [ ] . the expression of dc-signr in brain tissues is still unknown. thus, we guessed that the two molecular may have a difference mechanism to affect the brain metastasis of lung cancer and it can be investigated in further study. we also compared the serum levels of dc-signr in bone, lung, and lymph nodes metastasis with non-metastasis, respectively. but we found no significant difference between them. natural killer (nk) cells, as a major component of the innate immune system, can limit the growth and dissemination of several types of tumors [ ] . unlike t cells and b cells, nk cells can directly exert cellular cytotoxicity on tumor cells without prior sensitization and secrete immunostimulatory cytokines, which control both local tumor growth and metastasis [ ] . an epidemiologic survey showed that low nk cell activity is associated with increased cancer risk [ ] . numerous studies have demonstrated that the infiltration of nk cells appears to have prognostic value in gastric carcinoma [ ] , colorectal carcinoma [ ] , and lung carcinomas [ , ] , as a relatively higher level of nk cell infiltrate correlates with a better prognosis, thus suggesting relevant protective roles for nk cell infiltrate. in the present study, we detected a significantly negative correlation between serum levels of dc-signr and serum percentage of nk cells in lung cancer patients. the molecular mechanism for this negative correlation is not yet known. on the contrary, we found no significant correlations between serum levels of dc-signr and serum percentage of cd , cd , and cd in the present study. in order to know the dc-signr whether it has the ability to distinguish the benign and malignant diseases of the lung, we choose tuberculosis (tb) patients, which results in mycobacterium tuberculosis infections as a benign disease group. mycobacterium tuberculosis can targets dc-sign to inhibit the immuno-stimulatory function of dc through the interaction of the mycobacterial mannosylated lipoarabinomannan (manlam) to dc-sign [ ] . like dc-sign, dc-signr also can capture man-lam and rapidly internalizes it to lysosomes, and may be involved in the clearance of mycobacteria [ ] . tailleux l et al. [ ] showed dc-sign present in the alveolar cd b ? mus in the lungs of patients with tb. in present study, we found the dc-signr expression in lungs of patients with tb. however, there was no significantly difference between the dc-signr expression in lung cancer tissue and tuberculosis. strikingly, both of them are lower than those in normal lung tissues. in summary, we have shown that the serum level of dc-signr is a promising marker to help distinguish lung cancer patients from healthy individuals. additional studies are warranted to assess the potential value of dc-signr in vivo. these studies will most likely make dc-signr a useful biological molecule for clinical research on lung cancer mechanisms in the future. compliance with ethical standards the study was approved by the research ethics committee of dalian medical university, and fig. ihc for dc-signr expression in tissues of lung cancer tissues, tuberculosis patients, and normal tissues. ihc analysis was used to determine dc-signr expression and arrows indicate positive staining. areas in the black boxes of a, c, e, g, and i were enlarged below. dc-signr expression was detected in lung tissues from tuberculosis patients (a-b). dc-signr expression was detected in the lung tissues from lung cancer patients (c-d). dc-signr expression in alveolar epithelia cells of normal lung tissues (e-f). dc-signr expression in lymphatic endothelial cells exhibited strong positive (g-h). normal lymph node tissues were used as a negative control (i-j) fig. semi-quantitative image analysis of dc-signr expression in tissues. the graph displays a scatter plot of the levels of dc-signr expression in lung tissues from lung cancer patients, tuberculosis patients, and normal controls. there was a statistical significance in ihc for dc-signr expression between lung cancer tissues and normal lung tissues (p = . ), tuberculosis tissues and normal controls (p = . ). there was no significantly difference between lung cancer patients 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peripheral-blood lymphocytes and cancer incidence: an -year follow-up study of a general population prognostic value of intratumoral natural killer cells in gastric carcinoma the prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma prognostic significance of tumor infiltrating natural killer cells subset cd in patients with squamous cell lung cancer the prognostic value of natural killer cell infiltration in resected pulmonary adenocarcinoma identification of the mycobacterial carbohydrate structure that binds the c-type lectins dc-sign, l-sign and signr dc-sign induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis acknowledgments this study was supported by grants from the chinese national natural science foundation projects ( , , ) and science and technology planning project of liaoning province, china ( ). the authors declare that they have no conflict of interest. key: cord- - wv cb authors: matassov, demetrius; marzi, andrea; latham, terri; xu, rong; ota-setlik, ayuko; feldmann, friederike; geisbert, joan b.; mire, chad e.; hamm, stefan; nowak, becky; egan, michael a.; geisbert, thomas w.; eldridge, john h.; feldmann, heinz; clarke, david k. title: vaccination with a highly attenuated recombinant vesicular stomatitis virus vector protects against challenge with a lethal dose of ebola virus date: - - journal: journal of infectious diseases doi: . /infdis/jiv sha: doc_id: cord_uid: wv cb previously, recombinant vesicular stomatitis virus (rvsv) pseudotypes expressing ebolavirus glycoproteins (gps) in place of the vsv g protein demonstrated protection of nonhuman primates from lethal homologous ebolavirus challenge. those pseudotype vectors contained no additional attenuating mutations in the rvsv genome. here we describe rvsv vectors containing a full complement of vsv genes and expressing the ebola virus (ebov) gp from an additional transcription unit. these rvsv vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rvsv/human immunodeficiency virus type vaccine. one of these rvsv vectors (n ct -ebovgp ), which expresses membrane-anchored ebov gp from the first position in the genome (gp ), elicited a balanced cellular and humoral gp-specific immune response in mice. guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal ebov challenge, while control animals died in – days. subsequently, n ct -ebovgp demonstrated complete, single-dose protection of macaques following lethal ebov challenge. a single sham-vaccinated macaque died from disease due to ebov infection. these results demonstrate that highly attenuated rvsv vectors expressing ebov gp may provide safer alternatives to current ebov vaccines. previously, recombinant vesicular stomatitis virus (rvsv) pseudotypes expressing ebolavirus glycoproteins (gps) in place of the vsv g protein demonstrated protection of nonhuman primates from lethal homologous ebolavirus challenge. those pseudotype vectors contained no additional attenuating mutations in the rvsv genome. here we describe rvsv vectors containing a full complement of vsv genes and expressing the ebola virus (ebov) gp from an additional transcription unit. these rvsv vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rvsv/human immunodeficiency virus type vaccine. one of these rvsv vectors (n ct -ebovgp ), which expresses membrane-anchored ebov gp from the first position in the genome (gp ), elicited a balanced cellular and humoral gp-specific immune response in mice. guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal ebov challenge, while control animals died in - days. subsequently, n ct -ebovgp demonstrated complete, single-dose protection of macaques following lethal ebov challenge. a single sham-vaccinated macaque died from disease due to ebov infection. these results demonstrate that highly attenuated rvsv vectors expressing ebov gp may provide safer alternatives to current ebov vaccines. keywords. attenuation; rvsv vector; ebola vaccine; glycoprotein; challenge; nonhuman primates. the genus ebolavirus is classified within the filoviridae family and comprises closely related but antigenically distinct virus species: bundibugyo ebolavirus, reston ebolavirus, sudan ebolavirus, tai forest ebolavirus, and zaire ebolavirus [ , ] . in nature, all but reston virus have been isolated only in sub-saharan africa, where the likely virus reservoir is thought to be in bats [ , ] . all but the tai forest and reston viruses have caused sporadic, deadly outbreaks of disease in humans since the discovery of ebolavirus years ago, with case-fatality rates of %- % [ , ] . the ongoing ebola virus (ebov) outbreak in west africa is the largest recorded, with case-fatality rates of around %. hospitalization of patients seemingly improves clinical outcome and public health initiatives may slow the spread of disease, but mass vaccination programs in ebov-endemic regions will likely be required to extinguish the current outbreak and prevent such occurrences in the future. the ebov genome is composed of a single strand of negative-sense rna, approximately kb in length, encoding major viral polypeptides. one of these proteins is the ebov glycoprotein (ebovgp), which mediates virion attachment and fusion to susceptible cells [ ] . the ebovgp also contains the major virus neutralization epitopes [ , ] ; accordingly, the ebovgp has been the target for a variety of ebov vaccine designs. for a thorough review of vaccine approaches see falzarano et al [ ] . the vector in one such vaccine is recombinant vesicular stomatitis virus (rvsv), in which the gene encoding vsv g has been replaced with the gene encoding ebovgp [ , ] . this rvsv pseudotype replicates efficiently in cell culture and also propagates in vivo. vsv is a member of the rhabdoviridae family and, similar to ebov, has a single-strand, negative-sense rna genome, approximately kb long, encoding major viral proteins. in nature, vsv cycles between biting insects, which are the likely reservoir, and livestock [ ] [ ] [ ] . the virus replicates to high titer in virus-induced vesicles at insect bite sites on the nose, lips, teats, and coronary bands of hooves. disease in livestock is self-limiting and typically resolves in - days without significant consequences. vsv can also infect humans who have close contact with infected animals or purified virus [ , ] . infection results in mild influenza-like symptoms, which typically resolve in - days without complications [ , ] . the very low seroprevalence of vsv in humans favored development of vsv as a vaccine vector and became feasible when rose et al developed a system for recovery of rvsv from genomic complementary dna (cdna) [ ] . since then, rvsv vectors expressing antigens from human pathogens have demonstrated high levels of efficacy in the respective animal disease models [ ] [ ] [ ] [ ] . vsv exhibits a pronounced ′ to ′ gradient of gene expression due to discontinuous messenger rna (mrna) transcription across intergenic regions [ , ] , allowing modulation of virus protein or foreign antigen expression as a function of gene distance from the ′ transcription promoter [ , ] . a highly attenuated rvsv vector expressing human immunodeficiency virus type (hiv) gag from the first position in the genome was developed and has been demonstrated to be safe in stringent mouse and nonhuman primate (nhp) neurovirulence and biodistribution studies [ ] [ ] [ ] . this rvsv vector was attenuated by combining n gene translocation from position - (n ) in the genome and truncation of the g protein cytoplasmic tail (ct) from amino acids (aa) in native form to aa (ct ) [ , ] . this n ct -hivgag vector was subsequently shown to be safe and immunogenic in phase clinical trials (hvtn and hvtn ; available at: http://clinicaltrials. gov/), and after vaccination viremia was undetectable by an vsv infectivity assay or a vsv-specific polymerase chain reaction assay in blood, urine, or saliva specimens obtained from trial participants (unpublished data). here we describe the generation of attenuated rvsv vectors expressing ebovgp and their immunogenicity in mice. the vector that elicited the most balanced ebovgp-specific humoral and cellular immune response in mice was then evaluated for protective efficacy in guinea pig and nhp challenge studies. for murine studies, - -week-old female balb/c mice were used. mice were maintained according to the guide for the care and use of laboratory animals [ ] . in addition, procedures for the use and care of the mice were approved by profectus biosciences and new york medical college institutional animal care and use committees (iacucs). the guinea pig and macaque studies were carried out in strict accordance with the recommendations described in the guide for the care and use of laboratory animals of the national institutes of health, the office of animal welfare, and the us department of agriculture. all animal work was approved by the national institute of allergy and infectious diseases division of intramural research iacuc at the rocky mountain laboratories (rml). the facility is accredited by the american association for accreditation of laboratory animal care. all procedures were performed on animals after they were anesthetized by trained personnel under the supervision of veterinary staff. all efforts were made to minimize the pain and suffering of animals, with early end point criteria specified by the rml iacuc-approved score parameters used to determine when animals should be humanely euthanized. as described previously [ , ] , the n ct -hivgag vector was used as template for generating all rvsv/ebov vectors expressing ebovgp. the n ct -hivgag genomic cdna was modified by exchanging the gag gene located in position of the genome with the ebovgp gene ( , mayinga isolate), generating n ct -ebovgp cdna (gp ; figure a) . a vector expressing a third-position ebovgp (gp ; figure a ) was generated by inserting an expression cassette between the m and n genes. to insert the ebovgp gene in genome position (gp ; figure a ), the n and g genes were swapped and an expression cassette was positioned between vsv l and the trailer sequence. for adventitious agent testing of clinical trial material, large quantities of ebovgp-specific neutralizing antibody are required to neutralize vaccine virus infectivity. to avoid this requirement, a gene expressing secreted ebovgp was generated by deleting the sequence encoding the transmembrane region and cytoplasmic tail (aa - ). this modified ebovgp gene was inserted into position of n ct (gp Δtm; figure a ). the mucin-like domain of ebovgp spans aa - , and part of this domain (aa - ) was deleted (gp Δmuc; figure a ). this modified ebovgp gene was generated following observations that part of the mucin region was deleted upon multiple serial passage of n ct -ebovgp on vero cell monolayers and was included here to assess the effect of partial removal of the glycan "coat" on ebovgp immunogenicity. ebovgp expression was analyzed by western blotting for all vectors ( figure b ). vectors were rescued from genomic cdna as previously described [ ] . rescued virus was plaque purified and amplified on vero cell monolayers (atcc ccl- ). for animal studies, vectors were purified from infected cell supernatants by centrifugation through a % sucrose cushion. virus pellets were resuspended in phosphate-buffered saline (pbs), ph . , mixed with stabilizer ( mm k hpo , mm kh po , and mm sucrose), snap frozen, and stored at − °c. for the mouse immunogenicity study, rvsv/ebov vectors were administered at × plaque-forming units (pfu) by intramuscular injection of the tibialis anterior muscle (total injection volume, . ml). serum was collected at weeks , , and . ten days after final the inoculation, mice were euthanized by co inhalation, and serum and spleen cells were harvested. for the mouse neurovirulence study, groups of ten -weekold female swiss webster mice were anesthetized and inoculated intracranially with . ml of serial -fold dilutions of each vector as previously described [ ] . after inoculation, mice were weighed daily and assessed for signs of disease over weeks. neurovirulence was assessed by measuring morbidity and mortality as an end point. mice that were showing severe signs of disease or were moribund were promptly euthanized. cumulative deaths across all doses tested allowed the % lethal dose to be calculated for each vector [ ] . for guinea pig immunization, × pfu of each vector was administered intraperitoneally. on day after vaccination guinea pigs were challenged with focus-forming units (ffu) of guinea pig-adapted ebov (gpa-ebov) [ ] by intraperitoneal injection. all animals were weighed daily and monitored for signs of illness. for macaque challenge, healthy, ebov-naive, adult (age, - years; weight, - kg), male and female rhesus macaques (macaca mulatta) were randomly assigned to a vaccination group (n = ) and a nonvaccination control group (n = ). at study day − , macaques were anesthetized with ketamine and inoculated in the caudal thigh muscle with × pfu of rvsv/ebov vector (total volume, ml). at study day , macaques were anesthetized with ketamine and challenged with ffu of ebov [ ] by intramuscular injection of the caudal thigh muscle (total volume, ml). macaques were monitored for signs of illness (ie, abnormal temperature, weight loss, clinical examination findings, hematology findings, and blood chemistry findings) following vaccination and the ebov challenge portions of the study. for murine studies, vaccine-elicited ifn-γ elispot responses were determined using a mouse ifn-γ elispot kit (bd biosciences, san diego, california) as previously described [ ] . splenocytes were incubated with µg/ml con-a (sigma), peptide pools ( -mers overlapping by aa; final peptide concentration, µm [each]) spanning ebovgp, or medium alone. a murine enzyme-linked immunosorbent assay (elisa) previously described [ ] was modified by using elisa plates coated with ng/well of purified recombinant ebovgp (ibt bioservices) to determine ebovgp-specific serum immunoglobulin g (igg) titers. murine serum samples were added to elisa plates at a starting dilution of : and were further diluted -fold across the plates. antigen-specific antibody titers were defined as the reciprocal of the last serum dilution giving an od of > . . antigens for the macaque elisa were produced as previously described [ ] . macaque sera were inactivated by γ-irradiation ( mrad) according to a standard operating protocol approved by the institutional biosafety committee at rml. elisa with ebovgpΔtm was performed as described previously [ ] . to identify the most-effective rvsv/ebov vector design for induction of ebovgp-specific immune responses, the vectors outlined in figure a were compared for their ability to elicit ebovgp-specific cell-mediated immune and binding antibody responses in mice (figure ). groups of balb/c mice (n = ) were immunized intramuscularly at study week ( figure a ). ten days after primary immunizations, splenocytes were collected from mice/group and tested for ebovgp-specific ifn-γ secretion by elispot assay. the remaining mice/ group were boosted intramuscularly at study week with pfu of each rvsv/ebov vector. ten days after boosting, splenocytes were collected and tested as described above. after immunization, the highest mean ebovgp-specific ifn-γ elispot responses (±standard error of the mean [sem]) were detected in mice immunized with n ct -ebovgp Δtm ( ± spot-forming cells [sfcs]/ splenocytes) and n ct -ebovgp ( ± sfc/ splenocytes). interestingly, n ct -ebovgp Δmuc, elicited a significantly reduced response (mean ± sem, ± sfcs/ splenocytes) relative to n ct -ebovgp and n ct -ebovgp . notably, after immunization, gp responses were undetectable in mice immunized with vectors expressing ebovgp from genome positions or . after boosting, the highest mean ebovgp-specific ifn-γ elispot responses (±sem) were again detected in mice immunized with n ct -ebovgp Δtm ( ± sfcs/ splenocytes) and n ct -ebovgp (mean ± sem, ± sfcs/ splenocytes). mice immunized with n ct -ebovgp Δmuc showed an improved response (mean ± sem, ± sfcs/ splenocytes), and mice boosted with n ct -ebovgp or n ct -ebovgp still failed to demonstrate a measurable ebovgp-specific ifn-γ elispot response. immunized mice were also monitored for induction of serum antibody responses by elisa ( figure b ). ten days after boosting, mean serum ebovgp-specific igg titers (±sem) were highest in mice immunized with n ct -ebovgp Δmuc vector ( . ± . ), n ct -ebovgp ( . ± . ), and n ct -ebovgp ( . ± . ). interestingly, a statistically significant, approximately -fold lower ebovgp-specific igg titer was detected in mice immunized with n ct -ebovgp Δtm (mean ± sem, . ± . ) relative to mice immunized with n ct -ebovgp . a -fold lower titer was also detected in mice immunized with n ct -ebovgp ( . ± . ) relative to mice immunized with vectors expressing ebovgp from genome positions or . on the basis of the data presented in figure , the n ct -ebovgp vector expressing an anchored full-length ebovgp in position of the rvsv genome appeared to be the most immunogenic (with regard to cell-mediated and humoral immunity) in mice, so n ct -ebovgp was selected for guinea pig and nhp challenge studies. we next tested whether n ct -ebovgp could protect guinea pigs from a lethal challenge (figure ). in this experiment, guinea pigs were immunized intraperitoneally with × pfu of n ct -ebovgp . for controls, additional groups of guinea pigs were immunized with × pfu of wtvsv or medium alone. on day after immunization, all guinea pigs were challenged intraperitoneally with ffu of gpa-ebov. after challenge, guinea pigs were monitored for changes in body weight ( figure a) , signs of disease, and survival ( figure b ). guinea pigs immunized with medium alone or wtvsv began to lose weight on day after challenge and died from ebov infection during study days and as observed previously [ ] . in contrast, n ct -ebovgp immunized guinea pigs gained weight and were completely protected from disease during the course of the study. encouraged by findings from the guinea pig study, we determined whether n ct -ebovgp could protect macaques from lethal ebov challenge (table ) . three rhesus macaques were immunized intramuscularly in the caudal thigh muscle as follows: were immunized with × pfu n ct -ebovgp in a total volume of ml (ebov and ebov ), and macaque (ctrl) was mock immunized. at day after immunization, both n ct -ebovgp -immunized macaques demonstrated measurable ebovgp-specific serum igg responses (titer, : ), and these responses increased (titer, : ) by study day . in contrast, the sham-immunized macaque remained seronegative for ebovgp. at study day , all macaques were challenged with ffu ebov mayinga by intramuscular injection in the caudal thigh muscle. as shown in table , the single control animal failed to mount an ebovgp-specific response on day after challenge and quickly died from ebov infection. importantly, a rapid increase in the ebovgp-specific serum igg titer was observed on days , , and after challenge in both vaccinated macaques, and they survived without showing signs of disease. mice are exquisitely sensitive to intracranial instillation of wtvsv [ , ] , leading to lethal viral encephalitis. previously, it was shown that intracranial inoculation of -week-old swiss webster mice with pfu of the highly attenuated n ct -hivgag vector caused no significant illness, so a % lethal dose could not be achieved for this vector [ ] . to test the neurovirulence potential of n ct -ebovgp , groups of outbred swiss webster mice were inoculated intracranially with n ct -ebovgp in a dose-ranging study ( - pfu; figure ). two control groups were inoculated with pfu of a less attenuated rvsv (rvsv-hivgag ) or with pbs alone. following inoculation, mice were assessed for morbidity and mortality over weeks. consistent with previously published reports [ , ] , mice inoculated intracranially with pbs survived, and approximately half of the mice inoculated with rvsv-hivgag died - days after inoculation. importantly, all mice inoculated with n ct -ebovgp survived and were disease free. previous work demonstrated that vaccination with a single dose of replication-competent rvsv pseudotyped with ebovgps protected mice, hamsters, guinea pigs, and nhps from lethal ebov challenge [ , , ] . the rvsv vector used in those studies retained the natural vsv genome organization, except the vsv g gene was replaced with the ebovgp gene [ ] . here we demonstrate protection of guinea pigs and nhps with a highly attenuated, replication competent form of rvsv that retains the vsv g gene [ , , ] and expresses the ebovgp from an additional transcription unit inserted at position in the rvsv genome. the mouse immunogenicity study demonstrated that ebovgpspecific cell-mediated immune responses were more robust when the ebovgp gene was in the first position in the genome, compared with the third and sixth positions, consistent with greater relative abundance of ebovgp in infected cells due to the steep ′ to ′ transcription gradient present during vsv replication [ , , ] . the cell-mediated immune response was similar for secreted and anchored forms of ebovgp, indicating that both forms of ebovgp underwent a similar degree of processing and presentation of t-cell epitopes in antigen-presenting cells. interestingly, there was a significant reduction in the cell-mediated immune response to ebovgp when part of the mucin-rich region was deleted, possibly as a result of altered proteosomal processing of ebovgp or direct deletion of t-cell epitopes. after decay of the primary cell-mediated immune response, a second vaccine dose boosted elispot responses to levels seen after primary vaccination; however, there was still no detectable cell-mediated immune response elicited by vectors expressing ebovgp from position and , indicating the requirement for higher ebovgp levels in infected cells to generate detectable ebovgp-specific cell-mediated immune responses. as expected, the ebovgp-specific igg response in mice was greatest for n ct -ebovgp and n ct -ebovgp Δmuc vectors expressing the highest levels of ebovgp from genome position . interestingly, n ct -ebovgp Δtm elicited much lower levels of ebovgp-specific igg than n ct -ebovgp . we speculate this may reflect differences in the tertiary and quaternary structure of secreted ebovgp, compared with the trimeric conformation achieved by anchored ebovgp. notably, the secreted ebovgp described here is different from the naturally occurring secreted form of ebovgp that is translated from unedited ebovgp mrna [ ] . the corollary is that anchored trimeric ebovgp spikes on the surface of infected cells and virus particles present a dense array of structural epitopes that may interact more efficiently with b cells than soluble secreted ebovgp, as is the case for other particulate antigen compositions [ , ] . ebovgp-specific igg responses were highest for n ct -ebovgp Δmuc [ ] , possibly reflecting better exposure of ebovgp epitopes by removal of part of the carbohydrate cloak [ ] . the n ct -ebovgp vector also elicited a robust ebovgp-specific igg response, which contrasts with the undetectable ebovgp-specific cell-mediated immune response induced by this vector. the explanation for this anomaly is unclear but could be due to relative differences in ebovgp-specific cellular and humoral antigenicity. we believe the approximate % attenuation of expression across each gene junction could drop the ebovgp level below a threshold required to elicit cell-mediated immune responses (n ct -ebovgp ), while the ebovgp level is still adequate to induce a relatively robust humoral response. however, for n ct -ebovgp , the ebovgp-specific humoral response does decline markedly, presumably as a result of the calculated % reduction in ebovgp expression when in the sixth position in the genome. previous work demonstrated that the n ct vector backbone was highly attenuated in the mouse intracranial nv model [ , ] . however, western blot data demonstrated that ebovgp was present on the surface of virus particles when expressed by an rvsv vector that retained the vsv g gene (unpublished data). therefore, a mouse nv study was performed to assess any changes in virulence arising from the possible alteration of vector tropism mediated by ebovgp. results confirmed the highly attenuated nature of n ct -ebovgp in the mouse central nervous system, indicating that the presence of ebovgp on the virion surface, in addition to vsv g protein, did not enhance virus spread in the brain, presumably because cells in the central nervous system lack the ebovgpspecific receptor [ ] [ ] [ ] and because virus using vsv g receptors remained attenuated as previously described [ ] . because n ct -ebovgp elicited a desirable balance of humoral and cellular immune responses to ebovgp after a single vaccine dose in mice, it was selected for guinea pig and nhp challenge studies. one dose of this vector protected guinea pigs from signs of disease following challenge with a lethal dose of gpa-ebov. interestingly, there was a slight delay in onset of the first signs of disease after challenge of wtvsv-vaccinated guinea pigs. one possible explanation is that induction of ifns and other innate immune responses by rvsv [ , ] leaves the guinea pig innate immune system on high alert, even days after vaccination, resulting in a generalized antiviral state that may initially slow the gpa-ebov infection [ , ] . a single dose of n ct -ebovgp vector also protected nhps from disease following ebov challenge. an ebovgpspecific igg titer was detected in both immunized macaques prior to challenge and was expanded following ebov challenge, indicating that a preexisting ebovgp-specific antibody response is important for protection. unfortunately cell-mediated immune responses in nhps were not measured and may also contribute significantly to protection. the n ct -ebovgp vector used in these challenge studies is similar to the highly attenuated n ct -gag vector that demonstrated safety and immunogenicity in phase clinical studies. the studies described here are the first to demonstrate protection of guinea pigs and macaques with a single dose of highly attenuated rvsv expressing ebovgp, and we believe that the n ct -ebovgp vector has the essential safety and efficacy characteristics for use in a vaccine to prevent ebov infection in humans and the great apes. this highly attenuated vector platform will likely also have the potential to serve in a vaccine against other ebov strains that cause disease in humans and apes. ultimately, a multivalent ebov vaccine may be possible by combining ≥ vaccine vectors in a single vaccine formulation. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations serologic cross-reactivity of human igm and igg antibodies to five species of ebola virus fruit bats as reservoirs of ebola virus spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species ebola-a growing threat? case fatality rate for ebola virus disease in west africa structure of the ebola virus glycoprotein bound to an antibody from a human survivor protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus 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of alpha/beta interferon genes: interference by nonsegmented negative-strand rna viruses antiviral defense in mice lacking both alpha/beta and gamma interferon receptors natural killer cells in antiviral defense: function and regulation by innate cytokines financial support. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -ody u n authors: loh, so hee; park, jin-yeon; cho, eun hee; nah, seung-yeol; kang, young-sun title: animal lectins: potential receptors for ginseng polysaccharides date: - - journal: j ginseng res doi: . /j.jgr. . . sha: doc_id: cord_uid: ody u n panax ginseng meyer, belonging to the genus panax of the family araliaceae, is known for its human immune system-related effects, such as immune-boosting effects. ginseng polysaccharides (gps) are the responsible ingredient of ginseng in immunomodulation, and are classified as acidic and neutral gps. although gps participate in various immune reactions including the stimulation of immune cells and production of cytokines, the precise function of gps together with its potential receptor(s) and their signal transduction pathways have remained largely unknown. animal lectins are carbohydrate-binding proteins that are highly specific for sugar moieties. among many different biological functions in vivo, animal lectins especially play important roles in the immune system by recognizing carbohydrates that are found exclusively on pathogens or that are inaccessible on host cells. this review summarizes the immunological activities of gps and the diverse roles of animal lectins in the immune system, suggesting the possibility of animal lectins as the potential receptor candidates of gps and giving insights into the development of gps as therapeutic biomaterials for many immunological diseases. panax ginseng meyer is a well-known medicinal plant in the world. the ginseng is a deciduous perennial belonging to the family araliaceae and genus panax. the genus name of ginseng, panax, is derived from the greek pan (all) akos (cure), meaning "cure-all" or "all healing," which describes the traditional belief that ginseng has properties to heal all aspects of the body. the name ginseng comes from the chinese words "jen sheng," meaning "man-herb," because of the humanoid shape of the root or rhizome of the plant, which is the part of the plant most commonly used for extraction [ , ] . there are about different species of ginseng which have being identified all over the world. among them, the most commonly used species of ginseng are asian ginseng (p. ginseng meyer, renshen) and american ginseng (panax quinquefolius l., xiyangshen) which all belong to the panax genus of the araliaceae family [ ] . asian ginseng has been used for thousands of years as a tonic to improve overall health, restore the body to balance, help the body to heal itself, and reduce stress [ ] , and american ginseng has been used by native americans for at least hundreds of years [ , ] . ginseng is prepared and used in several ways as fresh ginseng (sliced and eaten, or brewed in a tea), white ginseng (peeled and dried), red ginseng (peeled, steamed, and dried), extract (tincture or boiled extract), powder, tea, tablet, or capsule [ , ] . it has been reported that ginseng exhibits a wide range of beneficial pharmacological effects including immunomodulation, antitumor, antioxidation, antidepression, hypoglycemic, inhibition of gastric lesions, attenuation of leptininduced cardiac hypertrophy, heart protection against ischemia and reperfusion injury, prevention of glucose-induced oxidative stress, prevention of diabetic nephropathy, retinopathy, and cardiomyopathy [ e ]. this broad spectrum of biological activity of ginseng has originated from its multiple bioactive components, namely ginsenosides, polysaccharides (pss), peptides, polyacetylenic alcohols, and gintonin [ e ]. ginsenosides were considered to be responsible for most of ginseng's pharmacological effects. however, recent studies indicate that ginseng polysaccharides (gps), one of the active components of ginseng [ ] , also possess a wide range of biological and pharmaceutical activities, including immune-modulation, antitumor, antiadhesive, antioxidant and hypoglycemic activities [ , ] . especially, gps are known for their immunostimulatory effects [ , , ] and a major contributor to the bioactivity of herbal medicines, providing great potential applications in food, pharmaceuticals, and other industries. therefore, gps were extensively studied for their constituents and chemical structures. gps are biopolymers formed from a complex chain of monosaccharides rich in l-arabinose, d-galactose, l-rhamnose, d-galacturonic acid, d-glucuronic acid, and d-galactosyl residue linked together through glycosidic bonds, resulting in complex macromolecular architectures [ , , ] . their molecular weights range from da to , , da [ ] , which contributes to their diverse physicochemical properties and biological activities [ , , , ] . gps include acidic and neutral pss. the pharmacological effects of gps, including immunomodulation, can be attributed to these acidic and neutral ps components [ ] . while the acidic gps contain different amounts of uronic acids and neutral sugars [ , ] , the neutral pss mainly contain different ratios of neutral sugar residues [ ] . so far, the studies about american gps have mainly been centered on acidic pss, resulting in relatively limited research that explores neutral pss. however, researchers also have interest in neutral pss of american gps, because neutral pss are also one of the important active components in the american ginseng roots. the pss from ginseng roots have many bioactivities, such as immunomodulation, antitumor, and hypoglycemic activities [ , ] , and contain % neutral starch-like pss, % arabinogalactans, and % pectins [ ] . similarly, the pss from ginseng leaves are also bioactive, and contain about % arabinogalactans and % pectins. gps enable enhancement of the production of cytokines and reactive oxygen species by stimulating macrophages [ , ] . in a recent study, gp was shown to stimulate dendritic cells (dcs) resulting in enhanced production of interferon-g (ifn-g) [ ] . it was reported that acidic gps promoted the production of cytotoxic cells against tumors and stimulated macrophages to produce helper types and (th and th ) cytokines [ , ] . an acidic gp from p. ginseng has been shown to display immunomodulatory effects either in an immunostimulatory or in an immunosuppressive manner, depending on timing of treatments and disease environments [ ] . this acidic gp was also shown to modulate the antioxidant defense systems such as superoxide dismutase and glutathione peroxidase enzymes, probably via inducing regulatory cytokines [ , ] . therefore, acidic gps have been considered as the major bioactive species for immune modulations. tomoda et al [ ] reported that two acidic pss of p. ginseng enhance the phagocytic activity of macrophages, and sonoda et al [ ] found that an acidic gp of p. ginseng was a potent inducer of interleukin- (il- ) production by human monocytes and thp- cells. shin et al [ ] reported that an acidic ps of p. ginseng shows immune modulatory activities via macrophage no production. recently, lemmon et al [ ] reported that the immunostimulatory effects of acidic gps of p. quinquefolius are mediated by ps with a molecular weight higher than kda. it was reported that acidic gps promoted the production of cytotoxic cells against tumors and stimulated macrophages to produce helper types and (th and th ) cytokines [ , ] . intravenous pretreatment of gp attenuated the production of serum proinflammatory and antiinflammatory cytokines after septic bacterial infection [ ] . in addition, ginsan, an acidic gp from of p. ginseng, is a wellknown medicinal herb and has been shown to have critical effects on immune cells, which shows an immunomodulatory acidic gp from p. ginseng [ ] . kim et al [ ] showed that ginsan induces th cell and macrophage cytokines. ginsan enhances the production of cytokines and reactive oxygen species by macrophages [ ] and stimulates the phagocytic activity of macrophages [ ] . also, ginsan induces the maturation of dcs [ ] , profoundly enhancing the production of il- , il- , and tumor necrosis factor alpha (tnf-a) by dcs and showed that ginsan may modulate dc function by altering cytokine levels [ ] . for neutral gps, it was reported that neutral gps of p. ginseng stimulate the proliferation of lymphocytes, increase the cytotoxicity of natural killer cell, enhance the phagocytosis and no production by macrophages, and increase the level of tnf-a in serum [ , ] . due to these results, many scientists have considered both acidic and neutral gps as stimulators in the immune system. x. zhang et al and kim et al reported that both acidic and neutral gps of p. ginseng (asian ginseng) may stimulate b cells, t cells and macrophages [ , ] . in addition, they considered the relation of acidic and neutral gps as the supporter, in which neutral gps help the enhancement of immunostimulatory effects of acidic gps. in fact, w. ni et al reported that neutral gps of p. ginseng enhance macrophage production of no [ ] . on the contrary to immunostimulatory effects of gps, recent studies showed that gps also suppress the proinflammatory responses. recently, it was reported that a novel neutral ps (ppqn, . kda) was isolated from american ginseng roots and could suppress inflammation by inhibiting the secretion of inflammatoryrelated mediator nitric oxide (no) and cytokines (tnf-a, il- , and il- b) compared to lipopolysaccharide (lps) treatment, implicating the therapeutic implications of ppqn in inflammatoryrelated diseases like tumors, atherosclerosis, and so on [ ] . as an example, one study reported that gps inhibit immunological responses associated with collagen-induced arthritis [ ] . other studies also suggest that cvt-e , a poly-furanosyl-pyranosyl polysaccharide-rich herbal and unique extract product of the root of american ginseng (p. quinquefolium), suppresses the inflammatory immune responses, reducing the activation of neutrophils [ ] , inducing the production of il- , ifn-g, tnf-a, and il- in spleen [ , , ] , and increasing the proliferation of splenic b lymphocytes, bone marrow, and natural killer cells. intravenous pretreatment of gp attenuated the production of serum proinflammatory and antiinflammatory cytokines after septic bacterial infection [ ] . also, this intravenous pretreatment of gps in mice enhances macrophage-mediated bactericidal activity by reducing the number of staphylococcus aureus which is present in the spleen, kidney, and blood and exerts a protective effect against infected septic mice by suppressing early acute inflammation [ , ] . in addition, recent studies reported that pretreatment with gp suppressed acute inflammatory responses at an early phase resulting in the enhancement of antimicrobial activities and protection of mice from staphylococcus aureus-induced sepsis as an antiinflammatory function [ , ] . as an example, cvt-e has been shown to be effective for preventing acute respiratory illness caused by influenza and respiratory syncytial virus [ , ] . another study revealed that intranasal administration of gps showed a protective effect on influenza viral infection by lowering the levels of inflammatory cytokines (il- ) and lung viral titers [ ] . because gps were reported to significantly increase the viability of peritoneal macrophage cells [ ] and ginseng was shown to inhibit degradation of long-lived proteins and to stimulate protein synthesis similar to polypeptide growth factors [ ] , it was suggested that maintaining the cell viability under the condition of viral infection-induced stress might be an another alternative mechanism for the protective effects of gp. it was reported that the recognition and binding of plant pss by toll-like receptor (tlr ) leads to the recruitment of various cytoplasmic toll/il- receptor (tir) domain-containing adaptors such as myeloid differentiation factor (myd ), tir domaincontaining adaptor protein (tirap), and tir (toll/interleukin- receptor)-domain-containing adapter-inducing interferon-b (trif)related adaptor molecule (tram) [ ] . it was also shown that the expression of tlrs including tlr , tlr , and the adaptor molecule myd is significantly reduced in murine macrophages by gp pretreatment in vitro, which were increased in murine macrophages with the stimulation of s. aureus [ , ] . on the contrary, friedl et al [ ] and lemmon et al [ ] showed that american gp extracts may mediate the immunostimulatory effect by the inducible nitric oxide synthase (inos), mitogen-activated protein kinase (mapk) kinases such as p , extracellular signal-regulated kinases / (erk / ), phosphoinositide -kinase (pi k), and nuclear factor kappa b (nf-kb) signaling pathways [ , ] . therefore, these results suggest that gps might be associated with the ability of the extract's ps fractions to bind to tlr receptor and upregulate or downregulate tlr receptor expression, which triggers or inhibits the intracellular signaling cascades and the production of proinflammatory mediators under basal or lps endotoxic conditions, respectively. animal lectins are carbohydrate-binding proteins which are highly variable in their amino acid sequences, widely distributed in microorganisms, viruses, animals and higher plants with different functions, structures, tissue localizations, and carbohydratebinding specificities [ ] . animal lectins were discovered before plant lectins, although many were not recognized as carbohydrate binding proteins for many years after first being reported [ ] . although plant and animal lectins do not have homologous primary structures, they have similar preferential binding to carbohydrates [ ] . animal lectins are neither immune origin nor catalyst, in contrast to antibodies or enzymes, and are able to detect or bind complex carbohydrate structures specifically through the carbohydrate-recognition domain (crd) [ , ] . each animal lectin possess its own crd which has an identical sequence motif of to amino acid residues and four cysteines that is thoroughly conserved and form two disulfide bonds [ , ] . animal lectin activity is found in association with an astonishing diversity of primary structures [ ] . at least structural families are known to exist and bind structures other than carbohydrates via protein-protein, protein-lipid or protein-nucleic acid interactions [ ] . their roles in glycol-recognition systems include complement activation, cell recognition, cell adhesion, cell migration, cell signaling, and morphogenesis. moreover, animal lectins are able to take part in defense mechanisms, importantly by recognizing a carbohydrate of pathogens [ , ] . animal lectins are means of attachment to various cells or viruses via the surface carbohydrate types of the cells to be attached [ ] . the function in recognition or cell surface interaction of animal lectins has been implicated in direct first-line defense against pathogens [ ] . for example, the mannose specific receptor, presented on the surface of macrophages, can bind to the infectious organisms which expose mannose-containing glycans on their surface, enabling them to ingest and kill the foreign organisms [ ] . in addition, animal lectins are involved in cell trafficking, immune regulation, and prevention of autoimmunity [ ] . animal lectins are classified into four families based on structure or function. these are the c-type (calcium-requiring) lectins, p-type (mannose- -phosphate binding) lectins, s-type (galectins) lectins, and i-type (immunoglobulinlike) lectins ( fig. ) [ ] . within each family, they have similar sequences and structural properties [ ] . recently, other lectin types have been found, including m-type, l-type, chitinase-like, and ftype lectins. in this review, four traditional families of animal lectins are introduced (table ). c-type lectins are endocytic receptors which are mostly expressed by macrophages, dcs, and some endothelial cells. c-type lectins require ca þ for activity and have common sequence motif of invariable and highly conserved amino acid residues [ , ] . as they have multi crds, c-type lectins are able to recognize a wide range of carbohydrate-based ligands from endogenous molecules to conserved structures found in bacteria, fungi, virusinfected cells, and parasites called pathogen-associated molecular patterns [ ] . after recognition, c-type lectins subsequently participate in the uptake for degradation in order to facilitate direct elimination by macrophages or antigen presentation by dcs and macrophages in major histocompatibility complex (mhc) molecules at the cell surface, resulting in stimulating the adaptive immune system [ , ] . for example, c-type lectins can recognize diverse bacterial pathogens and induce cytokine production and th responses in antibacterial immunity [ ] , and are critical in systemic infections with pathogens like cryptococcus neoformans in antifungal immunity of th effector cells [ ] . in addition, they are involved in clearance, homeostasis, and immunomodulation [ ] . many c-type lectins play primary roles in immunity. c-type lectins include dectins, dc-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign), sign-r , mannose receptor (mr), collectin (mbl, sp-a, sp-d) and selectins (l-, p-, e-) ( table ). dectin- specifically binds to b- , and b- , linked glucans of fungi, plant cell walls, and bacteria, including candida albicans, saccharomyces cerevisiae, coccidioides posadasii, and pneumocystis carinii, but cannot recognize monosaccharides or glucans with other linkages. however, c. neoformans, histoplasma capsulatum, and aspergillus fumigatus are not targeted by dectin- , in spite of the presence of b-glucans in their cell wall [ , ] . dectin- plays a primary role in inducing proinflammatory mediators like tnf-a in response to fungal pathogens. also, dectin- contains an immunotyrosine activation motif within its cytoplasmic tail, helping tlr signaling pathway by interaction with the immunotyrosine activation motif of dectin- [ ] . signaling by dectin- regulates various cellular responses including phagocytosis and the production of inflammatory cytokines such as ifns, il- , il- , and il- [ ] . dectin- expressed on dcs and macrophages can recognize n-glycans of the surface of tumor cells, following nuclear translocation, and the induction of several genes such as inam, which is known to induce tumor killing by nk cells by hemophilic interactions. dc-sign was originally identified as a receptor for intercellular adhesion molecule- (icam- ) that induces dc-mediated t-cell proliferation [ ] . it was subsequently unveiled to bind icam- on vascular endothelial cells, regulating dcs migration through interaction of n-linked high mannose structures consisting of from five to nine terminal mannose units [ ] . dc-sign, which is expressed by dcs, decidual and alveolar macrophages facilitates high-affinity binding to high mannose oligosaccharides through tetramerization [ ] . mannose-dependent interactions demonstrate the ability of dc-sign to bind human immunodeficiency virus (hiv) and various pathogens, including mycobacterium tuberculosis, c. albicans, leishmania mexicana, a. fumigatus, helicobacter pylori, and schistosoma mansoni [ ] . for instance, dc-sign recognizes mannosylated lipoarabinomannan, which is a mannose-capped glycolipid found in the cell wall of m. tuberculosis. this interaction can induce the secretion of the immunosuppressive cytokine, il- , from dcs which expresses dc-sign [ ] . dc-sign expression is mostly induced by il- , and is downregulated by ifn-g, tgfb, and dexamethasone [ , ] . the murine dc-sign homologues were reported to help to identify the roles of dc-sign in infection and inflammation and to play important roles in bacterial, fungal, and parasitic infections. there are seven mouse genes in the mouse dc-sign locus, containing sign-r - and sign-r , , and a pseudogene, sign-r [ , ] . the mrna of three sign-r genes encode type ii transmembrane proteins (sign-r , amino acids; sign-r , amino acids; sign-r , amino acids), but sign-r gene only encodes a crd without a cytosolic domain and a transmembrane domain (sign-r , amino acids) [ ] . amino acid sequence similarities between the crd of human dc-sign and the murine homologues are % for sign-r , % for sign-r , % for sign-r , and % for sign-r [ ] . sign-r , a murine homologous transmembrane of the dc-sign, is expressed by splenic marginal zone macrophages and peritoneal macrophages [ , ] . similar to dc-sign, sign-r is essential for the recognition and clearance of streptococcus pneumoniae-derived capsular pss. moreover, it can also recognize c. albicans, m. tuberculosis, s. pneumoniae cps, hiv, and yeast derived zymosan particles in a mannose inhibitable manner [ ] . sign-r directly binds to c q and dominantly regulates the immunoglobulinindependent classical complement pathway for c deposition of blood borne s. pneumoniae [ ] . in sign-r deficient mice, c deposition is abolished and innate resistance against pneumococci is reduced [ , ] . also, sign-r interacts specifically with , sialylated fc fragments of immunoglobulins, resulting in the antiinflammatory activity of intravenous immunoglobulin, which has been widely used to treat autoimmune diseases including immune mr (cd ) is described in myeloid cells and functions as a viral recognition receptor on the cell membrane for yeast, bacteria, hiv, and a wide variety of pathogens, such as c. albicans, leishmania donovani, p. carinii, klebsiella pneumonia, trypanosoma cruzi, m. tuberculosis [ e ] and capsular pss of s. pneumoniae through a mannose-type crd and pathogen-associated high mannose structures [ ] . mr which is mainly expressed in immune cells induces uptake and presents mannosylated antigens such as lipoarabinomannan on mhc class ii of metallophilic macrophages, resulting in influencing immune responses [ ] . for instance, the interaction between mr and hepatitis b virus (hbv) surface antigen (hbsag) enhances viral uptake by dcs, resulting in the impairment in the function of dcs and the ineffective antiviral response of chronic hbv [ ] . the recognition of viral surface glycoproteins by mr is also beneficial to influenza virus [ ] and hiv [ ] invasion into host cells. in addition, mr is able to mediate the clearance of endogenous inflammatory glycoproteins bearing ligands of the mannose-type crd [ ] . the expression of mr is upregulated by cytokines like il- , il- , and il- , but ifn induces a downregulatory effect to mr [ ] . selectins are cell adhesion molecules and have three groups, including e-selectin (endothelial) and p-selectin (platelet) on endothelium, and l-selectin (leukocyte) on leukocytes [ ] . selectins play roles in leukocyte recruitment from the bloodstream into sites of inflammation [ , ] . the recruitment of leukocytes proceeds initially by attachment leading to the rolling of leukocytes along endothelial vasculature via selectin-carbohydrate interaction. e-selectin (m.w. kda) is expressed by endothelial cells after stimulation with activators like tnf-a, il- , or bacterial lipopolysaccharide [ ] . these cytokines also upregulate the expression of p-selectin (m.w. kda), which is expressed by endothelial cells and platelets [ ] . also, p-selectin in released to the cell surface from storage vesicles in endothelial cells and platelets minutes after stimulation by a number of activators, such as thrombin or histamine [ ] . l-selectin (m.w. e kda) is expressed by leukocytes and aids in the homing of leukocytes. l-selectin has high expression on naïve t lymphocytes but, once t lymphocytes are activated, the expression of l-selectin is low or lacking [ ] . e-, p-, and l-selectin are composed of an n-terminal c-type lectin crd, an epidermal growth factor-like subunit, a number of short consensus repeat units, a membrane spanning region, and a c-terminal cytoplasmic tail [ ] . there is approximately % homology for analogous selectin crds across species, and w % homology between different selectins within a species [ ] . . . . p-type lectins p-type lectins are intracellular transmembrane glycoproteins with specificity for mannose- -phosphate (m p) to identify and route lysosomal enzymes to the lysosomal compartment (mpr signal) and they have two groups. one is the e kda cationdependent m p receptor (cd-mpr) which requires ca þ for activity and contains single extracellular domain, followed by a single transmembrane domain [ ] . the other is the e kda table a summary of the c-type lectin receptors dealt with in this review insulin-like growth factor ii/cation-independent m p receptor (igf-ii/ci-mpr) which does not require cation for activity and has a large extracellular domain containing two high-affinity binding sites [ ] . the crds of p-lectins for m p are located in the extracellular domain of cd-mpr and in extracellular repeats and with high affinity for igfii/ci-mpr [ ] . they are similar both in size and in sequence to the repeating units that consisted of short nterminal extracellular domain and c-terminal cytoplasmic tail [ ] . their function is the intracellular targeting of lysosomal enzymes (lysosomal hydrolases) in the trans-golgi network vertebrate animals and delivering them to prelysosomal compartments [ ] . also, the c-terminal cytoplasmic tail of the receptor which targets amino acid sequence plays a role in recognizing signal for transport to the endosomal compartment [ ] . igf-ii/ci-mpr has the capacity for endocytosis of ligands from the cell surface and serves to turn over igf by endocytosis, but not cd-mpr [ , ] . s-lectins, galectins (from to ), consist of globular galectintype crds which are specific for b-galactoside ligands and have conserved cysteine residues [ ] . they are found predominantly in mammals, but not in plants [ ] . s-lectins have a relatively simple structure and share a highly homologous domain named the scarbohydrate recognition domain (s-crd) [ ] . s-lectins mostly contain multiple sugar-binding sites, as the presence of two type of s-crd in a single polypeptide or its dimer [ ] . the function of slectins may be to crosslink n-acetyllactosamine-containing structures found at cell surfaces or in the extracellular matrix [ ] . s-lectins are conserved in unrelated organisms including frogs, birds, and mammals, meaning that b-galactoside binding to lectin may be important biologically [ ] . mammalian s-lectins are conserved in eight residues of the s-crds and involved in growth regulation, cell adhesion, cell migration, and immune responses [ ] . for example, galectin- mediates cell adhesion and apoptosis, and regulates cellular proliferation. galectin- mediates cell adhesion, regulates inflammation, pre-mrna splicing, and protects against induced apoptosis. galectin- and galectin- have a function in cellÀcell and cellÀextracellular matrix crosslinking and galectin- is involved in maturation and erythrocyte adhesion [ , ] . . . . i-type lectins i-type lectins are members of the immunoglobulin (ig) superfamily [ ] . they share the structural motif, the ig fold, with ig-like domains consisted of similar two planes of b-pleated sheets [ ] . the b-sheets are established about e amino acids, crosslinked by a disulfide bond and contain several hydrogen-bonded antiparallel chains [ ] . i-type lectins are classified into two domains according to the number and arrangement of b-strands present in the domains [ ] . one is the amino terminal, extracellular domain, which is similar to the variable region (v-type domain) of igg and is necessary for sialic acid-dependent binding [ ] . the other is the constant region (c-type domain) of igg that has various forms from to [ ] . i-type lectins function as not only cell adhesion molecules but also growth factor receptors and extracellular matrix molecules [ ] . the major subclasses of i-type lectins are the sialic acid-binding immunoglobulin superfamily lectins (siglecs) which contain an homologous n-terminal v-type domain with the sialic acid binding site and variable numbers of c-type domains [ ] . v-type domain and adjacent c-type domains of siglecs have conserved cysteine residues, resulting in formation of conventional intrasheet and interdomain disulfide bonds [ ] . the c-terminal cytoplasmic tail of most siglecs has immunoreceptor tyrosine-based motifs in the intracellular domain for signaling events [ ] . sialoadhesin found on the surface of macrophages is a member of the siglecs family [ ] . it contains a v-type domain and c-type domains [ ] . the n-terminal v-type domain of sialoadhesin enables binding of sialic acid in the ligands of neutrophils, monocytes, nk cells, b cells, and cytotoxic t cells with sialoadhesin [ ] . the diverse roles of botanical pss have been reported. the antitumor effect of botanical pss was first known more than years ago when it was found that pss could alleviate cancer in cancer patients [ ] . for example, lentinan from lentinus edodes and schizophyllan from schizophyllum commune have antitumor activities and have been used clinically for cancer therapy [ ] . through several experiments, it was suggested that the antitumor effects of botanical pss might be due to potentiation of the response of precursor t cells and macrophages to cytokines produced by lymphocytes after specific recognition of tumor cells [ ] . also, botanical pss of mushroom are known to stimulate natural killer cells, t cells, b cells, and macrophage-dependent immune system responses [ ] . in addition, arabinogalactans of botanical pss possess complement fixation activity and induce chemotaxis of human macrophages, t cells, and nk cells [ ] . although the roles of botanical pss have been continuously identified, the mechanism of action is not clear yet, because the receptors of botanical pss remain unknown. however, many scientists have proposed that various pattern recognition receptors (prrs) might be receptors for botanical pss. for example, shao et al [ ] suggested that the receptor of ps from the roots of astragalus membranaceus, a medicinal herb, might be mr, tlr , b-glucan receptor, etc. schepetkin and quinn [ ] also introduced prrs including tlr , mr, and dectin- as potential receptors of ps polymers. especially, it was reported that specific glycans of botanical pss act as immune stimulating agents and effective t cell immune adjuvants [ , ] . therefore, it was suggested that animal lectins are strong candidates for receptors of botanical pss among various prrs, because animal lectins are specialized in recognizing various pss [ ] . with extensive research for gps, the roles and chemical composition of gps have gradually been discovered. for instance, the gps of p. ginseng, the most common ginseng, are composed of sugars including mannose of . w . % by weight, glucose of . w . % by weight, galactose of . w . % by weight, and arabinose of . w . % by weight [ ] . in addition, cvt-e , an aqueous extract of the roots of north american ginseng, is composed of % poly-furanosyl-pyranosyl-saccharides including rhamnose, glucose, galacturonic acid, galactose, and arabinose [ ] . therefore, some of the animal lectins might recognize these glycan structures of gps, because there are many animal lectins to recognize glycan structures of galactose, glucose, rhamnose, and mannose [ e , , , , , ] . this could be speculated from the extensive research of specific receptors for botanical pss in the above section. in particular, clustering of animal lectins might enhance the recognition of gps, since clustering of simple binding sites in oligomers of the animal lectin polypeptides dramatically increases the affinity for diverse ps structures [ ] . for example, byeon et al [ ] expected dectin- as a receptor of red ginseng acidic ps, which are known to interact with ps fractions such as bglucan and zymosan [ , ] . gps which are isolated from a variety of traditional medicinal ginsengs involved in various innate immune responses, such as the production of cytokines and maturation of dcs in vivo and in vitro, show potential to be immunomodulators with wide applications [ e ]. in addition, most of them are relatively nontoxic and do not cause significant side effects, which are major problems of immunomodulatory bacterial pss and synthetic compounds. thus, gps are becoming ideal candidates for therapeutics for collageninduced arthritis, inflammation, and inflammatory-related diseases like tumors, atherosclerosis, and so on [ , , , , ] . although the roles of gps are being continuously identified, the detailed mechanisms of actions are not clear yet, because the receptors of gps remain largely unknown. therefore, it is tempting to speculate that animal lectins could be strong candidates of receptors for gps. to prove this possibility, extensive researches for the specific structures of gps and the interaction between gps and animal lectins is required in the near future. by unraveling receptors of gps in vivo, it is possible to specifically understand the detailed mechanism for the immunological activities of gps in the immune system, giving insights into the development of gps as therapeutic biomaterials for many immunological diseases. the authors declare no conflicts of interest. inflammation, cancer, and targets of ginseng recent methodology in the phytochemical analysis of ginseng structural and antiinflammatory characterization of a novel neutral polysaccharide from north american ginseng (panax quinquefolius) total fractionation and analysis of polysaccharides 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pectin and a pectic arabinogalactan from vernonia kotschyana sch a study on the immune receptors for polysaccharides from the roots of astragalus membranaceus, a chinese medicinal herb botanical polysaccharides: macrophage immunomodulation and therapeutic potential mushroom immunomodulators: unique molecules with unlimited applications immunomodulatory dietary polysaccharides: a systematic review of the literature specific medicinal plant polysaccharides effectively enhance the potency of a dcbased vaccine against mouse mammary tumor metastasis in-sung jung, investors; korea institute of radiological & medical sciences, assignee. composition comprising polysaccharide extracted from panax ginseng preventing and treating liver diseases. us patent diversity in recognition of glycans by f-type lectins and galectins: molecular, structural, and biophysical aspects structural basis of lectin-carbohydrate recognition the beta-glucan receptor dectin- functions together with tlr to mediate macrophage activation by mycobacteria molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from korean red ginseng this paper was supported by konkuk university in . key: cord- - y uyf authors: vashishtha, vipin m.; john, t. jacob; pothapregada, sriram title: correspondence date: - - journal: indian pediatr doi: . /s - - - sha: doc_id: cord_uid: y uyf nan however, the academy is capable of going much beyond merely reporting of the cases. we have the expertise to lead investigations and offer solutions regarding diagnosis and management of these 'mystery illnesses'. already, few iap members are involved in the investigations of the ongoing recurring outbreaks in muzaffarpur in their own individual capacity. the infectious disease chapter of iap should come forward and contribute to ongoing investigations. it can organize brain storming sessions on the problem involving all the stakeholders, including state and central agencies. the local pediatricians, usually the iap members, are keys to the success of this endeavor. in fact, the government of india is short of technical advice on many issues pertaining to outbreak investigations and usually depends on multiple agencies -some of their own and some from outsides -for solving the mystery and instituting preventive measures, which ultimately do not go beyond recommending mass vaccination against japanese encephalitis in affected areas [ ] . outbreak investigation in india is in a dismal state. once an outbreak is spotted, usually by the media, the regional and central investigating teams arrive, carry out field survey, collect few biological samples, perform virological investigations, and if no organism is identified, label the outbreak to be caused by an unidentified viral agent [ ] . the problem is each team starts with a fixed mindset and looks for some infective pathology behind every outbreak. there is lack of coordination and synchronization of efforts, and ultimately they waste their energy either duplicating the efforts of others or pursuing a different approach unmindful of other's accomplishment. individual experts start investigating these outbreaks according to their own areas of interests. for example, in an outbreak of aes amongst children in andhra pradesh, india in , the virology group concluded it to be an outbreak of acute encephalitis caused by chandipura virus [ ] and the neurology team claimed the outbreak was caused by a neurovascular stroke called as "epidemic brain attack", not by any encephalitis [ ] . similarly, in muzaffarpur outbreaks, one group claimed it to be caused by heat stroke, and another hinted towards a toxin contained in the litchi, a locally grown fruit [ ] . the current scenario is bit murky and resembles like five blind people describing an elephant. the need of the hour is to adopt a fresh systematic approach with an open mindset. every effort must be made to characterize the clinical entity, whether it is an encephalopathy, encephalitis or a multisystem disease. thorough clinical, biochemical, histopathological and microbiological investigations, and autopsies must be performed to reach at a correct clinico-pathological diagnosis. second stage of investigations should consist of proper epidemiological investigations to identify any risk factor. based on these investigations, further studies that may include detailed toxicology can also be planned. the team should include epidemiologists, pathologists, neurologists, toxicology experts, public health experts and pediatricians. they should report to one designated authority spearheading all these teams. it is definitely possible to crack the mystery behind these recurring outbreaks and put an end to the prolonged ordeal of innocent children. history repeats in bihar, as pointed out by iap president [ ] . the 'mystery disease' recurred annually for decades in the north-western districts, during premonsoon months; it was called 'muzaffarpur encephalitis' first and later acute encephalitis syndrome, as icmr/ncdc failed to find viral etiology. again, a volunteer team (t jacob john, arun shah and mukul das facilitated by nk sinha and guided by maya thomas) investigated the problem. we diagnosed hypoglycemic encephalopathy and have advised bihar health ministry how to investigate etiology and to mitigate the risk factor of undernutrition [ ] . these non-infectious encephalopathy cases can be prevented or treated. in up, public education that cassia occidentalis is poisonous was enough to prevent the disease [ ] . in bihar, early infusion of % dextrose saved lives [ ] . in healthcare, incorrect diagnosis or treatment is medical negligence. in public health, incorrect management is public health negligence -consequent deaths amount to homicide by public health negligence [ ] . state officials believe that outbreak investigation is the responsibility of the central government. in delhi, the view is that health is state subject; states are responsible for diagnosis and prevention. the unfortunate victims are people without voice. india's health management system lacks organization with clear lines of command and is in need of review and repair. iap can serve as advocate, advisor and guide in this regard. we read with interest, the recently published article on the atypical manifestation of dengue fever in children [ ] . the authors have highlighted the occurrence of atypical manifestations like splenomegaly, neurological abnormalities, acute respiratory distress syndrome (ards), disseminated intravascular coagulopathy (dic), diarrhea and myopathy. in this context, we would like to share our experience of the atypical manifestations during the epidemic of dengue fever at puducherry in - . during the dengue fever epidemic, atypical manifestations were seen in children ( . %) and out of them splenomegaly ( . %), biphasic fever ( . %) and diarrhea ( . %) was the most common; . % of children with severe dengue infection had bleeding. the common mode of presentation of severe dengue infection was with features of peripheral circulatory failure ( . %) and hypotension ( . %) without bleeding. ards, myocarditis and dic were seen in four children, five children had encephalopathy and refractory shock, and three children had myositis. ultrasound abdomen showed gall bladder wall edema in % of cases. there were six deaths; common causes for poor outcome were ards, multiorgan failure, dic and refractory shock. since many children of dengue hemorrhagic fever had features of peripheral circulatory failure without volume __ november , correspondence spontaneous bleed, we found it difficult to classify them according to the dengue hemorrhagic fever guidelines given by world health organization in [ ] . our clinical experience suggests a need to relook at the classification of dengue fever and its management guidelines. with recent epidemics showing the changing pattern of presentation, atypical manifestations occur more often than previously reported [ ] . the awareness regarding atypical manifestations of dengue fever is lacking among the health care personnel at primary health centers from where these cases are more often referred. since the case fatality rate in children with severe dengue infection is high, pediatricians have a very important role to play to reduce the disease burden, and the minimum we can do is to update the health care personnel and community at various forums, about the various atypical manifestations of dengue for prompt recognition and management. puducherry, india. psriram_ped@yahoo.co.in misery of mystery of muzaffarpur indian health ministry orders encephalitis vaccination in select districts after more than deaths inadequate research facilities fail to tackle mystery disease a large outbreak of acute encephalitis with high fatality rate in children in andhra pradesh role of chandipura virus in an "epidemic brain attack cassia occidentalis poisoning causes fatal coma in children in western uttar pradesh misery of mystery of muzaffarpur recurrent outbreaks of hypoglycaemic encephalopathy in muzaffarpur disappearance of a deadly disease, acute hepatomyoencephalopathy syndrome, from saharanpur homicide by neglect? uncontrolled pediatric infectious diseases atypical manifestations of dengue fever dengue hemorrhagic fever: diagnosis, treatment, prevention and control. nd edn. geneva: world health organization dengue viral infection in children -a perspective key: cord- -grjrf n authors: ihekweazu, chikwe; ncube, fortune; schoub, barry; blumberg, lucille; ruggles, ruth; salter, mark; madhi, shabir; kessel, anthony title: a north/south collaboration between two national public health institutes – a model for global health protection date: - - journal: j public health policy doi: . /jphp. . sha: doc_id: cord_uid: grjrf n rapid international spread of emerging infections has increased interest in strategic collaborations, as they may be the best way to protect populations. strategic collaborations can build capacity in less-resourced settings. as specialised institutions that provide a stable locus of expertise, continuity of experience, scientific knowledge, and appropriate human, technical, and financial resources, national public health institutes (nphis) are well-prepared to tackle public health challenges. we describe how a collaboration between the nphis of england and south africa built a mutually beneficial professional relationship to help implement the who international health regulations, build capacity for health protection, and promote the exchange of information, advice, and expertise. we illustrate how this can be achieved in a mutually beneficial way. recognition of the potential for rapid international spread of emerging infections led the th world health assembly to adopt revised international health regulations (ihr) on may . countries are to share relevant information with the world health organisation (who) . the ihr describe what all countries must be able to do to identify and respond to public health threats. to develop the expertise required to respond to public health threats and to adhere to the ihr, specialised institutions are needed. such expertise is often found within national public health institutes (nphis) and collaboration among existing nphis is vital for health security at the national and global levels . a nphi is a science-based organisation, or network of organisations, that provides national leadership and expertise to achieve substantive, long-term improvements in the public's health . nphis generally lead disease surveillance and outbreak investigations, provide reference laboratory services (specialist diagnostic services for rare organisms and confirmatory tests requiring specialised infrastructure and resources), and advise their governments on development and evaluation of interventions in public health. many nphis in low-and middle-income countries lack resources and expertise to deliver on all such responsibilities . collaboration among nphis is one way to ensure they fulfil these functions for their populations and contribute to global health security. in , in recognition of this, jeffrey koplan (former director of the us cdc) and pekka puska (former director general of finland's national institute of public health and welfare -thl), through initial grants from the rockefeller foundation and the bill and melinda gates foundation, formed the international association of national public health institutes (ianphi). key objectives included enlivening international advocacy, a scientific network for nphis, and building capacity of nphis in less well-resourced countries . the national institute for communicable diseases (nicd) in johannesburg is the nphi for south africa. from a central location in johannesburg, it employs about staff, provides reference microbiology, epidemiology, surveillance, and public health research to support government responses to communicable disease threats. nicd also reinforces public health responses in other african countries aided by its biosafety level (bsl ) laboratory, one of two on the continent. (bsl laboratories provide the safest environment for working with dangerous pathogens, such as ebola and marburg viruses.) nicd is organised into functional centres that bring together expertise in both reference microbiology and epidemiology. south africa has one of the first and best-resourced national infectious disease control institutes on the african continent. but the country faces africa's largest burdens of hiv (human immunodeficiency virus)/aids and tuberculosis (tb). economic migration between south africa and its neighbours creates additional challenges for the control of infectious diseases. england's nphi, public health england (phe), established by statute in april , replaced a predecessor organisationthe health protection agency (hpa)that had been responsible for the protection of population health and other smaller public health organisations. creation of phe in signalled intent to protect and improve the nation's health and well-being, and to reduce inequalities. phe has about staff working across local, provincial, and national levels of government. for ease of reading we refer below to the organisation as hpa/phe. in , the united kingdom (uk) government published a new strategy document, health is global. it made a commitment to 'increase uk and global health security' by strengthening surveillance and response capacity to infectious diseases . it called for establishing long-term links with equivalent institutions in other countries. when updated in , the strategy defined global health outcomes in three broad areas for action: global health security, international development, and trade . then governments of the uk, including northern ireland, signed a memorandum of understanding (mou) with the republic of south africa for a reciprocal exchange of healthcare professionals. it was intended to enhance clinical and technical skills in both countries, and explore best practices. the uk chose hpa as one of the institutions to implement the mou. hpa welcomed the opportunity to establish a partnership with nicd in johannesburg. as an institute with a similar mandate and relevant expertise, it could enable its neighbouring countries to develop these skills. to cultivate global health work, the department of health committed in a grant of £ . million over years to the hpa. from this global health fund the secondment to south africa was funded. the nicd, through its various training programmes, had a long history of supporting other african countries to build their capacity. in some countries, such as the democratic republic of the congo, nicd responded more directly by investigating sources of emerging disease threats . initially structured as the hpa/nicd collaboration, it evolved to become the phe/nicd collaboration. we describe the hpa/phe collaboration with nicd and assess its benefits as an example of a well-supported collaboration between two public health institutes with similar mandates. we embarked on the collaboration to build a mutually beneficial professional relationship to contribute to: • implementing the who ihr, • building capacity for health protection, and • promoting exchange of information, advice, and expertise. we also describe risks and benefits associated with planning similar nphis collaborations in the future. we exchanged personnel, resources, and expertise across both institutions. the executive director of public health strategy who was responsible for global health at hpa/phe and nicd's executive director provided leadership and oversight. to refine strategy jointly, leaders of both institutions met in person at regular intervals in both settings, and by teleconference. staff reported progress to management and presented results from joint projects at institutional conferences. the hpa's board technical committee on global health and nicd's management group served as the governing body. these boards were the highest decisionmaking settings in the respective institutions. hpa/phe and nicd organised collaboration in two broad and complimentary areas: (i) long-term secondment from hpa/phe to nicd of a senior consultant epidemiologist, and (ii) a series of shortterm exchanges between specific departments in the two institutes. long-term secondment of a consultant epidemiologist hpa/phe collaborators organised a competitive process for selection of a consultant epidemiologist for secondment from hpa/phe to nicd for years ( - ). both institutes participated in the selection process and agreed on objectives for the duration of the secondment. these included: • executing specific epidemiology projects, • building epidemiological capacity at nicd, • supporting short-term hpa/phe secondees visiting each institute, • providing, by the senior hpa/phe epidemiologist, public health leadership within nicd, • enabling exchange of resources across both institutes plus support for a sustainable relationship between them. the consultant epidemiologist reported to the director of nicd in south africa and to hpa's executive director of public health strategy (later phe's director of international public health). at the start of the collaboration the two institutes agreed on objectives for short-term secondees. hpa/phe intended to expose senior public health registrars (doctors and other health-care professionals participating in a -year specialisation programme in public health) and scientists in the hpa/phe to situations they would be unlikely to encounter in the uk. specific objectives included: (i) developing expertise in the management of infectious disease outbreaks uncommon in the uk, and (ii) acquiring skills and confidence to manage outbreaks of these rare diseases should occur in the uk. all parties emphasised the importance of building public health response capacity to protect populations in an increasingly connected and interdependent world. the registrars working in south africa were meant also to experience the impact of a different health-care system on disease control activitiesincluding practicalities of meeting surveillance priorities with reduced human, technical, and financial resources. uk public health registrars eligible for selection would have completed their professional examinations and acquired a high level of relevant competence in health protection. the nicd chose to build capacity in specific skill areas through staff secondments to specific departments in the hpa/phe. selection for the short-term secondments from nicd to hpa/phe entailed several steps: • a steering committee comprised of senior colleagues from both institutes drafted selection criteria for individuals who would benefit from exchanges and for expected outputs. they managed the selection processes. • candidates from various departments vied for participation in exchanges, and through interactive workshops identified areas of interest. • a steering committee evaluated these bids based on their objectives, feasibility, public health value, the personal development opportunity for the individual, and usefulness for the host department's future planning. the steering committee of the hpa/phe and nicd collaboration, made up of senior members of both institutions who drove the collaboration, assessed the secondments. they invited the consultant epidemiologist, seconded from hpa/phe to nicd, to join the review. they used information gathered from review meetings, secondment reports, presentation of outcomes at conferences, and interviews with staff who benefitted from the secondment opportunities. in addition, each secondee was obliged to submit a proposal before the trip and a report afterwards, both of which formed part of the review. over a period of . years ( - ), a senior consultant epidemiologist from hpa/phe worked in nicd and staff from both institutions participated in short-term exchanges at the other's institution of weeks- months duration. as epidemiologists were an uncommon resource in south africa, nicd invited the seconded senior consultant epidemiologist to co-lead the centre for tuberculosis, supporting its transformation from a reference laboratory to a public health focused centre, integrating specialist epidemiology into the existing specialist microbiology service for tb. he used his expertise in epidemiology to help implement several important projects for the instituteconducting a national tuberculosis drug resistance survey, establishing a surveillance system for tb, and integrating laboratory-based tb surveillance with the electronic tb register used to collate clinical data. ncid made building capacity for epidemiology the key objective of the secondment. the seconded epidemiologist supported activities of the south african field epidemiology and laboratory training programme (safeltp), an existing programme at the nicd, developed in partnership with the centers for disease control, atlanta, usa. he taught several short courses as part of safeltp. colleagues in both institutes benefitted from short-term exchanges as anticipated. hpa/phe registrars, working with colleagues from their nicd host institution: • investigated an outbreak of sporotrichosis among mine workers, • conducted a door-to-door household survey following a community outbreak of a diarrhoeal illness related to contaminated water supplies in a semi-rural village, • audited management of human exposures to rabies in rural clinics, this 'learning by doing' in south africa enabled uk public health registrars to expand their epidemiological knowledge, skills, and abilities, and to perform epidemiology work in the field for diseases they would not typically see in their home country. similarly, short-term exchanges provided opportunities for colleagues from nicd to develop areas of expertise during their exchanges, including: • specific laboratory methods such as molecular diagnostics for tb, • hemagglutination inhibition and micro-neutralization diagnostics assays for influenza, • multiple-locus variable number tandem repeats analysis (mlva) for salmonella typhimurium and salmonella enteritidis typing, • methods for the identification of pathogenic fungi. the acquired expertise in diagnostic techniques were taken back to the host institute, nicd, and used to enhance laboratory capacity. similarly, skills in epidemiology and data management around de-duplication of large data sets, and methods in data analysis and in geospatial analysis were used in the home institution. areas of benefit to nicd . microbiology methods required to diagnose public health relevant diseases evolve continually. hpa/phe's specialist microbiology services provide a comprehensive range of clinical diagnostic and public health microbiology tests and services. elements of the uk's system exist in eight regional laboratories across england and in national centres. fourteen scientists from nicd spent - weeks with their counterpart laboratories in hpa/phe, updating their skills in specific methods and learning new approaches to diagnostics. . epidemiology methods came to be recognised a specific need for nicd. to build capacity, nicd epidemiologists and data analysts worked for months with their counterparts in the epidemiology section of hpa/phe. visiting nicd scientists took home skills not yet in use at nicd, spurring completely new areas of work at nicd, such as geospatial analysis of epidemiological data. the visits also advanced collaboration in tb surveillance plus other areas. an epidemiologist from nicd who spent time with the health-care associated infections surveillance team in hpa/phe is now planning a similar surveillance programme in south africa. . management expertise advanced when senior members of nicd staff spent shorter periods ( - weeks) with hpa/phe counterparts in the uk exchanging ideas on management approaches in specific areas of work and exploring ideas for collaborative projects. . unexpected areas of engagement evolved. nicd established, for example, a biocontainment engineering management and support program with assistance from hpa/phe colleagues. this undertaking grew out of a visit by hpa/phe engineers and specialists to assess engineering capacity and gaps in critical containment equipment maintenance at nicd. one nicd engineer visited the bsl laboratory in london, after which his hosts developed a training curriculum and training strategy for biocontainment staff. areas of benefit to hpa/phe . hands-on experience for hpa/phe staff working in south africa equips registrars to manage similar situations in the uk. rapid spread of sars in toronto in revealed a need for clinical and public health capacity to respond, [ ] [ ] and heightened awareness in the uk of the value of local expertise about uncommon infectious diseases. exchanges with the nicd prepared uk public health registrars for risks that could emerge from an increasingly diverse uk population and from london as a major hub of global travel. . specific departments in hpa/phe, including the tb and hiv/ sexually transmitted infections sections, used staff exchanges to form departmental level continuing relationships. joint projects illustrate the value of these on-going collaborations, for example, exploring the use of whole genome sequencing to guide the use of public health responses to tb in low-and high-incidence settings. . formal collaboration offers on-going informal access to mutually beneficial resources across both institutions. colleagues from both institutes call their counterparts to discuss outbreak situations, or diagnostic options for rare pathogens. when someone visited the counterpart institution, colleagues at the home institution had to assume responsibility for the tasks usually performed by the travelling colleague. when some tasks depended on skills in short supply, substitution proved to be a noteworthy challenge. so too was the delicate matter of achieving balance between the needs and priorities of the institutionsand desires and needs for skills yielding benefits to the individuals seconded. as project funds came to an end, the steering committee realized how difficult it would be to fulfil the expectations raised by this collaboration over the long term. this collaboration has continued, albeit at a slower pace, using core budget at both institutes, rather than donor funds. in an interconnected world, relationships with strategic partners may provide the best protection for the health of the public as they build capacity in less-resourced settings. our collaboration between two nphis has led not only to important public health results for both countries, but also facilitated the exchange of public health expertise and information. as planners and participants found the relationship benefitted both countries, it may guide others to establish mutually beneficial north/south professional and institutional arrangements. the strong uk-south africa relationship may constitute a model for future public health collaboration and support implementation of ihr. collaboration among nphis globally can increase our collective wisdom about using medical and public health services to contribute to understanding preventable causes of ill health . the hpa/phe-nicd collaboration strengthened international networks critical for responding to public health emergencies as intended under ihr. nicd, by virtue of its role and capacity, provides the first response to emerging diseases in southern africa. when not adequately contained, such outbreaks may become multi-country problems that need larger responses, as was the case during a marburg virus outbreak in angola . close working relationships are critical for an efficient response and aided by good communication between partner institutes. to build capacity with short staff exchanges, trust and mutual respect needs to be established beforehand. in our collaboration, participants felt they and the work benefitted from such an environment. as the evolution of uk's hpa into 'phe' coincided with the uk collaboration with south africa's nicd, staff of the latter observed the broadening of public health responsibilities beyond communicable diseases and environmental hazards in the uk. south africa is now considering a similar expansion and is now discussing the potential benefits of widening the current mandate of nicdbased in part on lessons learnt by south african colleagues in the uk. factors that contributed to a successful collaboration included: • a steering committee of senior staff members of both organisations assured quick resolution of bottlenecks. • logistics for the secondment was eased by a dedicated international office within hpa/phe and a senior administrator who took on the project management responsibilities in nicd. they managed all travel requirements, from the processing of visas and travel to arranging specific meetings among colleagues. the ianphi was formed in with a us$ million, -year grant from the bill and melinda gates foundation through emory university, the host and coordinating institution . it recognised the need for nphis to work more closely together. the association wanted countries to begin to coordinate their national public health efforts. nphis exist for the public good; improvements in nphis contribute to improving population health . globalisation and threats of new and re-emerging diseases mean that nphis are needed to ensure competent and efficient responses. nphis provide a stable locus of expertise, continuity of experience, scientific knowledge, and appropriate human, technical, and financial resources to tackle public health challenges both within and among countries . knowledge and expertise gained by one institute protects the population of that country and other countries with which it is shared. we have illustrated a mutually beneficial way to share. we urge the nphis of other countries explore this approach. we view the mutual benefits of the collaboration between nicd and hpa/phe as a success for the health is global strategy of the uk government. the strategy was designed in recognition of the complexity of our globalised world, the changed perspective it demands of us, and the new alliances we need to build to meet its challenges . . buss, p., koplan, j.p., dusenbury, c., binder director at the national institute for communicable diseases oxon), msc, ffph is a consultant in public health medicine at public health england mfphm is global health consultant at public health england phd is infectious disease specialist and executive director at national institute for communicable diseases director of international public health at public health england and formerly director for public health strategy at hpa. notes and references global public health security framework for the creation and development of national public health institutes public health in africa -the role of national public health institutes national public health institutes: contributing to the public good health is global: an outcomes framework for global health studies of reservoir hosts for marburg virus early diagnosis of sars: lessons from the toronto sars outbreak public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto largest ever marburg haemorrhagic fever outbreak improving the world's health through national public health institutes key: cord- - y y authors: shoemaker, jason e.; fukuyama, satoshi; eisfeld, amie j.; zhao, dongming; kawakami, eiryo; sakabe, saori; maemura, tadashi; gorai, takeo; katsura, hiroaki; muramoto, yukiko; watanabe, shinji; watanabe, tokiko; fuji, ken; matsuoka, yukiko; kitano, hiroaki; kawaoka, yoshihiro title: an ultrasensitive mechanism regulates influenza virus-induced inflammation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: y y influenza viruses present major challenges to public health, evident by the influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin and monocyte chemotactic protein , exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatory-associated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. influenza viruses present major challenges to public health, evident by the influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin and monocyte chemotactic protein , exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatoryassociated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. invading pathogens induce acute inflammation when molecular signatures are detected by pattern recognition receptors (prrs; e.g., rig-i like receptors [rlrs] and toll-like receptors [tlrs] ) expressed on tissue-resident immune cells and non-immune cell types. prr ligation triggers innate immune responses and leads to the induction of inflammatory and antiviral gene expression, which together function to limit pathogen growth, activate the adaptive immune response, and ultimately resolve the infection [ , ] . precise regulation of prr-mediated signaling is necessary to both avoid inadvertent tissue damage in response to non-pathogenic stimuli, and to prevent immunopathology resulting from excessive expression of inflammatory molecules. in essence, the ideal inflammatory response must exhibit a balance between appropriate activation against a genuine threat and self-limiting behavior once that threat has been controlled. despite its importance in maintaining normal tissue homeostasis and limiting pathogen-associated diseases, the mechanisms underlying the regulation of this balance are poorly understood. influenza a viruses are recognized by both tlrs and rig-i-like receptors (rlrs) [ ] [ ] [ ] [ ] [ ] , and some strains are potent inducers of inflammatory and antiviral gene expression. generally, lung tissues infected with pathogenic isolates exhibit high virus titers and robust inflammatory gene expression, as has been documented in in vivo studies with the spanish influenza virus [ , ] , highly pathogenic h n avian influenza viruses [ ] [ ] [ ] , and the h n pandemic influenza virus [ , ] . in contrast, seasonal influenza viruses typically replicate less efficiently, elicit more restrained inflammatory responses, and are usually not associated with lethal infections. recent evidence has implicated the level of virus replication in infected lung tissues as the primary phenotypic variable driving inflammation and lethal outcomes [ , ] . other data indicate that influenza viruses that exhibit significant differences in pathogenicity stimulate qualitatively similar host responses that differ primarily at the level of magnitude and kinetics [ ] . however, these studies have not revealed the mechanisms that account for the different profile dynamics observed in infections by high and low pathogenic viruses. such information would aid in clarifying not only how some influenza viruses induce lethal disease, but also the general mechanisms that regulate inflammatory balance. to characterize the dynamics of influenza virus-induced inflammation, we developed a novel approach to infer gene regulatory models from dynamic gene expression data. referred to as systems inference microarray analysis, our method builds on current approaches that use co-expression analysis to isolate modules of functional signatures in gene expression data and then extends these methods by fitting the gene expression modules to mathematical equations (models) by using segmented regression analysis. models can be created to look for strain-dependent responses and, unlike traditional differential expression analysis, to predict gene expression under new experimental conditions. by using this method, we set out to determine how influenza viruses that exhibit variable pathogenicity profiles influence the dynamics of the inflammatory response. to characterize the dynamics of the host immune response to specific virus isolates, we infected mice with pfu of three virus isolates with distinct pathogenicity profiles and harvested lung tissues at ). an initial inoculation of pfu was used as previous studies indicated that a high virus dose was needed to invoke different pathologies in h n and ph n -infected mice [ ] . as expected, lung virus titers (virus titers determined by plaque assay and reported in plaque forming units [pfu] per gram lung; fig ) indicated a clear hierarchy of mild, moderate, and severe virus-induced disease. specifically, the h n virus produced the highest lung titers and between days and post-infection, this virus also caused mortality in the animals whose lungs were to be collected on day post-infection (i.e., 'severe' disease). in contrast, all animals infected with the h n or ph n viruses survived the duration of the time course study; however, ph n -infected animals were visibly sicker and exhibited higher lung titers relative to those infected with h n at all time points observed , , , , , , , , , and h and , , and days), and virus titers in lung tissues were determined by plaque assays in mdck cells. error bars illustrate the standard deviation from the mean. the gray boxes at each time point identify significant pairwise differences between the means of the viruses indicated at the right of the figure panel (significance was determined by anova followed by tukey's honestly significantly different test, p < . ). *, three h n -infected mice intended for collection on day succumbed to their infections prior to sample collection. after the first h post-infection (i.e., 'moderate' and 'mild' disease, respectively). histopathological analysis of tissue samples collected on days , , and post-infection (fig ) also showed that h n -infected tissue exhibited the earliest, most severe signs of inflammation and inflammatory immune cell infiltrates followed by ph n -infected tissue, whereas h n -infected tissue showed mild evidence of inflammation and was most similar to tissue from the control mice (mock-infected mice). we next used co-expression analysis to integrate inflammation-associated gene expression differences between influenza-infected and control lungs into a systems level context. we first asked whether the expression of inflammation-associated genes clustered into modules of coexpressed genes. tissues from the same animals that were used to determine virus growth were used to evaluate changes in global lung transcriptional profiles. a total of microarrays were developed (three per time point for h n -infected, ph n -infected, h n -infected and control mice). one microarray was removed after reviewing replicate quality. after filtering transcripts for minimally confident variation (we required at least one time-matched, infected condition compared with mock-infected absolute fold change and a false discovery rate [fdr]-adjusted p-value < . ), the log of the normalized intensity of the retained transcripts ( , ) for all samples were then clustered by using the weighted gene co-expression network analysis (wgcna) algorithm [ ] . in all, distinct co-expression modules were identified (referred to as n , n , etc.; s table provides the module assignments for all transcripts). to identify the biological role of the host response modules, we performed functional enrichment analysis on each gene module by using david [ ] and toppcluster [ ] . because each module was comprised of positively and negatively correlated transcripts, we used the module eigengene (i.e., the first principle component of the gene expression matrix) to divide each module into two submodules containing transcripts that were positively or negatively correlated with the parent module's eigengene, denoted as kme+ and kme-(referred to as module membership), respectively. this procedure allowed us to look for biological processes with similar but opposing dynamic responses to the virus infections. functional enrichment analyses were then applied to each submodule by using two bioformatics platforms to ensure robust results. we identified two submodules (n kme+ and n kme-, referred to as simply n and n in the remainder of the text) that were enriched for inflammatory response and inflammationassociated pathway signatures by using both bioinformatics platforms, and these two modules became the focus of our study (table summarizes the functional enrichment results for the immune and inflammatory related annotations. the complete enrichment results from toppcluster and david are available in s table and s and s files). the n module was uniquely enriched for cytokine activity and type i interferon (ifn) regulating tlr and rlr pathways [ ] , as well as transcriptional signatures associated with ifn-regulated activity (i.e., the transcription factor binding sites [tfbs] of irf , irf , irf , isre, and nf-κb). additionally, n was the only module that exhibited significant enrichment with a compendium of established ifn-stimulated genes ('isgs'; table ; see methods. the list of isgs is available in s file). a more recent study identified ifn stimulated genes in immortalized, human airway epithelial (calu ) cells [ ] . of these, mouse homologs were annotated on the microarrays and of the homolog probes were assigned to the n module (fisher's exact test; pvalue < - ; odds ratio = . ), further associating n interferon-stimulated gene activity. in contrast, the n module was only weakly enriched for some cytokine activity related annotations and not enriched for any of the binding sequences of transcription factors that are members of canonical inflammatory pathways (such as interferons, interferon regulatory factor proteins, or nfκb). instead, it was primarily associated with several annotations related to leukocyte and lymphocyte activity (see summary of toppcluster enrichment results in table ; see s and s files). further analysis with cten [ ] , a platform for associating clustered gene expression data with specific cell types, found n to be highly enriched for genes expressed in macrophages in various cellular states (e.g., bone marrow-derived macrophages exposed to lipopolysaccharide [lps]) ( fig a; additional details available in s table) . the remaining immune-associated submodules (the kme+ n , n , n , and n submodules; described in table ) were enriched for several key immune processes such as antigen presentation, and t cell and natural killer (nk) cell activity, but their further assessment would be beyond our focus and the scope of this study. thus, bioinformatics analyses robustly associated the n module with inflammation, cytokine production, and type i ifn pathway activity-likely activated in resident lung cells-whereas the n module is associated with migration and activation of macrophages in the lung. each module was divided into submodules in which each member gene's expression was positively or negatively correlated with the module's eigengene (denoted kme+ and kme-, respectively). for each module, we provide the number of transcripts assigned to each submodule, the top david annotation clusters for each submodule (parenthesis shows the-log of the average enrichment p value for all annotations in the cluster), the top enriched biological processes determined using toppcluster (parenthesis shows the-log of the fdr-adjusted p value), the enrichment of established transcription factor binding sites in each submodule (tfbs; parenthesis shows the-log of the fdr-adjusted p value; performed using toppcluster), and the enrichment score of a set of ifn-stimulated genes (see methods; enrichment score is the-log of the fdr-adjusted p value). doi: . /journal.ppat. .t a closer examination of the expression dynamics of each of the inflammation response-associated modules revealed patterns of expression that were consistent with the biological roles predicted by our bioinformatics analyses. we used the scaled difference of the module eigengene to characterize the expression of all genes within each module. we subtracted the mean of the eigengene of the control samples from the eigengene of time-matched, virus-infected dynamics of inflammation-associated co-expression modules. panel (a) shows the enrichment scores (-log of the fdr-adjusted p value) of the genetic signatures of cell types in the n module as determined using cten [ ] . data corresponding to macrophage signatures in different cellular states are colored red (see s table for within the inflammation-associated modules (n and n ), the h n virus induced the earliest gene expression changes and the highest peak expression levels, corroborating previous observations that h n viruses are strong inducers of inflammatory and ifn response signaling in vivo [ , , ] . consistent with the prediction that n is involved in detecting virus in infected tissues, the n module eigengene was the most highly correlated with virus titer (pearson pairwise correlation, ρ = . ). module gene expression dynamics further suggested that the n module gene is associated with lymphocyte infiltration. exudate macrophages [ ] and neutrophil [ ] have been identified as factors of severe disease during influenza infection. to associate gene expression dynamics with changes of immune cell counts, a new population of mice were infected with the three influenza viruses, five mice per infection group were sacrificed on days , , and and the changes in the number of macrophages and neutrophils was assessed (see methods). unlike the previous study by brandes, et al. [ ] , strong neutrophil infiltration was not specific to fatal infections but, instead, infiltration of both cell types had a clear hierarchical relationship with the severity of the infection (fig d and e ). the n module eigengene exhibited a lesser correlation to virus titer (ρ = . ), but was tightly correlated to macrophage influx into the lung (ρ = . ; p-value < . student's t-test). of the module identified in the studied, n had the highest correlation to both macrophage and neutrophil influx (the correlation of macrophage and neutrophil influx and all module eigengenes are provided in s table; ρ = . ; pvalue < . ). the n module on the other hand was weakly but significantly correlated with immune cell infiltration (ρ = . [p-value = . ] for neutrophils; ρ = . [p-value = . ] for macrophages), but its eigengene was not the most highly correlated ( other module eigengenes had a greater absolute correlation). these results further associate n with immune cell-specifically macrophage-infiltration, while the sum of the bioinformatics, virus titer correlations and immune cell infiltration evidences associated n with inflammation and type i ifn pathway activity. a further advantage of a network approach is that the functional relevance of genes might be inferred from their positions within the co-expression network [ ] . we used the module membership (the correlation between the gene's expression and the module eigengene, kme) to isolate potential regulators of the n module. among the top intramodular hub genes (i.e., genes with the highest module memberships, see s table) , we found mnda, herc and cd and several interferon regulated, virus replication inhibitory genes such as oas and oas [ ] . herc is involved in ubiquitination [ ] . mnda is significantly up-regulated in monocytes exposed to interferon α [ ] while cd is a transmembrane protein expressed on antigen presenting cells and modulates activation of t cells, b cells and myeloid cells. we also observed that interferon stimulated genes tended to have higher intramodular hub rankings, suggesting a regulatory role for interferon (wilcoxon rank sum test, p-value < - ). we then considered the module membership rankings of transcription factors known to regulate interferon. of the established interferon regulatory factors that are members of the n module (e.g., irf , irf , irf , irf , stat , and stat . nfkb and nfkb were not assigned to n ), irf and irf had the highest module memberships (kme = . and . ; ranking = and , respectively). irf expression was also several orders of magnitude greater than irf (s table) . together, these findings corroborate our bioinformatics analyses by suggesting that n is regulated by interferon, and n expression likely results in enhanced cytopatchic effects and regulation of the lymphocyte immune response. network analysis further suggests that irf may play a regulatory role upstream of interferon transcription. previous studies have suggested that highly pathogenic influenza virus infections induce an irregular or disproportionate inflammatory response relative to seasonal influenza viruses, and that these differences occur early in the host response [ ] . for this reason, we sought to further explore the possibility of isolate-specific or isolate-independent response patterns of the cytokine-associated n module. we wanted to infer mathematical relationships that could describe when inflammatory-associated gene expression occurs and what magnitude of expression is expected. by using the eigengene as a representation of the scaled gene expression dynamics, we attempted to infer simple mathematical models that can be related to common signaling mechanisms. surprisingly, when we plotted the n sde for each isolate against the corresponding virus titer, we observed a consistent profile for all three viruses; regardless of intrinsic virulence, the fold change in n gene expression remained initially low and rapidly increased only after a virus titer of approximately~ pfu/g (of lung) was reached (fig a) . following activation, n gene expression increased as a function of virus concentration at the same apparent rate for all infection conditions, and more complicated dynamics were observed only during the later phase of the infection when virus clearance was observed (i.e., when the virus titers began to decrease). these observations suggest that ifn-regulated (n ) gene expression was induced by an ultrasensitive response mechanism controlled at the level of the virus titer rather than the virus's intrinsic virulence. ultrasensitive responses characterize the dynamics of several signaling pathways that regulate essential and often toxic biological processes such as the cell cycle [ ] and apoptosis [ ] . as shown in fig b, ultrasensitive responses are typified by an attenuated response to low levels of stimulation but a strong response occurs once a threshold level of stimulus is reached. cooperativity [ ] and positive feedback [ ] are two mechanisms that produce ultrasensitive responses. to formalize the hypothesis that the inflammatory gene response follows an ultrasensitive response profile, we selected a segmented linear model (slm, defined in fig c) to be a simplified representation of the ordinary differential equations normally used to model ultrasensitive responses, and we fit the n sde to an slm that was strictly a function of the virus titer. the optimal fit showed a threshold of . ± . pfu/g is required for n module activation to occur, after which the sde's rate of activation (a ) was . ± . log (pfu/g) - with an intercept (b ) of - . (unitless) (see the methods and s fig for additional details) . below this threshold, the model predicted minimal gene expression (a = . ; b = . ). the slm goodness of fit on the training data was an adjusted r = . while an adjusted r = . was observed when the data was fit to a linear model. a davie's test confirmed that a segmented model was a significantly better fit than a linear correlation model (p-value < . e- ). while the h n -infected lung tissue did not exceed an average peak virus titer of . pfu/g (peak titer occurs at hpi in fig a) , we observed increased transcriptional activity in h n -infected mouse lung tissues after this time point, suggesting either that the actual peak virus titer occurred between hpi and the subsequent time point ( hpi), or that the model-predicted threshold was slightly over-approximated. we next sought to validate the threshold model by attempting to predict cytokine-associated gene expression in influenza virus-infected lung when only the virus titers are known. for this, we selected the h n virus, which has previously been associated with an excessive illustration of an ultrasensitive response to increasing stimulus. in our model, the response is the change in inflammatory-associated gene expression and the stimulus is the lung virus titers. a segmented linear model (slm) can approximate key aspects of the ultrasensitive response. in this study, the response is gene expression and the stimulus is the concentration of virus in the lung (c) the mathematical definition of an slm. below some threshold virus titer (thr), the scaled eigengene (se) is approximated by a linear model that is a function of the log of the virus titer, the slope (a ), and the intercept (b ). after the threshold virus titer is reached, the slope and intercept parameters change (a and b ) to describe the se after activation. the parameters were fit to the data shown in panel (a) and used to predict the n se in newly infected mice. (d) shows a comparison of the h n n eigengene from an infection at a dose of pfu (as shown in fig ) and an infection at a dose of pfu. panel (e) compares the predicted scaled eigengene based on the slm versus the measured eigengene behavior in mice infected with pfu of the h n virus. panel (f) shows gene expression changes for selected constituent genes of the n module as a function of virus titer. cytokine response [ ] . we infected mice with pfu of the h n virus (a -fold reduced dose compared with that used in the experiments to fit the model), determined lung virus titers at the same time points used for the initial experiment (s fig), and then evaluated the segmented linear model's ability to predict cytokine-associated gene expression. first, we confirmed that the original transcripts assigned to the n module were again co-expressed, and thus we used the same transcripts originally assigned to the n module to determine the eigengene (see s fig for an analysis of the conservation of the n module between the two experiments). in this experiment, as expected, the peak average virus titer ( . ± . pfu/g) for the pfu dose was delayed compared with that for the pfu dose (s fig, compare to fig ) . moreover, the sde exhibited an -h delay in activation and a -h delay in peak expression compared with the pfu n eigengene (fig d) . importantly, based on the virus titers alone, the fitted segmented linear model accurately predicted n -like sde behavior (r = . for all time points and r = . for time points up to peak expression; fig e and s fig), and this could be further demonstrated at the individual gene level for specific n -associated transcripts (e.g., herc , stat and irf ; fig f) . these observations provide strong evidence that activation of inflammatory-associated gene expression is dictated by a specific virus concentration in infected tissue, and further suggest the novel possibility that the pulmonary innate inflammatory response has a nonlinear, ultrasensitive-like activation profile that promotes tolerance to low concentrations of virus. the dynamics of key inflammatory cytokines are consistent with an ultrasensitive response although transcriptional activation of ifn-stimulated and inflammatory gene expression is a reasonable measure of the effects of inflammation response stimulation, we reasoned that a bona fide ultrasensitive mechanism that regulates this response should be reflected in other aspects of the associated signaling pathway(s). indeed, of the cytokines associated with the n module, -including key inflammatory proteins, such as interleukin (il- ) and monocyte chemoattractant protein- (mcp- )-were significantly correlated with the n module eigengene (pearson's ρ . ; fdr-adjusted p-value < . ; see s table) . in addition, when the protein expression levels of these cytokines were plotted against the corresponding titer data, we observed dynamics similar to that of the inflammatory-associated n gene module. initially, protein expression was low but strongly increased only after the virus titers exceeded the threshold of~ pfu/gram determined in the gene regulation model (fig a) . in contrast, most of the protein levels of the other measured cytokines with transcripts that were not assigned to the n module did not show any obvious relationship to the proposed threshold response (s fig). the only major exceptions were lif, rantes, and il (s fig). lif's gene transcript was not annotated on the arrays whereas il 's transcript was not identified as differentially expressed and therefore was not included in the clustering study. the rantes transcript was included in the clustering study and assigned by the wgcna algorithm to n although the transcript's correlation to the n eigengene suggests it could also have been assigned to the n module (pearson correlation = . and . to the n and n eigengene, respectively). some proteins appeared to conform to the threshold model in ph n -infected, but not h n -or h n -infected, mice. these may be cytokines that have strain-dependent responses and do not conform to the model. overall, changes in n -associated cytokine protein levels in influenza virus-infected mouse lungs were consistent with the proposed virus-titer regulated threshold-mechanism underlying the ifn-mediated response. threshold-like behavior is observed on upstream and downstream components of the ifn signaling pathway. (a) lung tissues from the same infected mice used for the original gene expression analysis were subjected to cytokine array analysis. thirty-two cytokine protein concentrations were measured and log scaled (data points lower than the limit of detection for each protein were set to . to allow scaling), and the average fold change relative to uninfected, time-matched lung samples was determined. the heat map illustrates protein expression values for the cytokines that had transcripts assigned to the n module (a blue-to-yellow scale shows expression levels, as indicated by the color key at the top of the panel; time points in which the change in the cytokine levels were not significant [fdr-adjust p < . ] were set to zero); of these, exhibited expression profiles that were highly significantly correlated with the corresponding transcript and the n module eigengene (pearson's ρ . and fdr < . ; eotaxin and tnf-α were the only two that did not exhibit a significant, direct correlation). non-n cytokines are shown in s (fig b) . for the h n data, significant levels of pstat were observed at hpi which-as noted previously-corresponds to the time immediately after the virus titers in h n -infected mice reached their peak (see previous discussion). on the other hand, significant increases in phosphorylated irf (pirf ) were observed only in ph n and h n infections, whereas significant increases in ifn-β were observed only in the h n infection. changes in the levels of ifn-β and pirf occurred after significant increases in pstat and ifn-α occurred. as such, the change in the percentage of pstat was more closely correlated to the n eigengene than was that of pirf (correlation = . ± . and . ± . respectively), and significant increases in pstat corresponded to time points at which the mean virus titer exceeded the threshold level identified in the gene expression analysis (~ . pfu/g). the greater correlation of ifnα and stat activation to the inflammation-associated, n module's gene expression dynamics and the enrichment of the n module for the irf binding sequence (table ) suggest that the primary driver of the threshold-regulated, inflammatory gene response originates along the irf ! ifn-α ! stat axis (fig c) . our data reveal that the activation of the ifn-associated inflammatory and antiviral response program in influenza-infected mouse lung is characterized by an ultrasensitive response driven by the virus load. the power of the threshold model is illustrated by its ability to accurately predict gene expression in infected mice, and the data further suggest that the molecular basis of threshold behavior originates upstream of ifn-α production. threshold-like and ultrasensitive mechanisms are hypothesized to be necessary for effective management of critical cellular machinery in noisy environments, and are recognized players in the activation of the cell cycle [ ] , mitogen-activated protein kinase signaling [ ] , and apoptosis [ , ] . however, while a role for threshold behaviors have been postulated to be essential for filtering noise or errant signaling in complex biomolecular environments [ ] , our study is the first to directly link threshold-like behavior to the virus-induced innate immune response. the ultrasensitive response observed in this study provides additional insight into the mechanisms that drive severe pathologies during influenza infection. several works have suggested that viral load is a key determinant of pathology [ , ] while other works suggest that highly pathogenic influenza viruses induce early, strong inflammatory responses that are independent of the viral load [ , , ] . recently, it was observed that fatal influenza infections in mice map, with titers surpassing the threshold level identified in the slm indicated in red. (b) another set of mice was infected with pfu of h n , ph n , or h n and lung tissues were harvested ( mice per infection per time point) for immunoblot (stat , phosphorylated stat , irf , and pirf ) or elisa (ifnα, ifn-β) analysis; virus titers were also determined. the heat map indicates the mean percentage of phosphorylated protein (purple) or the mean interferon concentration (pg/ml; green), and virus titers for each time point are indicated below. only significant changes are shown in the heat map (fdr-adjusted pvalue < . relative to mock-infected samples). titers highlighted in red indicate that the threshold level (as determined in the initial analysis) was exceeded. panel (c) shows a schematic of known canonical pathways that regulate ifn production in response to virus infection. black arrows denote induction whereas red lines denote points at which influenza virus is known to impair these pathways. ifn-α is primarily produced by antigen-presenting cells (apcs) whereas ifn-β is produced in virus-infected, non-apc cells. both ifns can induce stat phosphorylation and expression of isgs in responding cells. doi: . /journal.ppat. .g ultrasensitive mechanism regulates influenza-induced inflammation coincide with a strong influx of neutrophils in what the author's describe as a viral load-independent, "feedforward" inflammatory circuit [ ] . the ultrasensitive response suggested by our study consolidates these hypotheses by suggesting that viral load drives cytokine production (and in turn immune cell infiltration) in a nonlinear manner which is capable of producing states of high and low innate immune responses. characterization of key aspects of the inflammatory response, such as the onset and peak inflammatory gene expression, require a high temporal resolution of the virus growth and host response dynamics; an experimental design that was unique to our study. the ultrasensitive response model does not negate the significance of neutrophil infiltration [ ] in determining fatal infections but suggests that viral load drives the high and low innate immune states. the observed threshold may represent the transition to immunopathology; as indicated by the histopathology results (fig ) . moreover, the influenza virus' ns protein is crucial for inhibiting the interferon-mediated antiviral response [ ] . the ns protein of three viruses used in this study have the sumo acceptor site that indicates interferon antagonism capability [ ] [ ] [ ] . additional studies with ns -mutated viruses and other pathogens may better reveal strain-dependencies for the observed thresholding behavior. the ultrasensitive response further suggests that the innate immune response has a limited capacity to respond to influenza virus infection and supports the development of immunomodulatory therapies. interestingly, after the threshold was exceeded, the rate of activation for inflammatory and interferon-associated gene expression (n ) was conserved for a moderately pathogenic and deadly viruses (fig a) . the conserved rate of activation implies that the immune response detects the virus concentration but not the virus growth rate; suggesting the innate immune response is naturally limited in its ability to respond to high growth influenza viruses. additionally, studies in knockout mice indicate that type i ifn-associated pathways are essential for protection during primary infection [ ] and that earlier initiation of these pathways coincides with increased survival in mice infected with highly pathogenic isolates [ ] . in combination with these studies, the findings here suggest a novel means of protecting high risk groups by treating them with compounds that target the molecular mechanisms responsible for the threshold behavior. lowering the threshold required to induce the cytokine response may be a means of providing protection from severe influenza infection. since these compounds would target host proteins, such treatments would be effective against various influenza virus strains. data from the viruses studied here suggest that post-threshold, inflammatory gene expression primarily reflects interferon-regulated tissue damage, but time-course data from additional highly pathogenic viruses are needed to assess the degree to which interferon activity is associated with virus growth suppression. the a/california/ / h n virus (ph n ) was received from the centers for disease control and prevention. the a/kawasaki/utk- / h n virus (h n ) served as a reference for a seasonal influenza, whereas a fatal human isolate, a/vietnam/ / h n virus (h n ), served a highly pathogenic virus. all mouse experience were performed in accordance to the university of tokyo's regulations for animal care and use. these regulations were approved by the animal experiment committee of the institute of medical science, the university of tokyo (approval number: pa - ). the committee acknowledged and deemed acceptable the legal and ethical responsibilities for the animals, as detailed in the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology, . all experiments with h n viruses were performed in biosafety level containment laboratories at the university of tokyo, which are approved by the ministry of agriculture, forestry, and fisheries, japan. five-week old c bl/ j, female mice were obtained from japan slc. for all experiments, mice were anesthetized with isoflurane and intranasally inoculated with either or pfu of virus. initially, mice were inoculated with pfu of the h n , ph n , or h n virus or mock-infected with pbs (a total of mice). at time points, mice per group were humanely sacrificed and their lungs harvested. the lungs were sectioned and used to assess virus titers (right-upper lobe), cytokine levels (right lower), and the initial gene expression that was used to train the segmented linear model (left-lower). separately, mice were infected with pfu of the h n , sacrificed at the same time points, and lungs sections obtained as described previously to provide the model validation data. the same inoculation method was used for all mice in this study. the numbers of mice used for flow cytometry, western blot, and interferon protein assay experiments are specific in the corresponding sections. for cytokine and chemokine measurements, mouse lungs were treated with the bio-plex cell lysis kit (bio-rad laboratories, hercules, ca) according to the manufacturer's instructions. concentrations of other cytokines were determined with the bio-plex mouse cytokine -plex and -plex panels (bio-rad laboratories) array analysis was performed by using the bio-plex protein array system (bio-rad laboratories). virus titers were determined by plaque assay using mdck cells. mouse lung tissues were placed in rnalater (ambion, ca), an rna stabilization reagent and stored at - °c. all tissues were thawed together and homogenized for minutes at hz using a tissuelyser (qiagen, hilden, germany) as per the manufacturer's instructions. from the homogenized lung tissues, total rna was extracted with the rneasy mini kit (qiagen, hilden, germany) in accordance with the manufacturer's instructions. cy -labeled crna preparations were hybridized onto agilent- whole mouse genome x k microarrays for h at °c. feature extraction software version (agilent technologies) was used for image analysis and data extraction, and takara bio provided whole array quality control metrics. microarray normalization, replicate quality, annotation, and statistical analysis differential expression was assessed by using a linear regression model. by using the limma package [ ] version . . from bioconductor, probe intensities were background corrected by using the "norm-exponential" method, normalized between arrays (using the quantile method), and averaged over unique probes ids. replicate quality was assessed using hierarchical clustering, resulting in the removal of a single array (the array corresponded to a sample collected at hpi in h n -infected mice.) probe intensities were fit to a linear model that compared data from infected samples to time-matched data collected from uninfected mice. probes were annotated by matching to the probe names in the mgug a version . mouse annotation database available from bioconductor. all arrays in this study have been deposited on the geo expression omnibus (gse ). unsigned co-expression networks were constructed by using the blockwisemodules program from the wgcna package version . . [ ] in r. the analysis was performed with several different parameterizations to ensure robust clustering. for the results reported in this text, we removed all probes that did not have a confident fold-change greater than (fdr < . ) for at least one infected-tissue to control-tissue, time-matched comparison. we then clustered the log of the normalized intensities for all microarrays (corresponding the three samples for each time-point for each infected or control population with the exception of data from h n -infected mice at hpi which had two samples). based on the scale-free topology characteristics curve, a power of n = with no reassignment after clustering (reassignthreshold = ) and a maximum cluster size of probes was used. we generally observed that allowing gene reassignment between the modules led to poorer clustering based on the distribution the gene's module memberships (the correlation between a gene and the eigengene of the module to which it had been assigned. s fig illustrates the distribution of the gene kmes for modules n and n ). we then repeated the clustering using different powers (ranging from to ), allowing different cluster sizes, different subsets of the expression data (e.g., clustering data from each infection separately or together), or relaxing the differential expression condition. in all clusterings performed, the two modules discussed in the text were identifiable. fisher exact tests between each clustering run were used to determine whether the initial modules were significantly conserved under different parameterizations. we also considered if the n module would be isolated when using signed versus unsigned network construction. we constructed a signed co-expression network and found that % of the kme+ n genes are again clustered and confirmed that the gene expression dynamics were maintained (see s fig) . toppcluster [ ] and david [ ] were used for gene ontology and pathway enrichment, and toppcluster was also used for transcription factor binding site enrichment analyses. david uses clusters of related annotations constructed from several annotation databases (e.g., pathway and gene ontology annotations) to determine the function of a set of genes and scores the enrichment by averaging the unadjusted p-values (determined by fisher's exact test) of the annotations within the cluster. toppcluster uses hypergeometric tests to determine the enrichment between a set of genes and gene lists contained in categories (databases) detailed in the toppgene suite [ ] . the databases include cis-regulatory motif data [ , ] , referred to as transcription factor binding sites (tfbs) in the text. both tools were used with their default settings and the gene universe was considered to be all annotated mouse genes. for each module, we considered the enrichment of all genes assigned to the module and the kme+ and kmesubsets. generally, the enrichment analysis of the whole module gene set reiterated the enrichment results of the kme+ and kme-subsets albeit with slightly lower but still significant enrichment. since both tools returned similar go and pathway enrichment results, we summarized the functional and pathway enrichment results in table using the results from david. the enrichment of interferon stimulated genes was determined by using a list of interferon stimulated genes from the interferon stimulated gene database [ ] that was downloaded on may , (see s file). for each module, all module genes and the kme+ and kme-subsets were tested for enrichment using fisher's exact test in r. the p values were adjusted to control the false discovery rate. cten [ ] was used to determine enriched cell signatures in select co-expression modules. the enrichment score reported is the-log of the false discovery rate. model fitting and validation was performed in r using the 'segmented' package [ ] . five mice per time point per infection were infected with pfu of the described virus. five uninfected (naïve) mice served as negative controls. whole lungs were collected from mice, and incubated with collagenase d (roche diagnostics; final concentration: μg/ml) and dnase i (worthington; final concentration: u/ml) for minutes at °c. single-cell suspensions were obtained from lungs by grinding tissues through a nylon filter (bd biosciences). red blood cells (rbcs) in a sample were lysed with rbc lysis buffer (sigma). samples were resuspended with pbs containing mm edta and . % bovine serum albumin (bsa), and cell number was determined by using a disposable cell counter (onecell). to block nonspecific binding of antibodies mediated by fc receptors, cells were incubated with purified anti-mouse cd / (fc block, bd biosciences). cells were stained with appropriate combinations of fluorescent antibodies to analyze the population of each immune cell subset. the anti-f / (bm ; ebioscience) antibodies were used. all samples were also incubated with -aminoactinomycin d (via-probe, bd biosciences) for dead cell exclusion. data from labeled cells were acquired on a facsaria ii (bd biosciences) and analyzed with flowjo software version . . (tree star). three mice per time point per infection group were infected with pfu of the described virus. the primary antibodies of mouse anti-stat (phospho tyr ) mab (ab , abcam), rabbit anti-irf (phospho ser ) mab ( , cell signaling), and mouse anti-βactin (a ; sigma-aldrich) were used; the secondary antibodies were hrp-conjugated antimouse igg antibody (ge healthcare) and hrp-conjugated anti-rabbit igg antibody (ge healthcare). mouse lungs were collected and homogenized with ripa buffer (thermo scientific, rockford, il, usa) containing proteinase inhibitor (roche, mannheim, germany) and phosphatase inhibitor cocktails (sigma-aldrich, saint louis, missouri, usa). the lysates were then briefly sonicated and centrifuged. each sample was electrophoresed on sodium dodecylsulfate polyacrylamide gels (bio-rad laboratories, hercules, ca, usa) and transferred to a pvdf membrane (millipore, billerica, ma, usa). the membranes were blocked with blocking one (nacalai tesque, kyoto, japan) for min at room temperature, and then were incubated with the primary antibodies overnight at °c, followed by the secondary antibodies. they were then washed times with pbs plus tween (pbs-t) for min and incubated with secondary hrp-conjugated antibodies (as described above) for min at room temperature, followed by three washes with pbst. specific proteins were detected by using supersignal west femto maximum sensitivity substrate (thermo scientific, rockford, il, usa). photography and quantification of band intensity were conducted with the versadoc imaging system (bio-rad laboratories, hercules, ca, usa). the quantity of target bands from each sample was standardized by their respective β-actin. three mice per time point per infection group were infected with pfu of the described virus. half the lung of each mouse was dissolved in ml of ripa buffer. we measured the interferon-alpha and interferon-beta by using elisa kits (# , # , pbl assay science, nj, usa) according to the manufacturer's instructions. plates were read at an absorbance of nm using a versa max plate reader (moleculardevices, menlo park, ca). additional gene set overlap tests were performed in r with all of the genes annotated on the array as the reference (background) set. statistical tests to compare means within the western blot, flow cytometry, immune cell count and protein assay data sets were performed in r using the 'multcomp' package [ ] . supporting information s fig. the scaled difference of the module eigengene. the eigengene is the first principle component (equivalently the first eigenvector) of a matrix of gene expression data. in clustered data in which all genes are highly correlated (as in the case of wgcna), the eigengene is a scaled approximation of how gene expression changes for all genes in a module (i.e. cluster) across experimental conditions. the eigengene of module n is shown in (a) for the experimental conditions considered in this study ( infection conditions and time points). each point represents data from a single animal in each experimental condition ( animals per condition except for h n -infected animals at hpi which had ; total of samples). while the eigengene illustrates the changes in gene expression of module member genes, it is difficult to interpret the changes with respect to the control data. we therefore scaled the eigengene by subtracting from time-matched data the average of the eigengene from mock-infected animals and then dividing by the highest average eigengene for any experimental condition. the scaled difference of the eigengene (sde) is shown in (b). the sde now represents the fraction of greatest gene expression (i.e., the fraction of the largest log fold change in gene expression observed between all time-matched, infected-to-control samples). for example, the sde peaks in experimental condition (h n -infected animals at hpi), corresponding to the condition in which the log fold change was greatest for n member genes (see fig ) . the sde for ph n -infected animals at day pi (condition ) is~ . . we would expect the log fold change to be approximately half of that observed in h n -infected animals at hpi (see the heatmaps in fig ) . for completeness, we overlay the mean (lines) and the standard deviation to create the segmented linear model to describe the relationship between the n module eigengene and the virus titer data, we selected all titer data that occurred prior to and included the peak of the eigengene and then scaled the data such that the eigengene was bound between [ , ]. the model was then fit to the scaled data. panel (a) shows the time points selected as training data from each infection data set (indicated by the orange dots); and panel (b) shows the number of data points available for different ranges of the virus titer (top) and the segmented model's fit to the training data (bottom). in panel (c), we used the residuals (i.e., the difference between the predicted values and the actual values) to compare the slm's accuracy to that of a simple linear model. the line is the running average and the shaded region is the % confidence interval of the mean. the mean of the residuals of the segmented model was always near zero for the full range of virus titers and, thus, is a better fit than the linear model. two procedures were applied to determine whether the n genes that we identified as co-expressed in the original clustering analysis were also co-expressed in tissue from mice infected with pfu of h n virus. (a) compares the correlation between all , n gene transcripts and the eigengene (referred to as the kme) in the original gene expression data ( pfu infection conditions (referred to as ' pfu')) and the pfu infection condition (referred to as ' pfu'). more than % of the genes exhibited a kme > . . (b) the wgcna algorithm was repeated with all differentially expressed genes (fdr-adjusted p-value < . and fold change > for at least time point) from the pfu h n virus infection, and then the fisher's exact test was used to identify modules enriched for n genes. of the modules in the newly constructed co-expression network, only one module was enriched with the n transcripts. specifically, of the genes originally assigned to n , (benjimini-hochberg-adjusted p-value < . e- ) were differentially expressed and assigned to the same module in the pfu infection condition, as illustrated by the venn diagram. . the h n - pfu eigengene was constructed using the same set of probes assigned to the n module from the pfu infection condition and scaled to between [ , ]; and then the segmented linear model trained to the pfu h n , ph n , and h n data was used to predict h n - pfu scaled eigengene values. panel (a) shows a comparison of the predicted (black) and actual (pink arrows depicting the temporal evolution of the gene response) eigengenes, with error bars illustrating the standard deviation of the eigengene and of the log of the virus titer. panel (b) shows how the prediction residuals are distributed over time. for each time point, individual data points (black points) are shown, as well as the average (red points) and standard deviation (gray bars). the greatest deviations occurred at d and d , which was expected as the model was designed only to predict the onset of gene activation and the peak gene expression (peak of the eigengene). on days and post-infection, both the virus titers and gene expression has already peaked and are declining. panel (c) shows how the prediction residuals are distributed across the spectrum of observed virus titers, as compared to a linear model directly fit to the h n - pfu n eigengene. individual residuals are indicated by points, and the running average and the % confidence intervals are shown by the colored lines and the gray shading, respectively. as for the pfu infection condition, the segmented model performed well across the entire virus titer spectrum, and was significantly better than a linear model fitted directly to the data. (tif) s fig. protein concentrations of non-n module cytokines. as described in the fig leg end, cytokine concentrations were assayed in the lungs of mice infected with pfu of h n , ph n , and h n and compared with those in lung tissues from mock-infected animals. this heat map illustrates protein expression values for non-n -associated cytokines ( cytokine expression profiles are shown; il- (p )( ) was not detected at any time point and is not included), with a blue-to-yellow scale indicating expression levels (see the key to the right of the panel). the module to which the protein's mrna transcript was assigned during clustering is shown on the right hand side of the heat map (proteins whose gene transcripts were not de are labeled 'na'), and the average virus titers are shown below the heat map (red indicates that titers exceeded the threshold concentration predicted by the segmented linear model). while il- and leukemia inhibitory factor (lif) exhibited protein expression patterns consistent with n module behavior, the transcripts mapping to these proteins were not differentially expressed and were excluded from the co-expression network construction. (tif) s fig. virus titer and immunoblot data from an additional infection experiment with pfu of h n , ph n , or h n virus. as described in the fig legend (panel b) , an additional set of mice was infected with pfu of h n , ph n , or h n virus to determine total and phosphorylated levels of transcription factors by means of immunoblotting. here, virus titers (with standard deviation indicated by gray bars) for each infection condition are shown in panel (a), and immunoblot results are shown in panels (b) and (c). for immunoblot analyses, relative protein concentrations were determined by calculating the ratio of the gray intensity of the measured protein (iκbα, total irf , total irf ['irf '], phosphorylated irf [pirf '], total stat ['stat '], and phosphorylated stat ['pstat ']) relative to the gray intensity of actin (i.e., the loading control; referred to as the relative signal intensity, rsi) in each tissue sample. a linear model was used to compare the mean rsi of each protein at each time point to the mean rsi measured in uninfected animals (referred to as 'naïve'), and a significant difference was defined as having a false discovery rate (fdr) < . . the mean rsi of total irf , irf , and iκbα did not significantly deviate from the naïve data, but pstat , pirf and total stat significantly differed from the naïve data at several time points (signifi- the intramodular correlation (kme or correlation between each transcript and the eigengene) can be used to assess clustering quality. we show boxplots to illustrate the distribution of the kmes for all transcripts belonging to the n and n modules. (tif) s fig. applying signed co-expression network analysis also clusters the n kme+ transcripts. the n module presented in this work was identified by developing an unsigned co-expression network and then focusing on the kme-and kme+. therefore, the n kme+ set of genes should also cluster when developing a signed co-expression network. we confirmed this by repeating the wgcna procedure for signed network (power = ) . of the n kme+ transcripts, . % were assigned to the same cluster in the signed co-expression network (the new module is referred to as n signed). furthermore, . % if the n signed transcripts were n kme+ transcripts. we then confirmed that the same eigengene dynamics were observed. the scaled difference of the eigengene (sde) of the n signed module is shown versus time (a) and versus virus titers (b). (tif) s file. each of the kme+ submodules, labeled n , n ,. . .,n were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statisticis can be found at david.abcc.ncifcrf.gov (xls) s file. each of the kme-submodules, labeled n , n ,. . .,n were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statistics can be found at david.abcc.ncifcrf.gov (xls) s file. a list of interferon stimulated genes received from the interferon stimulated gene database. (xlsx) s table. the co-expression module to which each transcript was assigned. for each transcript we provide the entrez gene id, the gene symbol, the module it was assigned to and the correlation between the transcript's expression dynamics and the expression dynamics of its assigned eigengene (kme). module n is the set of transcripts which the algorithm did not identify as co-expressed. (xlsx) s table. enriched annotations identified using toppcluster. each kme+ or kme-submodule was analyzed in toppcluster for enriched domain, go biological process, go molecular function, go cellular component, mouse phenotype, pathway or transcription factor binding site annotations. columns a-d provide information on the annotation, including the category to which the annotation belongs, the database specific annotation id and the full title of the annotation. columns d-at contain the enrichment score for each annotation in each submodule. the enrichment score is the-log of the false discovery rate [fdr]-adjusted p value. a threshold enrichment score of (fdr-adjusted p < . ) was required to be considered as significantly enriched. (xlsx) s table. cten analysis of n module. the probes assigned to module n were analyzed in cten-a platform for identifying the genomic signatures of select cell type in microarray data. the enrichment score reported for each cell type is the-log of the false-discovery adjusted p value. (xlsx) s table. a heatmap of the gene expression of the probes assigned to the n and n modules. arrays were developed from the lungs of mice infected with h n , ph n , or hpai virus at time points. one array from the hpai infected data set was removed due to quality concerns. for each transcript, we provide annotation information (probe name, systematic name, entrez id, gene symbol, and gene name), identify to which module the transcript was assigned, the kme (the pearson correlation coefficient between the transcripts expression and the eigengene of the module it was assigned to), and the log fold change in the transcript's expression across all arrays used the study is illustrated by a heatmap. (xlsx) s table. correlation between the changes macrophage and neutrophil cell counts and each module eigengene. (xlsx) s table. correlation between changes in cytokine protein levels and cytokine gene transcript levels. (xlsx) regulation of toll-like receptor signaling pathways in innate immune responses immune signaling by rig-i-like receptors cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells recognition of single-stranded rna viruses by toll-like receptor differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates '-triphosphate rna is the ligand for rig-i aberrant innate immune response in lethal infection of macaques with the influenza virus genomic analysis of increased host immune and cell death responses induced by influenza virus lethal dissemination of h n influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection h n influenza viruses: outbreaks and biological properties fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia in vitro and in vivo characterization of new swine-origin h n influenza viruses integrated network analysis reveals a novel role for the cell cycle in pandemic influenza virus-induced inflammation in macaque lungs h n influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load viral replication rate regulates clinical outcome and cd t cell responses during highly pathogenic h n influenza virus infection in mice specific mutations in h n mainly impact the magnitude and velocity of the host response in mice wgcna: an r package for weighted correlation network analysis resources: expanded annotation database and novel algorithms to better extract biology from large gene lists toppcluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems snapshot: pathways of antiviral innate immunity pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses cten: a web-based platform for identifying enriched cell types from heterogeneous microarray data lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes integrated clinical, pathologic, virologic, and transcriptomic analysis of h n influenza virus-induced viral pneumonia in the rhesus macaque ccr + monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection transcriptomic analysis of autistic brain reveals convergent molecular pathology extracellular '- ' oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity systematic and quantitative assessment of the ubiquitin-modified proteome the transcriptional landscape of the mammalian genome ultrasensitivity in the regulation of cdc c by cdk bistability in apoptosis: roles of bax, bcl- , and mitochondrial permeability transition pores ultrasensitivity and positive feedback to promote sharp mitotic entry ultrasensitivity in the mitogen-activated protein kinase cascade identifying fragilities in biochemical networks: robust performance analysis of fas signaling-induced apoptosis the ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein d binding influenza a virus lacking the ns gene replicates in interferon-deficient systems identification of the non-structural influenza a viral protein ns a as a bona fide target of the small ubiquitin-like modifier by the use of dicistronic expression constructs modification of nonstructural protein of influenza a virus by sumo sumoylation affects the interferon blocking activity of the influenza a nonstructural protein ns without affecting its stability or cellular localization myd signaling is indispensable for primary influenza a virus infection but dispensable for secondary infection toll-like receptor pre-stimulation protects mice against lethal infection with highly pathogenic influenza viruses linear models and empirical bayes methods for assessing differential expression in microarray experiments toppgene suite for gene list enrichment analysis and candidate gene prioritization systematic discovery of regulatory motifs in human promoters and utrs by comparison of several mammals molecular signatures database (msigdb) . functional classification of interferon-stimulated genes identified using microarrays segmented: an r package to fit regression models with broken-line relationships simultaneous inference in general parametric models we would like to thank susan watson for editing the manuscript. key: cord- -di oqe authors: zhou, xianghui; li, qingling; zhou, xincan title: exacerbation of chronic obstructive pulmonary disease date: - - journal: cell biochem biophys doi: . /s - - - sha: doc_id: cord_uid: di oqe chronic obstructive pulmonary disease (copd) is a disease with high prevalence and substantial associated economical burden. a significant determinant of quality of life, long-term survival, and health care costs is an acute exacerbation of copd. acute exacerbations are provoked by respiratory viruses, altered airway microbiome, and environmental factors. the current treatment options are limited. in order to develop specific therapeutic measures, it is important to understand how acute exacerbations evolve. this review focuses on pathophysiology of stable and exacerbated copd. high prevalence and substantial associated economical burden set chronic obstructive pulmonary disease (copd) apart from other respiratory diseases. worldwide, million people were estimated by world health organization in to have copd. the estimates of the prevalence of this disease in different countries range between and % [ ] . in china, the prevalence of copd was estimated at . % by a recent study [ ] . being a sixth leading cause of deaths in the s, copd now ranks among top three leading causes of death worldwide, with more than million deaths in . the mortality rates of this disease are surpassed only by ischemic heart disease and stroke ( fig. ) . furthermore, world health organization estimates that the overwhelming majority (almost %) of copd-associated deaths occur in developing countries due to insufficient prevention and control. it is estimated that prevalence of copd will increase within the next years, while mortality will remain at the current rate. copd is substantial burden to both patients and the health care system. patients, especially those with severe disease, can be limited in their daily activities because of dyspnoea, cough, and fatigue. this constant handicap is further worsened during acute exacerbations of this disease. copd exacerbations deteriorate patients' quality of life and survival [ ] [ ] [ ] [ ] . a significant proportion of patients with copd experiences frequent exacerbations (''frequent exacerbators'') [ ] . frequent exacerbations are associated with increased likelihood of copd-associated death [ ] . furthermore, copd exacerbations are a very significant contributor to copd-associated health care costs, mostly due to hospitalizations. thus, the costs of copd hospitalizations were estimated to be $ . billion in the usa in , with an average cost of hospitalization of $ per patient [ ] . in china, the cost of inpatient care for acute exacerbation of copd has also been estimated high [ ] . given that copd exacerbations are a significant contributor to morbidity and mortality associated with this disease, and cause substantial financial costs, mainly due to hospitalization, it is crucial to better understand the factors leading to copd exacerbations to be able to develop effective measures to prevent and/or treat these exacerbations. the present review will focus on current understanding of the pathophysiology of copd exacerbations, and current and potential preventive or therapeutic measures. the global initiative for chronic obstructive lung disease (gold) defines copd as a disease that is ''usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases'' [ ] . these noxious particles or gases are inhaled with cigarette smoke or smoke from burning biomass fuel, the latter being more relevant for low-income countries [ ] . anatomically, the abnormal processes in the airways of patients with copd develop as poorly reversible or irreversible airway obstruction, or emphysema (rupture of the inner walls of alveolar sacs with creation of larger air spaces with trapped air), or a combination of both. the disease is frequently accompanied by mucus hypersecretion that leads to productive cough and may impair bacterial clearances. these abnormalities are a result of direct damaging effects of inhaled particles and gases, and subsequent cellular inflammatory processes in the airway. inflammation in the airways of these patients is excessive and persists even after smoke cessation. cellular inflammation is characterized by excessive numbers of macrophages, t and b lymphocytes, and neutrophils in airway lumen [ ] . airway epithelial cells are also prominently involved in perpetuation and amplification of airway inflammation in copd, as demonstrated by their pro-inflammatory phenotype under basal culture conditions in the in vitro experiments [ ] . airway epithelial cells and macrophages are the cells that are constantly present in the airways, and are, probably, the first responders to exposure to cigarette smoke or smoke from biomass fuels. cigarette smoke contains more than compounds, including carcinogens, oxidants, toxic metals, and even bacterial endotoxins [ ] [ ] [ ] these compounds have been shown in cell and animal studies to cause inflammatory responses, cell apoptosis and necrosis, and dysregulation of innate immunity. thus, numerous studies demonstrate that cigarette smoke stimulates production of inflammatory chemokines from airway epithelial cells. said chemokines attract haemopoetic inflammatory cells (e.g., neutrophils) which are normally not present in the airways and are capable of causing extensive damaging effects. it is plausible that inflammatory processes initiated by smoke inhalation are further perpetuated and amplified by the responses of cells resident to the airways (e.g., airway epithelial cells, macrophages, lymphocytes, fibroblasts). as ''biomass,'' one refers to organic materials (wood, charcoal, dried animal dung fuel, etc.) that are used for cooking or heating of a dwelling [ ] . the biomass combustion efficiency is lower than that of energy sources typically used in the western world (e.g., coal or natural gas). the burning of biomass releases gases and particulate matters, including carbon monoxide, pollutants, oxidants, cancerogens, etc. [ ] . these exert damaging effects on airway cells that are similar to those by cigarette smoke [ , ] . it is possible that the damaging effects are especially extensive in susceptible individuals, which is demonstrated by the fact that not all smokers develop copd. therefore, besides the exposure to damaging compounds in cigarette or biofuel smoke, it may also take additional environmental or genetic factors to develop copd [ ] . patients with copd often harbor increased numbers of commensal or pathogenic microorganisms in their airways. the total population of microorganisms that reside in a specific area (e.g., the airways) is called microbiome. the studies of copd microbiome have only recently commenced, and therefore, we presently have only a limited knowledge of potential microbiome abnormalities [ ] . traditionally, microorganisms have been identified via culturing, which led to believe that normal human airways are sterile. however, newer microbiome studies complement traditional culturing techniques with molecular biology tools, such as pcr or sequencing, and demonstrate that even healthy individuals harbor some non-pathogenic microorganisms in their airways. in contrast, patients with copd demonstrate either elevated numbers of the microorganisms found in healthy individuals or microorganisms not typically present in healthy airway microbiome. among different microorganisms typical for copd airways are haemophilus influenzae, streptococcus pneumoniae, moraxella catarrhalis, and, in more severe patients, pseudomonas aeruginosa (table ). it is suspected that the altered microbiome may also be a significant contributor to fig. the top causes of death worldwide (world health care data). the causes of death are shown as percentages. the copdassociated deaths account for . % worldwide, making copd among the top three causes of death. other causes of death showed without labels are lower respiratory infections ( . %), lung cancers ( . %), hiv/aids ( . %), diabetes mellitus ( . %), diarrheal diseases ( . %), road injuries ( . %), and hypertensive heart disease ( %). other causes account for . % of deaths worldwide perpetuation and amplification of airway inflammation in copd [ ] . in the past, atopic asthma was seen as a disorder very different from copd, with the research emphasized th and eosinophil-driven inflammatory mechanisms in atopic asthma and neutrophil-driven inflammation in copd. however, it is increasingly recognized that asthma and copd may be part of a long continuum of respiratory diseases with many disorders, such as atopic asthma in smokers or allergies in patients with copd. in latter patients, the inflammatory processes in the airways may be associated with eosinophilia [ ] . non-smokers can also develop copd [ ] [ ] [ ] . the disease development is of unclear pathogenesis in these patients and may involve genetic and/or environmental factors, including second-hand exposure to cigarette smoke. genetic factors predisposing to copd have not been revealed, possibly, due to multigenetic nature of abnormalities in the airways (e.g., antioxidant or inflammatory genes, genes encoding proteolytic enzymes, etc.). there is, however, mutation of the gene serpina , that encodes alpha- antitrypsin, that causes pathological abnormalities in the lung similar to those in emphysema. however, this genetic disorder is typically classified separately from typical copd. the consensus definition of acute exacerbation of copd is as follows: ''an acute event characterized by a worsening of the patient's respiratory symptoms that is beyond normal day-to-day variations and leads to a change in medication'' [ ] . as noted above, not only do copd exacerbations negatively affect the quality of life of these patients, the exacerbations also substantially increase risk of pre-mature death and cause substantial financial burden to the health care system due to hospitalization and need for specialized care. the most typical provoking factors for copd exacerbations are bacterial and viral infections. as discussed above, patients with copd are reported to harbor diverse bacteria, often including opportunistic microorganisms, such as p. aeruginosa. during exacerbations, the number of these bacteria (very commonly, h. influenzae) increases in many patients, which may be because of the outgrowth of the existing strains or acquisition of the new ones [ ] . bacteria-driven genesis of many copd exacerbations, especially those with increased sputum purulence, is confirmed by clinical efficacy of antibiotics [ ] . studies further demonstrate that acute exacerbations may be associated with acquisition of new bacterial strains [ , ] , against which the patients do not have specific antibodies [ ] . these observations further support etiological role of bacterial infection in acute exacerbation of copd. but what leads to acquisition of new strains or outgrowth of the existing strains? recent research data strongly suggest that the majority of copd exacerbations are associated with upper respiratory viral infections. first, respiratory viruses are detected in upper and lower airway secretions from patients with copd in a significant number of acute exacerbations [ , ] . second, experimental virus infections of patients with copd demonstrate a subsequent bacterial outgrowth [ ] , confirming a causative link between upper respiratory virus infections, subsequent bacterial infection, and acute exacerbation of copd. it is estimated that viral or bacterial infections or combination of both are responsible from two thirds to three quarters of all copd exacerbations [ , ] . the role of respiratory viruses as pathogens provoking the majority is increasingly recognized [ ] [ ] [ ] . owing to clinical availability of multiplex pcr, detection of respiratory viruses has been greatly facilitated in the recent years. respiratory viruses most commonly detected during acute exacerbations of copd are human rhinoviruses, respiratory syncytial virus, and influenza virus, while other viruses are detected less frequently ( table ) . among these viruses, vaccination is available for influenza virus, and there is a monoclonal antibody against respiratory syncytial virus (palivizumab). influenza vaccination is routinely used in patients with copd. it has been discussed that a widespread of influenza vaccination in the western countries decreased detection of this virus during copd exacerbations [ ] . as the cost of palivizumab is very high, the authors of this review are not aware of the use of this drug for copd. there is neither vaccine nor monoclonal antibodies available against human rhinoviruses. pivotal role of human rhinoviruses in copd exacerbations is demonstrated by both epidemiologic studies and by studies on experimental infection. specifically, it was shown that inoculation of patients with copd with a low-dose rhinovirus leads to lower airway symptoms typical for copd exacerbation (breathlessness, cough, increased mucus production, etc.), as well as secondary bacterial infection [ , ] . furthermore, the peak of rhinovirus load in the lower airway secretions precedes the rise in bacterial load [ ] . this confirms anecdotal evidence reported by many patients with copd that they have had an upper respiratory viral infection (''the common cold'') several days before developing an acute exacerbation of their disease. furthermore, in northern regions, the number of acute exacerbations of copd peaks during fall and winter months [ , ] , which are also the months with the highest prevalence of upper respiratory virus infections [ ] . therefore, efforts should be aimed at developing effective antiviral measures, also against human rhinovirus, to prevent these and secondary bacterial infections, since both appear to precipitate copd exacerbations. about one-third of copd exacerbations are provoked by factors other than bacterial or viral infections. these could be allergies (in patients with pre-existing allergies), environmental factors, stress, or other intrinsic factors. environmental factors, such as air pollution, are especially relevant for cities with large industrial base, such as many of those in china. the intrinsic factors may play a role in susceptibility of some copd individuals to frequent exacerbations (the so-called ''frequent exacerbators''), although it is also possible that these patients are prone to viral or bacterial infections. furthermore, poor patient compliance to therapies during stable disease has been shown as a risk factor for exacerbations [ ] . the current armamentarium of prophylactic and therapeutic measures to effectively control copd exacerbations is limited. as mentioned above, vaccination is currently available only for influenza virus. in addition, vaccination against endemic influenza strains is produced on empirical basis, following the outbreaks in south and far east asia. therefore, there is usually a lag time of several months between identification of the virus strain with a potential to cause an influenza endemic, production of the vaccine, and availability of the vaccine for general population and vulnerable subpopulation, including patients with copd. due to multiplicity of the human rhinovirus, the virus that causes the majority of upper respiratory viral infections, a universal vaccine has yet to come. antiviral anti-influenza drugs, such as amantadine, may be effective but have extensive adverse effects [ ] . the current guidelines for copd [ ] do not provide recommendations for or against the use of antiviral antiinfluenza drugs. there have been clinical trials on augmentation therapy with aerosolized interferon-b in asthma [ ] . interferon-b is an antiviral cytokine and may potentially facilitate virus clearance from lower airways of patients with copd; however, no clinical studies have been done to date to test antiviral efficacy and safety of interferon-b in these patients. with regard to altered microbiome, there is pneumococcal vaccine that boosts immunity against s. pneumoniae. this vaccination is currently recommended to all patients with copd, while there is still a lack of large randomized controlled trials to more objectively assess the benefit of pneumococcal vaccination [ , ] . there is also oral vaccine against h. influenzae, but the evidence for its efficacy to reduce the number or severity of copd exacerbations is mixed [ ] . the current guidelines for copd [ ] identify three major classes of pharmacological treatment (short-acting bronchodilators, systemic corticosteroids, and antibiotics), along with supporting therapies (oxygen therapy, non-invasive and invasive mechanical ventilation) that are commonly used, with more or less proven efficacy, to curb acute exacerbations of copd. the guidelines provide stepwise recommendations for the use of pharmacological measures for copd exacerbations. the guidelines further recommend to follow pharmacological treatment with early outpatient rehabilitation therapy to prevent subsequent exacerbations. exacerbations in copd are often aggravated by comorbidities which are common in these patients (e.g., ischemic heart disease, hypertension, pulmonary hypertension, heart failure, etc.). therefore, these conditions also require medical attention during treatment of acute exacerbations of copd. in stable disease, the copd guidelines recommend to treat co-morbidities as per treatment practices for patients without copd [ ] . there are currently no international guidelines specifically focusing on the management of co-morbidities during copd exacerbations. it has been noted, though, that copd exacerbations and co-morbidities appear to influence each other. thus, patients with copd and metabolic syndrome, a common co-morbidity in copd [ ] , were reported to have more frequent acute exacerbations of copd [ ] . vice versa, osteoporosis was more severe in patients with copd who had more frequent exacerbations [ ] . therefore, effective management of co-morbidities in copd will improve the overall patient well-being and contribute to preventing exacerbations. acute exacerbation of copd imposes a substantial burden on both the patients and health care system, and leads to increased risk of mortality. viral infections often precipitate secondary bacterial infections. both viral and bacterial infections are now recognized as provoking factors of the great majority of acute exacerbations of copd. newer therapies are needed to effectively control viral and bacterial infections to prevent copd exacerbations. continuing to confront copd international patient survey: methods, copd prevalence, and disease burden in - prevalence of chronic obstructive pulmonary disease in china: a large, population-based survey impact of copd exacerbations on patient-centered outcomes outcomes 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infection in patients with chronic obstructive pulmonary disease haemophilus influenzae oral vaccination for preventing acute exacerbations of chronic bronchitis and chronic obstructive pulmonary disease copd as a systemic disease metabolic syndrome is associated with increased risk of acute exacerbation of copd: a preliminary study impact of copd exacerbations on osteoporosis assessed by chest ct scan microbial communities in the upper respiratory tract of patients with asthma and chronic obstructive pulmonary disease analysis of the airway microbiota of healthy individuals and patients with chronic obstructive pulmonary disease by t-rflp and clone sequencing microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease a persistent and diverse airway microbiota present during chronic obstructive pulmonary disease exacerbations key: cord- -s otdtwq authors: mandal, nakul; lewis, geoffrey p.; fisher, steven k.; heegaard, steffen; prause, jan u.; la cour, morten; vorum, henrik; honoré, bent title: proteomic analysis of the vitreous following experimental retinal detachment in rabbits date: - - journal: j ophthalmol doi: . / / sha: doc_id: cord_uid: s otdtwq purpose. the pathogenesis of rhegmatogenous retinal detachment (rrd) remains incompletely understood, with no clinically effective treatment for potentially severe complications such as photoreceptor cell death and proliferative vitreoretinopathy. here we investigate the protein profile of the vitreous following experimental retinal detachment using a comparative proteomic based approach. materials and methods. retinal detachment was created in the right eyes of six new zealand red pigmented rabbits. sham surgery was undertaken in five other rabbits that were used as controls. after seven days the eyes were enucleated and the vitreous was removed. the vitreous samples were evaluated with two-dimensional polyacrylamide gel electrophoresis and the differentially expressed proteins were identified with tandem mass spectrometry. results. ten protein spots were found to be at least twofold differentially expressed when comparing the vitreous samples of the sham and retinal detachment surgery groups. protein spots that were upregulated in the vitreous following retinal detachment were identified as albumin fragments, and those downregulated were found to be peroxiredoxin , collagen-iα fragment, and α- -antiproteinase f. conclusions. proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications. rhegmatogenous retinal detachment (rrd) is characterized by the accumulation of subretinal fluid between the neurosensory retina and retinal pigment epithelium following the formation of a retinal break [ ] . the pathogenesis of rrd is complex and incompletely understood, involving age-related and/or inherited structural and molecular changes of the vitreous extracellular matrix and vitreoretinal interface, and the process of posterior vitreous detachment [ ] . the annual incidence of the condition has been estimated at . per , , and although primary surgical reattachment is successful in the great majority of cases, photoreceptor cell death, growth factors as a result of rrd significantly contributes to the pathogenesis of pvr, though the basic cause as well as a clinically effective therapeutic approach for this condition remains elusive [ , ] . proteomics studies proteins on a large scale in pursuit of a global and integrated view of disease processes at the protein level, which may potentially lead to the identification of novel biomarkers and therapeutic targets useful in clinical practice [ ] [ ] [ ] [ ] [ ] . rrd would likely be associated with alterations in the proteomic profiles of both the retina and vitreous. indeed, we initially undertook the first such retinal study from which a number of potentially important proteins were identified [ ] . the present study extends the proteomic investigation to the vitreous of this rabbit model of retinal detachment, building upon previous such analyses of human vitreous [ ] [ ] [ ] [ ] , in order to add further knowledge of the underlying pathophysiology [ , ] . inferior retinal detachment was created in the right eyes of six new zealand red pigmented rabbits. the eyes were normal with no evidence of disease on examination. combined injections of xylazine ( . mg/kg) and ketamine ( . mg/kg) were administered intramuscularly to induce anesthesia and analgesia. the pupils were dilated with topical drops of atropine and tropicamide ( % solutions). a pipette tip, with an external diameter of approximately m, was inserted into the eye through a pars plana incision. sodium hyaluronate (healon, . % in a balanced salt solution; pharmacia, piscataway, nj) was infused via a glass pipette between the neurosensory retina and retinal pigment epithelium. healon was necessary to prevent spontaneous retinal reattachment, and . % is the most dilute solution that maintains the detachment for extended periods. approximately % of the retina beneath the medullary rays, which included the central retina, was detached ( figure ). sham surgery was performed in the right eyes of five other rabbits that were used as controls, which involved surgical entry of the vitreous cavity without disruption of the retina. scleral incisions were closed with - nylon suture. seven days postoperatively the animals were euthanized by the administration of sodium pentobarbital ( mg/kg; butler schein, dublin, oh) and the eyes enucleated. after removal of the cornea and lens, the associated vitreous of the sham and detached retinas was extracted and immediately snap-frozen in liquid nitrogen within separate vials. there was no gross evidence of blood or other contamination of the vitreous samples at the time of tissue harvesting. the vitreous samples were stored at − ∘ c until further use. all of the animal experiments undertaken in this study were in accordance with the standards of the national institutes of health animal care and use committee protocols, the arvo statement for the use of animals in ophthalmic and vision research, and the guidelines of the animal resource center, university of california, santa barbara. the rabbit vitreous samples were homogenized and dissolved in a lysis buffer containing m urea, % (v/v) triton x- , % (v/v) immobilized ph gradient (ipg) buffer (ph - nonlinear), and % (w/v) dithiothreitol (dtt). the total protein content in each vitreous sample was determined with non-interfering protein assay (calbiochem, san diego, ca). the protein samples were stored at − ∘ c until further use. the extracted proteins were first fractionated by isoelectric focusing (ief) using ph - nonlinear cm ipg strips (ge healthcare, chalfont st. giles, buckinghamshire, uk). the ipg strips were rehydrated for h at room temperature in l lysis buffer each containing g protein from individual vitreous samples and l rehydration buffer ( m urea, % (w/v) -[( -cholamidopropyl)dimethylammonio]- -propanesulfonate (chaps), . % (w/v) dtt, and % (v/v) ipg buffer), using the immobiline drystrip reswelling tray (ge healthcare). the ief was undertaken on a multiphor ii electrophoresis system (ge healthcare) at v for h and v in two steps for h and . h in a gradient mode at ∘ c with the use of a multitemp iii thermostatic circulator (ge healthcare). before the second-dimension sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis (page), the ipg strips were equilibrated firstly for min with gentle agitation in ml of equilibration solution ( . % (w/v) tris-hcl, ph . , m urea, % (v/v) glycerol, % (w/v) sds, and . % (w/v) dtt) and secondly using . % (w/v) iodoacetamide and bromophenol blue. the ipg strips were then transferred to % polyacrylamide gels for electrophoresis, which was performed at a maximum voltage of v for approximately h to separate the proteins vertically on the basis of molecular mass. staining. the two-dimensional ( d) gels were silver stained using a protocol optimized for protein identification with mass spectrometry [ ] . in brief, the gels were fixed overnight in % (v/v) ethanol, % (v/v) acetic acid, and . % (v/v) formaldehyde. the gels were washed times for min in % (v/v) ethanol and pretreated for min in . % (w/v) na s o ⋅ h o. they were then rinsed in water and stained for min in . % (w/v) agno and . % (v/v) formaldehyde. following further rinsing with water, development was undertaken for approximately min in % (w/v) na co , . % (v/v) formaldehyde, and . % (w/v) na s o ⋅ h o. the development was arrested in a fixative solution of % (v/v) ethanol and % (v/v) acetic acid. the d gels were then dried between cellophane sheets and sealed in plastic envelopes. silver stained d gels were scanned on a gs- calibrated imaging densitometer (bio-rad, hercules, ca) using the quantity one program (bio-rad), and the pdquest software (bio-rad) was used to define, quantify, and match the protein spots on each of the d gels. all well-defined protein spots that were at least twofold (mann-whitney test, < . ) differentially expressed between the sham and retinal detachment vitreous groups were selected for identification with nanoliquid chromatographyelectrospray ionization tandem mass spectrometry (lc-ms/ms). the d gels were removed from their plastic envelopes and rehydrated in water. the selected protein spots were carefully excised from the gels with a scalpel and subjected to in-gel digestion with trypsin gold (mass spectrometry grade; promega, madison, wi). the peptide samples that were obtained were analyzed by lc-ms/ms as previously described [ ] . in brief, peptides generated by trypsin digestion were separated on an inert nano-lc system (lc packings, san francisco, ca) connected to a q-tof premier mass spectrometer (waters, milford, ma). the masslynx sp (waters) was used to obtain spectra and the raw data was processed using proteinlynx global server . (waters). the processed data were used to search the total part of the swiss-prot database using the online version of the mascot ms/ms ions search facility (matrix science, ltd.). the search was undertaken with doubly and triply charged ions with up to two missed cleavages, a peptide tolerance of ppm, one variable modification, carbamidomethyl-c, and a ms/ms tolerance of . da. contaminating peptides such as trypsin, keratin, bovine serum albumin, and all peptides originating from previous samples were disregarded. at least one "bold red" (matrix science ltd., http://www .matrixscience.com/) peptide match was required in the search for protein hits. individual peptide ions scores above approximately indicated identity or extensive homology giving a less than % probability that the observed match was a random event. all peptides for the protein hits are reported (table ) . blotting. in each case three micrograms of vitreous sample protein was separated on novex - % gradient tris-glycine polyacrylamide gels (invitrogen corporation, carlsbad, ca) and subsequently transferred to nitrocellulose hybond-c extra membranes (ge healthcare). the membranes were blocked overnight with % skimmed milk in mm na hpo , mm nah po , mm nacl, and . % tween buffer, ph . . membranes were incubated with anti-albumin (genway biotech, ca, usa; : ) and anti-peroxiredoxin (abcam, cambridge, uk; : ). no suitable antibodies were commercially available for the rabbit f isoform of - -antiproteinase or the rabbit collagen-i fragment that was identified with lc-ms/ms. following washing, the membranes were further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies: p sheep and p mouse (both : ; dako, glostrup, denmark). proteins were visualized with the enhanced chemiluminescence system (ge healthcare) and imaging system (fujifilm las- , tokyo, japan). up to approximately protein spots were clearly resolved on each of the d gels. ten protein spots were found to be significantly and at least twofold differentially expressed between the sham and detachment vitreous groups (figure ). three protein spots were upregulated and seven spots were downregulated. from the three upregulated protein spots, two were identified as fragments of albumin (spots and ), whilst spot could not be identified. four of the seven downregulated protein spots were identified as fragment of collagen-i (spot ), - -antiproteinase f (spots and ), and peroxiredoxin (spot ). protein spots , , and could not be identified ( figure ; table ). western blotting developed with anti-albumin showed a heavy band at approximately kda, which is likely to represent the full length protein, whilst multiple bands below this suggest the presence of several fragments, some of which may correspond with those identified with the d-page analysis (figure , left) . peroxiredoxin has a deduced molecular mass of approximately kda. however, spot containing peroxiredoxin migrates with a molecular mass around kda with d-page (figure ) , and this size was verified by western blot analysis (figure , right). though a single and specific band was achieved with anti-peroxiredoxin , western blotting could not be reliably used for quantification due to a weak signal near the detection limit and variable background reaction. analysis with d-page revealed fragments of albumin to be upregulated in the vitreous following retinal detachment. albumin is the most abundant protein in plasma, aqueous, and vitreous humor, where in the latter it constitutes around - % of total protein [ ] [ ] [ ] . serum proteins such as albumin are present in the aqueous and vitreous humor at a relatively lower level compared to the vascular circulation from where they may have in part originated [ , ] . western blot analysis showed an intense band at approximately kda corresponding with the full length albumin protein, with multiple lower molecular mass bands that are likely its fragments. increase of albumin and its fragments may signify increased proteolysis and the passage of albumin into the vitreous. indeed, the breakdown of the blood-retinal barrier that occurs with retinal detachment has also been implicated in the increase of other such proteins in the vitreous [ , [ ] [ ] [ ] [ ] [ ] [ ] . it is also possible that albumin in the vitreous may arise from de novo synthesis in the retina, similar to the reported increased gene and protein expression of albumin in the corneal epithelium during wound healing [ , ] . extraocular albumin is known to have diverse and important functions, which include maintenance of colloid osmotic pressure, transport of biomolecules, and inactivation of toxins through intermolecular binding [ , ] . albumin can also act as an antioxidant by scavenging reactive oxygen species and sequestration of metal ions and has anti-inflammatory and apoptotic regulatory abilities [ ] [ ] [ ] [ ] . vitreal albumin has been proposed to transport long chain fatty acids into the lens for biosynthesis of lenticular lipids [ , , ] . indeed, albumin is likely to have many such important roles in the eye, which requires further investigation. in the present study we observed peroxiredoxin to have a molecular mass above kda using both d-page and western blot analyses. however, the predicted molecular mass of the peroxiredoxin family of proteins is approximately kda- kda. this variation may represent the well-studied property of these proteins to undergo oligomerization, which can be promoted by a number of factors including overoxidation of cysteine residues of peroxiredoxin [ ] . although the present experiments were conducted in standard reducing conditions that aim to break cysteine bonds, we obtained a band well above kda. this is in keeping with another study, which also showed some peroxiredoxin western blot bands appearing at molecular mass much higher than kda that was suggested to result from oligomerization or posttranslational modification [ ] . our finding could also represent a novel alternative splicing variant of peroxiredoxin , as reported for peroxiredoxin [ ] . the peroxiredoxins are a group of ubiquitous antioxidant proteins that currently comprise six members in mammals [ ] . these proteins are primarily found at high levels intracellularly, mainly within the cytosol, but are also present in the mitochondria, peroxisomes, and nuclei, and they may be exported [ ] . furthermore, presence of peroxiredoxin has been shown in plasma, not only as a result of hemolysis but also possibly by secretion from the t lymphocytes [ , ] . these multifunction enzymes act as antioxidants by using redox active cysteines for the reduction and degradation of hydrogen peroxide, peroxynitrite, and organic hydroperoxides [ , ] . oxidative stress is thought to result from an imbalance between reactive oxygen species production and antioxidant ability and is recognized to be an important factor in the pathogenesis of a number of age-related and neurodegenerative diseases, which include age-related cataract, agerelated macular degeneration, glaucoma, diabetic retinopathy, retinal detachment, and pvr [ ] [ ] [ ] [ ] . indeed, the present study showed a decrease in the vitreal levels of peroxiredoxin following retinal detachment. this may be in keeping with reported reductions in the levels of other members of the antioxidant defense system such as glutathione and ascorbic acid both in vitreal and in blood samples of patients suffering from pvr [ , ] . furthermore, apart from their role as antioxidants, the peroxiredoxins can affect a diverse range of biological processes that include cellular proliferation, differentiation, and apoptosis by influencing signal transduction pathways that employ hydrogen peroxide as a secondary messenger [ , ] . recent studies on tears from patients with glaucoma have also identified peroxiredoxin as having a possible involvement in inflammation [ , ] . indeed, peroxiredoxin and other members of this family of proteins are liable to have a significant role in the pathophysiology of retinal detachment. a fragment of collagen-i was identified in the vitreous of the rabbit; however, type i collagen has not previously been identified as a natural component of the mammalian vitreous and is rather known to be a constituent of early pvr membranes [ ] [ ] [ ] and retinal blood vessels [ , ] . a mixture of type ii, ix, and v/xi hybrid collagen fibrils, which are separated out mainly by water and ions attracted to hyaluronan, characterizes the vitreous body [ ] . collagen, possibly with the aid of adhesive-like intermediate molecules, may provide the basis of vitreoretinal adhesion by connecting the vitreous with the retinal inner limiting membrane (ilm). this attachment is extremely strong in the vitreous base since the fibrils pass through the ilm to merge into underlying collagen networks and crypts [ , , ] . collagen is also a significant component of both epiretinal and subretinal pvr membranes [ , ] , and type i collagen is recognized to be a principal constituent during their early development [ ] [ ] [ ] . the presence of collagen in the subretinal space, a place normally devoid of this protein, suggests that certain cells, particularly the rpe and müller cells associated with membranes, are able to synthesize collagen under certain pathological conditions such as retinal detachment and pvr [ ] [ ] [ ] [ ] . however, the present analysis suggests collagen-i fragment to be found in sham vitreous, which furthermore showed a decreased concentration following retinal detachment that may indicate perturbed proteolytic activity. matrix metalloproteinases (mmp) and other proteolytic enzymes that are able to degrade and remodel vitreal collagen have been found to be increased with rrd and pvr [ ] [ ] [ ] [ ] , which could be in keeping with the decrease in - -antiproteinase shown in the present study. further studies will be necessary to confirm the source and nature of collagen-i in the vitreous and the possible mechanisms of collagen fragmentation that may be an important feature of vitreous liquefaction and rrd [ , , ] . alpha- -antiproteinase. d-page showed - -antiproteinase (also called - -antitrypsin or - -proteinase inhibitor) at two closely positioned spots, which were largely in keeping with their predicted molecular mass but differing by their charge. currently, four isoforms of - -antiproteinase have been identified in the rabbit, termed f, s , s , and e, which is a similar picture to the multiple variants identified in humans [ , ] . alpha- -antiproteinase is an acute phase protein and archetypal member of the superfamily of serine protease inhibitors (serpin), which are involved in a wide range of biological processes that includes inflammation, angiogenesis, blood coagulation, ecm remodeling, and tumor suppression [ ] . this protein has the ability to inhibit a large number of serine proteases though its principle target is neutrophil elastase [ ] . indeed, - -antiproteinase originally received much attention because its deficiency increases the risk of a variety of clinical conditions, such as chronic obstructive pulmonary disease, which can result from unrestrained elastase activity. we found the f isoform of rabbit - -antiproteinase to be downregulated in the vitreous following retinal detachment. the f isoform of - -antiproteinase is the only one of the rabbit isoforms so far identified that has been shown to have the oxidizable methionine residue site that is present in human - -antiproteinase [ ] . the oxidation of methionine to methionine sulfoxide, which can occur during episodes of inflammation as a result of oxygen-free radicals secreted by leucocytes, has an inhibiting effect upon - -antiproteinase function. this process is thought to enhance the ability of proteinases such as elastase to locally degrade tissue debris that occurs at sites of inflammation [ , ] . alpha- -antiproteinase is primarily produced in the liver and circulated to the rest of the body tissues via the blood; however, extrahepatic sites of its synthesis have been identified, which include blood monocytes, alveolar macrophages, bronchial and gastrointestinal epithelial cells, and the cornea [ ] [ ] [ ] [ ] . the protein has also been localized to the tear film, aqueous humor, and vitreous, where in the latter a phosphorylated form of - -antiproteinase has been suggested as a potential biomarker of idiopathic macular hole and rhegmatogenous retinal detachment [ ] [ ] [ ] . it has been postulated that one of the main functions of corneal - -antiproteinase is to protect against the damaging effects of neutrophil elastase produced during corneal inflammation [ ] , and it may be expected that a similar role in addition to others is applicable to vitreal - -antiproteinase, though this requires further investigation. this proteomic investigation of the rabbit vitreous has identified a set of proteins that assist our understanding of the pathogenesis of rhegmatogenous retinal detachment and its journal of ophthalmology complications. further studies will be necessary to clarify the role of these proteins. certain proteins, such as those of low abundance and at the extremes of molecular mass, together with membrane proteins, can be difficult to resolve and detect using the d-page technique. therefore, complementary proteomic methods such as gel-free mass spectrometry should be considered in future work in order to help address these limitations. the authors alone are responsible for the content and writing of the paper. recent trends in the management of rhegmatogenous retinal detachment pathogenesis of rhegmatogenous retinal detachment: predisposing anatomy and cell biology the epidemiology and socioeconomic associations of retinal detachment in scotland: a two-year prospective population-based study cellular effects of detachment and reattachment on the neural retina and the retinal pigment 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genetics of the major plasma serpin the serpins are an expanding superfamily of structurally similar but functionally diverse proteins. evolution, mechanism of inhibition, novel functions, and a revised nomenclature expression of the alpha -proteinase inhibitor gene in human monocytes and macrophages biosynthesis of -proteinase inhibitor by human lung-derived epithelial cells regulation of -proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells corneal synthesis of -proteinase inhibitor ( -antitrypsin) alpha- -antitrypsin in aqueous humour from patients with corneal allograft rejection identification of phosphotyrosyl proteins in vitreous humours of patients with vitreoretinal diseases by sodium dodecyl sulphate-polyacrylamide gel electrophoresis/western blotting/ matrix-assisted laser desorption time-of-flight mass spectrometry the authors appreciate the excellent technical support pro- the authors report no conflict of interests. key: cord- - d mz f authors: perkins, g.d.; handley, a.j.; koster, r.w.; castrén, m.; smyth, m.a.; olasveengen, t.; monsieurs, k.g.; raffay, v.; gräsner, j.-t.; wenzel, v.; ristagno, g.; soar, j. title: basismaßnahmen zur wiederbelebung erwachsener und verwendung automatisierter externer defibrillatoren: kapitel der leitlinien zur reanimation des european resuscitation council date: - - journal: notf rett med doi: . /s - - - sha: doc_id: cord_uid: d mz f nan für die erc-leitlinien wurde zusätzlich zu den ilcor-empfehlungen von den federführenden autoren die literatur zu den themenfeldern gesichtet, die bei ilcor keine berücksichtigung fanden. die leitlinien-verfasser waren sich bewusst, dass Änderungen gegenüber den empfehlungen von kosten und eventuell verunsicherung verursachen würden, und beschränkten sich daher auf Änderungen, die wirklich wichtig erschienen und durch neue evidenz belegt sind. die leitlinien wurden von den federführenden autoren konzipiert, von allen leitlinien-verfassern und den nationalen wiederbelebungsräten überarbeitet und schließlich vom erc-board verabschiedet. die leitlinien betonen die besondere bedeutung der interaktion des leitstellen-disponenten mit dem notfallzeugen, der mit der wiederbelebung beginnt, und der zeitnahen verfügbarkeit eines defibrillators. eine effektive, koordinierte gemeinsame aktion, die diese elemente zusammenführt, verbessert die Überlebenschancen des patienten nach einem kreislaufstillstand außerhalb eines krankenhauses (. abb. ). der leitstellendisponent spielt eine entscheidende rolle bei der frühzeitigen diagnose eines kreislaufstillstands, der leitstellengeführten reanimation (bekannt als telefonreanimation) und bei auffinden und einsetzen eines externen automatisierten defibrillators (aed). je früher der rettungsdienst alarmiert wird, desto eher wird eine adäquate behandlung begonnen und unterstützt. fertigkeiten, zutrauen und handlungsbereitschaft des ersthelfers hängen von den umständen des kreislaufstillstands, seinem trainingsstand und seinen vorkenntnissen ab. der erc empfiehlt dem notfallzeugen, der darin geschult und dazu in der lage ist, den zustand des patienten schnell zu beurteilen, indem er feststellt, ob der patient nicht ansprechbar ist und nicht normal atmet, und dann unmittelbar den rettungsdienst zu alarmieren. wenn irgend möglich, soll er währenddessen den patienten nicht verlassen. der patient, der nicht reagiert und nicht normal atmet, hat einen kreislaufstillstand und benötigt eine herz-lungen-wiederbelebung (cpr). unmittelbar nach dem kreislaufstillstand geht der blutfluss zum gehirn gegen null. das kann krampfanfälle auslösen, die möglicherweise mit einer epilepsie verwechselt werden. notfallzeugen und leitstellendisponenten sollen daher bei krampfenden patienten auch an einen kreislaufstillstand denken und sorgfältig klären, ob der patient normal atmet. die leitlinien-verfasser bekräftigen die ilcor-empfehlung, dass bei jeder wiederbelebung eine thoraxkompression durchgeführt werden soll. notfallzeugen, die ausgebildet und in der lage sind, eine atemspende durchzuführen, sollen herzdruckmassage und atemspende kombinieren. zusätzliche atemspenden können vorteilhaft für kinder bei asphyktischem kreislaufstillstand sein oder wenn sich das eintreffen des rettungsdienstes verzögert. da wir nicht davon überzeugt sind, dass eine wiederbelebung allein durch thoraxkompressionen einer standardwiederbelebung gleichwertig ist, empfehlen wir weiterhin die bisher praktizierte vorgehensweise. eine qualitativ hochwertige wiederbelebung ist entscheidend für eine verbesserung des ergebnisses (outcome). bei der herzdruckmassage soll eine adäquate drucktiefe sicher erreicht werden (etwa cm, jedoch nicht mehr als cm beim normalen erwachsenen), bei einer kompressionsfrequenz von − / min mit minimalen unterbrechungen. nach jeder kompression muss der brustkorb vollständig entlastet werden. atemspenden/beatmungen sollen eine sekunde dauern und zu einer deutlichen hebung des brustkorbs führen. das verhältnis von herzdruckmassage zu beatmung bleibt : . unterbrechen sie die thoraxkompressionen für die beatmung nicht länger als s. durch defibrillation innerhalb von − min nach dem kollaps können Überlebensraten von − % erreicht werden. eine frühzeitige defibrillation kann durch notfallzeugen unter verwendung von öffentlichen oder hauseigenen aeds durchgeführt werden. public-access-programme (öffentlicher zugang zu aeds) sollen an viel besuchten orten (flughäfen, bahn-oder busstationen, sportstätten, einkaufszentren, bürogebäuden und kasinos) etabliert werden. kreislaufstillstände an solchen orten werden häufig beobachtet, und ausgebildete helfer können schnell vor ort sein. das vorhalten eines aed gilt schon dann als kosteneffektiv, wenn sich an seinem standort alle jahre ein kreislaufstillstand ereignet, die kosten pro gewonnenes lebensjahr entsprechen denen anderer medizinischer interventionen. erfahrungen mit kreislaufstillständen in der vergangenheit und die art der umgebung können bei der wahl der aed-platzierung wegweisend sein. eine registrierung beim rettungsdienst ermöglicht es den disponenten, helfer zu einem nahe gelegenen aed zu führen und so die reaktionszeit zu verbessern. der ablauf der wiederbelebungsmaßnahmen für erwachsene kann auch bei kindern, die nicht ansprechbar sind und nicht normal atmen, sicher verwendet werden. helfer mit einer entsprechenden ausbildung können bei kindern die wiederbelebungschance mit initialen beatmungen verbessern. für den eher seltenen fall, dass ein helfer auf sich allein gestellt ist, ist es bei kindern und ertrunkenen auch besser, erst aktiv zu werden und verzögert hilfe zu holen. thoraxkompressionen atemspenden, elektroschocks sowie analysen der beatmung und des herzrhythmus führen zu unterbrechungen der herzdruckmassage. pausen von weniger als s vor und nach der abgabe eines schocks und ein anteil der thoraxkompressionen von mehr als % sind mit besserem outcome verbunden [ - ]. unterbrechungen der thoraxkompressionen sollen minimiert werden, wobei erkannt werden muss, wie wichtig die aufmerksamkeit und das wechselspiel der helfer ist, die zusammenarbeiten. die mund-zu-nase-beatmung stellt eine akzeptable alternative zur mund-zu-mund-beatmung dar [ ] . sie kann erwogen werden, wenn der mund des patienten schwer verletzt ist oder nicht geöffnet werden kann, der patient sich im wasser befindet oder bei der mund-zu-mund-beatmung keine abdichtung erreicht werden kann. aeds sind sicher und effektiv, wenn sie durch laien mit wenig oder ohne training verwendet werden [ ] . aeds ermöglichen eine defibrillation viele minuten, bevor professionelle hilfe eintrifft. helfer sollen thoraxkompressionen mit minimalen unterbrechungen durchführen, während der aed angelegt und verwendet wird. die helfer sollen sich darauf konzentrieren, der sprachführung unmittelbar zu folgen, insbesondere die herzdruckmassage sofort wieder aufzunehmen, wenn dazu aufgefordert wird, und unterbrechungen der thoraxkompressionen zu minimieren. pausen vor und nach einem schock sollten so kurz wie nur möglich sein [ , , , ]. standard-aeds können schon für kinder ab jahren verwendet werden [ ] [ ] [ ] . für kinder zwischen und jahren sollen spezielle klebeelektroden für kinder verwendet werden, wenn möglich mit einem kinderprogramm. stehen sie nicht zur verfügung, soll der defibrillator verwendet werden, wie er ist. die verwendung des aed wird nicht für kinder unter einem jahr empfohlen, obwohl es berichte über einen erfolgreichen einsatz in dieser altersgruppe gibt [ , ] . abgesehen von herzkranken kleinkindern ist eine defibrillierbare rhythmusstörung bei kleinkindern extrem selten [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ist in diesen wenigen fällen ein aed der einzige verfügbare defibrillator, sollte sein einsatz erwogen werden (vorzugsweise mit verringerter dosis). die bedeutung unmittelbarer defibrillation wurde immer in leitlinien und ausbildung betont, ihr wird großer ein-fluss auf das Überleben nach kammerflimmern zugesprochen. wurde dieses konzept infrage gestellt, da evidenz dafür vorlag, dass thoraxkompressionen von bis zu s vor einer defibrillation das Überleben verbessern können, wenn der rettungsdienst erst nach mehr als − min eintrifft [ , ] . drei jüngere studien konnten diesen vorteil nicht bestätigen [ ] [ ] [ ] . die analyse einer randomisierten studie deutete auf eine verschlechterung des Überlebens bis zur krankenhausentlassung durch eine längere periode cpr (reanimation ohne beatmung länger als s) mit dadurch verzögerter defibrillation eines defibrillierbaren rhythmus hin [ ] . allerdings war in rettungsdienstbereichen mit einer hohen ausgangsüberlebensrate bei krankenhausentlassung (definiert als mehr als % der initial defibrillierbaren fälle) eine wiederbelebung über s vor der defibrillation erfolgreicher als eine wiederbelebung über eine kürzere zeit ( − s) [ ] . der erc empfiehlt, dass cpr fortgeführt werden soll, während ein defibrillator oder aed gebracht und angelegt wird, aber dann soll die defibrillation nicht weiter verzögert werden. in der praxis werden aeds meist von ausgebildeten helfern eingesetzt, sodass die aed-sprachführung grundsätzlich auf ein kompressions-ventilations-verhältnis von : eingestellt werden soll. wenn -ausnahmsweise -aeds an einem ort platziert werden, wo es unwahrscheinlich ist, dass ausgebildete helfer dazukommen, kann der betreiber die einstellung auf herzdruckmassage ohne beatmung ändern lassen. hat ein vollautomatischer aed einen defibrillierbaren rhythmus erkannt, gibt er den schock ohne weiteres zutun des helfers ab. in einer studie an Übungsphantomen konnte gezeigt werden, dass ungeschulte krankenpflegeschüler mit einem vollautomatischen aed weniger fehler machten als mit einem halbautomatischen [ ] . eine simulierte studie an phantomen ergab, dass die sicherheit nicht gefährdet war, wenn ungeübte laienhelfer einen vollautomatischen statt einen halbautomatischen aed benutzten [ ] . Über die anwendung an menschen in einem klinischen bereich liegen keine daten vor. defibrillatoren in der Öffentlichkeit ("public access defibrillation", pad) [ , ] . die registrierung der aed-standorte erleichtert es dem leitstellendisponenten, einen notfallhelfer zum nächstplatzierten aed zu führen und somit die hilfeleistung zu beschleunigen [ ] . die frühzeitige defibrillation mit einem aed vor ort kann möglicherweise auch krankenhauskosten reduzieren [ , ] . das volle potenzial von aeds ist noch nicht ausgeschöpft, da sie meist im öffentlichen raum zum einsatz kommen sich aber bis % der kreislaufstillstände zu hause ereignen. der anteil der patienten, die mit kammerflimmern aufgefunden werden, ist zu hause geringer als in der Öffentlichkeit, wohingegen die absolute zahl zu behandelnder patienten zu hause höher ist [ ] . selten profitieren patienten zu hause von öffentlichen aed-programmen [ ] . wenn ein patient kollabiert, muss schnell ein aed verfügbar sein: ein klares, einfaches symbol muss auf seinen standort und den schnellsten weg dorthin hinweisen. ilcor hat ein solches aed-symbol entwickelt, das weltweit verstanden wird; daher wird dieses empfohlen [ ] . viele notfallzeugen beginnen nicht mit der wiederbelebung, weil sie befürchten, dass thoraxkompressionen bei einem patienten, der keinen kreislaufstillstand hat, ernste schäden verursachen. drei untersuchungen befassten sich mit den risiken einer herzdruckmassage bei personen, die keinen kreislaufstillstand hatten [ ] [ ] [ ] . in den gepoolten daten dieser drei studien, also von patienten, fanden sich knochenbrüche (rippen und schlüsselbein) mit , % ( %-ci, , - , %), schmerzen an der stelle der herzdruckmasage mit , % ( %-ci, , - , %), aber keine relevanten verletzungen innerer organe. ersthelfer sollten keine bedenken haben, mit einer wiederbelebung zu beginnen, da es nur in extrem seltenen fällen zu ernsthaften verletzungen kommt, wenn ein patient keinen kreislaufstillstand hat und von einem notfallzeugen wiederbelebt wird. eine systematische Übersicht zu skelettverletzungen durch manuelle thoraxkompression berichtet von - % rippenbrüchen und von - % sternumfrakturen [ ] . organverletzungen (lunge, herz, bauchorgane) sind sehr viel seltener und kommen mit und ohne knochenverletzungen vor [ ] . sie treten häufiger auf, wenn beim normalen erwachsenen tiefer als cm gedrückt wird [ ]. beobachtungsstudien zur ausbildung und tatsächlichen durchführung von wiederbelebungsmaßnahmen sowie fallberichte dokumentieren nur selten muskelzerrungen, rückenbeschwerden, kurzatmigkeit, pneumothorax, brustschmerzen, herzinfarkt oder nervenschäden [ , ] . die häufigkeit solcher ereignisse ist niedrig, und die ausbildung in wiederbelebungsmaßnahmen und deren tatsächliche durchführung ist unter den meisten umständen sicher [ ] . teilnehmer von wiederbelebungsschulungen sollten über art und ausmaß der körperlichen belastung während des trainingsprogramms aufgeklärt werden. lernenden und helfern, die während des trainings signifikante symptome entwickeln (z. b. brustschmerz oder starke atemnot), soll zum trainingsabbruch geraten werden. mehrere studien am Übungsphantom haben nachgewiesen, dass die drucktiefe bereits weniger als min nach beginn der thoraxkompressionen abnimmt [ ] . eine krankenhauspatientenstudie zeigte, dass auch bei echtzeit-feedbacks die durchschnittliche tiefe der herzdruckmassage zwischen , und min nach beginn der cpr nachließ [ ] . es wird daher empfohlen, dass sich ersthelfer etwa alle min abwechseln, um eine verschlechterung der druckqualität infolge der ermüdung des helfers zu verhindern. beim wechsel der helfer soll die herzdruckmassage nicht unterbrochen werden. viele studien zu öffentlich zugänglichen defibrillatoren ("public access defibrillation", pad) zeigen, dass aeds von laien und professionellen ersthelfern (first respondern) sicher angewendet werden können [ ] . eine systematische metaanalyse fand publikationen, die insgesamt unerwünschte ereignisse bei der defibrillation auswiesen [ ] . ursache waren zufälliger oder vorsätzlicher missbrauch des defibrillators, gerätefehlfunktion und versehentliche entladung während des trainings oder der wartung. in einzelfallberichten kam es durch die entladung implantierter herzschrittmacher (implantierbarer kardioverter-defibrillator, icd) zu schocks an helfern, was in einem fall zu einer schädigung peripherer nerven führte. es gibt keine berichte über schädigungen der ersthelfer durch defibrillationsversuche in feuchter umgebung. obgleich verletzungen der helfer durch defibrillationen extrem selten sind, konnte gezeigt werden, dass chirurgische handschuhe keinen ausreichenden schutz bieten [ ] [ ] [ ] [ ] . daher sollen helfer während der schockabgabe die thoraxkompressionen nicht fortsetzen, und der patient soll während der icd-entladung nicht berührt werden. direkter kontakt zwischen dem helfer und dem patienten während der schockabgabe soll vermieden werden. eine große prospektive studie zu pad berichtete von wenigen negativen psychologischen effekten in verbindung mit einer wiederbelebung oder dem einsatz eines aed, die eine intervention erforderten [ ] . zwei große retrospektive fragebogenstudien zur wiederbelebung stellten fest, dass notfallzeugen ihre wiederbelebung als positive erfahrung einstuften [ , ] . auch familienangehörige, die zeugen von wiederbelebungsmaßnahmen werden, können psychologisch davon profitieren [ ] [ ] [ ] . das seltene auftreten von nachteiligen psychologischen auswirkungen bei ersthelfern nach einer wiederbelebung soll dennoch registriert und angemessen behandelt werden. das risiko einer krankheitsübertragung während des trainings und der tatsächlichen wiederbelebung ist extrem niedrig [ ] [ ] [ ] . das tragen von handschuhen während der wiederbelebung ist sinnvoll, aber die wiederbelebung soll nicht verzögert oder gar unterlassen werden, weil keine handschuhe verfügbar sind. drei studien zeigten unter kontrollierten laborbedingungen, dass beatmungsfolien oder -ventile die Übertragung von bakterien verringern [ , ] die verlegung der atemwege durch einen fremdkörper ist eine seltene, aber potenziell behandelbare todesursache [ ] . da die meisten atemwegsverlegungen beim essen entstehen, werden sie üblicherweise beobachtet. da die betroffenen anfangs bei bewusstsein sind und reagieren, besteht oft die möglichkeit zur frühzeitigen intervention, die lebensrettend sein kann. atemwegsverlegung durch fremdkörper. fremdkörper können eine milde oder eine schwere atemwegsverlegung verursachen. es ist wichtig, den ansprechbaren patienten zu fragen: "haben sie einen erstickungsanfall?" ein patient, der antwortet, hustet und atmet, hat eine milde obstruktion. kann er nicht sprechen, nur schwach husten, ringt er nach luft oder kann nicht atmen, so liegt eine schwere obstruktion vor. husten erzeugt einen hohen und anhaltenden atemwegsdruck und kann den fremdkörper ausstoßen. eine aggressive behandlung mit schlägen auf den rücken, oberbauch-und brustkorbkompressionen kann schäden hervorrufen und die atemwegsverlegung verschlimmern. diese soll patienten vorbehalten bleiben, die zeichen einer schweren atemwegsverlegung aufweisen. patienten mit einer milden verlegung des atemwegs sollen unter kontinuierlicher beobachtung bleiben, bis es ihnen besser geht, weil sich eine schwere verlegung noch entwickeln kann. klinische daten zum ersticken sind größtenteils retrospektiv und anekdotisch. bei erwachsenen und kindern über jahr, die bei bewusstsein sind und bei denen eine komplette atemwegsverlegung durch fremdkörper erfolgt ist, haben fallberichte die effektivität von schlägen auf den rücken sowie oberbauch-und brustkorbkompressionen gezeigt [ ] . in ungefähr % der fälle kann die atemwegsverlegung nicht durch eine einzige maßnahme beseitigt werden [ ] . die erfolgsaussichten steigen bei der kombination von schlägen auf den rücken, oberbauchund brustkorbkompressionen [ ] . eine randomisierte studie an leichen [ ] und zwei prospektive studien an an-ästhesierten freiwilligen [ , ] haben gezeigt, dass mit brustkorbkompressionen im vergleich zu oberbauchkompressionen ein höherer atemwegsdruck erzeugt werden kann. herzdruckmassagen bei bewusstlosen oder nicht ansprechbaren patienten mit einer atemwegsverlegung durch fremdkörper sind mit einem guten neurologischen outcome assoziiert (odds ratio, , ; %-ci, - . , p < , ) [ ] . daher soll sofort mit thoraxkompressionen begonnen werden, wenn der patient nicht mehr reagiert oder bewusstlos wird. nach kompressionen versuchen sie zweimal zu beatmen. führen sie die reanimation fort, bis sich der patient erholt und normal zu atmen beginnt. nach erfolgreicher beseitigung einer atemwegsverlegung durch fremdkörper können immer noch fremdkörper in den oberen oder unteren atemwegen verblieben sein und später zu komplikationen führen. patienten mit anhaltendem husten, schluckbeschwerden oder dem gefühl, dass immer noch etwas im hals steckt, sollen daher einem arzt vorgestellt werden. oberbauchkompressionen und herzdruckmassagen können zu ernsthaften inneren verletzungen führen; daher sollen alle patienten, bei denen diese angewendet wurden, anschließend auf verletzungen untersucht werden. viele kinder werden nicht reanimiert, weil potenzielle helfer fürchten, schaden anzurichten, da sie nicht speziell in der wiederbelebung von kindern geschult sind. diese furcht ist unbegründet: es ist viel besser, ein kind nach dem bls-schema für erwachsene zu reanimieren, als nichts zu tun. um das lernen und erinnern zu vereinfachen, soll laien beigebracht werden, dass die erwachsenenmethode auch bei nicht reagierenden und nicht normal atmenden kindern angewandt werden kann. folgende geringe Änderungen an der erwachsenensequenz machen diese für kinder noch geeigneter: beatmen sie -mal, bevor sie mit den thoraxkompressionen beginnen. comparison of out-of-hospital cardiac arrest occurring before and after paramedic arrival: epidemiology, survival to hospital discharge and -month functional recovery. comparison of chest compression only and standard cardiopulmonary resuscitation for out-of-hospital cardiac arrest in singapore survival is similar after standard treatment and chest compression only in out-of-hospital bystander cardiopulmonary resuscitation cardiopulmonary resuscitation by bystanders with chest compression only (sos-kanto): an observational study effectiveness of bystander-initiated cardiac-only resuscitation for patients with out-of-hospital cardiac arrest evaluation of cardiopulmonary resuscitation (cpr) techniques. the cerebral resuscitation study group effectiveness of bystander cardiopulmonary resuscitation and survival following out-ofhospital cardiac arrest standard basic life support vs. continuous chest compressions only in out-of-hospital cardiac arrest conventional and chest-compression-only cardiopulmonary resuscitation by bystanders for children who have out-of-hospital cardiac arrests: a prospective, nationwide, populationbased cohort study impact of dispatcher-assisted bystander cardiopulmonary resuscitation on neurological outcomes in children with out-of-hospital cardiac arrests: a prospective, nationwide, population-based cohort study aed training and its impact on skill acquisition, retention and performance-a systematic review of alternative training methods effects of compression depth and preshock pauses predict defibrillation failure during cardiac arrest public access defibrillation improved the outcome after out-of-hospital cardiac arrest in schoolage children: a nationwide, population-based, utstein registry study in japan demographics, bystander cpr, and aed use in out-of-hospital pediatric arrests characteristics and outcomes of pediatric out-ofhospital cardiac arrest by scholastic age category first appropriate use of automated external defibrillator in an infant successful parental use of an automated external defibrillator for an infant with long-qt syndrome pediatric defibrillation after cardiac arrest: initial response and outcome outcomes of in-hospital ventricular fibrillation in children epidemiology and outcomes from out-of-hospital cardiac arrest in children: the resuscitation outcomes consortium epistry-cardiac arrest incidence, causes, and outcomes of out-of-hospital cardiac arrest in children. a comprehensive, prospective, population-based study in the netherlands influence of cardiopulmonary resuscitation prior to defibrillation in patients with out-of-hospital ventricular fibrillation delaying defibrillation to give basic cardiopulmonary resuscitation to patients with out-of-hospital ventricular fibrillation: a randomized trial cpr before defibrillation in out-of-hospital cardiac arrest: a randomized trial defibrillation or cardiopulmonary resuscitation first for patients with out-of-hospital cardiac arrests found by paramedics to be in ventricular fibrillation? a randomised control trial early versus later rhythm 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arrest european resuscitation council guidelines for resuscitation section cardiac arrest in special circumstances ilcor presents a universal aed sign a program encouraging early defibrillation results in improved in-hospital resuscitation efficacy automatic external defibrillators in the hospital as well? first responder for inhospital resuscitation: -year experience with an automated external defibrillator-based program automated external defibrillators and survival after in-hospital cardiac arrest: early experience at an australian teaching hospital automated external defibrillators and in-hospital cardiac arrest: patient survival and device performance at an australian teaching hospital automated external defibrillators and survival after in-hospital cardiac arrest automated external defibrillator use for in-hospital cardiac arrest is not associated with improved survival incidence and outcome of in-hospital cardiac arrest in the united kingdom national cardiac arrest audit should 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emergency response programs: the public access defibrillation trial experience rescuer fatigue under the erc guidelines, and its effect on cardiopulmonary resuscitation (cpr) performance rescuer fatigue during actual in-hospital cardiopulmonary resuscitation with audiovisual feedback: a prospective multicenter study is external defibrillation an electric threat for bystanders? will medical examination gloves protect rescuers from defibrillation voltages during hands-on defibrillation? handson defibrillation: theoretical and practical aspects of patient and rescuer safety do clinical examination gloves provide adequate electrical insulation for safe handson defibrillation? i: resistive properties of nitrile gloves do clinical examination gloves provide adequate electrical insulation for safe hands-on defibrillation? ii: material integrity following exposure to defibrillation waveforms bystander-initiated cardiopulmonary resuscitation out-of-hospital. a first description of the bystanders and their experiences factors surrounding cardiopulmonary resuscitation influencing bystanders' psychological reactions family presence during cardiopulmonary resuscitation offering the opportunity for family to be present during cardiopulmonary resuscitation: -year assessment presence during cardiopulmonary resuscitation is beneficial to family members in the out-of-hospital setting basic-cpr and aids: are volunteer life-savers prepared for a storm infections acquired during cardiopulmonary resuscitation: estimating the risk and defining strategies for prevention ethical and practical considerations in providing critical care to patients with ebola virus disease prevention of transmission of infection during mouth-to-mouth resuscitation prevention of oral bacterial flora transmission by using mouth-to-mask ventilation during cpr a randomised crossover comparison of mouth-toface-shield ventilation and mouth-to-pocketmask ventilation by surf lifeguards in a manikin mouth-to-mouth ventilation is superior to mouth-to-pocket mask and bag-valve-mask ventilation during lifeguard cpr: a randomized study comparison of mouth-to-mouth, mouth-to-mask and mouth-to-face-shield ventilation by lay persons international comparative analysis of injury mortality. findings from the ice on injury statistics. international collaborative effort on injury statistics cardiac arrest following foreign-body aspiration international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations the choking controversy: critique of evidence on the heimlich maneuver airway pressure with chest compressions versus heimlich manoeuvre in recently dead adults with complete airway obstruction airway obstructed by foreign material: the heimlich maneuver the treatment of food-choking relationships between pre-hospital characteristics and outcome in victims of foreign body airway obstruction during meals key: cord- -uqw ra authors: stenglein, mark d.; jacobson, elliott r.; chang, li-wen; sanders, chris; hawkins, michelle g.; guzman, david s-m.; drazenovich, tracy; dunker, freeland; kamaka, elizabeth k.; fisher, debbie; reavill, drury r.; meola, linda f.; levens, gregory; derisi, joseph l. title: widespread recombination, reassortment, and transmission of unbalanced compound viral genotypes in natural arenavirus infections date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: uqw ra arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. arenaviruses package a large (l) and small (s) genome segment in their virions. for segmented rna viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. from infected animals, we determined the complete or near complete sequence of genome segments that grouped into l and s genotypes. the majority of snakes were multiply infected, with up to distinct s and distinct l segment genotypes in individual animals. this s/l imbalance was typical: in all cases intrahost l segment genotypes outnumbered s genotypes, and a particular s segment genotype dominated in individual animals and at a population level. we corroborated sequencing results by qrt-pcr and virus isolation, and isolates replicated as ensembles in culture. numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. this diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential. several mechanisms generate viral genetic diversity [ ] [ ] [ ] . all work by producing populations of viral genomes, a small fraction of which may exhibit an adaptive advantage in a new host species, different tissue, or in the face of drug or immune pressure. replication of rna viral genomes by error-prone polymerases results in relatively high mutation frequencies, and rna viruses replicate as collections of closely related variant genotypes [ , [ ] [ ] [ ] [ ] . viral genomes can shed content or acquire new loci from their hosts by horizontal gene transfer. in coinfected cells, recombination between different viral strains or species can produce chimeric progeny [ ] [ ] [ ] [ ] . and in cells coinfected by segmented viruses, reassortment generates virions containing shuffled mixtures of segments from the parental genotypes [ , ] . understanding basic mechanisms of viral adaptation is essential in order to better combat, prevent, and predict viral diseases. for example, the ability of pandemic influenza virus strains to efficiently replicate in and be transmitted between humans frequently results from de novo mutation and reassortment [ ] . the continuous emergence of drug-resistant genotypes is a major hindrance to the effective treatment of human immunodeficiency virus- and other pathogens [ , ] . and, recombination between individually attenuated strains present in the oral poliovirus vaccine results in neuropathic progeny strains, and complicates eradication efforts [ ] . viruses in the family arenaviridae have bi-segmented single-stranded rna genomes with a characteristic organization and gene repertoire [ ] [ ] [ ] [ ] [ ] . the larger genome segment (l) is about kb in length and encodes the viral rna-dependent rna polymerase (l) and a small zinc-binding ring domain protein (z). the smaller segment (s) is about half as long and encodes the glycoprotein precursor (gpc) and nucleoprotein (np). on each segment, the two viral genes are in opposite coding orientations and are separated by an intergenic region (igr) that is predicted to form stable hairpin structures. two major lineages of arenaviruses have been described: those that primarily infect rodents and those that infect snakes. the rodent arenaviruses (proposed genus mammarenavirus) typically establish chronic mild infections in their natural hosts but can be transmitted to humans and other mammals [ ] . severe disease such as lassa hemorrhagic fever can result from these zoonotic infections. the snake arenaviruses (proposed genus reptarenavirus) were first identified in us cases of inclusion body disease, a progressive and sometimes fatal disease best described in members of the boidae and pythonidae families (boas and pythons) [ , ] . the identification and study of snake arenaviruses in captive snakes in europe corroborated and extended this finding [ ] [ ] [ ] . one major difference between the snake and mammalian arenaviruses is the provenance of their gpc genes, with the snake virus gene being more closely related to the glycoprotein gene of filoviruses and some avian retroviruses [ , ] . several mechanisms of arenavirus evolution have been described [ , [ ] [ ] [ ] . like all rna viruses, arenavirus genome replication is relatively error prone, and arenaviruses replicate as collections of related variants in vivo [ ] . recombination is thought to have given rise to the ancestral s segment of a clade of the new world rodent arenaviruses [ ] [ ] [ ] . recombination and reassortment between co-infecting arenaviruses has been observed in the laboratory [ ] [ ] [ ] . and, it has been suggested that arenaviruses detected in snakes in europe might have undergone recombination [ , ] . however, arenavirus recombination and reassortment have not been confirmed in natural infections involving extant species. in this study we document and investigate viral genetic complexity of an unanticipated extent and form in naturally infected captive snakes. we determined the complete or near complete sequences of viral genome segments using metagenomic sequencing. sequencing results were corroborated and extended by discriminating quantitative reverse transcriptase pcr (qrt-pcr) and by tissue culture isolation experiments. we detected widespread recombination and reassortment. we also observed an unbalanced accumulation of multiple distinct viral genotypes in individual infections. these findings provide an opportunity to study basic mechanisms of virus evolution and fitness through the identification of genetic determinants underpinning their action. in order to further characterize the genetic diversity of the snake arenaviruses, we gathered frozen case and control tissue samples from around the u.s.a. that were collected between and (s table) . we screened these samples for snake arenavirus rna using qrt-pcr with degenerate primers targeting the glycoprotein gene. a total of samples tested positive by qrt-pcr for viral rna. clinical data of varying detail was available for samples. histopathology was available for of the samples and detection of viral rna was well correlated with histopathology-based ibd diagnosis (table ) . however, many infected snakes displayed no overt clinical signs (s table) . thus, detection of viral rna was correlated with detection of inclusions in tissue sections, but not with obvious clinical measures in a straightforward fashion, consistent with previous reports [ , , , ] . we performed metagenomic sequencing to determine complete viral genome sequences. samples from animals, including the pcr-positive samples, were sequenced. samples from of the arenavirus-positive snakes were sequenced to a depth sufficient for assembly of complete or near complete viral genome segment sequences. in many cases, the assemblies included predicted terminal sequences (s fig, [ , ] ). in total, just over million reads contributed to the assembly of genome segment sequences ( l and s sequences) totaling . mb. genome segment assemblies were validated by re-mapping paired-end reads [ ] . assemblies were well supported, with -fold median coverage (s fig) . coverage levels were generally lowest in the intergenic regions (igr), likely as a result of the predicted hairpin-forming sequences in these regions (s fig) . in cases where coverage levels in igrs fell below reads, pcr was used to confirm assembly continuity. genome segments with less than % global pairwise nucleotide identity were classified into distinct genotypes (figs and and s fig) [ ] . a total of s and l genotypes were delineated by this criterion, and were designated s -s and l -l . within genotypes, l segment sequences shared a mean value of % pairwise nucleotide identity, and between genotypes, sequences shared % identity (s a fig) . for s segments, these values were % and % (s b fig). multiple alignments of the four coding sequences were used to create bayesian phylogenies to visualize the inferred evolutionary relationship of these genotypes (figs - and s fig) . in of infected snakes, viral genotypes consisted of a single s and a single l segment genotype (fig and s table, snakes # - ) . for example, snakes # - , the annulated tree boas from the california academy of sciences described in an earlier report, harbored s genotype and l genotype . this s /l genotype corresponds to the virus we referred to as "cas virus" (casv; [ ] ). in snakes # - , segments of genotype s and l were detected, a genotype corresponding to "golden gate virus" [ ] . numerous reassortant genotypes were evident among the singly infected snakes (fig ) . for example, the l segments present in snakes # and were nearly identical at . % pairwise identity, but their s segments shared only . % pairwise identity (genotypes s and s ), indicating a more distant common ancestor. similarly, the s segments in snakes # and were . % identical but the l and l segments in these snakes were only . % identical. it was not possible in most cases to determine which s/l pairs were ancestral, nor when precisely reassortment had occurred, but it was clear that reassortment had produced these permuted pairings. however, in the majority of animals ( of snakes), we observed viral genotypes that were substantially more complex than single s/l pairs. these snakes harbored multiple s and l genome segment sequences (fig ; snakes # - ). the sets of viral genotypes ranged from that which would be expected for a straightforward co-infection by virus strains in snake # , to more complex combinations such as that in snake # , which contained the sequences of s and distinct l segments. the animal with the most viral genotypes was snake # , which contained genotypes ( s and l) that could combine to produce virions with unique s/l pairs (assuming coinfection of individual cells). the distinct l and s segment sequences within individual snakes shared on average only % and % pairwise nucleotide identity respectively, a level of variation not consistent with sequencing error or intrahost diversification. snakes that were housed together often shared similar combinations of genotypes, and in these cases the sequences of these segments were closely related (figs and and fig ) . the accumulation of viral genotypes within individual animals was not balanced. in all cases there were more l than s segment genotypes (fig and s fig). on average, there were more than twice as many l segment genotypes as s genotypes per animal (mean values of . l and . s genotypes per multiply infected animal; s fig) . in fact, of animals with multiple l sequences harbored only a single s genotype. the s genotype was dominant in individual animals and at a population level (fig and fig ) . this genotype was first detected in a snake from collierville, tn, and a partial s sequence was reported previously [ ] . s segments of this genotype were detected in of snakes ( %). these sequences shared % average global pairwise nucleotide identity. re- individual snakes are infected by complex unbalanced sets of viral genotypes. these tables depict the fractional abundance of s and l genotypes detected in individual animals. each row corresponds to an individual animal. each column corresponds to a particular s or l segment genotype. phylogenies on top of the tables were created using representative sequences from each genotype and a neighbor joining clustering method. shading of cells indicates the fractional abundance of that genotype in the indicated animal, which was calculated as the proportion of sequencing reads mapping to that genotype divided by the total number of arenavirus-mapping reads from that animal. recombinant segments are depicted with a triangle. all shaded boxes correspond to coding-complete assemblies, except for those indicated with a circle. groups of animals harboring similar virus genotype combinations that were housed together are indicated with brackets. neg snake is a sample from an uninfected snake and hela is a sample from total hela cell rna. doi: . /journal.ppat. .g markably, the s segment genotype was found in combination with of the l segment genotypes described in this study. in addition to reassortant genotypes, we also identified recombinant s segments and recombinant l segments ( table) . we used the rdp software to detect and statistically evaluate support for recombination events ( table ; [ ] ). recombination events were well supported by rdp analysis and by read coverage levels over recombination junctions (s fig and table ). we also confirmed segment continuity by pcr amplification across junctions. while this analysis provided clear evidence of recombination, it was not always possible to determine which genotypes were parental and which were recombinant. however, it appeared that many of the recombinant segments coexisted in snakes with one of their parental genotypes. for example, in snake # , s segment sequences were evident. one of these was a canonical s genotype. the other segment, designated s , shared % average pairwise nucleotide identity with the s segment's gpc gene, but only % identity in the np gene ( fig a) . the s np appeared to derive from a segment similar to the s np found in snake # . similar patterns were observed for s segments in snakes # , , , , and , and for l segments in snakes # , , , , , , , , and (figs and and fig b) . these may be situations where the parental and progeny genotypes persistently replicated together following a recombination event. alternatively, the genotypes could have been acquired in independent transmission events. some recombination events resulted in unusual genome organizations. for example, the l genotype found in snakes # and # consisted of a full z coding sequence and a partial l coding sequence from one parental segment concatenated to a partial z and full l from a second segment (fig ) . this segment was predicted to contain intergenic hairpin-containing regions ( xigr) separating the l/z pairs. similar double-igr segments and partial cds were observed in recombinant s segments as well (fig ) . an offset template switching event during genome replication may have generated these xigr segments (s fig; [ ] ). we used several independent approaches to corroborate the original metagenomic sequencing. first, we completely re-sequenced samples, using independent library preparations, and derived essentially identical results. second, we developed a panel of pcr primer pairs that discriminated between distinct viral genotypes, and performed qrt-pcr on a subset of samples and genotypes. in all cases, qpcr-based genotyping mirrored sequencing results (s fig). third, we used tissue culture isolation as another means of determining viral genotype and to confirm that sequences corresponded to infectious virus (see below). the introduction of an already infected snake (# ) into the proximity of an uninfected snake (# ) in a private collection enabled us to monitor viral transmission (fig ) . a blood sample from snake # tested negative for snake arenavirus rna by qrt-pcr and deep sequencing. snake # was then not exposed to other snakes until september , when its owners obtained a second snake, # . snake # arrived with stomatitis and was anorexic. nevertheless, after a -week quarantine, snakes # and # were placed in the same enclosure. snake # continued to refuse to feed and died two weeks later. we obtained the body of snake # and a blood sample from snake # taken in november , and an additional blood sample from snake # from january . we determined that multiple genotypes were transmitted from snake # to snake # during their cohabitation (fig ) . snake # 's viral genotype consisted of s and l genotypes (s , / l , , , , , ; fig b) . the november snake # blood sample was arenavirus positive, but the only genotypes detected by sequencing were s and l . the january snake # sample was still positive, but now l genotypes , , and were also detectable in the blood. analysis of the viral sequences recovered from the two snakes revealed that they were closely related ( . - % identity). this data supports the transmissibility of compound unbalanced snake arenavirus genotypes in the context of cohabitation. we performed tissue culture isolation and passaging experiments to investigate whether compound viral genotypes were competent to initiate productive infections. we applied homogenates from samples to cultures of boa constrictor-derived jk cells and monitored levels of virus rna by qrt-pcr using genotype-discriminating primers. in all cases, we detected replication of all of the viral genotypes identified by our metagenomic sequencing (fig ) . for example, snake # contained viral sequences of genotype s /l , . when a liver homogenate from this snake was applied to a jk culture, replication of all of these segments was detected (fig a) . similarly, when a homogenate from snake # was used as inoculum, replication of all expected viral genotypes was observed (s and l , , , , ; fig b) . the distinct l segments exhibited approximately equal replication efficiencies in these experiments, and the populations could be passaged to uninfected cell cultures. thus, sequences of multiple viral genotypes recombinant genome segments with unusual organizations. recombinant s and l genome segments with unusual double-intergenic regions ( xigr) and/or partial coding sequences are depicted as cartoons. plotted below each cartoon are coverage levels (the number of sequencing reads supporting each base in the assembly) and predicted free energy of folding (i.e. predicted rna secondary structure; -Δg) of nt sliding windows. approximate locations of recombinant breakpoints are indicate with triangles and dotted lines. partial coding sequences are indicated. some of these segments, or very closely related versions thereof, were detected in multiple animals, as indicated. in these cases, cartoons and plots are based on the segment from the snake listed in bold font. doi: . /journal.ppat. .g corresponded to replication competent virus, and multiple viral genome segments replicated as stable ensembles in culture. we also investigated whether an l segment with an unusual x igr organization was competent for replication. snake # l contains a partial z region and predicted igr hairpins (fig ) . to track this segment during infection, we performed qrt-pcr using primer pairs: one pair that targeted the l gene of this segment and one pair that spanned the recombination junction (fig c) . throughout the experiment, near equivalent amounts of template were detected using these primer pairs, suggesting that most copies of this segment maintained their unusual structure. we performed endpoint dilution experiments to determine the genotypes of individual virus particles. we prepared dilution series from liver homogenates from snakes # and # and inoculated jk cells in well plates. after - days, we transferred supernatants to new plates and stained cells with anti-np ab to determine wells positive for the presence of virus. positive wells were then genotyped using discriminating qrt-pcr. in most cases, rna from a single s and a single l genotype were detected in individual wells infected with the most dilute inocula (fig ) . in of ( %) wells at these highest dilutions, more than one l genotype was detected. this could be the result of stochastic co-infection, clumped virus particles, or virus particles packaging more than one l segment. these results were consistent with the model that most virus particles packaged a single l segment, although we could not exclude the possibility that a minority of particles may package additional segments. intrahost variation for individual genotypes was also evident. for most genome segments, multiple sites with minor allele variants were detected (s fig). the frequency of variants in most of these cases was less than variant sites per kb (i.e., %; s fig) . in several cases, a higher frequency of variant sites was observed, for example the s segment of snake # , which averaged variant sites per kb ( . % variants sites). this could have resulted from a greater degree of intrahost variation and divergence or from infection by viruses with closely related genotypes whose sequences were too similar to separate using short read assembly. in this study, we surveyed the genetic diversity of arenaviruses infecting captive snakes in the usa. we used metagenomic sequencing and de novo assembly to determine genome sequences of viruses infecting snakes. we found that most snakes were multiply infected by unbalanced ensembles of s and l segment genotypes. in total, we assembled l and s segment sequences that grouped into l and s genotypes. this expands the known diversity within this group of viruses by several fold. the high level of multiple infection has apparently given rise to numerous recombinant and reassortant genotypes, altogether compromising hundreds of unique viral combinations. we also discovered recombinant genotypes with non-canonical genome organizations, including those harboring apparently superfluous content. metagenomic sequencing results were corroborated by pcr-based approaches, and extended by tissue culture isolation experiments. these findings highlight the utility of performing unbiased whole genome sequencing to determine pathogen genotypes. indeed, our initial pcr-based screening correctly identified infected animals, but completely failed to uncover the genetic complexity present in the infections. although natural infection by multiple arenaviruses has not been previously documented, this phenomenon has been reported for other viruses. for example, infection involving up to influenza viruses has been documented in humans and wild birds [ , ] . and, up to or distinct genotypes of torque teno virus or papillomavirus have been identified in individual human samples [ ] [ ] [ ] . in plants, a virus isolate from citrus trees persistently infected by citrus tristeza virus was found to include several genotypes [ ] . shared characteristics of hostpathogen interaction may enable such highly multiple infections. these include persistent, sub-clinical viral replication, the absence of barriers to superinfection, the lack of an immune response capable of clearing infection, and a high prevalence of infection. virus populations replicate as stable ensembles in culture: (a) liver homogenate from snake # was applied to cultures of jk cells and replication was monitored by measuring supernatant viral rna levels using qrt-pcr and genotype-specific primers. levels of distinct s and l genotypes detected are indicated and are normalized to the amount of s rna detected in the first time point. points and error bars represent mean and standard deviation of two independent experiments. (b) as in (a), but a liver homogenate from snake # was used as inoculum. (c) the xigr l segment detected in snake # replicates stably in culture. same experiment as (b), but qrt-pcr used primers that targeted two different regions of the l segment as depicted in the inset cartoon. doi: . /journal.ppat. .g although we detected many instances of snake tissues containing multiple viral genotypes, our results do not prove that individual cells in these animals were multiply infected. however, the detection of recombinant and reassortant genotypes suggests that at least in some cases cells are multiply infected. although multiple infection per se is not unprecedented, several aspects of these findings are. one is the apparent disconnect between the dynamics of the two viral genome segments, both in individual animals and at a population level. in individual animals, the accumulation of s and l segments was unbalanced: in all multiply infected animals, there were more l than s segments. in the most extreme case (snake # ), a single s genotype was paired with an most or all virus particles contain one l genotype. end-point dilution experiments were performed to determine the viral genotype of individual virus particles. serial dilutions were prepared and applied to jk cells in -well plates. after - days, supernatant was reserved and wells were stained with anti-np antibody to identify infected wells. qrt-pcr using discriminating primers was used to genotype the virus in individual well supernatant. each row corresponds to one well and each column to an s or l genotype. the fractional abundance of s and l genotypes detected in individual wells are indicated as in fig as are the dilution used to inoculate the genotyped well. negative wells ("neg"), not staining with anti-np antibody, from the highest dilutions served as negative controls. the amount of each genotype detected in the inoculum is also indicated. inoculum from snake # liver was used in (a) and from snake # liver in (b). doi: . /journal.ppat. .g ensemble of l genotypes. it is possible that within animals the s and l segments inhabit different fitness landscapes. another, not mutually exclusive, possibility is that differential replication kinetics or packaging efficiencies of the two segments may underlie the observed imbalance. additional experiments in vitro and in animals will clarify this issue. the population level dominance of the s genotype was also unexpected and is worthy of additional investigation. genotype s segments were present in / infections ( %) and in of these, no other s segment was detected. one possible explanation is that the s genotype replicates more efficiently within animals, or is more efficiently packaged and transmitted than other competing s segments. alternatively, the high frequency of this genotype may be a stochastic effect, or may be proportional to viral genotypes in natural virus populations, from which these viruses in captive snakes presumably originate. alternatively, it is possible that the l genotypes observed here were originally paired with s genotypes in free-ranging hosts. if this were the case, then s genotypes are unaccounted for. testing of wild-caught snakes could reveal the "missing" s genotypes and original s-l pairings and would reveal whether the s genotype has indeed risen to dominance in the context of captive animals. whether a similar degree of multiple infection is possible in mammalian arenaviruses is an open question, and one that may be relevant to the possible emergence of new mammalian arenavirus strains with pathogenic potential. it may be that there are larger species barriers for mammalian arenaviruses than there are for snake arenaviruses. another possibility is that an ecological situation analogous to captive snake breeding has never been created for rodents. alternatively, characteristics of the mammalian arenavirus host-pathogen interaction may prevent multiple infection. indeed, superinfection exclusion has been documented in mammalian arenavirus tissue culture experiments [ ] [ ] [ ] [ ] [ ] [ ] . and, cross-protection between mammalian arenaviruses has been documented in vivo [ ] [ ] [ ] . assuming that superinfection accounts for at least some of the genotype accumulation observed here, then no such mechanisms are operating in these snakes. laboratory experiments with mammalian arenaviruses and other segmented viruses could test the generality of this phenomenon. the discovery of " xigr" genome segment configurations was also unanticipated. it would be reasonable to predict that these segments would exhibit decreased fitness or be unstable during replication, given that they carry superfluous content. however, two lines of evidence suggested that these xigr segments are capable of transmission and are stable over multiple rounds of replication. first, several of these segments were detected in co-housed snakes (l in snakes # and ; l in # , , and ; l in # - ). presumably, each of these segments was initially generated via recombination in a single infected snake and then transmitted. second, in tissue culture these segments replicated stably and could be passaged and isolated (figs c and b) . more extensive passage experiments in animals and culture will reveal whether maintenance of the xigr configuration is disfavored over the long term. these novel segment configurations also raise the possibility for the creation of payloadcontaining arenavirus genome segments. for example, the l segment found in snakes # and # contain intergenic regions and bases of extraneous incomplete coding sequence. if a suitable reverse genetic system were developed, this regions could be replaced with an internal ribosomal entry site and the base nanoluc luciferase gene or other payloads [ ] . such a tagged virus could be used for example in in vivo pathogenesis studies as an alternative to trisegmented recombinant arenaviruses [ ] . the nomenclature and taxonomy of the snake arenaviruses will likely have to be reconsidered in light of these findings [ ] . we propose a nomenclature like that used for influenza a virus (iav) subtypes, where new s and l genotypes are simply enumerated [ ] . we would also propose following the taxonomic scheme for iav subtypes, which belong to a single species, influenza a virus. in this case, snake arenavirus genotypes could be grouped into one or possibly more species. recombinant genotypes were not limited to those described in this study. discordance between gpc-and np-based phylogenies including sequences from viruses detected in snakes in europe suggested possible s-segment recombination [ , ] . the increased phylogenetic resolution enabled by this study confirmed the recombinant nature of the s and l segments of boa av nl and the l segment of uhv- [ , ] (table , figs and ) . it is possible that snake importation and husbandry practices have inadvertently created an ecological context that has enabled this phenomenon. boa constrictors with different colorations ("color morphs") are highly valued by collectors and breeders. such colorations arise in nature as local adaptations and wild-caught snakes are commonly imported for breeding purposes. an estimated , boa constrictors per year were imported into the usa alone between - [ ] . mammalian arenavirus species have co-evolved with their distinct, geographically isolated rodent hosts, and it may be that snake arenavirus strains have coevolved similarly in the wild. it is plausible that apparently healthy snakes persistently infected by various arenavirus species have been imported and intermingled in high-density breeding operations. this possible anthropogenic disruption of pathogen ecology is reminiscent of the influenza virus diversity generated in live animal markets [ ] . sampling of viral diversity in free-ranging snake populations are needed to clarify the impact of human activities on the evolution of these viruses and to further assess the disease potential of the resulting recombinant and reassortant genotypes. in the absence of barriers to superinfection, an incalculable number of novel viral genotype configurations, made even more numerous by frequent intra-segment recombination and an error-prone polymerase, could rapidly evolve and accumulate within individual animals and in breeding facilities. in theory, this situation could be exacerbated by the introduction of mammalian arenavirus-infected rodents as feedstock [ ] , although it is unknown whether recombinant or reassortant mammalian/reptile arenaviruses are possible or viable. regardless, further investigation of high-density reptile breeding and feed rodent facilities should be considered. samples were collected between and from california, washington, arkansas, tennessee, louisiana, georgia, and florida. veterinarians in private practice or at university teaching hospitals collected samples. samples were submitted to the university of florida or the university of california san francisco for further processing and storage. blood samples were collected by cardiac or tail vein puncture and frozen until further processing. tissue samples were collected during necropsy and frozen until further analysis or placed in formalin for histopathology. for histopathology, samples were preserved in % formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin. board-certified pathologists blinded to infection status of samples examined h&e stained sections. rna extracted from tissues ( ng) or tissue culture supernatant was denatured for min at °c then cooled on ice and added to μl rt reactions containing pmoles random hexamer oligonucleotide (mds- ), × reaction buffer, mm dithiothreitol, . mm (each) deoxynucleoside triphosphates (dntps), and u superscript iii (life technologies). reaction mixtures were incubated for min at °c then for min at °c and then for min at °c. cdna was diluted : in mm tris ph , . mm edta. qpcr reactions contained μl diluted cdna, . μm each primer, mm tris ph . , mm kcl, . mm mgcl , . mm each dntp, % glycerol, . % np- , . % tween- , . μl taq dna polymerase, and x sybr green (life technologies) in each μl reaction. primer sequences are listed in s table. degenerate primers targeting the glycoprotein gene (mds- and - ) were used to screen for infection. qrt-pcr to screen for individual s and l genotypes was performed using panels of primers that were designed to discriminate between the different sequences. primer pair efficiencies were determined using template dilution series [ , ] . rna was extracted from tissues as previously described [ ] . sequencing libraries were prepared as previously described [ ] . paired-end x bp sequencing was performed on an illumina hiseq in the center for advanced technology at ucsf., producing an average of . x read pairs per sample. a stepwise pipeline was used to process sequencing data. first, data was demultiplexed. then, bases were trimmed of the end of reads and base off the end. next, low quality read pairs with any base window with an average quality score below were discarded. then, reads sharing > % global nucleotide identity (likely pcr duplicates) were collapsed using the cd-hit-est software version . [ ] . adapter sequences were trimmed from the ends of reads. then, host-derived sequences were filtered as previously described [ ] . viral genome sequences were assembled from the remaining reads an iterative strategy was used to assemble genome segment sequences. first, from each dataset, post-filtering reads were aligned using bowtie to a database composed of all alreadydescribed snake arenavirus genome segments sequences [ ] . alignment parameters were set stringently (minimum alignment score of in local mode alignment) so that only reads closely matching already described sequences aligned. alignments were converted into bam format using samtools software and inspected in geneious software [ , ] . sites differing from reference sequences were corrected to generate new draft genome sequences. remaining virus-derived reads (determined by blastx, as described below) that didn't align to an existing virus sequence were used to seed assemblies using the price targeted de novo assembler [ ] . price contigs were added to the set of genome segment sequences and the process was reiterated until all reads were accounted for as described below. once a complete set of genome segment sequences was assembled, we used bowtie to remap all reads from each dataset to the set of genome segment sequences derived from that dataset. these alignments were manually inspected and were used to generate coverage metrics. for each aligned base, coverage was only counted if the preceding and succeeding bases also aligned. this "continuous coverage" metric is more conservative than a simple coverage metric and was implemented to identify possible incorrect assemblies. bam files from these alignments, as well as fastq files of raw sequencing data for all snakes, have been deposited in the ncbi short read archive (sra; accession srp ). genome segment sequences have been deposited in genbank with accessions kp -kp . we employed the following analysis strategy to confirm that we were accounting for the full viral genetic complexity in our datasets, i.e. that we were not overlooking any viral genome segments. we used the blastx tool to align translated reads from each dataset to a database containing all available snake arenavirus protein sequences, including the ones from this study. because blastx alignments are based on protein sequence similarity, it is possible to use them to detect sequences with relatively distant homology. thus, the number of reads with blastx alignments to arenavirus protein sequences (with e-value - ) determined a minimum expected number of virus sequences in each dataset. then, we used the bowtie aligner to stringently map reads from each dataset to the coding sequences of the assemblies generated from that dataset as described above. this allowed us to confirm that the assemblies accounted for all of the arenavirus-derived sequences in each dataset. to calculate the fractional abundance of individual genotypes, we divided the number of read pairs mapping to that genotype by the total number of arenavirus-mapping reads from that dataset. we performed phylogenetic analyses to infer evolutionary relationships between viral genotypes. we created multiple sequence alignments of the coding sequences for each of the viral gene coding sequences, using mafft version v . with default parameters [ ] . these alignments were trimmed using the gblocks software version . b using default parameters except allowing up to half gaps in columns (parameters:-t = d-b = h [ ] ). we used these trimmed alignments and the jmodeltest software v . . to identify a best-fit nucleotide substitution model (gtr; [ , ] ). we ran this software with parameters:-s -f-i-g -aic-bi-c-aicc-dt-p-a-w. we used mrbayes . . to create bayesian phylogenies from these alignments, using commands lset nst = rates = propinv and mcmc ngen = [ ] . these phylogenies were visualized using figtree software (http://tree.bio.ed.ac.uk/software/ figtree/). to create phylogenies including representative snake and mammalian arenavirus sequences, we first downloaded all sequences from the ncbi nucleotide database w/ query: "txid [organism:exp]", i.e. all sequences annotated as being of arenavirus origin. we removed sequences that were not complete or not coding-complete. we extracted np and l cds from these sequences. we used cd-hit-est to create a set of representative sequences, with sequences sharing > % pairwise nucleotide identity collapsed (-c . ) [ ] . we then created and trimmed multiple alignments and phylogenies as described above. global multiple sequence alignments of all s and all l segments were created using mafft software version . with default parameters [ ] . full genome sequences for described european snake arenavirus isolates were included in these alignments (university of helsinki virus (uhv- ) and boa arenavirus nl; ncbi accessions nc_ . , nc_ . , nc_ . , and nc_ . ). alignments were analyzed with the rdp recombination detection program version . using default parameters except to specify linear molecule topology [ ] . we required that recombination events be detected by at least of the methods implemented in the software. putative recombinant segments were validated by examination of phylogenetic discordance and pairwise sequence alignments. genome segments sequences were divided into nt sliding windows (the approximate size of intergenic regions) offset by nt. centroidfold v . . was used to calculate the minimum free energy of folding for each window using parameters-g -e contrafold [ ] . variant sites were called using samtools version . . , using command mpileup-i. we required that variant sites be supported by at least reads in the context of a minimum coverage level of total reads. the number of variant sites per genome segment was calculated and normalized to the length of each segment. tissue culture ric for inoculation experiments, frozen tissue samples were thawed on ice and homogenized in mem + mm hepes (sf-mem) using a dounce homogenizer. homogenates were clarified by centrifugation at , g for minutes then passed through a . μm filter. filtrates were diluted : in sf-mem then added to cultures of near confluent jk cells. culture medium was replaced periodically and supernatants were stored at - °c until further analysis. we used qrt-pcr to measure viral rna levels in culture supernatant. we extracted rna from μl supernatant using the zymo viral rna kit (zymo research). μl rna ( % of eluate) was used as template in rt reactions as above. resulting cdna was diluted and used in qpcr reactions as above, with primers listed in s table. primer pair efficiencies were calculated as above and used to determine quantities of viral rna relative to the amount of s segment rna present in the first time point sample. jk cells were grown as described above and were plated at a density of cells per well in -well plates. one day later diluted virus stocks were added to cells. cells were incubated for - days and then supernatants were transferred to new well plates. then wells were stained with anti-ggv-np antibody, which cross-reacts with the nps of the viruses used in these experiments. staining and washing was performed as previously described [ ] . stained plates were scanned on an odyssey licor instrument to identify infected wells. supernatant from np-positive wells were transferred to -well plate wells plated the day prior with , jk cells per well. one day later cell culture supernatant was replaced. after an additional days of incubation, culture supernatant was collected and clarified by centrifugation at , g for minute. rna was isolated from these supernatants using the zr viral rna kit according to the manufacturer's protocol (zymo research). rna was used as template in qrt-pcr as described above to measure levels of viral rna of various genotypes. this study did not include experiments involving live animals. in some cases, samples (typically blood) were collected from live animals by attending veterinarians. in other cases, tissues were collected during necropsy. all samples were taken and used with owners consent. some samples were collected in the context of other, related studies: the acquisition of tissue samples at the university of florida was authorized under university of florida institutional animal care and use committee protocol a . the acquisition of samples at the university of california davis was authorized under iacuc protocol . supporting information s table. counts and fractions of viral reads in datasets. (xlsx) s fig. cartoons depicting genome segment organization, coverage levels, and predicted secondary structure. genome cartoons and features are drawn to scale. vertical lines at the end of genome segments indicate that the putative terminal sequences are included in the assembly for that segment. where applicable, partial coding sequences and the approximate location of recombination junctions are indicated. note that it was not possible to confidently identify the recombination breakpoint for the l genome segments so it is not depicted. below each cartoon are plotted coverage levels (the number of sequencing reads supporting each base in the assembly) and predicted free energy of folding (i.e. predicted rna secondary structure; -Δg) of nt sliding windows. displayed are fractional abundances of indicated s or l segment genotypes in individual samples as measured by qrt-pcr using a panel of genotype-discriminating primers (q) and sequencing (s). fractional abundance was measured for qpcr using standard curve-based quantitation and for sequencing using read mapping as in fig . neg snake is a sample from an uninfected snake and hela is total hela cell rna. asterisks ( Ã ) indicate the following issues related to template/primer compatibility: snake # l contains mismatches in the primer binding regions so doesn't amplify; primers targeting the l genotype also amplify recombinant genotype l in snake # and # ; primers targeting the l genotype also amplify recombinant genotype l in snake # . in these latter two cases, qpcr-measured abundance was split evenly between the two amplified genotypes. 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expressing two additional genes of interest a revision of the system of nomenclature for influenza viruses: a who memorandum the modern u.s. reptile industry wet markets-a continuing source of severe acute respiratory syndrome and influenza? the lancet lymphocytic choriomeningitis virus in employees and mice at multipremises feeder-rodent operation, united states a new mathematical model for relative quantification in real-time rt-pcr experimental validation of novel and conventional approaches to quantitative real-time pcr data analysis ball python nidovirus: a candidate etiologic agent for severe respiratory disease in python regius the sequence alignment/map format and samtools price: software for the targeted assembly of components of (meta) genomic sequence data. g genesgenomesgenetics mafft multiple sequence alignment software version : improvements in performance and usability improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments jmodeltest : more models, new heuristics and parallel computing a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood mrbayes : bayesian phylogenetic inference under mixed models prediction of rna secondary structure using generalized centroid estimators the authors thank jens kuhn and michael buchmeier for thoughtful comments, jennifer mann for logistical assistance, kelly crotty for preliminary analyses, and jessica lund, eric chow and the center for advanced technology at ucsf for assistance with sequencing. key: cord- -m fs f a authors: wang, mei; donovan, sharon m. title: human microbiota-associated swine: current progress and future opportunities date: - - journal: ilar j doi: . /ilar/ilv sha: doc_id: cord_uid: m fs f a gnotobiotic (gn) rodent models have provided insight into the contributions of the gut microbiota to host health and preventing disease. however, rodent models are limited by several important physiological and metabolic differences from humans, and many rodent models do not dependably replicate the clinical manifestations of human diseases. due to the high degree of similarity in anatomy, physiology, immunology and brain growth, the domestic pig (sus scrofa) is considered a clinically relevant model to study factors influencing human gastrointestinal, immune, and brain development. gnotobiotic piglet models have been developed and shown to recapitulate key aspects of gn rodent models. human microbiota-associated (hma) piglets have been established using inocula from infants, children, and adults. the gut microbiota of recipient hma piglets was more similar to that of the human donor than that of conventionally reared piglets harboring a pig microbiota. moreover, bifidobacterium and bacteroides, two predominant bacterial groups of infant gut, were successfully established in the hma piglets. thus, the hma pig model has the potential to be a valuable model for investigating how the gut microbiota composition changes in response to environmental factors, such as age, diet, vaccination, antibiotic use and infection. the hma also represents a robust model for screening the efficacy of pre- and probiotic interventions. lastly, hma piglets can be an ideal model with which to elucidate microbe–host interactions in human health and disease due to the similarities to humans in anatomy, physiology, developmental maturity at birth, and the pathophysiology of many human diseases. the human gut is colonized by a complex microbial community with a population approximately to times greater than the total number of host cells of which the body consists (björkstén et al. ) . germ-free (gf) animal studies have shown that gut microbiota and their hosts do not simply coexist, but rather form a mutualistic relationship . it is now clear that the structure and functions of the gut microbiota play a crucial role in human health through its contributions in fermentation of undigested carbohydrates, vitamin biosynthesis, regulation of energy storage, maturation of the immune system, pathogen colonization resistance, and brain development (douglas-escobar et al. ; li et al. ) . alteration in the composition of the gut microbiota has been associated with digestive tract diseases, including necrotizing enterocolitis (nec) (mai et al. ; wang et al. ) and inflammatory bowel diseases (ibd) (aomatsu et al. ; michail et al. ; schwiertz, jacobi et al. ; walker et al. ) . additionally, strong evidence from human studies and animal models links intestinal microbiota dysbiosis with a broad-range of immune, metabolic, and neurodevelopmental disorders , including asthma (vael et al. ) , eczema (gore et al. ; wang et al. ) , obesity (karlsson et al. ; ley et al. ; schwiertz, taras et al. ; turnbaugh et al. ), and autism (kang et al. ; parracho et al. ; wang et al. ) . defining the mechanistic underpinnings whereby the intestinal microbiota influences human health and disease has been hampered by individual variation in host genetics and microbiota and ethical concerns of using invasive procedures in human subjects, particularly infants and children. animal models, especially gn rodents, have been extensively employed for exploring the cross talk between the host and commensal bacteria (chow et al. ; gootenberg and turnbaugh ; leser and mølbak ; smith et al. ) . comparative studies of gf and conventional (cv) mice have demonstrated that the gut microbiota profoundly impacts host biology, ranging from intestinal morphology and motility, mucosal and systemic immunity, to absorptive and metabolic functions (smith et al. ). the term cv refers to an animal or human colonized by the microorganisms normally associated with its particular species. for example, gf mice have shorter ileal villi and crypt, and slower rate of small intestinal cell turnover than cv mice (smith et al. ; yi and li ) . furthermore, gf animals have fewer and smaller peyer's patches and mesenteric lymph nodes and greatly reduced fecal iga than animals raised under specific pathogenfree (spf) conditions (honda and takeda ; macpherson and harris ; round and mazmanian ) . moreover, gene expression profile of mouse ileal epithelium is altered in the absence of commensal bacteria . while gn rodent models have provided insight into host-microbe interactions, rodent models are limited by several important physiological and metabolic differences from humans (graham and aman ; heinritz et al. ). more importantly, many rodent models do not dependably replicate clinical manifestation observed in human diseases (lunney ) . therefore, more clinically relevant animal models are needed. nonhuman primates are good models for humans because they share significant physiological, metabolic, biochemical, and genetic similarity with humans; however, expensive housing, long lifespan, and ethical concerns limit their use (puiman and stoll ; shen ) . the domestic pig (sus scrofa) is closely related to the human in terms of anatomy, physiology, and genetics, and is considered the preferred nonprimate model for humans (dawson ; guilloteau et al. ; meurens et al. ; odle et al. ). in addition, the piglet is an excellent model for infectious diseases (meurens et al. ) . the goal of this review is to highlight the usefulness and limitations of the cv pig as a model for human gastrointestinal physiology, immunology, and neurodevelopment. in addition, findings of recent studies using gn and human microbiota-associated (hma) pigs, and future directions with the model will be discussed. pigs have served as biomedical models for decades. advantages of the swine model are highlighted in table . swine have high genome and protein sequence homology with humans, which facilitates understanding of gene-microbiome interactions and the availability of molecular probes and antibodies. for example, when porcine reagents are not available, antibodies and probes directed against human proteins and gene sequences often cross-react with porcine samples (lunney ) . from a nutritional perspective, pigs and humans are omnivorous, whereas rodents are granivorous. in terms of the gastrointestinal anatomy and physiology, pigs are also more similar to humans than are rodents (guilloteau et al. ; odle et al. ) . also, both pigs and human are colon fermenters, whereas fermentation take place in the cecum of rodents (heinritz et al. ) . pigs are also immunologically similar to humans. for example, porcine immune responses more closely resemble human responses than mouse responses with > % of parameters studied, whereas the immune response in mice was more similar to the human in < % of comparisons (dawson ) . humans and pigs also share similar brain growth and development patterns. the major brain growth spurt of the pig extends from late prenatal to the early postnatal period, resembling that of the human, which is different from other animals including rats (dobbing and sands ) . additionally, gross anatomical features such as gyral pattern and gray and white matter distribution of the piglet brain are comparable to those of human infants . furthermore, the possiblity of using pigs from the same litter and similar disease progression make the pig an excellent model for human gastrointestinal physiology, immunology, and neural development. due to similarities in immune function, pigs are also an outstanding model for infectious diseases and vaccine development (meurens et al. ) and have been used extensively to study infectious diseases relevant to human health, including respiratory (bordetella pertussis [elahi et al. ] , cornona virus [saif ], influenza viruses [khatri et al. ] , mycobacterium tuberculosis [gil et al. ] , pseudomonas aeruginosa, and staphylococcus aureus [nielsen et al. ]) and gastrointestinal pathogens (cryptosporidium parvum [vítovec and koudela ] , helicobacter pylori [nedrud ] , hepatitis e virus [krawczynski et al. ] , norovirus [cheetham et al. ], and rotavirus ). conventional piglets are extensively used for studies of early nutrition on gastrointestinal, immune and neural development (guilloteau et al. ; odle et al. ; rytych et al. ); however, a major limitation of the cv piglet model is that the gut microbiota of piglets differs from that of human infants. phyla-level gut bacterial composition of mother-fed or formula-fed (ff) month-old infants ) and -day-old piglets (unpublished observations) are compared in figure . while differences between mother-fed or ff neonates of both species can be appreciated, marked differences in the gut microbiota table advantages of the swine model • omnivorousnutritional requirement and physiology similar to human • high genome and protein sequence similarities with human • immune system more closely resembles human • brain growth and development patterns similar to human ○ the major brain growth spurt similar to human ○ gross anatomical features of the brain are comparable to that of human infants • body sizeallowing various surgical manipulation and collection of adequate quantity of samples. • large litter size ( - piglets/litter) • similar disease progression ○ metabolic diseases, such as obesity and heart disease ○ infectious diseases (e.g., influenza viruses, rotavirus, helicobacter pylori, and neisseria meningitides infection) conrad et al. ( ) ; dobbing and sands ( ) ; lunney ( ) ; meurens et al. ( ) between neonates of the two species exist. for example, actinobacteria (mainly bifidobacterium) predominates (> % of s rrna sequences) in both breastfed (bf) and ff infants, whereas little actinobacteria (< . % of s rrna sequences) is detectable in piglets. the predominant phyla in both sow-reared (sr) and fffed piglets are bacteroidetes and firmicutes, which is more similar to the adult human . additionally, both sr and ff piglets have greater microbial diversity than human infants. establishment of the intestinal microbiota after birth plays a vital role in development of the neonatal gastrointestinal and immune systems (adlerberth and wold ; sjögren et al. ). recent data have also shed light on the ability of microbiota to influence brain development and behavior (collins et al. ; desbonnet et al. ; diaz heijtz et al. ). however, differences in the native gut microbiota between the infant and the piglet complicate direct translation of results from piglets to humans. a solution to this problem is to develop piglets harboring a human gut microbiota. gnotobiotic animals are animals colonized with known strains of bacteria or microbiota. they are delivered by cesarean section (or sterile hatching of eggs) under aseptic conditions and are raised within sterile isolators and fed sterile water and food in order to control their exposure to microorganisms (butler ; gustafsson et al. ). germ-free animals are gnotobiotic animals that have been maintained free from microorganisms, including bacteria, fungi, viruses, and parasites throughout their life. gnotobiotic experiments take advantages of highly controlled, repeatable experimental design, which reduces interindividual variation. as of the writing of this review, over publications have used gn piglets. gnotobiotic pigs have been used to study the impact of bacterial colonization on the host, including organ growth, intestinal morphology, physiology, and immune development (table ) . relative to cv pigs, gf pigs have smaller thyroid and liver size, but larger spleen, lung, heart, and gall bladder mass at weeks of age (shurson et al. ). shirkey and colleagues ( ) investigated the effects of colonization of different bacterial species on small intestinal morphology and observed that the relative length of the small intestine (si) was smaller in gf and mono-associated (ma) piglets than in cv piglets at postnatal day . they also showed that gf and ma piglets had lower relative weights of proximal si regions than that of cv piglets. this is consistent with previous findings reporting that the si thickness of gf pigs was lower than cv pigs (shurson et al. ). in addition, gf and ma piglets had shorter crypt depths, longer villi height, reduced lamina propria cellularity, and smaller peyer's patches in their si compared to their cv counterparts (shirkey et al. ; willing and van kessel ) . the intestinal microbiota also affects brush border enzyme activities. aminopeptidase n and lactase phlorizin hydrolase activities were lower in si enterocytes of cv piglets in comparison with piglets maintained gf or mono- abbreviations: bf, breast-fed; ff, formula-fed; sr, sow-reared. associated with nonpathogenic escherichia coli or lactobacillus fermentum at days of age (willing and van kessel ) . shorter villus height was observed in cv pig enterocytes , thus the lower enzyme activity in cv pigs may be partly explained by reduced cell maturity or mature cell number. in addition, reduced enzyme activity in cv pigs could be due to microbial brush border enzyme deactivation (willing and van kessel ) . several studies have investigated the role of bacterial colonization on the host immune development. haverson and colleagues ( ) compared the immunological structure of the lamina propria in the jejunum of gf piglets with piglets associated with two strains of commensal e.coli between and days of age. by two days after transfaunation, they found that mono-association of gf piglets with e. coli increased the numbers of dendritic and t cells in diffuse lymphoid tissue of the jejunum. additionally, si expression of proinflammatory cytokines interleukin- β (il- β) and il- were higher in gf and ma piglets compared with cv piglets at postnatal day (shirkey et al. ) . other studies have shown effects on systemic immunity as well; relative to gf piglets, serum immunoglobulin level of piglets colonized with a mixture of defined bacteria was significantly greater on the first weeks of life (butler et al. ) . gene microarray profiling of the si epithelium in gf and cv piglets confirmed the essential role of a commensal microbiota for normal development of the host intestinal transcriptome. genes involved in transcription, cell proliferation and differentiation, nutrient transport and metabolism, xenobiotic metabolism and immune responsiveness were upregulated in gn piglets bearing a microbiota from cv piglets versus gf piglets (chowdhury et al. ) . despite the fact that gn piglets differ in aspects of gastrointestinal and immune development relative to cv piglets, gn pigs still provide a unique and powerful model for study of enteric diseases that affect both humans and pigs. for example, gn piglets have been used to investigate disease pathogenesis and/or immunity to rotavirus , enterohemorragic escherichia coli (brady et al. ) , clostridium difficile (steele et al. ) , and shigella dysenteriae type i (jeong et al. ) infections, among others (meurens et al. ) . furthermore, beneficial effects of probiotics (azevedo et al. ; liu et al. ) and the application of vaccination (jeong et al. ) have been tested with gn pig infection models. in most gn pig studies, pigs were colonized with single or multiple strains of bacteria. these studies are useful for delineating the physiological functions of specific microbes, but the effects of single or multiple bacteria on the host are not representative of a complex microbiota. recently, hma animal models have been developed, both in rodents and pigs. the hma rodent model has been used to investigate how the gut microbiome is influenced by dietary components and, in turn, influences host health and disease (gootenberg and turnbaugh ) . for example, production of equol from dietary soy isoflavone, the microbial reduction of cholesterol, the effects of a defined diet changes on the gut microbial community structure and functions, and the biogeography and assembly of the gut microbiota have all been studied in hma rodent models (gootenberg and turnbaugh ) . additionally, hma mice have been important for understanding the role of the microbiota in a variety of human diseases (gootenberg and turnbaugh ) , including the biological effects of microbiota obtained from obese adults or children with kwashiorkor (smith et al. ). these studies have definitively proven that the clinical signs and symptoms commonly associated with many human diseases could be recapitulated by transferring the microbiome and, in the case of organ growth gf pigs had a smaller thyroid and liver, but larger spleen, lung, heart, and gall bladder than cv at weeks of age. shurson et al. ( ) relative si length & weight in gf and ma pigs, the relative length of si was reduced compared with cv at postpartum day . shirkey et al. ( ) compared to gf and ma, relative weight of proximal si regions was higher for cv; while higher relative weight in the distal regions was reported in gf. shirkey et al. ( ) the si thickness of gf pigs was reduced compared with cv. shurson et al. ( ) gf pigs had fewer leukocytes and lower proportion of mature neutrophils in blood at weeks of age. shurson et al. ( ) mono-associated gf pigs with escherichia coli strains increased numbers of dendritic and t cells in diffuse lymphoid tissue of the jejunum days post-association. haverson et al. ( ) serum immunoglobulin level in piglets colonized with a mixture of defined bacteria was significantly higher than in gf piglets in the first weeks of life. butler et al. ( ) si expression of proinflammatory cytokines il- β and il- were higher in gf and ma pigs relative to cv at postnatal day . shirkey et al. ( ) abbreviations: cv, conventional; gf, germ-free; ma, mono-associated; si, small intestine. kwashiorkor, providing a similar diet (smith et al. ) . however, due to the differences in anatomy and physiology between rodents and humans, some important members of human gut microbiota, such as bifidobacterium do not readily colonize the rodent gut (raibaud et al. ). thus, results obtained from the use of rodent models may be difficult to extrapolate to humans, especially human infants, who are extensively colonized with bifidobacterial species. several studies have investigated the possibility of transfaunation of gut microbiota from humans to piglets (table ). in the study of pang and colleagues ( ) , piglets delivered by cesarean section were housed in an spf barrier system and were inoculated orally with a fecal suspension collected from a healthy -year-old boy. the culture-independent analysis of the gut microbiota of recipient piglets and the human donor revealed that the microbiota of hma piglet was more similar to that of human donor than to that of cv piglets. moreover, bifidobacterium and bacteroides, two predominant bacterial groups of the infant gut, were successfully established in the gastrointestinal tract of piglets. furthermore, introduction of solid food during the weaning period significantly altered the gut microbiota in hma piglets; this change in the gut microbiota is similar to that observed in human infants (table ) . in another study, piglets derived by cesarean section were inoculated with human infant or adult microbiota (table ). the piglets were housed in sterile isolators and maintained on infant formula or solid diet for swine. high throughput sequencing of the s rrna v region was used to monitor to what extent the transplanted human microbiota changed in piglets over time. when infant stool was transferred, the microbiota composition of the hma piglets converged toward that of the human donor. in contrast, the microbiota of hma piglets harboring the adult human microbiota did not converge toward the composition of the donor even days postinoculation. in a more recent study (zhang et al. ) , piglets derived by hysterectomy were inoculated with a suspension of fecal samples obtained from a bf infant between and days postpartum. the piglets were maintained in germ-free isolators and fed sterilized infant formula. sequencing the v region of s rrna genes showed that hma pigs harbored a microbiota similar to that of the infant donor. collectively, these studies demonstrate the feasibility of transplantation of a complex human gut microbiota to piglets. additionally, in comparison with the cv counterparts, the intestinal immunity of hma piglets is well developed (che et al. ), whereas that of hma rodents is not (imaoka et al. ). therefore, the hma piglet model provides a significantly improved system for research on gut ecology and host-microbe interactions, particularly when the human infant is the population of interest (pang et al. ; zhang et al. zhang et al. , . the hma pig model has been used in several recent publications to study dietary prebiotics and probiotics and for infection models. the first use of hma piglets was described by shen and colleagues ( ) , who studied the prebiotic activity of shortchain fructo-oligosaccharides (scfos). the piglets were inoculated with fecal suspension from a -year-old man and fed basal diets alone (control) or supplemented with scfos at . g/kg body weight daily for days after birth. the composition of the fecal microbiota was monitored by denaturing gradient gel electrophoresis and quantitative polymerase chain reaction (pcr). as demonstrated previously in human trials (bouhnik et al. ; , supplementation of scgos increased the abundance of bifidobacterium. the bifidogenic effect of gos ( g/l of formula) has also been examined in newborn cv piglets; however, no significant increase in fecal bifidobacteria abundance was detected after days of supplementation. differences in bifidogenic effects of fos observed in cv and hma piglets may partly due to differences in bifidobacterium species composition between hma and cv piglets. for example, hma piglets harbor bifidobacterium of human origin, such as b. longum, b. breve, b. catenulatum, and b. adolescentis, while bifidobacterium found in the gut of piglets are b. suis, b. globosum, and b. pseudolongum (harrman and knol ; heinritz et al. ) . previous studies have shown prebiotics stimulate bifidobacteria species differently. for example, a mixture of scgos and polydextrose in infant formula increased b. longum but not b. catenulatum counts (scalabrin et al. ) . because of the important role of bifidobacterium in preventing intestinal infection, promoting gut integrity, and modulating the host immune homeostasis (gibson and roberfroid ) , stimulating the growth of gut bifidobacterium is considered as a marker of prebiotic effect (roberfroid et al. ) . therefore, hma piglets provide a more attractive model than cv piglets for evaluation of potential prebiotics. another application of the hma piglet model is for testing therapeutic interventions, such as probiotics and vaccination on host immune response and gut microbiota. wen and colleagues ( ) tested dose-dependent effects of lactobacillus rhamnosus gg (lgg) on the immune response to human rotavirus (hrv) vaccination in the hma pig model. they observed that the human gut microbiota stimulated neonatal immune development, as evidenced by a significant increase in the frequencies of interferon (ifn)-γ producing t cells and a decrease in the frequencies of cd +cd -foxp + regulatory t cells (tregs), and il- -or tgf-β-producing tregs in hrv-vaccinated pigs. furthermore, the higher dose of lgg ( doses, up to a colony-forming-unit [cfu]/dose), but not the lower dose ( doses, up to cfu/dose), increased the lgg counts in the intestinal contents of hma pigs and significantly enhanced hrv-specific ifn-γ-producing t cell responses. moreover, oral supplementation of lgg prevented the changes in gut microbial composition caused by hrv infection (zhang et al. ). the hma pig model for studies of human gut microbiome because of the important role of human microbiota in the maintenance of health and causation of disease, several international efforts have designed to the study of human microbiota in recent years, including the human microbiome project (hmp) (http:// commonfund.nih.gov/hmp/index [human microbiome consortium ]) and the metagenomics of the human intestinal tract (metahit) (www.metahit.eu ]) initiatives. while much progress has been made by describing the composition of the gut microbiome in the human population and linking it to age-and health-related outcomes, much of the data at this point are associative. furthermore, confounding factors that influence the composition of the gut microbiota are difficult or impossible to control at the present time in human studies. these factors include individual variation in the host genetics and microbiota, current and past environmental exposures, and dietary nutrient composition and caloric load (gootenberg and turnbaugh ) . the hma pig model provides the ability to minimize many of the confounding variables mentioned above and, as such, will be valuable for studying microbiota composition change due to external factors, such as age, diet, viral infections, vaccination, and antibiotic use on the development of gut microbiota. human donors -y-old boy (n = ) adults (n = ; - y) -mo-old bf baby (n = ) adults (n = ; - y) - d-old bf infant (n = ) fecal inoculation ml of % fecal suspension ml of % fecal suspension ml of % fecal suspension ml of % fecal suspension ml of % fecal suspension • gn pigs carried a microbiota similar to the human donor's microbiota transplantation of gut microbiota from human to piglets is feasible. the pig intestine can be colonized with human fecal microbiota to generate a realistic model of human gi tract. human gut microbiota could be transplanted to and colonize gn pigs. abbreviations: bf, breast-fed; cv, conventional; d, day; eric-pcr, enterobacterial repetitive intergenic consensus sequence-pcr; gi, gastrointestinal; gn, gnotobiotic; hma, human microbiota-associated, qpcr, quantitative pcr; spf, specific pathogen free; ttge, temperature gradient gel electrophoresis; y, year. the hma pig model for investigating the role of microbiota on normal development establishment of the gut microbiota after birth plays an important role in stimulating the development of the neonatal gastrointestinal, immune and neural systems. studies in gf animals have shown that colonization of the commensal microbiota is required for normal intestinal epithelial cell proliferation and migration, and maintenance of villus morphology (shirkey et al. ; van kessel , ). gf animals do not develop normal lymph node architecture and have a reduced antibody production (macpherson and harris ; round and mazmanian ) . evidence for the role of gut microbiota in neural development is intriguing, and mechanistic data is rapidly emerging. diaz heijtz and colleagues ( ) investigated the impact of colonization of gut microbiota on the mammalian brain development and behavior and reported that gf mice displayed increased motor activity and reduced anxiety compared to spf mice with a normal gut microbiota. additionally, gf mice exposed to gut microbiota early in life showed characteristics similar to spf mice, including reduced expression of synaptophysin and psd- , two proteins that are specifically involved in synaptogenesis pathways (diaz heijtz et al. ) . studies of gastrointestinal, immune, and neural development often require tissue collection from the gastrointestinal tract, immune organs, and brain; however, due to ethical concerns and the limitation of invasive procedure, collecting tissue samples from human subjects is extremely difficult or impossible. therefore, clinically relevant animal models are needed. because of the high degree of similarity in anatomy, physiology, immunology, and brain growth and development patterns between pigs and humans, piglets are considered an ideal model for research on gastrointestinal, immune, and brain development. previous studies have shown environmental factors, such as diet and the use of antibiotics, pre-and probiotics, modify the composition of gut microbiota . germ-free piglets colonized with human intestinal communities provide a tool for examining the environmental factors on the establishment of gut microbiota and how the resultant microbiota impacts the development of gastrointestinal, immune, and neural systems. as previously discussed, symbiotic host-microbiota interactions play a key role in maintaining homeostasis. shifts in the bacterial composition of the human gut microbiota have been associated with several human disorders. table summarizes association between gut microbiota change and microbiota-associated diseases. much of the information regarding the role of gut microbiota in human diseases comes from cross-sectional studies in which microbial community structures are altered in subjects with disease compared to healthy controls. however, it remains unclear whether changes in gut microbiota composition are the cause or the consequence of the diseases. studies designed to access a causative role for the gut microbiota are critically needed. understanding dysbiosis in human subjects is challenging because of the extraordinary complexity of the gut ecosystem and the tremendous variability in microbiota between healthy individuals (gill et al. ) . hma pigs provide an excellent model for isolating microbiota as an environmental factor in disease models. for example, fecal samples could be collected from lean and obese humans or individuals suffering from microbiota-associated diseases, such as ibd and nec, and healthy controls and then used to colonize gf pigs. using hma pigs, together with metabolomics, metaproteomics, host gene expression profiling, and metatrascriptomics, we may be able to delineate the role of gut microbiota in diseases at the cellular and molecular level. using hma piglets to identify potential biomarkers of microbiota-associated diseases through the use of metabolomics and metaproteomics has implications for development of diagnostic and therapeutic strategies for both infectious and noninfectious conditions. however, a current limitation is the completeness of bioinformatics repositories for metabolomics and proteomics. pre-and probiotics have been studied in recent decades as a way to modulate gut microbial composition and functions (ducatelle et al. ). prebiotics are defined as "a selectively fermented ingredient that results in specific changes, in the composition and/ or activity of the gastrointestinal microbiota, thus, conferring benefit(s) upon host health" (roberfroid et al. , p. s ) . prebiotics have the potential to stimulate the growth of beneficial bacteria, such as bifidobacterium and lactobacillus. probiotics are "live microorganisms which when consumed in adequate amounts, confer a health benefit on the host" (fao/who , p. ). bifidobacterium and lactobacillus are the most commonly used probiotics (walsh et al. ) . other bacterial genera such as akkermansia and faecalibacterium have also been reported as potential probiotics (thomas et al. ) . pre-and/or probiotic intervention has been used successfully for promoting health and prevention or treatment of some microbiota-associated disorders, such as eczema, ibd, nec, and obesity in human studies (kadooka et al. ; li et al. ) ; however, the mechanisms underlying the beneficial effects of pre-or probiotics remain incompletely understood. understanding the impact of pre-or probiotics on the gut microbiota and host requires carefully controlled studies in which potential confounding variables such as host genotype, diet, and environmental exposure can be controlled. gnotobiotic animals can be reared under well-controlled conditions, representing one way to constrain some of these variables. recently, gn mice harboring a mixture of species of human gut microbiota were studied prior to and after gavage with five fermented milk strains (mcnulty et al. ). the results revealed only a minimal change in the composition of the microbiota, whereas significant changes in the expression of microbiome-encoded enzymes in numerous metabolic pathways, especially the pathways related to carbohydrate metabolism, were observed (mcnulty et al. ). compared to rodents, pigs colonized with human microbiota are more similar to humans in anatomy, physiology, microbiota, and genetics, providing a more attractive model for elucidating molecular bases of pre-and probiotic action. emerging studies have demonstrated the feasibility of generating and maintaining gn and hma piglets for relatively long periods of time. these models represent robust systems in which to dissect the intricacies underlying host-microbe relationships essential for maintaining health and preventing disease. to date, studies have not yet exploited hma piglets as recipients of microbiota associated with specific diseases, as has been effectively exploited in gn rodent models. the hma piglet is the optimal model for preclinical screening of novel pre-, pro-, and symbiotic preparations and elucidating the impact of these preparations on microbiota composition and host responses. establishment of the gut microbiota in western infants terminal restriction fragment length polymorphism analysis of the gut microbiota profiles of pediatric patients with inflammatory bowel disease lactobacillus acidophilus and lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus allergy development and the intestinal microflora during the first year of life shortchain fructooligosaccharides administration dose-dependently increases fecal bifidobacteria in healthy 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respiratory coronavirus infections in a swine model of enteric disease the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses new prebiotic blend of polydextrose and galacto-oligosaccharides has a bifidogenic effect in young infants microbiota in pediatric inflammatory bowel disease microbiota and scfa in lean and overweight healthy subjects primate models for cardiovascular drug research and development assessment of the modulating effects of fructo-oligosaccharides on fecal microbiota using human flora-associated piglets effects of commensal bacteria on intestinal morphology and expression of proinflammatory cytokines in the gnotobiotic pig physiological relationships between microbiological status and dietary copper levels in the pig influence of early gut microbiota on the maturation of childhood mucosal and systemic immune responses use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota gut microbiomes of malawian twin pairs discordant for kwashiorkor piglet models of acute or chronic clostridium difficile illness exploring the influence of the gut microbiota and probiotics on health: a symposium report a core gut microbiome in obese and lean twins an obesity-associated gut microbiome with increased capacity for energy harvest denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets high-throughput clone library analysis of the mucosaassociated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease beneficial modulation of the gut microbiota increased abundance of sutterella spp. and ruminococcus torques in feces of children with autism spectrum disorder reduced diversity in the early fecal microbiota of infants with atopic eczema s rrna gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis probiotic lactobacillus rhamnosus gg enhanced th cellular immunity, but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model enterocyte proliferation and apoptosis in the caudal small intestine is influenced by the composition of colonizing commensal bacteria in the neonatal gnotobiotic pig intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig the germfree murine animal: an important animal model for research on the relationship between gut microbiota and the host probiotics and virulent human rotavirus modulate the transplanted human gut microbiota in gnotobiotic pigs a pig model of the human gastrointestinal tract this work was supported in part by hatch funds (project illu- - ) distributed by the division of nutritional sciences at the university of illinois through its vision / program. key: cord- -mfl ey y authors: li, xiaoyu; wang, lili; zhen, yuhong; li, shuying; xu, yongping title: chicken egg yolk antibodies (igy) as non-antibiotic production enhancers for use in swine production: a review date: - - journal: j anim sci biotechnol doi: . /s - - - sha: doc_id: cord_uid: mfl ey y in recent years, the use of in-feed antibiotics for growth and disease prevention in livestock production has been under severe scrutiny. the use and misuse of in-feed antibiotics has led to problems with drug residues in animal products and increased bacterial resistance. chicken egg yolk antibodies (igy) have attracted considerable attention as an alternative to antibiotics to maintain swine health and performance. oral administration of igy possesses many advantages over mammalian igg such as cost-effectiveness, convenience and high yield. this review presents an overview of the potential to use igy immunotherapy for the prevention and treatment of swine diarrhea diseases and speculates on the future of igy technology. included are a review of the potential applications of igy in the control of enteric infections of either bacterial or viral origin such as enterotoxigenic escherichia coli, salmonella spp., rotavirus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus. some potential obstacles to the adoption of igy technology are also discussed. antibiotics have been widely used in the swine industry for more than years. the efficacy of antibiotics in increasing growth rate, improving feed utilization, and reducing incidence of disease is well documented [ ] . in general, sub-therapeutic levels of antibiotics in swine diets increase the growth rate by an average of % in weanling pigs, % in growing pigs, and % in growing-finishing pigs [ ] . in addition, antibiotics are also used for disease prevention (prophylactic doses) and treatment (therapeutic doses). however, serious concerns have arisen with regard to the potential risks for human health including drug residues in meat products and increased bacterial resistance due to the use and misuse of in-feed antibiotics [ ] . as a result, the european union has totally banned the use of antibiotics for growth promotion since january [ ] , while the us food and administration (fda) has been phasing out non-medical antibiotic use for livestock since december [ ] . diarrheal disease is a frequent cause of heavy economic losses for swine producers. a major challenge currently facing the swine industry is to develop alternative means for controlling diarrhea in young pigs (particularly neonatal and early-weaned piglets) that are not only cost effective, but also allow for sustainable pork production. in the past two decades, a variety of materials have been tested as effective alternatives to antibiotics to maintain swine health and performance. the most widely researched alternatives include enzymes [ ] , organic acids [ ] , pro-and prebiotics [ ] [ ] [ ] , herbal extracts [ , ] and neutraceuticals such as copper and zinc [ , ] . however, these alternatives produce limited growth promotion and protection against pathogens. recently, egg yolk antibodies, generally referred to as igy, have attracted considerable interest as an alternative to antibiotics for growth promotion in the presence of disease causing organisms [ ] . igy possesses a large number of advantages over mammalian igg such as cost-effectiveness, convenience, and high yield [ ] . oral administration of specific igy antibodies has been shown to be highly effective against a variety of intestinal pathogens which cause diarrhea in animals and human such as enterotoxigenic escherichia coli (etec), salmonella spp., bovine and human rotaviruses, bovine coronavirus, porcine transmissible gastroenteritis virus (tgev), and porcine epidemic diarrhea virus (pedv) [ , ] . following a brief description of igy technology and the advantages of igy, this review will focus on the potential to use specific igy as a production enhancer in swine production, and discuss the potential obstacles to the adoption of igy technology. as described more than years ago, klemperer [ ] first demonstrated that avian maternal antibodies are transferred from serum to egg yolk in order to protect the developing embryo from potential pathogens but at that time there was no scientific application for this knowledge. however, when animal welfare became a matter of serious ethical concern for the scientific community, the results of klemperer began to receive more interest, particularly since the s. since , igy technology (i.e. the production and use of igy) has become an internationally accepted practice [ ] . in , the european centre for the validation of alternative methods (ecvam) workshop strongly recommended that igy should be used as an alternative to mammalian antibodies [ ] . in , igy technology was approved as an alternative method for supporting animal welfare by the veterinary office of the swiss government (office vétérinaire fédéral). details concerning the production of specific igy have been reviewed by schade et al. ( ) [ ] . briefly, in order to produce specific igy antibodies, laying hens are immunized with specific foreign pathogens which induce immune responses, including the production of antibodies with activity against these specific disease conditions. these antibodies are then transferred to the egg yolk and deposited in large quantities. booster immunizations are usually given to ensure continued transfer of antibodies from the hen's serum to the egg yolk. these antibodies are then extracted from the egg yolk and processed to be administered directly to the animal or incorporated into diets. antibodies can be administered in several forms including whole egg powder, whole yolk powder, a water-soluble fraction powder or purified igy. antibody production and titer development as a result of immunization are not very predictable. variables influencing immunization include the antigen (dose, mw), type of adjuvant used, route of immunization, immunization frequency and the chicken itself (such as housing conditions, age, breed, egg laying capacity). the use of chickens as the immunization host for antibody production provide a number of advantages over production methods using mammals. the most significant advantage is that egg collection is non-invasive compared with the stressful bleeding of animals to obtain serum. in addition, the high and long-lasting titer produced in chickens reduces the need for frequent booster injections. another advantage is that the production of igy antibodies against highly conserved mammalian proteins is more successful in chickens than in other mammals [ ] and requires much less antigen to induce an efficient immune response due to the phylogenetic distance between chickens and mammals [ ] . a hen can be considered as a small "factory" for antibody production. one hen can produce more than . g of total igy per year of which to % are specific antibodies [ ] . this extraordinary quantity is equivalent to the production of . rabbits over the course of a year. the cost for maintaining laying hens are also lower than those for mammals such as rabbits [ ] . therefore, egg yolk offers a more hygienic, costefficient, convenient and rich source of antibodies compared with traditional production methods using mammals. in contrast to antibiotics, the use of igy is environmentally-friendly and elicits no undesirable side effects, disease resistance or toxic residues [ ] . in terms of function, unlike mammalian igg, igy does not activate mammalian complement and also does not interact with mammalian fc and complement receptors. as well, igy does not bind to protein a, protein g or rheumatoid factor [ ] . these differences provide great advantages for the successful application of igy technology in many research areas, diagnostics [ ] , antibioticalternative therapy [ ] and xenotransplantation [ ] . the exact mechanisms through which igy counteracts pathogen activity have not been determined. however, several mechanisms have been proposed by xu et al. ( ) [ ] including agglutination of bacteria, inhibition of adhesion, as well as opsonization followed by phagocytosis and toxin neutralization. among these, inhibition of adhesion is considered the primary mechanism by which specific igy functions. briefly, igy antibodies generated against intestinal disease causing organisms may reduce the incidence of disease by preventing the attachment of pathogens to the intestine, such as blocking the mucosal receptor, interfering with binding to mucins, or neutralizing the colonization factor (such as the outer membrane protein, lipopolysaccharide, fimbriae (or pili), and flagella) [ ] . the possible primary mode of action by which igy protects pigs against e coli k induced diarrhea is illustrated in fig. . diarrhoea in neonatal and post-weaning pigs can be caused by a number of causative agents and has become a serious problem in the swine industry due to the trend towards large intensive herds and early weaning (i.e. - rather than - days of age). the potential applications for using orally administered igy in the control of enteric infections of either bacterial or viral origin in piglets have been studied at length and are summarized in tables and . enterotoxigenic escherichia coli diarrhea due to enterotoxigenic escherichia coli (etec) is by far the most common enteric colibacillosis encountered in neonatal and post-weaned pigs [ ] [ ] [ ] . etec can cause diarrhea in piglets by colonization in the small intestine and thereafter through the production of either one or two enterotoxins namely heat-labile enterotoxin (lt) and heat-stable enterotoxins (st), which induce massive fluid and electrolyte secretion into the gut lumen [ ] . the strains of e. coli associated with intestinal colonization which cause severe diarrhea are those expressing the k (or f ), k (or f ), p (or f ), f and f fimbrial adhesions. these fimbrial adhesions mediate the adhesion of e. coli to the epithelial mucosa lining of the intestine and thereby contribute to their virulence. among the different etec, those expressing the k + fimbrial antigen are the most prevalent forms causing e. coli infection world-wide [ ] . it has been estimated that k + etec are responsible for more than half of the piglet mortality which occurs annually [ ] . oral administration of igy offers a potential prophylactic and therapeutic approach for controlling e. coli-induced diarrhea in piglets. igy has been shown to successfully control intestinal infections of e. coli k , k , p, and f in young pigs [ , , ] . yokoyama et al. [ ] showed that orally administered crude igy (the water-soluble fraction) generated against e. coli k , k , or p fimbriaes was protective against infection from three homologous strains of e. coli in a dosedependent manner in colostrum-deprived piglets. in vitro, e. coli k , k , and p strains adhered equally to porcine epithelial cells from the duodenum and ileum but failed to so in the presence of homologous antifimbrial igy [ ] . the group of marquardt [ , ] from the university of manitoba in canada carried out some excellent studies on the passive protective effects of igy against etec k fimbriae in neonatal and early-weaned piglets. in an animal feeding study, -day old pigs were orally challenged with high doses of etec k at and h of the experiment. they were then oral administered with crude igy (the water-soluble fraction) three times a day for two consecutive days after the first e. coli challenge. control piglets (treated with igy from non-immunized hens) had severe diarrhea within h and lost weight and % of the pigs died within h of infection. in contrast, the pigs given igy from immunized hens exhibited no signs of diarrhea and h after treatment, and had a positive weight gain. furthermore, this group performed studies on the practical use of igy in a field trial. their experiment showed suppression of the incidence and severity of diarrhea in - -day-old weaned [ ] piglets fed specific igy powder, which were much lower than those fed a commercial diet containing an antibiotic. the number of pigs in this study was not large and it would be desirable to repeat this study with a greater number of animals. in another study with f + e. coli, it was shown that the f antibodies diminished the incidence of diarrhea and death in weaned piglets infected with f + e. coli [ ] . zuniga et al. [ ] also reported that weaned pigs fed a basal feed plus % (w/w) egg powder with igy antibodies against the same fimbrial variant (f ab or f ac) were fully protected when the pigs were challenged with f + e. coli. in addition to reducing the incidence and severity of piglet diarrhea, several studies have shown that igy has growth promoting effects in early-weaned pigs, similar to spray-dried animal plasma and spray-dried porcine plasma [ ] [ ] [ ] [ ] . table shows the results of an experiment where the performance of pigs fed specific anti-k antibodies was compared with that of pigs fed diets supplemented with zinc oxide, fumaric acid or antibiotics after oral etec k challenge [ ] . all four feed additives successfully increased pig performance compared with unsupplemented pigs with significant reductions observed in scour score and piglet mortality. in this experiment, igy was equal to antibiotics in enhancing pig performance. table shows the effects of adding egg yolk containing specific anti-k antibodies (egg), egg-exchange, or spray dried porcine plasma to a corn-soybean meal-based (con) diet on performance, plasma urea and weight of pancreas of weaned pigs that were not challenged with etec k challenge [ ] . the results show that addition of egg to the con diets reduced plasma urea nitrogen concentration, and increased feed intake by % and tended to increase weight gain by % in phase ii. this study indicates that inclusion of egg in the diets for pigs immediately after weaning can significantly affect their future growth performance. unfortunately, limited numbers of pigs were used in this study and a repeat with greater numbers would certainly be welcomed. although adhesin-mediated colonization is a precondition for etec pathogenesis, enterotoxins are thought to be the central virulence determinants leading to diarrhea diseases and may also play a role in the colonization process [ , , ] . therefore, the ideal protective agent against etec infection should include protection against both adhesin antigens and enterotoxins [ ] . in contrast with ltb and sta, enterotoxins sta and stb are poorly immunogenic because of their small size, but they can attain immunogenicity when coupled chemically or genetically to an appropriate carrier [ , ] . in order to make useful toxoids, we constructed polyvalent enterotoxin sta-ltb-stb dna and protein vaccines endowing immunogenicity to both sta and stb [ ] . laying hens were immunized with dna vaccines and obtained antitoxic antibodies from egg yolks and this was confirmed by indirect elisa. the polyvalent dna vaccine pci-sta-ltb-stb expressed the sta-ltb-stb fusion peptide in vitro in cultured hela cells. these egg yolk antibodies were able to neutralize the natural toxicity of sta and ltb with the highest dilution of / and / in a suckling mouse assay [ ] . these results indicate that the recombinant sta-ltb-stb protein has the potential to serve as an effective and convenient polyvalent toxoid which can provide broad protection against etecinduced diarrhea. this study was conducted with mice and a piglet study should be conducted to confirm these findings. salmonella infection has been recognized as one of the most common foodborne diseases in humans, causing . million cases with an estimated economic impact of $ . billion each year in the united states [ , ] . this disease can occur via foodborne transmission, animal contact, or environmental spread [ , ] , and farm owusu-asiedu et al. [ ] animals are the most likely source of human salmonellosis [ , ] . therefore, it is important to control this disease, not only to reduce productive losses in domestic livestock, but also to prevent its transmission into the human food chain. salmonella species, s. enteritidis and s. typhimurium, in particular, are thought to be the major agents of human salmonellosis [ ] . they are non-host serotypes which can cause disease syndromes like gastroenteritis and systemic infectious in a wide range of animal species, including humans. studies by yokoyama et al. [ ] investigated the efficacy of igy antibodies specific for outer membrane protein (omp), lipopolysaccharide (lps), or flagella (fla) for controlling s. typhimurium or s. enteritidis. they treated mice orally with an appropriate placebo or purified igy following a challenge with s. enteritidis. antibody treatment resulted in survival rates of , and % using omp-, lps-, and fla-specific antibodies, in contrast to survival rates of only % in control mice. in the s. typhimurium trial, the survival rate was , and % using omp-, lps-, or fla-specific antibodies, in contrast to % in the control mice. these preliminary results suggest that antibodies against specific salmonella proteins can control salmonellosis when orally administrated to mice. studies with pigs have been reported [ , ] . unfortunately, reports where similar benefits of igy to those found in mice were not obtained with pigs. mathew et al. [ ] found that feeding egg yolk powder containing anti-salmonella igy antibodies may not be particularly effective in reducing salmonella shedding in pigs. in this experiment, specific egg yolk powder derived from chickens challenged with purified salmonella typhimurium antigens (fimbrial protein, omp, and lps) was included in swine feed. treatments were provided beginning on day of the experiment, and all pigs were challenged with salmonella typhimurium on day . fecal samples were collected on days , , , , , , , , and to determine shedding of salmonella. the results showed that in-feed igy antibodies did not diminish the shedding of salmonella, and was inferior to antibiotic treatment. the failure of igy to improve performance is suspected to be due to the fact that the invasive nature of salmonella allowed the organism to by-pass the gut and the dietary treatments by moving through vascular and/or lymphatic routes directly into the colon [ ] [ ] [ ] . however, available data are still too limited to allow reliable conclusions regarding the possible efficacy of igy to control salmonella infection in swine. porcine transmissible gastroenteritis virus (tgev), a porcine coronavirus, can cause a highly contagious enteric infection in swine of all ages [ ] . the infected piglets develop significant clinical signs, including vomiting, emaciation and severe diarrhea. the disease is especially severe in the animals less than weeks old with a mortality of nearly % [ , ] . with the lack of successful vaccines to prevent a tge outbreak, the disease occurs frequently in swine farms. it has been shown that specific igy has great potential as an alternative prophylactic approach like colostral antibodies against tgev [ , ] . in a prophylactic efficacy experiment, oral administration with igy significantly increased newborn piglet survival rate ( . %) after challenge exposure compared with the control ( %), whereas the therapeutic effects demonstrate that mortality was dramatically reduced by orally administered igy in two farms that showed tgev positive results [ ] . unfortunately, piglet performance was not monitored in this study. porcine epidemic diarrhea virus (pedv) is another important enteric viral pathogen that is responsible for neonatal piglet diarrhea [ , ] . although the clinical symptoms of pedv infection are similar to tgev infection, pedv is antigenically different from tgev. epidemiological observations have indicated that the spread of disease seems to be slower, but rather persistent compared with a tgev outbreak [ ] . it has been shown that igy can be an alternative method for conferring protection in piglets against pedv [ , , ] . igy was found to reduce mortality in piglets after challenge exposures. the field application of igy from three farms also revealed the survival rate was increased significantly in pigs treated with igy ( . %) compared with a control group ( . %). rotavirus is a major pathogen of infectious gastroenteritis, not only in children and infants, but also in domestic animals. in humans alone, it has been estimated that rotavirus infections result in several million deaths each year [ ] . animals are also seriously affected by this virus. rotavirus from calves, pigs, mice, foals, infant humans, lambs, chickens, and turkey are antigenically related [ ] . therefore, the appropriate anti-rotavirus antibodies will react with the virus present in any of these species. igy has been shown to successfully control intestinal infection of rotavirus in newborn calves and mice [ ] [ ] [ ] [ ] . studies with pigs have been reported and similar positive results were obtained [ ] . a passive treatment based on human rotavirus specific igy antibodies not only prevented gnotobiotic piglets from developing diarrhea caused by the prevalent strain of wa g p [ ] hrv, but also significantly reduced the amount of infectious virus shed compared with a negative control group. there are many obstacles that can limit the use of igy for the control of diarrhea diseases in swine. the most important issues which need to be addressed are as follows. the stability of igy in the gastrointestinal tract when they are fed to swine although the beneficial effects of pathogen-specific igy in animals have been known for about years, results on the experimental application of these antibodies in swine have not always been consistent. there are several reports where igy failed to improve pig performance [ , ] . the most likely explanation for the failure of the treatment is that the antibody failed to survive passage through the gastrointestinal tract as a result of its susceptibility to proteolysis [ ] . igy, being a glycoprotein, is sensitive to the same denaturing conditions as most proteins. it has been reported that igy is fairly resistant to digestion by intestinal proteases, but the activity of igy was decreased at ph . or lower and almost completely lost with irreversible change at ph [ ] . in addition, the inactivation at low ph is further enhanced by the presence of pepsin. in contrast to very young pigs, the stomach of older pigs has a low ph and pepsin, and therefore igy may not be effective in controlling post-weaning e. coli diarrhea in older animals. yokoyama et al. [ ] , in an excellent study, detected the rate of igy passage through the gastrointestinal tract of pigs and its ability to retain its activity in the different sections of the tract. their results demonstrated that igy is readily absorbed within h by the newly born pig. igy has a serum half-life of . days in newborn pigs, which is shorter than the reported serum half-life of to days for homologous igg (colostral antibodies). the amount of igy absorbed into the circulation when administered in pigs decreased with increasing age of pigs. since the primary target site of igy is the small intestine, in order for it to function, it must be able to survive passage through the harsh environmental conditions found in the stomach. microencapsulation techniques have been developed to protect igy from gastric inactivation [ , [ ] [ ] [ ] [ ] . table shows the results of an experiment where chitosan-alginate microcapsules were used for oral delivery of egg yolk immunoglobulin in weaned pigs challenged with enterotoxigenic e. coli k [ ] . the percentage of pigs with diarrhea h after treatment and the diarrhea score were improved in pigs receiving encapsulated igy compared with non- all pigs except negative control were orally challenged with ml of viable e. coli k organisms ( cfu/ml per pig) at time h. challenged pigs were left untreated (positive control) or treated three times (− , and h after bacterial challenge) on the first day and twice a day for two consecutive days each time with . g non-encapsulated igy, g of microencapsulated igy (equivalent to . g of igy) or . g of aureomycin. diarrhea and weight gain were assessed for days after challenge fc score is the mean fecal consistency score: , normal; , soft feces; , mild diarrhea; , severe diarrhea. pigs with a fecal score of < were considered not to have diarrhea means in a column followed by same or no letter do not differ (p > . ) encapsulated igy. in addition, weight gain over the three day period was significantly higher in pigs receiving encapsulated igy compared with non-encapsulated igy. both encapsulated and non-encapsulated igy treatments were numerically superior to an aureomycin treated group. however, this process will undoubtedly add additional costs. additional study will be needed before microencapsulation can be applied under commercial conditions. the stability of igy when subjected to feed processing egg-yolk antibodies can be administrated to young pigs either as a preventive or prophylactic treatment or as a therapeutic treatment after infection occurs. it appears that adding antibodies to swine feeds in the form of whole egg powder or whole yolk powder may be the most practical method of inclusion of igy for preventive or prophylactic treatment. it is not known if igy antibodies can tolerate heat-based feed processing techniques such as pelleting. presumably the stability of igy antibodies would be similar to that for enzymes and that stability would be greatly influenced by temperature, duration of heat treatment and cooling period as well as the moisture content of the diet. it is well known that a change in moisture content from to or % dry matter can dramatically decrease the stability of proteins when heated to high temperatures. preliminary studies by marquardt et al. [ ] have demonstrated that low temperature steam pelleting ( °c) did not reduce antibody titer. further studies need to be carried out on this problem igy is an attractive and effective alternative approach to antibiotics due to its high specificity. it should be noted that swine are exposed to many infectious agents in commercial operations and therefore the swine industry will benefit more from igy antibodies if they are produced against a mixture of common disease causing organisms rather than one specific disease. if this approach works well, it may help to justify, to some extent, commercial application of these antibodies. the efficacy of igy in the gastrointestinal tract is related to two factors namely the dose of antibodies and the concentration of pathogen. the results of yokoyama et al. [ ] demonstrated that antibodies pass through the digestive system within a relatively short period of time (slightly more than h). in addition, they can reach all sections of the gastrointestinal tract in less than h. the supply of antibodies in the intestine must be sufficiently high to prevent binding of pathogen to the intestinal receptors. the antibody must also be continuously or nearly continuously available in the intestine. studies by marquardt et al. [ ] showed that . g per day per piglet was sufficient to prevent diarrhea induced by infection with etec and the addition of . % of egg-yolk antibody in the diet was preventive against diarrhea in commercial farms. however, a considerable amount of additional research must be carried out to identify the best antigens to use and the appropriate prevention or treatment protocols for control of intestinal pathogens such as different strains of e. coli. at present, the production cost of high quality igy antibodies is higher than the cost of routine antibiotic inclusion [ ] . therefore, the development of methods for large-scale production of igy which produce a high recovery and purity of igy are needed. the method should be simple, economical and require few chemicals. in addition, there is no consensus on the most suitable igy extraction method for commercial application [ ] and this requires further study. oral administration of specific igy appears to have considerable potential as a means of controlling diarrhea diseases and exerting growth-promoting activity in swine. igy technology will probably provide the best alternative to antibiotics. some advantages of igy in the control of swine diseases are: ) they are highly effective. ) they are highly cost-effective with only a small amount of antibody required per pig. ) collection of eggs is non-invasive. ) the treatment is safe and live organisms are not used. ) the procedure is environmentally friendly. ) no toxic residues are produced and there is no development of resistance. ) the treatment can be used to control many different types of pathogens. why and how antibiotics are used in swine production agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria the european ban on growth promoting antibiotics and emerging consequences for human and animal health food and administration. phasing out certain antibiotic use in farm animals opportunities and challenges in using exogenous enzymes to improve nonruminant animal production organic acids as potential alternatives to antibiotic growth promoters for pigs feed additives for swine: fact sheets-prebiotics and probiotics, and phytogenics probiotics as a dietary additive for pigs: aa review mannan oligosaccharides in nursery pig nutrition and their potential mode of action use of phytogenic products as feed additives for swine and poultry review: chinese herbs as alternatives to antibiotics in feed for swine and poultry production: potential and challenges in application feed additives for swine: fact sheets-high dietary levels of copper and zinc for young pigs, and phytase reduced use of antibiotic growth promoters in diets fed to weanling pigs: dietary tools, part application of chicken egg yolk immunoglobulins in the control of terrestrial and aquatic animal diseases: a review peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections effect of chicken egg yolk antibodies (igy) against diarrhea in domesticated animals: a systematic review and meta-analysis ueber natürliche immunität und ihre verwerthung für die immunisirungstherapie egg yolk antibodies, state of the art and future prospects the production of avian (egg yolk) antibodies: igy. the report and recommendations of ecvam workshop chicken egg yolk antibodies (igy-technology): a review of progress in production and use in research and human and veterinary medicine efficient production of chicken egg antibodies against a conserved mammalian protein immune response in chicken with different amounts of antigen oral administration of chicken yolk immunoglobulins to lower somatic cell count in the milk of lactating ruminants adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens igy antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding in vitro inhibition of adhesion of enterotoxigenic escherichia coli k to piglet intestinal mucus by egg yolk antibodies passive protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic escherichia coli infection in neonatal piglets escherichia coli in domestic animals and humans. wallingford: cab international escherichia coli in domestic animals and humans. wallingford: cab international significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of escherichia coli-producing k pilus antigen aspects of genetic resistance to k e. coli in pigs chicken egg-yolk antibodies against f ab fimbriae of escherichia coli inhibit shedding of f positive e. coli by experimentally infected pigs passive protective effect of egg-yolk antibodies against enterotoxigenic escherichia coli k + infection in neonatal and early-weaned piglets reduced intestinal colonisation with f -positive enterotoxigenic escherichia coli in weaned pigs fed chicken egg antibody against the fimbriae response of early-weaned pigs to spray-dried porcine or animal plasma-based diets supplemented with egg-yolk antibodies against enterotoxigenic escherichia coli response of early-weaned pigs to an enterotoxigenic esherichia coli (k ) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody, zinc oxide, fumaric acid or antibiotic response of early-weaned pigs to an enterotoxigenic esherichia coli (k ) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody ileal amino acid digestibility in egg from hyperimmunized-hens fed to weaned pigs and piglet response to diets contain egg products relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic escherichia coli that produces multiple enterotoxins importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic escherichia coli strains construction of a nontoxic fusion peptide for immunization against escherichia coli strains that produce heat-labile and heat-stable enterotoxins a recombinant escherichia coli heat-stable enterotoxin b (stb) fusion protein eliciting neutralizing antibodies protection of mice against enterotoxigenic e. coli by immunization with a polyvalent enterotoxin comprising a combination of ltb, sta, and stb chicken egg yolk immunoglobulin (igy) developed against fusion protein ltb-sta-stb neutralizes the toxicity of escherichia coli heat-stable enterotoxins food-related illness and death in the united states multiresistant salmonella typhimurium dt infections of humans and domestic animals in the pacific northwest of the united states outbreaks of multidrug-resistant salmonella typhimurium associated with veterinary facilities-idaho, minnesota, and washington salmonella enterica infections in market swine with and without transport and holding epidemiological studies and proposed preventive measures in the fight against human salmonellosis oral passive immunization against experimental salmonellosis in mice using chicken egg-yolk antibodies specific for salmonella enteritidis and s. typhimurium effects of in-feed egg yolk antibodies on salmonella shedding, bacterial antibiotic resistance, and health of pigs assessment of different treatments to reduce salmonella in swine distribution of persistent salmonella typhimurium infection in internal organs of swine alternate routes of invasion may affect pathogenesis of salmonella typhimurium in swine interactions of the invasive pathogens salmonella typhimurium, listeria monocytogenes, and shigella flexneri with m cells and murine peyer's patches transmissible gastroenteritis and porcine respiratory coronavirus isolation of sc- strain of transmissible gastroenteritis virus of swine and characteristic analysis of gene comparsion of antigenicity between expressed proteins of the fragment including s gene whole antigenic sites and the deleted fragment in porcine respiratory coronavirus of transmissible gastroeritis virus prophylactic and therapeutic effects of egg yolk immunoglobulin against porcine transmissible gastroenteritis virus in piglets study and application of the hyperimmunized yolk antibodies against tgev and pedv in piglets an apparently new syndrome of porcine epidemic diarrhea experimental infection of pigs with a new porcine enteric coronavirus cv porcine epidemic diarrhea in a herd of breeding and finishing pigs immunoprophylactic effect of chicken egg yolk immunoglobulin (ig y) against porcine epidemic diarrhea virus (pedv) in piglets effect of igy treatment on porcine epidemic diarrhea virus (pedv) in piglets progress and barriers for the control of diarrhoeal disease rotavirus infection in calves, piglets, lambs and goat kids in trinidad passive protection against bovine rotavirus in calves by specific immunoglobulins from chicken egg yolk antibodies to rotavirus in chickens' eggs: a potential source of antiviral immunoglobulins suitable for human consumption prophylaxis of rotavirus gastroenteritis using immunoglobulin dose-dependent effects of specific egg-yolk antibodies on diarrhea of newborn calves igy antibodies protect against human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model evaluation effects of spray-dried egg protein containing specific egg yolk antibodies as a substitute for spray-dried plasma protein or antibiotics in weaned pigs the effect of dietary chicken egg-yolk antibodies on the clinical response in weaned pigs challenged with a k + esherichia coli isolate microencapsulation for the gastric passage and controlled intestinal release of immunoglobulin y anti-e. coli immunoglobulin y isolated from egg yolk of immunized chickens as a potential food ingredient detection of passage and absorption of chicken egg yolk immunoglobulins in the gastrointestinal tract of pigs by use of enzyme-linked immunosorbent assay and fluorescent antibody testing microencapsulation protects immunoglobulin in yolk (igy) specific against helicobacter pylori urease protective effect of microencapsulation consisting of multiple emulsification and heat gelation processes on immunoglobulin in yolk chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (igy) chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (igy): in vivo evaluation in a pig model of enteric colibacillosis control of diarrhea in young pigs using therapeutic antibodies bioactive components in egg yolk application of chicken egg yolk immunoglobulins against enterotoxigenic escherichia coli in piglets effect of oral egg antibody in experimental f + escherichia coli infection in weaned pigs research of hyperimmunized yolk antibodies for prevention and treatment of piglet dysentery preparation and application of chicken egg yolk immunoglobulins against enterotoxigenic escherichia coli in piglets the work from our laboratory that is reviewed in this manuscript was supported by funds from the national natural science foundation of china ( ; ; ). the authors declare that they have no competing interests.authors' contributions xl and yx carried out the literature review and drafted the manuscript. lw, yz, sl participated in literature review. all authors read and approved the final manuscript. key: cord- - qx tolv authors: lanski, steven l.; naga, osama title: emergency care date: - - journal: pediatric board study guide doi: . / - - - - _ sha: doc_id: cord_uid: qx tolv children less than years have the greatest risk for accidental ingestion and poisoning. • children less than years have the greatest risk. • jimson weed and deadly night shade produce anticholinergic toxins, e.g., atropine, scopolamine, and hyoscyamine • common garden vegetables in the solanum genus, including tomatoes, potatoes, and eggplants. • amygdalin is contained in seeds and produces hydrogen cyanide which is a potent toxin • inhibition of cellular respiration and can be lethal • ingestion of mushrooms also may have fatal consequences in species that harbor amatoxins (amanita) and related compounds products contain an aspirin • baby aspirin • regular aspirin at home includes: anti-diarrheal medications, topical agents, e.g., keratolytics and sport creams • refer to emergency departments for ingestions > mg/kg • ingestion of > mg/kg is generally considered toxic, > mg/kg is more significant toxicity, > mg/kg is potentially fatal • acute salicylism; nausea, vomiting, diaphoresis, and tinnitus • tachypnea, hyperpnea, tachycardia, and altered mental status can be seen in moderate toxicity • hyperthermia and coma are seen in severe acetylsalicylic acid toxicity • classic blood gas of salicylic acid toxicity is respiratory alkalosis, metabolic acidosis, and high anion gap • check serum level every h until it is consistently down trending • initial treatment is gastric decontamination with activated charcoal, volume resuscitation, and prompt initiation of sodium bicarbonate therapy in the symptomatic patients • goal of therapy includes a urine ph of . - . , a serum ph of . - . , and decreasing salicylate levels • an r wave in lead avr of > mm is independent predictor of toxicity • electrocardiography (ecg) parameter is superior to measured serum of tcas • stabilization of patient is the most important initial step specially protecting the airway, and ventilation support as needed, activated charcoal in appropriate patients • obtain ecg as soon as possible • ecg indication for sodium bicarbonate therapy include: qrs duration > ms, ventricular dysrhythmias and hypotension • strong acid and alkalis < or > ph can produce severe injury even in small-volume ingestion • patient can have significant esophageal injury without visible oral burns. • wash all exposed skin with soap and water and immediately remove all exposed clothing • laceration is a traumatic disruption to the dermis layer of the skin • the most common anatomic locations for lacerations are the face (~ %) and upper extremities (~ %) • an evaluation for life-threatening injuries is the first priority • ongoing bleeding that may cause hypovolemic shock • applying direct pressure usually is successful • sphygmomanometer may be used for up to h on an extremity • ring tourniquet on a digit for up to min to help control ongoing blood loss • lacerations of the neck should be evaluated for deeper structural injuries • if developmentally appropriate, two-point discrimination at the finger pads provides the best assessment of digital nerve function • it is critical to identify foreign material within the laceration anesthetics and anxiolysis • the use of the topical anesthetic let ( % lidocaine, : epinephrine, and . %tetracaine) has been shown to be effective and to reduce length of stay • let usually is effective - min after application to a laceration site on the face but often needs twice that amount of time to be effective elsewhere • blanching of the site after application most often indicates achievement of effective anesthesia • a local anesthetic also may be used to prepare for placement of sutures • dermabond: it is critical that the laceration be dry and well approximated to avoid application below the epidermal surface, which may cause the wound to gape open or lead to a "dermabond oma" • evenly spaced suture placement: the general rule is sutures should be spaced the same distance as they are placed from the wound edge. for irregular wound shapes, approximate the midpoint of the wound first and then work laterally • lip laceration require special care if the injury crosses the vermilion border • it is essential to approximate the vermilion border with a suture. failure to do so may result in a poor cosmetic outcome • an infraorbital or mental nerve block along the lower gum line may be considered to reduce tissue distortion for lip lacerations, including those through the vermilion border • it may be painful and produce anxiety for the child and parent • a digital nerve block should be applied to provide adequate analgesia for this injury • if the nail has been removed during the injury, the nail bed should be repaired with absorbable sutures by using a reverse cutting needle which they designed to protect • there is no benefit to administer antivenom to unrelated species due to risk of anaphylaxis and expenses as well • surgical assessment focuses on the injury site and concern for the development of compartment syndrome • fasciotomy is indicated only for those patients with objective evidence of elevated compartment pressure • bitten extremities should be marked proximal and distal to the bite and the circumference at this location should be monitored every min to monitor for progressive edema and compartment syndrome • black spider with bright-red or orange abdomen • neurotoxin acts at the presynaptic membrane of the neuromuscular junction, and decreased reuptake of acetylcholine and severe muscle cramping • pricking sensation that fades almost immediately • uncomfortable sensation in the bitten extremity and regional lymph node tenderness • a "target" or" halo" lesion may appear at the bite site • proximal muscle cramping, including pain in the back, chest, or abdomen, depending on the site of the bite • almost painless bite, and only rarely is a spider recovered • erythema, itching, and swelling begin to several hours after the bite • central ischemic pallor to a blue/gray irregular macule to the development of a vesicle • the central area may necrose, forming an eschar • induration of the surrounding tissue peaks at - h • lymphadenopathy may be present • the entire lesion resolves slowly, often over weeks to months • tetanus status should be assessed and updated • signs of cellulitis treated with an antibiotic that is active against skin flora • treatment is directed at the symptoms background • the only scorpion species of medical importance in the usa is the arizona bark scorpion ( centruroides sculpturatus). • toxins in its venom interfere with activation of sodium channels and enhance firing of axons. • local pain is the most frequent symptom • usually no local reaction • in small children -uncontrolled jerking movements of the extremities -peripheral muscle fasciculation, tongue fasciculation, facial twitching, and rapid disconjugate eye movements -may misdiagnosed as experiencing seizures • severe reaction -agitation -extreme tachycardia -salivation -respiratory distress • maintenance of a patent airway and mechanical ventilation in severe cases • victims may be managed solely with supportive care: -analgesia and sedation -airway support and ventilation -supplemental oxygen administration • antivenin therapy also may obviate or reduce the need for airway and ventilatory support • status epilepticus (se) is defined as a seizure that lasts more than min • treatment of se should be based on an institutional protocol, such as the following: • initial management -attend to the abcs before starting any pharmacologic intervention -place patients in the lateral decubitus position to avoid aspiration of emesis and to prevent epiglottis closure over the glottis -make further adjustments of the head and neck if necessary to improve airway patency -immobilize the cervical spine if trauma is suspected -administer % oxygen by facemask -assist ventilation and use artificial airways (e.g., endotracheal intubation) as needed -suction secretions and decompress the stomach with a nasogastric tube -carefully monitor vital signs, including blood pressure -carefully monitor the patient's temperature, as hyperthermia may worsen brain damage -in the first min of seizure activity, before starting any medications, try to establish iv access and to obtain samples for laboratory tests and for seizure medications -infuse isotonic iv fluids plus glucose at a rate of ml/ kg/h (e.g., ml d ns over h for a -kg child) -in children younger than fig. , p. s ) • bradycardia-most common pre-arrest rhythm in children with hypotension, hypoxemia and acidosis (fig. ) -sinus bradycardia • maybe non-pathologic in case of well conditioned individuals like athletes • causes include: hypothermia, hypoglycemia, hypoxia, hypothyroidism, electrolyte imbalance, toxic ingestion, head injury with raised icp • treatment-identify cause and treating that condition • hr < bpm in a child who is a well-ventilated patient, but showing poor perfusion, chest compression should be initiated • if hr remains below despite adequate ventilation and oxygenation, then epinephrine or atropine ( . mg/kg- . mg min and . mg max) should be given • symptomatic bradycardia unchanged by above may require pacing • av mode blocks -first degree-prolonged pr interval • generally asymptomatic -second degree- types • type -wenckebach ▪ progressive pr prolongation until no qrs propagated • type -regular inhibition of impulse ▪ usually every other p results in qrs -third degree-complete dissociation between p and qrs -reversible causes of cardiac arrest (fig. ) • fig. pediatric advance life support bradycardia algorithm. rosc return of spontaneous circulation, iv intravenous, io intraosseous, cpr cardiopulmonary resuscitation. (kleinman me et al. american heart association guideline for cardiopulmonary resuscitation and emergency cardiovascular care, part . circulation , , suppl , pp. s -s , fig. , p. s ) toxic plant ingestions. wilderness medicine nelson text book of pediatrics rattlesnake bites in southern california and rationale for recommended treatment clinical presentation and treatment of black widow spider envenomation: a review of cases clinical presentation and outcome of brown recluse spiderbite envenomation by the scorpion centruroides sculpturatus epilepsy foundation of america's working group on status epilepticus. treatment of convulsive status epilepticus. recommendations of the epilepsy foundation of america's working group on status epilepticus total burn care golden hour: handbook of pediatric advanced life support pediatric advanced life support key: cord- - sx tso authors: perez, a; ball, g d c title: are we overlooking the qualitative ‘look' of obesity? date: - - journal: nutr diabetes doi: . /nutd. . sha: doc_id: cord_uid: sx tso nan during the opening ceremonies of the th canadian obesity summit held recently in toronto, along with the traditional speeches and awards, a woman who formerly had obesity shared her personal story. emotional, heart-felt, and humanizing, her experience of living with obesity as a child, a professional, a wife, and an artist provided a detailed and personal view of her ongoing personal struggles with her weight, which set the tone for the meeting over the next few days. her story and others like it can provide rich insight into individuals' perspectives of obesity and weight management. in our view, these perspectives have been under-represented in the field of obesity research where numbers from quantitative research often take precedence over meanings derived from qualitative inquiry. qualitative research has proved important in many areas of clinical and health research, including understanding patients' and clinicians' decision making and enhancing quality of health services delivery related to utilization, feasibility and appropriateness of care. , despite being on the rise, the publication of qualitative studies in medical journals is still low, especially in high-impact journals. this pattern is of concern given the role that high-impact journals have in disseminating new evidence to academic and clinical audiences as well as to the public through knowledge translation activities that follow publication, including both traditional (newspaper, television and radio) and social (twitter, blogs) media outlets. obesity research is not immune to this tendency. recently, we completed an online search of original manuscripts published from january to december in five obesity journals (childhood obesity, clinical obesity, international journal of obesity, obesity, and pediatric obesity). of the total number of papers published (n = ), qualitative reports comprised . % (n = ). we also reviewed the authorship guidelines for all five journals and found no explicit statements regarding the exclusion of qualitative research or specific preferences for quantitative research, although some details (for example, testing hypotheses; including controls) were applicable to quantitative study designs only. a search of bibliographic databases including pubmed and scopus with obesity and qualitative research as key words yields hundreds of publications per year over the past several years, so a low total volume of qualitative studies related to obesity may not be a primary factor for the under representation of qualitative reports in obesity journals. that said, the poor quality and novelty of qualitative manuscripts submitted to obesity journals may be an issue; however, the extent to which this factor has a role is difficult to determine given that details regarding editors' and reviewers' familiarity and expertise in qualitative research are required along with the criteria used to gauge manuscript quality and appropriateness. as health research has been predominantly quantitative, the low proportion of qualitative studies published in obesity journals may not relate to poor quality, but to a lack of understanding, making it difficult for editors and reviewers to judge the value and quality of qualitative reports. the tension between qualitative and quantitative research approaches and different underlying epistemologies have been documented. in our experience leading qualitative, obesity-related research with clinical and health services foci, we have gained some experience in addressing potential challenges with publication. for instance, the submission and resubmission processes provide opportunities for authors to include additional rationale for key methodological decisions, especially in relation to issues including sample size, hypothesis testing, reliability of coding, data saturation and generalizability of findings. reviewers of our manuscripts have, more often than not, appreciated our explanations. this experience highlights the value of describing differences and addressing potential misperceptions between quantitative and qualitative research during the peer-review process. in addition, the inclusion of quality assessment and reporting checklists (for example, , ) that accompany our manuscript submissions has allowed us to provide a better understanding of the design, implementation, analysis and implications of our research. using checklists to explain methodological and reporting details of qualitative studies may also benefit from a halo effect as it is consistent with many journal requirements for quantitative research. we have also been flexible in accommodating editors' and reviewers' recommendations to edit our manuscripts in circumstances when changes have not compromised the rigor of our qualitative research, but may not necessarily be consistent with the original intent of the research method (for example, including frequency data in thematic analyses). we also believe that several steps can be taken to enhance the presence of qualitative research in obesity research. first, consistent with the approaches taken by other organizations (for example canadian obesity network; obesity action coalition), the inclusion and participation of individuals living with obesity at academic meetings can enable conversations about obesity in a manner that is less stigmatizing and biased, encouraging research (qualitative and quantitative) that is relevant to people living with obesity and providing a forum for their perspectives to inform research. second, obesity journals can establish dedicated sections to highlight excellence in qualitative research, an approach that has been used to organize other research areas (for example, clinical trials and investigations; epidemiology/genetics). finally, the inclusion of explicit instructions within authorship guidelines for obesity journals can highlight the range of research considered for publication, which can include requiring applicable reporting checklists and be accompanied by the inclusion of scientists, clinicians, and administrators at all stages of the peer-review process who possess methodological expertise in both quantitative and qualitative research. the publication of qualitative research has been positively linked with journals' policies, authorship guidelines, and editorial content, so a proactive approach can be beneficial. collectively, these steps can encourage more inclusive discussions about obesity as well as provide academic venues for publishing and disseminating research of greater epistemological breadth and relevance. qualitative research: adding drive and dimension to clinical research how do you modernize a health service? a realist evaluation of wholescale transformation in london is qualitative research second class science? a quantitative longitudinal examination of qualitative research in medical journals paucity of qualitative research in general medical and health services and policy research journals: analysis of publication rates journal prestige, publication bias, and other characteristics associated with citation of published studies in peer-reviewed journals using quantitative and qualitative data in health services research -what happens when mixed method findings conflict? bmc the elephant in the living room: or extending the conversation about the politics of evidence critical appraisal skills programme: making sense of evidence consolidated criteria for reporting qualitative research (coreq): a -item checklist for interviews and focus groups this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material key: cord- - x deimg authors: xu, jing-xiu; zhang, cong; cao, chang-yu; zhu, shi-yong; li, hui; sun, yan-chun; li, jin-long title: dietary selenium status regulates the transcriptions of selenoproteome and activities of selenoenzymes in chicken kidney at low or super-nutritional levels date: - - journal: biol trace elem res doi: . /s - - - sha: doc_id: cord_uid: x deimg to determine dietary selenium (se) status regulates the transcriptions of selenoproteome and activities of selenoenzymes in chicken kidney, -day-old chickens received low se ( . mg se per kg of diet) or super-nutritional se ( . or . mg se per kg of diet) in their diets for weeks. it was observed that dietary low or super-nutritional se did not make renal appearance pathological changes in chicken. low se significantly reduced total antioxidant capability (t-aoc), glutathione (gsh) content, but malondialdehyde (mda) content in the kidney increased and decreased glutathione peroxidase (gpx) and thioredoxin reductase (trxr) activity with changes in their mrna levels. super-nutritional se ( . mg/kg) increased t-aoc and gsh contents then made them reduce, but it reduced mda content significantly, elevated then reduced gpx activity, and decreased trxr activity with changes in their mrna levels. dietary low se downregulated the mrna expressions of gpx - , txnrd , sepn , selw, sepx , selh, and sepsecs. at super-nutritional se, most selenoproteins were upregulated in chicken kidney, but sepp and sep was only upregulated in se excess ( . mg/kg) bird. these results indicated that dietary se status stabilizes normal renal physiology function via regulation of the selenoprotemic transcriptions and selenoenzyme activities in avian. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. selenium (se) is an obligatory nutritional trace element and participates in many aspects of health such as chemoprevention, neurobiology [ , ] , immune function [ , ] , reproduction [ ] , aging [ ] , carcinogenesis [ ] , and muscle metabolism [ , ] . poultry is sensitive to se content of diet [ ] . se deficiency manifested as deformation and necrosis [ ] in sheep, cattle, horses, and chickens [ ] . the kidney can be ruled out as a product of metabolism in the organism and maintain electrolyte balance. it could also be influenced by se deficiency or excess, such as predominant pathological changes were convoluted tubule necroses. concentration of free se is the greatest in the renal cortex [ ] . se could protect the renal antioxidant function from oxidative damage [ ] , and se deficiency or excess causes a lot of selenoprotein resultant metabolic disorders in pig kidney [ ] . evidence of cellular damage (i.e., histopathologic and physiologic, including oxidative stress) was found in kidney systems of young snowy egrets [ ] . se can protect against oxidative damage which leads to the overproduction of free radicals that exert deleterious effects on the kidney [ ] . se is regulated in the body to maintain vital selenoproteins and to avoid toxicity, and it exerts its biological effects mainly through its direct incorporation into selenoproteins as the amino acid, selenocysteine (sec) [ ] ( fig. s a) . the mrna for a selenoprotein has two distinctive properties: uga in the open reading frame (orf) and a specialized stem-loop structure in the ′ untranslated region ( 'utr) . the stem-loop structure is known as a selenocysteine insertion sequence (secis) element [ ] .three unique trans-acting factors are vital for selenoprotein synthesis. trna[ser]sec has the anticodon for uga. it is initially charged with serine, which is then phosphorylated by o-phosphoseryl-trna[ser]sec kinase [ ] . the phosphoserine attached to trna[ser]sec is converted to selenocysteine by selenocysteine synthase, using monoselenophosphate as the se donor [ ] . selenocysteyl-trna[ser]sec binds to another vital trans-acting factor, the specific elongation factor efsec [ ] . efsec binds to the third vital trans-acting factor, secis-binding protein (sbp ). in addition to binding efsec, sbp has binding si tes for the secis element and t he ribosome. selenoproteins play an important role in the kidney function. there are intriguing relationships between se physiology and several derangements associated with acute and chronic kidney disease. selenoproteins and selenoenzyme can protect the normal function of the kidney [ ] . however, effects of dietary se on selenoproteome in chicken kidney are yet unclear. selenoproteome is a set of selenoproteins in eukaryotes and prokaryotes. avian selenoproteome consists of selenoproteins. several selenoproteins have been characterized as antioxidant enzymes, serving to alleviate damage caused by reactive oxygen species (ros) [ ] . some antioxidant enzymes contain se called selenoenzymes; the most important metabolic roles of se in cells are due to its function in the active site of these enzymes, for example glutathione peroxidase (gpx) and thioredoxin reductase (trxr) [ ] . gpx activity is essential for avoiding peroxide-generated oxidative damage and is provided by different families of enzymes, namely selenoglutathione peroxidases (gpx; ec . . . ; glutathione: hydrogen-peroxide oxidoreductase) [ ] . the thioredoxin (trx) system catalyzes the reduction of protein disulfides such as in ribonucleotide reductase, an enzyme essential for dna synthesis [ ] ; thioredoxin peroxidases (peroxiredoxins), critical enzymes in the defense against oxidative stress [ ] ; and protein disulfide isomerase (pdi). most selenoproteins in the kidney are intracellular enzymes with their activities relying upon a single selenocysteine residue in their main structures [ ] . although - selenoprotein genes were identified in mammals [ ] , only a few studies have determined collective responses of these genes to dietary se concentrations ranging from deficiency to moderately high levels in chicken kidney [ , ] . the transcriptional response of se is dependent selenoprotein genes. thus, systematic data are needed to link selenoprotein and relationship between se, selenoproteins, selenoenzymatic, and antioxidant function in the kidney, and associated study on poultry is still a vacancy. here, we tested gpx, total antioxidant capability (t-aoc), malondialdehyde (mda) content, selenoproteins, and related genes (table s ) . our results will describe changes for selenoproteome related to dietary se at low or supernutritional levels and the resultant metabolic disorders in chicken kidney which have yet to be determined before. expt. chickens (hy-line variety white, n = , day old) were fed four different diets, each holding chickens (cocks and hens were randomly distributed). all the groups were feed basal diet (table s ) except se. four experimental diets were prepared as . mg/kg se (low), . mg/kg se (standard), . mg/kg se (excess i), and . mg/kg se (excess ii). feed (corn and soybean meal) was from longjiang county, heilongjiang province, which is a typically se-deficient area in china. all other environmental conditions were maintained in accordance with existing norms for the facility. two identical feed troughs were attached side by side to each cage front. on the seventh day, inoculate newcastle disease virus and infectious bronchitis vaccines were given; on the th day, ibd vaccine; and on the st day, newcastle disease vaccine. mental state, diet condition, feathers, feces, and other ten indicators were observed with eye viewing and recorded. at the end of the second, fourth, sixth, and eighth week, experimental chickens were fasted for h before weighting and sacrificing. kidney samples were collected for analysis. frozen chicken kidney samples were homogenized, the homogenized samples were centrifuged, and the supernatant was transferred to fresh tubes. expt. chickens (n = , day old) were treated as expt. . at the end of the eighth week, chickens were sacrificed. the removed kidney tissue is stored in the locker as an extracted rna sample (cat , beijing tiandz, inc., p.r. china). chicks were fasted for h before weighting and sacrificing. every weeks, we randomly selected ten chickens in order to record their kidneys observing the changes with eye viewing and pathological tissue slice. kidney will be cut into small pieces on the ice packs and were fixed in % formaldehyde and embedded in paraffin for microscopic examination. sections ( μm thick) were cut and stained with hematoxylin and eosin (h&e). the pathological changes were observed under a microscope. se was determined in feed using inductively coupled plasma mass spectrometry (icp-ms, agilent cs-octopole reaction cell, agilent technologies, usa). the concentrations of se in the kidneys were determined with the calibration curve method and compared among the non-, h , and d reaction modes. the protein content was measured with a protein quantitative detection kit (a - , nanjing jiancheng bioengineering institute, p.r. china). bovine serum albumin (bsa) was used to construct the standard curve. the glutathione (gsh) content was measured with a gsh detection kit (a - , nanjing jiancheng bioengineering institute, p.r. china) according to the manufacturer's protocol. by measuring dihydronicotinamide adenine dinucleotide phosphate (nadph) oxidation in the presence of oxidized glutathione, and we used tert-butyl hydroperoxide as the peroxide substrate. the t-aoc was measured with a t-aoc detection kit (a , nanjing jiancheng bioengineering institute, p.r. china) according to the manufacturer's protocol by converting fe + into fe + , whereas the latter forms complexes with phenanthroline substances, which can be measured by a spectrophotometer. the content of mda in kidney was carried out with a mda detection kit (a - , nanjing jiancheng bioengineering institute, p.r. china) according to the manufacturer's protocol. by using the thiobarbituric acid method in which mda, the degradation product of lipid peroxidation, condenses with penthiobarbital and generates a red product with measurable absorbance by a spectrophotometer. the gpx activity was measured with a gpx detection kit (a , nanjing jiancheng bioengineering institute, p.r. china) according to the manufacturer's protocol. gpx catalyzed gsh into gssg, and glutathione reductase can use nadph to catalyze gssg into gsh; reduction of nadph can be calculated through testing the vitality of glutathione peroxidase level. trxr catalyzed nadph and made , ′-dithiobis ( nitrobenzoic acid) (dtnb) into trinitrobenzene (tnb). by measuring the increase of tnb at nm, activity of trxr can be calculated [ ] . total rna was isolated from the kidneys using the rnaout reagent (cat: , beijing tiandz inc., p.r. china) according to the manufacturer's protocol. the dried rna pellets were resuspended in μl water that was diethylpyrocarbonate treated. the content and purity of the total rna were determined spectrophotometrically at / nm. the total rna was immediately used to synthesize cdna. first-strand cdna was synthesized from μg of total rna using oligo(dt) primers and transcript reverse transcriptase (beijing transgen biotech co. ltd., p.r. china) according to the manufacturer's instructions. synthesized cdna was diluted ten times with sterile water and stored at − °c before use. primer analysis software (oligo . , molecular biology insights, inc. usa) designs specific primers (table s ). the relative mrna expressions of selenoproteins and factors associated with the regulation of the selenoprotein synthesis genes were assayed by qpcr array. to perform the qpcr array, gotaq® qpcr master mix (a , promega, usa), primers, template, and nuclease-free water (promega, usa) were added to form the reaction mixture (final volumes μl) in one plate according to the manufacturer's instructions. the qpcr array was performed on a real-time pcr system (applied biosystems, usa). four internal reference comparisons (β-actin (actb), glyceraldehyde -phosphate dehydrogenase (gadph), s ribosomal rna ( srrna), ribosomal protein l (rpl )) were designed (table s ). all data were normalized to the four internal control genes, and the relative expression levels were calculated using the −ΔΔct method [ ] . when the software analyzes the data, it uses the mean value of ct value of the selected four internal control genes to make the baseline correction. data analysis of mrna expression was analyzed using the genecopoeia-fulengen qpcr array data analysis system online (http://www.genecopoeia.com/product/qpcr/ analyse /index.php). the data were analyzed statistically using graphpad prism . (graphpad software inc., usa). one-way analysis of variance (anova) and the least significant difference (lsd) post hoc test were used to analyze the data. differences between the means of data were analyzed by the paired t test which was utilized to determine the effects of se supplementation. the results were expressed as mean ± s.d. of different groups. the significant differences of all data were showed by anova of each experiment. p < . is considered significant unless otherwise stated. as can be seen in fig. , the weight of chicken in the low se group was smaller than that in the standard group which was reported in previous paper [ ] and its feather was fluffy and messy. in se excess groups ( . and . se mg/kg), the size of chickens can be seen a little bigger than those in the standard group. dietary se content did not affect kidney performance, no significant differences observed in appearance, symptoms and pathological changes compared with the standard group with the other three groups. from the observation of pathological tissue section, kidney glomerulus, tubule, and interstitial showed no lesions (fig. ) . the levels of se greatly influence antioxidant defense. renal gsh contents in the low se, excess i, and excess ii groups were all significantly reduced compared with that in the standard group at the eighth week (p < . ) ( table ). however, lowered t-aoc activity can be seen significantly in the low se group compared with that in the standard group (p < . ), and it increased then decreased significantly in the excess i and the excess ii groups compared with that in the standard group (p < . ) ( table ) . meanwhile, the mda content showed the rising trend in the low se, the excess i, and the excess ii groups at the eighth week compared with that in the standard group (p < . ) ( table ) . the activity of gpx in the low-se group decreased significantly compared with that in the standard group all the weeks (p < . ). in the excess i group, it decreased significantly at the eighth week (p < . ). in the excess ii group, it decreased significantly at the eighth week (p < . ) ( table ) . gpx - mrna levels significantly increased in the excess i group (p < . ) and decreased significantly in the low-se and excess ii groups (p < . ) (fig. ) . the trxr activity decreased significantly in response to low se at the eighth week compared with that in the standard group (p < . ). in the excess i group, it went up significantly in the second week and eighth week compared with that in the standard group (p < . ), yet from the fourth week to sixth week it decreased significantly compared with that in the standard group (p < . ) ( table ) . txn mrna level increased significantly in all the groups compared with that in the standard group (p < . ) (fig. ) . meanwhile, txnrd and txnrd mrna levels all increased significantly in the low-se and the excess i groups (p < . ) compared with that in the standard group, whereas decreased significantly in the excess ii group (p < . ); nonetheless, txnrd mrna level has no significant changes in the low-se and excess i groups compared with that in the standard group; in se excess ii group, it decreased significantly (p < . ) (fig. ) . the effects of low and super-nutritional se on selenoprotein mrna abundance were determined by qpcr in chicken kidney. the levels of se greatly regulate the expression of several selenoproteins. all data for mrna expression of chicken renal selenoproteome was shown in table s , and the classifications of histogram were shown in figs. , , , . two kinds of trends can be seen: one was that iodothyronine deiodinases (dio , dio , dio ) mrna levels all increased significantly in the low-se and the excess i groups (p < . ) compared with that in the standard group, but decreased significantly in the excess ii group (p < . ), whereas dio mrna levels in the low-se group changed more obviously than that of dio and dio , over ten times (fig. ) . selenoprotein k (selk), selenoprotein u (selu), and selenoprotein p (sepp ) mrna levels showed the same trend with dios, and selk and selu altered more significantly than sepp (p < . ) (fig. ) . the other trend was that selenoprotein n (sepn ) and selenoprotein w (selw) mrna levels' variation was similar with each other in chicken kidney and decreased significantly in the low-se and the excess ii groups (p < . ) but increased significantly in the excess i group (p < . ) (fig. ) . the effects of low se and super-nutritional se on selenoprotein synthesis-related transcription factors were determined by qpcr in chicken kidney. seven selenoprotein synthesis-related transcription factors were studied (fig. ) . the trna selenocysteine-associated protein (secp ), phosphoseryl-trna kinase (pstk), and seryl-trna synthetase (sars) mrna levels were upregulated significantly under low-se condition (p < . ), whereas sep (ophosphoserine) trna:sec (selenocysteine) trna synthase (sepsecs) mrna had no expression. eukaryotic elongation factor, selenocysteine-trna-specific (efsec), sepsecs, secp , pstk, selenophosphate synthetase (sephs ), secis-binding protein (secisbp ), sars mrna levels were upregulated significantly in the excess i group (p < . ) (fig. ) . pstk, sephs , and secisbp mrna levels were downregulated significantly in the excess ii group (p < . ), and sepsecs mrna had no expression (fig. ) . we saw that secp and pstk altered more obviously, then sars, and finally efsec, sephs , and secisbp . we used selenoproteomic expression spectrum to indicate all selenoproteins and selenoprotein synthesis-related transcription factors [ ] . according to different colors, changes of low and super-nutritional se regulation of the selenoproteomic mrna expressions were demonstrated (fig. ) . partial avian selenoproteins including sepx , selo, selt, sels, sep , seli, selm, and selh mrna levels changed differently. sepx mrna level was downregulated significantly in the low-se and se excess i groups (p < . ). however, seli mrna has no expression in low-se and se excess ii groups; selo was the same with seli in se excess ii group (p < . ). meanwhile, in selt and sep , mrna levels were upregulated with low-se and se excess ( . mg/kg). moreover, sels mrna level was upregulated in se excess ( . mg/kg) but downregulated in se excess ( . mg/kg) (p < . ). in addition, selh and selm mrna levels were all downregulated with se excess ( . and . mg/kg), and this can also be observed on selh in low-se group (p < . ) (fig. ). in the present study, we observed that kidneys of chickens fed with low or super-nutritional se diet evidenced no appearance changes and the renal pathological section did not show differences among the four groups. the nutritional antioxidant se ameliorated oxidative stress and loss of cellular antioxidants and suggested that se efficiently protected the kidneys [ ] . it is reported that se can protect the kidney antioxidant defense system [ ] . se influences selenoproteins' and selenoenzymes' activity which is closely related to renal function [ ] . so we tested the antioxidant function of the kidney to find how se affected the organism. gsh and associated antioxidant enzymes are major combatants of oxidative stress that influence redox status [ ] . gsh also works as a specific substrate for the enzyme gpx [ ] . se supplementation was protective of kidney by providing substantial elevations of gsh levels [ ] . supernutritional se enhanced the endogenous antioxidant capacity, increasing gsh content. however, the lack of thiol response in the kidney might indicate that the redox cycling between the se compounds and the reduced intracellular gsh in tench did not occur or other cellular defenses against oxidative stress were induced to prevent oxidative damage [ ] , and gsh act both as a scavenger of various undesired compounds and their toxic metabolites [ ] . suitable content of se greatly improved t-aoc and decreased mda concentration in the kidney [ ] . our result is consistent with previous research. at low or super-nutritional se levels, chickens increased kidney content of mda, whereas decreased t-aoc. gsh level increased at first and then decreased. our results indicated that low or super-nutritional se increased mda content, which fig. the appearance of the chicken and histopathological examination. the pathology slices of kidney tissue of chicken at the eighth week by a microscope (× , he) caused an organism's gsh to protect itself from oxidative stress and in turn prevents it from being damaged in detoxification reactions. measurement of gpx activity has been used as a marker of adequacy of intake because activity of the enzyme is proportional to dietary intake [ ] . some studies previously indicated that low se can result in inactivation of gpx and development of oxidative stress in the kidneys [ ] , and induced gpx activity in the kidney of juvenile carp fed mg se/kg diet for days was the result of the enhanced detoxification ability [ ] . in this study, compared with those fed with a normal se diet, chicken fed with a low or super-nutritional se diet display significant decreases in renal gpx activities. it is interesting that gpx - mrna levels increased in excess i group, whereas gpx activity decreased. we suppose that super-nutritional se ( . mg/kg) could enhance mrna levels, but regulation of mrna translation can be affected by many factors, such as micrornas, or the precursor protein translation requires some modification and activation, and this process requires effects of other selenoproteins. trxr is a selenocysteine-containing enzyme which catalyzes the nadph-dependent reduction of trx and therefore plays a regulatory role in its metabolic activity [ ] . our results indicated that low or super-nutritional se made chicken renal trxr activity decrease. it may be because dietary insufficient se influences trxr directly or . and . mg/kg se excess indirectly as a result of damaging er. trxr is much more vulnerable to oxidative damage than gpx [ ] ; hence, its changes were different from gpx. txnrd and txnrd had different trends maybe because txnrd and are mostly cytoplasmic and mostly mitochondrial, respectively. txnrd is involved in sperm maturation [ ] . it is intriguing that txnrd , txnrd , and txn mrnas increased, whereas trxr activity decreased. it may be caused by the same reason as those of gpx we mentioned above. up to now, selenoproteins were identified in chicken. in this study, to obtain whether low or super-nutritional se would affect the renal selenoprotein transcription, mrna expressions of all avian selenoproteins were analyzed in chicken kidney. it reported that selenoprotein mrnas in mice kidney were identified, including gpx , selh, sepw , selm, txnrd , dio , sep , and sels that were significantly downregulated in se-deficient kidney. in this study, dio , dio , dio , selk, selu, and sepp had the same trend that increased significantly in low se and . mg/kg se excess but decreased significantly in . mg/kg se excess. these results suggested that dietary se status influenced the expressions of selenoprotein mrnas in chicken kidney. in low se diet, these selenoprotein mrnas' levels increased significantly but selenoenzyme activity and antioxidant capacity decreased. one possible explanation could be the mobilization of the se from the liver to the kidney [ ] . a selenoprotein hierarchy is when se supply is insufficient to support full expressions of all l stands for low-se group whose dietary se content was . mg/kg, s stands for standard group whose dietary se content was . mg/kg, ei stands for excess i group whose dietary se content was . mg/kg, and eii stands for excess ii group whose dietary se content was . the selenoproteins, the synthesis of some of them is downregulated so that others can be expressed more fully. moreover, we suggest although se deficiency made selenoprotein mrnas' expression higher, these selenoproteins did not exert function or too many selenoproteins accumulating in the kidney competed with each other leading to selenoproteins not used rationally in the kidney. a . mg/kg se excess can increase kidney function and promoted selenoprotein mrnas' expression; however, . mg/kg se excess might become a harmful accumulation, decrease selenoprotein mrna level, and then damage antioxidative function. we also suggest that too much se could cause organism disorder and lead to cell autophagy. even more noteworthy is sepp is a biomarker of se status, the kidney is dependent on hepatically derived sepp protein as a se source, and selu could potentially be a molecular biomarker of se status too. both of them may be near the top of the abundance hierarchy, so their mrna levels increased in low se diet. however, it needs to be further validated. we found the effects of dietary se status on avian sepn and selw expression were similar in chicken kidney, decreased significantly in low se and . mg/kg se excess, and increased significantly in . mg/kg se excess. gpx and selw regulation was in agreement with a previous study in rat's kidney that these two selenoproteins were highly regulated by se [ , ] ; selw downregulated by dietary low se was also associated with our research [ , ] . insufficient se influenced organism antioxidant capacity that decreased these selenoprotein mrna levels. se status on mrna regulation occurs at post-transcription; organism can reduce some selenoprotein mrnas to save more important selenoproteins in chicken kidney because of hierarchy. we noticed that . mg/kg se excess could improve kidney function and increase enzyme activity, as selenoprotein mrna levels increased. however, . mg/kg se excess might burden the kidneys, which caused decreased levels of selenoprotein mrna in chicken. some studies found that most selenoprotein mrna expressions were not significantly regulated by se status in mice. in rats' kidney, dio , dio , dio , gpx , gpx , seli, selm, selo, sepp , sels, selt, sep , txnrd , and txnrd were expressed but not regulated by dietary se levels. however, the authors disagree with these findings. in chicken kidney, sepx , txnrd , sels, and selt mrna expressions differed from one another. it was discovered that low or . mg/kg se excess levels did not affect selo or seli. sepp mrna levels kept on increasing in low-se and . mg/kg se excess groups. selh decreased all the time in low or super-nutritional se, and selm decreased in se excess. these results may be due to different selenoproteins in different animal disaffinity. insufficient se resulted in selenoenzyme activity decrease, but mrnas were not reduced in the meantime; . mg/kg se excess causes selenoenzyme activity decrease with mrnas decreasing. se deficiency may not decrease some mrna transcription, b u t p r e v e n t m r n a s ' t r a n s l a t i o n i n t o s o m e o f selenoproteins, and se excess directly affects the expression of mrnas. ultimately, it results in the decrease of selenoenzyme activity. these discrepancies from observation let us begin to further analyze on synthesis factors related to selenoprotein. selenoprotein synthesis requires translational recoding of the uga codon from a stop signal to a secis. any of these gene mrnas' change may influence selenoprotein expression. what we can see in our research is that dietary low se made pstk, secp , sps , and sar mrna levels increase significantly. all these gene mrna levels increased significantly in . mg/kg se excess. they, rows represent the probe sets. rna gene expression is shown using the indicated pseudo color scale from green (− ) to red (+ ) relative to values. green squares represent increased significantly (p < . ), red squares represent decreased significantly (p < . ), and gray squares represent no significant difference (p > . ) however, decreased in the . mg/kg se excess samples. some selenoproteins seemed to change as the same trend of these genes. the amounts of selenoprotein expression changes were not fully consistent with these factors. some selenoproteins could be expressed in other ways and this conjecture should be further discussed. it reported that sbp is potentially reduced by the thioredoxin and glutaredoxin systems, facilitating its relocation to the ribosomes and reinitiation of selenoprotein translation [ ] . secp plays a role in the formation or stabilization of the efsec-sbp -sec trna [ser]sec complex and promotes the formation and subcellular localization of the sps /sla/secp complex. secp may also assist in the decoding of multiple uga-sec codons in selenoprotein and in preventing degradation of selenoprotein mrnas by the nonsense-mediated-decay pathway [ ] . some genes can directly affect the function of selenoenzyme, resulting in kidney function to reduce, and these genes might influence each other, all linked with one another. one of these factors damaged cause of selenoprotein synthesis interruption, other related factors accumulated. we can also suggest that in low-se diet, description of selenoprotein synthesis regulation occurs at or before the beginning of the translation for polyclonal antibody protein or uga encoding selenocysteine terminal polypeptide; description of selenoprotein synthesis regulation occurs at or before the beginning of translation. numerous researches suggested that variational se intake regulated the expression of some selenoproteins more than others. from this observation, the concept of a selenoprotein hierarchy developed. in the present study, it was observed that dietary low or super-nutritional se did not make renal appearance pathological changes in chicken. however, dietary low se downregulated the mrna expressions of gpx - , txnrd , sepn , selw, sepx , selh, and sepsecs. at super-nutritional se, most selenoproteins were upregulated in chicken kidney, but selm and selh were downregulated. these results indicated that dietary se status stabilizes normal physiology function via regulation of the selenoprotemic transcriptions. we hypothesize that hierarchy of regulation of the transcriptions of selenoproteome makes an important role for renal se metabolism and transport in avian. an analysis of cancer prevention by selenium testicular toxicity induced by dietary cadmium in cocks and ameliorative effect by selenium the importance of selenium to human health the influence of selenium on immune responses effect of selenium-induced oxidative stress on the cell kinetics in testis and reproductive ability of male mice selenium metabolism in drosophila: selenoproteins, selenoprotein mrna expression, fertility, and mortality targeting selenium metabolism and selenoproteins: novel avenues for drug discovery skeletal muscle disorders associated with selenium deficiency in humans influence of organic selenium on hsp response of heat-stressed and enteropathogenic escherichia coli-challenged broiler chickens (gallus gallus) effects on liver hydrogen peroxide metabolism 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characterization of phosphoseryl-trna[ser]sec kinase biosynthesis of selenocysteine on its trna in eukaryotes decoding apparatus for eukaryotic selenocysteine insertion sex-specific transcriptional responses of the zebrafish (danio rerio) brain selenoproteome to acute sodium selenite supplementation selenium status highly regulates selenoprotein mrna levels for only a subset of the selenoproteins in the selenoproteome detoxifying response in juvenile tench fed by selenium diet comparison of different forms of dietary selenium supplementation on growth performance, meat quality, selenium deposition, and antioxidant property in broilers selenium, selenoproteins and human health: a review selenium inhibits renal oxidation and inflammation but not acute kidney injury in an animal model of rhabdomyolysis megalin mediates selenoprotein p uptake by kidney proximal tubule epithelial cells characterization of mammalian selenoproteomes the selenium deficiency disease exudative diathesis in chicks is associated with downregulation of seven common selenoprotein genes in liver and muscle transcript analysis of the selenoproteome indicates that dietary selenium requirements of rats based on selenium-regulated selenoprotein mrna levels are uniformly less than those based on glutathione peroxidase activity selenium regulation of the selenoprotein and nonselenoprotein transcriptomes in rodents marginal selenium deficiency down-regulates inflammation-related genes in splenic leukocytes of the mouse lipid peroxidative damage on cisplatin exposure and alterations in antioxidant defense system in rat kidneys: a possible protective effect of selenium effects of acute in vivo cisplatin and selenium treatment on hematological and oxidative stress parameters in red blood cells of rats regulation of selenium metabolism and transport selenium concentrations and enzyme activities of glutathione metabolism in wild long-tailed ducks and common eiders prevention of cadmium induced lipid peroxidation, depletion of some antioxidative enzymes and glutathione by a series of novel organoselenocyanates di( -ethylhexyl)phthalateinduced renal oxidative stress in rats and protective effect of selenium combined administration of oxalic acid, succimer and its analogue for the reversal of gallium arsenide-induced oxidative stress in rats biomarkers of selenium status effects of selenium diets on growth, accumulation and antioxidant response in juvenile carp the subcellular location of selenoproteins and the impact on their function mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase selenium as a feed supplement for heat-stressed poultry: a review the thioredoxin system protects ribosomes against stress-induced aggregation supramolecular complexes mediate selenocysteine incorporation in vivo acknowledgments this study was supported by the program for new century excellent talents in university (grant no. nect- - ), the program for new century excellent talents in heilongjiang provincial university (grant no. -ncet- ), and the doctor initial funding of northeast agricultural university (grant no. rbc ). we also acknowledge the valuable help provided by prof. shi-wen xu in northeast agricultural university and all involved workers. key: cord- -ui di qa authors: jung, kwangho; lee, sabinne title: a systematic review of rfid applications and diffusion: key areas and public policy issues date: - - journal: nan doi: . /s - - -z sha: doc_id: cord_uid: ui di qa rfid applicants called as e-id, smart tag, and contactless smart card are being applied to numerous areas in our daily life, including tracking manufactured goods, currency, and patients to payments systems. to review these various applications of rfid is important to exploring not only ongoing e-governance issues such as digital identification, delivery process, and governance but also business oriented application areas like supply chain. through a systematic review methodology from previous studies about rfid technology for public sector, we found six key areas of rfid applications: defense and security, identification, environmental applications, transportation, healthcare and welfare, and agriculture-livestock. we also suggest that the diffusion and applications of rfid can involve unexpected disadvantages including technological deficiency, uncertain benefits, dubious transparency, uncomfortable privacy issue, and unequal distribution of digital power and literacy. further research on rfid impact includes not only various theoretical issues of but also legal and managerial problems. rigorous research is required to explore what factors are critical to adopt and implement new rfid applications in terms of technology governance and digital literacy. massive data driven research is also expected to identify rfid performance in government agencies and various industry sectors. background rfid technology has been widely implemented all over the world and its impact on our daily life is very diverse and massive wyld, ) . those diverse areas of rfid application include logistical tracking, monitoring and maintenance of products, product safety and information, and payment process. today many governments around the world in both developed and developing countries are trying to apply it for various areas from tracking manufactured goods, currency, and patients to securing sagety of payments systems. massive rfid applications around all the industry sectors and countries are expected to generate a huge potential benefits for sustainable efficient energy infrastructure, transportation safety, and health care. over the past years, rfid technology went through innovations and progressions to become a more efficient and effective gadget for human beings as well as effective solutions of technical and organizational problems in various industry sectors. however, key issues of appropriate ict technology, governing networks among rfid domains, standardization requirement, and privacy still remain unsolved . we review previous literature about rfid technology used in public sectors in order to identify what has been done and found to suggest policy implications and further research agenda. more specifically, we discuss four aspects regarding rfid research issues and policy implications. first, we examine various competing concepts of rfid use by governments all over the world. second, we categorize numerous applications of rfid technology through analyzing previous literature. third, we try to figure out technological issues and governance problems that rfid technology faces today. last, we draw key public issues and suggest future research agenda. a brief history of rfid technology rfid technology was emerged as frederick hertz found existence of radio frequency during his experiment in (wyld, ) and developed for the purpose of defense during the second world war . during s and s, the rfid system attracted plenty of scholars and innovators, so efforts to register patents progressed (takahashi, ) . researchers like charles walton had registered a patent to use rfid. in the s, many us and european companies recognized the importance of developing rfid technology and started to manufacture rfid tags. soon scholars at mit university opened an auto-id center to promote the use and implementation of rfid technology. but most of the scholars report that the first commercialization of rfid technology was done by wal-mart as they launched rfid based material identifying system in (shahram and manish ) . wal-mart is now tracking merchandise including food, apparels, and electronic items with rfid technology in their supply chain. . rfid technology is a brand new policy tool that can ensure high transparency, efficiency and effectiveness not only in industrial areas but also in government service delivery. table describes a brief history of how rfid technology was developed and diffused. we searched online data base and expert based information to identify rfid publications between and . we categorized rfid applications and analyzed issues and concerns that rfid faces today by systematically reviewing published literature. we have the idea of using radio frequency to reflect waves from objects was started from frederick hertz's experiment. american navy research laboratories developed a system known as iff (identify friend or foe). the first application of rfid consisted of identifying allied or enemy planes during ww through the use of iff system. charles walton, a former ibm researcher registered patent using rfid technology, a radio-operated door lock. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] many us and european companies started to manufacture rfid tags. the auto-id center for mit became epc global, an organization whose objective is to promote the use and adoption of rfid technology. wal-mart launched and rfid pilot. collected literature we use for systematic review from two different resources. first, most of the studies are found by searching the e-database. we could access electronic databases, such as google scholar, world web of science (wws), proquest central, and science direct through seoul national university's main library homepage. we had set 'rfid technology' , 'rfid government, ' 'rfid application, and 'rfid issue' as keywords for searching literature. we found most of the research through this method of searching. the second method we used for collecting data was having discussions with experts. to do this, we first made a list of experts who specialize in it technology, science technology, and public administration. five experts agreed to help us and recommended some research papers that were known for their fluent flow of logic and plentiful contents. we chose relevant research papers from among experts' recommendations. in sum we had used previous literature collected from two methods we discussed above, searching e-database and asking experts, as our resource of searching. [ figure ] shows analytical frame that we use for this study. we have determined the literature for systematic review according to three stages shown on the flow chart. first, the original total number of studies we have found from the e-database was . also research papers were found from experts' recommendations and previous public papers. a total of , studies were chosen through the first stage. second, we excluded , following general eligibility criteria by screening title and abstract. more specifically, we excluded rfid studies only with one of the following criteria: ) studies focusing on private sector; ) studies without considering how public sector implemented rfid technology; ) studies that did not discuss any social scientific implications; and ) studies that only deal with rfid technology from pure scientific and engineering points of view. in sum, we included only papers that discussed rfid issues and their implications in public sector. third, we removed further studies fig. analytical frame too much focusing on private sector or rfid technology itself, rather on its applications in our society and social scientific implications. finally, articles were chosen for our systematic review. [ figure ] below showed descriptive statistics of collected literatures by published year. it shows studies were published in among literatures. as we already described above in history of rfid section, the popularization and commercialization of rfid technology was started in with wal-mart's adoption. it seems that after wal-mart's innovative footsteps hit the world, many scholars were started to recognize the potential of new technology and tried to understand and develop rfid technology. besides some governments from all over the world implemented new way of public service delivery using rfid technology. consequently, literatures were publishedbetween and and it forms almost % of our collected studies for this study, we categorized governments' way of using rfid technology in areas; agriculture and livestock, defense and security, environmental applications, healthcare and welfare, identification, and transportation. [ figure ] shows descriptive statistics of collected literatures categorized by applications. we categorized studies that did not focus on specific sector and analyze and introduce rfid technology from the general perspectives as 'rfid general'. 'rfid general' studies usually deal with various ways of using rfid technology in diverse sectors simultaneously. as we can see from [fig. ] , rfid general area had papers. that means still lots of rfid studies could not be fully specialized and remained in status of generally introducing rfid technology. identification sector scores secondly highest number of published literatures among areas. this result seems natural because e-id card or e-passport have most powerful force that can hurt privacy, one of the most serious and notorious issues that rfid technology face today key applied areas of rfid as we show in [table ], the history of rfid technology was started from the need for ensuring national security. almost years have passed since us army developed rfid based identification system to identify allies and enemies, rfid technology is still used for protecting people. for instance, weinstein ( ) and konsysnki & smith ( ) reported how the us army and navy implement rfid technology in cargo containers to identify materials. the us army and navy implement rfid not only to identify us troops' own weapons and containers but also to identify enemies in battle (tien ) . rfid systems are also important in terms of airport and port security. after the / terror attack on the united states, president george w. bush let all the airport and port in us adopt identification systems based on rfid technology to protect its nation from additional terrorist attacks (werb and sereiko ) . in , the taiwanese government decided to implement an rfid based e-seal system to increase security and efficiency (tsai and huang ) . in addition, rfid technology can be used effectively in prison management and child protection. in some countries like japan and republic of korea, the rfid tag is implemented in child protection monitoring (table ). electronic passports like 'e-passports' were adopted electrically after the / attack. after the terrible tragedy broke heart of united states, the american government became aware of the importance of checking visas and passports correctly. the us department of state soon let people who wanted to enter us to use rfid tag embedded electronic passports instead of traditional barcode based passports . the european union also endorsed the inclusion of biological information in e-passports. the eu justice and home affairs council decided to include fingerprints as a second mandatory identifier on passports in . in addition, rfid can be used in e-id cards in various countries. for example, in the united kingdom, prime minister tony blair and his labor party convinced the nation to adopt biometrically-enhanced national identification cards (ezovski and watkins ) . tony blair's administration announced its will to implement rfid tag embedded national identification card in late . china is another case where the e-id card is used today. as a matter of fact, china is the country where e-id card is widely and largely adopted today. the beijing olympics held in lit the fuse of adoption. the largest smart card project was implemented as a part of preparing the most prominent international sports event. in (table ) . rfid technology can be widely applied in environmental applications. adopting an rfid system in waste management is the most prominent way of using rfid to ensure efficient, eco-friendly waste management among lots of countries in the world. payt (pay-as-you-throw) program done by european union (eu) is the pioneer of this field. payt is an rfid based waste pricing model that allows each individuals or each household to pay for the tag along with the total amount of waste they throw. since each household and individual has a waste box in which rfid tags are embedded, the exact volume of waste can be calculated. in europe this incentive based system has been proven to be a powerful policy tool for reducing the total amount of waste and for encouraging recycling (schindler et al. ) . similar systems are broadly implemented in us (ransford et al. ) . in south korea, the ministry of environment introduced it to industry and urged them to use an rfid based waste management system, especially in medical waste management. rfid technology is implemented in waste management in developed and developing countries, but the purpose of adoption is somewhat different from europe to the us. india, the second-most populated country in the world, has adopted rfid technology to cope with the rapid increase of volume and types of waste (infotech ) . similarly in , what china faced were the world expo and huge amounts of construction waste that comprised % to % of the total urban waste. shanghai was chosen for a pilot project using an rfid based waste management system. all the waste dumping trucks had an embed rfid tag and volume of waste they carry was checked by the local government (ruan and hu ) . another interesting case of environmental application emerges from south korea. the south korean government operates u-street trees systems through which the exact location and status of street trees can be monitored. information about location and status of street trees are collected by an rfid tag that is attached to each tree is saved in a web information system, so trees can be managed effectively. kim et al. ( ) claim that this web based information system could manage information remotely with an interactive system (table ) . public transportation is another popular sector for rfid technology applications. rfid based electronic toll collection technology is one of the oldest and widespread rfid implementation (ulatowski ) . as soon as an rfid tag embedded car arrives at a toll booth, the rfid reader scans and reads the information that the rfid tag contains. the driver will pay debits according to the price that electronic reader suggests. in the us, electronic toll collection is thought as efficient and effective method that eliminates long lines of traffic at toll booth (ulatowski ) . rfid based toll collection is also adopted in criminal cases because it enables prosecutors to identify the exact location (smith ) . in south korea, the korean government has set credit card-linked electronic toll collection system called 'hypass' especially for collecting transportation tolls on express ways. if an rfid tag is embedded on their cars, drivers can pass the tollbooth without stopping the car because rfid reader scan the data immediately and handle the whole payment process in about s (kim ) . hong kong launched similar public transportation toll collection system in and the 'octopus card' is now internationally famous for its convenience. this system is able to handle million transactions per day and includes all modes of public transport (kovavisaruch and suntharasaj ) . south korea has set credit card-linked electronic toll collection system called 'hypass' especially for collecting transportation tolls on express ways. rfid technology is also implemented in railroad toll collection in india, where railroads are the most widely used form of public transportation. if an rfid tag is embedded on their cars, drivers can pass the tollbooth without stopping the car because rfid reader scan the data immediately and handle the whole payment process in about s (kim, ) . in addition, rfid has been used as a critical technology to promote efficiency and transparency for public transportation system in developing countries. for instance, the mexican government runs "creating traffic knowledge in mexico: applying rfid to prevent vandalism" and one of the purposes of this innovative project is to develop a transportation information system to acquire more subtle data necessary for government decision making (prado et al. ). analogous to mexican case, in bangladesh where brta (bangladesh road transport authority) was started in , the technology is operated mainly for control and supervision of the road transport systems ). rfid technology is also implemented in railroad toll collection in india, where railroads are the most widely used form of public transportation (table ) . rfid enables hospitals to manage their equipment more easily and save expenses in public health areas . the us government agencies like fda have also already used rfid tag in monitoring drug industry . since american hospitals handle almost , (table ) . rfid technology can be an effective tool for securing food safety and managing agriculture and livestock. another major advantage to this system is that animal disease tracking can be realized through innovative technology like rfid (hossain and quaddus ) . with the government support, researchers have developed the navigation system for appropriate pesticide use as a basic system for risk management in agriculture (nanseki et al. ) . rfid technology in agriculture was first introduced by the european union (eu) in the late s and shortly thereafter many countries, such as australia, japan and south korea, adopted the innovation. among those countries, the australian government was the most passionate in implementing rfid . for instance, all the livestock in australia have rfid embedded tags on their bodies immediately after they are born; information that enables farmers to identify each entity and its health status is registered in national livestock identification system (nlis). rfid technology in japan has been also adopted in agriculture especially to secure food safety and agricultural risk management that can occur by abusing pesticides (nanseki et al. , sugahara ). the japanese government planned to make a food traceability system by as a part of the "e-japan" plan . the united states is another case that applies mandatory rfid based identification system in managing livestock. according to rfid gazette ( ), the usda is pushing for rfid tagging of cattle to make tracing of disease patterns easier. with the formation of national institute for animal agriculture (niaa) in , the plan for setting the national animal identification system was started. what the us taiwan government fulfilled through this program was "to be able to identify all animals and premises that have had contact with a foreign or domestic animal disease of concern within h" (wyld, ) because "the sooner animal health officials can identify infected and exposed animals and premises, the sooner they can contain the disease and stop its spread (usda-aphis )" (table ) . public policy issues from rfid diffusion rfid applications and diffusion generate complex policy and governance problems. we address public policy issues such as technological gap and uncertainty of expecting potential benefits and costs from a rapid and massive rfid diffusion. uneasy governing issues in transparency, digital identification and power distribution are arising from inappropriate rfid applications. we discuss governance issues such as corruption, privacy problem, and digital monopoly and literacy in the following. technology is not still enough to satisfy all the elements that rfid is trying to perform various operational mechanisms. rfid technology deficiencies inevitably occur with the application of technology because there is niche space still left. for instance, rfid technology does not have a unified frequency standard yet. since there are no internationally agreed upon frequencies for rfid operations, permitted scanner/reader powers also differ between countries. there are still significant differences between the frequencies from the eu and the usa ). in addition, reichenhach ( ) pointed out the lack of storage capacity. in the eu, where rfid based waste management is common, there are technological barriers like a shortage of storage capacity . ema and fujigaki ( ) draw implications from a child monitoring case done in japan that being informed of children's exact location cannot guarantee their actual safety, but rfid tags often lead to that cherished illusion. vining ( ) warned about another possibility of niche space. according to his study about port security in the us, stealing goods without damaging rfid tag is possible because at ports, the container can be drilled into and contents can be removed. the rfid tag does not have to endure any damage through this whole process. in the us, as a response to continued pressure from various stakeholders, the us government even adopted the 'faraday cage' for privacy protection (table ) . . in reality, the rfid tag is much more expensive than a barcode, which was very popular in identifying materials before the rise of rfid technology (becker ) . purchasing rfid devices, hardware, and tags is not sufficient to drive system relevantly. to guarantee a better quality of service, the rfid system needs more additional things such as "circular process mechanism, the richness of consultant, project manager, programmers and plentiful project labors" . these elements for a better rfid performance may involve considerable costs. kuo and chen ( ) reported that rfid technology consumers and government should pay the extra hidden cost in the healthcare industry (table ) . rfid technology is expected to increase transparency and monitor corruption. however, rfid technology cannot ensure a high level of transparency than expected. as a matter of fact, rfid tags can be cloned and manipulated quite easily, and this kind of tag corruption can occur at every stage of rfid implementation. there are various examples to show an inappropriate use of rfid technology. for instance, armknecht et al. ( a, b) warned the possibility of tag corruption. lee et al. ( ) pointed out reader corruption of the rfid technology. an existing security model mainly focuses on the possibility of tag corruption, but reader corruption can hurt consumers' privacy as seriously as tag corruption can. jules ( ) reported one of the tag corruption cases that observed in united states. one of the staff members who worked in a dupyu store, an unscrupulous retailer, attached a cloned tag to counterfeit drugs. avoine et al. ( ) argued that internet based databases can also be directly attacked and emphasized the possibility of reader corruption. there are also unethical behaviors to avoid rfid monitoring process. in the eu where an rfid based waste management system is aggressively implemented, some people disposed waste that came from their house at work places in order to avoid exact calculation through the rfid system. not only this, bilitewski ( ) reported that some conscienceless people are burning or transferring (table ) . one of the most serious issues that rfid technology faces today is whether rfid technology is secure enough to protect privacy. privacy is the most important concern rfid users have to deal with (perakslis and wolk ) . rfid tag embedded chips often contain important personal information and usually this kind of private information can hurt one's privacy seriously if leaked. to prevent leakage of private information, engineers developed cryptography, but there remains criticism . the reason why these sorts of privacy concerns arise is because of the lack of security protection capacity of modern rfid technology. as we discussed above in the technological issues section, rfid technology today is not developed to secure perfect privacy. the technology itself has lots of deficiencies and people are smart enough to find niche spaces that can destroy the rfid security process. rfid itself can involve not only various hidden costs but also induces a serious privacy problem . however, despite these possibilities of attacks on privacy, there are lots of stakeholders and scholars who advocate the potential benefits of rfid. they claim that tracking and profiling consumers is solely for implementing rfid chips more effectively. eaward rerisi, one of the producers of early implementation of rfid technology argued that, "an rfid reader can read the number on a tag, but without knowing what the number means, there is no way to access personal information. the idea that the tags can be read by just anybody-that's pretty impossible" (murray ) (table ) . unequal distribution of rfid technology can generate unequal distribution of various resources such as information and digital literacy. especially in developing countries, active tag, which is more safe for securing privacy than passive tag is more expensive than passive tag the combination of unbalanced power distribution between stakeholders and a low level of digital literacy can cause serious problems. ketprom et al. ( ) emphasized that in developing countries like thailand , governments should provide education and training on how to use brand new technology to poor farmers whose digital literacy remains relatively low. but poor farmers in thailand are not the only stakeholders who are suffering from a lack of digital literacy. in bangladesh, where rfid toll collection is common, traffic policies have no interest in using rfid technology for managing public transportation systems. rather, they prefer traditional ways of toll collecting to information based technology . prasanth et al. ( ) found that the lack of digital literacy among the indian people hampered an effective process of railroad toll collection in india. another problem developing countries face is an unbalanced power distribution due to lack of democratic value embedded governance. chen et al. ( ) criticized the taiwanese government because it monopolizes most of the information collected by rfid technology. as we already discussed above, when rfid tag scanned, information saved in rfid tag is scanned by reader and then transmitted to an internet based database. if that data were available to the public, individuals and industry could make more reasonable decisions by analyzing them. we find another unbalanced power distribution case in china's waste management system. according to ruan and hu ( ) , the chinese government benefits most from the rfid system (table ) . we found, relying on a systematic review from rfid studies, six key areas of rfid applications. specifically in the defense and security section, we addressed how military and airports/ports manage rfid systems to ensure security. we also found that rfid is effectively implemented in prison management and child protection programs. numerous governments have introduced rfid identification tools such as e-passport and e-id. rfid systems for waste management and street tree management are widely used from rich to poor countries. in healthcare and welfare delivery, rfid based smart cards have turned out to be very efficient. rfid is now being used to monitor counterfeit drugs. rfid has been applied to delivering service for the impaired and to trace infection. however, despite potential benefits from rfid applications, various unexpected problems arise. rfid can still involve technological deficiencies, especially in securing cryptography techniques, international standards of frequency, and storage capacity. rfid technology is not still enough to be efficient and effective in some areas (becker , jensen et al. ). tag and reader corruption can hurt transparency and security. privacy issues are still the most serious issues that rfid faces today (naumann and hogben ) . rfid itself can generate new unequal digital literacy and power distribution, especially in developing countries such as thailand and bangladesh. even the most latest innovative technologies, like rfid, do not have perfect answers to securing efficiency, effectiveness, convenience, and transparency. rather, rfid technology itself creates unexpected problems. it should be noted that democratic governance and trust is still important to technological innovation and policy issues arising from a rapid rfid diffusion. our systematic review is incomplete to discuss all of the rfid issues from technology, market and management, e-government, and legal aspects. further research on rfid diffusion and impact include not only various theoretical issues of but also legal and managerial problems. for instance, both qualitative and quantitative research is required to explore what factors are critical to adopt and implement new rfid technology in terms of governance and digital literacy. both micro and macro approaches with massive data are also required to identify how rfid improve not only organizational performance in government agencies and various industry sectors but also quality of our life. endnotes for example, after serious attack by osama bin laden on / , the american government decided to implement an rfid tag embedded e-passport and visa waiver program. the us government asked their member countries to implement e-passport by it is essential to understanding how a rapid diffusion and massive applications of rfid generate conflict or harmony among human behaviors, digital literacy, institutional rules, and technology. us army and its allies could not only manage weapons and soldiers but also identify who was the enemy or not (castro and wamba, ) . this whole project of developing an rfid based identifying system was known as iff (identify friend of foe the new york city government also started an rfid e-seal pilot project in the new jersey port. once rfid read and scan the tag, it can identify the contents of the container box. also, the port of tacoma and seattle planned to adopt e-seals, made of metal bolts with embedded rfid devices to ensure its security (konsynski and smith ) . calpatria prison, located in los angeles, adopted a prisoner monitoring system using rfid chips in as the very first pilot using rfid in prison management in united states (kim ) . according to regulations of calpatria prison, all the prison inmates were issued bracelets in which rfid chips were embedded. since the pilot project at the calpatria prison was successful, the local government let other prisons in los angeles adopt the innovative bracelet. the la county prison started to use a brand new bracelet in response to the state government's order; it is reported that through its adoption the prison could increase efficiency and effectiveness and decrease crimes occurred between inmates simultaneously (nicholas ). for instance, city governments in the gifu and osaka prefecture provided rfid tags that can be attached in students' schoolbags to public elementary schools (ema and fujigaki, ) . similarly, in haewoondae beach, one of the most famous vacation spots in south korea, busan metropolitan city provides for parents rfid embedded bracelet that enables tracking exact location of their child by a smart phone (http://news.naver.com/ main/read.nhn?mode=lsd&mid=sec&sid = &oid= &aid= ). this project began in , but it took years to fully implement for all us passports (meingast et al. ) . the appearance of the e-passport is very similar to old passports, but woven into the paper of the passport, there is rfid tag that information about owner of the passport is included. information about nationality, sex, age, and so on is scanned, as airport staff members scan the passport through rfid reader (lorenc (kuo et al. ) with strong government support. one peculiar characteristic of this system as compared to other systems is that visually impaired people are both the reader carrier and the service beneficiaries, simultaneously. generally in government service delivery using rfid technology, the service provider usually carries rfid tag readers and service beneficiaries usually act more passive roles by attaching rfid tags. but in this pakistani case, the service beneficiaries can identify objects around them by operating rfid tag reader they have. the australian government passed legislation on mandatory use of rfid tags in the livestock industry, so hossain and quaddus ( ) categorized australian case as a very rare and special adoption case. according to trevarthen and michael ( ) 's case study, one of the australian farms where the rfid tag is implemented, farmers not only track the exact location of the cows but also check the condition, identify cows and even feed the new born cows automatically by using an rfid system. usually people throw waste in various places. they may throw it away at their houses or at work places, like an office. since the rfid tag is only attached to a garbage can in house, it is impossible to track all types of waste throwing behaviors. inevitably, this shortage of storage capacity leads to selective waste collection monitoring. the faraday cage is an object in metal; proponents of this device argue that faraday cage can prevent hacker's attack because electronic devices are prevented from passing through the object (ezovski et al. ) . but speculation about stability of this technology still remains. lorenc ( ) reported that if there is no additional technology, the faraday cage cannot preserve sensitive security. as a matter of fact, there are two kinds of rfid tags, the passive tag and the active tag. these two tags provide owners with different benefits and liabilities. active tags are implemented by a power source, such as small battery. active tags are more efficient and safe in protecting privacy than passive tags, so sensitive organizations like military prefer active tags to passive tags. but ordinary consumers have less accessibility to active tags because active tags are much more expensive than passive ones (jensen et al. ). according to laurie ( ) hwang et al. ( ) , technological deficiency enables hackers to engage in cloning, eavesdropping, replay attack, denial of service, forward security, tag tracing, individual data privacy, and data forging. specifically, the hacker can read the tag and then clone the tag (cloning), surreptitiously listen to all the communications between and the tag (eavesdropping), repeat or delay the message (replay attack), send large amount of message to break down rfid system (denial of service), compromise a tag (forward security), trace the exact location of the tag (tag tracing), find out shopping trend of the consumer (individual data privacy), and modify information saved on an rfid tag (data forging). in addition, numann and hogben ( ) categorized the privacy attacking cases more briefly. according to their research, the hacker can attack rfid tag in some ways. first, the attacker can open a connection to the chip and can steal the data inside (skimming). second, the attacker can intercept the communication between tag and reader (eavesdropping). last, the attacker can track the exact location of the tag or the person. in thailand, most of the farms are trying to adopt an rfid system in farm management, but rfid technology is widening the gap between poor and rich farmers. poor farmers are usually less educated people who have hardly had any experience using digital technology like rfid. on the other hand, well-educated wealthy farmers face low entry barriers and easily adopt the technology. rich farmers armed with innovative technology not only make enormous fortunes by increasing efficiency but also by replacing poor labors with rfid embedded devices. according to the thailand ministry of labor ( ), most farm labors are afraid of being replaced. unfortunately, this phenomenon eventually correlates to a serious gap between rich and poor. as readers, tags, hardware, and so on, but in the long run cost of management will decrease. on the other hand, the waste industry could carry a very heavy debt. the waste contractors have to deal with expensive rfid tag rental and as well as the cost of construction simultaneously. although this situation is totally unfavorable for them, the waste management industry cannot resist to this policy because the chinese government is the entity which has made the use of rfid policy and set prices. the industry has no other choice but consent. anonymous authentication for rfid systems on rfid privacy with mutual authentication and tag corruption time measurement threatens privacy-friendly rfid authentication protocols rfid and corporate responsibility: hidden costs in rfid implementation a new game of leapfrog? rfid is rapidly changing the product-tracking process. some say the technologyonce costs drop-could displace bar-coding from traditional to modern fee systems an inside look at rfid technology using rfid technology in food produce traceability rfid-based hospital real-time patient management system how far can child surveillance go?: assessing the parental perceptions of an rfid child monitoring system in japan the electronic passport and the future of government-issued rfid-based identification exploring the perceived measures of privacy: rfid in public applications an adoption-diffusion model for rfid applications in bangladesh designing and implementing rfid technology for vehicle tracking in bangladesh developing and validating a hierarchical model of external responsiveness: a study on rfid technology privacy and security requirements for rfid applications the impact of government trust perception on privacy risk perceptions and consumer acceptance of residual rfid technologies mitigating consumer perceptions of privacy and security risks with the use of residual rfid technologies through governmental trust rfid security and privacy: a research survey. selected areas rfid based waste management system closing digital gap on rfid usage for better farm management a divide-and-conquer technique 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that they have no competing interests.authors' contributions kj and sl carried out a systematic literature review not only from not only technological point of view but also from social scientific point of view. both authors read and approved the final manuscript. key: cord- -ekc p k authors: norbäck, d.; cai, g.‐h. title: dampness, indoor mould, fungal dna and respiratory health – molecular methods in indoor epidemiology date: - - journal: clin exp allergy doi: . /cea. sha: doc_id: cord_uid: ekc p k nan building dampness and indoor mould growth are recognized risk factors for respiratory health, including asthma, rhinitis and asthmatic symptoms [ ] . one metaanalysis on the prevalence of dampness and mould in the european housing stock, including published data from european countries, concluded that . % of the homes in europe had dampness, . % indoor mould and . % water damage [ ] . even higher prevalence of dampness and mould in european homes were found in the european community respiratory health survey (ecrhs), where . % of the participants reported that they had ever seen mould in their current home and . % reported water damage. researchers who visited a subset of the homes observed mould in . % and damp spots in . % of the homes [ ] . a number of review articles and meta-analysis have been published suggesting associations between dampness and indoor mould and rhinitis [ ] , bronchitis and airway infections [ ] and onset of asthma [ ] . these studies are mostly based on population samples and have not specifically studied exacerbation of asthma. one recent review on indoor environmental exposure has focused on exacerbation of asthma [ ] . they concluded that there is sufficient evidence of a causal association between outdoor culturable fungal exposure and exacerbation in asthmatics sensitized to fungi. they also concluded that there is limited or suggestive evidence of an association between indoor culturable penicillium exposure and exacerbation in asthmatic children with specific sensitization, any fungal sensitization, or unspecific sensitization. moreover, they concluded that there is limited or suggestive evidence of an association between indoor total culturable fungal exposure and exacerbation of asthma in children with any fungal sensitization. the study has no conclusions concerning exacerbation of asthma in adults in relation to indoor exposure to dampness or mould [ ] . most epidemiological studies on associations between building dampness and indoor mould have investigated respiratory symptoms [ ] , few have investigated associations for lung function. two prevalence studies in adults found lower forced expiratory volume in s (fev ) in damp homes [ ] and in a rehabilitation centre with dampness in the floor construction [ ] . another prevalence study found associations between airway obstruction and higher concentration of , -beta-glucan in homes, a marker of fungal exposure [ ] . one longitudinal european population study observed increased lung function decline (fev ) in adults living in homes with dampness and mould, equivalent to smoking - cigarettes per day [ ] . finally, one study found that asthmatic patients living in homes with confirmed dampness had lower fev than those living in dry homes [ ] . dampness in buildings has been defined broadly and most existing data on building dampness and mould but it is unclear which is exposure that is the causative agent in damp buildings [ ] . one consequence of building dampness is an increased growth of bacteria and mould on indoor surfaces and inside the building construction. lipopolysaccharide (lps, endotoxin) and peptidoglycan are the two most studied bacterial cellwall compounds. lps is a chemical marker for gramnegative bacteria [ ] . endotoxin is mostly measured by the biological limulus test [ ] but -hydroxy fatty acids from endotoxin can also be measured by chemical analysis [ ] . peptidoglycan is found in all bacteria but in the largest amounts in gram-positive bacteria [ ] . muramic acid (mua) is an amino sugar, is found exclusively in peptidoglycan and can be measured by similar chemical analysis [ , ] . associations between endotoxin concentration in indoor dust and respiratory health, including asthma and allergies, have been studied in a large number of studies. the effect can depend on exposure timing, dosage, environmental cofactors and genetics [ ] . radon has summarized the effects of endotoxin with respect to different phenotypes of asthma. the risk of atopic asthma, dominated by eosinophilic response, is decreased in those exposed to endotoxin. in contrast, the risk of non-atopic asthma, characterized by a neutrophilic response, is enhanced in subjects with higher endotoxin exposure [ ] . indoor mould is present everywhere and the issue of indoor exposure to mould is complex and most likely the adverse health effects depends on the amount of moulds as well as the species composition. moreover, there is some evidence of protective effects from fungal exposure on allergies from studies on children in farming environments [ ] . chemical analysis of ergosterol [ ] and analysis of beta- - glucan in dust by the limulus method [ ] has been used as markers of total fungal load. detection and quantification of indoor mould is now possible using mould-specific quantitative polymerase chain reaction (real time pcr) [ , ] . this molecular method can give quantitative data on the occurrence of the most common indoor moulds, irrespectively of viability. epa scientists have designed and tested primers and probes for over types of mould (http://www.epa.gov/microbes/moldtech.htm). the method is called mould-specific quantitative pcr (msqpcr) [ , ] . mould-specific quantitative pcr has been used to assess mould levels in indoor air and settled dust (surface contamination). the method can detect groups of mould (e.g. aspergillus/penicillium) [ ] as well as specific sequences (e.g. stachybotrys chartarum) [ ] . in a uk survey of moulds in homes, msqpcr analysis demonstrated that similar mould species were found in homes in the united states and great britain [ ] . researchers as well as commercial laboratories in the united states, canada and europe are currently using msqpcr. in epidemiological studies, data on fungal dna in indoor samples can be analysed in different ways. one way is to analyse health association between the concentrations of each fungal dna sequence in dust or air and the health parameter, mostly asthma or asthmatic symptoms. two prevalence studies in schools found associations between the concentration of certain fungal dna sequences in school dust (e.g. from aspergillus versicolor and streptomyces) and respiratory symptoms [ , ] as well as lower fev [ ] in the pupils. one case-control study reported that levels of aspergillus versicolor dna were higher in asthmatics homes as compared to controls [ ] . another study reported a positive association between levels of streptomyces dna in home dust and exhaled nitrogen oxide (no) in asthmatic children [ ] . vesper et al. [ ] have developed a concept called environmental relative moldiness index (ermi) to quantify the mould burden in homes. the ermi value is computed by quantifying the concentration of species-specific dna sequences from indicator mould species in home dust samples. the mould species are divided in two groups. the first group (group mould) consists of mould species that indicate water damage. the second group (group mould) consists of sequences from group species that can be from outdoor sources and these moulds are commonly found even without water damage [ ] . for each home, the mould burden is computed by taking the sum of log-transformed group mould species concentrations minus the sum of log-transformed group mould species concentrations. the ermi value does not measure the total fungal concentration in the dust or the total fungal exposure. it is used as a way to rank homes with respect to the relative mould burden in homes [ ] [ ] [ ] . environmental relative moldiness index has been used in epidemiological studies, and higher ermi levels have been found in home dust among children with asthma as compared to controls without asthma [ ] [ ] [ ] . one study found no significant association between ermi in home dust and infant wheeze [ ] . one longitudinal study found that early exposure to moulds as measured by ermi at year of age, but not years of age, increased the risk for asthma at years of age [ ] . in addition, one recent study found higher ermi values in school dust from schools with high prevalence of asthma as compared to schools with low asthma prevalence [ ] . finally, one study found lower lung function (fev ) among children who lived in homes with higher ermi values [ ] . few studies have used mould-specific quantitative pcr or the ermi-index in epidemiological studies on adult respiratory illness. one recent study found an association between ermi values in home dust and asthma and rhinitis in adults [ ] . moreover, few studies have investigated exacerbation of asthma from mould, assessed by the ermi-index. recently, in clinical and experimental allergy, mcsharry et al. [ ] have extended the use of the ermi-index and other microbial markers in the home environment to study exacerbation of asthma, measured as decreased fev % among non-smoking adult asthmatics in scotland. they also studied associations between fev % and corticosteroid use, asthma control questionnaire score (acq) and st. george's respiratory questionnaire score. fev % were negatively correlated with acq and sgrq scores and weakly with corticosteroid use. higher ermi values in home dust were associated with decreased fev % but there was no correlation between fev and other biological contaminants such as concentrations of endotoxin, , -beta-glucan or cat allergen (fel d ), dog allergen (can f ) or house dust mite allergens (der p or der p ) in home dust [ ] . the study adds evidence on the possible role of mould as a cause of exacerbation of asthma in adults and also links the ermi-index to airway obstruction measured as fev . the study supports the view that measurement of fungal dna in dust in epidemiological studies can be a useful indicator of fungal exposure in indoor environments. moreover, the study supports the view that the ermi-index has relevance for respiratory health and can be a useful indicator of relative fungal burden in indoor environments. the ermi-index may also be useful in patient investigations to identify patients that need to improve their home environment. however, more prospective studies are needed where ermi and other types of indoor biological contaminants are measured in parallel in dust samples collected prior to disease development. moreover, epidemiological studies on respiratory effects of indoor exposure should focus on disease development (e.g. asthma, rhinitis and lung function decline) as well as exacerbation of asthma. moreover, respiratory effects of different types of indoor biological contaminants, including fungal dna measured by mould-specific quantitative pcr and calculation of the ermi-index, should be extended from the home environment to other indoor environments such as day care centres, schools, hospitals and offices. moreover, as most epidemiological studies on respiratory effects, especially with ermi-index, are from united states or europe, similar studies need to be funded and performed in other parts of the world, including asia, where the current increase of asthma and allergy is high [ ] . world health organization (who) regional office for europe. guidelines for indoor air quality: dampness and mould prevalence of dampness and mold in european housing stock mould and dampness in dwelling places, and onset of asthma: the population-based cohort ecrhs association of indoor dampness and molds with rhinitis risk: a systematic review and meta-analysis respiratory and allergic health effects of dampness, mold, and dampness-related agents; a review of epidemiological evidence residential dampness and molds and the risk of developing asthma: a systematic review and meta-analysis indoor environmental exposures and exacerbation of asthma: an update to the review by the institute of medicine current asthma and biochemical signs of inflammation in relation to building dampness in dwellings norb€ ack d. dampness and -ethyl- -hexanol in floor construction of rehabilitation center: health effects in staff airways inflammation and glucan in a rowhouse area lung function decline in relation to mould and dampness in the home: the longitudinal european community respiratory health survey ecrhsii damp housing and asthma: a case-control study the two sides of the "endotoxin coin endotoxin, ergosterol, fungal dna and allergens in dust from schools in johor bahru, malaysia -associations with asthma and respiratory infections in pupils microbial exposure of rural school children, as assessed by levels of nacetyl-muramic acid in mattress dust, and its association with respiratory health endotoxin exposure in allergy and asthma: reconciling a paradox indoor fungi: companions and contaminants. indoor air quantitative pcr analysis of selected aspergillus, penicillium and paecilomyces species comparison of populations of mould species in homes in the uk and usa using mould-specific quantitative pcr (msqpcr) traditional mould analysis compared to a dna-based method of mould analysis quantification of stachybotrys chartarum conidia in indoor dust using real time, fluorescent probebased detection of pcr products comparison of populations of mould species in homes in the uk and usa using mold-specific quantitative pcr fungal dna, allergens, mycotoxins and associations with asthmatic symptoms among pupils in schools from johor bahru total viable moulds and fungal dna in classrooms and associations with respiratory health and pulmonary function of european schoolchildren indoor fungal contamination of moisture-damaged and allergic patient housing analyzed using real-time pcr microbial content of household dust associated with exhaled no in asthmatic children development of an environmental relative moldiness index for us homes correlation between ermi values and other moisture and mold assessments of homes in the american healthy homes survey screening tools to estimate mold burdens in homes geographic distribution of environmental relative moldiness index molds in usa homes quantitative pcr analysis of molds in the dust from homes of asthmatic children in north carolina higher environmental relative moldiness index (ermism) values measured in detroit homes of severely asthmatic children higher environmental relative moldiness index (ermi) values measured in homes of asthmatic children in boston environmental relative moldiness index and associations with home characteristics and infant wheeze high environmental relative moldiness index during infancy as a predictor of asthma at years of age mold contamination in schools with either high or low prevalence of asthma grimsey lf. decreased pulmonary function measured in children exposed to high environmental relative moldiness index homes higher environmental relative moldiness index values measured in homes of adults with asthma, rhinitis or both conditions decreased fev % in asthmatic adults in scottish homes with high environmental relative moldiness index values isaac phase three study group this logo highlights the editorial article on the cover and the first page of the article. the authors declare no conflict of interest. key: cord- - i x f authors: klotz, lynn c. title: danger of potential-pandemic-pathogen research enterprises date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: i x f nan t he research goal in a number of laboratories is to make highly pathogenic avian influenza (hpai) viruses contagious to humans via respiratory aerosols. for instance, the h n influenza virus has been made contagious to ferrets ( ), the animal model often used as a proxy for humans. concern over escape from a laboratory of a deadly human-contagious virus (e.g., influenza, severe acute respiratory syndrome [sars] , and middle east respiratory syndrome [mers] viruses) prompted the u.s. government to hold back funding for this research "until a robust and broad deliberative process ( ) is completed that results in the adoption of a new usg gain-of-function research policy." this discussion is now under way in the united states and is to be completed in . in relation to this discussion, mbio published three letters to the editor in a debate between dr. ron fouchier and me. defending the safety of his work in the first letter ( ), dr. fouchier calculated that it would likely take more than a million years for an escape from his lab through a laboratory-acquired infection (lai). intuitively, this million-year claim seems dubious. i questioned the equation used in the fouchier calculation and the extremely low probability of escape that he employed in the calculation, and i outlined an alternative approach ( ). fouchier's response to my comments was then published in mbio ( ) . it is clear that he did not understand my methodology. in calculations of the probability of a community lai ("e"), dr. klotz further assumes that transmission studies in the erasmus mc facility will be performed for a period ("y") of million years. i am hopeful that our research enterprise will have reached solid conclusions on determinants of airborne transmission a bit sooner. rhetorical quip aside, neither his million-year result nor my questioning of it implies or assumes in any way that research must be performed for a given number of years. my questioning and my alternative approach were simply a comment on his approach. elsewhere in my comments ( ) , i assumed that the research enterprise will be concluded in years, as does he. in order to respond to fouchier's misunderstanding and to expand on the general usefulness of my approach, i provide here two simple equations for estimating both the likelihood of escape and the elapsed time to an escape. the equations may be employed for a single lab and for a "research enterprise" of many labs. the conclusion is that the likelihood of an escape may be uncomfortably high and that the elapsed time to an escape may be uncomfortably short. dr. fouchier uses the simplistic formula y ϭ /p to calculate the likely number of years elapsed, y, before an escape occurs, where p is the probability of escape from his lab in year. his -million-year calculation ( ) is misleading, as it does not account for the fact that research will proceed for more than year, and it was not expanded to calculate the number of years that elapse before an escape occurs for the many labs in the research enterprise. my approach, embodied in equation , may be used to calculate the probability, e, that at least one escape will occur for an n-lab research enterprise conducting research for y years (at least one is likely exactly one, as the probability that two or more escapes will occur is extremely small, and if an escape does occur, the whole n-lab research enterprise would almost certainly be shut down), and equation may be used to calculate the number of years that elapse, y, before an escape. solving equation for y gives derivations of equations and may be found in the appendix. in equation , probability e may be viewed as how much escape risk we are willing to tolerate, that is, the value of e that is too high a risk for an n-lab y-year research enterprise. is an e of . %, %, or % too high? the level of risk that we are willing to tolerate is subjective. since a lab escape may result in an uncontrollable disease outbreak with thousands to millions of deaths, even a % chance, e ϭ . , seems much too high. following fouchier's focus on elapsed years as a measure of biosafety, only elapsed years will be calculated here. the results, presented in table , are for a -lab research enterprise; is the number of nih-funded labs that have been identified as subject to the research pause. (originally labs were identified for the funding pause. that number was subsequently reduced to .) while some of these labs do not focus on developing mammalcontagious influenza viruses, there are likely many labs throughout the world not funded by the nih that are, so the number seems a reasonable guess for the size of the research enterprise. presented in table are the same calculations but for a single lab (n ϭ ), such as fouchier's lab. three quite different probabilities for escape, p , are employed in the calculations in the tables. the highest probability (p ϭ . ) is a minimum estimate calculated ( ) from cdc statistics ( ) for undetected or unreported lais in biosafety level (bsl ) labs. this probability is likely higher than that for lais in bsl labs that have extra biosafety precautions in place (called bsl ϩ), such as fouchier claims for his lab. the probability (p ϭ . ) is times lower and is an estimate for differences between bsl and bsl ϩ labs. the final probability (p ϭ ϫ Ϫ ) is fouchier's estimate ( ) . to make his estimate, fouchier itemizes the various safety measures in his lab and generously reduces the probability for each safety measure but admits that it is a guess: "the magnitude of this increase in safety is not known." finally, the discussion here of probabilities has been restricted to lais, but there are other routes of escape, such as mechanical failure and removal of live virus from bsl ϩ containment accidentally or for hostile purposes. from table , it is clear that the research enterprise is unsafe when an intermediate small p equal to . and a risk tolerance (e) of % are employed. the number of years that we would need to wait to exceed the % chance of an escape is only . years, well within the estimated years for the research to be completed. if p is equal to . , as calculated from the cdc statistics, there would be a % chance of an escape in less than a year (y ϭ . year). if p is really as low as fouchier suggests, we would need to wait years to reach a % chance of escape, an elapsed time that would appear to make the research enterprise safe in some researchers' thinking, but risk equals likelihood times consequences, and consequences such as fatalities could be very high for a human-contagious influenza virus with a high case fatality rate. this would lead to an intolerable number of fatalities even using fouchier's low p estimate. potential fatalities for the enterprise and the fatality burden for each lab in the enterprise were quantified for his very low p of ϫ - in my published criticism ( ) of fouchier's million-year calculation. the conclusion there was that each lab in the enterprise would carry the potential burden of over fatalities per year. "to put this fatality burden number in perspective, no institutional review board tasked with assessing human subject research would approve a proposed research project with potential fatalities per year." turning to the number of years that elapse before an escape occurs for a single lab in table , for a % chance of escape with the intermediate p , it would take years of research to exceed the % risk tolerance. for fouchier's low p , it would take , years to reach a % chance of an escape, making the research seem quite safe. i suspect that most researchers in the enterprise use this reasoning to justify the safety of their own lab, but this kind of thinking is flawed, as argued over years ago by the philosopher immanuel kant for his "categorical imperative," which is the cornerstone of his moral reasoning: "act only according to that maxim whereby you can at the same time will that it should become a universal law without contradiction." the "it" in the quote is labs in the research enterprise. at the risk of trivializing kant's complicated moral reasoning, a few examples should make the categorical imperative argument clearer. is it really acceptable for a manufacturing company to dump mercury in the ocean, because that one factory's output would not be enough to pose any danger to us when we consume fish? is there nothing wrong if i buy a gas guzzler that gets miles per gallon and has faulty emissions control, since my car's individual contribution to climate destruction is minimal? "what if everyone researched live smallpox?" has been implicitly answered according to kant's categorical imperative by everyone agreeing to limit research to two places. clearly, the magnitude of the basic probability (p ) is critically important in assessing the risk of the research enterprise, and its magnitude is a point of contention. finding a good estimate of this basic probability should be a major focus in gryphon scientific's risk-benefit assessment. fouchier has other criticisms of my comments that i feel should be addressed. his response is problematic in several ways. in addressing the problems, i will quote frequently from my comments and from his response to make sure that it is clear what was said. the biggest problem is that dr. fouchier does not once address my calculation of potential fatalities and fatality burden that employs his low probability of an undetected or unreported lai escaping from his laboratory. instead, he chooses to argue against my peripheral comments that his probability is likely much too low. his focus unfortunately draws attention away from my calculation that finds intolerable numbers of potential fatalities and the fatality burden. i number specific comments below to keep each point separate. the cdc's shipping of an h n virus-contaminated sample to the usda and similar incidents show the importance of not underestimating human error, especially if one considers the influenza lab at the cdc to be one of the top federal labs in the country. although biosecurity measures have improved greatly over the years, human nature has not. laboratory accidents will happen, and laboratory workers will get infected, not realize it or not admit it, and so take the infection home. the achilles' heel in fouchier's argument is that no number of safety procedures can provide for human error. while the history of escapes should make us worry that the probability may be very high, here the difference between fouchier and me is moot since i employ his low probability in my calculations. . dr. fouchier writes ( ) dr. klotz proposes to multiply the low likelihood of lais by , based on an estimated laboratories involved in the "whole research enterprise" for years, and assumes that part of this research enterprise may lack the rigorous safety practices in place at erasmus mc. both assumptions are wrong, to the best of my knowledge; just over a handful of laboratories have worked on airborne transmission of avian influenza viruses, each of which has rigorous safety practices in place. our disagreement here is because we define "research enterprise" differently. i defined it as research on potential pandemic pathogens with nih funding (influenza and sars category pathogens) and labs otherwise funded. ( ) another key aspect is that dr. klotz estimates the likelihood of onward transmission from a case of lai as . ( %), in contrast to my justification for an adjusted likelihood of Ͻ ϫ - , based on the specific conditions under which the research is performed, without providing a rationale for that important deviation. i certainly do provide a rationale for the % likelihood of lai ( ) through references and (risk assessment studies). summarizing the literature, lipsitch and inglesby estimate the probability that a community lai leads to a global spread (pandemic) to be to %. this range is consistent with the to % range found by merler and coworkers ( ) and with the to % range found in a focused risk assessment ( ) for infection spread beginning on crowded public transportation. furthermore, there is a rather arcane subject in probability, branching theory, which allows prediction of the likelihood of uncontrolled spread of any pathogen based on its observed reproductive number (r o ) value and the variance to mean of the r o . a large variance to mean could occur due to superspreaders, for instance, some people infected with sars virus. for a wide range of r o values, lipsitch and coworkers have calculated the probability of uncontrolled spread (see fig. a in their study [ ] ). for a single infected individual with an r o of , the probability of an uncontrolled outbreak ranges from % (spread of r o s) to % (uniform r o ). thus, the pandemic likelihood from a single infected individual is potentially large. i suspect that future risk assessments will confirm that once a highly contagious potentially pandemic pathogen escapes, the probability of an uncontrolled outbreak is significant, leading again to a focus on the probability of a laboratory escape as the important factor. fouchier mentions vaccination and antivirals ( ) as factors that reduce onward transmission. antivirals would not be prescribed for undetected lais. vaccines may reduce viral replication in the index case, but active virus may still be present when the infected person leaves the laboratory, potentially infecting unvaccinated persons. the annual flu vaccine is sometimes less than % effective, so it is unclear if vaccinated laboratory workers are protected by the laboratory vaccine strain. i would classify vaccination and antivirals, effective or not, as inside laboratory measures. but if an lai escapes, clearly these measures were not effective in preventing the undetected or unreported lai. again, we come back to the probability of escape from a laboratory as a key challenge in this debate. once an undetected or unreported lai from a highly contagious pathogen escapes, it is out of fouchier's control. its global spread will depend on the reproductive number, r o , and other factors external to fouchier's laboratory. fouchier claims ( ) that "the viruses are ferret adapted rather than human adapted," which could lead to a lower r o in humans. among the different mutated viruses presumably under development in his laboratory, some could be highly transmissible and deadly in humans. we will never know, for testing them on humans is, fortunately, unethical. of course if one escaped. . . . the argument of being ferret adapted and not human adapted is misleading. first, it cannot be proved. second, fouchier's own work may have already brought an avian h n virus far closer to successful replication in humans. if such a virus escaped from his laboratory, it may well adapt within the individuals in the early transmission chain and then take off in a big way. dr. fouchier and the field do not have the knowledge to know just how short of a successful virus they have engineered. that is why they are doing this work. . dr. fouchier concludes ( ) the following. finally, dr. klotz describes the (apocalyptic) scenario of an influenza pandemic with million fatalities based on a % casefatality rate in % of the world's population. these numbers not only ignore the scientifically justifiable counterarguments raised before ( ) but also are at odds with the documented influenza pandemics of the past. in my view, the "gain-of-function" debate has suffered from the apocalyptic scenarios that are provided as factual whereas they provide estimates that are far beyond the observed worst cases ( ) . it is estimated that the pandemic influenza virus infected % of the world's population. the h n "spanish" flu killed perhaps % of its victims. the h n avian influenza virus, the subject of fouchier's research, kills about % of those who are infected through direct contact with poultry. the scenario i use as an example represents a combination of these three real events. while this scenario has not yet and may never occur in nature, it is a possible scenario perhaps more likely from a laboratory escape. since the consequences of most scenarios, even one on a par with seasonal influenza-several hundred thousand deathswould be catastrophic and unacceptable, it behooves us to be exceedingly careful in deciding which potential pandemic pathogen research should be allowed. for much of this research, the potential risk far outweighs the potential benefits. to a lab escape. let p be the yearly probability of escape of a pathogen from a single lab. the first question to be asked is "what is the probability of at least one escape from one of the n labs conducting research on the pathogen for y years?" the probability of no escapes in y years for a single lab is ( Ϫ p ) y . for y years and n labs, the probability of no escapes is ( Ϫ p ) yn . the probability of at least one escape in y years from one of the n labs (e) is solving equation for y will allow this question to be answered. log( Ϫ e) ϭ log( Ϫ p ) yxn ϭ y ϫ n ϫ log( Ϫ p ), where y ϭ ( ⁄ n) ϫ [log( Ϫ e) ⁄ log( Ϫ p )] checking the limit for equation , if there is no likelihood of escape, p is equal to , log( ) is equal to , and as expected, y is equal to ϱ. another observation about equation is that the number of years of research, y, which must elapse before we reach our risk tolerance is inversely proportional to the number of labs, n. airborne transmission of influenza a/h n virus between ferrets government gain-of-function deliberative process and research funding pause on selected gain-of-function research involving influenza, mers, and sars viruses. u.s. department of health and human services studies on influenza virus transmission between ferrets: the public health risks revisited comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory the consequences of a lab escape of a potential pandemic pathogen. front public health : monitoring select agent theft, loss and release reports in the united states- - categorical imperative, on wikipedia, the free encyclopedia transmission dynamics and control of severe acute respiratory syndrome acknowledgment i thank simon wain-hobson for comments and contributions to the text. key: cord- -gmujrso authors: castagnini, luis a.; goyal, meha; ongkasuwan, julina title: tonsillitis and peritonsillar abscess date: - - journal: infectious diseases in pediatric otolaryngology doi: . / - - - - _ sha: doc_id: cord_uid: gmujrso tonsillitis is one of the most common childhood infections. occasionally, it can lead to one of the most common deep space head and neck infections, peritonsillar abscess. the epidemiology, microbiology and treatment of tonsillitis and peritonsillar abscess are similar and crucial for the primary care physician, infectious disease specialist, otolaryngologist, and emergency medicine physician to understand. the routine use of tonsillectomy as a treatment option for recurrent tonsillitis and peritonsillar abscess has decreased over the last decade and clearer indications for surgery have emerged. this chapter provides an overview of the most recent literature regarding the epidemiology, microbiology, diagnosis, complications and management of tonsillitis and peritonsillar abscess. it also discusses the indications for tonsillectomy along with its complications. the tonsils are lymphoepithelial organs that function as secondary lymphoid organs. they contain specialized epithelial m cells that capture and transport antigens entering through the mouth and nose to extrafollicular regions or lymphoid follicles. the lymphoid follicles then release antibody-expressing memory b cells or plasma cells that migrate to the tonsils and produce antibodies. these antibodies are subsequently released into the tonsillar crypt lumen. all fi ve isotypes of immunoglobulins are produced in the tonsil. the most important of these isotypes is iga which functions as an important component of the mucosal immune system of the upper airway [ ] . the tonsils are at their largest size during the most active immunologic activity, which is estimated to be between the ages of and years. after this period they display spontaneous age-depended involution [ ] . chronic or recurrent tonsillitis alters the tonsillar immune system by causing shedding of the m cells and the tonsillar immunologic response to antigens weakens. the clinical signifi cance of this dysfunction is controversial. there are no data demonstrating signifi cant change in the systemic immune system after tonsillectomy [ ] . tonsillitis is infl ammation of the tonsils, specifi cally the palatine tonsils. acute pharyngitis is one of the most common illnesses seen in the primary care setting accounting for up to . % of all emergency department visits and up to % of offi ce visits for children and adolescents [ , ] . most cases in children are observed during winter and early spring when respiratory viruses are more common. during the summer months enteroviruses are responsible for the majority of cases [ ] . tonsillitis caused by group a β-hemolytic streptococcus (gabhs) most commonly occurs in children - years old, affecting less than % of children younger than years old, % of children less than years old, and % of school-aged children [ ] . the fi nancial burden of gabhs tonsillitis is estimated to be between $ and $ million per year with more than half being associated to non-medical costs [ ] . neisseria gonorrhoeae is an important pathogen in sexually active individuals or in victims of sexual abuse [ ] . repeated episodes of all-cause tonsillitis is reported in . % of children less than year old and . % of children between the ages of and years old [ ] . tonsillitis may be caused by a viral or bacterial infection of the tonsils, most commonly the palatine tonsils. viral etiologies are the most common cause of tonsillitis in the pediatric population. common viral pathogens include enteroviruses, particularly coxsackie virus, respiratory viruses (e.g. adenovirus, rhinovirus, infl uenza virus, coronavirus, parainfl uenza virus and respiratory syncytial virus), and viruses of the herpesviridae family like epstein-barr virus (ebv), cytomegalovirus (cmv) and herpes simplex virus (hsv) [ ] . the most common bacterial pathogen implicated in acute tonsillitis is gabhs, accounting for up to % of all episodes of acute pharyngotonsillitis in children. less frequent bacterial causes include staphylococcus aureus , streptococcus pneumoniae , group c streptococcus, mycoplasma pneumoniae , chlamydophila pneumoniae , corynebacterium diphtheriae , arcanobacterium haemolyticum , neisseria gonorrhea , francisella tularensis , yersinia enterocolitica , and mixed anaerobic fl ora from the oral cavity [ ] . fusobacterium necrophorum , a gram-negative aerobic bacilli, and the most common cause of lemierre's syndrome, has been cultured from adolescents and young adults with uncomplicated tonsillitis [ ] . patients with tonsillitis present with a variety of symptoms that include sore throat, fever, chills, odynophagia, cervical adenopathy, trismus, halitosis, erythematous and exudative tonsils and tonsillar pillars ( fig. . ) . the presence of conjunctivitis, coryza, cough, stomatitis, diarrhea and hoarseness strongly suggest a viral etiology. children younger than years of age may have an atypical presentation of gabhs infection called streptococcosis, which is characterized by fever, mucopurulent or serous rhinitis, and adenopathy, followed by irritability, loss of appetite and lethargy. exudative tonsillitis is rare in this age group. on physical exam, it is often diffi cult to distinguish between viral and bacterial tonsillitis, but some clinical fi ndings may provide important clues of the etiologic agent. for example, hsv typically presents with stomatitis, ebv may include lymphadenitis and coxsackie virus infections may present with throat ulcers (herpangina) or as part of hand-foot-mouth disease. complications of tonsillitis can be suppurative or non-suppurative in nature. suppurative complications include peritonsillar abscess, parapharyngeal or retropharyngeal space abscess, and suppurative cervical lymphadenitis. acute airway compromise, rheumatic fever, glomerulonephritis, and scarlet fever are non-suppurative complications of tonsillitis caused by gabhs. streptococcal toxic shock syndrome, an uncommon but rapidly progressive disease, can complicate cases of pharyngitis caused by a toxic-producing strain of gabhs [ ] . tonsillitis is primarily a clinical diagnosis. supportive tests include throat cultures, gabhs rapid antigen test, and anti-streptolysin-o (aso), anti-deoxyribonuclease b (anti-dnase b), antihyaluronidase and anti-streptokinase antibody titers [ ] . other tests may be helpful based on clinical suspicion, for example, ebv specifi c serology or monospot (heterophile antibody) test, ebv polymerase chain reaction (pcr) or hsv pcr as needed. the monospot test is particularly insensitive in young children, with only - % of children under the age of years infected with ebv having a positive monospot test [ ] . specifi c ebv serology to detect antibodies against viral capsid antigens (vca) that includes vca-igg and vca-igm in conjunction with antibodies against epstein-barr nuclear antigen or ebna are the preferred diagnostic method in this age group. a real-time ebv pcr assay is helpful in patients with immunocompromising conditions and to confi rm the diagnosis in patients with negative serology but strong clinical suspicion of infection [ ] . the most important step in diagnosis is distinguishing between viral and gabhs tonsillitis as anti-bacterial agents are not effective in the treatment viral tonsillitis. furthermore, with a few rare exceptions (e.g. arcanobacterium haemolyticum , neisseria gonorrhoeae and fusobacterium spp.) anti-microbial treatment is not benefi cial for bacterial causes of tonsillitis except gabhs given that there is not a signifi cant reduction in the rate of complications or in duration of clinical symptoms [ ] . seventy percent of patients presenting with sore throat are treated with antibiotics while only - % have documented gabhs tonsillitis. antibiotic treatment may be associated with adverse drug events that range from mild diarrhea to severe allergic reactions. thus, the utility of these drugs must be determined in order to avoid potential selection of resistant organisms, exposure to adverse events associated with anti-microbial use, and extra cost. treatment of gabhs is instrumental in preventing the potentially long-term and life-threatening complications associated with this pathogen, specifi cally and most importantly, arf. treatment also aids in the control of acute signs and symptoms, prevention of suppurative complications, and decreased transmission of gabhs to close contacts [ ] . throat pain and fever self-resolve by week and - days, respectively, after onset if left untreated; if treated, both symptoms resolve within days [ ] . the organisms are eradicated from the pharynx after days of treatment. arf can be prevented even if therapy is initiated after days of onset [ ] . of note, treatment does not prevent the development of psgn [ ] . the infectious disease society of america (idsa) recommends testing for gabhs unless a patient presents with symptoms strongly suggestive of a viral etiology; examples of such symptoms include cough, coryza, rhinorrhea, stomatitis or hoarseness. testing for gabhs is also not indicated in children less than years old. children in this age group do not present with classic symptoms of gabhs tonsillitis and the incidence of arf is rare, affecting approximately . % of children [ , ] . testing for gabhs in these children should only be pursued in the presence of other risk factors such as school-aged sibling with documented infection by gabhs, close household contact with diagnosis of symptomatic disease, or with personal or family history of a gabhs complication (arf) [ ] . one of the most commonly used in-offi ce diagnostic tests for gabhs is the rapid antigen detection test (radt). this test is done via throat swab of the surface of either tonsil or tonsillar fossa and posterior pharyngeal wall. swabs of other areas of the oropharynx or oral cavity may lead to false negatives. an enzyme immunoassay test with turn-around times as little as min is then done. it is % specifi c and - % sensitive based on the type or manufacturer of radt used. in the case of a positive radt, children should be treated with antibiotics. in the case of a negative radt, the idsa recommends a throat culture be done during the same offi ce visit. due to the variability in sensitivity of rdta based on manufacturer, the high rate of gabhs in children and implications of complications, a throat culture is recommended in order to capture any false negatives. the rapid turnaround time for radt makes it useful for rapid identifi cation and treatment of gabhs. rapid treatment decreases the risk of spread of gabhs among close contacts, the amount of time missed from school or work for caregivers, and the duration and severity of acute signs and symptoms of gabhs tonsillitis [ ] . throat cultures are recommended in children in the case of negative radt prior to the administration of antibiotics in order to avoid false negative results. a single throat swab has a - % sensitivity rate when done correctly. a throat swab similar to the radt test is done and is then either processed in an in-offi ce laboratory or sent to a microbiology laboratory. if the cultures are grown in-offi ce, specifi c instructions must be followed. the swab is processed on a sheep's blood agar plate and incubated at - °c for - h. while treatment decisions can be made based on growth patterns at h, a plate with no growth should be re-examined at h to ensure a correct diagnosis. two major disadvantages of using throat cultures for diagnostic purposes are the training and cost associated with accurate testing as well as delayed diagnosis due to processing time. however, even a delayed diagnosis can be benefi cial. studies show that treatment of gabhs tonsillitis can be delayed up to days from the onset of symptoms and still effectively prevent complications such as arf [ , , ] . therefore, regardless of the delay in treatment, throat cultures should be done in children with negative radt [ ] . other testing options include anti-streptococcal antibody titers; however, these titers are not helpful in the diagnosis of acute gabhs tonsillitis. rather, they are indicative of previous infection. antibody titers become positive - weeks after an acute infection and may persist for up to a year after the resolution of the infection. thus, they may be useful in determining the etiology of complications [ , , ] . children with recurrent tonsillitis are sometimes chronic carriers of gabhs with superimposed viral infections. up to % of asymptomatic school aged children can be carriers of gabhs in the winter and spring months [ , ] . the idsa does not recommend identifi cation or treatment of these chronic carriers for several reasons. distinguishing chronic carriers from recurrent acutely infected children is not possible with the current diagnostic modalities, chronic carriers of gabhs are unlikely to spread bacteria to close contacts and they are at minimal to no risk of developing complications of gabhs [ ] . moreover, eradication of gabhs from colonized tonsils and adenoids is much more diffi cult than treatment of acute gabhs tonsillitis. however, certain specifi c circumstances do call for treatment of chronic carriers of gabhs [ ] . these indications, along with treatment options, are discussed below in the section entitled "treatment of tonsillitis." routine post-treatment radt or throat cultures to confi rm eradication of gabhs are not recommended. post-treatment testing can be pursued in the case of a patient at high risk for developing arf (personal or family history of arf) or recurrent classic symptoms of gabhs tonsillitis shortly after the completion of treatment. testing or treatment of asymptomatic household contacts is not recommended as it has not been shown to decrease the incidence of subsequent gabhs tonsillitis [ ] . treatment of viral tonsillitis primarily consists of supportive measures including bed rest, hydration, analgesics, and oral hygiene. most cases of viral tonsillitis self-resolve in - days. recommended analgesics include acetaminophen and non-steroidal anti-infl ammatory drugs (nsaids). aspirin should be avoided due to the risk of reye's syndrome, a rare severe illness characterized by rapidly progressive encephalopathy with liver dysfunction and a mortality rate of up to % in children and adolescents suffering from a viral infection, especially varicella-zoster or infl uenza, in association with the use of salicylates [ ] . other nsaids such as ibuprofen or diclofenac can be used. nsaids and acetaminophen not only provide pain control but also act to reduce fever. corticosteroids have proven benefi cial in the reduction of the duration and severity of other signs and symptoms, but they do not affect pain levels. thus, they are not recommended for symptomatic control in acute tonsillitis [ , ] . acute bacterial tonsillitis is treated with anti-microbial therapy in addition to the supportive measures listed above. penicillins target the most commonly implicated pathogen, gabhs. they are narrow spectrum drugs with the greatest safety profi le and provide the highest effi cacy at a lower cost than other alternatives. furthermore, there have been no documented cases of penicillin resistant gabhs. a ten-day course of oral penicillin or amoxicillin or a one-time dose of intramuscular benzathine penicillin g is the treatment of choice. an amoxicillin suspension is preferred for younger children due to once a day dosing and better taste that facilitates improved compliance. while a clinical response should be achieved within - h of beginning antibiotic therapy, a day course of antibiotics has been shown to achieve the maximum rates of pharyngeal eradication of bacteria [ ] . patients with previous non-anaphylactic allergic reactions to penicillin can be treated with fi rst generation cephalosporins for days. narrow spectrum fi rst generation cephalosporins such as cefadroxil and cephalexin are preferred over broad spectrum cephalosporins such as cefaclor, cefuroxime, cefi xime, cefdinir, and cefpodoxime. approximately % of patients allergic to penicillins will also be allergic to cephalosporins. these patients can be treated with a day course of clindamycin, clarithromycin or a day course of azithromycin. erythromycin should be reserved for treatment resistant infections due to its high rate of gastrointestinal side effects. rate of gabhs antibiotic resistance in the united states are approximately % to clindamycin and - % to macrolides [ , ] . ampicillin and oral penicillin-based antibiotics can cause a generalized papular rash in the setting of infectious mononucleosis. thus, if infectious mononucleosis is suspected, treatment with antibiotics is not recommended. the idsa discourages the use of several antibiotics for the treatment of gabhs tonsillitis. given the high prevalence of resistant strains of gabhs, tetracyclines are not recommended and trimethoprim-sulfamethoxazole does not effectively eradicate gabhs in acute tonsillitis. newer fl uoroquinolones such as levofl oxacin and moxifl oxacin have proven active against gabhs in vitro but no in vivo effi cacy has been documented. fluoroquinolones are also expensive, broad-spectrum antibiotics with emerging resistance to streptococcus pneumoniae worldwide and are not recommended in children years of age or younger due to their potential for joint and cartilage toxicity [ , , ] . recurrent tonsillitis can be treated with penicillin, cephalosporins, macrolides, or clindamycin. if tonsillitis recurs shortly after the completion of a course of antibiotics, intramuscular penicillin should be considered. alternatively, a - week course of a penicillin coupled with a beta lactamase inhibitor such as amoxicillin plus clavulanate has been shown to be effective in treatment of recurrent tonsillitis [ ] . as discussed previously, routine treatment of chronic carriers of gabhs is not recommended. however, there are a few specifi c indications for treatment. according to the idsa and the american academy of pediatrics, chronic carriers should be treated in the following circumstances: ( ) during a local outbreak of arf, psgn, or invasive gabhs infection, ( ) outbreaks of gabhs pharyngitis in a closed community, ( ) personal or family history of arf, ( ) excessive family or caregiver anxiety about a gabhs infection, or ( ) if tonsillectomy is being considered only on the basis of chronic carriage of gabhs. patients meeting any of the above criteria should be treated with oral clindamycin, oral penicillin plus rifampin, oral amoxicillin plus clavulanate, or intramuscular penicillin plus oral rifampin [ , ] . tonsillectomy should be considered for patients suffering from chronic or recurrent tonsillitis whose frequency of infection does not decrease despite appropriate antibiotic treatment and with no other explanation for tonsillitis. specifi c indications for tonsillectomy are further discussed in the section entitled "tonsillectomy. " peritonsillar abscess is one of the most common deep space head and neck infections in children. this collection of pus is thought to be formed most commonly as a result of spread of infection from the tonsils or the minor salivary glands of weber, found on the superior tonsillar pole. the abscess forms deep to the tonsillar capsule between the tonsil, the superior constrictor muscle, and the palatopharyngeus muscle. the most common location is superior and medial to the tonsil; however it can occur lateral to the tonsil or even inferior [ ] . peritonsillar abscess comprises % of all soft tissue head and neck infections. in patients younger than years old, the incidence of peritonsillar abscess is . - . cases per , patients. it is most commonly diagnosed in adolescents and young adults, but can occur in any age group with an average age at diagnosis of . years old [ ] . peritonsillar abscesses are generally polymicrobial, representing the normal fl ora of the oral cavity and tonsillar area. aerobes such as gabhs, streptococcus viridans , staphylococcus aureus and haemophilus infl uenza , and anaerobes such as bacteroides spp., fusobacterium necrophorum and peptostreptococcus spp. that make up normal oral fl ora are frequently reported [ ] . the most commonly isolated pathogen is gabhs. findings at presentation commonly include fever, odynophagia, trismus, erythema, bulging of the soft palate with deviation of the uvula, unilateral otalgia, drooling, and "hot potato" voice ( fig. . ). trismus is a key fi nding in patients with peritonsillar abscess and is likely related to peritonsillar infl ammation of the pterygoid muscles. inability to swallow or signifi cant odynophagia usually results in dehydration in younger patients. the diagnosis of peritonsillar abscess is typically a clinical one; however, computed tomography (ct) can be utilized in atypical presentation such as when trismus limits the utility of a physical exam, or in uncooperative young children (fig. . ) . while the use of intra-oral ultrasound for diagnosis of peritonsillar abscess has been suggested, it is not yet widely used [ ] . an elevated white blood cell count and c-reactive protein are commonly found. throat culture and testing for infectious mononucleosis may be helpful to evaluate for other disease processes. complications of a peritonsillar abscess include airway distress, parapharyngeal or retropharyngeal abscess, aspiration pneumonia, and erosion into the carotid sheath. lemierre's syndrome, a severe disease characterized by thrombophlebitis of the internal jugular vein with metastatic septic emboli as a result of an acute oropharyngeal infection, is another potential complication [ ] . defi nitive treatment consists of incision and drainage or needle aspiration of abscess contents, antibiotics, and elective tonsillectomy after resolution of infection. in rare cases, quinsy tonsillectomy at the time of infection can be considered. indications for quinsy tonsillectomy are discussed below in the section entitled "quinsy tonsillectomy." drainage of the abscess leads to immediate improvement in pain and hastens recovery. drainage can be done with local anesthesia in the cooperative awake patient or in the operating room. children are more likely to undergo drainage in the operating room. when performing awake, transoral drainage, a pre-procedure dose of an opioid can be helpful with patient tolerance and the degree of trismus. needle aspiration or incision and drainage appear to have equal effi cacy [ ] . purulent material should be sent for aerobic and anaerobic culture. complications of drainage include bleeding, airway obstruction, and possible puncture of the carotid artery. ten to twenty percent of children undergoing incision and drainage or needle aspiration of a recurrent peritonsillar abscess will require a subsequent tonsillectomy for persistent symptoms or residual abscess contents [ , , ] . peritonsillar abscesses recur - % of the time depending on the defi nition of recurrence which varies by practitioner and system [ , ] . the use of tonsillectomy as a treatment for peritonsillar abscess remains controversial. it is favored by some practitioners in the setting of recurrent peritonsillar abscesses. a tonsillectomy at the time of infection (quinsy tonsillectomy) can be considered in rare cases (see section entitled "quinsy tonsillectomy"). an interval tonsillectomy - weeks after the resolution of infection may be performed in patients with recurrent tonsillitis. antimicrobials are used as adjunctive therapy for peritonsillar abscess. combination therapy with penicillin and metronidazole is - % effective [ ] . first generation cephalosporins can be used in patients with a non-anaphylactic penicillin allergy. patients with previous anaphylactic reactions to penicillin can be treated with clindamycin, clarithromycin or azithromycin. supportive therapy with hydration, pain control, and corticosteroids should also be administered [ , , , ] . tonsillectomy is one of the most common ambulatory surgeries performed in the pediatric population. recent studies show that , tonsillectomies are performed per year in children less than years old in the united states [ ] . a bimodal distribution of tonsillectomies is observed with the two most frequent age groups being - years old and - years old [ ] . a tonsillectomy entails the removal of the palatine tonsils with their capsule from the tonsillar fossa. the indications for tonsillectomy include recurrent infection and sleep disordered breathing (sdb) . the american academy of otolaryngology-head and neck surgery (aao-hns) recommends that children that suffered from greater than seven infections in the last year or greater than fi ve infections per year in the last years or greater than three infections per year in the last years and fulfi lled one or more of the following criteria should undergo a tonsillectomy with or without an adenoidectomy: temperature greater than . °c, cervical adenopathy, tonsillar exudate, or positive test for gabhs. children that do not meet these criteria but have multiple antibiotic allergies or intolerances or suffer from periodic fevers, aphthous stomatitis, pharyngitis and adenitis (pfapa syndrome) or with a history of peritonsillar abscesses may also be considered candidates for tonsillectomy. a signifi cant amount of missed school or work for patients and/or caregivers due to sdb or recurrent infections should also be considered when creating a treatment plan [ ] . the aao-hns emphasizes that children that do not meet this criteria may not signifi cantly benefi t from undergoing a tonsillectomy. guidelines suggest close observation and recording of frequency of episodes and symptoms instead of invasive intervention. the use of tonsillectomy as treatment of pfapa is still controversial. the aao-hns recommends consideration of tonsillectomy in these cases depending on the frequency of symptomatic illness, severity of infection and the patient's response to medical management, commonly steroid therapy [ ] . two randomized control trials showed statistically signifi cant benefi t of tonsillectomy to treat pfapa [ , ] . tonsillectomy is recommended in the case of sdb if caused by hypertrophic tonsils and there is signifi cant possibility of improvement of other co-morbidities caused by sdb. examples of such comorbidities include growth retardation, poor school performance, and behavioral problems. the decision to undergo surgery must be made in close communication with the child's caregiver(s) [ ] . complications of a tonsillectomy include throat pain, post-operative nausea and vomiting, dehydration due to delayed oral intake, post-obstructive pulmonary edema, velopharyngeal insuffi ciency and nasopharyngeal stenosis in the case of concurrent adenoidectomy, hemorrhage and death. the most common morbidity of tonsillectomy is throat pain. treatment includes over the counter analgesics and hydration. commonly used analgesics include acetaminophen and acetaminophen plus hydrocodone. the use of non-steroidal anti-infl ammatory drugs (nsaids) is generally not recommended due to a potential risk of post-operative bleeding. however, several studies show that nsaids do not signifi cantly increase the number of post-tonsillectomy bleeds requiring surgical or non-surgical intervention and that they decrease the incidence of post-operative vomiting [ ] . other studies show that while aspirin is associated with increased risk of post-tonsillectomy bleeding, non-aspirin nsaids do not signifi cantly increase this risk with one exception [ ] . intravenous ketorolac has been associated with post-tonsillectomy hemorrhage rates as high as % [ , ] . studies show that post-tonsillectomy pain in children is undertreated by caregivers, primarily due to caregiver attempt at balancing pain control with overtreatment [ ] . the aao-hns guidelines state that no specifi c medication or dosing interval (as needed versus scheduled) has been proven superior. it is most important that caregivers assess and re-assess a child's pain level even when the child does not spontaneously complain of pain [ ] . post-tonsillectomy hemorrhage is a much less common but the most concerning complication of tonsillectomy. it is the most common complication brought to the attention of medical personnel. post-tonsillectomy hemorrhage is stratifi ed based on time after surgery in order to help delineate the cause of bleeding. primary hemorrhage is bleeding occurring within the fi rst h after tonsillectomy and occurs in . - . % of patients. the most common cause is surgical technique or reopening of blood vessels. secondary hemorrhage is bleeding that occurs more than h after surgery, most commonly on post-operative days - . secondary hemorrhage is most commonly due to sloughing of the eschar as the tonsillar bed heals and occurs in . - % of patients [ ] . the incidence of post-tonsillectomy hemorrhage has been noted to range signifi cantly due to the defi nition of clinically signifi cant bleeding and the consideration of primary or secondary hemorrhage only. the use of specifi c surgical techniques to reduce the incidence of post-tonsillectomy hemorrhage is still under investigation [ ] . bleeding following a tonsillectomy requires clinical evaluation and profuse bleeding may be treated with cauterization, inpatient observation, transfusion, or surgery. the rate of mortality associated with tonsillectomy has been cited as less than in , [ ] . the most common causes of tonsillectomy-associated death include bleeding and opioid related respiratory depression [ ] . a quinsy tonsillectomy is done at the time of tonsillar infection. while tonsillectomy is generally recommended after the resolution of infection, it can be considered at the time of infection in a select few cases. indications include peritonsillar abscess in younger children; recurrent or unresponsive cases of peritonsillar abscess or in the setting of previous history of deep neck abscess, and peritonsillar abscess presenting with severe airway compromise [ ] . due to the infl ammation in an infected fi eld, the risk of intraoperative, and potentially post-operative, bleeding is increased. thus, candidates for quinsy tonsillectomy must be carefully and selectively chosen. tonsillitis and peritonsillar abscess are frequently seen in the pediatric population. antimicrobial management should be directed by radt or positive throat cultures. in the case of peritonsillar abscess, acute drainage of the pus is the defi nitive treatment in addition to the use of adjunctive antimicrobial therapy. quinsy tonsillectomy can lead to bleeding complications and is typically reserved for rare cases. oral cavity and oropharynx gray's anatomy for students clinical practice guideline: tonsillectomy in children national hospital ambulatory medical care survey: emergency department summary tables antibiotic prescribing by primary care physicians for children with upper respiratory tract infections principles and practice of pediatric infectious diseases clinical practice guideline for the diagnosis and management of group a streptococcal pharyngitis: update by the infectious diseases society of america burden and economic cost of group a streptococcal pharyngitis incidence and impact of selected infectious diseases in childhood detection of fusobacterium necrophorum subsp. funduliforme in tonsillitis in young adults by real-time pcr laboratory tests in the diagnosis and follow-up of pediatric rheumatic diseases: an update infectious mononucleosis clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary epstein-barr virus infection in children antibiotics for sore throat the role of the streptococcus in the pathogenesis of rheumatic fever prevention of rheumatic fever and diagnosis and treatment of acute streptococcal pharyngitis: a scientifi c statement from the american heart association rheumatic fever, endocarditis, and kawasaki disease committee of the council on cardiovascular disease in the young, the interdisciplinary council on functional genomics and translational biology, and the interdisciplinary council on quality of care and outcomes research: endorsed by the the human immune response to streptococcal extracellular antigens: clinical, diagnostic, and potential pathogenetic implications group a streptococci among school-aged children: clinical characteristics and the carrier state nonsteroidal anti-infl ammatory drugs exposure and the central nervous system oral dexamethasone for the treatment of pain in children with acute pharyngitis: a randomized, double-blind, placebo-controlled trial community-based surveillance in the united states of macrolide-resistant pediatric pharyngeal group a streptococci during respiratory disease seasons the use of systemic and topical fl uoroquinolones the use of fl uoroquinolones in children pediatric deep space neck infections in microbiology and management of peritonsillar, retropharyngeal, and parapharyngeal abscesses peritonsillar abscess in children: a -year review of diagnosis and management lemierre syndrome: two cases and a review an evidence-based review of the treatment of peritonsillar abscess medical and surgical treatment of peritonsillar, retropharyngeal, and parapharyngeal abscesses mosher award thesis. peritonsillar abscess: incidence, current management practices, and a proposal for treatment guidelines an evidence-based review of peritonsillar abscess pediatric peritonsillar abscess: an overview effectiveness of adenotonsillectomy in pfapa syndrome: a randomized study a randomized, controlled trial of tonsillectomy in periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome nonsteroidal anti-infl ammatory drugs and perioperative bleeding in paediatric tonsillectomy postoperative hemorrhage with nonsteroidal anti-infl ammatory drug use after tonsillectomy: a meta-analysis intraoperative ketorolac and posttonsillectomy bleeding tonsillectomy for recurrent sore throats in children: indications, outcomes, and effi cacy hemorrhage following tonsillectomy and adenoidectomy in , patients revisit rates and diagnoses following pediatric tonsillectomy in a large multistate population immediate tonsillectomy: indications for use as fi rst-line surgical management of peritonsillar abscess (quinsy) and parapharyngeal abscess key: cord- - zrnfaad authors: giese, matthias title: types of recombinant vaccines date: - - journal: introduction to molecular vaccinology doi: . / - - - - _ sha: doc_id: cord_uid: zrnfaad the original scientific strategy behind vaccinology has historically been to “isolate, inactivate, and inject,” first invoked by louis pasteur. new vaccines and vaccination strategies are being developed including the use of attenuated live mycobacteria, recombinant microorganisms, and subunits, prime-boost strategies based on the successive administration of a certain mycobacterial antigen under two different vaccine vectors, and dna vaccines [ ] . the various types of vaccines differ in eliciting an immune response. live attenuated vaccines (lavs) mimic a natural infection without being virulent and trigger the activation of the innate immune system through pamps. following injections, lavs rapidly disseminate throughout the vascular network to the draining lymph nodes. therefore, the route of application of lavs does not specifi cally infl uence the immune response. lavs also don't need an adjuvant; they possess a natural intrinsic adjuvancy. safety concerns exist because of the replication competence and the possibility of recombination with a wild type. non-live vaccines, inactivated and most recombinant vaccines, whether containing proteins or carbohydrates (−conjugates), are less effective. in the absence of replication, vaccine-induced immune reactions remain more limited, and therefore the route of vaccination infl uences the effi cacy and the duration of the immune reaction. nonlive vaccines induce a lower antibody response and generally no cytotoxic t lymphocyte activation. compared to lav, all non-live vaccines are regarded as biologically safe ( fig. . ). dna vaccines entail the direct, in situ inoculation of dnabased eukaryotic expression vectors that encode the sequence of a pathogenic protein antigen. the constructed plasmids are then subsequently grown in bacteria like e. coli and highly purifi ed via chromatographic methods. lps contamination of plasmids has to be prevented because of the immunotoxic properties of natural lps. after purifi cation the circular double-stranded dna plasmids are ready for vaccination. the de novo production of the encoded antigens in the host results in the elicitation of both the antibody and the cellular response by activating cytotoxic t lymphocytes (ctls). vaccine proteins made by the host are natural proteins and contain important posttranslational modifi cations such as the correct glycosylation. but like subunit vaccines, dna vaccines must be adjuvanted. naked dna does not work. the unique advantage of dna vaccines is their ability to mimic the effects of live attenuated vaccines without the risk associated with the administration of infectious albeit attenuated material. dna vaccines are able to stimulate a complete, humoral and cellular immune response. peptide fragments are processed via the endogenous pathway, resulting in the presentation of antigen on the cell surface by mhc class i molecules. plasmid dna is very stable also beyond a cold chain. therefore, the storage, transportation, and distribution of dna vaccines are more practical and also cheaper [ ] . mostly all plasmid dna constructs ( fig. . ) used for vaccination share fi ve main characteristics: • strong promoter/enhancer sequence for driving the incorporated foreign gene • convenient cloning site for insertion of foreign genes • origin of replication for initiation of plasmid replication • polyadenylation/termination sequence for production of mature mrna • resistance/antibiotic marker for selection • immunomodulators, e.g., cpgs, interleukins, ubiquitin, etc. • (on the same plasmid or on extra plasmids) uptake of plasmid dna. some biological barriers have to be overcome by dna vaccines on the way to the cell nucleus where the plasmid dna is translated into cellular mrna. after delivery of plasmid dna to the target tissue, e.g., skeletal muscle or skin, lots of tissue nucleases attack . dna vaccines and degrade a large amount of the applicated dna. also the extracellular matrix with collagen and hyaluronic acid infl uences the passage from the application site to the cell membrane. only a small portion ( % estimated) of the still intact plasmid dna will cross the cell membrane by phagocytosis or pinocytosis. inside the cell the route toward the nucleus is also spiked with exo-and endonucleases so that probably only . % (estimated) is successfully and actively transported through the nucleus pore membrane (npc). small particles (<~ kda) are able to pass through the nuclear pore complex (npc) by passive diffusion; larger particles need the support of carrier proteins for effi cient passage through the complex. because of this enormous loss of plasmid dna (up to . %), various tools were developed to protect the plasmid dna and thus increase the effi cacy such as encapsulation into liposomes or binding of dna to dendrimers. figure . illustrates the passage of plasmid dna from the extracellular matrix (ecm) to the nucleus. whereas in human medicine clinical trials with dna vaccines are still ongoing without any registered product on the market, the fi rst approved dna vaccines for the veterinarian medicine are available since and are discussed now. the fi rst veterinarian dna vaccines were developed for horses (davis b.s., for wnv [ ] ; giese m., for eav [ ] ). today the number of current clinical trials worldwide with veterinary dna vaccines is unmanageable and probably all species are hit. canine malignant melanoma (cmm) typically begins in the mouth or around the toes and can spread within the body to the heart, lungs, intestines, and other organs. canine malignant melanoma is known for being one of the most aggressive cancers in dogs and deadly. cmm is most commonly seen in golden retrievers, scottish terriers, dachshunds, labradors, and poodles ( fig. . ). metastases of the tumors will be found very often in distant parts of the body. the overall biology of cmm is similar to the biology of human melanoma. however the melanomas in dogs have diverse biologic behaviors due to the race and a variety of factors. standardized treatments such as surgery, radiation, and chemotherapy are the common tools to fi ght canine malignant melanoma. these traditional tools have afforded minimal to modest stage-dependent clinical benefi ts. xenogeneic dna vaccine. the plasmid dna contains a cdna for the human tyrosinase, hutyr, a tumor antigen (ta). this is a non-mutated differentiation antigen and specifi c to melanoma. tyrosinase is a glycoprotein and essential in the process of melanin synthesis ( fig. . ). like other tas tyrosinase is overexpressed in tumor cells and therefore an ideal target in cancer therapy. normally there is no strong immune reaction against the body's own protein. but immunization of dogs with xenogeneic hutyr cdna can break the immune tolerance against this self tumor differentiation antigen and induce antibody and cytotoxic t cell response against melanoma cells [ ] . tyrosinase is highly conserved from dog to mouse to man. radiotherapy in cases with positive surgical margins or positive regional lymph nodes. one dose contains μg dna given in a volume of . ml by the transdermal route via a needle-free vaccination device. booster immunizations were given at -month intervals. in march the drug manufacturer received a conditional license for oncept from the usda and a full license in . the results of the xenogeneic immunization of dogs with hutyr cdna as an adjunct therapy for cmm demonstrate a signifi cant increase of survival time compared to the control group. none of the dogs developed systemic adverse reactions; no toxicity was seen. the overall safety of this dna vaccine is confi rmed. this vaccine development represents a tremendous milestone in dna science and technology. virus. west nile virus (wnv) is a mosquito-borne member of the family flaviviridae , genus flavivirus , and was fi rst identifi ed in in uganda, africa. it is a positive-sense, single-strand rna virus, (+)ssrna, of about kb that encodes a single polyprotein with seven nonstructural proteins and three structural proteins. the rna strand is held within a nucleocapsid. wnv replicates in the cytoplasm of infected cells. wnv is a zoonotic virus. the primary reservoir is birds with a signifi cant impact to spread the infection across countries and continents. more than different species are described as carrier of this virus. wnv is spread from bird to bird by mosquitoes when they bite, or take a blood meal, from birds that are infected with the virus. birds from some species get ill and die; others have no clinical signs and survive. mosquitoes are also capable of spreading the virus to horses, dogs, cats, mice, alligators, and lots of other mammals but also to humans. one-third of all horses bitten by carrier mosquitoes develop the disease and die or are so affected that euthanasia is required. the incubation period ranges from to days. horses that do become ill vary in symptoms: muscle trembling, skin twitching, ataxia, sleepiness, dullness, and listlessness. wnv may cross the placenta from mother to gestating foal. horses cannot spread the disease to humans. wnv produces different outcomes in humans like in horses: fever, headache, chills, diaphoresis, weakness, swollen lymph nodes, drowsiness, and pain in the joints comparable to symptoms of infl uenza. more severe neuroinvasive infection includes meningitis and encephalitis. wnv-dna vaccine. the surface envelope protein e is the main target for the antibody response. there are more than copies of the e protein in a mature wnv virion. the e function is the interaction between the cell surface and the fusion between virus and cellular membrane. the premembrane protein prm is cleaved during viral maturation into a smaller membrane m peptide. the expression of prm and e protein in cells results in the formation of virus-like particles, vlp. these vlp share many of the antigenic and structural properties of fully mature viruses and are of special interest for a vaccine development ( fig. . ) . the fi nal expression plasmid for immunization of horses contains the human cytomegalovirus early gene promoter, signal sequences from japanese virus, and a fusion gene of part of the fi shing industry is aquaculture, also known as aqua farming, but it can be contrasted with commercial fi shing, which is the harvesting of wild fi sh. aquaculture involves cultivating freshwater and saltwater fi sh and other populations (shrimp, oyster) under controlled conditions. salmon is one of the main food-producing fi sh in the world. a dna vaccine for fi sh must be not only safe for the animal but especially safe for the fi sh consumer. salmon is the major economic contributor to the world production of farmed fi sh, representing over u$ billion annually in the united states. salmon farming is also very big in norway, scotland, canada, and chile and is the source for most salmon consumed in the united states and europe. like all other animals also fi sh is threatened by viruses, bacteria, and parasites. one major problem for salmons is the infectious hematopoietic necrosis (ihn) virus [ ] . virus. infectious hematopoietic necrosis (ihn) virus is a common viral pathogen of both wild and farmed salmonids, in particular pacifi c salmonids, rainbow trout, and atlantic salmon. ihn virus is enzootic to the pacifi c northwest; however it has varying effects on different pacifi c salmonids. it is a negative-sense single-stranded, (−)ssrna virus that is a member of the rhabdoviridae family, genus novirhabdovirus . the rna genome is , nucleotides long and contains a leader (l) and trailer (t) sequences at its ′-end and ′-end, respectively. the coding regions are n, p, m, g, nv, and l genes. g encodes the surface glycoprotein, so-called spikes, main target for the immune response. transmission. ihnv is transmitted following shedding of the virus in the feces, urine, sexual fl uids, and external mucus and by direct contact or close contact with surrounding contaminated water. the virus gains entry into fi sh at the base of the fi ns. salmons are carnivorous and are currently fed a meal produced from catching other wild fi sh and other marine organisms -a permanent origin of possible infections with ihnv. clinical signs of infection with ihnv include anemia, skin darkening, bulging of the eyes, fading of the gills, and abdominal distension. infected fi sh commonly hemorrhage in several areas, like the mouth, the pectoral fi ns, muscles near the anus, and the yolk sac of fry. diseased fi sh weaken, eventually fl oating on the surface of the water. necrosis is common in the kidney and spleen and sometimes in the liver. mortality rates in older fi sh ( - kg) tend to range from to %; in smolts the mortality rate often exceeds %. the average cumulative mortality following an outbreak is estimated at %. ihnv-dna vaccine. the antigen is the viral surface glycoprotein (g) capable of eliciting neutralizing antibody and the production of a protective immune response. the g gene was cloned into a eukaryotic expression vector by insertion of an intermediate-early promoter and a polyadenylation signal. but the speciality of this vaccine is to be prepared as a two-component vaccine in a single vaccine, one plasmid or more. the second component is a portion of the nucleic acid sequence encoding a second peptide, derived from a fi sh pathogen other than the said rhabdovirus resulting in a fusion. this second pathogen can be any fi sh pathogen, e.g., isav, ipnv, iridovirus, nnv, spdv, svcv, vhsv, koi herpes virus, and more. the rationale behind this is that the presence of the ihnv g protein boosts the immune response to the second protein, resulting in a protective effect against infection by this fi sh pathogen. the vaccine is given intramuscularly with a dosage of only μg in μl on the left dorsal fl ank, in the area just below the dorsal fi n [ , ] . this fi rst dna fi sh vaccine was licensed in in canada by the veterinary biologics section (vbs), animal health and production division, canadian food inspection agency (cfia) and is also used now in studies in norway. there are many environmental stressors and diseases which infl uence and seriously threat the life of european honey bees, apis mellifera. the european honey bee is professionally managed worldwide for honey production and pollination. the bee was imported to the united states years ago with the fi rst european settlers and called "white man's fl y" by the native americans, the indians. first reported in the united states, a mysterious socalled colony collapse disorder (ccd) decimated the bee colonies there between and %, fi rst observed during the winters of - and then - and without interruptions up to now. a similar situation is also given in europe. about , - , bees live in a colony. the fi rst description originated from the s. in the early nineteenth century, the colony losses were known in england as "isle of wight disease," and the americans called this phenomenon "disappearing disease" in the s, whereas these colony losses in france in the late s were called "mysterious bee losses." where have all the bees gone? economic value. the huge loss of honey bees as pollinators has a dramatic impact on agricultural pollination. about crops, nuts, fruits, and vegetables are pollinated by a. mellifera , with an overall value of more than $ billion in the united states and more than € billion for the eu in . a bee colony produces some kg/ lb honey per day. in return, these bees have to pollinate - million fl owers. one should keep in mind that besides european honey bees, wild insects, among them , species of wild bees, have also a very great impact on pollination and seem to be more effi cient in pollination as managed honey bees [ ] . the industrial farming threatens also the natural biotope of wild insect pollinators. ecologic value. the total global economic value of honey bee pollination was calculated in to more than € billion or $ billion. the food and agriculture organization (fao) of the united nations estimates that there are million managed honey bee colonies worldwide. beside this professional agriculture, honey bees are irreplaceable for the biodiversity. this organism appeared during evolution with the fi rst fl ower plants and exists since million years as described in chap. , fig. . . after swine and cattle, bees are in europe and north america the third important farm animal and since formally listed as farm animal in switzerland. therefore, ccd is not only an economical but also an essential global ecological problem which urgently must be solved in the future. "the bee is more than honey." what is causing ccd? the colony collapse disorder of the last years seems to differ from past outbreaks: the worker bees disappear instead of dying in place, leaving behind the queen and young bees. high levels of bacteria, viruses, and fungi are measured in the gut of the remaining bees. collapses can occur within days. a complex problem. different theories are discussed about what is causing ccd. pesticide contamination, hotly debated to interfere with the nerve system affecting foraging behavior of bees, lead them to abandon their hives. fungal diseases such as nosema spp. is known for big bee losses in spain. monocultures or gene-manipulated crops. electro smoke (radio waves) caused by cell phones destroys the bee's compass. the rigors of travelling in trucks from crop to crop in the usa. down from february professional us beekeepers travel with their colonies through the country until december. thereby the bees must relocate up to times. in europe the bee colonies begin the winter sleep around september. also the climate change, the temperature sensitivity is discussed to have an impact on crop pollination. ccd is likely caused by a combination of factors [ , ] . varroa destructor . but in all ccd cases, an overload of bloodsucking varroa mites is detectable and varroa is currently considered the major threat for apiculture. the infection and disease is called varroosis. varroa destructor is an ectoparasite, has a reddish-brown fl at shape, and is - . mm long and . - mm wide, with eight legs. v. destructor infest worker bees and drones and its brood. the mite develops inside the brood cells. varroa is a real colossus compared to the size of bees as can be seen in fig. . . varroa mites belong to the scientifi c class of arachnida, subclass acari. there are , species described alone from mites. some mites prefer carbohydrates as food such as meal or crops. the house dust mites feed fl akes of shed human skin. varroa mites prefer fresh "blood" and the hemolymph of bees and can feed . mg/ . lb within h. varroa is transported into the hives via piggyback by worker bees. the female mite enters broad cells, preferentially drone cells. once the cell is capped, varroa lays eggs on the larvae. the development from egg to insect takes days. bee larvae and mites hatch in about the same time and the newborn varroa mites spread to other bees [ , ] . the lifetime of summer mites are - weeks, whereas fall mites can live for several months. varroa can only reproduce in honey bees and thus are considered harmless to other insects. varroa is more than a disease. it is a global pest having devastating effects on bees ( fig. . ). varroa as vector. varroa may be not considered as isolated agent for the disease. the mortality of adult bees and its brood must be considered in the context with secondary viral infections. at least various viruses are able to infect honey bees, mostly ssrna viruses. eight viruses are known to be associated with varroa mites: acute bee paralysis virus (abpv), black queen cell virus (bqcv), chronic bee paralysis virus (cbpv), deformed wing virus (dwv), kashmir bee virus (kbv), sacbrood bee virus (sbv), cloudy wing virus (cwv), and slow bee paralysis virus (sbpv) [ - ] . varroa control. a number of natural and synthetic chemicals are commercially available for the control of varroa infestations. the fi rst compounds were bromopropylate, fl uvalinate, or other pyrethroid insecticides. and to make a long story short, varroa mites became resistant not only against one product of a given chemical class; the resistance was against the entire class with several related synthetic products. also the use of natural products, such as formic acid, mineral oil, or thymol, is only partially and temporally effective and show adverse effects [ ] . there is no successful chemical treatment. mites will quickly develop resistance to all chemicals. the immune system of insects. the basic difference between insect and vertebrate immunity is the missing highly specifi c antigen response of the acquired immune system in insects. nevertheless, in the million years of evolution, insects developed a powerful defense strategy against bacteria, fungi, viruses, and parasites. only protected by this "primitive" immune system insects were so successful that they colonized all terrestrial ecosystems. the insect innate immunity shows many similarities to the vertebrate and to the human innate immunity, is multifaceted, and involves both humoral and cellular components [ ] . most insights on insect immunity are provided by drosophila melanogaster research. the key mechanism is also observed in honey bees. the humoral and systemic response to bacterial and fungal infections is controlled by antimicrobial peptides (amps). there are circulating receptors sensing a danger signal and activating the toll pathway, whereas membranebound receptors activate the imd pathway. both pathways lead to the translocation of nf-kb-like transcription factors and the production of amps. nf-kb response elements can be detected in the promoter region of the diptericin gene. the cellular immune response is mediated by specialized blood cells, the hemocytes, plasmatocytes, crystal cells, and lamellocytes [ ] . plasmatocytes represent % of the majority of hemocytes. they express phagocytic receptors and patrol through the body, clear microorganism and cell a b c debris, and signal infections to the fat bodies. the bee genome was completely sequenced in [ ] . the bee dna vaccine. an expression plasmid was constructed with a cmv promoter. surprisingly, no bee or other insect specifi c promoter was essential to drive the expression of the protein. the enhanced green fl uorescent protein (egfp) was chosen as reporter gene and inserted into the multiple cloning site, together with an sv enhancer element. the plasmid construct was produced in e. coli and highly purifi ed by standard techniques. european honey bees ( apis mellifera ) were obtained from local beekeepers and cultivated under lab conditions. varroa mites were collected from infested bees. the oral vaccination of the egfp plasmid was operated by feeding the bees with a mixed solution of sugar and plasmid dna (vaccine sugar). standard sugar solutions made by the beekeeper are the normal food for winter bees. results. over days after onset of feeding, we measured the expression of egfp by immunofl uorescence and western blot analysis with egfp antibodies. between day and , a clear egfp signal was detected in the thorax and especially in the malpighian tubules. control bees fed with dna lacking the reporter did not show any signal. in parallel, control experiments with transformed e. coli were done to study the possibility of egfp expression in gut bacteria instead of bee cells. no egfp signal was detected in transformed bacteria. most surprisingly, we found the egfp signal after days in varroa mites sucking hemolymph of bees which were fed by the vaccine sugar solution and no signals in control mites of infested control bees. feeding of plasmid dna results in expression of a reporter gene in different bee tissues over a period of several days, and fi nally varroa absorbs this protein via bloodsucking. the bee blood is not carried by arteries and veins but fl ows loosely around the body. no egfp signals were detected either in the honey stomach or in the feces. figure . illustrates the egfp passage through the bee body and toward the varroa mite. we started with the simple idea that the biochemistry in eukaryotic cells remains the same, irrespective of the organism. a difference is given in the confi guration of the immune system. that means, an insect can successfully fi ght against parasites and infections but with different weapons. no t cells, no b cells, and consequently no antibodies and no memory. we are able to stimulate targeted immune genes of bees and measure an insect typical immune response. a standard plasmid dna vaccine, fi rst developed for horses, bridges the evolution from fi sh to insects to mammals. no other vaccine type is able to do this job. how fascinating biology is! a protein subunit is based on a single protein molecule and able to stimulate a humoral immune response, but usually not a cellular response. after phagocytosis proteins are degraded by acid-dependent proteases in endosomes (endosomal or exogenous pathway), resulting in an mhc ii presentation of the antigenic peptides. a peptide is one form of a subunit. carbohydrates are also used as subunits with a poor and age-dependent immunogenicity. carbohydrate antigens induce a t cell-independent b cell response as discussed in chap. . therefore carbohydrates are mainly linked to a protein (conjugation) to enhance toe immune reaction as discussed here with the hib conjugate vaccine. conjugate vaccines. the polyribosylribitol phosphate (prp) capsule of haemophilus infl uenzae type b (hib) is a major virulence factor for the organism. prp is a t cellindependent antigen characterized by, e.g., induction of a poor antibody response in less than -month-old infants and children and the inability to induce a booster response. polysaccharide vaccines based on prp alone were developed in the s. by covalent linkage of prp with t cell dependent protein antigens, a conjugated vaccine was created to overcome the t cell independent characteristics of prp. at present three different licensed protein carriers are linked to prp: • hboc: diphtheria crm protein , mutant corynebacterium -linkage: no spacer • hbomp: outer membrane protein, omp, neisseria meningitidis -linkage: spacer • prp-d: diphtheria toxoid, d -linkage: spacer these hib conjugate vaccines differ by protein carrier, polysaccharide size, and method of chemical conjugation, including use of a spacer between the prp and protein carrier. a standard chemical conjugation between a polysaccharide and a protein is illustrated in fig. . . subunit vaccines, while offering greater safety, are intrinsically poorly immunogenic and strong adjuvants are essential to boost the activation of immune responses. serotype variability is dictated by modifi cations of the o-antigen portion of lps. o antigens vary in the number of oligosaccharide unit repeats, the types and distribution of carbohydrates, and the intra-and intermolecular linkages [ ] . in s. fl exneri , these genes are encoded in the bacterial chromosome. in contrast, s. sonnei , which shows no serotype variability, expresses plasmid-encoded o-antigen modifi cation enzymes. the o antigen is one of the major immunogenic components of shigella and is a virulence factor, in part, due to masking the exposure of type three secretion apparatus [ ] . the inclusion of conserved proteins in vaccine compounds potentially solves the issue of serotype specifi city, thus allowing the generation of a highly desirable pan-shigella vaccine. in addition, recombinant proteins usually have increased safety profi les. another important impact in shigella epidemiology that prompts vaccine development is the increasing frequency of antibiotic-resistant strains. antibiotic resistance is continually rising for this pathogen [ ] . shigella spp. as causative agent of shigellosis. first defi ned as a causative agent of bacillary dysentery by shiga in japan, shigella is a gram-negative bacillus that is noncapsulated and nonmotile. diagnosis is generally based on symptoms [ ] since bloody, mucoid stools are indicative of shigella infections. however, because several diarrheal infections caused by other microorganisms share these symptoms (enteroinvasive e. coli and campylobacter , among others), the sole analysis of symptoms is insuffi cient for an accurate diagnosis. therefore, clinical diagnosis must be complemented with microbiological isolation from culture. shigella invasion and pathogenesis. shigella is transmitted through the fecal-oral route by consumption of contaminated food and water. following ingestion, the acid-tolerant shigella passes through the stomach and small intestine into the large intestine [ ] (fig. . ). here, they are taken up by m cells, transcytosed to the basolateral face of the colonic epithelium, and presented to resident macrophages wherein ipab of the type three secretion system (t ss) induces apoptosis by caspase activation, thereby escaping killing by the macrophage [ ] . shigella then invades epithelial cells using its t ss to create a translocation pore in the host cell membrane to initiate an orchestrated fl ow of effectors into the host cell cytoplasm to induce actin rearrangements that ultimately result in uptake of bacteria. once inside, shigella quickly escapes its vacuole, replicates, and moves about the cytoplasm via actin-based motility. in a t ssdependent manner, the shigella then forms a protrusion into a neighboring uninfected cell with the resulting vacuole being quickly lysed to complete the process of intercellular spread. the genes associated with the t ss are encoded on a -kb plasmid which is highly conserved among the shigella species. at the heart of the t ss is the type three secretion apparatus (t sa) which is composed of a basal body similar to that of fl agellar systems and an extracellular needle [ ] . invasion plasmid antigen d (ipad) is a kda protein that forms a pentameric ring at the tip of the needle. it controls secretion of effector proteins and is the environmental sensor for mobilization of ipab to the t sa tip complex. ipab is a kda translocator that forms a ring atop the ipad ring and is responsible for host cell contact. this contact is required for mobilization of ipac to the needle tip and formation of a complete unidirectional conduit from the bacterial cytoplasm to the host cell cytoplasm. the initiation of infl ammation and invasion processes occurs exclusively at the basolateral side of host cells, highlighting the importance of the previous steps of macrophage subversion in shigella colonization of the gut. animal models. shigellosis is strictly a human disease. while the basis of this restriction is unknown, it complicates the ability to investigate the pathogenesis of shigella . however, several animal models have been developed to study the pathogenesis of shigella , the resulting immune response against shigella antigens, and the protection efficacy of candidate vaccines against shigellosis: [ ] . nonhuman primate (nhp) models : nhp models have been used to defi ne the ability of vaccines to elicit immune responses and protection (rhesus and cynomolgus monkeys) [ ] . the main advantage of this model is that shigella is able to colonize the large intestine and generate symptoms that these bacteria generate in human infection. o antigen/proteosome : o antigen represents the variable portion of shigella lps ( fig. . ). administration of lps or o antigen alone in animal models is not enough to elicit immune responses, making them ineffective immunogens. to solve this limitation, these molecules have been used in conjunction with different proteins as carriers. several variants of lps/oantigen mixtures have been developed and characterized. one of these protein combination approaches uses s. fl exneri and s. sonnei lps complexed with neisseria meningitidis outer membrane protein proteosomes [ , ] . lps is extracted from s. fl exneri or s. sonnei by hot phenol extraction and mixed with detergent-extracted outer membrane proteins from n. meningitidis . the complex was then separated from free lps present in the mixture by gel fi ltration chromatography. the concept behind this vaccine is that the proteins present in the n. meningitidis proteosome are able to act as carriers for t cell stimulation, thus allowing the recognition of lps. shigella outer membrane vesicles. outer membrane vesicles (omvs) are particles composed of lps, proteins, and nucleic acids. in a proposed vaccine formulation, these particles were purifi ed from liquid cultures of s. boydii by centrifugation with subsequent fi ltering ( fig. . ). the precise identity and amount of the proteins included in this preparation is not currently known, although the presence of proteins having the same mass as ipab, ipac, and ipad suggests its composition includes these proteins. when these omvs are administered orally to mice, antibodies are generated against omv lysates. this vaccine has the advantage of heterologous protection (as shown by challenge against strains from each shigella serogroup) and the absence of adjuvant dependency. in addi-tion, immunity can be passively transferred to offspring, suggesting that the protective mechanism involves antibodies and raising the possibility that this vaccine can be used in infants, which is the main target population for a shigella vaccine. the use of live, fully virulent shigella during its formulation process, the presence of lps, and lot-to-lot consistency are possible downsides of this preparation. invaplex. another vaccine candidate that uses t ss proteins and lps as part of the formulation is the invaplex [ ] . these complexes are obtained by aqueous extraction followed by ion exchange chromatography (fig. . ). the precise composition of these extracts has not been completely characterized but includes lps, ipab, and ipac [ ] . these complexes are able a b d c fig. . depiction of lps/o-antigen-based vaccines. lps ( a ) extracted from shigella fl exneri or sonnei is admixed with protein preparations from n. meningitides and used as a carrier. when this complex is administered orally and intranasally to mice and guinea pigs [ ] , serum igg and mucosal iga in intestines and lungs are vaccine com-pound ( b ). o antigen purifi ed from lps is delivered in combination with exoprotein a from p. aeruginosa ( c ). finally, o antigen from different shigella serogroups is combined with ribosomes from shigella and is depicted in ( d ) to elicit igg and iga responses against ipa proteins as well as lps in both mice and guinea pigs. in addition, they are protective against the shigella species/serotype used for extract generation [ ] in the mouse and guinea pig challenge models. two phase one studies have been performed using the invaplex vaccine on adult volunteers [ ] and showed no major side effects to delivery of intranasal doses of up to μg. the highest dose employed in these studies generated an asc response to lps in % of the volunteers. an advantage of this approach is that, other than the invaplex itself, no additional adjuvants need to be administered. a drawback of this vaccine consists in a challenging production process that includes cultures of virulent shigella as well as the presence of bacterial lps products in the intermediate steps and fi nal formulation. another possible caveat is the uniformity of protein composition in these complexes through manufacturing lots. finally, this vaccine was not designed to protect against multiple serotypes. a solution for this possible drawback, however, is the generation of formulations that include invaplex complexes generated from more than one particular serotype, which increases an already diffi cult manufacturing process. this would allow the generation of vaccine formulations specifi c for the serotypes prevalent in a particular region. a vaccine candidate that targets conserved shigella virulence proteins includes some of the t ss ipa proteins (fig. . ) . recombinant ipab and ipad can be expressed in e. coli at high levels. ipad is then easily purifi ed from the e. coli cytosol while ipab must be purifi ed as a complex with its cognate chaperone ipgc. the chaperone is needed to maintain the hydrophobic ipab in a soluble state and to provide stability for ipab from proteolytic degradation. ipab can then be further purifi ed after separation from ipgc in low concentrations of detergent. analyses have indicated that ipab is greater than % pure following this scheme. in its fi nal formulation, this ipa-based vaccine also contains a double mutant of heat-labile enterotoxin from e. coli (dmlt) [ ] as an adjuvant. the mechanism of protection for this vaccine has not yet been worked out. nevertheless, it was tested in the mouse lethal pulmonary model [ ] where it exhibited over % homologous protection (against s. fl exneri ) and greater than % heterologous protection (using s. sonnei during the challenge experiments). igg and mucosal iga were generated after intranasal administration along with antigen-specifi c ifn-γ-secreting cells. ompa. a -kda outer membrane protein (omp) was purifi ed from s. fl exneri a using ion exchange chromatography. incubation of macrophages with this -kda protein induced the production of nitric oxide and increased production of il- and tnf-α. this protein was delivered parenterally fi ve times in rabbits, giving protection against challenge by s. fl exneri in the rabbit cecal ligation model [ ] . subsequent work using a recombinant protein purifi ed by affi nity chromatography identifi ed this -kda omp as ompa, part of a family of immunomodulating proteins present in numerous gram-negative bacteria. this protein showed high protective effi cacy in the mouse lethal pulmonary model [ ] where it elicited serum igg and mucosal iga. ticks are widely distributed throughout the world, affecting % of the world's cattle population [ ] . the economic importance of ticks and tick-borne diseases (tbds) has been estimated by a number of studies; however they most likely represent an underestimation of the real impact of these arthropod vectors and their transmitted diseases. tick feeding has devastating effects including disease transmission, paralysis, toxicosis, and secondary infections of the tick-feeding site [ ] . the effect of ticks and tickborne diseases is particularly pronounced in the livestock sector where it is repeatedly rated highly for its impact on the livelihood of farmers, particularly in countries of the south which are heavily dependent on agricultural production. there are six genera of ixodid ticks of importance, namely, amblyomma , dermacentor , haemaphysalis , hyalomma , rhipicephalus , and ixodes . historically, tick and tick-borne disease control has focused on the control of ticks at tolerable levels through acaricide use and treatment of disease with appropriate drugs. in some cases acaricide-based tick control is often the only method of reducing tick populations without sacrifi cing productivity [ ] . acaricides are commercially available in a number of formulations that are applied either directly onto livestock or in dipping vats where multiple animals can be passed through at regular time intervals. acaricide application relies heavily on correct formulation and administration to be effective. a large number of chemical compounds have been found to be effective against ticks including arsenic (introduced ~ ), ddt (~ ), cyclodienes and toxaphene (~ ), organophosphates-carbamate group (~ ), formamides (~ ), and macrocyclic lactones (~ ). the potency and usefulness of many of the abovementioned compounds is gradually eroding with resistance developing in many tick species of rhipicephalus , amblyomma , and hyalomma . multiple acaricide-resistant tick stocks have been identifi ed, limiting or entirely excluding the use of many acaricides [ ] . in addition to resistance, chemical control through the guiding principle for anti-tick vaccination stems from early studies conducted on acquired host resistance to tick infestations. repeated exposure of hosts to ticks or tick organ homogenates induced resistance to tick re-infestation. while the degree of resistance may vary between different tick and host species, evidence strongly suggests that natural resistance against tick infestation develops based on adaptive immune response mechanisms [ ] . ticks feeding from hosts vaccinated with tick components take up effector molecules during feeding that mediate deleterious effects on the ticks. this effect manifests as reduction of feeding time, tick mortality (during or after feeding), reduced engorgement weights, and reduced reproductive capacity of adult females. eggs laid from ticks fed on vaccinated hosts may also show reduced hatching rates. the overall result culminates in reduction of tick populations and tick-borne diseases. many of the anti-tick vaccine targets have been identifi ed using conventional immune-screening techniques. immunization of vertebrate hosts with tick homogenates or purifi ed tick extracts generates immune sera. these sera are used to screen for tick antigens detected by the host. the identifi cation of tick proteins essential for tick survival is a useful method for more targeted antigen discovery, which is made increasingly possible as information is gathered on tick biology. with the availability of genome sequences for a number of tick species, the number of candidates for discovery is expanding through reverse vaccinology. the use of other techniques such as rna interference (rnai) has been useful in confi rming the importance of antitick vaccine candidates and is likely to play a role in future anti-tick vaccine antigen discovery [ ] . exposed or concealed antigens. anti-tick vaccine candidates have been classifi ed into two categories: exposed or concealed antigens. exposed antigens are secreted in tick saliva during attachment and feeding on a host while concealed antigens are normally hidden from the host immune response. molecular mimicry by ticks of host components has been observed, and vaccination may induce host sensitivity and autoimmune reactions when exposed antigens are used [ ] . one advantage of using exposed antigens is that natural boosting occurs through tick feeding. mechanistically, vaccination with exposed antigens is thought to induce a focal hostile environment unsupportive for tick attachment and feeding. concealed antigens do not come into contact with the host immune response during natural tick feeding. although often contained within the thoracic cavity of the tick, some salivary gland proteins can be characterized as concealed if they are not secreted into the tick-feeding site. one diffi culty in the development of concealed anti-tick vaccines is that the antigen must be accessible to the induced humoral vaccine response. this often limits the number of candidates to those coming into prolonged and direct contact with the blood meal or where the humoral response can be transported over the gut barrier into the hemolymph [ - ] . the second limitation of concealed antigens relates to natural boosting of the immune response. as the antigens do not come into contact with the immune response within the host, suffi ciently high antibody levels must be induced through repeated vaccination. as the blood meal acts as the carrier for the effector immune responses, the anti-tick effect can take place over a longer period of time compared to exposed antigens. this effect may even extend beyond the mere feeding period into the inactive stages where digestion and molting/egg laying takes place. bm anti-tick vaccine. the bm -based anti-tick vaccine remains the only anti-tick vaccine commercially produced and has become the benchmark for future anti-tick vaccine development and evaluation. the gut-associated bm glycoprotein was fi rst identifi ed in r. microplus although homologues in other tick species have since been identifi ed [ - ] . the biological function of bm remains unknown although it is thought to play a role in the digestion of the blood meal [ ] . in r. microplus , expression of bm is increased during embryogenesis, reaching the highest level in unfed larvae. expression decreases during feeding and molting with lowest levels of expression detected during the resting stages of the tick. bm has a translated coding sequence of amino acids and a size of . kda. the protein contains four potential n-linked glycosylation sites and a leader peptide suggesting transport to the cell surface. localization studies have shown the molecule is located predominantly on the microvilli of gut epithelial cells. a single c-terminal transmembrane sequence is present in the unprocessed protein which is replaced by a glycosylphosphatidylinositol anchor in the mature protein. the protein also contains multiple predicted egf repeats rich in cysteine residues. vaccination has been performed mostly with the whole molecule, and protective epitopes for bm have not been well determined. the site of a protective b cell epitope was defi ned and additional epitopes are likely to exist. overlapping cross-reactive immune-reactive epitopes have been found between bm and the r. decoloratus homologue, bd . vaccine effi cacy is directly related to anti-bm antibody titer and the ability to control tick populations is directly related to achieving a strong antibody response. substantial animal-to-animal variation has been observed in the ability to generate anti-bm antibody titers which is likely related to the mhc class ii haplotypes expressed. antibodies to bm and cattle complement system are taken up during the blood meal. antibody binding results in lysis of the gut epithelial cells culminating in impaired blood meal digestion. strong antibody responses may induce tick mortality due to blood leakage from the gut into the hemolymph and ticks may turn reddish instead of gray. the development of the antibody response in cattle [ ] after immunization with rbm is demonstrated in fig. . . recombinant expression of bm has been attempted in several expression systems including escherichia coli , aspergillus nidulans , aspergillus niger , and pichia pastoris . vaccine trials showed that bm vaccination targeted mainly the adult stage of r. microplus , particularly the number of adult females fully engorging and post-engorgement mortality. reproductive capacity of adult r. microplus females was affected in terms of egg-laying capacity and hatching of eggs [ ] . under fi eld situations, vaccination of cattle reduced tick numbers by % within a single generation and reduced the reproductive capacity by %. reversal of negative effects of tick feeding on live weight of vaccinated animals by an average increase in live weight of . kg over a -month period was observed. extensive fi eld trials in cuba, brazil, argentina, and mexico showed between and % control of r. microplus ticks within a -week period [ ] . importantly, complete control of acaricide-resistant ticks could be accomplished by integrating bm vaccination with acaricide use [ ] , showing that integrated control systems are effective in controlling tick populations. vaccination also decreased the amount of acaricides required to control tick populations and prolonged the time interval between cattle dipping. bm vaccination has been extensively evaluated for its ability to control other tick species. almost complete cross-protection against rhipicephalus annulatus has been reported [ ] . signifi cant protection against hyalomma anatolicum , h. dromedarii , and r. decoloratus has been observed; however no cross-protection was seen against r. appendiculatus or amblyomma variegatum . genetically attenuated microorganisms, viruses and bacteria, can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system. some experimental vector systems are summarized in work with vectors classifi ed as bsl- does not require biosafety program approval. work with vectors classifi ed as bsl- or higher requires approval by the local biosafety committee. safety concerns. as demonstrated for adenovirus (ad ), following i.m. injection, the vector persisted mainly near the injection site and in draining lymph nodes for up to months. low levels of integration into chromosomal dna were observed, with a calculated mutation rate of × − mutations per cell. the spontaneous mutation rate of a cell is × − and therefore tenfold higher. ad is classifi ed as biosafety level (bsl- ). live vectors are able to stimulate the mucosal as well as a systemic humoral and cellular immunity. a severe drawback of the vector technology is that, once used, the vector cannot be effectively used in the patient again because it will be recognized by antibodies. repeated booster immunization will fail. also preexisting immunity in the patient against the vector could render the vaccination ineffective. a heterologous prime-boost and vector priming as described in chap. could circumvent this barrier. disease lactic acid bacteria have generally recognized as safe (gras) status and have been developed in the past decade as potent adjuvants for mucosal delivery of vaccine. a platform technology based on lactobacillus plantarum ( fig. . ) was developed to deliver antigens against plague disease caused by yersinia pestis , an aerobic, nonmotile, gram-negative bacillus belonging to the family enterobacteriaceae , which is transmitted to humans via fl eabite or via aerosol droplet, causing bubonic or pneumonic plague, respectively [ ] . most human plague cases present as one of three primary forms -bubonic, septicemic, or pneumonic. secondary plague septicemia, pneumonia, and meningitis are the most common complications. the pathogenicity of y. pestis results from its impressive ability to overcome the defenses of the mammalian host and to overwhelm it with massive growth. plague is enzootic in rodents in africa, asia, south america, and north america. y. pestis is transmitted from host to host by fl eas via blood feeding, through consumption or handling of infectious host tissues, or through inhalation of infectious materials. y. pestis infects an astonishingly broad range of mammals and uses rats, squirrels, mice, prairie dogs, marmots, or gerbils as reservoirs and several arthropod vectors for transmission [ ] (fig. . ) . humans acquire this zoonotic infection via an atypical bite from animal fl eas, sometimes prompted by an animal's death from plague, after which the fl ea seeks a new source of blood. most infected fl eas come from the domestic black rat rattus rattus or the brown sewer rat rattus norvegicus. y. pestis cells spread from the site of the infected fl eabite to the regional lymph nodes, grow to high numbers causing the formation of a bubo, and spill into the bloodstream where bacteria are removed in the liver and spleen. growth continues in these organs, spreads to others, and causes septicemia. fleas feeding on septicemic animals complete the infection cycle. in humans, bubonic plague can develop into an infection of the lung (secondary pneumonic plague) that can lead to aerosol transmission (primary pneumonic plague) [ ] . multiple antibiotic-resistant strains of y. pestis occur naturally and they can be easily bioengineered. thus, plague is a category a bioterrorism agent in need for novel strategies for its prevention. bubonic plague is the classic form of the disease. patients usually develop symptoms of fever, headache, chills, and swollen, extremely tender lymph nodes (buboes) within - days of contact with the organism either by fl eabite or by exposure of open wounds to infected materials. primary septicemic plague is generally defi ned as occurring in a patient with positive blood cultures but no palpable lymphadenopathy. patients are febrile, and most have chills, headache, malaise, and gastrointestinal disturbances. primary pneumonic plague is a rare but deadly form of the disease that is spread via respiratory droplets through close contact ( - ft) with an infected individual. it progresses rapidly from a febrile fl u-like illness to an overwhelming pneumonia with coughing and the production of bloody sputum. the incubation period for primary pneumonic plague is between and days. in general, patients who develop secondary plague pneumonia have a high fatality rate. fig. . schematic of the plague cycle with small mammals as hosts and fl eas as vectors. arrows represent connections affected by climate with a color coding depending on the most infl uential climate variable on this link (i.e., precipitation, temperatures, and other variables indirectly depending on them such as soil characteristics and soil moisture). grey rectangles somewhat arbitrarily delimit epizootic, enzootic, and zoonotic cycles. note that despite their location at the far end of the cycle, humans often provide the only available information on plague dynamics the laboratory diagnosis of plague is based on bacteriological and/or serological evidence [ ] . samples for analysis can include blood, bubo aspirates, sputum, cerebrospinal fl uid in patients with plague meningitis, and scrapings from skin lesions, if present. staining techniques such as the gram, giemsa, wright, or wayson stain can provide supportive but not presumptive or confi rmatory evidence of a plague infection. lactic acid bacteria (lab) are a group of gram-positive, nonpathogenic, non-sporulating bacteria that include species of lactobacillus (fig. . ) , lactococcus , leuconostoc , pediococcus , and streptococcus . they have limited biosynthetic abilities and require preformed amino acids, b vitamins, purines, pyrimidines, and a sugar as a carbon and energy source. these nutritional requirements restrict their habitats to those in which the required compounds are abundant. thus, these highly specialized bacteria occupy a range of niches including milk, plant surfaces, the oral cavity, the gastrointestinal tract, and the vagina of vertebrates [ ] . lab have been consumed for centuries by humans in fermented foods and have an extraordinary safety profi le. these intrinsic advantages turn lab into excellent delivery vectors of novel preventive and therapeutic molecules for humans. a number of studies of oral vaccines generated from genetically engineered pathogenic or commensal bacteria have been reported [ , ] . live attenuated pathogenic bacteria, such as derivatives of mycobacterium , salmonella , and bordetella spp., are the most popular live delivery vectors used currently. they are particularly well adapted to interact with mucosal surfaces as they have specialized machinery to initiate the infection process. the major disadvantages of live vaccines include inadequate attenuation and the potential to revert to virulence. lactic acid bacteria-based vaccines act as live attenuated vaccines but without the safety concern. lab have a generally recognized as safe (gras) status and thus are not likely to cause harm. the production of a desired antigen by lab can occur in three different cellular locations: • intracellular , which allows the protein to escape harsh external environmental conditions (such as gastric juices in the stomach) but requires cellular lysis for protein release and delivery • extracellular , which allows the release of the protein into the external medium, resulting in direct interaction with the environment (food product or the digestive tract) • cell wall anchored , which combines the advantages of the other two locations (i.e., interaction between the cell wall-anchored protein and the environment, in addition to protection from proteolytic degradation) in this context, several studies have compared the production of different antigens in lab, using all three locations, and evaluated the subsequent immunological impact. these studies demonstrated that the highest immune response was obtained with cell wall-anchored antigens exposed on the surface of lab. therefore, most of the recent lab vaccination studies have selected surface exposure of the antigen of interest, rather than intra-or extracellular production [ ] . dendritic cells (dcs) play a central role in bridging the innate immune system with the adaptive immune system. dcs are found throughout the body and are especially common at mucosal surfaces. with only a single layer of epithelial cells separating the external from the internal world amid the constant need for particle exchange, intestinal dendritic cells (dcs) play a key role in maintaining intestinal homeostasis as well as governing protective immune responses against invading pathogens. to avoid activation of selfreactive t cells and to limit unnecessary responses, such as those against commensal fl ora, dcs can imprint tolerance onto t cells (fig. . ). immature-type dcs are enriched underneath the epithelium of mucosal inductive sites and are poised to capture antigens. they extend protrusions between epithelial cells, enabling direct sampling of luminal antigens [ ] . through upregulation of mhc and co-stimulatory molecules, matured dcs convert into highly effi cient antigen-presenting cells. successful antigen presentation to cd + t cells requires recognition of cognate peptide in the context of mhc class ii molecules, whereas epitopes presented on mhc class i molecules stimulate ag-specifi c cd + t cells. when antigen uptake occurs, these dcs change their phenotype by expressing higher levels of co-stimulatory molecules and move to t cell areas of inductive sites for antigen presentation. thus, dcs and their derived cytokines play key roles in the induction of antigen-specifi c effector th cell responses. in this regard, targeting mucosal dcs is an effective strategy to induce mucosal and systemic immune responses. lab persistence. the ability of some lab to persist in the gastrointestinal tract may be critically important in the effectiveness of lab-based vaccines. a comparison of a persisting lab strain, l. plantarum , with a nonpersisting lab strain, l. lactis , identifi ed l. plantarum to be more effective at eliciting antigen-specifi c immunity, suggesting that persistence promoted immunogenicity [ ] . furthermore, it has been shown that particular lactobacillus species induced critical infl ammatory cytokines and induced activation and maturation of dendritic cells [ ] . it has also been shown that immature dcs effi ciently capture lactobacillus species, and these bacteria activated human dcs, resulting in the production of pro-infl ammatory cytokines like il- , increased proliferation of cd + and cd + cells, and skewed t cell response toward a th pathway believed to be involved in effective clearance of microbial pathogens [ , ] ( fig. . ). evidence suggests that the peptidoglycan layer of some lab promote natural immuno-adjuvanticity [ ] , and antigen localization on the cell wall makes it more accessible to the immune system as compared to intracellular or secreted proteins. leader peptides mark proteins for translocation across the cytoplasmic membrane, and lipid modifi cation is of major importance both for anchoring exported proteins to the membrane and for protein function [ ] . it has been shown that lipidation at the fi rst amino acid of the mature borrelia burgdorferi ospa protein is essential to induce an immune response via tlr [ ] . the leader peptide of ospa targets the protein to the cell envelope of lactobacillus and that the cys [ ] is recognized by the l. plantarum cell wall-sorting machinery that lipidates and anchors the protein to the cell envelope. the end result is a delivery system that exerts a potent adjuvant effect [ ] . virus-like particles (vlps) that mimic the antigenic architecture of authentic virions, however, can be produced in insect, mammalian, and plant cells by the expression of the capsid protein. the particulate nature and high-density presentation of viral structure proteins on their surface render vlps as a premier vaccine platform with superior safety, immunogenicity, and manufacturability. therefore, this chapter focuses on the development of effective nov vaccines based on vlps of capsid proteins. the expression and structure of nov vlps, especially vlps of norwalk virus, the prototype nov, are extensively discussed. the ability of nov vlps in stimulating a potent systemic and mucosal anti-nov immunity through oral and intranasal delivery in mice is presented. gastroenteritis (ge) is a worldwide health problem that affects people of all ages. as its name implies, ge is characterized by infl ammation of the gastrointestinal tract and often associated with symptoms of diarrhea, nausea, vomiting, and abdominal cramping and pain. ge and its associated diarrheal diseases remain as one of the top causes of death in the world especially in developing countries and in young children with an estimated death toll of four to six million per year [ ] . ge can be caused by a variety of pathogens including viruses, bacteria, and parasites and by ingestion of noninfectious toxins or medications, with viruses as the most common offending agents. norovirus (nov) and rotavirus are the most common viruses that cause viral ge, while adenovirus, astrovirus, coronavirus, and parechovirus are also known to cause ge in humans. novs are a group of genetically diverse rna viruses that belong to the genus of norovirus in the caliciviridae family [ ] . they were fi rst discovered and characterized in their prototype virus, the norwalk virus (nv), in [ ] . studies of nv revealed that novs are non-enveloped viruses with a rna genome surrounded by a round capsid protein shell approximately nm in diameter. novs are divided into genogroups and clusters with clusters in genogroup i (gi), in gii, in giii, and each in giv and gv. within the fi ve genotypes, gi and giv strains are found to infect humans exclusively and gii are found in both humans and pigs, while giii and gv strains are animal viruses that infect cattle and murine species, respectively [ ] . currently, strains in cluster of gii (gii ) are the most prevalent novs in human population [ ] . the genome of novs, which was fi rst characterized in nv, contains a single-stranded positive-sense rna of . - . kb with three open reading frames (orfs) and a poly a tail at its ′ end [ ] (fig. . ). orf encodes a polyprotein that is processed by viral protease clpro into the rnadependent rna polymerase and approximately fi ve other nonstructural proteins including p , the nucleoside triphosphatase, p , vpg, and clpro. the two structural proteins, the major (vp ) and minor (vp ) capsid proteins, are encoded by orf and orf , respectively [ ] . structural analysis of nov has revealed that each viral capsid is composed of dimers of vp in a t = icosahedral symmetry. vp folds into two domains: a shell (s) domain that is responsible for initiating capsid assembly and icosahedral contacts and a protruding domain (p), containing two subdomains of p and p , that enhance the stability of the capsid by providing intermolecular contacts between vp dimers. studies of nv also indicate that the vp protein enhances the expression level of vp and stabilizes the vp in the viral capsid. novs are highly contagious and spread rapidly, and their outbreaks commonly occur in various social places and settings where people share common food and water sources or are in close physical proximity, such as cruise ships, schools, military units, nursing homes, daycare centers, hospitals, restaurants, and catered events . the life cycle of nov has not been fully understood due to the lack of an in vitro cell culture system and a small animal model of infection. the failure of nov replication in mammalian cell cultures is not due to the lack of host factors to support intracellular expression of nov rna. instead, the problem may lie in the steps of viral binding to cellular receptors, virus entry into cells. nov [ ] . while serum antibodies to nov can be readily detected, this method has little clinical relevance due to the cross-reactivity of antibodies. since there is no culture system available for nov, the detection of virus in stool samples has become the preferred method of diagnosis. traditionally, nov infection was diagnosed by detecting the virus by immune transmission electron microscopy (tem). tem offers the advantage of direct visualization of any potentially responsible virus particles in stool samples. however, it does have the disadvantage of requiring sophisticated and expensive equipment and highly specialized technicians for its operation. several enzyme-linked immunosorbent assays (elisas) that detect nov antigens were later developed for nov diagnosis. studies have shown that nov antigen-detecting elisas have high specifi city ( - %) but poor sensitivity ( - %), most likely due to the antigenic diversity of nov strains [ ] . similar to elisa-based assays, rt-pcr is rapid and robust, because it can process large numbers of samples simultaneously and results can be obtained within a working day. however, it requires rna extraction from fecal samples and needs expensive equipment and skilled workers to operate. therefore, rt-pcr is more labor intensive and less economical than elisas. overall, tem, elisa, and rt-pcr-based methods all have their advantages and challenges. the three methods detect different components of the virus and therefore are complementary to each other. immunology of nov infection. the immunological knowledge of nov is mostly obtained from human challenge studies and natural outbreaks due to the lack of small animal models. observations of repeat infections in adults suggest the scarcity of long-term immunity against these viruses. however, other studies showed that close to % of the genetically susceptible subjects were not infected by nov challenge, which support the possibility of long-term immunity [ ] . nov. the lack of a tissue culture system also impedes the development of vaccines against nov. fortunately, the discovery of the spontaneous assembly of expressed vp into virus-like particles (vlps) that are morphologically and antigenically similar to the native viruses has facilitated vaccine development. vlps combine the best traits of wholevirus and subunit antigens for vaccine development. vlps are noninfectious, therefore, safer than inactivated or attenuated virus due to the lack of viral nucleic acid genome. importantly, vlps can induce potent cellular and humoral immune responses without adjuvants and are more effective vaccines than other subunit antigens because their architectures mimic infectious viruses. vlps can be produced by recombinant technology in heterologous expression systems without requiring the ability to support viral replication. this is particularly important for nov because no such culture system has been developed to support the growth of these viruses. studies have demonstrated that viruses and corresponding vlps have a particle size ideal for dc and macrophage uptake to initiate antigen processing [ ] . thus, the particulate nature of vlps favors their targeting to relevant apcs for optimal induction of t cell-mediated immune responses. vlps can also be presented effi ciently to b cells and induce strong antibody responses. like live viruses, the quasicrystalline surface of vlps, with its arrays of repetitive epitopes, presents a prime target that vertebrate b cells have evolved to specifi cally recognize. this recognition triggers the cross-linking of surface membrane-associated immunoglobulins (ig) on b cells and leads to their proliferation and migration, t helper-cell activation, antibody production and secretion, and the generation of memory b cells. thus, vlps can directly activate b cells at much lower concentrations than other subunit antigens and induce high titer and durable b cell responses in the absence of adjuvants. these inherent advantages of vlps have made them one of the most successful recombinant vaccine platforms. for example, fi ve vlp-based vaccines for hepatitis b virus characterization of nov vlps. vlps of novs were fi rst produced in insect cells using baculovirus vectors [ ] and then in plants using tobamovirus and geminivirus vectors and in mammalian cells using the venezuelan equine encephalitis (vee) replicon system [ ] . these studies demonstrated that expression of the major capsid protein vp alone can drive the self-assembly of vlps that morphologically and antigenically resemble native virus particles (fig. . ) . vlps generated by all three expression systems are similar to each other. the structure of nov vlps is exemplifi ed by the vlp of nv capsid protein (nvcp). studies of insect cellbaculovirus-derived nvcp vlps by cryo-electron microscopy and x-ray crystallography reveal that the nv capsid is a nm icosahedral arrangement of copies of the kda capsid protein vp organized into dimers in a t = symmetry. while all dimers are formed from two identical nvcp monomers, two different dimer confi gurations are required to correctly form the complete assembled capsid [ ] . as in native nv particles, the nvcp also folds into two distinctive domains in vlps, with s domain forming the inner core of the shell and p domain protruding out from the capsid [ ] . similarly, the p subdomain is also the most surface-exposed region in nv vlps and may contain hbga and neutralizing antibody-binding sites and determinants of strains specifi city [ , ] . the similarity between vlps of nv and other novs including gii. viruses has been demonstrated [ ] . insect cell-baculovirus vector-produced nvcp vlps. it was shown that four oral doses of as little as μg nvcp vlps without any adjuvant triggered serum nv-specifi c anti-igg response in the majority ( / ) of vlpfed icd outbred mice [ ] . systemic igg response was observed after two oral dosages and the highest titer was induced by four doses of μg vlps. moreover, mice in the μg dosage group developed nv-specifi c intestinal iga in a level up to . % of total iga. inclusion of the mucosal adjuvant cholera toxin (ct) did not signifi cantly change the number of positive responders of serum igg or intestinal iga but signifi cantly enhanced the amplitude of serum igg response, especially for higher doses of vlps. thus, nvcp vlp is clearly a potent oral immunogen and can induce both systemic and gut mucosal antibody responses. nanovaccines: gas infections group a streptococcus ( streptococcus pyogenes) (gas) is an important human mucosal pathogen that is responsible for a wide spectrum of diseases with varying clinical manifestations and severity [ , ] : pharyngitis (strep throat) is a common minor complication of gas infection but when left untreated can lead to life-threatening diseases including the autoimmune sequelae rheumatic fever (rf) and rheumatic heart disease (rhd). rhd results in permanent damage to the heart tissues and valves. gas infections cause > , deaths each year mostly in developing countries and indigenous populations within developed nations where poor socioeconomic conditions and overcrowding contribute to the high rates of gas diseases. in developing countries, rf is the leading cause of heart disease among children [ ] . there is currently no available vaccine to prevent infection with gas and consequently prevent gas diseases. a successful mucosal gas vaccine would need to stimulate the appropriate humoral and cellular immunity for protection against gas infection ( fig. . ). this is especially diffi cult due to a lack of human-compatible mucosal vaccine adjuvants that are essential to boost immune responses. researchers have therefore focused mainly on parenteral gas vaccine delivery approaches, for which suitable adjuvants are available, designed to provide protection against systemic infection via the induction of opsonic igg antibodies. antigen. an effective gas vaccine needs to have broad antigenic reach because of the many different gas strains (> different m types) circulating in a population fig. . gas vaccination approaches. gas that breaches the physical barrier of the mucosal epithelium of the nasal-associated lymphoid tissue, functionally analogous to human tonsils, is purported to be transported to the underlying lymphoid tissue via association with membranous (m) cells. cells of the innate immune system sense gas and produce cytokines and chemokines to contain the infection to the mucosa. gas antigens are delivered to antigen-presenting cells such as dcs and b cells. iga-committed b cells are activated and initiate antigen-specifi c iga responses. dcs play a fundamental role in the development of immunity to gas and present antigen to t cells to induce a th response that is integral along with iga for mucosal defense against pharyngeal gas colonization. mucosal vaccination is designed to mimic these responses and effectively clear gas from the mucosal surface upon infection to prevent gas colonization and carriage. gas that escapes the host's defense mechanisms can disseminate into the lymphatics and blood, leading to systemic infection. mucosal vaccination is also able to induce a systemic immune response characterized by the induction of opsonic igg antibodies, which destroy the pathogen by opsonophagocytosis. parenteral gas vaccination induces serum igg but is not able to induce mucosal immunity and should not induce immune responses that are potentially cross-reactive with self tissue proteins. the gas m protein is the major protective antigen and an ideal target for vaccine development; however it contains heart tissue cross-reactive epitopes particularly in the conserved region [ ] . evidence suggests that cross-reactive t cells especially play a pivotal role in the pathogenesis of rhd. the m protein is an α-helical coiled-coil surface protein consisting of a hypervariable amino-terminal region and a highly conserved (> % sequence identity) carboxyterminal c-repeat region [ ] (fig. . ) . functionally, the m protein is important in preventing bacterial clearance by complement-mediated phagocytosis, which limits host defense mechanisms. previous studies indicate that protective immunity to gas can be evoked by opsonic antibodies to serotypic epitopes at the amino-terminal region that are m-type specifi c [ ] . nanovaccine. combined vaccine/adjuvant delivery systems offer the potential of mucosal vaccine delivery. for example, the lipid-core-peptide (lcp) system is a novel, synthetic, selfadjuvanted vaccine delivery system that incorporates the adjuvant (prr agonist), carrier, and antigenic peptides of a vaccine into a single molecular entity ( fig. . ). this system has been previously shown to effi ciently deliver gas vaccines and induce immunity [ ] . evidence suggests the adjuvant activity of lcp involves the induction of dc activation. preclinical developments. three approaches are currently being investigated in the development of a subunit gas vaccine based on the m protein: . multivalent gas vaccine . a multivalent approach employs a combination of amino-terminal protein fragments representing different m types and is designed to target prevalent gas strains in a population. using this approach, a recombinant multivalent gas vaccine containing m protein peptides from different gas serotypes prevalent in north america was demonstrated to evoke opsonic antibodies in animals [ ] . from epidemiological data, the -valent vaccine would cover the majority of pharyngitis and invasive gas diseases, including rf, invasive fasciitis, and toxic shock syndrome. recently, a new -valent gas vaccine was shown to be immunogenic in rabbits and evoked opsonic antibodies against "non-vaccine" serotypes [ ] potentially creating a vaccine with much broader coverage. this type of vaccine is population specifi c and therefore may not be effective universally. it may also need to be re-designed periodically to refl ect changes in the epidemiology of gas infections. (ch ) (ch ) ch ch ch fig. . the lipid-core-peptide (lcp) system. the lcp system contains an adjuvant component (lipid-core made of lipoamino acids) and a polylysine carrier onto which peptide epitopes are attached. the example shows a gas vaccine candidate containing j and an amino-terminal serotypic epitope called . the adjuvant has three -amino-dodecanoic ( n = ) lipoamino acids separated by glycine amino acid spacers . j vaccine . a gas vaccine that employs peptide epitopes from the conserved c-repeat region of the m protein is the second approach and has the potential in theory for greater coverage of m types. immunization of mice with a c-region peptide gas vaccine candidate called j conjugated to the carrier protein diphtheria toxoid (dt) and co-delivery with an appropriate adjuvant led to protection against systemic and mucosal gas infection [ ] (fig. . ). j also elicited protective immunity against gas when linked to lipopeptides [ , ] . other studies have shown that intranasal immunization of mice with c-region peptides conjugated to the experimental mucosal adjuvant cholera toxin b subunit (ctb) evoked protective immunity against gas at the mucosal level [ ] . ctb could possibly enter olfactory regions of the central nervous system and cause neuronal damage following intranasal delivery [ ] and therefore is not suitable for human use. vector delivery approaches have included expressing the c-repeat region on vaccinia virus [ ] , the commensal bacterium lactococcus lactis [ ] , or streptococcus gordonii [ ] . . j . the third combination vaccine approach uses both serotypic and conserved m protein peptide epitopes. initially, a heteropolymer gas vaccine construct was synthesized by free radical-induced polymerization of acryloyl peptides to combine seven serotypic epitopes and a highly conserved c-region peptide epitope called j [ ] . the m types that were targeted in the heteropolymer represented gas infections prevalent in the northern territory of australia -a region highly endemic for gas. immunization of mice with the heteropolymer demonstrated excellent immunogenicity and protection fig. . preclinical evaluation of gas vaccine candidates. intranasal immunization of mice with the j -dt vaccine candidate led to significantly greater survival after intranasal challenge with gas versus control groups but the mucosal adjuvant ctb was essential for protection ( a ). a multiepitope lcp-based gas vaccine candidate elicited protective immunity against mucosal gas infection even in the absence of ctb ( b ) against homologous and heterologous gas strains, indicating its potential to provide broad coverage. however, batch-to-batch variation led to altered immune responses, which limited its applicability for human use. the vaccine also required the addition of an adjuvant to be effective, further limiting its use as a mucosal vaccine due to a lack of safe and effective mucosal adjuvants. later, multiepitope gas vaccine candidates were synthesized based on the lcp system that induced highly opsonic antibodies following parenteral delivery to mice [ ] , as well as protection against mucosal gas infection following intranasal immunization [ ] (fig. . ). safety and effi cacy. the main concern when using large regions of the m protein in a gas vaccine is the potential for inducing an autoimmune response due to immunological cross-reactivity with host proteins. it is therefore important to identify protective antigenic determinants and to separate the biological relevant epitopes from those that are host tissue cross-reactive and potentially harmful. epitope mapping studies were used to identify the conserved gas vaccine candidate j , which contains a conformational protective b cell epitope and was designed to lack a human heart cross-reactive t cell epitope [ , ] . tb vaccines -state of the art and progresses dna-antiviral vaccines: new developments and approaches -a review west nile virus recombinant dna vaccine 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ed invasin complex vaccine from shigella fl exneri a development and evaluation of a shigella fl exneri a and s. sonnei bivalent invasin complex (invaplex) vaccine safety and immunogenicity of an intranasal shigella fl exneri a invaplex vaccine characterization of a mutant escherichia coli heat-labile toxin, lt(r g/l a), as a safe and effective oral adjuvant broadly protective shigella vaccine based on type iii secretion apparatus proteins purifi cation and characterization of an immunogenic outer membrane protein of shigella fl exneri a outer membrane protein a (ompa) of shigella fl exneri a, induces protective immune response in a mouse model global aspects of the management and control of ticks of veterinary importance the global importance of ticks chemical control of ticks on cattle and the resistance of these parasites to acaricides factors that infl uence the prevalence of acaricide resistance and tick-borne diseases the molecular revolution in the development of vaccines against ectoparasites rna interference for the study and genetic manipulation of ticks proteins in the saliva of the ixodida (ticks): pharmacological features and biological signifi cance comparison of the proteins in salivary glands, saliva and haemolymph of rhipicephalus appendiculatus female ticks during feeding immunoglobulin-binding proteins in ticks: new target for vaccine development against a blood-feeding parasite amblyomma americanum : specifi c uptake of immunoglobulins into tick hemolymph during feeding synthetic vaccine (sbm ) against the cattle tick rhipicephalus (boophilus) microplus : preservation of immunogenic determinants in different strains from south america cloning, expression and immunoprotective effi cacy of rhaa , the homologue of the bm tick vaccine antigen, from hyalomma anatolicum anatolicum differential transcription of two highly divergent gut-expressed bm antigen gene homologues in the tick rhipicephalus appendiculatus (acari: ixodida) cloning and expression of a protective antigen from the cattle tick boophilus microplus bovine immunoprotection against rhipicephalus (boophilus) microplus with recombinant bm -campo grande antigen tick vaccines and the transmission of tick-borne pathogens vaccination against ticks (boophilus spp.): the experience with the bm -based vaccine gavac immunity against boophilus annulatus induced by the bm (tick-gard) vaccine developing live vaccines against plague yersinia pestis--etiologic agent of plague yersinia: strategies that thwart immune defenses plague . in: crc handbook series in zoonoses . section a. bacterial, rickettsial, chlamydial and mycotic diseases mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria the induction of hiv gag-specifi c cd + t cells in the spleen and gutassociated lymphoid tissue by parenteral or mucosal immunization with recombinant listeria monocytogenes hiv gag mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on lactobacillus casei induces neutralizing antibodies in mice lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and dna vaccines dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria protection against tetanus toxin after intragastric administration of two recombinant lactic acid bacteria: impact of strain viability and in vivo persistence lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells lactobacilli as natural enhancer of cellular immune response lactobacilli activate human dendritic cells that skew t cells toward t helper polarization instruments for oral disease-intervention strategies: recombinant lactobacillus casei expressing tetanus toxin fragment c for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. microbiol treponema pallidum and borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a cd -dependent pathway distinct from that used by lipopolysaccharide immune response to lactobacillus plantarum expressing borrelia burgdorferi ospa is modulated by the lipid modifi cation of the antigen a review of viral gastroenteritis noroviruses: a comprehensive review biological properties of norwalk agent of acute infectious nonbacterial gastroenteritis norovirus classifi cation and proposed strain nomenclature mechanisms of gii. norovirus persistence in human populations sequence and genomic organization of norwalk virus norwalk virus genome cloning and characterization methods for the detection and characterisation of noroviruses associated with outbreaks of gastroenteritis: outbreaks occurring in the north-west of england during two norovirus seasons european multicenter evaluation of commercial enzyme immunoassays human susceptibility and resistance to norwalk virus infection t cellindependent type i antibody response against b cell epitopes expressed repetitively on recombinant virus particles threedimensional structure of baculovirus-expressed norwalk virus capsids expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons conformational stability and disassembly of norwalk virus like particles: effect of ph and temperature structural studies of recombinant norwalk capsids e. coliexpressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability c-terminal arginine cluster is essential for receptor binding of norovirus capsid protein structural basis for the recognition of blood group trisaccharides by norovirus pathogenesis of group a streptococcal infections group: a streptococcal infections and acute rheumatic fever multiple cross reactive epitopes of streptococcal m proteins streptococcal m protein: molecular design and biological behavior localization of protective epitopes of the amino terminus of type streptococcal m protein towards the development of a broadly protective group a streptococcal vaccine based on the lipid-core peptide system immunogenicity of a -valent group a streptococcal vaccine new -valent m protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group a streptococci protection against group a streptococcus by immunization with j -diptheria toxoid: contribution of j -and diphtheria toxoid-specifi c antibodies to protection intranasal vaccination with a lipopeptide containing a minimal, conformationally constrained conserved peptide, a universal t-cell epitope and a self-adjuvanting lipid protects mice from streptococcus pyogenes and reduces throat carriage immunisation of mice with a lipid core peptide construct containing a conserved region determinant of group a streptococcal m protein elicits heterologous opsonic antibodies in the absence of adjuvant epitopes of group a streptococcal m protein that evoke cross-protective local immune responses cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues protection against streptococcal pharyngeal colonization with a vaccinia: m protein recombinant mucosal vaccine made from live, recombinant lactococcus lactis protects mice against pharyngeal infection with streptococcus pyogenes clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a streptococcus gordonii carrier strain intended for future use as a group a streptococcus vaccine new multi-determinant strategy for a group a streptococcal vaccine designed for the australian aboriginal population immunization with a tetraepitopic lipid core peptide vaccine construct induces broadly protective immune responses against group a streptococcus intranasal administration is an effective mucosal vaccine delivery route for self-adjuvanting lipid core peptides targeting the group a streptococcal m protein mapping a conserved conformational epitope from the m protein of group a streptococci mapping the minimal murine t cell and b cell epitopes within a peptide vaccine candidate from the conserved region of the m protein of group a streptococcus key: cord- -vjzfzshh authors: pereira-gómez, marianoel; sanjuán, rafael title: effect of mismatch repair on the mutation rate of bacteriophage ϕx date: - - journal: virus evol doi: . /ve/vev sha: doc_id: cord_uid: vjzfzshh viral mutation rates vary widely in nature, yet the mechanistic and evolutionary determinants of this variability remain unclear. small dna viruses mutate orders of magnitude faster than their hosts despite using host-encoded polymerases for replication, which suggests these viruses may avoid post-replicative repair. supporting this, the genome of bacteriophage ϕx is completely devoid of gatc sequence motifs, which are required for methyl-directed mismatch repair in escherichia coli. here, we show that restoration of the randomly expected number of gatc sites leads to an eightfold reduction in the rate of spontaneous mutation of the phage, without severely impairing its replicative capacity over the short term. however, the efficacy of mismatch repair in the presence of gatc sites is limited by inefficient methylation of the viral dna. therefore, both gatc avoidance and dna under-methylation elevate the mutation rate of the phage relative to that of the host. we also found that the effects of gatc sites on the phage mutation rate vary extensively depending on their specific location within the phage genome. finally, the mutation rate reduction afforded by gatc sites is fully reverted under stress conditions, which up-regulate repair pathways and expression of error-prone host polymerases such as heat and treatment with the base analog -fluorouracil, suggesting that access to repair renders the phage sensitive to stress-induced mutagenesis. mutation is the ultimate source of genetic variation and, therefore, a central evolutionary process. although mutations are required for adaptation, the short-term deleteriousness of most spontaneous mutations should generally favor low mutation rates (sniegowski et al. ) . in theory, the balance between these short-term costs and the long-term benefits for adaptation should produce an evolutionarily optimal, intermediate mutation rate which is dependent on selection strength (orr ; johnson and barton ) . other factors can also determine mutation rate evolution, including the costs of maintaining mechanisms of replication fidelity, population size, and structure, or the topology of the fitness landscape among others (andré and godelle ; clune et al. ; jiang et al. ; lynch ; sung et al. ) . despite this variety of factors, it has been noted that genomic mutation rates stay remarkably constant among dna viruses, bacteria, and unicellular eukaryotes (drake ; drake et al. ) . as a consequence, pernucleotide rates vary by , -fold and inversely with genome size, from - to - mutations per nucleotide per round of copying (m/n/r) (lynch ) . how evolutionary forces have shaped this inverse relationship in such widely different microbial systems and which molecular mechanisms allow for this mutation rate variation remain poorly understood questions. for dna viruses, mutation rates range from - m/n/r in double-stranded (ds) dna viruses such as herpes virus to - m/n/r in single-stranded (ss) dna viruses such as bacteriophage /x , whereas these rates range from - to - m/n/r in rna viruses (sanjuá n et al. ) . a primary determinant of viral mutation rates is replication fidelity, and polymerase variants with altered base-selection specificities have been described in several rna viruses including picornaviruses, alphaviruses, and retroviruses. however, fidelity variants that are not lethal typically alter mutation rates only slightly (pfeiffer and kirkegaard ; arias et al. ; mené ndez-arias ; coffey et al. ; graci et al. ) . the presence of exonuclease proofreading has a stronger effect on viral replication fidelity. all rnadependent polymerases except those of coronaviruses lack ' exonuclease activity, as opposed to virus-encoded dna polymerases, therefore providing a clear basis for the higher mutation rates of rna viruses compared with dna viruses (roberts, bebenek, and kunkel ; steinhauer, domingo, and holland ; mené ndez-arias ; denison et al. ; smith et al. ). in addition to polymerase fidelity, in dna viruses, mutation rates should be determined by their ability to access postreplicative repair. in ssdna bacteriophages such as /x or m , replication is carried out by the escherichia coli dna iii holoenzyme, which exhibits similar fidelity in phage and host templates (fersht ; fersht and knill-jones ) . however, these phages show a mutation rate approximately three orders of magnitude higher than the host (wickner and hurwitz ; raney, delongchamp, and valentine ; cuevas, duffy, and sanjuá n ). the high variability and fast evolution of small eukaryotic dna viruses such as parvoviruses and polyomaviruses similarly suggests elevated rates of spontaneous mutation (duffy, shackelton, and holmes ) . the efficiency of post-replicative repair can be higher than per cent (fijalkowska, schaaper, and jonczyk ) . in e. coli, strand-specific bidirectional methyl-directed mismatch repair (mmr) is performed by the dam/muthls system (jiricny ) . point mutations or small insertion/deletion loops are recognized by muts, which interacts with mutl, leading to activation of the muth endonuclease. the latter recognizes the parental strand by the presence of a methyl group in the adenosine of a gatc sequence motif located on either side of the mismatch, which has been previously added by dam methylase. muth then cleaves the non-methylated daughter strand, which is degraded and re-synthesized (modrich and lahue ; marti, kunz, and fleck ; schofield and hsieh ; li ; fukui ) . strikingly, though, the . kb genome of bacteriophage /x contains no gatc sites, whereas the randomly expected number of such sequences given the /x genome size and base composition is approximately . by impeding dam methylation, the lack of gatc sites therefore avoids a major repair pathway. however, the impact of gatc motifs on the phage mutation rate is still poorly understood. in a previous study (cuevas, pereira-gomez, and sanjuá n ) , we introduced four gatc sequence motifs in the /x genome and found no effects on mutation rate. however, by increasing the number of gatc motifs to seven, we obtained a thirtyfold reduction in mutation rate. furthermore, this effect was reverted in mmrdeficient mutd cells, indicating that the effects of gatc motifs were related to mmr. here, to better explore how mmr avoidance determines the mutation rate of /x , we constructed a /x variant encoding twenty randomly located gatcs with minimal effects on protein sequence. the engineered phage showed an eightfold reduction in spontaneous mutation rate compared with the wild type (wt), yet no obvious growth defects under standard conditions. however, the efficacy of gatc-driven mmr was curtailed by poor methylation of the phage dna, preventing recognition of the parental strand. furthermore, after constructing several mutants in which the number and location of gatc sites were varied, we found that their effects on mutation rate were non-additive and highly variable, with some combinations achieving an up to fiftyfold reduction in mutation rate while others having no effects. the highest efficiencies were shown by some intergenic gatcs, suggesting that steric constrains such as availability of the dna to muth may be important for mmr. finally, we found that the mutation rate reduction afforded by the twenty gatc motifs was fully reverted at c and in the presence of the base analog -fluorouracil ( -fu), two stress factors that promote overexpression of repair-associated error prone polymerases (layton and foster ; malkova and haber ) , thus suggesting that addition of gatc motifs renders the phage sensitive to stress-induced mutagenesis. the e. coli c strain ij was obtained from prof. james j. bull. the gro mutant was provided by prof. bentley a. fane (university of arizona). bacteriophage /x originally obtained from prof. james j. bull (texas university) was adapted to our laboratory conditions by long-term passaging in ij cells (domingo-calap, cuevas, and sanjuá n ) . gatc sites were engineered in the genetic background of this adapted virus, here denoted the wt, which contains no gatcs (genbank accession gq ). the /x dsdna replicative form was purified from infected cultures before lysis using a standard miniprep kit (macherey-nagel), and pg of this dna were used as template for polymerase chain reaction-based mutagenesis using phusion high-fidelity dna polymerase (thermo scientific) and contiguous, divergent, '-phosphorylated primers, of which the reverse primer carried the desired nucleotide substitution. polymerase chain reaction products were circularized with the rapid dna ligation kit (thermo scientific) and used for transfecting competent ij cells by the classical heat-shock method. a single plaque was picked, resuspended lysogeny broth medium, and stored at - c. the presence of each substitution was confirmed by sanger sequencing. this process was iterated until twenty gatc sites were introduced. full-length sequencing of the gatc virus was performed to verify that all mutations were present and that no other changes were introduced. each test consisted of twenty-four independent . ml ij cultures inoculated with the indicated initial number (n ) of plaque forming units (pfu) and incubated in a thermomix shaker (eppendorf) at rpm until n pfu were produced. this growth phase was done under standard conditions ( c), at high temperature ( c), or in the presence of ng/ml -fu ( c) by preincubating cells with -fu min before infection. all titrations were done under the same, standard conditions (ij cells with agar overlay, c, no -fu). n was determined by titrating six of twenty-four random cultures. to score mutants, . ml ( % of the total volume) was titrated on the restrictive e. coli gro strain, a rep mutant where only /x mutants with certain mutations in the n-terminal end of the viral protein a can form plaques (ekechukwu, oberste, and fane ) , the total number of different substitutions leading to the resistance phenotype being t ¼ under our assay conditions (cuevas, duffy, and sanjuá n ) . we estimated the rate m at which gro -resistant mutants appeared using the null-class method, which is based on counting the proportion of cultures showing zero versus at least one mutant. the number of mutations per culture should follow a poisson distribution with parameter k ¼ m(n -n ), such that the expected probability of no mutants in a culture is p ¼ exp[-m(n -n )]. mutation rates per nucleotide per round of copying (m/n/r) were then calculated as m ¼ m/t, where the factor stands for the fact that each base can mutate to three different bases. three independent tests were performed for each mutant, except for the wt, for which fifteen tests were performed. mutation rate estimates for the wt in the presence of -fu ng/ml were taken from a previous work (pereira-gó mez and sanjuá n ). for fluctuation tests performed under stress conditions, we applied a correction for bias in n estimation which may result from increased viral degradation relative to standard conditions. following previous work (bradwell et al. ) , the probability of observing no mutants was recalculated accordingly as where ź quantifies this bias. as an indicator of ź , we determined the relative plating efficiency of the wt virus under the two stress conditions. plating efficiency was slightly increased at c (ź ¼ . . ) and reduced in the presence of ng/ml -fu (ź ¼ . . ). we therefore used the corresponding ź values for calculating mutation rates at c and in the presence of -fu. since the relative plating efficiencies of the gatc virus did not differ significantly from those of the wt (t-test: p > . ), we used the same ź values. q-q plots showed that mutation rate estimates were not normally distributed, whereas normality was satisfied using log-transformed rates. all statistical tests were thus performed using log-transformed rates. the viral exponential growth rate was estimated as r ¼ ln(n / n )/t, where n and n were obtained from the fluctuation test assays, and t is the incubation time in hours. q-q plots indicated that growth rates were normally distributed. the /x dsdna replicative form was quantified using the quant-it picogreen dsdna broad range assay kit (life technologies), and all extracts were brought to the same concentration ( ng/ml). dna from each virus was split into three aliquots, which were treated with xhoi to linearize the genome, with xhoi and dpni to digest methylated gatcs, or with xhoi and mboi (i.e., dpnii) to digest non-methylated gatcs. double digestions were performed according to the manufacturer instructions (thermoscientific). a standard plasmid (pires, clontech) was used as a digestion control (not shown). a monochrome picture of the gel was transformed to an eight-bit image, and the pixel area and intensity of each band were quantified using imagej. given the size and base composition of the /x dna ( , bases, . % t, . % a, . % g, and . % c), the expected number of gatc sequence motifs in its genome is . Â . Â . Â . Â , ¼ . . the poisson probability of observing no gatc motifs is extremely low (p ¼ . Â - ), thus indicating a strong avoidance of these motifs. to restore gatc usage in /x , we created a mutant phage carrying twenty gatcs by sequential addition of these sites into the wt virus using site-directed mutagenesis (fig. ) . the twenty gatc motifs were evenly distributed throughout the phage genome, the greatest distance between any two consecutive of them being bases and, wherein possible, substitutions were made synonymous to minimize their effects on protein function. given that the dam/muthls system can perform mmr at a distance of up to kb from a gatc (modrich and lahue ) , the number and distribution of the introduced gatcs should allow for efficient mmr in the entire phage genome. to test the effect of gatcs on the viral mutation rate, we performed luria-delbrü ck fluctuation tests for the wt and gatc viruses. to score mutants phenotypically, we used the non-permissive e. coli c mutant gro , which carries a mutation in the dna helicase gene rep that blocks stage iii ssdna synthesis, preventing maturation of the wt phage (ekechukwu, oberste, and fane ) . the phage can overcome this restriction by changing certain amino acid residues in the n-terminal region of protein a, an endonuclease that nicks the negative strand of the supercoiled phage dna, and these protein changes can be conferred by at least seven different nucleotide substitutions (cuevas, duffy, and sanjuá n ) . by growing the virus in permissive cells and performing plaque assays in gro cells to score mutations, we obtained an estimated mutation rate for the wt of ( . . ) Â - m/n/r, a value consistent with previous studies (raney, delongchamp, and valentine ; cuevas, duffy, and sanjuá n ) . in contrast, the rate of the gatc virus was ( . . ) Â - m/n/r, revealing a . -fold reduction compared with the wt (t-test: p < . ; table ; fig. a ). the estimated growth rate was similar for the wt (r ¼ . . h - ) and the gatc viruses (r ¼ . . h - ; t-test: p ¼ . ; table ; fig. b ), indicating that these substitutions had no significant impact on short-term viral fitness. these findings confirm our previous results obtained with a mutant phage carrying seven gatc sites (cuevas, pereira-gomez, and sanjuá n ). since mmr relies on the presence of gatcs in the vicinity of the mismatch, addition of gatcs in this region should suffice to yield similarly low mutation rates. on the basis of this, we constructed a virus with four gatcs located between genome positions and , which were within . kb of known groresistance mutations (fig. ) . however, surprisingly, the mutation rate of this four gatc virus was significantly higher than that of the gatc virus (t-test: p ¼ . ) and similar to the wt rate (p ¼ . ). in light of this result and since dam methylation of gatc adenosines is required for mmr, we sought to determine the methylation status of the gatc /x dna. to do so, we purified the dsdna replicative form, linearized it, and treated it with the dpni restriction endonuclease, which selectively digests methylated and hemi-methylated gatcs. although dpni produced restriction bands of the expected size, digestion was only partial (fig. ) . to test whether this could be explained by incomplete dna methylation, we performed the same restriction analysis using mboi, which also recognizes gatc sites but digests them only in their non-methylated form. mboi also produced restriction bands, thus confirming that a fraction of the phage dsdna was not methylated. image analysis indicated that per cent of dna was digested by mboi and thus lacked at least one of the four possible methyl groups, whereas per cent was undigested by dpni, thus lacking all four methyl groups. overall, the similar efficiency shown by dpni and mboi suggests that roughly half of gatc motifs were methylated, although more detailed analyses would be required to reliably infer this fraction. we verified that under-methylation was not due to a dam defect in the host cell, since a standard plasmid grown in the same e. coli strain was fully digested by dpni and fully resistant to mboi (not shown). therefore, these results suggest that, as opposed to plasmid or chromosomal dna, gatc-mediated mmr is not fully efficient in /x because the phage dna is under-methylated. given that protein-coding regions represent approximately per cent of the /x genome, by chance one in twenty figure . /x genetic map and location of the gatc sequence motifs introduced in this study. open reading frames are represented by rectangles (b, k, and e are in different reading frames), and gray bars indicate intergenic regions. each gatc is represented by a dot, and its position is indicated on top. gatc motifs that were synonymous in all reading frames are indicated in blue, whereas those producing amino acid replacements in at least one frame are shown in green (a c produces a k q replacement in gene a and is synonymous in gene k; t g produces a v g replacement in gene a and is synonymous in gene b). mutations falling at intergenic regions are shown in red. the phage has circular dna but is represented linearly for convenience, where by convention the first position corresponds to the last nucleotide of the unique psti site. where t ¼ is the number of substitutions leading to gro resistance. g r ¼ lnðn =n Þ=t, where t is the incubation time in hours. table for details. gatc sites should fall at intergenic regions. to test how gatc location may influence mmr and the phage mutation rate, we created another mutant ( igatc) in which one of the four synonymous substitutions of the above gatc virus was replaced by substitution a g, which was located in the spacer region between genes h and a. the mutation rate of the igatc was ( . . ) Â - m/n/r, which represents a fiftyfold reduction compared with the wt (t-test: p < . ; table ). therefore, addition of this single intergenic substitution had a dramatic effect on the viral mutation rate, compared with the gatc virus. to further test the effect of intergenic gatcs on mmr, we first created the single mutant a g, which showed a mutation rate five times lower than the wt (t-test: p < . ; table ). this rate was significantly higher than for the igatc virus (t-test: p ¼ . ), showing that the effect of the a g substitution was enhanced by the presence of other, neighboring gatc sites, consistent with the lack of full methylation shown above. then, we constructed two additional intergenic single-gatc viruses located in the region between the h and a genes by introducing the appropriate nucleotide substitutions (fig. ) . the mutation rate of the virus carrying the c g/c a substitutions was fourteen times lower than the wt (t-test: p < . ; table ), whereas substitutions a g/t c were unable to reduce the mutation rate below the wt level (p ¼ . ) despite being located only five bases away from the previous substitutions. therefore, some but not all intergenic gatcs are able to promote mmr, and minute changes in their genome location lead to marked differences in mutation rate. induction of the chaperone-encoding groe operon in response to heat shocks up-regulates the expression of the error-prone dna polymerase iv, which participates in the repair of dsdna breaks under different types of cellular stress, leading to stress-induced mutagenesis (layton and foster ; malkova and haber ) . to address the effects of heat shocks on the /x mutation rate, we performed fluctuation tests at c for the wt and gatc viruses. the mutation rate of the wt was not significantly affected by the temperature shift (t-test: p ¼ . ; table ; fig. a ). in contrast, the mutation rate of the gatc virus was twenty times higher at c than at c (t-test: p < . ) and increased even above the wt level (p ¼ . ). therefore, the effects of gatc sites on the /x mutation rate observed at c were reverted at c. heat drastically reduced the viral growth rate but, whereas the wt and gatc viruses showed similar growth rates at c, the gatc virus grew significantly slower than the wt at c ( . . h - versus . . h - ; t-test: p ¼ . ; table ; fig. b ), suggesting that up-regulation of repair pathways under thermal stress slows down phage replication. to evaluate the effects of another stressor, we treated cells with -fu ( ng/ml) which, in addition to causing mutations directly by base mispairing, -fu inhibits thymidylate synthase, leading to deoxythymidine monophosphate deprivation and, subsequently, to dna strand breaks, induction of the sos dna damage response (ddr), and expression of error-prone dna repair enzymes (ahmad, kirk, and eisenstark ; fonville et al. ) . previously, we showed that this treatment increases the mutation rate of the wt virus by approximately tenfold (domingo-calap, pereira-gomez, and sanjuán ). fluctuation tests in the presence of ng/ml -fu showed that the drug had a more pronounced effect on the mutation rate of the gatc virus, which increased more than a -fold (table ; fig. a ). as a result, the difference in mutation rate between the gatc and wt viruses was fully abolished in the presence of -fu (t-test: p ¼ . ). we have shown that introduction of gatc sites in the /x genome can reduce the spontaneous mutation rate of the phage by up to fiftyfold, indicating that phage dna can undergo mmr if the required sequence motifs are present. the effect of gatc addition is greater than those reported previously in rna viruses, in which high-fidelity polymerase variants selected after serial transfers in the presence of nucleoside analogs typically reduce the viral mutation rate by threefold or less (pfeiffer and kirkegaard ; coffey et al. ) . a similarly modest effect was observed after serial passaging of /x in the presence of -fu (domingo-calap, pereira-gomez, and sanjuá n ). in that case, the anti-mutator phenotype was achieved by a delayed lysis, which increased the viral burst size per cell and thus allowed the phage to expand its population size in fewer rounds of copying (pereira-gó mez and sanjuá n ). in the dsdna bacteriophage t , a series of polymerase variants capable of strongly suppressing the action of chemical mutagens were isolated in early studies (drake et al. ; drake and greening ) . however, high fidelity variants of t polymerase tend to show diminished polymerization rates, therefore negatively impacting viral fitness (mansky and cunningham ) . in e. coli, figure . restriction fragment analysis of the /x replicative dsdna. phage dsdna was purified by standard miniprep as described in the materials and methods section and linearized with xhoi (ø), which recognizes a unique site at position , with xhoi and dpni to cleave methylated or hemi-methylated gatcs, or with xhoi and mboi to cleave non-methylated gatcs. expected (left) and observed (center) restriction fragments for the wt and gatc phage dsdna are shown. lower size fragments (< bp, fig. ) were expected but could not be visualized because the amount of input dna was low. the smear in lanes containing the purified phage dna probably results from degradation of host dna. the contrast of the gel image was enhanced to help visualize bands. right: percent abundance of each dpni and mboi restriction band ( - ). band in the dpni lane indicates the non-methylated dna fraction (i.e., none of the four gatc motifs was methylated), whereas in the mboi lane, this same band indicates the fully methylated fraction (i.e., the four gatc motifs were methylated). bands were quantified as detailed in the materials and methods section using the raw gel image with no contrast enhancement. changes in the a subunit of dna polymerase iii can increase replication fidelity between two-and thirtyfold (fijalkowska, dunn, and schaaper ) . a stronger anti-mutator phenotype was found in the adenine-dependent e. coli mud strain, but latter analyses suggested that this was probably due to poor detection of mutants (schaaper ) . in another study, e. coli clones were isolated with an up to fiftyfold anti-mutator phenotype, but the underlying mechanisms remained undetermined (quinones and piechocki ) . therefore, the magnitude of the mutation rate reduction afforded by the introduction of gatc motifs is similar or higher than those reported previously in other viruses and in bacteria and has a well-defined molecular basis. our results suggest that the /x mutation rate can be modified without significantly impacting viral fitness in the short-term, therefore allowing for evolutionary optimization of the viral mutation rate for long-term adaptability. however, our results also revealed constraints limiting mutation rate evolution, since the effects of gatc addition were lower than expected if mmr were fully efficient (fijalkowska, schaaper, and jonczyk ) . illustrating this, the lowest mutation rate achieved in this study ( Â - m/n/r) was still two orders of magnitude higher than that of e. coli (drake ; drake et al. ; lee et al. ) . our results suggest that inefficient mmr in /x is at least in part due to the fact that phage dna is under-methylated. full methylation may be impeded by the fast replication of the phage and the transient nature of the dsdna replicative form. cellular dam methylase levels must be tightly regulated, because hypo-and hypermethylation can compromise the ability of the mmr system to distinguish between the parental and daughter dna strands. showing this, both dam deficiency (bale, d'alarcao, and marinus ; marinus ) and overexpression (pukkila et al. ; mcclelland ) have been found to produce mutator phenotypes in e. coli. it is possible that dam methylation levels which are optimal for the host are too low for the phage, due to its fastest replication. this could be tested in future work by infecting dam-overexpressing e. coli c mutants with the gatc phage and determining phage dna methylation levels and mutation rates. assuming that the mmr system can use gatc sites at a distance of up to kb from the mismatch, for a dna showing the randomly expected density of gatcs, there should be approximately four such sites available for each mismatch. for a per cent methylation efficiency, the fraction of non-reparable mismatches would thus be on the order of . ¼ . , implying that the maximum mutation rate reduction achievable by mmr for this methylation efficiency would be / . ¼ seventeenfold. incomplete gatc methylation can hence account for the relatively limited efficacy of mmr in /x . we note that the fiftyfold mutation rate reduction observed for the igatc virus could be achieved with per cent methylation, a value experimentally undistinguishable from the per cent assumed above given that we could not finely quantify the fraction of methylated dna. however, for some gatcs, the efficacy of mmr was clearly below the upper-limit imposed by under-methylation, since the gatc and a g/t c viruses showed no change in mutation rate at all. our results indicate that intergenic gatcs tended to have stronger effects than those located in protein- table for details. coding regions, suggesting other factors curtailing mmr efficiency such as steric availability to muth. as we have shown, though, even two extremely close gatcs can have very different effects on mutation rate, and we lack a model for explaining these differences. the region in which the single intergenic gatcs were placed contains the a promoter and the h terminator. the c g/c a substitution, which had the strongest effect on mutation rate, was located farthest away from the a promoter. we can speculate that, if steric availability of the gatc motif to muth was limited by the transcription machinery, a more distal positioning from actively transcribed regions may allow for more efficient mmr. however, the a g and a g/t c substitutions were located approximately at the same distance of the a promoter. a g/t c was upstream of the promoter, whereas a g was downstream of the promoter and in a palindromic region with a relative high gþc content. as suggested previously, mismatch recognition may depend also on sequence context, increasing its efficiency in regions with higher gc content (jones, wagner, and radman ) . therefore, our results suggest that evolutionary optimization of the mutation rate may not be the sole factor driving gatc avoidance in /x or other enterobacteriophages, since we found that some gatcs had no effect on the viral mutation rate yet are also absent from the wt /x genome. mutation rate elevation can confer faster adaptation to new and stressful environments, and this has been shown to promote the spread of mutator strains in bacteria (leclerc et al. ; sniegowski, gerrish, and lenski ; jolivet-gougeon et al. ) . furthermore, bacteria have evolved the ability to up-regulate their mutation rates in response to stress by expressing of error-prone polymerases (rosenberg ; galhardo, hastings, and rosenberg ) . however, we have shown that gatc avoidance does not appear to increase the /x mutation rate under stress conditions, thus undermining the potential evolutionary advantage of such avoidance. it has been shown that phage yields tend to decrease in dam -muth þ suggesting that, in the absence of methylation, muth cleaves some gatc sites non-specifically and may also interfere with other stages of the infection cycle such as replication or encapsidation (deschavanne and radman ) . this would directly counterselect gatc sequence motifs in the phage. although in our experimental setting, we did not detect a significant deleterious fitness effect associated with gatcs in the absence of stress, such effects may potentially take place in other environments not assayed here. therefore, the evolutionary forces shaping gatc avoidance remain unclear, and may result from the joint action of several factors. interestingly, dna repair pathways may also be relevant to virus-host interactions in eukaryotes. vertebrate dna viruses have been shown to interact with the evolutionarily conserved ddr, which is aimed at detecting lesions in dna, initiating cell cycle arrest, and promoting repair. for instance, in hepadnaviruses, the synthesis of replication-competent covalently closed circular dna requires the participation of ku , a component of non-homologous end joining dna repair pathway (guo et al. ) . indeed, dna damage induction seems to be a common feature of many dna viruses including adenoviruses, herpesviruses, polyomaviruses, and papillomaviruses (luftig ) . most viruses degrade ddr components, but ddr activation and recruitment of some of its components into viral replication centers is also common. however, the outcomes of virus-host interactions at the ddr level are still poorly understood, and it remains at present unknown whether ddr activation is part of an antiviral cellular response or is exploited by the virus. in any case, repair pathways are activated following virus-induced dna damage, leading to the recruitment of error-prone host polymerases (malkova and haber ) . this suggests that changes in mmr efficiency following infection may determine the mutation rates of some dna viruses. thymine metabolism and thymineless death in prokaryotes and eukaryotes the evolution of mutation rate in finite asexual populations determinants of rna-dependent rna polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin characterization of dna adenine methylation mutants of escherichia coli k correlation between mutation rate and genome size in riboviruses: mutation rate of bacteriophage qb natural selection fails to optimize mutation rates for long-term adaptation on rugged fitness landscapes arbovirus high fidelity variant loses fitness in mosquitoes and mice mutation rate of bacteriophage /x modified through changes in gatc sequence context coronaviruses: an rna proofreading machine regulates replication fidelity and diversity counterselection of gatc sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system nucleoside analogue mutagenesis of a single-stranded dna virus: evolution and resistance a constant rate of spontaneous mutation in dna-based microbes suppression of chemical mutagenesis in bacteriophage t by genetically modified dna polymerases rates of evolutionary change in viruses: patterns and determinants host and /x mutations affecting the morphogenesis or stabilization of the s complex, a single-stranded dna synthesizing intermediate dna polymerase accuracy and spontaneous mutation rates: frequencies of purine.purine, purine.pyrimidine, and pyrimidine.pyrimidine mismatches during dna replication dna replication fidelity in escherichia coli: a multi-dna polymerase affair role of reca and the sos response in thymineless death in escherichia coli dna mismatch repair in eukaryotes and bacteria mutation as a stress response and the regulation of evolvability mutational robustness of an rna virus influences sensitivity to lethal mutagenesis characterization of the host factors required for hepadnavirus covalently closed circular (ccc) dna formation impacts of mutation effects and population size on mutation rate in asexual populations: a simulation study postreplicative mismatch repair the effect of deleterious alleles on adaptation in asexual populations bacterial hypermutation: clinical implications repair of a mismatch is influenced by the base composition of the surrounding nucleotide sequence error-prone dna polymerase iv is regulated by the heat shock chaperone groe in escherichia coli high mutation frequencies among escherichia coli and salmonella pathogens rate and molecular spectrum of spontaneous mutations in the bacterium escherichia coli as determined by whole-genome sequencing mechanisms and functions of dna mismatch repair viruses and the dna damage response: activation and antagonism the lower bound to the evolution of mutation rates mutations arising during repair of chromosome breaks virus mutators and antimutators: roles in evolution, pathogenesis and emergence dna methylation and mutator genes in escherichia coli k- dna mismatch repair and mutation avoidance pathways selection against dam methylation sites in the genomes of dna of enterobacteriophages mutation rates and intrinsic fidelity of retroviral reverse transcriptases mismatch repair in replication fidelity, genetic recombination, and cancer biology the rate of adaptation in asexuals delayed lysis confers resistance to the nucleoside analogue -fluorouracil and alleviates mutation accumulation in the single-stranded dna bacteriophage /x ' a single mutation in poliovirus rna-dependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity effects of high levels of dna adenine methylation on methyl-directed mismatch repair in escherichia coli isolation and characterization of escherichia coli antimutators', a new strategy to study the nature and origin of spontaneous mutations spontaneous mutant frequency and mutation spectrum for the accuracy of reverse transcriptase from hiv- evolving responsively: adaptive mutation' viral mutation rates antimutator mutants in bacteriophage t and escherichia coli dna mismatch repair: molecular mechanisms and biological function coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics evolution of high mutation rates in experimental populations of e. coli lack of evidence for proofreading mechanisms associated with an rna virus polymerase drift-barrier hypothesis and mutation-rate evolution conversion of /x viral dna to double-stranded form by purified escherichia coli proteins we thank silvia torres and pablo herná ndez for technical assistance. this work was supported by grants from the spanish mineco (bfu - ) and the european research council (erc- -stg- -virmut) to r.s., and by a ph.d. fellowship from the spanish ministerio de educació n to m.p.-g. data are available on request.conflict of interest: none declared. key: cord- -cxt rp authors: zheng, li-zhen; wang, xin-luan; cao, hui-juan; chen, shi-hui; huang, le; qin, ling title: src sirna prevents corticosteroid-associated osteoporosis in a rabbit model date: - - journal: bone doi: . /j.bone. . . sha: doc_id: cord_uid: cxt rp in an established steroid-associated osteonecrosis (saon) rabbit model we found recently that blockage src by sirna could improve reconstructive repair of osteonecrosis via enhancing osteogenesis and inhibiting bone resorption. the current study investigated if blocking src was able to prevent steroid-associated osteoporosis (saop) in the same saon animal model. rabbits were treated with pulsed lipopolysaccharide (lps) and corticosteroid methylprednisolone (mps). at , , and weeks after induction, src sirna, control sirna and saline were intramedullary injected into proximal femur, respectively. two fluorescent dyes xylenol orange and calcein green were injected before sacrificing the animals for in vivo labeling of the newly formed bone. at week after induction, proximal femora of rabbits were dissected for micro-ct and histological analysis. results showed significant bone loss in the metaphysis of femoral head in the control rabbits after saon induction. src sirna treatment was able to prevent steroid-associate bone loss in trabecular bone and increase cortical bone thickness at femoral neck. histomorphometry showed that src sirna increased the osteoblastic bone formation and decreased the eroded bone surfaces suggesting decreased osteoclastic bone resorption. this was the first study to report bone loss after saon induction in rabbit model that could be prevented by knocking down src by sirna. corticosteroids are widely indicated for many diseases attributed to its anti-inflammation effects. however, its long-term use could lead to adverse effects such as steroid-associated osteoporosis (saop) [ ] and/or steroid-associated osteonecrosis (saon) [ ] . saop patients with decreased bone mineral density (bmd) are reported at higher risk of bone fracture [ ] and the advanced saon patients often suffered from joint collapse [ ] . physiologically, the skeleton undergoes remodeling, with osteoclasts for resorbing old bone and osteoblasts for forming new bone towards maintenance of bone homeostasis [ ] . corticosteroids impair osteoblastic osteogenesis and increase apoptosis of osteoblasts and osteocytes. this has been regarded as common pathway or the underlying mechanism of corticosteroids-associated bone deterioration in both saop and saon [ ] . proto-oncogene tyrosine-protein kinase src (also known as c-src) participates in regulating a wide range of cell functions, including adhesion, motility, proliferation and survival [ , ] . in skeletal system, src inhibits osteoblast differentiation [ ] . it is reported that the decreased src expression enhances osteoblast differentiation and bone formation [ ] . src is also involved in regulating osteoclastic bone resorption [ ] as it is essential for the function of osteoclasts via regulating the osteoclast skeleton [ ] . transgenic expression of src can also rescue osteoclast function in src knockout mice [ ] . in a modified saon rabbit model, we found destructive repair at subchondral region of femoral head, i.e. a dominate old bone resorption without adequate new bone formation to maintain normal bone homeostasis; and knockdown of src expression by src specific sirna could enhance osteoblast differentiation, promote osteogenesis and inhibit the function of osteoclasts [ ] . in other words, src sirna may also have potential for treatment of saop. in this study, we investigated corticosteroid-associated bone loss at metaphysis of femoral head using an established saon rabbit model [ ] and potential effects of src sirna for prevention of saop. forty-eight -week-old male new-zealand white rabbits ( . ± . kg) were housed at the experimental animal center in prince of wales hospital in hong kong and received a standard laboratory diet and water ad libitum. all specified experimental protocols were approved by the animal experiment ethics committee of the chinese university of hong kong ( / /grf). based on our established protocol for inducing destructive repair saon, thirty-six rabbits were used and intravenously injected with lipopolysaccharide (lps) ( μg/kg body weight, escherichia coli o :b ; sigma-aldrich, st. louis, mo, usa) on day and day , and intramuscularly injected with corticosteroid methylprednisolone (mps) ( mg/kg body weight, pharmacia & upjohn, peapack, nj, usa) on days , , and again on days , and [ ] . twelve rabbits without any treatment were used as normal controls. at , and weeks after induction with pulsed lps and mps, rabbits were under general anesthesia with intramuscular injection of xylazine ( mg/kg body weight) and ketamine ( mg/kg body weight) for hair shaving on hip region and prepped with iodine tincture and % ethanol. lateral digital x-ray of hip was taken for monitoring the injection site. in order to prove the intervention concept, we adopted local delivery approach where a g needle was selected for local sirna administration and for controls as well by inserting it from the greater trochanter of proximal femur to the marrow cavity of the femur neck. the lps-mps treated rabbits were randomly divided into three groups (n = /group): ) s-sisrc group: nmol src sirna (synthesized by ribobio, guangzhou ribobio co., ltd., sense ′-ggu uca cca uca agu cag a dtdt- ′, antisense ′-ucu gac uug aug gug aac c dtdt- ′) was intramedullary injected into each proximal femur with μl in vivo-jetpei (polyplus transfection, strasbourg, france) as transfection reagent according to the manufactural protocol; ) s-nc group: nmol negative control sirna (synthesized by ribobio, sense ′-uuc ucc gaa cgu guc acg u dtdt- ′, antisense ′-acg uga cac guu cgg aga a dtdt- ′.) was injected with the same method described above; and ) s-vc group: vehicle control ( . ml saline) was injected. euthanasia was executed days after the week administration for each group and the femora were collected for further evaluations. . ml bone marrow was extracted from the injection site before injection for mrna isolation with rneasy® mini kit (qiagen, usa) and then cdna was reverse transcripted by quantitect® reverse transcription kit (qiagen, usa). real-time pcr was performed by abi prism sequence detection system (applied biosystem, foster city, ca, usa) using tf pack power sybr green pcr mas (applied biosystem, foster city, ca, usa). primers for rabbit are: src-forward ′-gcccatctacatcgtcacag- ′, src-reverse ′-tagttcatccgctccacgta- ′, gapdh-forward ′-tctggcaaagtggatgttgt- ′, gapdh-reverse ′-gtgggtggaatcatactgga- ′. at day and day before euthanasia, two fluorescent dyes, xylenol orange ( mg/kg body weight) and calcein green ( mg/kg body weight; both sigma-aldrich gmbh), were injected sequentially and subcutaneously into the rabbits (n = /group). after euthanasia, the proximal femora were embedded in methyl methacrylate (mma) without decalcification. after being fixed in buffered formalin for one week, the proximal femora were scanned using a micro-ct (μct- , scanco medical, brüttisellen, switzerland) with a spatial resolution of μm. in order to separate mineralized tissue from background signal, a low-pass gaussian filter (sigma = . , support = ) was used. the mineralized tissue was defined at a threshold of hu [ ] . -d sections of the scanned femoral heads were then reconstructed into -d structure. to evaluate metaphyseal trabecular architecture of femoral head, a predetermined bone cylinder was used as roi ( . mm diameter and . mm height) to quantify the volumetric bone mineral density (bmd), bone tissue volume fraction (bv/tv), connective density (conn. d.), trabecular number (tb. n), trabecular thickness (tb.th), and trabecular separation (tb. sp) [ ] . to evaluate cortical bone, femoral neck was used as roi ( . mm thick) after re-alignment of micro-ct images using built-in software ipl (image processing language) (fig. a) . a dual threshold technique was used for automatic segmentation of cortical ring of the femoral neck [ ] . the first threshold for outer contour and the second threshold for inner contour were both set at hu. using the micro-ct built-in evaluation methods for long bone midshaft evaluation, the cortical bone thickness (ct.th), periosteal diameter (ps.dm), endosteal diameter (es.dm) and polar moment of inertia (pmoi) were determined. the pmoi = ixx + iyy, where ixx is moi around x-axis, and iyy is moi around y-axis. for decalcified sections, the proximal femora were treated with % edta (ph . ) for weeks with weekly refreshing of the decalcifying solution (n = /group). the decalcification process was assessed by digital x-ray that the complete decalcification was confirmed without showing radiographic opacity. then the proximal femora were embedded in paraffin and cut into -μm-thick sections along the coronal plane. sections were stained with h&e for histomorphometry and tartrateresistant acid phosphatase (trap, sigma diagnostics, st. louis, missouri, usa) for identification of osteoclasts. the mma embedded samples were cut along the coronal plane using a saw microtome (leica sp , leica instruments, nussloch, germany) and polished to μm by a polisher (phoenix , buehler ltd. usa) for observation of fluorescence labeling and then cut into μm-thick sections using microtone (leica rm ) for dynamic histomorphometry and goldner's trichrome staining. histological images were digitalized with a microscopic imaging system (leica dm ; leica micro-systems, wetzlar, germany). histomorphometric analysis was carried out on the metaphysis of femoral head in a mm width area below the growth plate by using osteomeasure histomorphometry system (osteometrics, atlanta, ga, usa) and analyzed according to a standard histomorphometry protocol [ ] . all data were expressed as mean ± sd with one-way anova followed by bonferroni post-test to compare group differences. statistical analysis was performed using spss . software (chicago, il, usa). p b . was considered significant. quantitative real-time pcr showed that the src mrna level in bone marrow in s-sisrc group was consistently lower after week injection compared to that of the s-nc group throughout the experimental period (p b . ), while the src mrna level in bone marrow in s-nc was not different when compared to that of the s-vc group (fig. ) . we used micro-ct to analyze the metaphyseal trabecular architecture of femoral head. it revealed significant lower bone tissue volume fraction (bv/tv), trabecular thickness (tb.th) and connective density (conn. d.) in s-vc/s-nc group when compared to those of the normal control group (p b . for all). on the contrary, metaphyseal trabecular bone showed significant higher bone mineral density (bmd) (p b . ), bv/tv (p b . ), tb.th (p b . ) and conn. d. (p b . ) in s-sisrc group when compared to those of the s-vc/s-nc group. there was no significantly difference in each of above micro-ct parameters between s-vc and s-nc group (fig. ). micro-ct images of femoral neck showed that cortical thinning occurred in the s-vc group and s-nc group at the regions adjacent to greater trochanter and lesser trochanter when compared to the corresponding regions in the normal control group; and some thinning regions were even disconnected in the region adjacent to the greater trochanter (fig. ab) , while the average thickness of the whole ring cortical bone of the femoral neck (ct.th) did not show statistically significantly different between the s-vc/s-nc group and the normal control group. the ct.th and polar moment of inertia (pmoi) were significantly larger in the s-sisrc group when compared to those in the s-vc/s-nc group (p b . ). the periosteal diameter (ps.dm) and endosteal diameter (es.dm) were not significantly different among groups. (fig. c ). histomorphometric analysis was performed at the metaphysis of femoral heads (fig. ) . from the decalcified sections, we found that the osteoblast surface (obs/bs) of the s-vc/s-nc group was not different from that in the normal control group, while the ratio of eroded surface to total bone surface (es/bs), and the osteoclast number per bone surface (n.oc/bs) were significantly larger after induction when fig. ). there was no significant difference in n.oc/bs between s-sisrc group and s-vc/s-nc group, while the n.oc/es value was significantly larger in s-sisrc group when compared to that in the s-vc group (p b . , fig. ). from the undecalcified sections, the results showed that in the s-vc/s-nc group the mineral apposition rate (mar) and osteoid thickness (o.wi) were lower when compared to those in the normal control group (p b . ), while the mineralizing surface (ms/bs) did not show significant difference. the bone formation rate (bfr/bs) was significantly lower in the s-vc/s-nc group when compared to that in the normal control group (p b . ). src sirna treatment did not affect the mar and o.wi when compared to those in the s-vc/s-nc group, while the ms/bs and the bfr/bs were significantly higher in s-sisrc group when compared to those of the s-vc/s-nc group (p b . , fig. ). descriptive histological analysis on the femoral neck was summarized in supplemental fig. . the sequential fluorescence labeling showed newly formed bone on endosteal surface, including endocortical, intracortical, and trabecular surface, where we observed very little and scattered newly formed bone on periosteal surface at femoral neck in all the groups (supplemental fig. a ). goldner's trichrome staining showed some osteoid on the endocortical and intracortical surface in the normal control group and s-sisrc group. there was osteoclastic bone resorption in the s-vc and s-nc group, while in the s-sisrc group the osteoclastic bone resorption was hardly observed (supplemental fig. b) . this study investigated the bone loss induced by pulsed lps and mps using an established saon rabbit model and tested the therapeutic potential of src sirna, a bone anabolic and anti-resorption agent for prevention of corticosteroid associated osteoporosis (saop). our micro-ct analysis showed significant deterioration of trabecular bone at metaphysis of femoral head after lps-mps induction when compared with the normal control group, characterized with significant inferior values in bv/tv, tb. th and conn. d. of the metaphysical trabecular bone of the femoral head in the s-vc/s-nc group. the histomorphometry results showed that the bone loss was explained by the increased osteoclastic bone resorption with increased osteoclast number (n.oc/bs) and eroded surface, while with decreased osteoblastic bone formation accompanied by a decreased mineral apposition rate (mar) and bone formation rate (bfr/bs). it is well-known that corticosteroid induces bone resorption [ , ] , while long-term administration of corticosteroid would lead to the inhibition of osteoclastic bone resorption and reducing of bone turnover [ ] . our induction protocol with pulsed high dose of corticosteroid administration simulated the clinical situation, such as the one developed for treatment of severe acute respiratory syndrome (sars) patients in hong kong in that resulted in lower bmd at the hip of saon patients [ , ] , while around the osteonecrotic lesions the osteoclasts were also stimulated to resorb necrotic trabecular bone in the repair process [ ] . in this study, we investigated corticosteroid-associated bone loss using an established saon rabbit model [ ] . we found co-exist of osteonecrosis and osteoporosis in the same animal model. however, the rois of osteonecrosis and osteoporosis were different and this made our evaluation possible for comparison. the subchondral region, where with yellow bone marrow and low bone remodeling rate in normal rabbit but with elevated bone resorption and bone formation in model animal, was the roi for studying the repair of osteonecrosis; while the metaphysis, where with red bone marrow and normal bone remodeling, was the roi for assessing bone loss associated with corticosteroid treatment in the current study. although the apoptosis of osteoblasts and osteocytes induced by pulsed lps and mps inevitably occurred, it has been regarded as common pathway or the underlying mechanism of corticosteroids-associated bone deterioration in both saop and saon [ ] . this formed a structural basis for our novel findings of src sirna in prevention of saop. in the current study the pulsed lps and mps treatment down-regulated the metabolic activity of osteoblasts, as shown by a decreased mar in s-vc/s-nc group. similar findings were also reported in previous studies of saop [ , ] . however, the osteoblast surface (ob.s/bs) and the mineralizing surface (ms/bs) were not significantly different between the s-vc and the normal control. this inconsistence might be explained that there was osteonecrotic repair progress in our animal model while purely saop had decreased osteoblast differentiation and proliferation [ , , [ ] [ ] [ ] . micro-ct images of femoral neck showed cortical thinning after the pulsed lps-mps treatment when compared with the normal control group at particular regions adjacent to greater trochanter and lesser trochanter, where cortical thickness was originally thinner and comparable to trabecular bone thickness to make these regions more sensitive to lps-mps. the overall changes in cortical thickness between were not found statistically significant that might be explained by rather short effects on cortical bone using our current pulsed mps and lps treatment protocol. with regard to the saon and saop, apart from mps, lps was also administered in our rabbit model to simulate clinical indication for treatment systemic inflammation [ ] . it was reported that lps could inhibit osteoblast differentiation and induce osteoblast apoptosis [ , ] , and could promote osteoclast differentiation and survival [ ] , suggesting that lps had potential contribution to the pathogenesis of the bone loss in our rabbit model as well, although we could not delineate its effects from corticosteroid. it was also reported that lps could induce src expression and activation in macrophages [ ] , i.e. the lineage of osteoclasts, for their potential contribution to activating of bone resorption. the loss of trabecular bone at metaphysis of femoral head after pulsed lps and mps treatment was reversed by src sirna administration in the present study, with significantly better micro-ct indices, including bmd, bv/tv, tb. th and conn. d. our histomorphometry results confirmed that after src sirna administration, the ob.s/bs, ms/bs and bfr/bs were significantly higher, suggesting that sisrc could enhance osteoblast differentiation in rabbit model. however, the mar and o.wi in s-sisrc group were not different from that in the s-vc/s-nc group, implying that sisrc did not affect the function of the mature osteoblasts. this finding was also supported by an early work using src knockdown mice in vivo [ ] . it was reported that inhibition of src in calvarial osteoblasts from mice in vitro could lead to enhanced alp activity and nodule mineralization, and the enhanced alp activity was related to the enhanced alp positive cell numbers [ ] . it is known that src family inhibits osteoblasts through src-yap-runx signaling pathway by tyrosine phosphorylation of yes-associated protein (yap) and then suppressing runx transcriptional activity [ ] , where runx is the major transcription factor controlling osteoblastogenesis [ ] . it was reported that src stimulated il- and then induced insulin-like growth factor (igfbp ), a src activating factor only in immature osteoblasts to maintain osteoblasts in a less mature status. this suggested that there could be different pathways of src in regulating immature and mature osteoblasts, and this might be the reason of inhibiting src stimulated osteoblast differentiation and maturity but without stimulating osteoblast function [ ] . in the present study, we found n.oc/bs was not affected by sisrc, suggesting that src had no direct effect on the osteoclast differentiation; however, sisrc significantly decreased es/bs and increased n. oc/es, suggesting that sisrc could inhibit osteoclastic bone resorption by inhibiting the bone resorption function of the osteoclasts. higher n. oc/es means that if no change of the number of osteoclasts (n.oc), the eroded surface (es) is lower, or interpreted by lower average activity of osteoclasts. the function of src in osteoclasts was its role in regulation of cytoskeletal organization [ ] , and organized cytoskeleton in osteoclast showed its effect on synthesis and secretion of collagenolytic cysteine proteases, such as cathepsin k, while inhibition of src was reported to suppress the secretion of cathepsins and bone resorption in osteoclasts [ ] , implying src's involvement in activating bone resorption as well. it was reported that the lifespan of osteoclasts is about weeks in vivo, and the lifespan will be longer with glucocorticoid excess [ ] . this explained that the biweekly treatment with sirna should have sustaining therapeutic effects on osteoclastic bone resorption. in our current study, we also found that sisrc could enhance cortical bone thickness and the polar moment of inertia (pmoi), an indicator of torsional rigidity. micro-ct revealed that the femoral neck cortical thickness in s-sisrc group was significantly increased compared with that in the s-vc/s-nc group. clinically, the bmd measurement at the femoral neck is a standardized measurement to diagnose osteoporosis [ ] , and it is found that pmoi is also correlated to bone strength apart from bmd [ ] . the bone fractures occurred predominantly at cortical sites, so the quality of cortical bone at femoral neck played an important role to predict the fracture risk [ ] . our fluorescence labeling showed new bone formation on the endocortical surface and intracortical surface (supplemental fig. ). on the endosteal surface, including endocortical, intracortical, and trabecular surface, cells communicated in trabecular and cortical bone for bone modeling and remodeling [ ] . src sirna could enhance osteogenesis not only at trabecular surface but also the endosteal surface and intracortical surface to enhance the bone quality and decrease the bone fracture risk in the saop. because of the limitation of micro-ct resolution used for the current study, we did not determine the cortical porosity of femoral neck. however it was reported that drugs with anti-resorptive effect could reduce cortical porosity [ , ] , while anabolic agents could increase cortical porosity with increased cortical thickness and decreased risk of fracture because of the larger porosity of newly formed bone near the marrow cavity [ , ] . src sirna tested in the present study could inhibit src activity that had dual functions, including anti-resorptive and anabolic effect that resulted in increased cortical bone quality. on the periosteal bone surface there are also abundant number of osteoblasts and preosteoclasts (mononuclear, trap+) responsible for cortical bone remodeling [ ] and this suggested that knockdown src in these cells would also have anti-resorptive and anabolic effect to increase periosteal diameter. however, due to limitation of micro-ct resolution and also variations in animal or bone size, the current cross-sectional study (group comparison) did not shown statistical difference in cortical periosteal (ps.dm) or endosteal diameter (es.dm). longitudinal study (monitoring changes of bone size of same animal) with 'nano-scale' d analysis is desirable in future study for studying the biological and therapeutic effects of our designed src sirna on bone remodeling at cortical bone. as the current study was related to a local injection, the src inhibition effects were expected to show mainly in the bone marrow and endosteal surface. future study would focus on r&d of a systemic administration of src sirna for its knockdown efficiency on cortical bone, especially the periosteal region. as this is a very first study for exploring the therapeutic effect of src sirna for saop, local injection to the target organ or tissue is expected much more efficient, e.g. lower dose could already achieve therapeutic effect, such as in our current proof-of-concept study for a high risk skeletal site of osteoporosis and osteoporotic fracture as compared to the systemic administration with rather lower treatment efficiency. in vivo distribution of src sirna demonstrated that h after injection the most of the injected sirna was retained at the injection site, and a small part of sirna that did not transfect into the local injected site was catabolized by liver and excreted to gallbladder (supplemental fig. s ). however, concerning clinical application, future studies may focus on r&d of systemic bone-targeting delivery system for src sirna to treat systemic saop [ , ] . in conclusion, we found corticosteroid-associated osteoporosis in an established rabbit saon model and the knockdown of src by src sirna could increase bone formation and decrease bone resorption and therefore could become a potential novel therapeutic strategy to prevent corticosteroid-associated secondary osteoporosis. supplementary data to this article can be found online at http://dx. doi.org/ . /j.bone. . . . all authors declare no conflict of interest in the present study. prevalence of long term steroid treatment and the frequency of decision making to prevent steroid induced osteoporosis in daily clinical practice steroid-associated osteonecrosis: epidemiology, pathophysiology, animal model, prevention, and potential treatments (an overview) a metaanalysis of prior corticosteroid use and fracture risk pathogenesis and natural 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potential treatment with growth and differentiation factors glucocorticoid-induced osteoporosis glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells progression and cessation of collapse in osteonecrosis of the femoral head decrease in the mesenchymal stem-cell pool in the proximal femur in corticosteroid-induced osteonecrosis promotion of bone repair by implantation of cryopreserved bone marrow-derived mononuclear cells in a rabbit model of steroid-associated osteonecrosis role of macrophages in lps-induced osteoblast and pdl cell apoptosis lipopolysaccharide (lps) induces the apoptosis and inhibits osteoblast differentiation through jnk pathway in mc t -e cells lipopolysaccharide (lps) promotes osteoclast differentiation and activation by enhancing the mapk pathway and cox- expression in raw . cells chemical inhibition of src family kinases affects major lps-activated pathways in primary human macrophages tyrosine phosphorylation controls runx -mediated subnuclear targeting of yap to repress transcription transcription factors controlling osteoblastogenesis regulation of collagenolytic protease secretion through c-src in osteoclasts birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis standardising the descriptive epidemiology of osteoporosis: recommendations from the epidemiology and quality of life working group of iof prediction of bone strength at the distal tibia by hr-pqct and dxa intracortical remodelling and porosity in the distal radius and post-mortem femurs of women: a cross-sectional study skeletal stem/osteoprogenitor cells: current concepts, alternate hypotheses, and relationship to the bone remodeling compartment differing effects of denosumab and alendronate on cortical and trabecular bone microarchitectural deterioration of cortical and trabecular bone: differing effects of denosumab and alendronate anabolic effects of human biosynthetic parathyroid hormone fragment ( - ), ly , on remodeling and mechanical properties of cortical bone in rabbits pth(i- )] strengthens the proximal femur of ovariectomized nonhuman primates despite increasing porosity pdgf-bb secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis systemic drug delivery systems for bone tissue regeneration-a mini review a delivery system targeting bone formation surfaces to facilitate rnai-based anabolic therapy this work was supported by the hong kong general research fund (grf cuhk- ), the national natural science foundation of china (nsfc- ) and the natural science foundation (nsfc-gjhs ). key: cord- -d iuk authors: baquero-pérez, belinda; whitehouse, adrian title: hsp isoforms are essential for the formation of kaposi’s sarcoma-associated herpesvirus replication and transcription compartments date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d iuk kaposi’s sarcoma-associated herpesvirus (kshv) is an oncogenic herpesvirus associated with various aids-related malignancies. like other herpesviruses, multiple processes required for kshv lytic replication, including viral transcription, viral dna synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (rtcs). here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to kshv-induced rtcs and thus play a key role in kshv lytic replication. we show that several isoforms of the hsp chaperone family, hsc and ihsp , are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of kshv-induced rtcs. we demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed kshv rtcs, however during later time points the chaperones move within kshv rtcs and completely co-localise with actively replicating viral dna. the functional significance of hsp isoforms recruitment into kshv rtcs was also examined using the specific hsp isoform small molecule inhibitor, ver- . intriguingly, results highlight an essential role of hsp isoforms in the kshv replication cycle independent of protein stability and maturation. notably, inhibition of hsp isoforms precluded kshv rtc formation and rna polymerase ii (rnapii) relocalisation to the viral genome leading to the abolishment of global kshv transcription and subsequent viral protein synthesis and dna replication. these new findings have revealed novel mechanisms that regulate kshv lytic replication and highlight the potential of hsp inhibitors as novel antiviral agents. molecular chaperones represent a large group of proteins that are essential for maintaining cellular homeostasis and survival. as such, the roles of these proteins are numerous; facilitating correct protein folding or unfolding, assembly or disassembly of multimeric protein complexes, participating in translocation of proteins and vesicles into organelles, stabilising a wide range of signalling molecules and preventing aggregation of non-native proteins (reviewed in [ , ] ). heat shock proteins (hsp) are classified according to their molecular weight into several families: hsp , hsp , hsp , hsp , hsp , hsp and the small hsp (less than kda) [ ] . the functional importance of the hsp and hsp families of molecular chaperones is exemplified by their emerging implications in a variety of diseases, including cancer [ , ] , neurodegeneration [ ] or viral infection [ , ] . as such they have gained significant interest recently as potential drug targets. eukaryotes have multiple genes encoding for chaperones of the hsp family, which are amongst the most conserved proteins in evolution [ ] [ ] [ ] . the major hsp isoforms are the constitutively expressed hsc , the stress-inducible hsp (ihsp ), the endoplasmic reticulum resident (grp ) and the mitochondrial form (grp ). all hsp isoforms have an n-terminal domain which harbours a highly conserved atpase and a c-terminal substrate binding domain [ ] . hsp isoforms which comprise the inducible and constitutively-expressed isoforms (hsp α and hsp β respectively), the er resident (grp ) and the mitochondrial form (trap ), also possess a n-terminal atp binding domain, although this has no similarity to the atp-binding domain found in the chaperones of the hsp family [ ] . the presence of atpase pockets in both families of chaperones makes these proteins desirable targets for small molecule inhibitors [ , ] . the therapeutic potential of these compounds is especially evident for several hsp inhibitors, having already reached phase ii and iii clinical trials [ , ] . targeting of hsp isoforms has been more challenging [ ] , but recently specific inhibitors have also undergone clinical trials [ , ] . importantly, the development of highly specific inhibitors for hsp isoforms may have potential for the treatment of a diverse group of viruses as the functional importance of hsp isoforms in the life cycle of numerous viruses has been highlighted over the past few years [ ] . distinct hsp isoforms are usurped to aid in many stages of viral replication as varied as viral entry, uncoating, transcription, envelope protein maturation, morphogenesis or dna replication [ ] . therefore, the importance of these chaperones in the life cycle of such a wide range of viruses suggests the potential of these proteins as targets for broad-spectrum antivirals. kaposi's sarcoma-associated herpesvirus (kshv) is the causative agent of several aidsassociated malignancies, including kaposi's sarcoma (ks), a highly vascular tumour of ne cannot be purified completely due to its multiple subcellular connections. the outer nuclear membrane is continuous with the endoplasmic reticulum and interacts with the cytoskeleton [ , ] while the inner nuclear membrane binds to host chromatin [ ] [ ] [ ] . thus, we took advantage of its incomplete purity so that we could isolate not only the ne and embedded nuclear pore complexes, but also components found in the ne neighbourhood, such as rtcs. utilising this novel approach we demonstrate that cellular chaperones from the hsp family (hsc and ihsp ) are significantly increased in the ne-associated rtcs of reactivated cells. functional dissection further demonstrates that these chaperones were specifically recruited to the periphery of incipient rtcs coinciding in time with their formation. when actively replicated viral dna was synthesised the chaperones were recruited within rtcs. strikingly, inhibition of hsp isoforms precluded rtc formation, curtailed chaperone redistribution within rtcs and rnapii recruitment to viral promoters. importantly, abrogation of lytic replication occurred without affecting cell viability, suggesting that the cellular housekeeping functions carried out by these chaperones were not compromised. as such, hsp inhibitors may provide a novel therapeutic approach for the treatment of kshv-associated malignancies, in particular it would be interesting to determine the efficacy of combining the potential of inhibiting lytic replication using hsp inhibitors with the previous reported effect of hsp inhibitors to eradicate latent kshv reservoirs [ ] . the cellular chaperones hsc , ihsp and grp were enhanced in the ne-associated rtcs of reactivated hek- t rkshv. cells to investigate differential proteome changes which occur during kshv lytic replication in ne-associated rtcs, we utilised the hek- t cell line containing a latent recombinant kshv virus (rkshv. ) [ ] . this cell line can be reactivated into a full lytic replication cycle via chemical induction. unreactivated cells were grown in isotopically labelled media (r k ), while cells to be reactivated were grown in label-free media (r k ). after isotopic labelling was complete, cells were reactivated for h and nuclear envelopes (nes) were then successfully purified using a recently described method [ ] , with minor modifications. western blot analysis of the ne preparations demonstrated an enrichment of nucleoporins (nups), lamins and histones and a loss of cytoplasmic (gapdh) and nucleolar (b- , c- ) proteins compared with whole cell (wc) lysates (fig ) . the essential kshv mrna processing protein, orf [ ] , and viral rta served as markers for lytic viral replication and rtc enrichment, as both of these proteins are known to be recruited to kshv rtcs [ , ] . the monoclonal antibody (mab ), which recognises the phenylalanine-glycine (fg)-repeat motif present in numerous nups, and the polyclonal antibodies against nup and lamin b were used to assess enrichment of the ne region. ne preparations showed a higher number of nups (closed arrows) than their respective wc preparations; although some nups were lost following . m salt wash (open arrow). following lc-ms/ms analysis and using a minimum of three unique peptides assigned to a single protein, most proteins ( ) remained unchanged in their abundance irrespectively of kshv lytic infection and only five proteins had a significant reduction (ratio cut-off < . ) in nes of reactivated cells. in contrast, proteins showed a significant increase (ratio cut-off > . ) during lytic replication. importantly, multiple cellular proteins that are known or expected to localize within herpesvirus rtcs; such as those associated with kshv ori-lyt, the hcmv transactivator ie -p protein or the herpes simplex virus- (hsv- ) icp protein were found significantly increased in the ne regions of reactivated cells using the less stringent ratio cut-off of . (s table) . some of these cellular proteins included csnk a [ ] , blm [ ] , topoisomerases i and ii [ , , ] and dead box helicases ddx [ ] and ddx [ ] . thus, lc-ms/ms results confirmed the correct isolation of the ne region and accompanying rtcs. importantly, many of the identified proteins most likely represent novel cellular proteins hijacked by kshv, not only due to the subcellular fractionation carried out but also to the uncommon use of urea for protein extraction before lc-ms/ ms. all proteins identified by lc-ms/ms can be seen on s dataset. bioinformatical analysis revealed several upregulated pathways (ratio cut-off > . ) in reactivated cells (s table) . of particular interest was an upregulated pathway which related to protein folding. this included several hsp isoforms and their associated co-chaperones from the hsp (dnaj) family ( table ) . notably, the constitutively expressed chaperone hsc presented a . -fold increase with unique peptides assigned. this protein had the highest fold increase associated with the most unique peptide number of all the proteins identified by lc-ms/ms. this could be due to increased hsc expression or to the redistribution of hsc from the cytoplasm into the ne region during kshv lytic replication. therefore, due to the vital importance of hsp isoforms in the replication cycle of a wide range of viral families, we focussed our studies herein on the roles of the three main hsp isoforms (hsc , ihsp and grp ) during kshv lytic replication. hsc and ihsp are redistributed from the cytoplasm to both the periphery and within kshv-induced rtcs to verify the enrichment of hsc in the ne-associated rtcs of reactivated cells detected by our quantitative proteomic approach, indirect immunofluorescence was used to label endogenous hsc protein in trex bcbl -rta cells, a kshv latently infected b-lymphocyte cell line containing a myc-tagged version of viral rta under the control of a doxycycline-inducible promoter [ ] . hsc protein was equally distributed between the cytoplasm and nucleus of unreactivated cells in a fine punctuate pattern (fig ai) . similar hsc localization was seen during early lytic replication ( h reactivation), when rta protein was diffuse in the nucleus, successful enrichment of the nuclear envelope region and associated kshv rtcs in hek- t rkshv. cells. successful enrichment of ne regions in unreactivated (-) and reactivated for h (+) hek- t rkshv. cells was demonstrated by western blotting. equal amounts of total protein extracted from whole cell (wc) and nuclear envelope (ne) fractions were used. the monoclonal antibody mab specifically recognises the phenylalanine-glycine (fg)-repeat motif present in numerous nucleoporins (nups). closed arrows point to nups that were only detected by mab after ne enrichment. open arrow points to a nucleoporin lost during ne enrichment. several fg nups, together with nup , histone h and lamin b were all markedly enriched in the ne fractions compared with wc fractions. the nucleolar proteins b- and c- and the cytoplasmic gapdh protein were all decreased in nes fractions indicating correct subcellular fractionation. the viral rtc-associated orf and rta proteins were used to monitor lytic reactivation and assess rtc enrichment. orf antibody detected both full length orf protein (fl-orf ) and the caspase- -cleaved orf (cl-orf ). prior to rtc formation (fig aii) . in contrast, at later reactivation time points ( h), in which rta was organised into small viral rtcs peripherally located in the nucleus, numerous nuclear hsc foci that were predominantly adjacent to rtcs were observed (fig aiii) . during late reactivation time points ( h), cellular chromatin was marginalised to the nuclear periphery (fig aiv, nucleus highlighted in yellow) and larger hsc foci avidly accumulated within these fully-developed rtcs (fig aiv) . reduced levels of cytoplasmic hsc were also observed at this time point (fig aiv arrows) , suggesting that hsc is redistributed from the cytoplasm to the nucleus during kshv lytic replication. this is further supported by the fact that fractionation of trex bcbl -rta cells into nuclear (n) and cytoplasmic (c) fractions displayed an enrichment of hsc in the nuclei of reactivated cells which occurred without a noticeable increase in hsc protein levels in whole cell (wc) lysates ( fig b) . due to the observed redistribution of hsc to kshv rtcs, further co-localization studies between hsc and the sites of viral dna replication were performed in trex bcbl -rta cells. here, cells were triple-labelled with click-it edu alexa fluor and antibodies specific for rta and hsc . in unreactivated cells, newly synthesized cellular dna during mid-s-phase (edu incorporated) occurred mainly at the nuclear periphery as previously observed in other cell types [ , ] (fig ci) . during early reactivation, hsc was adjacent to rta which was present in small viral rtcs containing actively replicating viral dna (fig cii arrow) . at this stage, a proportion of hsc also co-localised with rta (fig cii') . during late reactivation, when cellular chromatin was marginalised, much larger rtcs were visible and newly synthesized cellular dna was not apparent, here hsc completely co-localised with newly synthesized viral dna and rta (fig ciii and ciii') . the location of the other two main hsp isoforms (ihsp and grp ) during kshv lytic replication was also investigated by indirect immunofluorescence microscopy. ihsp was cytoplasmic in unreactivated trex bcbl -rta cells (s ai fig), in contrast, an increase in nuclear ihsp labelling was observed in reactivated cells, which displayed similar chaperone foci as those seen during early reactivation for hsc (s aii fig). occasionally, cells displayed rtcs completely filled by ihsp (s aiii and s aiv fig, asterisks) . large ihsp foci positioned adjacent to rtcs were also seen in reactivated cells at later time points (s aiv fig arrows) . co-localisation of ihsp with actively replicating viral dna was also observed during late reactivation (s b fig). hsc and ihsp nuclear foci appeared at the same time as kshv rtcs were assembled, suggesting that these chaperones could be involved in rtc assembly. additionally, complete co-localization of hsc and ihsp with viral dna indicated that these chaperones could also participate in viral dna replication and/or capsid assembly. in contrast, the endoplasmic reticulum (er) hsp isoform, grp , was not redistributed in reactivated trex bcbl -rta cells (s fig) , consistent with its er retention signal [ ] . nevertheless, reactivated cells seemed to accumulate larger amounts of grp in the er. to confirm these results, immunoflourescence studies were also performed using hek- t prior to rtc formation, when rta was diffuse in the nucleus (ii). at h reactivation viral rta was assembled into incipient rtcs. hsc was not detected in the cytoplasm and instead numerous nuclear foci that were positioned predominantly adjacent to rtcs were seen (iii). at later reactivation times very large hsc foci were completely recruited within rtcs. hsc cytoplasmic depletion is indicated (iv arrows). nucleus highlighted in yellow shows a typical kshv fully developed-rtc with cellular chromatin marginalised to the nuclear periphery. (b) unreactivated (-) or reactivated for h (+) trex bcbl -rta cells were fractionated into whole cell (wc), cytoplasmic (c) and nuclear (n) fractions. equal amounts of total protein from each fraction were analysed by western blotting. nuclear fractions were characterised by enrichment of lamin b and absence of cytoplasmic gapdh, while cytoplasmic fractions showed the inverse profile. a small decrease in cytoplasmic hsc which correlated with a small increase in nuclear hsc was detected in reactivated cells, further supporting that hsc was redistributed from the cytoplasm to the nucleus. (c) trex bcbl -rta cells remained unreactivated (i) or reactivated for h (iiiii') followed by triple-labelling with antibodies specific for rta and hsc and click-it edu alexa fluor , the latter allowed visualization of newly synthesized dna. during early reactivation, a proportion of hsc protein was adjacent to small viral rtcs. arrow points to a small kshv rtc filled with viral dna (ii). hsc also partially co-localised with viral dna (ii) and rta (ii') in small rtcs. during later reactivation times, hsc completely moved within fully-developed rtcs strongly co-localising with viral dna (iii) and rta (iii'). rkshv. cells, in which the presence of the recombinant virus is tracked by expression of the green fluorescent protein (gfp) from the ef- alpha promoter and lytic reactivation levels are monitored by expression of the red fluorescent protein (rfp) from the kshv lytic noncoding polyadenylated nuclear (pan) rna promoter. unreactivated cells displayed cytoplasmic ihsp and hsc labelling, whereas~ % of reactivated cells ( h reactivation) revealed nuclear ihsp and hsc accumulations that appeared to assemble within small rtcs (s a fig arrows and s b fig arrows respectively) . the incomplete redistribution of ihsp and hsc foci into rtcs was likely due to a more asynchronous progression through the lytic cycle in induced cells by tpa and sodium n-butyrate than in doxycycline-induced trex bcbl -rta cells. similarly to trex bcbl -rta cells, grp was not redistributed in hek- t rkshv. cells, although larger amounts appeared to accumulate in the er of reactivated cells (s c fig), in agreement with the significantly increased amounts of grp detected in the ne region of these cells ( table ) . these results clearly demonstrate that kshv specifically redistributes the molecular chaperones, hsc and ihsp , from the cytoplasm to the nucleus, in contrast to grp , which coincides with the initial formation of kshv rtcs. treatment with the small molecule inhibitor ver- abrogated viral protein synthesis at non-cytotoxic concentrations members of the hsp chaperone family possess an n-terminal nucleotide binding domain with atpase activity which is essential for their function. to examine the implications of hsc and ihsp redistribution into kshv rtcs, a small molecule inhibitor, ver- , (a dibenzyl- -aminoadenosine analog) was utilised. this is the only inhibitor that has been demonstrated to specifically target the highly homologous atpase pocket present in the three main human hsp isoforms [ , , , ] , which is highly divergent structurally from the atpase pocket found in chaperones of the hsp family [ , ] . as such, ver- functions as an atp mimetic that specifically inhibits the atpase activity of members of the hsp family. initially, cytotoxicity of this compound was assessed in unreactivated trex bcbl -rta cells. following h inhibitor exposure, using a non-radioactive mts assay, which quantitatively assesses cell proliferation, a drastic reduction in cell metabolic activity was seen for inhibitor concentrations higher than μm (s fig) , thus concentrations ranging from to μm were used for further cytotoxicity characterization (fig ai) . the inhibitor triggered apoptosis in a dose-dependent manner as demonstrated by the caspase -mediated cleavage of full length poly [adp-ribose] polymerase (fl-parp ) protein into cleaved parp (cl-parp ) (fig aii) . small amounts of cl-parp were seen at μm and μm with a significant increase of this form after μm. these results were confirmed with apotox-glo triplex assay by quantitatively measuring viability, cytotoxicity and activation of effector caspases- / in the same sample well after h inhibitor treatment. a dose-dependent decrease in viability was evident from μm to μm while cytotoxicity and activation of caspases- / were only considerably increased at concentrations higher than μm ( fig b) . next, trex bcbl -rta cells were reactivated for h in the presence of drug vehicle dmso ( . %) or a range of increasing inhibitor concentrations. cells treated with the inhibitor at non-cytotoxic concentrations ( to . μm) revealed a drastic reduction in the levels of early orf and late minor capsid (mcapsid) proteins. a moderate reduction in the immediateearly rta protein was also seen ( fig c) . of note, when detecting the fusion protein rta-myc, which expression is not from the kshv genome, with anti-myc antibody, the decrease in rta-myc was not as dramatic as that seen for viral rta, suggesting that the decrease in viral proteins was not due to a general cytotoxic effect of the inhibitor on the cells, and that viral, but not cellular proteins were specifically affected. as an additional cellular control, protein levels of the large subunit of rnapii, which has a half-life of - h [ ] , was assessed with antibodies specific for the different phosphorylated forms of rnapii. protein levels of these forms were not significantly changed in the presence of the inhibitor, nor were those of hsc or hsp proteins. importantly, in the presence of ver- , ihsp levels were not upregulated. ihsp upregulation is a universal hallmark of hsp inhibition not only in vitro [ ] but also in clinical trials [ ] , pointing to selectivity for hsp isoforms by ver- . hsp and hsp chaperone machineries have been reported to be crucial for the stability and/or maturation of multiple viral proteins [ , , [ ] [ ] [ ] [ ] [ ] . therefore to ascertain whether hsp isoforms could stabilise the essential kshv lytic proteins rta and orf , trex bcbl -rta cells were reactivated for h to allow sufficient viral protein expression followed by addition of dmso control or ver- in conjunction with cycloheximide (chx) at μg/ml to block de novo protein synthesis. protein lysates were then collected at different times after addition of chx. the half-life of rta and orf proteins from inhibitor-treated cells were not altered compared with dmso-treated cells (fig d) . these results indicate that the observed decrease in viral protein synthesis was prior to translation and that neither viral rta nor orf protein were client proteins of the hsp isoforms. as such, this highlights a potentially novel role of hsp isoforms in the kshv replication cycle independent of viral protein stability and maturation. to further corroborate these results, experiments were also repeated in hek- t rkshv. cells. again, cell metabolic activity, parp cleavage, viability, cytotoxicity and activation of caspases- / in unreactivated cells were all assessed at a range of increasing inhibitor concentrations (fig a and b ). on this occasion, the inhibitor did not trigger apoptosis (fig aii and b ) but it caused a pronounced cell cycle arrest at h exposure at concentrations of μm demonstrated by a reduced number of metabolically active cells that exhibited no increased cytotoxicity [ ] (fig b) . it is known that the apoptotic potential of ver- is cell line-dependent and that ver- can cause cell cycle arrest in human colon, breast and lung tumour cell lines [ , ] . hek- t rkshv. cells were also reactivated for h in the presence of drug vehicle dmso ( . %) or increasing inhibitor concentrations. endogenous rta, orf and mcapsid protein levels were moderately reduced in cells treated at an inhibitor concentration of μm and severely reduced at μm while cellular proteins remain unaffected ( fig c) . these were relatively high inhibitor concentrations compared with trex bcbl -rta cells; nonetheless a concentration of μm has been shown before to be necessary for inhibition of hsp isoforms in human carcinoma cell lines [ ] . as previously seen in trex bcbl -rta cells, when blocking de novo protein synthesis with chx at μg/ml in hek- t rkshv. cells, the half-life of rta and orf proteins were not reduced even in the presence of ver- at μm ( fig d) . this supports the findings seen in trex bcbl -rta cells, suggesting that the decrease in viral protein production was due to a pretranslation event. using an mts assay. (ii) a dose-dependent cleavage of full length (fl) parp protein into cleaved (cl) parp was observed in the presence of ver- . (b) unreactivated cells were exposed for h to increasing inhibitor concentrations. concentrations higher than μm resulted in increased cytotoxicity and activation of effector caspases- / as demonstrated by quantification with apotox-glo triplex assay. (c) immunoblot analysis showing that reactivated cells treated with non-cytotoxic inhibitor concentrations ( to . μm) for h revealed a decrease in viral proteins compared with dmso-treated samples while cellular proteins remained unaffected. rnapiio denotes hypophosphorylated rnapii. ser and ser rnapii denote serine -and serine hyperphosphorylated rnapii forms respectively. (d) cells were reactivated for h to allow robust viral protein production. then, dmso control ( . %) or ver- was added in conjunction with cycloheximide (chx) at μg/ml to block de novo protein synthesis. protein lysates were collected at several times post-chx addition ( , , , and h) and analysed by western blotting. ver- did not alter the half-life of rta or orf protein, thus these proteins were not clients of hsp isoforms. the small molecule inhibitor ver- caused a significant reduction in viral transcripts, viral dna and progeny at non-cytotoxic concentrations as the block in kshv protein synthesis occurred pre-translationally, viral gene expression was quantified in the absence or presence of ver- . trex bcbl -rta cells were reactivated for h and two-step quantitative reverse transcription pcr (qrt-pcr) was carried out to quantify a range of viral transcripts. a significant decrease in early (pan, orf , k and vgpcr), late (gl and gb) viral transcripts and ori-lyt transcripts was observed in a dose-dependent manner, with all transcripts with the exception of vgpcr being significantly reduced at an inhibitor concentration of μm (fig a) . to determine whether cellular transcription was negatively affected in the presence of ver- , firstly the stability of gapdh transcript was determined in mrna decay assays using the transcriptional inhibitor actinomycin d (acd) ( . μg/ml) in trex bcbl -rta cells. after h of acd treatment, the amount of gapdh mrna was reduced by half (fig b) , indicating a short stability of gapdh mrna in this cell line. we then plotted the raw cycle threshold (c t ) for gapdh transcript from the same samples in which viral transcripts had been quantified after h of ver- treatment. as the same amount of total rna was converted into cdna for all samples, if cellular transcription was not compromised a very similar c t is expected for all samples. indeed, samples treated with up to μm ver- were all within . c t from the . c t of dmso-treated samples. only after concentrations higher than μm gapdh mrna levels were significantly reduced compared to dmso-treated samples as shown by a significantly higher c t value ( fig c) . this is consistent with the cytotoxicity profile of ver- in trex bcbl -rta cells (fig a and b ) and the clear decrease in rta-myc protein (which expression is not from the kshv genome) at inhibitor concentrations higher than μm ( fig c) . taken together, these results suggest that cellular transcription was compromised at concentrations of ver- higher than μm while at concentrations lower than μm transcription was occurring normally. interestingly, transcription of viral genes which also require host rnapii for their expression was negatively affected even at ver- concentrations lower than μm. next, we assessed whether the inhibitor also caused a reduction in viral dna replication. trex bcbl -rta cells were reactivated for h, total dna was isolated and realtime qpcr was performed using primers specific for orf . while dmso-treated cells reached~ -fold increase in viral dna load, inhibitor concentrations of μm or higher resulted in a significant reduction (> %) in viral dna ( fig d) . moreover, the production of infectious kshv virions in trex bcbl -rta cells was evaluated in the presence of ver- at . μm or vehicle drug dmso. for this, cells were reactivated and treated for h, culture medium was centrifuged and incubated for h with hek- t cells. total rna was then isolated and qrt-pcr carried out. a significant reduction (~ %) in the release of infectious viral progeny was observed in inhibitor-treated cells ( fig e) . viral transcripts were also quantified at h reactivation in hek- t rkshv. cells in the absence or presence of ver- . at non-cytotoxic concentrations of μm there was a drastic decrease for all parp was observed faintly. (b) unreactivated cells were exposed for h to increasing inhibitor concentrations. a reduced number of metabolically active cells accompanied by no increased cytotoxicity was observed in the presence of the inhibitor. there was no activation of effector caspases even in the presence of μm ver- . this phenotype is consistent with cell cycle arrest. results were quantified with apotox-glo triplex assay. (c) immunoblot analysis showing that reactivated cells treated with μm ver- for h resulted in abrogation of viral proteins compared with dmso-treated samples while cellular proteins remained constant. (d) cells were reactivated for h to allow robust viral protein expression. then, dmso control ( . %) or ver- was added in conjunction with cycloheximide (chx) at μg/ml to block de novo protein synthesis. protein lysates were collected at several times post-chx addition ( , , , and h) and western blotting was performed. as previously seen in trex bcbl -rta cells, ver- did not alter the half-life of rta or orf protein. hsp isoforms are essential for kshv lytic replication fig a) . it is intriguing that orf mrna levels did not show a clear dose-response with the inhibitor as seen for orf mrna in trex bcbl -rta cells; however the levels were decreased compared with dmso-control cells. this is the only transcript of all the viral transcripts tested in both cell lines that did not show a dose-response. however, western blotting did reveal a complete reduction of orf protein in cells treated with > μm ver- ( fig c) . this suggests that hsp isoforms may also play a role in the folding of orf protein. in fact, hsc has been previously reported to associate with at least - % of newly synthesized proteins during their biogenesis [ ] , thus, a role for hsp isoforms in folding viral proteins cannot be ruled out. in order to evaluate cellular transcription activity in the presence of ver- , we first determined gapdh transcript stability in mrna decay assays using acd ( μg/ml) in hek- t rkshv. cells. in contrast to trex bcbl -rta cells, gapdh transcripts were very stable, with no significant reduction in their levels after h of acd treatment ( fig b) . the half-lives of two cellular transcripts, srag (a cellular mrna export adapter) and histone h a (h a. ) were also determined in hek- t rkshv. cells. srag transcripts were reduced to % following h acd treatment (fig c) , while the h a. mrna was very unstable with nearly % reduction after h acd treatment ( fig d) . we then plotted the raw c t values for gapdh transcript from samples in which viral transcripts had been quantified after h of ver- treatment; these did not significantly change in the presence of ver- ( fig e) . next, the unstable srag and h a. mrnas were measured in the same samples in which viral transcripts had been quantified after h of ver- treatment. in contrast to viral transcripts, both cellular transcripts were not significantly reduced, indicating that transcription of cellular genes was occurring normally even in the presence of high ver- concentrations ( fig f) . taken together, these results demonstrate that inhibition of hsp isoform function abrogated the expression of viral genes from various temporal classes; however cellular rna-pii-mediated transcription was not compromised when using ver- at non-cytotoxic concentrations. following quantification of viral transcripts in both cell lines, it appeared that the reduction seen in viral gene expression, protein production and infectious virion production could be a consequence of a significant global reduction of viral transcripts in inhibitor-treated cells. this led to the possibility that hsp isoforms could be implicated in activation of viral promoters and subsequent transcription or alternatively hsp isoforms were required for kshv rtc formation. because viral rta and hsc co-localized in trex bcbl -rta cells and rta is (orange colour), late (red colour) and ori-lyt viral transcripts were all significantly reduced at μm ver- compared to the levels found in dmsotreated samples. all samples were normalised to gapdh. results show the mean of three biological replicates with error bar as standard deviation. (b) the stability of gapdh transcript was assessed in unreactivated trex bcbl -rta cells in the presence of the transcriptional inhibitor actinomycin d (acd) ( . μg/ml) or control dmso ( . %). cells were collected over the time points indicated and total rna was extracted followed by qrt-pcr. after h acd treatment, the amount of gapdh transcripts was reduced by more than half (c t = . ) compared with dmso-treated cells (c t = . ). a further reduction was observed at h treatment (c t = . ). the average of two biological replicates with error bar as standard deviation is shown. (c) the amount of gapdh transcripts did not significantly decrease when using ver- for h at concentrations lower than μm compared to dmso-treated samples. in contrast, concentrations higher than μm significantly showed a c t lower than dmso samples, indicating compromised cellular transcription at these inhibitor concentrations. the same samples in which viral transcripts had been quantified were used to plot all c t values. results show the mean of three biological replicates with error bar as standard deviation. (d) cells were reactivated for h, total dna was isolated and real-time qpcr was performed. viral dna load was significantly decreased at μm ver- . results show the mean of three biological replicates with error bar as standard deviation. (e) trex bcbl -rta cells were reactivated for h in the presence of ver- at . μm or vehicle drug dmso ( . %). the culture medium was then incubated for h with hek- t cells followed by total rna extraction and qrt-pcr. a significant decrease in viral progeny was seen in inhibitor-treated cells. results show the mean of three biological replicates with error bar as standard deviation. the master latent-lytic transactivator for multiple kshv immediate-early, delayed-early and late promoters [ ] [ ] [ ] [ ] [ ] , we further investigated the possibility that hsc was required for rta-mediated transactivation. initially we assessed whether an interaction occurred between hsc and rta in the absence of other viral proteins or dna. for this, hek- t cells were transiently transfected for h with control pegfp or prta-egfp and immunoprecipitations were carried out using a gfp-specific antibody. rta-egfp precipitated endogenous hsc in contrast to the control egfp protein (fig a) . in addition, hek- t cells were transfected with control pegfp or prta-egfp for h and examined by immunofluorescence. in cells expressing egfp protein, endogenous hsc remained cytoplasmic (fig bi) , while in egfp-rta-expressing cells hsc strongly co-localised with rta in the nuclei, suggesting rta expression alone is sufficient to redistribute hsc into the nucleus (fig bii) . similar nuclear redistribution was also seen for endogenous ihsp (s fig). next, we determined whether ver- was able to disrupt the interaction between egfp-rta and hsc in hek- t cells. hek- t cells exhibited a very similar cytotoxicity profile to that seen in hek- t rkshv. cells (s fig). hek- t cells were transiently transfected with prta-egfp or control pegfp. to allow maximal protein expression and avoid interference of the inhibitor with the transfection, the inhibitor was added at h post-transfection and incubated for a further h, prior to immunoprecipitations being performed. western blot analysis revealed that the inhibitor did not disrupt the interaction between hsc and rta even at high inhibitor concentrations ( μm) (fig c) , suggesting that the atpase function of hsc is not required for the interaction with rta protein. therefore, to investigate whether hsc was required for rta-mediated transactivation of the rta-responsive orf promoter, a dual-luciferase reporter assay system was utilised. hek- t cells were co-transfected with prta-egfp along with the renilla luciferase vector and either the orf promoter firefly luciferase reporter vector, or the empty reporter vector (pgl -basic). the same co-transfections were performed using pegfp, as a negative control. h post-transfection, cells were exposed for h to increasing concentrations of ver- and luciferase activities were measured. longer exposure times to the inhibitor affected the formation of the control renilla luciferase protein which has a half-life of h, suggesting that hsp isoforms may be required for the folding/maturation of this enzyme. in the presence of egfp-rta and the orf promoter reporter construct, the orf promoter activity was increased~ -fold, while the empty vector had a~ -fold increase. however, orf promoter activity was not significantly decreased in the presence of ver- ( fig d) . to confirm this result, hek- t cells were also transiently co-transfected with ppan-wt, a plasmid encoding the genomic region of wild type pan rna including its promoter region [ ] , and either prta-egfp or control pegfp. h post-transfection either vehicle drug dmso or ver- at μm was added and incubated for a further h followed by qrt-pcr. in quantification by rt-pcr of immediate-early (blue colour), early (orange colour), late (red colour) and ori-lyt viral transcripts showed that all transcripts were significantly decreased at μm ver- compared to the levels found in dmso-treated samples. all samples were normalised to gapdh. results show the mean of three biological replicates with error bar as standard deviation. (b-d) the stability of cellular gapdh, srag and h a. transcripts was determined in mrna decay assays using actinomycin d (acd) ( μg/ml) or control dmso ( %) in hek- t rkshv. cells. cells were collected over the time points indicated and total rna was extracted followed by qrt-pcr. the average of two biological replicates with error bar as standard deviation is shown. (b) gapdh transcripts were very stable, with no significant reduction in their levels after h of acd treatment. (c) at h acd treatment srag mrna levels were reduced by % showing a relatively short stability. (d) h a. mrna levels were very unstable, with nearly % reduction after h acd treatment. (e) gapdh transcript levels remained unchanged following h ver- treatment in reactivated hek- t rkshv. cells. the same samples in which viral transcripts had been quantified were used to plot all c t values. (f) the unstable cellular srag and h a. mrnas were not significantly decreased in the presence of ver- treatment compared with dmso treatment for h indicating that cellular transcription was not compromised even at high concentrations of ver- . the same samples in which viral transcripts had been quantified were used to quantify srag and h a. . all samples were normalised to gapdh. however, no significant decrease in the amount of pan rna was seen in inhibitor-treated cells (fig e) , indicating that rta-mediated promoter transactivation and subsequent synthesis of pan rna was occurring normally in the presence of ver- . these data demonstrate that hsp isoforms did not directly enhance rta-mediated transactivation. to assess the transcription activity of cellular rnapii in the presence of the inhibitor, the half-life of pan rna was determined in hek- t cells co-transfected with ppan-wt and prta-egfp. following h post-transfection, acd ( μg/ml) or dmso control ( . %) was added. after h of transcription inhibition, pan rna levels were reduced to % compared to dmso-treated cells (fig f) . this quick reduction in the stability of pan rna in the absence of orf protein is in agreement with previous reports [ ] . thus, if ver- was blocking general rnapii transcription, a significant reduction in pan rna levels should be observed after h of inhibitor treatment; however, pan rna levels were not reduced in cells treated with ver- for h (fig e) . a dramatic reduction in early, late and ori-lyt transcripts after h treatment with ver- was evident during kshv infection in both cell lines used. however, hsp isoforms were not required for rta-mediated transactivation of kshv promoters in transiently transfected cells. thus, we next monitored kshv rtc formation in the absence or presence of the inhibitor during kshv lytic infection. trex bcbl -rta cells were reactivated and treated with either control dmso or μm inhibitor. at h reactivation cells were fixed and immunofluorescence was performed using rta-and hsc -specific antibodies. dmso-treated cells displayed abundant rtcs and numerous nuclear hsc foci that partially co-localised with rtcs. hsc cytoplasmic depletion was also observed (fig a and a') . in contrast, inhibitortreated cells showed diffuse nuclear rta that was not able to assemble into rtcs (fig b and b' ). in these cells, hsc nuclear foci were still visible, but these were much less numerous and smaller compared with the foci seen in dmso-treated cells. significantly, following ver- treatment, hsc was observed in the cytoplasm of reactivated cells (fig b and b' ). hsc subcellular localization was also analysed in dmso-and inhibitor-treated cells by confocal profiling. profiling was performed by drawing a line long enough (~ μm) to cover the nucleus and cytoplasm at either side of the nucleus. if an hsc pixel intensity data point was equal or greater than to the data point in the previous and subsequent pixel and above the background noise, it was considered as an hsc peak, representing an hsc foci. analysed by immunofluorescence. endogenous hsc was cytoplasmic in cells expressing egfp (i). in contrast, hsc strongly co-localised with egfp-rta in the nucleus, but not the nucleoli (ii). (c) hek- t cells were transiently transfected with control pegfp or prta-egfp. h posttransfection, ver- was added and incubated for a further h. then, immunoprecipitations were carried out using a gfp-specific antibody. even in the presence of μm ver- the interaction between hsc and egfp-rta was not disrupted. (d) hek- t cells were co-transfected with prta-egfp along with the renilla luciferase vector and either the orf promoter firefly luciferase reporter vector, or the empty reporter vector (pgl -basic). the same co-transfections were performed using pegfp as a negative control protein. h post-transfection, cells were exposed for h to ver- and luciferase activities were measured. comparable activation of the rta-responsive orf promoter to that seen in dmso-treated cells occurred in cells treated with μm ver- . the results of three independent transfections were averaged with error bars as standard deviation. (e) hek- t cells were transiently co-transfected with ppan-wt and either prta-egfp or control pegfp. h post-transfection either vehicle drug dmso ( . %) or μm ver- was added and incubated for a further h. total rna was then extracted and qrt-pcr performed. rta-mediated promoter transactivation and subsequent synthesis of pan rna occurred at a similar rate in the presence of ver- or control dmso. the results of three independent transfections were averaged with error bars as standard deviation. (f) the stability of pan rna was determined in hek- t cells that had been co-transfected with ppan-wt and prta-egfp. following h post-transfection, actinomycin d (acd) ( μg/ml) or dmso control ( . %) was added. cells were collected over the time points indicated and total rna was extracted followed by qrt-pcr. the average of two biological replicates with error bar as standard deviation is shown. doi: . /journal.ppat. .g hsp isoforms are essential for kshv lytic replication dmso-treated cells predominantly showed hsc peaks only within the dapi boundaries, that is, within the nucleus (fig a") . inhibitor-treated cells displayed hsc peaks outside the dapi boundaries, that is, in the cytoplasm (fig b" asterisk) more often than control cells. a significant increase in cytoplasmic hsc peaks was seen in inhibitor-treated cells compared with dmso control cells (fig c) . fractionation of reactivated trex bcbl -rta cells in the presence or absence of ver- , also pointed to slightly higher levels of hsc in the cytoplasm and a decrease in nuclear hsc in inhibitor-treated cells compared with dmso control cells (fig d) . cells were also labelled with click-it edu alexa fluor and an antibody specific for hsc (s fig). the percentage of assembled rtcs in dmso-and inhibitor-treated cells was also calculated. in dmso-treated cells~ % of cells presented assembled rtcs while only % of inhibitor-treated cells showed assembled rtcs (fig e) . this demonstrates that chaperone recruitment to the nucleus is essential for the assembly of kshv rtcs and treatment with ver- was sufficient to impair nuclear chaperone recruitment and kshv rtc formation. the subcellular localisation of rnapii was also assessed by indirect immunoflourescent labelling in trex bcbl -rta cells with the monoclonal antibody ctd h , which specifically recognises unphosphorylated and serine- phosphorylated rnapii. in unreactivated cells, rnapii exhibited a nuclear localization, excluding the nucleolus (fig ai arrow) irrespective of the presence of dmso (fig ai) or the inhibitor (fig aii) . however, in reactivated and dmso-treated cells, rnapii was clearly hijacked to rtcs (fig bi) . in contrast, in reactivated cells treated with the inhibitor, rnapii was diffuse throughout the nucleus, but excluding the nucleoli, and formed very small foci (fig bii arrow) multiple kshv rtcs identified by rta-labelling were formed in dmso-treated cells to which large and numerous hsc foci were recruited. in these cells, hsc was depleted from the cytoplasm. (a') confocal images were subjected to profiling analysis using zeiss zen software. profiling was conducted for each cell in two representative confocal images taken with a -times objective; this included profiling a total of dmso-treated cells. a representative line profile (yellow line) and accompanying relative intensities of each channel at every pixel along the line is shown for a dmso-treated reactivated cell. the dapi intensity profile shows values below outside the nucleus boundary. rta profile shows a peak with intensity of that lies within the dapi boundaries corresponding to a kshv replication compartment. hsc peaks lie within the dapi boundaries demonstrating their nuclear location. an hsc intensity peak closely resembles the rta intensity peak (arrows), indicating co-localization of hsc with rta since both peaks occur at the same position along the line. (b) in cells reactivated and treated with μm ver- for h, rta was diffuse in the nucleus and not assembled into kshv rtcs. in addition, hsc nuclear foci were smaller and less abundant than in dmso-treated cells and hsc was not depleted from the cytoplasm. (b'). confocal profiling was performed for each cell in two representative confocal images taken with a -times objective; a total of inhibitor-treated cells were analysed. a representative line profile (yellow line) is shown for an inhibitor-treated cell. an hsc peak is seen outside the dapi boundaries corresponding to an hsc cytoplasmic peak (asterisk). (c) total hsc peaks and total nuclear hsc peaks per cell were combined for each confocal image and experimental condition. data was converted to percentage of nuclear and cytoplasmic peaks. cells exhibited very small rnapii foci and diffuse nuclear edu labelling ( s bii fig). to confirm that inhibition of hsp isoform function was able to abolish rnapii recruitment to viral genomes, we also utilised chromatin immunoprecipitation (chip) assays in trex bcbl -rta a polyclonal antibody against rta and a monoclonal antibody (ctd h ) against rnapii were used for immunoflourescence analysis. rnapii protein was nuclear excluding the nucleoli (arrows) regardless of inhibitor treatment. (b) trex bcbl -rta cells were reactivated and treated with either control dmso ( . %) or μm ver- for h. in dmso-treated cells rnapii and rta were recruited to viral rtcs (i arrow) while in inhibitor-treated cells rta was diffuse in the nucleus and rnapii formed numerous small foci that excluded the nucleoli and resembled prereplicative sites (ii arrow). (c) trex bcbl -rta cells were either reactivated (r) in the presence of dmso ( . %) or μm ver- for h. unreactivated (u) cells treated with dmso ( . %) were used to assess levels of lytic reactivation. chip assays were carried out with either monoclonal ctd h rnapii antibody or mouse control antibody (igg). in the presence of ver- , significantly reduced amounts of rnapii bound to viral promoters were detected. the average of three independent experiments is shown with error bars as standard deviation. cells that were either reactivated for h in the presence of dmso or μm ver- ( fig c) . in unreactivated control cells, there was a clear enrichment of rnapii at the promoter of gapdh gene, while rnapii occupancy at viral promoters was minimal. however, kshv reactivation in dmso-treated cells led to a drastic reduction of rnapii at the promoter of gapdh and a significant increase of rnapii at the viral promoters in agreement with the previous immunofluorescence results, showing rnapii recruitment to rtcs (fig bi) . conversely, upon treatment of the hsp inhibitor, the amount of rnapii bound at the promoters of ori-lyt, k and orf was decreased by~ %,~ % and~ % respectively compared with dmso-treated reactivated cells. importantly, these results indicate for the first time that inhibition of hsp isoforms leads to a severe impairment in rnapii recruitment at multiple viral promoters including that of ori-lyt. to further confirm the essential role of hsc and ihsp in the formation of kshv rtcs, specific individual sirna-mediated depletion of both isoforms was performed in hek- t rkshv. cells. following four days post-sirna transfection, cells were reactivated for h and rna and protein were extracted from the same sample. hsc depletion was evaluated by western blotting and by qrt-pcr, the latter showing~ % hsc mrna knockdown ( fig a) . in contrast, ihsp mrna levels were not affected confirming specificity of the hsc sirna. despite a successful knockdown at the mrna level, significant amounts of hsc protein remained in hsc -depleted cells (fig b) . however, even with this modest amount of depletion at the protein level, all viral transcripts (with the exception of pan) displayed a significant reduction in hsc sirna-treated samples compared with the scramble sirnatreated cells as demonstrated by qrt-pcr analysis (fig c) . orf , orf and gl mrna levels were decreased by~ % following hsc knockdown. ori-lyt and rta transcripts were reduced by~ % and gb levels by~ %. this suggests that depletion of hsc impaired the expression of viral genes from various temporal classes and thus hsc may be necessary for kshv rtc formation. viral dna replication was also assessed following hsc knockdown. for this, after four days post-sirna transfection, cells were reactivated for a further h. there were no significant differences between scramble and depleted cells (fig d) . the production of infectious kshv virions was also evaluated after h reactivation. again, no significant differences were seen between scramble and depleted cells ( fig e) ; however this result is not surprising due to incomplete hsc depletion even after seven days post-transfection ( fig f) . this highlights the remarkable stability of hsc protein in this cell line and it suggests that hsc depletion was enough to cause a reduction in viral transcripts but not enough to cause a reduction in the amount of viral proteins, thus kshv lytic replication remained unaffected. it is also possible that ihsp was able to functionally compensate for hsc . next, specific depletion of ihsp was performed in hek- t rkshv. cells. ihsp depletion at the mrna level reached~ % knockdown (fig a) which correlated with efficient depletion at the protein level ( fig b) . however, in ihsp -depleted cells, the majority of viral gene expression was unaffected, apart from gl and pan transcripts which were decreased bỹ % and~ % respectively (fig c) . importantly, taken together these results indicate that partial depletion of hsc at the protein level is sufficient to cause a reduction in viral transcription, suggesting an essential role of this chaperone in the formation of kshv rtcs, whereas ihsp may have a more subtle effect on viral gene expression. to further support the essential role of hsc during kshv rtc formation, hsc was specifically silenced in trex bcbl -rta cells. for this, nucleofection was carried out. transfection efficiency was monitored co-transfecting the hsc sirna together with pmaxgfp, which encodes maxgfp, a green fluorescent protein from the copepod pontellina p. (fig a) . note that higher transfection efficiency is expected for the sirna due to the smaller size of this compared with the plasmid dna. after four days post-nucleofection, hsc mrna levels showed~ % knockdown (fig b) with a minor depletion at the protein level (fig c) . due to the stability of hsc protein, cells were incubated for six days post-nucleofection followed by a further h reactivation and immunofluorescence for hsc and rta was carried out. rtc formation dramatically decreased after nucleofection. similar impairment in kshv lytic replication has previously been reported in electroporated trex bcbl -rta cells [ ] ; nevertheless, in scramble sirna-treated cells, groups of cells could still be seen displaying rtcs to which hsc was relocated (fig d) . in contrast, in hsc sirna-treated cells, fewer rtcs were visible and these exhibited nuclear hsc (fig e yellow arrow) while cells fully depleted of hsc , as identified by lack of hsc -labelling, did not form rtcs (fig d white arrows) . this result strongly suggests that hsc is an essential chaperone for the formation of kshv rtcs in trex bcbl -rta cells. current quantitative proteomics approaches have become an invaluable tool for large-scale, high-throughput identification of proteins in complex biological samples. moreover, advances in subcellular fractionation offer a way to further reduce the complexity of the samples to be analysed by lc-ms/ms, allowing identification of low abundance proteins. in this present study, we have developed a novel quantitative proteomic approach enhanced by subcellular fractionation that has enabled us to elucidate the cellular protein composition of kshv rtcs. this novel approach led to the identification of several upregulated pathways in reactivated cells associated with the ne fraction (s table) . the first scored pathway was rna post-transcriptional modification, the second highlighted pathway was protein synthesis with different ribosomal proteins identified and the third scored pathway was dna replication, recombination and repair. in addition, the isolation of the ne regions from unreactivated and reactivated cells followed by the uncommon use of urea for protein extraction led to the mass spectrometric identification of several hsp isoforms and their respective co-chaperones at significant levels in ne-associated rtcs of reactivated cells. immunoflourescence analysis confirmed that endogenous hsc and ihsp were redistributed from the cytoplasm to the periphery of kshv rtcs where they formed multiple nuclear foci during early lytic replication. the formation of rtcs coincided in time with the appearance of nuclear chaperone foci. similar virus-induced-chaperone-enriched (vice) domains that form adjacent to hsv- rtcs, have also been observed in hsv- -infected cells and contain sequestered hsc , ihsp , hsp and hsp [ , , ] . hsv- induced-vice domains also accumulate ubiquitinated proteins and components of the proteasome and function to sequester misfolded proteins away from rtcs and serve as protein quality control centers [ , ] . ihsp redistribution very similar to that seen in hsv- induced-vice domains was observed in multiple unreactivated and total rna and protein were extracted from the same sample. despite achieving~ % hsc knockdown at the mrna level (a), significant amounts of hsc protein remained in hsc sirna-treated samples (b) . despite this small knockdown at the protein level, in hsc -depleted cells there was a significant decrease in the amount of multiple viral transcripts from various temporal classes as quantified by rt-pcr analysis (c). viral dna replication was assessed by qpcr following hsc -knockdown. cells were reactivated for h after four days post-sirna transfection. no significant differences were observed between scramble and hsc -treated cells (d). similar virion production was detected in scramble and hsc -treated cells. cells were reactivated for h after four days post-sirna transfection, culture medium was centrifuged and immediately incubated with hek- t cells for h. total rna was then isolated and qrt-pcr carried out (e). incomplete hsc -knockdown at the protein level was observed by western blotting even after seven days post-sirna transfection in hek- t rkshv. cells. this demonstrates the remarkable stability of hsc in this cell line (f). the average of three independent transfections is shown with error bars as standard deviation (a, c-e). doi: . /journal.ppat. .g hsp isoforms are essential for kshv lytic replication fig . the majority of kshv lytic gene expression was unaffected following specific depletion of ihsp in hek- t rkshv. cells. cells were transfected twice with nm ihsp -specific sirna or nm scramble sirna. following four days post-sirna transfection, cells were either reactivated cells undergoing kshv lytic replication, moreover this labelling was also observed for hsc in some cells. hsc and ihsp remain positioned exclusively adjacent to hsv- rtcs. however, in contrast; these chaperones were very dynamic during kshv lytic replication and surprisingly very large chaperone foci were recruited within kshv rtcs when viral dna was actively synthesised (fig c and s b fig) . therefore, our results strongly suggest that these chaperones may also aid replication of kshv genomes during lytic replication. we therefore further suggest that hsp isoforms may have an important role in the assembly and activation of pre-initiation complexes on the origin of dna replication. this is supported by observations in prokaryotes, such as in plasmid p [ , ] , and eukaryotes, such as saccharomyces cerevisiae [ ] , human papillomavirus- [ , ] , hsv- [ ] and bacteriophage λ [ , ] . interestingly, hsp isoforms have also been identified in the kshv virion [ , ] and therefore an additional role of these chaperones in kshv capsid assembly may also be possible. cancer cells greatly rely on members of the hsp and hsp chaperone families for their growth and survival [ , ] , consequently, significant efforts have been invested to design small molecule inhibitors specific for these atpases as novel anticancer therapeutics. this has been successfully achieved for the hsp family with several inhibitors undergoing phase iii clinical trials [ ] , although the clinical efficacy of these inhibitors has been somewhat limited because of the inevitably upregulation of ihsp , when inhibiting hsp [ , ] . the development of hsp inhibitors has substantially lagged behind that of hsp inhibitors due to lack of natural product inhibitors specific for hsp isoforms, due to the highly polar nature of the hsp / hsc atp binding site and the high affinity for atp displayed by these atpases [ ] . nevertheless, in recent years several hsp inhibitors have been designed and tested in pre-clinical or clinical trials [ ] . however, a major challenge remains in finding inhibitors which can specifically discriminate between ihsp and hsc . to date, only peptide aptamers targeting ihsp are selective for a specific hsp isoform [ ] and also recently, methyl blue was reported to specifically inhibit ihsp by oxidizing a cysteine in its atpase domain, the same residue is absent in hsc allowing differential targeting of the isoforms [ ] . because both hsc and ihsp were specifically redistributed to kshv rtcs during lytic replication, we made use of a recently developed small molecule inhibitor, ver- , which targets the atpase pocket of the main three human hsp isoforms. this inhibitor used at non-cytotoxic concentrations was able to effectively abrogate early and late kshv transcription together with viral protein production, viral dna replication and viral progeny. therefore, our study highlights the potential of ver- or other novel hsp inhibitors to prevent kshv lytic replication in kshv-associated tumours. one could expect that hsp inhibition would be directly detrimental not only to cancer cell survival but also the virus-specific functions that are dependent on hsp isoforms. these results may have exciting implications in combination with the recently demonstrated efficacy of atp-competitive hsp inhibitors in blocking kshv latent cycle in vitro and in a xenograft kshv tumour model [ ] . it may be the case that combining hsp inhibitors with hsp inhibitors may lead to enhanced efficacy in eradicating latent kshv reservoirs. excitingly, in our cell culture models ver- abrogated lytic replication without severely affecting cell viability or triggering apoptosis. it is intriguing that inhibition of constitutively expressed hsc chaperone did not result in cell death. however, it is important to highlight previous studies carried out on the susceptibility of tumour cells versus normal cells to hsp inhibitors. in tumour cells, for h or remained unreactivated and total rna and protein were extracted from the same sample. efficient ihsp knockdown was achieved both at the mrna level (~ %) (a) and the protein level (b). a small decrease in the amount of gl and pan transcripts was observed in ihsp -depleted cells (c). the average of three independent transfections is shown with error bars as standard deviation. doi: . /journal.ppat. .g hsp is present entirely in multi-chaperone complexes with high atpase activity; in contrast, in normal cells hsp is in an uncomplexed conformation. these two distinct hsp presentations result in tumour hsp exhibiting a -fold higher binding affinity to an hsp inhibitor than normal hsp [ ] . thus, it is tempting to speculate that the multi-chaperone hsp foci that are recruited to kshv rtcs during lytic replication (fig and s fig) are more sensitive to ver- than the chaperones not assembled in vice domains which carry out the housekeeping functions for cell survival. this would explain why hsp inhibition had a profound effect on kshv lytic replication without affecting cell viability and support the idea that isoform specificity may not be a requirement for treatment of kshv-associated tumours. to establish the essential role of hsp isoforms during kshv lytic replication, we examined several possible functions. it could be hypothesised that hsp isoforms could be implicated in ) stabilization of essential viral proteins, ) clearing stalled rnapii during times of robust viral transcription, ) activation of viral promoters, ) formation of kshv rtcs. initially we hypothesised that hsp isoforms may be necessary for maintaining the stability of the key kshv viral proteins rta and/or orf . this was deemed a possibility as during kshv latency, the essential viral latency associated nuclear antigen (lana) protein has been recently shown to be a client protein of the hsp chaperone and several hsp inhibitors reduced the expression of lana [ ] . in addition, the lytic kshv k glycoprotein has also been reported to be a client protein of hsp [ ] . however, inhibition of hsp isoforms did not alter the half-life of rta or orf (figs d and d) . alternatively, hsc has been observed at the periphery of hsv- rtcs where it is also believed to aid in clearing stalled rnapii from viral genomes during times of active transcription [ ] . indeed, the serine- phosphorylated form of rnapii undergoes ubiquitination and robust proteasomal degradation during hsv- infection [ ] . in contrast, in kshv-reactivated cells, there was only a slight reduction of serine- phosphorylated rnapii in comparison with unreactivated cells even at late times ( h) post-reactivation ( fig c) and a significant decrease in the other rnapii forms was not evident (fig c) . this suggests that although robust rnapii degradation is a feature observed in virus infection, such as hsv- and influenza virus [ , ] , it may not be universally conserved. it is interesting to note that expression of a dominant-negative hsc (k m) that cannot hydrolyze atp during hsv- infection resulted in prevention of serine- rnapii degradation and rtcs formation [ ] . however, inhibition of hsp isoforms by ver- did not prevent the slight degradation of phospho-serine- rnapii protein ( fig c) . using transient transfections, we also demonstrated that hsp isoforms were not directly involved in the activation of viral promoters (fig d and e) . strikingly however, inhibition of hsp isoforms precluded kshv rtcs formation (fig ) and rnapii re-localization to viral promoters (fig ) , thus, blocking kshv rtcs formation led to abolishment of global viral transcription and subsequent protein synthesis and viral dna replication. these results, taken together with the differential hsc and ihsp labelling seen in hsv- and kshv-infected cells, suggest that these chaperones may be playing additional roles, such as participating in viral dna replication and/or capsid assembly, in kshv lytic infection compared with hsv- infection. importantly, in both viruses, hsv- and kshv, inhibition of hsc atpase function leads to a clear impediment in rtcs formation and presents a novel antiviral target for multiple herpesviruses. moreover, as hsv- and kshv belong to different subfamilies of the herpesviridae family (α and γ subfamily respectively), the key role of hsp isoforms in rtc formation may be conserved across all subfamilies. in support of this notion is the fact that hsc and ihsp have also been reported to be incorporated in the virion of the β-herpesvirus hcmv [ ] and the γ-herpesvirus ebv [ ] . our results also support that the finding of hsc and ihsp chaperones in the kshv virion [ , ] a decade ago was not casual, and that these chaperones are essential for kshv rtcs formation during lytic replication. ihsp has also been detected in the hsv- virion [ ] . to support the conserved role of hsp isoforms in herpesvirus infection, a recent study showed that cellular depletion of hsc protein significantly reduced hsv- viral output in cell culture without adversely affecting cell viability. depleting hsc from the hsv- virion also significantly reduced viral production by more than % [ ] . hsp isoforms may be recruited to rtcs for several reasons. firstly, the chaperone may sequester misfolded, modified or unwanted proteins away from rtcs. alternatively, hsp could produce a site for protein remodelling and/or degradation which may regulate or delay cellular pathways, such as the apoptosis cascade. finally, it may aid subtlety to clear stalled rnapii complexes during robust viral transcription and replication. an additional question which is yet to be addressed is the mechanism by which hsp isoforms are recruited to rtcs. one intriguing possibility is observations made during hsv- infection. here, hsv- icp has been shown to interact with hsc and is required for hsc nuclear foci formation [ ] . it will now be interesting to determine if the functional kshv homologue orf , also interacts with hsc and whether its nucleocytoplasmic shuttling ability is essential for hsc nuclear import. in summary, we have identified a new essential role for hsp isoforms during the formation of rtcs in kshv lytic replication. importantly, our results suggest that hsp inhibitors have the potential as novel kshv antiviral agents and it would now be interesting to test these in conjunction with other molecular chaperone inhibitors, specifically hsp inhibitors [ ] , which have the potential to eradicate latent kshv reservoirs in both in vitro and in vivo tumour models. trex-bcbl- -rta cells (kindly provided by dr. jae jung, university of southern california) are a bcbl- -based, primary effusion lymphoma (pel) b cell line that has been engineered to inducibly express exogenous myc-tagged rta by the addition of doxycycline, leading to a robust reactivation of the full kshv lytic cycle [ ] . the rkshv. cell line (kindly provided by dr. jeffery vieira, university of washington, seattle, usa) maintains kshv as a latent infection and was generated by infecting hek- t cells (atcc) with a recombinant kshv that contains a constitutively active puromycin resistance and gfp gene, and an rfp gene that is fused to an rta-responsive lytic cycle (pan) promoter; hence, expression of rfp can be used as a reporter of rta activity [ ] . hek- t rkshv. cells were grown in dmem (life technologies) supplemented with % foetal calf serum (fcs) (life technologies) and % penicillin/streptomycin (p/s). this cell line was kept under puromycin (sigma) selection ( . μg/ml). reactivation into the lytic cycle was achieved by addition of -o-tetradecanoylphorbol -acetate (tpa) ( ng/ml) and sodium n-butyrate (nab) (sigma) ( mm). the trex bcbl -rta cell line was grown in rpmi medium (life technologies) supplemented with % fcs and % p/s. this cell line was kept under hygromycin b (life technologies) selection ( μg/ml) and inductions were performed using μg/ml doxycycline hyclate (sigma) as previously described [ ] . all cells were maintained at °c in a humidified incubator with % co . plasmid transfections were carried out using lipofectamine (life technologies), as previously described [ ] . luciferase assay plasmids renilla luciferase vector prl-tk and firefly luciferase vector pgl -basic were purchased from promega, pegfp-n was obtained from clontech, prta-egfp and ppan-wt have been previously described [ , ] . the monoclonal mouse antibodies to anti-nuclear pore complex proteins (mab ) (ab ), gapdh ( c ), rabbit polyclonal anti-lamin b and anti-ser rnapii were purchased from abcam. the rabbit polyclonal anti-histone h c-terminus ( ) was purchased from active motif. the rabbit polyclonal anti-nup was obtained from bethyl laboratories. monoclonal antibodies to kshv orf ( . ), to hsc (b- ), to grp (a- ), to hsp ( f ), to b- ( ) and to c- (h ) were obtained from santa cruz. the mouse monoclonal (c f a- ) anti-ihsp was from enzo life sciences. the rabbit polyclonal anti-parp was purchased from cell signalling. the mouse monoclonal (ctd h ) anti-rnapii was purchased from millipore. the mouse monoclonal ( e ) anti-c-myc was from sigma. sheep anti-kshv minor capsid protein was purchased from exalpha biologicals, inc. the rabbit polyclonal anti-rta was a gift from professor david blackbourn (university of surrey, uk). the mouse monoclonal (jl- ) anti-gfp was supplied by clontech. the inhibitor for hsp isoforms (ver- ) was obtained from tocris bioscience. for silac, hek- t cells rkshv. were fed with either medium (r k ) or light (r k ) labelled medium (dundee cell products) containing % dialysed fcs (dundee cell products) for six passages to allow incorporation of the isotopes, as previously described [ ] . subsequently, to induce lytic replication three t flasks were reactivated with tpa ( ng/ml) and nab ( mm) for h, while another three t flasks remained unreactivated as control. to isolate nes a protocol published by korfali et al., [ ] was used with minor modifications. million cells were used per experimental condition. cells were washed with pbs and incubated in hypotonic lysis buffer ( mm hepes ph . , . mm mgcl , and mm kcl) for min followed by homogenization with a tight dounce homogenizer. to stabilise and avoid lysing the nuclei after the hypotonic swelling step, cells were resuspended in . m shkm ( . m sucrose, mm hepes ph . , mm kcl, and mm mgcl ) and m kcl. the resuspended cells were then underlayed with % shkm ( . m sucrose, mm hepes ph . , mm kcl, and mm mgcl ) and nuclei were pelleted at , xg for min at °c in a eppendorf centrifuge r. nuclei were resuspended in . m shkm, underlayed with . m shkm and transferred to . -ml ultracentrifuge tubes (beckman coulter). nuclei were then centrifuged at , xg for h at °c in a sorvall discovery se ultracentrifuge. pellets were resuspended in . m shkm ( mm hepes ph . , mm kcl, and mm mgcl ), treated with % triton-x in % shm ( . m sucrose, mm hepes, ph . , mm mgcl and . mm ca cl ) for min and centrifuged at , xg for min. nuclei were then treated with rnase a (thermo scientific) and dnase i (life technologies) for min, pelleted at , xg for min, resuspended in % shm and treated again with rnase a and dnase i for min. nuclei were centrifuged at , xg for min, resuspended and incubated for min in % shm containing . m nacl to remove nucleoplasmic contents. nuclear envelopes were then pelleted at , xg for min. insoluble nuclear envelope proteins were solubilised for min in pbs supplemented with . % triton-x and m urea. samples were centrifuged at , g for min to remove insoluble material and the supernatant containing nuclear envelope proteins was stored at - °c for further western blotting and mass spectrometry analysis. all solutions had freshly added x complete, edta-free protease inhibitors (roche). dtt ( mm) was also freshly added to the solutions specified on korfali's protocol. lc-ms/ms was performed as previously described [ ] . bioinformatical analysis was performed with the ingenuity systems software packet, ipa . (ingenuity systems, inc). protein samples were extracted using lysis buffer containing mm tris (ph . ), mm nacl, % np- and x complete, edta-free protease inhibitors (roche) for min on ice, as previously described [ ] . protein samples were run on sds-page gels and transferred to nitrocellulose membranes (amersham) via wet transfer. membranes were blocked with tbs + . % tween and % dried skimmed milk powder. membranes were probed with relevant primary and secondary antibodies, treated with ez-ecl (geneflow) and exposed to amersham hyperfilm ecl (ge healthcare). secondary antibodies were horseradish peroxidase (hrp)-conjugated polyclonal goat anti-mouse and polyclonal goat anti-rabbit (dako). hrpconjugated polyclonal rabbit anti-sheep was from santa cruz. gfp-trap (chromotek) experiments were performed as previously described [ ] . nuclear/cytoplasmic fractionations were performed as previously described [ ] , with the exception that nuclear pellets were solubilised for min in pbs supplemented with . % triton-x and m urea. vigorous pipetting and vortexing was applied to the nuclear pellet. after urea treatment, insoluble material was removed by centrifugation at , g for min and the supernatant kept for further analysis. cells were cultured overnight on poly-l-lysine (life technologies) coated glass coverslips in -well plates. cells were fixed with % formaldehyde (calbiochem) for min and permeabilised with . % triton x- for min as previously described [ ] . for labelling with grp antibody cells were fixed with ice-cold % methanol for five min. after permeabilization, cells were then incubated in blocking solution (pbs with % bsa) for h at °c. primary antibodies anti-hsc (diluted : ), anti-ihsp ( : ), anti-grp ( : ), anti-rnapii (ctd h ) ( : ) or rabbit rta ( : , ) were incubated for h at °c. coverslips were washed five times with pbs, incubated with appropriate secondary antibody for h at °c, washed five times with pbs again and mounted in vectashield with dapi (vector labs). images were obtained using a lsm meta confocal microscope (carl zeiss) and processed using zen imaging software (carl zeiss) as previously described [ ] . fluorescentlyconjugated secondary antibodies were all obtained from life technologies: alexa flour goat anti-mouse igg, alexa flour goat anti-mouse igg, alexa flour goat anti-rabbit igg, alexa flour donkey anti-mouse igg and alexa flour goat anti-rabbit igg. trex bcbl -rta cells were labelled using the click-it edu alexa fluor imaging kit (life technologies) according to the manufacturer's instructions with minor modifications as follows. cells were seeded onto poly-l-lysine treated coverslips in -well plates followed by induction and incubation at °c for hours. prior to cell fixation, μm edu ( -ethynyl- 'deoxyuridine) was added to each well for min. cells were then fixed for min in % formaldehyde and permeabilised in % triton x- for min. edu detection was carried out adding the click-it reaction cocktail for min and immunofluorescent labelling for rta and hsc was performed as above. cells were mounted in vectashield with dapi (vector labs). total rna from cells was extracted using trizol (life technologies) according to the supplier's protocol. dna-free dna removal kit (ambion) was used to remove any contaminating dna from rna samples. reverse transcription was performed with protoscript ii (neb) and oligo(dt) primers and . μg of total rna. negative control reactions were performed in the same manner but without reverse transcriptase. quantitative pcr (qpcr) reactions ( μl) included x sensimix sybr green master mix (bioline), . μm of each primer and μl template cdna (used at : dilution in rnase-free water). cycling was performed in a rotor-gene q machine (eppendorf). the cycling programme was a min initial preincubation at °c, followed by cycles of °c for sec, °c for sec and °c for sec. after qpcr, a melting curve analysis was performed between and °c (with . °c increments) to confirm amplification of a single product. relative expression compared to control cells was calculated using the ΔΔc t method as previously described [ ] . for each gene of interest and housekeeping gene (gapdh) a standard curve was constructed using a pool of cdna derived from unreactivated and reactivated cells. six different dilutions of the standards were quantified, these included : , : , : , : , : , and : , dilution. the slope of the standard curve was used to calculate the amplification efficiency (ae) of the primers using the formula: ae = ( − /slope ). the mean cycle threshold (c t ) was determined from three independent biological replicates. all genes of interest were normalised against the housekeeping gene gapdh (Δc t ). ΔΔc t was calculated subtracting Δc t of unreactivated cells from Δc t of reactivated cells and the fold change was then determined using ae (-ΔΔc t ) . statistical significance was validated by student's t-test. unreactivated trex bcbl -rta cells treated with dmso ( . %) were used as control to assess viral reactivation. reactivated cells were exposed to doxycycline for h. total dna was then isolated with the use of a qiaamp dna mini kit (qiagen) as per the manufacturer's instructions. qpcr was carried out as described above. ng of template dna and primers specific for the orf gene were used. quantification of gapdh gene was used to normalize between samples and the mean cycle threshold (c t ) was determined from three independent biological replicates. relative levels of viral dna compared with unreactivated cells were calculated using the ΔΔc t method as previously described [ ] . trex bcbl -rta cells that had been seeded on -well plates were reactivated and treated with control dmso ( . %) or ver- . unreactivated cells treated with dmso ( . %) were used as control to evaluate viral reactivation. after h reactivation, μl of the rpmi culture medium was centrifuged at g for five min, immediately mixed with μl of dmem supplemented with % fcs and % p/s and incubated for a further h with hek- t cells that had been seeded in -well plates the previous day. total rna was then extracted with trizol (life technologies) and qrt-pcr carried out as described above. relative expression compared to control cells was calculated using the ΔΔc t method as previously described [ ] . determination of the cellular metabolic activity was performed using a non-radioactive celltiter aq ueous one solution cell proliferation assay (mts) (promega), according to the manufacturer's manual. , cells trex bcbl -rta or , hek- t rkshv. cells were seeded in triplicate in a flat -well culture plate (corning). after h inhibitor exposure, celltiter aqueous one solution reagent was added and cells were incubated for h in a humidified incubator in % co at °c. absorbance was measured at nm using an infinite f (tecan) plate reader. background control had culture medium without cells and the signal from this was subtracted to all other absorbance values. this assay allows evaluation of viability, cytotoxicity and effector caspases activation within a single assay well. the assay was carried out as specified on the supplier's manual. , trex bcbl -rta cells or , hek- t rkshv. cells were seeded in triplicate in tissue culture treated black microplates (greiner bio-one). no-cell control (background) contained only culture medium and the signal from this was subtracted to all other absorbance and luminescence values. fluorescence and luminescence readings were collected using a glomax system (promega) (kindly provided by dr. john boyle, university of leeds, uk). luciferase activity was detected using the dual-luciferase reporter assay system (promega) as previously described [ ] . hek- t cells were seeded in triplicate in flat -well culture plate (corning) at a density of , cells per well. following the respective plasmid transfections and inhibitor exposure, media was removed from the culture wells and cells washed gently with μl pbs. μl x passive lysis buffer was added to the cell monolayer which was rocked for min and then μl of each lysate was transferred to tissue culture treated white microplates (greiner bio-one). luciferase measurements were carried out in a fluostar optima microplate reader (bmg labtech ltd), with injectors and being used to dispense μl of luciferase assay reagent ii and stop & glo reagent respectively. firefly luciferase activity was normalized to renilla luciferase activity. formaldehyde-crosslinked chromatin was prepared using the pierce chromatin prep module (thermo scientific) following the manufacturer's protocol. x cells were used per experimental sample and digested with six units of micrococcal nuclease (mnase) per μl of mnase digestion buffer in a °c water bath for min. these conditions resulted in optimal sheared chromatin with most chromatin fragments ranging from - base pairs. immunoprecipitations were carried out using ez-chip kit (millipore) according to the supplier's instructions and as previously described [ ] . immunoprecipitations were done overnight at °c and contained μl of digested chromatin ( x cells), μl of chip dilution buffer and . μg of rnapii antibody (clone ctd h ) (millipore) or isotype antibody, normal mouse igg (millipore). both antibodies were provided with the ez-chip kit. prior to qpcr analysis, eluted dna was subjected to a dna clean up step using ultraclean pcr clean-up kit (mo bio laboratories) according to the supplied protocol with the exception of using μl of spinclean buffer instead of μl. qpcr reactions were performed as described above and using either μl of chip'ed dna or μl of input dna as template. sirna knockdown hek- t rkshv. cells seeded on -well plates were reverse transfected with either nm of the specific silencer select sirna (life technologies) or nm allstars negative control sirna (qiagen) using μl of siport neofx transfection agent (life technologies) per transfection. the sirna id for hsc and ihsp were s and s respectively. s sirna targets the two major ihsp proteins (hsp - and hsp - ). two days post-transfection, cells were transfected again in the same manner. four days after the first transfection, cells were reactivated and incubated for the desired time. proteins and total rna were isolated with trizol (life technologies) and subsequent western blot and qrt-pcr were performed. × trex bcbl -rta cells were transfected once with μl of nucleofector solution v (lonza) to which μm sirna (scramble or hsc ) was added. in addition, to monitor transfection efficiency, μg of the control plasmid pmaxgfp was also co-transfected. cells were transfected using program t- of an amaxa nucleofector i (lonza). after nucleofection cells were maintained in six-well plates. medium was freshly replaced every day. confocal images were subjected to profiling analysis using zeiss zen software. this involved drawing a line in a confocal image to measure the relative intensity of each channel at every pixel along the line. profiling was conducted for each cell in two representative confocal images taken with a -times objective. these data were then analysed using microsoft excel . firstly, a function was used to define whether the relative intensity of a pixel could be defined as a "peak" and thus an hsc foci. this function asked whether the data point for one specific pixel of the rhodamine channel was , this was set as the arbitrary threshold to eliminate background noise. if this condition was met, the function then asked whether the data point was greater than or equal to the data point in the previous and subsequent pixel. if these conditions were true, then this data point was counted as a peak. this was performed for every data point measured in the line profile providing the total number of hsc peaks in the profile of one cell. next, another function was used to determine whether the relative intensity in the dapi channel was , a threshold determined from visualising the line profiling data as a graph. if a pixel was shown to exceed the threshold in the dapi channel and also in the rhodamine channel, then it was counted as a nuclear hsc peak. these measurements were conducted for each pixel in each profile allowing counting hsc nuclear peaks in each profile. hsc peaks outside the nucleus corresponded to cytoplasmic hsc peaks. oligonucleotide primer sequences are available upon request. all primers were purchased from sigma (uk). supporting information s table. cellular proteins previously reported to localise to herpesvirus rtcs and found significantly increased in the ne of reactivated cells. (xls) s fig. ihsp was redistributed from the cytoplasm to both the periphery and within kshv-induced rtcs. (a) trex bcbl -rta cells remained unreactivated or reactivated for either h or h. in unreactivated cells ihsp was cytoplasmic (i). in contrast, at h reactivation an increase in nuclear ihsp labelling was seen with numerous small ihsp foci found mainly adjacent to viral rtcs (ii). some cells displayed ihsp completely recruited within rtcs (iii and iv asterisks), while other cells accumulated large ihsp adjacent to rtcs (iv arrows). (b) trex bcbl -rta cells remained unreactivated (i) or reactivated for h (ii and iii) followed by triple-labelling with antibodies specific for rta and ihsp and click-it edu alexa fluor . complete co-localisation between ihsp , rta and actively replicated viral dna (edu-labelled) was observed in both incipient rtcs (ii) and in fully-developed rtcs (iii). note that in these cells ihsp was not depleted from the cytoplasm. trex bcbl -rta cells remained unreactivated or reactivated for h in the presence of control dmso ( . %) or μm ver- followed by labelling with click-it edu alexa fluor and an antibody specific for rnapii (clone ctd h ). (a) a high proportion of unreactivated trex bcbl -rta cells replicated their cellular dna (edu-labelled) in the presence of control dmso ( . %) or μm ver- . normal rnapii localization was observed in these cells, with nuclear rnapii excluding the nucleoli. (b) in contrast, reactivated cells entered cell cycle arrest as demonstrated by fewer edu-labelled cells. in the presence of dmso, multiple rtcs were formed with some replicating viral dna (white arrows). in cells treated with ver- , multiple pre-replicative sites were seen labelled by rnapii antibody and edu-labelling was more diffused in the nucleus compared with dmso-treated cells. 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protein to disrupt inflammatory signaling we are grateful to members of the whitehouse laboratory for helpful discussions, especially to alexander j. coleman for guidance with confocal profiling. the authors would like to thank key: cord- - ho av authors: abolnik, celia title: genomic and single nucleotide polymorphism analysis of infectious bronchitis coronavirus date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: ho av infectious bronchitis virus (ibv) is a gammacoronavirus that causes a highly contagious respiratory disease in chickens. a qx-like strain was analysed by high-throughput illumina sequencing and genetic variation across the entire viral genome was explored at the sub-consensus level by single nucleotide polymorphism (snp) analysis. thirteen open reading frames (orfs) in the order ′-utr- a- ab-s- a- b-e-m- b- c- a- b-n- b- ′utr were predicted. the relative frequencies of missense: silent snps were calculated to obtain a comparative measure of variability in specific genes. the most variable orfs in descending order were e, b, ′utr, n, a, s, ab, m, c, a, b. the e and b protein products play key roles in coronavirus virulence, and rna folding demonstrated that the mutations in the ′utr did not alter the predicted secondary structure. the frequency of snps in the spike (s) protein orf of . % was below the genomic average of . %. only three snps were identified in the s subunit, none of which were located in hypervariable region (hvr) or hvr . the s subunit was considerably more variable containing % of the polymorphisms detected across the entire s protein. the s subunit also contained a previously unreported multi-a insertion site and a stretch of four consecutive mutated amino acids, which mapped to the stalk region of the spike protein. template-based protein structure modelling produced the first theoretical model of the ibv spike monomer. given the lack of diversity observed at the sub-consensus level, the tenet that the hvrs in the s subunit are very tolerant of amino acid changes produced by genetic drift is questioned. coronaviruses (family coronaviridae, order nidovirales) are enveloped, single-stranded rna viruses with large genome sizes of $ - kb. the family is split into four genera: alpha-, beta, gamma and deltacoronaviruses, each containing pathogens of veterinary or human importance. a current evolutionary model postulates that bats are the ancestral source of alpha-and betacoronaviruses and birds the source of gamma-and deltacoronaviruses (woo et al., ) . the alphacoronaviruses infect swine, cats, dogs and humans. betacoronaviruses infect diverse mammalian species including bats, humans, rodents and ungulates. the sars coronavirus (sars-cov), which verged on a pandemic in with cases in humans and deaths is a betacoronavirus. another member of this genus, the recently-discovered middle east respiratory syndrome (mers) coronavirus (mers-cov) has claimed human lives from cases since april , and dromedary camels are the suspected reservoir (briese et al., ) . genus gammacoronavirinae includes strains infecting birds and whales (woo et al., ; mcbride et al., ; borucki et al., ) and deltacoronaviruses have been described in birds, swine and cats (woo et al., ) . the diversity of hosts and genomic features amongst covs have been attributed to their unique mechanism of viral recombination, a high frequency of recombination, and an inherently high mutation rate (lai and cavanagh, ) . infectious bronchitis virus (ibv) is a gammacoronavirus which causes a highly contagious respiratory disease of economic importance in chickens (cook et al., ) . ibv primarily replicates in the respiratory tract but also, depending on the strain, in epithelial cells of the gut, kidney and oviduct. clinical signs of respiratory distress, interstitial nephritis and reduced egg production are common, and the disease has a global distribution (cavanagh, ; cook et al., ) . the ibv genome encodes at least ten open reading frames (orfs) organised as follows: utr- a- ab-s- a- b-e-m- a- b-n- a- utr. six mrnas (mrna - ) are associated with production of progeny virus. four structural proteins including the spike glycoprotein (s), small membrane protein (e), membrane glycoprotein (m), and nucleocapsid protein (n) are encoded by mrnas , , and , respectively (casais et al., ; hodgson et al., ) . messenger rna (mrna) consists of orf a and orf b, encoding two large polyproteins via a ribosomal frameshift mechanism (inglis et al., ) . during or after synthesis, these polyproteins are cleaved into non-structural proteins (nsp - ) which are associated with rna replication and transcription. the s glycoprotein is post-translationally cleaved at a protease cleavage recognition motif into the amino-terminal s subunit ( kda) and the carboxyl-terminal s subunit ( kda) by the host serine protease furin (de haan et al., ) . the multimeric s glycoprotein extends from the viral membrane, and the globular s subunit is anchored to the viral membrane by the s subunit via non-covalent bonds. proteins a and b, and a and b are encoded by mrna and mrna , respectively and are not essential to viral replication (casais et al., ; hodgson et al., ) . a confounding feature of ibv infection is the lack of correlation between antibodies and protection, and discrepancies between in vitro strain differentiation by virus neutralization (vn) tests and in vivo cross-protection results. taken with the ability for high viral shedding in the presence of high titres of circulating antibodies, the involvement of other immune mechanisms are evident, and the roles of cell-mediated immunity and interferon have been experimentally demonstrated (timms et al., ; collisson et al., ; pei et al., ; cook et al., ) . dozens of ibv serotypes that are poorly cross-protective have been discovered and studied by vn tests and molecular characterisation of the s protein gene. most of these serotypes differ from each other by - % at amino acid level in s , but may differ by up to %. s contains the epitopes involved in the induction of neutralizing, serotype-specific and hemagglutinaton inhibiting antibodies (cavanagh, ; darbyshire et al., ; farsang et al., ; ignjatovic and mcwaters, ; meulemans et al., ; gelb et al., ) . most of the strain differences in s occur in three hypervariable regions (hvrs) located between the amino acid residues - (hvr ), - (hvr ) and - (hvr ) (moore et al., ; wang and huang, ) . monoclonal antibody analysis mapped the locations of many of the amino acids involved in the formation of vn epitopes to within the first and third quarters of the linear s polypeptide (de wit, ; kant et al., ; koch et al., ) , which is where closely-related stains (> % amino acid identity) also differ (bijlenga et al., ; farsang et al., ) . cavanagh ( ) proposed that these parts of the s subunit are very tolerant of amino acid changes, conferring a selective advantage. recently, the receptor-binding domain of the ibv m strain was mapped to residues - of the n terminus of s , which overlaps with hvr (promkuntod et al., ) . the s subunit, which drives virus-cell fusion, is more conserved between serotypes than s , varying by only - % at the amino acid level (bosch et al., ; cavanagh, ) . although it was initially thought that s played little or no role in the induction of a host immune response, it has since been shown that an immunodominant region located in the n-terminal half of the s subunit can induce neutralizing, but not serotype-specific, antibodies demonstrated by the ability of this subunit to confer broad protection against challenge with an unrelated serotype (kusters et al., ; toro et al., ) . ibvs are continuously evolving as a result of (a) frequent point mutations and (b) genomic recombination events (cavanagh et al., ; kottier et al., ; jackwood et al., ; zhao et al., ; kuo et al., ; liu et al., ) . multiple studies on ibv diversity have focused on inter-serotypic and inter-strain variation, and a few have focused on sub-populations within the s subunit in vaccine strains (gallardo et al., ; ndegwa et al., ) . the present study aimed to explore genetic variation across the entire viral genome at the sub-consensus level. it was anticipated, based on the published literature, that certain regions, and the s subunit hvrs in particular, would display significant sub-genomic variation. this study focused on a qx-like strain, a serotype currently causing significant poultry health problems across europe, asia, south america and south africa. . . origin and isolation of qx-like strain ck/za/ / twenty-eight-day old chickens in a commercial broiler operation presented with acute lethargy, reduced feed consumption and mortality. tracheitis and swollen kidneys were noted on post mortem, as well as a secondary escherichia coli infection. the worst affected houses had mortality rates of . %, . % and . %. ibv was isolated in specific pathogen free (spf) embryonated chicken eggs (ece) as described in knoetze et al. ( ) . after an initial two passages in ece, the virus was passaged twice further at the university of pretoria. rna was extracted from allantoic fluid using trizol Ò reagent (ambion, life technologies, carlsbad, usa) according to the manufacturer's protocol. the genome was transcribed to cdna and amplified using a transplex Ò whole transcriptome amplification kit (sigma-aldrich, steinheim, germany). illumina miseq sequencing on the cdna library was performed at the arc-biotechnology platform, onderstepoort, pretoria. illumina results were analysed using the clc genomics workbench v . . . paired-end reads were trimmed and a preliminary de novo assembly was performed. the larger segments were analysed by blast to identify the closest genomic reference strain (ita/ / , caz ) . this strain was retrieved and used as a scaffold for assembly-to-reference, generating a consensus sequence for / . trimmed paired-end reads were also mapped against other ibv serotype genomes, subsequently confirming that strain / was a pure culture of a qx-like ibv. the genome was deposited in genbank under the accession number kp . rna folding was predicted using the clc genomics workbench v . . . genetic recombination in the consensus sequence was evaluated using the recombination detection program rdp v . . coding sequence and orf prediction was carried out in vigor (wang et al., ) . trimmed paired-end reads were re-mapped against the / consensus sequence for snp detection. a snp detection table generated in the clc genomics workbench was manually edited to eliminate all snps with a frequency of < %. this conservative cutoff was selected to eliminate any nonspecific pcr errors introduced during preparation of the transcriptome library or deep sequencing, and excluded most of the point insertions producing gaps and frameshift mutations across the genome. nucleotide substitutions in coding regions were manually inspected for changes to the consensus amino acid (table , supplementary data). motifs were predicted using the elm eukaryotic linear motif resource for functional sites in proteins (dinkel et al., ) . protein structures for s and s were predicted in raptorx, a structure prediction server that predicts three dimensional ( d) structures for protein sequences without close homologs in the protein data bank (pdb) (kallberg et al., ) . s and s d structures were annotated and superposed in ccp mg v . . using the secondary structure (ssm) superposition method. this method superimposes pairs of structures by: ( ) finding the secondary structure elements (sses) and representing them as one simple vector spanning the length of the sse; ( ) finding equivalent sses in the two structures using graph-theory matching by geometric criteria of distances and angles between the vectors; ( ) superimposing vectors representing equivalent sses; ( ) finding the most likely equivalent residues in the superposed sses; ( ) superimposing ca atoms of equivalent residues; and ( ) iterating the last two steps. the genome sequence of qx-like strain ck/za/ / was assembled from , ibv-specific paired-end reads of bp each. the genome was , nt in length with the utr incomplete by $ nt. thirteen orfs were predicted by vigor in the order fig. ). this genome organisation including b, c and b was similar to that of turkey coronavirus (tcov; cao et al., ) , and the orfs b, c and b were also predicted in australian ibv strains (hewson et al., ) . when the sequences for a qx-like sequence (jq ) and arkdpi (eu ) were analysed using vigor, a similar genome arrangement was detected. mass (ay ) did not however contain the predicted b, c and b orfs (data not shown). orf b was amino acids (aa) in length and no smart domains were predicted, whereas orf c was aa in length and a low complexity region was identified. the b orf encoded a aa protein with a signal peptide predicted from residues to and two transmembrane domains from residues to and to . no recombination was detected across the genome of qx-like strain ck/za/ / . a gap was present between nucleotides and (table , supplementary data) ($aa in the a orf). although the gap was present in the majority ( . %) of reads, the sequence for strain / deposited in genbank contains the minority adenine residue because the gap introduced a frame shift, splitting orf a into two. it may be a legitimate mutation, but until further transcriptional analyses are conducted, the orf a gene has been reported intact here. two hundred and eight snps across the ibv qx-like genome were evaluated at the selected cut-off value. in table the consensus reference is juxtaposed with the allele variations, the relative frequencies of these point mutations, the actual number of counts and coverage at that position, the corresponding orf or region and the mutational effect. coverage ranged from -fold (position , ) up to fold (position , ). the relative frequencies of missense: silent snps in relation to orf length were calculated in order to obtain a comparative measure of variability in specific genes (table ) . results for the structural genes and polymerase are illustrated in fig. , and the results for the non-structural protein orfs and non-coding regions, which were much shorter in length, are presented in fig. . overall the most variable orfs in terms of total snps, in descending order, were: e, b, utr, n, a, s, ab, m, c, a, b (no snps were detected at the % cut-off in the a and utr regions). the most variable, as assessed by snps leading to missense mutations, in descending order, were: b, e, utr, a, n, m, a, ab, s, b, a/ c/ b. these mutations presumably did not affect the tertiary protein structure and might be advantageous to the virus. the orfs under the strongest positive selection pressure as indicated by the proportion of synonymous mutations, were, in descending order, c, ab, n, s, e, a/ b/m/ b/ a/ b. the e protein orf had significantly more missense mutations on average, at a frequency of . % of the orf, which is more than threefold higher than the average value ( . ) for the a, ab, s, m and n genes. the e protein gene was the most variable at the sub-consensus level, with missense mutations and only one silent mutation across its bp orf. despite its small size, the cov e protein drastically influences the replication of covs and their pathogenicity. in the sars-cov, it was experimentally demonstrated that the e protein is not essential for genome replication or subgenomic mrna synthesis, but it does affect morphogenesis, budding, assembly, intracellular trafficking and virulence. in fact, in sars-cov the e protein is the main antagonist associated with induction of inflammation in the lung, which causes the acute respiratory distress syndrome from which the virus derives its name (dediego et al., ) . no studies have been published for the ibv e protein, but the high variability demonstrated here suggests that it may be an important virulence factor in poultry, and that a higher mutation rate possibly provides an evolutionary advantage in overcoming host cellular immune responses. although the n protein gene contained one of the highest overall frequencies of snps ( . %), the n gene is evidently under greater selective pressure, since . % of these mutations ( . % as a total of the gene) were silent. the coronavirus n protein is multifunctional, playing vital roles in viral assembly and formation of the complete virion and is required for optimal viral replication. additionally, the cov n protein is implicated in cell cycle regulation and host translational shutoff, displays chaperone activity, activates host signal transduction and aids viral pathogenesis through the antagonism of interferon induction (reviewed by mcbride et al., ) . given its fundamental roles in rna binding, formation of the ribonucleoprotein complex and in the virion, it is not surprising that this structural protein is the most conserved, as evidenced by its gene having the highest ratio of silent mutations of all the genes analysed. the importance of maintaining the fig. . genome organisation of qx-like ibv strain / . sequence integrity in the n protein in ibv was demonstrated by kuo et al. ( ) , who reported that two residues within the nterminal domain of a taiwanese ibv strain were positively selected, and that mutation of either of these significantly reduced the affinity of the n protein for the viral transcriptional regulatory sequence. the glycosylated amino terminus of the m protein lies on the outside of the virion and m spans the membrane structure three times (collisson et al., ) . all four snps in the m gene resulted in missense mutations, two of which were located in the predicted transmembrane region. the m protein plays an important role in cov virion formation. ibv m protein co-expressed with s assembled into virus-like particles confirming its major role in virion formation, but cov m proteins also interact with other proteins and perform other roles in the infected cell. for example, m together with the accessory proteins a, b and were all found to prevent the synthesis of ifn-b through the inhibition of interferon promotor activation and irf- function, thus influencing disease outcome (yang et al., ) . coronavirus accessory proteins are generally dispensable for virus replication, but they play vital roles in virulence and pathogenesis by affecting host innate immune responses, encoding pro-or anti-apoptotic activities, or by effecting other signalling pathways that influence disease outcomes (susan & julian, ) . ibv was demonstrated to induce a considerable activation of the type i ifn response, but it was delayed with respect to the peak of viral replication and accumulation of viral dsrna (kint et al., ) . ibv accessory proteins a and b play a role in the modulation of this delayed ifn response, by regulating interferon production at both the transcriptional and translational levels. interestingly, ibv proteins a and b seem to have opposing effects on ifn production in infected cells: a seems to promote ifn production, and b is involved in limiting ifn production, antagonising each other to tightly regulate ifn production (kint et al., ) . field isolates lacking a and b displayed reduced virulence in vitro and in vivo (mardani et al., ) . orf a in strain / lacked snps, but orf b in had the highest frequency of snps relative to its size (n = ; . %). orf b is present in many international ibv strains (hewson et al., ; bentley et al., ) but is rarely mentioned in the literature since a canonical transcription regulatory sequence (trs-b) could not be identified upstream of the encoding rna. however, bentley et al. ( ) demonstrated that ibv was capable of producing subgenomic mrnas from noncanonical trs-bs via a template-switching mechanism with trs-l, the conserved trs in the leader sequence in the utr, which may expand the gammacoronavirus repertoire of proteins. they specifically demonstrated the transcription of the b orf by this mechanism. although no studies have been performed determining orf b s functional role in the pathogenesis of ibv, the homolog in mers-cov is a potent interferon antagonist (yang et al., ) . a single snp causing a missense mutation was present in . % of the sub-consensus population of the b orf in this study. the single mutation in orf c was silent, and the predicted protein contained a low complexity region. low complexity regions are regions of protein sequences with biased amino acid composition, and may be involved in flexible binding associated with specific functions (coletta et al., ) . orf b, a aa protein with a signal peptide and two transmembrane domains, was identified in the genome of strain / , and orf b was also reported in tcov and australian ibv strains (cao et al., ; hewson et al., ) . the homolog in sars-cov is aa in length and was identified as an endoplasmic reticulum/golgi membrane-localised protein that induces apoptosis. apoptosis may play an important role in promoting cov dissemination in vivo, minimising inflammation and aiding evasion of the host's defence mechanisms (ye et al., ) . protein from sars-cov accelerated the replication of murine cov, increasing the virulence of the original attenuated virus (tangudu et al., ) . presumably, this accessory protein plays a similar role in ibv pathogenesis, although this remains to be determined experimentally. the utrs of cov genomes contain conserved cis-acting sequence and structural elements that play essential roles in rna synthesis, gene expression and virion assembly, and each sub-genomic rna contains a leader segment that is identical to this utr region of the genome (goebel et al., ; sola et al., ) . no snps were detected in the utr in the sub-consensus sequences of strain / , which is consistent with the vital regulatory role that this region plays. conversely, the partial utr sequence of strain / was highly variable. the un-sequenced nucleotides from the end of the genome were extrapolated from the most similar genomic sequence, that strain ita/ / , and the secondary rna structure of the utr for / was predictively folded (fig. ) . the snps were then systematically substituted into the consensus sequence and rna folding repeated. delta g values for the predicted rna secondary structures in fig. (a) -(h) varied from À . kcal/mol to À . kcal/mol. apart from the c to g mutation (fig. (c) ), effects on rna secondary structure were minor and the structures in fig. (b) and (d)-(h) were similar. to assess the effect of combining mutations, an rna containing t(u), t(u), g and c was folded, and this resulted in a similar stem-loop structure to those in fig. (a), (b) and (d)-(h) (data not shown). apart from the mutation c to g, the snps had little effect on the secondary rna structure in the utr. twenty-three snps were identified in the bp spike protein orf; of these resulted in missense mutations at the amino acid level, and were silent mutations. the frequency of total snps in the s protein orf was below average, at . %, compared to the genome average of . %. it was anticipated that the majority of mutations in the s orf would be in the s gene, particularly in the hvrs, but, surprisingly, this was not the case. only three of these snps (two missense and one silent) were found in the s gene, and all three were located in the cooh-terminal half of the s protein (fig. ) . only one mutation, a missense mutation, . predicted structure of the spike protein monomer of qx-like ibv strain / . missense mutations in s (blue) and s (yellow) are indicated as coloured side chains. mapped to hvr . no snps were detected in hvr or hvr . the s subunit was considerably more variable, containing % of the polymorphisms detected across the entire s protein. two other notable features of s were detected: the first was a multi-a insertion site located between nucleotides , and , in the genome. the polymorphism involved the insertion of either one or two adenine nucleotides, possibly via a mechanism of polymerase stuttering. the second region of interest was located in close proximity, just downstream of the multi-a insertion site: a stretch of three consecutive mutated amino acids, namely c ? w, g ? d, s ? c followed by silent mutation g (fig. ) . template-based protein structure modelling was used to predict the secondary structure of the ibv spike monomer, based on the available crystal structure for the mers-cov s and s subunits (fig. ). s and s were modelled separately in raptor x and then superposed. the ibv s structure was arranged as two beta barrels and s formed packed a-helices. the s protein was not complete and the transmembrane domain was not represented since there were no sufficiently similar structures on which to model this region, but this is the first model of the spike protein monomer for ibv. hvr and the putative receptor binding domain maps to the apical beta barrel (fig. (a) ) and hvr is located on the flat plane on the base of the apical beta barrel and the peptide connecting it to the basal beta barrel (fig. (b) ). hvr maps to a region in the basal beta barrel of s that was predicted to contact or interact with s ( fig. (c) ). the locations of the missense mutations detected by snp analysis in the s and s subunits are indicated in fig. . many of these snps mapped to codons encoding amino acids on the surface of the predicted structure, but two regions were notable. firstly, the highly variable region in s spanning amino acids - was exposed on the s stalk, although folding of the remainder of the cooh domain may have influenced this conformation. secondly, ile was exposed on a projection at the top of the monomer. this residue precedes the second furin cleavage site in the s subunit with the sequence pisssgr/s . the cleavage of the s /s furin motif ( rrrr/s in strain / ) was found to be non-essential for attachment of ibv to the cell. rather, it promotes infectivity within the cell. in studies with the beaudette ibv strain, the second furin cleavage site in the s subunit was required for furin-dependent entry and syncytium formation, and the current hypothesis is that interplay between the s and s subunits determines virus attachment to specific receptors, determining tissue tropism of the virus (promkuntod et al., ) . the exact biological roles of these areas in s that are prone to mutation remain to be experimentally determined. archaeological remains of domestic chickens in northeast china and the indus valley date back $ years (west & zhou, ) . . the predicted locations of hvr ( a), hvr ( b) and hvr ( c) of qx-like ibv strain / , indicated as coloured side-chains on the s subunit. the cov group has been estimated to have arisen around bc, and the gammacoronaviruses diverged from the cov group around bc (woo et al., ) . covs have probably been co-evolving with their gallinaceous hosts for several thousand years. indeed, cook and co-authors ( ) state that ''ibv is found everywhere that commercial chickens are kept''. the implication is that although ibv was only discovered some years ago, the variety of serotypes we now observe are the results of hundreds if not thousands of years of genetic drift and recombination, accelerated by modern poultry farming practises where chickens are kept in high densities, and inter-regional trade in poultry and other avian species. studies on antigenic diversity of ibvs are heavily biased towards studies of the s gene, and the hvrs in particular (cavanagh, ; ducatez et al., ; kant et al., ; mork et al., ) . many of these studies cite frequent point mutations in the s gene, but this was not the finding of the present study. the discovery of a novel -to- exoribonuclease activity in cov nsp , which regulates replication fidelity and diversity in coronaviruses (denison et al., ) , lends weight to the theory that genetic drift is not primarily responsible for the degree of variation and serotypes we observe in poultry nowadays. instead, generation of variation by recombination is likely the main mechanism of serotypic diversity. the high frequency of rna recombination in coronaviruses is likely caused by their unique mechanism of rna synthesis, which involves discontinuous transcription and polymerase jumping (jeong et al., ) . sequencing of many field strains has provided convincing evidence that many, possibly all, ibv strains are recombinants between different field strains (cavanagh, ; kuo et al., ; liu et al., ; hewson et al., ) , driving ibv evolution at a population level. recombination of distinct ibv strains has been experimentally demonstrated in vitro, in ovo and in vivo (kottier et al., ; wang et al., ) . the s subunit hvr contains the ibv receptor-binding site. therefore despite the sequence variability in this region (which includes insertions and deletions), diverse strains must retain this critical biological function. all three hvrs may represent ancient artefacts of recombination, which have been perpetuated because they retain receptor-binding properties, with minimal permissive amino acid changes. this theory contrasts the tenet that the hvrs in the s subunit are very tolerant of amino acid changes produced by genetic drift, thereby conferring a selective advantage (cavanagh, ; de wit, ; kant et al., ; koch et al., ) . whereas s fulfils a primary role in receptor binding (promkuntod et al., ) , a broader role of s in antigenicity and attachment to receptors is emerging. chickens primed with a recombinant-expressed s subunit of a virulent arkdpi strain and boosted with a live mass-type vaccine were protected against challenge with live virulent arkdpi virus . although s subunits most likely do not contain an additional independent receptor-binding site, s in association with s forms part of a specific ectodomain which is critical to the binding of the virus to chicken tissues, which implies that both s and s contain determinants important to viral host range (promkuntod et al., ) . the results of the present study demonstrate that s is more predisposed to mutations than s , providing an adaptive advantage and at least one other study has reported higher variability in s compared to s (mo et al., ) . ibv has not been as extensively studied as other covs, and little progress has been made in effectively controlling or eradicating the disease in poultry. experimental and field studies provide substantial evidence that use of a homologous ibv vaccine is best, but sometimes, intriguingly, protection can be offered by an unrelated vaccine, or by the use of two heterologous vaccines (jones, ) . genotyping and phylogenetic analysis of ibv are typically focused on the s subunit sequence, and liu et al. ( ) caution against drawing conclusions based on a single gene sequence, particularly a partial gene sequence. the roles of the ibv e and accessory proteins and their roles in the pathogenesis of ibv have been completely overlooked, even when the roles of the homologs in other covs have been proven significant. accessory proteins of ibv and other covs may also offer a new generation of vaccine targets: the use of codon-deoptimization of non-structural virulence genes in influenza a virus and respiratory syncytial virus resulted in genetically stable viruses that retained immunogenicity but were attenuated (nogales et al., ; meng et al., ) . evidently virulence and immunogenicity in ibv is a multi-genic trait, and future studies must aim to pursue a better understanding and exploitation of the roles of various viral proteins in the host, if any advances are to be made in controlling the disease in poultry. identification of a noncanonically transcribed subgenomic mrna of infectious 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against challenge the structural and accessory proteins m, orf a, orf b, and orf of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists experimental confirmation of recombination upstream of the s hypervariable region of infectious bronchitis virus relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus vigor, an annotation program for small viral genomes did chickens go north? new evidence for domestication discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus analysis of a qx-like avian infectious bronchitis virus genome identified recombination in the region containing the orf a, orf b, and nucleocapsid protein gene sequences adrian knoetze and rainbow veterinary laboratory are thanked for providing strain / for this study. funding was provided by the poultry section, department of production animal studies. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.meegid. . . . key: cord- -r tl kq authors: nan title: dublin pathology . th joint meeting of the british division of the international academy of pathology and the pathological society of great britain & ireland date: - - journal: j pathol doi: . /path. sha: doc_id: cord_uid: r tl kq nan the christie nhs foundation trust, manchester, uk one controversy in preparing the rcpath "dataset for tumours of the urinary collecting system [ nd edition])", april was which who grading scheme ( or ) to use for urothelial carcinoma. since there is a split within grade ( ) between low grade & high grade, use of both schemes in parallel was recommended. this has the advantage of better indicating where a particular patient lies in the grading continuum & minimises the consequences of 'grading error' for cases close to the threshold between low & high grade in the scheme, (a critical distinction for management if who grading is used in isolation). the nice guidance on bladder cancer (http://www.nice.org.uk/guidance/ ng ) incorporates risk stratification tables for ta/t bladder cancer utilizing this parallel who grading recommendation in a multiparameter formula that also considers tumour size, pt classification and the presence/absence of cis or aggressive subtype(s). amongst the more aggressive bladder cancer subtypes is invasive micropapillary urothelial carcinoma and the nested variant. a bladder origin can be difficult to recognise at metastatic sites, especially for micropapillary and discohesive/plasmacytoid subtypes. uroplakin ii has recently become available, is more sensitive than uroplakin iii & may assist within a panel. spindle cell lesions of the bladder are often difficult & loss of cytokeratin expression is common in sarcomatoid carcinoma. there is potential to mistake inflammatory myofibroblastic tumour for a malignant tumour. two molecular pathways for bladder cancer are recognised. fgfr at p is the most frequently mutated oncogene in bladder cancer & is prevalent in low grade papillary tumours. p loss of function mutations and loss of rb are prevalent in cis. no prognostic molecular test is currently validated for clinical use in bladder cancer, though some (e.g. fish) are in use as an adjunct in diagnosis. indiana university school of medicine, indianapolis, usa some dogmas in testicular pathology do not hold up to scrutiny. the belief that all pure, postpubertal teratomas are malignant is invalid. within this group there is a small subset that is benign; these may be divided into dermoid and non-dermoid types that, however, share features that distinguish them from the usual teratoma of adults. these include absence of: atypia, regressive parenchymal changes, intratubular germ cell neoplasia, and i( p). also they usually show organoid arrangements, and prominence of ciliated epithelium, squamous cysts and smooth muscle. patients do not need further intervention beyond excision sufficient to establish the diagnosis. cases often interpreted as isolated testicular polyarteritis nodosa because of the presence of fibrinoid vascular necrosis are mostly attributable to chronic, intermittent torsion. they usually present as pain-associated, palpable or ultrasound-detected masses, with the "mass" corresponding to infarct and/or hemorrhage. there are associated chronic vascular changes, with frequent marked intimal hyperplasia of arteries, mural fibrosis of veins, dilated venules and arteriolar hyalinization, consistent with torsion-induced venous outflow obstruction and secondary arterial hypertension. these patients do not develop systemic vasculitis on follow-up. many "sarcomas" in patients with germ cell tumours, especially after chemotherapy, are more correctly regarded as sarcomatoid yolk sac tumours. they are reactive for cytokeratin and glypican . they often show characteristic features: nodular growth, myxoid and fibrous stroma, spindled and epithelioid cells, abrupt changes in cellularity, and tumor "ringlets." most are high grade and aggressive. most regressed germ cell tumours can be recognized through a combination of findings, although the only diagnostic ones are a scar with coarse intratubular calcifications or with intratubular germ cell neoplasia. alcohol consumption has doubled in ireland and the uk in the last years. the causes of this increase include increased affordability of alcohol and its widespread availability. as a consequence, the health harms associated with alcohol have dramatically increased. binge drinking and alcohol consumption among by women have risen especially dramatically. for example, the mortality from cirrhosis has doubled in the last years in both men and women. in response to this, the medical profession on both islands have led informal and later formal campaigns to encourage policy change regarding alcohol at a national level. in ireland, this was driven by rcpi. the involvement of the medical profession has had a powerful influence, as doctors do not have a conflict of interest in this matter, in contrast to the alcohol industry. the policy changes advocated include particularly minimum unit pricing (mup), which has been shown to be effective in reducing alcohol consumption, alcohol-related admission to hospital and crime in canada. modelling of the data suggests it would have similar benefits in uk and ireland. this is regarded as the single most important first step. other steps which will help include actions around alcohol labelling, availability and breaking the link between alcohol and sports and leisure promotion. turning off the tap of cheap alcohol will hopefully soon reduce alcohol health harms in uk and ireland  barrett's oesophagus: an evolving challenge for the gastroenterologist p dot o'toole cost-effective surveillance programmes for barrett's oesophagus (bo) needs to be focused at risk groups. it is not only a question of identifying more individuals with bo (initial screening) but screening and subsequent surveillance has to identify at-risk individuals with bo who can benefit most from surveillance or therapy. advances in endoscopic imaging (high resolution endoscopy ([hre] with dye-based chromoendoscopy, electronic chromoendoscopy, and autofluorescence,...) certainly prove beneficial in better detecting dysplasia within known bo and can guide sampling and subsequent therapy. dysplasia can be patchy and easily missed during routine biopsy sampling of bo and adequate training with high resolution instruments is needed to increase detection rates. once dysplasia is detected, endoscopic ablation is recommended. until recently, the standard treatment for hgd was oesophagectomy but endoscopic resection and ablation techniques are now available to eradicate dysplasia and mucosal adenocarcinomas. resecting visible lesions (using endoscopic mucosal resection [emr] or endoscopic submucosal dissection techniques) allows full pathological t staging. when invasive cancer is eliminated at multidisciplinary review (i.e., purely mucosal neoplasia confirmed with a nodal risk < %), further endotherapy to ablate residual metaplasia in bo can be performed using radiofrequency ablation (raf). in a tertiary centre use of staging emr is frequently necessary (> %) in patients referred for endotherapy and expert endoscopy is required to ensure safe and complete oncological resection. conversely pt submucosal cancers detected following emr are confidently triaged for oesophagectomy. combination of emr and rfa in expert groups exceeds > % for eradication of neoplasia and metaplasia. adverse events are quite low (stricture form healing, %; self-limited haemorrhage, very rare perforations ...) and recurrence of bo is also low (< % at years). careful follow-up endoscopies is necessary at to months initially; intervals theerafter probably yearly.  the pathologist's role in the diagnosis and management of neoplasia in barrett's oesophagus p c muldoon st. james's hospital, dublin, ireland the last decade has seen a revolution in the management of neoplasia in barrett's oesophagus. more detailed biopsy protocols, along with the advent of sophisticated local resection and ablation techniques, have radically altered the management of this expanding cohort of patients. these new treatment modalities, coupled with a massive increase in the numbers of cases of barrett's being diagnosed, have significantly altered the demands placed upon pathologists involved in this area. these changes have presented pathologists with an opportunity to challenge our existing practices, to improve the reproducibility of our analysis and to increase the clinical relevance of the way in which we report neoplasia in this setting, where the pathologist plays a critical role in the multidisciplinary management approach. this talk aims to outline a practical approach to the handling of these specimens and to provide clear guidelines as to how to report them in the most clinically useful way.  modern management in ibd p mwr vieth ; h neumann klinikum bayreuth, pathology, bayreuth, germany; university hospital erlangen, medical clinic i, erlangen, germany ulcerative colitis and crohn's make a distinct histological picture. around % of an ibd diagnosis is the clinical information and about % derives from histology, only. for routine purposes it is recommendable in case of a first manifestation of an ibd to make a diagnosis such as: "picture of ulcerative colitis or picture of crohn's disease" and to recommend a follow-up endoscopy with biopsies not prior to weeks after the first endoscopy and than confirm the diagnosis later to exclude mimickers of ibd. in crohn's disease it is helpful to take biopsies from the upper gi-tract to get further hints of crohn's disease. in case of neoplasia, the guidelines leave some room for local endoscopic treatment of low grade dysplasia whereas high grade dysplasia is still seen as an indication for operation since there is a high probability of detecting a carcinoma in the operation specimen afterwards. operation means on normal complete proctocolectomy. this has been indivdually questioned in the last time. there are exceptions for discussing the indication of an operation in ibd: cases with numerous pseudopolyps that cannot be searched for neoplasia, low grade lesions that cannot be completely removed, multiple neoplastic lesions and unresponsiveness to medical treatment.operation and endoscopic specimen in ibd need a subtile search for neoplastic lesions. a microscope with reverse light may help to identify suspicous lesions and may help to decide where exactely to cut a specimen. in conclusion a tight cooperation between clinical and histopathological partners is recommended to reach a high standard for patient care. second opinions may help to achieve and fuel the own learning process esp. in an institution with a lower number of ibd patients during the year. colorectal or bowel cancer screening (bcs) is commonplace and organised national screening programmes have been developed in many countries, most notably in western europe. traditionally, faecal occult blood (fob) detection has been the screening method of choice, those testing positive being selected for subsequent colonoscopy, but this is changing, with alternative or additional screening tests gaining favour. most of the problems in bcs pathology practice are particularly related to fob-based screening programmes, as these are enriched for large, bleeding sigmoid adenomas, in comparison to programmes utilising endoscopy as the primary screening modality. experience within the closely related uk bcs programmes to date has yielded several recurring problems: the diagnosis of stage pt or 'polyp' cancers, in particular distinguishing common epithelial misplacement from 'true' invasion; the management of stage pt cancers, in relation to indications for surgical intervention after such a diagnosis; and the minimum criteria for a biopsy diagnosis of colorectal adenocarcinoma. these issues will be discussed with illustrative examples, along with the somewhat more mundane but highly important practical issue of measuring various parameters related to bcs pathology. the importance of quality assurance measures to ensure high standards within bcs pathology is emphasised. with the introduction of colorectal cancer screening in various countries of the eu there is a sharp increase in the incidence of early colorectal cancer. a significant part of these early tumours presents in a pedunculated polyp. in most cases, these carcinomas are already completely removed by polypectomy. classic risk factors that suggest a high risk for lymph node metastases include haggitt level , positive resection margins, poor differentiation and lymphatic or vascular invasion. however, the evidence is rather thin. most pt studies are performed on sessile polyps. risk factors are more firmly established and include differentiation grade, lymphatic invasion, kikuchi level sm or the presence of budding. however, for a clinical useful decision model, we will need an integrated approach, and both specificity and sensitivity of the various factors should be taken into account. radical surgery seems overtreatment for a large number of polyp cancers. leeds university, leeds, uk minimum datasets have changed cancer reporting. this talk will explain the decision making processes behind the latest datasets both for cancer reporting and bowel cancer screening. it will also look at where we may be going in the future for staging and molecular reporting. the intestines play host to a broad spectrum of infective organisms ranging from viruses, through bacteria , fungi and unicellular parasites to worms. the spectrum of infections seen varies with geographical location, due to socioeconomic factors and due to changes in human behaviour. immunocompromisation due to infections (in particular hiv), malignancy (especially haematological tumours) and the use of immunosuppressive drugs also has an important role in determining the infections commonly seen in the gi tract. the typical pathological features of intestinal infections will be discussed together with suggestions on how to optimise the diagnosis of such pathologies. erasme university hospital, brussels, belgium pathologists are confronted with different types of colitis, most commonly infectious colitis and inflammatory bowel disease (ibd) followed by microscopic colitis and ischaemic colitis. several other forms of colitis, however, exist and might be underrecognised; these diseases include segmental colitis associated with diverticulosis, diversion colitis, eosinophilic colitis and behcet's colitis. clinical presentations of these rare types of colitis vary, and laboratory data are often non-specific; mucosal biopsy is essential in establishing the diagnosis. segmental colitis associated with diverticulosis (scad) is mainly characterised by the involvement of the sigmoid colon with sparing of the rectum and proximal colon. scad often mimics ibd at endoscopic and histological examination; since scad has a self-limited course that resolves without further recurrence or need for treatment, the implications of an inaccurate diagnosis are obvious. diversion colitis is a non-specific colonic inflammation following surgical diversion of the faecal stream. is is characterised by a chronic lymphoplasmacytic infiltrate, and the existence of lymphoid follicular hyperplasia is considered to be a hallmark feature. the development of diversion colitis is attributed to a lack of short chain fatty acids. eosinophilic colitis is etiologically obscure and can be associated with involvement of other sections of the gastrointestinal tract. an infiltrate of eosinophilic granulocytes is found to varying degrees in all wall layers. a history of food intolerance or allergy is present in most of the patients, and peripheral eosinophilia is present in % of cases. gastrointestinal involvement has been reported in up to % of patients with behcet's disease. in cases with ileocolonic involvement, it is often difficult to distinguish behcet's disease from other inflammatory bowel diseases. the diagnosis, therefore, often depends on clinical manifestations and intestinal ulcerative lesions. s·  how to write a paper and get it published p cs herrington ; p dm berney university of edinburgh, edinburgh, uk; barts health nhs trust, london, uk scientific papers have a predetermined structure, and writing in this way requires practice. most journals accept only a small fraction of submitted papers and it is important that any paper has something specific to say; and says it in a clear and concise way that can be understood by editors, reviewers and readers, all of whom play a role in assessment of its contribution. editors look for novelty and significance in the context of the aims and scope of their journal; and scientific rigour, which expert reviewers help them to assess. writing a paper and having it assessed by a journal is an iterative process. during the writing phase, the scientific rigour of the argument can be refined; and following submission and peer review, reviewers and editors often make constructive comments that help to improve it still further. the peer review process therefore acts not only as a quality filter but also as a mechanism for quality improvement. writing papers and submitting them for publication is therefore generally a positive experience, particularly if one remembers that the process is iterative and (inevitably) not all papers will be accepted for publication by the first journal that they are sent to.  large-scale routine diagnostics using whole-slide imaging in sweden -the linköping experience p c lundström cmiv, linköping university, linköping, sweden this presentation will describe the large-scale routine usage of wsi at linköping university hospital, sweden. since all histology slides are scanned, amounting to more than half a million slides to date. to a significant extent the digital images are used for primary review. the initial implementation led to several of the benefits foreseen with digital pathology, but it could also be concluded that further development was needed to unlock the full potential, in particular within the it solutions. therefore, a consortium led by cmiv, linköping university was formed in to create innovations for a new generation of digital pathology. this triple helix consortium also includes industry and more than half of sweden's health care providers, an engagement that reflects the dominating view in swedish pathology that large-scale adoption of wsi practice is possible and desirable. this talk covers the experiences made during the initial digitization, including laboratory process adjustments, and the later additions to the digital pathology toolbox accomplished by the ongoing innovation project. apart from obvious targets such as the pathologists' workstation, the developments also touch upon other areas including grossing and enterprise image management.  digital pathology -are we there yet? p sm hewitt national cancer institute, bethesda, maryland, usa the implementation of whole slide imaging for diagnostic histopathology is far more complex than connecting an instrument to a server, and placing a computer on a pathologist's desk. the technology is additive to the histology workflow, with additional cost beyond the current practice of review with a microscope. the adoption of digital pathology for histomorphologic diagnosis requires the restructuring of the workflow, additional technology advances beyond the imaging instrument, and development of new tools to assist the pathologist. the end goal is to improve pathologist's productivity and provide additional diagnostic information. digital pathology, to succeed must become a value-added proposition. the adoption of digital pathology requires: )improvements in scanner performance as measured by defined quality metrics. )advancement in server and networks to distribute images to the desktop efficiently. )software to facilitate review and diagnosis, beyond presenting only and image of the slide. evolution of the current technologies is required to provide an economic impetus for widespread adoption and use of digital pathology in the diagnostic setting. to a significant extent, the distinction between melanocytic naevi and malignant melanomas is based on tissue architecture. amongst the best known architectural features pointing to malignancy are absence of lesional symmetry and maturation, and presence of melanocyte ascent. however, each of these three features has significant pitfalls. as a rule, naevi are 'roughly symmetrical' and melanomas are not, but there are asymmetrical naevi (traumatized naevi, most larger congenital naevi; some combined naevi; some large acral and genital naevi) and symmetrical melanomas (including many small melanomas, especially small nodular melanomas; some spitzoid melanomas). in addition, it is not always clear whether a lesion should be considered 'roughly symmetrical' or not. i suspect that not uncommonly, a diagnosis is reached first, and the verdict regarding symmetry is adjusted according to that diagnosis. similar caveats relate to absence of maturation as an indicator of malignancy. it is seen in blue naevi and all its variants; deep penetrating naevi; some bap naevi. melanomas not uncommonly feature smaller cells in their deeper parts, or there may be an underlying naevus remnant with smaller cells. naevi with ascent include many spitz naevi; reed naevi; some naevi in early infancy; traumatized naevi; naevi of acral skin. over-interpretation of ascent may result from inexperience with melan-a and some other immune stains. melanomas devoid of ascending melanoma cells comprise a wide variety of subtypes including, desmoplastic melanomas and, vexingly, some spitzoid melanomas. these architectural features must, therefore, be evaluated in the context of all other findings, and with a 'splitter's' mind set, taking into account the individual characteristics of the specific naevus and melanoma variants that are of relevance to the case under study. most melanomas are fairly easy to diagnose on histological grounds. however, melanoma is a tumour that can histologically mimic almost any other tumour including epithelial and mesenchymal neoplasms. pathologists need to familiarize with the wide histological appearances of melanoma to avoid serious misdiagnoses. of crucial importance is the knowledge that a number of melanomas can closely mimic benign naevi. some variants of melanoma represent distinctive clinicopathological entities and these include desmoplastic melanoma, "malignant" blue naevus, pigment synthethizing melanoma, naevoid melanoma, spitzoid melanoma and epidermotropic metastatic melanoma. tumoral melanosis refers to complete regression of a melanoma, a diagnosis that it is often missed because of the absence of tumour cells within the regressed area. a small percentage of melanomas display focal or extensive histological changes that closely mimic other neoplasms and often a combination of histological features with immunohistochemistry is necessary to arrive to the correct diagnosis. microscopic variants of melanoma include adenoid (pseudoglandular), angiotropic and angiomatoid, signet ring cell, balloon cell, clear cell, rhabdoid and follicular (with exclusive involvement of hair follicles). some melanomas display heterologous differentiation also known as transdifferentiation. the latter should not be confused with the so-called collision tumour in which a melanoma co-exists with a neoplasm of different lineage. a wide variety of heterologous differentiation has been described in melanoma including osteosarcomatous and chondrosarcomatous (mainly seen in acral melanomas), meiomysarcomatous, rhabdomyosarcomatous, neuroendocrine, ganglioneuromatous and even epithelial. except for desmoplastic and pigment synthetizing melanoma, all other variants of the tumour have the same behaviour as ordinary melanomas. melanocytic tumours with spitzoid features represent one of the most challenging and controversial areas in dermatopathology. what is currently known as spitz naevus was initially reported as "juvenile melanoma" by sophie spitz on . she recognized the relatively indolent but somewhat unpredictable behaviour of these distinctive melanocytic lesions that are particularly common in young children. over the years, the histological spectrum of these tumours was expanded, and it has become clear that classical spitz naevi follow an entirely indolent disease course. the prognosis of tumours with atypical histological features remains somewhat unpredictable. this presentation will give an overview of the morphological spectrum of spitzoid melanocytic tumours, their behaviour and recent advances of their molecular characteristics. optimisation is a core tenet in radiography and involves the radiographer ensuring that images of diagnostic quality are produced with minimum radiation dose burden to patient and staff [ , ] . in paediatric practice this is particularly important due to the more radiosensitive nature of the child [ ] . in alignment with the isrrt world radiography day theme 'radiographers optimise dose', radiography students in an institution submitted clinical case study coursework that focused on paediatric radiation dose optimisation. the purpose of the current study was to analyse these case studies as examples of prevailing radiographic practice and to compare students' perception of optimisation with the evidence within each case. the evidence of optimisation was established through independent and objective image analysis along with thematic analysis of the case commentaries. the case study evidence demonstrated that optimised techniques were generally well implemented. the exception was collimation, which was sub-optimal in % (n= ) of the examinations, and on average irradiating an area % larger than necessary. students were generally able to correctly identify techniques as optimal or not. however, when appraising exposure, positioning and collimation, between % and % of students were inaccurate in their assessment of what is optimal. overall the study reflects positively on current irish paediatric radiography with regard to dose optimisation, although more accurate collimation needs to be practised. similarly student perceptions show good understanding of optimal techniques, although appreciation of exposure, positioning and collimation errors could be improved. the aim of this project was to design and prototype immobilisation devices for children who are unable to independently maintain upright sitting posture during radiographic investigations. while current market devices exist, they are seldom used by radiographers -particularly in europe as their methods of restraint have been deemed 'culturally unacceptable' with some claiming that they are in violation of the human rights of the child [ ] . the design challenge was to create devices that were functional (fit for purpose [ ], radio-lucent, compliant with infection control and easy to use) while minimising discomfort and intimidation. a search of the literature, prior art, patent landscape and current market devices was performed in order to identify product requirements. triz methodologies -a problem solving, analysis and forecasting tool derived from the study of patterns of invention in the global patent literature were used to identify the physical contradictions underlying the design challenge and generate potential solutions. eight unique concept designs were identified from these methods. these were then evaluated using pugh criteria -a ranking system of the relative merits of each concept based on design requirements identified. four of the eight concepts were chosen to be prototyped: a -d printed seat, a swing based template, an acrylic-based support and an adaptable wheelchair. the prototypes were made in collaboration with the ucd school of engineering and tested using paediatric phantoms in ucd radiography department. the final prototypes will be trialled in crumlin children's hospital with a view to future use and development. thrombotic microangiopathy (tma) is a pathology that results in thrombosis of capillaries and arterioles due to endothelial injury. it is usually characterized by an atypical haemolytic syndrome (ahus) or thrombotic thrombocytopaenic purpura (ttp). tma is considered to be caused by infections, drugs, autoimmunity, tumours, pregnancy, transplants and inherited abnormalities involving the alternate complement pathway. this presentation describes the pathology of tma. it includes a retrospective year study ( - ) of all renal biopsies reported by one pathologist. all renal biopsy request forms and reports, where cases included light (lm), fluorescence(fm) and electron microscopy(em), were reviewed. cases without all modalities (lm, fm, and em) were excluded. biopsies were reported in the study period ( in ( in to in . were transplant biopsies. biopsies were insufficient (lm, fm and em all not possible. this resulted in native renal biopsies as the study group. following review of the reports cases were reported as tma. were associated with thin membrane nephropathy, with minimal change disease and with plasma cell dyscrasia/ b cell malignancy. this resulted in cases with tma as the only pathology reported which represents % of all adequate native medical renal biopsies. clinical indications included proteinuria in %, nephrotic syndrome in %, increased creatinine in %, increased blood pressure in % and haematuria in % of the cases. acute renal failure was described in % and hus in just % of the cases. pathological changes were predominently arteriolar sclerosis and glomerular double contours on lm with chronic subendothelial injury on em. the conclusions of this presentation are . tma is overwhelmingly a chronic lesion as seen in renal biopsy pathology. . it is a very common pattern of injury. . it is not usually associated with clinical hus or ttp features at presentation. the era of targeted cancer therapeutics has brought forth new challenges for molecular diagnostic laboratories. the list of genes, and indeed specific mutations, that predict drug responses keeps growing, and with it grows the demand for molecular sub-classification of tumors. in colorectal carcinoma, for example, recent studies support expanding testing beyond kras to include nras and braf in predicting resistance to egfr-targeted therapies. similarly, in non-small cell lung carcinoma standard screening for egfr mutations and alk gene fusions may be insufficient when actionable alterations involving ros , ret, her , met, braf and other genes are being targeted (successfully) in ongoing clinical trials. fortunately, the introduction of next-generation sequencing (ngs) into the clinical laboratory is meeting the demand. due to its quantitative output, ngs not only provides precise mutant allele ratios, but it can also be used to detect gene gains and losses. furthermore, when applied to rna, ngs supports the detection of gene fusions and serves in assessing gene expression levels. while ngs is a powerful tool for molecularly characterizing solid tumors, the quality of the results in large part rests on the selection of appropriate input material; therefore, review by a pathologist prior to testing remains a cornerstone to success. other growing uses of ngs include monitoring minimal residual disease in the setting of hematologic malignancies, and in the detection of targetable mutations in cell-free dna within the plasma. the advent of next generation sequencing has ushered in an era of tremendous potential for identifying the molecular causation of simple and complex disorders, both rare and common. the successes of next generation sequencing reflect the combined interpretive skills of geneticists, bioinformaticians and clinicians working in close collaboration. in the realm of muscle disease, we have witnessed both the strengths and the weaknesses of next generation sequencing technologies. the use of exome sequencing in patients with rare muscle disease who have been carefully phenotyped has proven to be a successful strategy for identifying causative variants in new genes as well as in known genes. in fact, exome sequencing has significantly expanded both the clinical and the histological phenotypic spectra of muscle conditions associated with causative variants in known genes. in the absence of careful phenotyping or large genetic reference data sets for identifying variants of interest, causative variants may be missed, however. the use of rna sequencing-using rna extracted from muscle biopsy specimens-has proven to be a powerful tool for finding causative variants affecting gene splicing or expression, which may be missed with next generation sequencing. muscle pathology plays an essential role in complementing next generation sequencing. the deep phenotyping of patients with muscle disease relies heavily on muscle histological and immunohistochemical findings in combination with muscle imaging, clinical history and neuromuscular examination findings. examples of how next generation sequencing coupled with careful clinical and histological phenotyping has uncovered causative variants in new genes as well as in known genes will be discussed in this talk. medicine, diagnostic pathology, technology, and diagnostic tests are evolving at an extremely rapid pace. drug developers and diagnostic developers each face unique challenges. companion diagnostic development is a key component of pharma drug development strategy. we will discuss the importance of companion diagnostics in the success of personalized medicine, the fda position on companion diagnostics, and the role, advantages and disadvantages of tissue based companion diagnostics. other technologies, such as next gen sequencing, will increasingly be utilized in a complementary fashion along with traditional slide based immunohistochemical and in situ hybridization. the scope and limitations of available technologies will be reviewed. diagnostic technologies of all types will complement each other to provide the most accurate diagnostic information for clinicians and patients. following the who classification of haematological malignancies there has been a greater emphasis on the integration of molecular information with clinical and morphological data not just for diagnostic purposes but also to help convey both prognostic and therapeutic information. this talk will concentrate on routine testing in the work-up of common haematological malignancies focusing specifically on clonality and translocation analysis in lymphoproliferations and mutational testing in bcr-abl negative myeloproliferative neoplasms. using case studies to illustrate common indications for testing this talk will also highlight some of the practical points and pitfalls in the interpretation of these tests. beaumont hospital, dublin, ireland "should i keep the brain?" is one of the most frequent questions addressed to neuropathologists by surgical pathology colleagues. fears relating to inappropriate organ retention coupled with decreasing availability of expert neuropathology opinion and the widely held belief that advances in neuroimaging have replaced the brain autopsy, have all contributed to a decline in the post mortem study of human brain tissue. leaving aside the critical relevance of neuropathology to forensic medicine, the vital role played by careful examination of the post mortem brain extends far beyond pathology and has contributed greatly to science and medicine. in general, prolonged retention of entire brains may be avoided. in hospital practice it is uncommon for a patient to die without brain imaging. access to pre-mortem brain imaging will guide the surgical pathologist in careful and appropriate sampling of calvarial, dural, meningeal, vascular and parenchymal central nervous system components. spinal cord examination requires prior experience in spinal cord removal but most post mortem technologists are expert in cord extraction. sampling and appropriate processing of nerve and muscle requires prior experience or neuropathology advice. high quality photography obtained at all phases of post mortem brain examination including the coronally sectioned individual cerebral hemispheres, with retention of blocks from each of the brain lobes together with cerebellum, brain stem, vessels and dura -meninges will ensure that in the event of a neuropathology opinion benign required -that opinion will not be compromised. specific issues which will be addressed will include the death of patients with epilepsy, dementia, stroke and undiagnosed neurological disease. the key learning objective will be to ensure that pathology trainees approach post mortem examination of the nervous system with interest and excitement. following several high profile misdiagnoses in ireland a national quality assurance (qa) programme for cellular pathology in was initiated with a vision of establishing a patientcentred pathologist-led framework that would enhance the quality of patient care with timely, accurate and complete pathological diagnoses and reporting. national qa guidelines were developed based on key quality activities generating a total of key quality indicators (kqi). examples of the quality activities include turnaround time, monitoring of amended reports, frozen section correlation and various elements of peer review. all cellular pathology departments in the public state-funded hospitals participate in the programme in addition to laboratories within privately-run hospitals. each laboratory enters codes on individual cases designed to capture the relevant kqi and the anonymised encrypted qa data is then electronically extracted from the laboratory information system to a national database. this national central database is managed by a novel information technology system, the national quality assurance intelligence system (nqais)-histopathology, that was designed to process and display the qa data so that each individual laboratory can analyse their own data and also compare their performance to the national average for each kqi. since complete national data has been inputted into the nqais system and in initial qa targets were agreed for turnaround time, frozen section correlation and rate of intra-departmental consultation (idc). in additional targets for autopsy icd, frozen section deferral rate and turnaround time have been added. to our knowledge this programme has enabled ireland to be the first country to publically report national metrics on the quality of their pathology services.  the politics of eqa: the nhs england qa review and its consequences p de hughes in , a review of external quality assessment processes was carried out on behalf of nhs england. the main recommendations of this review related to the strengthening of governance of eqa, both nationally and within pathology provider organisations. recommendations specifically affecting cellular pathology were: (i) professional bodies, led by rcpath, should develop methodologies for assessing the performance of individuals in eqa schemes. (ii) all pathologists reporting pathology results and providing clinical advice should be participate in eqa schemes relevant to their practice, should achieve levels of performance determined by the professional bodies and this performance should be noted at annual appraisal. (iii) where a need to improve performance is identified, additional remedial training should be carried out, or practice in the area of concern should be stopped until appropriate retraining has been undertaken and revalidation achieved. this process should supported and resourced by the employing organisation, as should eqa scheme participation. (iv) interpretative eqa schemes are designed to assess and improve individual performance, and attempts at collusion are considered matters of professional probity. the professional response to these recommendations is expected to be led by the rcpath under the guidance of a newly-established national oversight group working on behalf of nhs england and should be more clear at the time of the pathsoc conference. a key part of this response will be to separately consider the implications of this review for technical schemes, affecting laboratories, and interpretative schemes, affecting individual practitioners. roche tissue diagnostics, tucson, arizona, usa tumour samples to guide treatment decisions have become of increasing significance. most importantly the results of companion diagnostic testing directly influences the management of individual patients as more drugs are approved for treatment of specific molecular distinct subgroups. reporting suboptimal quality test results may be harmful to the patient and cause the mismanagement of a prescribed companion drug. the consequences of unsatisfactory performance and measures for improvement are the responsibility of the laboratory. presently, there are number of eqa schemes for molecular testing available in europe however, their results clearly indicate the need for eqa since %- % of laboratories do not carry out according to the standard set by the eqa provider or utilize standardized procedures. continual improvement programs, internal quality control and validation program assist laboratories however; by using standardized quality practices such as iso and the use of external quality assurance schemes can provide essential feedback to the laboratory to assure accurate molecular testing results.  how to run a histopathology eqa in the digital age p nj mayer ;p jd oxley cork university hospital, cork, ireland; southmead hospital, bristol, uk interpretative eqa schemes in histopathology were first introduced in the uk in the mid- s, well before the advent of the internet and high resolution digital images. in this lecture we will outline key developments, as eqa schemes have evolved into the digital era, with particular emphasis on the national urological eqa scheme, which we have run since . we will outline the main practical issues involved in running an eqa scheme and share our personal experience of the development and introduction of the web-based eqalite software, which is being utilised by increasing numbers of schemes. we will address the pros and cons of traditional glass slide-based circulations versus virtual circulations using scanned digital images and show how the digital archive generated from old eqa circulations has become a valuable educational and teaching resource. we will also briefly explore, from an organiser's perspective, the major issues facing eqa schemes in the future, as eqa performance becomes more embedded into revalidation and fitness to practice.  molecular pathology: the future? p cs herrington molecular pathology is already central to stratified medicine. and the ability of pathologists to understand disease phenotype is essential for interpretation of the current explosion in '-omics' data. moreover, the future of stratified medicine will require integration of information from different sources, in the context of disease phenotype, to inform patient management: pathologists are ideally placed to lead this integration. this applies not only to data derived from ex vivo cells and tissues but also to molecular imaging data, which require accurate correlation with cell and tissue phenotype for accurate interpretation. molecular pathology is key to the future of pathology; and this future extends beyond the traditional light microscope the following plenary, oral and poster abstracts have been subjected to peer review. hypothesis: suppressor of cytokine signalling (socs) family members play a vital role in the activation of the jak/stat signalling pathway via a negative feedback loop and have been implicated in the development of cancers. in breast cancer (bc), socs mrna has been correlated with oestrogen receptor (er) positive tumours favouring a good prognosis (bmc cancer , : ) . this study aimed to determine whether socs at the protein level correlates with tumour morphology and low grade in bc. methods: differential expression analysis between tubular and grade matched nsts were undertaken in the metabric cohort. primary breast cancer tissue microarrays (n= ) were immuno-stained for socs and expression patterns correlated with clinico-pathological and molecular variables including outcome. results: differential gene expression analysis on the metabric data identified socs as the top gene with a significant overexpression in the tubular type as compared to low grade nsts (adjusted p value= . ). immunohistochemistry on the tenovus series showed positive nuclear socs expression to correlate with tumours of low grade (p< . ), low proliferation (ki p< . ), er/pr positive (p< . ) phenotype and tubular morphology (p< . ); as well as negative her status (p= . ) and non-triple negative status (p< . ). survival analysis revealed significant associations with long term breast cancer specific survival (p= . ). positive socs correlations were also observed with the expression of androgen receptor (ar) (p< . ) and stat (p= . ), further indicating its role in these two signalling pathways. conclusions: results from this study suggest socs to be a marker of favourable prognosis: identifying low grade, er positive breast tumours with particular correlations to the tubular histological tumour type. background: ovarian cancer is the fifth leading cause of cancer in women and has poor long-term survival, in part, due to chemoresistance. tumour hypoxia is associated with chemoresistance in ovarian cancer. however, relatively little is known about the genes activated in ovarian cancer which cause chemoresistance due to hypoxia. this study aimed to firstly identify genes whose expression is associated with hypoxia-induced chemoresistance, and secondly select hypoxia-associated biomarkers and evaluate their expression in ovarian tumours. methods: cisplatin-sensitive (a ) and cisplatin-resistant (a cis) ovarian cancer cell lines were exposed to combinations of hypoxia and/or cisplatin as part of a matrix designed to reflect clinically relevant scenarios. rna was extracted and interrogated on affymetrix human gene arrays. differential gene expression was analysed for cells exposed to hypoxia and/or treated with cisplatin. potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by rt-pcr. results: a wide range of genes associated with chemoresistance were differentially expressed in cells exposed to hypoxia and/or cisplatin. selected genes [angptl , her and hif- α] were chosen for further validation in a cohort of ovarian tumour samples, n= . high expression of angptl trended towards reduced progression-free and overall survival. high expression of her trended to increased progression-free but reduced overall survival, while high expression of hif- α trended towards reduced progression-free and increased overall survival. conclusion: this study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. we have also identified many potential biomarkers of hypoxia and platinum resistance and provide initial validation of a subset of these markers in ovarian cancer tissues. methods: analysis of affymetrix™ human exon . st microarray data revealed differentially expressed genes (degs) between a bc group and a control non-bc group. ingenuity pathway analysis (ipa; bioinformatics software) was used to identify networks of the degs. the expression of a micro-network was validated using immunohistochemistry. double immunofluorescence was undertaken to identify the lineage of cells expressing components of the network. results: we identified a network of interacting genes that were upregulated in fcdiib compared to normally formed cortex or fcd without balloon cells (fcdiia). some components of this network were expressed in bcs but others were expressed in novel cell populations. double immunofluorescence identified a cell with the phenotype of a glial progenitor that was only present in fcdiib but not in normally formed cortex. conclusions: we have identified a novel population of glial progenitors found frequently adjacent to bcs in fcdiib. paracrine signaling between bcs and the novel chi l positive cells is likely to be involved in the pathogenesis in fcdiib. further investigations into the role of these cells would give us a better understanding of the molecular abnormalities underlying fcd and possibly provide novel therapeutic targets. excellent anatomical knowledge of the anal sphincter complex (asc) is essential for the treatment and understanding of low rectal and anal pathology. some of the current descriptions of the asc are contradictory. in this study, the three-dimensional ( d) anatomy of the asc is described with relevance to low rectal and anal surgical pathology. six human adult cadaveric specimens (three males, three females) were obtained from the leeds gift research tissue programme. paraffin embedded mega-blocks containing the asc were serially sectioned at µm intervals. sections were stained with haematoxylin & eosin, masson's trichrome and millers' elastin, from which d reconstructions were developed. the asc is a complex structure, varying between individuals in the size and distribution of its layers with intermingling of fibres and inconsistency of the longitudinal smooth muscle affecting the creation of the surgical intersphincteric plane. longitudinal fibres penetrate the internal and external anal sphincter to anchor in the submucosa and ischiorectal fossa. striated muscle fibres from the external sphincter were identified in the submucosa in four of six specimens. the asc is highly complex due to the degree of variation in its structure and intermingling of smooth and striated muscle fibres and their penetration of major structures. this creates potential tissue planes for the spread of infection, fistula extension and tumour spread. the complex anatomy of the asc also impacts on the staging of low rectal cancers in this region, which requires further investigation. p h thorpe; a asiri; m akhlaq; d jackson; m ilyas cten is upregulated in a number of tumour types and in colorectal cancer expression is associated with advanced dukes stage, poor prognosis and distant metastasis. cten is localised at focal adhesions and regulates cell motility but knowledge of underlying signalling mechanisms is sparse. epithelial to mesenchymal transition (emt) is a process whereby cells acquire an invasive phenotype to aid cell migration and is found to occur in a number of biological processes including cancer metastasis. we investigated whether cten increases cell migration through emt pathways in colorectal cancer. cten was forcibly expressed in colorectal cell lines and snail expression determined by qpcr and western blot. the cycloheximide pulse chase assay was used to assess any changes in snail protein stability. further to this, the transwell migration assay was performed to investigate changes in cell motility. forced expression of cten was shown to increase snail protein expression in hct and caco cell lines. there was no change in the level of snail mrna suggesting that cten regulates snail at a post transcriptional level. inhibition of protein synthesis confirmed this and showed that cten regulates the stability of snail protein. simultaneous forced expression of cten and knockdown of snail demonstrated that this relationship was functionally active. forced expression of cten increased cell migration (p< . ) which was subsequently lost when snail was knocked down (p< . ). we are the first to identify snail as a downstream target of cten signalling. this finding advances the understanding of cancer cell motility regulatory networks and further highlights cten as a potential therapeutic target in colorectal cancer. work supported by a pathological society grant. treatment strategies for patients with advanced colorectal cancer p sd richman ; gj hemmings ; p chambers ; m taylor ; hm wood ; e tinkler-hundal ; k southward ; jm foster ; a ouime ; kg spink ; p quirke leeds institute of cancer and pathology, leeds, uk; affymetrix, high wycombe, uk treatment for advanced colorectal cancer is moving to combination therapies, targeting multiple signalling pathways. indeed, mrc focus has been designed to assess this. we determined pten protein expression, and assessed this in relation to other biomarkers associated with signalling downstream of the epidermal growth factor receptor. tissue microarrays were constructed from advanced colorectal cancer (acrc) clinical trials (focus and piccolo) for immunohistochemistry (ihc). mutation status of kras, nras, pik ca and braf was assessed by pyrosequencing. copy number variation was assessed on oncoscan® ffpe assay kit (affymetrix inc.). pten protein expression was correlated with mutation status, mmr status, primary tumour location and copy number. pten protein expression for patients showed complete loss of expression in / ( . %) -focus and / ( . %) -piccolo. braf mutation status was significantly different between the pten negative and pten positive populations (p< . ), with significantly more pten negative tumours having the braf v e mutation. loss of pten expression correlated with genomic deletions involving the pten gene. / ( %) of pten negative tumours exhibited loss of the pten region ( q), half of which were focal deletions. only / ( . %) pten positive tumours showed deletions of this region, and none were focal events. there was no significant difference in either primary tumour site or mmr status (p= . ) between the pten negative and pten positive populations. signalling pathways do not stand in isolation; they are interlinked in a complex signalling network. current treatment interventions must target the correct pathway combinations if patients are to benefit from targeted therapy. our data suggests a subset of patients may require dual akt and mek pathway inhibition, in addition to anti-egfr monoclonal antibody therapy and inhibition of braf. zonal differences in pd expression in centre of tumour versus periphery in microsatellite stable and unstable colorectal cancer p gm o'kane ; m lynch ; j aird ; s hooper ; c muldoon ; n mulligan ; c loscher ; dj gallagher st. james's hospital, dublin, ireland; dublin city university, dublin, ireland; mater misericordiae university hospital, dublin, ireland colorectal cancers (crc) that show evidence of microsatellite instability (msi-h) are marked by a high tumour infiltrating lymphocyte (til) population which is thought to be prognostic. programmed cell death (pd- ) is a negative regulator of the immune system and targeting the interaction with its ligand pd-l offers a potential therapeutic target. we aimed to characterize cd and pd- expression in both the tumour centre (ct) and tumour periphery (pt) of microsatellite stable (mss) and unstable crc. methods: paraffin-embedded tumour blocks were cut at um, prepared and stained using specific antibodies for cd and pd- . the pt was defined as the area within a x high power field (hpf) from the outline of the tumor. the ct was defined as the area at least one x hpf apart from the tumor outline toward centre of the tumor. images were taken at x, x, x and x. positive cells were averaged across high power fields and classified as high or low positivity. results: forty-two specimens have been analysed to date including msi-h and mss tumours. sixty-eight percent of msi-h were stage ii and % of mss were stage iii. in the msi-h group, a high cd count in the ct and pt correlated with and earlier tumour size and stage. pd- positivity was seen in % of msi-h ct compared to % positivity in the ct of mss tumours. the periphery of both mss and msi-h specimens showed significant pd- expression with % and % of samples showing positivity respectively. there was no association between high or low densities of staining and stage. conclusions: zonal differences exist in the expression of cd and pd- in microsatellite stable and unstable tumours. a high proportion of msi-h tumours show pd- activity in the centre of the tumour despite an improved prognosis. further profiling of other t cell populations may help to further understand this expression which may act as a biomarker or provide a therapeutic target biomarkers that are able to distinguish stage ii and iii colon cancer patients at high risk of developing disease recurrence, who may benefit from adjuvant chemotherapy, are still lacking. genome-wide profiling of somatic aberrations, including gene point mutations, dna copy number aberrations (cna) and structural variants (sv), is expected to provide better insight into the molecular pathology of tumour progression and clinical outcome. genome-wide analysis of cnas was performed using high-resolution comparative genomic hybridization for microsatellite stable (mss) stage ii and iii primary colon cancer samples (n= ). in addition, the prevalence of genes suffering from cna-associated chromosomal breaks, indicative for svs, was determined. the mutation status of commonly affected apc, tp , kras, pik ca, fbxw , smad , braf and nras genes was examined for samples using targeted massive parallel sequencing. associations of genomic aberrations with disease-free survival (dfs) rates were explored by log-rank tests using , permutations. disease recurrence and dfs rates differed significantly for several cna-regions (p< . ). a total of genes were recurrently affected by cna-associated chromosomal breaks (fdr< . ), among which genes ( %) that were also identified in a previously analysed cohort of metastatic colorectal cancers. gene point mutation frequencies were in concordance with literature. in a univariate analysis, none of the individual mutated genes appeared to be significantly associated with dfs. in summary, several associations are found between highly prevalent genomic cnas and disease recurrence in this cohort of mss stage ii and iii colon cancers. further in-depth analysis is required to unravel underlying biology that contributes to disease recurrence. km sutton ; d bottomley ; d morton ; p quirke ; p np west accurate and reliable methods for assessing the molecular profile of clinical tumour samples are important for the delivery of personalised medicine. when adopting a targeted amplicon sequencing method in combination with next generation sequencing (ngs), it is ideal to call mutations against a control sample to enable artefacts to be removed from the analysis. blood is considered the gold standard control but may not always be available. we compared the use of histologically normal mucosa to blood as a control in colon cancer. we examined mutations in colon cancers from the ncri foxtrot trial using the fluidigm access array for ngs library preparation. we assessed the use of both blood and normal colonic mucosa as a control for assessing mutations in genes. all samples were tested in duplicate. the work was partly funded by a pathsoc career development fellowship and is presented on behalf of the foxtrot collaborative group. mutation calls made using normal mucosa as a control compared to blood were in good agreement; a mathew's correlation coefficient above . was seen for all of the genes where agreement could be assessed. we found that false positive mutations were due to poorer amplification of the normal mucosa samples and false negatives were due to mutation calls in the normal mucosa. overall we found that when assessing mutations in hotspot oncogenes, testing in duplicate and the use of a normal control tissue is not required to make mutation calls. however, where a normal control is required, normal mucosa from the resection margin is a suitable alternative to blood where it is not available. we report a series of four unusual ovarian or extraovarian neoplasms composed of an admixture of adenosarcoma and a predominant component comprising a sex cord tumour. the neoplasms occurred in women aged to . three cases arose within the ovary and one was extraovarian (pelvis and abdomen) in location. in all four cases, there were minor areas with morphological features of adenosarcoma with a phyllodes-like architecture and periglandular increased cellularity with mitotic figures. in two cases, the stromal component was morphologically in keeping with a juvenile granulosa cell tumour. in one case, the stromal component had some features of both adult granulosa cell tumour and sertoli cell tumour within a fibromatous background. the fourth case morphologically could not be categorised as any of the usual types of ovarian sex cord tumour and was categorised as an unclassifiable sex cord tumour. in all four cases, there was immunohistochemical evidence of sex cord differentiation. in each case, we propose that the sex cord tumour arose from a pre-existing adenosarcoma thus representing an unusual form of sarcomatous overgrowth of sex cord elements which can occur within adenosarcomas. this phenomenon is not well described in the literature. background: human epididymis protein (he ) is a secreted protein that is overexpressed in some cancers. he is emerging as a useful biomarker in diagnosis and follow-up of endometrial cancers. the aim of this study was to evaluate the potential role of serum he in the diagnosis and management of endometrial cancer. methods: patients undergoing surgery for endometrial disease were recruited into this study and had pre-operative serum samples taken, n= . demographic, clinical, radiological and laboratory data were reviewed. he and ca serum levels were analysed using the fujirebio diagnostic elisa kits and results correlated with clinicopathological details. standard cut-off points of pmol/l for he and u/ml for ca were used. results: he showed a sensitivity of % and specificity of . % for detection of endometrial cancer. ca had a very low sensitivity of % for endometrial cancer diagnosis. he was elevated in all stages of endometrial cancer and demonstrated the ability to distinguish between benign and malignant groups. he also provided information about myometrial space invasion. conclusion: he has a role in endometrial cancer diagnosis and prognosis and has the potential to be used in a screening setting or as a triage marker in the primary care setting. for women diagnosed with endometrial cancer, he has the potential to stratify them into treatment regimens where the most appropriate treatment can be delivered resulting in improved quality of life and outcome for endometrial cancer patients. platelets drive metastatic changes in ovarian cancer cells p cd spillane ; nm cooke ; s o'toole ; d kenny ; o sheils ; jj o'leary histopathology department, trinity college dublin, dublin, ireland; department of molecular and cellular therapeutics, rcsi, dublin, ireland; department of obstetrics and gynaecology, trinity college dublin, dublin, ireland background: ovarian cancer is the th leading cause of cancer related deaths in women. previously we described a dynamic interaction between ovarian cancer cells and platelets in vitro, involving platelet adhesion, activation and induction of pro-survival and pro-angiogenic signals in the cancer cells. this study looked to further investigate this phenomenon in ovarian cancer cells by assessing the molecular changes it induced. methods: cell lines m and skov were used as in vitro models of metastatic ovarian cancer. platelet cloaking of cells was quantified by flow cytometry. cells co-cultured with/ without platelets for hrs were examined by rt-pcr for emt related changes and by affymetrix gene . st arrays for whole transcriptome changes. results: significantly more platelets adhered to skov cells than m cells. while there were different rates of adhesion, the platelets induced similar changes in emt related genes in both. there was a significant loss in expression of epithelial genes and an increase in mesenchymal genes, indicating the induction of emt. whole transcriptome analysis showed that there were a greater number of gene expression changes occurring in skov cells compared to m cells, correlating with the adhesion data. a gene panel of commonly affected genes in both cell lines was identified, many of which form part of an interlinking pathway that is regulated by tgfβ and associated with cell adhesion/ecm remodelling. though only genes overlapped, the biological processes affected in both cell lines were very similar, with of the processes enriched in the m data set also seen in the skov data set. conclusion: this study shows that platelets can enhance the metastatic potential of ovarian cancer cells through the induction of emt and ecm changes. in addition, it has identified a set of genes that hold potential to be in vivo markers of this interaction. background: during the metastatic cascade, circulating tumour cells rapidly and efficiently adopt a platelet cloak. platelet cloaking of tumour cells promotes metastatic disease by promoting cellular proliferation, angiogenesis and emt while inhibiting autophagy and apoptosis. the aim of this study is to examine whether the platelet cloak contributes to tumour cell evasion of nk cell mediated immune surveillance. methods: freshly isolated pbmcs were harvested from healthy donors and stimulated for hours with il- ( u/ml). pbmcs were co-incubated with ovarian ( m and skov ), melanoma (sk-mel- ) and cml (k ) cell lines that were either uncloaked, or cloaked with washed platelets from healthy donors. the nk-tumour cell receptor ligand systems, nkg d-mica/micb and cd /cd -cd were examined using nk cell cd a expression and interferon-gamma production to quantify nk cell mediated recognition and 'killing' of cancer cells. results: we first demonstrated that ovarian and melanoma cancer cell lines when cloaked with washed platelets strongly inhibited nk cell antitumor reactivity. platelet cloaking induced down-regulation of the stress ligands mica and micb on the tumour cell coupled with their release into the microenvironment, a known nk cell immune decoy strategy. in addition, platelets significantly down-regulated both cd (nk cell) and cd (tumour cell), inhibiting nk cell activity. both mechanisms occur in tandem to comprehensively incapacitate nk cells and promote tumour immune evasion. conclusions: ovarian and melanoma tumour cells are efficiently cloaked by platelets, which facilitates immune evasion by actively suppressing nk cell cytotoxicity and cytokine production. purpose of the study: glioblastomas (gbm) are the most common and most aggressive primary malignant brain tumours in adults. one of their histopathological hallmarks is the microvascular proliferation; these tumours are among the most angiogenic of malignancies by displaying the highest degree of microvascular proliferation. igfiir/man- -p is a receptor that belongs to the insulin-like growth factor (igf) system. the involvement of igf-iir/man- -p in the process of angiogenesis has been postulated in rare earlier studies. to our knowledge, the role of igf-iir/man- -p in the neovascularisation of human gbm has never been studied. methods: igf-iir/man- -p expression was evaluated in the vascular compartment from human gbm and from normal adult brain samples by means of quantitative immunohistochemistry on tissue microarray sections. in vitro cell line experiments were carried out in order to characterise the igfiir/man- -p role in angiogenesis. summary of results: igf-iir/man- -p was strongly expressed in the cytoplasm of endothelial cells in hyperplastic vessels and exhibited a dot-staining pattern. we found a higher expression of igf-iir/man- -p in gbm vessels compared to normal brain vessels (p= . ). furthermore, preliminary in vitro experiments suggest a role of igf-iir/man- -p in tube formation but not in growth of the ea.hy endothelial cell line. conclusions: this work shows a possible role of igfiir/man- -p in the process of neovascularisation of gbm angiogenesis. additional investigations are required to confirm the role of this receptor as a direct actor of angiogenesis in gbm. purpose of the study: vasa vasorum (vv) are microvessels which supply vessels that cannot be nourished by diffusion from their own lumina. vv are believed to be a key element in the pathogenesis of vascular diseases. a number of different imaging methods have been used to study the vv but there is still no definitive consensus on their structure. the aim was to describe the normal microvessel anatomy of temporal arteries. methods: human temporal artery, obtained following routine biopsy with ethical approval and patient consent. samples were embedded into paraffin blocks and serially sectioned at micron intervals. alternate sections were stained with h&e and scanned to create virtual slides. the slides were aligned, vv were segmented (annotated) and iso-surfaced to generate d reconstructions. summary of results: the reconstruction shows the structural arrangement of the vv as a complex plexus. no connection to the vascular lumen was visualised. in this segment a hierarchical branching structure was not observed. vv were almost exclusively restricted to the adventitia of the vessel wall. mean ± sd area of the vv (n = ) is . µm (± . ). the mean ± sd number of vessels per slide is . (± . ). these metrics are based on one arterial specimen. conclusion: this method allows us to study the three-dimensional spatial relationships of microvessels within arterial specimens. furthermore, metric data generated in the process can support the d images to study the microvasculature. this method will be applied to diseased arteries in future to generate novel hypotheses about the inflammatory process. acknowledgements: this research was supported by a pathsoc intercalated studentship. purpose of the study: it is controversial whether mesothelioma can be diagnosed with confidence in effusion cytology and therefore an ancillary marker of malignant mesothelial cells would be clinically valuable. brca- associated protein (bap ) is a tumour suppressor gene which shows biallelic inactivation in approximately half of all mesotheliomas. bap expression is commonly lost in mesothelioma. we investigated whether loss of bap expression can be used to support a diagnosis of mesothelioma in effusion cytology. methods: immunohistochemistry (ihc) for bap was performed on cell blocks from effusions associated with confirmed mesothelioma cases, effusions containing mesothelial cell atypia, benign effusions, and effusions from patients with lung adenocarcinoma. results: ihc for bap was performed on cases of confirmed mesothelioma. ( . %) showed negative staining in the presence of an internal positive control. in effusions considered to have atypical mesothelial cells in the absence of definitive diagnosis of mesothelioma, cases demonstrated negative staining for bap . on follow up, of these patients received a definitive diagnosis of mesothelioma in the subsequent months ( were lost to follow up immediately). only of consecutive benign effusions were interpreted as bap negative. patients with confirmed adenocarcinoma demonstrated positive staining for bap . conclusion: we conclude that loss of bap expression in effusion cytology is quite specific for mesothelioma. whilst it is not definitive, it can be used to support the diagnosis of mesothelioma in atypical effusions. we caution that interpretation of bap ihc on cell block may be difficult and that convincing positive staining in non-neoplastic cells is required before atypical cells are considered negative. we also note that bap loss is not a sensitive test and cannot be used to exclude mesothelioma. the south-east of scotland experience on the molecular detection of egfr, kras and alk mutations in lung adenocarcinomas p y kheng ; l williams ; k walsh ; j fairley ; s camus ; l gilroy ; k gilmour ; d stirling ; w wallace ; d harrison ; a oniscu royal infirmary of edinburgh, edinburgh, uk; the university of edinburgh, edinburgh, uk the approval of novel targeted treatments for egfr-positive and alk-positive non-small cell lung cancer (nsclc) has led to the increased requirement for mutation testing services in south east of scotland. egfr mutations are typically found in females, asians and never smokers whereas kras mutations are associated with smoking. alk rearrangements are commonly found in younger patients and never smokers. this study aimed to determine the prevalence of egfr, kras and alk mutations in south east of scotland and to evaluate our experience in testing of alk with ihc and fish. data of all patients tested were collected retrospectively from clinical records. from january to may , we reported mutation rates of egfr, kras and alk to be . % ( / ), . % ( / ) and . % ( / ) respectively. in our cohort, an increase in one pack years of smoking resulted in a decrease in the odds ratio of egfr-positivity (or . , % ci . - . , p< . ). kras-positivity was associated with a history of smoking, with rates in both former (or . , % ci . - . , p= . ) and current smokers (or . , % ci . - . , p= . ) significantly higher than in non-smokers. the number of smoking pack years had no influence on the rates of kras-positivity. alk-rearrangements were found to be associated with never smokers (p< . ) and younger patients (≤ years old) (p< . ). to date, no false positives were reported for parallel testing of alk with ihc and fish. we observed % sensitivity ( ihc+/ fish+) and . % specificity ( ihc-/ fish-) when comparing ihc with fish. in conclusion, the prevalence of egfr mutation in south east of scotland has reflected mutation rates reported in west of scotland. our findings further support the use of alk-ihc as a diagnostic screening tool. purpose of the study: the molecular mechanisms of metastasis and progression of penile squamous cell carcinoma (pscc) are unclear. nobody, to our knowledge, has investigated the expression of cell-cycle proteins in advanced or metastatic pscc. we aimed to determine the extent of hpv infection in patients with advanced pscc and its effect on the expression of the key cell-cycle proteins p , p ink a and retinoblastoma (rb). methods: archival paraffin embedded blocks were obtained from primary penile cancers, all patients having developed locally-advanced or metastatic disease. all patients were treated in the phase ii trial of docetaxel, cisplatin & -fluorouracil (tpf) chemotherapy cruk/ / (nicholson et al. bjc ; : - ) . samples were analysed immunohistochemically for p ink a, p and rb protein expression on a tissue microarray. all tumours were hpv typed using pcr. summary of results: hpv dna was detected in / ( %) with hpv present in / ( %). cases were not suitable for analysis. no association was found between hpv and expression of either p ink a (p= . ), p (p= . ) or rb (p= ) using fisher's exact test. conclusions: hpv dna is detected in less than half of progressive pscc, suggesting either the loss of hpv in advanced disease or that non-hpv related cancers progress more commonly. the lack of correlation between hpv and these cell-cycle proteins suggests that they may undergo somatic mutation that is not driven by hpv, leading to increased growth and invasiveness. treatment strategies may be hampered by this genetic diversity, which requires further investigation. prostate cancer is the second most common form of cancer in males, and the incidence of this disease is predicted to double globally by . more than . million new cases of prostate cancer are diagnosed each year and two thirds of these patients are from the western world. current diagnostic tests for prostate cancer are limited in both sensitivity and accuracy, and a method for accurate prognosis in these patients is yet to be developed; therefore, there is a need for a sensitive and specific prostate cancer test to implement early and appropriate therapy. the recent discovery of altered endosomal-lysosomal biogenesis in prostate cancer cells has identified a fundamental change in the cell biology of this cancer that holds great promise for the identification of novel biomarkers that can predict disease outcomes. investigation of the endosome compartment and endosome biogenesis revealed elevated gene and expression of critical machinery components that are required for endosome biogenesis and endocytosis. here we demonstrate significantly altered expression of endosomal and lysosomal genes in mrna microarrays of prostate cancer tissue compared to non-malignant tissue, and that specific endosomal and lysosomal genes are predictive of patient outcomes. two endosomal tri-gene signatures were identified that had a significant capacity to stratify patient outcomes. changes in the expression of these genes was further ascertained by qpcr in fresh-frozen prostate tissue specimens, which further implicated altered endosome biology during disease progression, with significant changes in expression observed between aggressive prostate cancer and indolent disease or normal prostate tissue. these findings support the initiation of a retrospective trial to determine if these new biomarkers can accurately predict clinical progression in prostate cancer patients. m craze; c joseph; c nolan; a green; ea rakha; io ellis; p a mukherjee university of nottingham, nottingham, uk introduction: lymphovascular invasion (lvi) is an important step in the metastatic cascade. identification of a molecular signature for the lvi positive phenotype will help identify relevant drivers and pathways. this study aimed to investigate determinants of lvi from a biomarker database. methods: biomarkers (n > ) from a well annotated series (n= ) were analysed for correlations with lvi [clinical/ihc (d - ) supplemented]. proteins with significant associations with lvi were interrogated for pathway enrichment analysis [corrected for false discovery rate (fdr)], using the string . platform incorporating gene ontology (go), kegg and nci. results: biomarker analysis related to both clinical/ihc determined lvi identified positively associated markers, in both clinical and ihc categories (e.g. ada , cd , foxp , kpna ). a further markers were negatively associated, in both categories (e.g. bcl , brca , mage and sox ). significant pathways (p< . ) unifying the positively associated proteins include metabolism, immune responses (t-cell regulation and differentiation), cell activation and transcription [go] ; t-cell receptor signalling pathways and pathways in cancer and haematopoietic cell lineages [kegg] . for negatively associated proteins, the following were significant: ubiquitination processes, regulation of the mitosis [go]; p pathways [kegg] and apoptotic and cell cycle pathways [go & kegg] . on cross-validating a subset included in the metabric cohort, there were overlapping enrichments for immune response regulation (go) and haematopoietic cell lineages (kegg). conclusions: these preliminary findings are the first to unify biomarkers for lvi pathway analysis in bc, using protein based data. within the constraints of selection bias, data mining from immunohistochemistry of multiple biomarkers in relation to biological processes hold promise. *am supported by the nihr and the academy of medical sciences abstracts s· assessment of her status on needle core biopsy of breast cancer: impact of histopathological concordance p m pigera; ahs lee; io ellis; ea rakha; z hodi nottingham city hospital, nottingham, uk one of the key recommendations introduced in the asco/cap update guideline recommendation on her testing is the novel concept of "histopathological concordance." it is proposed that certain tumour morphological features such as histologic type and grade should trigger repeating a molecular test in cases of "discordance". in this study we have we have reviewed breast cancer cases consecutively reported in routine practice in nottingham in the last years. data on her status was collected and cases with her assessed on resection specimens (rs) were analysed in details. results: of all cases, patients ( %) had her status assessed on core biopsy and the corresponding tumour rs. the main reasons for a repeat were tumour multifocality and morphologically different or heterogeneous tumours. a few cases were repeated because of borderline negative fish results or neoadjuvant therapy. cases were repeated due to insufficient tumour in the core biopsy. in this study the her status of the index tumour was changed in cases and both were in the borderline result category. her testing of different tumour foci of multifocal or morphological heterogeneous tumours was consistent with that of the index tumour assessed on the core biopsy apart from two cases; one positive and one negative. tumours were upgraded from grade on core to grade on excision and her status did not change. no contribution of hormone receptor or tumour type was identified. conclusion: there is excellent agreement between her assessed in core biopsy and rs. histopathological discordance seems to play a minor role which does not justify test repeat in routine practice. tamoxifen prevents breast cancer in a sub-set of high-risk women in a mechanism that appears to be dependent on reduction of md. animal model studies suggest that tamoxifen remodels the mammary stroma to a tumour-inhibitory phenotype. this study aims to analyse the effect of tamoxifen on breast fibroblast function and identify potential protumourigenic pathways contributing to density-associated risk. methods: primary human breast fibroblasts were treated with hydroxytamoxifen ( nm- µm). fibroblast function was analysed by measuring: proliferation; expression of stromal proteins fibronectin (fn), lox and collagen ; effects on tgf-β signalling via smad phosphorylation and upregulation of the myofibroblast marker sma. genome wide analysis was performed using rna-seq. summary of results: fibroblasts from patients were treated with tamoxifen. all patients showed reduced proliferation with treatment. in % of patients tamoxifen treatment resulted in reduced expression of fn. tgf-β-mediated upregulation of sma and fn were consistently inhibited by tamoxifen, as was fibroblast contraction of collagen gels. rna-seq analysis revealed modulation of a number of metabolic pathways by tamoxifen, including significant upregulation of dhcr , part of the microsomal antioestrogen binding site (aebs). conclusions:these data indicate that tamoxifen can directly remodel the stromal microenvironment, generating a less 'reactive' stroma. modulation of aebs activity has been proposed to be anti-tumourigenic, and also is implicated as a suppressor of hedgehog signalling. thus, tamoxifen impacts on multiple pathways to create a tumour inhibitory phenotype. this work was supported by the pathsoc small grant scheme. purpose of the study: the phenotypic features of basal like (bl) breast cancer (bc) resemble those occurring in brca -germline mutation carriers. several lines of evidence suggesting the overall tendency of basal-like/triple negative bc to spread through vascular rather than lymphatic routes. the latter has recently been attributed to the activation of cadherin switch, an emt-like phenomenon, in blbc. this study aims at studying the cadherin switch expression profile tgfb , a key emt-trigger, expression in brca mutated compared to sporadic bc. the expression of e-cadherin, n-cadherin and tgfb were studied in a subset of germline brca mutated bc (n= ) compared to non-selected cohorts of non-lobular sporadic invasive blbc (n= ) and non-basal bc (n= ) using ihc and tma. summary of results: compared to sporadic bc, brca mutated cases were of younger age, more grade , with more medullary-like tumours, and more lvi positive. e-cad was significantly less expressed in brca cases than in the sporadic non-basal and in the blbc. however, n-cad was not significantly expressed in brca , non-basal, and blbc. tgfb was significantly less expressed in sporadic bc, both non-basal blbc than brca mutated bc. e-cad/n-cad combinatorial expression phenotypes were significantly different between brca mutated and non-basal and blbc. higher proportions e-cad-/n-cad+ were significantly observed blbc than non-basal bc. brca mutated cases displayed the least e-cad+ expression and the highest e-cad-/n-cad+ in the studied series. conclusions: despite the known similarities between brca mutated and blbc, results of this study demonstrate the more occurrence of cadherin switch in brca mutated breast cancer. e-cad repression appears to contribute more than n-cad gain in blbc than non-basal bc. exploring molecular mechanisms underlying lymphovascular invasion in breast cancer p sn sonbul ; a mukherjee ; r russell ; om rueda ; m aleskandarany ; ar green ; e provenzano ; c caldas ; io ellis ; ea rakha the development of tamoxifen resistance (tr) in oestrogen-dependent breast cancer (bc) is a therapeutic challenge. insulin-like growth factor binding proteins (igfbps) may play a role in this process. we have investigated the role of igfbp proteins in tr bc. igf axis genes were evaluated in mcf- (wt) cells and tamoxifen-resistant (tamr) variants using qrt-pcr and confirmed by elisa, western, and ligand blotting. igfbp- & - were knocked down by shrna transfection, and subsequent sensitivity to -hydroxytamoxifen ( -ht) was determined via wst- . cell migration was investigated by using the incucyte system. igfbp- expression was evaluated in bc cases by tma immunohistochemistry. five out of genes of the igf axis (igf-ir, igf- r, igfbp- , - and - ) had the highest expression levels by both parental wt and tamr cells. igfbp- was down-regulated by ~ -fold while igfbp- was up-regulated by ~ -fold in tamr versus wt cells (mrna and protein levels). significantly, a knockdown of igfbp- in tamr cells restored sensitivity to ( -ht), reduced erα expression to ± . % and enhanced cell migration. expression of igfbp- was significantly (p< . ) associated with survival advantage in tr patients. igfbp- and igfpb- are reciprocally regulated in the acquisition of tr by mcf- cells. igfbp- may play a role in the development of tr in vitro and its high levels in clinical samples may predict tr. purpose of the study: several lines of evidence are currently suggesting that the morphologic heterogeneity of breast cancer is mirrored at the genetic level. understanding the molecular genetic evolution of bc would contribute further insights into the molecular derangements driving disease progression. moreover, varied clinical outcome and response to similar therapeutic regimen is attributed, at least in-part to intratumoural heterogeneity. ngs can reliably study the genetic events using miniscule amounts of genomic dna. methods: gdna was extracted from ffpe tissue sections from a case of invasive duct carcinoma ( primary tumour samples and samples from positive axillary lymph node metastases). sample preparation and exome enrichment was performed using nextera rapid capture exome kits (illumina, fc- - ). exome sequencing was performed using illumina miseq with x depth of coverage (following adapter/barcode trimming). exploratory analyses and data mining were executed regarding variant (s) concordance/ discordance between primary tumour samples and their respective metastatic variants. summary of results: initial findings revealed candidate indels common to all three axillary lymph node samples yet absent from the three primary tumour samples. several genes have been identified as having frameshift mutations caused by indels. molecular players previously linked to anti-angiogenesis are amongst the genes affected by indel mutations in their coding sequences that may lead to potential abrogation of protein function. conclusions: these initial findings provide the framework for detailed molecular analyses for assessing molecular evolutionary events in primary breast cancer and their corresponding metastases. c-myc is amplified in approximately % of breast cancers (bc) and is associated with poor outcome. c-myc protein is multi-faceted and participates in many aspects of cellular function and is linked with therapeutic response in bc. we hypothesised that the functional role of c-myc differs between molecular subtypes of bc. we therefore investigated the correlation between c-myc protein expression and other proteins involved in cell cycle control, proliferation, apoptosis and dna damage together with clinicopathological parameters, outcome and treatments in early invasive primary bc (n= , ) using immunuohistochemistry. the metabric bc cohort (n= , ) was evaluated for c-myc mrna expression. in whole series, there was significant association between c-myc protein expression with higher tumour grade, lymph node(ln) positivity and medullary-like tumours. c-myc showed differential association with other proteins in the molecular classes. in luminal a tumours, c-myc was associated with atm (p= . ), cyclin b (p= . ), pik ca (p= . ) and ki (p< . ). in contrast, in basal-like tumours, c-myc showed positive associated with cyclin e (p= . ) and p (p= . ) expression. c-myc was an independent predictor of a shorter distant metastases free survival in luminal a ln+ tumours treated with endocrine therapy (et; p= . ). c-myc expression did not predict patient outcome in the other molecular subtypes with respect to adjuvant treatment. high c-myc mrna expression was associated with higher grade and basal phenotype (p< . ). in luminal tumours treated with et, c-myc mrna expression was associated with bc specific survival (p= . ). c-myc function is associated with specific molecular subtypes of bc and confers resistance to et. the diverse mechanisms of c-myc function, particularly in luminal a bc, warrants further investigation. metasin axillary predictive score (maps): a measure of axillary nodal disease prediction to provide an informed choice for breast cancer patients and surgeons p pp gopinath ; d george ; p sai-giridhar ; s jader ; e arkoumani ; s holt ; g francis ; c yiangou ; s al ramadhani ; s el sheikh ; n agrawal ; v sundaresan purpose of study: intra-operative sentinel lymph node sampling and molecular analysis empowers the surgeon to carry out axillary clearance as a one-step process. we have recently completed the clinical validation of metasin, an intraoperative molecular assay for sentinel lymph node analysis in breast cancer patients ( cases). method: the assay uses positive predictive markers and is quantitative, enabling the prediction of tumour volume using markers ck and mammaglobin. in this study group, patients had positive sentinel nodes and cases underwent axillary clearance. of the axillary clearance cases, % contained positive lymph nodes. % were sentinel node (snb) macrometastases, % were snb micrometastases and % were snb negative or contained isolated tumour cells. informative data was available for sentinel nodes from positive cases. results: using the qpcr values (from metasin assays using standardised pre-mixes) and clinical axillary clearance data, the cases have been stratified on the basis of the involvement of other axillary nodes. we have shown a three-tiered predictive grouping exists: group a includes low tumour volume disease with a nodal positivity of % within the axilla (n= ): group b with a % positivity of other nodal involvement (n= ) and group c with positivity of % of axillary clearances (n= ). the clustering of the metasin data is dependent on the qpcr results and shows that the cases can be sub-grouped to provide a probability basis for prediction of axillary nodal involvement; dependent on the qpcr cut offs. this gives the patient and surgeon a statistical basis for determining the likelihood of other axillary nodal disease. sequencing of the brca and brca genes has long been used in genetics laboratories to identify cases of familial breast and ovarian cancer. however, the advent of chemotherapy for ovarian cancer based on parp inhibitors, which requires the presence of a brca or brca mutation, is turning this specialist test into a commonly-applied companion diagnostic. at the same time, the introduction of new dna sequencing technologies is posing challenges even for experienced genetics laboratories. emqn has been providing eqa of brca and brca gene sequencing world-wide for years. the rate of serious diagnostic errors has varied from year to year, but the mean has hovered stubbornly around %. in eqa, just samples per year are sent out, and the quality and experience of participating laboratories varies greatly. we recently carried out a collaborative study to measure the quality of brca gene sequencing by traditional and new methods in experienced, expert laboratories from countries. ten dna samples ( with pathogenic mutations, with normal dna sequence) were sent to each laboratory. ten labs used next-generation sequencing (ngs) alone, used sanger sequencing alone, and the others used combinations of sanger sequencing, ngs, mlpa and other technologies. seventeen ( %) of labs identified all clinically-significant variants on all samples. four false negative results were reported by labs. two were due to deficiencies in the bioinformatics pipeline of the ngs process, while were attributed to a sample swap, and incorrect interpretation of a melting profile. no significant trend was identified with respect to the genotyping accuracy of the different methodologies used. the observed error rate of % amongst expert laboratories indicates the complex and challenging nature of this kind of testing. caution will be required when applying these technologies to sub-optimal ffpe samples in pathology laboratories. p n wolstenholme ; sj patton ; z deans ; s abbs ; j coxhead ; k brugger ; p westwood ; k thomson ; h scheffer next generation sequencing (ngs) is increasingly being introduced into clinical diagnostic laboratories worldwide. the huge amount of data generated by ngs cannot be duplicated by alternative methods for laboratories to internally validate all results, therefore external assessment of data is required. the uk national external quality assessment scheme (ukneqas) for molecular genetics and the european molecular genetics quality network (emqn) have developed a joint eqa scheme for ngs, with the aims to: (a) assess and improve quality; (b) enable laboratories to benchmark their ngs service against others and against best practice; (c) work towards consistency of reporting clinical results generated by ngs; and (d) contribute towards best practice. emqn and ukneqas offer numerous disease-specific, molecular pathology and technical eqa schemes. the objectives for developing ngs eqa were to make it generic (independent of genes, diseases, platforms, and testing context (e.g,. somatic, germline etc)) and applicable all users. two pilot eqas have been run and labs from countries participated. these labs were sent a genomic dna sample and asked to sequence either their smallest gene panel or largest single gene which the lab tested, submit technical details, and genotypes at known snps. the results were compared against a "consensus eqa genome" established by multiple validations of the dna. different genes were tested. most labs are using small panel of - genes. % of all variants were detected by every lab which tested for them. a detailed summary of the key findings will be presented. both pilots have proved to be challenging to meet our objectives, however the results have enabled clinical diagnostic labs to start to address the quality of their ngs testing. tumours invade the vasculature, which transports circulating tumour cells (ctcs) to distant sites enabling growth of secondary tumours. ctcs hold the potential to monitor: therapeutic response, emergent mutations and act as a screening tool for the early detection of cancer. there are numerous methods to isolate ctcs. once isolated, epcam and/or panck positivity and cd negativity are used to verify ctc status. however, due to the metastasis associated process of epithelial-mesenchymal transition, epithelial markers may be ineffective at identifying all ctcs. to overcome such protein marker based limitations, we have developed a novel staining pipeline (ctc- ) that combines histochemical staining (giemsa) with immunofluorescene (dapi, epcam/panck, her and cd ) staining and whole slide imaging for robust identification, enumeration and characterisation of ctcs from cancer patients. ctcs are isolated from whole blood using screencell cyto devices. cyto devices are then slide mounted, giemsa stained and digitised. giemsa staining is washed out and slides are immunofluorescently stained for epcam/panck, cd , her and counter stained with dapi. fluorescently stained slides are digitised. giemsa stained and four colour immunofluorescent digital slides are processed in silico generating a single z-stacked digital slide for pathological assessment. the ctc- staining pipeline has been experimentally validated via ctc characterisation of peripheral blood from lung, breast and ovarian cancer patients, with respect to healthy donor and spiked-in controls. the ctc- pipeline overcomes recognised weaknesses in ctc characterisation. histochemical staining is added to the current gold standard of epcam/panck and cd staining, while also preserving a fluorescent channel for assessment of biomarker status (e.g. her , apoptosis or platelet cloaking). such advancements enable robust pathological assessment of ctcs in the clinic. thorough interrogation of diseased tissue requires the use of multiple biomarkers in order to investigate biological pathways. unless fluorescent technology is used, multiple sections are required from each tissue block as each section can only be tested for a limited number of markers. histogenic molecular mapping (hmm) is a technique which used digitized images to evaluate multiple biomarkers. although each section cut from a block is slightly different from the immediately preceding section, the similarity is sufficient to allow non-linear registration of images of successive sections. if the order is known, multiple sections can be mapped onto each other by registering each with the immediately preceding section. this allows several biomarkers to be mapped into a single "composite" section thereby giving a representation of the pathways activated/expressed in the tissue. we used hmm to investigate the mismatch repair pathway in colorectal cancer. sequential tissue sections were stained for mlh , pms , msh and msh and then scanned. bespoke computational algorithms were used for image registration and composite images were binned as either "mismatch repair proficient" or "mismatch repair deficient". validation of each category could be obtained by quantification of pixels in binarized images or pixel distribution using stereology. our data show that hmm can be used for interrogating biological pathways in tissue sections and, ultimately, automated diagnosis of disease states. personalising whilst pre-operative radiotherapy is the standard of care in locally advanced rectal cancer (larc), only half of patients respond. individualised treatment based on a predictive test could avoid unnecessary radiation exposure in poor responders. macrophages in the tumour microenvironment with tumoricidal m and tumour protective m phenotypes could be modulating this response. this study investigated the possible predictive value of m and m subpopulations in identifying the response to short-course radiotherapy (scrt). pre-treatment biopsies and post-treatment resection samples were taken from patients with larc given scrt. dual-staining immunohistochemistry was performed with cd , hla-dr (m marker), and cd (m marker). samples were scored for hot-and-random spots by nuance software (version . . ) and compared with tumour response measured by reduction in tumour-cell density. the work was partly funded by a pathsoc career development fellowship. samples showing a low score for hla-dr positive m macrophages exhibited a better response to scrt with a median % reduction in tumour cell density (iqr to ). those with a high score exhibited a poor response with only a % reduction (iqr to , p= · ). no such trends were observed for cd + m macrophages. the ratio of hla-dr+ to cd + macrophages for biopsy and resection samples was significantly different showing a drop in the hla-dr positive macrophages in the resection samples (biopsy median · , iqr . to . ; resection median · , iqr . to . ; p= · ). assessment of macrophage subpopulations in pre-treatment biopsies appears to predict the degree of response to scrt in larc. further investigation to validate these findings is now required prior to developing a predictive test for use in routine clinical practice. patients with a poor predicted response could avoid toxic and costly radiotherapy and undergo alternative strategies including chemotherapy. next-generation sequencing technologies (e.g. s profiling) are increasingly used to investigate complex bacterial communities. they have advantages over classical methods, as a significant proportion of bacteria are 'non-culturable'. however, they do not distinguish 'viable' and 'non-viable' populations, which may skew results, particularly following antibiotic exposure. here we report culture and s data from a clinically reflective human gut model, describing changes in the gut microbiota following exposure to multiple antibiotics. a triple-stage chemostat model was inoculated with pooled human faeces from healthy volunteers to establish gut microbiota populations. the model was sequentially exposed to clindamycin ( . mg/l, qds, days), vancomycin ( mg/l, qds, days) and fidaxomicin ( mg/l, bd, days). specific bacterial populations were enumerated daily on selective agars. periodically, s profiling of gut model samples was performed; dna was extracted on a qiaxtractor, s v pcr products were sequenced on an illumina miseq, and resulting data were analysed using qiime. both culture and s profiling demonstrated marked alterations in gut microbiota populations following antibiotic exposure. for many populations, notably bifidobacteria and enterobacteria, changes seen by culture correlated with s profiling. however, as culture describes numerical changes in populations, and s profiling describes proportional changes, results are not always directly comparable. s profiling greatly increased microbiome coverage, particularly for clostridia. population diversity (number of observed species and shannon index) decreased with sequential antibiotic exposure. use of culture and molecular methods in tandem can greatly increase understanding of changes occurring in complex microbial populations. barrett's oesophagus is the erosive replacement of the normal squamous oesophageal lining with a glandular epithelium and is the major precursor of oesophageal adenocarcinoma. barrett's patients are enrolled into active surveillance programmes in order to detect and treat oesophageal cancer at an early stage. surveillance however is costly and burdening to patients. to improve screening efficacy there is an acute need for accurate biomarkers of cancer progression risk in barrett's patients. understanding the pattern and pace of clonal evolution that occurs within the barrett's segment is a key step towards achieving this goal. opinion is divided over whether goblet cells (intestinal metaplasia) on oesophageal biopsy are required for a diagnosis of barrett's oesophagus. this is based on the unproven assumption that goblet cell differentiation marks increased cancer risk in barrett's oesophagus patients. we have investigated the clonal structure of non-dysplastic and neoplastic barrett's oesophagus by combining state-of-the-art d modeling and genetic lineage tracing. by tracing the clonal origin of an early oesophageal adenocarcinoma through whole-exome sequencing and mitochondrial dna sequencing, we find that this cancer developed from non-goblet columnar epithelium, whereas the adjacent goblet-bearing mucosa was free of oncogenic mutations. our results have important implications for the harmonization of the clinical diagnosis of barrett's oesophagus. long-course chemoradiotherapy (crt) is used to down-stage locally-advanced rectal cancer (larc) prior to resection. an interval period prior to surgery allows for tumour shrinkage to facilitate surgical removal. the optimal time interval remains unclear, with little high-quality evidence to guide clinical decisions about when to operate. this study explores the pathological outcomes from a pilot randomised controlled trial comparing an interval of weeks versus weeks between crt and surgery. thirty one patients were recruited from seven uk centres between june and may . photographs were taken of the specimens and assessed by a blinded histopathologist for the quality of the mesorectal dissection. rates of pathological complete response (pcr), down-staging, and circumferential resection margin (crm) involvement were determined. response was also assessed using novel tumour cell density (tcd) assessment where the slides from the resected specimen and baseline biopsy were scanned at x magnification, the tumour area selected and to data-points analysed by a blinded expert to describe the percentage of different tissue components. the work was partly funded by a pathsoc career development fellowship and is presented on behalf of the starrcat trial investigators. twenty three patients underwent surgery ( from the -week arm and from the -week arm). the mesorectal fascial plane was intact in specimens from the -week arm ( %) and from the -week arm ( %). three patients at -weeks and two patients at -weeks showed a pcr. only one patient (from the -week arm) had an involved crm. tcd was . % for the -week arm and . % for the week arm (p= . ). in this small randomised trial, rates of mesorectal quality, crm status, pcr and tcd were similar following either a or week interval after crt. further studies are now needed to clarify whether a longer interval does facilitate on going down-staging. the role of tissue factor pathway inhibitor (tfpi) in liver injury p g petts ; h kudo ; a dorling ; m thursz ; r goldin imperial college london, london, uk; kings college london, london, uk introduction: studies have demonstrated that inhibition of the coagulant cascade is associated with less advanced liver fibrosis and better outcome in acute liver injury. tfpi is a serine protease inhibitor that acts as a homeostatic inhibitor of the coagulation cascade and may be a target to modify outcome in liver disease. methods: transgenic mice carrying a genetic modification that allows cells expressing a-smooth muscle actin (asma; e.g. activated hepatic stellate cells) to simultaneously express tfpi were used in models of chronic liver injury (carbon tetrachloride, ccl ) or acute liver injury (paracetamol) and culled at set time points after dosing. results:chronic liver injury: at hours after the last dose of ccl the transgenic mice had significantly decreased asma expression and tissue inhibitor of metalloproteinase (timp) - gene expression but no difference in matrix metalloproteinase (mmp) - and - gene expression compared to wild types. this suggested a microenvironment that would promote fibrosis resolution. however after hours this difference was lost. at all time points there was no significant difference between fibrosis in transgenic and wild type mice as demonstrated by sirius red staining, hydroxyproline assay and collagen a gene expression. acute liver injury: in paracetamol induced liver injury there was a significant difference in parenchymal necrosis in transgenic mice compared to wild types at and hours after dosing ( hours: mean necrosis % vs. % respectively, mann-whitney test p= . . hours: mean necrosis % vs. % respectively, mann-whitney test p= . ). conclusion: these results suggest that tfpi is an unlikely therapeutic target in chronic liver injury. however in acute paracetamol induced liver injury tfpi appears to rescue the injured liver in a sustained manner from hours after the initial insult and suggests a role for tfpi in managing acute liver injury. (research funded by the pathological society). analysis of adenocarcinoma and non-small cell lung cancer (nos) for egfr mutations now forms part of the royal college of pathologists' lung cancer dataset. identification of patients harbouring these mutations facilitates delivery of targeted therapies with superior efficacy. testing of these tumours for alk has also been introduced in our centre. we assessed our compliance with the college guidelines in this area for and . in those tumours positive for egfr or alk mutations, we examined the original sections to assess any correlation between mutation status and morphological subtype. of the appropriate cases ( %) diagnosed histologically in were sent for egfr mutation analysis, increasing to / ( %) in . of the cases over this time ( %) were positive for an egfr mutation. of these, showed an acinar growth pattern, were solid, lepidic, papillary and micropapillary. it was not possible to characterise the growth pattern in two of the cases analysed as cell blocks. the most common mutation, a missense mutation at codon of exon , was most frequently associated with an acinar growth pattern. of the cases, ( %) were sent for alk mutation analysis, compared with ( %) in . both of the two cases with an alk translocation ( p rearrangement) showed an acinar growth pattern. our compliance with college guidelines in sending appropriate lung specimens for mutation analysis is improving. the correlations between mutation status and morphological subtype add to, and are in keeping with, the current body of evidence in this area. primary synovial sarcoma of the heart -an interesting case report and review of literature p s venkatesan ; p sloan ; s kendall ; m giles royal victoria infirmary, newcastle, uk; james cook university hospital, middlesbrough, uk seventy five percent of primary cardiac tumours are reported to be benign atrial myxomas. the remaining are malignant tumours with most of them being sarcoma, particularly angiosarcoma and malignant fibrous histiocytoma. synovial sarcoma of the heart is a very rare malignancy accounting for less than % of all primary cardiac tumours. most of them arise from the pericardium and the right side of the heart and is considered to be highly aggressive with reduced survival rates. diagnosis in these rare locations is also challenging. we report a -year-old gentleman who presented to us with productive cough, chest pain and paroxysmal nocturnal dyspnea. echocardiography revealed a calcified left atrial mass arising from the posterior leaflet of the mitral valve and radiologically was thought to be a benign atrial myxoma. excision was planned with histology showing a malignant biphasic spindle cell tumour exhibiting marked cellular atypia and numerous mitoses. on immunohistochemistry, the glandular component expressed diffuse positive staining for bcl- and ema with focal positive staining for pancytokeratins. the spindle cell component expressed cd and ema and was found to be negative for cd , s , desmin, melan-a and hmb- . cytogenetic testing revealed ss -ssx / gene fusion with ss rearrangement confirming the diagnosis of synovial sarcoma in this rare location. a postoperative computed tomography was performed which showed no evidence of metastasis or primary lesions elsewhere. there was excellent postoperative surgical recovery and adjuvant chemotherapy was considered in the multi disciplinary meeting. primary cardiac synovial sarcoma is an extremely rare malignancy especially when arising from the left atrium posing diagnostic difficulty mimicking atrial myxoma. in contrast to the poor prognosis mentioned in the literatures, there was excellent recovery in this gentleman. swyer-james-macleod syndrome (sjmls) is a rare lung condition that manifests radiologically as unilateral hemithorax lucency as a result of post-infectious obliterative bronchiolitis, leading to small airways obstruction and secondary emphysema. the histological features of sjmls are poorly and infrequently described. we present three cases of the syndrome that underwent lobectomies in our institution from to , in three women, aged, , and years, presenting with recurrent lower respiratory tract infection, shortness of breath and pleuritic chest pain. two underwent left upper lobectomies, one left lower lobectomy. the first case demonstrated hyperlucency of the affected lobe with markedly reduced blood vessel attenuation. the radiological findings of the second case were of extensive bronchiectasis, hyperlucency, mucus plugging and hypervascularity. the radiological findings of the third case were of an apical bulla and upper lobe cavitating lesion with lobar hypolucency and hypoperfusion. the main histological findings were bronchiolar changes with bronchiolectasis, mucus plugging, constrictive / obliterative bronchiolitis and various degree of peribronchiolar inflammation. emphysema was mild and diagnosed as loss of attachment of alveolar walls. in addition, case had dystrophic, hypoplastic or absent branches of the pulmonary arteries. case showed prominent bronchial arterioles and abnormal tortuous dilated pulmonary arteries and veins. case had established bronchiolar scars in the bronchovascular bundles, pleural arteries showed medial hypertrophy and the interlobular septa contained dilated prominent veins, as well as cystically dilated inflamed peripheral bronchus. these cases highlight the importance of vascular changes in sjmls, likely secondary to the bronchiolar inflammation and destruction leading to capillary bed destruction from secondary emphysema and reactive pulmonary and arterial changes. mediastinal nodal staging with ebus is recommended for patients with resectable non-small cell lung cancer and has emerged as a safe tool to establish granulomatous pathology in suspected sarcoidosis. we conducted a retrospective analysis of the outcomes of ebus performed in a large teaching university hospital with a rapid access lung clinic over a month period and correlation with endobronchial and ct guided biopsies, and surgical resections, when available, and compared the adequacy of ebus when performed with and without rapid on-site evaluation (rose background and aims: in interstitial lung disease (ild), when an aetiological factor appears absent and clinical-radiological correlation is non-contributory, histology is required. the traditional surgical lung biopsy (slb) is not without risks. cryotechnically obtained specimens contain more alveolated lung tissue and less crush artefact than conventional transbronchial biopsies and may offer an alternative to slb in selected cases. we aimed at studying the complications of cryoprobe transbronchial lung biopsy (cpbx) and the quality and pathological characteristics of the tissue obtained. methods: this is a prospective study of patients who were selected for cpbx including cases of possible/probable idiopathic pulmonary fibrosis (ipf). complications of the procedure as well as the quality and pathological characteristics of the tissue are studied. results: twenty-seven procedures were performed in patients, of which were radiologically ipf. a total of biopsies were obtained (average . biopsies per procedure). only one was inadequate initially. fibroblast foci and features consistent with usual interstitial pneumonia (uip) pattern were present in biopsies from patients ( . % of total; % of suspected ipf cases). granulomas were identified in patients ( . %), of which were radiologically suspected ipf ( % of suspected ipf cases). two patients ( . %) had organizing pneumonia; both were inconsistent with ipf radiologically. the findings in the remaining patients were nonspecific; two of these were radiologically ipf ( % of ipf cases). seven patients ( . % of procedures) developed pneumothorax, only of them ( . %) required chest tube drainage. five patients ( . %) developed bleeding (moderate in ( . %) and mild in ( . %)). conclusion: cpbx was useful in this cohort at potentially identifying features not typical of ipf and displayed an acceptable complication rate. cardiomyopathy zj van der klooster ; s sepehrkhouy ; m harakalova ; r goldschmeding ; n de jonge ; ajh suurmeijer ; ra de weger ; f asselbergs ; p a vink introduction: genetic dilated cardiomyopathy is a heterogenous group of diseases caused by mutations in various genes. several types of cardiomyocyte cell death have been implicated in dilated cardiomyopathy: (macro)autophagy-related cell death, apoptosis, necroptosis and oncosis. one plausible mechanism of genetic cardiomyopathy is proteotoxicity of accumulated protein aggregates. we investigated the association of such aggregates as sign of autophagy-related cardiomyocyte cell death with specific pathogenic mutations. methods: hearts from patients with a genetic dilated cardiomyopathy or a combined phenotype of dilated and arrhythmogenic cardiomyopathy were included. microscopic slices from regions were immunohistochemically stained for p , a marker for aggregated proteins destined for autophagy. results: sporadic p positive cells were seen in control hearts ( . % of cardiomyocytes, range . - . %). troponin mutations (tnnt and tnni ; . %, range . - . %, n= ) showed hardly any increase in p . titin ( . %, range . - . % ,n= ) and lamin a/c ( . %, range . - . %, n= ) mutations showed a threefold increase in p staining. a tenfold positive staining was found in desmosomal mutations (pkp and dsp; . %, range . - . %, n= ) and myosin mutations (myh and mybpc ; , % range . - . %, n= ). phospholamban mutations ( . %, range . - %, n= ) and desminopathies (desmin and alpha-b crystallin; % of cardiomyocytes, range . - %, n= ) showed the highest number of p positive cells. conclusion: accumulation of p positive protein aggregates is associated with the type of mutation underlying the dilated cardiomyopathy. titin, lamin a/c and troponin mutations revealed little protein aggregation, whereas desminopathies, phospholamban, desmosomal and myosin mutations show abundant aggregates. this suggests that the type of mutation plays an important role in determining distinct mechanisms of cardiomyocyte cell death. major trauma centre status and its impact on the department of cellular and anatomical pathology in a large tertiary referral centre p ra hadden background: major trauma has been centralised into major trauma centres which act as the focus of major trauma networks. in april , derriford hospital in plymouth, devon became operational as the regional major trauma centre for the south west peninsula. as a result, there was potential for an increased number of trauma-related deaths to be referred to the local coroner, as well as surgical specimens, potentially increasing the work load on pathologists. the case mix could include post-operative cases, neurosurgical cases, polytrauma cases and forensic cases. methods: on admission, all eligible trauma patients are recorded onto the trauma audit & research network (tarn) database. the tarn data was retrospectively analysed and cross referenced with the department of cellular and anatomical pathologies database to determine how many patients had died, how many had post-mortem examinations were performed and how many surgical specimens were sent, on patients from outside the region or transferred from smaller major trauma units. results: over the first two years, there was a small increase in workload from patients who, prior to trauma centre status would have gone to other centres. conclusions: in recieveing patients from elsewhere in the region, there was an increase in workload for both autopsy and non-autopsy work. this excluded some neurosurgical cases, which traditionally would have been referred (as derriford is the neurosurgical centre). there are several areas for implication including, apt time, mortuary space and non-autopsy surgical work. although the workload increase is small, at a time when services are being stretched it is important to ensure any increase in work will not be the "straw that broke the camels back" and can be dealt with accordingly. derriford hospital, plymouth, uk to attempt to streamline general pathologist's approach to the investigation of potentially asbestos-related deaths methods turnaround times, tissue sampling protocol and frequency with which samples were sent for formal fibre counts was investigated for consecutive coronial autopsies at the author's institution. colleagues at other institutions were questioned about their own practice when investigating cases of potential asbestosis, lung cancer or mesothelioma. the author found no consensus in opinion on methods of sampling of the lungs in potential asbestosis, lung cancer or mesothelioma. the most common indication for samples to be sent for asbestos fibre counts was for malignant mesothelioma. sending tissue for fibre counts led to considerable delays in the authorisation of postmortem reports and to significant cost implications. the author presents a pragmatic algorithmic guide to approaching potentially asbestosis-related deaths with suggestions for sampling the lungs and tumour in all cases. in general terms, malignant mesothelioma previously confirmed premortem with histology and immunohistochemistry should not require extensive postmortem histological sampling. lung cancer and asbestosis require widespread sampling of lung tissue to determine amphibole count according to helsinki criteria in the former, and in the latter, assessment of the distribution and degree of fibrosis in addition to fibre count. one or more of these tissue blocks can be sent for formal counts in equivocal cases after following the algorithmic approach. conclusions although predominantly intended as a pragmatic approach to assist the busy practicing autopsy pathologist, the author believes that the algorithm presented will help departments streamline their approach to these cases and help the relative of the deceased gain access to compensation when appropriate in a more timely fashion. purpose of the study: this is a case report of a three year old girl who died suddenly at home. an autopsy was performed in order to determine the cause of death. method: an autopsy was conducted which showed no gross abnormalities. microscopy of the main organs and microbiological samples were taken for further assessment. results: histological assessment of the heart showed multiple small foci of lymphocytes around vessels and within the interstitum of the epicardium, myocardium and subendocardium. these lymphoid aggregates consisted of - lymphocytes up to larger numbers of lymphocytes collectively. several foci were present within virtually all of the sections taken in both right and left ventricles. there was no evidence of myocyte necrosis. immunohistochemistry confirmed they were of t lymphocyte cell origin admixed with smaller numbers of macrophages. histology from the respiratory system showed a diffuse subepithelial lymphocytic infiltrate in the larynx and trachea, and the nasopharyngeal samples detected coronavirus, adenovirus and two types of parainfluenza virus. however, viral polymerase chain reaction (pcr) from the cardiac tissue was negative. conclusion: an unequivocal diagnosis of a myocarditis could not be made in this case due to the lack of myocyte necrosis and the absence of viral dna within the cardiac tissue. genetic testing was strongly advised as splenic material had been taken at autopsy and following molecular genetic techniques a mutation was detected in the sodium channel indicating an inherited ion channelopathy. further genetic counselling and testing of the remaining siblings and family members is being performed. varicose veins affect a third of the uk population. isolated case reports and small series of fatalities resulting from varicose vein haemorrhage appear in the literature infrequently. some of the earliest reports of fatality we have found appear in british medical journal ( ) and the lancet ( ), more recently they have appeared in journals of forensic pathology. our purpose is to establish and bring attention to the rarity of fatality resulting from varicose vein haemorrhage and the importance of the scene of death and autopsy findings. a literature review was undertaken, we obtained relevant office of national statistics (ons) mortality data for the years - , and reviewed our own post-mortem records for demographic, clinical and scene of death information in cases we have encountered. our findings confirm that fatality resulting from varicose veins remains a rare cause of death. some of these deaths are preventable and in nice (national institute of health and care excellence, uk) issued guidelines in which haemorrhage from varicose veins constitute a vascular emergency. importantly emphasis on first aid is required, simply elevating the limb stops bleeding and is life saving, whereas direct pressure and tourniquets do not. pathologists should be aware of potential findings at autopsy in these cases. in particular, awareness that even obscure minor injury to a varicose vein could have resulted in significant blood loss leading to death. blood lost at the scene will not be apparent at autopsy, and details of blood loss could be variably recorded on the scene of death information provided, therefore vigilance is required. histopathologists practice in an era of ever advancing medical treatments for a wide variety of oncological, neurological, haematological and rheumatological diseases. immune modulating therapies are taking a more prominent place in clinical practice. however, with such great advances in therapy comes great risk, with the potential of life threatening opportunistic infections in our patients. we present a series of immunosuppressed patients who acquired such infections and in whom the diagnoses were made by histopathological examination. the spectrum of these pathogens ranges from viral (cmv, ebv, herpes), parasitic (strongyloides) to fungal (p. jirovecii, cryptococcus), and the range of infections is diverse. our series includes males and females, with an age range of - (mean age = years). unsuspected infectious diagnoses were made at post mortem in of the cases. organs affected included lung (n = ), brain (n = ) and haematological system (n = ). in one case both colon and lung were affected (n= ) and in a further case both liver and lung were affected (n= ). immunohistochemistry and/or histochemistry was invaluable in making the diagnoses and was used in all cases (n= ). treatments leading to immunosuppression included chemotherapeutic agents, monoclonal antibodies, steroids and methotrexate. we believe that with the ever increasing use of immunosuppressive therapies (both new and old) for a wider number of disorders, vigilance should be paid to their potential to cause life threatening side effects. histopathologists play a pivotal role in the recognition of this risk and in the diagnosis of these diseases. audit of hospital-based adult autopsy practice in a university hospital from july - p d abu-sinn; f macsweeney the contribution of hospital-based autopsy practice to improvements in patient care is substantial; however, there remains a void in the processes of audit and raising quality of standards in autopsy services. we aim to assess the current autopsy practice compared to rcpath guidelines and identify areas for achieving a high quality autopsy service. all adult autopsy cases performed at a university hospital mortuary between july and were reviewed. a total of adult autopsies were performed by consultant histopathologists. ninety nine percent were coroners' cases. the median turnaround time was . days, with a range of - days, excluding outlier cases (complex timeconsuming cases). there was considerable variation in turnaround times in complex cases and between the various reporting pathologists. eighty five percent of cases were compliant with rcpath minimum dataset for autopsy practice. the remainder were lacking clinical information only. histology and toxicology contributed to cause of death in . % and . % respectively. no organs were retained. further review of the cases not compliant with rcpath guidelines ( %), identified that the possible reasons were the inaccuracy, and sometimes irrelevance to the cause of death, of the information received by the pathologists. in many instances, the clinical information given to the pathologist may be controversial, and a certain degree of caution needs to be implemented to avoid including misleading information in the autopsy report. the turnaround times could be improved if a preliminary report is issued within a set time frame, to be followed by the complete report when the histology and toxicology results are available. however, this practice is not acceptable to some coroners who prefer one complete final report. variations in autopsy practice are to be expected as each autopsy involves substantial case-specific information to which a case-specific answer to the cause of death is expected. prostate cancer is the second most common form of cancer in males, and the incidence of this disease is predicted to double globally by . more than . million new cases of prostate cancer are diagnosed each year and two thirds of these patients are from the western world. the current psa-based test for the diagnosis of prostate cancer lacks specificity, results in missed-diagnoses, over-diagnosis and unnecessary biopsies/treatment. there is an urgent need for a method that enables early accurate detection of prostate cancer. endosomes and lysosomes are cellular compartments that degrade and turnover macromolecules in order to maintain cellular homeostasis. these organelles are directly involved in the critical processes of energy metabolism, cell division, and intracellular signalling, which are all hallmarks of cancer pathogenesis. endosomes have a critical role in controlling the secretion of proteins into extracellular fluids, making them an ideal system to identify new biomarkers that are released from cancer cells. we have discovered that endosome biogenesis (formation and function of endosomes) is altered in prostate cancer. there were significant changes in the gene and protein expression for endosomal proteins and differential distribution of endosome subsets in prostate cancer cell lines. there were also changes to the endosomal traffic and signalling of the transferrin receptor in prostate cancer cells. these fundamental changes in the cell biology of prostate cancer have allowed us to identify a specific set of endosomal proteins that have diagnostic potential. we are developing elisa's to quantify these endosomal proteins in patient samples and antibodies for immune histology applications. the objective for this project is to develop an effective method for the early and specific diagnosis of prostate cancer, which is important as this will have a major impact on patient outcome and survival. the incidence of malignant melanoma has rapidly increased in recent times and melanoma currently represents the second most common cancer diagnosed in young adults. diagnosis is based predominantly on histological assessment; however, due to the wide spectrum of morphological characteristics and lack of firm diagnostic criteria, accurate diagnosis can be challenging. some atypical melanocytic lesions do not display clear-cut morphological features to allow distinction of benign from malignant tumours, making diagnosis and treatment difficult. among these atypical melanocytic lesions blue nevi, spitz nevi and dysplastic lesions are common. from histological features alone, it can be difficult to exclude a diagnosis of melanoma and therefore aggressive surgical strategies may be employed in cases were they are unnecessary, highlighting the need for improved diagnostic techniques. both mrna and mirna profiling have been shown to be able to distinguish benign nevi and primary melanoma tumours. studying mirna expression levels is an attractive strategy as mirnas are highly resistant to degradation and can be easily analysed in ffpe samples. we have studied mirna expression levels in a cohort of benign, blue, spitz and dysplastic nevi versus primary melanoma tumours and their derived metastases. expression levels of key melanoma mirnas, including mirna , mirna , mirna and mirna c can be used to distinguish between nevi and malignant melanomas. we propose an easy to implement, simple and robust molecular method based on mirna expression ratio that, in combination with histological assessment, allows diagnosis of difficult to classify atypical melanocytic lesions. background: diagnosis of lynch syndrome (ls) traditionally relies on clinical criteria to guide diagnostic genetic testing. mmr status of the patient's tumour can help detect lynch syndrome families as well as having other recognised applications for the patient's management including prognostic and predictive significance. as such, the 'dataset for colorectal cancer histopathology report' recommendations from the royal college of pathologists were updated in july to include screening of colorectal cancer patients under the age of and molecular testing for abnormalities in the mismatch repair genes. in south-east of scotland we introduced molecular testing to identify individuals at risk of ls. to widen our screening in line with revised guidelines set by european experts, we expanded our cohort criteria to include those between the age of and . methods: molecular analysis was carried out on individuals: via 'reflex testing' (newly diagnosed colorectal carcinoma ≤ yrs, or clinical/ pathological features associated with mmr defects, such as pre-menopausal endometrial carcinoma, multiple tumours and medullary-type carcinomas) and via 'request testing' (clinical criteria and referral dependent). 'molecular-positive' profiles for ls were identified for genetic pre-testing counselling/diagnostic testing. results: patients with potential ls were identified, ( . %) underwent genetic counselling/testing and cases were confirmed ls with germline pathogenic mutations in the mmr genes. eight of these were identified using reflex testing. conclusion: this is the first uk study to show that screening for ls in patients with colorectal cancer under the age of is effective at identifying families with ls. the testing protocol is in line with the recent recommendations. the human microbiome is rich and diverse, especially in the oral cavity and gastro-intestinal tract, where it has been shown to be more stable in adults, although various factors such as diet and antibiotics mays influence its composition. this pilot study aimed to examine and compare the oral and gut microbial composition in four individuals using a culture-independent approach. methods: saliva and faecal samples were collected from volunteers within the same day on two separate occasions. the v region of the s rrna gene was amplified in all samples and pcr products sequenced on an illumina miseq. unique barcodes were used to sequence multiplexed libraries together. the data were analysed using the quantitative insights into microbial ecology (qiime) software. a second series of samples of faeces from individuals were run to investigate consistency over time. operational taxonomic units (otus) were assessed and showed major phyla represented in the saliva samples: firmicutes, proteobacteria, bacteroidetes, fusobacteria and actinobacteria. similar phyla except forfusobacteria, were found in the stool samples. the weighted unifrac pcoa analysis displayed a clear separation of the sample groups, and also showed a more disperse bacterial profile for the saliva samples, based on population sizes, whereas rarefaction curves and unweighted analysis indicated higher bacterial diversity in the stool samples. each individual could be distinguished either by oral or faecal microbiome. one volunteer who had had previous radiotherapy to the mouth displayed a particularly distinct oral microbiota. the microbial community profiles of saliva and faecal samples of four individuals were found to be distinct from each other, despite sharing similar phyla. analysis of multiple samples from each volunteer clearly separated each sample by volunteer and by sample type. are current automated approaches for determining the phylogeny of multiple deposits capable of interpreting the complexity of cancer evolution? p tg palmer; hm wood; m taylor; w fateen; im carr; p quirke tumour heterogeneity is central to chemotherapy resistance and disease progression in advanced malignancy. this heterogeneity arises due to the evolution of clones within the tumour cell population; the advent of high throughput sequencing has allowed the detection of different tumour cell clones within and between primary tumours and their metastases, potentially allowing mapping of tumour evolution. several, automated bioinformatic approaches have been devised for determining tumour phylogeny from changes in genomic copy number (cn); either by the overall similarity of genomic changes between tumour deposits or by examining the occurrence of shared breakpoints. we have compared these automated approaches with a manual determination of phylogeny based upon shared breakpoints identified from four cases of metastatic colorectal cancer consisting of between and deposits. we illustrate several recurrent issues identified with the use of automated systems for the determination of tumour phylogeny associated with an inability to correctly identify and interpret changes in ploidy, an inability to identify heterogeneity within tumour deposits, the masking of smaller events by larger ones, overinterpretation of convergent, but unrelated events, over calling sequencing artefacts as changes in cn, and non-calling of genuine cn changes due to low tumour cell content or low sequencing depth. we conclude that manual interpretation of bioinformatics data is still required to determine the phylogeny of metastatic cancer within an individual. results: levels of agreement between each sample size and the 'gold standard' were evaluated using bland-altman plots. separate pairwise comparisons were performed. some small sample sizes were shown to have small mean difference and narrow limit of agreement. the ki- pis were then translated into grades and similar comparisons were performed by calculating the kappa score for categorical variables. additionally, the interobserver variation between the two independent researchers were calculated. conclusion: smaller sample sizes (below ) tend to overestimate the ki- pis, possibly due to the effect of concentric counting starting from the center of the hotspot. however, the ki- pis do start to stabilise closer to (e.g. ). the interpretation of whether a lower sample size can replace the current standard would be a subjective decision, but the kappa score gives a rough idea of how much it affects the clinical grading. updated data will be presented. purpose of the study: targeting the stem cell properties of tumor-initiating cells is an avenue through which cancer treatment may be improved. before this can be achieved, so-called cancer stem cell (csc) models must be developed and characterized in specific malignancies. methods: in this study, holoclone formation assays were used to characterize stem-like molecular signatures for prostate cancer (pca) cells. summary of results: lncap and pc parent cells were capable of responding to stem cell differentiation morphogen retinoic acid (ra), suggesting the presence of inherent stemlike properties. lncap cells, which represent early, androgen-responsive disease, formed holoclones after twenty six days. pc cells, which represent advanced, metastatic, castrationresistant disease, formed holoclones after only six days. holoclones displayed decreased expression of ra-genes, suggesting a more immature, less differentiated phenotype. gene and mircorna arrays demonstrated that holoclones downregulated a number of stem cell differentiation regulators while displaying enhanced regulation of g to m transition and the mitotic spindle checkpoint components of the cell cycle. pc holoclones displayed pronounced downregulation of known regulators of osteoblast differentiation from mesenchymal stem cells and epithelial mesenchymal transition. conclusion: our results suggest that some pca cells retain the ability to transition to a more immature state in which differentiation and metastatic mechanisms are changed. the highlighting of osteoblast differentiation regulators in this mechanism is particularly notable, considering the propensity of pca to metastasize to bone. we examined by flow cytometetry the interaction in vitro between platelets and human cancer cell lines of different origin and metastatic potential. the emt profile of cells hr post platelet exposure was assessed by morphology and gene expression analysis (rt-pcr). here we showed that platelet cloaking of cancer cells is universal, occurring across all tumour types examined. however, it is heterogeneous with adhesion rates varying both across and within tumour types, from % (pc -metastatic prostate cancer) to % (skmes -metastatic lung cancer). changes indicative of emt were seen in all cell lines. however, again they were heterogeneous in nature; with morphology changes akin to emt observed at varying degrees across the cancer types. also, there was no consistent pattern to the emt-like gene expression changes seen, with one exception a significant increase in the expression of plasminogen activator inhibitor (pai- )was observed in % of the cell lines examined. in this study we describe the universal nature of platelet cloaking and that even though the interaction is not inducing precisely the same molecular changes in all the cancer cells; overall it is driving these cells into a mesenchymal phenotype. giant cell tumours of bone (gct) are primary locally aggressive bone tumours with a recurrence rate of up to ~ %. the tumour is characterised by numerous osteoclasts and neoplastic stromal cells. making a diagnosis can be challenging because the differential diagnosis incudes an array of benign osteoclast-rich tumours but also osteoclast-rich osteosarcoma. recently the occurrence of h f a p.gly try (g w) and g l mutations was reported in % of gct, the latter occurring rarely. these mutations occur in less than % of > other benign and malignant bone tumours. it has been emphasised that a diagnosis of gct should be made with caution in the absence of detection of g w substitution. given the diagnostic importance of g w mutation in gct, we have developed a simple, quick and cost-efficient diagnostic test to detect this recurrent alteration in ffpe dna using droplet digital pcr (ddpcr). the ddpcr data from dna of > gct have been compared with previous 'genotype' data generated using sanger sequencing, and a number of next generation sequencing approaches (whole exome, whole genome and targeted panels). the g w and g l can both be detected in a single assay. we have demonstrated the sensitivity, specificity, repeatability and robustness of the test to be very high with a turnaround time of no more than working days. as ~ % of gct recur locally following curettage a blood test would be valuable to monitor patients. to this end the ddpcr test also shows that the mutation can be detected in plasma. typically resistant to chemotherapy and radiotherapy, high grade disease has been treated by surgery for more than years. it has recently been shown that idh and idh mutations are present ab initio in ~ % of chondrosarcoma cases and that these are retained throughout disease progression. this has opened up a number of new potential diagnostic, biomarker and therapeutic options. digital pcr is currently the most sensitive and accurate method for detecting and quantifying mutant dna molecules. the biorad qx digital pcr platform is also both cost effective and scalable. using the qx platform, we have developed assays for the common idh mutations and the common idh mutation. we have developed the idh assays both in singleplex and multiplex. we have optimised and validated all assays in tissue samples demonstrating both high sensitivity and specificity when compared to previously genotypes samples. we have demonstrated that the assays are quantitative over orders of magnitude and in high quality dna we can detect idh mutations at below mutant molecule in , wild type molecules. in a pilot study, we have used digital pcr to analyse circulating tumour dna levels in plasma taken pre-surgery from patients whose chondrosarcoma harbour an idh mutation. it was possible to detect idh mutant molecules in plasma of all grade iii samples, % of grade ii and none of the grade i samples. in of these cases where the ctdna was also measured post-operatively, the levels of ctdna dropped dramatically. tumour necrosis factor receptor, cd , gene functions as an oncogene and promotes cell proliferation in colorectal cancer cell lines p haa almasmoum; h thorpe; m ilyas introduction: cd is a tumour necrosis factor (tnf) receptor which regulates a range of cellular responses. cd is activated by its ligand cd l and may promote tumourigenesis in haematological cancers. however, cd functions as a tumour suppressor in solid cancers. cd maps to chromosome q , a region which is amplified in - % of colorectal cancer (crc). the functional activities of cd were tested in crc cell lines for cell proliferation and motility. methods: expression of cd was screened in crc cell lines by western blot. to define the role of cd in human crc, we knocked down cd using small interfering rna (sirna) and the knockdown was confirmed by qpcr and western blot. the prestoblue assay was used to study proliferation in colorectal cell lines, and flow cytometry to study the cell cycle. transwell migration and wound healing assays were performed to investigate the effect of cd on cell motility in crc. result: cd was expressed in crc cell lines hct , rko, dld and ht , and not expressed in sw and sw cell lines. knockdown of cd reduced cellular proliferation in hct (p= . ) and dld (p= . ) cell lines. knockdown of cd showed a higher number of cells in the sub g phase (dead cells) in the cell cycle analysis compared to the control. however, knockdown of cd in hct did not have an effect on cell motility in both the transwell migration (hct = p= . ) and wound healing assays. discussion: cd exhibited oncogenic activity in crc cell lines. cd enhanced cell proliferation but not cell motility in crc cell lines. the expression of cd (common leucocyte antigen) and cytokeratin is thought to be mutually exclusive with cd expression largely restricted to haematological malignancies and cytokeratin expression largely restricted to carcinomas. we report two clinically relevant cases. the first case is a urinary bladder biopsy showing a high grade malignant tumour with cells that had scanty cytoplasm, hyperchromatic stippled nuclei and high mitotic activity. nuclear molding was present. the tumour cells showed focal strong positivity for ck and diffuse positivity for cd and synaptophysin. focal positivity for cd was present and confirmed on repeat staining. the morphology and immunoprofile was consistent with a small cell carcinoma showing aberrant cd expression. the second case is a maxillary tumour biopsy composed of medium/large atypical lymphoid cells with hyperchromatic nuclei, small nucleoli and scanty cytoplasm. mitoses and apoptotic cells were noted. immunohistochemistry showed the atypical cells to express cd , cd a, bcl and mum but not cd , cd , cd , cyclin d , cd , tdt, alk , cd , cd , neuroendocrine or melanocytic markers. a high ki proliferation fraction was present. the appearances were consistent with a diffuse large b-cell lymphoma. the above cases highlight the possibility of aberrant expression as well as loss of expression of immunohistochemical markers by neoplastic cells in undifferentiated malignancies. attention to tumour morphology may provide diagnostic clues. interpretation of immunohistochemistry in the context of tumour morphology as well as awareness of aberrant expression/loss of expression can help avoid diagnostic error. accurate, timely diagnosis is the ultimate aim in surgical pathology. numerous histological pitfalls and lesional mimics exist, with the need to maintain an awareness of such entities vital if potentially serious misdiagnoses are to be avoided. this case report describes two distinct lesions within the same lymph node, both of which are potential mimics of each other. a year old female presented with a three week history of a left breast lump. she had no known previous breast disease or any associated risk factors. a needle core biopsy of this clinically and radiologically suspicious mass yielded a diagnosis of grade invasive lobular carcinoma. left axillary sentinel node biopsy was thus undertaken. two hot and blue sentinel lymph nodes were excised. one was free of neoplasia, the second contained benign naevus cell inclusions within the capsule together with a micrometastasis. immunocytochemistry confirmed the presence of two distinct cell populations; the benign naevus inclusion cells stained positively for s but not for ae / , the reverse pattern was observed in the invasive lobular carcinoma cells. heterotrophic benign inclusions within lymph nodes are an infrequent yet well recognised entity. ridolfi et al reviewed the lymph nodes from axillary surgery patients and found . % of lymph nodes contained benign naevus cell inclusions. small benign naevus cells within the capsule of a lymph node can resemble the 'indian file' pattern of classic invasive lobular carcinoma. this case is unusual in that both metastatic carcinoma and benign naevus inclusion cells were present within the same lymph node, enabling a clear comparison of the cytomorphology and immunoprofile of these two distinct lesions. an awareness of benign inclusions within lymph nodes helps to avoid the potential for misdiagnosis. the judicious use of immunocytochemistry can be useful in distinguishing benign inclusions from carcinoma. recently there has been increasing recognition of distinct breast cancer phenotypes. of these, basal phenotype breast cancer (bbc) has attracted particular interest since the majority are triple negative (tn); have an aggressive natural history and can be associated with brca germline mutation. this area is mired in difficulty, as a precise unifying definition of bbc remains elusive. several morphological features more prevalent in bbc have been identified. in our practise we noticed variable use of 'basal' in reports. given this, whilst no specific therapies to bbc currently exist, we felt it necessary to understand how accurate our designations have been and whether this is a worthwhile practise. method: the diagnostic database was searched for all malignant tn breast resections or reports containing the word 'basal' within -months. tn was defined as allred score er - / , pr - / and her , + or + negative on fish. for completeness, we considered including all breast cancers, but pragmatically this was not possible. we carried out ck and ck staining on all cases where not performed. results: of the invasive breast cancers, cases ( %) were identified, of which % were tn and % were designated bbc in the report. % were both tn and bbc. where a diagnosis of bbc was made, % of cases had additional markers requested. preliminary results showed % were ck + and ck +. this is higher than other studies, implying specificity but not sensitivity in suspecting bbc amongst reporting pathologists. discussion: a limitation of this review is that it cannot identify the rare non-tn bbc not diagnosed as such. it also represents current practise in a single institute and may not reflect national practise. we identified patchy use of the designation bbc, with overall under-reporting of this subtype. we recommend that if it becomes necessary to distinguish bbc lesions, additional markers studies, such as ck and be consistently performed. purpose: neoadjuvant chemotherapy (nact) is increasingly used for the management of large but operable, inflammatory, and locally advanced breast cancer (labc). little is known about predictors of response/survival following nact. the topoisomerase iiα (top a ) gene, a key regulator of dna repair and modelling, is thought to be target for anthracyclin and other chemotherapeutic agents. the aim of this study is to assess the role of top a as marker for response/resistance to nact and patient outcome. methods: patients who underwent nact, predominantly anthracyclin, for primary and operable invasive carcinoma or labc in the period between to at a single large tertiary referral breast unit were identified. comprehensive data on chemotherapy regimen, surgical treatment, pathological response and survival were collected. pre-treatment tumour samples were stained for standard predictive and prognostic markers and top a. results were correlated with pathological response (pr) and patient survival. results: patients fulfilled inclusion criteria. mean age was . ys. complete pr was achieved in . %. the mean expression level of top a in pre-treatment core biopsies was . %, range - %. there was significantly higher expression in high grade tumours (p= . ) and positive correlation with ki expression (r= . , p< . ). there was no correlation with nodal status, pr or her expression. cases with high expression (> %), had significantly worse overall survival (mean vs months, p= . ). this was also identified in the endocrine non responsive group (er allred score≤ ), mean vs months, p= . . on multivariate analysis, top a was not an independent factor for overall survival. conclusions: top a protein is expressed in high grade breast carcinoma with high ki proliferation index. its expression in pre-treatment biopsies predicted patient outcome in the neoadjuvant setting. this strong adverse effect on survival warrents further prospective investigation as a marker of outcome in nact patients. the risk of circumferential resection margin (crm) involvement is confined to tumours of the rectum with the risk of peritoneal involvement increasing the further a tumour is located above the peritoneal reflection. there is no internationally accepted definition of the upper limit of the rectum, and the term 'rectosigmoid' is frequently applied to tumours in this area leading to confusion around the risks and whether radiotherapy can be given. the photographs from abdominoperineal excision specimens were available for quantitation using aperio imagescope. both fresh and fixed specimen images were included where available. the position of the anal verge, top of the sphincters, anterior peritoneal reflection, mesorectal apex (defining the limit of the mesorectum) and high vascular tie were identified and the distances between each point measured. the work was supported by a pathsoc bursary. there was wide variation in the length of the mesorectum in both fresh (median mm, iqr to mm) and fixed ( mm, to mm) specimens. the length of the anal canal also showed variation (fresh mm, to mm; fixed mm, to mm). the height of the anterior peritoneal reflection was lower in females compared to males (fresh vs. mm, p= . ; fixed vs. mm, p= . ). there is marked variability in the anatomy of the rectum between individuals and genders. this potentially affects the risk of either crm or peritoneal involvement and whether radiotherapy could be offered. a fixed definition of the upper limit of the rectum for all patients is not helpful. this should be determined for individual patients on the basis of the mri findings. the term 'rectosigmoid' should be abolished and more accurate definitions based on the position of the mesorectal apex and commencement of the sigmoid mesentery should be used to define the boundaries of the rectum and sigmoid colon and determine subsequent risks to the patient. pre-operative chemoradiotherapy (crt) with anti-egfr antibodies may change the status of egfr pathway mutations. we assessed the mutational status of a number of egfr pathway genes before and after crt in the nwcog excite trial. patients with mri-threatened surgical margins were given pelvic radiotherapy ( gy) with capecitabine, irinotecan and cetuximab followed by surgery after weeks. dna was retrospectively extracted from the pre-treatment biopsy and resection specimen by macrodissecting areas of greatest residual tumour. the mutational status of kras (codons / / / ), nras ( / / ), pik ca ( / / / ) and braf (v e hotspot) were determined by pyrosequencing. the work is presented on behalf of the nwcog excite trial investigators and was part-funded by a pathsoc fellowship. patients commenced treatment and underwent surgery with pathological complete response in ( %) and near-complete in ( %). pre-treatment testing (n= ) detected mutations in kras (n= ), braf (n= ), nras (n= ) and pik ca (n= ). any egfr pathway mutation was detected in %. following crt, cases with residual tumour able to be tested (n= ) showed mutations in patients ( %). there was a discrepancy compared to pre-treatment biopsy in cases ( %): from wild-type (wt) to mutant (mut) in , from mut to different mut in and from mut to wt in . one patient changed in codons (mut to wt in kras /pik ca and wt to mut in kras ). in patients ( %) this changed their overall egfr pathway status ( x wt to mut and x mut to wt). intratumour heterogeneity may explain some of the differences in egfr pathway mutations reported between biopsies and resections presenting a challenge to personalised medicine. however, cetuximab may also drive the growth of undetectable mutant clones to detectable levels on pyrosequencing. further assessment using more sensitive sequencing technologies is currently being employed to investigate these differences. there is a vast amount of historical ffpe material held in archives, but due to variations in fixation and processing this presents several challenges when applying newer genomic technologies to it. in this study we compared the genomic information obtained with the oncoscan® ffpe assay kit (oncoscan) and next generation sequencing (ngs). samples from patients were obtained from centres taking part in the mrc cr trial of short course radiotherapy versus selective long course chemoradiotherapy in rectal cancer. dna was prepared using agilent sureselect kits and sequenced using illumina platforms in parallel to analysis using the oncoscan assay. for both methods, quality control (qc) data was generated and the sample classified as a 'pass' if it fell within the pre-defined qc boundaries. for the oncoscan assay, copy number (cn) and somatic mutation (sm) data was further investigated. this study was part funded by a pathsoc fellowship. in total, cases ( %) passed the ngs qc and ( %) passed the oncoscan qc. a total of ( %) passed qc on both platforms with marked variability in sample pass rates between the centres for the ngs (range % to %) and oncoscan (ranges % to %). when assessed manually, the oncoscan sm data was considered acceptable for cases ( %), which included initially classified as 'failed' by the qc data. similarly, the oncoscan cnv data was interpretable for the majority of cases. this study has shown that whilst historical dna held in the ffpe blocks of archival clinical trials like mrc cr can present challenges when using new genomic technologies, a large proportion of samples can still yield valuable genomic data. marked variation exists in the quality of genomic material between centres confirming that differences in specimen handling affect dna quality. prospective trials must address this by standardising fixation and processing protocols. the plane of colon cancer resection has recently been shown to predict survival. complete mesocolic excision (cme) with central vascular ligation (cvl) produces an oncologically superior specimen and appears to be related to optimal outcomes. we aimed to assess whether a regional educational programme in cme with cvl led to an improvement in the quality of colon cancer specimens. following a regional educational programme in cme with cvl in the capital and zealand areas of denmark, cases of primary colon cancer resected across six hospitals were assessed by grading the plane of surgery and undertaking tissue morphometry. these were compared to specimens resected prior to the educational programme. this work was partly supported by a pathsoc undergraduate bursary. across the region, the mesocolic plane resection rate improved from % to % (p< . ). hillerød hospital had implemented cme with cvl as standard prior to the educational programme and continued to produce optimal specimens. three of the other hospitals showed a significant improvement in the plane of surgical resection. hillerød specimens continued to be more radical with a greater distance between the tumour and the high tie, area of mesentery and lymph node yield compared to the other five hospitals. a multidisciplinary regional educational programme in cme with cvl has improved the oncological quality of colon cancer specimens as assessed by mesocolic planes, however, there has been no significant effect on the amount of tissue resected. surgeons at hillerød continue to produce more radical specimens suggesting that such educational programmes are not alone sufficient to increase the amount of tissue resected around the tumour. hillerød have recently published their long term outcomes with survivals being % higher when compared to other hospitals across the region. further engagement is now necessary to ensure that optimal outcomes are achieved across the region. investigating the faecal microbiome in formalin fixed paraffin embedded (ffpe) material p itr jobling ; m taylor ; c young ; hm wood ; p quirke university of leeds, leeds, uk; leeds institute of cancer and pathology, leeds, uk purpose: research into the faecal microbiome has shown a diverse population with a high level of variability between individuals. altered faecal microbiomes are present in a range of diseases but work remains to understand their role in gastrointestinal disease. current research into the microbiome makes use of fresh or frozen faecal samples. this restricts researchers to predominantly prospective study designs. one potential method for rapidly increasing and diversifying research is the retrospective study of ffpe material. we aimed to investigate the feasibility of typing the microbiome in ffpe faecal samples using next generation sequencing (ngs) technology. methods: material from six faecal samples was divided and stored as frozen or fixed and paraffin embedded creating two matched sub-groups. to assess assay sensitivity one sample was diluted to eight different concentrations before fixing and embedding. the v and v regions of the s rrna gene were amplified. primer pairs created approximately bp and bp targets in e.coli respectively. pcr products were multiplexed and sequenced on an illumina miseq. qiime software was used for analysis. results: analysis of alpha (within sample) diversity showed a significant difference between sub-groups when targeting v (p= . ) but not v . analysis of beta (between sample) diversity showed a significant difference between sub-groups when targeting v (p= . ) while the v region showed a reduced, but still significant (p= . ) difference. the sensitivity assay showed comparable results down to . % concentration levels. conclusion: to our knowledge this is the first feasibility study generating ngs data on the microbiome from ffpe faecal material. variation between matched frozen and ffpe faecal material was less when targeting v compared to v . we hypothesise this may be due to the shorter amplicon undergoing less dna fragmentation in ffpe material. there are several platforms available for dna mutation detection in formalin-fixed paraffin-embedded (ffpe) material, all with their relative strengths and weaknesses. we investigated the oncoscan® ffpe assay kit (oncoscan) in comparison to pyrosequencing in patients with operable colon cancer recruited to the phase ii component of the ncri foxtrot trial of pre-operative vs. post-operative chemotherapy. ffpe samples of tumour from the resection specimens of cases were tested for kras / / and braf v e mutations using pyrosequencing. the oncoscan assay allows for the interrogation of mutations across nine genes. pre-extracted dna was analysed on the oncoscan assay and quality control (qc) scores generated, indicating confidence in mutation calling results. the mutational status of all samples was automatically assessed in the affymetrix sm viewer, and then manually confirmed. this work is presented on behalf of the foxtrot collaborative and was part funded by a pathsoc fellowship. out of samples, failed oncoscan qc thresholds, however, only of these were deemed inconclusive by manual interrogation. samples were interpretable by pyrosequencing. of the samples that produced conclusive results on both platforms, the concordance rate was very high at . % when calling a mutated versus non-mutated kras/braf status. mutations were 'missed' by pyrosequencing in only case ( . %) and by oncoscan in cases ( . %). in addition, the oncoscan assay provides mutational data in additional genes along with copy number (cn) and loss of heterozygosity (loh) information. in patients with colon cancer recruited to the ncri foxtrot trial, the oncoscan ffpe assay shows good correlation with pyrosequencing when determining the mutational status of kras/braf. although pyrosequencing has a slightly lower failure rate, the oncoscan has the added advantage of targeting more mutations, producing genome wide cn, and loh information in one assay. excellent anatomical knowledge of the rectum and surrounding structures is essential for total mesorectal excision (tme). denonvilliers' fascia (dvf) has been frequently studied, though the optimal anterior plane in tme is still disputed. the relationship of the lateral edge of dvf to the autonomic nerves is also unclear. we studied whole-mount microscopic sections of en-bloc cadaveric pelvic exenteration specimens and describe implications for tme. four human adult cadaveric specimens (two males, two females) were obtained from the leeds gift research tissue programme. paraffin-embedded mega-blocks were produced and serially sectioned at and µm intervals. sections were stained with haematoxylin & eosin, masson's trichrome and millers' elastin. additionally, a developmental series of eleven human fetal pelvic specimens (embryonic age of - weeks) were studied. dvf consisted of multiple fascial condensations of collagen and smooth muscle fibres and was indistinguishable from the anterior mesorectal fascia and the capsule of the prostate or posterior vaginal wall. the lateral edges of dvf appeared fan-shaped, and the most posterior part was continuous with the mesorectal fascia. peri-rectal fasciae were not identified in fetal specimens. dvf is adherent to and continuous with the mesorectal fascia. optimal surgical dissection during tme should be carried out anterior to dvf to ensure radical removal, particularly for anterior tumours. autonomic nerves are at risk, but can be preserved by following the mesorectal fascia along the anterolateral mesorectum. the lack of evident fasciae in fetal specimens suggests that these might be formed in later developmental stages. the perineal body (pb) is poorly understood. in abdominoperineal excision (ape), there is no natural dissection plane through the pb. knowledge of the pb is essential to avoid straying in to incorrect planes leading to tumour perforation and unnecessary urogenital and anorectal injuries. this study describes the anatomy of the pb and the implications for ape. six human adult cadaveric specimens (three males, three females) were obtained from the leeds gift research tissue programme. paraffin-embedded mega-blocks containing the pb were produced and serially sectioned at and µm intervals. sections were stained to reveal collagen and elastin, and with an antibody against α-smooth muscle actin. the pb is formed of a fibromuscular mass, which was thicker and wider in female specimens compared to males, extending from the external anal sphincter to the rectogenital septum. muscles from the urogenital diaphragm and anterior rectal wall anchored into the pb. the longitudinal muscle (lm) of the rectal muscularis propria extended in anterolateral directions and intertwined with the somatic pelvic floor muscles to create strong fixation of the anorectum. the lm plays a dominant role in the formation of the pb. surgeons should be aware of the complex course of the lm through the pb to prevent injuries to the urogenital organs and perforation of the anterior rectal wall. the perineal phase of an ape starts with excellent exposure followed by proper tension on the pb to allow safe dissection through the densely-packed fibromuscular mass. introduction: ki is a proliferation marker that is exclusively present in dividing cells and absent in resting cells. its expression has already been studied in different cancers and used to understand the cellular organisation of barrett's epithelium (lavery ). however, very little is known about the cellular organisation based on ki expression patterns in upper gi sequence. this study aims to examine the cellular organisation as defined by ki expression patterns in upper gi cancer sequence. methods: ki expression within barrett's crypts was assessed in cases (nbde , lgd , hgd ) . the barrett's crypts were divided into three equal regions: crypt base (bottom third), middle region and the surface (upper third), respectively. ki was scored using the allred system and analysed using one-way anova with bonferroni post-hoc analysis. results: one-way anova showed significant difference across the three groups (p < . ). bonferroni post-hoc analysis showed significant difference in the surface architecture between nbde and hgd (p < . ) and lgd and hgd (p < . ). for the middle region, although there was no statistical significance between the groups, nbde and lgd and lgd and hgd showed statistical trends (p = . and p = . respectively). for the basal compartment there was significant difference between nbde and lgd (p = . ). this study showed for the first time a significant difference in the ki expression between nbde, lgd and hgd in the basal and surface regions. middle compartments showed trends but additional ndbe, lgd and hgd groups need to be analysed to increase the statistical power. the results warrant further molecular analysis between the various groups and show a clear role for proliferation in the maintenance of the cellular architecture and organisation across the upper gi groups which might help in the understanding of the origin and development of oac. • frequency of serosal involvement in rectal cancers (suggested contributing factors for this include effect of pre-operative therapy, tumour regression and recent changes in surgical practice). • turnaround times: suggested contributing factors include increased departmental workload, retirements and reduced reporting capacity. the following action plan was implemented to improve compliance with standards: • ensure all pathologists are aware of results via presentation/dissemination of audit report • identify issues affecting turn-around times and improvement strategies • support recruitment to increase reporting capacity. • maintain awareness of the need to recognise serosal involvement in rectal excisions. • re-audit in year. the design and maintenance of a pilot online digital archive of archetype colorectal polyps and gastrointestinal (gi) teaching cases for the national bowl screening programme (bowelscreen) in the republic of ireland. methods: suitable internal and referral cases were identified by bowelscreen consultants at saint vincent's university hospital. these cases were subject to both internal and external review, by the mater misericordiae university hospital, and represented typical examples of lesions seen in a national bowel cancer screening programmes (e.g adenomas, ssls, adenomas with misplacement, tsas). representative slides, including immunohistochemistry, were anonymised and digitised using the hamamatsu nanozoomer digital pathology (ndp) whole slide scanner platform and associated software packages (ndp scan and view). whole slide images (wsi) were uploaded to secure cloud storage using a generic file transfer protocol program. wsi were collated into cases and accessible via the pathxl gateway (pathxl.co.uk) by approved users via an online case referral and reporting system. users were notified of pending cases via email and the viewing of wsi occurred within the user's web browser utilising an online version of ndp view program and did not require local use of propriety software. each case was referred across the two participating sites and scored in four areas; diagnosis concordance, quality of wsi, web interface and the online referral and reporting system. conclusion: with the maturation of technology involved in digital microscopy a digital archive program is now a feasible approach to the standardisation of diagnosis and a useful adjunct to traditional optical microscopy in education within the national bowel screening programme. conclusions: a committed and conscientious bms can learn how to report histopathology cases. however, if this is to be achieved, the department in which he or she works must also be committed and supportive. carcinoid tumour of the appendix: a case report p aae shalaby; p aae shalaby a case of a years male operated on for acute appendicitis and an incidental finding of a carcinoid tumour at the tip is reported. the tumour was less than cm in greatest dimension but it infiltrates through the wall of the appendix into the surrounding fat. it stains positive for the neuroendocrine markers. carcinoid tumour of the appendix is unusual, but it has to be looked for during examination of appendectomy specimens done for appendicitis ( . %). women are more frequently affected than men ( : ) and the tumour is usually small less than cm in diameter and frequently located at the tip. it is usually diagnosed incidentally after an operation for acute appendicitis and sometimes during other procedures (colectomy, cholecystectomy and others). the tumour rarely metastasis to the liver and this is usually related to the tumour diameter) and can cause a "carcinoid syndrome": flush, diarrhea bronchoconstriction, cardiac valve disease. diagnosis is made by the pathologist and staging by conventional radiologic procedures (tac, us), dosage of neuroendocrine mediators such as hours urinary -hiaa. simple appendectomy is adequate treatment for appendicular carcinoids less than cm in diameter. adequate treatment for tumours greater than cm is right hemicolectomy. the mangement of tumours to cm range is controverisal, but generally, appendectomy alone is sufficient except when meso-appendix is invaded. carcinoid tumour of the appendix has a good prognosis with a -year-survival rate, of - %. the prevalence of epithelial changes in helicobacter pylori-associated gastritis in oman: a retrospective study p aae shalaby; a al saadi there is strong association between h. pylori gastric infection and epithelial changes and progression to cancer. it has been shown that h pylori infection is strongly associated with high proliferative activity and it could be a risk of initial step of gastric carcinogenesis. the aim of this study was to examine the association between epithelial changes in the gastric mucosa and gastric h pylori infection in oman by retrospective examination of the gastric biopsies for patients presented to sultan qaboos university hospital (squh) in . a total of biopsies were studied with a prevalence of h pylori infection in % with about % showing epithelial changes, mainly intestinal metaplasia in % out of the h pylori positive cases, a few cases with low grade dysplasia and reactive atypia. in cocnlusion intestinal metaplasia was the main epithelial change that was related to h pylori infection. further studues are required to investigate the relation between h pylori infection and the progression to gastric carcinoma. purpose of the study: low rectal carcinoma may require abdomino-perineal excision of the rectum (aper), which has been associated with higher rates of tumour perforation and circumferential margin (crm) involvement than anterior resection. this increases the risk of local recurrence and may necessitate adjuvant treatment. the extralevator abdominoperineal excision of rectum (elape) in the prone position has been found to improve these outcomes and has been encouraged by the low rectal cancer national development programme (lorec). we aimed to assess the effect of increasing the practice of elape on the histological and oncological outcomes in these cases in the mid-yorkshire nhs trust, a large district general hospital. in the number of surgeons routinely performing aper was reduced and all those performing the procedure had been trained in the cylindrical resection technique. joint operating and laparoscopic procedures were encouraged. a retrospective review of case notes and histological reports between and was performed (before and after sub-specialisation). patient demographics, histological findings and complications including local recurrence were recorded. summary of results: between and , apers were performed, with tumour perforation in ( %) and crm involvement in ( %) of cases. after sub-specialisation, were performed. none were perforated and cases ( %) showed margin involvement. local recurrence occurred in two cases before specialisation and none after at the time of follow-up. joint operating and subspecialisation increased the number of cases performed by each surgeon, and the number performed laparoscopically. conclusions: elape in conjunction with departmental restructuring significantly improves immediate oncological outcomes in a dgh setting, with no effect on day mortality. the technique may reduce local recurrence, although longer follow-up would be required. background: systems biology uses computational and simulation approaches to interrogate gene expression datasets and explore biological pathways. by employing systems biology and data mining tools we can identify new biomarkers. our objective was to ascertain the utility of a novel panel of systems biology derived biomarkers in cervical pre-cancer for more accurate grading and stratification of cin disease. methods: this project is conducted within the framework of an fp funded programme "systemcerv". gene pathways were analysed using matlab and sirene. along with accessing keggs online database for gene prediction and david for gene functional classification, we identified a novel panel of biomarkers. gephi software was used to visualise communities of genes related to cervical pre-cancer and cancer progression. clinical validation was performed by immunohistochemistry on a range of cervical lletz specimens (normal, cin , cin and cin ). all patients gave written informed consent. in parallel, p ihc was performed on all specimens as a benchmark stain. the mortality associated with cervical cancer can be reduced if the disease is detected at the early stages of development or at the pre-malignant stage. the pap smear is the current screening method, but is highly subjective and can often exhibit low specificity and sensitivity. for this reason, either a replacement or supportive technique is necessary to improve the quality of cervical cancer screening. raman spectroscopy is a powerful tool that can generate a biochemical fingerprint of a sample in a rapid and non-destructive manner. in this study, raman spectroscopy has been applied to the investigation of cervical cells from preservcyt specimens. raman measurements were taken from the nuclei of cervical cells from normal, cin , and cin samples. these spectra were processed, analysed and used to define a spectral signature for each grade of cervical disease. principal component analysis (pca) was used to discriminate between the two data sets. distinct raman spectral differences were detected between normal, cin and cin cells. notably, it was possible to observe spectral peak shifts representing fluctuations in guanine (dna/rna), ch deformation in proteins and carbohydrates, carbon-carbon double bonds in phenylalanine, tyrosine and tryptophan, and amide i. the pca showed an excellent discrimination between the data sets. this study has shown that raman spectroscopy can detect subtle changes between cervical cells, and may be a powerful tool for improved diagnosis of cervical dysplasia. background: myd and mad are two potential prognostic biomarkers that have been investigated in ovarian cancer. high myd and low mad ihc staining is associated with reduced pfs, both markers are also linked to paclitaxel chemoresistance. the main objective of this study was to assess the in vitro relationship between mad and myd , through alteration of mad , myd or its receptor tlr in two ovarian cancer cell lines using sirna targeting mad , tlr or myd and a myd overexpression plasmid vector. following overexpression/sirna knockdown procedures, myd , tlr and mad expression was assessed through qpcr and western blot analysis. mir- , mir- and mir- a gene expression was also assessed by qpcr. furthermore the effect of tlr / myd knockdown on chemoresponse was assessed in skov- cells using the cck- assay. results/discussion: it was found that knockdown or overexpression of myd in skov- or a cells respectively or knockdown of tlr in skov- cells had no effect on mad expression or the expression of mir- , mir- and mir- a. interestingly however knockdown of mad in both cell lines induced a fold increase in tlr expression, furthermore knockdown of tlr in skov- cells was shown to restore chemosensitivity to paclitaxel. the results demonstrate a potential in vitro link between tlr and mad and support a role for tlr in paclitaxel chemoresistance. background: the prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. recent evidence points to the toll-like receptor- (tlr ), and particularly its adaptor protein myd , as one potential mediator of this resistance. downregulation of mad , a key component of the spindle assembly checkpoint complex, has also been linked with paclitaxel resistance . both markers have individually been shown to be associated with poor outcome in ovarian cancer. high myd and low mad immunohistochemical staining is associated with reduced progression free survival. the main objective of this study was to assess the combined utility of mad and myd in predicting patient prognosis. methods: two tissue microarrays composed of cores from high grade serous epithelial ovarian cancers patients were constructed and stained for mad and myd . staining was scored based on previously derived scoring schemes for myd or mad staining. the mean overall score from triplicate cores was then used to classify patients into high and low staining categories. results: a trend towards reduced progression free and overall survival was observed in patients with high myd and low mad expression. the results demonstrate the combined utility of mad and myd as predictors of prognosis in ovarian cancer. purpose: ovarian cancer is characterised by high rates of terminal, chemoresistant recurrence. although chemoresistance is known to be a property of cancer stem cells (cscs), the mechanism is poorly understood. we have previously identified a novel four-member csc stem-progenitor cell hierarchy for ovarian cancer. the aim of this study was to characterise the contribution of each member of the ovarian csc hierarchy to chemoresistance. methods: the csc hierarchy was assessed for tolerance to chemotherapy drug cisplatin (mtt assay) both as components of the parent population (a cell line) and as isolated cell types. the hierarchy was additionally assessed in the long-term cisplatin-adapted 'a cis' cell line. cell types were analysed and isolated via flow cytometry and assessed for stem cell characteristic via single-cell asymmetric division and murine xenograft tumourigenicity assays, and molecularly characterised (whole transcriptome arrays). results: cisplain dose-response assays from a -derived csc sub-populations indicated that only one of the four populations within the hierarchy had a high cisplatin-tolerance (ic = um) compared to the other populations (ic = um). this was notable as the relative cisplatin ic s for the a and a cis parent cell lines are um and um respectively. treatment of the parent a cell line with the ic ( hours) resulted in a proportional % loss in each of the four cell types, suggesting that this specific csc subpopulation adapts to cisplatin over a longer period of time. conclusion: although cscs are known to be chemoresistant, the mechanism though which this is achieved is poorly understood. our data indicates that only some members of a csc hierarchy are responsible for chemoresistance. notably, this sub-population appears to possess inherent chemoresistance in pre-treatmentcells. as such, it should be possible to target these cscs in the primary malignancy to prevent chemoresistant recurrence. mixed sex cord-stromal tumours of the ovary are very rare. we report a case of mixed sex cord -stromal tumour (also referred to as gynandroblastoma) containing both sertoli-leydig cell tumour and adult granulosa cell tumour in a female years old who presented with postmenopausal bleeding. on histology, the majority of the tumour represented an unsual form of well differentiated sertoli-leydig cell tumour with a pseudoendometrioid appearance. minor foci of classic adult granulosa cell tumour were present. on immunohistochemistry, the tumour was diffusely positive for inhibin and sf and focally for calretinin, er and cd . ema, pax and ck were negative. as far as we are aware, this is the first report of an ovarian mixed sex cord-stromal tumour containing a component of pseudoendometrioid sertoli-leydig cell tumour. a year old female patient presented with bilateral painful warty lesions on the labia majora. the patient had had hiv for a long time and was on highly active antiretroviral therapy. she also suffered chronic renal failure requiring haemodialysis three times weekly. clinically, the lesions were highly suspicious of vulval cancer. the lesions increased significantly in size over a short period of time ( months) requiring surgical resection under general anaesthesia. histological examinations revealed polypoid lesions with prominent pseudoepitheliomatous hyperplasia and dense inflammatory infiltrate, composed mainly of lymphocytes and plasma cells, extending to the hypodermis. numerous abscesses with large numbers of eosinophils were present withinin the hyperplastic epithelium. the typical intranuclear inclusions of herpes simplex virus (hsv) were identified. hsv immunohistochemistry was positive. this is a rare case of vulval hsv warts mimicking cancer. oral acyclovir was administered following surgery and resulted in good control. literature review shows only previously described cases of verrucous hsv, types and , simulating neoplasia in patients with aids on antiretroviral therapy. primary mucinous eccrine adenocarcinoma of the skin is a rare adnexal neoplasm, typically involving the head and neck region in the elderly population. here we present a case of primary mucinous eccrine adenocarcinoma of the vulva; occurrence at this site is extremely rare, with only five cases published in english literature. a year old female presented with a mm vulval lesion, clinically suspected to be an inclusion cyst. the lesion was removed and sent for histopathological assessment. histological examination revealed a well circumscribed, partly encapsulated tumour composed of rounded and irregular nests of polygonal epithelial cells with scattered lumina, suspended in pools of extracellular mucin. the epithelial cell nuclei displayed a uniform chromatin pattern with small distinct nucleoli. the mucin pools stained positive for alcian blue and dpas. immunohistochemical staining demonstrated positivity for cea, ck , gcdfp, oestrogen receptor, progesterone receptor, synaptophysin and chromogranin. immunostaining was negative for ck , cdx , ca- , ttf- , ch / , ck , her- , wt- , cd and s . ki- proliferation fraction was approximately %. overall, the findings were those of a mucinous eccrine adenocarcinoma with neuroendocrine differentiation. following multidisciplinary discussion, and negative imaging of the breasts and gastrointestinal tract, a diagnosis of primary mucinous eccrine adenocarcinoma of the vulva was reached. only a handful of cases of primary mucinous eccrine adenocarcinoma of the vulva have been reported. metastatic disease, particularly from breast and colon, must be excluded. follow up data from patients with primary mucinous eccrine adenocarcinoma of the skin suggests a high local recurrence rate ( . %), necessitating close follow-up. however, risk of metastasis is low ( . %). royal shrewsbury hospital, shrewsbury, uk introduction: primary extraskeletal myxoid chondrosaroma (emc) of the vulva is a rare mesenchymal neoplasm. the myxoid tumour differential diagnosis on a core biopsy can be quite challinging. to date, few cases have been reported in the literature. case report: a -year old woman noticed a swelling on the right side of the labia, thought to be a bartholin's cyst in . she was managed conservatively. she had drainage and marsupialization under general anaesthesia. this resulted in extreme bruising of the vulva. this was managed with antibiotics and non-steroidal anti-inflammatory medication, and it resolved after weeks. six months later, the patient presented again with a persistent vulval mass. a biopsy was obtained under general anaesthesia, and it showed a myxoid tumour with differential diagnosis of low grade chondroid tumour. an mri was performed to assess the extent of the disease. the tumour was excised. at surgery, a x cm lobulated, extremely vascular vulval tumour was found. the tumour was inseparable from the inferior pubic ramus of the pelvic bone. a complete macroscopic resection was obtained. histology confirmed low grade myxoid chondrosarcoma. conculsion: vulval lesions with unusual characteristics or insidious evolution in the labia majora or bartholin's glands area should be carefully and promptly investigated. differential diagnosis of myxoid tumours in the vulva should include myxoid chondrosarcoma amongst other diagnoses. (fish) showed the presence of a bcl rearrangement in a proportion of cells. therefore this case is best regarded as a composite lymphoma of diffuse large b cell lymphoma with hairy cell leukemia rather than blastic transformation of hairy cell leukemia. to the best of our knowledge, simultaneous occurrence of diffuse large b cell lymphoma and hairy cell leukemia in a lymph node has not yet been reported in the literature. bone marrow examination by aspirate and trephine biopsy is an important haematological investigation. ideally, aspirate findings should inform examination of the trephine biopsy, but if the two modalities are separate the aspirate report can be delayed and histopathologists may assess the trephine biopsy without being aware of the aspirate findings. we audited the availability of aspirate results to the histopathologist examining trephine biopsies, over the period in which our department implemented an integrated haematopathology reporting system. the effects on diagnostic concordance, turnaround times and immunohistochemistry requesting were also assessed. the setting was a regional specialist haematopathology centre. prior to integration, a prospective audit of consecutive trephine biopsies received by a senior haematopathologist was carried out, against standards set by the international committee for standardisation in haematology. data were collected from hospital computer systems. the move to integrated reporting involved the installation of new software (hilis) to specifically handle integrated haematopathology data. ten months later, a retrospective analysis of a further cases was performed using hilis data. prior to integration, % of aspirates were reported within days, and access to the aspirate report was available at time of examination for % of trephine biopsies. after integration, % of aspirates were reported within days, and reports were available at time of examination for % of trephine biopsies. diagnostic concordance was % initially, and % after integration. the mean number of immunostains requested per case was unchanged ( . vs . ). our findings show integrated reporting has markedly increased the availability of aspirate reports to the histopathologist, and improved diagnostic concordance. this new model benefits the haematologist, histopathologist and patient. introduction: clonality studies are carried out when the diagnosis of lymphoma is particularly challenging. the detection of clonality in lymphoproliferative lesions suspicious for lymphoma can be a valuable supplementary tool as it has a high positive predictive value. clonal studies can also help to distinguish recurrent or residual disease from reactive inflammation. however false positive and negatives are common and can be attributable to several factors, including poor dna quality. methods: cases reported over a six month period (jun-nov ) were retrospectively reviewed, % of which were referral cases. we investigated various aspects of clonality studies; including dna quality, fixation method, clinical information provided and correlation between the morphological/immunophenotypical findings and clonality results using euroclonality/biomed- primers (igh, igk, tcr-Β and gamma-delta). results: ( %) had adequate dna quality, ( %) poor dna and ( %) had inadequate dna quality. external cases had better dna quality in the majority of cases. clinical information was provided in % of local cases and % of external cases. in % of cases clonality results supported the initial histological report. cases showed clonal expansion despite a benign process on histology. suspected cases lymphoma ( b-nhl and t-nhl) showed no clonality, of which yielded poor dna quality. skin cases although had good dna quality, usually had low number of neoplastic cells resulting in poor pcr products. conclusions: dna quality is very variable and clinical information is often not provided precluding adequate assessment of clonality findings. dna was worse locally (decalcified marrow trephines using formic acid and peloris system with high temperatures that can cause dna degradation). standardisation of fixation methods and interpretation of peaks/ bands in the clinical context of the patient is essential for clonality to be informative. kikuchi-fujimoto disease: a novel diagnosis by transbronchial biopsy of mediastinal lymphadenopathy p pm ellery; n archard; a ramsay ucl hospitals nhs foundation trust, london, uk objectives: kikuchi-fujimoto disease (kfd) is a rare, self-limiting form of necrotising lymphadenitis that most commonly affects young asian women, and classically presents with fever, malaise and lymphadenopathy. the cervical lymph nodes are involved in around % of cases, with other sites rarely involved. we report an unusual case in which an unexpected diagnosis of kfd was made via transbronchial biopsy of mediastinal lymph nodes. a year old boy of pakistani origin presented with a month history of lethargy, neck stiffness and weight loss, with fever (up to °c) and night sweats. chest x-ray, mantoux test, blood cultures and viral pcr were negative. lumbar puncture was normal, with no acid fast-bacilli. ct showed enlargement of the deep cervical lymph nodes (petpositive on further imaging), and mediastinal lymphadenopathy. he was transferred to our hospital for further management, with a differential diagnosis of tb, lymphoma, rare infection or autoimmune disease. he underwent transbronchial biopsy of the mediastinal lymph nodes. results: his biopsy showed blood clot and cores of lymph node, with focal collections of crescentic macrophages, admixed lymphocytes and prominent apoptotic debris. immunohistochemistry demonstrated a population of cd -positive plasmacytoid dendritic cells and granular mpo positivity in macrophage cytoplasm. the background lymphocytes were mainly cd -positive t-cells. the features were those of kfd. conclusion: involvement of deep lymph nodes is unusual in kfd, and to our knowledge, this is the first case diagnosed via transbronchial biopsy. such biopsies often produce scanty diagnostic material, and here the detection of the characteristic immunoprofile of kfd helped confirm the diagnosis. this case highlights that kfd should be considered at sites other than the cervical lymph nodes, and demonstrates the value of immunohistochemistry in reaching a definitive diagnosis. we describe a benign intravascular proliferation of atypical polytypic cd positive t cells, co-expressing follicular t helper cell lineage markers coincidental to local sepsis of the buttock. a year old female presented with a x cm buttock abscess at the site of a longstanding palpable lump. peripheral blood showed only a neutrophilic leucocytosis. immunohistological examination showed large aggregates of atypical cd positive, alk negative lymphoid cells expressing a pan t helper phenotype with cd partially downregulated. podoplanin proved that the atypical t cells were primarily, but not exclusively, localised within lymphatic channels. a t cell receptor clone was not detected using pcr. to find intravascular concentrations of atypical lymphoid cells is uncommon in skin biopsies and raises the possibility of leukaemia or intravascular lymphoma. intravascular lymphoma is a rare variant of non hodgkin lymphoma with a minority possessing t or nk cell lineage but frequently involving skin. cd is a transmembrane glycoprotein and a member of the tnf superfamily involved in regulating proliferation. it is considered a reliable marker of lymphoma. primary cutaneous cd positive tlpds encompass a spectrum of biological aggressiveness and include primary cutaneous anaplastic large-cell lymphoma and lymphomatoid papulosis (lyp). cd can also be up regulated in activated b and t cells and it has been proposed that cd positive ivtlpds are equivalent to an intravascular form of lyp. intravascular proliferations of atypical cd positive t cells have been linked with chronic inflammation and abscess formation. furthermore atypical cd positive tlpd expressing a cd positive t helper phenotype and exhibiting an indolent clinical course have been reported in the arm, trunk, neck and prepuce. ultimately ivtlpd may require follow up based upon clinical features and natural progression due to overlapping diagnostic features. p g laing; s craig; l moss; c crichton; p johnston the investigation of lymphoid neoplasia requires multiple sections, in our practice consisting of twelve antibodies and thirteen single stained slides. by selecting particular antibodies for double staining, spatial relationships between cell types and overall tissue organisation can be more easily visualised. this pilot study aimed to optimise the staining intensity and specificity to provide accurate diagnostic information, reduce slide number, material and consumables costs, preparation time in the laboratory and storage space to enhance costbenefit. twelve antibodies were chosen and paired: kappa/lambda, cd /cd , mum- /cd , cyclin d /cd , bcl- /cd and cd /pax- . firstly, these combinations were applied to normal tissue and then known tumours to optimise technique. once the staining protocols were finalised they were run on eleven consecutive cases with conventionally stained non-hodgkin lymphoma (nhl) panel requests. the slides were then reviewed by the lymphoma team for quality and diagnostic accuracy compared to the standard single stained slides. the results demonstrate that double staining is possible in the diagnosis of nhl. the combinations chosen have proved successful and have provided interpretable results; for example, the relationship of light chain staining in plasma cells, mum- and cd positive cells in diffuse large b-cell lymphoma proves positive. we feel time will be saved cutting sections to improve efficiency in lymphoma investigation and reduce panel storage space by around %. in conclusion the outcomes from this pilot study have been positive for medical and scientific staff, has shown that double staining in the diagnosis of nhl is possible and that optimising this protocol with a view to live diagnosis is worthwhile. in an age in which novel therapies are not necessarily defined by their ability to kill malignant cells, understanding the biology of malignant cells after treatment is extremely important. this is particularly true of ibrutinib (pcl- ) therapy in chronic lymphocytic leukaemia which is characterised by lymphocytosis. imagej/fiji image analysis software could therefore be used to analyse cell shape and grouping characteristics. we used a novel assay in which chronic lymphocytic leukaemia cells, cultured for days, were seeded onto fibronectin coated glass coverslips and then had their b cell receptors ligated with goat anti human igm. they were compared with cells simultaneously inhibited with ibrutinib. we tested multiple staining techniques and found that using either rose bengal or texas red phalloidin staining produced the most reproducibly analysable data when using imagej/ fiji. we demonstrated, using the assumption that the outline of interacting cells would be larger than cells which were alone that homotypic interactions were increased after b cell receptor cross linking ( groups larger than cells against group larger than cells per nm x nm field (p= . )). nuclei were also significantly larger when cross linked. (mean . (+- . ) pixels without cross linking and . (+- . ) (p=≤ . )) suggesting increased nuclear spreading. these effects were reversed by the addition of ibrutinib with groups larger than cells per the same field(p= . compared with cross linked sells) and nuclear size mean being . (p≤ . compared with cross linked cells). this study demonstrates a novel, easily reproducible assay to assess a variety of cellular responses to ibrutinib therapy and suggests a method of quantifying activity both when stimulated and inhibited. this technique could easily be scaled up to further investigate cellular behaviour following ibrutinib therapy. purpose: gliomas represent % of all solid intracranial tumours and are associated with a poor prognosis. recent studies indicated that the human cytosolic branched chain aminotransferase protein (hbcatc), which metabolises the branched chain amino acids (bcaa), was significantly upregulated in idh / wild type (wt) glioblastomas, correlated with methylation patterns in the bcat promoter and is associated with a worse prognosis compared with idh mutant gliomas. the diagnostic and prognostic significance of markers of bcaa metabolism is currently under investigation. methods: glioma tumour samples were compared for hbcatc, hbcatm and bckdc expression using western blotting and immunohistochemistry. dna was extracted from fresh frozen tissue. sanger sequencing of the p.arg region of idh and p.arg region of idh was undertaken using a dna analyser (applied bio-systems). summary: in idh wt tumours, like hbcatc (p= . ), the expression of the mitochondrial isoform (hbcatm) is significantly (p= . ) expressed relative to idh mutant gliomas. hbcatm additionally shows a more significant correlation with patient survival than hbcatc on kaplan-meier analysis. in idh wt tumours, low hbcatm expression is a positive prognostic factor (p = . ). hbcatm expression additionally correlated with who grade. although previous reports indicate that increased hbcatc occurs exclusively in idh-wt tumours, our studies demonstrate that % of idh mutant tumours express comparable levels of hbcatc. although hbcatc alone has been suggested as a putative therapeutic target, it is important to evaluate the expression of hbcatm in glioblastomas as its expression may impact the efficacy of new treatments targeting hbcatc. conclusions: idh wt high grade gliomas traditionally have a poor prognosis. however we demonstrate for the first time that relatively low hbcatm may select for a better performing clinical cohort and may be a possible candidate target for drug therapy. a year old female presented with an occipital mass, presumed to be a lymph node and underwent fine needle aspiration of the lesion. fna yielded two air dried slides, upon which a diagnosis of mesenchymal neoplasm was made. the patient underwent a subsequent incisional biopsy allowing a formal histological diagnosis of myxoinflammatory fibroblastic sarcoma to be made. myxoinflammatory fibroblastic sarcoma is a low-grade neoplasm usually occuring on the distal extremities and only rarely presents as a head and neck neoplasm. fna is a useful tool in the diagnosis and subsequent management of head and neck neoplasia and we describe here the cytological features and subsequent histological diagnosis of myxoinflammatory fibroblastic sarcoma occuring in the occipital scalp. objective: presentation of giant cell fibroblastoma (gcf). because of it has a dilemma of microscopic appearances; it is mandatory to differentiating it from atypical dermatofibroma (adf), fibrous hamartoma of infancy (fhi) and vascular lesions. methods: eighteen month-male egyptian child presented with painless slowly expanding subcutaneous back swelling at the left scapular area. results: histologically, the lesion is poorly circumscribed and range from cellular to myxoid in a dense to loose collagenous stroma. the tumours composed of mixture of spindle shaped or stellate cells admixed with multinucleated giant cells with occasional pleomorphism and very low mitotic index (< per high-power fields). these cells infiltrate around adnexal structures and through subcutaneous fat. a distinctive finding is cracking artifact of the stroma simulating angiectoid spaces. these pseudovascular spaces are lacking a true endothelial lining and lined by discontinuous layer of enlarged multinucleated giant cells. immunostains, including factor viii, cd , cd a, sma, s and cd were negative. ki labeling index is very low. all the cellular components show positive immunoreactivity for cd . conclusions: first case of gcf reported in egypt. we recommend a wide scaled study to categorize this tumour with molecularly similar lesions. the royal county sussex hospital, brighton, uk a year old man being investigated for obstructive hydronephrosis was incidentally found to have a . cm splenic mass on computed tomography (ct). no lymphadenopathy was present and the mass remained stable on sequential ct and ultrasound scans. on positron emission tomography (pet) the lesion had a low signal with moderate uptake. all haematological investigations were within normal limits including a negative epstein barr virus (ebv) test. his past medical history included previous immunosuppressive therapy for inflammatory bowel disease. a core biopsy under ct guidance was performed. the cores showed a paucicellular spindle cell lesion with bland, blunt ended nuclei, no cytological atypia and a sparse chronic inflammatory infiltrate. there was no necrosis. the spindle cells stained positive for smooth muscle actin (sma) and h-caldesmon indicating this to be a splenic leiomyoma. splenic lesions are uncommon and within their differential include, lymphoma, inflammatory pseudotumour, harmatomas and leiomyomas. ( ) a splenic leiomyoma is an unusual and rare benign smooth muscle tumour with an unknown pathogenesis. they are thought to arise from the capsule and blood vessel walls of organs. ( ) they have been documented in immunosuppressed states (constitutional or acquired), in those with ebv infection and in children with ataxia-telangiectasia. ( ) leiomyomas within the spleen are rarely reported in the literature, especially in those patients over the age of eighteen. in this case there was historical immunosuppression, however leiomyomas should be considered in the differential diagnosis of well-defined solitary splenic lesions purpose of the study: the zucker diabetic fatty (zdf) rat is extensively used as a model of diabetic kidney disease (dkd) associated with obesity and progressive insulin resistance ('diabesity'). this study aimed to validate qualitative ultrastructural parameters of glomerular injury in the zdf animal model and apply these criteria to an interventional study investigating the effects of roux-en-y gastric bypass (rygb) on dkd. methods: superficial renal cortices were immersion-fixed in . % glutaraldehyde, post-fixed in % osmium tetroxide, processed and embedded in epoxy resin prior viewing under a technai transmission electron microscope. glomerular basement membrane (gbm) thickness, podocyte foot process diameter (pfpd) and podocyte foot process frequency (pfpf) per unit length of gbm were determined for each group (sham and rygb operated zdf fa/fa diabetic animals vs non-operated non-diabetic zdf fa/+ lean controls). statistical analysis was performed using a mann whitney u test and an unpaired t-test where appropriate. summary of results: selected tem parameters (gbm thickness, pfpd and pfpf) demonstrated significant differences between specified sham-operated zdf fa/fa vs fa/+ samples, p= . . analysis of rygb interventional study samples still in progress. early post-operative glucose measurements showed a significant improvement in glucose homeostasis in the rygb group (rygb vs sham, p= . ) occurring independently of weight loss. urinary albumin:creatinine ratios were lower in the rygb group vs sham operated positive controls (p= . ) and were comparable with age-matched lean control fa/+ samples. conclusions: preliminary findings support a beneficial role for rygb in an animal model of 'diabesity'. validated ultrastructural parameters should assist in elucidating changes in podocyte activation and differentiation as mediators of the observed remission of albuminuria following rygb surgery. nottingham university hospital, nottingham, uk spitz naevus is a benign melanocytic lesion that shares many histological features with malignant melanoma. although the morphological criteria differentiating the two entities are well established however, some cases can be challenging. many isolated markers have been proposed to help in differentiating spitz naevus from melanoma, albeit none has been shown to be definitive. aim: this is a preliminary study looking at the immunohistochemical expression of markers that are known to have important role in cell cycle regulation, proliferation and melanocytic differentiation (p , ki , and hmb ). the aim is provide to a combination of proteins that can help in differentiating spitz naevus from malignant melanoma. the study included cases of spitz naevi, benign compound naevi and cases of malignant melanoma. immunohistochemical expression of p , ki , and hmb has been accessed and compared with the morphological features of these lesions. results: it is noted the mean p expression is higher in compound and spitz naevi than melanoma ( , , and respectively). proliferation activity as measured by ki index is higher in melanoma in comparison with compound and spitz naevi ( . , . , and . respectively). hmb shows only junctional positivity in out of cases of spitz naevi while in the other two it shows week dermal component. hmb is constantly positive at the deep dermal component of melanoma, albeit the staining intensity is variable. the immunoprofile of spitz naevus is different from that of a malignant melanoma. a combination of biological markers as (p , ki , and hmb ), can provide a potential tool to differentiate between the two entities. nevertheless, expanding the biomarker repertoire on a large number of cases is necessary to further establish a reliable panel to differentiate among difficult cases. direct immunofluorescence in a tertiary referral centre: an audit of local guidelines and usage p lj lumsden; l motta; r green salford royal hospital, salford, uk direct immunofluorescence (dif) forms an important and costly adjunct to conventional haematoxylin and eosin (h&e) histology in dermatopathology, particularly in bullous diseases and other immune-mediated diseases. we aim to assess the usage and diagnostic yield of dif in our dermatopathology department. requests for dif on skin biopsies received over a month period met the inclusion criteria. each individual report was assessed with regard to the indication for dif, whether dif was deemed to be indicated or not indicated on assessment of the clinical history supplied on the request card, the results of dif and whether dif was contributory to the final diagnosis. we also collected data on the usage of dif over the last years to assess changes in practice. all requests for dif were granted in line with current departmental policy. the indication categories were divided as follows: bullous , alopecia , lupus , vasculitis , dermatitis herpetiformis (dh) and 'other' . all requests for dif were deemed to be indicated in both the bullous and dh categories by our panel, but indicated requests varied from . % to % in the remaining categories. in . % ( out of ) of cases dif was deemed to be contributory to the final diagnosis. our analysis also showed that usage of dif in our department is escalating, with a . % increase in requests from to . our departmental policy with regard to dif is inclusive and operates solely on the basis of clinician request. with the increasing usage of dif, established departmental guidelines and/or a protocol for dif usage should be mutually agreed with dermatology colleagues in order to ensure effective use of this expensive test. overview of merkel cell carcinoma in an irish population p a cooper ; j thorne royal college of surgeons in ireland, dublin, ireland; beaumont hospital, dublin, ireland purpose of study: merkel cell carcinoma (mcc) is an uncommon but highly aggressive primary cutaneous malignancy of neuroendocrine cells with a propensity for regional and distal metastases. due to it's rarity, information relating to it's epidemiology in an irish population is limited, mainly owing to difficulty in gathering large patient series. our aim was to identify all cases of mcc in our institution in a defined year period and review the patient demographics compared to internationally available data. a search was carried out on the hospital laboratory system to identify all cases of mcc from / / to / / . all histology reports were reviewed and any information pertaining to patient demographics was recorded in an excel spreadsheet. a literature review was performed relating to the patient profile of mcc internationally and the results were compared. results: a total of reports pertaining to individual patients were recovered. all patients were of caucasian irish ethnicity. the incidence of mcc was higher in men ( % of cases, n= ) than women. the median age at diagnosis was years (range - ). men presented at an earlier age (median years) than women (median years). regarding the anatomic site of the tumours, % (n= ) were on the head or face, % (n= ) were on the lower limb and % (n= ) were on the upper limb. all were on sun-exposed sites. of note, the majority of tumours in the male population were on the head ( . %, n= ), while the female population showed an equal distribution between the head and the lower limbs ( %, n= for each sub-site). the subset of patients we identified show demographics consistent with published literature for us, australian and other european cohorts. merkel cell carcinoma is a disease of the elderly affecting sun-exposed sites. we note some variation in the dominant anatomic sites between genders and conclude this is due to differing environmental exposure. background: a novel cell-dispensing instrument referred to as a single cell manipulator (scm) device was developed with the following features: i) rapid optical and fluorescent detection of single cells ii) generation of picoliter sized droplets encapsulating the isolated single cell and iii) printing of the single cell in an "ink-jet" like manner onto a chosen substrate. this technology was used to isolate cells of interest from i) heterogeneous mixed populations of cells, ii) co-cultures of cells and iii) clinical patient samples for subsequent downstream biological analysis. methods: cells were injected into a reusable silicon dispenser chip that was coupled to a live cell camera for image capture and display of cells approaching the chip's exit nozzle. an optical detection mechanism determined the presence of single, fluorescent cells within the selected region of interest close to the chip exit nozzle. a sorting algorithm ensured that only droplets containing the single cells of interest were selected for printing to the prescribed location and user-chosen substrate. results: fluorescently labelled hpv caski cervical cells were spiked into a cervical liquid based cytology sample and printed onto a glass slide using the scm. undifferentiated ntera human embryonal cancer stem cells were isolated from a mixture of differentiated and undifferentiated cells based on fluorescent tagging of the cell surface receptor, stagespecific embryonal antigen (ssea ). the thyroid stimulating hormone receptor (tshr) was expressed in anaplastic v e mutated thyroid cancer cell lines that were treated with the mek inhibitor pd . treated cells were isolated using the scm. the scm pasca technology allows isolation of single cells from heterogeneous populations of cells and clinical samples for downstream analysis at a single cell level. this study aims at quantifying immunohistochemistry (ihc) stained human cell lines for protein biomarkers by manual pathologist review. staining analyses are used to calibrate tissue microarrays of tumour cores, against quantitative protein concentration allowing a systems-based data analysis. as a proof of concept, ffpe human cell line pellets (n= ) were ihc stained for smac protein and analysed using aperio image analysis software. staining quantification manually performed by pathologists provided parameters including average staining intensity, percent total cell positivity and h-score. these data were enriched by qualitative parameters pointing out possible histological artefacts. a calibration curve was plotted using h-score data and protein concentrations, previously determined by western blotting. the panel of cell lines provided a range of strong and weak/absent ihc staining using a highly specific smac antibody. the calibration curve showed a strong correlation between absolute protein concentrations and manual h-scores. expression amounts in cell lines correlate with ihc staining intensities determined by pathologist review. the linear correlation between manual h-score and absolute protein values provides an avenue to indirectly determine protein expression. further analysis will be performed on additional antigens and analysis outcomes will be then compared to digital results. this data will provide the basis for deterministic systems-biological data analysis approaches. purpose of the study: 'lean' is a management framework for maximising value and minimising waste. it originated in the automotive manufacturing industry and has been utilised successfully in non-manufacturing processes. one such application in our department was the 'leaning' of the molecular test requesting process using a smart-phone app. this study will look at the potential utility of this application within the national health service (nhs), wherein approximately twenty different molecular test request forms are currently in use. a mobile application to facilitate molecular test requesting was developed using xcode and the objective c programming language. the application was built around an email based system. patient anonymity was paramount in the design; nhs numbers are used as identifiers. the application generates a molecular test request form and can also generate a national cancer drugs fund application form for each request. administrative staff use colour coded flags to represent the progress of each email request through the workflow process to facilitate tracking. the app reduced the administrative staff workload by reducing the number of steps and paperwork involved in the molecular test requesting process. a threefold reduction in time taken by clinicians to request molecular tests was noted. a survey of staff involved with molecular test requesting revealed a % reduction in 'lost requests' after the introduction of the application. we present a 'lean' method for requesting molecular tests using a smart-phone app. this application can be used to standardise molecular test request forms within the nhs along with automatic generation of a national cancer drugs fund application form for each request. purpose of study: automated approaches for quantitative digital image analysis (dia) of tissues are becoming increasingly popular in pathology due to advances in whole slide scanning hardware and digital imaging technology. it is essential that dia is standardised to ensure accuracy and reproducibility of results. very limited published data exists on the effect of scanner hardware variations on the accuracy and reproducibility of dia results.the aim of this study was to test the following variables: variation in light source intensity during the day; presnap calibration & white balance of scanned images; variation in dia due to debris on peripheral parts of the section or coverslip edges. methods: immunohistochemistry stained sections from patient samples were scanned on the same aperio cs scanner, hourly, for consecutive days to generate scanned images of each sample, representing images in total. all scanned images were run through aperio software using a macro to quantify positive cell counts. for a subset of images, a region of interest was drawn around the tissue to exclude any debris/coverslip edges from peripheral parts of the slide in the subsequent dia. statistical analysis was performed to calculate the coefficient of variation between dia results from scans on different days. results: variation in light source intensity accounted for . % to . % variation in cell counts between repeat scans of the same slide. exclusion of debris/coverslip edges accounted for - % variation in cell counts between repeat scans of the same slide. subjective analysis revealed no significant difference in appearance of different scans of the same slide. conclusion: variation in light source intensity, presnap calibration and overall white balance, plus debris in peripheral areas of the section account for minimal variation in resultant dia results. technical advances in scanner hardware have reduced variability in scanning operations. further investigations are ongoing. the complexity of ovarian cancer resistance mechanisms: a novel, clinically relevant, in-vitro investigation p s busschots g blackshields ; bt hennessy novel carboplatin and taxol resistant cell lines were developed from upn oc cells in a clinically relevant selection strategy to better understand resistant mechanisms in oc. upn - c models carboplatin resistance and upn - t models taxol resistance. upn - calt and upn - talt were exposed to alternating treatments of both agents during development. affymetrix arrays were used to characterise gene/mirna signatures linked with the development of chemoresistance in oc cell lines upn - c and upn - t. bioconductor software, david v . and mirna-target interactions (mtis) analysis was carried out to identify de-regulated genes/mirnas, gene pathways and gene/mirna interactions involved in resistance. upn sublines developed using taxol were significantly resistant to taxol, vinblastine and olaparib (p-gp substrates), and reversible with elacridar (p-gp inhibitor) treatment. significant up-regulation abcb was seen in upn - t which was reflected at the protein level. srpx was highly up-regulated in upn - t. gli and ccl were up/down-regulated respectively in upn - c. gli had a validated interaction with mir- down-regulated in upn - c. lin b was highly deregulated in upn - c and upn - t and had a validated interaction with let- i, down-regulated in upn - c. p-gp over-expression is a dominant mechanism for taxol resistance in our cell lines. mechanisms for carboplatin resistance are more complicated. the top deregulated genes are involved in numerous pathways including apoptosis, cellular transformation, signal transduction, and cell migration cervical glandular neoplasia: the influence of excision procedure on margin status although cold knife cones (ckc) have been traditionally advocated for treatment of adenocarcinoma in situ (ais), large loop excisions of the transformation zone (lletz) are increasingly used. we analysed excisions from patients where ais was confirmed prior to procedure over years to assess the influence of excision procedure on final margin status. we tabulated whether margins were involved, close (lesional tissue less than mm from margin) or excised. results: lletzs were performed in % ( of patients), ckc excision in % ( of ). women who had lletzs were younger than those having ckc excision ( . years versus . years). positive margins were present in % ( of ) and close margins in % ( of ) lletz cases. for ckc, margins were positive in % of cases ( of ) and close in % ( of ) conclusion: although complete excision is more frequently observed when ckc is performed, compared to lletz, for the treatment of ais %) patients, discordant low-grade lymphoma was identified in the bone marrow during staging investigations. were classified as follicular lymphoma, marginal zone lymphoma, chronic lymphocytic lymphoma, lymphoplasmacytic lymphoma, non cll -like monoclonal b-cell lymphocytosis. five had an accompanying a low-grade component that could not be classified. none of the transformed high-grade lymphoma cases were ebv positive. in our cohort, we did not observe statistically significant difference in survival between the transformed and non-transformed cases. an equal proportion of cases transformed from follicular lymphoma and marginal zone lymphoma. . % of transformed patients had a previous history of another cancer, compared to . % of non-transformed cases. age, gender and a history of autoimmune disease were not associated with transformation. transformed chronic lymphocytic lymphoma an investigation into the presentation and nature of diffuse large b cell lymphoma within a large patient cohort p hr freer t cell/histoiocyte-rich large b cell lymphoma ( . %) was the next most common subtype, followed by primary dlbcl of the cns ( . %). a modified r-ipi (the patient performance score was unknown) was used to stratify the patients into four risk groups and was found to be predictive of patient outcome. the average ldh level was . iu/l, well above , the upper limit of the normal range. of the cohort of patients, . % achieved remission, . % were alive with disease at the end of the study and . % are now deceased. the majority of patients that did die did so within a year of diagnosis. in addition, bcl- negative patients were identified, with a mean age at diagnosis of . years. of these patients were female and male. the mean ldh level was some researchers included additional clinical features that were common when there was malignancy in the asymmetrical tonsils. in this study we aim to evaluate the cancer detection rate in this setting including those who possess high risk clinical features such as age (> ), pain and history of smoking. methodology: in total, consecutive tonsillectomy cases, clinically labelled as asymmetrical tonsils were analysed. out of these, cases (group ) were additionally labelled with investigation for unknown primary, obvious lesion or suspicious ulcer seen. the remaining cases with a sole clinical indication of asymmetry were stratified as group . results: in the first group, cases had a histological confirmation of tonsillar primary ( %). while, in the second group, the histological analysis showed cases with benign reactive pathology ( %) while, one case was diagnosed as malignant lymphoma ( %), cases with mild dysplasia ( %) conclusion: our results do correlate with the considerable agreement amongst otolaryngologists that the appearance of an asymmetrically enlarged tonsil in the presence of associated risk factors is considered an indication for tonsillectomy and histological examination given the significant rate of tonsillar malignancies in this group. anatomical difference in the depth of tonsillar fossa and asymmetry of the anterior tonsillar pillar may give a false impression of a clinically asymmetrical or unilateral enlarged tonsil metastatic adenoid cystic carcinoma to the lung and kidney: a single case report p rm doyle the tumour has three prognostically significant subtypes which form the basis for tumour grading; cribriform, most frequent, tubular and solid, associated with a more aggressive clinical course and metastasis. in the majority of cases ( %) the tumour has an insidious onset and patients have locally invasive disease at first presentation, which coupled with adjacent, important anatomical structures means a complete primary surgical resection is often not feasible. distant metastases are frequent and predominately involve the lung and bone with renal metastasis a rare occurrence. an optimal treatment regime for acc has yet to be established and while local management with combination surgery and adjuvant radiotherapy is currently favoured there is conflicting evidence regarding the use of radiotherapy and no formal surveillance guidance for local and regional recurrence or distant metastasis exists. we report a single case of a year old male who presented in with an asymptomatic right neck mass and underwent right radical neck dissection and adjuvant radiotherapy for acc. the patient represented in with aspiration pneumonia on a seven month history of dysphagia and dyspnea. xray and subsequent computed tomography (ct) imaging identified large, multiple right sided lung lesions, maximum cm, and a . cm left renal upper pole mass. ct guided biopsy of the right lung lesion and ultrasound guided biopsy of the renal mass were reported as metastatic adenoid cystic carcinoma regan ; cm martin ; cv timon cytokeratin (ck ) is a junctional biomarker with a seqika fragment which stabilises hpv- e transcripts. we assessed the expression pattern of ck protein in tumour specimens from patients diagnosed with oropharyngeal squamous cell carcinoma (scc) presenting at two major irish head and neck centres, within the last years. methods: archived tumour specimens together with epidemiological data were collected from patients presenting with new primary oropharyngeal scc at two main head and neck centres in ireland, within the last years. briefly, dna was extracted from tissue blocks and hpv testing carried out using spf hpv pcr immunohistochemical staining for ck [clone sp , ventana] was performed on tissue blocks following optimisation on the ventana benchmark ultra immunostainer. slides were analysed by light microscopy and scored using the h scoring system with % of these identified as hpv- subtype. ck expression was observed in the tonsillar crypt epithelium of both normal tonsils and tumour specimens. % of cases were positive for ck , with % of cases demonstrating h score of > . ck expression in the tumour cells was significantly linked to hpv status and our results suggest that the expression of ck in normal tonsillar crypt epithelial cells provides a selective advantage to hpv-related carcinogenesis at this site, possible due to the unique propensity of ck to bind and stabilise hpv- e transcripts regan ; cm martin ; cv timon methods: archived hpv-positive tumour specimens and epidemiological data were collected from patients presenting with new primary oropharyngeal scc at two head and neck centres in ireland over a one year period. briefly, dna was extracted from tissue blocks and hpv testing carried out using spf hpv pcr. the inno-lipa hpv genotyping extra test [fujirebio] was used to determine genotype. immunohistochemical staining for ck , gda, mmp- , agr- , pd- and pd-l was performed following optimisation. slides were analysed by light microscopy and scored using the h scoring system (junctional biomarkers) frozen section reporting of necrotising granuloma of the liver following percutaneous instrumentation of the biliary tree: a case series eh hadjimichael; p df fielding; mi ilyas; az zaitoun; dl lobo; pk kaye nottingham university hospital, nottingham, uk percutaneous transhepatic cholangiography (ptc) is an interventional radiological technique for both diagnostic imaging and therapeutic decompression of the proximal biliary tract in malignant distal obstruction when retrograde techniques fail. recognised complications of ptc include sepsis, haemorrhage and pneumothorax. we describe four cases where necrotising granuloma, apparently secondary to previous ptc, has resulted in frozen section examination at the time of subsequent planned cancer resection, to exclude tumour metastasis. four cases of necrotising granuloma in the liver have been identified between january and february . all cases were planned whipple's procedures for pancreatic cancer where initial intraoperative evaluation revealed solitary subcapsular liver lesions. biopsy and intraoperative frozen section examination were performed to exclude metastatic disease. all frozen sections except one were reported as showing benign necrotizing granuloma formation. the first case was initially reported as malignant and the operation was abandoned. a benign diagnosis was confirmed on paraffin sections in all four cases with the first patient undergoing successful surgical resection at a later date. to the best of our knowledge these are the first reported cases of necrotising granuloma in the liver secondary to prior instrumentation of the liver and leading to intraoperative histological assessment. we highlight this as a potential pitfall in frozen section interpretation undertaken ahead of planned potentially curative surgery which can lead to overstaging of otherwise resectable disease or to the interpretation of a potential diagnosis of tuberculosis. these risks can be reduced with greater surgical and pathological awareness of this entity. inclusion body fibromatosis, also known as infantile digital fibroma, is a benign, predominantly myofibroblastic tumour primarily found on the digits of infants. clinically, these lesions present as asymptomatic cutaneous nodules, rarely larger than cm in size, classically on the dorsal or dorsolateral aspect of the second, third and forth digits. they have a high recurrence rate, reported as between and %, although this can be reduced by undertaking complete wide local excision. we report a case of inclusion body fibromatosis in an month-old boy, presenting with an enlarging, firm lesion on his left second toe. following surgical excision, the lesion showed classical histological features of inclusion body fibromatosis -spindle cells arranged in interlacing fascicles in collagenous stroma and numerous pink intracytoplasmic inclusions. the lesion appeared incompletely excised and the patient will be kept under review on account of the high risk of recurrence.decreased expression of the mitochondrial bcat protein correlates with improved patient survival in idh wild-type gliomas me conway ; j hull ; m el hindy ; sc taylor ; f el amraoui ; c paton-thomas ; p white ; m williams ; p hr haynes ; sm hutson ; km kurian key: cord- - ip z authors: wang, denong; tang, jin; wolfinger, russell d.; carroll, gregory t. title: carbohydrate microarrays date: - - journal: polysaccharides doi: . / - - - - _ sha: doc_id: cord_uid: ip z carbohydrates, like nucleic acids and proteins, are essential biological molecules. owing to their intrinsic physicochemical properties, carbohydrates are capable of generating structural diversity in a multitude of ways and are prominently displayed on the surfaces of cell membranes or on the exposed regions of macromolecules. recent studies highlight that carbohydrate moieties are critical for molecular recognition, cell-cell interactions, and cell signaling in many physiological and pathological processes, and for biocommunication between microbes and host species. modern carbohydrate microarrays emerged in and brought in new high-throughput tools for “glyco code” exploration. in this section, some basic concepts of sugar chain diversity, glyco-epitope recognition, and the evolving area of glyco-epitomics and biomarker discovery are discussed. two complementary technologies, carbohydrate antigen arrays and photogenerated glyco-chips, serve as models to illustrate how to apply carbohydrate microarrays to address biomedical questions. cellular carbohydrates are present in multiple structural configurations, including mono-, oligo-, and polysaccharides, as well as various glycan-hybrid molecules. the latter include, but are not limited to, glycolipids, glycoproteins, proteoglycans, and glycosaminoglycans. unlike proteins, which are composed of amino acids that are connected solely by one possible peptide bond, carbohydrates utilize many possible glycosidic linkages so as to extensively diversify their structures. for example, two amino acid residues, such as two alanines, can produce only one possible dipeptide; however, two molecules of glucose have the potential to generate different disaccharides. a trimer of any of the nine common sugar residues of the human body theoretically can give rise to , different structural isomers; this is in striking contrast to the maximal construction of , tripeptides using different amino acid residues. theoretically, sugar chain structures can have unlimited variation. in human and virtually all animal species, cell display of specific complex carbohydrates is characteristically associated with the stages or steps of embryonic development, cell differentiation, and transformation of normal cells to abnormally differentiated tumor or cancer cells (feizi ; hakomori ; crocker and feizi ; focarelli et al. ) . in plants, a highly complex set of polysaccharides are associated with structural proteins and lignin to form cell walls (avci et al. ). even in a given tissue or cell, cell wall layers and domains may have very different carbohydrate structures and express different glyco-epitopes (albersheim et al. ; avci et al. ) . many microbial organisms also carry unique glycosylation systems and produce specific sugar signatures for almost every microorganism in the living world (dochez and avery ; heidelberger and avery ; ezzell et al. ; robbins and schneerson ; mond et al. ; wang and kabat ) . the term "glycome" has been recently introduced to cover the universe of carbohydrate moieties in all living organisms. importantly, multiple carbohydrate recognition systems are present in living species. for example, there are numerous anti-glycan antibodies produced by human and other animal species that play key roles in protecting a host from microbial infections (behring and kitasato ; wang and kabat ; lucas et al. ) and families of lectin-like glycan-binding proteins (gbps) that are evolved for carbohydrate-mediated cell-cell communication (drickamer ; sharon ; varki ). the well-known gbps that are associated with cell signaling and immunomodulation are the receptors of the innate immune system, such as dectin- that recognizes fungal β-glucans (brown et al. ) , mannose receptor that recognizes carbohydrate moieties on multiple pathogens and is involved in the clearance of inflammatory molecules in vivo (gruden-movsesijan and milosavljevic lj ) , and dc-sign (dendritic cell-specific icam- -grabbing non-integrin) that selectively detects viral glycoproteins, such as hiv- gp glycoprotein (curtis et al. ) . the interaction between hiv- gp and dc-sign plays an important role in the cd -independent association of hiv with human cells (curtis et al. ) . thus, carbohydrates are suitable for storing biological signals in forms that are identifiable by other biological systems. in the immunological and glycobiological literature, "glyco-epitope" is often used to specify the carbohydrate moiety that is recognized by an antibody or by a gbp. the antibody-binding glyco-epitopes are also classified as b cell epitopes or antigenic determinants. the term "glyco-epitome" was recently introduced to describe the entire repertoire of glyco-epitopes, including the b cell epitopes and those that are recognized by gbps. differing from the term "glycome," which covers all the existing carbohydrate molecules in living organisms, glyco-epitome refers to a unique subset of carbohydrates that serves as the sugar signatures for molecular recognition and biosignal transmission. "glyco-epitomics" is, thus, an area of glycomics research focusing on identifying, characterizing, and understanding the carbohydrate moieties that serve for multiple levels of biocommunication. it is noteworthy that glyco-epitope characterization requires not only carbohydrate structural analysis but also immunological studies. the structural aspects of glyco-epitomics focus on the elucidation of the glycan structures that display glycoepitopes. this research area has been substantially enhanced by the development of advanced profiling and structural characterization strategies. notably, these include high-resolution chromatography methods coupled with exoglycosidase digestions royle et al. ) , modern mass spectrometry (babu et al. ; goldberg et al. ; north et al. ) and nuclear magnetic resonance spectroscopy analyses (petrescu et al. ; wormald et al. ; petrescu et al. ) of carbohydrates and state-of-the-art methods of glycan structural modeling (woods and tessier ; jo et al. ) . however, availability of carbohydrate structural information alone is not sufficient in defining a glyco-epitope unless its specific binding by an antibody or a gbp is also demonstrated immunochemically and/or cryptographically. for example, chemical determination of a tetrasaccharide that decorates the spore of bacillus anthracis appears to be an important discovery in microbial glycomics (daubenspeck et al. ). based on past knowledge of immunogenic carbohydrate moieties, this structural glycomics progress may suggest that this unique sugar moiety may have potential in an immunological application (saksena et al. (saksena et al. , . however, whether such a carbohydrate moiety preserves a b cell epitope or a potent antigenic determinant must be determined immunologically, including at least demonstration of its antibody-binding specificity and capacity in eliciting immune responses in vivo (wang et al. ) . it was the integrated structural and immunological investigation of glyco-epitopes (wang et al. ; lucas et al. ) that has revealed anthrose tetrasaccharides as key immunological targets of b. anthracis. four research articles about carbohydrate microarrays first appeared in the scientific literature in (borman (borman , . these include polysaccharide and glycoconjugate microarrays, reported by denong wang's group at columbia university's genome center (now at sri international, ca, usa) (kiessling and cairo ; wang et al. ) ; monosaccharide chips, by milan mrksich and coworkers at the university of chicago (houseman and mrksich ) ; arrays of natural and synthetic neoglycolipids, by ten feizi's group at imperial college faculty of medicine, harrow, uk (fukui et al. ) ; and arrays of synthetic oligosaccharides in microtiter plates, by a scripps research institute group led by chi-huey wong (bryan et al. ) . a specialized book, "carbohydrate microarrays, methods and protocols (humana press)," recently edited by dr. yann chevolot of université de lyon in france (chevolot ) , provides a comprehensive summary of the emerging technologies for construction of carbohydrate microarrays. given the various structural characteristics of carbohydrates displayed on chips, carbohydrate microarrays are classified as monosaccharide chips (houseman and mrksich ; park and shin ) , oligosaccharide arrays (bryan et al. ; fukui et al. ; blixt et al. ; wang et al. ) , and microarrays of carbohydratecontaining macromolecules (wang et al. ; willats et al. ) . the latter includes polysaccharides and various glycoconjugates. these different sugar chips or arrays were developed to accommodate multipurpose applications in carbohydrate research. for example, mono-and disaccharide microarrays are suitable for screening and characterizing novel carbohydrate-binding proteins or carbohydrate-catalyzing enzymes and for identifying novel inhibitors of carbohydrate-protein interactions. a large class of carbohydrate-binding proteins, called lectins, was initially classified by their binding specificities to monosaccharides and recently by disaccharides. however, there are lectins and many antibodies with anti-carbohydrate reactivities that bind to larger and more complex carbohydrate ligands or antigenic determinants. mono-and disaccharide sugar chips are not sufficient for investigations involving such molecular targets. oligosaccharide, polysaccharide, and glycoconjugate microarrays fill this gap by displaying carbohydrates of complex structures or longer sugar chains on the chips. based on the technologies that are applied to immobilize carbohydrates on bioarray substrates, the various methods to construct carbohydrate microarrays can be classified as distinct technological platforms. these include technologies that directly utilize underivatized carbohydrates in microarray construction, technologies that require chemical modification of carbohydrates before microarray fabrication, methods of non-covalent immobilization of carbohydrates, and methods of covalent coupling of saccharides on array substrates. the use of underivatized saccharides for microarray construction has the advantage of preserving the native structures of the carbohydrate molecules. it requires, however, a ready-to-use microarray surface with appropriate surface chemistry that can be directly used to fabricate comprehensive carbohydrate microarrays with underivatized carbohydrates from a wide range of sources. methods currently in use include non-covalent binding of underivatized carbohydrate antigens by passive adsorption on a chip, such as nitrocellulose-coated glass slides (wang et al. ) or black polystyrene surfaces (willats et al. ) , and methods for covalently immobilizing underivatized carbohydrates on a slide surface by appropriate chemical linking techniques (angeloni et al. ; lee and shin ; carroll et al. ; zhou and zhou ; wang et al. ; zhou et al. zhou et al. , . carbohydrate microarrays can also be fabricated by using derivatized carbohydrates. due to their small molecular size and hydrophilic nature, most oligosaccharides cannot be directly immobilized onto nitrocellulose or black polystyrene surfaces for microarray applications. however, an oligosaccharide probe can be modified with a tag or coupled to a larger carrier molecule for non-covalent immobilization. methods include non-covalent immobilization of derivatized carbohydrates on microarray chips (fukui et al. ; palma et al. ) or on enzymelinked immunosorbent assay (elisa) microtiter plates (bryan et al. ) and covalent immobilization of derivatized carbohydrates on microarray chips. the latter includes, but are not limited to, the popular consortium for functional glycomics (cfg) printed glycan arrays (blixt et al. ; bochner et al. ) and various technologies of notable technical features that were developed independently (houseman and mrksich ; park and shin ; galanina et al. ; kohn et al. ; park et al. ; parthasarathy et al. ; gerland et al. a, b; morvan et al. ; goudot et al. ) . affinity immobilization is another class of approaches for coupling derivatized carbohydrates to solid surfaces. for example, biotin-derivatized carbohydrates can be immobilized on a streptavidin-coated substrate through the affinity interaction of the streptavidin-biotin pair to create carbohydrate microarrays. biotin-derivatized carbohydrates include carbohydrate ligands that are biotinylated via a short aliphatic spacer or at the peptide part of glycopeptides (guo et al. ; bochner et al. ; dyukova et al. ) . dna-directed immobilization (ddi) is another practical strategy for immobilization of oligonucleotide glycomimetic conjugates on a chip surface for the preparation of carbohydrate microarrays (gerland et al. a, b; morvan et al. ; goudot et al. ). despite technical differences among different platforms of carbohydrate microarrays, they are all solid-phase binding assays and share a number of common characteristics and technical advantages. for instance, they contain the capacity to display a large panel of carbohydrates in a limited chip space, they are highthroughput quantitative assays, they make an effective use of carbohydrate substances that are often difficult or cost-inefficient to synthesize, and, as discussed below, they are highly sensitive in monitoring carbohydrate-anti-carbohydrate interactions in multiplex manners. in a carbohydrate microarray, each carbohydrate is spotted in an amount that is drastically smaller than that required for a conventional molecular or immunological assay. this technical feature ensures a condition in which the binding of a molecule in solution phase to an immobilized microspot of ligand on the microarray substrate has minimal reduction of the molar concentration of the molecule in solution (ekins et al. ). thus, microarray-based assays are intrinsically optimized for binding equilibrium to take place, which is the basis for this class of hypersensitive binding assays (stoll et al. ) . carbohydrate microarrays have higher detection sensitivity than most conventional carbohydrate analytical tools, such as carbohydrate-specific elisa and the glycolipid-based "eastern blot" assays that were developed in the s by a number of early researchers in this field (wood and kabat ; tang et al. ) . historically, this situation is very similar to the relationship between conventional blotting methods for nucleic acids or proteins, such as southern, northern, and western blots, and nucleic acid-based or protein/peptide-based microarrays. carbohydrate microarrays constructed by various methods may differ in their technical characteristics and suitability for a given practical application. some platforms may be applied complementarily to solve a practical question. for example, the method of nitrocellulose-based immobilization of carbohydratecontaining macromolecules, including polysaccharides, glycoproteins, and glycolipids, is suitable for the high-throughput construction of carbohydrate antigen microarrays (wang et al. wang and lu ) to support the largescale immunological characterization of carbohydrate antigens and anticarbohydrate antibodies. however, the detection specificity of this carbohydrate microarray would be at the level of a carbohydrate antigen, not a glyco-epitope, if the native carbohydrate antigens were spotted. this is owing to the fact that many carbohydrate antigens display multiple antigenic determinants or glyco-epitopes (cisar et al. ; wang and kabat ; wang ) . examining the finer details of the binding properties would require the use of microarrays of defined oligosaccharide sequences. oligosaccharide array-based binding assays can be applied, in combination with saccharide competition assays, to decipher precise saccharide components of a specific antigenic determinant or glyco-epitope (fukui et al. ; blixt et al. ; wang et al. ; zhou et al. ). these technical features of carbohydrate microarrays are further discussed in two models, i.e., a carbohydrate antigen microarray platform and a technology of photogenerated oligosaccharide microarrays in subsequent sections. a practical approach for construction of carbohydrate microarrays is to print carbohydrate antigens onto nitrocellulose-coated glass slides. this was the first reported method for high-throughput production of carbohydrate microarrays (borman ; kiessling and cairo ; wang et al. ) . using this technology, carbohydratecontaining macromolecules of diverse structures, including polysaccharides, natural glycoconjugates, and mono-and oligosaccharides coupled to carrier molecules, can be stably immobilized on a glass chip without chemical modification. this approach was subsequently extended to production of lipids/glycolipid and liposome microarrays (fukui et al. ; wang et al. , . recently, this approach has been applied to produce integrated protein, lipid, and carbohydrate microarrays ). owing to the technical simplicity of this approach, anyone who has access to a standard cdna microarray facility would be able to explore this technology for his or her own research interests (fig. ). soluble antigen preparations are generally applicable for construction of microarrays in this platform. except certain antigens that require special solutions, proteins and carbohydrates for spotting are dissolved in phosphate-buffered saline (pbs; ph . ) and saline ( . % nacl), respectively. liposomes of various compositions, including homoand hetero-liposomes, are suitable for printing on this fig. a high-throughput platform for carbohydrate-based microarrays. a high-precision robot designed to produce cdna microarrays is utilized to spot carbohydrate antigens of various structural configurations onto a nitrocellulose-coated glass slide. the microspotting capacity of this system is approximately , spots per chip. the antibody-or gbp-stained slides were then scanned for fluorescent signals with a microarray scanner that was developed for cdna microarrays. toxins or viral pathogens can also be applied on this platform of carbohydrate microarrays to probe potential glycan-receptors of viruses or toxins substrate ). the former were produced via a single lipid preparation, e.g., phosphatidylcholine (ptc), cerebroside, and sulfatide. the latter contained two different lipid molecules with ptc as the support to display other lipid/glycolipids in desired ratios or epitope densities. for example, the heteroliposome of sulfatide is composed of sulfatide and ptc at a ratio of : (wt/wt), i.e., . mg sulfatide and . mg ptc per ml of liposome suspension in saline. methods employing sonication and extrusion (mechanical energy) to produce liposomes for microarray production were similarly described by a number of investigators palma et al. ). microarray printers that were designed for dna or protein microarrays, such as pixsys c (cartesian technologies, irvine, ca), are suitable for spotting carbohydrates onto glass slides pre-coated with nitrocellulose polymer (fast slides; schleicher and schuell, keene, nh) (wang ) . we often spot antigens in triplicate with spot sizes of μm and at μm intervals, center to center. the printed microarrays are air-dried and stored at either room temperature or c before application. immediately before use, the printed microarrays were rinsed with pbs, ph . , with . % (vol/vol) tween and then blocked by incubating the slides in % (wt/vol) bovine serum albumin (bsa) in pbs containing . % (wt/vol) nan at room temperature (rt) for min. they were then incubated at rt with antibodies at an indicated titration in % (wt/vol) bsa in pbs containing . % (wt/vol) nan and . % (vol/vol) tween . the secondary antibodies or streptavidin conjugates applied for microarray staining are specified in the figure legends. the stained slides were rinsed five times with pbs with . % (vol/vol) tween , air-dried at room temperature, and then scanned for fluorescent signals using a scanarray a microarray scanner (perkinelmer life science) following the manufacturer's manual. large-scale "repertory" microarrays containing thousands of microspots or larger are powerful means for discovering unexpected molecular targets. for example, microarray scanning of autoantibody responses allows one to "fish out" potential autoantigens in the glycome in autoimmune diseases. customized, smaller scale carbohydrate microarrays containing a few dozen antigens are, however, suitable for more defined purposes, such as antibody fine-specificity mapping, differential diagnosis among a number of known infectious diseases, measurement of autoantibodies for known targets in autoimmune diseases, etc. fig. shows a sub-array design where each chemically modified microglass slide contains eight separated sub-arrays. the microarray capacity is~ microspots per sub-array. a single slide is, thus, designed to enable eight microarray assays. similar sub-array designs with various array capacities are commercially available (schleicher and schuell, keene, nh; arrayit, sunnyvale, ca). • each microglass slide contains twelve sub-arrays of identical content. there is a chip space for about microspots per sub-array, with spot sizes of approximately μm and at -μm intervals, center to center. a single slide is, therefore, designed to enable detections. this design is typically for printing four -well plates of antigen preparations ( Â = ). • repeats and dilutions: our team usually prints carbohydrate antigens at the initial concentration of . - . μg/μl. the absolute amount of antigens printed on the chip substrate is in the range of . - . ng per microspot for the highest concentration. they are further diluted at : , : , and : , or as specified in each experiment. a given concentration of each preparation is repeated at least three times to allow statistical analysis of detection of identical preparations at given antigen concentrations. • positive controls and standard curves: fluorescent conjugates, such as bsa conjugates of fitc, cy , cy , or other dyes, are routinely applied for microarray printing to provide positive markers for each fluorescent channel. these markers are helpful for scanning calibration, alignment of microarray spots during data-capturing, the subsequent microarray data normalization, and cross-chip scaling of microarray detection. for serological studies, antibodies of igg, iga, and igm isotypes of corresponding species are spotted to produce standard curves in microarray format. these curves serve as reference standards for quantifying antibody signals of a specific igh chain isotype that are captured by spotted carbohydrate antigens. a number of nitrocellulose-coated glass slides with different technical characteristics are commercially available. given the structural diversity of carbohydrate antigens, examining each antigen preparation to determine the efficacy of its immobilization in a given type of substrate and the surface display of the desired glyco-epitopes in a microarray assay is essential. a practical approach is to incubate the printed microarrays with antibodies, receptors, or lectins known to react with the printed substance. figure is an example of such analysis where lectin galanthus nivalis agglutinin (gna) and antibody g were applied to examine specific glyco-epitopes on the spotted microarrays. inspection of both microarray images (fig. a ) and the quantitative datasets show that the gna-epitopes were presented by three glycoconjugates (fig. c) , i.e., man -cluster ( # ), m _ g -cluster ( # ), and man - rb ( # ). in contrast, g -glyco-epitopes were preserved only by one of the three, i.e., m _ g -cluster ( # ), on this microarray substrate (fig. d) . this carbohydrate microarray analysis demonstrates, therefore, an example that the same sugar chain may generate different glyco-epitopes when the sugar moiety is presented in different cluster configurations. in this case, the man glcnac asn moiety was coupled to the protein carriers in either (man glcnac asn) n-or [(man glcnac asn) ] n-configurations (fig. b) . the latter but not the former preserves well the g -defined broadly hiv- neutralizing epitope. a photogenerated glyco-chip technology carroll and colleagues ( ; wang et al. ; carroll and wang ) developed a photochemical method to covalently immobilize carbohydrates on chips. as illustrated in fig. , the method employs a self-assembled monolayer to present photoactive phthalimide chromophores at the air-monolayer interface. upon exposure to uv radiation, the phthalimide end-groups graft to surface-adsorbed carbohydrates to form a covalent bond. the amount of surface-grafted carbohydrate is enhanced when carbohydrate surface interactions are increased by the incorporation of amine-terminated molecules into the monolayer. one of the important applications of this technology is to identify immunogenic sugar moieties of microbial pathogens by screening the corresponding antisera obtained from vaccinated or infected subjects. the phthalimide chromophore used in the photogenerated glycan-chip was modified with a silane derivative in order to form a stable bond to glass. a . mmol portion of -bromoundecanetrimethoxysilane (gelest) was added to a solution of an equimolar amount of potassium phthalimide (aldrich) in ml of anhydrous dmf (aldrich). the solution was stirred overnight at room temperature (rt) under argon. chloroform ( ml) was added. the solution was transferred to a separatory flask containing ml of h o. the aqueous layer was separated and then extracted after synthesis of the phthalimide-silane, pam was prepared by immersing a clean glass slide into a toluene solution containing mm of the phthalimide-silane and mm of aminopropyltrimethoxysilane (gelest). the h o contact angle of the resulting surface was ae . note that the glass slide was cleaned with a : mixture of h so : % h o . extreme caution should be used when preparing and using such a solution, which can react violently and explosively if mixed with other chemicals. fig. photogenerated glycan arrays for rapid identification of pathogen-specific immunogenic sugar moieties. saccharide preparations were dissolved in saline ( . % nacl) at a given concentration and spotted using a high-precision robot (pixsys c, cartesian technologies, irvine, ca) onto the phthalimide amine (pam)-coated slides. the printed pam slides were subjected to uv irradiation ( nm) for h to activate the photocoupling of carbohydrates to the surface. pathogen-specific antisera were then applied on the glycan arrays to identify potential immunogenic sugar moieties of given pathogens (adapted from wang et al. ( )) . microarray spotting is performed as outlined in the above section. after spotting of carbohydrates, the pam slides were air-dried and placed in a quartz tube. the sealed tube was subsequently purged with argon or nitrogen before irradiation. uv irradiation was conducted by placing the quartz tube under a desktop lamp containing a nm rayonet bulb for h. precaution was made to avoid skin and eye contact with the radiation during the irradiation process. the photogenerated glycan arrays were applied to probe the potential immunogenic sugar moieties of bacillus anthracis spores (wang et al. ) . the rationale was that if b. anthracis spores expressed immunogenic carbohydrate structures, the spore antigen-immunized or b. anthracis-infected animals would be possible to mount antibody responses to these carbohydrates. this assumption was made on the basis of the fact that the host immune system is able to recognize subtle changes in sugar structures, especially those that are exposed on the surfaces of microbial pathogens that are foreign components of the mammalian hosts. figure below is an example of photo-chip characterization of the rabbit antisera elicited by b. anthracis spores. the photo-chips used were spotted with a large panel of saccharide structures, including synthetic fragments and derivatives of the anthrose-containing tetrasaccharide side chain of the b. anthracis exosporium and a number of control carbohydrate antigens. antibody staining was performed in the presence or absence of saccharide inhibitors. images (a-f) display a portion of the stained glycan arrays: (a) no saccharide inhibitor; (b) anthrose; (c) d-glucose; (d) α-anthrose trisaccharide; (e) α-anthrose tetrasaccharide; (f) β-anthrose tetrasaccharide. the locations of surface-bound anthrose-containing saccharides that are recognized by the antibody in the absence of inhibitor are highlighted by colored boxes: white, β-anthrose-trisaccharide; brown, β-anthrose-tetrasaccharide; yellow, α-anthrose-tetrasaccharide. microarray data sets are available upon request. this analysis confirmed that a tetrasaccharide of bcla glycoprotein bears a dominant antigenic determinant, which is composed of a terminal anthrose residue and three adjacent l-rhamnoses. the terminal trisaccharide unit is essential for the constitution of a highly specific antigenic determinant. given the fact that this carbohydrate moiety is displayed on the outermost surfaces of b. anthracis spores and its expression is highly specific for the spore of b. anthracis, the anthrosecontaining tetrasaccharide can be considered an important immunological target. its applications may include identification of the presence of b. anthracis spores, surveillance and diagnosis of anthrax infection, and development of novel vaccines targeting the b. anthracis spore. a unique technical advantage of this method is the ability to produce epitopespecific glycan arrays using unmodified mono-and oligosaccharides. the quality of the surfaces obtained depends in part on cleanliness. ideally, water with a resistivity of . mΩ-cm and total organic contaminant of less than five parts per billion should be used; however it is still possible to prepare the surfaces after rinsing with water of lower quality. self-assembly of trimethoxysilanes can be enhanced by adding small amounts of water and acid. such treatment was not used for the mixed surface described above but may possibly enhance the reaction times or surface coverage obtained. if the surface is allowed to dry upon removal from selfassembly solution, the aminosilane may polymerize, leaving white deposits that are difficult to remove. although specialized bioinformatics tools for carbohydrate microarrays are yet to be developed, some software packages developed for cdna microarrays are applicable for carbohydrate microarrays. this is owing to the fact that these different microarray assays are commonly based on the laser fluorescence detection systems regardless of the contents of the spotted microarrays. a number of advanced microarray software packages are currently available. these include, but are not limited to, significance analysis of microarrays (sam, http://wwwstat.stanford.edu/~tibs/sam/), prediction analysis for microarrays (pam http:// www-stat.stanford.edu/~tibs/pam/index.html), and jmp genomics (www.jmp. com/genomics). a straight forward way to present the raw results of a carbohydrate microarray assay is to illustrate the microarray raw datasets and/or images. for example, carbohydrate researchers often present microarray raw datasets with or without background substraction for the carbohydrate-binding profiles of monoclonal antibodies (palma et al. ) , lectins (blixt et al. ) , or viruses (childs et al. ). figure above illustrates two examples of binding profile presentations based on microarray raw data. in this experiment, the mannose-cluster-containing microarrays were stained with mab g ( μg/ml) and a biotinylated gna ( . μg/ml), respectively. figure a shows microarray images and fig. b illustrates the cluster configuration of the three man -conjugates. microarray detections are shown as the mean fluorescent intensities (mfis) of each microspot as captured by the scanarray a for the arrays stained with gna (fig. c) and g (fig. d) , respectively. results were compared using overlay plots of the mfis of the antigen-binding signals (red circles) versus those of local background signals surrounding the antigen microarrays (blue +) (fig. c, d) . analysis of such microarray raw data in association with visual inspection of the microarray image provides an initial evaluation of reproducibility and variation of this antigen microarray technology. in order to identify disease-associated biomarkers, such as antigen-specific antibodies with diagnostic and/or prognostic values, we compare microarray detection between a disease group and a normal control group or to more generally between multiple groups. proper statistical analysis is essential for making generalizable conclusions. typical steps include quality control, normalization, and statistical model fitting. in our general practice, carbohydrate array datasets are preprocessed and statistically analyzed using jmp genomics software from sas institute. antigen-specific antibody reactivities are represented as microarray scores, which are the log transformed values normalized by some method. we have explored several different normalization methods, including mean and/or variance centering, loess, and quantile normalization, and found the interquartile range (iqr) method to provide an appropriate degree of standardization (based on distribution and correlation plots) without overly correcting the data. the iqr method sets the th and th percentiles of the microarray distributions to be equal. after normalization, we utilize an antigen-by-antigen anova model to obtain statistically significant differences. data from triplicate spots for each antigen are included in the anova model for that antigen. a cutoff to detect significant differences is determined by applying a multiple testing correction to statistical results from the anova model. these procedures are further discussed below in an example of clinical sample analysis. here we present an example to illustrate general statistical approaches to carbohydrate microarray analysis of clinical samples. in this case, customized autoantigen microarrays were applied to characterize the cerebrospinal fluid (csf) of multiple sclerosis (ms) patients. ms is a complex neurological disorder in which an adaptive autoimmune response is thought to target myelin sheath in the central nervous system. we created a microarray displaying a panel of carbohydrate and lipid antigens to examine ms-associated autoantibody responses. a technical challenge to this study is the fact that the total ig concentrations in the csf of ms patients are higher than those in the csf of other neurological diseases (ond) subjects, which reflects one of the hallmarks of ms (kabat et al. (kabat et al. , steinman ; genain et al. ; raine et al. ; hueber et al. ) . however, it causes difficulty in identifying the disease-specific autoantigens and autoantibodies. in our microarray analyses, we reconfirmed this observation. figure a , b show the overlay plots of antibody profiles of the two groups. the colored needles that link the pairs of group mean values provide a global comparison of the antibody profiles between the ms group (red circles) and the ond group (blue crosses). this comparison reveals global differences in antibody profiles between the two groups. specifically, the microarray scores of csf-antibody activities in the ms group are generally higher than those seen in the csf of ond subjects. these include not only anti-lipid antibodies (right, - # ), as previously reported (ho et al. ), but also anti-carbohydrate antibodies (left, - # ). we further examined whether there is any selective enrichment of antigenspecific antibodies in the csf of ms patients. we reasoned that identifying such antibodies might provide clues to pinpoint key autoimmunogenic targets of ms. for this purpose, we introduced an approach to establish rar scores for microarray signals and then sought targets that capture the antibody signal with higher rar scores in ms patients. specifically, we normalized the microarray datasets by setting their iqr to be identical using the jmp genomics software package. this statistical operation effectively "quenches" the variation seen between subjects that are due to variable antibody concentrations in the csf. the two groups illustrate similar ig-rar profiles for both igg and igm antibody activities (fig. c, d) . however, a number of probes show higher igg-rar scores in the ms group than in the ond controls. these include two man -clusters ( # , and # ), three glucose polysaccharides, dextran n ( # ), b s ( # ), b s ( # ), and a bacto-agar ( c, extracted) antigen ( # ) (fig. c) . in fig. , an antigen-by-antigen analysis of variance (anova) model was applied to obtain statistically significant differences between groups in comparison. results are graphically presented as a volcano plot (zink et al. ) for a global comparison of all rar scores between the groups in comparison (fig. a) and as one-way analysis scatterplots for selected targets (fig. b, c) . in the volcano plot, each dot represents a statistically weighted and quantified difference between ms and ond groups. the x-axis is the normalized difference (log scale) and the y-axis uses -log ( p-value) for the difference. spots above the red-dashed line represent signatures that differ significantly between the groups after a multiple testing correction. for the one-way scatterplots in fig. b , c, each data point represents the mean of triplicate determinations. the means of the points are shown as horizontal green bars and standard deviations as green diamonds around the mean value. the comparison circles for the student's "t"-test appear to the right of the mean diamonds to illustrate the significance of the differences among the means. these circles allow visual inspection of the statistical significance of the differences. the more the circles intersect, the less significant their difference, and vice versa. of the antibody signatures captured in this assay (fig. c, d) , two were above the cutoff line [-log ( p-value) = . ] as highly significant fig. integrated lipid/carbohydrate arrays recognize globally elevated antigen-specific antibodies in the csf of ms patients as compared to those detected in the ond csf. csf samples from ms ( rrms and spms) and ond subjects were characterized using microarrays spotted with lipid and carbohydrate antigens. each preparation was spotted in triplicate at - dilutions. as illustrated in the overlay plots (a-d), this microarray supports detection of unique antibody signatures. these included igg (a, c) and igm (b, d) signatures. anti-human igg or igm secondary antibodies were used to reveal the antigen-specific antibodies detected by these microarrays. the captured igg were stained with an anti-human igg antibody conjugated with cy at μg/ml and the captured igm in the same array revealed by a biotinylated anti-human igm secondary antibody at μg/ml and developed with streptavidin-cy conjugate at μg/ml. microarray datasets in (a) and (b) were illustrated as microarray scores. in (c) and (d) microarray datasets were further processed using jmp genomics to produce rar* scores. the results are presented as overlay plots of the mean microarray scores for each group. the colored needles that link the group mean values of each pair of scores provide a global comparison of the antibody profiles of ms (red circles) and ond (blue crosses) groups (adapted from wang et al. ( ) ). *rar score: rar relative antibody reactivity, the value of log transformed and iqr-standardized microarray value (meanbackground). for each antigen in a given concentration, the mean value of triplicate array detections was calculated differentiators between ms and ond. these were m _ g _igg and man _igg. results of the one-way anova of the two man clusters are shown in fig. b . all other antibody signatures had -log p-values below the cutoff line (fig. a) , including four signatures that were variably higher in the ms group than in the ond group. these were n _igg (p = . ), b s_igg (p = . ), b s_igg (p = . ), and bacto_agar_igg (p = . ). this microarray analysis identified, therefore, oligomannoses as potential immunological targets in the volcano plot, each point represents a biologically unique feature captured by the microarray, i.e., a normalized difference for a specific antibody signature (rar ms -rar ond ). the x-axis is the normalized difference (log scale); the y-axis is the -log ( p-value), which weights the levels of significance of a difference. points above the red-dashed line (cutoff level ) represent signatures that differ significantly between the groups based on the bonferroni test. two signatures, m _ g _igg and man _igg, were identified by this critical statistical test as highly significant markers. in (b) and (c) one-way analysis was performed to compare group means among the selected carbohydrate antigens listed in each panel. each point in the panels represents the mean value (rar score) of triplicate array detections of a subject (adapted from wang et al. ( )) for further investigation. overall, jmp genomics is a valuable tool for statistical analysis of carbohydrate microarray data. modern carbohydrate microarrays emerged in (borman (borman , kiessling and cairo ; wang and collins ) and introduced new glycomics tools to decipher the biological information content in the glycome. these technologies are especially useful in exploring the repertoire of glyco-epitomes. a number of carbohydrate microarray platforms have now reached or are very close to the technical stage of the current nucleic acid-based or protein-based microarrays that are readily available for practical uses. technical issues that require further improvement may include, but are not limited to, optimization of existing technologies for array construction, quality control and technical standardization in both microarray production and application, and establishment of specialized bioinformatics tools to handle the massive amount of carbohydrate microarray data and to effectively extract diagnostic or research information from each microarray assay. nevertheless, exploring the repertoires of glyco-epitopes represents a long-term goal of glycomics research. it was estimated that the human glycome contains , - , minimal epitopes for glycan-binding proteins (cummings ). in considering the repertoires of the "hybrid" structures that are generated by protein posttranslational modification, including both nand o-glycosylation, the repertoires of carbohydrate-related antigenic structures can be much larger. furthermore, the conformational diversity of carbohydrates and microheterogeneity of carbohydrate chains substantially increases the repertoire of carbohydrate-based antigenic determinants or glyco-epitopes (wang and kabat ; wang ) . including carbohydrate structures of the microbial world, which are directly relevant to medicine, the sizes and diversity of the repertoires of glyco-epitopes are unpredictable. further development of carbohydrate microarrays requires libraries of carbohydrate antigens, including purified natural antigens and synthetic glycoconjugates, as well as anti-glycan mabs, lectins, and other glycan-binding proteins. naturally purified carbohydrate antigens have the advantage of preserving the native antigenic structures and often offer highly sensitive detection of antigen-specific antibodies. availability of synthetic oligosaccharides and glycoconjugates is, however, critical for epitope determination and fine-specificity studies of carbohydrateanti-carbohydrate interactions. anti-glycan mabs and gbps of known carbohydrate-binding specificities are required to characterize glyco-epitopes that are presented by carbohydrate microarrays. thus, collaborative efforts by both academic and industrial sectors are required to facilitate the establishment of large collections of glycan-targeting probes. this situation is similar to established flow cytometry technology and services. availability of specific 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was partially supported by nih grants, u ca and r ai , and sri international r&d funds (d. wang) and by an anhui institute of prenatal and postnatal care-sponsored research (j. tang). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. key: cord- -ghcwfcx authors: razanajatovo, norosoa h; nomenjanahary, lalaina a; wilkinson, david a; razafimanahaka, julie h; goodman, steven m; jenkins, richard k; jones, julia pg; heraud, jean-michel title: detection of new genetic variants of betacoronaviruses in endemic frugivorous bats of madagascar date: - - journal: virol j doi: . /s - - -y sha: doc_id: cord_uid: ghcwfcx background: bats are amongst the natural reservoirs of many coronaviruses (covs) of which some can lead to severe infection in human. african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks. a recent study in south africa isolated a genetic variant closely related to mers-cov from an insectivorous bat. though madagascar is home to bat species ( insectivorous and frugivorous) of which are endemic, no data exists concerning the circulation of covs in the island’s chiropteran fauna. certain malagasy bats can be frequently found in close contact with humans and frugivorous bats feed in the same trees where people collect and consume fruits and are hunted and consumed as bush meat. the purpose of our study is to detect and identify covs from frugivorous bats in madagascar to evaluate the risk of human infection from infected bats. methods: frugivorous bats belonging to three species were captured in four different regions of madagascar. we analyzed fecal and throat swabs to detect the presence of virus through amplification of the rna-dependent rna polymerase (rdrp) gene, which is highly conserved in all known coronaviruses. phylogenetic analyses were performed from positive specimens. results: from frugivorous bats, we detected coronaviruses from two endemic bats species, of which viruses were identified from pteropus rufus and one from eidolon dupreanum, giving an overall prevalence of . %. phylogenetic analysis revealed that the malagasy strains belong to the genus betacoronavirus but form three distinct clusters, which seem to represent previously undescribed genetic lineages. conclusions: our findings suggest that covs circulate in frugivorous bats of madagascar, demonstrating the needs to evaluate spillover risk to human populations especially for individuals that hunt and consume infected bats. possible dispersal mechanisms as to how coronaviruses arrived on madagascar are discussed. coronaviruses (covs) are enveloped viruses with singlestranded positive-sense rna belonging to the subfamily coronavirinae in the family coronaviridae (order nidovirales). genomes of covs range from to kb and show high genetic diversity [ ] . covs are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [ ] . in mammals and birds, covs are associated with upper and lower respiratory illnesses or gastroenteritis. in humans, covs infections are commonly caused by hcov- e and hcov-oc which generally cause mild respiratory illnesses [ ] . a new cov that causes severe acute respiratory syndrome (sars-cov) emerged in humans in - and infected more than , individuals with mortality rates estimated at around % [ ] . the emergence of sars-cov and its mortality rate have raised the risk of a new pandemic that could threaten public health. for this reason, the scientific community invested considerable interest in the identification and characterization of covs especially within mammal reservoirs. subsequently, two novel human covs were discovered: hcov-nl in [ ] and hcov-hku in [ ] . in june , a third novel coronavirus named hcov-emc/ (renamed mers-cov) was isolated from patients presenting with acute respiratory distress and pulmonary inflammation [ , ] . studies which aimed to identify potential reservoirs of emerging human covs have revealed that the betacoronavirus sars-cov was closely related to covs detected in bats, specifically members of the genus (rhinolophus), which brought the hypothesis of a spillover of this virus to several animal species (including civet cats and raccoons) sold in chinese markets as bushmeat for human consumption [ ] [ ] [ ] . bats have since become a particular focus and a number of alphacoronavirus and betacoronaviruses have been identified in many frugivorous and insectivorous bat species and in many countries worldwide in asia, the americas and europe (see review from drexler et al. ) [ ] . genomic characterization of the recently discovered mers-cov showed that this virus belongs to the genus betacoronavirus and seems to be closely related to bat coronaviruses hku and hku isolated from bats (tylonycteris and pipistrellus) [ ] . african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks [ ] [ ] [ ] . some authors have reported the detection of bat covs from mainland africa [ ] [ ] [ ] [ ] [ ] . a recent study in south africa detected a genetic variant closely related to mers-cov from neoromicia zuluensis, an insectivorous bat [ ] . the authors hypothesize that mers-cov may have a common ancestors with covs borne by bats from africa. though madagascar is home to bat species ( insectivorous and frugivorous) of which are endemic [ ] [ ] [ ] , no data exists concerning the circulation of covs in malagasy bats. certain bat species on the island can be frequently found in close contact with humans, particularly members of the family molossidae; these synanthropic species roost in human-occupied buildings, like houses, schools or hospitals [ ] , whilst frugivorous bats feed in the same fruit trees where people collect and consume fruits. moreover, hunting and consumption of bat bush meat, especially the larger frugivorous species of the family pteropodidae, is widespread on the island, bringing hunters, purveyors and consumers into contact with bats [ ] . in this study, we report the detection of covs amongst frugivorous bats in madagascar. our results demonstrated for the first time that covs belonging to the genus betacoronavirus are circulating amongst two endemic frugivorous bats species in madagascar. a total of bats belonging to endemic bat species of the family pteropodidae were captured and sampled: rousettus madagascariensis (n = ), pteropus rufus (n = ) and eidolon dupreanum (n = ) ( table ) . none of the throat swabs from any bat species (n = ) tested positive for cov, but . % ( / ) of fecal specimens tested positive for cov. prevalence within p. rufus, e. dupreanum and r. madagascariensis was respectively . % ( / ), . % ( / ) and % ( / ). all positive specimens originated from bats captured in the menabe region ( figure ). short amplicon sequences of bp in length of the rdrp gene were obtained for all pcr-positive animals, whereas larger fragment of bp sequences could only be obtained from seven of the pcr-positive animals. all amplicon sequences were aligned in-frame with a compilation of reference sequences from genbank for which collection-date data was available [ ] , giving final alignments containing different sequences of bp in length and different sequences of bp in length. gtr + i + g was identified as the optimal substitution model using jmodeltest v . . [ ] . multiple phylogenies were generated in beast using different combinations of model parameters, and the best models were selected using the tracer [ ] . bayes factor analysis employing marginal likelihoods, as detailed in [ ] . all parameter combinations produced identical, strongly supported tree topologies (data not shown). as has elsewhere been determined by lau et al. [ ] , bayesian skyline using a relaxed, exponentially distributed clock model was found to be the best fitting model for rdrp dated-tip phylogenies. the final phylogenetic analyses ( figure ) revealed that strains from madagascar are members of the betacoronavirus genus, rooting with hong kong strain btcov-hku (hm ) and kenyan strain ky (gu ), with posterior probabilities of . these lineages could be described as sars-like, and were uniquely affiliated with frugivorous bat hosts of the family pteripodidae. malagasy strains were sub-divided into three distinct clusters: two of which were closely related (clusters and ) and originating from p. rufus, and one more distantly related (cluster ) containing a strain detected from e. dupreanum and sequences previously detected from e. helvum in kenya [ ] . the malagasy covs were detected from bats captured in three different sites of the menabe region (west of madagascar). within cluster , strains were originated from bemanonga, mahabo and ankiliabo. within cluster , strains were originated from bemanonga and ankiliabo. the only virus detected belonging to cluster was detected in one bat captured in mahabo. overall, identities among malagasy covs ranged from to % at the nucleotide level and . to % at the amino acid level (data not shown). molecular dating estimates based on the bp fragment of the rdrp gene estimated the timescale of evolution of the coronavirus family to be thousands to tens of thousands of years, however dating estimates proved inaccurate, with broadly spanning hpd values at individual node positions. in the context of this study, we detected coronaviruses forming nine genetically distinct strains in two endemic malagasy frugivorous bat species. the overall prevalence ( . %) is consistent with those identified in studies elsewhere [ ] [ ] [ ] . thirteen viruses were detected from pteropus rufus and virus from eidolon dupreanum. we did not detect covs among the sampled r. madagascariensis. the detection of novel bat covs supports the observation that these viruses are diverse and have a nearly worldwide distribution [ , , , ] . we observed that all malagasy bat covs detected in the present study belong to a sars-related subgroup of the betacoronaviruses, with relatively close homology to btcov-hku [ ] . our strains displayed distinct clusters: clusters associated with p. rufus and cluster associated with e. dupreanum. it can be inferred from the results that multiple clusters of covs occurring within malagasy bat populations co-circulate and possibly in a syntopic manner. the high nucleotide and amino acid divergence between clusters and compared to the reference virus btcov-hku suggests previously undescribed genetic lineages. given the mobility of bats, and the especially long distances that can be travelled by colonies of fruit bats [ ] , these coronaviruses may be spread over a large region. however, host-genus-specific phylogenetic clustering (figure , inset) suggests likely host-specificity which may limit viral spill-over. thus, further molecular epidemiology studies would be required to fully understand the dispersal potential of covs amongst malagasy bats species. it is important to remember that, although all three of madagascar's fruit bat species were sampled, nothing is known of cov dynamics in tropical fruit bats, and many factors such as seasonality, bioclimate and the presence of other host species may have important influences on cov prevalence in these populations. more studies are needed in different locations including different species, particularly those with an insectivorous diet, to reveal a more comprehensive view of the diversity of these viruses in madagascar. since the strains of betacoronavirus identified from madagascar are closely related to those known from africa, some preliminary biogeographic considerations are in order. all three bat species analyzed herein are endemic to madagascar. the genus pteropus has a broad distribution from the australia-new guinea region west across the indian ocean to offshore islands of tanzania; it is unknown from the african continent. the genus eidolon is composed of two species: e. helvum occurring on the african mainland and offshore islands and e. dupreanum restricted to madagascar. based on a phylogeographical study, both species show broad panmictic population structure [ ] . further, these two taxa are estimated to have diverged from one another sometime in the late miocene or early pliocene [ ] . the genus rousettus is broadly distributed across africa and asia and the ancestral origin of the malagasy species, r. madagascariensis, is unresolved [ ] . as with the other two pteropodidae species occurring on madagascar, r. madagascariensis shows little genetic population structure and presumably broadly disperses across the island, which in turn has important epidemiological implications for these bats transmitting different zoonoses. although estimates of the most recent common ancestors (mrcas) proved inaccurate in our study, most likely as a result of a limited sequence availability from the identified viral strains, standard evolutionary analyses have estimated cov origins to date to somewhere between - , yrs. in the past [ ] [ ] [ ] . nevertheless, further investigations into the relevance of mrca prediction methodologies are required and a great level of caution must be employed in the interpretation of mrca data. . alternatively, viral lineages may have been imported to madagascar in recent history: while the vast majority of the island's bat fauna is endemic, a few species apparently disperse across the mozambique channel. probably the best example is the molossidae species mops midas, for which, based on genetic data, southern african animals are nested within malagasy populations [ ] . this bat makes its day roosts in rock crevices and may broadly occur synoptically at such sites with e. dupreanum and r. madagascariensis. these two fruit bat species are known to feed in the same fruiting trees with p. rufus [ ] , which would complete the cycle of how covs originated from africa mainland could be carried to madagascar and transmitted to different species of pteropodid bats. although we were not able to evaluate risk of human infection, the strains detected here may be considered as potential human pathogens, as bats are natural reservoirs of some pathogenic covs. isolation of malagasy covs using cell culture and molecular analysis of spike (s) gene could better evaluate risk of human infection. also, a longitudinal study amongst people who frequently handle live bats (e.g. bat hunters, bat bushmeat purveyors, and scientists), and who represent populations at higher risk of infection, would be interesting to establish possible cases of transmission to humans and public health risks. in our study, we confirm that covs are circulating in two species of endemic bats in madagascar. further work on cov diversity amongst the island's bat species, as well as aspects of the ecology and behavior of susceptible taxa, are needed to understand the origin, evolution and dispersal of these viruses across the island. to conclude, the results of our study demonstrate the need to develop research programs that aim at surveying viruses in the wild, especially in bats, in order to address possible emergence of zoonotic viruses within human populations. we sampled frugivorous bats in four different areas of madagascar: anjohibe, anosibe an' ala, menabe and toliara (table / figure ) based on known accessible colonies of roosting bats and sites where bats are frequently hunted and eaten by people. in the region of menabe, we selected different sites situated at a mean distance of km around mahabo to capture and collect specimens, while in the three other regions, sampling occurred at a single site. sampling was carried out under protocols approved and permitted by ministry of environment and forest (authorization # / , / , / , / and / ). fruit bats were captured by the use of mist-nets set near roosting sites (trees or caves) and with help of professional hunters [ ] . rectal and throat swabs were collected from each individual bat. all bats were identified according species specific morphological features well known by our field trained team (ecologist and veterinarian) and subsequently released. swabs were placed in viral transport media, almost immediately conserved in liquid nitrogen in the field and stored at − °c upon arrival at the laboratory in antananarivo. rna was extracted from specimens using the qiaamp viral rna minikit (qiagen, courtaboeuf, france) according to the manufacturer's protocol. briefly, total rna was extracted from μl of each sample and eluted in μl of qiagen ave elution buffer. the extracted rna was either immediately analyzed or stored at − °c until use. extracted rna was reverse transcribed to generate cdna by using the m-mlv reverse transriptase (invitrogen, california, usa) into a step reactions. first, μl of rna were mixed in a solution containing . μm of random hexamer primers (roche diagnostics, mannheim, germany), u/μl of rnase inhibitor ( units) and . μl of water, at °c for min, °c for min and °c for min. then, μl of rna issued from the first step was added to a mixture of . mm of each dntp (deoxynucleotide triphosphates), u/μl of reverse transcriptase m-mlv, x of buffer and . m of dtt (dithiothreitol), and incubated at °c for min and °c for min. pcr assay was performed to amplify the rna-dependent rna polymerase (rdrp) gene which is highly conserved in all known coronaviruses [ ] . the primers pair (forward ′-ggttgggactatcctaagtgtga- ′; reverse ′-c catcatcagatagaatcatcata- ′) was designed to amplify a bp fragment as described previously [ ] . reaction mixture was carried out using the gotaq/dntp mix, custom kit (promega corporation, madison, usa). briefly, μl of cdna was mixed with x of green gotaq flexi buffer, . mm of mgcl , . mm of each dntp, . μm of each primer, . u/μl of gotaq dna polymerase and . μl of water nuclease-free giving a final volume of μl. the thermocycling was performed under the following conditions: activation at °c for min and cycles of denaturation at °c for min, annealing at °c for min, extension at °c for min, and a final extension at °c for min. all negative samples were tested in a semi-nested pcr with the same pcr program and using the following pair of primers (forward ′-ggttg ggactatcctaagtgtga- ′; reverse ′-atcagata gaatcatcatagaga- ′). amplicons products were subsequently electrophorezed on a . % agarose gel and visualized using ethidium bromide under uv light. all specimens that showed a positive band at the expected size ( bp) were sequenced on both strands by beckman coulter genomics (essex, uk). from the sequences obtained from the bp, fragment, we designed new primers (reverse) that were strain specific for malagasy batcov ( ′-gatgacc tgtatattccca- ′ and ′-atgacctatacatacc catg- ). we then amplified a large fragment of the rdrp gene by using the consensus forward primer ′-gtgtacgctgctgatcctgctatgca- ′ [ ] . the following conditions were performed: °c for min and cycles of denaturation at °c for min, annealing °c for min, extension at °c for min, and a final extension at °c for min. the final size of sequences used for molecular dating was bp. sequences from the bp or bp fragments of the rdrp gene were cleaned and aligned with reference sequences collected from a literature search. alignment was performed using the translation alignment tool in geneious pro™ v. . . , created by biomatters (available from http:// www.geneious.com/), and the default clustalw cost matrix. the final alignment was respectively bp and bp in length for fragments bp and bp, and contained no free-end or internal gaps. from these alignments, the appropriate substitution model was identified in jmodeltest v. . . [ , ] . using the appropriate substitution model, * ^ iterations were run with or without the use of a base codon model, using different clock models, alternating between constant and bayesian skyline population size models, and seeding with uncorrelated log-normally distributed priors. trees were sampled every x ^ iterations, and analysis convergence was assessed in tracer v. . . [ ] (available from http://beast.bio.ed.ac.uk/tracer). all analyses converged after a % burn-in to give effective sample size values for all parameters superior to . bayes factor analyses were performed in tracer v . . , with bootstrap replicates to assess the relative performance of model selections on the generated phylogenies. after identification of an optimal model for phylogenetic classification and dating, two further independent analyses ( * ^ iterations, sampling every * ^ iterations) were run in beast, and all analyses were combined in logcombiner (beast package) with a burn-in of %, leaving only converged parameter estimates. the final phylogeny and mrca for fragments bp in length dates were extracted using treecombiner (beast package) and figtree v. . . 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methods for the study of bats identification of a novel coronavirus in bats genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was conducted in collaboration with the association madagasikara voakajy and the bangor university (darwin initiative project), a project examining aspects of emerging viruses in small wild mammals. the work in the toliara region was part of action concertée inter-pasteurienne (acip) research program. we would like to thank felicien herbert randrianandrianina of madagasikara voakajy and local hunters for their help in capturing bats. david a. wilkinson's post-doctoral fellowship was funded by "run-emerge", a european project funded by european commission under fp program. the authors declare that they have no competing interest.authors' contributions nhr and daw carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. lan coordinated the fieldwork, participated to the sampling collection, participated to molecular testing and drafted the manuscript. jhr participated in the design of the study and coordinated the fieldwork smg and rkj helped to draft the manuscript. jpgj participated in the design of the study and helped to draft the manuscript jmh: conceived, designed and coordinated the study and helped to draft the manuscript. all authors read and approved the final manuscript key: cord- -zz w ro authors: beermann, sandra; allerberger, franz; wirtz, angela; burger, reinhard; hamouda, osamah title: public health microbiology in germany: years of national reference centers and consultant laboratories date: - - journal: int j med microbiol doi: . /j.ijmm. . . sha: doc_id: cord_uid: zz w ro in , in agreement with the german federal ministry of health, the robert koch institute established a public health microbiology system consisting of national reference centers (nrcs) and consultant laboratories (cls). the goal was to improve the efficiency of infection protection by advising the authorities on possible measures and to supplement infectious disease surveillance by monitoring selected pathogens that have high public health relevance. currently, there are nrcs and cls, each appointed for three years. in , an additional system of national networks of nrcs and cls was set up in order to enhance effectiveness and cooperation within the national reference laboratory system. the aim of these networks was to advance exchange in diagnostic methods and prevention concepts among reference laboratories and to develop geographic coverage of services. in the last two decades, the german public health laboratory reference system coped with all major infectious disease challenges. the european union and the european centre for disease prevention and control (ecdc) are considering implementing a european public health microbiology reference laboratory system. the german reference laboratory system should be well prepared to participate actively in this upcoming endeavor. public health microbiology laboratories play a central role in detecting infectious disease, monitoring outbreak response and providing scientific evidence to prevent and control disease. they have important roles and responsibilities associated with accurate diagnosis, resistance testing and prevention of the spread of infectious disease. for example, outbreak investigations often depend on confirming cases by methods that are not commonly available in a routine laboratory setting. the scientific community, policy makers and pharmaceutical companies rely on advice and information from reference laboratories in order to adjust vaccine and antibiotic production (witze et al., ) . according to the european centre for disease prevention and control (ecdc), the five key activities of public health microbiology reference laboratories are reference diagnostics; reference material resources; scientific advice; collaboration and research; and monitoring, alerting and responding (european centre for disease prevention and control, ) . in the various european countries, microbiology reference laboratories are defined, organized, maintained and operated differently. we present an overview of germany's public health reference laboratory system. germany is a highly industrialized country with million inhabitants, and is made up of federal states ("länder"). the principal responsibility for public health lies with the states, or with their ministries of health, and with the almost local public health departments. since the s, the federal government ("bundesregierung"), federal assembly ("bundestag") and federal council ("bundesrat") have increasingly taken responsibility for healthcare reform and legislation. specific health issues, such as infectious diseases that threaten public safety and life cycle management of pharmaceuticals, are within the jurisdiction of the federal government. for example, the german protection against http://dx.doi.org/ . /j.ijmm. . . - /© elsevier gmbh. all rights reserved. infection act ("infektionsschutzgesetz," ifsg) as a federal law regulates the prevention and management of infectious diseases in humans. federated states are responsible for all primary aspects of public health, but there are also responsible for the implementation of federal laws, including federal social and labour laws. the robert koch institute (rki) is a federal institute within the portfolio of the federal ministry of health (bundesministerium für gesundheit, bmg). as such the rki is the central federal reference institution in the public health sector responsible for disease monitoring, control and prevention and conducting applied and response-oriented research in the field of disease control and prevention at the federal level. the research activities of the rki are partly directly related to the activity fields of a ministry. although robert koch and his contemporaries built a strong tradition for infectious disease epidemiology in germany in the late th and early th centuries, this tradition had all but disappeared in the s and s (allerberger, ) . in former west germany, the work of the rki as part of the then federal health office (bundesgesundheitsamt, bga) mainly focused on basic science research. the aids epidemic demanded a national public health response which resulted in the creation of the national aids centre in . in , when the bga was dissolved and the rki was assigned additional spheres of competence a combined aids center and infectious disease epidemiology division was created at the rki. in , representatives of the rki, the federal ministry of health and the federal ministry for education and research developed the concept of a network of collaborators whose goal was to intensify epidemiological research and improve infectious disease surveillance (fock et al., ) . as part of this concept, the rki implemented a weekly epidemiological bulletin, formed the committee for infectious disease epidemiology, trained epidemiologists for surveillance and outbreak investigation and set up a system of national reference laboratories: national reference centers (nrcs) and consultant laboratories (cls) (petersen et al., ) . they were responsible for laboratory surveillance of important pathogens and syndromes. these laboratories are considered national centers of excellence in the field of laboratory science for a particular pathogen or group of pathogens. nrcs establish and use reference methods, and can validate and verify test results from other laboratories (confirmatory testing). nrcs also produce and distribute reference materials for external quality control and assurance. owing to the high level of expertise, resources and infrastructure, nrcs and cls are involved in training and in providing expert advice to national health authorities and other laboratories. moreover, these laboratory scientists work closely together with their epidemiologist counterparts at the rki as well as those at the federal, state and local levels. the nrcs focus on outbreak detection and response and advice the rki in the preparation of case definitions according to the protection against infection act (ifsg). furthermore, the reference laboratories conduct or are involved in laboratory surveillance systems which provide additional information complementing statutory notifications. nrcs and cls are also involved in developing rki guidelines for physicians ("ratgeber für Ärzte") as well as investigating outbreaks and conducting epidemiological studies. the following are the basic tasks of nrcs and cls, which include detailed requirements referring to specific pathogens or syndromes as listed in the respective calls for tenders: general catalogue of nrc tasks ( ) developing or improving diagnostic procedures; coordinating standardization and distribution of generally accepted test procedures; initiating investigations for quality assurance. ( ) diagnosing and subtyping pathogens beyond routine measures, including molecular biological studies to elucidate the epidemiological context. ( ) maintaining a strain collection and distributing reference strains or diagnoses of specific reference strains, with the exception of commercially available isolates, such as from the american type culture collection (atcc) and the german collection of microorganisms and cell cultures (dsmz). ( ) organizing and coordinating the upkeep of a network of diagnostic facilities. ( ) providing a consulting service for public health services laboratories, practicing physicians, hospitals and research institutes; implementing continuing education and handling public relations. ( ) collaborating with reference laboratories of other countries as well as collaborating centers of the who, including participating in international ring trials. ( ) evaluating and interpreting data in coordination with the rki with the aim of best describing the epidemiological situation relevant for germany; initiating and participating in surveillance projects. ( ) monitoring incoming data with the goal of timely detection of outbreaks or outbreak hazards as well as immediate communication with the rki; support of public health services and the rki with complementary studies during outbreak investigations. ( ) epidemiological analysis and evaluating the development of resistance and virulence. ( ) reporting routinely to and consulting with the rki on relevant issues; participating in developing rki recommendations for diagnostics, therapies and prevention as well as for applied epidemiology of infectious diseases in general. general catalogue of cl tasks . consulting (especially with the public health services as well as laboratories, practicing physicians, hospitals and research institutes). . working within the framework of quality assurance (participating in studies and inter-laboratory tests, e.g., in cooperation with instand (german eqas), who, eu, and professional associations and participating in further education). . improving or developing diagnostic procedures. . participating in epidemiological evaluations of the current situation by the rki. . carrying out studies within the network of diagnostic facilities. . consulting with the rki in developing scientific materials concerned with pathogens or symptoms (e.g., case definitions, rki guidelines for physicians). the number of nrcs increased from in to in . presently, nrcs have been appointed (table ) . five laboratories are situated at the rki; the others are located at various universities and research facilities in germany. since , cls have decreased to designated cls, mainly devoted to providing scientific advice (table ) . currently a total of nrcs and cls located at universities, federal or state institutes and private laboratories are supported for this function by the rki. the high relevance of nrc and cl work for the surveillance of infectious diseases is evident by the wide range of national and international publications. for example, the nrc for mycobacteria and the rki performed analyses of routine laboratory diagnosis data of pediatric tuberculosis in the european union/european economic area (sanchini et al., ) . the nrc for helicobacter pylori and the rki examined h. pylori resistance to antibiotics in europe and its relationship to antibiotic consumption (megraud et al., ) . another example is the work of the streptococci nrc, which studied the epidemiology of streptococcus pneumoniae serogroup isolates from invasive pneumococcal disease in children and adults in germany (van der linden et al., ) . nrcs and cls are also involved in outbreak investigations and epidemiological studies. for instance, the cl for coronaviruses performed contact investigation for an imported case of middle east respiratory syndrome (reuss et al., ) , and the influenza nrc was involved in detecting local influenza outbreaks (schweiger and buda, ) . the rki and the nrc for surveillance of nosocomial infections examined the question, "how many outbreaks of nosocomial infections occur in german neonatal intensive care units annually?" (schwab et al., ) . additionally, the cl for legionella was involved in examining a legionnaires' disease outbreak associated with a cruise liner in august (beyrer et al., ) . dengue virus infections in a traveler returning from croatia to germany were analyzed by the nrc for tropical infection agents (schmidt-chanasit et al., ) . the nrc and the rki for meningococcal diseases and h. influenzae examined a cluster of invasive meningococcal disease in young men who have sex with men in berlin (marcus et al., ) . nrcs and cls are also involved in evaluating implemented vaccination recommendations and analyzing the effectiveness of the vaccines (kalies et al., ; ruckinger et al., ). for which pathogen a reference laboratory is to be established is decided based on the public health relevance of the pathogen as appraised by the rki and on the needs expressed by the national public health services ("Öffentlicher gesundheitsdienst," Ögd) . in addition, medical professional societies, the federal ministry of health and other third parties can approach the rki with perceived needs for additional reference laboratories. in the next step, the advisory board for public health microbiology (formerly called the committee for infectious disease epidemiology) assesses the proposal and provides the rki with a recommendation on whether to set up a new laboratory. in addition to the epidemiological relevance, and a declared need from national public health services, the availability of financial resources is another essential criterion. the decision to establish or continue an nrc or a cl is made by the rki, which considers recommendations given by the advisory board for public health microbiology, and must be confirmed by the federal ministry of health. appointments are restricted to three-year periods. the advisory board consists of up to experts, appointed by the rki for periods of three years. the members of this advisory forum are renowned experts in the fields of microbiology, virology, hygiene, epidemiology and public health. occasionally, other national and international professional societies and experts are consulted to achieve a solid appraisal of the candidate laboratories. from to , numerous important modifications were made to improve the transparency of the tendering and selection processes for the nrcs and the cls. a strict prioritization process, based upon necessity and not upon offer, was implemented. the evaluation process became more rigorous. essential evaluation criteria are public health needs and public health relevance, successful network activities, attestable quality assurance, publications as well as a positive appraisal of the advancement of diagnostic procedures. at the end of each appointment period, an evaluation of the laboratories is performed by the rki in cooperation with the advisory board for public health microbiology, which again consults national and international professional societies and experts. based on the evaluation results, the president of the rki, in cooperation with the federal ministry of health, appoints and reappoints the nrcs and cls. the evaluation of the cls resulted in the reappointment of cls and the shutdown of nine cls. reasons for closing were, for example, the retirement of the laboratory head (appointments are based on the combination of personal and institutional expertise), decreased public health relevance of the pathogen or an overlapping of the functional areas of responsibility with other cls or nrcs. in the evaluation of the nrcs, all nrcs were reappointed. in , the nrcs were supported with d , in total. in , the available funding increased to d , . the nrcs received between d , and d , per year. the decision on the level of funding of individual nrcs is made by the rki, based on criteria such as high consultation effort, high sample appearance and extraordinary public health relevance of the pathogen. in contrast to the nrcs, which have always been financially supported, the cls initially performed their work (mainly consultation) without any financial support. from april to december , the cls received basic funding of d per year (total amount in : d , ). in , the available funding increased to d , . the increase in funding was used to upgrade the cls' basic funding to around d , per year. beyond that, the funds had to cover new national networks. in , the nrcs received between d , and d , per year. thirty-three of the cls get d , , and seven cls with a high number of samples and extraordinary public health relevance of the pathogen received d , per year. the network projects were funded with approximately d , in . the nrcs have a more comprehensive work package than the cls and therefore the nrc receive a higher funding. public funding does not and cannot cover the total costs of the reference laboratories. since , the sphere of action and the workload of the laboratories have increased due to advancement of methods. at the same time costs in general have increased, but the grant total has remained unchanged. currently, funding increases for individual laboratories can occur only through money shifting from one laboratory to another or through giving up funding for existing nrcs or cls. in order to maintain the current quality and scope an increase in funding for nrcs and cls is urgently needed. since , the rki, in close cooperation with the advisory board for public health microbiology, has worked to foster collaboration between and among the nrcs and the cls. this concept was amended in a workshop with representatives of public health microbiology laboratories of other eu member states in . ten nrc networks were launched at a work conference of the standing working group of nrcs and cls ("ständige arbeitsgruppe nrz/kl") in stuttgart in . these networks covered the following topics: respiratory tract infections; enteral infections; infections in patients with immune deficiency or pregnancy; invasive bacterial infections; zoonoses; mycoses; sexual and blood transmitted infections; infections of the nervous system; antimicrobial resistance; and parasitoses, tropical and vector-borne infections. the aim of these networks was to facilitate the exchange of diagnostic methods among the nrcs and cls, to improve collaboration in planning and performing studies and to enlarge the geographic coverage of these services. furthermore, these networks should provide opportunities to work on issues beyond single pathogens. scientific coordination and administration are supervised by the rki. the advisory board for public health microbiology and external experts play a pivotal role in selecting the proposed network projects. essential selection criteria are public health relevance and the scientific quality of the proposal, the prospect of success and the cost efficiency of the planned network project. moreover, it is important that these projects contribute to expanding the network's characteristics. an exclusion criterion is if the project addresses established nrc or cl tasks. in , the network projects were funded with d , per year, allocated to ten projects (duration . years). as of , network projects ran for three years. in the funding period - , the rki supported eight projects. within the scope of the projects, common database infrastructures were set up, such as tissue material and serums. other projects performed cross-sectional studies to ascertain data on the prevalence and incidence of different pathogens. in , the rki evaluated the present composition and structure of the networks. the evaluations revealed that difficulties with ethical approval and with compliance with data protection and juridical aspects were the most commonly experienced hurdles during the study planning process. recruiting participating laboratories was also a challenge for some projects. it became clear that early involvement of epidemiological and statistical experts is necessary to further optimize study design and case number planning for the specific research questions and to raise the prospects of success for the projects. in addition to this evaluation, the rki organized a network meeting in , in which all nrcs and cls were represented. members of the advisory board for public health microbiology and representatives of the federal ministry of health participated. at this meeting, the networks shared their experiences. potential for improvement from the perspective of the members of the nrcs and cls as well as from the rki and the advisory board for public health microbiology was identified and discussed. as a consequence of this meeting, the rki initiated the following changes: ( ) regular network meetings with all nrcs and cls to address the stated need for regular face-to-face meetings, the rki will organize network meetings every three years. the meetings will take place one year before the start of the upcoming funding period for network projects, so that the nrcs, the cls and the rki can elaborate on the content and structure of project submissions. ( ) basic funding for the networks the rki will provide annual basic funding to allow for separate meetings of the respective networks, to facilitate exchange among network participants regardless of successful project applications. these meetings can be used for more intensive preparation of new project proposals and should strengthen network cohesion. ( ) stronger presence of the networks on the internet to satisfy network demand for the presentation of network projects to a larger professional audience, accepted projects will be presented on the rki's internet site. ( ) decrease in the number of funded projects in the past, the rki funded up to eight projects; in , the institute decided to decrease the number of projects funded per period in the future. in the current funding period four projects were selected for funding. in the following period only two projects will be financed. that implies higher financial support for single projects, which could be used to employ a study coordinator. ( ) two-stage application procedure for network projects the rki installed a two-stage application procedure for network projects. as the first step, the networks formulate short pre-applications. the rki screens these short concepts for network projects with the help of the advisory board for public health microbiology and external experts. in the case of positive assessment, the networks are asked to submit a detailed project proposal for final evaluation. during the last years, the field of public health microbiology has seen many changes. the everyday work of local public health agencies depends on the professional expertise of national reference centers (nrcs) and consultant laboratories (cls). meanwhile, the public often sees the relevance of public health microbiology only within the context of serious health events. during periods of restricted financial resources, the need for public health infrastructures is consistently questioned. the large ehec o outbreak in hamburg during provides an example of the importance of public health laboratory infrastructures (frank et al., ) . during the hamburg outbreak caused by fenugreek sprouts, the german public health system successfully investigated and controlled the outbreak, which would not have been possible without support from the nrc for enteral infections and the cl for hemolytic uremic syndrome (hus). this support would not have been possible without these highly specialized laboratory structures. the work of all other nrcs and cls is also highly relevant, since their work and expertise help in the efforts to contain and prevent higher levels of infectious disease. nevertheless, there is also room for improvement in germany. for example, the anticipating of new outbreak situations that might require cooperation with the responsible veterinarian and food authorities or with other national authorities should be the focus of optimization plans. the creation of a prospective network of eu-wide public health microbiology reference laboratories is currently being discussed within the european union, which will have consequences for the public health laboratory systems of each member state. from this perspective, considerable future challenges to the german public health laboratory system can already be foreseen. thus, the structures established during the past years should be adaptable so that the responding public health infrastructures can react adequately to the upcoming challenges. sandra beermann, franz allerberger, angela wirtz, osamah hamouda and reinhard burger have no financial disclosures to declare. structural requirements and conditions for effective microbiological diagnostics in disease outbreaks legionnaires' disease outbreak associated with a cruise liner core functions of microbiology reference laboratories for communicable diseases epidemiology of infection in germany large and ongoing outbreak of haemolytic uraemic syndrome prioritisation of infectious diseases in public health: feedback on the prioritisation methodology invasive haemophilus influenzae infections in germany: impact of non-type b serotypes in the post-vaccine era a cluster of invasive meningococcal disease in young men who have sex with men in berlin helicobacter pylori resistance to antibiotics in europe and its relationship to antibiotic consumption developing national epidemiologic capacity to meet the challenges of emerging infections in germany contact investigation for imported case of middle east respiratory syndrome reduction in the incidence of invasive pneumococcal disease after general vaccination with -valent pneumococcal conjugate vaccine in germany laboratory diagnosis of paediatric tuberculosis in the european union/european economic area: analysis of routine laboratory data dengue virus infection in a traveller returning from croatia to germany how many outbreaks of nosocomial infections occur in german neonatal intensive care units annually? detection of local influenza outbreaks and role of virological diagnostics epidemiology of streptococcus pneumoniae serogroup isolates from ipd in children and adults in germany scientific advice: crisis counsellors we thank all nrcs and cls for their excellent work during the last years. key: cord- -en d bj authors: witte, tobias title: ressourcenknappheit und verteilungsgerechtigkeit im seuchenfall date: - - journal: medizinrecht doi: . /s - - -x sha: doc_id: cord_uid: en d bj nan in ihrem grünbuch identifizierte die kommission diverse hindernisse für die ausschöpfung des entwicklungspotenzials von mhealth-lösungen. klärungsbedarf besteht insbesondere hinsichtlich medizinprodukterechtlicher abgrenzungsfragen und der sicherheitsanforderungen an apps. zur schaffung von investitionsanreizen ist ferner die kostenerstattungsfrage zu beantworten. die interoperabilität zwischen mhealth-produkten und gesundheitsnetzen ist eine technische wie rechtliche aufgabe. die stellungnahmen der interessenvertreter zum grünbuch zeigen unterschiedliche strategien hinsichtlich der regulierung auf. die vorschläge reichen von der schaffung verbindlicher abgrenzungsbestimmungen und sonderregelungen für lifestyle-und wellbeing-apps über die prüfung medizinischer apps durch eine benannte stelle bis hin zur regulierung von apps für das persönliche wohlbefinden . angelehnt an die us-regulierung wird ferner vorgeschlagen, die Überwachung von apps mit marginalem gesundheitsrisiko in das ermessen der behörden zu stellen . die sicherheit und das anwendervertrauen könnten private und öffentliche zertifizierungsprogramme stärken. beispielhaft ist eine sicherheitsprüfung durch die britische nhs health apps library, deren register mittlerweile über apps umfasst . hinsichtlich datenschutzrechtlicher anforderungen bietet der tÜv rheinland eine freiwillige zertifizierung an . zur sicherung gesundheitsbezogener daten wird die standardisierung technologischer verschlüsselungs-und authentifizierungsverfahren gefordert . der referentenentwurf zur umsetzung der telematikinfra struktur der gesetzlichen krankenversicherung spart rechtsfragen der einbindung von mhealth-apps in die regelversorgung der gkv noch aus. das gesetzgebungsverfahren bleibt abzuwarten. zu verfolgen bleibt auch, ob und in welchem ausmaß die ergebnisse der europäischen konsultation in ein gesetzgebungsverfahren einfließen und in wie weit das nationale fernbehandlungsverbot dem us-amerikanischen usus entsprechend den technischen möglichkeiten weichen wird. zu erwähnen ist schließlich, dass das aktuelle thema behördlicherseits aufgegriffen worden ist und das bfarm im märz im rahmen der "bfarm im dialog: medical apps"-veranstaltung chancen wie auch risiken gemeinsam mit vertretern aus anwendung, forschung, wirtschaft und politik diskutiert hat . in europa traten in den letzten jahren sars , die sog. schweinegrippe und ehec auf, in westafrika wütet seit monaten eine verheerende ebola-epidemie und madagaskar verzeichnet einen neuerlichen ausbruch der pest: die seuchen kehren zurück . hauptgrund dafür ist die mit der globalisierung einhergehende stärkere vernetzung der welt, die aufgrund der hohen menschlichen mobilität zu einer nie dagewesenen steigerung der weltweiten infektionsdynamik führt . dadurch werden infektionsrisiken, die immer schon bestanden, von der lokalen und regionalen auf die globale ebene getragen. zu diesen klassischen infektionsrisiken gehören zum einen erhebliche hygienedefizite, vor allem in südamerikanischen und asiatischen megacities . hinzu kommen zum anderen primär in entwicklungsländern fragwürdige tierzucht-und tierhaltungspraktiken . ein weithin unterschätztes problem ist ferner die traditionelle jagd auf "bushmeat", also auf solche tierarten in dschungelgebieten, durch die eine Übertragung neuartiger krankheitserreger vom tier zum menschen möglich ist . auch jenseits der ebola-epidemie stellt sich beispielsweise für infektionskrankheiten, zu deren bekämpfung bereits impfstoffe oder antivirale arzneimittel entwickelt wurden, zum einen die frage, in welchem umfang diese gegenmittel präventiv bevorratet werden. zum anderen ist in jeder seuchenbedingten knappheitssituation, sei es eine knappheit von impfstoffen oder von qualifizierten rettungswagen, das problem zu klären, anhand welcher priorisierungsregelung das kernproblem des allokationsmaßstabs de lege ferenda liegt darin, dass gesundheitsleistungen -beispielsweise antipandemische impfstoffe , zugang zu isolierstationen, antivirale arzneimittel -einer patienten-bzw. bevölkerungsgruppe aufgrund der knappheit zwingend versagt werden müssen, während eine andere gruppe sie erhält. im schlimmsten fall bedeutet das vorenthalten der seuchenschutzressourcen die der knappheit geschuldete faktische inkaufnahme des todes von personen der anderen gruppe. diesem grundproblem muss sich auf rechtsethischem und verfassungsrechtlichem wege genähert werden. fraglich ist, welches primärziel der staat mit der verteilung im katastrophenfall verfolgen darf und soll. als leitmotiv wird regelmäßig die maximierung genannt, also die möglichst umfassende erhöhung der Überlebendenzahl . dieses sog. "save the most lives"-prinzip erfreut sich allge-mein einer breiten, kritischen rechtsphilosophischen und medizinethischen diskussion , bedarf für den sonderfall einer schweren seuche aber spezifischer anpassungen. bei pandemien handelt es sich um soziale grenzsituationen mit potentiell extremen opferzahlen; entsprechend hoch ist auch die belastung des gesundheitswesens, insbesondere des krankenhaussektors , sowie der sonstigen infrastruktur. daher liegt im pandemiefall die vorrangige immunisierung des medizinischen personals nahe . diese "rettung der retter" ist wesentliches merkmal einer praktischen anwendung der maximierungsformel. andere priorisierungssysteme wie beispielsweise der sog. "birkenhead drill" ("frauen und kinder zuerst") oder das dringlichkeits-bzw. "save the worst off"-prinzip sind jedoch in knappheitssituationen ebenso denkbar . gegen die anwendbarkeit des "save the most lives"-prinzips spricht, dass eine erhöhung von menschenleben immer ein "gegeneinanderaufrechnen" bedeute, was aufgrund des verfassungsrechtlichen saldierungsverbots (art. abs. gg) und des grundsatzes der lebenswertindifferenz (art. gg) uneingeschränkt unzulässig sei . . grundrechte als subjektive rechte seien rechtstheoretisch hingegen strikt antiutilitaristisch konzipiert . für eine anwendung der maximierungsformel im seuchenfall spricht zunächst die fassung des art. abs. s. , gg. im wege der wortlautauslegung muss nüchtern festgehalten werden, dass auch das grundrecht auf leben einem einfachen gesetzesvorbehalt unterliegt. hiermit einhergehend hat das bverfg "das menschliche leben als ‚einen', richtigerweise aber nicht als ‚den' höchstwert klassifiziert" . es gibt güter von verfassungsrang, die auf gleicher ebene stehen wie das leben des einzelnen . taupitz nennt hier bezugnehmend auf einen beschluss des bverfg zur fortsetzung der strafvollstreckung gegen freigepresste straftäter beispielsweise die funktionsfähigkeit der strafrechtspflege . hieran zeigt sich, dass das lebensrecht, entgegen vielfachen anderslautenden Äußerungen, de facto nicht absolut geschützt ist. gesetzliche grenzen findet es beispielsweise bereits bei der notwehr oder dem notstand sowie im finalen rettungsschuss . als weiteres gut von verfassungsrang, das auf ebenso hoher ebene angesiedelt werden muss wie das leben, ist die funktionsfähigkeit des rechtsstaats an sich zu nennen. pawlik spricht in diesem zusammenhang von der "existenz bzw. der verfassungsrechtlichen identität des staates selbst" . um nichts anderes geht es im extremfall einer schwerwiegenden pandemie. die argumentationslinie gegen die maximierungsformel ist zu entkräften, indem man sich dem problem mit einem praktischen blick nähert: stellt der staat eine priorisierung für den zugang zu impfstoffen auf, wird damit festgelegt, welche gruppen eine begünstigung erhalten und damit aus dem gleichsam natürlichen lauf der pandemischen situation herausgehoben werden. unabhängig davon stehen die anderen, posteriorisierten personen: ihnen geschieht nichts anderes als ohne das aufstellen einer irgendwie gearteten rangfolge . der zweck der priorisierung infolge einer anwendung der maximierungsformel zielt einzig auf die damit zu gewährleistende einsatzfähigkeit der retter ab; eine instrumentalisierung der posteriorisierten ist damit nicht gegeben. diejenigen, die nicht (sofort, aber später) beispielsweise geimpft werden, werden gerade nicht zum staatlichen mittel degradiert. es findet gerade kein behördlich angeordneter abschuss unschuldiger statt, der unbestritten eine mit der menschenwürdegarantie des art. abs. gg unvereinbare staatliche handlung darstellen würde. vielmehr wird in einem schweren seuchenfall staatlicherseits in ausübung der schutzpflicht versucht, durch möglichst lange aufrechterhaltung der infrastrukturellen ordnung einen in der extremsituation noch maximal möglichen rechtsgüterschutz für die gesamtgesellschaft zu gewährleisten . ist die steigerung der Überlebendenzahl das leitmotiv der ressourcenverteilung im pandemiefall, lassen sich daraus konsequenzen für die dabei anzulegenden konkreten verteilungskriterien ziehen. verfehlt wäre es jedoch, aus der hier dargestellten verfassungsmäßigkeit der maximierungsformel abzuleiten, dass im seuchenfall unterschiedslos alle allokationskriterien, die die maximierung fördern, per se verfassungsgemäß seien . im juristischen und medizinethischen schrifttum wird eine große zahl an grundsätzlich denkbaren verteilungskriterien diskutiert , die von der "sozialen wertigkeit" über den zufall , das alter , die "qualitätsorientierte lebensjahrerwartung" bis hin zur "erwartbaren Überlebensdauer" reichen. bereits an den bezeichnungen wird deutlich, dass die meisten dieser kriterien zur rechtfertigung einer ungleichbehandlung i. s. des art. abs. gg keine sachlichen gründe darstellen können . anders ist die situation bei folgenden kriterien zu bewerten: die wichtigkeit für gesundheitswesen und öffentliche ordnung (schlüsselpersonal), die medizinische dringlichkeit (risikogruppen) und die förderung der infektionszahlen (ansteckungsmultiplikatoren) sind kriterien, die sowohl das erfordernis der Überlebendenzahlerhöhung umsetzen als auch verfassungsmäßigen anforderungen genügen. dabei handelt es sich um rahmenkriterien, deren gesetzliche normierung einen orientierungsmaßstab für die detaillierte ressourcenverteilung im konkreten katastrophenfall darstellen könnte. dem oben bereits angesprochenen schlüsselpersonal, also vor allem dem medizinischen personal sowie, im rang nachfolgend, sonstigem infrastrukturpersonal (polizei, feuerwehr, katastrophenschutz) müsste eine solche verteilungsregelung den höchsten rang einräumen. die priorisierung des schlüsselpersonals trägt bereits den keim der vermeidung des weiteren sterbens in sich. der not geschuldet muss der staat eine belastende auswahlentscheidung treffen, und die bestmöglich zu treffende entscheidung ist die, die die not am effektivsten behebt, um den normalzustand und die größtmögliche grundrechtsgewährleistung für alle schnellstmöglich wieder zu erreichen. effektiv bei der behebung der notlage durch sicherstellung elementarer gesundheitlicher und infrastruktureller dienste können nur diejenigen personen sein, deren profession diese dienste sind. es handelt sich daher nicht um eine unzulässige materiale lebensbewertung hinsichtlich einer irgendwie gearteten "sozialen wertigkeit", sondern vielmehr um eine sachlich begründete auswahl anhand des instrumentellen werts der priorisierten schlüsselpersonen . b) risikogruppen die risikogruppen, also diejenigen personengruppen, bei denen ein schwerer verlauf der infektionskrankheit medizinisch am wahrscheinlichsten ist, könnten an zweiter stelle der allokationsreihenfolge verortet werden. das dabei zugrunde liegende differenzierungskriterium ist die medizinische dringlichkeit, also die gefährdungslage . die staatliche schutzpflicht ist grundsätzlich umso intensiver berufen, je höher die gefährdungslage einzustufen ist . für gutmann ist daher die gefährdungslage des patienten das "primäre kriterium der priorisierung" in der allgemeinen gesundheitsversorgung . ein außergewöhnlicher katastrophenfall wie eine pandemie, bei der die öffentliche gesundheit und das staatswesen per se bedroht sind, vermag eine temporäre abkehr von der zugrundelegung der reinen medizinischen dringlichkeit zur erreichung des allokationsziels -steigerung der Überlebendenzahlzu rechtfertigen. vor diesem hintergrund stellt sich das dringlichkeitsprinzip als logische konsequenz des allokationsziels dar: wer möglichst viele leben retten will, muss da ansetzen, wo die zu rettenden leben am ehesten bedroht sind. die versorgung der risikogruppen nach dem schlüsselpersonal und vor den infektionsmultiplikatoren ist vor diesem hintergrund nicht willkürlich i. s. des art. abs. gg. den "personen in einrichtungen mit umfangreichem pu blikums verkehr sowie personen, die als mögliche infektionsquelle für von ihnen betreute ungeimpfte risikopersonen fungieren können" spricht der gemeinsame bundesausschuss (gba) bereits in normalzeiten einen anspruch auf Übernahme der kosten für schutzimpfungen gegen die saisonale influenza gem. § d abs. sgb v zu. wenn diese personengruppen bereits innerhalb der bekämpfung der alljährlichen saisonalen influenza gesondert behandelt werden, dann muss dies erst recht für die bekämpfung einer pandemischen infektionskrankheit gelten, potenzieren sich doch in diesem fall die gefahren. die epidemiologische wirksamkeit einer hochrangigen impfung der genannten multiplikatoren konnte durch mathematische modelle zudem für die "asia-pandemie" und für die "hong kong-pandemie" - in den usa nachgewiesen werden . dem allokationsziel der minimierung der krankheitslast und sterblichkeit ist durch eine vergabe von vakzinen dort, wo die seuchenspezifische dynamik zu einer erhöhten ausbreitung der krankheit führt, sehr viel besser gedient als in der normalbevölkerung. die unterschiede zwischen beiden gruppen sind wie gezeigt erheblich, sodass eine abwägung zwischen den gruppenunterschieden und der intensität der ungleichbehandlung vor dem hintergrund des art. abs. gg im ergebnis zur rechtfertigung dieser ungleichbehandlung führen muss. nach schlüsselpersonal, risikogruppen und infektionsmultiplikatoren wäre an vierter stelle die restliche bevölkerung zu versorgen. es handelt sich bei den obigen ausführungen um einen auf verfassungsrechtlichen erwägungen basierenden vorschlag für ein zulässiges allokationsschema. beinahe wichtiger noch als die detailfragen der priorisierung ist jedoch der umstand, dass -zur vermeidung von rechtsunsicherheit und länderübergreifendem verteilungschaos -überhaupt eine bundeseinheitliche gesetzliche normierung der allokation knapper lebensrettender ressourcen im seuchenfall erfolgt. aufgrund der oben angerissenen rechtsunsicherheit der pandemiepläne in bund und ländern sollte die zu schaffende verteilungsregelung nicht den rechtscharakter eines plans haben. eine verbindliche vorabnormierung der generellen ressourcenverteilung als orientierungsrahmen, vorzugsweise in einer anlage zum ifsg, ist zu favorisieren . dieser rahmen müsste dann durch die festlegung der risikogruppen für die jeweilige infektionskrankheit soweit nötig durch ein an selber stelle gesetzlich legitimiertes gremium aktualisiert werden . aufgrund erheblicher regelungsdefizite, vor allem in den bereichen bevorratung, verteilung und kostentragung ist die deutsche rechtsordnung auf einen schweren seuchenausbruch derzeit nicht vorbereitet. in einem solchen katastrophenfall wäre das verfassungsrechtlich zulässige und erforderliche gebot der stunde die maximierung der Überlebendenzahl. daraus ergeben sich wiederum folgeerwägungen zur kriteriengeleiteten verteilungsgerechtigkeit, an deren ende die normierung eines allokationssystems stehen sollte. festzuhalten ist, dass die ansteigende gefahr schwerer seuchen und die vernachlässigung, die das thema bisher in der katastrophenschutz-und medizinrechtlichen diskussion erfährt, in keinem verhältnis stehen. anmerkungen zum katastrophenrecht, s. ff.; hartleb, njw der begriff stammt aus der napoleonischen kriegsmedizin und meint die aussortierung verletzter mit dem ziel, die gesamtkampf kraft der truppe zu erhöhen grundlagen einer gerechten organverteilung wer bekommt die knappen arzneimittel?, in: fs f. fischer, , s. , bezugnehmend auf bverfge , der schutz des lebens ist nicht in dem sinne absolut geboten, daß dieses gegenüber jedem anderen rechtsgut ausnahmslos vorrang genösse ob im falle einer erpresserischen geiselnahme der forderung der entführer […] zum schutze des lebens der geisel entsprochen werden soll, haben die […] staatlichen stellen in eigener verantwortung zu entscheiden; die verfassung schreibt ihnen in solchen fällen prinzipiell keine bestimmte entschließung vor ff.; vgl. auch hartleb, njw zum rechtsethischen unterschied zwischen töten und sterbenlassen vgl. auch instruktiv green, american philosophical quarterly zum aus der staatlichen schutzpflicht fließenden gebot des maximalen rechtsgüterschutzes s. auch brech influenzapandemie: wer bekommt die knappen arzneimittel? zur kriterienbildung innerhalb verschiedener priorisierungssysteme vgl. schmitz=luhn, priorisierung in der medizin entschuldigung durch notstand schmitz=luhn/bohmeier (hrsg.), priorisierung in der medizin -kriterien im dialog schöffski/von der graf schulenburg (hrsg.), gesundheitsökonomische evaluationen, s. ff zur verfassungsrechtlichen bewertung verschiedener verteilungskriterien im seuchenfall vgl. witte, recht und gerechtigkeit im pandemiefall schlüssel-)person differenzierend white/katz/luce u. a., annals of internal medicine , f.; emanuel/wertheimer, who should get influenza vaccine when not all can? schmitz=luhn/bohmeier (hrsg.), priorisierung in der medizin -kriterien im dialog kerngedanke der zur skizze einer rechteorientierten theorie der gesundheitsversorgung anlage der richtlinie über schutzimpfungen nach § d abs zur umsetzung einer seuchenspezifischen verteilungsnorm vgl denkbar ist hier die beim rki angesiedelte ständige impf kommission (stiko) key: cord- -pdxm iiw authors: xiong, siping; tang, qi; liang, xudong; zhou, tingting; yang, jin; liu, peng; chen, ya; wang, changjun; feng, zhenqing; zhu, jin title: a novel chimeric anti-pa neutralizing antibody for postexposure prophylaxis and treatment of anthrax date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: pdxm iiw anthrax is a highly lethal infectious disease caused by the bacterium bacillus anthracis, and the associated shock is closely related to the lethal toxin (letx) produced by the bacterium. the central role played by the kda protective antigen (pa ) region of letx in the pathophysiology of anthrax makes it an excellent therapeutic target. in the present study, a human/murine chimeric igg mab, hmpa , was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmpa expressed in f cells could neutralize letx both in vitro and in vivo. at a dose of . mg/kg, it could protect all tested rats from a lethal dose of letx. even administration of . mg/kg hmpa h before letx challenge protected all tested rats. the results indicate that hmpa is a potential candidate for clinical application in anthrax treatment. the bacterium bacillus anthracis, which primarily affects livestock but can also infect humans, is the causative agent of anthrax, a zoonotic disease and bioterrorism threat , . b. anthracis spores can be used as bioterror agents in biological warfare. this threat has spurred significant efforts toward the development of countermeasures for anthrax, including anthrax vaccines and therapeutics . however, vaccines are effective only for prevention . currently, therapeutic antibodies that target the anthrax toxin are under development and are designed to protect against the disease. b. anthracis secretes a tripartite toxin comprising a protective antigen (pa), lethal factor (lf), and edema factor (ef) . this is an a-b (or "binary") bacterial toxin. pa is the "b" subunit, which is responsible for cell surface binding, while lf and ef are a subunits responsible for the enzymatic activity of the toxin , . pa combined with lf or ef constitutes the lethal toxin (letx) or edema toxin (edtx), respectively . the first step in cellular intoxication involves binding of an kd form of pa (pa ) to specific cell surface receptors (antxr and antxr ). following receptor binding, pa is cleaved after the arg-lys-lys-arg sequence at amino acid position by a furin-family protease. this results in a kda form (pa ) that spontaneously oligomerizes to either a heptamer or an octamer [ ] [ ] [ ] . dissociation of the kda form (pa ) from pa allows pa to bind to either or both ef and lf. then, oligomeric pa -receptor complexes translocate lf or ef into the cytosol, where they promote intoxication . previous studies have shown that pa inserts stably and irreversibly into lipid bilayers to form ion-permeable channels , . other research has shown that the protease cleavage site deletion or mutation in pa prevents ef and lf binding , , and that for cells treated with lysosomotropic agents, the ability of pa to mediate the actions of ef or lf is blocked . nasal immunization of mice with a mixture of pa , lf, and a poly-γ -d-glutamic acid conjugate have been shown to exhibit strong antibody responses against all three antigens . thus, pa seems to be an ideal target fragment for antibody generation and selection. since passive immunization with protective antibodies can provide immediate and extensive protection independent of the host response, it is an attractive option to enhance the current postexposure treatment of anthrax. especially with regard to biodefense, it is considered the primary available therapeutic measure . during the past years, extensive research has focused on development of therapeutic antibodies to target the main virulence factors of anthrax, namely, pa, lf, ef, and capsule [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among these, pa plays a central role in the pathophysiology of anthrax and is an excellent therapeutic target. further, pa is the most important part of pa. in the present study, we developed murine igg neutralizing antibodies that directly target pa . then, we selected an ideal antibody from among these and genetically recombined it to form human/murine chimeric igg (coded "hmpa "). hmpa could specifically bind to pa and protect j a. cells against letx challenge in vitro. further, it protected fischer rats (f ) from letx after challenge. elisa was performed to test the binding sensitivity of hmpa to pa . hmpa recognized rpa in a dose-dependent manner, and the graph of hmpa concentration and absorbance at nm was a hyperbolic curve (fig. ). western blot analysis showed that hmpa could specifically recognize rpa (fig. ) . no reaction was seen with the negative control. immunoprecipitation. immunoprecipitation was performed using pa , which could be split to active pa using trypsin. a protein of about kda was detected on sds-page, and its sequence matched that of the b. anthracis protective antigen in the swiss-prot database (fig. a,c,d) . a kda membrane protein was also detected using a commercial anti-pa antibody (fig. b) , and this protein did not reaction with any other antibodies. kinetics of binding. the equilibrium dissociation constant (kd) for hmpa was determined by biacorex analysis. the rate constants kon and koff were evaluated directly from the biacorex sensogram. the kd was also determined using the biacorex . one striking feature of hmpa is its very slow off rate, which may explain its high affinity of . × − m (fig. ) . in vitro letx neutralization assay. the ability of hmpa to protect against letx was assessed in j a. cells. hmpa , pa , and different concentrations of lf were simultaneously added to cells. cell viability test results indicated that hmpa could completely neutralize letx. at μ g/ml lf and . μ g/ml pa , > % of the hmpa -treated cells remained viable, while only % of the control igg antibody-treated cells remained viable. at . μ g/ml lf and . μ g/ml pa , % of the hmpa -treated cells were viable, while only % (fig. ) of the control cells were. protection of f rats. f rats were injected hmpa antibody via the tail vein either before or after letx injection. the survival time of group iii was significantly (p < . ) longer than that in groups i and ii. until the last observation, all group iv rats were alive (fig. a ). rats injected hmpa min before letx showed similar results. a dose of μ g antibody protected the rates from death ( fig. b) , although some symptoms, such as accelerated breathing and lethargy, were observed in one rat. hmpa injection before or after letx administration protected all rats from developing anthrax (fig. c) . the prophylactic function of hmpa was tested by injection of the antibody at different times before letx injection. in the groups that received prophylaxis min to h before letx injection, rats remained alive (fig. d ). when hmpa was injected before pa, it protected rats from anthrax death regardless of lf injection time (fig. e ). tissue pathology and immunohistochemical analysis. the lung of the rats were pathologically and immunohistochemically examined. h&e staining showed that the local tissue of rats injected only letx showed greater alveolar exudation (fig. c ) than untreated control rats (fig. a) . however, the group that received letx + μ g hmpa (fig. b) showed no significant differences from untreated control rats. since anthrax receptors were expressed in some cells including alveolar epithelial cells, cell binding to pa could be positive. when rats were injected with letx, the cells could dectect pa. further, the positive staining was mainly localized to the membrane. a strong positive reaction was found in the group injected only letx (fig. f) , while a weak positive reaction was found in the group injected letx + μ g hmpa (fig. e) . the untreated control group showed a negative reaction (fig. d ). this study revealed two major findings: first, the human/murine chimeric antibody hmpa can neutralize letx in vivo and can be used for prohylaxis before letx is released in the blood. second, since immunization with pa produces neutralizing antibody, it can be used to immunize animals. in the present study, we developed four murine mabs that could well neutralize letx in vitro, and one clone was selected to form a human/mouse chimeric antibody known as hmpa . this antibody showed excellent neutralization both in vitro and in vivo. the current status of therapeutic mabs was directed against the major virulence factors: pa, lf, ef and capsule. although lf could induce cell death, its effective result in vivo was unsatisfactory. pa was the critical factor which recognized cell membrane receptors. then pa was cleaved to active pa by furin which induced lf or ef into cells. our strategy was to get antibody against active pa . to immunize the mice, we used pa , which is formed by the dissociation of pa from pa , instead of pa , because although pa is a part of pa , these factors may have different structures and expose lf-or ef-binding sites. these differences may in turn lead to the production of completely different antibodies against anthrax in vivo. in the present study, we showed that pa could induce neutralizing antibodies, as proven through in vitro and vivo experiments. on the other hand, it was difficult to obtain active pa based on present methods. we screened out positive clones by active pa in a different way. based on traditional elisa, we coated plates with alive j a. cells overninght at cell culture condition. then pa was added into wells incubated about h. the negative control was cells without pa adding. the rest of elisa procedures was as normal. in this screening system, the active pa was more closed to in vivo state and could oligomerize to heptamer. in all, more diversity antibodies was generated in the immunization stage and more effective antibodies was obtained in the screening system. anthrax, whether resulting from natural or bioterrorist-associated exposure, is a constant threat to human health , and the low incidence of anthrax suggests that large-scale vaccination may not be the most efficient means of controlling this disease. passive immunization with protective antibodies is therefore considered the primary available biodefense measure , especially in bioterrorist-associated exposure. in the present study, hmpa was found to provide good protection in rats challenged with anthrax virulence factors. in the in vitro experiment, hmpa maintained % cell viability with . μ g/ml lf and . μ g/ml pa , while non-correlated igg maintained only % cell viability. further, with μ g/ml lf and . μ g/ml pa , hmpa maintained > % cell viability, while the control antibody maintained only %. moreover, in previous study, they often used f rats challenged with letx (table ) before tested with b. anthracis spores. therefore f rats was injected with letx vial tail vain. the hmpa with a kd of . nm protected all rats from death at a concentration of . mg/kg ( μ g per rat). however, in a previous study, . mg/kg raxibacumab (of human origin) with a kd of . nm was administered h before anthrax lethal toxin administration in f rats . we also tested an hmpa concentration of . mg/kg ( μ g per rat) administered h before letx injection; this dose also protected all rats. our findings indicate that hmpa may be used as a prophylactic. other humanized or chimeric mabs have been examined in other animal model, but the affinity found in the present study is the same as or better than those found previously , . murine mabs against pa have been developed, but our chimeric mab maintains a balance between the high-affinity murine component and the low-immunogenicity human component. other mabs of human origin are available but are difficult to produce and are expensive . the chimeric mab hmpa is produced in f cells, and the method of production is very convenient. in the in vivo test in the present study, irrespective of whether lf was injected before or after hmpa , the antibody protected the rats from death provided that it was administered before or simultaneously with pa. this finding indicated that hmpa could not prevent lf from binding pa . further, no morphological changes were observed in the rats of only injection letx, probably because the time between injection and sacrifice was very short (only about mins), and no histological changes occurred in this period. the ihc analysis showed a strong positive result in the group injected only letx. however, the group that received letx + μ g hmpa showed a weakly positive reaction. these findings indicate that hmpa prevents pa from binding to cell receptors. however, further experiments are required to validate this hypothesis. future studies should focus on detailed characterization of this mab (specificity, toxicity studies, autoantigen testing, etc.). second, epitope mapping and structure function analysis of hmpa should be performed. in the in vivo experiment in the present study, we demonstrated that hmpa could not interfere with lf binding to pa. further experimentation is needed to determine the exact mechanism by which hmpa neutralizes letx. lastly, more animal tests are required, for example, in which animals are challenged with anthrax spores. in summary, we reported a human/murine chimeric igg, namely, hmpa , which can specifically identify pa with high affinity, neutralize letx, and protect macrophages and f rats from anthrax-related death. we also showed that pa is a good immunogen. from our findings, we believe that once hmpa is further characterized, it can be used alone or in combination with other neutralizing mabs for treatment of anthrax. bl by using the pcoldii vector and purified by affinity chromatography. six balb/c mice aged weeks were intraperitoneally (ip) immunized with rpa and adjuvant as described previously . after immunization, the mouse spleen showing the best titer was removed, and splenocytes were extracted and fused with sp / myeloma cells using hybridoma technology . positive clones were screened by elisa using active pa -coated -well plates, and subcloning was conducted based on standard protocols. clonal expansion was conducted with hybridoma-sfm (gibco,usa). the cell supernatant was then removed and purified by affinity chromatography with protein g (ge, usa) according to the manufacturer's purification system. figure . in vivo letx neutralization assay in f rats. a. mean survival time. letx and the antibody were simultaneously injected via the tail vein. group i, μ g hmpa + μ g letx; group ii, μ g hmpa + μ g letx; group iii, μ g hmpa + μ g letx; group iv, μ g hmpa + μ g letx. ***p < . . b. different concentrations of the antibody were injected, and letx was injected min later via the tail vein. c. for each rat, μ g antibody was injected before (− min), after ( min), or simultaneously ( min) with letx. d. for each rat, μ g antibody was injected at different times before letx injection. e. group i, pa ( μ g) was injected min after lf ( μ g) + hmpa ( μ g); group ii, pa ( μ g) was injected min before lf ( μ g) + hmpa ( μ g). all experiments involving animals were performed in accordance with the protocols approved by the animal care and use committee of the national institute of allergy and infectious diseases, national institutes of health, usa. total rna was extracted from the pa hybridoma cells using the trizol reagent (invitrogen), and cdna was synthesized using reverse transcriptase superscript ii according to the manufacturer's instructions. eukaryotic vectors were constructed by separately cloning pa heavy and light variable regions into pth and ptl, which respectively include constant regions of igg heavy and light chains. murine variable regions of the heavy (v h ) and light chains (v l ) were first amplified by pcr using pa cdna as the template. to obtain v h and v l nucleotide sequences, these chains were cloned into the pmd- t vector. pcr primers were designed using the in-fusionr hd cloning kit (clontech), and v h and v l were amplified from right-sequenced pmd- t vectors by using these primers. finally, v h and v l were separately cloned into linearized pth and ptl vectors, respectively, by infusion pcr using the in-fusionr hd cloning kit. the recombinant elisa. ninety-six-well enzyme immunoassay plates were coated overnight at °c with μ l of rpa antigen ( μ g/ml) diluted in mm sodium carbonate buffer (ph . ). the plates were blocked and serial two-fold dilutions of hmpa were added to the wells ( wells for each concentration) as the primary antibody. the plates were incubated at °c for h and then washed times with μ l of pbs containing . % tween (pbst). subsequently, goat anti-human igg-hrp conjugate (sigma) was added as the secondary antibody and incubated at °c for min. after color development, the absorbance values of the wells were detected at nm. non-correlated igg was used as the control. the absorbance values at nm of hmpa were plotted using graphpad prism software version . (graphpad software, inc., la jolla, ca, usa). the cell lysates of rpa recombinant bacteria and e. coli bl were separately run on a % sds-page gel and then transferred onto a nitrocellulose membrane (bio-rad). the membrane was blocked with pbs containing % dry milk at °c overnight and then incubated for h at rt with : diluted hmpa from mg/ml stock. after it was washed times with pbst, the membrane was incubated with a : diluted secondary hrp-conjugated goat anti-human antibody (sigma) for an additional min at rt. following the same washing procedure, the signal was detected using ecl western blot substrate (pierce) according to the manufacturer's instructions. (ph . ) and mm nacl] with . μ g/ml trypsin (sigma) for min at °c, followed by addition of μ g/ml soybean trypsin inhibitor (sigma) . then, the mixture was incubated with μ g of hmpa at °c and rotated for h. next, μ l protein-a sepharose (invitrogen, usa) was added and incubated at °c. the immune complexes that formed were washed times with pbst. subsequently, μ l elution buffer was added to separate these antibody-antigen complexes from protein-a sepharose. as a negative control, another anti-tlr chimeric antibody (generated by our lab) was created using the same protocol. the protein complexes were isolated by running two % sds-page gels; one was transferred onto a nitrocellulose membrane, and the other was stained with coomassie blue. the nitrocellulose membrane was blocked at °c overnight, incubated with : diluted rabbit polyclonal anti-pa antibody (pierce, usa) for h at rt, washed with pbst times, and reacted with : diluted goat anti-rabbit igg-hrp conjugate (sigma) for an additional min at rt. the membrane was washed times with pbst, and the hybridization signal was detected using ecl western blot substrate. the target bands on sds-page gel were subjected to mass spectra identification with an abi proteomics analyzer and maldi-tof/tof mass spectrometer (applied biosystems, framingham, ma). the mass spectra were then searched within the swiss-prot database using the mascot search engine (http://www.matrix science.com; matrix science, uk). affinity and kinetic assay of antibody. the biacore x system (ge, usa) was used to analyze the affinity and kinetics of the hmpa antibody. pa was diluted to μ g/ml with acetate buffer ( mm naac, ph . ) and immobilized on the surface of a cm sensor chip (ge, usa) to capture purified mab, which was diluted in running buffer ( mm hepes, mm nacl, mm edta-na , . % p ; ph . ) to achieve different concentrations ranging from to nmol/l. the association time was set up at s and the dissociation time, at s, followed by regeneration with mm glycine-hcl (ph . ). sensograms were evaluated using the biacore x evaluation software. in vitro letx neutralization assay. the in vitro letx neutralization assay was performed as described previously injected via the tail vein with a mixture of pa + lf (letx) and different amounts of hmpa antibody prepared in sterile pbs. each rat was administered μ l of the mixture. further, the rats were also treated with different concentrations of the antibody min before exposure to letx. for this experiment, they were injected intravenously with pbs or , , or μ g of the antibody before receiving an intravenous injection of letx ( μ g pa + μ g lf). additionally, double the complete protection dose of antibody ( μ g) was injected to test its prophylactic ability. the rats were inoculated with μ g antibody followed by letx administration after different times, from min to h. two additional experiments were conducted with f rats. one group received pa ( μ g) injection min after lf + hmpa ( μ g + μ g, respectively), while the other received μ g pa min before lf + hmpa (at the same doses). after injection of letx, signs of malaise and death were checked for every min for the first h and then at h and h, followed by twice-daily checks for week. tissue pathology and immunohistochemical examination. the lungs of the f rats were embedded in paraffin wax at the department of pathology, nanjing medical university (jiangsu, china), using routine methods. sections ( μ m) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. some sections were treated with h&e staining and examined by light microscopy to determine the pathological features of the lung tissues. for the remaining sections, endogenous peroxidase activity was blocked by incubation with % hydrogen peroxidase in tris-buffered saline. some of these tissue sections were then incubated with rabbit polyclonal anti-pa primary antibody (pierce, usa), followed by the envision hrp complex (dako, carpinteria, ca). they were then counterstained with hematoxylin qs (vector laboratories, burlingame, ca). the results were analyzed according to the ihc score (ihs) as described previously . briefly, the ihs was determined by evaluation of both staining density and intensity. multiplication of the intensity and percentage scores yielded the final ihs. samples with ihs ≤ were considered weakly positive, while those with ihs ≥ were considered strongly positive. the ihc results were evaluated by two independent investigators blinded to the rat groups. in cases of conflict, a pathologist reviewed the cases, and a consensus was reached. survival data were analyzed using the graphpad prism version statistical analysis software (san diego, ca). a t-test was used to compare the mean survival time between groups. a two-tailed log rank test was used to determine the statistical significance of differences between groups. a p value of < . was considered statistically 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possible synergy with anti-protective antigen antibody this work is supported by the national natural science foundation (no. ).and science fund of jiangsu province, china (no. bk and no. cws j ) s.x., j.z., z.f. and q.t. wrote the main manuscript text and s.x. and x.l. prepared figures - and q.t. and s.x. prepared figures - . all authors reviewed the manuscript. competing financial interests: dr. zhu, dr. feng, qi tang and siping xiong have a common patent a issued in china. all other authors report no conflicts. key: cord- -pbahviaz authors: garg, shikha; jain, seema; dawood, fatimah s.; jhung, michael; pérez, alejandro; d’mello, tiffany; reingold, arthur; gershman, ken; meek, james; arnold, kathryn e.; farley, monica m.; ryan, patricia; lynfield, ruth; morin, craig; baumbach, joan; hancock, emily b.; zansky, shelley; bennett, nancy; thomas, ann; schaffner, william; finelli, lyn title: pneumonia among adults hospitalized with laboratory-confirmed seasonal influenza virus infection—united states, – date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: pbahviaz background: influenza and pneumonia combined are the leading causes of death due to infectious diseases in the united states. we describe factors associated with pneumonia among adults hospitalized with influenza. methods: through the emerging infections program, we identified adults ≥ years, who were hospitalized with laboratory-confirmed influenza during october through april , and had a chest radiograph (cxr) performed. pneumonia was defined as the presence of a cxr infiltrate and either an icd- -cm code or discharge summary diagnosis of pneumonia. results: among , adults hospitalized with influenza, ( %) had pneumonia. in multivariable analysis, factors associated with pneumonia included: age ≥ years, adjusted odds ratio (aor) . ( % confidence interval . – . ), white race aor . ( . – . ), nursing home residence aor . ( . – . ), chronic lung disease aor . ( . – . ), immunosuppression aor . ( . – . ), and asthma aor . ( . – . ). patients with pneumonia were significantly more likely to require intensive care unit (icu) admission ( % vs. %), mechanical ventilation ( % vs. %), and to die ( % vs. %). conclusions: pneumonia was present in nearly one-third of adults hospitalized with influenza and was associated with icu admission and death. among patients hospitalized with influenza, older patients and those with certain underlying conditions are more likely to have pneumonia. pneumonia is common among adults hospitalized with influenza and should be evaluated and treated promptly. electronic supplementary material: the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. influenza illness is generally characterized by acute onset of fever, mylagias, and respiratory symptoms, and while disease usually resolves without complications in healthy indiviudals, influenza is associated with an annual increase in hospital admissions for pulmonary, cardiovascular and neuromuscular compliations [ ] [ ] [ ] . the etiology of influenza-associated pneumonia may include primary influenza pneumonia, secondary bacterial pneumonia, or concomitant viral and bacterial pneumonia [ , , ] . pulmonary complications of influenza, including pneumonia and exacerbations of chronic pulmonary disease, are common and result in significant morbidity and mortality. oliveira and colleagues found that among all patients admitted to a large metropolitan hospital with influenza during the - season, % had pneumonia [ ] . further, in a study conducted over influenza seasons ( ) ( ) ( ) ( ) ( ) , murata and colleagues found that among patients hospitalized with influenza a, % had some type of acute findings on chest radiograph and % had definitive pneumonic infiltrates [ ] . although there is evidence that adult patients with underlying cardiac or pulmonary disease are more likely to develop influenza-associated pneumonia than those without underlying medical conditions [ , ] , much of the data describing factors associated with influenzaassociated pneumonia among adults comes from case series conducted at single sites and during a limited number of seasons. using data from a large multi-center, geographically diverse, population-based surveillance system, we describe factors associated with pneumonia among adults hospitalized with influenza over three consecutive years in which seasonal influenza viruses circulated. the emerging infections program (eip) network conducts active population-based surveillance for laboratory-confirmed influenza-associated hospitalizations. the network began adult surveillance in and covers over counties in states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, new york, oregon, and tennessee), representing approximately % of the adult u.s. population [ ] . patients were included in eip influenza surveillance if they resided and were hospitalized in an eip catchment area and were hospitalized within days of a positive influenza diagnostic test result. patients were excluded if the first positive influenza specimen was obtained > days after hospital admission because these patients might have had healthcare-associated influenza infection. influenza testing was performed at the discretion of health care providers. medical charts of hospitalized patients with laboratory-confirmed influenza were retrospectively reviewed [ , ] . the study period comprised influenza seasons, - to - . patients were included in this analysis if they were ≥ years of age, were hospitalized with laboratory-confirmed influenza during the - through - influenza seasons, and had a chest radiograph (cxr) performed during hospitalization. the following data were collected on patients: demographics, results of laboratory tests for influenza, influenza vaccination status for the current season, underlying medical conditions, bacterial coinfections, cxr data, antiviral treatment, clinical outcomes, and discharge diagnoses. laboratory confirmation of influenza was based on viral culture, direct or indirect immunoflourescence antibody staining, reverse-transcription polymerase chain reaction, or a rapid antigen test. surveillance staff completed medical record abstractions using check boxes to indicate whether or not a new infiltrate or consolidation was recorded on the official cxr transcript. discharge diagnoses were captured in two ways: ) the first nine international classification of diseases (icd- -cm) codes for each case were abstracted from the medical record; ) check boxes were marked for certain diagnoses, including pneumonia, if they were recorded by clinicians on the discharge summary. pneumonia was defined as the presence of a new infiltrate on cxr and either an icd- -cm discharge diagnosis code for pneumonia ( - . ) or a diagnosis of pneumonia recorded on discharge summary. information on the presence of selected bacterial infections was available only for patients who had a positive culture. a bacterial infection was recorded if bacteria other than those that are commonly considered to be contaminants grew from a sterile body site or a non-sterile respiratory site culture obtained within calendar days of hospital admission. sterile body sites for bacterial infections included blood, pleural fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, and deep tissue biopsy. non-sterile respiratory sites included sputum and endotracheal aspirates. use of influenza antiviral therapy was examined for all individuals. among those who were treated with antiviral agents, timing of treatment was assessed in relation to hospitalization date. early antiviral treatment was defined as initiation of antiviral treatment within days of hospital admission. we used bivariate analysis to compare adults hospitalized with influenza with and without pneumonia. we used χ and fisher exact tests for categorical variables and t-tests and wilcoxon-rank sum tests for continuous and ordinal variables. all variables significant in bivariate analysis, as well as biologically plausible variables, and potential confounders were included in a multivariable logistic regression model to identify factors independently associated with influenza-associated pneumonia. we used the breslow-day test for homogeneity to assess for effect modification of select variables. all tests were two-tailed and a p-value of . was considered significant. analyses were conducted using sas version . (sas institute inc., cary, nc). ethics statement eip adult influenza hospitalization surveillance activities during the - influenza seasons were determined by the centers for disease control and prevention (cdc) institutional review board (irb) not to involve research in accordance with the federal regulations for the protection of human subjects in research. starting with the - season, research questions were added to evaluate factors associated with severe outcomes during hospitalizations, and irb review was conducted at all surveillance sites and the cdc. the protocol was approved by the cdc irb and was either approved or received exempt status by all surveillance site irbs. because all surveillance data was analyzed anonymously, neither verbal nor written informed consent was obtained from participants. during the study period, of adults hospitalized with laboratory-confirmed influenza, ( . %) had an available cxr report and discharge diagnosis information and were therefore included in our study. of the adults, ( %) had pneumonia. the prevalence of pneumonia did not vary significantly over the influenza seasons included in the analysis. adults ≥ years of age represented the age group with the highest proportion of patients hospitalized with and without influenza-associated pneumonia (fig. ) . the median age of patients with pneumonia compared with patients without pneumonia was years versus years (p < . ) ( table ). the majority of patients hospitalized with and without influenza-associated pneumonia were white. white patients were older (median age years) than black patients ( years), hispanic patients ( years), and patients of other races including asian, pacific islander, american indian, alaskan native, and multi-race ( years) (p < . ). patients aged years and above had a higher proportion of underlying conditions ( %) compared to patients aged < years ( %) (p < . ). influenza was diagnosed by rapid test only in / ( %) patients with pneumonia and in / ( %) patients without pneumonia (p < . ). the median number of days from symptom onset to hospital admission was days for patients with and without pneumonia (table ) . patients with pneumonia were significantly more likely than patients without pneumonia to reside in a nursing home prior to hospital admission, to have received influenza vaccine, and to have the following underlying medical conditions: chronic lung disease, cardiovascular disease, and immunosuppression. patients with pneumonia were significantly less likely than patients without pneumonia to have asthma (table ) . a description of the most frequent discharge diagnoses (based on first listed icd- diagnosis code) among patients with and without pneumonia can be found in additional file : table s . except for influenza vaccination and cardiovascular disease, all factors included in a multivariable model remained independently associated with pneumonia including age ≥ years [adjusted odds ration (aor) . ], white race (aor . ), nursing home residence (aor . ) chronic lung disease (aor . ), immunosuppression (aor . ) and asthma (aor . ) ( table ) . sixty-one patients with pneumonia and patients without pneumonia had sterile site bacterial infections, % of which were cultured from the blood ( table ). the most common pathogens cultured from sterile sites in patients with pneumonia were staphylococcous aureus (s. aureus) and streptococcus pneumonia (s. pneumonia). patients with pneumonia had a longer median length of hospital stay than patients without pneumonia ( days versus days; p < . ). patients with pneumonia were also significantly more likely to have a hospital length of stay greater than one week (aor . ), require intensive care unit (icu) admission (aor . ), require mechanical ventilation (aor . ), and die (aor . ) ( table ) . among patients with pneumonia, factors independently associated with a poor outcome, defined as icu admission, need for mechanical ventilation or death, included nursing home residence (aor . ), chronic lung disease (aor . ), cardiovascular disease (aor . ), (table ). of note, older age was inversely associated with a poor outcome (aor . ) among patients hospitalized with pneumonia (table ) . patients with pneumonia [ / ( %)] were significantly more likely to receive influenza antiviral therapy than patients without pneumonia [ / ( %); p < . ]. through this large, population-based surveillance system, we found that pneumonia was present in almost one-third of u.s. adults hospitalized with laboratoryconfirmed influenza over three consecutive years in which seasonal influenza viruses circulated. patients with pneumonia were older and were more likely to have certain underlying medical conditions than patients without pneumonia. patients with pneumonia were also more likely to have a prolonged hospital stay, be admitted to an icu, require mechanical ventilation for respiratory failure, and die. while patients with pneumonia were more likely to receive antiviral therapy than those without pneumonia, treatment was more often delayed among patients with pneumonia. similar to findings from smaller inter-pandemic studies [ , ] pneumonia was common among adults hospitalized with influenza in this study. among those hospitalized with influenza, older adults and nursing home residents were at significantly increased risk for having influenza-associated pneumonia. respiratory viruses including influenza are a common etiology of pneumonia in older adults, and several factors may contribute to the development of severe lower respiratory tract disease in these individuals, including decreased respiratory muscle strength and lung compliance, and waning humoral and cell-mediated immunity [ ] [ ] [ ] . additional risk factors for lower respiratory tract disease among older nursing home residents include immobility and swallowing difficulties leading to aspiration [ ] . within closed settings such as nursing homes, large outbreaks of influenza and its subsequent complications, including severe pneumonia, may rapidly evolve and lead to significant morbidity and mortality [ , ] . influenza virus infection should thus be considered a potential cause of pneumonia in older individuals and nursing home residents during fall and winter months ( ) other streptococci c ( ) ( ) other pathogens d ( ) ( ) unknown pathogens ( ) ( ) [ ] and should be diagnosed and treated promptly. influenza vaccination is the most effective method to prevent influenza and its complications, and older adults, residents of nursing homes and other long-term-care facilities, and adults with underlying medical conditions should be considered high priority groups for receipt of annual influenza vaccination [ ] . similar to earlier studies conducted during periods of seasonal influenza virus circulation, patients with pneumonia in this study were more likely to have underlying medical conditions including chronic lung disease and heart disease [ , ] . an unexpected finding was that patients with asthma in our analysis were less likely to have a diagnosis of pneumonia than patients without pneumonia. our study results contrast with eip surveillance data in hospitalized children < years of age which has shown that children with influenza-associated pneumonia were more likely to have asthma than those without pneumonia [ ] . studies of the association between asthma and seasonal influenza-associated pneumonia among adults are lacking. a possible explanation for our finding is that respiratory distress caused by influenza-associated asthma exacerbation provided an alternate reason for hospitalization in adult patients in the absence of pneumonia. biases in hospital admission practices based on the presence of underlying conditions may have also contributed to admission of asthmatic patients with a less severe respiratory presentation compared to patients without underlying medical conditions. invasive bacterial infections, especially due to s. aureus and s. pneumoniae, were observed among patients with influenza-associated pneumonia in this study as well as other studies conducted during inter-pandemic [ ] and pandemic periods [ ] . among patients with pneumonia, s. aureus was the most common organism cultured from specimens collected from sterile sites. influenza virus and s. aureus co-infections are increasing [ ] [ ] [ ] and have been associated with particularly severe cases of community-acquired pneumonia during periods of seasonal influenza virus circulation [ ] . in patients hospitalized with influenza, sterile site cultures should be collected as early as possible for detection of bacterial infection and empiric antimicrobial coverage of the most likely bacterial organisms should be considered [ , ] . in our study, s. pneumoniae was the only organism to be cultured from a sterile site more frequently in patients with pneumonia that in patients without pneumonia. in addition to annual influenza vaccination, pneumococcal vaccine should be administered to adults aged - years with certain health conditions and to all persons aged ≥ years [ ] . patients with influenza-associated pneumonia had a significantly increased risk of icu admission, respiratory failure requiring mechanical ventilation, and death compared with patients without pneumonia. while case series conducted during the h n pandemic demonstrated elevated frequencies of icu admission ( - %) [ , ] , respiratory failure ( - %) [ , ] and death ( - %) [ ] [ ] [ ] [ ] among patients hospitalized with pandemic h n influenza-associated pneumonia, limited data is available on the association between seasonal influenzaassociated pneumonia and severe outcomes. in a small case series of patients hospitalized with influenza during the - season, ( %) of patients with pneumonia were admitted to the icu and ( %) patients died [ ] . in another observational study of patients hospitalized with influenza during - , ( %) of patients with acute pulmonary disease were admitted to the icu, ( %) required mechanical ventilation, and ( %) died [ ] . while pneumonia and acute respiratory distress syndrome (ards) have been shown to account for a majority of deaths associated with influenza virus infection during pandemics [ ] , data is limited on the association between seasonal influenza virus infection and death from pneumonia or ards. in our analysis, only % of patients hospitalized with laboratory-confirmed influenza received influenza antiviral treatment. when limiting the analysis to patients who presented to the hospital within days of symptom onset, only % of all patients received antiviral treatment; the majority received antiviral treatment within day of hospital admission. multiple studies have found early antiviral treatment to be associated with a reduction in serious influenza-associated outcomes including the development of lower respiratory tract infections [ ] [ ] [ ] . the advisory committee on immunization practices recommends empiric influenza antiviral treatment for all adults with suspected or confirmed influenza who are hospitalized, have severe, complicated, or progressive illness, or are at high risk for influenza-associated complications [ ] . several limitations to this study should be noted. influenza diagnostic testing was performed at the discretion of treating clinicians at the various eip hospital sites. while all hospitalized patients who tested positive for influenza were included in surveillance, data is unavailable for hospitalized patients who tested negative for influenza or who were not tested. thus, these data may not be representative of all individuals hospitalized with influenza who may not have been tested or have laboratory confirmation of influenza virus infection. it is possible that patients included in surveillance were more likely to be tested for influenza because they were more severely ill; thus a higher proportion of patients exhibiting pneumonia-like symptoms may have been tested for influenza than patients presenting with other symptoms. furthermore, in our analysis, patients with pneumonia were compared to patients without pneumonia but with a wide array of other diagnoses. clinican influenza testing practices based on patient diagnoses at presentation may have biased our findings. in one study conducted in an emergency department in australia, patients presenting with fever and respiratory diagnoses were more likely to be tested for influenza than patients presenting with cardiac or other diagnoses [ ] . this study assessed pneumonia specifically among adults hospitalized with laboratory-confirmed influenza, including those whose influenza virus infection preceded hospitalization by more than a few days, and findings are not generalizable to all hospitalized individuals with pneumonia of other etiologies or to non-hospitalized individuals. several of the findings in this study may have been biased by hospital admission practices. for example, the finding of an inverse association between asthma and pneumonia may have been due to more aggressive admission of asthmatic patients presenting with respiratory distress despite the absence of pneumonia, compared with patients without asthma. biases related to hospital admission practices were likely reduced by including patients from multiple hospital sites in geographically diverse settings. for certain underlying conditions such as chronic lung disease and cardiovascular disease, disease type and severity were not captured by the case report form. availability of detailed data on type and severity of underlying conditions may have helped to better identify factors more strongly associated with development of influenza-associated pneumonia. radiographic data were based on review of cxr reports by surveillance officers and not by actual review of radiographs by a designated study radiologist. as a result, some individuals may have been misclassified as having pneumonia based upon a report of infiltrates or opacities, when in fact they had a more chronic pulmonary condition or a transient episode of pulmonary edema or effusion. there was no requirement regarding timing of identification of radiologic abnormalities during the hospitalization, and the timing of chest radiographs during the hospitalization was not collected as part of eip surveillance; thus, some misclassification of communityacquired pneumonia versus nosocomial pneumonia may have occurred. using icd- -cm code data may also have led to misclassification if a diagnosis code was listed incorrectly or not listed at all. a joint case definition for pneumonia which used both radiographic data and discharge diagnosis data from icd- -cm codes or discharge summaries was utilized to minimize some of these biases. bacterial culture data was only available for patients with a positive culture result rather than for all specimens spent, thus limiting the interpretation of the culture data. pneumonia is common among adults hospitalized with seasonal influenza virus infection. among patients hospitalized with influenza, older adults and those with underlying medical conditions may be more likely to have pneumonia. further studies are needed to explore the association between influenza-associated pneumonia and asthma in adults. influenza-associated pneumonia can lead to severe outcomes including icu admission and death. adults hospitalized with suspected or confirmed influenza should receive early antiviral therapy, prompt evaluation for pneumonia, and appropriate management upon diagnosis of pneumonia. additional file : table s . the most frequent icd- diagnosis categories based on first icd- code listed among adults hospitalized with laboratory-confirmed influenza with and without pneumonia (n= ). abbreviations cxr: chest radiograph; aor: adjusted odds ratio; eip: emerging infections program; icd- -cm: international classification of diseases; cdc: centers for disease control and prevention; irb: institutional review board; icu: intensive care unit; s. aureus: staphylococcus aureus; s. pneumonia: streptococcus pneumonia. the authors declare that they have no competing interests. authors' contributions sg: contributed to conception and design of study, analysis of data and interpretation of results, and drafting of the manuscript; sj: contributed to conception and design of study, interpretation of data, and criticial review and revision of manuscript; fd: contributed to conception and design of study, analysis of data, and critical review of manuscript; mj: contributed to conception and design of study, interpretation of data, and criticial review of the manuscript; ap: contributed to analysis and cleaning of data, interpretation of results, and critical review of the manuscript; td: contributed to analysis and cleaning of data, interpretation of results, and critical review of the manuscript; ar: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; kg: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; jm: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; ke: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; mf: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; pr: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; rl: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; cm: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; jb: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; eh: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; sz: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; nb: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; at: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; ws: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; lf: contributed to conception and design of study, interpretation of data, and criticial review and revision of manuscript; all authors 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-recommendations of the advisory committee on immunization practices (acip) van hal sj. influenza a testing and detection in patients admitted through emergency departments in sydney during winter ; implications for rational testing we wish to thank the following individuals for their help with surveillance efforts: deborah submit your next manuscript to biomed central and take full advantage of: key: cord- -dftwz l authors: calvo, cristina; garcía-garcía, maría luz; pozo, francisco; paula, gallardo; molinero, mar; calderón, ana; gonzález-esguevillas, mónica; casas, inmaculada title: respiratory syncytial virus coinfections with rhinovirus and human bocavirus in hospitalized children date: - - journal: medicine (baltimore) doi: . /md. sha: doc_id: cord_uid: dftwz l it is not clearly established if coinfections are more severe than single viral respiratory infections. the aim of the study was to study and to compare simple infections and viral coinfections of respiratory syncytial virus (rsv) in hospitalized children. from september to august , a prospective study was conducted on children younger than years of age, admitted with respiratory infection to the pediatric department of the severo ochoa hospital, in spain. specimens of nasopharyngeal aspirate were taken for virological study by using polymerase chain reaction, and clinical data were recorded. simple rsv infections were selected and compared with double infections of rsv with rhinovirus (rv) or with human bocavirus (hbov). in this study, episodes corresponding to children were analyzed. at least virus was detected in % ( ) of the episodes. single infections ( rsv, rv, and hbov) were compared with rsv-rv and rsv-hbov double infections. the rsv-rv coinfections had fever ( % vs %; p < . ) and hypoxia ( % vs %; p < . ) more often than rv infections. hypoxia was similar between single or dual infections ( %). bronchiolitis was more frequent in the rsv simple group (p < . ). pediatric intensive care unit admission was more common in rsv simple or rsv-rv groups than in the rv monoinfection (p = . ). hospitalization was longer for both rsv simple group and rsv-hbov coinfection, lasting about day ( . vs . days; p < . ) longer than in simple hbov infections. there were no differences in picu admission. rsv single group was of a younger age than the other groups. coinfections between rsv-rv and rsv-hbov are frequent. overall viral coinfections do not present greater severity, but have mixed clinical features. a cute respiratory tract infections (aris) are common diseases among children and a major cause of hospitalization, mainly in infants. viral pathogens play an important role in infants who present aris, respiratory syncytial virus (rsv) being the most important virus associated. in addition, children with rsv infections are also exposed to a variety of other respiratory viruses with a similar seasonal pattern, mainly during winter months, such as influenza, rhinovirus (rv), human metapneumovirus (hmpv), and human bocavirus (hbov). despite the fact that numerous studies have revealed that an important number of ari pediatric patients become simultaneously infected with multiple respiratory viruses, there are few studies focused on analyzing viral coinfections. this issue usually becomes a marginal part of the studies. over the past few years, several groups, including ours, have described various viral coinfection combinations compared with single ones, with different methodologies, and some of them observed worse prognosis with multiple viral infections, [ ] [ ] [ ] [ ] whereas others revealed a very similar prognosis for single virus infections. , thus, there is a need of a carefully designed study to shed some light on this issue. we aimed to compare, in a prospective study, clinical characteristics and severity of single versus viral coinfections, defined as simultaneous detection of rsv with rv, hmpv, or hbov, in a large cohort of hospitalized children. the study population comprised of all children less than years of age with ari admitted to the secondary public hospital severo ochoa (leganés, madrid), between september and august . the study was approved by the medical ethics committee (carlos iii). informed consent was obtained from parents or legal guardians. exclusion criteria were refusal to participate. all patients were evaluated by an attending physician. clinical characteristics of patients were analyzed. during the hospital stay, and as part of the study, a physician filled out a study questionnaire with the following variables: age, sex, month of admission, clinical diagnosis, history of prematurity and underlying chronic diseases, need for oxygen therapy, evaluated via transcutaneous oxygen saturation, axillary temperature (! c), presence of infiltrates and/or atelectasis in chest radiographs, administration of antibiotic therapy, length of hospital stay, total white blood cell (wbc) count, c-reactive protein (crp) serum levels, and blood culture results (for those cases in which such tests had been performed). asthma or recurrent wheezing was not considered an underlying chronic disease. ''upper respiratory tract infection'' (urti) was diagnosed in patients with rhinorrhea and/or cough and no signs of wheezing, dyspnea, crackles, or bronchodilator use, with or without fever. ''asthma'' was diagnosed on the basis of the national asthma education and prevention program guidelines. all other episodes of acute expiratory wheezing were considered to be ''recurrent wheezing.'' acute expiratory wheezing was considered to be ''bronchiolitis'' when it occurred for the first time in children aged less than years. ''laryngotracheobronchitis'' was associated with inspiratory stridor and wheezing. ''laryngitis'' was associated with inspiratory stridor without wheezing. cases with both focal infiltrates and consolidation in chest radiographs were, in the absence of wheezing, classified as ''pneumonia.'' however, if wheezing were present, eventhough there was a radiographic infiltrate, the patient was classified as having episodes of wheezing. specimens consisted of nasopharyngeal aspirates (npas) taken from each patient at admission. each specimen ( for patient) was sent for virological investigation to the respiratory virus and influenza unit at the national microbiology center (isciii, madrid, spain). npas were processed within hours after collection. upon receipt, aliquots were prepared and stored at À c. both the reception and the npa sample processing areas were separate from those defined as working areas. three reverse transcription (rt)-nested polymerase chain reaction (pcr) assays were performed to detect a total of respiratory viruses. in these assays, the rt and first amplification round were carried out in a single tube using the qiagen onestep rt-pcr kit (qiagen, hilden, germany). influenza a, b, and c viruses were detected by using previously described primer sets only to amplify influenza viruses in a multiplex pcr assay. a second multiplex pcr was used to detect parainfluenza viruses to (piv), human coronaviruses (cov) e and oc , enteroviruses (evs) and rvs. presence of respiratory syncityal virus (rsv) a and b types, hmpv, hbov, and adenoviruses (adv) were investigated by a third multiplex rt-nested pcr-brq method. continuous variables were described using mean and standard deviation. categorical variables were described using absolute and relative frequencies. clinical characteristics of patients with single infections associated with rsv were compared with those associated with coinfections between rsv and hmpv, rsv or rv, and finally with rsv and hbov. to compare qualitative variables chi-square test or fisher exact test was used if there were items of data in a cell. for quantitative variables, as all of them followed a normal distribution, the means were compared using the student t test to compare the groups. when or more groups were compared, the test of analysis of variance (anova) was used, followed up with post hoc tests to identify which groups differ from the rest. a -sided value of p < . was considered statistically significant. results were adjusted for age. all analyses were performed using the statistical package for the social sciences (spss), version . . the study population consisted of children less than years of age who had a total of episodes of hospitalization for respiratory causes. a total of episodes ( %) had a positive respiratory viral identification ( . % were single infections). among them, episodes were caused by viral coinfection. out of multiple viral infections, dual infections between rsv and other viruses were analyzed. a comparison between single and dual infections was performed. viral isolations (single and multiple) are shown in figure . we detected rsv single infections and rsv multiple infections (fig. ) . we selected dual rsv infections and this group was analyzed and compared with single infections. out of them, we achieved a significant number of cases for comparing between dual rsv and rv (n ¼ episodes), and between rsv and hbov (n ¼ episodes). other dual infections were rsv plus hmpv (n ¼ episodes), rsv plus adv (n ¼ ), rsv plus influenza (n ¼ ), piv (n ¼ ), cov (n ¼ ), and ev (n ¼ ). a total of single rsv infections were compared with rsv plus rv infections and with rv single infections ( table ). the mean age of the children was different between the groups (p < . ), with . months on average for rsv, months for rv infections, and an intermediate age of . months in the coinfection group. the proportion of males ( %) was higher in the rv single group (p ¼ . ). fever was more frequent in children with rsv infection ( %) and coinfections with rv ( %) than in rv ones ( %) (p < . ). patients with coinfections had a risk of fever that was . (confidence interval [ci] . - . ) times higher than rv infections, and similar to rsv. also, hypoxia was more prevalent in children with rsv ( %), and rsv plus rv ( %), than in rv single infections ( %) (p < . ). risk of hypoxia was twice as high in infants with coinfection, than in rv alone, and similar than in infants with rsv. duration of hypoxia was day longer in children with rsv alone or in coinfection, than in rv single infections (p < . ). the clinical picture was also significantly different (p < . ). rv single infections were mainly associated with recurrent wheezing ( %), whereas rsv single infections were mostly diagnosed with bronchiolitis ( %). coinfections between them had an intermediate proportion of both clinical diagnoses with % of bronchiolitis and % of recurrent wheezing or asthma. pneumonia was more prevalent in children with rv single infections ( %) than in the other groups. mean crp (p < . ) and leukocyte count in blood (p ¼ . ) were significantly higher in children with rv than in infants with rsv or coinfections (table ) . hospitalization was shorter in rv infection children than in infants with rsv or in coinfection (p < . ). admission to pediatric intensive care unit (picu) was higher in children with single rsv infection or in coinfection, than in rv single infections (p ¼ . ). the coinfection rsv plus rv had a risk of admission in picu . times higher than the rv single infection (ci . - . ). nevertheless, there were no statistical differences between rsv alone or in coinfections to picu admission. monthly distribution of infections was also different among groups (p < . ), and it is shown in figure . coinfections between rsv and rv match up with rsv circulation, mainly in january, november, and december, whereas rv single infections occur throughout the whole year, with the highest proportion during september and october. a total of single rsv infections were compared with rsv plus hbov infections and with hbov single infections ( table ). total number of hbov detections was ( . % of viral detections), and of them ( %) were coinfections with other different viruses. mean age of the children was different between the groups (p < . ), with an average of . months for rsv, months for hbov infections, and an intermediate age of . months in the coinfection group. proportion of males ( %) was higher in hbov single group (p ¼ . ), and coinfections were more frequent in females ( %). coinfections of rsv plus hbov had fever more frequently ( %; p ¼ . ) than the other groups, and also a higher percentage of hypoxia ( %; p < . ), whereas the hbov group had only % of the latter and lasting about day less (p ¼ . ). clinical diagnosis for the coinfection group was recurrent wheezing and bronchiolitis in approximately equal figures ( % and %), and it was different to single infections (p < . ). bronchiolitis was the most frequent in rsv infections ( %) and recurrent wheezing was the most common in the hbov ones ( %). pneumonia was diagnosed in % of the hbov group. hospitalization stay was longer and similar in the rsv groups (single infection or coinfection) and nearly day more ( . vs . days; p < . ) than in hbov single infections. there were no differences in percentages of picu admission. mean crp (p < . ) and leukocyte count in blood (p ¼ . ) were significantly higher in hbov infections than in rsv single infections or in coinfections (table ) . antibiotic therapy was prescribed more frequently in hbov infections than in the other groups (p < . ). seasonality of both hbov infections and hbov plus rsv coinfections was observed in november and mainly in december (more than half of the coinfections) (p < . ) (fig. ). in this study, rsv coinfections were frequent. they were detected in % of the analyzed episodes of aris caused by this virus. although they were mainly dual infections, or more viruses were also detected simultaneously. the most frequent dual infection was between rsv and rv, and the second was between rsv and hbov. nevertheless, coinfections with all respiratory viruses were detected. adenovirus coinfections were the third most frequent, but the majority of them were found as multiple viral associations with inadequate dual cases for analysis. there are some systematic reviews about coinfections with different results. we focus our attention towards study conducted in the united states, with data corresponding to a single hospital, and the authors found that dual infections had more ratio of hospitalizations than single infections. however, recently, other reviews have been published and do not find evidences suggesting that there are any differences in disease severity between coinfections compared to single viral infections. , only one of them was performed in children. we agree with the appreciation that coinfections do not increase the severity, and in anyway, we think that studies should focus on dual versus multiple infections, since the different characteristics of the virus can cause a bias when analyzing the results all together. one of the strengths of our study is that we analyzed virus in pairs and only when the number of infected patients to compare was big enough to allow us to draw conclusions. to our knowledge, our study has the highest number of patients in pairs of virus to date. dual infections between rsv and rv have different characteristics to single infections of each virus. children with confections were around year old, whereas rsv single infections affected younger infants and rv was found in older children of almost years of age. in our series, coinfections had significantly more proportion of fever, and also more proportion of hypoxia than single infections of rsv o rv. the coinfections had a risk of fever . times higher than single infections, and the risk of hypoxia was twice as high. the presence of rsv implies a worse prognosis with increased duration of both hypoxia and days of hospitalization, whether in simple infection or in coinfection. the risk of admission in picu was . (ci . - . ) in patients with coinfections versus in those with rv single infections, but there were no differences in patients with rsv single infections. this supports the idea that rsv infection causes the severity. there are few studies focused on coinfections between rsv and rv, and the number of patients analyzed is still small. the most extensive evaluation of the role of rv infections, whether single or multiple in children, is the study by costa and queiróz. they analyzed inpatient and outpatient children with rv infections, and of them had coinfections, mainly with rsv. they found that rv single infections were less severe than coinfections rv plus rsv, requiring less proportion of hospital admission. patients with single rv were older than single rsv and coinfections, and the most frequent diagnosis was recurrent wheezing. similar to our series, there were no differences between rsv and coinfections of rv plus rsv with regard to severity. in our patients, the diagnosis of pneumonia was more common in single rv infection, and these children characteristically had leukocytosis and elevated crp more frequently than those with simple infections or coinfections of rsv plus rv. other authors have described the role of viral respiratory infections, and especially rv, as a risk factor to develop invasive pneumococcal pneumonia, , and we assume that this could explain the high frequency of pneumonia in our series. coinfections of rsv and rv in our series had a seasonality that exactly matches up with the rsv circulation during november and december, confirming the importance of rsv in this association. dual infections of rsv and hbov are also frequent and the interpretation of their role is interesting. up to % of the hbov infections are coinfections with one or more viruses, and have a prolonged viral shedding around days in outpatients, and up to . months in hospitalized children, which probably explains why hbov dna has often been found in asymptomatic cases. this is the reason that explains why some authors consider hbov as not a pathogenic virus. nevertheless, the severity of the illness was associated with hbov infections in children attending daycare, and reinfections contribute to long-term shedding. on the contrary, other studies have suggested that hbov is the most likely cause of respiratory tract disease if the patient has a single infection, high viral load in npa, and viremia. , a severe and life-threatening disease has recently been well documented in an -monthold child with acute respiratory distress in germany. also, hbov has been detected as the third agent causing confirmed pneumonia, , and so, all of them support the pathogenic role of this virus. and finally, serologic diagnosis of hbov has confirmed that it is a true pathogen in respiratory tract infections in children. unfortunately, this tehchnique is not yet available in many laboratories including ours. in our study, clinical features were different in children with hbov and rsv single infections. the most common clinical diagnosis in children with rsv was bronchiolitis. these children had hypoxia more frequently and a more prolonged stay in hospital than children with hbov, but there were no differences in terms of picu admission. in contrast, pneumonia was much more frequent in children with hbov; this could explain the higher wbc counts and crp levels in these patients. dual rsv and hbov shared clinical characteristics from both viruses, and the most frequent clinical diagnosis in these children was recurrent wheezing. we can show results with our series in which hbov infections have a pathogenic role. it can also be observed that the coinfections with rsv have different clinical aspects when compared to single rsv infections. a larger number of patients would be needed to conclude if there is any difference in severity between single and multiple infections. semple et al and greensill et al in the united kingdom found a strong association between coinfections of hmpv and rsv in infants with bronchiolitis and more severity, including admission to the icu. this series detected that out of ventilated infants had a coinfection in an epidemiologic season. other prospective studies performed in europe, and mainly in holland, did not find this association or any coinfections between rsv and hmpv. in our study, during consecutive seasons, we only found dual coinfections. the different seasonality pattern of rsv (mainly november and december) and hmpv (mainly in spring) might explain these results. probably, an atypical circulation of hmpv during the season studied in the united kingdom allowed the association, but it is not a common condition in respiratory infections in europe. therefore, we cannot evaluate if the coinfection rsv-hmpv increases the severity or not. other groups of viruses such as influenza, adenovirus, or parainfluenza, not included by us, should be analyzed in future prospective studies. other limitations of our study are that we have not studied shedding of hbov or performed serological studies. despite this, we think that our study provides evidence of the pathogenic role of hbov, as well as the characteristics of studied coinfections. regardless of the viruses studied, it is a common finding in the literature that coinfections present more fever than single infections. this is also the case in our series, both for rsv-rv and rsv-hbov coinfections. despite this fact, we cannot conclude that coinfections are more severe than single infections, and the severity in our patients was associated mainly with rsv infection either alone or in combination with other viruses. respiratory viral infections during the - winter season in central england, uk: incidence and patterns of multiple virus coinfections multiple simultaneous viral infections in infants with acute respiratory tract infections in spain dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis simultaneous infection with respiratory syncytial virus and other respiratory pathogens viruses in communityacquired pneumonia in children aged less than years old: high rate of viral coinfection the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis high rate of viral identification and coinfections in infants with acute bronchiolitis simultaneous multiple viral infections in childhood acute lower respiratory tract infections in southern taiwan expert panel report: guidelines for the diagnosis and management of asthma update on selected topics simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays detection of new respiratory viruses in hospitalized infants with bronchiolitis: a threeyear prospective study dual respiratory virus infections single and multiple respiratory virus infections and severity of respiratory disease: a systematic review clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis human rinovirus and disease severity in children high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia viruses and bacteria in sputum samples of children with community-acquired pneumonia clinical characteristics of human bocavirus infections compared with other respiratory viruses in spanish children frequent and prolonged shedding of bocavirus in young children attending daycare prolonged detection of human bocavirus dna in nasopharyngeal aspirates of children with respiratory tract disease clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study human bocavirus: primary infection and shedding in infants human bocavirus commonly involved in multiple viral airway infections high prevalence of human bocavirus in infants with lower acute respiratory tract disease in argentina severe human bocavirus infection impact of viral infections in children with community-acquired pneumonia: results of a study of respiratory viruses. influenza other respir viruses spectrum of respiratory viruses in children with community acquired pneumonia serologic diagnosis of human bocavirus infection in children human metapneumovirus in severe respiratory syncytial virus bronchiolitis human metapneumovirus and severity of rsv disease absence of human metapenumovirus co-infection in cases of sever rsv infection key: cord- -eozvlu z authors: britton, tom; juher, david; saldana, joan title: a network epidemic model with preventive rewiring: comparative analysis of the initial phase date: - - journal: nan doi: nan sha: doc_id: cord_uid: eozvlu z this paper is concerned with stochastic sir and seir epidemic models on random networks in which individuals may rewire away from infected neighbors at some rate $omega$ (and reconnect to susceptible individuals with probability $alpha$ or else simply drop the edge if $alpha= $), so-called preventive rewiring. the models are denoted sir-$omega$ and seir-$omega$, and we focus attention on the early stages of an outbreak, where we derive expression for the basic reproduction number $r_ $ and the expected degree of the infectious nodes $e(d_i)$ using two different approximation approaches. the first approach approximates the early spread of an epidemic by a branching process, whereas the second one uses pair approximation. the expressions are compared with the corresponding empirical means obtained from stochastic simulations of sir-$omega$ and seir-$omega$ epidemics on poisson and scale-free networks. without rewiring of exposed nodes, the two approaches predict the same epidemic threshold and the same $e(d_i)$ for both types of epidemics, the latter being very close to the mean degree obtained from simulated epidemics over poisson networks. above the epidemic threshold, pairwise models overestimate the value of $r_ $ computed from simulations, which turns out to be very close to the one predicted by the branching process approximation. when exposed individuals also rewire with $alpha> $ (perhaps unaware of being infected), the two approaches give different epidemic thresholds, with the branching process approximation being more in agreement with simulations. interactions among individuals in a population can be described by networks of who-contactswhom. studies of contact networks in sexually transmitted diseases have long revealed a high variability in the number of contacts per individual and highlighted the importance of those individuals described as "super-spreaders" for the onset of an epidemic [ , ] . similar conclusions about the importance of super-spread events were drawn from contact tracing data collected from recent epidemic outbreaks of airbone-transmitted diseases like those of the severe acute respiratory syndrome (sars) in and [ , ] . on the other hand, the risk perception among people during an epidemic outbreak triggers behavioural responses to lower the risk of contagion [ , ] , the avoidance of contacts with infected individuals being an example of such responses [ ] . this sort of social distancing led to the idea of disease-avoiding link rewiring and is one of the basis of the so-called adaptive or dynamic networks. such a preventive rewiring assumes transmission of information which allows people to gather knowledge about the disease status of their neighbours. therefore, in such networks the contact pattern is no longer static but evolves with the spread of an infectious disease according to the rules defining the rewiring process [ , , , , , ] . pairwise models have been the main approach used to analyse epidemic dynamics on adaptive networks [ , , , , , , ] . this class of models was initially developed to deal with processes defined on regular (random) networks and offers a good description of their dynamics. in their classic formulation and over heterogeneous networks, however, their accuracy is far from being satisfactory, especially for its prediction of the epidemic threshold. the so-called effective degree models are extensions of them with a higher accuracy in their predictions (but also with a higher complexity). in these models, in addition to the disease status of nodes, the number of neighbours for each status is also considered [ , , ] . in addition to the previous approach, stochastic models have been used to analyze epidemic outbreaks on dynamic networks. if the contact network has a large size and no cycles, it can be locally described as a tree and the initial phase of an epidemic can be approximated by a branching process [ ] . an example of a stochastic model defined on a dynamic network is the one developed in [ , ] . the model assumes that, at a given rate, the identities of neighbours change stochastically by means of an instantaneous edge swap between a randomly selected pair of links. so, this neighbour-exchange mechanism is independent of the epidemic dynamics because it does not depend on the disease status of the involved nodes. in other words, it is not an example of behavioural response against the presence of the disease. other models defined on dynamic networks whose architecture evolves by random edge swapping can be found in [ ] . this paper aims mainly at comparing the predictions from both modelling methodologies (pairwise/stochastic) for the initial phase of susceptible-infectious-recovered (sir) and susceptible-exposed-infectious-recovered (seir) epidemics with preventive rewiring among individuals (so, with an interplay between the spread of the disease and the rewiring process, that is, between disease's dynamics and network dynamics). in particular, in the sir model we will assume that susceptible individuals break off connections with infectious neighbors at a given rate ω and, in place of them, new connections to susceptible and recovered individuals are created with probability α. as for the seir model, we consider two alternative scenarios for the dynamics of exposeds (i.e., infected but not infectious individuals). in the first one, exposed individuals break off with their infectious neighbors at a rate ω ei and, with probability α, they reconnect to any non-infectious individual in the population. in turn, susceptibles can also reconnect, with the same probability α, to exposeds (in addition to other susceptibles and removed individuals) when breaking off with infectious neighbors. in the second scenario, exposed individuals do not rewire at all (ω ei = ), and susceptibles rewire away from both exposeds and infectives, and create new connections with probability α. from a modeling viewpoint, the rewiring scenario can depend on whether exposed individuals realize they have been infected (for instance, because they show symptoms) or not (they are asymptomatic). in both scenarios, the degree distribution changes over time and its mean degree is preserved only when α = . the introduction of a reconnection probability α allow us to consider different degrees of rewiring, ranging from a situation where each deleted link is replaced by a newly created one (α = ) to the limit case where no new connection is made and edges are simply deleted (α = ). in other words, α can be though of a measure of the intensity of social distancing of rewiring individuals. as it is claimed in [ ] , people value person-to-person contacts and are willing to accept some disease risk to gain contact-related benefits. so, different values of α could be considered according to the type of social relationship modeled by the network. the basic reproduction number r , namely, the average number of infections produced by a typical infectious individual when the fraction infected is still negligible, is one of the compared quantities. its predicted value will be checked against stochastic simulations carried out on contact networks with degree distributions that follow a poisson distribution and a power law, respectively. it is worth noting that, while the meaning of r in randomly mixing homogeneous populations is straightforward because any infectious individual is as likely as any other to infect a susceptible one, in heterogeneous networks it requires that one specifies the meaning of typical individual [ , ] . in most network models (in particular, for those without multiple levels of mixing), this definition implies that one has to compute r from the average number of infections per infective once the early correlations of disease status around infectious individuals have been formed, which takes a couple of generations after the occurrence of the primary cases. interestingly, this computation/redefinition of r has been obtained under both previous modelling approaches [ , , ] . in fact, it is well known that both pairwise models and branching process approximations lead to the same epidemic (or invasion) threshold in networks without rewiring [ ] . in section we present the sir and seir epidemic models with rewiring, here denoted by sir-ω and seir-ω respectively. in section we use the branching process approximation to analyse the early phase of an sir-ω epidemic. in particular, we compute r and the expected degree of infectives during this initial phase as a function of the rewiring rate ω. in section the same approximation is applied to the study of the early stage of an seir-ω epidemic under different types of rewiring processes. section contains the results of the initial phase obtained for both epidemic models using the pair approximation with the triple closure introduced in [ , ] for heterogeneous networks. in particular, the seir-ω pairwise model extends the one considered in [ ] to account for the rewiring of exposed individuals and the possibility that susceptibles reconnect to exposed ones after breaking off an infectious link. in section numerical estimates of r and the mean degree of infectives during the initial phase are obtained from continuous-time stochastic simulations on heterogeneous networks. finally, in section we discuss the analytical results obtained from both approximations and compare them to the output of the stochastic simulations. moreover, we comment about the new insight into the role of the rewiring process in the seir-ω epidemic model. let us define our stochastic network epidemic model. the population consists of a fixed number n of individuals and the stochastic network model is given by the configuration model with degree distribution d ∼ {p k } having finite mean µ and finite variance σ (e.g. [ ] ). this model is defined by all individuals having i.i.d. degrees d i and edge-stubs being pairwise connected completely at random with any loop or multiple edge being removed making the graph simple. we are primarily interested in the situation where n is large, and the approximations will rely on this. on this network we now define an epidemic model where susceptible individuals may rewire if they are neighbors of infectious individuals. we start by defining an sir epidemic where infected people immediately become infectious and later recover, and then extend the model to an seir model in which infected people are at first exposed (latent), then they become infectious, and eventually they recover. the latter model is a bit more complicated in that now the rate of rewiring could differ depending on whether the person rewiring is susceptible or exposed, and depending on whether the person he/she rewires away from is exposed or infectious. note that such a rewiring process causes the degree distribution to change over time. however, since we will focus our analysis on the early stage of an epidemic outbreak where only a tiny fraction of individuals is initially infected, we will assume that the degree distribution d does not change significantly during the initial phase of the epidemic. in this model individuals are at first susceptible. if an individual gets infected he/she immediately becomes infectious, and after some random time he/she recovers and becomes immune for the rest of the outbreak. sir hence stands for susceptible-infectious-recovered (e.g. [ ] for more on sir and seir epidemic models). the sir-ω model is defined on the network (described above) as follows. initially one randomly selected individual is infectious and the rest are susceptible. an infectious individual transmits the disease to each of its susceptible neighbors at a rate β, and the infectious periods are i.i.d. following an exponential distribution with rate parameter γ (so infectious individuals recover at a rate γ). further, susceptible individuals that are neighbors with infectious ones break off with such neighbors independently at rate ω and, with probability α, replace each lost connection by reconnecting to a randomly selected non-infectious (i.e., susceptible or recovered) individual in the community. therefore αω is the effective rewiring rate, i.e., the rate at which new links are created by susceptibles in substitution for those previously deleted. the sir-ω network epidemic has the following parameters: β (infection rate), γ (recovery rate), ω (rewiring rate), α (reconnection probability), and the degree distribution d with mean µ and variance σ . we will focus on what happens early on in the epidemic, before a substantial fraction of the community has been infected. in the seir-ω model an infectious individual transmits the disease to each of its susceptible neighbors at a rate β, but, when this happens, the neighbor first becomes exposed (or latent) and can transmit the disease only after a time delay. in other words, such an exposed individual becomes infectious at a rate φ, and at this moment can start infecting each of its susceptible neighbors at a rate β. as before, infectious individuals recover at a rate γ. as regards to rewiring, it can be modelled differently depending on when an individual starts and stops having a rewiring rate and also depending on which individuals it rewires away from. our model considers three different rewiring rates: a susceptible individual rewires away from each exposed neighbor at a rate ω se , a susceptible individual rewires away from each infectious neighbor at a rate ω si , and an exposed individual rewires away from each infectious neighbor at a rate ω ei . it is of course possible to also allow for rewiring from other states, e.g. that susceptible individuals rewire from recovered individuals, but since we are primarily focusing on the initial phase such rewiring would have negligible effects. if infected people are detected only when they become infectious (e.g. if the latent period is the same as the incubation period), and hence individuals rewire away from infected neighbors only when those become infectious and not while they are exposed, this would correspond to ω se = and ω si = ω ei > : susceptible individuals are not aware of exposed (latent) neighbors being infected and hence do not rewire, and susceptible but also exposed (latent but unaware) individuals rewire away from the infectious ones. we could obtain a second scenario if also exposed (latent) individuals are known to have been infected (e.g. by contact tracing or because they show some symptoms). we would then have ω se = ω si > and ω ei = : susceptible individuals rewire away both from exposed and infectious neighbors but exposed individuals do not rewire away since they know they have already been infected. in all cases, the reconnection probability α modulates the fraction of rewirings that are effectively done, and it is assumed to be the same both for susceptibles and for exposeds. therefore, αω ij is the effective rewiring rate of individuals in state i away from individuals in state i. the seir-ω network epidemic has all the parameters of the sir-ω epidemic except that the rewiring rate ω now becomes three different rates: ω se , ω si and ω ei , and there is a rate φ at which latent individuals become infectious. most stochastic epidemic models allow for a branching process approximation of the early stages of an outbreak, an approximation which can be made rigorous as the population size n tends to infinity (e.g. [ ] ). this applies also to network epidemics -we now describe the approximation of the current model. we derive expressions for the basic reproduction number r , here denoted by r ba to distinguish its expression from the one obtained using pair approximation. we also derive the exponential growth rate r (the malthusian parameter) for the situation that r > , and the average degree of infected individuals. since rewiring is a focus of this paper, we look at both the degree of newly infected individuals as well as on the average of all infectious individuals, the latter expected to be smaller than the former since individuals rewire away from infectious neighbors. recall that r is defined as the mean number of new infections caused by a typical infected individual during the early stage of the epidemic. individuals that get infected during the early stage will, at the time of infection, have the size biased degree distribution of neighbors, d ∼ {p k }, wherep k = kp k / j jp j = kp k /µ (e.g. [ ] ). one of the neighbors is its infector whereas the remaining neighbors, with large probability during the early stage of an outbreak, will be susceptible. when considering the disease progress it is only thed − susceptibles that are of interest since it is not possible to reinfect the infector. from the derivation above, the mean number of susceptible neighbours a typical new infectee has during the early stages equals (e.g. [ ] ). the probability to infect a given such neighbor is obtained by considering the competing events that may happen: there could be an infection (rate β), the neighbor could rewire away from the infectee (rate ω), or the infectee can recover (rate γ). the probability of infection hence equals β/(β + γ + ω). the basic reproduction number equals the mean degree multiplied by the transmission probability, i.e. if there is no rewiring (ω = ), the basic reproduction number equals e(d − )β/(β + γ) as is well known. therefore, the rewiring reduces r , as expected. we note that r ba is independent of α, so it has no effect on the beginning of an outbreak if rewired edges are dropped, always attached to new susceptible individuals or a mixture of two. during the early stage and assuming a large population, the number of infectives in the epidemic will asymptotically (as n → ∞) evolve like a branching process. r is the corresponding mean offspring distribution. another important quantity associated to this branching process is λ(t), the expected birth rate (rate of new infections) of an infectee having "age" t, where age corresponds to time since infection. we now derive λ(t) which in turn will help us derive the exponential growth rate of the epidemic. as derived earlier, the average number of susceptible neighbors upon infection is e(d − ) = µ − + σ /µ, and the infectee will infect each neighbor independently. the average rate of infection (="birth") for each neighbor is obtained by considering what must be fulfilled for infection to happen. in order to infect a neighbor t time units after infection, the infectee must still be infectious, the neighbor should not have rewired, and the infectee should not yet have infected the neighbor. given this, the infection rate equals β. since all events are assumed to follow an exponential distribution this gives us the following expression for λ(t): the average total number of births (infections) is hence ∞ λ(t)dt = e(d− )β/(β +γ +ω) = r as it should be. the mean birth rate λ(t) also determines the exponential growth rate of the epidemic, i.e. for which r, i(t) ∼ e rt (cf. [ ] ). this r, the malthusian parameter, is given by the solution of the lotka-volterra equation for our model this gives us, after a bit of algebra, also the exponential growth rate is independent of α. we now turn to the mean degree of infectives during the early stages of the epidemic. we consider two different means. the first one is for newly infected, which in fact has already been shown: during the early stages newly infected individuals will have degree distributiond (when considering the degree distribution we also count the non-susceptible infector), so the mean degree of newly infected individuals equals e(d) = µ + σ /µ. the second mean, e(d i ), denotes the average number of neighbors of all infectives during the early stages, not only that of the newly infected ones. as described above, during the early stage of an outbreak the degree distribution of newly infected equalsd. however, while still infectious, an individual loses susceptible neighbors by rewiring: each susceptible neighbor is lost at a rate ω. the probability that a susceptible neighbor v is still a neighbor (i.e. has not rewired) t time units after our individual x was infected and given that x is still infectious, is obtained by conditioning on the potential infection time of the neighbor (v only rewires if not yet infected). so, we have p (v still a neighbor at t) = t p (v still a neighbor at s | v infected at s)βe −βs ds note that we condition on that x remains infectious at t. when deriving the degree distribution of all infectives during the early stage, we have to take into account both this decrease of degree with age, but also the fact that, in the exponential phase of the epidemic (recall that i(t) ∼ e rt ), "young" infectives will be over-represented. the ratio of individuals infected s time units ago over the number of individuals infected at present equals e −rs due to the exponential growth rate. and only a fraction e −γs of them are still infectious at present. consequently, the fraction of infectives that were infected s time units ago or longer equals e −(r+γ)s , so the age distribution of infectives is exponential with parameter r + γ (this is the so-called stable age distribution of this branching process, [ ] ). the mean degree of all infectives during the early stage is obtained by conditioning on their age: where, as before, e(d) = µ+σ /µ, and r = βe(d − )−γ −ω was defined in equation ( ). this mean degree should be valid after a couple of generations and will then change as the depletion of susceptibles will start affecting things. as seen in ( ) also the mean degree of infectives is independent of α. at first this might seem surprising since the degree of infectious individuals are affected by rewiring. however, an infectious individual can loose edges (which reduces the degree) due to susceptible neighbours rewiring away from the infective, but it does not affect the degree of the infective whether these links are simply dropped or the rewiring susceptible connects to new individuals. we now study the extended seir model recalling that individuals who get infected are now first latent for an exponentially distributed time with rate parameter φ, after which they become and remain infectious according to earlier rules. individuals rewire away from infected neighbors. more precisely, a susceptible individual rewires from each exposed (latent) neighbor at a rate ω se and from each infectious neighbor at a rate ω si . moreover, exposed individuals rewire away from infectious neighbors at a rate ω ei . of course, some of these rewiring intensities may be zero (cf. sec . ). as before, upon each rewiring event the individual reconnects to a randomly chosen susceptible or recovered individual with probability α and with the remaining probability the edge is simply dropped. during the early stage of an outbreak, at the time of infection an exposed individual e has degree distributiond as before, one neighbor i being the infector and the remaining neighbors being susceptible. so, e has e(d) − expected susceptible neighbors. however, in the seir model this number can eventually increase by one, provided that e can rewire away from its infector i (if not yet recovered) and reconnect to a susceptible individual that will become a new neighbor. the probability for this to happen is αω ei /(φ + ω ei ). in consequence, the expected number of susceptible neighbors for e is e(d) − + αω ei /(φ + ω ei ). any such neighbor, say s, will get infected if e first becomes infectious (before s rewires away from e) and, then, an infection occurs (before e recovers or s rewires from e). hence, the probability for this to happen is the product of the probabilities of these two events, namely, φ/(φ + ω se ) · β/(β + γ + ω si ). to conclude, the expected number of neighbors infected by e equals: by studying eq. ( ) we make the following observations. if there is no rewiring from any state, r ba reduces to β/(β + γ) e(d − ), i.e. the same as for the sir case. further, r ba is decreasing in both ω se and ω si as expected. however, r ba increases with α and ω ei . if ω se = and ω si = ω ei = ω, the perhaps most realistic example discussed in sec. . , then r ba can be increasing in ω for some parameter set-ups, implying that the quicker individuals rewire the larger epidemic outbreak! the explanation to this is that, when αω ei > , the exposed (latent) individuals can rewire away from their infector to a susceptible neighbor, with the effect that they may later (once they become infectious) infect the new susceptible neighbor. as with the sir-ω model, in order to compute the malthusian parameter r we first derive an expression for λ(t), the average rate at which an individual, who was infected during the initial phase of the epidemic, infects new individuals t time units after his/her time of infection. at the time when an individual gets infected he has on average e(d) neighbors, one being infectious (its infector) and the remaining e(d − ) will, with large probability since we are in the beginning of the epidemic, be susceptible. at a rate β, the infected individual infects each of the e(d − ) initially susceptible neighbors t time units after infection if the following conditions are fulfilled: the infected individual must have terminated the latent period without the neighbor having rewired, and after that the infectious period should still be active, an infection should not yet have taken place, and the neighbor should not have rewired. as mentioned in the previous subsection, it is also possible that the infected individual infects through the link to the infector. this happens with a rate β at t time units after infection if the following holds: the infected individual rewired from its infector (and connects to a susceptible neighbor) while still latent, and after this the infected individual has become infectious, has not yet infected the neighbor nor has the new neighbor rewired. the above reasoning leads to the following expression for λ(t): λ(t) = βe(d − )p (infectious, neighbor did not rewire, neighbor not yet infected, at t) + βp (rewired while latent, infectious, neighbor not rewired, neighbor not infected, at t). by conditioning on the end of the latency period, the first probability equals the second probability, now conditioning both on the time of first rewiring and the end of the latent period, equals using these expressions in equation ( ) and solving the integrals results in the following expres-sion for λ(t): we now use λ(t) to derive the exponential growth rate r of the epidemic in case it takes off, and also to confirm our expression for r . the latter is easy. if we compute ∞ λ(t)dt using the expression above we get exactly r as defined in eq. ( ), as it should be. as for the malthusian parameter r this is given as the solution to the equation ∞ e −rt λ(t)dt = . for the expression of λ(t) above, this can be shown to be equivalent to for αω ei > , this is a third order equation, but for positive values of r (the relevant values as we assume r > ) the left hand side is decreasing in r, starting from a value larger than when r = and decreasing to as r → ∞ implying that there is a unique solution to the equation. equation ( ) is not explicit but it is still possible to see how various parameters affect the growth rate. for example, r is increasing in the infection rate β and the mean degree e(d). as regards to the rewiring rates, r decreases in ω si and ω se but increases with the "harmful" rewiring rate αω ei . finally, as we increase the rate to leave the latent state (i.e. making the latent state shorter), the effect depends on other parameter values, but if we increase φ towards infinity it can be shown that we obtain the expression for r of the sir-ω model (cf. eq. ( )) as expected. for the seir-ω model it is also possible to derive specific features of infected individuals during the initial phase of an epidemic. for example, as we did for the sir-ω case, we can compute e(d i ), the average degree of infectives during the early stage of the outbreak. however, other mean quantities might be equally relevant as, for instance, the mean degree e(d l ) of infected but still latent individuals, or the mean degree e(d i+l ) of either latent or infectives, or for that matter the expected number of susceptible neighbors while in one of these states. for brevity and because some of these quantities have even more complicated expressions, we compute e(d i ) without solving the integrals appearing in the derivation, and indicate how to modify the derivations if we want to compute another mean. to compute e(d i ), let us pick at random an infectious individual i during the early stage of an outbreak and let t denote how long ago this individual was infected. we first compute the expected degree of i conditional upon t = t, denoted by e(d i |t = t). this is done by conditioning on the duration of the latent period l = s, which must lie between and t since i is infectious t time units after infection: the second factor is given by f l (s|t = t) = φe −φs e −γ(t−s) / t φe −φu e −γ(t−u) du . as for the first factor, the individual has e(d) expected neighbours at the time of infection, one being infectious and the rest being susceptible. for the susceptible neighbors we compute the probability that they are still neighbors. for the infector, it could have lost a neighbor from this edge only if i rewired away from the infector to a susceptible neighbor and the new neighbor later rewired away from i. we hence get e(d i |t = t, l = s) = e(d − )p (susceptible neighbor did not rewire|l = s, t = t) ( ) + − p (i looses edge to infector |l = s, t = t). the first probability in ( ) is obtained by conditioning on whether the susceptible neighbor was infected or not, and, in the former case, whether the latent period ended before t or not: here there should be no α in front of ω ei because the event concerns an exposed neighbor and it is irrelevant for the degree of the infective whether this neighbor reconnects or not upon rewiring. the second probability in ( ) consists of two events. either i rewired away from its infector and dropped the edge while exposed, or else i rewired away from infector and reconnected while exposed, and the new neighbor rewired away from i. the first event has probability p (i rewired and dropped edge | l = s, the second event is obtained by conditioning on the time when i rewires away from the infector and reconnects to a susceptible neighbor, whether the second rewiring happens during the latency or infectious period of i, and in the latter case whether infection takes place or not: p (i rewired and reconnected, and new neighbor rewired away from i | l = s, t = t) note that α appears in front of ω ei only when it concerns the infective, because then it has to reconnect to make spreading to a new individual possible, whereas there is no α when the rewiring refers to exposed neighbors rewiring away from the infective. in the latter case it is irrelevant for the degree of the infective whether or not the exposed individual reconnects upon rewiring. it remains to derive the distribution for t , the time since infection. for this we know that due to the exponential growth rate r of infectives, there is a fraction e −rt to choose from t units earlier as compared to present time. however, we also require that the individual is infectious at present, an event which happens with probability t φe −φs e −γ(t−s) ds. the probability density of t is hence proportional to e −rt t φe −φs e −γ(t−s) ds, which after a bit of algebra gives the following density finally, the expected degree of a randomly chosen infective during the early stages is obtained by integrating with respect to this density: in order to compare this predicted e(d i ) with the one obtained from pair approximation (see next section), for each set of values of the parameters we obtain the value of r given by the positive solution of ( ) and evaluate the resulting expression of the previous integral. if we were to compute e.g. the average degree of a latent individual e(d l ), we would similarly condition on the time t since infection of the randomly chosen latent individual. this individual would have e(d) neighbours at the time of infection, one infectious and the rest susceptible, and we need to compute the probability that these neighbours would not have been lost similarly to what we did before. we would then integrate this expected value with respect to the probability density of t , which is proportional to e −rt e −φt . we now derive expressions for r and e(d i ) using an alternative deterministic approximation based on the closed-form equations for the dynamics of pairs of disease status, the so-called pairwise models. while the sir-ω pairwise model was already introduced in [ ] , the seir-ω pairwise model is a generalization of the one also introduced in [ ] that includes rewiring of exposeds and reconnection rules introduced in accordance with it. this extended model will allow for a better understanding of the impact of rewiring on r derived under this approach, here denoted by r p a . [ ] for details). note that, since we focus our analysis on the early epidemic stage with a very small number of initially infectious nodes, we approximate the expected degree of the susceptible central node of a triple by e(d), the mean degree of a node reached by following a randomly chosen link in a wholly susceptible population at t = , i.e., when the degree distribution is the initial one. upon introducing the triple closure into the original model, the initial dynamics of the sir-ω model is determined by [ir] [i] . it is interesting to observe that the reconnection probability α does not appear in the system. this means that disconnections from infectious individuals play a role in the early epidemic dynamics, but the way the new connections are created, or even if they occur at all (α = ), does not play any role at this stage. the first equation of ( ) is decoupled from the other two and has a unique positive equilibrium ([si]/[i]) * = e(d)− −ω/β, which is globally asymptotically stable. from this equilibrium and ( ), it immediately follows that which defines the same epidemic threshold r = as r ba (cf. eq. ( )), but overestimates r when it is larger than one. a graphical comparison of the expressions of r obtained from each modelling approach is shown in fig. using β as a tuning which is the same expression as the one obtained for e(d ba i ) (cf. eq. ( )). according to the rewiring processes described in section . , and using the same notation and the triple closure as before, the equations of the seir model at the initial phase of the and δ = otherwise. in the first case, susceptible individuals do not disconnect from exposed individuals and can reconnect to the latter when they rewire away from an infectious neighbor. in the second case (ω se > ), susceptible individuals recognize exposed ones, rewire away from them, and only reconnect (with probability α) to other susceptibles or to recovereds (so, δ = ). note that, in both cases, f ([s], [e], [r] ) → n at the early stage of an epidemic. if ω ei > , exposed individuals who break off a link with an infectious neighbor randomly reconnect to any susceptible, recovered, or exposed individual with a probability α , respectively. this corresponds to the situation where latent individuals are asymptomatic and, so, they do not know they have already got the infection. therefore, one can also assume that susceptible individuals do not know the disease status of the exposed neighbours and take ω se = . hence, susceptibles who break off a link with an infected neighbor reconnect to any susceptible, exposed or recovered individual with the same probabilities as the exposed ones, namely, α it is illustrative to check that the limit system for the dynamics of the local densities of disease status around an infectious individual (see appendix) only contains one term with the reconnection probability α, namely, the one with α ω ei as a prefactor. therefore, the other contributions from the remaining rewiring rates in ( ) do not appear when we restrict ourselves at the early stage of an epidemic. in other words, the precise rules for reconnecting susceptibles who have rewired away from an infectious neighbor, even if there is no reconnection at all of those individuals (α = ), does not affect the epidemic dynamics during the initial phase. this claim, however, is not true for exposed individuals. the last term of the equation for [se]/ [i] tells us that exposeds enhance the spread of the disease by rewiring away from infectious neighbors (because they will replace the latter with susceptible ones) and, hence, r p a must increase under this rewiring. the expression of r defined by ( ) , and computed from the corresponding positive equilibrium of the limit system for the local densities (see appendix) is given by is supercritical (i.e. larger than ) for smaller β than r p a . that is, both approaches predict the same epidemic threshold when exposed individuals do not rewire (α ω ei = ), but pair approximation predicts a higher epidemic threshold in terms of β than the one obtained from the branching process approximation when ω ei > (see the right panel in fig. ) . moreover, simulations show that r p a always overestimates the basic reproduction number when r ba > if ω ei = . finally, from eq. ( ) it follows that ξ * increases with ω ei , which implies that r p a also increases with ω ei as expected. on the other hand, from the positive equilibrium of the limit system for the local densities, note that, when there is no rewiring (ω si = ω se = ω ei = ), it follows that e(d p a i ) = e(d), as expected. moreover, when ω ei = , ξ * is given by ( ) and e(d p a i ) can be explicitly expressed in terms of the model parameters. for ω ei > and ω se = , the numerical evaluation of the previous expression and its comparison with that of e(d ba i ) show that both predictions are very close to each other (see fig. ). indeed, they are graphically distinguishable only for low values of β (left panel) or for high values of the rewiring rate ω (right panel), i.e., for those parameter values that give r close to . in these cases, differences occur at the second decimal place of the predicted mean degree. for ω ei = and ω se ≥ , both expressions give the same value for e(d i ) (at least until the twentieth decimal digit). to carry out continuous-time stochastic simulations we generated poisson networks with e(d) = and scale-free (sf) networks with characteristic exponent and minimum degree k min = , i.e. p(k) = k min k − . so, in both cases, e(d) = . all the networks had n = nodes. the sf networks were generated using the configuration model algorithm. for each network and each combination of parameters, we averaged the outputs over initial sets of individuals infected uniformly at random (primary cases). moreover, we take the reconnection probability α = in all simulations because it is when rewiring has the biggest effect. the stochastic time evolution of the infection spread was simulated by means of the gillespie algorithm [ ] . as mentioned in the introduction, since primary cases are selected at random regardless of their degree, a correct empirical computation of r relies on counting the mean number of infections produced by the secondary cases (individuals infected by the primary cases). so, for each experiment (that is, for each initial set of random primary cases) we let the epidemic evolve until all primary and secondary cases have recovered. in figs. and , corresponding respectively to sir-ω and seir-ω models, we compare the value of r predicted by eqs. ( ) and ( ) with that obtained from eqs. ( ) and ( ) respectively, and with the outputs of stochastic simulations carried out on a poisson and a scale-free network. since the variance of the theoretical sf degree distribution is quite high (it equals k m / = . ), there is a high variability among generated sf networks. therefore, in order to compare the results for both types of networks in the same figure, we have chosen a random sf network whose degree sequence leads to a value of e(d) very close to the expected one (µ = . , σ = . , and hence e(d) = . ). we have also tested the accuracy of the analytical predictions for e(d i ). recall that, for the sir-ω model, both approaches lead to the same value of e(d i ) (cf. eq. ( ) and ( ) is computed as the total number of links containing an infected individual (the edges joining two infected are counted twice) over i(t). the right panels of these figures show that the curve i(t) fits to an exponential function (initial phase) on an interval [ , t e ] with t e less than the time i(t) attains its maximum. it is precisely on this interval that the mean degree d i (t) on poisson networks keeps almost stationary around a value close to the predicted one (see left panels of fig. and ) . such a plateau in the profile of d i (t) is not so nicely observed when simulations are carried out on scale-free networks. it is known that pairwise models for the spread of sir-type diseases through static homogeneous networks predict the same epidemic threshold as the one obtained from the probabilistic computation of r when infectious periods are exponentially distributed [ , ] . by using a pairwise model with a triple closure introduced in [ , ] , and a branching process approximation of a stochastic network epidemic, we have seen that the same epidemic threshold is also predicted for dynamic networks whose topology evolves according to the preventive rewiring of susceptible individuals. as expected from a preventive rewiring, the higher the rewiring rate ω, the lower r is for both predictions, and this is true regardless of the value of the reconnection probability α. however, for any ω, the pair approximation overestimates r when it is larger than as compared to stochastic simulations and to the value r ba obtained from the branching process approximation. the reason is that the value r p a predicted by the pairwise model is a linear function of the infection rate β and, hence, an unbounded number of new cases is predicted as β increases (cf. eq. ( )). such a linear dependence on the infection rate is a common feature of deterministic epidemic models [ ] . in contrast, the hyperbolic dependence of r ba on β (cf. eq. ( )) reflects the saturation in the production of new infections for high infection rates, and leads to values of r that are closer to those obtained from the simulations. the same relationships between estimates of r , and between epidemic thresholds, also hold for seir-ω models when susceptible (but not exposed/latent) individuals break off connections with their infectious/exposed neighbours, and reconnect to randomly chosen susceptible or recovered individuals with a given probability α (ω ei = ). however, if exposed individuals also disconnect from infectious neighbours and reconnect to randomly chosen non-infectious individuals (α ω ei > ), then the epidemic thresholds from the two approaches differ from each other, with r ba > when r p a = . interestingly, as long as α > , this rewiring of exposeds is not preventive but harmful since it does not help to contain the disease: sooner or later exposed individuals will become infectious and, when an exposed replaces its infector with a susceptible one, the number of infections he/she can produce increases. this is why r increases with αω ei , in contrast to what happens with the other two rewiring rates, ω si and ω se . note that, because we are concerned with the initial stage of an epidemic, in large networks rewired links will, with a very high probability, point to susceptible individuals. therefore, as long as only susceptible individuals rewire, the initial propagation of the disease will not be particularly affected by the type and intensity (α) of the reconnection process. in fact, this is what follows from the computation of r under both approaches. for the sir-ω model, for instance, this can be easily seen from the derivation itself of r under the branching process approximation since the former does not depend on how new connections (if any) are made. similarly, from direct inspection of the limit system governing the dynamics of the local densities around infectious individuals (cf. eq. ( )), one sees that there is no term corresponding to reconnection of links (i.e., terms with αω as a prefactor). both estimates of r have been checked by obtaining, from stochastic simulations carried out on random networks, the mean number of infections produced not by the first infectious individuals landing in the population (primary cases), but by the second generation of infectives (secondary cases). it has been recognized elsewhere ( [ , , ] ) that this "redefinition" of r for epidemics on networks is the suitable one because it takes into account the local correlations of disease status developed around infectives during the epidemic exponential growth (initial phase). simulation results clearly indicate that the estimation of r obtained from the branching process approximation is much better than the one derived from pairwise models, and gives the correct epidemic threshold when ω ei > . in particular, for the seir-ω model there is an excellent agreement for all the shown values of β (fig. ) , whereas for the sir-ω model the agreement is not as good when β is not close to its critical value (fig. ) . on the other hand, for the sir-ω model we have also seen that both approaches predict the same expected degree e(d i ) of infectives at the early stage of an epidemic. in particular, it follows that e(d i ) is a linear decreasing function of the rewiring rate ω. its computation from stochastic simulations clearly shows that, for moderately large values of ω and β, the mean degree of the infectious nodes d i (t) remains quite constant during the exponential phase of the disease. moreover, the agreement between theoretical predictions and observations using both ω and β as tunable parameters is very good in poisson networks for low values of the rewiring rates and moderate values of β. for high values of β, the exponential phase is so fast that the time window where d i (t) is roughly constant is hardly noticeable. similarly, when rewiring is high, d i (t) decreases monotonously without any plateau during this initial phase. for scalefree networks and moderate values of β and ω, however, the predicted e(d i ) overestimates the observed d i (t) (cf. fig. and fig. ) . as for the seir-ω model, the values of e(d i ) computed from both approaches are very close to each other (see fig. ) and show a very good agreement with the simulations on poisson networks (see fig. ). however, the corresponding expressions are not easily manageable (both depend on the solution of a cubic equation when α ω ei > ) and, therefore, they have been evaluated numerically. from these evaluations, it follows that, when α > and ω ei = or, alternatively, when α = (dropping of edges), both approaches lead to the same values of e(d i ). for α ω ei > , predictions are almost graphically indistinguishable from each other (for α = and ω si = ω ei , the maximum differences occur at second decimal place of the expected degree). as with the sir-ω model, when the simulations take place on scale-free networks, the predicted e(d i ) overestimates the observed d i (t) (see fig. ). finally, it is important to note that, for dynamic heterogeneous networks whose degree distribution evolves in time, the value of r does not determine the final epidemic size. while the computation of r is based on the initial degree distribution, the final epidemic size depends on the whole evolution of the degree distribution. in particular, since reconnection is assumed to be uniform with respect to the degree of nodes, the variance of the degree distribution decreases over time whenever the initial network is highly heterogeneous (see [ ] ). determining an expression for the final epidemic size, using any approximation method, and studying how it [i] when individuals drop connections to infectious neighbours (α = ). in particular, this reflects the (expected) fact that whether exposed individuals break off with infectious neighbors (ω ei > ) or not (ω ei = ) does not affect the early dynamics of [si]/ [i] as long as they do not replace these links with new connections. for a given value ξ of ([e]/ [i] ) * , the equilibrium equations (i.e., the equations obtained by making the right-hand side (rhs) of the previous system equal to ) define a linear system for the remaining variables and, hence, the equilibrium can be easily expressed in terms of ξ. a simple inspection of the equations shows that there are two equilibria, p = ( , , , , , , , ) and p = ( , , γ/φ − , , , , , ), where the local densities around infectious nodes, ( such that ξ * > γ/φ − if γ > φ. note that, for ω ei = , eq. ( ) becomes a quadratic equation in ξ with a positive solution ξ * given by (see [ ] ) with the proviso that φβ(q − ) > (φ + ω se − γ)(β + ω si ). moreover, in this case (ω ei = ), when γ > φ it follows that ξ * > γ/φ − if φβ(q − ) > ω se (γ + β + ω si − φ). if γ ≥ φ and ω ei > , a necessary and sufficient condition for the existence of a unique solution ξ * of ( ) such that ξ * > γ/φ − is that the straight line defined by the left-hand side (lhs) of ( ) intersects the vertical line ξ = γ/φ − above the intersection with this vertical line of the cubic polynomial defined by the rhs of ( ) . this polynomial has two negative roots, ξ and ξ , and the third root ξ can be positive or negative depending on the sign of γ − φ − ω se . the resulting condition on the parameters is φβ((q − )γ + (q + α − ) ω ei ) > ω se (γ + β + ω si − φ)(γ + ω ei ). note that this condition is fulfilled when ω se = which was, indeed, what we assumed in the model if ω ei > . if γ < φ, then ξ < and a sufficient condition for the existence of a ξ * > is that the intersection of the lhs of ( ) with the y-axis is above the intersection with this axis of the rhs of ( ) . this condition leads to φβ((q − )φ + (q + α − ) ω ei ) > (φ − γ + ω se )(ω si + β)(φ + ω ei ). since we are assuming ω ei > , then ω se = , and from this inequality it follows a simpler sufficient condition for the existence of ξ * > , namely, (q − )β > ω si . relationship between the basic reproduction numbers r ba and r p a for the seir-ω model from ( ) , it follows that the only positive value of ξ * for which r p a = is ξ * = γ/φ. after replacing ξ * by this value, expression ( ) can be rewritten as φβ (φ + ω se )(γ + β + ω si ) e(d) − + α ω ei γ + φ + ω ei = . comparing this expression and that of r given by ( ) , it follows that r p a = ⇔ r ba = if α ω ei = , and that r p a = ⇒ r ba > (and r ba = ⇒ r p a < ) if α ω ei > . so, for α > , both approximations lead to the same epidemic threshold when ω ei = . note that only one positive term contains a rewiring rate, namely, the last one in the second equation which has α ω ei as a prefactor. since [se]/[i] increases with α ω ei and infectious diseases of humans: dynamics and control strong approximations for epidemic models graphs with specified degree distributions, simple epidemics and local vaccination strategies mathematical tools for understanding infectious diseases dynamics on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations random graph dynamics modeling dynamic and network heterogeneities in the spread of sexually transmitted diseases adaptive human behavior in epidemiological models stochastic simulation of chemical kinetics epidemic dynamics on an adaptive network insights from unifying modern approximations to infections on networks branching processes with biological applications outbreak analysis of an sis epidemic model with rewiring the effects of local spatial 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rewiring for adaptation epidemic spreading in evolving networks accounting for behavioral responses during a flu epidemic using home television viewing epidemic threshold and control in a dynamic network susceptible-infected-recovered epidemics in dynamic contact networks epidemic thresholds in dynamic contact networks infection spreading in a population with evolving contacts the limit system for the seir-ω model and its equilibriathe key: cord- -r yd i authors: huber-lang, markus; gebhard, florian title: inflammatory changes and coagulopathy in multiply injured patients date: - - journal: the poly-traumatized patient with fractures doi: . / - - - - _ sha: doc_id: cord_uid: r yd i severe tissue trauma leads to an early activation of several danger recognition systems, including the complement and the coagulation system, often resulting in an overwhelming almost synchronic pro- and anti-inflammatory response of the host. although the immune response is associated with beneficial effects at the site of injury including the elimination of exogenous and endogenous danger molecules as well as the initiation of regenerative processes, an exaggerated systemic inflammatory response significantly contributes to posttraumatic complications such as multiple organ failure (mof) and early death. besides pre-existing physical conditions, age, gender, and underlying comorbidities, surgical and anesthesiological management after injury is decisive for outcome. improvements in surgical intensive care have increased number of patients who survive the initial phase after trauma. however, instead of progressing to normal recovery, patients often pass into persistent inflammation, immunosuppression, and catabolism syndrome (pics). the characterization and management of pics will require new strategies for direct monitoring and therapeutic intervention into the patient’s immune function. in this chapter, we describe various factors involved in the inflammatory changes after trauma and aim to understand how these factors interact to progress to systemic inflammation, mof, and pics. multiple trauma results in a significant blood loss and accumulation of necrotic and/or devitalized tissue in an ischemic-hypoxic environment, both of which will become the origin of coagulatory and inflammatory changes. the inflammatory response after polytrauma is a major part of the host's molecular danger response. the acute posttraumatic phase of inflammation consists of two rather synchronically mounted columns: the pro-inflammatory response (systemic inflammatory response syndrome, sirs) and the anti-inflammatory response (compensatory anti-inflammatory response syndrome, cars) [ ] . sirs includes changes in the heart rate, respiratory rate, temperature regulation, and immune cell activation (table . ) [ ] . in the natural course of the inflammatory response after trauma, the balance of the pro-and antiinflammatory response is in equilibrium, which maintains the biological homeostasis and induces controlled regeneration processes, enabling the patient to recover normally without significant complications. however, the excessive inflammatory response after trauma seems to simultaneously and rapidly involve the induction of innate (both pro-and anti-inflammatory mediators) and suppression of adaptive immunity [ , , ] all of which decisively contribute to the development of the early multi-organ dysfunction syndrome (mods). furthermore, a prolonged and dysregulated immune-inflammatory state is associated with delayed recovery and complications, especially the development of late mods. based on improved intensive care and organ support, there is often a progress to the clinically evident persistent inflammation, immune suppression, and catabolism syndrome (pics) which might have replaced the late mods, but still is associated with a poor outcome, appearing as "silent death" [ ] . the steps of an inflammatory reaction to trauma involve fluid phase mediators (cytokines, chemokines, coagulation-and complement activation products, oxygen radicals, eicosanoids, and nitric oxide (no)) and cellular effectors (neutrophils, monocytes/macrophages, and endothelial cells) that translate the trauma-induced signals into cellular responses. these factors are closely interrelated and interconnected by upregulatory and down-regulatory mechanisms. the combination of these factors may cause severe sirs, acute respiratory distress syndrome (ards) and sepsis, acute kidney injury (aki), progressing to mods, depending on the type of injured tissue, the surgical and anesthesiological management after injury, age, gender, genetics, and most importantly, underlying comorbidities and physical conditions (exogenous and endogenous factors) ( fig. . ). patient survival after severe trauma requires an adequate molecular and cellular danger response. the injured tissues release cytosolic molecules (e.g., atp), organelles (e.g., mitochondria), histones, nucleosomes, dna, rna, matrix, and membrane fragments, all functioning as damage-associated molecular patterns (damps). furthermore, damage of external and internal barriers (e.g., skin, gut-blood barrier, air-blood barrier, brain-blood barrier) facilitates invasion of microorganisms, resulting in additional exposure to microorganisms-derived pathogenassociated molecular patterns (pamps). after multiple injury, the immune system of the injured patient is exposed to both damps (also termed alarmins) and pamps, which are summarized as danger-associated molecular patterns [ ] . the " -r-challenge" for the innate and adaptive immune system is to recognize, respond to, and resolve the "molecular danger". for recognition of the damage, there are effective fluid-phase "master alarm systems", such as the coagulation and complement cascade, and effective cellular "danger sensors", such as the pattern recognition receptors (prr). these systems transfer the damage/danger signals to the cells which in turn mount an acute phase reaction and inflammatory response to resolve the damaged tissue load [ ] . within an hour after trauma, inflammation resulting from tissue injury induces an increase in plasma concentration of a number of liverderived proteins (the acute phase proteins, app). pro-inflammatory cytokines (il- β, tnf, il- ) released locally by kupffer cells can systemically influence other cell types such as hepatocytes to synthesize more apps. proactive apps, sirs can be diagnosed when two or more of these criteria are present such as c-reactive protein (crp), procalcitonin (pct), serum amyloid a (saa), complement activation products (c a, c a), activated coagulation proteins (fviia, fxa, fiia), proteinase inhibitors, and metal-binding proteins, are increased during this phase [ ] , whereas the production of inhibitory apps, such as albumin, high-density lipoprotein (hdl), protein c, protein s, and atiii are decreased [ , ] . plasma concentrations of crp are normally below mg/l [ ] . hepatic synthesis of crp is regulated mainly by il- . serum levels of crp can be detected about h after systemic detection of il- . clinically, the plasma levels of crp are relatively non-specific and may not correlate with injury severity and are not predictive of posttraumatic complications such as infections [ ] . in the context of trauma, it is also still unclear whether the native pentameric or the denatured monomeric form of crp is responsible for the crp-induced cellular effects [ ] . pct is physiologically produced in the thyroid gland as the precursor molecule of calcitonin [ ] . during sepsis, stimulation by endotoxins or pro-inflammatory cytokines such as il- β or tnf dramatically increases the serum levels of pct up to -fold [ ] . in trauma patients, pct has been proposed as a practical biomarker for predicting posttraumatic complications such as severe sirs, sepsis, and mods [ ] [ ] [ ] [ ] . the biological immune response after trauma was considered in the past to be divided into an early innate phase and a late adaptive response. however, since multiple intensive interactions between both systems are known (e.g., via the complement cascade), a spatial-or timedependent discrimination of both systems in regard to pathomechanistic changes after multiple injury is irrational. both immune mechanisms contribute to effective recognition, activation, discrimination, regulation, and eradication of invading damage-and pathogen-associated signals [ ] . nevertheless, the innate immune response represents the "first line of defense", consisting of a barrier against exogenous nonself antigens and microorganisms. this includes the integrity of epithelial and mucosal cells: skin, respiratory tract, alimentary tract, urogenital tract, brain, and conjunctiva. exogenous pathogens that escape the first barrier are rapidly recognized and removed by the multiple components of innate immune cells such as neutrophils, monocytes/macrophages, natural killer cells, and dendritic cells [ ] . the innate immune response is closely accompanied by the specifically acquired immune response after the trauma impact. the adaptive immune response is conducted by the interaction of antigen-presenting cells (apcs), dendritic cells, monocytes/macrophages, t-lymphocytes, and b-lymphocytes. the apcs capture invading pathogens and create peptide-mhc (major histocompatibility complex) protein complexes. t-lymphocytes recognize the peptide-mhc protein complex via t-cells expressing antigen-binding receptors (tcrs) and are thereby activated. in turn, activated t-lymphocytes release cytokines to activate and amplify further cells of the immune system. t-helper lymphocytes (cd + t cells) differentiate into two phenotypes according to the cytokine release, the th and th lymphocytes. th cells promote the pro-inflammatory response through the release of il- , tnf, and interferon-γ (ifn-γ), while th cells produce anti-inflammatory cytokines (il- , il- , and il- ), which suppress macrophage activity [ ] . attention has been focused on the th /th -ratio. il- secreted from monocytes/macrophages promotes the differentiation of th cells by increasing the production of ifn-γ [ , ] . several studies have shown that a suppressed il- , il- , and ifn-γ, and elevated il- are observed after major trauma, which correlated with a shift of the th / th ratio towards the th -type pattern [ , ] . this imbalance in th /th -type cytokine response (from pro-to anti-inflammation) is not only a compensatory response but also increases the risk of infection by immune suppression [ ] . however, other reports do not support this view and question the clinical relevance of the th / th -shift after major tissue injury [ , ] . bleeding is a leading cause of death following polytrauma, and acute trauma-induced coagulopathy (atic) increases both the risk and severity of bleeding. clinically, there are several routine laboratory parameters which are indicative of coagulopathy development (table . ). around one third of severe polytrauma patients are already coagulopathic upon arrival in the emergency room [ ] and coagulopathy belongs together with acidosis and hypothermia to the "lethal triad" of polytrauma. thus, an important diagnostic and therapeutic strategy has been developed proposed as the "stop bleeding campaign" [ ] that addresses three major aspects of coagulopathy: fast detection and stopping of relevant bleeding sources; estimation and resuscitation of the lost blood volume; and rapid monitoring for coagulopathic conditions. the major mechanism of activation of the coagulation cascade following trauma is via the extrinsic coagulation system [ ] . the extrinsic cascade mediates inflammation by tissue factor (tf). exposure of the fvii to tf (e.g., from injured cells) results in the conversion of fvii to fviia. the fviia-tf-complexes activate fx to fxa, and fxa converts prothrombin to thrombin (fiia). thrombin activates fv, fviii, and fxi, which results in enhanced thrombin formation. thrombin also cleaves fibrinogen, and the fibrin clot is formed following polymerization and stabilization. in normal conditions, small amounts of tf are exposed to the circulating blood. however, under pathophysiological conditions, tf is upregulated on the surface of neutrophils, macrophages, and endothelial cells. endotoxin, activated complement (c a), and cytokines (il- β, tnf) induce tf expression [ ] . tf is highly thrombogenic, and its upregulation often results in hypercoagulability, leading to an increased tendency of thrombosis [ , ] . another phylogenetically ancient activation pathway is the rather unknown fsap (fvii activating protease) pathway that is activated by an autocatalytic mechanism promoted by factors released by necrotic or post-apoptotic cells such as nucleic acids, nucleosomes, and polyamines. fsap can regulate coagulation and fibrinolysis by activating factor vii and pro-urokinase, respectively. in polytrauma patients, an early and robust activation of fsap is seen which in turn contributes to the activation of both, the coagulation and complement system [ ] . in addition, coagulation mediators (fviia, fxa, and fiia) elicit inflammation with expression of tnf, cytokines, adhesion molecules (mcp- , icam- , vcam- , selectins, etc.), and growth factors (e.g., vegf) [ ] . inhibitors to prevent a hypercoagulable state include antithrombin iii (atiii), protein c, protein s and tf pathway inhibitor (tfpi). atiii inhibits fixa, fxa, and thrombin. tfpi suppresses the activity of tf/fviia/fxa complexes [ ] . protein c is activated by the thrombin-thrombomodulin complex on endothelial cells, and activated protein c, in combination with free protein s, cleaves and inactivates fv and fviii [ ] . therapeutically intervening with the production and/or activity of inhibitors could help to improve outcome by mitigating complications such as ards. for example, the crash trial has recently revealed that early application of tranexamic acid (a synthetic derivative of the amino acid lysine) that inhibits fibrinolysis by blocking the lysine binding sites on plasminogen significantly reduces the risk of death in bleeding trauma patients [ ] . almost synchronically to the coagulation response, there is an activation of the complement cascade immediately after multiple trauma [ , ] . the complement system consists of more than proteins. in the resting state, complement proteins circulate as inactive forms in plasma. the activation of the complement system can occur through four pathways (alternative, classical, lectin, and coagulation paths). the classical pathway of complement is activated by antigenantibody complexes (immune-globulin m or g) or crp. the alternative pathway is activated by modified from maegele et al. [ ] , brohi et al. [ ] , greuters et al. [ ] bacterial products such as lipopolysaccharides (lps). the lectin pathway is initiated by lectin binding to mannose, glucose, or other sugars of microorganisms. upon activation of the complement system, there is a generation of biologically active peptides. the cleavage of the central complement components c and c to the anaphylatoxins c a and c a, respectively, also induces the formation of opsonins and the membrane attack complexes (mac, c b- ) [ , ] . early after polytrauma, serum levels of the complement activation products c a and c a are significantly elevated and correlate with the severity of the injury (e.g., traumatic brain injury), septic complications, and mortality [ , ] . the circulating soluble mac is also enhanced within the first hours after polytrauma but almost not detectable between and h after polytrauma [ , ] . regulation of complement activation and protection against complement-mediated tissue destruction is provided by a selection of soluble-and membranebound complement regulatory proteins (cregs). the expression profile of cregs on leukocytes is specifically altered post polytrauma: cd (membrane co-factor protein) is significantly reduced in neutrophils, monocytes, and lymphocytes. in contrast, cd (decay accelerating factor) seems to be increased on neutrophils early after trauma. a delayed up-regulation of cd has been observed in monocytes from trauma patients. an initial enhancement of cd (mac inhibitor) expression was measured in neutrophils and monocytes at the time of admission. remarkably, c a receptor (c ar), cd and cd expression on neutrophils reversely correlated with injury severity [ ] . the anaphylatoxins c a and c a mainly play pro-inflammatory roles, which include the recruitment and activation of phagocytic cells (polymorphonuclear cells, pmns), monocytes/ macrophages, the enhancement of the hepatic acute-phase reaction, stimulation of the release of vasoactive mediators (such as histamine), and promoting the adhesion of leukocytes to endothelial cells and their permeation through injured tissues. c b forms a complex by the consecutive binding of proteins c -c , culminating in the formation of the mac (c b- ), which leads to the formation of pores in the cellular membrane causing lysis and death of the target cells [ ] . furthermore, the inflammatory response of complement activation leads to the production of free oxygen radicals and arachidonic acid metabolites and cytokines. the complement cascade bridges innate and adaptive immunity for defense against microbial pathogens. however, excessive consumption of complement proteins may also cause tissue damage of the host after trauma. within the first h after multiple injuries, there is a massive reduction in complement hemolytic activity (ch ), which recovers only around days after trauma, and can be used to discriminate between lethal and non-lethal outcome. this trauma-induced reduction of global complement function is referred to as trauma-induced "complementopathy" in analogy with "coagulopathy", both of which significantly participate to the impairment of the innate immune response after polytrauma (fig. . ). the kallikrein-kinin system involves a cascade of plasma proteases and is related to the complement and clotting cascade (intrinsic activation) [ ] . this contact system consists of plasma proteins factor xii (hageman factor; fxii), prekallikrein, high molecular weight kininogen (hmwk), and fxi. contact with negatively charged surfaces such as foreign bodies or the membrane fragments of stimulated platelets activates fxii [ ] . the active protein fxiia converts prekallikrein into the proteolytic enzyme kallikrein, which in turn cleaves the plasma glycoprotein precursor hmwk to form bradykinin [ ] . bradykinin increases vascular permeability and causes dilation of blood vessels by its action on smooth muscle cells. in turn, as a positive feedback loop, kallikrein itself accelerates the conversion of fxii to fxiia. kallikrein can also activate fibrinolysis to counterbalance the clotting cascade activated by fxiia. furthermore, kallikrein also exhibits chemotactic activity, converting c and c into the chemoattractant products c a and c a, respectively [ ] . pro-inflammatory cytokines play key local and systemic roles as intercellular messengers to initiate, amplify, and perpetuate the inflammatory response after trauma (table . ). cytokines are produced by many cell types in all organs. they have multiple targets and act in a pleiotropic manner. early after trauma, production and release of pro-inflammatory cytokines such as il- β, tnf, il- , and il- is initiated by monocytes and macrophages. il- β and tnf as well as il- and il- are released early after polytrauma [ , ] and predominantly function as pro-inflammatory mediators to repair damaged tissue. the release of il- β and tnf is mainly stimulated by bacterial endotoxins or other microbial products, immune complexes, and a variety of inflammatory stimuli. upon release, il- β and tnf usually return to baseline levels within h. tnf increases the activity of neutrophils and monocytes by activating the underlying endothelium. tnf promotes the expression and release of adhesion molecules such as icam or e-selectin, and increases the permeability of endothelial cells, which facilitates neutrophil migration into the damaged tissue [ ] . some studies have proposed tnf as a valid serum marker for complications after trauma. however, the results are inconsistent and to date, no data is available indicating whether tnf correlates to the severity of trauma or trauma outcome [ ] [ ] [ ] [ ] [ ] [ ] . many different cell types produce il- : in addition to immune cells such as monocytes, macrophages, neutrophils, t cells, and b cells, it is also produced by endothelial cells, smooth muscle cells, and fibroblasts. il- upregulates the hepatic acute-phase response, stimulating generation of c-reactive protein (crp), procalcitonin, serum amyloid a, fibrinogen, α -antitrypsin, and complement activation products (e.g., c a), which then promote neutrophil activation. there is strong evidence that serum il- level correlates with the severity of trauma, trauma pattern (especially in combination with chest trauma), and the risk of subsequent ards, mof, and lethal outcome [ , ] . therefore, il- may be considered as a clinically relevant and feasible parameter to estimate the severity of injury and prognosis after trauma [ , ] . in addition, for patients requiring second or subsequent surgeries following trauma, il- may prove to be an important biological marker in deciding the correct timing of surgery. in trauma patients with high initial levels of il- (> pg/dl), it is recommended to delay secondary procedures for more than days [ ] . the chemokine il- is secreted by monocytes/ macrophages, neutrophils, and endothelial cells. il- is mainly synthesized by t lymphocytes and monocytes/macrophages. it is the pivotal role of il- to inhibit the production of monocyte/macrophage-derived tnf, il- , il- , and free oxygen radicals [ ] . il- plasma levels are proportional to the severity of trauma and to posttraumatic complications [ ] [ ] [ ] [ ] [ ] ( table . ). in addition to its pro-inflammatory role, il- also has anti-inflammatory properties. as an immunoregulatory cytokine, il- stimulates macrophages to release anti-inflammatory cytokines such as il- receptor antagonists and soluble tnf receptors [ ] . moreover, il- induces macrophages to release prostaglandin e (pge ), the most powerful endogenous immune suppressant. pge regulates the synthesis of tnf and il- β by macrophages and induces the release of il- [ ] [ ] [ ] . overall, it has to be emphasized that almost all cytokines may not act strictly in either a pro-or anti-inflammatory manner, but rather may exhibit a "janus-faced behavior" depending on the underlying tissue, local environment, and trauma conditions. furthermore, the categorized pro-and anti-inflammatory cytokines follow not a specific temporal pattern but are rather synchronically and rapidly generated and released [ , ] , mounting the overall inflammatory response. when the simultaneous cytokine response is excessive, prolonged, and dysregulated, this may lead to severe complications, such as organ dysfunctions [ ] or persistent inflammation, immunosuppression, and catabolism syndrome (pics) [ ] (fig. . ). reactive oxygen species are released by leukocytes after exposure to pro-and antiinflammatory cytokines, chemokines, complement factors, and bacterial products. (fig. . ) . ros cause lipid peroxidation, cell membrane disintegration, and dna damage to endothelial and parenchymal cells [ , ] . furthermore, ros secreted by polymorphonuclear leukocytes (pmn) induce cytokines, chemokines [ ] , heat shock protein (hsp) [ ] , and adhesion molecules (p-selectin, icam- ) [ ] leading to cell and tissue damage. early after severe tissue trauma, neutrophils migrate along the chemoattractant gradient of complement activation products, interleukins, and ros to the site of tissue damage and to remote organ tissue. neutrophil mobilization is important for wound healing and protection against invading microorganisms, but their immigration to remote organ tissue contributes to sirs [ ] . neutrophil migration is composed of four steps: the first step, generation of leukocyte selectins (e.g., l-selectins) and e-and p-selectins on the endothelium is induced by anaphylatoxins (e.g., c a), cytokines (e.g., il- ), and toxins [ ] . these adhesion molecules are responsible for the rolling of neutrophils. the second step involves expression of integrins on neutrophils such as cd and cd , and intercellular adhesion molecules (icam- ) and vascular cell adhesion molecules (vcam- ) on the surface of endothelial cells, all of which are strongly induced by c a [ ] [ ] [ ] . the interaction of these upregulated molecules activate neutrophils to reinforce the contact between neutrophils and endothelial cells (sticking). in the next step, migration and accumulation into tissues occur, mediated by chemokines and complement anaphylatoxins. to migrate through cellular barriers, neutrophils undergo significant deformational changes to permeate through small cellular gaps with the help of locally released matrix metalloproteinases. in the final step, activation of neutrophils occurs to protect against dangerous molecules, microorganisms, and cells. neutrophils utilize a large arsenal for forming the "first line of defense" after trauma: chemotaxis, phagocytosis, oxidative burst reaction with release of ros and myeloperoxidase (mpo), generation of no, leukotriens, plateletactivating factor (paf), tissue factor (tf), proteases, and multiple pro-inflammatory cytokines. however, the active substances released from neutrophils may not only harm the invading microorganisms or injured cells but also healthy host cells, especially since neutrophils become "long-lived" after trauma by significant inhibition of neutrophil apoptosis. thus, neutrophils after trauma function as "friend and foe". monocytes/macrophages and neutrophils play a central role for the innate host defense, tissue repair, and remodelling, and for the intermediaries to the antigen-specific adaptive immune response. monocytes are circulating precursors of macrophages. monocytes migrate into the different tissues (liver, spleen, lungs, etc.) even in absence of local inflammation and become tissue macrophages. when monocytes/macrophages are activated by various phagocytotic events in response to trauma, they regulate the activation of t and b lymphocytes, which induce antigen presentation by the major histocompatibility complex ii (mhc ii). monocytes/macrophages also release chemokines, cytokines (il- , tnf, il- , il- , tgf-β), and various growth factors (fibroblast growth factor [fgf], epidermal growth factor [egf], and platelet-derived growth factors [pdgf] ) that initiate the formation of new extracellular matrix and promote angiogenesis and generation of new tissue at the site of injury. the functional phenotype shifts from a pronounced pro-inflammatory m type to a more anti-inflammatory and regenerative m -type macrophage. the monocyte/macrophage cellular response after minor trauma embodies several beneficial effects for the host. however, major trauma induces massive monocyte/macrophage activation. in this state, the effects of the monocyte/macrophage response become systemic and may also induce detrimental effects. systemically, the macrophagemodulated immune response influences microcirculation, metabolism, and triggering and progression of remote organ injury. deactivation of monocytes and decreased expression of mhc ii on their surface are observed after major trauma correlating with the severity of injury [ ] . natural killer (nk) cells are antigen-non-specific lymphocytes that recognize pathogen-associated molecular patterns (pamps) of invading microorganisms [ ] as well as damaged, transformed, or virus-infected host cells [ ] . since they are not dependent on pre-sensitization [ ] to mediate their cytotoxic effects and to release excessive amounts of pro-and anti-inflammatory cytokines within minutes of stimulation, nk cells are regarded as part of the "first line of defense" [ ] . their ability to release immune-modulatory cytokines may provide important regulatory functions during immune response, especially following severe injury. however, studies addressing the role of human nk cells after severe tissue trauma are rare and contradictory. some studies revealed an increase of nk cells in the early stage after severe trauma [ ] , whereas nk cell function is greatly depressed by traumatic injury. however, there was no correlation between the nk cell count or activity and injury severity [ , ] . concerning the effect of plasma samples from trauma patients on the cytotoxic activity of healthy nk cells in vitro, it has been shown that incubation times of more than h lead to suppressed nk cell function, suggesting that posttraumatic immune suppression is associated with suppression of nk cell activity [ ] . vice versa, murine experiments have collectively shown that nk cells as a key source of interferon γ exert harmful pro-inflammatory effects in the posttraumatic immune response and during the pathogenesis of sepsis [ , ] . in support, early depletion of nk cells results in reduction of liver il- expression and a % improved survival rate in a murine polytrauma model. lymphocyte apoptosis in spleen as well as neutrophil infiltration into lungs and liver is also attenuated [ ] . furthermore, in various mouse models of sepsis, depletion of nk cells leads to improved survival [ , ] suggesting that early posttraumatic activation of nk cells promotes amplification of the inflammatory response, and the subsequent loss of cellular functions might contribute to immune suppression manifested in later stages after trauma [ ] . mechanisms of the development of organ dysfunction the initial trauma insult activates an inflammatory cascade that stimulates the host immune system. massive initial trauma impact (first hit) causes severe sirs. in this situation, the overwhelming production and release of pro-and anti-inflammatory mediators result in rapid mods and early death. an initial trauma insult of lower severity induces a moderate state of sirs/cars. in this instance, inflammatory and immune cells undergo some "priming". however, some patients develop posttraumatic complications, such as sepsis, aki, ards, and mods. the development of these complications is regulated by various exogenous and endogenous factors. among these factors, it is important to understand the relationship between the biological changes and the anatomical region of initial injury. the central nervous system is a rich source of inflammatory mediators. traumatic brain injuries (tbi) with the disruption of the blood-brain barrier (bbb) allow immune cells to migrate into the subarachnoid space, leading to an accumulation of leukocytes from the periphery [ , [ ] [ ] [ ] . trauma to the chest area, particularly lung contusions, leads to an early increase in plasma mediators, which is associated with systemic inflammatory and anti-inflammatory reactions, such as pneumonia, ards, and mods [ ] [ ] [ ] . patients with severe soft tissue injuries to the extremities with resulting hemorrhagic shock or severe muscle crush syndrome are at risk of developing more serious remote organ injury (e.g., aki). ischemia/reperfusion injury (i/r) leads to the production of large quantities of ros. femoral fractures with soft tissue injuries usually result in alteration of hemodynamic parameters such as increased cardiac output, tachycardia, decreased systemic vascular resistance, and decreased hepatic blood flow [ ] . long bone fractures and unstable pelvic fractures are characterized by high blood loss and are associated with severe soft tissue injury, which initiate both a local and systemic inflammatory response [ , [ ] [ ] [ ] [ ] [ ] . these bodies of evidence suggest that the initial trauma itself predisposes trauma patients to posttraumatic complications. traumatized patients who survive the initial injury ("first hit") may still be at risk of death from sepsis and multiple organ failure. secondary insults following the initial injury amplify the systemic inflammatory response and upset the balance of pro-and anti-inflammatory mediators, pro-and anti-coagulatory factors, pro-and anti-apoptotic events, and pro-and anti-regenerative processes. secondary insults ("second hits") are compounded by endogenous and exogenous factors. endogenous secondary insults include respiratory distress, cardiovascular instability, ischemia and reperfusion injury, and infection. exogenous secondary insults include surgical and anesthesiological interventions [ ] [ ] [ ] , blood transfusions, and -not to forget -missed injuries. clinical studies have revealed that orthopedic surgical intervention can also cause major changes in the inflammatory response, and these changes are in proportion to the magnitude of surgery. for instance, femoral nailing induces an increase in systemic plasma levels of il- and il- . in these patients, human leukocyte antigen-dr expression on monocytes is reduced as well [ , ] . furthermore, reamed femoral nailing appears to be associated with greater impairment of immune reactivity than un-reamed nailing [ ] . blood transfusions are a paramount therapy in the management of trauma/hemorrhagic shock patients. however, various studies have demonstrated that blood transfusions are associated with infection, sirs, ards, and mods after trauma [ ] [ ] [ ] [ ] [ ] [ ] , also representing a "second hit" for the multiply injured patient. ischemia/reperfusion (i/r) injury is a common and important event in clinical situations such as trauma, hemorrhagic shock, cardiac arrest (hypoxemia, hypotension of systemic tissue), contusions, lacerations, vascular injuries, and compartment syndrome (increased pressure in a preformed anatomical compartment with resulting hypoperfusion and hypoxemia of local tissue). inadequate microvascular flow results in the activation of leukocytes and converts local endothelial cells into a pro-inflammatory and pro-thrombotic phenotype. i/r injury consists of two specific stages. during the first stage of ischemia and hypoxemia, oxygen and nutrients are deprived from tissues temporarily by the disruption of blood supply. during the ischemic phase, the lack of oxygen leads to decreased production as well as consumption of adenosine triphosphate (atp). as consumption of atp continues, it is degraded into adenosine diphosphate (adp) and adenosine monophosphate (amp), which is further degraded to inosine and hypoxanthine [ ] . atp depletion leads to an alteration in intercellular calcium and sodium concentration. it also results in the activation of cytotoxic enzymes such as proteases or phospholipases, all cumulating to reversible or irreversible cellular damage. the second stage of reperfusion is the revascularization or reestablished supply of oxygen to the ischemic tissue. the hallmark of the reperfusion phase is the generation of byproducts of neutrophil activation, which induces secondary tissue damage and organ dysfunction. on reperfusion with the reintroduction of molecular oxygen into the ischemic tissue, oxygen reacts with leukocytes and endothelial cells promoting the generation of reactive oxygen species and platelet-activation factor. the interactions of neutrophils and endothelial cells have been shown to contribute to massive interstitial edema caused by microvascular capillary leakage after reperfusion injury. the ischemia and reperfusion injury with atp depletion is a major cause for breakdown of physiological organ-blood barriers, such as bloodbrain, blood-gut, and blood-alveolus barrier. broken barriers characterized by diffuse microvascular leakage and tissue edema are thought to be main drivers of bacterial translocation (bt) and sepsis [ ] . bacterial translocation is defined as the phenomenon of both viable and nonviable bacteria as well as their products (bacterial cell wall components, lps, and peptidoglycan) crossing the intestinal barrier to external sites such as the mesenteric lymph nodes, liver, and spleen. bt occurs as a result of a loss of integrity of the gut barrier function after trauma, hemorrhagic shock, and burns [ ] , and may be associated with posttraumatic complications [ , ] . although most data on bt and its complications have shown consistent results in animal models of hemorrhagic shock, trauma, and severe burns, its importance in humans is questionable, with variable results in clinical studies. in addition, it is still debatable whether bt is an important pathophysiologic event or simply an epiphenomenon of severe disease [ ] . following trauma, acute inflammatory reactions may be triggered by infections (bacterial, viral, fungal, parasitic) and microbial toxins, or by any of several molecules released from necrotic tissue (hmgb , hyaluronic acid, etc.). pattern recognition receptors (prrs), including tolllike receptors, can detect these stimuli and trigger a signaling pathway that leads to the production of various mediators. in the acute phase of trauma, vasodilatation is induced by vasodilatatory mediators (no, prostaglandins), quickly followed by increased permeability of the microvasculature. vasodilatation and extravasation of plasma result in hemoconcentration, facilitating the peripheral migration of neutrophils. neutrophil migration from the blood stream into interstitial tissue is divided into several steps, which are mediated by endothelial cell adhesion molecules, cytokines produced by monocytes/macrophages and various other cells, chemokines, the complement system, and arachidonic acid. migrated neutrophils produce several mediators such as neutral protease, reactive oxygen species (ros), lipids (leukotriene, paf), and tissue factor (tf). these mediators act as secondary tissue damage mediators and pro-coagulatory factors depending on the degree of initial injury as well as additional insults. during inflammation, the plasmaic cascade, consisting of the complement cascade, the kallikrein-kinin system, and the coagulation cascade, is activated by toxins and inflammatory mediators. activation of the complement system induces generation and depletion 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transfusion in surgery transfusion of the injured patient: proceed with caution application of heme oxygenase- , carbon monoxide and biliverdin for the prevention of intestinal ischemia/reperfusion injury broken barriers: a new take on sepsis pathogenesis bacterial translocation: clinical implications and prevention bacterial translocation-related mortality may be associated with neutrophil-mediated organ damage the gut: the 'motor' of multiple organ dysfunction syndrome? curr opin clin nutr metab care bacterial [correction of baterial] translocation in humans key: cord- -xg bv gy authors: dayer, mohammad reza; dayer, mohammad saaid; rezatofighi, seyedeh elham title: mechanism of preferential packaging of negative sense genomic rna by viral nucleoproteins in crimean-congo hemorrhagic fever virus date: - - journal: protein j doi: . /s - - - sha: doc_id: cord_uid: xg bv gy the crimean-congo hemorrhagic fever (cchf) is an infectious disease of high virulence and mortality caused by a negative sense rna nairovirus. the genomic rna of cchfv is enwrapped by its nucleoprotein. positively charged residues on cchfv nucleoprotein provide multiple binding sites to facilitate genomic rna encapsidation. in the present work, we investigated the mechanism underlying preferential packaging of the negative sense genomic rna by cchfv nucleoprotein in the presence of host cell rnas during viral assembly. the work included genome sequence analyses for different families of negative and positive sense rna viruses, using serial docking experiments and molecular dynamic simulations. our results indicated that the main determinant parameter of the nucleoprotein binding affinity for negative sense rna is the ratio of purine/pyrimidine in the rna molecule. a negative sense rna with a purine/pyrimidine ratio (> ) higher than that of a positive sense rna (< ) exhibits higher affinity for the nucleoprotein. our calculations revealed that a negative sense rna expresses about . kj/mol higher binding energy per nucleotide compared to a positive sense rna. this energy difference produces a binding energy high enough to make the negative sense rna, the preferred substrate for packaging by cchfv nucleoprotein in the presence of cellular or complementary positive sense rnas. the outcome of this study may contribute to ongoing researches on other viral diseases caused by negative sense rna viruses such as ebola virus which poses a security threat to all humanity. cchfv is a tri-segmented negative sense rna virus with s (small), m (medium) and l (large) segments [ , ] . these segments of rna in cchfv encode nucleoprotein, glycoprotein and polymerase, respectively [ ] [ ] [ ] [ ] . as in other negative sense rna viruses, rna of cchfv is enwrapped by nucleoproteins forming a complex called ribonucleoprotein particle (rnp) which protects genomic rna against degradation by host cell nucleases and helps packaging the newly synthesized genomic rna to form virions [ , [ ] [ ] [ ] [ ] . the cchfv nucleoprotein comprises amino acid residues in its primary structure and alpha helices in the secondary structure as its predominant regular structure. the alpha helices include about % of amino acid residues whereas beta strand structures include only . % of amino acids [ , ] . in the three dimensional structure, nucleoprotein has two domains; a stalk and a head domains [ ] . sequence analyses indicated the existence of highly positively charged regions along the nucleoprotein that suggest its ability to bind negative back-bones of rna molecules [ , , ] . a region comprising residues lys , lys , lys , arg , lys , his and gln and a region with arg , arg and gln in the head domain, on the one hand, and a region with arg , his , lys , arg and arg in the stalk domain, on the other hand, provide suitable binding motifs for a genomic rna [ , ] . considering the low sequence similarity of nucleoproteins in bunyaviridae family, it is thought that the binding properties of nucleoproteins are not sequence specific [ - , , ] . the genomic rna of negative sense rna viruses, as in cchfv, is in opposite sense to mrna. therefore, it should be transcribed to complementary rna by viral polymerase prior to becoming suitable for translation by host cell machinery [ ] [ ] [ ] . it is well known that the viral polymerase specifically recognizes and binds the genomic rna encapsidated in rnps rather than the naked one and transcribe it to a positive rna [ ] [ ] [ ] . however, the presence of the nucleoprotein is essential for safe elongation of the transcription process as it likely plays a pivotal helicase activity to prevent pairing of the genomic rna and the newly synthesized complementary rna as a double stranded structure [ , [ ] [ ] [ ] . there are reports suggesting that the helicase activity results from the nucleoprotein different affinities for negative and positive sense rna strands i.e. with higher affinity for a negative (viral) rna and lower affinity for a positive sense (complementary) rna [ ] [ ] [ ] [ ] . in the present work, by analyzing genomic sequences of rna viruses either with negative or positive sense, performing different docking experiments and carrying out molecular dynamic (md) simulations, we undertook to study the mechanism conferring different affinities to cchfv nucleoprotein for negative and positive sense rnas'. the outcomes of this study may give a better understanding of packaging mechanism for cchfv negative sense genomic rna. the available two coordinate structures for monomeric and trimeric forms of cchfv nucleoprotein were obtained from protein data bank archive under pdb id numbers: u i, aqg and aqf respectively [ ] . these structures were constructed based on crystallographic data at - Å of resolutions prior to being used as starting structures to perform experiments. for the purpose of this study, the negative and positive sense rnas were prepared by dissociating a short double strand rna of nucleotides obtained from pdb (id: ke ) and a long double stranded rna of base pairs which had been constructed using arguslab, a free docking software [ ] . the sequence of the negative strand of the short rna was: aauuuaaaaauacaaucaagc with a purine/pyrimidine ratio of . , whereas that of positive strand was: gcuugauuguauuuuuaaau u with a purine/pyrimidine ratio of . . also, the sequence of the negative strand of the long rna was: gugacgugacgugacgugacgugagugacgug-acgugacgug with a purine to pyrimidine ratio of . , whereas that of the positive strand was: cacgucacgucacgucacucacgucacgucca-cgucacgucac with a purine to pyrimidine ratio of . . the coordinate structures of both short and long rnas (negative and positive senses) were hydrated and energy minimized by gromacs prior to being used in docking or md experiments. it should be noted that these positive and negative rnas were selected to have distant purine to pyrimidine ratio enough to magnify their difference in terms of their physicochemical properties in a bid to elucidate their differential behaviors. docking experiments were carried out on hydrated and optimized structures of cchfv nucleoproteins and rna molecules using hex software version . (http://www. loria.fr/*ritchied/hex/) [ ] . docking results were scored based on their energy and the first docked structures were averaged and used for energy calculations. for md experiments, the best docked structures of cchfv nucleoprotein in complex with short and long negative or positive sense rnas were placed separately in the center of rectangular boxes having dimensions of . . . , . . . , . . . and . . . nm respectively. the simulated boxes were filled and coverd with a water layer modeled by the spc/e model within a radiuos of . nm. setting up the systems: gromacs . . with double precision implemented on ubuntu- . were used for md simulations using amber sb-ildn force filed for parameterizations [ ] . systems charge neutralities were checked by gromacs machine and neutralized by adding sodium ions. systems were optimized for system constituents including solvent, ions and hydrogen atoms by more than , steps of energy minimization using steepest decent algorithm with the total energy below kj/mol. lincs and settle algorithms were used to constrain bond length and water geometry. short ps-md simulations with bond restraints were carried out prior to full length simulations [ ] . final ns-md simulations were performed at °c using berendsen thermostat and at atmosphere pressure using berendsen barostat for coupling temperature and pressure respectively. electrostatic interactions were treated with particle mesh ewald (pme) and the neutral ph was set using asp, glu, arg, and lys amino acids in ionized forms [ , ] . the results were analyzed statistically using the statistical package for the social science (spss-pc, version . spss, inc., chicago, il). the parameters were considered significantly different at p \ . . the transcription of viral negative sense rna by a polymerase to a complementary positive sense rna results in replacement of purine nucleotides (g and a) with their pyrimidine counterparts (c and u) and vise versa. depending on proportional nucleotide content of a parental rna, the complementary chain may differ in its overall purine to pyrimidine ratio when contrasted to the parental chain. in order to calculate purine/pyrimidine ratios of negative and positive rnas, we analyzed a large set of genomic information of well characterized negative and positive sense rna viruses in terms of their corresponding sequences. tables and list the negative and positive sense rna viruses obtained from www.ncbi.nlm.nih.gov/ nuccore for the same purpose. sequence analyses showed that the average purine/ pyrimidine ratios are . ± . and . ± . (average ± sd) for negative and positive sense rna respectively. although, the difference seemed trivial, but statistical analyses indicated significantly higher ratio for negative sense rna compared to positive sense one at p value \. . given their higher abundance, the larger purine bases bind stronger (via stacking and/or hydrogen bonding) to counter amino acids residues on cchfv nucleoprotein than pyrimidine bases. in order to test this hypothesis, we first constructed four mono nucleotides of g, a, c and u by arguslab software and optimized their chemical structures. the optimized nucleotides then were docked to cchfv nucleoproteins ( u i and aqg for monomeric and aqf for trimeric forms) using hex . and blind mode of docking. binding energy of the best docked structures then averaged as binding affinity. as expected, our data showed that the binding energy of mononucleotides to u i nucleoprotein were - . , - . , - . and - . kj/mol for g, a, u and c respectively. the same patterns were seen when using monomeric aqg and trimeric aqf nucleoproteins as targets (data not shown). as evidenced, purine nucleotides show higher binding energies than pyrimidine nucleotides. furthermore, the content of each nucleotides in the genomes of negative sense rna viruses calculated as percentage (mean ± sd) were . ± . , . ± . , . ± . and . ± . for a, g, t and c nucleotide respectively, showing prominently higher content for a nucleotide. the same calculation for genome composition of positive sense rna viruses indicated . ± . , . ± . , . ± . and . ± . % for a, g, t and c nucleotide contents respectively. interestingly, when the actual count of genomic nucleotides for negative and positive sense rna viruses (as listed in tables , ) are multiplied by their corresponding energies, the resulting total binding energy of negative sense rna become significantly higher than that of positive sense rna by . kj/mol per nucleotide. this energy builds up a significant barrier for positive sense rna to be packaged, as genetic material, by cchfv nucleoprotein instead of/or concomitantly with negative sense rna during virus assembly. this confers the negative sense rna with a greater affinity for cchfv nucleoproteins and hence preferential binding, as evidenced, this binding preference is driven by higher proportion of adenine rather than other nucleotides. what remains to be answered, at this stage, is whether the preferential binding of cchfv nucleoproteins viral nucleoproteins in cchfv to the negative sense rna is a sequence specific property or not. using single-stranded oligonucleotides with - nucleotides long, our serial docking experiments revealed that the binding interface of cchfv nucleoprotein and rna involves only - nucleotides and their counter residues. therefore, to study the effect of nucleotide sequence on binding energy, we constructed possible sequences of trimeric and possible sequences of tetrameric nucleotides using g, a, c and u nucleotides and optimized their chemical structures. these nucleotide structures were then docked to optimized structures of cchfv nucleoproteins ( u i and aqg for monomeric and aqf for trimeric forms). anova analysis of the best resulted structures from each docking experiments showed that the binding energies of trimeric and tetrameric sequences are significantly sequence independent (p value \. ). in other words, the binding energy of oligonucleotides is only dependent to the total content of purine bases but not to the sequence of nucleotides in rna string. in the next step, a short rna ( nucleotides) as well as a long rna ( nucleotide) both in negative and positive sense states were used and docked to monomeric ( u i and aqg) and trimeric ( aqf) forms of cchfv nucleoproteins. figure plots average binding energies obtained for the best structures of each negative and positive sense rnas with different nucleoprotein structures. as indicated, negative sense rna exhibited significantly higher binding energy than positive sense rna (p value \. ) with either monomeric ( u i and aqg) or trimeric ( aqf) nucleoproteins as docking targets. this finding suggests that cchfv nucleoproteins bind more tightly to negative sense rna than positive one. figure , also, shows that irrespective of their senses, long rnas have comparatively higher affinities to nucleoprotein than short rnas. based on the results of aforementioned docking experiments, we then selected cchfv nucleoproteins-rna complexes of maximum binding energies for positive and negative sense rnas (both short and long) to carry out md simulations. the complexes were then placed in cubic boxes filled with spce water at °c and atmosphere pressure and energy minimized to lower than kj/mol prior to md simulations. table summarizes simulation history for these systems. the simulation trajectories, all of ns duration, were then processed using gromacs commands. in all cases, the same outputs were obtained for short and long rnanucleoprotein complexes. figure a shows root mean square displacement (rmsd) curve of cchfv nucleoprotein during simulation of its complexes with both negative and positive sense rnas. the curve indicates that cchfv nucleoprotein undergoes similar patterns of structural alterations in the presence of either senses of rna which ultimately converge towards equilibrated states. as the structural alterations exerted by md force are similar during these simulations, therefore, both systems may be used for further comparative studies. figure b illustrates rmsd change of rna chains against protein back-bone for both negative and positive sense rna complexes. this curve provides a good tool to explore the movement of rna relative to protein backbone during simulation. the initial phase of rmsd curve ( - , ps) marks a sharper increase in rmsd for the negative sense rna in contrast to positive sense rna. towards formation of rna-cchfv nucleoprotein complexes, the faster movement of negative sense rna means that this docked rna chain has a closer position to that in the final conformation and so proceeds faster to reach the final state. after this initial phase has elapsed, the negative sense rna reaches a more stable state which restricts further movement, hence, its rmsd curve progresses with steeper slope than that of its counter rna (fig. b) . however, the positive sense rna does not reach a stable state until about , ps of simulation after which both systems of rna chains attain certain stable conformations with no further reasonable increase in rmsd values. the root mean square fluctuation (rmsf) of nucleoprotein alpha carbons during ns of simulation is shown in fig. . the rmsf curve is a very useful index for protein flexibility during simulation with hot (or more flexible) points being at the picks of the curve. as depicted, the total rmsf of the negative sense rna is significantly lower (* tenth) than that of the positive sense rna. the lower rmsf of cchfv nucleoprotein complex with negative sense rna compared to its complex with positive sense rna indicates lower flexibility and therefore more stable conformation of the former complex. this confirms the postulated preferential binding of the negative sense rna to cchfv nucleoprotein based on binding energy calculations (fig. ) . the trend of mean square displacement (msd) curve (fig. ) for the negative and positive sense rnas during simulation is a reliable index for the extent of movement or diffusion of rna chains inside cchfv nucleoprotein. this fig. binding energy for cchfv nucleoproteins (monomers of u i, aqg and trimer of aqf) to negative and positive sense rnas' with short and long length obtained from blind docking experiments using hex . software parameter indirectly reflects the stability of rna-nucleoprotein complex against applied md forces. the sharper increase of msd curve for the positive sense rna indicates reduced stability of its complex with cchfv nucleoprotein and its free movement inside protein during simulation. this finding is yet another indication on the stability of the negative sense rna-nucleoprotein complex. figure shows the hydrophobic solvent accessible surface (sas) of the nucleoprotein for complex formation with negative and positive rna during simulation. increase in sas during simulation could be interpreted as to be the result of structural alterations of nucleoprotein which leads to orientation of hydrophobic residues towards outside for rna binding. showing an elevated sas curve, the negative sense rna seems to exert more structural alterations in cchfv nucleoprotein during simulation than its counter rna does. this is again another indication of a stronger binding of the negative sense rna with the nucleoprotein. the stronger binding of the negative sense rna is also confirmed by a significant decrease in the content of the protein secondary structure of alpha helix (p value \. ) from . ± . to . ± . % (mean ± sd) as shown in fig. . this finding also is in agreement with increased changes in nucleoprotein structure caused by the presence of negative sense rna. figure a represents the final conformation of positive and negative sense rna complexes with cchfv nucleoprotein. in order to clarify the binding pattern, we only showed the secondary structure elements of the protein instead of showing all atoms. helices of a and a which are placed in the vicinity of cchfv binding site are shown in blue, while rna molecules are shown as balls and sticks. as seen, negative sense rna (right scheme) fits better to cchfv binding site in the region between head and stalk domains. in contrast, positive sense rna (left scheme) is placed somewhat far from binding site. figure a -b reconfirm the fact that the negative sense rna binds better and fits more effectively than the positive sense rna to its binding crevice throughout simulation. the agent of the crimean-congo hemorrhagic fever is a negative sense rna virus which causes one of most life threatening and lethal infectious diseases in human [ , ] . this virus is, therefore, worthy to be extensively [ ] [ ] [ ] . the main objective of the present study was to pick up some useful hints for better understanding of infection process of such a terrible disease. it seemed, therefore, sensible to start with the negative sense properties of cchfv and its ability to be enwrapped by viral nucleoprotein [ , ] . the preferential packaging of the negative sense rna by cchfv nucleoprotein in viral assembly seems to be assisted by specific structure recognition characteristics which differentiates between negative sense rna and positive sense one. this differentiation may be performed based on nucleotides composition and/or their assortment (sequence) within rna chains [ , [ ] [ ] [ ] [ ] . in the first run, we collected the complete sequences of genomic rna for positive and negative senses viruses listed in tables and [ ] . using microsoft excel, we calculated the total numbers of four nucleotides including g, a, c and u for each virus listed in the tables. then, we calculated the ratio of purines to pyrimidines for the negative and positive sense rna viruses. our results show that the negative sense rna has higher ratio of purines to pyrimidines (ratio [ ) than its counter positive sense rna which has a ratio of less than unity (ratio \ ). having bigger bases as well as polar groups, purine nucleotides such as g with highest binding energy and a with higher frequency amongst nucleotides interact stronger with their amino acid counterparts on cchfv nucleoprotein. our docking experiments confirm higher binding energies of . kj/mol per nucleotide for negative sense rna. this energy seems to be the main cause for preferential binding of the negative rna to cchfv nucleoprotein. our md simulations indicate that the negative sense rna makes a more stable complex with cchfv nucleoprotein ( fig. a-b) . the retained diffusion of the negative rna inside nucleoprotein with lowered slope of msd curve (fig. ) also confirms this claimed stability. attenuated curve of rmsf for cchfv nucleoprotein in complex with the negative sense rna compared to the positive sense rna (fig. ) indicates the formation of a more stable complex with lower flexibility in cchfv nucleoprotein. moreover, curves of the hydrophobic solvent accessible surface (sas) (fig. ) and the significant decrease in secondary structure (fig. ) confirmed stronger interactions between the negative sense rna and cchfv nucleoprotein during simulation hence inducing structural alterations of cchfv nucleoprotein. finally the extracted structures of binding site residues indicated the presence of more positive residues in cchfv for negative sense rna binding ( fig. a-b) . it was shown that lassa virus nucleoprotein binding site for rna is closed by a and a helices in closed conformation of trimeric structures. outward movement of these helices in gating mechanism open the binding site and permit rna entrance and binding to virus nucleoprotein [ ] . nevertheless the negative and the positive sense rnas here bind almost to the same binding site reported for lassa virus (fig. ) . however, our simulation trajectories indicate that there is neither a significant movement in a and a helices nor a significant disturbance in residues string of - to support the gating mechanism. instead, we conclude that cchfv nucleoprotein may act via different mechanism. cocrystallization of cchfv nucleoproteins with negative and positive sense rnas, x-ray crystallography and performing md simulations in conditions similar to those in virus seems to be the only way to elucidate the precise mechanism underlying rna binding to cchfv nucleoprotein. crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski- rna binding properties of bunyamwera virus nucleocapsid protein and selective binding to an element in the terminus of the negative-sense s 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structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation the glycoprotein cytoplasmic tail of uukuniemi virus (bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging electron cryo-microscopy and single-particle averaging of rift valley fever virus: evidence for gn-gc glycoprotein heterodimers crimean-congo hemorrhagic fever virus genomics and global diversity essential amino acids of the hantaan virus n protein in its interaction with rna crimean-congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses crystal structure of the borna disease virus nucleoprotein structural comparisons of the nucleoprotein from three negative strand rna virus families structure of the influenza virus a h n nucleoprotein: implications for rna binding, oligomerization, and vaccine design monomeric nucleoprotein of influenza a virus structure of influenza virus rnp. i. influenza virus 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characterization of inhibitor recognition molecular docking using arguslab, an efficient shape-based search algorithm and the ascore scoring function hexserver: an fft-based protein docking server powered by graphics processors whiskers-less hiv-protease: a possible way for hiv- deactivation the interpretation of protein structures: estimation of static accessibility characterization of the ph titration shifts of ribonuclease a by one-and two-dimensional nuclear magnetic resonance spectroscopy essential dynamics of lipase binding sites: the effect of inhibitors of different chain length crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding acknowledgments the financial support of shahid chamran university of ahvaz and tarbiat modares university, tehran, are acknowledged. there are no conflicts of interest to disclose. key: cord- - io ho authors: zwart, hub title: the art of living with nzt and ict: dialectics of an artistic case study date: - - journal: found sci doi: . /s - - - sha: doc_id: cord_uid: io ho i wholeheartedly sympathize conceptually with coeckelbergh’s paper. the dialectical relationship between vulnerability and technology constitutes the core of hegel’s master and slave (the primal scene of contemporary philosophy). yet, the empirical dimension is underdeveloped and coeckelbergh’s ideas could profit from exposure to case studies. building on a movie/novel (limitless) devoted to vulnerability coping and living with ict, i challenge the claim that modern heroism entails overcoming vulnerability with the help of enhancement and computers. himself to produce and process the items needed for his own subsistence (hegel (hegel / (hegel / . as a gesture of despair, he throws himself into a dramatic decisive struggle, putting his life at risk, and initially, this seems to work well. temporarily at least, he seems able to rely on the work of the slave (who is initially casted as the loser), on the tools and technologies the latter develops/deploys. whereas the master is allegedly autonomous, the slave is supposedly heteronomous (subjected to the master's will). gradually, however, due to the tools and technologies he develops and puts to use, the slave becomes increasingly powerful and autonomous, and the master increasingly vulnerable and dependent. we may read this as a simile for the human-technology relationship as such. initially, technologies (such as icts) are seen as instrumental (acquired and 'used' by us, for our own benefit). we decide to use them even if that means accepting the struggles and risks involved. gradually, we become increasingly dependent on the technologies which enable a particular way of being-in-the-world. they increasingly manage to re-sculpt our bodies and lives. while natural voices and ears, for instance, only allow us to communicate within the here and now, ict opens up a much broader realm, allowing us to communicate far beyond natural restrictions of space and time, enabling us to become who we are as hypermodern individuals, enabling us to function in a global environment. yet, once addicted to ict, new forms of vulnerability and risk are bound to proliferate. in short, i am basically in agreement with this paper (a piece of ''beautiful craftsmanship''). its basic 'poverty' for me resides in the 'empirical' dimension. inspiring ideas are developed on a fairly general level. they are not really tested/elaborated via systematic exposure to case studies. this is what i purport to do in my brief commentary: expose the author's arguments to a concrete artistic case of living-with-ict. in view of the convergence of the moral and the aesthetical dimension, 'genres of the imagination' (i.e. novels, movies, drama and the like) provide ample case material for such an exercise. i have selected one recent movie/novel for this purpose, which not only addresses (cognitive) enhancement, but also the various forms of living-with-itc associated with it. by doing so, it will become clear that, as soon as the empirical dimension is taken more seriously, the world is less straightforward than the (quietly theorizing) author presumes. in fact, at the point where his paper does become relatively concrete, i tend to disagree with him, namely where he speaks about modern cinematic heroes. according to the author, the typical modern hero ''is very powerful and has limited vulnerability, limited emotional involvement, and limited ties to others (think about protagonists in contemporary action films)''. building on a recent action movie, featuring a typical modern hero struggling in a world of ict, i will argue that this is not the case. quite the contrary, 'vulnerability' rather than 'power' remains the quintessence of hypermodern subjectivity, even in techno-thrillers. the focus, i will argue, is on the frantic (but inevitably faltering) efforts to cope with inherent, unsurmountable human vulnerability, notably in the face of ict-related risks. as a case study, i have chosen the movie limitless, based on the novel the dark fields by glynn (republished as limitless in ), telling the story of a middle-aged writer named eddie morra who, having finally secured a book-contract with a manhattan publisher, falls victim to a writers' block (glynn (glynn / . a nootropic wonder-drug named nzt allows him to lift his inhibitions, so that he not only finishes his book, but also becomes a stock market virtuoso. although limitless is primarily about brain enhancement, it addresses ict as well. or, rather, it unravels the various ways in which neuroenhancement and ict are intimately and mutually interconnected. from the very outset, eddie desperately tries to cope with human vulnerability. at the beginning of the movie, he struggles with language, with the first sentence of his unwritten novel: an alcohol-dependent artist in front on his laptop, unable to produce anything, while elsewhere in his room the telephone relentlessly sends out frightening signals (his publisher, reminding him of expiring deadlines). in other words, ict items meant to facilitate his productivity (laptop, telephone) become sources of anxiety. eddie is unable to live up to the personal ambitions and societal expectations mediated through these contrivances. suddenly, something unexpected happens. a former acquaintance offers him a wonderdrug, allowing him to mobilise his faltering creativity and to streamline his deficient brain. the drug completely alters his way of being-in-the-world, the tonality of his existence. it enables him to interact much more effectively with ict. his nzt-soaked brain increasingly mimics and merges with computers. he becomes so good with computers that he decides to play the stock-market. due to his ability to process huge amounts of information, he becomes the new hero of the trading scene (p. ). gradually, his electronic ruminations coalesce into ''an overwhelming vision of the vastness and beauty of the stock-market itself''. he sees it as ''a celestial firmament''. with its ''complexities and ceaseless motion, the -h global network of trading systems was nothing less than a template for human consciousness … a collective nervous system, a global brain'' (p. ). due to the sudden smoothness of the collaboration between machine and man, microchips and cells, circuits and synapses, he realizes ''a grand convergence of band-width and braintissue'', giving rise to the idea that his mind works ''like a living fractal'' (p. ). eventually, he becomes adviser and partner of the most powerful man of all manhattan: venture capitalist carl van loon. eddie masterminds the merger of two very large manhattan firms: a process of staggering complexity, beyond the grasp of ordinary individuals. so far, the movie/novel seems in complete agreement with coeckelbergh's verdict that modern heroes, notably in contemporary action films, are very powerful and have limited vulnerability. yet, on closer inspection, eddie's experience of invulnerability and power proves transitory and illusory. the risks involved in the living-with-ict which he enacts are transformed, significantly amplified even, rather than erased or reduced. very soon, his chronic and insurmountable vulnerability is painfully revealed. for a short period of time, eddie occupies the position of the master, accepting the life-threatening risks involved (addiction to an enigmatic neuro-pharmaceutical, and its increasingly detrimental sideeffects, but also the hazards involved in the world of high finance, such as blackmailing criminals). but in the end, his provision of wonder-pills proves finite. eddie's vicissitudes can be described in a topological manner. initially, he seems completely decentralised: a nameless writing atom adrift in manhattan. at a certain point, he moves towards the centre of the modern financial ict-dense world. but his performance within this global network is highly dependent on a combination of biopharmaceutical and information and communication technologies (zwart ) . interestingly, the novel and the movie opt for different endings. in the novel, eddie dies a miserable death as a fugitive suffering from paralyzing withdrawal symptoms, when the pill supply is discontinued for good. in the movie, he seems about to run for president. but would that change the picture? not really. it is not a coincidence, i guess, that the current us president is a president without a congress, disconnected from 'the system', governing via back-up systems (decrees), still attempting to live with vulnerability. open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the art of living with nzt and ict: dialectics of an artistic… limitless. london: faber & faber limitless as a neuro-pharmaceutical experiment and as a daseinsanalyse: on the use of fiction in preparatory debates on cognitive enhancement he was appointed as full professor of philosophy at radboud university's faculty of science. his research focus is on the philosophical dimensions of the life sciences, notably in areas such as synthetic biology and neuro-science. emerging issues are addressed from a 'continental' philosophical perspective, building on research traditions such as dialectics (hegel), phenomenology (heidegger) and psychoanalysis (lacan) key: cord- - aonqyub authors: royle, jamie; dobson, samuel john; müller, marietta; macdonald, andrew title: emerging roles of viroporins encoded by dna viruses: novel targets for antivirals? date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aonqyub studies have highlighted the essential nature of a group of small, highly hydrophobic, membrane embedded, channel-forming proteins in the life cycles of a growing number of rna viruses. these viroporins mediate the flow of ions and a range of solutes across cellular membranes and are necessary for manipulating a myriad of host processes. as such they contribute to all stages of the virus life cycle. recent discoveries have identified proteins encoded by the small dna tumor viruses that display a number of viroporin like properties. this review article summarizes the recent developments in our understanding of these novel viroporins; describes their roles in the virus life cycles and in pathogenesis and speculates on their potential as targets for anti-viral therapeutic intervention. as obligate intracellular parasites, viruses have evolved a myriad of strategies to manipulate the host cell environment to one that is conducive for virus replication. research over recent decades has identified a group of virus-encoded proteins able to mediate the passage of ions and solutes across cellular membranes, termed viroporins [ , ] . the majority of viroporins described are small (less than amino acids) and contain one or two transmembrane domains (tmd), although a small number of larger viroporins have been shown to encode up to three putative tmd [ ] . whilst high-resolution structural information is currently only available for a limited number of viroporins [ ] [ ] [ ] [ ] [ ] , a bio-informatic approach has often been successfully employed to identify key features in viroporins that lack any structural information [ , ] . their small size necessitates that viroporins must oligomerize in membranes to form an active channel complex. formation of these high-order complexes is often observed in mild detergents such as , -diheptanoyl-sn-glycero- -phosphocholine (dhpc) [ , ] and is likely to be mediated by hydrophobic interactions between the tmd of each monomer; although in some viroporins basic residues adjacent to the tmd may facilitate membrane binding and insertion [ , ] . known viroporins have been placed into distinctive classes based on the number of tmd and the orientation of their carboxyl termini relative to the endoplasmic reticulum (er) membrane [ ] . undoubtedly, this classification system is useful for cataloguing the expanding number of viroporins, however, it will need to adapt in order to accommodate those few viroporins encoding three tmd and it will also need to take into account the dynamic nature of membrane proteins in lipids, which are capable of altering the orientation of their termini depending on the lipid environment [ , ] . modulation of ionic homeostasis within specific cellular compartments allows for viroporins to manipulate a wide range of cellular processes from autophagy [ ] [ ] [ ] , trafficking [ , ] , inflammation [ , ] , transformation [ ] to cell survival [ ] . due to these broad perturbations to host cell physiology, it is not surprising that viroporin function has been shown to assist in all stages of the virus life cycle including entry, membrane penetration, genome replication and virus egress [ , ] . the existence of virus encoded pore-forming proteins was initially postulated nearly four decades ago [ ] . however, it was observations that the m protein of influenza a virus (iav) was able to form a tetramer [ ] and raise intracellular ph [ , ] that provided the first clues to its role as an ion channel. pioneering studies in xenopus oocytes demonstrated m -dependent currents that could be blocked by addition of the anti-viral compound amantadine [ ] . following this, viroporins were swiftly identified from a number of rna virus families. whilst iav m remains the paradigm, viroporins have now been described in a number of virus families including the flaviviridae, picornaviridae, retroviridae, coronaviridae, reoviridae and paramyxoviridae [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to date, the overwhelming majority of viroporins have been identified in rna viruses. however, given the myriad of cellular processes that they are able to modulate, it is logical to assume that all viruses might benefit from encoding such a protein. recent findings have now identified proteins that exhibit a number of viroporin characteristics encoded by members of the polyomaviridae and papillomaviridae. this review will summarize our understanding of these putative viroporins, describe their known functions and attempt to highlight how possible ion channel activity may aid the life cycles of these small dna tumor viruses. the polyomaviridae are small, non-enveloped, double-stranded dna viruses that infect a wide range of species [ ] . the family was named after the founding member, polyomavirus, which caused "many tumors" in mice [ ] , followed by the prototypic primate polyomavirus, simian vacuolating agent (sv ), from the rhesus monkey [ ] . the first two human polyomaviruses discovered in , jc and bk, were named after the index cases, and cause serious disease in the immunocompromised [ ] . the last decade has seen the discovery of several novel human polyomaviruses, including merkel cell polyomavirus, which causes an aggressive skin cancer [ , ] . these discoveries have led to resurgence in interest in polyomavirus biology and to the roles of virus encoded proteins in pathogenesis. in this regard, two members of the family have recently been shown to encode proteins with viroporin characteristics. in , progressive multifocal leukoencephalopathy (pml), a potentially fatal demyelinating disease of the brain, was discovered and later attributed to a novel polyomavirus termed jc [ ] . jc is widespread amongst the adult population, with studies suggesting infection rates are upwards of %, and possibly as high as % [ ] . despite the high prevalence, pml incidence is extremely low due to the tendency of jc to result in an asymptomatic latent infection of the kidneys, lymphatic system and bone marrow in immunocompetent individuals [ ] . activation of the virus occurs almost exclusively in immunocompromised patients and is characterised by a lytic infection of oligodendrocytes resulting in demyelination and development of pml, although it is not clear when the virus infects the central nervous system. before the introduction of antiviral therapies to combat the progression to aids, pml was a prominent feature of the hiv phenotype. since then, there has been a resurgence in pml cases as a wide range of immunosuppressant therapies are being applied to combat autoimmune diseases such as multiple sclerosis (ms) and aid in the acceptance of transplanted tissues [ ] . links between jc and a range of human cancers have also been reported, although these are disputed [ ] . similar to most polyomaviruses, jc encodes for early proteins, which constitute the small and large t antigens and their many splice variants, which engage with many host processes to ensure a cellular milieu is available that is conducive to virus replication. in addition to this, three late structural proteins are expressed. these include the major capsid protein, vp , and the vp /vp minor capsid proteins [ ] . jc encodes for an additional late protein termed agnoprotein [ ] . agnoprotein is only expressed by a limited number of polyomaviruses including the related bk and sv . the amino acid agnoprotein is highly basic and contains a central hydrophobic region capable of forming an amphipathic helix [ , ] . in concert with residues in the amino terminal region, this amphipathic helix is required for localization to the er and membrane insertion [ ] . biochemical analysis shows that residues - within the amphipathic helix are necessary and sufficient for dimer and oligomer formation [ ] . agnoprotein oligomers are stable in sds and do not depend on disulphide bridge formation [ ] . recent nuclear magnetic resonance (nmr) data confirmed the formation of an amphipathic helix between leu and phe [ ] . the basic nature of the protein may provide flexibility within the amino and carboxyl termini increasing the range of interactions with host partners. for a list of known jc agnoprotein binding partners see table . viroporin activity is associated with increased plasma membrane permeability, resulting in elevated cytosolic calcium levels [ ] . similar to several known viroporins, agnoprotein expression manipulates host trafficking pathways to allow transport of agnoprotein to the cell surface to mediate plasma membrane permeabilization [ ] . to achieve this, agnoprotein interacts with the δ sub-unit of adaptor protein complex (ap- ), inhibiting its function and as a consequence preventing trafficking of agnoprotein to the lysosome for destruction. substitution of two basic amino acids in the amino terminal region of agnoprotein (arg /lys ) prevented the interaction with ap- , perturbed sub-cellular localization and abrogated the plasma membrane permeabilization seen with the wild type protein. moreover, jc viruses containing either an agnoprotein deletion or alanine substitution of the basic residues resulted in a comparable defect in virion release from infected cells [ , ] . the conclusion of these studies is that the basic residues are a critical requirement for agnoprotein function, potentially by contributing towards viroporin activity. mutations of a similar basic motif in the hcv p protein also impaired viroporin activity, as assessed using a carboxylfluorescein dye release assay, however, further investigation in fact revealed that the mutant protein was no longer able to integrate correctly into membranes [ ] . it is plausible that by mutating the basic loop in agnoprotein, membrane integration has been perturbed which would be expected to have broader impacts on agnoprotein function beyond inhibiting viroporin function. agnoprotein has multiple roles within the jc life cycle, and some of these are known to require residues within the amphipathic helix, and as such may depend upon viroporin function. binding of the large t antigen to the viral origin of replication is enhanced in the presence of agnoprotein [ ] . agnoproteins containing mutations that would be expected to impair amphipathic helix formation display decreased large t binding and significantly reduced virus genome replication [ ] . some mutations within the amphipathic helix impact protein stability, resulting in reduced agnoprotein expression [ ] . as such the impact on virus replication might arise as a result of less agnoprotein rather than loss of putative viroporin activity. nevertheless, reduced jc replication is a feature observed in some agnoprotein deletion viruses, indicating that modulation of replication is a bona fide function of agnoprotein [ ] . as polyomavirus virions assemble in the nucleus, they are first required to exit this organelle into the cytoplasm prior to the egress from the infected cell [ ] . since complete lysis of the nuclear membrane would abolish the integrity of the infected cell and impair virus replication, jc has evolved to instead alter the nuclear envelope (ne) to mediate virus egress. jc infected cells show protrusions and invaginations in the ne, mediated through binding of agnoprotein to heterochromatin protein- (hp- ) [ ] . hp- normally interacts with the lamin-b receptor (lbr) to assist with the reassembly of the ne after cell division [ ] . however, the amino terminal domain of agnoprotein is capable of binding to hp- inducing disassociation from lbr and resulting in morphological changes to the ne, allowing escape of progeny virions [ ] . whether viroporin activity is required for this function is not clear, although binding to hp- requires the amino terminal residues, which are outside the putative tmd. agnoprotein has been shown to be subject to extensive post-translation modification and this is likely a key regulator of function. protein kinase c (pkc) can phosphorylate jc agnoprotein on three identified residues; ser , ser and thr [ ] . jc viruses containing alanine substitutions at these sites were less able to maintain an active infection, although they displayed enhanced agnoprotein expression levels at early time points during infection. subsequent studies identified an antagonistic role for protein phosphatase a (pp a) in agnoprotein phosphorylation [ ] . agnoprotein dephosphorylated by pp a was shown to inhibit jc replication to levels comparable with the alanine substitution mutants. mechanistically, jc small t antigen has been shown to bind to both pp a and agnoprotein, and is thought to reduce the ability of pp a to dephosphorylate agnoprotein [ ] . interestingly, depletion of pp a from jc infected cells using sirna also resulted in reduced virus replication [ ] . this raises the intriguing possibility that both phosphorylated and de-phosphorylated forms of agnoprotein have defined functions, and interplay between the two contributes to the successful replication and propagation of jc. these functions, however, have yet to be delineated. phosphorylation has been shown to be important in regulating the sub-cellular localization of proteins, as such it is plausible that the phosphorylation state of agnoprotein may determine its sub-cellular location and hence the role it plays in replication. whether it is also required to regulate viroporin function has not been shown. however, given the findings of sawa and colleagues that viroporin function is needed at the cell surface, it is a possibility that phosphorylation of agnoprotein may also contribute to localizing the channel to where it is required [ ] . regulation of viroporins by post-translational modifications would add another tier of control to these important proteins. jc agnoprotein shares significant identity with the agnoproteins encoded by the related viruses bk and sv ( % across the whole protein, rising to % in the amino terminal region) [ ] (figure ). as expected from such a high homology, agnoproteins of bk and sv appear to perform similar functions during the virus life cycle, although with some subtle differences. though less well studied, loss of agnoprotein appears to correlate with defects in the late stages of the virus life cycle and to perturb egress. in addition, agnoprotein may also play a role in packaging the sv genome [ ] . bk egress has recently been shown to be sensitive to the dids compound [ ] . given that dids is a broad-spectrum inhibitor of ion channels, it is possible that the target of its actions is an agnoprotein viroporin. it would be interesting to assess the impact of dids on virus egress from an agnoprotein knockout virus. the wide-ranging roles of agnoproteins identified from both jc and bk coupled with the deleterious effects seen in the agnoprotein deficient viruses warrants a serious analysis of the potential for agnoprotein as a target of direct acting antiviral therapeutics. groups have previously generated sirna to target jc agnoprotein and have had success of inhibiting viral replication in mice infected brains, justifying the premise of targeting this protein for inhibition [ ] . such studies would be expedited by a direct analysis of channel activity using recombinant agnoprotein, similar to the pioneering work on the hcv p protein [ ] . not only would this affirm channel function but would also provide a platform from which to screen the large libraries of compounds displaying viroporin inhibitory properties available. three late proteins have been shown to aid in sv entry and release by virtue of their viroporin-like properties. vp and vp are generated from successive met residues within the vp messenger rna so they share a common carboxyl-terminus. they are classed as minor constituents of the virus particle, being present at a stoichiometry of one copy of a vp or vp protein per vp pentamer [ ] . given that sv is a non-enveloped virus, it has evolved strategies to allow transport of the incoming virion through the membranous environment of the cytoplasm to the nucleus to initiate genome replication. a number of studies have shown that both vp and vp form pores in cellular membranes and may aid in delivery of the sv virion into the nucleus [ , ] . mutation of residues within putative tmd prevented membrane targeting and when engineered into sv genomes resulted in reduced infectivity [ ] . sv also encodes a unique very late protein termed vp , encoded by the same transcript as vp /vp . unlike other late proteins, vp is thought not to be incorporated into sv capsids. vp the wide-ranging roles of agnoproteins identified from both jc and bk coupled with the deleterious effects seen in the agnoprotein deficient viruses warrants a serious analysis of the potential for agnoprotein as a target of direct acting antiviral therapeutics. groups have previously generated sirna to target jc agnoprotein and have had success of inhibiting viral replication in mice infected brains, justifying the premise of targeting this protein for inhibition [ ] . such studies would be expedited by a direct analysis of channel activity using recombinant agnoprotein, similar to the pioneering work on the hcv p protein [ ] . not only would this affirm channel function but would also provide a platform from which to screen the large libraries of compounds displaying viroporin inhibitory properties available. three late proteins have been shown to aid in sv entry and release by virtue of their viroporin-like properties. vp and vp are generated from successive met residues within the vp messenger rna so they share a common carboxyl-terminus. they are classed as minor constituents of the virus particle, being present at a stoichiometry of one copy of a vp or vp protein per vp pentamer [ ] . given that sv is a non-enveloped virus, it has evolved strategies to allow transport of the incoming virion through the membranous environment of the cytoplasm to the nucleus to initiate genome replication. a number of studies have shown that both vp and vp form pores in cellular membranes and may aid in delivery of the sv virion into the nucleus [ , ] . mutation of residues within putative tmd prevented membrane targeting and when engineered into sv genomes resulted in reduced infectivity [ ] . sv also encodes a unique very late protein termed vp , encoded by the same transcript as vp /vp . unlike other late proteins, vp is thought not to be incorporated into sv capsids. vp encodes a single tmd and associates with membranes, where it generates a channel of defined pore size and increases membrane permeability. vp is expressed at least h later than other late proteins, indicating a potential role in virus release [ ] . vp channel activity is regulated by the lipid environment and shows a greater activity in liposomes mimicking the composition of the plasma membrane, and sv viruses lacking vp exhibit a significant defect in virus spread. together, these data indicate that vp is a viroporin that functions specifically during the latter stages of the sv life cycle [ ] [ ] [ ] [ ] . whether other mammalian polyomaviruses encode a vp protein is not clear. it is currently uncertain why sv might require vp when other polyomaviruses do not. jc agnoprotein has been shown to permeabilize the plasma membrane [ ] , and may therefore fulfil this role. however, given the sequence similarity it is likely that sv agnoprotein may also serve as a viroporin. it is possible that there is either redundancy or co-operation between agnoprotein and vp . these questions will remain unanswered until viroporin function is confirmed in sv agnoprotein. the papillomaviridae contains an extensive array of different papillomavirus (pv) types capable of infecting a variety of animal species, of which at least have currently been isolated from humans [ ] . like polyomavirus, they are small, non-enveloped double stranded dna viruses around nm in diameter [ ] . most pv encode six early (e , e , e , e , e and e ) and two late structural genes (l and l ). approximately hpv types, termed high-risk, are the causative agents of several ano-genital and oral malignancies [ ] . of these, hpv and hpv are the most important and are responsible for approximately % of the cervical cancer cases, and for the deaths of approximately , women in alone [ ] . low risk hpv are usually cleared by the body and vaccines are available for prophylactic treatment of high risk hpv, but there is as of yet no therapeutics for existing cases of hpv infection. three gene products e , e and e mediate the transforming potential of high-risk hpv. e and e are the major drivers of keratinocyte proliferation [ ] . they are necessary to maintain the cell cycle of differentiating keratinocytes and they achieve this by binding to and inactivating the retinoblastoma (prb) and p tumor suppressor proteins [ ] . repression of e and e in various cell lines has been shown to induce cellular senescence and halt differentiation. the e protein is the least understood of the three oncoproteins [ ] . hpv e is a highly hydrophobic, amino acid membrane protein that resides in the lumen of cytoplasmic membranes and interacts with a growing number of cellular partners [ , [ ] [ ] [ ] [ ] [ ] (figure a) . the triple membrane spanning topology of hpv e was determined by partial membrane permeabilization fluorescence studies in cells and these support the idea that the e amino terminus resides in the lumen of the er with a carboxyl terminus exposed to the cytosol [ ] . lack of specific antibodies preclude localization studies from virus infected cells, however, epitope tagged e has been shown by over-expression studies to predominantly localize to er and golgi membranes [ ] . cell surface localization has also been noted [ ] , although this has been disputed by others [ ] . sequence analysis demonstrates that a recognizable e gene is missing from several hpv types (beta, gamma and mu). further, viruses that do encode an e open reading frame show significant sequence divergence, with the resulting protein product ranging in size from approximately - amino acids. such significant divergence might indicate distinctive roles for e within the hpv life cycle, dependent on the specific virus type. importantly, all high-risk cancer causing hpv types encode an e protein similar to hpv , of approximately acids termed e alpha [ ] . hpv e oligomerizes in vitro and in cells, with oligomer formation driven not by the presence of disulphide linkages between cysteine residues, rather by hydrophobic interactions between individual monomers [ , ] . in our laboratory demonstrated that the e oligomer could mediate the controlled release of the small molecule carboxyflourescein using a liposome dye release assay [ ] . whilst some questions have been raised as to the validity of this indirect measure of channel activity [ ] , it has been widely used with multiple channels (e.g., hcv p , classical swine fever virus (csfv) p , respiratory syncytial virus (rsv) sh), and has provided important insights into their activity [ , , ] . the stoichiometry of hpv e was predicted to be hexameric using in silico modelling and subsequently confirmed by native page electrophoresis and transmission electron microscopy [ ] (figure b ). these complexes displayed channel-forming activity with a defined pore size, and activity was increased in acidic ph. sensitivity of e to the adamantane derivative rimantadine [ ] and the alkyl imino-sugar nn-dnj (our unpublished data) was demonstrated in vitro, as well as to a novel small molecule inhibitor generated using in silico modelling of the e channel and subsequent docking analysis [ ] . journal , volume, page-page fever virus (csfv) p , respiratory syncytial virus (rsv) sh), and has provided important insights into their activity [ , , ] . the stoichiometry of hpv e was predicted to be hexameric using in silico modelling and subsequently confirmed by native page electrophoresis and transmission electron microscopy [ ] (figure b ). these complexes displayed channel-forming activity with a defined pore size, and activity was increased in acidic ph. sensitivity of e to the adamantane derivative rimantadine [ ] and the alkyl imino-sugar nn-dnj (our unpublished data) was demonstrated in vitro, as well as to a novel small molecule inhibitor generated using in silico modelling of the e channel and subsequent docking analysis [ ] . e is predicted to have three transmembrane domains (tmds; boxes) based on the hydrophobic nature of its amino acids. membrane permeabilization assays demonstrate that the carboxyl-terminus extends into the cytosol while the amino-terminus is directed towards the endoplasmic reticulum (er) lumen. the first tmd gives rise to the subcellular localization of e and mediates binding to mhc class i molecules and calnexin. the second tmd facilitates the recently identified interaction with the transmembrane protein yipf as well as the k subunit of the h + -atpase. further functions of e such as the increase of koilocytosis, activation of egfr signaling and induction of endosome alkalinization are exerted via the third tmd; (b) the sequence of hpv e was obtained from uniprot and the secondary structure predicted using psipred and memsat- and energy minimized. the model for an e monomer contained three tmd and had the lowest energy and was used to build a hexameric model using the protocol described previously [ ] . each monomer in the model was minimized individually to restore the symmetry and was refined using prime (schrodinger inc). the oligomeric state of hpv e was confirmed by native page and transmission electron microscopy [ ] . e expression induces anchorage-independent growth in murine nih t cells and mitogenic effects in primary human foreskin epithelial cells [ , ] . transformation has been recapitulated in vivo using transgenic mouse models where e is expressed in epithelial cells under the control of the keratin- promoter. in these mice, e expression was associated with hyperplasia and tumor formation [ , ] . a number of studies have demonstrated the importance of the epidermal growth figure . hpv e is a tmd viroporin. (a) hpv e is predicted to have three transmembrane domains (tmds; boxes) based on the hydrophobic nature of its amino acids. membrane permeabilization assays demonstrate that the carboxyl-terminus extends into the cytosol while the amino-terminus is directed towards the endoplasmic reticulum (er) lumen. the first tmd gives rise to the subcellular localization of e and mediates binding to mhc class i molecules and calnexin. the second tmd facilitates the recently identified interaction with the transmembrane protein yipf as well as the k subunit of the h`-atpase. further functions of e such as the increase of koilocytosis, activation of egfr signaling and induction of endosome alkalinization are exerted via the third tmd; (b) the sequence of hpv e was obtained from uniprot and the secondary structure predicted using psipred and memsat- and energy minimized. the model for an e monomer contained three tmd and had the lowest energy and was used to build a hexameric model using the protocol described previously [ ] . each monomer in the model was minimized individually to restore the symmetry and was refined using prime (schrodinger inc). the oligomeric state of hpv e was confirmed by native page and transmission electron microscopy [ ] . e expression induces anchorage-independent growth in murine nih t cells and mitogenic effects in primary human foreskin epithelial cells [ , ] . transformation has been recapitulated in vivo using transgenic mouse models where e is expressed in epithelial cells under the control of the keratin- promoter. in these mice, e expression was associated with hyperplasia and tumor formation [ , ] . a number of studies have demonstrated the importance of the epidermal growth factor (egf) receptor (egfr) for e -induced transformation [ , ] , and these have since been reinforced by genetic studies which showed that transgenic mice expressing e failed to produce tumors when crossed with mice encoding for an inactive egfr [ ] . thus the contribution of e to host cell transformation appears dependent on manipulation of the egfr. studies aimed at understanding the role of e in the productive hpv life cycle show that e is required to maintain the proliferative status of infected cells as they undergo terminal differentiation, in order to allow virus genome replication. moreover, e causes a delay to the early stages of keratinocyte differentiation to achieve this. using small molecule inhibitors targeting egfr we have been able to show that these processes are also dependent on active egfr signalling (our unpublished data). the precise mechanism by which e manipulates the egfr is unclear. the current consensus model is that e expression is associated with a deacidification of endosomes [ ] . this prevents the normal degradative trafficking pathways, which would culminate in deposition of active egfr in the lysosome and termination of egfr signalling. in e expressing cells active egfr appears to be re-routed into recycling endosomes and returned to the plasma membrane, where it maintains mitogenic signalling [ ] . deacidification of endosomes might result from specific interactions with host binding partners [ ] , or, similar to iav m and hepatitis c virus p , might indicate a requirement for direct proton channel activity [ , ] . in support of this idea, enhanced egfr signalling observed in e expressing cells was reduced by the small molecule inhibitors that prevented carboxyfluorescein release in the in vitro dye release assays [ ] . these data suggest that the oncogenic activity of e may be directly linked to viroporin function. if this proves to be the case then e would represent the first example of an oncogenic viroporin. the development of specific mutant e proteins that lack channel activity will need to be tested in these assays in order to validate the small molecule inhibitor studies, which can be fraught with off-target effects. moreover, it will be of great interest to generate viruses containing these mutants in order to study at what stage of the hpv life cycle any putative viroporin function is necessary. the viroporin function was described for e from hpv [ ] . given that e proteins from the other high-risk hpv types are predicted to adopt a similar three tmd topology, it will be of interest to determine whether viroporin function is conserved amongst this group of viruses. whilst the presence of a defined pore size of < nm was confirmed in these studies [ ] , future work should focus on determining the ion selectivity of the e channel to conclusively determine whether there is a preference for protons or whether ion selectivity fluctuates dependent on the particular membrane environment, as has been shown for other viroporins [ ] . finally, although e is not thought to be expressed in cervical cancer cells due to the integrated nature of the genome [ ] , it is postulated to play a role in the early stages of cancer development. given the limited treatment options for those already infected with hpv, ongoing research into the development of e inhibitors with greater potency suited to drug development programs appears feasible given our findings [ ] . thus, there is potential for drug development targeting e , yet whether this will ultimately prove relevant in the post-vaccination era remains to be seen. viroporins are multi-faceted viral proteins shown to play key roles in the life cycles of a number of important human pathogens. the relatively recent identification of such proteins encoded by small dna tumor viruses offers the opportunity to understand their contribution towards the productive life cycle of these viruses and ultimately their pathogenesis. analysis of viroporin deletion mutants in these viruses will undoubtedly help to delineate their functions and may provide an important insight into how they manipulate critical host functions. the large economic and health burden associated with virus infection, alongside the rapid increase in resistance to existing therapeutic regimes, if available, demonstrates the need for new anti-viral drugs. given the low mutation rate of dna virus genomes, identifying virus encoded targets as exemplified by the viroporins presents an attractive target for future therapeutics. increasing understanding of viroporin structure and function, along with the advent of high throughput technologies will hopefully lead to the development of these much needed treatments. viroporins: structure, function and potential as antiviral targets viroporins: structure and biological functions high-risk human papillomavirus e oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors unusual architecture of the p channel from hepatitis c virus structural and functional properties of the hepatitis c virus p viroporin. viruses structure and inhibition of the sars coronavirus envelope protein ion channel insight into the mechanism of the influenza a proton channel from a structure in a lipid bilayer the small hydrophobic protein of the human respiratory syncytial virus forms pentameric ion channels determinants of hepatitis c virus p ion channel function and drug sensitivity identified in vitro a conserved basic loop in hepatitis c virus p protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria the human polyoma jc virus agnoprotein acts as a viroporin analysis of the processing and transmembrane topology of the e p protein of hepatitis c virus viroporin-mediated calcium-activated autophagy autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication viroporin activity of the foot-and-mouth disease virus non-structural b protein inhibition of cellular protein secretion by poliovirus proteins b and a intracellular proton conductance of the hepatitis c virus p protein and its contribution to infectious virus 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(jcpyv): virological background and clinical implications progressive multifocal leukoencephalopathy and other disorders caused by jc virus: clinical features and pathogenesis progressive multifocal leukoencephalopathy and other forms of jc virus disease jc virus: an oncogenic virus in animals and humans? semin agnoprotein of mammalian polyomaviruses essential roles of leu/ile/phe-rich domain of jc virus agnoprotein in dimer/oligomer formation, protein stability and splicing of viral transcripts nuclear magnetic resonance structure revealed that the human polyomavirus jc virus agnoprotein contains an α-helix encompassing the leu/ile/phe-rich domain viroporin activity of the jc polyomavirus is regulated by interactions with the adaptor protein complex infection by agnoprotein-negative mutants of polyomavirus jc and sv results in the release of virions that are mostly deficient in dna content virion assembly factories in the nucleus of polyomavirus-infected cells dissociation of heterochromatin protein from lamin b receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles domain-specific interactions of human hp -type chromodomain proteins and inner nuclear membrane protein lbr phosphorylation mutants of jc virus agnoprotein are unable to sustain the viral infection cycle dephosphorylation of jc virus agnoprotein by protein phosphatase a: inhibition by small t antigen anion homeostasis is important for non-lytic release of bk polyomavirus from infected cells an sirna against jc virus (jcv) agnoprotein inhibits jcv infection in jcv-producing cells inoculated in nude mice structure-guided design affirms inhibitors of hepatitis c virus p as a viable class of antivirals targeting virion release structure of simian virus at . -a resolution late proteins possess lytic properties that render them capable of permeabilizing cellular membranes sv vp and vp insertion into er membranes is controlled by the capsid protein vp : implications for dna translocation out of the er the viroporin activity of the minor structural proteins vp and vp is required for sv propagation the sv late protein vp is a viroporin that forms pores to disrupt membranes for viral release a very late viral protein triggers the lytic release of sv sv late protein vp forms toroidal pores to disrupt membranes for viral release the simian virus late viral protein vp disrupts the nuclear envelope for viral release classification of papillomaviruses (pvs) based on pv types and proposal of taxonomic amendments the natural history of cervical hpv infection: unresolved issues human papillomavirus oncoproteins: pathways to transformation human papillomavirus e oncoprotein: function and potential target for antiviral therapeutics koilocytosis: a cooperative interaction between the human papillomavirus e and e oncoproteins bap is a novel target of the human papillomavirus e protein karyopherin beta : a new cellular target for the hpv- e oncoprotein membrane orientation of the human papillomavirus type e oncoprotein characterization of the plasma membrane localization and orientation of hpv e for cell-cell fusion mucosal human papillomaviruses encode four different e proteins whose chemistry and phylogeny correlate with malignant or benign growth oligomerization of the e protein of human papillomavirus type occurs through multiple hydrophobic regions two different conformations in hepatitis c virus p protein account for proton transport and dye release the stability of secreted, acid-labile h /jfh- hepatitis c virus (hcv) particles is altered by patient isolate genotype a p sequences classical swine fever virus p protein is a viroporin involved in virulence in swine resistance mutations define specific antiviral effects for inhibitors of the hepatitis c virus p ion channel the e gene from human papillomavirus type is an oncogene which enhances growth factor-mediated signal transduction to the nucleus the e oncoprotein of human papillomavirus type transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes role for hpv e in cervical carcinogenesis requirement of epidermal growth factor receptor for hyperplasia induced by e , a high-risk human papillomavirus oncogene human papillomavirus type e gene stimulates the transforming activity of the epidermal growth factor receptor the e oncoprotein of human papillomavirus type inhibits the acidification of endosomes in human keratinocytes endoplasmic reticulum-localized human papillomavirus type e protein alters endosomal ph but not trans-golgi ph binding of human papillomavirus e to the kda subunit c (proteolipid) of the vacuolar h+-atpase can be dissociated from the e -mediated epidermal growth factor receptor overactivation inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment the authors declare no conflict of interest. key: cord- - iahlshj authors: loa, chien chang; wu, ching ching; lin, tsang long title: a multiplex polymerase chain reaction for differential detection of turkey coronavirus from chicken infectious bronchitis virus and bovine coronavirus date: - - journal: animal coronaviruses doi: . / - - - - _ sha: doc_id: cord_uid: iahlshj a multiplex polymerase chain reaction (pcr) method for differential detection of turkey coronavirus (tcov), infectious bronchitis virus (ibv), and bovine coronavirus (bcov) is presented in this chapter. primers are designed from the conserved or variable regions of nucleocapsid (n) or spike (s) protein genes of tcov, ibv, and bcov and used in the same pcr reaction. reverse transcription followed by pcr reaction is used to amplify a portion of n or s gene of the corresponding coronaviruses. two pcr products, a -bp band corresponding to n gene and a -bp band corresponding to s gene, are obtained for tcov. in contrast, one pcr product of bp corresponding to a fragment of n gene is obtained for ibv strains and one pcr product of bp corresponding to a fragment of s gene is obtained for bcov. turkey coronavirus (tcov) contributed to signifi cant economic losses and remains as a serious threat to the turkey producers. turkey coronaviral enteritis in areas with high concentrations of turkeys on a year-round basis is not easily eliminated and is encountered frequently in turkey poults [ ] . accurate and rapid method for diagnosis of tcov infection is the key to effective control of the disease. turkey coronavirus belongs to the family coronaviridae, which is a group of enveloped, positive-stranded rna viruses that infect a wide range of mammalian and avian species. the major structural proteins of coronavirus include phosphorylated nucleocapsid (n) protein, peplomeric spike (s) glycoprotein, and transmembrane or membrane (m) glycoprotein. spike protein contributes to the distinctive peplomers on the viral surface and contains neutralizing and group-specifi c epitopes. spike protein is highly variable among different coronaviruses while m and n proteins are more conserved among coronaviruses between different antigenic groups [ ] . there is a close antigenic and genomic relationship between tcov and infectious bronchitis virus (ibv) according to studies of immunofl uorescent antibody assay ( ifa ), enzyme-linked immunosorbent assay ( elisa ), and sequence analysis in our and other laboratories [ - ] . in addition, bovine coronavirus (bcov) was demonstrated to cause experimental enteric infection in turkey [ ] . therefore, close relationship between tcov and bcov was previously reported and tcov was placed in an antigenic group as bcov [ , ] . although the sequence data revealed divergence of s genes among tcov, ibv, and bcov [ - ] , there is still a need to detect and differentiate them accurately and quickly. polymerase chain reaction ( pcr ) assay has been an important approach for detecting many veterinary important microorganisms with the distinct advantages of high sensitivity and specifi city. this chapter describes a multiplex pcr assay to detect and differentiate tcov, ibv, and bcov in a single reaction [ ] . elmer centers corp., norwalk, ct, usa). . ethidium bromide, . μg/ml. stomacher with fi vefold volume of pbs solution. . the intestinal homogenates are clarifi ed by centrifugation at × g for min. the supernatants containing tcov are used as virus source for preparation of rna templates for reverse transcription (rt)-pcr reaction. . two hundred microliters of above supernatants are mixed with ml of rnapure reagent and incubated on ice for min ( see note ). . add μl of chloroform, mix the mixture, and vortex vigorously for s ( see note ). . centrifuge at , × g for min at °c. carefully take the upper aqueous phase into a clean tube and mix with equal volume of cold isopropanol by vortexing vigorously for s. incubate on ice for min. . centrifuge at , × g for min at °c. carefully discard the supernatant without disturbing rna pellet. . wash rna pellet with ml of cold % ethanol. incubate on ice for min. . the suggested ratio of rnapure reagent to sample is : . excess amount of rnapure reagent has no negative impact. the lower ratio ( : ) in this step is intended to obtain higher concentration of viral rna in the fi nal supernatants. if the upper aqueous phase after centrifugation at step is more than half of the total volume, there is not enough rnapure reagent added. the appropriate reagent amount may be adjusted. chloroform is applied at μl for every ml of lysate. . the sample mixture with chloroform at this step can be stored at − °c or lower than − °c before proceeding to the next step. . for routine practice in the laboratory for large number of samples, μl reaction can be applied with reduced cost of reagents. tcov positive: two pcr products, a -bp band, and a bp band. ibv positive: one pcr product of bp. bcov positive: one pcr product of bp. negative: no pcr product. one limitation of this multiplex pcr should be noted. when both tcov and ibv are present in the sample, the ibvpositive pcr product at bp is overlapping with one of the two tcov pcr products. the result of two pcr product bands at and bp refl ects a confi rmed diagnosis of tcov positive but does not rule out the presence of ibv. this concern is alleviated by tissue tropism of ibv. turkey enteric infection by chicken respiratory ibv has never been reported. accordingly, the presence of ibv in turkey intestines (the test sample) is not likely. any such concern should be further evaluated by a separate pcr specifi c for ibv [ ] . on the other hand, ibv-positive result is able to exclude the presence of tcov. coronaviral enteritis of turkeys (blue comb disease) coronavirus immunogens antigenic characterization of a turkey coronavirus identifi ed in poult enteritis and mortality syndrome-affected turkeys phylogenetic analysis of a highly conserved region of the polymerase gene from coronaviruses and development of a consensus polymerase chain reaction assay sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus sequence analysis of the turkey coronavirus nucleocapsid protein gene and ′ untranslated region identifi es the virus as a close relative of infectious bronchitis virus detection of antibody to turkey coronavirus by antibodycapture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen nucleocapsid protein gene sequence analysis reveals close genomic relationship between turkey coronavirus and avian infectious bronchitis virus experimental bovine coronavirus in turkey poults and young chickens antigenic and genomic relationships among turkey and bovine enteric coronaviruses sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus complete sequences of ′end coding region for structural protein genes of turkey coronavirus comparison of ′ end encoding regions of turkey coronavirus isolates from indiana, north carolina, and minnesota complete nucleotide sequence of polyprotein gene and genome organization of turkey coronavirus differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction redesign of primers and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de strain of infectious bronchitis virus the protocol "a multiplex polymerase chain reaction for differential detection of turkey coronavirus from chicken infectious bronchitis virus and bovine coronavirus" outlined in this chapter had been successfully carried out in the authors' studies on molecular diagnostics and molecular virology of turkey coronavirus infection in turkeys. those studies were in part fi nancially supported by usda, north carolina poultry federation, and/or indiana department of agriculture and technically assisted by drs. tom brien and david hermes, mr. tom hooper, and ms. donna schrader for clinical and diagnostic investigation, virus isolation and propagation, and animal experimentation. key: cord- -z pd v authors: yen, muh-yong; schwartz, jonathan; hsueh, po-ren; chiu, allen wen-hsian; armstrong, donald title: traffic control bundling is essential for protecting healthcare workers and controlling the ebola epidemic date: - - journal: clin infect dis doi: . /cid/ciu sha: doc_id: cord_uid: z pd v nan to the editor-a global health crisis, the ebola outbreak has now struck healthcare workers (hcws) at unprecedented levels. whereas historically, ebola epidemics spread via person-to-person transmission, the current outbreak in west africa has seen unexpectedly extensive spread of nosocomial disease, despite hcws' reliance on previously effective infection control procedures such as patient isolation, barrier nursing procedures, and required personal protective equipment (ppe) [ ] . indeed, infection struck even among hcws caring for patients with ebola virus disease (evd) in western hospitals equipped with modern facilities and procedures. this has sparked growing concerns regarding how to protect hcws [ ] , even those working outside the illprepared and overwhelmed regions of west africa now grappling with ebola [ ] . in our view, the most concerning examples include dr khan [ ] , a sierra leonean virologist who contracted ebola despite his extensive experience and careful adherence to procedures; dr spencer [ ] , a médecins sans frontières physician who became symptomatic upon returning to new york despite working in well-designed isolation units built specifically to protect hcws from evd infection; and dr sacra, an obstetrician who contracted ebola without having knowingly cared for any evd patients [ ] . based on these developments and the knowledge that ebola may remain viable to a certain degree on dry solid surfaces with fomites for approximately day [ , ] , we hypothesize that fomite transmis-sion of ebola may best explain some of these unanticipated cases. fomite transmission is facilitated by the practice of situating patients with acute symptoms and potentially extremely high viral loads outside isolation rooms in environments where adherence to routine disinfection practices is rare [ ] . taiwan's experience with severe acute respiratory syndrome (sars) in is instructive. we contend that during the height of the sars epidemic, hcws in institutions that failed to identify designated zones of risk simply assumed they were secure from risk as long as they were not in proximity to patients with highly contagious pathogens. however, their confidence in existing barrier precautions and ppe as providing sufficient protection when away from heavily contaminated areas proved unwarranted [ ] . as it turned out, consistent use of ppe and negative pressure isolation rooms was insufficient because the main cause of nosocomial sars transmission was casual contact with fomites in contaminated environments either outside of isolation zones or during removal of ppe [ , ] . unlike when dealing with contamination by nuclear or chemical spills, there exist no distinct boundaries delineating contaminated and clean zones when managing biological disasters. lacking clearly designated zones of risk, fomites far from patient rooms were still found to be positive for sars coronavirus rna [ ] . in the mistaken belief that they were well away from contaminated areas and because they encountered no visible contamination, hcws occasionally came into contact with these fomites after removing their ppe. in our view, this same scenario likely explains the infection with ebola suffered by drs khan, spencer, and sacra. realizing the threat of nosocomial infection, the taiwan centers for disease control responded by implementing traffic control bundling (tcb), which included triage and diversion of patients before they enter the hospital; clear delineation of zones of risk between contaminated and clean zones; and gloves-on hand disinfection at checkpoints between zones of risk ( figure ) [ ] . tcb proved critical (p < . ) for protecting hcws [ ] . indeed, infection rates among hcws caring for sars patients dropped to zero following its implementation, ultimately contributing to nationwide sars control [ ] . a key aspect of successful tcb is installation of alcohol dispensers in all zones to encourage hand disinfection. the dispensers not only demonstrate to hcws the significance of zones of risk, but also strengthen adherence to, and frequency of, hand disinfection. during the taiwan response to sars, alcohol dispensers were situated along the path from the contaminated zones through the intermediate zones and into the clean zones ( figure ). as a result, compliance by hcws with hand disinfection rose to % [ , ] . having already disinfected their hands, even when hcws touched their surroundings after leaving contaminated zones and while removing ppe, they were already decontaminated and thus did not make contact with nor left fomites. in conclusion, we suggest that tcb is a powerful, convenient, and economical tool for protecting hcws against highly contagious diseases. we recommend that it be implemented as part of ongoing efforts to contain and control the current ebola outbreak. figure . conceptual scheme of traffic control bundling. following triage outside the hospital entrance, all the way until being hospitalized in the isolation room, the patients remained contained inside a zone of risk (red arrow), which is distinguished from "clean zones" through the "intermediate zone." dispensers with % alcohol for gloves-on hand sanitation are installed at checkpoints positioned in between zones of risk. healthcare workers (hcws) in a clean zone are required to don personal protective equipment (ppe) before entering the zones of risk. when departing a zone of risk, but prior to entering a clean zone, hcws are required to undergo decontamination and remove ppe in an intermediate zone. here hcws disinfect their hands, gloved or not, between every single step of the decontamination process and removal of ppe to avoid casual contact of skin/mucosa with the virus. ebola and compliance with infection prevention measures in nigeria guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in u.s. hospitals, including procedures for putting on (donning) and removing (doffing) sierra leone's top ebola doctor gets virus nyc doctor craig spencer followed proper protocol after returning from ebolastricken west africa us doctor with ebola lands in nebraska for treatment persistence in darkness of virulent alphaviruses, ebola virus, and lassa virus deposited on solid surfaces assessment of the risk of ebola virus transmission from bodily fluids and fomites using an integrated infection control strategy during outbreak control to minimize nosocomial infection of severe acute respiratory syndrome among healthcare workers taiwan's traffic control bundle and the elimination of nosocomial severe acute respiratory syndrome among health care workers from sars in to h n in : lessons learned from taiwan in preparation for the next pandemic key: cord- - op yss authors: gordon, julian; gandhi, prasanthi; shekhawat, gajendra; frazier, angel; hampton-marcell, jarrad; gilbert, jack a. title: a simple novel device for air sampling by electrokinetic capture date: - - journal: microbiome doi: . /s - - - sha: doc_id: cord_uid: op yss background: a variety of different sampling devices are currently available to acquire air samples for the study of the microbiome of the air. all have a degree of technical complexity that limits deployment. here, we evaluate the use of a novel device, which has no technical complexity and is easily deployable. results: an air-cleaning device powered by electrokinetic propulsion has been adapted to provide a universal method for collecting samples of the aerobiome. plasma-induced charge in aerosol particles causes propulsion to and capture on a counter-electrode. the flow of ions creates net bulk airflow, with no moving parts. a device and electrode assembly have been re-designed from air-cleaning technology to provide an average air flow of lpm. this compares favorably with current air sampling devices based on physical air pumping. capture efficiency was determined by comparison with a . μm polycarbonate reference filter, using fluorescent latex particles in a controlled environment chamber. performance was compared with the same reference filter method in field studies in three different environments. for common fungal species by quantitative polymerase chain reaction (qpcr), there was % sensitivity and apparent specificity of %, with the reference filter taken as “gold standard.” further, bacterial analysis of s rna by amplicon sequencing showed equivalent community structure captured by the electrokinetic device and the reference filter. unlike other current air sampling methods, capture of particles is determined by charge and so is not controlled by particle mass. we analyzed particle sizes captured from air, without regard to specific analyte by atomic force microscopy: particles at least as low as nm could be captured from ambient air. conclusions: this work introduces a very simple plug-and-play device that can sample air at a high-volume flow rate with no moving parts and collect particles down to the sub-micron range. the performance of the device is substantially equivalent to capture by pumping through a filter for microbiome analysis by quantitative pcr and amplicon sequencing. understanding of the microbiology of air, the aerobiome, is an emerging field of discovery. high-throughput sequencing methods are being used to explore the spatiotemporal distribution of bacterial and fungal populations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a variety of sampling methods have been used for studying the air microbiome [ , [ ] [ ] [ ] [ ] . a variety of different sampling devices are currently available to acquire air samples of microbial and viral particles [ ] . these technologies include filters, impingers, impactors, and wet or dry cyclones. the underlying principle of impactors, impingers, and cyclones is the use of an abrupt change in direction of airflow so that aerosol particles will continue on to a surface by virtue of their momentum. filters are microporous membranes, impingers capture on to the surface of a nutrient agar plate for subsequent colony counts, and impactors capture on a solid surface for subsequent elution, as do dry cyclones. wet cyclones capture by vortexing into a liquid phase. aerosol particles may also be separated into size classes with multi-stage devices. for existing devices, capture efficiency falls off rapidly with particle size and there is considerable variability in performance [ ] [ ] [ ] . all of these devices require pumping against some resistance. different apparent microbial communities were found from the use of different air sampling techniques [ , ] . there is thus a need in aerobiome analysis for a sampling procedure that does not bias the measured biodiversity. here, we introduce the use of a very simple device for collection of samples and show equivalence to a reference method using filtration. in addition, a variety of air sampling methods have been applied to the airborne transmission of disease [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . brown first described the principle of ionic propulsion in us patents [ , ] . a corona wire is subject to a high voltage, creating plasma that imparts charge on particles in the vicinity. the charged particles are then propelled by the voltage gradient to electrodes at an opposing potential. the net flow of charged particles imparts forward momentum on the surrounding medium. the result is a net airflow with no moving parts. this principle has been used in commercial air-cleaning devices [ ] . custis et al. [ ] used such an air-cleaning device for collecting dust from the air for measuring allergens. we have developed a mini-scale device using the same principle with optimized airflow and an electrode cartridge that is optimized for sample collection (inspirotec sampler). we have demonstrated its use for detection of allergens by immunoassay [ , ] and viruses by quantitative pcr [ ] . the device is simple to operate, compact, and can be placed unobtrusively in any environment. here, we compare performance with a filter reference method for analysis of the aerobiome. for the purpose of this study, three environments were selected for side-by-side comparison of the inspirotec sampler, with an air filter as a reference device. the environments were a clean bathroom, a basement room with an exposed sump drain, and a hay storage room in a large equestrian facility. mold spores are ubiquitous in the environment but vary according to temperature, humidity, season, and other environmental conditions. table shows the results of -h samples in the three locations. the inspirotec sampler values have not been corrected for capture efficiency (approximately %, see "methods" section). if this table mold spores in three locations a inspirotec sampler, b filter. spore equivalent values were determined by multiplex qpcr (see "methods" section). green shading: detectable by both methods; yellow: detectable by inspirotec sampler only; no shading: below the limit of detection of the qpcr correction were applied, the values would all be consistent within the standard deviations achievable by the quantitative polymerase chain reaction (qpcr). those cases where the species was detected by both methods are indicated by green shading. in no instance was a species detectable by the filter and not detectable by the inspirotec sampler. in eight instances, the inspirotec sampler detected a species that was not detected by the filter, indicated by yellow shading in table . by this criterion, if the filter is considered a gold standard, the sensitivity is % and the specificity is %. however, these are likely to be true positives since the inspirotec sampler is sampling a larger volume of air in a given time. to address this concern, we examined the accumulation rate for eurotium amstelodami on both filters and the inspirotec sampler (fig. ). this shows that the inspirotec sampler processed both a larger volume, and correspondingly, a larger quantity of spore equivalents were captured, when compared to the filter. as with table , these numbers have not been corrected for capture efficiency. it is not clear why the quantity of spore equivalents captured appears to peak at h for both filter sampling and inspirotec sampler. nevertheless, this illustrates the advantage of the high sampling volume of the inspirotec sampler. this advantage is compounded by the inspirotec sampler's easier logistical set-up and silent performance. timed samples were run in the basement environment with the same schedule as in fig. . bacterial s rrna amplicon sequencing generated a total of , , sequences from samples. when rarified to sequences per sample, , operational taxonomic units (otus; % identity) were identified. no significant difference in microbial community structure was observed between the inspirotec samplers and the reference method with the use of the r project for statistical computing freeware (weighted or unweighted unifrac distance adonis, p > . , r = . ). false-discovery rate (fdr) and bonferronicorrected p values showed no significant differences in otu frequencies between platforms. the genus-level community profile generated by both technologies comprised predominantly acinetobacter, gordonia, methylobacterium, and pseudomonas (fig. ). differences in abundances in fig. are therefore not significant. interestingly, min produced a signal highly similar to the time zero (blank) suggesting that this time frame was insufficient to generate enough biomass for the detection threshold of the amplicon sequencing technology (fig. ) . however, by min, the community profiles were significantly different from time zero. reagent-based contamination is known to be an issue [ ] and explains the detected signal for blank and min. the table . volume sampled is computed from the individual inspirotec sampler flow rates and from the lpm setting for the filters. zero time samples were placed in the respective device, power was not turned on, and they were otherwise treated identically to the timed samples significance of the similarity between microbial profiles generated by the filter and inspirotec sampler technologies at each time point was assessed using procrustes analysis including the left and right electrode (technical replicates) of the inspirotec sampler as well as the pumpdriven filter. over the course of time, there was no significant difference between either technical replicate or the air filter, despite greater variability between samplers at the starting zero time (monte carlo, p > . ) (fig. ) . a prediction of the method of using ionic propulsion to capture the charge particles is that the capture should be independent of particle size of mass, unlike current aerodynamic-based sampling systems. the location of capture is dependent on the force vectors determined by the voltage gradient. mass may affect particle acceleration and velocity, but the final capture location is determined by a potential well. we therefore explored table . the top sequences were selected from the otu table, and relative abundance of bacterial genera was plotted across consecutive time points between samplers as described in the "methods" section. zero time samples were as in fig. sample capture with a random sample of air (bathroom of table ) and examined the size distribution of captured particles by atomic force microscopy. particles down to the nanometer range were captured (fig. ) , as demonstrated using a visual representation of particle size density (fig. a) , as well as a size distribution curve, with a significant fraction trailing into the lower range (fig. b) . the inspirotec sampler was run in the bathroom for -h (table ) and scanned (see "methods" section). the inset shows the lower end of the distribution curve and captured particles extending down into the -nm range and below. the sampler is thus able to capture particles going down to very low sizes and in the range that will penetrate the lungs and cause symptoms. this illustrates the range of sizes of not-identified aerosol particles that are captured. we have demonstrated the applicability of an electrokinetic air sampler for the molecular detection of microorganisms in air. all procedures are of wide applicability to any measurements in the aerobiome. this technology is easily deployed as it can be plugged into any electrical socket, silent, has low visual impact, and so could be readily applied to indoor settings for identifying and tracking emerging pandemics. the device performance was comparable to or exceeded that of the reference method. the device is inexpensive and requires no technical skill to operate, compared with any other competing technology. both sars and mers [ , ] epidemics may be traced back to human-animal interfaces, and early deployment in such emerging pandemics would facilitate the tracing of early stages and subsequent routes of transmission. in a separate study [ ] , we showed the capability of capturing venezuelan equine encaphilitis virus in a controlled environment chamber at the us army edgewood chemical biological center with aerosol particles down to μm. the viruses had been inactivated by gamma irradiation. the result was that a large proportion of the original virions had rna that was not amplifiable, so the capture efficiency was apparently very low based on the original virus titer. however, analysis by digital pcr using the poisson distribution at low amplicon concentrations showed that the capture efficiency was in the range of - % for these articles. here, the performance exceeded that of the reference method using a microporous filter and showed ability to detect mold spores using epa-accepted pcr technology [ ] . of the species for which primers and probes were used in the qpcr, acremonium strictum, alternaria alternata, aspergillus flavus, aspergillus fumigatus, aspergillus niger, aspergillus ochraceus, aspergillus sydowii, aspergillus ustus, aspergillus versicolor, chaetomium globosum, cladosporium cladosporioides, eurotium amstelodami, memnoniella echinata, paecilomyces variotii, penicillium aurantiogriseum, penicillium brevicompactum, penicillium chrysogenum (type ), penicillium purpurogenum, penicillium variabile, scopulariopsis brevicaulis, stachybotrys chartarum, trichoderma viride, ulocladium botrytis, all but aspergillus ustus, memnoniella echinata, and penicillium variabile were detectable as spores over the three locations tested. another feature of the electrokinetic propulsion is the ability to capture and measure particles down into the nanoparticle range. particles generated from respiratory activities in the range of . to μm are associated with infection [ ] . most commonly used air sampling devices have a cut-off at about μm [ ] . allergens may extend down to a size range that has been missed by (table ) and scanned (see "methods" section). a false color d representation of a × μm square. height above the plane is in the same micrometer scale. b particle size distribution analysis of same data. size distribution in bins and percent of particles in each bin plotted. inset: - . μm range shown on × expanded scale current sampling technology [ ] , and there is evidence that bacterial endotoxin, which exacerbates the effect of allergens, may exist in size ranges below the size of bacteria [ ] . the device described in this publication will have the capability of extending the size range of particles that can be collected from the aerobiome. fahlgren et al. [ ] found that the diversity of microbial communities captured by different samplers in external environments in norway and sweden were similar, whereas hoisington et al. [ ] have found significant inconsistencies between different sampler types at locations within a us retail store. in neither case was consideration made to the time of run needed to resolve reagent background. we showed that a minimum of h sampling was required regardless of the method. we demonstrate the application of ionic propulsion technology to capture a wider range of particle sizes than with traditional air filter sampling, with no significant bias in fungal and bacterial community recovery. inspirotec samplers and accompanying electrode cartridges were provided by inspirotec llc (glenview, il). a commercial air cleaner was modified to achieve a unidirectional flow. the disposable capture cartridges were designed to optimize capture of aerosol particles and for easy release into extraction tubes. they were made by d printing at exact prototyping, joliet, il, and stainless steel electrode strips by lakeshore cutting solutions, zeeland, mi. electrodes were finished by able electropolishing, chicago, il. cartridges were washed with % isopropanol and air dried, and electrodes were washed with isopropanol and dried in a vacuum oven for min at °c. assembled cartridges and electrodes were heatsealed into polyethylene bags until use. once the electrodes have been released, they cannot be re-used. there is no potentially non-sterile surface that physically contacts the electrodes between removal from the bag and release into the collection tube. an updated version of the inspirotec sampler has a block of honeycomb-structured mno catalyst covering the outlet. this modification removes traces of ozone from the effluent air but does not affect performance. samplers and cartridges can be purchased by sending an email to the senior author with heading "research request." the sampler will have a lower cost than any alternative system. flow rates were measured with a hot wire anemometer by averaging measured flow velocities across the width of a duct placed over the outlet [ ] . individual samplers' flow rates varied by ± lpm. capture efficiency (determined by alburtylabs, inc., drexel, mo) as judged by capture of μm fluoresbrite® yg carboxylate microspheres (polysciences, inc., warrington, pa) in a controlled environment chamber, compared with capture by a reference sampler consisting of a . -μm polycarbonate filter, was ( ± )% based on determinations. fluorescence of captured microspheres was with a turner quantech fluorometer. the reference samplers were run at lpm. thus, the capture efficiency is more than compensated by the higher volumes of air sampled. following their standard isolation procedure, captured mold spores were determined by qpcr for the most common mold species by emlab p&k, marlton, nj. primers and probes and amplification conditions used are in [ ] . electrodes were released into ml falcon tubes and shipped overnight for analysis. they were extracted by vortex mixing intermittently over min with . % tween , and spores were centrifuged down and extracted by bead-beating [ ] . results were computed as spore equivalents [ ] [ ] [ ] [ ] . these publications show that standard deviations of spore equivalent counts may be ± log or more. amplicon sequencing: each cartridge holds two electrodes. left and right electrodes were processed separately, and so were effective duplicates. individual electrodes were placed into sterile -ml conical tubes with ml of sterile water. samples were extracted for min on a vortex genie (mo bio laboratories, inc., carlsbad, ca). their standard powersoil extraction was performed according to the manufacturer's suggested protocol with the addition of a -min incubation at °c after addition of solution c , as suggested by the earth microbiome project. genomic dna was amplified using the earth microbiome project barcoded primer set, adapted for miseq (illumina inc., san diego, ca) by adding nine extra bases in the adapter region of the forward amplification primer that support paired-end sequencing. the v region of the s rrna gene ( f- r) was amplified with region-specific primers that included the illumina flow cell adapter sequences. the reverse amplification primer also contained a -base barcode sequence that supports pooling of up to different samples in each lane. each -μl pcr reaction contains μl of mo bio pcr water (certified dna-free), μl of prime hotmastermix ( μm concentration), μl of forward primer ( μm concentration, pm final), μl golay barcode-tagged reverse primer ( μm concentration, pm final), and μl of template dna. the conditions for pcr were as follows: °c for min to denature the dna, with cycles at °c for s, °c for s, and °c for s; and with a final extension of min at °c to ensure complete amplification. pcr amplifications were completed in triplicate and then pooled. following pooling, amplicons were quantified using picogreen (invitrogen) and a plate reader. once quantified, different volumes of each of the products were pooled into a single tube so that each amplicon was represented equally. this pool was then cleaned using the ultraclean® pcr clean-up kit (mo bio) and quantified using qubit (invitrogen). sequencing of the prepared library was performed on the illumina miseq platform, using the sequencing primers and procedures described in the supplementary methods of caporaso et al. [ ] . for the atomic force microscopy, a sampler was run for days in the clean bathroom location of table . it was examined with atomic force microscopy with a bruker dimension icon system which has the capability of providing sub-nanometer resolution. imaging was done in tapping mode with super sharp silicon probes. results were analyzed with the bruker analysis software. all sequence data will be made available through figshare, http://dx.doi.org/ . /m .figshare. competing interests julian gordon and prasanthi gandhi are co-founders and co-owners of inspirotec llc and plan to commercialize the inspirotec sampler. authors' contributions jg contributed to experimental design, performed the air sampling and mold spore data reduction, and had primary responsibility for drafting the manuscript. pg contributed to the experimental design and reviewing of the manuscript. gs performed the atomic force microscopy and interpretation of the results therefrom. af performed the pcr and amplicon analysis and wrote that part of the methods section. jh-m was responsible for the pcr and amplicon analysis experimental design and data reduction and contributed to the manuscript writing. jag contributed to the experimental design and manuscript review and writing. all authors read and approved the final manuscript. seasonal variability in airborne bacterial communities at a high-elevation site spatial variability in airborne bacterial communities across land-use types and their relationship to the bacterial communities of potential source environments sources of bacteria in outdoor air across cities in the midwestern united states microbiome of the upper troposphere: species composition and prevalence, effects of tropical storms, and atmospheric implications temporal variation in airborne microbial populations and microbially-derived allergens in a tropical urban landscape architectural design influences the diversity and structure of the built environment microbiome variability in 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airborne aspergillus contamination during hospital construction works: efficacy of protective measures monitoring aspergillus species by quantitative pcr during construction of a multistorey hospital building a study of air microbe levels in different areas of a hospital environmental sampling for aspergilli during building construction on a hospital site electrokinetic apparatus fluid flow control system reducing staphylococcus aureus bacterial counts in a dental clinic using an ionic breeze air purifier: a preliminary study quantitative measurement of airborne allergens from dust mites, dogs, and cats using an ion-charging device evaluation of a compact ionic capture device for airborne allergens with the use of a controlled environmental chamber evaluation of a compact ionic capture device for airborne allergens in inner city schools evaluation of an ion capture device for determination of aerosolized venezuelan equine encaphailitis virus and a novel method for absolute particle count determination. american association for aerosol research reagent and laboratory contamination can critically impact sequence-based microbiome analyses emerging diseases researchers scramble to understand camel connection to mers tracing the sars-coronavirus method of identifying and quantifying specific fungi and bacteria the role of particle size in aerosolised pathogen transmission: a review dust mite allergens are carried on not only large particles particle size distributions and concentrations of airborne endotoxin using novel collection methods in homes during the winter and summer seasons anonymous (traversing a duct to determine average air velocity or volume. application note tsi . tsi incorporated quantitative pcr analysis of selected aspergillus, penicillium and paecilomyces species quantitative pcr analysis of house dust can reveal abnormal mold conditions evaluation of a rapid, quantitative real-time pcr method for enumeration of pathogenic candida cells in water development of an environmental relative moldiness index for us homes house dust mite allergy: complete removal of the provoking allergen is a primary therapeutic approach this work was partly supported by breakout labs, a program of the thiel foundation, and partly from personal funds from julian gordon and prasanthi gandhi. the authors are grateful to ms diana schnell for making the equestrian facility available for this study. this work was supported in part by the us dept. of energy under contract de-ac - ch . • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - qxdgrq authors: d'silva, liesel; hassan, nesreen; nair, parameswaran title: serum procalcitonin and infective exacerbations of asthma date: - - journal: chest doi: . /chest. - sha: doc_id: cord_uid: qxdgrq nan www.chestpubs.org reports a -year and -year survival among patients with cwp that is similar to those with other indications for lung transplantation; six of the eight miners remained alive after a mean follow-up of , days after transplantation. enfi eld et al at the university of virginia also recently reviewed outcomes in patients at multiple centers who underwent lung transplantation for severe cwp since , using the database of the united network for organ sharing. in the university of virginia analysis, after account ing for age, lung allocation score, and type of transplant, -year survival after transplantation appeared to be signifi cantly lower in patients with cwp ( %) compared with non miner patients with copd ( %) or interstitial lung disease ( %). however, independent of the exact prognosis of cwp after transplant, the reality of advanced cwp is that medical treatment may ameliorate symptoms but does not reverse the lung damage or halt the progressive fi brotic process. because of this, we agree that lung transplantation must be considered an option, particularly for the younger miners who are now developing this disorder. dr diaz-guzman and colleagues encourage increasing awareness of advanced cwp and early referral to a transplant center, in recognition that massive fi brosis in coal miners often progresses even after removal from dust exposure. we concur that the medical community needs to be more effective in enhancing awareness of the continuing human toll from these dust diseases and in assuring optimal medical care and fair compensation for affected miners. progressive massive fi brosis is entirely preventable, since it is virtually only caused by excessive dust inhalation and does not occur from tobacco use or other causes. effective dust controls should have eliminated this type of lung disease in a modern mining industry, and the failure of the us industry to tackle this ongoing problem has been highlighted internationally. in addition to drawing attention to the role of lung transplantation to improve survival and functional status in these patients, we hope our report's fi ndings can motivate timely implementation of the necessary effective measures to reduce dust exposures and provide a healthful working environment for our country's coal miners. . wade aw , petsonk el , young b , mogri i . severe occupational pneumoconiosis among west virginian coal miners: one hundred thirty-eight cases of progressive massive fi bro sis to the editor: bafadhel and colleagues report in a recent issue of chest (june ) that serum procalcitonin levels are high in patients admitted to hospital with pneumonia but not in those admitted with exacerbations of asthma or copd. we examined the usefulness of serum procalcitonin in patients with moderate to severe exacerbations of asthma due to infections. we recruited patients ( men) with confi rmed diagnosis of asthma during what was considered an infective exacerbation (increased symptoms as measured by a seven-point likert scale, increased sputum volume and purulence) that was not severe enough to require hospitalization. none of the patients had radiologic evidence of pneumonia. spirometry was performed and nasopharyngeal swabs and sputum were obtained for virology, bacterial culture, and quantitative cell counts. measurements were repeated at , , and weeks until symptoms had completely resolved. procalcitonin was measured in serum at the time of exacerbation and at weeks. procalcitonin was measured in duplicate from m l of serum using a time-resolved amplifi ed cryptate emis sion technology assay (kryptor trace pct; brahms; berlin, germany ). the lower limit of detection is . ng/ml, and the assay functional sensitivity was . ng/ml. , . sputum e, % c . ( - ) . ( - ) . serum procalcitonin, ng/ml . ( . ) . ( . ) . values are given as mean (sd) unless otherwise noted. e eosinophil; n neutrophil; tcc total cell count. a symptoms of cough, chest tightness, wheeze, and shortness of breath were measured on a seven-point likert score ( best, worst). b median (interquartile range). c median (minimum-maximum ). to the editor: we thank dr d'silva and colleagues for their interest in our recent article in chest . most asthma exacerbations are considered to be associated with viruses, and current guidelines do not advocate the use of antibiotics for exacerbations of asthma. interestingly, dr d'silva and colleagues only identifi ed viruses in % of cases and bacteria in %. in copd, where bacterial infection is implicated as a major cause of exacerbations, current evidence does not support the use of antibiotics in the management of mild to moderate exacerbations. whether patients with asthma and bacterial-associated exacerbations benefi t from antibiotic therapy is uncertain; however, further detailed analysis of the microbiology of asthma exacerbations using both standard and molecular techniques is warranted. we and others have found that procalcitonin is not strongly associated with an exacerbation of copd or asthma, but is elevated in patients with pneumonia. it is, therefore, a good biomarker of a systemic infl ammatory response to pneumonia and may have potential clinical utility in directing antibiotic therapy. importantly, the value of procalcitonin might not be greater than the more widely available c-reactive protein. controlled trials of antibiotics directed by biomarkers such as c-reactive protein or sputum cell counts at exacerbations of asthma and copd are required. biomarker-guided therapy is commonplace in other medical specialties, such as cardiology. this approach needs urgent investigation and to be embraced by respiratory medicine if we are to make a change in the management of exacerbations of airways disease. leicester, england procalcitonin and c-reactive protein in hospitalized adult patients with community-acquired pneumonia or exacerbation of asthma or chronic obstructive pulmonary disease development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fl uid microbead-based assay measuring bronchitis in airway diseases: clinical implementation and application: airway hyperresponsiveness in asthma: its measurement and clinical significance role of sponsors: procalcitonin measurements were made by brahms gmbh, berlin, germany. none of the sponsors was involved in the development of the protocol, acquisition and interpretation of data, statistical analysis or preparation of the manuscript. other contributions: the procalcitonin measurements were facilitated by rudolf nothelfer, phd, brahms aktiengesellschaft, and virology by jim mahony, phd, mcmaster university. affi liations: from the institute for lung health, university of leicester . financial/nonfi nancial disclosures: the authors have reported to chest the following confl icts of interest: dr brightling is a wellcome senior clinical fellow; has received consultancy fees from medimmune, astrazeneca, glaxosmithkline, and roche; and has received research grants from astrazeneca, medimmune, and glaxosmithkline. dr bafadhel has reported to chest that no potential confl icts of interest exist with any companies/organizations whose products or services may be discussed in this article . symptoms and fev improved signifi cantly, and the sputum cell counts returned to normal at weeks ( table ) . however, there was no signifi cant difference in procalcitonin levels between the initial measurement and at weeks ( table ). there were also no differences between patients who had infective vs noninfective exacerbations or those with viral vs bacterial bronchitis.our data confi rm the observations of bafadhel and colleagues that serum procalcitonin is unlikely to be useful to identify infective exacerbations of asthma. elevated sputum total cell count with predominant neutrophilia is a more reliable indicator of an infective bronchitis. key: cord- -p vzmpg authors: gleason, a. e.; bolme, c. a.; lee, h. j.; nagler, b.; galtier, e.; milathianaki, d.; hawreliak, j.; kraus, r. g.; eggert, j. h.; fratanduono, d. e.; collins, g. w.; sandberg, r.; yang, w.; mao, w. l. title: ultrafast visualization of crystallization and grain growth in shock-compressed sio( ) date: - - journal: nat commun doi: . /ncomms sha: doc_id: cord_uid: p vzmpg nan gpa shots are shown here as panels (a) and (b)) and on ceo standard (panel (c)). pixels associated with the spaces in between asics of the cspads have been removed for refinement. show consistency in sample intensity over several runs and across -hour shifts (e.g., and ). examples of dark patterns are also shown to give ~ counts. note that some asics show an apparent, artificial offset in intensity for the dark patterns (e.g., - °). inset: graph of incident x-ray photon energy as a function of runs listed for the x-ray only fused silica patterns. on average, photons per pulse per shot were recorded for this experiment. velocity interferometer system for any reflector (visar) data was collected simultaneously for each drive intensity and xfel delay combination. the applied pressures listed in this paper are determined using the transit time through the sample package to calibrate a pressure-irradiance scaling law for the gdp ablator, and then using the scaling law exponent provided in swift and kraus to determine the sample pressure for the lower energy shots. this method is preferred to using the transit times at the lower stress states to directly determine the shock pressure from an inferred wave velocity because of the complicated compressive response of fused silica below ~ gpa. the variation in strain for the material involved in this style of compression is significant, however, the volume fraction is less than %. to determine the transit time through the entire sample, the drive laser was accurately co-timed with the streak cameras used for the visar diagnostic. possibly due to break-up of the fused silica material upon breakout of the shock-wave at the free surface, it was impossible to determine the free surface velocity of the fused silica. instead, we measure a total transit time through the gdp ablator and the fused silica sample. the hyades radiation hydrodynamics code was used to model the wave propagation through the gdp ablator and the fused silica sample. sesame and equations of state were used for the gdp ablator and fused silica, respectively considering the same drive conditions were used) and within the uncertainty of the visar derived stress value of . ± . gpa. no preferred orientation corrections in gsas were required to provide a match of ( ), ( ), ( ) and ( ) peak intensities to previously published powder data found in the crystallographic information file (cif) . this cif provided the starting phase information for gsas. from this comparison to ross et al. peak intensities, we find our diffraction data lack preferred orientation. note that the right most asics (at the highest θ) for each time slice show significant damage (e.g., not recording any intensities at all, damaged pixels, shadowing). therefore, only peak position is recorded for ( ) due to difficulties extracting reliable relative intensities in these quadrants. it should be noted that for our refinements, no amorphous component was considered when fitting the background. since the goal was to refine the crystalline peak position and intensity, we decided not to include the diffuse component in the refinement fitting and the background was fit with a chebyshev polynomial in gsas irrespective of the location of the shock front in the sample and percent shocked or unshocked material. we also note that the largest contribution to the reduced-χ value is derived from the ( ) peak straddling the edge of asics such that if we artificially fill in those intensities the reduced-χ value decreases significantly. to calibrate our sample-to-detector distances, detector tilts and determine instrumental broadening we used a nist standard reference material (srm) b ceo powder. a rietveld refinement (supplementary, fig. ) of ceo powder shows much narrower peaks than for the stishovite grown during shock compression. we find the compressed fused silica peak is centered at ~ . ( ) Å - . if we assume -fold coordination for this glass (i.e., comparing to work from sato and funamori for a low density amorphous (lda) phase), the pressure estimate is . (+/- . ) gpa. this contrasts the ( ) peak position of stishovite and visar data (in combination with the pressure-irradiance scaling) suggesting a much lower applied pressure of . ( ) gpa. we speculate the glass component is transitioning from a -fold to -fold coordination and a strict comparison to a pressure derived from only a -fold glass structure would be unfair and manifest as an inconsistency. in other words, a density or pressure comparison would only be valid if we: a) knew the structure of our glass phase, and b) found that structure to be comparable to the findings of sato and funamori . however, given the θ coverage of our data, we cannot assess the glass structure and therefore a pressure comparison is not appropriate. furthermore, the stress conditions we explore in this experiment are within the "mixed phase region" of fused silica which makes interpretation of these data complex and in no way are we indicating these findings are definitive, rather a starting place for exploring new physics. in an effort to provide a metric for identification (or classification) of behaviors as a function of pressure we decide to use the crystalline diffraction/visar applied pressure value of . gpa for the rest of the paper. the development of stishovite peaks at longitudinal stresses of . and . gpa is contradictory to the sio p-t phase diagram as determined from static compression experiments . in our experiments, diffraction peaks matching coesite are never seen. to the best of our knowledge, coesite has not been produced or recovered from shock wave experiments due to the quenching path necessary to form metastable coesite . we explain this by the sluggish nature of the transition quartz to coestite and the over-driven pressure conditions on a reduced timescale allowing the higher symmetry, metastable stishovite phase to form first. in other words, if the transition from fused silica is kinetically inhibited from going to coesite then it may be thermodynamically consistent to transition to stishovite in the coesite stability region. a similar phenomenon has been observed in water-ice. metastable ice vii forms from the liquid in the ice vi stability regime and metastable ice vii forms from the high density amorphous phase in the ice vi stability regime , . phases of simpler symmetry can be seen in the stability regime of less symmetric phases and indicates a kinetic-control process influenced by a low interface free energy. we cross-check our estimate of grain size against the commonly used scherrer equation which does not contain a strain broadening term : , where σ = grain size; k = dimensionless shape factor (commonly set to . ); λ = x-ray wavelength; β = line broadening at full width at half maximum (fwhm) minus instrumental broadening ( . °); θ = bragg angle. since this equation is suited for a single bragg peak, after calculating the grain size for each peak in a given trace, we average these values together to determine a single grain size per trace. the uncertainty extracted from each fit is also averaged to determine a total scherrer uncertainty for the trace. as expected, we find remarkable agreement (grain size values are identical within uncertainties) compared to the warren-averbach method using the prescription of hawreliak et al. . however, we note that the warren-averbach method does not discriminate between inter-grain and intra-grain strain broadening. in order to assume the simplest model possible, using the expression d = k(t-t ) /n , we fix n to single value and let k and t be free parameters. we tested a range of n values but found the curvature of the model trend did not match the data points well if we forced n ≤ . at n = or the t values were reasonable, but find the best fit for all datasets was at n = (± ). a table of our fitting parameters is as follows: for the . and . gpa data, the t values are indistinguishable with the uncertainty, therefore we group them together to give an average nucleation time of . ( ) ns. note that for the . gpa trend, we have only data points so the fitting is not well-constrained. general structure analysis system (gsas) expgui, a graphical user interface for gsas accurate measurements of laser-driven shock trajectories with velocity interferometry properties of plastic ablators in laser-driven material dynamics experiments hyades-a plasma hydrodynamics code for dense plasma studies sesame database: los alamos nat. lab. la- -ms p-v-t equation of state of stishovite up to mid-lower mantle conditions high pressure crystal chemistry of stishovite typical values of rietveld instrument profile coefficients high-pressure structural transformation of sio glass up to gpa the quartz-coesite transition equations of state and phase equilibria of stishovite and a coesitelike phase from shock-wave and other data shockwave compression of quartz crystallization of water in dynamic diamondanvil cell: evidence for ice vii-like local order in supercompressed water high density amorphous ice at room temperature scherrer after sixty years: a survey and some new results in the determination of crystallite size the effect of cold-work distortion on x-ray patterns high-pressure nanocystalline structure of a shock-compressed single crystal of iron key: cord- -dmbl emn authors: bonsor, daniel a.; beckett, dorothy; sundberg, eric j. title: structure of the n-terminal dimerization domain of ceacam date: - - journal: acta crystallographica section f structural biology communications doi: . /s x sha: doc_id: cord_uid: dmbl emn ceacam is a human cellular adhesion protein that is expressed on the surface of colon and rectum epithelial cells and is downregulated in colorectal cancers. it achieves cell adhesion through dimerization of the n-terminal igv domain. the crystal structure of the n-terminal dimerization domain of ceacam has been determined at . Å resolution. the overall fold of ceacam is similar to those of ceacam and ceacam ; however, there are differences, the most notable of which is an insertion that causes the c′′ strand to buckle, leading to the creation of a hydrogen bond in the dimerization interface. the k (dimerization) for ceacam determined by sedimentation equilibrium is tenfold tighter than that measured for ceacam . these findings suggest that the dimerization affinities of ceacams are modulated via sequence variation in the dimerization surface. carcinoembryonic antigen-related cell adhesion molecules (ceacams) belong to the immunoglobulin (ig) family and are expressed differentially on the surfaces of cells (gray-owen & blumberg, ; tchoupa et al., ) . there are ceacams found in humans: ceacam , ceacam -ceacam , ceacam and ceacam -ceacam (beauchemin & arabzadeh, ; tchoupa et al., ) . their functions and roles in cellular processes are diverse and include roles in phagocytosis, hearing, proliferation, signaling, tumor suppression and cell adhesion (oikawa et al., (oikawa et al., , benchimol et al., ; streichert et al., ; pils et al., ; singer et al., ; zheng et al., ) . ceacams are typically dysregulated in cancer and are found to be parasitized by bacteria (e.g. neisseria meningitidis, escherichia coli and haemophilus influenzae) and viruses in mice (e.g. coronavirus) during infection (dveksler et al., ; leusch et al., ; bos et al., ; schö lzel et al., ; virji et al., ; duxbury et al., ; litkouhi et al., ; obrink, ; singer et al., ) . ceacams contain an n-terminal immunoglobulin variable domain (igv), a variable number of immunoglobulin constant domains (igc ) and either a c-terminal transmembrane and cytoplasmic domain or a glycophosphatidyl-inositol (gpi) moiety by which they are anchored to the plasma membrane (tchoupa et al., ) . cell adhesion is achieved through the n-terminal domain of ceacams, which can undergo heterodimerization and homodimerization in a cis (on the same cell) or trans (across different cells) fashion (taheri et al., ; watt et al., ) . # international union of crystallography ceacam is expressed on highly differentiated epithelial cells of the colon and rectum and on the epithelial cells within the ducts of the pancreas (schö lzel et al., ) . the expression pattern of ceacam suggests a specialized function. in fetal tissues of the colon, ceacam is located at the base of epithelial cells and has been found to migrate to the apical surface a few days after birth (schö lzel et al., ) . ceacam contains three domains: an n-terminal igv domain, a single igc domain and a cell-surface gpi anchor domain (tchoupa et al., ) . ceacam expression is downregulated during the early development of colorectal tumors, unlike ceacam or ceacam , which are typically upregulated, suggesting a tumor-suppression function (schö lzel et al., ) . currently, it is unknown whether ceacam is involved in cell adhesion through homodimerization or if any structural differences exist that could potentially allow ceacam to function as a tumorsuppression molecule when compared with the two known structures of ceacam and ceacam (fedarovich et al., ; korotkova et al., ) . here, we report the . Å resolution x-ray crystal structure of the n-terminal domain of ceacam and have characterized its oligomeric state in solution. the n-terminal domain of ceacam was synthesized as a codon-optimized geneart string (life technologies), which was digested and ligated into an ncoi/xhoi-cut pet- d vector without a purification tag. ceacam was expressed in inclusion bodies in e. coli bl (de ) plyss cells. briefly, l of cells were grown in lb miller at k until an od nm of $ . was attained, prior to induction with mm isopropyl -d- -thiogalactopyranoside (iptg). cells were grown for a further h before harvesting ( g for min at k). the cells were resuspended in lysis buffer [ mm tris-hcl, mm nacl, %(v/v) triton x- ph . ] and lysed by sonication. inclusion bodies were isolated ( g for min at k), resuspended in lysis buffer, sonicated and isolated by centrifugation ( g for min at k). inclusion bodies were washed with a high-salt buffer ( mm tris-hcl, . m nacl ph . ) to remove dna, followed by lysis buffer without triton x- . inclusion bodies were dissolved in mm tris-hcl, mm nacl, . m urea ph . ($ ml per litre of grown cells), refolded by rapid dilution ( : ratio) at k into mm ches-hcl, mm l-arginine ph . and left for h. refolded ceacam was dialyzed against mm tris-hcl ph . and concentrated by anion-exchange chromatography (mono q, ge healthcare). a linear salt gradient from to mm was run at ml min À over min, with ceacam eluting at between and mm nacl. ceacam was further purified by size-exclusion chromatography (superdex , ge healthcare) in mm tris-hcl, mm nacl, mm edta ph . and fractions were stored at k. typically, mg of refolded protein per litre was obtained with a refolding efficiency of %. macromolecule-production information is summarized in table . ceacam was concentrated using a centricon centrifugal filter unit ( kda mwco, millipore) and subsequently dialyzed against mm tris-hcl, mm nacl ph . . ceacam at . mg ml À was screened against the jcsg+ suite screen (qiagen) using a crystal gryphon protein crystallography system (art robbins instruments) with sitting drops consisting of nl protein solution and nl reservoir solution equilibrated against ml reservoir solution. a shower of small crystals grew over d in condition d crystals of ceacam were washed and cryoprotected in mother liquor containing %(v/v) glycerol. data were collected on beamline -id-b at the advanced photon source (aps), argonne national laboratory, usa. data were processed using hkl- (otwinowski & minor, ) . data-collection and processing statistics are shown in table . f obs were obtained using scalepack mtz . molecular replacement was performed using molrep (vagin & teplyakov, ) and a chainsaw (stein, ) model of ceacam (pdb entry qsq; korotkova et al., ) , a protein with % sequence identity to ceacam . ceacam was refined with refmac (murshudov et al., ) and rebuilt in coot (emsley et al., ) . molprobity (chen et al., ) was used for ramachandran analysis. refinement statistics are shown in table . sedimentation-equilibrium measurements of ceacam were performed using a beckman-coulter xl-i analytical ultracentrifuge equipped with a four-hole an- ti rotor at c. prior to centrifugation, ceacam was dialyzed extensively against mm tris-hcl ph . , mm nacl. sednterp (http://sednterp.unh.edu) was used to calculate values for the protein partial specific volume and solvent density from the protein amino-acid sequence and buffer composition, respectively. ceacam at three different concentrations ( . , . and . mm) was loaded into cells equipped with six-hole charcoal-filled epon centerpieces ( . cm path length) with sapphire windows. centrifugation was carried out at , and rev min À and scans were acquired at nm with a step size of . and five averages per step. the data were globally analyzed using the winnonlin program (johnson et al., ) . a single x-ray diffraction data set was collected to a resolution of . Å . initial indexing of the data suggested that the crystal contained a primitive orthorhombic lattice with two molecules in the asymmetric unit. however, attempts to find a molecular-replacement solution using the ceacam monomer as a search model yielded no solutions that would refine in any of the orthorhombic space groups. the data were reprocessed in a primitive monoclinic lattice with a angle of . , which led to the correct solution in space group p with four copies of the search model found in the asymmetric unit. h|l|i tests of the data ( . ) show that the data are untwinned and no pseudo-merohedral twinning was detected. all residues in the final model were modeled except for the initial alanines of two of the four molecules in the asymmetric unit. the final model contained three chloride ions and waters. the final model was refined to an r cryst and r free of . and . , respectively. the structure factors and model have been deposited in the protein data bank (pdb entry y ). the closest homologs of ceacam in the pdb are ceacam and ceacam , which both share % sequence identity with ceacam , with residues differing between the proteins (fig. a) . the overall fold of ceacam is similar to those of the other ceacams that have been determined previously. the overall topology is that of the v-set fold of the immunoglobulin superfamily, comprised of two -sheets labeled abed and a gfcc c . the sheets are connected by the bc, ef, c d and aa loops (fig. b) . the r.m.s.d.s of the ceacam molecules to each other are low ($ . Å ), showing no structural differences within the asymmetric unit. the four molecules of ceacam form two pairs of dimers (fig. c) . the dimer interface is formed from the second -sheet, a gfcc c , specifically the gfcc c strands and the cc , c c and fg loops (fig. c) . dimerization buries Å of solvent-accessible surface area as calculated by pisa (krissinel & henrick, ) . this is similar to the ceacam and ceacam homodimers, which bury and Å of solvent-accessible surface area, respectively. the shape-complementarity value (sc; lawrence & colman, ) is . , which is smaller than those for the other ceacams, with values of . and . for ceacam and ceacam , respectively. ceacam forms nine hydrogen bonds in the dimerization interface. this is more than ceacam (six hydrogen bonds) but less than ceacam ( hydrogen bonds). of the residues in ceacam that differ from ceacam and ceacam , eight are found in the dimerization interface. the dimerization constant of ceacam was determined previously to be . mm by analytical ultracentrifugation (korotkova et al., ) . ceacam does dimerize but forms high molecular-weight oligomers (korotkova et al., ) . the dimerization constant of ceacam was estimated by sedimentation-equilibrium analysis using analytical ultra- in mm tris-hcl, mm sodium chloride ph . with rotor speeds of , and rev min À (blue, red and green curves, respectively). bottom, residuals of fitted data for each curve. centrifugation (fig. d ). an estimated average molecular weight of . ae . kda (the theoretical monomer molecular weight is da) and a k dimerization of nm (+ /À nm) were measured, a tenfold increase in affinity when compared with ceacam . we observed no higher molecular weight oligomers for ceacam other than the dimer. superposition of the ceacam dimer (a + b) onto the ceacam dimer (a + b) was achieved using one half of each dimer (molecule a) and two r.m.s.d.s were calculated: one for the first half of the dimer (molecule a), which results in an r.m.s.d. of . Å , and the second for the second half, which shows a larger r.m.s.d. of . Å (molecule b ; fig. a ). superposition of ceacam onto the ceacam dimer using the same method reveals similar r.m.s.d.s of . Å for molecule a and . Å for molecule b (fig. b) . closer comparison of ceacam with ceacam and ceacam shows two major regions of deviation. the first is the bc loop (residues - ), which is not involved in the dimerization interface (fig. c ). this region is highly conserved among members of the ceacam family; however, positions - of ceacam differ from those of the other ceacams. in all other ceacams these residues are leu and pro . however, in ceacam they are glu and ser . the loss of pro is likely to reduce the rigidity of the loop. the r.m.s.d. of this loop in ceacam is . Å relative to ceacam and the displacement of this loop causes a slight movement of the n-terminal -strand. notably, the preceding residue is asn , a unique n-linked glycosylation site found only in ceacam and ceacam . three other glycosylation sites are present in ceacam (asn , asn and asn ). these are highlighted in fig. (d) , showing that none are found in the dimerization interface. glycosylation of ceacam has been shown to be important for interaction with cd (roda et al., ) and therefore may also be important for ceacam function. the second region of deviation between ceacam , ceacam and ceacam is the c c loop and the c strand. ceacam is unique compared with other ceacams, as the c c loop contains a single amino-acid insertion (isoleucine) between residues and . to accommodate the insertion in the c c loop without altering the length of the c c or the c d loops, the c strand is found to be distorted relative to ceacam (fig. a) and ceacam (fig. b ). the c strand bulges in the center (residues - ), causing breakages of hydrogen bonds in the antiparallel -sheet between the c and c strands. the c strand of ceacam is held in place through a hydrogen bond from the o " atom of asn to the n atom of gly (fig. c) . in ceacam the carbonyl group of thr forms the hydrogen bond to gly (fig. d) . although the c strand does not buckle in ceacam , as in ceacam , it is found that thr (a) superposition of the ceacam dimer (cea a and cea b; red and pink, respectively) onto the ceacam dimer (cea a and cea b; light and dark cyan, respectively) through molecule a. (b) superposition of the ceacam dimer (cea a and cea b; red and pink, respectively) onto the ceacam dimer (cea a and cea b; blue and lilac, respectively) through molecule a. (c) alignment of ceacam and ceacam monomers, colored by r.m.s.d. dark blue is low r.m.s.d. and red is high r.m.s.d. ceacam is omitted for clarity. (d) potential n-linked glycosylation sites of the ceacam dimer. all residues are solvent-exposed and are not found in the dimerization interface. does form a hydrogen bond across the dimerization interface to asp (fig. e ). this buckling of the c strand creates an extra interaction in the dimerization interface, potentially explaining why ceacam forms such tight dimers and has not been found to form heterodimeric ceacam complexes such as ceacam -ceacam , ceacam -ceacam , ceacam -ceacam , ceacam -ceacam and ceacam -ceacam (oikawa et al., ; skubitz & skubitz, ; singer et al., ) . although ceacam does not buckle, it too creates an extra interaction across the dimerization through reorientation of asp . this also suggests that this hydrogen bond is important for a higher affinity interaction. the structure of ceacam reveals that the dimerization interface is comprised of the same face as other ceacams (gfcc c ) yet can accommodate different residues (eight from each monomer), suggesting that these sequence differences can modulate the homodimerization to achieve a tenfold increase in affinity. (a) the polypeptide backbone of the c and c strands of ceacam (red) and ceacam (cyan). (b) the polypeptide backbone of the c and c strands of ceacam (red) and ceacam (blue). (c) the insertion of ser in ceacam (red) results in breakage of the main-chain antiparallel hydrogen bonds and replacement with a hydrogen bond from the side chain of asn . this residue also forms a hydrogen bond across the dimer interface to asn (brown). (d) in ceacam (green), only the main-chain antiparallel hydrogen bonds exist. thr is too short to form a hydrogen bond across the dimer interface to asp (purple). (e) however, this is not the case for ceacam (blue). thr forms a hydrogen bond across the dimer interface to asp (gray). n-terminal dimerization domain of ceacam lung cancer proc. natl acad. sci. usa we thank the staff of the advanced photon source (aps) gm/ca cat beamline -id-b for their support. the authors declare no competing financial interests. key: cord- - px aq authors: griese, matthias; zarbock, ralf; costabel, ulrich; hildebrandt, jenna; theegarten, dirk; albert, michael; thiel, antonia; schams, andrea; lange, joanna; krenke, katazyrna; wesselak, traudl; schön, carola; kappler, matthias; blum, helmut; krebs, stefan; jung, andreas; kröner, carolin; klein, christoph; campo, ilaria; luisetti, maurizio; bonella, francesco title: gata deficiency in children and adults with severe pulmonary alveolar proteinosis and hematologic disorders date: - - journal: bmc pulm med doi: . /s - - - sha: doc_id: cord_uid: px aq background: the majority of cases with severe pulmonary alveolar proteinosis (pap) are caused by auto-antibodies against gm-csf. a multitude of genetic and exogenous causes are responsible for few other cases. goal of this study was to determine the prevalence of gata deficiency in children and adults with pap and hematologic disorders. methods: of patients with gm-csf-autoantibody negative pap, had no other organ involvement and had some form of hematologic disorder. the latter were sequenced for gata . results: age at start of pap ranged from . to years, patients were children. in half of the subjects gata -sequence variations were found, two of which were considered disease causing. those two patients had the typical phenotype of gata deficiency, one of whom additionally showed a previously undescribed feature – a cholesterol pneumonia. hematologic disorders included chronic myeloic leukemia, juvenile myelo-monocytic leukemia, lymphoblastic leukemia, sideroblastic anemia and two cases of myelodysplastic syndrome (mds). a year old child with mds and digeorge syndrome type was rescued with repetitive whole lung lavages and her pap was cured with heterologous stem cell transplant. conclusions: in children and adults with severe gm-csf negative pap a close cooperation between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor gata . treatment with whole lung lavages as well as stem cell transplant may be successful. pulmonary alveolar proteinosis (pap) is a rare disorder characterized by the progressive accumulation of surfactant in the alveoli of the lungs, leading to hypoxemic respiratory failure and, in severe cases, to death [ ]. pap is caused by (i) genetic diseases which result in dysfunction of surfactant or surfactant production (sftpc, sftpb, abca , ttf deficiency) mainly presenting during infancy, by (ii) disruption of gm-csf signaling from mutations in the receptor (gm-csfra, gm-csfrb) or from acquired autoantibodies against gm-csf, and by (iii) disorders that presumably impair surfactant clearance because of abnormal numbers or defective phagocytic functions of alveolar macrophages [ ] . the latter are caused by inhaled particles or by hematologic disorders affecting macrophage precursors. a broad spectrum of hematologic disorders have been associated with pap, most frequently myelodysplastic syndrome (mds), and more rarely acute (aml) and chronic myeloid leukaemia (cml), myelofibrosis, acute lymphoid leukaemia (all), adult t-cell leukaemia, aplastic anemia, lymphoma, multiple myeloma, plasmacytoma, essential thrombocytosis [ ] , congenital dyserythropoietic anemia [ ] , and status after unrelated stem cell transplant [ , ] . up to now only mutations in one specific gene -gata have been associated with this form of pap. gata is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells [ ] . among many other functions gata regulates the phagocytosis of alveolar macrophages [ ] . alveolar macrophages treated with the sense gata- expression construct show an increase in their phagocytic activity by up to % compared to the antisense construct [ ] . gata deficiency is a recently described disorder of hematopoiesis, lymphatics, and immunity, caused by heterozygous mutations leading to haplo-insufficiency of the transcription factor gata . the disease presents with a complex array of diagnoses and symptoms of varying extent including mds, aml, chronic myelomonocytic leukemia (cmml), severe viral, disseminated mycobacterial and invasive fungal infections, pulmonary arterial hypertension, warts, panniculitis, human papillomavirus (hpv) positive tumors, epstein-barr virus (ebv) positive tumors, venous thrombosis, lymphedema, sensorineural hearing loss, miscarriage and hypothyroidism [ ] . pap has been reported in about % of all subjects with gata deficiency [ , ] . however, not much is known on the severity and clinical course of pap in these subjects, which were exclusively adults. pneumologists treating patients with severe pap not caused by autoantibodies against gm-csf frequently do not know the cause of these rare conditions [ ] . here, we investigate a cohort of children and adults with severe and chronic pap and hematologic disease for the presence of gata mutations. gata mutations were identified in a minority of patients only. one child with a gata variant was successfully treated initially by therapeutic whole lung lavages (wll) and eventually by stem cell transplant. all subjects with pap diagnosed based on the characteristic phenotype by lung biopsy or bronchoalveolar lavage (bal) and ct scan findings were identified from the kids-lung-register (http://www.kinderlungenregister.de/ index.php/en/) within the child-eu project (european register and biobank on childhood interstitial lung diseases, european commission, fp , ga ). in order to differentiate subgroups of pap, all patients with gm-csf-ra or rb mutations [ ] and a high gm-csf autoantibody level were excluded. subjects were identified of whom ( adults, children) had pulmonary involvement only. subjects suffered from pap and a hematologic disease -diagnosed by standard proceduresand were selected for further analysis (fig. ) . the gata gene was analysed by sanger sequencing. genomic dna was isolated from edta blood samples using the qiaamp dna blood mini kit (qiagen) and pcr amplified using hotstartaq polymerase (qiagen). pcr products were purified with the minelute uf pcr purification kit (qiagen). primers used for pcr and sequencing reactions are shown in table . sequencing was performed by gatc biotech ag (konstanz, germany). in subject ng of genomic dna extracted from formalin fixed tissue were fragmented to an average size of bp and desired fragment sizes were obtained with agencourt® ampure® xp beads (beckman coulter). sequencing libraries were prepared using accel-ngs- s library kit (swift biosciences, ann harbor, mi), sequenced on the hiseq (illumina) and aligned to the human genome assembly (hg ). the gata locus was covered by reads. follow-up data of the patients were collected until october . the study was approved by the institutional review board, the ethics commission of the medical faculty of the ludwig-maximilians university, munich, germany (ek - ). all parents or guardians gave their written informed consent, the children gave assent. age at onset of pap ranged from . to years. half of the patients were children (table ). beside chronic respiratory insufficiency due to pap, all patients suffered from recurrent respiratory tract infections or exacerbations. these were mainly common cold viral infections, in some cases non-tuberculous mycobacteria, herpes simplex virus (hsv), and cytomegalovirus (cmv) were identified. all patients were immunodeficient: in three patients primary immunodeficiency syndromes were identified: two with a monocyte deficiency not further specified (no. , ) and one with a di george type ii syndrome with combined table sequences of primers used for amplification of the gata gene exon forward reverse # ccc gca aag tga tgt cg caa acg gac caa gcg att c # acc tcg tgg tgg gac ttt g gat cct aca tcc ggg the hallmark of all patients of this study was severe pap with continuous need of oxygen (table ). all but two subjects (no. , ) were treated symptomatically with repetitive therapeutic wll to improve respiratory insufficiency. treatment was eventually not successful in patients who died from respiratory failure, complicated by infection or acute respiratory distress syndrome (ards). autoantibodies against gm-csf were determined in order to characterize pap further, they were negative in all patients (table ) . serum levels of gm-csf were not significantly increased in the patients with serum available. of the patients had sequence anomalies in gata ( table ) . two of these, a synonymous variant and a missense variant were predicted not to be damaging by polyphen- [ ] and sift [ ] . the other two were heterozygous point mutations predicted to be deleterious, both of these patients had a phenotype characteristic for gata deficiency. the courses of cases , , and are reported in more detail to illustrate characteristic presentations. a year old male patient presented with disseminated mycobacterium avium intracellulare infection, including the lungs, chronic labial herpes including a positive serum hsv pcr, and m. bowen of the skin. cellular immunodeficiency was suspected based on absent monocytes; the patient was hiv-negative. years later the patient developed respiratory insufficiency, the ct scan showed consolidations suspicious of cryptogenic organizing or atypical pneumonia. several courses of antibiotic therapy were followed by two pulses of cyclophosphamide; the patient deteriorated further, ct scan showed bilateral interstitial thickening and crazy-paving pattern. open lung biopsy revealed pap. bone marrow aspiration was normal. he was treated symptomatically with wll under ecmo (extracorporeal membrane oxygenation) support, developed anuric acute renal failure, and died from acute cardiac failure. the patient carried the mutation p.y d in the gata gene. a year old female patient presented with recurrent bronchitis and sinusitis, breathlessness during exercise, and interstitial markings on chest x-ray. an open lung biopsy was performed, histology revealed pap associated with cholesterol pneumonia. liver and bone marrow biopsies were normal. in the following months she developed progressive peripheral edema and cardiac insufficiency with mild pulmonary hypertension. she further developed a pseudomembranous glomerulonephritis with nephrotic syndrome and a pseudomonas urosepsis at years. at years cellular immunodeficiency was demonstrated based on the absence of monocytes. she developed condylomatous lesions of the vulva and hpvassociated (types and ) vulva intraepithelial neoplasia grade - (vin -vin ), which was resected. her course was determined by chronic respiratory failure (at rest lo /min; pao mmhg (normal > ), normal pac ). however, no wll were performed as the patient was lost on follow up at years. the patient carried the mutation p.r w in the gata gene. case no. a . year old girl with di george syndrome type ii from deletion of p (omim ) with severe immunodeficiency, single right kidney, inner ear deafness, [ ] , with good clinical and radiological response over a period of one year (fig. ) . a hla-identical ( / ) unrelated-stem cell donor was identified at the age of years and she received allogeneic stem cell transplant. within a few weeks after transplant, pap and respiratory distress resolved (fig. ) . a full-term boy with normocytic anemia (hb . g/dl) presented after a few weeks with paleness, fatigue, tachydyspnea, and failure to thrive. at the age of four month the hemoglobin level had decreased to . g/dl and a sideroblastic anemia was diagnosed. no genetic cause was identified; pearson syndrome and mutations in alas and slc a were excluded. his bone marrow showed massive erythroid hyperplasia with dyserythropoiesis and marked depression of myeloid cells. pulmonary infiltrates were initially interpreted as pneumonia. at the age of months a cmv infection was successfully treated with ganciclovir. at months he developed respiratory failure (pao mmhg, paco mmhg) with bilateral pulmonary infiltrates. the diagnosis of pap was made by analysis of bal fluid and open lung biopsy. from the age to months the boy underwent therapeutic wlls, improving his general condition and gas exchange. currently he maintains oxygen-saturation levels above % under oxygen support . - l of o /min). the anemia is stable with hb levels between . and . g/dl. the molecular cause of very rare sideroblastic anemia was not revealed, the associated depression of myeloid cells was likely the cause of pap. gata deficiency was not causal, as the patient showed no gata variant in our investigations; potential intronic variations were not investigated. this study shows that gata mutations in patients with hematologic diseases and severe pap occur at a relatively low frequency. gata analysis may help to diagnose the underlying hematologic condition. severe pap in children due to mds can be cured by stem cell transplant. severe pap is a rare but serious complication of hematologic disorders. here we differentiated two groups of diseases having in common a presentation with significant chronic respiratory insufficiency and recurrent pulmonary tract infections or exacerbations. these groups included (i) patients with gata deficiency, a protean disorder of hematopoiesis, lymphatics, and immunity [ ] , and (ii) patients with functionally or numerically reduced alveolar macrophages and/or their respective mononuclear precursors due to cml, jmml, call, mds or sideroblastic anemia in the absence of disease causing gata mutations. gata deficiency has a broad phenotype encompassing immunodeficiency, mds/aml, pulmonary disease, and vascular/lymphatic dysfunction. a precise history and knowledge of the disease course may help to select potential candidates with high confidence for specific genetic diagnostic. gata gene mutations were associated with pap in two cases; p.y d is a novel missense mutation demonstrated here to present with the classical mono-mac syndrome [ ] . p.r w has previously been described in six patients, of whom had a pap [ ] . here we add the histopathological feature of cholesterol pneumonia to this phenotype. of interest, further heterozygous variations in gata were identified in two other subjects; however these were predicted to be non-disease causing ( table ) . as not all subjects with a specific gata mutation causing gata deficiency develop pap [ ] , additional factors are involved which determine pap. patients with the same gata mutation need to be investigated further and experimental models will help to better understand the molecular defect(s) leading to pap. similarly, in non-gata deficient patients with pap and hematologic abnormalities, the mechanism of pap development is unknown. currently, their pap is presumed to be due to reduced monophagocytic function in the alveoli to clear surfactant [ ] . best proof for the critical role of bonemarrow derived alveolar macrophages for surfactant homeostasis is provided by the correction of pap by sct. this was shown here for the first time in a young child. shortly, i.e. within days after take of sct, there was clearing of pap, superseding rapidly the need for therapeutic wlls (fig. ) . here we also show that wlls are feasible also for children, even at very young age, with small sized airways and in the presence of severe respiratory distress, using techniques we have established previously [ ] . wll can be used to symptomatically treat all types of pap and to bridge until the underlying condition can be cured or alternative treatments have been implemented. due to relative immune deficiency state and irrespective of the cause, the impact of infections should be considered fig. long term course of a child suffering from digeorge syndrome type ii and pap due to mds with monosomy , trisomy , and a gata missense variant. successful treatment by therapeutic wlls, and definitive treatment of the pap by sct. (a) clinical course (b,c) ct at presentation (d,e) ct after first whole lung lavages (f) cxr before sct (g) cxr weeks after sct and (h) cxr year after sct carefully. it is likely that with often severe respiratory tract infections pap may be triggered to deteriorate. thus we recommend early diagnosis and proper antimicrobial treatment, in particular of organisms characteristic for these often lethal conditions, including mycobacteria, nocardia, herpes viruses, and fungi [ , , ] . in addition and concordant with the immune deficiency the extensive usage of systemic corticosteroids should be cautioned, as there is so far no evidence of their beneficial effect in pap. extensive usage might enhance immune deficiency and weaken antimicrobial defense further [ ] . after confirming the diagnosis of pap either by a combination of characteristic ct scan and bal findings and trans-bronchial or open lung biopsy, it is important to determine the etiology of pap. as more than % of pap in adulthood is caused by autoantibodies against gm-csf [ , , ] , this condition has to be initially excluded by analysis of serum [ , ] . none of our patients had increased levels of gm-csf autoantibodies. serum levels of gm-csf are low in autoimmune pap [ ] , whereas elevated values can be found in congenital gm-csfra or gm-csfrb defects and possibly in other forms of pap. thus, particularly in children these other entities need to be excluded. in our cohort of patients with severe pap and hematologic diseases, serum gm-csf level were low to intermediate, supporting some up regulation of gm-csf. next, genetic analysis of gata is helpful in order to diagnose the underlying hematologic entity more precisely, in particular in patients who meet the broad but characteristic phenotype of gata deficiency [ ] . this diagnostic algorithm for pap will allow differentiating important subgroups and may help to identify further genetic abnormalities, known or suspected to be associated with pap. when investigating a patient with severe pap, the pneumologist should be aware of the wide range of diseases which can cause secondary pap and consider interdisciplinary involvement of a hemato-oncologist. conversely, hemato-oncologists should include pap in the differential diagnosis of respiratory failure associated with pulmonary interstitial changes in hematologic diseases. atb-binding cassette sub-family a member ; all: acute lymphoid leukemia; aml: acute myeloid leukemia; ards: acute respiratory distress syndrome; bal: bronchoalveolar lavage; call: common acute lymphoblastic leukemia; cml: chronic myeloid leukemia; cmml: chronic myelomonocytic leukemia ecmo: extracorporeal membrane oxygenation; gm-csf: granulocyte macrophage colony-stimulating factor; hpv: human papilloma virus hsv: herpes simplex virus; jmml: juvenile myelomonocytic leukemia mds: myelodysplastic syndrome; pap: pulmonary alveolar proteinosis sftpb: gene encoding for surfactant protein b; sftbc: gene encoding for surfactant protein c; ttf : thyroid transcription factor ; wll: whole lung lavage. references . seymour jf, presneill jj. pulmonary alveolar proteinosis: progress in the first years pulmonary alveolar proteinosis: diagnostic and therapeutic challenges secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis: a retrospective cohort study in japan pulmonary alveolar proteinosis in association with congenital dyserythropoietic anemia: a case report. case rep pediatr secondary pulmonary alveolar proteinosis after unrelated cord blood hematopoietic cell transplantation gata deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity master regulatory gata transcription factors: mechanistic principles and emerging links to hematologic malignancies downregulation of gata- transcription during pneumocystis carinii infection effect of transcription factor gata- on phagocytic activity of alveolar macrophages from pneumocystis carinii-infected hosts mutations in gata are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (monomac) syndrome secondary pulmonary alveolar proteinosis in hematologic malignancies characterization of csf ra mutation related juvenile pulmonary alveolar proteinosis a method and server for predicting damaging missense mutations predicting the effects of coding non-synonymous variants on protein function using the sift algorithm longterm follow-up and treatment of congenital alveolar proteinosis monomac syndrome in a patient with a gata mutation: case report and review of the literature the molecular basis of pulmonary alveolar proteinosis whole-lung lavage in infants and children with pulmonary alveolar proteinosis loss-of-function germline gata mutations in patients with mds/ aml or monomac syndrome and primary lymphedema reveal a key role for gata in the lymphatic vasculature gata haploinsufficiency caused by mutations in a conserved intronic element leads to monomac syndrome wash-out kinetics and efficacy of a modified lavage technique for alveolar proteinosis pulmonary alveolar proteinosis: new insights from a single-center cohort of patients anti-gm-csf antibodies in paediatric pulmonary alveolar proteinosis epidemiological and clinical features of idiopathic pulmonary alveolar proteinosis in japan granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects the work of mg was kindly supported by else-kroener-fresenius stiftung (mg _a ), by the german federal ministry of education and research (eupapnet project inside erare, number gm a), european register and biobank on childhood interstitial lung diseases (european commission, fp , ga , child-eu). the authors declare that they have no competing interests.authors' contributions mg designed the study, collected the cases, analysed the data and wrote the draft of the manuscript. he is the guarantor of the entire manuscript. uc, amh, jl, kk, bf, ml, cs, ck and mg contributed and evaluated patients and performed chart review, zr, as, bh, sk, ckr, and ja performed laboratory analyses, cu, ckr and bf critically revised the manuscript, dt provided tissue samples and performed histopathological analyses, as, tw, uc and fb contributed to the long term collection of subjects, patient recruitment and evaluation. all contributors read and agreed to the final version of the manuscript.author's information this paper is devoted to prof. maurizio luisetti's outstanding contribution to the field of human alveolar proteinosis. key: cord- -bbeznd r authors: gupta, garvita; lim, liangzhong; song, jianxing title: nmr and md studies reveal that the isolated dengue ns protease is an intrinsically disordered chymotrypsin fold which absolutely requests ns b for correct folding and functional dynamics date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: bbeznd r dengue genome encodes a two component protease complex (ns b-ns pro) essential for the viral maturation/infectivity, thus representing a key drug target. previously, due to its “complete insolubility”, the isolated ns pro could not be experimentally studied and it remains elusive what structure it adopts without ns b and why ns b is indispensable. here as facilitated by our previous discovery, the isolated ns pro has been surprisingly deciphered by nmr to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (pre). the disordered ns pro appears to be needed for binding a human host factor to trigger the membrane remodeling. moreover, we have in vitro refolded the ns pro in complex with either ns b ( – ) or the full-length ns b ( – ) anchored into the lmpc micelle, and the two complexes have similar activities but different dynamics. we also performed molecular dynamics (md) simulations and the results revealed that ns b shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. remarkably, the ns b cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the sars cl protease. indeed, a truncated ns b ( – ;Δ – ) with the flexible loop deleted is able to trap the ns b-ns pro complex in a highly dynamic and catalytically-impotent state. taken together, our study implies potential strategies to perturb the ns b-ns pro interface for design of inhibitors for treating dengue infection. dengue is the most prevalent mosquito-borne viral disease with over million human infections annually and . billion people at risk, particularly in tropical and subtropical regions [ , ] . the disease is caused by dengue virus (denv) belonging to the flaviviridae family, which also includes several other human pathogens such as the west nile virus, japanese encephalitis virus, and yellow fever virus [ ] [ ] [ ] [ ] . so far, four antigenically distinct denv serotypes have been identified: denv- to - , all of which cause dengue fever, dengue haemorrhagic fever and dengue shock syndrome. despite intense studies, currently there are neither vaccines nor other treatments available to treat this disease [ , ] . the denv genome is composed of an -kb single-stranded positive sense rna. upon infection, the rna genome is translated into a large polyprotein by the host-cell translation machinery, that is subsequently processed into proteins, including three structural proteins (capsid, membrane, and envelope) and seven nonstructural proteins (ns , ns a/b, ns , ns a/b, and ns ). the structural proteins form the viral particle while the nonstructural proteins participate in the replication of the rna genome, virion assembly, and attenuation of the host antiviral response. the cleavage of the polyprotein is carried out by host cell proteases including furin and signalaseas, as well as a virus-encoded serine protease ns b-ns pro, which has been established as a valuable target of therapeutic interest [ , ] . the protease domain ns pro consisting of the n-terminal part of ns adopts a chymotrypsin-like fold with two β-barrels, each composed of six β-strands, with the catalytic triad (his -asp -ser ) located at the cleft between the two β-barrels [ , ] . amazingly, unlike other proteases with a chymotrypsin-like fold, the flavivirus proteases including dengue protease, requires a stretch of approximately amino acids from the cytosolic domain of ns b for catalytic activity, thus called two-component protease [ ] [ ] [ ] . intriguingly, while the protease domains adopt highly similar structures in all crystal structures determined to date, the ns b cofactors have been found to assume two distinctive structures, namely open (inactive) and closed (active) conformations by x-ray crystallography and nmr spectroscopy [ ] [ ] [ ] [ ] [ ] [ ] . to understand why the ns b cofactor is indispensable for activating the dengue ns protease is not only of fundamental interest for enzymology, but also bears considerable implications for design of molecules with high affinity and specificity to inhibit the protease [ , ] . however, due to its "complete insolubility", it has been previously impossible to carry out any experimental studies on the isolated ns pro domain. in , we discovered that previously-thought "insoluble proteins" in fact could be solubilized in water with minimized salt ions [ ] [ ] [ ] [ ] , and therefore we have used this to study various previously-thought insoluble proteins including tdp- n-terminus [ ] . here with this approach, we have successfully characterized the solution conformations and dynamics of the isolated protease domain by cd, nmr and paramagnetic relaxation enhancement (pre). the results reveal that surprisingly the isolated ns pro domain with the native sequence is intrinsically disordered without any stable secondary and tertiary structures, as well as has no detectable activity. nevertheless, upon meeting the ns b cofactor, the disordered ns pro spontaneously folds into the well-structured and active enzyme highly similar to those co-expressed [ , ] . we have also successfully refolded the ns pro complexed with the full-length ns b anchored into the lmpc micelle. to further decipher the roles of the ns b cofactor in protein dynamics, we performed molecular dynamics (md) simulations which is very powerful in pinpointing the role of protein dynamics in the catalysis of proteases such as the sars c-like protease [ , ] . the correlation analysis reveals that the ns b residues play a central role in coordinating the correlated motion network in the ns b-ns pro complex. indeed, a truncated ns b is able to form a buffer-soluble complex with ns pro, but this complex is highly dynamic and catalytically-impotent. our results imply that the discovery/ design of molecules to block the correct folding of the ns b-ns pro complex or/and to decouple its correlated motion network might represent promising strategies to inhibit the dengue protease, thus holding the considerable potential to treat dengue infections. plasmid construction dna sequences encoding the ns protease and ns b from dengue virus type strain tsv (genbank accession number ay ) were optimized and synthesized by genscript (piscataway, nj). with designed primers, the optimized dna by genscript were used as templates for amplifying the dna fragments encoding residues - of ns pro and residues - of ns b, which have the same starting and ending residues as the constructs used in the previous nmr study [ ] , as well as the full-length ns b over residues - including the n-and c-terminal transmembrane domains. amplified dna fragments for ns ( - ), ns b ( - ) and were subsequently cloned into pet a vector with n-terminal his-tag (novagen) using ncoi and xhoi restriction sites, while fragments for ns b ( - ) and ns b ( - ; Δ - ) with thr -met replaced by three gly residues were cloned into pgex- t vector with gst-tag (ge healthcare) using bamhi and xhoi restriction sites. dna sequences of cloned constructs were verified by automated dna sequencing. competent escherichia coli bl (de ) star cells were transformed with pet a-ns pro or pgex- t -ns b plasmids. transformed single colony was inoculated overnight in ml luria-bertani broth containing μg/ml kanamycin for pet a and μg/ml ampicillin for pgex- t . then cells were transferred to lit lb media containing respective antibiotics and grown at °c with shaking until the a reached to . . cultures were induced with . mm isopropyl β-d-thiogalactopyranoside (iptg) for h at °c. cells were harvested and resuspended in cold phosphate buffered saline (pbs) ph . buffer for lysis by sonication. after centrifugation at g, gst-tagged ns b ( - ) protein was purified from supernatant using affinity chromatography. the gst-ns b ( - ) protein was cleaved with thrombin to remove the gst tag, and the released ns b peptide was further purified by rp-hplc on a vydac c column. the ns b ( - ) and ns pro proteins were completely insoluble and all found in inclusion body, which were solubilized with pbs buffer (ph . ) containing m urea. cell debris was removed by centrifugation at g, and supernatant containing his-tagged ns b ( - ) and ns pro were purified by ni-nta affinity chromatography under denaturing condition. eluted protein was further purified with rp-hplc on a vydac c column. ( nh ) so , [ c ]-glucose and d o were purchased from cambridge isotope laboratories (andover, ma). the generation of the isotope-labeled proteins for nmr studies followed a similar procedure except that the bacteria were grown in m medium with the addition of ( nh ) so for n labeling and ( nh ) so /[ c]-glucose for n-/ c-double labelling as previously described [ ] . the purity of the recombinant proteins was checked by sds-page gels and their molecular weights were verified by esi-ms and voyager str matrix-assisted laser desorption ionization time-of-flight-mass spectrometer (applied biosystems). the concentration of protein samples was determined by the uv spectroscopic method in the presence of m urea [ ] . was measured in the assay buffer containing mm tris-hcl (ph . ), . % triton x- , . mm egta, or mm sodium acetate (ph . ), . % triton x- , . mm egta. briefly, in the μl reaction mixtures containing . μm protease, μm protease specific fluorophore-tagged substrate benzoyl-nle-lys-arg-arg-aminomethylcoumarin (bz-nkrr-amc) was added and reaction mixtures were incubated at °c, and the liberated coumarin fluorophore was continuously monitored at λ ex of nm and λ em of nm on infinite m pro tecan microplate reader. for steady state kinetics, . μm refolded ns -ns b protease was incubated with various concentrations of bz-nkrr-amc substrate in assay buffer containing mm tris-hcl (ph . ), . % triton x- , . mm egta at °c. progression of enzymatic reaction was monitored as an increase in fluorescence at λ ex of nm and λ em of nm. initial fluorescence velocities (relative fluorescence units/sec) were calculated and curves were fitted to the michaelis-menten equation by nonlinear regression using graph-pad prism. steady-state kinetic constants were determined from triplicate measurements and reported as mean standard error. as the ns pro contains no free cys residue, three single-cys mutants were prepared: q c, e c, s c by use of the quikchange site-directed mutagenesis kit (stratagene, la jolla, ca, usa) as previously described [ ] . the mutated plasmids were confirmed by dna sequencing and their recombinant proteins were subsequently expressed and purified by the same procedures described above. h- n heteronuclear single quantum coherence spectroscopy (hsqc) experiments were performed on each mutant to validate that these mutations did not significantly perturb the conformation of the native ns pro sequence. the recombinant proteins of three single-cysteine mutants were cys-modified following the previous procedure [ ] , by the thiol-reactive nitroxide free radical probe, mtssl ( -oxyl- , , , -tetramethyl-Δ -pyrroline- -methyl) methanethiosulfonate (toronto research chemicals inc.). briefly, the hplc-purified recombinant protein of the each mutant was dissolved in the buffer containing m urea, mm phosphate (ph . ), which was pre-degassed with nitrogen gas for minutes. subsequently, the mtssl reagent was added from . mm stock solution in acetonitrile to reach a six-fold molar concentration of the protein, followed by incubation at room temperature with constant stirring for hours. to ensure a complete labeling, another dose of mtssl was added to a six-fold molar concentration of the protein for an overnight incubation. the mtssl-labeled protein was purified by reverse-phase hplc on a c column and lyophilized. based on the verification by the time-of-flight-mass spectrometer, the purity of the mtssl-modified proteins of all mutants was > % after the hplc purification. all circular dichroism (cd) experiments were performed on a jasco j- spectropolarimeter equipped with a thermal controller using -mm path length cuvettes. data from five independent scans were added and averaged [ ] . the ns b, ns pro and ns b-ns pro samples were prepared at a protein concentration of μm in either milli-q water (ph . ) and mm phosphate (ph . ) respectively. secondary structure contents of different samples were obtained by decovoluting cd spectra with continll program (http://lamar.colostate.edu/~sreeram/ cdpro/main.html). all nmr experiments were acquired on an mhz bruker avance spectrometer equipped with pulse field gradient units as described previously [ ] . for characterizing the conformation of the isolated ns pro in water, a pair of triple-resonance experiments hncacb, cbca (co)nh as well as n-edited hsqc-tocsy and hsqc-noesy were collected for the sequential assignment on a n-/ c-double labelled or n-labelled sample at a protein concentration of μm in % h o/ % d o (ph . ). for the refolded ns b-ns pro complexes, hsqc spectra were collected for n-labeled samples in either acetate buffer (ph . ), or phosphate buffer (ph . ) in the absence and in the presence of inhibitor p-nitrophenyl-pguanidino benzoate [ ] . for assessing the backbone dynamics on the ps-ns time scale, { h}- n steady-state noes were obtained by recording spectra on the n-labeled ns pro domain at μm in either milli-q water (ph . ), with and without h presaturation with duration of s plus a relaxation delay of s at mhz. all nmr data were processed with nmrpipe [ ] and analysed with nmrview [ ] . nh, n, cα and cβ chemical shifts of the isolated ns pro in water at ph . were further analyzed by both delta d [ ] to derive the secondary structure population. for each spin-labeled single-cysteine mutant, a pair of d h- n hsqc spectra were acquired at a protein concentration of μm in milli-q water (ph . ): one for the spin-labeled sample in the paramagnetic form, and another after adding ascorbic acid (to mm) to the sample to reduce the nitroxide, yielding the diamagnetic sample. we also acquired hsqc spectra for corresponding cysteine mutants without spin-labelling at the same conditions and only several hsqc peaks slightly shifted after spin-labeling, indicating that the spin-labeling would not significantly change the conformation. the spectra were subsequently analyzed to obtain intensity ratios of hsqc peaks in the paramagnetic and diamagnetic forms using the programs nmrpipe [ ] . protein structures were displayed by pymol molecular graphics system (w. l. delano, delano scientific llc, san carlos, ca). the crystal structure of the ns b-ns pro complex (pdb code: fom) with an open conformation [ ] was selected for molecular dynamics simulations. as ns b residues thr -met are missing in the crystal structure, those residues were added and the obtained structure was post-processed as previously described [ , ] . the simulation cell is a periodic cubic box with a minimum distance of Å between the protein and the box walls to ensure the protein would not directly interact with its own periodic images given the cutoff. the water molecules, described using the tip p model, were filled in the periodic cubic box for the all atom simulation. na + ions were randomly placed to neutralize the charge in md system. three independent -ns md simulations for either ns b-ns pro complex or ns pro alone were performed with the program gromacs [ ] with the amber- [ ] all-atom force field. the long-range electrostatic interactions were treated using the fast particle-mesh ewald summation method [ ] , with the real space cutoff of Å and a cutoff of Å was used for the calculation of van der waals interactions. the temperature during the simulations was kept constant at k by berendsen's coupling. the pressure was held at bar. the isothermal compressibility was . Ã − bar - . the time step was set as fs. all bond lengths including hydrogen atoms were constrained by the lincs algorithm [ ] . prior to md simulations, all the initial structures were relaxed by steps of energy minimization using the steepest descent algorithm, followed by ps equilibration with a harmonic restraint potential applied to all the heavy atoms of the protease. as the -ns simulations cannot reproduce the folding-unfolding event, so here we attempted to capture the low-frequency correlation motions by a recently established approach called mutinf [ ] . mutinf represents an entropy-based approach to analyze ensembles of protein conformers, such as those from molecular dynamics simulations by using internal coordinates and focusing on dihedral angles. in particular, this approach is even applicable for detecting conformational changes which are subtle in the short md simulations because the coupling is mostly entropic in nature [ ] . briefly, this approach utilizes second-order terms from the configurational entropy expansion, called the mutual information, to identify pairs of residues with correlated conformations, or correlated motions, in an equilibrium ensemble [ ] . in the present study, the normalized matrix values were used, and . was set up to be the threshold value to determine the pairs of highly correlated residues. the residue pairs with correlated values ! . make up the top % of the entire matrix. dna fragments encoding ns b ( - ) and ns pro ( - ) were sub-cloned into the expression vectors pgex- t and pet- respectively and subsequently expressed in e. coli bl (de ) star cells. the recombinant ns pro protein was found to be completely insoluble and all in inclusion body as previously reported [ , ] . as a consequence, the ns pro proteins were first purified by ni + -affinity column under denaturing condition in the presence of m urea, followed by purification with reverse-phase (rp) hplc. on the other hand, the gst-fused ns b ( - ) cofactor was found in supernatant and thereby purified under native condition, followed by the thrombin cleavage to release the cofactor which was also purified by rp-hplc. the lyophilized powder of the ns b ( - ) was soluble both in mill-q water and in buffers, while the ns pro protein was highly soluble at protein concentration of mm without any detectable aggregation for at least half a year in mill-q water. the ns pro protein also could be quickly diluted into mm phosphate buffer with a final protein concentration of μm and ph of . without visible aggregates for hr, but formed aggregates in nmr tube after hr, which has been commonly observed on a variety of "completely insoluble" proteins previously reported by us and other groups [ ] [ ] [ ] [ ] . as shown in fig a, ns pro has very similar far-uv cd spectra in milli-q water (ph . ) and mm phosphate buffer (ph . ), with the maximal negative signal at nm and no positive signal below nm, which is typical of a predominantly disordered protein without any stable secondary structure. this indicates that the isolated ns pro domain is highly disordered in aqueous solution. indeed, the deconvolution of its cd spectrum reveals that the isolated ns pro consists of % random coil, % turn, % extended strand and % helix secondary structures. moreover, it has a hsqc spectrum with very narrow spectral dispersions at both h (~ . ppm) and n (~ ppm) dimensions (fig b) , further indicating the absence of tight tertiary packing. interestingly, on the other hand, ns b ( - ) has a cd spectrum with the maximal negative signal at nm and positive signal at nm, as well as an additional negative signal at nm. further deconvolution shows that it contains % random coil, % turn, % extended strand and % helix secondary structures. previously ns b has been shown to have slightly different secondary structures in the open [ ] and closed [ ] states of the ns b-ns pro complexes. in the open (inactive) state (pdb id: fom), ns b has a short helix over residues glu -gly while in the closed (active) state (pdb id: u i), the ns b only has β-strands but no helical segment. therefore, it is possible that upon losing tertiary contacts with the ns pro domain, the free-state ns b has more helical conformations populated over residues glu -gly , or/and even has helical conformations populated over residues which adopt βstrands in the ns b-ns pro complexes. indeed, we previously observed that upon disrupting the all β-barrel native fold, the mutants of a sh domain became highly helical even over the residues which adopt β-strands in the native fold [ ] . unfortunately, hsqc peaks of the ns b ( - ) are much more broadened than those of ns pro, implying that the isolated ns b ( - ) has dynamic aggregation or/and conformational exchanges on the μs-ms time scale, thus preventing from further high-resolution nmr studies. despite its narrow spectral dispersions, we have successfully assigned nmr resonances of almost all non-proline residues of the -residue ns pro ( - ) by analyzing nmr spectra including hn(co)cacb/cbca(co)hn and hsqc-tocsy/hsqc-noesy. fig c pres ents the obtained (Δcα-Δcβ) chemical shifts, which is a sensitive indicator of the residual secondary structures in disordered proteins [ ] . previously, by analyzing nmr chemical shifts, the ns b ( - )-ns pro ( - ) complex was characterized to adopt the same structure in solution and crystal [ ] . here we downloaded the chemical shifts of ns pro in complex with ns b (bmrb id of ) and included them (grey bars in fig c) in parallel to those of the isolated ns pro (red bars in fig c) for comparison. the ns pro domain in complex with ns b has very large (Δcα-Δcβ) deviations characteristic of a well-folded protein. by contrast, the isolated ns pro has dramatically decreased (Δcα-Δcβ) over the whole sequence ( fig c) . for example, many ns pro residues in the complex have the absolute values of (Δcα-Δcβ) > ppm, while all isolated ns pro residues have the absolute values of (Δcα-Δcβ) < ppm. interestingly, many residues of the isolated ns pro still have the (Δcα-Δcβ) patterns similar to those in the complex, implying that similar secondary structures might be weakly populated over these residues. indeed, we further analysed the nh, n, cα and cβ chemical shifts by delta d program [ ] and the results indicate that the isolated ns pro adopts highly-populated random coil conformations over the whole sequence. nevertheless, over some short segments, the extended strand conformation is also populated to some degree but the helix conformation is almost lacking (fig d) , completely consistent with the deconvolution result of the cd spectrum. furthermore, as seen in fig e, only sequential noes d nn(i,i+ ) and d αn(i,i+ ) manifest over the majority of the sequence, clearly indicating that the isolated ns pro has no stable secondary structures and its nmr conformation represents an average of an ensemble of different structures. taken together, cd and nmr results define the -residue ns pro domain to be an intrinsically disordered protein which is lacking of both stable secondary and tertiary structures in the absence of the ns b cofactor [ , [ ] [ ] [ ] [ ] [ ] [ ] . to pinpoint the backbone flexibility, we measured the { h}- n heteronuclear steady-state noe (hnoe) of the isolated ns pro, which is a measure of the backbone motions on the psns time scale [ , , , , ] . previously, all except for several c-terminal residues of the complexed ns pro were shown to have positive hnoe values, with many even close to , clearly indicating that the backbone of the ns pro in the complex is very rigid [ ] . by contrast, residues of the isolated ns pro have small or even negative hnoes (fig a) , with an average hnoe of only . . more specifically, all residues have hnoe values < . , while many residues even have negative hnoe, such as n-/c-termini, ser -gly , arg -arg , gln -ile and arg -gly (fig a) . this strongly suggests that the isolated ns pro has largely unrestricted backbone motions on the ps-ns time scale, consistent with its absence of any stable secondary and tertiary structures. nevertheless, residues over ile -leu have relatively large hnoe, implying that this region might have transit tertiary packing to a certain degree. previously many intrinsically disordered proteins such as α-synuclein have been shown to have transient long-range interactions by measurements of paramagnetic relaxation enhancement (pre), which is a powerful tool in detecting transiently existing contacts in highly disordered proteins with distances up to~ Å [ , , ] . here, by site-directed mutagenesis, we introduced cys residue into ns pro one by one at three locations: gln , glu and ser (s fig). three single cys mutants were subsequently labelled with the nitroxide free radical probe, mtssl whose pre were measured by hsqc (s fig) as we previously described [ ] . fig b shows the intensity ratios of hsqc peaks from the oxidized (paramagnetic) and reduced (diamagnetic) spectra of q c mutant. interestingly, the residues which were significant affected (ratio < . ) are all located in the first β-barrel of the chymotrypsin fold adopted by the ns pro domain in the native ns b-ns pro complex (fig c) . this suggests that these affected residues have long-range contacts with cys with distances < Å. interestingly, although residues thr -glu assuming a short helix and β-sheet have very close contacts with cys in the native structure, they were not significantly perturbed by the spin-label at cys . this strongly implies that the tertiary packing is non-native in the isolated ns pro domain. the spin-label at the position significantly affected the residues on both β-barrels (fig d and e) , suggesting that these residues have long-range contacts to cys with distances < Å. again, many residues have very close contacts with cys in the native fold but were not significantly perturbed by the spin-label at cys . the residues affected by the spin-label at the position are mostly located on second β-barrel, the loop connecting two βbarrels, and the last β-sheet over residues ser -gly (fig f and g) . the pre measurements revealed that despite lacking of stable secondary and tight tertiary structures, the isolated ns pro domain is not completely extended, but instead has a loose tertiary packing. however, this packing is highly dynamic and non-native, in which the residues over the middle region of the ns pro sequence are slightly less dynamic than the n-and c-terminal ones, completely consistent with hnoe results (fig a) . the isolated ns pro ( - ) and ns b ( - ) are largely disordered in solution (fig a) and the ns pro alone showed no detectable catalytic activity even with a protease concentration up to μm in buffers at ph . ( fig b) . however, upon mixing them at an equal molar ratio in milli-q water at ph . , the mixture has a cd spectrum for a protein with a substantial amount of secondary structures, which has a large positive signal at nm and the maximal negative signal at nm ( fig a) . the deconvolution shows that this refolded complex between ns b ( - ) and ns pro in water at ph . contains a large portion of β-strand and βturn structures: % random coil, % turn, % extended strand and % helix secondary structures, which is consistent with its three-dimensional structure (fig ) . this sample was subsequently split into two: one at the same condition at ph . , and another with buffer added to reach ph . . as shown in fig a, the cd spectrum of the mixture in buffer at ph . has slight changes: the maximal positive signal shifted to nm as well as became larger; and the maximal negative signal shifted from to nm, almost identical to that previously reported on the co-expressed ns b-ns pro complex [ ] . this change appears to result from the slight increase of β-strand and reduction of random coil conformations. the slight variations in cd spectra of the refolded ns b-ns pro complex at ph . and . suggest that the complex may have some conformational differences at two ph values. indeed, the complex at ph . has a hsqc spectrum with many hsqc peaks too broad to be detected (fig c) , indicating that the complex undergoes significant dynamic aggregation, or/and conformational exchanges on the μs-ms time scale. by contrast, the refolded ns b-ns pro complex at ph . has a hsqc spectrum typical of a well-folded protein, which has very large spectral dispersions at both h (~ . ppm) and n (~ ppm) dimensions (fig d) . in particular, although in the present study the ns b peptide is unlabelled, a large portion of the hsqc peaks of the present ns pro domain are almost superimposable to those of the previous complex with both ns b and ns pro n-labelled and co-expressed [ , ] , as exemplified by the characteristic hsqc peaks (green cycled), furthermore, the addition of the protease inhibitor p-nitrophenyl-p-guanidino benzoate previously used [ ] triggered dramatic shifts of many hsqc peaks, indicating the refolded ns b-ns pro is active in binding the inhibitor (fig e) . we measured the enzymatic activity of the complex refolded at ph . in two buffers: one at ph . and another at ph . . interestingly, it has no detectable activity in the acetate buffer at ph . even with the protein concentration reaching μm. nevertheless, if the sample refolded at ph . was measured in the buffer at ph . , it is highly active with no detectable difference from that refolded in the buffer at ph . (fig b) . the enzymatic parameters of the refolded ns b-ns pro complex in the buffer at ph . were obtained with km = . ± . μm and kcat = . ± . s - , which are very similar to the previous results with a co-expressed ns b-ns pro complex [ ] . the structure and enzymatic activity revealed that once ns b and ns pro meet, they will bind to each other and initiate the folding process to form the two-component complex. however, it seems that if at ph . , the ns b-ns pro complex exists as an intermediate which already has substantial secondary structures and tertiary packing. however, the tight tertiary packing is not completely achieved and consequently the complex undergoes μs-ms conformational exchanges, very similar to what we previously observed on a molten globule formed by a small protein at ph . [ , ] . the intermediate of the ns b-ns pro complex at ph . is enzymatically inactive, but nevertheless, once it is transferred into the buffer at ph . , the tightly-packed structure will immediately form and the complex can reach the fully active state. to study the structure and activity of the ns pro in complex with the full-length ns b ( - ), which is expected to be anchored into the membranes by forming the transmembrane helices at both n-and c-termini, the dna fragment encoding the full-length ns b ( - ) was cloned into pet a vector and the ns b ( - ) protein was purified under denaturing condition. the full-length ns b ( - ) was reconstituted in the lmpc micelle at a molar ratio of : (ns b:lmpc) in buffer (ph . ). furthermore, ns pro and ns b ( - ) were also refolded together at the equimolar concentrations in presence of lmpc in buffer (ph . ). interestingly, the far-uv cd spectrum of the full-length ns b ( - ) reconstituted in the lmpc micelle have the maximal negative signal at nm and positive signal at nm, as well as an additional negative signal at nm ( fig a) . the deconvolution reveals that the ns b ( - ) reconstituted in the lmpc micelle contains % random coil, % turn, % extended strand and % helix secondary structures. the large portion of the helix conformation is anticipated to result from the formation of the transmembrane helices at both of n-and c-terminal termini. interestingly, the ns b ( - ) in the lmpc micelle has~ % higher β-strand conformation than the ns b ( - ) in buffer, implying that they only have a slight difference in secondary structures if considering the error in cd deconvolution. on the other hand, only a small set of broad peaks could be detected in its hsqc spectrum (fig b) , indicating that the ns b ( - ) in the lmpc micelle undergoes significant conformational exchanges on μs-ms time scale, or/and dynamic aggregation, which thus prevents from further high-resolution nmr studies. the observed line-broadening cannot mainly result from the increased molecular weight in the lmpc micelle as much more narrow peaks for almost all residues could be detected for the helical superoxide dismutase (sod ) with residues in the dpc micelle [ ] . we also refolded the ns pro with the full-length ns b ( - ) in the lmpc micelle. as shown in fig a, the far-uv cd spectrum of the complex between ns b ( - ) and ns pro in the lmpc micelle has the maximal negative signal at nm and positive signal at nm, as well as an additional negative signal at nm (fig a) . the deconvolution shows that its secondary structure contents are very similar to those of the complex between ns b ( - ) and ns pro in buffer, only with~ % increase of β-strand conformation and~ . % reduction of the helix conformation. however, very different from the complex between ns b ( - ) and ns pro in buffer (fig d) , for the hsqc spectrum of the complex between ns b ( - ) and ns pro in the lmpc micelle (fig c) , only a small portion of the hsqc resonance peaks could be observed, which are also much more broad. this is partly due to the increased molecular weight of the complex upon being anchored into the lmpc micelle. a closer examination of the spectra revealed that a small set of the ns pro hsqc peaks of the ns b ( - )-ns pro complex in the lmpc micelle is almost superimposable to those of the ns b ( - )-ns pro complex. these residues were identified to be the c-terminal residues glu -lys of ns pro, which were not visible in crystal structures of the ns b-ns pro complexes, thus suggesting that they are similarly flexible in both complexes ns b ( - )-ns pro in buffer and ns b ( - )-ns pro in the lmpc micelle). on the other hand, some hsqc peaks of the ns pro domain complexed with the ns b ( - ) in the lmpc micelle are not superimposable to those in the ns b ( - )-ns pro complex. this implies that these ns pro residues may have different structures, or/and dynamics, or/and chemical environments in the ns b ( - )-ns pro and ns b ( - )-ns pro in the lmpc micelle. furthermore the fact that most well-dispersed hsqc peaks disappeared (fig c) implies that the ns pro may undergoes significant conformational exchanges on μs-ms time scale upon being anchored in the lmpc micelle. nevertheless, the ns b ( - )-ns pro in the lmpc micelle is similarly active, with km = . ± . μm and kcat = . ± . s - , which are very similar to the activity of the ns pro in complex with the ns b ( - ) with transmembrane regions deleted. molecular dynamics simulation is a powerful tool to gain insights into protein dynamics and folding/unfolding that underlies protein functions. although it remains extremely challenging to simulate the folding/unfolding for large proteins such as ns pro, which usually occurs on the ms-s time scale, short md simulations in conjunction with the correlation analysis are able to capture the low-frequency correlated motions, which have been recently found to be essential for the catalysis of the sars c-like protease [ , ] . therefore, to understand the role of the cofactor in the dynamics of the dengue ns protease, we conducted -ns md simulations for both ns b-ns pro complex and isolated ns pro respectively. here we used the crystal structure of the ns b-ns pro complex (pdb code: fom) with an open conformation [ ] for md simulations, because this complex has sequences almost identical to what we studied here, with only one and two conserved residue variations respectively as compared to our ns pro and ns b constructs. furthermore, in this structure, the ns b cofactor assumes an open conformation, thus has a minimal contact surface with the ns pro domain as compared to other structures with the closed conformation. this would facilitate the identification of the most important residues of the ns b in mediating the dynamics of the protease complex. fig a and b present the structure snapshots in the first md simulations for the ns pro alone and the ns b-ns pro complex, showing that within ns, both isolated ns pro domain and that in the complex remains dynamically stable, in particular over the regions with regular secondary structures. by contrast, the ns b cofactor has large structural fluctuations, particularly over the residues thr -met which are invisible in all previous crystal structures with an open conformation. fig c- e show the root-mean-square deviations (rmsd) of cα atoms (from their positions in the energy minimized structures) for three independent simulations of the isolated ns pro, the ns b and ns pro in the context of the complex respectively, and fig fig . overall dynamic behaviors in the md simulations. structure snapshots (one structure for -ns interval) of the first md simulations respectively for the isolated ns pro (a) and ns b-ns pro complex (b). root-mean-square deviations (rmsd) of the cα atoms (from their positions in the energy minimized structures) for three independent md simulations of the isolated ns pro (c), the ns pro (d) and ns b (e) in the context of the ns b-ns pro complex. (f) rmsd trajectories averaged over three independent md simulations of the isolated ns pro (blue), the ns pro (red) and ns b (cyan) in the context of the ns b-ns pro complex. f shows their averaged rmsd trajectories. the averaged rmsd values are . ± . , . ± . and . ± . Å respectively for the isolated ns pro, the ns pro and ns b in the context of the complex. this clearly indicates that upon removing ns b, the isolated ns pro domain will have higher conformational dynamics even within the -ns simulations. as such, it is expected that the isolated ns pro domain may become unfolded if the simulations could reach ms-s time scale. on the other hand, even in the context of the complex, the ns b cofactor has much higher structural fluctuations than ns pro, implying that the bound ns b cofactor is still capable to sample a large ensemble of conformations. the high flexibility of ns b uncovered by md simulations here is completely consistent with the invisibility of many cofactor residues in crystal structures and also provides a dynamic basis for the conformational exchange of ns b between open and closed conformations as previously observed [ ] [ ] [ ] [ ] [ ] . noticeably, similar dynamic behaviours are also reflected by the root-mean-square fluctuations (rmsf) of the cα atoms in the md simulations (fig a and b) . fig c presents the averaged rmsf of three trajectories for the isolated ns pro and ns b-ns pro complex. interestingly, only three regions of the isolated ns pro have slightly higher fluctuations than the corresponding ones in the complex. the highest structural fluctuations are observed on the ns b residues. more specifically, the ns b residues with rmsf > the average are over n-/cterminal residues g -ala and leu -gly , as well as ser -glu which include the missing residues thr -met in all crystal structures of the open form [ ] . strikingly, the ns pro residues with rmsf > the average are not only over loop/turn residues and c-terminal residues which include g -leu , glu -thr , his -gly , leu -val , thr -gly , gly -val and arg -gly ; but also over the short helix val -lys , and β-strands gly -gln , lys -ile , asp -ile hosting the catalytic triad residue asp , thr -val and gly -val (fig d and e) . recently, we have deciphered that a global correlation motion network exists in the sars c-like protease [ , ] . remarkably, a mutation n a on the extra domain which is far away from the active site is sufficient to decouple the correlated motions and consequently leads to the inactivation of the enzymatic catalysis. here, the correlation analysis of the md simulation trajectories revealed that like the sars c-like protease, a global correlation network does exist in the ns b-ns pro complex. most strikingly, in this network, the majority of the significant correlated motions are established between the cofactor and ns pro residues ( fig a) . surprisingly, although the cofactor residues ser -glu , ser -thr are highly dynamic in the md simulations, and thr -met are even missing in all crystal structures of the open form, they were revealed to play a key role in coordinating the correlated motions with the ns pro residues asp -gln , gly -gly , met -glu , ly -val , pro -gln , and lys -gly , which cover not only the residues having direct contacts with the cofactor, but also residues located far away from the cofactor (fig b and c ). furthermore, the global correlation network is largely eliminated as uncovered by the correlation analysis of the md trajectories of the isolated ns pro with the ns b cofactor removed (fig d) , which is similar to what was observed on the inactivated n a mutant of the sars c-like protease [ , ] . therefore, as implied by our previous results with the sars c-like protease, slight manipulations of the ns b-ns pro interface may be sufficient to decouple the correlation network to inactivate the activity of the dengue protease. the conformation and activity of the refolded ns pro in complex with ns b ( - ;Δ - ) although residues thr -met are missing in all crystal structures of the open form, they were revealed by md simulations to play a key role in coordinating the correlated motions with the ns pro residues. therefore, we generated a truncated ns b ( - ;Δ - ) in which the residues thr -met were replaced by three gly residues and subsequently conducted the refolding of ns b ( - ;Δ - ) with ns pro using the same protocol for refolding of ns b . interestingly, although the isolated ns pro is completely insoluble in buffer, the ns pro became highly soluble in buffer at ph . in the presence of ns b ( - ;Δ - ). this suggests that ns pro forms a complex with ns b ( - ;Δ - ) and the complex is soluble in buffer. indeed, as shown in, the decovolution analysis of the far-uv cd spectrum (fig a) reveals that the ns b ( - ;Δ - )-ns pro complex contains % helix, much higher than those of the isolated ns pro ( %), and ns b ( - )-ns pro complex ( %). on the other hand, the ns b ( - ;Δ - )-ns pro complex contains much lower strand ( %) but higher ( %) turn conformations than the isolated ns pro and ns b ( - )-ns pro complex. interestingly, the ns b ( - ;Δ - )-ns pro complex has ~ % unstructured conformation, slightly higher than that of the ns b ( - )-ns pro complex ( %), but much lower than that of the isolated ns pro ( %). these results indicate that the ns b ( - ;Δ - )-ns pro complex has a very different structure from either isolated ns pro or ns b ( - )-ns pro complex. indeed, very different from the hsqc spectra of the isolated ns pro ( fig b) or ns b ( - )-ns pro complex (fig d) , only a small set of hsqc peaks could be detected for the nlabeled ns pro in complex with ns b ( - ;Δ - ) ( fig b) . furthermore, a large portion of detectable hsqc peaks are superimposable to those of the ns b ( - )-ns pro complex (fig b) , implying that some residues of the ns b ( - ;Δ - )-ns pro complex have conformations similar to those of the ns b ( - )-ns pro complex. however, hsqc peaks of the majority of residues were not detected, indicating that the ns b ( - ;Δ - )-ns pro complex undergoes conformational exchanges on μs-ms, or/and dynamic oligomerization, thus retarding further nmr characterization of its high-resolution conformation. most strikingly, the ns b ( - ;Δ - )-ns pro complex had no detectable catalytic activity even with a concentration up to μm in buffers at ph . , suggesting that this complex is trapped in a highly-dynamic and catalytically impotent state. as virus-encoded proteases have been shown to be essential for the replication and infectivity of many viruses, consequently they become important targets for design of anti-viral drugs [ , ] . indeed, drugs have been successfully developed to treat hiv and hcv infections by targeting their proteases [ , ] . the dengue ns protease has its catalytic machinery hosted by a chymotrypsin fold, which has been extensively shared by a variety of proteases. on the other hand, unlike most other chymotrypsin-like proteases, only flavivirus proteases including dengue ns protease need an additional cofactor ns b to form the enzymatically active complex. noticeably, even within the flavivirus proteases, the ns b cofactors are very divergent, only with sequence identity of~ % among different members [ ] . so the ns b-ns pro interface may represent an attractive target for developing molecules which specifically inhibit flavivirus proteases. however, due to its "complete insolubility", the isolated ns pro domain has never been experimentally characterized so far. in the present study, as facilitated by our discovery in [ ] [ ] [ ] [ ] [ ] , for the first time, the isolated ns pro domain has been extensively characterized in solution by cd, nmr and pre. the results decipher an unexpected fact that despite owning a high-complexity sequence which is very different from classic iups [ ] [ ] [ ] [ ] [ ] [ ] , the ns pro with the native sequence is intrinsically disordered without the ns b cofactor, with no stable secondary structures and tight tertiary packing. this indicates that the isolated ns pro becomes completely insoluble in buffers by following the same mechanism as we previously established for other "completely insoluble" proteins [ ] [ ] [ ] [ ] [ ] . nevertheless, in the presence of the ns b cofactor, the disordered ns pro domain folds into the well-structured chymotrypsin-like fold hosting the active catalytic machinery by the "binding-coupled folding" mechanism [ ] [ ] [ ] [ ] [ ] . previously, the chymotrypsin fold has been found to be an autonomous folding unit even in the context of the sars clike protease with an extra domain [ ] . therefore, to the best of our knowledge, the ns pro domain represents the first intrinsically-disordered chymotrypsin-like fold which absolutely requests the additional cofactor to achieve its correct folding. so an interesting question is whether being intrinsically disordered for the isolated ns pro bears any in vivo relevance? interestingly, a recent study has revealed that the dengue ns pro domain without ns b was capable of binding human fatty acid synthase (fasn) to trigger the membrane remodelling [ ] . as such, our biophysical results imply that this interaction needs the dengue ns pro to be largely disordered. indeed, we found that the well-folded ns b-ns pro complex only had weak binding to the fasn domain. furthermore, denv and other members of the flaviviridae family are dependent on the host er to translate, replicate and package their genome, and their infection has been found to induce significant rearrangements of intracellular membranes [ ] [ ] [ ] [ ] . in particular it has been recently revealed that the rearrangement and expansion of the er appear to be driven by viral but not host protein synthesis early after denv infection, independently of the upr or srebp pathways [ ] . therefore, on a speculative note, we propose that the highly disordered ns pro itself might also play a role in triggering the early rearrangement of the er before activating specific pathways [ ] . previously, we have shown that the als-causing p s mutation of the er-anchored vapb protein rendered the well-structured β-barrel fold of its msp domain to be highly disordered and also become "completely insoluble" in buffer [ ] , similar to what we found here on the isolated ns pro. remarkably, the p s-vapb suddenly gained the novel capacity to remodel the er structure which was not observed for the wild-type vapb [ ] . therefore, the disordered human p s-vapb and dengue ns pro may use similar mechanisms to remodel the er structure. in fact, the degue ns protein itself is also anchored into the er membrane before ns forms the catalytically-active complex with ns b to cleave itself from the polyprotein. in this study, with our previous discovery that "insoluble proteins could be solubilized in water with minimized salt ions, we also successfully refolded the ns pro protease with the fulllength ns b ( - ) anchored into the lmpc micelle. interestingly, although nmr characterization deciphers that the ns pro domains have different dynamics on the μs-ms time scale in the contexts of being complexed with ns ( - ) in buffer and with ns b ( - ) in the lmpc micelle, they have very similar enzymatic activities. this membrane-anchored complex may provide a platform for screening the inhibitors for the dengue protease. in this regard, despite challenging, it is of both fundamental and therapeutic interest in the future to explore how the different structures and dynamics upon being anchored into membranes affect the affinity and specificity of the dengue ns pro in binding substrates and inhibitors. further md simulations provide critical insights into the indispensable role of the ns b cofactor. even within -ns simulations, the isolated ns pro domain already showed higher dynamic instability than the ns pro domain in the ns b-ns pro complex (fig f) . it is thus expected that on the long time scale, the isolated ns pro would become unfolded as experimentally demonstrated here. strikingly, the ns b residues show much higher structural dynamics than ns pro even in the complex, suggesting that the cofactor is capable of sampling a large ensemble of conformations, thus providing the dynamic mechanism for the observed conformational exchange of ns b between open and closed conformations [ ] [ ] [ ] [ ] [ ] . the correlated motions in proteins have been extensively recognized to be critical for their diverse functions [ , , , ] . in particular, we have recently revealed that a global correlated motion network was essential for the catalysis of the sars c-like protease [ , ] , which also utilizes the chymotrypsin fold to harbour its catalytic machinery. more specifically, without altering the three-dimensional structure of the enzyme, the n a mutation on the extra domain is sufficient to inactivate the catalytic machinery by globally decoupling the correlation network, while the sti/a mutations also on the extra domain enhance the catalytic machinery by altering the correlation network pattern [ , ] . in the present study, a similar scenario has been observed for the dengue ns b-ns pro complex. a global correlation network does exist in the ns b-ns complex. most intriguingly, this global correlation network is mostly coordinated by the ns b residues which have very high structural dynamics. as such, the md results not only rationalizes the central role of the ns b cofactor in maintaining the dynamic stability of the ns pro domain, but further implies that a slight perturbation of the ns b-ns pro interface may be sufficient to decouple the correlation network to inactive the catalytic machinery of the dengue ns b-ns pro protease, as we found on the n a mutation of the sars clike protease [ , ] . indeed, by deleting the ns b residues highly flexible in md simulations, we obtained a truncated ns b ( - ;Δ - ), which is able to trap the ns b-ns pro complex in a catalytically-impotent state with significant μs-ms dynamics. in the further, it would be of significant interest to test whether this truncated ns b can act as a specific inhibitor of the dengue protease in vivo. despite the success in treating viral infections with inhibitors to target the active sites of hiv and hcv proteases [ ] [ ] [ ] , many challenges/difficulties still remain for this approach. for example, the structural architecture and catalytic mechanism of viral proteases are also largely shared by many human proteases. this makes it extremely challenging to design inhibitors which only specifically bind to viral proteases but not to human ones. moreover, there is an addition challenge associated with the dengue ns pro protease. it has been proposed that the flat and charged nature of its active site may at least partly contribute to the current failure in developing effective inhibitors [ , ] . to overcome these difficulties, alternative strategies are requested to targets sites other than the active sites of the viral proteases, such as to inhibit the dimerization required for activity [ ] , to trigger allosteric inhibition [ ] , or even to block the folding of the protease [ ] . in this regard, our present study successfully implies potential strategies to perturb the ns b-ns pro interface for future developing effective and specific inhibitors for the dengue protease. more specifically, inhibitory molecules might be developed to: ) trap the ns b-mediated folding into an inactive intermediate as exemplified by the truncated ns b; and ) decouple the global correlation network of the ns b-ns pro complex. furthermore, our success in in vitro refolding of the active ns pro in complex with both ns b ( - ) in buffer and ns b in the micelle provides experimental platforms for implementing the above-proposed approaches of inhibitor design as well as to decode the underlying mechanism for inhibitors obtained by high through-put screening. for example, recently the dengue protease inhibitors were identified by high through-put screening, which were implied to target the ns b-ns pro interface by in silico prediction or mutagenesis [ , ] . it would be of significant interest to delineate how these molecules achieve the inhibitory effect: by trapping the folding or modulating the dynamics of the protease complex, if they directly target the ns b-ns pro interface. in summary, our study decrypted that the dengue ns pro domain is the first intrinsicallydisordered chymotrypsin-like fold which absolutely requests the ns b cofactor to coordinate the correct folding as well as correlated motions of the ns b-ns pro complex to achieve its catalytic function. in light of a recent study [ ] , the disordered ns pro is needed for interacting with the human host factor to initiate the membrane remodeling. furthermore, our results also imply potential strategies to manipulate the ns b-ns pro interface for design of molecules in the future, which may effectively and specifically inhibit the protease activity for treating the dengue infection. best practices in dengue surveillance: a report from the asia-pacific and americas dengue prevention boards the global distribution and burden of dengue the dengue viruses dengue: a continuing global threat structural proteomics of dengue virus structural biology of dengue virus enzymes: towards rational design of therapeutics structural basis for the activation of flaviviral ns proteases from dengue and west nile virus ligand-bound structures of the dengue virus protease reveal the active conformation binding of low molecular weight inhibitors promotes large conformational changes in the dengue virus ns b-ns protease: fold analysis by pseudocontact shifts nmr analysis of a novel enzymatically active unlinked dengue ns b-ns protease complex binding mode of the activity-modulating c-terminal segment of ns b to ns in the dengue virus ns b-ns protease the dengue virus ns b-ns protease retains the closed conformation in the complex with bpti mechanism of ns b-mediated activation of ns pro in dengue virus: molecular dynamics simulations and bioassays new binding site conformations of the dengue virus ns protease accessed by molecular dynamics simulation resurrecting abandoned proteins with pure water: cd and nmr studies of protein fragments solubilized in salt-free water insight into "insoluble proteins" with pure water intrinsically insoluble proteins" and "dark mediators" revealed by studies on "insoluble proteins resolving the paradox for protein aggregation diseases: nmr structure and dynamics of the membrane-embedded p s-msp causing als imply a common mechanism for aggregation-prone proteins to attack membranes tdp- n terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssdna dynamically-driven inactivation of the catalytic machinery of the sars c-like protease by the n a mutation on the extra domain dynamically-driven enhancement of the catalytic machinery of the sars c-like protease by the s -t -i /a mutations on the extra domain intrinsically unstructured domain of hepatitis c virus ns a forms a "fuzzy complex" with vapb-msp domain which carries als-causing mutations how to measure and predict the molar absorption coefficient of a protein nmrpipe: a multidimensional spectral processing system based on unix pipes nmrview: a computer program for the visualization and analysis of nmr data determination of secondary structure populations in disordered states of proteins using nuclear magnetic resonance chemical shifts algorithms for highly efficient, load-balanced, and scalable molecular simulation a point-charge force field for molecular mechanics simulations of proteins based on condensedphase quantum mechanical calculations lincs: a linear constraint solver for molecular simulations quantifying correlations between allosteric sites in thermodynamic ensembles nmr evidence for forming highly populated helical conformations in the partially folded hnck sh domain unfolded proteins and protein folding studied by nmr intrinsically unstructured proteins and their functions intrinsically unstructured proteins classification of intrinsically disordered regions and proteins extended disordered proteins: targeting function with less scaffold backbone dynamics of a free and phosphopeptide-complexed src homology domain studied by n nmr relaxation distance information for disordered proteins from nmr and esr measurements using paramagnetic spin labels a gradual disruption of tight side-chain packing: d h-nmr characterization of acid-induced unfolding of chabii molecular mechanism underlying the thermal stability and ph-induced unfolding of chabii mechanism for transforming cytosolic sod into integral membrane proteins of organelles by als-causing mutations viral proteases the design of drugs for hiv and hcv new merck and vertex drugs raise standard of care in hepatitis c allosteric inhibition of the ns b-ns protease from dengue virus dissection study on the severe acute respiratory syndrome c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis modification of intracellular membrane structures for virus replication organelle-like membrane compartmentalization of positive-strand rna virus replication factories early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the upr and srebp- pathway elimination of the native structure and solubility of the hvapb msp domain by the pro ser mutation that causes amyotrophic lateral sclerosis a vapb mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum structured crowding and its effects on enzyme catalysis hiv- protease folding and the design of drugs which do not create resistance novel dengue virus-specific ns b/ns protease inhibitor, bp , discovered by a high-throughput screening assay. antimicrob agents chemother a small compound targeting the interaction between nonstructural proteins b and inhibits dengue virus replication key: cord- -fl llkoj authors: meltzer, martin i.; gambhir, manoj; atkins, charisma y.; swerdlow, david l. title: standardizing scenarios to assess the need to respond to an influenza pandemic date: - - journal: clin infect dis doi: . /cid/civ sha: doc_id: cord_uid: fl llkoj nan an outbreak of human infections with an avian influenza a(h n ) virus was first reported in eastern china by the world health organization on april [ ] . this novel influenza virus was fatal in approximately onethird of the confirmed cases detected in the months following its initial identification [ ] , and limited humanto-human h n virus transmission could not be excluded in some chinese clusters of cases [ , ] . there was, and still is, the possibility that the virus would mutate to the point where there would be sustained human-to-human transmission. given that most of the human population has no prior immunity (either due to natural challenge or vaccine induced), such a strain presents the danger of starting an influenza pandemic. in response to such a threat, the joint modeling unit at the centers for disease control and prevention (cdc) was asked to conduct a rapid assessment of both the potential burden of unmitigated disease and the possible impacts of different mitigation measures. we were tasked to evaluate the following interventions: invasive mechanical ventilators, influenza antiviral drugs for treatment (but not large-scale prophylaxis), influenza vaccines, respiratory protective devices for healthcare workers and surgical face masks for patients, school closings to reduce transmission, and airport-based screening to identify those ill with novel influenza virus entering the united states. this supplement presents reports on the methods and estimates for the first listed interventions, and in this introduction we outline the general approach and standardized epidemiological assumptions used in all the articles. given that there had not yet been (and subsequently has not been to date) a pandemic caused by the h n virus, there are no relevant large-population data concerning transmission and clinical impacts of h n . we therefore had to consider the potential impacts of disease and interventions for a not fully defined pandemic (ie, a pandemic caused by a generic influenza strain hxny). thus, any model that we built had to allow for a wide range in virus transmissibility and resulting clinical impact. the models had to also fully consider a range of effectiveness of interventions-for example, influenza antiviral drugs could be less effective against the next influenza strain causing a pandemic. given these uncertainties, and the need for a rapid assessment of a large number of factors, the models produced had to meet a number of specifications: had to be produced in a manner that would allow the models to be easily transferred to other units in government and to public health officials, and subsequently used by people who did not build them; had to provide easy identification of all input variables, their values, and ability to rapidly change those values; can be easily stored and resurrected for future use and reference at some unspecified time in the future; and, the results from each model can be readily compared to each other. in response to these specifications, we decided to require that each model be built in a spreadsheet format, and that we would essentially have model for each intervention considered. meeting these specifications had the added value of producing models that readily fit into the existing cdc emergency operations response structure. in this structure, groups called task forces are formed to focus on particular aspects of a response to a public health emergency. for example, for an influenza pandemic response, there are usually task forces that focus on vaccines (eg, recommendations regarding prioritization of vaccine supplies, issues related to distribution), medical countermeasures (eg, recommendations regarding use of drugs for treatment and prophylaxis, use of personal protective equipment such as face masks), and nonpharmaceutical interventions (eg, recommendations regarding school closures, border security, and screening). to allow easy comparison between results (a specification), we standardized a risk space defined by using ranges of transmission and clinical severity from a previously published influenza severity assessment framework ( figure ) [ ] . the framework can be used to plot, and compare to historical data, the relative severity of an influenza pandemic (or nonpandemic influenza season). the framework uses scales: a scale of clinical severity, and a scale of transmissibility. the severity scale has a number of components in it, including case-fatality ratio and caseto-hospitalization ratio (table ) [ ] . the transmissibility scale is assessed by considering factors such as the clinical (symptomatic) attack rate in various locales, such as school, community, and workplace (table ) [ ] . we defined and chose a risk space that has a transmission scale that runs from approximately a scale of (eg, comparable to a community attack rate of %- %) to a scale of (community attack rate of > %) ( figure , table ). our defined risk space has a low-end clinical severity scale of , with a case-fatality ratio of . %- . % and a death-to-hospitalization ratio of %- % ( table ). the upper range of severity in our risk space was defined as a scale of , with a case-fatality rate of . %- . %, and a death-to-hospitalization ratio of %- % ( table ). note that the defined risk space encloses the and pandemics ( figure ). it is essential to note that this chosen risk space is illustrative, not definitive. until there are data defining the epidemiological elements of the next pandemic, such as rate of transmission, and case-fatality rate, other risk spaces could be chosen for planning purposes. the models presented in this collection, built to the specifications listed here, allow for rapid alterations in input values. the size and shape of the epidemic curve could impact the effectiveness of interventions. for example, the impact of influenza vaccines depends upon the start of deliveries of large amounts of vaccine compared to the timing of the pandemic peak. thus, we included in the standardized epidemiological scenario epidemic curves, produced using a simple simulation model (see below). we configured the model using clinical attack rates of approximately % and %. these clinical attack rates represent the aggregated attack rate across the entire us population. within the population, subpopulations will typically experience different attack rates (eg, children will experience a higher attack rate than adults - years old-see description later in paper). furthermore, for each attack rate, we assumed starting (seeding) scenarios. we used one scenario in which the pandemic started with the arrival of infectious cases and the other when the pandemic started with infectious cases ( figure ) . to model the epidemic curves, we built a simple, nonprobabilistic (ie, deterministic) model that simulates the spread of influenza through a population by moving the population into groups of susceptible, exposed, infectious, and recovered or death ( table provides values used). we divided the population into age groups ( - , - , - , or ≥ years of age). we table . see main text for additional details. note that the - , - , and - seasons were nonpandemic seasons. they are included to provide reference points regarding the impact of nonpandemic seasons. adapted from reed et al [ ] . modeled the probabilities of daily contact (and thus risk of disease transmission) by constructing a contact matrix using data from the united kingdom (see table a in technical appendix a). we thus produced notably different epidemic curves (figure ). for example, the two % attack rate scenarios peak in weeks and , whereas the % attack rate scenarios peak in weeks and ( figure ). the clinical attack rates by age group are presented in table . obviously, the largest numbers of cases occur in the largest age group of -to -year-olds; however, children in both the - and - age groups have the highest attack rates, indicating a potentially greater degree of vulnerability (table ) . perhaps one of the greatest strengths of the simple models presented in this collection of articles is that they highlight what is for case-fatality ratio and case-hospitalized ratio, scale shows low severity, and scale shows high severity (in bold). source: adapted from reed et al [ ] . a these estimates related to the framework for assessing the impact of influenza pandemics, shown in figure . table ). seeding refers to the number of infectious cases, either or , that arrives nearsimultaneously in the united states to start the pandemic. and is not known about the burden of disease and the potential impact of a planned intervention. to find the weaknesses of what is currently known, a reader need only consult table in each article. these tables list inputs, their assumed values, and data sources. an example of an important unknown is as follows: when estimating the number of respiratory protection devices (eg, face and surgical masks) needed by first responders ( police officers, firefighters, emergency medical technicians), one could assume that first responders will need mask per person whom they encounter with influenza-like illness. the problem is that there are no readily available data that report on the measurement of such [ ] . similarly, when considering the potential use and impact of influenza antiviral drugs, o'hagan et al had to assume that existing influenza antiviral drugs would have the same level of effectiveness against the strain causing the next influenza pandemic as they do with existing influenza strains [ ] . despite these limitations, these simple models make it fairly straightforward to rapidly assess the relative importance of each of the input variables. one assumption that may not be readily appreciated is the impact of the shape of the standardized epidemiological curves used in all the models (figure ). previous influenza pandemics have produced different shapes of deaths over time (figure ). such differences in deaths over time can greatly influence the success of some of the interventions. for example, when considering the number of mechanical ventilators needed at the peak of the pandemic, meltzer et al initially assumed that the peak demand for ventilators would equal approximately % of all patients needing mechanical ventilation [ ] . however, in the % attack rate epidemiological curve (figure ), the number of cases that figure ). note that the data for were recorded once every weeks, whereas all other plots used weekly data. see technical appendix b for further details. occur in the peak days is approximately % of all cases. thus, the authors of the ventilator study conducted a sensitivity analysis by changing from % to % the assumed number of mechanically ventilated patients that occurs at the peak of a pandemic. the articles in this supplement also incorporate other important implicit assumptions. one of the more important is that each article essentially assumes that the healthcare system can absorb and/ or successfully execute any of the interventions so modeled. for example, biggerstaff et al provide some estimates of the impact of influenza vaccination in which it was assumed that million persons could be vaccinated each week [ ] . the us private and public health systems, collectively or separately, have never previously achieved such a rate (though the authors clearly demonstrate that achieving such a rate would have very positive public health outcomes). furthermore, the successful deployment and ultimate impact of each intervention is likely to have a wide variation. schools can close for different lengths of time, antiviral drug prescription and distribution may not be equally efficient in all areas, and healthcare workers and patients may have different levels of compliance in wearing protective gear. finally, readers will note that there are no reports in this collection that consider the simultaneous deployment of ≥ interventions. it is realistic to assume that, during the next influenza pandemic, public health officials, healthcare providers, and other policy makers are likely to enact several interventions at once (eg, close schools, start dispensing antiviral medications, recommend use of protective personal gear). the problem arises in that such multi-intervention models become very scenario specific. for example, different locales are likely to face different unmitigated epidemic curves ( figure ) . thus, researchers who estimate the potential impact of combining several interventions at once have to make a very large increase in the number of assumptions. this makes it more difficult to both generalize the results and to rapidly understand what assumptions are relatively more important. despite these limitations, we believe that the benefits of using these models outweigh the limitations. this assessment is based on our experience of using the models and results produced to help public health leadership reassess us influenza pandemic planning and preparedness. in the response to the h n threat, the most important outcome from policy makers seeing the results from these models was the intense debate concerning the inputs and assumptions. we thus believe that the methodology used here to develop and guide the building of the models in this collection, and the subsequent interpretations and use of the results, can be a useful part of future public health responses. supplement sponsorship. this article appears as part of the supplement titled "cdc modeling efforts in response to a potential public health emergency: influenza a(h n ) as an example," sponsored by the cdc. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. standardized epidemiological curves-contact matrix: to model the epidemic curves (figure ), we built a simple, nonprobabilistic (ie, deterministic) model in which we divided the population into age groups ( - , - , - , ≥ years). to measure the risk of contact and possible onward spread between and within each age group, we used the contact matrix shown in table a . for the contact matrix, we used, in the absence of relevant data from the united states, data from the united kingdom [ ] , collected as part of a study called the polymod study that collected contact data from approximately persons living in european countries [ ] . because the uk data are split into -year age groups, we had to aggregate the data into the age groups used in our model. during this aggregation, we ensured that the total number of contacts between any age groups is "equal in any direction" (eg, the number of contacts between - years and - years is the same as those between - years and - years). we used, for this aggregation process, the age distribution of the us population (www. censusscope.org). as noted above, the uk contact data [ ] are split into -year age groups, which we had to aggregate into the age groups used in our models. furthermore, the matrix that we constructed had to meet the condition of being symmetrical. that is, the number of contacts from age group a to age group b should equal the number of contacts in the reverse direction. we begin the explanation of how we built our contact matrix by introducing some notation: the mixing matrix elements of the published matrix [ ] are denoted by u ij , i, j = , . . . , m, where i, j refers to rows and columns, respectively, and m is the number of age groups in the mixing matrix. as the mixing matrix required has fewer age groups than that of the published polymod matrix [ ] , indexed by f, g = , . . . , n, then we let age group u contain narrower age groups i ¼ lð f Þ to uð f Þ. . the contact rate between someone in group i and another in group g is given by we then proceed according to the following steps: . if the us population distribution is such that the population in age group i is n i , we can calculate the population-weighted means of each of the elements d, to obtain contact rates between groups f and g. for f = g, this calculation is simple: . for the elements that are off the diagonal, the calculation becomes more complicated because we need to sum up the correct number of contacts made between each age group. the total reported rates of contact from f to g and g to f are: . theoretically, these values should be equal to one another; however, they differ from one another when calculated from actual reported contact rates (from self-administered surveys such as those conducted by mossong et al [ ] ), and so, to ensure that they are equal, we can average them before calculating the final mixing-matrix elements e fg and e gf : here then, e fg is the rate at which an individual in age group f makes contacts with anyone in age group g, per unit time, as reported in the original data (ie, per day for the original mossong et al [ ] data). an example of this procedure is given below, following the steps above, outlined theoretically: . we begin with the "all contacts" (ie, both conversational and physical) matrix for great britain from the polymod study. the elements of this matrix denote the daily number of contacts between an individual in one -year age group with those in another -year age group. element ( , ) , for example, is the daily number of contacts a person aged - years has with someone aged - years. the fill matrix is as follows: summing the columns of the -year group matrix according to the desired group widths (eg, the first columns are summed to give a -year age group column) gives the following intermediate -group by -group matrix: . next, we obtain a vector whose elements are the numbers of individuals in each of the age groups of the original matrix (here -year width groups, taken from the great britain census; the age distribution should correspond closely with the distribution that held at the time when the contact survey was performed), and we perform a sum of the total number of contacts to produce an aggregated age group (ie, two year age groups are aggregated into one -year age group). elements of the total contact matrix. note that the numbers in the above matrix in these positions differ slightly from those in the y , y calculations outlined above; this is because daily polymod contacts were rounded for the calculations illustrated. . once we have completed this procedure for the whole aggregated total contact matrix, we need to divide our total contact numbers by the correct number of individuals in each age group, to ensure we end up with a matrix that gives the number of contacts per person per day in the relevant age group. for example, the ( , ) element of the final matrix is the ( , ) element of the matrix produced by step ( ) divided by the total number of individuals in the first age group (ie, the sum of the individuals in the first two -year age groups = + = ); and the ( , ) element is divided by the same number, whereas the ( , ) element is divided by the number in the second age group = + = . dividing through gives the final matrix below (which is similar to table a , accounting for rounding in the illustrative calculation). to model the curves shown in figure , we used the estimated number of deaths from previous pandemic seasons ( , , and ). we compared those to the estimated clinical cases from the epidemiological model built for this exercise, using attack rates of both % and % (ie, the curves shown in figure , main text). all deaths were based on the clinical data reported during the specific pandemic season. however, in an effort to obtain current death estimates, we extrapolated the seasonal case values (either clinical data or number of deaths) into currentyear us cases at a total population of million. • influenza pandemic: we obtained from the source [ ] the weekly number of deaths in ( per people) for the reported different us geographic locations (west, east, and midwest/south). we then adjusted those number of deaths, per , to the approximate current us population of million persons (ie, multiplied each data point by ). this gave us the equivalent number of deaths for the us population. • influenza pandemic: we obtained the total, all ages biweekly (ie, reported every weeks) number of respiratory illnesses per from figure in the report of the cdc (then known as the communicable disease center) [ ] . we then adjusted those number of cases to the approximate current us population of million persons (ie, multiplied each data point by ). this gave us the equivalent number of cases for the us population. to obtain estimates of deaths in equivalent us population, we multiplied the estimates of cases by a case-fatality ratio of . (ie, . % of all cases result in death). this case-fatality estimate was taken from table in the main text [ ] . • influenza pandemic: we obtained the weekly reported number of pneumonia-influenza deaths in us cities from figure in sharrar et al [ ] . however, the total number of deaths recorded by sharrar et al was only , which is notably lower than what may be expected. we therefore used a multiplier of . to adjust upward their estimates. we constructed this multiplier by noting that meltzer et al's figure [ ] showed approximately deaths for a -type influenza pandemic occurring in the us population (ie, / = . ). • attack rates: we took the curves plotting the % and % clinical case attack rates shown in figure- of the main text (the plots assuming infectious persons start, or "seed," the pandemic in the united states). we then used a case-fatality rate of . (ie, . % of all cases result in death), taken from table in the main text [ ] . for simplicity, we assumed a low severity (scale of ) of . % for both attack rates to generate the number of deaths. human infection with influenza a(h n ) virus in china human infection with avian influenza a (h n ) virus-update epidemiology of human infections with avian influenza a(h n ) virus in china probable person to person transmission of novel avian influenza a (h n ) virus in eastern china, : epidemiological investigation novel framework for assessing epidemiologic effects of influenza epidemics and pandemics potential demand for respirators and surgical masks during a hypothetical influenza pandemic in the united states estimating the united states demand for influenza antivirals and the effect on severe influenza disease during a potential pandemic estimates of the demand for mechanical ventilation in the united states during an influenza pandemic estimating the potential effects of a vaccine program against an emerging influenza pandemic-united states social contacts and mixing patterns relevant to the spread of infectious diseases nonpharmaceutical interventions implemented by us cities during the - influenza pandemic the epidemiology of asian influenza. - . a descriptive brochure national influenza experience in the the economic impact of pandemic influenza in the united states: priorities for intervention for example, to construct the matrix element pertaining to the total number of contacts between the - -year age group and the - -year age group (ie, itself ), we perform the following sum: Ã : þ Ã : ¼ : this is the first diagonal element of the "total contacts" matrix and, again, it represents the total number of contacts made per day between those in the - -year age group.because diagonal elements are of course the same as their off-diagonal counterparts, there is no problem. . however, corresponding pairs of off-diagonal totals should be the same; that is, the total number of contacts between those in the - -year and - -year groups should be the same as the total number between those in the - -year and - -year age groups. y = n *d + n *d = * . + * . = y = n *d + n *d = * . + * . these total contact numbers are not the same and so we take the average of them and they become the ( , ) and ( , ) key: cord- - p bpt authors: sapmak, ariya; boyce, kylie j.; andrianopoulos, alex; vanittanakom, nongnuch title: the pbrb gene encodes a laccase required for dhn-melanin synthesis in conidia of talaromyces (penicillium) marneffei date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: p bpt talaromyces marneffei (basionym: penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in southeast asia. t. marneffei cells have been shown to become melanized in vivo. melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. the synthesis of the two most commonly found melanins in fungi, the eumelanin dopa-melanin and the allomelanin dhn-melanin, requires the action of laccase enzymes. the t. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrb, during growth and development. a strain carrying a pbrb-gfp fusion shows that pbrb is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. the pbrb gene is required for the synthesis of dhn-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type. talaromyces marneffei (basionym: penicillium marneffei) is an opportunistic human fungal pathogen endemic to southeast asia and southern china [ ] [ ] [ ] . most cases of t. marneffei infection occur in immune-deficient hosts, especially those infected with human immunodeficiency virus (hiv), however, infections have also been reported in non-hiv children with the underlying immune defects (e.g. severe combined immunodeficiency (scid), congenital lymphopenia, hyper-igm syndrome, and hyper-ige syndrome). failure to treat infections is fatal, especially in children with primary immunodeficiency [ , ] . t. marneffei is a dimorphic fungus with a thermally regulated dimorphic switch. as such, t. marneffei is capable of undergoing a transition from the saprophytic filamentous multicellular hyphae found in the environment (or in vitro at °c) to a unicellular yeast growth form in vitro at °c and during infection. during growth at °c, hyphae can also undergo asexual development (conidiation) to produce conidia, the infectious propagules. hyphae differentiate by sequential production of an aerial stalk followed by the budding of the metula and phialide cell types from the stalk tip plos one | doi: all transformants were produced from a uracil auxotrophic strain (g , Δpkua pyrg -) derived from talaromyces marneffei frr (cbs . , atcc ) [ , ] . strain g lacks the pkua gene encoding the ku protein which functions in non-homologous dna end joining repair [ ] . therefore, genetic transformation is mediated via homologous recombination only. g harbors a spontaneous, loss-of-function mutation in pyrg (orotidine- '-phosphate decarboxylase encoding gene) selected by growth in the presence of -fluoroorotic acid [ ] . all pyrg + transformants and the t. marneffei g (Δpkua::pyrg + ) strain used as a control, were maintained on anm medium containing % (w/v) glucose, mm ammonium sulfate [ ] . the t. marneffei f strain isolated from an aids patient (cbs ) [ ] was maintained on malt extract (me) agar. conidial suspensions of transformants and the g strain were harvested from conidiating colonies growing on anm solid medium at °c for week. colonies on petri dishes were flooded with sterilized phosphate buffer saline, gently scraped, filtered through miracloth (calbiochem), and recovered by centrifugation. conidia were harvested from t. marneffei f colonies cultured on me agar at °c for week. a suspension of conidia was spread on me agar with and without μg/ml tricyclazole in -well plate. the plate was incubated at °c for week. the expression of pbrb was investigated by rt-pcr. rna samples were extracted from t. marneffei f cultured in brain heart infusion (bhi) broth at °c and °c for days. rna was extracted and used as a template to produce cdna using omniscript reverse transcription kit (qiagen). the pbrb cdna was then amplified using specific primers pmlac -f and l re. the s rrna was amplified as a control using pm and pm primers [ ] . primers used in this study are listed in table . talaromyces (penicillium) marneffei pbrb gene to construct the Δpbrb plasmid, pmlac u and pmlac l primers were used to amplify the pbrb gene containing approximately . kb of upstream and downstream untranslated regions (utrs) from t. marneffei strain g . purified pcr product ( , bp) was ligated into pgem-t easy (promega) to produce the pbrb plasmid (plac ). this plasmid was digested with bglii and stui to remove the pbrb coding region ( bp before atg to bp before stop codon). the bglii/stui pbrb plasmid was ligated to a bamhi/ecorv fragment containing the pyrg blaster selectable marker cassette (pab [ ] ) to generate pplilac . the linearized insert from pplilac was excised by noti and purified before transformation. for the complementation construct, p n , the noti fragment from plac was ligated into a noti-digested/ dephosphorylated pab vector. in order to generate the pbrb(p)::pbrb::gfp strain, plac was amplified by inverse pcr using ill ec /iul ec primers. the pcr product was digested with ecorv and acli and then ligated with ecorv/clai fragment containing egfp to produce plac gfp. the egfp coding region was inserted at the ' end of pbrb, bases after the start codon. the egfp coding region was amplified from the pegfp-c vector (kindly provided by dr. amornrat o'brien [ ] ) using fpegfpc /rpegfpc primers. the whole pbrb(p)::pbrb::gfpfragment was excised from plac gfp by noti digestion and cloned into noti-digested/dephosphorelated pab to generate pgfplac n. t. marneffei strain g was used to generate the Δpbrb::pyrg + mutant and pbrb(p)::pbrb:: gfp strain. dna-mediated transformation was performed using peg-mediated transformation protocol described previously [ ] . the pyrg + transformants were selected on medium without uracil. to generate a Δpbrb pyrgstrain, the Δpbrb::pyrg + strain was inoculated onto anm agar containing mg/ml -fluoroorotic acid to select for loss of the pyrg cassette via homologous recombination between the inverted cat repeats that flank pyrg in the construct [ ] . to generate the Δpbrb pbrb + complementation strain, the Δpbrb pyrgstrain was transformed with p n and pyrg + transformants were selected. genetically modified t. marneffei transformants were examined by pcr and/or southern blot analysis [ ] . the pbrb amplification was performed using pmlac f/l re primers, while fpegfpc /l re primers were for egfp-pbrb amplification. the t. marneffei fetc gene (pmaa_ ) was amplified as an internal control of pcr using pmlac f/pmlac r primers. the hybridization probe for southern blot analysis was the pbrb gene amplified by pcr using pmlac u/pmlac l primers. for germination assays, conidia ( ) of each strain were inoculated in a six well microtitre plate containing bhi broth and the plate was incubated at °c or °c for hours. aliquots of the broth culture were placed on slides to count germinating conidia. three replicates were performed counting at least cells each time. for growth rate assays, approximately conidia were spread onto plates of anm agar and incubated at °c for days. the diameter of individual colonies was measured daily over a period of days. mean and standard error of the mean (sem) values were calculated and analyzed for statistical significance using graph-pad software (http://www.graphpad.com/). conidia ( ) of the pbrb(p)::pbrb::gfp strain were inoculated into two flasks containing ml of bhi broth. each flask was incubated in a shaking incubator at °c or °c for days. mycelia from the °c culture were poured onto miracloth and rinsed with cold pbs before microscopic examination. fungal cells from the °c culture were harvested by centrifugation, washed with cold pbs, placed on a microscope slide and examined using an olympus provis ax fluorescence microscope. to examine conidiating fungal cells, a piece of anm agar block (about x x mm) was placed on a sterile glass slide. conidia from the pbrb(p)::pbrb::gfp strain were inoculated on each side of agar block. a cover glass was placed on the top of the agar block. the slide culture was incubated in a humidified chamber at °c for days. the agar block was removed and fungal cells that remained attached to the slide were fixed with absolute ethanol before air drying in a biosafety cabinet. fungal cells on the slide were incubated with mg/ml trichoderma harzianum cell wall lysing enzyme (sigma-aldrich, uk) dissolved in osmotic buffer [ ] at °c for minutes and then the solution was poured off. fungal cells were permeabilized by the addition of . % triton x- in pbs for minutes. slides were washed with cold pbs solution. immunofluorescence staining was performed as previously described [ ] . briefly, slide cultures were blocked with superblock blocking buffer (pierce, usa) at °c for hours. the slides were incubated with μg/ml of anti-melanin igm antibody, kindly provided by dr. sirida youngchim [ ] , at °c for . hours. slides were washed with pbs and then incubated with a dilution : of rhodamine-labeled goat anti-mouse igm antibody (jackson immunoresearch laboratories, usa) at °c for . hours. after washing, cells were observed under a fluorescence microscope. the negative control samples were produced by omitting the antimelanin igm binding step in order to assess the background caused by non-specific binding of secondary antibody. the superblock blocking buffer used to dilute anti-melanin igm was added, instead of using anti-melanin igm, before incubating with secondary antibody. to examine chitin deposition, calcofluor white staining was performed as previously described [ ] . three-day-old fungal cells were harvested from bhi broth culture as described above and the cells were washed with pbs before staining. the green pigment synthesized during conidiation in t. marneffei is dihydroxynaphthalene melanin to examine if the green coloration of conidia was attributed to dhn-melanin, t. marneffei conidia were inoculated onto medium with or without the inhibitor tricyclazole. tricyclazole inhibits the two hydroxynaphthalene reductases functioning in dhn-melanin synthetic pathway ( fig a) [ ] [ ] [ ] . in the absence of tricyclazole, t. marneffei conidia appeared green ( fig b) . in contrast, in a presence of tricyclazole, the t. marneffei conidia appeared yellow (fig b) . this suggests that the synthesis of dhn-melanin contributes to the green coloration of conidia during asexual development. the t. marneffei genome encodes ten multicopper oxidases laccases are members of the multicopper oxidase (mco) family, which also includes ferroxidases and ascorbate oxidases. to identify laccase encoding genes in t. marneffei the c. neoformans lac and a. fumigatus abr (fig a) encoding genes were used as a query sequence in blast searches of the t. marneffei atcc genome sequence (genbank, ncbi). this identified two highly homologous genes (pmaa_ and pmaa_ ) (cnlac homology of % and %, respectively) that were then also used for additional blast searches to retrieve related sequences in the t. marneffei genome. these homology searches identified genes encoding putative multicopper oxidases (pmaa_ , pmaa_ , pmaa_ , pmaa_ , pmaa_ , pmaa_ , pmaa_ , pmaa_ , pmaa_ , and pmaa_ ). generally, laccases contain four copper atoms including type cu, type cu, and a pair of type cu. the patterns of conserved amino acids coordinated with each type of copper are hxhg, hxh, hchxxxhxxxm/f/l, and hxxhxh, which occupy the l -l signature sequences starting from the n-terminus [ ] [ ] [ ] . we found that of the t. marneffei mcos (excluding pmaa_ ) have these conserved patterns (see s fig) . previous phylogenetic studies reveal that mco sequences are often clustered with other genes according to function, fungal division, and source organism [ , , ] . to predict which t. marneffei mco participates in conidial dhn-melanin synthesis, we combined fungal mco sequences and performed alignments using clustalw (http://www.genome.jp/ tools/clustalw/). tree construction was conducted in mega version [ ] using the neighbor--joining method. the fungal mco sequences were separated into clades with wellsupported branches (> % bootstrap support) (fig a) . each clade was highlighted and named according to sequences with known functions and fungal divisions. the ascomycete laccase lineage can be divided into clades supported with very high bootstrap values ( % and %). focusing on the second clade containing t. marneffei pbrb, this clade comprises of characterized laccases functioning in conidial dhn-melanin synthesis. t. marneffei pbrb is more closely related to a. fumigatus abr than a. nidulans ya [ ] [ ] [ ] . pbrb is expressed during asexual development and localizes to all cell types of the conidiophore the expression of pbrb was investigated by rt-pcr. rna samples were extracted from t. marneffei f grown in bhi broth at °c and °c for days. a transcript was not detected under these conditions suggesting there is little or no pbrb expression in vegetative hyphal or yeast cells (data not shown). to analyze pbrb expression further, a strain was generated that expresses a fusion construct in which pbrb, expressed from the native promoter, is fused to gfp (pbrb(p)::pbrb::gfp). conidia of the pbrb(p)::pbrb::gfp strain were inoculated into bhi broth and cultured at °c and °c for days. in support of the rt pcr analysis, gfp fluorescence was not detected in vegetative hyphal cells at °c or vegetative yeast cells at °c nlm.nih.gov/protein/) and used to build sequence alignments in clustalw. the alignment was then used to construct a relatedness tree using mega software. phylogenetic relationships of the mcos were inferred using the neighbor-joining method and bootstrap tested ( replicates). branch lengths of the tree under standard conditions (fig a and b ). in addition, vegetative cells were grown at °c and °c in a variety of other types of medium (anm, synthetic dextrose and malt extract) and in the presence of copper with low glucose ( . %) or under acidic condition ph . , which are known laccase inducing conditions [ ] . no signal from pbrb::gfp could be detected under any of these conditions after , and days of incubation (data not shown). a slide culture of the pbrb(p)::pbrb::gfp strain was prepared to observe expression of pbrb during conidiation. compared to the negative control (fig c) , strong gfp fluorescence was observed in conidiophores of the pbrb(p)::pbrb::gfp strain, suggesting that pbrb is expressed during asexual development (fig d) . the pbrb::gfp protein was localized as distinct spots in metulae and phialides (fig d) . to investigate if pbrb co-localizes with melanin, melanin was detected in the pbrb(p)::pbrb:: gfp strained by immunolabeling with an anti-melanin antibody. immunostaining detected strong fluorescence in phialide cells with some weaker staining in the stalk, metulae and conidial cell types. there was also some punctate melanin staining in the various cell types. this staining are drawn to scale and bootstrap support indicating at the branch sites. fungal mcos that share a common ancestry with more than % bootstrap value are shaded in gray. functions are defined at each clade based on characterized function of mco members. (b) a partial alignment representing the conserved motifs of copper binding sites and l -l signature sequences found in t. marneffei pbrb (pmaa_ ) and its orthologs a. fumigatus abr (afua_ g ) and a. nidulans ya (an ). the numbers in front of each sequence indicate the amino acid position of a particular protein. cu binding motifs are identified above the sequences, numbers including , , and are type cu, type cu, and type cu. the asterisk indicates the potential proton donor for the reaction intermediates. was highly co-localised with the fluorescence form the pbrb::gfp fusion protein (fig ) . this supports the hypothesis that pbrb is likely to be involved in melanin biosynthesis during conidiation. to characterize the role of pbrb in t. marneffei, Δpbrb mutants were generated. two independent mutants were characterized with respect to conidiation, germination and growth. in order to examine morphogenesis during conidiation, Δpbrb conidia were grown on anm plates. in contrast to the wild type control, which exhibits green colored conidiation at °c after days, conidiation of the Δpbrb mutants appeared a light brown color (fig ) . reintroduction of a wild type copy of pbrb at the native locus (materials and methods) in the Δpbrb mutant complemented the conidiation phenotype (fig ) . slide cultures of the Δpbrb and Δpbrb pbrb + complemented strains growing on anm agar at °c for days were prepared to examine the morphology of conidiophores. deletion of pbrb did not result in any morphological defects in any of the conidiophore cell types (fig c and d ). the change in phenotype indicates that pbrb has a unique function that cannot be compensated by other t. marneffei laccases. to assess if deletion of pbrb and the consequent change in pigmentation of the conidia affected germination, conidia of the Δpbrb and Δpbrb pbrb + strains were inoculated in bhi broth and incubated at °c or °c for hours then the number of germinated conidia were counted. there was no difference in the germination of the Δpbrb and Δpbrb pbrb + strains at °c (fig a and b ) or °c (data not shown). hyphal cells from the Δpbrb and Δpbrb pbrb + strains were grown at °c for days and examined microscopically. staining of hyphae with μg/μl calcofluor white showed no defects in chitin deposition nor was any morphological difference detected (data not shown). growth rates of the Δpbrb and Δpbrb pbrb + strains were determined by measuring colony diameters daily over the course of days on anm medium at °c. mean and standard error of the mean (sem) were calculated and statistical differences were assessed using the t-test. the differences in growth rates between Δpbrb and Δpbrb pbrb + strains at and days were found to be statistical significant (p = . and . , respectively) (fig ) . deletion of pbrb results in brown conidiation color, in contrast to the green coloration of wild type. this suggests that like a. fumigatus and a. nidulans, pbrb in t. marneffei is required for the synthesis of dhn-melanin. as melanins protect fungal cells against environmental assaults, the susceptibility of the Δpbrb mutant to a variety of stresses was determined. ten-fold dilutions of Δpbrb and Δpbrb pbrb + conidial suspensions were dropped on anm and bhi agar containing % h o (oxidative stress), μg/ml sds (cell wall stress), μm congo red (cell wall stress), m sorbitol (osmotic stress), . m nacl (salt stress), mm nano (nitrosative stress) or antifungals ( . μg/ml amphotericin b, . μg/ml clotrimazole, μg/ml fluconazole, and . μg/ml itraconazole) and incubated at either °c and °c for days. in addition, susceptibility to antifungal activity of macrophage (j ) was examined. however, no differences in stress susceptibility were observed (data not shown). talaromyces (penicillium) marneffei pbrb gene cytoplasmic protein extracts from the wild type and Δpbrb mutant cultured in brain heart infusion broth at °c for days were capable of catalyzing l-dopa (data not shown). in addition, Δpbrb cells growing in bhi medium at °c for days showed equivalent staining as the wild type using the anti-melanin antibody. these results suggest that melanization of vegetative cell types is not dependent on pbrb and this is consistent with the results from the susceptibility tests using the various stress agents. the multicopper oxidase (mco) family comprises enzymes that typically contain four copper atoms classified into three types (type cu, type cu and type cu). members of mco family include laccases, ferroxidases, ascorbate oxidases, bilirubin oxidases, cueo, and ceruloplasmin [ , , , ] . among mco members, laccases can be found in various organisms (e.g. plants, fungi, bacteria and insects), but not in humans. a large number of laccases are produced in many basidiomycete and ascomycete fungal species and a variety of physiological roles have been reported including morphogenesis, stress defense, fungal pathogen/host interaction and delignification [ , , ] . in pathogenic fungi, laccases have attracted attention due to their involvement in melanization and the correlation with host invasion and protection against host immunity. laccases contribute to the melanization pathways producing the most commonly isolated fungal melanins; dopa-and dhn-melanin [ , ] . generally, melanins are deposited in the cell wall of conidia and fungal cells [ , ] . this study has determined the role of a developmentally regulated laccase, encoded by pbrb, during dhn-melanin synthesis in conidia. neither the pbrb transcript nor the gfp-tagged protein in a pbrb(p)::pbrb::gfp strain could be detected during vegetative hyphal or yeast growth. however, gfp fluorescence was observed in conidiophores of the pbrb(p)::pbrb::gfp strain, suggesting that pbrb is only expressed during asexual development in t. marneffei. likewise in a. fumigatus and a. nidulans, expression of the orthologous abr and ya genes characteristically occurs during conidiophore development but not during vegetative growth [ , ] . phylogenetic examination showed that t. marneffei pbrb is more closed to a. fumigatus abr than a. nidulans ya (fig a) . moreover, t. marneffei Δpbrb colonies displayed brown-pigmented conidia that resemble those of the Δabr strain [ , ] . our data suggests that t. marneffei pbrb is polymerizing polymers of , -dhn to form dhn-melanin, as has been shown in a. fumigatus [ ] . t. marneffei pbrb is located within a genomic cluster of genes with homology to those required for conidial pigment biosynthesis in other systems and includes pmaa_ (conidial biosynthesis oxidase abra), pmaa_ (conidial biosynthesis protein ayga), pmaa_ ( , , , -tetrahydroxynaphthalene reductase arpa), pmaa_ (conidial pigment biosynthesis scytalone dehydrogenase arpa) and pmaa_ (conidial pigment polyketide synthase alba) (see s a fig) . this cluster is conserved in a. fumigatus (s b fig). the biochemical pathway for dhn-melanin production was first described by wheeler and bell ( ) and has been well characterized both biochemically and genetically in many ascomycetes. the genus aspergillus comprises many species and pigmented conidia appear in various colors among species. effects of inhibitors on dhn-melanin synthesis (using tricyclazole and phthalide) and dopa-melanin synthesis (using kojic acid and tropolone) in a range of aspergillus species demonstrate that differences in the amounts and types of pigments and melanins synthesized by related species are likely to be a common theme [ , ] . among aspergillus and penicillium species, most exhibit green to bluish green conidial pigments and the use of inhibitors of melanization pathways has revealed that most of these pigments are made from pentaketide metabolites [ ] . the starting carbon units are acetate [ , ] , malonyl coa and/or acetyl coa [ , , ] and are catalyzed by a polyketide synthase (pks) in the first step before subsequent processing through enzymatic steps to produce dhn-melanin [ , , , ] . mutation of the gene encoding the pks in a. nidulans (wa), a. fumigatus (pksp) and t. marneffei (wa) similarly results in white, non-melanized conidia [ , , ] . while this early part of the pathway seems to be conserved amongst these fungi, differing results have been shown when tests are conducted with the dhn-melanin synthesis inhibitor tricyclazole. tricyclazole specifically affects the reductases that reduce , , , -tetrahydroxynaphthalene to scytalone and , , -trihydroxynaphthalene to vermelone [ ] [ ] [ ] ] . tricyclazole alters the conidial color phenotype of a. fumigatus but it does not affect conidial coloration of a. nidulans, a. flavus, and a. parasiticus [ , ] . further studies showed that the conidial pigment biosynthesis in a. fumigatus and a. nidulans possess some steps that are distinct from the general model of dhn-melanin biosynthetic pathway (fig ) [ , ] . instead of producing tetrahydroxynaphthalene (thn) from the starter units, a. nidulans and a. fumigatus utilize malonyl coa and acetyl coa to produce heptaketide naphthopyrone ywa by heptaketide synthase (hks) [ , ] . although ywa is a precursor for green conidial pigmentation in both a. nidulans and a. fumigatus, the downstream metabolic steps are different in the two organisms. in a. nidulans, ya laccase catalyzes ywa to produce the green pigment of conidia. in contrast, a. fumigatus ywa is catalyzed through a hydrolytic polyketide-based shortening step to produce , , , -thn by ayg [ ] . a homolog of a. fumigatus ayg is also presents in t. marneffei genome (see s fig) . as the addition of tricyclazole affected conidial color of t. marneffei, it suggests that hydroxynaphthalene reductases function in dhnmelanin synthetic pathway (fig ) . similar to a. fumigatus, ywa should be converted to , , , -thn in t. marneffei. then , , , -thn is processed through additional steps to produce , -dhn. finally, , -dhn is polymerized by pbrb laccase to form dhn-melanin, which appears as the green color of t. marneffei conidia (fig ) . melanization of t. marneffei has been described previously, however the type of melanin was not determined [ ] . in this study, we found that pbrb encodes a laccase enzyme expressed during conidiation. pbrb is required for the synthesis of dhn-melanin in conidia and when deleted results in brown conidiation, in contrast to the green conidiation of wild type. the existence of additional uncharacterized multicopper oxidase-encoding genes in the t. marneffei genome suggests that in addition to dhn-melanin, t. marneffei may also have the capacity to produce dopa-melanin depending on growth conditions and supply of precursors. [ , , ] . processing steps of the well-known dhn pathway are presented with dark arrows. pks, polyketide synthase; hnr, tetrahydroxynaphthalene reductase; sd, scytalone dehydratase; hnr, trihydroxynaphthalene reductase; vd, vermelone dehydratase; , , , -thn, tetrahydroxynaphthalene; , , -thn, trihydroxynapthalene; , -dhn, dihydroxynaphthalene. tc, tricyclazole, can inhibit both reductases ( hnr and hnr) presented in a model pathway. distinctive steps described in a. nidulans (an), a. fumigatus (af), t. marneffei (tm) are shown with dashed arrows. asterisks, * and **, refer to data from previous study [ ] and this study, respectively. doi: . /journal.pone. .g phylogeny and nomenclature of the genus talaromyces and taxa accommodated in penicillium subgenus biverticillium penicilliosis in children without hiv infectionare they immunodeficient? insights into the pathogenicity of penicillium marneffei penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects control of morphogenesis in the human fungal pathogen penicillium marneffei melanin synthesis in microorganisms-biotechnological and medical aspects fungal laccases-occurrence and properties color me bad: microbial pigments as virulence factors synthesis and assembly of fungal melanin function and molecular evolution of multicopper blue proteins phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences laccase: microbial sources, production, purification, and potential biotechnological applications multiple multi-copper oxidase gene families in basidiomycetes-what for? curr genomics effect of the laccase gene, cnlac , on virulence of cryptococcus neoformans biosynthesis of fungal melanins and their importance for human pathogenic fungi molecular characterization of the aspergillus nidulans ya locus a developmentally regulated gene cluster involved in conidial pigment biosynthesis in aspergillus fumigatus characterisation of the laccase-encoding gene abr of the dihydroxynaphthalene-like melanin gene cluster of aspergillus fumigatus cryptococcus neoformans laccase catalyses melanin synthesis from both d-and l-dopa. microbiol melanization of penicillium marneffei in vitro and in vivo. microbiology detection of dopa-melanin in the dimorphic fungal pathogen penicillium marneffei and its effect on macrophage phagocytosis in vitro strategies for the molecular genetic manipulation and visualization of the human fungal pathogen penicillium marneffei melanins and their importance in pathogenic fungi melanin biosynthesis inhibitors (mbis) for control of rice blast the second naphthol reductase of fungal melanin biosynthesis in magnaporthe grisea: tetrahydroxynaphthalene reductase area controls nitrogen source utilisation during both growth programs of the dimorphic fungus penicillium marneffei an ste homolog from the asexual, dimorphic fungus penicillium marneffei complements the defect in sexual development of an aspergillus nidulans stea mutant new tools for the genetic manipulation of filamentous fungi phagocytosis and killing of human pathogenic, penicillium marneffei and non-pathogenic, penicillium citrinum by mouse macrophage j . cells rapid identification of penicillium marneffei by pcr-based detection of specific sequences on the rrna gene membrane topology of murine coronavirus replicase nonstructural protein a basic helix-loop-helix protein with similarity to the fungal morphological regulators, phd p, efg p and stua, controls conidiation but not dimorphic growth in penicillium marneffei the fungal type ii myosin in penicillium marneffei, myob, is essential for chitin deposition at nascent septation sites but not actin localization combined sequence and structure analysis of the fungal laccase family basic and applied features of multicopper oxidases, cueo, bilirubin oxidase, and laccase laccases: a never-ending story mega : molecular evolutionary genetics analysis version . induction and transcriptional regulation of laccases in fungi melanin is an essential component for the integrity of the cell wall of aspergillus fumigatus conidia dopa and dhn pathway orchestrate melanin synthesis in aspergillus species the effects of tricyclazole, pyroquilon, phthalide, and related fungicides on the production of conidial wall pigments by penicillium and aspergillus species the melanin biosynthesis gene of alternaria alternate can restore pathogenicity of the melanin-deficient mutants of magnaporthe grisea , -dihydroxynaphthalene (dhn)-melanin biosynthesis inhibitors increase erythritol production in torula corallina, and dhn-melanin inhibits erythrose reductase isolation and molecular characterization of the aspergillus nidulans wa gene identification of a polyketide synthase gene (pksp) of aspergillus fumigatus involved in conidial pigment biosynthesis and virulence site of inhibition by tricyclazole in the melanin biosynthetic pathway of verticillium dahliae characterization of melanin pigment produced by aspergillus nidulans the developmentally regulated alb gene of aspergillus fumigatus: its role in modulation of conidial morphology and virulence pentaketide melanin biosynthesis in aspergillus fumigatus requires chain-length shortening of a heptaketide precursor hydrolytic polyketide shortening by ayg p, a novel enzyme involved in fungal melanin biosynthesis the authors acknowledge members of the andrianopoulos lab, department of genetics, university of melbourne, and vanittanakom lab, department of microbiology, chiang mai university for helpful discussions and providing the necessary facilities and research training. competing interests: the authors have declared that no competing interests exist.supporting information s fig. patterns of copper binding sites in t. marneffei mcos. sequence alignments showing the general copper signature sequences (l -l ) found in fungal laccases [ , ] . site of the first amino acid of each sequence is indicated next to pmaa number. the copper binding residues are highlighted and note the type of copper ( , , and ). asterisk is a potential proton donor. type copper ligand (m/l/f) superscripted with i/ii/ii refers to redox potential class from low to high [ ] . genomic region in t. marneffei (tm) from gene pmaa_ to pmaa_ , which encompasses a cluster of genes, predicted to be required for conidial pigment synthesis. genes required for conidial pigment biosynthesis are colored brown (pmaa_ oxidase, abra), blue (pmaa_ ayga), orange (pmaa_ , , , -tetrahydroxynaphthalene reductase, arpb), aqua (pmaa_ scytalone dehydratase, arpa), red (pmaa_ laccase, pbrb) and green (pmaa_ polyketide synthase, wa). (b) a. fumigatus gene cluster involved in dhn melanin synthesis. this gene cluster is conserved in a. fumigatus (af) (afug_ g , abr ; afug_ g , abr ; afug_ g ayg ; afug_ g arp ; afug_ g arp and afug_ g alb ).(tif) key: cord- -f hjy authors: morgan, b. paul; harris, claire l. title: complement, a target for therapy in inflammatory and degenerative diseases date: - - journal: nat rev drug discov doi: . /nrd sha: doc_id: cord_uid: f hjy the complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. in this review, we describe the history, current landscape and future directions for anti-complement therapies. supplementary information: the online version of this article (doi: . /nrd ) contains supplementary material, which is available to authorized users. the complement system includes more than component proteins, regulators and receptors, present in plasma and on cells, which collaborate to provide defence against infection and to clear toxic materials. complement is a key part of innate immunity and a modulator of the adaptive immune response; inherited deficiencies in complement proteins predispose individuals to bacterial infection and/or immune complex disease. the powerful cell-targeting and cell-killing properties of complement can be turned against self, a scenario that is prevented by the abundant expression of regulator proteins. in health, these regulator proteins maintain a fragile truce in which complement is continuously activated in what is known as tickover. bacteria or other foreign surfaces lacking regulator proteins provoke massive activation and amplification of complement, generating activation products that label the invader, attract and activate phagocytic cells and directly cause lytic pathogen killing. complement comprises a cluster of activation pathways that are triggered in various ways and progress through several amplifying enzymes to converge on a final common cell-killing pathway , . surface-bound antibodies trigger the classical pathway, bacterial sugars initiate the lectin pathway and amplification is provided by the alternative pathway, regardless of initiating route (fig. ) . fluid-phase regulators prevent uncontrolled activation in plasma, whereas membranebound regulators restrict activation on cells. the protective (and pathological) effects of complement are mediated by its activation products -active fragments or complexes of complement proteins. c , the most abundant complement protein, has numerous activities; some c fragments (for example, c b, ic b and c dg) can covalently bind to targets and act as flags to instruct phagocytes to bind to and engulf the targets, or they can enhance humoral immunity by facilitating b cell activation. the small c a fragment, among its many roles, can attract and activate phagocytes. downstream of c , the terminal pathway releases the proinflammatory fragment c a and assembles a pore in target membranesthe membrane attack complex (mac) -that effects the lysis of bacteria and other activating cells. the list of diseases in which complement has a role, either primary or secondary to other triggers, is long and growing (table ) . genetic deficiencies in complement components are relatively rare and associated with infections and immune complex diseases (box ) ; however, excessive or dysregulated complement activation contributes to many inflammatory, autoimmune and degenerative diseases, and is a much larger problem , . in a minority of these diseases, genetic changes, mutations or polymorphisms in complement proteins, regulators or receptors are the direct cause of dysregulation and disease; in most of these diseases, complement is not the primary cause but is recruited in response to tissue damage and serves to amplify and exacerbate inflammation and injury. crucially, a small but growing group of conditions are now widely recognized as diseases caused by complement dysregulation, presenting obvious targets for complement-inhibiting drugs , . lessons from figure | targets for inhibition in the complement pathway. the figure shows a highly simplified view of the complement system and highlights the targets for pathway inhibition. activation is triggered through classical (antibody) or lectin (sugar) pathways that rapidly converge to form a complement c -cleaving enzyme (c convertase), c b a. the alternative pathway can be independently activated to generate its own c convertase (c bbb) but, more importantly, amplifies activation regardless of trigger. c fragments, both soluble and surface-attached, engage specific receptors on expressing cells to mediate key activities. the c convertase, formed by the recruitment of an additional c b into the c convertase, cleaves c to release a small fragment, c a, which binds to receptors on expressing cells to mediate activation events. formation of c b initiates the membrane attack pathway; sequential recruitment of components c , c , c and c creates a pore in the target membrane -the membrane attack complex (mac) -that can activate or kill the targeted cell. the complement system presents many targets for inhibition with drugs. in the activation pathways these include the initiating complexes and enzymes, the initiators of the alternative pathway loop and the c convertases. in the c -c axis, potential targets include the individual components (for example, c and c ), the activation fragments (for example, c a and c a) and the c convertases. in the terminal pathway, agents might target individual components (such as c , c or c ) or intermediates (such as c b and c b ), block the functional mac pore or inhibit the downstream signalling events that mediate cell activation or destruction. proteins and protein fragments that coat pathogens and dead or dying host cells to label them for recognition and elimination by phagocytes. the principle complement opsonins are c b and ic b. disease-associated polymorphisms and highly penetrant disease-causing mutations in complement proteins have pinpointed the pathways and components driving pathology and guided the development of therapies for the numerous disease indications in which complement has an important role (table ) . the merits of anti-complement therapy in preclinical models of disease have been explored in hundreds of papers; the earliest examples date back more than years and describe the use of cobra venom factor (cvf) to inhibit disease in animal models , . despite this long history, few drugs have entered clinical trials, and fewer still have progressed beyond phase i. the recent interest in complement and disease has been fuelled by a number of factors -the demonstration of genetic associations that firmly link complement to common diseases was a key finding, but the biggest factor has been the success of a single drug, eculizumab (soliris; alexion pharmaceuticals), in an orphan application: treatment of paroxysmal nocturnal haemoglobinuria (pnh) (boxes , ) . pnh was among the first diseases to be treated with anti-complement therapeutics (box ) ; however, pnh is rare, and the identification of a much more common disease with complement dysregulation at its root was required to catapult complement into the spotlight. agerelated macular degeneration (amd) is the commonest cause of blindness in the western world; a perfect storm of genetic, pathological and clinical evidence has demonstrated beyond doubt that complement dysregulation is a crucial driver of inflammation and tissue damage in amd and has catalysed an explosion of interest in controlling complement , . as a consequence, what began as a small industry targeting pnh and a few other orphan diseases has, over the past decade, grown into a major endeavour involving numerous companies ranging from small biotechnology companies to large pharmaceutical organizations. as the field has grown, it has become increasingly clear that there will be no 'one size fits all' solution to complement therapies; agents that are effective in one disease might do nothing in, or even exacerbate, another. rational design and selection of therapies will require in-depth understanding of the way complement works in each disease. in this review, we consider the diverse disease targets, the many intervention points, the challenges associated with drugging these targets, and the multiple approaches to inhibition that are emerging as complement moves into the clinical mainstream. below, before exploring pathology, we briefly revisit physiology. complement is a key part of innate immunity because it interacts with many other processes; for any therapy that seeks to block or modulate complement, the potential effects on these physiological roles must be considered to avoid iatrogenic injury. patients deficient in complement proteins usually present with recurrent bacterial infections. c is a crucial source of opsonins, which label bacteria for removal by phagocytes; deficiency of c is rare and always marked by severe recurrent infections . patients deficient in classical pathway or lectin pathway proteins (c , c , mannose-binding lectin (mbl), mblassociated serine protease (masp ) and masp ) may also present with recurrent bacterial infections, and deficiencies of alternative pathway components or the positive regulator properdin also predispose to infection, although these deficiencies are associated with a strong bias towards gram-negative bacterial infections. deficiencies in terminal pathway proteins specifically predispose to infections with neisseria species, typically those causing meningococcal meningitis or sepsis, as a consequence of the peculiar sensitivity of these pathogens to lysis by the mac. protection against immune complexes. immune complexes comprise antibodies bound to target antigens (cell debris) in a multi-molecular complex. unhindered, immune complexes grow by acquisition and aggregation, eventually becoming large, insoluble aggregates that lodge in capillary beds and trigger inflammation and tissue damage. in healthy individuals, immune complexes activate the classical pathway and become coated with c and fragments of c and c ; these proteins mask antigens in the immune complex and disrupt the lattice, thereby limiting growth of the aggregates and simultaneously providing ligands for the receptors (complement receptor type (cr )) on erythrocytes, which sequester immune complexes, and for the receptors (cr , cr and cr ) on phagocytic cells, which engulf and destroy immune complexes . patients with deficiencies in classical pathway components (in particular c and c ) usually present with an immune complex disease that closely resembles systemic lupus erythematosus (sle), itself a disease of complement dysregulation . priming adaptive immunity. the role of complement in stimulating the adaptive immune response was first demonstrated in complement-depleted mice years ago . antigens coated with complement activation fragments provoke markedly greater antibody responses than do those that are uncoated; the c dg fragment is crucial to this effect, ligating cr on b cells to deliver a powerful co-stimulatory signal that reduces the threshold for b cell receptor triggering and increases the amplitude of the response . the smaller fragments of complement activation, c a and c a, enhance the ability of antigen presenting cells (apcs) to present antigens and stimulate t cell proliferation; together, complement products in the inflammatory microenvironment markedly influence t cell activation and the balance between effector and regulatory t cells . other physiological roles. many other roles of complement continue to emerge, including directing haematopoietic stem cells to marrow niches, regulating triglyceride uptake and storage in adipose tissue and the regeneration of damaged tissue after injury and during wound healing . each role in itself could warrant a review; therefore, here we merely highlight that these many roles may be affected by complement inhibitors. anti-complement drugs. anti-complement drugs have the potential to affect each and all of the physiological roles discussed above (fig. ) . it is inevitable that a drug that blocks any of the complement pathways will increase the risk of infections, either non-selectively or for certain groups of organisms. any drug that stops activation of the classical pathway will affect the clearance of immune complexes and apoptotic cells. inhibition of the activation pathways may disrupt an individual's capacity to mount an adaptive immune response, although therapeutic inhibition later in life is less detrimental as adaptive immunity is established and developed in older individuals. anti-complement drugs may also disrupt normal lipid metabolism or interfere with the healing and resolution of injuries. all of these potentially damaging effects need to be considered when deciding whether and when to use an anti-complement drug and which part of the system to target. with appropriate prophylactic measures such as immunization and antibiotic therapy, and with careful consideration of the target, most of these effects can be managed. a limiting factor in the use of anti-complement drugs has been an understandable concern regarding the harmful consequences of blocking the important physiological effects of complement described above, which are graphically illustrated in individuals with complement deficiencies. deficiencies in the classical pathway prevent efficient clearance of immune complexes and apoptotic cells, leading to a high penetrance of lupus ( % penetrance in individuals with a c q deficiency and % penetrance in individuals with a c deficiency) , whereas individuals who are deficient in c or components of the alternative pathway or the terminal pathway have recurrent infections that can be life-threatening . although these risks can be managed with good clinical care, a guiding principle for any drug should be to cause as little disruption of physiological roles as possible, particularly if treatment is to be continued in the long term. researchers have devised several strategies to inhibit complement without risking iatrogenic injury: complement can be inhibited late in the pathway, wherein the only infection risk is from neisseria species; complement can be inhibited transiently, which is sufficient for acute conditions, with minimal risk of immune complex disease; or therapy can be delivered directly to disease sites such that systemic inhibition is avoided. these strategies have spawned drugs that have entered the clinic, in some instances with dramatic effect. the c -specific monoclonal antibody (mab) eculizumab prevents mac formation; opsonization is thus unaffected by eculizumab treatment and the risk of meningococcal infection can be mitigated through vaccination and antibiotics. the c blocker compstatin and an antibody antigen-binding fragment (fab) directed against factor d (fd), administered intravitreally, have undergone clinical trials for amd , . the compstatin derivative apl- (apellis pharmaceuticals; a long half-life form) has just entered phase i trials for add-on to standard-of-care in pnh; it will be interesting to see how systemic administration affects the risk of infection. chimeric agents, comprising a complement ligand-targeting modality linked to a complement inhibitor, localize activity at the disease site. this concept has shown promise in murine models and has been developed as a therapy for pnh , . for any anti-complement drug, short-term inhibition for acute indications in a hospital setting is unlikely to incur serious risk of immune complex disease, and infection risk can be managed with tailored antibiotic cover. specific issues for complement-targeted drugs dosing. experience gained from the first few drugs entering the clinic has added knowledge and identified issues that are specific to complement-targeted therapies. one major issue is dosing; most complement proteins are abundant in plasma and turn over rapidly, so adequate dosing of an inhibitor can be challenging. for example, c is present in plasma at ~ g per l, and ~ - % of total plasma c (~ - mg) is consumed and replaced daily simply through the normal tickover process . it is therefore obvious that large doses of complement-targeting drugs and frequent administration will be needed to block complement at the level of c . dosing is an issue for current drugs; the cinryze (a plasma-derived c inhibitor; shire pharmaceuticals) dose for prophylaxis in hereditary post-infection hus clinical and therapeutic *the table comprises an incomplete list of the many diseases in which complement has a role; the diseases are grouped by process, although these groupings are porous and many diseases fit more than one category. ‡ clinical indicates evidence from human studies including biomarker and pathological findings; genetic indicates evidence from gene linkage studies; models indicates preclinical evidence of complement involvement from animal models; and therapeutic indicates evidence from use of a therapeutic (in humans or in animal models). ahus, atypical haemolytic uremic syndrome; anca, anti-neutrophil cytoplasmic antibody; ards, adult respiratory distress syndrome; copd, chronic obstructive pulmonary disease; iga, immunoglobulin a; mpgn, membranoproliferative glomerulonephropathy; pnh, paroxysmal nocturnal haemoglobinuria; ttp, thrombotic thrombocytopenic purpura. , , whereas in atypical haemolytic uremic syndrome (ahus) the maintenance dose is , mg every weeks. these huge and frequent doses contrast starkly with agents targeting cytokines, which are released de novo in disease and at much lower levels; for example, the tumour necrosis factor (tnf)-specific mab adalimumab (humira; abbvie) is effective in rheumatoid arthritis at a dose of mg every weeks . the plasma c concentration is ~ mg per l and the turnover rate is ~ hours ; as a consequence, even with such high doses, breakthrough activity can occur in some patients treated with eculizumab and monitoring of complement activation in plasma is required . alexion have recently launched two new agents that are in phase i trials (alxn and alxn ; 'next-generation' eculizumab molecules) that were probably developed to overcome some of the issues with eculizumab. c is not limiting in the complement cascade; therefore, inhibition at the c stage requires near-complete blockade or depletion of c ; these threshold kinetics necessitate large doses to achieve an effect. approaches to circumvent the problem of high levels of protein turnover have been described, using drugs that only bind to activated forms of complement proteins, which are present at much lower concentrations in plasma , ; these approaches show potential but have not been tested in humans. apart from the problems arising from the sheer abundance of complement proteins, dosing is complicated because plasma complement levels vary widely in the population and because many are acute phase reactants, with synthesis increasing markedly in inflammation, which sometimes causes plasma levels to rise even in the face of increased consumption . the plasma concentration of fd is relatively low (~ mg per l) but it is turned over extremely rapidly (~ . mg per kg per day) ; it can therefore be predicted that large amounts of a drug will be needed to effectively inhibit fd in vivo. studies in nonhuman primates support this -a mg per kg intravenous dose of a fd-specific fab (lampalizumab, formerly called fcfd s, roche/genentech) inhibited the alternative complement pathway for only hours; in that time the plasma levels of fd increased -fold, which is probably reflective of the retention of fd-lampalizumab complexes in the plasma . lampalizumab, administered intravitreally, is currently in phase iii trials in amd. owing to their chemical nature, small-molecule anticomplement agents tend to have short half-lives in vivo. this is not necessarily a limitation, as full coverage can often be achieved through repeated dosing in situations of long-term therapy. daily subcutaneous dosing is manageable and is likely to be acceptable for patients with life-threatening diseases; oral bioavailability can often be achieved with these agents, which is a key advantage. in some conditions -such as traumatic brain injury, myocardial infarction, transplantation, stroke and other ischaemia-reperfusion injuries -transient complement inhibition for a few hours may be sufficient to prevent tissue damage. in these cases, small molecules have an added benefit in that once dosing is ceased, rapid clearance leads box | genetics dictate the risk of complement-mediated injury complement comprises a large number of interacting components and regulators, and common genetic variation in these proteins dictates whether a given individual possesses a complement system that is more or less active. we have reviewed this complotype concept previously , and here we focus on its relevance to complementmediated diseases and therapy. put crudely, individuals who have inherited a more active complotype are better able to resist infection but are at greater risk of complement-driven inflammation. some variant proteins directly affect the activity of the amplification loop (for example, complement c r g (c -r g) and factor b r q (fb-r q)) , and this is the primary reason for disease association, whereas others influence control local to the disease site, and risk is thus disease-or tissue-specific. this concept is well-illustrated by considering age-related macular degeneration (amd): the most important genetic risk factor in amd is a common polymorphism in the alternative pathway regulator fh that changes a single amino acid in the middle of the molecule (y h). the risk of developing amd is at least threefold greater in his-variant homozygotes than in tyr homozygotes , . the y h variant influences the localization of fh to retinal ligands, whereas other complement polymorphisms (including those in fb and c ) influence amd risk by increasing complement alternative pathway activity [ ] [ ] [ ] ; common variants in just these three proteins together alter amd risk up to -fold. in deciding whether and how to inhibit complement for therapy in an individual, the complotype should be an important consideration. mutations in complement components and regulators can also affect complement dysregulation and disease risk. again, fh provides the best example. fh is a large, elongated molecule comprising a chain of short consensus repeats. the th short consensus repeat (scr ) is a hotspot for mutations that may be single amino acid substitutions, premature stops or more complicated changes; many of these mutations are strong risk factors for another disease of complement dysregulation, atypical haemolytic uremic syndrome (ahus), which is typified by thrombosis, haemolysis and renal injury , . onset of ahus typically occurs in childhood or young adulthood and the frequency and severity of episodes is highly variable, with some patients having an indolent course requiring no therapy and others rapidly progressing to end-stage renal failure. all of the mutations in the scr domain of fh that are associated with ahus disrupt the capacity of fh to bind to and regulate complement on cells and other surfaces; regulation in the fluid phase is unaffected. numerous other complement protein mutations have been described and shown to influence the risk of ahus and/or other diseases in which complement plays a part. many of these are coding mutations that change the amino acid sequence and affect protein activity, others are non-coding mutations and probably affect the biosynthesis and expression of the protein; some mutations trigger fh-specific autoantibody production . an understanding of the influence of polymorphisms and mutations in complement proteins on complement activity in a particular disease is essential for the intelligent design and selection of therapeutic approaches. the figure summarises the effect of gain-of-function and loss-of-function polymorphisms and mutations in complement proteins and regulators that alter the risk of disease. to speedy recovery of complement activity, restoring its infection-fighting and tissue-repairing properties. when developing small-molecule drugs for specific targets, their mode of action is crucial and target tractability becomes a key consideration; in some instances, the small size of the drugs may become a limitation, for example in blocking protein-protein interactions (see below). when a drug with a longer half-life is needed, agents such as antibodies come to the fore. alternatively, small molecules may be modified to increase their mass by pegylation or by coupling to domains that extend their half-life in vivo: for example, albumin-binding or antibody crystallisable fragment (fc) domains , . in some diseases, complete inhibition of complement may not be needed for efficacy; agents such as soluble cr (scr ; also known as tp , celldex therapeutics) that downmodulate the complement enzymes through dynamic activities (such as accelerating their decay or increasing cofactor availability) can mediate dosedependent partial inhibition of complement rather than the sharp threshold effect typical of blocking antibodies , . such agents may enable therapy at scalable doses and in a safe and effective manner; however, the fact that even large biological agents such as scr have relatively short half-lives will affect the choice of indication. getting to where the action is. many pharmaceutical companies are now focused on the development and manufacture of biologics because of their historical success, good economic return and low rate of attrition. as a consequence, most anti-complement agents developed to date are biologics -either antibodies or recombinant proteins -that deliver inhibitory activity. essential to all drugs is their ability to get to the sites of disease; some complement-mediated diseases manifest in the vasculature (for example, pnh and vasculitis), whereas in other diseases (such as amd, neuromyelitis optica and arthritis) complement attacks tissues at sites remote from systemic circulation. the ability of systemically-administered drugs to diffuse to the site of tissue damage and inflammation will thus be essential for treatment success. the liver produces most of the complement components found in the circulation, and if diffusion of circulating components to disease sites drives inflammation and disease then systemic depletion or blocking of specific complement components may ameliorate the pathology. however, locally produced complement is known to have key roles in some indications. for example, in renal allograft rejection, locally produced and activated c drives the t cell response and immune rejection ; in this case, systemic anti-c therapy will be ineffective unless the drug gets into the tissues. similar challenges exist in organs protected by tight barriers, such as the nervous system and the eye. in some diseases these barriers become damaged and leaky with disease progression, but it is likely that complement activation contributes to the early, pre-inflammatory stages of eye and brain diseases, so to be of value complement --inhibiting drugs are required in the relevant organ early in the disease when the barriers are still intact. assuming that they have good bioavailability, small-molecule drugs may have advantages over biologics in these contexts because of their higher tissue penetrance; however, of the numerous small-molecule anti-complement agents that have been developed and tested in humans, none has yet progressed to the market [ ] [ ] [ ] . getting in the way. complement is a good target for biologics because the cascade involves multiple proteinprotein interactions and conformational changes, exposing large surfaces that lend themselves to large blocking box | the development of eculizumab for the treatment of paroxysmal nocturnal haemoglobinuria the first description of functional blocking of complement c with a monoclonal antibody (mab) dates back almost years; a c -specific mab, bb . , generated in c -deficient mice, blocked haemolysis in mouse serum . bb . provided a powerful proof of principle for inhibition of the membrane attack complex (mac) that was effective in numerous mouse models ; bb . also provided a rationale for the development of blocking mabs specific to human c . one such mab, g . , was selected by alexion, humanized and further manipulated to generate fab (antigen-binding) and scfv (single-chain variable) fragments , . by , humanized g . , termed eculizumab, was in early clinical trials in a range of joint, kidney and skin conditions ; however, its remarkable efficacy in paroxysmal nocturnal haemoglobinuria (pnh) focused efforts in that area . pnh is a clonal haematological disorder that involves chronic intravascular haemolysis, with complement lysis of glycosylphosphatidylinositol (gpi)-anchor-deficient erythrocytes as its principal feature. patients with pnh are anaemic with resultant lethargy and at risk of thrombosis. prior to the use of eculizumab, treatment involved frequent blood transfusions and symptomatic support. eculizumab transformed the outlook for patients with pnh, preventing haemolysis, reversing anaemia and removing transfusion dependence in most patients, thereby improving quality of life . eculizumab therapy is effective over the long term (> years), and substantially reduces mortality . the only other disease for which eculizumab is currently approved by the us food and drug administration (fda) is atypical haemolytic uremic syndrome (ahus). as with pnh, there were no specific therapies for this potentially catastrophic disease and the evidence implicating complement was overwhelming. in patients with ahus, eculizumab treatment increased platelet number and improved renal function over the course of year and some patients were no longer dependent on dialysis . numerous other clinical trials in various conditions are in progress, and these are summarized in table . a major concern of governing bodies in the united kingdom and elsewhere is cost; at the time of writing, standard pnh maintenance therapy ( mg eculizumab delivered intravenously every weeks) costs approximately uk£ , per patient per year; therefore, even for a rare disease with only ~ cases in the united kingdom, the estimated cost to the national health service (nhs) is £ million per year. a concise and up-to-date summary of the clinical data can be found on the electronic medicines compendium website (see further information). it should be noted that an eculizumab biosimilar is already being developed by epirus biopharmaceuticals. drugs . by contrast, for a small molecule to block a protein-protein interaction it must target crucial sites and specific surface features -for example, charged or hydrophobic pockets . the wealth of structural information now available in the field, including snapshots of convertase enzymes and mac precursor complexes captured in active conformations, unmasks the precise nature of these protein-protein interactions and identifies sites that are key to the interaction that can be targeted with small molecules or biologicals using structure-based drug design , . alternatively, the expansive small-molecule libraries that are now ubiquitous in pharmaceutical companies have been successfully screened to reveal agents that bind to and inhibit interactions within the cascade through competitive or allosteric mechanisms , . small peptides selected from such libraries for binding components or inhibiting complexes are now numerous in the literature; for example, the cyclic peptide compstatin, which blocks c from binding to the convertase, has already entered clinical trials , . target validation in models. validation in rodent models of disease can be challenging or even misleading, and care needs to be taken to ensure that the disease mechanism in the chosen model is the same as that in the human disease. to illustrate the differences in complement systems between mice and humans, consider the receptors for c fragments. in humans, cr and cr are encoded by separate genes and have different roles: cr binds to c dg and enhances humoral immunity whereas cr acts as a cofactor for the fi-mediated cleavage of c b and has a key role in transporting immune complexes . in mice, cr and cr are generated from the splicing of one gene and have limited expression; the main cofactor for c b cleavage and generation of c dg in the mouse is cr -related gene/ protein y (crry), a broadly expressed protein absent in humans . therefore, mice and humans respond to c fragments through different receptors and with different outcomes that will probably alter the effects of c -targeted drugs. nevertheless, in many models, disease mechanism and response to complement inhibition translate well and proof-of-principle is readily obtained. with current efforts to reduce animal use in scientific research, initiatives are emerging to better validate drug targets at an early stage of development without the use of animals, for example, using technologies such as microfluidic organs-on-chips . what does the current toolbox contain? before describing specific tools, we first provide some guiding principles and suggestions for the classification of anti-complement agents. there are several discrete ways in which an agent might act to influence complement (fig. ). an agent might inhibit progression in the activation or terminal pathways, act as an antagonist of an activation product, or cause activation and consumption of complement. another broad distinction can be drawn between those drugs that inhibit complement systemically and those that are directed to specific sites of pathology to localize their inhibitory activity. anti-complement drugs can also be divided into categories based upon their molecular characteristics: purified or recombinant forms of naturally occurring human regulators; modified protein copies or mimics of natural regulators; copies of pathogen-derived regulators; mabs or fragments of mabs; naturally occurring or synthetic small-molecule inhibitors; or antisense or similar nucleic acid constructs. finally, anti-complement agents can be grouped according to the part of the system they target, a convention that we follow in this review. targeting the binding and assembly of the initiating complex. initiation of classical or lectin pathway activation involves the recognition of an activating surface; in the classical pathway this activation occurs through a surfacebound antibody that ligates c q, whereas in the lectin pathway this occurs via specific sugars on the surface that bind to mbl or collectins (fig. ) . agents that block these events would switch off the respective activation pathways at the very first step, an attractive prospect given the amplifying nature of the complement system . a potential classical pathway inhibitor might block c q-binding sites on the antibody or the antibody-binding sites on c q. a lectin pathway inhibitor might mask surface mbl-binding sugars or the sugar-binding sites on mbl. in either case, the agent would need to bind the target with higher affinity than the physiological ligand to compete effectively. alternatively, an agent could disrupt or prevent the assembly of the multi-molecular complex, which comprises the recognition unit (c q and mbl in the classical and lectin pathways, respectively) and the associated enzymes (c r and c s in the classical pathway and masp and masp in the lectin pathway) that are essential for pathway activation . both complexes circulate preassembled in plasma, so any agent targeting the complex would need to dislodge the bound enzyme to be effective; surprisingly, the c s-and masp -targeting mabs from true north and omeros, respectively (described below), both achieve this effect. another approach to inhibiting activation of the complexes would be to develop a molecular lock that restricts the conformational changes in c q or mbl that occur upon ligand binding and are essential for activation of the associated enzymes. such approaches have been used successfully to develop small-molecule inhibitors of chemokine receptor activation , , but have not yet been reported for c or the mbl-masp complex. several c q function-blocking mabs have been reported and were recently shown to be effective in a murine model of the antibody-mediated demyelinating disease, neuromyelitis optica . numerous peptide blockers of antibody-c q interactions have been described, derived from the sequences of c q binding sites in the antibody, c q receptors or c q itself, or selected from libraries. one such peptide blocker, termed peptide j, is a powerful inhibitor of the classical activation pathway in multiple species ; however, there have been no further reports of this agent. other peptides have been described that interfere with the binding of c r and c s in the c complex, but none has progressed beyond in vitro studies. the binding of mbl to specific sugar residues on surfaces presents a potential drug target, but no agents acting in this manner are reported. a recent publication describes a virus-derived peptide, peptide inhibitor of complement c (pic ), that binds to the collagenous regions in both c q and mbl and inhibits both the lectin and classical pathways in vitro and in vivo in rodents . targeting the enzymes of the initiating complexes. the enzymatic activity of the classical or lectin pathwayinitiating complexes is provided by the serine proteases c s and masp , respectively. in the c complex, immunoglobulin binding induces conformational changes in c q, triggering auto-activation of c r that in turn cleaves and activates c s in the same complex. the precise steps involved in activation of the mbl-masp complex are less clear but probably involve a similar multi-step process . the serine protease enzymes in these complexes are potential targets for pharmacological inhibition. both the pharmaceutical industry and nature itself have invested heavily in the development of serine protease inhibitors (spis), and numerous spi drugs have been made, of differing specificities and compositions, to target the myriad serine proteases essential for digestion, haemostasis, immunity, reproduction and many other physiological and pathological processes. the biggest problem in designing an spi drug to inhibit a particular protease is specificity -almost all spi drugs have some 'off-target' effects that can limit their use. even nature struggles -the sole natural inhibitor of c r and c s is an spi, c inhibitor (c inh; also known as serping ), but c inh also controls masps and proteases in the coagulation and kinin systems. indeed, deficiency of c inh leads to dysregulation in all these proteolytic cascades that, in aggregate, cause the disease hae , . c inh removes activated c r and c s from c q to form a stable complex in which c inh itself is cleaved and inactivated. c inh has a long history of use as a drug; indeed, it can claim to be the 'first-in-man' complement drug and the first of the natural inhibitors to be recognized as a potential therapeutic. more than years ago, patients with hae were shown to be deficient in c inh and to respond to plasma replacement, provoking efforts to purify c inh for treatment of acute attacks , . remarkable successes in these early studies rapidly led to the adoption of plasma-derived c inh as the standard of care for acute episodes in hae. the obvious advantage of this approach is that a plasma-derived molecule should be low risk (once infectious agents are eliminated) and non-immunogenic. the widespread availability of industrial-scale protein-fractionation facilities -established for the production of immunoglobulins, albumin and other plasma products -made this a viable approach for purifying c inh. unfortunately, concerns about viral transmission in plasma-derived proteins have led to its removal from the clinic in several countries, including the united states. recent methodology improvements and new approaches to production of c inh have changed this unsatisfactory situation; two ultra-pure plasmaderived c inh products, cinryze and berinert (csl behring) are now approved by the us food and drug administration (fda) and other regulatory agencies. some companies invested in better methods of purifying plasma c inh, whereas others set about making it. a fulllength recombinant c inh was first reported a decade ago. this agent, termed ruconest (salix pharmaceuticals), has been approved for therapy of acute attacks in europe and, very recently (july ), also in the united states. early administration of c inh reduces the duration and severity of acute attacks, findings confirmed in the large impact study of over , attacks . its use in prophylaxis is more controversial; a subset of hae patients have frequent and severe attacks despite therapy with danazol (an anabolic steroid that increases c inh synthesis) and/or ε-aminocaproic acid (a protease inhibitor) or cannot tolerate these agents. regular intravenous administration of c inh (every - days) to restore plasma levels is an effective therapy in this group , ; however, in the united states it is now becoming accepted practice to treat all hae patients in this manner, a practice limited elsewhere by cost and lack of evidence base . berinert has been approved for on-demand, patientadministered therapy in the united states and europe. given its long history and known safety and efficacy in hae, it is surprising that c inh has not been more widely used in other conditions characterized by complement dysregulation. there are a number of published studies, mostly small, demonstrating that administration of c inh reduces mortality in sepsis, and in some studies the effect is startling , . other studies have reported positive effects of c inh in myocardial infarction and in ischaemia-reperfusion injuries in both animal models and humans, but no large-scale clinical trials in these other applications have been published , . several broad-spectrum protease inhibitors already in clinical use have c r, c s and masps among their targets; perhaps the best example of these is nafamostat mesilate (also known as futhan or fut- ), a small-molecule blocking protein-protein interactions offers numerous targets for drugs in the complement system; approaches may involve blocking the formation of an essential complex or receptor-ligand interaction, preventing a key conformational change or inhibiting a crucial enzymatic cleavage. such activities underpin many of the current and evolving therapeutics, particularly the monoclonal antibody (mab)-based agents. several of the mabs that target c convertase work in this way; either they prevent the complex from forming at all or they lock it in an inactive conformation. similarly, the staphylococcus aureus-derived inhibitor scin (staphylococcal complement inhibitor) holds the convertase in an inactive state . the c -specific mab eculizumab binds to c remote from the cleavage site, at a site in the α-chain that prevents c from binding to c convertase , , . human experimental proof was recently provided: a c polymorphism, present in ~ . % of the japanese population, caused a single residue change (r h) in the putative eculizumab binding site and rendered the protein refractory to inhibition . in r h heterozygotes treated with eculizumab, half of the circulating c remained active and paroxysmal nocturnal haemoglobinuria (pnh) therapy was ineffective. blocking protein-protein interactions using small molecules has attracted less interest because of the perceived difficulties of successfully interfering with large protein-protein interfaces ; for example, the footprint of the factor h regulatory domains on the c convertase extends over an enormous , Å (ref. ). nevertheless, some success has been achieved with peptides or other small-molecule inhibitors; the best example is that of compstatin -a amino acid cyclic peptide that inhibits c -which, over the course of almost two decades, has been modified into a highly potent high-affinity inhibitor of c cleavage , , [ ] [ ] [ ] . compstatin binds to native c and prevents its interaction with the c convertase, thus blocking further complement activation. in vivo testing of compstatin has been limited by its specificity for human and primate c ; in primates, compstatin reduced drusen load and inhibited disease in an age-related macular degeneration (amd) model . alcon (a novartis company) has taken a compstatin analogue, pot- (al- a), through phase i trials; phase ii trials in amd, which started in , are yet to report any results (see the alcon website in further information) . for their compstatin analogue, amy- , amyndas has gained european medicines agency (ema) and us food and drug administration (fda) orphan drug status for use in pnh. apellis pharmaceuticals are developing a compstatin derivative in an inhaled short-acting form (apl- ) for the treatment of asthma and chronic obstructive pulmonary disease (copd) (currently in phase i as a disease modifying therapy for copd). this derivative is also being developed in a long-acting form (apl- ) for the treatment of haemolytic disorders and amd; apl- is currently in phase i for pnh as an add-on to eculizumab therapy (clinicaltrials.gov identifier: nct ), and a phase i trial has recently started for apl- as an intraocular therapy for amd (nct ). protease inhibitor used to treat disseminated intravascular coagulation and acute pancreatitis that affects multiple plasma protease systems . however, the many off-target effects and resultant toxicity of nafamostat mesilate make it a poor candidate for regulating complement in vivo. other small-molecule inhibitors of c s of varying degrees of specificity and strength have been described, including c s-inh- (basf pharma) and bcx- (biocryst pharmaceuticals), both no longer in development, and recently a family of biphenylsulphonyl thiophene derivatives that, when pegylated, displayed strong c s inhibition and promising pharmacokinetics . modern methods of structure-based design offer the prospect of much more selective smallmolecule inhibitors of the initiating complex enzymes, as demonstrated for other proteases . indeed, the 'directed evolution' of a sunflower-derived peptide, trypsin inhibitor , created specific inhibitors of masp and masp that await testing in vivo . an inhibitory mab against c r and c s or masps might address the specificity issue that limits the clinical use of small-molecule agents. a mab against c s, tnt (true north therapeutics), prevented erythrocyte haemolysis in an ex vivo study of autoimmune haemolytic anaemia ; a humanized analogue of this mab, tnt (true north therapeutics), is being fast-tracked to clinical trials in this disease. a recombinant mab fab was recently described that bound c s with nanomolar affinity and potently inhibited classical pathway activation, but so far this has only been tested in vitro . a masp -blocking human mab (oms ), which is an effective and long-lived (up to a week) lectinpathway inhibitor that is delivered intravenously or subcutaneously, was recently awarded us fda orphan drug status for use in ahus and other thrombotic angiopathies and is currently in phase ii/iii trials (nct ). a recent (august ) omeros press release described positive outcomes in ahus from this trial. targeting the c convertases. the c -cleaving enzymes of the classical, lectin and alternative pathways, c b a and c bbb, are assembled in quite different ways despite their functional and structural similarities (fig. ) . in the classical and lectin pathways, c is cleaved by activated c s (or masp ) in the surface-bound c (or mbl-masp) complex; the large c b fragment binds to adjacent surfaces via a nucleophilic attack on a thioester group, captures c and presents it for cleavage by c s or masp as above. the c b a complex is the c convertase of the classical and lectin pathways. c a is a serine protease that cleaves c to form c a and the opsonin c b; c a can also cleave c (when properly presented) to form c a and c b. these two complement proteins are the sole substrates of the c a serine protease. assembly of the alternative pathway c convertase requires c b, generated through classical and lectin pathway activation or from tickover activation on surfaces. c b binds to fb, triggering major conformational changes that expose fb to cleavage by fd, a unique plasma serine protease that has fb as its sole substrate. the c bbb complex is the c convertase used in the alternative pathway; the bb fragment, generated from fb cleavage, is a serine protease that cleaves c and c in precisely the same manner as c a in the classical pathway. the key amplifying role of the alternative pathway makes it a very attractive source of drug targets that can be targeted in a variety of ways (fig. ) . it is obvious from the above description that the c convertases present a dizzying array of opportunities for regulation. agents might target the assembly of one or more of the complexes, preventing complex formation and/or causing complexes to break up. they might target one or more of the serine proteases responsible for formation of the convertase (c r, c s, masp or fd), convertase activity (c a or bb) or convertase regulation (fi). alternatively, they might target the substrates of these enzymes, c or c , by blocking binding to or cleavage by the convertase or acting as molecular locks to prevent conformational changes essential for activation. agents might also target active fragments (c a, c a, c b or c b) to block their downstream effects. indeed, nature has adopted some of these strategies to keep complement in check. the convertases are naturally labile and break up in a matter of minutes; regulatory proteins that accelerate this decay exist both in plasma and on membranes, and they function by binding to the complex and displacing the enzyme. some of these regulatory proteins remain bound after decay and act as cofactors for the further cleavage and irreversible inactivation of c b or c b by the plasma serine protease fi. using the endogenous c convertase regulators as drugs or leads. the plasma regulators of the c convertases were obvious targets for drug development given the success of c inh. to date, none of the other plasma regulators has entered the clinic despite a large amount of research and preclinical activity. in vitro studies more than years ago showed that modest (around % above baseline) increases in the concentrations of the complement regulators fh and fi markedly reduced plasma alternative-pathway activity, provoking the suggestion that augmentation of these proteins might be of therapeutic benefit ; however, large (and probably frequent) doses will be required to substantially alter the levels of these abundant proteins. despite this, there has been considerable recent interest in using plasmaderived fh as a therapy, particularly in those diseases strongly linked to fh polymorphisms and mutations. deficiency of fh is rare and always associated with severe c dysregulation in plasma or on cell surfaces that leads to a range of renal diseases, including c glomerulopathy and ahus; fh deficiency has been successfully treated with plasma exchange, strongly suggesting that fh would be an effective therapy in these diseases . indeed, in fh-deficient mice, therapy with human fh reversed renal injury . amd is strongly linked with the fh y h polymorphism, and this has provoked intense interest in giving at-risk individuals the protective y form of fh to prevent or treat amd. several companies established programmes to produce plasmaderived or recombinant fh with this as the primary aim, but none of these programmes survive. of note, some evidence from mouse models and human studies suggest that fh would need to be administered intra-ocularly in amd to control complement dysregulation locally and alter the course of disease , . in one study, instead of expressing full-length fh for use in therapy, this large molecule was stripped down to its key functional units and re-assembled to create a minimized fh, mini fh, which comprised four amino-terminal, complement-regulatory short consensus repeats and two carboxy-terminal, surface-binding short consensus repeats joined by a short linker. mini fh proved to be an effective complement inhibitor in the fluid phase and on surfaces in multiple in vitro assays . , and it is now in preclinical development for therapy of pnh (amy- ; see the amyndas website). other approaches aimed at targeting fh activity to surfaces are described in later sections. c bp, the fluid-phase regulator of the classical pathway c convertase, is a complex, oligomeric molecule that to date has received little attention in terms of therapeutic applications. c bp deficiencies are extremely rare, restricted to a single case report . in mouse models of arthritis, administration of plasma-derived c bp reduced severity of disease , but no human studies have been described. fi is the key enzyme for regulation of the c convertases and deficiency causes complement consumption and consequent susceptibility to bacterial infections. individuals with an fi deficiency present with repeated infections that may be life-threatening, and even heterozygotes may have an increased infection risk . plasma therapy to replace fi would, by analogy with fh deficiency, be expected to help, although a single small study found no clinical benefit . a recent report showed that fi supplementation reduced complement activity in sera from individuals with more active complotypes, provoking the suggestion that fi therapy would reduce the risk of developing diseases linked to complement dysregulation . properdin is a plasma protein that binds to and stabilizes the alternative pathway convertase; in other words, it acts as a positive regulator . properdin may also act as a pattern-recognition molecule, binding targets and initiating activation of the alternative pathway. deficiency of properdin (x-linked) predisposes individuals to infections by bacteria, particularly neisseria species. supplementation of properdin in individuals who have a deficiency would be expected to reduce infection risk. a recent study suggested a broader role for properdin therapy -low-dose recombinant properdin markedly reduced susceptibility to bacterial infection in a mouse model, raising the possibility that supplementation might be effective in sepsis and other severe infections in humans . the stabilizing role of properdin on the alternative pathway c convertase means that blocking this positive regulator may be therapeutic in inflammatory disease. a mab targeting properdin (nm ) and a small-molecule properdin antagonist have been reported by novelmed therapeutics for use in pnh and rheumatoid arthritis, respectively (see the novelmed therapeutics website). using the membrane c convertase regulators as drugs or leads. the membrane regulators of the c convertases present a different challenge compared to the plasma regulators in that they cannot readily be purified for testing as potential drugs. the only way to test whether a membrane regulator might work as a therapeutic is to make a recombinant soluble form. the first of these, described in , was a soluble recombinant form of cr (known as scr or tp ) comprising the entire extracellular sequence of thirty short consensus repeats (> , amino acids). the protein was a powerful inhibitor of both classical and alternative pathway convertases in vitro in human and rodent sera; pretreatment with scr protected against reperfusion injury in a rat model of myocardial infarction . over the ensuing two decades, this agent provided an excellent proof-of-principle for complement inhibition in diverse models of human disease, effective in a wide range of disease models and in rodents, pigs and primates. after initial development work by smithkline beecham, avant immunotherapeutics took scr into clinical trials in adult respiratory distress syndrome, acute lung injury and cardiopulmonary bypass; although some positive results were described , there were also many failures, notably in large sepsis trials, and scr has not progressed for these indications. more recently, guided by success in animal models, scr entered the amplification (c b feedback) loop is a positive-feedback cycle that consumes complement c to generate more enzyme and activation products; if unregulated, it cycles until all available c is consumed. tight regulation is provided in the plasma by enzymes and cofactors that remove the c fragment c b from the feedback cycle for breakdown into smaller fragments. as a consequence, the c b feedback cycle normally operates at a very low rate (tickover). the balance between activation and regulation is disturbed in disease; a healthy balance can be restored by providing extra control (for example, increasing regulation, such as that provided by the complement regulatory protein factor h (fh), thereby increasing 'feed out' from the amplification loop) or by preventing the formation of c b in the feedback cycle (for example, by blocking convertase enzyme, thereby decreasing 'feed in' to the amplification loop). agents that target amplification of complement can have major therapeutic effects. ba, non-catalytic fragment of fb; c b-fh, complex between c b and fh; cr , complement receptor type ; ic b-fh, complex between inactive c b and fh. adapted with permission from ref. , elsevier. clinical trials in dense deposit disease (cdx- ; celldex therapeutics); regrettably, this study terminated in because of recruitment issues. numerous modified forms of scr have been created to improve its therapeutic potential and/or simplify its production. tp incorporated multiple oligosaccharide sialyl lewis x epitopes to bind to selectins, which are upregulated on endothelia at inflammatory sites. this simple change improved efficacy compared to unmodified scr in rat ischaemia-reperfusion models . the minimum complement regulatory unit from cr was known to be the three n-terminal short consensus repeats. a drug that comprised these three short consensus repeats attached to a nonspecific membrane-binding motif (apt- ; also known as mirococept; adprotech) proved remarkably effective as a complement inhibitor when delivered locally or systemically in rodent and pig models , . the agent was inexpensive to produce and proved safe in human studies. the mrc-funded empirikal trial (uk clinical research network (ukcrn) identifier: ), which is due to commence in , tests the effect of mirococept pretreatment of donor kidneys on their survival and function post-transplant. although recombinant soluble forms of the other membrane c convertase regulators, cd and cd , have been produced and tested in animal models, most have stalled at the early preclinical stage. the exception was a chimeric cd -cd molecule (cab- ; also known as mln- ; millenium pharmaceuticals) that entered phase i trials in cardiopulmonary bypass but was not progressed. there are eight protease targets that could be used for convertase regulation. the classical and lectin pathway initiating-complex enzymes (c r, c s, masp and masp ) are discussed above. generation of the alternative pathway convertase requires fd, and deficiency of fd results in the absence of alternative pathway activity and marked susceptibility to bacterial infections . the alternative pathway has a crucial role as an amplifier regardless of the initiating trigger, making it an attractive target for therapeutic modulation and bringing fd centre stage. the first fd-targeted therapeutic to reach the clinic, lampalizumab, is a humanized immunoglobulin g (igg ) fab fragment that inhibits fd activity by binding to the exosite to prevent substrate binding . a published phase i study of intraocular administration in patients with a severe form of amd termed geographic atrophy (ga) demonstrated safety . phase ii results of monthly treatments, announced but unpublished (mahalo study), describe a % reduction in ga area compared to controls at months; these startling results were even better in patients selected based on an undisclosed fi genetic biomarker, who showed a % reduction at months. phase iii trials of therapy ( mg intravitreal every - weeks) in patients with ga are ongoing. several broad-spectrum small-molecule protease inhibitors, including dichloroisocoumarin, bind to and inhibit fd; structures obtained from these complexes have identified unique properties of fd that offer the prospect of designing specific inhibitors , . novartis recently reported the evolution of small-molecule inhibitors of fd that are active in the nanomolar range . very recently, achillion have announced the development of orally active, potent and specific small-molecule inhibitors of fd that they propose for therapy in pnh. it remains to be seen whether the high natural turnover of fd will limit the use of these small molecules in humans. a number of mabs or mab fragments have been described that inhibit fb cleavage in a variety of ways. one of these, termed mab , has been developed as a therapeutic by taligen ; ta is a fab fragment of mab that blocks formation of the alternative pathway convertase. taligen was acquired by alexion in for us$ m, and the agent is currently under going humanization for further use in humans, with amd and asthma as primary targets. another fb-specific antibody, bikaciomab (nm ; novelmed), is also in development for use in amd, and small molecule inhibitors of fb have been reported (in novartis patents) although there is no evidence yet of their use in preclinical models of disease. isis pharmaceuticals have described the use of fb antisense oligonucleotides for therapy of murine lupus nephritis, but progression of this technique to humans has not been reported . the functional homologue of fb in the classical pathway is c ; despite a more limited role in pathology, c a-specific mabs that block the classical pathway c convertase have been developed and patented by tanox (now part of genentech; patent ref. wo a ) ; no trials are reported on these interesting drugs. an intriguing agent, a linear peptide (rh d) derived from a schistosoma species protein, bound c and inhibited c convertase formation, but has not been further developed to date . individual convertase components and regulators can be targeted in various ways to influence complement activity: components may be depleted in the plasma by increasing their consumption or decreasing their synthesis; regulator concentrations may be increased by supplementation or an increase in synthesis; components and/or regulators may be prevented from undergoing essential conformational changes or enzymatic cleavage; or components may be rendered more susceptible to spontaneous or accelerated decay. one example of altering consumption is illustrated by cvf, a c b analogue present in cobra venom that, when injected into mammals, binds to fb to form a stable c convertase that is resistant to normal regulatory processes and thus activates the system to exhaustion. this activity of cobra venom was recognized over a century ago, and purified cvf was first used as a tool to render the complement system of rodents deficient almost years ago, making it the first anti-complement 'therapeutic' (refs , ) . although the empiric use of cobra venom in therapy has a long history in everything from cancer to cosmetics, cvf as a drug has severe limitations: first because it is immunogenic, which renders repeat injections ineffective, and second because rapid and complete activation of complement by cvf generates high concentrations of c a, causing circulating neutrophils to activate and aggregate in the lungs and other organs . it is also unclear whether cvf can deplete complement locally in tissues. attempts to address immunogenicity through humanization of cvf have created a hybrid that is % identical to human c ; small stretches of cvf-derived sequences in the c α-chain are sufficient to retain cvf-like complement consumption . although immunogenicity may be low, concerns over the toxic effects of continuous consumption of complement by cvf ensure that use will be restricted to acute conditions. supplementation of the plasma convertase regulators offers therapeutic possibilities above and beyond the restoration of plasma levels in rare cases of deficiency. systemic modulation of the cascade using endogenous soluble regulators (or soluble versions of membraneassociated control proteins) is likely to cause downmodulation rather than complete blockade and effect therapy in a manner that is more measured. efforts to modulate complement by administering extra fh or fi are described above. however, the amounts of protein needed to raise plasma levels sufficiently to affect convertase activity may be prohibitive; the feasibility of this approach will require careful dose prediction, and dosing will probably be disease-specific and perhaps individualspecific. gene-therapy approaches to increase local synthesis are also now attracting attention -for example, the introduction of a construct that increases the production of fh or another complement regulator in the retina might prove effective in amd, a disease in which other gene-therapy approaches have already been effective in models and have reached phase i/ii clinical trials . decreased synthesis is the aim of a variety of antisense and related agents targeting complement components either systemically or locally, and these agents are the subject of a number of published patents. however, to date these strategies have only been tested in vitro and (for a minority) in animal models; for example, c -targeted antisense oligonucleotides markedly reduced hepatic synthesis and plasma c levels, and ameliorated disease in a mouse model of traumatic brain injury . c antisense is described in a number of patents over the past years but has not progressed in vivo. alnylam described an rna interference (rnai) molecule targeting c , aln-cc , that reduced plasma c levels by > % when administered monthly by subcutaneous injection to non-human primates; this agent, developed for therapy of pnh, is now in phase i/ii trials (as announced in a recent alnylam press release). targeting the anaphylactic peptides. the polypeptides c a and c a, released owing to the action of the c and c convertase enzymes, are biologically active, and they bind specific receptors on phagocytes and many other cell types to exert effects. the range of cell targets and activities is large and reviewed well elsewhere , but their pro-inflammatory activities are particularly relevant to disease. c a is a powerful inflammatory trigger implicated in many diseases, whereas c a exhibits a complex mix of pro-and anti-inflammatory activities . both c a and c a (and their metabolites) bind to seven-transmembrane-domain g-protein-coupled receptors (gpcrs), which are members of the large cytokine and chemokine receptor family. similar to other gpcrs, those that bind c a and c a are attractive drug targets; many specific small-molecule blockers have been developed for cytokine receptors, although few have been developed for chemokine receptors, which more closely resemble the c a anaphylatoxin chemotactic receptor (c ar) and c ar (ref. ). dompé pharmaceutical have described small-molecule c ar blockers and claim potential for therapy of a wide panel of diseases; however, no trials are yet reported. perhaps the most studied c ar blocker is the cyclic hexapeptide pmx , invented by taylor and woodruff and developed first by peptech and then arana therapeutics . this agent, modelled on the c terminus of c a, showed high specificity and nanomolar affinity for c ar and worked across many species; this latter property enabled the testing of pmx in a broad sweep of rodent models of inflammatory disease, and in many of these models this peptide had good efficacy. a number of phase i and iia clinical trials with pmx were initiated in rheumatoid arthritis, osteoarthritis, psoriasis and amd; the molecule proved safe, but all trials were terminated because of poor efficacy. one possible reason for these failures was the poor oral bioavailability and tissue penetrance of pmx (ref. ) . a modified version of pmx , pmx , shows improvements in these parameters and may re-surface in future trials, perhaps in combination with other agents. an orally active small molecule that blocks c ar has also been developed by chemocentryx; this agent, ccx- , has completed phase ii studies in anti-neutrophil cytoplasmic antibody (anca) vasculitis, in which it was successful in enabling the reduction and elimination of high-dose corticosteroids and demonstrated improvement in renal health parameters. in , ccx- was granted orphan drug status in both anca-associated vasculitides and ahus. emerging evidence for the role of c a as a 'modulator' of the c a-driven inflammatory response highlights the potential for c ar agonism in acute neutrophil-driven pathologies such as ischaemia-reperfusion injury , . an alternative approach, again by analogy to the chemokine and cytokine field, is the use of blockerseither antibodies or others -that bind to c a or c a and prevent receptor engagement. cytokine-specific antibodies and decoy receptors have evolved into blockbuster drugs. inflarx have developed a blocking mab against c a (ifx- ) that is currently in a phase ii trial for early sepsis and septic shock. noxxon described a series of l-rna aptamers (spiegelmer; nox-d to nox-d ) that block c a; these agents were effective in models of sepsis and transplant rejection , , but have not yet progressed to humans. all of the above suggests that pharmacological blockade of c a and/or c a activities might be relatively straightforward; however, such an approach will only work in those situations in which these molecules are the major drivers of pathology, such as in neutrophilmediated disorders including anca-vasculitis, chronic obstructive pulmonary disease (copd) and lupus. targeting c . as described above, c is activated to create two moieties with distinct activities. the small fragment, c a, is a powerful chemoattractant, and therapeutic blockade of its activities is described above. the larger fragment, c b, forms the foundation of the mac. assembly of the mac involves the nonenzymatic aggregation of the five terminal pathway components (c b and c -c ) into a pore that inserts into and through the cell membrane. c b, while still attached to the convertase, binds sequentially to c and c ; the c b complex is released from the convertase and binds to the membrane via a labile hydrophobic site. c and multiple copies of c are recruited in turn to build the pore. mac activity is naturally regulated by inefficiencies built into the assembly system and by regulators policing the membrane to block complexes that reach the surface. the hydrophobic binding site in c b is intrinsically unstable, decaying by interaction with water in a fraction of a second. additionally, there are several proteins in the plasma that bind to the mac to further restrict its capacity to bind to the membrane: clusterin and vitronectin (also known as s protein) are multifunctional proteins that, among their many roles, inhibit mac assembly in this way, and c is an efficient inhibitor if it binds to c b in the fluid phase. once bound to the membrane, c b is protected from these plasma inhibitors and recruits c and c to form the mac. the last line of defence is the membrane regulator cd , a small glycosylphosphatidylinositol (gpi)anchored protein that binds to the forming complex and prevents the completion of the mac pore. opportunities for therapeutic inhibition are legion. the first obvious target is the first mac component, c b. cutting off the supply of c b by targeting the c and c convertases will inhibit mac production; alternatively, c itself can be targeted to render it resistant to cleavage, which is the mode of action of the c monoclonal eculizumab, the most successful complement-specific drug to date. the success of eculizumab (box ) has triggered interest in other ways of preventing c cleavage. novartis have taken a c -specific mab, lfg , into phase ii trials of intraocular therapy in advanced amd (completed but unpublished) and, more recently, in choroiditis and panuveitis . in august , novartis announced phase i trials to assess the safety and efficacy of lfg in patients with pnh (nct ). a recombinant human c -specific mab fragment, mubodina (adienne pharma & biotech), that prevents cleavage of c has undergone early-stage trials in ahus and other renal diseases, although no development has been reported recently. ornithodoros moubata complement inhibitor (omci), a small ( kda) lipocalin present in the saliva of soft ticks, is a potent c inhibitor that probably protects the tick from complement in its blood meal . recombinant omci (coversin; volution immuno pharmaceuticals) blocked c in numerous species and inhibited disease in rodent models , . the drug bound c at a site remote from the convertase cleavage site, suggesting that it functioned by either blocking the interaction of c with the convertase or preventing conformational change essential for cleavage. coversin completed phase i trials in and is in development for pnh and other mac-driven diseases. sobi is a c -blocker that is based on affibody technology and developed by swedish orphan biovitrum. inclusion of an albumin-binding motif extends the plasma halflife of sobi . this small (~ kda) protein, derived using phage display, avidly binds to c and prevents cleavage; the simple structure of sobi makes synthesis easy and inexpensive. sobi is currently in a phase i trial, which was recently listed as terminated (nct ). various non-biologic approaches have also been developed and are either soon to be tested or already in clinical trials. ophthotech have developed a c -specific aptamer (arc , also known as zimura) that, when given intraocularly in a phase iia study in combination with an antibody targeting vascular endothelial growth factor (vegf), improved outcomes in patients with wet amd. the rnai approach taken by alnylam is described above; at the american society of haematology (ash) meeting, alnylam reported effective knockdown in non-human primates using aln-cc (see further information), and phase i trials are now underway with pnh as the primary indication. interim phase i data demonstrate substantial knock-down of c in singleascending-dose studies in humans (as announced in a recent alnylam press release). rapharma have developed a cyclic peptide that binds to c and prevents cleavage and activation; promising preclinical data were reported at ash (see further information), and phase i studies are proposed for the near future. as with many of the c -targeting agents reported here, the primary indication is likely to be pnh. the crowded 'landscape' in the c space (fig. ) clearly illustrates the activity generated by the success of eculizumab and the desire to block the activities of this key component in the cascade. targeting the terminal pathway: beyond c . blocking c cleavage by any of the above strategies will stop the production of c a as well as the mac and this may have important consequences if continued long term. to specifically target the mac, agents with activities downstream of c cleavage are needed. agents that mimic the ability of the natural inhibitors clusterin and vitronectin (and c ) to compete for the membrane binding site on c b might further reduce the efficiency of membrane attack, whereas mimics of cd that block the final steps of mac assembly might be effective blockers of lytic pore formation. soluble recombinant cd (scd ) has been generated and shown to inhibit lysis in vitro; however, in vivo, scd binds to plasma proteins, markedly reducing its inhibitory effects . recombinant cd , attached to the same membranebinding motif as in mirococept, has been generated and shown to protect pnh erythrocytes from lysis ex vivo and ameliorate arthritis development in a rat model when administered intra-articularly at the time of disease induction , . alternatively, any one of the terminal pathway components, each of which is essential for figure | progress of complement therapeutics towards clinical use. the figure graphically illustrates a current 'snapshot' of the different areas of complement that are being targeted and the progress of the drugs -both marketed and en route to approval. it is a rapidly moving field and, inevitably, some compounds will progress, others will fail and new drugs will emerge to take their positions in the landscape in the coming months and years. one certainty is that this already crowded field will become much more so in the near future. mac formation, could be targeted. regenesance bv has targeted c with both blocking c -specific mab and antisense approaches, and the latter was effective in animal models . regenesance also described a smallmolecule inhibitor of c in development for guillain-barré syndrome, but further development was not reported. a recent publication describes a small molecule (aurin tricarboxylic acid) that inhibits mac assembly by blocking c binding; the agent prevented the lysis of pnh erythrocytes by serum ex vivo . localizing therapeutics to sites of pathology inhibition of systemic complement activity is a major concern with most anti-complement drugs as it renders the recipient susceptible to infection. delivery of the drug specifically to the site of disease will not only reduce risk but also enhance efficacy. at its simplest, local delivery could mean direct administration to the site of disease -into the inflamed joint, eye or brain, for example. more interesting are the many modifications that have been made to ensure the homing of an agent where needed. modification of scr to favour its accumulation on inflamed endothelia (tp ) increased efficacy in models , and addition of a membrane-binding tag to a truncated form of scr (mirococept) converted an inactive fragment into a viable therapeutic , . similarly, a recombinant truncated fh comprising the minimal active site (short consensus repeats - ) is essentially inactive in vivo, but adding in the fh membrane-binding short consensus repeats creates a powerful inhibitor (amy- ) , . localising anti-complement agents to sites of complement activation has attracted abundant attention. homing of the drug not only avoids systemic inhibition but also enhances efficacy by concentrating the drug in one site and by placing active sites in the correct orientation to inactivate target enzymes. these approaches tend to focus on the homing of agents that have regulatory functions, such as decay acceleration or cofactor activity, rather than the blocking activities typical of antibodies. it is important that the drug that is delivered to the site is able to 'recycle' and release its target once inactivated, otherwise the target surface will rapidly become saturated with useless agent. more than years ago, we described a prodrug approach whereby recombinant forms of natural regulators were generated as fc fusion proteins in which regulatory activity was absent because of steric hindrance. engineered enzyme-cleavage sites enabled the release of active agent by matrix metalloproteinases and related enzymes that are abundant at inflammatory sites . similar strategies have proved successful in anticancer therapy . complement fragments bind covalently to tissues at sites of activation and provide a potential ligand for localizing an anti-complement drug where needed. a hybrid molecule comprising the rodent c convertase regulator crry coupled to the c dg-binding short consensus repeats of cr provided proof-of-principle of this approach; the hybrid molecule localized to sites of pathology and was -fold more active compared to crry alone in mouse models of ischaemia-reperfusion . the same group also described a cr -fh hybrid protein that was effective in several murine models ; this provided the impetus to create a human cr -fh hybrid, tt , which is currently under development by alexion for therapy of pnh (alxn and alxn ) , . potential advantages of this agent over eculizumab include a reduced risk of infection, a lower dose requirement and protection of pnh erythrocytes from c deposition and the resultant extravascular haemolysis. progress towards the clinic has been slow, perhaps because of commercial rather than scientific considerations. a phase i clinical trial was initiated (nct ), although no data have been reported. looking to the future although the many roles of complement in driving pathology in diverse diseases have long been recognized, the capacity to treat these diseases by controlling complement activation has lagged behind. today, we boast just two anti-complement drugs that are being marketed, approved only in rare orphan diseases (hae, pnh and ahus), which is a rather disappointing outcome given the investments made by researchers and companies. however, the future looks much brighter; new therapeutics are entering the clinic, targeting different parts of the complement system, with different half-lives, different routes of administration and each with its own range of activities and side effects (fig. ) . importantly, although the emphasis on orphan applications was pivotal in getting the first agents to the market, many of the new agents target common acute and chronic inflammatory diseases, for example amd, arthritis and cardiovascular disease. hurdles to the commercialization of complement therapeutics still exist, including cost, routes of administration and infection risk. short-duration complement inhibition, particularly in a controlled clinical environment, carries the least risk, and anti-complement drugs will probably first become mainstream for acute indications, such as reperfusion injuries. for chronic conditions, cost, ease of administration and safety may be substantial issues. cost will become less of an issue as increased competition (the emergence of small-molecule inhibitors) and increased use should, theoretically, bring prices down rapidly. the experience of chronic therapy with agents that block complement is still relatively short-term, and it is possible that the elimination of molecules, such as c a, may affect other activities of this multi-functional molecule, such as modulation of adaptive immunity, in the long term . however, eculizumab was approved for use in pnh in and for ahus in and, although the long-term consequences of c blockade remain uncertain, it is encouraging that after almost a decade of use no substantial adverse effects have been noted in carefully monitored and compliant recipients. frequent intravenous administration is not clinically acceptable for chronic use, and so agents will need either very long half-lives or to be delivered by routes not requiring medical intervention (for example, subcutaneous or oral). it may be that a therapy could be titrated to give partial inhibition of complement, sufficient to inhibit disease but sparing key protective functions. alternatively, a drug may be engineered to deliver complement inhibition where it is needed at the site of disease. inevitably, different diseases will require different approaches to complement inhibition -some may be entirely mac-driven, others c a-dependent, and most involve a complicated interplay between the various complement products and other inflammatory mediators. selecting the right agent or agents will be crucial to success in trials, and here a comprehensive understanding of the mechanisms by which complement drives pathology in the target disease is a sine qua non. biomarkers that reflect complement activation in fluids and tissues can inform the selection of the right drug for the right patient, demonstrating engagement of the target and efficacy of the drug, and monitoring response to therapy. a lack of good biomarkers often handicaps early stage trials and simple readouts of effectiveness are crucial. in some cases this is obvious; for example, blockade of mac formation can be demonstrated in serum through simple haemolytic assays. when activation fragments such as c a, c a, c d, bb and terminal complement complex (tcc) are present in the disease their levels can be monitored to demonstrate response to therapy and confirm target engagement. in some diseases, such as pnh, there is an obvious clinical readout of target engagement -eculizumab treatment prevents intravascular haemolysis in pnh. however, for diseases restricted to specific sites, for example, the retina, central nervous system or kidney glomerulus, plasma complement biomarkers may not reflect the response to therapy and are poor tools for assessing target engagement. there is thus a need for effective, non-invasive methods to detect complement activation and deposition within affected tissues without resorting to biopsy. methods for imaging complement activation in vivo may fill this need; agents comprising the c -binding domain of cr have been used to detect c fragments deposited in the kidney by magnetic resonance imaging in mouse models . antibodies against c dg have also been developed as imaging agents and shown to identify retinal lesions in a mouse model of amd . further development of the capacity to image complement activation in vivo will inform the conduct of future clinical trials by providing measurable outcomes and early proof of 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carboxypeptidase b serves as a protective mediator in osteoarthritis innate immunity and the etiology of late-onset alzheimer's disease inflammation in alzheimer disease a brief review of the basic science and clinical literature this paper describes a novel role of complement in synaptic pruning relevant to development and disease diabetic angiopathy, the complement system and the tumor necrosis factor superfamily complement involvement in neovascular ocular diseases soluble form of membrane attack complex independently predicts mortality and cardiovascular events in patients with st elevation myocardial infarction treated with primary percutaneous coronary intervention targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia c inhibitor protects from brain ischemia-reperfusion injury by combined antiinflammatory and antithrombotic mechanisms the role of complement in trauma and fracture healing the complement system in ischemiareperfusion injuries targeted complement inhibition as a promising strategy for preventing inflammatory complications in hemodialysis therapeutic c inhibitor cp abrogates complement activation induced by modern hemodialysis filters stec-hus, atypical hus and ttp are all diseases of complement activation systemic complement inhibition with eculizumab for geographic atrophy in age-related macular degeneration: the complete study terminal complement inhibition decreases antibody-mediated rejection in sensitized renal transplant recipients epirus biopharmaceuticals: www.epirusbiopharma.com ifx- omeros press release the authors thank k. miller of glaxosmithkline, for her thoughtful review and discussion of the manuscript. the authors declare competing interests: see web version for details. key: cord- - g ov t authors: kurpiers, laura a.; schulte-herbrüggen, björn; ejotre, imran; reeder, deeann m. title: bushmeat and emerging infectious diseases: lessons from africa date: - - journal: problematic wildlife doi: . / - - - - _ sha: doc_id: cord_uid: g ov t zoonotic diseases are the main contributor to emerging infectious diseases (eids) and present a major threat to global public health. bushmeat is an important source of protein and income for many african people, but bushmeat-related activities have been linked to numerous eid outbreaks, such as ebola, hiv, and sars. importantly, increasing demand and commercialization of bushmeat is exposing more people to pathogens and facilitating the geographic spread of diseases. to date, these linkages have not been systematically assessed. here we review the literature on bushmeat and eids for sub-saharan africa, summarizing pathogens (viruses, fungi, bacteria, helminths, protozoan, and prions) by bushmeat taxonomic group to provide for the first time a comprehensive overview of the current state of knowledge concerning zoonotic disease transmission from bushmeat into humans. we conclude by drawing lessons that we believe are applicable to other developing and developed regions and highlight areas requiring further research to mitigate disease risk. amplifi er hosts from which spillovers to humans have been documented (childs et al. ; daszak et al. ) . not surprisingly, the most devastating pandemics in human history, the black death, spanish infl uenza, and hiv/aids, were all caused by zoonoses from wildlife (morens et al. ) . zoonotic diseases can spill between animal hosts and humans in a variety of ways, including through (a) shared vectors, such as mosquitoes for malaria, (b) indirect contact, such as exposure to rodent feces in a peridomestic setting, or (c) direct contact with an animal through consumption, animal bites, scratches, body fl uids, tissues, and excrement (wolfe et al. a ) . most pathogens infecting animals fail to make the jump into humans, but % of zoonotic pathogens (~ out of zoonotic pathogen species studied) that have spilled over are known to be transmissible between humans (taylor et al. ) . of all eids, zoonotic spillovers from wildlife have been identifi ed as the most signifi cant, growing threat to global health (cleaveland et al. ; jones et al. ) . recent evidence highlights the link between infectious diseases and biodiversity loss, land use changes, and habitat fragmentation (cleaveland et al. ; maganga et al. ; gottdenker et al. ) . although additional research on the relationship between habitat degradation and eids is needed, gottdenker et al. ( ) reviewed studies incorporating a broad variety of diseases and found that the most common land use change types related to zoonotic disease transmission were deforestation, habitat fragmentation, agricultural development, irrigation, and urbanization. functionally, the mechanisms infl uencing disease spillover include disruption of food web structures, changes in host-pathogen interactions, and mixing of pathogen gene pools resulting in increased pathogen genetic diversity (jones et al. ) . many studies have shown that habitat fragmentation and biodiversity loss correspond to an increase in disease and pathogen abundance and diversity within a host species (allan et al. ; gillespie et al. ; keesing et al. ; salzer et al. ; cottontail et al. ; young et al. ) . specifi cally, the emergence or re-emergence of many zoonotic diseases including yellow fever, lyme disease, hantavirus pulmonary syndrome, nipah virus encephalitis, infl uenza, rabies, malaria, and human african trypanosomiasis have been linked to anthropogenic habitat changes (jones et al. ) . many of these human environmental changes are occurring in sub-saharan africa where human bushmeat activities have been linked to numerous virulent disease outbreaks, including ebola (leroy et al. a ) , hiv (van heuverswyn and peeters ) , and monkeypox . pathogen spillover from bushmeat can occur through consumption; however, the main risks are associated with exposure to body fl uids and feces during handling and butchering (kilonzo et al. ; paige et al. ) . historically, when a spillover occurred, the likelihood of an epidemic was limited because hunter-gatherer tribes were generally small and widely dispersed, hampering disease transmission between groups of people. once agricultural expansion occurred, human population densities increased, and people became better connected, diseases could spread more easily. as a result, transmissions of infectious diseases from animals to humans have led to devastating outcomes across the globe (lebreton et al. ) . eids cause hundreds of thousands of deaths annually (bogich et al. ) . some outbreaks have spread across large regions and became pandemics, costing the global economy tens of billions of dollars (e.g., sars, h n , the - west african ebola outbreak) and bringing entire nations to the brink of economic collapse. in this review, we explore the links between bushmeat-related activities and eids in sub-saharan africa, where the vast majority of african emerging infectious zoonotic diseases occur (jones et al. ) . the recent ebola outbreaks have highlighted the potential role of bushmeat as a source of pathogens, but a comprehensive review of the different pathogens that may emerge from wildlife through bushmeatrelated activities is lacking. although we are in no way suggesting that this issue is more important than other pressing health crises in sub-saharan africa (such as malaria prevention/treatment and improving healthcare infrastructure), we argue that a better assessment of the public health threats associated with this humanwildlife interaction is warranted and necessary to improve management of future disease outbreaks. the term "bushmeat" refers to the meat derived from wild animals for human consumption (milner-gulland and bennett ) ( fig. . ). it includes a wide range of animals, such as invertebrates, amphibians, insects, fi sh, reptiles, birds, and mammals, including as many as species in sub-saharan africa (ape alliance ). although research has focused largely on mammals and, to a lesser extent, birds, theoretically any wildlife species harvested for bushmeat could be a potential source of zoonotic disease that can spillover during the hunting, butchering, and preparation process (wolfe et al. ; karesh and noble ) . hunters face risk of injury from live animals, which might allow animal blood to enter the hunter's bloodstream through open wounds. while small animals can be carried in bags, large animals are commonly carried on the shoulder or back, bringing the hunter in close contact with the animal and facilitating transfer of body fl uids (lebreton et al. ) . the highest risk of disease transmission occurs during the butchering of animals, e.g. skinning, opening of the body cavity, removal of organs, and cutting of meat. more people butcher than hunt animals ( % and %, respectively, lebreton et al. ) and butchering involves the use of sharp tools, which may lead to cuts during the process. subramanian ( ) found that % of respondents cut themselves on a regular basis during butchering. women are especially at risk of disease transmission as they engage more often in butchering and in food preparation than men. in discussing the links between bushmeat and disease, we refer to this all-encompassing suite of risky behaviors as "bushmeat-related activities." nonhuman primates, rodents, and bats have all been linked to the spillover of zoonotic diseases into humans (cleaveland et al. ; jones et al. ; kilonzo et al. ) . a review of the west and central african bushmeat literature including market, offtake, and consumption surveys documented a total of species from orders that were harvested for bushmeat, including ( %) mammal species, ( %) bird species, ( %) reptile species, and (< %) amphibian species (taylor et al. ) . among mammals, the largest group was primates ( species) including western gorillas ( gorilla gorilla ), bonobos ( pan paniscus ), and common chimpanzees ( pan troglodytes ), followed by ungulates ( species), carnivores ( species), and rodents ( species). in terms of biomass offtake, however, ungulates are generally the most prominent group. although the taylor et al. ( ) study is very comprehensive, it only included studies that: ( ) provided a quantitative measure of bushmeat offtake, consumption, and/or market availability/sales; ( ) used non-biased data collection methods and systematically sampled settlements/hunters to prevent selection bias; ( ) identifi ed carcasses to the species level; and ( ) recorded either the number of carcasses or the total biomass (kg). for a more inclusive and general review of existing central african bushmeat studies, see wilkie and carpenter ( ) , and for west african studies, see schulte-herbrüggen ( ). fa et al. ( ) found that of the approximately one million carcasses traded in the cross-sanaga region of nigeria and cameroon, % were mammals; of which around % were ungulates, % rodents, and nearly % primates. however, as wildlife populations become depleted, such as near urban areas and intensively used agricultural landscapes, smaller bodied mammals comprise a larger share of hunters' offtake (bowen-jones et al. ; schulte-herbrüggen et al. a ). humans have hunted wild animals for consumption and to protect their crops for millennia (shipman et al. ; grubb et al. ; davies et al. ) , and it remains an important source of food and income security among rural communities today (de merode et al. ; brashares et al. ) . bushmeat is an important source of animal protein in many west and central african countries, with up to % of total animal protein consumption coming from wild animals (fa et al. ) . overall, the contribution of bushmeat to protein and food security is generally lower in urban than rural areas and is highest among remote rural communities . for example, the relative importance of bushmeat in the diet of rural gabonese households ranged from % of total household consumption value in a village near a town to % in a remote community (starkey ) . similarly, for rural equatorial guinea, allebone-webb ( ) showed that bushmeat consumption contributed % to total protein consumption in a village with poor transport links, but only % in a village with good connections. in remote cameroonian communities with very few opportunities for purchasing alternative protein sources, bushmeat comprised - % of animal protein consumption (muchaal and ngandjui ) . in rural communities with relatively good market access and low levels of bushmeat consumption, the importance of bushmeat for food has been shown to increase seasonally during the agricultural lean season (e.g. the planting season between harvests) when farming households receive little income (dei , de merode et al. , schulte-herbrüggen et al. b ) and during the dry season when fi sh is not available (poulsen et al. ). bushmeat is also an important source of nutrients, especially among children. evidence from rural madagascar shows that removing bushmeat consumption would result in a % increase in the number of children suffering from anemia and triple the cases of anemia among children in the poorest households most hunters sell at least part of their harvest making it an important source of income, especially where alternative income-generating activities are lacking. the importance of bushmeat in household economies varies across sites and individual hunting households, ranging from % to more than % of the total cash income earned (reviewed in schulte-herbrüggen ). in rural gabon, hunting accounts for up to % of household incomes, with the proportion rising in poorer, more remote communities (starkey ) . hunters are also more likely to sell large animals and keep small animals for their own consumption, because the latter fetch a lower price per animal and may be less marketable (van vliet and nasi ) . finally, households facing income shortages during the agricultural lean season and requiring cash income to pay for urgent expenditures, such as hospital bills, are more likely to sell bushmeat than keep it for own consumption (de merode et al. ) . overall, income from bushmeat sales can be lucrative and compare favorably with alternative work in many rural places. vega et al. ( ) found that commercial hunters in equatorial guinea generated a mean of us$ per year from bushmeat sales. hunters supplying markets in central african logging concessions earned twice the income of junior technicians working at a logging company (tieguhong and zwolinski ) . rural kenya hunters can earn . times the average salary in the area (fitzgibbon et al. ) , and ghanaian hunters can earn income similar to that of a graduate entering wildlife service, and up to . times the government minimum wage (ntiamoa-baidu ) . very successful zambian hunters have been reported earning just below the mean annual income in a single hunting trip (brown ) . the sale of bushmeat historically occurred at a local level, but with increased transportation routes and globalization , the bushmeat trade is expanding to supply urban and international demand. in the past, novel pathogens entering the rural communities may not have spread beyond the community, but this is no longer the case as remote rural areas are connected to urban areas, and increased global trade networks and air travel increases the risk of disease transmission worldwide . this expanding trade network links hunters to consumers, and with many people along this commodity chain coming into contact with bushmeat, the opportunity for disease spillover can occur at many points. for example, the commodity chain supplying bushmeat to an urban market in ghana includes hunters, wholesalers, market traders, restaurant owners, and consumers (mendelson et al. ) . the bushmeat commodity chain supplying an urban market in democratic republic of the congo is comprised of hunters, porters who carry the meat to the road, the bicycle traders who transport the meat into town, and the market-stall owners who sell the bushmeat to consumers (de merode and cowlishaw ) . a recent study from ghana estimates that a minimum of , bats are sold each year through a commodity chain that stretches up to km and involves multiple vendors (kamins et al. a ) . in zambia, mozambique, and malawi, well-developed and complex rural-urban trade supply networks link rural hunters to urban consumers who are willing to pay high prices for bushmeat (barnett ) . understanding commodity chains is important, as pathogens likely remain viable for some period after an animal is killed. for example, prescott et al. ( ) demonstrated that ebola virus remains viable on monkey carcasses for at least seven days, with viral rna detectable for weeks. bushmeat has become a multi-million dollar business due to a growing human population and is now serving both subsistence and trade objectives. harvest volumes have been estimated at , tones per year in the cross-sanaga rivers region of nigeria and cameroon (fa et al. ) , , tones per year in côte d'ivoire (caspary ) , , tons per year in ghana (ntiamoa-baidu ) , and at total of - . million tons per year in central african forests (wilkie and carpenter ; fa et al. ) . however, it is important to recognize that our understanding of the scale of bushmeat harvest is limited by the availability of information and hence current regional harvest estimates might underestimate actual harvest volumes. despite substantial effort in recent years, our knowledge is still site-specifi c and data are lacking from many regions. most surveys have been restricted to relatively small areas or market catchments from which national estimates were extrapolated. research efforts have focused on central africa with some data available for % of countries compared to % of west african countries (taylor et al. ) . a large number of sites with detailed bushmeat data are concentrated in the cross-sanaga region of nigeria and cameroon, where fa et al. ( ) collected market data at sites, hence presenting a geographical bias in our understanding of bushmeat harvest . furthermore, the majority of available data samples ( . % and . %, in west and central africa, respectively) identifi ed by taylor et al. ( ) come from market surveys with poorly defi ned catchment areas, compared to offtake and consumption surveys. strong variation between individual estimates highlights the problems with extrapolation of survey data to national or regional levels and the effects of sampling strategies (hunter versus market surveys), timing of survey (open season versus lean season), survey location, and extrapolation methods. individual fi gures should therefore be treated with caution, but the overall message remains: bushmeat is harvested at an enormous scale exposing those involved in the bushmeat commodity chain to zoonotic diseases. the current scale of bushmeat hunting is primarily the result of socio-demographic changes (wilkie and carpenter ) . africa's human population has risen from . billion in to . billion in and is expected to rise to . billion by (united nations ). where alternative sources of animal protein and income are scarce, human population growth has been linked to increasing hunting intensity (brashares et al. ) . bushmeat has been and remains a staple source of animal protein among the rural poor, yet recent attention has focused on urban consumers of bushmeat as a driver of increased hunting. urban consumers generally have a range of meat sources from which to choose, but value bushmeat for its taste, cultural connotations, and as a luxury food item (fa et al. ). while urban consumers generally consume less bushmeat than rural consumers , urban populations in africa have increased dramatically from about % of the total population in to % in (united nations ) and have created a strong demand for bushmeat and hence market for rural hunters. the increasing demand for bushmeat has been accompanied by changes in hunting technology and improvements in hunting effi ciency. traditional hunting tools , such as nets and bow and arrow, have been replaced with more modern tools of guns and snares. modern guns have an up to -times higher rate of return compared to traditional weapons (wilkie and curran ) , substantially increasing the ease and cost-effectiveness of hunting (alvard ) . this enables hunters to catch more animals and sell a larger part of their catch (bowen- jones and pendry ; bowen-jones et al. ; nasi et al. ) . hunting effi ciency has also improved as remote forests have become more accessible through the construction of logging roads and improved transportation (wilkie et al. ; auzel and wilkie ) . for example, after the construction of km of logging roads in northern congo, the average time for a hunting trip was reduced from to hours (wilkie et al. ) . development of rural businesses , such as timber companies, attracts workers and their families to remote locations, increasing bushmeat demand, especially when no hunting regulations are in place and alternative protein sources are not provided bennett and gumal ; poulsen et al. ). the effect of logging company presence on hunting pressure was documented in gabon where ape populations decreased % between and as a result of hunting (walsh et al. ). in addition, agricultural expansion and mining have exerted a strong force in changing the african landscape and infl uencing human migration patterns (norris et al. ). due to increased access, people are brought into closer contact with wildlife, which facilitates accessibility to bushmeat hunting and makes transportation of bushmeat from rural to urban areas easier and more cost-effective (wolfe et al. a ) . along with increased ease of transportation comes the opportunity for bushmeat to be traded on the international market. the international trade in bushmeat has recently gained attention as both a driver of bushmeat hunting and the cross-border spread of zoonotic diseases. illegal wildlife trade is the second-largest black market worldwide, involving millions of animals and estimated to be worth us$ - billion per year (united nations environment programme ). case studies at airports screening passenger luggage for bushmeat estimated that approximately tons of bushmeat per week arrive at paris roissy-charles de gaulle airport (chaber et al. ) and . tons per year at zurich and geneva airports (falk et al. ) . as bushmeat hunting, globalization, and human interconnectedness increase, the potential for zoonoses leading to eids also increases. this risk was highlighted when retroviruses (e.g., simian foamy virus) and herpesviruses (cytomegalovirus and lymphocryptovirus) were found in confi scated primates at us airports (smith et al. ). indisputable evidence of the transmission of pathogens from wildlife to humans exists only for relatively few cases because the standard of proof is very high. nevertheless, the evidence for spillovers is very strong and many pathogens can be classifi ed as very likely to spillover (jones et al. ; kilonzo et al. ) . furthermore, countless pathogen species of zoonotic potential will likely be discovered as surveillance increases (taylor et al. ; jones et al. ). our close phylogenetic relationship with nonhuman primates increases the likelihood that pathogen spillover from these animals to humans will cause infection (childs et al. ). moreover, it is not surprising that many studies have focused on spillover events from nonhuman primates to humans given the high prevalence of these largely diurnal mammals in the bushmeat trade (taylor et al. ) . for instance, nonhuman primates of the family hominidae include the gorillinae and paninae, which show a genetic difference of only % or less with humans (gonzalez et al. ) , and members of these subfamilies share many morphological, physiological, and ecological features that may have a direct role in the transmission of infectious diseases (davies and pedersen ) . cleaveland et al. ( ) , in their assessment of the risk of disease emergence by taxa, found that the relative risk of disease emergence was highest for bats, followed closely by primates, then ungulates and rodents. there have been surprisingly few studies of the connection between hunting of birds or other vertebrates and eids, especially in africa, but surveillance for zoonotic pathogens in african birds is strongly needed (e.g., for avian infl uenza tracking see simulundu et al. simulundu et al. , . the characteristics of different species may render them more or less susceptible to hunting. behavioral traits such as communal nesting, large-group living, loud acoustic performances, and a diurnal lifestyle-which are found in many primate species-may facilitate the detection and harvesting of several individuals at one time (bodmer ) . taste preferences for certain species infl uence hunters' decisions as do attempts to maximize returns by preferring large-bodied animals that provide more food or fetch a higher price when sold than small-bodied species (bodmer ) . bats, especially the larger fruit bats popular in the bushmeat trade, are susceptible to hunting because they are often found in large, sometimes vocal groups that are visible during the day or in high concentrations in caves (mickleburgh et al. ). increased human encroachment in recent decades (kamins et al. b ) has driven some bat species to become peridomestic (o'shea et al. ; plowright et al. ) , which renders them easy targets for hunting. finally, sick animals may be less successful in evading hunters and hence more easily hunted, thereby increasing the risk of disease transmission to hunters. in addition to the behavioral traits that may infl uence which species are hunted, physiological traits of these species may make them more likely to harbor and transmit diseases. for example, bats, which are present in the bushmeat trade and comprise the highest risk among all wildlife for harboring emerging diseases (cleaveland et al. ) , present unique traits that suit them to hosting pathogens. these traits include: ( ) relatively long lifespans for their body size (munshi-south and wilkinson ), which may facilitate pathogen persistence for chronic infections; ( ) fl ight, which allows movement and dispersal over long distances and which creates high body temperatures that may select for co-evolution with viruses that can live at febrile temperatures and are therefore highly virulent in humans (o'shea et al. ); ( ) physiological similarity across sympatric species that roost together in high densities enabling pathogens adapted to any of the sympatric species to spillover to others (streicker et al. ) ; and ( ) regulation of their immune systems in such a way as to make them more likely to host, but remain unaffected by viral pathogens, serving as the reservoir host for emerging and highly virulent viruses . despite the fact that pathogens are common and often occur in high numbers in basically all animals, only a relatively small proportion of these pathogens will spillover to humans (cleaveland et al. ) . that said, when spillover events do occur, they can be not only deadly but costly. for example, the united nations development program ( ) has estimated that west africa as a whole may lose us$ . billion per year between and due to the - ebola outbreak. this loss stems from the cumulative effects of closed borders, decreased trade, decreased foreign direct investment, and decreased tourism, resulting in increased poverty levels and food insecurity. to understand the dynamics of spillover events and risks in relation to the pathogen, a number of factors must be considered, including: ( ) the evolutionary history of the pathogen, ( ) how the pathogen is maintained among its wildlife host(s), ( ) how the pathogen is transmitted across a species barrier, ( ) whether a productive infection is produced in the new host, ( ) whether that infection produces signifi cant disease in that host, and ( ) whether morbidity and/or mortality levels in the secondary host are suffi cient to be considered signifi cant (childs et al. ) . from this, it follows that emerging pathogens are not an arbitrary selection of all pathogens. becoming established in a human host typically requires adaptations, often for increased virulence, as has been documented in hiv (wain et al. ; etienne et al. ) . generalist pathogens have the ability to infect more than one host species and have higher relative emergence risk than pathogens that are very hostspecifi c (cleaveland et al. ); this is especially true for pathogens that can infect species in more than one taxonomic order. one example of this generalist "broad" host range is found in the newly described african henipavirus, which can enter and infect cells of nonhuman primates, bats, and humans (lawrence et al. ) . of particular importance for understanding bushmeat-related spillover events is whether a wildlife species is a natural or incidental pathogen host. natural or reservoir hosts are a natural part of the pathogen life cycle and may maintain the infectious pathogen for prolonged periods of time, often without showing symptoms. in contrast, an incidental or dead-end host may be infected by the pathogen and may even transmit it, but it is not a part of the normal maintenance cycle of the pathogen and is more likely to be affected by it than natural hosts. for example, contact with sick common chimpanzees and western gorillas has been tightly linked to ebola virus spillover in several outbreaks (leroy et al. b ) . like their human cousins, these great apes are largely considered incidental or dead-end hosts for this virus and do not maintain it long-term in nature. in the case of this deadly fi lovirus, understanding what species are true reservoirs (likely fruit bats in the family pteropodidae; pourrut et al. pourrut et al. , hayman et al. hayman et al. , and the spillover events between these reservoirs and other mammals (including apes, carnivores, and ungulates; leroy et al. a ) will prove critical to mitigating the components of disease transmission that are due to bushmeat-related activities. unfortunately, it is often diffi cult to defi nitively determine the natural host(s) of a particular pathogen as it requires, in descending order of importance, isolation of the agent from individuals of the target species, detection of pathogen-specifi c nucleic acid sequences from individuals, and serological evidence that an individual has been exposed previously. indeed, the study of reservoir systems and how infectious agents move between and within them can be complex, requiring rigorous and sophisticated analyses of multiple interrelated variables (gray and salemi ; viana et al. ) . descriptions of the types of pathogens potentially encountered through bushmeatrelated activities can be found below, with several important and well-studied examples described in more detail. in their review of global trends in eids, in which they separately listed each antimicrobial pathogen strain that has recently emerged, jones et al. ( ) report that the vast majority of pathogens involved in eids are bacterial or rickettsial, followed by viral or prion, then protozoa, fungi, and helminths. other studies have ranked viruses as more prevalent (taylor et al. ; woolhouse et al. ; cleaveland et al. ). in jones et al.'s ( ) analysis of eid events between and , only four eids list bushmeat as the driver; other signifi cant drivers were socioeconomic factors such as human population density. these four bushmeat-related eid events were all signifi cant events; all due to viruses (ebolavirus, human immunodefi ciency virus- , monkeypox virus, and sars), suggesting that viruses are the most important pathogens in regard to spillover due to bushmeat-related activities (see also kilonzo et al. ) . we review the literature from sub-saharan africa in relation to bushmeat species by pathogen type (viruses, bacteria, helminths, protozoa, fungi, and prions), noting the signifi cant potential for pathogens not yet associated with bushmeat-related activities to also be involved. very few studies have considered all of the potential zoonotics in a region or in a taxonomic group. magwedere et al.'s ( ) comprehensive study of zoonotics in namibia is an exception. table . summarizes these pathogens by bushmeat host taxonomic group, conservatively listing only those species/pathogen combinations that have been tied strongly to spillovers from wildlife to humans via bushmeat-related activities and recognizing that this link is often putative and diffi cult to establish. thus, table . does not include some of the potential but not demonstrated spillover risks of poorly studied groups such as helminths and protozoans. furthermore, due to their close genetic relationship with humans, common chimpanzees and western gorillas may share many pathogens of all varieties with humans, but the direction of spillover is not always clear (e.g. tourist interactions may spread disease from humans to apes) and much of these data are not discussed herein. also not included in the table are studies where pathogens are not determined to species and, consequently, the bushmeat host-human link is unclear, or where exposure would be via an insect vector, which could be encountered when handling bushmeat. while we have attempted a very thorough treatment of pathogens that meet our criteria for inclusion in the table, it is possible that some relevant studies have been missed. viruses are obligatory intracellular parasites characterized primarily by the nature of their nucleic acids (dna or rna; single or double stranded, etc.). they are the most abundant form of life on earth; many viruses are recognized as important disease-causing agents, and they are subject to frequent mutation and thus evolution. the advent of modern molecular techniques has advanced our understanding of viral diversity and pathogenesis in both animal and human hosts. for example, in relation to bushmeat, it is now clear that many virus variants are present in hunted nonhuman primate species, which have received most of the research attention, and that these variants have crossed between nonhuman primates and humans on multiple occasions (peeters and delaporte ; table . ). bats and rodents are also major zoonotic virus carriers (meerburg et al. ; baker et al. ); other taxonomic groups are less studied, at least in sub-saharan africa. several sub-saharan african viruses of importance are vector-borne, including rift valley fever and crimean-congo hemorrhagic fever. while one presumes that this would make them unlikely to spread via bushmeat-related activities, the possibility remains that animal handling could present a risk (magwedere et al. ). however, no signifi cant links between vector-borne viruses and bushmeat hunting have been made, and we will not include a discussion of these viruses here. . evidence suggests that siv crossed over to humans by blood contact when hunters had an exposed open wound or injured themselves during the butchering of nonhuman primates (hahn et al. ; wolfe et al. a , b ; karesh and noble ) . the closest relatives of hiv- found among nonhuman primates are sivcpz and sivgor, from common chimpanzees and western gorillas in west central africa (gao et al. ; sharp et al. ; keele et al. ; van heuverswyn et al. , takehisa et al. ) and at least four separate spillovers have occurred (peeters et al. the potential for future and continued spillovers from sivs is high, and multiple species-specifi c variants exist. for example, peeters et al. ( ) and peeters ( ) estimated that more than % of nonhuman primates hunted for food are infected with a variant of siv; locatelli and peeters ( ) and peeters et al. ( ) noted that at least species-specifi c variants of siv from at least primate species are currently recognized. aghokeng et al. ( ) sampled nonhuman primate carcasses from species found in bushmeat markets in cameroon. they documented low overall prevalence of siv (only . % of carcasses), with the lowest prevalence found among the most common species in the market. however, they did fi nd siv variants in about % of the tested primate species. in total, serological evidence of siv infection has been documented for at least different primate species (aghokeng et al. ; liégeois et al. liégeois et al. , . cross-species transmission of strains and co-infection with more than one strain have been documented, sometimes followed by genetic recombination (hahn et al. ; bibollet-ruche et al. ; aghokeng et al. ; gogarten et al. ), a recipe for future spillovers into humans (locatelli and peeters ) . human t-cell lymphotropic virus (htlv) : similar to hiv, human t-lymphotropic viruses (htlv) are related to simian viral lineages in which signifi cant diversity has been found (ahuka-mundeke et al. ; peeters and delaporte ) . all three sub-saharan great apes and additional nonhuman primates have been documented to have stlv/htlv variants and a variety of htlv viruses have been documented in wildlife and in central african hunters (calattini et al. (calattini et al. , courgnaud et al. ; sintasath et al. a , b ; wolfe et al. b ; zheng et al. ; locatelli and peeters ) . similar to hiv/siv, dual infections with more than one variant have been documented in nonhuman primates (agile mangabeys, cercocebus agilis ; courgnaud et al. ) and in humans (calattini et al. ; wolfe et al. b ) . simian foamy virus : simian foamy retroviruses ( sfv ) are endemic in most african primates (hussain et al. ; switzer et al. ; peeters and delaporte ) and are known to transmit to humans (sandstrom et al. ; switzer et al. ; calattini et al. ; mouinga-ondémé et al. , . like the other retroviruses discussed above (hiv and htlv), sfv is genetically diverse and relatively host species-specifi c. in cameroon, wolfe et al. ( b ) documented three geographically independent sfv infections, which could be traced to de brazza's monkey ( cercopithecus neglectus ), mandrill ( mandrillus sphinx ), and western gorilla. likewise, in gabon, mouinga-ondémé et al. ( , documented human spillover events involving multiple strains of sfv, with infected humans having been bitten by common chimpanzees, western gorillas, or mandrills infected with their respective variant of sfv. ebola and marburg viruses : there are seven species of fi loviruses currently identifi ed, fi ve of which occur in sub-saharan africa-genus ebolavirus : tai forest ebolavirus (tafv), sudan ebolavirus (sudv), zaire ebolavirus (ebov), bundibugyo virus (bdbv); genus marburgvirus: marburg virus ( marv ) . these pathogens are periodically emerging viruses, typically from single spillover events, which cause hemorrhagic fevers (reviewed by olival and hayman ; rougeron et al. (but note that rougeron's listing for a single case of sudv in sudan in is erroneous)). the - west africa outbreak of ebov is still ongoing at the time of this writing (labouba and leroy ) . while the zoonotic source of this outbreak is unknown, three initial outbreaks of the ebola virus in the democratic republic of the congo from to involved victims who were reported to have handled western gorilla or common chimpanzee carcasses or to have had physical contact with people who touched the animals (leroy et al. a , b ) . similarly, marburgvirus was fi rst identifi ed in laboratory workers who had dissected imported grivet ( chlorocebus aethiops ) (martini et al. ; siegert et al. ). both western gorillas and common chimpanzees have suffered signifi cant mortality from fi lovirus outbreaks (walsh et al. ; leroy et al. a , b ; bermejo et al. ; rizkalla et al. ) and antibodies to ebov were documented in several other primate species by leroy et al. ( b ) . the single case of tafv occurred in an ethnologist likely infected while performing a necropsy of a dead common chimpanzee following a rash of common chimpanzee deaths in the tai national park in côte d'ivoire (le guenno et al. ; wyers et al. ) . beyond primates, other incidental hosts in the wild are possible, as was demonstrated for duikers ( cephalophus spp.) (leroy et al. a ; rouquet et al. ) . as reviewed by weingartl et al. ( ) , both dogs (naturally) and pigs (at least experimentally) can also be infected. during the - ebov outbreak in gabon, allela et al. ( ) found over % seroprevalence in dogs living in villages with ebov human and animal cases. those dogs appeared to be asymptomatic and were presumed to be exposed by scavenging wild animals. although incidental hosts likely play important roles in the ecology of these viruses, especially when moribund or dead animals are consumed, strong evidence suggests that bats are the natural reservoir hosts for at least marburgvirus and ebov. for marburgvirus, the cave dwelling and densely packed egyptian rousette fruit bat ( rousettus aegyptiacus ) is now well-documented as a reservoir host amman et al. ), but antibodies against the virus and/or the presence of viral rna have been found in several other species (see table . ). the strong association of marburgvirus with the egyptian rousette makes sense in light of the outbreaks of this virus in people visiting tourist caves or working in mines (adjemian et al. ; timen et al. ; towner et al. ; amman et al. ). the picture for ebov is less clear, but evidence of infection has been found in at least eight sub-saharan bat species (pourrut et al. , hayman et al. hayman et al. , table . ). of the ten bat species listed in table . for marburgvirus and ebov, seven are fruit bats, which are relatively larger and more visible, and thus targets of bushmeat hunters. that said, bushmeat hunting of these bats is not ubiquitous throughout their range and cannot solely explain fi lovirus spillovers. mari saéz et al. ( ) unconvincingly suggested the non-fruit bat, mops condylurus, might have been the source of the - west african ebola outbreak. pourrut et al. ( ) found evidence of antibodies against zebov in this species, but there is no real evidence that this free-tailed bat played a role in the - outbreak. to date, no bat host has been identifi ed for bdbv, sudv, or tafv and broader surveillance for indications of these viruses in bats and other hosts should be conducted. henipaviruses and other paramyxoviruses : hendra virus and nipah virus (hnvs) are paramyxoviruses in the genus henipavirus that emerged in australia and southeast asia, respectively, with fruit bats in the genus pteropus (family pteropodidae) as reservoir hosts (reviewed by croser and marsh ) . however, recent studies have identifi ed henipavirus and henipa-like viruses in sub-saharan african fruit bats, which are a phylogenetically distinct clade of pteropodid bats that do not overlap distributionally with any pteropus species. documentation of henipavirus and related rna (drexler et al. ; muleya et al. ; baker et al. ) and anti-henipavirus antibodies (hayman et al. ; pernet et al. ) in the african straw-colored fruit bat ( eidolon helvum ) clearly show that this deadly and diverse viral group is present in sub-saharan africa. this bat species is a frequent target of hunters and a signifi cant protein source where it is found (kamins et al. b ). weiss et al. ( ) documented the presence of this group of viruses in these bats found live in bushmeat markets. strong evidence of spillover to humans was documented by pernet et al. ( ) who found antibodies against hnvs in human samples from cameroon. these seropositive human samples were found almost exclusively in individuals who reported butchering these bats. this bat is also a long-distance migrator with signifi cant panmixia across the continent, which could facilitate viral transmission between bats (peel et al. ) . the paramyxovirus story in sub-saharan africa is still unfolding. both drexler et al. ( ) and baker et al. ( ) describe great diversity in paramyxoviruses from sub-saharan bats. in their comprehensive study of the evolutionary history of this virus family, drexler et al. ( ) found that the henipavirus lineage originated in africa and identifi ed bats as the likely origin of this large family of viruses. a precautionary tale from sub-saharan africa comes from the recent discovery and naming of the sosuga virus from a wildlife researcher who became very ill after handling and dissecting hundreds of bats and rodents in uganda and south sudan (albariño et al. ). this virus is most closely related to tuhoko virus , a rubulalike virus recently isolated from the leschenault's rousette fruit bat ( rousettus leschenaultii ) in southern china. amman et al. ( ) subsequently found sosuga virus in r. aegyptiacus captured from multiple locations in uganda; the researcher infected by this virus handled this species extensively in her studies. lyssaviruses : rabies is the oldest known zoonotic eid, recorded as early as the twenty-third century bc (steele and fernandez ) . an estimated , people die in africa each year from rabies (dodet et al. ) , some portion of which may be from exposure that occurs in bushmeat-related activities, although most human cases can be attributed to domestic dogs. rabies virus (rabv) is in the lyssavirus genus. it is joined in africa by at least fi ve additional species: lagos bat virus (lbv), mokola virus (mokv), duvenhage virus (duvv), shimoni bat virus (shibv), and the newly proposed ikoma lyssavirus (ikov). these viruses have bat(s) as their reservoir host ) with two exceptions. the mokola virus is found in shrews ( crocidura spp.), rusty-bellied brush-furred rat ( lophuromys sikapusi ; saluzzo et al. ) , and companion animals (delmas et al. ; kgaladi et al. ). the ikoma virus has thus far only been documented in african civets ( civettictis civetta ; table . , marston et al. ) . a variety of wildlife species can be secondary hosts of rabies (e.g., in botswana, see moagabo et al. ) and rabies has been documented to occur in a number of nonhuman primate species, including those encountered in the bushmeat trade (gautret et al. ) . lyssaviruses are found worldwide, but the greatest genetic diversity is in africa and lagos bat virus may be more than one species (delmas et al. ; markotter et al. ; kuzmin et al. a ) . while most human cases are due to rabies virus, duvenhage virus has been documented in human fatalities associated with bat scratches that likely transmitted the virus (van thiel et al. ; paweska et al. ) . mokolo virus has been detected in two human cases without mortality (kgaladi et al. ) . the lyssavirus story in africa will continue to emerge due to increased surveillance and improved molecular techniques. the discovery of ikoma virus in an african civet in serengeti national park in tanzania, where domestic dogs are largely absent and detection in bat hosts is nonexistent (marston et al. ; horton et al. ) , highlights the likelihood that many more lyssaviruses exist in a variety of host species. the true diversity of lyssaviruses in africa, and the potential for human spillover via bushmeat-related activities, remains to be discovered. lassa and other arenaviruses : arenaviruses include a number of zoonotic species, typically transmitted from rodents to humans. lassa virus is the best known of the viral hemorrhagic arenaviruses in africa and is well-documented in west africa, especially guinea, sierra leone, nigeria, and liberia. as with some of the bacterial pathogens described below, the primary risk comes from peridomestic exposure to the rodent host, the natal mastomys ( mastomys natalensis ), via exposure to urine or fecal materials. however, ter meulen et al. ( ) found a strong association between hunting of peridomestic rodents and antibodies to and symptoms of lassa virus, tying bushmeat-related activities to the spillover of this virus to humans. human monkeypox virus : contrary to its moniker, the reservoir hosts of human monkeypox virus (mpx) are neither monkeys nor humans, but rather rodents. the fi rst case of human monkeypox was identifi ed in in the democratic republic of the congo, with subsequent outbreaks in liberia, sierra leone, côte d'ivoire, nigeria, and democratic republic of the congo (reviewed by reynolds et al. ; rimoin et al. ) . recent mpx increases in the democratic republic of the congo and elsewhere have been attributed to cessation of the human smallpox vaccine, which conferred some immunity to other pox viruses . human and nonhuman primate infections are suspected to result from wildlife exposure such as would occur in bushmeat-related activities; infected species include squirrels (e.g., thomas's rope squirrel, funisciurus anerythrus ; khodakevich et al. ; african ground squirrels; xerus sp.; reynolds et al. ) , dormice ( graphiurus sp.; reynolds et al. ) , and giant pouched rats ( cricetomys sp.; reynolds et al. ) . the outbreak that occurred in the usa in after exposure to rodents in the illegal pet trade also linked human monkeypox to rope squirrels, dormice, and pouched rats (hutson et al. ). while dormice are small and not likely to be the target of hunting, the diurnal and highly visible squirrels and the giant pouched rats are routinely hunted (taylor et al. ) , making the spillover to humans highly plausible. jones et al. ( ) list . % of eid events as being caused by bacteria and there is good evidence to suggest that bacterial pathogens have the potential to be just as important as viruses when it comes to those that may spillover due to bushmeat-related activities, but in this capacity they have received far less attention (cantas and suer ) . transmission pathways for bacterial pathogens can occur through direct exposure to body fl uids or feces, but they can also possibly be transferred indirectly through exposure to disease vectors such as fl eas and ticks when handling animals. in a rare survey of bacterial pathogens that might spillover via bushmeat-related activities, bachand et al. ( ) sampled muscle from bushmeat carcasses from multiple species at markets in gabon for the presence of campylobacter , salmonella , and shigella . while they only recorded the presence of salmonella , the potential for contamination and thus spillover of enteric pathogens from carcass handling remains high, especially in the days after purchase when pathogens continue to replicate. bacteria in the genus leptospira are endemic sub-saharan african pathogens that have a high risk of spillover during bushmeatrelated activities as they are shed in urine. jobbins and alexander ( ) documented their widespread presence in wild mammals, birds, and reptiles, highlighting the role that wildlife may play in leptospirosis. the bushmeat interface may also play a role in human cases of anthrax, caused by bacillis anthracis , which is largely a disease of grazing herbivorous mammals, but to which common chimpanzees are also susceptible (leendertz et al. ). if bushmeat includes not only the hunting of apparently healthy animals but also sick animals or salvage of contaminated carcasses, the risk of human outbreaks increases (hang'ombe et al. ) . a number of bacterial pathogens are vector-borne, which at face value would make them unlikely to spread via bushmeat-related activities. however, especially for bacteria with fl ea or tick as vectors, as opposed to mosquitoes for example, one can envision that animal handling could present a risk. the most frightening among the vector-borne bacterial pathogens is plague, caused by the bacteria yersinia pestis and transmitted through the infected fl eas of rodents. africa remains an endemic region of importance for this pathogen (world health organization ; davis et al. ) . fleas and ticks are also responsible for transmitting rickettsial pathogens, such as rickettsia africae , which causes african tick-bite fever ( atbf ) . mediannikov et al. ( ) collected ticks from duikers and a pangolin that were living in close proximity to humans in guinea and found r. africae in % of ticks collected from the tree pangolin ( manis tricuspis ), suggesting the potential for spillover with the close handling of these animals. further research is clearly and urgently needed to fully assess the potential for bacterial disease spillovers via bushmeat-related activities. the helminths or "worm-like" animals include many parasites of zoonotic potential, although taylor et al. ( ) found helminthes less likely to cause eids. humans engaging in bushmeat-related activities are likely exposed to these pathogens via exposure to fecal material in which eggs are shed, from transcutaneous exposure to infectious larvae, or from consumption of uncooked meat (mccarthy and moore ) . several studies have examined the prevalence of helminths in animals from bushmeat markets and found high rates of multiple species. for example, adejinmi and emikpe ( ) collected fecal samples from greater cane rats ( thryonomys swinderianus ) and bush duikers ( sylvicapra grimmia ) in bushmeat markets in nigeria and documented high prevalence rates ( . % and . %, respectively) of helminth ova in feces as well as larvae from fecal cultures. likewise, magwedere et al. ( ) and mukaratirwa et al. ( ) reviewed the evidence for trichinella infection in humans, livestock, and wildlife in sub-saharan africa and noted that bush-pigs ( potamochoerus spp.) and desert warthogs ( phacochoerus aethiopicus ) are a source for human infection. as is the case with many other pathogens, humans and nonhuman primates share susceptibility to many parasitic helminth species (pedersen et al. ; pourrut et al. ). pourrut et al. ( sampled gastrointestinal parasites from wild monkeys of species collected from bushmeat markets in cameroon and documented high helminth loads, including species known to infect humans. gillespie et al. ( ) had similar fi ndings from common chimpanzee fecal samples. overall, the available evidence suggests that spillover of many of these pathogens during bushmeat-related activities is likely. protozoans are a paraphyletic group of eukaryotic organisms that are neither animals, plants, nor fungi and include amoebas and giardia. the risk of protozoan spillover from bushmeat-related activities is similar to that for helminths and bacteria in that exposure to feces, bodily fl uids, and even potentially to meat could transmit disease to a permissive human host (pourrut et al. ) . a number of protozoans are important pathogens with zoonotic potential (taylor et al. ). perhaps the best example are the amoebozoa, which cause diarrheal disease and which are documented in a variety of animals, including bushmeat species such as nonhuman primates (gillespie et al. ; pourrut et al. ) . gillespie et al. ( ) documented the amoeba entamoeba histolytica and the ciliated protozoan balantidium coli in common chimpanzees; both are human pathogens (although the direction of spillover is uncertain, as common chimpanzees and other primates may have obtained this parasite from humans). indeed, lilly et al. ( ) documented both protozoans in common chimpanzees, western gorillas, agile mangabeys, and humans living in the same region in central african republic. a number of other nonhuman primates have had documented e. histolytica infections as well (see table . ). other protozoan examples include toxoplasma gondii , which causes the disease toxoplasmosis, but could not be detected during a recent, albeit small scale, survey of bushmeat (prangé et al. ) and water/foodborne parasites such as giardia. recent studies have documented giardia in a variety of species that exist in the bushmeat trade, including western gorilla and african buffalo ( syncerus caffer ) (hogan et al. ). fungi are increasingly being recognized as important pathogens that may emerge, even in humans (jones et al. ; fisher et al. ) , and a number of fungi are considered medically important. in particular, fungal infections are problematic for people who are immunosuppressed (e.g., from hiv infection), in which case their immune systems are unable to adequately fi ght the infection. nonetheless, we have uncovered no examples of eids in africa caused by fungal pathogens not related to human immunosuppression, as even the s outbreak of cryptococcal meningitis in the democratic republic of the congo has been likely linked to co-infection by hiv (molez ; jones et al. ). only % of prion diseases are acquired (as opposed to inherited), but these include the well-publicized outbreaks of scrapie, bovine spongiform encephalopathy (bse, or "mad cow disease"), and chronic wasting disease (cwd) in ungulates from europe and north america. of these, only bse has been detected in humans and in captive-held primates (imran and mahmood a , b ; bons et al. ; lee et al. ) , likely due to consumption of contaminated meat products. the authors have found no descriptions of infectious prion diseases in africa, but this poorly studied pathogen type may well be present in the world's second largest continent. as it relates to bushmeat-related practices, prions can be found in nearly all tissues and are resistant to degradation, even by cooking, rendering them a potential pathogen worth watching. the risk of disease spillover from bushmeat to hunters is highest during butchering and especially if no precautions are taken. whether hunters take precautions may depend on their knowledge and perception of disease risk. there is increasing evidence that the perception of and knowledge about zoonotic diseases is generally low but varies strongly between sites. a survey among rural bushmeat hunters and traders in sierra leone showed that % reported knowledge of disease transmission from animals to humans (subramanian ) . similarly, % of rural-urban hunters and traders in ghana perceived a disease risk from a bat-bushmeat activity, with signifi cantly more respondents associating risk with bat consumption than bat preparation or hunting (kamins et al. ) . individuals who participate in butchering wild animals typically associate less risk to meat preparation and consumption than those who do not participate in butchering (kamins et al. ) (fig. . ) . lebreton et al. ( ) found that hunters and butchers who perceived personal risks were signifi cantly less likely to butcher wild animals, but that risk perception was not associated with hunting and eating bushmeat. thirty-three percent of bushmeat consumers in a ghanaian market were not aware that zoonotic diseases could be transmitted from bushmeat to humans. those who were aware gave ebola ( %) and anthrax ( %) as examples of zoonotic diseases (kuukyi et al. ). in contrast, a large-scale survey among rural central african population showed that the majority ( %) of respondents perceived contact with bushmeat blood or body fl uids as dangerous (lebreton et al. ) . unfortunately, studies in this fi eld can be challenging, as reported perceptions may differ from actual or 'revealed' behaviors and beliefs (wilkie ) . although there seems to be some level of risk awareness in certain human populations, several studies report a distinct lack of precautionary behavior, resulting in hunters, butchers, and consumers exposing themselves to zoonotic diseases. lebreton et al. ( ) found that only % of hunters and % of people reporting butchering indicated that they took precautions against contact with animal blood and fl uids while hunting and butchering. furthermore, the few that took precautions may not have protected themselves adequately, as the most common response was "generally being careful." this was followed by "washing hands," and the least number of participants reporting "avoiding contact with blood, draining blood from carcasses and wearing suitable clothing." paige et al. ( ) examined human-animal interactions in western uganda and found that nearly % of participants reported either being injured by an animal or having contact with a primate. the most commonly reported animal injuries were bites ( . %) and scratches ( . %). in a separate study, it was also shown that although ghanaian hunters generally handle live bats, they do not typically use protective measures such as gloves, and thereby come into contact with blood through scratches and bites (kamins et al. ) . given the lack of awareness and precautionary measures taken among people who come into contact with bushmeat, the opportunity for new zoonotic pathogens to spillover into humans remains high (lebreton et al. ) . this is especially true, since the current rate of hunting wild animals will likely continue-at least until domestic animal production increases and can support the protein needs of the local people. current global disease control efforts focus almost exclusively on responding long after a spillover event has occurred, which increases the risk of a single spillover event causing an epidemic or pandemic. this retroactive response to emerging disease outbreaks is often costly economically and in terms of human well-being (childs and gordon ; united nations development program ) . increased pre-spillover surveillance measures along with quantifi cation of spillover risk is critically needed. for example, wolfe et al. ( b wolfe et al. ( , b found that % of rural cameroonians are infected with wild primate variants of t-lymphotropic viruses and another % are infected with wild primate variants of simian foamy virus. these sorts of data are simply lacking for most emergent disease systems. here we will discuss the regulatory and educational measures that could be taken to mitigate the risk of a zoonotic spillover event and spread. such efforts should be undertaken as a part of a comprehensive response to other sub-saharan public health crises so as to not divert scarce resources. for example, increases in eid surveillance efforts and in post-emergence management go hand in hand with the improved healthcare infrastructure that must become a priority for sub-saharan africa. at face value, the risk of disease transmission would be reduced if people stopped harvesting bushmeat; however, this scenario is not realistic given the importance of bushmeat in many communities in africa for which there is limited affordable access to alternate protein sources (pike et al. ; gebreyes et al. ) . a more practical option may be to restrict hunting of nonhuman primates, as many zoonotic eids have come from them, and instead allow communities to hunt smaller-bodied mammals with higher reproductive rates. any intervention aiming to restrict access to wildlife should involve community leaders and stakeholders during public outreach to reduce the risk of alienating communities (monroe and willcox ) . the education and enforcement necessary to implement such a restriction must consider the cultural and economic contexts surrounding individual communities. consider, for example, the problems with enforcement of access restrictions and the history of antagonistic relationships due to exclusion from protected areas between conservationists and local communities. without proper educational outreach, this could result in backlash from local communities. furthermore, using zoonotic diseases to enforce hunting restrictions runs the risk of demonizing species considered to be the main disease carriers. nonhuman primates could then become targets and their populations could be decimated (pooley et al. ) . a more realistic strategy may be to concentrate on preventing future zoonotic spillover events through culturally appropriate education and preventing the spread of diseases through better disease surveillance. in that effort, it would also be important to incorporate collaborative and interdisciplinary approaches between veterinary researchers, ecologists, microbiologists, public health researchers, and anthropologists to develop surveillance and research approaches that will be both culturally appropriate and improve detection of zoonotic diseases tied to bushmeat hunting (kilonzo et al. ). the risk of disease transmission could be reduced through community education that focuses on people with high levels of exposure to wild animals (wolfe et al. ). communicating with hunters and butchers about the risks associated with bushmeat and promoting awareness of safer techniques may reduce current levels of pathogen exposure and transmission. to enhance the effectiveness of prevention campaigns, it is particularly important to reinforce the potential for infections during hunting and butchering as this may be overlooked by some hunters (lebreton et al. ) . because the risk perception of hunters and those engaging in butchering wild animals has a negative association with the level of participation in meat preparation and consumption (kamins et al. ) , this may reduce current levels of pathogen exposure and transmission, if not by discouraging individuals to participate in preparation and consumption, then by encouraging those individuals to more proactively consider safety and preventative measures. global viral forecasting (gvf; now "global viral" and "metabiota") has been pivotal in educating vulnerable populations in rural central africa by providing information on the risk of zoonotic disease transmission from hunting wild animals (lebreton et al. ) . hunters are informed about disease risks associated with different species, what steps can be taken to avoid infections, and how they can reduce their contact with blood and body fl uids of wild animals. hunters are urged to redirect hunting efforts away from apes and monkeys and towards less risky species such as antelope and rodents, while also being discouraged from butchering animals when there are cuts or injuries on their hands and limbs. of course, a common aspect of such attempts at social outreach and education is that even when it is possible to promote awareness, individuals may not believe the hazard is important or that it could affect them. some authors have even found that when people do believe the risk is real and relevant, there is often little evidence that this knowledge promotes a change in behavior (mccaffrey ) . for example, a pilot education program among ghanaian hunters resulted in substantially improved understanding of disease risk, yet largely failed to change peoples' behavior (kamins et al. ) . when asked about what would change their behavior, participants responded; becoming ill from zoonotic disease followed by alternative livelihoods and stricter laws. because awareness is not directly related to behavior, monroe and willcox ( ) suggest that campaigns should not rely on the threat of infection to change behavior, but should rather use community leaders to change cultural norms associated with hunting and educate people involved in butchering about best practices of how to protect themselves. with the increasing prevalence of zoonotic disease emergence and the associated risk for public health, we have to improve our understanding of the dynamics of spillover events of pathogens from animal to human hosts (rostal et al. ) and improve systematic global monitoring efforts. this could help detect, defi ne, and control local human emergence while it is still locally confi ned and before it has a chance to spread globally. improved detection and surveillance will lead to a better prioritization of public health efforts. one of the most effective strategies in terms of early detection of an emergent pathogenic threat would be to focus surveillance efforts among people who are highly exposed to at-risk animals and on the animal populations to which they are exposed (lebreton et al. ) . bushmeat hunters would be an important target group, as they are in contact with bodily fl uids from animals and are at risk for transmission and infection from novel pathogens. as an example, gvf has established monitoring programs at multiple sites throughout central africa to detect the moment of a pathogen spillover, which can then be used to predict and ultimately prevent zoonotic disease emergences (evans and wolfe ) . in order to track and provide data for eids, this effort coordinates the collection of fi lter-paper blood samples from both hunted animals and people who hunt and butcher wild animals. early results have shown that this type of surveillance can assist in early detection of new diseases by offering insight into pathogen origin. it would also help describe the spillover dynamics of new or existing diseases. such data are valuable for developing a detailed, mechanistic understanding of the processes that drive disease emergence and prevent spillovers from spreading in early stages of an outbreak. contextualizing the relative or actual risks of spillovers would be vital for the preferential allocation of resources to high-risk regions or humans who perform high-risk activities (daszak et al. ). as part of these efforts, improved knowledge of how anthropogenic environmental changes and sociological or demographic factors affect the risk of disease emergence will likely be a cost-effective and sustainable mechanism to reduce or control disease spillover risks (daszak et al. ). the social and environmental issues surrounding bushmeat represent a complex problem for conservation, global public health, and sustainable development, as it is often the poorest and most vulnerable populations who depend on bushmeat for income or food security. accordingly, the challenge should be addressed in a holistic manner, by integrating multiple efforts to achieve common objectives. although much progress has been made not only in addressing the problems concerning bushmeat harvest and zoonotic disease spillover, there is much work to be done. research that would pave the way for future efforts would include the quantifi cation of social response to environmental policy change (e.g., in the context of harvest restriction), development of a more representative picture of bushmeat consumption in africa, a broader exploration of the many classes of pathogens within wildlife, and more thorough understanding and quantifi cation of the dynamics behind spillover events and the risks to humans. such efforts could facilitate the development of policy and infrastructure that would help curb the dependency on bushmeat, reduce risks associated with bushmeat harvest, and help understand in what circumstances zoonotic disease spillover events occur. there is still uncertainty as to how education should be implemented in different regions and what features of such education would be most valuable for local people. such an effort might consist of surveying rural bushmeat-harvesting populations across africa and using the resulting data to contextualize priorities and goals in a way that could help standardize education approaches. while some locations in africa have had extensive research in the scope and impact of bushmeat harvest, much of africa has been neglected in those efforts. a more developed understanding of the location, scale, and structure of bushmeat harvest throughout the continent would help researchers and policy-makers prioritize efforts related to disease surveillance, education, or 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in africa emergence of a novel and highly divergent htlv- in a primate hunter in cameroon key: cord- -l mx lp authors: mansbach, jonathan m.; clark, sunday; piedra, pedro a.; macias, charles g.; schroeder, alan r.; pate, brian m.; sullivan, ashley f.; espinola, janice a.; camargo, carlos a. title: hospital course and discharge criteria for children hospitalized with bronchiolitis date: - - journal: j hosp med doi: . /jhm. sha: doc_id: cord_uid: l mx lp background: for children hospitalized with bronchiolitis, there is uncertainty about the expected inpatient clinical course and when children are safe for discharge. objectives: examine the time to clinical improvement, risk of clinical worsening after improvement, and develop discharge criteria. design: prospective multiyear cohort study. setting: sixteen us hospitals. participants: consecutive hospitalized children age < years with bronchiolitis. measurement: we defined clinical improvement using: ( ) retraction severity, ( ) respiratory rate, ( ) room air oxygen saturation, and ( ) hydration status. after meeting improvement criteria, children were considered clinically worse based on the inverse of ≥ of these criteria or need for intensive care. results: among children, the median number of days from onset of difficulty breathing until clinical improvement was (interquartile range, – . days). of the total, ( %) met clinical improvement criteria, with % worsening ( % required intensive care). children who worsened were age < months (adjusted odds ratio [aor]: . ; % confidence interval [ci]: . ‐ . ), gestational age < weeks (aor: . ; % ci: . ‐ . ), and presented with severe retractions (aor: . ; % ci: . ‐ . ), inadequate oral intake (aor: . ; % ci: . ‐ . ), or apnea (aor: . ; % ci: . ‐ . ). readmissions were similar for children who did and did not worsen. conclusions: although children hospitalized with bronchiolitis had wide‐ranging recovery times, only % worsened after initial improvement. children who worsened were more likely to be younger, premature infants presenting in more severe distress. for children hospitalized with bronchiolitis, these data may help establish more evidence‐based discharge criteria, reduce practice variability, and safely shorten hospital length‐of‐stay. journal of hospital medicine ; : – . © society of hospital medicine although bronchiolitis is the leading cause of hospitalization for us infants, there is a lack of basic prospective data about the expected inpatient clinical course and ongoing uncertainty about when a hospitalized child is ready for discharge to home. this lack of data about children's readiness for discharge may result in variable hospital length-of-stay (los). [ ] [ ] [ ] one specific source of variability in discharge readiness and los variability may be the lack of consensus about safe threshold oxygen saturation values for discharge in children hospitalized with bronchiolitis. , in , the scottish intercollegiate guidelines net-work recommended a discharge room air oxygen (rao ) saturation threshold of %. the same year, the american academy of pediatrics (aap) bronchiolitis clinical practice guideline stated that oxygen is not needed for children with rao saturations % who are feeding well and have minimal respiratory distress. there is a need for prospective studies to help clinicians make evidenced-based discharge decisions for this common condition. we performed a prospective, multicenter, multiyear study [ ] [ ] [ ] to examine the typical inpatient clinical course of and to develop hospital discharge guidelines for children age < years hospitalized with bronchiolitis. we hypothesized that children would not worsen clinically and would be safe to discharge home once their respiratory status improved and they were able to remain hydrated. we conducted a prospective, multicenter cohort study for consecutive years during the to winter seasons, as part of the multicenter airway research collaboration (marc), a program of the emergency medicine network (www.emnet-usa.org). the number of participating sites varied over the years: in year , in year , and in year . each month from november until march , site investigators across us states used a standardized protocol to enroll a target number of consecutive patients from the inpatient wards and the intensive care unit (icu). we aimed to enroll % of our total sample from the icu. to over sample children in the icu, the ward and icu enrollments were separate. once the site reached their target enrollment for that month, the investigators would stop enrollment until the beginning of the following month. all patients were treated at the discretion of the treating physician. inclusion criteria were an attending physician's diagnosis of bronchiolitis, age < years, and the ability of the parent/guardian to give informed consent. the exclusion criteria were previous enrollment and transfer to a participating hospital > hours after the original admission time. therefore, children with comorbid conditions were included in this study. all consent and data forms were translated into spanish. the institutional review board at each of the participating hospitals approved the study. of the enrolled children, we excluded ( %) children with a hospital los < day due to inadequate time to capture the required data for the present analysis. among the remaining children, ( %) had daily inpatient data on all factors used to define clinical improvement and clinical worsening. thus, the analytic cohort was comprised of children hospitalized for bronchiolitis. investigators conducted detailed structured interviews. chart reviews were conducted to obtain preadmission and daily hospital clinical data including respiratory rates, daily respiratory rate trends, degree of retractions, oxygen saturation, daily oxygen saturation trends, medical management, and disposition. these data were manually reviewed, and site investigators were queried about missing data and discrepancies. a follow-up telephone interview was conducted with families week after discharge to examine relapse events at both hours and days. we used the question: "how long ago did the following symptoms [eg, difficulty breathing] begin [for the] current illness?" to estimate the onset of the current illness. pulse was categorized as low, normal, or high based on age-related heart rate values. presence of apnea was recorded daily by site investigators. nasopharyngeal aspirate collection and virology testing as described previously, site teams used a standardized protocol to collect nasopharyngeal aspirates, which were tested for respiratory syncytial virus (rsv) types a and b; rhinovirus (rv); parainfluenza virus types , , and ; influenza virus types a and b; novel h n ; human metapneumovirus; coronaviruses nl- , hku , oc , and e; enterovirus, and adenovirus using polymerase chain reaction. , [ ] [ ] [ ] defining clinical improvement and worsening clinical improvement criteria were based on the aap guidelines. for respiratory rate and oxygen saturation, clinicians estimated average daily respiratory rate and oxygen saturation based on the recorded readings from the previous hours. this estimation reflects the process clinicians use when rounding on their hospitalized patients, and thus may be more similar to standard clinical practice than a calculated mean. the respiratory rate criteria are adjusted for age. , for daily estimated average oxygen saturation we used the aap criteria of rao saturation of %. considering that oxygen saturation is the main determinant of los, healthy infants age < months may have transient oxygen saturations of around %, and that errors in estimation may occur, we included a lowest rao of % in our improvement criteria. by combining the dichotomized estimated oxygen saturation ( % or not) with the lower limit of %, there was little room for erroneous conclusions. a child was considered clinically improved on the earliest date he/she met all of the following criteria: ( ) none or mild retractions and improved or stable retractions compared with the previous inpatient day; ( ) daily estimated average respiratory rate (rr) < breaths per minute for age < months, < breaths/minute for age to months, and < breaths/minute for age months with a decreasing or stable trend over the course of the current day; ( ) daily estimated average rao saturation %, lowest rao saturation % ; and ( ) not receiving intravenous (iv) fluids or for children receiving iv fluids a clinician report of the child maintaining oral hydration. children who reached the clinical improvement criteria were considered clinically worse if they required intensive care or had the inverse of of the improvement criteria: moderate/severe retractions that were worse compared with the previous inpatient day, daily average rr with an increasing trend over the current day, need for oxygen, or need for iv fluids. all analyses were performed using stata . (stata-corp, college station, tx). data are presented as proportions with % confidence intervals ( % cis), means with standard deviations, and medians with interquartile ranges (iqr). to examine potential factors associated with clinical worsening after reaching clinical improvement, we used v , fisher exact, student t test, and kruskall-wallis tests, as appropriate. adjusted analyses used generalized linear mixed models with a logit link to identify independent risk factors for worsening after reaching clinical improvement. fixed effects for patient-level factors and a random site effect were used. factors were tested for inclusion in the multivariable model if they were found to be associated with worsening in unadjusted analyses (p < . ) or were considered clinically important. results are reported as odds ratios with % cis. we performed several sensitivity analyses to evaluate these improvement criteria: ( ) we excluded the lowest rao saturation requirement of %, ( ) we examined a % daily estimated average rao saturation threshold, ( ) we examined a % daily estimated average rao saturation threshold, and ( ) we examined children age < months with no history of wheeze. there were children hospitalized with bronchiolitis with data on all factors used to define clinical improvement and clinical worsening. the median number of days from the beginning of difficulty breathing until admission was days (iqr, - . days; range, - days) and from the beginning of difficulty breathing until clinical improvement was days (iqr, - . days; range, - days) ( figure ). the variance for days to admission was significantly less than the variance for days to clinical improvement (p < . ). in this observational study, clinicians discharged ( %) of the children before meeting the definition of clinical improvement. thus, ( %; % ci: %- %) children reached the clinical improvement criteria, had a los > day, and had data on all factors ( figure ). of the children who met the clinical improvement criteria, there were children ( %; % ci: %- %) who worsened ( figure ). the worsening occurred within a median of day (iqr, - days) of clinical improvement. forty-six ( %) of the children required transfer to the icu ( required intubation, required continuous positive airway pressure, and had apnea), ( %) required oxygen, and ( %) required iv fluids. eight percent of children met multiple criteria for worsening. a comparison between children who did and did not worsen is shown in table . in general, children who worsened after improvement were younger and born earlier. these children also presented in more severe respiratory distress, had moderate or severe retractions, oxygen saturation < % at hospitalization, inadequate oral intake, and apnea documented during the hospitalization. neither viral etiology nor site of care influenced whether the children worsened after improving. however, stratified analysis of children based on initial location of admission (ie, icu or ward) showed that among the children admitted to the icu from the emergency department (ed), % met the improvement criteria and % clinically worsened. in contrast, among children admitted to the ward from the ed, % met the improvement criteria, and only % clinically worsened. stratified multivariable models based on the initial location of admission from the ed (ie, icu or ward) were not possible due to small sample sizes after stratification. none of these children had relapse events requiring rehospitalization within either hours or days of discharge. on multivariable analysis (table ) , independent risk factors for worsening after reaching the clinical improvement criteria were young age, preterm birth, and presenting to care with more severe bronchiolitis represented by severe retractions, inadequate oral intake, or apnea. to further evaluate the improvement criteria in the current analysis, multiple sensitivity analyses were conducted. the frequency of clinical worsening after reaching the improvement criteria was stable when we examined different ra criteria in sensitivity analyses: ( ) excluding ra as a criterion for improvement: % met improvement criteria and % experienced clinical worsening, ( ) changing the average ra threshold for clinical improvement to %: % met improvement criteria and % experienced clinical worsening, and ( ) changing the average ra threshold for clinical improvement to %: % met improvement criteria and % experienced clinical worsening. furthermore, stratifying by age < months and restricting to more stringent definitions of bronchiolitis (ie, age < year or age < year no history of wheezing) also did not materially change the results (see supporting figure in the online version of this article). we compared the children who were discharged prior to reaching clinical improvement with the children who reached the clinical improvement criteria. the children were less likely to be age < months ( % vs %; p . ). these groups ( vs ) were similar with respect to in this large, multicenter, multiyear study of children hospitalized with bronchiolitis, we found that children present to a hospital in a relatively narrow time frame, but their time to recovery in the hospital is highly variable. nonetheless, % of children continued to improve once they had: ( ) improving or stable retractions rated as none/mild, ( ) a decreasing or stable rr by age, ( ) estimated average rao saturation % and lowest rao saturation of %, and ( ) were hydrated. the % of children who worsened after clinically improving were more likely to be age < months, born < weeks, and present with more severe distress (ie, severe retractions, inadequate oral intake, or apnea). based on the low risk of worsening after clinical improvement, especially among children admitted to the regular ward ( %), we believe these clinical criteria could be used as discharge criteria for this common pediatric illness with a predominantly monophasic clinical course. variability in hospital los for children with bronchiolitis exists in the united states and internationally. , cheung and colleagues analyzed administrative data from over , children admitted for bronchiolitis in england between april and march and found sixfold variation in los between sites. they concluded that this los variability was due in part to providers' clinical decision making. srivastava and colleagues addressed variable clinician decision making in bronchiolitis and other common pediatric conditions by embedding discharge criteria developed by expert consensus into admission order sets. they found that for children with bronchiolitis, the embedded discharge criteria reduced the median los from . to . days. in contrast to the single-center data presented by white and colleagues, the prospective, multicenter marc- data provide a clear understanding of the normal clinical course for children hospitalized with bronchiolitis, determine if children clinically worsen after clinical improvement, and provide data about discharge criteria for children hospitalized with bronchiolitis. although there is a lack of rigorous published data, the lower tract symptoms of bronchiolitis (eg, cough, retractions) are said to peak on days to of illness and then gradually resolve. in the present study, we found that the time from the onset of difficulty breathing until hospital admission is less variable than the time from the onset of difficulty breathing until either clinical improvement or discharge. although % of children have clinically improved within . days of difficulty breathing based on the iqr results, the remaining % may have a more prolonged recovery in the hospital of up to weeks. interestingly, prolonged recovery times from bronchiolitis have also been noted in children presenting to the ed and in an outpatient population. it is unclear why % to % of children at different levels of severity of illness have prolonged recovery from bronchiolitis, but this group of children requires further investigation. given the variability of recovery times, clinicians may have difficulty knowing when a child is ready for hospital discharge. one of the main stumbling blocks for discharge readiness in children with bronchiolitis is the interpretation of the oxygen saturation value. , , , , however, it should be considered that interpreting the oxygen saturation in a child who is clinically improving in the hospital setting is different than interpreting the oxygen saturation of a child in the ed or the clinic whose clinical course is less certain. in the hospital setting, using the oxygen saturation value in in the aap guideline, % of children clinically worsened after they met the improvement criteria, a clinical pattern observed previously with supplemental oxygen. this unpredictability may explain some of the variation in providers' clinical decision making. the children who worsened, and therefore deserve more cautious discharge planning, were young (< months), premature (< weeks gestational age), and presented in more severe distress. those children admitted to the icu from the ed worsened more commonly than children admitted to the ward ( % vs %). interestingly, the viral etiology of the child's bronchiolitis did not influence whether a child worsened after reaching the improvement criteria. therefore, although children with rv bronchiolitis have a shorter hospital los than children with rsv bronchiolitis, the pattern of recovery did not differ by viral etiology. in addition to unsafe discharges, clinicians may be concerned about the possibility of readmissions. although somewhat controversial, hospital readmission is being used as a quality of care metric. [ ] [ ] [ ] one response to minimize readmissions would be for clinicians to observe children for longer than clinically indicated. however, shorter los is not necessarily associated with increased readmission rates. given that the geometric mean of hospital charges per child with bronchiolitis increased from $ in to $ in , the potential for safely reducing hospital los by using the discharge criteria proposed in the current study instead of other criteria may net substantial cost savings. furthermore, reducing los would decrease the time children expose others to these respiratory viruses and possibly reduce medical errors. our study has some potential limitations. because the study participants were all hospitalized, these data do not inform admission or discharge decisions from either the ed or the clinic; but other data address those clinical scenarios. also, the sites that participated in this study were large, urban teaching hospitals. consequently, these results are not necessarily generalizable to smaller community hospitals. although numerous data points were required to enter the analytic cohort, only % of the sample was excluded for missing data. there were children who did not meet our improvement criteria by the time of discharge. although the inability to include these children in the analysis may be seen as a limitation, this practice variability underscores the need for more data about discharging hospitalized children with bronchiolitis. last, site teams reviewed medical records daily. more frequent recording of the clinical course would have yielded more granular data, but the current methodology replicates how data are generally presented during patient care rounds, when decisions about suitability for discharge are often considered. we documented in this large multicenter study that most children hospitalized with bronchiolitis had a wide range of time to recovery, but the vast majority continued to improve once they reached the identified clinical criteria that predict a safe discharge to home. the children who worsened after clinical improvement were more likely to be younger, premature infants presenting in more severe distress. although additional prospective validation of these hospital discharge criteria is warranted, these data may help clinicians make more evidence-based discharge decisions for a common pediatric illness with high practice variation, both in the united states and in other countries. , infectious disease hospitalizations among infants in the united states a hospital is no place to be sick variation in inpatient diagnostic testing and management of bronchiolitis watson ph international variation in the management of infants hospitalized with respiratory syncytial virus. international rsv study group population variation in admission rates and duration of inpatient stay for bronchiolitis in england impact of pulse oximetry and oxygen therapy on length of stay in bronchiolitis hospitalizations pulse oximetry in pediatric practice in: nhs quality improvement scotland. edinburgh, scotland: scottish intercollegiate guidelines network diagnosis and management of bronchiolitis prospective multicenter study of children with bronchiolitis requiring mechanical ventilation prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis apnea in children hospitalized with bronchiolitis evaluation of the cardiovascular system: history and physical evaluation apnea in children hospitalized with bronchiolitis respiratory viral infections in patients with chronic, obstructive pulmonary disease evaluation of real-time pcr for diagnosis of bordetella pertussis infection evaluation of three real-time pcr assays for detection of mycoplasma pneumoniae in an outbreak investigation normal ranges of heart rate and respiratory rate in children from birth to years of age: a systematic review of observational studies development of heart and respiratory rate percentile curves for hospitalized children effect of oxygen supplementation on length of stay for infants hospitalized with acute viral bronchiolitis. pediatrics longitudinal assessment of hemoglobin oxygen saturation in healthy infants during the first months of age. collaborative home infant monitoring evaluation (chime) study group prospective multicenter study of bronchiolitis: predicting safe discharges from the emergency department delays in discharge in a tertiary care pediatric hospital using quality improvement to optimise paediatric discharge efficiency bronchiolitis in infants and children: treatment; outcome; and prevention wolters kluwer health duration of illness in infants with bronchiolitis evaluated in the emergency department duration of illness in ambulatory children diagnosed with bronchiolitis a clinical pathway for bronchiolitis is effective in reducing readmission rates measuring hospital quality using pediatric readmission and revisit rates pediatric readmission prevalence and variability across hospitals preventability of early readmissions at a children's hospital. pediatrics hospital readmission: quality indicator or statistical inevitability? children's hospitals with shorter lengths of stay do not have higher readmission rates trends in bronchiolitis hospitalizations in the united states preventable adverse events in infants hospitalized with bronchiolitis key: cord- -q s authors: van hoek, b.; verkade, h.j.; porte, r.j. title: levertransplantatie date: - - journal: leverziekten doi: . / - - - - _ sha: doc_id: cord_uid: q s in verrichtte thomas starzl in denver de eerste levertransplantatie bij de mens. in werden in nederland de eerste twee (auxiliaire, zie par. . . ) levertransplantaties verricht in leiden en arnhem, in startte cambridge. helaas resulteerden de eerste levertransplantaties niet in langetermijnoverleving als gevolg van niet-optimale operatietechniek, matige immuunsuppressie en onbekendheid met complicaties. eindstadium chronische leverziekte betreft met name levercirrose, maar ook bijvoorbeeld het budd-chiari-syndroom, in een eindstadium. dat wil zeggen dat er sprake is van leverfalen (zie hoofdstuk ). de complicaties die hierbij optreden zijn vastgelegd in prognostische scores. de bekendste is de child-pugh-classificatie (tabel . ). bij een child-pugh klasse c, of b met progressie is er veelal een indicatie voor levertransplantatie. [ ] tegenwoordig wordt de mayo end-stage liver disease-score (meld-score) veel gebruikt. deze correleert invers met de driemaandsoverleving bij cirrose, ook bij hogere scores -waar de child-pughscore geen onderscheidend vermogen meer heeft. [ ] bij een meld-score tussen en is de kortetermijnoverleving bij cirrose zonder en met levertransplantatie gelijk (figuur . ). bij ontvangst van een lever met een hoge donor-risico-index [ ] is er op korte termijn alleen nadeel en pas later voordeel van de levertransplantatie. [ ] meestal vindt levertransplantatie dus pas plaats bij een meld-score van meer dan . [ ] de meld-score is opgebouwd uit het totaal bilirubinegehalte, de inr en het creatininegehalte (te berekenen op www.mdcalc.com en www.eurotransplant.nl). sommige ziekten zijn wel een indicatie voor levertransplantatie, maar hebben een lage meld-score, zoals polycysteuze leverziekte. daarvoor zijn er in eurotransplant uitzonderingen geformuleerd zodat deze patiënten extra punten krijgen, waardoor levertransplantatie toch vaak op tijd mogelijk is. voor een deel van de patiënten met een meld-score onder is mogelijk meld gecombineerd met het serumnatrium een betere voorspeller voor overleving, zoals in de uk-eld die in groot-brittannië wordt gebruikt. [ levertransplantatie voor levertumoren betreft met name het hepatocellulair carcinoom (hcc). aangezien hcc veelal in een cirrotische lever ontstaat, is resectie met voldoende behoud van functie vaak niet mogelijk. ook een centrale ligging van de tumor of portale hypertensie kunnen resectie te riskant maken. resectie is dus de eerste optie, maar indien dit niet mogelijk is wordt levertransplantatie overwogen. er moet dan geen extrahepatische tumor aanwezig zijn. ter beoordeling van de uitbreiding worden een ct-thorax, ct-of mri-abdomen (vierfasencontrast) en een skeletscan verricht. soms is een laparoscopie nodig. bij ascites wordt deze geanalyseerd op maligne cellen. een tumor mag niet te groot zijn in omvang of aantal en er mag geen macrovasculaire invasie zijn, daar anders de kans op terugkeer van hcc na levertransplantatie te groot is. veelal worden daarbij de 'milaan-criteria' aangehouden: een tumor van maximaal cm of maximaal drie van ieder maximaal cm. [ ] de tumorvrije overleving na levertransplantatie is echter niet slechter met de zogenaamde ucsf-criteria, die iets ruimer zijn: een tumor van maximaal , cm of maximaal drie tumoren van maximaal , cm, met totale diameter van maximaal cm. downstaging tot binnen de milaan-criteria maakt soms levertransplantatie mogelijk. bij een fibrolamellair hcc be-hoeven genoemde grenzen niet aangehouden te worden. uit analyse van patiënten die getransplanteerd zijn terwijl ze buiten de milaan-criteria vielen, blijkt dat met name micro-angio-invasie een slechte prognostische marker is en dat de overleving bij grotere of meer dan drie tumoren nog goed kan zijn als er geen micro-angio-invasie is. er wordt momenteel hard gezocht met onder andere micro-arrays naar andere merkers die het biologisch gedrag van de tumor weergeven, zodat de selectie voor levertransplantatie kan verbeteren. bij een deel van de patiënten keert hcc namelijk terug na levertransplantatie, met name in lever, longen of botten. om de wachttijd te overbruggen wordt zo mogelijk lokale ablatieve therapie van de tumor(en) gedaan met radiofrequente ablatie (rfa), percutane ethanolinjectie (pei), transkatheter arteriële chemoembolisatie (tace), lokale intrahepatische radiotherapie met yttrium (sirt) of combinaties daarvan. er zijn aanwijzingen dat door deze aanvullende multimodale therapie op de wachtlijst de tumorvrije overleving na levertransplantatie verbetert. [ ] mogelijk verkleint immuunsuppressie met sirolimus ook de kans op recidief. waarschijnlijk is het ook goed om in ieder geval een halfjaar te wachten met levertransplantatie na het starten van genoemde aanvullende therapie: bij patiënten met een biologisch agressieve tumor wordt dan onterechte levertransplantatie vermeden. de multi-tyrosinekinase-en angiogeneseremmer sorafenib verlengt de overleving bij anders onbehandelbaar hcc; wellicht gaan deze of andere systemische behandelingen een rol spelen als (neo)adjuvante therapie bij hcc. het cholangiocarcinoom is in principe een contra-indicatie voor levertransplantatie, omdat deze tumor een zeer hoge recidiefkans na levertransplantatie heeft: er blijft vrijwel altijd tumorweefsel achter ten gevolge van microscopische lokale doorgroei. recentelijk is in onderzoekssetting levertransplantatie ingepast als eindstation na intensieve voorbehandeling met chemoradiotherapie en stagiëring, waarbij vele patiënten voor levertransplantatie afvielen. de patiënten die uiteindelijk levertransplantatie ondergingen, hadden een acceptabele tumorvrije overleving. [ ] wellicht is er dus in een dergelijk strict protocol in de toekomst toch plaats voor levertransplantatie bij geselecteerde patiënten met cholangiocarcinoom. overige tumoren waarvoor bij geselecteerde patiënten levertransplantatie verricht wordt, zijn het hemangio-endothelioom, neuro-endocriene tumoren, hepatoblastoom (naast chemotherapie) en nog enkele zeldzame tumoren met een goede prognose na levertransplantatie. soms is levertransplantatie een optie bij grote goedaardige tumoren, al wordt dan meestal naar andere oplossingen gezocht. voor levertransplantatie bij metastasen van bijvoorbeeld een coloncarcinoom, is momenteel geen plaats wegens de hoge recidiefkans en de schaarste aan donorlevers. bij patiënten zonder voorafgaande leverziekte die hyperacuut, acuut of subacuut leverfalen ontwikkelen (zie hoofdstuk voor kliniek, oorzaken en overige behandeling) is er vaak een indicatie voor hoogurgente levertransplantatie. [ ] [ ] [ ] bij de indicatiestelling voor levertransplantatie worden meestal de criteria van het king's college hospital (kch-criteria) aangehouden (kader). bij virale etiologie (hepatitis b) werden in het verleden de clichycriteria gehanteerd, maar die zijn minder accuraat. de kch-criteria hebben een goede negatief voorspellende maar matige positief voorspellende waarde. dit laatste kan mogelijk verbeterd worden door het toevoegen van serumlactaat (> , mmol/l bij opname of > , mmol/l na resuscitatie met infuus - uur na opname). [ de positief en negatief voorspellende waarde van een meld-score onder of boven is ongeveer gelijkwaardig aan de kch-criteria. ook de apache ii-score en de sofa-score zouden bruikbaar zijn. volgens een recente publicatie zou aanwezigheid van drie van de volgende zes criteria de noodzaak van een levertransplantatie beter voorspellen dan de kch-criteria en de meld-score: [ ] -leeftijd jaar of ouder; -interval tussen icterus en encefalopathie > dagen; -encefalopathie graad - ; -hersenoedeem; -pt > seconden; -creatinine > mmol/l ( , mg/dl). totdat de overige scores goed gevalideerd zijn wordt aangeraden de kch-criteria, eventueel aangevuld met het serumlactaat te gebruiken. voor sommige indicaties, zoals de ziekte van wilson, is een aparte score ontworpen. [ ] [ ] vaak is het geïndiceerd om de preoperatief gestarte koeling, conti-nue venoveneuze hemofiltratie en antibiotica peroperatief en postoperatief enige tijd voort te zetten. met name moet niet alleen vóó r maar ook tijdens de levertransplantatie gewaakt worden voor toename van hersenoedeem en hypoglykemie. [ ] j . . enzymdeficiënties die gelokaliseerd zijn in de lever, die niet op andere wijze te behandelen zijn en die aanleiding geven tot irreversibele, levensbedreigende aandoeningen kunnen een indicatie zijn voor levertransplantatie. deze indicaties spelen vooral op de kinderleeftijd (zie par. . ). bij volwassenen gaat het bijvoorbeeld om familiaire amyloïdotische polyneuropathie. in zulke gevallen waarbij de lever op één enzym na normaal is, kan soms een dominolevertransplantatie plaatsvinden (zie par. . . ). bij hemochromatose, de ziekte van wilson of alfa- antitrypsinedeficiëntie is het leverfalen bij cirrose meestal de reden van levertransplantatie, al kan de ziekte van wilson zich als acuut leverfalen presenteren. [ ] j . . ongeveer procent van de levertransplantaties is een retransplantatie. dit kan nodig zijn bij falen van een transplantatielever of bij recidiverende sepsis vanuit de donorlever, bijvoorbeeld bij galwegstricturen met cholangitis. in de eerste twee weken na de eerste levertransplantatie komt dit vooral door primaire non-functie of bij een arteria hepatica-trombose. later na transplantatie kan dit komen door bijvoorbeeld non-anastomotische stricturen in de galwegen of terugkeer van de oorspronkelijke ziekte. bij een vroeg falen van een transplantaat is er een hoge mortaliteit op korte termijn en mag een hoogurgente retransplantatie plaatsvinden; meer dan twee weken na een eerste levertransplantatie kan dit alleen in uitzonderingsgevallen. de eenjaarsoverleving van relevertransplantatie is lager (ongeveer %) dan van eerste levertransplantatie; dit komt vooral door de slechtere overleving bij vroege retransplantaties. levertransplantatie is tijdelijk of soms definitief niet mogelijk bij systemische infectie waarbij de infectiefocus niet in de lever ligt, bij extrahepatische maligniteit -tenzij follow-up genezing waarschijnlijk maakt -en bij andere aandoeningen die de prognose negatief beïnvloeden of kunnen leiden tot complicaties bij levertransplantatie (bijv. aandoeningen van hart of longen, zoals acute respiratory distress-syndroom, of extreme ondervoeding). ook indien een patiënt medicatie niet juist kan innemen, medische voorschriften niet begrijpt of niet in staat is de risico's van de behandeling en eisen die gesteld worden te begrijpen, is een levertransplantatie niet mogelijk. ook bij ernstige psychiatrie, actieve verslaving (uitgezonderd methadon), hersen-dood (met name bij comateuze patiënten, bijv. door inklemming van de hersenstam bij acuut leverfalen) en ernstige niet-reversibele portopulmonale hypertensie wordt geen levertransplantatie uitgevoerd. [ ] obesitas kan levertransplantatie moeilijker maken, maar heeft in afwezigheid van comorbiditeit geen invloed op de overleving en is dus geen absolute contra-indicatie. [ ] coronairlijden, nierinsufficiëntie, diabetes mellitus, obstructief longlijden (copd) en bindweefselziekten verkleinen de overlevingskans na levertransplantatie. [ ] ook bij een meldscore van meer dan daalt de overlevingskans na levertransplantatie vandaar dat levertransplantatie zo mogelijk voor dat moment moet plaatsvinden. seropositiviteit voor hiv is een relatieve contra-indicatie (met name bij hepatitis c). uitgebreide veneuze trombose tot in de vena mesenterica superior was vroeger een contra-indicatie, maar tegenwoordig zijn vaak oplossingen mogelijk in de vorm van renoportale anastomose of cavoportale transpositie. voordat iemand op de wachtlijst geplaatst wordt, is een uitvoerig vooronderzoek verricht en wordt alles in een multidisciplinair team besproken om de indicatie, de contra-indicaties en de timing goed af te wegen. de patiënt en zijn of haar naasten worden uitgebreid voorgelicht. ook tijdens de wachtlijstperiode wordt de patiënt geregeld onderzocht om veranderingen waar te nemen, medicatie bij te sturen en alles te optimaliseren voor de transplantatie. helaas overlijdt in nederland momenteel ongeveer procent van de patiënten op de wachtlijst voordat er een donorlever beschikbaar is. de donorlever wordt volgens een standaardprocedure door een hiervoor gecertificeerde chirurg verwijderd bij de donor. dit is meestal een hersendode, heart-beating (hb) donor, soms een non-heart-beating (nhb) donor -ook deceased from cardiac death (dcd) donor genoemd. de tijd tussen het bij de donor spoelen van de lever met koude bewaarvloeistof en het van het ijs halen van de lever bij de ontvangeroperatie heet de koude ischemietijd, de tijd tussen dit moment en de reperfusie met bloed van de ontvanger is de warme ischemietijd. de totale ischemietijd moet onder de uur -en liefst minder -blijven; onder de uur is optimaal. bij nhb-donoren is er per definitie een hartstilstand, dus een extra eerste warme ischemietijd, die kan leiden tot extra ischemie en/of reperfusieschade aan de lever en galwegen. door strenge selectie van donoren en het kort houden van ischemietijden kunnen even goede resultaten als met hb-donoren gehaald worden. [ ] [ ] voor implantatie wordt een donorlever uitgebreid nagekeken en voorbereid op implantatie. ook vindt controle van de donor plaats op onder andere hiv en hepatitis b en c. de kwaliteit van donorlevers verschilt en kan bijvoorbeeld worden weergegeven in een donor risico-index. [ ] eurotransplant alloceert levers op naam naar bloedgroep en gewicht en in volgorde van de meld-score, eerst binnen de regio (nederland en belgië zijn ieder een regio) en daarna erbuiten. bij hoge urgentie (acuut leverfalen of retransplantatie binnen twee weken) wordt direct volgens de eurotransplant-wachtlijst (dus ook over de grenzen heen) gealloceerd. het transplantatieteam beslist van tevoren of een door eurotransplant aangeboden lever geaccepteerd kan worden voor deze patiënt, op grond van gegevens van zowel donor als ontvanger. zo leidt een sterk vervette lever voor een oude ontvanger met hepatitis c tot een grote kans op complicaties. meestal wordt bij de ontvanger de zieke lever verwijderd en vervangen door een gehele donorlever op dezelfde plaats (orthotope levertransplantatie, olt). [ ] de operatie bij de ontvanger bestaat uit drie fasen: ( ) resectie van de eigen lever (met galblaas) via een bilaterale subcostale incisie, ( ) de anhepatische fase (zonder lever, waarin bloedingen gestopt worden en de nieuwe lever ingehecht wordt) en ( ) de fase na reperfusie van de donorlever. de eerste fase kan moeilijk zijn, met name bij ernstige portale hypertensie. soms is het nodig een tijdelijke portocavale shunt aan te leggen. vroeger werd de vena cava inferior, die immers door de lever loopt, met de lever verwijderd; er was dan een perfusiecircuit nodig om het bloed van de onderste helft van het lichaam naar het hart terug te brengen (figuur . ). tegenwoordig wordt de eigen lever van de vena cava 'afgepeld', waardoor deze cavo-cavale bypass niet meer nodig is. dit scheelt operatietijd en leidt tot minder bloedverlies. bij het inhechten wordt de onderzijde van de vena cava van de donor 'afgestapled' en de bovenzijde van de vena cava wordt side-to-side of end-to-side op de vena cava van de ontvanger gezet, de zogenaamde piggy-back-methode (figuur . ). na de cavo-cavale en de porto-portale anastomose volgt reperfusie. in deze fase kunnen grote hemodynamische schommelingen en verschuivingen in elektrolyten (bijv. kalium), het zuur-base-evenwicht en de stolling en kan fibrinolyse optreden. na de reperfusie wordt de arteriële anastomose gemaakt, meestal op de eigen leverarterie, soms via een conduit op de aorta. de ductus choledochus wordt vervolgens op de eigen ductus choledochus gezet, ofindien dat niet mogelijk is -op een roux-en-ydarmlis. bij de resectie is de galblaas verwijderd; de ontvanger krijgt geen galblaas terug, daar dit leidt tot infecties en steenvorming. tijdens de operatie worden vele laboratoriumwaarden frequent bewaakt. de stolling wordt integraal bewaakt met trombo-elastografie (teg) op de operatiekamer. zonodig wordt medicatie tegen hyperfibrinolyse gegeven. ook is een rapid infusion system aangesloten om bij veel bloedverlies in korte tijd veel bloed te kunnen transfunderen. zonodig worden met de cell-saver de cellen uit het verloren bloed gecentrifugeerd en met plasma van de bloedbank als autotransfusie teruggegeven. tegenwoordig is er meestal slechts weinig transfusie nodig. aan het eind van de vier tot tien uur durende operatie is te zien of de donorlever functioneert aan de stolling, het zuurstofverbruik en de functietesten. bij metabole ziekten en acuut leverfalen kan het bij hemodynamisch stabiele jongere patiënten aantrekkelijk zijn de eigen lever niet geheel te verwijderen en er een halve of hele donorlever naast te zetten. met een dergelijke auxiliaire levertransplantatie, worden bij geselecteerde patiënten goede resultaten bereikt. [ ] [ ] de oudere heterotope auxiliaire levertransplantatie (halt), waarbij een partieel transplantaat onder de eigen lever werd gezet, is grotendeels verlaten. tot nu toe wordt bij auxiliaire levertransplantatie in veel centra de auxiliair partieel orthotope levertransplantatie (apolt) gebruikt, met arteriali-satie van de vena portae, waarbij een deel van de eigen lever verwijderd wordt. recentelijk werd een verdere verbetering van de techniek ontwikkeld: de auxiliaire levertransplantatie met reno-portale anastomose (repalt) (figuur . ). [ ] hierbij komt de portaalveneuze inflow van het transplantaat uit de niervene van de ontvanger, en is vrijwel geen resectie van de eigen lever nodig, wat een aantal voordelen heeft. het blijkt dat bij twee derde van de overlevenden van auxiliaire levertransplantatie regeneratie van de eigen lever optreedt. [ ] de donorlever kan dan weer worden verwijderd -en soms zelfs hergebruikt -of de donorlever atrofieert door de afweeronderdrukkende medicatie uit te sluipen. [ ] het voordeel van auxiliaire levertransplantatie voor de patiënt is dat deze zonder de riskante immuunsuppressie verder door het leven kan. [ ] bij een hemodynamisch instabiele patiënt kan olt -of auxiliaire levertransplantatie met een grote resectie van de natieve lever [ ] -beter zijn omdat resectie van de necrotische lever het toxic liver-syndroom -door cytokines -kan tegengaan. soms kan een lever van een donor die ouder is dan jaar, meer dan kg weegt en minder dan vijf dagen op de icu gelegen heeft, gesplitst worden voor een volwassene (rechterkwab, figuur . ) en een kind (de kleinere linkerkwab, figuur . ). [ ] dit wordt een split levertransplantatie genoemd. ook kan in sommige gevallen een grote lever gesplitst worden voor twee volwassen ontvangers. [ ] een nadeel van split levertransplantatie is dat de extra ischemietijd en het resectievlak leiden tot meer complicaties en een iets lagere overlevingskans dan bij olt. als dit overlijden op de wachtlijst te- als iemand wegens een stofwisselingsdefect in de lever een levertransplantatie moet ondergaan, kan de verwijderde lever soms hergebruikt worden voor een andere -meestal urgente -ontvanger. dit kan bijvoorbeeld bij familiaire amyloïdotische polyneuropathie; het duurt dan ongeveer tien jaar voor de nieuwe ontvanger verschijnselen van polyneuropathie krijgt en geretransplanteerd moet worden. [ ] j . . donatie bij leven is in japan ontwikkeld, aanvankelijk als donatie van de linkerkwab van een ouder aan een kind. later werd ook de grotere rechterkwab gedoneerd van volwassene aan volwassene. uiteraard moeten potentiële donoren een uitgebreide beoordeling ondergaan. [ ] dan nog gaat donatie van een rechterkwab gepaard met ongeveer à procent mortaliteit en procent morbiditeit. bij donatie van segment en , van bijvoorbeeld volwassene aan kind, ligt het donatierisico een factor lager. het is bij volwassen ontvangers meestal nodig de rechterkwab te doneren omdat zowel de ontvanger als de donor voldoende levermassa over moeten houden (dat is als de lever > , % van het lichaamsgewicht is). is dit niet het geval, dan treedt het smallfor-size-syndroom (sfs) op, met icterus en leverfalen. dit is voor een deel het gevolg van hyperperfusie wat leidt tot beschadiging van sinusoïdaal endotheel en een belemmerde afvloed. ook als de grote levervenen niet op de circulatie van de ontvangerworden aangesloten, kan een belemmerde afvloed en daardoor een functioneel sfs ontstaan. sinds kort worden ook twee linkerkwabben van twee donoren samen in een ontvanger getransplanteerd. [ ] dit garandeert voldoende levervolume bij de ontvanger én de donoren. in azië, de verenigde staten en sommige centra in europa maakt levertransplantatie met een levende donor een substantieel percentage van het aantal levertransplantaties uit. in nederland is levertransplantatie met een levende donor begonnen, al is er vooralsnog terughoudendheid vanwege de risico's voor de donor. [ ] de resultaten bij de ontvangers zijn zeker even goed als bij olt met een postmortale donor en mogelijk beter indien de overleving op de wachtlijst meegeteld wordt. [ ] j . . tijdens de operatie en daarna levenslang wordt immuunsuppressie gegeven om afstoting van de donorlever te voorkomen. [ ] dit treedt nog maar bij ongeveer procent van de patiënten op. de dosering kan na ongeveer drie maanden omlaag naar een onderhoudsdosis. meestal wordt begonnen met corticosteroïden, een calcineurineremmer (tacrolimus of cyclosporine) en eventueel mycofenolaatmofetil. ook wordt vaak in de eerste dagen na levertransplantatie een infuus met anti-cd , bijvoorbeeld basiliximab, gegeven. verder is het gebruikelijk de corticosteroïden tussen drie en zes maanden verder af te bouwen en zo mogelijk te stoppen. rond de operatie geven de meeste centra intraveneus antibiotica, bijvoorbeeld gedurende uur. sommige geven selectieve darmdecontaminatie met niet-resorbeerbare antibiotica -eventueel aangevuld met norfloxacine. in de eerste dagen wordt frequent laboratoriumonderzoek verricht en echografie van de buik verricht, onder andere om arteriahepaticatrombose uit te sluiten. ook wordt gelet op vochtcollecties (eventueel diagnostische punctie met granulocytgetal en microbiologisch onderzoek) en op galwegdilatatie (al is dat een laat optredend fenomeen bij afvloedbelemmering). meestal wordt in de eerste week een ct-abdomen verricht met dezelfde doelstellingen. de medicatie wordt ingesteld en aangepast op bloedspiegels; eventuele rejectie nabloeding of gallekkage kunnen redenen zijn voor relaparotomie. zo mogelijk wordt geprobeerd de patiënt eerst te stabiliseren. leverschade door ischemie en/of reperfusie leidt meestal tot een asat-en alat-piek in de eerste drie dagen als gevolg van necrose in zone ; meestal dalen de aminotransferasen snel in de eerste dagen en herstelt deze schade. aminotransferasen boven in de eerste drie dagen zijn vaak een uiting van ernstige leverschade door ischemie en/of reperfusie en zo'n aminotransferasepiek kan gevolgd worden door een periode van intrahepatische cholestase. behalve de bekende complicaties van grote operaties zoals nierfalen, acuut respiratoir distress syndroom (ards) en hemodynamische instabiliteit, zijn er een aantal specifiek aan levertransplantatie gekoppelde problemen. primaire non-functie (pnf) komt bij tot procent van de levertransplantatie voor, primaire graft-disfunctie (pgd) of initiële slechte functie (ipf) bij procent. manifestaties zijn: slechte stolling, bloeden uit buikdrains, hoge aminotransferasen, oplopend bilirubinegehalte, hypoglykemie en geen galuitvloed (al wordt dit niet vaak meer gezien nu er meestal geen externe galdrain gebruikt wordt). systemische uitingen zijn icterus, encefalopathie, hemodynamische instabiliteit, acidose en nierfalen. het wordt veroorzaakt door ischemie en/of reperfusieschade, soms bovenop al aanwezige schade in de donorlever door onder andere steatose, celoedeem, hersendood van de donor en shock. [ ] er zijn enkele bekende risicofactoren voor pnf en pgd, zoals een hogere leeftijd van de donor, een sterk vervette donorlever, een hoog serumnatrium van de donor en de duur van ic-verblijf. bij de ontvanger zijn dit het creatinine-en bilirubinegehalte, de life support en de urgentiestatus op de wachtlijst. verder zijn er intraoperatieve factoren zoals de koude ischemietijd, de hoeveelheid bloedtransfusie en de warme ischemietijd. [ ] aanvankelijke berichten dat infusie van prostaglandine e tegen pnf en pgd zou beschermen, konden niet worden bevestigd. bij nhb-donoren geven een leeftijd onder jaar, warme ischemie bij de donor van minder dan minuten en een koude ischemietijd van minder dan uur bij een niet-urgente ontvanger een uitkomst die vergelijkbaar is met die bij hb-donoren. [ ] j . . trombose van de arteria hepatica (hat) komt bij ongeveer procent van de levertransplantaties voor, met name bij aanwezigheid van arteriële varianten en vooral in de eerste dagen na transplantatie. hat uit zich meestal doordat asat en alat plotseling zeer fors stijgen. vervolgens ontstaan necrotische gebieden in de lever en/of galwegstricturen. een aneurysma ter hoogte van de arteriële anastomose kan het gevolge zijn van een lokale (mycotische) infectie. hat of schade aan de arteriële plexus kan ook leiden tot intrahepatische cholestase door downregulatie van galzuurtransporters in de hepatocytmembraan, alvorens stricturen van de grotere galwegen optreden. [ ] bij een deel van de patiënten kan een hat die maanden tot jaren na levertransplantatie optreedt met opmerkelijk weinig symptomen gepaard gaan, maar in de acute fase kan alleen snelle interventie een retransplantatie voorkomen. soms is er een arteriële stenose die met een stent behandeld kan worden. [ ] bij de donor wordt zonodig een arteriële reconstructie verricht. de noodzaak voor zo'n reconstructie en een hogere donorleeftijd zijn risicofactoren voor hat. zonodig wordt via een conduit een anastomose rechtstreeks op de aorta gemaakt. meestal wordt een end-to-endanastomose op de arteria hepatica gemaakt. direct na levertransplantatie en de volgende dag -en bij verdenking op hat -wordt de doorgankelijkheid van de arteria hepatica met echo-doppler gecontroleerd. als geen arterieel signaal gezien wordt, moet direct een ct-angiografie verricht worden. hat en trombose van de vena portae (pvt) kunnen gepaard gaan met infectie of uitingen van leverfalen zoals genoemd in paragraaf terwijl vroeger een acute cellulaire rejectie bij procent van de patiënten na levertransplantatie voorkwam, is dat nu -met de verbeterde immuunsuppressie -nog maar procent. vroeger trad rejectie meestal in de eerste twee weken na levertransplantatie op, tegenwoordig ook wel later na het verlagen van de immuunsuppressie, na conversie naar een ander immuunsuppressivum of bij onjuiste inname van de medicatie. rejectie uit zich eerst door oplopende leverenzymen, daarna door vermoeidheid, drukgevoel in de rechterbovenbuik, en soms laat door koorts en icterus. [ ] bij verdenking op rejectie moeten andere oorzaken uitgesloten worden en wordt een leverbiopsie verricht die deze diagnose ondersteunt: een portaal lymfocytair infiltraat, endotheliitis en even-tueel cholangitis. meestal worden daarvoor de banff-criteria aangehouden. behandeling met hoge doses corticosteroïden gedurende enkele dagen, in combinatie met het optimaliseren van de onderhoudsimmuunsuppressie, waarbij tijdelijk serumspiegels als vlak na levertransplantatie gehanteerd worden, is bijna altijd succesvol. onbehandelde rejectie leidt tot chronische rejectie met verlies van galgangen in de portadriehoekjes, ductopenie. waar rond nog bij procent van de levertransplantaties chronische ductopene rejectie optrad, is dat nu nog bij procent van alle levertransplantaties het geval, vooral bij niet-herkende late rejectie onder te lage immuunsuppressie of soms bij interferontherapie voor hepatitis c. chronische ductopene rejectie uit zich als cholestase en meestal is retransplantatie de enige optie. j . . infectie bij een grote buikoperatie kunnen gemakkelijk infecties optreden, zeker bij een zieke patiënt die zware immuunsuppressie krijgt. infecties zijn belangrijke veroorzakers van morbiditeit en mortaliteit na levertransplantatie. [ ] [ ] bacteriële en soms mycotische infecties vlak na levertransplantatie veroorzaken vooral peritonitis, pneumonie, cholangitis en urineweginfecties. geïnfecteerde vochtcollecties of hematomen in de bovenbuik komen met name geregeld voor. de natuurlijke immuniteit van donor en ontvanger spelen een rol bij de kans op infecties na levertransplantatie. [ ] bij koorts of andere tekenen van infectie is snelle diagnostiek (soms inclusief ascitespunctie of bronchoscopische lavage) en behandeling nodig. [ ] door selectieve darmdecontaminiatie is waarschijnlijk een verschuiving van gramnegatieve naar meestal minder heftig verlopende grampositieve infecties te bewerkstelligen en door gerichte profylaxe is het aantal gistinfecties te verminderen. [ ] ook virale infecties kunnen optreden, bijvoorbeeld primo-infectie met of reactivatie van het cytomegalovirus (cmv). in het eerste jaar vindt geregelde controle van cmv-dna in het bloed plaats, met pre-emptieve therapie met (val)gancyclovir zodra deze test positief wordt. bij combinatie van een seropositieve donor en een seronegatieve ontvanger met betrekking tot igg-anti-cmv wordt (val)gancyclovir profylactisch gegeven gedurende drie maanden. [ ] het epstein-barr-virus (ebv) kan (bij % van de levertransplantaties) leiden tot posttransplantatielymfomen (ptld); die zijn tegenwoordig vaak curatief te behandelen en voor een deel mogelijk te voorkomen door ebv-dna-metingen in het eerste jaar, met zonodig aanpassen van de immuunsuppressie. [ ] als een patiënt zich na levertransplantatie presenteert met koorts moet onmiddellijk diagnostiek op de spoedeisende hulp plaatsvinden en wordt direct gestart met intraveneuze antibiotica (bijv. combinatie van cefuroxim en gentamycine). als na uur de kweken negatief blijven, kan daarmee vaak gestopt worden en anders worden de antibio-tica daarna gericht op geleide van de kweek. als een cholangitis de oorzaak kan zijn, kan een ercp nodig zijn ter uitsluiting en behandeling van galwegstricturen. j . . galwegcomplicaties uiten zich vaak door cholestase en cholangitis. bij galwegstricturen zijn er bij echografie vaak geen uitgezette galwegen zichtbaar, mogelijk doordat de patiënt zich onder immuunsuppressie snel met cholangitis presenteert. diagnostiek vindt plaats met behulp van mrcp of ercp. kort na levertransplantatie kan er een gallek op de anastomose zijn, meestal met een galcollectie ernaast, een biloom. een klein gallek is vaak via ercp met papillotomie en het plaatsen van een endoprothese te behandelen (figuur . ). soms, met name bij een biliodigestieve anastomose, is percutane transhepatische cholangiografische (ptc) galwegdrainage nodig; deze kan tegenwoordig gedeeltelijk door ercp met de dubbelballonendoscoop vervangen worden. bij een biloom wordt dan meestal tevens een percutane drain in het biloom geplaatst, met kweek van de inhoud en bij infectie tijdelijk behandeling met antibiotica. een groot gallek vereist soms chirurgie (reanastomose of conversie naar biliodigestieve anastomose met een roux-en-y-lis). de oorzaak van een gallek is vaak de matige doorbloeding van het onderste deel van de ductus choledochus van de donor. dit is ook de oorzaak van anastomotische stenosen. die treden vooral op in het eerste halfjaar na levertransplantatie en zijn meestal goed via ercp of ptc met dilatatie en endoprothese of percutane drainage te behandelen. ook hiervoor is soms reoperatie nodig. [ ] niet-anastomotische galwegstricturen kunnen alleen de donorcholedochus betreffen (type a), de bifurcatie (type b), ook het gebied daarboven (type c) of de gehele lever (type d). ze worden ook wel ischemic-type biliary lesions (itbl) genoemd, omdat de meeste van deze niet-anastomotische stricturen in het eerst halfjaar ontstaan en vooral aan ischemie en/of reperfusieschade gerelateerd lijken te zijn. latere stricturen lijken meer aan cmv en bijvoorbeeld terugkerende scleroserende cholangitis gerelateerd te zijn. [ ] stricturen van type a en type b zijn vaak wel via ercp of ptc te behandelen, bij type c en type d kan retransplantatie nodig zijn, al kunnen sommige patiënten met onderhoudsantibiotica langdurig vrij van cholangitis blijven. bij olt met een lever van een hartdode donor treden meer galwegstricturen op, al kan de patient-en transplantaatoverleving bij goede selectie van donoren hetzelfde zijn als bij olt met een lever van een hersendode donor. [ ] j . . tumoren vaker dan posttransplantatielymfomen komen huidtumoren voor na levertransplantatie. als de immuunsuppressie een mtor-remmer (sirolimus of everolimus) bevat, komen minder huidtumoren voor. patiënten die levertransplantatie hebben ondergaan wegens door alcoholgebruik veroorzaakte cirrose, hebben een verhoogde kans op orofarynxen slokdarmtumoren. na levertransplantatie wegens primaire scleroserende cholangitis en bij inflammatoire darmziekte is de kans op colonkanker sterk verhoogd en wordt jaarlijkse colonoscopie met biopsieën ter uitsluiting van dysplasie aangeraden. ook van andere tumoren is de incidentie enigszins hoger na levertransplantatie dan in de gewone populatie. j . . het hepatitis b-virus (hbv) moet bij een hbsagpositieve patiënt onderdrukt worden tegen terugkeer van actieve hbv-infectie in de nieuwe lever. hiervoor wordt een combinatie van anti-hbs-antistoffen en replicatieremmers gebruikt, waarmee dit meestal goed lukt. het is wel nodig de hbv-dnaload te vervolgen en zonodig de hbv-medicatie aan te passen. als hepatitis c nog aanwezig is ten tijde van levertransplantatie, kan dit in procent van de gevallen leiden tot enige mate van chronische hepati-tis na levertransplantatie; ongeveer procent van de patiënten krijgt actieve hepatitis c en ontwikkelt versneld cirrose in het transplantaat, leidend tot een verminderde overleving en retransplantatie. ook auto-immuunhepatitis, primaire biliaire cirrose en psc kunnen in het transplantaat terugkeren. mogelijk kan de activiteit daarvan met veranderde immuunsuppressie en ursodeoxycholzuur verminderd worden. ook een aantal andere aandoeningen, waaronder hepatocellulair carcinoom, kan terugkeren in de getransplanteerde lever. de calcineurineremmers cyclosporine en tacrolimus kunnen leiden tot chronisch nierfalen en hypertensie. om deze reden staan schema's waarbij in het eerste jaar van een calcineurineremmer naar een mtor-remmer gegaan wordt momenteel in de belangstelling. bij cyclosporine kan haargroei in het gelaat optreden, bij tacrolimus -vooral in combinatie met prednison -komt vaker diabetes mellitus voor. corticosteroïden kunnen osteopenie en osteoporose verergeren of doen ontstaan en leiden tot cosmetische bezwaren, water-en zoutretentie, hypertensie en soms psychische klachten. azathiopri- ne en mycofenolaat-mofetil kunnen leiden tot maagklachten en de laatste ook tot diarree en beide kunnen cytopenie geven. mtor-remmers (sirolimus, everolimus) kunnen leiden tot huidklachten of -bij hoge doses -mondulcera. dit zijn slechts enkele voorbeelden. vaak zijn bijwerkingen de reden om de immuunsuppressie te wijzigen, soms moet medicatie bijgegeven worden wegens bijwerkingen (antihypertensiva, orale antidiabetica, insuline). verhoogde gevoeligheid voor infectie blijft een van de belangrijkste bijwerkingen van immuunsuppressie. immuunsuppressie verhoogt de kans op maligniteiten, meestal van de huid, in ongeveer à procent van de olt's treden lymfomen op. dit zijn vaak aan het ebv-virus gerelateerde lymfomen waarbij met verlagen van immuunsuppressie, anti-cd en eventueel chemotherapie vaak volledige remissie te bereiken is. keel-en slokdarmkanker bij ex-alcoholisten en colonkanker bij olt voor psc zijn reeds genoemd. wellicht zijn in de toekomst schema's die immuuntolerantie opwekken mogelijk, waardoor minder immuunsuppressie benodigd zal zijn. j . in figuur . is de verbetering van de patiëntoverleving na levertransplantatie in de loop er jaren weergegeven. momenteel is er een goede patiëntoverleving in gespecialiseerde centra: een jaar na levertransplantatie is dat procent en na vijf, tien en vijftien jaar respectievelijk , en procent. [ ] de overleving van het transplantaat (graft survival) ligt meestal ongeveer procent lager. de grootste risico's op overlijden liggen in de eerste drie maanden na levertransplantatie, en dit houdt verband met de genoemde complicaties. veel patiënten voelen zich vanaf drie maanden na de levertransplantatie veel beter dan gedurende lange tijd daarvoor, al is de kwaliteit van leven niet altijd optimaal. zo komt vermoeidheid vaak voor na levertransplantatie waarvoor diverse oorzaken kunnen zijn, zoals bijwerkingen van medicatie en stress. velen kunnen sporten, de helft van de patiënten gaat na levertransplantatie werken. als mensen zich beter voelen is het belangrijk het nut van goede inname van medicatie, onthouding van alcohol, geregelde controles en het direct ondernemen van actie bij koorts nog eens te onderstrepen. levertransplantatie bij kinderen verschilt van die bij volwassenen met betrekking tot indicaties, pretransplantatiezorg ten aanzien van optimaliseren van groei en ontwikkeling, operatietechnische aspecten en epidemiologie van postoperatieve complicaties, zoals posttransplantatie lymfoproliferatieve ziekte (ptld). j . . cholestatische leverziekte is bij tot procent van de kinderen de indicatie voor levertransplantatie. andere belangrijke indicaties zijn metabole ziekten en acuut leverfalen (elk - %). eindstadium cholestatische leverziekte ten gevolge van biliaire atresie is wereldwijd verantwoordelijk voor ongeveer de helft van alle levertransplantaties bij kinderen. biliaire atresie (galgangatresie) heeft een incidentie van op . - . , hetgeen voor nederland betekent dat de diagnose jaarlijks bij tien nieuwe patiënten wordt gesteld. andere cholestatische leverziekten die bij kinderen een indicatie voor levertransplantatie kunnen betekenen, zijn onder andere de ziekte van alagille (een autosomaal dominante aandoening met gelaatsafwijkingen (dysmorfie), congenitale hartafwijkingen, cholestatische geelzucht en botafwijkingen), congenitale leverfibrose, vormen van progressieve familiaire intrahepatische cholestase (pfic) en neonatale hepatitissyndromen. j . . . galgangatresie als indicatie voor levertransplantatie galgangatresie is een ernstige, potentieel fatale leverziekte. de aandoening is gekenmerkt door een progressieve, inflammatoire destructie van de extrahepatische en soms ook van de intrahepatische galwegen. meestal wordt de ziekte op de leeftijd van tot weken ontdekt bij overigens vaak (nog) goed gedijende zuigelingen die worden onderzocht wegens geelzucht, ontkleurde ontlasting en donkere urine. een belangrijke valkuil is het klinisch sluipende beloop waarbij er in de eerste fase geen ontkleurde ontlasting en/of donkere urine behoeven te zijn. onbehandeld leidt galgangatresie tot terminaal leverfalen op de leeftijd van maanden tot jaar. de oorzaak is onopgehelderd. bij tot procent van de kinderen gaat de aandoening gepaard met een belangrijk verschil tussen levertransplantaties bij kinderen en bij volwassenen is het feit dat de meeste postmortale orgaandonoren volwassenen zijn. hierdoor kan bij kinderen slechts zelden gebruikgemaakt worden een volledige (kinder)donorlever. als alternatief werd daarom eind jaren tachtig van de vorige eeuw in parijs de leverreductietechniek (reduced size-levertransplantatie) geïntroduceerd, door bismuth en houssin. [ [ ] voor het splitsen van donorlevers wordt gebruikgemaakt van chirurgische technieken die ook worden gebruikt bij leverresecties en die ervaring met leverchirurgie en goede kennis van de leveranatomie vereisen. behalve transplantatie van gereduceerde en gesplitste levers bestaat er sinds een aantal jaren ook de mogelijkheid van donatie door een levende donor. [ ] vaak is de donor een van de ouders van het zieke kind. bij de levende donor wordt een resectie verricht van de linkslaterale segmenten van de lever, die vervolgens gebruikt worden voor transplantatie bij het kind. j . . de postoperatieve situatie bij kinderen verschilt op een aantal punten van die bij volwassenen. in de eerste plaats is er bij jonge kinderen die een cholestatische leverziekte hadden vaak sprake van een ernstige groeiachterstand. in de jaren na transplantatie kan meestal een forse inhaalgroei bereikt worden. desondanks blijkt uit eigen historische gegevens dat ongeveer de helft van de getransplanteerde kinderen uiteindelijk een lengte bereikt die lager is dan - , sd van hun 'targetlengte'. [ ] het is aannemelijk dat een deel van deze achterblijvende groei kan worden toegeschreven aan het gebruik van corticosteroïden als immuunsuppressie. de laatste jaren is het corticosteroïdengebruik in de verschillende immunosuppressieve schema's sterk verlaagd. een tweede specifieke situatie na kinderlevertransplantatie is de verhoogde kans op een primoinfectie met het epstein-barr-virus (ebv) en het hieraan gekoppelde verhoogde risico op posttransplantatie-immunoproliferatieve ziekte (ptld). de meeste kinderen worden op jonge leeftijd getransplanteerd, op een moment dat zij immunonaïef zijn tegen ebv. zij lopen dan ook een hoge kans op het ontwikkelen van een primo-infectie met ebv onder immuungesupprimeerde omstandigheden. [ ] de ebv-bron kan het getransplanteerde orgaan zijn, het virus kan afkomstig van ebv-positieve oudere kinderen of volwassenen of er kan sprake zijn van een normale nosocomiale besmetting. ptld wordt in eerste instantie behandeld met het verlagen of volledig staken van de immuunsuppressie, met de hierbij horende risico's op rejectie van de lever. bij onvoldoende effect kunnen andere middelen noodzakelijk zijn, inclusief monoklonale antilichamen (anti-cd ) en chemotherapie. een derde aandachtspunt na levertransplantatie bij kinderen is de ontwikkeling. enerzijds kan de ontwikkeling vertraagd zijn geraakt door een langdurige ziekteperiode voor de transplantatie. anderzijds blijkt postoperatief de periode van puberteit en adolescentie tot psychologische druk te leiden in het gezin. in deze fase kan extra begeleiding nodig zijn om de autonomie van zorg en medicijngebruik succesvol van de ouders aan het kind over te dragen. j . . de langetermijnoverleving na levertransplantatie bij kinderen is goed. het overlevingspercentage een jaar na transplantatie bedraagt tot procent en dit percentage blijft in de daaropvolgende jaren redelijk stabiel. de tienjaarsoverleving bij kinderen na levertransplantatie bedraagt tot procent. uit groningse ervaringen is gebleken dat circa procent van de kinderen binnen tien jaar na de (eerste) levertransplantatie een retransplantatie nodig heeft vanwege fibrose en uiteindelijk cirrose van het transplantaat. de pathogenese van dit transplantaatverlies is niet geheel duidelijk en onderwerp van nader onderzoek. tot slot kan het niet genoeg benadrukt worden dat levertransplantatie bij kinderen vaak niet alleen maar levensreddend is; de kwaliteit van overleven maakt het meestal mogelijk om 'gezonde' levensdoelen te realiseren in termen van relaties, gezinsvorming, opleiding en werk. hepatocellular carcinoma: latest developments successful liver transplantation for hilar cholangiocarcinoma intensive care management of acute liver failure 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polymorphisms confer a major risk for severe infections after liver transplantation infectious complications limit the outcome of liver transplantation in medical urgency code patients risk stratification and targeted antifungal prophylaxis for prevention of aspergillosis and other invasive mold infections after liver transplantation impact of targeted oral ganciclovir prophylaxis for transplant recipients of livers from cytomegalovirus-seropositive donors posttransplantation lymphoproliferative disorder -the great mimic in liver transplantation: appraisal of the clinicopathologic spectrum and the role of epstein-barr virus a prospective study of standardized nonsurgical therapy in the management of biliary anastomotic strictures complicating liver transplantation nonanastomotic biliary strictures after liver transplantation, part : management, outcome, and risk factors for disease progression similar graft and patient survival in liver transplantation with brain death and controlled cardiac death donors with restrictive acceptance criteria long-term results after liver transplantation reduced size orthotopic liver graft in hepatic transplantation in children orgaan-en weefseldonatie en transplantatie. nederlandse transplantatie vereniging transplantation einer spenderleber auch bei zwei empfänger (splittingtranspantation): eine neue methode in der weiterentwicklung der lebersegementtransplantation levertransplantatie met een levende donor growth and final height after liver transplantation during childhood the value of prospective monitoring of ebv dna in blood samples of pediatric liver transplant recipients key: cord- -s qnhswe authors: shu, panpan; wang, wei; tang, ming; do, younghae title: numerical identification of epidemic thresholds for susceptible-infected-recovered model on finite-size networks date: - - journal: chaos doi: . / . sha: doc_id: cord_uid: s qnhswe epidemic threshold has always been a very hot topic for studying epidemic dynamics on complex networks. the previous studies have provided different theoretical predictions of the epidemic threshold for the susceptible-infected-recovered (sir) model, but the numerical verification of these theoretical predictions is still lacking. considering that the large fluctuation of the outbreak size occurs near the epidemic threshold, we propose a novel numerical identification method of sir epidemic threshold by analyzing the peak of the epidemic variability. extensive experiments on synthetic and real-world networks demonstrate that the variability measure can successfully give the numerical threshold for the sir model. the heterogeneous mean-field prediction agrees very well with the numerical threshold, except the case that the networks are disassortative, in which the quenched mean-field prediction is relatively close to the numerical threshold. moreover, the numerical method presented is also suitable for the susceptible-infected-susceptible model. this work helps to verify the theoretical analysis of epidemic threshold and would promote further studies on the phase transition of epidemic dynamics. epidemic threshold, which is one of the most important features of the epidemic dynamics, has attracted much attention recently. the existing studies have provided different theoretical predictions for epidemic threshold of the susceptible-infected-recovered (sir) model on complex networks, while the numerical verification of these theoretical predictions is still lacking. as a result, it is very necessary to develop an effective numerical measure for identifying the sir epidemic threshold. in this paper, the numerical identification of the sir epidemic threshold is systematically studied. we present a numerical method by analyzing the peak of the epidemic variability to identify the epidemic threshold. to understand the effectiveness of the variability measure, the distribution of outbreaks sizes is investigated near the epidemic threshold on random regular networks. based on the analysis of the cutoff hypothesis of the outbreak size distribution, we find that the variability measure can provide an excellent identification of the epidemic threshold. we further use the variability measure to verify the existing theoretical predictions on scale-free and real networks. the results show that the heterogeneous meanfield (hmf) prediction agrees very well with the numerical threshold, except the case that the networks are disassortative, in which the quenched mean-field (qmf) prediction is relatively close to the numerical threshold. the numerical method presented can effectively identify sir epidemic thresholds on various networks, and could be extended to other dynamical processes such as information diffusion and behavior spreading. this work provides us a deep understanding of epidemic threshold and would promote further studies on phase transition of epidemic dynamics. models for disease propagation are at the core of our understanding about epidemic dynamics on complex networks. , two epidemic models of particular importance are the susceptible-infected-susceptible (sis) and susceptibleinfected-recovered (sir) models. at each time step, an infected node can transmit a disease to each of its susceptible neighbors with probability b. at the same time, the infected nodes become susceptible again in the sis model or recover in the sir model with probability l. in the sis model, a critical value of the effective transmission rate k ¼ b=l separates the absorbing phase with only healthy nodes from the active phase with a stationary density of infected nodes. differently, no steady state is allowed in the sir model, but a threshold still exists above which the final fraction of recovered nodes is finite. the traditional theoretical study on the epidemic threshold of the sis model is based on the heterogeneous meanfield (hmf) theory, which means that all the nodes within a given degree are considered to be statistically equivalent. , according to the hmf theory, the epidemic threshold of sis model , is given by k hmf c ¼ hki hk i , where hki and hk i are the first and second moments of degree distribution p(k), respectively. as the quenched structure of the network and dynamical correlations between the state of adjacent nodes is a) neglected in the hmf theory, researchers have proposed an important improvement over the hmf theory-quenched mean-field (qmf) theory. the qmf theory fully preserves the actual quenched structure of the network described as its adjacency matrix, and the epidemic threshold is predicted to be [ ] [ ] [ ] where k n is the maximum eigenvalue of the adjacency matrix of a given network. considering that the existing theories more or less have some limitations (e.g., the hmf theory neglects the quenched structure of the network; qmf theory ignores dynamical correlations ) , some numerical methods such as the finite-size scaling analysis, susceptibility, and lifetime measure have been proposed to check the accuracies of different theoretical predictions for the sis model. among these existing methods, the relatively common one for a network with finite size n is the susceptibility measure where q denotes the outbreak size. in ref. , the susceptibility measure has been shown to be very effective for identifying the sis epidemic thresholds on various networks. for another paradigmatic epidemic model, the sir model, there have been a lot of theoretical studies on its epidemic threshold. the earliest theoretical study on the sir epidemic threshold is based on the assumption of homogeneous mixing, showing that the sir epidemic threshold is inversely proportional to the average connectivity hki. at the hmf level, the epidemic threshold of sir model takes the value on networks with power-law scaling pðkÞ $ k Àc , , where c is the degree exponent, the hmf approach predicts a vanishing threshold for scale-free networks with c and the finite threshold for c > . mapping the sir model to a bond percolation process, the epidemic threshold coincides with the result of eq. ( ) for a sir model with unit infection time (e.g., l ¼ ), and when the infection times vary among infected nodes (e.g., l < ), the epidemic threshold is given by according to the qmf theory, the epidemic threshold of the sir model has the same expression as eq. ( ). however, the qmf result is even qualitatively not correct, as the qmf predicts a vanishing threshold for power-law distributed networks with c > that is in conflict with the visually numerical results. although the numerical threshold of the sis model has attracted much attention, [ ] [ ] [ ] [ ] the systematic study of the numerical identification of the sir epidemic threshold is still insufficient. it is well known that the outbreak size becomes finite above the threshold k c . however, as the value of k increases, the outbreak size continuously changes from an infinitesimal fraction to a finite fraction in the sir model, and thus, it is difficult to determine the value of k at which the outbreak size turns to be finite. to our knowledge, there has no effective numerical method for identifying the sir epidemic threshold in previous studies. in this work, we perform extensive numerical simulations of the sir model on networks with finite size, and present a numerical identification method by analyzing the peak of the epidemic variability , (i.e., the maximal value of the epidemic variability) to identify the epidemic threshold. the effectiveness of the numerical measure is checked on random regular networks (rrn), where the hmf theory is exact. to get a deep understanding of the validity of the numerical method, we investigate the distribution of outbreaks sizes near the epidemic threshold. the robustness of the variability measure is confirmed by the analysis of cutoff hypothesis of the outbreak size distribution. we further employ the variability measure to verify the theoretical predictions on scale-free networks and real-world networks, where the results indicate that the hmf prediction agrees very well with the numerical threshold, except the case that the networks are disassortative, in which the qmf prediction is relatively close to the numerical threshold. in this section, we give the detailed description of the simulation of sir model, propose the numerical identification measure of the epidemic threshold, and make deep analysis of the effectiveness of the numerical measure. in the sir model, each node can be one of three states which are the susceptible state, infected state, and recovered state, respectively. at the beginning, one node is randomly selected as the initial infected (i.e., seed), and all other nodes are susceptible. at each time step t, each susceptible node i becomes infected with probability À ð À bÞ n i if it has one or more infected neighbors, where n i is the number of its infected neighbors. at the same time, all infected nodes recover (or die) at rate l and the recovered nodes acquire permanent immunity. time increases by dt ¼ , and the dynamical process terminates when all infected nodes are recovered. in this paper, l is set to , unless otherwise specified. the susceptibility measure can not only identify an effective sis epidemic threshold, but can also be used to determine the critical point of the percolation process. since the connection between sir and bond percolation is made through the assimilation of the size of the percolating giant component with the final number of recovered individuals, we check the effectiveness of susceptibility measure v for the sir model on rrn, where all nodes have exactly the same degree k. on these networks, the hmf prediction we compare the hmf prediction with the numerical threshold identified by the susceptibility measure k v p in fig. (a) , where the result shows that the numerical threshold of the sir model identified by v is significantly larger than =ðk À Þ. considering that the fluctuation of the outbreak size is large near the epidemic threshold, we try to identify the epidemic threshold by the variability measure , which is a standard measure to determine the critical point in equilibrium phase on magnetic system. the inset of fig. (a) shows that the variability d exhibits a peak over a wide range of k, so we estimate the epidemic threshold from the position of the peak of the variability k d p . on rrn with different values of k, we find that k d p is always consistent with the hmf prediction. when the degree k is given, we further consider the relationship between the epidemic threshold and the network size n in fig. (b) , where the numerical thresholds k v p and k d p do not change with n. k d p is very close to the hmf prediction, while there is an obvious gap between k v p and hmf prediction. from the above, we know that the variability d can identify an effective sir epidemic threshold, while the epidemic threshold identified by the susceptibility v is overestimated on rrn. thus, a new problem has arisen: why the variability d performs well but the susceptibility v goes awry for the sir model? b. analysis of the effectiveness of numerical identification measure next, we make an analysis of the effectiveness of the numerical identification measures above, by investigating the distribution of outbreak sizes which has the strong heterogeneity near the epidemic threshold. on rrn with k ¼ , where the epidemic threshold k c ¼ =ðk À Þ ¼ = , fig. (a) shows the distribution of outbreak sizes near k c . the outbreak sizes follow approximately an exponential distribution at k ¼ : that is smaller than k c . at k ¼ k c , the outbreak sizes follow a power-law distribution pðqÞ $ q a with a cutoff at some values, where a ' À : . [ ] [ ] [ ] since the disease may die out quickly or infect a subset of nodes when k > k c , the distribution of outbreak sizes is bimodal, , with two peaks occurring at q ¼ =n and q ' : for k ¼ : , respectively. moreover, the theoretical distribution of outbreak sizes (see appendix for details) is compared with the results obtained by numerical simulations in fig. (a) , where the theoretical probability from eq. (a ) is consistent with the numerical results for relatively small outbreak sizes (q < : ). at the epidemic threshold, the theoretical results also show that the outbreak sizes obey a power-law distribution with the exponent of about À . . when k > k c , some large outbreak sizes constitute a lump in the numerical scattergram, but the probability of large outbreak sizes cannot be solved from eq. (a ). we thus speculate that the nonignorable lump may affect the numerical identification of sir epidemic threshold for the susceptibility measure. to verify the speculation, fig. (b) investigates the effectiveness of the susceptibility measures under some cutoff hypotheses. we set the cutoff value of the outbreak size as r c , which means that only the outbreak sizes with q r c are used to numerically count the susceptibility v under the cutoff hypothesis. three kinds of r c are considered, where r c ¼ : corresponds to the maximum value of small outbreak size before the lump appears in the numerical scattergram, r c ¼ : means that the numerical scattergram consists of a part of the lump, and r c ¼ : means that there is a complete lump in the numerical scattergram. as shown in fig. (b) , the susceptibility measure can indeed give a quite effective estimate of the sir epidemic threshold when the whole lump is ignored (i.e., r c ¼ : ). with the increase of r c , the position of peak value of susceptibility v gradually shifts to the right for large outbreak sizes are considered. this shows that the susceptibility v loses its effectiveness in identifying the sir epidemic threshold due to the existence of the lump. in fig. (c) , the robustness of the variability d is further checked in theory. as the numerical distribution of the large outbreak sizes is concentrated, we assume that there is a lump located at r c with pðr c Þ ¼ À r q<r c pðqÞ in the theoretical probability distribution diagram of outbreak sizes while k > k c . based on such theoretical distribution, we plot the variability measure as a function of k for different values of r c in fig. (c) . since the variability d measures the heterogeneity of the outbreak size distribution, which is strongest at the epidemic threshold, - the peak position of the variability measure does not change with the position of the lump. from the above analysis, we can conclude that the variability d is effective in identifying the epidemic threshold of sir model, while the bimodal distribution of outbreak sizes for k > k c leads to the overestimation of the sir epidemic threshold when using the susceptibility v. we further verify the theoretical predictions on scalefree and real networks, by comparing them with the numerical thresholds from the variability d. we build scale-free networks (sfn) with degree distribution pðkÞ $ k Àc based on the configuration model. the so-called structural cutoff k max $ n = and natural cutoff k max $ n =cÀ (ref. ) are considered to constrain the maximum possible degree k max on sfn. differently, the degreedegree correlations vanish on scale-free networks with structural off, while the disassortative degree-degree correlations exist when c < for scale-free networks with natural cutoff, because high degree vertices connect preferably to low degree ones in this case. we consider the sir model on sfn with structural cutoff in figs. (a) and (c) , where the sir epidemic threshold increases monotonically with the degree exponent c, and the variation of epidemic threshold with network size n is approximately linear in logarithmic relationship. the hmf prediction k hmf c is very close to the numerical threshold k d p , while there is an obvious difference between the qmf prediction k qmf c and k d p . the sfn with natural cutoff are considered in figs. (b) and (d) , where the variations of epidemic threshold with c and n are similar to the results on sfn with structural cutoff. when c > , the hmf prediction is close to the numerical threshold, while there is a gap between the qmf prediction and the numerical threshold. since the disassortative degreedegree correlations exist for c < , there is a slight difference between k hmf c and k d p . in fig. (d) , the distinction between k hmf c and k d p becomes large with the increase of n for c ¼ : , while in such case the qmf prediction is always close to the numerical threshold since the principle eigenvector is delocalized when < c = . it can been seen from the above analysis that the numerical method presented provides quantitive indexes for the observations in ref. , where the hmf theory is relatively accurate for the sir model. to check the performance of the variability d on realworld networks, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] fig. depicts d as a function of k for hamsterster full network, which contains friendships and family links between users of the website hamsterster.com, and facebook (nips) network, which contains facebook user-user friendships. the numerical results intuitively show that the variability d always reaches a maximum value near the value of k above which the outbreak size q becomes finite. the theoretical predictions of the hmf theory and of the qmf theory are quite close to the numerical threshold identified by d on hamsterster full network, but they become poor on facebook (nips) network. considering the difference in the behaviors of the theoretical predictions on the two networks above, the detailed comparisons between the numerical and theoretical thresholds on other real networks are presented in table i . the results indicate that although the hmf prediction and the numerical threshold k d p ðsirÞ are nearly the same for assortative networks, there is an obvious difference between them for the networks showing significant disassortative mixing. the qmf prediction is relatively worse than the hmf prediction for assortative networks, but the former is close to k d p ðsirÞ for some disassortative networks (e.g., router views, caidi, and email contacts ). these findings numerically show the accuracies of existing theoretical predictions on real-world networks. in summary, we have studied the numerical identification of sir epidemic threshold on complex networks with finite size. we have checked the effectiveness of the susceptibility measure for sir on rrn, where the hmf is exact. the results showed an obvious gap between the numerical threshold identified by the susceptibility v and the hmf prediction. then, we proposed the numerical identification method by analyzing the peak of the epidemic variability, and found that the numerical threshold identified by the variability measure d agrees very well with the hmf prediction on rrn. in order to get a deep understanding of the effectiveness of the two numerical measures above, we have analyzed the distribution of outbreak sizes near the epidemic threshold k c . the outbreak sizes follow approximately an exponential distribution when k < k c . at the epidemic threshold, the outbreak sizes follow a power-law distribution with the exponent of about À . . when k > k c , the numerical distribution of outbreak sizes is bimodal with two peaks occurring at q ¼ =n and o( ), respectively. the probability of small outbreak sizes in theory is consistent with that obtained by numerical simulations, but the probability of large outbreak sizes that constitute a lump in the numerical scattergram cannot be obtained theoretically. based on the analysis of the cutoff hypothesis of the outbreak size distribution, we found that the susceptibility measure can give a fairly effective sir epidemic threshold when the lump is ignored. since the variability measure reflects the heterogeneity of the outbreak size distribution, it is always effective in identifying the epidemic threshold, where the distribution of outbreak sizes has the very strong heterogeneity. we further employed the variability measure to verify the theoretical predictions on scale-free and real networks. the hmf prediction is close to the numerical threshold on most of the networks, but on sfn with natural cutoff and degree exponent c < = , it becomes poor due to the existence of disassortative mixing. similarly, the hmf prediction agrees well with the numerical method on real networks with assortative mixing, while it becomes very poor for disassortative networks, where the qmf prediction is relatively close to the numerical threshold. these findings provide quantitive indexes for the accuracies of existing theoretical predictions from the perspective of simulation. as part of the discussion, we have considered the epidemic threshold for l < in fig. . the results on rrn and sfn all show that the numerical thresholds for l ¼ : are a little larger than those for l ¼ : . as shown in the inset, l ! leads to an epidemic threshold close to eq. ( ), while l ! leads to an epidemic threshold close to eq. ( ). it should be pointed out that the numerical threshold of sir model is inclined to the theoretical prediction k c ¼ hki hk iþðlÀ Þhki from the edge-based compartmental theory , when < l < . these findings could be complementary to some existing results. moreover, we have tried applying the variability measure to the identification of the sis epidemic threshold. as shown in fig. and table i , the numerical threshold k d p from the variability measure agrees very well with the threshold k v p identified by the susceptibility measure, whose validity for the sis model has been confirmed in ref. . this shows that the variability measure can also provide an effective estimate of the sis epidemic threshold. we have put forward a numerical method for identifying the epidemic threshold for sir model, which is also suitable for the sis model. this method can effectively identify epidemic thresholds on various networks, and could be extended to other dynamical processes such as information diffusion and behavior spreading. further work should be done to check the effectiveness of this method on more complicated networks (e.g., temporal networks and multilayer networks ). besides, the accurate analytic approximation of the epidemic threshold for general networks remains an table i. characteristics and epidemic thresholds of real-world networks. n is the network size, k max is the maximum degree, r is the correlation coefficient of the degrees, k hmf c and k qmf c are the hmf and qmf predictions for sir, respectively, k d p (sir) denotes the numerical threshold of sir identified by d, and k d p (sis) and k v p (sis) represent the numerical thresholds of sis identified by d and v, respectively. the threshold k c vs. c for l ¼ : (c) and l ¼ : (d) on sfn, where solid and empty symbols denote sfn with structural cutoff k max $ n = and natural cutoff k max $ n =cÀ , respectively. "circles" and "squares" denote k hmf c and k d p , respectively. inset: k c as a function of l on rrn with n ¼ and k ¼ . the results are averaged over  independent realizations on networks. important problem. this work helps to verify theoretical analysis of epidemic threshold and would promote further studies on phase transition of epidemic dynamics. shu et al. chaos , ( ) dynamical processes on complex networks infections diseases in humans networks: an introduction nonequilibrium phase transitions in lattice models monte carlo simulation in statistical physics proceedings of the th acm sigkdd international conference on knowledge discovery and data mining advances in neural information processing systems for the case of the sir model and similar models with no steady-state, the static properties (e.g., the final outbreak size and the epidemic threshold) of the epidemic outbreak can be mapped into a suitable bond percolation problem. in this framework, the distribution of occupied cluster sizes is related to the distribution of outbreak sizes. to get the distribution of small outbreak sizes in the sir model with a fixed value of k when recovery rate l ¼ , we will present the derivation of the distribution of small occupied cluster sizes in bond percolation with bond occupation probability k. after the percolation process on a general network with arbitrary degree distribution p k , the average degree of the occupied network a , which composes of vertices and occupied edges, is hk t i ¼ khki, where hki is the average degree of the original network a . and, the size distribution of the small subgraphs of network a iswhere s is the small subgraphs size and g ðzÞ is the generating function of the excess degree of network a . in addition, the generating function of degree distribution of a isand we thus havein a random regular network, which has an unique degree k with p k ¼ , we can easily obtain thatandsubstituting eq. (a ) into eq. (a ), we can obtain the distribution of small outbreak sizeswhere cðx þ Þ ¼ x!; a ¼ ðs À Þ; a ¼ sðk À Þ À ðs À Þ, and a ¼ sðk À Þ À . key: cord- -a sbdhhn authors: xiaokaiti, yilixiati; wu, haoming; chen, ya; yang, haopeng; duan, jianhui; li, xin; pan, yan; tie, lu; zhang, liangren; li, xuejun title: egcg reverses human neutrophil elastase-induced migration in a cells by directly binding to hne and by regulating α -at date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: a sbdhhn lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. egcg has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. in this study, we demonstrated a novel mechanism by which egcg reverses the neutrophil elastase-induced migration of a cells. we found that neutrophil elastase directly triggered human adenocarcinoma a cell migration and that egcg suppressed the elevation of tumor cell migration induced by neutrophil elastase. we observed that egcg directly binds to neutrophil elastase and inhibits its enzymatic activity based on the cdocker algorithm, md stimulation by gromacs, spr assay and elastase enzymatic activity assay. as the natural inhibitor of neutrophil elastase, α -antitrypsin is synthesized in tumor cells. we further demonstrated that the expression of α -antitrypsin was up-regulated after egcg treatment in neutrophil elastase-treated a cells. we preliminarily discovered that the egcg-mediated induction of α -antitrypsin expression might be correlated with the regulatory effect of egcg on the pi k/akt pathway. overall, our results suggest that egcg ameliorates the neutrophil elastase-induced migration of a cells. the mechanism underlying this effect may include two processes: egcg directly binds to neutrophil elastase and inhibits its enzymatic activity; egcg enhances the expression of α -antitrypsin by regulating the pi k/akt pathway. signaling pathways, such as the activator protein (ap), nuclear factor-κ b (nf-κ b), and phosphatidylinostitol- -oh kinase pathways (pi k/akt) [ ] [ ] [ ] . however, the molecular mechanisms underlying the anti-tumor metastatic properties of egcg in the specific context of inflammation remain elusive. in this study, we demonstrated additional contributions of inflammation to the progression of lung cancer metastasis and a novel molecular target of egcg, human neutrophil elastase, which induces lung cancer cell migration. neutrophils, as a component of the tumor microenvironment, have only recently been thought to play an important role in tumor growth and invasiveness . the presence of increased infiltrated neutrophils within tumors was significantly associated with a poorer clinical outcome in patients with bronchioloalveolar carcinoma . neutrophil elastase, which is released by activated neutrophils, is the most potent neutrophil protease. the potential substrates of this protease include cytokines and cytokine receptors, indicating that neutrophil elastase may regulate the inflammatory process . recent reports have shown that neutrophil elastase directly induces uncontrolled tumor proliferation in a lung adenocarcinoma mouse model and in lung epithelial tumor cells. alpha- antitrypsin (α -at, aat) is a serum glycoprotein that contains three potential glycosylation sites. as an acute-phase protein, aat is thought to play an important role in limiting host tissue injury triggered by proteases, particularly human neutrophil elastase (hne). the clinical relevance of aat is demonstrated in individuals with an inherited deficiency in circulating aat, who exhibit increased susceptibility to early-onset pulmonary emphysema and liver and pancreatic diseases . neutrophil elastase and aat are a protease and protease inhibitor counterpart pair . it has been assumed that in aat-deficiency, the protease/ anti-protease balance is shifted toward hne, which leads to extensive tissue damage, particularly by causing emphysema. xu et al. demonstrated that the natural polyphenol product curcumin inhibits tumor proliferation induced by neutrophil elastase via the upregulation of aat in lung cancer. altering the balance between aat and hne may represent an innovative form of lung cancer treatment. cell culture and treatment. the human lung adenocarcinoma cell line a was used for in vitro experiments. the cells were maintained in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) containing % fetal bovine serum (fbs), u/ml penicillin and μ g/ml streptomycin in a humidified incubator containing % co in air at °c. the cells were treated with different concentrations of neutrophil elastase (merck group, darmstadt, germany) and/or egcg (sigma-aldrich co., st. louis, mo, usa) for h and then evaluated as described below. cell viability assay. cell viability was determined using a cell titer ® aqueous one solution cell proliferation assay (mts assay, promega, madison, wi, usa). a cells were seeded on -well plates at a density of cells/well and were allowed to adhere for h. after incubation in neutrophil elastase and/or egcg for h, μ l of the mts solution was added to each well, followed by incubation for h at °c. the resulting color was assayed at nm using a microplate reader (bio-rad laboratories, inc., hercules, ca, usa). in vitro wound healing assay. the wound healing assay was performed as previously described (liang et al., ) to assess the capacity for cell migration. briefly, the a cells were seeded on a -well plate. when the cells reached - % confluence, scratches were applied using a sterile -μ l pipette tip. the cells were washed three times with phosphate buffered saline (pbs), and the initial wounds were examined under an olympus microscope. then, the cells were incubated in medium containing neutrophil elastase and/or egcg for h, and the wounds were photographed again. the rate of cell migration was evaluated based on the rate of wound closure. transwell migration assay. cell migration was further investigated using the transwell migration assay. a cells were suspended in serum-free dmem ( × cells) and placed in the upper chamber. the plate was incubated at °c for h, followed by fixation of the cells in % formaldehyde. the upper chamber was gently wiped with a cotton swab to remove the non-migrated cells, and the migrated cells on the underside of the polycarbonate filter were stained with crystal violet and counted under an olympus microscope (× magnification). immunofluorescence assay. a cells were seeded on glass coverslips in an environment containing % co in air at °c and then incubated in nm neutrophil elastase and μ m egcg for an additional h. the coverslips were gently washed in pbs, fixed in . % paraformaldehyde for min, permeabilized in pbs containing . % triton x- for min and blocked in pbs containing % bsa for min. the fixed cells were incubated in an anti-human zinc finger e-box-binding protein- (zeb- ) antibody (cell signaling technology, beverly, ma, usa) overnight at °c in a humidified chamber. then, the cells were washed and incubated in the corresponding dylight -conjugated secondary antibody (jackson immunoresearch laboratories, west grove, pa, usa) for h at °c. after washing in pbs, the cells were incubated in hoechst ( μ g/ml, sigma-aldrich) for min at room scientific reports | : | doi: . /srep temperature. after several washes, the immunostained specimens were observed under a tcs-sp laser scanning confocal microscope (leica microsystems, wetzlar, germany). . (accelrys software, inc., san diego, ca) using the charmm engine was used in this study to evaluate the potential molecular binding mode between egcg and hne. the program was run using a dell optiplex server (round rock, tx). the cdocker algorithm employed a strategy involving the generation of the initial ligand orientation in the active site of the target protein followed by molecular dynamics (md)-based simulated annealing and final refinement via energy minimization. the crystal structure of hne (pdb id: z f) was obtained from the protein data bank (http://www. rcsb.org/pdb/). the water molecules in the protein were removed, the protein was refined based on the root mean square deviation (rmsd), and egcg was docked to the protein according to the appropriate parameters. beginning from the egcg configuration, a set of different orientations was randomly generated. the co-crystalized ligand sei was used as the positive control ligand, and the binding site was defined based on the binding of sei and hne. the interaction energy was calculated to analyze the interaction between the ligand and the receptor. once the ligand was docked to the active site, a molecular dynamics (md) simulation was performed using the gromacs . . program. the gromos a force field and the spc/e water condition were selected during the topology process. water was position-restrained using a force constant (kpr) of kj mol − ·nm − . the protein was placed in the box at least . nm from the box edge, and the box was defined as a cube. during the genion process, cl − were added to the system for charge equilibration. a -ns md simulation was performed. the gromacs program was used to constrain the bonds involving hydrogen atoms, allowing for an integration interval of fs. the particle mesh ewald method using a threshold of nm was employed to account for the electrostatic interactions. a threshold of nm was used for the van der waals interactions. the non-bonded pair lists were updated every . ps. the coordinates were saved every ps. accuracy testing was performed by calculating the rmsd after re-docking the internal ligand to the protein using the algorithm. binding free energy calculations. the trajectories obtained from molecular dynamics were used for binding free energy calculations. the calculations were performed using gromacs molecular mechanics poisson boltzmann surface area (g_mmpbsa) method, implemented in gromacs . . the binding free energy for a protein-ligand complex is given as: where, △g binding is an estimate of absolute free energy of binding and g is the average free energy of complex, receptor and ligand respectively. the average free energy on the other hand is defined as: where, ts is the solute entropic contribution to the system at temperature t. e mm is the molecular mechanical energy obtained from bonded and non-bonded interactions within the system and can be represented by following equation: g solv is the average solvation free energy and is equal to the sum of electrostatic and non-polar terms and represented as: g pb gb is the electrostatic contributions to solvation free energy which is evaluated by finite differential poisson boltzmann (fdpb) in case of pbsa method. − g non polar is the hydrophobic non polar contributions to solvation free energy, calculated as: where sasa is the solvent accessible surface area and, γ and b are constants. for the g_mmpbsa calculations the molecular dynamic trajectories were split into "snapshots". a total , snapshots were extracted from , frames obtained in ns of production run. this allows not only to sample the flexibility of the binding site, but also to obtain a more reliable free energy estimate of binding than compared to a single snapshot calculation. surface plasmon resonance (spr) biosensor analysis. the binding affinity of egcg to hne in vitro was assayed using the spr-based biacore instrument (biacore ab, uppsala, sweden) as previously reported , . recombinant hne protein (molecular mass, kda; pl . in pbs) with a purity of greater than % was purchased from merck group (darmstadt, germany). the manufacturer indicated that this material could be used for in vitro binding assays. the hne protein was dissolved in coupling buffer ( μ g/ml, in mm sodium acetate, ph . ), and response units (ru) of the hne protein, which was used as the ligand, were immobilized on a cm sensor chip using n-ethyl-n-( -dimethylaminopropyl) carbodiimide and n-hydroxysuccinimide according to the standard primary amine-coupling procedures; hbs-ep ( mm hepes, nm nacl, mm edta, and . % (v/v) surfactant p , ph . ) was used as the running buffer. equilibration of the baseline was performed by continuously flowing hbs-ep across the chip surface for - h. the biacore data were collected at °c using hbs-ep as the running buffer at a constant flow rate of μ l/min. egcg was serially diluted into the running buffer to create a series of concentrations. the samples were injected into the channels at a flow rate of μ l/min, followed by washing with running buffer. the binding responses were continuously recorded in ru at a frequency of hz as sensorgrams and were expressed as a function of time. the association (k on ) and dissociation (k off ) rate constants and the equilibrium dissociation constant (k d = k off ) were calculated as concentrations of egcg using bia evaluation software version . (biacore) and the : langmuir binding fitting model. the curve-fitting efficiency was evaluated according to the statistical parameter χ . western blot analysis. total protein was extracted using ripa lysis buffer, and equal amounts of proteins were subjected to % sds-page and then transferred to polyvinylidene difluoride membranes (millipore corp., ma, u.s.a). the membranes were blocked and then incubated in primary antibodies against vimentin, β -catenin, aat), insulin receptor substrate- (irs- ) ( : , cell signaling technology, beverly, ma), pakt and akt ( : , cell signaling technology), ppi k and pi k ( : , cell signaling technology), and gapdh ( : , sigma-aldrich) overnight at °c with gentle agitation, followed by incubation in the anti-rabbit ir-dye -or cw-labeled secondary antibody for h at room temperature. two -min washes in tbst were performed after secondary antibody labeling; then, the membranes were placed in tbst. the membranes were imaged using a licor odyssey scanner. boxes were manually placed surrounding each band of interest to measure the raw near-infrared fluorescence intensity values, and the intra-lane background intensity was subtracted using odyssey . analytical software (licor, lincoln, ne, usa). hne activity assay. the assessment of hne activity was performed according to löser the reaction was terminated via the addition of μ l of soybean trypsin inhibitor solution ( . mg/ ml in tris-hcl buffer, ph . ). the samples were then vortexed, and the absorbance was measured at nm. the remaining activity of hne (as a % of the control) was calculated relative the control in the absence of the inhibitor, considering the influence of the buffer, the substrate, the solvent and the extract. the inhibitory effect of the selective hne inhibitor sivelestat sodium ( μ mol/l), which was previously established using the same assay, was considered as a positive control. to evaluate the anti-metastatic effect of egcg on lung cancer with neutrophil elastase involvement in vitro, we first treated the metastatic a cells with various concentrations of neutrophil elastase ( - nm), egcg (up to μ m) or egcg (up to μ m) and neutrophil elastase ( nm). the results from the mts proliferation assay showed that neutrophil elastase at a concentration of at least nm enhanced tumor cell viability; among all of the concentrations tested, nm neutrophil elastase displayed the strongest effect (fig. b) . thus, we selected nm neutrophil elastase as the intervention condition. the mts proliferation assay results showed similar cell viability between the control group and the groups treated with various concentrations of egcg (fig. c ) and also showed a non-significant change in cell viability between the group treated with neutrophil elastase ( nm) and the groups treated with egcg and neutrophil elastase ( nm). however, in the wound healing assays, the cells treated with or μ m egcg migrated at a much slower rate than the control cells and the cells treated with neutrophil elastase ( nm) ( fig. a,b) , suggesting that egcg displays anti-migratory properties. to confirm this result, we performed further cell migration assays using the transwell chamber model. as shown in fig. c ,d, the group treated with nm neutrophil elastase displayed significantly more migrating a cells than the control group. alternatively, treatment with either or μ m egcg (after exposure to nm neutrophil elastase), resulted in dramatically fewer migrating a cells than the control treatment and treatment with neutrophil elastase ( nm) alone. these results indicated that treatment with egcg at a concentration between and μ m induces a substantial anti-migratory effect without affecting the proliferation of a cells exposed to neutrophil elastase. to determine whether egcg regulates the hne-induced emt process, the expression of emt marker scientific reports | : | doi: . /srep proteins was detected via western blot assays and confocal microscopy. the results showed that vimentin expression was significantly altered after hne treatment. however, as the specific inhibitor of hne sivelestat sodium significantly up-regulated the expression of vimentin. nevertheless, the vimentin protein levels were clearly down-regulated in response to egcg treatment in the presence or absence of hne (fig. b,c) . the expression of β -catenin was increased after hne induction but was significantly decreased after treatment with μ mol/l egcg (fig. b,c) . we also detected the expression and sub-cellular localization of zeb- because a recent study suggested that zeb- plays a role in lung cancer invasiveness and metastasis development. the results showed that zeb- expression was significantly increased after treatment with hne, and this effect was ameliorated after the addition of egcg (fig. a) . additionally, the sub-cellular translocation of zeb- from the cytoplasm to the nucleus was detected after treatment with hne. the expression of zeb- in the nucleus was decreased after treatment with egcg in the neutrophil elastase-treated a cells. we analyzed the binding pattern between egcg and hne. first, a crystal structure of hne bound to a selective inhibitor (pdb id: z f) was selected. the interaction energy between egcg and hne is - . kj/mol, which is similar to the interaction energy (− . kj/mol) between hne and its endogenous inhibitor sei . the active binding site between egcg and hne (pdb id: z f) was defined as the ligand-binding site with a -Å radius. as anticipated, egcg stably docked to the ligand-binding domain (lbd) (fig. b) . at least residues in the lbd were involved in the interaction between egcg and the hne protein. egcg potentially formed hydrogen bonds with ser , thr , arg and arg . moreover, egcg possessed a potential aromatic interaction with arg (fig. d) . the binding mode of egcg in the lbd of hne provided detailed structural insight into the interaction between this compound and the hne protein. furthermore, we performed a -ns md process using gromacs . . software. the time-averaged normalized ratio of the gyration radius, the rmsd, hydrogen bonds and the gromacs energy were analyzed to reflect the distribution of egcg molecules surrounding hne. the gyration radius and the rmsd of the ligand egcg and the receptor hne trended toward stability as time progressed (fig. a,b) . the interacting h-bond number was shown to change with in a stable range as time progressed (fig. c) . additionally, the gromacs energy did not fluctuate as time progressed changed (fig. d ). g_mmpbsa method calculated results showed that ∆g binding = − . ± . .kj/mol ( table ). to verify the prediction from the cdocker-based analysis that egcg directly binds to the hne protein, the binding affinity of egcg to hne was determined using spr biosensor technology. the ability of egcg to bind to hne was reflected by the ru values that were directly recorded from the biacore instrument. as shown in fig. a , the ru increased with increasing egcg concentration, indicating that egcg bound to hne in a concentration-dependent manner. the association (k on ), dissociation (k off ), and equilibrium dissociation constants (k d ) of egcg binding to hne were . × m − ·s − , . × − s − , and . × − m, respectively. the curve-fitting efficiency was evaluated according to the Χ , a statistical parameter in the spr assay. the Χ value was calculated to be . . the results indicated that egcg displayed specific binding affinity for hne (fig. a) . we first tested the enzyme activity of hne by adding the indicated concentrations of hne to the substrate solution system. the results showed that hne reacted with the substrate in a concentration-dependent manner (fig. b) . after establishing the steady enzyme-substrate reaction system, we tested the inhibitory effect of egcg at the indicated concentrations. sivelestat sodium was used as the positive control to evaluate the inhibitory effect of egcg. images of zeb- in a cells exposed to egcg, sivelestat sodium and neutrophil elastase. a cells were grown to % confluence and then incubated in μ m egcg, μ m sivelestat sodium and/or nm neutrophil elastase for h. the cells were labeled with an anti-human zeb- antibody, a dylight -conjugated secondary antibody (green) and the hoechst stain (blue). (b, c) the protein levels of vimentin were clearly down-regulated in response to egcg treatment with or without hne. (b, d) the expression of β -catenin was increased after hne induction but was significantly decreased after treatment with μ mol/l egcg. the gels were electrophoresed using the identical experimental conditions. the results showed that egcg inhibited hne activity in a concentration-dependent manner; the ic of egcg was . μ m (fig. c) , which was higher than that of sivelestat sodium (ic = . μ m) (fig. d) . aat is a serine protease inhibitor (serpin) and is the natural inhibitor of hne. the imbalance between aat and hne plays an important role in lung cancer progression . based on this understanding, we predominantly focused on the regulation of aat by egcg to investigate the mechanisms by which egcg inhibits hne-induced cell proliferation. western blot assays were used to assess whether the aat protein levels were altered by egcg in a cells. when co-incubated with neutrophil elastase ( nm), egcg ( μ m) enhanced aat expression in a cells (fig. a,b) . neutrophil elastase directly induced lung tumor cell proliferation by degrading irs- , which is an adapter protein of pi k, and subsequently activating the pi k pathway in the tumor cells . upon neutrophil elastase exposure, the protein level of irs- was decreased, and the phosphorylation of akt (thr ) and pi k were elevated. egcg ( μ m) enhanced the protein levels of irs- (fig. a,c) and reduced the phosphorylation of akt and pi k in the neutrophil elastase-stimulated a cells (fig. a,d-f ). in recent years, egcg has been reported to inhibit tumor proliferation and metastasis and to induce the apoptosis of lung cancer cells in vitro and in vivo . egcg suppresses proinflammatory cytokines and chemokines induced by toll-like receptor agonists in prostate cancer cells . in addition, egcg suppresses lung cancer cell growth via ras-gtpase-activating protein sh domain-binding protein- . many molecules are involved in the anti-tumor activity of egcg, including jak/stat, mapk, pi k/akt, wnt, notch, nf-κ b and ap- . because cancer progression is a complex process, its pathogenic mechanism remains somewhat elusive. egcg is considered as a natural multi-targeting chemopreventive agent. thus, the possibility that egcg inhibits tumor development via alternative mechanisms cannot be excluded. the purpose of this study was to investigate the novel inflammation-related mechanisms by which egcg mediates anti-cancer migratory activity in lung cancer. recent studies suggested that chronic inflammatory pulmonary diseases such as emphysema are highly associated with an increased risk of lung cancer, independent of smoking . hne, among the most potent proteinases released by neutrophils, is considered to be responsible for the elastolytic damage in emphysema . to some extent, neutrophil elastase may represent the link between emphysema and lung cancer. recent reports showed that modest levels of neutrophil elastase led to the uncontrolled proliferation of a lung epithelial tumor cells . we found that hne treatment ( nm) enhanced the , the pakt (thy )/ akt ratio (d), the pakt (ser )/akt ratio (e) and the ppi k/pi k ratio (f) in a cells treated with neutrophil elastase, egcg and sivelestat sodium. the data are presented as the means ± sem; n = . **p < . ; ***p < . . the images for irs- , aat and gapdh were cropped from the same gel. the images for p-pi k, p-akt (t ) and gapdh were cropped from the same gel. the images for pi k and p-akt (s ) were cropped from the same gel. the images for akt and gapdh were cropped from the same gel. all gels were electrophoresed using the identical experimental conditions. scientific reports | : | doi: . /srep models of breast cancer . therefore, the role of hne, which is generally considered as the major effector of human neutrophil function, particularly merits investigation. thus, we provide the first evidence for the proliferation-promoting effect of hne on a cells. next, we explored whether egcg directly binds to hne using the cdocker algorithm. cdocker is an efficient technique used to predict interactions between small molecules and proteins by modeling and rmsd analysis. the binding site was defined based on the definition of the positive control ligand (sei ) binding site. the analysis showed that arg a: and thr a: formed hydrogen bonds with the hydrogens on the phenolic hydroxyl groups of the small molecule egcg. additionally, arg a: formed a pi bond with egcg. applying minimization and md to egcg and hne enabled us to perform a ligand-protein interaction analysis, as depicted in fig. a-d. to determine the specific binding site, we directly transformed the cdocker binding results in the pdb file to an md preparation gro file. this analysis showed that the interaction between egcg and hne may not involve instantaneous binding but rather may be a long term interaction. based on the results of md simulation, we further calculated the binding free energy. according to study of sulea et al. , we verified that egcg could bind with hne protein. to verify these results concerning the interaction between egcg and hne, we further evaluated the binding affinity of egcg to hne using spr biosensor analysis. the results confirmed the computer modeling prediction. we are the first to discover this novel target of the multi-targeting molecule egcg. interestingly, the inhibitor of hne sivelestat sodium displayed a lower affinity to hne than egcg. we found that the low water solubility of sivelestat sodium led to the appearance of the lipid solvent as an interfering factor. because of equipment limitations, we could not resolve this issue in the present study. to confirm the inhibitory effect of the interaction between egcg and neutrophil elastase on the activity of this enzyme, we measured the enzymatic activity of neutrophil elastase after treatment with egcg or sivelestat sodium. alasbahi had verified the method of the elastase enzymatic activity assay. egcg exerted a concentration-dependent inhibitory effect on the enzymatic activity of neutrophil elastase. as the positive control, sivelestat sodium exerted a greater inhibitory effect than egcg. as aat is the specific and primary inhibitor of neutrophil elastase, we assumed that aat might mediate the inhibitory effect of egcg on neutrophil elastase-induced cell migration in vitro. aat is predominantly synthesized in liver but is also expressed in extra-hepatic tissues and cells, including carcinoma cells . the role of aat in cancer development has been supported by some laboratory data; however, the molecular mechanism underlying the role of aat in tumor cell migration is poorly understood. the primary function of aat is the inhibition of neutrophil elastase. our results indicated that the protein levels of aat in a cells were clearly increased after treatment with egcg. however, the contribution of aat to the tumor development is somewhat controversial. in contrast to its effects against tumor growth, aat has been shown to be correlated with poor prognosis in lung adenocarcinoma and with the inhibitory effect of polymorphonuclear neutrophils (pmns) on the proliferation and invasiveness of lung cancer hcc cells . upon binding to neutrophil elastase, aat activates cleavage within the reactive site loop (rsl) in a suicidal action . evidence has indicated that the aat levels are significantly elevated in the skin of ne −/− mice compared with ne +/+ mice in a murine model of the autoimmune disease bullous pemphigoid . it has been reported that the deficiency of circulating aat is highly associated with lung inflammation and, especially, the early onset of pulmonary emphysema . additionally, clinical findings have indicated that aat, which is elevated in the serum of cancer patients, is a serum biomarker for the diagnosis of lung cancer and prostate cancer . previous studies showed that egcg inhibits pi k/akt/mtor signaling in various tumor cells , . our previous data demonstrated that the inhibition of neutrophil elastase-induced cell proliferation using curcumin (a type of polyphenol similar to egcg) is also dependent on the pi k pathway in a cells . recently, it was shown that neutrophil elastase released by activated neutrophils within the lung is absorbed by adjacent epithelial tumor cells and degrades irs- , which is a binding partner of the p regulatory subunit of pi k, thereby inducing the hyperactivity of the pi k pathway, which leads to uncontrolled tumor cell proliferation . our results showed that the neutrophil elastase-induced decrease in irs- expression was significantly inhibited by egcg in a cells. the suppression of akt and erk / has been thought to mediate the anti-tumor migration property of egcg . therefore, we explored whether egcg treatment remarkably suppressed neutrophil elastase-induced phosphorylation and activation of pi k/akt. our results showed that the expression of ser -phosphorylated akt was increased in neutrophil elastase-induced a cells and was down-regulated after co-treatment with egcg and neutrophil elastase. moreover, we observed the phosphorylation of akt at thy . compared with the regulation of ser phosphorylation, the phosphorylation state of thy was not significantly changed after treatment with neutrophil elastase. these results indicated that the migration-promoting effect of neutrophil elastase was not mediated by akt phosphorylation at thy but might be mediated by the regulation of p -akt. in the positive control group, neither p -akt nor p -akt expression was significantly changed after treatment with the inhibitor of neutrophil elastase sivelestat sodium. we speculated that sivelestat sodium, as a selective neutrophil elastase inhibitor, cannot suppress the metastasis of lung cancer, in contrast to the multi-targeting natural product egcg, via the pi k/akt signaling pathway. taken together, these data suggest that the direct binding of egcg to neutrophil elastase and the stimulation of aat by egcg are crucial for the inhibition of the neutrophil elastase-induced migration of a cells by egcg. however, the molecules and signaling pathways that modulate the expression of aat remain unclear, and it is important to explore the mechanisms that regulate aat, which is a serpin that is highly related to lung inflammation and lung cancer. additionally, the role of egcg in the neutrophil elastase-induced induction of tumor metastasis and growth in other xenograft murine models of lung cancer remains to be investigated. this study demonstrates that egcg, an inhibitor of neutrophil elastase, modulates the migration of nsclc a cells and blocks the neutrophil elastase-induced migration of a cells by up-regulating aat expression. these results provide a novel inflammation-related mechanism by which egcg prevents tumor metastasis and suggests the potential of egcg for the treatment of other inflammation-related diseases in the lung, such as emphysema (fig. ) . global cancer statistics cancer-related inflammation green tea catechin, epigallocatechin- -gallate, attenuates the cell viability of human non-small-cell lung cancer a cells via reducing bcl-xl expression pi k/akt/mtor signaling is involved in (− )-epigallocatechin- -gallate-induced apoptosis of human pancreatic carcinoma cells inhibition of carcinogenesis by tea cancer chemoprevention with dietary phytochemicals targeting multiple signaling pathways by green tea polyphenol (-)-epigallocatechin- -gallate epigallocatechin- -gallate inhibits epidermal growth factor receptor signaling pathway. evidence for direct inhibition of erk / and akt kinases tumor-associated neutrophils: new targets for cancer therapy neutrophil alveolitis in bronchioloalveolar carcinoma: induction by tumor-derived interleukin- and relation to clinical outcome the dual role of neutrophil elastase in lung destruction and repair alpha -antitrypsin deficiency. : epidemiology of alpha -antitrypsin deficiency role of imbalance between neutrophil elastase and alpha -antitrypsin in cancer development and progression curcumin inhibits tumor proliferation induced by neutrophil elastase through the upregulation of alpha -antitrypsin in lung cancer open source drug discovery, c. & lynn, a. g_mmpbsa--a gromacs tool for high-throughput mm-pbsa calculations cinanserin is an inhibitor of the c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro severe acute respiratory syndrome coronavirus c-like proteinase n terminus is indispensable for proteolytic activity but not for enzyme dimerization. biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations inhibition of neutrophil elastase activity by cinnamic acid derivatives from cimicifuga racemosa neutrophil elastase-mediated degradation of irs- accelerates lung tumor growth the green tea polyphenol egcg potentiates the antiproliferative activity of c-met and epidermal growth factor receptor inhibitors in non-small cell lung cancer cells epigallocatechin- -gallate suppresses proinflammatory cytokines and chemokines induced by toll-like receptor agonists in prostate cancer cells epigallocatechin gallate suppresses lung cancer cell growth through ras-gtpase-activating protein sh domainbinding protein the role of the green tea component egcg in chemoprevention chronic obstructive pulmonary disease and altered risk of lung cancer in a population-based case-control study inflammatory proteinase slips into tumor cells tumor entrained neutrophils inhibit seeding in the premetastatic lung solvated interaction energy (sie) for scoring protein-ligand binding affinities. . benchmark in the csar- scoring exercise screening of some yemeni medicinal plants for inhibitory activity against peptidases effects of native and cleaved forms of alpha -antitrypsin on me tumor cell functional activity an evaluation of the prognostic significance of alpha- -antitrypsin expression in adenocarcinomas of the lung: an immunohistochemical analysis s. alpha -antitrypsin and its c-terminal fragment attenuate effects of degranulated neutrophilconditioned medium on lung cancer hcc cells, in vitro inhibitory mechanism of serpins. mobility of the c-terminal region of the reactivesite loop the serpin alpha -proteinase inhibitor is a critical substrate for gelatinase b/mmp- in vivo pulmonary emphysema and alpha -antitrypsin deficiency the importance of alpha- antitrypsin (alpha -at) and neopterin serum levels in the evaluation of non-small cell lung and prostate cancer patients green tea catechin, epigallocatechin- -gallate (egcg): mechanisms, perspectives and clinical applications epigallocatechin gallate inhibits growth and epithelial-to-mesenchymal transition in human thyroid carcinoma cell lines this work was supported by grants from the national natural science foundation of china (no. , , , , and ) and the research fund from the ministry of education of china ( project, no. b ). the funders played no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript. key: cord- -ceatyj o authors: huang, yong; xing, na; wang, zengguo; zhang, xiujuan; zhao, xiaomin; du, qian; chang, lingling; tong, dewen title: ultrasensitive detection of rna and dna viruses simultaneously using duplex undp-pcr assay date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ceatyj o mixed infection of multiple viruses is common in modern intensive pig rearing. however, there are no methods available to detect dna and rna viruses in the same reaction system in preclinical level. in this study, we aimed to develop a duplex ultrasensitive nanoparticle dna probe-based pcr assay (duplex undp-pcr) that was able to simultaneously detect dna and rna viruses in the same reaction system. pcv and tgev are selected as representatives of the two different types of viruses. pcv dna and tgev rna were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for tgev and pcv were added to form a sandwich-like complex with nucleic acids released from viruses. after magnetic separation, dna barcodes specific for pcv and tgev were eluted using dtt and characterized by specific pcr assay for specific dna barcodes subsequently. the duplex undp-pcr showed similar sensitivity as that of single undp-pcr and was able to detect copies each of pcv and tgev in the serum, showing approximately -fold more sensitivity than conventional duplex pcr/rt-pcr assays. no cross-reaction was observed with other viruses. the positive detection rate of single mmps- and duplex mmps-based duplex undp-pcr was identical, with . % for pcv , . % for tgev and . % for pcv and tgev mixed infection. this duplex undp-pcr assay could detect tgev (rna virus) and pcv (dna virus) from large-scale serum samples simultaneously without the need for dna/rna extraction, purification and reverse transcription of rna, and showed a significantly increased positive detection rate for pcv ( %) and tgev ( . %) preclinical infection than conventional duplex pcr/rt-pcr. therefore, the established duplex undp-pcr is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. along with the development of large-scale and intensive swine production, mixed infections of multiple pathogens are increasingly becoming common in swine farms. porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus type (pcv ), classical swine fever virus (csfv), porcine pseudorabies virus (prv), transmissible gastroenteritis virus (tgev), porcine parvovirus (ppv) and porcine epidemic diarrhea virus (pedv) are major pathogens causing heavy economic losses in swine industry [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . on the basis of clinical signs, it is difficult to determine whether sick pigs are infected by single or multiple viruses [ ] . therefore, it is imperative to establish an effective and rapid method to detect multiple dna and rna viruses simultaneously in single sample. traditional diagnostic methods of dna and rna viruses are mainly dependent on detection of viral proteins and nucleic acids. currently, common methods for detecting viral antigen in solution is enzyme-linked immunosorbent assay (elisa), but elisa shows some shortcomings that are difficult to overcome. for example, elisa requires at least one highly specific antibody for a particular viral antigen, but a specific antibody is not able to detect all viral strains of a kind of virus due to genotype difference; a elisa kit is usually for one kind of virus, leading to that elisa detection are complicated, time-consuming and expensive, and it is difficult to achieve scale detection when we need to detect a variety of viruses at the same time. in addition, it is difficult to find viral infection by elisa when viral load is below a certain level because the sensitivity base line of elisa is relatively higher [ , ] . the nucleic acids-based detection methods include conventional pcr, rt-pcr, loop-mediated isothermal amplification (lamp) and real-time pcr, which have been used in the diagnosis of virus infection [ ] [ ] [ ] [ ] [ ] . although lamp and real-time pcr are more sensitive than conventional pcr or rt-pcr, lamp can only detect one pathogen at a time and lamp products are difficult to identify, while real-time pcr requires special or expensive instruments and easily shows false positive results [ , ] . however, more critical is that all the existing pcr-based assays need rna/dna extraction. it is known that the extraction and detection procedures of dna and rna are different from each other. rna is easily degradable as compared to dna, so in pcrbased methods of detecting rna viruses, viral genomic rna extracted from field samples should be utilized to synthesize complementary dna (cdna) first, which are time-consuming and labor-intensive. undp-pcr is an ultrasensitive nanoparticle dna probe-based pcr method, in which magnetic microparticles (mmps) coated with virus specific dna probes and gold nanoparticles (aunps) coated with virus specific oligonucleotides are used to enrich virus genomes from samples and form an aunp-rna/dna-mmp complex. then the specific oligonucleotides are released and characterized by pcr after the complex is washed. in the previous study, we established this method to detect dna virus pcv , which exhibited a detection limit of copies of pcv genomic dna and copies of pcv in serum that is -fold more sensitive than conventional pcr [ ] . however, it is still needed to test whether this method can be used in the detection of rna virus. in this study, we aimed to develop a method for simultaneous detection of preclinical dna and rna virus mixed infection in the same reaction system based on undp-pcr method. tgev and pcv , as the representatives of rna and dna viruses respectively chosen from a variety of viruses related to porcine diseases, were used to establish duplex undp-pcr assays. pcv , a dna virus with a circular genome of . kb, has been reported to cause wide infection throughout the world and serious damage to pig producers, while tgev, an enveloped virus with a positive-stranded rna genome, has been recognized as a principal causative agent of enteric disease [ , ] . firstly, the undp-pcr assay for tgev was developed and identified. then, single mmps-based or duplex mmps-based duplex undp-pcr assays for both pcv and tgev was developed. mmps coated with specific dna probes for either tgev or pcv (single mmps), or for both tgev and pcv (duplex mmps) were used to enrich tgev and pcv viral genomes from serum samples, and aunps coated with optimal oligonucleotides (oligo) specific for either tgev or pcv were used to magnify weak signals from very low level of tgev/pcv virus enriched by mmps from serum samples. the duplex undp-pcr assay is suitable for simultaneous detection of rna and dna viruses in early viral infection, providing an effective approach for diagnosis of swine diseases. viruses and cells tgev strain (genbank no. hq ), pcv strain (genbank no. eu ), ppv yl strain (genbank no. jn ), pedv strain (genbank no. af ) and prrsv shaanxi strain (genbank no. hq ) used in this study were isolated and purified previously by our team and stocked in our laboratory [ ] [ ] [ ] [ ] [ ] . the csfv shimen strain (genbank no. ay ) was provided kindly by professor yanming zhang [ ] . these virus strains were maintained at - °c and used as standard viruses for this study. tgev, pcv and ppv were propagated in pk- cells. csfv was propagated in st cells. pedv was propagated in vero cells. prrsv was propagated in marc- cells. four types of cells (pk- , st, vero, and marc- ) were maintained in dulbecco's modified eagle medium (dmem) (gibco, gaithersburg, md, usa) supplemented with % heat inactivated fetal calf serum (gibco). during the period from nov, to jun, , serum samples were collected from healthy pigs in pig-producing farms near xianyang and baoji of shannxi province, china. the pigs were humanely euthanized by injecting with mg/kg of ketamine in the jugular vein, then - ml of blood samples were collected from each pig by jugular venipuncture. the serum samples were tested using the conventional pcr/rt-pcr assay and single or duplex undp-pcr. the experiment was approved by the ethical committee for animal experiments of the northwest a&f university and performed according to the animal ethics procedures and guidelines of the people's republic of china. no other specific permissions were required for this study. purification of viral dna or rna viral rna/dna kit (omega, usa) was used to extract and purify viral genomic dna or rna from virus-infected cell cultures or serum samples according to the manufacture's protocol. then the extracted viral rna was reverse transcribed into the complementary dna (cdna) using the reverse transcriptase kit (takara corp., japan) according to manufacturer's instructions. in this study, specific tgev dna probes and oligonucleotides used in undp-pcr were designed via multiple sequence alignment of complete genomes of orf a from various tgev strains published on national center for biotechnology information using vector nti and dnastar software package. primers for conventional rt-pcr method of detecting tgev and pedv were designed using primer-blast software on the basis of tgev and pedv highly conserved region of orf a. primers for conventional pcr/rt-pcr detection of pcv , ppv, prrsv and csfv were designed as described in previous studies [ , ] . all the primers, probes and oligonucleotides designed for this experiment owned higher specificity to make sure diagnosis more precise. the primers, probes and oligonucleotides presented in table were synthesized by sangon (shanghai, china). preparation of mmps coated with tgev and / or pcv specific probes for undp-pcr assay in the presence of water-soluble n-ethyln - [ -dimethylaminopropyl] carbodiimide hydrochloride (edc), tgev and/ or pcv specific oligonucleotide probes modified with ' amino (nh ) were bond to the carboxylated-modified myone dynabeads to form a peptide bond. the detailed steps of the assay were according to manufacturer's instruction [ ] . then the functionalized mmps were resuspended in tris-edta (te) buffer to a concentration of mg/ml and stored at °c until required. preparation of aunps coated with tgev or pcv specific oligos aunps coated with tgev or pcv specific oligo were prepared as described in previous study [ ] , briefly, ml of nm-diameter aunps ( nmol/l) were washed and resuspended in μl sterile deionized water. then, the ' sulfydryl (sh)-modified tgev/pcv specific oligonucleotides were added and mixed with aunps to establish covalent au-s bond. in the binding process of thiolated aunps and sh-modified oligonucleotides, a final concentration of . m pbs ( . m nacl in . m of phosphate buffer, ph . ) was developed by adding . m pbs to the reaction tube three times to incubate for more than hours at room temperature. then, unbound dna probes were removed by washing twice with . m pbs. finally, the prepared functionalized aunps were stored in . m pbs at °c until used. viral serum samples were mixed with same volume of lysis buffer ( . m tris-hcl, mm edta, . m nacl, . % sds, ph . ), and then boiled for minutes to release viral rna or dna. the products were mixed with μl of probe-coated mmps and × hybridization buffer ( ×ssc, . % tween- and % sds in h o). the mixture of these components was incubated for minutes by stirring. subsequently, μl of functionalized aunps were added, followed by incubation at °c for minutes. the sandwich-like aunp-rna/dna-mmp complexes in the tube were separated using magnetic wells and washed twice with ml of hybridization buffer and twice with ml te buffer to remove remaining hybridization buffer and unbound probes-functionalized mmps and oligos-functionalized aunps. the oligonucleotides on the surface of gold nanoparticles were eluted using μl of elution buffer ( . m dtt, mm tris-hcl, mm edta, ph . ) for minutes at room temperature. then, the eluted oligonucleotides were precipitated with naac and absolute alcohol. the precipitated oligonucleotides were mixed with specific capture ssdna and detected by pcr using specific detect-pcr primers for pcv and/or tgev in table . the pcr assay was performed as described in the previous study [ ] . the pcr products were separated by electrophoresis through . % agarose gel stained with ethidium bromide and were photographed under uv light. the sensitivity, specificity, and reproducibility of the undp-pcr assay for tgev tgev genomic rna was extracted using rna kit and reverse transcribed to synthesize cdna. then, the part of tgev orf a gene was amplified from cdna using the primers ( '-cgtaatggtgacagccgat- '/ '-agcagcatcacggaaaccat- '). the bp amplified products were cloned into pmd- t simple vector (takara, japan) and sequenced. the concentration of the plasmid was measured by nanodrop spectrophotometer (thermo scientific, usa). the formula: amount (copies/μl) = . × (copies/mol) × concentration (g/μl)/mw (g/mol) was used to calculate the plasmid copy number. the viral copy number in samples per ml was tested by real-time pcr with serially diluted plasmid from to copies/ml. to test the sensitivity, the quantitative serum samples were diluted serially from to copies/ml, and then detected by undp-pcr and conventional rt-pcr. the specificity of undp-pcr was tested through comparing with pcv , ppv, prrsv, pedv and csfv. inter-assay and intra-assay were performed in three replicates by testing different concentrations of diluted serum samples ( × copies/ml, × copies/ml, copies/ml) for three consecutive days to test reproducibility of the undp-pcr assay for tgev. to test the sensitivity of duplex undp-pcr assay for tgev and pcv , the serum samples containing tgev or pcv were diluted serially from to copies/ml respectively. the diluted samples containing same viral copy numbers of tgev and pcv per ml were mixed, and then tested by single mmps-based and duplex mmps-based duplex undp-pcr, or conventional pcr/rt-pcr. the specificity and reproducibility of duplex undp-pcr assay for dna and rna viruses in the study of evaluating the specificity of duplex undp-pcr, ppv, pedv, prrsv and csfv were tested by the established method. the inter-assay and intra-assay tests were carried out in triplicate by detecting three different concentrations of mixed serum containing serial diluted tgev and pcv ( , , copies/ml) to evaluate the reproducibility of this assay. to find a rapid and ultrasensitive diagnosis method for preclinical mixed infection of dna and rna viruses, a series of related experiments were performed to establish two kinds of protocol for duplex undp-pcr assay as schematically depicted in fig . in the previous study, we have optimized and established a undp-pcr method for dna virus pcv , which can detect copies/ml of pcv serum sample and exhibited high specificity and reproducibility [ ] . in the present study, pcv and tgev are selected as representative dna and rna viruses, respectively. therefore, a undp-pcr method for rna virus tgev needs to be established first. tgev, an enveloped virus of the coronaviridae family, contains a single-stranded positivesense rna genome of . kb. the first two-thirds of the viral genome encodes two replicases and only exists in genomic rna, while the last third exists in both genomic rnas (grna) and subgenomic mrnas (sgmrnas) to encode structural and nonstructural proteins of virus [ , , ] . in this assay, we first quantified viral number using primers targeted to tgev grna and different sgmrnas and found that viral numbers were significantly different when the primers were targeted to tgev grna and different sgmrnas (data not shown). therefore, we designed probes targeted to replicase protein-encoding region orf a to quantify the amount of tgev grna. six targeted regions were selected from the ' end, middle and ' end of the orf a for designing probes to ( table ). all the targeted sequences of these probes are highly conserved in variety of tgev strains. probes to were coated to magnetic microparticles to prepare functional magnetic beads mmp-p , -p , -p , -p , -p and -p , respectively. to select the optimal probes for capturing of tgev genomic rna, capture efficiency of these designed probes were determined by conventional rt-pcr assay. the results showed that all six probes could capture tgev rna, however mmp-p and mmp-p exhibited higher capture ability for tgev rna (fig a) . next, we assessed capture efficiency of functionalized gold nanoparticles coated with oligonucleotides (oligo) to that shared same targeted sequence with probe to . the prepared functionalized au-nps were precipitated by centrifugation and were detected by rt-pcr assay. the results showed that oligo and -coated au-np possessed higher binding ability with tgev nucleic acid (fig b) . therefore, in the undp-pcr assay for tgev, probe -functionalized mmps and oligo -functionalized au-nps are optimal for capture of tgev rna and formation of sandwich complex. serial -fold dilutions of tgev in serum samples were tested to assess the sensitivity of undp-pcr for rna virus. tgev genomic rna was released by boiling with lysis buffer with rnase inhibitors and was used to form aunp-rna-mmp complexes, followed by magnetic separation and oligonucleotide elution. the oligonucleotides were then purified and detected by undp-pcr. as shown in fig a, visible targeted bands around bp could be seen in simultaneous detection of dna and rna virus by duplex undp-pcr lanes representing serum samples with viral concentrations ranging from copies/ml to copies/ml respectively, but it could not be detected in the negative control serum without tgev and the tgev serum sample below copies/ml, indicating that the detection limit of undp-pcr assay for tgev was copies/ml in serum sample. however, at least copies/ ml of tgev serum sample was able to be detected by conventional rt-pcr assay (fig b) , suggesting that the sensitivity of undp-pcr specific for tgev was -fold that of the conventional rt-pcr for tgev. the reproducibility of undp-pcr for tgev was estimated by three independent runs for three consecutive days, with triplicates of each concentration ( × copies/ml, × copies/ ml, copies/ml). consistent results of undp-pcr were obtained in the inter-assay and intra-assay test (fig a) . ppv, pcv , prrsv, csfv, pedv, tgev and the serum collected from healthy pigs were used to evaluate the specificity of the established undp-pcr for tgev. in the fig b, the undp-pcr detection only appeared to be positive with tgev, whereas no specific pcr products of bp were obtained from the assay using pcv , csfv, prrsv, pedv, ppv or healthy pigs serum as pending samples, which indicated that undp-pcr assay had high specificity for tgev and did not show cross-reactivity with pcv , csfv, prrsv, pedv and ppv. to compare the sensitivity of single mmps-based and duplex mmps-based duplex undp-pcr assay for simultaneous detection of tgev and pcv in same reaction assay system, qualified serum samples of tgev and pcv were diluted serially with the range from to copies/ml. then the diluted samples containing tgev and pcv were mixed as the template for testing the sensitivity of single mmps-based and duplex mmps-based duplex undp-pcr assay. as shown in fig a and b , the detection limits of single mmps-based and duplex mmps-based duplex undp-pcr were identical with copies/ml for tgev and pcv in serum. however, at least copies/ml of pcv and tgev serum sample was able to be detected by conventional duplex pcr/rt-pcr assay (fig c) . these results suggested that the sensitivity of duplex undp-pcr specific for pcv and tgev was -fold that of simultaneous detection of dna and rna virus by duplex undp-pcr the conventional duplex pcr/rt-pcr for these two viruses, and that mmps coated with one virus probe or two virus probes did not affect the capture efficiency of functionalized mmps and the sensitivity of undp-pcr assay. the specificity and reproducibility of duplex undp-pcr for both dna and rna viruses to evaluate the specificity of single mmps-based and duplex mmps-based duplex undp-pcr for both dna and rna viruses, magnetic beads coated with specific probes for tgev and pcv alone or together and gold nanoparticles coated with specific oligos for tgev and pcv alone were added to one single reaction tube. as shown in fig a and b , both assays were specific to the target viruses, producing specific amplified products ( bp for tgev and bp for pcv ). no amplicons were yielded with ppv, prrsv, csfv, pedv and samples from health pigs. three independent replicates assay of both assays gave highly consistent results respectively (fig c and d ). pre-clinical serum samples from epidemic farms without diseased pigs were detected for tgev and pcv using duplex undp-pcr assay and conventional duplex pcr/rt-pcr. conventional duplex pcr/rt-pcr detection showed that among samples, samples were pcv positive, samples were tgev positive, but none of samples were found to be positive for both pcv and tgev. whereas duplex undp-pcr assay showed that samples were pcv positive, samples were tgev positive, samples were positive for both pcv and tgev ( table ). the positive samples detected by the duplex undp-pcr method included all of the samples found to be positive by the conventional duplex pcr/rt-pcr. the positive detection rate of duplex undp-pcr was . % for pcv , . % for tgev and . % for pcv and tgev mixed infection, which increased % and . % for pcv and tgev preclinical infection than that of conventional duplex pcr/rt-pcr (p < . ). as shown in table , the results were consistent when single mmps-based and duplex mmps-based duplex undp-pcr were used to detect these samples, suggesting two kinds of duplex undp-pcr possessed same detection efficiency. next, the relative band intensities of the unknown samples were compared with that of standard virus samples ( , and copies/ml pcv and tgev) to evaluate the viral loads of pcv and tgev in all of positive samples. among pcv positive samples, samples were over copies/ml, samples were between and copies/ml, samples were between and copies/ml, and samples were below copies/ml in viral load (data not shown); among tgev positive samples, samples were over copies/ml, samples were between and copies/ml, samples were between and copies/ml, and samples were below copies/ml (fig a) . among pcv and tgev double positive samples, the pcv viral loads of samples were between and copies/ml, sample was between and copies/ml, and sample was below copies/ml, the tgev viral loads of samples were between and copies/ml, sample were between and copies/ml, and sample was below copies/ml (fig b) . taken together, this undp-pcr assay was found to be the best method for detecting dna and rna viruses in preclinical samples simultaneously. all the results indicated that undp-pcr-based assay was a rapid and economical approach with high specificity and high sensitivity. to date, traditional approaches to detect mixed infection of rna and dna viruses present some limitations, such as complex virus genome isolation procedures, limited sensitivity, narrow detection range and lack of specific antibodies for different viruses, which lacks the [ , ] . in addition, low virus titer in early stage of infection is not able to be detected by conventional pcr or rt-pcr, failing in timely diagnosis of infection. viruses are more likely to spread across piggeries and cause more sickness and death in piglets, which will bring huge threat to pig industry [ ] . with the development of nanotechnology, the undp-pcr based on nanoparticle and dna probe makes it possible to detect dna and rna viruses simultaneously especially in subclinical infection without the need for nucleic acid extraction separately [ ] . duplex undp-pcr assay gains more merits over established traditional approaches for rna/dna virus diagnosis. the first advantage that should be mentioned is that duplex undp-pcr is more time-saving and cost-effective than other routine pcr-based methods. in this study, field samples collected from pig-producing farms were boiled with lysis buffer for min to release viral genome, so there is no need to extract nucleic acid using dna/rna extraction kits. particularly, unlike other pcr-based assays, extracted and purified rna need to be reverse transcribed into cdna. hence, the whole detection process could be completed in short period of time. secondly, this assay could detect dna and/or rna virus in serum samples with an extreme low concentration in preclinical infection. accordingly, functionalized magnetic beads and gold nanoparticles which were coated with tgev and/or pcv specific probes and oligonucleotides targeting two distinct virus genomic sequences were added to form a sandwich complex. in each binding events, the gold nanoparticles carried with large number of oligonucleotides, which could be used as templates for subsequent pcr assay. thus, weak signals from extreme low concentration of samples were highly amplified. in addition, the duplex undp-pcr could detect dna and rna virus from large-scale serum samples simultaneously. in the reaction system, magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for tgev and pcv were added to capture and enrich viral nucleic acid, so this approach could detect infection of tgev and pcv alone or together in a single reaction tube. the results showed that duplex undp-pcr (single mmps-based and duplex mmps-based) developed in the study was able to detect copies for pcv and tgev. the sensitivity of duplex undp-pcr was approximately -fold that of conventional duplex pcr/rt-pcr. in terms of specificity, specific probes and oligonucleotides for tgev and pcv were assessed through testing capture efficiency and specificity of different probes-or oligonucleotidescoated magnetic beads or gold nanoparticles. as a consequence, duplex undp-pcr showed high specificity with tgev and pcv , yielding different size of pcr products ( bp and bp respectively). the results of detection for preclinical samples indicated that duplex undp-pcr assay ( . % for pcv , . % for tgev and . % for pcv and tgev mixed infection) described here was more sensitive than conventional detection methods ( . % for pcv , . % for tgev and % for pcv and tgev mixed infection). the duplex undp-pcr assay developed in this study provided a useful tool for simultaneous detection of rna (tgev) and dna viruses (pcv ) without the need for viral nucleic acid extraction, purification and reverse transcription. this assay could increase positive detection rates of virus infection and is useful to evaluate the viral loads in pre-clinically infected samples. in summary, the duplex undp-pcr assay is an economical and rapid detection approach with high specificity and sensitivity. preliminary study on prevalence, risk factor and genetic homogeneity of porcine reproductive and respiratory syndrome virus in registered pig farms in heilongjiang, china. transboundary and emerging diseases journal of veterinary diagnostic investigation: official publication of the american association of veterinary laboratory diagnosticians classical swine fever in china: a minireview molecular epidemiology of outbreak-associated pseudorabies virus (prv) strains in central china prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type in china molecular epidemiology of porcine epidemic diarrhea virus in china occurrence and investigation of enteric viral infections in pigs with diarrhea in china the survey of porcine teschoviruses, porcine circovirus and porcine transmissible 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evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases coronavirus genome structure and replication. current topics in microbiology and immunology isolation and identification of porcine transmissible gastroenteritis virus shaanxi strain and sequence analysis of its n gene evidence for different patterns of natural intergenotype recombination between two pcv parental strains in the field sequence analysis of porcine parvovirus yl strain and prokaryotic expression of vp porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression. veterinary microbiology cloning, sequence analysis and prokaryotic expression of orf gene of prrsv sx strain in vitro inhibition of classical swine fever virus replication by sirnas targeting npro and ns b genes a method for the high efficiency of water-soluble carbodiimide-mediated amidation the structure and functions of coronavirus genomic ' and ' ends. virus research structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous rna synthesis establishment and application of a multiplex pcr for rapid and simultaneous detection of six viruses in swine a transmissible gastroenteritis in pigs nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins we thank professor yanming zhang (northwest a&f university) for providing csfv. we also would like to thank benxiang and hengda for providing preclinical samples. conceived and designed the experiments: yh nx dt. performed the experiments: nx zw xz qd xz lc. analyzed the data: yh nx. contributed reagents/materials/analysis tools: yh dt. wrote the paper: yh nx. key: cord- -kz b wq authors: gantt, soren; gachelet, eliora; carlsson, jacquelyn; barcy, serge; casper, corey; lagunoff, michael title: nelfinavir impairs glycosylation of herpes simplex virus envelope proteins and blocks virus maturation date: - - journal: adv virol doi: . / / sha: doc_id: cord_uid: kz b wq nelfinavir (nfv) is an hiv- aspartyl protease inhibitor that has numerous effects on human cells, which impart attractive antitumor properties. nfv has also been shown to have in vitro inhibitory activity against human herpesviruses (hhvs). given the apparent absence of an aspartyl protease encoded by hhvs, we investigated the mechanism of action of nfv herpes simplex virus type (hsv- ) in cultured cells. selection of hsv- resistance to nfv was not achieved despite multiple passages under drug pressure. nfv did not significantly affect the level of expression of late hsv- gene products. normal numbers of viral particles appeared to be produced in nfv-treated cells by electron microscopy but remain within the cytoplasm more often than controls. nfv did not inhibit the activity of the hsv- serine protease nor could its antiviral activity be attributed to inhibition of akt phosphorylation. nfv was found to decrease glycosylation of viral glycoproteins b and c and resulted in aberrant subcellular localization, consistent with induction of endoplasmic reticulum stress and the unfolded protein response by nfv. these results demonstrate that nfv causes alterations in hsv- glycoprotein maturation and egress and likely acts on one or more host cell functions that are important for hhv replication. human herpesvirus (hhv) infections are ubiquitous and are responsible for substantial morbidity and mortality worldwide, particularly among people infected with human immunodeficiency virus (hiv). herpes simplex virus (hsv) and cytomegalovirus (cmv) infections can be recurrent and difficult to treat in hiv coinfected individuals [ ] . moreover, genital hsv infection has been associated with greater risks of hiv acquisition, transmission, and progression of disease [ ] . hhv- and epstein-barr virus infections cause the most common aids-defining malignancies, kaposi sarcoma and non-hodgkin lymphoma, respectively [ ] . although greatly reduced by effective antiretroviral therapy (art), complications of hhv infections remain among the most common medical problems in people infected with hiv worldwide [ ] [ ] [ ] [ ] [ ] . currently available antiviral drugs to treat or prevent complications of hhv infections all directly or indirectly target the viral polymerase [ ] . each of these drugs has one or more important limitations, including selection of drugresistant viral mutants, significant toxicities, and/or poor bioavailability requiring intravenous administration. for example, treatment of acyclovir-resistant hsv or ganciclovirresistant cmv infections requires the use of intravenous foscarnet or cidofovir, both of which are associated with nephrotoxicity. as such, new agents that are effective for hhv infections are needed that are safe, orally bioavailable and have a high barrier to resistance. nelfinavir (nfv) is a first-generation hiv aspartyl protease inhibitor recently found to block production of multiple hhvs [ ] . furthermore, because it also has potent antitumor and antiangiogenic properties, clinical trials are ongoing to evaluate nfv for the treatment of several cancers [ ] [ ] [ ] [ ] [ ] [ ] . the mechanisms by which nfv acts on tumor cells are multifactorial and include inhibition of cellular proteases, akt activation, and nf -b signaling, as well as induction of the endoplasmic reticulum (er) stress, the unfolded protein response (upr), and autophagy [ , , ] . in contrast to the aspartyl protease required for hiv maturation, hhvs utilize a serine protease, which for hsv- is the gene product of ul open reading frame (orf) [ ] . as such, we hypothesized that nfv does not inhibit hhv replication by acting on the viral protease. furthermore, we speculated that nfv acting on a nonprotease viral target would be improbable and that its antiviral activity is more likely due to one or more of its effects on host cells, which could impair efficient viral replication. we therefore investigated mechanism of action of nfv on hsv- , by attempting to identify host cell or viral targets of the drug in vitro. human fibroblasts (hf) were cultivated in dulbecco's modified eagle's medium (dmem; gibco) containing % fetal bovine serum (fbs) and units per ml penicillin g and g per ml streptomycin (pen-strep) and maintained at ∘ c in a humidified % co atmosphere, as previously described [ ] . hek -t cells were maintained in dmem supplemented with % fbs and pen-strep. hsv- (strain f) was a gift from keith jerome (fred hutchinson cancer research center). drugs. nfv and indinavir (idv) were obtained through the aids research and reference reagent program, division of aids, niaid, nih. nfv was solubilized in dimethyl sulfoxide (dmso). idv and acyclovir (sigma-aldrich) were solubilized in water. the primary antibodies used were as follows: mouse monoclonal anti-hsv gb (virusys), mouse monoclonal anti-hsv gc clone g (abcam), rabbit polyclonal anti-lc b (cell signaling technology), mouse monoclonal anti--actin, anti-flag m monoclonal, and anti-ha monoclonal antibody, clone ha- (sigma-aldrich). for western blotting, the secondary antibodies used were peroxidaseconjugated affinipure f(ab ) fragment of goat anti-mouse igg(h + l) or peroxidase-conjugated affinipure f(ab ) fragment of goat anti-rabbit igg(h + l) (jackson immunoresearch). secondary antibodies for immunofluorescence were alexafluor f(ab ) fragment of goat anti-mouse igg(h + l) or alexafluor f(ab ) fragment of goat anti-rabbit igg(h + l) (life technologies). blotting. biotinylated lectins used in the eastern blots (vector laboratories, inc.) were as follows: peanut agglutinin (pna), ricinus communis agglutinin i (rca i), wheat germ agglutinin (wga), and concanavalin a (cona). total cellular proteins ( . - . g loaded per lane) were separated by sds-page and transferred to a pvdf membrane. the membranes were blocked with % bsa in tris-buffered saline with . % tween- (tbs-t), incubated with - g lectin/ml in blocking buffer for - hours, washed three times in tbs-t, and then incubated hour with horseradish-peroxidase-conjugated avidin d (vector labs) at - g/ml in tbs-t. the blots were washed three times in tbs-t following the avidin incubation. detection was performed using an enhanced chemiluminescence method (pierce ecl plus). hf were infected with hsv- at an moi of . for hour at ∘ c and then incubated at ∘ c with either m nfv, m acv, or no drug for - days until most cells were lysed. four passages were made for each group. virus was harvested with freeze-thaw cycles followed by the addition of nonfat milk. virus titers were determined by plaque assay previously described [ ] . . . hsv- protease activity assay. the effect of nfv on hsv- maturational protease activity was assayed by transfecting hek -t cells with vectors expressing the protease (vp ; n-terminal amino acids of the ul orf gene product), as well as two substrates: the hsv- capsid scaffold protein (amino acids - of the ul orf gene product; full-length product of ul . ; icp ) and a catalytically inactive mutant (s a) of the full-length protease-scaffold protein transcript (amino acids - of the ul orf gene product) [ ] . the protease coding sequence contained an n-terminal ha tag and each substrate contained an nterminal flag tag; all vectors were synthesized by blue heron biotech, llc. each substrate was expressed alone or in combination with the protease, in the presence of nfv or a vehicle control. expression was under the control of the cmv promoter. the plasmid pegfp-n , which expresses a gfp variant from the cmv immediate-early promoter, was used to monitor transfection efficiencies. transfection was performed using mirus transit- reagent (mir ) according to the manufacturer's instructions. nfv ( m final concentration) or dmso ( . % final concentration) was added to the cells concurrently with the dna and transfection reagent. cultures were incubated hours with no change of medium, at which time the cells were harvested on ice by scraping into ripa buffer containing protease inhibitors (roche complete mini edta-free protease inhibitor cocktail tablets). protein concentrations were determined by bca assay (pierce). extracted proteins ( g/lane) were separated by sds-page and transferred to pvdf membranes by tank transfer overnight. the blots were blocked with % nonfat dry milk and probed with anti-flag m mouse monoclonal antibody (sigma), recognizing the tag on the scaffold protein, anti-ha monoclonal antibody, clone ha- (sigma), and recognizing the tag on vp . human foreskin fibroblasts (hffs) were mock-infected or infected with hsv- (strain f) at an moi of or . after hour, the inoculum was replaced with medium containing . % dmso or m nfv. at or hpi the medium was replaced with a : mixture of dmem and / karnovsky's fixative [ ] and the culture was returned to the incubator for minutes. the mixture was replaced by / karnovsky's, and the cells were incubated a further - minutes at room temperature. the cells were scraped, transferred to a microcentrifuge tube, and pelleted at ×g. the pellet was resuspended in ml of fixative and then dehydrated, embedded, sectioned, and affixed to grids according to standard methods. the grids were examined on a jeol or a jeol jem transmission electron microscope at the electron microscopy lab at the fred hutchinson cancer research center. hffs were seeded in well chamber slides to a confluence of approximately %. they were mock-infected or infected with hsv- at an moi of . after h, the inoculum was replaced by medium with . % dmso or m nfv, and the cultures were returned to the incubator. sixteen hours after infection, the cells were fixed in % paraformaldehyde in phosphate-buffered saline. cells were then permeabilized with . % tween- . endogenous peroxidase activity was inhibited with % h o . the cells were then incubated with % nonfat dry milk containing % normal goat serum (blotto/ngs; jackson) to block nonspecific binding. primary antibody binding was revealed with anti-mouse igg-hrp (jackson) followed by a -minute tsa amplification with tsa- (life technologies). the nuclei were stained with to-pro . slides were mounted after addition of slowfade (life technologies) and analyzed by confocal microscopy. confocal images were generated on an lsm pascal system (zeiss). we passaged hsv- in the presence of nfv to determine if resistant mutants could be selected in an attempt to elucidate the mechanism of action of nfv on hhv production. as expected, [ ] multistep passage under drug pressure was readily selected for high-level resistance to acyclovir ( figure ). in contrast, no significant change in susceptibility to nfv was observed despite parallel passaging in the presence of nfv. of note, the inhibitory activity of nfv was not different between acyclovir-resistant and wild type isolates, further suggesting a distinct antiviral mechanism. to determine if nfv, an aspartyl protease inhibitor, could act on the essential hsv- ul serine protease, we tested whether nfv could inhibit the activity of the hsv- protease on its scaffold protein substrates using a cotransfection assay. at nfv concentrations that potently block production of infectious hsv- , there was no effect on the activity of the hsv- protease expressed in hek t cells (figure ). attributed to a decrease in akt activation. one of the prominent effects of nfv on human cells that has been described is inhibition of the akt signaling pathway by reducing the phosphorylation of akt by phosphatidylinositol- kinase [ ] [ ] [ ] . hsv- infection results in an increase in the level of akt phosphorylation (figure ), as has been previously described [ ] . the akt inhibitor ly completely suppressed akt phosphorylation in hsv- infected cells, but nfv did not reduce the levels of phosphorylated akt even at drug concentrations that potently block virus production ( figure ). furthermore, ly treatment of hsv- infected hf cells did not reduce the production of infectious virus by plaque assay (not shown), consistent with published data [ ] . therefore, it is unlikely that the documented inhibition of nfv on akt activation plays a role in the drug's inhibition of hsv- . as shown by kalu et al. [ ] and supported by our preliminary experiments (data not shown), late hsv- gene expression in hf cells was not reduced by nfv treatment. we therefore explored the effects of nfv on hsv- infected hf cells by transmission electron microscopy. normal numbers of virus particles appeared to be produced in nfv-treated cells. similar numbers of capsids were observed in the nucleus of untreated and nfv-treated cells (approximately and , resp., figures (a) and (b) ). however, compared with untreated cells, capsids in nfv-treated cells appeared to be disproportionately retained within the cytoplasm (∼ versus protease: figure : nfv does not inhibit activity of the hsv- protease. the coding sequence of the full-length protein product of ul orf (pul ) and the expression proteins employed are diagramed in panel (a). transfection vectors were constructed to express the vp protease with an n-terminal ha tag ("protease"), the pul . with an n-terminal flag tag (substrate ), or the s a pul mutant inactive protease-scaffold protein with an n-terminal flag tag (substrate ). tags are depicted in grey at the left end of each construct. the release (r) and maturation (m) cleavage sites are shown. after transfection into hek -t cells, construct expression and substrate cleavage were evaluated by western blot using anti-ha and flag antibodies. each protein expressed alone was of the expected size ( . , . , and . kd, resp., including tag). similarly, coexpression of protease with each of the substrates resulted in the n-terminal cleavage products of the expected sizes. the generation of substrate cleavage products was not affected by the addition of nfv at concentrations that inhibit hsv- replication in vitro. , resp.), in which virus particles were more often located outside the plasma membrane (∼ versus ), suggesting a block in virus maturation or egress. in addition, in contrast to those in the cytoplasm of untreated cells, cytoplasmic capsids in nfv-treated cells were rarely observed to be enveloped (∼ versus in the fields shown; figures (c) and (d)). interestingly, although endoplasmic reticulum (er) stress, the unfolded protein response (upr), and autophagy are well known effects of nfv [ , [ ] [ ] [ ] [ ] , neither er dilation nor the abundance of double-membrane bound vesicles consistent with autophagosomes appeared consistently different between nvf-treated and untreated hsv- -infected cells. are impaired by nfv. during evaluation of hsv- glycoproteins gb and gc expression by western blotting, it was apparent that nfv treatment of infected cells resulted in increased electrophoretic mobility (figure (a) ) compared to untreated controls or idv-treated cells. the apparent change in molecular weight of these viral proteins was estimated to be consistent with the reduction in glycosylation [ ] [ ] [ ] . to assess the effect of nfv on protein glycosylation in hsv- infected cells, eastern blotting was performed using lectins pna, rca-i, wga, and cona ( figure (a) ). compared to untreated or idv-treated cells, nfv resulted in a marked reduction in staining by pna, rca-i, and wga indicating decreased addition of galactose, n-acetyl-d-galactosamine, and n-acetyl-d-glucosamine [ , ] . in contrast, cona staining was not appreciably reduced, suggesting relatively normal levels of oligomannose-type n-glycans. nfv resulted in a clear alteration in the subcellular localization of hsv- gb ( figure (b) ) by immunofluorescent antibody staining. compared with control treatments in which viral envelope glycoprotein staining uniformly delineated the plasma membrane of hsv- -infected hf cells, with nfv treatment staining appeared predominantly perinuclear, suggesting improper trafficking to the cell surface. consistent with the electron microscopy results, using immunofluorescence, no increase in lc -ii staining, a marker of autophagy, was apparent (data not shown). nfv is an hiv aspartyl protease inhibitor that, in addition to its antiretroviral activity, has complex effects on numerous human cellular functions, including on akt signaling, inhibition of cellular proteases, and induction of er stress, many of which might contribute to its ability to broadly inhibit tumor cell growth as well as replication of several non-hiv viruses [ , , , , , ] . in this study, we specifically explored the mechanism(s) by which nfv might inhibit production of infectious hsv- in vitro. we found that nfv acts on hsv- late in virus production, without a detectable effect on late viral gene expression. furthermore, abundant virus particles were observed within infected cells, though envelopment and release of virus appeared to be substantially diminished. this is consistent with a recently published study by kalu et al., which also reported that nfv blocked hsv- maturation and egress [ ] . nfv showed no activity on the vp protease, which was expected given that it is a serine protease that lacks structural or functional similarity with the hiv (aspartyl) protease. this was shown using a transfection system to examine enzymatic activity in trans and supports the finding that scaffold protein cleavage appears unaffected in hsv- -infected cells treated with nfv [ ] . no resistance to nfv could be selected under conditions that readily resulted in acyclovir-resistant hsv- , which is again consistent with findings by kalu et al. [ ] . though nfv-resistant hsv- may well be isolated using other conditions, this finding suggests a relatively high barrier to resistance in vitro and suggests a mechanism of action on a host cell function required for virus production, rather than a direct effect on a viral target [ ] [ ] [ ] [ ] [ ] . indeed, many of the cellular functions affected by nfv have similarly been described to play a role in hsv- replication. nfv inhibits cellular proteases and the proteasome, which leads to accumulation and inefficient removal of misfolded proteins in the er and golgi [ , , ] . the finding that nfv resulted in impaired viral protein glycosylation and trafficking is consistent with these processes and again validates the recent findings by kalu et al. [ ] . of note, based on cona staining, n-linkage of immature (high mannose) carbohydrates appeared relatively normal [ ] . these mannose structures are largely assembled in the cytoplasm, whereas trimming and modification of more complex sugar residues occur in the er and golgi. we found that the impairment of viral glycoprotein processing is at least one mechanism by which nfv reduces infectious hsv- production. agents that induce er stress, such as thapsigargin, similarly interfere with hsv- glycoprotein posttranslational processing and production of infectious virus [ ] . numerous studies have reported that tunicamycin, which blocks the synthesis of the n-acetylglucosamine-lipid intermediates, and other inhibitors of protein glycosylation decrease the infectious yield of hsv- in vitro [ ] [ ] [ ] . furthermore, tunicamycin does not affect the level of late viral gene product expression, and normal appearing capsids were noted within the cytoplasm, similar to the effects we observed with nfv. it is unclear, however, whether impaired hsv- envelope protein glycosylation would block virus egress based on studies using cell lines deficient in n-acetylglucosaminyl transferase activity, in which virus yield was only mildly reduced [ ] . this work has several important limitations. the effects of nfv are highly pleiotropic, and we stress that nfv might affect the production of infectious hsv- through multiple mechanisms. in addition, based on what is known about nfv's effects on tumor cells [ , ] , the most relevant mechanism(s) of action may differ with respect to individual hhv, cell type, and drug concentration. by necessity, all of the possible mechanisms by which nfv might affect hsv- replication were not evaluated. autophagy, a catabolic process that maintains cellular homeostasis under conditions of stress, is a prominent effect of nfv [ , ] . hsv- encodes genes to block autophagy in infected cells, including infected cell protein (icp) . , which is required for neurovirulence [ ] . increased levels of autophagy can reduce production of infectious hsv- similarly to nfv, with relatively normal viral gene expression and formation of viral particles that are retained within cells [ , ] . we were not able to formally show that nfv increased autophagy in hsv- infected cells under the conditions used. nfv treatment appeared to result in retention of cytoplasmic virus particles within single membranebound vesicles that could be late autophagosomes as in other reports [ , ] . however, pathognomonic doublemembrane structures were not convincingly observed in greater numbers in nfv-treated versus untreated hsv- infected cells nor were we able to show that nfv increased lc -ii staining during hsv- infection. autophagy can be difficult to demonstrate [ ] and cannot currently be excluded as a potential contributor to the effect of nfv on hsv- or other hhvs [ ] . nfv is an orally bioavailable, fda-approved treatment for hiv infection and is well tolerated 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immune escape dysregulation of autophagy in murine fibroblasts resistant to hsv- infection autophagy is involved in antiviral activity of pentagalloylglucose (pgg) against herpes simplex virus type infection in vitro guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes nelfinavir: a magic bullet to annihilate cancer cells? this work was supported by grants from the nih: kl rr - , ul rr , and university of washington center for aids research p ai . the authors declare that they have no conflict of interests regarding the publication of this paper. key: cord- -p mbmfaq authors: alfonso-morales, abdulahi; rios, liliam; martínez-pérez, orlando; dolz, roser; valle, rosa; perera, carmen l.; bertran, kateri; frías, maria t.; ganges, llilianne; díaz de arce, heidy; majó, natàlia; núñez, josé i.; pérez, lester j. title: evaluation of a phylogenetic marker based on genomic segment b of infectious bursal disease virus: facilitating a feasible incorporation of this segment to the molecular epidemiology studies for this viral agent date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: p mbmfaq background: infectious bursal disease (ibd) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. the current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment b. this marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent ibd virus (vvibdv) strains worldwide. methodology/principal findings: sequences of the segment b gene from ibdv strains isolated from diverse geographic locations were obtained from the genbank database; cuban sequences were obtained in the current work. a phylogenetic marker named b-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. this last parameter is based on the ability of b-marker to reconstruct the same topology as the complete segment b of the viral genome. from the results obtained from b-marker, demographic history for both main lineages of ibdv regarding segment b was performed by bayesian skyline plot analysis. phylogenetic analysis for both segments of ibdv genome was also performed, revealing the presence of a natural reassortant strain with segment a from vvibdv strains and segment b from non-vvibdv strains within cuban ibdv population. conclusions/significance: this study contributes to a better understanding of the emergence of vvibdv strains, describing molecular epidemiology of ibdv using the state-of-the-art methodology concerning phylogenetic reconstruction. this study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvibdv strains. therefore, it highlights the need to obtain information about both genome segments of ibdv for molecular epidemiology studies. infectious bursal disease (ibd), or gumboro disease, is a viral infection that was described for the first time in the 's in gumboro, delaware, united states [ ] and now occurs worldwide. the most important lesions observed in affected animals are lymphoid tissue damage found in the bursa of fabricius [ ] . ibd is caused by the ibd virus (ibdv), a non-enveloped virus belonging to the birnaviridae family with a genome consisting of two segments of double-stranded rna (segments a and b) [ ] . segment a ( . kbp) encodes a precursor polyprotein in a major open reading frame (orf) that is cleaved by auto-proteolysis to yield the mature vp (outer capsid), vp (protease) and vp (inner capsid) proteins [ ] . segment b ( . kbp) encodes the virus rna-dependent rna polymerase (rdrp) vp [ ] which exists in the virus particle both as a free protein and as a genome-linked protein; it interacts with the viral genome and the carboxy-terminal region of vp [ , ] . two different serotypes of ibdv have been reported, which can be differentiated by virus neutralisation test [ ] . all pathogenic isolates belong to serotype- strains [ , ] . additionally, the serotype- strains, based on in vivo studies, molecular and phylogenetic analyses, have also been classified as: attenuated ibdv (atibdv), classical virulent ibdv (cvibdv), antigenic variant ibdv (avibdv) and very virulent ibdv (vvibdv) [ ] . in particular, vvibdv strains appeared in belgium during the early s associated with high mortality in young chickens [ , ] . since vvibdv strains emerged, they have been the source of great economic losses in the poultry industry in many countries. whereby the scientific community has focused special attention related to the emergence and expansion of vvibdv strains [ , , ] . on the one hand, phylogenetic analyses have revealed independent evolutionary histories for the two genome segments (a and b), suggesting that a reassortment event may have played a role in the emergence of vvibdv [ ] . on the other hand, analyses based on reverse genetic have evidenced the fact that both genome segments influence in vvibdv's pathogenicity [ ] . hence, to conduct molecular epidemiology studies, including sequence analysis for both genomic segments, is an important step to explain the links between emergence, spreading and maintenance of the vvibdv strains around the world. most of the phylogenetic studies based on segment a use the hyper-variable region of vp (hvr-vp ) as phylogenetic marker [ , , ] , whereas those few studies including segment b use the complete segment [ , ] . however, sequencing the full genome of ibdv is still expensive, from both computational and laboratory perspectives. moreover, for many computationally intensive analyses, utilizing the full genome is unfeasible. it would be, therefore, beneficial to use only those genomic regions that contain the highest phylogenetic signal to reduce cost without losing valuable information [ , ] . in the current study, the reliability of a phylogenetic marker included into segment b (bmarker) was assessed. this b-marker could be applied in feasible molecular epidemiology studies involving both genome segments of ibdv. in addition, based on the reliability of the bmarker results and using phylogenetic inference based on hvr-vp , the presence of a novel ibdv natural reassortant between segments a and b was reported. the present work also highlights the need to obtain information about the genetic structure of both genome segments of ibdv, to elucidate the causes of the emergence and spreading not only of the vvibdv strains but also of the novel ibdv natural reassortant (between segments a and b) strains. novel ibdv natural reassortant strains have been described in few countries [ , ] and their effect on the epidemiological behavior of ibdv around the world remains unknown. ethics statement. international standards for animal welfare were used for all animal samples collected, following the regulations for animal sampling of the institute of veterinary medicine (imv), ministry of agriculture (minagri) of the republic of cuba. the protocol was approved by the committee on the ethics of the minagri of the republic of cuba and all efforts were made to minimize suffering of the animals. birds were euthanized using cervical dislocation to collect the samples. the samples were sent directly from the imv to the animal virology laboratory at censa. the imv is the official regulatory body of the republic of cuba; therefore, additional permits were not required. collection, selection and processing of samples. forty-one bursa of fabricius samples previously confirmed ibdv positive, which had been selected for molecular studies [ ] were used in the current study. additionally, the atibdv strain used as vaccine was also included in the current work: the commercial vaccine strain "gumboro" (labiofam, s.a., cuba) (http:// www.labiofam.cu/productos/vacuna-gumboro.html) which is currently applied in the vaccination program conducted by cuban veterinary services (reviewed in [ ] ). the bursal samples and the vaccine strain were printed on fta cards, suited for the preservation of genetic material and adequate transportation [ ] . the fta cards were sent to centre de recerca en sanitat animal, barcelona, spain (cresa) where the laboratory procedures were conducted. the rna of all samples was extracted from fta cards using the method described by moscoso et al. [ ] with modifications. briefly: one gram from the spotted areas in the fta cards was cut by a disposable scalpel blade and placed in . ml microcentrifuge tubes. for each fta paper portion, μl of nuclease free water were added, vortexed and incubated for min at room temperature. the final suspensions were centrifuged at g for min at °c. rna was extracted from μl of supernatant recovered using nucleospin rna virus kit (macherey-nagel, düren, germany) following the manufacturer's instructions. rna was eluted in a final volume of μl of nuclease free water. the rna extracted from fta cards was used to amplify a region of the segment b using the primer pair gb f: ´-gaccaggagtacttcccaaar- ´/gb r: ´-gtccacttgat gacttgaggt- ´, previously described by lojkic et al. [ ] . within this region, the b-marker of bp of length was selected, which is framed between n-terminal and f domains (s fig) . both domains have a functional role in vp more than structural constrains [ ] . the amplification products were visualized by electrophoresis on . % agarose gels stained with ethidium bromide and were cleaned by qiaquick pcr purification kit (qiagen inc., valencia, ca) following manufacturer's instructions. the resulting products were submitted to bi-directional dna sequencing using a bigdye terminator v . cycle sequencing kit following the manufacturer's directions (applied biosystems). sequencing products were read on an abi prism- genetic analyzer (applied biosystems). the sense and antisense sequences obtained from each amplicon were assembled, and a consensus sequence for each gene was generated using the chromaspro v . program (technelysium pvt. ltd., ). nucleotide blast analysis (http://www.ncbi.nlm.nih.gov/ blast/blast.cgi) was initially used to verify the identity of each fragment sequence obtained. the sequences were submitted to the genbank database under accession numbers (lk -lk ). different sequence datasets were organized: i) to assess the reliability of the b-marker, a set of complete segment b sequences of ibdv available at genbank database (http://www.ncbi. nlm.nih.gov/) were used (table , sequences denoted). from this sequences dataset two subsets were prepared: one containing the alignment of the entire b segments and the other one containing only the b-marker region. ii) to conduct the classification study, the cuban sequences obtained were used together with reference strain sequences selected (table , sequences denoted). for this study, sequences from the hypervariable region of segment a (hvr-vp ) were also used (s table) . finally, iii) to estimate rates of nucleotide substitution per site, per year and the time to the most recent common ancestors (tmrca) of specific groups, only sequences with a known year of collection were included. in all cases, the alignments of the sequences were performed using the algorithm clustalw method included in the program bioedit sequence alignment editor [ ] . the software jmodeltest . [ ] was used to estimate the best-fit model using the akaike and bayesian information criteria (aic and bic). the best-fit models for the complete segment b and phylogenetic marker were selected and used for phylogenetic analysis. to remove sequences with a possible recombinant event from the alignment of all sequence datasets, searches for recombinant sequences and crossover regions were performed using geneconv [ ] , rdp [ ] , maxchi [ , ] , chimera [ ] , bootscan [ ] , siscan [ ] , seq [ ] and lard [ ] , all implemented in rdp beta . [ ] . programs were executed with modified parameter settings determined according to the guidelines in the rdp manual for the analysis of divergent sequences (available upon request). recombinant sequences were tested with a highest acceptable p value of . , and bonferroni's multiple comparison correction was used. analyses were conducted twice to ensure the repeatability of results. phylogenetic relationships of the ibdv strains were analyzed using the bayesian inference (bi) and maximum likelihood (ml) methodologies. bayesian inference analyses were performed with the software mrbayes . [ , ] . the markov chain monte carlo (mcmc) searches were run with four chains for million generations, with sampling of the markov chain every generations. at the end of the run, the convergence of the chains was inspected through the average standard deviation of split frequencies and the first % of the trees were discarded. after discarding the burn-in, the four mcmc chains were combined and summarized on a majority rule consensus tree. the convergence was again assessed on the basis of the effective sampling size (ess) using tracer software version . (http://tree.bio.ed.ac.uk/ software/tracer/). only a log-likelihood with ess's of > was accepted. a tree with clade credibility was constructed using the posterior probability distribution. the trees yielded from segment b and b-marker were unrooted. the tree yielded from segment a analysis was rooted using the sequence of ibdv serotype with accession number at genbank database m . ml trees were computed using the phyml v . [ ] , and confidence levels were estimated by bootstrap replicates. all topologies were tested by the kishino and hasegawa test (k-h) [ ] and the shimodaira-hasegawa test (s-h) [ ] , which computed the log-likelihoods per site for each tree and compared the total log-likelihoods for each proposed topology, using the pamlv . program [ ] . ten thousand replicates were performed using the k-h and s-h topologies tests by re-sampling the estimated log-likelihoods for each site (rell model) [ ] . finally, the trees selected were visualized by figtree v . . [ ] . evaluation of the substitution saturation. the loss of phylogenetic information due to substitution saturation was evaluated comparing the complete segment b and the phylogenetic marker. the level of saturation was studied by plotting the pairwise number of observed transitions and transversions versus genetic distance. in addition, substitution saturation was evaluated with xia's test [ ] . all these studies were performed using the dambe program [ ] . likelihood mapping. the phylogenetic signal of each sequence dataset was investigated by the likelihood mapping analysis of , random quartets generated using treepuzzle [ ] . in this strategy, if more than % of the dots fall in the center of the triangle, the data are considered unreliable for phylogenetic inference purposes. evaluation of the phylogenetic-relationship reconstruction. to assess if the b-marker selected contains the adequate phylogenetic signal to reduce cost without losing valuable information, topologies using complete segment b and the phylogenetic marker were constructed following the methodology described above. all topologies obtained using both datasets were compared as described above the dataset of sequences previously selected, was used to generate the beast input file by beauti within the beast package v . . [ ] (freely available at http://beast.bio.ed.ac.uk). rates of nucleotide substitution per site, per year and the tmrca were estimated employing a bayesian mcmc approach. the model selection was performed by estimating model marginal log-likelihood through the path sampling (ps) and stepping-stone (ss) sampling methods described by baele et al. [ ] . the estimation of model marginal log-likelihood through the ps and ss for the twelve coalescent demographic models including parametric models (constant population size, exponential growth and logistic growth) and nonparametric models (bayesian skyline plot, bsp) with strict, uncorrelated lognormal distribution (ucld) and uncorrelated exponential distribution (uced) relaxed molecular clocks were calculated (s table) . rates of nucleotide substitution per site, per year and the tmrca were also estimated. in addition, a bayesian skyline plot to infer the population dynamics in terms of changing levels of relative genetic diversity (neτ) through time was performed. for bsp analysis data were collected and plotted using graphpad prism software . ( - , graphpad prism software inc.). an unpaired t test with whelch's correction was performed for statistical analysis of comparison of neτ between groups. in all cases the mcmc chains were run for million generations, in order to obtain an ess > , and the first % trees were discarded as ''burn-in", as recommended by the beast package manual [ ] (freely available at http://beast.bio.ed.ac.uk). convergence was assessed by estimating the effective sampling size (ess) after a % burn-in, using tracer software version . (http://tree.bio.ed.ac.uk/software/tracer/). the tree with maximum log clade credibility was selected and visualized by figtree v . . [ ] . saturation effects were investigated plotting the absolute number of transitions and transversions versus genetic distance for complete segment b and b-marker (fig ) . the number of observed transversions relative to that of transitions gradually increased with growing divergence, and both datasets resembled a line, indicating that transitions and transversions were not saturated. moreover xia's test did not support saturation for complete segment b and the b-marker (iss < iss.c, p < . ) (fig ) . the phylogenetic noise in each data set was investigated by means of likelihood mapping. the percentage of dots falling in the central area of the triangles ranged from . % for the complete segment b to . % for the b-marker (fig ) . none of the datasets showed more than % noise, which enabled the use of the b-marker to deduce the phylogenetic signal. the phylogenetic relationships among the ibdv strains were reconstructed based on complete segment b and b-marker sequences by means of ml and bi analyses. both algorithms yielded congruent results showing the same topologies, which was supported by moderate to high confidence values given by the bootstrap percentage and the posterior probability (fig ) . even though the ml tree yielded from complete segment b was the best, the statistical support for this tree was not significantly different from the bi tree from the complete segment b or the ml/bi trees from b-marker (s table) . thus, all topologies obtained from both datasets yielded two highly divergent lineages, corresponding to both recognized vvibdv and non-vvibdv clades (fig ) . each lineage was strongly supported by the maximum posterior probability value of . and the highest bootstrap value of % (fig ) . the phylogenetic relationships among the ibdv strains were reconstructed based on b-marker sequences using ml analysis. moderate to high confidence values given by the bootstrap percentage supported the topology yielded (fig , left panel) . the ml tree that estimated the phylogenetic relationships between the cuban ibdv sequences and other reference ibdv strains is shown in fig (left panel) . all cuban ibdv field strains, excepting the cuban vaccine strain and the / pir strain, were grouped in the defined lineage corresponding to the vvibdv strains (fig , left panel) . therefore, the cuban vaccine strain and the / pir strain were grouped in the defined lineage corresponding to the non-vvibdv strains (fig , left panel) . at the same time, the topology yielded from hvr-vp showed that the cuban vaccine strain was grouped within the defined cluster corresponding to atibdv strains (fig , right panel) . whereas, the remaining cuban field strains were grouped within the defined cluster corresponding to vvibdv strains (fig , right panel) . these cuban field strains included the / pir ibdv, which had been grouped within the non-vvibdv strain lineages for segment b (fig ) . thus, the cuban / pir ibdv strain can be classified as natural segment-reassortant vvibdv-like segment a and non-vvibdv-like segment b. nucleotide and amino acid deduced comparisons were carried out among the sequences of the ibdv cuban field strains obtained in the current study. the nucleotide sequence identities of the b-marker sequences among the ibdv cuban field strains ranged . - % and the deduced amino acid identities ranged . - % (table ) . ps and ss analyses based on b-marker showed an exponential coalescent and an exponential, uncorrelated clock best fitted to our data (s table) . the estimated mean ( % hpd) for the substitution rate of the segment b of all populations assessed of ibdv was . x - ( . x - - . x - ) substitutions/site/year (table ) . nevertheless, both lineages showed different substitution rates for segment b, the estimated mean ( % hpd) of the substitution rate for non-vvibdv lineage was . x - ( . x - - . x - ) substitutions/site/year, and the mean of (table ) . thus, the substitution rate of segment b for vvibdv lineage was approximately times higher than the substitution rate of segment b for non-vvibdv lineage. the date bayesian phylogenetic tree obtained for the global ibdv strains was characterised by a clear temporal structure; the oldest samples tended to fall closer to the root of the tree, while the most recent samples were located at the most distal tips. the mean tmrca for diversification of both lineages for segment b of ibdv was framed in different dates. the diversification of the non-vvibdv lineage was located at approximately ( % hpd from to ), while the mean of tmrca for vvibdv lineage was ( % hpd from - ) (fig ) . in this context, the ancestor for the cluster in which cuban strain / pir was located was framed around the year , whereas the ancestors for the remaining cuban strains were defined between the years and (fig ) . demographic inference using the bsp model is summarised in fig , which essentially plots neτ as a function of time. neτ can be considered a measure of relative genetic diversity that reflects the number of effective infections established by the virus. the bsp for both ibdv lineages showed different patterns for neτ, indicating different epidemiological behaviours for both viral populations (fig ) . for non-vvibdv lineage, a decrease in neτ from its emergence to early ' s was observed, with a subsequent maintenance in the neτ, suggesting stability in the diversity of this population. on the contrary, an abrupt increase in neτ from the emergence of vvibdv lineage (approximately in ) to was observed (fig ) , which proves an epidemic behaviour of this viral population during this period. subsequently, a mild increase in neτ was observed until year followed by a maintenance until , suggesting a stability in diversity of this population. despite of the stability of neτ for vvibdv lineage, the genetic diversity was statistically higher for this lineage than for the non-vvibdv lineage (fig ) . the sudden and dramatic emergence of vvibdv strains, which have caused high mortality rates in birds and considerable economic losses in different parts of the world since more than two decades [ ] , still represents a considerable threat to the poultry industry. the polygenic nature of ibdv pathogenicity, in particular the clear role of both segments (a and b) in vvibdv's pathogenesis [ , ] , has shown that the characterization of ibdv strains based on both genome segments is essential to understand the epidemiological behavior of this viral agent. nonetheless, the complete sequencing of both segments is impractical in routine practice in diagnostic laboratories. in addition, utilizing the full genome for molecular epidemiology studies would result in costly computational analyses, which would be also unfeasible [ ] . evaluation of a phylogenetic marker based on genomic segment b of ibdv therefore, the use of phylogenetic markers for molecular epidemiology studies of ibdv is a useful and needful approach. in the present study, a novel phylogenetic marker (b-marker) included into segment b of ibdv's genome is proposed. the initial selection of b-marker was based on two main aspects: length of the fragment and location. being the b-marker less than bp ( bp) of length, this fragment can be easily obtained and sequenced in laboratories with limited resources. likewise, the size of the b-marker facilitates the computational analyses, avoiding delayed results generated by large size fragments (reviewed in: [ ] ). in addition, b-marker is framed between nterminal domain and partial f domain of vp [ ] . the n-terminal domain of ibdv vp folds into a mixed α/β structure. this domain has been associated with the protein priming process and interacts with the fingers and thumb domains [ ] . the f domain has been linked to the nucleotide recognition and binding process as well as to the rna template binding mechanism [ ] . other domains such as domain d, starting from residue (end of b-marker) are involved in structural integrity, and c-terminal domain is highly conserved not only among ibdv strains but also for all members of birnaviridae family [ ] . thus, the structural domains in which b-marker is framed have a functional role in vp more than structural constrains; therefore, this region could be more influenced by evolutionary changes than possible negative selective restrictions. the reliability of this b-marker to conduct feasible molecular epidemiology studies involving both genome segments of ibdv was also evaluated. furthermore, the global phylodynamic of the ibdv strains was studied based on the consistency of the results obtained for this bmarker. in this context, the genetic diversity of the cuban ibdv strains was also analyzed. besides, the current work revealed the presence of a novel ibdv natural reassortant between segment a from vvibdv strains and segment b from non-vvibdv strains in cuban ibdv population. reliable reconstruction of phylogenies using molecular data can be affected by several factors; one of these critical factors is the saturation of substitutions [ ] . when a dataset is saturated, phylogenetic reconstruction may be misled by homoplasious signal [ ] , which is evidenced by no further increase in transitions observed despite increasing genetic distance, indicating that multiple substitutions at nucleotide positions have occurred. the gradual increase of transitions/transversions observed with respect to genetic distance for b-marker suggests a lack of saturation for this selected region. correspondingly, xia's test supported the same outcome. xia's test is based on the ratio of observed entropy to the entropy of full substitution saturation defined as the index of substitution saturation (i ss ). if this value is not significantly smaller than the critical i ss (i ssc ) (value at which the sequences start to fail to recover the correct tree), the sequences have experienced severe substitution saturation and should not be used for phylogenetic reconstruction [ ] . from the results obtained for b-marker we can infer that this region does not experience substitution saturation and can be used for phylogenetic studies. an important aspect for a successful phylogenetic experimental design is to predict the power of a dataset. phylogenetic noise from fast-evolving sites misleads phylogenetic inference [ ] . therefore, identification of optimal levels of noise exclusion reduces the number of topologies that are not significantly worse than the optimal tree and allows a more robust inference of phylogeny and stronger conclusions about character evolution [ ] . in our study, the phylogenetic noise associated to the b-marker proposed was just around % less, in comparison with whole segment b. this result strongly supports the use of the region proposed as phylogenetic marker since a value less than . % for phylogenetic noise is accepted as reliable in phylogenetic inference [ , ] . a high consistency between some particular regions and their whole genome in referring phylogenetic relationships can be considered as a good signature of a phylogenetic marker [ ] . as a whole, we consider that the b-marker is a reliable phylogenetic marker for ibdv strains since it was able to reconstruct the same tree as the complete segment b of the viral genome. this outcome was also supported by s-h test, which has been proved as powerful indicator of the optimal level of phylogenetic noise reduction and topologies exclusion [ ] . thus, the b-marker was able to discriminate between the defined lineages corresponding to the vvibdv and non-vvibdv strains. taking advantage of the reliability of this phylogenetic marker, the cuban ibdv strains were classified for this segment of the viral genome. previously classified vvibdv strains [ ] were also classified as vvibdv for segment b. using the b-marker we also estimated the emergence of vvibdv-lineage in the global scenario around . this date matches the range of years proposed by hon et al. [ ] , who determined the emergence of tmrca for the vvvp sequences in the same year. in our previous work, the expansion of strains carrying hvr-vp sequences linked to high virulence of ibdv was fixed starting from iran in [ ] . these results clearly suggest that around , two important events took place. firstly, the emergence of the genetic background very virulent for both segments (a and b) of ibdv. subsequently, these two genetic backgrounds very virulent were put together into a reassorted strain that we know today as vvibdv. the demographic history of the segment b of the ibdv genome for non-vvibdv lineage, showed a trend toward a decrease in genetic diversity, possibly generated by the introduction of effective vaccination programs against classical and low virulent strains from early stages of the discovery of the disease [ , ] . taking into account that most commercially available conventional live ibdv vaccines are based on classical virulent strains (reviewed in [ ] ), the use of vaccine strains with similar genetic background to the field strains could have subjected ibdv's population to repeated bottleneck effects leading to a loss of fitness through a process of natural selection [ , ] . on the contrary, the demographic history of the segment b of ibdv genome for vvibdv lineage showed a trend toward an initial growth of genetic diversity, possibly generated by the initial emergence of these strains. conventional live ibdv vaccines based on classical virulent strains exhibit only poor efficacy against vvibdvs [ ] . thus, ibdv strains with this new genetic background had the possibility to express the potential advantages of their large quasispecies cloud making expedite their establishment and spreading around the world, especially during the first years of their emergence. the maintenance in genetic diversity of segment b for this lineage, suggesting stability in diversity of this population during - , could be associated to the application of more strict control measures for these emergent strains during this period. in fact, novel vaccines were developed to be more effective against vvibdv strains, such as subunit vaccines, genetically engineered live ibdv vaccines, dna vaccines and others [ ] . however, different factors including reversion to virulence of the vaccine strains [ ] , non-sufficient induction of protective immune response [ ] as well the intrinsic property of ibdv to evolve quickly, have made the task of controlling ibd by vaccination even more challenging. the expansion of vvibdv strains was linked to a reassortment event of the genome (segment b) of ibdv with a mutant vp background, which caused a sudden increase in virulence of these kind of strains [ ] . thenceforth, vvibdv strains have been kept antigenically and genetically homogeneous, spreading to most of the countries (with the exception of australia) at least for two decades. however, the recent isolation of vvibdv strains with rare natural segment-b-reassorted [ , ] have evidenced a possible change in the genetic structure and stability of vvibdv strains. thereby, ingrao et al. [ ] suggested that it is probably only a matter of time until vvibdvs are replaced by an emerging strain with new antigenic or pathotypic properties. in fact, he et al. [ ] have currently reported that reassortant ibdv strains were dominantly prevalent in southern china during - [ ] . in the current work, the strain / pir was defined as a natural reassortant between segments a from vvibdv and b from non-vvibdv. the date of the emergence of this natural reassortant in cuba was estimated to be , after the introduction of vvibdv strains to cuba [ ] . in fact, the ancestor for the cluster in which cuban strain / pir was located was framed around the year , coincident with the first introduction of the virus in the country [ ] . therefore, the reassortment event that originated the strain / pir seems to have occurred between the novel vvibdv strains introduced in cuba around and the atibdv strains, which had been circulating among the cuban poultry population since . the pathogenicity and antigenicity of the strain / pir reassortant is unknown. even though, several studies have confirmed that ibdv reassortants and vvibdv strains possess different biological characteristics [ , ] . the pathogenicity and antigenicity of the strain / pir reassortant need to be further investigated. additional pathogenicity and antigenicity studies would also be required for bf hab and bf hab strains. on the one hand, bf hab strain showed a unique pattern of triplet amino acids - , which has been associated with pathogenicity of ibdv [ ] . on the other hand, bf hab strain showed the signature d present in non-vvibdv strains suggesting a possible attenuation [ ] . therefore, both strains could be useful to better understand the signatures and virulence factors for ibdv. regarding the evolution process of ibdv, our results for segment b showed that the substitution rate for non-vvibdv lineage was lower than vvibdv lineage ( . x - per . x - ). these different rates could be explained by several factors. on one hand, segment b from non-vvibdv lineage has been concomitant with a host for a longer period than vvibdv lineage. hence, non-vvibdv lineage may have a fitness advantage by keeping a narrow population rather than undergoing frequent changes, therefore facilitating a better host-virus equilibrium [ ] . on the other hand, the application of different types of vaccines that did not fully protect chickens against infection by vvibdv strains could have induced a "selective noise" with greater chance for beneficial mutations to accumulate. this "selective noise" allows occasional slips from the first lightest mutational loads (originated by the immune response of the host induced by the vaccination) towards an increase of the weight of mutational loads (given by the quasispecies cloud) (reviewed in [ ] ) with a consequent increase in the substitution rate. in this study, a powerful assessment of a phylogenetic marker included into segment b of the genome of ibdv was performed. this phylogenetic marker showed to be useful for classification and phylodynamic analyses for molecular epidemiology studies regarding segment b of ibdv strains. evolutionary rates and phylodynamic analyses from b-marker for ibdv showed difference in the mutation rates and the expansion pattern for non-vvibdv and vvibdv lineages. framed in cuba, the present work revealed the presence of a novel ibdv natural reassortant between segment a from vvibdv strains and segment b from non-vvibdv strains in cuban ibdv population. this study also contributed to a better understanding of the emergence of vvibdv strains, describing molecular epidemiology of ibdv using the state-of-the-art concerning to phylogenetic reconstruction approaches. the present work also proved the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvibdv strains. therefore, it also highlights the need to incorporate the phylogenetically useful information from segment b in the molecular epidemiology studies of ibdv. supporting information s fig. structural visualization of the residues translated from b-marker genome region on vp crystal. x-ray crystal structures of vp , crystal structure r was downloaded from protein data bank; chimera software v . . was used for visualization. the residues translated from b-marker genome region were expanded from vp . residues belonging to n-terminal domain are denoted in yellow. residues belonging to f domain are denoted in cyan. the remaining residues translated from b-marker genome region are denoted as heteroatoms. the remains of vp structure is maintained in gray. (tif) s fig. molecular analysis of the deduced amino acid sequences for the b-marker of the cuban field and reference strains. the pattern of the triplet amino acids - was framed in red rectangle, the position associated with virulence was also framed in red rectangle. each main lineage (vvibdv and non-vvibdv) and cuban sequences were denoted. (tif) s table. ibdv sequences of the hypervariable region of segment a used for classification. table. comparison of topologies obtained for the complete segment b and the b-marker of ibdv using ml and bi methods. li: log-likelihoods, pkh: p value for kh normal test (kishino & hasegawa ) , prell: rell bootstrap proportions (kishino & hasegawa ) , psh: p value with multiple-comparison correction (mc in table infectious bursal disease history of infectious bursal disease in the usa-the first two decades avian dis biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded rna genomes biochemistry and immunology of infectious bursal disease virus vp of infectious bursal disease virus is an rna-dependent rna polymerase the last c-terminal residue of vp , glutamic acid , controls capsid assembly of infectious bursal disease virus autoproteolytic activity derived from the infectious bursal disease virus capsid protein isolation and serological studies 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resolve phylogeny phylogenetic signal detection from an ancient rapid radiation: effects of noise reduction, long-branch attraction, and model selection in crown clade apocynaceae phylogeography and epidemiological history of west nile virus genotype a in europe and the mediterranean basin molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene calculation of evolutionary correlation between individual genes and full-length genome: a method useful for choosing phylogenetic markers for molecular epidemiology attenuation of infectious bursal disease virus and vaccination trials under laboratory and field conditions standard requirements for vaccines against infectious bursal disease current status of vaccines against infectious bursal disease how does the genome structure and lifestyle of a virus affect its population variation? positive selection pressure on the b/c domains of the e -gene of classical swine fever virus in endemic areas under c-strain vaccination dna vaccination with vp gene of very virulent infectious bursal disease virus (vvibdv) delivered by transgenic e. coli dh alpha given orally confers protective immune responses in chickens infectious bursal disease: a complex host-pathogen interaction genomic sequencing and molecular characteristics of a very virulent strain of infectious bursal disease virus isolated in china reservoir host immune responses to emerging zoonotic viruses the relation of recombination to mutational advance key: cord- -pemva authors: shi, shu jing; li, hui; liu, meng; liu, ying mei; zhou, fei; liu, bo; qu, jiu xin; cao, bin title: mortality prediction to hospitalized patients with influenza pneumonia: po( )/fio( ) combined lymphocyte count is the answer date: - - journal: clin respir j doi: . /crj. sha: doc_id: cord_uid: pemva introduction: community‐acquired pneumonia (cap) severity scores perform well in predicting mortality of cap patients, but their applicability in influenza pneumonia is powerless. objectives: the aim of our research was to test the efficiency of po( )/fio( ) and cap severity scores in predicting mortality and intensive care unit (icu) admission with influenza pneumonia patients. methods: we reviewed all patients with positive influenza virus rna detection in beijing chao‐yang hospital during the – influenza seasons. outpatients, inpatients with no pneumonia and incomplete data were excluded. we used receiver operating characteristic curves (rocs) to verify the accuracy of severity scores or indices as mortality predictors in the study patients. results: among hospitalized patients with influenza pneumonia, ( . %) died. among those who were classified as low‐risk (predicted mortality . %– . %) by pneumonia severity index (psi) or confusion, urea, respiratory rate, blood pressure, age ≥ year (curb‐ ), the actual mortality ranged from . to . %. multivariate logistic regression indicated that hypoxia (po( )/fio( ) ≤ ) and lymphopenia (peripheral blood lymphocyte count < . × ( )/l) were independent risk factors for mortality, with or value of . ( % confidence interval . – . ) and . ( % confidence interval . – . ), respectively. po( )/fio( ) combined lymphocyte count performed well for mortality prediction with area under the curve (auc) of . , which was significantly better than current cap severity scores of psi, curb‐ and confusion, respiratory rate, blood pressure, age ≥ years for mortality prediction (p < . ). the scores or indices for icu admission prediction to hospitalized patients with influenza pneumonia confirmed a similar pattern and po( )/fio( ) combined lymphocyte count was also the best predictor for predicting icu admission. conclusion: in conclusion, we found that po( )/fio( ) combined lymphocyte count is simple and reliable predictor of hospitalized patients with influenza pneumonia in predicting mortality and icu admission. when po( )/fio( ) ≤ or peripheral blood lymphocyte count < . × ( )/l, the clinician should pay great attention to the possibility of severe influenza pneumonia. three global influenza pandemics of the th century, ranging from spanish flu of to the hong kong pandemic, proved to be disasters in the history of mankind and caused death toll more than that of the first world war ( ) . the recent swine origin pandemic influenza a h n virus led more than million laboratory confirmed cases in countries and over deaths until august , resulting in substantial human admission rate and mortality ( ) . most patients with severe influenza infections present with pneumonia ( ) ( ) ( ) ( ) . as we know that, at the time of a pandemic, one of the most important decisions is to decide the appropriate site of care, which needs not only physician's clinical judgment, but also the objective severity scores or indicators to help the physicians, especially the physicians in the emergency department, to make a right decision. a variety of pneumonia severity scores have been used to help the physicians to evaluate the mortality or severity of patients with community-acquired pneumonia (cap), including pneumonia severity index (psi) ( ) , confusion, urea, respiratory rate, blood pressure, age year (curb- ) ( ) , confusion, respiratory rate, blood pressure, age years (crb- ) ( ) , systolic blood pressure, multi-lobar chest radiography involvement, albumin level, respiratory rate, tachycardia, confusion, oxygenation, arterial ph (smart-cop) ( ) and lung injury score (lis) ( ) . psi and curb- are valid scores in predicting -day mortality with cap patients ( ) . smart-cop can predict patients who might need vasopressoror ventilatory support interventions with more than % accuracy ( ) . lis is a useful tool to diagnose acute lung injury/ adult respiratory distress syndrome ( ) . but current cap severity scores (psi, curb- and crb- ) in predicting mortality in patients with influenza pneumonia is unpersuasive. several studies ( ) ( ) ( ) indicated that current cap severity scores failed to predict mortality in patients due to influenza pneumonia. only one research pointed out that smart-cop presented the best performance to indicate intensive care unit (icu) admission in patients with h n pneumonia ( ) . to our knowledge, there is no relative research about lis in predicting mortality or severity with influenza pneumonia. the current cap severity scores are mainly developed for cap patients with the major pathogens of streptococcus pneumonia, haemophilus influenza and atypical bacterial pathogens ( , ( ) ( ) ( ) , which are different from pneumonia caused by influenza virus. as a kind of interstitial pneumonia, pathologic characteristics ( ) of influenza pneumonia includes interstitial inflammation, hyaline membranes formation, intra-alveolar hemorrhage, edema and necrotizing bronchitis that affecting pulmonary diffusion function ( ) . taking these into considerations, it may be more possible that severity indices reflecting pulmonary diffusion functions may perform well in predicting prognosis with hospitalized patients due to influenza pneumonia. according to our previous study ( ) and those from korea ( ) , hypoxia (po /fio ) was an independent risk factor for death in patients with influenza a (h n ) pneumonia. in this study, we want to test the efficiency of po / fio and cap severity scores in predicting mortality and icu admission with influenza pneumonia patients. during the - influenza seasons (from november to the next february in beijing area of china), we screened patients with positive influenza virus rna detection of respiratory specimen from the microbiology lab in beijing chao-yang hospital. then we excluded outpatients and inpatients with no pneumonia. cases with unavailable data to calculate severity scores were also excluded from this analysis. patients with influenza pneumonia: during the influenza seasons, patients with respiratory symptoms and a new pulmonary infiltrate on the chest radiograph, combined with positive influenza virus a rt-pcr testing and negative for other kinds of viruses as below. patients with bacterial co-infection: a patient who fulfilled the above criterion of influenza pneumonia, at the same time, with culture positive for bacterial pathogen from blood/sputum/bronchoalveolarlavage fluid or positive urinary antigen or positive mycoplasma pneumonia, chlamydia pneumonia rt-pcr detection before or within the initial h of hospital admission. microbiological evaluation was performed according to our previous reports ( , ) , (i) qualified sputum (defined as an adequate quality sputum sample with > leukocytes and < epithelial cells per magnification field) were sent for gram staining and culture according to standard methods; (ii) urinary antigen for legionella pneumophila (binax now l. pneumophila urinary antigen test; trinity biotech, bray, ireland) and urinary antigen for s. pneumoniae neoplastic disease: defined as any cancer except of basilar or squamous cell cancer of the skin that was active at the time of presentation or diagnosed within year of presentation. liver disease: defined as a clinical or histological diagnosis of cirrhosis or another form of chronic liver disease, such as chronic active hepatitis. cerebrovascular disease: defined as a clinical diagnosis of stroke or transient ischemic attack or stroke documented by magnetic resonance imaging or computed tomography. renal disease: defined as a history of chronic renal disease or abnormal blood urea nitrogen and creatinine concentrations documented in the medical record. cardiac disease: defined as systolic or diastolic ventricular dysfunction documented by history, physical examination and chest radiograph, echocardiogram, multiple gated acquisition scan or left ventriculogram. obesity: a patient with the body mass index more than kg/m was considered obesity. mortality: the outcome variable of mortality was defined as all-cause mortality at the time of hospital discharge. according to psi and curb- , we also calculated the number of deaths and actual mortality in each class or score. predicted mortality rate was acquired from the original publications of the severity score ( , ) . severity of influenza pneumonia was evaluated using po /fio , psi, curb- , crb- , smart-cop and lis. the severity scores or indices were calculated within h of hospital admission, which were compared between deceased and survival groups. statistical analysis was performed using statistical software package spss (version . ). patients were grouped as dead vs alive. continuous variables were described as means ( standard deviation). vchisquared test or fisher exact test was used to compare categorical variables and t-test for continuous variables. we used multivariate logistic regression to identify independent predictors of mortality. receiver operating characteristic curves (rocs) were generated to compare the total predictive accuracy of po /fio , psi, curb- , crb- , smart-cop and lis for mortality and icu admission, and the area under the curves (aucs) were calculated. a p value < . was considered statistically significant, % confidence intervals were calculated. during the study period, patients with positive influenza virus rna detection were screened. five hundred fifty-eight outpatients, inpatients without pneumonia and patients with incomplete data were excluded. at last, a total of hospitalized patients with influenza pneumonia were enrolled in the study (fig. ) . among patients, cases of h n , one case of h n h n , one of h n and of influenza a, but not typed. bacterial co-infection was documented in seven ( . %) patients, with three cases of pseudomonas aeruginosa, two with acinetobacter baumannii, one with staphylococcus aureus and one of klebsiella pneumonia. all the other pathogens (including m. pneumonia and c. pneumonia, l. pneumophila) were negative. mortality was . % ( / ). baseline characteristics of the hospitalized patients with influenza pneumonia were shown in table . the variables associated with mortality in hospitalized patients with influenza pneumonia were decreased lymphocyte count, decreased hemoglobin, decreased platelet, decreased albumin, decreased po /fio , elevated respiratory rate, elevated blood urea nitrogen, elevated lactate dehydrogenase, elevated serum glucose, multilobar infiltrates and pleural effusion. multivariate logistic regression indicated that hypoxia (po / fio ) and lymphopenia (peripheral blood lymphocyte count < . /l) ( ) were independent risk factors for mortality, with or value of . ( % confidence interval . - . ) and . ( % confidence interval . - . ), respectively. the result of multivariate logistic regression was showed in table . current cap severity scores underestimate mortality with low-risk patients the deceased patients and the total number of patients stratified by psi and curb- with their actual and predicted mortality rates were shown in table . predicted mortality in the range of . %- . % was defined as low risk. according to psi and curb- , low risk was determined in ( . %) patients and ( %) patients, respectively, which accounted for a majority part of hospitalized patients with influenza pneumonia. the actual mortality in these patients ranged from . to . %. that was to say, psi and curb- underestimated the mortality in a significant number of hospitalized patients with influenza pneumonia. as we mentioned above, lymphopenia was also an independent risk factor for mortality. so, we finally used eight severity scores or indices (psi, curb- , crb- , smart-cop, lis, po /fio , lymphocyte count and pao /fio combined lymphocyte count) to compare aucs for mortality prediction in hospitalized patients with influenza pneumonia ( table ). the rocs for mortality prediction was shown in supporting information e- fig. . pao /fio combined lymphocyte count performs best in mortality prediction with hospitalized patients due to influenza pneumonia with auc of . ( % confidence interval, . - . ), and the auc of po /fio was . ( % confidence interval, . - . ), which was lower than pao / fio combined lymphocyte count. the aucs of psi, curb- and crb- were less than . . pao /fio combined lymphocyte count was significantly better than current cap severity scores of psi, curb- and crb- (p < . ). the aucs of lis, smart-cop and lymphocyte count were respectively . ( % confidence interval, . - . ), . ( % confidence interval, . - . ) and . ( % confidence interval, . - . ), which were all less than the auc of pao / fio combined lymphocyte count. the aucs of the eight severity scores or indices for icu admission prediction in hospitalized patients with influenza pneumonia confirmed a similar pattern to mortality prediction ( table ). the rocs for predicting icu admission was shown in supporting information e- fig. . pao /fio combined lymphocyte count was also a good predictor for icu admission prediction with auc of . ( % confidence interval . - . ), which was higher than pao /fio alone. the auc of pao /fio combined lymphocyte count was also greater than current cap severity scores of psi, curb- and crb- (p < . ). the auc of lis, smart-cop and lymphocyte count were all less than that of pao /fio combined lymphocyte count. this study demonstrated that po /fio combined lymphocyte count is simple and reliable severity predictor of hospitalized patients with influenza pneumonia, which is significantly better than current cap commons and denholm ( ) investigated patients of h n influenza infection and found that the common used cap severity scores (psi and curb- ) had insufficient predictive ability to lowrisk patients in icu admission. other researches ( ) ( ) ( ) also showed that routine prediction rules underestimated severity of influenza a (h n ) pneumonia, but no effective severity score had been put forward. in our study, psi and curb- underestimated a significant number of hospitalized patients. but the influenza pneumonia patients with high risk (psi class of v and curb- score of - ) are scarce, so the conclusion is inadequate and we need to increase the sample size in our further research. the severity scores of psi and curb- heavily weight on advanced age and complication ( , ) , but h n influenza a virus infection has been reported to occur in young, previously healthy individuals ( ) ( ) ( ) ( ) , which may be one reason that the scores fail to predict mortality. another possible explanation may be due to the facts that current cap severity scores are mainly developed for cap patients with the typical bacterial and atypical bacterial pneumonia. concerning on pao /fio and the prognosis of influenza pneumonia, a study in washington ( ) identified different mean pao /fio values in survivors ( ) and non-survivors ( ) (p . ) in patients with influenza pneumonia. ho et al. ( ) indicated that pao /fio was a useful parameter in predicting mortality with influenza pneumonia, but the study only evaluated cases and there was no comparison with other severity scores. the pathologic changes of influenza pneumonia are characterized by diffused alveolar damage and altered pulmonary diffusion function ( , ) . the clinical and radiological characteristics of cap caused by influenza a (h n ) differed markedly from cap caused by bacterial agents, with high frequency of dyspnea, hemoptysis and bilateral interstitial infiltrate ( ) . these above features of influenza pneumonia are closely associated with low po /fio . therefore, there was no surprise that the index of po /fio performed so well in predicting mortality of hospitalized patients with influenza pneumonia. and it is also understandable that severity prediction scores including the variable of pao /fio , such as smart-cop and lis, presented better performance than psi, curb- and crb- , which have no variables associated with pao /fio . but the severity scores of lis and smart-cop are more complex to calculate, and some variables (e.g. respiratory system compliance) in the scores are difficult to obtain, which confine its application. our study also found that decreased lymphocyte count was an independent predictor of mortality. previous studies pointed out that lymphopenia were an early and reliable laboratory finding of adult severe influenza a infection ( , , ) . concerning on the mechanism of lymphopenia, it needs further research. some limitations of this study should be noted. first, our study only included patients from one centre, the conclusion generalized to be widely used still need a large-scale clinical validation. second, our study consists of only hospitalized patients with influenza pneumonia, we are not able to define severity scores or indices in ambulatory patients. despite the above limitations, we believe that our study has shown important and novel findings about the death prediction in hospitalized patients with influenza pneumonia. to our knowledge, this is the first study that compared po /fio and lymphocyte count with current cap severity scores. our findings that po /fio combined lymphocyte count is a useful predictor for severity of influenza pneumonia may help clinicians more accurately predict prognosis, and triage place to improve outcome. in conclusion, we found that po /fio combined lymphocyte count is simple and reliable predictor of hospitalized patients with influenza pneumonia in predicting mortality and icu admission. when po / fio or peripheral blood lymphocyte count < . /l, the clinician should pay great attention to the possibility of severe influenza pneumonia. additional supporting information may be found in the online version of this article at the publisher's website: figure (e-fig ) . the rocs for mortality prediction in hospitalized patients with influenza pneumonia pao /fio combined lymphocyte count performs best in mortality prediction with hospitalized patients due to influenza pneumonia with auc of . ( % confidence interval, . - . ), and the auc of po / fio was . ( % confidence interval, . - . ), which was lower than pao /fio combined lymphocyte count. the aucs of psi, curb- and crb- were less than . . pao /fio combined lymphocyte count was significantly better than current cap severity scores of psi, curb- and crb- (p < . ). the aucs of lis, smart-cop and lymphocyte count were, respectively, . ( % confidence interval, . - . ), . ( % confidence interval, . - . ) and . ( % confidence interval, . - . ), which were all less than the auc of pao /fio combined lymphocyte count. figure (e-fig ) . the rocs for predicting icu admission in hospitalized patients with influenza pneumonia pao /fio combined lymphocyte count was also a good predictor for icu admission prediction with auc of . ( % confidence interval . - . ), which was higher than pao /fio alone. the auc of pao /fio combined lymphocyte count was also greater than current cap severity scores of psi, curb- and crb- (p < . ). the auc of lis, smart-cop and lymphocyte count were all less than that of pao /fio combined lymphocyte count. mortality prediction to hospitalized patients with influenza pneumonia shi et al. world health organization (who) weekly epidemiological record. ten things you need to know about pandemic influenza swine influenza a (h n ) infection in two children-southern california a report of influenza of pneumonia pneumonia complicating asian influenza severe influenza virus pneumonia in the pandemic of - complications of viral influenza a prediction rule to identify low-risk patients with community-acquired pneumonia defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study bts guidelines for the management of community acquired pneumonia in adults: update smart-cop: a tool for predicting the need for intensive respiratory or vasopressor support in community-acquired pneumonia an expanded definition of the adult respiratory distress syndrome severity assessment tools for predicting mortality in hospitalized patients with community-acquired pneumonia. systematic review and meta-analysis computed tomography in established adult respiratory distress syndrome correlation with lung injury score predicting mortality in hospitalized patients with h n influenza pneumonia severity assessment tools in icu patients with influenza a (h n ) pneumonia mortality and severity evaluation by routine pneumonia prediction models among japanese patients with pandemic influenza a (h n ) pneumonia the role of pneumonia scores in the emergency room in patients infected by h n infection infectious diseases society of america/american thoracic society consensus guidelines on the management of communityacquired pneumonia in adults chinese society of respiratory diseases. guide for diagnosis and treatment of community-acquired pneumonia the jrs guidelines for the management of community-acquired pneumonia in adults: an update and new recommendations fatal diffuse influenzal pneumonia: premortem diagnosis by lung biopsy acute respiratory distress syndrome: the berlin definition clinical features of pneumonia caused by influenza a (h n ) virus in beijing a prediction rule to identify severe cases among adult patients hospitalized with pandemic influenza a (h n ) emergence of communityacquired adenovirus type as a cause of community-onset pneumonia accuracy of igm antibody testing, fq-pcr and culture in laboratory diagnosis of acute infection by mycoplasma pneumonia in adults and adolescents with community-acquired pneumonia diagnostic importance of relative lymphopenia as a marker of swine influenza (h n ) in adults triaging pandemic flu: pneumonia severity scores are not the answer severity of influenza a (h n ) pneumonia is underestimated by routine prediction rules. results from a prospective, population-basedstudy pandemic influenza (h n ) pneumonia: curb- score for predicting severity and nasopharyngeal sampling for diagnosis are unreliable usefulness of curb- and pneumonia severity index for influenza a h n v pneumonia pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico intensive-care patients with severe novel influenza a (h n ) virus infection-michigan intensive care adult patients with severe respiratory failure caused by influenza a (h n )v in spain critically ill patients with influenza a (h n ) in mexico influenza pneumonia: a descriptive study prognostic factors for fatal adult influenza pneumonia depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans clinical features of the initial cases of pandemic influenza a (h n ) virus infection in china all work was performed at the beijing chao-yang hospital, capital medical university. the authors thank all the authors that contribute to the study. the authors also thank ning chen and yang li gao in the department of radiology for their help with radiologic evaluation. this work was supported by a grant from the national science for distinguished young scholars key: cord- -b kbyp s authors: zadrazilova, iveta; pospisilova, sarka; pauk, karel; imramovsky, ales; vinsova, jarmila; cizek, alois; jampilek, josef title: in vitro bactericidal activity of - and -chloro- -hydroxy-n-[ -oxo- -(phenylamino)alkan- -yl]benzamides against mrsa date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: b kbyp s a series of nine substituted -hydroxy-n-[ -oxo- -(phenylamino)alkan- -yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant staphylococcus aureus (mrsa) and s. aureus atcc as the reference and quality control strain. the minimum bactericidal concentration was determined by subculturing aliquots from mic determination onto substance-free agar plates. the bactericidal kinetics of compounds -chloro- -hydroxy-n-[( s)- -methyl- -oxo- -{[ -(trifluoromethyl)phenyl]amino}butan- -yl]benzamide ( f), n-{( s)- -[( -bromophenyl)amino]- -methyl- -oxobutan- -yl}- -chloro- -hydroxybenzamide ( g), and -chloro-n-{( s)- -[( , -dichlorophenyl)amino]- -methyl- -oxobutan- -yl}- -hydroxybenzamide ( h) was established by time-kill assay with a final concentration of the compound equal to x, x, and x mic; aliquots were removed at , , , , and h time points. the most potent bactericidal agent was compound f exhibiting remarkable rapid concentration-dependent bactericidal effect even at x mic at , , and h (with a reduction in bacterial count ranging from . to . log( ) cfu/ml) and at x mic at , , , and h ( . log( ) cfu/ml reduction in bacterial count) after incubation against mrsa . reliable bactericidal effect against other strains was maintained at x mic at h. the antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems [ ] . methicillin-resistant staphylococcus aureus (mrsa) has become the most common clinically relevant multiresistant pathogen [ ] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to % [ ] . the prevalence of mrsa is increasing worldwide and, according to the latest information of the european centre for disease prevention and control from [ ] , can be considered alarming in some european countries, especially in portugal and romania, where ≥ % of all s. aureus isolates from invasive infections were identified as mrsa in (although, e.g., in romania the prevalence of mrsa was - % in ), followed by italy, greece, and poland with - % isolates being mrsa in (for comparison, in poland mrsa isolates constituted - % from all s. aureus isolates in ). the treatment failure of vancomycin, the therapeutic anti-mrsa agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration (mic) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [ , ] . thus, the emergence of mrsa (and vancomycin-resistant s. aureus in the recent years as well [ ] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [ ] . however, for the treatment of s. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [ ] . this fact should be considered during the development of effective and safe treatment options for mrsa infections. the history of clinical usage of salicylanilides ( -hydroxy-n-phenylbenzamides) dates back to the s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid s [ ] . nowadays, salicylanilides (sals) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [ ] , antibacterial [ , ] , antimycobacterial [ ] , antifungal [ ] , and antiviral [ , ] , among others. despite being studied since the s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. sals have been found to inhibit the two-component regulatory systems (tcs) of bacteria [ ] . the latest studies specified them also as selective inhibitors of interleukin- p production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [ ] . furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase a from s. aureus [ ] , d-alanine-d-alanine ligase [ ] , or transglycosylases from s. aureus (but not from m. tuberculosis) [ ] . these enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [ ] . thus, sals seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. such new agents could be a solution to the resistance challenges. this study is a follow-up paper to a recently published article [ ] . the synthesis of the series of novel derivatives of salicylamides, -and -chloro- -hydroxy-n-[ -oxo- -(phenylamino)alkan- -yl]benzamides, called diamides due to their skeleton (for general structure see table ), was described previously [ , ] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [ ] . as these compounds expressed very significant antibacterial activity with low mic values against clinical isolates of mrsa as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against mrsa. the aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of mrsa as representatives of multidrug-resistant bacteria. to the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of sal analogues and revealing their bactericidal effect. the synthetic pathway of the series of novel diamides was described recently [ , ] , and their structures (see table ) were confirmed by ir, nmr, and ms spectrometry, and the purity of the compounds was checked by chn analysis [ , ] . [ ] ; and mrsa sa [ ] (national institute of public health, prague, czech republic) both of human origin. suspected colonies were confirmed by pcr; a bp fragment specific for s. aureus was detected [ ] . all isolates were tested for the presence of the meca gene encoding methicillin resistance [ ] . these three clinical isolates were classified as vancomycin-susceptible (but with higher mic of vancomycin equal to g/ml (va -mrsa) within the susceptible range for mrsa ) methicillinresistant s. aureus (vs-mrsa). for the mics of vancomycin, see table . vancomycin-susceptible methicillin-susceptible staphylococcus aureus (vs-mssa) atcc , obtained from the american type culture collection, was used as the reference and quality control strain. the bacteria were stored at − ∘ c and were kept on blood agar plates (columbia agar base with % ovine blood) between experiments. (mbcs) . the mbcs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots ( l) from wells with no visible bacterial growth and from control wells of mic determination onto substance-free mueller-hinton agar (mha) plates. the plates were incubated aerobically at ∘ c for h for colony count. the mbc was defined as the lowest concentration of substance, which produced ≥ . % killing table : chemical structures and in vitro mic and mbc [ g/ml] values of tested -and -chloro- -hydroxy-n-[ -oxo- -(phenylamino)alkan- -yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after h of incubation as compared to the colony count of the starting inoculum [ ] . to ensure reproducibility, each mbc assay was performed in at least triplicate on separate occasions. n h o h n o oh r r r comp. r r r mic [ g/ml] mbc [ g/ml] a -cl -ch (s)-ch > > > > > > > > b -cl -ch (s)-ch(ch ) > > > > > c -cl -ch (s)-benzyl > > > > > > > > d -cl -ch (r)-ch -indolyl > > > > > > > > e -cl -och (s)-ch(ch ) > > > > > > > > f -cl -cf (s)-ch(ch ) g -cl -br (s)-ch(ch ) h -cl , -cl (s)-ch(ch ) i -cl , -cl (s)-benzyl . . amp - - - > > > . > > > . cpx - - - > > > . > > > . van - - - time-kill assays were performed by the broth macrodilution method according to previously described methodology [ ] with some modifications. briefly, flasks containing sterile fresh mueller-hinton broth (mhb) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately . × cfu/ml (actual inoculum concentrations ranged from . × to . × cfu/ml) and a final concentration of the antibiotic equal to x, x, and x mic in ml volume. for the determination of viable counts, aliquots were removed at , , , , and h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots ( l) were plated on mha plates in duplicate. colony counts were performed on plates yielding to colonies, and the mean was calculated. antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. the plates were incubated at ∘ c for to h, and the number of colonies was determined. to ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. the growth control without the addition of antimicrobial agents and the control containing dmso without any antimicrobial agent to exclude antibacterial activity of this solvent were included. time-kill curves were constructed by plotting the log cfu per millilitre versus time (over h), and the change in bacterial concentration was determined. the results were analysed by evaluating the numbers of strains that yielded Δ(log cfu/ml) values of − (corresponding to % killing), − ( % killing), and − ( . % killing) at , , , and h compared to counts at h. bactericidal activity was defined as a reduction of at least . % (≥ log ) of the total count of cfu/ml in the original inoculum. diamides seem to be promising candidates for antibacterial agents with very strong anti-mrsa activity, as it was published recently [ ] . in the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of mrsa, and staphylococcus aureus atcc (methicillin-susceptible) as the reference and quality control strain. since sals and their analogues are known as compounds with bacteriostatic effect [ ] , this is the first study where sal-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. recently mic values of diamides expressed as molar concentrations in mol/l were published [ ] . to allow comparison with mbc values of the present study, mics in g/ml were calculated and are recorded in table along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. potential bactericidal activity of diamides was assessed using mbc assay [ ] . mbc values of all tested compounds are recorded in table as well. based on the obtained results, all compounds assessed as active according to mic values in our previous study ( f-i) showed low or moderate mbc values against all four strains. the mbc values of these compounds did not exceed the highest tested drug concentration and ranged from to g/ml. in all cases, there were comparable mbc values for the clinical isolates of mrsa and the s. aureus reference strain. bactericidal activity is defined as a ratio of mbc to mic of ≤ [ ] . table bactericidal activity is expressed in bold. as mentioned above, sals are known to exhibit a bacteriostatic effect [ ] , so it was very interesting to discover that diamides possess bactericidal activity. the amide bond (-conh-) can cause interactions with a variety of enzymes [ ] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against mrsa. the activity of sals and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that sals are capable of inhibiting transglycosylases in later stages of s. aureus (including mrsa) cell wall biosynthesis [ ] . these enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid ii, which occurs at the outer face of the membrane [ ] . since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [ , ] , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against mrsa. based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. -oxobutan- -yl}- -hydroxybenzamide ( h) were tested in time-kill studies at x, x, and x mic against all mrsa isolates and the s. aureus reference strain. the antibacterial effect of dmso [ ] used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. the extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from to log cfu/ml in viable cell count at different times after incubation. a summary of these data is presented in table . based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: f > h > g. no bactericidal activity (i.e., ≥ log cfu/ml decrease) was observed at x mic for any strain and time after incubation tested. at x mic from the four strains, compounds f, g, and h killed , , and strains, respectively, at h after incubation and , , and strains, respectively, at h after incubation. the findings of time-kill studies for each of the four staphylococci strains at exposure to compounds f, g, and h are summarized in table . bactericidal activity (i.e., ≥ log cfu/ml decrease) is expressed in bold. for compound f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of mrsa . time was not the predictive factor influencing the antibacterial activity because log differences in cfu/ml from the starting inoculum were the same for x mic (with the highest efficiency with a reduction in bacterial count of . log cfu/ml) or very similar for x mic (with a moderate regrowth after h causing a loss of bactericidal activity) over h. the bactericidal effect was maintained even at x mic at h after incubation for this strain (reduction of . log cfu/ml). for the remaining strains, clinical isolates of mrsa sa , mrsa sa , and s. aureus atcc , reliable bactericidal effect was recorded at x mic at h after incubation for all these strains with a reduction in bacterial count of . , . , and . log cfu/ml, respectively. for compound g bactericidal effect against mrsa was noticed at x mic at and h after incubation and at x mic at , , and h after incubation with a reduction in bacterial count ranging from . to . log cfu/ml. the most effective killing was achieved at h for both concentrations. as in the case of compound f, a regrowth was observed after h after incubation. for the remaining isolates of mrsa, sa and sa , bactericidal effect occurred only at x mic at h after incubation with a reduction in bacterial count of . and . log cfu/ml, respectively. the highest bactericidal effect was recorded for mrsa sa at x mic at h after incubation. a reduction consistent with bacteriostatic effect ( . to . log cfu/ml) was observed at other concentrations over time for both isolates. no bactericidal effect was observed for the s. aureus reference strain; compound g demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count ranging from . to . log cfu/ml at x mic over time. in other cases, a slight increase in bacterial counts (i.e., overgrowth) compared with the starting inoculum was observed with values ranging from . to . log cfu/ml for this reference strain. for compound h bactericidal effect against mrsa was maintained at x mic at and h after incubation with a reduction in bacterial count of . and . log cfu/ml, respectively. the same as for g, the most potent bactericidal effect was maintained at h after incubation. regrowth at h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. the reason for regrowth of the test organism at h in the experiment is unknown. most probably, selection of resistant mutants is responsible for this phenomenon [ ] ; degradation of the drug in the growth medium is not assumed, as regrowth was number of strains showing the following log cfu/ml decrease a at the designated incubation time not observed for any other tested strain. for mrsa sa concentration-dependent killing was recorded at x mic at , , and h after incubation with log differences in cfu/ml from the starting inoculum being very similar over time (ranging from . to . log cfu/ml). for mrsa sa reliable bactericidal effect was maintained only at x mic at h after incubation with a reduction in bacterial count of . log cfu/ml. as for compound g, bacteriostatic activity against s. aureus reference strain was observed with a reduction in bacterial count ranging from . to . log cfu/ml at x and x mic. overgrowth (values ranging from . to . log cfu/ml) was recorded at x mic for this strain. it is of note that in all staphylococci strains with similar mics and mbcs for compounds g and h the responsiveness to antibacterial activity of these compounds varied with clinical strains of mrsa being effectively killed and the reference strain remaining unaffected at x mic. there is a discrepancy between bactericidal results of mbc assay compared with time-kill kinetics. this difference could be caused by comparing microtiter (mbc assay) to macrobroth (time-kill assay) dilutions [ ] . moreover, although time-kill assays are more labour intensive and time consuming than mbc assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents [ ] . concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then [ ] . however, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load [ ] . moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, aids patients, etc.) bactericidal agents are unequivocally indicated. considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds [ ] . the clinical outcome of mrsa bacteraemia is significantly influenced by vancomycin mic. treatment failure exceeding % for s. aureus with vancomycin mic of g/ml resulted in the change of susceptibility breakpoint from g/ml to g/ml by the clinical and laboratory standards institute (clsi) in [ ] as well as by the us food and drug administration (fda) in [ ] . it has been recommended that for infections caused by mrsa strains with elevated vancomycin mics ( g/ml), alternative therapy should be considered [ ] . it is of note that based on time-kill assays in the present study, all tested diamides (particularly compound f exhibiting rapid bactericidal concentration-dependent effect even at x mic) were most effective against isolate mrsa , which is the strain with elevated vancomycin mic of g/ml. the activity against the remaining isolates with vancomycin mic of g/ml was lower. considering the emergence of decreasing vancomycin susceptibility of mrsa isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against mrsa in vitro. based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. the present study is the first evidence of bactericidal effect of sal analogues. against other strains, reliable bactericidal effect was maintained at x mic at h after incubation. considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future. antimicrobial resistance prevention initiative-an update: proccedings of an expert panel on resistance skin and soft-tissue infections caused by community-acquired methicillin-resistant staphylococcus aureus influence of antimicrobial regimen on decreased in-hospital mortality of patients with mrsa bacteremia proportion of methicillin resistant staphylococcus aureus (mrsa) isolates in participating countries in relationship of mic and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant staphylococcus aureus bacteremia in vitro efficacy of antimicrobial agents against high-inoculum or biofilm-embedded meticillin-resistant staphylococcus aureus with vancomycin minimal inhibitory concentrations equal to g/ml (va -mrsa) vancomycin-resistant staphylococcus aureus in the united states mrsa new treatments on the horizon: current status vancomycin in vitro bactericidal activity and its relationship to efficacy in clearance of methicillin-resistant staphylococcus aureus bacteremia salicylanilide ester prodrugs as potential antimicrobial agents-a review the role of combination anthelmintic formulations in the sustainable control of sheep nematodes high-throughput identification of antibacterials against methicillin-resistant staphylococcus aureus (mrsa) and the transglycosylase new derivatives of salicylamides: preparation and antimicrobial activity against various bacterial species salicylanilide diethyl phosphates: synthesis, antimicrobial activity and cytotoxicity identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of hcv ns b polymerase inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide novel inhibitors of bacterial two-component systems with gram positive antibacterial activity: pharmacophore identification based on the screening hit closantel salicylanilides: selective inhibitors of interleukin- p production identification of novel inhibitors of bacterial surface enzyme staphylococcus aureus sortase a atp competitive inhibitors of d-alanine-d-alanine ligase based on protein kinase inhibitor scaffolds salicylanilide derivatives block mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase synthetic route for the preparation of -hydroxy-n-[ -( -hydroxyphenylamino)- -oxoalkan- -yl]benzamides performance standards for antimicrobial susceptibility testing ; sixteenth informational supplement performance standards for antimicrobial susceptibility testing: twenty-fourth informational supplement breakpoint tables for interpretation of mics and zone diameters methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-ninth edition a timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant staphylococcus aureus evaluation of different methods to detect oxacillin resistance in staphylococcus aureus and their clinical laboratory utility species-specific and ubiquitous-dna-based assays for rapid identification of staphylococcus aureus antimicrobial susceptibility testing protocols multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems in vitro biofilm formation and bactericidal activities of methicillin-resistant staphylococcus aureus clones prevalent in korea acetylcholinesterase-inhibiting activity of salicylanilide -alkylcarbamates and their molecular docking in vitro antimicrobial activity of dimethylsulfoxide activity of telavancin compared to other agents against coagulase-negative staphylococci with different resistotypes by time kill methods for determining bactericidal activity of antimicrobial agents; approved guideline mrsa bacteraemia fda lowers vancomycin breakpoints for staphylococcus aureus vancomycin therapeutic guidelines: a summary of consensus recommendations from the infectious diseases society of america, the the authors would like to thank marie slavikova for her help and excellent laboratory cooperation. they thank also helena zemlickova from national institute of public health, prague, czech republic, for providing clinical isolates of mrsa. this study was financially supported by iga vfu brno, projects nos. / /fvl and / /faf, and by project "ceitec-central european institute of technology" (cz. . / . . / . ) from european regional development fund. the authors also wish to acknowledge the institutional support of the faculty of chemical technology, university of pardubice to the ministry of education, youth and sports. the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- -afg nmib authors: saksena, sumeet; fox, jefferson; epprecht, michael; tran, chinh c.; nong, duong h.; spencer, james h.; nguyen, lam; finucane, melissa l.; tran, vien d.; wilcox, bruce a. title: evidence for the convergence model: the emergence of highly pathogenic avian influenza (h n ) in viet nam date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: afg nmib building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (eid), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. the model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. we developed and tested a model of the emergence of highly pathogenic avian influenza (hpai) h n based on suspected convergence factors that are mainly associated with land-use change. building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher hpai h n emergence risk. the work highlights that peri-urban areas of viet nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. we also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. these results support the convergence hypothesis in general and demonstrate the potential to improve eid prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. two decades after the institute of medicine's seminal report [ ] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [ ] . highly pathogenic avian influenza (hpai) (subtype h n ) is the most significant newly emerging pandemic disease since hiv/aids. its eruption in southeast asia in - and subsequent spread globally to more than countries fits the complex systems definition of "surprise" [ ] . in this same year that iom had published its final report on microbial threats which highlighted h n 's successful containment in hong kong in [ ] , massive outbreaks occurred in southeast asia where it remains endemic, along with egypt's nile delta. since , hpai h n has killed millions of poultry in countries throughout asia, europe, and africa, and humans have died from it in sixteen countries according to who data as of january . the threat of a pandemic resulting in millions of human cases worldwide remains a possibility [ ] . lederberg et al. [ ] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [ ] . the model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. joshua lederberg, the major intellectual force behind the studies summed-up saying "ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [ ] . combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. one approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (cas) [ , ] . the convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. these associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. the initial hpai h n outbreaks in vietnam represent an ideal opportunity to adapt and test a cas-convergence model. emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. specifically we hypothesized a positive association between the presence of hpai outbreaks in poultry at the commune level and: ) peri-urban areas, as defined by saksena et al. [ ] , ) land-use diversity, and ) co-location of intensive and extensive systems of poultry. we used the presence or absence at the commune level of hpai h n outbreaks in poultry as the dependent variable. vietnam experienced its first hpai h n outbreak in late , since then, there have been five waves and sporadic outbreaks recorded over the years [ , ] . we chose to study the first wave (wave ) that ended in february and the second wave (wave ) that occurred between december and april . we used data from the viet nam agricultural census to develop an urbanicity classification that used data collected at a single point in time ( ) but across space ( , communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [ ] . the provinces in vietnam (not counting the urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. a commune in viet nam is thus the third level administrative subdivision, consisting of villages/hamlets. for the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. we included risk factors documented in previous work. we also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. for this purpose we chose to study the red river and mekong river deltas, well known hot spots of the disease. hence we conducted two sets of analyses (waves and ) for three places (nation, red river delta, and mekong delta) producing a total of wave-place analyses. data on outbreaks were obtained from the publicly available database of viet nam's department of animal health. given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. we used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [ ] . very few empirical studies have attempted to determine whether urbanization is related to eid outbreaks or whether urbanization is associated primarily with other factors related to eid outbreaks. one immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [ ] . other studies prioritized human population density as a satisfactory surrogate [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. spencer [ ] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. he found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of hpai. these studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [ ] [ ] [ ] [ ] . while these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". perhaps the best known model of peri-urbanization is mcgee's concept of desakota (indonesian for "village-town") [ ] . mcgee identified six characteristics of desakota regions: ) a large population of smallholder cultivators; ) an increase in non-agricultural activities; ) extreme fluidity and mobility of population; ) a mixture of land uses, agriculture, cottage industries, suburban development; ) increased participation of the female labor force; and ) "grey-zones", where informal and illegal activities group [ ] . saksena et al. [ ] built on mcgee's desakota concepts and data from the viet nam agricultural census to establish an urbanicity classification. that study identified and mapped the , communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. this project used the saksena classification to assess associations between urbanicity classes, other risks factors, and hpai outbreaks. researchers have estimated that almost % of zoonotic diseases are associated with landcover and land-use changes (lcluc) [ , ] . lcluc such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). the importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [ , ] . landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [ ] furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [ ] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [ ] . rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with hpai h n outbreaks. human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. as theorized by von thünen in [ ] , much of this demand is met by farms near cities [ ] , many in areas undergoing processes of peri-urbanization [ ] . due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in southeast asia and compete with existing small backyard farmers [ ] . large, enterprise-scale ( , - , birds) operations are still rare in viet nam (only communes have such a facility). on the other hand, domestic and multinational companies frequently contract farmers to raise between , and , birds. recent studies have examined the relative role of extensive (backyard) systems and intensive systems [ , [ ] [ ] [ ] ] . in much of asia there is often a mix of commercial and backyard farming at any one location [ ] . experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [ ] . intensive systems allow for virus evolution (e.g. low pathogenic avian influenza to hpai) and transformation, while extensive systems allow for environmental persistence and circulation [ ] . previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [ , [ ] [ ] [ ] ] . in isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. intensive and extensive systems in viet nam have their own fairly well defined flock sizes. a diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of hpai h n outbreaks at the commune level. this study investigated for the , communes of viet nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the hpai virus. many of these variables were identified based on earlier studies of hpai (as reviewed in gilbert and pfeiffer [ ] ). three novel variables were included based on hypotheses generated by this project. all variables were measured or aggregated to the commune level. the novel variables were: • degree of urbanization: we used the urbanicity classification developed by saksena et al. [ ] to define the urban character of each commune. the classification framework is based on four characteristics: ) percentage of households whose main income is from agriculture, aquaculture and forestry, ) percentage of households with modern forms of toilets, ) percentage of land under agriculture, aquaculture and forestry and ) the normalized differentiated vegetation index (ndvi). the three-way classification enabled testing for non-linear and non-monotonous responses. • land-use diversity: we measured land-use diversity using the gini-simpson diversity index [ ] . the gini-simpson diversity index is given by -λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. in situations with only one class (complete homogeneity) the gini-simpson index would have a value equal to zero. such diversity indices have been used to measure land-use diversity [ ] . we used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the agricultural census. the area under the last class was calculated as the difference between the total area and the sum of the first four classes. the following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • human population related transmission. human population density [ , - , , , , ] . • poultry trade and market. towns and cities were assumed to be active trading places [ , , , , ] . so, the distance to the nearest town/city was used as indicator of poultry trade. trade is facilitated by access to transportation infrastructure [ , , ] . so, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • disease introduction and amplification. the densities of chicken were calculated based on commune area [ , , , ] . • intermediate hosts. duck and geese densities were calculated using total commune area [ , , ] . as previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • agro-ecological and environmental risk factors. previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [ ] . we used percentage of land under rice cultivation as a measure of extent. rice cropping intensity is also a known risk factor [ , , ] . we used the mean number of rice crops per year as a measure of intensity. the extent of aquaculture is a known risk factor [ ] , possibly because water bodies offer routes for transmission and persistence of the virus. the percentage of land under aquaculture was used as a metric. proximity to water bodies increases the risk of outbreaks [ , [ ] [ ] [ ] , possibly by increasing the chance of contact between wild water birds and domestic poultry. we measured the distance between the commune and the nearest: a) lake and b) river. climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [ , ] . elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in vietnam [ ] . compound topographical index (cti, also known as topographical wetness index) is a measure of the tendency for water to pool. studies in thailand and elsewhere [ ] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. in the absence of reliable and inexpensive data on the extent of surface water we used cti as a proxy. cti has been used in ecological niche models (enm) of hpai h n [ , ] . however, given the nature of enm studies, the effect of cti as a risk factor has been unknown so far. cti has been used as a risk factor in the study of other infectious and non-infectious diseases [ ] . some studies have shown that at local scales, the slope of the terrain (a component of cti) was significantly correlated with reservoir species dominance [ ] . cti is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. cti is computed as follows: cti = ln (a s / (tan (β)) where; a s = area value calculated as ((flow accumulation + ) à (pixel area in m )) and β is the slope expressed in radians [ ] . though previous studies have indicated that normalized difference vegetation index (ndvi) is a risk factor [ , , , , ], we did not include it explicitly in our models, as the urban classification index we used included ndvi [ ] . we obtained commune level data on hpai h n outbreaks from the publicly available database of the department of animal health [ ] . viet nam experienced its first major epidemic waves between december and february [ ] . we chose to study the first wave (wave ) that ended in february and the second wave (wave ) that occurred between december and april . in wave , % of the communes and in wave , % of the communes experienced outbreaks. we used data from the population census of viet nam to estimate human population per commune. we relied on data from two agriculture censuses of viet nam. this survey is conducted every five years covering all rural households and those peri-urban households that own farms. thus about three-fourths of all of the country's households are included. the contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. detailed economic data are collected for large farms. we used the agriculture census for most variables because the first three epidemic waves occurred between the agricultural censuses of and but were closer in time to the census [ ] . however, for data on poultry numbers we used the agriculture census data set because between and the poultry population grew at an average rate of % annually. however, in , after the first wave of the h n epidemic, the poultry population fell %. only by mid- did the poultry population return close to pre-epidemic levels. thus, we considered the poultry population data from the census to be more representative. we aggregated census household data to the commune level. a three-way classification of the rural-to-urban transition was based on a related study [ ] . raster data on annual mean temperature and precipitation were obtained from the world-clim database and converted to commune level data. the bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at m spatial resolution [ ] . this public database provides data on the average climatic conditions of the period - . elevation was generated from srtm meter digital elevation models (dem) acquired from the consortium for spatial information (cgiar-csi). compound topographical index (cti) data were generated using the geomorphometry and gradient metrics toolbox for arc-gis . . prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. illogical values occurred mainly (less than % of the cases) for land-related variables such as percentage of commune land under a particular type of land use. next we tested each variable for normality using the bestfit software (palisade corporation). most of the variables were found to follow a log-normal distribution and a log-transform was used on them. we then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). correlations were analyzed separately for each place. certain risk factors were then eliminated from consideration when |r| ! . (r is the pearson correlation coefficient). when two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and cti), c) chicken density (only at national level, correlated with cti), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and cti), e) annual temperature (correlated with elevation and cti) and f) cropping intensity (correlated with percentage land under paddy). considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: ) multi-level generalized linear mixed model (glmm) and ) boosted regression trees (brt) [ , ] with an autoregressive term [ ] . glmm is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while brt is a 'space' oriented approach that accounts for the effects of physical proximity. we began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the euclidean distance [ ] . the limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [ ] . to determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. we finally included only those variables whose coefficient had a significance value p . (in at least one wave-place combination) and we noted the sign of the coefficient. this choice of p value for screening risk factors is common in similar studies [ , , , ] . we used a two-level glmm (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. we used robust standard errors for tests of fixed effects. boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of hpai h n occurrence and determine the relative influence of each risk factor to the hpai h n occurrence. this method was developed recently and applied widely for distribution prediction in various fields of ecology [ , ] . it is widely used for species distribution modeling where only the sites of occurrence of the species are known [ ] . the method has been applied in numerous studies for predicting the distribution of hpai h n disease [ , , [ ] [ ] [ ] . brt utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [ , ] . the advantage of brt is that it applies stochastic processes that include probabilistic components to improve predictive performance. we used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. the sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. two important parameters specified in the brt model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [ , ] . in our model we used sets of training and test points for cross-validation, a tree complexity of , a learning rate of . , and a bag fraction of . . other advantages of brt include its insensitivity to co-linearity and non-linear responses. however, for the sake of consistency with the glmm method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. in the glmm models we used p . to identify significant risk factors. the predictive performances of the models were assessed by the area under the curve (auc) of the receiver operation characteristic (roc) curve. auc is a measure of the overall fit of the model that varies from . (chance event) to . (perfect fit) [ ] . a comparison of auc with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [ ] . we used the corrected akaike information criteria (aicc) to compare each glmm model with and without its respective suite of fixed predictors. we used spss version (ibm corp., new york, ) for glmm and r version . . (the r foundation for statistical computing, ) for the brt. for calculating the spatial correlogram we used the spdep package of r. the fourteen predictor variables we modeled (see tables) were all found to be significantly associated with hpai h n outbreaks (p . ) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( table ) . land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the glmm models, as measured by the auc, is very good with auc values ranging from . to . (tables - ). the predictive power of the national models was higher than that of the delta models. the predictive power of the brt models is good, with aucs ranging from . to . . the brt models also had a better predictive power at the national level than at the delta level. these values are higher than those reported for wave (auc = . ) and wave (auc = . ) by gilbert et al. [ ] . both gilbert et al. [ ] and this study found that at the national level the predictive performance for wave was higher than that for wave . wave mainly affected the mekong river delta. previous studies indicated the duck density was an important predictor [ ] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. both the glmm and brt models found annual precipitation to be a significant factor. the glmm model indicated a negative association; similar to what was found by studies in china [ ] and in the red river delta [ ] . a global study of human cases also found occurrence to be higher under drier conditions [ ] . generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. the unadjusted relative risk (rr) of peri-urban areas in comparison with non-peri-urban areas was . and . for waves and , respectively. in terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the compound topographical index (cti) to be highest in peri-urban areas (fig a- e) . we also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (fig f) . the urbanicity variable alone, however, was not found to be significantly associated with hpai h n in any place according to the glmm model except for the urban level in red river delta for wave and in the mekong river delta for wave . the brt model ranked urbanicity as one of the least influential variables. land-use diversity was found to be significantly associated with hpai h n in both waves for viet nam according to the glmm model, but at the delta level the association was significant only for wave in the mekong river delta. the brt model indicated that land-use diversity highly influenced hpai h n at the national level in wave . for the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. both the glmm and brt models indicated that the diversity of chicken flock-size had a strong association with hpai h n for both waves at the national level. this was generally found to be true at the delta levels with some exceptions. the diversity of duck and goose flock size was also significantly associated with hpai h n in all places, but the associations were much stronger in wave than in wave . the glmm model indicated that the cti had a very strong association with hpai h n at the national level in both waves although this was not true in the two deltas. the cti is a steady state wetness index commonly used to quantify topographic control on hydrological processes. accumulation numbers in flat areas, like deltas, are very large; hence the cti was not a relevant variable in the glmm model in these areas. the brt model however indicated that cti had middle to low influence in all waves and places. we found very high spatial clustering effects as indicated by the fact that in all waves and places the brt model found the spatial autocorrelation term to have the highest rank of influence. as expected, the relative influence of the autocorrelation term at the national level was higher ( - %) than at the delta levels ( - %). in the glmm models we found the akaike information criterion (aic) using the entire set of variables to be much lower than the aics of a glmm model without fixed effects. this indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. a limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [ ] . under-reporting/detection in rural areas as compared to peri-urban areas is possible. we believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. previous studies have tended to use human population density as a proxy for this purpose. in our study we found a strong association between human population density and urbanicity. but we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. this study explored the validity of a general model for disease emergence that combined the iom 'convergence model' [ ] and the social-ecological systems model [ , ] , for investigating the specific case of hpai in vietnam. we sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. our results generally support the hypothesis that social-ecological system transformations are associated with h ni outbreaks in poultry. the results presented here highlight three main findings: ) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; ) high land-use diversity landscapes, a variable not previously considered in spatial studies of hpai h n , are at significantly greater risk for hpai h n outbreaks; as are ) landscapes where intensive and extensive forms of poultry production are co-located. only one other study has explicitly examined urbanicity in the context of hpai h n . loth et al. [ ] found peri-urban areas in indonesia were significantly associated with hpai h n cases, even based on multivariate models. our study, however, attempted both to associate hpai h n with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. when those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. we found in the main river deltas in viet nam (red river and mekong), urbanization had no significant association with hpai h n . this may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. this is the first study to examine land-use diversity as a risk factor for hpai h n . measured by the gini-simpson diversity index of the five land-use classes on which data were collected in the viet nam agricultural census, and the presence or absence of hpai outbreaks at the commune level, our results indicate a strong association between land-use diversity and hpai h n at the national level and in the mekong river delta. this metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. our results are similar to what has been observed by studies of other eids using fragmentation metrics (e.g. [ ] [ ] [ ] . this is one of the few studies, however, to link landscape fragmentation to an eid disease in poultry and not just to the vector and/or hosts of the eid. previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [ , [ ] [ ] [ ] . this study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. future studies need to examine the biological causal mechanisms in this context. we suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). mapping land-use classes at the national scale for local administrative units (i.e., the , communes in viet nam) is not an insignificant task. future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [ ] . vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. while other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. another important contribution of this study was the discovery of the importance of cti. so far cti had been used only in ecological niche modeling studies of hpai h n ; the specific role and direction of influence of cti had has so far been unknown. our study, the first to use cti as a risk factor, found it had a large positive influence on hpai h n risk at the national level. previous studies have highlighted the role of surface water extent in the persistence and transmission of the hpai h n virus. these studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. cti on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a gis database. the national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. in the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. this suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the mekong delta [ ] . though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled ndvi median may-october, buffalo density and sweet potato yield. another study in the red river delta [ ] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. we speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. the differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. our study has the potential to inform the design of future research related to the epidemiology of other eids in viet nam and elsewhere. for example, we speculate that in southeast asia, japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [ ] , may also show a strong association with peri-urbanization. in some areas of asia these ecological conditions occur near, or occasionally within, urban centers. likewise, hantaan virus, the cause of korean hemorrhagic fever, is associated with the field mouse apodemus agrarius and rice harvesting in fields where the rodents are present [ ] . our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, cti, and distance to infrastructure. our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as newcastle disease. finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates iom's convergence model with the subsequently proposed social-ecological systems and eid framework. thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices. committee on emerging microbial threats to health in the st century. emerging infections: microbial threats to health in the united states emerging infectious diseases in : years after the institute of medicine report navigating social-ecological systems: building resilience for complexity and change committee on emerging microbial threats to health in the st century. microbial threats to health: the threat of pandemic influenza avian influenza virus (h n ): a threat to human health 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in china identifying risk factors of highly pathogenic avian influenza (h n subtype) in indonesia risk factors and clusters of highly pathogenic avian influenza h n outbreaks in bangladesh freegrazing ducks and highly pathogenic avian influenza modelling the ecology and distribution of highly pathogenic avian influenza (h n ) in the indian subcontinent the urban health transition hypothesis: empirical evidence of an avian influenza kuznets curve in vietnam? urbanization and the spread of diseases of affluence in china defining the "urban" in urbanization and health: a factor analysis approach understanding community context and adult health changes in china: development of an urbanicity scale quantifying the urban environment: a scale measure of urbanicity outperforms the urban-rural dichotomy the emergence of desakota in asia: expanding a hypothesis risk factors for human disease emergence global trends in emerging infectious diseases pathogenic landscapes: interactions between land, 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statistical artefact? seasonal patterns in human a (h n ) virus infection: analysis of global cases integrated mapping of establishment risk for emerging vector-borne infections: a case study of canine leishmaniasis in southwest france fragmentation analysis for prediction of suitable habitat for vectors: example of riverine tsetse flies in burkina faso the impact of habitat fragmentation on tsetse abundance on the plateau of eastern zambia spatial pattern analysis program for quantifying landscape structure risk factors of highly pathogenic avian influenza h n occurrence at the village and farm levels in the red river delta region in vietnam factors in the emergence of infectious diseases we thank nargis sultana, university of hawaii, manoa for assistance with compiling a gis database. we thank the following for giving us advice and suggestions on the statistical model- key: cord- -tp o fxx authors: oliveira, cláudia c.; van hall, thorbald title: alternative antigen processing for mhc class i: multiple roads lead to rome date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tp o fxx the well described conventional antigen-processing pathway is accountable for most peptides that end up in mhc class i molecules at the cell surface. these peptides experienced liberation by the proteasome and transport by the peptide transporter tap. however, there are multiple roads that lead to rome, illustrated by the increasing number of alternative processing pathways that have been reported during last years. interestingly, tap-deficient individuals do not succumb to viral infections, suggesting that cd t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tap-independent peptides have been identified in the grooves of different mhc class i alleles. some of these peptides are not displayed by normal tap-positive cells and we therefore called them teipp, for “t-cell epitopes associated with impaired peptide processing.” teipps are hidden self-antigens, are derived from normal housekeeping proteins, and are processed via unconventional processing pathways. per definition, teipps are presented via tap-independent pathways, but recent data suggest that part of this repertoire still depend on proteasome and metalloprotease activity. an exception is the c-terminal peptide of the endoplasmic reticulum (er)-membrane-spanning ceramide synthase trh that is surprisingly liberated by the signal peptide peptidase (spp), the proteolytic enzyme involved in cleaving leader sequences. the intramembrane cleaving spp is thereby an important contributor of tap-independent peptides. its family members, like the alzheimer’s related presenilins, might contribute as well, according to our preliminary data. finally, alternative peptide routing is an emerging field and includes processes like the unfolded protein response, the er-associated degradation, and autophagy-associated vesicular pathways. these data convince us that there is a world to be discovered in the field of unconventional antigen processing. defective ribosomal product (drip) proteins generates small peptides. potentially, a multitude of proteolytic systems may generate antigenic peptides, but the proteasome is responsible for the liberation of majority of them. inhibition of proteasome activity strongly decreased the pool of mhc class i-binding peptides ( ) . proteasomal cleavage typically creates a peptide's c-terminus compatible with mhc class i binding, and peptides are typically extended at their n-terminus ( , ) . peptides generated in the cytosol are translocated into the endoplasmic reticulum (er) by the tap /tap peptide transporter, where they have access to the peptide loading complex (plc), which is located within the er. tap is a heterodimeric member of the atp-binding cassette (abc) family of transporters, and peptide binding induces atp hydrolysis and transport across the er membrane. once in contact with the plc, the er-amino peptidase eraap (also known as erap ) trims n-extended peptides to a length appropriate for mhc class i binding ( ) ( ) ( ) . chaperone tapasin promotes the formation of stable mhc class i/peptide complexes and acts as an editor. additionally, calnexin facilitates the early folding of mhc class i heavy chains, whereas calreticulin and erp are involved in peptide loading ( ) ( ) ( ) . this pathway is also known as the proteasome-tap pathway and is considered as the conventional processing route because it is the mainstream pathway operating in cells under normal conditions ( ) ( ) ( ) . however, cells are equipped with alternative routes leading to liberation and loading of peptides into mhc class i molecules. these routes are independent of one or more molecules from the conventional pathway such as the proteasome, tapasin, or tap. this has become apparent from studies on cells with deficiencies in the conventional processing pathway. in this review, we will discuss what is known to date regarding alternative enzymes and routes to peptide loading compartments of endogenously generated peptides that feed the direct mhc class i pathway, especially important in cases of failure of the conventional route. we have not included interesting literature on cross-presentation pathways for mhc class i peptides. more than years ago, several papers made the important discovery that a large oligopeptidase, called tripeptidyl peptidase ii (tppii), participates in endoproteolytic activity in the cytosol and partially compensates for a deficient proteasomal activity ( ) ( ) ( ) ( ) . increased tppii activity even allowed for cell survival in lethal conditions of proteasome inhibition ( , ) . in these conditions, tppii activity also partially restored peptide presentation in mhc class i molecules and it was speculated that it could account for the generation of some epitopes independently or in cooperation with the proteasome ( ) . in fact, a paper from seifert et al. showed that tppii was involved in the generation of an epitope from the human immunodeficiency virus (hiv) protein negative factor (nef) ( ) . after that, an increasing number of proteolytic enzymes have been implicated in the generation of peptide-epitopes independently of the proteasome. insulindegrading enzyme (ide) generates an epitope from the human melanoma antigen mage-a ( ) . thimet oligopeptidase (top) and nardilysin are required for the generation of three other clinically relevant ctl epitopes: the tumor-antigen prame, an epitope from epstein-barr virus (ebv) protein ebna c, and an epitope from the melanoma protein mart- ( ) . these enzymes are part of an array of cytosolic endo-and exo-proteases that complement proteasomal activity and degrade proteasome products ultimately into amino acids. importantly, the process of peptide liberation from the protein context is inevitably coupled to the destruction pathway and all proteases mentioned above also destroy some antigenic peptides ( ) ( ) ( ) ( ) . peptides that are "rescued" from total destruction are transported by tap into the er and can potentially bind mhc class i molecules. in eukaryotic cells, secretory and membrane proteins contain a signal sequence essential for protein targeting to the er, the entrance for the secretory pathway ( , ) . these signal sequences are typically composed of three domains: a hydrophobic core (h region) of - amino acids, a polar c-terminal end (c region) with small uncharged amino acids, and a polar nterminal region (n region) with a positive net charge ( ). after insertion into the protein-conduction channel, signal peptides are usually cleaved from the preprotein by signal peptidase (sp) ( ). thereafter, signal peptides, which are small domains and trapped in the er membrane, can undergo intramembrane proteolysis by cleavage within their transmembrane region by the presenilintype aspartic protease signal peptide peptidase (spp) ( , ). peptide ligands suitable for mhc class i binding are thought to be generated after the intramembrane proteolysis by spp that promotes the release of signal peptide fragments from the er membrane ( , ). the spp-cleaved fragments in the vicinity of the cytosol can get access to the cytosol again and be further processed by the proteasome and transported by tap into the er. most hla class i molecules donate their leader sequences for binding to the non-classical hla-e, and the cleavage of these signal sequences is mediated by sp and spp ( - ). these leader peptides are even the most dominant source of peptides for hla-e. proper surface expression of hla-e prevents cytotoxic action of natural killer cells that continuously sense the presence of peptide/hla-e complexes through their cd /nkg receptors ( - ). the absence of these complexes at cell surface results in failure of interaction with cd /nkg a receptors, which activate nk cells for killing their targets. pioneering studies by peter cresswell and victor engelhard in revealed that most peptides presented at the surface of tap-deficient cells were derived from signal sequences, specific protein regions at the n-terminus of proteins ( , ). indeed, the parts of the leader peptide within the er membrane that are closest to the er lumen are released there and can get access to mhc class i grooves independent of proteasomes or tap (figure ) . after the cleavage of the transmembrane sequence by spp, the peptide fragments are released and can associate with mhc class i/β m nascent molecules. this intramembrane proteolysis by spp is thought to be important for the clearance of the er membrane by removing small protein remnants anchored at figure | classical and alternative pathways for mhc class i presentation. cells with deficiencies in components of the mhc class i processing pathway, such as tap, can present a repertoire of peptides derived from alternative processing pathways. different "housekeeping" cell functions such as signal peptide cleavage, protein maturation in the golgi, and protein/organelle disposal via autophagy can provide peptide ligands for mhc class i loading. the membrane rendering them susceptible for subsequent degradation. the intramembrane cleavage by spp is favored by spp topology that conceals the catalytic center within the plane of the membrane. the two aspartic residues required for the proteolytic activity of spp are located within conserved (y/f)d and g(l/i/f)gd amino acid motifs in two adjacent transmembrane domains ( , ). regarding a cleavage motif for spp, no consensus cleavage site has been described. however, spp demonstrates a strong preference for substrates with helix-destabilizing residues in their transmembrane domain ( - ). amino acids like asparagine, serine, and cysteine disturb a perfect alfa-helical conformation of the transmembrane domain and are therefore referred to as "helix-bending" or "helix-breaking" residues. signal peptides have been shown to contain amino acids with "helixbreaking" capacity within their h region, which critically influence their proteolytic processing by spp ( , ( ) ( ) ( ) . the disturbance of the α-helix caused by these residues is thought to facilitate intramembrane proteolysis. this and other issues would be clarified with the atomic resolution of this protease but this is still lacking due to its technical difficulties. we can have an approximation of that by looking at the crystal structure of presenilin/spp homologs recently published ( ) ( ) ( ) ( ) . jr is an spp homolog from the archaeon methanoculleus marisnigri that harbors nine transmembrane helices, similar to what is predicted for spp, with tmd and tmd containing the yd and gxgd motif, respectively. the two catalytic aspartate residues are located close to each other and approximately Å into the lipid membrane. proteolytic activity occurs in the presence of water molecules that gain access to the catalytic aspartates through a large cavity between two terminal domains ( ) . the three-dimensional structure of a human presenilin comprised into the γ-secretase complex has also been described ( ) . for the near future, we can expect more information on the catalytic activity of this family of proteases, including spp, which is definitely an important contributor of peptides for mhc class i presentation. our recent data revealed an additional role for intramembrane proteolysis by spp. regardless its name, spp also appeared to liberate a c-terminal peptide, independent of proteasome activity. the processing of c-terminal regions of a type ii protein inserted in the er membrane leads to the presentation of peptides independently of proteasome and tap ( , ) . the cterminal region from the ceramide synthase trh , which is a multiple membrane-spanning protein in the er, contains a mer peptide-epitope that is located at the very c-terminal end of the protein and protrudes into the lumen of the er ( - ). the trh protein has a housekeeping function and is ubiquitously expressed. inhibition of spp activity blocked the generation of the trh peptide. experiments with mutant forms of the trh protein indicated that the intramembrane cleavage by spp occurs at the direct upstream region of the t cell epitope within the lipid bilayer ( ) . we speculated that spp activity in the er membrane is sufficient to liberate the minimal c-terminal -mer peptide and release of this peptide into the er lumen. other proteolytic enzymes, such as amino-peptidases, were dispensable. further carboxy-terminal processing was not needed since the epitope is located at the very c-terminal end of the protein. direct release of the liberated peptide into the er lumen is very likely, due to the type ii transmembrane orientation of the trh protein tail (nterminus to c-terminus orientation). the exact peptide loading mechanism of the trh membrane peptide, however, remains to be determined. what triggers the cleavage of trh by spp is not known yet. in addition to its liberation function of small transmembrane substrates, spp has been shown to associate with misfolded membrane proteins in complexes where spp is represented as a monomer, dimer, or multimer ( ) . it was suggested that such high molecular weight complexes act as chaperones to dispose of membrane aggregates ( , ) . the role of spp in this degradation machinery might be to liberate such aggregates from the er membrane. these discoveries were based on the viral us and us proteins, which successfully labels mhc class i molecules in the er for retrograde transport to push this protein back to the cytosol for degradation by proteasomes ( , ) . interestingly, the spp family member, presenilin , seems to be associated with this membrane proteolysis as well ( ) . since spp and presenilin have opposing preference for type i and type ii transmembrane orientations, such a "degradome" machinery might be responsible to clear the er membranes. this type of machinery is called the erassociated degradation (erad) pathway. erad is an er quality control system, which monitors the integrity of nascent or erresident proteins and targets incorrectly folded or misassembled proteins to degradation. the disposal of misfolded mhc class i heavy chains also occurs through the erad pathway in the absence of viral interference ( ) . clearly, viruses take advantage of these existing pathways and hijack them in order to evade immune recognition by cytotoxic t cells ( , ) . another role for spp in erad has come from a recent paper describing the cleavage of the unfolded protein response (upr) regulator xbp u by spp ( ) . xbp u is a type ii membrane protein and undergoes intramembrane cleavage within a conserved type ii tm domain while integrated in a complex containing spp, the protease derlin- , and the e ligase trc , which prime spp for xbp u cleavage. an ectodomain shedding of spp substrates prior to spp cleavage is thought to be required and normally performed by sp in signal sequences but such cleavage was unnecessary in the case of xbp u, similarly to what we have described for trh ( , ) . in general, the upr induces a strong downregulation of mhc class i molecules at the cell surface ( , ) . thus, spp activity seems to have a direct impact on mhc class i peptide presentation by cleavage of leader sequences from nascent proteins and the liberation of some c-termini for mhc class i loading. a more indirect role that impacts on mhc class i presentation has been revealed and occurs through an erad pathway. this is also supported by a recent study using a systemslevel strategy reporting the involvement of spp in the network that coordinates the erad response ( ). the group of yewdell showed more than a decade ago that peptides located at the c-terminus of er-targeted proteins are very efficiently generated and presented on mhc class i ( ) ( ) ( ) ( ) . the location of the peptide was essential to be at the very end of the c-terminus of the protein, not requiring c-terminal trimming, in line with the fact that there is poor carboxypeptidase activity in the er ( ) . they described the presentation of tap-independent peptides from one er-resident protein, jaw , and proteins in the secretory pathway, like ovalbumin and cd . in each case, the peptides were efficiently liberated from the very c-terminus by the activity of yet unidentified endo-proteases to be generated as class i ligands. based on this pathway of peptide liberation, the authors provided the term "c-end rule" to highlight the capacity of er-resident proteases to liberate class i ligands from the cterminal ends of er-targeted proteins. the liberation of peptides without the intervention of the proteasome can also occur in the trans-golgi network ( , ) . the main proteolytic enzyme involved was shown to be furin, a known protease of the trans-golgi network normally required for the maturation of secreted proteins (e.g., growth factors and neurotransmitters) by cleaving at precise stretches of three to four basic residues ( ) . furin is part of a family of proprotein convertases that comprises nine members ( ) . three members (pc / , pace , and pc ) including furin are widely expressed and together they take part in a variety of processes occurring in the trans-golgi network, cell surface, or endosomes. this leads to the activation or inactivation of receptors, ligands, enzymes, viral glycoproteins, or growth factors ( ) . furin also has important functions during development by processing substrates like bone morphogenetic protein (bmp ), a member of the tgf-β superfamily that plays a critical role in heart development ( ) . furin processes a wide variety of precursor proteins after the c-terminal arginine residue in the preferred consensus motif -arg-x-arg/lys-arg↓-x-(x is any amino acid and "↓" indicates the cleavage position) ( ) . initially, this pathway was studied with the use of a model peptide at the cterminus of the secreted hepatitis hbe protein. furin-processed antigens targeted to the secretory route were presented by mhc class i at the cell surface and could elicit functional cd t-cell responses in vivo in a tap-independent fashion ( , ) . the ctermini of secretory or er-localized proteins thus appear to be processed for presentation to cd t cells (figure ). the generation of mhc class i ligands described above defines several alternative ways to generate peptide ligands without the intervention of the proteasome. these represent unusual pathways for peptide generation. now, we will discuss a different constraint in the conventional antigen presentation pathway related to the blockade of peptide entrance in the er due to tap deficiency. in human beings, tap-deficiency syndrome has been described in several independent families and results from mutations in either one of the subunits of the peptide transporter, tap and tap ( , ) . interestingly, these tap-deficient individuals do not succumb to viral infections, suggesting that cd t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tapindependent peptides have been identified in the grooves of different mch class i alleles. importantly, these tap-deficient patients harbor a polyclonal cd t-cell repertoire that is capable of recognizing peptides from the ebv virus, like protein lmp , presented on tap-deficient cells ( ) . the tap-independent processing pathway is capable of generating enough mhc class i/peptide complexes in order to keep immunosurveillance and control of viral infections. studies with tap -knockout mice have shown that surface expression levels of mhc class i are indeed lower, but the remaining complexes do induce a broad and polyclonal repertoire of cd t-cells ( ) ( ) ( ) . the tcr usage was shown to be very comparable to that of wild-type mice and tap -knockout mice were capable of mounting anti-viral cd t cell responses. together, these data show that, although crippled, the mhc class i-presented peptide repertoire in the absence of tap is sufficient to support cd t cell immunity. interestingly, peptides emerging from alternative tapindependent routes appeared to be immunogenic. following immunizations in mice with tap-deficient tumor cells, specific cd t-cells were induced that recognize tap-deficient cells, but not normal cells ( , ) . these t-cell epitopes seemed to be selectively presented by tap-deficient cells but not under normal conditions. this alternative peptide repertoire emerges due to processing defects and therefore these peptides were named "t cell epitopes associated with impaired peptide processing" (teipp). the molecular identification of some teipp peptides revealed that they can be diverse in length (from -mer to -mer), amino acid composition, and mhc class i binding, as some are presented by classical mhc class i molecules and others by the non-classical mhc molecule hla-e and the mouse homolog qa- b ( table ) ( , [ ] [ ] [ ] [ ] . they are derived from normal housekeeping proteins with ubiquitous expression, but are surprisingly not loaded on mhc class i in cells with an intact antigen-processing machinery. they constitute normal self-peptides (non-mutated, not pathogen-or tumor-specific) and can be regarded as real neo-antigens. the immunogenicity of teipp peptides exists because they are not presented by normal cells including the thymus. during thymic development, t cells are subjected to two subsequent processes called positive and negative selection ( , ) . negative selection is necessary for the maintenance of self-tolerance as it induces the deletion or inactivation of potentially autoreactive thymocytes ( ) . we recently demonstrated that teipp-specific cd t-cells indeed do not undergo negative selection and are thereby available for therapeutic exploitation. since the peptides recognized by teipp-specific ctl are derived from housekeeping proteins, we tried to understand why teipps are not presented by processing intact cells. collectively, our data show that teipp peptides are actually produced within processing proficient cells, but somehow are not or not sufficiently presented by their surface mhc class i molecules. taking the trh -derived teipp peptide as a model, we have analyzed the expression of the trh gene in several epithelial populations isolated from wild-type and tap -ko mice ( ) . this analysis revealed the same level of rna transcripts between the normal and knockout populations, suggesting comparable protein levels in both cell types. the liberation of the trh peptide is performed by spp, which is active in tap-positive as well as tap-negative targets ( ) . overexpression of the trh gene in tap-positive cells leads to surface presentation in mhc class i ( ), still in a proteasome-independent way. moreover, proteasome inhibition in tap-positive cells results in presentation of the endogenous trh peptide ( ) , indicating that, indeed, this teipp peptide is generated in all cells but loses competition with the overwhelming amount of tap-imported peptides in tappositive cells. some alternative peptides, like the ones derived from ebv proteins were shown to be presented on tap-positive cells to comparable extent, although using alternative pathways. moreover, our data suggest that the limited quantity of the trh peptide-epitope in the er is the main cause of selective presentation in tap-deficient cells. interestingly, gradual increase of overexpression correlated with the degree of recognition by the teipp-specific ctl clone, implying that tap transport actually constitutes a strong barrier for teipp peptides. the study of human teipp antigens corroborates these findings. one antigenic peptide is encoded by the human calca gene and derives from the signal sequence of preprocalcitonin (ppct) protein. this peptide is liberated in the er lumen by sequential cleavage with sp and spp, independently from proteasomes ( table ) ( ) . the presentation of the ppct peptide to specific ctl was found in human lung and medullary thyroid carcinomas that had very low expression of tap. presentation of the ppct peptide occurred also in normal non-transformed cells, such as dendritic cells (dcs), after knockdown of tap. overexpression of the calca gene in dcs and tap-positive tumor cells resulted in recognition by the specific ctl clone ( ) . identification of additional human teipp antigens at the molecular level will enable cd t cell targeting of otherwise ctl-resistance tap-negative tumor variants ( , ) . together, these findings support the model of peptide competition in the er as a factor that prevents presentation of peptides from alternative sources, and shape a picture of alternative processing pathways that emerge upon defects in the conventional one (figure ). the precise loading mechanism of tap-independent signal peptides into mhc class i molecules is not known, since processing by sp and spp is thought to take place outside of the plc. this sophisticated machinery for optimizing ligand length and quality and facilitating peptide loading onto nascent mhc class i molecules greatly facilitates peptide loading by physical bridging transporters to chaperones for loading and also "edits" the repertoire of bound peptides to maximize their affinity ( ) . the plc molecule tapasin tethers mhc class i molecules to the peptide transporter acting together with the chaperone calreticulin and the oxidoreductase erp ( ). tapasin can sense the quality of peptide bound by mhc class i complexes, and allows successive rounds of peptide binding until a certain affinity threshold is met. trimming of incoming peptides by eraap can be necessary for obtaining a good peptide length before selection by a defined mhc class i allele. a recent paper states that mhc class i molecules initially bind a variety of peptides, including some lowaffinity or n-terminally extended ones but then quickly dissociate from the molecule followed by the selection of the best fitting candidates ( ) . however, tap-independent leader peptides do not arrive in the er via tap and thus lack these editing and optimizing chaperones. in the absence of tap transporters, the loading of peptides into mhc class i occurs without a fully functional plc at hand. in that respect, inefficient loading of leader peptides in tap-positive cells can be expected, whereas in cells devoid of tap, this alternative er entrance mechanism allows emergence of these peptides. so here, the plc floats in the er membrane and is dispatched from the entry site of peptides. indeed, differential mass spectrometry analysis showed an enhanced presentation of leader peptides in cells lacking the peptide transporter ( ) . after all, mhc class i molecules get loaded with the available repertoire: from conventional or unconventional sources. but how do these "untapped" peptides find their way to empty mhc class i molecules at all? it has been shown that loading of peptides in mhc class i can occur with the minimal components of tapasin-erp -mhc class i complexes ( ), but we have to assume that the chances of a peptide to find the plc machinery in the er are scarce. on the other hand, tap-deficient cells harbor more peptide-receptive class i molecules compared to normal cells ( ) . these peptides might actually be actively chaperoned toward these open grooves. chaperones with high peptide binding capacity in the er are heat shock proteins (e.g., hsp ) and pdi, an isomerase that efficiently binds free peptide ( ) . it is possible that tap-independent peptides are captured by these molecules and chaperoned to mhc class i. recent studies suggest that the tapbpr molecule ("tapasin-like") binds those mhc class i proteins that are not bound to tapasin in the plc, so we might hypothesize that this pool of peptide-receptive grooves may function to load leader peptides ( ) . however, this still needs to be investigated. other ways to access peptide-receptive mhc has been proposed and are related to the intracellular traffic of hydrophobic peptides. studies with several epitopes from the lmp protein of the ebv showed that peptides, which possess a high hydrophobicity index, were presented in a tap-independent manner ( ) . the proposed mechanism describes the generation of these peptides outside the er, since proteasome activity is needed for presentation, and subsequently free diffusion across the er membrane possible due to their high hydrophobicity. these peptides might transgress membranes spontaneously or via alternative membrane transporters. a recent study by tey et al. showed that the processing of a peptide antigen from the human cytomegalovirus (hcmv) latency associated protein, pul , occurs entirely in the vesicular pathway and is mediated by autophagy ( ) . also other examples of autophagy enhanced mhc class i presentation of viral antigens were reported ( , ) . during autophagy, large portions of cytoplasmic content, including proteins and organelles, are encapsulated in double membrane vesicles called autophagosomes ( ) ( ) ( ) ( ) . the autophagosomal membrane has been proposed to originate from the er ( , ) and peptide-receptive mhc class i molecules might be present in autophagosomes, allowing for loading of peptides in these vesicular compartments ( ) . in addition, recirculating mhc class i molecules from the cell membrane end up in endosomes and can have contact with autophagosomes before returning to the endocytic network. transit for membrane-associated proteins between autophagosomes and endosomes has been observed by live cell imaging ( ) . moreover, peptides generated by the proteasome seem to get access to the endocytic vesicular pathway as well. we recently found at least one example for this in our teipp repertoire proportions and combinations. the mhc class i peptide repertoire can therefore be compared to a painter's palette where the different "colors" (peptides) are mixed and used to create a colorful and complex "picture." that is generated by the proteasome but is tap-independently presented ( table ) ( ) . the presentation of this peptide was surprisingly enhanced by blockade of the proton pump in endosomes, indicating that this antigen most likely crosses the vesicular pathway ( ) . finally, mass spectrometry analysis of the tapindependent peptide repertoire pointed at proteins located in the vesicular compartment as an important source ( ) ( ) ( ) . though these alternative processing and loading compartments have not fully been unraveled, these data strongly support the notion that the vesicular pathway and autophagy contributes to antigen presentation by mhc class i molecules. on summarizing, we can conclude that, even though the conventional proteasome-tap pathway represents the major source of the mhc class i peptide repertoire, alternative processing pathways clearly complement the total pool. this multitude of mhc class i processing pathways can be compared to a colorful palette where the painter combines the different colors that will end up in different proportions and combinations in the final picture (figure ) . in situations of viral infections, cellular transformation, or other initiators of stress, the contribution of these alternative pathways might gain importance. the spp and maybe their family members are convincing examples of alternative proteolytic systems that feed the alternative routes of antigen presentation. the precise molecular identification of alternative loading mechanisms unto peptide-receptive mhc class i molecules, whether it be in the er or in autophagosomes, still needs indepth investigation. in that respect, peptides can walk on multiple different paths before ending up in the grooves of mhc class i and therefore illustrate the old expression that multiple roads lead to rome. the protease inhibitor, n-acetyl--leucyl--leucyl-leucyl--norleucinal, decreases the pool of major histocompatibility complex class i-binding peptides and inhibits peptide trimming in the endoplasmic reticulum s proteasomes and immunoproteasomes produce mainly n-extended versions of an antigenic peptide the sizes of peptides generated from protein by mammalian and s proteasomes. implications for understanding the degradative mechanism and antigen presentation an ifngamma-induced aminopeptidase in the er, erap , trims precursors to mhc class i-presented peptides eraap customizes peptides for mhc class i molecules in the endoplasmic reticulum the er aminopeptidase erap enhances or limits antigen presentation by trimming epitopes to - residues mechanisms of mhc class i-restricted antigen processing and cross-presentation recent advances in antigen processing and presentation proteasome and peptidase function in mhc-class-i-mediated antigen presentation towards a systems understanding of mhc class i and mhc class ii antigen presentation proteases in mhc class i presentation and cross-presentation the known unknowns of antigen processing and presentation a giant protease with potential to substitute for some functions of the proteasome a proteolytic system that compensates for loss of proteasome function antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic t cell epitopes integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity an essential role for tripeptidyl peptidase in the generation of an mhc class i epitope production of an antigenic peptide by insulin-degrading enzyme a major role for tppii in trimming proteasomal degradation products for mhc class i antigen presentation pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases the efficiency of human cytomegalovirus pp ( - ) cd + t cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases the cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of mhc class i antigen presentation the translocon: a dynamic gateway at the er membrane approaching the mechanism of protein transport across the er membrane requirements for signal peptide peptidasecatalyzed intramembrane proteolysis on the mechanism of spp-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane structural analysis of hepatitis c virus core-e signal peptide and requirements for cleavage of the genotype a signal sequence by signal peptide peptidase maturation of hepatitis c virus core protein by signal peptide peptidase is required for virus production signal peptide peptidase cleavage of gb virus b core protein is required for productive infection in vivo the crystal structure of gxgd membrane protease flak structure of a presenilin family intramembrane aspartate protease crystal structure of bacillus subtilis signal peptide peptidase a structure of signal peptide peptidase a with c-termini bound in the active sites: insights into specificity, self-processing, and regulation three-dimensional structure of human gamma-secretase new role of signal peptide peptidase to liberate c-terminal peptides for mhc class i presentation peptide transporter tap mediates between competing antigen sources generating distinct surface mhc class i peptide repertoires mammalian ceramide synthases two mammalian longevity assurance gene (lag ) family members, trh and trh , regulate dihydroceramide synthesis using different fatty acyl-coa donors selective cytotoxic t-lymphocyte targeting of tumor immune escape variants signal peptide peptidase (spp) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes signal peptide peptidase is required for dislocation from the endoplasmic reticulum a high-coverage shrna screen identifies tmem as an e ligase involved in er-associated protein degradation the human cytomegalovirus us gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol interactome analyses of mature gamma-secretase complexes reveal distinct molecular environments of presenilin (ps) paralogs and preferential binding of signal peptide peptidase to ps er quality control in the biogenesis of mhc class i molecules a membrane protein required for dislocation of misfolded proteins from the er the hcmv gene products us and us target mhc class i molecules for degradation in the cytosol signal peptide peptidase functions in erad to cleave the unfolded protein response regulator xbp u the unfolded protein response (upr)-activated transcription factor x-box-binding protein (xbp ) induces microrna- expression that targets the human antigen peptide transporter (tap ) mrna and governs immune regulatory genes stimulation of an unfolded protein response impairs mhc class i expression defining human erad networks through an integrative mapping strategy two novel routes of transporter associated with antigen processing (tap)-independent major histocompatibility complex class i antigen processing promiscuous liberation of mhc-class i-binding peptides from the c termini of membrane and soluble proteins in the secretory pathway trimming of antigenic peptides in an early secretory compartment tap-independent delivery of antigenic peptides to the endoplasmic reticulum: therapeutic potential and insights into tap-dependent antigen processing peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by mhc class i major histocompatibility complex class i viral antigen processing in the secretory pathway defined by the trans-golgi network protease furin furin-processed antigens targeted to the secretory route elicit functional tap -/-cd + t lymphocytes in vivo furin at the cutting edge: from protein traffic to embryogenesis and disease the multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions the activation and physiological functions of the proprotein convertases furin is the major processing enzyme of the cardiacspecific growth factor bone morphogenetic protein proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells postendoplasmic reticulum rescue of unstable mhc class i requires proprotein convertase pc description of hla class i-and cd -deficient patients: insights into the function of cytotoxic t lymphocytes and nk cells in host defense clinical and immunological aspects of hla class i deficiency human peptide transporter deficiency: importance of hla-b in the presentation of tap-independent ebv antigens positive selection of self-and alloreactive cd + t cells in tap- mutant mice tap -deficient mice select a cd + t cell repertoire that displays both diversity and peptide specificity tap mutant mice are deficient in antigen presentation, surface class i molecules, and cd - + t cells generation of cd + t cells specific for transporter associated with antigen processing deficient cells alternative peptide repertoire of hla-e reveals a binding motif that is strikingly similar to hla-a cd + t cell responses against tap-inhibited cells are readily detected in the human population the nonpolymorphic mhc qa- b mediates cd + t cell surveillance of antigen-processing defects nonclassical mhc class ib-restricted cytotoxic t cells monitor antigen processing in the endoplasmic reticulum antigen presentation in the thymus for positive selection and central tolerance induction selection of the t cell repertoire the thymic medulla: a unique microenvironment for intercellular self-antigen transfer preprocalcitonin signal peptide generates a cytotoxic t lymphocyte-defined tumor epitope processed by a proteasome-independent pathway different expression levels of the tap peptide transporter lead to recognition of different antigenic peptides by tumor-specific ctl strategies to counteract mhc-i defects in tumors a novel category of antigens enabling ctl immunity to tumor escape variants: cinderella antigens tapasin-the keystone of the loading complex optimizing peptide binding by mhc class i molecules in the endoplasmic reticulum the first step of peptide selection in antigen presentation by mhc class i molecules features of tap-independent mhc class i ligands revealed by quantitative mass spectrometry selective loading of high-affinity peptides onto major histocompatibility complex class i molecules by the tapasin-erp heterodimer peptide-receptive class i major histocompatibility complex molecules on tap-deficient and wild-type cells and their roles in the processing of exogenous antigens tap-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp , protein disulfide isomerase and calreticulin the binding of tapbpr and tapasin to mhc class i is mutually exclusive processing of a multiple membrane spanning epstein-barr virus protein for cd (+) t cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway autophagy mediates transporter associated with antigen processing-independent presentation of viral epitopes through mhc class i pathway alternative pathways for mhc class i presentation: a new function for autophagy autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv- infection autophagy and adaptive immunity when autophagy meets viruses: a double-edged sword with functions in defense and offense unveiling the roles of autophagy in innate and adaptive immunity endoplasmic reticulum and golgi complex: contributions to, and turnover by eating the endoplasmic reticulum: quality control by autophagy generation of mhc class i ligands in the secretory and vesicular pathways the itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes dominant contribution of the proteasome and metalloproteinases to tapindependent mhc-i peptide repertoire allele-dependent processing pathways generate the endogenous human leukocyte antigen (hla) class i peptide repertoire in transporters associated with antigen processing (tap)-deficient cells diversity of natural self-derived ligands presented by different hla class i molecules in transporter antigen processing-deficient cells a transporter associated with antigen-processing independent vacuolar pathway for the mhc class i-mediated presentation of endogenous transmembrane proteins financial support was received from the portuguese foundation for science and technology (grant sfrh/bd/ / to co). gamblin artists colors (portland, or, usa) is acknowledged for the free use of figure . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -p ikd ns authors: hansra, satyender; pujhari, sujit; zakhartchouk, alexander n title: exploration of new sites in adenovirus hexon for foreign peptides insertion date: - - journal: open virol j doi: . / sha: doc_id: cord_uid: p ikd ns adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. there are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. the first includes an insertion of the foreign gene expression cassette into the e region. the second strategy is antigen incorporation into the viral capsid proteins. to extend this methodology, we have searched for new sites at the human adenovirus serotype major capsid protein hexon for a vaccine antigen insertion. to this end, we utilized sites in the hexon hypervariable region (hvr) , and to display a -mer peptide containing the main neutralizing epitope of porcine reproductive and respiratory syndrome virus. however, we could not rescue the viruses with the insertions of the peptide into hvr and , consistent with the viruses being unable to tolerate insertions at these sites. in contrast, the virus with the insertion of the peptide in hvr was viable - growing well in cell culture and the inserted peptide was exposed on the virion surface. human adenovirus type (had ) vector has been shown to be an excellent vaccine delivery system in humans and animals [ , ] . the replication-defective (e -deleted) and replication-competent (e -deleted) had have both been used as vaccine delivery vectors. for biosafety concerns, a replication-defective had is considered to be more suitable for vaccination. replication-defective vectors may also have deletion in the e region to increase the amount of foreign dna that can be inserted. conventional strategy for the expression of a vaccine antigen by adenoviral vector includes insertion of the foreign gene expression cassette into the e region. this antigen is expressed as transgene after the infection of permissive cells with recombinant adenovirus. apparent drawbacks of the conventional transgene expression of antigen include an inability of the had -based vectors to produce a potent humoral response against certain antigens and, in some cases, the inability of the vector to express a foreign gene [ ] . to overcome such hurdles, the "antigen capsid-incorporation" strategy was developed [ ] . incorporating an immunogenic peptide into the had capsid offers several potential advantages. importantly, the processing of capsid-incorporated antigen via the exogenous pathway could result in a strong humoral response, similar to that generated by native had capsid proteins, and this strategy may also be useful in boosting antigen-specific antibody immune responses. adenoviral capsid consists of the hexon, penton base and fiber [ ] . antigenic epitopes could be incorporated into these proteins as well as into the minor capsid protein ix [ ] . importantly, hexon is the most abundant of the capsid proteins, accounting for more than % of the capsid protein [ ] . furthermore, hexon is shown to be a vaccine adjuvant [ ] . early analysis of the hexon protein sequences revealed that, in addition to the conserved regions, there were discrete hypervariable regions (hvrs) [ ] . in a subsequent study, they are reclassified as regions to [ ] . these hvrs do not appear to be involved in the binding of any other viral proteins, and the loops at the top of the hvrs are the most amenable to modifications. it was also shown that short heterologous peptides can be incorporated within the hvrs of hexon without affecting the viability of the virus [ , ] . incorporating antigens into hexon is applicable towards vaccination for several pathogens including the poliovirus, pseudomonas, b. anthracis, influenza, malaria and hiv [ ] [ ] [ ] [ ] [ ] [ ] . in selected studies, protective immune responses in a mouse model are documented [ , ] . in had , hvr through , and have been used for the incorporation of foreign peptides [ , ] . in this study, we explored different sites in hvrs , and of had hexon for the insertion of a peptide corresponding to the porcine reproductive and respiratory syndrome virus (prrsv) main neutralizing epitope b [ ] of the gp protein. our results provide potentially applicable information for adenovirusvectored vaccine development involving such hexon modifications, particularly in hvr . in order to access new sites in had hexon, tailored for the incorporation of foreign peptide sequences, we genetically inserted a amino acid (aa) residue peptide into hvr , hvr or hvr region of the had hexon. the peptide consisted of embedding a aa residue sequence containing the prrsv major neutralizing epitope flanked by the lgs spacer sequences (fig. ) . fig. ( ) . sites in the hvrs that were modified with a peptide containing the prrsv major neutralizing epitope b. the arrows mark the position where the peptide was inserted. the numbers show the positions of the amino acid residues in had hexon. peptide sequence corresponding to the prrsv epitope is underlined. insertion of the dna encoding peptide into the had hexon gene was achieved by pcr followed by bacteriumbased homologous recombination. the linearized dna of the plasmid containing the right portion of the had genome with modified hexon and the bp deletion in the e region was co-transfected into human embryo kidney (hek) cells together with a plasmid containing the left portion of the had genome with an e -deletion. homologous recombination in hek cells led to generation of the recombinant virus, termed ad hvr epb. multiple attempts to rescue the other two recombinant viruses (ad hvr epb and ad hvr epb) were not successful. this inability to rescue recombinant viruses with the foreign peptide insertion into hvr and hvr of hexon suggested that the insertion interferes with the formation of viral particles. interestingly, in a previous study [ ] , hexon-modified virus with incorporation of the his peptide into hvr was reported to be viable. our result with hvr is contradictory, evidently due to the differences in the peptides sequences and their lengths. further, while the two studies are made with changes within hvr , the specific locations of the incorporations differ as well. notably our study uses a foreign sequence inserted between asn and gly ( fig. ) , while wu et al. [ ] placed his between pro and trp substituting for aa residues of hexon. to corroborate the insertion of the peptide encoding dna into the hexon gene of had , the viral genome fragment was amplified using pcr from the viral dna extracted from ad hvr epb-infected cells, and the pcr product was digested with avrii since the peptide encoding dna contained the single avrii site. predictably, a bp pcr fragment was amplified from ad hvr epb dna (fig. , lane ), and its site specific cleavage by avrii provided two fragments: bp and bp (fig. , lane ) . the results of this analysis suggested that recombinant ad hvr epb contained the epitope b encoding sequence in the hvr of hexon. the growth kinetics of the recombinant virus ad hvr epb was analyzed in hek cells. cultures of the cells were infected with ad hvr epb or ad -empty (e -deleted and partially e -deleted had with unmodified hexon), and the cells were harvested at , , , , and hr post infection intervals. virus in each sample was released by freeze-thawing and titered on monolayers of hek cells. fig. ( ) shows the insertion of the peptide in hvr , resulting in similar virus yields to unmodified virus with maximal titers ranging from x to tcid per ml in a culture of x cells. we also determined the viral particle/infectious particle (vp/ip) ratio for cscl gradient purified preparations of the control virus ad -empty, as well as the hexon-modified virus ad hvr epb. a . fold increase was observed in the vp/ip ratio of ad hvr epb preparations as compared to the unmodified control ad -empty ( table ) . a similar observation is reported for hexon-modified had with peptide incorporation into hvr or [ ] . a typical vp/ip ratio of unmodified adenoviruses ranges from to [ ] . we next examined whether prrsv epitope was presented on the surface of the virion, using an elisa binding assay. different amounts of cscl gradient purified viruses (ad hvr epb and ad -empty) were immobilized on the wells of -well plates and incubated with peptide antisera. the binding of antibodies to the virions was detected with alkaline phosphatase (ap)-conjugated secondary antibody, followed by ap color reaction hvr : ……. development. evidently, the anti-peptide antibodies bind well to adhvr epb virions and not to ad -empty (fig. ) . this suggests that the prrsv epitope inserted into hvr of hexon is exposed on the virion surface. while a previous study showed that epitopes were exposed on the virion surface when incorporated in hvr , hvr and hvr , and not exposed in hvr and hvr , as well as poorly exposed when a peptide was placed in hvr [ ] . in the current study, we find a new site in hvr for incorporation of foreign peptide into had hexon, and determine that the peptide was also exposed on the virion surface making it readily accessible for antibody binding and be potentially useful for vaccination. . ( ) . the prrsv epitope incorporated in hvr of hexon is accessible in the context of an intact virion. in the assay, varying amounts of purified viruses were immobilized in the wells of elisa plate and incubated with anti-peptide antibody. the binding was detected with an ap-conjugated secondary antibody. values are expressed as the mean and standard deviation. * indicates a p value of < . . herein, we report a novel adenovirus vector (ad hvr epb) with the insertion of the prrsv main neutralizing epitope b in different hvr regions of the major capsid protein hexon. while the insertion of the peptide into hvrs and were not tolerated by the adenovirus, the -mer epitope placed within hvr is determined to be exposed on the virion surface. plasmid puc-ad -hex was used as a template for pcr and as a cloning vector. this plasmid contained a . kb dna fragment of had genome encoding a portion of the hexon gene. to insert the dna sequence encoding the main neutralizing epitope b of prrsv in hvr and obtain the dna fragment for cloning, four consequent pcrs were performed using phusion high-fidelity dna polymerase (neb). first, a kb fragment was amplified using primers bl f and nhe-r, and puc-ad -hex as a template. second, a kb fragment was amplified using primers blf and nhe-r, and the product of pcr as a template. third, a bp fragment was amplified using primers bl r and nde-f, and puc-ad -hex as a template. fourth, a . kb fragment was amplified using primers nde-f and nhe-r, and the mixture of the products of pcr and from above as a template. the final . kb pcr product was digested with ndei and nhei and cloned into ndei and nhei sites of puc-ad -hex, creating puc-ad hex-hvr epb. similar strategy was used to construct puc-ad hex-hvr epb and puc-ad hex-hvr epb. specific sequences of the primers are presented in table . the plasmid ph r-hexhvr epb was generated by homologous dna recombination in e. coli bj [ ] between a . kb dna fragment excised by kpni and ndei from puc-ad hex-hvr epb and bamhi-linearized ph r [ ] . similar strategy was used to construct ph r-hexhvr epb and ph r-hexhvr epb. fig. ( ) shows the sites of the insertion of the peptide in the had hexon protein sequence. human embryonic kidney (hek) cells were transfected with paci-digested dnas of the plasmids ph r-hexhvr epb, ph r-hexhvr epb or ph r-hexhvr epb and paci-digested ph -l [ ] using lipofectamine- (invitrogen). homologous recombination in the transfected cells led to generation of the recombinant adenovirus, and the virus was detected by developing cytopathic effect (cpe) in the cell monolayer. if cpe was not detected by the th day after transfection, the cells and the cell culture media were collected and freeze-thawed. this material was used to infect a fresh monolayer of hek cells. the virus was amplified by passaging in hek cells and purified by cscl gradient centrifugation [ ] . the concentration of virus particles (vp) in purified virus preparations was determined by measuring the optical density at nm as previously described [ ] , whereas numbers of infectious particles (ip) were determined by the tissue culture infective dose (tcid ) method [ ] . hek cells grown in wells of -well plates were infected with ad -empty or ad hvr epb viruses at a multiplicity of infection (m.o.i.) of . infected cells were harvested at the indicated time intervals post-infection. cells were lysed in the growth medium by freezing-thawing three times. the titres of infectious viral progeny were determined by tcid method in monolayers of hek cells. hek cells grown in t flasks were infected with the virus ad hvr epb. after hours post infection, when full cpe was observed, the cells and cell culture media were harvested followed by cycles of freezing and thawing. cell debris was removed by centrifugation at , rpm for min, and the supernatant ( µl) was used for viral dna isolation. briefly, the supernatant was incubated with µl of dnase i ( mg/ml) at c for min, followed by addition of µl of . m ethylenediaminetetraacetic acid (edta) (ph . ), . µl of % sodium dodecyl sulfate (sds), . µl of proteinase k ( mg/ml) and incubation at c for h. total dna was further extracted using a geneclean spin kit (mp biomedicals) according to the manufacturer's instructions. the isolated dna was subjected to pcr using fermentas ( x) pcr master mix and primers ( '-atcatgcagctgggagagtc- ' and '-cattgcccagcaacattgag- ') that were designed to anneal in the sites flanking the hexon hvrs. amplification product was digested by avrii and size-separated by electrophoresis in % agarose gel. the peptide, containing amino acid residues of the prrsv neutralizing epitope b (shlqliynl), was synthesized by genscript inc. the peptide was conjugated to keyhole limpet hemocyanin (klh) or bovine serum albumin (bsa) as carrier molecules. two new zealand white rabbits were immunized with the conjugated peptide ( µg/rabbit) emulsified with freund's complete adjuvant (fca) followed by two injections of conjugated peptide ( µg/rabbit) in freund's incomplete adjuvant (fia) at four weeks apart. serum was collected ten days after the third injection and tested for ability to bind to the peptide by elisa. the enzyme-linked immunosorbent assay (elisa) binding assay was used to test if the peptide incorporated into the hexon protein is accessible to anti-peptide antibody at the virion level. different amounts of purified virions ranging from to viral particles were immobilized on the wells of a -well plate (immulon, thermo scientific) by overnight incubation in ul/well of mm carbonate buffer (ph . ) at c. after washing with pbs, containing . % tween , the immobilized virus was incubated with : diluted anti-peptide rabbit serum for h at c, followed by ap-conjugated goat anti-rabbit antibody (cedarlane) incubation. color reaction was performed with p-nitrophenyl phosphate (sigma-aldrich), and optical density at nm was measured with a microplate reader. new insights on adenovirus as vaccine vectors use of adenoviral vectors as veterinary vaccines alternative codon usage of prrs virus orf gene increases eucaryotic expression of gp( ) glycoprotein and improves immune response in challenged pigs capsid-incorporation of antigens into adenovirus capsid proteins for a vaccine approach adenovirus structure tropism-modification strategies for targeted gene delivery using adenoviral vectors structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response analysis of adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues identification of sites in adenovirus hexon for foreign peptide incorporation optimization of capsidincorporated antigens for a novel adenovirus vaccine approach protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing p. aeruginosa oprf expression of a foreign epitope on the surface of the adenovirus hexon hiv antigen incorporation within adenovirus hexon hypervariable for a novel hiv vaccine approach epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity replacing adenoviral vector hvr with a malaria b cell epitope improves immunogenicity and circumvents preexisting immunity to adenovirus in mice characterization of a permissive epitope insertion site in adenovirus hexon using multivalent adenoviral vectors for hiv vaccination identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain production and release testing of ovine atadenovirus vectors generation of recombinant adenovirus using the escherichia coli bj recombination system severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice preparation and titration of cscl-banded adenovirus stocks evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy construction of capsidmodified recombinant bovine adenovirus type this work was supported by grants from the canadian swine health board and the saskatchewan ministry of agriculture. we thank the staff of the vido-intervac animal care unit for immunizations of rabbits. this paper was published with the permission of the director of vido-intervac, journal series no. . the authors confirm that this article content has no conflict of interest. key: cord- -dtvzwin authors: jeong, joojin; cho, sang-yun; lee, wang-hyu; lee, kui-jae; ju, ho-jong title: development of a rapid detection method for potato virus x by reverse transcription loop-mediated isothermal amplification date: - - journal: plant pathol j doi: . /ppj.oa. . . sha: doc_id: cord_uid: dtvzwin the primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. reverse transcription loop-mediated isothermal amplification (rt-lamp) has been used to detect viral rna molecules because of its simplicity and high sensitivity for a number of viruses. rt-lamp for the detection of potato virus x (pvx) was developed and compared with conventional reverse transcription polymerase chain reaction (rt-pcr) to demonstrate its advantages over rt-pcr. rt-lamp reactions were conducted with or without a set of loop primers since one out of six primers showed pvx specificity. based on real-time monitoring, rt-lamp detected pvx around min, compared to min for rt-pcr. by adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. rt-lamp was conducted using simple inexpensive instruments and a regular incubator to evaluate whether rna could be amplified at a constant temperature instead of using an expensive thermal cycler. this study shows the potential of rt-lamp for the diagnosis of viral diseases and pvx epidemiology because of its simplicity and rapidness compared to rt-pcr. lamp will be useful not only for the detection of infected plants but also quarantine. the reactions are easily monitored by detecting the turbidity caused by the production of a large amount of target dna (webster et al., ) . the reaction time for rt-lamp is less than min and this time can be further reduced by adding two more loop primers (ju, ; nagamine et al., ) . in addition, if a fluorescent dye like sybr green is present in the reaction, pcr products in the reaction tubes can be seen with the naked eye under a uv lamp (cardoso et al., ) . viruses infecting potatoes including plrv and pvy have been detected by rt-lamp (ju, ; nie, ) . the rt-lamp method has not been reported for the diagnosis of pvx. the purpose of this study was to develop a system to diagnose pvx by rt-lamp based on its unique nucleotide sequences encoding a coat protein. the specificity, sensitivity, and rapidity of rt-lamp were also assessed to optimize the detection of pvx. preparation of pvx-infected plant material. a pspvxp binary vector, containing pvx full-length cdna, was kindly provided by dr. kim from seoul national university, seoul korea (park and kim, ). an agro-infiltration method previously described by english et al. ( ) has been applied for pvx infection to nicotiana benthamiana. after two or three weeks, the treated-leaves were collected from healthy and diseased plants. total rna extraction. total rna was extracted from the leaves of pvx-infected or non-infected n. benthamiana using the easy-blue rna extraction kit (intron, republic of korea) as directed by the manufacturer's instructions. primer design. primer sets were designed from the coat protein (cp) gene of pvx (fig. ) . three primer sets were designed using primer explorer v (eiken chemical co. ltd., japan), and the other three primer sets were designed using lamp designer (premier biosoft, usa). all primers were purified by hplc. lamp. the rt-lamp reaction was conducted by incubating a total reaction mixture of μl at o c for min. a homemade mixture (table s ) and two commercial mixtures (loopamp rna amplification kit (eiken chemical co. ltd., japan) and isothermal master mix (imm kit) (optigene ltd., england)) were used. in order to optimize the rt-lamp mixtures, different concentrations of dntps and primers were tested as follows: dntps from . to . mm, fip and bip (inner primers) from to pmole, and f and b (outer primers) from to pmole. real-time monitoring. rt-lamp amplification was spectrophotometrically monitored by recording the optical density at nm with a real-time turbidimeter (eiken co. ltd., tokyo, japan). visualization under uv. rt-lamp amplification products were visualized by adding μl of fluorescent detection reagent (eiken co. ltd., tokyo, japan) to the reaction mixture before incubation. the products were examined under natural light and under uv light. using the maxime tm rt-pcr premix kit (in-tron biotechnology, korea), an one-step rt-pcr reaction was conducted. the mixture was incubated at o c for min and denatured at o c for min, followed by cycles of o c for sec, . o c for sec and o c for selection of specific primer sets over pvx. in order to examine the specificity of six primer sets, three replicates of the experiments were conducted. when rt-lamp assays were conducted using the rt-lamp mixture, primer sets a, c, and f showed ladder-like bands on the agarose gel only for the pvx rna containing samples (fig. s ). by repeating the experiments with primer sets a, c, and f, we confirmed true positive reactions for primer sets a and f. to evaluate the sensitivity of the two primer sets, a dilution series of pvx rna was used for rt-lamp. the detection limits of primer sets a and f were observed at . ng and . µg, respectively ( fig. a and b ). in addition, the detection limit of rt-pcr was . ng (fig. c ). in comparison, rt-lamp conducted with primer set a ( fig. a) showed higher sensitivity (about times) than conventional rt-pcr ( fig c) . finally, primer set a was selected and used for rt-lamp pcr. the rt-lamp products of rna from leaves infected by pvx showed ladder-like bands on the agarose gel, but not rom rt-lamp products of rnas from healthy plant leaves and plrv-infected plant leaves, indicating that primer set a is specific for pvx (fig. s ). to determine the optimal rt-lamp reaction, various parameters including ampli- fication temperature and the concentration of dntps and outer (f and b ) and inner (fip and bip) primers were tested. rt-lamp reactions were carried out using gradient pcr setting the temperature range from o c to o c. fig. a shows that all temperatures were capable of producing rt-lamp amplicons. a similar amount of product was also generated at temperatures ranging from to . o c. however, temperatures under o c or above o c showed decreased yield. fig. b shows that the concentration of dntps affected the amount of rt-lamp products. a minimum concentration of . mm dntps was needed to acquire a positive rt-lamp result. the greatest amount of product was for both . and . mm suggesting that the optimum concentration of dntps was either . or . mm. the concentrations of primers also affected the amount of rt-lamp products, but the effect of primer concentration depended on the types of primers (fig. c) . a greater amount of amplification products were generated from reactions at both and pmole (fig. c - ) of inner primers compared to pmole of inner primer. using the same inner primers (fip and bip), a similar amount of rt-lamp products was obtained regardless of the concentrations of outer primers (f and b ); thus, showing that the concentrations of fip and bip were more effective than those of f and b . effect of loop primers. rt-lamp reactions were performed using primer set a with or without loop primers to examine the effect of loop primers on shortening the reaction time according to the turbidity of reaction mixtures with or without loop primers. the results showed that the time required for the initiation of rt-lamp amplification was or min with or without loop primers, respec-tively (fig. ) . evidently, the use of loop primers accelerated the amplification of pvx, reducing the reaction time to almost half. the rt-lamp amplification products in the reaction tubes were visualized using fluorescent dyes under ambient light or uv light, which also allowed for determination of the detection limit of pvx rna. rt-lamp products showing positive results had a light green color under ambient light, whereas the negative control was light yellow-orange (fig. ) . under uv light, positive tubes emitted bright fluorescent light, but negative samples showed dim or no fluorescent light (lane and ). in addition, rt-lamp amplification products could be visualized when µg to . ng of pvx rna was used as the template, whereas no products were visualized when . ng of pvx rna was used (lane ), indicating that . ng of pvx rna is the detection limit in this study. plant diseases have been increasing due to globalization of trade, increased human mobility, global warming, pathogen's evolution, and improper crop management (anderson et al., ; garrett et al., ) . therefore, it is essential to diagnose disease agents for proper disease control strategies. specifically, accurate, rapid, and efficient diagnostic tools are crucial to control viral diseases because agrochemicals to cure plant viral diseases have not been commercialized. many techniques have been developed and used for the diagnosis of potato viral diseases including pvx (el-araby et al., ; khan et al., ) . elisa has been accepted as the most common and reliable detection method for viruses including pvx because it has long history of use and it is also rapid and inexpensive (clark and adams, ; makkouk and kumari, ) . in general, elisa methods have drawbacks such as long reaction time, less specificity caused by cross reactivity, fewer numbers of antibodies available, and less sensitivity (el-araby et al., ). since molecular-based assays are known to have higher sensitivity, rt-pcr may reduce shortcomings caused by less sensitivity. rt-pcr-based assays have also been shown to remedy additional defects of elisa (agindotan et al., ; el-araby et al., ). however, one of the limitations of rt-pcr is that the assay needs sophisticated and expensive instruments because it depends on thermal cycling for denaturation of double stranded dna into single stranded dna and enzymatic replication of the dna (saiki et al., ) . lamp pcr is relatively new and does not require thermal cycling because it is an assay based on isothermal amplification (notomi et al., ) . rt-lamp pcr, one of the variants of lamp pcr, has been used for the diagnosis of plant rna viruses (ju, ; nie, ) . the specificity of lamp is generally high because the rt-lamp pcr assay uses four primers that perceive six regions on the target gene. however, primer design seems to be an important limiting factor for the general use of rt-lamp pcr in pvx diagnosis, as previous studies reported that more nonspecific reactions among primers are often found in lamp pcr compared to conventional pcr (boubourakas et al., ; notomi et al., ; wei et al., ) . in order to determine the rt-lamp primer sets that could be used for detection of pvx, six sets of primers were designed. only one primer set (set a) was selected based on its specificity ( fig. s and s ) and sensitivity (fig. ) for highly successful detection of pvx. although the sensitivity for detecting classical swine fever virus (yin et al., ) is slightly lower for rt-pcr compared to rt-lamp, most rt-lamp assays are more sensitive than regular rt-pcr or nested pcr. the rt-lamp pcr for pvx that was developed in this study showed , -fold greater sensitivity than conventional rt-pcr assays, similar to most previous reports (fig. ) (ju, ; kuan et al., ; parida et al., ; venkatesan et al., ) . the optimum temperature for different rt-lamp assays might be different because of different primer sets, for example . and o c for peach latent mosaic viroid and to o c for squash leaf curl virus (boubourakas et al., ; kuan et al., ) . in fig. a , the optimum temperature of the rt-lamp to detect pvx seemed to range from to . o c. as shown in many studies, increasing or decreasing the reaction temperature beyond this tempera-ture range results in decreased yield (fukuda et al., ; wei et al., ) . this might be because of inactivation of enzymes or reaction instability caused by too high or low temperatures. different target genes may require different concentrations of dntps when those were amplified by lampbased methods. this study showed that amplification products were obtained using a concentration of dntps ranging from . to . mm, but no reaction products at . mm. the concentration of dntps required to detect roundup ready soybeans by lamp ranges from . to . mm (wang et al., ) . in general, most rt-pcr amplification takes a couple of hours, including min of an additional reverse transcription step. however, rt-lamp amplification takes less than min, even with the reverse transcription step (fukuda et al., ) . many studies have revealed a reaction time of less than min for rt-lamp (kuan et al., ; soliman and el-matbouli, ) . in addition, virus can be detected within min by real-time rt-lamp when two loop primers are applied (fukuta et al., ; ju, ; parida et al., ) . this study showed similar results in that the rt-lamp assay included two loop primers and took only min for detection of pvx. in conclusion, the rt-lamp assay developed to diagnose pvx in this study is rapid, cost-effective, specific, and sensitive for the detection of pvx. moreover, this assay can be extended many other applications for diagnostic purposes. diagnosis and control of cereal viruses in the middle east a sensitive and reliable rt-nested pcr assay for detection of citrus tristeza virus from naturally infected citrus plants simultaneous detection of potato viruses, plrv, pva, pvx and pvy from dormant potato tubers by taqman real-time rt-pcr emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnological drivers sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye characteristics of the microplate method of enzyme linked immunosorbent assay for the detection of plant viruses biological, serological and molecular diagnosis of three major potato viruses in egypt requirement of sense transcription for homology-dependent virus resistance and trans-inactivation development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum climate change effects on plant disease: genomes and ecosystems immunocapture reverse transcription-polymerase chain reaction combined with nested pcr greatly increases the detection of prunus necrotic ring spot virus in the peach enhanced detection of prune dwarf virus in peach leaves by immunocapture-reverse transcription-polymerase chain reaction with nested polymerase chain reaction (ic-rt-pcr nested pcr) simple and rapid detection of potato leafroll virus (plrv) by reverse transcription loop-mediated isothermal amplification (rt-lamp) further studies on resistance -breaking strains of potato virus x detection of important plant viruses in in vitro regenerated potato plants by double antibody sandwich method of elisa rapid detection of squash leaf curl virus by loop-mediated isothermal amplification molecular diagnosis of plant viruses estimation of vector propensity for lettuce mosaic virus based on viral detection in single aphids a single tube, quantitative real-time rt-pcr assay that detects four potato viruses simultaneously accelerated reaction by loop-mediated isothermal amplification using loop primers reverse transcription loop-mediated isothermal amplification of dna for detection of potato virus y loop-mediated isothermal amplification of dna new device and method for capture, reverse transcription, and nested pcr in a single closed-tube rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay a new generation of innovative gene amplification technique perspectives in clinical diagnosis of infectious diseases agroinfiltration-based potato virus x replicons to dissect the requirements of viral infection primerdirected enzymatic amplification of dna with a thermostable dna polymerase reverse transcription loo-mediated isothermal amplification (rt-lamp) for rapid detection of viral hemorrhagic septicaemia virus (vhs) loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of product development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples development of a rapid detection method for pvx by rt potato viruses in china detection of roundup ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick detection of mix-infected potato viruses with multiplex rt-pcr diagnosis of plant viral pathogens one-step detection of bean pod mottle virus in soybean seed by the reverse-transcription loop-mediated isothermal amplification this paper was supported by rural development administration (rda) fund pj , republic korea. key: cord- -algzczs authors: theuns, sebastiaan; conceição-neto, nádia; christiaens, isaura; zeller, mark; desmarets, lowiese m. b.; roukaerts, inge d. m.; acar, delphine d.; heylen, elisabeth; matthijnssens, jelle; nauwynck, hans j. title: complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium, january date: - - journal: genome announc doi: . /genomea. - sha: doc_id: cord_uid: algzczs porcine epidemic diarrhea virus (pedv) is a member of the family coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. here, we report the complete genome sequence ( , nt) of a pedv strain isolated during a novel outbreak in belgium. diarrhea in europe from the s to the s, but has since been reported only sporadically in the last few decades ( , ) . from , severe outbreaks with mortality have been identified in asia, and since the spring of , the virus was detected for the first time in u.s. swine herds ( ) ( ) ( ) ( ) ( ) ( ) ( ) . genetically, these american isolates were highly related ( . % nucleotide similarity) to asian pedv strains ( , ) . in january , a novel pedv variant strain oh was isolated in the united states, which was associated with milder disease symptoms ( ) . similar strains contained several deletions and insertions in their spike genes and were therefore called indel strains ( ) . in may , an outbreak of diarrhea occurred in fattening pigs on a german farm, and the isolated strains were genetically almost identical to u.s. strain oh ( ) . an outbreak of diarrhea in fattening pigs occurred on a belgian pig farm at the end of january , and the presence of pedv rna in the feces of these pigs was demonstrated using an in house rt-qpcr assay targeting the nucleocapsid gene. this was the first confirmed pedv case in belgium in decades. therefore, the complete genome of this novel isolate, bel/ v / , was unraveled by next-generation sequencing, in order to assess its genetic relation to other pedv isolates circulating around the globe. viral particles were purified, rna was extracted using the qiaamp viral rna mini kit (qiagen), and whole transcriptome amplification was performed (wta kit, sigma aldrich) to generate cdna. the sequencing library was prepared using the nextera xt dna library preparation kit (illumina). sequencing was performed on a hiseq platform (illumina) for cycles ( -bp pairedend reads). raw reads were trimmed for quality and adapters using trimmomatic ( ) , and reads were mapped using bwa against the german pedv strain l (genbank lm ) ( ) . remaining gaps were closed using sanger sequencing. isolate v contained a genome with a size of , nucleotides (nt), excluding the poly-a tail. open reading frame (orf ), encoding the replicase polyprotein, was located between nt and and between nt and , with a ribosomal frameshift between both parts. the other genes were arranged as follows: spike (s), nt to ; accessory membrane protein (mp, orf ), nt to ; envelope (e), nt and ); membrane (m), to ; and nucleocapsid (n), nt to . at the whole-genome level, strain v was most closely related to strain l ( . % nucleotide similarity) isolated in germany in , and the prototype u.s. indel strain oh ( . %). strain v was less closely related to the former prototype european strain cv ( . %). in conclusion, a pedv strain from a novel outbreak in belgium was characterized and showed high relatedness to recent german pedv isolates and u.s. pedv indel strains. our findings indicate that pedv is still or again circulating in europe. surveillance and development of prophylactic measures are urgently needed. nucleotide sequence accession number. the complete genome sequence of pedv bel/ v / has been deposited at genbank under the accession number kr . epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy a new coronavirus-like particle associated with diarrhea in swine complete genome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in south china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china complete genome sequence of a novel porcine epidemic diarrhea virus in south china distinct characteristics and complex evolution of pedv strains pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/ from the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states new variant of porcine epidemic diarrhea virus comparison of porcine epidemic diarrhea viruses from germany and the united states trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform key: cord- - epvg authors: kearney, alexis; pettit, catherine title: introduction to biological agents and pandemics date: - - journal: ciottone's disaster medicine doi: . /b - - - - . - sha: doc_id: cord_uid: epvg nan alexis kearney and catherine pettit biological agents have been used as weapons since antiquity. in bc solon of athens poisoned the wells of his adversaries with hellebore-a purgative herb-during the siege of krissa. similarly the assyrians contaminated the wells of their enemies with rye ergot. [ ] [ ] [ ] in the fourteenth century corpses of plague victims were hurled over walls to infect enemies, and in the seventeenth and eighteenth centuries smallpox-laden blankets were used to target native americans. biological agents played a role in military offenses into the twentieth century and have been used in terrorist actions around the world. in president richard nixon halted offensive biological and toxin research and production in the united states. stockpiles of various biological agents and toxins, including bacillus anthracis, botulinum toxin, and francisella tularensis, were subsequently destroyed. in the united states, the united kingdom, the ussr, and more than other nations ratified the biological weapons convention (bwc). the bwc prohibits the development, production, and stockpiling of weapons of mass destruction. , despite this, during the last years, multiple signatory nations have violated the pact set forth by the bwc. additionally, there has been a rise in the use of biological agents in terrorist attacks, including the anthrax attacks in , which resulted in few deaths but widespread fear. the u.s. centers for disease control and prevention have organized biological weapons into three categories (table - ) . category a, or high-priority agents, include organisms that can be easily disseminated, result in high mortality, and have the potential to cause significant public panic. anthrax, botulism, smallpox, tularemia, and the viral hemorrhagic fevers are included in category a. category b agents, including food and water safety threats, are moderately easy to disseminate. although mortality rates due to these agents are lower they may result in significant morbidity. finally, category c agents are considered emerging pathogens. these agents may be adapted in the future to take full advantage of their pathogenicity, availability, and lethality. in general, biological weapons are characterized by low visibility, high potency, and relative ease of delivery and dissemination. the agents must also be easily obtained, cultured, or reproduced and be relatively stable in the environment. surveillance a good surveillance system is essential to any public health effort and is recognized as the single most important factor in identifying events of global concern. historically, surveillance systems relied on manual reporting of notifiable diseases or suspicious cases from clinicians, hospitals, and laboratories. there has been a shift to focus more on automated surveillance of readily available data to improve the timeliness, sensitivity, and specificity of the system. the exponential increase in social media use and availability of web-based applications has added another potential surveillance domain, which is being utilized for research and communication. in an effort to better identify and track potential outbreaks related to infectious diseases, both naturally occurring and those related to biowarfare and terrorism, public health practitioners developed surveillance systems designed to analyze routinely collected health information. syndromic surveillance, as it has come to be known, includes a wide range of surveillance activities, from monitoring over-the-counter medication purchases to tracking discharge diagnoses from emergency departments and analyzing internet search queries. , true syndromic surveillance monitors syndromes-or constellations of symptoms-that may represent the prodromes of biological agents or emerging epidemics. it relies on the automated analysis of routinely collected data to detect aberrancies in expected trends in near real-time. this process has been streamlined with the increased availability of electronically collected and exchanged data. it is frequently used in conjunction with alternative surveillance methods and verification techniques to improve outbreak detection. the goal of syndromic surveillance systems is to enable more timely detection of outbreaks by identifying trends before these patterns are recognized clinically and a formal diagnosis is made. this allows a more rapid response, ultimately decreasing morbidity and mortality. once an outbreak is suspected public health responders must proceed with a thorough epidemiological investigation to further describe the outbreak and implement control measures. although syndromic surveillance complements the more timeconsuming and burdensome conventional surveillance systems that rely on physician and laboratory reporting, there are significant limitations, including frequent false alarms. if the system is sensitive enough to detect small outbreaks, it may result in false alarms, which consume resources and make it difficult to separate true outbreaks from daily variation. , , additionally, the ability of a surveillance system to detect an outbreak depends on a variety of factors, including the size of the outbreak, pattern of population dispersion following exposure to the agent, and data sources and syndrome definitions used in the analysis. methods have been developed to analyze data using time and time-space relationships to take into account baseline variability; however, these methods have not been standardized across surveillance systems. [ ] [ ] [ ] each community utilizing syndromic surveillance must ultimately set its own threshold level for activation. these thresholds should be set using historical data, hazard vulnerability analysis, and risk-benefit calculations for each syndrome. environmental surveillance systems rely on the remote detection of aerosol clouds or point detection systems to collect and analyze data. remote detection systems identify and analyze the components of clouds, subsequently transmitting that information to public health personnel on the ground. point detection systems sample an environmental area using high speed particle concentration methods and rapid diagnostic modalities to detect and identify potential agents. in the department of homeland security launched biowatch, an environmental air sampling program currently under way in more than u.s. cities, with the goal of facilitating detection of specific agents that could be aerosolized and used in a biological attack. bio-watch is intended to complement current surveillance activities at the state and local levels. however, in its current design, it is unlikely that the biowatch system will result in more timely detection of biological agents unless there is a large-scale aerosol attack in a location monitored by biowatch using biological agents detectable by the system. since the turn of the century we have faced numerous outbreaks-both naturally occurring and intentional-that have changed the landscape of public health surveillance and preparedness. in b. anthracis spores were sent to various locations around the united states, resulting in cases and deaths. this was followed by the epidemic of severe acute respiratory syndrome (sars) in , an outbreak of novel influenza a h n in , and in middle east respiratory syndrome (mers-cov), which continues to spread. since the anthrax attacks, a significant amount of money and resources has been poured into improving public health infrastructure. however, despite this focus, it is unclear if improvements have actually been achieved. in part this stems from conflicting goals, shifting priorities, and the lack of a clear definition of what it means to be prepared. , ultimately reactionary response programs have less impact than hardening all hazards public health infrastructure, which has been neglected for decades. in a diverse expert panel convened by the rand corporation developed the following definition of public health emergency preparedness (phep) in order to strengthen accountability and streamline preparedness efforts. they define phep as the capability of the public health and health care systems, communities, and individuals, to prevent, protect against, quickly second highest priority agents include those that are moderately easy to disseminate, result in moderate morbidity rates and low mortality rates, and require specific enhancements of the cdc's diagnostic capacity and enhanced disease surveillance. brucellosis (brucella species) epsilon toxin of clostridium perfringens food safety threats (e.g., salmonella species, escherichia coli o :h , shigella) glanders (burkholderia mallei) melioidosis (burkholderia pseudomallei) psittacosis (chlamydia psittaci) q fever (coxiella burnetii) ricin toxin from ricinus communis (castor beans) staphylococcal enterotoxin b typhus fever (rickettsia prowazekii) viral encephalitis (e.g., venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis) water safety threats (e.g., vibrio cholerae, cryptosporidium parvum) category c third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of availability, ease of production and dissemination, and potential for high morbidity and mortality rates and major health impact. emerging infectious diseases, such as nipah virus and hantavirus respond to, and recover from health emergencies, particularly those whose scale, timing, or unpredictability threatens to overwhelm routine capabilities. preparedness involves a coordinated and continuous process of planning and implementation that relies on measuring performance and taking corrective action. the panel further argued that phep must cover a full range of activities, including prevention, mitigation, response, and recovery. additionally, it must take into account not only capacity (i.e., infrastructure, trained personnel), but also capability-the ability to implement preparedness plans in real time. large-scale public health emergencies occur infrequently; as a result, it is difficult to execute, assess, and refine preparedness plans based on experience. furthermore there is no universally agreed upon standard of preparedness. federal, state, and local organizations have all established their own conflicting requirements, and there are few data to support one set of standards over another. written assessments and exercises have frequently been used to assess preparedness. although written assessments are easily administered to large groups of people and the data obtained are generally easier to analyze, they frequently focus on the capacity as opposed to the capabilities of a system. although important, these factors do not ensure an effective emergency response. exercises, in contrast, may be discussion based or operations based and generally provide a more realistic view of an organization's capability to mobilize resources and infrastructure. however, real-time exercises are rarely evaluated with standard metrics to identify and address performance gaps. moving forward it is essential to incorporate evaluations into routine public health functions. in public health practitioners in both los angeles and new york city embedded assessments into influenza a h n vaccination campaigns. as a result, invaluable information was gained about the optimal placement of points of dispensing influenza vaccines within a community and the potential for scaling up electronic immunization information systems to better track immunization progress and manage supply and distribution of vaccines in a pandemic. , although it may be difficult to identify questions and develop research protocols in the midst of an emergency, the time between events can serve as an opportunity to engage leaders, develop template protocols, and prioritize areas for investigation. summary ultimately, leaders must make decisions regarding public health responses with imperfect, limited data. the information provided by surveillance systems, used in conjunction with clinical data, will ultimately help public health practitioners identify an etiologic agent. as preparedness strategies become more standardized and evidence based, our ability to respond to public health emergencies, including biological attacks, will improve. each chapter in section will cover a specific biological agent. the authors will outline what is known about the agent currently and, from this, attempt to extrapolate how this agent might be used in a bioterrorism attack. medical management of biological casualties handbook biological warfare and bioterrorism: a historical review biologic warfare: a historical perspective current concepts in the management of biologic and chemical warfare casualties biosurveillance: a review and update systematic review: surveillance systems for early detection of bioterrorism-related diseases syndromic surveillance and bioterrorism-related epidemics what is syndromic surveillance? real-time public health surveillance for emergency preparedness biowatch and public health surveillance: evaluating systems for the early detection of biological threats infectious agents of bioterrorism: a review for emergency physicians conceptualizing and defining public health preparedness assessing public health emergency preparedness: concepts, tools, and challenges research as a part of public health emergency response incorporating research and evaluation into pandemic influenza vaccination preparedness and response efficiency of points of dispensing for influenza a(h n )pdm vaccination distribution of pandemic influenza vaccine and reporting of doses administered key: cord- -e zhty authors: tassier, troy; polgreen, philip; segre, alberto title: network position and health care worker infections date: - - journal: j econ interact coord doi: . /s - - - sha: doc_id: cord_uid: e zhty we use a newly collected data set coupled with an agent-based model to study the spread of infectious disease in hospitals. we estimate the average and marginal infections created by various worker groups in a hospital as a function of their network position in order to identify groups most crucial in a hospital-based epidemic. surprisingly, we find that many groups with primary patient care responsibilities play a small role in spreading an infectious disease within our hospital data set. we also demonstrate that the effect of different network positions can be as important as the effect of different transmission rates for some categories of workers. vaccines are a primary way to stop or slow the spread of many infectious diseases, perhaps most notably, influenza. the lack of appropriate vaccination levels is a major health problem. for instance, influenza is a major cause of morbidity and mortality throughout the world despite the availability of a highly effective and inexpensive vaccine. in the us alone, influenza causes an estimated , deaths and , hospitalizations annually yet only around / of healthcare workers are vaccinated each year (thompson et al. ) . efficient provision of vaccinations poses a difficult problem in that the positive externality associated with a vaccination is the product of the probability of infection, the cost of the infection, and the marginal infections generated by an agent if infected (all of which may vary across agents). there is great concern over the spread of infectious diseases in hospitals, but little knowledge is available to identify healthcare workers who are most likely to acquire and transmit infectious diseases in hospitals. the problem is especially difficult because the transmission of many infectious diseases is not observable. for instance if someone in your household acquires influenza, you likely do not know which of the potentially hundreds of people you come in contact with each day that may have caused the infection. further if a vaccine is available for an infectious disease and the vaccine is in short supply or is expensive, it is imperative to know which individuals should have the highest priority in vaccine campaigns. in this paper we use a newly collected data set on hospital worker contacts in order to identify hospital worker groups that have the potential to create the largest number of infections based on their location in a hospital contact network. to achieve this goal, we have collected person-to-person contact information on individuals belonging to one of types of healthcare workers at the university of iowa hospitals and clinics (uihc). the data contain information on the contacts between healthcare workers and patients and between healthcare workers and other healthcare workers at the hospital. with this information we develop a network model describing the spread of an infectious disease in a hospital. we estimate, using an agent-based model, the effect of network position of different hospital worker groups on the spread of infectious diseases in a hospital. through this model we are able to identify the hospital worker groups that create the largest externality if removed from the network (perhaps through a vaccination or a quarantine). we argue that methods such as those used in this paper can help hospitals, health care professionals, and epidemiologists to design efficient programs for healthcare worker vaccinations. of importance, we note that we only study the externality in terms of network position within the hospital. in this paper we do not consider other potential heterogeneity among agents such as differences in transmission rates across workers, or differences in behavior outside the hospital that may play a role. while these effects are also important, the large differences across classes of workers shown below are worthy of independent study. this is the first paper to use specific micro-level contact data within a hospital that can be used to help guide policy makers and public health officials in the problem of efficiently allocating vaccines within hospitals. the data used in this paper is unique and detailed in comparison to other studies. the data consists of shadow data (where a research assistant follows a specific, randomly chosen hospital worker for an entire shift) for the major groups of healthcare workers at the uihc, a -bed major medical center. this results in over h of direct hospital worker observations and the notation of over specific worker to worker or worker to patient contacts throughout the hospital. to the best of our knowledge, the data that we have collected comprises the most detailed micro-level healthcare worker contact data set in existence. as a comparison, ueno and masuda ( ) collect data on contacts between doctors, nurses, and patients. their data is based on two calendar days from a small, room, community hospital in tokyo. they model contacts between nurses, physicians and patients. their data does not consider contacts with and between other healthcare worker groups (other than nurses and doctors). based on our data at the uihc, these assumptions would ignore over % of hospital staff, including most of the groups we identify as most crucial to the spread of an infection disease. we begin by discussing the background and motivation for our study and then move to develop a simple model of infectious disease transmission. in the model, we initially assume homogeneous contacts as in traditional epidemiological models. we then discuss a similar model with heterogeneous contacts and discuss the difficulties of achieving efficient vaccination levels. following the theoretical discussion, we use our newly collected data on healthcare worker and patient contacts to model the spread of an infectious disease in a hospital setting. the model allows us to identify the healthcare worker groups that would be expected to play the largest role in the spread of infectious diseases, in terms of network position, in this hospital setting. traditionally, epidemiology research has focused on well-mixed (randomly mixed) populations where agent contacts are homogeneous. in these models, every agent in a population may "bump into" any other agent with equal probability, much as a gas molecule may bump into any other gas molecule with an equal probability over a fixed time period. only recently have epidemiologists and other researchers begun to study the heterogeneous contact structures between people over which infectious diseases spread (early studies include comins et al. ; grenfell and harwood ; wallinga et al. ) . we focus this study on healthcare workers and a particular class of infectious disease, of which influenza is an example. healthcare workers are at especially high risk of contracting influenza. one study of healthcare workers with a low rate of influenza vaccination demonstrated that % of healthcare workers had evidence of influenza infection during a single influenza season (elder et al. ) . two features of influenza make its spread difficult to control in hospitals. first, people with influenza are infectious - days before the onset of symptoms. thus, they can spread the virus when they are still feeling well and are unaware of their own infectious state. second, only about % of infected persons develop classic influenza symptoms (cdc (cdc , consequently, restricting healthcare workers with influenza-like symptoms from the workplace will not completely prevent transmission of influenza because healthcare workers with atypical symptoms could continue working and spreading the virus. furthermore, studies show that healthcare workers are more likely than other workers to return to work early or to keep working when they have influenza-related symptoms (weingarten et al. ) . because of the ease with which influenza may be contracted and spread by healthcare workers, the centers for disease control and prevention (cdc) have, for the past two decades, recommended influenza vaccination for all healthcare workers. yet, in the us, only % of workers who have direct patient contact are immunized against influenza annually (smith and bresee ) . outside of concerns about traditional influenza, there are additional reasons to study the spread of infectious diseases in hospitals. first, healthcare-associated infections affect about million patients in us hospitals each year (jarvis ). second, there is a growing fear that hospitals could become a breeding ground for new strains of influenza such as the recent h n influenza outbreak, the potential emergence of person-to-person transmission of avian flu, or other "new viruses." much as sars spread widely in hospitals to begin the sars epidemic in toronto (chowell et al. ) , person-to-person transmission of avian flu may start in hospitals as well, and, if a more lethal version of h n were to develop, hospitals again could be a breeding ground for new infections. this last point is of particular salience. with the recent h n outbreak and the subsequent work to develop a vaccine, controversies arose concerning which individuals to vaccinate first. healthcare workers were high on the list. but, as we show below, not all healthcare workers are equal in terms of their importance in spreading infectious diseases. thus, one primary focus of this paper is identifying the individual hospital workers who are most important to vaccinate should a similar outbreak occur again. there is a growing literature in economics on the vaccination choices of individuals and of the externalities associated with vaccinations. but scant attention is paid to the network effects determined by heterogeneous contacts that we focus on in this paper. for example, francis ( ) solves for the optimal tax/subsidy policy for influenza in an sir model with a constant contact rate and random mixing among the population. geoffard and philipson ( ) examine how the individual incentives for vaccination decrease as disease incidence decreases and thereby argue that relying exclusively on private markets is unlikely to lead to disease eradication. boulier et al. ( ) , the most similar paper to ours, investigate the magnitude of the externality associated with a vaccination as a function of the number of vaccinations in the population, the transmission rate of the disease, and the efficacy of the vaccination. they find nonmontonic relationships between each of these items and the magnitude of the vaccine externality. more specifically, the externality and the number of infections prevented per vaccination initially increases before eventually decreasing. however, like francis, they do not consider the case of heterogenous contact number or heterogenous sorting among the population. finally, much of the recent literature on the economics of infectious disease is summarized in philipson et al. ( ) . we begin by describing a simple model where agents in a population have contacts with each other with a uniform probability. this is the traditional random mixing model in epidemiology pioneered by kermack and mckenrick ( ) . the important results in this paper describe exceptions to this homogeneous contact assumption, but we use the simplified model to develop intuition before describing a richer model with heterogeneous contacts. in this simple model, we assume that all agents are homogeneous in that all agents have the same number of contacts with other agents and that these contacts are randomly drawn with uniform probability from the population at large. suppose that agents are assigned to one of three states: susceptible (s), infected (i), or recovered (r). a susceptible agent can transition to being infected with probability α if she is in contact with an infected agent. once infected, an agent transitions to the recovered state according to a parameter κ. once recovered, the agent is immune to the possibility of future infection. this is a classic susceptible-infected-recovered (sir) model for infectious diseases such as influenza (kermack and mckenrick ) . the description and parameters yield the following differential equations describing the flows of agents among the various states, assuming a constant population of size n and contact rate of γ . each equation describes the rate of growth for one of the three populations in the sir model. equation describes how susceptible agents contact γ other agents in the population, of which i t /n are infected, and how, of these contacts with infected agents, a percentage α cause the susceptible agent to transition to being infected. equation describes the previously mentioned flows from susceptible into infected and that each infected agent moves to being recovered at rate κ. finally, eq. describes the flows from infected to recovered. we can write these equations in terms of population shares by dividing each of the above equations by the population size n and using lower case letters to denote these population shares, s t = s t /n , i t = i t /n , and r t = r t /n , yielding the following population share equations: the number of infected agents will increase in the population if the flows into the infected state from the susceptible state exceed the flows out of the infected state into the recovered state, di t dt > . this condition is equivalent to αγ s t > κ or s t > κ αγ . if this inequality holds then we say that the population is above the epidemic threshold. note that we cannot remain above the epidemic threshold forever without an introduction of new susceptible agents since ds t dt < : eventually the population will run out of susceptible agents to infect unless the susceptible population is replenished at a sufficient rate. one goal of healthcare policy is to attempt to place a population below the epidemic threshold so that the number of infectious agents in a population does not grow subject to some cost constraint. a population is most vulnerable to being above the epidemic threshold when the infectious disease first enters a population because s t ≈ . this implies that each infected agent infects approximately αγ κ new agents in the population. this fraction is sometimes referred to as the initial reproduction number in the population and is commonly denoted as r ≡ αγ κ . without new individuals entering a population in the susceptible state this reproduction number can only decline as the infectious disease spreads. a successful vaccination moves an agent from state s to state r without incurring the costs of infection. if we reduce the initial population of susceptible individuals, s , by enough we can push the population below the epidemic threshold. specifically, if s < κ αγ then the infectious disease dies out of its own accord without further action. thus an epidemic is prevented whenever s < κ αγ which occurs when ( − κ αγ )n agents are successfully vaccinated. vaccinating enough agents to produce this effect is called herd immunity (smith ) ; once enough people are vaccinated, the entire population (herd) is effectively protected without everyone being vaccinated. the question then becomes, given a cost of vaccination, c(v), what is the efficient level of vaccinations to provide in a population and how do we obtain this efficient level? we begin to approach this question by introducing standard value function notation. in this initial model, once an agent enters state r she remains there forever. thus the value of being in state r is simply the lifetime discounted utility received in state r. we also introduce the possibility of having heterogenous contacts at this stage by indexing agent j's contacts (γ j ) and other terms that we allow to vary across agents. where u j ( ) = utility of agent j from the specified state and β is the discount rate. if an agent is in state i, she will remain in state i for /κ periods, on average, until recovered and then enter state r. if an agent is in state s, she receives the same utility as she would if she was recovered, unless she becomes infected. the value to an agent of being in state s is the value of being in state r less the product of the probability that the agent becomes infected and the cost of the infected period. where π(γ j , α, i) is the probability of becoming infected over the course of the epidemic as a function of the contacts of the agent and the transmission rate of the infectious disease. the cost of being infected is the difference in utility between states s and i during the time spent in state i , with the value functions specified we can now specify the vaccination problem for the individual and the social planner. to simplify the vaccination choice of individuals we assume that an agent can only be vaccinated at time period . at this time the agent will choose to be vaccinated if the value of being in the recovered state less the cost of the vaccination is greater than the value of being in the susceptible state. thus the agent will choose to be vaccinated if which implies the agent will choose to be vaccinated if c(v) < π(γ j , α, i)c j . the social planner's vaccination problem is more difficult than the individual vaccination problem. essentially, the social planner's problem is to vaccinate agent j if the cost of the vaccination is less than the expected dis-utility of the increase in infections created by agent j if agent j is infected weighted by the probability that agent j is infected. we define the term "marginal infections" of agent j to be the additional infections that occur if j is infected that would not occur if j was not infected. note that this is not simply the number of infections that agent j creates. as an example, agent k maybe infected by agent j. but, if k would have been infected by another agent had she not been infected by j then this would not be a "marginal infection" of agent j. k will be infected regardless of the vaccination choice of agent j. along these lines of thinking, measuring marginal infections is a difficult problem for epidemiologists for at least three reasons: . as mentioned earlier, many infectious disease transmissions are not observable. thus it is not easily known how many infections a given agent causes. . even if transmissions are observable, the marginal infections created by agent j are not simply the number of other agents that j infects. this is because some agents that j infects may get infected by agents other than j even if j does not infect them herself. further one needs to know how many additional agents are infected by those that j infects and any additional infections created by these agents and so forth. thus one needs to know information on the dynamics of the entire epidemic to measure the true marginal infections of a given agent. . the marginal value of vaccinating an agent depends on the behavior and vaccination choices of other agents. it eventually must be decreasing in the number of other vaccinations that are performed. in the extreme, if there are enough vaccinations in the population to produce herd immunity the marginal value of vaccinating an additional agent only involves the probability that the agent is infected from outside the agent population. in effect, the only value is preventing a single agent from infection because she will not, on average, infect anyone else. because of these difficulties we use a simulation approach to help us measure the average and marginal effects of individuals belonging to different worker groups in our hospital contact data. with simulations one can monitor the various infections that occur and also perform controlled experiments to sort out the effects of various groups on potential hospital epidemics. define m j (γ j , α, κ, i, v) as the true marginal infections created by agent j if infected, where v is the number of agents vaccinated in the population. for the majority of the paper we will suppress the notation that does not differ across agents and simply refer to marginal infections as m j (γ j ) since the primary focus of the paper will be on the effect of heterogeneous contacts on the spread of infectious diseases. as shown in boulier et al. marginal infections may be increasing in v for sufficiently small v. but, marginal infections must eventually decrease in v; at the extreme, marginal infections are for any level of v above the point at which herd immunity is reached. thus marginal infections may be increasing or decreasing in the number of vaccinations depending on the specifics of disease transmission, contact patterns, and the number of vaccinations performed. we can now state the social planner's vaccination problem. vaccinate agent j if: note that the individual and social planner's vaccination problems differ by the term π(γ j , α, i)m j (γ j )c j (i). this is the positive externality created by a vaccination when a vaccinated agent j protects other agents which he contacts from acquiring the disease from him. the key terms to investigate in this externality are the probability agent j gets infected, and the marginal infections created by agent j if infected. note that if these marginal infections, m j (γ j ), go to , the social planner's problem and the individual problem converge, and the externality is removed. similarly the externality is removed if enough vaccinations are performed to reach herd immunity, again because m j (γ j ) = when herd immunity is achieved because the epidemic never occurs. the main focus of this paper concerns the network position of hospital workers. as such, we assume throughout the paper that the cost of an infection is equal across all agents, c j (i) = c k (i) for all j and k. further, without loss of generality, we can normalize c j (i) = . thus the externality above is the product of the probability of infection and the marginal infections produced by the agent if infected. one question then emerges: how are π(γ j ) and m(γ j ) related? at a simple level, if contacts only vary in degree, that is if the only difference in contacts between two agents is the number of contacts and not other, qualitative, aspects of the contacts, then you would expect π(γ j ) and m(γ j ) to be highly correlated. if an agent has a high likelihood of being infected because he has many contacts then he also has many contacts to pass on the infection. suppose that each agent pair is connected with a fixed probability. then, by chance, some agents will have a higher than average number of contacts and some a lower than average number of contacts. in this case, any agent who has a large number of contacts will also generate a large number of secondary infections since there is a lack of structure within the network population. thus any agent with a high probability of infection, π(γ j ) will also be expected to generate a high level of marginal infections m(γ j ). example one is fairly direct. however, various relationships are possible as we show below. in this case each agent in a population is directly connected to every other agent in the population. if this is the case, then any agent that becomes infected is directly tied to all other agents and can infect anyone in the population. thus once someone is infected each agent has a high probability of becoming infected (either from the original agent or from secondary infections). but, since the first agent contacts everyone in the population, and many agents will be infected from him, the other agents in the population may have a low m(γ j ). thus it is possible to have a high probability of infection π(γ j ) and low marginal infections generated m(γ j ) from the same agent. imagine that there are three groups of agents in a population. two of these groups, call them a and b, are separate fully connected graphs containing equal numbers of agents, who do not have any connections to the other group. in other words an agent a ∈ a is connected to every agent a ∈ a but no agent a ∈ a is connected to any agent b ∈ b. suppose that group b is formed in a similar manner. the third group is composed of one agent, j. agent j has only two contacts: one to agent x ∈ a and one to agent y ∈ b. in this example it may be unlikely that agent j gets infected, especially if there is a low transmission rate, because he only has two contacts in the populations. but, if agent j is infected, he may be integral to spreading the disease to the second fully-connected group. suppose an agent in a becomes infected and subsequently infects agent x (or any of the other agents in a) as well as several other agents in a. agents in group b are safe from infection as long as agent j is not infected. but, if j becomes infected, then it is possible that a large fraction of agents in b may become infected as well. thus agent j may have a low probability of being infected, π(γ j ), but create a large number of marginal infections, m(γ j ), if he does become infected. note that each of these three examples offer different implications for public policy approaches to encouraging vaccinations. in the first example, each agent has a probability of being infected that is in line with the number of marginal infections generated. in the other two examples, the infection rate and the number of marginal infections generated may not have a simple relationship with each other. this is an important observation if one considers using subsidies or other means to encourage increased vaccination rates. in the remaining portion of the paper we examine a data set on contacts of and between healthcare workers and patients in a large hospital on the university of iowa campus. we discuss the data and use agent-based models to identify the healthcare workers whose position in the hospital contact network has the potential to create large numbers of infections in the hospital. observational data on contacts between healthcare workers and patients was collected during the winter and early spring of - (the - "flu season") at the university of iowa hospitals and clinics (uihc). the uihc is a -bed comprehensive academic medical center and regional referral center in iowa city. data were collected by randomly selecting uihc employees from each of job classifications (specified below) and then using research assistants to "shadow" the selected employees. the research assistants manually recorded every human contact of the subjects (within approximately three feet) over a work shift. note that these observed contacts include anyone contacted within the hospital, not just with the other workers in the shadow sample. this resulted in a total of recorded contacts over h of observation. additionally, the ra recorded the worker or patient group category for each observed contact (patient or category of healthcare worker) in our data set, and the location in the hospital where the contact occurred. the job categories and number of observed subjects in the data set are as follows: floor nurse ( ), food service ( ), housekeeper ( ), intensive care nurse ( ), nurse assistant ( ), pharmacist ( ), phlebotomist ( ), physical/occupational therapist ( ), resident/fellow/ medical student ( ), respiratory therapist ( ), social worker ( ), staff physician ( ), transporter ( ), unit clerk ( ), and x-ray technician ( ). the data for each group contain approximately h of shadowing. the data were summarized into tallies of contacts over -min intervals and then aggregated into contacts per h shift by the authors. table lists the average number of non-repeated contacts per h that occur between the various worker (and patient) categories. note that we were not allowed to choose patients as subjects in our shadow data directly because of privacy concerns. we were only able to observe patient contacts as a result of shadowing other members of the hospital. thus they do not appear as a row in the table. we use this contact data to model the spread of an infectious disease across the uihc hospital. with this data we create a probabilistic contact network for the hospital worker groups. the network is constructed to match the distribution of worker groups at the university of iowa hospitals and clinics. this totals employees. the distribution of workers across the categories is given in table . we create a contact network among these agents. in the model, each worker in a given group connects to other workers according to the rates observed in our shadowed subjects given in table . as an example, all floor nurses in the model create contacts to other randomly selected floor nurses on average, contacts to food service workers, etc. we assume that the contacts are symmetric in our model, that is, a contact from a given floor nurse to a given housekeeper is also a contact from the housekeeper to the floor nurse. there are at least two reasons for this assumption. first, if our subject is in close enough proximity to pass on the influenza virus to a second agent, the second agent is also within close enough proximity to pass on the influenza virus to our subject. thus the ability to acquire or to pass on virus is a symmetric relationship. second, the reader may note in the table that the matrix of observed contacts is not symmetric because of randomness in the observation of subjects. for instance one notices that the subject floor nurses were not observed to contact food service workers, but a small number of food service worker to floor nurse contacts were observed. thus by assuming that all contacts that occur in the matrix are undirected, we create a symmetric contact matrix where the total number of contacts from a member of group x to group y (and from group y to group x) is one-half the sum of the observed average contacts from group x to group y and from group y to group x. we create the contacts in a uniform random manner within groups. let ρ i j be the ratio of the average contacts between a member of group i and j (taken from table ) to the total number of group j employees (taken from table ). we then take each pair of employees across each group and create a contact with probability ρ i j . specifically, let agent a be a member of group i and agent b be a member of group j. then the probability that a and b are connected is average number of contacts observed between members of groups i and j and n j is the number of employees belonging to group j. before moving to the computational model, we mention two limitations of our data set. human contact networks frequently have a small number of individuals with a much larger than average number of contacts, perhaps differing by orders of magnitude. these individuals are often called hubs. these hubs have the potential to significantly influence disease transmission because they are highly likely to be infected, and if so, to pass on infections to a large number of individuals. because our sample includes approximately . % of the hospital worker population, we may be missing hubs in our sample if they exist in this setting. however, we note two things in relation to this: first, the relatively homogeneous workday responsibilities of workers within categories, likely limit the variation of contacts within a category. for instance two physicians or two floor nurses are likely constrained to see a relatively similar number of patients each day. this is unlike many other social network data sets, (such as general friendship or online networks) where there are not these work responsibility constraints on individual contacts. thus the possibility of hubs with orders of magnitude differences in numbers of contacts is more limited in our data context. second, our data set is more comprehensive than any other within hospital contact data set in existence in terms of the worker categories included. recall from earlier in the paper that the ueno and masuda data set only includes physicians, nurses, and patients in a hospital much smaller than is studied here. our results below suggest that many of the most important groups in the hospital are not included in their study. thus, at a minimum, our study highlights the importance of funding for studies that aim to collect even more comprehensive data sets that include individuals with nonpatient care responsibilities and more comprehensively cover a larger share of hospital workers. in addition, we note that we consider all contacts in our data set to be equivalent, or "equally weighted." there may be some concern that not all contacts are created equal in our context. for instance, a contact between a physician and a patient that occurs during a physical examination, may be more likely to spread an infectious disease than other contacts in our data set. we attempt to control for this possibility later in the paper when we consider heterogeneous transmission rates and repeated contacts. as mentioned above, transmissions of infectious disease are not usually observable. thus studying infectious disease transmission using an agent-based model can be a useful tool. in the remainder of the paper we model the spread of an infectious disease across the simulated hospital contact networks described above. once created, we use the contact network in a model of the spread of an infectious disease in the hospital as follows: agents can be in one of three states, susceptible to being infected (s), infected and able to infect others (i), or recovered (and therefore immune) (r). we assume that each infected agent recovers after periods which is in-line with the infectious period for influenza. once recovered the agent enters state r and is therefore immune to further transitions to the infected state. initially, all agents in the model are in state s. agents may be vaccinated against infection. vaccinations occur only in the initial period of the model. once vaccinated, an agent moves immediately from state s to state r and is thus immune for the remainder of the model. in the initial period of the model, each agent in state s (all agents that have not been vaccinated) is subject to infection with probability α = . . these are the agents of our model that seed the potential epidemic. once these initial infections occur we assume that each contact in our network occurs once in each subsequent period of the model. if a contact occurs between a state i and a state s agent, the state s agent transitions to state i with probability α, which we vary across experiments. we continue the model until no agents remain in state i. in each period of the model we calculate the fraction of agents in each worker category in each state (s, i, or r). for each of the results reported below we run replications of each parameter set or computational experiment reported. the results reported are averages over these replications. in addition to the results reported below, we have studied a wide range of parameters for our model and find the results reported below to be robust to changes in all of the parameters within reasonable bounds. the purpose of the model is to estimate m(γ j ) and the externality generated for the network of contacts in the uihc shadow data and, in turn, to identify the classes of workers most important to vaccinate. this is a two step process. first we perform a series of base-line models as described above with none of the healthcare workers and patients vaccinated. from this baseline, we observe the rate of infection for each class of agents in the hospital population ( worker groups and patients for a total of groups). we denote the infection rate of group k in the base model as a function of the transmission rate α as π k b (α) and the overall infection rate in the entire population as a function of α as π b (α). second, we want to calculate the average and marginal infections generated by each group. to do so, we change the vaccination rate for each group, one group at a time, and re-run the model. as an example, we run the model with all floor nurses vaccinated and no other vaccinations and observe π (α). then we run the model with all housekeepers vaccinated and no one else and observe π (α) and so on for each group. we then compare the change in the average number of infections between the models, δ(b, k) = (π b (α)−π k (α))n , which is the difference between the overall infection rate in the base model with no vaccinations and the overall infection rate in the model with all of group k vaccinated, multiplied by the total population size n . now, using the notation described above, the change in the number of infections δ(b, k) is equal to the change in number of people vaccinated, n k , multiplied by the probability that each of these agents becomes infected if not vaccinated, multiplied by the number of additional infections each infected agent would generate. simplifying, if we assume each agent infects the same number of individuals, we can write the average number of secondary infections generated per infected person in group k, as a k and write: one can then find an estimate of the average secondary infections per infected person as: effectively, this process removes each group from the hospital, one at a time. we then can observe the effect of each individual worker group on the size of the modeled epidemic. instead of vaccinating all agents of a group at once, we can vaccinate a fraction of a group. as we change this fraction at intervals n k we can view the effect of increases in vaccination rates for each group (one at a time). we then have an estimate of the marginal infections prevented per vaccination as: we now proceed in two steps. first we investigate the effect of each group in total on the epidemic process by vaccinating an entire group one at a time and calculatinĝ a k for each group. we then choose a sample of interesting groups and study the details of the epidemic process as we vary the number of vaccinations performed in each of these groups, at specified intervals between and %. interestingly, as we vary the percent of each group vaccinated, we will see that there are different outcomes across these different groups in terms of marginal infections generated, probability of infection and the overall effect of a vaccination (in terms of reducing the number of infections). we begin by varying the transmission rate, α, over the range [ . , . ] and observing the base infection rate π b (α). the results are displayed in fig. . as one can see in the figure, a sufficiently large transmission rate is needed to generate an epidemic of reasonable size. further, as expected, the number of infections generated monotonically increases as a function of the transmission rate, α. our primary interest is in intermediate ranges of epidemic outbreaks. if the transmission rate is too high then almost everyone in a population needs to be vaccinated in order to reach herd immunity. and, if the transmission rate is too low, then there is not a large need to worry about vaccine priority. thus, we concentrate on two intermediate levels of the transmission rate α = . and α = . . with no vaccinations, these levels yield an epidemic where between one-third and one-half of the population is infected over the course of the epidemic. we now find the average effect of vaccinations across the hospital worker groups using the procedure described above for α = . and α = . . we present results for the average "secondary infections" generated per infected person, k , and the product of average infections and probability of infection, which yields the "decrease in infections per vaccination," δ(b,k) n k in tables and . from the decrease in infections per vaccination we have an indication of how much the vaccination of an individual group member is contributing to preventing the spread of an epidemic. the results of this experiment suggest where efforts should be directed in the event of an influenza vaccine shortage or in the event of the development of new disease for which a vaccine may be developed (e.g., avian flu, swine flu, etc.) but is initially in short supply until mass quantities may be made available. note that some of the groups have vaccinations that prevent less than one infection per vaccination. this occurs because these groups have a low probability of infection and sufficiently low number of average infections that each member generates if infected. groups with large decreases in infections per vaccination are the ones to prioritize in times of a vaccine shortage, assuming equal costs of infection. in these experiments we see three clear groups that stand above the others in terms of the effect of vaccinations. for the parameters of the experiments, each vaccination of a unit clerk, social worker, and phlebotomist, results in a decrease of . infections or more on average for α = . and of . or more for α = . . in addition vaccinating unit clerks is extremely effective; each unit clerk vaccination results in a decrease of over infections for α = . and over infections for α = . . somewhat surprisingly, some of the groups that are seen as central to the functioning of a hospital play a very small or moderate role in spreading an infectious disease. vaccinating staff physicians results in a lower than average decrease in infections. we revisit this result in our discussion of transmission rates later in the paper. also of note, as one would expect, as the transmission rate increases, the probability of infection increases. but, this has the effect of making individual vaccinations less beneficial on average. note that k is smaller for each group for a higher transmission rate. this has the effect of lowering the variance of average infections. for the α = . case above the standard deviation is . , and for the α = . case, the standard deviation is . . as the transmission rate increases, a larger fraction of individuals are infected throughout the population. thus there are more opportunities for each individual to be infected if she has not already been infected. vaccinating a given person in the population will only prevent one of these multiple channels for infection. so, as the infection rate increases, the effectiveness of a vaccination becomes more uniform across the groups. this has direct policy applications. an infectious disease that is highly contagious could best be met with a uniform vaccination strategy since each individual in the population will create a similar level of infections on average. but an infectious disease with a low level of contagiousness could most effectively be met with a targeted vaccination campaign (bansal and pourbohloul ) . we next look at the marginal effect of a vaccination as the number of vaccinations increase. we present results in figs. , , , , and for five interesting worker group categories for the same two transmission rates discussed above. unit clerks, social workers and phlebotomists are chosen because of their large number of secondary infections generated. we also choose floor nurses and staff physicians because of interest in the effect of worker groups with primary patient care responsibilities. in fig. we plot the marginal infections prevented per vaccination as a function of the number of vaccinations performed for the five groups. recall that the number of marginal infections is the additional number of infections that an agent generates if the agent becomes infected. in fig. we plot the probability of infection for the five groups. and, in fig. we plot the product of marginal infections and probability of infection which yields the total number of infections prevented per vaccination. these figures all consider a transmission rate of . . figures , , and plot the same relationships for a transmission rate of . . we begin with marginal infections. recall that marginal infections may be increasing or decreasing in the number of vaccinations performed for small numbers of vaccinations (boulier et al. ). (here the number of vaccinations performed is small relative to the entire population as we are only vaccinating some members of one of the groups. so, even if we vaccinate an entire group, this is a small number relative to the entire population.) here, we see two interesting outcomes. first, we see that marginal infections for both unit clerks and floor nurses increase as more vaccinations are performed. for these two groups, each additional vaccination prevents a larger and larger number of infections. this is particularly extreme for a transmission rate of . and the case of unit clerks where a small number of vaccinations results the product of the two previously discussed statistics yields the number of infections prevented per vaccination. again, unit clerks display a unique relationship in that the number of infections prevented per vaccination dramatically increases in the number of vaccinations performed. the other four groups result in much flatter plots. thus again there is little difference between the marginal and the average for these groups. there are two interesting points to be made from these results. the first is that the effect of vaccinating unit clerks in our data is most important both from a marginal and an average perspective regardless of how many vaccinations have been performed. particularly, the marginal effect of vaccinating a unit clerk increases at a greater rate than the probability of infection for a non-vaccinated unit clerk falls. thus, it is always more beneficial to vaccinate one more unit clerk as opposed to a worker from another group (assuming transmission rates are equal). the second is that there is little difference between the average and the marginal effect of a vaccination for the other groups considered here. this second point can be interpreted as good news from a policy making perspective in the sense that the optimal allocation of vaccinations does not switch as more of a group is vaccinated. in other words it is not the case that group a is the optimal group to target up to some vaccination percentage, after which group b should be targeted. switching such as that would indicate a much more complicated solution to the optimal vaccine allocation problem. here, because there is little difference between the marginal and the average effect of a vaccination for most groups, one can pragmatically target the groups with the largest average effect of a vaccination. we now move to discuss the important features of the contact network that creates the externality. as we will see below, it is not just the number of contacts that an agent has but also which specific agents and groups the agent contacts, as well as who the agent's contacts connect to in turn. we begin by looking at some basic statistics of the contacts in our data in table . for each group, the table displays the total number of contacts, the percentage of total contacts that are with members of an agent's own group, and the number of patient contacts. total contacts and contacts with patients could be directly correlated with the likelihood of being infected and with passing on infections. the percentage of contacts within one's own group can indicate how varied one's network is and how widespread one's connections are. for instance having few contacts within one's own group provides the possibility of introducing an infection to other groups within the hospital. in addition the table also contains a common network characteristic measure, betweenness centrality, which we discuss below. on a network, the geodesic distance, g a,b , between nodes a and b is the length of the shortest path between the two nodes that traverses connections on the network measured as the number of connections traversed (or "hops" required) to reach node b from node a. as one measure of the centrality of a node on a graph one can calculate the average distance to all other nodes on the graph. thus if a graph g is composed of n nodes, average distance for node a is calculated as: a short average distance indicates that a node is close to other nodes on average an thus may be likely to be infected and to pass on infections. thus it is sometimes considered a measure of the centrality or importance of a node in a network. betweenness is another measure of the centrality of a node in a network. let c i jk be the proportion of all geodesics linking node j and node k which pass through node i. let c i be the sum of all c i jk for i = j = k. letc i be the maximum possible value for c i . (normalized) betweenness for node i, b i is then: betweenness for node i is therefore a measure of the proportion of shortest paths between nodes that go through node i. in the the table below we report group level values for betweenness centrality. the values are created in the following manner. first we create a network using the same methods as described above. second, we then calculate the betweenness centrality measurement for each agent in the simulated network. third, we calculate the average value for each hospital worker group and report the result in the table below. this network variable is likely to be an indicator of importance for disease transmission in the network. if a hospital worker group has a high average betweenness value, then the nodes in this group are potentially important in passing infections on to other nodes as it plays a crucial role in location along many of the shortest paths between nodes in a network. as such, this measure should be closely related to the marginal infections that a group generates. what is most interesting in table is the lack of a clear relationship between any of the variables in the first three columns and our previously-listed most important groups (highlighted in bold). the only measure that consistently aligns well is the betweenness measure. for a moment concentrate on the values in the first three columns. each of these three plausibly important characteristics fail to display a meaningful relationship with the average or marginal infections generated. if we concentrate on the top three most important groups, some have relatively large numbers of contacts (unit clerks), although not the largest, while others have contacts significantly below the average (phlebotomists). some have large numbers of patient contacts (phlebotomists) while others have some of the smallest number of patient contacts (unit clerks and social workers). one interesting thing that appears in the table is that phlebotomists have almost all of their contacts with patients and other phlebotomists. thus the network position of phlebotomists, is in some sense, very similar to that of patients. overall, what these observations imply is that there is not likely to be a simple relationship (or a small set of simple relationships) indicating which individuals are most important to vaccinate by looking at easily observed worker interaction patterns. instead the relationship depends on the intricate and complex web of relationships that make up the entire contact network of the hospital. this is what is captured in the betweenness centrality measure. betweenness measures the percentage of shortest paths in the entire network on which an agent is located. if we remove agents with high betweenness measures (by vaccinations) from the disease propagation network, we disrupt the flow of an epidemic. for concreteness, the correlation between the betweeness measure and the average infections generated is about . for both α = . and α = . . as mentioned above, the primary focus of this paper concerns the effect of network position on the marginal infections generated within a hospital. here, we consider two robustness checks to the results presented above. first, we discuss a comparison of the magnitude of the above described network effects and the effect of transmission rates on marginal infections for an interesting example group, staff physicians. second, we do an additional set of experiments using an additional data set that includes observed repeated contacts. we perform these robustness checks for two main reasons: first, because of the job responsibilities of different worker groups, the different worker groups may have different transmission rates, durations of contacts, or frequency of contacts creating another source of heterogeneity in infections. as a few examples, a staff physician may be more likely to transmit an infection over the course of a patient exam that includes a series of physical contacts, compared to a nurse who has a brief arm's-length conversation with a hospital transporter. a floor nurse may have multiple interactions with the same patient during the course of the day. we begin this analysis by varying the transmission rate of staff physicians. (recall that staff physicians created a lower than average number of infections in our earlier model.) again, this is primarily to account for the fact that physicians may have longer duration contacts with patients and the contacts may more frequently involve physical touch. we now assign a special transmission rate to staff physicians that we denote as α p . this will be the transmission rate for any contact between a physician and another agent where one of the agents is infected. we use α = . for all other contacts in the population and vary α p from α p = . and α p = . . as we do this we again measure average infections as described earlier and show the resulting average infections for unit clerks and staff physicians in fig. . before we present the results we note that there are alternative ways to model this scenario. for instance, we could re-weight our contact matrix. if we knew, for instance, that physician to patient contacts lasted twice as long as other pairs of contacts in the hospital worker population, we could re-weight each physician to patient contact by a factor of two. but note that, on average, this is equivalent to increasing the transmission probability by a factor of two because the expected number of new infections is the number of susceptible to infected contacts in a period multiplied by the transmission rate. a doubling of the number of contacts is equivalent to a doubling of the transmission rate. recall from our results above that when the transmission rates are equal the average secondary infections created by unit clerks are slightly greater than and slightly greater than . for staff physicians. as we increase the transmission rate for staff physicians we see several things: first, as you would expect, the average infections for staff physicians increases. but the change is not large. for α p = . the average secondary infections created by staff physicians is . , slightly less than a two-fold increase and still well below the level of average infections noted for unit clerks when the transmission rates are equal. for unit clerks, as α p increases, the average infections of unit clerks drops rapidly. this occurs for at least two reasons: one is that unit clerks become less important relative to staff physicians as α p increases. another is that, as α p increases, the overall infection rates increase, and as we reported earlier, this causes average infections to become more uniform across groups. overall, the level of average infections between these two groups does not become similar until the α p increases to about . , a five-fold increase in the staff physician transmission rate. and, the average infections of staff physicians does not become greater than that of unit clerks until α p = . . most interesting about these results is the magnitude of the network effects relative to the magnitude of the transmission rates. in the case of staff physicians and unit clerks it takes a - times increase in the transmission rate of staff physicians to "make up" the difference in network position. this suggests that the network effect differences in average infections are very important in understanding overall transmission patterns. as a second robustness check we use an additional cut of our data that includes observed repeated contacts. in some instances members of the hospital population were observed to have come in contact more than once during the observation period. we use this data as one way to include the weighting of contacts mentioned above into the analysis. table displays the observed contacts that were observed and had occurred previously in our data. as you see in the table many of these contacts involved repeated interactions with patients (often by members of the nursing staff). we re-perform the analysis above with these additional contacts added into the data. the only additional change is that we modify the transmission rate to α = . so that the total number of infections in the population remains nearly constant compared to the non-repeat contact data. (recall from earlier in the paper that a larger epidemic smooths out differences in the population groups and makes the average and marginal values more similar across groups. thus we control for epidemic size by varying the transmission rate.) we present the results in table . you will notice in the table that most of the groups that previously had the largest effect still do. unit clerks are still the most important group to vaccinate, but the difference in magnitude between unit clerks and other important groups is less than in the the previous model. further, as one would expect, groups that have more repeated contacts, such as all types of nurses, become more important. the important point though, is that the relative ranking of most of the groups changes very little. of course this is partially due to there being relatively few repeated contacts in the data set. taking these two robustness checks in combination, they demonstrate two important things. first, network structure is at least as important as transmission rate in determining the course of an epidemic in our data set. second, while the data we have collected is not perfect in terms of comprehensiveness, the relative ranking of group importance appears relatively robust to alternative measures of network structure. a a minimum, taken in combination, these results suggest a need for a greater emphasis on network based data collection in order to better understand both micro level and macro level epidemiology. we now make a small shift in focus. generally, a hospital's primary goal is to restore or improve patient health. thus prioritizing healthcare worker vaccinations so as to best protect patients may be a legitimate goal of a hospital. in other words hospital administrators may care about protecting patients from infection as much, or more, than they do about protecting the entire hospital population from infection. of course these may be closely related goals. in addition, in a large scale epidemic, it may be of great importance to have a healthy staff of physicians to treat patients. thus protecting physicians may be another important goal in vaccine priority within a hospital. in tables and we display the same relationships as displayed in our initial results section above but this time only with regard to patient infections generated and staff physician infections generated, not infections in the entire hospital. in this analysis we see very similar results to the overall population results. beginning with patients, the four groups (unit clerk, social worker, physical and occupational therapist, and phlebotomist) that played the most important role in transmitting to the hospital population as a whole also play an important role in their effect on patients specifically. however, we see some difference in the two groups of models. first, groups that have more direct patient contacts increase in importance. for instance, phlebotomists replace unit clerks as the most important group. second new groups emerge as important for transmitting to patients. for instance, hospital transporters are among the top four groups in transmitting to patients but are significantly below the average in terms of transmissions to the general population. with this in mind it seems that giving vaccination priority to health care workers with direct patient contacts is more important for protecting patients than it is for protecting the general population, as one would expect. but still, some of the groups with the largest impact on infecting patients have few direct patient contacts (unit clerks and social workers, for example). for staff physicians we see similar results. again, the four most important groups remain important, but other groups such as floor nurses increase in importance when considering staff physicians specifically. to summarize the results of this section, the same groups that create infections in the general population also create infections in the patient and staff physician population. but, groups that have direct patient or staff physician contacts have increased importance. still, one should not ignore other groups central to the network of the hos- pital that have only few direct patient or staff physician contacts (e.g., social workers and unit clerks). we utilize a newly collected data set on contacts of health care workers at a large university hospital to estimate network effects for infectious disease transmission. interestingly the most important groups to vaccinate tend to have heterogeneous contacts throughout the hospital. groups such as social workers and unit clerks are very important to vaccinate even though they have been given low priority in past vaccine campaigns because of their relatively limited number of patient contacts. for instance, the cdc recommended in their interim influenza vaccination recommendations in - that influenza vaccine priority be given to "health-care workers involved in direct patient care" and further stated, "persons who are not included in one of the priority groups described above should be informed about the urgent vaccine supply situation and asked to forego or defer vaccination." this mismatch of scientific results and past policy decisions suggests that future research in this area is warranted especially when one considers the public health dangers associated with the emergence of avian flu, a more lethal version of swine flu, or recent dangers such as sars. with that stated, we want to be careful to recognize that one reason to vaccinate primary care providers is to assure individuals are available to care for the sick. this important incentive is outside the scope of our model. the results of this paper lead to important public policy considerations. specifically, hospital workers with a low probability of infection may be likely to ignore recommendations for vaccination even if they are central to the spread of an infectious disease. one way to increase the overall vaccination level is with a subsidy program. but, as the results in this paper show, not all hospital workers are equal in terms of the positive externality generated by a vaccination. because of the heterogeneous contacts throughout the hospital, some workers are more important to the spread of an infectious disease than others. thus if hospitals and other public health organizations want to efficiently distribute vaccines they need to target specific worker groups, perhaps by allocating subsidies, on the basis of discrepancies in probability of infection and marginal infections generated. this paper is the first to use specific micro-level contact data within a hospital to guide policy makers and public health officials in this endeavor. to be clear, these results are not meant to be specifically calibrated to measure the exact effect of vaccinations in these groups. instead our hope is that the orderings of the hospital worker groups (which are robust across the parameters that we have explored) indicate where public health officials can effectively intervene in order to prevent widespread epidemics within hospitals. and these experiments reveal interesting and surprising groupings. prior to this study, as quoted above, it had been argued that groups like unit clerks be excluded from influenza vaccine campaigns, in times of vaccine shortages, because of their minimal patient contacts. the results of this study suggest that decisions such as these need to be more fully explored. a comparative analysis of influenza vaccination programs article cdc ( ) epidemiology and prevention of vaccine-preventable diseases, th edn. public health foundation prevention and control of influenza: recommendations of the advisory committee on immunization practice (acip) model parameters and outbreak control for sars the spatial dynamics of host parasitoid systems incidence and recall of influenza in a cohort of glasgow healthcare workers during the - epidemic: results of serum testing and questionnaire optimal tax/subsidy combinations for flu season disease eradication: private versus public vaccination meta)population dynamics of infectious diseases selected aspects of the socioeconomic impact of nosocomial infections: morbidity mortality, cost, and prevention a contibution to the mathematical theory of epidemics epidemiology economic, diseases infectious prospects of the control of disease prevention and control of influenza: recommendations of the advisory committee on immunization practices, morbidity and mortality weekly report mortality associated with influenza and respiratory syncytial virus in the united states controlling nosocomial infection based on the structure of hospital social networks perspective: human contact patter ns and the spread of airborne infectious diseases barriers to influenza vaccination acceptance. a survey of physicians and nurses key: cord- -wmmbkmrg authors: wang, de-guo; brewster, jeffrey d.; paul, moushumi; tomasula, peggy m. title: two methods for increased specificity and sensitivity in loop-mediated isothermal amplification date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: wmmbkmrg the technique of loop-mediated isothermal amplification (lamp) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional pcr methods. the high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. in this study, a set of lamp primers were designed targeting the prfa gene sequence of listeria monocytogenes, and dimethyl sulfoxide (dmso) as well as touchdown lamp were employed to increase the sensitivity and specificity of the lamp reactions. the results indicate that the detection limit of this novel lamp assay with the newly designed primers and additives was fg per reaction, which is ten-fold more sensitive than a commercial isothermal amplification kit and hundred-fold more sensitive than previously reported lamp assays. this highly sensitive lamp assay has been shown to detect strains of listeria monocytogenes, and does not detect other listeria species (including listeria innocua and listeria invanovii), providing some advantages in specificity over commercial isothermal amplification kits and previously reported lamp assay. loop-mediated isothermal amplification, developed and reported by notomi et al., in [ ] , can specifically, sensitively and rapidly amplify nucleic acids with two pairs of primers recognizing independent sequences of a target gene under isothermal conditions. moreover, nagamine et al., has advanced the method by putting forward loop primers that accelerate the lamp reaction [ ] . therefore, the lamp assay theoretically has the advantage of specificity, selectivity, and rapidity over polymerase chain reaction (pcr) [ , ] , nucleic acid sequence based amplification (nasba) [ , ] , strand displacement amplification (sda) [ ] , rolling circle amplification (rca) [ ] , helicase dependent amplification (hda) [ ] , and cross-priming amplification assay (cpa) [ , ] . for practical application of lamp as well as reduction of the rate of false positive results in lamp reactions, most researches currently focus on development of closed-tube detection to reduce aerosol pollution and cross pollution, which include the use of turbidity [ ] , sybr green i [ ] , picogreen [ ] , gelred tm [ , ] , lateral flow dipstick [ ] , hydroxynaphthol blue dye [ ] , malachite green [ ] , microfluidic chips and gmr sensors as well as calcein used by eiken chemical co., ltd [ , ] . however, there is still no report studying non-specific amplification and cause of false positive results in lamp reactions at present. the objective of this paper is to study the cause and limit the rate of false positive results in lamp reactions targeting l. monocytogenes as well as to increase the specificity and sensitivity of these lamp reactions using dmso and touchdown lamp. although there were only two primers, non-specific amplification occurred in the isothermal amplification of four primer combinations out of seven combinations, and it was more obvious in the reaction of three primer combinations (hlya-fip + hlya-lf, hlya-fip + hlya-lb, hlya-fip + hlya-b ), as table shown. analysis on the three combinations indicated that they had the common situation that - bases at ' end of both primers had two complementary sequences on a same primer, and such situation had been avoided when lamp primers for prfa of l. monocytogenes were designed and screened in this study. moreover, it was proved by the experiment that non-specific amplification caused by primer dimers was one reason for false positive results of lamp. the lamp reaction mixtures containing varying concentrations of dmso were heated at °c for min ( s per cycle), as indicated in figure . when % dmso was added, the detection time for pg l. monocytogenes genomic dna was less than min; however, one of the two negative controls amplified as well. the tm value and melt curve were obviously different from those of the positive controls, and were therefore as attributed to non-specific amplification, which may be caused by partial complementation among primers of lamp. the detection time with % dmso was slightly longer than with . %. overall, the results showed that the lower concentration of dmso does not inhibit non-specific amplification while the higher concentration of dmso may inhibit the activity of bst . warmstart dna polymerase, and therefore, . % dmso was determined to be the optimal among these three concentrations. . % dmso was added to lamp reaction mixtures and the reactions were carried out at varying temperatures for min, as shown in table . with a reaction temperature of °c, only one of two positive controls ( pg l. monocytogenes genomic dna) was detected. the threshold time obtained using a reaction temperature of °c was shorter than that obtained using °c reaction temperature and slightly shorter than that obtained using the other temperatures. therefore, °c was chosen as the most suitable reaction temperature. the optimized lamp reaction conditions were used with the conventional lamp methodology with a serial dilution of l. monocytogenes dna template was and these mixtures were heated at °c for min. as shown in table , the detection limit of the optimized reaction mixture using the conventional lamp technique was found to be fg l. monocytogenes dna template. the optimized lamp mixture was used with the touchdown methodology to detect a serial dilution of l. monocytogenes dna template. after the mixtures were preheated at °c for min and bst . warmstart dna polymerase (new england biolabs, beverly, ma, usa) were added, they were heated at °c for min, at °c for min, at °c for min and then at °c for min, and, as indicated in table , the sensitivity of touchdown lamp was found to be fg of l. monocytogenes dna. therefore comparing these identical reaction mixtures in the conventional lamp assay and the touchdown lamp assay shows that the touchdown lamp method increases the overall sensitivity of lamp assay. the detection limit of the original reported lamp method by tang, et al (tang method) tested using fg l. monocytogenes dna template, as well. only one of positives controls amplified using these conditions. moreover, one of four negative controls showed non-specific amplification, as reported in table . sensitivity of both the isothermal master mix using our own designed lamp primers as well as the loopamp ® listeria monocytogenes detection kit to detect l. monocytogenes was tested. the results indicate that the detection limit of both commercial lamp kits is fg l. monocytogenes dna template per reaction, as shown in table . therefore, the sensitivity of the newly developed lamp assay presented here was ten-fold higher than that obtained using the commercial isothermal amplification kits and hundred-fold higher than the originally reported tang lamp assay. this newly developed lamp assay was tested with listeria monocytogenes strains ( stereotypes) and as shown in table , all were successfully detected. the assay was also tested with five other listeria species. in the initial experiment, listeria invanovii atcc was falsely detected and one of three reactions amplified, while the other species had negative results. the experiment with l. invanovii atcc was repeated four times and all four repeated reactions were negative. therefore, the initial false positive result may have been caused by slight aerosol pollution of dna templates. [ ] ; however, the optimized lamp assay required an extended amplification time of min, and even with the lengthy reaction time, two strains (listeria monocytogenes j - (stereotype: / b) and listeria monocytogenes atcc (stereotype: b) were not detected, as shown in table . extending the amplification time further to h, led to non-specific amplification of negative controls. the two commercial lamp kits were able to distinguish listeria monocytogenes from other listeria species. there were, however, two negative controls that exhibited non-specific amplification, and, sometimes, l. monocytogenes j - (stereotype: a) was not detected, as shown in table . therefore, the newly developed lamp assay presented here can detect stereotypes of listeria monocytogenes selectively while not detecting other listeria species (including listeria innocua and listeria invanovii), and had some advantages over commercial isothermal amplification kit and the original tang lamp assay in specificity. the lamp primers targeting specific gene hlya of listeria monocytogenes reported by tang, et al., are used for studying non-specific amplification of lamp [ ] , as shown in table . targeting the specific gene prfa (genbank locus: ay . ) of l. monocytogenes, a set of lamp primers were designed and selected with primerexplorer and oligo according to the reported methodology [ ] , and are listed in table . the isothermal amplification was performed in a total μl reaction mixture containing . [ , ] . %, . % and % dmso were added into different reaction tubes. lamp was carried out at °c for min and a melt curve was obtained using a stepone tm system. lamp was performed as above in a μl reaction mixture containing pg l. monocytogenes dna template as well as the optimized concentration dmso at , , , and °c for min, and a melt curve was obtained using a stepone tm system. the optimized lamp mixture was combined with serial dilutions of dna template of listeria monocytogenes ranging from to fg, and the reaction mixtures were heated at selected temperature °c for min in stepone tm system, and the detection limit of conventional lamp was determined. in the case of touchdown lamp, the reaction mixture was preheated at °c for min. after min, bst . warmstart dna polymerase (large fragment) was added and the reaction mixture was heated at temperatures °c higher than selected temperature for min, at temperature of °c higher than selected temperature for min, at temperature of °c higher than selected temperature for min and then at selected temperature for min, and the sensitivity of touchdown lamp was determined and compared with that of conventional lamp. for comparison, the detection limit of the reported method by tang, et al., (tang lamp assay) was determined by carrying out lamp reactions according to the conditions specified in their publication [ ] . isothermal master mix and loopamp ® listeria monocytogenes detection kits were purchased from optigene limited (west sussex, uk) and eiken chemical co., ltd (tochigi, japan), respectively, and lamp was carried out according to the manufacturers' instructions using a set of serially diluted dna template of l. monocytogenes ranging from to fg. eleven strains of l. monocytogenes (different stereotype) and other listeria species (including l. innocua and l. invanovii) were used for the specificity study (table ) . listeria strains were cultured overnight at °c in difco tm buffered listeria enrichment broth base (becton, dickinson and company, san jose, ca, usa) and the others in luria-bertani (lb) broth. dna from these pure cultures was extracted according to the manufacturer's handbook of dneasy ® blood and tissue kit (qiagen ltd, north manchester, uk), and these dna templates was used for determining the specificity of the optimized touchdown lamp assay, the tang lamp assay and the lamp assay utilizing the commercial isothermal amplification kit. the amount of dna template used is pg per reaction. it is difficult to avoid primer dimers and non-specific amplification when couple numerous sets of primers are used in lamp assays. this is especially true when the concentrations of primers, mg + , dntps and dna polymerase in reaction mixtures are as high as those used in real-time pcr. the concentrations of these factors must be strictly controlled to avoid non-specific amplification in real-time pcr [ ] as well as lamp reactions. there are instances in which standard pcr amplification reaction conditions do not produce acceptable results. addition of dmso and use of touchdown temperature conditions have been used improve pcr results. we investigate these approaches for the first time for optimization of lamp reactions. unfortunately, with the information presently available it is not possible to predict which enhancing agent is best for any particular target. but dmso has been frequently used in this capacity [ ] . the results presented here using dmso in lamp reaction mixtures indicate that, dmso can increase amplification with lamp at low concentration and can inhibit activity of bst . warmstart dna polymerase. we had tried to enhance the reaction of lamp with betaine, tetramethylammonium chloride, tetramethylene sulfoxide, and formamide, but their effect was not as good as that of dmso, because of the limitation of length, no more tautology here. while dmso may not necessarily be the best enhancing agent [ , ] , i.e., the manufacturer of the commercial isothermal amplification kit used in this experiment may have found some favorable enhancing agent, but their reagents are proprietary, and dmso served to decrease non-specific amplification in the specific experiments presented here. touchdown pcr offered a simple and rapid means to optimize pcrs, increasing specificity, sensitivity and yield, without the need for lengthy reaction times and/or the redesigning of primers [ , ] . touchdown lamp, compared to conventional lamp methods, results in increased sensitivity and yield of lamp. this improvement may be due to the high temperature inhibiting the formation of primer dimers and promoting the correct combination of primers and template. the biggest advantage of lamp is that the reaction can be performed isothermally, and people argue all the time that all we need is a simple water bath for the rapid detection, so the advantage may be compromised by the developed touchdown lamp assay, we made such efforts here just to reveal or verify the main cause for false positive results of lamp and inspire people to modify and improve lamp technology, my colleagues and i have also been looking for a more suitable method, which can not only keep the advantage but also improve the sensitivity and specificity of lamp. in summary, non-specific amplification was a limiting factor in the applicability of the lamp methodology. a few different options to eliminate this issue have been reported here to successfully selectively and sensitively detect l. monocytogenes. designing ideal primers, additives such as dmso, and method modifications such as touchdown lamp may be favorable alternatives for increased specificity and sensitivity in lamp in other applications as well. loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia primer-directed enzymatic 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isothermal amplification for rapid detection of porcine epidemic diarrhea virus loop-mediated isothermal amplification for the detection of goose circovirus a loop-mediated isothermal amplification (lamp) method for the identification of species within the echinococcus granulosus complex the development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of vibrio parahaemolyticus visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for leishmania infection a novel hbv genotypes detecting system combined with microfluidic chip, loop-mediated isothermal amplification and gmr sensors a new surveillance and response tool: risk map of infected oncomelania hupensis detected by loop-mediated isothermal amplification (lamp) from pooled samples rapid and sensitive detection of listeria monocytogenes by loop-mediated isothermal amplification simple and rapid method for detecting foodborne shigella by a loop-mediated isothermal amplification kinetic pcr analysis: real-time monitoring of dna amplification reactions betaine and dmso: enhancing agents for pcr a specificity enhancer for polymerase chain reaction. nucl. acid res improvement of pcr amplified dna sequencing with the aid of detergents touchdown pcr for increased specificity and sensitivity in pcr amplification touchdown" pcr to circumvent spurious priming during gene amplification this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to acknowledge the china scholarship council. this work was supported by natural science foundation of china ( ), nsfc-henan talent the authors declare no conflict of interest. key: cord- -s knxdne authors: perra, nicola; gonçalves, bruno title: modeling and predicting human infectious diseases date: - - journal: social phenomena doi: . / - - - - _ sha: doc_id: cord_uid: s knxdne the spreading of infectious diseases has dramatically shaped our history and society. the quest to understand and prevent their spreading dates more than two centuries. over the years, advances in medicine, biology, mathematics, physics, network science, computer science, and technology in general contributed to the development of modern epidemiology. in this chapter, we present a summary of different mathematical and computational approaches aimed at describing, modeling, and forecasting the diffusion of viruses. we start from the basic concepts and models in an unstructured population and gradually increase the realism by adding the effects of realistic contact structures within a population as well as the effects of human mobility coupling different subpopulations. building on these concepts we present two realistic data-driven epidemiological models able to forecast the spreading of infectious diseases at different geographical granularities. we conclude by introducing some recent developments in diseases modeling rooted in the big-data revolution. historically, the first quantitative attempt to understand and prevent infectious diseases dates back to when bernoulli studied the effectiveness of inoculation against smallpox [ ] . since then, and despite some initial lulls [ ] , an intense research activity has developed a rigorous formulation of pathogens' spreading. in this chapter, we present different approaches to model and predict the spreading of infectious diseases at different geographical resolutions and levels of detail. we focus on airborne illnesses transmitted from human to human. we are the carriers of such diseases. our contacts and mobility are the crucial ingredients to understand and model their spreading. interestingly, the access to large-scale data describing these human dynamics is a recent development in epidemiology. indeed, for many years only the biological roots of transmission were clearly understood, so it is not surprising that classical models in epidemiology neglect realistic human contact structures or mobility in favor of more mathematically tractable and simplified descriptions of unstructured populations. we start our chapter with these modeling approaches that offer us an intuitive way of introducing the basic quantities and concepts in epidemiology. advances in technology are resulting in increased data on human dynamics and behavior. consequently, modeling approaches in epidemiology are gradually becoming more detailed and starting to include realistic contact and mobility patterns. in sects. . and . we describe such developments and analyze the effects of heterogeneities in contact structures between individuals and between cities/subpopulations. with these ingredients in hand we then introduce state-of-the-art data-driven epidemiological models as examples of the modern capabilities in disease modeling and predictions. in particular, we consider gleam [ , ] , episims [ ] , and flute [ ] . the first model is based on the metapopulation framework, a paradigm where the inter-population dynamics is modeled using detailed mobility patterns, while the intra-population dynamics is described by coarse-grained techniques. the other tools are, instead, agent-based model (abm). this class of tools guarantees a very precise description of the unfolding of diseases, but need to be fed with extremely detailed data and are not computationally scalable. for these reasons their use so far has been limited to the study of disease spread within a limited numbers of countries. in comparison, metapopulation models include a reduced amount of data, while the approximated description of internal dynamics allows scaling the simulations to global scenarios. interestingly, the access to large-scale data on human activities has also started a new era in epidemiology. indeed, the big-data revolution naturally results in real time data on the health related behavior of individuals across the globe. such information can be obtained with tools that either require the active participation of individuals willing to share their health status or that is mined silently from individuals' health related data. epidemiology is becoming digital [ , ] . in sect. . we introduce the basic concepts, approaches, and results in this new field of epidemiology. in particular, we describe tools that, using search queries, microblogging, or other web-based data, are able to predict the incidence of a wide range of diseases two weeks ahead respect to traditional surveillance. epidemic models divide the progression of the disease into several states or compartments, with individuals transitioning compartments depending on their health status. the natural history of the disease is represented by the type of compartments and the transitions from one to another, and naturally varies from disease to disease. in some illnesses, susceptible individuals (s) become infected and infectious when coming in contact with one or more infectious (i) persons and remain so until their death. in this case the disease is described by the so-called si (susceptible-infected) model. in other diseases, as is the case for some sexual transmitted diseases, infected individuals recover becoming again susceptible to the disease. these diseases are described by the sis (susceptible-infected-susceptible) model. in the case of influenza like illnesses (ili), on the other hand, infected individuals recover becoming immune to future infections from the same pathogen. ilis are described by the sir (susceptible-infected-recovered) model. these basic compartments provide us with the fundamental description of the progression of an idealized infection in several general circumstances. further compartments can be added to accurately describe more realistic illnesses such as smallpox, chlamydia, meningitis, and ebola [ , , ] . keeping this important observation in mind, here we focus on the sir model. epidemic models are often represented using chart such as the one seen in fig. . . such illustrations are able to accurately represent the number of compartments and the disease's behavior in a concise and easily interpretable form. mathematically, models can also be accurately represented as reaction equations as we will see below. in general, epidemic models include two type of transitions, "interactive" and "spontaneous." interactive transitions require the contact between individuals in two different compartments, while spontaneous transitions occur naturally at a fixed rate per unit time. for example, in the transition between s to i, susceptible individuals become infected due to the interaction with infected individuals, i.e. sci ! i. the transition is mediated by individuals in the compartment i, see fig. but how can we model the infection process? intuitively we expect that the probability of single individual becoming infected must depend on ( ) the number of infected individuals in the population, ( ) the probability of infection given a contact with an infectious agent and, ( ) the number of such contacts. in this section we neglect the details of who is in contact with whom and consider instead individuals to be part of a homogeneously mixed population where everyone is assumed to be in contact with everyone else (we tackle heterogeneous contacts in sect. . ). in this limit, the per capita rate at which susceptible contract the disease, the force of infection , can be expressed in two forms depending on the type of population. in the first, often called mass-action law, the number of contacts per individual is independent of the total population size, and determined by the transmission rateǎ nd the probability of randomly contacting an infected individual, i.e. dˇi=n (where n is the population size). in the second case, often called pseudo massaction law, the number of contacts is assumed to scale with the population size, and the transmission rateˇ, i.e. dˇi. without loss of generality, in the following we focus on the first kind of contact. the sir framework is the crucial pillar to model ilis. think, for example, at the h n pandemic in , or the seasonal flu that every year spread across the globe. the progression of such diseases, from the first encounter to the recovery, happens in matters of days. for this reason, birth and death rates in the populations can be generally neglected, i.e. d t n Á for all times t. let us define the fraction of individuals in the susceptible, infected, and recovered compartments as s; i, and r. the sir model is then described by the following set of differential equations: where dˇi Áˇi n is the force of infection, and d t Á d dt . the first equation describes the infection process in a homogeneous mixed population. susceptible individuals become infected through random encounters with infected individuals. the second equation describes the balance between the in-flow (infection process, first term), and the out-flow (recovery process, second term) in compartment i. finally, the third equation accounts for the increase of the recovered population due to the recovery process. interestingly, the sir dynamical equations, although apparently very simple, due to their intrinsic non-linearity cannot be solved analytically. the description of the evolution of the disease can be obtained only through numerical integration of the system of differential equations. however, crucial analytic insight on the process can be obtained for early t t and late times t ! . under which conditions a disease starting from a small number, i , of individuals at time t is able to spread in the population? to answer this question let us consider the early stages of the spreading, i.e. t t . the equation for the infected compartment can be written as d t i d i.ˇs /, indicating an exponential behavior for early times. it then follows that if the initial fraction of susceptible individuals, s d s =n, is smaller than =ˇ, the exponent becomes negative and the disease dies out. we call this value the epidemic threshold [ ] of the sir model. the fraction of susceptibles in the population has to be larger than a certain value, that depends on the disease details, in order to observe an outbreak. typically, the initial cluster of infected individuals is small in comparison with the population size, i.e. s i , or s . in this case, the threshold condition can be re-written asˇ= > . the quantity: is called the basic reproductive number, and is a crucial quantity in epidemiology and provides a very simple interpretation of the epidemic threshold. indeed, the disease is able to spread if and only if each infected individual is able to infect, on average, more than one person before recovering. the meaning of r is then clear: it is simply the average number of infections generated by an initial infectious seed in a fully susceptible population [ ] . for any value of > , the sir dynamics will eventually reach a stationary, disease-free, state characterized by i d d t i d . indeed, infected individuals will keep recovering until they all reach the r compartment. what is the final number of recovered individuals? answering this apparently simple question is crucial to quantify the impact of the disease. we can tackle such conundrum dividing the first equation with the third equation in the system . . we obtain d r s d r s which in turn implies s t d s e r r t . unfortunately, this transcendent equation cannot be solved analytically. however, we can use it to gain some important insights on the sir dynamics. we note that for any r > , in the limit t ! , we must have s > . in other words, despite r , the disease-free equilibrium of an sir model is always characterized by some finite fraction of the population in the susceptible compartment, or, in other words, some individuals will always be able to avoid the infection. in the limit where r we can obtain an approximate solution for r (or equivalently for s d r ) by expanding s d s e r s at the second order around r . after a few basic algebraic manipulations we obtain in the previous sections we presented the basic concepts and models in epidemiology by considering a simple view of a population where individuals mix homogeneously. although such approximation allows a simple mathematical formulation, it is far from reality. individuals do not all have the same number of contacts, and more importantly, encounters are not completely random [ ] [ ] [ ] [ ] . some persons are more prone to social interactions than others, and contacts with family members, friends, and co-workers are much more likely than interactions with any other person in the population. over the last decade the network framework has been particularly effective in capturing the complex features and the heterogeneous nature of our contacts [ ] [ ] [ ] [ ] [ ] . in this approach, individuals are represented by nodes while links represent their interactions. as described in different chapters of the book (see chaps. , , and ), human contacts are not heterogeneous in both number and intensity [ ] [ ] [ ] [ ] ] but also change over time [ ] . this framework naturally introduces two timescales, the timescale at which the network connections evolve, g and the inherent timescale, p , of the process taking place over the network. although the dynamical nature of interactions might have crucial consequences on the disease spreading [ ] [ ] [ ] [ ] [ ] [ ] , the large majority of results in the literature deal with one of two limiting regimens [ , ] . when g p , the evolution of the network of contacts is much slower than the spreading of the disease and the network can be considered as static. on the other hand, when p g , the links are said to be annealed and changes in networks structure are much faster than the spreading of the pathogen. in both cases the two time-scales are well separated allowing for a simpler mathematical description. here we focus on the annealed approximation ( p g ) that provides a simple stage to model and understand the dynamical properties of epidemic processes. we refer the reader to chap. face-to-face interactions for recent approaches that relax this time-scale separation assumption. let us consider a network g .n; e/ characterized by n nodes connected by e edges. the number of contacts of each node is described by the degree k. the degree distribution p .k/ characterizes the probability of finding a node of degree k. empirical observations in many different domains show heavy-tailed degree distributions usually approximated as power-laws, i.e. p .k/ k ˛ [ , ] . furthermore, human contact networks are characterized by so-called assortative mixing, meaning a positive correlation between the degree of connected individuals. correlations are encoded in the conditional probability p .k jk/ that a node of degree k is connected with a node of degree k [ , ] . while including realistic correlations in epidemic models is crucial [ ] [ ] [ ] they introduce a wide set of mathematical challenges that are behind the scope of this chapter. in the following, we consider the simple case of uncorrelated networks in which the interdependence among degree classes is removed. how can we extend the sir model to include heterogeneous contact structures? here we must take a step further than simply treating all individuals the same. we start distinguishing nodes by degree while considering all vertices with the same degree as statistically equivalent. this is known as the degree block approximation and is exact for annealed networks. the quantities under study are now i k d i k n k ; s k d s k n k , and r k d r k n k , where the i k ; s k , and r k are the number of infected, susceptible, recovered individuals in the degree class k. n k instead describes the total number of nodes in the degree class k. the global averages are given by i d using this notation and heterogeneous mean field (hmf) theory [ ] , the system of differential equations ( . ) can now be written as: the contact structure introduces a force of infection function of the degree. in particular, k d k k where is the rate of infection per contact, i.e.ˇd k, and k describes the density of infected neighbors of nodes in the degree class k. intuitively, this density is a function of the conditional probability that a node k is connected to any node k and proportional to the number of infected nodes in each class in the simple case of uncorrelated networks the probability of finding a node of degree k in the neighborhood of a node in degree class k is independent of k. in this case k d d p k .k / p .k / i k =hki where the term k is due to the fact that at least one link of each infected node points to another infected vertex [ ] . in order to derive the epidemic threshold let us consider the early time limit of the epidemic process. as done in sect. . . . let us consider that at t t the population is formed mostly by susceptible individuals. in the present scenario this implies s k i k and r k k. the equation for the infected compartment then becomes d t i k d k i k . multiplying both sides for p .k/ and summing over all values of k we obtain d t i d hki i. in order to understand the behavior of i around t let us consider an equation built by multiplying both sides of the last equation by .k / p .k/ =hki and summing over all degree classes. we obtain d t d . hk i hki hki / . the fraction of infected individuals in each value of k will increase if and only if d t > . this condition is verified when [ ] : giving us the epidemic threshold of an sir process unfolding on an uncorrelated network. remarkably, due to their broad-tailed nature, real contact networks display fluctuations in the number of contacts (large hk i) that are significantly larger than the average degree hki resulting in very small thresholds. large degree nodes (hubs) facilitate an extremely efficient spreading of the infection by directly connecting many otherwise distant nodes. as soon as the hubs become infected diseases are able to reach a large fraction of the nodes in the network. real interaction networks are extremely fragile to disease spreading. while this finding is somehow worrisome, it suggests very efficient strategies to control and mitigate the outbreaks. indeed, hubs are central nodes and play a crucial role in the network connectivity [ ] and by vaccinating a small fraction of them one is able to quickly stop the spread of the disease and protect the rest of the population. it is important to mention that in realistic settings the knowledge of the networks' structure is often limited. hubs might not be easy to easily known and other indirect means must be employed. interestingly, the same feature of hubs that facilitates the spread of the disease also allows for their easy detection. since high degree nodes are connected to a large number of smaller degree nodes, one may simply randomly select a node, a, from the network and follow one of its links to reach another node, b. with high probability, node b has higher degree than a and is likely a hub. this effect became popularized as the friend paradox: on average your friends have more friends than you do [ ] . immunizing node b is then much more effective than immunizing node a. remarkably, as counter-intuitive as this methodology might seem, it works extremely well even in the case of quickly changing networks [ ] [ ] [ ] . the next step in the progression towards more realistic modeling approaches is to consider the internal structure of the nodes. if each node in the network represents a homogeneously mixed sub-population instead of a single individual and we consider the edges to represent interactions or mobility between the different subpopulations, then we are in the presence of what is known as meta-population. this concept was originally introduced by r. levins in [ ] for the study of geographically extended ecological populations where each node represents one of the ecological niches where a given population resides. the metapopulation framework was later extended for use in epidemic modeling by sattenspiel in . in a landmark paper [ ] sattenspiel considered two different types of interactions between individuals, local ones occurring within a given node, and social ones connecting individuals originating from different locations on the network. this idea was later expanded by sattenspiel and dietz to include the effects of mobility [ ] and thus laying the foundations for the development of epidemic models at the global scale. metapopulation epidemic models are extremely useful to describe particle reaction-diffusion models [ ] . in this type of model each node is allowed to have zero or more individuals that are free to diffuse among the nodes constituting the network. in our analysis, as done in the previous section, we follow the hmf approach and consider all nodes of degree k to be statistically equivalent and write all quantities in terms of the degree k. to start, let us define the average number of individuals in a node of degree k to be w k d where n k is the number of nodes with degree k and the sum is taken over all nodes i. the mean field dynamical equation describing the variation of the average number of individuals in a node of degree k is then: where p k and p kk represent, respectively, the rate at which particles diffuse out of a node of degree k and diffuse from a node of degree k to one of degree k . with these definitions, the meaning of each term of this equation becomes intuitively clear: the negative term represents individuals leaving the node, while the positive term accounts for individuals originating from other nodes arriving at this particular class of node. the conditional probability p .k jk/ encodes all the topological correlations of the network. by imposing that the total number of particles in the system remains constant, we obtain: that simply states that the number of particles arriving at nodes of degree k coming from nodes of degree k must be the same as the number of particles leaving nodes of degree k. the probabilities p k and p kk encode the details of the diffusion process [ ] . in the simplest case, the rate of movement of individuals is independent of the degree of their origin p k d p for all values of the degree. furthermore, if individuals that are moving simply select homogeneously among all of their connections, then we have p kk d p=k. in this case, the diffusion process will reach a stationary state when: where w d w=n, w is the total number of walkers in the system, and n the total number of nodes. the simple linear relation between w k and k serves as a strong reminder of the importance of network topology. nodes with higher degree will acquire larger populations of particles while nodes with smaller degrees will have proportionally smaller populations. however, even in the steady state, the diffusion process is ongoing, so individuals are continuously arriving and leaving any given node but are doing so in a way that maintains the total number of particles in each node constant. in more realistic settings, the traffic of individuals between two nodes is function of their degree [ ] : in this expression  modulates the strength of the diffusion flow between degree classes (empirical values are in the range : Ä Â Ä : [ ] ), where w is a constant and t k d w hk c i=hki is the proper normalization ensured by the condition in eq. ( . ). in these settings, the diffusion process reaches a stationary state when: note that for  d this solution coincides with the case of homogeneous diffusion [eq. ( . )]. combining this diffusion process with the (epidemic) reaction processes described above we finally obtain the full reaction-diffusion process. to do so we must simply write eq. ( . ) for each state of the disease (e.g., susceptible, infectious, and recovered for a simple sir model) and couple the resulting equations using the already familiar epidemic equations. the full significance of eq. ( . ) now becomes clear: nodes with higher degree have higher populations and are visited by more travelers, making them significantly more likely to also receive an infected individual that can act as the seed of a local epidemic. in a metapopulation epidemic context we must then consider two separate thresholds, the basic reproductive ratio, r , that determines whether or not a disease can spread within one population (node) and a critical diffusion rate, p c , that determines if individual mobility is sufficiently large to allow the disease to spread from one population to another. it is clear that if p d particles are completely unable to move from one population to another so the epidemic cannot spread across subpopulations and that if p d all individuals are in constant motion and the disease will inevitably spread to every subpopulation on the network with a transition occurring at some critical value p c . in general, the critical value p c cannot be calculated analytically using our approach as it depends non-trivially on the detailed structure of the network and the fluctuations of the diffusion rate of single individuals. however, in the case of uncorrelated networks a closed solution can be easily found for different mobility patterns. indeed, in the case where the mobility is regulated by eq. ( . ) we obtain: interestingly, the critical value of p is inversely proportional to the degree heterogeneity in the network, so that broad tailed networks have very low critical values. this simple fact explains why simply restricting travel between populations is a highly ineffective way to prevent the global spread of an epidemic. the mobility patterns considered so far are so-called markovian: individuals move without remembering where they have been nor they have a home where they return to after each trip. although this is a rough approximation of individuals behavior, markovian diffusion patterns are allowed to analytically describe the fundamental dynamical properties of many systems. recently, new analytic results have been proposed for non-markovian dynamics that include origin-destination matrices and realistic travel routes that follow shortest paths [ ] . in particular, the threshold within such mobility schemes reads as: the exponent Á, typically close to : in heterogeneous networks, emerges from the shortest paths routing patterns [ ] . interestingly, for values of Â Ä : , fixing Á d : , p c in the case of markovian mobility patterns is larger than the critical value in a system subject to non-markovian diffusion. the presence of origindestination matrices and shortest paths mobility lower the threshold facilitating the global spreading of the disease. instead, for values of  > : the contrary is true. in these models the internal contacts rate is considered constant across each subpopulation. interestingly, recent longitudinal studies on phone networks [ ] and twitter mention networks [ ] point to the evidence that contacts instead scale super-linearly with the subpopulation sizes. considering the heterogeneity in population sizes observed in real metapopulation networks, the scaling behavior entails deep consequence in the spreading dynamics. a recent study generalized the metapopulation framework considering such observations. interestingly, the critical mobility thresholds, in the case of mobility patterns described by eq. ( . ), changes significantly being lowered by such scaling features of human contacts [ ] . despite their simplicity, metapopulation models are extremely powerful tools in large scale study of epidemics. they easily lend themselves to large scale numerical stochastic simulations where the population and state of each node can be tracked and analyzed in great detail and multiple scenarios as well as interventions can be tested. the state of the art in the class of metapopulation approaches is currently defined by the global epidemic and mobility model (gleam) [ , ] . gleam integrates worldwide population estimates [ , ] with complete airline transportation and commuting databases to create a world wide description of mobility around the world that can then be used as the substrate on which the epidemic can spread. gleam divides the globe into transportation basins. each basin is defined empirically around an airport and the area of the basin is determined to be the region within which residents would likely use that airport for long distance travel. each basin represents a major metropolitan area such as new york, london, or paris. information about all civilian flights can be obtained from the international air transportation association (iata) [ ] and the official airline guide (oag) [ ] that are responsible for compiling up-to-date databases of flight information that airlines use to plan their operations. by connecting the population basins with the direct flight information from these databases we obtain the network that acts as a substrate for the reaction diffusion process. while most human mobility does not take place in the form of flights, the flight network provides the fundamental structure for long range travel that explains how diseases such as sars [ ] , smallpox [ ] , or ebola [ ] spread from country to country. to capture the finer details of within country mobility further information must be considered. gleam uses census information to create a commuting network at the basin level that connects neighboring metropolitan areas proportionally to the number of people who live in one are but work in the other. short-term short-distance mobility such as commuting is fundamentally different from medium-term long-distance airline travel. in one case, the typical timescale is work-day ( h) while in the other it is day. this timescale difference is taken into account in gleam in an effective, mean-field, manner instead of explicitly through a reaction process such as the one described above. this added layer is the final piece of the puzzle that brings the whole together and allows gleam to describe accurately the spread from one country to the next but also the spread happening within a given country [ ] . in fig. . we illustrate the progression in terms of detail that we have undergone since our initial description of simple homogeneously mixed epidemic models in a single population. with all these ingredients in place we have a fine grained description of mobility on a world wide scale on top of which we can finally build an epidemic model. within each basin, gleam still uses the homogeneous mixing approximation. this assumption is particularly suited for diseases that spread easily from person to person through airborne means such as ili. gleam describes influenza through an seir model as illustrated in fig. . . seir models are a modification of the sir model described above that includes a further compartment, exposed, to represent of the remaining symptomatic individuals, one half is sick enough to decide to not travel or commute while the remaining half continue to travel normally. despite their apparent complexity, large scale models such as gleam are controlled by just a small number of parameters and ultimately, it's the proper setting of these few parameters that is responsible for the proper calibration of the model and validity of the results obtained. most of the disease and mobility parameters are set directly from the literature or careful testing so that as little as possible remains unknown when it is time to apply it to a new outbreak. gleam was put to the test during the h n pandemic with great success. during the course of the epidemic, researchers were able to use official data as it was released by health authorities around the world. in the early days of the outbreak there was a great uncertainty about the correct value of the r for the /h n pdm strain in circulation so a methodology to determine it had to be conceived. one of the main advantages of epidemic metapopulation models is their computational tractability. it was this feature what proved invaluable when it came to determine the proper value of r . by plugging in a given set of parameters one is able to generate several hundreds or thousands of in silico outbreaks. each outbreak contains information not only about the number of cases in each city or country as a function of time but also information about the time when the first case occurs within a given country. in general, each outbreak will be different due to stochasticity and by combining all outbreaks generated for a certain parameter set we can calculate the probability distribution of the arrival times. the number of times that an outbreak generated the seeding of a country, say the uk, in the same day as it occurred in reality provides us with a measure of how likely the parameter values used are. by multiplying this probability for all countries with a known arrival time we can determine the overall likelihood of the simulation: where the product is taken over all countries c with known arrival time t c and the probability distribution of arrival times, p c .t/ is determined numerically for each set of input values. the set of parameters that maximizes this quantity is then the one whose values are the most likely to be correct. using this procedure the team behind gleam determined that the mostly likely value of the basic reproductive ratio was r d : [ ] , a value that was later confirmed by independent studies [ , ] . armed with an empirical estimate of the basic reproductive ratio for an ongoing pandemic, they then proceeded to use this value to estimate the future progression of the pandemic. their results predicting that the full peak of the pandemic would hit in october and november were published in early september [ ] . a comparison between these predictions and the official data published by the health authorities in each country would be published several years later [ ] clearly confirming the validity of gleam for epidemic forecasting in real time. indeed, the model predicted, months in advance, the correct peak week in % of countries in the north hemisphere for which real data was accessible. in the rest of cases the maximum error reported has been weeks. gleam can also be further extended to include age-structure [ ] , interventions and travel reductions. the next logical step in the hierarchy of large scale epidemic models is to take the description of the underlying population all the way down to the individual level with what are known as abm. the fundamental idea behind this class of model is a deceptively simple one: treat each individual in the population separately, assigning it properties such as age, gender, workplace, residence, family structure, etc: : : these added details give them a clear edge in terms of detail over metapopulation models but do so at the cost of much higher computational cost. the first step in building a model of this type is to generate a synthetic population that is statistically equivalent to the population we are interested in studying. typically this is in a hierarchical way, first generating individual households, aggregating households into neighborhoods, neighborhoods into communities, and communities into the census tracts that constitute the country. generating synthetic households in a way that reproduces the census data is far from a trivial task. the exact details vary depending on the end goal of the model and the level of details desired but the household size, age, and gender of household members are determined stochastically from the empirically observed distributions and conditional probabilities. one might start by determining the size of the household by extracting from the distribution of household size of the country of interest and selecting the age and gender of the head of the household proportionally to the number of heads of households for that household size that are in each age group. conditional on this synthetic individual we can then generate the remaining members, if any, of the household. the required conditional probability distributions and correlation tables can be easily generated [ ] from high quality census data that can be found for most countries in the world. this process is repeated until enough synthetic households have been generated. households are then aggregated into neighborhoods by selecting from the households according to the distribution of households in a specific neighborhood. neighborhoods are similarly aggregated into communities and communities into census tracts. each increasing level of aggregation (from household to country) represents a decrease in the level of social contact, with the most intimate contacts occurring at the household level and least intimate ones at the census tract or country level. the next step is to assign to each individual a profession and work place. workplaces are generated following a procedure similar to the generation of households and each employed individual is assigned a specific household. school age children are assigned a school. working individuals are assigned to work places in a different community or census tract in a way that reflects empirical commuting patterns. at this point, we have a fairly accurate description of where the entire population of a city or country lives and works. it is then not entirely surprising that this approach was first used to study in detail the demands imposed on the transportation system of a large metropolitan city. transims, the transportation analysis and simulation system [ ] , used an approach similar to the one described above to generate a synthetic population for the city of portland, in oregon (or) and coupled it with a route planner that would determine the actual route taken by each individual on her way to work or school as a way of modeling the daily toll on portland's transportation infrastructure and the effect that disruptions or modification might have in the daily lives of its population. episims [ ] was the logical extension of transims to the epidemic world. episims used the transims infrastructure to generate the contact network between individuals in portland, or. susceptible individuals are able to acquire the infection whenever they are in a location along with one or more infectious individuals. in this way the researchers are capable of observing as the disease spreads through the population and evaluate the effect that measures such as contact tracing and mass vaccination. more recent approaches have significantly simplified the mobility aspect of this kind of models and simply divide each h period into day time and nighttime. individuals are considered to be in contact with other members of their workplace during the day and with other household members during the night. in recent years, modelers have successfully expanded the large scale agent based approach to the country [ ] and even continent level [ ] . as the spatial scale of the models increased further modes of long-range transportation such as flights had to be considered. these are important to determine not only the seeding of the country under consideration through importation of cases from another country but also to connect distant regions in a more realistic way. flute [ ] is currently the most realistic large scale agent-based epidemic model of the continental united states. it considers that international seeding occurs at random in the locations that host the largest international airports in the us by, each day, randomly infecting in each location a number of individuals that is proportional to the international traffic of those airports. flute is a refinement of a previous model [ ] and it further refines the modeling of the infectious process by varying the infectiousness of an individual over time in the sir model that they consider. at the time of infection each individual is assigned one of six experimentally obtained viral load histories. each history prescribes the individuals viral load for each day of the infectious period and the infectiousness is considered to be proportional to the viral load. individuals may remain asymptotic for up to days after infection during which their infectiousness is reduced by % with respect to the symptomatic period. the total infectious period is set to days regardless of the length of the symptomatic period. given the complexity of the model the calibration of the disease parameters in order to obtain a given value of the basic reproductive ratio, r requires some finesse. chao et al. [ ] uses the definition of r to determine "experimentally" its value from the input parameters. it numerically simulates instances of the epidemic caused by a single individual within a person fully susceptible community for each possible age group of the seeding individual and use it to calculate the r a of each age group a. the final r is defined to the average of the various r a weighted by age dependent attack rate [ ] . the final result of this procedure is that the value of r is given by: where is the infection probability per unit contact and is given as input. flute was a pioneer in the way it completely released its source code, opening the doors of a new level of verifiability in this area. it has successfully used to study the spread of influenza viruses and analyze the effect of various interventions in the los angeles county [ ] and united states country level [ ] . the unprecedented amount of data on human dynamics made available by recent advances technology has allowed the development of realistic epidemic models able to capture and predict the unfolding of infectious disease at different geographical scales [ ] . in the previous sections, we described briefly some successful examples that have been made possible thanks to high resolution data on where we live, how we live, and how we move. data availability has started a second golden age in epidemic modeling [ ] . all models are judged against surveillance data collected by health departments. unfortunately, due to excessive costs, and other constraints their quality is far from ideal. for example, the influenza surveillance network in the usa, one of the most efficient systems in the world, is constituted of just providers that operate voluntarily. surveillance data is imprecise, incomplete, characterized by large backlogs, delays in reporting times, and the result of very small sample sizes. furthermore, the geographical coverage is not homogeneous across different regions, even within the same country. for these reasons the calibration and test of epidemic models with surveillance data induce strong limitations in the predictive capabilities of such tools. one of the most limiting issues is the geographical granularity of the data. in general, information are aggregated at the country or regional level. the lack of ground truth data at smaller scales does not allow a more precise selection and training of realistic epidemic models. how can we lift such limitations? data, data and more data is again the answer. at the end of almost billion of people had access to the internet while almost billion are phone subscribers, around % of which are actively using smartphones. the explosion of mobile usage boosted also the activity of social media platforms such as facebook, twitter, google+ etc. that now count several hundred million active users that are happy to share not just their thoughts, but also their gps coordinates. the incredible amount of information we create and access contain important epidemiologically relevant indicators. users complaining about catching a cold before the weekend on facebook or twitter, searching for symptoms of particular diseases on search engines, or wikipedia, canceling their dinner reservations on online platforms like opentable are just few examples. an intense research activity, across different disciplines, is clearly showing the potential, as well as the challenges and risks, of such digital traces for epidemiology [ ] . we are at the dawn of the digital revolution in epidemiology [ , ] . the new approach allows for the early detection of disease outbreaks [ ] , the real time monitoring of the evolution of a disease with an incredible geographical granularity [ ] [ ] [ ] , the access to health related behaviors, practices and sentiments at large scales [ , ] , inform data-driven epidemic models [ , ] , and development of statistical based models with prediction power [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the search for epidemiological indicators in digital traces follows two methodologies: active and passive. in active data collection users are asked to share their health status using apps and web-based platforms [ ] . examples are influenzanet that is available in different european countries [ ] , and flu near you in the usa [ ] that engage tens of thousands of users that together provide the information necessary for the creation of interactive maps of ili in almost real time. in passive data collection, instead, information about individuals health status is mined from other available sources that do not require the active participation of users. news articles [ ] , queries on search engines [ ] , posts on online social networks [ , [ ] [ ] [ ] [ ] , page view counts on wikipedia [ , ] or other online/offline behaviors [ , ] are typical examples. in the following, we focus on the prototypical, and most famous, method of digital epidemiology, google flu trends (gft) [ ] , while considering also other approaches based on twitter and wikipedia data. gft is by far the most famous model in digital epidemiology. launched in november together with a nature paper [ ] describing its methodology, it has continuously made predictions on the course of seasonal influenza in countries around the world. the method used by gft is extremely simple. the percentage of ili visits, a typical indicator used by surveillance systems to monitor the unfolding of the seasonal flu, is estimated with a linear model based on search engine queries. this approach is general, and used in many different fields of science. a quantity of interest, in this case the percentage of ili visits p, is estimated using a correlated signal, in this case the ili related queries fraction q, that acts as surrogate. the fit allows the estimate of p as a function of the value of q: logit .p/ dˇ cˇ logit .q/ c ; ( . ) where logit .x/ d ln x x ,ˇ andˇ are fitting parameters, and is an error term. as clear from the expression, the gft is a simple linear fit, where the unknown parameters are determined considering historical data. the innovation of the system lies on the definition of q that is evaluated using hundreds of billions of searches on google. indeed, gft scans all the queries we submit to google, without using information about users' identity, in search of those that ili related. this is the paradigm of passive data collection in digital epidemiology. in the original model the authors measured the correlation of millions search queries with historic cdc data, finding that of them were enough to ensure the best correlation between the number of searches and the number of ili cases. the identity of such terms has been kept secret in order to avoid changes in users' behavior. however, the authors provided a list of topics associated with each one of them: were associated with influenza complications, to cold/flu remedies, to general terms for influenza, etc. although the search for the terms has been performed without prior information, none of the most representative terms were unrelated to the disease. in these settings gft showed a mean correlation of : with real data and was able to predict the surveillance value with - weeks ahead. gft is based on proprietary data that for many different constraints cannot be shared with the research community. other data sources, different in nature, are instead easily accessible. twitter and wikipedia are the two examples. indeed, both systems are available for download, with some limitations, through their respective apis. the models based on twitter are built within the same paradigm of gft [ , [ ] [ ] [ ] ] . tweets are mined in search of ili-related tweets, or other health conditions such as insomnia, obesity, and other chronic diseases [ , ] , that are used to inform regression models. such tweets are determined either as done in gft, or through more involved methods based on support vector machine (svm) or other machine learning methods that, provided an annotated corpus, find disease related tweets beyond simple keywords matches [ , [ ] [ ] [ ] ] . the presence of gps information or other self-reported geographical data allows the models to probe different granularities ranging from countries [ , , , ] to cities [ ] . while models based on twitter analyze users' posts, those based on wikipedia focus on pages views [ , ] . the basic intuition is that wikipedia is used to learn more about a diseases or a medication. plus, the website is so popular that is most likely one of the first results of search queries on most search engines. the methods proposed so far monitor a set of pages related to the disease under study. examples are influenza, cold, fever, dengue, etc. page views at the daily or weekly basis are then used a surrogates in linear fitting models. interestingly, the correlation with surveillance data ranges from : in the case of ebola to : in for ilis [ , ] , and allows accurate predictions up to weeks ahead. one important limitation of wikipedia based methods is the lack of geographical granularity. indeed, the view counts are reported irrespective of readers' location but the language of the page can be used as a rough proxy for location. such approximation might be extremely good for localized languages like italian but it poses strong limitations in the case of global languages like english. indeed, it is reported that % of pages views for english pages are done in the usa, % in the uk, and the rest in australia, canada and other countries [ ] . besides, without making further approximation such methods cannot provide indications at scales smaller than the country level. despite these impressive correlations, especially in the case of ilis, much still remains to be done. gft offers a particular clear example of the possible limitations of such tools. indeed, despite the initial success, it completely failed to forecast the h n pandemic [ , ] . the model was updated in september to increase the number of terms to , including the terms present in the original version. nevertheless, gft missed high out of weeks in the season - . in gft predicted a peak height more than double the actual value causing the underlying model to be modified again later that year. what are the reasons underlying the limitations of gft and other similar tools? by construction, gft relies just on simple correlations causing it to detect not only the flu but also things that correlate strongly with the flu such as winter patterns. this is likely one of the reasons why the model was not able to capture the unfolding of an off-season pandemic such as the h n pandemic. also, changes in the google search engine, that can inadvertently modify users' behavior, were not taken into account in gft. this factor alone possibly explains the large overestimation of the peak height in . plus, simple auto-regressive models using just cdc data can perform as well or better than gft [ ] . the parable of gft clearly shows both the potential and the risks of digital tools for epidemic predictions. the limitations of gft can possibly affect all similar approaches based on digital passive data collection. in particular, the use of simple correlations measures does not guarantee the ability of capturing the phenomena across different scales in space and time with respect to those used in the training. not to mention that correlations might be completely spurious. in a recent study for example, a linear model based on twitter simply informed with the timeline of the term zombie was shown to be a good predictor of the seasonal flu [ ] . despite such observations the potential of these models is invaluable to probe data that cannot be predicted by simple auto-regressive models. for example, flu activity at high geographical granularities, although very important, is measured with great difficulties by the surveillance systems. gft and other spatially resolved tools can effectively access to these local indicators, and provide precious estimates that can be used a complement for the surveillance and as input for generating epidemic models [ , ] . the field of epidemiology is currently undergoing a digital revolution due to the seemingly endless availability of data and computational power. data on human behavior is allowing for the development of new tools and models while the commoditization of computer resources once available only for world leading research institutions is making highly detailed large scale numerical approaches feasible at last. in this chapter, we present a brief review not only of the fundamental mathematical tools and concepts of epidemiology but also of some of the state-of-the-art and computational approaches aimed at describing, modeling, and forecasting the diffusion of viruses. our focus was on the developments occurring over the past decade that are sure to form the foundation for developments in decades to come. essai dune nouvelle 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analysis global capacity for emerging infectious disease detection web-based participatory surveillance of infectious diseases: the influenzanet participatory surveillance experience assessing vaccination sentiments with online social media: implications for infectious disease dynamics and control you are what you tweet: analyzing twitter for public health forecasting seasonal outbreaks of influenza forecasting seasonal influenza with stochastic microsimulations models assimilating digital surveillance data the use of twitter to track levels of disease activity and public concern in the us during the influenza a h n pandemic validating models for disease detection using twitter national and local influenza surveillance through twitter: an analysis of the - influenza epidemic towards detecting influenza epidemics by analyzing twitter messages detecting influenza epidemics using search engine query data detecting epidemics using wikipedia article views: a demonstration of feasibility with language as location proxy wikipedia usage estimates prevalence of influenzalike illness in the united states in near real-time guess who is not coming to dinner? evaluating online restaurant reservations for disease surveillance satellite imagery analysis: what can hospital parking lots tell us about a disease outbreak? public health for the people: participatory infectious disease surveillance in the digital age google flu trends using twitter to estimate h n influenza activity a content analysis of chronic diseases social groups on facebook and twitter. telemedicine and e-health reassessing google flu trends data for detection of seasonal and pandemic influenza: a comparative epidemiological study at three geographic scales predicting consumer behavior with web search acknowledgements bg was partially supported by the french anr project harms-flu (anr- -monu- ). key: cord- -kbiwze z authors: zhang, huimin; ravcheev, dmitry a.; hu, dan; zhang, fengyu; gong, xiufang; hao, lina; cao, min; rodionov, dmitry a.; wang, changjun; feng, youjun title: two novel regulators of n‐acetyl‐galactosamine utilization pathway and distinct roles in bacterial infections date: - - journal: microbiologyopen doi: . /mbo . sha: doc_id: cord_uid: kbiwze z bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d‐galactosamine (galn)/n‐acetyl‐d‐galactosamine (galnac) catabolism pathway in bacterial pathogenesis. here, we report the genomic reconstruction of galn/galnac utilization pathway in streptococci and the diversified aga regulons. we delineated two new paralogous agar regulators for the galn/galnac catabolism pathway. the electrophoretic mobility shift assays experiment demonstrated that agar (agar ) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and rna‐seq suggested that agar (not agar ) can effectively repress the transcription of the target genes. removal of agar (not agar ) from streptococcus suis zyh augments significantly the abilities of both adherence to hep‐ cells and anti‐phagocytosis against raw . macrophage. as anticipated, the dysfunction in agar ‐mediated regulation of s. suis impairs its pathogenicity in experimental models of both mice and piglets. our finding discovered two novel regulators specific for galn/galnac catabolism and assigned them distinct roles into bacterial infections. to the best of our knowledge, it might represent a first paradigm that links the galn/galnac catabolism pathway to bacterial pathogenesis. amino sugars are referred to a variety of diversified/complex monosaccharides in which a hydroxyl group is chemically replaced with the amine group. most of current knowledge on the metabolism of amino sugars comes from studies with escherichia coli (reizer et al. ) , bacillus subtilis (freymond et al. ; gaugue et al. ; plumbridge ) and streptomycetes coelicolor (rigali et al. ) . relative to the best-known examples of amino sugars, glucosamine (glcn) and n-acetylglucosamine (glcnac), the investigations on the other two amino sugar derivatives of galactose, n-acetyl-d-galactosamine (galnac) and d-galactosamine (galn), are relatively limited, but increasingly accumulated (abu-qarn et al. ; leyn et al. ) . it seemed very likely that galn/galnac amino sugars as common components participate in the formation of various cell structures/constitutes in three domains of life (abu-qarn et al. ; plumbridge ) . in general, not only does galnac act as an element of lipopolysaccharide displayed on bacterial cell wall (bernatchez et al. ; leyn et al. ) , and but also connects carbohydrate chains in mammalian mucins (carraway and hull ) . additionally, it functions as the substrate in n-acetyl βgalactosidation, a new type of post-translational modification of protein in organisms, including bacterial pathogens (sadler et al. ; barr and nordin ; davis et al. ; abu-qarn et al. ) . given the multiple roles played by galn/galnac, we therefore anticipated a hypothesis that galn/galnac metabolism might be linked to bacterial infectivity. the paradigm pathway for galn/galnac utilization/ catabolism, which was initially proposed for e. coli in (reizer et al. ) , contained the following five steps ( fig. ): ( ) the transport and phosphorylation of galn/galnac substrates (catalyzed by phosphortransferase system [pts] systems agabcd and agavwef, respectively) (brinkkotter et al. ) , ( ) the agaa-mediated deacetylation of galnac- -p (reizer et al. ) , ( ) the deamination/isomerization of galn- -p by the bi-functional enzyme agas into tag- -p (reizer et al. ) , ( ) the agaz kinase-aided phosphorylation of tag- , -p from tag- -p (reizer et al. ) , and ( ) cleavage of tag- , -pp by the class ii aldolase, agay (brinkkotter et al. ) , to produce glyceraldehyde -phospahte and glycerone phosphate (pep). interestingly, a recent comparative genomics-based study suggested an extensive diversity in galn/galnac utilization pathways of proteobacteria such as shewanella (leyn et al. ) . in particular note the first two steps of this pathway exhibit of high variability, while the latter three steps are largely conserved (leyn et al. ) . in e. coli, not only does agar regulator that belongs to the deor family of transcriptional factors, act as an autoregulator, but also negatively controls the expression of two aga genes (agaz and agas) of galn/ galnac catabolism pathway via direct binding of the specific palindromes in front of these target genes (ray and larson ) . although the fact that both galn and galnac can induce activities of these aga genes-encoding protein products is aware, the physiological ligands for agar repressor remain unclear (leyn et al. ) . streptococcus suis, a gram-positive bacterium, is a zoonotic agent with the ability to infect both its natural host swine and human individuals with close contact with swine/porkrelated products . according to the differentiation in their bacterial capsule structure, this streptococcus species is categorized into serotypes (feng et al. , b . among them, serotype (ss ) is generally believed to be most virulent, in that it is frequently isolated from clinical diseased swine (ma et al. ; feng et al. b ) and human sporadic cases (feng et al. b) and/or big-scale outbreaks (tang et al. ; chen et al. ) . we are aware that ss has spread to more than countries/regions and claims nearly cases of human infections worldwide (feng et al. b ). the molecular mechanism underlying bacterial pathogenicity has been partially delineated thus far (feng et al. b) , and these identified virulence determinants include the previouslyknown virulence factors exemplified with capsule (benga et al. ; seitz et al. ) and suilysin (jacobs et al. ; lun et al. ; takeuchi et al. ). in addition to the regulatory networks amongst the newly identified factors (e.g., the two-component systems salk/salr , nisk/nisr [xu et al. ] , ciarh [li et al. ], etc.) , it would be of particular interest to note the contributions of enzymes from central metabolisms (enolase [eno] [esgleas et al. ; feng et al. a; zhang et al. a; lu et al. ] , glutamine synthase [glna] [si et al. ], inosine -monophosphate dehydrogenase [impdh] zhou et al. ], etc.) to bacterial pathogenesis (feng et al. b) . however, nothing is aware regarding the potential relevance of bacterial galn/galnac metabolism and/or its regulation to s. suis infection. in this work, we employed the integrative approaches combining the bioinformatics and comparative genomics to conduct the genomic reconstruction of the galn/galnac utilization pathway in lactobacillaceae, including the zoonotic pathogen s. suis. also, we are the first to report the functional definition of two new gntr-type transcription factors (referred to agar and/or agar ) with involvement of galn/galnac catabolism. more intriguingly, we observed that agar -dependent regulation of galn/galnac utilization pathway is required for bacterial virulence of s. suis serotype . to the best of our knowledge, it represents the first example that the genetic control of galn/galnac catabolism is linked to bacterial infectivity of streptococcus. suis are highlighted with yellow and light-blue background arrows, respectively. galnac, n-acetyld-galactosamine; galn, galactosamine; pts, phosphotransferase system; bgac, βgalactosidase; agaa, galnac- -p deacetylase; agas, galn- -p deaminase/isomerase; tag, tagtose; agaz, tag- -p kinase; agay, tag- , -pp aldolase. here corresponded to the human laryngeal epithelial cell hep- (cctcc gdc ) and the mouse macrophagocyte raw . (atcc tib- , rockville, md), respectively (table s ) (hu et al. ) , and cultivated at °c in the presence of % co in dulbecco's modified eagle's medium with % fetal bovine serum (roche, indianapolis, in, usa), μg/ml gentamycin, and μg/ml penicillin g . the s. suis agar (ssu _ ) gene was amplified using polymerase chain reaction (pcr) with primers ssu _ -f plus ssu _ -r (table s ) and ligated into the bamhi and xhoi sites of pet a(+) expression vector , resulting in the recombinant plasmid pet - (table s ) . similarly, the other expression plasmid pet - was given through direct cloning of s. suis agar (ssu _ ) gene carrying bamhi and sali sites introduced by primers (ssu _ -f plus ssu _ -r) into the expression vector pet a(+) with the same cuts (tables s , s ). the above two plasmids (pet - and pet - ) are designed to prepare in vitro proteins of agar and agar , respectively. for functional complementation, the two genes (ssu _ and ssu _ ) were separately inserted into the low-copy shuttle vector pva (romero et al. ) , giving the plasmids pva - and pva - , respectively (table s ) . all the recombinant plasmids involved in this study were confirmed with both pcr assays and direct dna sequencing. figure . discovery of the genome-wide regulons encoding the galnac/galn utilization pathways in lactobacillales arrows represent the galnac/ galn catabolism-related genes, and circles denote the predicted agar-recognizable sites. the genes are variably colored according to differential functions assigned in figure and labeled with the last letter of the corresponding protein. genes from the same genetic loci that are not adjacent/ neighbored each other are separated by a slash. similarly, genes from different genetic loci are separated by a double slash. given the two types of putative agar-binding palindromes shown on the top (details in table s ), agar and agar sites are accordingly colored with red and blue. sequence logos were given using the weblogo package (http://weblogo.berkeley.edu/logo.cgi). the detailed information on the displayed loci is listed in table s and figure s . galnac, n-acetyld-galactosamine; galn, galactosamine. the two recombinant plasmids (pet a- and pet a- ) were separately transformed into bl (de ), giving the engineered strains fyj , and fyj , respectively (table s ). the two versions of hexahistidine-tagged agar protein (referred to agar [ssu _ ] and agar [ssu _ ]) were produced using the above two engineered strains fyj and fyj , respectively. in brief, when bacterial optical density at wave-length of nm (od ) reached . - . , the bacterial cultures with an appropriate plasmid (table s ) were induced with . mmol/l isopropyl-βdthiogalactopyranoside (iptg) at °c for - h. as we described before (feng and cronan ) , two rounds of french pressure-based lysis were conducted for release of the recombinant protein agar (and/or agar ), and subsequent procedures of protein purification included the nickel column-based affinity purification followed by fast phase liquid chromatography (fplc) feng and cronan ) . consequently, the protein of interest was concentrated via ultrafiltration , and the purity was judged by % sodiumdodecyl sulfate polyacrylamide gel electrophoresis (sds-page). western blot was performed routinely to further verify the hexahistidine-tagged agar (and/or agar ) protein. the protein samples were separated with % sds-page and thereafter transferred to a nitrocellulose membrane (amersham, ge healthcare, piscataway, nj, usa). the primary antibody was an anti-hexahistidine mouse monoclonal antibody, and the secondary antibody was a peroxidase-conjugated goat anti-mouse immunoglobulin g. the signal of target protein band was captured by an exposure to the high-performance chemiluminescence ecl film (amersham, ge healthcare, piscataway, nj, usa). to verify the identity of the recombinant agar (and/ or agar ), the resultant peptides by digestion with sequencing grade trypsin (g-biosciences, st. louis, mo, . ng μl − in mmol/l ammonium bicarbonate) were subjected for analyses of a waters q-tof api-us quad-tof mass spectrometer linked to a waters nano acquity uplc (feng et al. b ). the mass data acquired were assayed through waters protein lynx global server . . , mascot (matrix science, boston, ma, usa) combined with blast against ncbi nr database (feng et al. a ). the nickel column-based purified agar (and/or agar ) protein was further assayed by gel filtration chromatography using a superdex column (amersham, ge healthcare, piscataway, nj, usa) run on an Äkta fast protein liquid chromatography system (ge healthcare) as described figure . maximum-likelihood phylogenetic tree of agar proteins. agar (ssu _ ) is given in red, and agar (ssu _ ) is highlighted in blue. the nagr proteins from bacillus subtilis, streptococcus suis, and lactobacillus plantarum are used as outtree. the logos for the predicted palindromes recognized by agar (agar ) is presented on the right hand. all the homologs of agar (agar ) is accessed to table s . nagr proteins from b. subtilis (bertram et al. ) , l. plantarum wcfs , and s. suis zyh were used as an outgroup. nagr was selected because, as like agar and agar , this protein is a member of the hutc subfamily of the gntr family and a regulator of aminosugar metabolism. previously cronan , ) . the column effluent was evaluated at a flow rate of . ml/min in pbs buffer ( mmol/l na hpo , mmol/l kh po , mmol/l tris-hcl, mmol/l nacl, . mmol/l kcl, ph . ). the protein peak at the position of expected elution volume was sampled and verified by % sds-page. to further elucidate the solution structure of agar (and/or agar ) protein from s. suis, we conducted chemical cross-linking experiments in which ethylene glycol bis-succinimidylsuccinate (egs) (pierce, rockford, il, usa) was added . in each reaction system ( μl in total), the interested protein (~ mg/ ml) was separately incubated with the egs cross-linker (at different levels [ . , . , . , . , . , , , μmol/l for agar protein; , . , , , μmol/l, for agar protein]) for h at room temperature. finally, the reaction products were visualized via % sds-page followed by commassiee brilliant blue staining. the function of the predicted agar and agar binding sites were proved using electrophoretic mobility shift assays (emsa) as we established earlier (feng and cronan ; feng et al. a,b) with minor modifications. all the doublestrand dna probes were produced in vitro by annealing two complementary oligonucleotides in ten buffer ( mmol/l tris-hcl, mmol/l ethylene diamine tetraacetic acid (edta), mmol/l nacl; ph . ), and labeled with dig-ddutp (roche, indianapolis, in, usa) by the terminal transferase cronan , ) . three agar specific dna probes used here included ssu _ / site probe ( bp), ef site probe ( bp), and ef site probe ( bp). in addition to ssu _ probe ( bp) as the negative control, the other four tested agar recognizable sites corresponded to ssu _ probe ( bp), ssu _ probe ( bp), ssu _ / site ( bp), and ef site probe ( bp) (table s ). after min of incubation of the dig-labeled dna probes ( . pmol) with or without agar (and/or agar ) protein in the binding buffer (roche) at room temperature, the dna-protein complexes were separated by the native % page gel and transferred onto an equilibrated, positively charged nylon membrane (roche) by contact blotting followed by uv cross-linking ( mj for sec) (feng and cronan ; feng et al. b ). finally, the signals were captured by exposure to the high-performance chemiluminescence film (amersham hyperfilm ecl) cronan , ) . mid-log phase cultures of s. suis strains (wild type [wt], Δagar , Δagar , cΔagar and cΔagar ) grown in thb media (with/without galnac- p) were collected to prepare the total bacterial rna using an rneasy bacterial rna isolation kit (qiagen, hilden, germany) li et al. ). as we performed before cronan , ) , rna integrity/quality was validated by separation of . % agarose gel electrophoresis. the possible contamination of trace genomic dna in the rna samples was ruled out by pcr-based detections, using the total rna as the template (feng and cronan . using the qualified rna preparations, complementary dnas (cdnas) were synthesized by reverse transcription (rt). then, the real-time quantitative pcr (qpcr) combined with the sybr green method feng and cronan ) was carried out to probe possible relevance of agar (and/or agar ) to the altered expression profile of genes encoding galnac utilization pathway. the method of −ΔΔct ( ) was applied to determine the relative level of the target genes associated with galnac utilization in which the s rrnaencoding gene s rdna as the internal reference (table s ) . target genes tested here are agas (ssu _ ), figure . binding of agar to the predicted cognate sites. alignment of the agar -binding sites from species of lactobacillales (a) and the resulting sequence logo (b). in (a) the identical residues are white letters in red background, similar residues are black letters in yellow background, and varied residues are in black letters. names and locus tags for genes from streptococcus suis are shown by red font. in (b) the sequence logo is generated using weblogo (http://weblogo.berkeley.edu/logo.cgi). the detailed information of the cognate sites is seen in table s . (c) agar does not bind its own promoter. (d) binding of agas (ssu _ ) promoter to agar proteininteraction of agar protein with the promoter regions of both agaa (ssu _ ) (in e) and bgac (ssu _ ) (in f) agar protein from streptococcus suis cannot bind to the agar site of ef of enterococcus faecalis v (in g), whereas it binds to its agar site (in h). the minus sign denotes no protein of agaa added. the protein levels of agar (in the right hand four lanes of each panel [left to right]) were . , , and pmol. the protein samples were incubated with . pmol of dig-labeled probe in a total volume of μl. a representative result from three independent gel shift assays ( % native page) is given. agaay (ssu _ / ), bgac (ssu _ ), and gad-vwef (ssu _ , ssu _ , ssu _ and ssu _ ), respectively. the agar (or agar ) gene from the s. suis strain zyh was replaced with the spectinomycin resistance (spc r ) cassette by homologous recombination hu et al. ) . briefly, the spc r cassette from pset (takamatsu et al. ) was cloned into the puc vector (invitrogen) to give the intermediate plasmid puc -spc (table s ) , and then the two dna fragments adjacent to the agar (or agar ) gene were separately inserted into the puc -spc vector, giving the knockout plasmid puc:: and puc:: , respectively (table s ). as we did before li et al. ) , the plasmid of puc:: (or puc:: ) was electroporated into the competent cells of s. suis zyh to acquire positive transformants with spc r . the multiplex-pcr techniques were adopted to screen the Δagar (and/or Δagar ) mutant (table s ) . consequently, the mutants we acquired for functional experiments were further proved by direct dna sequencing. the two plasmids of pva - and pva - were separately transformed into Δagar and Δagar mutants to give the complemented strains cΔagar and cΔagar , respectively (table s ) . as hytönen et al. (hytonen et al. ) reported, s. suis bacteria (wt, Δagar and cΔagar ) grown in the midlog phase were subjected to cell lines-based analyses. the two cell lines are hep- (human laryngeal epithelial cell line) and murine macrophage raw . cells hu et al. ). bacterial adherence was tested with hep- cell line, and the evaluation for ability of antiphagocytosis was based on the raw . cells. to reveal the role of agar (and/or agar ) in bacterial pathogenesis/virulence, two different kinds of experimental animals were employed, including balb/c ( -week old, female) mice and spf-piglets. in the infection experiment of mice, totally animals were challenged that are classified into six groups ( mice/group). except that thb acted as a negative control, the other five groups infected with s. suis (at a dose of × cfu per mouse) corresponded to wt, Δagar , cΔagar , Δagar , and cΔagar , respectively. the result obtained from the experiment of mice infection was further checked using the infection test of piglets, its natural host of s. suis. given the fact that only agar (not agar ) plays a role in bacterial infectivity, three groups of piglets (six piglets/group) were rechallenged by wt, Δagar , and cΔagar , respectively. clinical syndromes of the infected mice/piglets were monitored for h. of particular note, deaths were recorded and moribund animals were humanely killed. all experiments on live vertebrates in this study were approved by the ethics figure . galnac/galn utilization pathway is repressed by agar and induced by addition of galnac. (a) schematic diagram for agar and its cognate target genes.circles denote the agar -recognizable sites, and the arrows represent the genes of the galnac utilization pathway. note: ssu _ and ssu _ constitutes an operon agaay, whereas the four ordered genes (ssu _ , ssu _ , ssu _ , and ssu _ ) might form an operon of bgac-gadvwef. (b) real-time qpcr assays for effects of agar on expression profile of genes of galnac/galn utilization pathway in streptococcus suis. (c) addition of galnac increases expression level of galnac/galn utilization pathway-encoding genes in s. suis.the data are expressed as averages ± sd, and error bars mean sd. no less than five independent experiments were carried out here. the minus sign denotes no addition of galnac into growth media, whereas the plus sign denotes addition of galnac in vitro. note: p < . . galnac, n-acetyld-galactosamine; galn, galactosamine; qpcr, quantitative pcr; sd, standard deviations. command and performed in accordance with the relevant guidelines and regulations (cao et al. ; hu et al. ) . genome sequences were downloaded from the microbesonline genomic data base (dehal et al. ). identification of orthologs was performed using the blastp search in the non-redundant database (altschul et al. ) and microbesonline tree browser. for functional protein annotations by distant homology to characterize proteins, blast search in the swissprot/uniprot database was used. analysis of chromosomal gene clustering was performed by microbesonline and seed web resources (dehal et al. ; disz et al. ). the galnac utilization subsystem curation and analysis were conducted, using the seed platform (disz et al. ) . protein domains were determined by protein similarity search tools in the pfam database (sonnhammer et al. ) . multiple sequence alignments were constructed by either clustalw (http://www.ebi.ac.uk/tools/clustalw /index.html) (thompson et al. ) or muscle (edgar ) . phylogenetic trees were constructed using the maximum likelihood algorithm implemented in the phylip package (felsenstein ) and visualized via the dendroscope tool (huson et al. ). sequences logos were constructed using weblogo package (crooks et al. ) . for genomic reconstruction of the regulons, we used the well-established comparative genomics approach (rodionov ) . the approach includes inference of transcriptional factor-binding sites (tfbss), construction of nucleotide positional weight matrices (pwms) for tfbss motifs, and reconstruction of regulons in complete genomes on the basis of prediction of putative tfbss in the promoter gene regions. first, in the studied lactobacillales genomes, we revealed orthologs of previously known genes for galnac utilization (table s ). second, we predicted possible transcriptional regulators for the uncovered galnac utilization. candidate regulators were attributed to the regulons by using a genomic colocalization and co-occurence of a putative regulator with the identified galnac utilization genes. such analysis defined two groups of transcriptional regulators belonging to the hutc subfamily of the gntr family (fig. ) . for each group of agar proteins, we identified putative binding motifs analyzing upstream regions of presumably regulated gene by the discover profile tool implemented in the regpredict web server (novichkov et al. ) . in this approach, putative tfbss were determined as overrepresented words in upstream regions of putatively coregulated genes. based on the genomic co-occurrence and co-presence of genes, it has been proposed that agar binding sites should be located upstream hydrolase and pts genes while agar sites ought to be upstream of deacetylase, isomerase, and aldolase genes. for the prediction of putative tfbss, we analyzed upstream regions of probably related operons, expanding − to + bp relative to start codon of the first gene of the operon. both agar and agar belong to hutc subfamily. tfbss for hutc subfamily proteins have structure of an even palindrome (rigali et al. ; suvorova et al. ) . thus, for prediction of the agar and agar tfbss motifs, we searched for even palindromic dna motifs of - bp. among all motifs found for each set of upstream regions, we selected the longest motif with the highest information content. the selected motif was quality controlled by two approaches, ( ) consistency check and ( ) phylogenetic footprinting. consistency check (mironov et al. ; rodionov ) was done, that is, motif was checked for the presence in multiple number of genomes. high quality motif should find predicted tfbss in upstream regions of predicted regulated operons in genomes having the analyzed regulator, but not in genomes lacking the regulator. phylogenetic footprinting technique is based on analysis of multiple alignments for upstream regions of orthologous genes (shelton et al. ). high quality motif should find predicted tfbss that are located inside conserved islands of multiple alignments. when the high quality of the motif was confirmed by both consistency check and phylogenetic footprinting, the identified regulatory motifs were used for the construction of the pwms (profiles) and the obtained matrices were used to determine additional candidate regulatory sites in the analyzed genomes, using the run profile tool in the regpredict web server. the scores of sites were calculates as a sum of nucleotide weights for each position. the data used here were expressed as mean ± sd. unless specified, data were analyzed by two-tailed, unpaired t test, and all assays were repeated no less than three times. the threshold for significance refers to the p < . . the galnac utilization pathway has been previously reconstructed by the comparative genomics approach in a large number of proteobacteria species (leyn et al. ) and experimentally analyzed in e. coli (brinkkotter et al. ) and shewanella sp. ana- (leyn et al. ). here we used the same approach for reconstruction of the galnac utilization pathway in the zoonotic agent, s. suis ( fig. and table s ). for integrated genomic reconstruction of galnac utilization pathways and concordant transcriptional regulation, we searched orthologs of known galnac utilization genes (leyn et al. ) in complete lactobacillales genomes. as a result, the genes encoding, this metabolic pathway was identified in genomes representing four families, streptococcaceae, lactobacillaceae, enterobacteraceae, and carnobacteriaceae (table s ). the minimal number of genes in the reconstructed regulons was detected in lactobacillus helveticus and streptococcus pyogenes. in these organisms, only one or two genes from the galnac utilization pathway were found because these genes were insufficient for galnac utilization. most probably, these organisms cannot utilize galnac. the minimal gene set allowing utilization of galnac is present in a group of closely related genomes including streptococcus gordonii, streptococcus mitis, and streptococcus pneumonia. this gene set contains genes for regulator (agar), galactosamine- -phosphate deaminase/isomerase (agas), glycoside hydrolase (bgac), and pts (gadvwef). the other well-studied streptococcus genomes also contain genes for the tagatose- , -diphosphate aldolase (agay). in contrast, all the analyzed lactobacillus lack agay gene but contain the gene for n-acetylgalactosamine-phosphate deacetylase (agaa). amongst streptococcaceae, agaa gene was identified only in the s. suis genome. the gene for tagatose- -phosphate kinase (agaz) was found only in enterococcus faecalis and carnobacterium sp. - . previously agay gene was shown to be not obligatory for the galnac utilization and function of agaz was proposed to be actualized by other genes, such as lacc (ec . . . ) or pfk (ec . . . ). orthologs of these genes were found in the studied genomes, for example in s. suis lacc is encoded by gene with locus tag ssu _ and pfk is encoded by ssu _ . the presence or absence of agaa gene is the crucial point for the ability or disability to utilize galnac. thus, the absence of this gene in the aga regulons points to the ability of the organism to utilize only galn, but not galnac utilization. thus, organisms lacking agaa gene should have galn-specific transporters whereas organism having agaa gene should be able to transport galnac (leyn et al. ) . to predict the specificity of the lactobacillaceae transport systems, we compared all the identified ptss to the previously described ones. the phylogenetic analysis revealed that all the lactobacillaceae ptss are orthologous to the transport system from haemophilus parasuis (fig. s ). this pts was previously proposed to be specific to galn-containing oligosaccharides. co-occurrence and co-localization of this pts with the hydrolase genes in the most studied genomes confirms its specificity to the oligosaccharides. on the other hand, pts genes are co-localized also with the agaa gene in the large number of studied genomes (table s ) that designate to the possibility of this system to transport galnac or both galnac and galn containing oligomers. however, we cannot have success in predicting the precise specificity of the gad-vwef encoded pts, using only the comparative genomics approach. for the integrated genomic reconstruction of galnac utilization pathways and the concordant transcriptional regulation, we searched orthologs of known galnac utilization genes (leyn et al. ) in complete lactobacillales genomes. as a result, the genes encoding this metabolic pathway were identified in genomes representing four families, streptococcaceae, lactobacillaceae, enterobacteraceae, and carnobacteriaceae (table s ). analysis of conserved chromosomal loci with the galnac utilization genes detected the presence of one or two suggested transcriptional regulators per genome ( fig. and table s ). all predicted regulators belong to the gntr family of transcription factors (hoskisson and rigali ). phylogenetic analysis revealed that the predicted regulators form two separate orthologous groups that we named agar and agar (fig. ) . a strong tendency of agar and agar genes to cluster onto the chromosome with the galnac utilization genes suggests conservation of their function (fig. ) . two lactobacillaceae and four streptococcaceae genomes have both agar and agar genes, whereas in other genomes only one of the regulator genes was present ( fig. and table s ). to infer the agar /agar regulons in lactobacillales, we used the comparative genomics approach applied in the regpredict web server. this approach combines prediction of candidate regulator-binding sites with crossgenomics comparison of regulons. since agar and agar protein orthologous groups are distantly related to each other ( % identity), we propose that their binding motifs should also be different. in most analyzed genomes, agar is co-localized with genes for glycoside hydrolase and pts, whereas agar tends to be clustered onto the chromosome with the genes for deacetylase, isomerase, and aldolase ( fig. and table s ). thus, we proposed that agar -binding sites should be located upstream hydrolase and pts genes while agar sites ought to be upstream of deacetylase, isomerase, and aldolase genes. upstream regions of genes presumably regulated by each transcriptional factor were analyzed using discover profile tool of the regpredict web resource. after the identification of putative-binding motif, we searched for additional regulatory sites in the analyzed genomes and finally reconstructed agar and agar regulons. the candidate motifs for both the studied regulators have an even palindrome structure (figs. , a and table s ) that is in good agreement with previous observations on binding motifs for proteins of the hutc subfamily of gntr family (hoskisson and rigali ). predicted agar -binding sites have a length bp, whereas the predicted-binding sites for agar are bp sequences ( fig. a and table s ). both agar and agar -binding motifs have a palindrome at-rich central part. the agar -binding motif is at-rich at all times and quite degenerated, that is, has moderate information content. on the other hand, the agar -binding motif is more cg-rich and conserved and has a higher information content than agar -binding motif. additionally, agar -binding motif demonstrate similarities with the motifs of some regulators, previously characterized experimentally or in silico, such as gnbr of lactobacillus casei bl (bidart et al. ) and nagr proteins of b. subtilis (bertram et al. ; leyn et al. ) and various lactoacillales (ravcheev et al. ) . composition of the agar and agar regulons varies between species (fig. ) . in lactobacillus rhamnosus (l. rhamnosus), enterococcus faecalis (e. faecalis), and carnobacterium sp. - , all genes for galnac utilization are organized in a single operon that is regulated by a single regulator, agar in the first two genomes and agar in the last one ( fig. and table s ). in the most part of the streptococcaceae and lactobaciilaceae genomes, we observed a strong "division of labor" between the two regulators (fig. ) . thus, agar -binding sites were found upstream of hydrolase/pts genes, whereas agar looks to regulate genes for deacetylase/isomerase/aldolase (fig. ) . in the genomes of s. suis, lactobacillus gasseri (l. gasseri) and lactobacillus johnsonii (l. johnsonii), overlapping of regulons was detected (fig. ) . in all these genomes, genes for the transport system seemed likely to be under double regulation by agar and agar . also, in the vast majority of the analyzed genomes autoregulation was identified for both agar and agar genes (table s ) . to probe the putative function of the two new members (s. suis agar and agar ) of the gntr family of transcription factors, we employed bl (de )/pet (a) a prokaryotic expression system to prepare the above two proteins in vitro. consequently, the n-terminal hexahistidine tagged s. suis agar protein was purified to homogeneity and gave a single-protein band of appropriate molecular mass (~ kda for monomer) (fig. a) . the xhis tagged version of this recombinant s. suis agar protein was also determined by western blot using the anti- xhis tag primary antibody (fig. b) . chemical cross-linking assays with the egs cross-linker visualized clearly the egs dose-dependent dimerization of agar , indicating its predominant solution structure of this protein is a dimer (fig. c) . liquid chromatography mass spectrometry-based determination of tryptic peptides of the recombinant agar protein band excised from an sds-page gel (fig. a ) verified its identity, in that the peptides matched s. suis ssu _ protein with % coverage of the expected peptides (fig. d) . somehow different from the agar protein, two forms of agar protein (monomer, the predominant form, and trace amount of dimer) still can be detected by the separation with % sds-page (fig. s a) . given that two possibilities are present (either the contaminated protein with the molecular mass at the dimeric position, or the dimeric form of agar ), western blotting with the anti- xhis tag primary antibody was conducted. as anticipated, it clearly showed that two protein bands with an appropriate molecular mass (~ kda for monomer and ~ kda for dimer), ruling out the possibility of protein contamination (fig. s b ). this observation is unexpected, but not without precedent. in fact we recently encountered a similar scenario in the case of the brucella bior regulator that is also a member of the gntr family transcription factor (feng et al. a ). the essence of bior forming a dimer was further proved by chemical cross-linking assays (fig. s c) . ms-based analyses of two protein bands of agar (one is cut from a dimer, the other is collected from a monomer) validated exactly the identity wherein the two forms of peptides covered s. suis ssu _ protein at the level of no less than % ( fig. s d and e). it seemed very likely that the genome of s. suis zyh (accession no.: cp . ) encodes a fully functional galnac utilization machinery and most of the genes encoding this pathway are regulated by agar (and/or agar ) (fig. ) . thereby, we employed emsa to probe the binding of the agar (and/or agar ) protein to the cognate palindromes (table s , fig. a and b) . on the s. suis chromosome, totally three putative agar -binding sites (ssu _ probe, ssu _ probe and ssu _ / site probe) were localized, whereas only one possible agar -recognizable site (ssu _ / site probe) was detected. because of the absence of the predicted agar binding site upstream of the agar gene, we expected that this gene is not autoregulated. indeed, emsa experiments suggested that agar protein cannot bind to its own promoter region (ssu _ probe), ruling out the possibility of autoregulation by agar regulator (fig. c ). by contrast, emsa tests revealed clearly that agar protein bound the other three agar -binding palindromes in a dose-dependent manner (fig. d-f) . additionally, we also used s. suis agar protein to evaluate the function of the two agar-binding sites in front of the ef locus of e. faecalis v , a close relative of s. suis. as a result, agar physically interacted with the ef site , ttgtggttataaccagtt (fig. h) , but not the ef site , tttattgacaaaataaaaaa (fig. g) further validated the specificity of agar binding. different from the scenario with agar , s. suis agar was only found to have the ability to interact with the predicted ssu _ / site , tctattaatatactaacact (fig. s a ). as anticipated from the comparative genomic analysis of the regulons, no interplay was observed between the agar -specific ssu _ / site , ttgtggttatgtccagta and s. suis agar protein (fig. s b) , ruling out the possibility of cross-regulation by agar and agar . more importantly, the two putative agar recognizable sites from e. faecalis v (referred to ef site , aaattcaatatattaagata and ef site ) were demonstrated to be functional, using gel shift assays with the s. suis agar protein ( fig. s c and d) . given the observation that agar efficiently binds to the promoter regions of the three genes/operons (agas (ssu _ ), agay (ssu _ ), and bgac (ssu _ )) encoding galnac utilization pathway in s. suis (figs. , a), we therefore attempted to elucidate its possible function in modulating streptococcus galnac utilization machinery. the Δagar (Δ ) isogenic mutant was constructed, using an approach of homologous recombination, and its complementary strain cΔagar (cΔ ) was generated through the low-copy plasmid pva -borne expression of agar gene (table s ). we noted that the deletion of agar does not affect its growth (fig. s a) . additionally, no obvious alteration of capsule was observed in the Δ agar mutant in comparison with the wt strain zyh (fig. s b and c) . the qpcr-based transcriptional analyses showed that removal of agar gene gave at least five-fold increment of agas (agaay and bgac-gadvwef operon) expression (fig. b) . whereas the transcription of the above target genes was restored to the level seen in its parental strain s. suis zyh (fig. b) . similar results were also observed in altered expression profile of the Δagar mutant revealed by the rna-seq (e.g., expression of the three genes ssu _ (agas), ssu _ (agaa), and ssu _ (agay) are elevated upon removal of agar , table ). therefore, it seemed true that agar is a functional repressor for the streptococcus galnac utilization pathway. in contrast, we failed to visualize any significant alteration of the bgac (ssu _ ) expression level in the Δagar mutant in comparison with the wt strain and its complementary strain cagar (not shown). the suggested possibility that agar might play an uncovered role or be an evolutional relic with loss of physiological demand and/or advantage, in that ( ) there is no in vivo regulatory role attributed to its protein product agar although it retains dna-binding activity to its own promoter, ( ) the fact that all the other galnac metabolism-related genes are without control by agar is due to the lack of the predicted agar -recognizable palindromes, ( ) no physiological function can be present in galnac metabolism, even agar can autoregulate itself. thus, our subsequent interests focused on agar regulator. considering the direct relevance of agar to galnac utilization in s. suis, it is reasonable to probe the possibility whether this regulator acts to be responsive to the presence of galnac. we thereby grew wt strain s. suis zyh in the medium with/without the supplementation of galnac, and compared the expression profile of relevant target genes like agas. as anticipated, the expression level of all the tested galnac catabolism pathway-encoding genes (including agas and bgac) was given a -to -fold increment upon the addition of mmol/l galnac into growth conditions (fig. c) . together, we concluded that the galnac/galn utilization pathway is repressed mainly by the agar regulator, whereas induced by an addition of galnac. contribution of agar (agar ) to bacterial pathogenesis as we knew that amino sugars (galn and/or galnac) constitute common residues for surface structure of various bacterial cell walls (bernatchez et al. ; freymond et al. ) , we thus ambitiously reasoned that ( ) such a bacterial surface structure might be involved in mutual communication/crosstalk between bacterial pathogens and the inhabited/infected hosts, ( ) the maintenance and regulation of galn/galnac catabolism is probably implicated into successful infections of some bacterial pathogens. to probe possible interference between galn/galnac catabolism and the formation of virulence-associated surface structures, we systemically employed two lines of approaches including cell lines-based tests and infections of experimental animals. first, hep- cell line was used to evaluate the ability of bacterial adherence ( fig. a and b) , and raw . macrophage was subjected to dissecting its capability of anti-phagocytosis ( fig. c and d) . fluorescence-activated cell sorting (facs)-based experiments elucidated that around threefold increment of bacterial adherence potential to hep- cells was given in the Δagar mutant relative to the wt zyh strain (fig. a and b) . furthermore, the deletion of agar gene was found to give nearly fivefold increment of anti-phagocytosis ability against raw . macrophage ( fig. c and d) . this finding was in much similarity to scenarios seen with both Δneub and Δcps b mutants of s. suis (note: the two genes are involved in bacterial surface architecture) . thereby, we anticipated that the enhancement in abilities of bacterial attachment and anti-phagocytosis might be partially due to the altered carbohydrates with galn/ galnac residues on bacterial cell wall surface and this kind of alteration could be attributed to the dysfunction in agar -mediated regulation of galn/galnac utilization pathway. in contrast, no obvious difference between the Δagar mutant and its parental strain zyh was observed on either bacterial adherence to hep- cell line or bacterial anti-phagocytosis against raw . macrophage (not shown). in fact, such a different effect exerted by agar (and agar ) is not very surprising, in that only aga (not agar ) evolved to possess a regulatory role in controlling galnac catabolism in s. suis. given the fact that not only the two mutants (Δneub and Δcps b) of s. suis feature the potential of both increased adherence/anti-phagocytosis, but also exhibit dramatically reduced virulence in infection models of both mice and piglets , it is of much interest to further probe a possible role of agar in bacterial virulence. in the mice infection assays, all the balb/c ( week old, female) mice infected with the wt strain zyh were sick shortly, and most of the rats died within h (fig. e ). by contrast, mice of the negative control group inoculated with thb survived (not shown). in particular note, nearly all the mice infected with the Δagar mutant survived, while most of animals injected with the complementary strain cΔagar died within days. to verify the results obtained from the above model of mice, we repeated the infection tests using the model of spf-piglets, natural hosts for s. suis. as a result, we observed that the six spf-piglets inoculated with wt virulent strain developed most of the typical disease symptoms (high fever, limping, swollen joints, etc.), and most of them died on day (fig. f) . as expected, all the piglets infected with the Δagar mutant survived during the period of entire experiment. however, the re-introduction of agar gene into the Δagar mutant restored fully its strong virulence (fig. f) . collectively, we believed that the prevalent regulator agar for galn/galnac utilization pathway contributes to bacterial infectivity of s. suis, although we failed to note an apparent role of the other secondary regulator agar in bacterial pathogenicity (not shown). the data shown here represents a first report that illustrated the genomic reconstruction of galn/galnac catabolism pathway in streptococci and firmicutes. different from the scenarios described in e. coli (reizer et al. ) and shewanella (leyn et al. ), a significant variation of this pathway was proposed for streptococci ( fig. and table s ). as you can see from the working model, agaz is absent in s. suis. it seemed likely that our continued genomic analyses pointed out that s. suis actually lacks any ortholog of this gene. thereby, we speculated that the loss of agaz function in s. suis might be compensated by two other genes that are not in aga regulon: ( ) ssu _ (lacc ; tagatose -phosphate kinase, ec . . . ), ( ) ssu _ (pfk, -phosphofructokinase, ec . . . ). this hypothesis required further experimental verification. through extensive analyses for genomic contexts from firmicutes, we dissected two galn/galnac pathwayspecific regulators namely agar and agar . the agar gene is divergently transcribed into genes forming an operon and encoding a βgalactosidase (bgac) (hu et al. ) and a pts sugar uptake system (named agabcde that could be a galn uptake system). the agar gene in many streptococcus species is located in front of the genes encoding the predicted galactosamine- -phosphate isomerase and tagatose , -diphosphate aldolase, two key enzymes in the aga catablic pathway. in s. suis, the locus ssu _ is annotated to be agar , whereas ssu _ is referred to agar . somewhat evolutionally distinct from the paradigm regulator (the e. coli agar protein product) that belongs to the deor family of transcription factor, the two newly identified regulators agar and agar are classified into the gntr family of transcription repressors (figs. , s ) . additionally, it seemed likely that the two gntr-like regulators (agar and agar ) themselves fit into two different subgroups and might possess the varied preference in their dnabinding sites (figs. , s ) . the fact that agar -recognizable palindrome is predicted to be present in front of multiple target genes whereas agar has only one site located in its own promoter region (fig. ) raised the possibility that agar could have been evolved into a prevalent/ major regulator for galn/galnac utilization pathway (agar might be a minor/and even a cryptic regulator). our in vitro gel shift assays validated the direct interaction between the agar (and/or agar ) protein and its binding sites (figs. , s ). given the facts that a significant regulated expression of galn/galnac utilization-related genes (such as agas, agaay , and bgac) by agar was observed, however, no in vivo role could be attributed to agar , we came to be more confident that agar relative to agar acts as a prevalent/leading player in modulating expression of genes encoding bacterial galn/ galnac catabolism in most firmicutes. however, we did not obtain evidence for physiological ligand/molecular effector of agar repressor, although we observed that galnac does induce the expression of galn/galnac catabolism-related genes such as agas (fig. c) . the essence that amino sugars (galn and/or galnac) are common components on the surface of bacterial cell walls (bernatchez et al. ; freymond et al. ) has driven us to test possible roles for the regulation of galn/ galnac catabolism in the infectivity of bacterial pathogens. as expected, an interference of galn/galnac catabolism/ utilization by disrupting agar encoding a prevalent regulator, significantly altered abilities of both bacterial adherence to hep- cells and anti-phagocytosis against raw . macrophage (fig. ) . given the fact that in this alteration we observed a similar scenario seen with the two virulence-associated determinants (neub and cps b) with biological roles in constitution/development of bacterial surface architecture , we reasoned that regulation/maintenance of galn/galnac catabolism pathway is essential for full virulence of s. suis. in fact, this proposal was subsequently proved in that the dysfunction in agar -mediated regulation impairs bacterial infectivity of s. suis in both mice and piglets (fig. ) . also, we are not surprised to establish the relevance of galn/galnac catabolism to bacterial pathogenicity, in that the alteration of carbohydrates with galn/galnac residues on bacterial cell surface interferes with the bacterial pathogen-binding host cells. taken together, we are first to report the genomic reconstruction of the galn/galnac utilization pathway in s. suis and its novel regulatory network with variations. more intriguingly, we revealed that the interference of galn/galnac utilization pathway by the inactivation of the agar regulator attenuates greatly the bacterial infectivity of the zoonotic pathogen s. suis. our finding might provide a metabolic basis for design of small molecule drugs (inhibitors)-based therapeutics against s. suis infection through targeting the regulatory network of galn/ galnac utilization pathway. additional supporting information may be found in the online version of this article: table s . strains and plasmids used in this study. table s . dna primers used in this study. table s . agar regulons in firmicutes. table s . agar (agar ) binding sites. figure s . multiple sequence alignments of ssu _ (agar ) with two other bacterial homologs. the three homologous proteins used here included bacillus subtilus nagr (nc_ . ), ssu _ (agar ) of streptococcus suis zyh (nc_ . ), and escherichia coli agar (nc_ . ). the program of clustalw (http://www. ebi.ac.uk/tools/clustalw /index.html) was applied to conduct the multiple alignment of protein sequences, and the final output is generated by the espript . program (http:// espript.ibcp.fr/espript/cgi-bin/espript.cgi). identical residues are in white letters with a red background, similar residues are in red letters with a white background, varied residues are in black letters, and dots represent gaps. the predicted protein secondary structure is given on the top. designations: nagr, n-acetylglucosamine repressor; agar, acetyl-galactosamine repressor; bs, bacillus subtilus; ec, e. coli, α, αhelix; β, βsheet; t, βturns/coils. figure s . purification, verification, and characterization of the agar (ssu _ ) protein. (a) % sds-page profile of the purified agar (ssu _ ) protein from streptococcus suis. (b) western blot analyses for the nterminal x his tagged agar protein, using the anti- xhis tag primary antibody. the monomeric protein with expected size of ~ kda is indicated with an arrow, whereas the dimer form (~ kda) is highlighted with an asterisk. designations: m, protein standard marker; wb, western blot. (c) determination for the solution structure of the agar protein, using chemical cross-linking assays. the chemical cross-linker used here is ethylene glycol bis-succinimidylsuccinate (egs). the triangle on the top represents the addition of the egs cross-linker in varied concentrations ( . , . , . , . , . , , , μmol/l in the right-hand eight lanes [left to right]). the minus sign denotes no addition of egs. the protein sample was separated with % sds-page. the ms-based identification of the purified agar protein with the solution structure of both monomer (in d) and dimer (in e). the tryptic peptides that match the agar protein are given in bold and under-lined type. figure s . binding of agar to the predicted palindromes. the predicted agar -binding site (in a) of ssu _ / gene bound agar (ssu _ ) protein, whereas the agar -binding site (in b) of this locus does not interact with agar protein. (c) streptococcus suis agar protein bound to the predicted agar site of ef gene from enterococcus faecalis v . (d) s. suis agar protein bound to the putative agar site of ef gene from e. faecalis v . the minus sign denotes no addition of agaa protein. the protein levels of agar (on the right-hand four lanes of each panel [left to right]) were . , , , and pmol. the protein samples were incubated with . pmol of the dig-labeled probe in a total volume of μl. a representative result from three independent gel shift assays ( % native page) is given. figure s . phylogenetic analyses of phosphotransferase system (pts). in total, the pts system is classified into five sub-groups, one of which is pts-v (highlighted in blue). locus tag of pts system is showed here for agac, ssu _ is indicated in red. not just for eukarya anymore: protein glycosylation in bacteria and archaea gapped blast and psi-blast: a new generation of protein database search programs biosynthesis of glycoproteins by membranes of acer pseudoplatanus. incorporation of mannose and n-acetylglucosamine polysaccharide capsule and suilysin 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serotype this work was supported by the start-up package of zhejiang university (youjun feng), the zhejiang provincial natural science foundation for distinguished young scholars (grant no. lr h ), the national natural science foundation of china (grant no. , , , , , and ), the russian academy of sciences (program "molecular and cellular biology".), the national research fund (# ), luxembourg, and was co-funded under the marie curie actions of the european commission (fp -cofund). dr. feng is a recipient of the "young talents" award. none declared. key: cord- -gqbtkf x authors: lee, sunhee; kim, youngnam; lee, changhee title: isolation and characterization of a korean porcine epidemic diarrhea virus strain knu- date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: gqbtkf x severe outbreaks of porcine epidemic diarrhea virus (pedv) have re-emerged in korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. despite the availability of pedv vaccines in the domestic market, the disease continues to plague the korean pork industry, raising issues regarding their protective efficacy and new vaccine development. therefore, pedv isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. in the present study, one korean pedv strain, kor/knu- / , was successfully isolated and serially propagated in vero cells for over passages. the in vitro and in vivo characteristics of the korean pedv isolate were investigated. virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time rt-pcr. the infectious virus titers of the viruses during the first passages ranged from ( . ) to ( . ) tcid( ) per ml. the inactivated knu- virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. animal studies showed that knu- virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain knu- is highly enteropathogenic in the natural host. in addition, the entire genomes or complete s genes of knu- viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. our genomic analyses indicated that the korean isolate knu- is genetically stable during the first passages in cell culture and is grouped within subgroup g b together with the recent re-emergent korean strains. porcine epidemic diarrhea (ped) is a devastating disease in pigs that is characterized by acute enteritis and lethal watery diarrhea followed by dehydration with high mortality in suckling piglets (debouck and pensaert, ; saif et al., ; pijpers et al., ) . the disease was initially recognized in england in and has then spread to swine-producing european countries (oldham, ; pensaert et al., ) . since the s, ped has become rare in europe and is more often associated with post-weaning diarrhea in adult pigs (saif et al., ) . ped was first reported in asia in and has since had a great economic impact on the asian pork industry (chen et al., ; kweon et al., ; li et al., ; puranaveja et al., ; takahashi et al., ) . in may , ped outbreaks suddenly appeared in the united states and have rapidly spread nationwide as well as to canada and mexico, causing high mortality in newborn piglets and significant financial concerns (mole, ; stevenson et al., vlasova et al., . the etiological agent of ped, ped virus (pedv), was identified as a coronavirus in , which is a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales (lai et al., ; pensaert and de bouck, ; saif et al., ) . pedv is a large, enveloped virus possessing a single-stranded positivesense rna genome of approximately kb with a cap and a polyadenylated tail (pensaert and de bouck, ; saif et al., ) . the spike (s) protein of pedv is the major envelope glycoprotein of the virion and plays pivotal roles in interacting with the cellular receptor for virus entry and mediating neutralizing antibodies in the natural host (jackwood et al., ; lai et al., ; lee et al., ) . therefore, the pedv s glycoprotein is known to be an appropriate viral gene for determining the genetic relatedness among pedv isolates and for developing diagnostic assays and effective vaccines (chen et al., ; gerber et al., ; lee et al., ; lee and lee, ; oh et al., ) . the first ped epizootic in korea was confirmed in (kweon et al., ) . however, a retrospective study revealed that pedv already existed as early as (park and lee, ) . since the emergence, ped outbreaks occurred every year, resulting in substantial economic losses to the korean swine industry until early . after severe outbreaks of foot-and-mouth disease (fmd) during to , however, the prevalence of pedv infections was occasional with only sporadic outbreaks in korea. this epidemic situation probably resulted from the mass culling of more than one-third of the entire domestic pig population in korea during the - fmd outbreaks. however, starting in november , severe ped epidemics re-emerged in korea and swept more than % of pig farms lee et al., a,b) . although both modified live and killed vaccines against ped are commercially available in korea, continuous ped epidemics indicate a low effectiveness of the domestic vaccines. this result appears to be due to genetic and antigenic differences between s proteins of vaccine and field strains (lee et al., ; oh et al., ; lee and lee, ) . thus, the lack of effective vaccines enhances the need for the development of next-generation vaccines to control ped. pedv isolation in cell culture is critical for developing effective vaccines for ped prevention as well as performing various pedv research. however, the cell culture isolation of pedv has shown to be difficult and even the isolated virus may be unable to maintain infectivity upon further passages in cell culture (chen et al., ) . to date, there have only been two reports in more than two decades on the cultivation of the korean pedv strain that is genetically divergent from field pedvs (kweon et al., ; song et al., ) , while a number of pedv strains have been recently isolated in the us and successfully grown in cell culture for a year (chen et al., ; oka et al., ) , in the present study, we attempted to isolate pedv from various pedv-positive samples using vero cells. at this time, one highly virulent korean strain kor/knu- / has been successfully isolated and serially propagated in cell culture for over passages. we aimed to characterize the growth and titer of the pedv isolate knu- during the serial passages and the pathogenicity of the virus in suckling piglets. our in vivo assessment demonstrated that knu- is highly entero-pathogenic in piglets, exhibiting severe clinical symptoms as well as macroscopic and microscopic lesions typical for pedv infection. in addition, the complete genome or full-length s gene sequences of knu- were determined at selected passages to study the genetic stability and relationship. our data indicated that knu- isolate is relatively stable during the first passages in cell culture and is classified into subgroup g b that includes pedv strains responsible for recent severe outbreaks in korea and the us. vero cells (atcc ccl- ) were cultured in alpha minimum essential medium (␣-mem; invitrogen, carlsbad, ca) with % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions ( ×; invitrogen) and maintained at • c in a humidified % co incubator. seven small intestinal homogenates and stool specimens that tested positive by rt-pcr using an i-tge/ped detection kit (intron biotechnology, seongnam, south korea) were selected for virus isolation experiments. intestinal homogenates were prepared to % (wt/vol) suspensions with phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of s at a speed of rpm. fecal samples were diluted with pbs to be % (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for min at × g (hanil centrifuge fleta , incheon, south korea). the supernatant was filtered through a . -m-pore-size syringe filter (millipore, billerica, ma) and stored at − • c as an inoculum for virus isolation until use. virus isolation of pedv was attempted on vero cells as described previously with some modifications (hofmann and wyler, ) . briefly, prior to inoculation, inocula were prepared by adding trypsin (usb, cleveland, oh) to intestinal or fecal suspensions prepared above to give a final concentration of g/ml. confluent vero cells grown in -well plates were washed with pbs and inoculated with l of each inoculum containing trypsin. after incubating at • c for h, ml of virus growth medium [␣-mem supplemented with antibioticantimycotic solutions, . % tryptose phosphate broth (tpb; sigma, st. louis, mo), . % yeast extract (difco, detroit, mi), mm hepes (invitrogen), and g/ml of trypsin] was added. the inoculated cells were maintained at • c under % co and monitored daily for cytopathic effects (cpe). when % cpe appeared, inoculated cells were subjected to three rounds of freezing and thawing. the culture supernatants were then collected and centrifuged at × g for min. the clarified supernatants were aliquoted and stored at − • c as 'passage (p )' viral stocks until use. one hundred millimeter diameter tissue culture dishes were used for serial passages of the isolate. if no cpe was shown in inoculated cells for days, the plates were frozen and thawed three times, and the supernatants were harvested by centrifugation and inoculated on fresh vero cells for the next passage. if cpe and rt-pcr results were negative after blind passages, the virus isolation was considered negative. the pedv n protein-specific monoclonal antibody (mab) was obtained from choogang vaccine laboratory (cavac; daejeon, south korea). vero cells were infected with each passage knu- virus stock in the presence of trypsin as described above. the culture supernatants were collected at or h postinfection (hpi) at which a % cpe is commonly developed. for growth kinetics experiments, the supernatants were harvested from cells infected with each selected passage virus at different time points ( , , , , and hpi) and stored at − • c. virus titers were measured in -well plates by -fold serial dilution of the samples in triplicate per dilution to determine the quantity of viruses required to produce cpe in % of inoculated vero cells and calculated as tcid per ml using the reed-muench method (reed and muench, ) . the pedv titer was also determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. vero cells grown on microscope coverslips placed in -well tissue culture plates were mock infected or infected with pedv at a multiplicity of infection (moi) of . . the virus-infected cells were subsequently propagated until hpi, fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with n-specific mab for h. after being washed five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor (invitrogen), followed by counterstaining with , -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer and cell staining was visualized using a fluorescence leica dm il led microscope (leica, wetzlar, germany). viral rna was extracted from virus isolates or fecal samples prepared as described above using an i-tge/ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step sybr primescript rt-pcr kit (takara, otsu, japan) as described elsewhere (kim et al., ; sagong and lee, ) . the reaction took place using a thermal cycler dice real time system (takara) and the results were analyzed by the system software as described previously (sagong and lee, ) . eight - month-old guinea pigs (weighing - g) were randomly allocated into inoculated (n = ) and control (n = ) groups. six guinea pigs were immunized subcutaneously with . ml of binary ethylenimine (bei)-inactivated knu- -p virus in the presence of freund's complete adjuvant (sigma) and boosted once with a freshly prepared emulsion of the inactivated virus and freund's incomplete adjuvant (sigma) at a -week interval. two sham-inoculated guinea pigs were administered and boosted with cell culture media in the presence of the respective adjuvant. pre-immune sera were collected before starting the immunization and antisera were collected at weeks after the final boost. the presence of pedv-specific neutralizing antibodies in serum samples collected from guinea pigs in all groups was determined using a serum neutralization (sn) test in -well microtiter plates using pedv isolate knu- or vaccine strain sm - as previously described (oh et al., ) with minor modifications. briefly, vero cells were grown at × /well in -well tissue culture plates for day. the knu- -p virus stock was diluted in serum-free ␣-mem to make tcid in a l volume. the diluted virus was then mixed with l of -fold serial dilutions of individual inactivated sera in -well plates and incubated at • c for h. the mixture was inoculated into vero cells and incubated at • c for h. after removing the mixture, the cells were thoroughly rinsed times with pbs and maintained in virus growth medium at • c in a % co incubator for days. for the sn test using the vaccine strain, pedv strain sm - propagated in the absence of trypsin was diluted in serum-free ␣-mem to make pfu in a l volume. the diluted virus was then mixed with each serum sample in -well plates as described above and incubated at • c for h. subsequently, approximately × vero cells in l of ␣-mem containing % fbs were added to each well and the mixture was maintained at • c in a % co incubator for days. the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all of the duplicate wells. the in vivo swine studies described here were performed at the improah animal facility under the guidelines established by its institutional animal care and use committee. a total of suckling piglets of days of age were obtained from a commercial pig farm with no known prior ped outbreak or vaccination with pedv. all animals were determined to be free of antibodies to pedv as well as to transmissible gastroenteritis virus (tgev) and porcine reproductive and respiratory syndrome virus. pigs were randomly assigned to experimental groups housed in separated rooms: pedv-inoculated (n = ) and contact control (n = ) in room and sham-inoculated control (n = ) group in room . following a day acclimation period, only piglets in the pedv-inoculated group orally received a ml dose of tcid /ml of knu- -p virus. two piglets were exposed to the virus by direct contact with inoculated piglets in the same room. the sham-inoculated pigs were administered with cell culture media as a placebo. clinical signs of diarrhea and mortality were monitored daily for the duration of the study. stool samples from all groups were collected prior to inoculation and daily with inch, cotton-tipped swabs and subjected to rt-pcr using an i-tge/ped detection kit and real-time rt-pcr as described above. pedv-inoculated piglets were necropsied daily (days , , , and ) after challenge for post-mortem examinations throughout the study, whereas all pigs from the contact and control groups were euthanized at days post-challenge for post-mortem examinations. small intestinal tissue specimens (< mm thick) collected from each piglet were fixed with % formalin for h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut at - m thick on a microtome (leica), floated on a • c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for min and washed in decreasing concentrations of ethanol ( %, %, %, %, and %) for min each. the deparaffinized intestinal tissues sections were stained with hematoxylin and eosin (sigma) for histopathology or subjected to immunofluorescence assay using pedv n-specific mab as described above. the paraffin-embedded tissue sections were deparaffinized, treated with . m citrate buffer (ph . ) in a microwave oven for min, chilled at rt for min, and then incubated with . % hydrogen peroxide in dw for min to block endogenous peroxidase. after being washed three times in pbs, the sections were blocked with normal horse serum (vectastain abc kit; vector laboratories, burlingame, ca) and incubated h at rt with n-specific mab. after rinsing in pbs, the samples were reacted for min at rt with a horse anti-mouse secondary antibody (vectastain abc kit), incubated with avidin-biotin peroxidase complex for min (vectastain abc kit), and developed using the dab substrate kit (vector laboratories) according to the manufacturer's instructions. the slides were then counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted on microscope glass slides in mounting buffer and tissue staining was visualized using a microscope. the complete genomic sequences of the original fecal sample as well as those of the p and p isolates were determined by nextgeneration sequencing (ngs) technology. total rna was extracted from the feces as well as p and p isolates using a rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions and used as a template to amplify cdna fragments as described elsewhere lee et al., b) . ten overlapping cdna fragments were generated to encompass the entire genome of each sample, pooled in equimolar amounts, and subjected to ngs using the ion torrent personal genome machine (pgm) sequencer system (life technologies, carlsbad, ca) and a v sequencing chip (life technologies) as described previously lee et al., b; rothberg et al., ) . the single-nucleotide variants (snvs) were analyzed using the clc genomic workbench version . (clc bio, cambridge, ma) and the individual ngs reads were assembled using the complete genome of pedv reference strain kor/knu- / (genbank accession no. kj ). the and ends of the genomes of the original feces and the p and p isolates were determined by rapid amplification of cdna ends (race) as described previously . the full-length genomic nucleotide sequences of the knu- -feces, knu- -p , and knu- -p were deposited in genbank under accession numbers kr , kr , and kr , respectively. the s glycoprotein gene sequences of the virus isolates were also determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were rt-pcr amplified as described previously (lee et al., ) . the individual cdna amplicons were gel-purified, cloned into pgem-t easy (promega, madison, wi), and sequenced in both directions using two commercial vector-specific t and sp primers and the s gene-specific primers. in addition, the complete structural gene sequences of the virus isolate at selected passages (knu- -p , knu- -p , knu- -p , and knu- -p ) were determined by the sanger method as described above and deposited to the gen-bank database under their accession numbers shown in fig. . the sequences of fully sequenced s genes and complete genomes of pedv isolates were independently used in sequence alignments and phylogenetic analyses. the multiplesequencing alignments were generated with the clustalx . program (thompson et al., ) and the percentages of nucleotide sequence divergences were further assessed with the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences by using the neighborjoining method and subsequently subjected to bootstrap analysis with replicates in order to determine percentage reliability values on each internal node of the tree (saitou and nei, ) . all tree figures were produced using mega . software (tamura et al., ) . student's t test was used for all statistical analyses, and p-values of less than . were considered statistically significant. we attempted to isolate pedv from pcr-positive clinical samples, including feces and intestinal homogenates on vero cells. one pedv isolate designated knu- was successfully isolated from the feces of a naturally infected piglet from a commercial farm located in kyungpook province obtained on september , . the knu- virus was capable of producing distinct cpes typical for pedv infection, such as cell fusion, syncytium, and detachment, in infected vero cells from passage (p ). we then investigated whether the isolate can be efficiently cultivated and maintained in cell culture. thus, the isolated pedv strain knu- was further serially passaged in vero cells for a total of passages (p to p ). the time of cpe onset was at hpi and, accordingly, prominent cpe was observed within hpi in the first productive passages (p and p ). in the later passages, visible cpe appeared at hpi and became predominant by hpi. virus propagation was confirmed by detecting pedv antigens by ifa using a pedv n protein-specific mab. the distinct staining was distributed in the cytoplasm of typical syncytial cells. in contrast, no cpe and n-specific staining was evident in mock-inoculated vero cells. examples of cpe and corresponding ifa images in selected passages are shown in fig. . the level of viral genome in each selected passage was further assessed by real-time rt-pcr and the mean cycle threshold (ct) value was determined to be . , ranging from . (p ) to . (p ). the infectious titer of the isolate ranged from . to . tcid /ml up to p , whereas it was determined to be approximately tcid /ml in the later passages. the peak viral titer reached . tcid /ml (equivalent to . pfu/ml) or more since passage ( fig. a) . the growth kinetics study further showed that knu- replicated rapidly and efficiently in vero cells, reaching a maximum titer > tcid /ml by dpi (fig. b) . guinea pig antisera were collected before immunization (preimmune) and at weeks after the final boost and tested for their neutralizing activity against the isolate knu- or vaccine strain. as shown in fig. , the guinea pig antisera were highly effective in inhibiting knu- infection with mean neutralizing antibody (na) titers of : , whereas the antisera at relatively low dilution fully protect vero cells from sm - infection with mean na titers of : . in contrast, none of the pre-immune and nonimmunized sera showed neutralizing activity against either strain. taken together, our data indicated that the isolate knu- elicits potent antibody responses in immunized animals. however, the antisera strongly recognized the homologous field isolate, but inefficiently the heterologous pedv vaccine strain, suggesting the antigenic variations between the vaccine strain and field pedvs. four piglets were challenged orally with the knu- virus, while control animals were inoculated with cell culture media. two piglets were housed together with inoculated piglets in the same room for direct contact exposure. during the acclimation period, all piglets were active and had normal fecal consistency. pedv-challenged piglets exhibited clinical signs including lethargy and diarrheic feces by day post-inoculation (dpi) and experienced severe watery diarrhea with vomiting thereafter. pedv-associated mortality occurred in one of the inoculated pigs at dpi. contact piglets housed with the inoculated group exhibited diarrheic feces with vomiting by dpi and the progression of clinical signs was similar to that of the inoculated animals. furthermore, all inoculated and contact piglets were positive for pedv, as determined by rt-pcr, by or dpi and shed pedv in feces with mean ct values of . (range . - . ) and . (range . - . ), respectively. negative control pigs remained active with normal feces and continued to be undetectable for fecal shedding of pedv throughout the study period. one piglet died from pedv and was subsequently necropsied at dpi and the remaining inoculated piglets were randomly selected for necropsy thereafter. all contact and control animals were euthanized at the end of the study for postmortem assessments. all inoculated and naïve contact pigs macroscopically displayed typical ped-like lesions. the small intestine was dilated and accumulated with yellow fluid and its wall was thin and transparent, due to the villous atrophy (fig. , panel a) . the stomach was distended and filled with curdled and undigested milk. in contrast, the other intestinal organs appeared grossly normal. microscopic intestinal observations consistent with viral enteritis were developed in all inoculated and contact piglets, which included vacuolation of small intestinal enterocytes and shortening and fusion of small intestinal villi (fig. , panels b and c) . similar microscopic lesions were continuously present in the challenged and contact pigs through dpi. furthermore, both ifa and ihc staining revealed that the viral antigen was predominantly detected in the cytoplasm of epithelial cells on atrophied villi in all segments of the small intestines (fig. , panels d-f). these finding are comparable to those in pigs naturally or experimentally infected with virulent strains of pedv (debouck et isolates. pedv-specific cpes were observed daily and were photographed at hpi using an inverted microscope at a magnification of × (first panels). for immunostaining, infected cells were fixed at hpi and incubated with mab against the n protein, followed by alexa green-conjugated goat anti-mouse secondary antibody (second panels). the cells were then counterstained with dapi (third panels) and examined using a fluorescence microscope at × magnification. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) ). neither macroscopic nor microscopic intestinal lesions were observed in the negative control piglets during the experiment. entire genome sequence data of the original fecal sample (knu- -feces) and cell culture-passaged knu- -p and -p viruses were determined using the ngs technology. the full-length genome of kor/knu- / pedv was used as the initial reference for each ngs read and the individual complete genome sequences were successfully obtained by the assembly of respective ngs reads. the and ends of their genomes were also sequenced by race. all three genomes were , nucleotides (nt) in length, excluding the poly(a) tail and exhibited the genomic organization typical of all previously sequenced pedv strains, consisting of the -nt utr, the , -nt orf a/ b (nt to , for a and nt , to , for b), the -nt s gene (nt , to , ), the -nt orf (nt , to , ), the -nt e gene (nt , to , ) , the -nt membrane (m) gene (nt , to , ), the -nt n gene (nt , to , ), and the - fig. . virus-neutralizing antibody titers in the sera of guinea pigs inoculated with a pedv knu- isolate. guinea pigs were immunized twice with inactivated knu- -p virus by subcutaneous injection. blood samples were collected prior to immunization and at weeks after the second immunization and subjected to the virus neutralization assay using homologous (knu- ; solid circles) and heterologous (sm - ; solid diamonds) strains. neutralizing antibody titers for individual infected animals were spotted as a log scale. values are representative of the mean from three independent experiments in duplicate and error bars denote standard deviations. nt utr. the slippery heptamer sequence, tttaaac, followed by a stem-loop structure was present at the end of orf a in the pedv genome, which is a potential signal for a ribosomal frame shift during translation to generate the pp ab. the complete genome sequences of all three viruses were compared, and the results are summarized in table . compared to the knu- -feces, knu- -p showed one different nt at position , resulting in one amino acid (aa) change (leu to phe) in the s protein, while knu- -p gained two additional nt changes at positions , and , causing two aa mutations located in the s protein. the full-length s genes of the original feces and knu- viruses at selected passages (p , p , p , p , p , and p ) were also sequenced using the traditional sequencing method. the s protein sequences of knu- -feces, -p , and -p were completely identical to those determined by ngs. one nt change present in the s gene of knu- -p had been acquired since passage (knu- -p ). a total of three nt mutations, identified at passage compared to the feces, were sustained through passage (knu- - ) . in addition, compared to knu- -p , we were able to detect two independent mutations at positions , and , , resulting in amino acid changes in orf (asp to tyr) and e (pro to ser) of knu- -p , respectively, which were maintained at passage . altogether, our results revealed that the pedv isolate knu- is genetically stable during serial passages in cell culture. the entire genome sequences of pedvs determined in this study were further compared to those of other korean and non-korean pedv strains available in genbank, and the nucleotide homology and difference data are described in table . knu- isolates (feces, p , and p ) had the highest nucleotide identity ( . %) with the korean re-emergent strain kor-knu- and us strains, in and mn, showing to different nt at the genomic level. all three viruses were genetically distinct from the korean vaccine strains, sm - and dr- , and the prototype cv strain and exhibited relatively low nucleotide identity ranging from . % to . %. by alignment of the global pedv strains, a single-nucleotide insertion between nucleotides , and , has been previously identified in one chinese ah and three us strains, table nucleotide and amino acid changes of knu- during serial passages in cell culture. nucleotide amino acid position feces p p p p p p position feces p p p p p p utr ( ) -a -n d b nd --nd nd --nd nd --nd nd orf a ( , ) --nd nd --nd nd --nd nd --nd nd orf ab ( , ) --n d nd --nd nd --nd nd --nd nd s ( , ) a a a a c c c k k k k t t t c t t t t t t resulting in the shorter pp ab protein in length (chen et al., ) . however, none of the genomes of the three pedvs included such an insertion, and this result was further confirmed by the traditional sanger sequencing method (data not shown). in agreement with previous results (lee et al., ; lee and lee, ; lee et al., a,b) , the full-length s gene-based phylogenetic tree revealed that the global pedv strains were clearly defined into separate clusters, designated genogroup (g ; classical) and genogroup (g ; field epidemic). each genogroup can be further divided into subgroups a, b, a, and b (fig. a ). all original feces and passaged pedv viruses through passages were classified into subgroup b along with the recent korean field isolates, which were most closely clustered together with the emergent us strains in an adjacent clade with the same subgroup. subsequent phylogenetic analysis of the s region showed the same grouping structure as the s gene-based tree (data not shown). in addition, phylogenetic analysis based on the entire genome sequences demonstrated that strain knu- is grouped within the same cluster with the recent korean and us stains (fig. b ). in korea, three pedv strains, sm - , dr- , and chinju , were initially isolated almost two decades ago in korea. genetic and phylogenetic analyses revealed that sm - and dr- belong to the classical group , whereas chinju is classified into the field epidemic group . of these, only sm - and dr- isolates can be grown in cell culture and, accordingly, have been used as commercial vaccine seeds. since , we have been monitoring the genetic diversity of pedv prevalent in the field based on the s gene. our data demonstrated the existence of antigenic and genetic variations between vaccine and field strains, exhibiting a more than % amino acid difference in the s domain (lee et al., ; lee and lee, ; oh et al., ) . this may be one of the reasons for the incomplete efficacy of current vaccines in korea, suggesting that the isolation of field pedv is required for the development of a next-generation vaccine. although virus isolation in cell culture from clinical samples of naturally or experimentally infected pigs is fastidious, recent studies reported the successful isolation and propagation of several us original pedv strains using vero cells (chen et al., ; oka et al., ) . in this study, we initially sought to isolate pedv efficiently propagated in vitro from intestine homogenates and fecal samples of naturally infected piglets (field cases) and were able to obtain only isolate from feces in vero cell culture. the virus isolation rate, in the present study, was less than % and was relatively lower than that in recent us studies ranging from to % (chen et al., ; oka et al., ) . in previous studies, all isolated pedv strains originated from intestinal contents of naturally infected or experimentally inoculated pigs, suggesting that intestine samples may be a better source for virus isolation (chen et al., ; oka et al., ) . however, we failed to isolate pedv from intestine homogenates, which may be due to the number of intestinal contents (n = ) included in our study and be responsible for the low isolation rate. although pedv isolation might be affected by multiple factors, it appears to depend on the number of samples with good quality rather than the type of samples (intestinal contents or feces). further studies are needed to improve the isolation methodology or to determine the contributing factor(s) to enhance the success rate of pedv isolation in cell culture. the pedv isolate, knu- , was cytopathogenic in vero cells from passage and after passage , exhibited more severe and rapid cpe characterized by fusion of infected cells (syncytium or polykaryon formation). the initial viral infectious titers ranged from approximately to log tcid /ml and increased after several more passages reaching to log tcid /ml or more. these growth characteristics, including cytopathology, infectious titer, and growth kinetics were unchanged or even more efficient throughout the experiment ( passages), indicating that the isolate knu- is phenotypically stable during serial passages in vero cells. since the antibody response is a critical indicator to prove the cause of viral infection, we immunized guinea pigs twice with the inactivated isolate (knu- -p ) and determined whether the animals developed humoral immunity using an sn test. the guinea pig sera raised against the isolate contained high levels of na ( . log ), demonstrating the ability of knu- to elicit immune responses. on the other hand, the antisera showed a relatively weak neutralizing response (an almost -log reduction), when the heterologous vaccine strain sm - was used for an sn test. this weak neutralizing activity of the anti-knu- guinea pig sera against sm - was somewhat expected because of a high degree of genetic diversity between the s proteins of the vaccine strain and field isolates (lee et al., ; lee and lee, ; oh et al., ) . experimental oral inoculation of nursery pigs with pedv isolate knu- induced severe clinical disease typical of acute enteritis throughout the study, demonstrating that the isolate was highly enteropathogenic in neonatal piglets. onset of clinical signs including lethargy and watery diarrhea in inoculated pigs was found as early as dpi. the pedv genome was detected in feces in % of challenged pigs on dpi and virus quantities from fecal shedding were maintained thereafter in all challenged pigs throughout the study period, leading to the source for direct transmission of virus to other animals. this result is inconsistent with a previous study using a us pedv strain that demonstrated virus fecal shedding on dpi in only one-fourth of the challenged pigs (madson fig. . phylogenetic analyses based on the nucleotide sequences of the spike genes (a) and the full-length genomes (b) of pedv strains. a putative similar region of the spike protein and the complete genome sequence of tgev was included as an outgroup in each panel. multiple-sequencing alignments were performed using clustalx program and the phylogenetic tree was constructed from the aligned nucleotide sequences by using the neighbor-joining method. numbers at each branch represent bootstrap values greater than % of replicates. names of the strains, countries and years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site. et al., ) . the difference between the current and previous studies is the age of the pigs: -week-old neonatal pigs and -week-old weaned pigs used in the present and previous experiments, respectively. therefore, younger piglets in this study were more sensitive to pedv infection shedding virus in feces earlier than older pigs, probably due to an age-dependent disease severity as previously described (shibata et al., ) . ihc and ifa revealed that viral antigen in villous enterocytes were observed at dpi in all segments of the small intestine of inoculated piglets. the onset of clinical signs and viral fecal shedding and the detection time of viral antigen in the target tissue were similar to recent independent reports using different us pedv strains jung et al., ) . two non-challenged piglets were included in this study for direct contact to inoculated piglets in the same space. all contact piglets displayed ped-like symptoms within h after the onset of clinical signs in inoculated piglets. the presence of pedv in feces and infected tissues was further verified in contact piglets, showing % morbidity in our study. mortality averages % in suckling piglets up to week of age, often approaching % in -to -dayold piglets, and decreases to % thereafter (saif et al., ) . in our study, mortality was observed only in one out of four inoculated piglets, resulting in % mortality in the current study involving -day-old piglets. for reproducible challenge studies in vivo using the isolate in the future, a neonatal swine bioassay will be needed to determine either the median pig diarrhea dose or lethal dose as a standardized dose and this aspect is currently under investigation. whole-genome sequences of korean pedv strains (knu- -feces, p , and p ) were determined using ngs approaches coupled with race experiments. regions covering the structural genes were also sequenced at the selected passages by the sanger method for confirmation. compared to the original feces (knu- -feces), only one nt difference at position , was identified for the first passages (knu- -p ) in cell culture, which led to a non-synonymous mutation at the corresponding position of the s protein. this nt change was initially found at passage and further maintained through passage . however, we were unable to investigate whether this mutation had been acquired at the beginning of the vero cell culture since infectious virus was not obtained during the first two passages. interestingly, the identical c t mutation at the whole-genome level (l f at the aa level of s) has been previously reported in a us pedv isolate isu- e during cell culture passage (chen et al., ) , suggesting its potential importance for adaption of the field virus to growth in vitro. at passage , two more nt differences at positions , and , of the genome (a c and g c) were detected when compared to knu- -feces. all of the changes acquired in knu- -p led to non-synonymous mutations at the respective positions and of the s protein (k t and e q). these findings were similar to recent data reported by chen et al. ( ) that two us isolates individually gained the mutations located in orf b, s, and e through passage in cell culture. however, no nucleotide changes were identified in orf b and e for the first passages in vero cells. the mutations in the s protein were persistent for passages in cell culture. celladapted pedv vaccine strains, sm - and dr- , are known to contain a -nt deletion spanned from the end of s to the start of orf and a -nt deletion in the middle of orf . thus, we further sequenced structural genes of knu- -p and -p and identified two additional nucleotide changes (g t and c t) acquiring non-synonymous amino acid mutations in orf and e, respectively. except for those nucleotide substitutions, the isolate had no extra change, including such large deletions in structural genes through passage , indicating the genetic stability of knu- during serial passages in vero cells. sequence comparisons with other pedv strains showed that knu- isolates are genetically most similar to recent korean and us epidemic strains with . - . % identities, but most distinctly related to classical strains, cv , sm - , and dr- , with . - . % identities at the genomic level. all phylogenetic analyses based on the complete genome, the full-length s gene, and the s portion formed the similar tree structure, revealing that the korean knu- strains are clustered together with the re-emergent korean strains in subgroup b that also includes emergent us strains and - field epidemic chinese strains. in conclusion, we isolated and serially propagated pedv in cell culture that is phenotypically and genotypically identical to field strains responsible for the recent severe ped outbreaks in korea. to our knowledge, this is the first report describing the isolation and in vitro and in vivo characterization of korean pedv associated with the field epidemic. with the availability of the korean isolate, we are now able to spur the development of new effective and safe vaccines for ped prevention. indeed, our pedv isolate has been used for the development of an inactivated vaccine that is currently being evaluated under experimental and field conditions. furthermore, we are continuing to passage the isolate in vero cells to develop a live attenuated vaccine that generally prove to provide a more efficient protective immunity than killed viral vaccines. for this purpose, pathogenic and molecular characterization of the isolates at selected passages will be assessed to determine their phenotypes in pigs and to identify the genetic changes involved in pedv attenuation. molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv the pathogenesis of an enteric infection in pigs, experimentally 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, and several coronaviruses porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus a simple method for estimating fifty percent endpoints an integrated semiconductor device enabling non-optical genome sequencing porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-b production by inhibiting irf activation in immortalized porcine alveolar macrophages coronaviruses the neighbor-joining method: a new method for reconstructing phylogenetic trees isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega : molecular evolutionary genetics analysis (mega) software version . the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains we gratefully thank gun-seok park and jae-ho shin from kyungpook national university for their help in ngs analysis. this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology(nrf- r a a ). key: cord- -idmj d p authors: onabajo, olusegun o.; porter-gill, patricia; paquin, ashley; rao, nina; liu, luyang; tang, wei; brand, nathan; prokunina-olsson, ludmila title: expression of interferon lambda is associated with reduced proliferation and increased cell death in human hepatic cells date: - - journal: j interferon cytokine res doi: . /jir. . sha: doc_id: cord_uid: idmj d p interferon lambda (ifn-λ ) is a novel type-iii interferon that can be generated only in individuals carrying a Δg frame-shift allele of an exonic genetic variant (rs -Δg/tt). the rs -Δg allele is strongly associated with decreased clearance of hepatitis c virus (hcv) infection. here, we further explored the biological function of ifn-λ expressed in human hepatic cells—a hepatoma cell line hepg and fresh primary human hepatocytes (phhs). we performed live confocal imaging, cell death and proliferation assays, mrna expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. not only did we observe significant intracellular retention of ifn-λ but also detected secreted ifn-λ in the culture media of expressing cells. secreted ifn-λ induced strong activation of the interferon-stimulated genes (isgs) in ifn-λ -expressing and surrounding cells in transwell assays. specifically, in phhs, secreted ifn-λ induced expression of the cxcl transcript and a corresponding pro-inflammatory chemokine, ip- . in ifn-λ -expressing hepg cells, we also observed decreased proliferation and increased cell death. all ifn-λ -induced phenotypes—activation of isgs, decreased proliferation, and increased cell death—could be inhibited by an anti-ifn-λ -specific antibody. our study offers new insights into biology of ifn-λ and its possible role in hcv clearance. w ith more than million infected individuals, hepatitis c virus (hcv) infection represents a significant healthcare burden worldwide (mohd hanafiah and others ) . hcv infection is treated with interferon (ifn)-a-based regimens and recently approved ifn-a-free direct-acting antiviral agents (daa) (liang and ghany ) . genome-wide association studies identified a single nucleotide polymorphism rs , located upstream of the ifnl (il b) gene and thus initially referred to as the ''il b marker,'' as one of several genetic variants strongly predictive of hcv clearance (ge and others ; thomas and others ) . further studies showed that rs is located in the intronic region of a novel gene, ifnl , and is in high linkage disequilibrium (ld) with an ifnl exonic frameshift polymorphism rs -tt/dg, initially designated as ss (prokunina-olsson and others ). the rs -dg allele, which creates an open reading frame for a novel human interferon, interferon lambda (ifn-l ), is associated with decreased hcv clearance (prokunina-olsson and others ) [reviewed in o'brien and others ( ) ]. the rs -dg has allele frequency of * % in individuals of african ancestry, * % in europeans, while only %- % in asians (prokunina-olsson and others ). in individuals of african ancestry, rs is more predictive of hcv clearance than rs (prokunina-olsson and others ; aka and others ); while in europeans and asians, these markers are in high ld and thus provide comparable predictive information (prokunina-olsson and others ). a genetic polymorphism rs -c/t, which introduces an amino-acid substitution p s in the ifn-l protein (prokunina-olsson and others ), is associated with reduced biological activity of ifn-l and increased hcv clearance (terczynska-dyla and others ), thereby supporting the critical role of ifn-l in this process. recent clinical trials showed that ifnl variants rs and rs are predictive of treatment efficacy even for daa therapies (fujino and others ; meissner and others a; o'brien and pfeiffer ) and these markers, possibly together with p s, could be used to optimize treatment regimens and duration in resource-limited settings. the functional importance of ifn-l is evidenced by the strong positive selection that favored elimination of ifn-l from human populations (key and others ) . although this selection cannot be explained by any known viral infection, it may reflect antiviral response to some extinct highly deadly infection. previously, we showed that transient transfection of an expression construct that generates ifn-l protein induced interferon signaling, with activation of interferon-stimulated genes (isgs) and generation of antiviral response in hepg , a human hepatoma cell line (prokunina-olsson and others ). however, the function of ifn-l and its role in impaired hcv clearance remained unclear. here, we further explored this question by performing additional functional analyses of ifn-l transiently and stably overexpressed in human hepatic cells-fresh primary hepatocytes and hepg cells. the human hepatoma cell line hepg (atcc hb- ) was purchased from the american tissue culture collection (atcc) and maintained in dulbecco's modified eagle's medium (dmem) supplemented with % heat-inactivated fetal bovine serum (fbs). the custom isre-luc-hepg cell line stably expressing a luciferase reporter under control of the interferon-stimulated response element (isre) was previously described (prokunina-olsson and others ); the cells were maintained in dmem supplemented with % fbs and mg/ml puromycin. fresh primary human hepatocytes (phhs) were purchased from bioreclamation ivt. the cells were received in suspension within h after isolation and were maintained in in-vitrogro hi culture media with torpedo antibiotic mix. the liver donor was a -year-old woman who died of cardiac arrest, and the liver donor was a -year-old man who died of anoxia. both were hcv-free caucasians. on arrival, phhs had % and % viability for donor and , respectively. dna extracted from the phhs was genotyped with a custom taqman assay (prokunina-olsson and others ) for the exonic ifnl genetic variant rs (dg/tt). both donors were homozygous for the rs -tt allele, which is associated with higher hcv clearance (prokunina-olsson and others ). the rs -tt allele introduces a frame shift within the first exon of ifnl gene and eliminates the ifn-l protein. thus, no ifn-l protein could be endogenously produced in these phh samples. the expression constructs (ifnl -halo, p -halo, and control-halo) based on the pfc a vector (promega) were previously described (prokunina-olsson and others ). the control-halo construct generates only a halo-tag protein ( kda), the ifnl -halo construct generates the ifn-l -halo protein ( kda) consisting of the ifn-l protein ( kda) c-terminally fused with the halo-tag protein ( kda), and the p -halo generates a protein of kda, consisting of the halo-tag c-terminally fused with a splicing protein isoform of ifn-l ( aa, kda, designated as p ), which lacks exon and is nonfunctional in induction of interferon response (prokunina-olsson and others ). all constructs have been validated by western blotting with an a-halo antibody (promega) and/or an a-ifn-l antibody (mabf ; emd millipore). the ifnl -halo construct generating the secreted ifn-l -halo protein was created and validated the same way. hepg cells and phhs were transfected for or h with corresponding constructs; hepg cells were transfected using lipofectamine/ltx and opti-mem with standard protocols (life technologies), while phhs were transfected using a nucleofection protocol optimized for primary cells (lonza). briefly, for each transfection, · fresh phhs were resuspended in ml of optimized nucleofection buffer l and transfected with mg of corresponding constructs using d-nucleofector (lonza). immediately after transfection, dead cells were separated by centrifugation ( min, rpm) of the transfection mix overlaid with ml of ficoll (ge healthcare) and ml of supernatant with dead cells was removed. the remaining cells were resuspended in invitro-gro cp media with torpedo antibiotic mix (bioreclamation ivt), plated onto collagen-coated plates (bd biosciences), and incubated overnight. unattached and dead cells were removed h post-transfection by replacing the transfection media with invitrogro hi culture media with torpedo antibiotic mix (bioreclamation ivt). tetracycline-inducible hepg cell lines for ifn-l -gfp, p -gfp, and control-gfp were generated using the tet-on g expression system (clontech). first, a stable hepg cell line expressing the tet-on g transactivator was generated by lentiviral transduction and blasticidin selection ( mg/ml). corresponding cdnas were cloned into a ptreg g plasmid (clontech) to generate tetracyclineinducible ifn-l -gfp, p -gfp, and control-gfp constructs, which were transfected into the stable tet-on g-hepg using lipofectamine/ltx. positive clones were identified by limiting dilution under neomycin selection ( mg/ml) over weeks. expression of corresponding proteins was induced by treatment with mg/ml doxycycline for indicated experiment-specific time periods. hepg cells were transfected with corresponding constructs in -well coverslip chambers ( · cells/well; labtek). after h, transfection media was replaced with live cell imaging solution (life technologies), supplemented with mm glucose. cell-permeant halo-tag ligands (tmr red or oregon green; promega) were added to live cells ( : , for min), and live imaging was performed using a lsm confocal laser scanning microscope (carl zeiss) fitted with a temperature and co -controlled chamber. when applicable, nuclei were visualized using fluorescent hoechst dye (life technologies). for cell death analysis, live cells were imaged using inverted oil lens at · magnification, with s scanning per minute per field of view for h, generating , images per field; fields were imaged in sequence using stage-positioning software. each field of view had at least transfected cells. signs of cell death were scored visually by counting membrane rupture events in the videos representing the -h imaging periods. cell death rates were determined as the percentages of ruptured cells of the total counts of fluorescent cells in each field of view. hepg cells were transfected with corresponding constructs and imaged live as described earlier using oregon green halo-tag ligand. the mobility of the ifn-l -halo and control-halo proteins was evaluated with fluorescence recovery after photobleaching (frap) method (snapp and others ) . briefly, after prebleach scans, a selected region was bleached at % of imaging power with iterations and ms per iteration, with . s per scan. imaging was continued for a total of s. fluorescence intensity was plotted with curve fitting to evaluate fluorescence recovery in the bleached areas over time. half-time fluorescence recovery (t½) and the ratio of immobile fraction were averaged for individually scanned cells for each construct (ifnl -halo and control-halo). recovery data were normalized to unbleached regions inside the cells and adjusted for background fluorescence; data acquisition and analyses were performed using lsm frap software. the stable hepg -isre-luc cells were seeded in -well plates that were either untransfected or transfected with corresponding constructs. after h, the cells were treated for h with the following antibodies: mg/ml of a blocking a-il r antibody (r&d systems), mg/ml of a goat isotype igg control (abcam), a range ( , , , . , or . mg/ml) of a custom rabbit monoclonal a-ifn-l antibody (prokunina-olsson and others ), or mg/ml of a rabbit isotype igg control (cell signaling). recombinant human interferons- ng/ml of custom ifn-l (prokunina-olsson and others ), ifn-a and ifn-b (pbl assay science), or ifn-g (r&d systems) were added to the untransfected cells pretreated with the antibodies. negative controls included nontreated cells, phosphate-buffered saline (pbs) in media, and mock transfection with control-halo constructs. the cells were assayed for luciferase expression of the isre-luc reporter h post-transfection. all transfections and treatments were done in biological replicates. for other experiments, inducible stable hepg cells were seeded into -well plates and protein expression was induced for h. antibodies ( mg/ml of rabbit monoclonal a-ifn-l or mg/ml of rabbit igg control) were added to shared culture media for h. hepg and phhs were transfected with corresponding constructs and seeded onto -well plates. separately, transwell inserts with . mm pores (corning) were seeded with untransfected cells and placed into -well plates with culture media, with insert per well. for phhs, both the plates and transwell inserts were collagen coated (corning). after h, media was replaced and transwell inserts with growing cells were added to the plates containing transfected cells. the pore size of transwell inserts allowed free circulation of the media but not of the cells. for phhs from donor , the transwell experiment was expanded to include antibody treatment. phhs transfected with ifnl -halo, ifnl -halo, and control-halo constructs were incubated with transwell inserts seeded with untransfected phhs. antibodies ( mg/ml of rabbit monoclonal a-ifn-l or mg/ml of rabbit igg control) were added to shared culture media for h. after h of coincubation, cells from both chambers that shared culture media (transfected vs. untransfected transwell cells) were collected separately for rna extraction and stored in rlt buffer; shared culture media was collected for protein analysis and stored at - °c until use. for transient transfections, hepg cells were transfected with corresponding constructs for h and media was changed after overnight incubation. expression of halo-tag in live cells was detected using cell-permeant halo-tag tmr red ligand. for stable hepg cells, expression of proteins was induced for h. in both systems, dead cells were identified using near-ir fixable live/dead marker (life technologies). for analysis of apoptosis, cells were additionally stained for annexin v (bd biosciences). for proliferation assays, the cells that were induced for h were treated with mm -bromo- ¢-deoxyuridine (brdu) for h. dead cells were excluded by removing nonadherent cells, and the remaining cells were stained using the apc brdu flow kit (bd biosciences). multiparametric flow cytometry analysis was performed on facs aria iii (bd biosciences) with flowjo. software (tree star). for viability assays, the protein expression was induced for days in -well plates and viability was evaluated with the multi tox-glo assay (promega). in some experiments, viability assays were performed after protein induction for h, with similar results. quantitative reverse-transcriptase-polymerase chain reaction expression analysis total rna was isolated using an rneasy kit with oncolumn dnase i treatment (qiagen). rna quantity and quality were evaluated by nanodrop (thermo scientific). cdna was prepared from to ng of total rna with the rt first-strand cdna kit and random hexamers with an additional dna-removal step (qiagen). quantitative reverse-transcriptase-polymerase chain reaction (qrt-pcr) mrna expression analysis was performed using sybr green antiviral response qrt-pcr plates (qiagen). the plates included expression assays for target genes, as well as positive, negative, and endogenous normalization controls (supplementary tables s -s ; supplementary data are available online at www.liebertpub.com/jir). a custom expression assay for ifnl (prokunina-olsson and others ) and predesigned taqman expression assays for ifnl (hs _g ) and ifng (hs _m ; life technologies) were not included on the predesigned plates and were used separately (supplementary table s ). the qrt-pcr reactions ( or ml) included expression master mixes (qiagen or life technologies), cdna, and corresponding expression assays; the quantification was performed in - technical replicates on the quantsudio instrument (life technologies). expression was measured in c t values (pcr cycle at detection threshold), which are distributed on log scale. expression was normalized by the geometric means of endogenous controls (actb, b m, gapdh, hprt , rplp ) included on the qrt-pcr plates or of endogenous controls used separately (assay e for gapdh, and assay e for actb; life technologies). differences in expression were calculated according to the relative quantification method, as dc t = c t (control) -c t (target). fold differences between expression of any samples or groups of samples were calculated as (dct -dct ) . heat-map analysis of auto-scaled expression data for a panel of selected isgs was performed with the genex software (multid). protein levels of ip- (encoded by cxcl ) in culture media from hepg and phhs transfected with corresponding constructs were measured with an elisa kit (r&d systems), which has a protein detection range of . - pg/ml. culture media was collected h after transfection media change ( h post-transfection). media samples were diluted : or : , and each sample was measured in technical triplicates. a standard curve was generated using the protein provided with the kit, with a correlation coefficient > . . the plates were analyzed using glomax luminometer (promega). ifn-l was detected with a custom-developed electrochemical elisa on the meso quickplex sq instrument [mesoscale discovery (msd)]. briefly, standard capacity multi-array -well plates (msd) were coated overnight at °c with ml of mg/ml custom monoclonal rabbit a-ifn-l antibody. blocking was performed with bovine serum albumin (msd blocker a) for h at room temperature with rotation at rpm, followed by washes with pbs and . % tween. standards, controls, and experimental samples were prepared using diluent (msd). culture media samples were diluted : and incubated at room temperature for h with rotation at rpm. after additional wash steps, ml of the mg/ml detection antibody [mouse monoclonal a-ifn-l antibody (buffer exchanged into pbs, mabf ; emd millipore)], conjugated with a sulfo-tag (msd) was added to the wells and incubated for h at room temperature with rotation at rpm. after additional washing, the plates were scanned in a · read buffer (msd) on the meso quickplex sq and the results were analyzed with the msd discovery workbench software. purified recombinant ifn-l protein (prokunina-olsson and others ) was used as a standard at concentrations in the range of pg/ml to ng/ml, and the detection range for the standards was determined as pg/ ml to ng/ml. hepg cells were transfected for h with ng of corresponding constructs with/without cotransfection with . ml of the endoplasmic reticulum stress response element (erse)-luc cignal reporter (qiagen). transfections were done in a -well plate, with replicates per transfection. media was changed h post-transfection, and the plate was assayed for luciferase and renilla using the glomax luminometer. data were presented as relative luciferase units, which correspond to luciferase/renilla ratio. the stable hepg -isre-luc cells were transfected with corresponding constructs in -well plates; untransfected cells were treated with human recombinant interferons-ifna ( ng/ml; pbl assay science) or custom ifn-l ( ng/ ml). all experiments were represented by biological replicates. the media was replaced h post-transfection by ml of full culture media with or mm jak inhibitor [active against jak , jak , and jak , # ; emd millipore, in . % dimethyl sulfoxide (dmso)]. for il r blocking experiment, the transfection culture media was replaced by ml of media with mg/ml of an a-il r blocking antibody (mab ; r&d systems) or mg/ml of a goat isotype igg control (abcam). for treatment experiments, cells were pretreated for h with the jak inhibitor or for h with the a-il r or igg control antibodies and then treated with ifn-l ( ng/ml) or ifn-a ( ng/ml). negative controls included untreated cells, cells treated with . % dmso in media, and cells transfected with control-halo construct. the cells were assayed for isre-luc reporter h post-transfection, which corresponds to h posttreatment with jak inhibitor and antibodies. sirna silencing of ifnlr in hepg -isre-luc cells the stable hepg -isre-luc cells were transfected with corresponding constructs in identical -well plates. the cells were co-transfected with pmol/well of a scrambled negative control sirna (am ; ambion) or a set of sirnas against ifnlr (m- - ; thermo scientific). after h, the cells were treated with culture media, ifn-l ( ng/ml), or ifn-a ( ng/ml). after h, the first plate was assayed for the isre-luc reporter on the glomax luminometer. the second plate was used for rna extraction (zymo research) and mrna expression analysis. cdna was synthesized using super script iii reverse transcriptase (life technologies). specific taqman assays for ifnlr (hs _m ) and endogenous control actb (assay e) from life technologies were used for qrt-pcr analysis on the quantstudio instrument (life technologies). expression of ifnlr was normalized to expression of actb and then expression in samples treated with ifnlr sirna was compared with untreated samples (no sirna) or treated with scrambled sirna. all samples were represented by biological replicates. compared with sirna-untreated samples ( %), the expression of ifnlr was decreased to % in si-scr samples and to % in si-ifnlr samples ( supplementary fig. s d ). unless specified, data plotting and statistical analyses were performed with prism (graphpad), p values are for -sided unpaired t-tests. shown are means and standard errors of the mean. previously, by performing western blot analysis, we were unable to detect ifn-l in culture media of hepg cells transiently transfected with an ifn-l -producing construct, even though this transfection resulted in strong activation of interferon signaling (prokunina-olsson and others ). however, ifn-l was detectable at low levels in culture media of transfected cells by western blot analysis after acetone precipitation (hamming and others ) . now, we developed a mesoscale assay (an electrochemical elisa) and were able to detect ifn-l in culture media of transfected hepg cells (fig. a) . treatment of these cells with a custom a-ifn-l antibody decreased the interferon signaling by % (fig. b) , suggesting that ifn-l generated in this experimental system is a secreted biologically active interferon. simultaneously, the a-ifn-l antibody did not affect signaling of the main interferons (ifn-a, ifn-b, ifng, and ifn-l , fig. c ). signaling induced by ifn-l was strongly attenuated by the jak inhibitor and by blocking of the ifn-l family receptors (ifnlr and il r , supplementary fig. s a -c), confirming these canonical components of the jak/stat pathway as essential elements for the ifn-l signaling. we evaluated the biological activity of the secreted ifn-l by its ability to induce expression of a set of isgs (supplementary tables s -s ). we analyzed mrna ex-pression in cells transiently transfected with ifnl -halo or control-halo constructs and in bystander nontransfected cells exposed to media from the corresponding transfected cells in transwell assays (ifnl -trans and halo-trans, fig. a ). in both hepg and phhs, cells transfected with ifnl -halo or exposed to media from those cells (containing secreted ifn-l ) showed strong induction of isgs, such as ddx (rig-i), dhx , ifih (mda ), isg , mx , oas , and stat and chemokines cxcl and cxcl ( fig. a) . expression of cxcl and cxcl was much higher in phhs compared with hepg , highlighting cell-specific differences ( fig. a) . ifn-l was detectable in culture media of ifnl -transfected phhs and hepg cells, but not in corresponding halo-transfected cells (fig. b) . we did not detect expression of other interferons (ifna , ifna , ifnb, ifng, ifnl , ifnl ) in any of the experimental conditions (supplementary table s ). cxcl encodes ifn-g inducible protein (ip- ), which is a chemotactic factor for neutrophils. high levels of ip- have been associated with inflammation and pathogenesis of chronic hcv infection (harvey and others ; lagging and others ) . we measured the levels of ip- in culture media of phhs and hepg transfected with different constructs. in phhs, ip- was detectable in the media from samples transfected with ifnl -halo and ifnl -halo (fig. b) , while it was undetectable in hepg cells transfected with ifnl -halo (data not shown), in accordance with a much lower cxcl mrna expression observed in hepg compared with phhs (supplementary tables s -s ) . importantly, the a-ifn-l antibody added to the shared media strongly decreased mrna expression of isgs, including cxcl , in nonexpressing transwell phhs (fig. a ) and the amount of ip- secreted to the shared media (fig. a, b) . even though some of ifn-l gets secreted, as described earlier, ifn-l was also detected as a cytoplasmic protein by confocal imaging in fixed cells-in hepg cells transiently expressing ifn-l -halo, and in phhs induced to express endogenous ifn-l (prokunina-olsson and others ). we next performed live imaging of intracellular movements of ifn-l -halo and control-halo proteins (supplementary video s ). we also monitored frap (fig. a ). this method does not detect secreted proteins such as ifn-l , but it helps characterize intracellular proteins based on their mobility (fig. b) , which is proportional to the speed of fluorescence recovery (t½, in seconds). the control-halo protein showed very high mobility, with a t½ of . - . s (fig. c and supplementary video s ), while ifn-l -halo showed a much lower mobility, with a t½ of . - . s (fig. c and supplementary video s ). intracellular mobility of proteins could be limited by their attachment to structural elements, involvement in proteinprotein interactions, or confinement within vesicles (lippincott-schwartz and others ; snapp and others ). therefore, we also estimated the fraction of immobile expression of mrna transcripts was quantified using an antiviral response qrt-pcr plate. the cells were transfected with corresponding constructs for h, and transwell cells shared culture media with the corresponding transfected cells for h. all samples were represented by biological replicates. expression of each target assay on the plate was measured using the same amount of cdna; expression was normalized to a geometric mean of endogenous controls included on the plate. the results are presented as dc t values for targets normalized by endogenous controls, on a log scale; less negative dc t values correspond to higher levels of expression. full expression data for hepg cells and phh are available as supplementary tables s and s . protein, which is represented by unrecoverable fluorescence-nearly % of ifn-l -halo ( . % - . %) compared with * % of control-halo protein ( . % - . %) was estimated to be immobile (fig. d) . we conclude that in our experimental system the control-halo was mostly present as a mobile unattached cytoplasmic protein, in agreement with other studies that used halo-protein (huybrechts and others ) . in contrast, we detected ifn-l -halo both as an intracellularly retained protein with limited mobility and as a mobile protein potentially available for secretion or release. while conducting live confocal imaging in transiently transfected hepg cells, we observed dying cells that underwent morphologic changes of swelling, membrane blebbing, and rupture, followed by release of cytoplasmic content (fig. a, b and supplementary videos s -s ), suggesting necrotic-type cell death (fiers and others ; festjens and others ; galluzzi and others ) . cell rupture events over the course of h of imaging were significantly more frequent in ifn-l + compared with control-halo + hepg cells (fig. c) . flow cytometry analysis showed that necrotic cells (defined as annexin v + and live/dead + ) were significantly more common in cells transfected with ifnl -halo compared with p -halo (a nonfunctional protein isoform of ifn-l ), ifnl -halo, and control-halo constructs (fig. d , e). transfection efficiency was comparable for all these constructs (fig. g , but could not be determined for the secreted ifnl -halo), and thus could not explain the observed differences in cell death. we also evaluated cell death in phhs transiently transfected with ifnl -halo or control-halo constructs. in general, in phhs, cell death rates were very high even in untransfected cells and similar for both constructs (data not shown), possibly due to physiologically high baseline cell death rates in primary cells unrelated to the effect of ifn-l . cell death can also be triggered as a result of an endoplasmic reticulum response to unfolded recombinant proteins generated in vitro. previously, we showed that transient expression of ifnl -halo and its splicing forms-p , p , and p -induced the erse-luc reporter in hepg cells (prokunina-olsson and others ). we next repeated this experiment for the erse-luc reporter cotransfected with ifnl -halo or p -halo. transfection with p -halo did not induce cell death (fig. d , e), despite considerable induction of the esre-luc reporter (fig. f ). we conclude that potential activation of the unfolded protein response to in vitro generated ifn-l cannot explain the observed ifn-l -induced cell death. in an effort to define the mechanism of ifn-l -induced cell death, we assayed for ripk -dependent necrosis (necroptosis) and caspase- -dependent cell death (pyroptosis), using specific inhibitors (necrostatin and yvad). however, we saw no evidence of the activation of these pathways after transient transfection with the ifnl -halo construct (data not shown). treatment with zvad, a pan-caspase inhibitor, caused a significant decrease in ifn-l -induced cell death, which is suggestive of apoptosis, but this effect was also ) . graphs represent of independent experiments, n = . (c) cell viability was analyzed with multi tox-glo assay and presented as the percentage of induced to uninduced cells, n = . **p < . , ***p < . , based on t-tests. onabajo et al. observed in controls (data not shown), implying that transfection itself might be contributing to apoptosis in this experimental system. to further explore our observations done in transient expression system, we developed inducible stable hepg cell lines expressing ifn-l and p fused with c-terminal green fluorescent protein (gfp) and a control-gfp cell line, all of which were under control of a tetracycline-inducible promoter. stable clones showed %- % intracellular expression ( fig a) . secreted ifn-l -gfp was detectable in media after h of induction (fig. b ) and was biologically active (fig. c ). to assess cell death, protein expression was induced for h and cells were stained for live/dead + and annexin v + markers. the ifn-l -gfp-expressing cells showed a significant increase in cell death compared with cells expressing p -gfp or control-gfp (fig. a, b) . in addition, cell viability analysis showed that days of induced ifn-l expression resulted in a % reduction in the counts of viable cells compared with uninduced cells, with no change observed for the controls (fig. c) . although we observed increased annexin v + staining in the induced ifn-l -expressing cells (fig. a) suggesting apoptotic cell death, we did not detect any caspase- activation (data not shown), which is expected in apoptosis. we reasoned that cell death might be associated with decreased proliferation, and we evaluated cell proliferation after h of induced ifn-l expression by measuring brdu incorporation. compared with p -gfp and gfp control, induction of ifn-l was associated with a significant reduction of proliferation by % (fig. a, b) . we induced ifn-l expression in the presence or absence of the rabbit monoclonal a-ifn-l antibody, which we previously found to be able to block the ability of ifn-l to induce interferon signaling (fig. ) . the a-ifn-l antibody treatment attenuated ifn-l -induced cell death (fig. a) , while it increased proliferation (fig. b ) and improved cell viability (fig. c ). since the antibody is not expected to enter the cells and can only block the secreted protein, these results indicate that the observed cell death and decreased viability and proliferation are caused by the secreted ifn-l . interestingly, this effect was localized to ifn-l -expressing cells and was undetectable in nonexpressing bystander cells in the transwell assays (supplementary fig. s ) . thus, the effect of ifn-l may be concentration dependent and primarily affecting the ifn-l -expressing cells. fig. . a-ifn-l antibody inhibits ifn-l -induced cell death and proliferation defect. protein expression of ifn-l -gfp, p -gfp, or control-gfp in stable hepg cells was induced with mg/ml doxycycline for h. at h, cells were treated with mg/ ml of rabbit a-ifn-l antibody or mg/ml of rabbit igg control (rigg). cells in different treatment conditions were assessed for cell death (a), cell proliferation (b), and cell viability (c). graphs represent of independent experiments, n = , *p < . , **p < . , ***p < . based on t-tests. we suggest that ifn-l may have at least functions in human hepatic cells-activation of interferon signaling, inhibition of cell proliferation, and induction of cell death. these roles may be overlapping, synergistic, or independent, and some or all of them may be relevant for hcv clearance. activation of isgs by ifn-l was expected based on our previous observations in transiently transfected hepg cells (prokunina-olsson and others ). early activation of a panel of isgs has also been reported in both in vitro hcvinfected and adjacent uninfected phhs (sheahan and others ) . however, we now determined the following about ifn-l -induced isg activation: it is specifically caused by the ifn-l secreted from cells at low but detectable amounts; it has an effect on both primary hepatocytes and hepatoma cells; it similarly affects the expressing and nonexpressing bystander cells; and it can be specifically blocked by the a-ifn-l antibody. anti-proliferative and cell-death-inducing anti-tumor effects are, in general, characteristic of interferons (wang and others ) , including in hepatoma cell lines (murata and others ) . however, we now present the first evidence of these effects caused by ifn-l , the newest addition to the interferon family. while activation of isgs was observed in both the ifn-l -expressing and nonexpressing bystander cells, the decrease of proliferation and induction of cell death were detectable only in the expressing cells. this could mean that these effects require different concentration thresholds. the dose-dependent anti-proliferative effect in hepatic cells has already been shown for ifn-a (lim and others ) , and the same might be true for ifn-l . anti-proliferative activity of ifn-l is isg dependent as it was blocked by the same a-ifn-l antibody that blocks ifn-l induced activation of isgs. some of the isgs induced by ifn-l , including ¢ ¢oas and ip- , are known for their contribution to the anti-proliferative and pro-apoptotic effects of interferons (rysiecki and others ; hassel and others ; aksoy and others ; liu and others ) . at this point, we are unable to determine the mechanism of ifn-l -induced cell death. although we did not detect activation of caspase- by ifn-l , therapeutic anti-tumor mechanisms of interferons have been related to cell growth arrest and induction of caspase- -independent apoptosis via the p pathway (vogelstein and others ; takaoka and others ; , and this pathway should be tested for ifn-l as well. the effect of ifn-l on activation of interferon signaling and cell death might be complex and intertwined, and further studies are warranted. the genetic association between the ability to generate ifn-l (in carriers of the rs -dg and rs -t alleles) and impaired spontaneous and treatment-induced clearance of hcv is well established, but the molecular understanding of this association is still limited. recently, it has also been reported that the same individuals who are more likely to clear the hcv infection, in fact, are significantly more likely to develop liver fibro-proliferative disease (fibrosis), which is associated with increased mortality (eslam and others ) . our new findings regarding ifn-l function may help reconcile these counterintuitive connections. we suggest that the induction of interferon signaling by ifn-l may have both positive and negative implications. the ifn-l -induced isgs might provide some antiviral protection, explaining the lower pretreatment hcv loads in individuals with the rs -dg allele, compared with those who do not carry this allele (uccellini and others ) . on the other hand, the increased pretreatment expression levels of these isgs have been associated with refractoriness to ifn-a therapy (dill and others ), especially in carriers of the ifnl -rs -dg or rs -t alleles (urban and others ; prokunina-olsson and others ) . this could be because ifn-a and ifn-l compete for induction of the same set of isgs, and, once activated by ifn-l , these isgs do not respond to ifn-a. the fact that despite having lower pretreatment hcv levels, individuals with rs -dg allele are more likely to develop chronic hcv and fail ifn-a-based treatment, suggests that either negative effects of ifn-l outweigh its positive effect on lowering hcv viral load or antiviral effects induced by ifn-l are insufficient for complete viral clearance. the ifn-l effect on proliferation and cell death might also be positive and negative. increased cell death has been correlated with inflammation during hcv infection and was suggested as a factor contributing to liver damage (bantel and schulze-osthoff ) . high rates of cell death in ifn-l -expressing cells could amount to substantial cell loss and inflammation, contributing to the development of liver disease. simultaneously, the anti-proliferative effect of ifn-l might limit the tissue remodeling ability that leads to liver fibro-proliferative disease (fibrosis), as has been reported by the largest study on this subject to date (eslam and others ) . individuals who are genetically unable to produce ifn-l or in whom ifn-l is not sufficiently induced by specific stimuli may be more likely to clear the infection, spontaneously or after treatment, especially with ifn-abased therapies. however, they may not have the benefit of an anti-proliferative effect also contributed by ifn-l , and will be more likely to develop fibrosis. since in our system the a-ifn-l antibody efficiently blocked activation of isgs (including ip- ), and ifn-l induced cell death, blocking of ifn-l with therapeutic agents in carriers of the rs -dg allele might be a plausible therapeutic option before or in conjunction with other treatments. blocking of ifn-l might boost mechanisms of endogenous host immunity, which are important for efficient clearance of hcv and prevention of posttreatment relapse (meissner and others b) , and also decrease liver damage due to inflammation and cell death. although the antiviral role of ifn-l has been shown for a number of viruses (hamming and others ; lu and others ) , it remains to be found what other biologically relevant factors can trigger endogenous ifn-l expression. identification of these triggers in hepatic and other human cell types could help explore the role of ifn-l in other relevant clinical conditions. so far, expression of endogenous ifnl has only been detected in liver biopsies from patients infected with hcv, with significant correlations between hcv titers and expression levels of ifnl and isgs (amanzada and others ; konishi and others ; meissner and others b; murakawa and others ). however, ifnl was undetectable in liver biopsies from patients infected with hepatitis b virus or affected by onabajo et al. unrelated liver diseases (amanzada and others ) . in vitro, ifnl expression could be induced in phhs by hcv infection or treatment with polyi:c, but polyi:c treatments failed to induce ifnl expression in hepg cells. the ifnl region is absent in the genomes of mouse and rat, precluding the development of relevant rodent models. we acknowledge the limitations of our experimental system, which is based on expression of ifn-l in human hepatic cells-a hepatoma cell line hepg and phhs. the observed effects might differ from the physiologic conditions in the hcv-infected hepatic cells in the complex whole-organ environment by the magnitude, duration, and efficiency of ifn-l induction. we also did not address the mechanism of intracellular retention and the function of the nonsecreted ifn-l . however, we provide a comprehensive functional analysis of ifn-l , a novel human interferon, and suggest its role in activation of interferon signaling, inhibition of cell proliferation, and induction of cell death in human hepatic cells. association of the ifnl -deltag allele with impaired spontaneous clearance of hepatitis c virus cxcr surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation interferon-lambda (ifnl ) transcript expression in human liver tissue samples apoptosis in hepatitis c virus infection interferon-induced gene expression is a stronger predictor of treatment response than il b genotype in patients with hepatitis c interferon-lambda rs genotype and liver fibrosis in viral and non-viral chronic liver disease necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response more than one way to die: apoptosis, necrosis and reactive oxygen damage predictive value of the ifnl polymorphism on outcome of telaprevir, peginterferon, and ribavirin therapy for older patients with genotype b chronic hepatitis c molecular definitions of cell death subroutines: recommendations of the 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ifn-lambda : the paradoxical new member of the interferon lambda family a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus constitutive expression of a ¢, ¢-oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness measuring protein mobility by photobleaching gfp chimeras in living cells integration of interferon-alpha/beta signalling to p responses in tumour suppression and antiviral defence new aspects of ifn-alpha/beta signalling in immunity, oncogenesis and bone metabolism reduced ifnlambda activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes genetic variation in il b and spontaneous clearance of hepatitis c virus hcv rna levels in a multiethnic cohort of injection drug users: human genetic, viral and demographic associations il b genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis c surfing the p network interferon: current status and future prospects in cancer therapy the work has been supported by the intramural research program ( key: cord- -rzdxdzp authors: jenks, christopher l.; uysal, askin; papacostas, michael f. title: drug hypersensitivity causing organizing eosinophilic pneumonia in a pediatric patient date: - - journal: heart lung doi: . /j.hrtlng. . . sha: doc_id: cord_uid: rzdxdzp objective: to describe a relatively rare hypersentivity reaction with pulmonary manifestations in a pediatric patient. data sources: electronic medical records. study selection: patient treatment in the pediatric critical care unit. data extraction and synthesis: electronic medical records. conclusions: eosinophilic pneumonias are rare in the pediatric population. peripheral eosinophilia is not necessary to make the diagnosis. bronchoalveolar lavage is the diagnostic study of choice. lung biopsies are rarely needed to make the diagnosis. the treatment of choice is steroids. if steroids fail to improve the patient's condition, consider ivig, and cyclosporine a. a year old female with no significant past medical history presented to our emergency department for evaluation of dyspnea and fever. she is normally an active girl, with no smoking history, no second hand smoke exposure at home, and no history of illicit drug use. about weeks prior to her presentation, she was seen by her primary care physician for an abscess on her thumb. she started a course of sulfamethoxazole/trimethoprim. shortly thereafter, she developed a fever, cough, and a diffuse rash. she stopped taking the sulfamethoxazole/trimethoprim after days due to decreased oral intake. she returned to her primary care physician for re-evaluation, and was diagnosed with an acute otitis media (although no ear symptoms were present at the time). she was started on amoxicillin. she stopped the amoxicillin after one day due to nausea and vomiting. her amoxicillin was then changed to azithromycin. she promptly stopped taking it due to profuse vomiting and an urticarial type rash. she then presented to an urgent care facility. her work up included a rapid flu and strep both of which were negative. she was given a prescription for topical hydrocortisone and oral diphenhydramine. she started to improve so her mother re-started the original sulfamethoxazole/trimethoprim. shortly after re-starting the sulfamethoxazole/trimethoprim, she developed diffuse myalgias, dyspnea, and fever to . c. she then went to an emergency department. work up included another rapid flu (negative), a rapid strep (negative), wbc thousand/ mm , and a chest roentgram (cxr) which was consistent with a "viral process." she was discharged on oral prednisone mg a day. her dyspnea worsened necessitating ambulance transport to our facility. on presentation, she complained of cough, dyspnea, upper abdominal pain worse with deep inspiration, and anxiety to the point that she was afraid to go to sleep. she had no problems with confusion, seizures, emesis/diarrhea, chest pain, or swollen joints. there was no history of recent travel, tick bites, or sick contacts. her blood pressure was / , heart rate , respiratory rate , temperature . c, height cm, weight . kg, body mass index . , pulse oximetry % on l nasal cannula. physical exam was significant for a clear oropharynx, coarse breath sounds which were decreased in the bases bilaterally, tachypnea and tachycardia. she was admitted to the general pediatric floor. her oxygen was continued. cxr showed bibasilar interstitial prominence. heart & lung j o u r n a l h o m e p a g e : w w w . h e a r t a n d l u n g . o r g a cxr revealed pneumomediastinum, pneumopericardium, and bilateral pneumothoraces. oxygen saturations noted to be %. intubation, pleural decompression followed by bilateral chest tube placement helped stabilize her condition. oxygen saturations returned to normal. a peripherally inserted central catheter (picc) line was placed in her left upper extremity. the following day a computerized tomography (ct) scan of the chest with pulmonary embolism protocol revealed saddle thrombus located in the right pulmonary artery seen within the middle and lower lobe lobar, segmental, and subsegmental arteries with a probable tiny amount of thrombus within the left lower lobe segmental and subsegmental arteries, also noted to have ground glass appearance of the lower lobes bilaterally. hypercoagulable work up included homocysteine level, protein c and s, antithrombin iii, factor ii, factor v leiden, and factor viii all which were normal. lupus anticoagulant anticardiolipin antibodies, b glycoprotein were all negative. ultrasound of her left upper extremity showed an occlusive thrombus associated with the picc line involving the axillary and basilica vein. she did not have any thrombosis in her lower extremities. she was placed on a continuous heparin drip with goals to keep her anti xa levels . e . . blood and urine cultures remained negative. tests for tularensis, rickettsi, erlichia, hanta virus, human immunodeficiency virus, herpes simplex virus (hiv), pneumocystis were negative. bronchoscopy with bronchoalveolar lavage (bal) was negative for cytomegalovirus, legionella, ebstein barr virus, yeast, and acid fast bacilli. her bal showed % eosinophils. an open lung wedge biopsy was negative for mycobacterium, mycoplasma, and herpes simplex virus. the surgical pathology returned as organizing eosinophilic pneumonia consistent with a drug hypersensitivity reaction. no evidence of infectious organisms, vasculitis, or evidence of aspiration seen. we re-started her on corticosteroids after which she showed continuous improvement over the next weeks. her pneumomediastinum and pneumothoraces resolved, and her chest tubes were removed. we discharged her on enoxaparin for her pulmonary embolism. the eosinophilic pneumonias encompass a wide variety of different interstitial lung disease, but all typically have eosinophilic infiltration of the lungs. the exact role of the eosinophil is these disease processes are unknown, but may be related to tissue injury from inflammation and eosinophilic degranulation. the eosinophilic pneumonias are generally placed into two large categories: idiopathic and secondary. the idiopathic group contains eosinophilic granulomatosis with polyangiitis, idiopathic chronic eosinophilic pneumonia, and idiopathic acute eosinophilic pneumonia. the secondary group includes parasites, drugs, toxins, allergic bronchopulmonary aspergillosis and neoplasms. many drugs have an association with eosinophilic pneumonia, but not all have a proven causal relationship. some of the associated drugs include the following: antibiotics (beta lactams, tetracycline, sulfonamides, nitrofurantoin, isoniazid, ethambutol, pentamadine), antiepileptics (carbamazapine, phenytoin), illicit drugs (heroine, cocaine), non-steroidal anti-inflammatory drugs (ibuprofen, naproxen, indomethacin, diclofenac, piroxicam), antidepressants (amitriptyline, trazodone, venlafaxine), chemotherapeutic agents (bleomycin, camptothecin), cardiovascular drugs (angiotensin converting enzyme inhibitors, amiodarone, b-blockers), biologics (infliximab, interferon alpha, granulocyte e macrophage colony stimulating factor), and nutraceuticals (l-tryptophan). eosinophilic pneumonia is not common in the pediatric population. the mean age is typically in the late s. the exact incidence is unknown. there appears to be an association with hiv, smoking, and hypersensitivity to drugs or toxin exposure. our patient seemed to have a causal relationship with sulfamethoxazole/ trimethoprim. the presentation is typically rapid over the course of e days, and generally involves fever, myalgias, pleuritic chest pain, crackles on lung exam, plus or minus peripheral eosinophilia as was the case in our patient. a cxr typically shows non-specific findings but includes consolidation, hilar adenopathy, pleural effusions, and reticulonodular densities. ct scans of the chest usually show ground glass opacities, nodules, or irregular lines. the ct chest in our patient demonstrated a ground glass appearance as well as a pulmonary embolism. bronchoalveolar lavage is the diagnostic study of choice to diagnose an eosinophilic lung disease as it may be the only clue revealing a high eosinophil count (typically > % when the normal in bal fluid is < %). our patient had a slight elevation in her bal eosinophils likely due to the fact that she had been partially treated with corticosteroids prior to her hospital presentation. a recent search of the available literature has revealed only a few papers that describe an association with a pneumothorax related to an eosinophilic pneumonia (in the setting of a paragonimiasis infection). there have been very few reported cases of organizing eosinophilic pneumonia being associated with pulmonary embolism or a pneumomediastinum. our patient's condition was associated with a pneumothorax, pneumopericardium, and pneumomediastinum. the exact etiology of the pulmonary embolism is unclear. some possible etiologies could include the picc line, an undiagnosed hypercoagulable disorder, or related to the inflammation of the eosinophilic disorder. her hypercoagulable work up was negative but an undiagnosed mutation is always a possibility. eosinophilic pneumonia has no obvious association with pulmonary embolism but still could be the possible etiology. the picc line was present for less then hours, but theoretically could have been the cause. an interesting component of our patient is that she did not readily improve on her initial corticosteroid treatment, but did clinically respond to the corticosteroids later on in her hospital course. corticosteroid refractory eosinophilic lung disease has been previously reported in the pediatric literature. ronald mortan et al reported a neonate at weeks of life that had a protracted course of pneumonia, received several courses of corticosteroids with no improvement. at weeks of life the patient had a lung biopsy which showed the eosinophilic pneumonia. the patient was then treated with an empiric course of intravenous immunoglobulin (ivig). the ivig was ineffective and a trial of cyclosporine a was instituted resulting in rapid clinical improvement. eosinophilic pneumonias are rare in the pediatric population. peripheral eosinophilia is not necessary to make the diagnosis. when this diagnosis is entertained, a bal should be performed as early as possible because it is the diagnostic study of choice. lung biopsies are rarely needed to make the diagnosis, but may be necessary if the primary diagnostic study of choice is unrevealing. the treatment of choice is corticosteroids. if corticosteroids fail to improve the patient's condition, other treatment options could include ivig, and cyclosporine a. eosinophilic lung diseases. immunol allergy clin north am eosinophilic lung diseases pleuropulmonary infection by paragonimus westermani in the united states: a rare cause of eosinophilic pneumonia after ingestion of live crabs steroid-refractory neonatal eosinophilic pneumonia responsive to cyclosporin a. am j respir crit care med key: cord- - fmouoal authors: tong, pearl shuang ye; kale, anita sugam; ng, kailyn; loke, amelia peiwen; choolani, mahesh arjandas; lim, chin leong; chan, yiong huak; chong, yap seng; tambyah, paul anantharajah; yong, eu-leong title: respiratory consequences of n -type mask usage in pregnant healthcare workers—a controlled clinical study date: - - journal: antimicrob resist infect control doi: . /s - - -z sha: doc_id: cord_uid: fmouoal background: outbreaks of emerging infectious diseases have led to guidelines recommending the routine use of n respirators for healthcare workers, many of whom are women of childbearing age. the respiratory effects of prolonged respirator use on pregnant women are unclear although there has been no definite evidence of harm from past use. methods: we conducted a two-phase controlled clinical study on healthy pregnant women between to weeks gestation. in phase i, energy expenditure corresponding to the workload of routine nursing tasks was determined. in phase ii, pulmonary function of subjects was measured whilst at rest and exercising to the predetermined workload while breathing ambient air first, then breathing through n -mask materials. results: exercising at met while breathing through n -mask materials reduced mean tidal volume (tv) by . % ( % ci − . % to − . %, p < . ) and lowered minute ventilation (ve) by . % ( % ci − . % to − . %, p < . ), with no significant change in breathing frequency compared to breathing ambient air. volumes of oxygen consumption (vo( )) and carbon dioxide expired (vco( )) were also significantly reduced; vo( ) by . % ( % ci − . % to − %, p = . ) and vco( ) by . %, ( % ci − . % to − . %, p = . ). although no changes in the inspired oxygen and carbon dioxide concentrations were demonstrated, breathing through n -mask materials during low intensity work ( met) reduced expired oxygen concentration by . % ( % ci: − . % to − . %, p < . ), and increased expired carbon dioxide by . % ( % ci: . % to . %; p < . ) suggesting an increase in metabolism. there were however no changes in the maternal and fetal heart rates, finger-tip capillary lactate levels and oxygen saturation and rating of perceived exertion at the work intensity investigated. conclusions: breathing through n mask materials have been shown to impede gaseous exchange and impose an additional workload on the metabolic system of pregnant healthcare workers, and this needs to be taken into consideration in guidelines for respirator use. the benefits of using n mask to prevent serious emerging infectious diseases should be weighed against potential respiratory consequences associated with extended n respirator usage. trial registration: the study was registered at clinicaltrials.gov, identifier nct . lessons learnt on infection control from the severe acute respiratory syndrome (sars) pandemic in have been used to formulate strategies [ , ] to manage the recent middle eastern respiratory syndrome (mers) [ ] and h n influenza outbreaks [ ] . these infection control measures include recommendations for increased use of protective filtering face-piece respirators (ffr), such as n -masks [ ] especially during aerosol generating procedures. existing influenza pandemic control plans in many countries have also incorporated recommendations for more widespread use of ffr [ ] [ ] [ ] . when the influenza a h n pandemic was declared in , there were guidelines for universal use of n -masks despite a lack of scientific evidence for its appropriateness in different health care settings [ , ] . the n ffr has also been recommended for the novel mers coronavirus. little is known about the effects of n -masks on the respiratory function of pregnant healthcare workers, who can be subjected to prolonged usage of ffr because of their vulnerability to complications from influenza, varicella, and other pathogens transmitted via the respiratory tract [ ] . it is also known that pregnant women have a significantly greater respiratory burden due to factors such as increased oxygen (o ) demand, increased nasal airway resistance, decreased functional residual capacity due to diaphragmatic splinting; all these contributing to the "physiologic" dyspnea of pregnancy [ ] . there are also robust data linking respiratory compromise and adverse perinatal outcomes in women who have chronic respiratory conditions, from large scale studies on women with conditions such as asthma and obstructive sleep apnea. these outcomes include preterm labour, impaired fetal growth, and pre-eclampsia [ , ] . balancing the potential benefits of respiratory protection against the possible discomfort [ ] and potential additive adverse effects on the respiratory functions of pregnant healthcare workers is difficult in the absence of clear data although there is no definite evidence of harm from decades of use of such respirators [ ] . a recent study comparing a cohort of pregnant women between to weeks gestation and non-pregnant women showed no differences in respiratory rate, oxygen saturation and transcutaneous carbon dioxide levels in pregnant compared with non-pregnant subjects wearing the n ffr during exercise and sedentary activities for over a -hour period [ ] . however, that study did not specifically examine the impact on busy healthcare workers. pregnancy has been reported to be the most common cause for denying medical clearance for n -mask use in a non-medical setting but the specific adverse effects of the respirator itself have not been documented [ ] . our study was performed to address the limited data on n -mask usage in pregnancy with the aim of investigating the effects of breathing through the n mask materials on respiratory functions at rest, during low intensity work, and recovery thereafter in pregnant healthcare workers. the differences in work of breathing and potential adjustments in respiration that are contributed by pregnancy may provide guidance on the use of n -masks by pregnant health care workers in high-risk environments. this controlled clinical trial was carried out in phases in the investigational medicine unit of national university hospital (nuh), singapore. study procedures were approved by the national healthcare group domain specific review board in july (reference number: / ), and written informed consent was obtained from all participants. healthy women with singleton pregnancies between to weeks gestation were recruited on a voluntary basis from amongst hospital staff and clinic patients. eight pregnant health care workers were recruited in phases i and pregnant women were recruited in phase ii to participate in the study. subjects were instructed to have adequate rest and to avoid strenuous activity prior to the study to ensure that the tests were conducted under normal lifestyle conditions. all subjects were told to have their meals at least h before start of study. a screening questionnaire was administered to each subject, who then had baseline medical and obstetric examinations prior to participating in the study. subjects had spontaneously conceived singleton pregnancies and were between to years old. they had no history of cardiorespiratory illness, influenza-like illness in the week prior to the trial, or any pregnancyrelated complications such as gestational diabetes, hypertension, intrauterine growth restriction, placenta praevia, ruptured membranes, or threatened preterm labor. they were also free of any neuromuscular conditions that would preclude them from using the treadmill. their hemoglobin levels were ≥ g/dl, and they did not have any haemoglobinopathies such as thalassemia that could interfere with oxygen carriage in the blood. in phase i, the volume of o uptake (vo ) corresponding to the workload of routine nursing tasks in the ward was determined. these healthcare workers wore, and breathed through, a tight-fitted respiratory mask (hans rudolph, v-mask, kansas) that was attached by a harness to a portable telemetric metabolic cart [ ] [ ] [ ] (k b , cosmed s.r.l, rome, italy) while moving about freely performing simulated routine nursing tasks in a specific order, such as walking around the ward, sponging and transferring mannequins from beds to chairs with another assistant (fig. ). their average work intensity was determined with vo (ml/kg/min) measurements and converted to the corresponding metabolic equivalents (met) to gauge the energy expenditure ( met is equal to . ml/kg/min of o consumed). in phase ii, the respiratory effects of wearing n masks were examined. each subject underwent two minute exercise cycles on a treadmill. each subject wore a hans rudolph mask, similar to that in phase i, attached to a laboratory-based metabolic cart (cortex metalyser br , leipzig, germany) in order to obtain real time respiratory parameters during exercise. in the first (control) cycle, subjects wore the hans rudolph mask with the outlet opened to ambient air. in the second (n ) cycle, outlets of the hans rudolph masks were covered by materials obtained from representative supplies of n masks ( m, st. paul, mn, usa). n mask materials were trimmed to form an airtight seal over the hans rudolph mask outlet so that the air flow resistance on inspiration and expiration would come from the mask material, simulating the actual wearing of an n respirator (fig. ) this experimental design allowed each subject to act as her own control. the fine adjustments were made to the treadmill speed every min to maintain energy expenditure at met. similar treadmill speed profile was repeated in the second exercise cycle for each subject. for both control and n cycles, respiratory parameters were measured during an initial -minute rest period, followed by a -minute exercise period, and subsequently a -minute rest period. there was a -minute break between control and n cycles. prior to the n cycle, an additional -minute conditioning period was allowed to enable patient to adapt fig. determination of average work intensity of health care workers: in phase i pregnant subjects performed simulated patient care activities while breathing through a tight fitting mask with a pneumotachometer. oxygen content was sampled at every breath and measured with a portable telemetric metabolic cart to n -respirator conditions. the subjects breathed through the n mask materials continuously throughout the n cycles (fig. ) . a cardiotocography (ctg) was performed prior to the study and after each exercise-cycle. finger-prick lactate concentrations were measured immediately before and after each exercise-cycle with lactate pro (arkray global business inc). the borg scale questionnaire [ ] was administered after each exercise cycle to measure the rating of perceived exertion. the borg scale ranges from for "no feeling of exertion," to which corresponds to "very, very hard exertion". a fully equipped resuscitation cart was present throughout the study with trained medical personnel available to immediately address any medical concerns of the subjects. in both phases, the participants wore a tight fitting mask (hans rudolph) that was attached to the metabolic cart through an air sampling tube. inspired ambient air and expired air were channeled through a pneumotachometer that was attached to the front of the mask which calculated air volume by the rate of rotation of a rotor turbine located within it. the turbine had zero resistance to air flow and the rate of rotation of the turbine, sensed by infrared light within the pneumotachometer, corresponds directly to inspired and expired air volume for each breath. multiple air samples from each expired-breath was drawn into the metabolic carts through a sampling line for the measurement of oxygen and carbon dioxide content by the respective gas sensors within the metabolic carts. from this data, the following parameters were calculated: volumes of oxygen (vo ) and carbon dioxide (vco ) exchanged, breathing frequency (bf), tidal volume (tv), minute ventilation (ve), forced expired o (feo ), forced expired co (feco ), forced inspired end-tidal o and co concentrations (fi et o , fi et co ). the calibration procedures for both portable and laboratory-based metabolic carts were carried out according to the manufacturer's instructions and were performed daily to ensure uniformity in their measurements. the study was conducted in a standardized air-conditioned room similar to a hospital ward with constant humidity and temperature. the study was to be terminated if: ( ) the ctg revealed that the fetus was adversely affected by the mother's activity on the treadmill either by a suspicious or pathological trace as defined by the national institute for health and care excellence [ ] ,( ) the subject was unable to complete the trial for any reason including breathlessness or pain, ( ) injury was sustained as a result of the exercise, or ( ) maternal heart rate was > beats/min [ , ] . since there were many respiratory variables of interest, the sample size calculations were performed on an overall picture that there was at least a % difference between breathing through n mask materials vs control for any of respiratory variable of interest. postulating that breathing through n mask materials would have an at least % variation (with standard deviation %) from the control respiratory variables, recruitment of subjects would have a % power and a sided p-value of % to show a statistically significant result. a mixed linear model analysis (to handle paired observations) adjusting for relevant covariates was performed. all analyses were performed using ibm spss version . (armonk, ny) and statistical significance was set at p < . . twenty-eight pregnant women were recruited between september and september . in phase i, the mean and sem of vo was . (± . ) ml/kg/min, which was equivalent to about~ met. all subjects enrolled in phase i fulfilled the inclusion criteria and completed the study. subjects in phase ii were then subjected to this workload. for phase ii, subjects were screened. two subjects were excluded because of maternal anemia, and a third was excluded because the fetus exhibited ventricular bigeminy on ultrasound examination prior to starting the study. one subject experienced uterine contractions and did not complete the study, bringing the total number of subjects who completed phase ii of the study to . there were no other trial terminations due to adverse events. the mean age of the subjects in phase ii was . (± . ) years, their average gestation was . (± . ) weeks, and mean bmi was . kg/m (± . ). of the cases, were primigravidas and were multigravidas. there were nurses, homemakers, and women doing administrative work. during the pre-exercise rest period, breathing through n -mask materials lowered tv by a mean of . l compared with controls ( % ci: − . , − . ; p < . ) (fig. a) . tv during both exercise cycles increased rapidly to reach a plateau which was about % higher than that observed at rest, within a minute. however, compared with controls, exercising with n -masks reduced mean tv by . l ( % ci: − . , − . ; p < . ), a % decrease (fig. a, table ). mean bf increased by % during exercise for both control and n cycles compared to the rest period, but there was no difference in bf with and without wearing n -masks (fig. b, table ). wearing n -respirators lowered v e by . %, a mean difference of . l/min ( % ci: − . , − . ; p < . ) (fig. c , table ). significant differences in tv and v e with n cycles persisted in the post-exercise rest period (table ) . there was a tidal volume reduction of . l (p = . ) and minute ventilation reduction of . l/min (p = . ). forced expired o concentration (feo ) during the pre-exercise rest period decreased by . % ( % ci: − . , − . ; p = . ) with n -mask use versus controls (fig. a, table ). wearing of n -masks during exercise reduced feo by . % compared with controls ( % ci: − . , − . ; p < . ). reduction in feo with the use of n -masks persisted in the post-exercise rest period. concomitantly, forced expired co concentration (feco ) was significantly elevated with n -mask use in the pre-exercise, exercise and post exercise periods compared with no mask usage (fig. b, table ). during the exercise period, the wearing of n -masks resulted in increase in feco of . % ( % ci: . , . ; p < . ) compared with controls. (fig. b, table ). in contrast, no significant differences were observed in the inspired oxygen (fio ) or carbon dioxide (fico ) concentrations of inspired air before, during or after exercise (fig. , table ). when performing work on the treadmill equivalent to met, vo and vco increased by about two-fold for all subjects compared to the rest periods. strikingly, wearing of the n -mask during exercise resulted in lowering vo by . %, a mean of . ml/ min/kg ( % ci: − . , − . ; p = . ) (fig. a , table ). similarly, vco was lowered by . %, a mean of . ml/min/kg ( % ci: − . , − . ; p = . ) (fig. b, table ). for all subjects, overall maternal heart rate increased from ± . to ± . beats/min with exercise. there were no significant difference in heart rate between the n -masks and control cycles. there were also no changes in basal fetal heart rates (mean heart rate of beats per minute) or variability ( - beats per minute) in all the ctgs. there were no significant differences in there were no differences in finger-tip capillary oxygen saturation levels with the mask and without the mask; . ± . % and . ± . % respectively. the borg scale indicated that exercise induced a borderline increase in perceived effort from being . (± . ) to . (± . ) after the n cycles. these parameters did not reach statistical significance (table ). we found that in women in mid-pregnancy, breathing through the n respirator material when performing low intensity work significantly reduced vo ( . %) and vco ( . %), which was due to a corresponding decrease in v e ( . %) and tv ( %), without a compensatory increase in bf. this decrease in air intake volume, together with unchanged concentrations of inspired o and co imply a decrease in overall amount of o and co inspired. coupled with a . % decrease in feo and an . % increase in feco , these results suggest an increased consumption of o and production of co , leading to possible concerns regarding prolonged usage of n -masks on respiratory functions in pregnant women performing physical work. the decrease in v e , tv, feo and the increase in feco were also significant during the rest periods. these results suggest that breathing through the n mask material can limit the overall volume of amount of oxygen intake and also increase the rate of metabolism. when performing work equivalent to routine bedside nursing with n -masks, non-pregnant subjects have been previously reported to maintain their v e compared with controls, with non-significant changes in the tv and bf [ ] . other studies in both non-pregnant and pregnant subjects have shown, on the contrary, an increase or decrease in bf but the tv and v e were not measured in these trials [ , , ] . in contrast, our pregnant subjects were unable to proportionately increase both tv and bf to maintain their v e during rest and in response to exercise while breathing through n -masks materials. a significant % reduction in v e, with a % reduction in mean tv was noted during exercise, possibly due to diaphragmatic splinting. this decrease in v e led to a corresponding decrease in vo ( . %) and vco ( . %) (fig. ) . the decrease in feo and increase in feco are likely due to a stimulation of the respiratory drive resulting in greater efforts required to breathe through the n mask materials and a concomitant greater extraction of o for aerobic metabolism. these results also suggest that the n mask may impede gaseous exchange resulting in hypoventilation. although the subjects appeared to adapt to the increased workload with no changes in the other maternal and fetal physiological parameters with no evidence of hypoxia found on finger-tip capillary oxygen saturation nor increased lactic acid production to suggest a shift toward anaerobic respiration, our results suggest that performing physical work with the n -mask appears to stress the aerobic metabolism and increase the co load within the circulation. in non-pregnant subjects, it has been shown that use of n -respirators can increase co levels within the masks by . - %, suggesting that the increase in expired co concentration could also be due to the accumulation of expired co trapped in the dead space of the n mask [ , ] . our results do not support such a view because fico did not increase even with the use of n materials and total co intake was reduced due to the corresponding decrease in v e . these results further affirm that the increase in expired co mainly arose from increased rate of aerobic metabolism. the higher circulating co concentration observed in our study was in agreement with increase in transcutaneous co observed in pregnant and non-pregnant women after a -minute exercise wearing a respirator as compared to not wearing the respirator [ ] . it must be borne in mind that co levels in the blood of pregnant women are usually lower due to physiological hyperventilation. an increase in forced expired co which reflects a rise in blood co levels hence contributes to impair elimination of fetal co as arterial co is normally reduced in pregnancy to allow for a steeper diffusion gradient of co from fetal blood across to the mother. our study was limited in that we were unable to evaluate the effects of n -mask usage at higher work intensities and over longer durations because of ethical concerns. to ascertain safety, parameters such as maternal and fetal heart rate changes, lactate and fingertip capillary oxygen saturation levels were monitored to ensure that no significant hypoxemia was induced in our subjects and their fetuses. wearing the hans rudolph mask with aperture occluded by the mask material allows accurate measurement of respiratory parameters despite its use not being exactly the same as the standard use of the n mask. however this is the closest way of obtaining accurate physiologic data to best characterize the impact of the materials used in the respirators. the other limitation of our study was the narrow window of gestation studied (between to weeks). these group of women were deemed to be representative of pregnant women as they would have undergone marked respiratory adaptations to pregnancy, especially diaphragmatic splinting from the enlarging uterus. however, it is postulated that, in situations of exercising at a higher intensity, prolonged n -mask usage or at more advanced gestations, a greater degree of oxygen deficit due to a corresponding decrease in v e can have more marked effects on the respiratory function of pregnant women. we also focused exclusively on pregnant women without using nonpregnant controls to better define the risks in this group which are the most controversial. our study demonstrates, for the first time, that pregnant women in mid-pregnancy are unable to maintain their minute ventilation while breathing through n -mask materials. there is also a decrease oxygen uptake and increase carbon dioxide production as a result of the increased workload on breathing imposed by mask use, both at rest and low work intensity. this supports the view of some that pregnant healthcare workers should probably refrain from prolonged n -mask use towards the third trimester. the complications to the women and her fetus that can result from a prolonged decrease in v e and increased work of breathing, are unknown. while there is a substantial negative change in tv, v e and the vo and vco exchanged, there was no impact of breathing through n mask materials, to finger-tip oxygen saturation, maternal or fetal heart rate and no drive to increase bf in pregnancy compared to breathing ambient air at the level of exercise in our study. this study shows important descriptive findings of changes to respiratory physiology with mask use, which do not appear to have sufficient significant clinical impact based on the parameters monitored, that had been deliberately kept within the normal ranges to ensure safety of the subjects. although harm was not demonstrated in the context of this experimental protocol, the significant changes to respiratory physiology caused by breathing through n mask materials raise the concern regarding prolonged use of n -masks by pregnant healthcare workers. our results suggest that pregnant women may experience more fatigue and require more rest breaks from mask use. scheduled work breaks should be considered for pregnant healthcare workers working in high risk areas which require prolonged use of n respirators. in face of the imminent threat of pandemic airborne respiratory diseases it should be emphasized that the benefit of using n mask to prevent serious emerging infectious diseases should be weighed against possible respiratory consequences associated with n mask usage. use of alternative protective methods such as surgical masks with lesser airway resistance, should be considered in appropriate settings [ ] [ ] [ ] . these have been shown to be equally effective for the prevention of droplet infections such as influenza [ ] , although they are insufficient for protection against airborne pathogens. innovative interventions to improve the design of ffr, are urgently needed in the face of the imminent threat of pandemics of acute airborne respiratory infections. the key is to ensure healthcare worker 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obstetrics and gynecology for exercise during pregnancy and the postpartum period effects of physical exercise on pregnancy outcomes: a meta-analysis review physiological impact of the n filtering facepiece respirator on healthcare workers the physiological cost of wearing a disposable respirator pulmonary and heart rate responses to wearing n filtering facepiece respirators physiologic effects and measurement of carbon dioxide and oxygen levels during qualitative respirator fit testing short-term measures of carbon dioxide levels, physiological indicators and subjective comfort of healthcare workers wearing n masks. influenza other respir viruses surgical mask vs n respirator for preventing influenza among health care workers: a randomized trial protecting healthcare workers from pandemic influenza: n or surgical masks? quantifying exposure risk: surgical masks and respirators we would like to extend our gratitude to the defence science organisation for their technical support during the study, and to the department of obstetrics and gynaecology, national university health system for funding the study.author details department of obstetrics and gynecology, national university hospital, mandalay road, singapore , singapore. lee kong chian school of medicine, nanyang technological university, singapore, mandalay road, singapore , singapore. biostatistics unit, national university of singapore, singapore, republic of singapore. medicine, national university of singapore, e kent ridge road, level , singapore , singapore. the authors declare that they have no competing interests.authors' contributions pst, mac, ysc, cll, pat, ely designed the trial. pst, apl, kn, ask, cll, ysc, ely were responsible for recruitment of subjects, organisation and conduct of the trial. yhc analysed the data and advised on statistical issues. pst wrote the first draft of the report. mac, ysc, cll, pat, ely contributed to redrafting. all authors read and approved the final manuscript. key: cord- -zvb bxix authors: connolly, john title: the “wicked problems” of governing uk health security disaster prevention: the case of pandemic influenza date: - - journal: disaster prev manag doi: . /dpm- - - sha: doc_id: cord_uid: zvb bxix purpose: the purpose of this paper is to examine the governance and policy-making challenges in the context of “wicked problems” based on the case of pandemic influenza. design/methodology/approach: the case study research is based on an analysis of official documentation and interviews with policy elites at multiple levels of uk governance. findings: results of this study show that policy actors regard risk communication, the dynamics of international public policy and uk territorial governance as the main governance challenges in the management of influenza at a macro-level. the paper also serves to identify that although contingencies management for epidemiological issues require technical and scientific considerations to feature in governance arrangements, equally there are key “wicked problems” in the context public policy that pervade the health security sector. practical implications: the study indicates the need to build in resources at a national level to plan for policy coordination challenges in areas that might at first be seen as devoid of political machinations (such as technical areas of public policy that might be underpinned by epidemiological processes). the identification of the major governance challenges that emerge from the pandemic influenza case study is a springboard for a research agenda in relation to the analysis of the parallels and paradoxes of governance challenges for health security across eu member states. originality/value: this paper provides a novel interrogation of the pandemic influenza case study in the context of uk governance and public policy by providing a strategic policy lens from perspective of elites. in april the british public were reminded that pandemic influenza (flu), in the context of health security, continues to be a major concern for uk policy-makers. there is recognition by the uk government that a pandemic remains the top health risk for the population (nhs england, ) . the risk of pandemic flu has come into the spotlight recently due to the fact that key measures to prevent such a diseaseinduced disaster may not be effective. a review of the effectiveness of tamiflu [ ] (the main contingencies measure to manage a pandemic) produced by the cochrane collaboration ( jefferson et al., ) , referred to in the rest of this paper as "cochrane review", in the uk concluded that tamiflu had no significant impact on reducing hospital admissions and does not serve to prevent the person to person spread of influenza. this led to pharmaceutical companies retorting by questioning the reliability and validity of the review. uk health authorities responded by insisting that investing in tamiflu remained "a good insurance policy for the population" . the debates around the cochrane review (which are often scientific and epidemiological in nature) also served to highlight the public policy-orientated challenges to managing health security threats such as a pandemic. this paper seeks to delve into the public policy aspects of contingencies management for disease threats from the perspective of policy elites based on the case study of the influenza pandemic in the uk. the paper addresses a lacuna in uk disaster and crisis management literature by contributing to the "governance" aspects managing health security. there are studies which consider crisis management, resilience and risk in the context of uk public policy (e.g. mcconnell, ; drennan and mcconnell, ; brassett et al., ) , however, there are very few case-based research studies which illustrate crisis and disaster governance challenges from the perspective of those institutions and policy actors that are responsible for managing such "wicked problems" from a macro-level policy position. from a public policy point of view, pandemics represent challenges that are unstructured, relentless and cross-cuttingall hallmarks of a wicked problem (weber and khademian, , p. ) . first, as weber and khademian ( , p. ) note, issues that can be viewed in this way are unstructured given that there are high informational demands, there is not always a clear solution to the problem and they have multiple ripple effects. second, the relentlessness of such problems emerges as a result of the fact that the fight is never over and a line cannot be drawn under themthere is no finality. third, cross-cutting refers to the fact that wicked problems involve multiple stakeholders who have a range of views and knowledge within a complex political and economic context. the complexities of disease threats and their transcendence of systems elucidates the fact that health security and pandemic prevention involves trade-offs, require flexibility, resource sharing and collaboration to ensure policy success (durant and legge, ) . other examples of wicked problems include responding and managing climate change, social justice, drug trafficking, immigration, and epidemics. pandemic influenza fits with the notion of a wicked problem given that they call for multi-level and multi-actor responses across territories requiring a high degree of resilience to deal with the contours of the disease (unstructured and cross-cutting). at a population level the risk of a pandemic endures over time as a result of the mutation of disease strains and the impact of pandemics can traced over many years (relentless). indeed, policy-makers are well aware that in the last century alone the , and influenza pandemics have contributed to millions of fatalities as well as vast economic and social disruption (kamradt-scott and mcinnes, , pp. - ) . the case of pandemic influenza is a "way in" to understanding the policy dynamics of the health security sector in the context of disaster research. the issues to emerge from pandemic influenza are likely to have consequences for the governance of other diseases (and, naturally, reference to other diseases will be made in this paper given policy responses to specific diseases do not exist in a vacuum). the question that underpins the paper, therefore, is: what does the case study of pandemic influenza tell us about what policy elites regard as the key "wicked problems" of contingencies management in the context of uk governance? the case study has been interrogated by deploying mixed qualitative methods. the case study method is effective in public policy research for exploring, assessing, conceptualising, and refining explanations for the characteristics and dynamics of social realities or events (lowi, , p. ; eckstein, , pp. - ; flyvbjerg, , p. ) . the approach included a thematic analysis of secondary sources and official government documentationincluding the and house of lords reports into pandemic influenza (hl- , ; hl- , ), the department of health (doh) national framework for responding to an influenza pandemic (doh, ) , the independent review of the influenza pandemic (cabinet office, ), the uk influenza pandemic preparedness strategy (doh, ) and reports of the european commission. the research also involved undertaking in-depth semi-structured elite interviews with policy actors who occupy strategic-level governmental positions in scotland, the uk and in the european commission (the commission being the main policy initiator at the eu level). the analysis of documentation served to support the identification of interviewees and contributed to the themes that structured interview schedules. the interviewees were purposely sampled due to their positions at different tiers of government in the health security policy area. the interview breakdown is as follows: scottish government ¼ interviews (subnational/devolved level); uk government (national) level: public health england (phe) (agency of the uk civil service) ¼ ; westminster/uk parliament ¼ ; uk cabinet office (central ministerial department/headquarters for government) ¼ ; and european commission (health threats unit: ). interviews lasted on average . to hours in length. the scottish and uk level interviews were face-to-face in governmental or parliamentary premises and the interviews with european commission officials took place via teleconference facilities (with the interviewer being present on university premises). there were inconsistencies in terms of the medium by which the interviews were conducted and the necessity for teleconference interviewing was borne out of resource limitations for travel (particularly the high costs for travel between scotland and luxembourg -where the officials were based in the commission). however, these inconsistencies should not necessarily been seen as limitations given that the interviews conducted via teleconference were equally rich in depth and exploratory. interviews were also sought from individuals who work in the area of public health and disease risk management in the doh, however, there was a lack of preparation by such individuals to engage with this research via an interview. the position of the department was that their institutional approach had been detailed sufficiently in strategy documentation (cited earlier in this paper). digital recordings of all the interviews were fully transcribed within hours of the interviews taking place. the interview data was thematically coded around the most significant strategic policy challenges to emerge from the data. it should be noted that the majority of the interviewees felt it to be impossible to "rank" the extent of whether one challenge outweighs another but regarded these challenges to be equally placed on a continuum of policy challenges that were present in the case of pandemic influenza. the quality of the interviews is borne out of the fact that the individuals are situated in strategic-level positions within the institutional environment under investigation (less senior officials, of which there would likely be higher numbers, would be less able to discuss strategic relations across territories). in this respect the quality of the interviews outweighs the need for quantity given the low number of individuals that are in such senior positions in this policy sector (i.e. ten interviews represents a strong sample from a small pool of available data). this approach also serves to highlight the uk health security contribution that emerges from the study in that the interviewees where able to discuss matters of macro, strategic level concern from their perspectives. the interviewees were made up of a senior uk parliamentarian (with a remit for health security) and senior officials working within the areas of contingencies and crisis management for public health at multiple levels of governance (the european commission, uk government departments/agencies and in the scottish government). in short, the paper seeks to identify where the "politics" fits in the area of contingencies and crises management from the perspective of elites in relation to health security within a multilevel governance context. the study accommodates all study protocols which were approved by the author's university ethics committee and by the funding body for the research (carnegie trust, scotland). viewing health security in the context of "disaster" in a similar vein to "crisis", no definition of disaster has been agreed upon in the literature (hood and jackson, ; perry and quarantelli, ) . alexander ( , p. ) has described disaster research as being embedded in a "definitional minefield". analyses have ranged from explaining disasters as the collapse of cultural protections (carr, ) , unique events (rubin and popkin, ), a form of collective stress (barton, ) , systemic events, and a form of a social catalyst (kreps, ) . disasters tend to be events which lead to large-scale damage to human life, damage to the physical environment and have vast economic and social costs. there is a considerable body of literature that has focused on the impact that physical (sometimes expressed as environmental) disasters have had on human activities (alexander, ; steinberg, ) . indeed, human vulnerabilities have been an important defining factor in the classification of an event as a disaster (smith, ) . again, however, there is an analytical grey area between what constitutes a crisis or disaster due to shifting identities and contextual change. for example, analyses have described a shift from threats to disasters (rijpma and van duin, ) and from crises to disasters (davies and walters, ) . the definition(s) applied to analyses seem to be dependent on the discipline using the term and the aims of the researcher because "actors create a definition with different ends in mind" (perry, , p. ) . in this respect, the "securitisation" of health, particularly around pandemic disaster prevention and management in the context of global governance, is now generally accepted by key supranational authorities (who, ; european commission, ) and in the academic literature in the context of global governance (kay and williams, ; connolly, ) . it is argued that health security can be studied and framed in the context of disaster research given that if threats of disease pandemics are not managed effectively, and safeguards are weakened, then a pandemic, and thus pandemonium, will ensueleading to far-reaching damage to human life and produce vast economic social and environmental costs on a global scale. the case of pandemic influenza in the uk the implications of pandemic influenza are "feared by politicians, health practitioners and security experts alike" (kamradt-scott and mcinnes, , p. ). significant concern of a pandemic occurring was heightened as a result of the threat of the avian flu virus in asian countries (h n virus) in and . more recently, fear about human to human transmission of influenza was particularly acute as a result of the h n "swine flu" virus. the threat emerged when the world health organisation (who) declared that there was an outbreak of swine flu following confirmation of human cases in the usa and mexico in april . two confirmed cases of pandemic influenza subsequently emerged which involved a couple who had returned to scotland from mexico (connolly, ) . this led the uk government to increase their stockpile of antivirals (tamiflu) to million (from million). the government's approach was to maintain the policy of containment e.g. through contract tracing and antiviral treatment (cabinet office, , pp. - ) . by june/july the cases of swine flu has reached almost , in countries. the cases of swine flu in the uk reached , and there were pockets of concentration of the disease in birmingham and greater glasgow (cabinet office, , p. ). the who declared the outbreak had moved to pandemic levels which triggered the uk government to procure vaccines to cover per cent of the population. in late november modellers concluded that the pandemic had peaked and a gradual reduction in cases followed (cabinet office, , p. ) . the pandemic led to [ ] deaths during the pandemic in the uk (cabinet office, , p. ). the uk response to the pandemic relied on cooperation between supranational, national and subnational jurisdictions (with uk state level being the "core" crisis management actor through the department of health phe and the uk cabinet office). following the pandemic the government produced a uk influenza pandemic preparedness strategy (doh, ) which, building on the "national framework" for responding to an influenza pandemic (doh, ) , sets out in some detail the key planning assumptions and presumptions for planning for a pandemicincluding a summary of the key roles of government departments and agencies as well as the control strategies in order to mitigate against the impact of a pandemic influenza crisis. an important point, made rather passively in the strategy, is that preparedness and response to the threat is coordinated at local, national and international levels (doh, , pp. - ; connolly, ) . the case of pandemic influenza in highlighted that disease threats, such as pandemic influenza are trans-boundary which can penetrate integrated political and economic systems (such as the european union) and, therefore, call for a large number of organisational actors, at different governance levels, to be engaged in crisis management processes (allison, ; 't hart et al., ) . the resilience literature recognises the importance of considering the implications of when crises outweigh local capacity and there is a need for a multi-level response across borders and tiers of governance (see, e.g. brassett et al., ) . there is general agreement in the literature that local-or state-centric studies of crisis, emergency management, security and risk ('t hart et al., ) need to adapt their analytical frames to consider multi-level systems (coaffee, , p. ). the study "resilience" is characterised by its inexactitude given that, as anderson ( , p. ) , notes it has been referred to in academic circles, amongst other things, as "ethos", "programme", "ideology", "concept", "term", "governing rationality", "doctrine", "discourse", "epistemic field", "logic", "buzzword", "normative or ideal concept", and "strategy of power". however, in the context of health security, we are reminded by the - global ebola outbreaks of the need for international systems to be resilient in terms of being flexible and multi-partnership focused whilst, at the same time, having clarity over institutional roles and responsibilities. this is in order to avoid disorganised and belated responses (as demonstrated by the global response to ebola). in this regard, successful resilience is dependent on being able to navigate complexity given the context of interdependences between governmental and non-state actors across multi-level uk health security and cross-cutting jurisdictional boundaries (bevir, , p. ) which necessitate inter-organisational coordination (perry and lindell, ) . as a result, this warrants the need for contingency planning needs to take place at multiple levels of governance given that internal failures can have implications for the integrated system and have disastrous consequences (see turner and pidgeon, ; boin and mcconnell, ) . these requirements are not always matched by the characteristics of bureaucratic contexts which are known for their conservatism when it comes to institutional change. what is more, conflictual and political behaviours can manifest at different levels or governance and, as a result, a lack of contestation between political and bureaucratic actors cannot be assumed (rosenthal et al., , pp. - ) . yet evidence of this is often masked by the tendency of official governmental documents (such as strategies and contingency planning documents) to be read as if contingency processes (particularly for scientific or epidemiological issues) are in some way non-political in that such documents tend to focus on a range of "manual-like" sequential steps that should be taken in the event of an incident. clarke ( ) discusses the symbolic and political nature of such crisis contingency planning by the use of "fantasy documents". it is the contention that governmental documents that attempt to tame crises or disasters are "little more than vague hopes for remote futures and have virtually no known connection with human capacity or will" (clarke, , p. ) . it is within this context that questions about the "politics" of contingencies and crisis management functions across multiple levels of governance are pursued and, specifically, how this relates to the issue of health security. the case study data indicates that balancing the activities of risk communication with pharmaceutical interests is a major governance challenge in policy-making for pandemic flu (and, arguably, for other diseases). the conclusions of the aforementioned cochrane review questioned whether government investment in a stockpile of "tamiflu" as a contingency measure to safeguard the population matched any potential benefits of taking the medication. the evidence presented by jefferson et al. ( ) suggested that tamiflu moderately reduced the period that individuals would have flu symptoms. this led to pointed media reporting of the issue which included headlines such as "drugs given for swine flu were waste of £ million" (knapton, ) and "what the tamiflu saga tells us about drug trials and big pharmaceuticals" (goldacre, ) . the response from the industry was that the review underestimated the benefits of tamiflu and that they disagreed with the statistical analyses and therefore disagreed with all of the conclusions (gallagher, ) . the uk department of health confirmed that they would not change their public health advice in relation to the use of tamiflu as part of its preparedness planning despite the findings of the cochrane review. it is with key reference to the issue of tamiflu that elite actors suggest that there are challenges with regards to risk communication and, revealingly, the chief medical officer (cmo) for scotland pointed out that "we are not very good in government at conveying the full arguments and why we have decided to continue with the current policy" (keel, ) . for policy-makers the former secretary of state for health, and latterly the chair of the uk parliamentary committee for health, highlighted the difficulties in "communicating the subtleties" of scientific evidence (dorrell, ) . this chimes with the perspectives of the cmo for scotland and the chair of phe who considered that the challenges of "coordinating and digesting advice" (keel, ) that is "unadulterated, clear, properly analysed and packaged for the policy-makers" ) are significant. the wider point here is that scientific evidence is just one consideration by policy-makers and it would almost be impossible for this to not be the case in a politically driven society . a further challenge in terms of risk communication is getting across the message that it will take at least four to six months after a novel virus has been identified and isolated before pharmaceutical manufacturers can make an effective vaccine available (doh, , p. ) . although it could be said that the provision of scientific products by pharmaceutical companies are essential, because they are the only place where new drugs and vaccines and biotechnologies are being developed, the risk for many politicians is that because they are profit-making organisations "it is easy to be accused of favouring a pharmaceutical company because of vested interests" . however, contingency planning for health security requires the need for a stockpile of drugs and vaccines but this undoubtedly remains a political decision because the opportunity cost would be that policy-makers could be accused, in the event of a crisis, of not having appropriate measures in place thus endangering the health of the public (dorrell, ) . pandemic flu served to highlight that risk communication, coupled with industry interests, need to be taken into account when it comes to the management of science-or medical-based areas of public policy. political leaders can have their fate determined by how they respond to crises (boin et al., ) , and investing in a stockpile (even if they are never used), symbolises government readiness ('t hart, ) . the uk department of health has highlighted that in a globalised world it is not possible to prevent, manage and eradicate a new virus in neither the country of origin nor when it penetrates uk borders (doh, , p. ) . it is for this reason that the current chair of ohe, writing back in , noted that reporting and responding to infectious diseases requires collaboration across territories (heymann, , p. ) . heymann ( , p. ) suggested that "[t]his phenomenon is a potential infringement on national sovereignty that compromises the concept that states reign supreme over their territories and peoples". the twin threats of sars and avian influenza served to seal a new approach to health security for disease threats within a globalised world whereby the norms to responding to public health threats are such that reporting is much more the norm in order to eradicate diseases as efficiently and effectively as possible. the rise of surveillance using the internet and international standards and agreements, such as the global influenza surveillance network (of which there are member states), overseen organisations such as the who, has now replaced a situation whereby individual states provide information on disease threats and outbreaks on a voluntary basis (heymann, , p. ) . the relationship between state actors and international policy regimes in the process of contingencies management for health security becomes ever more apparent in the context of eu public policy. the legislative competence of the eu in coordinating contingencies and crisis management arrangements for public health threats has increased after a series of serious disease episodes, such as sars, avian flu and pandemic influenza, which have presented opportunities for closer policy integration and europeanisation (ryan, ) . health security within the eu is coordinated by the directorate general for health and consumer protection of the european commission. the role of the commission has become legally enshrined since the sars outbreak which ended the voluntary arrangement in place for member states to provide data to supranational institutions uk health security (as was the case in terms of providing national level information to the who). there is now a system in place for eu level surveillance in that member states are legally required to statistically report on cases of communicable diseases through the eu health security committee on an annual basis and second, states are obliged to inform each other using an electronic system of outbreaks of one of these communicable disease which could have effects on other member states. yet, enthusiasm for closer integration between states cannot be taken for granted given that given that larger member states (such as the uk, france and germany) have their own systems of contingencies management that have strengthened over time and, therefore, "carrying smaller member states" can be a distraction (phe official, ) . there have also been difficult tensions (which is a key hallmark of "wicked problem") when it comes of the sharing of what some member states would describe as "sensitive data" with each other through the route of the health security committee when it comes to contingency planning. the reason for this is articulated by a senior european commission official: we have member states that plan to vaccinate percent of their population in the event of a pandemic. in other words, percent will not be vaccinated and then you have countries that are providing for percent. clearly if this information becomes public the citizens of the concerned countries -the percent who won't get the vaccine might have some questions to ask of their politicians. the health security committee does not oblige member states to vaccinate everybodythat's not the purpose of the exercisethis is a national decision for how many people they consider suitable or requiring a vaccination. for example it could be public service workers, it could be medical personnel, the armed services, it could be the security services. in germany it was the politicians for some reason. so this is a very political and national decision. what we have done is to take the figures from each member state and formulate a joint tender. so we now have the figures for the different member states which are quite confidential i can tell you and we will make a tender to industry and the industry will be able to apply to supply this vaccine. for senior policy-makers, the governance challenges are grouped around managing public policy dynamics around ensuring closer integration and europeanisation in the knowledge that diseases transcend borders and therefore requires collaboration across territories. however, the complexities come from the politics of using resources to support "weaker" member states and the sensitivities around sharing classified information about who will be prioritised when it comes to implementing a vaccination programme in the event of a pandemic. the wicked problem of uk territorial governance uk policy actors (i.e. in scottish and uk governments) in the area of health security have highlighted the domestic state-level challenges of managing planning for pandemic disease within uk borders and the political dimensions to this process. the uk constitutional arrangements are such that we have devolved governments (with limited powers) in scotland, wales and northern ireland. the governance challenges are complex and this is partly due to the fact that control over health policy in the uk is not straightforward. for example, in the case of scotland public health (i.e. nhs and wider healthcare) is a devolved matter, however, matters of health security that have public health implications (such as bio-terrorism) are reserved matters for the uk government. such challenges are exacerbated when administrations are headed by different political parties at different tiers of governance. for example, the scottish government is headed up by a different political administration to the uk level (the scottish nationalist party) and the case study data has shown there to be evidence that nationalist politics has impacted on approaches to multi-level contingencies management for health security (although, for pandemic influenza specifically, both uk and scottish level elites highlighted that there were strong relationships between public health officials). a scottish government official indicated how the nationalist public health minister, michael matheson, was concerned that documentation on cross-border contingencies management pertaining to health (such as radiation protection and nuclear monitoring) had the word "england" in the title as a result of the creation of "phe" as an executive agency of the uk department of health at uk level in april . this had serious implications for civil servants in scotland in that they had to research and scope out the reasons why there is not the capacity to undertake this type of work at the subnational level in scotland: the current policy arrangements are such that the devolved administrations in the uk contribute to national strategies for preparedness planning and crisis simulations, however, as one senior scottish government official noted, "it is not always the case that the devolved administrations are there and creating plans with the uk government" (scottish government official, ). interviewees from different triers of uk governance agreed that there is mutual interest in maintaining strong relationships across levels of governance both in terms of managing the spread of diseases and ensuring that clear communication channels are in place. this is not to say, however, that there are no political tensions when it comes to multi-level contingencies management for national health and security. a senior official in scottish government gives the example of counterterrorism efforts as part of the operations of the commonwealth games in glasgow . as noted above although counter-terrorism measures have implications for public health, contingencies management arrangements for terrorism are a reserved issue for westminster. this leads to multi-level tensions in terms of information sharing (even to the officials of the host country of the games) given the sensitivities around responses to terrorism: we had an exercise for the commonwealth games and cobra [of the uk cabinet office] was involved. one of the issues that people mentioned quite a lot was the counter-terrorism aspect as that is a big issue. there is an issue in terms of sharing information between parts of the administration. it is clear from such insider perspectives that there are political interests that infiltrate the approach of policy-makers even when it comes to so-called "technical" areas of contingencies and crisis management. the cmo was clear about the fact that there are interests on both sides of the border between scotland and england in terms of maintaining the current arrangements even if the devolution of more powers continues given that diseases and organisms do not stop at the border (keel, ) . in terms of the experience of managing the influenza pandemic the independent review of crisis noted that strong sub-national and national relations were not taken for granted given that "the h n pandemic was the first uk-wide crisis in a devolved policy area, and therefore there could have been inconsistencies and disagreements between the four uk nations during the response" (cabinet office, , p. ). yet the report concluded that "the willingness of the devolved administrations and the department of health to work closely together within a common uk framework was fundamental to the overall success of the response" (cabinet office, , p. ). notwithstanding this encouraging narrative, as noted above, there have been examples of nationalist fervour impacting on the public policy process in scotland which have placed demands on civil servants north of the border. there are also intriguing inter-institutional dynamics here if one considers the fact that reserved areas of public policy (which have health security implications) are legislated for in england (such as counter-terrorism policy which include measures to manage biosecurity) and the uk government can become protectionist when it comes to sharing information with subnational government despite public health being fully devolved to scotland. this serves to demonstrate that the case of public health threats fit with the perspectives of those who consider there to be an "unequal plurality" and a "predominantly asymmetric imbalance" (marsh et al., , p. ) in uk governance. this is certainly the case for the context of contingencies management processes for uk health security. the paper has provided key insights into strategic-level relationships across multiple levels of governance in relation to contingencies management policy-making for health security. it has sought to unpack some of the political multi-level policy complexities associated with managing pandemic influenza as a "wicked problem". contingency management processes in relation to this case study highlights the considerable public policy and political challenges, articulated by for policy elites, in terms of risk communication, the internationalisation and europeanisation of national contingencies management processes and uk national-subnational relations. the lens adopted by this paper, in terms of identifying the perspectives of policy elites, has emerged out of the desire to address a lacuna in uk disaster and crisis research in that there is a dearth of case-based analyses of the challenges and paradoxes of contingencies management processes from a "macro" governance position. by interrogating the case of pandemic influenza the paper highlights that the recent high profile debates over the efficacy of tamiflu instigated by the cochrane review is but one example of the governance challenges that face policy elites. from a practical point of view it is important that risk management registers (i.e. organisational systems for identifying levels of risks and countermeasures) at different tiers of governance address the management of policy and political relations across such levels and that this is continually evaluated as a result of bureaucratic coordination and conflict challenges (which are likely to emerge, in part, by constitutional reforms). this research also presents opportunities for comparative research in terms of the multi-level governance processes for contingencies management in the context of health security. this includes whether the findings of the uk experience are translatable to other state contexts with 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new answers to old questions from accident to disaster: the response to the hercules crash the bureau-politics of crisis management disaster recovery after hurricane hugo in south carolina, natural hazards research and applications information center interview with the author in the eyes of the beholder? making sense of the systems(s) of disaster(s) acts of god: the unusual history of natural disasters in america man-made disasters wicked problems, knowledge challenges, and collaborative capacity builders in network settings foreign policy and health security an economic theory of democracy uk devolution and the european union: a tale of cooperative asymmetry pandemic influenza preparedness in the asia-pacific region serious cross-border threats to health risk and crisis management in the public sector crises and crisis management: toward comprehensive government decision making the changing world of crises and crisis management about the author the project, funded by the economic and social research council, concerned analysing the dynamics of policy and organisational change in relation to the uk animal health security sector (with particular emphasis on foot and mouth disease and avian flu). the doctoral thesis was awarded the sir walter bagehot prize by the uk political studies association for its contribution to research in public policy and administration. after completing his phd dr connolly worked as a policy adviser in the scottish public sector (nhs health scotland) and returned to academia on a full-time basis in for instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm or contact us for further details: permissions@emeraldinsight key: cord- -k eo c authors: hendaus, mohamed a; jomha, fatima a; alhammadi, ahmed h title: virus-induced secondary bacterial infection: a concise review date: - - journal: ther clin risk manag doi: . /tcrm.s sha: doc_id: cord_uid: k eo c respiratory diseases are a very common source of morbidity and mortality among children. health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. the reason expressed by many clinicians is the trouble to confirm whether the fever is caused by a virus or a bacterium. the aim of this review is to update the current evidence on the virus-induced bacterial infection. we present several clinical as well in vitro studies that support the correlation between virus and secondary bacterial infections. in addition, we discuss the pathophysiology and prevention modes of the virus–bacterium coexistence. a search of the pubmed and medline databases was carried out for published articles covering bacterial infections associated with respiratory viruses. this review should provide clinicians with a comprehensive idea of the range of bacterial and viral coinfections or secondary infections that could present with viral respiratory illness. viral respiratory tract infections (vrtis) are very common in children and their presentations vary from simple colds to life-threatening infections. [ ] [ ] [ ] [ ] [ ] the detection of a respiratory virus does not necessarily infer that the child has only a viral infection, since outbreaks of vrtis are being linked to increased incidence of bacterial coinfections. the human body is usually capable of eliminating respiratory viral infections with no sequelae; however, in some cases, viruses bypass the immune response of the airways, causing conceivable severe respiratory diseases. robust mechanical and immunosuppressive processes protect the lungs against external infections, but a single respiratory tract infection might change immunity and pathology. health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. the reason expressed by many clinicians is the challenge to confirm whether the fever is caused by a virus or bacterium. acute otitis media (aom) is a usual bacterial coinfection that occurs in %- % of cases of vrtis. [ ] [ ] [ ] [ ] in addition, almost % of children with vrti have changes in the maxillary, ethmoidal, and frontal sinuses. , moreover, in the year , it was estimated that - million individuals died from the influenza pandemic, many of which were due to secondary bacterial pneumonia with streptococcus pneumoniae. a search of the pubmed database and google was carried out, using different combinations of the following terms: virus, induced, bacteria, pathogenesis, prevention, vaccine, and children. in addition, we searched the references of the identified articles for additional articles. we then reviewed abstracts and titles and included studies that were submit your manuscript | www.dovepress.com hendaus et al relevant to the topic of interest. finally, the search was limited to studies of disease in humans that were published in english and spanish from to the end of ( figure ). the epithelium ( figure ) is usually covered by a layer of mucus that functions as a boundary. mucins, which are charged glycoproteins, are the main components of mucus. , muc ac and muc b are the most common mucins in the human sputum, and they assist the innate immune system through their anti-inflammatory and antiviral properties. , in addition, they facilitate trapping and clearance of viruses; however, overproduction of those mucins might have a paradoxical effect. , the airway epithelium not only functions as a physical barrier but also recognizes microorganisms through pattern recognition receptors such as toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), and retinoic acid-inducible gene (rig)like helicases. , tlrs are single, noncatalytic, membrane-spanning receptor proteins used by the innate immune system. respiratory viruses collaborate with tlr lanes, leading to extended bacterial load in the lungs. , in comparison, nlrs and rig-like helicases activate innate immune responses through cytosolic sensing of viral and bacterial components. , nod and nod , which are family members of nlrs, are induced by molecules synthesized during the production and/or degradation of bacterial peptidoglycan. [ ] [ ] [ ] [ ] in addition, many epithelial cells express the classical antiviral interferons (infs), especially ifn-α and ifn-β. , moreover, the respiratory virus-infected epithelia facilitates the attraction of inflammatory cells, including natural killer cells, neutrophils, macrophages, and eosinophils from the bloodstream into the infected site. finally, the airway epithelium consists of many molecules including intercellular adhesion molecule (icam- ), carcinoembryonic antigen-related cellular adhesion (ceacam- ), and platelet-activating factor receptor (paf-r). viruses have an effect in modulating these receptors, leading to an increase risk of bacterial adherence; for example, rhinovirus upregulates the expression of paf-r, leading to the binding of s. pneumoniae to bronchial epithelial cells. different mechanisms might contribute to the debilitation in host defense of the respiratory tract against bacteria following viral infection. some of the mechanisms have been extrapolated from studies conducted in animal models of sequential infections by respiratory viruses and several bacterial pathogens. mammalian cells are prone to bacterial attachment during a viral illness. , viruses can debilitate the mucociliary clearance structure, leading to the increased attachment of bacteria to mucins and colonization; moreover, the condensed mucus will impede the penetration of antibacterial material and immune cells. viruses like the respiratory syncytial virus (rsv) can damage ciliated cells, resulting in ciliostasis and, therefore, deterioration of mucociliary clearance. the same concept applies to an influenza virus infection, leading to decreased tracheal mucociliary velocity and clearance of s. pneumoniae. , moreover, virus-induced cell death debilitates the mechanical elimination of the attached pathogens and displays novice receptors for bacterial adherence. studies have shown that the rsv virus induces the adherence of s. pneumoniae, pseudomonas aeruginosa, and haemophilus influenza to airway epithelial cells. [ ] [ ] [ ] [ ] in addition, adenovirus and rhinovirus play the same role in the adherence of s. pneumoniae to the airway epithelial cells; , however, the measles virus decreases the risk of adherence of streptococcal bacteria, implying that every virus has a specific mode of changing the host cell membrane. moreover, bacterial adhesion might also be a result of the upregulation of surface receptors including paf-r, which is involved in pneumococcal invasion. , in patients with cystic fibrosis, bacterial adherence forms a biofilm, creating permanent airway colonization with p. aeruginosa. viruses such as rsv, rhinovirus, and influenza virus also lead to pneumococcal biofilm formation on the airway lining. furthermore, rsv increases the risk of adherence of staphylococcus aureus and bordetella pertussis to hep- (human epidermoid cancer) epithelial cells. , virus effect on the immune system post-viral sustained desensitization of lung sentinel cells to tlr signals may be one possible contributor to the common secondary bacterial pneumonia associated with viral infection. for instance, tlr and tlr pathways are altered after influenza virus infection, resulting in decreased neutrophil attraction, thereby leading to increased attachment of s. pneumonia and p. aeruginosa to the airway epithelial cells. the interrelation between host cells and microorganisms during an infection induces immune responses that include the generation of proinflammatory molecules. despite their crucial role as a bactericidal, proinflammatory cytokines such as tnf-α produced in response to infection could be detrimental to the host cells. during a viral infection, tlr and rig-i-like receptor activation induces production of type i ifns, which can augment the inflammatory response to tlr ligands including lipopolysaccharide (lps). , in addition, certain bacteria such as s. aureus integrate into the a respiratory epithelial cells (adeno-carcinomic α β hendaus et al human-alveolar basal-epithelial cells) during a respiratory viral infection by increasing the expression of icam- . rsv differs from influenza virus in that the former upregulates cellular receptors including ceacam- and icam- , which eventually leads to bacterial infection. finally, interaction between type i ifns and nod /nod signaling leads to bacterial recognition, but indicts harmful effects in the virally infected host. another study showed that the rates of bacteremia and om were % and %, respectively, in children with viral-induced bronchiolitis. the highest incidence of aom is usually - days after an upper respiratory infection. , isolation of viruses alone from sinus aspirates or in concomitance with bacteria proposes the role of viruses in the induction of bacterial sinusitis, with rhinovirus and parainfluenza viruses being the culprits. the rate of bacteremia in children with acute bronchiolitis ranges from . % to . %. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in addition, the rate of bacterial urinary tract infection (uti) in children with bronchiolitis can be as high as . %. in a recent study, hendaus et al the human myxovirus resistance protein (mxa) is an important intermediary of the ifn-induced antiviral response against a variety of viruses. mxa expression is firmly modified by type i and type iii ifns, which also requires signal transducer and activator of transcription signaling. additionally, mxa has many characteristics similar to the superfamily of large guanosine triphosphatases. mxa analysis could be beneficial to differentiate between bacterial and viral infections. engelmann et al conducted a prospective, multicenter cohort study in different pediatric emergency departments in france on the role of mxa in the diagnosis of viral infections. mxa blood values were calculated in infants and children with verified bacterial or viral infections, uninfected controls, and infections of unknown origin. a receiver operating characteristic analysis was used to verify the diagnostic performance of mxa. the study, which included children, showed that mxa was significantly higher in children with viral versus bacterial infections and uninfected controls (p . ). additionally, mxa levels were significantly higher in children with clinically diagnosed viral infections than in those with clinically diagnosed bacterial infections (p . ). other authors have also reported the usefulness of blood mxa testing in patients with viral infections. , the use mxa in diagnosing viral infection is very promising, especially in patients who are at risk of infectious complications. two separate studies have shown that blood mxa is beneficial in differentiating between viral illness and acute graft-versushost disease after allogenic stem cell transplantation. , it has been recommended that treatment or prevention of a viral disease may be a superior method for diminishing it has also been published that live attenuated influenza vaccine is effective in reducing the incidence of all-cause aom [ ] [ ] [ ] and pneumonia compared to placebo in children. in addition, the intranasal influenza vaccine can reduce om by %. moreover, studies have shown that a combined influenza/pneumococcal vaccine is efficient in the prevention of om in children and pneumonia. , however, the credit of protection was awarded to the influenza vaccine since studies have shown that pneumococcal vaccine has no benefit in the reduction of aom. , in addition, the pneumococcal polysaccharide vaccine showed no efficacy in the prevention of pneumonia in adults. treatment of viral infection is anticipated to prevent bacterial superinfections. currently, the only respiratory virus that is pharmacologically treatable is the influenza viruses (type a and b). neuraminidase inhibitors can potentially diminish the morbidity related to influenza. oseltamivir can reduce the incidence of aom in preschool children, and the reduction rate can be up to %. a meta-analysis review showed that oral oseltamivir reduces the rate of hospitalization by % and morbidity by %. in addition, its use can reduce the use of antibiotics by up to %, , the same concept of protection applies to vaccines that prevent against rsv infections. the vaccine available for rsv is palivizumab (medimmune, gaithersburg, md, usa), a humanized monoclonal antibody that perceives the fusion protein of rsv. the other monoclonal antibody that is under clinical trials is motavizumab (medimmune), which has a higher affinity for rsv fusion protein than palivizumab and can prevent against medically attended lower respiratory tract infection. the rate of concurrent serious bacterial infections with viral illness is appreciable. similar emphasis must be given to the prevention and treatment of viral illnesses, especially in young children. furthermore, health care providers should emphasize to parents on the importance of clinical follow-up of infants and young children diagnosed with vrti. moreover, the introduction of mxa in the diagnosis of viral illnesses in children is promising. the authors declare no conflicts of interest in this work. respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study respiratory viral infections in adults emerging respiratory agents: new viruses for old diseases? respiratory pathogens in children with and without respiratory symptoms asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection how do viral infections predispose patients to bacterial infections? the airway epithelium: soldier in the fight against respiratory viruses influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology occult serious bacterial infection in infants 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in children oral oseltamivir treatment of influenza in children impact of neuraminidase inhibitor treatment on outcomes of public health importance during the - influenza a(h n ) pandemic: a systematic review and meta-analysis in hospitalized patients impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial: us oral neuraminidase study group motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial publish your work in this journal submit your manuscript here: http://www.dovepress.com/therapeutics-and-clinical-risk-management-journal therapeutics and clinical risk management is an international, peerreviewed journal of clinical therapeutics and risk management, focusing on concise rapid reporting of clinical studies in all therapeutic areas, outcomes, safety, and programs for the effective, safe, and sustained use of medicines. this journal is indexed on pubmed central, cas, embase, scopus and the elsevier bibliographic databases. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- -v vv o authors: shen, mingwang; xiao, yanni; rong, libin title: modeling the effect of comprehensive interventions on ebola virus transmission date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: v vv o since the re-emergence of ebola in west africa in , comprehensive and stringent interventions have been implemented to decelerate the spread of the disease. the effectiveness of interventions still remains unclear. in this paper, we develop an epidemiological model that includes various controlling measures to systematically evaluate their effects on the disease transmission dynamics. by fitting the model to reported cumulative cases and deaths in guinea, sierra leone and liberia until march , , we estimate the basic reproduction number in these countries as . , . and . , respectively. model analysis shows that there exists a threshold of the effectiveness of isolation, below which increasing the fraction of latent individuals diagnosed prior to symptoms onset or shortening the duration between symptoms onset and isolation may lead to more ebola infection. this challenges an existing view. media coverage plays a substantial role in reducing the final epidemic size. the response to reported cumulative infected cases and deaths may have a different effect on the epidemic spread in different countries. among all the interventions, we find that shortening the duration between death and burial and improving the effectiveness of isolation are two effective interventions for controlling the outbreak of ebola virus infection. the ebola virus disease (evd) affecting multiple countries in west africa in has an unprecedented magnitude, which is far larger than all previous evd outbreaks combined. by march , , a total of , cases (including , confirmed, , probable, and , suspected cases) of evd, as well as , deaths from the infection, have been reported in guinea, sierra leone, liberia . ebola virus spreads primarily via contact with body fluids of infectious people, and with those dead but not buried who can still transmit the virus in traditional west african funeral practices. because of lack of licensed therapeutic treatment regimens and vaccines , major control measures are a combination of early diagnosis, case isolation, contact precaution, awareness campaigns, and sanitary burial practices. identifying infected people quickly by the polymerase chain reaction (pcr) assay and isolating them to break chains of evd transmission may be effective to control the outbreak. the effect of reducing the time between symptoms onset and diagnosis with rapid testing has also been investigated in the literature [ ] [ ] [ ] . chowell et al. used a mathematical model to study the effect of early diagnosis of pre-symptomatic individuals and found that the basic reproduction number always decreases as the fraction of early diagnosed individuals increases. the result may be affected by the effectiveness of isolation. although the isolated class has a lower transmission rate than the non-isolated class, isolated individuals may live longer due to supportive clinical care and have more chance to infect others. whether the relationship between the fraction of early isolation and the effectiveness of isolation affects the ebola epidemic has not been explored. in this paper, we will use a model to systematically evaluate the effect of a number of factors, including isolation of pre-symptomatic individuals and the time between symptoms onset and isolation, on both the basic reproduction number and the final epidemic size in the above three african countries. media report on the numbers of infected cases and deaths can greatly affect social behavior and play an important role in defining health issues in the evd epidemic [ ] [ ] [ ] [ ] . mathematical models have been used to explore the effect of mass media on infectious disease outbreaks such as sars in [ ] [ ] [ ] and h n in [ ] [ ] [ ] [ ] [ ] , assuming that media coverage depends on the number of infected individuals and reduces the incidence rate. very few models have considered the impact of media coverage on the transmission dynamics of evd. recently, the first large-scale trials that aim to assess the safety and efficacy of two ebola vaccines (cad -eboz and vsv-zebov) were initiated in liberia on february , . the effectiveness of these vaccines still remain unclear. we will include the effect of vaccination in our model. the objective of this paper is to assess the effect of all possible intervention strategies (isolation, media impact, safe burial, and vaccination) on controlling the spread of ebola virus in guinea, sierra leone, liberia. we develop a mathematical model on the basis of the model in ref. and fit to epidemiological data of reported cumulative numbers of infected cases and deaths . using the model, we evaluate the potential effect of increasing the fraction of latent individuals prior to symptoms onset, shortening the duration between symptoms onset and isolation, improving media coverage, following restrict burial procedures, and administrating timely vaccine on the epidemic of ebola infection. we develop a mathematical model (eq. ) to study the impact of comprehensive interventions including isolation, media impact, safe burial and vaccination. the population is divided into eight classes: s (susceptible individuals who can be infected by ebola virus following a contact with infectious cases), v (vaccinated individuals), e (latent undetectable individuals), e (latent detectable individuals), i (infectious individuals with symptoms), j (isolated individuals), d (individuals who are dead but have not been buried; they can still transmit the disease during funerals), and r (recovered individuals). let n denote the total population size, i.e., n = s + v + e + e + i + j + r. susceptible individuals become infected through contact with infectious individuals and enter into the latent class at rate where β is the mean human-to-human transmission rate per day,   ( ≤ ≤ ) quantifies the relative transmissibility of isolated individuals compared to infectious symptomatic patients who are not isolated. therefore,  ( − ) × % (reduction in transmissibility) provides a measure of the effectiveness of isolation. β d is the transmission rate during funerals. in the model, , where c i and c d are the cumulative infected cases and deaths, respectively. m and m are non-negative parameters that measure the effect of media reported cumulative numbers of infected cases and deaths on the contact transmission. when m i (i = , ) is or relatively small, the transmission rate β is equal or close to the constant β . for m i > , the public is more aware of the disease so that the transmission rate could be decreased to β (< β ) as the number of accumulated infected cases c i or deaths c d increases. similarly, the transmission rate β d is assumed to be less than β d during funerals. we assume that susceptible individuals receive the vaccine at a rate ξ. the effectiveness of immunization is assumed to be η where ≤ η ≤ with η = meaning that the vaccine is perfectly effective and η = meaning that the vaccine has no effect. latent undetectable individuals (e ) progress to the latent detectable class e at a rate k , and subsequently move to the infectious symptomatic class i at a rate k . let f t be the rate at which latent detectable individuals are detected and isolated. thus, θ = f t / (f t + k ) represents the fraction of isolated patients among latent detectable individuals exiting the class. infectious individuals are isolated at a rate α, or removed from the class at a rate γ due to recovery (with probability − δ) or disease-induced death (with probability δ). similarly, isolated individuals are removed at a rate γ r because of recovery (with probability − δ) or disease-induced death (with probability δ). a flow diagram of the model (eq. ) is shown in fig. and parameters are described in table . the dynamical model is as follows: figure . a schematic flow diagram of ebola infection with isolation, media impact, post-death transmission and vaccination. the description of parameters can be found in table . it should be noted that we keep track of the cumulative number of infected cases c i and deaths c d (c i and c d are not epidemiological states) by applying the solutions of eq. ( ) to the following equations in the absence of effective vaccine (i.e. ξ = η = ), we obtain the basic reproduction number r which is the spectral radius of the matrix ∼ −  fv by using the next generation matrix approach given in where ρ denotes the spectral radius and the matrices  f and ∼ v are note that each term of the aforementioned expression for r has clear epidemiological interpretation. − θ = k /(f t + k ) is the fraction of latent detectable individuals becoming symptomatic among those exiting the e class. /(α + γ) is the mean infectious period of symptomatic cases (not isolated). α/(α + γ) is the fraction of symptomatic cases that are isolated among those exiting the i class. /γ r is the mean infectious period of isolated cases and /γ d is the mean duration of the infectious period between death and burial. r i , r j and r d reflect the contribution to new infection from the symptomatic class (i), the isolated class (j), and the dead class (d), respectively. when vaccine is included, i.e., ξ > , η > , the reproduction number can be calculated as r v = ηr , where r is given by ( ). data fitting and parameter estimation. we obtained data of the total cumulative cases and deaths (as the sum of confirmed, probable and suspected cases) for guinea, sierra leone, liberia from the world health organization (who) . we used t (see table ) as the starting date for each country when the first infectious case was reported . we compiled publicly available time series of reported cases and deaths from who beginning from march , may , and june for guinea, sierra leone, liberia, respectively , . the data as of march were used to fit the model equation ( ) to estimate the unknown parameters. the latest data from march to may were used to assess how well our forecasts match additional data points. based on previous studies, the mean incubation time for evd is days ( /k + /k = ) with days from the latent undetectable class to latent detectable class ( /k = ) and days from the latent detectable class to symptomatic class ( /k = ) , . the mean time from symptoms onset to isolation is days ( /α = ). the mean time that infectious individuals are removed from the class after recovery or disease-induced death is days ( /γ = ) , . the average duration from death to burial is chosen as days ( /γ d = ) . as for the total population size n, we used the number of for guinea, for sierra leone, and for liberia, provided by the world bank . the other parameters were estimated by fitting the equation ( ) to the data on cumulative cases and deaths of each country and using an adaptive metropolis-hastings (m-h) algorithm to carry out the bayesian markov chain monte carlo (mcmc) procedure implemented by matlab. the estimated parameter values and their % confidence intervals are listed in table . model prediction and comparison with data. we first consider the situation without vaccination because of the unavailability of vaccine before february , , and then explore the effect of vaccination in liberia where the first large-scale vaccine trials were implemented since then . figure shows that the model provides a very good fit to the reported data of both infected cases and deaths in all three countries. using our parameter estimates (table ) , we calculated the basic reproduction number r in guinea, sierra leone and liberia as . , . and . , respectively. they are all within the range of estimated values in the literature (see table ). in guinea, the symptomatic component of we plotted the simulated newly infected individuals in fig. (red solid lines) and compared with the reported weekly confirmed cases in the three countries. most of the reported cases in guinea were confirmed ( / ), while only / and / (the numerator and denominator denote the confirmed and total cases on march , respectively) of cases were confirmed in sierra leone and liberia, respectively. there is a small difference between the simulated new infection and reported data in guinea ( fig. (a) ), while the simulated results are higher than confirmed cases in sierra leone and liberia ( fig. (b,c) ) due to a lower confirmation rate in these two countries. moreover, the simulated trend of evd outbreak and predicted peak time are in good agreement with what the who data and other references [ ] [ ] [ ] [ ] indicated. the solution of model ( ) is shown in fig. . the number of infected individuals is decreasing and tends to vanish gradually at the end of ( fig. (a-c) ). effect of isolation on r and the final epidemic size. to explore the effect of early and broad diagnosis on the basic reproduction number r , we calculate the derivative of r with respect to the parameter θ (i.e. the fraction of latent individuals diagnosed prior to symptoms onset) as follows the derivative is greater than when  > for guinea, sierra leone and liberia respectively (see table blue lines in fig. ) , respectively, which are all less than their respective threshold. thus, increasing the fraction θ of detecting pre-symptomatic cases results in a decline in the basic reproduction number. if the effectiveness of isolation increases to %, i.e., the relative transmissibility  of isolated individuals decreases to % (magenta lines in fig. ), then about %, %, % of pre-symptomatic patients need to be detected in guinea, sierra leone and liberia to control the disease. if the isolation is perfect  ( = ) , then the disease could be controlled easily (see yellow lines in fig. ). next, we study the effect of the fraction θ of latent individuals diagnosed prior to symptoms onset on the final epidemic size, which is defined as z = c i (t ) = c d (t ) + r(t ) . the time t is min{t > ,e (t) + e (t) + i(t) + j(t) + d(t) = }. although the explicit formula between the final size z and θ cannot be obtained, there exists a threshold which decides whether the final epidemic size increases as θ increases. the relationship between z and θ is similar to that between r and θ. for example, it follows from fig. (a) that if the effectiveness of isolation is lower than . %, i.e., the relative transmissibility  of isolated class is greater than . %, then the final epidemic size increases as θ increases. this suggests that strict measures must be taken to limit the contact of isolated individuals. otherwise, low effectiveness of isolation would lead to more infected individuals because isolated people live longer due to improved medical care and have more chances to infect other people. the benefit of having a reduced transmission rate would be counteracted by a longer infectious period of the isolated class. fig. (d-f) show the situation in which the final epidemic size decreases as θ increases. using the estimated value of θ (see table , θ = . , . , . in guinea, sierra leone and liberia, respectively), the simulated final size is , and in these three countries respectively (blue lines in fig. ). this simulated result in liberia where the outbreak of evd was considered ended by the who on may is very close to the actual final size with a relative error . %. similarly, to investigate the effect of shortening the time ( /α) between symptom onset and isolation (i.e, increasing the isolation rate α of infectious symptomatic individuals) on the basic reproduction number r , we plot fig. , which shows a similar relationship to that between r and θ when the the relative transmissibility  of isolated class varies. this is not surprising because the derivative of r with respect to α is it has the same threshold  = γ γ ⁎ r that decides whether the basic reproduction number increases with an increasing α. if the relative transmissibility  of isolated individuals decreases to % (see magenta lines in fig. ) , then the disease will be under control if the isolation rate α exceeds . (i.e., the time between symptom onset and isolation is less than days) in sierra leone and liberia. in this case  ( = %) , evd will always be controlled no matter how long the time ( /α) is in guinea. we study the effect of media coverage m (response to the reported cumulative number of infected cases) and m (response to the reported cumulative deaths) on the final epidemic size. let q ( ≤ q ≤ ) be the percentage of increase of m and m from its baseline estimate (table ) . we examine how the final epidemic size changes with an increasing media impact q in fig. . for example, increasing m (or m ) by % from its baseline value (while keeping other parameters fixed) can reduce the final epidemic size by . % (or . %) in guinea, table . scientific reports | : | doi: . /srep . % (or . %) in sierra leone, and . % (or . %) in liberia, respectively. this indicates that the response to the reported cumulative deaths (m ) may be stronger than the response to reported cumulative cases (m ) in guinea and liberia. a possible explanation of this result is that the case fatality rate in these two countries is relative high (see table ). thus, people had increased attention to evd-induced deaths. in sierra leone, an opposite scenario was observed. in conclusion, the analysis of media impact indicates that massive news coverage on cumulative cases and deaths would greatly curb the spread of the ebola disease. effect of post-death transmission on r . to assess the effect of reducing post-death transmission on the basic reproduction number r , we plot the variation in r with the efficacy of intervention, z d , at funerals (i.e., the post-death transmission rate β d is decreased to β d ( − z d )) for various durations of the traditional burial /γ d in fig. . it shows that increasing the efficacy of intervention leads to a decline in r in all three countries. in particular, the epidemic in guinea could be controlled by following very restrict burial procedures (a large z d ). for example, when /γ d = (blue line in fig. (a) ) and the efficacy of interventions z d exceeds about %, the basic reproduction number r will become less than . however, reducing transmission during funerals is insufficient to control the disease in sierra leone or liberia no matter how large z d is ( fig. (b-c) ). this is because the burial-related transmission contributes less to the spread of the disease in sierra leone (r d /r = . %) and liberia (r d /r = . %) than in guinea (r d /r = . %). figure also demonstrates that r decreases as the duration of funeral decreases. this suggests that the duration of funeral should be as short as possible for the control of the disease. sensitivity analysis. in order to identify which parameters the basic reproductive number r and the final epidemic size are most sensitive to, we perform sensitivity analysis using the latin hypercube sampling (lhs) technique and calculate the partial rank correlation coefficients (prccs) , in fig. . the magnitude of these prccs shows the sensitivity of these parameters and the sign of the prccs indicates a positive or negative correlation between the inputs (i.e. parameters) and outputs (i.e. r and table . the final epidemic size). it follows from fig. (a-c) that the two parameters with the most significant impact on r are the relative transmissibility  of isolated classes and duration of the burial /γ d . a smaller relative transmissibility  of isolated classes or a shorter duration of burial results in a smaller r , which is consistent with the results shown in figs and . these two parameters  ( and /γ d ) also have the most significant impact on the final epidemic size, as shown in fig. (d-f) . these results suggest that increasing the effectiveness of isolation (decreasing ) and shortening the duration of funerals ( /γ d ) are of crucial importance to reduce the evd infection. the effect of media (m or m ) has no effect on r (see the formula of r in ( )), which is in agreement with the findings in refs. - , that the media table . table . impact does not affect the epidemic threshold. however, the media impact leads to a decline in the final epidemic size, as shown in fig. . effective vaccination, if used before the epidemic peaks, would be projected to prevent tens of thousands of deaths. in liberia, the evd epidemic reached the peak around mid-september , , which is in good accordance with our simulated peak time, sep , as shown in fig. table ). after initiating the vaccine, the reproduction number is reduced from . to . (table ). using the estimated values of ξ and η, we plotted how the simulated cumulative cases would vary with different timing of vaccination. it shows that the earlier the vaccination is initiated, the lower the cumulative cases ( fig. (a) ). it follows from fig. (b) that the final epidemic size grows with delayed vaccination. there would be more cases if vaccination had started one week later ( fig. (b) ). this indicates that the timing of vaccine administration does not have a big effect on the final size when the epidemic declines. in this study, we developed a mathematical model to study the transmission dynamics of ebola virus. the model includes the effect of case isolation, media impact, post-death transmission and vaccination. by fitting the model to the who reported data of infected cases and deaths (fig. ) , we obtained reasonable estimates of the parameters ( table ). the basic reproduction number in guinea, sierra leone and liberia is estimated as . , . and . , respectively. they are all in good agreement with previous estimates ( table ). the simulated results indicate that the outbreak in the above countries reaches the peak on oct , oct , sep , respectively (see fig. ), which also agrees with the observations in sierra leone , , and in liberia [ ] [ ] [ ] . we found that isolation does not always contribute to the control of the evd transmission. whether this intervention is beneficial depends on the effectiveness of isolation (or the relative transmissibility  of isolated individuals). if the infectious period of isolated individuals is less than that of non-isolated, i.e., < γ γ , then isolation is always beneficial to disease control because of a lower transmission rate and a shorter infectious period of isolated individuals. on the contrary, if the infectious period of isolated individuals is longer than that of non-isolated, then when the isolation effectiveness is relatively low (e.g.  > ) γ γ r , there will be more infected cases as the fraction θ of latent individuals prior to symptoms onset increases (figs and (a-c) ) or the duration /α between symptoms onset and isolation table . scientific reports | : | doi: . /srep decreases (fig. ) . when the isolation effectiveness is high (e.g.  < ) γ γ r , then enhancing isolation can greatly reduce new infection (figs , (d-f), and ). these results suggest that isolation may not always have a positive effect on disease control as shown in refs. - . sensitivity analysis also shows that the relative transmissibility  of isolated classes has the most significant impact on both the basic reproduction number and the final epidemic size, which further explains why the isolation effectiveness determines whether the disease can be controlled successfully. therefore, strict measures should be taken to limit the contact with isolated individuals. a few reasons may explain why the isolated class contributes more to the basic reproductive number r than the symptomatic class and the dead class. one is that the infectious period of isolated individuals (i.e. /γ r ) is longer than that for non-isolated individuals because of improved clinical care. this is consistent with the parameter estimates shown in table . the other reason is that a majority of patients (θ) are isolated but the isolation may not be effective in reducing the transmission of the disease. ebola virus is very contagious and the transmission is also rapid, which makes isolation, as a containment strategy, usually inefficient . this agrees with the estimate of  in table , which is not small, implying that isolated individuals still have a high capacity to transmit the virus. in our model, we did not discriminate isolated individuals from the latent detectable class (e ) and from the infectious class with symptoms (i). isolated cases from the e class may have a different duration ( /γ r ) staying in the isolated class from the isolated cases coming from the i class. however, the above conclusion that the isolated class contributes more to r should still be valid as long as the infectious period of isolated individuals is longer than that of their corresponding preceding class. this is usually true in view of the improved health care received for isolated individuals. we also found that the estimate of the case fatality rate δ in guinea ( . ) is almost as twice as that in sierra leone ( . , see table ). one reason is a low level of preparedness, as well as poor availability and quality of medical care in guinea . another important reason is that the outbreak in guinea was caused by the zaire strain , which induces an average fatality rate of %, the highest death rate of the five known ebola strains . media impact can significantly affect the ebola infection, as shown in fig. . however, the responses to the reported cumulative deaths and cumulative cases may have different effect on the infection. we find that the response to the reported cumulative deaths (m ) is more sensitive than the response to the reported cumulative cases (m ) in guinea and liberia. it can be explained by a higher case fatality rate in these two countries (table ) . post-death transmission is an important factor that induces more infection and hence can not be ignored during evd outbreaks , . our simulation shows that increasing the efficacy of intervention at funerals can control the disease in guinea. however, this measure is insufficient to eliminate the disease in sierra leone and liberia (shown in fig. ) because the burial-related transmission does not contribute much to the spread of the disease in these two countries. sensitive analysis indicates that the duration of the burial /γ d is the second parameter which most affects r and the final epidemic size. thus, shortening the duration between death and burial and reducing transmission before burial would effectively reduce the infection. because the interventions we considered mainly address isolation of infected people within evd treatment centres, one limitation of our model is that we do not explicitly account for hospital bed capacity, which plays an important role in affecting both the outbreak dynamics and the intervention efforts . additionally, the conventional homogeneous mixing assumptions used in our model may be over-simplified, especially in countries where infection always occurs in households due to the structure of the community . taking into consideration the effect of social networks structure on ebola infection would also be interesting for future research. in summary, we used an epidemiological model to study the effect of various measures on evd transmission dynamics. depending on the effectiveness of isolation, early and massive diagnosis of pre-symptomatic individuals with rapid testing may remain beneficial to reduce the transmission of the disease. shortening the duration between death and burial and improving the effectiveness of isolation are two effective interventions for controlling the evd outbreak. world health organization, ebola situation report- ebola vaccination: if not now, when? modelling the effect of early detection of ebola ebola control: rapid diagnostic testing transmission dynamics and control of ebola virus disease (evd): a review sample nlpde and nlode social-media modeling of information transmission for infectious diseases: case study ebola ebola 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methodology for performing global uncertainty and sensitivity analysis in systems biology american ebola quarantine zones will be genocidal death traps how many people infected with ebola die? emergence of zaire ebola virus disease in guinea-preliminary report modeling post-death transmission of ebola: challenges for inference and opportunities for modeling surveillance and interventions in the ebola epidemic a three-scale network model for the early growth dynamics of west africa ebola epidemic who ebola response team. ebola virus disease in west africa: the first months of the epidemic and forward projections strategies for containing ebola in west africa impact of ebola disease progression on transmission and control in liberia spatiotemporal spread of the outbreak of ebola virus disease in liberia and the effectiveness of nonpharmaceutical interventions: a computational modelling analysis dynamics and control of ebola virus transmission in montserrado, liberia: a mathematical modelling analysis m.s. and y.x. designed the study and carried out the analysis. m.s. performed numerical simulations. m.s., y.x. and l.r. contributed to writing the paper. competing financial interests: the authors declare no competing financial interests. key: cord- -sd n w authors: madalli, shimona; beyrau, martina; whiteford, james; duchene, johan; singh nandhra, inderpal; patel, nimesh s. a.; motwani, madhur p.; gilroy, derek w.; thiemermann, christoph; nourshargh, sussan; scotland, ramona s. title: sex-specific regulation of chemokine cxcl / controls neutrophil recruitment and tissue injury in acute inflammatory states date: - - journal: biol sex differ doi: . /s - - - sha: doc_id: cord_uid: sd n w background: tissue infiltration by neutrophils during acute inflammatory states causes substantial tissue injury. while the magnitude of tissue neutrophil accumulation in innate immune responses is profoundly greater in males than females, fundamental aspects of the molecular mechanisms underlying these sex differences remain largely unknown. methods: we investigated sex differences in neutrophil stimulation and recruitment in ischemia/reperfusion (i/r; mesenteric or renal) or carrageenan pleurisy in rats or mice, as well as skin injury in human volunteers. the induction of potent chemoattractive mediators (chemokines) and neutrophil adhesion molecules were measured by real-time pcr, flow cytometry, and protein assays. results: mesenteric i/r in age-matched wistar rats resulted in substantially more neutrophil accumulation and tissue injury at h reperfusion in males than females. using intravital microscopy, we show that the immediate (< min) neutrophil response to i/r is similar in males and females but that prolonged neutrophil recruitment occurs in males at sites local and distal to inflammatory insult partly due to an increase in circulating neutrophil populations with elevated surface expression of adhesion molecules. sex differences in neutrophil kinetics were correlated with sustained induction of chemokine cxcl in the tissue, circulation, and bone marrow of males but not females. furthermore, blockade of cxcl in males prior to ischemia resulted in neutrophil responses that were similar in magnitude to those in females. conversely, administration of cxcl to males in the absence of i/r was sufficient to increase levels of systemic neutrophils. cxcl treatment of bone marrow neutrophils in vitro caused substantial induction of neutrophil-mobilizing cytokine granulocyte colony-stimulating factor (gcsf) and expression of β integrin that accounts for sexual dimorphism in circulating neutrophil populations in i/r. moreover, male cxcl -stimulated bone marrow neutrophils had an increased capacity to adhere to β integrin ligand icam- , implicating a greater sensitivity of male leukocytes to cxcl -mediated activation. differential induction of cxcl (human cxcl ) between the sexes was also evident in murine renal i/r, rat pleurisy, and human skin blisters and correlated with the magnitude of neutrophil accumulation in tissues. conclusions: our study reveals that sex-specific induction of chemokine cxcl /cxcl contributes to sexual dimorphism in neutrophil recruitment in diverse acute inflammatory responses partly due to increased stimulation and trafficking of bone marrow neutrophils in males. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. neutrophils are essential for early innate immune responses in infection and sterile injury such as ischemia/ reperfusion (i/r). however, due to their high destructive potential, regulation of accumulation of neutrophils in tissues is critical for limiting tissue injury and loss of function in acute inflammatory conditions (for review, see [ ] ). while it is established that women are more susceptible to chronic inflammatory autoimmune diseases (for review, see [ ] ), profound sex differences also exist in severity and mortality from disorders that are characterized by prolonged or excessive innate immune responses (for review, see [ ] ). for example, the severity and incidence of i/r-induced organ failure and acute respiratory distress syndrome (ards) are substantially higher in men compared to those in women (for reviews, see [ ] [ ] [ ] ). while differential lifestyle/environmental factors between men and women influence outcome of these conditions, sex differences are also evident in experimental mammalian models of disease, intimating that this sex bias is an inherent feature in males. elucidation of the male and female patterns of innate immune responses has important implications for understanding disease progression in men and women and therefore appropriate targeting of antiinflammatory therapies [ ] . we have previously demonstrated that acute exposure to bacteria and other irritants in male mice and rats result in increased severity of inflammation compared to females, associated with enhanced recruitment of neutrophils into tissues [ ] . similarly, other studies indicate increased neutrophil accumulation following cytokine treatment or i/r in males [ , ] . however, many fundamental aspects of this differential regulation of neutrophils remain unclear, and thus, attempts to exploit such sex differences therapeutically have not yet been successful. for example, it remains unclear whether neutrophil responses in females involve distinct molecular pathways, or whether female responses are delayed or resolve earlier than in males. thus, to understand the regulatory mechanisms that underlie differential neutrophil responses between the sexes, we have investigated the temporal regulation and molecular pathways of neutrophil recruitment in males compared to those in females. trafficking of neutrophils from the circulation into tissues is coordinated by a network of chemokines, particularly those harboring the elr (glu-leu-arg) motif (e.g., cxcl , cxcl ) [ ] , which establish chemotactic gradients between blood and tissue as well as within tissues [ ] . neutrophils are recruited via the leukocyte adhesion cascade through multiple steps (e.g. rolling adhesion and emigration), mediated by adhesion molecules including selectins and integrins [ ] . the bone marrow is considered to be the primary site of neutrophil production and a store of neutrophils that can be rapidly mobilized to increase the number of circulating neutrophils available for recruitment at sites of inflammatory insult. therefore, regulation of neutrophil release from the bone marrow is a key step that dictates the magnitude and duration of neutrophil recruitment. similar to recruitment from the blood into the tissues, trafficking of neutrophils out of the bone marrow into the circulation is also directed by elr+ chemokines. the actions of elr+ chemokines in bone marrow mobilization are a combined effect of direct interaction with neutrophil cxcr receptors together with the negative regulation of the predominant bone marrow neutrophil retention pathway, i.e., neutrophil cxcr and its ligand cxcl (for review, see [ ] ). we hypothesized that differential regulation of the chemokine network underlies sex differences in the magnitude of neutrophil recruitment. the aim of our study was to investigate sexual dimorphism in neutrophil kinetics and identify the specific chemokines responsible for bringing about these effects. we found that the onset of neutrophil infiltration into tissues was similar between the sexes but that the levels continued to increase with time in males due to substantial increases in circulating neutrophils. the predominant endogenous chemotactic mediator generated by i/r in males was elr+ chemokine cxcl (human cxcl [ ] ), which had a unique temporal profile compared to other chemokines that accounts for sustained neutrophil stimulation, mobilization, and recruitment during reperfusion. the greater induction of cxcl in males was associated with more neutrophilia and consequently a higher magnitude of neutrophil recruitment and tissue injury than that in females. therefore, these studies provide the first evidence for sex-specific regulation of cxcl / in acute inflammatory states. experiments were conducted on age-matched male and female wistar rats ( - g; - weeks, charles river) or c bl mice ( - g; - weeks, charles river) and approved by animals scientific procedures act (uk). for intravital microscopy, rats were kept on a restricted diet for h to reduce intestinal motility during imaging. anaesthetized rats (pentobarbitone mg/kg, ip) were prepared for intravital microscopy (ivm), as previously described [ ] . rats were placed on a heated ( °c) viewing stage, and a loop of intestine was exposed to visualize the mesenteric microcirculation. the mesentery was superfused with tyrode's solution ( % co , ph . ). images were recorded using pinnacle studio software (v. ). in each animal, a single un-branched postcapillary venule (diameter - μm, length > μm) was selected for the study. the following parameters were measured: leukocyte flux, rolling velocity, adhesion, and emigration. rolling leukocytes were observed as cells moving visibly slower than red blood cells and were measured by counting the number of rolling leukocytes passing a fixed reference point (flux) on the vessel segment over a -min period. leukocyte rolling velocity was determined from the time required for a randomly chosen leukocyte to roll across μm. rolling velocities of six leukocytes were averaged and expressed as micrometer per second. adherent leukocytes were identified as cells that remained stationary within the vascular lumen for a period of at least s and were counted in four consecutive -μm vessel segments. leukocyte emigration from the postcapillary venule into the tissue was quantified by counting the number of cells up to , - , and - μm away from the vessel wall in parallel with -μm vessel segments. four readings were taken for each vessel and quantified on one side of the vessel wall. red blood cell centreline velocity was measured in venules using an optical doppler velocimeter (microcirculation research institute, texas a&m university, usa). mean red blood cell velocity was calculated from centreline velocity/ . , and venular shear rate was determined based on the newtonian definition ( × mean red blood cell velocity / venular diameter). in some experiments, male rats were treated with cxcl ( μg/kg, ip, r&d systems) and ivm conducted at h. rats from the same litter were alternately assigned to sham or i/r group, with experiments conducted on males and females on alternate days. ischemia was induced by occluding the superior mesenteric artery (sma). after min of ischemia, reperfusion was permitted for up to h and leukocyte dynamics were measured by ivm every min. sham-operated animals underwent identical surgical procedures without occlusion of the sma. blockade of endogenous cxcl was achieved by -h pre-treatment with cxcl monoclonal antibody ( μg/kg, iv, r&d systems). the intestinal wall was removed and portions of mesentery were fixed ( % paraformaldehyde, min) and dried onto slides overnight. tissues were immersed in absolute alcohol ( min) and washed with distilled water prior to staining with hematoxylin and eosin. following reperfusion, a -cm segment of the exposed intestinal wall was purged, cut into - mm cubes and incubated with p-nitroblue tetrazolium dye (nbt, . mg/ ml, min, °c). necrotic unstained portions were separated from viable stained tissue and expressed as a percentage of total wet weight of the segment. lung tissues were gently rinsed, homogenized in . % (w/v) hexadacyl trimethylammomium bromide and centrifuged ( , g, min, °c). peroxidase activity of the supernatant was measured as the rate of h dependent oxidation of , ′, , ′-tetramethylbenzidine relative to purified myeloperoxidase (mpo), by optical density ( nm). tissue mpo levels were normalized to total protein content. blood leukocytes were collected into . m edta. peritoneal leukocytes were collected by lavage ( ml pbs/ . % bsa/ mm edta). bone marrow (bm) cells were isolated from the femur by flushing with ice-cold pbs (rats, ml; mice, ml) and passing through a μm cell strainer. plasma and supernatants were collected by centrifugation ( g, min, °c) and stored at − °c for protein analysis. for all cell samples, erythrocytes were lysed and leukocytes were either snap frozen for rna analysis or prepared for flow cytometry. mesenteric tissues were isolated by separating the mesentery from the intestinal wall, snap frozen, and stored at − °c for rna analysis. leukocytes were resuspended in pbs/ % goat serum ( × cells/ml). flow cytometry was conducted on bd facscalibur™ with data analyzed by flowjo . . . leukocytes were fixed and permeabilized (leucoperm, abd serotec) and incubated with antibodies ( min, °c) to leukocyte subset markers, integrins, or l-selectin (in live cells), using respective isotype antibodies as controls and compensated as appropriate for multiple labeling. surface integrin and l-selectin expression was calculated as fold expression compared to isotype (relative fluorescence intensity: rfi). for additional details about antibodies, see additional file : table s . neutrophil shape change was assessed by increased ability of rp + leukocytes to scatter light in flow cytometer and expressed as percentage of neutrophils exhibiting high forward scatter (fsc hi ), as previously described [ , ] . rna was extracted from leukocyte pellets and mesenteric tissue (nucleospin, macherey-nagel), reverse transcribed (mouse moloney leukaemia virus reverse transcriptase) and ng cdna submitted to quantitative realtime pcr (applied biosystems ht), and quantified using sybr® green (for primer sequences, see additional file : table s ). rna levels of target genes were assessed by threshold cycle number (ct) and normalized to ct of house-keeping gene for s and calculated as fold expression relative to the mean ct value of the control group, using ΔΔct method [ ] . chemokines in plasma, cell-free bm washouts, or blister fluid were measured by enzymelinked immunosorbent assay (elisa) according to the manufacturer's instructions (r&d systems: cxcl , cxcl , cxcl ; ebioscience: ccl ). in vitro stimulation of leukocytes bm leukocytes ( × ) in phenol red-free rpmi medium ( % fetal calf serum, mm l-glutamine, u/ml penicillin/streptomycin) were plated in -well plates and stimulated with cxcl or cxcl ( ng/ml, h, r&d systems). cells were collected using edta cell dissociation buffer (invitrogen). bm leukocytes ( cells) were prepared in serum-/phenol red-free rpmi medium and stimulated with cxcl ( ng/ml, h, r&d systems) prior to plating in black opaque -well plates coated with bsa ( % w/v) or icam ( μg/well) for min ( °c). plates were prepared by coating wells with protein overnight and treated with bsa ( % w/v, h) to inhibit non-specific interactions prior to addition of leukocytes. non-adherent leukocytes were removed by washing with ca + /mg + -free pbs. adherent leukocytes were labeled with calcein am ( μm, h), quantified by spectrophotometry (absorbance nm), and expressed as a percentage of absorbance of labeled cells. pleurisy was induced in wistar rats by injection of . ml of % carrageenan (w/v) into the pleural cavity. pleural leukocytes were collected at h by lavage ( ml pbs/ . % citrate (w/v). edema was assessed by the weight of excess fluid recovered from the pleural cavity. male and female c bl mice were anesthetized with ketamine/xylazine ( mg/kg, mg/kg, ip), and renal pedicles were occluded using microvascular clamps. after -min bilateral ischemia, the clamps were removed and the skin was sutured. analgesic buprenorphine ( . mg/ kg, s.c.) was administered, and mice were allowed to recover for h prior to harvesting samples. ethical approval was obtained from university college london ethics board. blisters were elicited on the ventral aspect of the forearms of healthy volunteers (eight men, six women, aged - years) by applying μl of . % cantharone (dormer labs, inc.). volunteers did not take any medications for weeks before commencement of the study and abstained from exercise, alcohol, and caffeine for at least h prior to induction of blister. blister fluid was collected at h and leukocytes were prepared for cytometry, as previously described [ ] . after exclusion of cd + lymphocytes, neutrophils were identified as cd hi /hla-dr − and monocytes as cd hi /hla-dr + . data are expressed as mean ± sem. comparisons between two groups were made by two-tailed unpaired student's t test. for comparisons between multiple groups, a one-way anova was performed followed by bonferroni's posttest. comparisons between time-response curves were made using a two-way anova, followed by bonferroni's post-test. blister samples were analyzed by nonparametric mann-whitney test. statistical analysis was performed using prism . (graphpad software inc.). to understand whether sex differences exist in the temporal regulation of leukocytes in acute inflammatory responses, we subjected male and female rats to -min complete mesenteric ischemia followed by -h reperfusion. histology of the mesenteric vasculature revealed substantially more cell infiltration at the end of reperfusion in male tissues than that in females (fig. a) . facs analysis identified recruitment of rp + neutrophils into the peritoneal cavity in both sexes, but levels were significantly greater in males than those in females (fig. b) . in addition to increased neutrophil recruitment in males, substantial redness and edema was evident in the small intestine at the end of reperfusion in males but not females (additional file : figure s a ). quantification of intestinal wall necrosis using nbt [ ] confirmed that the extent of tissue injury was significantly more in males at both -min and -h reperfusion (fig. c) . to directly examine the events that precede enhanced i/r-stimulated neutrophil sequestration in male tissues, we used intravital microscopy to visualize leukocyte/vessel wall interactions throughout the reperfusion period. the rate of leukocytes interacting with mesenteric venules (leukocyte flux) increased from the onset of reperfusion in both sexes, but the increase was less in females and plateaued at h whereas leukocyte flux continued to increase with reperfusion time in males (fig. d ). dampened leukocyte flux in females was also accompanied by fewer adherent leukocytes to the vessel lumen and subsequent emigration into the mesenteric tissue (additional file : figure s b -c). these differences in leukocyte dynamics were not a consequence of differences in i/r-induced changes in hemodynamics (mean arterial blood pressure, red blood cell velocity, or wall shear rate) since no significant change from basal values was evident during reperfusion or between the sexes (additional file : figure s a -c). while leukocyte flux in the mesenteric vasculature was substantially more in males, the velocity of rolling leukocytes was similar in both sexes implicating a difference in the availability of leukocytes rather than in their capacity to interact with venules (fig. e) . consistent with this, neutrophil adhesion molecule l-selectin expression (rfi) at -h reperfusion was . ± . and . ± . (n = , p > . ) in males and females, respectively. therefore, we hypothesized that a key difference in the response to i/ r in males and females involves the regulation of release of neutrophils from the bone marrow. indeed, reperfusion stimulated a time-dependent surge in circulating neutrophils in males but had little effect on blood neutrophil populations in females (fig. f) . in contrast, the modest i/ r-induced changes in circulating monocyte numbers were similar in both sexes, suggesting a selective differential regulation of neutrophil mobilization in males (additional file : figure s d ). moreover, the levels of circulating neutrophils were directly correlated (r = . , fig. g ) with the extent of i/r-induced tissue necrosis in both sexes, distinct temporal regulation of neutrophil recruitment protects against i/r injury in females. male and female rats were subjected to min mesenteric ischemia followed by up to -h reperfusion. a representative images of segments of male and female mesentery at h of reperfusion, stained with hematoxylin and eosin, demonstrating fewer nucleated cells within and around female venules. dashed lines approximately demarcate venule lumen. b accumulation of peritoneal rp + neutrophils, measured by cytometry. c proportion of intestinal necrosis, measured by nitroblue tetrazolium. sham n = rats/group, i/r n = rats/group. d, e leukocyte/vessel wall interactions throughout reperfusion in mesenteric venules, measured by intravital microscopy: d leukocyte flux and e leukocyte rolling velocity (sham n = rats/group, i/r n = rats/group). f circulating rp + neutrophils, measured by flow cytometry. g direct correlation between number of circulating neutrophils and extent of intestinal necrosis in both males and females, measured at -min and -h reperfusion. sham n = rats/group, i/r n = rats/group. data are presented as mean ± sem. ***p < . by one-way anova. §p < . by two-way anova, and ‡p < . or #p < . by bonferroni's post-test compared to male i/r. ns denotes p > . supporting a causative link between mobilization of neutrophils and i/r injury. the finding that circulating neutrophil numbers are elevated during reperfusion indicates that i/r triggers bone marrow neutrophil stimulation and mobilization in males but not females. acute granulopoeisis and neutrophil maturation/mobilization during infection are controlled by granulocyte colony stimulating factor (gcsf) [ ] . accordingly, i/r induced an early and sustained synthesis of gcsf by bone marrow cells and ischemic mesenteric tissue in males but not females (fig. a) . these results imply that increased gcsf levels in males stimulate enhanced bone marrow neutrophil motility compared to those in females during reperfusion. indeed, we noted a significant increase in the size of individual bone marrow neutrophils in males at -h reperfusion, indicative of neutrophil shape change [ , ] (fig. b) . furthermore, surface integrin expression (β : fig. c ; β , α , αl, αm: additional file : figure s ) on the bone marrow and circulating neutrophils at -h reperfusion was significantly elevated in males but remained unchanged in females. a possible functional consequence of increased circulating neutrophils with high integrin expression is that neutrophils can infiltrate other tissues remote to the site of inflammatory insult. in particular, secondary injury of the fig. increased bone marrow neutrophil stimulation in males. male and female rats were subjected to -min mesenteric ischemia followed by up to -h reperfusion. a expression of mesenteric tissue and bone marrow cell gcsf mrna, normalized to levels of s and calculated as fold expression relative to mean value of male sham group. b bone marrow neutrophil shape change measured by cytometry as the proportion of rp + cells with high forward scatter (fsc hi ), at -h reperfusion. c bone marrow and blood neutrophil β integrin expression at -h reperfusion, measured by cytometry and expressed as relative fluorescence intensity (rfi) compared to isotype control antibody. d neutrophil accumulation in the lungs, assessed by tissue mpo activity. sham n = rats/group, i/r n = rats/group. data are presented as mean ± sem. *p < . , **p < . , ***p < . , and ns p > . by one-way anova lung due to inappropriate neutrophil recruitment is a common consequence of i/r (for review, see [ ] ). myeloperoxidase (mpo) activity in lung tissue, an index of neutrophil accumulation, remained unaltered in the lungs of female rats but was elevated fivefold in males by the end of reperfusion (fig. d) . thus, these studies strongly indicate that more bone marrow neutrophils are mobilized in males during i/r and that neutrophils circulating during reperfusion exhibit a greater adhesive phenotype compared to females. together, this differential regulation of neutrophils may protect against local and remote tissue injury in females. while gcsf is essential for regulation of neutrophil number in the bone marrow, egress of mature neutrophils into the circulation in acute inflammatory states is partly dependent on engagement of neutrophil chemokine receptor cxcr with elr+ chemokines such as cxcl [ ] . in keeping with a central role of cxcr for exit of neutrophils from the bone marrow, expression of cxcr mrna by bone marrow cells was elevated throughout reperfusion in males but not females (fig. a) . fig. sustained release of tissue-derived cxcl throughout reperfusion in males but not females contributes to i/r injury. a-e male and female rats were subjected to -min mesenteric ischemia followed by up to -h reperfusion. a-c expression of bone marrow neutrophil mobilization pathways: a bone marrow cell cxcr mrna, b bone marrow total cxcl protein, and c bone marrow total cxcl protein. sham n = rats/group, i/ r n = rats/group. d mesenteric tissue mrna levels of cxcl and cxcl . levels of mrna are normalized to s and calculated as fold expression relative to mean value of male sham group. sham n = rats/group, i/r n = rats/group. e plasma protein levels of cxcl and cxcl , at -min and -h reperfusion. sham n = rats/group, i/r n = rats/group. f-h male rats were treated with either anti-cxcl ( μg/kg, iv) or control igg ( μg/ kg, iv) h prior to mesenteric ischemia (n = rats/group). f leukocyte/vessel wall interactions in mesenteric venules throughout reperfusion. g circulating rp + neutrophils and cd + monocytes. h mesenteric necrosis, measured by nitroblue tetrazolium at -h reperfusion. data are presented as mean ± sem. *p < . , **p < . , ***p < . , ns p > . by one-way anova. §p < . by two-way anova and ‡p < . by bonferroni's post-test most important cxcr ligands that promote neutrophil mobilization from bone marrow stores during acute infections. however, the predominant endogenous chemokines that are released during vascular injury, such as i/r, which regulate bone marrow neutrophil trafficking are not known. surprisingly, protein levels of cxcl within the bone marrow were not modulated by i/r in either sex (fig. b) whereas cxcr ligand cxcl was upregulated by i/r and increased with reperfusion time in males, with no discernible change in cxcl in females (fig. c) . pro-mobilization signals during infection typically arise from local tissue production of cytokines and chemokines at the site of infection. to verify the primary cellular source of i/r-induced chemokine production, we measured chemokine mrna in mesenteric tissue at -min and -h reperfusion. in males, tissue cxcl mrna was rapidly induced upon reperfusion and increased with reperfusion time whereas induction of chemokines cxcl and ccl were greater at min and had declined by h (fig. d and additional file : figure s a ). the profile of circulating levels of chemokine protein mirrored the profile of tissue mrna levels ( fig. e and additional file : figure s b ), implicating the ischemic tissue as a principle source of these chemokines. importantly, levels of both tissue and circulating chemokines were less in females with the greatest fold difference being release of cxcl (fig. d ). sex differences in chemokine levels were specific to ligands for cxcr or ccr since induction of chemokines ccl /mip α or ccl /rantes (ligands for ccr + and ccr + leukocytes) was similar in both sexes (additional file : figure s c-d) . the unique profile of cxcl synthesis and its differential induction between the sexes indicates that this particular chemokine may be a pivotal endogenous mediator of i/r-induced neutrophil responses and tissue injury. neutralization of cxcl in males reduced plasma cxcl at -h reperfusion from . ± . to . ± . ng/ml (p < . , n = ) and dampened i/r-induced leukocyte dynamics (fig. f , additional file : figure s c -d) to similar levels as those seen in females (fig. d) . the effect of anti-cxcl on leukocyte recruitment was partly due to a reduction in neutrophilia (fig. g) , which was also limited to a similar magnitude as that in females (fig. f ) . collectively, these studies demonstrate that augmented levels of cxcl in males account for increased neutrophilia during i/r compared to females. to recapitulate the impact of tissue-derived cxcl on bone marrow neutrophil stimulation and mobilization, we administered cxcl ( μg/kg) locally into the peritoneal cavity of male rats and measured neutrophil populations at h. this dose of cxcl increased circulating levels at h from . ± . to . ± . ng/ ml (p < . , n = ), bone marrow levels from . ± . to . ± . ng/femur (p < . , n = ), and stimulated similar levels of leukocyte recruitment into mesentery (additional file : figure s a -c) as i/r (fig. d , additional file : figure s b&c ). administration of cxcl , in the absence of ischemia, increased circulating neutrophils, but not monocytes, as well as neutrophil β integrin (fig. a,b) and was sufficient to induce significant tissue injury at the site of administration and in the lungs (fig. c,d) . to identify the molecular pathways in cxcl -induced bone marrow neutrophil stimulation/mobilization, rat bone marrow cells were treated with cxcl ( ng/ml, h) in vitro. the proportion of rp + neutrophils isolated from femurs was similar (p > . , n = ) in both sexes ( . ± . and . ± . % in males and females, respectively). cxcl , but not cxcl , increased gcsf mrna in both sexes (fig. e) , implicating that cxcl can influence bone marrow neutrophil numbers and stimulation. consistent with this notion, cxcl promoted expression of β integrins in rp + neutrophils (fig. f ) . notably, this effect of cxcl was significantly greater in male neutrophils compared to females, as confirmed by the increased ability of cxcl -stimulated male leukocytes to adhere to β integrin ligand icam (fig. g) . cxcl , but not equivalent amounts of cxcl , stimulated expression of cxcr as well as cxcl in leukocytes from both sexes (fig. h,i) . these studies confirm the hypothesis that elevated cxcl activity in males is sufficient to directly increase bone marrow neutrophil stimulation and mobilization pathways. renal dysfunction and neutrophil accumulation following i/r is more severe in males than that in females [ , ] . therefore, to understand whether sex-specific regulation of cxcl in i/r occurs in other organs and mammals, we measured tissue and circulating chemokines following acute bilateral renal i/r in male and female mice. renal ischemia of min followed by -h reperfusion resulted in greater levels of tissue cxcl mrna in males than females (fig. a) as well as plasma cxcl protein ( ± pg/ml and ± , p < . , n = , respectively). similar to mesenteric i/r, this chemokine profile in males was also associated with more circulating neutrophils than that in females (fig. b) . next, we examined whether disparate induction of cxcl also dictates sex differences in neutrophil responses induced by an exogenous stimulus. injection of carrageenan into the pleural cavity of rats results in greater accumulation of fluid (fig. c) and neutrophils in the cavity in males than in females [ ] . this sex difference in pleurisy was also associated with higher levels of cxcl in the lung tissue and bone marrow of males compared to females (fig. d,e) . to examine the role of cxcl in the magnitude of neutrophil responses in humans, we used the cantharidin skin blister model of leukocyte trafficking in healthy volunteers. the mean age of participants was ± . (males, n = ) and ± . (females, n = , p > . ). at h following application of cantharidin, edema (blister volume) was substantially greater in men than women (fig. f ). similar to responses in rodents, the number of neutrophils (but not monocytes) recruited to the site of injury was also greater in men compared to that in women (fig. g) . levels of cxcl , the human ortholog of rodent cxcl , tended to be higher in the blisters of men than women and were correlated with the number of neutrophils recovered from the blister (fig. h,i) . thus, together, our findings indicate that increased induction of cxcl /cxcl in males in the early phases of innate immune responses is a common response to diverse pro-inflammatory stimuli in male mammals that contributes to elevated neutrophil stimulation and tissue infiltration. regulation of neutrophil accumulation in tissues is a critical feature of acute inflammation. here, we provide (e-f, h-i) bone marrow cells ( × ) were isolated from male and female rats (n = rats/group) and treated with cxcl ( ng/ml) or cxcl ( ng/ml) for h. e bone marrow cell gcsf mrna expression. f expression of neutrophil β integrin, measured by cytometry. g isolated male and female bone marrow cells ( ) were stimulated with cxcl ( ng/ml, h) prior to incubation in -well plates for min. adhesion of cells to wells coated with bsa ( %) or icam ( μg/well), quantified using calcein-am (n = rats/group). h-i bone marrow cell mrna expression of h cxcr , i cxcl . levels of mrna are normalized to s and calculated as fold expression relative to mean value of male control group. data are presented as mean ± sem. *p < . , **p < . , ***p < . , ns p > . by one-way anova novel insight into the nature and molecular mechanisms that determine sexual dimorphic regulation of neutrophil responses in i/r. specifically, we provide evidence that cxcl / is the key molecular signal whose greater induction in males primarily affects circulating neutrophil numbers and adhesion molecule expression, thereby enhancing tissue infiltration. tissue injury during i/r involves multiple complex interacting mechanisms and several cell types (for review, see [ ] ). in this study, we focused on understanding sex differences in the initial inflammatory response during i/r and the early stages of neutrophil recruitment. we, and others, have demonstrated increased total neutrophil infiltration of tissues in males compared to females in acute inflammatory states [ ] [ ] [ ] . in agreement with these studies, we observed substantially greater neutrophil influx into the peritoneal cavity following mesenteric i/r in male rats compared to age-matched females. migration of blood neutrophils to sites of inflammatory insult is a multi-step process involving leukocyte rolling, adhesion, and emigration that is regulated by a network of chemokines, particularly elr+ chemokines. in the current study, we aimed to understand the primary differences in neutrophil dynamics between males and females. using ivm to monitor changes in i/r-induced leukocyte responses within the same animal in the mesenteric vasculature in real time, we reveal that the initial (< min) increase in leukocyte flux during reperfusion is similar between the sexes. in males, these responses continue to increase with reperfusion time, leading to accumulation of substantial number of neutrophils within the peritoneal cavity by h. in contrast, the extent of leukocyte/vessel c excess volume of fluid recovered from pleural cavity (lung edema), d lung tissue cxcl mrna, e bone marrow cxcl protein. levels of mrna are normalized to s and calculated as fold expression relative to mean value in males. data are presented as mean ± sem. *p < . and **p < . by two-tailed unpaired student's t test. f-i cantharidin-induced skin blisters in healthy male and female volunteers. f blister volume (edema), g blister neutrophils and monocytes, measured by cytometry, h blister fluid cxcl protein, and i correlation between blister cxcl and neutrophils. individual data points represent one volunteer. blister samples were analyzed by non-parametric mann-whitney test wall interactions in females remains relatively constant between -min and -h reperfusion. however, the velocity of rolling male leukocytes is similar to female cells, suggesting little differences in leukocyte motility and their ability to interact with blood vessels. sex differences in neutrophil dynamics appear to be predominantly due to the greater neutrophilia that is evident in males throughout reperfusion. therefore, the male neutrophil response does not appear to be an earlier onset or slower clearance of recruited cells compared to females but rather a difference in the absolute magnitude of the response brought about, in part, by more neutrophils available in the circulation at later stages of reperfusion. indeed, the extent of tissue injury in the intestine is directly correlated with the number of circulating neutrophils and is therefore substantially greater in males. similarly, several lines of evidence indicate that severity of inflammatory diseases are correlated with the extent of circulating neutrophils [ ] [ ] [ ] , pathologies that are also considered to be more severe in age-matched men compared to women. we believe that distinct regulation of neutrophil trafficking in males may be a fundamental feature of acute inflammatory responses. circulating neutrophil populations are rapidly increased in inflammatory responses, predominantly via elevated rates of trafficking out of bone marrow stores. our study provides the first evidence for differential regulation of neutrophil pro-mobilization pathways in males and females. gcsf is a major mobilizing cytokine that directly regulates granulopoiesis and bone marrow neutrophil shape change, motility, and release [ ] under homeostatic [ ] and inflammatory conditions [ , ] . mesenteric i/r induces greater gcsf expression at the site of injury and within bone marrow cells, neutrophil shape change, and an increase in neutrophil integrin expression in males than in females. these findings are consistent with increased rates of trafficking of stimulated neutrophils into the circulation during reperfusion in males but not females. trafficking of neutrophils is predominantly guided by chemokines. in particular, archetypal elr+ chemokines generated at the site of inflammation are not only direct neutrophil chemoattractants but also induce neutrophil mobilization from bone marrow stores, primarily through interaction with leukocyte receptor cxcr . for example, elr+ chemokines cxcl and cxcl are required for bone marrow neutrophil motility and mobilization following administration of gcsf [ ] or thioglycollate [ ] in mice. in the current study, we identified cxcl as the predominant elr+ chemokine released throughout i/r in males but not females. circulating and bone marrow levels of cxcl are upregulated by i/r and increase with reperfusion time in males, with little induction of cxcl evident in females. indeed, blockade of cxcl in males results in an identical neutrophil response as that seen in females. as such, leukocyte/vessel wall interactions within -min reperfusion are unaffected by cxcl inhibition but leukocyte recruitment and tissue injury at later stages of reperfusion are substantially reduced. thus, cxcl has a predominant role in driving sustained neutrophil recruitment during i/r. at early time points, neutrophil infiltration is most likely mediated by other chemokines such as cxcl and ccl that are transiently induced in tissues of both sexes and can recruit neutrophils already in the circulation. the functional effect of cxcl production during i/r in males is partly to increase circulating neutrophils since inhibition of cxcl also reduces i/r-induced neutrophilia to similar levels seen in females. conversely, administration of cxcl to males in the absence of ischemic insult is sufficient to account for sex differences in i/r-induced changes in circulating neutrophil populations. therefore, cxcl has a non-redundant role in release of neutrophils into the circulation during i/r, which may be a general phenomenon during acute resolving/selflimiting inflammation. previous studies indicate an important homeostatic role of cxcl in maintaining bone marrow neutrophil numbers [ ] . however, our study shows no apparent sex difference in homeostatic functions of cxcl since the levels and cell populations are similar in untreated male and female controls (data not shown) and in shamoperated animals. we only observe disparities in the functional role of cxcl in males and females in response to pro-inflammatory stimuli where a temporal relationship between production of cxcl and extent of neutrophil infiltration into tissue intimates a causal relation. cxcl is an upstream regulator of gcsf production within bone marrow in vivo [ ] . similarly, in the current study, application of cxcl to isolated bone marrow cells in vitro induced substantial expression of gcsf and neutrophil β integrin. surprisingly, these effects on cell surface β integrin were greater in male than those in female cells despite similar basal levels of receptor cxcr expression as confirmed by greater capacity of male cells to adhere to purified integrin ligand icam- . thus, male bone marrow cells have increased sensitivity to cxcl through disparate cxcr signaling; the precise mechanism is not elucidated in the current study but merits further investigation. nonetheless, this important finding indicates that even in situations where cxcl levels are elevated in females, circulating neutrophils are likely to have a less adhesive phenotype compared to neutrophils in males. the effect of cxcl is clearly more potent than cxcl in either sex, in agreement with previous reports indicating that truncated forms of cxcl have greater affinity for cxcr than other ligands [ ] . administration of cxcl also induces its own expression as well as its receptor, which suggests that cxcl appears to work in a positive feedback loop to enhance and prolong synthesis at a transcriptional level. this may underlie differential kinetics of this chemokine whereby cxcl has a longer half-life than other elr+ chemokines. for example in acute endotoxemia in mice, cxcl expression remains elevated longer than cxcl and cxcl in almost all tissues [ ] . moreover in severe inflammation, cxcl production prolongs the activity of cxcl and cxcl by establishing local chemokine gradients and thereby promoting tissue injury [ ] . previous reports have demonstrated greater renal injury, neutrophil accumulation, and mortality rates in males than in females following renal i/r in rats [ ] . therefore, to assess whether sex-specific regulation of cxcl -mediated neutrophil responses occurs during i/r in other organs and species, we investigated sex differences in renal i/r in mice. similar to our findings in rat mesenteric i/r, greater cxcl expression was evident in the kidneys of male mice compared to females at the end of reperfusion and associated with elevated numbers of circulating neutrophils with higher β integrin expression. it is important to note that the induction of ischemia itself may be different in females since sex hormones, in particular estrogens, can limit the production of tissue-damaging mitochondrial reactive oxygen species and thereby increase tolerance to ischemia and reduce the inflammatory response (for review, see [ ] ). however, we observed the same divergent regulation of cxcl -dependent neutrophil responses in males and females following administration of equivalent amount of a chemical stimulus (carrageenan) into the pleural cavity of rats. increased expression of cxcl in the lung tissue and bone marrow of males was also associated with greater lung edema. overall, our data imply that distinct regulation of cxcl induction in males is a general phenomenon during i/r and other acute inflammatory responses that result in enhanced neutrophil accumulation and tissue injury. in humans, cxcl exists in two forms, namely cxcl / ena and cxcl /gcp . recent evidence indicates that cxcl /gcp is the closer ortholog to rodent cxcl [ ] . our study in human volunteers supports the thesis that sex-specific regulation of cxcl underlies increased neutrophil recruitment in males. topical application of cantharidin in healthy volunteers disrupts intercellular connections between keratinocytes and stimulates the influx of leukocytes, predominantly neutrophils, within h [ ] . while there is variation in the magnitude of leukocyte recruitment into blisters between individuals, the extent of neutrophil (but not monocyte) influx and edema is greater in men. despite the small sample size, in agreement with our studies in rodents, local amounts of cxcl within the blister fluid are correlated with neutrophil accumulation and tend to be greater in men. therefore, together, our findings indicate that reduced production and sensitivity to cxcl / may underlie a fundamental tissue-sparing mechanism in acute inflammatory responses in women. furthermore, cxcl / may be an important therapeutic target in perturbing acute inflammatory diseases as well as a biochemical marker of disease progression. sex differences in regulation of cxcl may have important consequences for progression of acute inflammatory disorders. few studies of human disease have investigated the biological role of cxcl ; however, ortholog cxcl /ena is involved in a variety of inflammatory diseases such as ards [ , ] , acute coronary syndromes, inflammatory bowel disease, asthma, and psoriasis, diseases known to have a neutrophil component. serum levels of chemokine cxcl have also been associated with intima-media thickness of the common carotid artery, a marker of preclinical atherosclerosis, and cxcl levels are reduced by anti-inflammatory statin therapy [ ] . whether cxcl activity underlies sex differences in acute inflammatory disorders is not known. however, polymorphisms in cxcl gene are correlated with acute coronary syndrome [ ] , a disease where more numerous plaque-destabilizing neutrophils have been observed in lesions of men compared to women. we suggest that our findings indicate a role for increased cxcl / induction in males in contributing to sex differences in inflammatory disorders that are characterized by excessive neutrophil recruitment. further investigations will be required to elucidate the upstream mechanisms that lead to greater induction of cxcl / in males compared to females. our study does not distinguish whether male factors increase cxcl / or whether female factors inhibit cxcl / . current evidence indicates complex and interacting influences of sex on inflammatory responses including multiple sex hormones, x-linked and y-linked genes (for reviews, see [ , , ] ), although no unifying explanation exists since these factors may paradoxically be pro-or antiinflammatory depending on cell type, stimulus, stage of response, and age. thus, to fully understand how sex differences in neutrophil responses are brought about, future studies need to examine the impact of individual sex hormones and chromosomes on regulation of chemokine production and neutrophil trafficking. moreover, although our data demonstrate that the male neutrophil response can be made "female" by acutely blocking endogenous cxcl , it remains to be shown whether cxcl alone is sufficient to make a female neutrophil response identical to that in males. in the current study, we primarily focused on the role of chemokine production on sex differences in neutrophil dynamics in the onset phases of leukocyte recruitment. points of control in inflammation sex differences in autoimmune disease sex and inflammation in respiratory diseases: a clinical viewpoint gender dimorphism following injury: making the connection from bench to bedside gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury race and gender differences in acute respiratory distress syndrome deaths in the united states: an analysis of multiple-cause mortality data ( - ) sex bias in trials and treatment must end sex-differences in resident immune cell phenotype underlies more efficient acute inflammatory responses in female mice suppression of endothelial p-selectin expression contributes to reduced cell trafficking in females: an effect independent of no and prostacyclin gender difference and sex hormone production in rodent renal ischemia reperfusion injury and repair chemokines: a new classification system and their role in immunity intravascular danger signals guide neutrophils to sites of sterile inflammation breaching multiple barriers: leukocyte motility through venular walls and the interstitium neutrophil kinetics in health and disease the evolution of mammalian chemokine genes interleukin- -induced leukocyte extravasation across rat mesenteric microvessels is mediated by plateletactivating factor a flow cytometric method to measure shape change of human neutrophils multiparameter flow cytometric analysis of polymorphonuclear leucocytes in whole blood from patients with adult rapidly progressive periodontitis reveals low expression of the adhesion molecule l-selectin (cd l) analysis of relative gene expression data using real-time quantitative pcr and the -ΔΔct method characterisation of leukocytes in a human skin blister model of acute inflammation and resolution quantitating intestinal ischemia with nitroblue tetrazolium salts granulocyte colony-stimulating factor: molecular mechanisms of action during steady state and 'emergency' hematopoiesis pathophysiology of ischemia-reperfusion injury the coordinated action of g-csf and elr + cxc chemokines in neutrophil mobilization during acute inflammation cellular and molecular mechanisms of sex differences in renal ischemia-reperfusion injury cell biology of ischemia/reperfusion injury bcl prevents acute inflammatory lung injury in mice by restraining emergency granulopoiesis hypercholesterolemia promotes bone marrow cell mobilization by perturbing the sdf- :cxcr axis neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on cxcl signaling g-csf-mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of cxcr ligands increased granulopoiesis through interleukin- and granulocyte colonystimulating factor in leukocyte adhesion molecule-deficient mice g-csf downregulation of cxcr expression identified as a mechanism for mobilization of myeloid cells phagocytosis of apoptotic neutrophils regulates granulopoiesis via il- and il- cxcl regulates chemokine scavenging and pulmonary host defense to bacterial infection cxcr and cxcl regulate the il- /g-csf axis and neutrophil homeostasis in mice posttranslational modification of the nh -terminal region of cxcl by proteases or peptidylarginine deiminases (pad) differently affects its biological activity the murine neutrophil-chemoattractant chemokines lix, kc, and mip- have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regulation in endotoxemia estrogen and mechanisms of vascular protection cantharidin blisters: a technique for investigating leukocyte trafficiking and cytokine production at sites of inflammation in humans inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome human endothelial cells synthesize ena- : relationship to il- and to signaling of pmn adhesion serum cxc ligand is a new marker of subclinical atherosclerosis in type diabetes epithelial neutrophil-activating peptide (ena- ), acute coronary syndrome prognosis, and modulatory effect of statins the complex role of estrogens in inflammation y genetic variation and phenotypic diversity in health and disease sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone this study is supported by barts the data suggest that a primary component of the sex difference in neutrophil trafficking is due to a difference in the number of circulating neutrophils and cxcl -stimulated recruitment. however, several other components of the inflammatory response may also be differentially regulated between the sexes and contribute to greater neutrophil accumulation in male tissues. for example, it is possible that differences in leukocyte cytoskeleton integrity or function may affect leukocyte motility and impede the recruitment of female leukocytes into tissues or their release from the bone marrow due to differences in signaling of adhesion molecules such as psgl- and l-selectin or a difference in the ability of neutrophils to deform and transmigrate. previous studies also indicate that female sex hormones can influence the life span of neutrophils [ ] , a finding that may have implications for the number of neutrophils in the circulation and tissue, particularly in later stages of inflammation. our data suggest greater cxcl / production in males in different inflammatory models in three species; however, further investigations with larger sample size will be needed to fully establish the existence and consequence of differential cxcl / production between the sexes in inflammatory disorders. detailed understanding of fundamental differences in male and female neutrophil responses may lead to improved anti-inflammatory therapies. in conclusion, acute inflammatory insult initiates distinct neutrophil responses in males and females. a salient feature of this difference comprises an increased production and sensitivity to chemokine cxcl / in males. the data demonstrate a novel and predominant role for cxcl / in sustaining neutrophil recruitment into tissues partly through mobilization of tissue-damaging neutrophils from bone marrow. these findings may have important implications for understanding the basis of sex differences in neutrophil trafficking in diverse acute inflammatory pathologies. additional file :tables s and s . table s . details of antibodies used for flow cytometry. table s . sequence of primers used for realtime quantitative pcr with sybr green. (pdf kb) additional file : figure s . distinct temporal regulation of neutrophil recruitment increases tissue i/r injury in males. male and female rats were subjected to -min mesenteric ischemia followed by up to -h reperfusion. the authors declare that they have no competing interests.authors' contributions sm conducted and analyzed most of the experiments. mb designed and interpreted flow cytometry for rodent studies. jw designed, conducted, and analyzed adhesion assays. jd established real-time pcr protocols. nsap conducted renal i/r. isn conducted and analyzed skin blister study. mpm created skin blisters. jd, ct, dwg, sn, and rss provided essential reagents, contributed to design of studies, analysis and interpretation of data. rss wrote the manuscript. all authors read and approved the final manuscript. key: cord- -xzm og authors: valdez, anna maria title: are you covered? safe practices for the use of personal protective equipment date: - - journal: j emerg nurs doi: . /j.jen. . . sha: doc_id: cord_uid: xzm og nan e mergency nurses frequently encounter patients with a known or suspected infectious illness. to prevent the spread of infection and injury, emergency nurses must be well prepared to appropriately select and use personal protective equipment (ppe). furthermore, emergency nurses must have readily available access to ppe, as well as effective and timely training, including routine fit testing for respiratory protection. , according to the occupational safety & health administration (osha), when ppe is required, training for health care personnel must include the identification of the correct ppe; how to properly put on (don), wear, and remove (doff) equipment; limitations of ppe; and how to appropriately maintain and dispose of ppe. personal protective equipment ppe is defined by osha as "specialized clothing or equipment worn by an employee for protection against infectious materials." in health care, ppe includes a range of items including but not limited to gloves, gowns/aprons, masks and respirators, goggles, face shields, and foot/leg covers. when selecting ppe, emergency nurses must consider the type of anticipated exposure and be knowledgeable about current standards set forth by the centers for disease control and prevention (cdc) and organizational policy. , the cdc sets the standard for ppe selection and use in health care settings within the united states. these standards are based on the type of precautions required to prevent the spread of infection and include standard precautions (formerly termed universal precautions), as well as categories of expanded precautions: contact, droplet, and airborne infection isolation. standard precautions are required any time an infectious agent may be present in a patient's blood or body fluids. this type of precaution is used for contact with all patients, hence the term "standard precautions." the amount and type of ppe used for standard precautions depends on the expected exposure that the health care provider will have with the patient. for example, gloves must be worn when contact with blood or bodily fluids is anticipated. during procedures when bodily fluids may splash or spray, health care providers should also be wearing fluid-resistant gowns, a mask and goggles or face shield, and shoe covers. contact precautions contact precautions are required as an expanded transmissionbased precaution when infectious agents may be spread through touch either directly with the patient or indirectly with objects in the environment of care. examples of illnesses that require contact precautions are norovirus, rotavirus, and clostridium difficile. patients who are suspected or known to have an illness that can be spread through touch contact need to be placed in a private room or in a room with other persons who have been colonized with the same organism. they also need to be treated with dedicated equipment that can be left in the room. anyone entering the room who may come in contact with the patient or objects in the room must wear a gown and gloves. ppe, including gowns and gloves, should be removed before leaving the room or in an anteroom if available. as with all patient contact, thorough hand hygiene is critical to prevent the spread of infection. droplet precautions are necessary when infectious pathogens can travel from the respiratory tract of the patient over short distances (usually less than ft, but the distance can extend up to ft). transmission of infection through droplet exposure generally occurs when a patient sneezes, coughs, talks, or undergoes invasive procedures such as endotracheal intubation or suctioning. examples of infections that can be spread through droplet exposure include influenza, bordetella pertussis, respiratory syncytial virus, and severe acute respiratory syndrome-associated coronavirus. , in addition to taking other appropriate standard precautions, emergency nurses should be wearing a face mask when in close proximity to patients requiring droplet precautions. according to the cdc, "airborne transmission occurs by dissemination of either airborne droplet nuclei or small particles in the respirable size range containing infectious agents that remain infective over time and distance." in addition to proper use of ppe, controlling the spread of infectious agents that can be transmitted via an airborne route requires special air handling and ventilation systems. examples of infections spread through airborne transmission include mycobacterium tuberculosis, rubeola virus (measles), and varicella-zoster virus (chickenpox). patients with suspected or known infection that can be transmitted via the airborne route should be placed in an airborne isolation infection room, and health care providers must wear respiratory protection certified by the national institute for safety and health at n or higher when they enter the patient's room. complex transmission precautions infectious agents may fall into several transmission categories and require a combination of precautions to prevent the spread of infection. one example of an infectious illness that requires health care providers to adhere to multiple levels of precautions is the ebola virus disease (evd). health care providers who are caring for a patient with known or suspected evd must adhere to standard, contact, and droplet precautions. recently the cdc issued revised standards for evd precautions, which include detailed guidance on the types of ppe required during patient care and strategies for ensuring safe practice. because of the complex and detailed nature of the guidance on caring for a patient with known or suspected evd, emergency nurses should seek information about precautions and ppe standards directly from the cdc web page at http://www.cdc.gov/vhf/ebola/hcp/index.html. emergency nurses can also gain current and accurate information about how to safely triage and screen patients for evd, manage their care, and select and utilize ppe by accessing the ena ebola resource page located at http://www.ena.org/about/media/ebola/pages/default.aspx. the intent of ppe is to prevent harm to the health care provider; however, the use of ppe is not without risk, especially when wearing ppe that limits movement or when wearing respirators. risk of injury from the use or misuse of ppe can be addressed by implementing safety strategies in the emergency setting. examples of strategies that can be used to prevent injury include strict adherence to infection control precautions, hands-on and in situ training, and staffing that supports safe care. to prevent the spread of infection, emergency nurses must follow appropriate infection control precautions and use ppe as recommended by the cdc and organizational policy. in a study by nichol et al, it was found that fewer than half of the nurses involved in the study met adherence standards for the recommended use of ppe. this finding is also supported by a literature review conducted by gammon et al, who found that compliance with infection control precautions is unacceptably low among health care providers. in addition, nichols et al found that emergency nurses were % less likely to report adherence with infection control procedures than were critical care nurses. this finding is supported by an observational study by creedon et al, who found that compliance with hand washing was lower in the emergency setting than in other areas of the hospital. known barriers to achieving optimal compliance include lack of training, time constraints, and lack of readily available ppe. , emergency nurses and organizational leadership should explore ways to improve adherence to infection control procedures in the emergency setting. considering that the total number of infectious illness outbreaks have been increasing since , and with the recent emergence of infectious illnesses in the united states, including evd and enterovirus d , the need for strict adherence to infection control protocols cannot be overstated. comprehensive training on infection control protocols and the proper use of ppe is a critical component of safe care delivery in the emergency setting. nichol et al found that fewer than half of the nurses observed in their study demonstrated competence when using an n respirator. another finding in this study was that only half of the participants reported having received recent training or fit testing. participants that had been trained and fit tested in the prior years were . times more likely to report adherence with recommended use of ppe. the cdc recommends that health care providers receive repeated training and demonstrate competency in performing all ebola-related infection control practices and procedures, including donning and doffing proper ppe before engaging in patient care activities. this guidance can be applied for all types of infection control precautions and is particularly important when n or powered air-purifying respirators (paprs) are used. the use of respirators, particularly paprs, requires comprehensive training to ensure competency, and failure to properly use this equipment could place the emergency nurse at risk for acquiring an airborne infection. according to the cdc, another area of ppe training that should receive special focus is the doffing procedure. historically, this part of the ppe sequence may not have been emphasized in the training process, but recent cases of the acquisition of evd by nurses have demonstrated that doffing ppe is a high-risk period that requires careful attention to detail, monitoring by a trained observer, and a designated space for equipment removal. in addition to understanding how to properly select and use ppe, emergency nurses need to have practice in using ppe in realistic patient care situations. the use of ppe can affect clinical performance by limiting manual dexterity, impairing hearing and communication, and causing discomfort for the user. [ ] [ ] [ ] [ ] hands-on practice allows the user to be better prepared to provide safe patient care while wearing ppe. a strategy that can be used to prepare health care providers to provide safe care in the emergency setting is the use of in situ simulation training sessions. , in situ simulation is a team-based training process that involves the use of a standardized scenario in the practice environment using actual unit staff, equipment, and resources. regular practice using ppe in realistic simulation scenarios will aid members of the emergency care team in developing and maintaining competency. safe staffing patterns should be considered when planning care for patients with infectious illness. the cdc recommends that health care providers have adequate time to properly don and doff equipment before engaging in patient care. when engaged in the care of a patient with evd, this donning and doffing procedure should be monitored by a trained observer. radanovich et al conducted a study on respirator tolerance in health care providers and found that a significant portion of the study participants were unable to tolerate wearing a respirator for an -hour shift, even with break periods. shenal et al also found that health care workers experienced increasing discomfort when wearing respiratory protection over a prolonged period. when health care providers are wearing paprs and fullbody coverage, especially when they are engaged in complex patient care activities, the period in ppe that is tolerable may be even shorter. planning should be in place to ensure that health care providers have adequate periods of rest without wearing ppe. in complex care situations, such as the care of a patient with evd, providers may require extended rest periods to prevent heat stress, fatigue, and dehydration. the united states department of health and human services lists psychological stress, heat stress, and dehydration as risks associated with the use of ppe during chemical emergencies. to minimize the risk of injury, first responders are advised to obtain baseline vital signs and weight prior to donning equipment and after doffing equipment, hydrate before and after using ppe, monitor total time in ppe, and minimize time in ppe when possible (especially when wearing the highest levels of protection). given the similarities in ppe that are recommended for the care of patients with evd and the types of ppe used during chemical and biological hazards, it may be prudent to integrate these safety measures into organizational policies and procedures for the use of paprs in the care of patients with evd. this step would require additional staff to assess providers before and after use of ppe; however, the "trained observer" recommended by the cdc could be used in this capacity. emergency nurses work in a hazardous environment. to minimize the risk of injury, emergency nurses must maintain competency in infection control measures and the use of ppe. the cdc and john hopkins medicine have created a web-based training program for the safe donning and doffing of ppe that can be accessed online at http://www.cdc.gov/vhf/ebola/hcp/ppe-training/index. html. although this type of training is important, emergency nurses should also advocate for regular handson training with ppe to ensure competency with equipment and supplies available in their work setting. emergency nurses should ensure that they have been fit tested for an n respirator and know the appropriate size to use when providing patient care. finally, emergency nurses can minimize risk of injury by committing to strict adherence to infection control standards. guidance for the use and selection of personal protective equipment (ppe) in healthcare settings guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings occupational safety & health administration. personal protective equipment basic infection control and prevention plan for outpatient oncology settings: transmission-based precautions ebola (ebola virus disease): when caring for suspect or confirmed patients with ebola ebola (ebola virus disease): information for healthcare workers and settings behind the mask: determinants of nurse's adherence to facial protective equipment a review of the evidence for suboptimal compliance of healthcare practitioners to standard/universal infection control precautions hand hygiene compliance: exploring variations in practice between hospitals global rise in human infectious disease outbreaks guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in u.s. hospitals, including procedures for putting on (donning) and removing (doffing) personal protective equipment for care of pandemic influenza patients: a training workshop for the powered air purifying respirator respiratory tolerance in health care workers respiratory and facial protection: a critical review of recent literature discomfort and exertion associated with prolonged wear of respiratory protection in a health care setting highreliability emergency response teams in the hospital: improving quality and safety using in situ simulation training in situ simulation: detection of safety threats and teamwork training in a high risk emergency department united states department of health and human services key: cord- -c elsnag authors: fusco, marnie l.; hashiguchi, takao; cassan, robyn; biggins, julia e.; murin, charles d.; warfield, kelly l.; li, sheng; holtsberg, frederick w.; shulenin, sergey; vu, hong; olinger, gene g.; kim, do h.; whaley, kevin j.; zeitlin, larry; ward, andrew b.; nykiforuk, cory; aman, m. javad; berry, jody; saphire, erica ollmann title: protective mabs and cross-reactive mabs raised by immunization with engineered marburg virus gps date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: c elsnag the filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. antibodies against the filovirus surface glycoprotein (gp) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. many monoclonal antibodies (mabs) have been described against ebola virus. in contrast, relatively few have been described against marburg virus. here we present ten mabs elicited by immunization of mice using recombinant mucin-deleted gps from different marburg virus (marv) strains. surprisingly, two of the mabs raised against marv gp also cross-react with the mucin-deleted gp cores of all tested ebolaviruses (ebola, sudan, bundibugyo, reston), but these epitopes are masked differently by the mucin-like domains themselves. the most efficacious mabs in this panel were found to recognize a novel “wing” feature on the gp subunit that is unique to marburg and does not exist in ebola. two of these anti-wing antibodies confer and % protection, respectively, one hour post-exposure in mice challenged with marv. filoviruses are filamentous, enveloped viruses that can cause highly lethal hemorrhagic fever in both humans and non-human primates. the filovirus family includes the major genera ebolavirus and marburgvirus and the newly discovered cuevavirus. in the ebolavirus genus are five known species: ebola virus (ebov), sudan virus (sudv), bundibugyo virus (bdbv), reston virus (restv), and taï forest virus (tafv). in the marburgvirus genus, there is one species, the eponymously named marburg virus (marv) [ ] . marv is further subdivided into different strains, including ci , musoke, ravn and angola. ravn is the most divergent strain of marv, differing by % in genomic sequence from other marburg strains [ ] , and is sometimes referenced as a separate filovirus species. marburg virus was the first filovirus to be identified when it sickened laboratory workers handling infected animals originating from uganda in [ ] [ ] [ ] . marburg virus has since reemerged at least times, and has been imported to the united states and europe by travelers who became infected in africa [ ] [ ] [ ] [ ] . angola, the most lethal strain of marburg virus [ ] , emerged in and caused the largest marv outbreak known to date with an extremely high case fatality rate of % [ ] . the emergence of ebola virus in west africa in has caused an outbreak unprecedented in magnitude, and is a grim reminder of the devastation that can be caused by filoviruses. the filoviruses present a single viral protein on their envelope surface, the glycoprotein (gp), which is responsible for attachment and entry of viruses into target cells. gp is expressed as a precursor that is cleaved by furin in the producer cell to yield two subunits: gp and gp , which remain linked by a disulfide bond [ , ] . gp contains the putative receptor-binding region [ ] , as well as two heavily glycosylated domains: a glycan cap which sits immediately atop the putative receptor-binding site and a larger, largely unstructured mucin-like domain [ , ] . the mucin-like domains contain a dense clustering of n-and o-linked glycans and likely mask the gp from immune surveillance [ , ] . the second subunit of gp, termed gp , possesses the transmembrane domain that anchors gp into the viral surface and the hydrophobic fusion peptide required for fusion. in ebolaviruses, the furin cleavage site lies at residue and the entire mucin-like domain is attached to the gp subunit. in marburg virus, however, the furin cleavage site lies at residue , splitting the mucin-like domain so that a portion of it remains attached to the gp subunit [ ] . we have termed this amino-acid n-terminal gp extension the "gp wing". after cell entry by macropinocytosis [ , ] filovirus gp undergoes additional cleavage by host cathepsin proteases in the endosome [ , ] . this cleavage event removes the glycan cap and the mucin-like domain, resulting in a loss of over % of the molecular mass of gp [ ] [ ] [ ] . endosomal cleavage renders gp competent for receptor binding [ , , ] , allowing the exposed gp head to bind a shared filovirus receptor, niemann-pick c (npc ) [ , ] . although antibodies that broadly cross-react among ebola-and marburgvirus gps would be highly desirable, only one such antibody, mr , has been described [ ] . recent work in non-human primates has demonstrated that passive administration of monoclonal antibody (mab) cocktails against gp can provide highly effective post-exposure therapy for ebov infection [ ] [ ] [ ] [ ] [ ] . polyclonal sera against marburg virus has shown similar efficacy, suggesting that antibodies could also be a viable treatment option for marv infection [ ] . however, fewer monoclonal antibodies, from which such cocktails could be developed, are currently available for marv. one human survivor panel has recently been described; most of these mabs compete for the same site on the gp core [ , ] . antibodies targeting other epitopes on marburg gp would be desirable in order to form a treatment cocktail. in general, monoclonal antibody cocktails are most effective when the component antibodies display synergistic effects. combining mabs with non-overlapping epitopes can significantly increase the overall potency of the cocktail over the individual mabs alone [ ] , and can mitigate antigenic escape by the virus [ , ] . anti-viral antibodies are often selected based on neutralization, or the ability of the mabs to prevent viral entry into target cells in vitro. however, for filoviruses as well as other viruses, neutralization in vitro does not necessarily correlate with protection in vivo [ , ] . non-neutralizing antibodies are known to confer protection by antibody-dependent cellular cytotoxicity (adcc), phagocytosis, prevention of virus budding, and other mechanisms [ , ] . indeed, one successful anti-ebov oligoclonal cocktail is composed entirely of antibodies that are not potent neutralizers [ , ] . in this study we produced a diverse panel of antibodies against marburg virus by immunization of mice with different strains of the surface gp antigen. immunogens included gp -mucin-deleted ectodomains (gpΔmuc) from marburg strains ci , musoke, angola, and ravn. mucin-deleted immunogens were used to direct the immune response away from the highly variable mucin-like domains. ten antibodies were chosen and analyzed for in vitro neutralization, in vivo efficacy, and biochemical recognition of marv and ebov gps. antibodies against multiple epitopes were found. four antibodies target a novel marv-specific "wing" epitope on gp ( g , g , g and g ), and confer - % protection in mice challenged with marv. a separate marv-specific antibody, a , directed against an epitope in gp , confers % protection. another mab directed against gp , g , confers % protection and was found to be broadly cross-reactive among the core of filovirus gps, including both marburg-and ebolaviruses. to generate marv gp-specific mabs, balb/c mice were immunized with gpΔmuc antigens from either marv strain ci , musoke, angola, or ravn ( fig a) . mice for each subset were immunized and boosted with the same antigen with the exception of the series ( g , g , g ). eight of the ten mabs in the panel are mouse igg . the remaining two mabs, a and d , are igg a ( fig a) . to characterize the binding of mabs, we performed enzyme-linked immunosorbent assays (elisas) with recombinant gps from four marv strains, and determined ec values for binding with different forms of marv ravn gp: gp, gpΔmuc, gpcl (fig a) . all ten mabs exhibit medium binding (ec between ng/ml and ng/ml, colored dark yellow) to high binding (ec < ng/ml, colored orange) against ravn gp and gpΔmuc ( fig b) , but only seven of the mabs cross-react with gp from other marv strains ( fig c) . all mabs bind the protease-cleaved ravn gp core, termed gpcl, as well as gpΔmuc, with the exception of a . antibody a exhibits an -fold decrease in binding to gpcl as compared to gpΔmuc ( fig b) . additionally, to evaluate whether the mabs have the capacity to bind cell-surface gp, eli-sas were performed with virus-like particles (vlps) bearing full-length wild-type marv ravn gp. eight mabs bind as well (or nearly as well) to vlps as purified recombinant ravn gp. in contrast, a exhibits nearly -fold weaker binding to vlps than to gp ectodomain, and g binding to vlps is lost at the highest concentration tested (fig b) . to determine antibody epitopes, we performed western blotting with ravn gp and pepscan analysis with overlapping -mer pins of peptides from ravn or musoke gp. five of the mabs bind gp , and five bind gp by western blot (s fig panel a) . pepscan identified linear epitopes for only four mabs, g , g , g and g , all of which overlap within residues - ( fig b) . this shared region lies in an extension of gp that is specific to marv (as a result of the furin cleavage site shift from in ebov to in marv), which we have termed the gp wing (fig a) . in order to confirm the pepscan results, we engineered a gpΔmuc with an additional deletion of residues - , termed gpΔmucΔw (fig a) . indeed, binding to gpΔmucΔw is lost for only the four anti-wing mabs, whilst the remaining six mabs against different epitopes do bind gpΔmucΔw (s fig). no definitive epitope information could be identified by pepscan for the remaining antibodies, suggesting that these mabs bind conformational epitopes. sequence alignment of marv gp residues - reveals that while ci , musoke and angola are completely conserved in this region, ravn has unique residues. the most notable change is residue , which is a glu (e) in ravn but a lys (k) in the other strains ( fig b) . wing mabs g and g are specific for marv ravn. correspondingly, elisa data comparing binding of wild-type ravn gpΔmuc to e k ravn gpΔmuc confirm that the presence of lys at position (as exists in other strains of marv) likely hinders binding of g and g . g , however, still retains some binding to e k, while g is unaffected by this mutation, retaining binding at and . ug/ml ( fig c) . these results agree with pepscan results (based on -mer peptides overlapping by amino acids) which define the epitope for g as slightly shifted away from position , towards the n-terminus of gp ( fig b) . this shift may explain why g is the most cross-reactive of the anti-wing mabs ( fig c) . antibodies were screened for in vitro neutralization using a vsv-pseudovirus containing marv ravn gp on the surface. six of the ten mabs exhibit partial neutralization at the highest concentration tested ( ug/ml), reducing entry by - %. the remaining four mabs do not neutralize (fig ) . notably, all five gp -directed mabs produced in this study exhibit some neutralization, while only one gp -directed mab, a , inhibits entry of ravn gp pseudovirions. polyclonal sera from mice that yielded the series mabs ( g , g and g ) reduces entry by only about %, suggesting that mabs g , g , and g represent the maximum potency of the polyclonal population (fig ) . human survivor mab mr was used as a positive control and reduces pseudovirion entry by almost %. all mabs were evaluated for in vivo protection using balb/c mice challenged with a lethal dose of marv virus [ ] . one hour after challenge with , pfu mouse-adapted marv ravn, mice were treated ip with μg purified mab. two separate studies were performed, with half of the mabs repeated in both studies. control animals in study # , treated with pbs, exhibited / survival (fig a) . both control groups in study # , treated with pbs or anti-ha mab, exhibited / survival (fig b) . marv mab treatment groups varied widely in efficacy, ranging from - % protection. all four mabs against the gp wing were found to be moderately or highly protective: mab g conferred % survival ( / ) , mab g % survival ( / ), mab g % survival ( / ) , and mab g % survival ( / ) . monoclonal antibody a , against gp , conferred % survival ( / ) (fig a and b) . other mabs against the gp core exhibited - % survival; of these, only g offered strongly significant protection (p value . ). the only mab against a gp epitope other than the wing, mab g , exhibited zero protection ( / ) (fig a) . in both studies, mice in all treatment groups displayed an elevation of disease score by day (fig a and b) , and there were no significant differences in weight loss between treatment groups and control groups. in study # , g -treated mice faired only modestly better than the other groups, reaching a disease score maximum of and fully recovering by day (fig a) . two of the highly cross-reactive marv antibodies, mabs g and d , also exhibit binding to ebola, sudan, bundibugyo and reston virus mucin-deleted gps by elisa (fig a) . binding curves show that the affinity of g and d for mucin-containing ebov gp is weak, affinity for gpΔmuc is stronger, and binding to ebov gpcl (the receptor-binding competent core) is strongest and equal to that of marv gpcl ( fig b) . hence, the g and d epitopes are conserved across the filovirus family, exposed on all versions of marburg virus gp, but masked on ebolavirus gp by the mucin-like domain and the glycan cap. single particle electron microscopy of the most protective anti-gp ( a ) and anti-gp ( g ) antibodies was performed in complex with purified antigen. negative stain d class averages of a fabs in complex with marv ravn gpΔmuc show one, two, or three fabs bound to the dense trimeric gp core (fig a) . in contrast, d class averages of the anti-gp wing mab g in complex with marv ravn gpΔmuc show a single fab bound to gp, at a distance further away from the high density gp trimer (fig b) . deuterium exchange mass spectrometry (dxms) studies suggest this gp wing region is unstructured and likely flexible (s fig). to ensure that the wing epitope is not artificially positioned in gpΔmuc as compared to the biologically relevant mucin-containing gp, we also performed em with g fab in complex with the complete ectodomain of marv ravn gp. images obtained were similar to those with gpΔmuc, with only one fab binding per trimer (fig c) . likely footprints of fabs a and g are drawn onto the marv ravn gpcl crystal structure (fig d) . in this study, a small panel of mabs targeting marv gp were isolated from immunized mice. those that conferred the greatest in vivo protection are directed against a novel "wing" domain on marv gp . this wing region is a marv-specific portion of the mucin-like domain attached to gp . such an epitope does not exist in ebolaviruses because the entire mucin-like domain is attached to gp . although this study size was small, we note that gp wing-directed mabs were only obtained when mice were immunized with mucin-deleted ravn gp. it may be tempting to assume that this epitope is masked by the mucin-like domain; however, anti-wing mabs are able to access their epitope on mucin-containing gp, neutralize pseuodviruses bearing mucin-containing gp and provide in vivo efficacy when challenged with marburg virus. we believe that the elicitation of anti-wing antibodies when using ravn gpΔmuc may instead result from the greater homogeneity and stability of ravn gpΔmuc over other marv antigens. a seven-year protein engineering effort in our laboratory to identify crystallizable versions of marv gp indeed found that gps produced from strain ravn are the most homogenous, and have a lesser tendency to aggregate than those from other strains of marv [ ] . the homogeneity may have lead to improved presentation of this protective epitope within this study. it is interesting to note that among this panel of murine mabs and the recently published panel of human survivor mabs [ ] , no antibodies that bind the gp -and gp -containing base of marv gp were identified. the "base" of gp is a common site of neutralization for the ebolaviruses and is the epitope target of anti-ebov neutralizing antibodies kz [ ] , g and g [ ] , as well as the anti-sudv mab f [ ] . perhaps the presence of the flexible gp -wing in marv blocks access to this site on the gp core. nonetheless, antibodies directed against the gp wing itself do have the potential to be fully protective, and represent a novel epitope in marv for therapeutic cocktail design. the most protective of these mabs, g , is promising but only binds with high affinity to the gp from ravn, and hence, protection by g against other marv strains may be limited. in contrast, monoclonal antibody g only confers % efficacy, yet cross-reacts with mucin-containing gps from four strains of marv. however, g is a murine igg , an isotype that typically exhibits weaker immune effector activity than murine igg a [ ] . replacement of the constant domain framework may improve its in vivo efficacy. in this panel, two mabs against gp were identified which also bind the gp cores of ebolaviruses. these antibodies, g and d , bind all marv gps, but only bind ebola, sudan, bundibugyo and reston gp from which the mucin-like domain is deleted (fig ) . hence, these highly conserved epitopes are exposed on marburgvirus gps, but masked on ebolavirus gps. these observations parallel those obtained from a panel of anti-marv gp antibodies isolated from a human survivor [ ] , and support structural observations that the orientation of the mucin-like domains differs between ebov and marv [ ] . indeed, no cross-filovirus anti-gp antibody (reactive to both ebola and marburg) has yet been elicited by an ebolavirus gp immunogen, nor has any such antibody yet been isolated from an ebolavirus survivor. although the filovirus cross-reactive mab g confers only % survival, g or another antibody like it [ ] may be useful in an immunotherapeutic cocktail because a highly conserved epitope would likely be less subject to antigenic escape. antibody a is also directed against gp but its pattern of binding is distinct from g and d . a is the only mab in this panel that has a lower affinity to gpcl than gp or gpΔmuc. this suggests that the epitope of a is partially lost upon cleavage and that a could be similar to a glycan cap binder like c or h for ebov gp [ ] . unfortunately, due to the single preferred orientation of gpΔmuc + a fab particles on negative stain em grids, a high-resolution reconstruction could not be determined, and better understanding of the a epitope awaits further study. a affords % protection in vivo and is highly crossreactive among marv ectodomain gps. for ebola virus, in vitro neutralization is not necessarily an effective predictor of in vivo protection. one anti-ebov cocktail is composed entirely of non-neutralizing or weakly neutralizing mabs, yet still confers in vivo protection, presumably by recruiting immune effector function [ , ] . more recent cocktail formulations have included a mix of neutralizing and non-neutralizing antibodies [ ] . in this study of mabs against marv, none of the mabs offered significant in vitro neutralization, yet several did confer partial to complete in vivo protection against marv one hour after challenge. although this study is limited in scope, we note that among this set of antibodies, those that exhibited in vitro neutralization also conferred the best in vivo protection. (there was only one mab that weakly neutralized but offered no protection, mab g ). future studies, performed at longer time periods after challenge and with lower treatment doses, will test the limits of efficacy of the individual mabs. promising mabs could then be evaluated in non-human primates (nhps) to predict therapeutic potential in humans. in this work, we provide biochemical and structural mapping of antibody epitopes on marv gp, and analyze the conservation of these epitopes among different strains of marv. we find antibodies against a novel gp "wing" epitope that confer - % protection in vivo, and two mabs against different sites in gp that confer % and % protection. mab cocktails are thought to be most effective when the component antibodies display synergistic effects. combining mabs with non-overlapping epitopes can significantly increase the overall potency of the cocktail over the individual mabs alone [ , ] , and can mitigate antigenic escape by the virus [ ] . the panel of antibodies described here, although limited in number, provides three possible components of an anti-marv immunotherapeutic cocktail: an anti-gp core mab such as g (or a neutralizing mr mab), the anti-gp mab a , and an anti-gp wing mab such as g or g . future studies will determine the limits of protection and therapeutic potential of these antibodies when delivered in combination. this study was approved and carried out in accordance with protocols provided by the institutional animal care and use committee (iacuc) at tsri, emergent biosolutions, niaid, and usamriid. research at usamriid was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals, and adhered to principles stated in the guide for the care and use of laboratory animals, national research council, . marburg angola, ravn and ci gpΔmuc (tsri). marburg virus gp immunogens used to raise antibodies at emergent were designed and produced at tsri. dna encoding the marv gpΔmuc ectodomain (residues - with a mucin deletion of residues - ) was cloned into a derivative of the invitrogen pdisplay vector. in this derivative vector, the pgdfr sequence is replaced by a c-terminal purification tag (either an ha or strep tag). large-scale production was performed by pei transfection (polysciences, inc mw , ) of plasmid into % confluent hek gnti-/-cells (atcc) in corning -layer cellstacks. supernatants were harvested four days post-transfection, concentrated with a centramate tangential flow system, and affinity purified using streptactin (qiagen) or anti-ha f (roche) affinity resin. trimeric gpΔmuc was then isolated by s size exclusion chromatography (sec) in mm tris, mm nacl, ph . ( x tbs). in order to improve furin cleavage processing during expression, which decreased aggregation and improved yield of purified trimers, bulky hydrophobic residues near the furin cleavage site were mutated in the gpΔmuc constructs. ravn gpΔmuc mutations included f l, w a, f g, f n and angola gpΔmuc mutations included w v, m a, f g. marburg musoke and angola gpΔmuc (ibt). marburg virus gpΔmuc antigens used to raise antibodies at ibt were produced at ibt. musoke gpΔmuc ( - Δ - ) was produced by pei transfection of hek t cells with the derivative pdisplay plasmid containing a c-terminal ha-tag. protein was purified by anti-ha f affinity (roche) followed by lectin affinity and superose size exclusion chromatography (ge healthcare). angola gpΔmuc ( - Δ - ) was produced by baculovirus infection (bac-to-bac, invitrogen) of sf cells (invitrogen) according to the manufacturer's protocol, and purified via a c-terminal hisx tag on ni +-nta sepharose resin (ge healthcare). production of g , g , g , g , g , g , and g . six week-old balb/c mice were injected subcutaneously (sc) with μg (in μl volume pbs) of purified marv gps in freund's complete adjuvant (cfa; brenntag biosector). additional boosts were injected intraperitoneally (ip) on day and with μg of the same gp in incomplete freund's adjuvant (ifa; brenntag biosector). thereafter mice received a final push of μg purified gp (in pbs by ip) before conducting fusions. standard protocols were used to produce hybridoma cell lines [ ] , and monoclonal antibodies specific to gpΔmuc antigen were purified on protein g resin. mice immunized with ravn gpΔmuc raised antibodies g , g and g . mice immunized with ravn gpΔmuc, then boosted two times with a complex of ravn gpΔmuc bound to g fab, raised antibodies g , g and g . mice immunized with angola gpΔmuc yielded antibody g . immunization of mice at emergent was performed according to animal use protocols (aup) approved by the protocol management and review committee (pmrc), university of manitoba. production of a and d . six week-old balb/c mice were immunized intramuscularly (im) three times with μg purified marv gps in glucopyranosyl lipid adjuvant (gla) adjuvant at week intervals, and boosted intravenously (iv) with μg antigen days before harvest of spleen/lymph nodes for fusions. standard protocols were used to produce hybridoma cell lines [ ] , and monoclonal antibodies specific to gpΔmuc antigen were purified on protein g resin. mice immunized with musoke gpΔmuc yielded antibody a . mice immunized with angola gpΔmuc yielded antibody d . immunization of mice at ibt was performed according to aup approved by noble life sciences iacuc. production of a . immunization of mice was performed by bio-quant inc (san diego, ca). six week-old balb/c mice were injected subcutaneously (sc) with μg of purified ci gpΔmuc in cfa followed by additional boosts at week intervals with μg antigen ( μg in ifa by ip and μg in ifa by sc) before conducting fusions. standard protocols were used to produce hybridoma cell lines, and mab a was purified on protein g resin. purified filovirus gp antigen preparation. all filovirus ectodomains for elisa, western blot, em, or dxms were produced at tsri in drosophila s cells [ ] , with the exception of musoke gp, which was produced by ibt in sf cells (as described above for musoke gpΔmuc). briefly, effectene reagent (qiagen) was used to transfect s cells with pmtpuro plasmids containing a strep-tagged filovirus gp gene of interest, followed by stable selection of transfected cells with μg/ml puromycin in insect xpress protein free medium (lonza). secreted gp ectodomain expression was induced with . mm cuso and supernatants harvested after days. proteins were affinity purified using streptactin resin (qiagen), followed by purification via superdex sec in x tbs. the cleaved "core" ectodomain for marv (marv gpcl) was produced by incubating mg ravn gpeΔmuc with . mg trypsin (sigma) at °c for hour in tbs ph . , followed by s sec purification. the cleaved "core" ectodomain of ebov (ebov gpcl) was produced by incubating mg ebov gpeΔmuc with . mg thermolysin (sigma) overnight at room temperature (rt) in tbs buffer plus mm cacl , followed by s sec purification. sds-page gels comparing purity and molecular weight of several antigens in shown in s panel b. vlp preparation. virus-like particles were produced by co-transfection of hek t cells with pcaggs plasmids expressing full-length marv ravn gp or marv vp . supernatants were harvested after sixty hours, vlps pelleted down at , xg for minutes, washed with pbs, and re-pelleted. vlp pellets were then gently resuspended in pbs containing . % np and . % triton x , diluted : with pbs, and used as coating antigen for elisa. western blotting. purified marv ravn gp reduced and non-reduced samples were run on - % sds-page gels and transferred onto pvdf immobilon membranes (millipore). membranes were blocked overnight in % milk (biorad blotting grade) pbs- . % tween (pbs-t), incubated for hour at room rt with anti-marv mabs at a concentration of ug/ml in % milk pbs-t, washed with pbs-t, then incubated with goat anti-mouse (or anti-human for mr ) alkaline phosphatase (ap) conjugated antibody at a : dilution. ap activity was detected with sigmafast bcip/nbt substrate. recognition of various forms of gp and cross-reactivity by elisa. to determine half maximal effective concentrations, or ec s, mabs were tested for binding to gp ( - ), gpΔmuc ( - Δ - ), and gpcl of marv ravn at a concentration range of μg/ml to . ng/ml using -fold serial dilutions. data was analyzed using graphpad prism . software. to determine cross-reactivity, mabs were tested for binding to marv angola, musoke, ci , and ebov gp ( - ) at , , . and . μg/ml. because mucin-containing musoke and ci gps readily aggregate, binding in fig c is reported as high, medium, or low rather than as a quantitative ec value. antibodies d and g were further analyzed for binding to the gpΔmuc antigens of ebov ( - Δ - ), sudv ( - Δ - ), bdbv ( - Δ - ) and restv ( - Δ - ), as well as to ebov gpcl. binding curves for d and g were determined at a concentration range of μg/ml to . ng/ml using -fold serial dilutions. elisas were performed as follows: corning -well high-binding microtiter plates were coated with filovirus gp antigens, blocked with % bsa in pbs hour at rt, and incubated with anti-marv mabs in . % bsa hour at rt. plates were then incubated with : goat anti-mouse igg (h+l) hrp conjugated secondary (thermo scientific) in . % bsa hour at rt. (plates were washed between each step with pbs containing . % tween ). color development was produced with tmb substrate (thermo scientific), stopped with n sulfuric acid, and quantified by measuring absorbance at nm. pepscan and gp wing analysis. in an attempt to map linear epitopes, all mabs were tested by elisa pepscan against synthetic -mer peptides designed from ravn gp sequences, overlapping by amino acids. pepscan was repeated for mabs a , a , d and g against peptides designed from musoke gp sequences. as a control for gp wing pepscan defined epitopes, additional elisas were performed with a marv gp lacking both the gp mld (Δ - ) and the gp wing (Δ - ), termed gpΔmucΔw. gp wing-directed antibodies were further evaluated for binding to both wild-type ravn gpΔmuc and ravn gpΔmuc containing a point mutant (e k). coating antigens for the point mutant elisas were produced in hek t cells to represent a mammalian glyco-profile in and around the gp wing. pseudovirus neutralization assays. vesicular stomatitis virus (vsv) pseudovirions containing a gfp gene in place of the vsv envelope glycoprotein gene (vsvΔg) and bearing the full-length glycoprotein of marv ravn were generated as previously described [ ] . pseudovirions were incubated with anti-vsv g mab (a gift from a. takada) for hour at rt, then incubated with μg/ml of each anti-marv gp mab in dmem- % fbs (gibco) for an additional hour at rt. pseudovirion/mab complexes were added to vero cell (atcc) monolayers in -well plates at a multiplicity of infection (moi) between . and . . after hours, infection was evaluated by counting gfp-expressing cells. experiments were performed in triplicate and standard deviations displayed. all mammalian cell lines used in this study tested negative for mycoplasma contamination at tsri. animal work. all procedures with infectious marburg viruses were performed in a biosafety level (bsl ) facility at usamriid. male and female balb/c mice between and weeks of age were challenged intraperitoneally (ip) with plaque-forming units of mouseadapted marv ravn [ ] in two separate studies. one hour post-exposure, the mice were treated ip with μg of purified monoclonal anti-marv gp antibody in pbs ( . mg/ml) or pbs alone. study two included an additional negative control group treated with μg of anti-ha igg in pbs ( . mg/ml). each test group consisted of animals for a total of mice. all antibodies were blinded by ibt before submission to usamriid researchers. animals were weighed and monitored daily over a day period post-challenge, at which point mice were euthanized in accordance with an iacuc-approved protocol. once animals were symptomatic, they were examined twice per day. health was scored using the following parameters: = normal, = reduced grooming/ruffled fur, = subdued, = lethargic/hunched posture (provide dietgel for hydration), = unresponsive; euthanize. health scores for the g and a treatment groups in fig are shown for the one or two animals that survived, respectively. no animals were excluded from analysis and the experiments were not randomized. all balb/c mice used in these experiments were obtained from the frederick cancer research and development center, national cancer institute (frederick, md). statistical analysis. graphpad prism . software was used to calculate p values using the log-rank mantel cox test. each treatment group was compared to the corresponding pbs control for either study # or # . p values > . are considered non-significant (ns). purified ravn gpcl was evaluated by deuterium exchange mass spectrometry (dxms) as previously described [ ] . for negative stain em analysis, marv ravn ectodomains were produced in drosophila s cells as described above. fab g and a fragments were generated by standard papain digestion (sigma) of igg and purified by mono q (ge healthcare) ion-exchange chromatography. five molar excess fab was added to trimeric gpΔmuc or gp and allowed to bind overnight at °c. complexes were diluted to . mg/ml in tbs buffer and deposited onto to carbon-coated copper mesh grids which had been plasma cleaned for sec (gatan) and stained for sec with μl of % uranyl formate. the stain was blotted off the edge and the grid was allowed to dry. data were automatically collected with leginon [ ] using a fei tecnai f electron microscope operating at kev with an electron dose of e -/Å and a magnification of , x that resulted in a pixel size of . Å at the specimen plane when collected with a spirit k x k ccd camera (for g ) and . Å at the specimen plane when collected with a tietz k x k ccd camera (for a ). images were acquired at a constant defocus value of - . μm at various tilt angles from to °. particles were picked automatically using dog picker [ ] and placed into a particle stack using the appion software [ ] . reference-free d class averages were calculated by using particles binned by with the xmipp clustering d alignment software [ ] and sorted into~ - particles per class. supporting information s fig. (a) western blot of anti-gp marv mabs incubated at μg/ml, against reduced (+ dtt) and non-reduced (-dtt) purified ravn gp. (b) non-reducing - % sds-page gels of several purified marv gp and ebola antigens from s cells. note ravn gpcl runs larger than ebola gpcl due to extra mass of the gp wing. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations phylogenetic assessment of filoviruses: how many lineages of marburg virus? on the etiology of an unknown human infection originating from monkeys fatal human disease from vervet monkeys agent of disease contracted from green monkeys forty-five years of marburg virus research response to imported case of marburg hemorrhagic fever, the netherland imported case of marburg hemorrhagic fever-colorado outbreak of marburg virus disease in johannesburg marburg virus angola infection of rhesus macaques: pathogenesis and treatment with recombinant nematode anticoagulant protein c marburgvirus genomics and association with a large hemorrhagic fever outbreak in angola biochemical analysis of the secreted and virion glycoproteins of ebola virus the glycoproteins of marburg and ebola virus and their potential roles in pathogenesis conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor structure of the ebola virus glycoprotein bound to an antibody from a human survivor structural basis for marburg virus neutralization by a cross-reactive human antibody ebolavirus glycoprotein gp masks both its own epitopes and the presence of cellular surface proteins steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein proteolytic processing of marburg virus glycoprotein cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection mechanism of human antibody-mediated neutralization of marburg virus sustained protection against ebola virus infection following treatment of infected nonhuman primates with therapeutic intervention of ebola virus infection in rhesus macaques with the mb- monoclonal antibody cocktail protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques reversion of advanced ebola virus disease in nonhuman primates with zmapp postexposure antibody prophylaxis protects nonhuman primates from filovirus disease potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology development of a highly protective combination monoclonal antibody therapy against chikungunya virus an update on the use of antibodies against the filoviruses protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts preventing infectious disease with passive immunization inhibition of marburg virus budding by nonneutralizing antibodies to the envelope glycoprotein epitopes involved in antibody-mediated protection from ebola virus development and characterization of a mouse model for marburg hemorrhagic fever structures of protective antibodies reveal sites of vulnerability on ebola virus a shared structural solution for neutralizing ebolaviruses divergent immunoglobulin g subclass activity through selective fc receptor binding delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques reversion of advanced ebola virus disease in nonhuman primates with zmapp human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants production of monoclonal antibodies a system for functional analysis of ebola virus glycoprotein automated molecular microscopy: the new leginon system dog picker and tiltpicker: software tools to facilitate particle selection in single particle electron microscopy appion: an integrated, databasedriven pipeline to facilitate em image processing a new generation of the imagic image processing system key: cord- -b s stvz authors: guimarães, luísa eça; baker, britain; perricone, carlo; shoenfeld, yehuda title: vaccines, adjuvants and autoimmunity date: - - journal: pharmacological research doi: . /j.phrs. . . sha: doc_id: cord_uid: b s stvz abstract vaccines and autoimmunity are linked fields. vaccine efficacy is based on whether host immune response against an antigen can elicit a memory t-cell response over time. although the described side effects thus far have been mostly transient and acute, vaccines are able to elicit the immune system towards an autoimmune reaction. the diagnosis of a definite autoimmune disease and the occurrence of fatal outcome post-vaccination have been less frequently reported. since vaccines are given to previously healthy hosts, who may have never developed the disease had they not been immunized, adverse events should be carefully accessed and evaluated even if they represent a limited number of occurrences. in this review of the literature, there is evidence of vaccine-induced autoimmunity and adjuvant-induced autoimmunity in both experimental models as well as human patients. adjuvants and infectious agents may exert their immune-enhancing effects through various functional activities, encompassed by the adjuvant effect. these mechanisms are shared by different conditions triggered by adjuvants leading to the autoimmune/inflammatory syndrome induced by adjuvants (asia syndrome). in conclusion, there are several case reports of autoimmune diseases following vaccines, however, due to the limited number of cases, the different classifications of symptoms and the long latency period of the diseases, every attempt for an epidemiological study has so far failed to deliver a connection. despite this, efforts to unveil the connection between the triggering of the immune system by adjuvants and the development of autoimmune conditions should be undertaken. vaccinomics is a field that may bring to light novel customized, personalized treatment approaches in the future. vaccines have been a preventive treatment option available for over years. they have been proven to be effective in preventing infections that previously had high morbidity and mortality. an example of this is the eradication of small pox, which was mainly attributed to successful vaccination programs. preventing a high burden disease has since proven to be a cost effective measure and, as such, vaccines have become a part of multiple national health programs. these promising results led to the development of more and more vaccines and to the study of its applicability in other fields such as cancer prevention and treatment. vaccines are drugs administered to healthy individuals, and much like other drugs, vaccines are associated with adverse events. usually the described adverse events are transient and acute, but may rarely present with hypersensitivity and induction of autoimmunity that may be severe and fatal. these adverse events play an important role in the life of the vaccinated patients. immune mediated diseases arise from various different sources; these include environmental, genetic, hormonal and immune defects. the combination of these defects can be described as the mosaic of autoimmunity [ ] . patient background can be used as a clue to determine the response that may be elicited following drug administration. it has been proven that infectious agents may elicit an autoimmune disease in a prone subject through various mechanisms, including, but not limited to, molecular mimicry, epitope spreading and polyclonal activation [ ] . scientific findings suggest that autoimmunity may be triggered by vaccine adjuvants, of which aluminum compounds (aluminum hydroxide and phosphate) have been the most studied and the most widely used. adjuvants are molecules, which, in combination with antigens, enhance immunological response. this enables an easier and more effective recognition of "non self", which in turn permits the triggering of adaptive and innate immune responses [ ] . recently a new syndrome was described: "autoimmune/inflammatory syndrome induced by adjuvants" (asia). this embodies a spectrum of reactions, which are usually mild but may also be severe. these reactions are attributed to adjuvant stimulation, which can include chronic exposure to silicone, tetramethylpentadecane, pristane, aluminum, infectious components and other adjuvants. all of these environmental factors have been found to induce autoimmunity and inflammatory manifestations by themselves both in animal models and in humans. the mechanisms of this disease will be described in further detail [ ] . this review will focus on general mechanism of vaccines, adjuvant-induced autoimmunity, and on vaccines and the specific autoimmune diseases that they may trigger. adjuvants approved to date for human vaccines are: aluminum, mf in some viral vaccines, mpl, as , as b and as a against viral and parasitic infections, virosomes for hepatitis b virus (hbv), human papilloma virus (hpv), hepatitis a virus (hav), and cholera toxin for cholera. adjuvants may be composed of several different compounds. currently, oil based adjuvants, virosomes, toll-like receptors (tlrs) related adjuvants, mpl, adjuvants made of unmethylated cpg dinucleotides and tuftsin have all been described. it is of great interest the understanding of the mechanisms related to the adjuvant effect, as well as to aluminum salts. aluminum acts through multiple pathways, which do not depend solely on tlrs signaling. each of these pathways leads to an enhanced host immune response [ ] . there are many oil based adjuvants. one is incomplete freund adjuvant (ifa), which contains water in oil emulsion. another is complete freund adjuvant (cfa), which is the same as ifa, except that it also contains killed mycobacteria in addition to water in oil emulsion. usually, cfa is used for primary vaccination and ifa for boosting. recent oil based adjuvants that have been developed are mf (novartis ® ), as (glaxosmithkline ® ), advax tm which are based on inulin compounds (vaxine tm pty) and qs- /iscoms, which are immune stimulating complexes composed of cholesterol and phospholipid with or without antigen (table ) . virosomes are adjuvants that contain a membrane-bound hemagglutinin and neuraminidase obtained from the influenza virus. both components facilitate the uptake into antigen presenting cells (apc) and mimic the natural immune response [ ] . leucocyte membranes have membrane bound pattern recognition receptors (prrs) called tlrs, which are responsible for detecting most (although certainly not all) antigen-mediated infections. their activation leads to adaptive immune responses. for this reason, many adjuvants that are used today are directed to prrs. these adjuvants are called tlrs related adjuvants [ ] . mpl is a series of 'monophosphoryl lipid a obtained from the purification of a modified lipopolysaccharide (lps) of salmonella minnesota. bacterial deoxyribonucleic acid (dna) is immunostimulatory due to unmethylated cpg dinucleotides. vertebrate dna has relatively low amounts of unmethylated cpg compared to bacterial dna. the adjuvant effect of cpg is enhanced when conjugated to protein antigens. this adjuvant is being tested in vaccines directed at infectious agents, allergens and tumor cells [ ] [ ] [ ] . another type of adjuvant is tuftsin. tuftsin is an auto adjuvant, which is a natural self-immunostimulating tetrapeptide (thr-lys-pro-arg). this tetrapeptide is a fraction of the igg heavy chain molecule produced by enzymatic cleavage in the spleen [ ] . its functions include: binding to receptors on neutrophils and macrophages to stimulate their phagocytic activity, increasing tumor necrosis factor alpha (tnf␣) release from human kupffer cells enhancing secretion of il by activating macrophages, activation of macrophages expressing nitric oxide (no) synthase to produce no and enhancement of murine natural cell mediated cytotoxicity in vitro [ ] [ ] [ ] . in summary, it is an adjuvant with minor side effects with a promising effect in restoring innate immune mediated response. adjuvants may exert their immune enhancing effects according to five immune functional activities: . translocation of antigens to the lymph nodes where they can be recognized by t cells. . antigen protection enabling longer exposure. . enhanced local reaction at the injection site. . induction of the release of inflammatory cytokines. . interaction with prrs, specifically tlrs [ ] . a adjuvant effect the term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigenspecific immune response in vivo. the microbial components of adjuvants activate apcs to produce pro-inflammatory cytokines ("non-specific" signal ) and to up-regulate molecules essential for antigen presentation. these molecules include major histocompatibility complex (mhc) class ii (antigen-specific signal ) and b - / . these innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of t cells [ ] . in accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [ ] . it has been shown that auto-reactive t-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [ ] . infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [ ] . an example of this is the causality between viral infection and myocarditis. half the cases of myocarditis are preceded by an acute viral infection. infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [ ] . it has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. an example of this is that bladder administration of bcg (bacille calmette-guérin) has been shown to be effective against superficial bladder cancer development [ ] . it can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". prr-pamp (pattern recognition receptor-pathogen-associated molecular patterns) interactions activate the apcs to promote antigen-specific lymphocytic responses [ ] . the definition of pamps has now broadened, in that the recognized structures do not need to be pathogens. thus the concept of "microbe-associated molecular patterns" (mamps) and of "danger/damage-associated molecular patterns" (damps) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [ ] . tlrs are single-transmembrane prrs localized on cell surface and endosomal membranes. from all the prrs, these are the most studied. tlrs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. the interaction between the four known tlrs adapters: myd , tirap/mal, tram and trif, in tlr signaling, shape the innate immune response. besides prrs the innate immune system also detects proteolytic enzymes generated during infection [ ] . merging the response to different prrs signaling may be the pathway for developing customized responses to different aggressions [ ] . b experimental models of adjuvants many animals have been used in experimental models of adjuvant-related autoimmune conditions [ ] . these include primates, salmons, rabbits and swine; however, the most common are murine models. murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( table ). an interesting model is that described by lujan et al. the authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. an excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. this was suggested to be part of the spectrum of asia syndrome. moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [ ] . c toxicity of aluminum adjuvants aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (bbb) and of eliciting immune inflammatory responses. these are probably the reasons why aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [ , ] . aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. it has been described as a neurotoxin because even when a relatively small amount of aluminium reaches the brain [ ] , is can act as a genotoxin [ ] , a prooxidant [ ] , it can be proinflammatory [ ] , act as an immunotoxin [ ] and also as an endocrine disruptor [ ] . aluminum interferes with many essential cellular processes. memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. moreover, alzheimer's [ ] , amyotrophic lateral sclerosis, parkinsonism dementia [ ] , multiple sclerosis [ ] , and neurological impairments in children have been linked to aluminum neurotoxicity [ ] . brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [ ] . aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. one of the most significant is the activation of the nlrp inflammasome pathway [ ] . nlpr activation has been shown to trigger type diabetes. by using nlpr knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [ ] . on the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines il- ␤ and il- are strongly implicated in the development of several central nervous system (cns) disorders [ ] . the vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. upper limits set by us food and drug administrations (fda) for aluminum in vaccines are set at no more than g/dose. these values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [ ] . the quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the fda. however the scientific basis for this recommendation does not take into account aluminum persistence in the body. the concern about aluminum in dietary intake has been reinforced by the food and agriculture (fao) who expert committee, which lowered the provisional tolerable weekly intake of aluminum from mg/kg/bw ( mg/week, for an average kg human) to mg/kg/bw ( mg/week) [ ] . the amount of dietary intake of aluminum has risen in urban societies to up to mg/day considering the widespread use of processed convenience foods. however, only about . % of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [ ] . absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. moreover, the presence of aluminum in breast tissue was associated with breast cancer [ ] . aluminum compounds persist for up to - years post vaccination in human body. this fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [ ] . the clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: . it can persist in the body. . it can trigger pathological immunological responses. . it can pass through the bbb into the cns where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. there is a link between allergies and autoimmunity since both are the result of an abnormal immune response [ , ] . metals such as mercury, aluminum, nickel and gold are known to induce immunotoxic effects in humans. the immunologic effects of these metals include immunomodulation, allergies and autoimmunity. they may act either as immunosuppressants or as immune adjuvants. metals bind firmly to cells and proteins and thus have the ability to modify autologous epitopes (hapetenization). t-cells then recognize the proteins as foreign and trigger an autoimmune response [ ] . hypersensitivity caused by metals may be referred to as type iv delayed hypersensitivity. the reaction is considered delayed because the first symptoms appear - h after exposure, because it is mostly t-cell mediated and the gold standard for diagnosis of delayed type hypersensitivity is patch testing [ ] . in mercury-sensitized patients, even mercury concentrations within the normal range might provoke neuroallergic reactions in the brain [ ] . identifying metal sensitivity and removal of the sensitizing metals, such as dental amalgam, have been proved successful by showing symptom improvement in patients with previous autoimmune diseases. these diseases included fibromyalgia, autoimmune thyroid diseases and orofacial granulomatosis [ ] [ ] [ ] [ ] (table ). the timeline regarding the field of vaccinology has been divided in two generations, the first regarding the administration of inactivated pathogens in whole or live attenuated forms (e.g., bacillus calmette guerin (bcg), plague, pertussis, polio, rabies, and small- pox) and the second regarding vaccines assembled from purified microbial cell components, also referred as subunit vaccines (e.g., polysaccharides, or protein antigens) [ ] . this latter approach there are obstacles to conventional vaccine development methods such as non-cultivable in vitro pathogens (e.g., hepatitis c, papilloma virus types and , and mycobacterium leprae), antigen hypervariability (e.g., serogroup b meningococcus, gonococcus, malaria), opportunistic pathogens (e.g., staphylococcus aureus) and rapid evolving pathogens such as human immunodeficiency virus (hiv) [ ] . vaccine research gained a new perspective as the genomics field emerged over the last decades. bacterial genomes have been sequenced and analyzed making it possible to choose the best candidate vaccine antigens by using the concept of reverse vaccinology [ ] . the main known factors influencing the observed heterogeneity for immune responses induced by vaccines are gender, age, ethnicity, co-morbidity, immune system, and genetic background. the interaction between genetic and environmental components will dictate the response to vaccines. studying the vaccine and the host will enable the development of customized treatment options. the combination of genetics, epidemiology and genomics in vaccine design has been denominated "vaccinomics" [ ] . the importance of genetic influence is supported by twins and siblings studies, which show familial aggregation. this suggests that genomics is crucial in inter-individual variations in vaccine immune responses [ ] . both human leukocyte antigen (hla) and non-hla gene markers have been identified as markers for immune response to vaccines. multiple studies have shown connections between hla gene polymorphisms and non-responsiveness to the hbv vaccine [ ] . hla region is divided in three sub regions: class i is associated with the induction and maintenance of cell-mediated immune response, class ii is associated with presentation of exogenous antigens to helper t cd + cells and class iii, where immune non hla related genes are located. normal human tissue has at least hla antigens, and although new recombinant haplotypes may occur, it is inherited mostly intact from progenitors [ ] . hla allelic differences are associated with different responses to vaccines, either by hyper or hypo responsiveness. we can infer that a similar response may be associated with different safety in relation to the development of autoimmune reactions to vaccines, particularly in the patients with genetic predisposition to an enhanced response to vaccine inoculation [ ] . furthermore, patients that share the same hla, for instance siblings, have been diagnosed with asia following similar environmental stimuli [ , ] . autoantibodies help to diagnose certain autoimmune diseases, however, they can also be found in healthy individuals. thus, autoimmune diseases cannot be diagnosed based solely on antibody detection [ ] . inoculation of vaccines triggers autoimmune responses that result in the development of autoantibodies. many studies have been carried out in animals, healthy subjects and patients with autoimmune diseases to understand if this development is of clinical significance [ ] [ ] [ ] [ ] . a difference in eliciting the production of autoantibodies in healthy humans has been observed between adjuvanted and non-adjuvanted influenza vaccines [ ] . the annual influenza vaccine has been the most heavily researched vaccine, along with hpv and pneumococcal vaccines as far as their relationship with patients who have previously been diagnosed with an autoimmune disease [ ] [ ] [ ] . autoantibody induction after hpv vaccination was also shown in adolescent girls with systemic lupus erythematosus (sle) [ ] . although induction of autoantibodies was proven following vaccine administration, there have been no proven relation with disease diagnosis in either of the specific groups studied so far [ , ] . it has been widely demonstrated that autoantibodies can develop years before the manifestation of a full-blown autoimmune disease [ ] . moreover, the development of a specific autoantibody is also genetically determined, and the link between genetic, autoantibodies and vaccines may become an even more intriguing area of research [ ] . silicones are synthetic polymers that can be used as fluids, emulsions, resins and elastomers making them useful in diverse fields. they were thought to be biologically inert substances and were incorporated in a multitude of medical devices such as joint implants, artificial heart valves, catheters, drains and shunts. of all the silicone-containing products, the most famous are most likely breast implants. silicon is one of the substances suspected to induce asia [ ] . it is currently believed that exposure alone is not enough to trigger the disease but that it requires the presence of additional risk factors (e.g., genetic susceptibility, other environmental factors) [ ] . silicone exerts local tissue reactions. some of these reactions are considered para-physiological, such as capsular tissue formation around an implant. other reactions are viewed as abnormal, like when capsular contractures and allergic reactions to silicone or platinum (catalyst used in silicone polymerization found in minute concentrations in implants) occur [ ] . cutaneous exposure to silicone with cosmetics or baby bottles could potentially sensitize patients [ ] . there is also a systemic component of silicone exposure related to diffusion of silicone through the elastomer envelope, commonly termed "bleeding". it may arouse systemic effects as it degrades and fragments in tissue, it can also spread throughout the body and lead to the development of cancer or autoimmune phenomena [ ] . patients with ruptured implants complain more frequently of pain and chronic fatigue when compared to patients with intact implants [ ] . anti-silicone antibodies were found to be present in human sera more frequently in patients who have undergone silicone breast implants, however, their pathological significance remains uncertain [ ] . the same was seen for other antibodies such as autoantibodies directed against dsdna, ssdna, ssb/la, silicone and collagen ii, which were found to be present in increased levels in patients after exposure to silicone [ ] . it has also been shown that the formation of autoantibodies is directly related to implant duration. several autoimmune diseases have been linked to silicone exposure including rheumatoid arthritis (ra), systemic lupus erythematosus (sle), polymyositis, systemic sclerosis (ssc) and fibromyalgia. although asia symptoms may arise years after the onset of exposure to silicone implants [ ] , most of the follow-up periods are short and concluding evidence is yet to come regarding this causality. there have been published case reports, epidemiologic and research studies that suggest a connection between several vaccines and certain autoimmune conditions, notwithstanding that, overall the benefits of vaccination outweigh the risks. thrombocytopenia has been reported as the main adverse event following mmr vaccine. after mmr vaccine the onset of immune thrombocytopenic purpura (itp) usually occurred within weeks at a risk rate of : , - , mmr vaccine doses, while the incidence of itp following infections is : for measles and : for rubella [ ] . as the risk of thrombocytopenia is higher in patients who experience natural infection with measles, mumps or rubella than in those receiving the vaccine, vaccination is encouraged. arthralgia complaints have also been reported and they may present as transient arthralgia, acute arthritis and rarely chronic arthritis [ ] . some risk factors have been found to be associated with the development of arthritis in vaccinated patients such as: female gender, older age, prior seronegativity and specific hla alleles [ ] . yf vaccine is only advisable to people in, or going to endemic areas. the risk of developing yf vaccine-associated neurologic disease (yel-and) is inversely proportional to age [ ] . this is why children aged < months cannot be vaccinated and < months, except during epidemics [ ] . vaccination is not advisable to people > years because of possible higher risk of severe adverse effects (saes) even though the incidence remains low [ ] . besides being a vaccine for mycobacterium tuberculosis (tb), the bcg has proved effective as immunotherapy for bladder cancer. although the mechanism is yet to be fully understood, it is thought that bcg binds to fibronectin forming complexes that enable the recognition as "non-self" by the innate immune response of th cells. ultimately the pathways result in the apoptosis of tumor cells [ ] . because of its effect in treating non-muscle-invasive urothelial carcinoma, as well as superficial bladder tumors, it was expected that bcg could play a role in treating other types of cancer, despite data having not corroborated this hypothesis so far. adverse events vary according to the site and method of administration. intradermal administration of bcg has been reported to elicit arthritis [ ] , dermatomyositis [ ] and takayasu's arteritis (ta) [ ] among others. intravesical treatment for bladder cancer can cause reactive arthritis (rea) [ ] . the risk relies on a systemic reaction composed of an early infective phase (pcr positive and response to anti-tb treatment) and a late hypersensitivity reaction [ ] . hbv is a dna virus of the hepadnaviridae family, responsible for acute and chronic liver disease. hbv vaccines are considered the first efficient vaccines against a major human cancer. hbv vaccines have reduced the risk of developing chronic infection and they also have proved to reduce the incidence of liver cancer in children [ ] . the vaccine has been associated mainly with autoimmune neuromuscular disorders. they include, but are not limited to: optic neuritis, guillain-barre syndrome (gbs), myelitis and multiple sclerosis (ms), systemic lupus erythematosus (sle), arthritis, vasculitis, antiphospholipid syndrome (aps) and myopathy [ ] . hbv vaccine is the most common immunization associated with acute myelitis. there are studies that indicate that the pathogenicity behind such vaccine and autoimmunity might be based on cross-reactivity between hbv antigen (hbsag) epitopes, yeast antigens, as well as other adjuvants contained in the vaccine itself [ ] . up to % of cervical cancer deaths, occur in developing countries that lack the ability to fully implement the papanicolau (pap) screening programs. hpv poses a special challenge in vaccine safety. hpv is necessary for the development of cervical cancer. however, most women infected with hpv will not develop the disease since % of infections will resolve within a year and up to % within years without specific treatment. over the course of decades, cancer may result in a small proportion of the remaining infected women. death rate from cervical cancer in - year old girls is zero and long-term benefits are yet to be proven. in this specific case, short term risks to healthy subjects can prove to pose a heavier burden than cervical cancer [ ] . there are at least types of hpv strains, of which have been pathologically associated with cancer. two vaccines, gardasil tm and cervarix tm , are commercially available against hpv. both contain the l capsid proteins of several hpv strains as antigens. gardasil tm contains serotypes , , , . these antigens are combined with aluminum (al) hydroxyphosphate sulphate as an adjuvant. cervarix tm contains a combination of the oil-based adjuvant monophosphoryl lipid a (mpl) and al hydroxide (aso ) as adjuvant and is directed at strains and [ ] . there have been several reports of post-licensure adverse events, some of which have even been fatal [ ] . compared to other vaccines, an unusually high proportion of adverse drug reactions has been reported associated with hpv vaccines [ ] . in , australia reported an annual adr rate of . / , , the highest since . this increase was almost entirely due to adrs reported following the commencement of the national hpv vaccination program for females aged - years in april ( out of a total of adrs records). the numbers only decreased after the cessation of the catch-up schedule. although the percentage of convulsions attributable to the hpv vaccine decreased, the overall report remained comparable between and ( % and % respectively). these reports do not prove the association, but show that there is a higher frequency of adrs related to hpv vaccines reported worldwide, and that they fit a consistent pattern (i.e., nervous system-related disorders rank the highest in frequency) that deserves further investigation [ ] [ ] [ ] . indeed, several autoimmune diseases have been linked to hpv immunization. examples include gbs, ms, acute disseminated encephalomyelitis (adem), transverse myelitis (tm), postural orthostatic tachycardia syndrome (pots), sle, primary ovarian failure (pof), pancreatitis, vasculitis, immune thrombocytopenic purpura (itp) and autoimmune hepatitis (ah) [ ] . influenza is an acute viral infection that affects the respiratory tract and is caused by influenza type a-c viruses of the orthomyxoviridae family [ ] . h n mortality rates in the outbreak showed high risk in those aged years and older, presence of chronic diseases and delayed admission. risk of infection was lower in those who had been vaccinated for seasonal influenza with / trivalent inactivated vaccine [ ] . studies have demonstrated that influenza vaccine is safe and immunogenic in patients with sle or rheumatoid arthritis (ra), diminishing the risk of respiratory infections [ ] . it has been shown that adjuvanted vaccine had more local reactions but did not increase systemic adverse reactions [ ] . molecular mimicry has been suggested as a mechanism to explain an autoimmune response following influenza vaccination. however, a causal relationship between influenza vaccines and induction of autoimmune diseases remains unproved [ ] . diseases or symptoms reported after influenza vaccination include mostly neurological syndromes such as gbs [ref] . nonetheless, influenza vaccines should be recommended for patients with ms, because influenza infection is associated with increased risk of exacerbations. that being said, influenza vaccinations showed increased risk of autoimmune responses suggestive of asia [ ] , vasculitis [ ] and aps [ ] among others. meningococcal disease is caused by neisseria meningitidis. one of the following five serogroups causes almost every invasive disease: a-c, y, and w- . vaccines available so far for its prevention encompass either pure polysaccharide vaccines that use purified bacterial capsular polysaccharides as antigens, or protein/polysaccharide conjugate vaccines, which use the polysaccharide molecule plus diphtheria or tetanus toxoid as tcell-stimulating antigens. n. meningitidis serogroup b (menb) menb glycoconjugate vaccines are not immunogenic and hence, vaccine design has focused on sub-capsular antigens [ ] . menb capsular polysaccharide is composed of a linear homopolymer of ␣( → ) n-acetyl-neuroaminic acid (polysialic acid; psa). menb psa and psa found on neural cell adhesion molecules are structurally identical. as a result of this, it has been proposed that infection with menb or vaccination with psa may be associated with subsequent autoimmune or neurological disease [ ] . no evidence of increased autoimmunity was found to be associated with meningococcal serogroup b infection [ ] . regarding vaccination, the inoculation does not cause autoimmune diseases but may unmask autoimmune phenomena in genetically predisposed individuals. local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines. the intramuscular administration of the conjugate vaccine (versus subcutaneous for that of polysaccharide) may, in part, explain the higher reactivity [ ] . diseases previously associated with meningococcal vaccines are gbs [ ] , henoch-schönlein purpura (hsp) [ ] and bullous pemphigoid (bp) [ ] . streptococcus pneumoniae (pneumococcus) is the main cause of bacterial community-acquired pneumonia and meningitis in western countries, as well as the cause of more than , children deaths in developing countries [ , ] . there are three anti-pneumococcal vaccines commercially available. two of these are conjugated to a protein carrier (pcv and pcv ) and one is not conjugated (ppv ). ppv was licensed in and consists of the capsular polysaccharides of twentythree different streptococcus pneumoniae serotypes ( - , b, f, , n, v, a, a, f, , b, f, c, f, a, , f, f, and f). it does not elicit immunological memory because the immune response it triggers is t-cell independent. it is usually administered to the elderly (above years), as it is believed to be less effective in children. pcv is composed of the most frequent serotypes , b, v, , c, f, and f. pcv is directed at serotypes , - , a, b, f, v, , c, a, f, and f. contrary to ppv both pcv and pcv have an aluminum adjuvant in their composition that elicits a t-cell mediated response [ ] . ever since vaccines were introduced in the healthcare system, prevalence, fatality and admissions for invasive pneumococcal disease have decreased significantly [ ] . vaccine adverse events vary depending on whether the vaccine is adjuvanted or not. in a non adjuvanted vaccine, local reactions are present in % of people vaccinated intra muscularly and in % of those immunized sub-cutaneously [ ] . in conjugated vaccines, this percentage rises to % [ ] . systemic reactions such as fever, irritability, decreased appetite and sleep disturbances occur in - % of recipients of pcv or ppv. symptoms like arthralgia, arthritis, myalgia, paresthesia and fatigue are more frequent in patients post ppv. this may be related to the fact that the vaccines are administered to different age groups. autoimmune risk following ppv vaccine is very low. only case reports were found after ppv vaccine. six of these referred to reactivation of a previous autoimmune disorder. studies directed to access vaccine safety in subjects with autoimmune diseases showed immunization was safe [ , ] . tetanus toxoid (tt) is a potent exotoxin produced by the bacteria clostridium tetani. the toxin has a predominant effect on inhibitory neurons, inhibiting release of ␥-aminobutiric acid (gaba). when spinal inhibitory interneurons are affected the symptoms appear [ ] . the vaccine against c. tetani contains deactivated tetanus toxoid plus an adjuvant (usually aluminium hydroxide). the most studied and prevalent disease associated with tt is antiphospholipid syndrome (aps), but cns complications have also been reported such as optic neuritis, acute myelitis and encephalomyelitis [ ] . in mice, the immune response to tt depends on genetic background and to the specific adjuvant used for immunization. naive balb/c mice, immunized with tt, developed antibodies directed to tt, dsdna and ␤ gpi and were extremely sick [ ] . aps is an autoimmune disease characterized by the occurrence of thrombotic events. patients suffering from this condition have recurrent fetal loss, thromboembolic phenomena, thrombocytopenia as well as neurological, cardiac and dermatological involvement [ ] . the serological marker of aps is the presence of antiphospholipid antibodies (apl), which bind negatively charged phospholipids, platelets and endothelial cells mainly through the plasma protein beta- -glycoprotein-i (b gpi). the presence of igg and igm anti-cardiolipin antibodies (acl) and lupus anticoagulant is associated with thrombosis in patients with aps [ ] . ␤ gpi was identified as the most important antigen in aps. ␤ gpi has several properties in vitro which define it as an anticoagulant (e.g., inhibition of prothrombinase activity, adenosine diphosphate-induced platelet aggregation, platelet factor ix production) [ ] . passive transfer of anti-␤ gpi antibodies induce experimental aps in naïve mice and thrombus formation in ex vivo model [ ] . evidence suggests that the molecular mimicry mechanism between ␤ gpi and tt is one of the possible causes for aps. besides tt, aps has also been reported following hbv and influenza virus vaccination, although data are scarce [ , ] . sle is a multisystem autoimmune disease characterized by the production of a variety of autoantibodies. igg isotype antibodies to double-stranded dna (dsdna) are thought to be diagnostic markers and their presence correlates with disease pathogenesis. several factors including genetic, hormonal, environmental and immune defects are involved in the induction of autoantibodies in this disease [ ] . post vaccination manifestations of sle or lupus like syndrome have been reported and range from autoantibody induction to full blown clinical disease. reports have been published associating sle to hbv, mmr, dtp, hpv, influenza, bcg, pneumococcal and small pox vaccinations [ ] . vaccination in sle diagnosed patients is associated with disease exacerbation and decreased antibody response, which may be due to the underlying disease and the frequent use of immunosuppressive drugs [ ] . a temporal link between sle and hbv vaccination is the only relation that has been demonstrated [ ] . several studies have demonstrated an increased prevalence of hpv in individuals with lupus compared to the general population, which has increased awareness for the need to vaccinate this high-risk population [ ] . to do so, the association between immunization with hpv vaccines and sle like symptoms, as well as the higher incidence of flares in known lupus patients must be taken into account. vasculitis is the name given to a group of autoimmune mediated diseases, which involve blood vessels of different types and sizes. they can be categorized according to several disease features indluding: the type of vessel affected, organ distribution, genetic predisposition and clinical manifestation [ ] . so far, cases of large vessel vasculitis have been detected. this includes cases of giant cell arteritis (gca) following influenza vaccination, cases of takayasu disease (td), and one case of large cell arteritis involving subclavian and renal arteries following hbv vaccines. two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (pmr)-like illness [ ] . one case of polyarteritis nodosa (pan) following the administration of tetanus and bcg vaccine is described. all other cases of pan in adults follow the administration of hbv vaccine [ ] [ ] [ ] . case reports of medium vessels vasculitis -both polyarteritis nodosa and kawasaki disease (kd) -have also been published in pediatric patients. kd has been described one day after the second dose of hbv vaccine and following yellow fever vaccine [ , ] . two cases of pediatric patients with pan have been reported two months after receiving the hbv vaccine [ , ] . eosinophilic granulomatosis with polyangiitis (egpa) after tetanus vaccination [ ] and following hbv vaccine [ ] have been reported. there are also cases of microscopic polyangiitis (mpa) and cases of granulomatosis with polyangiitis (gpa) following influenza vaccines in the literature [ , ] . henoch schönlein purpura (hsp) is the most common vasculitis of childhood. it is generally benign and self-limited. it is mediated by iga immune complex deposition in various tissues as well as in small-sized blood vessels. genetic risk factors play an important role in the pathogenesis of the disease: it is associated with hla-drb* , and . hsp was associated with seasonal influenza, influenza a (h n ), pneumococcal and meningococcal disease, hepatitis a virus (hav), hbv, anti-human papilloma virus (hpv) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [ , [ ] [ ] [ ] . leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [ ] , hav vaccine [ ] , hbv vaccine [ ] , pneumococcal vaccine [ ] , varicella [ ] , rubella, smallpox [ ] and the anthrax vaccine [ ] . dermal vasculitis with pan uveitis has also been described following mmr vaccine [ ] . ra is the most prevalent chronic inflammatory arthritis affecting the synovial membrane of multiple diarthrodial joints. although its etiology has not been completely clarified, deregulation of the immune system is evident with a preponderance of inflammatory cytokines and immune cells within the joints. ra has an estimated heritability of %, leaving a substantial proportion of risk to environmental factors. immunizations have previously been proposed as potential environmental triggers for ra. in the norfolk arthritis register database, of the first patients reported receiving a tetanus vaccination within weeks prior to the onset of arthritis. similarly, a transient rise in rf titer was recorded in out of military recruits - weeks after receiving concomitant immunization against tetanus, typhoid, paratyphoid, mumps, diphtheria, polio and smallpox. however, only showed a persistent elevation in titer and none developed arthritis [ ] . several mechanisms have been proposed to explain the putative association between vaccination and the initiation of ra, the most prominent of which are molecular mimicry and non-specific immune system activation [ ] . vaccines who have been associated with ra include rubella vaccine in which reactive arthritis occurs in % of recipients. controlled studies failed to show persistent arthritis or arthralgia in these patients [ ] . patients following hbv vaccine showed an increase of arthritis in a vaers study, but this was not seen in a large retrospective epidemiological study [ ] . data so far suggest that vaccines carry an insignificant role in the pathogenesis of ra. several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as tnf, aiming to induce high titers of endogenous neutralizing anticytokine antibodies with the goal of breaking natural th tolerance to auto antigens. other cytokines, namely il- il- , mif, rantes, il- , mcp- are also being tested [ ] . another vaccine related therapy uses autologous t cell lines to induce a specific immune response by the host's t cells directed against the autoimmune (vaccine) t cells [ ] . this strategy has been successful in mouse models and has shown encouraging results in a small pilot study of ra patients, where patients showed a clinical response, defined by acr improvement criteria [ ] . uctd is a clinical condition characterized by signs, symptoms and laboratory tests suggestive of a systemic autoimmune disease but that does not fulfill the criteria for any defined connective tissue disease (ctd). such patients with clinical manifestations suggestive of systemic connective tissue disease but not fulfilling any existing criteria are quite frequent: - % of the patients initially asking for a rheumatologic evaluation may at least temporarily be diagnosed as affected by 'undefined' or 'undifferentiated' connective tissue disease. comparing studies on these diseases is unfeasible because of the inexistence of defined criteria for diagnosis [ ] . within years of follow-up, patients usually evolve to defined ctds, which include sle, systemic sclerosis (ssc), primary sjögren's syndrome (pss), mixed connective tissue disease (mctd), systemic vasculitis, poly-dermatomyositis (pm/dm) and ra. maintaining an undefined profile for years makes evolving into ctds less probable and the diagnosis of "stable uctd" reliable [ ] . disease etiology is a concern and it has been associated with vitamin d deficiency and silicone implants, both of which lead to an imbalance in proinflammatory and anti-inflammatory cytokines [ ] . vaccines have also been associated with this disease, namely the hbv vaccine [ ] . etiopathogenesis of uctd is unknown and it has been suggested it might fall on asia spectrum since symptomatic similarities are striking and uctd etiopathogenesis has been associated with adjuvants [ ] . aa is an autoimmune disease, characterized by one or more well demarcated oval and round non-cicatricial patches of hair loss. the disease may affect any hair bearing part of the body and has a great impact on a patient's self-esteem and quality of life. depending on ethnicity and location, aa is the most prevalent skin disease. aa prevalence varies and is estimated to be between . - . % in the united states and . % in singapore [ , ] . as with any other autoimmune disease, the development of aa encompasses genetic and environmental factors. environmental factors associated with aa development are emotional and/or physical stress, infections and vaccines [ ] . secondary syphilis is one of the most well studied examples, however epstein barr virus [ ] and herpes zoster [ ] infections have also been related to the development of the disease. as far as vaccines go, hbv vaccine has been associated with aa development. in one study of patients, developed aa after vaccination with hbv vaccine. of those patients, were rechallenged, and the reappearance of disease was witnessed [ ] . in mice this association failed to be established [ ] . one case of aa was witnessed following tetanus toxoid, as well as two case reports following hpv and mmr vaccine [ ] [ ] [ ] . itp is an autoimmune disease defined by a platelet count of less than platelets/l without overlapping diseases. it can present with or without anti-platelet-antibodies. thrombocytopenia is relatively common and the overall probability of developing itp was , % in a cohort of patients. it was also found that % of patients developed an overlapping aid other than itp [ ] . the etiology of the disease is yet to be fully understood but it has been detected following infectious diseases, such as helicobacter pylori, hepatitis c virus (hcv), novel influenza a infection, rotavirus infection and human immunodeficiency virus (hiv) [ ] . itp onset has also been reported, although rarely, as a severe adverse event following vaccine administration. this was more often observed after measles-mumps-rubella (mmr), hepatitis a and b, diphtheria-tetanus-acellular pertussis (dtap), and varicella vaccinations [ ] . molecular mimicry has been suggested as a possible mechanism for the development of itp, namely following helicobacter pylori infection. its eradication has been shown to increase platelet count and diminish the levels of anti-caga antibody in a subset of h. pylori infected subjects with itp [ ] . these data point towards a beneficial role of h. pylori eradication in chronic itp. two cases of itp following anti-rabies vaccine have been reported and one after hpv vaccine. reactivation of itp was reported two weeks after a tick-borne encephalitis vaccination [ ] . the most consistent association with itp is with the mmr vaccine [ ] . however, it should be emphasized that the number of cases are fewer than expected without vaccination. t d is due to antigen specific reactions against insulin producing beta cells of the pancreas. much like other autoimmune diseases, t d results from a combination of genetic, environmental, hormonal and immunological factors. environmental factors such as pathogens, diet, toxins, stress and vaccines are believed to be involved in the beginning of the autoimmune process [ ] . although the mechanisms by which viral infections cause autoimmune diabetes have not been fully clarified, there is some evidence to suggest a role for natural infections in the pathogenesis of t d mellitus in susceptible individuals [ ] . it has been hypothesized that vaccination could trigger t d in susceptible individuals. although post-vaccination t d may be biologically plausible, cumulative evidence has not supported an increased risk of t d following any vaccine [ ] . several experimental data have suggested that, depending on the timing, vaccination might exert a protecting or aggravating effect on the occurrence of diabetes [ ] . a study suggests that haemophillus influenza type b vaccine might be a risk factor in the induction of islet cell and anti-gad antibodies measured at one year of age [ ] but there are previous studies that show no association between hib and t d [ ] . in a cohort of american military officers diagnosed with t d, there was no association found between vaccination and t d diagnosis [ ] . available data about a relation between the mumps vaccine and t d are still incomplete and their interpretation is difficult because of miscellaneous confounding factors associated with the development of t d [ ] . association between hemagglutinin neuraminidase (h n ) vaccines and t d is so far unproven [ ] . in humans, it has been hypothesized that early-age bcg vaccination is associated with the risk of t d. the few studies conducted to date provided no consistent evidence of an association. there are, however, studies showing a possible temporary boost of the immune function after vaccination [ ] . studies also show that among bcg-vaccinated children who test positive for islet autoantibodies, there is a higher cumulative risk of t d [ ] . in animal experiments it has been observed that bcg seems to have a protective effect against diabetes, however researchers have yet to translate this benefit to humans [ ] . in all, studies results do not support any strong association between vaccination and t d. narcolepsy is a sleep disorder described as excessive sleepiness with abnormal sleep pattern characterized by uncontrollable rapid eye movement (rem) events which occur at any time during the day. these event and may or may not be accompanied by a loss of muscle tone (cataplexy) [ ] . a plethora of data indicates that narcolepsy is caused by the lack of orexin (also known as hypocretin), an important neurotransmitter, which is involved in the regulation of the sleep cycle. in narcolepsy patients, a loss of orexin producing neurons in the hypothalamus and low levels of orexin in the cerebrospinal fluid (csf) has been reported [ ] . narcolepsy has been shown to have an autoimmune background. antibodies against tribbles (trib ) have been found in these patients, which may be related to the pathogenesis of disease. an experimental model of narcolepsy in mice has been made by passive transfer of total igg from narcolepsy patients into the animal's brains through intra ventricular injection [ ] . environmental factors like influenza a virus and streptococcal infections have been associated with disease onset. interestingly, fever by itself without the diagnosis of an infectious etiology was found to be a risk factor for narcolepsy [ ] . several groups have studied and found an increase in the incidence of narcolepsy diagnosis following the introduction of influenza vaccination, specifically, aso -adjuvanted pandemrix tm vaccine. this association was shown in finland especially in [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year-olds, but also in case reports from other countries [ ] . other studies failed to find an association. the actual infection with h n has been associated with disease development in china, however no such relationship has been noted in europe [ ] . the above-mentioned associations are specifically related to the aso -adjuvanted pandemrix tm vaccine. the same association has not been reported for other h n adjuvanted or non-adjuvanted vaccines. the major difference between the aso and the mf adjuvants is the presence of the ␣-tocopherol. ␣-tocopherol is unique in that it can achieve the highest and longest antibody response by producing an enhanced antigenspecific adaptive immune response. in vitro it was shown that ␣-tocopherol could increase the production of orexin as well as increase the proteosome activity. this increased production of orexin fragments may facilitate antigen presentation to mhc class ii, thus triggering an autoimmune process [ ] . all these data together support the relationship between the h n pandemrix tm vaccine and the development of narcolepsy. gluten induced enteropathy, gluten sensitive enteropathy, or more commonly called celiac disease (cd) is a life-long autoimmune condition mainly of the gastrointestinal tract, specifically affecting the small intestine. the abnormal immune response crates autoantigens which are directed towards tissue transglutaminase (ttg). the two main autoantibodies and the most widespread serological markers to screen for the disease are anti ttg and anti endomysium. two additional auto-antibodies, namely: anti deaminated gliadin peptide and anti-neoepitope ttg were found recently to be reliable for cd screening as well [ ] . cd is an autoimmune disease induced by well-known nutritional environmental factors. the non-dietary ones are less studied and established. several infectious disease have been linked to its development, the so-called infectome [ ] . a clear cause-effect relation is yet to be established for most of the pathogens associated with cd. what has been shown, however, is that in countries with low economic status, inferior hygiene conditions and higher infectious load, cd prevalence is lower [ ] . an epidemiologic relationship was established in between rotavirus infection and cd. data showed that in genetically predisposed individuals, rotavirus infection was related to childhood cd development [ ] . in subsequent research studies, a celiac peptide was recognized and proved to share homology with rotavirus major neutralizing protein vp and with the cd autoantigen ttg. the antibodies directed against the viral protein vp were shown to predict the onset of cd and induce typical features of cd in the intestinal epithelial cell-line t [ ] . it has also been suggested that rotavirus vaccine alters b and t behavior, as the percentage of b-cells was higher in the vaccinated infants [ ] . rotavirus vaccine as an inducer of cd is still in discussion and warrants further study. pmr is an autoimmune inflammatory rheumatic disease characterized by raised inflammatory markers with pain and morning stiffness of shoulders and pelvic girdles and synovitis of proximal joints and extra-articular synovial structures. its diagnosis is clinical and it is typically a disease of the elderly occurring mainly in subjects above . etiopathogenesis of pmr remains unknown, but genetic and environmental factors play a role [ ] . a close temporal relationship has been ascertained concerning epidemics of mycoplasma pneumoniae, chlamydia pneumonia, parvovirus b and peaks of cases of pmr and giant cell arteritis, however this is not clearly proven [ ] . cases of pmr following vaccination have rarely been reported. however, it is believed that post vaccination pmr may be underreported due to its symptomatic similarities with the transient effects of vaccines, namely: arthralgia, myalgia and low-grade fever. this leads to failure in establishing a chronological relationship when the disease is diagnosed. most of the reported cases are associated with seasonal influenza vaccine (inf-v). often, the time interval between vaccine administration and symptoms onset varies from one day, to three months. three cases were reported with associated giant cell arthritis. a case report of relapsing pmr after four years of remission following tetanus vaccination has also been reported [ , ] . acute disseminated encephalomyelitis (adem) is an inflammatory demyelinating disease of the central nervous system (cns). adem is usually poly-symptomatic with encephalopathy (i.e., behavioral change or altered level of consciousness). it affects mostly children and young adults and has higher prevalence in males. its incidence is . - . per per year [ ] . although there is no concrete evidence of a clear pathogenic association, adem has been associated with immunization or previous viral infection. post-vaccination adem accounts for only - percent of all cases, while post-infectious adem accounts for percent of all cases of adem [ ] . the hypothesis that better describes these associations is molecular mimicry. t-cells targeting human herpesvirus- (hhv- ), coronavirus, influenza virus and epstein-barr virus (ebv) have been shown to cross-react with myelin basic protein (mbp) antigens. anti-mbp t-cells were detected in patients following vaccination with simple rabies vaccine [ ] [ ] [ ] . in a post experimental therapy for alzheimer's disease with a vaccine that contained aggregates of synthetic a␤ fragments of amyloid precursor protein, adem was shown to develop in mice [ ] . the experimental model of ms, eae mice, may be induced with injection of a␤ , but only when the latter is administered together with the complete freund's adjuvant [ ] . this observation points to the importance and central role of the adjuvants in induction of adem and autoimmunity in general [ ] . the overall incidence of post vaccination adem is estimated to be . - . per and a higher risk has been reported following immunization against measles. other vaccines accountable for post-vaccination adem include vaccines against the varicella zoster, the rubella, the smallpox and the influenza viruses [ ] . surprisingly, certain vaccines such as anti-tetanus vaccine were shown to have a negative correlation with adem (statistically significant decreased risk) [ ] . hbv immunization has been studied as a possible cause for adem but was later associated with clinically isolated syndrome (cis) (a first time occurring demyelinating episode that may, or not develop to ms) and complete conversion to ms [ ] . as far as case reports are concerned, adem was associated with vaccination with influenza, hepatitis a and b, mmr, hpv and tetanus [ , , ] . bullous dermatoses are characterized by the presence of blisters and autoantibodies against structural components of the skin: desmosomal proteins (in pemphigus), adhesion molecules of the dermal-epidermal junction (in pemphigoid diseases), and epidermal/ tissue transglutaminase (in dermatitis herpetiformis). the most frequent autoimmune bullous diseases are bullous pemphigoid (bp) and pemphigus vulgaris (pv). bp is more frequently observed in the elderly, while the age of onset of pv is between and years. neither of the diseases have any gender preference [ ] . bp and pv etiology is, so far, poorly understood. both diseases have been associated with various environmental factors, which include emotional and/or physical stress, infections and vaccinations [ ] . genetic predisposition has also been studied with overexpression of certain hla class ii alleles. these include hla-dqb * , drb * , drb * , and dqb * . these alleles have been found to be more prevalent in bp patients than in the general population [ ] . pv is associated with certain hla class ii loci such as hla-dr and hladr alleles (drb * and drb * , which is prevalent in ashkenazi jews, iranian and sardinian patients). other loci include drb * (common among japanese and italian patients) and two dqb alleles (dqb * and dqb * ), which are strongly associated with pv. bp and pv patients' sera were found to have significantly higher prevalence of antibodies to hepatitis b virus, hepatitis c virus, helicobacter pylori, toxoplasma gondii and cytomegalovirus [ ] . as far as vaccination is concerned, bp developed in patients following influenza, diphtheria, tetanus, pertussis, hepatitis b, bcg, polio and herpes zoster vaccines [ , , ] furthermore, reactivation of bp following influenza vaccination was reported in one case report [ ] . new onset pv was associated with: influenza vaccine, hepatitis b vaccine, anthrax vaccine, typhoid booster and rabies vaccination. in addition, exacerbation of pv after vaccination was also reported following influenza vaccine and tetanus vaccine [ ] . iim compose a group of skeletal muscles diseases in which myositis without a recognized cause occurs. iim is usually subdivided in entities: dermatomyositis (dm), polymyositis (pm), inclusion body myositis (sibm) non-specific myositis (nsm) and immune mediated necrotizing myopathy (iam) [ ] . iim prevalence is around . × − cases, with a bimodal age of distribution that peaks in childhood and again between and years. dm is the most common inflammatory myopathy while pm is the least frequent. despite exhibiting similar clinical symptoms, the subsets of iim exhibit significant immunopathological variation. dm begins with the activation of the complement and formation of membrane attack complexes (mac). in pm and sibm the fundamental process is related to cd + t cells mediated cytotoxicity [ ] . it is unclear what breaks the tolerance and drives the immune response to induce iim. so far, dm, pm and sibm have been linked to vaccination. several cases have been reported in the literature associating different vaccines with the development of idiopathic inflammatory myopathies. cases of iim had been reported to vaers database up to june . out of these cases, were classified as pm, as dm and an only one as a sibm. dm has been reported after almost any vaccine, however only a few studies have attempted to clarify the possible relationship between dm and vaccination. pm is a frequent misdiagnosed disorder. some reports have associated previous immunization, especially hepatitis b vaccine with pm [ ] . despite being recently differentiated from other iim, sibm has already been related to hbv vaccine [ ] . some vaccines associated with myositis are mmr vaccine, smallpox vac-cine, poliomyelitis (ipv), diphtheria and tetanus toxoid, influenza, hpv and bcg [ ] . fms is an entity that is related to the inability of the cns to modulate pain. the conditioned pain modulation process in the cns appears to be compromised among many fms patients, which might explain the enhanced pain sensation experienced by these patients [ ] . the etiology of fms is yet to be unveiled. genetic predisposition, physical trauma (particularly to the cervical spine), emotional stress (to various stressors) as well as a variety of infections have been linked with fms. vaccines have been associated with the triggering of fms namely rubella and lyme disease vaccines [ ] . there are several reports of fibromyalgia-like disease after vaccination, specifically hpv (martinez-lavin journal of clinical rheumatology ). the medical community and regulatory agencies should be aware of these possible adverse effects aiming at defining their magnitude. chronic fatigue syndrome (cfs) is a disease characterized by disabling fatigue, headaches, concentration difficulties and memory deficits ( %). other symptoms such as sore throat ( %), tender lymph nodes ( %), skeletal muscle pain and feverishness ( %), sleep disruption ( %), psychiatric problems ( %) and rapid pulse ( %) are often observed. it more frequently affects women and has a prevalence of . - . % [ ] . although disease etiology is still unknown, there are several pathogens, such as epstein-barr virus (ebv), which have been associated with cfs. patients often have higher titers of igm to the ebv viral capsid antigen. cytomegalovirus and human herpes virus antibodies were also detected more often in cfs patients, although other reports failed to replicate these results. parvovirus b infection has also been suggested as a trigger to cfs [ ] [ ] [ ] . vaccine inoculation has also been appointed as a probable cause. vaccinations against rubella, q fever and hepatitis b were found to be associated with higher risk of developing cfs while meningococcal vaccine, poliovirus and influenza vaccine were not. surprisingly, staphylococcus toxoid vaccine appeared to have a protective effect [ , , ] . defined in by shoenfeld and agmon-levin asia syndrome is characterized by hyperactive immune response to adjuvants [ ] . as previously stated, asia incorporates four known medical conditions: siliconosis, gws, mmf, and post-vaccination phenomena [ ] . recently, the sick building syndrome (sbs) was proposed as a candidate for the asia spectrum [ ] . all of these diseases satisfy several criteria for fms and seid [ ] . a macrophagic myofasciitis (mmf) mmf has been described as an emerging condition of unknown cause characterized by a pathognomonic lesion in muscle biopsy mixing large macrophages with submicron to micron-sized agglomerates of nanocrystals in their cytoplasm and lymphocytic infiltrates. these lesions were related to aluminum deposits in muscle following immunization with aluminum containing vaccines [ ] . mmf lesion is now universally recognized as indicative of a long-lasting persistence of aluminum adjuvant at the site of prior intramuscular immunization. the long-lasting mmf lesion should be considered as a biomarker of aluminum bio persistence in a given individual. patients with mmf have higher reported myalgia with incidence being up to %. its etiology is not clear but genuine muscle weakness is rare and the diagnosis of fibromyalgia is also rare. higher prevalence of chronic fatigue syndrome (cfs) in patients with mmf has been reported as well. cognitive impairment has been associated with mmf: in one series of mmf patients, up to % had attention and memory complaints and neuropsychological tests were abnormal in % [ ] . b gulf war syndrome (gws) gws is a clinical entity specifically related to a certain time and place in history. it was described among veterans of the military conflict occurring in - in the persian gulf. the syndrome is characterized by chronic fatigue, musculoskeletal symptoms, malaise and cognitive impairment. it clinically overlaps with post traumatic stress disorder (ptsd), fms, cfs and other functional disorders [ ] . the unique conditions that have been associated so far with disease development are the exposure to extreme climate in the persian gulf, exposure to various chemicals (pesticides, depleted uranium), stress provoked by prolonged waiting without actual combat and the intense exposure to vaccinations of the soldiers for fear of biological weaponry [ ] . comparing gulf war veterans and veterans of the bosnian conflict, multiple vaccinations administered to servicemen in the gulf war was identified as a unique exposure [ ] . the mechanism through which vaccination exposure may lead to the development of functional symptoms is not completely understood. the possibility that a shift from th to th type reactions could be of pathogenic significance was raised and is supported by an increased frequency of allergic reactions, low natural killer cell activity and low levels of interferon ␥ and il- in these patients [ ] . one study with gws patients showed a connection between anti-squalene antibodies and symptoms development. this was refuted by a larger study that found no association between antisqualene antibodies and chronic multi-symptom illness [ ] . c asia registry a registry is a collection of data related to patients with the same specific characteristic. it is often the first approach in the study of an area of inquiry. in rare diseases, registries are often the way to get a sufficiently sized sample of patients which can be used either for epidemiological or research purposes. asia syndrome may be underreported because of unawareness and failure to connect the syndrome with the exposure. this registry was created to fully understand the clinical aspects of disease and compare patients from all over the world in order to have fully validated criteria for disease diagnosis and also to define demographic and environmental history of disease. the asia syndrome registry website can be found on the following link: https://ontocrf.costisa.com/en/web/asia. only cases reported by physicians are accepted. to make an informed decision in medicine, there is always a need to weigh the pros and cons. ards may play an important role in deciding whether vaccination is or is not appropriate to a patient. in these cases, patients are immunosuppressed on account of their diagnosis and even more so if they are under specific immunomodelatory medication [ ] . if the efficacy of vaccination is reduced, there is a potential for development of disease flares following vaccination. in the case of live vaccines, its inoculation may even be enough to trigger disease in the host. for these specific reasons, live vaccines are generally contraindicated in patients receiving immunosuppressant medication. there is a need for screening and treatment of latent tuberculosis infection (ltbi) before starting anti-tnf-alpha therapy. the same is true for vaccination. preferably, even recommended vaccination (see table ) should be administered before the initiation of disease-modifying anti-rheumatic drugs (dmards) because these may reduce vaccine efficacy [ ] . immunosuppression equals high risk of infection and lower vaccine efficacy. taking into account safety concerns and efficacy, the eular recommendations for immunizations in aiird patients are: • assess vaccination status in initial investigation. • administer vaccines in a stable disease phase. • live attenuated vaccines are to be avoided especially if immunosuppressive agents are being administered. bcg is not recommended. • administer vaccines ideally before starting dmards and anti-tnf␣ agents. • influenza and -valent polysaccharide pneumococcal vaccination is recommended. • tetanus toxoid vaccination is recommended following recommendations of general population, in case of major and/or contaminated wounds in patients receiving rituximab in the previous weeks tetanus ig is indicated. • hpv and herpes zoster should be considered. • in hyposplenic/asplenic patients, influenza, pneumococcal, haemophilus influenza b and meningococcal c are advisable. • hepatitis a and b is recommended in patients at risk. • travel patients should be immunized according to general population guidelines except for live attenuated vaccines, which are to be avoided [ ] . vaccines have many beneficial effects in combating infectious diseases and preventing mortality and morbidity. they have also proved to be effective cancer treatments by immunomodulation, as demonstrated by the intravesical administration of bcg to treat superficial bladder cancer [ ] . vaccines are however, linked to autoimmunity. beneficial outcomes, like the adjuvant effect are based on immunity triggering and enhanced immunity mechanisms. these same responses account for autoimmunity exertion. vaccines induce the production of autoantibodies, but their pathologic effect is yet to be unveiled. although vaccines are widely considered safe, there are subjects with predispositions to whom vaccines pose a bigger threat. an example is the fact that animal models with autoimmune predispositions develop autoimmune disease following adjuvant exposure. as many as % of recipients of aluminum containing adjuvants may be sensitized to future exposure [ ] . silicon-induced inflammatory fibro proliferative response is irrefutable and well documented. the presence of anti-silicone antibodies and silicone-associated autoimmune phenomena seems very plausible. asia syndrome and aluminum safety studies show that the use of aluminum containing "placebo" in control groups in vaccine safety studies should be carefully evaluated. new studies must be performed using a proper placebo to adequately test vaccine safety. another evident failure in vaccine safety studies are the short-term periods which are evaluated. continued immune system activation has been observed to be a potential mechanism of disease. a disease which is poorly understood so far. vaccine recommendations should be reassessed frequently in different subsets of the population. this does not invalidate the need for vaccines, however, the lower the possibility of exerting adverse events, the easier it will be for the potential benefits to outweigh the risks. vaccinomics represents a major breakthrough in vaccine development and can lead to the development of targeted vaccines to peptides most likely to be immunogenic [ ] . a predictable response to vaccine can be achieved by differentiating the host variability. this can be achieved namely in genetics and pathogen variability. developing a vaccine accordingly will lead to increased specificity in treatment and leave less room for adverse events. by using immunomodulation, vaccinomics can also give rise to novel therapies for autoimmune diseases. there are several reports of cases of autoimmunity diseases following vaccines but despite in vitro positive results and due to both the limited number of cases and the long latency 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patients with auto-immune inflammatory rheumatic diseases: a systematic literature review for the european league against rheumatism evidence-based recommendations for vaccination in adult patients with auto-immune inflammatory rheuma eular recommendations for vaccination in paediatric patients with rheumatic diseases vaccination against yellow fever among patients on immunosuppressors with diagnoses of rheumatic diseases updated consensus statement on the use of rituximab in patients with rheumatoid arthritis safety and efficacy of meningococcal c vaccination in juvenile idiopathic arthritis unexpectedly high incidence of persistent itching nodules and delayed hypersensitivity to aluminium in children after the use of adsorbed vaccines from a single manufacturer key: cord- -ulq xqma authors: li, jing; rao, yuhan; sun, qinglan; wu, xiaoxu; jin, jiao; bi, yuhai; chen, jin; lei, fumin; liu, qiyong; duan, ziyuan; ma, juncai; gao, george f.; liu, di; liu, wenjun title: identification of climate factors related to human infection with avian influenza a h n and h n viruses in china date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ulq xqma human influenza infections display a strongly seasonal pattern. however, whether h n and h n infections correlate with climate factors has not been examined. here, we analyzed cases of h n infection and cases of h n infection. the spatial characteristics of these cases revealed that h n infections mainly occurred in the south, middle, and northwest of china, while the occurrence of h n was concentrated in coastal areas of east and south of china. aside from spatial-temporal characteristics, the most adaptive meteorological conditions for the occurrence of human infections by these two viral subtypes were different. we found that h n infections correlate with climate factors, especially temperature (tem) and relative humidity (rhu), while h n infections correlate with tem and atmospheric pressure (prs). hence, we propose a risky window (tem – °c and rhu – %) for h n infection and (tem – °c and prs - kpa) for h n infection. our results represent the first step in determining the effects of climate factors on two different virus infections in china and provide warning guidelines for the future when provinces fall into the risky windows. these findings revealed integrated predictive meteorological factors rooted in statistic data that enable the establishment of preventive actions and precautionary measures against future outbreaks. influenza viruses with high infectivity and widespread outbreak patterns, these two aivs often cause sporadic and small outbreaks, despite the lack of human-to-human transmission ability, so other more complex reasons were involved. because these two viruses are associated with both wild and domestic bird populations, it is likely that environmental factors play a significant role in the spread of the viruses. generally, wild birds may act as the primary spreading agent for the outbreak of the influenza virus . after the movement of the virus from wild birds to poultry, the virus may spread from flock to flock and farm to farm through several ways . these include wild bird migration, poultry-wild bird interaction, and human proximity to poultry. for example, poultry trade areas that are in proximity to wild waterfowl habitats, such as wetlands or lakes, can facilitate virus exchange between wild birds and poultry , . animal model data shows that the transmission of iav is more likely at low temperatures and relative humidity conditions . however, the prevention of secondary spread can be achieved through restricting bird movement and proper biosecurity procedures. given the mobility of wild birds and the challenges of sustained control, it is of great importance to identify which environment factors correlate with the occurrence of these two viruses. both the h n and h n influenza viruses are of avian origin and have similar infectivity to humans, but their transmission region, outbreak timing, and host range are diverse. outbreaks of h n in wild birds are concentrated in the central part of europe, while outbreaks in poultry are mainly found in eastern europe, with human infections mainly in southeast asia and eastern europe . human infections with h n are found in china, corresponding to its high prevalence in certain lpms (live poultry markets) , while the occurrence of h n in wild birds is rare . previously, data have indicated that environmental factors affect the prevalence of h n and h n , with the infection and spread of the two viruses being closely correlated with bird habitats, migration, and local climate factors (e.g., temperature and relative humidity) . however, we found that both viruses have species-specific manifestations; the outbreak patterns of these two viruses in humans are not coincident with infections in wild birds or waterfowl. the reasons for this discrepancy could be due to the prevalence of the viruses in wild birds and waterfowl, the proximity of humans to these animals, and/or characteristics of the viruses themselves. the susceptibility of humans to the viruses due to host immune function and inhibition of mucociliary clearance by the inhalation of cold, dry air could also be a factor. in the present study, we compared the physical environmental factors and anthropogenic environmental factors that were present during h n and h n aiv outbreaks, as these viruses have similar endemic areas, host ranges, and are the main concerns to public health in recent years in china. understanding the outbreak patterns of these viruses is important to identify high-risk populations and areas, as well as to establish preventive actions and precautionary measures against future outbreaks. spatial characteristics of h n cases and h n cases. as the temperature varied heterogeneously in gradients in different provinces, we classified the affected provinces into the three commonly used geographical sectors that have similar meteorological characteristics: south (south area of the yangtze river), central (the yellow river to the south and the yangtze river to the north), and north china (north area of the yellow river). south china is comprised of guangxi, guangdong, and fujian provinces, central china is comprised of sichuan, guizhou, hubei, hunan, jiangxi, anhui, jiangsu, shanghai, and zhejiang provinces, and north china is comprised of shaanxi, shanxi, henan, hebei, shandong, and liaoning provinces, as well as beijing and tianjin (municipal cities). as shown in table , the h n influenza infection cases mainly occurred in south, central, and northwest china, with the most (eight cases) in hunan province. in contrast to h n , the occurrence of h n in mainland china was concentrated in the coastal areas of south china. climatic characteristics of h n cases and h n cases. to understand the discrepancies in climate factors between h n and h n infections, the null hypothesis was tested using the t-test. as shown in table , the p-values for rhu.mean, prs.mean, and win.mean were < . . these results imply that there were significant differences between h n and h n infections in terms of humidity, pressure, and wind speed. h n infections occurred with the highest frequency in a humidity range of - %, while h n infections occurred in the - % rage (fig. b) . in terms of atmospheric pressure, the cases of h n were concentrated at - kpa, while the occurrences of h n infection were found at - kpa (fig. c ). in addition, as the composition of meteorological factors, the wind speed during h n cases was concentrated at - m/s, while the occurrences of h n infection were found at - m/s wind speed (fig. f ). temperature and specific humidity were the best individual influence factors for influenza infection. according to null hypothesis , both the mean value of the air temperature and the mean value of the ground surface temperature displayed no significant difference comparing h n and h n infections. h n influenza occurred with the highest frequency in a temperature range of - °c, with a second peak in the range of - °c. h n influenza occurred with the highest frequency in a temperature range of - °c, with a second peak in the interval of - °c. consistent with the tem value, the h n influenza infection cases were relatively more prevalent when the ground surface temperature was - °c, with a second peak at - °c; h n influenza infection cases peaked at - °c and - °c, respectively (fig. a,d) . to determine the correlation between climate factors and influenza infection, we established a null hypothesis that states that h n /h n infection is independent of climate factors. the chi-squared test was used to test this hypothesis. as shown in table , we found that the p-value for all of climate is < . . these results demonstrate that there were significant relationships between climate factors and h /h influenza infection. to further understand the relationships among these factors with h /h infection, we conducted principal component analysis (pca) for all climate factors employed in our study. as shown in table , fig. a and supplementary video s , the results from the h n influenza cases indicate that the first principle component, whose dominant contributors are temperature variables (i.e., tem.mean, tem.high, tem.low, and gst.mean) explain . % of the total variance; the second principle component, which is dominated by relative humidity variables (i.e., rhu.mean and rhu.low), explains . % of the total variance. the third principle component was dominated by wind speed (win.mean) and other variables and only explains . % of the total variance. the total contribution of these three sets of climatic variables accounted for . % of the total contribution. similar to the h n influenza cases, the first h n principle component was led by temperature variables (i.e., tem.mean, tem.high, tem.low, and gst.mean) and explains almost half of the total variance ( . %, see table , fig. c and supplementary video s ). in contrast to h n , the second principle component of h n was dominated by pressure (i.e., prs.mean), which explains . % of the total variance, and the third principle component included humidity and other variables, explaining . % of the total variance. the total contribution of these three sets of climatic variables accounted for . % of the total contribution. from the above analysis, we can see that among all of the climatic variables, temperature and humidity were the key factors for h n infection. for h n infection, temperature and atmospheric pressure were the key factors, though humidity also had significant effects. a heat map was generated to evaluate the factors conducive to the spread of h n (fig. b ). based on the heat map, we created a climate high-risk window that we consider represents a high risk for the spread of h n (temperature - °c and rhu - %) and encompassed h n infections ( . %), as well as a broader climate moderate-risk window that we consider represents a moderate risk for the spread of h n (temperature - °c and rhu - %), encompassing infections ( . %). according to these criteria, weeks - in north china, - and - in central china, and - and - in south china commonly satisfy the criteria of the climate high-risk window. furthermore, in weeks furthermore, based on the mean temperature-humidity and temperature-atmospheric pressure during - , the predictions of city distribution and month distribution were calculated ( supplementary fig. s , table s and s ). we found that the occurrence of h n influenza virus was concentrated in march, april, october, and november, while h n influenza peaked in january and february. darker color in the figure corresponds to a higher occurrence ratio of influenza in a specific province. to determine whether the risky windows were statistically meaningful, we used the t-test to examine the differences in case numbers within and outside risky windows. for each province i, we counted the cases that met the criteria of the risky windows (i.e., n r,i ) and did not meet these criteria (i.e., n n,i ), respectively. then, t-tests were performed to examine the mean value difference between n r,i and n n,i across all provinces. the null hypothesis is that the means value of n r,i and n n,i are the same, indicating that the risky window is not statistically meaningful. the alternate hypothesis is that the mean values are different, suggesting that the risky window is statistically significant. the t-test was performed on both h n and h n , yielding p-values of . and . , respectively. this suggests that case numbers within and outside risky windows are significantly different for both h n and h n . both avian h n and the newly emerged h n influenza viruses are highly pathogenic aiv subtypes that can directly infect humans and cause severe diseases with high mortality. unlike seasonal influenza, human infection with these two subtypes is sporadic, and little is known about the factors underlying the occurrence of human cases and the environmental factors favoring the occurrence of human infections. herein, we identified that temperature, humidity, and atmospheric pressure are potential warning meteorological factors for the incidence of both h n and h n subtype infections. the dependence of influenza virus transmission on environmental factors, including temperature, humidity, and atmospheric pressure, has been documented by many previous studies [ ] [ ] [ ] [ ] . however, we found that the most adaptive meteorological conditions for the occurrence of the human cases of these two aiv subtypes are different. for example, most h n cases appeared when the atmospheric humidity was - %, whereas h n cases predominantly occurred when the humidity was between - %. in addition, differential atmosphere pressure and wind speed favor the occurrence of h n and h n cases, respectively. however, what underlies these differences is unknown and deserves further investigation. several lines of evidence indicate that the high incidence of the featured seasonal pattern of h n virus infection in poultry in china , , consistent with the human cases of h n influenza virus in our study. furthering the understanding of the correlation between the survival ability of the viruses relative to climatic factors should be considered. aside from meteorological factors, many other factors also impact the occurrence of human cases of h n and h n infection , . the migration of wild birds and live poultry transactions play an important role in spreading h n and h n viruses. the occurrence of human h n cases has been suggested to overlap with wild bird flyways . although it is also reported that h n virus is found only in chickens, ducks, and pigeons at live poultry markets and that no migratory birds have tested positive for the virus, migratory birds are implicated in the transmission of the virus. the regions with high incidences of h n and h n are places located along migratory bird flyways and also have surrounding poultry farms serving the densely populated areas that have many live poultry markets . to assess the different roles that various climatic factors play in h n and h n infections, we applied pca to patient-specific meteorological data, and the results suggest that the first two principle components (pc and pc ) can be used to estimate correlations. temperature factors and relative humidity are the dominant variables for pc and pc (respectively) for h n , while temperature and pressure are the key factors for h n cases. these results suggest the possibility of using temperature and relative humidity to approximate the effects that the climate factors have upon human h n infection. just as compared with highly pathogenic aiv (hpaiv), the h n virus shows differential environmental factors. although climatic factors exhibit a strong correlation with h n or h n infection, other crucial factors doubtlessly include the density of the pathogen virus, human behavior, and population density. several previous studies depict risk maps in terms of h n /h n infection in birds , , [ ] [ ] [ ] . there are similar spatial distributions with h n infections in humans but differential spatial distribution with h n infections in humans. the risk areas for human h n infection were more concentrated in south and central china in our analysis. hpaiv viruses are generally thought to arise in poultry after domestic birds become infected by lpai h and h viruses from the wild-bird reservoir . the probable long co-existence of multiple viruses in multiple hosts provides the driving force for the virus to adapt to infect humans. furthermore, it is difficult to identify humans, birds, or pigs infected with subclinical h n /h n viruses from clinical signs, and it is extremely difficult to conduct large-scale surveillance to identify virus infections in humans, birds, or pigs for an extended period. therefore, we must pay more attention to overlap areas. the lpms act as a melting pot for a variety of viruses at a range of densities, and market networks with numerous trade connections should permanently be closed down to avoid future avian influenza virus infections. however, there is vast number of small-scale poultry producers, supplying a strong demand for live poultry in china. consequently, permanently closing lpms would likely prove unpopular, and therefore, their periodic closure may represent a more viable compromise. the climate risk windows delineated in this study should be considered as important references for the guidance of any such solution. this strategy of metrological factor-based prediction can avoid large-scale live poultry culling and blindly closing live poultry markets to save a large amount of social resources. outcome data. we collected the h n and h n influenza data reported on the chinese mainland from the climate dataset contained the air temperature, ground surface temperature, relative humidity, wind speed, and atmospheric pressure of all meteorological stations in the reported provinces at the reported dates for each record. there is more than one meteorological station in each province, and the site-level climate datasets were aggregated into the province-level (average for the climate factors), which are able to represent the weather situation in the study areas. furthermore, several indices were derived from the province-level climate data for further analysis, i.e., the maximum, minimum, and mean value of the air temperature (tem.mean, tem.high, and tem.low), the minimum and mean value of the relative humidity (rhu.mean and rhu.low), the mean value of the ground surface temperature (gst.mean), the pressure (prs.mean), and the wind speed (win.mean) on each reported date. briefly, the average daily data from each weather station in the province, observed four times on a daily basis ( : , : , : , and : ), were used after removing outliers. according to province-level climate data, the daily mean data were selected to average total weather stations of each province from january through december . the monthly mean data were calculated from the daily reports of each province. first, we employed eda to summarize the data distribution and statistical characteristics of our datasets for both h n and h n infection cases. we analyzed the distribution of all potential meteorological factors for the h n and h n datasets using histograms, respectively. to further determine whether the climatic factors differ between the h n events and h n events, a series of two-sample t-tests were employed in our research due to the unequal sample sizes of the two groups. all hypothesis tests had a similar null hypothesis that the mean values of specific factors (x) are equal. then, a t statistic was obtained by the equation below, where x i , s i , and n i represent the mean value, variance, and sample size of a specific factor of the ith group (i.e., group : h n ; group : h n ), respectively. statistically, this statistic denotes a t distribution with the degree of freedom of df, which was calculated as below, given the significance level of a p-value (i.e., . in this study), the null hypothesis should be rejected, which indicates that the mean values of the specific factors differ between the h n events and h n events, if the calculated t statistic meets the following critical region, where t −a/ ,df is the critical value of the t distribution with the degree of freedom of df. pearson's chi-squared test. in addition to the t-tests, pearson's chi-squared tests were used to examine the dependence of h n events and h n events on the occurring month and climatic factors. the null hypothesis of the pearson's chi-squared test is that a specific factor is statistically independent from the occurrence of h n and h n . considering that the pearson's chi-squared test could only be applied to the categorical variables, all continuous variables, including age, and all climate factors were converted into categorical variables by dividing observations into several groups using pre-defined thresholds. specifically, all observations of h n and h n were divided into: ) three groups for occurring month, (group : january to march; group : april to june; and group : july to december); ) nine groups for air temperature, from - to °c with a step of °c; ) eight groups for relative humidity, from % to % with a step of %; and ) eight groups for ground surface temperature, from - to °c with a step of °c, separately. after the grouping procedure, the counts of all observations in each group could be exhibited as in the table , accordingly, a χ statistic was calculated based on the following equations, where e i,j is the expectation value of each group at the ith row and jth column, and n is the total sample size of these two datasets. statistically, this calculated χ statistic denotes a chi-squared distribution with the degree of freedom (df) equal to r- . therefore, if the calculated χ meets the following critical region, we rejected the null hypothesis, which indicates that this factor is dependent on the occurrence of h n and h n , where χ -a/ ,df is the critical value of the chi-squared distribution, a is the significance level (i.e., . in this study), and df is the degree of freedom. pca. in addition to the descriptive analysis applied for our datasets, we also deployed pca for both datasets to determine relative contributions of each meteorological variable for both h n and h n cases and to reduce the dimensions of meteorological factors for further analysis. this quantitative analysis yielded better insight into the correlation between h n /h n infection cases with dominant meteorological factors. group influenza in tropical regions influenza in the tropics influenza surveillance in pune, india, - epidemiology of human influenza a and b viruses in myanmar from 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viral genome historical prevalence and distribution of avian influenza virus a(h n ) among wild birds comparative epidemiology of human infections with avian influenza a h n and h n viruses in china: a population-based study of laboratory-confirmed cases distribution and risk factors of pandemic influenza a (h n ) in mainland china the influence of meteorology on the spread of influenza: survival analysis of an equine influenza (a/h n ) outbreak a climatologic investigation of the sars-cov outbreak in beijing, china mapping spread and risk of avian influenza a (h n ) in china genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia dynamic patterns of avian and human influenza in east and southeast asia epochal evolution shapes the phylodynamics of interpandemic influenza a (h n ) in humans synchrony, waves, and spatial hierarchies in the spread of influenza bird migration and risk for h n transmission into qinghai lake the emergence of influenza a h n in human beings years after influenza a h n : a tale of two cities environmental factors contributing to the spread of h n avian influenza in mainland china predicting the risk of avian influenza a h n infection in live-poultry markets across asia weather variability and influenza a (h n ) transmission in shanghai, china: a bayesian spatial analysis airborne transmission of influenza a/h n virus between ferrets predicting unprecedented dengue outbreak using imported cases and climatic factors in guangzhou j.l. analyzed data and wrote the paper. y.r., q.s., j.j. and x.w. collected data. j.c., y.b., f.l., d.l. and g.f.g. modified the paper. q.l., z.d. and j.m. analyzed data. w.l. designed the experiments. key: cord- -k s iy u authors: khalafalla, abdelmalik i.; lu, xiaoyan; al-mubarak, abdullah i.a.; dalab, abdul hafeed s.; al-busadah, khalid a.s.; erdman, dean d. title: mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia, – date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: k s iy u to assess the temporal dynamics of middle east respiratory syndrome coronavirus (mers-cov) infection in dromedary camels, specimens were collected at – month intervals from independent groups of animals during april –may in al-ahsa province, saudi arabia, and tested for mers-cov rna by reverse transcription pcr. of live camels, ( . %) nasal swab samples were positive; of camel carcasses, ( . %) lung tissue samples were positive. positive samples were more commonly found among young animals (< years of age) than adults (> years of age). the proportions of positive samples varied by month for both groups; detection peaked during november and january and declined in march and may . these findings further our understanding of mers-cov infection in dromedary camels and may help inform intervention strategies to reduce zoonotic infections. m iddle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected persons ( , ) . saudi arabia is the country most severely affected by the virus and is where the first recognized case was identified in . the origin of mers-cov remains a mystery. bats seem to be the reservoir host of the virus ( ) but are probably not the source of the ongoing mers-cov outbreak because of limited contact with humans in the arabian peninsula. early observations that some mers-cov-infected persons had been exposed to camels suggested a possible role of these animals as intermediate reservoir hosts ( , ) . serologic surveys subsequently conducted in several countries in the arabian peninsula and africa identified high rates of mers-cov-specific antibodies in dromedary camels ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . furthermore, mers-cov infection in dromedary camels was definitively proven by the detection of virus and virus sequences in respiratory specimens, feces, and milk collected from camels in qatar ( , ) , oman ( ) , saudi arabia ( , , ) , and egypt ( ) . the few published studies that looked for mers-cov in the respiratory tract of naturally infected dromedary camels examined nasal or ocular swab samples but not samples from the lower respiratory tract. moreover, several studies relied on only a few specimens or collected specimens at only time point ( , ( ) ( ) ( ) . to address these limitations and to clarify the dynamics of mers-cov infection in these animals, we conducted a year-round study in which we collected a large number of specimens from the upper respiratory tracts of live dromedary camels and from the lungs of dromedary camel carcasses. this study was approved by the institutional review board of the camel research center, king faisal university, al-ahsa, saudi arabia. respiratory specimens were collected from independent groups of mixed-age dromedary camels (camelus dromedaruis). the first collection was obtained during april -may at the al omran abattoir, al omran city, in al-ahsa province in the eastern region of saudi arabia. livestock slaughtered at this abattoir include cattle, goats, sheep, and camels originating from al-ahsa and neighboring provinces. animals selected for slaughter were mainly from the livestock market and from herds located around al-ahsa province. at the livestock market in al-ahsa, dromedary camels are housed in small groups ( - animals), where they may stay for no more than days. they are then transported in vehicles to the abattoir, where they are kept for no more than hours before slaughter. samples were taken from slaughtered dromedary camels on occasions (every - months). on each particular collection date, tissue specimens were collected from the lungs of all slaughtered dromedary camels. a total of animal carcasses were sampled; had been young animals (< years of age) and had been adults (> years of age). lung lobes that showed pulmonary lesions were sampled; if both lobes showed lesions or if no lesions were visible, the left lobe was sampled because of its close proximity to the person collecting the sample. the tissue samples (≈ - g) were collected aseptically from inside the lung lobes by using sterile surgical instruments (scalpels, forceps, and scissors). to avoid cross-contamination, lungs were moved to a clean room adjacent to the slaughtering hall and examined on a freshly disinfected table by a person wearing a newly donned gown, face mask, and sterile gloves and using a new set of sterile surgical instruments. collected tissue samples were immediately deposited in labeled sterile plastic bags and placed in a cooler containing ice packs for transport to the laboratory. a second sample was collected from age-matched animals over the same period and consisted of nasal swab specimens ( young animals and adults), from visually healthy dromedary camels and from camels with nasal and lachrymal discharge. nasal swabs were collected from animals at locations in al ahsa province (al omran abattoir, al ahsa livestock market, and the veterinary hospital of king faisal university). for this procedure, a long sterile flexible swab was inserted into nostril until slight resistance was felt; the swab was then rotated, held in place for seconds, withdrawn, and placed in ml of cold viral transport medium containing antibiotics (this medium was chosen to enable future attempts to isolate the virus). both swab and lung specimens were transported on ice to the laboratory within - hours of collection and stored at − °c until testing. collection dates and numbers of samples are listed in table . swab specimens in transport media were mixed and then clarified by centrifugation at × g for minutes; the supernatants were recovered for extraction. lung samples were thawed and homogenized by using a tissueruptor homogenizer (qiagen, hilden, germany), and % suspensions were prepared in ml of transport medium. the resulting homogenates were subjected to centrifugation as above, and the supernatants were recovered for extraction. total rna was extracted from μl of each nasal swab or lung sample by using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. extracted rna was tested by using a gel-based pancoronavirus reverse transcription pcr (rt-pcr) assay according to the protocol of vijgen et al. ( ) . realtime rt-pcr (rrt-pcr) was performed by using an assay kit provided by the centers for disease control and prevention (cdc; atlanta, ga, usa). this assay panel targets the mers-cov nucleocapsid protein gene ( ) and a region upstream of the envelop protein gene described by corman et al. ( ) . all samples were screened by using gel-based rt-pcr and rrt-pcr assays and were considered positive for mers-cov if a positive result was obtained with at least of the tests following world health organization recommendations (http://www.who.int/csr/disease/coronavirus_infections/ who_interim_recommendations_lab_detection_mer-scov_ .pdf). all rt-pcrs included no-template negative controls and quantified mers-cov transcript as positive control. cdna was prepared from positive samples and shipped to cdc for independent confirmation and sequencing. to assess the genetic variability of mers-cov, we sequenced the spike protein gene coding region ( , nt) on the positive samples. sequencing was performed on an applied biosystems xl genetic analyzer (thermo fisher scientific, grand island, ny, usa) by using sequencher version . software (gene codes, ann arbor, mi, usa) for sequence assembly and editing. sequence alignments were performed by using clustalx version . implemented in bioedit version . . (http://www.mbio. ncsu.edu/bioedit/bioedit.html). phylogenetic analyses were performed by using mega version . (http://www. megasoftware.net). the neighbor-joining method (tree algorithm inferred with the kimura -parameter substitution model of sequence evolution) was used to construct phylogenetic trees, and bootstrap resampling analyses were performed ( , replicates) to test tree reliability. during the study, a total of lung tissue samples and nasal swabs were obtained from the groups of camels (table ) (table ) . all animals from both groups appeared healthy on visual inspection except for . these -month-old dromedary camel calves, located outside of the al omran abattoir, exhibited purulent nasal and lachrymal discharge; mers-cov rna was detected in nasal swab specimens from these calves (figure ). mers-cov rna was more often detected in the lung and nasal cavity of young camels than adult camels (table ) our results confirm previous reports documenting wide circulation of mers-cov in dromedary camel populations in the middle east. in other studies, rt-pcr detection of mers-cov in nasal swab specimens from these animals has ranged from . % to . %. studies conducted in qatar detected mers-cov in ( . %) of ( ) and ( . %) of ( ) animals tested; in saudi arabia, ( %) of ( ) and ( %) of ( ); in oman, ( . %) of ( ) ; and in egypt, ( . %) of ( ) . a recent large study of , dromedary camels in the united arab emirates identified mers-cov rna in only . % of animals ( ) . of note, these authors found proportionately more positive animals near the border with saudi arabia and detected > fold more among animals sampled from slaughter houses. overall, we detected mers-cov in the upper respiratory tract of a higher proportion of animals tested in al-ahsa, but this proportion was within the upper range previously reported. in contrast, alagaili et al. ( ) , in a comprehensive survey conducted in november and december , sampled regions of saudi arabia (gizan in the south, taif in the west, tabuk in the north, uniza in the center, and hofuf [al-ahsa] in the east) and reported % positivity by rrt-pcr in animals from taif versus only % from al-ahsa, despite seroprevalence of % in the latter. during the same period and in the same region, we detected mers-cov in . % of nasal swab samples. this difference may be because of differences in the numbers and ages of animals sampled, time of specimen collection, or even between geographically proximate dromedary camel herds where rates of mers-cov detection can vary dramatically ( ) . of note, detection of mers-cov rna by rt-pcr does not necessarily indicate active virus replication. when dromedary camels were experimentally inoculated, infectious mers-cov was detected in the upper respiratory tract for only days, but rna could be detected by rt-pcr for up to days after inoculation ( ). we were unable to perform virus isolation studies because of lack of suitable biosafety infrastructure. we also found that a high proportion of lung tissues from slaughtered dromedary camels at the al omran abattoir were mers-cov positive by rt-pcr. in their experimental inoculation study, adney et al. ( ) observed histologic lesions in the epithelium of the upper and lower (trachea, bronchi, and bronchioles) respiratory tract and recovered viable virus from these tissues and from of lung lobes of an animal euthanized days after inoculation; viable virus was not recovered from tissues of other animals at and days after inoculation. although that limited study found infection extending to the lung of animal, the authors found that the upper respiratory tract was the predominant site of virus replication and offered that finding as an explanation for the lack of observed systemic illness among naturally infected dromedary camels. an alternative hypothesis posits that, in the natural setting, subclinical mers-cov infection of the lower respiratory tract also occurs, possibly enhanced by crowding and stress endured during transport and corralling before slaughter. although we did not collect matching premortem nasal swab samples from slaughtered animals to determine how many were also positive for mers-cov in the upper respiratory tract, our findings raise the possibility that testing upper respiratory tract samples alone may underestimate the true number of actively infected animals. in humans, mers-cov was detected in the lower respiratory tract of infected patients for ≈ month while oronasal swab samples were negative ( ) . likewise, mers-cov detection has been found to be enhanced from lower respiratory tract specimens, and therefore these specimens are recommended by the world health organization for diagnosis of mers-cov infection ( , , ) . although great care was taken to avoid contamination with ambient mers-cov present in the abattoir, the possibility that sample contamination occurred cannot be entirely ruled out. further studies that include immunohistologic examination and virus isolation from the lower respiratory tract of naturally infected dromedary camels will be needed to substantiate these findings. our detection of mers-cov rna in camel calves with purulent nasal discharge was consistent with those of hemida et al. ( ) , who also observed mild clinical signs characterized by nasal discharge in some naturally infected young dromedary camels, and of adney et al. ( ) , who documented appearance of purulent nasal discharge in the experimentally infected adult dromedary camels. we also detected mers-cov rna in a higher proportion of specimens from younger than from older adult dromedary camels, consistent with findings of previous studies that mers-cov infection is more common among young camels ( , ) . our study also investigated temporal variation in mers-cov infection in dromedary camels. although data interpretation was complicated by discontinuity in the months sampled and sampling from only animal group in some months, a temporal pattern in mers-cov prevalence was apparent. for both animal groups, peak detection occurred during november -january , followed by a steady decline, reaching the lowest point in may . although we observed no clear temporal differences in the geographic origins or ages of dromedary camels brought to slaughter, which might bias these results, our data are nevertheless limited and should not be used to imply a general pattern of mers-cov circulation in dromedary camels in saudi arabia. nevertheless, these findings would not be unexpected. increased circulation of mers-cov among dromedary camels during the cool season is consistent with the prevailing cooler ambient temperatures, which have been shown to enhance coronavirus survivability outside the host ( , ) , and the cool season is the period of peak circulation of other respiratory viral pathogens of humans in saudi arabia ( ) ( ) ( ) . this period also corresponds with the peak calving season for dromedary camels in saudi arabia ( ) ; higher rates of mers-cov infections among a greater proportion of young animals with higher virus loads may increase opportunities for virus spread ( , ) . whereas the link between dromedary camels and mers-cov infection of humans is well established ( , ) , the overall contribution of zoonotic infections to community-acquired mers-cov remains unclear. serologic studies of animal handlers in saudi arabia who work emerging infectious diseases • www.cdc.gov/eid • vol. , no. , july in close proximity to dromedary camels have shown limited evidence of mers-cov infection ( ) ( ) ( ) . alghamdi et al. ( ) , who examined patterns of mers-cov infections among humans in saudi arabia between june and may , did not find a concomitant temporal increase in human infections that corresponded with our findings in dromedary camels. those authors observed a slight, temporary increase in cases among humans in june and september and few cases from october through february, after which cases and deaths sharply increased beginning in april . the authors concluded that lower relative humidity and higher temperatures during these months might have contributed to the dramatic surge in reported cases. however, more recent data from the world health organization ( ) show a sharp decline in mers-cov cases among humans in may ; low numbers of cases were reported from june through august , when mean temperature was highest and relative humidity was lowest in saudi arabia ( ) . moreover, a recent increase in numbers of mers-cov cases in humans from september through february corresponds more closely with the temporal pattern we found in dromedary camels the preceding year. further studies conducted over multiple years are needed to better understand the ecology of mers-cov, which might help inform intervention strategies to reduce zoonotic infections. isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus in bats, saudi arabia recovery from severe novel coronavirus infection middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia seroprevalence in domestic livestock in saudi arabia seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralization assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan 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coronavirus by real-time reverse-transcription polymerase chain reaction structure of mers-cov spike receptor-binding domain complexed with human receptor dpp prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels mers-cov study group. clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission severe respiratory illness caused by a novel coronavirus stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions t he effects of temperature and relative humidity on the viability of the sars coronavirus viral aetiology and epidemiology of acute respiratory infections in hospitalized saudi children respiratory viruses in children attending a major referral centre in saudi arabia viral agents causing acute lower respiratory tract infections in hospitalized children at a tertiary care center in saudi arabia evidence for camel-to-human transmission of mers coronavirus investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during . influenza other respir viruses lack of middle east respiratory syndrome coronavirus transmission from infected camels the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment we thank isam al jalii and khalid borsais for assistance with sample collection and marzooq m. al eknah for financial support.dr. khalafalla is professor of veterinary virology at king faisal university, al-ahsa, saudi arabia. his research focus is on viral diseases of dromedary camels. key: cord- - mzr s authors: kanazawa, nobuo title: c-type lectin receptors date: - - journal: immunology of the skin doi: . / - - - - _ sha: doc_id: cord_uid: mzr s c-type lectins, originally defined as proteins binding carbohydrates in a ca( +)-dependent manner, form a large family containing soluble and membrane-bound proteins. among them, those expressed on phagocytes and working as pathogen pattern-recognition receptors were designated as c-type lectin receptors (clrs), in accordance with toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i–like receptors (rlrs). most of the genes for clrs are clustered in human chromosome close to the natural killer gene complex. similar to the killer lectin-like receptors whose genes are clustered in this complex, most of the clrs induce activating or regulatory signal cascades in response to distinct pathogen- or self-derived components, through the immunoreceptor tyrosine-based activating or inhibitory motif, respectively. in this chapter, some representative clrs are picked up and their structural features leading to the functional consequences are discussed, especially on the signaling cascades and pathogen interactions, including some impacts on cutaneous pathophysiology. these clrs should provide targets to develop effective vaccination and therapeutics for distinct infectious and autoimmune/inflammatory diseases. sugar-binding capacity, whose amino acid sequence shows incomplete conservation of the common residues in the crd [ ] . it should be noted that the term ctld is also used for the broad definition containing all ctls irrespective of carbohydrate recognition [ ] . based on the domain structure, vertebrate ctls were divided into (i-vii) or, more recently, (i-xvii) groups including the traditional seven groups [ , ] . among them, group i (lecticans: aggrecan, brevican, versican, and neurocan), iii (collectins: mannose-binding protein and pulmonary surfactant proteins), and vii (reg proteins) are soluble, whereas group ii (asialoglycoprotein receptor family), iv (selectins), v (natural killer receptor family), and vi (mannose receptor family) are bound to the cell membrane ( fig. . ). the natural killer (nk) receptor family (group v) contains a series of type ii transmembrane proteins with a single ctld expressed on nk cells, whose genes are clustered in the nk gene complex (nkc) in chromosome [ ] . they are collectively called killer lectin-like receptors (klrs) and are equipped to regulate the killer activity of their expressing cells. among them, heterodimers of cd and activating/inhibitory nkg receptors, as well as the activating nkg d homodimer, recognize major histocompatibility complex (mhc) class i or related molecules, to distinguish target cells appropriately. the ctlds included in these receptors have no capacity to bind carbohydrate. phagocytes, such as macrophages and dendritic cells (dcs), also express various kinds of ctl receptors on their cell surface for antigen capture. the mannose receptor (mr) family (group vi) contains type i transmembrane proteins with multiple ctlds, such as the mr (cd ) and dec- (cd ) [ ] . they commonly consist of an n-terminal cysteine-rich domain and a fibronectin type ii domain as well as eight or ten ctlds. in contrast, the asialoglycoprotein receptor family (group ii) contains type ii transmembrane proteins with a single ctld, such as dc-specific icam -grabbing nonintegrin (dc-sign, cd ), dectin- , dectin- , dc immunoreceptor (dcir), and macrophage-inducible c-type lectin (mincle) [ , , , , ] . notably, expression of some receptors is specific and they can be markers for distinct dc subsets; langerin (cd ) on langerhans cells (lcs) and blood dc antigen (bdca)- (cd ) on plasmacytoid dcs (pdcs) [ , ] . most of the genes for these group ii receptors are also clustered in or next to the nkc ( fig. . ) [ ] . in contrast to klrs, most receptors of both groups contain fully conserved crds binding carbohydrates, and are considered to work as pattern recognition receptors (prrs) recognizing various pathogen-associated molecular patterns (pamps). especially, dectin- is critical for recognition of fungal β-glucan and has a collaborative effect with toll-like receptor (tlr) on yeast-induced activation signals, whereas dc-sign is involved in the recognition of a variety of microorganisms including viruses, bacteria, fungi, and parasites, and suppresses the immune activation signals induced by these pathogens [ , , ] . for these pathogen-recognizing ctl receptors, "clrs" have been designated analogous to other prrs, such as tlrs, nod-like receptors (nlrs), and rig-i-like receptors (rlrs) [ ] . notably, most of the clrs are linked to the signaling cascade through the immunoreceptor tyrosine-based activating or inhibitory motif (itam or itim, respectively), which was first identified in paired klrs [ , ] . [ , ] . in addition, some of them are also able to bind endogenous self-molecules and are involved in some pathophysiological aspects. for example, dc-sign have a role in cellular trafficking, dc-sign and dectin- can mediate an interaction between dc and t cells, and dectin- is involved in ultraviolet (uv)-induced tolerance [ , , , ] . furthermore, dectin- and dcir are involved in the development of autoimmunity in mice, and snps in clec a and clec e encoding dcir and mincle, respectively, are reportedly associated with autoimmune arthritis [ , , , , ] . in this chapter, some representative clrs are picked up and their structural features leading to the functional consequences are discussed, especially on the signaling cascades and pathogen interactions, including some impacts on cutaneous pathophysiology. regarding the nomenclature, genes for the clrs have formally been designated by the symbol "clec (c-type lectin domain containing)", which is approved by the human genome organization gene nomenclature committee (hgnc) (http://www.genenames.org/genefamilies/clec). langerhans cells (lcs) are immature dcs resident in the epidermis (see chap. ). as sentinels, they form a honeycomb-like network and spread dendrites to the skin surface through epidermal tight junctions. once they capture and internalize antigens, they modulate expression of adhesion molecules and chemokine receptors to migrate through dermal lymphatics into the draining lymph node and there, after maturation, they present processed antigens to the specific t cells. lcs have been defined electron-microscopically by the presence of birbeck granules (bgs) with a "tennis-racket"-like appearance, and immunohistochemically with bg-specific lag antibody [ , ] . langerin has been designated as the first surface lc marker recognized by the lc-specific monoclonal antibody (mab) dcgm , and subsequently isolated by the expression cloning strategy [ , ] . recent structural analysis has revealed that langerin stands as a stable homotrimer via the coiled-coil interaction of its neck region [ ] . langerin has no signaling motif other than the proline-rich domain for internalization in its intracellular domain. however, the presence of such an intracellular domain provides a possibility that langerin interacts with other proteins to modulate or mobilize cellular machanisms required for defense against pathogens, such as interfering viral tlr signaling [ ] . as predicted by the presence of epn (glutamate-proline-asparagine) motif in its crd, langerin can bind mannose, fucose, and n-acetylglucosamine structures and this binding leads to internalization and transfer of the antigen into bgs, which consist of superimposed and zippered plasma membrane [ , ] . langerin deficiency by the w r point mutation in humans abolished the bg formation without apparent immunodeficiency [ ] . notably, langerin expression is not specific in lcs but is also detected in a part of dermal dcs in mice [ , , ] . by analysis of langerin-dtr mice, in which langerin-expressing cells were transiently depleted by diphteria toxin administration, distinct ontogeny of langerin þ dermal dcs from migrating lcs have been revealed. langerin recognizes human immunodeficiency virus (hiv)- through highmannose structures in its envelope glycoprotein gp [ ] . in contrast to dermal/submucosal dcs, which transmit hiv- to t cells through dc-sign, epidermal/mucosal lcs clear hiv- and prevent its infection to t cells through langerin without inflammation. immunoelectron-microscopic analysis showed that hiv- was captured by langerin and internalized into bgs, to be finally degraded. therefore, langerin on epidermal lcs is considered a natural barrier to hiv- infection. as inhibition of langerin caused hiv- infection in lcs and its subsequent transmission to t cells, anti-hiv therapeutics should not interfere with langerin expression or functions. langerin also recognizes candida and malassezia species through cell wall mannose structures and β-glucans [ , ] . mycobacterium species are also recognized and the langerin binding to their components such as lipoarabinomannan (lam) leads to its internalization into bgs and efficient loading to cd a for presentation to t cells [ ] . therefore, bgs might be an organelle specialized to load glycolipid antigens to cd a. although dc-sign has been designated by its pivotal role on dc-t cell interaction, it is well-known as a prr for a variety of pathogens. indeed, it was first cloned from placenta as a hiv- gp -binding protein independent of cd [ ] . dc-sign stands as a tetramer and contains dileucine (ll) and yxxl internalization motif in its intracellular domain [ ] . its binding to gp was inhibited by mannan, d-mannose, and l-fucose and, after binding, gp was immediately internalized [ ] . dc-sign does not allow hiv- entry into dcs, but promotes its efficient infection in trans of cells expressing cd and chemokine receptors [ ] . dc-sign also functions for capturing dengue virus, a mosquito-mediated flavivirus that causes hemorrhagic fever, and is indispensable for its infection to dcs [ , ] . interestingly, the cytoplasmic tail of dc-sign is not essential for, but enhances dengue virus infection to dcs. dc-sign is also involved in infection of other viruses such as ebola, cytomegaloviruses, human c-type hepatitis virus (hcv), measles, and severe acute respiratory syndrome (sars) coronavirus, bacteria such as helicobacter pylori and klebsiela pneumonae, fungi such as candida albicans, and parasites such as leishmania pifanoi and schistosoma mansoni [ , ] . dc-sign further works for capturing and internalizing mycobacterium species (m. tuberculosis and bcg) through the mannose-capped mycobacterial cell wall lam (manlam) [ ] . in immature dcs, internalized mycobacteria or manlam are transported into lysosome, where they are colocalized with lamp . notably, targeting dc-sign with soluble manlam inhibits mycobacteria-or lipopolysaccharide (lps)-induced dc maturation and induces il- production, suggesting that dc-sign signaling interferes with the tlr signaling to cause an antiinflammatory effect. such an effect has been shown to be mediated by a serine/ threonine kinase raf signaling, which does not require the cytoplasmic yxxl motif of dc-sign but involves formation of a distinct protein complex (lsp , ksr, cnk, larg, and rhoa) with dc-sign [ ] . raf signaling does not induce dc activation but modulates tlr-mediated nuclear factor (nf)-κb activation, resulting in increased and prolonged transcription of il- and some other cytokines. by analysis of bronchoalveolar lavage cells, alveolar macrophages from tuberculosis patients specifically expressed dc-sign, and its expression was induced by m. tuberculosis independently of tlr , il- , and il- [ ] . indeed, accumulation of m. tuberculosis in dc-sign-expressing alveolar macrophages was immunohistochemically shown. by the case-control studies, a single nucleotide polymorphism (snp) in the promoter region of dc-sign, À c has reportedly been involved in a risk for parenteral hiv- infection in european americans [ ] . it has also been reported that À snp is associated with severity of dengue diseases, including severe dengue fever and dengue hemorrhagic fever [ ] . in tuberculosis, association of À snp has further been reported by analysis of a south african population living in the areas showing a high incidence rate of tuberculosis [ ] . À g and À a are associated with protection against tuberculosis, and their combination showed a higher frequency in non-african populations, possibly as a result of genetic adaptation to a longer history of exposure to tuberculosis. recently, it has been reported that the anti-inflammatory capacity of intravenous immunoglobulin (ig) is recapitulated in mice expressing human dc-sign, by transfer of bone-marrow-derived macrophages or dcs treated with the fc fragment of igg containing glycans terminating in sialic acids (igg-sfc) [ ] . treatment with igg-sfc induced il- production in macrophages/dcs and caused expansion of il- -producing basophils in their administered mice, which in turn increased expression of an inhibitory fc receptor, fcγriib, on effector macrophages. such a novel igg-sfc-induced dc-sign-mediated th /regulatory pathway might be an endogenous mechanism for immunological homeostasis and provide a line of evidence for therapeutic effects of intravenous ig administration on various inflammatory diseases such as kawasaki disease, autoimmune dermatomyositis/vasculitis/neuropathy, myasthenia gravis, pemphigus vulgaris, and severe drug eruptions [ ] . dectin- was originally reported as a dc-specific lectin that had been cloned from a cdna library of murine xs cells after subtraction of j macrophage library [ ] . at first, dectin- was considered a costimulatory molecule because soluble recombinant dectin- stimulated t-cell proliferation. however, subsequently, dectin- was identified as a β-glucan receptor on macrophages with a different binding site from that for t cells [ ] . dectin- is capable of binding a major yeast cell wall component zymosan and a variety of β-glucans from fungi and plants, however, it does not recognize carbohydrates with other linkages. although the clec a gene is located within the nkc, dectin- expression is not observed in nk cells, but in myeloid cells including monocytes, dc, macrophages, and neutrophils [ , ] . dectin- has a cooperative role with tlr on zymosan-induced macrophage activation [ ] . dectin- and tlr are colocalized on the surface of zymosantreated macrophages and the zymosan-induced activation signals require dectin- as well as tlr . notably, the yxxl hemi-itam (hemitam) in the cytoplasmic tail of dectin- is shown to be essential for this activation. upon zymosan-binding, this hemitam directly recruits and activates spleen tyrosine kinase (syk), probably dependent on the receptor dimerization, which induces nf-κb activation through interaction with caspase recruitment domain (card) nine coupled with the bcl -malt complex [ , ] . recently, protein kinase (pk) c-δ was identified to link syk activation and card signaling [ ] . this syk activation also induces il- β secretion, through pro-il- β transcription and processing, mediated by the card -bcl -malt and the malt -asc-caspase complex, respectively [ ] . the latter complex is called noncanonical caspase -dependent inflammasome. additionally, zymosan-induced dectin- -dependent signals reportedly include phospholipase (pl) cγ -dependent ca þ influx leading to activation of mitogenactivated protein kinases (mapks) as well as nf-κb [ ] . furthermore, nlrp inflammasome is activated through dectin- -syk signaling in mycobacterium abscessus (mabc)-activated human macrophages, whereas both tlr and dectin- are required for mabc-induced il- β secretion [ ] . a cytoplasmic scaffold protein p /sqstm mediates this dectin -syk-nlrp pathway, at least partly through production of reactive oxygen species (ros). finally, raf signaling, which regulates nf-κb activation independently of syk, is also involved in the dectin- activation, as observed in case of the dc-sign activation [ ] . among syk-induced cytokines, expressions of il- β, il- , il- , and il- are enhanced but the il- expression is impaired by raf signaling, and, therefore, th responses become dominant by dectin- activation. notably, although both soluble monomer and particulate polymer of β-glucan can bind dectin- , only the polymer can activate dectin- signaling by forming a so-called "phagocytic synapse," by not only clustering the receptor but also excluding inhibitory receptors with tyrosine phosphatase activities, cd and cd [ ] . interestingly, zap -deficient skg mice reportedly developed autoimmune arthritis only in the presence of dectin- agonist, indicating a role of dectin- signaling as a trigger for manifestation of subclinical autoimmune phenotype [ ] . furthermore, zymosan-mediated dectin- signaling induced il- production and exacerbated autoimmunity through th /th responses in tlr -deficient mice, whereas zymosan-mediated tlr signaling activated foxp þ regulatory t (treg) cells through induction of il- and raldh , an essential enzyme in retinoic acid metabolism [ ] . thus, antifungal dectin- signaling is considered rather aggressive and regulated by the simultaneous tlr signaling to prevent autoimmunity. notably, dectn- -deficient mice were shown to be impaired in zymosan recognition and increased in susceptibility to candida albicans infection, whereas, in another report, they were rather more susceptible to pneumocystis carini infection [ , ] . in , a nonsense mutation (y x) in the clec a gene was identified in a dutch family with chronic mucocutaneous candidiasis (omim# ) [ ] . the mutation was homozygous in three sisters with vulvovaginal candidiasis and/or onychomycosis and heterozygous in their parents with onychomycosis. the mutated dectin- , which lacks c-terminal amino acids, showed defective β-glucan binding and induction of cytokine productions after stimulation with β-glucan or candida albicans. however, fungal phagocytosis and killing were shown to be unaffected and, therefore, invasive fungal infection was not observed in the patients. the allele frequency of the y x variant is reportedly . in the healthy dutch population and identified carriers are all heterozygous, and the variant has never been found in the chinese population [ ] . in addition, the same variant was reported to be a risk factor of increased susceptibility to invasive aspergillosis in hematopoietic stem cell transplantation, with the highest risk in cases where the variant is present in both donors and recipients [ ] . dectin- was reported as another dc-specific lectin that had been isolated by subtraction cloning from murine xs cells, whereas its human homologue was originally cloned by the database search of human chromosomal sequences homologous to the exon sequences of murine dc immunoactivating receptor (dcar) [ , ] . dcar was identified as the putative activating partner of dc immunoreceptor (dcir), and the overall amino acid sequence of murine dcar is highly homologous ( % identity) to murine dectin- [ ] . although the gene for dectin- (clec a) is clustered with those for dcir (clec a) and murine dcar (clec b) and located next to the nkc, dectin- expression is observed in various myeloid cells including monocytes, macrophages, neutrophils, and dc subsets such as lcs [ ] . indeed, murine dcar is the first reported dc/macrophage clr that associates with an yxxlx - yxxl itam-bearing adaptor molecule, the γ chain of fcr (fcrγ), whereas the direct human orthologue of dcar is absent and the functional significance of this molecule is still unknown [ ] . similar to murine dcar, dectin- with a short cytoplasmic domain associates with fcrγ, a type i membrane protein with a short ectodomain that contains itam intracellularly and provides a docking site for syk [ ] . dectin- stimulation activates the card -bcl -malt pathway through pkcδ and induces various cytokines and chemokines mediated by nf-κb activation [ , ] . additionally, dectin- signaling activates a mapks pathway through plcγ , as is the case with dectin- [ ] . most importantly, through malt -mediated selective c-rel nf-κb subunit activation, dectin- , but not dectin- , signaling leads to th responses [ ] . as predicted by the presence of epn motif in its crd, dectin- binds structures with high mannose [ ] . actually, a number of pathogens including fungi such as candida albicans, saccharomyces cerevisiae, aspergilus fumigatus, cryptococcus neoformans, paracoccidioides brasiliensis, microsporum audouinii, and trichophyton rubrum, as well as parasites such as histoplasma capsulatum and schistosoma mansoni, and mycobacterium tuberculosis are recognized by dectin- [ ] . notably, the yeast and hyphal forms of candida albicans induce different responses through dectin- [ , ] . most impressively, it has been shown by analyses of dectin- -deficient mice that dectin- is essential for protective th response to intravenous infection with candida albicans [ ] . by stimulation of murine bone-marrow-derived dcs with soluble schistosomal egg antigens following pretreatment with tlr ligands, nlrp -dependent il- β secretion was increased through dectin- -fcrγ-syk signaling and ros production [ ] . furthermore, house dust mites are also recognized by dectin- and induce eicosanoid production, leading to the th responses [ , ] . interestingly, murine dectin- is reportedly involved in ultraviolet (uv)-induced immune regulation [ ] . although injection of soluble ectodomain of dectin- (sdec ) has no effect on induction of contact hypersensitivity (chs), it inhibits uv-mediated suppression of chs and tolerance induction. sdec -binding cd þ cd þ t cells were shown to produce il- , il- , and tgfβ upon hapten stimulation and to transfer the uv-induced tolerance. dcir was originally identified as a molecule homologous to hepatic asialoglycoprotein receptors and macrophage lectin, by selecting sequences in dc libraries following the database search of human genomic sequences using the tblastn algorithm [ ] . dcir is unique among known dc/macrophage clrs because of the presence of i/vxxyxxv/l immunoreceptor tyrosine-based inhibitory motif (itim) in its cytoplasmic portion. as the itim-related signaling capacity of dcir, sh domain-containing tyrosine phosphatase (shp)- and shp- can be recruited to human dcir upon stimulation [ , ] . furthermore, using murine b cells expressing a chimeric receptor containing a cytoplasmic portion of dcir and ectodomain of fcγriib (dcir-fcr), coligation of dcir-fcr and b cell receptor (bcr) showed dcir-itim-dependent inhibitory effects on bcr-mediated calcium influx and tyrosine phosphorylation of cellular proteins [ ] . on the other hand, murine dcar was identified as a molecule whose crd shows % amino acid identity with that of dcir [ ] . dcar interacts with fcrγ to stand efficiently on the cell surface and ligation of the chimeric dcar-fcr induced activation signals into its expressing cells dependent on the itam in fcrγ. thus, dcir and dcar are equipped as structurally and functionally paired immunoreceptors in mice, although the biological significance of their pairing has been undefined [ ] . among in vitro generated human dc subsets, a higher expression of dcir was observed in cd þ -derived dermal-type dcs than in cd a þ -derived lc-type dcs [ ] . in monocyte-derived dcs (modcs), its expression did not change during differentiation from monocytes to immature dc, but remarkably decreased after stimulation with lps or cd ligand. notably, dcir expression was detected not only in myeloid dcs, but also in monocytes, macrophages, b cells, neutrophils, and plasmacytoid dcs (pdcs) [ , ] . by reverse transcription (rt)-pcr, the only reliable method to distinguish expression of dcir and dcar, dcar was rather limitedly observed, strongly in spleen and lung and weakly in skin and lymph nodes, whereas dcir was detected ubiquitously. both increased during the bonemarrow-derived dc development, but increase of the dcar expression preceded that of dcir [ ] . notably, murine dc-specific mab d recognized dcir , which can be a specific marker for cd -dcs located in the red pulp and marginal zone of the spleen [ ] . despite the presence of an eps (glutamate-proline-serine residues) motif conserved in dcir and dcar, which is postulated to recognize galactose residues [ ] , their recognizing carbohydrates have not been clarified. notably, dcir reportedly participates in capturing hiv- and its transmission to cd þ t cells [ ] . hiv- captured by surface dcir is stored as intact virion and replicates de novo intracellularly in infected dcs and, subsequently, is transmitted to cd þ t cells through induced dcir expression on them, where hiv- replication is enhanced [ , ] . these processes have been shown to be mediated by the itim signaling. furthermore, dcir can act as an attachment factor for hcv on pdcs [ ] . notably, the binding of hcv to dcir on pdcs induced specific inhibition of tlr -induced ifnα production, in contrast to the case of dcir activation on dcs by anti-dcir monoclonal antibody (mab), which induced specific inhibition of tlr -induced il- and tnfα production [ , , ] . as predicted by the presence of a tyrosine-based internalization motif overlapping with the itim, targeting dcir on dcs with antigen-conjugated abs induced clathrin-dependent receptor internalization, its trafficking into lysosomal compartments, and efficient antigen cross-presentation to cd þ t cells [ , ] . in contrast, targeting murine dcir induced class ii presentation to cd þ t cells, including stimulation of foxp þ treg cells [ , ] . the most impressive results on in vivo significance of dcir have been obtained by analyses of dcir -deficient mice. they spontaneously developed sialadenitis and enthesisis and exacerbated collagen-induced arthritis with marked expansion of dcs [ ] . furthermore, in humans, by the case-control study, a snp in the clec a gene was shown to be associated with the susceptibility to anticyclic citrulinated peptide ab (acpa)-negative rheumatoid arthritis (ra) [ , ] . among bdcas, which were designated for a series of antigens recognized by mabs raised against human cd þ lineageblood dcs, bdca- is unique by its restricted expression on cd c À cd (il- receptor) bright pdcs in fresh human blood without expression on immature modcs or cd þ -derived dcs [ ] . although murine dectin- was once proposed as the murine homologue of human bdca- , its proper murine orthologue is still undefined and murine dcar, which lacks a human orthologue, is rather considered closely related to human bdca- on structural and functional aspects [ ] . in dc subpopulations, bdca- expression was detected strongly in pdcs and very weakly in immature modcs, and its expression level was downregulated after culture with il- in pdcs and after maturation with lps in modcs [ , , ] . in tonsil, bdca- expression was observed on cd þ pdcs that were primarily present in t-cell areas but not in the germinal center. similar to dcar and dectin- , bdca- associates with fcrγ and cross-linking of bdca- on pdcs results in intracellular ca þ mobilization depending on src-family protein tyrosine kinases and on tyrosine phosphorylation of cellular proteins [ , ] . however, unlike other fcrγ-coupled clrs, bdca- signaling does not lead to nf-κb activation through the syk-pkcδ-card pathway, but passes through the syk-blnk-plcγ pathway [ ] . indeed, contrary to its predicted immunoactivating property, ligation of bdca- suppressed the production of type i ifn and the secretion of tnf-related apoptosis-inducing ligand (trail) in pdcs induced by various tlr agonists [ , ] . furthermore, inhibition of type i ifn production enhances il- production in pdcs to polarize immune responses towards th [ ] . bdca- -mediated suppression of type i ifn production by cpg oligonucleotides or by influenza virus suggests that viruses target bdca- for immune escape. on the other hand, the same phenomenon induced by antidouble-stranded dna mab plus plasmid dna or by sera from patients of systemic lupus erythematosus (sle) suggests that targeting bdca- is beneficial for treatment of sle. as predicted by the presence of the triglutamate (eee) late endosomal sorting motif in the cytoplasmic portion of bdca- , it was reported that targeting bdca- with mab induced internalization of the ab complexes through phosphorylation of actin, tubulin, and clathrin, resulting in antigen presentation to cd þ t cells [ ] . however, more recently, bdca- signaling has rather been shown to inhibit antigen processing and presentation to t cells by pdcs, and, furthermore, to suppress the induction of costimulatory molecules on cpg-stimulated pdcs [ ] . although bdca- contains the epn motif in its crd similar to dectin- , it has recently been reported that bdca- recognizes asialo-galactosyl-oligosaccharides with terminal β-galactose residues and that pdcs bind to subsets of cd þ monocytes, modcs, and several tumor cell lines via recognition of asialo-galactosyloligosaccharides by bdca- [ ] . accordingly, these cells can introduce the suppression signal into their binding pdcs through bdca- , especially on type i ifn secretion. furthermore, bdca- can bind gp of hiv- and hcv glycoprotein e , both of which can also suppress the type i ifn secretion by pdcs [ , ] . mincle was originally identified from murine peritoneal macrophages by subtraction cloning as a molecule specifically induced by activation of nf-il transcription factor [ ] . mincle is expressed on monocytes, macrophages, neutrophils, myeloid dcs, and subsets of b cells, but not on t cells, pdcs, and nk cells [ ] . mincle associates with fcrγ dependently on the positively charged arginine residue in the transmembrane portion of mincle, as is the case with murine dcar but is not the case with dectin- [ ] . mincle-fcrγ activation induces signaling through syk-pkcδ-card -nf-κb and mapk pathways, resulting in the induction of various cytokines and chemokines. as predicted by the presence of the epn motif in its crd, mincle can bind carbohydrates containing α-mannose or other structures, and fungal, mycobacterial, and necrotic cell components have been identified as ligands for mincle [ , , ] . the candida albicans cell wall component was one of the first identified ligands and mincle was shown to mediate the protective antifungal immunity [ ] . malassezia is also recognized by mincle and induces cytokine and chemokine production and inflammatory cell recruitment through mincle [ ] . mincle also recognizes fonsecaea pedrosoi, which was revealed to cause chromomycosis in the skin due to insufficient costimulation of tlr on mincle signaling [ ] . mincle also recognizes mycobacterial trehalose dimycolate (tdm), which is an immunomodulatory glycolipid component and has been called mycobacterial cord factor, and mediates tdm or synthetic trehalose dibehenate-induced inflammatory responses including production of cytokines and nitric oxide, nlrp inflammasome activation, th and th activation, and granuloma formation [ , , , , ] . however, in mincle-deficient mice, variable inflammatory responses were observed dependent on the species of infected mycobacteria, probably because they contained different combinations of pamps [ , ] . interestingly, mycobacteriainduced granuloma formation was unaffected irrespective of mycobacteria species. also interesting is that the significance of mincle expression was reportedly different in monocytes and neutrophils. mincle expression was associated with cytokine production in monocytes, whereas it was rather associated with fungal uptake and killing in neutrophils [ ] . furthermore, the expression of mincle was reciprocally detected between monocytes and neutrophils in individuals. mincle also works as a sensor for necrosis by recognizing spliceosomeassociated protein (sap) released from necrotic cells [ ] . notably, mincle has been shown to use a different binding site for sap from that used for carbohydrate recognition. although it is predictable that mincle is involved in the regulation of autoimmunity, an autoimmune phenotype of mincle-deficient mice has never been reported. in contrast, it has recently been reported that a snp in the clec e gene is associated with acpa-positive ra [ ] . as described in this chapter, critical roles of the representative clrs have been revealed in the responses for viral, fungal, and mycobacterial infections, as well as in the maintenance of immunological homeostasis (table . ). as a surface barrier to environmental stimuli including invasive pathogens, skin provides the first battlefield and dermatologists should prepare more and better ways to combat pathogens and keep homeostasis. clrs should certainly provide targets to develop effective vaccination and therapeutics for distinct infectious and autoimmune/ inflammatory diseases. intravenous gammaglobulin suppresses inflammation through a novel th pathway involvement of dectin- in ultraviolet radiation-induced tolerance molecular and genomic characterization of human dlec, a novel member of the c-type lectin receptor gene family preferentially expressed on monocyte-derived dendritic cells cloning of a second dendritic cell-associated c-type lectin (dectin- ) and its alternatively spliced isoforms identification of a novel, dendritic cellassociated molecule, dectin- , by subtractive cdna cloning promoter variation in the dc-signencoding cd is associated with tuberculosis dectin- recognition of house dust mite triggers cysteinyl leukotriene generation by dendritic cells dectin- mediates th immunity through the generation of cysteinyl leukotrienes apcs express dcir, a novel c-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif role of mincle in alveolar macrophagedependent innate immunity against mycobacterial infections in mice card mediates dectin- -induced iκbα kinase ubiquitination leading to activation of nf-κb in response to stimulation by the hyphal form of candida albicans electron microscopy of melanocytes a new receptor for beta-glucans identification of a novel population of langerin þ dendritic cells bdca /fc epsilon ri gamma complex signals through a novel bcr-like pathway in human plasmacytoid dendritic cells dectin- y x polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient-and donor-dependent mechanism of antifungal immunity sequence and expression of a membraneassociated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp c-type lectin langerin is a beta-glucan receptor on human langerhans cells that recognizes opportunistic and pathogenic fungi langerin is a natural barrier to hiv- transmission by langerhans cells two distinct classes of carbohydrate-recognition domains in animal lectins evolution of ca þ -dependent animal lectins drickamer k ( ) c-type lectin-like domains differential antigen processing by dendritic cell subsets in vivo bdca- , bdca- , and bdca- : three markers for distinct subsets of dendritic cells in human peripheral blood bdca- , a novel plasmacytoid dendritic cellspecific type ii c-type lectin, mediates antigen capture and is a potent inhibitor of interferon α/β induction plasmacytoid dendritic cells: from specific surface markers to specific cellular functions the mannose receptor family trimeric structure of langerin human dectin- deficiency and mucocutaneous fungal infections hcv glycoprotein e is a novel bdca- ligand and acts as in inhibitor of ifn production by plasmacytoid dendritic cells dcir deficiency causes development of autoimmune diseases in mice due to excess expansion of dendritic cells collaborative induction of inflammatory responses by dectin- and toll-like receptor identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells dc-sign-icam- interaction mediates dendritic cell trafficking mycobacteria target dc-sign to suppress dendritic cell function sef-and nonself-recognition by c-type lectins on dendritic cells immunoreceptor tyrosine-based inhibition motif-bearing receptors regulate the immunoreceptor tyrosine-based activation motif-induced activation of immune competent cells blood-derived dermal langerin þ dendritic cells survey the skin in the steady state activation of the innate immune receptor dectin- upon formation of a 'phagocytic synapse' phospholipase cγ (plcγ ) is key component in dectin- signaling pathway, mediating anti-fungal innate immune responses c-type lectin dc-sign modulates tolllike receptor signaling via raf- kinase-dependent acetylation of transcription factor nf-kappab dectin- directs t helper cell differentiation by controlling noncanonical nf-κb activation through raf- and syk selective c-rel activation via malt controls anti-fungal t(h)- immunity by dectin- and dectin- dectin- is an extracellular pathogen sensor for the induction and processing of il- -beta via a noncanonical caspase- -inflammasome card controls a non-tlr signaling pathway for innate anti-fungal immunity a replication study confirms the association of dendritic cell immunoreceptor (dcir) polymorphisms with acpa-negative ra in a large asian cohort cloning and characterization of a novel itim containing lectin-like immunoreceptor llir and its two transmembrane region deletion variants langerhans cells utilizes cd a and langerin to efficiently present nonpeptide antigens to t cells direct recognition of the mycobacterial glycolipid, treharose dimycolate, by c-type lectin mincle bdca- signaling inhibits tlr -agonistinduced plasmacytoid dendritic cell activation and antigen presentation dcir acts as an inhibitory receptor depending on its immunoreceptor tyrosine-based inhibitory motif dendritic cell immunoactivating receptor, a novel c-type lectin immunoreceptor, acts as an activating receptor through association with fc receptor γ chain molecular cloning of human dectin- dendritic cell immunoreceptors: c-type lectin receptors for pattern recognition and signaling on antigen-presenting cells letterer-siwe disease: immunopathologic study with a new monoclonal antibody the dectin- family of c-type lectin-like receptors: an update animal lectins: a historical introduction and overview cross-priming cd þ t cells by targeting antigens to human dendritic cells through dcir the c-type lectin surface receptor dcir acts as a new attachment factor for hiv- in dendritic cells and contributes to trans-and cis-infection pathways hiv- induces dcir expression in cd þ t cells dcir-mediated enhancement of hiv- infection requires the itim-associated signal transduction pathway mycobacterium abscessus activates the nlrp inflammasome via dectin- -syk and p /sqstm neutrophils promote mycobacterial treharose dimycolate-induced lung inflammation via the mincle pathway association of arthritis with a gene complex encoding c-type lectin-like receptors toll-like receptor -dependent induction of vitamin a-metabolizing enzymes in dendritic cells promotes t regulatory responses and inhibits autoimmunity hiv- go inhibits tlr -mediated activation and ifn-α secretion in plasmacytoid dendritic cells association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection a novel lps-inducible c-type lectin is a transcriptional target of nf-il in macrophages the carbohydrate-recognition domain of dectin- is a c-type lectin with specificity for high mannose targeting dcir on human plasmacytoid dendritic cells results in antigen presentation and inhibits ifn-alpha production dcir is endocytosed into human dendritic cells and inhibits tlr -mediated cytokine production dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infectionof human dendritic cells by mosquito-cell-derived dengue viruses myeloid c-type lectin receptors in pathogen recognition and host defense the dermis contains langerin þ dendritic cells that develop and function independently of epidermal langerhans cells engagement of bdca- blocks trailmediated cytotoxic activity of plasmacytoid dendritic cells human c-type lectin domain family , member c (clec c/bdca- /cd ) is a receptor for asialo-galactosyl-oligosaccharides granulocyte macrophage-colony stimulating factor reduces the affinity of shp- for the itim of clecsf in neutrophils: a new mechanism of action for shp- schistosoma mansoni triggers dectin- , which activates the nlrp inflammasome and alters adaptive immune responses dectin- is a syk-coupled pattern recognition receptor crucial for th responses to fungal infection bdca- ) signals in plasmacytoid dendritic cells via a bcr-like signalosome involving syk, slp and plcγ syk-dependent cytokine induction by dectin- reveals a novel pattern recognition pathway for c type lectins dectin- is required for host defense against pneumocystis carinii but not against candida albicans dectin- recognition of alpha-mannans and induction of th cell differentiation is essential for host defense against candida albicans dectin- is a pattern recognition receptor for fungi that couples with the fc receptor gamma chain to induce innate immune responses a variant in the cd promoter is associated with severity of dengue disease mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog treharose-dibehenate intravenous immunoglobulin therapy: how does igg modulate the immune system? the mycobacterial cord factor adjuvant analogue treharose- , -dibehenate (tdb) activates the nlrp inflammasome dc-sign; a related gene, dc-signr; and cd form a cluster on p restoration of pattern recognition receptor costimulation to treat chromoblastomycosis, a chronic fungal infection of the skin characterization of carbohydrate recognition by langerin, a c-type lectin of langerhans cells syk kinase-coupled c-type lectin receptors engage protein kinase c-δ to elicit card adaptor-mediated innate immunity dectin- is required for beta-glucan recognition and control of fungal infection dc-sign (cd ) mediates dengue virus infection of human dendritic cells dual specificity of langerin to sulfated and mannosylated glycans via a single c-type carbohydrate recognition domain dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells the monoclonal antibody dcgm recognizes langerin, a protein specific of langerhans cells, and is rapidly internalized from the cell surface langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules langerin functions as an antiviral receptor on langerhans cells dc-sign: escape mechanism for pathogens conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts a lack of birbeck granules in langerhans cells is associated with a naturally occurring point mutation in the human langerin gene mincle polarizes human monocyte and neutrophi responses to candida albicans drickamer k ( ) the c-type lectin superfamily in the immune system the macrophage-inducible c-type lectin, mincle, is an essential component of the innate immune response to candida albicans adjuvanticity of a synthetic cord factor analogue for subunit mycobacterium tuberculosis vaccination requires fcrgamma-syk-card -dependent innate immune activation macrophage-inducible c-type lectin is associated with anti-cyclic citrullinated peptide antibodies-positive rheumatoid arthritis in men phospholipase cγ is critical for dectin- -mediated ca þ flux and cytokine production in dendritic cells mincle is an itam-coupled activating receptor that senses damaged cells c-type lectin mincle is an activating receptor for pathogenic fungus cd þ cd þ splenic dendritic cells are specialized to induce foxp þ regulatory t cells immune functions encoded by the natural killer gene complex identification of a human homologue of the dendritic cell-associated c-type lectin- , dectin- a role for fungal beta-glucans and their receptor dectin- in the induction of autoimmune arthritis in genetically susceptible mice the c-type lectin-like domain superfamily key: cord- -vaygaqe authors: cheng, ming soon; lau, suk hiang; chan, kwai peng; toh, chee-seng; chow, vincent t. title: impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses date: - - journal: biosens bioelectron doi: . /j.bios. . . sha: doc_id: cord_uid: vaygaqe we describe an impedimetric cell-based biosensor constructed from poly-l-lysine (pll)-modified screen-printed carbon electrode for real-time monitoring of dengue virus (denv) infection of surface-immobilized baby hamster kidney (bhk- ) fibroblast cells. cytopathic effects (cpe) induced by denv- new guinea c strain (including degenerative morphological changes, detachment, membrane degradation and death of host cells), were reflected by drastic decrease in impedance signal response detected as early as ~ hours post-infection (hpi). in contrast, distinct cpe by conventional microscopy was evident only at ~ hpi at the corresponding multiplicity of infection (moi) of . a parameter that describes the kinetics of cytopathogenesis, cit( ), which refers to the time taken for % reduction in impedance signal response, revealed an inverse linear relationship with virus titer and moi. cit( ) values were also delayed by . h for each order of magnitude decrease in moi. therefore, based on the analysis of cit( ), the virus titer of a given sample can be determined from the measured impedance signal response. furthermore, consistent impedance results were also obtained with clinical isolates of the four denv serotypes verified by rt-pcr and cycle sequencing. this impedimetric cell-based biosensor represents a label-free and continuous approach for the dynamic measurement of cellular responses toward denv infection, and for detecting the presence of infectious viral particles. in recent decades, outbreaks of emerging and re-emerging diseases caused by infectious viruses such as dengue virus (denv), ebola virus, influenza virus, severe acute respiratory syndrome coronavirus and west nile virus pose ongoing threats to global biosecurity. in confronting these infectious diseases that spread well beyond the initial affected regions, their surveillance and control often create serious challenges for public health organizations. hence, the development of rapid, effective and safe virus detection tools has become a major priority of the global community. current clinical laboratory virus detection tests based on real-time and multiplex pcr techniques, provide a promising platform for simultaneous nucleic acid amplification and detection of multiple target sequences in a single test (caliendo, ; gullett and nolte, ) . however, these molecular techniques are incapable of identifying live infectious viral particles since they detect the nucleic acids originating from both infectious and noninfectious viruses. virus samples usually comprise high viral particle-to-plaque-forming unit or pfu ratio , which may indicate that the minority of viruses in a given sample is infectious, or the presence of non-infectious viruses with mutated or damaged genomes, or failure of most of the viruses to successfully infect due to the complexity of the infection cycle (van der schaar et al., ) . proof of infectious viral particles is highly important and can only be accomplished by conventional cell culture assays, which is time-consuming and labor-intensive. clearly, it is essential to develop an effective and simple virus detection tool for the assessment of virulence and identification of infectious viral particles to aid in the control of an epidemic. the electrochemical impedance spectroscopic (eis) technique has recently gained popularity in cell-based assays in view of advantages such as high sensitivity, non-invasive measurement, accessibility of time-dependent and quantitative data. cell-based assays using adherent mammalian cell cultures such as fibroblast and endothelial cells have been utilized in toxicological and virological studies (peters et al., ; hofmann et al., ) . since the first reported application of eis in cell culture study (giaever contents and keese, ) , this technique has been widely employed for in vitro monitoring of dynamic responses of cells toward drugs, toxic agents and pathogens (kiilerich-pedersen et al., ; asphahani et al., ; xu et al., ) . principally, the eis cell-based analytical system involves quantitative monitoring of the spreading, morphology and viability of adhered cell cultures in real-time, by applying a constant alternating current (ac) electric field. due to the low conductivity of cellular membrane, formation of a confluent cell monolayer on the electrode surface often constricts current flow. the corresponding change in impedance signal response can be continuously monitored to obtain information on cellular growth or coverage on the electrode surface (cheung et al., ) . ultrasensitive impedimetric methods have recently reported non-invasive cell-based analyses of viral infections including herpes simplex virus infection of vero cells (cho et al., ) , human cytomegalovirus infection of human fibroblasts (kiilerich-pedersen et al., ) , and bluetongue virus infection of bovine endothelial cells (drew et al., ) . the direct, real-time investigation of denv-induced cytopathogenesis in mammalian fibroblast cells using the eis cell-based analytical system is first reported in this study. mosquito-borne dengue infections are caused by single-stranded rna-containing denv which is transmitted by aedes mosquitoes. denv exists as four antigenically distinct serotypes designated denv- , - , - and - (teles, ) . among the serotypes, secondary infection with denv- has proven to be responsible for the more severe symptoms of dengue fever (vaughn et al., ) . invasion of host cells by lytic viruses such as denv will ultimately result in degenerative changes and damage to host cells, known as cytopathic or cytopathogenic effects (cpe). such effects are generally characterized by degenerative morphological changes, detachment, membrane degradation and eventual death of cells. in general, decreased tight junctions between cells, and increased separation between cells and electrode arising from cpe may lead to decreased impedance signal response. in comparison to conventional inspection methods such as microscopy and plaque assay that rely on observable changes in the morphology and surface coverage of cells, the eis technique offers a more promising platform for studying virus-host interactions based on the real-time measurement of cpe-induced impedance response (cho et al., ) . here we report an impedimetric cell-based biosensor constructed from poly-l-lysine (pll)-modified screen-printed carbon electrode (spce) for real-time monitoring of denv infection of surface-immobilized baby hamster kidney (bhk- ) fibroblast cells. this in vitro method in which the cell-based biosensor is immersed in the culture medium, detects viral-induced cpe by gradual decrease in the impedance response. the sensitivity of the biosensing system is enhanced using spce, which constricts the current to flow within a small area of working electrode (campbell et al., ; alonso-lomillo et al., ) . furthermore, adherence of a cationic polymeric film (pll) onto spce significantly improves the attachment and spreading of cells (mazia et al., ; sanders et al., ; frey and corn, ; carrier and pézolet, ; khademhosseini et al., ; sitterley, ) . the excellent performance in terms of rapid detection time, automated analysis, high sensitivity and capability of indirectly identifying infectious viral particles are some desirable features of this impedimetric cellbased approach in real-time biosensing. spce, carbon working electrode, silver pseudo-reference electrode, and platinum auxiliary electrode on ceramic substrate were purchased from dropsens. pll (mw , - , at . % w/v) and potassium hexacyanoferrate (iii) (k fe(cn) , $ %) were purchased from sigma-aldrich. fetal bovine serum (fbs), and dulbecco's phosphate-buffered saline (dpbs) without calcium and magnesium were obtained from biowest. trypsin-edta (  ) and penicillin-streptomycin (  ) were obtained from paa laboratories. rpmi- medium was purchased from thermo scientific hyclone. avicel rc- ( . % solid) was purchased from fmc biopolymer. μl of . % (w/v) sterile-filtered aqueous solution of pll was applied onto the carbon electrode surface (with an area of . mm ) and the tip of pipette was used to spread the solution evenly over the entire electrode surface. the electrode was inverted and placed in a drying oven at °c for at least min to ensure the transformation of pll film into the stable β-sheet configuration, and adherence of pll (at a thickness of . nm) to the electrode surface (jordan et al., ) . excess pll solution was aspirated, and the electrode surface was thoroughly rinsed with sterile ultrapure water. the pll-modified spce was characterized using cyclic voltammetry (cv) in the presence of . mm fe(cn) À / À in  pbs, ph . at a scan rate of mv s À and potential range from À . to . v. bhk- cells (american type culture collection) were cultured in a t tissue culture flask containing ml of growth medium (rpmi- medium supplemented with % fbs and  penicillin-streptomycin solution). cells were grown in a humidified incubator at °c with % co . when the cells were - % confluent (after - days), growth medium was removed from the flask. cells were washed once with ml of  dpbs, ml of  trypsin-edta was added, and incubated at °c with % co for min until cells detached. the detached cells were flushed, trypsin-edta was removed, the cells were subsequently resuspended in ml of growth medium, and centrifuged at rpm for min. the supernatant was discarded, and the cell pellet was resuspended in ml of growth medium. bhk- cells were propagated at a sub-cultivation ratio of : . denv- new guinea c (ngc) strain and four clinical isolates each of denv serotypes , , and were propagated in bhk- cells supplemented with maintenance medium (rpmi- supplemented with % fbs,  penicillin-streptomycin). virus at multiplicity of infection (moi) of . was inoculated, and the tissue culture infectious fluid (tcif) was harvested - days upon the observation of cpe. moi represents the ratio of the number of infectious agents (viruses) to the number of infection targets (cells) adsorbed on the electrode surface. uninfected cells served as the control. tcif was subsequently centrifuged at rpm for min. the supernatant containing viruses was collected and diluted with rpmi- to achieve the desired moi for viral infection monitoring experiments. plaque assay using avicel ( . %) and crystal violet staining was performed to determine the virus titer expressed as pfu ml À . the experimental set-up consists of a chi d potentiostat-galvanostat (ch instruments) with data acquisition software, and a sterilized polypropylene vessel integrated with a pll-modified spce ( fig. ). total cell count was ascertained using tc automated cell counter (bio-rad). cell suspension with viable cell count of % was harvested to obtain a cell concentration of  cells ml À . bhk- cells (in μl) were seeded on the pll-coated carbon electrode surface, and allowed to attach and spread on the electrode surface for approximately h. thereafter, ml of growth medium was gently added to the vessel. cells were allowed to proliferate in an incubator at °c with % co for h. subsequently, μl of denv- stock solution was added and mixed gently with cell medium. bhk- cells infected with denv- at different moi were incubated at °c and % co for another days. proper sterilization of all apparatus and preparatory work were carried out in an antiseptic environment. the impedimetric properties of cell monolayers before and after viral infection were monitored at °c using the eis technique. electrochemical impedance spectra were recorded at open circuit potential, frequency of khz, and excitation amplitude of mv. . . rna isolation, reverse transcription (rt), polymerase chain reaction (pcr), and analysis of rt-pcr products of clinical isolates of dengue viruses the qiagen rneasy kit was employed to isolate total cytoplasmic rna from tcif collected from each denv serotype (at moi of . ) propagated in bhk- cells cultured with rpmi- medium supplemented with % fbs, and incubated at °c with % co for days. synthesis of cdna (rt) was carried out using ng of rna, u of moloney murine leukemia virus (m-mlv) reverse transcriptase (promega), u of rnase inhibitor, mm of dntps, and . mm each of four downstream primers dsp , , , and , and subjected to °c for min. pcr was carried out using ng of each cdna and . mm each of primers, dsp , , , , and dv . the pcr cycles consisted of an initial denaturation at °c for min, followed by cycles each of denaturation at °c for s, annealing at °c for s, extension at °c for s, and a final extension at °c for min (seah et al., a) . pcr products ( ml each) were electrophoresed for h in % agarose gels (in  tbe) containing sybr safe dna gel stain (life technologies). images were obtained using the chemidoc mp system (bio-rad). the specific pcr products corresponding to denv serotypes , , , and were excised and subjected to cycle dna sequencing using bigdye terminator cycle sequencing kit v . and xl genetic analyzer (applied biosystems). initially, we added cells to the bare spce and observed that the impedance signals were negligible and fluctuating between experiments compared with the baseline signals from the bare spce without cells added. the former signals remained unintelligible even when cells on the bare spce were infected with denv (data not shown). these findings alluded to poor attachment of cells to the bare spce, which led us to modify the electrode by coating it with pll to facilitate cell attachment. the carbon working electrode surface of spce was modified with a cationic polymeric film (pll) using a similar method described previously (anson et al., ) . the configuration of pll in solution changes from random coil to α-helix at about ph . subsequent heating to °c promotes the transformation of the pll film into the β-sheet configuration, which is believed to comprise chains that are partially cross-linked by hydrogen bonding. the cyclic voltammograms of the pll-modified spce obtained in the presence of . mm fe(cn) À / À in  pbs, ph . ( fig. (a) ) indicated fairly constant cv peak current responses for repetitive potential cycles with rsd values of . % and . % for anodic and cathodic peak currents, respectively. this excellent stability of current response reflected strong adherence of the pll film to the carbon working electrode surface. in comparison to other electrode surfaces such as platinum and glassy carbon, pll has been shown to be more resilient and strongly adhered onto the rough surface of screen-printed carbon (fogg et al., ) . the contrasting behavior of a bare spce and a spce modified with . % (w/v) pll toward the redox couple fe(cn) À / À is shown in fig. (b) . the cyclic voltammograms of the pll-modified spce exhibited a more reversible current response. coating of electrode surface with a layer of cationic polymer pll reduces the electrode overpotential and facilitates the redox reaction of anionic redox probes such as fe(cn) À / À at the electrode surface. this enhanced anionic exchange capability of the cationic polymer-modified electrode is indicated by a . % increase in cv peak current response compared to a bare electrode (fig. (b) ). electrode surface coating methods such as gelatin coating (campbell et al., ) and equilibration with culture medium (mccoy and wang, ; muller et al., ) are commonly employed to promote the attachment and spreading of cells on the electrode surface. despite their good biocompatibility, the utility of these methods is limited by the instability, weak adherence to the electrode surface, and poor cell capture capability. in this study, the positively charged pll coating was adopted owing to its excellent cell capture capability and strong adherence to carbon electrode surface. various studies have documented that adherent cell lines such as fibroblast and endothelial cells possess an overall negative surface charge, which facilitates their adhesion to positively charged surface (somosy et al., ; singh et al., ; hamdan et al., ) . this negative surface charge may be attributed to the carboxylic groups of aspartic and glutamic acid (pka ¼ . - . ) of proteins, carboxylic groups of sialic acid (pka ¼ . ) of glycoproteins, phosphate groups (pka¼ . ), and sulfate groups (pka¼ . ) of sulphated proteoglycans or glycoproteins (singh et al., ) . another explanation is that their extracellular matrix is rich in negatively charged glucosaminoglycans (somosy et al., ) , thus suggesting that the observed cell adhesion behavior is mainly electrostatic in nature. the enhanced cationic properties of pll-modified carbon electrode are therefore responsible for the strong adhesion of bhk- cells. in this study, the virus-host interactions between denv and bhk- cells were investigated by the eis technique. bhk- cells constitute a type of adherent fibroblast cells, derived by single-cell isolation from the kidneys of five syrian golden hamsters, mesocricetus auratus (stoker and macpherson, ) . this cell line is susceptible to infection by denv, vesicular stomatitis virus, reovirus, and human adenovirus. the bode diagram shown in fig. (a) was generated from eis spectra recorded at a frequency ranging from hz to mhz. in the absence of electron mediator or redox probe, the electrode impedance of cells is limited by stray capacitance at high frequency and electrode polarization at low frequency. moderate frequency ( - khz) is well-suited for probing the cellular morphological changes induced by viral infection or cytotoxicants (mccoy and wang, ; campbell et al., ; cho et al., ) . the intermediate frequency at which the impedance signal was most affected by the change in cellular morphology was $ khz ( fig. (a) ), where the impedance signals measured between cells prior to virus inoculation and during day of viral infection (at moi¼ ) were considerably different from each other. thus, the eis data recorded at khz appear to provide meaningful results which may correlate with the morphological dynamics of cells and the kinetics of denv-induced cytopathogenesis. this finding was further extended to the infection monitoring experiments where the impedance readings of cell adhesion and subsequent denv infection were monitored at khz every min up to h. due to the variation of the coating of cells on the electrode surface, the impedance signals of four different cell-based biosensors at hours post-infection (hpi) varied between . Ω and . Ω with a rsd of . %. normalization of the impedance signal response in the presence of virus (z virus ) against the response derived from the same biosensor in the absence of virus at hpi (z virus ¼ ) yielded a relatively low rsd of . %. normalization of the impedance signal against the blank signal enhances the reproducibility, precision of results, and avoids the need to calibrate each individual biosensor. the denv infection process involves several stages: attachment of viral envelope glycoprotein to cellular receptors such as dendritic cell-specific icam- grabbing nonintegrin (dc-sign), heparin sulfate or cell surface immunoglobulin (chen et al., ; tassaneetrithep et al., ) . after fusion with the plasma membrane, viruses are enveloped in an acidified endocytic vesicle known as endosome, and subsequently release rna into cytoplasm. virus replication would eventually trigger membrane degradation and release viral progeny into the extracellular environment. in general, viral invasion of host cells induces a series of intra-and inter-cellular remodeling processes, which ultimately lead to changes in tight junctions between cells, and the distance between cells and electrode. fig. (b) shows the normalized impedance signal response as a function of viral infection time at different moi. this plot reveals information on the attachment and growth of cells on the electrode surface, morphological dynamics of cells, and kinetics of viral-induced cytopathogenesis. due to the low conductivity of cell membranes, the flow of moderate frequency current at the cellelectrode or cell-cell interface is sensitively influenced by nanometer changes in the adhesion regions or tight junctions between cells (cheung et al., ) . thus, a gradual increase in the electrode impedance observed during cell culture indicates that cells are actively propagating and densely populating onto the pll-modified carbon electrode surface. impedance signal keeps on increasing after virus inoculation as cells continue to grow until cytopathogenesis induces a series of cellular remodeling processes that influence the morphology and adhesion of cells on the electrode surface. viral-induced cytopathogenesis often results in severe morphological and physical changes, detachment of cells from surface, and eventual cell death (campbell et al., ) . in fig. (b) , fluctuation of the normalized impedance signal signifies the onset of cpe induced by denv- ngc strain, followed by a drastic reduction in impedance signal response, which could be detected as early as $ hpi at moi of . moreover, after the observed cpe, the normalized impedance signal of the cell-based biosensor declined by % at $ hpi. technically, this particular time taken for % reduction in the cell impedance response is termed cit , a kinetic parameter typically used to characterize the viral-induced cytopathic events (fang et al., ) . to further characterize the viral-induced cytopathogenesis, cit was regressed as a function of virus titer in the logarithm of moi. fig. (c) displays an inverse linear relationship between virus titers and cit values (r ¼ . ). the resultant regression indicates that cit was delayed by . h for each order of magnitude decrease in moi. therefore, the dependence of the cpe-induced impedance signal response on the viral infectious dose permits the estimation of virus titers of viral samples based on the measured impedance signals. several control experiments were carried out to assess the performance of impedimetric cell-based biosensor for the investigation of virus-host interactions. a control experiment using bhk- cells in the absence of denv (represented by the black line in fig. (b) produced a gradual increment in impedance signal with time. clearly, bhk- cells cultured in growth medium remain viable and actively propagate on the electrode surface. the normalized impedance signal reaches its maximum after $ days of incubation with growth medium where the electrode surface is fully covered with cells. another control experiment using a pllmodified spce immersed in growth medium in the absence of bhk- cells and denv gave a fairly constant impedance signal over a period of days (data not shown). fig. shows the control experiments using different host cells infected with denv- ngc strain at moi of . in the control using biosensor prepared with vero cells, a commonly used mammalian cell line, the electrode impedance increased initially as the vero cells grew and reached a plateau from days to . another control using the c / aedes albopictus mosquito cell line produced a fairly constant impedance signal from days to , thus revealing poor attachment and spreading of c / cells on the pll-modified carbon electrode surface. in summary, denv-induced cpe could not be observed with vero and c / cell lines even at a relatively high moi of . this suggests that the impedance response is specific to bhk- cells and may not relevant to other cell lines. in summary, decrease in impedance signal could only be detected with the occurrence of denv-induced cytopathogenesis of the bhk- fibroblast cell line. for comparison, denv- ngc-infected bhk- cells seeded in a pll-coated culture flask were observed for cpe using an evos fl digital fluorescence microscope (life technologies). microscopic images were taken using a sony high-sensitivity monochromatic ccd camera, with contrast and brightness adjusted to improve the quality of images. uninfected bhk- cells served as control. microscopic imaging of bhk- cells after days of infection with denv- ngc strain at moi of displayed typical cpe including considerable changes in cellular morphology. detachment of cells from the surface was indicated by the presence of many empty spaces and rounded cells instead of the original completely confluent monolayer of spindle-shaped cells as depicted in fig. (a) . on the other hand, the monolayer of densely populated uninfected bhk- cells remained virtually intact after days of incubation with growth medium (fig. (b) ). to study the kinetics of cpe of bhk- cells infected with denv- ngc at moi of and , monitoring by conventional microscopy was performed at , , , , , , , , , , , , , , and hpi. bhk- cells mock-infected with heat-inactivated denv- ngc at "moi" of , and bhk- cells treated with denv- ngc viral rna served as additional controls. as expected, no cpe was observed for both negative controls up to hpi. however, the earliest distinct microscopic evidence of cpe was observed at and hpi for moi of and , respectively (supp. fig.) . in direct comparison to the optical method, the more sensitive cell biosensor could "sense" cpe much earlier as reflected by the drastic reduction in impedance signal response at $ and $ hpi for moi of and , respectively. the potential application of cell-based biosensor in analyzing clinical isolates was examined against four different dengue serotypes, denv- , - , - , and - . these viruses were isolated from the respective denv-infected patients. the four clinical denv isolates were verified to be denv- , - , - , and - strains by rt-pcr and cycle sequencing of amplified products (seah et al., a) , and their sequences deposited in the genbank database under accession numbers km , km , kp . fig. (a) shows agarose gel electrophoresis of the rt-pcr products of denv- , - , - , and - . their nucleotide sequences exhibit high identities to isolates rr ( %), dn/ ( %), tb ( %), and a ( %), respectively. infection of bhk- cells with the four clinical isolates of denv was monitored by using the procedures described for the ngc strain. fig. (b) displays similar cpeinduced impedance signals for the four clinical isolates (at moi of ). slight variation in cpe detection time may possibly be attributed to the varying degrees of cpe induced by these clinical denv strains. in summary, cpe induced by the four clinical isolates of denv could be detected within - days post-infection. the performance of this impedimetric cell-based biosensor underscores its utility for the analysis of clinical denv isolates. future studies are warranted to evaluate this biosensor for the analysis of actual clinical specimens from dengue patients, e.g. blood samples (seah et al., b; phanthanawiboon et al., ) . in conclusion, an impedimetric cell-based biosensor constructed from pll-modified spce has proven to be useful for realtime monitoring of denv-induced cytopathogenesis on surfaceimmobilized fibroblast cells. based on this platform, cpe reflected by the drastic decrease in impedance signal response could be detected at $ hpi at moi of . this impedimetric biosensing system outperforms conventional microscopic inspection that requires - days of denv infection to conclusively observe the changes in morphology and surface coverage of cells. other attractive features of this cell-based biosensor include the possibility of automation, non-invasive measurement, reduced timeframe for the diagnosis of live denv infection, mediatorless and label-free monitoring procedure, and capability of detecting the presence of infectious dengue viral particles to complement existing diagnostic tests such as rt-pcr and serology. in addition, virus titers of given viral samples can be estimated from the measured impedance signals by analysis of cit values. the utility of this impedimetric cell-based biosensor for detecting clinical infectious viral isolates also demonstrates its potential application in the clinical setting. proc. natl. acad. sci. usa the authors thank the singapore immunology network, agency for science, technology and research for research grant funding. cms acknowledges nanyang technological university for supporting his ph.d. scholarship. we thank dr. justin chu for providing chikungunya virus as control. supplementary data associated with this article can be found in the online version at http://dx.doi.org/ . /j.bios. . . . key: cord- - ib yws authors: xie, xing; lin, yan; pang, maoda; zhao, yanbing; kalhoro, dildar hussain; lu, chengping; liu, yongjie title: monoclonal antibody specific to ha glycopeptide protects mice from h n influenza virus infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ib yws canine influenza virus (civ) subtype h n is a newly identified, highly contagious respiratory pathogen that causes cough, pneumonia and other respiratory symptoms in dogs. data indicate that the virus is responsible for recent clinical cases of dog disease in china. however, therapeutic options for this disease are very limited. in this study, seven monoclonal antibodies (mabs) against civ js/ (an h n subtype virus) were produced and characterized. among them, mab d , which is specific for the ha glycopeptide (gp), induced the highest neutralization titers. the protection provided by mab d was evaluated in balb/c mice challenged with homologous or heterologous strains of h n influenza virus, including two strains of civ and one strain of swine influenza virus (siv). the data show that mab d protected the mice from infection with the three viral strains, especially the homologous strain, which was indicated by the recovery of body weight, reduction of viral load, and reduction of tissue damage. moreover, the levels of ifn-γ and tnf-α in the lungs, as detected by elisa, were reduced in the infected mice treated with the mab d compared with those without mab d treatment. thus, our findings demonstrate, for the first time, that a mab could reduce the release of ifn-γ and tnf-α associated with tissue damage by civ infection and that the mab might be of great therapeutic value for civ infection. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. influenza a virus, a highly contagious pathogen, can infect both birds and mammals. it has undergone significant genetic variation to adapt to different hosts [ ] . its interspecific transmission is achieved by the recombination or direct transfer of genetic material [ ] . the first case of dog infection with h n canine influenza virus (civ) was reported in the usa in [ , ] , followed by a report of civ in south korea, which subsequently demonstrated that civ was able to transmit directly from dog to dog [ , ] . recently, the first case of h n civ infection was reported in guangdong province in [ ] . over recent years, infection with h n civ in dogs has developed from scattered cases to wide distribution across the country [ ] [ ] [ ] . dogs have no natural immunity to this virus, thus a number of preventive and therapeutic measures against civ have been attempted to control the prevalence of this virus. among them, vaccination is an important method to prevent and control influenza virus infection [ ] [ ] [ ] . current vaccine research against civ has made some progress. in , the u.s. department of agriculture (usda) approved a list of vaccines against h n civ, which could effectively reduce viral shedding [ ] . in , the patent for an h n civ vaccine in south korea was also approved [ ] . preventive vaccination is historically the primary measure to control influenza virus infection, but it has some limitations [ ] . for example, influenza vaccines may not be effective enough to prevent against divergent viral strains, or may be less immunogenic and effective in certain groups, such as the very young, the old, and the immunocompromised [ ] . therefore, it is crucial to develop other measures to protect animals from infection/disease [ ] . for example, passive immunity by transferring a specific antibody to a recipient could protect animals from infection [ ] . monoclonal antibodies (mabs) can neutralize viruses, thus preventing virus attachment to, or fusion with, the host cell [ ] . many studies have demonstrated that mabs are an effective and preventive treatment against human-origin [ ] [ ] [ ] or avian-origin influenza virus infection [ , , ] . however, to date, there are no neutralizing mabs available to prevent and control h n civ infection. in this study, we identified seven mabs against h n civ, and tested one of them, the d mab, against three different h n subtype virus strains in animal experiments. this is the first description of a neutralizing mab against h n civ. three viral strains of the h n subtype, including a/ canine/jiangsu/ / (js/ ), a/canine/guangdong/ / (gd/ ) and a/swine/shandong/ / (sd/ ) were used in this study. the genbank accession numbers of js/ , gd/ and sd/ are jn to jn , kf to kf and eu to eu , respectively. the three viral strains were adapted to mice by passaging times. they were propagated in -day-old specific-pathogen free (spf) embryonated chicken eggs and stored at − °c before use. madin-darby canine kidney (mdck) cells were cultured in dulbecco's modified essential medium (dmem) containing % (v/v) fetal bovine serum (hyclone, tah, usa) and maintained at °c and in a % (v/v) co atmosphere. balb/c mice ( weeks old, female) were purchased from the animal experiment center, yangzhou university. all animal experiments complied with the guidelines of the animal welfare council of china, and the animal ethics committee of nanjing agricultural university approved the study. fifty-percent tissue culture infective dose (tcid ) assays one day before infection, a -well dish containing a monolayer of mdck cells was prepared. the next day, serial dilutions of the three influenza virus strains were made, and the cell monolayers were laterally inoculated; each dilution had three replicates. the cytopathic effect (cpe) was observed daily and the numbers of wells for a virus dilution that showed more than and less than % pathological changes were recorded. tcid titers were calculated in accordance with the reed-muench method [ ] . canine influenza virus js/ was grown in -day-old spf embryonated chicken eggs at °c for h. allantoic fluids were harvested and the hemagglutination (ha) activity of the allantoic fluids was tested at room temperature using % chicken red blood cells (rbc). ha titers more than or equal to : were selected, and the virus was purified using differential centrifugation and sucrose density gradient centrifugation. preparation of anti-h mabs followed standard hybridoma technology, as previously described [ ] . six-week-old female balb/c mice were injected intracutaneously with μg of purified virus js/ using complete freund's adjuvant (sigma, beijing, china) as the primary adjuvant, followed by incomplete freund's adjuvant. three days before harvesting the splenocytes, μg of js/ were inoculated intravenously. isolation and screening of the hybridomas was performed as described previously [ ] . mabs were prepared by injecting hybridoma cells into the peritoneal cavities of pristane-primed balb/c mice. the ascetic fluid was collected after - days and inactivated at °c for min. the hi test was performed to assess antibody reactivity against three h n strains, js/ , gd/ and sd/ , as previously described [ ] . briefly, μl of serial twofold dilutions of the purified -fold diluted ascetic fluid of the mab were mixed with ha units of virus in disposable hemagglutination plates and incubated at °c for min. then, μl of % chicken rbc were added to each well and incubated at room temperature for min. to rule out non-specific inhibition, in the hi assay, we used the ascetic fluid produced with the injection of sp / myeloma cells as a negative control. the hi titer was expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination of ha units of the virus [ ] . cell-based neutralization assays were performed as previously described [ ] . a dose of tcid of viruses was used in the assays. supernatants of ascites were tested at a starting dilution of : . briefly, two-fold dilutions of hybridoma supernatants were mixed with virus suspension containing tcid of purified h n virus and incubated at °c in a % co incubator for h before their addition to a monolayer of mdck cells in -well plates. one hundred microliters of serum-free dmem was added to each well and incubation at °c continued for h. the cytopathic effect was observed every h for to h. to determine the recognized ha domain of the mabs, we recombinantly expressed the ha, ha and ha proteins of virus js/ . the recombinant proteins were subjected to sds-page under reducing conditions. the proteins were then electro-transferred and immobilized on a nitrocellulose membrane. the membrane was blocked with % nonfat milk in phosphate buffered saline (pbs) containing . % tween (pbst) at °c for h. the membrane was subsequently incubated with the mab prepared in this study, rinsed in pbst, and incubated with horseradish peroxidase (hrp)-conjugated rabbit anti-mouse immunoglobulin (bio-rad, shanghai, china), followed by incubation with chromogenic reagents (tiangen, beijing, china) [ ] . cross-protection by h -specific mab balb/c mice were used to determine the protective efficacy of mab d . intranasal inoculations with tcid of virus strains js/ , gd/ and sd/ were given to experimental groups i (n = ), ii (n = ) and iii (n = ), respectively; the control group received pbs (n = ). each experimental group was divided into three subgroups (n = for each subgroup), which were the virus-infected, mab d and irrelevant mab igg subgroups, respectively. mice injected with pbs or irrelevant mab igg were considered as blank and negative controls, respectively. for the mab d and irrelevant mab igg subgroups, mice were pretreated intraperitoneally with mab d ( μg/ml) or irrelevant mab igg ( μg/ml) against igm from chinese breams developed in our laboratory, at a dose of mg per kg of body weight in μl of pbs before the viral challenge [ , , ] . after h, mice were challenged with three different h n strains. mice were observed daily to monitor body weight and clinical symptoms for up to days. three mice from each subgroup were euthanized humanely according to a pre-designated schedule. at , , , and days post-infection (dpi), blood samples and tissues including heart, spleen, lung, brain and intestine, as well as feces were collected. virus shedding was detected by screening fecal samples. detection of viral rna was used to determine tissue distribution and virus shedding. tissues and feces were homogenized in lysates at a ratio of : (g/ml), respectively, centrifuged at × g for min, and the supernatants were collected for the extraction of viral rna using the virus nucleic acid extraction kit ii (geneaid, taiwan). all tissues collected above, including blood, were used for virus titration; the lung, brain and heart were also used for histological and immunohistochemical analysis at dpi. quantitative assays were carried out to measure viral loads in the blood and main organs [ ] . total rna from tissues and blood samples was reverse transcribed using the primescript™ rt reagent kit with gdna eraser (perfect real time) (takara, dalian, china) and then run on an abi real time pcr system using the sybr premix ex taqtm (perfect real time) kit (takara). reverse transcription and cdna amplification were carried out as described previously [ ] . the primers used were designed against a region of the matrix gene: ′-tctatcgtcccatcaggc/ggtcttgtctttag ccattc- ′. a reference standard was prepared using pmd -t simple vector ( ng/μl; takara) that contained the corresponding target virus sequences. a series of eight -fold dilutions equivalent to × - × copies per reaction were prepared to generate calibration curves and were run in parallel with the test samples [ ] . rna of the amount of the two avian-origin civ and human-origin influenza viruses was calculated from the standard curve by real-time rt-pcr. the detection limit of this assay was copies of rna per ml. after euthanasia at dpi, the heart, brain and lung from the mice inoculated with js/ , gd/ , sd/ or pbs were collected and placed into % neutral buffered formalin. after fixation the tissues were embedded in paraffin, sectioned at μm and stained with hematoxylin and eosin for histological evaluation. sequential slides were stained using an immunoperoxidase method [ ] . expression of hemagglutinin in tissues was examined by immunohistochemical staining of histological sections. in brief, sections were blocked with % bovine serum albumin/pbs, stained with mab d at a dilution of : for one hour at °c, followed by biotin conjugated goat anti-mouse immunoglobulin (bio-rad) at a dilution of : for min at °c. the sections were subsequently incubated with hrp conjugated streptavidin (bio-rad) at °c for min. sections were then developed with hrp-dab chromogenic substrate kit (tiangen) for min and counterstained with hematoxylin. the lungs were assigned a grade of to based on the histological character of the lesions. score criteria of different grades were in accordance with a previous study [ ] . we further investigated if there was a correlation between severe disease and inflammatory cytokine production in virus-challenged mice, and ascertained whether passive immunization with antibodies affected the levels of cytokines involved in defense against three different influenza virus infections. sections of the lungs (alternating right and left lungs) from all the mice were homogenized in ml of pbs per g of lung tissue. the homogenates were centrifuged, and the supernatants were frozen at − °c until tested. the supernatants were assayed for gamma interferon (ifn-γ) and tumor necrosis factor (tnf-α) using elisa kits (sigma-aldrich, beijing). the minimum detection limits of such assays were as follows: pg/ml for tnf-α and pg/ml for ifn-γ, as previously determined by the manufacturer. data were collected and analyzed using ms excel and spss statics v . software. weight loss, viral titers, cytokine levels and histological score were analyzed by analysis of variance (anova), followed by turkey's multiple comparison test with p < . considered to be a significant difference, while p < . was considered to be statistically extremely significant. civ was propagated in mdck cells and the titers of three viral strains, js/ , gd/ and sd/ were determined to be . tcid /ml, . tcid /ml and tcid /ml, respectively. after fusion between spleen cells from h n virusimmunized mice and sp / myeloma cells, we obtained seven mabs against the js/ virus. isotyping tests showed that all of these mabs were igg b isotypes, except for one that was igg a. of the seven mabs identified, four mabs reacted with ha. among these four mabs, mab d reacted with ha and three other mabs reacted with ha ( figure ), as demonstrated by western blotting. hi and neutralization titers of the seven mabs showed that mab d had the highest neutralization activity, but had no hi activity (table ). further analysis indicated that mab d could react with virus strains js/ , gd/ and sd/ , and produce high neutralization activities against the three viral strains, especially against the homologous strain js/ ( table ) . to access the protective efficacy of mab d , we inoculated mice with three different h n strains one day after treatment with mab d . three days after the inoculation, all mice challenged with the three virus strains exhibited clinical signs of infection, including depression, decreased activity and huddling. similar clinical signs were observed in irrelevant mab igg pretreated groups. however, similar to the pbs control group, mice in mab d pretreated groups seemed to be energetic and had a good appetite during the infection. in terms of body weight, mice challenged with js/ after treatment with d showed a similar increase in body weight compared with the pbs control group and there was no significant difference between the two groups ( figures a, b and c). at dpi, mice treated with mab d showed a body weight increase of nearly %. although the body weights in the virus-infected group and irrelevant mab igg group both demonstrated an upward trend, the growth rate was slower than that in the mab d group. the extent of the increase in body weight was significantly slower compared with that of the mab d group at , and dpi (p < . ) (figure a ). in the group of mice infected with gd/ , the body weights of the mice in the three experimental groups all showed an upward trend, but the growth rate of the mice treated with mab d was much higher than in the other two groups. in addition, the body weight changes of mice in the mab d group at , and figure antigen identification of mab d by western blotting using recombinant proteins ha, ha and ha . lane m, protein pre-stained mass markers; lane , mab d reacted with expressed viral protein ha; lane , mab d reacted with recombinant protein ha (not visualized here); lane , mab d reacted with expressed protein ha . viral proteins were identified by dab staining with hrp-labeled goat anti-mouse secondary antibody. dpi were significantly different from those of the other two experimental groups (p < . ). the mice in the mab d group showed weight gains of nearly %, which was not significantly different from the pbs control group ( figure b ). however, after infection with sd/ , mice in the virus-infected group showed a slight , the results are expressed in terms of percent body weight. *p < . , or **p < . , indicates a significant difference in weight data for the mab d treated groups compared with the virus-infected groups and irrelevant mab igg groups. # p < . , indicates a significant difference in weight data for the pbs group compared with the mab d treated group. for viral loads in the lungs (d, e, f), the results are expressed in terms of mean log number of copies/g of rna standard deviation. *p < . , or **p < . , indicates a significantly different virus titer for the mab d group compared with the other two groups. / , and / , indicate the proportion of the lungs in which virus could be detected. decrease in body weight at dpi and mice in the mab igg group displayed a slight decline at dpi. by contrast, mice in the mab d group continued to grow at (p < . ), (p < . ) and dpi (p < . ). the growth rate in the mab d group was significantly higher than that in the virus-infected group and mab igg group, but slightly lower than in the pbs control group at dpi; mice body weight gain in the mab group reached approximately % ( figure c ). real-time pcr was used to evaluate the kinetics of viral rna loads in the lung, heart, brain, spleen, intestine, feces and blood of the infected mice. the viral titers were expressed as the number of copies of viral rna. the dynamic changes of viral titers in the lungs of mice in the virus-infected group, mab d group and irrelevant mab igg group were similar: peak viral titers were observed at , and days after infection, after which the viral titer declined at and dpi (figures d, e and f). however, viral titers in the lungs of mice treated with mab d were significantly lower than in the mice in the other two groups at specific time points. after challenge with js/ , viral loads in lungs of mice treated with mab d were significantly lower than those in the other two groups at , and dpi (p < . ) ( figure d ). for strain gd/ , viral titers of the lungs in the mab d group were significantly lower compared with the other two experimental groups at (p < . ) and dpi (p < . ). in addition, at and dpi, viral rna could not be detected in some lung samples ( / and / , respectively) in the mab d group ( figure e ). for virus sd/ , the viral titer of mice in the mab d group was significantly lower than in the other two groups at (p < . ), (p < . ) and dpi (p < . ) ( figure f ). for mice in the virus-infected group, peak viral titers of three virus strains js/ , gd/ and sd/ were . , . , . copies/g, respectively, while for mice in the mab d group, peak values were . , . and . copies/g, respectively. considering that mab d resulted in a significant reduction in viral titers in the lungs of mice infected with three different virus strains at dpi, we chose that time point to determine the viral rna loads in different tissues and fecal samples. we found that viral loads in collected feces, blood and other tissues at dpi in the mab d group were also lower than those in the other two groups (figure ). after mice were challenged with virus js/ , viral titers of the lung, heart, intestine, feces and blood in the mab d group decreased by (p < . ), (p < . ), (p < . ), (p < . ) and (p < . ) fold, respectively, compared to the other two groups ( figure a ). for mice challenged with virus gd/ , viral titers of the lung, heart, spleen, feces and blood of mice treated with mab d were found to be reduced by (p < . ), (p < . ), (p < . ), (p < . ) and (p < . ) fold in comparison with the other two groups ( figure b ). for virus sd/ , mab d resulted in a reduction in viral titers of the lung, spleen, intestine, feces and blood by (p < . ), (p < . ), (p < . ), (p < . ) and (p < . ) fold, respectively ( figure c) . generally, mab d could reduce viral loads of the three virus strains in the infected mice. notably, in the virus-infected group and irrelevant mab igg group, after infection with virus js/ , even till dpi, virus rna was detected in most tissues, while for the mice treated with mab d , virus rna could not be detected in the brain and almost all the other tissues, except for the lung and blood, at dpi (additional file ). for virus gd/ , at dpi, viral titers in all the detected tissues were much lower compared with the other two viruses, in all three groups. viral titers in some tissues were undetectable. mice in the mab d group showed a much faster virus clearance rate than the other two groups. at dpi, no virus was detected in the intestines and feces of three mice in the mab d group and at dpi, virus was undetectable in all the other tissues in mice treated with mab d , except for the lungs in one mouse (additional file ). however, in mice infected with sd/ , the virus showed the longest retention time. at dpi, nearly all tissues from mice in virus-infected and irrelevant mab igg groups showed detectable virus rna. even in the mab d group, all mice showed positive virus rna in the detected tissues, except for the intestines and feces (additional file ). to compare the above results with pathological findings in mice infected with three different virus strains, and treated with mab d and irrelevant mab igg, we chose the heart, brain and lung from different treatment groups at dpi to perform histopathological and immunohistochemical analysis. all the sampled tissues from mice in the virus-infected group showed significant lesions and viral antigen staining, while those from the control group did not show any lesions. mice treated with mab d had markedly fewer lesions compared with the virus-infected group (figure ) . histological lesions in the mab igg group showed similar results with those in the virus-infected group (data not shown). regardless of virus strain, the lung interstitial space was obviously widened, and the bronchial lumen became narrow, with the alveolar septum thickened by the infiltration of a number of inflammatory cells (figures a and c) . large areas of the lung appeared consolidated, with symptoms of pulmonary congestion ( figure e ). interstitial pneumonia was also obvious, with the alveolar figure viral loads in collected tissues and fecal samples of mice at dpi. mice were pretreated with mg/kg of mab d , mab igg or pbs day before viral challenge with virus js/ (a), gd/ (b) or sd/ (c), respectively. in each virus group, the lung, heart, brain, spleen, intestine, feces and blood of mice were collected for determination of viral loads using real-time pcr at days post-challenge. *p < . , or **p < . , indicates significantly different virus titers compared with the other two groups. septum and proliferation of connective tissue infiltrated with numerous macrophages around the bronchioli and blood vessels (figures c and e) . in brief, histological lesions were characterized by multifocal to coalescing reddish consolidation in mice infected with the virus js/ , gd/ or sd/ in both the virus-infected and irrelevant mab igg groups. however, the degree of histological lesions observed for sd/ was the most severe, and gd/ showed the least severe lesions among these three viruses. mice treated with mab d showed only mild necrotizing bronchiolitis and ciliated tracheal epithelium with mild hyperplasia ( figure f ). in addition, very small gaps were observed between the alveoli and there were no excessive amounts of alveolar macrophages in the lung (figures b and d) . for virus strain gd/ , mab d demonstrated the best protective efficacy compared with the other two viruses. mice in the pbs group showed bronchia with a simple ciliated columnar epithelium and the alveolar cavity as a vacuolated thin-walled structure ( figure g ). viral antigen staining was present in almost all bronchiolar epithelial cells and some alveolar cells at dpi after virus challenge ( figures a, c and e) , while mice in mab d showed only a little virus antigen staining surrounding vessels near the alveoli (figures b, d and f) . the pbs control group had no viral staining ( figure g ). lung grades for degree of injury at dpi are shown in figure . there was significantly less injury to the lungs in the mab d group than in the virus-infected group and mab igg group for all virus strains. similar to the lung, all mice in the virus-infected groups showed histological lesions in the brain. the extent of histological lesions infected was the most severe with sd/ and was the least severe for gd/ . the severity of the infection may depend on the differences in virus titer. in the cerebrum, congestion and hemorrhage were evident. nerve fibers were dissolved and neurons had necrolysis-like vacuoles; glial nodules and neuronophagia were also observed ( figures o and q) . dilation and hyperemia were found in the capillaries ( figure s ). moreover, microglial cells and nerve cells showed a satellite phenomenon. the cytoplasm of the neurons was basophilic because of contraction ( figures o and q) . viral antigens could be detected in glial nodules and microglial-gathered areas (figures o, q, and s) . the brains of mice treated with mab d had only mild lesions surrounding microglial cells and nerve cells ( figures p and t) , and almost no antigen staining was found (figures r and t) . the pbs control group showed no histological findings ( figure u ) and no viral staining ( figure u ). in the heart, for all mice in virus-infected groups, the cardiac striated muscle was disordered and full of vacuoles, characterized by myocarditis, and lymphocyte infiltration was observed ( figure h ). lymphoproliferation was also found among the muscle fibers ( figure j ). the nuclei showed pyknosis, with some myocardial cells showing coagulative necrosis ( figures j and l) to gain a better understanding of the effect of civ on the innate immune response and to ascertain whether passive immunization with monoclonal antibody affected the levels of cytokines, we examined the levels of ifn-γ and tnf-α in the lungs of mice in the virus-infected and mab d groups. as shown in figure , the level of ifn-γ in response to all three virus strains showed an identical trend, higher at , and dpi than at and dpi. the ifn-γ levels in mab d group in all three virus strains were significantly lower than the virus-infected group or mab igg group, especially at , and dpi. however, the cytokine levels differed with the various virus strains. for virus strain js/ , the ifn-γ level of all groups reached its peak at dpi, and then exhibited an overall downward trend. the cytokine levels in the mab d group at (p < . ) and dpi (p < . ) were significantly lower than that in the virus-infected or mab igg group; however, the ifn-γ level did not show any significant difference among the three groups at other time points ( figure a ). the ifn-γ level in the mab d group returned to normal after dpi, while the other two groups returned to the normal level at dpi. for virus strain gd/ , the ifn-γ levels of the three experimental groups were significantly higher than in the mice in the pbs group at (p < . ), (p < . ) and dpi (p < . ), sustaining a relatively high level until dpi, after which it decreased. the peak level of ifn-γ in the mab igg group was lower compared with the other two virus strains at dpi ( figure b ). although the ifn-γ levels in the three experimental groups challenged with gd/ did not show much difference compared with each other, the ifn-γ level in the mab d group at dpi returned to a normal level, which was significantly lower (p < . ) compared with the other two groups; all the three groups showed normal levels at dpi. for virus strain sd/ , this virus induced the largest rise in ifn-γ levels. the level in the sd/ -infected the degree of lung injury after infection with virus js/ , gd/ and sd/ at dpi. the lungs were assigned a grade to based on the histological character of the lesions. *p < . , or **p < . , indicates a significantly different score for the mab d group compared with the other two groups. group was significantly higher than that of the other two viruses at dpi. in addition, mice in virus-infected and mab igg groups both demonstrated the same trend in the period of to dpi (p < . ), i.e., significant elevation followed by a downward trend ( figure c ). although the ifn-γ level in the mab d group at to dpi was relatively high compared with the pbs group, the ifn-γ levels were significantly lower than those of the other two groups at (p < . ) and dpi (p < . ), and then declined to the normal level at dpi. figure characterization of ifn-γ and tnf-α secretion from lung tissues of mice challenged with virus. cytokine concentrations were measured by elisa in supernatants of homogenates from the lungs infected with three virus strains. mice were pretreated with mg/kg of mab d , mab igg or pbs day before challenged with virus js/ (a, d), gd/ (b, e) or sd/ (c, f), respectively. the cytokine levels were measured in the infected mice on days , , , and post challenged. the results are expressed in terms of pg/ml. *p < . , or **p < . indicates significantly different changes for mab d group compared with the virus-infected group or the mab igg group. # p < . , or ## p < . , indicates a significant difference between this group and the pbs control group. changes in tnf-α level were not the same as those for ifn-γ. after infection with the three virus strains, tnf-α levels of mice in all groups were only slightly higher than those of the pbs group at dpi and increased at and dpi. levels reached their maximum at dpi and decreased at dpi, however, the cytokine level was still higher than that of the control group at dpi which was quite different compared with the ifn-γ level. the increase in tnf-α level was lower compared with the ifn-γ level. in addition, the tnf-α level in the mab d group was apparently lower than the virusinfected group or mab igg group, especially at and dpi. for virus strain js/ , tnf-α levels in the virusinfected group and mab igg group showed a small rise at dpi, reaching its peak at dpi, and then declined; however, the cytokine level was still significantly higher (p < . ) than that of the pbs group ( figure d ). the tnf-α level in the mab d group at dpi (p < . ) was remarkably lower than that in the virus-infected group and mab igg group, and decreased to a similar level as the pbs group. for virus strain gd/ , the tnf-α level of all three experimental groups increased from to dpi, and then returned to normal at dpi ( figure e ). the tnf-α level of the mab d group at dpi was significantly lower (p < . ) than in the other two groups, while the cytokine level did not show much difference in these three groups at the other time points. for virus strain sd/ , the tnf-α levels in the virusinfected group and mab d group were markedly higher than those of the pbs group from to dpi ( figure f ). although the tnf-α level in the mab d group was also significantly higher (p < . ) than the pbs group, except at and dpi, the level in the mab d group was significantly lower (p < . ) compared with the virus-infected and irrelevant mab igg groups at and dpi. h n civ is a newly identified avian influenza virus (aiv) subtype that can infect dogs and transmit directly from dog to dog [ , ] . civ infection has been reported in several countries, including south korea and china [ , , ] . it is important to develop a set of measures to prevent and control civ infection in dogs. antibody-mediated passive immunity can provide protection against invading pathogens [ , ] . in this study, we developed seven mabs against js/ , whose pathogenicity has been characterized both in mice [ ] and dogs [ ] . among them, four mabs reacted with ha. the ha glycoprotein is the primary target of antibodies that confer protective immunity to influenza viruses [ ] . therefore, the generation of neutralizing antibodies against antigenic sites on the ha glycoprotein is regarded as a criterion for evaluating immunity to influenza viruses and is believed to constitute the main correlate of protection [ , ] . anti-ha globular head mabs have potent neutralizing activity against homologous strains, but have very limited breadth of reactivity because of the high variability of amino-acid changes in the ha globular head [ ] . ha , which is the ha stalk, however, is a conserved region of ha among all influenza a virus subtypes [ , ] and is responsible for the fusion of the virus and the endosomal membrane during the entry of the virus into the cell [ ] . here, western blotting showed that mab d recognized the ha domain of h , and had highest neutralization activities. although mab d lacked hi activity, some previous studies reported that a lack of in vitro hi activity of anti-ha mabs does not rule out protective activity in vivo [ , ] . therefore, we selected the anti-ha mab d for further evaluation in regards to protection against different influenza virus strains. to investigate the protection of mab d against homologous and heterologous strains of h n influenza viruses, we selected three virus strains to perform the challenge experiment in mice, including two strains of civ (js/ and gd/ ) and one strain of swine influenza virus (siv) (sd/ ). considering that almost all h n civ isolates reported were not lethal to mice or dogs in challenge experiment [ , , , , ] , we evaluated the protection efficacy by body weights, viral loads and histological lesions. body weight loss is the parameter most commonly used to evaluate influenza viral pathogenicity in mice [ ] . our study shows that all three virus strains could remarkably reduce the growth rate of the mice after infection, while pretreatment with mab d helped to control the declination to some extent. from the pathological point of view, the lung, heart and brain in mab d treated groups showed markedly fewer lesions compared with the virus-infected group and mab igg group, with all virus strains. the pathological scores of the lungs in mab d group were lower than those in the virus-infected group and mab igg group, suggesting that mab d could mitigate the damage caused by influenza virus. these results suggest that mab d could offer a protective effect against the three virus strains. to further evaluate the effects of the anti-influenza virus mab, we monitored viral loads by real-time pcr. the mab d decreased the viral loads in the lungs to significantly lower levels, relative to those in the virusinfected group and mab igg group. similar results were also found in other tissues. for js/ , the virus in the brain and other organs of all three mice treated with mab d had been cleared by dpi, except for the lungs. for virus gd/ , the application of mab d caused virus clearance from the intestine and feces days earlier than that in the other two groups; moreover, there was no detectable virus rna at dpi. for virus sd/ , a slower rate of clearance of viral load was observed. this virus could persist and be detected in most tissues in the mab d group until dpi, but virus in the intestine and feces had been cleared by dpi. these results indicate that protection against the virus strains provided by mab d might be caused by earlier clearance of the virus from the tissues or shortening the time of virus shedding. a previous study has reported that mabs could reduce the period of virus clearance [ ] . our study indicates that mab d could provide good protection against challenge with homologous, as well as heterologous, virus strains of h n influenza virus. this finding was in accordance with a previous report [ ] that ha mabs are highly cross-reactive among strains of the same subtype, and even within different subtypes. in spite of this, we found that this mab was relatively less effective against the swine-lineage than caninelineage h virus strains. sequence analysis of amino acids showed that js/ had higher sequence identity to canine-lineage gd/ ( . %) than to swine-lineage sd/ ( . %), with and different amino acids, respectively, in ha (data not shown). the difference in amino acid sequence may affect antigen-antibody recognition. this may explain why the mab induced by canine-lineage influenza virus strain js/ is not strong enough to react against more distantly related strains within the same subtype. cytokines are important in establishing an innate immune response, as well as in determining the magnitude of the inflammatory response to influenza virus infection. the most important feature of the mechanism of immune suppression with influenza virus h n is the cytokine storm [ , ] . here, to gain a better understanding of how virus infection and mab treatment affected host immune response, we analyzed the levels of ifn-γ and tnf-α in the lung. an important finding in this study is that the tnf-α levels peaked two days later than ifn-γ levels in all groups. a previous study showed similar results: ifn-γ and tnf-α in the lungs of pigs infected with human h n influenza virus peaked at dpi, and dpi respectively [ ] . ifn-γ has important immune-regulatory functions and antiviral activity, and is primarily produced by natural killer (nk) and t cells. nk cells are a major player in innate immune responses. we speculated that early production of ifn-γ during infection probably arises from nk cells, whereas tnf-α functions relatively late in the inflammatory cycle induced by infection, at a time when virus is already being contained and the response is centered on resolution of the inflammation [ , ] . tnf-α in mice challenged by js/ and sd/ maintained higher levels until dpi. a previous study demonstrated that the depletion of tnf-α in influenza or respiratory syncytial virus-infected animals significantly reduced pulmonary inflammation and cytokine production, without compromising viral clearance [ ] . we speculated that the elevated level of tnf-α at dpi might be correlated with the uncleared virus loads in the lungs. in the present study, three strains of h n civ were shown to induce elevated levels of cytokines in the lungs. this was in agreement with previous reports on h n civ [ , ] . during h n influenza virus infection, the elevated pro-inflammatory cytokine response has been proposed as the main cause of the increased severity of the disease [ ] . the study of lee et al. [ ] reported that the levels of ifn-γ and tnf-α in the lungs of dogs infected with h n influenza virus increased quickly, while the infected dogs developed severe bronchointerstitial pneumonia accompanied with massive infiltration of immune cells. this result suggests that the dysregulation of chemokines during h n civ infection might contribute to viral pneumonia characterized by extensive immune cell infiltration. in support of this hypothesis, we observed that elevated levels of cytokines accompanied the clinical manifestations in the civ infected dogs. we found that ifn-γ and tnf-α levels in the mab d group were significantly lower than in the virus-infected group or mab igg group, while the histopathological findings showed more significant lesions in lungs of mice from the latter two groups than in the mab d group. these observations indicate that mab d treatment may reduce the virus-induced cytokine production and pathological lesions caused by virus infection. the effect is likely to be mediated by inhibition of civ replication by the mab. fritz et al. [ ] reported that active influenza virus replication is required for the induction of potent proinflammatory, regulatory and chemotactic factors. our study and a previous report [ ] demonstrate that active replication of civ in the canine respiratory system results in intense inflammatory responses. considering that it is important for the host to maintain a balance of the cytokine levels, we speculated that inhibition of the inflammatory cytokine response might offer a therapy for civ infection. however, salomon et al. [ ] demonstrated that inhibition of the cytokine response during h n influenza virus infection is not sufficient to protect against death, and proposed that therapies targeting the virus would be preferable. in conclusion, our results suggest that the ha -specific mab d could contribute to early recovery from influenza infection with different h n virus strains. this mab will further our understanding of the antigenic properties of h n virus and might contribute to the prevention and control of h n virus epidemic in dogs. pandemic and seasonal human influenza virus infections in domestic cats: prevalence, association with respiratory disease, and seasonality patterns host range restriction and pathogenicity in the context of influenza pandemic transmission of equine influenza virus to dogs influenza a virus (h n ) in dogs with respiratory disease transmission of avian influenza virus (h n ) to dogs experimental infection of dogs with avian-origin canine influenza a virus (h n ) 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pathology in dogs with experimental canine h n influenza virus infection role of host cytokine responses in the pathogenesis of avian h n influenza viruses in mice severe canine influenza in dogs correlates with hyperchemokinemia and high viral load inhibition of the cytokine response does not protect against lethal h n influenza infection additional file : viral rna detection in collected samples of mice inoculated with virus strain js/ . numbers , and represent the three mice euthanized from each group at different dpi. + represents viral loads in terms of mean log number of copies/g of rna. numbers of +'s represent the magnitude of rna copies.additional file : viral rna detection in collected samples of mice inoculated with virus strain gd/ . numbers , and represent the three mice euthanized from each group at different dpi. + represents viral loads in terms of mean log number of copies/g of rna. numbers of +'s represent the magnitude of rna copies.additional file : viral rna detection in collected samples of mice inoculated with virus strain sd/ . numbers , and represent the three mice euthanized from each group at different dpi. + represents viral loads in terms of mean log number of copies/g of rna. numbers of +'s represent the magnitude of rna copies. the authors declare that they have no competing interests. key: cord- -tnlyk wz authors: rodrigues, anderson messias; fernandes, geisa ferreira; araujo, leticia mendes; della terra, paula portella; dos santos, priscila oliveira; pereira, sandro antonio; schubach, tânia maria pacheco; burger, eva; lopes-bezerra, leila maria; de camargo, zoilo pires title: proteomics-based characterization of the humoral immune response in sporotrichosis: toward discovery of potential diagnostic and vaccine antigens date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: tnlyk wz background: sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans. methodology: we explored the presence and diversity of serum antibodies (igg) specific against sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. antigen profiling included protein extracts from the closest known relatives s. brasiliensis and s. schenckii. enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = ), healthy cats (n = ), and cats with other diseases (n = ). principal findings: enzyme-linked immunosorbent assay-based quantitation of anti-sporothrix igg exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, . ; % confidence interval, . – ; p< . ) versus controls. the two sets of sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. one-dimensional immunoblotting indicated that -carboxymuconate cyclase (a -kda protein in s. brasiliensis and a -kda protein in s. schenckii) is the immunodominant antigen in feline sporotrichosis. two-dimensional immunoblotting revealed six igg-reactive isoforms of gp in the s. brasiliensis proteome, similar to the humoral response found in human sporotrichosis. conclusions: a convergent igg-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of sporothrix and its warm-blooded hosts. we propose that -carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans. sporothrix schenckii was originally described in as the causal agent of a subcutaneous disease in humans in the mid-atlantic usa [ ] . subsequently, the disease was reported in rats naturally infected in southeastern brazil [ ] and later in a wide range of animals including dogs, cats, horses, cows, camels, dolphins, goats, mules, birds, pigs, and armadillos. several sporothrix spp., previously reported to be closely related to s. schenckii, are known to establish infections in various hosts, with dissimilar virulence traits [ , ] and responsiveness to antifungal treatment [ ] . the s. schenckii complex consists of at least four closely-related species [ , ] , ranging from geographically restricted agents such as s. brasiliensis [ , ] to cosmopolitan pathogens such as s. schenckii s. str. and s. globosa [ , , ] . sporothrix spp. are endowed with an extraordinary ecological diversity [ ] [ ] [ ] [ ] ; they are frequently recovered from soil, plants debris, and insects (coleoptera: scolytidae). phylogenetic data support a recent habitat shift within sporothrix from plants to cats [ ] that culminated in the largest epizootic transmission in southeastern brazil [ ] [ ] [ ] [ ] . feline sporotrichosis emerged in the s, with s. brasiliensis recovered from many outbreaks [ , ] . more recently, s. brasiliensis has been recognized as a threat to humans [ ] [ ] [ ] due to the massive zoonotic transmission in southeastern brazil that affects thousands of patients regardless of whether they are immunocompetent or immunocompromised [ , [ ] [ ] [ ] . cats have been a source of sporothrix spp. infection transmitted to humans and other animals [ , , ] . most human cases occurred in housewives and professionals who had contact with infected animals and a history of scratches or bites [ , ] . the largest epidemic of sporotrichosis due to zoonotic transmission was reported in the state of rio de janeiro, brazil [ , , , , ] ; since then, the incidence of sporotrichosis among animals, particularly cats, has increased [ , , ] . more than , humans and , cats were diagnosed at instituto nacional de infectologia (ini) evandro chagas /fundação oswaldo cruz by [ ] . pereira et al. [ ] observed that the majority of cats with sporotrichosis in rio de janeiro between and were male, mongrel, and unneutered, had a median age of months, and presented with three or more cutaneous lesions in non-adjacent locations. this mycosis in cats is hard to treat and generally requires a long period of daily care; these animals do not always respond well to antifungal treatment [ ] . sporothrix is widely distributed in nature, and traumatic inoculation of plant organic matter is classically associated with infection [ ] . feline habits render cats more susceptible to the fungal agent because they are constantly in contact with soil, plant debris, and other cats that may be infected. in cats, a broad spectrum of clinical presentation ranges from single lesions to fatal systemic forms. after monitoring the feline epidemic for years, schubach et al. [ ] observed that the lymphocutaneous form occurred less frequently than did involvement of the mucous membranes of the respiratory tract and upper digestive tract and multiple cutaneous lesions. some animals present involvement of skin and various internal organs [ ] . cats are susceptible to a variety of fungal infections, including blastomycosis [ ] , histoplasmosis [ ] , cryptococcosis [ ] , candidiasis [ ] , dermatophytosis [ ] , aspergillosis [ ] , coccidioidomycosis [ ] , and sporotrichosis. misdiagnosis results in ineffective treatment, which further worsens outcome. major contributors toward misdiagnosis include the small number of affordable and effective treatment techniques as well as other social issues. definitive diagnosis of feline sporotrichosis is based on mycological culture, micromorphological characterization, and mold-to-yeast conversion. histopathological methods and cytopathological examination are useful tools for the presumptive diagnosis of sporothrix infection in cats [ ] . detection of specific anti-sporothrix antibodies offers a rapid alternative for accurate diagnosis [ ] [ ] [ ] . we recently proposed a serological approach that employs an enzymelinked immunosorbent assay (elisa) to diagnose feline sporotrichosis [ ] using purified antigen (the s. schenckii cona binding fraction) and crude antigen, with high sensitivity and specificity. there is a lack of information about feline sporotrichosis and the antigenic components involved in infection; therefore, the present study aimed to explore the diversity of molecules expressed by closely related species (s. brasiliensis and s. schenckii) and that are recognized by immunoglobulin g (igg) in sera from cats naturally infected with s. brasiliensis. we found remarkable cross-reactivity among s. brasiliensis and s. schenckii antigens, and we identified an immunodominant molecule that is an important biomarker in feline sporotrichosis, irrespective of clinical manifestation. here, we show that, although s. brasiliensis and s. schenckii may infect different warm-blooded hosts, infection result in highly similar igg-mediated response in cats compared to humans, what is important for serodiagnosis and to the development of prophylactic or therapeutic vaccine against the enormous health burden of sporotrichosis in endemic areas. this knowledge may enable selection of potential biomarkers that can be used in seroepidemiological studies, diagnosis, and vaccine development, and may contribute to understanding of the pathogenesis of this infection in cats and humans. this study was performed in strict accordance with recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol was approved by the ethics in research committee of the fundação oswaldo cruz, rio de janeiro, brazil, under license number l- / . this study was also approved by the institutional ethics in research committee of the federal university of são paulo under protocol number / . s. brasiliensis and s. schenckii s. str. from cats and humans were used for protein extraction ( table ). the dimorphic nature of sporothrix spp. was demonstrated by converting the fungus to its yeast form at °c in brain-heart infusion medium (difco) and observing typical oval multibudding yeast cells. molecular identification was performed and confirmed via dna sequencing of the gene encoding calmodulin [ ] . isolates were selected because they had been previously characterized at the molecular level [ , , , ] ; crude exoantigen (cbs = ss ; s. schenckii s. str.) was successfully used to diagnose feline sporotrichosis via elisa [ ] . sporothrix spp. was grown on sabouraud medium (difco) agar slants at room temperature ( - °c) for days. approximately x conidia ( % viable cells) were used to inoculate -ml flasks containing ml of brainheart infusion broth. cultures were incubated at °c in a rotary shaker (multitron ii, infors ht) with constant orbital agitation at rpm for days. whole extracts of sporothrix yeast cells were obtained as described elsewhere [ ] . briefly, yeast cells ( ml of each culture) were collected via centrifugation at x g for min at °c and washed three times in ultrapure water. pellets were frozen in liquid nitrogen and disrupted by gridding. cells were macerated with a pestle until a fine powder was obtained. this cellular powder was vortexed for min at °c in tris-ca + buffer ( mm tris-hcl ph . , mm cacl ) containing a commercial cocktail of protease inhibitors ( : ; ge healthcare), rnase and dnase ( : ; ge healthcare), and -μm glass beads ( : ; sigma). cell debris and glass beads were removed via centrifugation at x g for min, and dithiothreitol (final concentration mm) was added to the supernatant [ ] . protein concentrations were determined using the bradford method [ ] . cell extracts were kept at - °c until use. sera from cats with definitive diagnoses of sporotrichosis (via s. brasiliensis isolation in culture; n = ) were obtained from ini/fundação oswaldo cruz, rio de janeiro, brazil. the distribution and number of skin lesions of the cats were classified as l (cutaneous lesion in only one place), l (cutaneous lesion in two non-adjacent places), or l (cutaneous lesions in three or more non-adjacent places). during examination, blood was collected via vein puncture and stored in an incubator for h; serum was obtained via centrifugation and stored at - °c until use. sera from cats with no evidence of sporotrichosis or other diseases (the control group) were obtained from são paulo as described elsewhere [ ] . sera from cats with other diseases were also studied to verify cross-reactions with feline infectious peritonitis/coronavirus ( sera), feline leukemia virus ( sera), feline immunodeficiency virus ( sera), leptospirosis ( sera), rickettsiosis ( sera), erlichiosis ( sera), and leishmaniasis ( sera) as previously described [ ] (s diagram). all sera were stored at - °c until use. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and d immunoblotting s. brasiliensis and s. schenckii protein extracts ( μg) were analyzed via sds-page with % gels [ ] and silver-stained [ ] . the relative molecular weights of the fractions were estimated using standard broad-range molecular weight markers (protein benchmark, invitrogen). for immunoblotting, proteins ( μg) from strains cbs , cbs , cbs , and cbs were resolved with sds-page and transferred onto . -μm polyvinylidenedifluoride membranes (bio-rad) at v for min with transfer buffer ( mm tris base, mm glycine, % methanol, ph . ) [ ] using a trans-blot sd semi-dry device (bio-rad). electrotransference was confirmed by staining with . % ponceau s and % acetic acid [vol/vol]. membranes were destained and free binding sites were blocked overnight in phosphate-buffered saline blocking buffer ( % bovine serum albumin supplemented with . % [vol/vol] tween , % [wt/vol] skim milk, ph . ) at °c. to determine the best dilution of serum, one sample was tested at four dilutions ( : , : , : , and : ) against yeast extracts. afterward, for all sera, membranes were probed individually with primary antibody diluted : at °c for h. membranes were washed three times with tris-buffered saline (ph . ) containing . % [vol/vol] tween- for min and incubated with horseradish peroxidase-conjugated goat anti-feline igg ( : ) for h at room temperature. membranes were then washed with tris-buffered saline (ph . ) containing . % [vol/vol] tween- and signal was detected with an enhanced chemiluminescence detection kit (ge healthcare). blots were imaged in a transilluminator (uvitec cambridge). allience . software was used to take several images at different time exposures, from s each to a total of images over s. proteins were separated via d gel electrophoresis as previously described [ , ] . briefly, proteins ( μg) were precipitated using the d clean-up kit (ge healthcare) and resuspended in rehydration buffer ( m urea, m thiourea, % chaps, . % destreak, % vol/vol isoelectric focusing buffer ph - , and trace amounts of bromophenol blue) to a final volume of μl. immobilized ph gradient strips (ph - , cm; ge healthcare) were rehydrated at v for h. isoelectric focusing was performed at °c using a multiphor iii system (ge healthcare) as follows: v for h, v for h, v for h, and a gradient applied from to v for h. finally, the voltage was set to v for , vhr. after d isoelectric focusing, the ipg strips were reduced for min with . % dithiothreitol and alkylated for min with . % iodocetamide in equilibration buffer ( m urea, mm tris-hcl ph . , % glycerol, and % sodium dodecyl sulfate). second-dimension separation was carried out by placing equilibrated ipg strips onto % polyacrylamide gels, sealing them with . % [wt/vol] low-melting-point agarose, and separating the proteins at °c using a hoefer se unit ( ma/gel for min and then ma/gel until the dye front reached the bottom of the gel). proteins were developed with silver staining [ ] or were directly transferred for immunoblotting. for d immunoblotting, proteins were transferred onto . -μm polyvinylidenedifluoride membranes at v for h with transfer buffer [ ] using the trans-blot sd semi-dry system. the success of electrotransference was evaluated by staining with . % ponceau s and % acetic acid % [vol/vol]. membranes were destained and free binding sites were blocked overnight in phosphate-buffered saline blocking buffer ( % bovine serum albumin supplemented with . % [vol/vol] tween , % [wt/vol] skim milk, ph . ) at °c. membranes obtained from d gels were probed with : primary antibody (gold standard pooled feline sera; n = ) under the conditions used for d immunoblotting. immunoreactive antigens were detected using an enhanced chemiluminescence detection kit (ge healthcare). d immunoblots were imaged using the method used for d immunoblots. diagnostic values included sensitivity, specificity, positive predictive value, and negative predictive value. receiver operating characteristic (roc) curves were prepared and analyzed to determine the sensitivity and specificity of each antigen preparation for elisa. the area under the curve (auc) for roc analysis was calculated to evaluate the diagnostic value of elisa. we assumed that a test lacked diagnostic power when the roc curve was linear with an auc of . (the roc curve coincided with the diagonal). a powerful test was assumed to yield an auc of~ . , indicating the absence of both false-positives and false-negatives (the roc curve reached the upper left corner of the plot). to measure the degree of concordance of the results from preparations from strains cbs , cbs , cbs , and cbs , we calculated the kappa statistic and its % confidence interval (ci). kappa values were interpreted as follows: . - . , poor agreement; . - . , fair agreement; . - . , moderate agreement; . - . , good agreement; . - . , very good agreement [ ] . p-values . were considered statistically significant. all calculations were performed with medcalc statistical software version . . (medcalc software bvba; http://www.medcalc.org). findings are reported in line with the stard checklist for studies of diagnostic accuracy (s checklist). we previously reported a high prevalence of s. brasiliensis in feline sporotrichosis outbreaks [ , , ] . based on this information, the main goal of the present investigation was to evaluate the presence and diversity of serum-derived antibodies against s. brasiliensis antigens in naturally infected cats. further, we previously proposed the existence of a convergent humoral response in human sporotrichosis against antigens from s. brasiliensis, s. schenckii, and s. globosa [ ] . to establish whether s. brasiliensis and s. schenckii express different antigens, we assessed whole cellular protein extracts from two strains of s. brasiliensis plus two strains of s. schenckii s. str. that were previously characterized by molecular [ , , , ] and serological [ , [ ] [ ] [ ] [ ] methods. remarkably, and in support of our hypothesis that immunological distance increases with phylogenetic distance, sera from these cats reacted similarly, with no significant differences in titer between elisa plates coated with proteins from s. brasiliensis or s. schenckii (fig ) . elisa . further information about elisa statistics appears in s table. doi: . /journal.pntd. .g s. schenckii cbs , median . od, % ci . - . od (s table) . when using the assay to diagnosis cats with sporotrichosis, the area under the roc curve was . ( % ci . - . ; p< . ; fig ) , indicating excellent performance. the control group of non-sporothrix infected animals was associated with lower medians and smaller ranges: s. brasiliensis cbs , median . od, % ci . - . od; s. brasiliensis cbs , median . od, % ci . - . od; s. schenckii cbs , median . od, % ci . - . od; and s. schenckii cbs , median . od, % ci . - . od (s table) . differences between the absorbance values for the infected and noninfected groups were statistically significant (p< . ). sera from cats with other infections were non-reactive. similar cutoff values yielded % specificity and sensitivity: s. brasiliensis cbs , . od; s. brasiliensis cbs , . od; s. schenckii cbs , . od; and s. schenckii cbs , . od (s fig). elisa results showed very good agreement for the antigens assayed (kappa = . ). to diagnosis feline sporotrichosis via elisa, we recommend the use of antigen preparations of s. brasiliensis, since this is the most prevalent species in feline sporotrichosis outbreaks. antigen diversity was assayed with d immunoblots using the four antigen preparations of s. brasiliensis and s. schenckii tested via elisa. proteins extracts were evaluated according to the amount of protein extracted, the diversity of bands, the integrity of the samples, and the reproducibility of extraction. approximately μg of sporothrix yeast whole-cell extracts were resolved by sds-page; silver staining revealed numerous proteins ranging from kda to kda in size, with different intensities. the tris-ca + extraction protocol [ , ] was suitable for the study of sporothrix antigenic molecules during feline sporotrichosis, yielding samples with high amounts of protein and no degradation. as expected, antibodies from cats with sporotrichosis reacted with a wide variety of s. brasiliensis and s. schenckii proteins kda to > kda in size (fig ) . cat-to-cat variation resulted in characteristic banding patterns for each animal (fig ) ; supporting the hypothesis that in a genetically diverse population, the antibody repertoire is expected to vary among individual cats. on the other hand, we detected minor or no differences in igg-reacting banding patterns between antigen preparations (fig ) , consistent with the close genetic distance between s. brasiliensis and s. schenckii [ , ] . despite this variation, all cats produced antibodies against a -kda molecule in the s. brasiliensis proteome and a -kda molecule in the s. schenckii proteome. the major antigenic s. brasiliensis molecules (cbs and cbs ) recognized by feline igg consisted of the following sizes: kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), and kda ( % and %, respectively) (fig a and c ). minor molecules recognized by feline igg had sizes of kda, kda, kda, kda, kda, kda, kda, kda, kda, and kda (fig a and c) . the major antigenic s. schenckii molecules (cbs and cbs ) recognized by feline igg had sizes of: kda ( % and %, respectively), kda ( % and %, table. doi: . /journal.pntd. .g respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), kda ( % and %, respectively), and kda ( % and %, respectively) (fig b and d) . the minor s. schenckii molecules recognized by feline igg had sizes of kda, kda, kda, kda, kda, kda, kda, kda, kda, and kda (fig b and d ). sera from uninfected cats did not react with s. brasiliensis or s. schenckii antigens. sera from cats with other infections were also non-reactive in the immunoblot assay. the frequencies at which sporothrix molecules were recognized in the antigen preparations are presented in s table. there was no association between the number of bands recognized by each serum and the distribution and number of skin lesions on cats with sporotrichosis. we previously reported that the -kda and -kda proteins in s. brasiliensis and s. schenckii, respectively, are related to virulence profiles and are the main antigenic molecules during murine [ ] and human [ ] sporotrichosis. we also determined that this protein undergoes post-translational modification and is present as isoforms and glycoforms in the s. brasiliensis and s. schenckii proteomes [ ] . we therefore investigated whether antibodies present in cat sera recognize all six isoforms in the s. brasiliensis proteome, as previously shown with human antibodies [ ] . s. brasiliensis proteins were therefore resolved via d electrophoresis and immunoblotted with pooled sera from cats with sporotrichosis (n = ) and optimal antibody titers according to elisa. serum-derived antibodies in naturally infected cats mainly recognized all six isoforms of gp (fig ) . the present results confirm that s. brasiliensis carboxymuconate cyclase is a highly polymorphic protein [ ] with sizes of - kda and with isoelectric points of . - . . d immunoblotting revealed less diversity and more weakly reacting spots than d immunoblotting, perhaps due to protein loss during sample preparation for d electrophoresis (compared to the crude extracts used in d immunoblotting and elisa) and serum dilution during pooling. in the past, sporotrichosis was reported in horses more frequently than in other animals [ , ] . although the disease has been reported in several animal species, cats are currently the most frequently affected domestic animal. outdoor cats are exposed to the fungus through contact with natural environmental sources or other sick cats. cats have gained importance in the zoonotic transmission of sporothrix to humans [ , - , , ] . presently, the all-time high number of feline sporotrichosis cases reported in brazil has reached epidemic proportions [ , , , ] . unlike humans, cats are highly susceptible to this fungus due to the low frequency of granulomas and the richness of fungal elements observed during skin histopathology [ ] . moreover, unlike human-disseminated sporotrichosis, which classically affects immunocompromised individuals [ , ] , systemic disease in cats occurs frequently and is not associated with immunodeficiency caused by feline immunodeficiency virus and/or feline leukemia virus [ ] . in this scenario, absence of an efficient host immune response is a key factor in disease progression. the low frequency of granulomas and uncontrolled fungal growth suggest that a lack of adequate cellular immunity underlies disease severity and pathology. elisa has been achieved with various antigen preparations, thus enabling serodiagnosis of human [ - , , ] and feline [ ] sporotrichosis. the present investigation suggests that elisa-based quantitation of anti-s. brasiliensis igg is remarkably sensitive for the detection of feline sporotrichosis (cutoffs of . - . od); as expected, this strategy does not differentiate between s. brasiliensis or s. schenckii as the agent of infection. despite its high prevalence in south and southeast brazil, s. brasiliensis infection in cats is not an exclusive host-pathogen association, since the sibling agent s. schenckii s. str. also occurs in cats in brazil [ , , ] and malaysia [ ] , albeit with significantly lower frequency. our serology-based observations of a convergent antigenic response are similar to previous d immunoblotting results for human sporotrichosis [ ] . interestingly, in other thermally dimorphic fungi such as paracoccidioides brasiliensis and p. lutzii (onygenales), antigen composition seems to vary considerably between species, an observation that supported development of a differential diagnosis system based on titration of serum-derived antibodies from humans infected with distinct strains of paracoccidioides [ ] . it is likely that similarities in antigen profiling among clinical sporothrix spp. (s. brasiliensis, s. schenckii, and s. globosa) could be related to a recent speciation event and therefore be less susceptible to variability; however, this hypothesis requires further investigation. in addition, factors related to environment or to host association may impose strong selection pressures on sporothrix antigen profiles. to date, no information about the humoral response in feline sporotrichosis has been reported in the literature; it is a completely unknown area in veterinary medicine that merits exploration. here, sera from cats with sporotrichosis displayed immunoblotting patterns of sporothrix-specific immunogenic molecules that were markedly different from patterns from sera from uninfected cats. these immunogenic components were - kda in molecular weight. the main molecules recognized by cat sera were a -kda protein in the s. brasiliensis proteome and a -kda protein in s. schenckii s. str., followed by molecules weighing kda, kda, kda, and kda. the variety of antigenic components recognized by each serum may be due to specific antigens secreted by individual fungal strains [ , ] as well as to different mechanisms of activation of each host's immune system [ , ] . however, variation in antibody repertoire seems reasonable in a genetically diverse host population [ ] . we interpreted published data lacking taxonomic information or protein identification via matrix-assisted laser desorption/ionization time of flight (maldi-tof)/mass spectrometry (ms) and identified an immunodominant fraction that oscillated between kda and kda in various studies (table ). in this scenario, the humoral immune response may be influenced by the infection strain and the antigen preparation used to detect the humoral response. regarding human sporotrichosis, mendoza et al. [ ] observed that -kda, -kda, -kda, -kda, and -kda molecules are commonly recognized by sera from patients with sporotrichosis, and scott & muchmore [ ] showed that -kda, -kda, -kda, and -kda molecules are immunodominant in human sporotrichosis. a -kda molecule was previously highlighted as immunodominant during murine sporotrichosis [ ] [ ] [ ] , with igg and igg predominant [ ] . this -kda molecule was originally described as an adhesin molecule for fibronectin and laminin in s. schenckii s. str. [ , ] that localized to the fungal cell wall [ ] . more recently, we identified this molecule via d immunoblotting followed by maldi-tof/ ms as -carboxymuconate cyclase (genbank: kp ), the major antigen of human sporotrichosis [ ] . based on d electrophoresis [ , ] and d electrophoresis [ ] , the molecular weight ( - kda) and isoelectric point ( . - . ) of -carboxymuconate cyclase vary intraand interspecifically [ , , , ] . variation in gp /gp may be related to differential glycosylation patterns and amino-acid substitution along the protein core, since all glycoforms/isoforms display identical maldi-tof/ms spectra [ ] (table ) . here, the intensity of the recognition of distinct molecules differed among sera from different cats, but serum from an individual cat displayed little variation when probing different antigen preparations. these data suggest that the antibody response differs between cats and that there are few qualitative variations in the expression of cellular antigens by s. brasiliensis and s. schenckii s. str. in this study, the clinical presentation of sporotrichosis in cats corresponded mainly to multiple skin lesions; we observed no association between the distribution and number of skins lesions and the number or type of molecules recognized by antibodies in the sera. although it remains to be clarified whether the antibodies produced during active infection in feline sporotrichosis are protective, antibodies against -carboxymuconate cyclase appear to inhibit fungal adhesion to the host in a dose-dependent manner [ , ] . in another dimorphic fungus, p. brasiliensis, passive administration of monoclonal antibodies against the immunodominant antigen gp or against the recombinant protein before and after intratracheal or intravenous infections reduced fungal burden and decreased pulmonary inflammation in mice [ , ] . gaining insight into host-parasite interplay in the immunological context is essential for understanding the emergence of feline sporotrichosis and is critical to serodiagnosis and the development of vaccines. here, we demonstrated that antigens derived from yeast cell extracts of s. brasiliensis and s. schenckii s. str. yielded excellent results in elisa and immunoblotting. the variety of molecules recognized by sera may be related to certain characteristics of the isolate, such as virulence, or even related to immune-system activation in each individual host. during infection, sporothrix antigens elicit an igg-mediated response; -carboxymuconate cyclase (gp in s. brasiliensis and gp in s. schenckii) is the immunodominant molecule in feline sporotrichosis, similar to murine and human disease. therefore, this molecule may also be useful as a marker in the diagnosis of feline sporotrichosis and is a promising candidate for the development of therapeutic vaccines to tackle sporotrichosis in highly endemic areas. supporting information s checklist. stard checklist. (doc) s table. summary statistics for antigen detection via elisa with four antigenic extracts. (doc) s table. frequencies of antigen recognition on immunoblotting with serum-derived antibodies against s. brasiliensis and s. schenckii proteins in naturally infected cats (n = ). on refractory subcutaneous abscesses caused by a fungus possibly related to the sporotricha on a mycosis observed in men and mice: contribution to the knowledge of the socalled sporotrichosis characterization of virulence profile, protein secretion and immunogenicity of different sporothrix schenckii sensu stricto isolates compared with s. globosa and s. brasiliensis species different virulence levels of the species of sporothrix in a murine model genetic diversity and antifungal susceptibility profiles in causative agents of sporotrichosis molecular phylogeny of sporothrix schenckii global its diversity in the sporothrix schenckii complex phylogenetic analysis reveals a high prevalence of sporothrix brasiliensis in feline sporotrichosis outbreaks emerging sporotrichosis is driven by clonal and recombinant sporothrix species phylogeography and evolutionary patterns in sporothrix spanning more than , human and animal case reports sporothrix globosa, a pathogenic fungus with widespread geographical distribution phylogeny of the ophiostoma stenoceras-sporothrix schenckii complex multi-gene phylogenies define ceratocystiopsis and grosmannia distinct from ophiostoma emerging lineages in the ophiostomatales sporothrix chilensis sp. nov. 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the head of bacteriophage t improved silver staining of plant proteins, rna and dna in polyacrylamide gels electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications practical statistics for medical research. london: chapman and hall genotyping species of the sporothrix schenckii complex by pcr-rflp of calmodulin systemic mycoses in dogs and cats sporotrichosis in a horse feline sporotrichosis: transmission to man sporotrichosis in man and animal feline sporotrichosis in the southern region of rio grande do sul, brazil: clinical, zoonotic and therapeutic aspects a comparative serological study of the sscbf antigenic fraction isolated from three sporothrix schenckii strains development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis molecular typing of sporothrix schenckii isolates from cats in malaysia serology of paracoccidioidomycosis due to paracoccidioides lutzii heterogeneity of proteins expressed by brazilian sporothrix schenckii isolates differential induction of th -prone immunity by human dendritic cells activated with sporothrix schenckii of cutaneous and visceral origins to determine their different virulence identification of the feline humoral immune response to bartonella henselae infection by protein microarray production of culture filtrates of sporothrix schenckii in diverse culture media immunoblot analysis of antibody responses to sporothrix schenckii cell surface expression of adhesins for fibronectin correlates with virulence in sporothrix schenckii passive immunization with monoclonal antibody against a -kda putative adhesin of sporothrix schenckii induces protection in murine sporotrichosis humoral immune response against soluble and fractionate antigens in experimental sporotrichosis host organism defense by a peptide-polysaccharide extracted from the fungus sporothrix schenckii isolation and some properties of a glycoprotein of kda (gp ) from the cell wall of sporothrix schenckii involved in fungal adherence to dermal extracellular matrix detection of immunoreactive antigens in the cell wall of sporothrix brasiliensis and sporothrix globosa the monoclonal antibody against the major diagnostic antigen of paracoccidioides brasiliensis mediates immune protection in infected balb/c mice challenged intratracheally with the fungus saccharomyces cerevisiae expressing gp protects mice against paracoccidioides brasiliensis infection key: cord- -rhi hi m authors: wilkes, rebecca p.; hartmann, katrin title: update on antiviral therapies date: - - journal: august's consultations in feline internal medicine, volume doi: . /b - - - - . - sha: doc_id: cord_uid: rhi hi m nan update on antiviral therapies rebecca p. wilkes antiviral chemotherapy use is still relatively uncommon in veterinary medicine. controlled studies evaluating the efficacy of antiviral drugs in cats are lacking, or, if studies have been done, in many cases, the data are insufficient to determine effective dosing for these drugs. with the exception of the recombinant feline interferon (rfeifn)-omega, so far no antiviral drugs are specifically licensed for veterinary medicine, which leaves the veterinary community with the option to use off-label antivirals made for humans to combat viral diseases in feline patients. the goal of research in antiviral chemotherapy is the discovery of antiviral agents that are specific for the inhibition of viral multiplication without affecting normal cell division; however, because viruses are dependent on host cell machinery for replication, drug targets are often nonspecific. this makes antivirals inherently more toxic than antimicrobials are because the antiviral drugs are damaging to not only the virus but also the host cells as well. in addition, agents considered safe for human use are not always safe when administered to cats. antivirals made for systemic use often require host and/ or viral metabolism to be active. therefore, agents designed for use in humans are neither reliably nor predictably metabolized by cats or their viruses. thus antiviral agents should always be tested first in vitro for efficacy and safety, and then followed by pharmacokinetic studies in cats. systemic antivirals often have a relatively narrow safety margin, and special considerations should always be given to patients with reduced hepatic or renal function. well-designed blinded, placebo-controlled studies in client-owned animals should follow studies in laboratory-bred, experimentally infected cats to confirm results in genetically diverse cats. most of the human antivirals are specifically intended for treatment of human immunodeficiency virus (hiv) or human herpesvirus infections. therefore, feline immunodeficiency virus (fiv) and feline herpesvirus type (fhv- ) infections have been the most important indications for antiviral chemotherapy in veterinary medicine. topical antiviral therapy has been mainly used for herpetic ocular disease, but studies have evaluated a systemic antiviral compound (famciclovir) for treatment of multiple clinical syndromes associated with fhv- infections. even though combination antiviral therapy has been successful in slowing disease progression in people with hiv, similar therapy has not been thoroughly evaluated in cats. recent studies have focused on combination therapy and evaluation of additional hiv drugs that have not been previously evaluated in feline cells. it is hoped that expanding the number of drugs that are shown to be effective for fiv will lead to effective combination therapy for feline patients. some additional feline infections that have been the focus of current antiviral studies are feline leukemia virus (felv) infection and feline infectious peritonitis (fip). some of the hiv antivirals, such as raltegravir, are nonspecific, and they display activity against additional retroviruses, including felv, in in vitro studies. identification of the human coronavirus that causes severe acute respiratory syndrome (sars) has led to evaluation of antivirals for treatment of various coronaviruses, including the feline coronaviruses (fcovs) that cause fip, although testing is mainly in in vitro stages. several studies have also evaluated the use of rfeifn-omega for treatment of multiple feline viruses. a review of the literature for antiviral treatment in cats, including current recommendations for drug dosages and use, is given in table - . feline immunodeficiency virus infects lymphocytes, cells of the monocyte-macrophage lineage, and cells of the central nervous system causing a variety of clinical signs (figure - ) . the viral replication cycle of fiv is highly similar to hiv. feline immunodeficiency virus binds to host cells by an initial interaction of the fiv envelope (env) glycoprotein with the cd molecule on the host cell, resulting in subsequent interaction with the co-receptor cxcr on the host cell, followed by viral envelope fusion with the host cell membrane. this allows entry of the viral nucleocapsid into the cytoplasm. the viral rna is released into the cytoplasm and transcribed to complementary dna (cdna) by the reverse transcriptase (rt) enzyme, which is specific to retroviruses. the cdna is subsequently synthesized to double-stranded dna, transported to the nucleus, and integrated into the host genome by another virus-specific enzyme, the integrase. viral messenger ribonucleic acid (mrna) and genomic rna are then transcribed and transported to the cytoplasm. viral proteins are translated and processed by a third virus-specific enzyme, the protease. the immature virion moves to the cell membrane and acquires the viral envelope and glycoproteins and then is finally released from the cells. antiretroviral drugs studied extensively in hiv infection have targeted the three virus-specific enzymes (protease, rt, and integrase), as well as some additional targets, interfering with different steps of the virus replication cycle. as of , approximately compounds are approved by the u.s. food and drug administration (fda) for treatment of different stages of hiv infection. some of these drugs can also be used for fiv, and steps that can be inhibited include: ( ) virus entry into susceptible cells by blocking attachment to the host cell co-receptor cxcr ; ( ) reverse transcription of viral genomic rna; ( ) viral dna integration into host genomes; and ( ) proteolytic processing of precursor viral proteins into mature viral proteins (figure - ). , dose indication close similarities exist between the rt of hiv and fiv, and it has been shown that several rt-targeted antiviral compounds active against hiv are also effective in inhibiting fiv replication in vitro. the rt of hiv is actually the target for three classes of inhibitors: nucleoside rt inhibitors (nrti), nucleotide rt inhibitors (ntrti), and nonnucleoside rt inhibitors (nnrti). nucleoside rt inhibitors and ntrti interact with the catalytic site (the substrate-binding site) of the rt enzyme, whereas nnrti interact with an allosteric site located at a short distance from the catalytic site. for the nrti and ntrti to interact with the substrate-binding site, they need to be phosphorylated. all , and emtricitabine) can be considered as nucleoside analogues, and they act in a similar fashion. after they have been taken up by the cells, they are phosphorylated three times to the active triphosphate form, and they act as competitive inhibitors of the normal deoxynucleoside triphosphate (dntp) substrates, which are used by the cell to make dna. unlike dntp substrates, nrti lack a ′-hydroxyl group on the deoxyribose moiety. once incorporated into the dna chain, the absence of a ′-hydroxyl group, which normally forms the ′-to ′-phosphoester bond with the next nucleic acid, blocks further extension of the dna by rt, resulting in dna chain termination. the analogues cannot be cleaved from the active center and thus block the rt enzyme. nucleoside analogues are not only accepted as false substrates by viral enzymes, but also by cellular enzymes, and infectious diseases mononuclear cells. six of these drugs (abc, ddi, emtricitabine, tc, d t, and azt) had been previously evaluated in feline cells, and three (amdoxovir, racivir, and dexelvucitabine) had not. significant differences among the drugs were not found, but based on the data obtained, amdoxovir, dexelvucitabine, and racivir appear to be options for future studies investigating their potential use in fiv-infected cats. though pharmacological data for cats are not available for these drugs, cytotoxic properties of these compounds suggest they could likely be used in vivo at dosages comparable to that for azt. nucleotide rt inhibitors are distinguished from nrti as they are nucleotide analogues (not nucleoside analogues), which means that they only need two (not three) phosphorylation steps to be converted to their active form. most importantly, they contain a phosphonate group that cannot be cleaved by hydrolases (esterases), which would make it more difficult to cleave off these compounds, once incorporated at the ′-terminal end, compared with their regular nucleotide counterparts. use of these compounds also results in dna chain termination. one of these drugs, cidofovir, is active against virtually all dna viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. cidofovir has been used for treatment of fhv- (see feline herpesvirus type ). adefovir ( -( -phosphonylmethoxyethyl)adenine [pmea]) has a spectrum of activity that partially overlaps with cidofovir, in that both are active against herpesviruses, but adefovir is also active against hepadnaviruses (hepatitis b) and retroviruses, including fiv and felv. the antiviral activity spectrum of tenofovir (pmpa) is narrower than that of pmea, in that it no longer extends to herpesviruses but is confined to hepadna-and retroviruses. this drug has been tested in vitro against felv (see feline leukemia virus). adefovir has been tested in fiv-infected cats in a -week placebo-controlled, double-blinded, clinical trial; cats received adefovir ( mg/kg subcutaneously [sc] twice weekly) and cats received a placebo. there was no decrease in the proviral or viral loads in treated cats, and the cats developed a progressive, life-threatening anemia. this is a common adverse effect of some nucleotide analogues. adefovir was also tested in combination with the co-receptor inhibitor plerixafor (see co-receptor inhibitors) in the same study, producing the same outcome as seen with use of the adefovir alone. a related drug, (r)- -( -phosphonylmethoxypropyl)- , diaminopurine (pmpdap), has been shown previously to be a potent inhibitor of fiv replication in cell culture and has reduced the viral load in three of four cats experimentally infected with fiv when treated at mg/kg sc three times per week for weeks. there were no changes in the red blood cell counts or hemoglobin values with treatment. a recent study evaluated the efficacy of this drug in a placebocontrolled, double-blind study with a population of cats naturally infected with fiv. no significant differences were found between pmpdap-treated ( mg/kg sc twice weekly for weeks) and placebo-treated cats, although cats treated with pmpdap showed a tendency for improvement this is the major cause of their toxicity. zidovudine is the nrti most studied in cats, including in vivo studies evaluating the clinical response of experimentally and naturally fivinfected cats treated with the drug. zidovudine can increase the cd +/cd + ratio and improve clinical condition scores in fiv-infected cats; however, it can result in adverse effects, such as dose-dependent nonregenerative anemia and neutropenia. , in addition, mutations producing resistance against the drug can develop. , therefore, a study evaluated nine nrti to inhibit fiv replication in feline peripheral blood receptor and co-receptor proteins dna a significant decrease in the provirus load but did not lead to improvement of clinical or immunological variables. a statistical decrease in serum magnesium levels was observed in the treatment group, without clinical consequences. no development of resistance of fiv isolates to plerixafor was found during the treatment period, making it a potential treatment for fiv-infected cats. limited oral bioavailability and short half-life preclude clinical use of plerixafor in hiv infection, , but additional cxcr antagonists are under development and should be tested for efficacy against fiv when available. integrase catalyzes strand transfer ( ′-end joining), which inserts both viral dna ends into a host cell chromosome. integrase inhibitors are used to treat hiv infection. one of the integrase inhibitors (raltegravir) has been shown to be effective for inhibition of felv (see feline leukemia virus). administration of a combination of drugs from different classes, termed highly active antiretroviral therapy (haart), to hiv-infected patients has turned an invariably fatal disease into a chronic but manageable condition. , the goals associated with the use of combinations of three (or more) anti-hiv compounds are: ( ) to obtain synergism among different compounds acting at different molecular targets; ( ) to lower the individual drug dosages to reduce their adverse side effects; and ( ) to diminish the likelihood of development of drug resistance. combination therapy has not been thoroughly investigated for treatment of fiv infection in cats, and use of multiple classes of drugs is more difficult in cats because some of the drug classes that are effective for hiv do not work for fiv. , however, the need for combination antiretroviral therapy for feline patients has been the focus of recent studies. the goal of antiviral therapy should be improvement of the cat's clinical status. this is not always correlated with virus replication, as measured by a plasma viral load. it has been suggested that antiretroviral therapy should be administered to fiv-infected cats in the later stages of the asymptomatic phase of infection, during which the cat does not show clinical signs and the immune system is relatively normal and more likely to respond to treatment. after experimental infection, when the cd +/cd + ratio decreases, viral load increases markedly, and clinical signs of immunosuppression begin to appear. however, the situation in naturally infected cats is different, and the quality of life is not associated with the viral load. therefore, it is debated at which time point antiviral therapy should be started and whether it should be administered to asymptomatic cats. in a recent study, antiretroviral therapy was initiated during the later stages of the asymptomatic phase of infection in naturally infected cats. the cats were defined as being in the later stages of the in their clinical signs and cd +/cd + ratios. mild hematological side effects (slight decline in packed cell volume and hemoglobin values) were seen in the treatment group. compared with other ntrti, pmpdap seems to be slightly less toxic. unlike the nrti and ntrti, nnrti are an active form, with no dependence on intracellular metabolic pathways. nnrti inhibit the rt by binding to the enzyme in a hydrophobic pocket that is located away from its catalytic site. the interaction of the compounds with the rt induces conformational changes that affect the catalytic activities of the enzyme. nonnucleoside rt inhibitors are considered highly specific inhibitors of hiv- , and thus not active against other retroviruses, including fiv. this is due to differences in the structure and/or flexibility of fiv rt that prevent nnrti from interacting with the fiv rt. protease inhibitors are based on the "peptidomimetic" principle, that is, they contain a hydroxyethylene scaffold that mimics the normal peptide linkage (cleaved by the hiv protease) but which itself cannot be cleaved. they thus prevent the hiv protease from carrying out its normal function, which is the proteolytic processing of precursor viral proteins into mature viral proteins. despite similarities between the hiv and fiv proteases, all but one of the currently available hiv protease inhibitors have failed to inhibit the protease of fiv. the one compound of interest, tipranavir, has only been tested against fiv in vitro so far. however, studies have demonstrated that these compounds can be used to inhibit fcov replication (see feline coronavirus). co-receptor inhibitors block viral attachment by binding to receptors on the host cell membrane to obscure the site of interaction of env with the receptor. most of the receptor homologues or antagonists are highly selective for hiv and not useful for veterinary medicine. one exception can be used in cats with fiv infection, the class of bicyclams (e.g., plerixafor). plerixafor ( , ′-[ , -phenylenbismethylene]bis( , , , -tetraazacyclotetradecane)-octachloride dehydrate, [amd ], [ jm ]), is the prototype compound among the bicyclams. bicyclams are dimeric low-molecular weight nonpeptidic compounds that bind selectively to the chemokine receptor cxcr . this is the cell surface co-receptor used by both hiv and fiv for attachment and infection of susceptible cd + lymphocytes, and the amino acid sequences of human and feline cxcr are highly similar. drug binding inhibits attachment of the viral envelope to the host cell. the efficacy of plerixafor against fiv was recently investigated in naturally fiv-infected cats that were treated in a placebo-controlled, double-blind clinical trial. plerixafor was administered at . mg/kg sc every hours. treatment of fiv-infected cats with plerixafor caused infectious diseases agriculture (usda) as a treatment aid for cats infected with fiv or felv. the primary therapeutic effect is activation of progenitor cd t-cells to mature cells, which then produce cytokines, including interleukin (il)- and interferon (ifn). a few studies performed by the manufacturer are highlighted in a review article. the studies suggest reduced virus load, improved clinical signs, and improved hematological parameters with treatment. however, the data for placebocontrolled studies were not shown, and a field study with naturally infected cats lacked a control group. independent placebo-controlled, blinded studies are warranted. additional information about immunomodulators and immunostimulants is provided in the feline herpesvirus type and feline coronavirus sections. feline leukemia virus, like fiv, is a member of the family retroviridae, but unlike fiv, felv is a gammaretrovirus and not a lentivirus. feline leukemia virus causes a wide variety of clinical signs in infected cats (figure - ). structural differences affect the susceptibility of gammaretroviruses to anti-hiv drugs, but the similarities in mechanism of replication suggest that some of these drugs can also inhibit felv. this is true of most nrti. zidovudine effectively inhibits felv replication in vitro, and in vivo in experimental infections. however, in naturally felv-infected cats, it did not reduce plasma virus load, improve immunological and clinical status, increase quality of life, nor prolong life expectancy. its bone marrow toxicity can also cause adverse side effects (e.g., nonregenerative anemia) that are more pronounced in felv-infected cats than in fiv-infected cats. therefore, it is not recommended as a first line of therapy for felv infection. asymptomatic phase of infection when the cd +/cd + ratio reached . , because at this stage of infection, the viral load increased markedly, and clinical signs of immunosuppression began to appear. the ratios were calculated every months for to years prior to initiation of the antiviral therapy, and viral loads of all cats were quantified once a year. the cats were randomly assigned to treatment groups of eight cats each. treatment included combination therapy, but no placebo group was used, and the study was not blinded. the follow-up was performed over year, through clinical evaluation and the determination of viral loads and cd +/cd + ratios. comparisons of pretreatment and post-treatment values from the cats were performed, as well as comparison of values between treatment groups. a combination of two nrti (azt + tc, mg/kg every hours orally [po]) was compared to treatment with azt alone ( mg/kg every hours po). the combination of azt and tc is often used in hiv-infected patients, given that both drugs show a synergistic effect. treatment with azt alone or in combination with tc induced a significant increase in the cd +/ cd + ratio and a significant decrease in viral load within and among groups, with an even greater reduction with combination therapy than with azt alone. only mild side effects, including vomiting in one of eight cats, anorexia in two of eight cats, and anemia in one of eight cats, were seen with this treatment combination, but therapeutic interventions resolved the problems, and treatment did not have to be stopped. however, the lack of a control group and lack of blinding make the results of the study very difficult to interpret. therefore, treatment of asymptomatic fiv-infected cats with antivirals cannot be generally recommended based on the currently available data. an earlier in vivo study was performed in experimentally fiv-infected cats that were treated with a high-dose azt and tc combination ( or mg/kg/day po for each drug). the combination had no anti-fiv activity in these chronically infected cats. severe side effects, which included fever, anorexia, and marked hematologic changes, were observed in some of the cats with such high-dose dual-drug treatment, but the toxic effects were reversed when the dose was lowered to mg/kg every hours. ideally, combination therapy for feline patients will contain at least two to three drugs from at least two different classes, as recommended for human patients. as previously mentioned, pmea (an ntrti) was tested in combination with the co-receptor inhibitor plerixafor; however, because of the toxicity associated with the pmea, this combination cannot be recommended. therefore, use of plerixafor in combination with other ntrti that are less toxic than pmea or compounds of other drug classes are should be further investigated in the future. lymphocyte t-cell immunomodulator (ltci), a protein produced by a bovine-derived thymic stromal epithelial cell line, is conditionally licensed by the united states department of alphaherpesviruses, such as herpes simplex virus (hsv- ). they have been investigated for treatment of fhv- . members of this group of antiviral agents include acyclovir (and its prodrug, valacyclovir), ganciclovir, and penciclovir (and its prodrug, famciclovir). all require three phosphorylation steps for activation. the first of these steps must be catalyzed by the fhv- viral enzyme, thymidine kinase. this makes the drugs less toxic in vivo compared to many of the other antiviral drugs. however, the activity of the thymidine kinase in fhv- is not equivalent to the enzyme of human herpesviruses. the second and third phosphorylation steps must be performed by host enzymes, which are not as effective in cats as they are in humans. this knowledge helps explain why the acyclic nucleoside antiviral agents developed for humans infected with hsv- are not predictably effective when administered to cats infected with fhv- and why pharmacokinetic and efficacy studies are always needed to establish appropriate dosing in cats. acyclovir has been adequately tested in cats for the treatment of fhv- , but it has a relatively low antiviral potency and poor bioavailability. a very high dose is required for effective treatment, which is associated with unacceptable toxicity, with signs related to bone marrow suppression and nephrotoxicity. a prodrug of acyclovir, valacyclovir, was developed for increased bioavailability in humans, but use for fhv- treatment in experimentally infected cats induced fatal renal and hepatic necrosis and bone marrow suppression, and did not reduce viral shedding or clinical disease severity. , therefore, despite its superior pharmacokinetics, valacyclovir should not be used in cats. ganciclovir appears to be at least -fold more effective against fhv- than acyclovir in vitro. ganciclovir is available for systemic as well as topical use in the form of a . % ophthalmic gel formulation in humans. ganciclovir holds promise for feline fhv- infection and currently available formulations warrant safety and efficacy studies in cats. tenofovir, an ntrti used for treatment of hiv, has been shown to be effective against felv in vitro. the anti-felv mechanism of tenofovir is probably similar to what has been described for hiv- . tenofovir is given in the form of a prodrug, which is converted to an acyclic nucleoside phosphate. once converted to the active diphosphate form, tenofovir is incorporated by rt into viral dna, where it acts as a chain terminator to inhibit further elongation of the viral dna. however, in vivo studies in felv-infected cats are lacking. another compound currently used for human hiv therapy, raltegravir, could be considered for the treatment of felv-infected cats. the high degree of conservation across lentiviruses, betaretroviruses, gammaretroviruses, and alpharetroviruses of integrase active sites suggests that felv might be highly sensitive to integrase inhibitors. the mechanism of action against felv is the same as for fiv, inhibition of integration of the viral dsdna that is produced by reverse transcription of the viral rna genome. an in vitro study evaluated the effective % inhibitory concentration (ec ) for felv inhibition of raltegravir in several feline cell lines and found these values are in the range of that observed for hiv and a related gammaretrovirus, xenotropic murine leukemia virus, and are well below the minimal plasma concentrations found in humans. the effective concentration of raltegravir had no appreciable effect on cell viability nor induced apoptosis, suggesting that this could be an effective and safe drug also in vivo. however, raltegravir is partly eliminated as glucuronide, a metabolic pathway that is not very efficient in cats, and it would increase the risk of toxicity resulting from drug accumulation. as of , no in vivo studies have been published. feline herpesvirus type is a member of the subfamily alphaherpesvirinae, order herpesvirales. herpes simplex viruses and and varicella zoster virus are also members of this subfamily, and antivirals developed for the treatment of these human viruses have been used for treatment of fhv- in cats. feline herpesvirus type typically infects epithelial and mucosal surfaces and travels retrograde along sensory axons to establish latency in the trigeminal ganglia. reactivated virus travels down those same axons to infect similar tissues to those that were originally infected, potentially resulting in recurrent or chronic sequelae, including keratitis, conjunctivitis, rhinosinusitis, dermatitis (figure - ) , and potentially blindness. whereas drug combinations have become standard procedure for the treatment of hiv infections, the treatment of other virus infections, including herpesviruses, is routinely based on the use of a single antiviral drug. a group of antiviral drugs known as acyclic nucleoside analogues are used for the systemic treatment of human effective against fhv- . this is potentially an alternative therapy to the use of topical drugs, the majority of which require multiple daily applications. however, an implantable silicone polymer device impregnated with penciclovir has been developed that holds promise for long-term, steadystate subconjunctival delivery of the drug for the treatment of ocular herpetic disease. although herpetic ocular disease is commonly treated with topical antiviral ophthalmic solutions or ointments (including idoxuridine, vidarabine, or trifluridine), these antivirals do not require a virus-specific phosphorylation step for activation. moreover, they damage host cells, specifically resulting in bone marrow suppression. therefore, they should not be used systemically. for good reviews of these topical drugs, see the reports of maggs and gould. cidofovir, a member of the ntrti class of drugs, has been tested for topical treatment of fhv- ocular disease but not for systemic use. it appears to be efficacious topically and is a newer drug (therefore it is included in this section). cidofovir requires the typical two host-mediated phosphorylation steps without virally mediated phosphorylation. its safety when given topically arises from its relatively high affinity for hsv dna polymerase compared with human dna polymerase. it is commercially available only in injectable form in the united states for treatment of a human betaherpesvirus. when applied topically as a . % solution twice daily to cats experimentally infected with fhv- , it led to reduced viral shedding and improvement of clinical disease compared to the placebo group. its efficacy with only twice daily administration (despite being virostatic) is believed to be due to the long tissue half-lives of the metabolites of this drug. there are reports of its experimental topical use in humans and rabbits being associated with stenosis of the nasolacrimal duct, but this has not been shown in cats. the fact that a twice-daily topical treatment is sufficient, whereas all other topical antivirals require application every to hours, makes cidofovir a useful alternative for ocular topical treatment. , , small interfering rna small interfering rnas (sirnas) designed to target the fhv- dna polymerase and glycoprotein d have been used in vitro to induce rna interference in an immortalized cell line and in primary feline corneal epithelial cells to inhibit fhv- replication. rna interference is a posttranscriptional, rna-guided gene-silencing mechanism present in eukaryotes. interference of the fhv- essential genes resulted in reduction of virus replication up to ± %. this type of therapy is intended for topical treatment of chronic herpetic disease. however, a preliminary in vivo study evaluating topical delivery of sirnas to feline corneas was unsuccessful. the lack of delivery was likely the result of sirna dilution and rapid removal by tear film and blinking. studies are ongoing to identify a means of increasing the most promising systemic drug for the treatment of fhv- is famciclovir, a prodrug of the active compound penciclovir, which has been shown to be highly efficacious in inhibiting fhv- replication in vitro. penciclovir is absorbed poorly when given orally, so the oral form famciclovir was developed with increased bioavailability and uptake from the intestinal tract. famciclovir requires di-deacetylation, mainly in the blood, and oxidation by a hepatic aldehyde oxidase for conversion to the active compound penciclovir. unfortunately, hepatic aldehyde oxidase activity is basically absent in cats, which makes the pharmacokinetics of this drug complex and results in lower than expected plasma penciclovir concentrations despite administration of relatively high doses of famciclovir. , despite this, studies evaluating famciclovir in vivo have shown it to be safe and efficacious for use in feline patients. , cats experimentally infected with fhv- and receiving famciclovir mg/kg po three times daily for days had significantly improved outcomes for systemic, ophthalmic, clinicopathologic, virologic, serologic, and histologic variables when compared with placebo-treated cats. treatment was initiated on day zero, the same day the cats were infected. even though this study did not mimic how cats with natural infection would be treated, results from a clinical case study suggested this drug is likely effective for treatment of clinical cases, though it was not blinded and placebo controlled. clinical cases with primary ocular disease, rhinosinusitis, and dermatitis each attributed to fhv- (though not definitively diagnosed), were treated with famciclovir at doses of . mg po once or twice daily for ocular herpetic disease or rhinosinusitis or up to mg po three times daily for dermatitis. famciclovir was well tolerated with each dose and had a positive effect on each clinical condition. a definitive dose rate has not been established for famciclovir. however, penciclovir has no appreciable in vitro effect if present for hours prior to infection, suggesting that famciclovir should be administered more frequently than once every hours to ensure exposure to penciclovir as additional epithelial cells become exposed to viral infection. current pharmacokinetic data suggest that dosing three times daily is required, and mg/kg po three times daily has been suggested for treatment of cats infected with fhv- , based on effective concentrations obtained in in vivo studies , and determination of new in vitro %-inhibitory concentrations. the most commonly reported adverse effects of famciclovir treatment in humans include urticaria, hallucinations, headaches, and confusion (especially in elderly humans), which would likely be more difficult to detect in animals. for these reasons, judicious use of this drug is recommended in client-owned cats, especially those with preexisting hepatic or renal insufficiency. pharmacokinetic studies have also evaluated the concentration of penciclovir in tears, and treatment with an oral dose of mg of famciclovir/kg three times daily achieves a penciclovir concentration at the ocular surface likely to be a bolus and not added to food. any benefit from lysine therapy is likely only possible with daily, lifelong treatment of cats with chronic herpetic disease, rather than use of lysine as a treatment during acute or recrudescent episodes. potentially, daily therapy would reduce episodes of viral recrudescence. however, clinical studies in pet cats are lacking. the cost of this therapy should be weighed against the potential benefits. owners should be made aware that this is only an adjunctive therapy and that administration of antiviral drugs might be necessary to gain better control of signs. polyprenyl immunostimulant (pi) is an immunomodulator that has a conditional license in the united states for treatment of fhv- infection. in blinded, placebo-controlled, experimental challenge studies, pi started on the day of virus exposure significantly reduced the severity and duration of rhinitis and conjunctivitis associated with acute fhv- disease (legendre and kuritz, manuscript in preparation). according to the manufacturer, pi upregulates the innate immune system and modulates the immune response toward a cellular response. this activity was attributed to positive effects associated with treatment of fhv- , which requires a cell-mediated immune response for control. viral titers were not compared between treatment and control groups in the studies, but based on the reduced signs associated with treatment, clinical studies are warranted. feline infectious peritonitis is associated with clinical signs that can affect almost any body system (figure - ) . currently, there is no effective treatment for fip despite its importance as the leading infectious cause of death in young cats. following the discovery that sars is caused by a coronavirus (sars-cov), efforts to find an antiviral drug for coronaviruses increased. a few antiviral agents that target different steps in the replication cycle have been tested against feline fcov. coronavirus spike proteins on the viral envelope initially bind to receptors on the host cell membrane. the spike protein mediates fusion of the viral envelope with host cell membranes. during this process, heptad repeats and (hr and hr ) of the spike protein assemble to form a complex, resulting in a conformational change that is necessary for fusion. peptides have been used as antivirals by inhibiting the hr -hr interaction, thus preventing membrane fusion. the spike protein must be cleaved for entry of the virus into the cytoplasm. feline coronavirus infection is dependent on cathepsin b, a host cysteine protease found within the cell, making this the likely protease responsible for spike protein cleavage. therefore, cathepsin b can serve as a potential target for the development of therapeutic drugs against fcov. following entry into the cell, fcov produce viral polyproteins that are processed into contact time between the corneal cells and sirnas to allow delivery. twice-daily oral l-lysine bolus administration, initiated prior to experimental infection, reduced the severity of conjunctivitis in cats undergoing primary infection. l-lysine bolus administration also reduced viral shedding in latently infected cats experimentally infected with fhv- , following changes in husbandry and housing but not following corticosteroid administration. in vitro, lysine supplementation led to reduction of fhv- replication. arginine exerts a substantial growth-promoting effect on fhv- and is an essential amino acid for viral protein synthesis, and lysine antagonizes this effect. lysine and arginine competitively inhibit transport of each other by using a common transport system, and lysine induces arginase, an enzyme that causes the degradation of arginine. arginine deficiency inhibits synthesis of infectious viral particles and downregulates synthesis of viral proteins. however, unlike the protocol for hsv- -infected humans, owners of cats receiving lysine for fhv- should not be advised to restrict their cat's arginine intake because feeding a diet lacking l-arginine is associated with a severe risk of hyperammonemia and encephalopathy. it has been suggested that the ratio of l-lysine to l-arginine, rather than the concentration of each amino acid, is critical in achieving an inhibitory effect on viral replication. dietary supplementation increases mean plasma concentrations of l-lysine without reducing l-arginine concentrations and has been shown to be safe for use in cats, up to g/kg of diet. supplementation with higher doses has been shown to result in reduced food intake. , despite promising initial in vitro data and in vivo results from experimental studies, current studies question whether viral inhibition with increased lysine concentrations, in the absence of decreased arginine concentrations, can be biologically important. a new study evaluating the effect of various ratios of l-lysine and l-arginine on fhv- dna replication in vitro demonstrated only a modest reduction in viral dna (less than log) at ratios considered difficult to obtain in vivo in healthy cats. a lack of efficacy of l-lysine supplementation has also been demonstrated in vivo in shelter settings. , dietary supplementation was unsuccessful, likely because the cats were anorexic during peak disease and were not ingesting the lysine when they needed it the most. bolus administration was also unsuccessful, likely because of stress associated with the lysine administration. , the stress of bolus administration in shelter situations could negate its effects and even cause transfer of pathogens among cats by shelter workers administering the lysine. however, data do not support dietary supplementation. , unfortunately, no studies to date have been conducted on client-owned cats; however, anecdotal evidence suggests that there is a benefit from administration of lysine in individuals. dosing is mg po twice daily, which should be given as infectious diseases ment to the host cell. the antiviral effects were concentration dependent, and nelfinavir displayed cellular toxicity at higher doses. gna was a better inhibitor of fcov, and when the two agents were added together, a synergistic antiviral effect was produced. the results suggest that the combined use of gna and nelfinavir could have therapeutic potential in the treatment of cats with fip. viral fusion has also been targeted effectively with a synthetic peptide based on the putative hr sequence of fcov. virus replication was significantly inhibited in vitro compared to controls, and the peptide was nontoxic. this peptide was also used in combination with human ifn-alpha. the two displayed a synergistic effect, but the cells were pretreated with ifn prior to infection by the virus. see the section on interferon for further information about interferon treatment for fip. immunomodulators have been considered because fip is an immune-mediated disease. a drug that has shown promise for immunomodulation is pi. this drug has a conditional license in the united states for treatment of fhv- infection. in a case series of three cats, pi was associated with prolonged survival in cats with noneffusive fip. no placebo group was included for comparison, so definitive conclusions about the effectiveness of this drug for treatment of fip cannot be drawn. additional immunostimulants such as immunoregulin (propionibacterium acnes), an inactivated bacterin, and a t-cell receptor peptide (manufactured by imulan biotherapeutics), have been suggested for treatment of fip. these are not antiviral drugs; instead, each of these products is reported to stimulate the immune response toward a cell-mediated response or to reduce an overactive type helper t-cell (th ) response. an imbalance in t-cell versus b-cell immune response has been suggested to contribute to the development of fip. it has been proposed that a strong cell-mediated immune response protects a cat from the development of fip, whereas the production of antibodies is counterproductive, enhancing the uptake and replication of feline infectious peritonitis virus (fipv) in macrophages and contributing to the pathology by producing a type iii hypersensitivity vasculitis. however, this hypothesis has been questioned. therefore, even though the use of these types of drugs for stimulation of a cell-mediated response might seem a logical approach for the treatment of fip, there is currently no data to support their use. an additional non-antiviral drug that has been evaluated for treatment of fip is propentofylline. this drug appears to downregulate proinflammatory cytokines, which in turn can reduce vasculitis. vasculitis, as stated earlier, is responsible for pathology associated with fip. however, in a placebocontrolled, double-blind study in cats with late stage fip, mature proteins by viral-specific proteases, the main protease ( c-like [ cl] protease) and the papain-like protease. because the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is also an attractive target for therapeutic intervention. in an in vitro experiment in crandell-rees feline kidney cells, cl protease inhibitors and cathepsin inhibitors were tested for their ability to inhibit fcov replication. both types of drugs produced effective inhibition with ec values in the nanomolar range and each drug tested was nontoxic to the cells at effective concentrations. the cl protease inhibitors were more effective than the cathepsin inhibitors and when used in combination, these drugs had strong synergic effects. there have not been any in vivo studies with these drugs in cats to date. in one in vitro study, antiviral compounds, including nucleoside analogues used to treat herpesviruses, nrti used for hiv, and protease inhibitors also used for hiv, were tested for their ability to inhibit fcov in cell culture. among the drugs tested, two showed significant inhibition of fcov when compared to the untreated cells. these were nelfinavir and galanthus nivalis agglutinin (gna). nelfinavir is a hiv protease inhibitor that has been shown to target the cl protease of sars-cov by interacting with residues of the protease. the drug was slightly less effective against fcov than against sars-cov, likely because only seven of the corresponding residues of the cl protease of fcov are identical to the sars-cov protease. gna, a carbohydrate-binding agent, exhibits its antiviral effect by binding to coronavirus glycosylated envelope glycoproteins, thereby inhibiting viral attach- ineffective within a few weeks because of the development of neutralizing antibodies that limit its activity. , rhuifn-α can be given orally for a longer period because no antibodies will develop during oral treatment. unlike rhuifn, rfeifnomega, being a feline recombinant product does not induce neutralizing antibodies when administered sc. this means that the high-dose parenteral protocol can be used safely and efficiently even if repeat administration is required. this is an important factor to consider in a condition where management needs to be lifelong. given po, ifns are inactivated by gastric acid and destroyed by trypsin and other proteolytic enzymes in the duodenum and therefore are not absorbed and cannot be detected in the blood after oral administration. direct antiviral effects are unlikely after oral application; however, ifn still seem to have immunomodulatory activity. type i ifns likely bind to mucosal receptors in the oral cavity, stimulating the local lymphoid tissue, leading to cytokine release from lymphatic cells in the oropharyngeal lymphoid tissues, triggering a cascade of immunologic responses that act systemically. interferons have been used for the treatment of feline retrovirus infections. treatment with ifn improved the clinical condition scores of cats infected with felv and fiv, but not because of a reduction in viral load. this suggests that the improved clinical condition seen with treatment is not specific to an antiviral effect, at least not for fiv and felv, but instead is a result of immunomodulation, potentially associated with the innate immune response. , , some clinical signs in fiv-infected cats are caused by immunopathological reactions, such as gingivostomatitis and uveitis. immunomodulation might be the cause of improvement of some clinical signs associated with ifn treatment, probably the result of an effective control over inflammatory cytokines in diseased organs. it has been suggested that a nonspecific stimulation of the immune system with ifn therapy might be contraindicated in fiv-infected cats because it could lead to a rise in viral replication produced by the activation of lymphocytes and macrophages harboring latent infections and therefore accelerate disease progression in these cats, and the use of ifn in hiv-infected humans is controversial. however, use of low-dose oral huifn (natural, not recombinant in this study) in ill fiv-infected cats ( iu per kg on the oral mucosa daily for days on alternating weeks for months, followed by a -month break, and then repetition of the -month treatment) resulted in improvement of clinical signs in a placebo-controlled, doubleblind study. parenteral rfeifn-omega used according to the licensed protocol (table - ) resulted in decreased mortality rates in felv-infected cats, compared with the control group in another placebo-controlled study. , in another study evaluating fiv-and/or felv-infected cats housed in a shelter, hematologic values remained within reference ranges, and there were no biochemical abnormalities there was no statistically significant difference in the survival time, the quality of life, or any clinical or laboratory parameter in cats treated with this drug versus cats receiving a placebo. of the cats in the study, of cats displayed effusion at the start of the study. the drug might be more useful in cats without effusion because it may have a chance to prevent vasculitis and therefore effusions, but studies are lacking. interferons are molecules produced by vertebrate cells in response to viral infections or certain inert substances, such as double-stranded rna, and other microbial agents. there are three types of ifns. type i ifns comprise the largest subfamily and include ifn-α, ifn-β, and ifn-omega. type i ifns are produced by various cell types, such as leukocytes and fibroblasts, in direct response to virus infection. there is only one member of the type ii ifn subfamily, ifn-γ, that is an immunomodulatory cytokine, produced in response to recognition of infected cells by t lymphocytes and natural killer cells of the host's immune system. type iii ifns, which contain three ils, (il- a, il- b, and il- ), are identified. this subfamily also has the ability to interfere with virus replication and has been suggested to be the ancestral antiviral system of vertebrates. interferons are not virucidal; rather, they trigger expression of various antiviral proteins and thus induce an antiviral state within the host cell to limit replication and spread of viruses. further, type i ifns have been shown to potently enhance innate and adaptive immune responses in vivo, through various immunomodulatory effects, such as activation of dendritic cells (dcs), amplification of antibody response, and enhancement of t-cell and natural killer cell cytotoxicity. viruses causing lysis of their target cell are most effectively inhibited by ifn through their antiviral activity in noninfected cells. therefore, ifns have their highest utility in the prophylaxis or early postexposure management for virus infections. given that ifns are not specific for a particular virus, they have been tested for the treatment of multiple feline viruses, including fhv- , fiv, felv, feline calicivirus (fcv), and fcov. two molecules of type i ifns are currently being used for therapy in cats: human recombinant interferon alpha (rhuifn-α), and rfeifn-omega, which is licensed for use in cats and dogs in europe, australia, and some asian countries. ifns are not strictly species-specific in their effects; however, their biologic activity and toleration are greater in cells of genetically related species. in vitro results suggest that rfeifn-omega would likely be more effective than rhuifn-α in vivo, although both ifns have been shown to have therapeutic value in cats. there are two common treatment regimens for use of rhuifn-α in cats: injection of a high dose ( to international unit per kg sc every hours) or oral application of a low dose ( to international unit [iu] per kg every h). when given parenterally to cats, rhuifn-α becomes feline coronavirus shedding was reduced but not significantly, and there was not enough fpv detected in the population to draw any significant conclusions. however, there was no placebo group used for this study, and without a placebo group, it is difficult to determine definitively if the results are due to antiviral effects of ifn or are just consistent with the natural resolution of viral shedding. in a separate study, cats with naturally acquired upper respiratory tract disease housed in a humane society facility were treated with one drop of rfeifn-omega solution ( unit/ml), rhuifn-α solution ( iu/ml), or saline ( . % nacl) solution ( cats/group) in each eye twice daily for days for the treatment of keratoconjunctivitis. there was no statistical difference between the treatment groups and the placebo group with regard to clinical scores or viral shedding (fhv- and fcv), determined by real-time quantitative pcr from oropharyngeal and conjunctival swabs. feline herpesvirus type shedding was lower, though not statistically significant, on day compared with day for all groups (including the placebo group), and clinical scores were significantly decreased on day compared to day , again for all groups including the placebo group. therefore, comparing results between days and in the treated cats without the inclusion of the placebo group would have resulted in a different conclusion for this study. these cats were not infected with felv, and even though the fiv status was unknown for all the cats, the ones that were tested were negative. however, this study highlights the need for a placebo group for accurate evaluation of the effect of ifn therapy on fhv- and fcv shedding and associated disease. oral and sc ifn therapy has been associated with an improvement in oral ulcers and gingivitis and gingivostomatitis in cats infected with fiv, , a condition that is common in cats with fcv infections. feline calicivirus is also associated with chronic gingivostomatitis in cats not infected with fiv or felv, and a study evaluated the efficacy of rfeifnomega ( iu/day for days by topical oromucosal administration) for the treatment of fcv-associated feline chronic gingivostomatitis (fcgs) and caudal stomatitis in fiv-/ felv-negative cats. cats were included in the study if they continued to show persistence of clinical signs of fcgs at least months after periodontal treatment (scaling, subgingival débridement, and polishing), tooth extraction, and weeks of antibiotics with analgesic and anti-inflammatory drugs as needed. twenty-four cats were treated with the ifn, and the effect was compared with a positive control group that received a standard corticosteroid therapy. the results suggested that the ifn therapy was as effective as the corticosteroid treatment for this condition for improvement in clinical signs. feline calicivirus viral loads were not evaluated in this study, and there was no placebo group used for comparison. however, assuming the ifn therapy was the cause of the improvement seen in these cats, the results add to the hypothesis that improvement in oral lesions in fivand/or felv-infected cats likely is associated with the effect of ifn on opportunistic viral infections. differences in the outcome of the different studies could be due to different associated with rfeifn-omega treatment used according to the licensed protocol. these findings suggest that ifn treatment is safe for treatment of fiv-and felv-infected cats, , , , but further studies are required to clearly demonstrate its efficacy against fiv and felv in vivo. a recent study evaluated the use of oral administration of rfeifn-omega for the treatment of symptomatic naturally infected, client-owned fiv-infected cats. the treatment protocol was iu/cat po every hours for consecutive days, administered by the cats' owners. a historical group that was treated sc with the licensed protocol was used as a control for comparison, but no placebo group was included. treatment resulted in significant improvement of clinical scores between pretreatment and post-treatment values, and there was no significant difference between the sc historical control group and the po group, suggesting that po administration of rfeifn-omega could be used effectively as an alternative to the licensed protocol, at a significantly reduced cost. an additional benefit of using ifn therapy for fiv and felv treatment could be the effect of ifn on opportunistic infections by other viruses, including fhv- and fcv. in fact, the effect of ifn on these additional viral infections might be the cause of the improved clinical scores associated with ifn treatment. , both fiv and felv replicate in lymphoid and monocytoid cell subsets and cause immunosuppression. in fiv-infected cats, most of the clinical signs are not directly caused by the fiv itself but are the result of secondary infections, as well as neoplasia. , although felv causes more severe clinical syndromes than fiv does, diseases secondary to immunosuppression account for a large portion of the syndromes seen in felv-infected cats as well. considering that ifn therapy seems to have no effect on fiv and felv virus load but it is immunomodulatory, it would seem advisable to treat retrovirus-infected cats with ifn when they have clinical signs, as they would benefit from its effects in improving their clinical condition. a recent study attempted to evaluate the hypothesis that improvement in clinical scores with ifn treatment in fivand felv-infected cats might be a reflection of reduction in viral shedding of secondary viruses in these cats. sixteen naturally infected fiv-and/or felv-infected cats (seven fiv, six felv, and three coinfected) were followed during rfeifn-omega therapy (used according to the licensed protocol) to monitor clinical signs and to correlate them with excretion of concomitant viruses (fcv, fhv- , fcov, and feline parvovirus [fpv] ). shedding of these viruses was evaluated by real-time quantitative polymerase chain reaction (pcr) (fhv- and fcov) or conventional pcr (fcv and fpv). pretreatment and post-treatment samples were compared. feline calicivirus shedding was detected in of cats on day and not detected on day . the amount of fhv- shedding was significantly reduced in the cats at the end of the study (day ), compared with the beginning. before euthanasia with a mean survival time of days. there was only one long-term survivor (> months) in the rfeifnomega group. interferon treatment might be more effective if started earlier, but this is not of relevance in the treatment of cats with fip in the field. however, ifn therapy might be useful for treatment of cats with chronic fcov shedding, but further studies are required. as previously mentioned, treatment with rfeifn-omega (licensed sc protocol) was associated with a decrease in fcov shedding in fiv-and/ or felv-infected cats; however, the results were not compared with a placebo group. in conclusion, antivirals are still in their infancy for the treatment of feline diseases. however, as new drugs are produced for human viral diseases that can be used for feline patients, and testing of currently available drugs continues, it is hoped that determination of effective protocols for treatment of feline viral diseases will be possible in the future. application methods (e.g., ocular versus oral); however, definitive conclusions cannot be drawn without additional studies that also evaluate viral load in naturally infected cats that are randomized, placebo-controlled, and double-blinded. ifn has also been evaluated for treatment of fip. in a randomized placebo-controlled, double-blinded treatment trial, cats with fip were treated with rfeifn-omega or placebo. in all cats, fip was confirmed by histology and/or immunohistochemical or immunofluorescence staining of fcov antigen in effusion or tissue macrophages. all cats received glucocorticoids, either as dexamethasone in case of effusion ( mg/kg intrathoracic or intraperitoneal injection every hours) or prednisolone ( mg/kg po every hours). cats also received either a placebo or rfeifn-omega at iu/kg sc every hours for days and subsequently once a week. there was no statistically significant difference in the mean survival time of cats treated with rfeifn-omega versus the placebo. cats survived for a period of to days antiviral therapy for feline herpesvirus infections pharmacological inhibition of feline immunodeficiency virus (fiv) anti-hiv drugs: compounds approved within years after the discovery of hiv antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells evaluation of different antiretroviral drug protocols on naturally infected feline immunodeficiency virus (fiv) cats in the late phase of the asymptomatic stage of infection acyclic nucleoside phosphonates: past, present and future bridging chemistry to hiv, hbv, hcv, hpv, adeno-, herpes-, and poxvirus infections: the phosphonate bridge efficacy and adverse effects of the antiviral compound plerixafor in feline immunodeficiency virus-infected cats -phosphonylmethoxypropyl) - , -diaminopurine (pmpdap) in the treatment of feline immunodeficiency virusinfected cats their discovery, development, and use in the treatment of hiv- infection: a review of the last years susceptibility of feline immunodeficiency virus/ human immunodeficiency virus type reverse transcriptase chimeras to non-nucleoside rt inhibitors clinical aspects of feline immunodeficiency and feline leukemia virus infection is azt/ tc therapy effective against fiv infection or immunopathogenesis? lymphocyte-cell immunomodulator (ltci): review of the immunopharmacology of a new veterinary biologic discovery of drugs that possess activity against feline leukemia virus a trial with ′-azido- ′, ′-dideoxythymidine and human interferon-a in cats naturally infected with feline leukaemia virus inhibition of feline leukemia virus replication by the integrase inhibitor raltegravir controlled release delivery of penciclovir via a silicone (med- ) polymer: kinetics of drug delivery and efficacy in preventing primary feline herpesvirus infection in culture a -year journey in search of selective antiviral chemotherapy effects of valacyclovir in cats infected with feline herpesvirus evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type- in vitro cytotoxicity and antiviral efficacy against feline herpesvirus type of famciclovir and its metabolites treatment of feline herpesvirus- associated disease in cats with famciclovir and related drugs pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study feline herpesvirus- : ocular manifestations, diagnosis and treatment options effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus- infection in cats evaluation of the effects of small interfering rnas on in vitro replication of feline herpesvirus- use of interfering rnas targeted against feline herpesvirus glycoprotein d for inhibition of feline herpesvirus infection of feline kidney cells therapeutic sirna: principles, challenges, and strategies evaluation of delivery agents used for introduction of small interfering rnas into feline corneal cells excess dietary lysine does not cause lysinearginine antagonism in adult cats effects of physiologic concentrations of l-lysine on in vitro replication of feline herpesvirus oral supplementation with l-lysine did not prevent upper respiratory infection in a shelter population of cats potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus c-like protease peptides corresponding to the predicted heptad repeat domain of the feline coronavirus spike protein are potent inhibitors of viral infection synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus effect of polyprenyl immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis an update on feline infectious peritonitis: virology and immunopathogenesis randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis interferons: signaling, antiviral and viral evasion use of recombinant interferon omega in feline retrovirosis: from theory to practice relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter oral recombinant feline interferon-omega as an alternative immune modulation therapy in fiv positive cats: clinical and laboratory evaluation comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, doubleblind study in cats lowdose interferon-treatment for feline immunodeficiency virus infection therapeutic effects of recombinant feline interferon-omega on feline leukemia virus (felv)-infected and felv/feline immunodeficiency virus (fiv)-coinfected symptomatic cats effects of topical ocular administration of high doses of human recombinant interferon alpha- b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis key: cord- - rlh or authors: litterman, nadia; lipinski, christopher; ekins, sean title: small molecules with antiviral activity against the ebola virus date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: rlh or the recent outbreak of the ebola virus in west africa has highlighted the clear shortage of broad-spectrum antiviral drugs for emerging viruses. there are numerous fda approved drugs and other small molecules described in the literature that could be further evaluated for their potential as antiviral compounds. these molecules are in addition to the few new antivirals that have been tested in ebola patients but were not originally developed against the ebola virus, and may play an important role as we await an effective vaccine. the balance between using fda approved drugs versus novel antivirals with minimal safety and no efficacy data in humans should be considered. we have evaluated molecules from the perspective of an experienced medicinal chemist as well as using simple molecular properties and have highlighted compounds that have desirable qualities as well as those that may be less desirable. in addition we propose that a collaborative database for sharing such published and novel information on small molecules is needed for the research community studying the ebola virus. viruses remain a constant threat to global health, with new infections from human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) killing more than million people annually , . the flavivirus that causes dengue fever infects up to million people each year, leading to death in . % of cases , . other viral outbreaks including severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory coronavirus (mers-cov), affect far fewer people but have high mortality rates and the potential to spread to epidemic size , , . thus, even with the development of vaccines and other treatments, viruses lead to a large burden on human health. more than small molecule drugs have been developed that have activity against individual viruses, including hiv, influenza, hbv, and more recently hcv , . however, a large number of virus types remain without any effective therapeutics, and there are few broadspectrum anti-virals available. thus, when viruses emerge that cause life-threatening infections, such as the recent ebola virus epidemic in west africa, there are no treatment options. since it is likely that these and other types of infectious agents will emerge in the future, an important goal is to identify inhibitors to be available to contain such outbreaks. a large array of drug discovery efforts have proven that despite their small size, viral genomes represent suitable targets for drugs. direct-acting antivirals, which target viral proteins rather than the host's, have been the subject of extensive investigation . targets in this class fall into multiple categories: virus adsorption inhibitors, inhibitors of viral dna or rna synthesis, viral protease inhibitors required for virus maturation, and viral neuraminidase inhibitors required for virus elution . in addition, cellular targets exist that are required for viral replication, including inosine monophosphate (imp) dehydrogenase, which is required to supply the pool of guanosine triphosphate (gtp) that serves as a substrate for rna and dna, and s-adenosylhomocysteine (sah) hydrolase, which is required for the methylation and hence maturation of viral dna. while finding specific inhibitors to target each viral threat individually may be ideal, the cost-savings and feasibility of finding drugs that act as broad-spectrum antivirals, or those that target specific viral genus or family, is an important goal given the expense associated with developing any one drug for one disease . there are important individual aspects of each virus, and viral family to consider, but nonetheless, many mechanisms of the viral life cycle are mirrored across families, and thus represent opportunities to learn from the many experimental studies that have already been performed. like many around the world, we have been watching the devastating effects of the ebola virus in west africa. we have been impressed by the important contributions of the health organizations and the personal sacrifices the medical workers are making to serve patients and hamper the spread of disease. and yet we began to wonder, why has there been relatively little focus on small molecules apart from a recent review of ebola virus therapeutic strategies in general ? small molecules have several advantages over other therapeutic approaches including the ability to be produced at a large scale and stability necessary for broad distribution. we felt it was time to therefore focus more on small molecules while we await a vaccine. we have found that indeed there is much prior knowledge regarding small molecules that have been shown to be active against the ebola virus in vitro or in animal models - , including a number of fda-approved drugs - . a thorough literature search of pubmed, and cas scifinder tm (cas, columbus oh) using terms including "ebola" identified molecules suggested to have activity against ebola virus in vitro and/or in vivo (supplemental table ). fda approved small molecules with activity against the ebola virus recently, a pharmacophore was generated from four fda approved compounds for other diseases (non-antivirals) resulting from two high throughput screens against the ebola virus , and closely matched the receptor-ligand pharmacophores for the ebola viral protein (vp ) . follow-up docking studies suggested that these compounds may have favorable inhibitory interactions with this receptor. vp is a cofactor in the rna polymerase transcription complex, and helps the virus evade the immune response by blocking activation of the interferon regulatory factor , which is required for the induction of interferons alpha and beta. thus, blocking vp should allow for an enhancement of the host immune response to the ebola virus. it is proposed that similar compounds may be acting via a closely related mechanism, though there has been no experimental evidence to directly prove this yet. another recent study has highlighted the ability of three clinically approved ion channel blockers to inhibit the ebola virus cellular entry. the drugs amiodarone, dronedarone, and verapamil, were given at concentrations that are possible in human serum, and were effective against a number of filoviruses. the authors hypothesized that these drugs may act by disrupting late endosomal processing or by disrupting calcium signaling that is required for viral entry. of course, none of these aforementioned fda approved drugs were designed to target the ebola virus. amodiaquine and chloroquine are antimalarials, clomiphene and toremifene are selective estrogen receptor modulators. amiodarone, dronedarone, and verapamil are anti-arrhythmics. interestingly, all of these compounds have a common tertiary amine feature, which may suggest they could act through similar mechanism , . however, they are all orally bioavailable and generally safe for humans. thus these repurposed drugs may represent a fast track to potential evaluation and approval as a feasible option for preventing the spread and mortality associated with the ebola virus in a large population. small molecules tested in humans with the ebola virus several small molecules have actually been tested in very small numbers of humans for activity against the ebola virus. for example there has been some press on favipiravir, which is undergoing phase clinical trials in the us for influenza and is approved in japan, as it has shown promising efficacy against the ebola virus in mice . faviparavir is thought to act by inhibiting the viral rna-dependent rna polymerase selectively and has demonstrated activity against a number of other viruses. at least one ebola patient, who has since recovered, was given favipiravir , and japan offered to supply it to the world health organization. a second experimental drug, brincidofovir , in phase clinical trials for treatment of cytomegalovirus and other dna viruses has shown efficacy against the ebola virus in vitro and animal studies are ongoing . brincidofovir is thought to mimic cytidine, a building block of dna, and thereby inhibit viral dna polymerases, and its mechanism of action against the ebola virus, an rna virus, is yet unknown. brincidofovir, which has demonstrated safety in humans, has been given to at least two ebola virus patients, one in dallas and one in nebraska. while unfortunately the dallas patient died, the nebraska patient survived . it is of course too early to know the effect of this molecule on the progression of the disease. this compound is a pro-drug that is converted into the active antiviral, cidofovir diphosphate. brincidofovir has higher oral bioavailability, intracellular concentrations of drug and increased antiviral potency . this compound only appeared in the literature in and there is very little published information. beyond these early stage drugs, there are a number of other compounds that have been identified as active against the ebola virus as summarized by erik de clercq . while many are not ready for in human use, they may present an attractive starting point to be refined in a future drug discovery effort. for example, a novel nucleoside analog, bcx demonstrated efficacy in mice and nonhuman primates against the ebola virus . this compound targets viral rna polymerase activity by inducing early termination of transcription and thus blocking replication. bcx is not only active against ebola virus, but also targets other members of the filovirus family as well as other rna virus families. because of the potent and efficacious effects, bcx is being fast-tracked for clinical trials in humans. medicinal chemistry analysis of small molecules active against the ebola virus we have recently described an expert's medicinal chemistry analysis of the over nih probe compounds using public and commercial sources of chemical structures and the issues related to doing this type of analysis . the likely chemistry quality of these probes was scored based on a number of criteria including literature related to the probe and potential chemical reactivity. through a series of machine learning models, we also computationally predicted the scores which were being identified through a painstaking manual process . external validation and comparison with other measures of drug-likeness and filtering rules suggested a comparable level of accuracy . we have now carefully analyzed in a similar manner the small molecules with activity against the ebola virus identified from our literature search. the chemist's (c.a.l.) decisions on compound quality are summarized in supplemental table . in contrast to the previous work in scoring compounds as chemical probes, the current aim is very specific -to treat a very serious viral disease for which there is a phenotypic readout. all the known drugs in clinical use were rejected as the chemist looked for compounds that looked more interesting in his opinion. only out of were selected as desirable in this analysis with no potential problems based on medicinal chemistry experience. in addition to this manual approach, we applied computational filters such as pan assay interference compounds (pains) to identify potentially problematic compounds from structures , (supplemental table ). pains analysis was enabled using the mmds mobile app . based on this approach we have identified several molecules that appear problematic and agreed with the medicinal chemistry analysis. for example molecules appear to fail the pains filters, including the rhodanine compound lj- which was found to be active against numerous enveloped viruses and was found through a screen of inhibitors of nipah virus entry (ic μm) . in vivo a steady state plasma concentration could not be maintained at a therapeutic level. rhodanines are known to be problematic pains compounds , . interestingly amodiaquine was not scored favorably by pains or the medicinal chemist, yet this is a successful antimalarial drug. in addition we analyzed the simple chemical properties (calculated in the collaborative drug discovery (cdd) vault (collaborative drug discovery, inc) using the chemaxon toolkit (chemaxon, budapest, hungary)) of the molecules and compared these to their medicinal chemistry classification ( table ). the mean calculated molecular property values for compounds were compared using the t-test and anova with jmp v. . . (sas institute, cary, nc). significant differences were noted in logp and lipinski rule of violations between desirable and undesirable compounds although one molecule sara- skewed the data with a molecular weight over ( table ) . removal of this compound leads to significant differences for molecular weight, number of hydrogen bond table . mean ± sd molecular properties calculated in cdd vault using chemaxon software for the molecules with activity against the ebola virus. * statistically significant p < . using the t-test and anova. ** statistically significant p < . using the t-test and anova. note data are skewed by sara- . when this molecule is removed the mean values and significance data are shown in parentheses. collaboration for the ebola virus drug discovery the research described for small molecule inhibitors against the ebola virus has occurred in a disconnected manner and there have been few efforts to summarize the total medicinal chemistry efforts to date. we think this research can learn from other areas in which there are currently efforts to improve collaboration and screening. in the area of tuberculosis research, funding from the bill and melinda gates foundation and the european commission have enabled the tb drug accelerator and the more medicines for tuberculosis, as large-scale collaborations between academia, research institutes and industry. these collaborations have promoted the selective sharing of related data in a secure environment between collaborators using the cdd vault (figure ). the centralized availability of publicly available data on compounds screened for activity against mycobacterium tuberculosis enables researchers to leverage the existing literature alongside their private data. this knowledge can be used for building of validated computational models that can help in selecting additional compounds or lead optimization , , saving time and effort. by organizing the data on small molecules tested against the ebola virus similarly in a central database and using machine learning models based on public data may help identify additional compounds for testing. such an effort may also prevent duplication of efforts as we have seen with the screening of multiple libraries of fda approved compounds against the ebola virus - . in order to catalyze this we have made the compounds (supplemental table ) freely available as a dataset in cdd public (https://app.collaborativedrug. com/register). the compound representation in cdd vault can be accessed via the text compound descriptors (as in the supplemental table ) or in a batch connection table format as an mdl format structure data file (*.sdf) that is universally read by all chemistry aware software. being able to easily retrieve the chemical structures in machine retrievable form has considerable value. identifying compounds by name or company code number does not per se allow direct access to chemical structure and often fails entirely to link compound identifier with the chemistry structure of the compound. inchikey has the great advantage of being searchable on the web (e.g. via google) but requires a lookup table (e.g. embl's unichem) to get back to the chemical structure. smiles and inchi representations allow direct access to chemical structure and are compatible with structure searching in most public chemistry databases as well as the proprietary chemistry acs cas scifinder database. the iupac name is universally used in naming chemical compounds in patents and software exists for going from iupac name to chemical structure. the compounds which are fda approved drugs for other diseases - but with activity against ebola virus in vitro or in vivo may represent useful starting points with the advantage that much is known regarding their adme and tox properties. however they may not be ideal in the opinion of a medicinal chemist. by bringing together all compounds described in the literature with activity against the ebola virus, this body of evidence may be more convincing than each study in isolation. in addition it allows us to consider structural features and molecular properties, which may provide insights into the target or mechanism of action. it is unclear whether any of the compounds might also have activity when used as combination therapy as is the current standard of care for hiv, in order to overcome drug resistance, and this needs experimental evaluation. clearly, we are a considerable distance from having an fda approved drug for the ebola virus, but this analysis illustrates that there are already drugs on the shelf that can be potentially repurposed in a shorter time period and at a lower cost and yet they do not appear to have been tested in patients with the ebola virus. in addition there are at least molecules which in the opinion of an experienced medicinal chemist are possibilities for further optimization. while novel compounds are likely more commercially viable they also will require considerable effort to assess safety. resources will need to be allocated to this effort and administrators and scientists should perhaps consider some of the medicinal chemistry insights we have provided as well as using a collaborative database to share molecules that are active amongst all scientists. it is hoped these efforts could inspire further drug discovery efforts around small molecules. to illustrate the level of interest in repurposing efforts for the ebola virus, the following studies were identified upon submission that describe additional compounds as well as those already described herein - . all authors contributed to the collaborative writing of this project. antiviral drug discovery: broadspectrum drugs from nature the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry a common feature pharmacophore for fdaapproved drugs inhibiting the ebola virus publisher full text lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (fa n- cells) pubmed abstract | publisher full text | free full text a high content screening assay for identifying lysosomotropic compounds successful treatment of advanced ebola virus infection with t- (favipiravir) in a small animal model french nurse cured of ebola contracted in liberia development of cmx (brincidofovir) for the treatment of serious diseases or conditions caused by dsdna viruses chimerix's brincidofovir has in vitro activity against ebola potential and emerging treatment options for ebola virus disease protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx computational prediction and validation of an expert's evaluation of chemical probes parallel worlds of public or commercial bioactive chemistry data new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays chemistry: chemical con artists foil drug discovery cheminformatics workflows using mobile apps a broad-spectrum antiviral targeting entry of enveloped viruses acknowledgments se acknowledges several discussions with dr. megan coffee, dr. joel s. freundlich, dr. nancy connell and dr. peter madrid. the author(s) declared that no grants were involved in supporting this work. the authors have collected small molecules reported to have activity against the ebola virus from the literature and organized them into a dataset that is easily searchable for those who may be interested in pursuing further research on the design of inhibitors of this virus. no competing interests were disclosed. the authors have searched a collection of compounds for a common pharmacophore directed against ebola proteins, and detected hits. these were filtered by an experienced medicinal chemist using well-established techniques and intuition to provide legitimate compounds that may serve the ebola research community as a publicly available resource. this is an excellent example of the power of shared, collaborative research databases providing valuable resources to the research community. no competing interests were disclosed. competing interests: key: cord- - xg guk authors: lecailtel, sylvain; broucqsault-dedrie, céline; vanbaelinghem, clément; nyunga, martine; colling, delphine; herbecq, patrick title: how unclogging a sink can be lethal: case report of an accidental methyl bromide poisoning leading to a multiple organ failure date: - - journal: j intensive care doi: . /s - - - sha: doc_id: cord_uid: xg guk methyl bromide (ch br) is a colorless and odorless volatile gas, used as an insecticide, fire extinguisher, fumigant, and refrigerant. although forbidden since for domestic use, it is still used in industry, for example, to fumigate agricultural fields which are for importation in the united states. here is the case of a -year-old man who was accidentally exposed to methyl bromide after using an old fire extinguisher. even though he finally survived, he developed a severe multiple organ failure and spent months in intensive care unit. we present in this report all the difficulties we had to diagnose this unusual poisoning. methyl bromide (ch br) is a colorless and odorless volatile gas, used as an insecticide, fire extinguisher, fumigant, and refrigerant. however, as every halogen gas, it is toxic both to environment and to humans. as a consequence, the montreal agreements have forbidden its use since . unfortunately in many countries, especially developing countries, this gas is still used. thirty-eight cases of methyl bromide poisoning were reported in the literature. ch br poisoning is usually accidental, and related to chronic, or acute exposure. we present here a case report of acute accidental methyl bromide poisoning, responsible for a severe multiple organ failure (mof). a -year-old man with an unremarkable medical history was referred to the roubaix hospital emergency department on november th . the reasons for his consultation were headaches, vomiting, walking trouble, and dysarthria of sudden onset a few hours ago. his vital signs were blood pressure: / mmhg, heart rate: /min, and body temperature: °f ( °c). the first examination of the patient revealed cerebellar signs such as ataxia, apraxia, and dysarthria. the initial level of consciousness was normal (glasgow coma score (cgs) ). the head ct scan was normal, without ischemic or hemorrhagic stroke. because of meningeal signs, a lumbar puncture was performed and found only cells per mm . the neurological investigations were completed by a brain magnetic resonance imaging (mri), confirming the absence of ischemic or hemorrhagic stroke. a brainstem stroke was also discarded, and the basilar artery was permeable. few hours later, the patient deteriorated and presented coma (gcs ), requiring intubation and mechanical ventilation. he was then transferred to our intensive care unit. the first lab results revealed a nonspecific inflammatory syndrome (white blood cells: , /μl, c-rp: mg/l) and renal failure (creatinine: μmol/l, urea: . mmol/l). all standard toxic substances were negative (alcohol, barbiturates, benzodiazepines, tricyclic antidepressants, and carbon monoxide). the chest x-ray revealed an alveolar infiltrate of the right lung. the renal ultrasound was normal. initial management included mechanical ventilation, sedation, volemic expansion, broad spectrum antibiotics to treat a possible meningitis, and intravenous acyclovir to treat a possible herpetic encephalitis. early evolution was marked by the persistence of shock requiring vasopressive support (norepinephrine). a few hours later, the patient presented an acute coronary syndrome, complicated by severe arrhythmias, namely ventricular fibrillation, responsible for a cardiogenic shock with a left ventricular ejection fraction (lvef) at %. because of multiorgan failure, including cerebral, renal, pulmonary, and cardiac dysfunctions, several diagnoses were suspected. normal levels of complement system's proteins and plasma protein electrophoresis were not in favor of an autoimmune disease. c-anca antibodies, anti-glomerular basement membrane antibodies, antinuclear, and lupus anticoagulant antibodies were negative, allowing to rule out wegener's granulomatosis, goodpasture's syndrome, and lupus erythematosus. finally, a thrombotic microangiopathy was also suspected, but adamst activity was normal. in addition, there was no hemolytic anemia, neither thrombopenia. it should be noted that viral serologies such as hiv and b and c hepatitis were also negative. after discussion with the radiologist and the neurologist, it appeared that the first mri might have been realized too early. a second one was then performed on november th (day ). it showed several abnormalities as follows: hyperintense lesions of thalami (figure a at day , the son of the patient brought us the answer: an old empty extinguisher ( figure ). the patient used it in the morning of his consultation to unclog his sink and he inhaled the content of the extinguisher, which contained methyl bromide. a blood sample was sent to specialized toxicology laboratory in paris. the methyl bromide blood level was over mg/l (normal level < . mg/l). assuming a -day half-life for methyl bromide [ ] , his serum bromide was probably much higher at his admission ( days before the blood sample). four weeks later, methyl bromide was still positive in the patient's blood ( figure ). several authors have studied methyl bromide's neurotoxicity and reported similar brain mri results. bilateral strikingly symmetrical high signal intensity on t weighted and flair sequences have been reported in the bilateral dentate nuclei of the cerebellum, periventricular area of the fourth ventricle, dorsal medulla (inferior olivary nuclei, restiform bodies, vestibular nuclei), dorsal pons, periaqueductal gray matter, superior and inferior colliculi, posterior putamen, subthalamic nuclei, and corpus callosum. these lesions were hypointense on t -weighted images and devoid of mass effect. postgadolinium studies demonstrated no enhancement [ ] [ ] [ ] [ ] [ ] . these lesions were thought to be related to local injury due to ischemia resulting from vasospasm, to increased axonal and interstitial water content, and also to a direct effect of the methyl bromide on purkinje cells' nucleic acid [ , ] . as regards to the cardiovascular system, the patient presented an acute coronary syndrome at day , responsible for a cardiogenic shock. the diagnosis was made based on the combination of an elevated troponin up to μg/l and changes in the -lead electrocardiogram. there was indeed a de novo st elevation and a pardee wave in the inferolateral territory, with mirror images in the anterior territory. the transthoracic echocardiography revealed kinetics abnormalities in the corresponding territories and a decreased left ventricle ejection fraction at %. the patient also presented ventricular fibrillation, reversed after three external electric shocks. given the fact that the patient was highly unstable, presented acute renal failure, and in the hypothesis of a septic shock, we decided not to perform an angiocoronarography immediately. the cardiogenic shock was controlled after days of cardiotropic treatment by dobutamine, and the electrocardiogram was back to anterior state within h, under cover of a treatment by double platelet anti-aggregation. one month later, the lvef increased at %. the angiocoronarography performed around day found normal coronary arteries. the assumption of a myocardial infarction induced by the methyl bromide himself was then not confirmed. the major etiological hypothesis for this cardiac and/or coronary distress is a side effect of the severe initial shock with low cardiac output. indeed, initial cardiac ultrasound showed localized kinetics abnormality, well correlated with electrocardiogram, which did not suggest a stress cardiomyopathy such as takotsubo (no contrast between apex and basis of the left ventricle). a direct cardiotoxicity of the methyl bromide is quite unlikely because no similar case has been described in the literature. as regards to renal failure, the urinary balance showed a proteinuria at . g/day. the renal ultrasound showed no obstruction of the urinary tract, two kidneys of normal size, and a good corticomedullary differentiation. the renal function got worse to reach a real anuria with creatininemia at μmol/l and ureamia at , mmol/l. extrarenal epuration was required for days. we used a continuous venovenous hemofiltration instead of intermittent dialysis because of hemodynamic unstability. a normal renal function was finally reached in days, with a creatininemia at μmol/l, and a normal urinary output. this renal failure seems to be of renal type. on the first blood sample from the emergency ward, creatinine level is already more increased than the urea is. the natriuresis was never locked. moreover, the early volemic expansion and hemodynamic stabilization had no effect on renal function and diuresis. this renal failure can easily be explained by the methyl bromide himself [ ] . as regards to respiratory failure, ventilator-associated pneumonia was suspected at day . the patient presented hyperthermia at . °f ( . °c), an important inflammatory syndrome with c-rp at mg/l and a right alveolar infiltrate on the chest x-ray. two days later, in spite of a broad-spectrum antimicrobial treatment (tazocilline), it progressed to acute respiratory distress syndrome (ards) with a pao /fio ratio at and bilateral diffuse alveolar syndrome on the chest x-ray. there was no argument in favor of a cardiogenic pulmonary oedema, in spite of the concomitant ventricular dysfunction, because the left ventricular filling pressures were low. the patient required specific ards ventilation and curarization. finally, ards quickly resolved, and the patient did not require prone positioning. the pulmonary evolution was also marked by the occurrence at day of a pneumothorax under mechanical ventilation, which had to be been drained by a chest tube. all the respiratory events were obviously not the result of direct injury of the intoxication but were induced by the neurological injury, requiring intubation and mechanical ventilation. delayed weaning from the ventilator was partly related to ventilator-associated pneumonia. in addition, the patient presented a polyneuropathy with tetraparesia. icu-acquired weakness or "critical illness polyneuropathy" was confirmed by an electromyogram showing a peripherical neurogenic disturbance. it was certainly favored by days of mechanical ventilation and the severity of the multiple organ failure [ ] . however, this kind of peripheral neuropathy has also been frequently described in cases of methyl bromide poisoning [ ] [ ] [ ] [ ] . on top of this, the methyl bromide intoxication resulted in central neurological effects, such as delayed awakening, and alertness disorders. moreover, the patient experienced swallowing disorders, probably due to the methyl bromide intoxication itself [ , ] . a percutaneous tracheotomy was performed because of weaning difficulties. finally, the patient was decanulated after days of mechanical ventilation. a brain mri, performed months later, showed that most of the lesions had improved. it remained only sequels on the left internal capsule and specifically the thalamus and the dentate nucleus. according to the literature, brain mri lesions due to methyl bromide toxicity are frequently reversible [ , , ] . he has been transferred to a rehabilitation department specialized in rehabilitation of neurological patients. to date, he is back home. after hours of physical and speech therapy, he still faces sequels such as dysarthria and ataxia and needs a zimmer to walk by himself. as far as pathophysiology is concerned, several mechanisms seem to be the cause of the methyl bromide toxicity. most of the authors have described a cellular toxic effect. indeed, following a methylation reaction, the methyl bromide conjugates itself with glutathione to make methylglutathione, which is highly toxic for the respiratory chain [ ] [ ] [ ] [ ] . a second cause often described is the modulation of the inhibitory neurotransmitter γ-aminobutyric acid receptor within the cerebellar and vestibular systems [ , , ] . a third hypothesis attributes toxicity to methyl bromide metabolites such as methanethiol and formaldehyde [ , ] . another suggested mechanism is that methyl bromide rapidly inhibits creatine kinase activity in all regions of the brain and in other target organs. creatine kinase is an enzyme catalyzing the conversion of adenosine triphosphate and creatine to adenosine diphosphate and phosphocreatine [ , ] . cases of methyl bromide poisoning have become rare since his prohibition in thanks to the montreal agreements. these cases being rare, it makes it even harder to establish the proper diagnosis. the clinical presentation of the acute methyl bromide poisoning can be tricky. however, common clinical signs have been reported, such as headaches, vomitings, cerebellar signs, and seizures [ ] . our patient presented all of them, except for seizures, which are however quite frequent in this intoxication [ ] . several complications have been described such as ards and acute kidney injury. our patient experienced all these complications. some authors reported the use of extrarenal epuration [ , ] , but they highlight the importance of the earliness of this treatment. in chronic intoxication, signs are quite different. neuropathies, liver insufficiencies, kidney insufficiencies, and neuropsychiatric disorders were reported [ ] . however, no antidote is available. many treatments have been tested such as glucagon or n-acetylcysteine but are ineffective [ , ] . written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor in chief of this journal. illness associated with exposure to methyl bromidefumigated produce-california the neurological effects of methyl bromide intoxication neurological manifestation of methyl bromide intoxication diffuse lesion in the splenium of the corpus callosum in patients with methyl bromide poisoning a case of chronic methyl bromide intoxication showing symmetrical lesions in the basal ganglia and brain stem on magnetic resonance imaging methyl bromide intoxication causes reversible symmetric brainstem and cerebellar mri lesions case report: mri of the brain in metronidazole toxicity reversible resonance imaging findings in metronidazole induced encephalopathy methyl bromide poisoning critical illness polyneuropathy. a review of the literature, definition and pathophysiology central and peripheral neurotoxic effects of chronic methyl bromide intoxication central nervous system toxicity and early peripheral neuropathy following dermal exposure to methyl bromide peripheral neuropathy induced by acute methyl bromide skin exposure: a case report methyl bromide induced neuropathy: a clinical, neurophysiological, and morphological study the neurological effects of methyl bromide poisoning mr imaging of metronidazole induced encephalopathy: lesion distribution and diffusionweighted imaging findings determination of acute toxic effects in mice following exposure to methyl bromide studies on the mechanisms of methyl bromide induced olfactory toxicity effect of methyl bromide on regional brain glutathione, glutathione-stransferases, monoamines, and amino acids in rats inhibition of the acute toxicity of methyl chloride in male b c f mice by glutathione depletion national pesticide information center magnetic resonance for evaluation of toxic encephalopathies: implications from animal experiments inhibition of creatine kinase activity in rat brain by methyl bromide gas a cluster of neurological signs and symptoms in soil fumigators three cases of acute methyl bromide poisoning in a seedling farm family treatment of methyl bromide poisoning with haemodialysis exposure of the skin to methyl bromide: a study of six cases occupationally exposed to high concentrations during fumigation glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning we would like to particularly thank the toxicology laboratory of paris which performed all the methyl bromide blood tests, and more precisely doctor antoine villa: centre antipoison et de toxicovigilance de paris, hôpital fernand widal, rue du faubourg saint denis, paris cedex . the authors declare that they have no competing interests. all authors were involved in the patient care at bedside. cbd and sl grafted the manuscript. all authors read and approved the final manuscript. key: cord- -yyh kikb authors: hossain, liaquat; karimi, faezeh; wigand, rolf t.; crawford, john w. title: evolutionary longitudinal network dynamics of global zoonotic research date: - - journal: scientometrics doi: . /s - - -y sha: doc_id: cord_uid: yyh kikb at global and local levels, we are observing an increasing range and rate of disease outbreaks that show evidence of jumping from animals to humans, and from food to humans. zoonotic infections (i.e. hendra, swine flu, anthrax) affect animal health and can be deadly to humans. the increasing rate of outbreaks of infectious diseases transferring from animals to humans (i.e. zoonotic diseases) necessitates detailed understanding of the education, research and practice of animal health and its connection to human health. these emerging microbial threats underline the need to exploring the evolutionary dynamics of zoonotic research across public health and animal health. this study investigates the collaboration network of different countries engaged in conducting zoonotic research. we explore the dynamics of this network from to based on large scientific data developed from scopus. in our analyses, we compare several properties of the network including density, clustering coefficient, giant component and centrality measures over time. we also map the network over different time intervals using vosviewer. we analyzed publication records. we found united states and united kingdom as the most collaborative countries working with and other countries in and cases, respectively. our results show increasing close collaboration among scientists from the united states, several european countries including united kingdom, italy, france, netherland, switzerland, china and australia with scientists from other parts of the world. united states, several european countries including united kingdom, italy, france, netherland, switzerland, china and australia with scientists from other parts of the world. background zoonosis can be referred to as the transmissible diseases between vertebrate animals and humans (who ) , which comprises % of emerging infectious diseases (taylor et al. ) . therefore, successful management of zoonotic diseases risks and outbreaks require the understanding of the complex interaction network of humans, animals and their living environments (who b) . previous bibliometric studies on relevant topics either investigated specific infectious diseases such as acquired immune deficiency syndrome (aids) (patra and chand ; uthman ) , tuberculosis (ramos et al. ) , and malaria (garg et al. ) or examined infectious diseases in general (bliziotis et al. ; ramos et al. ramos et al. , takahashi-omoe and omoe ) . the latter studies examined the research productivity and contribution of different countries and regions of the world in infectious diseases showing a gradual increase in research on infectious diseases in the us, the eu and other regions in the world. our investigation focuses on the contribution and collaboration of countries in exploring the intersection between animal and human health. we provide an investigation of the dynamics of zoonotic research networks over years by constructing and using large scientometric data. the study first explains the process of developing scientometric data for exploring research collaboration on this topic. these data are based on the extracted publication information from elsevier's scopus in the span of - . it proceeds with exploring these data by extracting a bibliometric networks (i.e. countries network). several social network measures such as network density and centrality are employed to analyze this network. the countries collaboration trend, network maps and measures, and their dynamics over this period of time are then discussed. elsevier's scopus (www.scopus.com) as one of the main sources of bibliometric data covering the greatest number of journals romo-fernández et al. ) is used to build the database of this study. the search for publications has been carried out with search queries using combinations of keywords including ''coordination, collaboration, cooperation, communication, preparedness, surveillance, emergency response, crisis management, containment, recovery, zoonotic, zoonosis, animal human, disease outbreak, illness outbreak, epidemic, pandemic and social network'' occurring in the articles' titles, abstracts and keywords. this initial set of keywords was selected after consulting with two experts in the field. the focus of the keywords was on three concepts including coordination, zoonotic diseases, and disease outbreaks at the various stages of disease prevention, detection, effective response and elimination. the publications information [e.g., author(s), document title, year, source title, citation count, source and document type, affiliations, publisher] were extracted using scopus export option. the publications used in the subsequent analysis were restricted to the ones in english. it is theoretically possible to miss out certain publications in the search process explained earlier. in order to minimize any missed out document and to account for any important keyword that was not included in the first stage, another set of keywords were identified to run a second round of search. as such, in the second stage, the keywords used in the extracted publications from the first stage were analyzed for their frequency. the frequency analysis of the keywords included identifying the most frequent ''single word'' and ''multiple words'' keywords. the latter keywords were the original keywords used by the authors and the former keywords were produced by splitting the ''multiple words'' ones. table shows the top ten most frequent ''single and multiple words'' keywords. another set of keywords including ''avian influenza, west nile virus, h n , control, risk'' were used in combination with ''coordination, collaboration, cooperation, communication, preparedness, surveillance, emergency response, crisis management, containment, recovery, outbreak, epidemic, pandemic and social network'' for a second round of search for publications ( search queries). the keywords that were too generic or used in the previous stage such as virus were not included in this round of search. the extracted publication data from this round was added to the previous results. the search span in both stages consisted of the period from to . the search for the publications was conducted in july . the two rounds of search resulted in publications of different types (e.g., article, conference paper, review) after filtering the publications with the same title. an application program was developed to extract bibliometric networks from these data, discussed in the next section. at least four bibliometric networks can be built using the database of publications developed in this study including networks involving authors, keywords, countries, and affiliations. figure shows these networks. in order to build these network (i.e. identify the links between the nodes) from the information available in the database, an application program is written in matlab. the algorithm of this application is explained here for the co- authorship network. the process of building the other networks including co-word, countries, and affiliation uses the same algorithm. there is a co-authorship relation between two authors if they wrote a document together. as such, in social network terms, there is a link (edge) from node a (author a) to n ode b (author b). to map the data exported from scopus into a co-authorship network, the following algorithm was developed where its variables are presented below: • max-no-papers maximum number of papers in the database with known authors • max-no-aut-paper maximum number of authors in a paper in the database • max-no-authors maximum number of authors in the database • list-all-authors a list containing the name of all the authors in the database • list-max-no-authors-per-paper a list indicating the maximum number of authors for each paper in the database • list-all-papers-with-all-their-authors a list of all authors for each paper in the database • co-authorship-initial a matrix with the size of (max-no-papers, max-no-aut-paper) that its cell (i,j) indicates whether author j in list-all-authors participated in writing paper i in the database • weighting a matrix with its cell (i,j) indicating the number of papers wrote by authors i and j in list-all-authors • co-authorship a matrix with its cell (i,j) indicating whether authors i and j in list-all-authors wrote a paper together or not in order to build the network of collaborating countries, the affiliation records of each publication were processed to extract the countries cited. in table below, we show some examples of the affiliation records in the database. the name of the countries, when reported, appears at the end of the record. in order to extract the countries associated with each publication, the steps explained below were followed: • reverse the affiliation record string, • count the number of characters till the first occurrence of a space (e.g., x characters), • extracting the first ''x'' characters identified in the previous step from the reversed string, • reverse the string extracted in step three which gives the name of the country reported. publications contained no affiliation information. these publications were excluded from the countries network analysis. further data cleaning process in this stage included identifying the variations in the name of the countries reported (e.g., united states, usa, us, united kingdom, uk) and unifying them. if an affiliation entry did not include the name of the country or useful information (e.g., the name of an institution) to search for the correct country of origin, it was excluded from further analysis ( publications). some other cases of data cleaning included finding the relevant country associated with a university, institution, company, state, or city where the name of the country was missing. this resulted in publications to carry out the data analysis. in this study, we apply the following network measures to perform our analysis: the density measure ''describes the general level of linkage among the points in a graph'' (scott , p. ) . in social network analysis terms, this is the number of links in a network, expressed as a proportion of the maximum possible number of links (scott ) . the density of a network increases as the number of linkages between its nodes grows. the densest network (with all its nodes linked together) has a density of and the least dense network (with no node linkage) has a density of . degree centrality- freeman ( ) defined degree centrality of a node as the number of its adjacent nodes. two nodes are adjacent if an edge links them together. therefore, degree centrality of a node counts the number of other nodes that are directly connected to it. the closeness centrality of a node is the sum of the graph-theoretic distances of that node to all other nodes in the network. the distance of a node from another is the length of the shortest path (geodesic path) between them (borgatti ; freeman ). normalized closeness centrality value of a node is calculated by dividing the number of all other nodes in the network by the sum of the distances of the node to all others (freeman ; leydesdorff ). the betweenness centrality of a node is defined as the frequency with which it settles in the shortest path connecting any other pair of nodes in the network (freeman ) the giant component in many cases, large and complex networks are seen to have a connected component that includes a substantial portion of the nodes in those networks. this connected component is referred to as the giant component. if a network has a giant component, it is usually only one (easley and kleinberg ) . the clustering coefficient of a node (e.g., node a) refers to the probability that two randomly selected adjacent nodes of a are adjacent to each other. in other words, it is the fraction of the pairs of a's adjacent nodes that are linked together (easley and kleinberg, ) . clustering and mapping vosviewer . . is used for displaying the structure of countries network. vosviewer provides both mapping and clustering of networks (especially bibliometric networks) in a unified approach as an alternative to combing the mapping and clustering techniques with different assumptions. its clustering technique is based on a weighted and parameterized variation of modularity-based clustering, and it uses visualization of similarities (vos) as its mapping technique . vos is a distance-based mapping technique rather than a graph-based one. while in the latter technique the distance between two nodes is not necessarily meaningful, in the former technique this distance represents the strength of their relationship (van eck and waltman ). vosviewer provides different visualizations of networks. in the label views, the size of a nodes' circle and label portray its importance. larger circles and labels represent more important nodes in terms of their weight (van eck and waltman ). the weight of the nodes in the countries networks in our study are determined based on the number of other countries associated with them and the strength of the associations. the color of the circles also depicts the cluster the node belongs to (van eck and waltman ) . the density view is helpful for identifying the most important areas of a map. in this view, the nodes are represented with the same label structure as the label view. the color of the point a node is placed in depends on the number of nodes around that point and their weights (i.e. their density). more nodes with greater weight neighboring a node lead to greater density for that node. according to the default color scheme used by vosviewer, three color (redgreen-blue) represent density where red and blue are assigned to the highest and lowest densities respectively (van eck and waltman ). as shown in fig. , the trend of publications on zoonotic research has been increasing since . while before , the number of extracted publications is constantly low, an increasing trend starts after that. this increase in the number of publications continues gradually and accelerates after . this observation provides three time intervals to examine detail changes in the collaboration networks including - , - , and - . the dynamic analysis in this study focuses on the last two time intervals as the number of publications in the first period is limited. another interesting point of time in fig. is . up to this year the number of publications is increasing but this upward trend halts here with occasional rises. to have a better understanding of the possible underlying reasons for such a trend in the zoonotic research output, the frequency of zoonotic research publications and who's disease outbreak news per year (who a) since are depicted in fig. . three highest points of disease outbreak news occurred in [due to suspected severe acute respiratory syndrome (sars) pandemic], (due to avian influenza pandemic) and (due to h n pandemic). after , with sars and avian influenza pandemics, the publications on zoonotic research grew rapidly, and then started to decline after , but again raised in with the spread of the h n pandemic to decrease again with the reduction in the disease outbreak incidences. it seems that the output of scientific research in the zoonotic disease outbreaks have reached a saturation level since and only occurrence of global disease outbreaks triggers increases in quantity of the related publications. as such, the changes of zoonotic research collaboration networks will also be examined for another two periods including - and - . table also shows the top ten journals publishing on this topic over the years examined in this study, in which emerging infectious diseases, veterinary record, euro surveillance: european communicable disease bulletin, and plos one hold the first three positions. countries collaboration trend figure below illustrates the changes in collaboration among the countries over time in terms of the number of collaborating countries, distinct collaboration links between them, and total number of collaboration links per year. the first instances of international collaboration start from and steadily increase although the number of collaboration links among countries experiences some rises and falls over the years. the highest amount of collaborations ( distinct links and total occurrences) takes place in among countries. in , the amount of collaborations drops to distinct collaboration ties while the number of participating countries increases to . since - the collaborating countries consist of - countries which are nearly % of all the countries ( ) in our database. in other words, the countries collaboration network at its most collaborative status comprises half of the publishing countries. in addition, the trend of recruiting more collaborating countries each year although is overall growing; its pace slows down after . however, the number of collaboration links between the present countries and the frequency of such collaborations shows fast increase (with occasional declines). as such, it seems that after the community of collaborating countries is more focused on having more collaboration with other existing countries in the community and strengthening these collaborative relationships. countries network measures and maps over time table demonstrates the measures of countries network in different periods. in the first period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , the density of the network is very low ( . %) indicating that a limited number of all possible collaboration links among countries are realized (see fig. a ). in addition, the high clustering coefficient ( . %) implies the high possibility of collaboration among two adjacent countries of a third country. figure a , pertaining to this period, illustrates this implication in the form of several triangles in the network. the network also has a giant component and several other small components. the network's degree of centrality is average ( . %). as shown in fig. a , the countries are gathered around a few central nodes in the network including the united states and united kingdom. they also possess the largest fig. countries network map during ( - ) and ( - ) . a label view of countries network ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b label view of countries network ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) labels. south africa, germany, and italy (overlaid by germany in the map illustration), and france are the next countries with large labels which indicate their importance in the zoonotic research in this period. there is also a split evident in the map which separates countries around united states from countries around united kingdom. in other words, there is low density between the two areas. this is an implication of less collaboration among these two important areas of the network. in the second period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , the density of the network has improved (almost doubled) although it is still low. there is also a slight improvement in the clustering coefficient value ( . %). the degree centrality shows considerable improvement ( . %). as shown in fig. b , the countries are coming closer together compared to the previous period. the network closeness and the betweenness have decreased. the high value of closeness ( . %) and low value of betweenness ( . %) measures implies the low distance between the countries. the network measure for the whole period of time is similar to the second time period, which is expected given that most of the publications belong to this period. similar trend in the values of the network measures are observed in the other two periods ( - and - ) . the interesting observation here together with the information from fig. is the high value of clustering coefficient ( . %), density ( . %) and degree centrality ( . %) in - period which indicates the high probability that two collaborators of a country will be collaborating with each other and countries having more collaborations and collaborators. these values are higher than the seven years before . in other words, in the recent years, the countries were putting more effort in strengthening their relationship with more other countries (see figs. a, b) . the label views of the network in the whole period (fig. a) shows that united states and united kingdom are the major collaborating countries. italy, france and netherlands are the next main countries. as evident in the density view (fig. b) , united states and united kingdom are depicted in red indicating their high density; although united kingdom is nearer to other moderate density areas of the map while united states is placed farther from this part of the map. these countries as well as the others shown in red or orange comprise the important part of the world in terms of research, publication, and collaboration on zoonotic research. top collaborating countries table lists the countries with or more total collaborations and the corresponding number of countries they collaborated with. according to this list, united states and united kingdom are the first and second most collaborative countries working with and other countries in and cases, respectively. in addition, the number of countries they collaborate with exceeds the others. italy, france and netherland are the next most collaborative countries; although france's collaborating countries ( ) are slightly more than italy ( ). this result complements previous studies on the output of infectious disease research community. previous studies show that the united states and the western european countries productivity exceed the other countries in terms of publication on infectious disease research (bliziotis et al. ; takahashi-omoe and omoe ) , and united kingdom, france, and germany lead the european countries in terms of number of publication on this topic (ramos et al. (ramos et al. , ). our results show a similar pattern in terms of collaboration efforts on zoonotic research. the united states, united kingdom, france, italy and netherlands are the leading collaborative countries in zoonotic research. table presents the list of the strongest collaboration links among the countries. strength of a collaboration link is defined as the frequency of its occurrence during the years. - and - - - dynamic analysis during the period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , united states, united kingdom, germany, italy, and south africa are the top collaborating countries. in the second period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , the number of collaborators and frequency of collaborations increase. while the united states and united kingdom still hold their position at the top of the list of most collaborative countries, some changes are observed in the countries that follow them. south africa is no longer among the top countries. italy, france and netherlands have progressed further up the list. china, australia and switzerland also managed to appear among the top ten collaborating countries (see table for further details). to identify their collaboration growth rates, first the average amount of collaboration for each country in each of two time periods (av = total collaborations/years) are calculated which is presented in table . the growth rate is then obtain as the quotient of the second and first periods' average collaborations (growth rate = av /av ). if a country did not have any collaboration in the first period its average collaboration for the second period is considered as the growth rate. the collaboration growth rates of the countries are identified for the - and - periods as they represent more recent data compared to the other time periods ( - and - ) examined in this study. nigeria, mexico, portugal, egypt and singapore collaboration growth rates exceed the other countries. this study provides longitudinal analysis of countries networks of zoonotic research during - based on large scientific data developed from scopus. the overall properties of these networks as well as their dynamics over these years are examined in detail. the countries network shows steady increase in collaboration among different countries. the united states is the most collaborative country having links with countries and total instances of collaborations. several european countries including united kingdom, italy, france, netherlands, switzerland, germany and belgium are among the top ten collaborating countries. china and australia are also among the ten most collaborating countries. nevertheless, the density of the network is still low which means that limited numbers of countries are collaborating. however, this trend is improving such that almost half of the countries had at least one collaboration link in the recent years. in addition, in spite of the recent lower rate of addition of more countries into the network, within the current network, the countries are increasingly initiating new collaborations as well as enhancing these collaborating relations (strengthening them). conflict of interest none. investigating attachment behavior of nodes during evolution of a complex social network: a case of a scientific collaboration network evolutionary dynamics of scientific collaboration networks: multi-levels and cross-time analysis worldwide trends in quantity and quality of published articles in the field of infectious diseases networks, crowds, and markets: reasoning about a highly connected world centrality in social networks conceptual clarification bibliometrics of global malaria vaccine research betweenness centrality as an indicator of the interdisciplinarity of scientific journals hiv/aids research in india: a bibliometric study. library and information science research publication of european union research on infectious diseases a bibliometric overview of infectious diseases research in european countries a bibliometric analysis of tuberculosis research indexed in pubmed co-word based thematic analysis of renewable energy social network analysis: a handbook worldwide trends in infectious disease research revealed by a new bibliometric method risk factors for human disease emergence software survey: vosviewer, a computer program for bibliometric mapping vosviewer manual a unified approach to mapping and clustering of bibliometric networks zoonoses and the human-animal-ecosystems interface no. key: cord- - kddituy authors: shirbaghaee, zeinab; bolhassani, azam title: different applications of virus‐like particles in biology and medicine: vaccination and delivery systems date: - - journal: biopolymers doi: . /bip. sha: doc_id: cord_uid: kddituy virus‐like particles (vlps) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. vlps can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and e. coli were used to express recombinant proteins, especially for vlp production. recent studies are also generating vlps in plants using different transient expression vectors for edible vaccines. vlps and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. herein, we describe vlp production in different systems as well as its applications in biology and medicine. © wiley periodicals, inc. biopolymers : – , . v irus-like particles (vlps) known as viral "empty shells" maintain the same structural properties of virions, without genome. these constructs are considered very efficient as vaccine platforms and therapeutic delivery systems. many antigens can readily be displayed on the surface of vlps. these antigens can be genetically or chemically fused to the vlp. regarding to the reports, the immune stimulation by vlps contains: (a) stimulation of innate immunity through tlrs and pattern recognition receptors (prrs) due to the expression of multivalent structures; (b) induction of strong humoral response and also igm in t-cell independent way; and (c) enhancement of the uptake, processing and presentation by apcs through mhc i and mhc ii cross-presentation pathway due to the particulate nature of vlps. vlps can be subcutaneously or intramuscularly injected. their small diameter facilitates entry into lymphatic vessels and direct drainage into local lymph nodes. once in the lymph node, vlps are taken up by lymph node resident dendritic cells (dcs). this uptake is enhanced by the size and form of vlps. vlps stimulate cd t cells via the mhc ii pathway, as well as highly efficient cross-presentation on the mhc class i pathway. generally, viral-like particles, are considered as vaccine candidates because their natural properties such as multimeric antigens and their specific structures are suitable for the stimulation of efficient humoral and cellular immunity. currently, the development of recombinant subunit vaccines (suvs) has been significantly increased using heterologous expression systems. antigens derived from many bacterial, viral, fungal and parasitic pathogens were used for safe and effective vaccination. five vlp-based vaccines have been already approved including three for hbv and two for hpv, while in the veterinary field; a vlp-based vaccine against porcine circovirus type (pcv ) has been approved. some vlp-based vaccines targeting human and animal diseases are recently in late stages of clinical trials. vlps have a positive value as academic, industrial, and commercial systems especially in gene therapy and design of nanomaterials. however, the study of the vlp-based applications (vaccination, gene and drug delivery, and imaging) must be followed to show the reliability, and cost efficiency of this technology. furthermore, the expression systems would be improved to achieve the best strategy for vlp production from different viral genes. this review will focus on vlp characteristics and its applications especially as vaccines or delivery systems for dna, sirna and drugs. it should be noted that in the vaccination field whenever a viral-like particle carries genetic material is called "vectored vaccines " and in gene therapy, they are called viral vectors. however, for simplicity in this review, we called all particles entitled as viral-like particles (vlps). viral-like particles (vlps) have been generated for over thirty various infectious viruses in animals and humans. vlps are composed of one or more structural (/capsid) proteins possessing natural properties for self-assembly, and are morphologically similar to authentic viruses. , comparing to live viruses, vlps are non-replicating and non-infective due to the lack of infectious genetic material. virus-like particles have the potential to be used as safe vaccine candidates without the need for any adjuvant. , different viruses present different structures for generation of viral-like particles such as: a. simple viral capsids with one or two major proteins (e.g., parvoviruses, papillomaviruses, circovirses, calciviruses, hepatitis e virus (hev) and polyomaviruses). b. complex viral capsids with various protein layers, encoded by many distinct mrnas, or generated from a single polyprotein (e.g., picornaviruses). c. viral capsids with lipid envelopes including a lipid bilayer obtained from the host cell, as well as viral glycoprotein spikes (e.g., influenza, hiv and hepatitis c). , figure shows the general model of vlp along with its applications. the selection of expression vector is one of the major factors in vlp generation. the reports showed the successful production of vlps indicating that bacterial systems, yeast and insect systems are used in %, %, and % of the cases. in addition, mammalian cells ( %) and plants ( %) were usually applied to produce vlps with special properties. bacterial systems are often included the commercial e.coli strains and expression vectors, to produce non-enveloped vlps in high levels compared to other systems (table i) . in addition, bacterial cells have been applied for generation of vlps which need several types of structural proteins, such as the avibirnavirus ibdv vp , vp , and vp -polyproteins. the reports indicated that the expression of the hepatitis b virus (hbv) capsid protein in e. coli leads to the formation of structures similar to the hbv core (hbc) particle. bacterial figure general model of vlp along with its applications: the picture shows the recombinant hpv l pentamers assembled in vitro into capsid-like structures. self-assembly of recombinant viral coat proteins into empty capsids is a promising strategy for production of virus-like particles (vlps) in vaccine design. the resulting vlps can induce a protective immune response by mimicking the authentic epitopes of virions. systems are not always a desired plan for vlp production due to several factors, such as (a) lack of ability to generate recombinant proteins with mammalian-like post-translational modifications (ptm), (b) failure to produce the correct disulfide bonds, (c) drawbacks of protein solubility, and d) the existence of lipopolysaccharides (lps)/or endotoxins in production of recombinant proteins (rp). viral coat proteins (cps) can be efficiently produced as insoluble inclusion bodies, purified under denaturing conditions, refolded, and selfassembled, as indicated in the parvovirus b and the ccmv and cmv plant viruses. a simple change in the cultivation conditions such as low-temperature can solve the problem of inclusion bodies and induce the formation of soluble vlps, as performed for two viral systems, the densovirus ihhnv, and the potyvirus pvy. some factors including the resistance markers of the expression plasmids and the composition of the cultivation medium can also change the vlp assembly (e.g., bacteriophage qb). another strategy applied to increase expression levels and solubility involves the use of different fusion protein systems, e.g., glutathione-s-transferase (gst) fusion proteins such as the papillomavirus l , the polyomavirus mupyv, and the picornavirus fmdv. [ ] [ ] [ ] [ ] other prokaryotic hosts have been recently used to generate vlp, e.g., lactobacillus. the intracellular assembly of hpv l vlp was reported in lactobacillus casei, a lactose-inducible expression strain. furthermore, the production of l vlps using lactobacillus developed new live mucosal prophylactic vaccines (table i) . , a pseudomonas fluorescens (p. fluorescens) expression system is an efficient choice against e. coli, because of simple manipulation, high yields of active and soluble proteins, and largescale cultivation. some differences between p. fluorescens and e. coli including the various sizes of genome, and diverse metabolic approaches can influence the generation of recombinant proteins. the capsid protein of a plant bromovirus, the cowpea chlorotic mottle virus (ccmv), has been recently expressed as a soluble form in p. fluorescens, and assembled into vlps in vivo. this construct was structurally similar to the natural viral particles provided from plants. eukaryotic expression systems are a striking alternative to bacteria, especially for solving the problem of bacterial endotoxins in vaccine development. some structural genes of mammalian viruses expressed in yeast are able to form the vlp. this expression host has been efficiently applied to generate the first licensed hbv vaccine. hbsag is one of the antigens commonly utilized for production of vlp-based hbv vaccine. hbsag has been expressed in pichia pastoris (p. pastoris), sac-charomyces cerevisiae (s. cerevisiae) and hansenula polymorpha (h. polymorpha) (table i) . , it is critical to consider that the viral-like particles are not always formed during the cultivation procedure of the yeast cells. these studies showed that the selfassembly of the vlps in pichia system should be completed during the protein purification. , [ ] [ ] [ ] the expression and selfassembly of recombinant bacteriophage q coat protein (q-cp) was indicated in saccharomyces cerevisiae and pichia pastoris. the yeast-derived q-vlps were greatly immunogenic in mouse similar to that in e.coli-derived q-vlps. ms vlps produced in saccharomyces cerevisiae could package functional heterologous mrnas. for example, the linkage of the ms packaging sequence to the human growth hormone mrna allowed the packaging of the mrna in ms vlps. indeed, the high stability of ms vlps suggests them as an efficient delivery system for rna-based vaccines. the p. pastoris system was also utilized as a potent alternative for expression of ccmv coat protein vlps due to easy manipulation and high expression levels. in addition, this system has been utilized to express efficiently the premembrane and envelope glycoproteins of dengue virus type (denv- ), hbsag, , hccag resulting in the generation of vlps. the major advantage of yeast systems is the ptm including phosphorylation or glycosylation, as indicated in hbv vlps. the studies indicated that hbc phosphorylation plays a major role in viral replication and capsid formation. such yeast-derived hbc vlps are valuable for vaccination and diagnostics. furthermore, the potent multigene expression systems have been constructed in yeasts. for example, the expression of three rotavirus structural genes from a single plasmid vector led to the generation of triple layered vlps in saccharomyces cerevisiae. , however, the multimerization of protein into vlps is not supported for the enveloped viruses (e.g., gag vlps of hiv- ), suggesting that yeast does not have the essential factors of host. thus, the generation of enveloped hiv- pr gag vlps has been performed using s. cerevisiae spheroplasts, morphologically similar to immature viral particles. , in general, the construction of yeast expression systems, especially hansenula and pichia strains, are more difficult than bacterial vectors. in addition, the yield of vlp production is less than that in e.coli. other limitation of yeast system is its dissimilarity with mammalian cells in the ptm of proteins, especially glycosylation. , therefore, this system is more suitable for the generation of non-enveloped viral-like particles. another attractive system utilized broadly for production of vlp is the baculovirus-insect cell expression system, due to shirbaghaee and bolhassani some advantages, such as the rapid growth ratios, the culture preparation in large-scale, and the ptm of the target proteins similar to mammalian cells. [ ] [ ] [ ] the results showed that both yeast and insect cells were previously used for the vp expression of several polyomaviruses, and its assembly into viral-like particle. in addition, insect cells were used to provide vlpbased vaccines, e.g., the approved hpv vaccine, cervarix. indeed, insect cells are able to generate both vlp types (i.e., enveloped and non-enveloped). there are enveloped vlps in clinical trials. the main limitation of insect cell system is protein contamination with the enveloped baculovirus particles, suggesting the development of efficient plans for purification of vlps. recently, co-expression of four genes of human influenza h n virus (i.e., ha, na, m , and m ) in insect cells led to generate influenza vlps which protected mice against h n virus challenge. these data suggested that viral-like particles are a hopeful vaccine candidate for h n influenza and probably other subtypes of virulent avian influenza viruses. the non-infectious viral-like particles of the alphavirus sav was also generated using the recombinant baculoviruses expressing sav capsid protein and two major immunodominant viral glycoproteins (e and e ) in insect cells. moreover, baculovirus expression system was utilized to generate vlps from cowpea mosaic virus (cpmv), tomato bushy stunt virus, and entorovirus (ev ). , , recently, non-replicative baculovirus have been developed to cope with the problem of baculovirus contamination. stable systems using insect cells have been also tested. moreover, silkworm expression systems were efficiently applied to generate vlps and the surface of vlps could be changed by some strategies, irrespective if their constructs are enveloped or not. silkworms show a high capability for production of recombinant proteins, in comparison with insect cells, and also easy and inexpensive protein preparation similar to e.coli expression system. for over two decades, different mammalian cell lines have been developed as a source of commercial therapeutic proteins for clinical applications, because of their ability for proper protein folding, assembly and ptm (e.g., the correct glycosylation pattern). , however, high costs of production and potential safety concerns remained a challenge for these systems. the mammalian cells were progressively utilized to produce vlpbased vaccines , , e.g., for influenza viruses. for instance, the generation of a stable mammalian cell line (e.g., vero cells) expressing four influenza structural proteins (ha, na, m , and matrix (m )) led to form hybrid vlps containing matrix proteins, and surface glycoproteins of h n and h n influenza types, respectively. , another examples are the produc- and hiv- vlp in cos- /vero cells, and hbv vlp in cho cells. [ ] [ ] [ ] plant systems plants were successfully used to express specific gene products. the feasibility of recombinant plants for generation of vaccine antigens were shown in tobacco plants, potato tubers, and others. this approach develops vaccine strategies which can stimulate mucosal as well as systemic immune responses. in addition, it can be delivered orally as part of a normal biologic function in human. the antigen expressed in plant systems shows extensive disulphide crosslinking and oligomerization for formation of virus-like particles. for example, the hepatitis b major surface antigen has been expressed in several plant systems. plants are able to express and assemble both types of vlps (i.e., enveloped and non-enveloped) as multimeric and chimeric proteins. the high expression of vlps in plant is easy and rapid (e.g., - weeks) using a tobacco mosaic virus (tmv) rna replicon system and/or a bean yellow dwarf virus (beydv) dna replicon system. another advantage of plants is the use of plant virus particles as a delivery system to present foreign epitopes. furthermore, the problem of plant-specific glycans has been partially solved using the development of transgenic plants with "humanized" glycosylation pathways. plantderived vlps can be used for oral delivery of vaccines. virallike particles are more resistant to digestive enzymes than soluble proteins in body, because of their highly ordered and packed structures. for example, the gastrointestinal virusesderived vlps including noroviruses and rotaviruses were utilized orally as potent candidates for mucosal immunization. plant-derived vlps showed the same structures with vlps generated in other expression systems accompanied by a comparable or higher immunogenicity. some plant-derived vlps could induce protective humoral and cellular immunity and also safety in clinics. the studies showed that the level of protein expressed in the recombinant plants is variable and often low. therefore, further increase in expression will be necessary for practical and efficient products. recent progress in the glyco-engineering of plants allows human-like glycol-modification and optimization of desired glycan structures for increasing safety and functionality of recombinant pharmaceutical glycoproteins. some plantbased systems can stabilize antigen and thus reduce storage and distribution costs. different applications of virus-like particles toxoplasma gondii (t. gondii) is an obligate intracellular parasite infecting the nucleated cells of warm-blood vertebrates. this parasite is able to stimulate strong humoral, cellular and mucosal immunity, and thus it can be used as an efficient delivery system for heterologous antigens. t. gondii was applied as a vector for live vaccination against infectious pathogens. [ ] [ ] [ ] [ ] [ ] [ ] recently, a non-pathogenic kinetoplastida, leishmania tarentolae, was utilized to express heterologous proteins. the studies showed that expression of mammalian glycoproteins in this parasite leads to their modification with mammalian-like oligosaccharides. [ ] [ ] [ ] [ ] recently, our group has focused on its use as a live vector or killed vaccine, [ ] [ ] [ ] and also generation of viral coat proteins and their assembly as vlp in this system. virus-like particles show an efficient strategy to deliver antigens to the immune system, inducing both arms of the adaptive immunity. indeed, vlps present antigenic epitopes in the proper conformation, leading to induce humoral responses. for example, preclinical trials with influenza vlps indicated their capacity to induce both humoral and cellular immune responses at low antigen doses. several authors have reported antibody response to parenterally or orally administered plant-derived antigens. , as exogenous antigens, vlps are taken up by professional antigen presenting cells (apcs), especially dcs, followed by antigen processing and presentation via mhc class ii molecules, dc activation and maturation through up-regulation of co-stimulatory molecules and cytokine production, and stimulation of cd t helper cells. all these events can efficiently induce both humoral and cellular immunity. in addition, the exogenous vlps can enter the cytosol of dcs, be processed and presented by mhc class i molecules to cytotoxic t lymphocytes (ctls) using cross-presentation. , , furthermore, the b-cell activation using vlps is robust enough to induce t cell-independent igm antibodies. , dcs loaded with yeast-derived hiv vlps can alter gag-specific memory cd t cells into effector cells through cross-presentation in chronically hiv-infected individuals, although some gag-specific t cells in these patients did not show any response. the reports showed that the expression system used for generation of vlp might significantly affect direction, type and outcome of immune responses. for example, potent and specific immunomodulatory effects were assigned to yeast-derived hiv vlps in comparison with other expression systems. on the other hand, plant-or insectderived vlps, consisting of the l capsid protein of hpv, were both immuno genic to an equal degree. half of mice fed trans-genic potatoes expressing hpv vlps developed l -specific antibodies. the studies indicated that the vlps are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. vlp-derived peptides are loaded onto mhc i that have been recycled from the cell surface. a study showed that uptake and activation of dc by vlp involves proteoglycan receptors, tlr and nf-kb, and can be inhibited by heparin. several data suggest different routes of vlp uptake by dc and langerhans cells (lc). for example, lcs and dcs internalize similar amounts of hpv-vlps in vaccine design, albeit through different uptake mechanisms. , vlp uptake by dcs results in activation and cross-presentation of mhc i-restricted peptides with co-stimulation to t-cells. on the other hand, vlp uptake by lc leads to cross-presentation in the absence of costimulation. efficient vlp cross-presentation by lcs with costimulation can be achieved by addition of cd ligand. the lack of a protective immune response after viral contact with lcs may explain why some women fail to induce an immune response against the virus. lcs endocytose hpv vlps via a non-clathrin, non-caveolae, actin-independent pathway, whereas dcs take up hpv vlps both by a clathrin-mediated mechanism and via macropinocytosis in an actin-dependent manner. this difference in endocytosis resulted in processing and presenting hpv vlp peptides by lcs similar to that by dcs on their surface, but in the absence of co-stimulation. with the addition of cd l, lcs incubated with hpv vlps generated the efficient amounts of the pro-inflammatory cytokine (il- ) and could stimulate a hpv-specific immune response after incubation with t cells. despite these differences, vlps taken up by dc and lc were able to prime naive cd t cells and induce cytolytic effector t cells in vitro. furthermore, hiv- pr gag virus-like particles could stimulate strong humoral and cellular immune responses. vlp expressed by recombinant baculoviruses activated human pbmc to release pro-inflammatory (il- , tnf-a), antiinflammatory (il- ) and th -polarizing (ifn-c) cytokines as well as gm-csf and mip- a in a dose-and time-dependent manner. furthermore, vlp-induced monocyte activation was shown by up-regulation of molecules involved in antigen presentation (mhc ii, cd , and cd ) and cell adhesion (cd ). exposure of vlp to serum inactivated its capacity to stimulate cytokine production. the linking of vlps to adjuvant molecules was also shown to improve the immunogenicity of the nano-bioparticles. adjuvanted vlps [e.g., cpg odn or poly (i: c) adjuvants] elicited a higher titer of total specific igg compared to vlps alone. furthermore, while vlps alone induced a balanced th pattern, vlps formulated with adjuvant elicited a th -biased igg subclasses (igg a and igg ), with poly (i: c) more potent than cpg odn in shirbaghaee and bolhassani animal model. in addition, mice immunization with chimeric simian immunodeficiency virus (siv) vlps containing gm-csf significantly induced siv env-specific antibodies as well as neutralizing activity at higher levels than those elicited by standard siv vlps, siv vlps containing cd l, or standard vlps mixed with soluble gm-csf. on the other hand, the incorporation of immunostimulatory molecules showed significantly increased cd and cd t-cell responses to siv env, compared to standard siv vlps. formulation of vlps with rough lps (r-lps) adjuvant as well as dna primed-vlp boosted regimen were led to increase specific immune responses as compared to vlps alone, but among them the vlp/r-lps highly enhanced immune response. recent studies demonstrated the potential of the hbc vlps as an oral immunogen. intraperitoneal immunization with the hbc vlp induced a strong, mixed th /th response. in contrast, oral administration of the hbc vlp generated a high humoral response with mainly igg a antibodies, directing toward a th response which is essential in the control of intracellular pathogens. in addition, the intranasal monovalent adjuvanted norwalk vlp vaccine was well tolerated and highly immunogenic. the studies showed that chimeric hpv-vlps are able to elicit potent ctl responses in mice against hpv transformed tumors; however, the mechanism of t cell priming has remained obscure. hpv vlp could bind to human mhc class ii-positive apcs through interaction with fccriii, and immature dcs were activated after incubation with hpv vlp. it was shown that binding and uptake of vlp by dc from fccrii, fccriii, and fccrii/iii deficient mice are reduced by up to % compared with wild-type mice. in addition, maturation of murine dc from fccrii/iii-deficient mice by vlp is also reduced, indicating that dc maturation, and thus ag presentation, is diminished in the absence of expression of fccr. poor immunogenicity of mucosally administered proteins has been a major barrier for development of efficient oral vaccines. one way to overcome this obstacle is the use of appropriate adjuvants. also, delivery of antigen to mucosal surfaces as vlp provides an efficient way of inducing mucosal immunity. after oral or intranasal immunization with norwalk vlp, or rotavirus vlp without adjuvant, intestinal iga was detected in immunized mice, which were protected from virus challenge. in addition, the plasma cell precursors that migrate to the genital tract are derived primarily from mucosal lymphoid tissues and often secrete iga. the studies indicated that immune responses generated by mucosal administration of vlp were generally weaker than systemic administration. vlp specific iga was higher in intestine washes following intrarectally (i.r.) than intravaginally (i.va.) immunization, and higher in vaginal washes following intramuscularly (i.m.) than i.r. or i.va immunization. some studies suggested that the immunogenicity of virus particles at mucosal surfaces is probably a property of particulate antigens assembled as multimers of subunits. indeed, vlp might be actively taken up by mucosal apc through the integrin receptors. lipoparticles are stable, highly purified, homogeneous, and specialized vlps containing high concentrations of an integral membrane protein. integral membrane proteins are involved in different biological functions and are targeted by % of existing therapeutic drugs. however, because of their hydrophobic domains, membrane proteins are difficult to manipulate outside of living cells. lipoparticles can incorporate a wide variety of the membrane proteins, including g proteincoupled receptors, ion channels, and viral envelopes. lipoparticles provide a platform for different applications such as antibody screening, production of immunogens, and ligand binding assays. [ ] [ ] [ ] during the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, known as lipid rafts, frequently function as a natural target for viral proteins. the role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and t cell signaling is extensively recognized. on the other hand, in order to improve the immunogenicity of hiv- envelope glycoproteins, the fusion of gp was performed to a carrier protein, hepatitis b surface antigen (hbsag) which is capable of spontaneous assembly into viruslike particles. the hbsag-gp hybrid proteins assembled efficiently into - nm particles. the particles resembled native hbsag particles in size and density, consistent with a lipid composition of about % and a gp content of about per particle. particulate gp folded in its native conformation and was biologically active, as shown by high affinity binding of cd . because the particles are lipoprotein micelles, an array of gp on their surface closely mimics gp on the surface of hiv- virions. these gp -rich particles can enhance the quality, and also quantity of antibodies elicited by a gp vaccine. virus-like particles show an expanding spectrum of applications such as gene therapy, nanotechnology, vaccination, and diagnostics. , recently, the studies showed a pattern of direct conjugation of some ligands, including nucleic acids and proteins attached to vlp surface. , in addition, because of the superior accessibility of cysteine and lysine residues on vlps, bio-conjugation has been performed by commercial homo-or hetero-bifunctional linkers. [ ] [ ] [ ] [ ] for example, three foreign proteins were chemically conjugated to the vlp surface of cpmv by proper bifunctional cross-linkers. on the other hand, the researchers could produce an alphavirus vlp surrounding a functional gold nanoparticle. vlps have been also used to stimulate immune responses and generate antitumor responses, e.g., alphavirus-based virus-like replicon particles (vrp) expressing various melanoma antigens. [ ] [ ] [ ] it is interesting that the first viral-associated cancer vaccines were founded on hbv vlp and hpv vlp to prevent hbvassociated hepatocellular carcinoma (hcc) and hpvassociated cervical carcinoma, respectively. , it should be noted that these vlp formulations are viral vaccines that prevent a viral infection that may progress to carcinoma after a long time. we indicate some applications of vlps against viral diseases as following: in several studies, specific vaccine antigens were generated by various expression systems to induce protective immune responses and apply in licensed recombinant viral vaccines. , some examples of preventive vlp-based vaccines are recently commercialized worldwide including glaxos-mithkline's engerixv r (hbv) and cervarixv r (hpv), and merck and co., inc.'s recombivax hbv r (hbv) and gardasilv r (hpv). other vlp-based vaccines undergo preclinical evaluation or clinical trials, including parvovirus-, influenza-, norwalk-derived vlps and also different chimeric vlps. for generation of immunogenic vlps, eukary otic expression hosts including yeast (s. cerevisiae, p. pastoris and h. polymorpha) and mammalian cells (chinese hamster ovary cell line [cho]) were used. the studies indicated that the recombinant hbsag generated in cho and h. polymorpha have significant differences in size, molecular weight (mw), and monomer number. furthermore, the cho-derived viral-like particles include a combination of glycosylated and non-glycosylated hbsag proteins, similar to those in patients' sera, while yeastderived antigens were reported to be non-glycosylated. chobased vaccines were provided by pasteur-m erieux aventis in france (genhevac bv r ) and scigen in israel (sci-b-vac tm ). both vaccines contained not only the hbsag s pro tein but also the m protein (genhevac b) or the m and l protein (sci-b-vac). on the other hand, gardasil approved by the fda in is a quadri valent hpv types / / / l vlp vaccine produced in s. cerevisiae. cervarix is the other licensed hpv vaccine approved by the fda in . cervarix is a bivalent hpv types / l vlp vaccine expressed via a recombinant baculovirus vector. , different experiments have been concentrated on hpv vlp vaccination in mouse and human models including: (a) activation of immature human dcs by chimeric hpv vlps, (b) determination of systemic cytokine pattern elicited by hpv l vlps, (c) identification of gene expression signatures in hpv l vlp-induced human pbmcs, (d) generation of potent and prolonged neutralizing l antibodies using a single intramuscular (im) mice injection with recombinant adenoassociated virus encoding hpv l protein (raav- l ), (e) augmentation of immunogenicity of hpv l dna vaccines using genetic linkage to a chemokine and secretory signal peptide sequences, (f) potent stimulation of systemic and mucosal immune responses to vlp vaccines using the encapsulation of a genetic cytokine adjuvant (e.g., il- ), (g) improvement of hpv vlp immunogenicity by linkage to the modified adjuvant, and m) nasal immunization of mice with hpv vlps. hpv l -e chimeric virus-like particles (cvlp) could induce e -and l -specific ctls. the therapeutic potential of the cvlp also indicated a considerable safety in high grade cervical intraepithelial neoplasia patients (cin / ). several improvements in vaccine design by vlp are still in preclinical trials. some main examples are referred as following: a. co-injection of the hpv l vlp with e. coli heatlabile enterotoxin (lt) as an adjuvant significantly increased the levels of serum igg and vaginal iga after nasal or bronchial mice immunization. antigens (hiv- p /p : ty vlp) was also immunogenic and well-tolerated in phase i clinical trials. , g. several groups have focused on improving bacteriophagebased vlp vaccines, e.g., rna bacteriophage qb. these chimeric vlp vaccines were targeted against noninfectious diorders including hypertension, allergy, neurodegenerative and autoimmune diseases (e.g., diabetes mellitus type ii and alzheimer), cancer (e.g., melanoma). the vaccine candidate against alzheimer (cad- ) was constructed to display chemically coupled amyloid beta (ab - ) peptide derived from the n-terminal b cell epitope of ab protein, on the surface of qb-cp vlps. this vaccine could stimulate ab-specific igg and decrease amyloid accumulation in animal models expressing ab precursor protein, without eliciting t-cells or inflammatory reactions in brain tissue. , in addition, the angiotensin ii vaccine was synthesized by covalently conjugation of a peptide derived from angiotensin ii to the rna bacteriophage qb vlp capsid. this modified vlp could decrease blood pressure in spontaneously hypertensive rats. table i shows preclinical and clinical studies of vlps in vaccine development. generally, a major application of vlps is the stimulation of immunity against foreign protein epitopes by genetically fusing or chemically conjugating them to vlps entitled as chimeric vlp (cvlps). antigens can be fused to vlps through either covalent or non-covalent bonds. the most common covalent bond is generated by the heterobifunctional chemical cross-linkers with amine and sulfhydryl-reactive arms. for instance, cysteine-containing antigens can be conjugated to lysine residues of vlps surface at a high density (e.g., three peptides per coat protein molecules). the non-covalent conjugation strategy contains the use of streptavidin as linkers to attach biotinylated antigens and vlps through their efficient and specific interactions. sv vlps can also encapsulate various materials such as dna ( kb) and proteins as antigens. insertion of a special exogenous peptide into the surface loops of vp produced sv vlps with the ability of cell targeting. moreover, sv vlps stimulated innate immunity as a natural adjuvant. indeed, sv vlps may be a promising vaccine candidate to deliver heterologous antigens followed by the induction of ctls without synthetic adjuvants. several chimeric vlp vaccines have entered clinical trials, such as the anti-influenza a m -hbcag vlp vaccine (hbcag vlps displaying m epitope of influenza a), the anti-hiv p /p : ty vlp, two anti-malaria vaccines (hbcag vlps displaying malaria epitopes), the nicotine-qb vlp and the anti-ang ii qb vlp. genetic linkage contains a stable bond between vlp and antigen. the studies showed that only peptides shorter than amino acids (small peptides) can be presented without interfering with the correct assembly of vlps. other limitations contain the improper folding of displayed antigens and the formation of cvlps with heterogeneous size. to prevent these issues (e.g., assembly problems), structural studies have identified domains for different vlps such as hbcag, hbsag and hiv gag that were not necessary for vlp assembly as well as allowed insertion of foreign antigens. the simplest way for generation of single component cvlps, is the insertion of peptides at the n-or c-terminal regions of chimeric vlps. multiple fusion positions should be identified to produce multicomponent cvlps inducing broad immune responses. the direction and intensity of the immune responses are significantly influenced by the vlp type, the foreign antigen density, and its accessibility on or within the vlp. furthermore, preexisting immunity against the epitopes of the vlp as a delivery system may importantly change the response against the heterogenous antigen. for example, hbcag was also utilized to display a neutralizing epitope of hpv l protein. the nasal delivery of hbcag-hpv l epitope cvlps expressed in tobacco induced antigen-specific antibody responses in mouse model. on the other hand, an hpv l -based chimeric vlp was generated in transgenic tomato to present several t-cell epitopes from hpv e and e proteins. the hpv l -e /e vlps elicited a neutralizing antibody response similar to that from an equal amount of the commercial vaccine (gardasil) in preclinical study. moreover, the chimeric vlps induced ctl responses against the e and e epitopes. chimeric hpv l vlps were also designed using genetic fusion to display epitopes of influenza m protein. to overcome the problems associated with genetic fusion including the antigen size, conformation and vlp assembly, different applications of virus-like particles chemical conjugation approaches were applied to construct cvlps. in this strategy, target antigens and native vlps were generated individually and coupled together by attachment of the antigen to the surface of the pre-assembled vlps. two main advantages of this strategy include: (a) various sizes and types of antigens can be exposed, and (b) the antigen-vlp binding site can be manipulated for further presentation of the conjugated antigen. for example, vlps were used to display full-length and correctly folded proteins, such as interleukin- (il- ). generally, vlps were used for delivery of protein/peptide, dna, sirna and drugs as a brief description in following: viral-like particles were used as a peptide/protein carrier, in vitro and in vivo. there are several examples for delivery of protein/peptide using vlps as following: a. chimeric vlp vaccines have been improved based on rna bacteriophage ap , presenting peptides of selfantigens or pathogens fused to either the n-or cterminal regions of ap coat protein. ap -derived vlps were highly immunogenic in mice. furthermore, influenza m vlps stimulated an efficient m -specific antibody response and full protection against lethal influenza virus challenge. b. vlps containing flt ligand (fl-vlps), a dc growth factor, could effectively increase immunogenicity in mice. dcs exposed to vlps also produced high levels of il- . c. a plant vlp-based approach was used to develop respiratory syncytial virus (rsv) vaccine. a target peptide displaying amino acids - of the rsv g protein was delivered on the surface of recombinant alfalfa mosaic virus (almv) particles. this construct induced high pathogen-specific immune responses in immunized animals. , d. in a recent study, a peptide from an external loop of mouse ccr protein was inserted into a neutralizing epitope of hpv l . the particles generated by this chimeric l could elicit high levels of ccr antibodies that specifically recognized the surface of ccr transfected cells and blocked in vitro infection of an mtropic hiv strain in mice. in addition, chimeric vlps containing the full length hpv e oncoprotein linked to l , or the n-terminal region of e fused to l , could induce antigen-specific protection of mice from lethal challenge with e -expressing tumor cells. - e. a pre-s epitope of hbv was also inserted into the ef loop of hpv vlp recognized by hbv-specific antibody. chimeric vlps produced in e.coli carried a virus-neutralizing hbv pre-s epitope in the major immunodominant region (mir) and a highly conserved n-terminal hcv core epitope (aa to ) at the c-terminal region of the truncated hbv core vlps (hbc). the presence of two different foreign epitopes within the hbc molecule did not interfere with its vlp-forming potential, with the hbv pre-s epitope exposed on the surface and the hcv core epitope buried within the vlps. mice vaccination showed a specific t cell activation by both foreign epitopes and a highlevel antibody response against the pre-s epitope, whereas an antibody response against the hbc carrier was inhibited. f. the researchers have shown that the nanosized hbc-vlps bearing mycobacterial antigen cfp- (hbc-vlp: cfp- fusion protein) induced an increased immune response in balb/c mice compared to mixtures of native antigen. g. chimeric papillomavirus vlps based on the bovine papillomavirus type (bpv- ) l protein were designed by replacing the -carboxyl-terminal amino acids of the bpv major protein l with a synthetic "polytope" minigene, containing known ctl epitopes of human pv e protein, hiv iiib gp p , nef, and reverse transcriptase (rt) proteins, and an hpv e linear b epitope. the chimeric l protein assembled into vlps in insect cells. polytope vlps could deliver multiple b and t epitopes as immunogens to the mhc class i and class ii pathways. this study has demonstrated that hybrid vlps can be used as an efficient antigen delivery system to transfer more than one ctl epitope through mhc class i pathways. h. the chimeric hpv vlps were generated in which hpv l neutralization epitopes (l residues - or - ) are inserted within an immunodominant surface loop (between residues and ) of the l major capsid protein of bpv . immunization of rabbits with assembled particles elicited high l -specific serum antibody responses. l. the studies showed that the c-terminal region of gag fused by t cell epitopes from human cytomegalovirus pp led to the formation of hybrid vlps activating antigen-specific cd memory t cells ex vivo. regarding to previous studies, the gag polyprotein is the only retroviral protein required for vlp formation. [ ] [ ] [ ] vlps, derived from an avian retrovirus, were applied to deliver proteins to cells, either as part of gag fusion proteins (intracellular delivery) or on the surface of vlps. the construct is an effective system because the vlps are completely made of the gag fusion protein, and a single vlp will deliver - copies of gag fusion protein into a transduced cell. delivery of foreign genes to the digestive tract mucosa by oral administration of non-replicating gene transfer vectors would be a very useful method for vaccination and gene therapy. the studies indicated that plasmid dna could be packaged in vitro into a vlp composed of open reading frame (orf ) of hev, which is an orally transmissible virus. these vlps could deliver this foreign dna to the intestinal mucosa in vivo, eliciting high mucosal and systemic immunity in mice, without the use of adjuvants. an orally administered hiv dna vaccine encapsulated in hev-vlps could induce mucosal and systemic cellular and humoral immune responses. moreover, the ability of hpv vlps was examined to mediate delivery and expression of dna plasmids in vitro and in vivo. hpv pseudoviruses were provided by disrupting hpv-vlp, mixing them with dna plasmids and reassembling them into the pseudoviruses (vlps with plasmids inside). the pseudovirus induced more potent immune responses than dna vaccines. the pseudovirus could be used in gene therapy by transferring the therapeutic genes into lymphoid tissues in human. in addition, the recombinant hpv l vlps, produced in insect cells, could efficiently encapsulate a plasmid harboring either a gene for the gfp or b-galactosidase during in vitro disassembly-reassembly of vlps. vlp-mediated delivery of a gfp reporter construct in vitro showed to be highly dependent on the presence of full-length l protein within the vlps. similarly, expression of gfp and luciferase reporter plasmids in vivo was efficiently enhanced by co-administration of l /l vlps. in addition, co-administration of vlps with a hpv e -expressing plasmid increased significantly e -specific cellular immune responses. the reports indicated that the recombinant major structural protein of the bk polyomavirus (bkv vp ) was shown to self-assemble into vlps with a diameter of - nm. the potential of bkv vp vlps was investigated to transfer gene into cos- cells using three methods for the formation of pseudovirions: disassembly/reassembly, osmotic shock and direct interaction between vlps and plasmid dna. the most efficient method is the direct interaction between vlps and linearized plasmid dna. the findings generally demonstrated that bkv vlps have exogenous dnabinding activity, as a promising vehicle for gene transfer studies. sirna delivery there is a major challenge to identify novel approaches for specific and effective delivery of new types of drugs like sirnas and peptides. systemic delivery of small interfering rna (sirna) was restricted by its poor stability and low cellpenetrating properties. to overcome these limitations, an efficient sirna delivery system was designed using polyethyleneimine (pei)-coated vlps derived from adeno-associated virus type (pei-aav -vlps). generally, one of the strategies to integrate sirna into nanoparticles was to coat these particles with positively charged polymers, including pei, poly b-amino different applications of virus-like particles ester, or poly l-lysine. electrostatic coating could increase the efficiency of systemic sirna delivery due to its protective effects and improved cellular uptake. an insect/baculovirus expression system was used to generate aav -vlps. pei-aav -vlps could condense sirna, protect it from enzymatic degradation, transfer it with high efficiency and induce cell death in mcf- breast cancer cells, for breast cancer therapy. furthermore, micrornas (mirnas) play an essential role in immunoregulation and may be involved in the pathogen esis of systemic lupus erythematosus (sle). among these sle-related mirnas, mir- a, acts as a significant inhibitor of autoimmunity, myeloproliferation, and cancer. a novel mirna-delivery approach was described via bacteriophage ms vlps for evaluation of the therapeutic effects of mir- a, in bxsb lupus-prone mice. treatment with ms -mir- a vlp increased the level of mature mir- a, leading to a significant reduction in the expression of autoantibodies and total igg. furthermore, the levels of inflammatory cytokines, including ifn-a, il- b and il- were decreased in mice. the stimulation of dysregulated mirnas by an ms vlp-based delivery system may be considered as a novel therapy. [ ] [ ] [ ] the use of ms vlps was reported for selective delivery of nanoparticles, chemotherapeutic drugs, sirna cocktails, and protein toxins to human hcc. in addition, the researchers used jc virus (jcv) vlps as a vector for delivering rnai in silencing the il- cytokine gene. jcv vlps were non-toxic, and showed the therapeutic use as a gene therapy approach for autoimmune diseases (aid) including sle. , drug delivery a major challenge in pharmacology is to find methods that drugs (especially anti-cancer drugs) can be delivered specifically to target tissues. a potential strategy would be to package or encapsulate the drug molecules inside a particle which is bound to the cancerous tissue. such encapsulation would protect the drug from degradation in blood. for this purpose, it will be necessary to develop particles which can be modified on their outer surface to carry drug molecules into the target cells. novel nanocarriers such as dendrimers, liposomes, polymersomes, micelles, and vlps indicated high potency in improving drug delivery, and targeting strategies. all of these delivery systems make drugs more biocompatible, watersoluble, or colloidal, indicating low toxicity and high uptake in cells. different virus-based materials were studied for drug delivery such as: the ccmv, the cpmv, the red clover necrotic mosaic virus (rcnmv), ms rna-containing bacteriophage, the bacteriophage qb, m bacteriophage, the tmv. drug cargo can be loaded through covalent attachment of drugs or their analogs to particular reactive residues on the capsid pro-teins. several cancer cell targeting ligands were attached to different types of vlps, including small molecules, antibodies, peptides and proteins, as well as dna aptamers. folic acid (fa) was broadly used in drug delivery targeted to cancer cells. uptake of fa into cells is mediated by the folate receptor (fr). recently, lactobionic acid (la) was applied for the specific targeting of a rotavirus capsid vp to hepatocytes or hepatoma cells bearing asialoglycoprotein receptors (asgprs). human holo-transferrin (tfn) is essential for iron homeostasis. tfn is especially recognized by the tfn receptor (tfnr), which is over-expressed on the surface of various tumor cells and efficiently taken up by cells in the clathrinmediated endocytosis. , tfn has been conjugated to cpmv and bacteriophage qb. the cellular uptake of the qb-tfn particles was relative to the tfn density; while the internalization was prevented by comparable concentrations of free tfn. antibodies contain another group of targeting proteins that could be chemically linked to vlps. for instance, a single-chain (scfv) antibody that recognizes the carcinoembryonic antigen (cea) over-expressed in a variety of tumor cells, has been attached to cpmv. an important strategy to improve cellular uptake of therapeutic molecules is the use of cell-penetrating peptides (cpps). the hiv- tat peptide is one of the cpps that were extensively used in the delivery of vlps. , in general, virus-like particles represents an attractive system for drug delivery in vitro. the efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is a critical issue in the biological field. recently, the intracellular delivery of hydrophobic dyes or drugs encapsulated in vlps through cyclodextrins (cds) showed high efficiency. as a model anticancer drug, paclitaxel (ptx)-cd complexes encapsulated inside vlps exhibited a dose-dependent cytotoxic effect with a -fold smaller ic than that of free ptx dissolved in dmso. cell targeting is aimed to effective uptake of therapeutic and/ or diagnostic reagent in a special location such as a tumor. targeting can also be achieved using proteins (mainly antibodies), peptides, nucleic acids (aptamers), small molecules, vitamins and carbohydrates. by attachment of targeting ligands, specificity for cell targeting was obtained by receptor-mediated endocytosis. for instance, bacteriophage ms vlps, were chemically conjugated to a targeting peptide (sp ) for the selective delivery of nanoparticles, chemotherapeutic drugs, sirna cocktails and protein toxins to human hcc. [ ] [ ] [ ] recently, the chemical conjugation of human epidermal growth factor (egf) to simian virus vlps allowed for cell shirbaghaee and bolhassani selective targeting. simian viruses vlps have attracted a great attention in gene delivery due to their high stability and low toxicity in blood. in design of polymeric nanoassemblies, chemical modification is necessary to conjugate the dye or probe for in vitro and in vivo imaging. however, in the case of nanobioassemblies, chemical or genetic modification can be applied for bioconjugation of fluorescent dyes or other probes. another advantage of nanobioassemblies such as vlps for bioimaging is their biological compatibility. quantum dots (qds) and gfp were used broadly for in vitro and in vivo imaging as alternatives to labelling. for example, fluorescent chimeric vlps of canine parvovirus were expressed in insect cells. to create the fluorescent chimeric vlps of canine parvovirus, gfp was genetically engineered onto the n-terminal region of the viral protein vp , as a visualization tool to understand mechanisms of viral infections. gfp was also used to design chimeric hiv vlps allowing protein to be followed during assembly and transmission using live-cell imaging. , advantages of vlps include: (a) no need to propagate pathogenic organisms, (b) repetitive and ordered surface structures, (c) multivalent as well as particulate in nature, (d) safer than other vaccines because of non-infectious and non-replicating properties: the studies showed that there is no risk of disease progress in vaccinated groups with vlp-based vaccines as compared to attenuated viral vaccines, because they lack the genomic material needed for the replication and the spread of the viruses, (e) stable in extreme environmental conditions, depending on vlp structure (i.e., envelope or non-envelope), and (f) as carrier to express foreign antigen. the potential of vlps to target dcs is a main advantage of vlp vaccines, for activating the innate and adaptive immune responses. they have a special benefit against other delivery systems in size, stability, and capacity to transfer biological molecules across cell barriers. particles in the - nm range can stimulate cd , cd cells and especially generate th responses. in addition, despite a limited number of vlp vaccines approved for human use, they represent a promising platform for the development of novel mucosal vaccine strategies. indeed, vlps are sufficiently small, and the composition of their surface chemistry can be designed to minimize hydrophobic and electrostatic adhesive interactions with mucus. they can also be engineered for recombinant expression of multiple antigenic epitopes and for incorporation of co-stimulatory and immuno-regulatory proteins. however, vlp technology can be limited by difficulties of scale-up and the need for purification from the expression systems. other limitation in chimeric vlp vaccine is to determine the compatibility of peptide with assembly of vlp and its immunogenicity property. under the host immune defence, pathogens undergo mutation which render the vlp vaccine ineffective and will be effective for only highly conserved b or t cell epitopes. the major challenge is to develop novel production platforms that can deliver vlp vaccines while significantly reducing production times and costs. viral-like particles (vlps) have shown high ability for the improvement of vaccines against infectious and non-infectious diseases. several recombinant expression systems were successfully applied for vlp production, with different efficiency. the use of vlps in vaccine development showed that they are considered safe. in addition, nano-sized vlps, can act as an adjuvant as well as antigen delivery system through increasing the antigen uptake by apcs. thus, it is not necessary for the use of adjuvants along with vlps to stimulate potent immune responses. vlps have shown a natural affinity to target host cells, and this property has been used for cell-targeting applications. regarding the advantages of vlps, it is necessary for further studies in various aspects especially easy and low-cost purification of vlps as well as their application as a delivery system in vivo. different applications of virus-like particles hum vaccine hepatitis b virus vaccine ip recombinant (genetically engineered): enivac hb hepavax-genev r . summary of product characteristics revac-b tm. available at prescribing information. merck prescribing information. glaxosmithkline medicago to present additional positive clinical data at the eswi influenza conference medicago inc. news release exp rev vaccine hum vaccine immunother different applications of virus-like particles antimicrob agents chemother intranasal norwalk vaccine hum alves, p. m. exp rev vaccine exp rev vaccines different applications of virus-like particles curr top microbiol immunol the authors are grateful to elnaz agi and negar zohrei (dept. of hepatitis and aids, pasteur institute of iran) for technical assistance. key: cord- -kb mez authors: calvillo batllés, p.; carreres polo, j.; sanz caballer, j.; salavert lletí, m.; compte torrero, l. title: hematologic neoplasms: interpreting lung findings in chest computed tomography() date: - - journal: nan doi: . /j.rxeng. . . sha: doc_id: cord_uid: kb mez lung disease is very common in patients with hematologic neoplasms and varies in function of the underlying disease and its treatment. lung involvement is associated with high morbidity and mortality, so it requires early appropriate treatment. chest computed tomography (ct) and the analysis of biologic specimens are the first line diagnostic tools in these patients, and sometimes invasive methods are necessary. interpreting the images requires an analysis of the clinical context, which is often complex. starting from the knowledge about the differential diagnosis of lung findings that radiologists acquire during training, this article aims to explain the key clinical and radiological aspects that make it possible to orient the diagnosis correctly and to understand the current role of ct in the treatment strategy for this group of patients. hematologic neoplasms (hn) are characterized by being frequently disseminated at the moment of diagnosis, with bone marrow affectation. they are especially sensitive to chemotherapy or radiotherapy, therefore the patients usually receive aggressive chemotherapy and, in certain cases, hematopoietic precursors transplantation (hpt). the disease per se and its treatments cause prolonged pancytopenias, predisposing to very serious infections that make up a diagnostic and therapeutic emergency. non-infectious pulmonary complications secondary to treatment are also frequent and determine prognosis. pulmonary tumor disease includes infiltration due to hn, pulmonary neoplasm and post-hpt lymphoma. chest computed tomography (ct) narrows down the differential diagnosis of these diseases. their indications are reviewed, as well as the main clinical information that should be recorded in the radiologic application and interpretation of the findings based on the clinical context. performing helical chest cts in patients with hn pursues two objectives: early detection of lesions not visible in the chest x-rays which require urgent treatment, and better characterization of the findings to outline diagnostic and therapeutic possibilities. reconstructions being < . mm cut thick and high resolution computed tomography (hrct) are required since many of the pulmonary complications kick in as interstitial patterns. the etiologic diagnosis of fever manifestations in patients with neutropenia and/or hpt requires the search for microorganisms and infectious markers. chest hrct plays a fundamental role---urgent when there are clinical signs of severity and early (< h) in the absence of a response to antibiotics therapy in --- h because treatment of a possible invasive fungal infection (ifi) requires an early administration, a determinant factor for prognosis. in patients clinically classified as being in high risk of ifi , antifungal drugs are administered empirically, while in subgroups of lower risk it is possible to delay treatment in cases of very likely clinical manifestations or early positive specific infection markers, which reduces the high costs and toxicity of these drugs. the markers usually used are the serum galactomannan test while the chest hrct is performed early in a serial way. , galactomannan, a component of the aspergillus cell membrane, is falling into disuse as a marker due to its loss of sensitivity associated to antifungal prophylaxis. the hrct gains more importance still as an urgent diagnostic test that allows starting an early therapy when pulmonary lesions characteristic of ifi are visualized. in addition, it can give rise to other etiologies and guide the acquisition of bronchoalveolar lavage (bal) through bronchoscopy, thus speeding up the diagnosis of germs not covered by the initial empirical therapy. in other respiratory manifestations hrct is necessary to identify and characterize non-infectious complications, relapse and secondary neoplasms that can go unnoticed in radiographic tests or show similar patterns. when studying the hrct of a patient with hn we need to know the clinical information and other fundamental data about the underlying condition as well as therapies and complications (table ) . patients with hodgkin lymphomas (hl) and non-hodgkin lymphomas (nhl) receive a less intense polychemotherapy than those with leukemias, with shorter neutropenias so if pulmonary lesions are seen the possibility of tumor affectation should be taken into consideration. it is not odd to find pulmonary neoplastic spread in autopsies of leukemias, but its radiologic manifestation is exceptional and the respiratory disease is mainly marked by infections. acute myeloid leukemia (aml) deserves special attention, since pulmonary manifestations occur in all the stages of the disease, and it is possible to observe added to infections, more cases of hemorrhages due to thrombocytopenia and toxicity due to chemotherapy. multiple myeloma (mm) occurs mainly with bacterial infections due to humoral immunity deficiency and hypoventilation due to bone affectation. pulmonary edema is very common while findings of pulmonary infiltration due to amyloids, plasma cells or light chain deposits are rare. chemotherapeutic drugs do not only depress immune function, but some of them are responsible for pulmonary toxicity, suspected by the radiologic pattern and its temporal relation with the treatment. other therapeutic agents used can cause respiratory failure, often with a radiologic expression similar to alveolar damage, edema or hemorrhage. in especially chemosensitive tumors (lymphomas and mm), the goal of autologous hpt is to allow the administration of high doses of chemotherapy with subsequent corticotherapy graft vs host disease (gvhd) (immunosuppressant therapy) actual infectious prophylaxis wide-spectrum antibiotics antifungal prophylaxis p jirovecii rescue of hematopoiesis thanks to the infusion of hematopoietic precursors obtained previously from the same patient. this prevents irreversible medullary failure and shortens the time of neutropenia. pulmonary complications will be fundamentally the consequence of intensive chemotherapy not the consequence of the transplant per se. the allogenic hpt is mainly used in patients with acute leukemias to re-establish hematopoietic and immunity functions. the cells are obtained from a family member with an identical hla (human leukocyte antigen) or else from someone unrelated but with a compatible hla and they may come from the bone marrow, peripheral blood or the umbilical cord. before the transplant the patient receives conditioning with high doses of chemotherapy, with or without full-body radiotherapy, to suppress the bone marrow, destroy the malignant cells and prevent the host's rejection against the graft cells. also a strong immunosuppressant treatment is later administered to avoid reverse rejection and when the hematopoietic function is reinstated the donor's cells recognize the recipient's tissues as foreign and they cause graft-versus-host disease (gvhd). yet despite of this some patients develop gvhd and require a more extended immunosuppressant therapy. all of this explains why the allogenic hpt recipient suffers from a deep and prolonged immune deficit that leads to death in nearly --- % of patients. based on at what specific moment in his history the patient is he will be included in one of these groups to approach diagnosis (tables and see tables and . in the onset of the disease, before treatment, infectious nodes are uncommon. they are usually bacterial or viral and exceptionally, at the very moment of the diagnosis of aml with severe cytopenia or iron overload they can be fungal. in severely immunologically depressed patients, the nodes can be fungal, viral or bacterial (fig. ) . filamentous fungi are common in prolonged deep neutropenias (over two weeks) especially during chemotherapy of an aml and in the early post-hpt period. in % of invasive aspergillosis it is possible to observe nodes that are usually multiple-bilateral and at least one > cm. the ground-glass attenuation halo distinguishes them from bacterial nodes , but it usually disappears during the first five days. mucormycosis is suspected before aspergillosis if nodes or more are observed as well as sinus affectation, pleural effusion and/or the inverted halo sign, especially if the antifungal therapy did not cover that fungus. the hrct is useful because there is no serological test available today for the diagnosis of mucormycosis. viral nodes often have poorly-defined edges and/or halos but they are distinguished by their small size (< cm) and the absence of cavitation. bacterial nodes usually have betterdefined edges. centrilobular nodes often represent unspecific infectious bronchiolitis, especially if accompanied by sprouting-tree images; however, diffuse symmetrical bilateral centrilobular nodes with predominance in upper fields and ground-glass attenuation, in the absence of other infectious findings, should lead to respiratory bronchiolitis by tobacco or pneumonitis due to hypersensitivity secondary to the treatment. in nhl and hl infiltration is observed more often in relapses, increasing due to the greater survival of the patients with the current therapies. it usually manifests itself as multiple pulmonary nodes < cm with irregular edges, consolidations and/or masses, which are more frequent in nhl and have worse prognosis. primary pulmonary lymphoma occurs in patients of --- years of age, it is usually non-hodgkin of the b malt type (mucosa-associated lymphoid tissue) and it shows as a nodule or peribronchovascular consolidation at the bronchogram test. these lesions can be single or multiple bilateral. , they are characterized by the absence of mediastinal adenopathies at the moment of diagnosis and up to months later, they grow slowly. identical presentations to those of lymphocytic interstitial pneumonia have also been described (fig. ) . myeloid leukemias and myelodysplastic syndromes can make up a blastic tumor called granulocytic sarcoma---extremely rare in the lung, where it can occur as a node, mass or consolidation. exceptionally, mm can present nodes and/or masses, associated with adenopathies that simulate a lymphoma or a pulmonary cancer with a bad prognosis. lymphomatoid granulomatosis is an extranodal lymphoproliferative process associated with the epstein---barr virus (ebv) mainly affecting the lung and the central nervous system. it occurs as multiple pulmonary nodes in more than % of the cases, they are variable in number and size, and of basal predominance. nodes can progress rapidly, coalesce and cavitate, as well as disappear spontaneously, migrate or evolve making up the sign of the inverted halo sign. , secondary neoplasms lung cancer has increased in survivors of hl, nhl and cll, and its etiopathogenesis is being studied. its appearance ranges from one to several years after the diagnosis of hn. patients receiving allogenic hpt can develop a posttransplant lymphoproliferative disorder (ptld), usually during the first six ( ) months, and up to a year, with a second peak at --- years. in ptld and the hiv-associated lymphoma (most attributed to ebv) pulmonary nodes are usually bilateral, well-defined, and may associate a halo. it is important not to take them for an ifi by taking into account the clinical context and the evolution of the lesions. ifi nodes typically cavitate --- weeks after the disease onset accompanying the recovery from neutropenia (neutrophiles > ), making up the growing air image (crescent moon). previously nodes can show low central attenuation (fig. ) . low attenuation nodes. invasive fungal infection (ifi) due to aspergillus. acute lymphoid leukemia and prolongued neutropenia. high-resolution computed tomography (ct). upper cut (upper side images) and lower cut (lower side images). the nodes low attenuation can be seen in images with filter while the mediastinal window (arrows in a and d) is very typical of ifi due to necrosis prior to cavitation seen two ( ) weeks later (c and f). serum gactolamannan was positive one ( ) month later. fungal and bacterial consolidations (especially staphylococcal or tuberculous ones) can also cavitate and less commonly nodes and consolidations due to lymphoma. the appearance of cavitated nodes corresponding to langerhans cell histiocytosis has been described in patients with hl and more rarely in leukemias; it is not known whether there is an actual association, but in any case tobacco plays a role in the etiopathogenesis. areas of attenuation in groud glass. consolidations (fig. ) see tables and infections at the disease onset and in the course of mm, it is possible to observe bacterial and viral bronchopneumonias similar to the rest of the population while in patients with severe immunodepression they are due to different germs (see tables and ) . segmental or lobar consolidations are most often bacterial. an ifi is suspected when they have a pleural base and halo, , but the most sensitive diagnostic sign ( %) is vascular occlusion in the sinus of the lesions shown through pulmonary angio-ct. patched bilateral ground-glass opacities, whose size is very variable, are more typical of viruses and they can be accompanied by small nodes and consolidations. --- in patients with hpt the paved pattern is of viral origin mainly and less commonly of a non-infectious cause. infection due to pneumocystis jirovecii causes perihilar bilateral ground-glass attenuation areas in upper fields, usually after the th day post-hpt---a moment in which prophylaxis is usually withdrawn. respiratory colonization due to candida is common in these patients, but pneumonia is exceptional except for in situations of spread candidiasis. emerging ''non-candida'' yeasts do cause infectious pulmonary consolidations and nodes. pulmonary edema is frequent in the course of mm (due to pet, cardiac and/or renal amyloidosis) and aml (mainly as a consequence of treatment). at the onset and in the course of aml it should be differentiated from diffuse alveolar hemorrhage (dah) due to thrombocytopenia and/or infection. secondary pulmonary lymphoid infiltration in leukemias is rare. it can be seen as ground-glass attenuation areas , or consolidations similar to an organizing pneumonia, , with adenopathies. in patients with cll of years of evolution and adenopathies, the appearance of peribronchovascular consolidations, even endobronchial occupations, and clinical worsening, will make us suspect transformation into a high-grade lymphoma. leukostasis (aggregation of blasts in pulmonary microvascularization) is exceptional and it will only be suspected in the diagnosis or relapse of an aml due to a rapid increase of the percentage of leucocytes in blood; it is characterized by respiratory and neurological failure kicking in with con pulmonary consolidations partly due to alveolar damage and hemorrhages due to plateletpenia. , in mm, consolidations due to pulmonary infiltration of plasmatic cells causing respiratory distress of poor prognosis have also been described. , noninfectious complications secondary to treatment in the h following the transfusion of blood products, the sudden appearance of consolidations simulating an edema and accompanying a respiratory failure usually reflect acute pulmonary damage (apd) called trali (transfusion-related alveolar lung injury) which associates high mortality rates. any courses of chemotherapy can cause pulmonary toxicity, although the most usual patterns are: organizing pneumonia (op), unspecific interstitial pneumonia (uip), pneumonitis due to hypersensitivity, eosinophilic pneumonia and diffuse alveolar damage. carmustine (used in lymphomas and mm), which usually gives a uip pattern ; cytarabine (administered in high doses for the treatment of aml), that can cause non-cardiogenic pulmonary edema and bleomycin (lymphoma treatment), causing diffuse alveolar damage and op are among the most common drugs used. expiration before-hpt rituximab is an anti-cd monoclonal antibody used in the treatment of lymphoproliferative b syndromes that have been associated to pulmonary disease due to a mechanism that has not been clarified yet after a median of four cycles and with a median time of appearance of weeks after the last infusion. the ground-glass areas or bilateral diffuse pulmonary consolidations appear in weeks or months, and less commonly in hours or days, and they correspond histologically to inflammatory and lymphocytic infiltrates, among others. most of them show hypoxemia of lethal evolution in %. the granulocyte colony-stimulation factor (g-csf) used in neutropenia recovery can rarely induce an apd during the first days of treatment displaying images similar to those of edemas or alveolar hemorrhages. all-trans retinoic acid (atra) and arsenic trioxide, used for the treatment of acute promyelocytic leukemia, can cause ( %) a ''differentiation syndrome'' consisting of respiratory failure manifestations during the following --- days probably due to the release of cytokines. pleural effusion, ground-glass areas and bronchial wall thickening due to edema and hemorrhage can be seen. the pre-graft early post-hpt period can become complicated with: • pulmonary edema-hydrostatic or due to increased patency (the most common non-infectious complication). • dah of multifactor etiology, characterized by large ground-glass attenuation areas that can evolve into paved patterns, in the absence of cardiomegalia and effusion; hemoptysis is only observed in % of the cases, but a drop in the levels of hemoglobin and the high rate of macrophages in bal help us in the diagnosis. • ''graft syndrome'' or pre-graft respiratory distress---the consequence of an endothelial damage possibly due to the release of pro-inflammatory cytokines; it is similar to edema but without cardiomegalia and with clinical manifestations of fever and cutaneous exanthem. • ''idiopathic pneumonia syndrome'' defined by the american thoracic society as an acute respiratory failure and diffuse alveolar damage in the absence of cardiac or renal disease, iatrogenesis and responsible microorganism. it is an exclusion diagnosis, probably of multifactor etiology or due to non-affiliated infections, such as it would happen with the recently discovered metapneumovirus. , in the late post-hpt period, peribronchovascular reticulation and ground-glass areas, and on occasion paved pattern ones, can represent an unclassifiable interstitial pneumonia, associated with rejection. histologically, it corresponds to a bronchioloalveolar lymphoplasmocytic infiltration that spreads toward peripheral regions and evolves into a fibrosis with traction and hive bronchiectasis. op can be secondary to infection, toxicity by drugs, radiotherapy or rejection; peripheral peribronchovascular and perilobular consolidations, and the inverted halo sign are characteristic findings in the appropriate clinical context. in a situation of neutropenia the inverted halo should lead us to consider mucormycosis in the first place which is different from the op in that the thickness of its ring is > cm and also presents internal reticulation (fig. ) . acute pneumonitis due to radiation can occur --- weeks after mediastinal lymphoma radiotherapy and cause clinical and radiologic manifestations similar to those of a pneumonia. it is located in the irradiated area with a marked delimitation of the healthy parenchyma; it evolves into paramediastinal fibrosis. acute pulmonary rejection to hpt and pulmonary alveolar proteinosis due to leukemic infiltration or lately to treatment are extremely rare. , these images represent bronchioles filled with mucous, liquid or pus; they usually correspond to an infectious bronchiolitis that can be due to many different germs. it can be due to unspecific respiratory infection, smoking, bronchiolitis obliterans, lymphoid infiltration or other bronchial conditions. liquid retention and inflammatory or tumor infiltration of perilymphatic distribution translate into thickening of the bronchovascular axes, interlobular septa and the subpleural interstice. the straight interstitial thickening can correspond to an interstitial pulmonary edema and a graft syndrome in the early post-graft period. in the --- months following the hpt, pulmonary veno-occlusive disease can be a rare complication leading to pulmonary hypertension with pulmonary congestion in the absence of left cardiac disease. secondary lymphatic tumor pulmonary infiltration can kick in as a straight or nodular thickening of the perilymphatic interstice , and make up peribronchial consolidations ( fig. a) . in most cases dissemination is retrograde from the hilar and mediastinal affectation, and can get to occlude the airways, especially in the cll. in mm, pulmonary amyloidosis can be seen radiologically as a septal interstitial thickening. chest computed tomography (ct) and mild mosaic patterns in expiration due to air entrapment (asterisks). heart failure and pleuropericardial effusion due to cardiac and serous amyloidosis. although the most frequent cause is pulmonary carcinoma we should not forget that they are a rare form of hl and nhl presentation. also in the course of cll, it is possible to see lymphoid growth in the lumen or bronchial wall making up obstructive endoluminal masses and cases of extra-osseous plasmacytoma as endoluminal masses have been published. it is a characteristic finding of bronchiolitis obliterans (bo), the most common ( --- %) and relevant complication --- months post-hpt. it is considered a chronic gvhd defined by functional criteria of airway obstruction in the absence of active respiratory infection and confirmed through transbronchial and/or surgical biopsy. the ''bo syndrome'' can be diagnosed without histology when there is air trapping and/or bronchiectasis in hrct and gvhd in at least one other organ. , the images in forced expiration show air entrapment in --- % of the cases; on the other hand and yet despite the fact that this is the finding with the most diagnostic sensitivity, it is not very useful for the detection of early preclinical stages. bronchiectasis appears in more advanced stages and its progression has been associated with the forced expiratory volume in one second. hrct is a very useful addition to rule out airway infection or tumor infiltration, which can alter respiratory tests. this disease causes slow functional worsening, especially if not detected early. its treatment with corticoids and immune depressants causes infectious complications that will be the main cause of death. it can be secondary to distress, toxicity, infection or radiotherapy (fig. ). in the late period after hpt it can correspond to an unclassifiable interstitial pneumonia or to a pleuroparenchymatous fibroelastosis likely as a consequence of chemotherapy, radiotherapy and gvhd. the history, distribution and evolution of the findings will guide the etiological diagnosis. they can be seen in a transitory way in the sinus of infectious consolidations and be due to organizing pneumonia. irreversible dilations (bronchiectasis) in areas of air entrapment are a characteristic finding of bronchiolitis obliterans unlike those observed in areas of fibrosis due to traction. interstitial pulmonary emphysema: air leak syndrome (fig. f) it is a typical complication of advanced post-hpt bo and a marker of poor prognosis. the increase of alveolar pressure leads both to its rupture and air leak around the ( ) week later (normal evolution not meaning non-responsiveness). (c) cavitation two ( ) weeks later coinciding with a recovering neutropenia, air crescent sign (arrows). (d---f) a different patient. consolidation (asterisk) with an initial halo (d); two ( ) weeks later (e) size increase and disappearance of the halo; further size decrease with persistence of peribronchovascular consolidation with bronchial dilations (f) without resolution. the resection of lesion to guarantee the control of the infection before the hematopoietic precursor transplantation (hpt) showed organized pneumonia without micro-organisms or malignant cells. bronchovascular axes due to retrograde dissection; it can reach the mediastinum, the subcutaneous tissue and the pleural cavity. the treatment is usually conservative except if the pneumothorax requires drainage. , pulmonary infiltration, radiotherapy and infection postulate as causes for a greater incidence of pneumothorax in hodgkin's disease (fig. ). in the absence of radiotherapy or infection, the possibility of pleura-pulmonary infiltration described in lymphomas and mm should be taken into consideration. , in the late post-hpt period, the air leak syndrome due to bo or the rupture of apical vesicles due to a pleuroparenchymatous fibroelastosis should be taken into consideration. small cysts in the upper fields can correspond to pneumatoceles due to infection by pneumocystis jirovecii. bilateral cysts, isolated or associated with nodes and adenopathies should make us think of the possibility of a pulmonary disease due light chains deposit disease in patients with mm or macroglobulinemia and obstructive functional pattern (fig. ) . in patients with a fever and active infection findings in the hrct it is recommended that the examination be repeated until resolution of the findings: a) in cases of good clinical response, it is possible to wait for several weeks and even for or months---the estimated time for the resolution of the findings. during the first week of treatment the ifi lesions can grow worse which does not mean poor evolution (fig. ) . also it is convenient to recognize in any infection an immune reconstruction syndrome not to be taken for an absence of response. it is an inflammatory exacerbation secondary to neutrophil recovery or to the withdrawal of immunosuppressant therapy which in turn translates into clinical worsening and lesion growth. b) if suspicion that there is not a good response (due to clinical and/or radiographic findings) it is important to repeat the diagnostic tests to rule out initially unidentified mixed infections. if nodes are detected without infectious clinical manifestations, they should be compared with previous images to find out whether they are residues of an infection that occurred during periods of immune suppression. indeterminate nodes can be followed up according to the recommendations made by the fleischner society, while the fnpa or the biopsy should be considered in lesions of suspected malignancy because of their characteristics, due to history of lymphoma and/or allogenic hpt, or because the lesions did not evolve as expected after therapy (fig. ). preferentially the sample will be obtained through cutaneous (guided by ct or ultrasound) or transbronchial access, and surgery will be considered as a last resort or preferably videothoracoscopy if available. the diffuse pulmonary disease of unclear etiology usually ends up requiring surgical or videothoracoscopic pulmonary biopsy due to its greater diagnostic yield. -fdg pet-ct is not only useful for the staging and follow up of metabolically-active lymphomas but also a pathological uptake in the absence of pulmonary findings in the hrct can raise early suspicion of drug toxicity , or leukemic infiltration and it can be useful to differentiate active lesion scarring (whether toxic, infectious or tumoral). in patients with hp the thoracic hrct helps us come close to the differential diagnosis of infectious and non-infectious pulmonary complications by integrating image findings and clinical data. the hrct needs to be performed in cases of acute clinical presentations and quickly when suspicion of ifi. it allows us to assess the response to treatment, detect malignancies and optimize the obtention of both the bal and the pulmonary biopsy. protection of people and animals. the authors declare that no experiments with human beings or animals have been performed while conducting this investigation. data confidentiality. the authors declare that in this article there are no data from patients. right to privacy and informed consent. the authors declare that in this article there are no data from patients. the authors declare no conflict of interests. manager of the integrity of the study study idea: pcb study design: pcb data mining data analysis and interpretation: - . statistical analysis pulmonary infections in the late period after allogeneic bone marrow transplantation: chest radiography versus computed tomography prevention of fungal infections in the immunocompromised host evidence-based approach to treatment of febrile neutropenia 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[ f]-fluorodeoxyglucose positron emission tomography combined with computed tomography detection of asymptomatic late pulmonary toxicity in patients with non-hodgkin lymphoma treated with rituximab-containing chemotherapy. leuk lymphoma the authors wish to thank ms. lucía flors, mr. carlos muñoz, ms. laura trilles, and mr. carles fonfría for their collaboration in the gathering of cases used to illustrate this article. key: cord- -e zojanb authors: lieberoth, andreas; pedersen, mads kock; marin, andreea catalina; planke, tilo; sherson, jacob friis title: getting humans to do quantum optimization - user acquisition, engagement and early results from the citizen cyberscience game quantum moves date: - - journal: nan doi: . /hc.v i . sha: doc_id: cord_uid: e zojanb the game quantum moves was designed to pit human players against computer algorithms, combining their solutions into hybrid optimization to control a scalable quantum computer. in this midstream report, we open our design process and describe the series of constitutive building stages going into a quantum physics citizen science game. we present our approach from designing a core gameplay around quantum simulations, to putting extra game elements in place in order to frame, structure, and motivate players' difficult path from curious visitors to competent science contributors. the player base is extremely diverse - for instance, two top players are a year old female accountant and a male taxi driver. among statistical predictors for retention and in-game high scores, the data from our first year suggest that people recruited based on real-world physics interest and via real-world events, but only with an intermediate science education, are more likely to become engaged and skilled contributors. interestingly, female players tended to perform better than male players, even though men played more games per day. to understand this relationship, we explore the profiles of our top players in more depth. we discuss in-world and in-game performance factors departing in psychological theories of intrinsic and extrinsic motivation, and the implications for using real live humans to do hybrid optimization via initially simple, but ultimately very cognitively complex games. when online participants are used as workhorses for difficult problems such as eterna's needle-inhaystack-like rna model selection task (lee et al, ) or eyewire's formidable challenge of mapping neural connectivity in the mouse retina (marx, ) , conclusive results may lie months and years into the future. in the rapidly growing field of human computation the design of new initiatives is often based on intuition rather than proven design hypotheses. the citizen cyberscience community is, however, a remarkably open scientific group, which allows us to capitalize on a unique and generous culture to learn from each other at each step of the journey -not just in the end, when all is securely tested and published. the citizen cyberscience game quantum moves was designed to help build a quantum computer -a computer more powerful than any other in the world based on moving atoms around under the principles governing quantum physics. this delicate process involves a constant risk of losing hold on the volatile atoms, if they are not moved precisely and quickly. the simulations in which our algorithms tried to optimize this process bear a remarkable similarity to side scrolling casual games, and so the notion of human quantum optimization was hatched: what if real humans would do things differently than the logical step-by-step nature of the algorithms? would explicit understandings of the counterintuitive quantum problems help people solve our problem more intelligently? would human physical and cognitive fallibility add an interesting random factor? and even if people in general could not beat the ai, would play trajectories resulting from certain lucky punches or persistent quantum heroes be enough to help the ais learn in new ways? this is the premise of human-computer hybrid optimization: helping ais learn through real people's blooming buzzing mess of solutions, when problems can be represented as engaging game levels. quantum moves places itself with eyewire, galaxy zoo, foldit and eterna in a small category of large-scale resource demanding online citizen cyberscience endeavors where real problem solving is taking place beyond pure data gathering. with this midstream paper, we want to share our experiences from this first year of beta design and player recruitment, and make our reflections and learning curves available. we believe that our findings about the engagement process -although preliminarycontain a series of conclusions pertinent to the design-and engagement processes hidden behind human computation. we first give a brief introduction to the physics behind the game, and the method of turning its core tenets into a playable game. this paper does not focus on the actual game results and the reader with no interest in physics can safely skip the first part. we then turn to the main focus of the paper, the description of the design considerations from the first year, especially relating to user acquisition and the structural gameplay surrounding the game's core loop. finally, we present data about participation for the beta year, noting how different properties like recruitment source and physics interest predict tenacity and performance in the game. we conclude by looking closer at our most dedicated "heroes" and discussing future perspectives for human computation and quantum moves. quantum mechanics originated in the beginning of the th century when the physicists of the time realized that the known laws of physics were not capable of describing the structure of atoms. experiments made by ernest rutherford showed that the atom had to consist of a positively charged nucleus orbited by negatively charged particles called electrons (longair, ) . in , niels bohr showed that only certain orbits were allowed, and that the electron could only jump from one obit to another by absorbing or emitting a quantum of light with the correct amount of energy. even more remarkably the electron was allowed to be in two different orbits simultaneously until a measurement was performed to determine in which of the orbits it was. from this, quantum mechanics evolved through the work of heisenberg (born, ), de broglie ( , schrödinger ( ) and many others. they showed that atoms should be described by a wave function: a distribution describing the probabilities of measuring the atom in every point in space. computer technology has also progressed rapidly over the past decades. moore's law states that available computer power doubles every months (moore, ) , due to the ability to fabricate ever-smaller transistors. however, the miniaturization has a lower limit due to quantum effects. this led to the proposal of the quantum computer (feynman, ) , in which the computer bits which are traditionally only allowed to be either or are replaced by quantum bits (qubits) that are allowed to be and simultaneously. a quantum computer holds the potential for huge calculation power since qubits would be capable of representing numbers (larger than the total number of atoms in the universe), in contrast to the normal computer where bits can represent just number. quantum computers have already been created in a very small system of qubits (vandersypen, ) , but they have yet to be implemented in a scalable system capable of outperforming traditional computers. many proposals for such scalable architectures exist in various systems and in one of them, atoms are contained in an egg-tray like trap made of interfering light beams (weitenberg, a , weitenberg, b . the quantum computation is then performed by concatenating a sequence of individual qubit flip operations (jørgensen, ) and two-qubit operations consisting of picking up an atom in one well with a focused tweezer of light and transporting it into contact with another atom somewhere else in the computer (weitenberg, b) . this is non-trivial because any fast movement of the tweezer causes the (probability distribution of the) atom to slosh around and this sloshing will result in an error in the calculation because a sloshing atom contains kinetic energy, which means that it is not in the ground state. the challenge of quantum moves consists in finding algorithms describing how the laser in the physical machine should be controlled to move atoms quickly from one location to another without introducing sloshing at the end. in quantum moves, players help build a powerful quantum computer by finding ways of moving a simulated atom from one location in the game interface to another, without inducing sloshing. the movement of the atom is guided and constrained by a so-called potential landscape spanning the screen (black line in figure ). the probability distribution of the atom (green in figure ) resembles a liquid, but will slosh and distribute itself in smaller waves at the slightest wrong movement according to the rules of quantum physics. we call the collective shape of the atom at any given instance of time its state. each level describes a unique problem represented by the potential landscape's line combined with pre-specified beginning-and target states. success is measured by the degree of overlap between the final state of the atom and that of the target area. a game always consists of controlling the simulated tweezer with your computer mouse for a given amount of time (by moving the bottom of an indentation in the potential landscape). dragging the mouse horizontally changes the position, whereas a vertical move increases or decreases the depth of the trapping indentation (physically realized by turning the power of the laser up or down). a game consists of one complete trajectory of the mouse through a particular level. this can be characterized as the game's "core loop" (fields, ) or (fittingly) "game atom" (elias, garfield, & gutschera, ) , as it recurs in every game level, variably modified with new obstacles, potential landscapes, goal-states and bonus-points to challenge players or simulate particular physics problems. the levels are organized according to an overall structural gameplay, or metagame, where they get unlocked in stepwise fashion and players receive rewards through different symbolic feedback such as highscores, - "stars" according to the degree of success, and acquisition of skill-and achievement badges. the entire solution for each game played is stored on our servers for potential future use in our laboratory. computer algorithms used to optimize this type of problems typically exist in two variants. in local algorithms, an initial trajectory is slightly perturbed in a stepwise fashion, and if a change proves beneficial, further in that same direction. this step -although it sounds simple -can be very sophisticated in state-of-the-art algorithms. unfortunately, this deterministic update often causes it to get stuck in what is called a local maximum. in contrast, algorithms with a random component such as genetic optimization algorithms can jump erratically from one solution to the next. this randomness ensures that they will never get stuck and eventually find the best solution -called the global maximum. the disadvantage with this type of optimization is the fact that the steps are random and therefore only beneficial in a small fraction of the times. this makes algorithms with random components exceedingly slow, often in fact so sluggish that in practice they will never converge to the optimal solution. the aim of quantum moves is to combine the best of both worlds in our gamified human quantum optimization: optimization that is rational most of the time, but sometimes makes seemingly random errors or leaps of intuition to rapidly find the sought after solutions. one example of a gameplay trajectory which requires a certain critical breakthrough (iacovides, aczel, scanlon, & woods, , is the level bring home water fast (see figure ). the aim is to fetch an atom from the well on the far right of the screen, and ferry it carefully back to your beginning position on the left. here, a good solution consists in utilizing the principle of quantum tunneling by bringing the two wells close together without merging them into one. the atom will the tunnel through the classically forbidden intermediate region, and appear in the well created by the moveable tweezer. specifically, we compare the players to the computer algorithms in two ways. first, for problems which can eventually be solved by the computer, we compare the score after each optimization to the equivalent high score of players after having played an equal amount of times. as long as the player score is higher than the computer score, the human result really represents the fastest way of getting results at that particular junction. of course, since the specialty of the computer is to make minute adjustments and improvements, if the particular problem is of a nature that can be solved by the computers, eventually it will overtake the players. in such cases it is an extremely interesting question, to which extent player results can be used as a starting point for a computer optimization that will yield faster convergence rate than the computer optimization alone. finally, for some of the problems posed the computer fails to find good solutions and it is of course extremely interesting to which extent players can find these solutions. although the analysis of the more than , unique play trajectories generated so far is not complete yet, a pattern seems to emerge that the human optimization is indeed superior to the computer in many problem spaces investigated in all of the three areas discussed above. even more surprising, it seems that the fraction of the players actually outperforming the computer is quite large. whether the players succeed by fluke, by building in-game skills, or possibly through a simple theoretical understanding of the quantum physics principles represented in the game is still an open question, which will be the topic of future research. a central challenge to achieve the optimization described above is that quantum moves needs to recruit engageable players and hone their in-game skills over time. player acquisition is a central part of online game launch strategies. the industry metric user acquisition cost (uac) is an aggregate of advertisement cost, development cost, back-end expenditures and similar prices used to describe how much money a social game developer will spend, on average, to get a new user (fields, ) . developers thus expect to spend quite a bit of time and money to get click-through and installations of their games. this is both an ongoing process of marketing and design, and a focused enterprise to build critical mass at launch. however, while the commercial industry needs to balance this effort and expenditure against each player's average lifetime value (ltv) in dollars and cents, as well as their ability to make the game a social place and recruit friends to join them (lifetime network value, lnv), citizen science games need to consider the time and relative price of a different kind of payoffnamely tangible science contribution from each player and his network. we can label the average direct player contribution user science value (usv). player contributions on our sister-game galaxy zoo have been aggregated into four main clusters, or participation profiles. these reveal that some contribute a lot early, usually on their first login, never to return, while others establish a stable pattern of play over a prolonged period of time (brasiliero, ; ponciano, brasiliero, simposon & smith, ) . each galaxy pattern recognized is a worthwhile addition of data. in more complex games like eyewire (robinson, personal communication, / . ) and quantum moves, however, players need to build a modicum of skill before they can reliably contribute to the core scientific challenges (barring flukes arising from the random factor that is human cognitive and behavioral processing, i.e. bob, ) . we could call the average point at which players start doing anything directly useful, the game's user contribution threshold (uct). we have come to call the small percentage of players who tenaciously acquire the necessary skills and persist far beyond the uct "heroes". our notion of heroes mirrors that of whales from commercial games that rely on free-to-play strategies where only a small group of players are ever really monetized through in-game purchases, premium memberships and the like. while citizen science games with low cognitive complexity can benefit from every minute any player spends, games with high cognitive complexity like quantum moves and eyewire only usually gain direct value from a player if he/she becomes a hero. players who just visit out of curiosity and then drop out can be called flâneurs, while average participants explore the structural gameplay deeper, and might contribute by fluke. to that calculation we must then add a player's network value, which means that it seems like a viable strategy to make the game fun and attractive for everyone, even if only a few manage to contribute to the core scientific challenge. generating sustained engagement at less complex levels of game participation may also be a way to gradually build the loyalty and skills needed for a flâneur to transition into a hero. a real concern, which our empirical work aims to address, is, however, if the average hero's preferences and play trajectories diverge substantially from average players. from a psychological standpoint, it is important to mention the difference between intrinsic and extrinsic motivation, as they apply to participant retention in citizen science projects. there are competing schools of thought on the matter (deci, ; malone, ) , but they agree on core tenets: in extrinsic motivation, action is based on outside rewards like money or socially valued praise, or avoidance of unpleasant states like scolding. this entails a tendency to act half-heartedly and cease the behavior when the outside factors dry out (deci, koestner, & ryan, ) . in a state of intrinsic motivation, on the other hand, the user is driven by desires to participate, explore, learn and master the activity in itself. extrinsic design-logics are commonly found in vulgar points-badges-leaderboards (pbl) gamification, where mainstream ideas (e.g. bowser, preece, & hansen, ; marczewski, ) resemble diluted versions of behaviorist token economy (e.g. kazdin, ; skinner, ) and th century utility-based economics brought into question by behavioral economists like kahneman ( ) . extrinsically informed motivational design places great importance on the magnitude and temporal distribution of rewards, as well as their psychological framing. intrinsic motivation theories, on the other hand, prescribe design structures that support self-determination via a sense of competence, autonomy and social relatedness (deci & ryan, ; rigby & ryan, ) , or challenge, fantasy and curiosity (malone, ) plus interpersonal factors like recognition, competition and cooperation (malone & lepper, ) . for an overview, see below figure . fogg and eckles' ( ) "behavior chain for online participation" can be used to conceptualize phases of user involvement. the discovery phase "learning about the service" and "visiting the site. once they have arrived on the site, users have plenty of opportunities to explore the information available and get the chance "to be educated and influenced". for example, the quantum moves site has videos and photos integrated. also, the website hosts a forum, where visitors can read discussions of registered players and get the chance to be exposed to the game. in the superficial involvement stage, users are influenced to "decide to try" and "get started" with the game. at this stage the structural gameplay will fluently guide them through the tutorial levels and hopefully prompt them to create a profile and validate an email activation link, so they can save their progress. it is only in the final phase, called true commitment, users generate large added value. in quantum moves this translates to users making a valuable contribution to either the science, game development, or through their lifetime network value. such examples range from creation of posts and video materials to creating levels, but contributors' core commitment to quantum moves is measured in play counts and scores. this -phase process mirrors the conceptual difference between interest (berlyne, (berlyne, , , motivation (grant & dweck, ; jensen & buckley, ; prestopnik & crowston, ; raddick et al., ; ryan, rigby, & przybylski, ) and sustained engagement (kular, gatenby, rees, soane, & truss, ; rigby & ryan, ; skinner, seddon, & postlethwaite, ) . interest can be understood as the immediate psychological allure of something encountered in the world, usually as a property of perceptual processing (berlyne, (berlyne, , ) exacting a motivational pull of curiosity to investigate. engagement (kahn, ) includes both motivation, behavioral change, and persistence in an activity. to acquire engaged players across the beta period, we used four separate recruitment strategies. the early parts of the game (see below) were tested in high-school science classrooms and our own university lectures, where large numbers of students were forced to sign up. we also had the opportunity to speak at several high-profile events, such as a couple of public lectures with over people combined, which we used for an a/b-test of certain game elements (see below). the project has additionally garnered a good deal of attention in traditional media and online, which has generated substantial influxes in identifiable jolts. finally, a fourth group of ongoing clickthrough can be attributed to community efforts and general buzz arising online and in-world, making their origin essentially unknown. since many players cease participating quite quickly (a common pattern in games and citizen science projects alike, but especially prevalent for students forced to participate), we also announced several "featured challenges", where existing players were prompted to come back and knuckle down on particular levels. we awarded extra in-game badges for participation, and offered combinations of books, logo mugs, t-shirts, and even a lab visit with all expenses paid (see below) to top contributors. we can thus envision a two-dimensional space for motivational devices, with one axis constituted by in-game (points, progress, good core gameplay, community, etc.) versus in-world (our talks and teaching efforts, prizes, recommendations from friends) (lieberoth & roepstorff, ; stevens, satwicz, & mccarthy, ) situation, and the other ranging from intrinsic (science participation, challenging gameplay, fun, fascination with physics, etc.) to extrinsic (physical prizes, mandatory high school participation) motivational flavors. game designs based on intrinsic motivation are thought to satisfy the criteria through gameplay processes alone (the core loop and the structural gameplay surrounding it), while our recruitment strategies in educational settings and prizes in featured challenges can be said to be classically extrinsic. extrinsic reward has been found to exact a detrimental effect on intrinsic motivations (deci et al., ) , but it is worth noticing that points, leaderboard placements and even physical prizes for top-performance may act as intrinsically motivating feedback, social devices and signs of mastery. but people may also already have interests and preferences in the real world, that makes citizen science participation intrinsically motivating, as for instance seen in player data from galaxy zoo where the most prevalent motivators were not related to the site's superficial gamification devices, but rather a fascination with outer space or a sense of participating in something greater (raddick et al., ) . as such, creating engagement is usually a question of balancing design strategies rather than relying on a single approach, such as vulgar pbl-gamification. recruitment activities in-world and online, an engaging in-game core loop, a structural gameplay to frame, structure and motivate the player's continual progression through the levels, as well as an active community where participants get a sense of continually contributing to science, are all central components of the strategy laid out to hopefully realizing the scientific goals of quantum moves. in the remaining sections, we describe the game design process as it has unfolded in a sometimes stumbling but always-creative fashion with its many different ideas and theoretical rationales. we then report statistics about users, recruitment and retention, and the attributes that characterize our small crop of heroes so far. we developed quantum moves as the first game under the scienceathome.org umbrella, one of the first citizen science projects in the quantum physics segment. the initiative is developed within our cross disciplinary centre for community driven research (coder) , where we aim to bridge theoretical and experimental research with online community efforts. the early development of quantum moves, previously known as the quantum computer game, started in december with the first coding iterations deployed in matlab -an algebra program widely used in physics. the decision was based on the program's plotting flexibility and the programming experience available in the student pool. an early version of the game was ready in february (see figure a . in the appendix) and subsequently tested in several danish high schools. this served as an overall proof of concept, yet we noticed that several challenges hindered the game experience, mainly due to matlab's limited portability and graphic support. over the following months, we improved various aspects of the initial prototype. however, a test session with volunteers in the summer of made it clear that, if we wanted the game to have a large public appeal, we had to abandon matlab and rewrite the code in a flexible, end-user accessible programming language. the best solution at that time was java, as it addressed most of our concerns of end-user accessibility, robustness, flexibility in programming and variety in graphics. a preliminary java edition came out in early october (see figure a. ), and a testing version made available to a small group outside the coder team in dec (see figure a. , a. , a. ). the first, fully-fledged beta version was publicly launched and advertised on the th of june (see figure a . ). the game was structured around levels: tutorial, arcade (a series of games where players could practice) scientific (where we included the games we considered most relevant for our lab research) and user space (a sandbox environment, where players could design their own games or try those created by other users in the community). when the tutorial games were successfully completed, a user was allowed to roam around and try any of the other games found in the arcade and scientific levels. the launch of this beta version was covered in several national media outlets, e.g. national geographic and videnskab.dk. the media attention played a definite role in making the game known to an audience beyond the usual high school students. yet, we experienced several glitches with the university server which slowed down our initial success. a brief follow-up survey sent personally to the top players after launch pointed out that the technical issues were doubled by frustrations with the abrupt increase in difficulty, starting with the tutorial. also, looking at the data gathered in the days after the release, we noticed, that once players got past the tutorial, they predominately chose to play in the scientific level with insufficient time spent in the arcade level intended to help them acquire necessary core skills. this became a premise for our discussions and, as the team expanded to include a business school graduate and a psychology researcher, we focused on reevaluating the game design. in august , the current beta version was introduced ( figure a. ) . from this point on, we refer to that version in this paper. early, we were confronted with two pressing aspects that needed to be addressed: one was redesigning the tutorial to create a lower entry barrier, while also ensuring an effective learning curve. the other was to create a game structure that would enable players to hone the skills needed to perform with high effect in the scientific levels, hopefully turning some into heroes. we started by operationalizing the main physics concepts applied in the game and their equivalent in-game operations into a set of core skills that players would need to acquire: deceleration of the atom speed, tunneling the atom into the target state and stabilization of the atom state. these became our guiding references for the structural redesign of both the tutorial and the advanced levels. firstly, we reorganized the tutorial into a set of games, which gradually introduced the main physics elements and core game loop: the atom as a ball, continuing with the atom as a wave, and finishing with the insertion of a static obstacle. to ensure that players had appropriate visual scaffolding and understood the goals of each challenge, we added video animations preceding each level, presenting one possible trajectory along with written hints. the successful completion of the last tutorial level allows players to access the main menu, with the option of playing in separate skill labs: cool, tunneling and control. we decided to create predetermined paths, presented in a tree structure (see figure a. ) . by doing so, we aimed to push players to go through the skill training levels before the scientific levels where the main contributions to our current citizen science problems are made. since individual levels differ in difficulty and require refinement of particular in-game operations, each skill lab was divided into a bachelor and master section. the user has to successfully complete - levels to acquire skill badges on their profile, which would unlock specific new levels. once the bachelor level was completed, players would gain partial access to both the master section and the scientific labs: qcomp and beat ai. to achieve full scientific access, the master's would have to be completed at some point. beyond this, the structural gameplay tree consisted of a few more nodes: created as a more theoretically grounded, finetune contains a set of tools that makes it easy for a player to manually change points on the path of his previously played levels, adjust the timing, stretch or shrink the total time or smooth the path. since the tool functions are not automated, it is ultimately up to the user to decide which parameters are to be changed in order to optimize the path. the user-defined space is open to all players, regardless of skills. it includes construction yard, a sand box type of environment where users can build their own levels with goals and obstacles and playground, a space where users can play each other's' creations. compared to the predefined games, these are not connected to the main tree structure, and can be played immediately after completing the tutorial. therefore, we decided not to set any skills requirements for creating games. furthermore, since the user games created are primarily supposed to be used as community building drivers, they are neither checked for resolvability, nor included in any of our data analysis processes. based on the number of plays, the user-created games are ranked on the "the most popular games in the community" leaderboard. the last and newest branch in early combined predefined user-created games in challenges with the intention to enhance competition and the community feeling. for instance the newest game type is called "quantum quests". here we catered to achievement oriented players (heeter, lee, medler, & magerko, ; sherry & lucas, ) , who crave more traditionally "gamy" elements such as finite super mario-like lives. building on the iron man type of game principles, this game consisted of a series of selected games, where a player has to progress as much as possible through the level with only a limited number of attempts, called "lives". it is currently being used as a base for creating more immersive d levels using the unity game development platform. to create a sense of progress in quantum moves with minimal effort, we implemented the simple points-badges-leaderboards (pbl) mechanic recurrent in casual games like candy crush saga and shallow marketing gamification. in quantum moves, the score is calculated on the basis of various parameters, such as time penalty, overlap with target state, points collected while avoiding the obstacles, and is presented to players in the end screen triggered by the finalization of a game sequence. based on the obtained score, a player would move up or down on the leaderboard, presented in the lower right corner of the game window (e.g. fig. a ). to mark a player's progress, his score on the leaderboard is shown in relation to similar scores in his range. this is similar to the techniques found on social games, where the scores of a player are presented in relation to others in his/her social network. we made use of two feedback techniques found in gamified applications (groh, ) and mobile games to introduce visual cues that could enhance a player' sense of progression (deterding, ) . the first is a bar presented at the top of the game interface, which provides players with real time feedback on their performance. the bar changes its color from red to yellow and finally to green to suggest the effectiveness of a player's trajectory. mirroring the wellknown concept of stars found in games like candy crush saga, we also introduced "atoms" as an alternative three-tier grading system reflecting acceptable, good and excellent performance thresholds for each levels, indirectly challenging players to revisit levels and perfecting their collection. the potential downside is that once a game is completed with three stars, players might lose motivation to further optimize his/her score. the last technique to indicate progress is by acknowledging specific achievements in the form of badges on the player profile (antin and churchill, , denny, ) . we designed a series of possible badges for two types of achievement: performance and engagement. depending on performance at various junctions of the game, and cumulative time spent logging and interacting with the game universe, a player could receive a recognition for reaching a specific milestone, "bachelor degree" or a particular threshold of play counts like "quantum frenzy ". to test the effectiveness of the pbl framework and constrained paths of play on player engagement, we also set up a randomized a/b-test surrounding a major event in august , where around people would attend talks about quantum moves and the quantum computer. the literature on gamification is rife with pundits clamoring about the efficacy of badges in motivational design, but there is not much quantitative data to back them up, so we jumped on the opportunity to test whether giving badges to players at various junctions would be a significant motivator for repeated visits and engaged gameplay. at the time, we needed to figure out how to best guide players on their path to the scientific levels, without creating barriers to progress and the feeling of competence. we had conflicting hypotheses about how to build this into structural gameplay: on one hand, the central psychological usability (norman, ) and choice architecture (thaler & sunstein, ) principle of guiding constraints recommends that a system should have a certain amount of openness, but the basic interface for core operations should be limited to the most preferable options. locking levels until the necessary skills are accumulated fits this view. on the other hand, we want players to follow their curiosity, maximizing autonomy, motivation, and access to the science levels. in this view, the structural gameplay would not need stringent locks based on skills, but just the tree-like lab structure laid out as an open map, with friendly hints about where to go, if a level proves too difficult. to this end, an "a/b test" was conceived as a x factorial design, randomly assigning the expected new players to one of four conditions: locked levels or open levels with or without badges. unfortunately, the test was crippled by both time constraints and technical difficulties. our programmers were working overnight to implement the system to automate a/b-test cell assignment, but also had a lot of more pressing design issues to address before launch. in the end, only a fraction of the badges we had designed were implemented, and players only got weak cues when achieving them. since we did not have time to test it, the skill system unlocking levels seemed also somewhat counterintuitive. some old server issues also came back to haunt us on the day, so many invitees were probably unable to log on to the game after creating their account. with these limitations, the usable data were only a fraction of the people assigned to each condition (down to n= player in the "open levels with no badges" cell). the results were statistically insignificant, and will not be reported below. we are awaiting new opportunities to gather this kind of data, which we believe is central to both our own work and adding a much-needed controlled evidence-base to the academic discussion of gamification in general. as these game design choices were being implemented, we collected data on user acquisition, play trajectories, and the few dedicated players we call heroes from beta launch in to writing this paper in april . the results are reported in the next section. between the th of november and th of april , there were approximately users visiting the www.scienceathome.org website, . % of these being new unique visitors. average visits last minutes, where users visit an average of pages on the website. ip addresses came from at least countries (see figure ); yet, the country tally could be even higher, as we could not place a country of origin to approximately users. at this point, most registrations originate from denmark, followed then by germany and the united states. in the following analysis we present results derived from data collected over a period of months, from the beta version launched on th of june until th of april . the sample relevant for the demographic and human computation data is equal to a cohort of people. % of all players actually finish the tutorial (see figure ) , which is an encouraging number. figure illustrates early drop-off, as people move from superficial involvement to commitment via the mandatory tutorial levels. these data were collected from all users registered between th of july to th of march , which is equivalent to a total sample of players using the newest version of the tutorial. as it can be noticed on figure , the drop-off mainly happens between play during the first six levels (m= %, % ci[ %, %]). however, the th tutorial level display a large discrepancy between the numbers of tried versus completed levels. a one-way anova was performed, to see if the th tutorial level (m= %) belongs to a different normal distribution of completion ratios than the first six levels (f( , ) = . , p < . **). this indicates that the th level has a significantly lower completion ratio than the first six tutorial levels, suggesting that the th tutorial level, which is the first level that includes the zones of death, which the atom is not allowed to touch, and which constitutes a significant barrier to an otherwise fluid flow of progress, most likely proving too difficult or disrupting competence motivation. an alternative explanation could, however, be that noncommitted visitors simply realize that they have now seen all the game has to offer, and either stop because their flanêurs' curiosity has been satisfied, or because they are not sufficiently attracted by the gameplay to engage further. as detailed at the top of section , during the beta period we used four strategies to recruit players. the classifications online/media and voluntary by talks were based on all registration in a ± days' time span before and after a registered major online or in-world event. the days before the event was included to account for the users that wanted to checkout the game in anticipation of the event. to avoid overlap with online/media, we attributed the voluntary by talks tag only to the users registered in the same country as where the event took place. the classification of forced by talks was given by a tag added at the point of registration. the results of these recruitment efforts are summarized in figure , which displays increases in unique users attributable to one of the main groups. a large percentage of our registered beta users originate in the online/media group. the steep initial rise of this curve can be attributed to national media coverage of the game launch in june , while the stepwise increases beginning around day is due to ongoing publicity. from mid-september , we increased our communication efforts by setting up a content strategy for the blog and forum on scienceathome.org, where players ask and answer questions related to the game, report bugs or request new game features. these efforts were complemented by a social media presence, with the launch of a facebook page and twitter account, and an increased focus on producing video content for vimeo. also, more attention was directed towards a proactive public relations approach, in order to ensure the recognition of the game on established human computation websites and blogs like scistarter and citizen science centre. following this, we noticed an increase in the number of other sources mentioning the game or giving positive reviews referring back to our website. to analyze if any of our data could indicate a predictor for the number of play counts (see figure ), a one-way anova test for differences in the number of play counts generated per active day for each player was performed between the four registration origin groups (see table ). this showed a significant between-groups difference (f( , )= . , p=< . **). in order to discover which groups are different from each other a tukey-kramer post-hoc comparison was performed and it shows that both forced by talks and voluntary by talks differed significantly from online/media (p= . * and p< . ** respectively), unknown (p= . ** and p= . ** respectively) and each other (p< . **). online/media and unknown did not differ significantly from each other (p= . ) suggesting that these two large player groups were qualitatively similar, revealing a pattern where people who actually went out the door in-world on their own accord are highly motivated to play, compared to those who found their way to us online, and especially the poor souls forced by teachers who only played a little. a similar procedure was conducted for physics interest, years of physics education beyond th grade, and finally between male and female (all summarized in table ). the one-way anova shows significant differences for both physics interest (f( , )= . , p< . **), gender (f( , )= . , p< . **) and physics education (f( , )= . , p< . **). tukey-kramer post-hoc comparisons show that high interest in physics leads to significantly more play that low and middle interest (both p< . **), while the latter two do not differ significantly (p= . ). for physics education beyond the th grade the post-hoc comparisons show that the very large group with - years of schooling played much more than those with - years (p= . **) and - years (p< . **) of additional schooling behind them, which did not differ significantly from each other (p= . ). the game framing of our citizen science project thus seems to speak to people with a notable casual interest in physics, but not a too professional background. we were also interested in who performed well after having completed the tutorial levels, so we calculated the average score on the tutorial levels (the most stable figure, since players may have chosen many different paths after the th tutorial level) calculated for the four registration groups, interest, physics education beyond th grade, and gender (see table a one-way anova reveals that the registration groups differed significantly on average score reached on the tutorial levels (f( , )= . , p< . **). the tukey-kramer post-hoc comparisons show that forced by talks differed significantly from online/media (p< . **) and unknown (p= . **), but not from voluntary by talks (p= . ). voluntary by talks surprising differ from online/media (p= . *) but not unknown (p= . ), which do not differ from each other (p= . ). interestingly, the forced by talks group performed best, perhaps because they received better in-world instructions than those tackling the tutorial only with in-game cues as scaffolding. but it is also worth noticing, that these players forced by extrinsic means were much less likely to complete the tutorial levels the first place, so the population registered here is likely to reflect only the most tenacious and intrinsically motivated members of the cohort, who might also have engaged fully in the game on their own time. no significant effect on tutorial scores was found for interest (f( , )= . , p= . ), nor education f( , )= . , p= . ), but there was a significant difference between the genders, with females significantly outperforming their male competition (f( , ) = . , p= . **). to assess how quantum moves retained players recruited by the four different means, we calculated the number of active days, defined as a day with at least one play count, for each user. a score of can thus mean someone who plays intensely for days on end never to return, or someone who returns a day per months for half a year. a look at the drop-off curves in figure indicates that % of the users drop out within days and never return to the game. a one-way anova comparing the drop-off rates did not show a significant difference between the four registration groups. however, players that stayed for more than active days were all recruited at public events, and subsequently signed up voluntarily. these players are the ones we refer to in our paper as "heroes". sadly, demographic data is available only for of them. even though the sample is small, we present a comparative analysis of the heroes versus more casual players in quantum moves. in figure , it can be noticed the results derived are based on a sample of "heroes" and casual users. a considerable part of our work has converged on heroes: that is, identifying players who significantly contribute in the science levels based on existing personal attributes like talent, interests and cognitive surplus, and in turn designing structural gameplay to help more casual players cross the uct through combination of motivation and fluid skill acquisition. a wilcoxon rank sum test for equal medians was conducted to compare heroes to more casual participants interest in physics (reported on a likert scale ranging - ) (hero mean= . sd= . ; casual mean= . sd= . ) and years of education related to physics (e.g. high school science) (hero mean= years, sd= . ; casual player mean= . years sd= . ), revealing no significant predictive power for who becomes a hero on either of these. firmly believing in the power of mixed methods triangulation, however, we also have some interesting qualitative data to illuminate this. out of the identified heroes, we had direct contact with three, namely sus, shb and meilby. for privacy reasons, we will refer to them by their quantum moves user name. due to their early contributions, sus and shb were invited as special guests to an offline community event and lab visit on the th of november , where they gave brief interviews to help us understand player motivations and promote quantum moves. the semi-structured chat was based on questions related predominantly to their motivation to play quantum moves and their preferred game elements. contact to meilby was established via email. his response was among those we received as a reply to the email sent out after the beta version launch in june . in this email, the questions were formulated around the strategies top players used to obtain above average scores in particular games, as well as to give their impressions with regard to the game structure and the newly launched features. sus (f, years old) was working as an accountant, based in copenhagen, denmark, when we met her. she signed up as a player in quantum moves after a public talk on the topic of information security and quantum computers, held by jacob sherson at the experimentarium on - august . since that date, sus had active days and play counts. this is noteworthy as, according to her own testimony, sus had never played a computer game until she signed up for quantum moves. when we asked sus what motivates her to keep playing the game, she mentioned that what motivated her to play quantum moves was the "knowledge that the results will be used for real, scientific purposes" (recorded interview with sus, th of nov ). sus' case helps us to understand, that quantum moves does not necessarily acquire its heroes from a traditional gamer demographic (although women + are the quickest expanding gamer group, software entertainment association (esa), ), and should take this into account for both recruitment, structural and core game design, and framing purposes. the drive to help science by playing a computer game was also what shb (f, ) mentioned as main motivation to sign up for quantum moves. shb is a danish high school student and member of the unf, a youth organization which aims to attract and develop young talents into natural sciences. just as sus, shb signed up as a player after the public talk at the experimentarium on - august . she has had active days and play counts. when asked what motivates her to keep playing quantum moves, she answered "it is fun to play, among others because i know (that by doing so) i help science" (danish-english translation of an excerpt from recorded interview with shb, th of nov. ). shb's case suggests that in-world membership in scientific communities of interest may provide a fertile ground for future recruitment. meilby joined the quantum moves community early and was part of testing several previous editions of the game before the beta version launch in august . in the selected time span, he had a total number of active days and a cumulative level plays. meilby drives a taxi and has no physics education beyond the standard level obtained by attending high school. his case becomes interesting because he reported being able to replicate, through several iterations, the correct sequences of actions needed to perform an operation known in physics as "quantum tunneling" based on theoretical reasoning rather than trial and error (for full description, see appendix . ). based on his formal education, he would not have the sufficient knowledge to analytically translate the physics concepts used in the game. meilby thus exemplifies the contrast between several hypotheses we have about how a hero manages to move to the top of the leaderboards: through explicit theoretical knowledge about quantum principles (as expected by the physicists in our group, who advocate focusing on explicit semantic knowledge in player skill acquisition), through implicit familiarity and complex processes of predictive coding in the core handeye coordination used to play (friston, ; scott & dienes, ) , or some combination of these higher-and lower-order cognitive processes, unique to the human mind/brain/body complex, allowing human players to explore, tinker and strategize their way through difficult and counterintuitive physics problems via more or less conscious trajectories that no computer algorithm would ever fathom. in this paper, we have tried to open up our design process for quantum moves, and reported significant findings about factors predicting play activity and performance, ranging from gender to recruitment. notably, we have devoted time to understand the most tenacious and talented kind of players, who we label heroes. quantum moves gets most of its participants online, but our beta data reveal a much more interesting pattern: people who were very interested in physics but had little formal background in the field, and who actually at some point went out the door to meet us, played much more tenaciously than those who clicked their way to www.scienceathome.org online -or were forced by extrinsic means. thus, genuinely interested amateurs reached in-world seem to be the most fertile ground for recruiting intrinsically motivated citizen cyberscientists to games like quantum moves. having attended talks with real physicists also seems to help people achieve better scores once they start playing. for citizen science, the relationship between in-world and in-game motivation is nontrivial. as for the actual human computation, the first year of quantum moves has been devoted to "calibrating our users" to solve various challenges (first scientific results will be published elsewhere shortly). we purposely abstained from helping the users beyond teaching them the basic game mechanics, even when we knew of efficient solutions to a problem. this choice provided an unbiased ensemble of solutions, which has two advantages: first it contains solutions we would never have thought of, and second, it provides a measure of what the users can realistically contribute. we use this to compare user learning curves with computer algorithms as they both found solutions for the same level. the score of a user after each try is compared with the score the computer would have after the same number of iterations. this has shown that users are fast at obtaining a good score, but they will never obtain the perfect score, whereas the computer algorithms need a lot more iterations to obtain a good score, but they are capable of finding the solution with a level of precision which yields the perfect score that we need. however, for a level like bring home water fast the users try out solution-patterns which the computer algorithms would never find by themselves, and they discover solutions which are faster than the computer algorithms' more linear computational trajectory. getting to know our player-base and especially the heroes reveals that citizen cyberscience games like quantum moves cater to a nontraditional demographic compared to both science and gaming. for instance, female players significantly outscore males after having played the tutorial, even though males play more per day. our data comparing those forced to play via schoolwork to players who came to us via in-world events or online channels also suggest that users are much more inclined to play if the action springs from curiosity or if players are intrinsically motivated to contribute to science, supporting earlier findings by raddick and colleagues ( ) as well as the central tenets of self-determination theory. what is less clear, however, is what game elements help engage and retain players once they have been recruited, and begin to move from superficial to true commitment. while quantum moves is unique compared to other citizen science games in having an engaging and challenging core game loop that by itself lives up to prominent definitions of (casual) games (juul, ; salen & zimmerman, ) , we also expect that a well-designed structural gameplay (sometimes called metagame) is central to frame, structure and motivate the play experience, both helping and goading players to move from level to level along appropriate learning curves balanced between boredom and anxiety. in the end, we accept that the high level of cognitive complexity in quantum moves compared to citizen science games that rely on players as simple pattern-hunters or "mules" carrying data gathering devices into the real world , means that we will lose players at a higher rate due to the difficulty, but we believe that it is a viable strategy to work towards a game that is fun and learnable for everyone: on one hand to capitalize on each player's lifetime network value, and on the other in the hope of helping players hone the three skills central to succeed at the scientific levels, moving them beyond the user contribution threshold. we were vague about the difference between a hero and a player who crosses the uct. this is because we view hero-status as a significant individual property combining sustained engagement and a high level of skill, while crossing the uct can happen to anyone by a fluke or one good gameplay. in this sense, we conceive of our heroes less as loincloth clad conan-types who individually topple empires and more alike the ww trench-heroes who made up the bulge with grit, skill, and intrinsic determination, and together make a difference worth reverence. however, even though many players may be truly engaged and come back for several days, we still need deeper analysis of the scientific results to tell how many actually manage to cross the uct by accident and/or by attaining hero status -that is, who actually manages to contribute to the quantum computer, as our beta population has mainly guided us in designing an effective game interface, and test simple hybrid optimization against only specific algorithms. the limits to the current study are manifold, as we have compiled this contribution as an open midstream report rather than waiting for the best possible data years into the future. centrally, the n of active players, and especially heroes, is limited, and mainly stem from a danish context. as such, new patterns are likely to emerge when we establish an even wider international profile. also, the many anovas run are almost certain to show some kind of statistically significant results, even though they may not be practically significant. mancovas would have been preferable, but since the population for each test differed slightly, this is the right kind of statistical test available at this point. further analysis and data gathering is clearly needed. in this instance, our a/b test was unable to show a statistically significant role for challenging constraints, openness to explore or the infamous. the lesson learned is, that large-scale tests should only be conducted after a satisfactory alpha-test of the gameplay in each cell, and ideally supported by using qualitative measures of player engagement and interaction-patterns to allow triangulation of causal mechanisms shaping gameplay trajectories. the games industry might not have time for these kind of detailed studies with most casual games being developed by small expert teams in less than months, but as scientists, we should. so much the wiser, we still hope to one day lead the citizen cyberscience games community in methodologically sound testing of design hypotheses about recruitment, engagement and different kinds of motivation. the relationship between curiosity on one hand, and intrinsic versus extrinsic motivation on the other, goes some way toward explaining the steep and permanent drop-off in player engagement as they make their way through the tutorial, and is especially seen after quantum moves teaching and recruitment events, where people may have been forced to register and thus driven primarily by external factors. a steep drop-off curve is in no way unusual for online games (fields, ) , so a full % tutorial playthrough suggests that the levels encountered early in the behavior chain actually keep players interested for a good while. future design perspectives include building better just-intime feedback into the core game loop, making it more 'juicy' and helping players understand exactly what is going well or wrong at any moment. after a year of stalling due to programming pool limitations, we also finally have the capacity to integrate the game with social media such as facebook, which will add an entirely new social dimension to both gameplay and recruitment. finally, we have yet to narratively structure the structural gameplay and implement truly juicy graphics. as our community manager noted, we do not need to become the next candy crush saga in terms of juiciness and engagement -just the candy crush saga of physics. we are now in a position to deploy more advanced psychological methods in mapping player trajectories and trying to predict who might be born heroes and who can become ones, which should significantly inform our future design of the structural gameplay. our centre's unique crossdisciplinary nature allows us access to eye-tracking and other tools that will soon help us test interaction design, and conflicting hypotheses about the usefulness of explicit understanding of the physics issues (as expressed my meilby) versus implicit learning of the hand-eye coordination and establishment of predictive coding (bubic, von cramon, & schubotz, ; friston, ) to account for the counterintuitive movement of the 'liquid' wave. as another exiting step we will soon be able to report the first comprehensive psychometrics battery collated especially for citizen science gaming, in collaboration with other major players. the revolutionary bit will, however, not be the our data collection, but the analysis of complex relationships between hosts of in-game and in-world variables via our next game: a title both for teaching statistics and engaging our community in analyzing data with advanced techniques like path analysis, that require intelligent human causal propositions and critical evaluations of other user's solutions -something no algorithm can ever do. human optimization is superior to the computer in many instances, but we have yet to understand the exact cognitive nature of these solutions as they apply to the varying problem spaces in quantum moves. this, along with more work on motivation, will be the subject of extensive future research. contrary to commercial casual games, we cannot change or abandon our core game loop, which represents the real quantum optimization processes needed to operate a scalable quantum computer. we can, however, change the look, feel, usability, and structural gameplay surrounding our game to frame, structure and motivate more engaging play trajectories, and ensure a sufficient learning curve to help people cross our high science contribution threshold. we have opened up that messy and experimental design process in this inaugural issue, hoping that others will be able to learn from our experiences. we encourage other researchers to publish future human computation pieces along the same lines. the present study would not have been possible without all the citizen scientists and casual players on http://www.scienceathome.org/. this is your research as much as ours. "merge merge was a pain (…) and since i did not really figured out how to get a single atom to the excited state i gave up kind of fast. i got the best score i think it was , , and i was a little mad about being so bad at the challenge because the score went to as max, and . is just not even near it. well i had some time off, and i tried again a, or some days after. with no luck. then i said to myself, since i was not doing any progress, and did not know how is should get the first atom to the excited state, i started doing the excited challenge again, and found the only way i could do it. since the background was with lines i started using them, i started raising the curve with the first atom to just above the second atom (according to the lines) and figured i was letting then merge too fast because the first atom was what i would say too excited, and not going to relax. then i had to be patient, when they were merging, and with some patience letting it flow in itself when they got near each other. the first times it did not work out, instead of the st atom splitting into it spilt into , and i knew i had it i just needed more tries. then it happened after some cursing it split into two, and i got a score points, which i was chocked about, i was like wtf, i just did it, and i am getting nothing! i think it is too hard to do, and the fact that you cant do it fast is giving me stress, the fact i have to wait all seconds every time to make it happen is a reason why it's not so funny to do. (i guess i played too much, and hate the fact i can't get it done right, pisses me off j) then i started finetune my score point, i knew already it was done right because it spilt into and the nd atom was really relaxed. it took me some time to be familiar with the finetune part, but after spending some time in there, i finally got something, by zoom and keep moving small parts and watching the score i got to points. i used the smooth tool early on, because it was flickering the line in the mittle graph, and after i had dragged the line around and around, it started to flicker (jeg mener linjen bliver zig zagget), i believe it is not good for the score, but i'm not able to remove it, i tried the locking tool to lock the line around the part and the use the smooth tool on the not locked line but it fucked it all up. i've added some pictures to show you what happened." badges in social media: a social psychological perspective a theory of human curiosity novelty, complexity, and hedonic value chaos, cognition and the disordered brain gamifying citizen science : lessons and future directions volunteers engagement profiles in volunteer thinking systems prediction, cognition and the brain the psychology of self-determination a meta-analytic review of experiments examining the effects of extrinsic rewards on intrinsic motivation self-determination theory: a macrotheory of human motivation, development, and health the effect of virtual achievements on student engagement gamification: designing for motivation a universe of consciousness characteristics of games mobile & social game design: monetization methods and mechanics mobile persuasion: perspectives on the future of behavior change predictive coding, precision and synchrony clarifying achievement goals and their impact gamification: state of the art definition and utilization beyond player types: gaming achievement goal what can breakdowns and breakthroughs tell us about learning and involvement experienced during game-play? making sense of game-play : how can we examine learning and involvement ? why people attend science festivals: interests, motivations and self-reported benefits of public engagement with research half-real: video games between real rules and fictional worlds psychological conditions of personal engagement and disengagement at work thinking, fast and slow the token economy: a decade later employee engagement : a literature review rna design rules from a massive open laboratory deep and shallow gamification: the thin evidence for effects in marketing and forgotten powers of good games engaging consumers through branded entertainment and convergent media mixed methods in games research -playing on strengths and countering weaknesses toward a theory of intrinsically motivating instruction* making learning fun -a taxonomy of intrinsic motivations aptitude, learning, and instruction gamification -a simple introduction. tips, advice and thoughts on gamification the design of everyday things volunteers' engagement in human computation astronomy projects gaming for (citizen) science: exploring motivation and data quality in the context of crowdsourced science through the design and evaluation of a social-computational. e-science workshops (esciencew) galaxy zoo: motivations of citizen scientists glued to games -how video games draw us in and hold us spellbound the motivational pull of video games: a self-determination theory approach rules of play: game design fundamentals. leonardo the conscious, the unconscious, and familiarity video game uses and gratifications as predictors of use and game preference playin computer fames: motives, responses and consequences the free and happy student creating a model to examine motivation for sustained engagement in online communities. education and information technologies essential facts about the computer and video game industry in-game , in-room , in-world : reconnecting video game play to the rest of kids ' lives nudge: improving decisions about health, wealth, and happiness ).scienceathome, home key: cord- -rvxxv dn authors: wang, mingyang; veit, michael title: hemagglutinin-esterase-fusion (hef) protein of influenza c virus date: - - journal: protein cell doi: . /s - - -x sha: doc_id: cord_uid: rvxxv dn influenza c virus, a member of the orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. humans are the main reservoir of the virus, but it also infects pigs and dogs. very recently, influenza c-like viruses were isolated from pigs and cattle that differ from classical influenza c virus and might constitute a new influenza virus genus. influenza c virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein hef that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of hemagglutinin (ha) and neuraminidase (na) of influenza a and b viruses. here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. the main focus is on the structure of the hef protein as well as on its co- and post-translational modification, such as n-glycosylation, disulfide bond formation, s-acylation and proteolytic cleavage into hef and hef subunits. finally, we describe the functions of hef: receptor binding, esterase activity and membrane fusion. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. influenza c virus was first isolated during an epidemic of respiratory illness in . since the virus showed no cross reactivity with antisera against influenza a and b viruses it was classified as a new genus of the orthomyxoviridae, named influenza c virus (francis et al., ; taylor, taylor, , . influenza c virus usually causes inflammation of the upper respiratory tract, especially in children from two to six years of age. clinical symptoms, such as cough, fever, malaise are typically mild. only occasionally the virus spreads to the lower respiratory tract and causes bronchitis, bronchiectasie and broncho-pneumonia (gouarin et al., ; kauppila et al., ; matsuzaki et al., ; matsuzaki et al., ; muraki and hongo, ) . although influenza c virus infections occur primarily in a pattern of sporadic cases or in limited outbreaks (joosting et al., ; minuse et al., ) , serological studies indicated that this virus is widely distributed around the world and that the majority of humans develope antibodies against the virus early in life (matsuzaki et al., ; salez et al., ) . in a serological study carried out in france, %- % of the population was found to be previously exposed to the virus, the highest rates of positive samples was found in the - years age group . in a -year tracking study in hospitalized children in spain, influenza c infections accounted for % of influenza-positive cases (calvo et al., ) . the results indicate intense circulation of influenza c virus in the human population. the primary host and reservoir of influenza c virus are humans, but there is evidence that this virus possesses the ability to also infect animals (muraki and hongo, ) . serological studies showed that antibodies against influenza c virus are widely present in dogs and especially in pigs (brown et al., ; horimoto et al., ; manuguerra et al., ; ohwada et al., ; yamaoka et al., ; youzbashi et al., ) . in , a number of influenza c virus strains were isolated from pigs in beijing and these strains could be transmitted from pig to pig under experimental conditions (guo et al., ) . in , an influenza c-like virus was isolated from clinically ill pigs exhibiting influenza-like symptoms (c/oklahoma/ / ) and also from cattle (d/bovine/oklahoma/ / ) which subsequently turned out to be the main reservoir of this newly discovered virus (collin et al., (collin et al., , hause et al., ) . phylogenetic analysis showed that these strains have only % overall amino acid homology to human influenza c viruses, a divergence similar to that described between influenza a and b viruses (hause et al., ) . accordingly, no cross reactivity was observed between these strains and human influenza c virus antisera. this new strain has a broader cell tropism than human influenza c virus and is capable of infecting and transmitting by direct contact in both pigs and ferrets. it also encodes a novel mechanism for generating the m protein and, importantly, is unable to reassort with human influenza c virus and generate viable progeny. based on these differences to influenza c virus it was suggested that this virus warrants classification as a new genus of influenza virus, named influenza d virus (collin et al., ; hause et al., ) . influenza c virus particles exhibit two morphologies, either spherical with a diameter of - nm or filamentous with the same diameter but with lengths in µm range (waterson et al., ) . already during the budding process at the plasma membrane, filamentous particles may aggregate via their long axes into μm long cord-like structures, which are all covered by a layer of surface projections (nishimura et al., ) . studies using reverse genetics showed that an amino acid exchange at residue of the m protein (ala to thr) that reduces membrane association of this intrinsically hydrophobic protein eliminates cord formation and also affects virus morphology (muraki et al., ; nishimura et al., ) . another unique characteristic of influenza c virus particles observed by electron microscopy is a reticular hexagonal structure, which is formed by the hef protein and discussed in more detail below (apostolov and flewett, ; flewett and apostolov, ; herrler et al., ) . the influenza c virus genome consists of negative-sense, single-stranded rna (desselberger et al., ) , but in contrast to influenza a and b virus only seven (not eight) gene segments are present in virus particles (see fig. for the structure of a virus particle and fig. for the structure of viral genome segments). the longest three segments encode the proteins pb , pb and p that form the heterotrimeric polymerase complex (yamashita et al., ) . the protein encoded by the third segment is named p (instead of pa as in the case of influenza a virus) since it does not contain negative charges at neutral ph. the fourth segment encodes the glycoprotein hef, the only spike of the viral membrane (herrler et al., a) . the fifth segment encodes the nucleoprotein np that associates with the viral genome segments along its whole length and builds, together with the polymerases the viral ribonucleoprotein complex (vrnps) (nakada et al., b) . the sixth segment encodes two proteins, the matrix protein m , a peripheral membrane protein that covers the viral envelope on its inside, and cm , a short transmembrane protein supposed to exhibit proton-channel activity required for dissociation of ribonucleoprotein (rnp) complexes from m and thus release of uncoated rnps into the cytoplasm where they are imported into the nucleus to start viral replication. m and cm are generated by alternative splicing, but in a different manner as described for influenza a virus. whereas in influenza a virus m is translated from a unspliced mrna, m of influenza c virus is generated from a spliced mrna. removal of an intron generates the stop codon uga such that a protein containing residues is translated (yamashita et al., ) . the unspliced mrna encoding cm translates into a long precursor protein ( residues), named p . p contains an internal signal peptide (residues - ) which co-translationally targets the protein from the cytosol to the er and presents it to the translocon. here residues c-terminal to the signal peptide are translocated into the lumen of the er until translocation is stopped by a second hydrophobic region (residues - ) that functions as the transmembrane region (tmr) of cm . the signal peptide is then cleaved by signal peptidase yielding the cm protein ( residues) and the p protein ( residues). p , which is identical in sequence to m (except the c-terminal amino acids), is rapidly degraded after cleavage from p suggesting that it does not plays any functional role for the viral life cycle (hongo et al., ; pekosz and lamb, ) . whether cm is a proton channel has not been directly demonstrated by biophysical assays, but it alters the intracellular ph in transfected cells and its transmembrane domain can substitute for that of the influenza a virus m protein (stewart and pekosz, ) . the seventh vrna encodes the two non-structural proteins ns and ns that are also generated via mrna splicing (nakada et al., ; nakada et al., ) . the unspliced mrna is translated into the ns protein ( residues) and the spliced mrna translates the shorter ns protein ( residues). the n-terminal residues of ns and ns are identical in sequence, splicing then generates a shift in the orf such that the remaining residues are translated from a different reading frame (alamgir et al., ) . all orfs are flanked by non-coding (nc) sequences, which are more variable in length than those of influenza a and b virus (crescenzo-chaigne et al., ) . non-coding sequences are divided into conserved and non-conserved sequences. the first twelve nucleotides at each ′ end ( ′-ucguu/cuucgucc- ′) as well as the last eleven nucleotides at each ′ end ( ′-agcaguagcaa- ′) are conserved between genome segments (desselberger et al., ; robertson, ) and are partially complementary to each other, which enables the single stranded rna to form a "panhandle" structure (cheong et al., ; desselberger et al., ) . this peculiar structure serves as the promotor for transcription of crnas and vrnas, and is required for the endonuclease activity of the viral polymerase complex figure . scheme of influenza c virus and influenza a/b virus particles. proteins having the same function are depicted with the same symbol. note that influenza c virus has only one spike protein, the hemagglutinin-esterase-fusion glycoprotein hef that combines the functions of both hemagglutinin (ha) and neuraminidase (na) from influenza a and b virus. pb , pb , p and pb , pb , pa are the polymerase proteins of influenza c virus and influenza a/b virus, respectively, that build together with the nucleoprotein np and the viral rna-segments the ribonucleoprotein complexes (vrnp). m is the matrix protein and m and cm the proton-channel. segment six and seven encode two proteins which are generated by splicing. influenza c virus possesses minus-senses, singlestrand and segmented rna. each segment possesses conserved nucleotides at ′ terminal and conserved nucleotides at ′ terminal. a poly u motif is close to ′ terminal and it transcripts into mrna poly a tail. each of the longest segments possesses only open-reading-frame (orf) and encodes pb , pb , p , hef and np, respectively. (crescenzo-chaigne et al., ; fodor et al., ; hsu et al., ) . a uridine-rich region located at position to at the ′ end of each segment is the template for the poly a tail present at the ′ end of each mrna (desselberger et al., ) . while influenza a and b virus contain the two glycoproteins hemagglutinin (ha) and neuraminidase (na) inserted into the viral membrane, influenza c virus possesses only one spike designated hemagglutinin-esterase-fusion (hef) protein which combines the functions of both ha and na (herrler et al., a; herrler and klenk, ) . like ha, it recognizes and binds to a receptor on the cell surface to initiate virus entry. however, the receptor determinant is not n-acetyl-neuraminic acid, but another derivative of neuraminic acid, namely n-acetyl- -o-acetylneuraminic acid (rogers et al., ) . hef also catalyzes fusion of the viral envelope with endocytic vesicles by a mechanism that is believed to be similar to the well characterized fusion activity of ha. finally, hef is the receptor-destroying enzyme, which is the function of the neuraminidase (na) in influenza a and b virus. hef does not cleave the terminal sialic acid residue from carbohydrates, but has an esterase activity that removes the acetyl group from position c- of n-acetyl- -oacetylneuraminic acid (herrler et al., ) . this function is probably required to release freshly budded virus particles from infected cells, which would otherwise be trapped at their plasma membrane if the receptor would still be present. interestingly, hef can substitute for both ha and na to support influenza a virus replication if its gene is equipped with the packaging signals from influenza a virus (gao et al., ) . after a description of the structure and the modifications of hef its three functional activities will be discussed in more detail below. all full-length hef protein sequences present in the influenza virus database (http://www.ncbi.nlm.nih.gov/genomes/ flu/database/nph-select.cgi) contain amino acids (aa, excluding signal peptide), except hef from one strain which is one amino acid shorter. hef (like ha) is a typical type transmembrane protein with a short n-terminal, cleavable signal peptide ( amino acids), a long ectodomain ( aa), a transmembrane region ( aa) and a very short cytoplasmic tail (three aa). hef present in infectious virus particles is composed of two subunits, the n-terminal amino acids are the hef polypeptide, the remaining sequence including the hydrophobic fusion peptide, the transmembrane domain (tmd) and the cytoplasmic tail is called hef (nakada et al., a; pfeifer and compans, ) . hef proteins of the novel influenza c-like viruses (influenza d virus) contain a very similar number of amino acids ( including signal peptide) as hef from influenza c virus, are predicted to also adopt a type i membrane topology, but the amino acid identity with hef is only ∼ % (hause et al., ) . according to a phylogenetic analysis of their hef genes the existing strains are divided into six genetic and antigenic lineages, taylor/ / , aichi/ / , sao paulo/ / , kanagawa/ / , yamagata/ / and mississippi/ (matsuzaki et al., ; muraki and hongo, ; muraki et al., ; speranskaia et al., ) . however, there is very little sequence variation (table s ) and thus influenza c virus was considered to be monosubtypic and stable in evolution, but reassortment between strains within the influenza c genus occur frequently which leads to the appearance of new strains better adapted to their host (matsuzaki et al., ; peng et al., ) . we aligned the hef sequences from the six influenza c virus lineages to reveal amino acid identity and other common characteristics (table s ). all residues important for the structure of hef, such as glycosylation sites, cysteine residues (with one exception, cys in the taylor lineage, numbering of hef excluding the signal peptide), the n-terminal region of hef containing the hydrophobic fusion peptide and the amino acids of the receptor-binding and receptor destroying domain of hef are invariant. in general (and in contrast to the highly variable ha proteins of influenza a and b virus), only a few amino acid residues are not conserved through all lineages of hef. of them are located in hef and seven in the smaller subunit hef . there are three small regions in hef where many of the variable amino acids are clustered; residues - contain four amino acid substitutions, residue - six exchanges and residues - five substitutions (table s ). in the crystal structure of hef the variable regions are located in loops at the surface of the trimer; residue - and - near the receptor binding site at the top of the molecule and residues - near the esterase domain (fig. ) . these amino acids have been shown to be antibody epitopes that gradually change due to antigenic drift (matsuzaki et al., ) . initial studies using electron microscopy showed that the hef spike forms a mushroom-shaped trimer consisting of a membrane-near stalk and a globular head (herrler et al., ; hewat et al., ) . x-ray crystallography of the bromelain-cleaved ectodomain of hef then revealed the high resolution structure ( . Å) of the hef trimer. although there is only % amino acid identity between ha and hef, the overall structure of both molecules as well as folds of individual segments are quite similar, except an additional bulge, which is located at the lower part of the globular domain and contains the esterase region that is not present in ha (fig. ) . similar to ha, the receptor-binding region is located at the top of the head domain, which consists only of hef residues. the stalk is formed by three Å long αhelices that contain the whole hef sequence and n-terminal residues - and c-terminal residues - of hef . the fusion peptide at the n-terminus of hef is located around Å above the membrane, but in contrast to ha, the first four residues are exposed at its surface and not buried within the trimer (rosenthal et al., ; zhang et al., ) . the detailed structure of the receptor binding site and the catalytic center of the esterase activity will be discussed in more detail in the last paragraph. hef is synthesized on membrane-bound ribosomes and the primary translation product is subjected to a series of co-and post-translational modifications, most of them are required for proper folding and/or functioning. already during translocation of hef into the lumen of the er the n-terminal signal peptide is cleaved, carbohydrates are attached and intramolecular disulfide linkages are formed and probably remodeled. these co-translational modifications affect folding of the molecule and its trimerization, processes which are (at least in ha and other viral glycoproteins) a prerequisite for exit of cargo from the er (doms et al., ) . later on a long chain fatty acid is attached to a cysteine located at the end of the transmembrane region and hef is proteolytically cleaved into the subunits hef and hef , a process that is essential for virus replication. timing of both modifications has not been analyzed for hef, but in ha palmitoylation occurs prior to proteolytic cleavage (veit and schmidt, ) . hef, like ha, contains only asparagine-linked carbohydrates; o-glycosylation does not occur (herrler and klenk, ; hongo et al., a) . the composition of the carbohydrate chains has not been precisely determined, but apparently, some of them are not terminally glycosylated since they are not processed to an endo-h resistant form (pekosz and lamb, ) . the location of the individual glycosylation sites in the crystal structure of hef is depicted in fig. . seven of the eight highly conserved n-glycosylation sequons (asn-x-ser/thr) are used. one is located in hef and six in hef , three in the globular head and two in the hinge region that connects the stalk with the head. the site at position is not glycosylated, probably because it is located too close to the membrane-spanning region and cannot be accessed by the oligosaccharide transferase (herrler and klenk, ; nakada et al., a; pfeifer and compans, ) (fig. and (h n )) and flc (hef from influenza c virus strain c/jhb/ / strain). hef and hef subunits of one monomer are drawn in red and green, respectively. the other two monomers are labelled in yellow and blue, respectively. evolution (skehel and wiley, ) , their distribution is quite similar to that of hef, i.e. the majority is located in the larger subunit. glycosylation of hef is crucial for proper folding of the glycoprotein by protecting it from proteolytic degradation and hence important for the presentation of antigenic epitopes (hongo et al., b) . there are cysteine residues in hef , twelve of them form six intrachain disulfide linkages that stabilize the globular head domain. their location is depicted in the crystal structure of hef in fig. . cysteine is not required for proper folding and functioning of hef since it is exchanged by a tyrosine in the taylor lineage of influenza c virus. two cysteines, cys and cys that do not form a disulfide linkage in the mature protein are located at the hinge that connects the globular head with the stalk region. the remaining cysteine in hef forms an interchain disulfide bond with the only cysteine residue in the ectodomain of hef , which is located at the bottom of the trimer. a similar distribution of disulfide bonds is present in ha of influenza a virus, i.e. one disulfide bond connects ha with ha ; the majority are intrachain bonds, three or four in ha and just one in ha (segal et al., ; skehel and wiley, ) . the rare occurrence of disulfide-bonds in hef and ha allows this subunit to perform the large conformational changes that catalyze membrane fusion. hef (at least from the strain c/jhb/ / ) is subject of a complicated folding procedure involving the formation and remodeling of intramolecular disulfide bond (szepanski et al., ) . in virus-infected cells freshly synthesized hef has an apparent molecular weight (mw) of kda as demonstrated by both reducing and non-reducing sds-page. this corresponds to the predicted molecular weight of the glycosylated form of the protein (sugawara et al., ) . subsequently hef is converted to a form with a mw of kda, which appears after non-reducing, but not after reducing sds-page, indicating that intramolecular disulfidebond formation causes the decrease in electrophoretic mobility (herrler et al., ) . besides reduction of disulfide bonds, proteolytic cleavage also converts the kda into the kda form suggesting that the kda form possesses a strained conformation (herrler et al., ; szepanski et al., ) . when hef from c/jhb/ / was expressed from cdna in the absence of the other viral proteins, conversion into the kda form was either very inefficient or not observed at all suggesting that the interaction of hef with other viral proteins is required for folding (szepanski et al., ) . expressed hef is not transported to the cell surface, which is in line with the established paradigm that proper folding is a prerequisite for exit of proteins from the er and hence transport to the plasma membrane (doms et al., ) . the defect in disulfide bond formation and surface transport was partially overcome by either deleting its short cytoplasmic tail (arg-thr-lys), replacing it by the longer cytoplasmic tail of influenza a virus ha or exchanging the two basic amino acids to acidic or hydrophobic residues (oeffner et al., ; szepanski et al., ) . in contrast, hef proteins from the strains c/california/ , c/ann arbor/ / and c/taylor/ / were efficiently transported to the plasma membrane in the absence of other viral proteins (pekosz and lamb, ; vlasak et al., ) . in addition, conversion of an kda to a kda band was not obvious by sds-page, although heterogeneity of bands after non-reducing sds-page suggests that remodeling of (table s ). threonine residue , which is exchanged by isoleucine in a virus variant that has acquired the ability to grow in mdckii cells, is also marked as sticks. (this residues is termed thr in the publication (szepanski et al., ) since the amino acid long signal peptide was included in the numbering). figure was created with pymol from pdb file flc. hef and hef subunits are drawn in red and green, respectively. disulfide bonds also occurs (pekosz and lamb, ) . the reason for this strikingly different behavior of hef proteins is unknown, but either subtle amino acid differences between hef proteins of different virus strains or between the cloned hef-gene and the gene present in virus particles of c/jhb/ / have been discussed (pekosz and lamb, ) . there are also other indications that folding of hef is more complicated than folding of ha. whereas has from several influenza a virus strains have passed the medial-golgi (determined as acquisition of endo-h resistant carbohydrates), around min after synthesis and are rapidly (t / : min) and completely transported to the cell surface (engel et al., ) , intracellular transport of hef is slow and incomplete. half times of more than min for acquisition of endo-h resistant carbohydrates and for transport to the plasma membrane have been reported and only a fraction ( %) of all synthesized molecules appear at the cell surface (pekosz and lamb, ) . in addition, hef exhibits intrinsic temperature sensitivity. expression levels of hef at the plasma membrane are two times higher at °c compared to °c and, probably as a consequence, membrane fusion is more efficient at °c than at °c. since trimerization of hef is also reduced at °c the underlying cause of reduced cell surface exposure is slower and less efficient folding of hef at higher temperatures (takashita et al., ) which is reminiscent of temperature sensitive mutants of ha of influenza a virus . the temperature sensitivity of hef is probably an adaption of the virus to replicate only in the upper respiratory tract that has, due to contact with inhaled air before it is warmed up, a lower temperature than the lower respiratory tract. in the lab (cell culture and chicken embryos) influenza c virus is also amplified at °c where it grows to higher titers than at °c (crescenzo-chaigne and van der werf, ; o'callaghan et al., ; pachler et al., ; wagaman et al., ) . however, other proteins also influence the temperature preference for virus replication since the polymerase also exhibits a higher activity at °c than at °c (nagele and meier-ewert, ). another common modification of viral glycoproteins is the covalent attachment of fatty acids, usually palmitate (c : ) in a thioester-type linkage to cysteine residues located either at the cytosol-facing end of the transmembrane region or in the cytoplasmic tail (veit, ; veit et al., ) . hef of influenza c virus is unique in this aspect, since it contains mainly stearic acid linked to cysteine (veit et al., ; veit et al., ) (fig. ) . this longer chain fatty acid (c : ) was initially identified by chromatographic determination of hef-bound, [ h]-labelled fatty acids, but results were recently confirmed by mass-spectrometry with c-terminal anchoring fragments of hef purified from virus particles (kordyukova et al., ) . these studies revealed also that influenza b virus ha possessing two cytoplasmic cysteines contains only palmitate, whereas has of influenza a virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate, but the latter is exclusively attached to the cysteine positioned in the transmembrane region (kordyukova et al., ; naeve and williams, ; naim et al., ; steinhauer et al., ; veit et al., ; veit et al., ) (fig. ) . it was originally proposed that the different length of the cytoplasmic tails of ha ( aa) and hef ( aa) could be the reason for different fatty acid selection (veit et al., ) , but a recent comprehensive mutagenesis study with ha revealed that the location of a cysteine relative to the transmembrane region is the decisive factor for selective attachment of stearate (brett et al., ) . enzymes that attach palmitate and stearate to ha or hef (or to other viral glycoproteins) have not been identified so far, but likely candidates are members of the family of dhhcproteins, polytopic membrane proteins with the glu-his-his-cys motif in one of their cytoplasmic loops, that are known to acylate cellular proteins (greaves and chamberlain, ) . acylation of ha of influenza a virus is essential for virus replication, since (depending on the virus strain) either virus mutants with more than one acylation site deleted show drastically impaired growth or could not be created at all by reverse genetics (chen et al., ; wagner et al., ; zurcher et al., ) . recombinant virus lacking the acylation site of hef could be rescued, but viral titers were reduced by one log relative to wild type flu c (our unpublished results). the resulting virus particles have a regular protein composition and no changes in their morphology were obvious by electron microscopy, but their hemolytic activity is reduced indicating a defect in membrane fusion. this is in accordance with results on several ha subtypes showing that (i) the stearoylated cysteine at the end of the transmembrane span is less important for virus replication compared to the two cytoplasmic palmitoylated cysteines and that (ii) acylation affects opening of a fusion pore (sakai et al., ; ujike et al., ; wagner et al., ) . however, for h subtype ha it was reported that acylation does not influence ha's membrane fusion activity, but plays an essential role for virus particle assembly (chen et al., ; zurcher et al., ) . several studies illuminated the essential role of palmitoylation for association of ha with rafts, cholesterol and sphingolipid-enriched nanodomains on the cellular plasma membrane that serves as the viral assembly and budding site (engel et al., ; levental et al., ; melkonian et al., ; veit and thaa, ) (fig. ) . interestingly, hef is apparently not a component of rafts, at least it does not associate with detergent-resistant membranes, their controversial biochemical correlate indicating that virus particles buds from the bulk phase of the plasma membrane (zhang et al., ) . after synthesis of the precursor hef a yet unknown protease hydrolyses the peptide bond between arg and ile , which is located in the stem region of the trimeric spike and thus in a similar position as the cleavage site in ha (klenk et al., ; lazarowitz and choppin, ; rosenthal et al., ; sugawara et al., ) linkage, which is also located in the stalk region of the protein. proteolytic cleavage is an essential prerequisite for the membrane fusion activity of hef (and also of ha) since it enables the protein to get activated by low ph (herrler et al., ; kitame et al., ; ohuchi et al., ) . hef proteins from all influenza c virus strains contain a monobasic cleavage site and are in this respect similar to has from human, porcine, equine and low pathogenic avian influenza a viruses (herrler and klenk, ; neumann and kawaoka, ) . polybasic cleavage sites that are present in ha of highly pathogenic avian influenza a viruses and processed by the ubiquitous protease furin are not found in any hef protein. consequently, replication of influenza c virus is limited to the site of virus infection, the respiratory tract. spread to other tissues or even systemic infection, as observed for highly pathogenic avian influenza virus having a multibasic cleavage site between ha and ha , does not occur with influenza c virus (horimoto and kawaoka, ; stieneke-grober et al., ) . multiple replication cycles of influenza c virus in tissue culture are enabled by addition of trypsin, whereas embryonated eggs produce infectious virus with cleaved hef (herrler et al., ; sugawara et al., ) . the enzyme catalyzing proteolytic cleavage of hef has not been identified so far, but since both ha and hef can be cleaved by trypsin at similar concentrations in vitro ( ∼ µg/ml) it seems likely that they are also activated by the same (or very similar) enzymes inside cells (herrler et al., ; nerome et al., ) . likely candidates are members of the family of type ii transmembrane serine proteases, such as hat (human airway trypsin-like protease), tmprss and tmprss (transmembrane protease serine s , members and ). these enzymes are expressed at the plasma membrane of human bronchial/ tracheal epithelial cells where they cleave ha from various, but not all subtypes (bottcher-friebertshauser et al., ) . electron microscopy revealed another unique feature of influenza c virus particles not observed for influenza a and b virions. hef trimers on the surfaces of both spherical and filamentous particles are arranged in a reticular structure that has been described to consist mainly of hexagons (compans et al., ; flewett and apostolov, ; waterson et al., ) . the regular polymeric reticular structure can be observed not only on the surface of intact viral particles, but also when hef is removed from the membrane, either by limited proteolytic digestion or by spontaneous release (herrler et al., ) . these results indicate (i) that the hexagonal arrangement is an intrinsic feature of hef and does not require other viral proteins such as m and (ii) its formation likely involves lateral interaction between the ectodomains of hef; the tmr and cytoplasmic tail are not required to maintain the structures. which amino acids form lateral interactions between hef trimers and which function it serves for virus replication has not been investigated. one might speculate that the formation of a regular arrangement of hef trimers on the plasma membrane might induce membrane curvature, i.e. it acts like an extrinsic coat that might help to sculpt a virus particle out of the membrane. however, the lateral arrangement of exclusively hexagons would result in the formation of a flat structure without any curvature. thus, in order to create and cover a spherical particle, hef must form a precisely defined arrangement of pentagons and hexagons. virus budding might be reinforced by the matrix protein m that has been shown to form virus-like particles when expressed in the absence of other viral proteins (muraki et al., ) . m might execute a pushing force by oligomerization at the inner site of the plasma membrane. how these two assumed activities of hef and m are coupled is not obvious since the cytoplasmic tail of hef is very short, only three amino acids, and might thus not be able to bind to m with high affinity. ha of influenza a and b virus and hef of influenza c virus use different neuraminic acid derivatives as receptor (fig. ) . hef binds to n-acetyl- -o-acetylneuraminic acid ( -o-ac-neu ac), which can be present on both glycolipids and glycoproteins to function as viral receptor (herrler et al., a; rogers et al., ) . likewise, hef binds to its receptor regardless of whether -o-ac-neu ac is attached via an α- , or α- , linkage to the following galactosyl residue (rogers et al., ) . in contrast, ha uses terminal n-acetylneuraminic acid (neu ac) and the glycosidic bond of neu ac influence the host specificity. avian influenza viruses usually bind to neu ac-α , -gal while mammalian influenza viruses usually bind to neu ac-α , -gal (thomas and noppenberger, ; trebbien et al., ) . the unique receptor specificity of influenza c virus has been used as an efficient tool to detect -o-ac-neu ac on the surface of various cells (martin et al., ; muchmore and varki, ; zimmer et al., ) . there is some evidence that the abundance of -o-ac-neu ac in cultured cells influences the tropism of influenza c virus. influenza c virus is usually grown in mdck i cells, whereas another subline of madin-darby canine kidney cell, mdck ii cells are (due to insufficient number of receptors) resistant against virus infection. a mutant of influenza c virus with the ability to replicate in mdck ii cells has an amino acid exchange from threonine to isoleucine at position (see fig. for the location of thr in the crystal structure of hef) that apparently increases the affinity of hef for its receptor (szepanski et al., ) . using reverse genetics it was recently confirmed that the exchange from threonine to isoleucine is necessary and sufficient to enable influenza c virus to grow in mdck ii cells (crescenzo-chaigne and van der werf, ). the crystal structure shows that hef binds to -o-ac-neu ac in a similar pattern as ha binds to neu ac. the binding elements consist of an α-helix, a loop and an extended strand (fig. a) . the key residues for binding hef to -o-ac-neu ac are shown in fig. b . tyr , thr , gly , tyr and arg form hydrogen bonds with hydroxyl-groups of the ligand, and some other residues form the structural support of the receptor binding site. the hef binding site also contains a unique hydrophobic pocket that accommodates the acetyl methyl group (rosenthal et al., ) . some coronaviruses, such as the prototype member mouse hepatitis virus (mhv) and human and bovine coronavirus, contain a hemagglutinin esterase (he) protein that also uses -o-ac-neu ac as receptor (mayr et al., ; schwegmann-wessels and herrler, ; vlasak et al., ) . the crystal structure of he from bovine coronavirus revealed that the ligand is bound in an opposite orientation compared to hef and ha (zeng et al., ) (fig. c ). in accordance with its receptor binding specificity, hef is an esterase that cleaves acetyl from the c position of terminal -o-ac-neu ac residues to release virus particles from infected cells (herrler et al., a; herrler et al., ; mayr et al., ) (fig. ) . the esterase activity of hef belongs to the class of serine hydrolase, where the −oh group of a serine residue performs a nucleophilic attack on the carbonyl-group of the substrate. since the −oh group is not sufficiently nucleophilic it is activated by two other amino acids that together build the typical catalytic triad of serine hydrolases, the amino acids serine, histidine and aspartic acid. the base histidine polarizes and deprotonates the −oh-group of serine to increase its reactivity whereas aspartic acid aligns and polarizes the histidine (charge relay system) (herrler et al., b; kraut, ; pleschka et al., ) . crystallography in the presence of two non-hydrolysable receptor analogues of hef revealed that serine , aspartic acid and histidine are the key residues for the acetylesterase activity of hef (rosenthal et al., ) (fig. d ). prior to that it has already been shown that mutation of ser and his completely abolished the enzymatic activity of hef, essentially confirming the data from crystallography, but mutation of other residues in the vicinity, i.e. asp , asn and his also affected the hydrolytic activity of hef (pleschka et al., ) . ser is positioned for nucleophilic attack on the carbonyl carbon of the -o-ac-neu ac group. the carbonyl oxygen of the substrate points into an 'oxyanion hole' formed by the . cellular receptors and receptor-destroying activity of influenza c virus and influenza a and b virus. the structure of cellular receptors for hef from influenza c virus (n-acetyl- -o-acetylneuraminic acid) and ha from influenza a and b virus (nacetylneuraminic acid) are shown. both neuraminic acid derivatives are the terminal sugars in carbohydrate chains attached to glycolipids or glycoproteins located at the cellular surface. subtypes of influenza a virus ha discriminate between an α - and α - linkage to the second galactosyl residue, a property that (partially) explains species specificity. hef of influenza c virus apparently recognizes n-acetyl- -o-acetylneuraminic acid independent of its linkage to the next sugar. hef has also esterase activity that cleaves acetyl from the c position. in influenza a and b virus the receptor-destroying activity is performed by the na protein, which hydrolyzes the glycosidic bond between sialic acid and galactosyl residues. the cleaved bonds are indicated by a red line. side chain of asn and the nh -groups of gly and ser (rosenthal et al., ) . arg of hef forms two hydrogen bonds with the sialoside carboxylate group (fig. d) . the structure of the esterase site is quite similar between hef and coronavirus he. the catalytic triad of he consists of the same amino acids, i.e. ser , his and asp ; ser also forms an oxyanion hole with the side chains of gly and the nh group of asn (zeng et al., ) . membrane fusion between the viral envelope and endocytic vesicles is the crucial step to release the viral genome into the cytoplasm of the cell (hamilton et al., ; skehel and wiley, ) . there are two essential requirements for both hef and ha to catalyze membrane fusion: (i) the precursor proteins hef and ha , must be cleaved into the subunits hef (ha ) and hef (ha ). (ii) the proteins must then be exposed to acidic ph to become fusogenic. this was initially demonstrated for influenza viruses by a simple membrane fusion assay, hemolysis of erythrocytes that occurs only if virus particles containing cleaved ha or hef are exposed to acidic ph (formanowski et al., ; huang et al., ; kitame et al., ; lenard and miller, ; maeda and ohnishi, ; ohuchi et al., ) . biochemical assays subsequently revealed that low ph initiates a conformational change since molecules become susceptible to proteolytic (rosenthal et al., ) and (c) from reference (zeng et al., ) with permission. digestion (formanowski et al., ) . the low ph is thought to cause protonation of specific amino acids that triggers the following large scale rearrangement of the proteins. histidines might play this role since their pkas match the ph of endosomes ( . - ). for ha of influenza a virus specific histidine residues have been identified (mair et al., ) , but similar studies have not been performed with hef. in both influenza a and c virus threshold ph values that initiate membrane fusion differ from strain to strain by about . ph units. this does not necessarily mean that different histidines are the relevant target of protonation, but that (between strains) variable amino acids in the vicinity of a specific histidine affect its pka. for influenza c virus ph values required to cause hemifusion (measured as lipid mixing, range of . - . ) are . - . ph units higher than ph values for full fusion (measured by hemolysis, range of . - . ) (formanowski et al., ) . interestingly, kinetic studies with influenza c virus revealed a lag phase before onset of fusion that is not observed with influenza a and b virus (formanowski et al., ) . it is likely that the lag phase reflects dispersion of the lateral arrangement of hef spikes on the viral membrane that might hinder hef's conformational change. accordingly, when virus particles are treated with low ph before electron microscopy, hef spikes are less well ordered and the typical hexagonal structure disappeared (hewat et al., ) . the molecular details of the subsequent refolding of hef have not been revealed, but it is believed that they are similar to the well characterized conformational changes of ha that were elucidated by a comparison of the crystal structure of ha at neutral ph with the structure of a ha fragment after low ph treatment (bullough et al., ) . the first conformational change removes the fusion peptide from its buried location at the bottom of the stalk and exposes it at the surface of the molecule such that it can insert into the endosomal membrane ("jackknife mechanism"). a second conformational change then bends the ectodomain thereby drawing the fusion peptide towards the transmembrane region. this leads to a close apposition of viral and endosomal membranes, hemifusion with exchange of lipids, opening of a fusion pore and eventually complete merger of both lipid bilayers. the second conformational change requires so-called "heptad repeats", amphipathic helices which interact to form a stable -helix coiled coil domain (cross et al., ; harrison, ; kemble et al., ; skehel and wiley, ) (fig. a and b ). with the software "multicoil scoring form" (http://groups.csail.mit.edu/cb/ multicoil/cgi-bin/multicoil.cgi), a highly probable heptad repeats domain was found between amino acids and , residues that encompass the long α-helix of hef . they are thus in a similar position as the heptad repeats that form the six-helix bundle coiled-coil in the low ph structure of ha (fig. c ). this region of hef thus might convert from a long, uninterrupted helix into two smaller, antiparallel helices, which are connected by a loop and form the coiled-coil domain that stabilizes the fusion conformation (bullough et al., ) (fig. a and b) . the fusion peptides of ha and hef have similar, but also different features. the first amino acids of ha (glfgaiagfieggwtgmidgwyg, sequence of h subtype) is highly conserved between subtypes and contains hydrophobic, aromatic, but also some negatively charged residues. it is also characterized by gxxg and gxxxg motifs that are known to mediate interactions between transmembrane segments. in a lipid environment the fusion peptide of ha forms a boomerang-like structure (aa - ) or a (tighter) helical hairpin (aa - ) (han et al., ; lorieau et al., ) . the sequence at the n-terminus of hef (ifgid-dliigllfvaiveagigg) is not conserved to that of ha . however, if the first six residues, which are not buried within the trimeric stalk, are not taken into account some sequence homology between ha and hef is apparent. the fusion peptide of hef has a similar amino acid composition as that of ha, but glycine residues do not form gxxg or gxxxg motifs. the structure of hef's fusion peptide in lipid micelles is not known, but in the hef trimer it already adopts a looplike structure. although influenza c virus is currently not a serious threat to humans, it might be nevertheless fruitful and revealing to study its biology. whereas the receptor-binding and receptor-destroying activities of hef are now well characterized and its fusion activity is likely to be similar to that of ha, the mechanism of virus assembly and budding is largely unexplored and might be different for influenza a and c virus. if it is confirmed by more sophisticated methods that hef does not associate with membrane rafts (zhang et al., ) , it is likely that influenza a and c virus bud at different sites of the apical plasma membrane, membrane rafts in the case of influenza a virus (gerl et al., ; rossman and lamb, ) and the bulk phase or other domains in the case of influenza c virus. since rafts are believed to enrich viral proteins and deplete many cellular proteins they represent the first concentration step in the assembly of a virus particle that contains very little cellular proteins (veit and thaa, ) . one might speculate that the regular arrangement of hef trimers might substitute for the concentration of ha in rafts, i.e. its formation might displace cellular proteins from the viral assembly site. a regular arrangement of hexagons and pentagons might then help to shape a virus particle out of the plasma membrane. for influenza a virus it has been demonstrated that virus scission is achieved by the m protein that is targeted to the edge of the assembly site and inserts an amphiphilic helix into the inner leaflet to induce membrane curvature (rossman et al., ) . whether cm plays a similar role for release of influenza c virus has not been investigated, but the bioinformatic tool heliquest (http://heliquest.ipmc.cnrs.fr/) predicts the presence of an amphiphilic helix at the beginning of the cytoplasmic tail of cm . the recent developments of reverse genetics systems for influenza c virus make some of the mentioned questions amenable to experimental verification (crescenzo-chaigne and van der werf, ; muraki et al., ; pachler et al., ) . thus, further studies might reveal common and different principles of influenza virus budding that might be helpful to combat the disease. . probable mechanism for hef-mediated membrane fusion. (a) probable conformational change of hef during membrane fusion. left part: structure of the hef subunit at neutral ph. the hr is located in the long α-helix of hef and thus in a similar position as the hr in ha. right part: hypothetical structure of the hef subunit at acidic ph. (b) hypothetical scheme for the hef-catalyzed membrane fusion mechanism. upper left part: hef binds to its receptor via its hef subunit (brown) and is endocytosed. upper right part: acidification of the endosome causes a conformational shift in hef . the fusion peptide, which was (partially) buried in the stalk is exposed and inserts into the endosomal membrane. lower, left part: the middle part of the hr domain (green) changes its conformation from a helix to a loop, which causes bending of the molecule and close apposition of viral and cellular membrane allowing the exchange of lipids (hemifusion). lower, right part: interactions between the fusion peptide and the tmd of hef might cause opening of a fusion pore. 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infection of dogs by influenza c virus: a serological survey in spain recombinant influenza c hemagglutinin-esterase as a probe for sialic acid -o-acetylation location of neutralizing epitopes on the hemagglutinin-esterase protein of influenza c virus frequent reassortment among influenza c viruses clinical features of influenza c virus infection in children a nationwide epidemic of influenza c virus infection in japan in influenza c virus and bovine coronavirus esterase reveal a similar catalytic mechanism: new insights for drug discovery role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. many raft proteins are acylated, while few are prenylated type c influenza virus. i. studies of the virus and its distribution selective inactivation of influenza c esterase: a probe for detecting -o-acetylated sialic acids the molecular virology and reverse genetics of influenza c virus evolution of the haemagglutinin-esterase gene of influenza c virus identification of an amino acid residue on influenza c virus m protein responsible for formation of the cordlike structures of the virus a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein fatty acids on the a/japan/ / influenza virus hemagglutinin have a role in membrane fusion influenza-c-virion-associated rna-dependent rna-polymerase activity effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins complete nucleotide sequence of the influenza c/california/ virus nucleoprotein gene influenza c virus rna codes for a nonstructural protein the influenza c virus ns gene: evidence for a spliced mrna and a second ns gene product (ns protein) established cell line sensitive to influenza c virus host range restriction and pathogenicity in the context of influenza pandemic characterization of the cordlike structures emerging from the surface of influenza c virusinfected cells the ability of influenza c virus to generate cord-like structures is influenced by the gene coding for m protein properties of influenza c virus grown in cell culture the cytoplasmic tail of the influenza c virus glycoprotein hef negatively affects transport to the cell surface demonstration of hemolytic and fusion activities of influenza c virus distribution of the antibody to influenza c virus in dogs and pigs in yamagata prefecture a seven plasmid-based system for the rescue of influenza c virus influenza c virus cm integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence cell surface expression of biologically active influenza c virus hef glycoprotein expressed from cdna genetic reassortment of influenza c viruses in man structure of the influenza c glycoprotein gene as determined from cloned dna the catalytic triad of the influenza c virus glycoprotein hef esterase: characterization by site-directed mutagenesis and functional analysis ' and ' terminal nucleotide sequences of the rna genome segments of influenza virus influenza c virus uses -o-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus influenza virus assembly and budding influenza virus m protein mediates escrt-independent membrane scission fatty acids on the a/ussr/ influenza virus hemagglutinin facilitate the transition from hemifusion to fusion pore formation influenza c virus high seroprevalence rates observed in different population groups sialic acids as receptor determinants for coronaviruses disulfide bond formation during the folding of influenza virus hemagglutinin receptor binding and membrane fusion in virus entry: the influenza hemagglutinin genetic diversity and evolution of the influenza c virus deacylation of the hemagglutinin of influenza a/aichi/ / has no effect on membrane fusion properties the influenza c virus cm protein can alter intracellular ph, and its transmembrane domain can substitute for that of the influenza a virus m protein and support infectious virus production influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease effects of various proteases on the glycoprotein composition and the infectivity of influenza c virus a single point mutation of the influenza c virus glycoprotein (hef) changes the viral receptor-binding activity post-translational folding of the influenza c virus glycoprotein hef: defective processing in cells expressing the cloned gene intrinsic temperature sensitivity of influenza c virus hemagglutininesterase-fusion protein studies on survival of influenza virus between epidemics and antigenic variants of the virus a further note on influenza c virus avian influenza: a review distribution of sialic acid receptors and influenza a virus of avian and swine origin in experimentally infected pigs influence of acylation sites of influenza b virus hemagglutinin on fusion pore formation and dilation palmitoylation of virus proteins timing of palmitoylation of influenza virus hemagglutinin association of influenza virus proteins with membrane rafts the hemagglutinating glycoproteins of influenza b and c viruses are acylated with different fatty acids site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins palmitoylation of influenza virus proteins the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses detection of influenza c virus by using an in situ esterase assay acylationmediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity the fine structure of influenza a, b and c viruses prevalence of antibody to influenza c virus among pigs in hyogo prefecture evidence that the matrix protein of influenza c virus is coded for by a spliced mrna comparison of the three large polymerase proteins of influenza a, b, and c viruses distribution of influenza c virus infection in dogs and pigs in bavaria structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution x-ray crystallographic determination of the structure of the influenza c virus haemagglutinin-esterase-fusion glycoprotein influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins modification of sialic acids by -o-acetylation is detected in human leucocytes using the lectin property of influenza c virus mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation the work in the authors' laboratory on influenza virus is supported by the german research foundation (sfb , tp c ). mingyang wang is a recipient of a phd fellowship from the china scholarship council.abbreviations -o-ac-neu ac, n-acetyl- -o-acetylneuraminic acid; c, cys, cysteine; cm , influenza c ion-channel matrix protein ; ct, cytoplasmic tail; er, endoplasmic reticulum; ha , hemagglutinin precursor; ha , ha , hemagglutinin subunits; hef, hemagglutininesterase-fusion protein; hef , hef , hemagglutinin-esterase-fusion protein subunits; kda, kilodalton; mdck i, madin-darby canine kidney cell type i; mdck ii, madin-darby canine kidney cell type ii; m , matrix protein ; na, neuraminidase; neu ac, n-acetylneuraminic acid; orf, open reading frame; rnp, ribonucleoprotein complex; s, ser, serine; sds-page, sodium dodecyl sulfate polyacrylamide gel electrophoresis; tmd, transmembrane domain; tpck-trypsin, tosylphenylalanine chloromethyl-ketone treated trypsin; wt, wild type. mingyang wang and michael veit declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/ licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -ij dn h authors: cho, hae-wol; chu, chaeshin title: outbreak of middle east respiratory syndrome in korea? date: - - journal: osong public health res perspect doi: . /j.phrp. . . sha: doc_id: cord_uid: ij dn h nan it was a total surprise to everybody! since late may , we have observed an imported pathogen make a huge impact on the general public and economy of the republic of korea, which has one the most advanced medical and public health systems in the world. nobody expected korea to be in second place on the list of countries with the highest incidence of middle east respiratory syndrome (mers), right after saudi arabia. the korea centers for disease control and prevention (kcdc) has been at the center of this unprecedented event. we have seen the incredible commitment of the members of the division of epidemic intelligence service in the kcdc, and of the mers outbreak investigation team. this editorial is a record of what the virus fighters have experienced in the past months. before we review the whole picture, we would like to give our sincere homage to their professional attitude and heroic sacrifices. we would also like to acknowledge the efforts of medical staff in treating infected patients and of public health officials in tracing contacts. the mers-coronavirus (cov) outbreak in korea started with a -year-old male case who visited bahrain, saudi arabia, and the united arab emirates from april to may , . the symptom onset date of this index case was may , and this patient visited two small clinics and two hospitals: pyeongtaek st. mary's hospital in pyeongtaek, samsung medical center (smc) in seoul. whether he also visited saudi arabia from may to may was not known until after laboratory confirmation results were released to smc on may . he was immediately transferred to the national medical center for isolation. since august , cases, including case exported to china (patient ), were laboratory-confirmed. of those cases, ( . %) have recovered, have died ( . %), and , including serious cases, are still being treated. eighty-two ( . %) were patients in hospitals, ( . %) were caregivers, and ( . %) were healthcare workers. the average incubation period was estimated to be . days [ ] . the clinical presentation of those laboratoryconfirmed patients has ranged from mild illness to severe disease and death. their symptoms include fever or chills ( . %); myalgia ( . %); cough ( . %); nausea, vomiting, diarrhea, or gastric discomfort ( . %); headache ( . %); sputum ( . %); and sore throat ( . %). pneumonia was initially seen in . % of the cases. overall, . % (n z ) were male, and the median age was years (range, e ). fifteen of the death cases had underlying diseases, such as chronic pulmonary diseases, cancer, and cardiovascular diseases. of the death cases, . % were aged > years old [ ] . the first epi curve ( figure ) shows the progression of the outbreak based on the laboratory-confirmed cases up to june , . the horizontal axis represents the date when a person showed symptoms after exposure. the vertical axis is the number of persons who became ill on each date. the colors represent the hospitals, index cases, and deaths with case number. the index cases in red are the four identified clusters in four hospitals. blue, yellow, and green indicate the number of patients from pyeongtaek st. mary's hospital, smc, and gun-yang hospital, respectively. gray squares indicate death cases [ , ] (see figure ). as of june , , smc-related cases have been confirmed. patient , who visited the smc emergency department on may e , played a role in exposing many people to mers-cov, however, spread related to this patient is being controlled. among the smc-related cases, have been hospitalized, and are dead. of the , close or casual contacts related to smc, are hospital staff members, and , are patients. one hundred and forty-six close contacts are quarantined at the hospital, , are under home quarantine, , casual contacts are under active monitoring, and , have been released from quarantine or monitoring [ ] . smc has enhanced the personal hygiene and education of its healthcare workers and the daily cleaning of rooms with confirmed cases. thorough cleaning was conducted following the detection of case . the patients and healthcare workers' risks of exposure were grouped by the location and duration of exposure using electronic medical records, telephone interviews, and closedcircuit television (cctv). group-specific risk was highest at zone . smc is revising the contact tracing list to identify additional contacts using the data from the health insurance review and assessment service. cctv analysis is ongoing to confirm the contacts of case within the hospital [ ] . to detect mers-cov, we are using real-time reversetranscription polymerase chain reaction (rt-pcr) following the protocol recommended by the world health organization (who). after the confirmation of the first mers case in kcdc, we expanded the screening capacity by including provincial-level laboratories and major private diagnostic centers (n z ) on may and june , respectively. all participating laboratories are using the same screening method of real-time pcr, and the laboratories in the network can process at least samples/d in case of a surge of samples. a total of , samples have been tested and cases have been confirmed as mers positive since may . we have also successfully isolated mers- after the confirmation of the mers cases on may , we implemented several measures to better control this mers outbreak. case definition for case detection and management protocol for hospitalized patients has been developed and revised for the early detection and better management of cases in alignment with the guidance for mers-cov prevention and control from the kcdc. among the confirmed cases since june , confirmed cases are from the close contact list and one from the casual contact list. forty-nine confirmed cases ( . %) were not included in the contact list. in the early stage of this outbreak (may e ), the list of the contacts of confirmed cases was not accurate. from may to june , most of the confirmed cases were from the contact list, and the proportion of confirmed cases among nonlisted contacts decreased. however, because of some missing contact information from smc, the percentage of confirmed cases among nonlisted contacts increased on june e . as a result of the strong control measures implemented on june , the percentage of missed contacts has decreased again since june [ ] . as of june , : am, , contact persons are under quarantine: close contacts ( . %) are in health facilities, , close contacts ( . %) are under home quarantine, and , people have been released from quarantine; , casual contacts are under active monitoring, and , contacts have been released from monitoring [ ] . patients that show no symptom and obtain two consecutive negative pcr results (at a -hour interval) are discharged, whereas contacts with two negative pcr results ( -hour interval) within days of monitoring are released from isolation after that period. a travel ban for close contacts was imposed on june , and one for casual contacts was added on june . however, the prohibition of casual contacts from travel was lifted on june . currently, we are monitoring close contacts that travel, at the airport. as of june , , people are under the travel ban. the list of contacts under the travel ban is provided to the ministry of justice, and a short message service (sms) is sent to each person to notify them of their travel ban status [ ] . in healthcare facilities, an enhanced infection prevention and control (ipc) strategy against mers has been implemented for case isolation, the management of hospitalized patients, and the monitoring of exposed healthcare personnel. in addition to standard precautions, including hand hygiene, appropriate personal protective equipment, including gloves, gowns, and respiratory and eye protection, is used when seeing confirmed or suspected mers cases. each patient is placed in a negative-pressure room with a high-for contact tracing, contact lists are obtained by interviewing cases, viewing cctv surveillance, and global positioning system (gps) tracking of mobile phones. close contacts with confirmed or suspected cases are quarantined at home or at health facilities and monitored actively twice a day by phone call, checking for fever or any new symptoms for days from the last-exposure date. law enforcement has been in place for noncompliant persons. casual contacts with confirmed or suspected cases are monitored actively twice a day by telephone for fever or new-symptom development for days from the last-contact date. if any suspected case is detected during the quarantine period, he/she is isolated, and respiratory specimens are immediately obtained for laboratory confirmation. editorial efficiency particulate air filter or a minimum of air changes/h. also, aerosol-generating procedures are recommended to be performed in a negative-pressure isolation room by fully protected healthcare personnel. visits to mers-cov patients are restricted, and facilities maintain a record of all visitors. healthcare workers are trained in the ipc guidelines. all healthcare workers are monitored for fever and all respiratory symptoms twice a day for days after the last known contact with a mers-cov patient to minimize exposure to and transmission of mers-cov in hospital settings [ ] . since june , strict pneumonia surveillance has been implemented in hospitals in four areas with major outbreaksdseoul, gyeonggi province, daejeon city, and chungnam province. as of june , severe pneumonia cases have been identified, among which respiratory specimens were tested for mers-cov and two tested positive. the two positive cases were already on the contact list and were isolated in a cohort isolation hospital in pyeongtaek. a cross-sectional nationwide survey of hospitalized pneumonia cases who had ever visited affected hospitals was conducted between june and june . as of june , six suspected cases among , pneumonia patients in , hospitals have been identified. among those cases, three tested negative for mers-cov, and the other three were already under appropriate management [ ] . the government has designated referral hospitals for mers-cov patient management, and hospitals are currently caring for mers patients. also, over triage hospitals for suspected cases with a temporary space for safe triage of respiratory-illness patients and isolation of suspect mers cases have been designated nationwide. guidelines for ipc and education, and resources such as personal protective equipment have been provided to the hospitals. financial and human resources support has also been provided [ ] . as the whoekorea joint mission team concluded, this outbreak was unexpected, and mers-cov was unfamiliar to all doctors and the general public in korea. although we may need more weeks to control this unexpectedly large and complex outbreak, the number of new cases seems to be declining. as the whoekorea joint mission team summarized, several factors contributed to the spread of mers-cov in korea: ( ) suboptimal ipc measures in hospitals; ( ) crowded emergency and multibed rooms; ( ) the practice of "doctor shopping," which involves visits to multiple hospitals; and ( ) the custom of having many family members or other visitors in the patient's room, which leads to the secondary spread of infection among contacts [ ] . to date, virus sequencing results show no strong evidence to suggest the change in virus transmissibility and all cases are associated with hospital-related infection. currently, there is no evidence of ongoing community transmission of mers-cov in korea. the role of environmental factors, such as inadequate ventilation in hospitals, and other factors, is being investigated. the decline of new cases indicates that the implementation of much stronger contact tracing and monitoring and quarantine measures might be working. however, we need to be vigilant to end this outbreak because sporadic cases related to smc are still occurring. we also want to invest in strengthening medical capacity and human resources to deal with new infectious diseases. finally, we would like to thank all the international experts who participated in the whoekorea joint mission and other experts with whom we collaborated in the virus sequence analysis. the views and advice of the experts from the joint mission were extremely useful to fully understand the situation and effectively control the outbreak. kcdc is willing to further collaborate with the who, the us centers for disease control and prevention and its saudi arabian and chinese counterparts, and other international partners to share its experiences related to these mers-cov cases and fill gaps in knowledge about mers-cov. the ministry of health and welfare will make full efforts for the early identification of cases, the quarantine/isolation and monitoring of all contacts and suspected cases, the full implementation of ipc measures, and risk communication to the public and national and international partners [ ] . middle east respiratory syndrome coronavirus outbreak in the republic of korea ministry of health and welfare, mers portal statement for the th meeting of international health regulation emergency committee on mers-cov outbreak from the ministry of health and welfare, the republic of korea intensified public health measures help control mers-cov outbreak in the republic of korea editorial hae-wol cho * osong public health and research perspectives hwcho@eulji.ac.kr chaeshin chu* osong public health and research perspectives key: cord- - bt x authors: crocchiolo, r.; bramanti, s.; vai, a.; sarina, b.; mineri, r.; casari, e.; tordato, f.; mauro, e.; timofeeva, i.; lugli, e.; mavilio, d.; carlo‐stella, c.; santoro, a.; castagna, l. title: infections after t‐replete haploidentical transplantation and high‐dose cyclophosphamide as graft‐versus‐host disease prophylaxis date: - - journal: transpl infect dis doi: . /tid. sha: doc_id: cord_uid: bt x background: recently, a platform of t‐cell replete haploidentical hematopoietic stem cell transplantation (haplo‐hsct) using post‐transplant cyclophosphamide (cy) has shown high reproducibility and acceptable safety profile. method: this prospective cohort analysis allowed us to collect data on infections among consecutive recipients of haplo‐hsct affected by various hematologic malignancies. results: after a median follow‐up of months, cumulative incidence of viral infections was % ( % confidence interval [ci] – ) at days and % ( % ci – ) at year; of patients at risk had cmv reactivation ( %) and the rate of polyomavirus‐virus‐associated cystitis was % ( / ). cumulative incidence of bacterial and fungal infections at year were % ( % ci – ) and % ( % ci – ), respectively. of note, only invasive fungal infection occurred beyond year after transplant (day + ). conclusion: in conclusion, despite a high rate of viral infections in the early period, present data suggest a satisfactory infectious profile after t‐cell replete haplo‐hsct using post‐transplant cy. these results may help clinicians to improve both prophylactic and therapeutic antimicrobial strategies in this emerging haploidentical setting. one of the major limitations of hematopoietic stem cell transplantation from haploidentical donor (haplo-hsct) is the impaired immune reconstitution owing to extensive immunosuppression necessary to overcome human leukocyte antigen disparity. despite important advances over the last decades, infections are still mostly responsible for toxicity and non-relapse mortality among transplanted patients, owing to prolonged immunosuppression related, or not, to chronic graft-versus-host disease (gvhd) ( , ) . a platform for t-cell replete haplo-hsct using posttransplant cyclophosphamide (cy) ( ) showed a low treatment-related mortality (trm) and a high feasibility with an acceptable safety profile. this type of haplo-hsct seems to compare favorably with t-cell depleted methods, in terms of infectious complications ( , ) . to further explore this topic, we report herein the incidence of infections in a single-center cohort of consecutive patients receiving t-cell replete haplo-hsct with post-transplant cy at our center, in order to provide useful information about the post-transplant period after this type of emerging transplant platform. data on patients with hematologic malignancies who underwent haplo-hsct between april and april at humanitas cancer center (milan, italy) were prospectively collected using electronic patients' charts. conditioning regimens were myeloablative, reducedintensity, or non-myeloablative. non-myeloablative conditioning consisted of cy . mg/kg/day intravenously (i.v.) on days À and À , fludarabine mg/ m /day i.v. on days À to À , and total body irradiation (tbi) cgy on day À . two reduced-intensity regimens were administered: (i) thiotepa mg/kg/ day i.v. on day À , fludarabine mg/m /day i.v. on days À to À , cy mg/kg/day i.v. on days À and À , and tbi cgy on day À ; or (ii) thiotepa mg/ kg/day i.v. on days À and À , fludarabine mg/m / day i.v. on days À to À , and busulfan . mg/kg/day i.v. on days À and À , as modified from sanz et al. ( ) . the myeloablative regimen consisted of thiotepa mg/ kg/day i.v. on days À and À , fludarabine mg/m / day i.v. on days À to À , and busulfan . mg/kg/day i.v. on days À to À . all patients received unmanipulated bone marrow or mobilized peripheral stem cells. gvhd prophylaxis was performed with cy mg/kg/day on days + and + ; tacrolimus mg/day i.v. from day + (to reach a concentration of - ng/ml), or cyclosporine mg/ kg/day i.v. from day + (to reach a concentration of - ng/ml), both up to day + and then tapered up to day + , unless gvhd occurred; and mycophenolate mofetil mg/kg a day orally from day + to + . infections were defined according to european society for blood and marrow transplantation (available at: http://www.ebmt.org/contents/about-ebmt/who-we-are/scientificcouncil/documents/idwpdefiniti ons.pdf), including microbiologically documented viral, bacterial, or fungal infections with or without laboratory and/or radiologic features consistent with organ involvement. cytomegalovirus (cmv) reactivation (or de novo infection) and disease were diagnosed as reported elsewhere ( ) , and invasive fungal infections (ifis) were classified according to the definitions of the eortc/msg consensus group ( ); only proven or probable ifis were recorded. data were recorded as of june , for all patients. the study was approved by the local institutional review board. antimicrobial prophylaxis was started during the conditioning regimen and consisted of acyclovir mg/ m² times in a day; levofloxacin mg/day; and cotrimoxazole tablets per day until day À , and then tablet every other day was resumed after hematologic reconstitution. antifungal prophylaxis was performed with an echinocandin (either caspofungin or micafungin mg/day) until day + , when itraconazole ( mg/day i.v.) was administered, unless contraindicated; otherwise the echinocandin was maintained. after september , the echinocandin was maintained for all patients. acyclovir, levofloxacin, and antifungal prophylaxis were administered until engraftment occurred. twice weekly blood polymerase chain reaction (pcr) cmv monitoring was started at day + until day + and weekly until day + , or when clinically indicated. weekly epstein-barr virus (ebv) monitoring by pcr was started at day + up to day + , or when clinically indicated. all other tests were performed whenever indicated. piperacillin-tazobactam alone or in combination with an aminoglycoside was administered as empirical therapy for febrile neutropenia, unless previous colonization for resistant bacteria was documented; in this case, an appropriate antibacterial agent was delivered. the same was true when a suspected bacterial infection occurred, with the exception of pneumonia for which linezolid was added, in combination with the abovecited antibacterial drug(s). first-line preemptive therapy for cmv infection/reactivation was with intravenous ganciclovir, whereas foscarnet was administered if the patient was in aplasia. ebv reactivation and polyomavirus-related hemorrhagic cystitis were treated by rituximab and cidofovir, respectively. threshold of cmv viremia for the initiation of therapy was copies/ml; threshold of ebv viremia was , copies/ml. analysis of circulating lymphocytes was performed at regular intervals whenever available, at days + , + , + , and + , and every month afterward. the following transplant infectious disease : : - monoclonal antibodies and combinations were used: cd , cd /cd , cd /cd , cd , cd /cd (beckman coulter, fullerton, california, usa), to quantify t, b, and nk cell compartments at the different time points studied. categorical variables were expressed as absolute numbers with respective percentage and continuous variables as the median with the respective range. cumulative incidence of viral, bacterial, or fungal infections was calculated using competing risk analysis ( ) starting from the day to the day of the first infection; death was considered as the competing event. infection incidence was also expressed as the events/ patient-days (pt-days) for each time period within year after transplant, with intervals defined as follows: days - , - , - , and - . owing to the paucity of late events, data beyond day + were collected singularly and classified according to pathogen group. neutrophil engraftment was defined as the first of consecutive days with a persistent count > . /l; platelet engraftment was defined as the first of consecutive days with a persistent count > /l (https://portal. ebmt.org/sites/clint /clint/documents/statguidelines_ oct .pdf). the kaplan-meier method was used to compute overall survival ( ); cumulative incidence of trm and acute and chronic gvhd were calculated using competing risk analysis ( ) . death because of documented infection was defined as infection-related death. logrank test was used to compare the incidence of infections with flow cytometry results. seventy consecutive adult patients undergoing haplo-hsct were identified. the main patient and transplant characteristics are shown in table . fifty-five patients ( %) were affected by lymphoma, and % of patients underwent haplo-hsct in partial or complete remission. only patient/donor pairs were cmv dÀ/rÀ. as concerns antifungal prophylaxis, patients did not receive itraconazole owing to moderate increase in pre-transplant liver function tests (n = ) or reduction in left ventricular ejection fraction (n = ); more patients did not receive itraconazole because haplo-hsct was performed after september . three patients received secondary antifungal prophylaxis with voriconazole because of previous pulmonary aspergillosis. median follow-up of living patients was months (range - ) from day of stem cell infusion. at last follow-up, a total of documented infectious events occurred among of patients, with a median of events/patient (range - ); % were of viral origin (n = ), % bacterial (n = ), % fungal (n = ). cumulative incidence of first viral infection was % ( % confidence interval [ci] - ) and % ( % ci - ) at day + and + , respectively; at day + , the incidence of bacterial infections was % ( % ci - ), and that of ifis was % ( % ci - ) (fig. ) . in % ( of patients at risk) at least cmv reactivation developed; of these, patients had cmv reactivation, and , , , and patients had a total of , , , and cmv reactivations, respectively. two non-fatal ( colitis, pneumonia) and fatal (pneumonia) cmv diseases occurred. no primary cmv infections occurred in the cmv dÀ/rÀ patient/donor pairs. polyomavirus-related hemorrhagic cystitis was observed in patients ( %): were caused by bk virus and by jc virus. importantly, no ebv-related lymphoproliferative disorders occurred so far. forty-five patients ( %) presented with at least documented bacterial infection: ( %), ( %), and ( %) patients had an infection by grampositive, gram-negative, or both types of bacteria, respectively. eleven ifis were detected in patients: n = probable invasive aspergillosis (pneumonia in patients and sinusitis in ), n = invasive candidiasis, all by non-albicans candida ( candidemias, colitis, and hepatosplenic candidiasis); median of occurrence was days from haplo-hsct (range - ). among the patients receiving secondary antifungal prophylaxis, only non-albicans candida colitis was observed at day + in patient. no ifis occurred under active gvhd. notably, only ifis occurred beyond day + : pulmonary aspergillosis at day + , and candidemia at day + , this latter in a patient who was under salvage treatment for post-transplant relapse. details of etiologies are reported in table . we did not observe significant differences of infectious events according to conditioning regimen administered (data not shown). when considering the timing of all episodes, bacterial infections occurred mostly between day and + , whereas viral infections/ reactivations between days + and + , with . bacterial events/ pt-days between day and + , and . viral events/ pt-days between days + and + (fig. ) . the overall incidence of viral events between day and day + was . events/ ptdays. a total of bacterial and viral infections were observed after year from transplant. engraftment rate was % ( of patients), with a median of days (range - ) and days (range - ) for neutrophil and platelet recovery, respectively. four patients died before engraftment (on days + , + , + , and + because of gram-negative sepsis, multiorgan failure, progressive disease, and bacterial pneumonia, respectively) and presented primary antibody-linked graft failure, and are alive at last follow-up, after autologous reconstitution (days + and + ). cumulative incidence of acute grade - and - gvhd was % ( % ci - ) and % ( % ci - ) respectively; chronic gvhd was % ( % ci - ). two-year overall survival was % ( % ci - ) and trm was % ( % ci - ); patients ( %) relapsed or progressed after haplo-hsct. infection-related deaths were % ( / , occurring between days + and + ; bacterial pneumonia = , gram-negative sepsis = , cmv pneumonia = , h n pneumonia = , and jc virus-related progressive multifocal leukoencephalopathy = ). the other, non-infectious, causes of trm were heart failure (n = ), secondary malignancy (n = myelodysplastic syndrome, n = esophageal cancer), multiorgan failure (n = ), thrombotic microangiopathy (n = ), and acute hepatitis (n = ). at last follow-up, patients are alive and of them are in complete remission. immunophenotypic analysis reveals a progressive increase in all lymphocyte subset counts from day + through later post-transplantation time points. we found a trend toward less viral infection incidence among those patients who have a total lymphocyte count > /mm at day + : hazard ratio . , p = . . no other associations were observed. lymphocyte subsets and number of patients analyzed are shown in figure . in the present analysis, we described infectious complications after unmanipulated, t-cell replete haplo-hsct using post-transplant cy in consecutive patients and found, aside from a high incidence of viral infections/reactivations, especially in the early posttransplant period, a quite low incidence of late bacterial infections, together with a very low incidence of ifis after day + ( events in the overall observed). present findings confirm that the infectious profile is better in t-cell replete vs. t-cell depleted haplo-transplantation ( ); the lower incidence of infections observed after day + may reflect a partial and quite effective restoration of antimicrobial immunity during the post-transplant period in this type of haplo-hsct. importantly, the low rate of chronic gvhd seen in our cohort is likely to contribute to this phenomenon, as chronic gvhd is known to be a major risk factor of late morbidity and mortality ( ) . nevertheless, we found an unexpected % trm incidence, higher than that originally reported with post-hsct cy ( ); this may be a result of the inclusion of patients with more advanced disease in the haplo-hsct program at our center. as concerns viral infections, our results are in line with previous publications in the setting of t-cell replete haploidentical transplants. ciurea et al. ( ) reported . events/ pt-days within the first months from transplant (vs. . events/ pt-days between day + and + in our hands), and raiola et al. ( ) found that % of patients presented with a viral infection in the first year. the % of cmv reactivations found here was comparable to the - % reported in similar haploidentical settings ( , , ); the slightly higher incidence that we found here may be explained by the longer follow-up in our series. the polyomavirus-associated cystitis rate of %, which is lower than that reported in the myeloablative setting ( ) , is likely because of the different conditioning regimens in our cohort, although a role played by the different gvhd prophylaxis cannot be excluded; indeed, bk virus nephropathy was found to be more frequently associated with tacrolimus than with cyclosporine in recipients of kidney allografts ( ) . here, a quarter of the patients ( / ) received cyclosporine as gvhd prophylaxis. interestingly, we confirm the lack of ebv-related lymphoproliferative disorders, as recently reported also by kanakry et al. ( ) . we found a % incidence of fungal infections, none of them fatal; it is important to note that fatal episodes of fungal infections are among the major limiting toxicities associated with t-cell depletion in haplo-hsct ( , ) ; we cannot exclude a role played by the use of anti-mold prophylaxis during marrow aplasia, although it is difficult to draw definitive conclusions owing to the lack of a true control arm in our study, and to the low number of ifis. of note, we observed only ifis months after transplant. concerning bacterial infections, we may explain the low prevalence of late bacterial infections (i.e., beyond year) by the surprising % incidence of chronic gvhd; in fact, the risk of bacterial events remained low in the absence of late immunosuppressive therapy ( ) . with a median follow-up of months, we observed late bacterial events in a total of patients having at least year of observation (last observation day is months after haplo-hsct). all this information argues in favor of the fact that giving a t-cell replete graft without deep in vivo t-depletion (i.e., with anti-thymocyte globulin or alemtuzumab) and with post-transplant cy allows a satisfactory infectious profile after transplant. the posttransplant high-dose cy permits naive and non-activated memory cells to reconstitute the immune system later on ( , ) , enabling patients to be protected from late infectious events. the same mechanism probably also explains the high viral reactivation incidence found in the first months, owing to the low number of adoptively transferred memory t cells in the early phase after transplantation (lugli e. et al., unpublished data). we acknowledge that potential selection bias may be present in the study, as we cannot exclude the possibility that some non-severe or very late infections were not captured because of incomplete reporting. however, all patients were followed at the same institution; therefore, it is unlike that clinically relevant infectious complications were missed; moreover, diagnostic procedures and prophylactic measures were similar for all patients, thus contributing to the accuracy of diagnosis of the infectious events. in conclusion, the present single-center data on consecutive patients receiving t-cell replete haplo-hsct with post-transplant cy confirm a high rate of viral infections before day + and a lower incidence of infections afterward, suggesting a satisfactory although non-optimal immune reconstitution after this type of transplantation. future comparisons with other haploidentical platforms and/or other alternative stem cell sources (i.e., cord blood), as well as investigations of novel strategies of transfer of immunity are warranted. furthermore, the present data may provide useful information in an attempt to improve control of infections by adequate prophylaxis and/or antimicrobial therapy in the early post-transplant period, after use of the emerging transplant platform of haplo-hsct. reduced mortality after allogeneic hematopoietic cell transplantation long-term survival and late deaths after allogeneic hematopoietic cell transplantation hla-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide outcomes of related donor hla-identical or hla-haploidentical allogeneic blood or marrow transplantation for peripheral t cell lymphoma cord blood transplantation from unrelated donors in adult with high-risk acute myeloid leukemia definitions of cytomegalovirus infection and disease in transplant recipients revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group estimation of failure probabilities in the presence of competing risks: new representations of old estimators nonparametric estimation from incomplete observations infectious complications in cord blood and t-cell depleted haploidentical stem cell transplantation risk factors for late infections after allogeneic hematopoietic stem cell transplantation from a matched related donor improved early outcomes using a t cell replete graft compared with t cell depleted haploidentical hematopoietic stem cell transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical transplantation using t cell replete peripheral blood stem cells and myeloablative conditioning in patients with high-risk hematologic malignancies who lack conventional donors is well tolerated and produces excellent relapse-free survival: results of a prospective phase ii trial ast infectious diseases community of practice. bk polyomavirus in solid organ transplantation absence of post-transplantation lymphoproliferative disorder after allogeneic blood or marrow transplantation using posttransplantation cyclophosphamide as graft-versus-host disease prophylaxis full haplotype-mismatched hematopoietic stem-cell transplantation: a phase ii study in patients with acute leukemia at high risk of relapse infusion of suicidegene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the tk trial): a non-randomised phase i-ii study high-dose cyclophosphamide for graft-versus-host disease prevention thymic t-cell development in allogeneic stem cell transplantation thanks: we thank all personnel working in the hematology and transplantation unit at humanitas cancer center for their remarkable contribution in patients' care and assistance to their families.author contributions: r.c. designed the study, performed data analysis, and wrote the manuscript; s.b., b.s., f.t., and e.m. provided clinical care; a.v. collected data and performed statistical analysis; r.m., e.c., and i.t. provided laboratory and microbiological data; e.l. and d.m. critically revised the manuscript; c.c-s., a.s., and l.c. provided clinical care and critically revised the manuscript.conflict of interest: all authors declare no financial conflict of interest. key: cord- -rquefe l authors: lemmens, pieter title: social autonomy and heteronomy in the age of ict: the digital pharmakon and the (dis)empowerment of the general intellect date: - - journal: found sci doi: . /s - - - sha: doc_id: cord_uid: rquefe l ‘the art of living with icts (information and communication technologies)’ today not only means finding new ways to cope, interact and create new lifestyles on the basis of the new digital (network) technologies individually, as ‘consumer-citizens’. it also means inventing new modes of living, producing and, not in the least place, struggling collectively, as workers and producers. as the so-called digital revolution unfolds in the context of a neoliberal cognitive and consumerist capitalism, its ‘innovations’ are predominantly employed to modulate and control both production processes and consumer behavior in view of the overall goal of extracting surplus value. today, the digital networks overwhelmingly destroy social autonomy, instead engendering increasing social heteronomy and proletarianization. yet it is these very networks themselves, as technical pharmaka in the sense of french ‘technophilosopher’ bernard stiegler, that can be employed as no other to struggle against this tendency. this paper briefly explores this possibility by reflecting upon current diagnoses of our ‘technological situation’ by some exemplary post-operaist marxists from a stieglerian, pharmacological perspective. this paper briefly looks at the impact of the new digital network technologies (dnts) on today's workers, consumers and citizens 'living' with these technologies within the context of today's cognitive and consumerist capitalism, diagnosing it as predominantly heteronomizing (decreasing the autonomy of subjects) and therefore disempowering, a loud choir of ideological proponents of a bright digital utopia notwithstanding. it also explores, however, how these technologies can nevertheless be recruited to increase social autonomy. more specifically, it explores how these technologies can foster the empowerment of what is called the 'general intellect' in the italian post-marxist movement of post-autonomism or post-operaism, one of the most vital movements of marxist thought in recent times. although this tradition of thinkers, whose ranks include antonio negri, michael hardt, paolo virno, maurizio lazzarato and franco berardi, is explicitly concerned, among other things, with studying the shifting relationships between technological change on the one side and the class struggle of workers against capital and vice versa on the other, what is generally absent in their analyses is an explicit account of technology and the various ways it mediates, constitutes and conditions social relationships and relations to the self. in short, it lacks an explicit philosophy of technology. in this paper, therefore, i will use bernard stiegler's pharmacological conception of technology and see how it can be used to elaborate and enrich the analyses of today's general intellect by the operaist-autonomist tradition, which always revolve around the issue of recomposition (an autonomist term referring to the unification, politicization and becoming-conscious of itself-i.e., für sich and not just an sich-of the labor force) or the empowerment of the general intellect as a process of collective political subjectivation (the process of becoming a political subject) of workers/citizens. given the exploratory nature of this paper, i will only provide some suggestions in that direction. cognitive capitalism, a term coined by the french economist yann moulier-boutang, refers to the current mode of capitalism in which not labor power but cognitive and affective capacities are the crucial determinants of productivity and the main sources of surplus value. it is a capitalism that thrives on the exploitation of cognition, attention, affection and what moulier-boutang calls attention-power and invention-power (moulier-boutang , ) . it involves the capturing of monetary gains from knowledge production and innovation by a 'collective intelligence' or a 'general intellect' described by lazzarato in terms of 'cooperation between brains' (lazzarato ) . using the metaphor of bees, moulier-boutang writes that the wealth produced under cognitive capitalism derives from the constant 'pollination' of society by the multitudes on the wings of the digital networks (moulier-boutang , ). cognitive capitalism submits cognition to the economic imperatives of profit maximization and competition, and replaces industrial capitalism, which was mainly based on the exploitation of labor power as muscular capacity. in the context of cognitive capitalism, and as a result of the deployment of dnts, the human cognitive apparatus is increasingly incorporated into the digital cognosphere. another central concept within post-autonomist discourse, derived from the famous 'fragment on machines' in karl marx' grundrisse and reinterpreted in the context of today's cognitive capitalism, is that of the general intellect. marx uses this notion of the general intellect in the grundrisse only once, to designate the objectified labor incorporated in machines or as the power of knowledge and science, incarnated in machines as dead labor or as fixed capital subsuming and controlling variable capital or living labor (i.e. workers). for marx, the notion of general intellect refers to the system of machinery as the scientifically objectified power of labor that is transformed as such into the power of capital. in the machinery of industrial capitalism, the laborers' knowledge appears as something alien and external to him and living labor appears as subsumed under selfactivating objectified labor. as marx writes: 'it belongs to the concept of capital that the increased productive force of labor is posited rather as the increase of a force outside itself, and as labor's own debilitation' (marx , ) . in post-autonomism, however, the concept of the general intellect is understood quite differently, not as external machinery opposing living labor as fixed capital but as fixed capital inside living labor, i.e., as an attribute of living labor. in cognitive capitalism, the general intellect comes to refer to the information, the communication, the languages, codes, skills and competences of the immaterial laborers. as antonio negri emphasizes, cognitive capitalism's labor force carries its means of production once again inside itself, in its brains (negri b, ) . it is fixed capital residing in the nervous systems of living labor and is referred to in the hr departments of companies and public institutions as 'human capital'. according to negri and many other post-autonomists, this general intellect as the fixed capital residing in human brains grants labor a relative yet increasing independence with respect to capital and allows it a growing margin of autonomy (ibid.). it would also represent a break with the process of proletarianization, since it involves a certain reappropriation of the means of production by labor, thereby putting an end to the dialectics of the instrument of labor first theorized by hegel (negri a, ) . as will be shown, however, this assertion is highly doubtful. post-autonomists generally applaud the digital revolution and the chances it provides for the struggle of labor against capitalist domination. moulier-boutang, and in particular hardt and negri in their empire trilogy, affirm that dnts are ultimately favorable to the expansion of the autonomy of the multitude vis à vis capital, although they enable new and highly effective forms of labor control as well (moulier-boutang, ch. , hardt and negri , - ) . both labor and capital have to operate under the network condition today, the digital networks providing the matrix for both the new figure of sovereignty known as empire and of immaterial production by the multitude, the living prey of and alternative to empire. for hardt and negri, given its globally networked condition, the multitude as the assemblage of singularities forming the new proletariat is principally capable of self-rule and autonomy, since it is able to produce its own means of production (that is to say: cooperation and communication as the substance of the common) all by itself, independent of capital (hardt and negri , f) . in fact, they suggest that the immaterial production and decision-making via dnts by the multitude provides, by itself, the model for a genuine multitudinous and absolute democracy (hardt and negri , ) . so, for hardt and negri, dnts are ultimately empowering and autonomizing, providing, in principle, the socio-technological condition for a global radical democracy as the self-rule and selfmanagement of the multitude. in stark contrast to hardt and negri, franco berardi fails to perceive the emancipatory potential of dnts. his term for the contemporary form of capitalism is semiocapitalism: a capitalism thriving on the exploitation of linguistic, symbolic and cognitive, that is to say sign-producing or semiotic labor (berardi a, ) . berardi points to the fact that cognitive or immaterial labor, far from gaining more and more autonomy with respect to capital, is becoming increasingly captured and controlled inside the digital networks, through the generalized implementation of what he calls technical and financial automatisms operating exclusively according to the logic of competition and surplus extraction and engendering more and more psychological and social automatisms (ibid., ; berardi b, ) . what he sees emerging in the context of cognitive capitalism is an increasing integration of the human mind into the digital circuits of capital, the organic nervous systems of the cognitariat more and more incorporated into and submitted to the operational logics and rhythms of the digital nervous system, shaping a genuine 'assembly line of netproduction' (berardi , ) . the cognitariat's consciousnesses are exposed to an ever more expanding and accelerating cogno-and infosphere, a situation that leads to a growing discrepancy between cyberspace, the ever growing and virtually infinite mass of semiotic and informational commodities circulating on the net, and cybertime, the finite mental time necessary for processing and elaborating this information (berardi b, - ) . this discrepancy induces all kinds of psychopathologies-like depression, anxiety, panic and attention disorders of all kinds-and leads to the exhaustion and ultimately destruction of psychic and mental energies (berardi a, ) . it erodes subjects' sensibility and affectivity due to the constant pressure to adapt one's psychic apparatus to the codes and rhythms of the network. moreover, the constant mobilization of attention as well as the fragmentation and fractalization of workers' time prevents the formation of collectivity and solidarity as a necessary basis for effective resistance and action against the domination of capital. it is due to these paralyzing and sterilizing labor conditions, berardi claims, that the political recomposition of the general intellect, and the possibilities for political subjectivation and the creation of solidarity, is systematically frustrated (berardi , ) . the necessity of always 'being connected' for participating in cognitive production supposes the adjustment of one's psychic apparatus to the codes, the logic, the speed and the rhythm of the infosphere and engenders the loss of reflexivity and the erosion of sensibility and receptiveness. a central theme in the admittedly quite gloomy analyses of berardi is that of the exhaustion of libidinal energies as a defining dimension of contemporary cognitive labor, a theme that also runs through the work of stiegler, as we will see. the minds of the cognitarians, glued to demanding screens from the morning until the evening, are more and more disconnected from their bodies as well as from the social body. berardi states that it cannot be denied that the intellectual capacity of today's general intellect is potentially boundless-and here he is largely in agreement with hardt and negri and moulier-boutang-but that it currently lacks any consciousness of itself (ibid., ). the creation of such a self-consciousness is precisely the emancipatory political task of the future. berardi's work is important in that it shows that transformations in the techno-cognitive environment change the conditions for political subjectivation and redefine the possibilities as well as the constraints for collective struggle. however, he only seems to perceive the negative side of current transformations. for him, the big political question is how to create a self-consciousness out of the general intellect so as to gain a common ground of understanding for collective action that can be effective and truly autonomizing. his suggestion is that the kind of politics necessary for such a task of commonization and autonomization should have the character of a therapy and not of political resistance and action in the traditional, say leninist sense of the term, which would only deepen our exhaustion and depression, as he argues (ibid., - ). an effective and vitalizing future emancipatory politics should concern itself instead with re-directing the social investments of desire, from the pathogenic investment in competition, accumulation and work to an investment in community, friendship and love. at some point he even proposes the embracement of a 'radical passivity' as the only way to counter the neoliberal ethos of relentless competition and productivity (ibid., ). thus, for berardi, desire and affect are crucial. important to note here is that berardi conceives of desire not anymore (as marcuse, foucault and also deleuze and guattari were inclined to do in their days) as a positive force that is inherently emancipatory but, inspired by jean baudrillard, as a field upon which conflicting forces intersect, principally mediated by technology (berardi a, ) . the problem with berardi, however, is that no effort is made of developing a strategy for confronting and dealing with the diagnosed situation in terms of that situation itself, i.e., on the terrain of dnts, the inevitable condition for political engagement in our time. berardi keeps silent when it comes to the question of how to transform the situation from within, so to speak. he does not seem to perceive any chance of changing the currently hegemonic disempowering efficacy of the dnts for the better, or at least he does not make any effort in theorizing such a possibility. this might be explained from the fact that he lacks a theory of human-technology interaction and of technological change. neither does he possess any conception of a technopolitics. for this, we have to turn our attention to the work of stiegler. for stiegler, the dnts represent nothing less than an epochal technological transmutation, that is to say a mnemotechnological (concerning the technologies of memorization and information and communication more generally) transmutation no less decisive and disruptive than the invention of writing at the dawn of western civilization and printing at the dawn of modernity. it represents a new, that is to say third phase in what the french linguist sylvain auroux calls grammatization, as the process of formalization and externalization of human language, the two earlier phases being that of alphabetization and writing (stiegler a, ) . as such, dnts usher in a wholly new episteme in the sense of michel foucault and allow for totally new epistemologies in the sense of gaston bachelard (stiegler b, ) . they will totally reconfigure the human mindset, like writing did in ancient greece at the time of plato, as classicists like ong, goody and havelock have shown (ong ; goody ; havelock ) , or like printing did from the early fifteenth century onwards, as elisabeth eisenstein has pointed out (eisenstein ) . they are also on the way of reconfiguring our neuronal structures, changing our brain structure, as neurologist and psycholinguist maryanne wolf has argued, from 'reading brains' to 'digital brains', just like writing turned the 'oral brains' of our pre-literate ancestors into 'reading brains' (wolf ) . more directly, they also furnish a new 'political organology' (see below) that has yet to be 'appropriated' politically. stiegler has developed an organological theory of human, technology and society interaction, which i will sketch very briefly here. this general organology conceives of human evolution and history as technogenic processes involving three organ systems: ( ) psychosomatic organs, ( ) social organizations and ( ) technical organs (stiegler a, b, c, - ) . changes in the technical organs always induce de-functionalizations and subsequent re-functionalizations in the psychosomatic and social organs, which-in the course of evolution and later during history-are involved in a constant process of adoption of (or adaptation to) new technologies. the relations between these three organ social autonomy and heteronomy in the age of ict: the… systems are of a transductive nature, meaning that they only take shape within and from out of their relation to each other. what is most important in the current context is that the relations between these three organ systems constitute circuits of desire, of libidinal energy, given that the technical organs and the social organizations give shape to the drives residing in the psychosomatic organs (stiegler b, ) . now, the technical organs, stiegler argues, can both intensify the binding of drives into libidinal energy, supporting processes of sublimation and psychic elevation, and cause their decomposition, which leads to desublimation and psychic regression. as such, every organology constitutes a libidinal economy. a political organology would consider the conditioning effects of the technical organs, or in other words the technical milieu, on the formation of political affects, most importantly on what aristotle in his political writings called philia, or collective desire, as the conditio sine qua non of all political life. now, for stiegler, the fact that technical organs, i.e., technologies of all sorts but most explicitly so-called mnemo-or psychotechnologies, can both positively and negatively condition libidinal economies, is related to the fact that they are what he calls pharmaka, a greek word that means both poison and medicine. as compensations for the 'original lack' [défaut originaire] of intrinsic attributes characteristic of human beings, technical pharmaka can both aid and impede, both support and undermine aspects of those beings. they are both toxifying and curative. that is to say: given the fact that they are constitutive of the human being's thrown and projective being-in-the-world, they have an autonomizing as well as a heteronomizing potential (stiegler a, - ) . this pharmacological conception of technologies has affinities with andrew feenberg's idea of technical ambiguity (feenberg , ) but is more firmly rooted in an 'onto-anthropological' understanding of what the human life form as a technical life form is. as instruments for political subjectivation, technical pharmaka like dnts can both support politicization and depoliticization, both emancipation and docilization. they can both elevate and 'dumb down' collective intelligence by supporting either short circuits ('egoistic' circuits of drive) or long circuits of individuation (producing desire and sociality). what dnts 'are'-and here stiegler is in agreement with feenberg-depends to a large extent on how they are adopted by individuals and collectives. it may be argued that berardi's diagnosis exclusively emphasizes the proliferation of short circuits that exhaust libidinal energy and induce drive-based behavior (i.e., capturing libidinal energy into short loops of immediate gratification that tendentially induce frustration in subjects), thereby acknowledging only the toxic tendency and disregarding or at least neglecting any thought of a therapy-other than that of creating zones of human resistance outside of the 'system'-that would precisely be based on an emancipatory and autonomizing adoption or appropriation of dnts that would counter its toxic, heteronomizing tendencies. stiegler stresses that autonomy exists only in an intimate relation with technical heteronomy, on the condition, that is, that it is adopted intelligently, reflexively and with care, which assumes a practice, that is to say a therapy, a sociotherapy, or in short: a politics (stiegler c, , ) . dnts, as stiegler argues again and again in all of his writings, can truly function as a technical milieu creating long circuits and supporting a new social autonomy. this presupposes, however, a collective appropriation of that milieu, so that it becomes an associated milieu instead of a dissociated milieu, the latter being the dominant situation today. in fact, dnts are ideally suited for this, certainly so if compared to the older analog information and communication technologies like radio and television. to give some idea of that: dnt's are characterized by multilinearity, which allows for demassification and desynchronized access (think of podcasts for instance), by annotability, which allows for tagging and the bottom-up creation of metadata, by hypermediality, which means that they can handle all levels of grammatization from texts to genomic sequences, and most importantly by bidirectionality, which allows receivers to be senders and vice versa (stiegler , ) . stiegler's assessment of the current impact of dnts on the general intellect, however, is closer to the view of berardi than to those of hardt and negri, for instance. he is much less pessimistic however. in fact, the dnts are for stiegler nothing less than the way out of the state of deep intoxication that our current mnemotechnical milieu suffers from as a result of its annexation by consumerist and cultural capitalism. it sometimes even looks as if dnts represent, for him, a kind of heideggerian saving power, if only we were able to succeed in carrying through what he refers to sometimes as a 'pharmacological turn' of this pharmakon. concurring with the analysis of berardi and fundamentally disagreeing with the views of hardt and negri, however, stiegler contends that instead of tendentially autonomizing and empowering the multitude because of the increasing independence of the general intellect from capitalist control, in reality the general intellect appears to be the object of an intense process of proletarianization today that seems to disempower it and rob it of its autonomy, producing a regression of the collective intelligence of the general intellect that he refers to as a state of 'systemic stupidity' (stiegler , ) . instead of fixed capital migrating to living labor (the immaterial means of production increasingly residing in the brains of cognitive laborers), thereby granting it more autonomy as hardt and negri claim, stiegler states that through generalized automation as exteriorization and short-circuiting of psychic and cognitive functions in the dnt, we cannot but admit that the multitude's collective intelligence is becoming increasingly proletarianized (ibid., ). instead of a growing commonization and autonomization of the multitude, what seems to be the rule nowadays is cognitive proletarianization and psychic heteronomization of the singularities that make up the multitude. so-called cognitive capitalism in fact destroys cognition, forcing workers to adapt their cognitive apparatus and capacities to a digital technical milieu that ruthlessly imposes the imperatives of profit and competition. it is true that dnts enable the 'cooperation between brains' (lazzarato) but so far they have predominantly served capital in controlling this cooperation and capturing its fruits (ibid., ). however, this is a pharmacological situation and as such it is anything but hopeless. while still functioning largely as technologies of capture and control (as analyzed by berardi) , at the same time the dnts are gradually beginning to be transformed into the conditions for the emergence of a wholly new associated milieu that can function as the support of a therapeutic countermovement able to fight the proletarianizing tendency. phenomena like free software, peer-to-peer, wikipedia, wikileaks, anonymous, and many other commons-based collaborative projects can be understood as being the first movements in that direction, movements that are characterized by stiegler in terms of deproletarianization. the hackers and their anti-protestant-although not necessarily anticapitalist-ethics of voluntary cooperation, sharing, passion and care with respect to digital work (as expounded by pekka himanen in his well-known bestseller the hacker ethic) are the exemplary heroes of this countermovement (himanen ) . social autonomy and heteronomy in the age of ict: the… what is lacking in stiegler, however, is a class-based analysis of this pharmacological situation in terms of an account of the possibilities for recomposition of the general intellect. such an account should be developed, i claim, because it seems unrealistic if not totally illusory to put too much trust, as stiegler does in his writings, in the initiative of governments or representatives of today's public institutions, to promote and support the emergence of alternative modes of production and collaboration beyond the consumerist model, that can break with the current hegemony (stiegler ) . in an all-out class war of capital and the state against the laboring multitudes, which is currently waged with an unprecedented brutality and cynicism, calling for a public power to positively intervene in the situation and prescribing a sociotherapy in terms of a positive pharmacology seems more than a bit naive. such a therapeutic project should and can only emerge from 'below', i.e., from the multitude itself, or more realistically from its dissident and least proletarianized factions. unfortunately, there is no space here to embark on a compositionalist pharmacological analysis of today's dnts. for that reason i will necessarily have to limit myself here to proposing a few questions that could guide such an analysis. it is clear that dnts by themselves, left within the confines of the market economy, will never bring about true social autonomy, but only enforce the toxic, desublimatory, heteronomizing tendency that berardi and also stiegler rightly diagnose. what is needed is a political project of adoption in terms of a therapy and this 'therapy' must intervene at a systemic level and aim for a new organization of society, no less! a society that would be truly intelligent and autonomous in the sense that it, as the late andré gorz put it nicely in one of his books, paraphrasing a famous dictum by karl marx, should create the conditions 'in which the full development of each person's ability is everyone's aim' (gorz , ) and in which production would serve human development instead of human development serving the production of surplus value (ibid., ). in light of the critical diagnoses offered by berardi and stiegler, the following questions could lead a pharmacological analysis of class composition in the current situation. first of all: how can workers and 'deconsumerizing' citizens employ the digital pharmakon itself against the hegemonic tendencies it now supports? how can dnts be appropriated or maybe newly designed so that they can be used to struggle against the constant acceleration of online life-and as a consequence also of offline life-that they now everywhere support? how can they be repossessed for the purpose of 'recapacitation' (amartya sen) of the cognitariat as the subject of the general intellect? how can the digital pharmaka be changed so as to slow down life and shape the conditions and the time for reflection, critique and a more free and autonomous disposition of our individual and collective time? how can the digital pharmaka be changed so as to foster commonality and solidarity instead of atomizing and individualizing us (think also of social networks like facebook and myspace here)? how can we change the underlying algorithms-the technical codes so to speak (not necessarily but possibly in the sense of feenberg)-that constitute the technofinancial automatisms servant to capital that berardi writes about? how can the digital pharmaka be appropriated or redesigned so as to become the supportive technologies of what stiegler has called an 'otium of the people', i.e., as technologies of the self and others, of care of the self and care of the other in the sense of michel foucault, instead of promoting competition and increasing automation, heteronomization and carelessness? how can these pharmaka be creatively re-appropriated to struggle against the disaffection, disaffectation and libidinal exhaustion and how can they become the instruments of new modes of critical and 'deep' attention? how can they become inducive to/ engaged in processes of commonization and the formation of 'collective desire for the collective', instead of increasing individualization? how can they be remade to reconnect subjectivities with the social fabric they still remain part of yet hardly experience anymore? all these questions suppose that we should not only learn the 'art of living with icts (information and communication technologies)', but that we should also learn to fight, produce, commonize and live in common, i.e., collectively manage our collective existence, with icts. 'the art of living with icts' should in any case not be understood in an adaptationist sense, as the necessity of having to cope with something inevitable, of having to get along with it, of having to adapt ourselves to it. instead we should try to adapt those technologies to what we, as socio-technical creatures, collectively decide to be a life worth living-i.e., in the sense of adopting and creatively appropriating them as weapons (in the sense of gilles deleuze) for (re-)conquering our collective autonomy in the face of an increasingly totalitarian capitalism bent on controlling subjects more and more through what lazzarato has most recently described as 'machinic enslavement' (lazzarato , - ) . that is the most urgent question, i would like to suggest, with respect to the question of the 'art of living with icts'. open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the soul at work precarious rhapsody. semiocapitalism and the pathologies of the post-alpha generation after the future the printing revolution in early modern europe the immaterial. knowledge, value and capital. calcutta: seagull books multitude. war and democracy in the age of empire the muse learns to write: reflections on orality and literacy from antiquity to the present the hacker ethic and the spirit of the information age puissance de l'innovation. la psychologie économique de gabriel tardre contre l'économie politique signs and machines. capitalism and the production of subjectivity grundrisse. london: penguin cognitive capitalism. cambridge and malden the porcelain workshop. for a new grammar of politics. los angeles: semiotext(e) orality and literacy. the technologizing of the word la télécratie contre le démocratie social autonomy and heteronomy in the age of ict: the… for a new critique of political economy pharmacologie du front national. suivi du vocabulaire d'ars industrialis par victor petit de la misère symbolique what makes life worth living. on pharmacology the hyperindustrial epoch per toeval filosoferen proust and the squid. the story and science of the reading brain he currently teaches philosophy and ethics at the department of philosophy and science studies, part of the institute for science, innovation and society (isis) at the radboud university. he has published on themes in the philosophy of technology and innovation key: cord- -itiz mqv authors: christoffersen, s. title: the importance of microbiological testing for establishing cause of death in forensic autopsies date: - - journal: forensic science international doi: . /j.forsciint. . . sha: doc_id: cord_uid: itiz mqv abstract microorganisms have always been one of the great challenges of humankind, being responsible for both high morbidity and mortality throughout history. in a forensic setting microbiological information will always be difficult to interpret due to lack of antemortem information and changes in flora postmortem. with this study we aim to review the use of microbiological procedures at our forensic institute. in a retrospective study including autopsies performed at our institute, where microbiological test had been applied, analyses were made with regard to: type of microbiological tests performed, microorganisms found, histological findings, antemortem information, c-reactive protein measurement and cause of death. fiftyone different microorganisms were found distributed among cases, bacteria being the most abundant. nineteen of the cases were classified as having a microbiological related cause of death. c-reactive protein levels were raised in cases of the cases, histological findings either supported or were a decisive factor for the classification of microbiologically related cause of death in cases. as a multitude of abundant microorganisms are able to cause infection under the right circumstances, all findings should be compared to anamnestic antemortem information, before conclusions are drawn. a definite list of true pathogens is nearly impossible to compile. microorganisms have always been one of the great challenges of humankind, being responsible for both high morbidity and mortality throughout history. in a forensic setting microbiological information will always be difficult to interpret due to lack of antemortem information and changes in flora postmortem. with this study we aim to review the use of microbiological procedures at our forensic institute. in a retrospective study including autopsies performed at our institute, where microbiological test had been applied, analyses were made with regard to: type of microbiological tests performed, microorganisms found, histological findings, antemortem information, c-reactive protein measurement and cause of death. fiftyone different microorganisms were found distributed among cases, bacteria being the most abundant. nineteen of the cases were classified as having a microbiological related cause of death. creactive protein levels were raised in cases of the cases, histological findings either supported or were a decisive factor for the classification of microbiologically related cause of death in cases. as a multitude of abundant microorganisms are able to cause infection under the right circumstances, all findings should be compared to anamnestic antemortem information, before conclusions are drawn. a definite list of true pathogens is nearly impossible to compile. ß elsevier ireland ltd. all rights reserved. and/or swaps are collected once suspicion is raised by gross pathology findings during the autopsy. tissue samples were analyzed at the department of clinical microbiology, university hospital of odense. samples are routinely analyzed for bacteria and fungi, using agar culturing. pcr analysis for viruses is performed per request. crp was measured in full blood using ''quickread crp'' produced by orion diagnostica. the ''quickread crp'' supplies results in the range of - mg/l, results below or above this interval will be noted as < and > respectively. though an exact cut off value for crp on postmortem material is not easy established [ ] , values above mg/l are considered raised. the age ranged from ½ months to years, cases were children under the age of , one was between and years old, were between and , and were older than years. twenty-eight were male and were female. in five cases, no microorganisms were found. bacteria were found in ( . %) of the cases, amounting to of ( . %) samples taken. viruses were found in of cases ( . %). fungi were found in eight cases ( . %), amounting to of ( . %) samples taken. no other named fungi than candida albicans was found. in one case c. albicans was the only microbiological finding. bacteria were found in cases where they were not related to cod. virus was only found in one case which was not classified as an infectious cod. in the autopsies cod was classified as infectious in cases ( . %), distributed among: three cases of viral pneumonia ( . %), three cases of bacterial pneumonia ( . %), four cases of sepsis ( . %), two cases of meningitis ( . %), two cases of peritonitis, one due to accidental perforation of the intestine during surgery and one due to perforated appendicitis ( . %), one case of pericarditis ( . %) and three cases of combined opiate poisoning and infection ( . %), table . the cod of the remaining cases, which were not related to microbiological findings, are listed in table . in all, different microorganisms were found including one report of ''normal flora'', table shows the distribution. testing in one case, classified as viral pneumonia cod, did not yield any positive viral findings. however the histological findings were consistent with viral pneumonia (interstitial pneumonitis), and the person had been treated with antiviral antibiotics before death which could cause the negative viral findings [ ] . crp was measured in cases. in nine cases crp was below mg/l. regarding the cases classified as infectious cod, crp values were below mg/l in two cases, cases had elevated crp values (p < . ), and in three cases no measurement was performed. of the cases with an infectious cod, histological examination was of importance in cases ( . %). in two of the cases no microorganisms were found but the histological changes, crp values and other information lead to the conclusion (one case of viral pneumonia and one case of neutrophilic pneumonia in an opiate poisoning case). in four cases, histological findings did not show inflammation to support the microbiological findings, however crp levels above mg/l and the bacteriological results were sufficient to verify the microbiologically related cod. culturing bacteria on agar plates is an old invention whereas polymerase chain reaction (pcr) used for viral detection is a fairly new technique and is still costly compared to agar cultures. hence bacteria and their colonization of the body is the most thoroughly investigated group of pathogens. a single study has used pcr for bacterial detection with excellent results [ ] . though this might be the way to proceed in the future, the skill and equipment required for pcr, compared to using agar plates, could mean that it will not be the investigation of choice in the near future on a worldwide basis. the key point in interpreting postmortem microbiological results is to determine whether detected microorganisms arise from postmortem spread or are the result of true antemortem infection. larsen et al. suggest that to be sure a bacteria was present in the bloodstream before death, it must be cultured from at least two different sites [ ] . however authors do not agree on the most reliable sites for sampling, blood, lung and spleen are generally mentioned, as well as cerebrospinal fluid [ , ] . tuomisto et al. [ ] suggest liver and pericardial sampling as the most reliable within the first five days after death. preferably, cultures should be verified by gross pathologic or histologic findings [ , ] . when bacteria are cultured postmortem, one must consider several mechanisms which could lead to bacteria in the samples. four lines of spread have been suggested [ , ] . a microorganism has genuinely invaded the bloodstream or target organ during life leading to bacteriaemia/ infection/sepsis, the group of cases which could be classified with a microbiological cod. . agonal spread: according to this theory the ischemia in the agonal period or during resuscitation lead to mucosal damage allowing bacteria to invade through the surface in question. . postmortem translocation: the movement of naturally present bacteria across the mucosal barriers, into the surrounding tissue, bloodstream and body. one should expect a mixed growth and not a single isolate bacteria [ ] . . contamination: microorganisms are introduced into the tissue at time of sampling, and were not present in life. the theory of agonal spread is according to a review by morris et al., considered unlikely [ ] . several papers find that elapsed time from death to sampling point does not have a significant influence on false positive bacterial results provided that bodies are kept refrigerated [ , [ ] [ ] [ ] . however animal experiments performed by heimesat et al. [ ] do show a rather large increase in bacteria in extra-intestinal tissue especially up to h after death. perhaps even more interesting, they observe a rise in extraintestinal bacteria already five minutes after death. maujean et al. report an interesting case in which neisseria meningitidis was detected in a putrefied body after days using molecular analyses [ ] . pryce et al. compared antemortem vs. postmortem sampling and concluded that postmortem sampling is as valuable as antemortem, when related to an infectious cod [ ] . the risk of contamination from the autopsy room seems to be small [ ] . table shows the vast diversity of microorganisms found in our cases, and illustrates the large number of possible pathogens. the majority of bacteria could be considered postmortem translocation, however as many of the microorganisms contributing to our normal flora can become pathogens under the right circumstances, all findings must be carefully considered. in our study bacteria were found in cases, of these cases were not classified as having infectious cod, adding to the presumption of a significant postmortem spread. the vast majority of the microorganisms found in these cases are part of our natural flora, and are to be considered opportunistic pathogens, several being able to cause both pneumonia and bacteremia. in six of these cases no clear cod was found, four were sids and two unexplained under the age of two (table ). histology did not show inflammation in any of the samples, and none of the cases had elevated crp values, though in two cases crp measurement was not performed. not surprisingly, these problematic cases are all infants. as several of the bacteria are possible pathogens, infection cannot be completely ruled out due to the fact that a not fully developed immune system could be responsible for the lack of both inflammatory response and rise in crp values. several more or less commonly encountered microorganisms, such as n. meningitidis, neisseria gonorrhoeae, haemophilus influenzae, salmonella species, staphylococcus aureus, streptococcus pneumoniae, b-hemolytic streptococci, klebsiella species, escherichia coli, mycobacterium tuberculosis, members of the enterobacteriaceae and c. albicans should, according to some authors, always be considered pathogens [ , , ] . several of the above are however part of our natural flora, and may be considered opportunistic pathogens [ ] , only causing disease in persons with an underlying illness, immune deficiency, or are perhaps only known to cause disease in children etc. [ ] . generally all cases should be correlated to anamnestic information, if any present, both medical and socio-economic [ ] before a microorganism is classified as a true infection. hence a definite list of true pathogens is impossible to comprise. considering that microbiological testing amounts to only . % of the autopsies in the investigative period versus the yield of cases which were classified as microbiologically related cod ( . %), this inexpensive testing seems to be rather well founded. perhaps microbiological testing should be applied in even more cases. in denmark around % of autopsies do not yield a viable cause of death, and though microbiological testing would be performed on most, the possibility remains that a few of these deaths could be due to infections/sepsis. in this study of cases with a microbiologically related cod had elevated crp values (p < . ). considering that result and the literature on the subject [ , ] , one would suggest that crp measurement is carried out at the beginning of the autopsy. this would give an early indication of a possible infection in cases where antemortem information does not suggest such, allowing for microbiological sampling before significant handling of the body and a hence reduced risk of mechanical contamination, both by direct transfer of bacteria and by displacement of bowel-near blood in arteries and veins. besides the possibility of improved sampling methods we also need to consider other methods to improve the microbiological testing in relation to autopsy. pcr analyses have been proved useful [ ] , and although it seems to be excellent with regards to virus investigations, it does not bypass the biggest pitfall in bacterial analysis, the postmortem spread. since it is even more sensitive than culturing [ ] it might possibly yield an even higher number of positive results. pcr analysis targeting specific bacterial toxins or the toxin-genes might be of some use [ ] , investigations into pm bacterial toxin production could be an interesting area for future work. microbiological sampling remains an important part of the autopsy yielding the cause of death in . % of the cases in which it was performed. optimized storage of the body and precautions to avoid contamination greatly enhance the value of the results. the decision to classify a cod as microbiological rests on a combination of factors, including crp levels, histology and an analysis of the microbiological agents. a raised crp level is correlated with microbiologically related cod and if performed at the beginning of the autopsy could be used as a guideline to initiate a microbiological autopsy, before mechanical manipulation leads to contamination. histology is an important part of the microbiological investigation in almost / of the cases with a microbiologically related cod, verifying inflammation in the cultured material, and in a few cases being the decisive factor, though supported by other information, in the diagnosis where the microbiological results were negative. therefor all sites sampled for microbiological testing should also be histologically examined. in very few of our cases only one single microbiological agent was found or an agent which is without doubt the sole cause of death. several of the microorganisms contributing to our natural flora, colonizing human skin, respiratory tract or other inner surfaces or which are found in abundance in our surroundings can lead to life-threatening infections under the right circumstances, i.e. persons with underlying illness, immune suppression etc. conversely widely found microorganisms known to cause sepsis could be the result of postmortem spread, considering their established virulence, target age group and point of infection. investigators should always take into account all findings and anamnestic information in each case compared to the detected microorganisms before making any conclusions. hence a definite list of ''true pathogens'' is nearly impossible to compile, and we suggest consulting microbiological literature on each microorganism, once it has been deemed of interest due to the above factors. accuracy of cause of death determination without forensic autopsy examination postmortem diagnosis of sepsis the importance of the autopsy in emerging and reemerging infectious diseases determinants for autopsy after unexplained deaths possibly resulting from infectious causes, united states a study of pneumococci and allied organisms in human mouths and lungs after death the routine use of c-reactive protein in forensic investigations particularities regarding the etiology of sepsis in forensic services evaluation of postmortem bacterial migration using culturing and real-time quantitative pcr ii: comparison of a group of cancer patients with a control group procurement, interpretation, and value of postmortem cultures bacteriological investigationsignificance of time lapse after death postmortem bacteriology: a re-evaluation practical and theoretical aspects of postmortem bacteriology sterile site infection at autopsy in sudden unexpected deaths in infancy postmortem interval and bacteriological culture yield in sudden unexpected death in infancy (sudi) comprehensive postmortem analyses of intestinal microbiota changes and bacterial translocation in human flora associated mice the interest of postmortem bacteriology in putrefied bodies microbiological findings in sudden unexpected death in infancy: comparison of immediate postmortem sampling in casualty departments and at autopsy bacteriological sampling of postmortem rooms principles and practice of infectious diseases sudden death caused by fulminant bacterial infection: background and pathogenesis of japanese adult cases postmortem bacteriology in forensic pathology: diagnostic value and interpretation autopsy cases of fulminant bacterial infection in adults: clinical onset depends on the virulence of bacteria and patient immune status cerebrospinal fluid pcr analysis and biochemistry in bodies with severe decomposition staphylococcal toxins in sudden unexpected death in infancy: experience from a single specialist centre key: cord- -lrz bdx authors: nayyar, gaurvika m. l.; attaran, amir; clark, john p.; culzoni, m. julia; fernandez, facundo m.; herrington, james e.; kendall, megan; newton, paul n.; breman, joel g. title: responding to the pandemic of falsified medicines date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: lrz bdx over the past decade, the number of countries reporting falsified (fake, spurious/falsely labeled/counterfeit) medicines and the types and quantities of fraudulent drugs being distributed have increased greatly. the obstacles in combating falsified pharmaceuticals include ) lack of consensus on definitions, ) paucity of reliable and scalable technology to detect fakes before they reach patients, ) poor global and national leadership and accountability systems for combating this scourge, and ) deficient manufacturing and regulatory challenges, especially in china and india where fake products often originate. the major needs to improve the quality of the world's medicines fall into three main areas: ) research to develop and compare accurate and affordable tools to identify high-quality drugs at all levels of distribution; ) an international convention and national legislation to facilitate production and utilization of high-quality drugs and protect all countries from the criminal and the negligent who make, distribute, and sell life-threatening products; and ) a highly qualified, well-supported international science and public health organization that will establish standards, drug-quality surveillance, and training programs like the u.s. food and drug administration. such leadership would give authoritative guidance for countries in cooperation with national medical regulatory agencies, pharmaceutical companies, and international agencies, all of which have an urgent interest and investment in ensuring that patients throughout the world have access to good quality medicines. the organization would also advocate strongly for including targets for achieving good quality medicines in the united nations millennium development goals and sustainable development goals. malaria is a devastating illness, particularly to young children and pregnant women in tropical countries. a recent review reported that the active pharmaceutical ingredient (api) was absent in over one-third of close to , antimalarial drug samples tested from pharmacies in seven southeast asian and sub-saharan african countries ; over % of the alleged artemisinin-containing drugs were falsified, outright fakes. a wide variety of falsified brand name and generic medicines and even falsified raw ingredients for several essential pharmaceuticals have been found in rich and poor countries. [ ] [ ] [ ] [ ] [ ] [ ] such drugs are often used for acutely ill patients, many of whom would die or suffer prolonged illness without proper treatment. in addition to patients' loss of confidence in the health-care delivery system, microbial resistance to the drug may develop and spread if medicines contain subtherapeutic doses or no api. the increasing global scientific and public awareness and epidemic proportions of the spreading problem are reflected in the number of articles on "fake drugs" cited in pubmed: until recently, there were a paucity of reports from pharmaceutical companies on the type and quantity of drugs that were fraudulently compounded or transferred by criminals. data are emerging from the pharmaceutical security institute (psi), a not-for-profit membership organization of pharmaceutical security directors, indicating that a large number of companies, products, and countries are targeted. for instance, since , pfizer pharmaceuticals (pfizer global security, new york, ny) has identified a rapidly increasing number of falsified products, countries reporting falsified drugs, and breaches of the legitimate supply chain national entry points (table ) ; the increases have been from % to over %. of pfizer products, those for erectile dysfunction are most frequently falsified ; other such products target patients with alzheimer's disease, cancer, high cholesterol, hypertension, malaria, and anxiety disorder. facilities where fake drugs were made or compounded were discovered with moldy walls, dirty equipment, and infested with rodents and insects ( figure ). falsifiers have created products that are visually indistinguishable from the genuine product, clearly demonstrating criminal intent to deceive. this increasingly recognized problem on virtually all continents is a pandemic defined as "an epidemic occurring over a very wide area, crossing international boundaries, and usually affecting a large number of people." definitions despite increasing awareness of the fraudulent drug epidemic, efforts to quantify and stop this peril have been stymied by multiple obstacles, not the least of which is agreement on definitions. , , poor quality drugs include substandard/ spurious/falsely labeled/falsified/counterfeit (ssffc) medical products. falsified (also commonly called fake or counterfeit products are intentionally and fraudulently produced and contain no api, the incorrect dose of the api, or the incorrect api. substandard medicines are caused by unintentional or negligent errors of manufacturing or by degradation after manufacturing resulting in insufficient api, poor dissolution properties, or degradation products. the nomenclature used by the world health organization (who), the world trade organization, the united nations (u.n.) office on drugs and crime, interpol, and others can be confusing; hence, we are using terms agreed upon by who. there are also properly manufactured medicines that are unlawful for reasons apart from their quality. these can be unregistered with company branded or generic medicines that, for reasons of theft or accidental or intentional diversion, do not have the legally required marketing authorization of the country's regulators to be imported or sold there, and medicines that infringe the trademark of a legal product. relatively little is known about medicines that have expired and are repackaged with a new date; these topics along with diverted products are beyond the realm of this article. this article focuses mainly on medical and public health considerations of falsified medicines that are particularly widespread in low-and middle-income countries. [ ] [ ] [ ] although there are increasing reports of detection of a variety of fake drugs from around the world, paradoxically, there are virtually no reports from middle-or high-income countries quantifying the state of poor quality medicines, only anecdotal case citations. governments have been hampered by a confusing array of expensive detection technologies. few functional national regulatory authorities exist in low-income nations that lack trained staff and suitably equipped laboratories to test drug quality centrally or in peripheral pharmacies or markets. , furthermore, the variability or absence of national and international criminal statutes, lack of an international agreement against trafficking of poor quality medicines, and inadequate punishments for convicted offenders reflect the weak legal framework for confronting drug fraud. one of the biggest obstacles in provision of quality-assured pharmaceuticals is the lack of effective manufacturing, regulatory, and quality processing in india and china. in , global public health agencies including providers, foundations, and research institutions contributed to developing an advocacy campaign to address falsified medicines, particularly in china. this campaign called fight the fakes is a step toward raising awareness about the problem, but legal action has to follow along with more public and political awareness. the u.s. institute of medicine (iom) has published a report "countering the problem of falsified and substandard drugs." the iom recommendations to "stem the global trade" in such products are laudable in advising that the u.s. food and drug administration (fda), the national institute of standards and technology, and other u.s. and international pharmaceutical and financing agencies be more actively involved in setting standards and financing improvements; yet this report falls far short of making a strong call for standardized, agreed-upon quality assessment technologies; an international law convention; and a more activist, internationally recognized lead organization, all three of which are essential for stopping the many health threats of fake drugs. global leadership to date has devolved in parts to the who, the u.n. office on drugs and crime, and interpol, each with diverse missions, responsibilities, limited authorities, and their own collaborations, funding networks, cultures, and languages. no organization is leading assertively. of the three areas listed, an international convention and improved national regulations are likely to have the most enduring value in concert with effective leadership and other innovations. the focus of all actions tied to drug quality must be on public and individual health, and strengthening national capacities to improve the health of their citizens. detection methods and technology. a major hindrance to understanding the types, names, extent, and amount of poor quality drugs nationally and globally has been ) the lack of agreed-upon field survey approaches and ) available lowcost tools to detect and classify bad drugs quickly at points of entry into countries, at public and private pharmacies, and in health units. in , the who published draft guidelines for surveys of medicine quality that are currently being revised. two or more levels of drug quality tests exist: ) methods useable in the field that are quick, inexpensive, and easy to use and teach; these methods are targeted mainly to examine packaging and detect drug contents and ) technologies requiring a laboratory equipped for exhaustive chemical analysis. these approaches are summarized in table , deriving from the iom report and a recent analysis by green and others from the centers for disease control and prevention reference laboratory. within each method there are numerous tools and prototypes being used and new ones tested. current technologies for field use rely on visual packaging inspection, lot number reporting via mobile phones, thin-layer chromatography, colorimetric tests, and simplified spectroscopic methods. gas and liquid chromatography and mass spectrometry are some of the more advanced and complex techniques for investigating drug quality in central laboratories. qualitative or semiquantitative tests for an api are not substitutes for proper manufacturing control, dissolution studies, pharmacokinetics equivalence, and supply chain integrity. a very promising recent development has been the u.s. pharmacopeia promoting the quality of medicines (pqm) program in several african, asian, and latin american countries using the "minilab" (global pharma health fund e.v., giessen, germany). [ ] [ ] [ ] this training program supported by the u.s. agency for international development (usaid) and the president's malaria initiative (pmi) has trained several hundred persons in rapid chemical analysis of drugs taken from public and private pharmacy stocks. a major reference training center has recently been opened in accra, ghana, with usaid and u.s. pharmacopeia support as a referral testing and regional training center. important also is the development of the counterfeit detection (cd)- (us food and drug administration, forensic chemistry center, cincinnati, oh), a promising handheld electronic device for peripheral use that detects fake packaging at point of sale with images and videos of the suspect samples. , the fda, skoll foundation, and other partners are supporting expansion of testing and use of this device. we recommend that a precertification of essential diagnostics, drugs, and vaccines should be required for specific regional and global control, elimination, and eradication programs and campaigns. more information is needed to confirm that precertification of products is occurring for the pmi and the global fund to fight aids, tuberculosis and malaria, and products purchased by u.n. international children's emergency fund (unicef) and who. essential drugs designated by who should also be targeted for special vigilance by quality assurance mechanisms. no independent agency has inventoried and performed comparative quality assessments of these packaging and drug-testing devices and made recommendations to countries for their use. objective comparisons are needed of the diversity of field methods in terms of accuracy, reliability, costs of equipment and supplies, level of training needed, ease of use, spare part availability, and maintenance requirements. simplified standard survey protocols and methods for sampling drugs at country entry points (seaports, airports, and roads); at major pharmacy depots; in health units (public and private hospitals and clinics); and at more peripheral distribution sites (district and village pharmacies and individual vendors) are also needed. low-cost, portable detection tools would empower pharmaceutical inspectors in numerous countries that have oversight of the medicine supply. results would be available promptly rather than delayed when samples are sent to national or international laboratories as occurs now; lamentably, intervals of several years have occurred from the time specimens were collected to the time the results were available to those needing to take action. ideally, central reference laboratories vetted by who, fda, or another agency would back up spot checks and random sampling of pharmaceuticals at the periphery. good quality medicines by law. falsified medicines are ultimately a problem that impacts public health. the solution needs to reflect various incentives, either via financial gain, avoidance of punishment or both. a multi-sectorial effort is essential for taking into account how this illegal market is interwoven with world trade agreements, business models, and associated legal ramifications. globalization has enlarged the international trade in medicines. for example, india exports over us$ . billion in pharmaceuticals, which are among their most important exports. as of , % of drugs and % of apis for drugs in the united states are imported from foreign countries. an international law convention against substandard and falsified medicines would address both regulatory and criminal international governance challenges simultaneously through technical, legal, and financial mechanisms. how would the convention work and what national benefits would it bring? a convention would provide four legal underpinnings that do not exist, that together would advance patient safety and access to quality medicines. first, a convention would define the various sorts of wrongful medicines accurately and thereby avoid misunderstandings caused by today's problematic or vague terminology (e.g., where countries seized good quality generic medicines as "counterfeit"). second, a convention would promote the requirement that signatory countries enact national laws to designate wrongful actssuch as the intentional manufacture, trafficking, or selling of falsified medicines-as criminal offences, with attendant obligations to alert health-care workers and to prosecute or extradite the offenders to justice promptly. third, a convention would provide the legal and institutional framework for participating countries to agree, implement, and evolve convergent standards of medicine regulation, so as to reduce poor quality medicines in international trade. fourth, and for lower income countries particularly, a convention would contain mechanisms for financial and technical assistance, and, to join local and regional networks. these actions would help build national and regional medicine regulatory authorities (mras) to a point where patients' access to quality medicines is protected. some have said that establishing recommended codes of practice that are nonbinding (soft law) are better than international norms and regulations that are binding (hard law). we disagree with soft law in regard to controlling the current fake-drug pandemic. there are precedents for using international law in this way. a treaty that internationally criminalizes counterfeit banknotes provides an analogy for falsified medicines. in the health field, there are treaties specifically addressing the illicit traffic of certain narcotic drugs and treaties to prevent harm-particularly, the framework convention on tobacco control and its associated protocols to stanch illicit trade. that convention has brought over us$ million new funding to global tobacco-control efforts, demonstrating that international law need not compete for resources, but can increase them. the u.n. office on drugs and crime has been developing "draft model legislative provisions on fraudulent medical products" for several years but there has been no agreement on final text; the focus appears to be on criminal and judicial issues. challenges ahead. information is accruing that large quantities of falsified drugs are being manufactured in asian countries. china and india are two of the largest producers of good quality drugs and vaccines, many of which are purchased or funded by the usaid; unicef; global fund to fight aids, tuberculosis and malaria; who; and other organizations, charities, and national agencies for global disease control and eradication programs. however, according to the world customs organization, in , % and % of unlawful drugs of all sorts confiscated worldwide were manufactured in india and china, respectively. the circuitous travel itineraries of fake medicines have been traced across continents, such that the unsuspecting recipient countries assume a bona fide origin. a particularly heinous example is that of multiples instances of the production, marketing, and international travel of falsified bevacizumab (avastin ), a cancer medicine; the fake drug closely matched the appearance of the real medicine, but tests indicated salt, starch, and various cleaning solvents instead of the active ingredient with resulting endophthalmitis. the internet has opened up an unregulated opportunity for criminals to promote and sell fake drugs to unsuspecting vulnerable populations, often the aged and others seeking convenience and low cost. a recent survey of over , online pharmacies found that % operated outside legal regulations and a large percentage closed operations within years of operation. , the fda and other organizations participate with interpol in annual international actions (operation pangea) to shutdown illegal pharmacy websites selling potentially counterfeit and illegal medical products. more than , such illegal websites were closed during one week in with seizure of us$ . million of pharmaceuticals worldwide. leadership, collaboration, and national strengthening. arguably, the major obstacle to solving the problem of poor quality medicines has been the lack of a clearly identified lead organization with a plan of action developed in concert with countries, pharmaceutical companies (multinational corporations and innovator/biotechnology enterprises), and national and international agencies-and a sense of urgency to implement the plan with resources and partners-including pharmaceutical companies in low-income countries. who has estimated that % of countries have inadequate medicines regulation authorities (mras) or none at all. moreover, who has found that % of african mras lack the capacity to undertake medicine regulatory functions and therefore cannot guarantee the quality, efficacy, and safety of medicines, , the new partnership for africa's development has found that there is either limited or declining government funding for mras in the east african community partner states. many have looked to who for this leadership, given its successful implementation of the public health treaty on tobacco control. however, some argue that the u.n. system, including the who, is poorly suited to be in a leadership role because of sparse technical expertise in products, manufacturing, and quality systems. u.n. agencies are beholden to member states and cannot regulate or enforce anything easily, especially, in india and china. in this regard, who could serve the role of a partner rather than a leader. the recently revitalized rapid alert program at who has begun to "track and trace" poor quality drugs as reported voluntarily by member countries. rapid alert notices are published periodically by who indicating the fake drug type, lot number, quantity of product, and place detected. strong action by countries can stem the tide as shown in rwanda and cambodia, although unique situations and major multi-sectorial engagements exist in these countries. one solution is creation of regional harmonization networks, addressing some elements of drug registration tied to regional economic communities; the african medicines registration harmonization initiative is one example of such a network. the u.n. office on drugs and crime (unodc) has also made recent attempts at facilitating international cooperation against falsified medicines. one proposal has been a trilateral coalition of the unodc, who, and interpol. still, active and transparent support from the fda, drug companies, individual countries, and other partners will be needed; the fda may be the most qualified as a leadership organization based on their technical expertise and global influence. mechanisms for training technical staff, regulating products, improving manufacturing practices, and stopping criminal production are needed to assure a good supply of medicines. given that the problem of substandard and falsified medicines should be approached primarily from a public health and equity perspective, it is important that the negotiations on the way forward be led by the ministries of health along with the ministers of finance and trade, while respecting legitimate intellectual property rights. could and should who be the lead organization in curbing the spreading epidemic of falsified pharmaceuticals? who's ability to take more assertive action is strengthened by the revised international health regulations. who's director general can convene emergency committees in response to public health emergencies as has been done recently for the influenza a (h n ) pandemic in , the middle east respiratory syndrome (mers-cov) in , the polio crises in , and the ebola epidemic in - . illicit drug trafficking is an emergency. the drug quality and security act, signed into law by president obama in , outlines steps to build an electronic system to identify and trace certain prescription drugs in the united states. the results of this system are awaited. finally, the millennium development goals (mdgs), under revision, should include measurable objectives for good quality drugs. this will encourage national establishment of baseline status and achievable targets, particularly for essential drugs. establishment of mdg targets and sustainable development goals (sdgs) will help greatly to solve the poor quality drug epidemic by application of available technology and good pharmaceutical vigilance and governance. one incentive that would transform the current system is applying a "universal quality standard" to drug products. for example, if india allows a substandard manufacturer to sell products in africa, the fda could ban import of products from india. although difficult to develop and implement, a combination of incentives and penalties driven at the political and economic levels is needed. the major urgent needs to improve the quality of the world's medicines fall into three main areas: ) research to develop and compare the most accurate and affordable tools to identify high-quality drugs at point of sale and deployment of the best methods; ) an international convention and national legislation to facilitate production and use of high-quality drugs and protect all countries from the criminal and the negligent who make, distribute, and sell life-threatening products; ) designation of a highly qualified, well-supported international organization, possibly the fda or who, that will establish standards, training programs, drug quality surveillance, and authoritative guidance for countries in cooperation with national medical regulatory agencies, pharmaceutical companies, and international agencies, all of which have an urgent interest and investment in ensuring that patients throughout the world have access to good quality medicines. the organization would also advocate strongly for including targets for achieving good quality medicines in the mdgs and sdgs including certification of pharmaceutical products entering countries that request such services, 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detection device cd- india aims to clock usd . bn pharma exports fy import of human drugs and human drug components global health and the law follow the money: how the billions of dollars that flow from smokers in poor nations to companies in rich nations greatly exceed funding for global tobacco control and what might be done about it counterfeiting, a global spread, a global threat counterfeit bevacizumab and endophthalmitis progress report for state and federal regulators medicines counterfeiting is a complex problem: a review of key challenges across the supply chain fda takes action against thousands of illegal internet pharmacies. food and drug administration news release effective medicines regulation: ensuring safety, efficacy and quality availability of drug regulatory and quality assurance elements in member states of the who african region the african medicines regulatory harmonization initiative: rationale and benefits international medical products anti-counterfeiting taskforce (impact) combating substandard and falsified medicines: a view from rwanda quality of antimalarials at the epicenter of antimalarial drug resistance: results from an overt and mystery client survey in cambodia why the mdgs need good governance in pharmaceutical systems to promote global health this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- - w avwo authors: koenig, kristi l.; alassaf, wajdan; burns, michael j. title: identify-isolate-inform: a tool for initial detection and management of measles patients in the emergency department date: - - journal: west j emerg med doi: . /westjem. . . sha: doc_id: cord_uid: w avwo measles (rubeola) is a highly contagious airborne disease that was declared eliminated in the u.s. in the year . only sporadic u.s. cases and minor outbreaks occurred until the larger outbreak beginning in that has become a public health emergency. the “identify-isolate-inform” tool will assist emergency physicians to be better prepared to detect and manage measles patients presenting to the emergency department. measles typically presents with a prodrome of high fever, and cough/coryza/conjunctivitis, sometimes accompanied by the pathognomonic koplik spots. two to four days later, an erythematous maculopapular rash begins on the face and spreads down the body. suspect patients must be immediately isolated with airborne precautions while awaiting laboratory confirmation of disease. emergency physicians must rapidly inform the local public health department and hospital infection control personnel of suspected measles cases. the ebola outbreak, a public health emergency originating in west africa, as well as the emergence of middle east respiratory syndrome and pandemic influenza, are stark reminders that we must prepare our healthcare systems for emerging infectious diseases (eids). the public health threat includes not only novel diseases, but also the reemergence of existing diseases that were previously well controlled, such as measles in the u.s. in , cases of measles from states were reported to the u.s. centers for disease control (cdc), the greatest number reported since the endemic disease was declared eradicated in the year . from january to march , , cdc reported people from states and the district of columbia to have measles, with most related to a multi-state outbreak linked to an amusement park in southern california. measles is one of the most transmissible diseases in existence, with at least a % infection rate in susceptible populations. the incubation period ranges from - days, and humans are the only natural host. measles can be contagious four days prior to the onset of rash and, in fact, is most contagious prior to rash manifestation. measles can mimic influenza, croup, bronchiolitis or pneumonia before the rash occurs. it can be transmitted from surfaces and air for up to two hours after an infected person leaves a room. yet most clinicians in industrialized countries have never seen even a single case of measles. following a brief review of measles, this paper describes the novel i tool, initially developed for ebola virus disease, as adapted for use in the initial detection and management of measles patients in the emergency department (ed). measles classically presents with a high fever (often > o f [ o c]), generally of - days in duration. this initial sign occurs after an incubation period of - weeks following exposure (average - days). during this prodromal phase, a classic triad of cough, coryza and conjunctivitis (the " cs") is often present. patients may have photophobia. the eyes have a characteristic appearance, typically showing erythema of the palpebral conjunctiva with nonpurulent discharge ( figure ) and sometimes periorbital edema. patients may also report malaise, myalgias, anorexia, and diarrhea. adults often develop transient hepatitis. a tool for initial detection and management of measles patients koenig et al. koplik spots, when seen, are pathognomonic of measles ( figure ). if present, they manifest - days prior to the rash and last for - days. they appear as bluish-gray enanthema ("small grains of sand") on a red base and are typically seen on the buccal mucosa opposite the second molars. therefore, it is essential to have proper lighting to visualize them. during a measles outbreak, after donning appropriate respiratory protection, emergency physicians (ep) should carefully assess the oropharynx in patients presenting with non-specific viral syndromes and assess for the presence of koplik spots. the rash of measles generally erupts about days after exposure, which is usually - days after onset of symptoms. unlike rashes of some infectious diseases that start on the lower extremities or trunk, the rash of measles begins on the face and progresses cephalocaudally to the torso and extremities. thus, assessing the pattern of rash evolution is essential to identify measles patients. erythematous macules and papules coalesce into patches and plaques within about hours ( figure ). petechia and ecchymosis can also be seen. by the time a rash develops, within - days, patients will be ill appearing. after - days, the exanthem begins to fade, forming coppery-brown hyperpigmented patches that may desquamate. the rash initially disappears at the location where it first appeared. the rash can be more difficult to detect on dark-skinned patients ( figure ). disease manifestations are often more severe in children under five and adults over years of age. patients who are immunocompromised may present atypically and may not develop a rash. during a measles outbreak, clinicians should advise patients with viral syndromes who are being discharged from the ed to monitor for appearance of a rash, especially one that first appears on the face. if a rash develops, children or adult patients should avoid public places and seek immediate medical advice. people who received vaccinations between and with the original killed-virus measles vaccine may have incomplete immunity and present with milder symptoms. during those years, this vaccine was only administered to u.s. children at about one year of age, so persons presenting in with "atypical measles" would be - years of a tool for initial detection and management of measles patients age. a prodrome of fever, headache, abdominal pain and myalgias can be subclinical. in these atypical presentations, the rash can be macular, vesicular, petechial, or urticarial and can begin on the hands and feet and spread centripetally. when atypical measles was first reported in the late s and early s, it was often mistaken for rocky mountain spotted fever. in patients who receive post-exposure prophylaxis with serum immunoglobulin, a modified variety of measles can occur with similar but milder signs and symptoms and an incubation period of up to days. the population most at risk for measles are those persons who are exposed, but not immunized, or with inadequate immunity. this includes infants too young to receive immunizations (under months), children whose parents have declined immunization, travelers from countries where immunization rates are low, and immunocompromised patients. when measles is suspected, the clinician should collect separate swabs of the throat and nasopharynx using viral culture swabs and contact the local health department for realtime polymerase chain reaction (rt-pcr) testing. serum can also be obtained and sent for measles-specific igm antibody, but the test may be negative early in the course of disease. some local health departments may also request additional testing of serum and urine for rt-pcr testing. according to the cdc, there are no data supporting routine checking of serum antibody titers, particularly given the fact that they have notable false negative result rates. pregnant women and other persons with deficiencies in cell-mediated immunity are at increased risk for serious complications, including primary measles giant cell pneumonia with respiratory failure. spontaneous abortion and premature delivery have been described. however, there is no increased risk for congenital anomalies as has been reported in other diseases contracted during pregnancy, such as toxoplasmosis, rubella, cytomegalovirus, herpes simplex, and human immunodeficiency virus. the vast majority of patients with measles who are well-nourished (not vitamin a deficient) recover; however, measles can lead to several complications and even death. , while certain groups of patients are at high risk for complications, even previously healthy children can become severely ill and require hospitalization. the following are well-described complications of measles: • in every children develops bacterial otitis media (can lead to permanent hearing loss) • in every children develops bacterial pneumonia • in every children develops acute encephalitis (often resulting in permanent brain damage) • unknown frequency of measles giant cell pneumonia in pregnant and other immunocompromised patients • in every children dies (from respiratory and neurologic complications) • febrile seizures are also seen in addition, a rare late complication ( to years after measles infection) is subacute sclerosing panencephalitis (sspe), a fatal degenerative disease of the central nervous system characterized by behavioral and intellectual deterioration and seizures. permanent blindness may also result from measles infection. measles is transmitted by the airborne route. it can be a tool for initial detection and management of measles patients koenig et al. contracted for up to two hours from the air or from airborne particles on surfaces in a room that was occupied by a measles patient. in addition to standard precautions, all practitioners, even if immunized, who enter a room with a suspected or confirmed measles patient should wear fittested n respirators or equivalent respiratory protection. prior to onset of rash, measles can mimic influenza, croup, bronchiolitis, other viral illnesses, and pneumonia. once the rash develops, particularly with accompanying fever, other entities, including common childhood diseases, are in the differential diagnosis. characteristics of these diseases help to distinguish them from measles ( figure ). in addition to alternate infections, eps must distinguish the rash of measles from other acute rash presentations with systemic symptoms, including allergic drug reactions. treatment for measles is primarily supportive care. hydration and antipyretics, such as in other viral illnesses, are the mainstays of therapy. if a secondary bacterial infection such as otitis media or pneumonia develops, appropriate antibiotics are indicated. vitamin a is a measles-specific treatment that is critical in any patient with low levels; it can be administered orally. this treatment can lessen severity and even prevent mortality. the dose of vitamin a for measles patients is large, typically , international units (iu) for two days ( , iu if under months and , iu for - months). measles is a major cause of death in refugee camps where vitamin a deficiency is common, immunity is poor, and conditions are crowded. for every case of measles, more persons in the camp are thought to be incubating disease. vitamin a supplementation significantly reduces mortality. the vast majority of cases of measles in patients with intact cellular-mediated immunity are uncomplicated and resolve by about - days after onset of illness. in fact, in prevaccination times, mothers used to purposely expose their children so they would all get sick at once and develop immunity before adulthood. nevertheless, in modern days, disease prevention should be the goal. all persons should be vaccinated against measles (as part of the measles, mumps and rubella [mmr] vaccine) unless there is a medical contraindication to live virus immunization. vaccination is highly effective in preventing disease, but it is not % protective, even in persons who have received two doses of vaccine at appropriate intervals. also, some patients are too young to be vaccinated, i.e., those under months of age. there has been a movement in the u.s. by some parents to avoid or delay vaccination based on personal, philosophical beliefs that are not medical or religious in origin. while this is permitted in some states, it results in decreased herd immunity, placing persons with medical contraindications to vaccination and others at greater risk. this reluctance to vaccinate is based in part on a study that reportedly found a link between the mmr vaccine and autism. this study has been discredited, the researcher accused of providing fraudulent data, and the paper has been retracted. infants under six months of age and nonimmune/ nonimmunized pregnant women, if exposed, should receive passive immunity with intramuscular immune serum globulin. when administered within six days of virus exposure, these antibodies can prevent measles or reduce illness severity. if they are over six months, infants should receive a standard vaccination. other nonimmunized persons who are exposed to measles should receive vaccination as well, ideally with hours of the exposure. while vaccination might not prevent the disease, if illness develops it is generally less severe and for a shorter duration than in completely unvaccinated persons. household contacts should be advised that measles is highly contagious and infected family members should be isolated from four days before to four days after the rash manifests. anyone at risk who is not fully vaccinated should receive vaccine as soon as possible. most people born or living in the u.s. before have had measles and are therefore immune. as with any other patient presenting to the ed, it is important for eps to be familiar with admission vs. discharge criteria. admission criteria for measles patients are similar to those for others. however, there are special considerations for patients being discharged. as measles is highly contagious, there are public health considerations as well as individual concerns for patients who do not meet hospitalization parameters. infected patients must be isolated from others and public health must be notified so that contact tracing and community protective measures can be instituted. eps should provide return precautions. in addition to routine instructions, these should include the following: ) monitor for occurrence of rash if one is not already present, and ) pay special attention to the development of respiratory distress and neurologic symptoms. for someone being discharged, clinicians should document that the patient is well nourished and that the family is not in poverty (therefore not suspected to be vitamin a deficient), and that there are no neurological or respiratory risks. the identify-isolate-inform tool initially developed for figure . pediatric exanthems that may mimic measles, continued. ebola virus disease can be modified for the ed evaluation and management of patients under investigation for measles virus ( figure ). while patients typically present to the ed with symptoms, during an outbreak, concerned but asymptomatic patients and parents of potentially exposed children may seek care. therefore, the first branch of the algorithm involves determination of whether the patient is symptomatic or asymptomatic. for asymptomatic patients, the goal is prevention of disease in both the individual and the population. this is accomplished by assessing exposure history and patient risk and, if it exists, providing post-exposure prophylaxis (with vaccine or with immune serum globulin if the patient is immunocompromised). the patient must then undergo public health monitoring for days to monitor for the development of signs and symptoms. home quarantine should be strongly considered, as patients with measles may be contagious for a few days prior to onset of symptoms. conversely, patients with signs and symptoms of measles (prodrome of fever, cough/coryza/conjunctivitis, koplik spots followed by rash), should be immediately masked and isolated using airborne precautions. to assist in risk assessment, eps should inquire about immunization status, sick contacts, and travel to a region with measles. all healthcare providers, including those who have been vaccinated, should don n respirators or equivalent respiratory protection prior to caring for suspected measles patients. as with other airborne diseases, clinicians must be current on their "fit testing" requirements (typically renewed annually) in order to properly use an n respirator. isolated patients should have samples obtained urgently and sent to the local public health department laboratory for disease confirmation. whether patients are symptomatic and immediately isolated or asymptomatic and exposed/at risk, public health authorities must be immediately notified / . in addition, clinicians should promptly inform the hospital infection control and prevention practitioner on duty, regardless of time of day. measles is a highly contagious preventable viral disease that had been declared eradicated in the u.s., but made a resurgence starting in . identify-isolate-inform is a tool for eps to apply when patients who may have measles present to the ed. in addition, several pearls a tool for initial detection and management of measles patients are helpful when managing measles patients ( figure ) . as emergency physicians working on the front lines of clinical medicine, we must be prepared for emerging and reemerging infectious diseases. • check immunization status • ask about travel and exposure history • essential history: rash should begin on face and neck and spread distally, with delayed onset of rash after illness began • check for koplik spots on patients presenting with severe viral respiratory syndromes • advise patients discharged with viral syndromes to monitor for later development of rash • remember that immunocompromised patients may not develop a rash the ebola virus outbreak and other emerging infectious diseases centers for disease control and prevention. measles cases and outbreaks identify, isolate, inform: a -pronged approach to management of public health emergencies s principles and practice of infectious diseases measles associated hepatobiliary disease: an overview determination of immunity to measles virus in young adults: comparative evaluation of a commercial enzyme immunoassay and the hemagglutination inhibition techniques vitamin a for treating measles in children ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children johns hopkins abx guide is there a case for quarantine? perspectives from sars to ebola key: cord- -i tkdgy authors: suo, siqingaowa; wang, xue; zarlenga, dante; bu, ri-e; ren, yudong; ren, xiaofeng title: phage display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: i tkdgy the spike (s) protein of porcine transmissible gastroenteritis virus (tgev) is located within the viral envelope and is the only structural protein that possesses epitopes capable of inducing virus-neutralizing antibodies. among the four n-terminal antigenic sites a, b, c, and d, site a and to a lesser extent site d (s-ad) induce key neutralizing antibodies. recently, we expressed s-ad (rs-ad) in recombinant form. in the current study, we used the rs-ad as an immobilized target to identify peptides from a phage-display library with application for diagnosis. among the phages selected that specifically bound to rs-ad, the phage bearing the peptide tlnmhlfpfhtg bound with the highest affinity and was subsequently used to develop a phage-based elisa for tgev. when compared with conventional antibody-based elisa, phage-mediated elisa was more sensitive; however, it did not perform better than semi-quantitative rt-pcr, though phage-mediated elisa was quicker and easier to set up. transmissible gastroenteritis virus (tgev) is a member of the coronaviridae family and is a major cause of enteric disease in pigs where it threatens swine production and triggers substantial economic losses in the industry [ ] [ ] [ ] [ ] . its genome is composed of positive-stranded rna approximately . -kb in length. the virus consists of four structural proteins: envelope (e), membrane (m), spike (s), and nucleocapsid (n) proteins [ , , ] . non-structural proteins, which comprise two-thirds of the -proximal end, are encoded by open reading frames a and ab as well as the replicase. in contrast, the end of the genome encodes both non-structural and structural proteins ( -s- a- b-e-m-n- - ) [ ] . the s protein, which induces neutralizing antibodies, is important in the initiation of infection [ ] [ ] [ ] and has been further delineated into four antigenic sites a, b, c, and d which are located within the n-terminal region of the s protein [ ] . among these, only site a and to a lesser extent site d (herein defined as s-ad) are involved in eliciting neutralizing antibodies. recent work demonstrated that recombinant s-ad (rs-ad) was able to induce antibodies capable of neutralizing tgev infection in vitro [ ] . edited attenuated or inactivated tgev vaccines are less than optimal because they are capable of reverting back to virulent phenotypes and generally do not prevent viral shedding. therefore, effective diagnostic tests have become important in virus management and control. phage display is a proven technology for identifying small peptide ligands that can bind specific target proteins [ ] [ ] [ ] [ ] . it has been utilized in antibody engineering [ ] , drug discovery [ ] , vaccine development [ ] , and molecular diagnosis. in virology, phage display has been used to identify peptides that interact with several viruses such as bovine rotavirus [ ] , adenovirus type [ ], andes virus [ ] , sin nombre virus [ ] , coronavirus [ ] , and avian h n virus [ ] . herein, we use similar technology and advance previous work by using the rs-ad as an immobilizing target to select phages from a peptide display library, with diagnostic potential for tgev. our results indicate that phages bearing peptide ligands that bind rs-ad can be used to develop a phage-mediated elisa with high sensitivity and specificity to distinguish tgev from other common swine viruses. biopanning swine testis (st) cells were purchased from atcc and used to propagate tgev strain pur -mad [ ] . the rs-ad was produced and purified as described elsewhere [ ] . a -mer phage-display library was purchased from new england biolabs for panning according to published protocols [ , , ] using the rs-ad as a target at a concentration of lg/well. the -well plates coated with rs-ad, were initially incubated with the phage library ( . pfu/ml; ll/well) suspended in tbst ( mm tris-hcl [ph . ], mm nacl, . % tween- ) for min. subsequent pannings , , and were performed using incrementally higher concentrations of tween- . the phage titers of the input, output (elution), and amplified phages were determined as defined by the manufacturer. indirect elisa was used to assess the phages that remained after four rounds of biopanning. either tgev ( . mg/ml) or rs-ad ( lg/well) in . m nahco ph . was used to coat -well plates at °c for h. the next day, the plates were blocked with % bovine serum albumin (bsa) in tbs (tbsb) for h, washed ( ) with tbst, and then incubated with phage ( . pfu/ml in . m nahco , ph . ; lg/well) for h at °c. the plates were again washed with tbst, then incubated for h at °c with rabbit anti-m antibody ( : in tbsb; abcam), followed by horseradish peroxidase (hrp)-conjugated goat anti-rabbit igg antibody (garp; : in tbsb, sigma). the od nm was determined in triplicate as previously described [ ] . ten phages with the highest affinity for binding rs-ad as determined by elisa were amplified, precipitated with polyethylene glycol-nacl, and then used for dna extraction according to the manufacturer's instructions (new england biolabs). amplification of the genes encoding the exogenous peptides was performed using sense ( -tcacctcgaaagcaagctga) and anti-sense ( -ccctcatagttagcgtaacg) m primers followed by dna sequencing [ , ] . the pcr conditions were as follows: °c for min, cycles of °c for s, °c for s, °c for s, and a final extension at °c for min. to compare the sensitivities of phage-mediated elisa to antibody-mediated elisa, tgev serially diluted in . m nahco (ph . ) was coated onto duplicate elisa plates overnight at °c followed by blocking with % skim milk for h at rt. the selected phages or unbound phage complexes (negative control) diluted in pbs ( . pfu/ml) were added to one set of plates, followed by anti-m antibody ( : in pbs ? bsa). to the second set of duplicate plates, rabbit anti-tgev polyclonal antiserum serially diluted in pbs ? bsa, and normal rabbit serum were added as the primary and control antibodies, respectively. after incubating both sets of plates for h at °c followed by extensive washing, garp ( : ) was added as described above. the od values were read on all plates; od ratios where od (sample-negative standard) (p)/od (positive control-negative standard) (n) [ were judged as positive. all experiments were performed in triplicate. the tcid of tgev was determined using the reed-muench method, and tgev was adjusted to . mg/ml in pbs. total rna was extracted from ll of virus (fastgene, china) and dissolved in ll of sterile water. reverse transcription was performed in ll using ll of rna ( ng/ll), oligo dt as primer, and m-mlv reverse transcriptase as recommended by the manufacturer (takara, china). the resulting cdna ( ll) was used as a template for pcr in ll which included . ll of easy taq polymerase (takara, china), ll of dntp ( . mm), pcr buffer ( ll), and . ll each of sense ( -cttagtagtaatattttgcatac) and antisense ( -tatagcagatgatagaattaaca) primers. amplification conditions were as follows: °c for min, then cycles of °c s, . °c s, and °c s followed by a final extension at °c for min. the amplified fragment was confirmed by dna sequencing. phage specificity was evaluated against the following panel of porcine viruses: tgev, strain hr/dn [ ] , porcine epidemic diarrhea virus (pedv; strain hljby) [ ] , porcine reproductive and respiratory syndrome virus (prrsv; strain jilintn ) [ ] , porcine circovirus type ii (pcv ; strain pcv -ljr) [ ] , porcine parvovirus (ppv; strain ppv ) [ ] , porcine pseudorabies virus (prv; strain kaplan) [ , ] , and porcine rotavirus (prov; isolate dn ) [ ] . all viruses were initially coated at lg/ml then serially diluted in . m nahco (ph . ) and subjected to phage-elisa as described above. average od values were obtained from three independent experiments. data were collated and the mean ± sd values were determined. arithmetic means were compared between treatment groups using anova (spss . ; spss inc., chicago, illinois, usa) followed by duncan's multiplerange test. values of p \ . and p \ . were defined as statistically significant (''*'') or highly significant (''**''), respectively. in this study, we used phage display to select -mer peptides that bind rs-ad [ ] and that may function for diagnosing tgev infections. after four rounds of panning, rs-ad-specific phages increased from . in the first round to . in the fourth round (table ) . following the last screen, we selected phage clones from the original that bound both rs-ad and tgev. this subset was characterized by elisa with respect to their binding efficiencies (fig. ) . pcr amplification and sequencing indicated that nine distinct -mer peptides were identified among the phages that were selected ( table ). in contrast to previous reports [ , , ] , these peptides exhibited substantial sequence diversity in the number of peptides that bound to rs-ad. it is not known if this relates to the length of the target protein or to changes made in the panning process to enhance binding specificity. as shown in fig. , we selected four (phtgev-sad- , phtgev-sad / , phtgev-sad , phtgev-sad ) of the ten phages with the highest binding affinity to tgev for further testing. the lowest detectable quantity of tgev for the above defined phages was . , . , . , and . mg, respectively, suggesting that phtgev-sad was the most sensitive when used in a phage-based elisa. binding directly to tgev was uncharacteristically better than binding to the rs-ad used in the selection process (figs. , ) . this is likely attributable to more complete folding of the native protein or to better accessibility of the binding epitope in the native form. the minimum quantity of tgev required for detection via antibody-based elisa was . lg (p/n value [ ) (fig. ) , whereas the minimum quantity of tgev required for phtgev-sad -based elisa was . lg. this is consistent with the phage-mediated elisa being more sensitive than conventional antibody elisa. a number of elisa-based assays have been developed over the years for detecting tgev, many of which have been directed at differentiating tgev from prcv-infected animals. among the earlier ones, sestak et al. [ ] targeted the s glycoprotein of tgev in a competition elisa where recombinant s protein was coated onto plates and used to capture host antibodies. using a monoclonal ab to epitope d and which is specific for tgev, the investigators were able to differentiate the infectious agents. liu et al. [ ] cloned and expressed the nucleoprotein (n) to develop an elisa. compared to the virus neutralization assay, they demonstrated % sensitivity and specificity; however, they did not characterize or address the lower level of sensitivity in vitro or in vivo. in , elia et al. [ ] used the recombinant s protein to develop an elisa to assess swine-like tgev coronaviruses in canine hosts. given the novelty of the virus, they were unable to compare it to other assays currently in use. zou et al. [ ] use techniques similar to those developed here, i.e., peptide display, to target the m protein of tgev in developing an elisa-based diagnostic test. in this case, the sensitivity of the elisa exceeded that when the phage-mediated elisa and antibody elisa were compared to rt-pcr which targeted a -base pair fragment of the s gene, the rt-pcr was most sensitive of all assays tested. this is not unexpected given the higher sensitivity of pcr assays in general. pcr amplification was positive using cdna equivalents of . lg of tgev (data not shown). real-time pcr and/or nested pcr would clearly have generated even more sensitive results. in addition, phages expressing peptide that bind to tgev s-ad did not bind to other selected viruses (fig. ) . table sequences of tgev rs-ad peptides. predicted amino acid sequences were generated for ten selected phages in summary, we identified peptides that specifically bind to tgev and can form the basis of new diagnostic tests where the sensitivity of phtgev-sad was . lg of tgev. this sensitivity fared quite well when compared to the antibody-mediated elisa which had a sensitivity of . lg but fell short of the sensitivity of rt-pcr; however, phtgev-sad- provides a quicker and less costly alternative to rt-pcr. diseases of swine th ed the authors declare no conflicts of interest. key: cord- -b wlr t authors: engstrom-melnyk, julia; rodriguez, pedro l.; peraud, olivier; hein, raymond c. title: chapter clinical applications of quantitative real-time pcr in virology date: - - journal: methods in microbiology doi: . /bs.mim. . . sha: doc_id: cord_uid: b wlr t abstract since the invention of the polymerase chain reaction (pcr) and discovery of taq polymerase, pcr has become a staple in both research and clinical molecular laboratories. as clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has pcr performance. through optimisation, the present-day uses of real-time pcr and quantitative real-time pcr are enumerable. the technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time pcr provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. all of these serve vital roles in the continuum of care to enhance patient management. beyond this, quantitative real-time pcr facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. processes, small sample volumes and can be utilised in a wide variety of applications, making it the method of choice in today's molecular laboratories. through the aid of fluorescent signalling probes to measure amplification of dna at each pcr cycle, at the point of exponential dna accumulation, real-time pcr is able to provide broader linear dynamic ranges and increased assay performance as determined by sensitivity, specificity, precision, and reproducibility. due to the consistency in signal intensity changes during the exponential growth phase of pcr, it is also easily adaptable for quantitative reporting. however, there are three properties that are uniquely associated with quantitative real-time pcr: quantification, standardisation, and lower limit resulting. the accumulation of fluorescence signal is measured at each pcr cycle of the reaction and the cycle at which this signal exceeds a predetermined background fluorescence threshold during the logarithmic phase of amplification is referred to as the cycle threshold (c t ). the c t value is inversely proportional to the viral copy number in the specimen, and through comparisons of this value to an external calibration curve or an internal quantitation standard, the initial nucleic acid target concentration can be calculated (heid, stevens, livak, & williams, ; livak & schmittgen, ) . however, accurate quantitation within each sample is hindered when relying solely on an external standard as amplification efficiencies for each individual sample may be variable and inconsistent. by utilising a standard internal reference template, with the rationale that any variable influencing amplification efficiency should example of amplification and detection of target nucleic acid by real-time pcr. affect both template and target similarly, inhibition and amplification effects are compensated for which allows for more accurate quantitation ( figure ). this control can be further enhanced when incorporating an internal reference that utilises the same primer sequence as the target since any potential additional effects on pcr efficiency for each of the two targets is eliminated. thus, the competitive real-time pcr strategy is the most reliable approach for nucleic acid quantitation (diviacco et al., ; gilliland, perrin, & bunn, ; stieger, demolliere, ahlborn-laake, & mous, ; wang, doyle, & mark, ; zentilin & giacca, ) and is the basis for the majority of present-day virology assays. it is equally important to utilise appropriate quantitation standards, when available, to ensure accurate quantitative results, inter-laboratory correlation, and overall standardisation. standardisation of reported viral loads ensures not only interlaboratory consistency but also high clinical utility of viral load monitoring, sets the foundation for establishing clinical correlations and critical thresholds leading to better management of infections and treatments, and are critical for the development of clinical guidelines (miller et al., ) . with the wide availability of assay methods, viral targets, specimen type, and lack of standard reference material (hayden et al., ) , viral load variability across laboratories can range significantly, as high as . log copies/ml . specifically, results from proficiency testing/external quality assessment programmes as well as interlaboratory specimen exchange studies have demonstrated that there is significant variability in quantitative results for assays that lack appropriate standards quantitation of viral target using competitive quantitation standard (qs). the qs compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen. the competitive qs contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons. the competitive qs is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous pcr amplification, and detection. viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the qs signal (dashed line) for each specimen and control (a, b) . in the presence of inhibitors, both qs and viral target are equally suppressed and yield accurate viral load calculations (c). introduction (hayden et al., ; pang et al., ; preiksaitis et al., ; wolff, heaney, neuwald, stelrecht, & press, ). findings such as these reinforce the fact that with this high degree of variability and discrepancy, clinicians are unable to compare test results between two different laboratories and, further, clinically relevant cut-offs set by one test would not apply to results of another . without standardisation, the quality of patient care is dramatically impacted, preventing meaningful inter-laboratory comparison of patient results and influencing disease prevention and management programmes (kraft, armstrong, & caliendo, ) . this is especially critical for transplant patients, who may be initially monitored at one institution and then transferred to another for longer-term follow-up receiving results that no longer correlate. therefore, whenever possible, viral load monitoring tests must report results in iu/ml and be fully traceable to the higherorder first who international standard. they must generate highly accurate and reliable results based on a robust calibration methodology and have excellent reproducibility across the dynamic range of the test with demonstrated co-linearity to the who standard. lastly, there exist two distinct end-points with quantitative real-time pcr, which should be of consideration for result interpretation and reporting: the lower limit of detection (llod/lod) and the lower limit of quantitation (lloq/loq). these two limits are assessed differently and are not equivalent in either definition or, in some cases, their assigned values. the lod (also referred to as analytical sensitivity) represents the lowest viral load level at which ! % of tested samples are detected (clsi ep -a, ); theoretically, viral levels at or below the lod are not detected ! % of the time. it differentiates between 'detectable' and 'undetectable' results. the lloq, on the other hand, is the lowest viral level that is within the linear and analytically acceptable range of the assay (clsi ep -a, ) . in other words, the lloq is the lowest point at which an accurate viral load can be assigned and determines which 'detectable' sample will have a reported viral load. a common misconception is that the lod of the assay is the minimum viral level for a 'detected' result but 'undetectable' and 'detectable' viral levels are never differentiated by a single theoretical viral threshold as viral levels less than the lod may still have a high probability of being detected. this probability spans a broad range in which the lower the viral titre, the more likely the 'undetectable' result. ultimately, the statistical probability will favour the 'undetectable' result ( figure ). and because the lloq can be equal or greater than the lod on some viral load assays, it is not unusual for 'detectable but below the loq' (detectable/bloq) result reporting (cobb et al., ) . further, the 'detectable/bloq' results should not be inferred that the actual viral concentration of the sample is between the lod and loq. the clinical demand has driven and shaped the evolution of pcr and continues to do so as we gain a greater understanding of the infections we monitor and treat. through the study of the natural history and disease progression attributed to specific viral infections, the need for sensitive, accurate, precise, reproducible, and reliable quantitative measurements of viral levels has become a necessity. with the deeper understanding of the natural history of human immunodeficiency virus (hiv) infections, it is now well understood that progressive immunosuppression and the onset and development of clinical disease are strictly associated with increasing viral burden (furtado, kingsley, & wolinsky, ; ho, moudgil, & alam, ; mathez et al., ; nicholson et al., ; schnittman et al., ) . thus, quantitative real-time pcr is critical for monitoring patients infected with hiv (hufert et al., ; mellors et al., ) and those undergoing antiretroviral therapy (art) to ensure viral replication is sufficiently and effectively suppressed and to monitor potential for viral resistance to the medication (dhhs hiv, ) . this monitoring and maintained viral suppression is absolutely necessary not only to maintain progression-free survival of hiv-infected patients but also to reduce subsequent hiv transmission (cohen et al., ; diffenbach, ) . due to the significance of viral load monitoring and maintaining viral suppression, the demand for likelihoods of different test results given different viral concentration. when the viral concentration tends to , the proportion of 'target not detected' increases to (dotted line), increasing the likelihood of 'not detected' results. as the concentration tends to lloq (dashed line), the likelihood of 'detected but <lloq' results peaks. when the concentration tends to infinity, the proportion of quantitative results tends to (solid line), resulting in a continual increase in the likelihood of 'detected: quantitative' results. at any concentration, the sum of the three types of reported results is always % and throughout the concentration continuum, variations in result reporting exist. as the concentration of viral levels approaches the lloq, near equal likelihood of 'detected: quantitative' and 'detected but <lloq' results are possible, as are 'not detected' results. decreasing concentrations will further shift the likelihoods and increase the chance of 'detected but <lloq' or 'not detected' results. diagram assumes lloq ¼ llod. increasingly more sensitive assays for hiv has driven the innovation of diagnostic tests and continues to push the limits of the lod and loq ever lower (glaubitz et al., ; sizmann et al., ) . additionally, the amount of viral dna in samples from either hepatitis b virus (hbv) or cytomegalovirus (cmv) chronic carriers is indicative of active viral replication in liver cells and is correlated with liver disease progression (chen, lin, et al., ; chen, yang, et al., ; emery et al., ; humar et al., ; humar, kumar, boivin, & caliendo, ; iloeje et al., ; wursthorn, manns, & wedemeyer, ) , highlighting the need for quantitative viral levels in chronic disease monitoring. similarly, hepatitis c virus (hcv) levels have been linked to prognosis and treatment outcomes (trepo, ) , treatment response (pearlman & ehleben, ; zeuzem et al., ) , as well as assessing sustained virologic response (svr), or 'cure', following treatment (pearlman & traub, ) . more recently, as new antiviral therapies for hcv treatment rely on targeting specific biological steps of the viral replication cycle, reliable quantitative monitoring of the viral burden at specific time-points to ensure treatment compliance and resistance emergence during treatment is recommended (au & pockros, ; aasld/idsa/ias-usa, ). alongside the changing clinical needs, several instrument and manufacturing innovations have been introduced to meet these newer requirements. the first pcrbased diagnostic test and the first automated system were both introduced in the early s, allowing for improved standardised technique, increased efficiency, and reduction in error and contamination. in , to meet the clinical needs of monitoring patients infected with hiv, the first 'personalised healthcare' diagnostic test was fda approved, that monitored whether art was working and whether a patient was on optimal treatment. entire systems were introduced in the early s that automated the up-front sample preparation step, leading to further reduction of hands-on time, increased reliability and reproducibility. with the introduction of real-time pcr techniques to further enhance assay performance, the first pcr tests that allowed for simultaneous amplification and detection were introduced in . throughout the decade, improvements in assays and instrumentation began pushing the boundary for hiv- detection, achieving greater and greater sensitivity, a feat that has proven to be incredibly relevant in understanding risks of viral load rebound and virologic failure (doyle et al., ; estevez et al., ; pascual-pareja et al., ) . more recently, shortly after the release of two new higher-order standards for cmv (first who international standard and nist cmv standard), fully automated real-time quantitative pcr assays were fda approved to meet laboratory and clinical demands for standardisation. not only does the instrumentation reduce assay design variability and inconsistency across laboratories, but also the assays are standardised to the who and report in international units, providing accuracy across the entire dynamic range that is only achieved by calibration and traceability to these higher-order standards. but it is not until these fda-approved tests gain widespread use will inter-laboratory agreement for cmv viral load results truly improve (kraft et al., ) . the objective of test innovation is to evolve and adapt to clinical and laboratory needs. as patient outcome gaps are identified, and as we gain greater understanding and insight into the clinical progression of disease and disease management, technological achievements facilitate advancements in quality of diagnostics and patient outcomes. life-threatening illnesses convert to chronic/manageable disease with the aid of viral load monitoring. and pressure exists to develop more precise, accurate, sensitive assays, which, in turn, drives the development of more efficacious drugs. this synergy demonstrates the value of diagnostics: it is an integral part of the patient care continuum. applications of quantitative real-time pcr for virology are extensive. it is especially necessary when antibody seroconversion is delayed after an acute infection and early diagnosis are essential (dibiasi & tyler, ; thomson et al., ) , in immunocompromised patients that may not have an optimal antibody response (kadmon et al., ) , and for the diagnosis of congenital or perinatally acquired viral infections (park, streicher, & rothberg, ; young, nelson, & good, ) . additionally, it is critical in maintaining a high level of patient care at each stage of the disease and infection (figure ). screening tests are designed with one key parameter in mind: exceptional sensitivity (herman, gill, eng, & fajardo, ) . they must ensure that disease is not missed in a population that is generally free from risk to allow for appropriate early intervention, thereby effectively reducing mortality and morbidity. although traditional applications of real-time pcr in the continuum of care and patient management. population-based screening programmes and recommendations do not often utilise nucleic acid testing (nat) for virology targets, relying more on immunoassays and antigen testing, nats and real-time pcr assays are still integral components. donor-eligibility determination ensures that a donor is eligible to donate cells or tissues to be used as human cells, tissues, and cellular and tissue-based products (hct/ps), which can include haematopoietic stem/progenitor cell, organ, semen, and other types of donations. in part, living and cadaveric hct/p donor eligibility is granted if screening shows that the donor is free from risk factors for, and clinical evidence of, infection due to relevant communicable disease agents and diseases such as hiv type and , hbv, hcv, and west nile virus (wnv) (fda testing, ) . the fda testing recommendations stipulate that hct/ps tests for hiv and hcv may include fda-licensed nat blood donor screening and that, specifically, an fda-licensed nat should be used to assess infection with wnv. despite the fact that these nat tests provide qualitative as opposed to quantitative results, the molecular technology utilised is often real-time pcr due to its enhanced accurate and reliable resulting (fda assays, ) . additionally, pre-emptive virology screening post-transplant may reduce subsequent complications and provide a more cost-effective management strategy (evers, ) . pre-emptive therapy utilises routine viral screening to initiate therapy at the first indications of viraemia, prior to clinical manifestation of disease. this strategy reduces the overall morbidity and mortality post-transplant compared to strategies in which treatment is initiated at the onset of clinical disease (sch€ onberger et al., ). the pre-emptive treatment model utilising routine screening by quantitative realtime pcr of asymptomatic post-transplant patients has been shown to be especially effective for paediatric patients undergoing haematopoietic stem cell transplant, who are uniquely at a high risk of cmv and epstein-barr virus (ebv) infections (evers, ) . this strategy and prospective screening utilising a quantitative real-time pcr assay for bk polyoma virus (bkv) is recommended as part of routine posttransplant follow-up of all kidney transplants since early identification and management of bkv infection may prevent future incidence of polyoma virus-associated nephropathy (hirsch et al., ; kdigo, ) . nats and real-time pcr are also utilised as vital parts of certain screening algorithms. the center for disease control (cdc) currently recommends that hcv rna testing be utilised for anyone who may have been exposed to hcv within the preceding months. in addition, nats would identify active hcv infection among persons who have tested anti-hcv positive or those with an indeterminate antibody test indicating need for referral for further medical evaluation and care (cdc hcv, ). viral diagnostic tests are used to determine presence or absence of current or previous infection. because many viral infections present with similar symptoms, accurate diagnosis is critical as each requires unique and vastly different interventions and/or management strategies. historically, diagnosis of viral infections has relied on viral growth in cell culture, immunoassays, antigen assays (elisa), haemagglutination testing, and electron microscopy (krishna & cunnion, ) . the introduction of molecular methods including nats and real-time pcr assays has vastly improved viral diagnosis with their superior sensitivity, specificity, and rapid result reporting (emmadi et al., ) . nat-based infectious disease testing, providing rapid results, aids in outbreak detection (ebola), genotype identification (hcv), and identification of possible drug resistance (hiv- ), which can lead to rapid clinical therapeutic decisions and early infection control to prevent spread of disease (espy et al., ) . viral culture was traditionally considered to be the 'gold standard' for viral diagnosis because of increased sensitivity compared to rapid antigen-testing methods. however, a significant limitation of viral culture was a long time to result, reaching days for cmv (gleaves, smith, shuster, & pearson, ) . improvements in culture included the introduction of shell viral culture, which reduced the result turnaround-time (tat) for cmv to h. although considered to be a vast improvement, certain viruses require immediate treatment intervention to prevent life-threatening infection and therefore, a -h tat is simply much too long. further, reliance on viral culture for diagnostics testing introduces other pronounced drawbacks, the most noteworthy being that not all routine viruses grow in culture and that virus viability, and thus its culture ability, could be impacted by sample collection, transport conditions, or prior patient treatment. with these limitations in mind, nat testing has tremendous advantages and has replaced culture for most viruses due to its greatly reduced tat (typically less than h) while still retaining high sensitivity. the applications of nat testing, and more specifically, real-time quantitative pcr technology, in viral diagnostics and confirmatory testing continue to expand. the cdc-updated recommendations for hiv testing state that specimens that are reactive on the initial antigen/antibody combination immunoassay and non-reactive or indeterminate on the hiv- /hiv- antibody differentiation immunoassay should be tested with an fda-approved hiv- nat test (branson, ; branson et al., ) . additionally, nats have demonstrated utility in high-risk populations, in which antibody testing alone might miss a considerable percentage of hiv infections that are otherwise detectable by nat virologic tests (priddy et al. ; stekler et al., ) . particularly, immunoassays for hiv diagnosis are limited by the marked delay between infection and seroconversion, a time when hiv viral levels are at their peak ( figure ). for this reason, nats are the recommended method for diagnosis of hiv during the acute phase of infection ( - days post-infection). hiv viral rna is the first marker to manifest itself approximately days post-infection at initiation of the acute phase of infection (lindback et al., ) . not until - days after initial detection of hiv rna do the hivp antigen levels rise to detectable levels using fourth-generation immunoassays. this marked delay and the need for rapid diagnosis by the identification of hiv rna is especially critical when an early diagnosis is medically warranted. during the acute phase of the hiv infection, patients typically present with flu-like symptoms, which can often lead to a misdiagnosis. misdiagnosing those at the highest risk of infection such as prison inmates, iv drug users, and men who have sex with men (msm), also increases the risk of further disease spreading (chu & selwyn, ) . therefore, the use of a real-time pcr testing method that is able to identify the presence of the hiv virus at this initial stage is critical. additionally, nats are critical for early diagnosis of congenital or perinatally acquired viral infections, especially infants born to hiv-infected mothers (tang & ou, ) . the maternal antibodies against hiv can persist in exposed infants for up to months of age, which, in turn, prevents the use of antibody-based assays for early diagnosis of infection. there is a high level of morbidity and mortality during the first years of life for infected infants; therefore, it is of high importance to determine infection status quickly in the exposed infant to implement the appropriate art therapy early (van rossum, fraaij, & de groot, ) . in addition to hiv, additional viruses have been demonstrated to exhibit perinatal transmission including respiratory virus, cmv, herpes simplex virus (hsv), varicella zoster virus (vzv), hbv, enterovirus, rotavirus, and human papilloma virus (prober et al., ) . for all these, nat testing may serve as a valuable tool for early intervention, especially in this critical neonate population that lacks fully developed immune systems. in addition to their applications for hiv diagnosis, nat testing is also utilised as a diagnostic for hcv. although diagnostic hcv rna real-time pcr tests are predominately used as confirmation of a positive hcv antibody result (cdc hcv, ), nat testing is recommended for the confirmation of a non-reactive hcv antibody test for patients suspected of having recent hcv exposure. hcv rna can be detected as early as weeks post-exposure, whereas hcv antibodies are not detectable until - weeks post-infection (ghany, strader, thomas, & seeff, ). therefore, similar to strategies outlined for hiv, nat testing is the most suitable for detection and diagnosis during the acute phase of hcv infection. further, immunocompromised patients, those with weakened immune systems can include those that are hiv positive, on immunosuppressive drugs, or undergoing chemotherapy, may lack the ability to generate an appropriate immune response required for an anti-hcv test. in these special circumstances, the cdc recommends considering hcv rna testing for diagnosing viral infections. rapid diagnostic testing is also very important for viruses with high potential to create an epidemic or outbreak. throughout history, influenza has caused many such outbreaks, the most notable being the spanish flu thought to be responsible for an estimated million deaths (taubenberger & morens, ) . in response to the possibility of future outbreaks, the world health organization (who) has established that pcr-based influenza testing is now the first-choice diagnostic test for both humans and animals (who influenza, ) . the conversion to molecular tests was based on the fact that these assays are more sensitive and specific for detecting influenza viruses compared to other non-molecular methods. nat methods also have a lower likelihood of false positive or false negative results, and therefore, result interpretation is less impacted by community-based influenza prevalence (cdc influenza, ) . the introduction of rapid molecular testing, which can provide results in as little as min, is extremely beneficial especially in hospitals, nursing homes, and chronic care facilities where early influenza identification can prevent outbreaks. the number of molecular-based diagnostic tests has expanded even further with the introduction of tests for wnv, respiratory syncytial virus, hsv, rotavirus, and even more recently, ebola. as development and improvements continue to be made to real-time pcr technology that will reduce both cost and time to result even further, the number of nat diagnostic tests will continue to increase and the applications for real-time pcr in diagnostics will also expand. once a patient is appropriately diagnosed and linked to care, partly through the aid of quantitative real-time pcr, clinicians will often consider several baseline factors, which collectively help to guide therapeutic decision. these decisions are based on a series of questions including: are treatment options available? what treatment regimen should be prescribed? for how long will the patient need to be on therapy? factors influencing these therapeutic decisions include disease complications, patient predisposition, prior treatment experience, viral genotype, viral resistance profile, and host genetic profile, among others. and depending on the viral infection, a quantitative baseline viral load may also serve an important role in helping to guide treatment decision (table ) . over the past years, pharmaceutical development and clinical trials investigating cutting-edge antiviral treatments have relied heavily on the data generated from pcr-based-and eventually quantitative real-time pcr-based-technology to determine safe and effective drug use (cobb et al., ; mackay, arden, & nitsche, ) . practice guidelines continue to reference registrational and nonregistrational studies utilising quantitative real-time pcr to guide both clinicians and laboratories in the proper implementation of treatment and testing in order to deliver the most effective personalised care to patients (aasld/idsa/ias-usa, kotton et al., ) . because of this extensive co-utilisation of real-time pcr, it is widely accepted as the gold-standard technology for measuring a patient's quantitative viral load before, during, and after the course of treatment. once the decision to initiate treatment for chronic hcv infection is made, several baseline factors are routinely considered (aasld/idsa/ias-usa, ). among these are complications like liver fibrosis stage, co-infection with hiv, hepatocellular carcinoma, end-stage liver disease, genetic variations like hcv genotype, subtype and resistance markers, and prior treatment experience and outcome. the association of baseline viral load, measured by quantitative real-time pcr, to chronic hcv treatment outcome has been well documented with earlier therapies (pegylated-interferon plus ribavirin) but, given the lack of alternative therapeutic options, has not been recommended as a therapeutic decision factor (jensen et al., ; pawlotsky, ) . historically, practice guidelines have recommended the measurement of baseline viral load to serve only as an initial time-point required for effective monitoring of treatment without any prognostic indication (yee, currie, darling, & wright, ) . currently, tremendous advancements in the treatment of chronic hcv infection that employ direct-acting antivirals (daas) reported cure rates (as determined by svr) of > % for even the once most difficult to treat hcv genotype- patients, the most predominant in the united states. because of this high potency of these drugs across patient populations and the greater importance of numerous other factors, including hcv genotype and prior treatment experience, in determining the appropriate course of treatment, the most recent aasld/idsa practice guidelines still do not recommend a baseline quantitative viral load as a therapeutic decision factor. however, in the rapidly evolving field of hcv treatment, the recent fda approval of a fixed-dose combination drug consisting of two daas (sofosbuvir and ledipasvir) for the treatment of hcv genotype- , the manufacturer's drug label now includes a new indication for quantitative real-time pcr. it is indicated that treatment naïve and non-cirrhotic patients with a specific baseline viral load are eligible for shortened therapy, an indication with tremendous implications. according to the prescribing information, patients with a baseline viral load below million iu/ml are eligible to have shorter therapy duration of weeks, much shorter than the -or -week duration for other patient populations (harvoni, ) . this therapeutic decision practice is the first of its kind in treatment of chronic hcv infection and is likely to be a recurring theme as daa manufacturers strive to develop high efficacy regimens requiring shorter treatment durations. additionally, shorter treatment durations are more favourable to patients and payers when considering the cost of achieving svr with daas and may improve patient drug adherence and completion of therapy (hep c online, ) . as much as quantitative real-time pcr helped to develop this claim for this particular regimen, this technology will also be employed by numerous laboratories to aid in this part of therapeutic decision. in contrast to chronic infection, treatment of patients presenting in the acute phase of hcv infection, within the first months after exposure, is not recommended by aasld/idsa for patients in whom hcv infection spontaneously clears (aasld/ idsa/ias-usa, ). therefore, careful monitoring of hcv rna by a sensitive nucleic acid test is required in order to confirm spontaneous clearance, defined as hcv rna negative at two specific measurements. quantitative and qualitative real-time pcr assays are both widely used for this purpose, given their comparable sensitivity. factors influencing art decision for hiv-infected patients include determination of pregnancy, aids-defining conditions, acute opportunistic infections, low cd counts, hiv-associated nephropathy, potential drug interactions, co-infection with hcv or hbv, hiv resistance testing, and prior treatment experience (dhhs hiv, ). plasma hiv rna viral load, performed widely by quantitative real-time pcr, is also recommended as a pre-art decision factor specifically for treatment naïve patients. the department of health and human services (dhhs hiv) recommends that only art-naïve patients with a plasma hiv viral load below , cp/ml can be prescribed various regimen options, which they otherwise should be restricted from taking with higher viral load. this is primarily due to inferior virologic responses in patients with higher viral loads observed in clinical studies (sax et al., ) . these clinical trial studies employed quantitative real-time pcr in order to help determine this cut-off and many labs have utilised the same technology to help guide hiv-treating clinicians in this decision. in the case of chronic hbv infection, several studies have shown that hepatitis b 'e' antigen (hbeag) and high levels of hbv dna are independent risk factors for the subsequent development of cirrhosis and hepatocellular carcinoma (chen, lin, et al., ; chen, yang, et al., ; iloeje et al., ) . however, due to the fluctuating nature of chronic hbv infection, the prognostic utility of one high hbv dna level at a single time-point is limited. thus, hbv baseline dna viral load, along with hbeag, alanine aminotransferase (alt) levels, and fibrosis, collectively aids in the decision to treat with antiviral agents as well as which hbv antiviral regimen to choose and duration of treatment (lok & mcmahon, ). typically, patients with an hbv dna viral load > , iu/ml, signs of liver disease (i.e. high alt levels and/or significant fibrosis), and loss of hbeag are considered for immediate treatment with antivirals, whereas patients < iu/ml are closely monitored for viral load changes prior to treatment. patients who fall in between this range are monitored for persistent viraemia and signs of liver disease before deciding to treat. quantitative real-time pcr, therefore, plays a crucial role in the care of chronic hbv patients who, if not treated at the appropriate time with the appropriate regimen and duration, are at greater risk of liver complications. unlike treatment guidelines for hcv, hiv, and hbv, management of cmv after solid organ transplant is not associated with specific quantitative cmv viral load cutoffs in order to make therapeutic decisions (kotton et al., ) . this is partly due to the historical lack of an international standard and varying assay designs, which has led to poor inter-institutional correlation of quantitative nats. in addition, the widespread practice of universal prophylaxis, where cmv antiviral medication is administered to patients early in the post-transplant period and continued for a finite period of time, has diminished the clinical utility of baseline viral loads for making therapeutic decisions. however, with the recent availability of the who cmv international reference standard, the establishment of viral load cut-offs that can be applied to pre-emptive monitoring of patients prior to treatment initiation may soon become more widely accepted . until then, institutions are required to determine their own test performance characteristics and clinical cut-offs. several studies have shown that a low cmv virologic threshold (e.g. detectable viraemia) using quantitative real-time pcr should be used for starting pre-emptive therapy especially in high-risk cases where the organ donor screens positive and the receptor screens negative for cmv serology (atabani et al., ; couzi et al., ; sun, cacciarelli, wagener, & singh, ) . among a variety of baseline risk factors that may indicate longer cmv treatment duration, significant predictive value has been demonstrated with higher baseline viral loads where longer treatment duration may prevent cmv disease relapse (kotton et al., ; sia et al., ) . clinical trial studies supporting the recent fda approval of a quantitative real-time pcr cmv test calibrated to the who international standard also demonstrated clinical value for baseline testing of patients with cmv disease who are undergoing treatment with the anti-cmv drugs ganciclovir or valganciclovir (razonable et al., ) . data from this study suggested that patients with a baseline cmv viral load < , iu/ml are likely to resolve cmv disease more rapidly than those who have a higher baseline viral load. further studies are needed to determine universal thresholds for pre-emptive therapy initiation and predictive value for cmv baseline viral load in defining optimal treatment duration. there exists a clear application for quantitative real-time pcr technology in baseline determination of patients with significant viral infections, and in fact, quantitative viral load determination plays a critical role in therapeutic decision for many other viral infections. high baseline viral load has been shown to correlate with advanced disease during infection with numerous viruses such as bkv, hsv- , ebv, and adenovirus and may potentiate the need for longer duration therapies in certain scenarios (cincinnati children's hospital medical center, ; domingues, lakeman, mayo, & whitley, ; gustafson et al., ; randhawa et al., ) . after the patient's baseline assessment or pre-emptive monitoring suggests if treatment is available, which treatment regimen to choose and perhaps the duration of therapy, the patient can move on to therapeutic administration. quantitative realtime pcr has helped and continues to set the stage for decisions that potentially saves lives, reduces complications, decreases morbidity, and lessens the economic burden to both the patient and the healthcare system. serial measures of viral load serve as an individualised map of a viral infection through the estimation of the amount of virus found within an infected person. tracking viral load in the continuum of care is a vital tool used predominantly to monitor treatment response and its effectiveness, early signs of resistance emergence during therapy of chronic viral infections, and viral activation or reactivation in immunocompromised patients following bone marrow or solid organ transplantation. while the goal of treatment for chronic hcv infection is svr, patients may fail therapy due to non-response, on-treatment breakthrough, or post-treatment relapse ( figure ). the early change in quantitative viral load over time may be predictive of treatment efficacy and a shorten therapy for patients who respond rapidly to treatment (yee et al., ) . this 'response-guided therapy' (rgt) is best exemplified during treatment of chronic hcv patients. specifically, the sooner a patient becomes hcv rna undetectable during treatment, the lower the relapse rate when treatment is shortened. conversely, the longer it takes for a patient to become hcv rna undetectable, the longer they need to remain on treatment to limit relapse. however, given the poorer efficacy of earlier regimens, not all patients who received therapy achieved svr. for this reason, 'futility rules' or 'stopping rules' were also developed, which required that failure of a patient to respond (target not detected or viral load cutoff ) by a given time-point indicated the need to immediately discontinue therapy. monitoring hcv viral loads during treatment. despite advances in treatment for hcv patients, failure to achieve svr is still a reality. patients who do not achieve svr fall into four categories: ( ) null responders (black line) achieve less than -log decrease in hepatitis c viral load upon treatment; ( ) partial responders (red line; light grey in the print version) experiences at least a -log decrease in hepatitis c viral load during hcv treatment but fail to proceed to an undetectable viral load level; ( ) breakthrough patients (orange line; light grey in the print version) have an undetectable hcv viral load, but the virus rebounded during treatment; ( ) relapsers (blue line; dark grey in the print version) have had an undetectable hcv viral load, but the virus rebounded after they completed hcv treatment. although these rgt notions were originally developed from observations made during treatment with the older therapies, peg-ifn and ribavirin, rgt was also required during treatment with the much more potent first-generation daas, telaprevir and boceprevir, and stopping rules were put in place during treatment with the second-generation daa, simeprevir (aasld/idsa/ias-usa, ; ghany et al., ; yee et al., ) . newer ifn-free daa regimens targeting hcv, which are better tolerated by patients and by virtue of the targets they inhibit, have a higher barrier to resistance, yield more rapidly declining viral kinetics, and, thus, do not contain treatment indications for rgt in their prescribing information (harvoni, ; olysio, ; sovaldi, ; viekira, ) . while rgt was a major driver for regular viral load monitoring during antiviral therapy, it is not the only reason to monitor hcv viral load. in the interval between baseline measurement and assessment of svr, the aasld/idsa guidelines also include recommendations for monitoring initial response (week on treatment with a repeat at week if detectable) and end of treatment in order to provide an assessment of drug compliance/early efficacy and predict treatment outcomes, respectively (aasld/idsa/ias-usa, ). in the most recent revision to these web-based guidelines, it is recommended that an hcv viral load increase of greater than -fold on repeat testing at week (or thereafter) should prompt a discontinuation of hcv treatment. many clinicians also closely monitor and report the declining viral loads to their patients in order to demonstrate treatment efficacy, motivating patients to continue treatment and remain adherent to the drug regimen until the next follow-up appointment (fusfeld et al., ) . regardless of monitoring during hcv treatment for rgt, adherence/compliance, patient motivation, early treatment efficacy, etc., quantitative real-time pcr is widely used by laboratories due to its sensitivity, accuracy, and reproducibility of each consecutive viral load test. for patients infected with chronic viral infections, such as hiv, the lifelong regimen of highly active art aims to suppress hiv viral levels to near undetectable levels, ensuring progression-free survival (delay or all together prevention of the progression to aids) and reducing potential transmission. alongside monitoring immune function and immunologic efficacy through cd t-cell count, hiv viral levels are critical in the clinical evaluation and assessment of hiv-infected patients undergoing art. determining a patient's hiv viral load is indicated prior to entry into care, at the initiation of art, at - weeks after art initiation, and then typically every - months while on treatment: ( ) to establish a baseline level of hiv viral load; ( ) to establish viral response to the therapy to assess the virologic efficacy of art; and ( ) to monitor for abnormalities that may be associated with antiretroviral drugs (dhhs hiv, ) . the baseline hiv viral load is not only linked to treatment options (sax et al., ) but also helps to establish the magnitude of viral load decline after initiation of art and provides prognostic information about the probability of progression to aids or death (marschner et al., ; murray, elashoff, iacono-connors, cvetkovich, & struble, ; thiebaut et al., ) . once treatment is initiated, the goal is to reach and maintain suppressed hiv replication as determined by undetected viral levels utilising highly sensitive nat tests, which is generally achieved within - weeks after art initiation. the need for sensitive assays to effectively assess viral suppression hinges on the need to suppress hiv replication to the extent that viral evolution and drug resistance mutations do not emerge, which typically do not occur in patients whose hiv rna levels are maintained below the llod of current real-time quantitative pcr assays (kieffer et al., ) . due to the introduction of more sensitive real-time pcr assays, which can detect as few as viral copies/ml, natural variability in hiv viral levels over time, even in patients with effective suppression, is much more evident (lima, harrigan, & montaner, ; gatanaga et al., ; willig et al., ) . although controversy exists between the clinical significance of viral loads between llod and < copies/ ml, there are reports suggesting that this low-level viraemia is predictive of virologic rebound (doyle et al., ; eron et al., ; laprise, de pokomandy, baril, dufresne, & trottier, ) , virologic failure (estevez et al., ) , and indication of drug resistance (taiwo et al., ) , signifying the need for highly sensitive assays. viraemic blips, a single detectable hiv viral load (< copies/ml) in an otherwise seemingly suppressed patient (figure ) , however, do not indicate subsequent virologic failure or development of resistance mutations (castro et al., ; lee, kieffer, siliciano, & nettles, ; nettles et al., ) . blips are not unusual (havlir et al., ) and appear to be more common in winter, suggesting that host-related and seasonal factors are associated with the occurrence of viraemia (van sighem et al., ) . on the other hand, persistent hiv rna levels ! copies/ml are often evidence of viral evolution and accumulation of drug resistance on-treatment hiv patient monitoring. (a) hiv viral loads will fluctuate as patients are on treatment, and, in most instances, will remain 'undetectable' (at or below dotted line); viral 'blips' are not uncommon and will result in transient 'detectable' and even quantifiable results (above the dashed line). (b) virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time. mutations (aleman, soderbarg, visco-comandini, sitbon, & sonnerborg, ; karlsson et al., ) . once treatment failure is confirmed, immediate intervention is recommended to avoid progressive accumulation of resistance mutations and effective response of new regimen (dhhs hiv, ), which is benefited by low hiv rna levels and/or higher cd cell counts (eron et al., ) , and even a brief interruption in therapy may lead to a rapid increase in hiv rna and a decrease in cd cell count and increases the risk of clinical progression (deeks et al., ; lawrence et al., ) . with the development and administration of newer drugs that target specific biological processes of hiv, routine and clinical monitoring of viral loads using a real-time quantitative pcr assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success. viral load monitoring is also essential when the recipient of a solid organ transplant is cmv seropositive and the decision is made to initiate treatment only once the cmv levels predictive of disease are reached. this strategy, known as pre-emptive therapy, utilises intensive monitoring for cmv activity by sensitive real-time quantitative pcr methods and short-term antiviral treatment is given only to those with significant viral counts before symptoms occur. cmv is one of the most common opportunistic pathogens that infect solid organ transplant recipients (fishman, ) and is associated with increased morbidity and mortality (sagedal et al., ; schnitzler et al., ) . following primary infection, the virus establishes a lifelong latent infection in several sites of the body and may reactivate in the presence of immunosuppression, such as in transplant recipients. once reactivated, cmv is able to modulate the immune system and is known to be a potent upregulator of alloantigens (razonable, ) , increasing the risk of chronic allograft dysfunction (reischig, ; sagedal et al., ; smith et al., ) and acute rejection (sagedal et al., ) . pre-emptive therapy reduces the incidence of cmv disease (khoury et al., ; reischig et al., ) , which has been documented as a serious problem in randomised trials upon completion of universal antiviral prophylaxis therapy (kalil, levitsky, lyden, stoner, & freifeld, ; lowance et al., ; paya et al., ) . long-term studies have demonstrated that patients receiving preemptive therapy, when compared to prophylaxis therapy, were less likely to develop moderate-to-severe kidney scaring and atrophy and significantly better survival of the transplanted organ (reischig et al., ) . however, challenges still exist around defining appropriate thresholds to initiate pre-emptive therapy (humar & snydman, ) . but with new standardised real-time pcr assays, widespread adoption, and utilisation of these tests, pre-emptive therapy relying in intensive viral load monitoring may become the standard for certain at-risk patients. test of cure, or end of treatment response, is assessed following a given therapeutic regimen for signs of treatment efficacy. in few cases, a quantitative viral load measurement serves as a way to establish a cure rate, but, in others, may only be used as a confirmation of virologic suppression as clinical cure may not yet be possible with current therapies or technical limitations by real-time pcr that limits the overall sensitivity of viral detection. regardless of the clinical utility for measuring a virologic suppression, quantitative real-time pcrs with their current limits of detection and limits of quantitation are valuable tools in measuring low-level viraemia and establishing undetectable viral loads. utilisation of quantitative real-time pcr to assess virologic cure is perhaps best exemplified by treatment of patients with chronic hcv. according to the aasld/ idsa guidelines, patients who have 'undetectable' hcv rna in the serum, when assessed by a sensitive pcr assay, or more weeks after completing treatment, are deemed to have achieved a sustained virologic response . achieving an svr is considered a virologic cure of hcv infection since, in these patients, hepatitis c-related liver injury stops and recurrence of infection is marginal, detected in < % of patients after years post-treatment (aasld/idsa/ias-usa, ; manns et al., ) . in agreement with these guidelines, the fda recommendation to pharmaceutical daa manufacturers also stipulates that viral rna clearance at svr- be measured in clinical trials using an fda-approved sensitive and specific quantitative hcv rna assay (fda hcv, ) . according to prescribing information accompanying the current daas, the threshold of svr- is defined as a quantitative threshold of hcv rna < iu/ml at weeks after the end of treatment (feld et al., ; kowdley et al., ; lawitz et al., ) . this is somewhat dissimilar to the aasld/idsa guidelines as 'undetected' viral levels are not equivalent to 'detected but below the limit of quantitation' (figure ). but, with the benefit of high sensitivity and reproducibility, quantitative real-time pcr has a clear established role in assessment of hcv virologic cure in both clinical trials and clinical practice and is able to meet the needs for assessing svr. quantitative real-time pcr may also play a critical role in the assessment of cmv disease resolution. the consensus guidelines recommend that two consecutive negative samples be obtained with a minimum treatment course of weeks before treatment is discontinued, which is thought to minimise the risk for development of resistance and disease recurrence (asberg et al., ; chou, ; sia et al., ) . still, some transplant centres may extend treatment (secondary prophylaxis) in patients with compartmentalised disease for as long as necessary to reduce the likelihood of recurrent cmv infection (kotton et al., ) . resolving cmv disease has the long-term benefits of reducing mortality, potential allograft rejection, and the risk of bacterial, fungal, or viral opportunistic infections, among many other transplantand non-transplant-specific effects (arthurs et al., ; fishman, ) . although there is currently no cure for hiv infection, highly sensitive quantitative and qualitative real-time pcr tests targeting total hiv dna and rna have been used in clinical studies for both sterilisation (elimination of hiv-infected cells) and functional (controlled hiv in the absence of art) cures (kibirige, ; lewin & rouzioux, ) . improvements in real-time pcr technology may lead to profound increases in assay sensitivity and the ability to achieve single-copy detection ( cp/ml) may lead us to a better understanding of hiv virology and what may be needed therapeutically to achieve a cure (alidjinou, bocket, & hober, ) . if therapeutic strategies are one day able to achieve an hiv cure, these highly sensitive tests will no doubt play a key role in the continuum of care for patients and, most importantly, in the confirmation of cure. clinical laboratories have undergone changes to become more efficient and flexible while delivering the same high-quality results. when choosing to implement new testing, even beyond viral targets, laboratories have to consider first and foremost the performance and medical value of the test and then factors such as tat, ease of use, and cost. real-time pcr with its wide dynamic range, high specificity, and high sensitivity is considered the gold standard for the quantification and identification of a variety of targets including bacteria, fungi, viruses, or oncological mutations (klein, ) . furthermore, the multiplexing capability of real-time pcr increases the number of targets and information gathered from the same test, further improving laboratory workflow, tat, and costs (deshpande & white, ) . while novel technologies have entered clinical laboratories including mass spectrometry and next-generation sequencing, real-time pcr remains a staple and an attractive option for clinical laboratories aiming to create molecular laboratory-developed tests (ldts). in addition, pcr can quickly be adapted to provide a robust test for the identification of emerging disease and molecular testing is now able to reach beyond the clinical laboratory and further enhance healthcare (farrar & wittwer, ; foudeh, didar, veres, & tabrizian, ) . most molecular tests used in clinical laboratories are fda-approved and commercially available. there are instances, however, when a test may not be available for a specific virus or the sample type and/or clinical indication used by the laboratory differs from those of the fda-approved assay, typically leading a laboratory to design its own pcr-based test or modify existing assays. fda defines an ldt as 'a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory' and recognises that 'ldts are important to the continued development of personalised medicine' (fda ldt, ) . laboratory developed tests can be grouped into three categories, fda-cleared or approved test that have been modified, tests that are not subject to fda clearance or approval, and tests for which no performance specifications have been provided by the manufacturer (e.g. analytespecific reagents or asrs) (burd, ; code of federal regulations, ). with alternative sample types or applications, fda-approved tests are often modified to fit the testing needs of laboratories, including alternative collection media and sample types or expanded clinical applications. as an example, a recent gap was created in the hcv-screening algorithm for the confirmation of a positive enzyme immunoassay result following the discontinuation of the only fdaapproved confirmatory test (alter, kuhnert, & finelli, ) . in response, the cdc published recommendation for the use of fda-approved tests detecting hcv viraemia (cdc hcv, ), despite the fact that most of these assays did not have specific claims for confirmatory testing; as a result, several laboratories chose to validate these assays as ldts to meet the screening needs for hcv. additionally, ldts are the only option for the identification of the aetiologic agents of viral infections that can occur in transplant patients, such as ebv, adenovirus, vzv, and bk virus, that often present with non-specific clinical manifestations (razonable, ) and for which fda-approved assay options are lacking. ldts are an integral part of molecular laboratory testing. whether created from the ground-up or modified from fda-approved assays, ldts are answering the clinicians' needs for information as an aid for diagnosis or treatment of patients. as with any clinical tests, ldts have to meet the minimum standards set forth by clia prior to report patient results (code of federal regulations, ). in july , fda informed congress of the agency's ldt regulatory oversight framework (fda ldt, ) . fda aims to address concerns over high-risk ldts with inadequately supported claims, lack of appropriate controls, and falsification of data that may lead to inadequate treatment, possible harm to patients, and unnecessary healthcare cost. presently, there is still a high degree of uncertainty as to what the final regulation scope will be and the possible impact on molecular laboratories will have to be seen. palaeopathology confirmed the truism that humanity, since its inception, has been exposed to genetic and infectious diseases with early documentation of trachoma ( b.c.e.), tuberculosis ( b.c.e.), and pneumonia (ca. b.c.e.) (aufderheide & rodreguez-martin, ; hershkovitz et al., ; roberts & manchester, ; webb, ) . even today, emerging infectious diseases (eids) continue to appear unpredictably driven by changes in human demographic, land use, and population behaviour (lederberg, hamburg, & smolinski, ; sehgal, ; taylor, latham, & woolhouse, ) . these infections can be classified as either newly emerging/a previously unknown disease or re-emerging infectious diseases/a previously known disease, that reappears after a significant reduction in incidence or elimination (morens & fauci, ) . eids are a threat not only to human health but also to global stability and economy. efforts to monitor these eids are in place both at the global level spearheaded by the who and at the national level. in the united states, governmental agencies (department of health and human services, united states agency for international development, department of defense) are supporting activities to detect, assess, and respond to potential outbreaks. specifically, pcr and real-time pcr are easily adaptable to detect nucleic acid targets that are unique to each given pathogen, and as such, they play essential roles in the identification and detection of infectious pathogens and have been routinely used by health organisation agencies during epidemic outbreaks such as severe acute respiratory syndrome (sars), h n , h n , and ebola (shuaib et al., ; who influenza, ) . the sars epidemic appeared in november , in the chinese province of guangdong before reaching the adjacent hong kong in (who sars, . this sars eventually spread to countries and resulted in more than cases. in response, the cdc triggered its emergency operations centre and issued a draft genome in april , days after the initial who global alert (cdc sars, ) . soon after, real-time quantitative pcr assays were described and put in use for the diagnosis of sars (drosten et al., ; peiris et al., ) . a host of measures were taken in order to contain this epidemic, and the molecular identification and diagnosis of the infectious agent by pcr played a key role in providing critical information to address the situation and contributed to the care of the patients infected. additionally, the re-emerging ebola epidemic (cdc ebola, ; who ebola, ) started in guinea in march of before spreading to nearby west african countries and eventually reaching the united states and europe (who ebola, ) . at the height of the epidemic, fda issued an emergency use authorisation (eua) for the use of the first real-time rt-pcr assay (fda eua, ) and less than months later, five additional realtime pcr tests were authorised under an eua (fda eua, ) to provide an early diagnosis of the ebola viral disease (cdc ebola, ). eids remain a constant and unpredictable threat to human health. the flexibility of real-time pcr technology continues to show how promptly it can be used for the detection of infectious agents. by providing a rapid diagnostic, real-time pcr can help in starting the appropriate treatment right away and maximise the chances of a positive outcome. the goal of point-of-care testing (poct) is to quickly obtain a test result that will be used to implement the appropriate treatment for an improved clinical outcome. by definition, poct is laboratory testing that takes place at or near the site of patients (cap poc, ) . the advantages of poct are an improved tat and result availability regardless of normal core laboratory hours, access to care in remote areas, and greater patient involvement. the fight against aids largely contributed to the development of poct devices with viral load capabilities (hong, studer, hang, anderson, & quake, ; lee et al., ; marcus, anderson, & quake, ; tanriverdi, chen, & chen, ; unitaid, ; vulto et al., ) . originally developed to meet the difficult conditions associated with remote places, far from any core laboratory facility often found in the developing world, the design and convenience of a portable poct device with fast turnaround and accurate results extends the reach of healthcare. with this in mind, these poct systems could easily be used in developed nations at hospitals, within clinics a physicians' offices, pharmacies, correction facilities, or mobile health units, to target pathogens that benefit from immediate actionable results, for which not only accurate but also quick results are critical (kiechle & holland, ). ultimately, test menu available on these platforms will drive its implementation as a complement for the clinical laboratory core testing. the ideal molecular poct system that includes medical value, simplicity, fast tat, and ruggedness remains an ongoing engineering challenge. however, the latest advances in microfluidics are a great example of the potential of these devices and brings the real-time pcr lab-on-a-chip closer to mainstream diagnostic use. this is an exciting time for molecular poct and the upcoming years should bring new systems and perhaps a paradigm change in the world of healthcare. as the needs of the clinicians, laboratory, and patients continue to evolve, so do the applications of molecular diagnostics and pcr. over the past decade, quantitative real-time pcr technology has been increasingly phased into clinical practice and all of the potential present-day applications of real-time pcr-based methods are enumerable. they serve to advance experimental approaches within biological fields, pushing the boundaries of what we know and what we can learn, as well as to diminish empiric medical identification and management of viral diseases. the high sensitivity of the technology has reduced risks of the most commonly transmitted transfusion illnesses and has become an integral part of managing a variety of viral infections by providing pretreatment prognostic information, therapeutic effectiveness through monitoring, and end of treatment response assessment. quantitative real-time pcr complements serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses and are able to predict therapeutic failures sooner than traditional methods, allowing for a more timely management response. real-time pcr assays can be rapidly developed in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. the next few years are likely to see an even further increase in the expansion of the clinical applications of nucleic acid quantification, particularly following bone marrow and solid organ transplantation for which the newest standardised assays may provide an avenue for the development of consensus management guidelines for initiating pre-emptive anti-cmv treatment. further, with the drive towards hiv eradication and complete elimination of the virus from within cells of infected patients, innovations in quantitative real-time pcr assay design will continue to push the boundaries of detection and 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syndrome cost ramifications of increased reporting of detectable plasma hiv- rna levels by the roche cobas ampliprep/cobas taqman hiv- version . viral load test multi-site pcr-based cmv viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology natural history: the importance of viral load, liver damage and hcc management and treatment of hepatitis c viral infection: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program office discordant human immunodeficiency virus infection in dizygotic twins detected by polymerase chain reaction. the pediatric infectious competitive pcr for precise nucleic acid quantification expert opinion on the treatment of patients with chronic hepatitis c key: cord- - ki pd authors: reis, veronica massena; teixeira, kátia regina dos santos title: nitrogen fixing bacteria in the family acetobacteraceae and their role in agriculture date: - - journal: j basic microbiol doi: . /jobm. sha: doc_id: cord_uid: ki pd for centuries, the acetobacteraceae is known as a family that harbors many species of organisms of biotechnological importance for industry. nonetheless, since representatives of this family have also been described as nitrogen fixing bacteria able to plant growth promotion by a variety of mechanisms. nitrogen fixation is a biological process that guarantees that the atmospheric n( ) is incorporated into organic matter by several bacterial groups. most representatives of this group, also known as diazotrophic, are generally associated with soil rhizosphere of many plants and also establishing a more specific association living inside roots, leaves, and others plants tissues as endophyte. their roles as plant growth‐promoting microorganisms are generally related to increase in plant biomass, phosphate and other mineral solubilization, and plant pathogen control. here, we report many of these plant growth‐promoting processes related to nitrogen fixing species already described in acetobacteraceae family, especially gluconacetobacter diazotrophicus and their importance to agriculture. in addition, a brief review of the state of art of the phylogenetics, main physiological and biochemical characteristics, molecular and functional genomic data of this group of acetobacteraceae is presented. bacteria belonging to the alphaproteobacteria, order rhodospirillales, are known for their agricultural applicability. this order is represented by two bacterial families: rhodospirillaceae and acetobacteraceae. in general, the etymology of members of the acetobacteraceae family derives from the latin acetum or acidum gluconicum þ bacter due to their peculiar main characteristic to produce organic acids during many biotechnological processes, such as vinegar and wine productions. bacteria of the genus acetobacter are known since with the description of a. aceti [ , ] . however, only at , it means years after the first species description in this family, is that a diazotrophic species was described in this family [ ] . at that time, this discovery raised the possibility that bacteria of many other species could also present nitrogen fixing and plant growth promotion properties for agricultural purpose, similar to the rhizobia inoculant for soybeans. this document brings the trajectory of accumulated knowledge about the species gluconacetobacter diazotrophicus and other diazotrophs genera and species that were described later in this family. the acetobacteraceae family bacteria belonging to the acetobacteraceae family (ex henrici, ) [ ] are classified as rods (coccus or ellipsoidal), gram-negative, mobile, aerobic that conduct an incomplete oxidation of sugars and alcohols to produce organic acids as final product of their metabolism. they have the ability to grow in very acidic environments with ph close to . - . , but the optimum range is . - . [ ] . they are commonly peculiarities of some acetobacteraceae genera some peculiarities that deviate from the classical description of aab can be highlighted on the genera and their species described over these last years. the genus rhodopila was proposed by imhoff et al. [ ] to accommodate a group of purple non-sulfur bacteria that presents vesicular intracytoplasmic membranes, similar to rhodobacter species, but grows at low ph. the genus acidiphilium was described after isolation and characterization of heterotrophic, mesophilic bacteria with requirements for high acidity and unable to use elemental sulfur or ferrous [ ] . later on, isolates from acidic hot springs and mine drainage were characterized on the basis of molecular and phenotypic traits, especially related to ion-chelated chlorophyll a type, in the genus acidisphaera, which differs from other aerobic bacteriochlorophyll-containing (abc) bacteria by producing zinc-chelated bacteriochlorophyll a (zn-bchl) [ ] [ ] [ ] . in addition to them, other genera known to present bacteriochlorophyll a type were also phylogenetically clustered into the family acetobacteraceae, and they are as follows: roseomonas [ , ] , roseococcus [ ] , craurococcus, paracraurococcus [ ] , rubritepida [ ] , humitalea [ ] , and rhodovastum (proposed but not yet recognized genus) [ ] . the genus acidocella was proposed to accommodate two previously described acidiphilum species that do not present bchl a and that have been classified as monophyletic unit apart from other acidiphilium species according to s rdna sequence [ , ] . the genus stella is known as a polyprosthecate bacteria characterized by having numerous appendage (from the greek prostheca) [ ] . although it has being classified in this family, further studies based on polar lipids and s rrna genes of the two type strains species indicated its close relationship to members of rhodospirillaceae [ ] . the characterization of a group of acidophilic methanol-utilizing bacteria leads to reclassification of acetobacter methanolicus into a new genus, acidomonas [ ] . afterwards, an emendation of the genus description stated that acidomonas presents acid tolerance, instead of acidophily [ ] . representatives of the genus asaia do not grow in presence of methanol, are characterized by poor or nonexistent production of acetic acid from ethanol, and by the absence of growth in presence of . % acetic acid (w/v) [ ] . the genera asaia, kozakia, swaminathania, and neoasaia are related phylogenetically to each other; however, tsp i and mboii restriction of the s- s rdna its can differentiate asaia species from kozakia and neosaia-type strains [ ] . the genera muricoccus and theichococcus were proposed to accommodate isolates that do not produce bacteriochlorophyll a under aerobic conditions and were obtained from building material of a children's day care center, specifically from gypsum liner walls of a children's sleeping room [ ] . however, sanchez-porro et al. had suggest that t. ludipueritiae and m. roseus, the single species of each genera, should be placed within the genus roseomonas based on pigment color, carbon metabolism, and fatty acids profiles [ ] . the genus saccharibacter corresponds to isolates able to grow in the presence of high concentrations of glucose ( - % w/v-optimum at % w/v) but that present negligible or very weak productivity of acetic acid from ethanol. the species type strain s. floricola was isolated from pollen collected in kanagawa, japan [ ] . the genus acidisoma comprises two species, a. tundrae and a. sibiricum, that were isolated from acidic sphagnumdominated tundra and siberian wetlands in russia [ ] . as general characteristic, they are chemoorganotrophic strict aerobes, psychrotolerant, do not possess bacteriochlorophyll a, produce poly-b-hydroxy-butyrate, and are oxidase-and catalase-positive. representative of the genus nguyenibacter, n. vanlangensis, was isolated from rhizosphere of rice plants in vietnam [ ] . the main characteristics of this genus are oxidation of acetate to carbon dioxide and water but not lactate, no production of acetic acid from ethanol; growth is weakly positive either on % d-glucose (w/v) or in the presence of . % acetic acid (w/v). as isolation procedure, the authors used lgi n-free-medium prior to cultivation onto another medium containing glucose and ethanol as carbon source and ph . but nitrogen fixation was not assessed. two genera were proposed during classification of isolates obtained from a flower sample from thailand, the genus neokomagatea that comprises n. thailandica and n. tanensis [ ] and the genus swingsia that comprises the species s. samuiensis, but is not formally recognized [ ] . endobacter medicaginis, the first acetobacteraceae found as legume nodules endophytes, was isolated from surface sterilized nodules of medicago sativa grown in an acidic soil at the province of zamora, spain [ ] . several phenotypic and genotypic studies served as the basis for reclassification of several species previously described in the gluconacetobacter genus at the generic level, including nitrogen fixing species [ ] [ ] [ ] [ ] [ ] . the genus komagataeibacter were proposed during separation of gluconacetobacter xylinum group from the gluconacetobacter liquefaciens group. to date, among all acetobacteraceae genera only some representatives of the genera gluconacetobacter, acetobacter, komagataeibacter, swaminathania, asaia, and acetobacter are reported as nitrogen fixing bacteria and the strategies used in order to obtain these new species are described in table [ , , [ ] [ ] [ ] [ ] [ ] ] . the gluconacetobacter genus until late s, there was not any report of aab capable to fix nitrogen but cavalcante and d€ obereiner [ ] isolated on n-free semi-solid media and described the first n fixing aab. they succeed to isolate from sugarcane plants a group of acid-tolerant bacteria able to fix nitrogen even at ph below . using a minimal medium based on lg medium [ ] , named lgi-p medium, that presents % of raw sugar as carbon source and ph around . . afterwards, during phylogenetic study of representatives of the genus acetobacter, a new genus was proposed by yamada et al. [ ] leading to the elevation of the subgenus gluconoacetobacter to the generic level. two species previously classified as acetobacter were then renamed into this new genus, g. liquefaciens and g. xylinus, based on s rna sequence, predominance of q- quinone type, flagella, pigment production, cellulose production, and fatty acid profile. later on, during the validation, the name has been corrected to gluconacetobacter in accordance with rule of the bacteriological code [ ] . since then, a plethora of species isolated from various environments were described into this genus. nonetheless, after detailed phylogenetic analysis some of them were reclassified into another genus [ , , , [ ] [ ] [ ] [ ] [ ] . after , new nitrogen fixing species were described in the genus gluconacetobacter, g. johannae, and g. azotocaptans [ ] . g. swingsii and g. rhaeticus are cellulose-producing acetic acid bacteria isolated from apple juice of fruits cropped in the south tyrol region of italy [ ] . g. sacchari was isolated from sugarcane and insects [ ] . g. saccharivorans and g. nataicola were proposed based on a reclassification study of gluconacetobacter hansenii strains [ ] . g. tumulicola and g. asukensis were isolated from biofilms growing on the surface of the plaster walls of the mural paintings of the kitora tumulus in japan [ ] . g. tumulisoli, g. takamatsuzukensis, and g. aggeris were isolated from the burial mound soil collected at takamatsuzuka tumulus in asuka village, nara prefecture, japan [ ] . as previously occurred to g. kombuchae, the species g. kakiaceti [ ] , g. medellinensis [ ] , and g. maltaceti [ ] were reclassified into the genus komagataeibacter [ ] based on previous phylogenetic studies using s rrna sequences [ , ] . the reasons that corroborated the existence of two phylogenetic groups in the genus gluconacetobacter was discussed by yamada and yukphan [ ] . according to previous observations, the group included g. liquefaciens, g. diazotrophicus, g. sacchari, g. johannae, and g. azotocaptans, while group included g. xylinus g. hansenii, g. europaeus, g. entanii, g. oboediens, g. intermedius, g swingisii, g rhaeticus, g. saccharivorans, and g. nataicola. group species differentiate from group by many physiological and morphological traits, such as flagella and motility, water-soluble brown pigment production, production of g-pyrone compounds, and , di-ketogluconic. species clustered into these groups also showed other common features such as biotechnological applications and habitats. ecologically, the species of group were found associated with plants, fruits, or flowers, noteworthy group were generally isolated from fermentation process and processed food, such as vinegar, kombucha tea, and nata de coco [ ] . nitrogen fixation agents of biocontrol and associated to several plant growth promotion were related to species of group , while most species in group were related with industrial applications. cleenwerck et al. [ ] also contributed with genotypic data to reinforce the separation of gluconacetobacter species at generic level. recently, g. xylinum group (group ) were separated from the g. liquefaciens group (group ) leading to reclassification of several species in the genus komagataeibacter, including g. kombuchae, a nitrogen fixing species later considered heterotypic synonym of previously named g. hansenii [ , ] . in the present scenario, the ability to fix nitrogen has being observed in the following gluconacetobacter species: g. diazotropicus [ , ] , g. johannae, and g. azotocaptans [ ] . the species gluconacetobacter diazotrophicus (former acetobacter diazotrophicus) was isolated from roots, stems, and leaves of sugarcane not only in brazil but also in argentina, uruguay, mexico, cuba, united states, india, canada, egypt, beside others [ , [ ] [ ] [ ] and then from other agricultural crops such as sugar beet, rice, pineapple, coffee, carrot, and many others [ ] [ ] [ ] . it was also isolated from bugs, such as mealybugs commonly found associated to sugarcane crops [ , ] . this species has been considered as one of the most important diazotrophic bacteria found in high numbers ( - cfu g À plant fresh tissues) and colonizing the inner parts of roots, stems, and leaves of sugarcane [ ] . it is a nitrogen-fixing bacterium originally classified as acetobacter diazotrophicus but later renamed to the genus gluconacetobacter based on the s rdna sequence and the predominant type of ubiquinone [ , ] . physiological characteristics. it is gram-negative, aerobic but fixes nitrogen in microaerobic conditions. it does not use tricarboxylic acids to grow and is adapted to conditions of high osmolarity and sucrose content ( - %). in addition, its cultivation in presence of high sugar content revealed that nitrogenase activity is only partially inhibited by the addition of ammonium to the culture medium [ ] . it shows high acidity tolerance, fixes nitrogen in presence of nitrate concentrations greater than mm, and reduces the deleterious effect of oxygen concentration to the nitrogenase activity using oxidative metabolism in the periplasmic space at membrane level [ , ] . genetic traits. studies of genetic characterization in g. diazotrophicus started in the early s. the first report of the chromosomal localization of nitrogen fixation genes and presence of plasmids in g. diazotrophicus strains was presented during the th international nitrogen fixation with non-legumes in egypt [ ] . later on, the nif genes, transcription regulatory genes (nifa and ntrbc) and others related to ammonium sensing and transport (glnb, glnd, and amtb) were sequenced and expression of some of them were studied using transcriptional gusa fusion [ ] [ ] [ ] [ ] [ ] . the nif-fix gene organization found in g. diazotrophicus was similar to that of azospirillum brasilense, but their products were homologous to those observed to representatives of rhizobiaceae family and r. capsulatus, considering the genbank data available at that time [ , ] . in addition, accessory genes related with nitrogen sensing and metabolism were also studied by sequencing and insertion mutagenesis. at least three copies of genes coding pii-like protein were identified. the copy located upstream to glna (that codes for glutamine synthetase) was named glnb, because of its homology and conserved organization on many others nitrogen-fixing bacteria. the others pii-like coding genes, glnk and glnk , were located upstream of copies of genes that codes for ammonium or methylammonium transportes, amtb and amtb , respectively [ ] . further characterization of single and double mutants containing gus-fusion suggested that glnb and glnk are required as positive signals to efficiently relieve repression by glnk . in addition, the authors showed that glnk protein clearly has a function different from those of glnb and glnk , since nif gene expression were repressed in glnb glnk double mutant under all conditions tested. based on these studies, the authors suggested that none of the three pii homologs is required for nif gene expression indicating novel regulatory features of g. diazotrophicus pii proteins. the glnk protein acts primarily as an inhibitor of nif gene expression while glnb and glnk control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by glnk [ ] . some years ago a lab consortium, named riogene, announced that the genome of this microorganism was sequenced [ ] . its genome is composed of a . mb chromosome and plasmids of . and . kb, respectively. the genome size is in the average of others aab genome already published [ ] [ ] [ ] [ ] [ ] [ ] [ ] . further studies of this microorganism functional genomics have been underway by several researchers group and are leading to a comprehension of the role of its gene contents to its general metabolism, nitrogen fixation, and others plant growth mechanisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, several mutants have already been obtained and used for functional genomic characterization [ , [ ] [ ] [ ] [ ] [ ] [ ] ] . the expression of genes related to reactive oxygen species (ros) detoxification (sod, kat, and gor) was evaluated in g. diazotrophicus grown under nitrogen non-fixing (nfix) and fixing conditions to elucidate the paradox of oxygen consumption during respiration protection and ros inhibitory effect on nitrogenase activity and nif gene expression [ ] . they observed that growth of this microorganism under nitrogen fixing condition leads to reduction of ros accumulation and a strong induction of soda, kate, kat, katc, and gora genes in comparison to cells grown under non-fixing condition. in addition, soda and kate gene expression correlates with nifd suggesting that reduction of ros is essential for the homeostasis during respiratory protection and nitrogen fixation in g. diazotrophicus. later on, the participation of g. diazotrophicus detoxifying genes during the first step of root colonization was investigated using rice plants [ ] . it was shown that ros accumulates during the first steps of inoculation of g. diazotrophicus wild type and mutant strains as response of a plant defense mechanism. noteworthy, they found out that gr (glutathione reductase) and sod (superoxide dismutase) mutants of g. diazotrophicus could not reduce efficiently the ros accumulated in the period of - h after inoculation. in addition, they measured pr genes expression and detected that expression of ja/et pathway gene increased during wild type-plant interaction, but not to both mutants. these data suggest that g. diazotrophicus alters its redox metabolism during bnf by increasing antioxidant transcript levels to circumvent ros inhibitory effect to nitrogenase activity. probably this transcriptional regulatory mechanism protects nitrogenase activity during initial stages of plant colonization. genes homologous to those of alternative asparagine biosynthesis pathway and its role during nitrogen fixation were investigated since free asparagine, as well as others amino acids, inhibits nitrogenase activity [ , ] . further, genome analyses revealed that genes of asparaginyl-trna and asparagine synthetase orthologs are absent in the g. diazotrophicus genome. however, the correlation between repression of nifd expression and increase of asparagine level indicated that genes for an alternative route that converts asp-trna asn into asn-trna asn by a glutamine-dependent asp-trna asn amidotransferase b, encoded by the operon gatcab, might be present. in fact, in g diazotrophicus the presence of gatcab operon indicates that the orf gdi encodes an aspartyl-trna synthetase of nd-asprs type [ ] . the role of asparagine and glutamine as n storage molecule in plant tissues, including sugarcane, is a general rule; however, their role in plant bacteria interaction is scarce and requires more investigation. the content of amino acids in sugarcane sap, apoplast, and symplast has been evaluated and shows that asn is one of the most abundant [ , ] . it is known that asn and gln are amino acids that repress nitrogen fixation in vitro. noteworthy, the presence of orfs coding for putative lasparaginase precursor (gdi ), l-asparaginase ii protein (gdi ), and orfs coding putative (aspartate) aminotransferase in g. diazotrophicus genome may represent an adaptive advantage to its endophytic behavior. nonetheless, a detailed study is necessary to justify this assumption. kerby and roberts [ ] identified in g. diazotrophicus genome the presence of two orfs coding predicted proteins similar to r. rubrum cown (co weal-nitrogenase) and transcriptional regulator cooa, which belongs to the crp/fnr family. the orfs gdi_ and gdi_ , previously annotated as hypothetical protein [ ] , share common features of organization and motifs to cown and cooa, respectively. interestingly, the characterization of these genes in r. rubrum using psi-blast searches revealed that they are widespread and generally presents similar organization in nitrogenfixing bacteria. the importance of cooa and cown for mo-nitrogenase-dependent functioning in the presence of co was shown to r. rubrum and r. capsulatus, but not to the fe-dependent nitrogenase of the latter [ , ] . the authors suggested that cooa and cown may act as a two component system that senses and modulates the modependent nitrogenase activity by protecting it in the presence of co in r. capsulatus and also in many others nitrogen fixing organisms. however, how this protection mechanism works and its relevance to nitrogen fixation is not yet understood. here, we presented some examples of the functional genomics studies that have already been published about g. diazotrophicus, but many others have already been thoroughly reviewed or are underway [ , ] . colonization. it is considered an endophytic bacterium because it has low rate of survival in the soil and was found colonizing the intercellular space of plant tissues of sugarcane [ , , , ] . this bacterium is located in different parts of the plant as described by reis et al. [ ] and james et al. [ ] . they demonstrated during "in vitro" inoculation studies under controlled conditions, that g. diazotrophicus enters into sugarcane micropropagated plants through the tissue of secondary roots then the bacteria penetrated inner tissues and colonize the intercellular spaces (apoplast). other possible points of infection are wounds and the stomata of sugarcane plants [ ] . it also colonizes tip of roots and root hairs of other plants such as wheat, sorghum, and rice as showed using reporter genes [ , ] . at field conditions, the main route of transmission is by vegetative multiplication of stem pieces of sugarcane, although the trash should also serve as an alternative inoculum source when incorporated into the soil [ ] . another possibility to introduce this bacterium in plants appears to be related to the phloem sap sucking by the insects (mealybugs) presenting this species in the lymph and living within sugarcane leaves sheath pocket [ ] . caballero-mellado and mart ınez-romero [ ] hypothesized that these insects and also micorrhyzal spores could be responsible for local dispersion of this species within short distance while sugarcane setts and bud chips used to propagate sugarcane could carry the bacteria to further distant geographic regions. no further evidence that these insects are responsible for the dispersal of g. diazotrophicus species is reported, although it is plausible that this occurs. unfortunately, ecological studies are underemphasized nowadays and this data is not available to a great number of newer described species. oliveira et al. [ ] observed colonization of micropropagated sugarcane using this species in combination with four other strains of diazotrophs. promising results were obtained when micropropagated sugarcane plants were inoculated with the type strain pal in combination with small doses of nitrogen as shown by moraes and tauk-tornisielo [ ] . oliveira et al. [ ] showed that the combined inoculation of five endophytic diazotrophs promotes a synergistic effect when compared with the individual bacterial inoculation in micro-propagated plants of sugarcane in pots and later at field conditions where increases of up to % in the accumulation of n via bnf were observed in two varieties of sugarcane planted in three soil types [ ] . plant growth-promoting strategies. sevilla et al. [ ] described the contribution of inoculation with g. diazotrophicus in the nutrition of sugarcane and found that other factors influence on plant growth, such as growth regulators production. the ability of g. diazotrophicus to fix nitrogen and growth promotion of sugarcane was evaluated by comparing plants inoculated with the wild type (pal t ) and an nif mutant (mad acarry a nifd mutation) in two experiments [ ] . both, the type strain and the mutant, colonized sugarcane plants and persisted in mature plants. under conditions of nitrogen deficiency, plants inoculated with pal t generally grew better and had a higher content of total nitrogen days after planting when compared to plant inoculated with the nif mutant. these results indicate that the transfer of fixed nitrogen from g. diazotrophicus to sugarcane may be an important mechanism for the growth promotion in this association. when nitrogen was not limiting, the stimulation of growth was also observed in plants inoculated with both bacteria suggesting an additional effect of g. diazotrophicus inoculation related to growth promotion [ ] . this contribution to growth was also observed by riggis et al. [ ] ] g. diazotrophicus was inoculated into maize plants. among plant-growth substances produced by g. diazotrophicus, the indole acetic acid and gibberellins a and a are phytohormones that act on plant root growth and development of aerial part tissues [ , ] . g. diazotrophicus synthetize gluconic acid by the extracellular oxidation of the glucose by the action of the enzyme glucose dehydrogenase (gdh-pqq), localized in the perisplasmic space [ , ] , leading to the production of gluconic acid. this mild non-corrosive acid can, besides lowering the ph, promote chelation and exchange reactions and has been associated with phosphate and zinc solubilisation/chelation by g. diazotrophicus [ ] [ ] [ ] [ ] [ ] [ ] . saravanan et al. [ ] observed that g. diazotrophicus grown in zn-amended broth suffers deformation leading to pleomorphic, aggregate-like cells. noteworthy, characterization of a mutant unable to grown in the presence of zn, co, and cd salts revealed that the product of czca gene is responsible for g. diazotrophicus resistance to these heavy metals [ ] . biological control. another promising effect of g. diazotrophicus inoculation is related to the biological control of other microrganisms, such as xanthomonas albilineans [ , ] , colletotrichum falcatum [ ] , helminthosporium spp. [ ] , and fusarium spp. [ ] . by cdna-aflp analysis, some plant genes (using leaf tissue) involved in biocontrol activity was identified [ ] . these results indicate that inoculation stimulates genes involved in plant defense such as genes controlling the ethylene defense pathway. this pathway is activated when sugarcane micropropagated plants are inoculated with endophytic bacteria [ ] [ ] [ ] . even nematodes can be controlled by inoculating g. diazotrophicus as demonstrated by chawla et al. [ ] that used the isolate number - to control meloidogyne incognita in cotton. in contrast to g. diazotrophicus, which inhabits inner plant tissues as an endophyte, g. johannae and g. azotocaptans were only found colonizing the rhizosphere of coffee plants [ ] . later on, g. azotocaptans was also isolated from the rhizosphere of corn [ ] . little information is available about other aab-n fixing plant interaction. in the case of g. azotocaptans, mehnaz and lazarovits [ ] conducted a trial inoculating plants of four varieties of maize in a greenhouse experiment using sterile soil substrate. the inoculation consisted of g. azotocaptans, azospirillum lipoferum, and pseudomonas putida. at days after planting, the authors observed greater root growth and dry mass of the aerial part of the inoculated pots and also observed that some of the strains isolated from the rhizosphere of maize in canada presented significant plant growth expressed as increased root/shoot mass compared with non-inoculated plants in sand and/or soil, depending on the combination of bacteria and maize variety tested. the genus komagataeibacter was proposed to group members of gluconacetobacter species that cluster closely to g. xylinus [ ] . most of representatives of this new genus are known to be of industrial application but two of them are also nitrogen-fixing bacteria. the species gluconacetobacter kombuchae considered a heterotypic synonym of previously named g. hansenii [ ] was lately reclassified as kamagataeibacter hansenii. it was isolated during a survey of bacteria associated to kombucha tea together with acetobacter nitrogenifigens [ ] . kombucha tea is a fermented beverage that contains an association of yeast and bacteria that takes - days to be prepared. the authors utilized aliquots of the final preparation of the tea to inoculate solid plates containing lgi medium described by cavalcante and d€ obereiner [ ] but with final ph . for its isolation. this bacterium also grows in the presence of % of glucose or sucrose and can produce cellulose. sequences deposited at genbank and described as partial nifa (ef ) and nifh (dq ) coding regions do not share identities/similarities to deduced amino acids of others nitrogen fixing bacteria, as indicated by blast analysis. komagataeibacter (gluconacetobacter) kakiaceti was isolated by iino et al. [ ] from traditional kaki vinegar (produced from fruits of kaki, diospyros kaki thunb). recently, the genome of the k. kakiaceti jcm was sequenced and revealed presence of genes homologous to nif and other regulatory proteins related to nmetabolism. its whole genome sequence (wgs) is available at genbank (scaffolds accession number nz_baio -nz_baio ). however, up to date no further experimental evidence of n-fixation by this species was reported. swaminathania salitolerans was classified as a new genus and new species by loganathan and nair [ ] . these authors identified new isolates tolerant to salinity stress using rhizosphere, roots, and stems of mangroveassociated wild rice plants (porteresia coarctata tateoka). the medium used to obtain these new isolates was a semisolid lgi culture medium without the addition of nitrogen, final ph . with the addition of mm nacl. samples were collected in the city of tamil nadu in india, where isolates were obtained and identified as rods, gram-negative, mobile, and with peritrichous flagella. strains grew well in the presence of increasing concentrations of acetic acid ( - %) in a very acid ph, . and also could grow in the presence of % nacl and % kno . isolates were able to fix nitrogen and solubilized phosphate in the presence of this level of salt, mimicking the location of its isolation. the colonies grown on lgi medium are initially yellow orange but become darker after aging, smooth, and raised margin, characteristics commonly found in this bacterial family. in this case, there is a description of fixing species using acetylene reduction assay (ara) to estimate nitrogenase activity during growth in semisolid lgi medium, besides pcr amplification of nifd. but no further works using this bacterial species were published and therefore its agricultural importance is unknown. the asaia genus was first described with a single species a. bogorensis and then six more species were included: a. siamensis, a. krungthepensis, a. lannaensis, a. platycodi, a. prunellae, and a. astilbes [ , , , ] . these strains produce low quantities of acetic acid from ethanol and grew in medium with dulcitol as the sole carbon source, indicating that they belong to the genus asaia [ ] . the isolates also grow on lgi containing % sucrose as carbon source (lgi-p), but in this case it is acidified to ph . with acetic acid. interestingly, this genus includes species described from samples taken from the interior of insects as mosquito anopleles and plasmodium that are vectors of malaria and dengue fever and cause of its sanitary importance, many studies describe the presence of various species of mosquitoes in association with this group of bacteria [ ] . in india, a study was performed in order to isolate diazotrophs from three different samples: one flower called michalia champaca, from the anopheles mosquitoes, and from ants, such as in tetraponera rufonigra, pseudomyrmex, cephalotes, and paraponera [ , ] . it is important to emphasize that these isolates where obtained using n-free lgi medium as described by magalhães et al. [ ] to isolate azospirillum amazonense modifying the final ph of the medium from . to . replacing sulfuric acid by acetic acid. the same medium was used to characterize the g. diazotrophicus species in [ ] . samaddar et al. [ ] isolated asaia spp. as endophytes based on the surface disinfection of the plant tissue and confirmed their ability to fix nitrogen by using ara to estimate nitrogenase activity, amplification, and sequencing of nifh-like gene. asaia bogorensis (mtcc t ), a. siamensis (mtcc t ), and a. platycodi as strains were positive to these tests. they formed pink colonies, shiny, smooth, with entire margin in agar plates containing ag medium composed of d-glucose ( . %), glicerol ( . %), peptona ( . %), yeast extract ( . %), malt extract ( . %), caco ( . %), and agar ( . %) as described by yamada et al. [ ] . this pigmentation increased after prolonged incubation at °c. all of these isolates were classified as aerobic grow at ph . and °c. acetate and lactate are oxidized to carbon dioxide and water but the activity was considered low. partial sequences of nifh-like genes from several isolates and asaia type strains were obtained and, as well as their genomes [ ] , are deposited at genbank [ ] . noteworthy, up to date blast analysis revealed that these sequences blast only among them and with several partial nifh-like sequences from unculturable clone or chlorophyllide reductase subunit x (bchx) partial gene sequences from several microbial genome or unculturable clones (fig. ) . although this genus is the oldest of acetobacteraceae family, the description of diazotrophs representatives was first raised by the description of acetobacter diazotrophicus in the s. however, based on detailed taxonomic and phylogenetic studies, this species was reclassified and renamed into the genus gluconacetobacter. no other species of the genus acetobacter had been described as diazotrophic until when muthukumarasamy et al. [ ] presented various isolates belonging to a. peroxydans that fix nitrogen. most of the isolates were obtained from samples of flooded rice cultivated in india but studies of nifh amplification and ara confirmed that even in the type strain of a. peroxydans lmg t these characteristics were present [ ] . shortly thereafter, another study presented the description of the second species of nitrogen-fixing acetobacter named a. nitrogenifigens based on isolates obtained from kombucha tea in india [ ] . the nitrogen fixing acetobacter species a. nitrogenifigens shows polar flagella similar to those of gluconobacter [ ] . it also produces brown pigment and g-pyrone compounds, suggesting that -ketogluconate and , -diketogluconate are also produced as found in gluconobacter and gluconacetobacter [ , , ] . although claimed as positive for ara, the a. nitrogenifigens rg partial nifh sequence deposited at genbank (ay ) do not blast with any nifh coding protein as also observed to k. hansenii (rg ). an overview of the source of isolation and data about nitrogenase activity and nif genes to all these species are shown in table . figure . maximum likelihood tree of partial nifh and bchx proteins selected from protein sequences blast analysis using nifh gene deduced protein from g. diazotrophicus and asaia bogorensis as query. maximum likelihood method was based on the dayhoff matrix model in mega [ ] . the percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap values). the agricultural application of species and strains belonging to acetobacteraceae family will be based almost entirely in a single species, g. diazotrophicus. this species is the oldest described and characterization of its agricultural potential to important crops like sugarcane was quite widespread in brazil and other countries. for the other nitrogen fixing species descriptions of use are rare or no report of agricultural application is available. the use of diazotrophs in agriculture has been explored for over years and its apex in the past century, the decade of - , then the description of g. diazotrophicus. one of the first reports that populations of diazotrophs could be affected by increasing doses of n-fertilizer was made by vose et al. [ ] in sugarcane which showed that high levels of mineral n caused a significant reduction in the acetylene reduction activity, very popular method which measures the indirect activity of the nitrogenase enzyme acting as a competitive inhibitor. this effect was believed to inhibit this enzyme synthesis. in , after the description of g. diazotrophicus, studies conducted in mexico by fuentes-ram ırez et al. [ ] reported that the association between g. diazotrophicus and sugarcane could be severely limited by high n-fertilization, which would explain the decrease in acetylene reduction activity. in their study, the crops fertilized with kg n ha À showed higher number of isolates than the plots fertilized with kg n ha À , levels not applied in brazil. muthukumarasamy et al. [ , ] obtained similar results in india for g. diazotrophicus. they suggest that this effect was not directly related to the presence of high levels of nitrogen fertilizer in sugarcane crop since this bacterium is able to grow and fix nitrogen "in vitro" in presence of high concentrations of no ( mm). it is more likely that at these high n doses the physiological state of the plant undergoes changes and subsequently influences negatively the population of this organism. muñoz-rojas and caballero-mellado [ ] observed a negative effect on g. diazotrophicus population in the presence of high doses of nitrogen appled in sugarcane planted in mexico. these results were confirmed by reis jr. et al. [ ] using two sugarcane varieties planted in a sand soil fertilized with kg de n ha À in comparison with the control without n application. only the variety sp presented plant with high levels of total n in the fertilized plots and lower numbers of g. diazotrophicus. medeiros et al. [ ] utilized different sources of nitrogen and observed that g. diazotrophicus reduced acetylene reduction activity in the presence of high levels of n. studies conducted in india with the application of g. diazotrophicus were repeatedly evaluated by suman et al. [ ] [ ] [ ] . they reported that the population of g. diazotrophicus was influenced by increased doses of n-fertilizer and that n efficiency in sugarcane increased in the presence of g. diazotrophicus inoculation in greenhouse experiments [ ] . later on, suman et al. utilized one strain of g. diazotrophicus, named is , besides strains of a. brasilense and azotobacter chrococcum to evaluate nitrogen efficiency applied in increased doses on sugarcane planted in india [ ] . g. diazotrophicus showed the best results of crop yield, followed by its combination with a. chrococcum and a. brasilense. application of g. diazotrophicus was also evaluated in the germination of stem pieces of sugarcane by de la cruz et al. [ ] in philippines. these authors tested inoculation with different cell densities ( , , and cells ml À ) and methods of application (spray, immersion for h and dipping during min). they observed that inoculation led to increase in percentage survival plant height and shoot/root biomass when compared to the control at days after planting. introduction of microbial inoculant in ml À cells by immersion method produced taller plants with greater biomass and root compared to other treatments and uninoculated control. strains of gluconacetobacter diazotrophicus, azospirillum amazonense, herbaspirillum seropedicae, herbaspirillum rubrisubalbicans, and burkholderia tropica species were applied in sugarcane using pots filled with kg of soil and also field experiments planted in three different soil types in são paulo and rio de janeiro states of brazil showing contributions of the biological process with higher crop yields of different varieties sp - , sp - , rb , rb [ , , , ] . oliveira et al. [ ] used the technique of d n (natural abundance of n in the soil) and tested seven types of inoculants and found that the inoculant containing five strains described above showed the best results. these authors also quantitated the contribution of bnf showing that the mixture of five strains obtained . % of the accumulated n derived from the air. schultz et al. [ ] utilized the same five strains and modifications of the d n method of bnf quantification of soil applied in the sugarcane yield of rb and rb varieties and showed that plant biomass increased, but found no contributions of nitrogen fixation process by the inoculation. in order to understant how the sugarcane was colonized by this mixture, a fluorescent in situ hybridization (fish) analysis based on rrna-targeted oligonucleotide probes confirmed that in micropropagated sugarcane inoculated with this mixture of five species reached the endophytic habitat of micropropagated sugarcane plantlets through active infection of the root cap and emerging zone of secondary roots, although with different efficiencies due to apparently different competitiveness for colonization [ ] . maheshkumar et al. [ ] observed that this species was able to solubilize rock phosphate "in vitro," and it could be one of several effects that can promote plant growth after inoculation. sugar beet has also been used to check the response of g. diazotrophicus inoculation as described by jambukar and wange [ ] . in , tian et al. [ ] observed the effect of g. diazotrophicus inoculation in different maize genotypes, hybrids, and sweet corn varieties planted in canada. colonization of hybrids and sweet corn varieties by g. diazotrophicus was confirmed using species specific primers, but populations were quantified only in the order of - cells g À of plant tissue. g. diazotrophicus is known worldwide for nitrogen fixation but this is only one of its mechanisms of interest for agriculture and other industrial processes. for example, g. diazotrophicus strain srt has genes for the production of levan-sucrase both endo and exo levanases which are expressed under stress conditions [ , ] . another product of its growth is bacteriocins that may act to control growth of other microorganisms or even other strains of the same species [ ] . this bacteriocin is constitutively expressed in different conditions of culture medium and dependent on the strain tested. however, is there g. diazotrophicus as commercial product for use in agriculture? the answer is yes. descriptions of products containing g. diazotrophicus can be found elsewhere. in argentina, the ene- endophyte-plus sold by the company arbo srl laboratory, is recommended to be applied as an inoculant for wheat, maize, soybean, and tomato (http://www.arbolab.com.ar/ es/productos/ lvl/prom.html). in mexico, the company agro organics gaia sells a product containing a mixture of g. diazotrophicus and the fungi penicillium called glubac (http://www.organicosgaia. com.mx/biotransferentes-de-nutrientes.html). in brazil, origin of the g. diazotrophicus description, a patent based on a microorganism species or strain is not allowed, but to several other countries it is. in the united states, there is a patent for use of several nitrogen fixing bacteria, including g. diazotophicus, to enhance plant growth in cereals (us b ), and other claiming its use to reduce n fertilization in sugar rich plants, especially sugar beet ( ), showing that it can be part of a product for agricultural use. in brazil, bioprocess can be a matter of patent claims, such as the growth conditions of this bacterial to the production of biomass and fermentation products (pi - a ). however, a good product for the industry needs to possess a long shelf-life in order to reduce the costs and facilitates the distribution. unfortunately, a few studies have developed vehicles and protective substances that increase the longevity of cells of this species. nita et al. [ ] tested several substances such as cell protective for g. diazotrophicus under different temperatures ( and °c). efficacy was evaluated in tests of wheat inoculation under greenhouse and field conditions. the best method tested was the application of molasses (cane syrup) with . % (w/v) of nh cl. trehalose, arabic gum, and polyethylene glycol (peg ) presented the best results. addition of l-ascorbic acid ( . % w/v) to the preservation medium also enhanced the efficacy of the substances used as protectors. after - months of stock ay °c, g. diazotrophicus (strain l ) showed the best results of shelflife in the presence of arabic gum ( % w/v) and peg ( % w/v), respectively, and also keeping the growth promotion effect. silva et al. [ ] tested a polymer based on carboxymethylcellulose on the survival of g. diazotrophicus strain pal t with a shelf life of cell ml À for days. to date, data about agricultural application of other nitrogen fixing acetobacteraceae are restricted to asaia and it is based on a single report of weber et al. in [ ] that utilized a single strain of a. bogorensis (ab ) as inoculant for pineapple and monitored colonization by using agar plates of jnfb medium (malate as a carbon source and final ph . ) and population by the most probable number (nmp). the growth and fruiting of pineapple were benefited from the inoculation of a. bogorensis (strain ) associated with irrigation and increasing doses of organic fertilizer (compost). since the discovery of gluconacetobacter diazotrophicus in , many other diazotrophs belonging to the family acetobacteraceae were described as nitrogen fixing species, but this number can increase. the strategy to isolate and identify new species generally is not based on criteria of biological nitrogen fixation ability and this character is not a discriminatory one. interestingly, we observed that some researcher groups used the nitrogen free lgi medium as a strategy to obtain new isolates. it is expected that using n-free medium during isolation process can enrich populations of nitrogen fixing bacteria leading to recovery of many of them from environmental samples. in addition, the original ph of lgi was . , but several new species were described with a simple modification of the final ph to levels lower than . . since representatives of this family are adapted to acidic environment, lowering ph can be considered another strategy used by many authors to isolate and describe new species of nitrogen fixing acetobacteraceae. it is noteworthy that as many as new species have been described over the last years, publications containing studies of their ecology and distribution diminished considerably. g. diazotrophicus is the most studied nitrogen fixing bacteria of this family for agricultural application. since the beginning, several studies describing its survival, habitats, mode of plant colonization, and transference to new hosts have accumulated in the literature. based on them, the description of g. diazotrophicus as a true endophyte was proposed and accepted. the endophytic behavior of g. diazotrophicus is based on many ecological surveys and studies while these data are lacking to other species described. actually, nowadays publications of ecological research are an exception when we compare with the increasing numbers of species description mainly based on a set of physiological and molecular data, small numbers of specimens or even only one representative. nitrogen fixation is a biological processes well characterized and understood, at to some points, in pure culture and in vitro that occurs when an appropriate energy source is available in combination with the optimal temperature, ph and controlled o concentration. nonetheless, it is not an easy task to really prove that a single strain is responsible for part of the assimilated nitrogen in plant, especially under field conditions. in general, the main effects that are easily identified in plants that establish association with non-symbiotic nitrogen fixing bacteria are root surface enhancement, increased grain production, and early maturation. besides nitrogen fixation ability, g. diazotrophicus also produces growth hormones such as auxins and gibberellins and also can be considered pgpb when compared to a. brasilense. in agricultural perspective the application of acetobacteraceae species can contribute to plant growth and improve nitrogen assimilation of the host plant. however, under field conditions the effect of the number of bacteria in the plant versus the contribution of biological nitrogen fixation and/or plant growth promotion is not clearly established. it is already reported that introduced populations can undergo changes not only on their physiological aspects, but also in genomic aspects. so far, over more than years of studies most of the knowledge of these aspects of the bacterium-plant interaction is based on analysis conducted under controlled laboratory conditions. although the centennial knowledge of this versatile bacterial family to industrial application, a lot has to be done about the potential of the nitrogen fixing acetobacteracea to agricultural application and even to many other industrial biotechnological processes is limited yet. to improve agricultural use or even to broaden the industrial purpose of these nitrogen fixing acetobacteraceae species depends on development of new biotechnological data. for development of new biotechnological application and products, it will be necessary to increase knowledge and exploit the genomic potential for adaptation, competition, and survival of these bacteria. introdution of certain species to different plants and/or environmental conditions has to be explored. besides, the demand of development of methods, easily applied under field conditions, for bacterial inoculation, monitoration, and validation must be constantly considered. in addition, further efforts to characterize the ecology related to these microorganisms and plant relationship is essential. selection or development of genetically modified bacteria adapted to field competition, stress, and interaction with other components of the microbiota will be one of the goals to improve the inoculation technology worldwide. . m emoire sur la fermentation ac etique € uber die arten der essigbakterien a new acid-tolerant nitrogen-fixing bacterium associated with sugarcane intra-and intergeneric similarities of the ribosomal ribonucleic acid cistrons of acetobacter and gluconobacter the family acetobacteraceae: the genera acetobacter biotechnological applications of acetic acid bacteria roseomonas, a new genus associated with bacteremia and other human infections a novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family acetobacteraceae comparison of two bacteremic asaia bogorensis isolates from europe acetobacter cibinongensis bacteremia in human acetobacter indonesiensis pneumonia after lung transplant genera and species in acetic acid bacteria taxonomic studies on acetic acid bacteria and allied oxidative bacteria isolated from fruits. a new classification of the oxidative bacteria the flagellation and taxonomy of genera gluconobacter and acetobacter with reference to the existence of intermediate strains essai sur la systematique des acetobacters gluconoacetobacter, a new subgenus comprising the acetate-oxidizing acetic acid bacteria with ubiquinone- in the genus acetobacter the phylogeny of acetic acid bacteria based on the partial sequences of s ribosomal rna: the elevation of the subgenus gluconoacetobacter to the generic level validation list no. : validation of publication of new names and new combinations previously effectively published outside the ijsb lpsn-list of prokaryotic names with standing in nomenclature nguyenibacter vanlangensis gen. nov., sp. nov., an unusual acetic acid bacterium in the a-proteobacteria rhodovastum atsumiense gen. nov., sp. nov., a phototrophic alphaproteobacterium isolated from paddy soil sediminicoccus rosea gen. nov., sp. nov., isolated from the sediment of a eutrophic lake swingsia samuiensis gen. nov., sp. nov., an osmotolerant acetic acid bacterium in the a-proteobacteria rearrangement of the species and genera of the phototrophic "purple nonsulfur bacteria acidiphilium cryptum gen. nov., sp. nov., heterotrophic bacterium from acidic mineral environments acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments bergey's manual of systematic bacteriology: volume two: the proteobacteria, part a: introductory essays transfer of teichococcus ludipueritiae and muricoccus roseus to the genus roseomonas, as roseomonas ludipueritiae comb. nov. and roseomonas rosea comb. nov., respectively, and emended description of the genus roseomonas phylogenetic positions of novel aerobic, bacteriochlorophyll a-containing bacteria and description of roseococcus thiosulfatophilus gen proposal of craurococcus roseus gen. nov., sp. nov. and paracraurococcus ruber gen. nov., sp. nov., novel aerobic bacteriochlorophyll a-containing bacteria from soil rubritepida flocculans gen. nov., sp. nov., a new slightly thermophilic member of the a- subclass of the proteobacteria humitalea rosea gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium of the family acetobacteraceae isolated from soil acidiphilium aminolytica sp. nov.: an acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment transfer of acidiphilium facilis and acidiphilium aminolytica to the genus acidocella gen. nov., and emendation of the genus acidiphilium stella, a new genus of soil prosthecobacteria, with proposals for stella humosa sp. nov. and stella vacuolata sp. nov phylogenetic relationships of the genera stella, labrys and angulomicrobium within the "alphaproteobacteria" and description of angulomicrobium amanitiforme sp. nov acidomonas gen. nov., incorporating acetobacter methanolicus as acidomonas methanolica comb. nov emendation of the genus acidomonas urakami, tamaoka, suzuki and komagata asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the alpha-proteobacteria neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the a-proteobacteria teichococcus ludipueritiae gen. nov. sp. nov., and muricoccus roseus gen. nov. sp. nov. representing two new genera of the a- subclass of the proteobacteria saccharibacter floricola gen. nov., sp. nov., a novel osmophilic acetic acid bacterium isolated from pollen acidisoma tundrae gen. nov., sp. nov. and acidisoma sibiricum sp. nov., two acidophilic, psychrotolerant members of the alphaproteobacteria from acidic northern wetlands neokomagataea gen. nov., with descriptions of neokomagataea thailandica sp. nov. and neokomagataea tanensis sp. nov., osmotolerant acetic acid bacteria of the a-proteobacteria endobacter medicaginis gen. nov., sp. nov., isolated from alfalfa nodules in an acidic soil nitrogen-fixing and cellulose-producing gluconacetobacter kombuchae sp. nov., isolated from kombucha tea differentiation of species of the family acetobacteraceae by aflp dna fingerprinting: gluconacetobacter kombuchae is a later heterotypic synonym of gluconacetobacter hansenii subdivision of the genus gluconacetobacter yamada, hoshino and ishikawa : the proposal of komagatabacter gen. nov description of komagataeibacter gen. nov., with proposals of new combinations (acetobacteraceae) novel nitrogen-fixing acetic acid bacteria, gluconacetobacter johannae sp. nov. and gluconacetobacter azotocaptans sp. nov., associated with coffee plants novel nitrogen-fixing acetobacter nitrogenifigens sp. nov., isolated from kombucha tea swaminathania salitolerans gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice (porteresia coarctata tateoka) nitrogen fixation in asaia sp. (family acetobacteraceae) transfer of gluconacetobacter kakiaceti, gluconacetobacter medellinensis and gluconacetobacter maltaceti to the genus komagataeibacter as komagataeibacter kakiaceti comb. nov., komagataeibacter medellinensis comb. nov. and komagataeibacter maltaceti comb. nov diversity of acetic acid bacteria in indonesia, thailand, and the philippines natural association of gluconacetobacter diazotrophicus and diazotrophic acetobacter peroxydans with wetland rice asaia siamensis sp. nov., an acetic acid bacterium in the alpha-proteobacteria isolation and nitrogen fixing efficiency of a novel endophytic diazotroph gluconacetobacter diazotrophicus associated with saccharum officinarum from southern districts of tamilnadu complete genome sequence of the sugarcane nitrogen-fixing endophyte gluconacetobacter diazotrophicus pal asaia astilbes sp. nov., asaia platycodi sp. nov., and asaia prunellae sp. nov., novel acetic acid bacteria isolated from flowers in japan gluconacetobacter kakiaceti sp. nov., an acetic acid bacterium isolated from a traditional japanese fruit vinegar draft genome sequence of asaia sp. strain sf . , an important member of the microbiome of anopheles mosquitoes soil bacteriological studies. further contributions to the physiology and morphology of the members of the azotobacter group acetobacter europaeus sp. nov., a main component of industrial vinegar fermenters in central europe description of gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug description of gluconacetobacter swingsii sp. nov. and gluconacetobacter rhaeticus sp. nov., isolated from italian apple fruit gluconacetobacter tumulicola sp. nov. and gluconacetobacter asukensis sp. nov., isolated from the stone chamber interior of the kitora tumulus gluconacetobacter tumulisoli sp. nov., gluconacetobacter takamatsuzukensis sp. nov. and gluconacetobacter aggeris sp. nov., isolated from takamatsuzuka tumulus samples before and during the dismantling work in reclassification of gluconacetobacter hansenii strains and proposals of gluconacetobacter saccharivorans sp. nov. and gluconacetobacter nataicola sp. nov gluconacetobacter medellinensis sp. nov., cellulose-and non-cellulose-producing acetic acid bacteria isolated from vinegar gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium genera and species in acetic acid bacteria phylogeny and differentiation of species of the genus gluconacetobacter and related taxa based on multilocus sequence analyses of housekeeping genes and reclassification of acetobacter xylinus subsp. sucrofermentans as gluconacetobacter sucrofermentans (toyosaki et al. ) sp. nov. , comb. nov acetobacter diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium associated with sugarcane a nitrogen-fixing endophyte of sugarcane stems (a new role for the apoplast) recent advances in bnf with non-legume plants gluconacetobacter diazotrophicus: a natural endophytic diazotroph of nile delta sugarcane capable of establishing an endophytic association with wheat improved methodology for isolation of acetobacter diazotrophicus and confirmation of its endophytic habitat coffea arabica l., a new host plant for acetobacter diazotrophicus, and isolation of other nitrogen-fixing acetobacteria natural endophytic occurrence of acetobacter diazotrophicus in pineapple plants acetic acid bacterial biota of the pink sugar cane mealybug, saccharococcus sacchari, and its environs limited genetic diversity in the endophytic sugarcane bacterium acetobacter diazotrophicus technical approaches to inoculate micropropagated sugar cane plants were acetobacter diazotrophicus effect of high sugar concentration on nitrogenase activity of acetobacter diazotrophicus physiology and dinitrogen fixation of acetobacter diazotrophicus plasmid contents and nif genes detection in acetobacter diazotrophicus strains analysis of nif and regulatory genes in acetobacter diazotrophicus characterization of genes involved in regulation of nitrogen fixation and ammonium sensing in acetobacter diazotrophicus, an endophyte of sugarcane molecular analysis of the chromosomal region encoding the nifa and nifb genes of acetobacter diazotrophicus characterization of a major cluster of nif, fix, and associated genes in a sugarcane endophyte, acetobacter diazotrophicus identification of three genes encoding pii-like proteins in gluconacetobacter diazotrophicus: studies of their role(s) in the control of nitrogen fixation whole-genome analyses reveal genetic instability of acetobacter pasteurianus genome-wide phylogenetic analysis of gluconobacter, acetobacter, and gluconacetobacter: genome-wide phylogenetic analysis of aab genome sequences of the high-acetic acid-resistant bacteria gluconacetobacter europaeus lmg t and g. europaeus lmg (reference strains), g. europaeus p , and gluconacetobacter oboediens bp (isolated from vinegar) draft genome sequence of komagataeibacter rhaeticus strain af , a high producer of cellulose, isolated from kombucha tea complete genome sequence and comparative analysis of acetobacter pasteurianus b, a strain well-adapted to the cocoa bean fermentation ecosystem acetic acid bacteria genomes reveal functional traits for adaptation to life in insect guts draft genomic dna sequence of the facultatively methylotrophic bacterium acidomonas methanolica type strain mb gluconacetobacter diazotrophicus pal strain: selection and characterization of mutants deficient in nitrogen-fixation ability protein expression profile of gluconacetobacter diazotrophicus pal , a sugarcane endophytic plant growth-promoting bacterium a comparative proteomic analysis of gluconacetobacter diazotrophicus pal at exponential and stationary phases of cultures in the presence of high and low levels of inorganic nitrogen compound validation of a tn transposon mutagenesis system for gluconacetobacter diazotrophicus through characterization of a flagellar mutant proteome of gluconacetobacter diazotrophicus co-cultivated with sugarcane plantlets exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogenfixing endophyte gluconacetobacter diazotrophicus identification and characterization of an iron abc transporter operon in gluconacetobacter diazotrophicus pal structural studies of an exopolysaccharide produced by gluconacetobacter diazotrophicus pal gluconacetobacter diazotrophicus pal possesses an active quorum sensing regulatory system transcriptional regulation and signalpeptide-dependent secretion of exolevanase (lsdb) in the endophyte gluconacetobacter diazotrophicus antioxidant pathways are up-regulated during biological nitrogen fixation to prevent rosinduced nitrogenase inhibition in gluconacetobacter diazotrophicus the bacterial superoxide dismutase and glutathione reductase are crucial for endophytic colonization of rice roots by gluconacetobacter diazotrophicus pal influence of carbon and nitrogen sources on growth, nitrogenase activity, and carbon metabolism of gluconacetobacter diazotrophicus transfer rna-dependent asparagine biosynthesis in gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation nitrogen compounds in the apoplastic sap of sugarcane stem: some implications in the association with endophytes inoculation of sugarcane with pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium sustaining n -dependent growth in the presence of co nifa-and cooa-coordinated cown expression sustains nitrogen fixation by rhodobacter capsulatus in the presence of carbon monoxide recent advances in nitrogen-fixing acetic acid bacteria research progress and perspectives of nitrogen fixing bacterium, gluconacetobacter diazotrophicus, in monocot plants infection of sugar cane by the nitrogen-fixing bacterium acetobacter diazotrophicus infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs further observations on the interaction between sugar cane and gluconacetobacter diazotrophicus under laboratory and greenhouse conditions colonization of sorghum and wheat by seed inoculation with gluconacetobacter diazotrophicus monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium gluconacetobacter diazotrophicus marked with gfp and gusa reporter genes: gfp marked g. diazotrophicus on rice response of micropropagated sugarcane varieties to inoculation with endophytic diazotrophic bacteria efeito da inoculas cão de acetobacter diazotrophicus em cana-de-as c ucar (saccharum spp) variedade sp - , a partir de cultura de meristemas yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria comparison of benefit to sugarcane plant growth and n incorporation following inoculation of sterile plants with acetobacter diazotrophicus wild-type and nif-mutant strains enhanced maize productivity by inoculation with diazotrophic bacteria production of indole- -acetic acid and gibberellins a and a by acetobacter diazotrophicus and herbaspirillum seropedicae in chemically-defined culture media ecological occurrence of gluconacetobacter diazotrophicus and nitrogen-fixing acetobacteraceae members: their possible role in plant growth promotion evidence for a membranebound pyrroloquinoline quinone-linked glucose dehydrogenase in acetobacter diazotrophicus mineral phosphate solubilizing activity of acetobacter diazotrophicus: a bacterium associated with sugar cane solubilization of insoluble zinc compounds by gluconacetobacter diazotrophicus and the detrimental action of zinc ion (zn þ) and zinc chelates on root knot nematode meloidogyne incognita identification and characterization of gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc assessing the zinc solubilization ability of gluconacetobacter diazotrophicus in maize rhizosphere using labelled zn compounds mineral phosphate solubilization activity of gluconacetobacter diazotrophicus under p-limitation and plant root environment assessing the in vitro zinc solubilization potential and improving sugarcane growth by inoculating gluconacetobacter diazotrophicus zinc metal solubilization by gluconacetobacter diazotrophicus and induction of pleomorphic cells essential role of the czc determinant for cadmium, cobalt and zinc resistance in gluconacetobacter diazotrophicus pal gluconacetobacter diazotrophicus elicits a sugarcane defense response against a pathogenic bacteria xanthomonas albilineans antagonism of gluconacetobacter diazotrophicus (a sugarcane endosymbiont) against xanthomonas albilineans (pathogen) studied in alginate-immobilized sugarcane stalk tissues antagonistic potential of n -fixing acetobacter diazotrophicus against colletotrichum falcatum went., a causal organism of red-rot of sugarcane inoculation effects of pseudomonas putida, gluconacetobacter azotocaptans, and azospirillum lipoferum on corn plant growth under greenhouse conditions in vitro suppression of soil borne pathogenic fungi and pyoluteorin production by gluconacetobacter diazotrophicus expression of sugarcane genes induced by inoculation with gluconacetobacter diazotrophicus and herbaspirillum rubrisubalbicans signalling pathways mediating the association between sugarcane and endophytic diazotrophic bacteria: a genomic approach members of the ethylene signalling pathway are regulated in sugarcane during the association with nitrogen-fixing endophytic bacteria colonization behaviour of gluconoacetobacter diazotrophicus in root-knot nematode (meloidogyne incognita) infected and healthy cotton plants validation list : list of new names and new combinations previously effectively, but not validly, published asaia krungthepensis sp. nov., an acetic acid bacterium in the a-proteobacteria asaia lannaensis sp. nov., a new acetic acid bacterium in the alphaproteobacteria acetic acid bacteria, newly emerging symbionts of insects bacterial infections across the ants: frequency and prevalence of wolbachia, spiroplasma, and asaia field studies on response of sugar beet to microbial inoculants under graded nitrogen levels meg a : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods glucose metabolism and gluconic acid production by acetobacter diazotrophicus potential n -fixation by sugarcane, saccharum sp. in solution culture-i. effect of nh þ vs. no À , variety and nitrogen level acetobacter diazotrophicus, an indoleacetic acid producing bacterium isolated from sugarcane cultivars of mexico influence of n fertilisation on the isolation of acetobacter diazotrophicus and herbaspirillum spp. from indian sugarcane varieties effect of inorganic n on the population, in vitro colonization and morphology of acetobacter diazotrophicus (syn. gluconacetobacter diazotrophicus) population dynamics of gluconacetobacter diazotrophicus in sugarcane cultivars and its effect on plant growth influence of nitrogen fertilisation on the population of diazotrophic bacteria herbaspirillum spp. and acetobacter diazotrophicus in sugar cane (saccharum spp nitrogen source effect on gluconacetobacter diazotrophicus colonization of sugarcane (saccharum spp improving sugarcane growth and nutrient uptake by inoculating gluconacetobacter diazotrophicus nitrogen use efficiency of sugarcane in relation to its bnf potential and population of endophytic diazotrophs at different n levels effects of diverse habitat biofertilizers on yield and nitrogen balance in plant-ratoon crop cycle of sugarcane in subtropics sprouting, survival and growth of young sugarcane (saccharum officinarum l.) treated with diazotrophic bacteria (gluconacetobacter diazotrophicus) the effect of inoculating endophytic n -fixing bacteria on micropropagated sugarcane plants avalias cão agronômica de variedades de cana-de-as c ucar inoculadas com bact erias diazotr oficas e adubadas com nitrogênio colonization of the nitrogen-fixing bacterium gluconacetobacter diazotrophicus in a large number of canadian corn plants isolation and enzymic properties of levansucrase secreted by acetobacter diazotrophicus srt , a bacterium associated with sugar cane functional production and secretion of the gluconacetobacter diazotrophicus fructose-releasing exo-levanase (lsdb) in pichia pastoris antagonism among gluconacetobacter diazotrophicus strains in culture media and in endophytic association liquid formulations of acetobacter diazotrophicus l and herbaspirillum seropedicae j and their field trials on wheat survival of endophytic bacteria in polymer-based inoculants and efficiency of their application to sugarcane effect of diazotrophic bacterium inoculation and organic fertilization on yield of champaka pineapple intercropped with irrigated sapota the authors wish to thank the coordination of improvement of higher education personnel-capes, the national council for scientific and technological development-cnpq and the carlos chagas foundation for research support of the state of rio de janeiro-faperj for the scholarships. finantial support and also scholarships from cnpq, project number / - and also cnpq/inct-fbn (process no. / - ). key: cord- -e wz qb authors: mehra, neelesh kumar; jain, keerti; jain, narendra kumar title: pharmaceutical and biomedical applications of surface engineered carbon nanotubes date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: e wz qb surface engineered carbon nanotubes (cnts) are attracting recent attention of scientists owing to their vivid biomedical and pharmaceutical applications. the focus of this review is to highlight the important role of surface engineered cnts in the highly challenging but rewarding area of nanotechnology. the major strength of this review lies in highlighting the exciting applications of cnts to boost the research efforts, which unfortunately are otherwise scattered in the literature making the reading non-coherent and non-homogeneous. in the current scenario, cnts are one of the forefronts in a diverse field of medicine, which are continuously being explored by researchers, scientists and academicians for the development of new, safe and effective nanomedicine. the cnts are promising quasi one-dimensional nanomaterial with well ordered flat networks of fused benzene rings or hollow graphitic cylindrical tubular structure consisting of hexagonal arrangement with sp hybridized carbon atoms [ ] [ ] [ ] [ ] . cnts are mainly classified into four types, based upon the structure and diameters including single-, double-, triple-, and multi-walled carbon nanotubes. the length of the tubes can be extended depending on the type of production method [ ] [ ] [ ] [ ] . cnts are one of the attractive tools in nanotechnology with surface engineered carbon nanotubes (f-cnts), emerging as a new family of carbon nanovectors that enabled numerous biomedical applications. functionalization plays a pivotal role in the design of cnts-based nanomaterials by rendering them more biocompatible and less toxic with increased dispersibility in comparison to pristine cnts. the methodology of functionalization of cnts can be classified broadly into two categories: (a) covalent attachment of active biochemical moieties through chemical reactions onto cnts skeleton, and (b) non-covalent adsorption or wrapping of different functional molecules [ , ] . the various types of nanocomposites have been summarized in fig. . the study of cellular uptake mechanism of the surface engineered cnts has been intensely addressed in the last two decades [ , ] . lacerda et al. have demonstrated that the surface engineered cnts have propensity to enter the cells via an energy-dependent, endosomally-mediated cellular internalization approach and direct cytoplasmic translocation through insertion or passive diffusion in a non-invasive manner (tiny nanoneedle mechanism) [ ] [ ] [ ] . the f-cnts have been easily cross the blood brain barriers (bbb) without requirement of any external transporter device owing to nano-dimension and tiny nano-needle tubular structure morphology ( fig. a) . the main pathways speculated by scientists for cellular uptakes of f-cnts are: (i) phagocytosis, (ii) cnts piercing into membrane by passive diffusion, (iii) caveolae-mediated endocytosis, (iv) clathrin-mediated endocytosis, and (v) caveolaeclathrin mediated endocytosis. the intracellular uptake mechanisms of cnts are crucial for the controlled uptake and devising new strategies in the field of novel drug delivery [ , ] . reports have shown that the nanotubes may enter cells rapidly with free trafficking into the cytoplasm in the first hour of internalization. this penetration of surface engineered cnts into phagocytic and non-phagocytic cells is mediated by three alternative pathways: i) via membrane wrapping as individual tubes; ii) via direct membrane translocation of individual nanotubes; and iii) in bundles within vesicular compartments (fig. b) [ ] . these exciting facts about intracellular trafficking of cnts have opened a new door for the development of a promising delivery system for therapeutic and diagnostic agents [ ] . the potential of surface engineered cnts and possible biomedical applications are discussed below and various cnts conjugates employed in bioactive delivery shown in fig. . transdermal drug delivery system (tdds) represents a convincing as well as promising alternative to oral or injectable delivery of drugs. recently, cnts have been investigated as tdds due to their tiny nano-needle shape structure and superior adsorption capacity. im and co-workers prepared an electro-sensitive tdds of cnts using electrospinning method to control drug release with polyethylene oxide and pentaerythritol triacrylate polymers. in this tdds, cnts facilitated the electro-sensitive-transdermal drug delivery [ ] . the advantages of cnts in tdds include high loading efficiency and enhanced transdermal penetration of drugs by application of a small electrical bias to create a programmable drug delivery system. the future explorations of f-cnts in tdds present a feasible solution to limitation of current smoking cessation and opioid withdrawal symptom treatments [ , ] . several approaches have been exploited in the development of safe, biocompatible and comfortable nanovehicle for controlled ophthalmic drug delivery with enhanced patient compliance and reduced toxicity. ophthalmic drugs have a short residence time (< min) and only - % of applied therapy may penetrate the cornea and enter into the intraocular tissues. the cnts-based ocular drug delivery has been found to enhance the bioavailability of few ophthalmic medications overcoming the limitation of traditional ophthalmic drug delivery methods [ ] . cnts could also be used for ocular targeting of different therapeutic agents to the different ocular sites. unfortunately only very few reports are available in this area [ ] . application of 'kajal' (also known as kohl or surma) is a common practice in indian families. kajal has been claimed for prevention and treatment of various eye diseases (blepharitis, cataract and conjunctivitis etc.) and also said to ward off an 'evil eye'. cosmetic application of cnts is well documented in various literatures [ , , ]. surface engineered cnts based delivery systems have been explored as a new platform to repair the damaged cns tissue/cells in the field of neuroscience. the cnts have been claimed to be able to cross the blood brain barrier (bbb) by different targeting mechanisms and act as safe and effective targeted drug delivery system [ , ] . since the last one-decade researchers have continuously explored and assessed the cnts as substrate for neuronal tissue growth, regeneration and its application in neuroscience research. cnts have numerous salient features with a simple and inert molecular tubular nanoneedle structure and porosity beneficial to neural interface for trafficking of cells, sensing of microenvironments, delivery of transfection agents, and scaffolding for incorporating within host body, to provide the support for adhesion, proliferation and migration of cells. ren and co-workers developed a dual targeted delivery system based on the oxidized pegylated mwcnts modified with angipep- (a targeting ligand of lowdensity lipoprotein receptor related-protein (lrp) receptors) loaded with doxorubicin (dox) and systematically evaluated their in vitro and in vivo anti-glioma effect by c cytotoxicity and median survival time (mst) of intracranial c glioma bearing balb/c mice. the combination of o-mwcnts-peg with angiopep- as targeting ligand was claimed to play a role of active dual-targeting and combination of o-mwcnts-peg and angiopep- constituted an ideal dual-targeting drug delivery to brain glioma [ ] . photodynamic therapy (pdt) is an effective, alternative, noninvasive, non-toxic therapy used in oncology [ ] . till date only few numbers of reports are available on the applications of surface engineered cnts in pdt. photosensitizer [ -aminolevulinic acid ( -ala)] loaded pamam dendrimers modified mwcnts (dmnts) showed good biocompatibility with significant increase in the accumulation of -ala in mgc- tumor cells leading to a potential photodynamic anti-tumor effect [ ] . chitosan wrapped chlorine (ce ), which is a photosensitizer, loaded swcnts revealed high cellular uptake (high anticancer effects) and low toxicity against hela cancer cells than free ce [ ] . cnts hold promise in photothermal therapy (ptt) due to extraordinary photon-to-thermal energy conversion efficiency with high absorption in the near infra-red (nir) light [ ] [ ] [ ] . the swcnts have the ability to absorb - nm nir radiation and convert it into heat, which kills cancer cells [ ] . moon and co-workers demonstrated the photothermal effect of pegylated swcnts upon nir irradiation on kb tumor bearing nude mice and observed that sufficiently high thermal energy was generated from optically excited peg-swcnts to destroy tumor in a non-invasive manner. the mice treated with peg-swcnts and nir irradiation cnts conjugates employed in bioactives delivery. www.drugdiscoverytoday.com reviews post screen folic acid (fa)-chitosan (chi)/alginate (alg)-swcnts. hela cell line doxorubicin through p-p stacking. fluorescence cell viability and cell uptake studies cnts complex found to be more selective and effective than free dox due to targeting based on fa and release of dox at lysosomal ph. [ ] mab-bsa adsorbed-swcnts. wpdr colon cancer cell line doxorubicin through p-p stacking. electron microscopy (sem, hrtem) raman spectroscopy uptake study revealed that delivery efficiency was %. all cells could take up the swcnt complexes. [ ] diaminotriethylene glycol-mwcnts. -hydroxycamptothecin (hcpt) through p-p stacking. in vitro single photon emission computed tomography (spect showed the complete eradication of solid malignant tumor devoid of any toxicity or abnormal behavior days post treatment. the issue concerned with residual swcnts accumulation inside the body after the treatment was resolved as nanotubes were excreted from mice's body in about months through the biliary or urinary pathway in biodistribution study [ ] . hashida and co-workers reported the photothermal ablation activity of a novel swcntspeptide composite with a designed peptide having a repeated structure of h-(-lys-phe-lys-ala-) -oh[(kfka) ] against tumor cells. the obtained results suggest the remarkable suppression of tumor growth compared with only swcnt-(kfka) injection alone or nir irradiation alone [ ] . currently viral vector system based gene delivery is most widely used to achieve high efficiency of gene expression, which concerns the safety, as viral vector can be immunogenic, oncogenic, and may be responsible for inflammation. further, gene therapy may suffer rapid degradation of exogenous nucleic acid. now-a-days some effective multiple non-viral delivery systems like polymeric nanoparticles, liposomes, cell penetrating peptides, cationic lipids, dendrimers and f-cnts are being employed to efficiently deliver nucleic acids including sirna with reduced immunogenicity. the efficiency of gene expression mediated by these nanovectors is lower as compared with the viral vector but they provide the advantages of easy up-scaling, flexibility and reduced immunogenicity. the applications of cnts as new vectors for delivery of nucleic acids is another area of research and development which could offer several advantages for nucleic acid, sirna, gene and protein delivery owing to their thin and long architecture that offers a large surface area to which sirna can be bound and delivered to target site. further, cnts may have protective capacity, which stabilize and lead to more efficient cellular transfection and prevent the lysosomal degradation of encapsulated nucleic acids [ ] . kawaguchi and co-workers synthesized dna-functionalized swcnts to improve the dispersibility as compared to acid treated swcnts. the binding ratio of the acid-treated swcnts and dna was calculated to be : (dna/swcnts) from the phosphorous content in dna-swcnts [ ] . in context of the enhanced gene delivery sanz and co-workers were first to develop and demonstrate the enhanced gene delivery using lysosomotropic anti-malarial drug chloroquine loaded polyethyleneimine (pei)-coated dwcnts to enhance the cell transfection efficiency [ ] . infectious diseases such as tuberculosis, leishmaniasis, severe acute respiratory syndrome (sars), flu (swine, bird and avian) and anthrax infection indicate that infectious diseases have emerged as a critical public health issue with global concerns. surface engineered cnts have been used in the treatment of infectious diseases due to their ability to easily conjugate drugs like amphotericin b (amb) and dapsone. leishmaniasis is a microbial disease in which macrophages and other cells of reticuloendothelial system (res) serve as host as well as replication site for the parasite [ ] . conjugation of amphotericin b (amb) to f-cnts can modify its properties in terms of toxicity and anti-mycotic efficiency [ , ] . drug discovery today volume , number june cell sorter analysis the f-swcnts-cos-gtx-p was found to be most effective delivery vehicle with a controlled release and enhanced cytotoxicity rendered through apoptosis in hela cells. [ ] gl-mwcnts-dox wu and co-workers explored the toxicity and antifungal activity of amb loaded cnts (amb-cnts) functionalized with fluorescein isothiocynate (fitc) for efficient nuclear localization in human jurkat lymphoma t cells and observed that amb-cnts could be easily taken up by mammalian cells precluding any specific toxic effect but preserving antifungal activity [ ] . cnts surface engineered with mannose showed promising targeted delivery of amb to j macrophages cells [ ] . cancer is one of the leading causes of death worldwide. more than % cases of chemotherapy destroy cancer cells along-with the normal healthy cells, with serious side effects and hence strategies based on nanotechnology are being explored as 'safe and effective' strategy to achieve selective uptake of chemotherapeutic agent by target cancer cells [ ] . a significant decrease in toxicity with preferential killing of cancer cells was achieved with dual targeted nanocarrier combining mwcnts and iron oxide magnetic nanoparticles (mns) conjugated with fa for antineoplastic agent, dox [ ] . jain and coworkers observed tumor targeted delivery of dox and reduced toxicity with dox loaded dexamethasone mesylate anchored mwcnts to a- lung epithelial cancer cells [ ] . further, cnts surface engineered with folic acid and d-alpha tocopheryl polyethylene glycol succinate (tpgs) showed tumor targeted delivery of dox with improved cytotoxicity toward cancer cells and enhanced cellular uptake speculated to be mediated by endocytosis and tiny nano-needle mechanism [ , ] . fig. presents the different conjugates based on cnts employed in the delivery of anticancer bioactives varying from pristine cnts to drug loaded cnts. from the aforesaid account it may be concluded that the surface engineered cnts could have promising potential in cancer therapy. reports showed that cnts may emerge as promising nanocarrier in targeting of macrophages owing to their exceptional physicochemical characteristics. amb conjugated cnts showed improved antileishmanial activity as compared to free amb in j a. macrophage cells [ ] . later, amb loaded mannose conjugated cnts showed efficient macrophage targeting in j macrophage cell lines with significantly reduced toxicity toward rbcs and kidney cells showing considerable reduction in the nephro-and hemolytic-toxicity, which are the major constraints in clinical use of amb [ ] . a brief summary of various bioactives delivered through surface engineered cnts is shown in table [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cnts alone as well as in combination with other nanocarriers like dendrimers, nanoparticles and quantum dots are emerging as promising novel drug delivery and diagnostic nano-vector tool with minimal toxicity and immunogenicity. delogu and co-workers assessed the echogenic property of nanotubes in vitro and found that the ultrasound signal of f-mwcnts is higher than the pristine mwcnts, graphene oxide and f-swcnts. the authors found that the mwcnts are highly echogenic in liver and heart as examined on pig as an experimental animal model. thus the nanotubes could show enormous potential as ultrasound contrast agents and theragnostics moiety combining diagnostic and therapeutic modalities [ ] . the smart/intelligent surface engineered carbon nanotubes are still having controversy about their biodegradable/non-biodegradable nature. previous literature suggested non-biodegradable nature of cnts and excretion through biliary pathway, especially via urine and feces. however, few investigations observed that cnts are degraded in specific environments via natural, enzymatic catalysis, natural horseradish peroxidase (hrp) with low concentration of h o (approximately mm) at c over weeks or in the presence of human neutrophil enzyme, myeloperoxidase. importantly biodegraded nanotubes did not produce any inflammatory response when aspirated in to the lungs of mice [ ] [ ] [ ] . bianco and co-workers investigated the biodegradation of swcnts and mwcnts under different conditions and concluded that the oxidized mwcnts were highly degrading in nature as compared to swcnts [ ] . these few recent literature reports support the biodegradable nature of cnts, otherwise previously, cnts were considered non-biodegradable and excreted out as examined by electron microscopic studies. additionally biocompatible nanotubes could be prepared via conjugation of phospholipids (pl) and polyethylene glycol (peg) chains on to them, which may be a promising alternative candidate in biomedical applications [ ] [ ] [ ] . the toxicities associated with the cnts are yet unclear. several research studies demonstrated the toxicity of pristine cnts, which could fortunately be minimized via surface engineering that renders them more biocompatible and non-immunogenic. the specific structural properties of cnts like needle-shape, long and thin and biopersistent nature (insoluble) may produce adverse pulmonary effect but this potential risk could be avoided by use of surface engineering of cnts. however, till date cnts' eco-toxicological data are limited and controversial and also lack reliable information of exposure as per occupational health and safety regulations. in contrast to ecotoxic point of view, surface of cnts can often be modified with the different chemical reactions or by 'grafting to' and 'grafting from' make cnts more biocompatible with better stability. additionally, there is a significant need for research and development in the field of analytical and detection methods. strict norms and regulations are needed in the development of new drug products for being safe, effective and economic in generally regarded as safe (gras) prominence. cnts' potentials as novel carriers in bioactives delivery make them promising candidate for the development of new pharmaceutical products in clinical relevance. before the clinical use of cnts based products; their pharmacological and toxicological parameters must be proved to be safe and effective. surface of cnts could be easily engineered with targeting and imaging agents, peptides, proteins, sirna, nucleic acids, antibodies and chemotherapeutic drugs for aforementioned pharmaceutical and biomedical applications. these surface engineered cnts are considered promising nanomaterial for use in many biomedical applications, including biocompatible modules for the delivery of bioactives. in conclusion, before realizing the full potential of cnts, it is very important to generate data on their safety, efficacy, and feasibility. although the scientists are optimistic about the possible application of cnts yet learning the lesson from past (like thalidomide tragedy), the safety and efficacy of cnts/cnts based products/devices etc. must be established beyond doubt. we have witnessed the emergence of several materials/devices/products, which could not make their way to market but then that's the research. let's keep our fingers crossed and wait for the conclusive proof regarding the possible application of cnts, particularly in biomedical arena. no conclusion should be drawn either in haste or without conclusive evidence of safety. carbon nanotubes: an emerging drug carrier for targeting cancer cells carbon nanomaterials: multi-functional agents for biomedical fluorescence and raman imaging selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery fucosylated multiwalled carbon nanotubes for kupffer cells targeting for the treatment of cytokine-induced liver damage biotinylated amphiphile-single walled carbon nanotubes conjugate for target-specific delivery to cancer cells receptor based targeting of therapeutics a review of ligand tethered surface engineered carbon nanotubes the cancer targeting potential of d-a-tocopheryl polyethylene glycol succinate tethered multi walled carbon nanotubes potential and emerging trends in nanopharmacology carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction how do functionalized carbon nanotubes land on, bind to and pierce through model and plasma membranes carbon nanotubes cell translocation and delivery of nucleic acids in vitro and in vivo translocation mechanisms of chemically functionalized carbon nanotubes across plasma membranes the effect of carbon nanotubes on drug delivery in an electrosensitive transdermal drug delivery systems carbon nanotubes for transdermal drug delivery carbon nanotubes membranes for use in the transdermal treatment of nicotine addiction and opioid withdrawal symptoms advancement in carbon nanotubes: basics, biomedical applications and toxicity interactions between cultured neurons and carbon nanotubes: a nanoneuroscience vignette kajal (kohl)-a dangerous cosmetic kohl along history in medicine and cosmetics the targeted delivery of anticancer drugs to brain glioma by pegylated oxidized multi-walled carbon nanotubes modified with angiopep- dendrimers and derivatives as a potential therapeutic tool in regenerative medicine -a review dendrimers in oncology: an expanding horizon photosensitizer-loaded dendrimer-modified multi-walled carbon nanotubes for photodynamic therapy photodynamic effects of chlorine e attached to single wall carbon nanotubes through noncovalent interactions in vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes photothermal ablation of tumor cells using a single-walled carbon nanotubes-peptide composite carbon nanotubes-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field internalization of mwcnts by microglia: possible application in immunotherapy of brain tumors dispersion stability and exothermic properties of dnafunctionalized single-walled carbon nanotubes chloroquine-enhanced gene delivery novel therapeutic strategies for treatment of visceral leishmaniasis targeted delivery of amphotericin b to cells by using functionalized carbon nanotubes antifungal activity of amphotericin b conjugated to carbon nanotubes macrophages targeting of amphotericin b through mannosylated multi walled carbon nanotubes dual targeted delivery of doxorubicin to cancer cells using folate-conjugated magnetic multi-walled carbon nanotubes development and characterization of dexamethasone mesylate anchored on multi walled carbon nanotubes targeted killing of leishmania donovani in vivo and in vitro with amphotericin b attached to functionalized carbon nanotubes supramolecular chemistry on water soluble carbon nanotubes for drug loading and delivery soluble single-walled carbon nanotubes as longboat delivery systems for platinum (iv) anticancer drug design multiwalled carbon nanotube-doxorubicin supramolecular complexes for cancer therapeutics drug delivery with carbon nanotubes for in vivo cancer treatment carbon nanotubes filled with a chemotherapeutic agent: a nanocarrier mediates inhibition of tumor cell growth targeted delivery and controlled release of doxorubicin to cancer cells using modified single wall carbon nanotubes drug loading, dispersion stability, and therapeutic efficacy in targeted drug delivery with carbon nanotubes covalently combining carbon nanotubes with anticancer agent: preparation and antitumor activity the response of peritoneal macrophages to dapsone covalently attached on the surface of carbon nanotubes enhanced anticancer activity of multi-walled carbon nanotubes-methotrexate conjugates using cleavable linkers distribution and clearance of peg-single-walled carbon nanotubes cancer drug delivery vehicles in mice reversible targeting and controlled release delivery of daunorubicin to cancer cells by aptamer wrapped carbon nanotubes sirna delivery with functionalized carbon nanotubes hyaluronate tethered smart multi walled carbon nanotubes for tumor-targeted delivery of doxorubicin polymer functionalized single walled carbon nanotubes mediated drug delivery of gliotoxin in cancer cells glycyrrhizin conjugated dendrimer and multi-walled carbon nanotubes for liver specific delivery of doxorubicin functionalized multiwalled carbon nanotubes as ultrasound contrast agents biodegradation of single-walled carbon nanotubes through enzymatic catalysis carbon nanotubes degraded by neutrophil myeloperoxidase induce less pulmonary inflammation oxidative biodegradation of single-and multi-walled carbon nanotubes promises, facts and challenges for carbon nanotubes in imaging and therapeutics the authors report no conflict of interest related to manuscript. key: cord- -zshjrc authors: to, kelvin kai-wang; zhou, jie; chan, jasper fuk-woo; yuen, kwok-yung title: host genes and influenza pathogenesis in humans: an emerging paradigm date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: zshjrc the emergence of the pandemic influenza virus a(h n )pdm in and avian influenza virus a(h n ) in provided unique opportunities for assessing genetic predispositions to severe disease because many patients did not have any underlying risk factor or neutralizing antibody against these agents, in contrast to seasonal influenza viruses. high-throughput screening platforms and large human or animal databases from international collaborations allow rapid selection of potential candidate genes for confirmatory functional studies. in the last years, at least seven new human susceptibility genes have been identified in genetic association studies. integration of knowledge from genetic and phenotypic studies is essential to identify important gene targets for treatment and prevention of influenza virus infection. influenza virus is one of the most common seasonal respiratory viruses affecting humans, leading to - deaths every year [ ] . the pandemic a(h n ) virus (a[h n ]pdm virus) was estimated to cause respiratory deaths in the first months, with most deaths occurring in patients aged < years [ , ] . the avian influenza viruses a(h n ), a(h n ), a(h n ), a(h n ), together with the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses, are the most virulent respiratory viruses affecting humans [ , - ] . most patients with influenza virus infection develop mild upper respiratory tract infection. life-threatening complications include severe viral pneumonia or secondary bacterial pneumonia, acute respiratory distress syndrome, pulmonary embolism, myocarditis, encephalopathy, reye's syndrome, hemophagocytic syndrome, multiorgan dysfunction and exacerbation of underlying chronic cardiovascular and respiratory diseases [ , ] . currently available antivirals and vaccines have limited efficacy in the treatment and prevention of influenza virus infection [ ] . understanding the pathogenesis of influenza virus infection in human is important in designing novel strategies to improve the management of influenza. to cause infection, influenza virus needs to evade the host immunity, enter and replicate inside host cells, and disseminate to other cells or organs. host damage can be a result of direct virus-induced damage, immune-mediated damage and/or secondary bacterial infection [ ] [ ] [ ] . virologists have extensively studied the role of viral components in the viral life cycle and in the pathogenesis of influenza virus infection in humans [ ] . specific amino acid changes in the viral proteins have been associated with increased disease severity in humans or adaptation of avian influenza viruses in humans [ ] . clinical risk factors for severe influenza have been well described ( figure ) [ ] . however, specific human genes regulating the influenza virus life cycle or virus-induced inflammatory responses are less well described. traditionally, the study of host genes depends on prior knowledge of a candidate gene. the importance of the candidate gene is verified using single gene knockout/ knockdown or gain-of-function studies with reverse genetics in vitro or in vivo. for example, chinese and japanese patients with influenza-associated encephalopathy (iae) were found to have elevated (c : + c : )/ c acylcarnitines ratios. carnitine palmitoyltransferase (cpt ) variants f c and v i were found to be overrepresented in these iae patients when compared with the general population. these mutations render this enzyme susceptible to inactivation at high temperature occurring in febrile patients [ , ] . high-throughput screening platforms have allowed researchers to systematically screen a large number of genes associated with influenza virus infection in vitro, in animals or in humans. the availability of deep sequencing data of the human genome allows researchers to compare genetic variations between influenza patients and the general population [ ] . these technological advances, combined with in vitro or bioinformatics analysis, have revealed several genes associated with severe influenza ( figure and table ). in this review, we focus on specific host genes that have been shown to be directly related to the pathogenesis of human influenza identified or with updated knowledge in the past years. four types of surfactant proteins are found in human pulmonary surfactant. both surfactant protein a (sftpa ) and surfactant protein d (sftpd) are collectins and exhibit antiviral activity against influenza virus [ ] . sftpa variants (rs -c, rs -a, and haplotype a[ ]) have been associated with more severe respiratory deterioration [ ] . surfactant protein b (sftpb) is a hydrophobic protein responsible for the structural integrity of the pulmonary alveoli [ ] . in a cohort of chinese patients with severe and mild a(h n )pdm infection who were matched for age, sex and underlying conditions, the snp rs -c was significantly associated with severe disease. the association between rs -c and severe disease was verified in a second cohort patients with a(h n )pdm infection, in which multivariate analysis was performed to adjust for potential confounding factors [ ] . the snp rs -c has been associated with glycosylation at the amino acid residue of sftpb, which reduces secretion of sftpb [ ] . it remains to be determined whether sftpb has direct antiviral activity against influenza virus. surfactant protein c (sftpc) is a lipoprotein with antiviral activity against respiratory syncytial virus [ ] , but its antiviral activity against influenza virus or its genetic variants associated with severe influenza has not yet been reported. lectin, galactoside-binding, soluble, (lgals ), also known as galectin , can bind to influenza viruses and inhibit viral replication [ ] . carriage of lgals rs /rs haplotype gg was associated with protection from a(h n ) virus infection in humans. furthermore, rs /rs haplotype gg was correlated with higher levels of lgals mrna and protein expression in lymphoblast cell lines [ ] . therefore, the differential lgals expression may contribute to the distinct susceptibility of some individuals to human a(h n ) influenza. upon influenza virus infection, type i interferons are produced by host cells to limit viral replication. the host factors -age -pregnancy, obesity, underlying conditions -pre-existing level of immunity -genetic susceptibility function of type i interferons is mediated via the expression of many interferon stimulated genes (isgs). several isgs are especially important in the pathogenesis of influenza virus infection. interferon-induced transmembrane proteins (ifitm), including ifitm , ifitm and ifitm , were identified to have direct antiviral activity against influenza virus in a genomewide sirna screen [ ] . a human long noncoding rna, which reduces ifitm gene expression, enhanced influenza virus replication in a transgenic mouse model [ ] . ifitm , which is located in endosome, inhibits viral replication by blocking the fusion of the viral and the host membrane [ , ] . interestingly, amphotericin b, an antifungal, was found to increase viral replication by interfering with the blockage of membrane fusion by ifitm [ ] . the c/c genotype of rs is associated with a -amino-acid truncation at the n-terminal of the ifitm protein, which alters the localization of the ifitm from endosomal compartment to the cell periphery [ ] . several studies have looked into ifitm snp rs in the susceptibility to severe influenza in humans. the genotype rs -c was over-represented in caucasian and chinese patients with severe a(h n )pdm virus infection [ , ] . in patients with a(h n ) infection, those carrying rs -c/c genotype had higher mortality [ ] . however, the importance of rs -c allele in severe influenza has been challenged. firstly, in a study using pseudotyped influenza a viruses, transfection of a cells with plasmids expressing the truncated form of ifitm could reduce viral replication similarly to those with plasmids carrying the full length ifitm [ ] . secondly, in a study comparing patients with severe a(h n )pdm virus infection and > controls, no significant difference in the rs genotype frequency was found [ ] . interferon regulatory factor (irf ) is a transcription factor regulating the expression of type i interferons [ ] . influenza a virus replicates to high titers in madin-darby canine kidney cells with knockdown of the irf gene [ ] . murine tracheal epithelial cells deficient in irf have impaired expression of interferons after influenza virus infection [ ] . in a study comparing the genetic variants in healthy individuals, irf snp rs host genes and influenza pathogenesis to et al. host genetic determinants of influenza virus disease severity identified in humans. host genes that have been associated with severe influenza are highlighted in red. was found to be associated with the induction of antiviral genes in dendritic cells in response to influenza virus infection [ ] . in a -year-old girl without known immunodeficiency who suffered from severe influenza virus infection, irf mutation was identified using whole exome sequencing [ ] . the induction of type i and type iii interferon genes in dendritic cells and pulmonary epithelial cells were found to be impaired in this girl. post-translational cleavage of influenza virus hemagglutinin by host protease is a prerequisite for the virus-host membrane fusion and thereby, for virus infectivity, tissue tropism and virus pathogenicity [ ] . transmembrane protease, serine (tmprss ), a type ii transmembrane serine protease, cleaves and activates the viral hemagglutinin during influenza virus infection [ ] . three independent studies assessed the role of tmprss in influenza-infected mice. tmprss knockout mice were resistant to influenza a(h n ) and a(h n ) virus infection, but there were discrepant results regarding susceptibility to a(h n ) virus infection [ ] [ ] [ ] . by integration of a pilot genomewide association study and the lung expression quantitative trait loci (eqtl) dataset, a tmprss intronic snp rs was prioritized for further studies [ ] . the genetic predisposition of rs to severe a(h n )pdm was validated in a(h n )pdm patients including severe cases and mild controls. in the functional study, a regulatory snp rs in high linkage disequilibrium with rs was uncovered as the causal variant underlying the genetic association. genetic predispositions of rs and rs to severe influenza were also validated in an a(h n ) patient cohort. serpine , an isg, encodes plasminogen activator inhibitor (pai- ). a cells over-expressing serpine inhibited the spread of influenza a virus when compared to cells without serpine overexpression [ ] . pai- inhibits the extracellular cleavage of hemagglutinin from ha into ha and ha . serpine knockout mice had higher viral titers and more severe disease than wild type mice. in human fibroblast cell lines derived from patients carrying rs -a allele which results in intracellular retention of pai- , influenza virus replication was enhanced when compared with infection in cell lines deriving from patients carrying rs -t/t genotype. cytokines are important in the defense against influenza virus infection. however, excessive cytokine response is associated with severe influenza [ , ] . tumor necrosis factor-a (tnf-a) has been considered a proinflammatory cytokine, and treatment with anti-tnf-a improved the survival of a(h n )-infected mice [ ] . however, tnfknockout mice had more severe disease [ ] . furthermore, the soluble form of tnf-a has been shown to be required for the control of cd + t cell response [ ] , which suggests that tnf-a is required for the control of infection. a network based approach combining mouse, human and in vitro data showed that the tnf pathway is important in influenza virus infection [ ] . tnf- a and tnf- g alleles have been associated with severe influenza in studies comparing a(h n )pdm patients and healthy controls [ , ] . however, these associations were not found in another study comparing fatal cases and the general population [ ] . interleukin- (il- ) is persistently elevated in patients with severe influenza virus infection [ ] . il- knockout mice were shown to have more rapid viral clearance and improved survival [ ] . blockage of il- b could ameliorate inflammation of influenza-virus infected human pulmonary endothelial cells and lung fibroblasts [ ] . il- - c, il- - a allele and il- - a/a genotype have been associated with severe disease [ ] . in another study involving patients with a(h n )pdm virus infection and healthy controls, rs of il a and rs of il b gene were associated with susceptibility to infection [ ] . however, the associations of snps in il- , il- a and il- b genes with influenza virus disease severity were based on single cohorts, and further studies are necessary to confirm the significance of these polymorphisms in influenza. the interaction between the host antibody fc region and fc receptors is important in the immune defense against influenza virus infection. fc fragment of igg, low affinity iia, receptor (fcgr a) gene encodes fcg receptor iia (fcgriia), which binds immune complexes. the association between fcgr a polymorphism and severe a(h n )pdm virus infection was first identified in a european cohort [ ] , although such association was not found in a chinese cohort [ ] . fcgriia signaling was found to be important in immune complex-mediated platelet activation during influenza virus infection [ ] . interestingly, in recent studies of broadly neutralizing antibodies against influenza viruses, the ability to form immune complex by the interaction between fc region of the antibody and fcg receptors may mediate antibodydependent cellular cytotoxicity, which is important for in vivo efficacy of the antibody [ , ] . tlr is a major sensor of viral double stranded rna. f s mutation of tlr was found to be associated with influenza-associated encephalopathy [ ] , and snp rs -c/t genotype was associated with a(h n )pdm patients with pneumonia [ ] . the importance of tlr has been subsequently confirmed in a knockout mice study [ ] . the future on the study of host genes in humans this review has summarized host genes that have been shown to be important for the pathogenesis of influenza in humans. with high-throughput assays, a large number of genes or genetic variants were found to be associated with viral replication or disease severity in animals or in humans. the current challenge is how we can select specific host genes that are important for the treatment or prevention of influenza virus infection. discrepancies in the results from similar studies also raise the suspicion whether certain genes are indeed important. for example, out of host factors which affect influenza virus replication that were identified during in vitro genomewide sirna knockdown screening, only genes were present in at least two of these screens [ ] . in human genetic association studies, many genetic variants identified to be associated with disease severity in one study could not be reproduced in other studies (table ) . several strategies can be used to improve our ability to find genes that are relevant to influenza pathogenesis in humans. in human genetic association studies, it is important to limit confounding factors as much as possible. for example, the cases and controls should be matched for age, sex, ethnic group and comorbidities, and multivariate analysis can be utilized to assess whether the genetic variant is an independent risk factor. the identified genetic variants should be validated in other independent cohorts. the functional significance of these genetic variants can be refined by eqtl analysis [ ] . pathway analysis helps to identify pathways that may not be apparent when analyzing individual genes. interactome screens, which identify host genes that interact with viral proteins, may increase the likelihood of identifying genes that are potential host targets for treatment [ ] . the establishment of genetically diverse mice population has allowed the identification of susceptibility genes by correlating genetic variations to disease phenotypes [ ] . in addition to genetic data, host response to influenza has also been assessed using transcriptomic, proteomic, metabolomic and lipidomic analysis of cell cultures, animals or humans [ , ] . these data reflect biological regulation of host factors at different levels. an integration of these 'omics' data will improve the accuracy of identifying the susceptibility genes [ ] . although studies have identified several genetic polymorphic genes that predispose to severe influenza, it is possible that these susceptible individuals may have very different susceptibility gene combinations. in the future, it is possible that whole genome sequencing will allow more accurate identification of susceptibility gene combinations in any particular individual (figure ). in addition to comparing severe and mild influenza cases, another approach to identify host susceptibility genes is to examine patients who are resistant to influenza virus infection. in a study of a patient with a congenital disorder of glycosylation but without serological evidence of influenza virus infection, virus production was only found in of macrophage cultures derived from this patient [ ] . the authors of this study postulated that the lack of glycosylation of viral surface proteins may affect virus production. understanding the role of host factors in virus infections have led to host-targeted antivirals. das removes sialic-acid containing receptors from respiratory epithelial cells and prevents virus attachment to host cells. in a phase ii clinical trial, das has been shown to reduce nasal or pharyngeal viral load in influenza patients [ ] . antivirals and anti-inflammatory agents targeting host factors and vaccine strategies improving host response have shown promise in animal models. for example, sphingosine- -phosphate agonist, protectin d and anti-leptin antibody have been shown to protect mice against lethal influenza virus infection [ , , ] . imiquimod, a tlr agonist, has been shown to expedite and augment antibody response after influenza virus vaccine in both animal and human studies [ , ] . an integration of in vitro, animal and clinical data will allow researchers engineering for viral resistance papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest world health organization: influenza (seasonal) estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study two years after pandemic influenza a/ /h n : what have we learned? viral lung infections: epidemiology, virology, clinical features, and management of avian influenza a(h n ) emergence in china of human disease due to avian influenza a(h n ) -cause for concern? middle east respiratory syndrome coronavirus: another 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compared to the general population interferon-induced transmembrane protein- genetic variant rs -c is associated with severe influenza in chinese individuals early hypercytokinemia is associated with interferon-induced transmembrane protein- dysfunction and predictive of fatal h n infection ifitm polymorphism rs -c restricts influenza a viruses ifitm and susceptibility to respiratory viral infections in the community the irf family transcription factors at the interface of innate and adaptive immune responses high yield production of influenza virus in madin darby canine kidney (mdck) cells with stable knockdown of irf type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia common genetic variants modulate pathogen-sensing responses in human dendritic cells this is the first report of the use of whole exome sequencing in identifying genes associated with susceptibility to severe influenza the hemagglutinin: a determinant of pathogenicity influenza and sars-coronavirus activating proteases tmprss and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts the host protease tmprss plays a major role in in vivo replication of emerging h n and seasonal influenza viruses tmprss is a host factor that is essential for pneumotropism and pathogenicity of h n influenza a virus in mice tmprss is essential for influenza h n virus pathogenesis in mice the identification of tmprss as the susceptible gene for severe illness of pandemic a(h n ) influenza and infection of a(h n ) influenza a serpin shapes the extracellular environment to prevent influenza a virus maturation this study demonstrated the importance of serpine gene in influenza virus pathogenesis by showing the differences in the phenotype of cells isolated from patients with different genetic variant in serpine gene inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal h n influenza infection in mice negative regulation of lung inflammation and immunopathology by tnf-alpha during acute influenza infection soluble, but not transmembrane, tnf-alpha is required during influenza infection to limit the magnitude of immune responses and the extent of immunopathology prioritizing genes responsible for host resistance to influenza using network approaches this study incorporated in vitro, animal and human data to prioritize genes for future research on genetic susceptibility to severe influenza role of tumor necrosis factor gene single nucleotide polymorphisms in the natural course of influenza a h n virus infection plasma cytokine levels and cytokine gene polymorphisms in mexican patients during the influenza pandemic a(h n )pdm a pilot study of host genetic variants associated with influenza-associated deaths among children and young adults a detrimental effect of interleukin- on protective pulmonary humoral immunity during primary influenza a virus infection induction of interleukin- beta (il- beta) is a critical component of lung inflammation during influenza a (h n ) virus infection genetic variants in il a and il b contribute to the susceptibility to pandemic h n influenza a virus genetic variants associated with severe pneumonia in a/h n influenza infection the lower serum immunoglobulin g level in severe cases than in mild cases of pandemic h n influenza is associated with cytokine dysregulation influenza virus h n activates platelets through fcgammariia signaling and thrombin generation broadly neutralizing influenza hemagglutinin stem-specific antibody cr targets residues that are prone to escape due to host selection pressure broadly neutralizing hemagglutinin stalk-specific antibodies require fcgammar interactions for protection against influenza virus in vivo a missense mutation of the toll-like receptor gene in a patient with influenza-associated encephalopathy toll-like receptor gene 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hypogammaglobulinemia, and resistance to viral infections a phase ii study of das , a novel host directed antiviral for the treatment of influenza infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection leptin mediates the pathogenesis of severe pandemic influenza a(h n ) infection associated with cytokine dysregulation in mice with dietinduced obesity recombinant influenza a virus hemagglutinin ha subunit protects mice against influenza a(h n ) virus infection immunogenicity of intradermal trivalent influenza vaccine with topical imiquimod: a double blind randomized controlled trial ifitm limits the severity of acute influenza in mice proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium human h-ficolin inhibits replication of seasonal and pandemic influenza a viruses a functional variation in cd increases the severity of pandemic h n influenza a virus infection thermal instability of compound variants of carnitine palmitoyltransferase ii and impaired mitochondrial fuel utilization in influenza-associated encephalopathy tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells enrichment of variations in kir dl /s and kir dl /l among h n / icu patients: an exploratory study killer-cell immunoglobulin-like receptors (kir) in severe a (h n ) influenza infections chemokine receptor big up tri, open allele in patients with severe pandemic (h n ) the ccr delta allele is not a major predisposing factor for severe h n pdm infection characterization in vitro and in vivo of a pandemic h n influenza virus from a fatal case key: cord- -t ghng authors: santos, roberto parulan; tristram, debra title: a practical guide to the diagnosis, treatment, and prevention of neonatal infections date: - - journal: pediatric clinics of north america doi: . /j.pcl. . . sha: doc_id: cord_uid: t ghng neonatal infections continue to cause morbidity and mortality in infants. among approximately , infants followed nationally, the incidence rates of early-onset sepsis infection within days of life are . cases per live births. newborn infants are at increased risk for infections because they have relative immunodeficiency. this article provides evidence-based practical approaches to the diagnosis, management, and prevention of neonatal infections. neonatal infections continue to cause morbidity and mortality in infants. among approximately , infants followed nationally, the incidence rates of early-onset sepsis (eos) infection within days of life were . cases per live births. more than two-thirds of the frequently isolated organisms were associated with group b streptococcus (gbs) ( %) and escherichia coli ( %). although % of the term infants were treated in the newborn nursery, % of the infected infants required intensive care management. of those who survived beyond days of life, about % had an episode of late-onset sepsis (los) infection after days of life. the overall mortality rate of infected infants was %. newborn infants are at increased risk for infections because they have relative immunodeficiency. this may be due to decreased passage of maternal antibodies in preterm infants and to immaturity of the immune system in general. , the innate immune functions in infants are impaired with decreased production of inflammatory markers (interleukin and tumor necrosis factor) and with decreased dendritic and neutrophil functions. the adaptive immune system is less than optimal with decreased cytotoxic functions, decreased cell mediated immunity, and delayed or lack of isotype switching. , complement is important in opsonization and bacterial killing. in term infants, complement levels are approximately half compared with adults. taken together, these predispose infants to severe, prolonged, or recurrent infections associated with bacterial, viral, or fungal infections. suspected sepsis, presumed infection, and ruling out sepsis remain the most common diagnoses in the nursery intensive care unit (nicu). the american academy of pediatrics (aap) committee on fetus and newborn has published a clinical report extensively discussing clinically relevant challenges: identifying newborns with signs of sepsis with high likelihood of eos requiring antimicrobial regimen and identifying healthy-appearing newborns with high likelihood of eos requiring antimicrobial regimen. the committee concluded that, although these guidelines are evidencebased, they may be modified by the clinical judgment of the provider. the primary reason is that the clinical presentation of neonatal infection may be subtle and nonspecific, and may overlap with noninfectious causes. , many clinicians empirically start broad spectrum antimicrobial regimen for infants considered at risk for sepsis but antibiotics are occasionally continued despite a negative blood culture. this practice may be detrimental to the infant because it increases the risk of invasive fungal infections, necrotizing enterocolitis (nec), or death, , which increases the pressure for selecting multidrug-resistant organisms and even the risk of los. the purpose of this article is to provide evidence-based practical approaches to the diagnosis, management, and prevention of neonatal infections. the timing of transmission is one of the factors contributing to the cause of neonatal infections. different pathogens may be acquired during pregnancy (prenatal), during delivery (perinatal), or after delivery (postnatal). table shows the different periods of transmission of various neonatal pathogens. the introduction of new molecular-based assays, such as quantitative real-time polymerase chain reaction (pcr), has paved the way for increasing recognition of respiratory viral infections contributing to ruling out sepsis in late-onset infections. table includes respiratory viral infections (coronavirus, enterovirus, human metapneumovirus, influenza, parainfluenza virus, respiratory syncytial virus [rsv], and rhinovirus) as possible causes of postnatal infections in infants. [ ] [ ] [ ] clinical presentations early-onset infections eos is arbitrarily defined as infection within the first days of life. the most common organisms associated with eos include gbs and e coli. , in general, the risk of bacterial infection in a healthy-appearing newborn remains relatively low. the most common clinical findings include hypoglycemia (< mg/dl, %) and hypothermia (< . c, %), followed by hyperglycemia (> mg/dl, %) and apnea ( %). edwards and baker summarized that newborn infants with sepsis manifest similar clinical signs as those with meningitis, including hyperthermia; hypothermia; respiratory distress; anorexia or vomiting; jaundice; and lethargy. hypotension may be more frequently found in infants with sepsis, whereas irritability, convulsions, and bulging or full fontanel is found in those with meningitis. however, they cautioned that absence of any of the aforementioned signs do not exclude central nervous system involvement. furthermore, it was suggested to evaluate infants for various foci of infections such as acute otitis media, conjunctivitis, osteomyelitis, pyogenic arthritis, and skin soft-tissue infections. los is arbitrarily defined as infection after days of life. the most common organisms isolated with los include coagulase-negative staphylococci in more than a third of the cases, which may or may not be associated with a medical device. yeast or candida spp infection is another important pathogen. also, there is increasing recognition of viral respiratory infections as a possible cause in los. the most common clinical findings include hypothermia ( %), hyperglycemia ( %), apnea ( %), and bradycardia ( %). there are several factors that may increase the risk for los. there are significantly more infants with los who have an indwelling central vascular catheter at the time of infection than those infants with eos ( % vs %, p<. ). additionally, there are more infants with los who had a surgical procedure before infection ( % vs %, p<. ). the clinical presentations of infections may overlap with noninfectious causes in newborns. it has been previously demonstrated that relying on symptoms alone may not be sufficient in diagnosing neonatal infections. bacteremia has been reported in infants without clinical signs of sepsis. there are several diagnostic tests and principles that may guide clinicians in evaluating infants with infections. the aap committee on fetus and newborn have published a clinical report on the evaluation of asymptomatic infants (< and ! week gestation) with risk factors for sepsis. evaluation of asymptomatic preterm infants (< -week) with risk factors for sepsis is shown in fig. . similar algorithms for the evaluation of asymptomatic term infants (! week gestation) are available from the aap committee on fetus and newborn. (http://www.ncbi.nlm.nih.gov/pubmed/ ). additional principles in the evaluation of infants with risk factors for sepsis follow: major risk factors for neonatal sepsis include chorioamnionitis, prolonged rupture of membrane or more hours, and colonization of gbs with inadequate intrapartum antimicrobial prophylaxis (iap). chorioamnionitis usually presents as maternal fever greater than c ( . f) and its diagnosis should be discussed with the obstetric providers. maternal fever may be the only abnormal finding in chorioamnionitis. adequate iap means maternal treatment with penicillin, ampicillin, or cefazolin at or earlier than hours before delivery. at least ml of blood may be sufficient for a single blood culture from a peripheral vein. blood culture from umbilical artery catheter or umbilical vein may be a reliable alternative following aseptic techniques screening blood cultures have not been proven of value and are not recommended. complete blood count with differential has poor positive predictive value and it is suggested waiting to hours after birth to avoid falsely normal values at birth. platelet counts remain low days to weeks after sepsis; thus this cannot be used in following response to treatment. the sensitivity of c-reactive protein (crp) improves if done to hours after birth. bacterial sepsis is unlikely if crp remains normal. lumbar puncture may be indicated in infants whom sepsis is highly suspected, those infants with bacteremia, and in infants who fail to respond to antimicrobial therapy. urinary tract infection in newborns is associated with episodes of bacteremia; thus urine culture should not be part of routine sepsis workup. microbiologic evaluation using gastric aspirates, tracheal aspirates, or superficial body sites cultures are of limited value and are not routinely recommended for sepsis. several acute-phase reactants or biomarkers (neutrophil cd [ncd ], procalcitonin [pct], or crp) may be used adjunctively in the evaluation and management of neonatal infection. the diagnostic usefulness of the various surrogate markers depends on the phases of neonatal sepsis: early phase or to hours (ncd ), mid phase or - hours (pct), and late phase or greater than hours (crp). ncd is a high-affinity fc receptor that increases with exposure to bacterial or fungal agents. , the usefulness of ncd is related to its high negative predictive value as well as decreasing concentration on serial determinations on infants undergoing antimicrobial treatment of bacteremia. however, there is a scarcity of medical evidence to recommend ncd for routine evaluation of neonatal infection and this may not be readily available. procalcitonin released from tissues increases with infection at around hours and peaks at hours. it may also increase with noninfectious causes such as in respiratory distress syndrome and a physiologic increase during the first hours of birth. pct may not be readily available and the turnaround time varies in different institutions from minutes to hours. crp increases around hours associated with an inflammatory response with release of interleukin- and peaks at hours. crp has been used in the algorithm-based guideline from the aap committee on fetus and newborn for the evaluation of asymptomatic term and preterm infants with a risk factor for sepsis. it is best used as part of a group of diagnostic tests together with blood culture and white blood cell with differential in the evaluation of neonatal infection. however, there is not enough medical evidence at this time to recommend serial determinations of crp in guiding duration of antimicrobial therapy in infants. , further studies are needed to evaluate the usefulness of sequential determination of crp and biomarkers for an antimicrobial stewardship program (asp) in the nicu setting. in , the infectious disease society of america, in collaboration with the american society for microbiology, affirmed the importance of close collaboration and positive working relationships between clinicians and microbiologists to better serve patients. the most up-to-date edition of the red book provides contact information for expert advice and national collaborative study groups that give guidance on diagnostic assays regarding specific agents causing mother-to-child transmission. it is important to know the various microbiologic resources available locally, which include but are not limited to pcr and matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof). rapid antigen tests for respiratory viruses may lack sensitivity, which is important in the nicu setting in controlling local outbreaks. there are several nucleic acid amplification test platforms currently available that differ in the number of analytes detected. it is important to obtain adequate specimens and to use suitable viral transport media following manufacturer instructions. maldi-tof is a valuable alternative to the conventional microbiologic assays; however, it may not be a readily available resource for diagnostic testing in most institutions. however, if it is available, it has several practical applications that may benefit clinical management even in the nicu settings: earlier and accurate diagnosis of neonatal sepsis due to various bacteria rapid identification of highly virulent gbs that causes meningitis and los in infants identification of maternal-to-child transmissions (chorioamnionitis and neonatal infections) of opportunistic pathogen accurate identification of bloodstream infection associated with fungal infections in the nicu identification and monitoring the spread of nosocomial outbreak (eg, methicillinresistant staphylococcus aureus [mrsa] and candida parapsilosis in the nicu). when appropriate specimens for diagnostic evaluations are collected in clinically stable patients, then empirical antimicrobial therapy should be initiated for neonatal sepsis. it is recommended to discuss complicated cases, such as multidrug resistant organisms and infants not improving while on therapy or those requiring unconventional dosing regimens and antimicrobial agents, with pediatric infectious disease specialists. ampicillin and gentamicin remains the cornerstone of initial antimicrobial regimen for early-onset neonatal infections. the combination of such broad-spectrum antibiotic regimens cover the most common cause (gbs and e coli in more than %) of eos and has synergistic activity (against gbs and listeria monocytogenes). , the dosing regimen for ampicillin may change over time based on the chronologic age of the infant and body weight. for example, an -day-old infant weighing greater than g may need dosing adjustment of ampicillin from mg/kg/d intravenous (iv) divided every hours to mg/kg/d iv divided every hours. once-daily dosing of gentamicin ( mg/kg iv qd) has been used in the term newborn for more than a decade. the pharmacodynamic characteristics of aminoglycosides that allow the use of once-daily dosing include concentration-dependent killing (peak concentration to minimal inhibitory concentration [peak/mic] ratio), , postantibiotic effect with leukocyte enhancement, , and prevention of adaptive resistance. third-generation cephalosporins should be used judiciously. there is significant association between the use of third-generation cephalosporins and invasive candidiasis in preterm infants. cefotaxime has excellent penetration to the cerebrospinal fluid and its therapeutic use should be limited to gram-negative meningitis. routine use of cefotaxime for eos may lead to rapid development of drug-resistant organisms. ceftriaxone is contraindicated in neonates for reasons: ( ) it is highly protein bound and may displace bilirubin progressing to hyperbilirubinemia and ( ) concurrent administration with calcium-containing solutions may produce insoluble precipitates (ceftriaxone-calcium salts) leading to cardiorespiratory complications. the aap periodically updates the dosing regimens and recommended therapy for selected neonatal infections through nelson's pediatric antimicrobial therapy. it provides various antimicrobial regimens (antibiotic, antiviral, and antifungal agents) based on body weight of infants and their chronologic age or gestational and postnatal age. between new editions, a monthly update of short and interesting reports related to pediatric antimicrobial therapy is posted at www.aap.org/en-us/aap-store/nelsons/ pages/whats-new.aspx. suggested durations of antibiotic therapy for eos adapted from nelson's pediatric antimicrobial therapy and the aap committee on fetus and newborn are shown in table . there are several antiviral agents that can be used for the treatment of neonatal viral infections. acyclovir ( mg/kg/d iv divided every hours) is the treatment of choice for term infants with herpes simplex virus (hsv) and varicella-zoster infections. there are several topical agents ( . % ganciclovir ophthalmic gel, . % iododeoxyuridine, or % trifluridine) that may be added to systemic antiviral regimen if there is eye involvement. after parenteral therapy with acyclovir, it is recommended to give hsv suppressive regimen ( mg/m /dose po tid), which improves neurodevelopmental outcomes of infants with central nervous system involvement. there is currently no dosing regimen for valacyclovir in infants younger than months of age. the aap committee on infectious diseases and the committee on fetus and newborn recently published an algorithm-based guideline on the evaluation and treatment of asymptomatic infants born to mothers with active herpes lesions (http:// www.ncbi.nlm.nih.gov/pubmed/ ). oral valganciclovir ( mg/kg/dose po bid) is the drug of choice for infants with symptomatic congenital cytomegalovirus (cmv) disease with or without central nervous system involvement. , the treatment of congenital cmv should be initiated in the first month of life. kimberlin and colleagues concluded from the phase iii randomized double-blind placebo-controlled multinational study that months of valganciclovir regimen for symptomatic congenital cmv disease significantly improves hearing and neurodevelopmental outcomes. there is significant improvement in language and receptive communication at years of age. there was less grade to neutropenia at weeks oral valganciclovir (w %) compared with weeks of iv ganciclovir ( %) reported previously. iv ganciclovir ( mg/kg/dose bid) can be used initially for infants with symptomatic congenital cmv disease if oral valganciclovir is contraindicated due to extreme prematurity or nec. the same dosing regimen is the treatment of choice for perinatally or postnatally acquired cmv disease associated encephalitis, hepatitis, pneumonitis, or persistent thrombocytopenia. oral oseltamivir ( mg/kg/dose bid) remains the treatment of choice for term infants with influenza infections. , oral suspension formulation is available ( mg/ml) and should be offered to young infants with suspected or confirmed influenza infection regardless of severity because they are at higher risk for complications. limited data are available for the weight-based dosing regimen for preterm infants using postmenstrual age (ie, gestational age plus chronologic age): less than weeks postmenstrual age, mg/kg/dose po bid to weeks, . mg/kg/dose po bid greater than weeks, mg/kg/dose po bid. there is currently no dosing regimen for inhalational zanamivir for young infants. suggested durations of antiviral therapy, prophylaxis, and suppressive regimen for congenital and perinatal or postnatally acquired viral infections adapted from nelson's pediatric antimicrobial therapy and the aap committee on infectious diseases and the committee on fetus and newborn are shown in table . fig. shows an extensive cutaneous aspergillosis on a preterm infant who was a poor surgical risk successfully treated with combination antifungal agents. fluconazole prophylaxis ( mg/kg/d twice a week) may be indicated in high-risk infants with birth weight of less than g from institutions with high incidence of candidiasis (ie, above %). fluconazole prophylaxis ( mg/kg once weekly) may be offered to young infants younger than months old on extracorporeal membrane oxygenation. suggested durations of antifungal treatment of candidiasis and aspergillosis adapted from nelson's pediatric antimicrobial therapy are shown in table . surgical interventions may be indicated for the source control of neonatal infections. in a single-center -year retrospective study, nec-associated blood stream infection (bsi) occurred within days of nec diagnosis and was noted in approximately % ( out of infants with one episode of bsi). infants with nec-associated bsi had higher odds (adjusted odds ratio . ; % ci . - . ) of having surgical interventions compared with those without bsi. it is of utmost importance to correspond with pediatric surgery regarding source control of infection if clinically indicated because nec-associated bsi had higher odds of death (adjusted odds ratio . ; % ci . - . ). the following includes disease-specific conditions that may require surgical interventions for adequate source control of infections if the infant is clinically stable. pediatric providers are encouraged to discuss with their surgical colleagues the following surgical treatment options : early debridement of cutaneous lesions with disseminated aspergillosis surgical drainage of peritonitis with bowel rupture wound cleaning and debridement rapidly spreading cellulitis (s aureus), necrotizing fasciitis (group a or b streptococci), tetanus neonatorum surgical drainage of pus in osteomyelitis and suppurative arthritis thoracostomy drainage of empyema surgical drainage of breast abscess may be needed to minimize damage to breast tissue. surgical interventions for primary diseases in infants may also increase the risk for neonatal infections. higher rates of surgical site infection defined as superficial, deep, and organ infections within days of surgical procedures were noted among infants following closure of gastroschisis. it is important to closely monitor infants with surgical site infection because they require significantly longer hospital stay. there are various measures that can be used, depending on the availability of local resources, to prevent neonatal infections. these include but are not limited to gbs prophylaxis, hand hygiene, immunization and immunoprophylaxis, asp, probiotics and prebiotics, and care bundles. iap is the only preventive strategy that substantially reduces the incidence of earlyonset gbs. , the following are indications for iap: previous infant with invasive gbs disease gbs bacteriuria during the current pregnancy positive gbs vaginal-rectal screening (at - week gestation) except for cesarean delivery without labor or ruptured membrane unknown maternal gbs status with delivery at less than weeks, rupture of membrane at or before hours, or fever equal to or greater than . f (! c). adequate iap means receiving penicillin, ampicillin, or cefazolin for at least hours before delivery. cefazolin may be used if with nonserious b-lactam allergy. if there is history of serious b-lactam allergy (anaphylaxis, angioedema, respiratory insufficiency, or urticarial rash) and if gbs isolate is susceptible, clindamycin may be used. otherwise, vancomycin is an alternative. because of high resistance rates, erythromycin is not recommended. the center for disease control and prevention has an extensive online resource on gbs for clinicians, including the algorithm-based guidance on secondary prevention of eos in newborns. the web page also provides an application, prevent group b strep, which includes guidance on various patient scenarios in collaboration with different medical societies, such as the aap and the american college of obstetricians and gynecologists (http://www.cdc.gov/groupbstrep/guidelines/index.html). there is no doubt that hand hygiene remains the cornerstone in decreasing health care-associated infections in different hospital settings, including the nicu. in fact, there are various educational programs, multidisciplinary quality-improvement teams, and guidelines on the proven effectiveness of hand hygiene in decreasing infection; however, this is significantly affected by compliance. , the center for disease control and prevention has a web site (http://www.cdc.gov/handhygiene/) containing resources for hand hygiene in health care settings including an application, iscrub, for monitoring hand hygiene compliance using an iphone or ipod touch. thus, hand hygiene guidelines are effective in reducing infections only if we use it. soap and water is recommended for decontaminating visibly soiled hands by rubbing hands together vigorously for seconds. , alcohol-based gel or foam or an antiseptic soap may be used for routine hand hygiene if not grossly contaminated. , hand hygiene compliance is improved if with available alcohol-based products at the infant's bedside. antimicrobial-impregnated towelettes or wipes are considered alternatives but not substitutes for washing with soap and water or alcohol-based gel or foam. the development of a safe and effective vaccine is arguably one of the greatest medical interventions in the last century. hepatitis b vaccine is the only agent in the united states recommended to be administered at birth. the various brands available in the united states have an efficacy of % to % in preventing hepatitis b virus infection and disease. additional information regarding recommended dosages of hepatitis b vaccines are available in the most recent edition of the red book and in the annual publication from the advisory committee on immunization practices. the first dose in the primary series of the subsequent vaccines (diphtheria and tetanus toxoids and acellular pertussis vaccine [dtap], haemophilus influenza type b, inactivated polio, pneumococcal, or rotavirus vaccine) can be administered at a minimum age of weeks. care givers at home should be advised on the importance of immunizing family members to protect infants who are too young to be vaccinated. this is called cocooning and prevents vaccine-preventable diseases, such as pertussis and influenza, in young infants. educational materials on cocooning for parents and clinicians are available at the aap web site http://www .aap.org/immunization/families/ cocooning.html. in october , the use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) during every pregnancy was recommended because of increasing cases of pertussis in young infants in the recent years. the mother's protective antibodies against pertussis are short-lived and a dose of tdap in a previous pregnancy may not be protective to the infants of subsequent pregnancies. young preterm infants (< year of age born at < weeks and -day gestation) should receive palivizumab during the rsv season for immunoprophylaxis. five monthly doses of palivizumab at mg/kg given intramuscularly will provide adequate protection for months. other indications during the rsv season include preterm infants (< year of age born at < weeks and -day gestation) with chronic lung disease of prematurity who required greater than % oxygen for at least the first days of life and young infants (< year of age) with certain hemodynamically significant heart disease. clinicians should consult the most current guidelines or policy statement from the aap regarding palivizumab prophylaxis among young children at increased risk for hospitalization for rsv infection. injudicious use of antibiotics can alter the neonates' microflora that increases exposure and pressure that leads to antimicrobial resistance. the nicu milieu and interventions are permissive for the development of antibiotic-resistant organisms. , the aap committee on fetus and newborn have listed asp strategies that may be useful in the nicu setting based on the guideline from the infectious diseases society of america and the society for healthcare epidemiology of america (box ). there is some medical evidence supporting the use of probiotics in the prevention of nec in preterm infants. probiotic is an oral supplement containing sufficient amount of viable microorganisms that alters the host microflora with potential for health benefits. a meta-analysis based on randomized control trials involving approximately infants born before or at weeks gestation and/or weighing less than or equal to g at birth showed that enteral use of probiotic significantly decreased the incidence of severe nec and mortality. there were no severe adverse events or systemic infections directly related to the probiotics used were reported. the aap committee on nutrition, however, cannot recommend the use of all probiotics in young infants until further studies are done to resolve problematic issues. they noted the large heterogeneity of the studies included in the review, the different mixture of probiotics used, and that the combinations of probiotics used in the studies are not available in the united states. further, there remains some gap in knowledge on which probiotic bacteria species to use, the microbial dose, as well as the duration of administration. in , an updated review of the aforementioned meta-analysis of randomized controlled trials continues to support a change in practice of supplementing preterm infants with probiotics. the review provided similar results involving more than infants in whom probiotics significantly reduced severe nec and mortality. however, the previously mentioned gap in knowledge remains, as well as the need for comparative studies. there is scarcity of medical evidence to recommend the addition of prebiotics such as oligosaccharides in infant formula. prebiotics are nondigestible food ingredients that occur naturally or as dietary supplements that enhance growth of probiotic bacteria such as bifidobacterium spp. several studies had reported that the addition of prebiotics in infant formula significantly increased the bifidobacteria counts in their stool without adverse effects noted. however, clinical efficacy as well as cost-benefit analyses regarding the addition of oligosaccharides to infant formulas is lacking. for infants, human milk remains the best source of naturally occurring prebiotics and probiotics, and immunoprotective compounds known to decrease the incidence of respiratory and gastrointestinal infections. , nursery intensive care unit care bundles there are invasive procedures that may increase the infant's risk of health care-associated infections in the nicu setting. these infections include central line-associated bsis (clabsis), pneumonia, skin, and soft tissue infections; and, occasionally, vaccine-preventable diseases and outbreak of respiratory viral infections. care bundles are sets of interventions aimed at reducing health care-associated infections in the nicu. the most common cause of clabsi and los are coagulase-negative staphylococci. several randomized clinical trials on the use of low-dose vancomycin in parenteral solutions in preterm infants did not show significant decrease in the length of stay and mortality. there is an antibiotic-lock therapy done in neonates that significantly decrease clabsi however it was not powered to answer whether vancomycin resistance occurred. both are currently not recommended because of the lack of long-term efficacy evidence as well as concern for development of drug-resistant organisms. infection control intended to decrease clabsi in the nicu should include measures to decrease extraluminal and intravascular catheter-related infections. various techniques and guidelines in the prevention of clabsi in infants adapted from the aap committee on fetus and newborn are shown in box . there are specific practices that may be adapted in the local setting for preventing vaccine-preventable diseases and outbreaks of respiratory viral infections. these include but are not limited to vaccination of health care providers against influenza and pertussis (tdap), visitation guidelines to screen ill or symptomatic visitors, and cohorting in cases of clustering of infections or in outbreak situations. cohorting may only be possible if early screening procedures, such as the use of pcr-based assays, are in place if available in cases of clustering of respiratory viral infections. [ ] [ ] [ ] further, appropriate isolation (eg, contact precautions for mrsa, droplet precautions for influenza, and airborne precautions for measles) should be observed if the infant is clean the umbilical insertion site using an antiseptic such as povidone-iodine before catheter insertion. avoid using topical antibiotic ointment or creams on insertion sites to prevent fungal infections and antimicrobial resistance. use low doses of heparin ( . - . u/ml) to the fluid infused through umbilical arterial catheter. remove umbilical catheters as soon as no longer needed or if signs of vascular insufficiency to the lower extremities (for umbilical artery access) are present; they may be replaced if malfunctioning. umbilical artery catheters should not be left in place for more than days. umbilical venous catheters may be used up to days if managed aseptically. colonized or infected with a pathogen requiring additional protection beyond standard precautions. neonatal infections continue to cause morbidity and mortality in infants. gbs and e coli are the most common agents of eos, whereas coagulase-negative staphylococcus is the predominant cause for los. there is increasing recognition of respiratory viral infections contributing to ruling out sepsis in very young infants whose presentations are similar to bacterial infections. blood culture at birth and white blood cell with or without crp has been used in the algorithm-based guideline for the evaluation of asymptomatic term and preterm infants with risk factors for sepsis. ampicillin and gentamicin remains the cornerstone of initial antimicrobial regimen for neonatal infections. third-generation cephalosporins should be used judiciously. the use of antiviral (acyclovir, ganciclovir, valganciclovir, and oseltamivir) and antifungal (fluconazole, amphotericin b, and voriconazole) treatment and prophylactic regimens may reduce mortality and morbidity to specific viral and fungal disease in infants. there are various strategies, such as gbs prophylaxis, hand hygiene, immunization, and immunoprophylaxis, asp, probiotics, and prebiotics, and nicu care bundles, which may be used in preventing infections in infants. early onset neonatal sepsis: the burden of group b streptococcal and e. coli disease continues neonatal infectious diseases: evaluation of neonatal sepsis the many faces of b cells: from generation of antibodies to immune regulation neonatal innate tlr-mediated responses are distinct from those of adults innate immunity of the newborn: basic mechanisms and clinical correlates the neonatal adaptive immune system committee on fetus and newborn. management of neonates with suspected or proven early-onset bacterial sepsis antibiotic use and misuse in the neonatal intensive care unit the association of third-generation cephalosporin use and invasive candidiasis in extremely low birth-weight infants prolonged duration of initial empirical antibiotic treatment is associated with increased rates of necrotizing enterocolitis and death for extremely low birth weight infants prolonged initial empirical antibiotic treatment is associated with adverse outcomes in premature infants duration of empiric antibiotics for suspected early-onset sepsis in extremely low birth weight infants clinical utility of pcr for common viruses in acute respiratory illness viral respiratory tract infections in the neonatal intensive care unit: the virion-i study unrecognized viral respiratory tract infections in premature infants during their birth hospitalization: a prospective surveillance study in two neonatal intensive care units nosocomial rhinovirus infection in preterm infants principles and practice of pediatric infectious diseases successful medical treatment of cutaneous aspergillosis in a premature infant using liposomal amphotericin b, voriconazole and micafungin seventy-five years of neonatal sepsis at yale: - neonatal sepsis workups in infants >/ grams at birth: a population-based study principles and practice of pediatric infectious diseases culture negative sepsis and systemic inflammatory response syndrome in neonates utility of complete blood count and blood culture screening to diagnose neonatal sepsis in the asymptomatic at risk newborn effective biomarkers for diagnosis of neonatal sepsis a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: recommendations by the infectious diseases society of america (idsa) and the american society for microbiology (asm)(a) laboratory medicine in neonatal sepsis and inflammation group b streptococcus st- and emerging st- clones by maldi-tof mass spectrometry capnocytophaga species and perinatal infections: case report and review of the literature bloodstream infections by malassezia and candida species in critical care patients first outbreak of pvl-positive nonmultiresistant mrsa in a neonatal icu in australia: comparison of maldi-tof and snp-plus-binary gene typing maldi-tof mass spectrometry and microsatellite markers to evaluate candida parapsilosis transmission in neonatal intensive care units nelson's pediatric antimicrobial therapy introduction to paediatric and perinatal drug therapy the therapeutic monitoring of antimicrobial agents in vitro postantibiotic effect and postantibiotic leukocyte enhancement of tobramycin once-daily versus multiple-daily dosing of aminoglycosides pharmacodynamic factors of antibiotic efficacy gentamicin vs cefotaxime for therapy of neonatal sepsis. relationship to drug resistance intravenous ceftriaxone and calcium in the neonate: assessing the risk for cardiopulmonary adverse events oral acyclovir suppression and neurodevelopment after neonatal herpes committee on infectious diseases, et al. guidance on management of asymptomatic neonates born to women with active genital herpes lesions six months versus six weeks of oral valganciclovir for infants with symptomatic congenital cytomegalovirus (cmv) disease with and without central nervous system (cns) involvement: results of a phase iii, randomized, double-blind, placebo-controlled, multinational study. id week. infectious disease society of america committee on infectious diseases. recommendations for prevention and control of influenza in children concurrent bloodstream infections in infants with necrotizing enterocolitis surgical site infections in infants admitted to the neonatal intensive care unit prevention of perinatal group b streptococcal disease-revised guidelines from cdc group b strep (gbs) strategies for prevention of health careassociated infections in the nicu healthcare infection control practices advisory committee, et al. guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hic-pac/shea/apic/idsa hand hygiene task force hygiene in healthcare settings how to communicate with vaccine-hesitant parents recommended childhood and adolescent immunization schedule-united states committee on practice and ambulatory medicine, et al. immunizing parents and other close family contacts in the pediatric office setting updated recommendations for use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) in pregnant women-advisory committee on immunization practices (acip) importance of timing of maternal combined tetanus, diphtheria, and acellular pertussis (tdap) immunization and protection of young infants updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection neonatal sepsis: the gut connection infectious diseases society of america and the society for healthcare epidemiology of america guidelines for developing an institutional program to enhance antimicrobial stewardship probiotics and prebiotics in pediatrics probiotics for prevention of necrotizing enterocolitis in preterm infants probiotics for prevention of necrotizing enterocolitis in preterm infants transmission-based precautions key: cord- -jh dyyz authors: alenquer, marta; amorim, maria joão title: exosome biogenesis, regulation, and function in viral infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jh dyyz exosomes are extracellular vesicles released upon fusion of multivesicular bodies (mvbs) with the cellular plasma membrane. they originate as intraluminal vesicles (ilvs) during the process of mvb formation. exosomes were shown to contain selectively sorted functional proteins, lipids, and rnas, mediating cell-to-cell communications and hence playing a role in the physiology of the healthy and diseased organism. challenges in the field include the identification of mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles and the understanding of the underlying processes directing mvbs for degradation or fusion with the plasma membrane. the investigation into the formation and roles of exosomes in viral infection is in its early years. although still controversial, exosomes can, in principle, incorporate any functional factor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation. this review initially focuses on the composition and biogenesis of exosomes. it then explores the regulatory mechanisms underlying their biogenesis. exosomes are part of the endocytic system, which is tightly regulated and able to respond to several stimuli that lead to alterations in the composition of its sub-compartments. we discuss the current knowledge of how these changes affect exosomal release. we then summarize how different viruses exploit specific proteins of endocytic sub-compartments and speculate that it could interfere with exosome function, although no direct link between viral usage of the endocytic system and exosome release has yet been reported. many recent reports have ascribed functions to exosomes released from cells infected with a variety of animal viruses, including viral spread, host immunity, and manipulation of the microenvironment, which are discussed. given the ever-growing roles and importance of exosomes in viral infections, understanding what regulates their composition and levels, and defining their functions will ultimately provide additional insights into the virulence and persistence of infections. multivesicular bodies (mvbs) or late endosomes are components of the endocytic pathway that range from to nm in diameter. within mvbs are vesicles called intraluminal vesicles (ilvs) that range from to nm in diameter. mvbs can either be degraded or can fuse with the plasma membrane, releasing the ilvs into the extracellular space. the ilvs are called exosomes following their release from the mvb [ ] . the formation of the ilvs within the mvb and the budding of enveloped virions share many features. both processes require induction of membrane curvature, inclusion of specific cargo, and membrane fission for release. what is most striking is that evolutionarily unrelated viruses, with dramatically different genomes, have converged in their use of the host machinery for ilv formation to promote their own budding. important human pathogens viruses , , - more detail below [ ] . secretion of exosomes requires maturation of early endosomes into mvbs (a process reviewed in [ ] ), with concomitant formation of ilvs, and fusion of mvbs with the cell surface to release exosomes. at any point, material can be further internalized to the tgn and integrated in canonical secretory pathways [ , ] . viruses , , -x figure . the endocytic and secretory pathways. cargo binds to the plasma membrane, is endocytosed by a plethora of processes and, independently of the entry route, is transported to early endosomes (ee). from this sub-compartment, cargo is sorted to one of three destinations: recycling, degradation, or secretion. these routes require maturation of the ee into recycling endosomes or multivesicular bodies (mvbs), which can either fuse with lysosomes (l) to generate endolysosomes (el) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. the membranes of the sub-compartments of the endocytic pathway have different compositions. specific members of the rab gtpase family, for example, are differentially enriched in each sub-compartment: rab is enriched in ee; rab in mvbs; rab , rab , rab , and rab in the slow and rapid recycling routes; and rab a/b in mvbs. rab is present in vesicles destined for retrograde transport to the trans-golgi network (tgn). in uninfected cells, interfering with these rabs affects exosome release. many viruses use these rabs in diverse steps of the viral life cycle, although whether this usage impacts in exosomal release has not been investigated. for example, at late stages of infection, viruses such as iav, rsv, sendai virus (sev), and andes virus (andv) were shown to hijack rab vesicles to transport their progeny rna to the cell surface. hiv, hsv , and human cytomegalovirus (hcmv) were shown to require rab a/b vesicles for assembly. human herpes (hhv- ) virions were shown to be secreted upon fusion of mvb with the plasma membrane, together with exosomes. the endocytic pathway is a convoluted web of interconnected sub-compartments with distinct cell localization, lipid and protein composition, and ph, which operates as follows: cells internalize ligands by endocytosis concomitantly with membrane proteins and lipids [ , ] . irrespectively of figure . the endocytic and secretory pathways. cargo binds to the plasma membrane, is endocytosed by a plethora of processes and, independently of the entry route, is transported to early endosomes (ee). from this sub-compartment, cargo is sorted to one of three destinations: recycling, degradation, or secretion. these routes require maturation of the ee into recycling endosomes or multivesicular bodies (mvbs), which can either fuse with lysosomes (l) to generate endolysosomes (el) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. the membranes of the sub-compartments of the endocytic pathway have different compositions. specific members of the rab gtpase family, for example, are differentially enriched in each sub-compartment: rab is enriched in ee; rab in mvbs; rab , rab , rab , and rab in the slow and rapid recycling routes; and rab a/b in mvbs. rab is present in vesicles destined for retrograde transport to the trans-golgi network (tgn). in uninfected cells, interfering with these rabs affects exosome release. many viruses use these rabs in diverse steps of the viral life cycle, although whether this usage impacts in exosomal release has not been investigated. for example, at late stages of infection, viruses such as iav, rsv, sendai virus (sev), and andes virus (andv) were shown to hijack rab vesicles to transport their progeny rna to the cell surface. hiv, hsv , and human cytomegalovirus (hcmv) were shown to require rab a/b vesicles for assembly. human herpes (hhv- ) virions were shown to be secreted upon fusion of mvb with the plasma membrane, together with exosomes. no underlying mechanism has yet been reported to differentiate mvb formation for degradation or fusion with the cell membrane. there are, however, reports suggesting the existence of subpopulations of mvbs, depending on their fate. using a biotinylated derivative of the cholesterol-binding toxin perfringolysin o from clostridium perfringens to localize cholesterol at the subcellular level, möbius et al. identified two types of mvbs of similar morphology, one cholesterol-rich that was destined for secretion, and the other cholesterol-poor, which was destined for degradation [ ] . conversely, two sub-populations of mvbs differing in the presence or absence of lysobisphospatidic acid have been described. for example, egf and its receptor (egfr) are only present in mvbs negative for lysobisphospatidic acid [ ] . this supports the previously described role of phosphatidyl inositol (pi) in the formation of mvbs containing egfr [ ] , but not lysobisphospatidic acid [ ] . another study showed that a full-length p receptor was only released from sympathetic neurons and pc cells upon kcl-mediated cell depolarization. under this stimulus, p -containing mvbs escaped endolysosomes and degradation [ ] . whether this reflects distinct steps in mvb trafficking or even distinct mechanisms in mvb formation that mark them for degradation or fusion with the membrane is still unclear. ilv formation is characterized by inward budding of membranes, a process that starts in early endosomes but greatly augments as endosomes mature [ ] . evidence indicates that exosomes correspond to secreted ilvs of mvbs. relative to the composition of the cytoplasm, exosomes are enriched in components such as lipids, rnas, and proteins. lipids include cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylcholine with saturated fatty acids [ ] [ ] [ ] . enriched rnas are specific mirnas, non-coding rnas, trnas, rrnas, and mrnas [ ] [ ] [ ] [ ] . finally, proteins found in higher concentration than in the cytosol include specific factors of the immune system, those of the escrt apparatus, those involved in trafficking, and lipid-rafts residents. the latter are, for example, cytokines, tetraspanins, major histocompability complex (mhc) class i and ii molecules, glycosylphosphatidylinositol-anchored proteins, rabs, snares, and flotillin [ , , , [ ] [ ] [ ] . importantly for the context of this review, cells infected with viruses were shown to release exosomes containing viral proteins and rnas [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (reviewed in [ ] ). some viral proteins, including the hiv nef, seem to have exosomal localization signals [ , ] . however, in the majority of cases, it is unclear whether the inclusion of viral components in exosomes results from direct sorting or from hijacking the machinery for exosome biogenesis, trafficking, and/or release. nevertheless, the specific composition of exosomes derived from cells, regardless of their infectious state, suggests that soluble and membrane-bound cargo are selectively incorporated into ilvs [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the formation of ilvs is accomplished by several molecular mechanisms. proteins belonging to the escrt family are, by far, the best characterized and have been extensively reviewed [ , , , [ ] [ ] [ ] . there are, however, ubiquitin-and escrt-independent pathways [ ] , including the oligomerization of the tetraspanin complexes [ ] , the sphingomyelinase pathway that catalyzes ceramide synthesis [ ] , or phospholipase d and adp rybosylation factor- -mediated ilv budding [ ] . one way for viral modulation of exosome release is by directly interfering with the machinery involved in exosome biogenesis; this has been reviewed elsewhere for the escrt machinery [ ] . however, in principle, anything that hinders mvb formation might impact, even if indirectly, on exosome biogenesis. for a clearer exploration of the latter hypothesis, we will briefly summarize some regulatory mechanisms operating in the endocytic pathway, the system in charge of exosome formation. there has been much debate on whether vesicles that deliver material from a donor compartment to a specific acceptor sub-organelle contain information on where to go [ , ] . in the s, it was found that different compartments, and the vesicles they produced, were populated by distinct rab gtpases. the rab gtpases belong to a large family of highly conserved proteins with members, which were discovered to regulate vesicular trafficking in eukaryotes [ ] . the description of the endocytic pathway in figure can be explained with a set of rabs as follows: after endocytosis, sorting in rab -positive early endosomes [ ] delivers cargo to return to the plasma membrane along fast (rab , rab ) [ ] or slow (rab a, rab b, rab ) [ , ] recycling processes. alternatively, rab endosomes acquire rab and release rab by a process called endosome maturation [ , ] . rab -containing endosomes sort material to ilv, decrease ph, and acquire hydrolytic proteases able to degrade their internal contents [ ] , or instead acquire rab a/b and fuse with the plasma membrane-releasing exosomes [ ] . at any point, vesicles might acquire rab to enter a retrograde transport to the tgn [ ] . in recent years, it has been shown that interfering with the levels and activation of rab gtpases influences exosome release. depending on the cell type, rab , rab , rab , rab , and rab were all implicated in the release of vesicles. rab overexpression was shown to inhibit progression of endocytosed material from early endosomes, impacting negatively on exosomal release of markers such as syndecan, cd , and alix, and this reduction was rescued by the overexpression of rab [ ] . in agreement, rab depletion severely impaired exosomal release of the same factors [ ] . the lack of a functional active rab reduced the secretion of exosomes in the erythroleukemia cell line k , drosophila s cells, and retinal epithelial cells, as evaluated by the following exosomal proxies: transferrin and hsc- , wingless and evi, or flotillin and anthrax-toxin, respectively [ ] [ ] [ ] [ ] .rab was identified in a screen performed in oligodendroglial cancer and primary cells to analyze phospholipase d -containing exosomes [ ] . rab has been found to facilitate the release of the exosomal markers mhc ii, cd , and cd in many cancer types, including hela cells [ , , ] . interestingly, rab and rab did not influence the release of wingless in drosophila s cells [ ] and rab did not affect the extracellular levels of flotillin or of anthrax toxin in retinal epithelial cells [ ] . the roles of these rabs in the endocytic pathway allowed for speculation that there might be extracellular vesicles derived from different routes such as recycling and mvb, but this awaits confirmation [ ] . in conclusion, altering the levels of any of the referred rabs has the potential to interfere with the progression of cargo at specific endocytic locations. consistently, it has been shown that switching off rab and repopulating the endosome with other rabs is a prerequisite for the maturation of early endosomes into other types of endosomes [ , ] . conversion to rab forms mvbs and endolysosomes. mvb acquisition of rab is, in some cases, required for exosome release [ , ] . additionally, exchanging rab for rab or rab allows progression from early endosomes to the slow recycling route and exchanging rab for rab or rab allows progression from early endosomes to the fast recycling route [ ] . many viruses were shown to use the rabs mentioned above to assist several steps of their replication. there is no evidence yet relating the viral usage of these proteins with exosome biogenesis and function. the picture of the viral usage of endocytic proteins has been built during years of research, making of viruses excellent tools to understand the crosstalk between different endocytic sub-compartments. in this sense, we provide an overview of identified interactions between viruses and endocytic proteins that regulate exosome biogenesis. examples of viruses using these rabs are identified in red in figure and in table . the orthomyxovirus iav, the paramyxoviruses sev and rsv, and the bunyavirus andv all share negative strand rna genomes, infect the lung epithelia, and use rab a pathway in their infectious cycle [ , [ ] [ ] [ ] [ ] [ ] [ ] . in the case of sev, rsv, and iav, progeny rna (in the form of viral ribonucleoproteins, vrnps) attach, facing the cytoplasm, to rab vesicles as a way to facilitate their transport to the apical side of the plasma membrane. for these three viruses and conversely to andv, viral interaction with the recycling endosome occurs via activated (gtp-bound) rab a [ , [ ] [ ] [ ] [ ] [ ] [ ] . the impact of rab viral hijacking in the recruitment and activation of other rabs and exosome biogenesis has not been investigated. however, as mentioned above, interfering with rab levels can inhibit or promote the release of exosomes containing transferrin, hsc- , flotillin, and anthrax toxin [ ] [ ] [ ] [ ] . during iav infection, the total levels of rab remain fairly constant, but it is possible that the amount of the activated form suffers viral-induced fluctuations. mechanistically, in uninfected cells, vesicular transport is promoted by binding of molecular motors to activated rab . amongst many of the rab effectors reported [ ] , the members of the rab -interacting family proteins (fips) have been well characterized in facilitating vesicular movement [ ] . reduction in the levels of some fips was reported to interfere with sorting of recycling vesicles [ , ] . for iav, our unpublished results suggest that the vrnp hijacking of the rab pathway "slows down" recycling efficiency. this system could then be used to assess the effects of impairing rab in exosome biogenesis. the effects in recycling efficiency might differ for rsv, as utley et al. [ ] have shown that several members of the recycling machinery were required for rsv vrnp transport. in the case of andv, viral replication and assembly is thought to occur in the lumen of a membranous delimited sub-compartment, followed by budding to the cytoplasm and transport to the periphery. rab depletion was shown to reduce over -fold the levels of produced virions [ ] . in this case, the viral structural protein n was shown to co-localize mainly with the gdp-bound rab near the tgn, at a perinuclear location, although it has not yet been addressed whether andv inhibits rab activation or affects mvb formation and exosome release [ ] . such analysis has also not been performed for rsv and sev. rab a regulates secretion of lysosome-related organelles, including mvbs [ , ] . it was reported that rab a levels increased in hcmv-infected cells [ ] , a dna virus member of the betaherpesvirinae subfamily. this rab was found in association with viral envelopes of hcmv in an undefined compartment related to the tgn or in vesicles being transported between the tgn and endosomes [ ] . the molecular mechanisms leading to increased levels of rab a in hcmv infection are still unclear, as well as their interference with exosome release. however, given the positive role of rab a in extracellular mhc ii-containing exosomes and the regulation of immune responses in glial cells, this role of rab a in hcmv infection should be further evaluated [ ] . another virus that hijacks rab a to promote assembly is hiv (figure ) , by a mechanism that is described below [ ] . hiv also uses the escrt pathway to facilitate budding [ ] and additionally viruses , , - increases the transcription of genes involved in mvb formation and exosome release [ ] . this viral interference with many different steps of mvb/exosome formation suggests a high dependency on the host ilv machinery. in conclusion, much work has been done in understanding how different rabs regulate particular steps in the life cycle of specific viruses. how the usage of various rabs impacts the overall regulation of the endocytic pathway, including exosome release and their still controversial associated functions, remains to be evaluated. as mentioned above, it was shown by many independent groups and using several models that specific rabs interfere with exosome release. however, rabs are themselves subjected to tight regulation and are not the only factors controlling membrane identity [ , ] . figure depicts the complexity behind the composition of each sub-compartment membrane, with many factors required to create the correct microenvironment. of these, seven phosphoinositide phosphates (pip) are crucial to recruit specific rabs, albeit indirectly (for a comprehensive review on pip chemistry and biology please refer to [ , ] ). these phospholipid derivatives are enriched in specific cellular membranes. for example, the plasma membrane contains mostly phosphoinositol- , -bisphosphate (pi( , )p ) and phosphoinositol- , , -triphosphate (pi( , , )p ), whilst the membranes of the endocytic pathway are decorated with phosphoinositol- -phosphate (pi p). during maturation of endosomes, mvbs acquire phosphoinositol- , -bisphosphate (pi( , )p ), a form that becomes prevalent in lysosomes [ ] . the pip isoforms are recruited by kinases and phosphatases that also occupy precisely defined sub-compartments [ ] . viruses have been shown to alter this pip equilibrium and by doing so alter the content of rabs involved in exosome formation. for example, phosphatidylinositol -phosphate (pi p) was shown to be recruited by flavivirus and picornavirus to sites near the endoplasmic reticulum. mechanistically, such enrichment was shown to be mediated by specific phoshoinositide (pi) kinases [ , ] , one of them able to recruit rab to the golgi [ ] . another recent paper explored the mechanisms of hiv- particles' assembly at the plasma membrane, a process that occurs in micro-domains enriched in pi( , )p and the viral protein pr gag . it was found that in t cells, rab a (and some of its effectors) boosted pi( , )p production by delivering the mvb-associated kinase pi kiiα to the cell surface [ ] . rab a was also implicated in exosome release in t cells; therefore, these hiv and exosome release could, in principle, be intertwined, although such relation has not been investigated yet. other factors that control rab delivery to specific membranes include the proteins that directly activate/deactivate them. being gtpases, rabs suffer cycles of gtp/gdp binding, which are a function of the protein families' rab guanine exchange factors (gef) and guanine-activating proteins (gap), respectively ( figure ) (reviewed in [ , , , [ ] [ ] [ ] ). there is also a clear cross-talk between rab proteins and other regulators of vesicular trafficking called adp-ribosylation factors (arfs) [ , ] . arfs are also gtpases, recruited by gefs and gaps that reside in specific membranes according to their pip composition. the complete picture of the regulators controlling the location and levels of the rabs mentioned above is far from complete and these factors have not been explored in the context of viral infection. however, it is widely accepted that cells react to stimuli to adjust the distribution and levels of intracellular pips as well as their cycles of degradation/secretion/recycling [ ] . all the above considerations raise important questions concerning the interplay between regulators of the endocytic process, exosome release and viral usurpation of specific steps in the endocytic pathway. first, can viral modulation of any of these regulators alter the content and levels of released exosomes? and second, would control of the levels and composition of secreted exosomes have associated functions? these are interesting questions that deserve more attention in the near future, especially given the ever-growing functions of exosomes in viral infections. concerning the interplay between regulators of the endocytic process, exosome release and viral usurpation of specific steps in the endocytic pathway. first, can viral modulation of any of these regulators alter the content and levels of released exosomes? and second, would control of the levels and composition of secreted exosomes have associated functions? these are interesting questions that deserve more attention in the near future, especially given the ever-growing functions of exosomes in viral infections. regulators of membrane identity. membrane composition is important to maintain the integrity of endocytic process. phosphoinositide (pi) kinases (and phosphatases) ensure the levels of specific pips in distinct membranes. these operate as docking platforms for guanine exchange and activator factors (gefs and gaps) able to recruit and turn on/off gtpases. gtpases involved in membrane integrity and vesicular biogenesis are mainly of two kinds: adp ribosylation factors (arfs) and arf-like proteins (arls); and rabs. arfs and arls are involved in early steps of vesicular biogenesis such as recruiting coating proteins and cargo, membrane curvature, and neck formation. rabs operate at later stages by recruiting effectors such as molecular motors, which are able to generate pulling forces and move released vesicles. vesicle scission and release are mediated by highly specialized proteins that recognize, encircle, and cut the membrane neck, using gtp or atp hydrolysis to drive the reaction. in normal circumstances, cells show remarkable processes to communicate with the exterior, including the release of exosomes. as mentioned throughout this review, exosomes can transfer functional proteins, lipids, and distinctive sets of rnas from cell to cell in homeostatic conditions [ , , ] . in the last decades, exosomes were also shown to facilitate cell-to-cell transport of disease-related proteins involved in neurodegenerative disorders, such as prions [ ] and betaamyloid peptides [ ] . this machinery could, in principle, also contribute to viral spread. for this to happen, two prerequisites would be necessary. first, viral rna and proteins would need to access ilv. indeed, vesicular stomatitis virus (vsv), dengue virus (and other flavivirus members), and hepatitis c virus (hcv) components were found in these sub-compartments [ , ] . a relevant question is: how are soluble viral rnas sorted into ilv? rna incorporation into exosomes has been suggested to operate via a selective and conserved mechanism linked to the lipid content of vesicles. for a comprehensive review on specific mechanisms, readers are directed to [ ] . nevertheless, the contribution of rna-binding proteins has been recognized. the hnrnpa b rna binding protein, for example, was shown to bind to a four triplet motif on mirnas and transport them into exosomes [ ] . in the case of viral transmembrane proteins, the most common mechanism of targeting molecules to ilvs is via ubiquitination and recruitment of the escrt machinery [ , , ] . for hcv, it was shown that the escrt component hrs is critical for release of nucleocapsid [ ] , and for hepatitis a virus (hav) the escrt protein vps b and the accessory proteins alix [ ] were deemed crucial for ilv budding. second, exosomes would need to enter a recipient cell and release their infectious content into the cytoplasm. this mechanism was recently reported for hcv [ ] , where exosomes derived from infected human hepatoma cells containing full-length viral rna, along with core and envelope figure . regulators of membrane identity. membrane composition is important to maintain the integrity of endocytic process. phosphoinositide (pi) kinases (and phosphatases) ensure the levels of specific pips in distinct membranes. these operate as docking platforms for guanine exchange and activator factors (gefs and gaps) able to recruit and turn on/off gtpases. gtpases involved in membrane integrity and vesicular biogenesis are mainly of two kinds: adp ribosylation factors (arfs) and arf-like proteins (arls); and rabs. arfs and arls are involved in early steps of vesicular biogenesis such as recruiting coating proteins and cargo, membrane curvature, and neck formation. rabs operate at later stages by recruiting effectors such as molecular motors, which are able to generate pulling forces and move released vesicles. vesicle scission and release are mediated by highly specialized proteins that recognize, encircle, and cut the membrane neck, using gtp or atp hydrolysis to drive the reaction. in normal circumstances, cells show remarkable processes to communicate with the exterior, including the release of exosomes. as mentioned throughout this review, exosomes can transfer functional proteins, lipids, and distinctive sets of rnas from cell to cell in homeostatic conditions [ , , ] . in the last decades, exosomes were also shown to facilitate cell-to-cell transport of disease-related proteins involved in neurodegenerative disorders, such as prions [ ] and beta-amyloid peptides [ ] . this machinery could, in principle, also contribute to viral spread. for this to happen, two prerequisites would be necessary. first, viral rna and proteins would need to access ilv. indeed, vesicular stomatitis virus (vsv), dengue virus (and other flavivirus members), and hepatitis c virus (hcv) components were found in these sub-compartments [ , ] . a relevant question is: how are soluble viral rnas sorted into ilv? rna incorporation into exosomes has been suggested to operate via a selective and conserved mechanism linked to the lipid content of vesicles. for a comprehensive review on specific mechanisms, readers are directed to [ ] . nevertheless, the contribution of rna-binding proteins has been recognized. the hnrnpa b rna binding protein, for example, was shown to bind to a four triplet motif on mirnas and transport them into exosomes [ ] . in the case of viral transmembrane proteins, the most common mechanism of targeting molecules to ilvs is via ubiquitination and recruitment of the escrt machinery [ , , ] . for hcv, it was shown that the escrt component hrs is critical for release of nucleocapsid [ ] , and for hepatitis a virus (hav) the escrt protein vps b and the accessory proteins alix [ ] were deemed crucial for ilv budding. second, exosomes would need to enter a recipient cell and release their infectious content into the cytoplasm. this mechanism was recently reported for hcv [ ] , where exosomes derived from infected human hepatoma cells containing full-length viral rna, along with core and envelope proteins [ ] , were shown to be infectious and a major route of transmission. interestingly, the non-enveloped virus hav has been reported to acquire a host-derived membrane for cell-to-cell transmission and the virus is still able to replicate in the recipient cell. the exosomes containing hcv rna and exosome-like vesicles containing whole hav capsids were less susceptible to antibody neutralization, and consequently this transmission mechanism has been reported to operate as an immune evasion strategy [ , , ] . upon reaching their destinations, exosomes containing viral proteins and rna would only be infectious provided they could enter target cells and reach the cytoplasm. exosomes containing vsv and other flaviviruses seem to enter the cell by being taken up by the endocytic pathway [ , ] . these two viruses were shown to escape late endosomes and reach the cytosol by a "back-fusion" process, a phenomenon by which ilvs fuse with the external late endocytic membranes [ ] . alternatively, exosomes containing viral antigens can induce signaling cascades at the surface upon binding their receptors to control host immune responses [ ] . research on exosome-mediated viral spread is still very limited; however, exosome modulation of the immune responses has been explored in some detail and will be discussed next. one of the main functions assigned to exosomes is the mediation of intercellular communication during innate and adaptive immune responses. in fact, many different cells of the immune system, including dendritic cells and b and t lymphocytes, have been shown to release exosome vesicles with immune modulatory properties. these exosomes can be found in bodily fluids (reviewed in [ ] [ ] [ ] ). in , raposo et al. demonstrated that b lymphocytes infected with epstein-barr virus (ebv), a human gammaherpesvirus associated with a variety of lymphoblastoid and epithelial cancers, released exosomes containing mhc ii molecules, and that these vesicles were capable of activating specific cd + t cell clones in vitro [ ] . two years later, zitvogel et al. published a study showing that exosomes released by dendritic cells had the ability to suppress the growth of tumors in vivo. this led to the interpretation that exosomes could be used as therapeutic agents modulating immune responses [ ] . subsequent studies with ebv-infected lymphoblastoid and nasopharyngeal carcinoma cells have shown that the exosomes secreted by these cells also harbor ebv-encoded latent phase mrnas [ ] , proteins [ , , [ ] [ ] [ ] , and mature mirnas [ , ] . accumulating evidence suggests that these exosomes exert immune inhibitory effects on tumor-infiltrating lymphocytes. latent membrane protein (lmp ), the major viral oncogene expressed in most ebv-associated tumors, has been detected in exosomes and was shown to inhibit immune response, namely t lymphocyte activation and proliferation, nk cytotoxicity, and the ability of cells to produce interferon gamma [ , , ] . exosomes secreted by ebv-infected nasopharyngeal carcinoma cells also contained high amounts of the immunoregulator protein galectin- , which is able to induce apoptosis of ebv-specific cd + t cells [ , ] . ebv mirnas present in the exosomes are internalized by dc where they downregulate specific immunoregulatory genes [ ] . in contrast, exosomes released from ebv-infected b lymphocytes were found to exert a stimulatory effect on non-infected b lymphocytes, driving their proliferation, class-switch recombination, and differentiation into plasmablast-like cells [ ] . it was recently reported that exosomes also regulate innate immunity. this was illustrated with the identification of important innate immune effectors (ifi , caspase- , interleukin b (il- b), il- , and il- ) in exosomes released from ebv-infected cells. such a strategy removes these effectors from infected cells to reduce innate immunity activation [ ] . another interesting approach was proposed for hsv infection. in this case, cells export the innate immune sensor sting (stimulator of ifn genes), viral mirnas, and mrnas through exosomes that are delivered to uninfected cells [ ] . the functional significance of this strategy is still not clear, but the fact that some mirnas are able to suppress reactivation of latent virus suggests that, in specific circumstances, hsv has evolved mechanisms to restrict, rather than expand, the spread of infection. exosomes harboring hcv rna are transferred from infected cells to non-permissive plasmacytoid dcs, where viral rna can trigger a type i ifn response [ ] . it is clear that in viral infections, exosomes play a dual role in the modulation of the immune system, both serving as a host program to induce innate and adaptive immunity and as a viral strategy to evade those same responses. viral infection is thought to be responsible for % to % of all human cancers, which makes the understanding of how pathogens modulate host cell functions during their transformation program seminal, both from a scientific and a clinical perspective [ ] . seven human tumor viruses have been identified, human papillomavirus (hpv), merkel cell polyomavirus (mcv), hcv and hepatitis b virus (hbv), the members of the herpes family ebv and kaposi's sarcoma associated herpesvirus (kshv), and the retrovirus human t-lymphotropic virus- . hiv is also tumorigenic, although indirectly, since the decrease of host immunity it provokes allows cell transformation, mostly by other viruses such as kshv. it is now well established that tumor cells secrete exosomes [ , , ] , but the cancer types in which exosomes are quantitatively, qualitatively, and functionally different from healthy tissues are incompletely characterized. many studies reported differential composition of exosomes in healthy organisms versus those infected with several tumor viruses [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nasopharyngeal carcinoma cells infected with ebv produced exosomes containing lmp [ , ] . these exosomes were shown to be taken up by neighboring cells [ ] and the transcription profiles of the recipient cell were subsequently altered [ , ] . the mechanism underlying transcriptional alterations occurred via lmp -mediated increase of egfr release by exosomes that lead to activation of erk and pi k/akt pathways in epithelial, endothelial, and fibroblast cells [ ] . erk and pi k/akt are renowned factors able to promote cell growth and migration. a specific mirna composition has been found in tumor viruses' derived exosomes [ , , , , [ ] [ ] [ ] [ ] [ ] . for example, there are several types of hpv-some tumorigenic, each associated with a different mirna exosomal profile [ ] [ ] [ ] . in the case of exosomes isolated from cancer patients infected with tumorigenic hpv, the mirna content was enriched for species controlling cell proliferation, senescence, and apoptosis. the exosomal mirna compositions were dependent on the expression of the viral oncogene e /e , suggesting that this is one mechanism by which the oncogene contributes to the growth of hpv-positive cancer cells [ ] . regardless of the infection status, the significance of exosomes in tumor development was demonstrated in breast cancer, where normal cells became immortalized when incubated with exosomes derived from tumor cells [ ] . another example was the injection of rab a depleted breast carcinoma cell lines in immunocompetent mice. the lack of rab a was associated with reduced release of exosomes [ ] and poor tumor development when compared to cells containing rab a, where tumor progression was normal and formed metastasis [ ] . one of the major difficulties clinicians face is the lack of efficient methods to diagnose tumors, especially in early stages. in the case of tumors that result from viral infection, the identification of biomarkers for poor prognosis of infection would greatly benefit patients. research needs to be done to identify clear populations of exosomes involved in infection and tumors, and to clearly define functions associated to exosomes in both conditions. these topics warrant investigation, as exosomes show great promise as biomarkers for cancer and/or infection, as therapeutic agents, and have the additional advantage of being accessible without the use of invasive techniques. the mechanisms regulating the levels, content, and function of exosomes in viral infection remain poorly characterized. there are four areas that need detailed investigation. first, it is unclear how the endocytic system controls the percentage of mvbs that fuse with the plasma membrane. we hypothesize that signaling events regulate the levels of activated rabs at each step of the endocytic pathway, and that this fluctuation allows for an adjustment of the levels of biomolecules sent for degradation and/or secretion. viruses provide excellent tools to better answer these questions as many efforts have been made to understand how each virus modifies distinct endocytic compartments. the next challenge is to better understand how these changes affect the endocytic process, including exosome release. second, little is known about the biological processes sorting material to ilvs of mvbs. the inclusion of viral proteins and rnas in exosomes offers a unique system to identify signals and sorting mechanisms into ilvs. third, the functional relevance of exosomes in infection and disease remains incompletely characterized. although exosome modulation of adaptive immunity has been extensively researched, its role in innate immunity remains largely unexplored. the recent identification of rna transfer that might affect host gene expression has been an important discovery that has renewed interest in this field. the role of exosomes in viral spread is far less explored, namely its overall contribution to viral infection. interestingly, using exosomes could be a means to mitigate exposure of viral antigens and operate as an immune evasion strategy. clearly a lot has to be done in identifying viruses able to use this pathway and the mechanisms leading to the inclusion of viral proteins/rna/capsids in ilvs-this also feeds into question two. finally, exosomes and many viruses share size, shape, and molecular characteristics. technical improvements in methods to separate and obtain pure exosomal fractions will facilitate the understanding of their role in infection, 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transfected cells human papillomaviruses modulate microrna expression to directly control genome amplification dependence of intracellular and exosomal micrornas on viral e /e oncogene expression in hpv-positive tumor cells exosomes derived from epstein-barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells the authors would like to acknowledge fundação para a ciência e a tecnologia, portugal, key: cord- -z hh c authors: cotogni, paolo; trombetta, antonella; muzio, giuliana; brizzi, maria felice; canuto, rosa angela title: polyunsaturated fatty acids and cytokines: their relationship in acute lung injury date: journal: diet and nutrition in critical care doi: . / - - - - _ sha: doc_id: cord_uid: z hh c acute lung injury (ali) and acute respiratory distress syndrome (ards) are inflammatory diseases whose clinical severity depends on the grade of inflammatory response. inflammatory cytokines are key elements in the pathogenesis of ali/ards, and the occurrence of an imbalance between pro- and anti-inflammatory cytokines leads to additional non-pulmonary organ dysfunction which contributes to excess mortality rates. treatment of these patients includes nutrition support with lipids, usually soybean oil-based lipid emulsions, which are rich in omega (n)- polyunsaturated fatty acids (pufas) and deficient in n- pufas; however, too much n- pufas are detrimental due to their pro-inflammatory effects. conversely, a large amount of experimental studies and some randomized clinical trials showed the benefits of the n- pufa administration in the context of ali because of their anti-inflammatory properties. based on these data, several scientific societies recommended in their guidelines, with an a or b grade of recommendation, the use of n- pufas in ali/ards patients. however, at present, the issue of lipid therapy in ali/ards is still controversial due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n- pufas. acute lung injury (ali) and acute respiratory distress syndrome (ards) are inflammatory diseases whose clinical severity depends on the grade of inflammatory response. inflammatory cytokines are key elements in the pathogenesis of ali/ards, and the occurrence of an imbalance between pro-and antiinflammatory cytokines leads to additional non-pulmonary organ dysfunction which contributes to excess mortality rates. treatment of these patients includes nutrition support with lipids, usually soybean oil-based lipid emulsions, which are rich in omega (n)- polyunsaturated fatty acids (pufas) and deficient in n- pufas; however, too much n- pufas are detrimental due to their pro-inflammatory effects. conversely, a large amount of experimental studies and some randomized clinical trials showed the benefits of the n- pufa administration in the context of ali because of their anti-inflammatory properties. based on these data, several scientific societies recommended in their guidelines, with an a or b grade of recommendation, the use of n- pufas in ali/ards patients. however, at present, the issue of lipid therapy in ali/ards is still controversial due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n- pufas. acute respiratory distress syndrome (ards) is considered a form of acute diffuse lung injury (ali). according to the berlin definition (ferguson et al. ) , each subcategory of ards (mild, moderate, and severe) is defined by mutually exclusive ranges of the ratio between arterial oxygen partial pressure (pao ) and fractional inspired oxygen (fio ) ( mm hg<pao /fio mm hg, mm hg<pao /fio mm hg, and pao / fio mm hg, respectively). the acute phase of this syndrome is manifested by the early onset of respiratory failure (characterized by hypoxemia refractory to oxygen supply within week of a known clinical insult or new/worsening respiratory symptoms) related to several triggers and clinical disorders generally distinguished in pulmonary [pneumonia (bacteria or virus), microbial products (e.g., endotoxins), pulmonary aspiration of gastric contents] and extrapulmonary (trauma/hemorrhagic shock, extensive burns, ischemia/reperfusion injury, acute pancreatitis, cardiopulmonary bypass, transfusions of blood products) (leaver and evans ) . ards is frequently progressive and it is characterized by distinct phases with different clinical, radiographic, and histopathological findings. radiographic findings are similar to those observed in the case of cardiogenic pulmonary edema, mainly bilateral infiltrates (irregular and asymmetric) and sometimes pleural effusions (fig. ) . histopathological findings of ali include diffuse alveolar epithelium damage (principally, denudation of the basement membrane of the alveolus and necrosis of type i cells) with activated neutrophils, alveolar macrophages, hyaline membranes, and protein-rich edema fluid in the alveolar spaces (fig. ) . the pathophysiology of ali is complex and multifaceted. the lung inflammatory response involves various interactions among many cell types, and it is characterized by the production and release of several chemical mediators. figure is a simplified graphic description of how the anatomy and physiology of alveolar space, the main site of inflammatory reactions occurring in ali, change. the lung is a complex organ with many different cell types, i.e., pneumocytes (alveolar type i and ii), fig. anteroposterior chest radiograph from a patient with the acute respiratory distress syndrome who was receiving mechanical ventilation. irregular and asymmetric infiltrates are present throughout both lung fields pulmonary capillary endothelial cells, alveolar macrophages, and fibroblasts. several studies suggest that the lung itself can be an important cytokine-producing organ. cytokines are a diverse group of inflammatory mediators, produced by a wide variety of cell types, which initiate and orchestrate the host response to different injuries. intercellular communication through cytokine networking plays a key role for homeostasis. cytokines seem to have concentration-dependent effects. at lower concentrations, they modulate tissue response to injuries; at higher concentrations, they induce proportionally more severe local and systemic inflammatory responses. if this cytokine overproduction is not regulated, it may remarkably amplify the release of further pro-inflammatory mediators and, at least, the overall inflammatory response. both ali and ards are characterized by an intense inflammatory response. in ards early phase, the alveolar space is the site of significant inflammatory reactions (pugin et al. ) . ali/ards are characterized by alveolar infiltration with neutrophils and macrophages, both able to release inflammatory cytokines. however, recent theories suggest that it is not the neutrophil and macrophage number but their the normal alveolus and the alveolus in acute lung injury. neutrophil is shown migrating through a gap in the capillary endothelium into the alveolar space (activated neutrophil). in the alveolar space, an alveolar macrophage is secreting cytokines: tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , il- , and il- . the alveolar type i cells are damaged or necrotic, while alveolar type ii cells are intact and release cytokines. the tissue architecture is disrupted with the formation of hyaline membranes on the denuded basement membrane. the alveolar space is filled by protein-rich edema fluid fig. alveolar damage in acute lung injury/acute respiratory distress syndrome. the tissue architecture is disrupted, alveoli are collapsed, alveolar type ii (atii) cells are hyperplastic, and alveolar type i cells are necrotic. alveoli contain protein-rich edema fluid and macrophages. collagen deposition in the interstitium has begun. hematoxylin and eosin staining,  magnification. the picture was kindly given by luisa delsedime, md, anatomia patologica, aou città della scienza e della salute, turin, italy pro-inflammatory cytokine release that produces lung injury. in this setting, cytokines have a central position as signaling mediators that initiate, increase, and maintain inflammatory responses on a local basis. indeed, in ali/ards, an accumulation of both pro-and anti-inflammatory cytokines within the alveolar spaces is demonstrated. many cytokines were detected at elevated levels in bronchoalveolar lavage (bal) fluid in patients with ards, i.e., tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , il- , and il- . tnf-α is the main pro-inflammatory cytokine and it is important in driving the initial lung inflammatory response, principally indirect by the stimulation of other cytokines (i.e., il- and il- ). il- occupies a crucial place in the ards inflammatory response. chemokine il- is the major neutrophil chemotactic factor into the alveolar space, and it is an early marker for the development of ards. finally, il- , which has anti-inflammatory effects, plays a pivotal role in closing down the inflammatory response once it achieves its primary purpose. the pivotal role of a hyper-inflammatory response, characterized by overproduction of tnf-α, il- , and il- , in the evolution of the disease from ali to ards is documented. indeed, many studies report a higher mortality in patients who have increased pro-inflammatory cytokine concentrations in the bal at the onset of ards or persistent increased concentrations in days - . physiologically, the critical dependency on the presence of the inflammatory ligand (e.g., platelet-activating factor) and free arachidonic acid (aa) may help to confine the outbreak of lipid-mediator production to sites of initial inflammatory process. conversely, compartmentalization of alveolar cytokines can be lost in the case of a damaged alveolar barrier. cytokines generated in the alveoli may go into the systemic circulation leading to the beginning of a systemic inflammatory response syndrome. the reason why a patient with ali develops ards while another recovers is still not completely clear. in ards-developing patients, it is hypothesized that, at some level, regulation of the inflammatory response must be inadequate, but why or when this regulation is insufficient is a matter of debate. the study of the balance between pro-and anti-inflammatory cytokines is of greater importance to understand the pathophysiology of ali than a single cytokine concentration, because the degree of cytokine imbalance is a contributing element to disease severity (park et al. ) . persistently high levels of pro-inflammatory cytokines, as well as a reduced production of anti-inflammatory cytokines, have been demonstrated to be correlated with the severity of lung injury. the degree of this cytokine imbalance has a pivotal role in inducing other non-pulmonary organ dysfunctions and increasing mortality rates in ards patients. in particular, the pro-inflammatory activity depends on the balance between the cytokine and its inhibitor(s). in ards early phase, the finding of low levels of anti-inflammatory cytokines (i.e., il- and il- receptor antagonist) in the bal is associated with an increase in patient mortality. the ratio tnf-α to il- in the bal fluid was significantly higher ( . vs. . ) in patients with ards than in those at risk of developing ards. among ali ventilated patients, malnutrition has been associated with adverse outcomes due to increased infection risk, prolonged mechanical ventilation dependency, prolonged intensive care unit (icu) length of stay (los), and higher mortality. in general, critically ill patients receive nutrition support including lipids. administration of lipid emulsions offers many advantages in patients with acute respiratory failure, because compared with glucose they provide a richer source of calories in a small volume with a lower carbon dioxide load and less hyperglycemia. moreover, lipid supply is mandatory to avoid essential fatty acid (fa) deficiency. accepted guidelines recommend providing - % of nonprotein calories as lipids in patients with ali/ards (mizock ) . lipids were introduced into parenteral nutrition (pn) formulations in the s. however, there is a lack of consensus about the optimal source and composition of lipids (long-vs. medium-chain triglycerides, soybean oil vs. olive oil and fish oil) both in parenteral and enteral formulas for the patient with ali/ards. commonly used parenteral lipid emulsions (i.e., soybean oil-based fat emulsions) contain large amounts of n- polyunsaturated fatty acids (pufas) (i.e., more than % of linoleic acid) that form aa [omega (n)- pufa], while only % α-linolenic acid can be metabolically converted to eicosapentaenoic acid (epa, n- pufa) and docosahexaenoic acid (dha, n- pufa). therefore, intravenous fat emulsions have a very low n- /n- pufa ratio, ranging between : and : . likewise, the n- /n- ratio in enteral products may be as low as : . thus, ali/ards patientseither on standard enteral nutrition (en) or pnare generally exposed to a large quantity of linoleic acid. however, administration of high quantities of linoleic acid is not desirable in ali/ards patients because it causes a production of high amounts of aa, whose main functional role is to be a substrate for the synthesis of bioactive pro-inflammatory mediators known as eicosanoids [above all, prostaglandin (pg)-e , thromboxane (tx)-a , and leukotriene (lt)-b ] and platelet-activating factor, which modulates severity and duration of inflammatory responses. the impact of parenteral lipid supply on the lung function of patients with acute respiratory failure has been contradicting. potential harmful effects have been related to type and/or administration rate of lipid emulsions. in particular, some authors thought that these negative effects might correlate to the infusion of soybean oil-based lipid emulsionsbecause they are rich in linoleic acidand suggested that linoleic acid-derived synthesis of eicosanoids may worsen gas exchange and pulmonary hemodynamics in critically ill patients. eicosanoids are major pathogenetic factors of ards, and their negative effects on pulmonary function (e.g., increased permeability, edema formation, polymorphonuclear activation, thrombocyte aggregation, vasoconstriction, and bronchoconstriction) have been widely investigated. as early as , the immunomodulatory properties of lipids were displayed. over the past years, there has been an improved understanding of pufa pathophysiology, and several mechanisms for the interaction between pufas and inflammation or immune response have been demonstrated. indeed, after pufa supply (diet, as well as en or pn), many cell properties and related functions are modified, mainly the inflammatory and immunity responses (calder ) . briefly, n- pufas are more regarded as anti-inflammatory, whereas n- pufas as proinflammatory. more recent studies suggested that n- pufas may be involved in the resolution of inflammation (calder a) . since a network of factors regulates the relationship between n- and inflammation, the mechanisms underlying the effects of n- pufas on cell event modulation are complex. to summarize the mechanisms of n- pufa modulation of the inflammatory response, it can be said that the main and well-demonstrated anti-inflammatory actions of n- pufas include the following: (a) a decreased production of aa-derived mediators such as -series pg and ltb as a result of a reduction in aa content of cell membranes and of inhibition of aa metabolism. (b) an increased production of eicosanoid mediators from epa (e.g., -series pg and ltb ), since these mediators have weak biological potency compared with those produced from aa; for example, ltb is -to -fold less potent as a neutrophil chemotactic agent than ltb , or pge is a less potent inducer of cyclooxygenase- gene expression in fibroblasts and of il- production by macrophages compared with pge . (c) a decreased t-cell proliferation and production of il- induced by epa and dha providing. (d) a decreased expression of some adhesion molecules on the surface of monocytes, macrophages, lymphocytes, and endothelial cells induced by epa and dha providing. (e) a decreased production of pro-inflammatory cytokines, due to decreased activation of pro-inflammatory transcription factors such as nuclear factor κ-b and increased activation of anti-inflammatory transcription factors such as peroxisome proliferator-activated receptor-γ. (f) an increased production of resolvins (e-series from epa and d-series from dha) and protectins (from dha) having potent antiinflammatory or inflammation-resolving properties. pufas and lung injury: nutrients or drugs? over the last years, the discovered ability of n- pufas to downregulate many different responses of the inflammatory process has suggested to several researchers that these fas might have a key role in determining the grade of severity of some inflammatory diseases. consequently, n- pufas have been widely used not only as nutrients but mainly as pharmacological agents. briefly, the rationale of the therapeutic use of n- pufas was based on several well-established issues: ( ) pufas are key structural and functional elements of the phospholipids in cell membranes; ( ) cells involved in the immunity and inflammation (neutrophils, monocytes, macrophages, dendritic cells, and endothelial cells) are richer in n- pufas than n- , but the contents of aa, epa, and dha can be modified by n- pufa (i.e., epa and dha) administration (oral, enteral, and parenteral); ( ) pufa challenge, as well as the fa composition of cell membrane of human inflammatory cells, influences the functions of those cells; and, last but more important, ( ) n- pufas and n- pufas have opposing influences on inflammation. ali/ards are inflammatory diseases too; therefore, numerous studies have been carried out to evaluate strategies (drugs or interventional procedures) to reduce the severity of lung inflammatory processes by lowering the production of pro-inflammatory mediators (eicosanoids and cytokines). nonetheless, few studies have found a significant improvement on mortality in those patients. since , many editorials have stressed the possibility to manipulate the inflammatory response in ali/ards patients using n- pufas as drugs (the so-called pharmaconutrition) (mayer and seeger ) . the research on the influence of fas on immunity started in the s. subsequently, a great number of works with cell or animal models have been carried out with the aim to demonstrate efficacy in modifying the inflammatory responses of fish oil or their main active components (i.e., epa and dha). likewise, there have been some clinical trials of enteral or parenteral administration of fish oil-enriched nutrition formulas in critically ill patients with ali/ards. the experimental models used were animal isolated lungs or, mainly, cell line models. frequently, a human lung carcinoma cell line (a cells) was used. a are alveolar epithelial cells with type ii pneumocyte properties that retain many of the characteristics of normal human type ii epithelial cells and could synthesize lecithin and phosphatidylcholine with a high percentage of desaturated fas. several authors used alveolar type ii cells because it was shown to have a central position in the pathophysiology of the alveolar space. after in vitro administration, fas were rapidly incorporated in the phospholipid spectrum of alveolar cell membranes; in fact, as pulmonary surfactant (the surface-active phospholipoprotein complex) producers, alveolar type ii cells have a higher lipid metabolism, e.g., a significant increase in dha concentration in the lung phospholipid fraction was observed h after dha infusion. several experimental studies with isolated lungs and culture of different pulmonary cells clearly showed a pivotal role of aa and its metabolites as mediators of injury. in contrast, supplementation of n- pufas demonstrated to reduce alveolar release of pro-inflammatory mediators and to reduce organ failure in animal lung models. rapid changes in cell membrane phospholipid fa composition and lipid-derived inflammatory mediator generation were observed both after short cell exposure to n- pufas and short-term intravenous administration of n- pufas. it was observed that pg was produced within min of a cell exposure to epawith a peak at hand this rapid increase of pg was correlated with the metabolism of epa by cyclooxygenases in the a cells. experimental data suggested that epa and dha are both able to inhibit pge production. a short-term n- pufa supplementation induced a significant increase in the n- pufa composition of perfusate (after min) and lung tissue (after h). after as little as h of lung perfusion with an n- pufa-enriched emulsion, significant alterations in the spectrum of eicosanoid generation were found in the isolated lung model. likewise, after min in a model of septic lung injury, it was found that the vascular leakage provoked by bacterial exotoxin in rabbit lungs was differentially influenced by aa versus epa, related to the generation of -versus -series lts. early studies demonstrated that epa and dha inhibited endotoxin-stimulated production of il- and il- by cultured human endothelial cells and n- pufas inhibited endotoxin-induced tnf-α production by cultured monocytes. the differential impact of manipulation of cell membrane phospholipid composition due to n- pufa versus n- pufa administration on cytokine production provoked by pro-inflammatory mediators was demonstrated in a cells. although dha is not able to give rise to lts, it has a pivotal role since it can be incorporated unchanged into membrane lipids or retroconverted into epa. evidence suggests that the antiinflammatory and immunosuppressive effects of n- pufa supply may be attributed mainly, or even exclusively, to dha supplementation. in the past two decades, many authors have highlighted that the n- /n- pufa ratio in nutrition support may influence inflammation since optimal n- administration was not only dose related but was also independently affected by the n- /n- pufa ratio. different n- /n- pufa ratios from : to : have been proposed, but the question of the most favorable n- /n- pufa ratio in ali/ards patients is still to be defined. the impact of the n- /n- pufa ratio on cytokine release by a cells exposed to an endotoxin (i.e., lipopolysaccharide -lps) challenge was investigated in a recent experimental study (cotogni et al. ) . as in many studies, the inflammatory process was elicited by administration of lpsa component of gram-negative bacteria cell wallbecause lps is an important mediator in the pathogenesis of ards and a cells are able to produce the acute phase protein lps-binding protein. in particular, it was determined the time-and dose-dependent effect of lps on the tnf-α response in a cells (fig. ) . the fa composition in phospholipids of a cell membranes was also analyzed, and the n- /n- pufa ratio was reported to be : , with aa as the most prevalent pufa (table ) . the administration of different ratios of aa and dha on the lps-induced cytokine response from a cells showed that the supply of : and tnf-α concentrations were measured in cell culture media by elisa assay at the time points indicated. the results were expressed as picograms of released cytokines per adherent cells (pg/ cells) and as mean ae sd (n = experiments). lps lipopolysaccharide, tnf-α tumor necrosis factor-α. * p < . lps and versus baseline, at and h; ** p < . lps versus baseline, at h; *** p < . lps versus baseline, at h; # p < . lps h versus lps h : dha/aa ratios reversed the baseline predominance of n- over n- in the n- /n- pufa ratio of cell membranes. particularly, the release of pro-inflammatory cytokines (tnf-α, il- , and il- ) was reduced by : and : dha/aa ratios; on the contrary, it was further increased by : and : dha/aa ratios if compared to lps challenge alone (fig. ) . moreover, the : and : dha/aa ratios were effective in increasing antiinflammatory il- release and in modifying the balance between pro-and anti-inflammatory cytokines (fig. ). this finding confirmed previous studies showing that il- protects against the lethal effects of lps by significantly reducing the production of pro-inflammatory cytokines in table percentage content of fatty acids in phospholipids of a cell membranes fatty acid c : (saturated) . c : (saturated) . c : (monounsaturated) . c : (saturated) . c : (monounsaturated) . c : (linoleic acid, n- ) . c : (arachidonic acid, aa, n- ) . c : (docosahexaenoic acid, dha, n- ) . n- /n- pufa ratio lipopolysaccharide-stimulated tnf-α, il- , il- , and il- release from a cells treated with different dha/aa ratios. interleukin concentrations were measured in cell culture media by elisa assay at h. the results were expressed as picograms (pg) of released cytokines per adherent cells and as mean ae sd (n = experiments). aa arachidonic acid, bl baseline, c control, dha docosahexaenoic acid, il interleukin, tnf-α tumor necrosis factor-α. * p < . versus control; ** p < . versus all; # p < . versus control and : human monocytes and murine peritoneal macrophages. these data suggested that inflammatory cytokine release was dependent on the proportion of n- in the n- /n- pufa ratio of alveolar cell membranes, being reduced with the supply of dha and increased with a high proportion of aa. according to several other data, shifting the pufa supply from n- to n- with a ratio of : may be a good means to dampen pro-inflammatory responses in ali. experimental studies with animal models demonstrated that production of aa-derived eicosanoids and cytokines decreases with a diet containing fish oil in ali and septic murine models. macrophages isolated from mice fed with a diet containing fish oil produced less pge and il- in response to lps stimulation than those isolated from mice fed with other lipid-enriched diets. feeding rats with an n- pufa-enriched diet, as compared with an n- pufa-enriched one, was associated to: ( ) a reduced severity of lung microvascular protein permeability and hypotension in a model of endotoxin-induced ali, ( ) a reduced synthesis of pro-inflammatory eicosanoid (ltb , pge , and txb ) released from stimulated alveolar macrophages, and ( ) a decrease in aa and an increase in epa and dha in cell membrane phospholipids of alveolar macrophages. after intraperitoneal injection of lps, peak plasma tnf-α, il- β, and il- levels were more reduced in mice fed with a fish oil diet than in those fed with a safflower oil diet (i.e., a diet rich in n- pufas). using either a model of ali, in which lps was instilled in the trachea, or a model of intraperitoneal inflammation, in which lps was injected intraperitoneally, it was demonstrated that a -day course of intravenous lipid emulsion infusions was sufficient to modify inflammatory responses. fish oil-based lipid emulsions reduced alveolar leukocyte transmigration, protein leakage, and cytokine production, as well as cytokine concentrations in the intravascular compartment. conversely, soybean oil-based lipids lead to a further increase in the inflammatory response. tnf-α /il- (log pg/ cells) il- /il- (log pg/ cells) il- /il- (log pg/ cells) clinical data showing benefits associated with the administration of nutrition formulas enriched with n- pufas in ali/ards patients came initially from three randomized controlled trials (rcts) using the enteral route. these rcts showed that en with a study formula containing epa, γ-linolenic acid, and antioxidants (vitamins c and e, β-carotene, and taurine), as compared with high-fat formula (control formula), reduced alveolar inflammatory mediators and improved clinical outcomes in patients with ali, ards, or sepsis. the first rct showed the ability of an enteral formula with a high n- /n- pufa ratio ( : ) to reduce pulmonary inflammation and improve clinical outcomes, i.e., better oxygenation, shorter requirement for mechanical ventilation, shorter icu-los, and less incidence of new organ failure; however, no difference in mortality was observed in ards patients (gadek et al. ) . similarly, an rct in ali/ards mechanically ventilated patients showed that an n- pufaenriched enteral diet may be more beneficial for gas exchange, respiratory dynamics, and length of mechanical ventilation if compared with a control formula, but no difference both in icu or hospital los and mortality was found between the two formulas (singer et al. ) . another rct was able to demonstrate that the administration of the same enteral diet contributed to guarantee an improved oxygenation and an independence from mechanical ventilation, less incidence of new organ dysfunction, and more icu-free days and, mainly, was associated with lower mortality rates in ards mechanically ventilated patients with severe sepsis and septic shock (pontes-arruda et al. ) . in , a meta-analysis of the pooled outcomes from these three rcts concluded that the n- pufa-enriched enteral formula could reduce mortality, rate of new organ failure, icu-los, and length of ventilation in ali/ards patients (pontes-arruda et al. ) . nevertheless, in , three further rcts were published that addressed the issue of the potential positive effects of an enteral n- pufa-enriched nutrition in ali/ards patients showing mixing results. the first rct analyzed the effect of an enteral n- pufa-enriched diet in septic patients with ali or ards showing that the administration of the study formula, compared to a control formula with less lipids than in the previous three studies, was associated to a shorter icu-los but not to an improvement in gas exchange or in a lower incidence of novel organ failures (grau-carmona et al. ) . a phase placebocontrolled rct in ali patients investigating an enteral administration of fish oil for up to days did not demonstrate a decrease in cytokine or other inflammatory marker concentrations both in bal fluid from baseline to day or and in plasma, as well as a decrease in ventilator-free days, icu-free days, organ failure score, or -day mortality (stapleton et al. ). the last rct tested the effects of enteral feeding with n- pufas, γ-linolenic acid, and antioxidants on clinical outcomes in ali patients in a phase trial (omega trial) using a twice-daily bolus administration (rice et al. ) . contrary to all other rcts, the authors reported that enteral administration of this formula was associated to significantly fewer ventilator-free days, icu-free days, and non-pulmonary organ failure-free days, as well as more days with diarrhea. the trial was stopped early for futility at the first interim analysis. the interpretation of the omega trial presents some difficulties (lev and singer ) . firstly, it was used a different approach of twicedaily bolus administration of enteral formulas instead of continuous enteral infusion. secondly, the enteral diets administered to the study group (i.e., megadoses of n- pufas and lower protein content) and to the control group (i.e., higher protein and carbohydrate content) are notably different from those used in the first three rcts. enteral administration of n- pufas might require more time before effectively influencing the cellular fa composition and the lipidmediated inflammatory response. moreover, the daily volume of enteral formula prescribed is calculated such as to meet patients' requirement of calories rather than a scheduled "pharmacological" dosage of n- pufas. it is well known that icu patients exhibit different degrees of tolerance to en, as it is well documented that the more patients are critically ill, the less they tolerate en. thus, an unpredictable number of critical patients might not receive the "pharmacological" dosage of n- pufas. an alternative approach to provide n- pufas to icu patients unable to meet their nutrition requirements by oral or enteral route is to administer a parenteral formula prepared from fish oil. differently to oral/enteral feeding, parenteral administration of pufas can induce more rapid changes in lipid-derived responses. indeed, in ali, there is the need of promptly active treatments to early modulate the net inflammatory response in the alveolar spaces. in septic patients, intravenous infusion of n- pufas modified lipid mediator synthesis and reduced endotoxin-stimulated monocyte proinflammatory cytokine production, while cytokine production was markedly amplified by n- pufa administration (mayer et al. ) . conversely, pn administered for days with a : mixture of medium-chain triglycerides and longchain triglycerides, with the n- /n- pufa ratio of : compared with a same mixture supplemented with fish oil with the n- /n- pufa ratio of : , did not affect inflammatory marker production or measures of clinical outcomes in an rct in unselected critically ill medical patients (friesecke et al. ) . in the last years, three rcts have investigated the effects of parenteral administration of n- pufa-enriched lipid emulsions in ali/ards patients. the first rct investigated the effects of an n- pufa-enriched lipid emulsion in ards patients with en intolerance showing no significant changes in hemodynamic and gas exchange parameters (sabater et al. ) . previously, the same authors had showed that parenteral administration of an n- -enriched lipid emulsion was accompanied by the synthesis of a smaller amount of pro-inflammatory lipid mediators compared to long-chain triglyceride % emulsion in ards patients. in a second trial, patients with systemic inflammatory response syndrome or sepsis were randomized into two groups to receive an intravenous lipid emulsion enriched or not in fish oil (barbosa et al. ) . the authors reported that the pao :fio ratio at day was significantly higher and plasma il- concentration significantly decreased in the group treated with fish oil; however, fish oil administration did not improve the length of ventilation or icu-los and mortality. the last rct investigated the effects of an intravenous administration of n- pufas with the n- /n- pufa ratio of : as supplementation of en for days in ards patients finding that fish oil administration alone did not improve ventilation, icu-los, or survival (gupta et al. ). ards is the most severe life-threatening manifestation of ali. both remain a significant health burden for intensivists and critical care physicians because of their considerable morbidity and mortality. in , in the usa, the crude incidence of ali was . per , person-years; each year, there are , cases of ali associated with , deaths and . million hospital days. in , in icus of ten european countries, ali occurred in ( %) out of , admissions, and in the % of the whole mechanically ventilated patients, % cases occurred on icu admission. pathologically, ali and ards are inflammatory diseases; clinically, their severity depends on the grade of inflammatory response. thus, numerous studies have been carried out investigating new strategies to reduce the grade of this inflammatory response, mainly by reducing the release of pro-inflammatory mediators. however, pharmacological therapies such as corticosteroids, ketoconazole, antioxidants, nitric oxide, prostacyclins, exogenous surfactants, and β agonists failed to show positive results in human studies. supplementation with n- pufas was demonstrated to exert beneficial effects in a number of inflammatory diseases. the rationale for their use in ali/ards patients is consistent and based on several issues. firstly, a large amount of experimental studies in lung models supports the antiinflammatory properties of n- pufas. secondly, patients at risk for ards and with established ards have plasma epa concentrations about % and %, respectively, as compared with normal, hypothesizing a potential benefit for n- pufa administration in those patients. finally, in three level i studies involving patients with ali, ards, and sepsis, the use of an enteral diet enriched with n- pufas (in the form of epa), γ-linolenic acid, and antioxidants was shown to significantly reduce icu-los, mechanical ventilation length, organ failure incidence, and mortality if compared to the use of a standard enteral formula. based on these data, the european society for clinical nutrition and metabolism, american society of parenteral and enteral nutrition, society of critical care medicine, and canadian guidelines recommended, with an a or b grade of recommendation, in their guidelines published between and , the use of n- pufas in ali/ards patients. however, nowadays, the clinical application of these recommendations has been lagging behind in critical or intensive care, which is due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n- pufas. over the last years, the data coming out from a great number of studies have left us an improved knowledge of how n- pufas and their derivate mediators modulate inflammation/resolution and immunity. evidence that supports both the antiinflammatory and antitumor effects of n- pufas has been gathered; moreover, the molecular pathways underlying these effects have been clarified both in vitro and in vivo. in particular, there is now a better understanding of various mechanisms generating the production of inflammatory cytokines in several inflammatory, metabolic, and neurodegenerative diseases, as well as in inflammation-related cancers. the capacity of n- pufas to determine a shift from a strongly pro-inflammatory environment to one of reduced inflammation supports the hypothesis that these fas might be useful as a component of the therapy in various inflammatory diseases. thus, epa and/or dha have been evaluated in some inflammatory conditions. for the potential or in-use applications of n- pufas to clinical conditions different from ali, see a recent review (calder b) . briefly, fish oil or its derived epa and dha have demonstrated the efficacy in animal models of rheumatoid arthritis, inflammatory bowel diseases, and asthma. there have been a number of clinical trials evaluating the effects of administration of n- pufas in those diseases with contradicting results. for example, there is some pretty good evidence of the efficacy of n- pufas in rheumatoid arthritis. conversely, despite positive results in some studies, there is only weak evidence that n- pufas have clinical benefits in inflammatory bowel diseases. finally, clinical studies on fish oil in adult patients with asthma do not show benefit. the "european society for clinical nutrition and metabolism guidelines on enteral nutrition: intensive care" (kreymann et al. ) stated that "patients with ards should receive en enriched with n- fatty acids and antioxidants" as a b grade recommendation. however, this recommendation was not evaluated in the "espen guidelines on parenteral nutrition: intensive care" (singer et al. ), although the authors stated that "addition of epa and dha to lipid emulsions has demonstrable effects on cell membranes and inflammatory processes; fish oil-enriched lipid emulsions probably decrease length of stay in critically ill patients" both as b grade recommendations. the american society for parenteral and enteral nutrition "guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient" (mcclave et al. ), also being co-published by the society of critical care medicine, stated that "patients with ards and severe ali should be placed on an enteral formulation characterized by an antiinflammatory lipid profile (i.e., n- fish oil, borage oil) and antioxidants" (a grade recommendation). this recommendation was based on the three positive trials published at that time. the canadian guidelines (updated june ) stated that "based on level studies and level studies, the use of an enteral formula with fish oils, borage oils and antioxidants in patients with ali and ards should be considered. when pn with intravenous lipids is indicated, lipids that reduce the load of n- fa/soybean oil emulsions should be considered. however, there are insufficient data to make a recommendation on the type of lipids to be used that reduce the n- fa/soybean oil load in critically ill patients receiving pn." ali/ards patients are at high risk of malnutrition; therefore, they need nutrition support, including lipids. soybean oil-based lipid emulsions, rich in n- pufas and commonly used for these patients, may aggravate the inflammatory response. in contrast, fish oil-based lipid emulsions, rich in n- pufas, may exert an anti-inflammatory effect. these data suggest that shifting the pufa supply from n- to n- could be an important element affecting the alveolar cytokine and eicosanoid release and consequently ameliorates the clinical outcome in ali/ards patients. in summary, there are good experimental evidence and convincing rationale according to the n- pufas used in ali/ards patients. however, recent rcts using n- pufas in these patients did not confirm these positive effects. further studies are necessary to offer more precise indications, timing, and modalities for n- pufa administration. • acute lung injury (ali) and acute respiratory distress syndrome (ards) are inflammatory diseases whose clinical severity depends on the grade of inflammation. • cytokines are key elements in the pathogenesis of ali/ards. • soybean oil-based lipid emulsions, rich in omega (n)- polyunsaturated fatty acids (pufas), may aggravate the inflammatory response. in contrast, fish oil-based lipid emulsions, rich in n- pufas, may exert an antiinflammatory effect. • experimental and clinical data suggested that shifting the pufa supply from n- to n- decreases the alveolar cytokine and eicosanoid release and consequently ameliorates the clinical outcome in ali/ards patients. • based on these data, several scientific societies recommended in their guidelines the use of n- pufas in ali/ards patients. effects of fish oil containing lipid emulsion on plasma phospholipid fatty acids, inflammatory markers, and clinical outcomes in septic patients: a randomized, controlled clinical trial n- polyunsaturated fatty acids, inflammation, and inflammatory diseases n- fatty acids, inflammation and immunity: new mechanisms to explain old actions omega- polyunsaturated fatty acids and inflammatory processes: nutrition 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profibrotic reactions in the early phase of acute respiratory distress syndrome enteral omega- fatty acid, γ-linolenic acid, and antioxidant supplementation in acute lung injury effects on hemodynamics and gas exchange of omega- fatty acidenriched lipid emulsion in acute respiratory distress syndrome (ards): a prospective, randomized, double-blind, parallel group study benefit of an enteral diet enriched with eicosapentaenoic acid and gammalinolenic acid in ventilated patients with acute lung injury espen guidelines on parenteral nutrition: intensive care a phase ii randomized placebo-controlled trial of omega- fatty acids for the treatment of acute lung injury key: cord- -cxi cr authors: yoshida, asuka; kawabata, ryoko; honda, tomoyuki; tomonaga, keizo; sakaguchi, takemasa; irie, takashi title: ifn-β-inducing, unusual viral rna species produced by paramyxovirus infection accumulated into distinct cytoplasmic structures in an rna-type-dependent manner date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: cxi cr the interferon (ifn) system is one of the most important defensive responses of mammals against viruses, and is rapidly evoked when the pathogen-associated molecular patterns (pamps) of viruses are sensed. non-self, virus-derived rna species have been identified as the pamps of rna viruses. in the present study, we compared different types of ifn-β-inducing and -non-inducing viruses in the context of sendai virus infection. we found that some types of unusual viral rna species were produced by infections with ifn-β-inducing viruses and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. one of these structures was similar to the so-called antiviral stress granules (avsgs) formed by an infection with ifn-inducing viruses whose c proteins were knocked-out or mutated. non-encapsidated, unusual viral rna harboring the ′-terminal region of the viral genome as well as rig-i and typical sg markers accumulated in these granules. another was a non-sg-like inclusion formed by an infection with the cantell strain; a copyback-type di genome, but not an authentic viral genome, specifically accumulated in the inclusion, whereas rig-i and sg markers did not. the induction of ifn-β was closely associated with the production of these unusual rnas as well as the formation of the cytoplasmic structures. eukaryotic cells are equipped with various defense mechanisms to detect and respond to viral infections rapidly. the interferon (ifn) system is one of the most important natural defenses of mammalian cells in the early phase of viral infection. host cells sense the invasion of viruses by recognizing their pathogen-associated molecular patterns (pamps), including the structural characteristics of viral rnas that differentiate them from cellular rnas . viral rnas are detected by non-self rna sensors such as toll-like receptors (tlr), and a family of cytosolic rna helicases termed rig-i-like receptors (rlrs), including retinoic-acid inducible gene-i (rig-i), melanoma differentiation-associated gene (mda ), and laboratory of physiology and genetics gene (lgp ). this is followed by the subsequent induction of ifn-β (gitlin et al., ; kaisho and akira, ; saito et al., ) . autocrine or paracrine ifns bind to ifn receptors on the cell surface, leading to the expression of s of ifn-stimulated genes (isgs) through the jak/stat signaling pathway, which ultimately exerts various antiviral effects . translational arrest is one of the ifn responses of host cells triggered by viral infections. various eukaryotic translation initiation factor (eif ) kinases such as protein kinase r (pkr) are activated in response to ifns, and the accumulation of phosphorylated eif α inhibits the translation of both cellular and viral mrnas kedersha and anderson, ; holcik and sonenberg, ) . cytoplasmic stress granules (sgs), which are the foci of concentrated s translation preinitiation complexes and defined by certain marker rna binding proteins such as t-cell intracellular antigen- (tia- ), tia- -related protein (tiar), and ras-gap-sh domain-binding protein (g bp ), are formed under these conditions kedersha and anderson, ) . because they contain stable inert mrna, sgs are believed to serve as temporary sites at which mrnaprotein complexes are stored to pause active translation or be decayed in adjacent processing bodies (anderson and kedersha, ; balagopal and parker, ; buchan and parker, ) . a number of viruses have been shown to induce the formation of sgs in infected cells, and this may be related to the virus-induced shut-off of cellular protein translation. for example, hepatitis c virus, poliovirus, semliki forest virus, and mammalian orthoreovirus promote the shut-off of cellular proteins and the assembly of sgs at the early phase of infection, and this is inhibited as the infection progresses (mcinerney et al., ; white et al., ; qin et al., qin et al., , ariumi et al., ; white and lloyd, ; garaigorta et al., ; panas et al., ; ruggieri et al., ; fitzgerald and semler, ; pager et al., ; carroll et al., ) . two rna viruses, respiratory syncytial virus and coronavirus, are also known to utilize sgs as part of the machinery to inhibit host cellular protein translation (raaben et al., ; lindquist et al., lindquist et al., , . sgs were not detected during infection by influenza a virus (iav); however, a recombinant iav lacking non-structural protein (ns ), an inhibitor of pkr, efficiently induced the formation of sgs and the production of ifn-β in a pkr-dependent manner (khaperskyy et al., ; mok et al., ; onomoto et al., ) . in this case, sgs were suggested to play an important role as the sites of viral rna sensing and subsequent anti-viral responses, because rlrs localized together with viral nucleoproteins and rna as well as anti-viral proteins in sgs (onomoto et al., ; ng et al., ) . the rna species produced during the course of rna viral replication, such as mrna, dsrna, and -triphosphate ( -ppp) rna including leader, trailer, genome, and antigenome rnas as well as defective interfering (di) genomes, have been shown to trigger the production of type i ifns (yoneyama et al., ; hornung et al., ; pichlmair et al., ; strahle et al., ; hausmann et al., ; baum et al., ; baum and garcia-sastre, ; kato et al., ; marq et al., ; davis et al., ; bowzard et al., ; weber et al., ; runge et al., ; schmolke et al., ) . we and other groups have recently reported that recombinant viruses of sendai virus (sev), a prototype of the family paramyxoviridae, in which the c proteins are knocked-out or mutated, generate dsrna in infected cells at levels similar to the production of ifn-β (takeuchi et al., ; irie et al., ) . previous studies also reported that, in the cases of sev and iav, copyback (cb)-and internal deletion (id)-type di genomes, respectively, rather than full-length viral genomes, preferentially associated with rig-i and strongly induced the production of ifn-β (baum et al., ; baum and garcia-sastre, ) . in spite of the large number of studies conducted in this field, it remains unknown what kinds of viral rna species are recognized by rlrs and where the sites of recognition are in real infections by rna viruses. in the present study, we compared some types of ifn-β-non-inducing and ifn-β-highly inducing viruses in the context of sev infection. one of the biggest advantages of this study is that the comparison can be performed within the context of the same viral species. we found that some types of unusual rna species that were distinguishable according to specific detectability by the anti-dsrna antibody or fish analysis were produced during the viral replication of ifn-inducing sevs, but not ifn-non-inducing sev strains, and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. these unusual rnas exhibited distinct properties in infected cells in terms of encapsidation with the viral n protein and subcellular distribution with sg marker proteins and rlrs. our results suggest that rna-typedependent mechanisms recognize and accumulate virus-derived, ifn-β-inducible, unusual rnas into specific compartment to trigger the production of ifn-β, and that sev may evade detection by the host innate immune system by preventing the production of these rna species. llc-mk cells (macaque monkey kidney-derived cells, described in kiyotani et al., ) and hela cells (ccl- ; purchased from atcc) were maintained as described previously (irie et al., ) . all of the sevs, even the wt of strain z and hamamatsu (hmt), used in this study were recovered from cdna using a reverse genetics technique as described previously (kato et al., ; fujii et al., ) , except for the cantell (cnt; vr- ; atcc), fushimi (fsm), and nagoya (ngy) strains. the sev recombinants, c /c(-), c(-), and v(-), were kindly provided by kato (national institute of infectious diseases, japan; kato et al., ; kurotani et al., ) . all of the sevs as well as virulent and avirulent newcastle disease virus (ndv) miyadera and d strains (toyoda et al., ) , respectively, were propagated in embryonated chicken eggs. sev and ndv titers were determined by an immunofluorescent infectious focus assay in llc-mk cells and expressed as cell infectious units (cius)/ml, as described previously (kiyotani et al., ) . one-step growth kinetics of the viruses was determined as described previously (irie et al., ) . plasmids encoding the p, c, and v proteins of sev strain z in the pcaggs. mcs vector have been described previously (sakaguchi et al., (sakaguchi et al., , irie et al., b irie et al., , . the polyclonal antibodies (pabs) against whole virions of sev and ndv were described previously (kiyotani et al., ) . the pabs against the sev p and c proteins were kindly provided by kato. the monoclonal antibody (mab) against the sev n protein was kindly provided by suzuki (national institute of infectious disease, japan). the mab and pab against g bp (sc- , santa cruz biotechnology; and ab , abcam, respectively), pab against rig-i ( ; immuno-biological laboratories, japan), mab against tiar, and dsrna (# , cell signaling technology; and j , scicons, hungary, respectively) were used according to the protocols of the suppliers. hela cells cultured on glass coverslips were infected with the indicated viruses or transfected with the indicated plasmids. the culture medium was replaced by serum-free dmem h postinfection (p.i.) or post-transfection (p.t.), and cells were treated with sodium arsenite (naaso ; sigma) at a final concentration of . mm for min or with ifn-α ( , iu/ml; r&d systems) for h. hela cells cultured on glass coverslips were transfected with the indicated plasmids using fugene hd transfection reagent (promega) or infected with the indicated viruses at an moi of . at h p.t. or p.i., cells were fixed and immunostained as described previously using appropriate combinations of primary and secondary antibodies (irie et al., ) . to detect rig-i, and dsrna, the tyramide signal amplification (tsa) kit with hrp-goat anti-mouse igg and alexa fluor tyramide (molecular probes) were used to increase the detection sensitivity. coverslips were mounted on glass slides with the slowfade gold antifade reagent with or without dapi (molecular probes) and observed using an lsm confocal microscope (carl zeiss). hela cells were infected with the indicated viruses at an moi of . at h p.i., total rna was prepared using the high pure rna isolation kit (roche diagnostics). viral rna in the working viral stocks was prepared using the high pure viral rna kit (roche diagnostics). quantitative (q) rt-pcr was performed as described previously (irie et al., (irie et al., , . qrt-pcr samples were analyzed using the eco real-time pcr system (illumina). for direct comparison, all of the indicated samples were analyzed in the same experiments. to detect the genome-length viral rnas and cbdi genomes of sev stocks, qrt-pcr was performed using the primer sets of sevz + sevz , as described previously (irie et al., a (irie et al., , , and cbdidetect , - , + cbdidetect , - , , which were complementary to the regions of the indicated positions of sev genome rna, as described recently by baum et al. ( ) . hela cells were lysed in ripa buffer ( . % np- , mm tris-hcl [ph . ], mm nacl) after h of infection by the indicated viruses or min of the arsenite treatment, and the insoluble fraction was removed by high-speed centrifugation. the viral proteins in the lysates were removed by three consecutive immunoprecipitation steps using anti-sev or anti-ndv pabs and protein g sepharose beads (ge healthcare life sciences). supernatants were harvested from the final immunoprecipitation samples, and were then subjected to rna preparation, as described above. hela cells cultured on glass coverslips were transfected with μg of the indicated rna samples prepared above, together with . μg of an empty puc vector, using the fugene hd transfection reagent. cantell samples ( . × ciu/ml) that were ∼ -fold serially diluted were inoculated into the allantoic cavity of -days-old embryonated chicken eggs, and incubated for h at • c. allantoic fluid was harvested from the eggs, and the titers of each fluid stock were determined as described above. rna samples were prepared from μl of each fluid stock, and the ratios of the cbdi genomes to viral genomes were determined by qrt-pcr as described above. hela cells cultured on glass coverslips were infected with the indicated viruses. at h p.i., cells were fixed with % paraformaldehyde solution in pbs, and then subjected to fish analysis using the fish tag rna green kit with alexa fluor dye (molecular probes) according to the protocol of the supplier. the rna probe was designed to be complementary to the region of , - , in sev genome rna. after fish, some samples were further subjected to fluorescent immunodetection as described above using the indicated antibodies. final samples were observed using an lsm confocal microscope. we first examined whether g bp -positive granular structures were formed during infections by a series of c knockedout and mutated sev recombinants and the parental z strain by immunofluorescence microscopy (figure ; supplementary figure s ). treating hela cells with sodium arsenite (naaso ) caused the formation of granular structures in the cytosol, which were figure | subcellular distribution of g bp and tiar in hela cells treated with or without sodium arsenite (a), and infected with a series of c-deficient rsevs, dy , dy , d y, c /c(-), and c(-), and a v-deficient rsev, v(-) (b). hela cells treated with ethanol (etoh) or sodium arsenite (naaso ) or infected with the indicated virus were immunostained with anti-g bp mab, anti-tiar mab, and anti-sev pab. (c) the g bp foci in the samples of (b) were observed under a fluorescent microscope, and the number of g bp granule-positive cells was counted and is presented as percentages against the number of sev antigen-positive cells (closed bars). the relative amounts of ifn-β mrna in cells infected with the indicated viruses were determined by qrt-pcr, and normalized to those of β-actin mrna (open bars). (d) similar experiments were performed for a series of c-mutated sev recombinants, and the results are presented as bar graphs, similarly to (c). defined as sgs based on the expression of related proteins such as g bp and tiar. g bp and tiar are well-established sg-associated proteins that are typically and diffusely present throughout the cytoplasm and dominantly present in the nucleus, respectively. however, treating cells with arsenite markedly changed the localization to form sgs containing these proteins in nearly all cells ( figure a) . when cells were infected with c(-), g bp -positive granular structures were observed in almost % of sev antigen-positive cells, and were considered to be sg-like structures since tiar was also detected in the majority of the granules (figures b,c) . in this situation, tiar was mostly in the cytoplasmic structures, whereas a larger part of tiar was still observed in the nucleus in the arsenite-treated cells. this difference is probably due to the different exposure time to the stimuli: min. for the treatment with arsenite and h for the infection. in contrast, the percentages were only % or less in cells infected with the parental z-wt as well as the dy , dy , and d y recombinants, which lacked the smaller c proteins, y , y , and both y and y , respectively ( figure c; supplementary figure s a ). an infection by c /c(-) and v(-), lacking the larger c proteins, c and c, and the v protein, respectively, resulted in a slight increase in the number of granules ( figure c ; supplementary figure s a ). of note, unlike the viruses reported previously, such as ns -deficient iav and vesicular stomatitis virus (mok et al., ; onomoto et al., ; dinh et al., ) , the fluorescence of the sev antigen was not colocalized with that of the representative sg marker g bp in the granules (figure b ; supplementary figure s ). ifn-β mrna levels in the infected cells were also compared between the viruses ( figure c) . strong correlations were observed between ifn-β mrna levels and the percentages of granular structure-forming cells against infected cells. similar strong correlations were observed for a series of c mutant viruses that possessed single-to triple-amino-acid substitutions of highly conserved, charged amino acids within the c proteins, which diminished their ability to antagonize the host ifn system to various degrees (figure d ; supplementary figure s b ; irie et al., ) . the marked difference observed in granular formation and ifn-β mrna levels among the viruses was not due to their different growing abilities. the one-step growth kinetics of all the recombinants used above was previously reported to be similar to that of the parental z-wt, except for c(-) and c /c(-), the titers of which were - logs lower than that of z-wt throughout the time course, and viral protein synthesis in the infected cells did not significantly differ among the viruses examined (kurotani et al., ; irie et al., a irie et al., , . treatment of hela cells with ifn-α did not induce g bp -positive granules, indicating that the formation of sglike structures observed by sev infection was triggered by sev infection, but not by sev-induced type i ifns (supplementary figure s c) . taken together, these results strongly suggested that a relationship may exist between the formation of g bp -positive granules and the induction of ifn-β in the c recombinants, and also that c proteins may suppress the formation of granules. we examined whether expression of the c protein alone and infection of the non-granule-forming sev could inhibit the formation of granules in cells treated with arsenite or infected with ndv (figure ; supplementary figure s ). ndv, another prototypic paramyxovirus, which was considered to be an ifn-β-inducing virus, could induce g bp -positive granules in nearly all cells infected with virulent as well as avirulent strains (miyadera and d strains, respectively; figure b ). the c protein alone failed to inhibit the formation of granules in cells treated with arsenite (figure a) as well as infected with ndv ( figure b) , although the number of granules was slightly reduced in the c-expressing cells treated with arsenite compared to that observed in the neighboring non-c-expressing cells (figure a) . in addition to c, the other p gene products, p and v proteins, also failed to inhibit the formation of both types of granule (supplementary figure s ) . infection by sev-z-wt and c-deficient recombinant c(-) also failed to inhibit the arsenite-induced formation of granules (figures c,d) . small granules dispersed in the cytoplasm were induced by arsenite in both cases of infection by wt and c(-). of note, the g bp -positive granules induced by arsenite and viruses, such as c(-) and ndv, differed in size; the granules induced by the infection were apparently larger than those induced by arsenite (figures and ) , implying a possible difference in cellular pathways leading to these two types of granular structure. these results demonstrated that sev did not have the ability to inhibit the formation of both types of granules. a marked difference was noted in the abilities of the sev strains to induce ifn-β. although most of the sev strains including z have been characterized by their strong ability to counteract the innate immune system, the cnt strain has been widely used as a virus that induces high levels of ifn-β (baum et al., ; tapia et al., ) . therefore, we compared the abilities of some sev strains to induce ifn-β and sg-like structures (figure ; supplementary figure s ). in all cases, g bp -positive granular structures were only detected in less than % of sev antigen-positive cells (figure ; supplementary figure s a ). ifn-β mrna was not highly induced in cells infected with the sev strains, except for cnt, whereas cnt induced ifn-β mrna at a level that was -fold higher than that by z (figure ) . regarding the sev strains, unlike that observed in the c recombinants, a correlation was not observed between ifn-β mrna levels and the percentage of granular structure-forming cells against infected cells (figure ) . the different levels of ifn-β mrna induced by the strains could not be attributed to differences in viral growth (supplementary figure s b) . these results suggested that the formation of g bp -positive granules was not necessarily required to sense the sev cnt infection, followed by the production of ifn-β, unlike the c recombinants. we attempted to identify the reason for the difference in granular formation between these two types of ifn-β-inducing virus, c(-) and cnt. to address this issue, we first examined the ability of total rna prepared from virus-infected as well as arsenite-treated cells to induce g bp -positive granules ( figure a; supplementary figure s a ). total rna samples prepared from cells infected with any of the ifn-β-inducing viruses, sev-cnt, c(-), and ndv, were able to induce the granules as well as ifn-β in hela cells despite no formation of the granules by the cnt infection, whereas those from cells infected with the ifn-β-non-inducing sev-z-wt or treated with arsenite were not (figure a) . we then examined the content rates of cbdi genomes, a potent ligand for rig-i, against viral genome-length rnas in the rna samples used above ( figure b ). the cnt sample contained cbdi genomes at a level that was ∼ , -fold higher than that in the z-wt sample, although the sample of c(-) contained similar levels to the z-wt sample ( figure b) . we further examined the effect of removal of encapsidated viral rna species, such as viral full-length and di genomes, by three consecutive rounds of immunoprecipitation using antisera against the whole virions of sev and ndv prior to the preparation of rna samples (supplementary figure s b) . total rna prepared from the postimmunoprecipitation samples of c(-) and ndv was still able to induce the granules as well as ifn-β, but that of cnt lost these abilities (figure c ). since the naked cbdi genomes have been reported readily to form an ideal structure as the rig-i ligands of -triphosphated, blunt-ended dsrna (kolakofsky, ) , these results indicated that the major ifn-β-inducing viral rna species produced in the cells infected with cnt was encapsidated cbdi genomes, whereas those for sev- c(-) and ndv were not. the results above suggested that there may be at least two types of ifn-β-inducing viral rna species with a marked difference in the formation of sg-like granules. to elucidate this difference, we examined the subcellular distribution of unusual viral rna species produced by c(-) and cnt (figures and ) . we and other groups previously reported a strong correlation between the production of dsrna detected by the anti-dsrna antibody j (j -dsrna) and the induction of ifn-β in infections of the c-mutated sev recombinants and ndv (takeuchi et al., ; irie et al., ) . therefore, we first examined the production and subcellular distribution of j -dsrna in the cells infected with the ifn-β-inducing viruses by immunofluorescence microscopy (figure ) . dsrna fluorescent signals were absent in cells infected with ifn-non-inducing z-wt as well as in those with the ifn-β-inducing strain cnt ( figure a ). in contrast, dsrna fluorescent signals were clearly observed in the cytoplasm of cells infected with ifn-β-inducing sev- c(-) and ndv with dispersed and granular distributions, respectively ( figure b) . a previous study reported that ns -deficient iav infections caused rig-i to form granular aggregates that contained sg markers as well as viral rna (onomoto et al., ) . therefore, the cells infected with c(-) and ndv were co-stained with anti-rig-i and anti-g bp antibodies ( figure c ). similar to iav, rig-i almost perfectly colocalized with the virus-induced g bp -positive granules in both cases of infection ( figure c) ; however, unlike iav, these granules did not colocalize with the viral antigen or viral j -dsrna ( figure b, arrowheads) , as shown in figures and . together with the results of figure , j -dsrna appeared to be not or less encapsidated by viral nucleoproteins because the access of the antibody to and the formation of dsrna by tightly encapsidated viral rna, such as viral genomes, was unlikely. we further attempted to visualize the subcellular distribution of the unusual viral rna species that were not fully encapsidated, unlike the genome-length viral rnas, by fish analysis (figure ) . infected cell samples were prepared without a protease treatment to exclude fully encapsidated viral rna species, and were then stained with an rna probe complementary to the , - , region of the (-)-sense viral genome rna, designed by referring to two wellcharacterized cbdi genomes of sev (calain et al., ; -gil et al., ) . fluorescence-positive cytoplasmic inclusions were observed in the c(-)-as well as in the cntinfected samples, while an apparent signal was not detected in z-wt-infected or uninfected samples ( figure a ). when the z-wt-infected sample was treated with proteinase k, apparent signals were observed throughout the cytoplasm ( figure a) . these results strongly suggested that fully encapsidated viral genomes were not detected, whereas nonor partially encapsidated viral rna species were detectable in this system. the fish-positive inclusions observed in cntinfected cells were no longer detected in the cells infected with the cnt-lowdi (figure a) , which contained ∼ -fold fewer cbdi genomes than the original cnt sample ( figure b) . together with the results of figure , the fish signals observed in the cnt-infected cells were considered as the cbdi genomes. the fish samples of c(-) and cnt-infected cells were further immunostained to examine the subcellular colocalization of fish signals, the sev n protein, rig-i, and g bp (figures b-d, respectively) . the sev n protein was detected in the fish-positive inclusions of cnt-infected cells, suggesting that the rna species detected in the cnt samples were only partially, not fully, encapsidated, unlike the fully encapsidated, full-length, intact viral genomes ( figure b ). in contrast, the n protein was not detected in the inclusions of c(-)-infected cells, which suggested that the rna species detected in the c(-) samples were not encapsidated ( figure b) . the fish-positive inclusions of cnt-infected cells were not apparently colocalized with rig-i or g bp , whereas most of those in the c(-)-infected cells colocalized obviously with rig-i and g bp , unlike the j -dsrna (figures c,d) . these results indicated that unusual viral rna species harboring the -region of (-)-sense sev genome rna, which was not produced during infection by ifn-β-non-inducing z-wt, was produced in infections by , and were selectively formed into distinct cytoplasmic inclusions in an rna-type-dependent manner. the inclusions in the c(-)-infected cells could be identified as avsgs, and may be the site to detect viral rna by rig-i, whereas those in cnt-infected cells were not avsgs, but as-yet-unidentified structures. although a number of studies have examined host innate immune responses against pathogenic microbes including rna viruses, the virus-derived rna species that serve as pamps in real viral infections and the sites at which pamps are recognized by rlrs have yet to be clarified in detail. two important insights were reported recently. regarding the real pamps in infections by rna viruses, the cb and id types of di genomes were identified as the ligands of rig-i in sev-and iav-infected cells, respectively, both of which could form ideal structures as the rig-i ligands (baum et al., ; baum and garcia-sastre, ; martinez-gil et al., ) . the sg-like structures have been suggested to serve as the sites at which the rlrs encounter viral rna and subsequently activate the ifn signaling pathways in infections by rna viruses (onomoto et al., ; yoo et al., ) . in order to establish what and where viral rna species were detected by rlrs, in the present study, we compared two types of ifnβ-inducing sev, a recombinant c(-) and a strain cnt, with a non-ifn-β-inducing z strain, in terms of the formation of sglike granules and the production of unusual viral rna species. a major advantage of our study is that the comparison can be performed within the context of the same viral species. several types of unusual viral rna species were found to be generated in cells infected with the ifn-inducing sevs but not those with the ifn-non-inducing sevs (summarized in table ). one was a dsrna (j -dsrna) that was detected by the anti-dsrna antibody, j (type i in table ) . as for sev, the generation of j -dsrna may have been restricted by the c proteins because it was only detected in cells infected with c-deficient or mutated recombinants, but not in those with intact sevs (figure ; takeuchi et al., ; irie et al., ) . we and other groups demonstrated that j -dsrna activated pkr, and this was followed by the phosphorylation of eif and the production of ifn-β, both of which resulted in antiviral effects in the host cells. the activation of pkr has been reported to induce the formation of sg-like structures during infections by some rna viruses, such as iav and measles virus (mev; mok et al., ; onomoto et al., ; okonski and samuel, ) , and this also appears to be the case for the sev c recombinants. unlike these viruses, the sg-like structures formed during c(-) infections did not include the j -dsrna, which was dispersed in the cytoplasm, although they contained rig-i (figure ) . this may lead to the assertion that the sg-like structures induced during infections by c recombinants are not the sites at which to detect sev infections. however, the sg-like structures formed by c(-) were revealed to contain another type of unusual viral rna species by fish analysis, in which an rna probe targeting the nt region of the -end of the (-)-sense sev genome was used (type ii in table ; figure ). this now strongly suggests that the sg-like structures found in the c(-) infection are defined as avsgs. unlike the iav infection, the type i and ii rna species seemed to be not or less encapsidated, given that they were not colocalized with viral antigens and were not removed from the infected cell lysates by immunoprecipitation using anti-sev antibody (figures and - ) . sev trailer rna, which is transcribed from the -ends of (+)-sense antigenome rna, was previously reported to interact with tiar to inhibit apoptosis and the formation of sgs induced by infection (iseni et al., ) . the c(-) virus was shown to induce apoptosis more quickly and severely in infected cells than the wt virus (irie et al., ) . although appearing to contain the nonencapsidated -end of a (-)-sense genome rna, at least in the part that is concordant with the trailer rna, it is unlikely that the type ii rna observed in the c(-)-infected cells has the ability of the trailer rna to inhibit apoptosis and sg formation. although details of the type i and ii rnas remain to be solved, given the cytoplasmic replication of sev rna without forming inclusion bodies and the differences of the rna species in subcellular distribution and reactivity with the j and fish probe, the type i dsrna somewhat unwound actively or incidentally into the type ii rna might be accumulated into the avsgs. although the sg-like structures and j -dsrna were not detected during cnt infections, fish-positive non-granularshaped inclusions were observed (type iii in table ; figure ). these cnt inclusions were markedly different from those of c(-). the inclusions did not include rig-i or g bp , but contained the n protein (figure ) , suggesting that the inclusions were not avsgs, and that the type iii rna was at least partially encapsidated. the cnt stock used in the present study contained a larger amount of cbdi genomes than the other viral stocks ( figure b) . the sev cbdi genomes have been shown to be partially and/or more loosely encapsidated than intact genomes (kolakofsky, ; strahle et al., ) . the sev cbdi genomes were recently identified as strong ligands for rig-i (baum et al., ; tapia et al., ) . indeed, the cnt-lowdi had lost the ability to induce ifn-β and the inclusions had not been found in the infected cells (figures b and ) . taken together, the type iii rna was identified as the cbdi genome. these results indicated that the process of detecting infections and the subsequent induction of ifn-β differed largely between c(-) and cnt: avsg-dependent and -independent mechanisms for c(-) and cnt, respectively. most viruses have been shown to possess the ability to antagonize host ifn pathways in order to avoid activating host antiviral actions, and this strategy is mostly based on a counteraction against the molecules involved in these pathways (versteeg and garcia-sastre, ) . however, a recent study reported that encephalomyocarditis virus (emcv) has the ability to disrupt sgs by cleaving g bp in order to avoid the innate immune detection of its infection and subsequent induction of ifn-β (ng et al., ) , suggesting that another effective strategy for viral evasion from the ifn system by preventing avsg formation exists. generation of the unusual viral rna species triggering the production of ifn-β seems to be suppressed during intact rna viral replication. similarly to sev, another paramyxovirus mev c protein was recently shown to impair the production of j -dsrna and the activation of pkr coupled with the formation of sg-like structures (okonski and samuel, ; pfaller et al., ) . unlike the case of sev, in which the knockout of c resulted in the production of j -dsrna (figures and ), but not cbdi genomes, the j -dsrna produced during the infection by a c-deficient mev recombinant was reported to be a cbdi genome (pfaller et al., ) . although the cbdi genomes were more dominantly produced by sev-cnt than by the other strains and c-recombinants tested ( figure b and data not shown), this unique property of cnt might be attributed to its c protein that possibly have a functional difference with those of the other sevs. the c proteins of both sev and mev have been shown to play critical roles in modulating viral rna synthesis and maintaining its integrity by potentially stabilizing the ribonucleoprotein (rnp)-polymerase complex (tapparel et al., ; reutter et al., ; bankamp et al., ; irie et al., a irie et al., , ito et al., ) . dysfunctions in the c proteins may result in a disturbance in integrity, leading to the production of the unusual, ifn-β-inducing rna species. the results of the present study indicate that several types of ifn-β-inducible, unusual viral rna species may be produced during sev infections and included in avsg-like and non-avsg-like cytoplasmic inclusions, which suggests that rna-typedependent mechanisms recognize and accumulate such unusual viral rnas in specific compartments. in addition, the production of these unusual rna species may be restricted during intact viral replication in order to avoid detection by host innate immunity. pathogen recognition and innate immunity visibly stressed: the role of eif , tia- , and stress granules in protein translation rna granules hepatitis c virus hijacks p-body and stress granule components around lipid droplets polysomes, p bodies and stress granules: states and fates of eukaryotic mrnas identification of naturally occurring amino acid variations that affect the ability of the measles virus c protein to regulate genome replication and transcription differential recognition of viral rna by rig-i preference of rig-i for short viral rna molecules in infected cells revealed by nextgeneration sequencing rig-i goes beyond naked recognition eukaryotic stress granules: the ins and outs of translation molecular cloning of natural paramyxovirus copy-back defective interfering rnas and their expression from dna amino acids and of mammalian orthoreovirus protein microns are necessary for stress granule localization, core protein lambda interaction, and de novo virus replication the ' untranslated regions of influenza genomic sequences are 'ppp-independent ligands for rig-i induction of stress granule-like structures in vesicular stomatitis virus-infected cells poliovirus infection induces the colocalization of cellular protein srp with tia- , a cytoplasmic stress granule protein involvement of the leader sequence in sendai virus pathogenesis revealed by recovery of a pathogenic field isolate from cdna hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus rig-i and dsrna-induced ifnbeta activation translational control in stress and apoptosis '-triphosphate rna is the ligand for rig-i inhibition of interferon regulatory factor activation by paramyxovirus v protein conserved charged amino acids within sendai virus c protein play multiple roles in the evasion of innate immune responses paramyxovirus sendai virus c proteins are essential for maintenance of negative-sense rna genome in virus particles recruitment of alix/aip to the plasma membrane by sendai virus c protein facilitates budding of virus-like particles sendai virus c proteins regulate viral genome and antigenome synthesis to dictate the negative genome polarity clustered basic amino acids of the small sendai virus c protein y are critical to its ran gtpase-mediated nuclear localization sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis measles virus nonstructural c protein modulates viral rna polymerase activity by interacting with host protein shcbp toll-like receptor function and signaling the paramyxovirus, sendai virus, v protein encodes a luxury function required for viral pathogenesis initiation of sendai virus multiplication from transfected cdna or rna with negative or positive sense rig-i-like receptors: cytoplasmic sensors for non-self rna stress granules: sites of mrna triage that regulate mrna stability and translatability influenza a virus inhibits cytoplasmic stress granule formation immediate protection of mice from lethal wild-type sendai virus (hvj) infections by a temperaturesensitive mutant, hvjpi, possessing homologous interfering capacity isolation and characterization of sendai virus di-rnas sendai virus c proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication short double-stranded rnas with an overhanging ' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a sendai virus-derived rna agonist of rig-i as a virus vaccine adjuvant importance of eif alpha phosphorylation and stress granule assembly in alphavirus translation regulation the ns protein of influenza a virus interacts with cellular processing bodies and stress granules through rna-associated protein (rap ) during virus infection encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity modulation of hepatitis c virus rna abundance and virus release by dispersion of processing bodies and enrichment of stress granules sequestration of g bp coupled with efficient translation inhibits stress granules in semliki forest virus infection measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif alpha phosphorylation and pkr mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies mutations in the measles virus c protein that up regulate viral rna synthesis dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection in vivo ligands of mda and rig-i in measles virus-infected cells regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene aip /alix is a binding partner of sendai virus c protein and facilitates virus budding rig-i detects mrna of intracellular salmonella enterica serovar typhimurium during bacterial infection sendai virus defective-interfering genomes and the activation of interferon-beta activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity inhibition of sendai virus genome replication due to promoterincreased selectivity: a possible role for the accessory c proteins structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of newcastle disease virus viral tricks to grid-lock the type i interferon system incoming rna virus nucleocapsids containing a '-triphosphorylated genome activate rig-i and antiviral signaling inhibition of cytoplasmic mrna stress granule formation by a viral proteinase poliovirus unlinks tia aggregation and mrna stress granule formation the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses dhx enhances rig-i signaling by facilitating pkrmediated antiviral stress granule formation we thank the staff of the analysis center of life science, hiroshima university, for the use of their facilities. we also thank dr. k. takeuchi (university of tsukuba) for fruitful discussions. this work was supported by jsps kakenhi (grant numbers and ). the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . key: cord- -x z o gs authors: artusi, sara; nadai, matteo; perrone, rosalba; biasolo, maria angela; palù, giorgio; flamand, louis; calistri, arianna; richter, sara n. title: the herpes simplex virus- genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: x z o gs guanine-rich nucleic acids can fold into g-quadruplexes, secondary structures implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. the remarkably high guanine content of the herpes simplex virus- (hsv- ) genome prompted us to investigate both the presence of g-quadruplex forming sequences in the viral genome and the possibility to target them with g-quadruplex ligands to obtain anti-hsv- effects with a novel mechanism of action. using biophysical, molecular biology and antiviral assays, we showed that the hsv- genome displays multiple clusters of repeated sequences that form very stable g-quadruplexes. these sequences are mainly located in the inverted repeats of the hsv- genome. treatment of hsv- infected cells with the g-quadruplex ligand braco- induced inhibition of virus production. braco- was able to inhibit taq polymerase processing at g-quadruplex forming sequences in the hsv- genome, and decreased intracellular viral dna in infected cells. the last step targeted by braco- was viral dna replication, while no effect on virus entry in the cells was observed. this work, presents the first evidence of extended g-quadruplex sites in key regions of the hsv- genome, indicates the possibility to block viral dna replication by g-quadruplex-ligand and therefore provides a proof of concept for the use of g-quadruplex ligands as new anti-herpetic therapeutic options. g-quadruplexes are nucleic acids secondary structure that may form in g-rich sequences. they are based on the formation of g-quartets, which are stabilized by hoogsteen hydrogen bonds between guanines (sen and gilbert, ) . g-quartets stack on top of each other to give rise to g-quadruplexes that are further stabilized by the interaction with monovalent cations. dna strands in g-quadruplex may be oriented in anti-parallel, parallel, or hybrid configuration, and the nucleotide linkers between g-quartet stacks can adopt a multitude of loop structures (patel et al., ; phan, ; phan et al., ; simonsson, ) . g-quadruplexes occur in functionally important regions of the genome (huppert, ; neidle, ) : they have been identified in the promoters of a wide range of genes that are important in cell signaling (balasubramanian et al., ; duquette et al., ) , suggesting the possibility that g-quadruplexes behave as structural switches of cellular processes, therefore providing a basis for therapeutic intervention (neidle and parkinson, ; zhang et al., ) . one of the most potent g-quadruplex ligands is braco- (fig. a) , a , , -trisubstituted acridine derivative designed to stabilize the quadruplex dna structures formed in human telomeres (read et al., ) . has been shown to inhibit telomerase activity (harrison et al., ) and to display in vitro and in vivo antitumor activity (burger et al., ; gowan et al., ) . besides humans, other mammals (verma et al., ) , yeast (hershman et al., ) and prokaryotic cells (beaume et al., ; rawal et al., ; wieland and hartig, ) showed enrichment of putative g-quadruplex forming sequences. in viruses, evidence of significant g-quadruplex regions is increasingly being recognized. in the epstein-barr herpes virus (ebv), g-quadruplexes modulate ebv nuclear antigen (ebna ) activity and translation (murat et al., ) ; in particular, braco- inhibited ebna -dependent stimulation of viral dna replication http://dx.doi.org/ . /j.antiviral. . . - /Ó elsevier b.v. all rights reserved. (norseen et al., ) . in hiv- , we have demonstrated a g-quadruplex-mediated transcriptional silencing activity in the sp binding region of the long terminal repeat promoter (perrone et al., a) and in the nef coding gene (perrone et al., b) . in both cases, g-quadruplex ligands were able to stabilize the g-quadruplex conformations and to exert antiviral activity (perrone et al., ) . in the sars coronavirus, g-quadruplexes targeting by a key viral protein was proposed to control host's cell response to viral infection (tan et al., ) . the herpes simplex virus type (hsv- ) kbp genome has % guanosines (gs) and cytosines (cs) composition, which reaches . % in simple sequence repeats (ssrs), ubiquitously distributed in both coding and non-coding regions with similar relative frequency (ouyang et al., ) ; in contrast, gc content in the human genome is %. the hsv- genome is composed of two covalently linked regions of unique sequences, the unique long (u l ) and unique short (u s ) sequences, flanked by large inverted repeated sequences, tr l -ir l and ir s -tr s (fig. b) (hayward et al., ) . hsv replication is temporally regulated in rounds of transcription that yield immediate-early, early and late proteins. hsv- is a neurotropic virus: after the first lytic infection within mucosal epithelial cells, the virus enters sensory neurons where latency is established. the virus can later reactivate resulting in the generation of new virions that cause recurrent disease (roizman et al., ) . hsv- most commonly causes vesicular lesions affecting the mucous membranes; however, it can also cause infrequent but serious diseases, such as encephalitis, disseminated neonatal infections and visceral infections in immunocompromised patients (frangoul et al., ; genereau et al., ) . the presence of hsv increases sexual transmission of hiv- (freeman et al., ) . hsv- symptoms are not cured but treated with nucleoside analogues, such as acyclovir (acv) and its derivatives (vere hodge and field, ). however, the virus remains in the body for life, since no cure that eradicates herpes has yet been developed. in addition, emergence of resistance to current anti-herpetic drugs has long created an obstacle for the treatment of hsv- (bacon et al., ) . therefore new antiviral approaches able to suppress both lytic and possibly also latent infections are highly required. earlier work by roizman indicated the presence of an alternative conformation of a viral g-rich dna and rna sequence around the origin of dna replication in the hsv- genome (mccormick et al., ; roller et al., ) ; however no systematic analysis on the presence of g-quadruplex structures in the hsv- nucleic acid has ever been provided. here we present the first comprehensive genome analysis and evidence that extended g-rich sequences mainly located in repeats of the hsv- genome can fold in stable g-quadruplex structures. we demonstrate that treatment with the g-quadruplex ligand braco- greatly stabilizes these sequences resulting in decrease of infectious viral particles, reduction of late viral transcripts, inhibition of taq polymerase processing at the hsv- genome, specifically affecting viral dna replication at g-quadruplex regions. a detailed description of this section is provided in supporting information. . . highly conserved putative g-quadruplex forming sequences are present in repetitive regions of the hsv- genome we identified nine regions in the hsv- genome with highly repeated putative qgrs (quadruplex forming g-rich sequences) ( table ) . six of them (named gp a-f) were found in the leading strand of the gp gene ( fig. b) which encodes ul , the largest viral protein and essential viral tegument component (mcnabb and courtney, ) (table ). the gp series sequences displayed the g xtg xtg xtg xt common motif, where x was either a t or a c base, and covered about bps. four more putative qgrs were clustered at the terminal and internal repeats (both long and short) (fig. b, table ): they covered about bps in the leading strand and bps in the lagging strand. the exact role of these regions is as yet unknown and thus these positions were named ''un''. all identified sequences were highly conserved among different hsv- strains ( table ). the only exception was the unb sequence, which was identical to gp b, and was not conserved in the kos strain. to note that all identified sequences, apart from un , were characterized by four gggg-tracts, therefore possessing the ability to fold into -tetrad stacked g-quadruplex. this kind of tetraplex is in principle very stable, as denoted by the high g-scores, which indicate the propensity of a sequence to fold into unimolecular g-quadruplex, obtained by these oligonucleotides (table ). the un sequence displayed four ggg-tracts therefore gaining a lower g-score. folding of the hsv- putative qgrs was determined by circular dichroism (cd) spectroscopy. in physiological concentrations of k + , all tested oligonucleotides displayed cd g-quadruplex signatures that included three different conformations (vorlickova et al., ) : hybrid (gp ), antiparallel (un ) and parallel (un , un , fig. a) . usually, g-quadruplex folding occurs in the presence of k + , while in the absence of the monovalent cation oligonucleotides are mostly unfolded. the hsv- oligonucleotides, however, displayed peaks characteristic of the g-quadruplex conformation even in the absence of k + (fig. s ). stability of hsv- g-quadruplexes in the presence of k + was assessed by thermal denaturation experiments monitored by cd and uv spectroscopy. un and un were the most stable g-quadruplexes with t m above °c, table sequences of putative g-quadruplex forming oligonucleotides in the genome of hsv- . sequence repeats within hsv- main strains and g-scores are shown. followed by gp oligonucleotides. (fig. b , table ). because of the very low solubility of un in k + , t m values for this sequence were not obtained. in the absence of k + , un was the least stable sequence, followed by un . un and gp oligonucleotides showed comparable stability, with the exception of gp d, which was extremely stable even in the absence of k + ( table ). the ability of hsv- qgrs to fold into g-quadruplex was further assessed by emsa analysis. even in the absence of k + , all gp sequences run markedly faster than their bp-length control marker, indicating folding in these conditions (fig. c) . in particular, gp d displayed the fastest mobility, confirming its inherent property to fold into g-quadruplex in the absence of k + . un ( -bp) and un ( -bp) also migrated faster than the -long control marker (see * symbol in fig. c ), indicating effective folding. however, a band corresponding to the unfolded un was also observable (see § symbol in fig. c ). in contrast, the bp-long un migrated as a totally unfolded oligonucleotide (compare lane with lane m, fig. c ), confirming its lower tendency to fold in the absence of k + (see lower g-score, table , and lower stability, table ). in the presence of k + (fig. d) , all sequences displayed increased migration indicating effective folding. for the gp sequences, slower migrating species, ascribable to intermolecular dimeric g-quadruplex forms, were also observed (lanes a-f, fig. d) . the g-quadruplex ligand braco- was next tested by cd analysis for its ability to bind the hsv- qgrs in the presence of k + . braco- converted the gp hybrid conformation to an antiparallel-like g-quadruplex (fig. s ) and further stabilized all hsv- oligonucleotides, increasing t m to above °c (table ) . additional evidence of the intrinsic stability of the identified hsv- g-quadruplexes within a longer dna context and on the increased stabilization imparted by braco- was provided by the taq polymerase stop assay. all g-quadruplex forming sequences except un stopped the polymerase at the first g-rich region encountered by the polymerase even in the absence of k + (lanes , un , gp a, gp d, fig. ). when k + was added, strong stops were observed in all g-quadruplex templates (lanes , fig. ). upon addition of braco- the intensity of the initial stop sites dramatically increased (lanes , fig. ) ; moreover, an additional intense stop site corresponding to a second g-rich region was observed in the un template, possibly indicating the presence of multiple g-quadruplex structures. to note that braco- stopped the processing of the polymerase to such an extent that the full-length product did not form in all templates, with the exception of un , which resulted the sequence less susceptible to the activity of the compound (lanes , fig. ). in contrast, tmpyp , a porphyrin derivative which shares chemical features with braco- , i.e. a large aromatic surface and cationic moieties, but displays a different g-quadruplex binding mode and markedly lower g-quadruplex binding affinity (han et al., ; le et al., ) , used here for comparison, did not induce any stop at g-rich regions and it did not modify the amount of the fulllength amplicons (lanes , fig. ). moreover, a control sequence devoid of g-tracts did not show any stop site in the presence of k + and braco- (lanes - , non-g template, fig. ) , indicating that the observed polymerase inhibition was g-quadruplex dependent and specific. given the ability of braco- to recognize and stabilize hsv- g-quadruplexes, we tested the possibility that it displayed antiviral activity. tmpyp was used for comparison. cytotoxicity of both compounds was assessed on vero cells, a monkey kidney epithelial cell line which sustains hsv- infection. both compounds were non-cytotoxic up to lm, the highest tested dose (cc > lm). in hsv- infected vero cells, braco- demonstrated a statistical significant antiviral effect at lm, which increased up to % at lm (ec = lm) (fig. a) . in contrast, tmpyp displayed ec = lm. since both braco- and tmpyp , as most g-quadruplex binding molecules, are polycationic agents, we first excluded that the observed antiviral activity was due to the interaction of these molecules with the negatively charged heparan sulfate (campadelli-fiume et al., ; herold et al., ) , thus preventing virus attachment to the cells. braco- or tmpyp were added at different times relative to infection until the virus had completed its entry into cells (i.e. h.p.i.) (sodeik et al., ) . in this time range we did not observe any difference to the activity obtained by treating the cells h pre-infection (fig. s ) , indicating that neither braco- nor tmpyp act by inhibiting viral entry. one cluster of hsv- qgrs was embedded in the gp gene, which encodes the essential tegument protein ul ; we thus and tmpyp (t). extended un , un , gp a and gp d dna templates (table s ) in the absence and presence of k + mm (lanes and , respectively) and in the presence of tmpyp (t) and braco- (b) ( . lm) (lanes and , respectively) were used as templates for the taq polymerase. a non-g-quadruplex template (non-g lanes) was also used as negative control. p indicates primer. or h.p.i. total rna was isolated, retrotranscribed into cdna and expression of specific genes determined by rt-pcr (table s ). the mrnas of the immediate-early us , icp and ul , early ul dna polymerase and late proteins ul and ul were analyzed. rq are relative quantities. in all data sets: n p , mean ± s.d., student's t-test, ⁄⁄ p < . . investigated if treatment with braco- impaired ul transcription. transcript levels of a second tegument protein, ul , were also assessed as control for a protein whose coding sequence lacks important qgrs clusters. both viral transcripts were decreased to - % at h.p.i. (fig. b) and this effect was specific for the hsv- transcripts (fig. s a) . the mrna levels of representative immediate-early and early proteins, whose coding sequences lacked important g-quadruplex forming regions, were also analyzed: braco- did not affect transcription of these earlier proteins (fig. b) . moreover, tmpyp reduced hsv- transcript levels to a low extent ( - %) and it did not differentiate among the different kinetic groups of proteins. the fact that both analyzed late transcripts were affected by braco- to a similar degree indicates that the observed effect is independent of the presence of qgrs in the ul coding sequence; the absence of effect on earlier genes indicates that the compound likely exerts it activity against g-quadruplex-controlled viral mechanisms that mostly influence transcription of late proteins. since the identified clusters of qgrs were mostly present in non-coding repetitive-element regions, we tested the possibility that qgrs folding induced by braco- inhibited polymerase processing at the viral dna level. hsv- dna was extracted from a viral stock and treated with increasing concentrations of braco- and tmpyp . genome regions containing g-rich tracts were amplified by standard pcr using taq polymerase, which was used as a model dna polymerase enzyme. a non-g-rich/non-g-quadruplex forming sequence was also amplified as negative control sequence. amplified dna corresponding to g-quadruplex regions decreased in a concentration dependent manner in the presence of braco- (fig. a, un and gp , braco- ). in contrast, braco- was not able to disrupt polymerase processing in the non-g-quadruplex region (fig. a , non-g , and tmpyp had no effect in both g-rich and non-g-rich regions (fig. a, un, gp , non-g , tmpyp ). real-time pcr was employed for quantification purposes. amplification of the g-quadruplex region was inhibited up to % (with respect to the non-treated control, %) in the presence of braco- ( lm), while no effect was detected in the non-g-quadruplex region (fig. b) , confirming lack of activity of the compound against the polymerase enzyme. no activity of tmpyp was observed. this data demonstrate that braco- specifically interacts with g-quadruplex regions in the hsv- genome in vitro and inhibits polymerase processing likely due to the steric hindrance caused by multiple tetraplex structures stabilized by the ligand. we next tested the effect of braco- on viral dna amounts in infected cells. hsv- infected cells were treated with increasing concentrations of braco- or tmpyp and intracellular viral dna was extracted and quantified by real-time pcr. dna extraction was performed at different time points, before ( h.p.i.) and after ( h.p.i.) the viral dna was replicated in cells. braco- reduced intracellular viral dna levels to % of the untreated control ( %) at the highest concentration at h.p.i. (fig. c ). in contrast, at h.p.i. braco- and tmpyp did not affect viral dna amounts. moreover, addition of braco- did not modify cellular dna amounts (fig. s b) . to define the viral step targeted by braco- in infected cells, a time-of-addition experiment was set up (daelemans et al., ; pauwels et al., ) . this assay indicates the last viral step targeted by a compound. acv was used as reference compound with established mode of action in the time frame of replication events (james and prichard, ) . braco- was added to infected cells every h up to h.p.i. (fig. d) . a sharp increase in virus amounts (pfu/ml) was observed between and h.p.i., indicating that braco- was active at events that occur in this time range. acv showed a similar increase between and h.p.i. since acv acts by inhibiting the viral dna polymerase during dna replication, these data confirm the activity of braco- during viral dna replication. in this work we have found that the gc-rich hsv- genome presents multiple extended repeated clusters of g-quadruplex forming sequences, covering bp on the leading strand and bp on the lagging strand (fig. b, table ) . a first intriguing aspect is that these sequences display a remarkably high stability: in physiological conditions all but one sequence (un ) displayed t m around °c and were capable of folding into tetraplex even in the absence of k + . in particular, among the gp oligonucleotides, gp d standed out for its improved stability. interestingly, this is the only sequence that presents one c base in the loop and displays identical ct loops. it has been reported that c can also be involved in g-quartet formation (lim et al., ), therefore c bases may augment the folding stability of this sequence. to our knowledge the hsv- g-quadruplex structures reported in this work are the most stable ever reported in an organism. hsv- g-quadruplexes will likely form in the viral dna when the duplex is stimulated to unwind, i.e. during replication and transcription events. in eukaryotes g-quadruplex formation at the dna level has been shown to slow down replication and increase the likelihood of chromosomal breakage, genetic instability (ribeyre et al., ) and genomic rearrangements (cahoon and seifert, ); moreover, g-quadruplexes accumulation during the s-phase of the cell cycle, the phase at which replication occurs, has been reported (biffi et al., ) . dna processing is allowed by the activity of helicases (anand et al., ) , which disentangle the tetraplex structures. hsv- probably exploits similar cellular or viral enzymes to unwind its g-quadruplex sequences and allow replication/transcription events to proceed. moreover, g-quadruplex motifs have been associated with over % of dna replication origins (besnard et al., ) . here we have used braco- , a tri-substituted acridine characterized by excellent g-quadruplex binding properties, to investigate the effect of hsv- g-quadruplex stabilization. we have shown that braco- stabilized all tested sequences and that it was able to arrest viral dna processing in vitro. in infected cells, this activity resulted in less viral dna being synthesized in treated cells, shut down of late, but not immediate-early and early protein mrnas and overall, in inhibition of viral production. the activity of braco- was compared to that of tmpyp , a molecule that shares the chemical features of a g-quadruplex ligand, but has a much lower binding affinity toward g-quadruplexes (han et al., ) . at the highest concentrations, tmpyp displayed antiviral activity, which, based on the observation that tmpyp was essentially inactive in the polymerase assays and it did not modify the amount of intracellular dna, likely exploits a non-g-quadruplex related mechanism. the time-of-addition assay indicated an early time range of activity ( - h.p.i.) of braco- , similarly to acv, which targets viral dna polymerase. altogether these data indicate that braco- inhibits viral dna replication by stabilizing the extended clusters of g-quadruplex mainly present in non-coding regions of the viral genome. it has to be noted that ( ) the time-of-addition indicates only the last step affected by the antiviral molecule, therefore earlier steps may also be affected by ( ) only large clusters of g-quadruplex sequences have been considered in the present work. because of the abundance of g nucleotides in the hsv- genome, additional single tetraplexes in key regions of the hsv- genome may form to promote multiple yet non-identified functions required for the viral biology. since its first introduction in the s, acv has been the antiviral drug of choice for the treatment of hsv- infections (vere hodge and field, ) . however, the emergence of resistance to acv has created an obstacle for the treatment of hsv- (bacon et al., ) . because of the inherent different mechanism of action, g-quadruplex ligands could be envisaged as therapeutic options against hsv- strains resistant to current anti-herpetic drugs. in conclusion, this work provides a proof of concept for the development of selective anti-hsv- agents with an innovative mechanism of action. none to declare. (b) real-time pcr on a gp g-quadruplex sequence and a control non-g-quadruplex region was performed in the presence of braco- or tmpyp ( -fold dilutions from lm to . lm). quantification of amplified products was obtained by sybr Ò green detection. hsv- dna was extracted from hsv- -infected vero cells. (c) quantification of intracellular dna amounts obtained from infected cells at h and h.p.i., treated with increasing concentration of braco- (b ) and tmpyp (p ), with n p , mean ± s.d., student's t-test, ⁄⁄ p < . . rq are relative quantities. 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replication in vitro by a novel series of tibo derivatives a dynamic g-quadruplex region regulates the hiv- long terminal repeat promoter formation of a unique cluster of g-quadruplex structures in the hiv- nef coding region: implications for antiviral activity anti-hiv- activity of the g-quadruplex ligand braco- human telomeric g-quadruplex: structures of dna and rna sequences dna architecture: from g to z genome-wide prediction of g dna as regulatory motifs: role in escherichia coli global regulation structure-based design of selective and potent g quadruplex-mediated telomerase inhibitors the yeast pif helicase prevents genomic instability caused by g-quadruplex-forming ceb sequences in vivo herpes simplex viruses cellular proteins specifically bind single-and double-stranded dna and rna from the initiation site of a transcript that crosses the origin of dna replication of herpes simplex virus formation of parallel four-stranded complexes by guanine-rich motifs in dna and its implications for meiosis g-quadruplex dna structures-variations on a theme microtubule-mediated transport of incoming herpes simplex virus capsids to the nucleus the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes antiviral agents for herpes simplex virus genome-wide computational and expression analyses reveal g-quadruplex dna motifs as conserved cis-regulatory elements in human and related species circular dichroism and guanine quadruplexes investigation of mrna quadruplex formation in escherichia coli g-quadruplex structures and their interaction diversity with ligands supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.antiviral. . . . key: cord- - q jbcl authors: coppola, damon p. title: participants – multilateral organizations and international financial institutions date: - - journal: introduction to international disaster management doi: . /b - - - - . - sha: doc_id: cord_uid: q jbcl multilateral organizations are composed of sovereign governments. they may be regional, organized around a common issue or function, or global. international financial institutions (ifis) are international banks composed of sovereign member states that use public money from the member states to provide technical and financial support for developing countries. the united nations is the organization most involved in the mitigation of, preparedness for, response to, and recovery from disasters around the world. it is considered the best equipped to do so because of its strong relationships with most countries, especially the developing countries where assistance is most needed. when disasters strike, the un is one of the first organizations to mobilize, and it remains in the affected countries during the recovery period for many years after. the consolidated appeal process is one way the un garners international support for relief and reconstruction. in many regions, governments have formed smaller international organizations, many of which address risk, as well. the ifis provide nations with low capital reserves funding in the aftermath of disasters recovery reconstruction. the world bank is regarded as one of the largest sources of development assistance. a multilateral organization is an organization composed of the central governments of sovereign nations. multilateral organizations are also called intergovernmental organizations and international organizations. member states come together under a charter of rules and responsibilities they have drawn up and agreed on. multilateral organizations may be regionally based (e.g., the european union [eu] , the association of south east asian nations [asean]), organized around a common issue or function (e.g., the north atlantic treaty organization [nato] , the organization of the petroleum exporting countries [opec]), or globally based (e.g., the united nations [un] ). like sovereign states, they are recognized as having an established legal status under international law. the un is the most well-known and largest of all of the multilateral organizations because its membership draws from nearly every nation, and because it covers a wide range of issues. the first international organization to address the topic of disaster management was the international relief union (iru), which was founded in italy in and later integrated into the league of single vote, with key issues decided by two-thirds majority. (less significant matters are decided by simple majority.) as mentioned earlier, the general assembly cannot force its decisions on a sovereign state, although they generally receive wide support. the assembly holds regular sessions from september to december, and special/emergency sessions may be called at any time. when not in session, the assembly's work is carried out by its six main committees, other subsidiary bodies, and the secretariat. the un security council's primary responsibility is maintaining international peace and security in accordance with the un charter. this council, which convenes at will, consists of members, five of which are permanent members (china, france, the russian federation, the united kingdom, and the united states). all un member states are obligated to carry out the council's decisions. decisions require nine affirmative votes, including all five votes of the permanent members. when the council source: un, a. considers threats to international peace, it first explores peaceful settlement options. if fighting is under way, the council attempts to secure a cease-fire, and it may send a peacekeeping mission to help the parties maintain the truce and keep opposing forces apart. the council can take measures to enforce its decisions, such as imposing economic sanctions or arms embargoes. on rare occasions, the council has authorized member states to use "all necessary means," including collective military action, to see that its decisions are carried out. these are referred to as "peacemaking operations." the economic and social council is the central mechanism by which international economic and social issues are addressed and by which policy recommendations are created. it also consults with non-governmental organizations (ngos) to create and maintain working partnerships between the un and civil society. the council has members, elected by the general assembly for three-year terms. it meets throughout the year, but its main session is held in july, during which major economic, social, and humanitarian issues are discussed. the council has several subsidiary bodies that regularly meet to address issues such as human rights, social development, the status of women, crime prevention, narcotic drugs, and environmental protection. the trusteeship council originally provided international supervision for trust territories administered by seven member states and ensured that adequate steps were taken to prepare the territories for self-government or independence. by , all trust territories had attained self-government or independence. its work completed, the trusteeship council now consists of the five permanent members of the security council. it has amended its rules of procedure to allow it to meet as and when the occasion may require. the international court of justice, also known as the world court, is the un's main judicial organ. the world court consists of judges elected jointly by the general assembly and the security council. it serves to settle disputes between countries. participation is voluntary, but when a state agrees to participate, it must comply with the court's decision. the court also provides advisory opinions to the general assembly and the security council on request. the secretariat carries out the day-to-day work of the un as directed by the general assembly, the security council, and the other organs. at its head is the secretary general, who provides overall administrative guidance. the secretariat is made up of various departments and offices and maintains a total staff of about , people throughout the world. duty stations include the un headquarters in new york and offices in geneva, vienna, and nairobi, as well as other locations. the secretariat's functions are diverse, ranging from "administering peacekeeping operations to mediating international disputes, from surveying economic and social trends and problems to preparing studies on human rights and sustainable development" . the secretariat staffs also work to publicize the un's work through the world media and to organize conferences on issues of global concern. secretariat staffs are considered international civil servants and answer only to the un for their activities. disaster-response-oriented projects to disaster mitigation, the un adopted the international strategy for disaster reduction to promote disaster reduction and risk mitigation as part of its central mission. this initiative continues to evolve in its pursuit of disaster risk reduction, promoting global resilience to the effects of natural hazards, and reducing human, economic, and social losses by: • increasing public awareness of the hazard risks faced and the options to address them • obtaining commitment from public authorities to mainstream risk reduction into their work • stimulating interdisciplinary and intersectoral partnership and expanding risk-reduction networking at all levels • enhancing scientific research on the causes of natural disasters and the effects of natural hazards and related technological and environmental disasters on societies these strategies are integrated into the work carried out by each un country office and promoted to the national and local governments in each member country where the un works. hazard mitigation and disaster preparedness strategies are communicated to members of all levels of society via public awareness campaigns, and promoted by obtaining commitment from public authorities, facilitating cooperation and communication between various government and non-governmental sectors, and enabling the provision or transfer of technical knowledge. because the un is such a complex organization, it can be difficult to illustrate the myriad ways in which it addresses disaster management other than to describe the role of each organization and agency in this area. the un general assembly does not partake in any operational disaster management activities. however, as the main deliberative organ of the un, it is responsible for launching many influential and effective disaster management programs that are ultimately carried out by the various un offices and by the un member state governments. examples include the endorsement of the undp capacity for disaster reduction initiative (cadri) and the launching of the international decade for natural disaster reduction and its subsequent international strategy for disaster reduction. the general assembly is also responsible for organizing and reorganizing the un system to maximize its disaster management capabilities, as in under the un program for reform ( ) , which created the office for the coordination of humanitarian affairs (ocha) and the un office for disaster risk reduction (unisdr). the un secretariat is the international working staff of un employees located at duty stations throughout the world. the un secretariat employees carry out the diverse day-to-day work of the various un offices. it services the principal un organs and administers the programs and policies laid down by them. at its head is the secretary-general, who is appointed by the general assembly on the recommendation of the security council for five-year renewable terms. the secretariat has approximately , employees. as international civil servants, staff members and the secretary-general answer only to the un and take an oath not to seek or receive instructions from any government or outside authority. under the charter, each member state agrees to respect the appointed valerie amos of guyana to replace mr. john holmes of the united kingdom as under-secretary-general for humanitarian affairs/un emergency relief coordinator. ocha regional offices monitor the onset of natural and technological disasters. staff are trained in disaster assessment and post-disaster evaluation methods before disasters strike. once an impending or actual disaster event is identified, ocha initiates response and generates a situation report to provide the international response community with detailed information, including damage assessment, actions taken, needs assessment, and current assistance provided. if necessary, ocha may then deploy a un disaster assessment and coordination (undac) team to assist relief activity coordination and assess damages and needs. if a disaster appears inevitable or is already unfolding, the erc in consultation with iasc may designate a humanitarian coordinator (hc), who becomes the most senior un humanitarian official on the ground for the emergency. the hc is directly accountable to the erc, increasing the likelihood that the humanitarian assistance provided is quick, effective, and well-coordinated. the hc appointment generally signals that the event merits a long-term humanitarian presence. the criteria used by the erc to determine whether to appoint an hc center on the need for intensive and extensive political ocha organizational chart source: ocha, a. management, mediation, and coordination to enable the delivery of humanitarian response, including negotiated access to affected populations; massive humanitarian assistance requiring action by a range of participants beyond a single national authority; and a high degree of external political support, often from the un security council. an on-site operations coordination center (osocc) may be set up in the field to assist local firstresponse teams to coordinate the often overwhelming number of responding agencies. the osocc has three primary objectives: ( ) to be a link between international responders and the government of the affected country; ( ) to provide a system for coordinating and facilitating the activities of international relief efforts at a disaster site; and ( ) to provide a platform for cooperation, coordination, and information management among international humanitarian agencies. finally, ocha can set up communications capabilities if they have been damaged or do not exist at an adequate level, as required by the un responding agencies. ocha generally concludes its responsibilities when the operation moves from response to recovery. overall, ocha coordination is performed to maximize the response and recovery capabilities that converge on the disaster scene, and to minimize duplications and inefficiencies. the structures and policies that have been established to support this function include (adapted from ocha ): • developing common strategies. humanitarian assistance is most effective when common priorities and goals exist among stakeholders and responders agree on tactics and jointly monitor progress. ocha works with its partners to develop a common humanitarian action plan and to establish clear divisions of responsibility. • assessing situations and needs. ocha staff assume responsible for assessing damages and identifying needs, developing a plan of action to meeting those needs, and monitoring progress. responses are adjusted, if necessary, using ongoing analysis of political, social, economic, and military environments and by assessing humanitarian needs to help the responding agencies better understand the situation. • convening coordination forums. in its role as coordinator, ocha holds a wide range of meetings to bring together the various disaster management players for planning and information exchange. these meetings help the participants to more accurately analyze the overall status of humanitarian relief efforts as well as network and share lessons learned and best practices. • mobilizing resources. through the cap, ocha leads the drive to get governments to commit funding and resources necessary to address the identified needs. allocation of funds has been found to be more efficient within this centralized system. • addressing common problems. every crisis is unique, and both new and old problems arise. as coordinator, ocha analyzes and addresses problems common to humanitarian actors, such as negotiating with warring parties to gain access to civilians in need, or working with un security officials to support preparedness and response measures in changing security situations. • administering coordination mechanisms and tools. ocha, and the un in general, have several tools with which they can better address the humanitarian needs of disaster victims. these include the iasc; rapid-response tools, such as the un disaster assessment and coordination teams and the international search and rescue advisory group; and smaller forums such as the geographic information support team. ocha also assists with civil-military cooperation, ensuring a more efficient use of military and civil defense assets in humanitarian operations. the field coordination support unit in geneva manages ocha's human, technical, and logistical resources. these resources are primarily provided by the danish and norwegian refugee councils, the danish emergency management agency, the swedish rescue services agency, and the emergency logistics management team of the united kingdom overseas development administration. the under-secretary-general for humanitarian affairs/emergency relief coordinator advises the un secretary-general on disaster-related issues, chairs the executive committee on humanitarian affairs (echa), and leads the iasc. the coordinator is assisted by a deputy, who holds the position of deputy emergency relief coordinator (derc) and is responsible for key coordination, policy, and management issues. the inter-agency standing committee (iasc) was established in under un resolution / . it serves as a platform within which the broad range of un and non-un humanitarian partners (including un humanitarian agencies, the international organization for migration, three consortia of major international ngos, and the red cross movement) may come together to address the humanitarian needs resulting from a disaster. the iasc's primary role is to formulate humanitarian policy that ensures a coordinated and effective response to all kinds of disaster and emergency situations. the primary objectives of the iasc are to: • develop and agree on system-wide humanitarian policies • allocate responsibilities among agencies in humanitarian programs • develop and agree on a common ethical framework for all humanitarian activities • advocate common humanitarian principles to parties outside the iasc • identify areas where gaps in mandates or lack of operational capacity exist • resolve disputes or disagreement about and between humanitarian agencies on system-wide humanitarian issues (ocha ) iasc members (both full members and standing invitees) include: • the ocha donor relations section (drs), separated from the cap in , is the focal point for all relations with donors, particularly for funding-related issues. drs advises the senior management team on policy issues related to interaction with donors and resource mobilization. in addition, it plays a key role in facilitating the interaction of all ocha entities with donors, both at headquarters and in the field level. the coordination and response division (crd) was created in by joining the former new yorkbased humanitarian emergency branch and the geneva-based response coordination branch. crd is responsible for providing disaster-related direction, guidance, and support to the erc, the un resident/humanitarian coordinators, and ocha's field offices (including the deployment of extra personnel as necessary, or providing emergency cash grants). based in geneva, the ocha emergency services board (esb) was created to expedite the provision of international humanitarian assistance. esb develops, mobilizes, and coordinates the deployment of ocha's international rapid response "toolkit"-the expertise, systems, and services that aim to improve humanitarian assistance in support of disaster-afflicted countries. esb's humanitarian response activities include the coordination of disaster response and assessment (undac; see in the following section), the setting of international urban search and rescue standards (insarag; see in the following section), and the establishment of osoccs. esb supports ocha field offices through the following: • surge capacity and standby partnerships • military and civil liaison and mobilization of military and civil defense assets • dispatch of relief supplies and specialized assistance in environmental emergencies • dissemination of disaster-related information by means of reliefweb, the central register of disaster management capacities, and the virtual onsite operations coordination center. within the esb are seven separate sections, established to manage particular aspects of disaster response: . civil-military coordination section . emergency preparedness section . environmental emergencies unit . emergency relief coordination centre . field coordination support section . logistics support unit . surge capacity section established by the iasc in , the civil military coordination section (cmcs), previously named military and civil defense unit (mcdu), is the focal point for the efficient mobilization of military and civil defense assets for use in humanitarian emergencies and for liaison with governments, international organizations, regional organizations, and military-civil defense establishments deploying these assets. it also coordinates un agency participation and participates in major military exercises comprising significant humanitarian scenarios. this section is responsible for the overall management of the ocha central register of disaster management capacities, with specific maintenance of the mcda directory of military and civil defense assets and expertise. cmcs acts as a facilitator and secretariat to the development of documents involving the broad international humanitarian community and is custodian of the "oslo" and "mcda" guidelines detailing the use of mcda in support of un humanitarian operations in natural, technological, and environmental disasters and complex emergencies, respectively. the emergency preparedness section (eps) helps to maintain ocha's operational readiness and to reinforce disaster preparedness work. eps works with stakeholders at the national government level in un member countries in order to help build disaster response and recovery capacity in advance of disasters. much of the work performed by this unit is guided by the hyogo framework for action, which recommends the strengthening of disaster preparedness for effective response at all levels. the environmental emergencies unit, or the joint un environmental programme (unep)/ocha environment unit, serves as the integrated un emergency response mechanism that provides international assistance to countries experiencing environmental disasters and emergencies. this joint unit can rapidly mobilize and coordinate emergency assistance and response resources to countries facing environmental emergencies and natural disasters with significant environmental impacts. the unit performs several key functions geared toward facilitating rapid and coordinated disaster response: • monitoring. the unit performs continuous monitoring and ongoing communication with an international network of contacts and permanent monitoring of news services and websites for early notification of environmental occurrences. • notification. when disasters strike, the unit alerts the international community and issues "information and situation" reports to a comprehensive list of worldwide contacts. • brokerage. the unit is able to quickly establish contact between the affected country and donor governments ready and willing to assist and provide needed response resources. • information clearinghouse. the unit serves as an effective focal point to ensure information on chemicals, maps, and satellite images from donor sources and institutions are channeled to relevant authorities in the affected country. • mobilization of assistance. the unit mobilizes assistance from the international donor community when requested by affected countries. • assessment. the unit can dispatch international experts to assess an emergency's impacts and to make impartial and independent recommendations about response, cleanup, remediation, and rehabilitation. • financial assistance. in certain circumstances, the unit can release ocha emergency cash grants of up to $ , to meet immediate emergency response needs. the emergency relief coordination center (ercc) is the physical facility where ocha centralized coordination activities are focused. the facility enables closer collaboration between internal and external humanitarian stakeholders and has the capacity to serve as an ocha situation centre, providing updates on humanitarian relief activities worldwide. the centre consists of a main task force room, a small conference room that can also be used for a second task force, and a technical room to control all facility capabilities. the ercc allows ocha to coordinate two response teams simultaneously. the field coordination support section (fcss) was established within esb in to support national governments and the un resident coordinators in developing, preparing, and maintaining "standby capacity" for rapid deployment to sudden-onset emergencies to conduct rapid needs assessments and coordination. fcss manages several programs and offices to improve international disaster coordination and cooperation, including: • the united nations disaster assessment and coordination (undac) team. the undac team is made up of disaster management specialists selected and funded by the governments of un member states, ocha, undp, and operational humanitarian un agencies (such as wfp, unicef, and who). it provides rapid needs assessments and supports national authorities and the un resident coordinator in organizing international relief. undac teams are on permanent standby status so that they can deploy within hours. • the international search and rescue advisory group (insarag) . insarag is an intergovernmental network within the un that manages urban search and rescue (usar) and related disasterresponse issues. it promotes information exchange, defines international usar standards, and develops methodologies for international cooperation and coordination in earthquake response. • the virtual on-site operations coordination centre (virtual osocc). the internet has made it possible for humanitarian relief agencies to share and exchange disaster information continuously and simultaneously, and between any locations where internet access can be obtained. the virtual osocc is a central repository of information maintained by ocha that facilitates this exchange of information with ngos and responding governments. the information is stored on an interactive web-based database, where users can comment on existing information and discuss issues of concern with other stakeholders. the logistics support unit (lsu) manages stocks of basic relief items that can be dispatched immediately to disaster-or emergency-stricken areas. the stockpile, which is located at the un humanitarian response depot in brindisi, italy, includes nonfood, nonmedical relief items (such as shelter, water purification and distribution systems, and household items) donated by un member governments. the lsu is also involved in other logistical challenges, such as designing contingency plans for the rapid deployment of emergency relief flights and providing interface on logistical matters with other humanitarian agencies (such as wfp, who, unhcr, ifrc, and icrc). the lsu participates in the operation of a un joint logistics center (see exhibit . ) and has co-sponsored an effort to adopt a un-wide system for tracking the un joint logistics center (unjlc) is an interagency facility reporting to the humanitarian coordinator [within a che], and overall to the iasc. its mandate is to coordinate and optimize the logistics capabilities of humanitarian organizations in large-scale emergencies. unjlc operates under the direction of the world food programme (wfp), who is responsible for the administrative and financial management of the centre. the unjlc is funded from voluntary contributions channeled through wfp. the requirement to establish [the unjlc] was born out of the humanitarian response to the eastern zaire crisis, which demanded intensified coordination and pooling of logistics assets among unhcr, wfp, and unicef. the interagency logistics coordination model was applied on subsequent unjlc interventions in somalia, kosovo, east timor, mozambique, india, and afghanistan. in march , unjlc concept was institutionalized as a un humanitarian response mechanism, under the aegis of wfp, by the inter-agency standing committee working group (iasc-wg). the unjlc core unit was subsequently established in rome. in case of major disaster with substantial humanitarian multi-sector involvement during the immediate relief phase, the un agencies involved may consider that the establishment of a joint logistics centre would contribute to the rapid response, better coordination, and improved efficiency of the humanitarian operation at hand. . . . a standby capacity will be developed for facilitating, if required, the timely activation and deployment in the field of a united nations joint logistics centre-unjlc. the unjlc will support the united nations agencies and possibly other humanitarian organisations that operate in the same crisis area. the capacity includes the option to establish satellite joint logistic centres (jlc) dispersed at critical locations in the [affected area] and offering logistics support on a reduced scale. . . . upon [unjlc] activation, agencies will establish a deployment requirements assessment (dra) team to carry out a quick evaluation of the logistics situation and determine the requirements to deploy the unjlc in the crisis area. this dra team will work in close coordination with the humanitarian authorities and, if deployed, with the united nations disaster assessment and coordination (undac) team. it will take all necessary measures for installing the unjlc and draft ad hoc terms of reference (tor) for endorsement by the relevant humanitarian authorities. in case of peacekeeping operations or in a complex environment, the unjlc activation will be coordinated with the department of peacekeeping operations (dpko) or the relevant military entities. • the role of the unjlc will be to optimise and complement the logistics capabilities of cooperating agencies within a well-defined crisis area for the benefit of the ongoing humanitarian operation. • the unjlc will provide logistics support at operational planning, coordination, and monitoring levels. unless specified otherwise, the un agencies and other humanitarian bodies, which are established in the area, will continue (continued) relief supplies and common procedures for air operations. finally, the lsu contributes information related to stockpiles and customs facilitation agreements (which helps speed up the delivery of relief items). the surge capacity section (scs) works to ensure ocha always has the means and resources to rapidly mobilize and deploy staff and materials to address the needs of countries affected by suddenonset emergencies. scs operates using a number of distinct surge capacity resources, which include: • the emergency response roster (err). err, which became active in june , aims to rapidly deploy ocha staff to sudden-onset emergencies to conduct assessments and establish initial coordination mechanisms. the staff included in the err are deployable within hours of a request for their services through a deployment methodology based on the undac model. staff serve on the roster for about six months. • the stand-by partnerships programme (sbpp). sbpp is structured on legal agreements with partner organizations that provide short-term staffing to field operations free of charge when gaps arise. partners maintain their own rosters of trained and experienced humanitarian professionals, many of whom have ocha or other un humanitarian experience. sbpp staff can usually be deployed within four weeks of the formal request, and an average deployment lasts five to six months. • associates surge pool (asp). asp, which was created in late , helps to bridge the gap between the immediate response surge and the arrival of regular staff. asp comprises external disaster management staff who can be deployed for up to six months upon the issuance of a temporary appointment. contracting and deployment preparations take an average of three to four to exercise their normal responsibilities. as a result, the unjlc will not be involved in policy and establishment of humanitarian needs and priorities. • responsibilities will be defined as per the requirements on a case-by-case basis but will, in principle, be limited to logistic activities between the points of entry and distribution in the crisis area. detailed responsibilities . . . would be: • collecting, analysing, and disseminating logistics information relevant to the ongoing humanitarian operation; • scheduling the movement of humanitarian cargo and relief workers within the crisis area, using commonly available transport assets; • managing the import, receipt, dispatch, and tracking of non-assigned food and nonfood relief commodities; • upon specific request, making detailed assessments of roads, bridges, airports, ports, and other logistics infrastructure and recommending actions for repair and reconstruction. • the scope of the unjlc activities may vary with the type of emergency, the scale of involvement of the cooperating partners, and the humanitarian needs. the reso and roso positions were created following a need to have senior surge staff available to deploy to new and escalating emergencies for up to three months to provide leadership and stability to ocha operations. they spend percent of their time in the field and percent at headquarters. when not in the field, resos and rosos work with the surge staff development team to develop and deliver trainings and support lesson learning and other exercises to improve ocha emergency response during non-deployment periods. although ocha's efforts primarily focus on coordinating the response to major disasters, the agency also performs various tasks related to disaster risk reduction. for instance, ocha representatives work with disaster management agencies to develop common policies aimed at improving how the wider stakeholder community of responders prepare for and respond to disasters. it also works to promote preparedness and mitigation efforts in member states to decrease vulnerability. crd and esb work closely with the un development programme, other un programs as necessary, and outside organizations on various projects and activities to increase working relationships with national governments and apply lessons learned from completed disaster responses. ocha's geneva offices are continually monitoring geologic and meteorological conditions, as well as major news services, for early recognition or notification of emerging disasters. working with un resident coordinators, country teams, and regional disaster response advisers, ocha maintains close contact with disaster-prone countries in advance of and during disaster events. ocha's regional disaster response advisers work with national governments to provide technical, strategic, and training assistance. they also provide this assistance to other un agencies and regional organizations to improve international disaster management capacity. • it facilitates the negotiations of member states in many intergovernmental bodies on joint courses of action to address ongoing or emerging global challenges. • it advises national governments on translating un-developed policy frameworks into countrylevel programs and, through technical assistance, helps build national capacities. this final area is where desa addresses disaster management activities within its division for sustainable development. as part of this effort, desa launched a plan of action during the world summit on sustainable development in johannesburg, south africa, that included commitments to disaster and vulnerability reduction. see exhibit . for more information on this plan of action. the un center for regional development (uncrd) is another component of desa that addresses disaster management issues. through its headquarters in nagoya, japan, and its regional offices in nairobi, kenya, and bogotá, colombia, uncrd supports training and research on regional an integrated, multi-hazard, inclusive approach to address vulnerability, risk assessment, and disaster management, including prevention, mitigation, preparedness, response, and recovery, is an essential element of a safer world in the twenty-first century. actions are required at all levels to: . strengthen the role of the international strategy for disaster reduction and encourage the international community to provide the necessary financial resources to its trust fund; . support the establishment of effective regional, subregional, and national strategies and scientific and technical institutional support for disaster management; . strengthen the institutional capacities of countries and promote international joint observation and research, through improved surface-based monitoring and increased use of satellite data, dissemination of technical and scientific knowledge, and the provision of assistance to vulnerable countries; . reduce the risks of flooding and drought in vulnerable countries by, [among other things], promoting wetland and watershed protection and restoration, improved land-use planning, improving and applying more widely techniques and methodologies for assessing the potential adverse effects of climate change on wetlands and, as appropriate, assisting countries that are particularly vulnerable to those effects; . improve techniques and methodologies for assessing the effects of climate change, and encourage the continuing assessment of those adverse effects by the intergovernmental panel on climate change; . encourage the dissemination and use of traditional and indigenous knowledge to mitigate the impact of disasters and promote community-based disaster management planning by local authorities, including through training activities and raising public awareness; . support the ongoing voluntary contribution of, as appropriate, ngos, the scientific community, and other partners in the management of natural disasters according to agreed, relevant guidelines; . develop and strengthen early warning systems and information networks in disaster management, consistent with the international strategy for disaster reduction; . develop and strengthen capacity at all levels to collect and disseminate scientific and technical information, including the improvement of early warning systems for predicting extreme weather events, especially el niño/la niña, through the provision of assistance to institutions devoted to addressing such events, including the international center for the study of the el niño phenomenon; . promote cooperation for the prevention and mitigation of, preparedness for, response to, and recovery from major technological and other disasters with an adverse impact on the environment in order to enhance the capabilities of affected countries to cope with such situations. development issues and facilitates information dissemination and exchange. uncrd maintains a disaster management planning office in hyogo, japan, that researches and develops communitybased, sustainable projects for disaster management planning and capacity-building in developing countries. the hyogo office also runs the global earthquake safety initiative, designed to improve risk recognition and reduction in cities around the world. five regional economic commissions are within the economic and social council. the secretariats of these regional commissions are part of the un secretariat and perform many of the same functions (including the disaster management functions listed earlier). the five commissions promote greater economic cooperation in the world and augment economic and social development. as part of their mission, they initiate and manage projects that focus on disaster management. while their projects primarily deal with disaster preparedness and mitigation, they also work in regions that have been affected by a disaster to ensure that economic and social recovery involves adequate consideration of risk reduction measures. the five regional commissions are: • in response periods of disasters, the united nations development programme (undp) sees that development does not cease during emergencies. if relief efforts are to contribute to lasting solutions, sustainable human development must continue to be vigorously supported, complementing emergency action with new curative initiatives that can help prevent a lapse into crisis. (un, ) the undp was established in during the un decade of development to conduct investigations into private investment in developing countries, to explore the natural resources of those countries, and to train the local population in development activities such as mining and manufacturing. as the concept and practice of development expanded, the undp assumed much greater responsibilities in host countries and in the un as a whole. the undp was not originally considered an agency on the forefront of international disaster management and humanitarian emergencies because, while it addressed national capacities, it did not focus specifically on the emergency response systems (previously considered to be the focal point of disaster management). however, as mitigation and preparedness received their due merit, undp gained increased recognition for its vital risk reduction role. capacity building has always been central to the undp's mission in terms of empowering host countries to be better able to address issues of national importance, eventually without foreign assistance. international disaster management gained greater attention as more disasters affected larger populations and caused greater financial impacts. developing nations, where the undp worked, faced the greatest inability to prepare for and/or respond to these disasters, largely as a result of the development trends described in chapter . undp's projects have shifted toward activities that indirectly fulfill mitigation and preparedness roles. for instance, projects seeking to strengthen government institutions also improve those institutions' capacities to respond with appropriate and effective policy, power, and leadership in the wake of a disaster. undp fully recognizes that disaster management must be viewed as integral to their mission in the developing world as well as to civil conflict and che scenarios. there are implicit similarities between undp ideals and those of agencies whose goals specifically aim to mitigate and manage humanitarian emergencies. undp work links disaster vulnerability to a lack of or a weak infrastructure, poor environmental policy, land misuse, and growing populations in disaster-prone areas. when disasters occur, a country's national development, which the undp serves to promote, can be set back years, if not decades. even small-to medium-size disasters in the least developed countries can "have a cumulative impact on already fragile household economies and can be as significant in total losses as the major and internationally recognized disasters" (undp ) . it is the undp's objective to "achieve a sustainable reduction in disaster risks and the protection of development gains, reduce the loss of life and livelihoods due to disasters, and ensure that disaster recovery serves to consolidate sustainable human development" (un ) . in , as part of the un's changing approach to humanitarian relief, the emergency response division (erd) was created within the undp, augmenting the organization's role in disaster response. additionally, percent of undp budgeted resources were allocated for quick response actions in special development situations by erd teams, thus drastically reducing bureaucratic delays. the erd was designed to create a collaborative framework among the national government, un agencies, donors, and ngos that immediately respond to disasters, provide communication and travel to disaster management staff, and distribute relief supplies and equipment. it also deploys to disaster-affected countries for days to create a detailed response plan on which the undp response is based. in , under the un programme for reform, the mitigation and preparedness responsibilities of the ocha emergency relief coordinator were formally transferred to the undp. in response, the undp created the disaster reduction and recovery programme (drrp) within the erd. soon after, the undp again reorganized, creating the bureau of crisis prevention and recovery (bcpr) with an overarching mission of addressing a range of non-response-related issues: • disaster risk reduction and climate change management • conflict prevention • rule of law, justice, and security in countries affected by crises • women in conflict prevention, peacebuilding, and recovery • immediate crisis response • livelihoods and economic recovery • crisis governance bcpr helps undp country offices prepare to activate and provide faster and more effective disaster response and recovery. it also works to ensure that undp plays an active role in the transition between relief and development. undp's disaster management activities focus primarily on the development-related aspects of risk and vulnerability and on capacity-building technical assistance in all four phases of emergency management. it emphasizes: • incorporating long-term risk reduction and preparedness measures in normal development planning and programs, including support for specific mitigation measures where required; • assisting in the planning and implementation of post-disaster rehabilitation and reconstruction, including defining new development strategies that incorporate risk-reduction measures relevant to the affected area; • reviewing the impact of large settlements of refugees or displaced persons on development, and seeking ways to incorporate the refugees and displaced persons in development strategies; • providing technical assistance to the authorities managing major emergency assistance operations of extended duration (especially in relation to displaced persons and the possibilities for achieving durable solutions in such cases). undp spends between $ and $ million each year on disaster risk reduction projects. the focus of these projects has included the establishment or strengthening of early warning systems, the conduct of risk assessments and drafting of hazard maps, and the establishment of national disaster management agencies. through their projects, undp staff help to strengthen national and regional capacities by ensuring that new development projects consider known hazard risks, that disaster impacts are mitigated and development gains are protected, and that risk reduction is factored into disaster recovery. following conflict, crises, and disasters, countries must transition from response to recovery. many countries are unable to manage the difficult and widespread needs of recovery on their own, as they may have experienced widespread loss of infrastructure and services. displaced persons and refugees may have little to return to, and economies may be damaged or destroyed. bcpr operates during the period when the response or relief phase of the disaster has ended but recovery has not fully commenced (sometimes referred to as the "early recovery period"). sustainable risk reduction is central to the undp recovery mission. the bureau recognizes that local expertise in risk management and reduction may not be available, and that the technical assistance they provide may be the only option these communities have to increase their resilience to future disasters. this program has proved effective in many countries' recovery operations, including cambodia after three decades of civil war, afghanistan after the conflict, and gujarat, india, after the earthquake. the top recipients of undp crisis prevention and recovery funding include: to meet these recovery priorities, five support services have been developed to assist the undp country offices and other undp/un agencies to identify areas where bcpr can provide assistance. these support services include: • early assessment of recovery needs and the design of integrated recovery frameworks. this includes assessing development losses caused by conflict or natural disaster, the need for socioeconomic and institutional recovery, identification of local partners, and the need for capacity building and technical assistance. • planning and assistance in area-based development and local governance programs. area-based development and local governance programs play key roles in recovery from conflict because they tailor emergency, recovery, and development issues across a country area by area, based on differing needs and opportunities. area-based development helps bring together different actors at the operational level, promoting enhanced coordination, coherence, and impact at field level. areabased development is often seen as the core mechanism that most benefits reintegration. • developing comprehensive reintegration programs for idps, returning refugees, and ex-combatants. internal displacement, returning refugees, and demobilized former combatants create a huge need for in-country capacity building on different levels. protection and security become serious issues, and efforts to sustainably reintegrate these populations into their host communities are critical. bcpr provides expertise on reintegration of idps, returnees, and ex-combatants, including capacity building benefiting both the returnees and the formerly displaced, as well as their host communities, through activities such as income generation, vocational training, and other revitalization activities. • supporting economic recovery and revitalization. one main characteristic of disasters and conflict is their devastating impact on the local and national economies. livelihoods are destroyed through insecurity, unpredictability, market collapse, loss of assets, and rampant inflation. for recovery to be successful, these issues need to be well understood from the outset and addressed accordingly. • supporting capacity building, coordination, resource mobilization, and partnerships. protracted conflict and extreme disasters tend to create political stressors that temporarily exceed the capacities of un country offices and other ngo partners. however, many recovery needs must be addressed right away to ensure that recovery sets out on a sustainable course. bcpr offers several services to accommodate the needs of this intense phase through the provision of surge capacity and short-to medium-term staff, assistance in resource mobilization within specific fundraising and coordination frameworks (such as the cap), and partnership building. when required to assist in recovery operations, bcpr may deploy a special transition recovery team (trt) to supplement undp operations in the affected country. the focus for these teams varies according to specific needs. for instance, when neighboring countries have interlinked problems (such as cross-border reintegration of ex-combatants and displaced persons), the trt may support a subregional approach to recovery. it is important to note that the undp has no primary role in the middle of a che peacekeeping response, only a supportive one in helping to harmonize development with relief. during recovery and reconstruction, together with others, they take the lead. in addition to the previously mentioned roles and responsibilities, the undp leads several interagency working groups. one such group (which consists of representatives from the wfp, who, the food and agriculture organization [fao] , the un populations fund, and unicef) develops principles and guidelines to incorporate disaster risk into the common country assessment and the un development assistance framework. the international strategy for disaster reduction working group on risk, vulnerability, and disaster impact assessment sets guidelines for social impact assessments. undp also coordinates a disaster management training programme in central america, runs the conference "the use of microfinance and micro-credit for the poor in recovery and disaster reduction," and has created a program to elaborate financial instruments to enable the poor to manage disaster risks. the undp has several reasons for its success in fulfilling its roles in the mitigation, preparedness, and recovery for natural and man-made disasters. first, as a permanent in-country office with close ties to most government agencies, activities related to coordination and planning, monitoring, and training are simply an extension of ongoing relationships. the undp works in the country before, during, and long after the crisis. it is able to harness vast first-hand knowledge about the situations leading up to a crisis and the capacity of the government and civil institutions to handle a crisis, and can analyze what weaknesses must be addressed by the responding aid agencies. in addition, its neutrality dispels fears of political bias. second, the undp functions as a coordinating body of the un agencies concerned with development, so when crisis situations appear, there is an established, stable platform from which it may lead. from this leadership vantage, it can (theoretically) assist in stabilizing incoming relief programs of other responding un bodies, such as the wfp, unicef, the department of humanitarian affairs, and the unhcr. once the emergency phase of the disaster has ended and ocha prepares to leave, undp is in a prime position to facilitate the transition from response efforts to long-term recovery. and third, the undp has experience dealing with donors from foreign governments and development banks, and can therefore handle the outpouring of aid that usually results during the relief and recovery period of a disaster. this contributes greatly to reducing levels of corruption and increasing the cost-effectiveness of generated funds. in several recent events, the undp has established formalized funds to handle large donor contributions, which have been used for long-term post-disaster reconstruction efforts. (see exhibits . and . ). when a major disaster operation requires extended efforts, the undp may accept and administer special extra-budgetary contributions to provide the national government with both technical and material assistance, in coordination with ocha and other agencies involved in the un disaster management team (dmt). an example of such assistance includes the establishment and administration of a un dmt emergency information and coordination (eic) support unit. special grants of up to $ . million also may be provided, allocated from the special programme resources funds for technical assistance to post-disaster recovery efforts following natural disasters. see exhibit . for information about the undp capacity for disaster reduction initiative (cadri). like most major un agencies, unicef (formerly known as the united nations international children's emergency fund) was established in the aftermath of world war ii. its original mandate was to aid children suffering in postwar europe, but this mission has been expanded to address the needs of women and children throughout the world. unicef is mandated by the general assembly to advocate for children's rights, to ensure that each child receives at least the minimum requirements for survival, and to increase children's opportunities for a successful future. under the convention on the rights of on may , , the government of sri lanka declared military victory over the rebel liberation tigers of tamil eelam, formally ending a decades-long armed conflict. in the wake of the war, undp demonstrated that developing and building on strong partnerships is key to ensuring a fast and well-targeted response. an estimated , idps gathered in camps during the first half of . many of them lacked basic documentation, making it difficult to access basic services and prove claims to land and assets. undp assisted the registrar general to establish a temporary office inside one of the largest camps with capacity to process birth and marriage certificates per day, complemented by additional staffing capacity in colombo to handle the increased number of document requests. between july and december the camp office processed close to , requests, prioritizing those from children who needed identification to sit for national school exams. undp also supported mine action coordination and management. survey and clearance activities advanced rapidly, and by the end of a total of square kilometers of land had been released for resettlement. this allowed the pace of returns and resettlements to increase exponentially in the fourth quarter of , with over , idps returning or resettling. in the eastern province, fao, ilo, wfp, unhcr, and undp continued to champion the "delivering as one" approach to support community-based recovery and contribute to the stability of returnees in selected divisions of the east. as the funding conduit, undp was in charge of the overall coordination of project implementation while also directly implementing small-scale infrastructure construction such as roads, wells, and community centers (which provided a space for cooperatives and trading groups to come together). the selection of target communities was informed through village profile maps and data generated by unhcr, while wfp provided six months' worth of food supply rations, until the foundations for agricultural self-reliance and food security for resettled families were laid. undp also launched a new initiative in to foster partnerships between sri lanka's manufacturers and resettled communities. undp, with its presence in the field, played a catalytic role, identifying the resettled communities, facilitating meetings with the large consumer companies, securing fair and long-term contracts, and supporting training as well as supply of equipment to improve production. through this project, farming and fishing families in the north and the east have secured income for the next two to three years. on may , , cyclone aila hit southern bangladesh, resulting in widespread tidal flooding and the destruction of large parts of the region's protective embankment network. economic losses were estimated at $ million and more than , families were affected in satkhira, the district that had also suffered the most from cyclone sidr in . many of the affected were still recovering from the impact of the earlier disaster. the government of bangladesh provided emergency relief and planned for the reconstruction of the damaged embankment network, but many of the most vulnerable families have been unable to return to their homes, which remain submerged. with funding from the undp bureau of crisis prevention and recovery (bcpr), an early recovery program focused on livelihoods was developed, covering all villages in the worst-affected part of satkhira. the program included a cash-forwork component that built on self-recovery efforts of affected families. this resulted in the creation of an estimated , work days devoted to road repair and ground elevation. the program also included support for the restoration of essential community infrastructure; support to local small enterprises through working capital grants for carpentry tools, sewing machines, and tea stall equipment; and assistance for home-based income-generating activities, such as vegetable cultivation, crab fattening, handicrafts, poultry rearing, and fish drying. this effort benefited more than , families. the child (crc), a treaty adopted by countries, the unhcr holds broad-reaching legal authority to carry out its mission. as of late , unicef maintains country offices in more than different nations. this is probably its greatest asset in terms of the agency's disaster management capacity. preparedness and mitigation for disasters among its target groups is a priority, with programs able to address both local-level action and national-level capacity building. in keeping with the recommendations laid out by the yokohama strategy and plan of action for a safer world, unicef incorporates disaster reduction into its national development plans. it also considers natural hazard vulnerability and capacity assessments when determining overall development needs to be addressed by un country teams. through public education campaigns, unicef works to increase public hazard awareness and knowledge and participation in disaster management activities. unicef country offices include activities that address these pre-disaster needs in their regular projects. for example, they develop education materials required for both children and adults, and then design websites so educators and program directors can access or download these materials for use in their communities. in situations of disaster or armed conflict, unicef is well poised to serve as an immediate aid provider to its specific target groups. its rapid-response capacity is important because vulnerable groups are often the most marginalized in terms of aid received. unicef works to ensure that children have access to education, health care, safety, and protected child rights. in the response and recovery periods of humanitarian emergencies, these roles expand according to victims' needs. (in countries where uni-cef has not yet established a permanent presence, the form of aid is virtually the same; however, the timing and delivery are affected, and reconstruction is not nearly as comprehensive.) the unicef office of emergency programmes (emops), which has offices in new york and geneva, maintains overall responsibility for coordinating unicef's emergency management activities. cadri was created in as a joint program of the undp bureau for crisis prevention and recovery (undp/bcpr), the united nations office for the coordination of humanitarian affairs (ocha), and the secretariat of the international strategy for disaster reduction (isdr). recognizing that capacity development is a cross-cutting activity for disaster risk reduction as stipulated in the hyogo framework (hf), cadri's creation is designed to support all five priorities of the hf. cadri was formally launched by the three organizations at the global platform for disaster risk reduction meeting, june , geneva. cadri succeeds the un disaster management training programme (dmtp), a global learning initiative, which trained united nations, government, and civil society professionals between and . dmtp is widely known for its pioneering work in developing high-quality resource materials on a wide range of disaster management and training topics. more than trainers' guides and modules were developed and translated. cadri's design builds on the success and lessons learned from the dmtp and reflects the significant evolution in the training and learning field since the start of the dmtp, particularly regarding advances in technology for networking and learning purposes. cadri's design also reflects the critical role that the un system plays at the national level in supporting governments' efforts to advance disaster risk reduction. in the context of the un's increasingly important role, cadri provides capacity enhancement services to the un system at the country level as well as to governments. these include learning and training services and capacity development services to support governments to establish the foundation for advancing risk reduction. emops works closely with the unicef programme division, managing the unicef emergency programme fund (epf; see the following section) and ensuring close interagency coordination with other participating humanitarian organizations. in this role, unicef is also in the position to act as coordinator in specific areas in which it is viewed as the sector leader. for instance, unicef was tasked with leading the international humanitarian response in the areas of water and sanitation, child protection, and education for the asia tsunami and earthquake response. (in aceh province alone, more than agencies addressed water and sanitation issues.) unicef maintains that humanitarian assistance should include programs aimed specifically at child victims. its relief projects generally provide immunizations, water and sanitation, nutrition, education, and health resources. women are recipients of this aid as well, because unicef considers women to be vital in the care of children. (see exhibit . .) to facilitate an immediate response to an emergency situation, unicef is authorized to divert either $ , or $ , from country program resources (depending on whether the country program's annual budget is above or below $ million, respectively) to address immediate needs. if the disaster is so great it affects existing unicef programs operating in the country, the unicef representative can shift these programs' resources once permission is received from the national government and unicef headquarters. unicef also maintains a $ million global epf, which provides funding for initial emergency response activities. by the end of the three weeks of fighting in early in gaza, children had been killed and , injured, and much of gaza's infrastructure, including schools, health facilities, and vital infrastructure for water and sanitation, had been damaged. unicef was on hand to provide humanitarian support. it led the collective efforts of un agencies on the ground to restore education, provide emergency water supplies and sanitation, maintain nutritional standards, and protect children from further harm. from the early days, unicef made sure that first aid and emergency medical kits, essential drugs, and water purification tablets flowed into gaza. emergency education supplies such as classroom tents and school-in-a-box kits maintained some sense of continuity and normalcy for children. unicef and its partners were able to reach more than , school-age children. unicef raised global awareness of the harm being done to children through extensive media coverage and advocacy. attention was also raised by the visits of the special representative of the secretary-general for children and armed conflict, radhika coomaraswamy-who called for the protection of children-and unicef executive director ann m. veneman, as well as goodwill ambassadors mia farrow and mahmoud kabil. unicef also extended psychosocial services, including in-depth counselling and structured recreational activities, across gaza. training reinforced the capacities of psychosocial workers to protect children and help them heal. radio programmes and , leaflets designed for children warned of the risks of mines and unexploded ordnance left behind. unicef water tankers ensured a steady supply of clean drinking water to schools with , students, while desalination units were installed to rid water of dangerous concentrations of chlorides and nitrates. to thwart the risk of acute malnutrition, unicef worked through health clinics for mothers and children to offer supplements of micronutrients and fortified food. the quality and supply of teaching materials were improved through unicef's provision of math and science teaching kits. programmes for vulnerable adolescents concentrated on supporting remedial learning, relieving stress, and providing life skills-based education and opportunities to engage in civic activities. through unicef's systematic advocacy with partner organizations, almost half the attendees were girls. the world food programme (wfp) is the un agency tasked with addressing hunger-related emergencies. it was created in by a resolution adopted by the un general assembly and the un fao. today, the program operates in countries and maintains eight regional offices. in the year alone, the wfp provided . million metric tons of food aid to . million people in countries through its relief programs. over the course of its existence, the wfp has provided more than million metric tons of food to countries worldwide. wfp was an early member of the former inter-agency task force for disaster reduction (see below) and maintains disaster risk reduction as one of its priority areas, focusing on reducing the impact of natural hazards on food security, especially for the vulnerable. the wfp policy on disaster risk reduction and management, approved in , highlights this role as being central to the organization's work. wfp drr programs seek to build resilience and reduce risk through such activities as soil and water conservation, rehabilitating infrastructure, and training community members in disaster risk management and livelihood protection. the meret project in ethiopia is one example. this program targets food-insecure communities in degraded fragile ecosystems prone to drought-related food crises. other programs maintained by wfp include: • r resilience initiative: the rural resilience initiative (r ) is a partnership between wfp and oxfam america, with support from global reinsurance company swiss re, to test a new, comprehensive disaster risk reduction and climate change adaptation approach. the program allows cash-poor farmers and rural households to pay for index insurance with their own labor, so they can both manage and take risks to build resilient livelihoods. • livelihoods early assessment and protection (leap): wfp has been assisting the government of ethiopia to develop an integrated risk management system through the livelihoods early assessment and protection (leap) project. leap provides early warning data on food security that allows a rapid scale-up of the "national productive safety net programme" by activating contingency plans. when a serious drought or flood is detected, resources from a us$ million contingency fund are made immediately available to ensure early and more effective emergency response, thereby protecting livelihoods and saving lives. • the joint wfp/ifad weather risk management facility (wrmf): wrmf supports the development of innovative weather and climate risk management tools, such as weather index insurance (wii). the goal of these programs is to improve quality-of-life issues and to reduce the incidence of food shortages. this program was launched in through funding from the bill and melinda gates foundation. it has been piloted in china and ethiopia. wfp has established a steering committee for disaster mitigation to help its offices integrate these activities into regular development programs. examples of mitigation projects that focus on food security include water harvesting in sudan (to address drought), the creation of grain stores and access roads in tanzania, and the creation of early warning and vulnerability mapping worldwide. because food is a necessity for human survival and is considered a vital component of development, a lack of food is, in and of itself, an emergency situation. the wfp works throughout the world to assist the poor who do not have sufficient food so they can survive "to break the cycle of hunger and poverty." hunger crises are rampant-more than billion people across the globe receive less than the minimum standard requirement of food for healthy survival. hunger may exist on its own, or it may be a secondary effect of other hazards such as drought, famine, and displacement. the wfp constantly monitors the world's food security situation through its international food aid information system (fais). using this system, wfp tracks the flow of food aid around the world (including emergency food aid) and provides the humanitarian community with an accurate inventory and assessment of emergency food-stock quantities and locations. this database also includes relevant information that would be needed in times of emergency, such as anticipated delivery schedules and the condition and capabilities of international ports. in rapid-onset events such as natural disasters, the wfp is a major player in the response to the immediate nutritional needs of the victims. food is transported to the affected location and delivered to storage and distribution centers. (see figure . .) the distribution is carried out according to preestablished needs assessments performed by ocha and the undp. the wfp distributes food through contracted ngos that have the vast experience and technical skills to plan and implement transportation, storage, and distribution. the principal partners in planning and implementation are the host rice donated by japan is loaded by the world food programme onto wfp trucks to feed survivors of the asia tsunami and earthquake events sources: skullard, ; wfp, . governments, who must request the wfp aid, unless the situation is a che without an established government, in which case the un secretary-general makes the request. the wfp works closely with all responding un agencies to coordinate an effective and broad-reaching response, because food requirements are so closely linked to every other vital need of disaster victims. during the reconstruction phase of a disaster, the wfp often must continue food distribution. rehabilitation projects are implemented to foster increased local development, including the provision of food aid to families, who, as a result, will have extra money to use in rebuilding their lives; and food-for-work programs, which break the chains of reliance on aid as well as provide an incentive to rebuild communities. wfp administers the international emergency food reserve (iefr), which was originally designed to store a minimum of , tons of cereals. this program has not enjoyed the full support of donors as agreed in its creation, however, and as such, annual funding levels have fluctuated significantly. if supported, iefr would manage separate resources provided by donors to address long-term operations such as ches, and would dedicate $ million from its general resources for emergency assistance in addition to $ million for long-term emergency assistance. the program's immediate response account is a cash account maintained for rapid purchase and delivery of food in emergency situations. resources would be purchased from local markets (whenever possible), thereby ensuring food arrives sooner than other aid, which must move through regular channels. wfp response begins at the request of the affected country's government. . in the early days of an emergency, while the first food supplies are being delivered, emergency assessment teams are sent in to quantify exactly how much food assistance is needed for how many beneficiaries and for how long. they must also work out how food can best be delivered to the hungry. . equipped with the answers, wfp draws up an emergency operation (emop), including a plan of action and a budget. [the emop] lists who will receive food assistance, what rations are required, the type of transport wfp will use, and which humanitarian corridors lead to the crisis zone. . next, wfp launches an appeal to the international community for funds and food aid. the agency relies entirely on voluntary contributions to finance its operations, with donations made in cash, food, or services. governments are the biggest single source of funding. [more than governments support wfp's worldwide operations.] . as funds and food start to flow, wfp's logistics team works to bridge the gap between the donors and the hungry. [in , the agency delivered . million metric tons of food aid by air, land, and sea.] (wfp ) ships carry the largest wfp cargo, their holds filled to the brim with , tons or more of grain, cans of cooking oil, and canned food; the agency has ships on the high seas every day, frequently rerouting vessels to get food quickly to crisis zones. in extreme environments, wfp also uses the skies to reach the hungry, airlifting or airdropping food directly into disaster zones. before the aid can reach its country of destination, logistics experts often need to upgrade ports and secure warehouses. trucks usually make the final link in wfp's food chain, transporting food aid along the rough roads that lead to the hungry. where roads are impassable or nonexistent, wfp relies on less conventional forms of transport: donkeys in the andes, speedboats in the mozambique floods, camels in sudan, and elephants in nepal. at this stage, local community leaders work closely with wfp to ensure rations reach the people who need it most: pregnant mothers, children, and the elderly. the world health organization (who) was proposed during the original meetings to establish the un system in san francisco in . in , at the united health conference in new york, the who constitution was approved, and it was signed on april , (world health day). who proved its value by responding to a cholera epidemic in egypt months before the epidemic was officially recognized. who serves as the central authority on sanitation and health issues throughout the world. it works with national governments to develop medical and health care capabilities and assist in the suppression of epidemics. who supports research on disease eradication and provides expertise when requested. it provides training and technical support and develops standards for medical care. who was an early member of the former interagency task force for disaster reduction (see below), and continues to assist local and national governments as well as regional government associations with health-related disaster mitigation and preparedness issues. it does this primarily by providing education and technical assistance to government public health officials about early detection, containment, and treatment of disease and the creation of public health contingency plans. who activities address primary hazards, such as epidemics (e.g., avian influenza, malaria, dengue fever, sars, swine flu, and mers/cov), and the secondary health hazards that accompany most major disasters. through their website and collaboration with various academic institutions, who has also worked to advance public health disaster mitigation and preparedness research and information exchange. the who director-general is a member of the iasc and the iasc working group. in those capacities, the who recommends policy options to resolve the more technical and strategic challenges of day-to-day emergency operations in the field. to incorporate public health considerations in un interagency contingency planning and preparedness activities, the who also participates in the iasc task force on preparedness and contingency planning. the who emergency risk management and humanitarian response department was created to enable who to work closely with member states, international partners, and local institutions in order to help communities prepare for, respond to, and recover from emergencies, disasters, and crises. the emergency response framework (erf) was developed in to clarify the who role and their responsibilities in emergency situations (who ). in the event of a disaster, who responds in several ways to address victims' health and safety. most important, it provides ongoing monitoring of diseases traditionally observed within the unsanitary conditions of disaster aftermath. who also provides technical assistance to responding agencies and host governments establishing disaster medical capabilities and serves as a source of expertise. it assesses the needs of public health supplies and expertise and appeals for this assistance from its partners and donor governments. per the erf, who is obligated to respond to emergencies under several conventions and agreements, including the international health regulations and the interagency steering committee. the key functions of hac in times of crises are: • measure health-related problems and promptly assess health needs of populations affected by crises, identifying priority causes of disease and death; • support member states in coordinating action for health; • ensure that critical gaps in health response are rapidly identified and filled; and • revitalize and build capacity of health systems for preparedness and response. when other government agencies, private medical facilities, or ngos cannot meet the public health needs of the affected population, who's country-level emergency response team and international emergency support teams bring together expertise in epidemics, logistics, security coordination, and management, collaborating with un agencies participating in response and recovery. who has several bilateral agreements with other un agencies and ngos (including the red cross and red crescent movement) and coordinates the interagency medical/health task force (imtf), an informal forum that provides guidance on technical and operational health challenges in humanitarian crises. the who global emergency management team (gemt) was created in to lead the planning, management, implementation, monitoring, and evaluation of who's emergency work (including national preparedness, institutional readiness, and emergency response for disasters that exhibit public health consequences.) the gemt is made up of staff from both who headquarters and regional office directors responsible for disaster risk management issues (e.g., preparedness, surveillance, alert, and response). as needed, other relevant staff are invited to join gemt efforts. gemt focuses on all-hazards emergency risk management, notably that of leadership on the health cluster. when technical expertise beyond that held by the team's members is needed, the global emergency network (gen), comprising directors (or delegates) of departments and programs that have various emergency management functions, is consulted. the gemt continuously tracks global health events and the organization-wide use of internal and external resources in all emergencies, and reports on all major emergencies. during an actual emergency or disaster, a subset of the gemt, known as the gemt-response (gemt-r), is mobilized to grade and manage the response to a specific emergency. for larger-scale emergencies, the gemt-r is responsible for making recommendations to executive management on the best use of who resources given the event's scale, scope, duration, and complexity (given other existing requirements in ongoing events). since its inception, six regional offices have been established. these offices focus on the health issues in each region: • regional office for africa • pan american health organization • regional office for south-east asia • regional office for europe • regional office for eastern mediterranean • regional office for the western pacific the food and agriculture organization (fao) was established as a un agency in in quebec city, canada. the organization's mandate is to "raise levels of nutrition, improve agricultural productivity, better the lives of rural populations and contribute to the growth of the world economy" (fao ) . it provides capacity-building assistance to communities that need to increase food production. in , fao pledged to help current and future generations achieve food security by . in spite of their work, more than million people worldwide continue to suffer the effects of food shortages, including more than million children under five years of age who show signs of malnutrition-based growth stunting. fao is headquartered in rome, italy. the organization also maintains five regional, five subregional, and country offices, each of which works with un member countries and other partners to coordinate various activities, including disaster management. it has member nations, two associate member nations, and one member organization (the european union). fao was an early member of the interagency task force on disaster reduction prior to its becoming the global platform for disaster risk reduction. the world food summit mandated fao to assist un member countries in developing national food security, vulnerability information, and specialized mapping systems to cut worldwide malnutrition. a key component of this strategy is strengthening the capacity of communities and local institutions to prepare for natural hazards and respond to food emergencies during disasters and crises. this objective focuses on: • strengthening disaster preparedness and mitigation against the impact of emergencies that affect food security and the productive capacities of rural populations; • forecasting and providing early warning of adverse conditions in the food and agricultural sectors and of impending food emergencies; • strengthening programs for agricultural relief and rehabilitation and facilitating the transition from emergency relief to reconstruction and development in food and agriculture; and • strengthening local capacities and coping mechanisms by guiding the choice of agricultural practices, technologies, and support services to reduce vulnerability and enhance resilience. in , fao released strategic objective i, which guided the organization in conducting "preparedness for, and effective response to, food and agricultural threats and emergencies." this strategy laid out three specific results that were sought, including: . countries' vulnerabilities to crises, threats, and emergencies is reduced through better preparedness and integration of risk prevention and mitigation into policies, programs, and interventions; . countries and partners respond more effectively to crises and emergencies with food-and agriculture-related interventions; and . countries and partners have improved transition and linkages between emergency, rehabilitation, and development. within fao, the emergency coordination group is the organizational mechanism for the overall coordination of emergency and disaster reduction issues. this group strengthens fao's capacity to perform food-based disaster management activities in support of member countries and partners in a more integrated way. ecg is chaired by the director of the office for coordination of normative, operational and decentralized activities and has a secretariat provided by the office of the special advisers to the director-general. key units of this group include: the investment centre division prepares investment programs and projects for funding by major multilateral development banks during the rehabilitation and reconstruction and the recovery phases. fao field offices are in developing countries. regional and subregional offices are also maintained. at any time, fao is involved in some , agricultural projects in the developing world. experts working on these projects in affected countries are frequently called upon to help with emergency needs assessments and field operations. in a disaster, fao representatives in developing countries respond by coordinating with the government and other partners. in countries with ches, fao coordinates actions that address emergency agricultural needs and assists in the development and implementation of strategies for creating conditions conducive to recovery and sustained development. fao's approach is to set up coordination units that: • provide technical assistance to help the impacted government and its citizens to manage agricultural relief; • monitor the ongoing crisis relative to food; • advise ngos and other organizations involved in food and agriculture; • help build the necessary national capacity to transition from response to recovery; and • establish information collection and database management systems. examples of countries where fao emergency coordination units have been set up include bosnia, tajikistan, rwanda, burundi, liberia, sierra leone, somalia, iraq, and angola. fao also maintains a website of disaster reduction information through its world agricultural information center (waicent). this online portal provides access to the global information and early warning system, information on crop prospects, and other relevant documents and data. fao also works to help countries adopt sustainable agricultural and other land-use practices. its land and water division has helped to reverse land loss, thus increasing disaster resilience, by promoting the development of disaster-resistant agro-ecosystems and the sound use of land and water resources. in times of disaster, the emergency operations and rehabilitation division helps communities recover. while other agencies, such as wfp, address immediate food needs by providing the actual food aid to victims, fao provides assistance to restore local food production and reduce dependency on food aid. the fao's first action following disasters, in partnership with wfp, is to send missions to the affected areas to assess crops and food supply status. the emergency operations service of the emergency operations and rehabilitation division leads these missions, sending fao experts to consult with farmers, herders, fisheries, and local authorities to gather disaster and recovery data. using their assessment, fao designs an emergency agricultural relief and rehabilitation program and mobilizes the funds necessary for its implementation. the emergency operations and rehabilitation division distributes material assets such as seeds, fertilizer, fishing equipment, livestock, and farm tools. in a che, fao helps affected communities bolster overall resources and restore and strengthen agricultural assets to make them less vulnerable to future shocks. for example, fao has been working in regions outside government authority in the sudan to conduct community-based training of animal health workers aimed at keeping their livestock-a vital part of local livelihoods-from dying. when a disaster occurs, the emergency operations and rehabilitation division of fao establishes an emergency agriculture coordination unit consisting of a team of technical experts from a wide range of fields (including crop and livestock specialists). this field-level team provides information and advice to other humanitarian organizations and government agencies involved in emergency agricultural assistance in the affected area. fao coordination units also facilitate operational information exchange, reducing duplications of and eliminating gaps in assistance. fao's primary beneficiaries include: • subsistence farmers • pastoralists and livestock producers • artisan "fisherfolk" • refugees and internally displaced people • ex-combatants • households headed by women or children and/or afflicted by hiv/aids the special emergency programmes service (tces), also within the emergency operations and rehabilitation division, is responsible for the effective implementation of specially designed emergency programs. these programs require particular attention because of the political and security context surrounding their interventions and the complexity of the institutional setup. tces was responsible for fao's intervention in the framework of the oil for food program in iraq and fao's emergency and early rehabilitation activities in the west bank and gaza strip. the rehabilitation and humanitarian policies unit (tcer) is the final component of the emergency operations and rehabilitation division. tcer is responsible for making recommendations regarding disaster preparedness, post-emergency, and rehabilitation initiatives. the unit coordinates fao's position on humanitarian policies and ensures that fao addresses the gap between emergency assistance and development. tcer also liaises with other un entities dealing with humanitarian matters. the fao's disaster-and emergency-related projects are funded by contributions from governmental agencies, ngos, other un agencies, and by the fao technical cooperation programme (tcp). each year, approximately percent of fao emergency funds are raised through the cap. fao expenditure on emergency efforts has grown significantly during the past few years, indicative of the greater role the organization has assumed in disaster management. current emergency-related projects include: • improved food security for hiv/aids-affected households in africa's great lakes region • rehabilitation of destroyed greenhouses in the west bank and gaza strip • land-tenure management in angola • emergency agricultural assistance to food-insecure female-headed households in tajikistan • consolidation of peace through the restoration of productive capacities of returnee and host communities in conflict-affected areas in sudan • rehabilitation of irrigation systems in afghanistan • rehabilitation of farm-to-market roads in the democratic republic of the congo the position of united nations commissioner for refugees (unhcr) was created by the general assembly in to provide protection and assistance to refugees. the agency was given a three-year mandate to resettle . million european world war ii refugees. today, unhcr is one of the world's principal humanitarian agencies, operating through the efforts of more than , personnel and addressing the needs of . million people in more than countries. unhcr promotes international refugee agreements and monitors government compliance with international refugee law. unhcr programs begin primarily in response to an actual or impending humanitarian emergency. in complex humanitarian disasters and in natural and other disasters that occur in areas of conflict, there is a great likelihood that refugees and idps will ultimately result. the organization's staffs work in the field to provide protection to refugees and displaced persons and minimize the threat of violence many refugees are subject to, even in countries of asylum. the organization seeks sustainable solutions to refugee and idp issues by helping victims repatriate to their homeland (if conditions warrant), integrate in countries of asylum, or resettle in third countries. unhcr also assists people who have been granted protection on a group basis or on purely humanitarian grounds, but who have not been formally recognized as refugees. unhcr works to avert crises by anticipating and preventing huge population movements from recognized global areas of concern ("trouble spots"). one method is to establish an international monitoring presence to confront problems before conflict breaks out. for example, unhcr mobilized a "preventive deployment" to five former soviet republics in central asia experiencing serious internal tensions following independence. unhcr also promotes regional initiatives and provides general technical assistance to governments and ngos addressing refugee issues. in times of emergency, unhcr offers victims legal protection and material help. the organization ensures that basic needs are met, such as food, water, shelter, sanitation, and medical care. it coordinates the provision and delivery of items to refugee and idp populations, designating specific projects for women, children, and the elderly, who comprise percent of a "normal" refugee population. the blue plastic sheeting unhcr uses to construct tents and roofing has become a common and recognizable sight in international news. unhcr maintains an emergency preparedness and response section (eprs), which has five emergency preparedness and response officers (epro) who remain on call to lead emergency response teams into affected areas. the epros may be supported by a range of other unhcr human resources, including: • emergency administrative officers and emergency administrative assistants, for quickly establishing field offices • the members of the emergency roster, which includes staff with diverse expertise and experience, are posted throughout the world and are available for rapid emergency deployment • staff (by existing arrangement) from the danish refugee council, the norwegian refugee council, and un volunteers, to provide specialized officials on short notice as needed (more than people are available at any given time) • individuals registered on a roster of "external consultant technicians," who are specialized in various fields often required during refugee and idp emergencies (including health, water, sanitation, logistics, and shelter) • select ngos that have been identified as capable of rapid deployment to implement assistance in sectors of need (e.g., health, sanitation, logistics, and social services) unhcr has the capacity to respond to a new emergency impacting up to , people. the agency can also mobilize more than trained personnel within hours. these experts come from its emergency response team (ert) roster. unhcr has also developed mechanisms for the immediate mobilization of financial resources to help meet the response to an emergency without delay. unhcr staff may be supported under an agreement with the swedish rescue services agency, which is prepared to establish a base camp and office in affected areas within hours' notice. other supplies and resources, such as vehicles, communications equipment, computers, personal field kits, and prepackaged office kits are maintained for rapid deployment to support field staff. unhcr maintains stockpiles of relief aid, including prefabricated warehouses, blankets, kitchen sets, water storage and purification equipment, and plastic sheeting. these are stored in regional warehouses or may be obtained on short notice from established vendors that guarantee rapid delivery. unhcr also maintains agreements with stockpiles outside the un system from which they may access items, such as the swedish rescue board and various ngos. unhcr developed a quick impact project (qip) initiative. qips are designed to bridge the gap between emergency assistance provided to refugees and people returning home and longer-term development aid undertaken by other agencies. these small-scale programs are geared toward rebuilding schools and clinics, repairing roads, and constructing bridges and wells. unhcr is funded almost entirely by voluntary contributions from governments, intergovernmental organizations, the private sector, and individuals. it receives a limited subsidy of less than percent of the un budget for administrative costs and accepts "in-kind" contributions, including tents, medicines, trucks, and air transportation. as the number of people protected or of concern by unhcr has reached record highs, its annual budget has likewise jumped several fold in just a few years. in , the unhcr budget was a record $ . billion, yet by that number rose to over $ . billion-a rate that has been maintained ever since. (see figure . .) in , unhcr established a new global emergency stockpile in dubai, united arab emirates. the new stockpile is the largest of several unhcr global stockpiles. it is used to store relief items such as tents, blankets, plastic sheeting, mosquito nets, kitchen sets, and jerry cans, among other items, for up to , people. prefabricated warehouses and other safety and security equipment for staff and office support are also available. in , items from the stockpile were sent to countries, including syria, jordan, lebanon, turkey, and others in africa and asia. the items shipped included , , blankets, , buckets, , jerry cans, , kitchen sets, , sleeping mats, , mosquito nets, , plastic tarpaulins, and , family tents (unhcr ). although unhcr does not often become involved in natural disaster response, rather focusing on areas of conflict, its expertise and assistance were required in the aftermath of the october earthquake that severely impacted south asia. during the response phase of this disaster, unhcr provided flights loaded with supplies from its global and regional stockpiles and contributed , family tents, , blankets, , plastic sheets, and thousands of jerry cans, kitchen sets, stoves, and lanterns. the aid items were drawn from its existing warehouses in pakistan and afghanistan, as well as other locations throughout the world. because of the earthquake, roads used to access , afghan refugees affected by the earthquake were severely damaged, but unhcr was able to quickly assess damages and needs and meet those needs through their existing networks (unhcr ) . in the event of a large-scale disaster, the un may form a disaster management team (dmt) in the affected country. if the disaster clearly falls within the competence and mandate of a specific un agency, that organization will normally take the lead, with the un dmt serving as the forum for discussing how other agencies will work to support that lead agency. the un dmt is convened and chaired by the un resident coordinator and comprises country-level representatives of fao, undp, ocha, unicef, wfp, who, and, where present, unhcr. specific disaster conditions may merit participation by other un agencies. the leader of the undac team, assigned by ocha, automatically becomes a member of the un dmt. a undp official called the disaster focal point officer often serves as the un dmt secretary, but the team is free to choose another person, if necessary. undp is also responsible for providing a venue for the team and any basic administrative support needs. the un dmt's primary purpose is to ensure that in the event of a disaster, the un is able to mobilize and carry out a prompt, effective, and concerted response at the country level. the team is tasked with coordinating all disaster-related activities, technical advice, and material assistance provided by un agencies, as well as taking steps to avoid wasteful duplication or competition for resources by un agencies. the un dmt interfaces with the receiving government's national emergency management team, from which a representative may, where practical, be included in the un dmt. the central emergency response fund (cerf) was created in through un general assembly resolution / to allow for faster operational action by un agencies. the fund, which was originally called the central emergency revolving fund but renamed in under resolution / , is administered on behalf of the un secretary-general by the emergency relief coordinator. during times of disaster, cerf provides agencies involved in the humanitarian response with a constant source of funding to cover their activities. its purpose is to shorten the amount of time between the recognition of needs and the disbursement of funding. agencies that have received pledges from donors but have not yet received actual funds, or agencies that expect to receive funds from other sources in the near future, can borrow equivalent amounts of cash, interest free, through cerf. voluntary contributions from donor nations and private-sector donors have raised billions since the inception of cerf, of which more than $ billion has been allocated in the form of grants to almost countries. the program's goal is to have $ million replenished annually. (see table . for a full list of donors.) at the outset, cerf was designed only for ches, but in the general assembly voted . the lending agency submits a request for an advance to the erc, which includes a descriptive justification on the project or activities to be funded. if a future pledge for funding has been promised by a donor or if the agency has other means for repaying the loan, this information is included in the request. . an ocha officer reviews the request. if it is accepted (statistics show that the majority are accepted), the erc informs the agency and sets out the loan use and repayment terms. . disbursement usually occurs within hours. payment is made through an internal un "voucher." . loans must be repaid within six months. this entire process is conducted at ocha's new york office. figure . illustrates patterns of use by the various un agencies. the consolidated appeals process (cap), which began in , allows humanitarian aid organizations to plan, implement, and monitor their activities. these organizations can work together to produce a common humanitarian action plan (chap; see the following section) and an appeal for a specific disaster or crisis, which they present to the international community and donors. the cap fosters closer cooperation between governments, donors, aid agencies, and many other types of humanitarian organizations. it allows agencies to demand greater protection and better access to vulnerable populations, and to work more effectively with governments and other actors. the cap is initiated in three types of situations: . when there is an acute humanitarian need caused by a conflict or a natural disaster . when the government is either unable or unwilling to address the humanitarian need . when a single agency cannot cover all the needs on november , , typhoon haiyan (yolanda) hit the philippines. the humanitarian situation in the areas devastated by the typhoon was catastrophic. an estimated million people were affected, including million children. close to million people were displaced and in dire need of humanitarian assistance. in response, the united nations emergency relief coordinator, valerie amos, released us$ million to seven united nations agencies and the international organization for migration (iom) on november. • the united nations children's fund (unicef) received $ , , to ensure water, sanitation, and hygiene facilities. unicef also provided child protection, including protective learning environments, and reduced the risk of outbreaks of vaccine-preventable diseases among children aged to months. finally, unicef provided nutrition interventions to children aged to months as well as to pregnant and lactating women. • with an allocation of $ , , the food and agriculture organization (fao) provided emergency food assistance. • the united nations population fund (fpa) received $ , to ensure access to reproductive health services and to prevent gender-based violence. • to support the internally displaced persons (idps), the united nations high commissioner for refugees (unhcr) provided emergency shelter assistance through an allocation of $ , , . • the world food programme (wfp) received $ , , to provide emergency food assistance. wfp also coordinated the humanitarian operations in the areas affected. • the world health organization (who) provided health services through an allocation of $ , , . • the united nations development programme (undp) received $ , , to manage time-critical debris disposal. • through an allocation of $ , , , iom supported evacuation centers and idp sites by procuring and distributing emergency shelter kits and essential non-food items. the cerf allocations were expected to ultimately benefit more than . million people. the cap is led by the hc, who triggers the interagency appeal and collaborates with the iasc country team at the local level and the erc at headquarters. participants in the process include: • iasc. although all team members are encouraged to participate in chap development, some members may make appeals for funding outside of the un and its cap (as is often the case with the red cross). • donors. donors participate in chap development by committing to "good humanitarian donorship principles." • host government(s). the cap is best prepared in consultation with the host government, particularly the ministries the un operational agencies are working with on a day-to-day basis. • affected population(s). whenever possible, it is always advantageous to include the affected populations' perspective into relief and recovery planning. a consolidated appeal (ca) is a fundraising document prepared by several agencies working to outline annual financing requirements for implementing a chap. although governments cannot request funding through the ca, ngos can make a request as long as their proposed project goals are in line with chap priorities. the ca is usually prepared by the hcs in september or october, and then launched globally by the un secretary-general at the donor's conference held each november. the ca lasts as long as is necessary for funding purposes, usually a year or more. the sectors that may be considered by the ca include: • agriculture • coordination and support services the process for filing a ca is as follows: . at the onset of the emergency, a situation report is issued (can cover from day to week ). . in the meantime, a flash appeal may be prepared and launched (covers week to month ). . finally, a ca may be issued. if the situation and needs in the field change, a revision to any part of an appeal can be issued at any time. additionally, projects can be added, removed, or modified within the appeal at any time. approximately percent of cap and flash appeal funding comes from a small group of wealthy nations, including canada, the european community humanitarian office (echo) and the european commission, germany, japan, the netherlands, norway, sweden, the united kingdom, and the united states. in high-profile events, private donors may constitute a large percentage of donations, such as occurred in the case of the tsunami disaster in asia. the chap is a strategic plan developed by agencies working together at the field level that assesses needs in an emergency and coordinates response. it acts as the foundation for a ca, and includes the following information: . common analysis of the context for humanitarian assistance . needs assessment . best, worst, and most likely scenarios . identification of roles and responsibilities (who does what and where) . clear statement of long-term objectives and goals . framework for monitoring strategy and revising as necessary a flash appeal is a special kind of ca, designed for structuring a coordinated humanitarian response for the first three to six months of an emergency. whenever a crisis or natural disaster occurs, the un hc may issue a flash appeal in consultation with all stakeholders involved in the humanitarian response (including the affected government). it is normally issued between the second and fourth weeks of the response and provides a concise overview of urgent lifesaving needs. it may also include early recovery projects if they can be implemented within the appeal's time frame. in , as a part of the inter-agency standing committee (iasc) transformative agenda, the united nations changed the way that the cap was issued. the appeal, which addresses the emergencies, was the largest appeal to date, calling for $ . billion (more than $ . billion greater than the appeal) to support million people in countries. the increase was primarily due to a $ . billion request for the complex humanitarian emergency in syria, as well as another che in the central african republic and typhoon haiyan in the philippines. the changes begin with the document's name, which is now called the "overview of global humanitarian response" rather than the former "overview of consolidated appeals process." the overall goal of the change is to ensure that the cap process is needs-based and funds are adequately monitored. the change is explained in the appeals document as follows: "now, instead of one overweight cap document trying to present all elements of the program cycle, for the key elements appear in a series of documents produced in sequence: humanitarian needs overview; strategic response plan (comprising the country strategy plus cluster plans); and periodic monitoring bulletins reporting on basic delivery and outputs compared to targets. discussions are ongoing about the possible production of end-of-year reports on achievements versus objectives" (un b). (see exhibit . ). since , more than consolidated and flash appeals have been launched, collectively raising more than $ billion for ngos, the international organization for migration (iom), and un agencies. in addition to the un agencies discussed previously, which tend to be the primary agencies involved in all forms of disaster management, a handful of organizations provide more focused assistance as deemed necessary in most disasters that require international participation. as illustrated in figure . , which details un assistance to the various countries affected by the december asian tsunami and earthquake events, a different mix of un assistance is needed in each country, even within the same international disaster scenario. several of these organizations are detailed in the following list. • international labour organization (ilo). the ilo works with the affected population to address issues related to employment, including job creation, skills training, employment services, small business assistance, and other functions. (see exhibit . .) • international organization for migration (iom) . the iom provides rapid humanitarian aid to displaced populations by supplying emergency shelter, transporting relief materials, and assisting in medical evacuations. the organization stabilizes populations through the provision of short-term community and microenterprise development programs. iom also actively supports governments in the reconstruction and rehabilitation of affected communities by being the lead service provider of : haiti cannot afford to become a forgotten crisis. important progress has been made in recent years, but the country is still one of the most exposed to risk from disaster and climate change. multiple disasters combined with high unemployment, increased inequality, and poor access to basic social services have prolonged the vulnerability of an estimated three million haitians to displacement, food insecurity, and fragile living conditions. haiti suffers the world's largest cholera epidemic, which has affected over , people and killed , . although the humanitarian situation in south sudan has stabilized on several fronts, needs remain high-driven primarily by violence and displacement, persistent food insecurity, and chronic poverty. national capacity to deliver basic services is low, with aid agencies the main providers of health care, clean water, livelihoods support, and other services in many parts of the country. while needs are expected to remain high in - , in some areas such as food insecurity, there are opportunities for innovative and more targeted approaches to break recurring cycles of hardship. the strategy for - has three objectives: responding to immediate needs, enhancing communities' resilience against shocks and stresses, and building national capacity to deliver basic services. alongside core programmes to save lives and ease suffering, partners are increasingly integrating actions to reduce the risk of natural disasters, strengthen and diversify livelihoods, and address the long-term needs of vulnerable groups, including refugees and children. the strategy also emphasizes the importance of delivering aid in partnership with line ministries and national ngos to ensure humanitarian relief has a long-term positive impact. the strategy spans three years, to better address deep-rooted challenges and measure the impact of relief actions. requirements: $ . billion funding received against requirements: % people in need: . million people to receive help: . million in yemen, more than half the population needs some form of humanitarian aid. the collapse of basic services in - , endemic food insecurity, destroyed or damaged livelihoods and under-development, along with displacement resulting from conflict, have combined to plunge the country into a humanitarian emergency which may persist into . inflows of refugees and migrants from the horn of africa and returning yemeni migrants count among the vulnerable. ten and a half million people are food-insecure or severely food-insecure, and , , children under five suffer from acute or severe malnutrition. about half the population has no access to adequate water sources or sanitation facilities, and a further . million have insufficient access to health services. an estimated , returnees need assistance to rebuild their lives, while , refugees, mostly from somalia, and tens of thousands of mainly ethiopian migrants are stranded in the country. it is expected that the number of returning yemeni migrants, estimated to be , people, will double in . the weakness of rule-of-law institutions has been identified as a serious protection risk. according to the latest ilo estimates, . million out of the . million workers who have either temporarily or permanently lost their livelihoods were working in the service sector. over one third, or . million, were in agriculture and around per cent in the industry sector. "service sector includes people working in shops, public markets, restaurants, vendors, tricycle and jeepney drivers, mechanics, clerks, teachers, . . . who, like farmers and fisherfolks, have seen their source of income wiped away," said ilo philippine office director lawrence jeff johnson. "at least . million affected workers were already in a vulnerable situation before the typhoon struck, often living at or near the poverty line, doing whatever work they could find to survive and provide for their families. these people have lost the little they had to begin with. they have no home, no income, no savings and no one to turn to for help," said director johnson. "as the reconstruction efforts gather pace, the number one priority is to ensure that these workers have access to decent jobs, which include at least minimum wage, social protection, and safe working conditions," johnson said. the department of labor and employment (dole) and the department of social welfare and development (dswd) are rolling out emergency employment programmes to respond to the enormous reconstruction and livelihoods needs. the ilo is working closely with them as well as with local governments, business' and workers' organizations, and international partners. "these programmes comply with philippine regulation and international labour standards, ensuring that people are not exploited while they help to rebuild their communities and local economies," johnson explains. workers under the emergency employment programmes receive the minimum wage prevailing in the area and are employed for a minimum of days. they also have access to social protection benefits. "this is a very first step to jump start the economy and quickly put the affected communities back in the driver's seat in rebuilding their lives. ensuring minimum wage and social protection will help stimulate economic growth and speed the recovery process." johnson said. source: ilo, . unaids works with victims to protect them from the kinds of violence and activity that spreads hiv. in , the un general assembly held a special session on hiv/aids and declared that through unaids: [the un would] develop and begin to implement national strategies that incorporate hiv/aids awareness, prevention, care, and treatment elements into programs or actions that respond to emergency situations, recognizing that populations destabilized by armed conflict, humanitarian emergencies, and natural disasters, including refugees, internally displaced persons, and in particular, women and children, are at increased risk of exposure to hiv infection; and, where appropriate, factor hiv/ aids components into international assistance programs. (un ) • united nations population fund (unfpa). unfpa works to promote basic human rights throughout the world, and to increase the possibilities of women and young people to lead healthy and productive lives. their work focuses specifically on reproductive health and safe pregnancies and deliveries. during humanitarian crises, there is often a demand for reproductive health services even though distribution and health care systems have broken down. unfpa works closely with its humanitarian relief partners to support early and effective action to meet the reproductive health needs of refugees, idps, and others caught in crisis situations. supply shortages compound health risks in already dangerous situations and are a major obstacle to reproductive health in emergencies. existing supplies may fall far short of demand when large numbers of people move into a safer location. supplies, equipment, and medicine are organized and stored by unfpa for immediate distribution when an earthquake, flood, violent conflict, or other crisis arises. a rapid-response fund enables unfpa to mount a quick response to emergencies, especially in the initial stages. supplies are packaged in different emergency reproductive health kits, including a "clean birthing kit." once an emergency situation stabilizes, the procurement of reproductive health materials becomes a regular part of a more comprehensive healthcare program. • united nations human settlement programme (un-habitat) . un-habitat is mandated by the un general assembly to "promote socially and environmentally sustainable towns and cities with the goal of providing adequate shelter for all" (un ). un-habitat is mandated through the habitat agenda (a global settlement plan adopted in june by the international community) to take the lead in mitigation, response, and post-disaster rehabilitation capabilities in human settlements. the habitat agenda clearly outlines the link between human settlement development and vulnerability to disasters. in addition, it emphasizes the need for coordination and close partnerships with national and local governments, as well as civil society. finally, the habitat agenda recognizes the strong impact disasters have on women, and affirms the need for women's active involvement in disaster management. these steering principles underpin all normative and operational activities of the un-habitat disaster management programme (dmp). dmp operates under the disaster, post-conflict and safety section (dpcss), urban development branch. it was created to marshal the resources of un-habitat and other international agencies to provide local government, civil society, and the private sector with practical strategies for mitigating and recovering from conflicts and natural disasters in the context of human settlements. specific areas of attention include: • protecting and rehabilitating housing, infrastructure, and public facilities • providing technical and policy support to humanitarian agencies before and after crisis in the context of human settlements • building partnerships and providing complementary expertise in resettlement of displaced persons and refugees • restoring local social structures through settlement development • rehabilitating local government structures and empowering civil society • land and settlements planning and management for disaster prevention un-habitat launched the city resilience profiling programme (crpp) to support local government efforts to build capacity to reduce disaster risk. through their guidance, governments are assisted in the development of comprehensive and integrated urban planning and implementation of a resilient management approach. the city resilience profile is a baseline assessment of a city-system's ability to withstand and recover from potential hazards. examples of cities that have participated in the program include balangoda, sri lanka; barcelona, spain; beirut, lebanon; dagupan, philippines; dar es salaam, tanzania; lokoja, nigeria; portmore, jamaica; talcahuano/concepcion, chile; tehran, iran; and wellington, new zealand. un-habitat plays an important role in disaster recovery, given the impact on housing so many disasters have. the organization has lead agency status within the united nations system for coordinating activities related to human settlement. it is mandated in this role through the habitat agenda. the organization's responsibilities in this regard are to support national governments, local authorities, and civil society in ensuring that risk is not retained in the reconstruction housing that follows the event. housing reconstruction often begins soon after the disaster has occurred, and un-habitat seeks to deploy quickly to ensure that resilient building practices are incorporated into the recovery planning process. • united nations environmental program (unep). unep is the un agency focused on the protection of the environment and wise use of natural spaces. unep has several divisions that address global emergency and disaster management needs. • unep's disasters and conflicts sub-program was created to assess and address the environmental impacts of disasters and conflicts, especially as they relate to human health, livelihoods, and security. since , this program has responded to crises in more than countries. their assistance is provided to other un agencies responding as well as directly to the host country government. the disasters and conflicts sub-program has four overarching objectives: • perform post-crisis environmental assessments; • support post-crisis environmental recovery; • foster environmental cooperation for peacebuilding; and • promote disaster risk reduction. as the focal point for environment within the un crisis response system, unep also works to integrate environmental considerations within humanitarian and peacekeeping operations. coordinated by unep's post-conflict and disaster management branch, the disasters and conflicts sub-program is delivered through several key actors and partners, including the joint unep/ocha environment unit, the environment and security (envsec) initiative, and the apell (awareness and preparedness from emergencies on a local level) programme. apell, which is based out of the unep industry and environment office in paris, supports disaster risk reduction and disaster preparedness. it seeks to minimize the occurrence and harmful effects of technological accidents and emergencies resulting from human activity or as the consequence of natural disasters, particularly in developing countries. understandably, unep plays a major role in climate change activities, including climate change adaptation. the organization supports developing countries in their efforts to identify and address risk specifically related to changing temperature and precipitation that are associated with global climate change patterns, including sea level rise. one of the primary functions of this office is to help governments to integrate climate change adaptation policy throughout all sectors of government, such that it becomes a major policy goal rather than a distinct, stove-piped component of government. finally, unep promotes sustainable land-use management and helps countries identify opportunities to reduce carbon emissions, which are often blamed for the bulk of climate variability. • united nations educational, scientific, and cultural organization (unesco). unesco's goal is to contribute to the peace and security of the world through education, science, and culture. unesco has been involved in disaster management for decades. this organization advocates for the need for a shift in emphasis from relief and emergency response to prevention and increased preparedness and education of potentially affected populations. it strongly supports the design and dissemination of mitigation measures, as well as public education and awareness. unesco works to increase the role of academic and research sectors in creating risk and vulnerability reduction measures, and supports existing and new institutions through financial and material support. unesco proclaims that their function regarding disaster management is: to promote a better understanding of the distribution in time and space of natural hazards and of their intensity, to set up reliable early warning systems, to devise rational land-use plans, to secure the adoption of suitable building design, to protect educational buildings and cultural monuments, to strengthen environmental protection for the prevention of natural disasters, to enhance preparedness and public awareness through education and training communication and information, to foster post-disaster investigation, recovery and rehabilitation, to promote studies on the social perception of risks. (unesco ) • in , the un general assembly created un women, which merged four existing un organizations that focused exclusively on gender equality and women's issues. these included the united nations development fund for women (unifem), the international research and training institute for the advancement of women (instraw), the office of the special adviser on gender issues and advancement of women (osagi), and the division for the advancement of women (daw). un women works to ensure that the needs of women are considered in disaster planning and preparedness efforts, as well as in the aftermath of disasters and in the recovery from them, when women face extraordinary vulnerabilities. un women provides financial and technical assistance to innovative programs and strategies to foster women's empowerment and gender equality. (see exhibit . .) • united nations institute for training and research (unitar). unitar was created to provide training and research within the un system with the goal of increasing the effectiveness of all un programs. in recent years, more of these efforts have focused on the four phases of disaster management, addressing many related topics such as climate change, hazardous materials and pollution, land use, and biodiversity. the global platform for disaster reduction was established in as a forum for information exchange. the platform meets every two years and allows participants to discuss innovations and developments in drr as well as to share existing knowledge and build partnerships among the various stakeholders. the goal in creating the program was to improve drr implementation by fostering better communication and coordination among stakeholders, to serve as a way for un members to voice their concerns and needs, and to share their best practices and lessons learned. the global platform replaces the former interagency task force for disaster reduction (iatf/ dr), which was led by the un under-secretary-general for humanitarian affairs and composed of representatives from un agencies, international organizations, ngos, and other civil society for millions of people in rural viet nam, the impacts of climate change are mounting and sometimes deadly. as weather patterns change, many of viet nam's women in particular are paying a high price. "the weather becomes more extreme and erratic. storms, heavy rains, and floods destroy fields and houses, kill animals and people every year," said ranh nguyen, , a farmer and the head of the women's union group in an dung commune, in binh dinh province, central viet nam. there, ranh and her neighbours have joined the viet nam women's union and are working with un women to strengthen the role of women in disaster risk-reduction and disaster-reduction management. some kilometers from the city of binh dinh, an dung commune is always at high risk of flooding, as it only has one road connecting it to other communes,and landslides often occur during the storm season. almost every year, the commune suffers at least one severe flood that damages crops and houses heavily. and women are often the most affected. however, things are starting to change. "thanks to good preparation and detailed mapping that we developed in the meetings before each storm, nobody in the village was killed or injured severely in the last year storm season. crops, fowl, and cattle were saved," explains ranh, now an official member of the committee for flood and storm control in her commune. prior to the project, there were few women on the committees for flood and storm control (cfsc) in the village. through the training of women in disaster management, as well as national lobbying-supported by un women, undp and other stakeholders-the contribution of women has been recognized. a government decree issued in september now provides an official space for the women's union in decision-making boards of the cfsc at all levels. "after being involved in the project, i am more aware of the situation of climate change and its impacts on us. last year, we participated in the training and exchanged experiences with other women. we prepared better for our families and our village before the storm came," ranh said. she said that she talked to the other members of the communal committee for flood and storm control. as a result, before the flooding began, they had plans ready to evacuate people living in lowland areas and near the river. "the mapping we did together in the training was really helpful. we discussed how to encourage people to harvest earlier, before the storm season started." in the end, she said, no lives were lost. last year, a four-year-old boy was saved from drowning because his mother performed cpr on him. she and another women and girls learned this technique from the rescue and first aid training provided by the project. "i could not swim before and used to be frightened by the flooded river. but now i am no longer afraid of water thanks to the swimming classes. i will teach my children how to swim and tell other people to learn how to swim too," ranh said. this project continues to be implemented in four new provinces including thua thien hue, quang binh, ca mau and dong thap, all of which face a high risk of flooding. this project is financed through core funding to un women and from the government of luxemburg. stakeholders. the global platform is organized by the un office for disaster risk reduction (unisdr; see below). the global platform for disaster risk reduction is considered the most significant gathering of disaster risk reduction and disaster management stakeholders worldwide. every ten years, the world conference on disaster reduction is held. the first was held in . the second world conference, held in in kobe, japan, led to the launching of the hyogo framework for action (hfa). the world conference is to be held in sendai, japan, which was significantly impacted by the great east japan earthquake and tsunami in . the focus of the conference is on the follow-up to the hyogo framework for action, termed the post- framework for disaster risk reduction in the lead-up to the conference. the united nations office for disaster risk reduction (unisdr) is the secretariat of the international strategy of disaster reduction and the global hub of the disaster risk reduction community, which includes national governments, ngos, intergovernmental organizations, financial institutions, technical bodies, and others. unisdr serves as the focal point for the implementation of the hyogo framework for action (hfa), the ten-year plan to address global disaster risk that commenced in and is set to expire in . unisdr was created in , at the end of the international decade for natural disaster reduction. the organization functions as a clearinghouse for disaster reduction information; campaigns to raise hazard awareness; and produces articles, journals, and other publications and promotional materials related to disaster reduction. unisdr maintains an organizational vision that is guided by the three strategic goals of the hfa for which it is tasked to oversee. these include: integrating disaster risk reduction into sustainable development policies and planning; . developing and strengthening institutions, mechanisms and capacities to build resilience to hazards; and . incorporating risk reduction approaches into emergency preparedness, response, and recovery programs. the organization describes the four key functions that guide its efforts as follows: • we coordinate international efforts in disaster risk reduction and guide, monitor as well as report regularly on progress of the implementation of the hyogo framework for action. we organize a biennial global platform on disaster risk reduction with leaders and decision makers to advance risk reduction policies and support the establishment of regional, national and thematic platforms. • we campaign and advocate to create global awareness of disaster risk reduction benefits and empower people to reduce their vulnerability to hazards. our current campaigns focus on safer schools and hospitals as well as resilient cities. • we encourage for greater investments in risk reduction actions to protect people's lives and assets including climate change adaptation, more education on drr, and increased participation of men and women in the decision making process. • we inform and connect people by providing practical services and tools such as the risk reduction website preventionweb, publications on good practices, country profiles and the global assessment report on disaster risk reduction, which is an authoritative analysis of global disaster risks and trends. (unisdr ) unisdr is led by the un special representative of the secretary-general for disaster risk reduction. margareta wahlstrom currently holds this post. the position was created in to lead and oversee all drr activities mandated by the un general assembly (ga), the economic and social council (ecosoc), and the hyogo framework for action (hfa), as well as policy directions by the secretary-general. other responsibilities include the ongoing and arduous process of facilitating the development of the post- framework for drr that will follow the hfa, overseeing the management of the trust fund for the international strategy for disaster reduction, and carrying out highlevel advocacy and resource mobilization activities for risk reduction and implementation of the hfa. one of the most significant functions of unisdr is monitoring the progress achieved by nations and global regions per the hyogo framework for action. monitoring is an almost ongoing process, with reports on progress produced every two years (as well as interim reports on off years, in some instances). the hfa monitor is an online reporting system that nations and regional organizations use to assess their capabilities and progress according to the indicators outlined in the hfa. the hfa monitor template, found on the hfa monitor website, defines the areas of assessment. the result of this process is a national or regional hfa progress report. not all countries produce the reports, and for those that do, reports are not necessarily submitted for each reporting period. critics note that it is a self-reporting and ranking system, but in the absence of any other system on the scale of the hfa monitor, the information it provides is highly informative and very useful in estimating capacity. the information is also used to produce papers and reports on various thematic issues, such as gender in disaster management, integration of drr and climate change adaptation, early warning, and others. the world bank and unisdr work closely together on a number of key disaster risk reduction issues, notably those related to development and disaster reconstruction, through a unisdr/world bank global facility for disaster reduction and recovery (wb/gfdrr) partnership. other similar drr-and disaster risk management-focused partnerships have been formed with various regional international organizations, including the association of southeast asian nations (asean), organization of the islamic conference (oic), pan american health organization (paho), applied geoscience and technology division of the secretariat of the pacific community (sopac), economic community of west african states (ecowas), and african union (au). unisdr is headquartered in geneva and has representation at the un headquarters in new york city. the organization also has regional offices in africa (nairobi), the americas (panama and brazil), asia/pacific (bangkok, japan, and korea), the pacific (sub-regional office in suva), the arab states (cairo), europe (brussels and bonn), and central asia (sub-regional office in almaty). (see figure . .) unisdr also works with and advises a number of key thematic platforms on disaster risk reduction issues, including: response (un-spider) the un is the only global international organization of its kind. it is not, however, the only governing organization made up of several national governments. many of the world's regions have pooled their collective resources and services to create large, influential organizations. like the un, these organizations address issues of regional and global importance, many of which focus on or peripherally address disaster management. in times of disaster, both within and outside of their regions of concern, they bring much of the same financial, technical, and equipment resources discussed throughout this book. this section identifies and briefly describes the largest of these organizations. the north atlantic treaty organization (nato) is an alliance of countries from north america and europe formed by a treaty signed on april , . its fundamental goal is safeguarding its members' freedom and security using political and military means. over the years, nato has taken on an increasing role in international disaster management and peacekeeping missions. nato maintains a military force made up of member countries' troops. although they work in concert, troops always remain under the control of their home nation's government. nato has helped to end violent conflicts in bosnia, kosovo, and the former yugoslav republic of macedonia. nato's disaster and crisis management activities, which extend beyond its typical military operations, are geared toward protecting populations. as part of the worldwide civil protection drive described in chapter , nato began developing measures to protect member nation citizens from nuclear attack as early as the s. as elsewhere, nato member countries soon realized that these capabilities could be used effectively during disasters induced by floods, earthquakes, and technological incidents and during humanitarian disasters. nato's first involvement in disaster operations came in , following devastating floods in northern europe. in , it established detailed procedures for the coordination of assistance between nato member countries in case of disasters. these procedures remained in place and provided the basis for nato's civil emergency planning in subsequent years. in , nato established the euro-atlantic disaster response coordination center to coordinate aid provided by member and partner countries to a disaster-stricken area in a member or partner country. it also established a euro-atlantic disaster response unit, which is a non-standing, multinational mix of national civil and military elements volunteered by member or partner countries for deployment to disaster areas. civil emergency planning has become a key facet of nato involvement in crisis management. in recent years, nato has assisted flood-devastated albania, czech republic, hungary, romania, and ukraine, supported the unhcr in kosovo, sent aid to earthquake-stricken turkey, helped to fight fires in the former yugoslav republic of macedonia and in portugal, supported flood response in pakistan, and supported ukraine and moldova after extreme weather conditions destroyed power transmission capabilities. nato has taken an active role in the response to the south asia earthquake, as described in exhibit . . nato also regularly conducts civil emergency planning exercises. the european union (eu) originated in may of , when six european countries (belgium, germany, france, italy, luxembourg, and the netherlands) joined together to address common issues related to the coal and steel industries. since that time, the scope of their work has expanded significantly, as has their membership. the eu is now a major regional international organization representing member states and is in the process of admitting several other eastern and southern european countries in a push toward greater inclusion. the eu considers itself a "family of european countries, committed to working together for peace and prosperity" (bbc ). like the un, it is not a government, nor does it have any authority over its members; it is an organization established for increased regional cooperation. regional international organizations the devastating october earthquake in pakistan is estimated to have killed , people and left up to three million without food or shelter just before the onset of the harsh himalayan winter. on october , , in response to a request from pakistan, nato launched an operation to assist in the urgent relief effort. nato airlifted supplies donated by nato member and partner countries as well as the unhcr via two air bridges from germany and turkey; flights delivered almost , tons of relief supplies. the supplies provided included thousands of tents, stoves, and blankets necessary to protect the survivors from the cold. in addition, nato deployed engineers and medical units from the nato response force to assist in the relief effort. the first teams arrived on october , . in just three months of operations, nato achieved the following: • nato's air bridges flew almost tons of aid to pakistan with flights. these flights carried in nearly , tents, , blankets, nearly , stoves/heaters, more than , mattresses, , sleeping bags, tons of medical supplies, and more. • nato's field hospital treated approximately , patients and conducted major surgeries. mobile medical units treated approximately , patients in the remote mountain villages; they also contributed significantly to the who immunization program that has helped to prevent the outbreak of disease. • in the cities of arja and bagh, nato engineers repaired nearly kilometers of roads and removed over , cubic meters of debris, enabling the flow of aid, commerce, and humanitarian assistance to the inhabitants of the valley. nine school and health structures were completed and tent schools erected. the engineers distributed cubic meters of drinking water and upgraded a permanent spring water distribution and storage system to serve up to , persons per day. • nato engineers also supported the pakistani army in operation winter race, by constructing multipurpose shelters for the population living in the mountains. • nato helicopters transported more than , tons of relief goods to remote mountain villages and evacuated over , disaster victims. • nato set up an aviation fuel farm in abbottabad, which carried out some , refueling missions for civilian and military helicopters. during the mission some , engineers and supporting staff, as well as medical personnel, worked in pakistan. nato was part of a very large effort aimed at providing disaster relief in pakistan. the pakistani army provided the bulk of the response, with the support of nato, the un, and other international organizations and several individual countries. on october , nato received from pakistan a request for assistance in dealing with the aftermath of the october earthquake. the next day, the north atlantic council approved a major air operation to bring supplies from nato and partner countries to pakistan. the airlift began on october and the first tons of supplies arrived in pakistan on october. on october, nato opened a second air bridge from incirlik, turkey, to deliver large quantities of tents, blankets, and stoves donated by the unhcr. on october, in response to a further request from pakistan, nato agreed to deploy engineers and medical personnel from the nato response force to pakistan to further assist in the relief effort. a nato headquarters was deployed to pakistan on october to liaise with pakistani authorities and pave the way for the incoming troops. the first troops, the advance elements of the medical team, began arriving on october, and immediately began treating hundreds of people a day. engineering teams followed and began working in the area around bagh in support of pakistani efforts to repair roads and build shelters and medical facilities. nato engineers also supported the pakistani army in operation winter race, by constructing multipurpose shelters for the population living in the mountains. on november, nato opened a sophisticated -bed field hospital, which provided a wide range of care including complex surgical procedures. on the same day, heavy-lift transport helicopters assigned to nato for the operation began flying and delivering supplies to remote mountain villages and evacuating victims. nato also set up an aviation fuel farm in abbottabad, which carried out refueling for civilian and military helicopters, which were essential to the relief effort. on october, additional foreign secretary of pakistan tariq osman hyder addressed a meeting of the euro-atlantic partnership council at nato headquarters in brussels, asking for further assistance. he said that nato could provide continued airlift, funds, logistic and airspace management, mobile fuel tanks, spare parts for helicopters and tactical aircraft, command and control, and winterized tents and sleeping bags. that same day, nato's euro-atlantic disaster response coordination center (eadrcc) received an urgent request from the unhcr for the transport of additional shelter and relief items stored in turkey to pakistan before the winter sets in. nato's relief mission came to an end, on schedule, on february . nato's short-term relief mission was based on the following five elements: . coordination of donations from nato and partner countries through the eadrcc in brussels; . the air bridge from turkey and germany for the transport of relief goods to pakistan; . five helicopters operating in the earthquake-affected area for the transport of supplies to remote mountain villages and evacuation of victims; . medical support with a field hospital and mobile medical teams in the area of bagh; . engineer support operating in the area around bagh in support of pakistani efforts for the repair of roads and building of shelters, schools, and medical facilities. humanitarian assistance has been a part of the eu mission since , and since that time the organization's work in that area has grown such that today it is the world's most significant humanitarian aid donor. taken together, its members represent a sizeable piece of the global economy, thus enabling them the ability to provide more than percent of all humanitarian aid worldwide. the eu has also structured itself to be an active stakeholder in international disaster management. their work in this regard is not limited to europe and in fact has a global presence. since taking on disaster management responsibilities, the eu has responded through one or more of its various departments to disasters in more than countries. in , the eu restructured its global hazard risk and disaster management capacities. these changes resulted in the merging of two former divisions: one that handled humanitarian assistance and another that centered on civil protection. together these units formed the combined directorate general for humanitarian aid and civil protection (echo). the acronym is a carryover from a former component of the eu's response mechanism called the european community humanitarian office. the move effectively integrated these two functions, which, over time, saw duplicative missions. exhibit . is drawn from an eu factsheet describing how the eu responded to typhoon haiyan in the philippines in using this combined function. through its humanitarian aid and civil protection department (echo), the european commission made available us$ . million to help the survivors of the typhoon with food assistance, shelter, water and sanitation, health and nutrition, short-term livelihood support, reconstruction of schools, emergency logistics, and coordination of relief efforts. within hours after the disaster struck, the european commission's experts had been deployed to identify priority needs. the commission implemented its assistance primarily through the following partner organizations: assistance supported by echo reached approximately . victims in the areas affected by the typhoon. the eu civil protection mechanism was activated to ensure coordination of european relief efforts. participating member states supplied personnel and material to support the operation. the eu civil protection mechanism, coordinated by the commission's emergency response and coordination centre (ercc), also supported the transport of civil protection assets to the region with around us$ . million. in addition to humanitarian funds, the european commission has released $ . million from the eu's development funds to help rebuild people's lives by assisting in recovery and rehabilitation. examples of eu-funded humanitarian projects are described in the following section. • to address food insecurity among the affected population who had little to eat and little to no access to markets, the eu funded the efforts of the world food programme (wfp). wfp provided general distribution of food, including highenergy biscuits during the emergency phase, and then provided supplementary feeding for children and pregnant and lactating women. "food-for-work" and "cash-for-work" initiatives were established. • the eu provided funds to the international committee of the red cross (icrc) and national red cross societies to provide thousands of families with shelter repair kits and to support the livelihoods recovery and wash clusters. the national red cross societies projects supported the delivery of non-food items, including blankets and water storage containers. many families were provided with unconditional cash grants, and communities were given assistance in improving sanitation facilities, restoring primary healthcare services (including medicines), disease prevention, and hygiene awareness. • the eu supported a consortium that includes plan international and oxfam. funding helped to provide relief for the most significantly affected households by enabling livelihoods recovery, distribution of cash-for-work vouchers, and rehabilitation of public service infrastructure, including child-friendly spaces, classrooms, day care centers, and health stations. • to help the approximately million people left homeless by the typhoon, the eu funded the international organization for migration (iom) efforts to improve the well-being and living conditions of those who were displaced, who have returned, or who are planning to go back to their places of origin. special attention was given to persons with disabilities and other special needs. the project provided shelter repair kits to the affected populations and ensured quality management of displacement sites and timely information on communities' return and relocation processes. finally, vulnerable groups targeted by the initiative received health services, psychosocial support, and non-food items such as blankets. based on: ec, . echo enables the eu to respond to most major crises regardless of where they are in the world. at the time of this publication, the eu was involved in the response to ches in syria, south sudan, and the central african republic and was working in several other countries that were no longer entrenched in conflict but nonetheless faced humanitarian needs (e.g., côte d'ivoire). in recent years, the eu annual budget allocation for humanitarian operations has remained at around us$ . billion, or about us$ per person from the combined population of the eu member countries. through this funding, the organization has reached on average about million people each year. echo maintains a staff of more than at its brussels headquarters and more than dispersed throughout field offices in countries worldwide. when a disaster strikes, and presumably upon request, echo staff deploy in order to conduct a needs assessment. if it is determined that assistance is warranted, staff will remain and monitor the situation as it progresses and oversee the implementation of the humanitarian aid projects that echo supports. echo has established relationships with more than other disaster management stakeholders, including un agencies and ngos. echo humanitarian assistance can come in several forms, including food aid; clothing; healthcare supplies; and materials for shelter, water, and sanitation. echo also supports relief work, such as infrastructure repair, removal of mines, psychological support, and education, among many others. echo has a special program, called the "forgotten crisis assessment," that focuses on less salient events. through this program, echo tries to raise the profile of serious incidents it finds are receiving too little attention among the humanitarian community, for the purpose of increasing the funds available to impacted victims. in , echo distributed humanitarian aid worth us$ . billion (which amounts to less than percent of the eu budget, yet is, in gross terms, a significant amount in total funding when compared to most other donors). this funding assisted million people in more than countries outside the european union. echo also oversees the eu civil protection mechanism, which comprised states ( eu member states, plus former yugoslav republic of macedonia, iceland, liechtenstein, and norway). this mechanism enables these nations to coordinate and cooperate in the event of a disaster in one or more eu countries or elsewhere in the world. civil protection agencies from member countries provide inkind assistance, equipment, and teams, or experts that perform damage and needs assessments. echo civil protection relies on the resources of member governments and, if assistance is required in non-eu countries, it typically works in parallel with the humanitarian aid component of echo. for european countries, the coordination and cooperation provided under echo is, in essence, a highly formalized mutual assistance compact that increases the capacity of all nations involved. nations pool their resources and maximize their collective efforts. the key instrument for european civil protection is the civil protection mechanism (cpm), which was established in . the operational heart of cpm is the european commission's monitoring and information centre (mic), which will soon become the european emergency response centre (erc). any country inside or outside eu affected by a disaster and overwhelmed by its magnitude can make an appeal for assistance through the mic/erc. to provide formalized mitigation and preparedness assistance, echo launched its disaster preparedness program, disaster preparedness echo (dipecho), in . dipecho attempts to reduce population vulnerability in disaster-prone regions. between and , dipecho provided more than $ million for hundreds of projects worldwide. dipecho-funded projects are implemented by aid agencies working in the region of concern, and support training, capacity building, awareness raising, and early warning projects, as well the organization of relief services. echo disaster preparedness efforts, however, extend beyond dipecho. many of echo's major humanitarian financing decisions, for example, include disaster preparedness or prevention as an objective. even post-disaster emergency responses can seek to reduce future risk. examples of echo risk-reduction activities include livestock shelters built after extreme cold snaps to protect against further herd depletion (peru), training and equipping of community-based fire brigades in forest fire risk zones (indonesia), cholera preparedness and health information (malawi), and antirust measures to prevent water pollution and protect pipes from the effects of volcanic ash (ecuador). the organization of american states (oas) was established in by nations located in north, central, and south america and the caribbean that wished to strengthen cooperation and advance their common interests in the western hemisphere. through the oas charter these nations committed to a set of common goals. respect for each other's sovereignty has always played a central role in oas affairs. today, all independent nations in the region have ratified the oas charter and serve as members of the organization (though the cuban government was excluded from participation in oas from to , and has yet to rejoin since the lifting of its ban). the oas is heavily involved in disaster risk reduction and preparedness efforts in the region. the vast majority of such projects are facilitated by the oas office for sustainable development and environment (osde), which supports activities in both individual countries and those that involve multiple countries. the more prominent of these activities focus on the following goals: • supporting the management of trans-boundary water resources • improving information for decision making in biological diversity • establishing land-tenure reform and property rights • supporting the exchange of best practices and technical information in environmental law and enforcement, renewable energy, water management, and biodiversity • improving management systems to reduce the impacts of natural disasters • understanding climate-related vulnerabilities affecting small island states the following is a list of projects that illustrates the range of disaster risk reduction and preparedness activities carried out by oas: mitigation capacity building program. the three-year program assisted countries in the caribbean region to develop comprehensive, national hazard vulnerability reduction policies and associated implementation programs, and develop and implement safer-building training and certificate programs. improvement program with assistance from oas to offer hurricane-resistant home improvement options to low-income families. this program trains local builders in safer construction, offers small loans to families wishing to upgrade their homes, and provides the services of a trained building inspector who approves materials to be purchased and checks minimum standards. in addition to the osde, oas supports disaster risk reduction through its inter-american committee for natural disaster reduction (iacndr). iacndr is the organization's main forum for integrating disaster risk reduction into sustainable development practices. the oas general assembly established the iacndr to strengthen its role in natural disaster reduction and emergency preparedness. the southern african development community (sadc) began in , when a loose alliance of nine southern african states formed (then known as the southern african development coordination conference, or sadcc). the organization's aim was to coordinate development projects to decrease economic dependence on south africa. in , it shifted from a "coordination conference" to a development community known as the sadc. sadc member states are angola, botswana, the democratic republic of congo, lesotho, madagascar, malawi, mauritius, mozambique, namibia, seychelles, south africa, swaziland, united republic of tanzania, zambia, and zimbabwe. sadc's primary mission is to help define regional priorities, facilitate integration, assist in mobilizing resources, and maximize regional development. it approaches problems and national priorities through regional cooperation and action. several sadc programs address the region's safety and security, primarily through risk-reduction mechanisms that include disaster preparedness and mitigation. the following are some examples of sadc disaster-related programs: • food, agriculture, and natural resources directorate • regional early warning unit • regional remote sensing unit the coordination center for natural disaster prevention in central america (cepredenac) was established in as a coordination center to strengthen the central american region's ability to reduce their population's vulnerability to natural disasters. in may , cepredenac became an official organization to foster regional cooperation among the governments of costa rica, el salvador, guatemala, honduras, nicaragua, and panama. the organization's headquarters are in guatemala city, guatemala. since its founding, the organization has been instrumental in securing region-wide commitment to disaster risk reduction through the passing of several resolutions and the creation of several plans and strategies signed by participating countries. the organization's agenda parallels and coordinates with other specialized regional entities in areas including hydrological resources, agriculture, nutrition, and food security. the cepredenac regional disaster reduction plan (prrd) was created to foster disaster reduction as an integral part of the sustainability of central american societies. its strategic objectives are: • promoting the incorporation of disaster risk reduction in legislation, policies • enhancing and developing greater resilience of the population to disaster risk • promoting the incorporation of disaster risk analysis in the design and implementation of prevention, mitigation, response, recovery, and reconstruction in the countries of the region a participating state may request disaster response assistance once its capabilities have been overwhelmed. cdema solicits and coordinates the assistance offered by other governments, organizations, and individuals, both within and outside the region. this is cdema's primary function. other functions include: • securing, collating, and channeling disaster information to interested governmental organizations and ngos as needed • mitigating disaster consequences affecting participating states • establishing and maintaining sustainable disaster response capabilities among participating states • mobilizing and coordinating disaster relief from governmental organizations and ngos for affected participating states the cdema participating states are structured into four subregions, each of which is headed by an operation unit known as a sub-regional focal point. the functions of each focal point relevant to the recovery effort are to: • acquire and maintain comprehensive emergency management capacity information • test and maintain communications with the coordinating unit and with national disaster management agencies • ensure subregion continuity of operations membership in cdema requires the participating state to establish or maintain a national disaster organization (ndo) or a national relief organization capable of responding swiftly, effectively, and in a coordinated manner to disasters in participating states (typically the government body tasked with domestic emergency management). ndos are headed by the national disaster coordinator (ndc), who is a government official responsible for the day-to-day management of the organization; ndos are the national focal points for cdera's activities in the participating state. the participating states are, in addition, required to: • establish planning groups and define national policies and priorities to address disasters • provide national relief organizations with adequate support, including named emergency coordinators, liaison officers with key ministries, emergency services, utilities, etc. • define the disaster role and functions of government agencies • establish and equip a suitable emergency operations center (eoc) • develop and maintain an appropriate emergency telecommunications system • perform disaster operations planning and associated drills and exercises • review and rationalize disaster-related statutory authorities • develop an emergency shelter policy program involving local participation • develop and implement a comprehensive disaster public awareness program • develop and implement appropriate training programs for disaster management staff in , twenty-four countries in eastern and southern africa established a drought monitoring centre, with its headquarters in nairobi (the dmcn) and a sub-center in harare (dmch), in response to a series of devastating weather-related disasters. in october , the heads of state and governments of the intergovernmental authority on development (igad) held their th summit in kampala, uganda, where dmcn was adopted as a specialized igad institution. the name of the institution was changed to igad climate prediction and applications centre (icpac) in order to better reflect its expanded mandates, mission, and objectives within the igad system. a protocol was signed in april , integrating the institution fully into igad. icpac is responsible for seven member countries (djibouti, eritrea, ethiopia, kenya, somalia, sudan, and uganda) and three other countries (burundi, rwanda, and tanzania). the centre's vision is "to become a viable regional centre of excellence in climate prediction and applications for climate risk management, environmental management, and sustainable development," while its mission is "provision of timely climate early warning information and support specific sector applications to enable the region to cope with various risks associated with extreme climate variability and change for poverty alleviation, environment management and sustainable development of the member countries" the objectives of the centre are: . to provide timely climate early warning information and support specific sector applications for the mitigation of the impacts of climate variability and change for poverty alleviation, management of environment, and sustainable development; . to improve the technical capacity of producers and users of climatic information, in order to enhance the use of climate monitoring and forecasting products in climate risk management and environment management; . to develop an improved, proactive, timely, broad-based system of information/product dissemination and feedback, at both sub-regional and national scales through national partners; . to expand climate knowledge base and applications within the sub-region in order to facilitate informed decision making on climate risk related issues; and . to maintain quality controlled databases and information systems required for risk/vulnerability assessment, mapping and general support to the national/ regional climate risk reduction strategies. (icpac n.d.) the centre has several functions relative to these objectives, which are: • acquisition of climate and remotely sensed data; • develop and archive national and regional climate databanks including calibration of remote sensing records; • process data and develop basic climatological statistics required for baseline risk scenarios and other applications; • monitor, predict, and provide early warning information of the space-time evolutions of weather and climate extremes over the sub-region; • hazards and climate risk mapping of the extreme climate events thresholds; • networking with wmo, the national meteorological and hydrological institutions as well as regional and international centers for data and information exchange; • capacity building in the generation and applications of climate information and products; • applications of climate tools for specific climate sensitive sector risk reduction, environment management , and sustainable development, including integration of indigenous knowledge; • monitor, assess, detect and attribute climate change and associated impacts, vulnerability, adaptation and mitigation options; • develop relevant tools required to address the regional climate challenges through research and applications in all climate sensitive socio-economic sectors including addressing linkages with other natural and man-made disasters; and • networking and exchange of information regarding disasters in the sub-region. (icpac n.d.) the centre offers a number of informational products, including periodic climate and weather bulletins, updates on climate and el niño, and annual climate summaries. to date, the centre has been instrumental in increasing drr in the sub-region through the provision of capacity enhancement, informational products, networking assistance, and more. the league of arab states (las) is a regional igo based in cairo, egypt and encompassing north africa and southwest asia. las was formed in following the adoption of the alexandria protocol, with a stated goal to "draw closer the relations between member states and co-ordinate collaboration between them, to safeguard their independence and sovereignty, and to consider in a general way the affairs and interests of the arab countries." member states include algeria, bahrain, comoros, djibouti, egypt, iraq, jordan, kuwait, lebanon, libya, mauritania, morocco, oman, state of palestine, qatar, saudi arabia, somalia, sudan, syria, tunisia, united arab emirates, and yemen. in response, and as a follow-up to the first arab summit on socio-economic development, the council of arab ministers responsible for the environment adopted specific actions relating to disaster risk reduction through a decision in may of to develop an arab strategy for disaster risk reduction. this strategy, entitled the arab strategy for disaster risk reduction , adopted in december of , has a two-fold purpose: . to outline a vision, strategic priorities, and core areas of implementation for disaster risk reduction in the arab region, and . to enhance institutional and coordination mechanisms and monitoring arrangements to support the implementation of the strategy at the regional, national, and local level through preparation of a programme of action. the arab strategy for disaster risk reduction is designed to complement existing and ongoing efforts in disaster risk reduction by national institutions and regional technical organizations in the las region. implementing partners of the strategy are to focus on multi-sectorial approaches with the purpose of reducing emerging risks across the arab region by , in line with the global priorities outlined by the hyogo framework for action (hfa) and the millennium development goals. the five priorities of the las strategy directly mirror those of the hfa, including the desire to increase nations' capacity to incorporate drr into disaster recovery. specific commitments detailed under these priorities, which pertain to recovery planning actions in the region, include: • ensuring that disaster risk reduction measures are integrated into post-disaster recovery and rehabilitation processes • establishing disaster preparedness plans, contingency plans, and recovery and reconstruction plans at all administrative levels with the participation of women, the aged, children, idps, and people with special needs • ensuring that national/ local financial reserves and contingency mechanisms are in place and well understood by all stakeholders to ensure effective response and recovery when required • addressing national trans-boundary cooperation on disaster response, preparedness and recovery among arab states in the arab region, funding remains the main challenge faced by national and local authorities, civil society organizations, and humanitarian workers implementing disaster risk reduction measures targeting communities at risk. las encourages its members to dedicate at least percent of national development funding and development assistance toward disaster risk reduction measures. specifically, it was recommended that member states assess the possibility of utilizing existing regional funds and mechanisms (including, among other mechanisms, socio-economic development funds and national disaster relief and response budgets) by allocating a dedicated budget for disaster risk reduction and recovery activities at the subregional, national, or local level. the las regional centre for disaster risk reduction (rcdrr) was established in by a partnership between the kingdom of saudi arabia, the united nations international strategy for disaster reduction (unisdr), and the arab academy for science, technology and maritime transport (aas-tmt), as an intergovernmental organization of the league of arab states targeting the achievement of sustainable development in the arab region. the centre seeks to address risk through knowledge, research, and training of scientific and technical cadres in various disciplines on drr. the main objectives of rcdrr, as per the rcdrr statutes, are: • integration of drr into regional and national sustainable development policies, strategies, and plans • enhancing regional and national capacities in the field of drr research, education, and training • contributing to the development and harmonization of regional drr methodologies and tools, including database and guidelines • promoting partnership building with a multi-stakeholder approach to accelerate the implementation of the hyogo framework of action the south asian association of regional cooperation (saarc) was officially established in . the objectives of the organization are to: • promote the welfare of the people of south asia and to improve their quality of life • accelerate economic growth, social progress, and cultural development in the region and to provide all individuals the opportunity to live in dignity and to realize their full potential • promote and strengthen selective self-reliance among the countries of south asia • contribute to mutual trust, understanding, and appreciation of one another's problems • promote active collaboration and mutual assistance in the economic, social, cultural, technical, and scientific fields • strengthen cooperation with other developing countries • strengthen cooperation among themselves in international forums on matters of common interest • cooperate with international and regional organizations with similar aims and purposes. the saarc member countries include afghanistan, bangladesh, bhutan, india, maldives, nepal, pakistan, and sri lanka. after • establish and strengthen the regional disaster management system to reduce risks and to improve response and recovery management at all levels • identify and elaborate country and regional priorities for action • share best practices and lessons learnt from disaster risk reduction efforts at national levels • establish a regional system to develop and implement regional programs and projects for early warning • establish a regional system of exchanging information on prevention, preparedness, and management of natural disasters • create a regional response mechanism dedicated to disaster preparedness, emergency relief, and rehabilitation to ensure immediate response • create a regional mechanism to facilitate monitoring and evaluation of achievements toward goals and strategies. the saarc disaster management centre (sdmc) was established in october of at the facilities of the national institute of disaster management in new delhi to serve as a center of excellence for knowledge, research, and capacity building in disaster management. the centre has the mandate to serve the saarc member countries by providing policy advice and facilitating capacity building services, including strategic learning, research, training, system development, and exchange of information for effective disaster risk reduction and management in south asia. sdmc conducts studies and research, organizes workshops and training programs, publishes its reports and documents, and provides various policy advisory services to the member countries. the secretariat of the pacific community (spc) was founded in australia in under the canberra agreement to restore order in the region following world war ii. in , the spc applied geoscience and technology division (sopac) was created as a undp regional project, and in it became an independent igo. in , sopac became a new division under spc, dedicated to promoting sustainable development in its member countries, and its work is carried out through its secretariat based in suva, fiji. sopac members include australia, cook islands, fiji islands, guam, federated states of micronesia, kiribati, marshall islands, new zealand, papua new guinea, samoa, solomon islands, tonga, tuvalu and vanuatu, niue, nauru, and palau. associate members (local administrations of nonself-governing territories) include american samoa, french polynesia, new caledonia, and tokelau. the purpose of sopac is to ensure the earth sciences (inclusive of geology, geophysics, oceanography, and hydrology) are utilized fully in the fulfillment of the spc mission. to fulfill this purpose, the division has three technical work programs: • ocean and islands • water and sanitation • disaster reduction these three programs share common technical support services: the sopac disaster reduction programme (drp) provides technical and policy advice and support to strengthen disaster risk management practices in pacific island countries and territories. the program carries out this responsibility in coordination and collaboration with other technical program areas within sopac and also with a range of regional and international development partners and donors. the overarching policy guidance for drp is the hfa-linked pacific disaster risk reduction and disaster management framework for action - (pacific drr and dm framework for action), which supports and advocates for the building of safer and more resilient communities. the other significant regional policy instruments that help to guide the efforts of the drp are the pacific plan and the pacific islands framework for action on climate change - . the sopac disaster risk management policy and planning team (ppt) is responsible for the drm mainstreaming initiative, which sopac spearheads on behalf of the pacific disaster risk management partnership network. in fulfilling this responsibility, the ppt provides the following services to pacific island countries and territories (picts): • leads and coordinates high level advocacy at cabinet/political level to garner support for drm mainstreaming in national, sectorial, local, and community planning and budgetary processes • leads and coordinates the development and implementation of drm national action plans with the support of other members of the pacific drm partnership network • supports the integration of drm and climate change adaptation initiatives at the national level within picts • analyzes budgeted drm investment in annual appropriations of picts • analyzes the economic impact of disaster events • analyzes the cost-benefit of drm measures a major focus of the ppt is to build member country resilience by facilitating the creation of disaster risk management national action plans (naps). the partnership network continued to provide strong support in terms of the realization of drm initiatives linked to nap exercises and also for other risk reduction and disaster management-related activities. in the past several years, this support has shifted to development of joint national action plans (jnaps) that integrate policy on disaster risk reduction and climate change adaptation. several of the countries in the region have established jnaps at the national level. the region is also moving toward a regional-level integrated joint strategy for disaster risk reduction and climate change. at present, regional-level disaster risk reduction and climate change adaptation policies remain separate. sopac led the development of the pacific disaster risk reduction and disaster management framework for action (rfa), signed in , and the secretariat of the pacific community environmental programme (sprep) led the development of the pacific islands framework for action on climate change (pifacc), also signed in . however, these organizations initiated an effort in to establish a more integrated solution to coincide with the year expiration of both frameworks. an ongoing process named "the roadmap" is marked by wide stakeholder involvement via a steering committee and broad technical support provided by a technical working group (which includes spc/sopac, sprep, and unisdr). the roadmap process has to date resulted in a draft-integrated strategy entitled the strategy for disaster and climate resilient development in the pacific (srdp). the draft strategy is designed to promote action that is harmonized with existing member state institutional arrangements for climate change adaptation and disaster risk reduction "[to] ensure that efforts are nested within the context of countries' national development strategies and reflected in their budgets, encourage the participation of multiple stakeholder groups, strengthen countries' capacities for risk governance and support the development of well-coordinated innovative funding mechanisms" (spc ). drp supports the strengthening of disaster management governance, which has included the development of institutional, policy, and decision-making processes such as disaster management legislative and planning frameworks, and national focal points (ndmos) and guidelines or models of good practice for national application. the emergency management preparedness, response, and coordination capabilities within countries will be critically assessed to determine the level of resources and capacity that is available to protect vulnerable communities. a priority will be to ensure that effective emergency response, communication, and coordination processes are established, and that existing resources are utilized in the most effective way. the drp disaster management team provides the following services to picts: • technical advice and support to review and update national drm governance arrangements and legislation, operational plans and procedures • support for the design and conduct of operational and table-top exercises to test emergency response plans and procedures • support for the conduct of disaster risk management training in collaboration with the pacific drm program of the asia foundation/office of us foreign disaster assistance • design and development of professional training courses in collaboration with taf/ofda and the fiji national university in , sopac established the pacific disaster risk management partnership network to provide a collaborative and cooperative mechanism to support disaster risk management capacity building in the region and help pacific island countries and territories adapt and implement the pacific drr and dm framework for action. the partnership is an "open-ended, voluntary" membership of international, regional, and national government and non-government organizations, with comparative advantages and interests in supporting pacific countries toward mainstreaming drm through addressing their disaster risk reduction and disaster management priorities. the members of the partnership network agree that: • disaster risk reduction and disaster management are sustainable development issues within the broader context of economic growth and good governance; • national governments have a critical role in developing disaster risk reduction and disaster management national programs and plans that reflect the needs of all stakeholders in a whole-ofcountry approach; • a regional effort must be responsive to and support and complement national programs and plans to strengthen resilience to disasters; • as regional partners, we commit to coordinating our activities and to work cooperatively and collaboratively under the guidance of the pacific plan and regional framework for action - ; and • we can build safer and more resilient nations and communities to disasters if we work in unison and accept this disaster risk management charter as a basis for future action. international financial institutions (ifis) provide loans for development and financial cooperation throughout the world. they exist to ensure financial and market stability and to increase political balance. these institutions are made up of member states arranged on a global or regional basis that work together to provide financial services to national governments through direct loans or projects. in a disaster's aftermath, nations with low capital reserves often request increased or additional emergency loans to fund the expensive task of reconstruction and rehabilitation. without ifis, most developing nations would not have the means to recover. several of the largest ifis are detailed in the following section, including the world bank; one of its subsidiaries, the international monetary fund (imf); the asian development bank; and the inter-american development bank. the world bank was created in to rebuild europe after world war ii. in , france received the first world bank loan of $ million for post-war reconstruction. financial reconstruction assistance has been provided regularly since that time in response to countless natural disasters and humanitarian emergencies. today, the world bank is one of the largest sources of development assistance. in the fiscal year, it provided more than $ . billion in loans, breaking all previous lending records for the organization. in fiscal year , the amount of loans had fallen to $ . billion, but the bank remains one of the largest development lenders. the world bank is owned collectively by countries and is based in washington, dc. it comprises several institutions referred to as the world bank group (wbg): • international bank for reconstruction and development • international development association the world bank's overall goal is to reduce poverty, specifically to "individually help each developing country onto a path of stable, sustainable, and equitable growth, [focusing on] helping the poorest people and the poorest countries" (wagstaff ) . as disasters and ches take a greater and greater toll on the economic stability of many financially struggling countries, the world bank is taking on a more central role in mitigation and reconstruction. developing nations, which are more likely to have weak disaster mitigation or preparedness capacity and therefore little or no affordable access to disaster insurance, often sustain a total financial loss. in the period of rehabilitation that follows the disaster, loans are essential to the success of programs and vital to any level of sustainability or increased disaster resistance. the world bank lends assistance at several points along this cycle. for regular financial assistance, the world bank ensures that borrowed funds are applied to projects that give mitigation a central role during the planning phase. it utilizes its privilege as financial advisor to guide planners, who otherwise might forego mitigation measures in an effort to stretch the loaned capital as far as possible. ensuring that mitigation is addressed increases systems of prediction and risk analysis in projects funded by the world bank. once a disaster occurs, the world bank may be called on for help. because it is not a relief agency, it will not take on any role in the initial response; however, it works to restore damaged and destroyed infrastructure and restart production capabilities. (see exhibit . .) a world bank team may assist with initial impact assessments that estimate financial losses resulting from the disaster and estimated costs of reconstruction, including raised mitigation standards. the world bank also could restructure the country's existing loan portfolio to allow for expanded recovery projects. in addition, world bank projects that have not yet been approved but are in the application process can be redesigned to account for changes caused by the disaster. finally, an emergency recovery loan (erl) can be granted to specifically address recovery and reconstruction issues. erls restore affected economic and social institutions and reconstruct physical assets such as essential infrastructure. it is important to note that erls are not designed for relief activities. they are most appropriate for disasters that adversely impact an economy, are infrequent (recurrent disasters are accommodated by regular lending programs), and create urgent needs. erls are expected to eventually produce economic benefits to the borrowing government; they are usually implemented within three years and are flexible to accommodate the specific needs of each unique scenario. construction performed with erls must use disaster-resistant standards and include appropriate mitigation measures, thus providing overall preparedness for the country affected. once an erl has been granted, the world bank coordinates with the imf, the undp, ngos, and several other international and local agencies to create a strategy that best utilizes these funds within the overall reconstruction effort. the two lending arms of the world bank are the international bank for reconstruction and development and the international development association. international bank for reconstruction and development (ibrd) . established in , the ibrd reduces poverty in middle-income and creditworthy poorer countries. the ibrd attempts to promote sustainable development activities through its loans. it also provides guarantees and other analytical and advisory services. following disasters, countries with strong enough credit can borrow or refinance their existing loans from the ibrd to pay the often staggering costs of reconstruction. international development association (ida). the ida lends to the world's poorest countries, classified as those with a income of less than $ , per person. sixty-four countries currently are eligible to borrow from the ida. it provides interest-free loans and grants for programs aimed at boosting economic growth and improving living conditions. this need is almost always present in the aftermath of disasters, including those caused by violent conflict. in , the global facility for disaster risk reduction, or gfdrr, was created, with the world bank designated as facility manager on behalf of the countries and eight international organizations that make up its membership. gfdrr has a secretariat, based in the washington dc world bank headquarters, which carries out its day-to-day operations. the purpose of gfdrr is to help developing countries address disaster vulnerability and vulnerability to the effects of climate change. its work is primarily driven by the hyogo framework for action, and its programs focus on mainstreaming exhibit . world bank disaster assistance to bosnia and herzegovina washington, june , - the world bank group's board of executive directors today approved a us$ million credit for the floods emergency recovery project for bosnia and herzegovina (bih), to meet critical needs and restore the functionality of infrastructure essential for public services and economic recovery in affected areas in the aftermath of the worst flooding to hit the country in documented history. the project was prepared in record time in view of the dire situation in the country and will be financed from the international development association's (ida) crisis response window resources. this project will target areas that were hit hardest by the devastating floods. preliminary evidence shows that the largest impact from this disaster was on livelihoods, housing, transport, agriculture, and energy. given the magnitude of the damage caused by flooding and subsequent landslides, the project is designed to support efforts by local and entity governments to quickly re-establish public services to pre-flood levels. the project will also support the government's on-going economic recovery initiatives, in particular in the agriculture sector. in addition to this project, the world bank is working on several other fronts to ensure the provision of a comprehensive package of support for bih as it recovers and rebuilds from the physical and economic devastation. notably, the bank is participating in a systematic recovery needs assessment, led by the bosnia and herzegovina (bih) authorities and supported also by the european union and the united nations. the assessment will provide a basis for developing effective rehabilitation measures for infrastructure and services in the affected areas. the world bank is also considering the restructuring of existing projects in its bih portfolio to meet reconstruction needs. while immediate recovery needs are the top priority of this project, the world bank also stands ready to work with the bih authorities to scale-up flood protection and implement early warning systems. the recently approved drina flood protection project is a good example of the type of work that could be scaled-up, as it addresses the need to prevent future flooding. as emphasized by laura tuck, world bank vice president for europe and central asia, "the floods emergency recovery project will finance critical goods, such as fuel and electricity imports, as well as the reconstruction of local infrastructure. this immediate response, combined with the drina river flood protection project, will support economic recovery in the affected areas, and will help restore bosnia and herzegovina to a growth path following the floods." the world bank portfolio of active projects in bih now includes operations totaling approximately us$ . million. areas of support include agriculture, environment, energy efficiency, health, social safety and employment, local infrastructure, and private sector development. release, . disaster risk reduction and climate change adaptation throughout all government sectors in member countries. gfdrr organizes its efforts according to three "business lines," which include: • track i: global and regional partnerships -track i supports unisdr in helping countries to leverage resources to perform pre-disaster investments and activities related to prevention, disaster risk reduction, and disaster preparedness. the key objectives of track i are to: ) enhance global and regional advocacy, strategic partnerships, and knowledge management for mainstreaming disaster risk reduction; and ) promote the standardization and harmonization of hazard risk management tools, methodologies, and practices. • track ii: mainstreaming disaster risk reduction in development -track ii provides pre-disaster assistance to developing countries to mainstream and expand disaster risk reduction and climate change adaptation activities. work in this track is performed in conjunction with world bank regional teams, un agencies, and national governments, and is aimed at integrating disaster risk reduction into poverty reduction and development efforts. there are also several sub-programs that include risk assessment, risk reduction, risk financing, and climate change adaptation. • track iii: sustainable recovery -track iii is aimed at early post-disaster recovery in low-income countries through its standby recovery financing facility (srff). track iii is less programmatic than track i and track ii because it is deployed for post-disaster situations, but it does work to build national capacity and facilitate knowledge management with the long term in mind. srff support includes two financing windows: ) the technical assistance (ta) fund, which supports damage, loss, and needs assessments and develops national capacity for recovery planning and implementation; and ) the callable fund for accelerated recovery, which provides speedy access to financial resources for disaster recovery and reconstruction. the international monetary fund (imf) was established in to "promote international monetary cooperation, exchange stability and orderly exchange arrangements; to foster economic growth and high levels of employment; and to provide temporary financial assistance to countries to help ease balance of payments adjustment." it carries out these functions through loans, monitoring, and technical assistance. since , the imf has provided emergency assistance to its member countries after they were struck by natural disasters, and, in a great many cases, when affected by complex emergencies. the assistance provided by the imf is designed to meet the country's immediate foreign-exchange financing needs, which often arise because earnings from exports fall while the need for imports increases (among other causes). imf assistance also helps the affected countries avoid serious depletion of their external reserves. in , the imf began to provide this type of emergency assistance to countries facing post-conflict scenarios in order to enable them to reestablish macroeconomic stability and to provide a foundation for recovery, namely in the form of long-term sustainable growth. this type of assistance is particularly important when a country must cover costs associated with an "urgent balance of payments need, but is unable to develop and implement a comprehensive economic program because its capacity has been damaged by a conflict, but where sufficient capacity for planning and policy implementation nevertheless exists" (imf ) . the imf maintains that their support must be part of a comprehensive international effort to address the aftermath of a conflict in order to be effective. its emergency financing is provided to assist the affected country and to gather support from other sources. it is not uncommon for a country to severely exhaust its monetary reserves in response to an emergency situation. in the event of a natural disaster, funding is directed toward local recovery efforts and any needed economic adjustments. the imf lends assistance only if a stable governing body is in place that has the capacity for planning and policy implementation and can ensure the safety of imf resources. after stability has been sufficiently restored, increased financial assistance is offered, which is used to develop the country in its post-emergency status. when a country requests emergency assistance, it must submit a detailed plan for economic reconstruction that will not create trade restrictions or "intensify exchange." if the country is already working under an imf loan, assistance may be in the form of a reorganization of the existing arrangement. it can also request emergency assistance under the rapid financing instrument (rfi). the rapid financing instrument (rfi) is the vehicle that the imf uses to meet disaster-impacted countries' financing needs. the rfi provides funding quickly and with few requirements in instances where it is determined that a disaster or emergency situation has resulted in urgent balance-of-payments needs. emergencies need not be related to a natural or technological hazard-they can also be the result of rapid increases in the price of certain commodities or because of an economic crisis. unlike other imf assistance, there does not need to be a full-fledged financing program in place. prior to the creation of the rfi, the imf used a number of separate programs to address emergency needs, including the emergency natural disaster assistance (enda) program and the emergency post-conflict assistance (epca) program. the creation of the rfi program combines all emergency needs. rfi financial assistance is provided in the form of outright purchases without the need for a full-fledged program or reviews. however, when a country does request assistance under rfi, they must cooperate with the imf to make every effort to solve their balance-of-payment problems, and must explain the economic policies it proposes to follow to do so. the imf makes the rfi program available to all of its members, though oftentimes very poor countries are more likely to seek assistance under a different program called the rapid credit facility (rcf), which provides similar assistance but has economic-based requirements that many wealthier countries cannot meet. funds access under the rfi program is limited to percent of a nation's quota per year and percent of quota on a cumulative basis. under the rcf program, the access limits are percent of a nation's quota per year and percent of quota on a cumulative basis. the level of access in each case depends on the country's balance-of-payments need. financial assistance provided under the rfi is subject to many of the same financing terms that nations would see in other imf programs, and the funds borrowed are ideally paid back within to months (imf ). in certain cases, as decided by the imf and according to specific criteria, recipients of emergency funding may benefit from the imf poverty reduction and growth facility (prgf). the prgf is the imf's low-interest lending facility for low-income countries. prgf-supported programs are underpinned by comprehensive country-owned poverty reduction strategies. under this program, the interest rate on loans is subsidized to . percent per year, with the interest subsidies financed by grant contributions from bilateral donors. this program has been available for post-conflict emergencies since , but in january , following the south asia tsunami events, the imf executive board agreed to provide a similar subsidization of emergency assistance for natural disasters upon request. the government of a country devastated by disaster often requires technical assistance or policy advice because it has no experience or expertise in this situation. this is especially common in post-conflict situations, where a newly elected or appointed government has been established and officials are rebuilding from the ground up. the imf offers technical assistance in these cases to aid these countries in building their capacity to implement macroeconomic policy. this can include tax and government expenditure capacity; the reorganization of fiscal, monetary, and exchange institutions; and guidance in the use of aid resources. the asian development bank (adb) is a multilateral development financial institution whose primary mission is reducing poverty in asia and the pacific. adb was established in by countries from both within and outside the region, and has grown to include members as of . forty-eight are from the region and are from other regions. its clients are the member governments, who are also the adb's shareholders. the adb provides emergency rehabilitation loans to its member countries following disasters. adb determined that its assistance in this critical phase of recovery would allow an affected developing country to maintain its development momentum. bank analysts found that, without such assistance, the affected country may reallocate its scarce budgetary resources away from development issues to cover disaster-related expenses, sidetracking development progress. additionally, they found that the production of goods and services would quickly suffer or fail completely if the country could not perform adequate rehabilitation following a disaster. adb assistance in emergencies began in , but was initially extended only to smaller developing countries (e.g., the maldives, papua new guinea, and the smaller pacific island states). loans were limited to $ , (increased to $ million in ), with funded projects to be completed within months of disbursement. the funding was designed to address only simple repair and rehabilitation activities as needed in the immediate aftermath of a disaster, with more comprehensive repair being covered by regular bank lending programs. lending was designed to be provided within six weeks of being requested. in , emergency lending was extended to all developing member countries regardless of their size. this change included a fundamental shift in what the emergency loans would cover, from simple repairs to more comprehensive, informed rehabilitation activities. most important, adb wanted to ensure that projects funded by its loans reduced overall risk to the affected nation and its population. other major changes in adb emergency lending policy are included in the following list: • introducing a typology of the causes and effects of disasters • more clearly defining the adb's response during various phases of post-disaster situations • identifying the nature, focus, and coverage of rehabilitation projects • introducing detailed, yet simplified, guidelines for processing rehabilitation projects • targeting rehabilitation loans toward restoring infrastructure and production activities, including capacity building and modernization • mandating that risk analysis and disaster prevention measures be included in all adb projects in disaster-prone developing member countries • closely coordinating disaster responses at all levels (local, national, and international) with those of other external funding agencies, ngos, and community groups • specifying that disaster prevention and mitigation activities were to be promoted along with regional cooperation • including non-natural disasters, for example, wars, civil strife, and environmental degradation (adb ) between and , adb provided $ . billion to disaster-affected countries in the form of loans at a rate of approximately one loan per month. the vast majority of the adb emergency loan services during this period were provided in response to natural disaster events, with the remaining dedicated to post-conflict situations. these loans rarely averaged more than percent of the total annual lending by adb and were concentrated primarily in south asia. the project comprises two components: (i) reconstruction and upgrading of damaged roads and bridges in sichuan and shaanxi provinces, and (ii) reconstruction and improvements of damaged schools in shaanxi province. the project will rehabilitate and reconstruct high-priority earthquake-damaged roads in the worst affected counties of sichuan province and subprojects in the four worst affected counties of shaanxi province. the project will rehabilitate and reconstruct highpriority earthquake-damaged education facilities in the three worst affected counties in shaanxi province. these components are designed to be mutually supporting in achieving the overall objective of restoring the affected communities' access to infrastructure to pre-earthquake levels, and ensuring restored infrastructure is in strict compliance with the latest seismic code. based on the government's damage and needs assessment and the request of the prc government, the project identifies specific sectors that require emergency assistance in two of the worst earthquake-affected provinces (i.e., sichuan and shaanxi). the project seeks to (i) build on the immediate relief provided by the government in the earthquake-affected provinces; (ii) contribute to coordinated rehabilitation and reconstruction by different development partners and the government; and (iii) specifically address sustainable recovery priorities by providing indirect livelihood support through public infrastructure rehabilitation and reconstruction, which generates public employment and underpins the restoration of livelihood activities by rehabilitating roads, bridges, and schools. the project design draws on the adb experience in delivering emergency assistance acquired in different developing member countries over the past two decades, and complements relief and other rehabilitation and reconstruction assistance provided by the government, united nations agencies, ngos, bilateral development partners, and the world bank. by meeting the earthquake reconstruction needs of the next three years, the project is consistent with adb's disaster and emergency assistance policy ( ) . the project supports the state overall plan for post-wenchuan earthquake restoration and reconstruction approved by the government on september . the impact of the project is accelerated restoration of education and transport infrastructure in earthquake-affected areas of sichuan and shaanxi provinces. the project will support the government's efforts to (i) restore the livelihoods and economic activities of the affected population; (ii) accelerate poverty alleviation in the earthquake-affected counties, many of which have a high incidence of poverty; and (iii) rehabilitate and reconstruct public and community-based infrastructure that is vulnerable to natural disasters. the outcome of the project is restoration of people's access to transport and education infrastructure to preearthquake levels in counties of sichuan and four counties of shaanxi provinces. the total project cost is estimated at $ . million equivalent. a loan of $ million from adb's ordinary capital resources will be provided under adb's london interbank offered rate (libor)-based lending facility. the loan will have a grace period of years with a maturity period of years, an interest rate determined in accordance with adb's libor-based lending facility, a commitment charge of . % per annum, and such other terms and conditions set forth in the draft loan and project agreements. until june , . adb also provides mitigation-related project loans and regional technical assistance (reta) aimed at reducing member countries' overall disaster vulnerability. between august and december , adb approved $ . billion for more than disaster risk management-related projects (in addition to the $ . billion provided in disaster-related financing). mitigation and preparedness projects are not considered "emergency" in nature and are therefore funded through the bank's regular lending activities. because mitigation and preparedness activities are most often included as components within larger development projects, adb does not maintain records of its total financial risk reduction-based lending. projects may include resilience-increasing activities such as reforestation, watershed management, coastal protection, agricultural diversification, slope stabilization, and land-use planning, although the project's overall goal is more development oriented. reta and single-country technical assistance activities have included hazard management and disaster preparedness software programs and infrastructure protection assistance. adb has december , . as the project is for emergency assistance, implementation will start immediately after approval and be completed within months. sichuan provincial communications department in sichuan province; and hanzhong city government and baoji city government in shaanxi province implementing agencies sichuan highway administration bureau in sichuan province; and county-level highway administration bureaus for roads and bridges, and county-level education bureaus for schools in shaanxi province. the project will bring benefits to the project area by (i) reconstructing and improving road conditions and accessibility in townships and in villages in the sichuan and shaanxi provinces, (ii) reconstructing and improving schools in shaanxi province, and (iii) creating local employment opportunities from project construction and related activities. the project will provide equal benefits to females and males. the economic benefits of the rural roads and bridges include (i) savings in vehicle operating costs as a result of improved traffic and road conditions, (ii) time-savings for rural road users, (iii) savings in road accident costs as a result of fewer accidents, and (iv) economic benefits from generated traffic. the reconstruction and upgrading of rural roads in sichuan and shaanxi provinces will benefit about . million people, three-quarters of whom are rural and one-third of whom are poor. as reliable transport to markets becomes more readily available, cash crop farming in remote or isolated areas will be stimulated and access to off-farm employment opportunities will be broadened. the project will focus on reconstruction of and improvements to model schools to appropriate design standards, including six junior secondary and six primary schools. this will bring immediate benefits to the schools' , students (including more than female students), and long-term benefits to future students drawn from the , residents of the areas serviced by the schools, about % of whom are from rural areas of remote counties. the project will contribute to the government's efforts to rebuild the economy, rehabilitate public infrastructure and utilities, reinstate seismic code compliance, and generate employment. the rehabilitation and reconstruction of damaged schools will enable education services to be restored and will offer long-term benefits for affected persons by supporting opportunities for employment and participation in economic activities. finally, adb assists countries in restarting rehabilitation and overall development in the aftermath of armed conflict. in the past, adb post-conflict intervention focused almost exclusively on infrastructure rehabilitation, an area in which the adb has extensive experience. its focus in this area began to shift in the s to preventing conflicts and helping post-conflict countries move along a solid path of economic and social development. adb is now committed to assisting affected member countries develop mechanisms to effectively manage conflict, including addressing the problems of poor governance and corruption. in , adb established the asia pacific disaster response fund (apdrf) to provide quick funding in the aftermath of a disaster to help governments meet urgent life-saving disaster-response needs. between and , grants were approved under the fund. apdrf assistance is provided as a grant that may be no larger than us$ million per event. the size of the grant is determined by: . the geographical extent of the disaster's damage; . initial estimates of fatalities, injuries, and displaced persons; . the country's disaster response capacity; and . the date and magnitude of the last disaster to have impacted the country (thereby taking into account the cumulative effect of disasters on the country's ability to respond) (adb ). in , adb approved a pilot asian development fund disaster response facility for countries eligible for low-interest loans in the event of a disaster. the pilot program, which runs from to , is being conducted to strengthen adb's ability to respond to disaster-impacted member countries in a manner that is less ad hoc. the drf will require countries that are eligible to borrow from the asian development fund (adf countries) to contribute a small fraction of their allocations for the benefit of accessing the drf in case of a disaster. the drf will be available to these countries in the case of natural disasters, and will support relief, response, recovery, and reconstruction needs. per the pilot program, the size of the drf will be percent of the total performance-based allocation (pba) received. in case of a disaster, an adf country can get up to percent of its annual pba, or us$ million per disaster, whichever is higher, from the drf. a blend country, which is a country eligible for both the adf and adb ordinary capital resources, can receive up to percent of its annual pba from the drf if affected by a disaster. established in december , the inter-american development bank (iadb) is the oldest and largest regional multilateral development institution. it was first created to help accelerate economic and social development in latin america and the caribbean. the iadb has been a pioneer in supporting social programs; developing economic, social, educational, and health institutions; promoting regional integration; and providing direct support to the private sector, including microenterprises. the iadb addresses disaster and risk management through its sustainable development department. through the efforts and actions of this department and its disaster risk management policy, the iadb addresses the root causes of the region's high vulnerability to disasters. building on its mandate to promote sustainable development in latin america and the caribbean, the iadb works with countries to integrate risk reduction into their development practice, planning, and investment, and to increase their capacity to manage risk reduction. it also provides funding that directly or indirectly supports disaster mitigation and preparedness. in their "plan of action: facing the challenge of natural disasters in latin america and the caribbean," the iadb outline their six strategic areas of assistance: . national systems for disaster prevention and response: building national legal and regulatory frameworks and programs that bring together the planning agencies, local governments, and civil society organizations; developing national strategies for risk reduction; and assessing intersectoral priorities, backed by separate budgets. . a culture of prevention: developing and disseminating risk information and empowering citizens and other stakeholders to take risk-reduction measures. . reducing the vulnerability of the poor: supporting poor households and communities in reducing their vulnerability to natural hazards and recovering from disasters through reconstruction assistance. . involving the private sector: creating conditions for the development of insurance markets, encouraging the use of other risk-spreading financial instruments where appropriate, and designing economic and regulatory incentives for risk reduction behavior. . risk information for decision-making: evaluating existing risk assessment methodologies; developing indicators of vulnerability, and stimulating the production and wide dissemination of risk information. . fostering leadership and cooperation in the region: stimulate coordinated actions and to mobilize regional resources for investments in risk mitigation. (iadb ) the iadb created two mechanisms to allow for rapid loan disbursement in times of disaster: the disaster prevention sector facility and the facility for the immediate response to natural and unexpected disasters (formerly the immediate response facility). in the iadb established the natural disaster network, represented by each of its borrowing member countries. network members meet annually to discuss topics related to disaster management, such as "national systems for risk management" ( ) the iadb revised its disaster risk management policy in . the new policy is designed to improve the iadb's ability to assist member countries in reaching their development goals by supporting their disaster risk management efforts. (see appendix . for the full text of the iadb disaster risk management policy guidelines.) . iadb supports disaster risk reduction through the disaster prevention sector facility, which provides up to $ million to assist countries in taking an integrated approach to reducing and managing their risk. the iadb also provides loans to help countries cope with financial or economic crises and natural or other disasters through its emergency lending program. in the case of a financial or economic crisis, the iadb requires that the emergency loan fits within an imf-approved and monitored macroeconomic stabilization program. emergency loan disbursement periods are much shorter than other non-disaster loans, ranging up to months in duration. they may be used to support national, provincial, state, and municipal governments and autonomous public institutions. they have a five-year term and a three-year grace period. in the case of natural or other disasters, the emergency lending program is known as the emergency projects, in order to improve project viability. whenever significant risks due to natural hazards are identified in project preparation, appropriate measures will be taken to secure the viability of the project, including the protection of populations and investments affected by bank-financed activities. the bank has nonreimbursable resources that may be used to cover the transaction costs incurred with the implementation of these guidelines. . . these guidelines will also recommend ways to evaluate the benefits and opportunity costs of loan reformulations and give guidance on how to ensure adequate transparency and effective monitoring, auditing, and reporting on the use of redirected funds. in addition, the guidelines describe precautions to be taken to avoid rebuilding or increasing vulnerability during rehabilitation and reconstruction. . . the guidelines are designed to be flexible in their application to the various situations that borrowing member countries and the bank may experience, in the face of natural hazards and disasters affecting their development prospects and performance. . . the present guidelines apply to all natural hazards, including the hydrometeorological hazards-windstorms, floods, and droughts-that are associated with both the existing climate variability and the expected change in long-term climate conditions. of note for risk assessments, climate change is expected to change some countries' disaster risk (their probable losses) by changing the characteristics of the hydrometeorological hazards. . although uncertainty persists, recent advances in downsizing climate models are allowing disaster managers to better calibrate their risk assessments to understand potential impacts due to climate change at the subnational level. tools for identifying such climate risk at the country and project levels, and measures for mitigating these increased risks to bank investments (climate change adaptation) will be developed under pillar of the bank's sustainable energy and climate change initiative (secci) action plan. purpose and scope . . the purpose of this section is to provide guidance to bank teams on the implementation of directive a- of the disaster risk management policy, particularly for countries classified as having high disaster risk, as well as for those sectors that are associated with a high vulnerability to natural disasters and in which the bank has identified opportunities for financing. in accordance with this policy, the bank will encourage countries to include proactive drm in programming activities in those countries, as indicated in directive a- of the policy: . . a- . programming dialog with borrowing member countries. the bank will seek to include the discussion on proactive disaster risk management in the dialog agenda with borrowing member countries. the bank will give due consideration to vulnerability associated with natural hazards and risk management in relation to the priority areas of intervention discussed and agreed with the borrowers for the development of country and regional strategies, and operational programs. the bank will identify countries according to their level of exposure to natural hazards based on existing indicators and bank experience. for countries that are highly exposed to natural hazards, the bank will identify their potential vulnerability as a major development challenge and propose a country level disaster risk assessment. when the assessments identify that potentially important disruptions in the country's social and economic development could be caused by disasters resulting from natural hazards, the bank will encourage the inclusion of disaster risk management activities in the country strategy and operational program agreed with the borrower. these may include policy reforms, specific institutional strengthening and land-use planning activities, measures of financial protection such as through risk transfer, and investment projects conducive to reducing vulnerability at the national, regional, and municipal levels. where the natural hazards may affect more than one country, the bank will encourage a regional approach within the existing programming framework. the bank will promote the use of the disaster prevention sector facility and the disaster prevention fund, described in section v of this policy, and other means it offers to finance the recommended actions resulting from the assessment process. to meet the requirement of the drm policy to identify countries according to their level of risk exposure, a provisional country classification has been developed. the provisional classification will be subject to change, based on expert knowledge, and eventually on the complete data set of risk information derived from the implementation of the bank's indicators for disaster risk and risk management program in its borrowing member countries. the indicators program has been completed in countries to date. as indicated in directive a- , countries that have been identified as being highly exposed to natural hazards will be encouraged by the bank to include drm as a priority area for bank assistance. in those cases, the bank will propose that a country disaster risk assessment be carried out. the assessment would give an overview of the risks facing a country; identify the sectors and geographical areas that should receive priority attention; and provide initial policy orientation, reviews of relevant institutional capacities, and assistance needs. these assessments may already exist, or may be put together from country and secondary sources. . . the evaluation of the macroeconomic impacts as part of the country disaster risk assessment may allow for the identification of risk reduction needs and the quantification of possible resource gaps between available resources and funding needed for disaster response and recovery. recommendations will be prepared concerning opportunities for the bank to contribute to financial protection against disasters, as appropriate, such as direct funding for risk identification and support for risk transfer in financial markets in order to improve the effectiveness of the country's development efforts in the areas and sectors of bank involvement. . . identification of opportunities for bank financing. in line with the new country development risk framework, a more detailed disaster risk assessment will be recommended when disaster risks faced by certain areas/sectors of bank involvement could significantly jeopardize the achievement of a country's development objectives. these sector-specific or areaspecific assessments would analyze how these risks could affect specific areas/sectors and make recommendations on how best to address the risks identified. for this purpose, loans, technical cooperations, and nonfinancial bank products for proactive drm may be proposed within the country programming activities. . implementation of the country strategy: programming dialogue and portfolio management . . when deemed necessary by the bank and if the borrower agrees, drm activities will be included as in the implementation of the country strategy. the bank will give due consideration to the following: in the programming and portfolio reviews, the bank and the borrower may seek to implement risk reduction investments in the priority sectors and geographical areas through disaster prevention and mitigation measures. these investments may be financed with free-standing loans or as part of larger investment programs, policy based loans (pbl), or private sector operations. technical assistance may be considered for carrying out area-or sector-specific risk evaluations, strengthening risk management through policy reforms, organizational design, land-use planning activities, the preparation of new prevention loan programs, and supporting the implementation of financial protection schemes such as through insurance to cover disaster losses. loan portfolio modifications will likely be necessary due to the occurrence of major disasters during the regular programming cycle. borrowers may request new emergency or reconstruction financing and will have access to either new resources, for instance, through the immediate response facility for emergencies caused by disasters (gn- - and gn- - ), or "existing" resources, through loan reformulations (see directive b- ). . . the results of the drm implementation in-country programming will be evaluated using the monitoring system defined in the country strategy document. the bank may recommend activities of a regional nature whenever it is known that a particular disaster could affect several borrowing member countries simultaneously. examples of this situation are the enso (el niño southern oscillation) phenomenon, and the hurricanes and tropical storms in the caribbean and central america. . . the regional activities that possibly involve bank financing will be agreed beforehand with the affected borrowing member countries and may involve coordination with other international entities. the resulting operations to be included in the regional portfolio of the regional strategy document could be funded through bank instruments, such as technical cooperation of the regional public goods program or disaster prevention fund, or loans prepared in parallel, in close cooperation with the countries interested in a regional program. . . the purpose of this section is to provide guidance to project teams on the implementation of the bank's disaster risk management policy directive a- : risk and project viability. this directive is designed to promote the incorporation of drm in a systematic manner during project preparation and execution. the objective is to reduce risk to levels that are acceptable to the bank and the borrower, as indicated in directive a- of the policy: . . identification and reduction of project risk. bank-financed public and private sector projects will include the necessary measures to reduce disaster risk to acceptable levels as determined by the bank on the basis of generally accepted standards and practices. the bank will not finance projects that, according to its analysis, would increase the threat of loss of human life, significant human injuries, severe economic disruption, or significant property damage related to natural hazards. during the project preparation process project teams will identify if the projects have high exposure to natural hazards or show high potential to exacerbate risk. the findings will be reported to the bank through the social and environmental project screening and classification process. project teams should consider the risk of exposure to natural hazards by taking into account the projected distribution in frequency, duration, and intensity of hazard events in the geographic area affecting the project. project teams will carry out a natural hazard risk assessment for projects that are found to be highly exposed to natural hazards or to have a high potential to exacerbate risk. special care should be taken to assess risk for projects that are located in areas that are highly prone to disasters as well as sectors such as housing, energy, water and sanitation, infrastructure, industrial and agricultural development, and critical health and education installations, as applicable. in the analysis of risk and project viability, consideration should be given to both structural and nonstructural mitigation measures. this includes specific attention to the capacity of the relevant national institutions to enforce proper design and construction standards and of the financial provisions for proper maintenance of physical assets commensurate with the foreseen risk. when significant risks due to natural hazard are identified at any time throughout the project preparation process, appropriate measures should be taken to establish the viability of the project, including the protection of populations and investments affected by bank-financed activities. alternative prevention and mitigation measures that decrease vulnerability must be analyzed and included in project design and implementation as applicable. these measures should include safety and contingency planning to protect human health and economic assets. expert opinion and adherence to international standards should be sought, where reasonably necessary. in the case of physical assets, the bank will require that, at the time of project preparation, the borrower establish protocols to carry out periodic safety evaluations (during construction as well as during the operating life of the project) and appropriate maintenance of the project equipment and works, in accordance with generally accepted industry norms under the circumstances. the bank's social and environmental project screening and classification process will evaluate the steps taken by project teams to identify and reduce natural hazard risk. . . under the bank's new risk management development effectiveness framework, a common approach to the management of project risks is proposed. disaster risk is one of several project risks. these guidelines are an input to the bank's approach on project risk management. they apply to bank-financed investment loans and technical cooperation projects in the public and private sector as well as to operations supported by the multilateral investment fund. . . during the assessment, management, and monitoring of disaster risk at the project level, the disaster risk is reviewed at various stages of project preparation and implementation. on this basis, appropriate actions are taken to protect project benefits and outcomes. . . directive a- requires that the bank's social and environmental project screening and classification process provide for project teams to identify and reduce disaster risk. the recommended drm steps are as follows: project screening and classification outcome: identifies those projects where the drm policy is applicable and classifies as high, moderate or low risk. document: report of the social and environmental safeguards policy filter (spf) and social and environmental safeguards screening form. document: disaster risk profile in the environment and social strategy. disaster risk assessment (dra), including disaster risk management plan outcome: provides a detailed evaluation of the impacts of the significant natural hazards identified during project classification on project components; and outlines appropriate risk management and mitigation measures. document: dra report, prepared by the borrower (this may be a stand-alone report or it may be incorporated into the environmental impact assessment report). disaster risk management summary outcome: provides information on the specific disaster risks associated with the project and the risk management measures proposed by the borrower. document: drm summary, for inclusion in the environmental and social management report (esmr), prepared by project teams. project implementation, monitoring and evaluation outcome: identifies the approaches which the executing agency applies during project implementation; and which project teams apply during project monitoring and evaluation. . . the bank's social and environmental screening and classification system of projects will be used to filter and classify those projects for which disaster risk is likely to be an issue for project viability and effectiveness. . . there are two possible types of disaster risk scenarios: type : the project is likely to be exposed to natural hazards due to its geographic location. type : the project itself has a potential to exacerbate hazard risk to human life, property, the environment or the project itself. . . the purpose of this step is to establish, early in the project preparation process, whether natural hazards are likely to pose a threat to the project area during the execution (construction) period and/or the operational life of the project, due to type and type risk scenarios. project classification . . type risk scenario: the level of disaster risk associated with a given project is dependent on the characteristics of the natural hazards as well as on the vulnerability of the sector and project area. the project is classified on the basis of an estimate of the impacts/losses due to the significant hazards associated with type risk scenario. project teams classify their projects in terms of high, moderate, or low disaster risk on the basis of the (i) projected frequency of occurrence and magnitude or intensity of the hazard and (ii) estimated severity of the impacts associated with the hazard, i.e., the magnitude and extent of the likely social, economic, and environmental consequences of the hazard on the various project components and on the general zone of influence of the project. the classification process also provides project teams with a preliminary indication of the hazards likely to be of greatest significance, as well as their likely impacts on project components. and reported as part of the disaster risk profile presented in the environment and social strategy document. the project team will report its findings to the bank unit responsible for social and environmental screening and classification of projects, as part of the bankwide safeguards and risk management procedure. high-risk projects . . the project will typically be classified as high-risk if one or more of the significant natural hazards may occur several times during the execution (construction) period and/ or the operational life of the project and/or the likely severity of social, economic, and/or environmental impacts in the short to medium term are major or extreme. these impacts are of sufficient magnitude to affect project viability and may affect an area broader than the project site. as such hazards may affect project viability, a more detailed investigation of disaster risk, in the form of a dra, is required. moderate-risk projects . . the project will typically be classified as moderate risk if one or more of the prevalent natural hazards are likely to occur at least once during the execution (construction) period and/or the operational life of the project and/or the likely severity of impact in the short to medium term is average. these impacts are typically confined to the project site and can be mitigated at reasonable costs. projects associated with a moderate disaster risk do not typically require a dra. however, a more limited dra may be required, depending on the complexity of the project and where the anticipated vulnerability of a specific project component may compromise the achievement of project outcomes. low-risk projects . . the project will typically be classified as low risk if natural hazards are not likely to occur during construction and/or the operational life of the project and/or associated with a low severity of impact in the short to medium term. those impacts that occur do not lead to a disruption in the normal functioning of the operation and can be corrected as part of project maintenance. the occurrence of the hazard event does not impact on the achievement of project outcomes. a dra is not required. . . type risk scenario: the impacts associated with type risk scenario are addressed under directive b- of the bank's environment and safeguards compliance policy (op- ). such impacts are thus considered and included in the categorization of environmental impacts. . . the unit responsible for environmental and social risk mitigation reviews the classification of all operations and may recommend a new classification based on the review of the disaster risk profile presented in the environment and social strategy. the unit and line divisions will need to agree on the final classification of the operations, the level of disaster risk assessment required, and a proposed strategy to address and manage the anticipated impacts. for projects that are identified as high-risk, a dra is required and is prepared by the borrower. the objective of the assessment is to evaluate in greater detail the impacts of the significant natural hazards identified during project classification on project components. the results of the risk assessment will guide the selection of appropriate risk management and mitigation measures. evaluates the frequency, intensity, and severity of previous hazard events that have affected the project area, as well as those predicted to affect the site over the project's operational life. identifies the vulnerability and probable losses of project components, i.e., the nature and magnitude of the probable social, economic, and environmental impacts due to each hazard; this includes both direct and indirect impacts. provides a disaster risk management plan, including proposals for the design of disaster prevention and mitigation measures, including safety and contingency plans to protect human health and economic assets, and their estimated costs; an implementation plan; a monitoring program and indicators for progress; and an evaluation plan. the implementation plan includes protocols to undertake periodic safety evaluations from project implementation up to project completion and maintenance of project equipment and works. project teams include a summary of the dra report in the environmental and social management report, which is reviewed by both the bank unit responsible for environmental and social risk mitigation screening and the sector divisions chiefs will sign off on the esmr and safeguard compliance plan, including the drm activities. the drm summary provides information on the specific disaster risks associated with the project and the risk management measures proposed by the borrower. . . the project's proposed management and mitigation measures should comply with international standards of good practice and relevant national laws and regulations, such as national planning policies, laws and regulations, as well as national building codes and standards. . . project teams will analyze the impact of the disaster risk prevention and mitigation elements in their assessment of project viability, verifying that identified hazard impacts on project components are reduced to acceptable levels. . . the executing agency is responsible for ensuring that all drm activities ( including prevention and mitigation measures) associated with the project are implemented in accordance with the provisions of the loan agreement. this includes periodic safety evaluations and appropriate maintenance during project implementation and through project completion. project teams will monitor implementation to verify that the drm actions in the project risk management plan are carried out effectively; they shall use standard monitoring (project performance monitoring report; ppmr) procedures. purpose and scope . . the loan reformulation addressed by these guidelines provides financing for postdisaster response to the impacts of natural hazard events and physical damage (such as structural collapse and explosions) caused by technological accidents or other types of disasters resulting from human activity. loan reformulation includes the diversion of existing loan resources to specific analysis needs to determine performance indicators, based on the possible revisions and reformulations being considered. analysis of loans used as a source of funding . . the impact of redirecting loan resources from existing loans will be estimated taking into account the intended uses and project objectives of the loan or loans to be used as a source relative to the new proposed use of the funds, thereby creating the conditions for more informed decisions. resource transfers could be done between cost categories within a project (in which case more streamlined approval procedures will apply), or between separate loans as stipulated by bank procedures. . . for choosing existing projects as origin of resources, following factors (in order of priority) would be considered: a. public sector projects. only public sector loans would be considered. loans to the private sector should not be included in the package of loans for possible reformulation as a result of disasters. b. development impact in the reformulated operations. the loans that are having a relatively low economic/financial impact in the country should be considered first as a source for redirecting resources from existing loans toward emergency funding. redirecting resources that are within a loan generally have a smaller effect than those involving several operations. the original development objectives may not be achieved due to the new social or economic situation created by the disaster or it could be considered too expensive to reorient the resources within the old operation. recommendations regarding the redirection of resources will be based on project performance indicators used by the bank. c. level of execution. operations with a low level of physical execution or disbursements and commitments could be chosen for redirection, except for those loans with a very high development impact. the selection should not only be based on a low disbursement rate of the existing loans alone, but also on an analysis of the underlying causes of the poor performance and any remaining opportunities for attaining project goals. d. loans in affected sectors. resource transfers within an affected sector will be preferred due to the greater similarity of their respective objectives compared with those of loans in different sectors. e. loans in affected region(s). in general, existing projects in the disaster area will not be used to provide resources to be transferred to other programs in the same area. however, when damage is so severe that the attainment of the original development objectives is in jeopardy, or the continuation of a certain component of the project as a whole is unjustifiable on account of excessive costs, parts or all of the undisbursed balances may be re-channeled toward emergency or rehabilitation and reconstruction projects in the same area. factors to be considered in projects receiving funding . . the following are the recommended actions to be considered by project teams, while preparing the funding analysis: . i. technical analysis. for emergencies, the technical analysis will be aimed at re-establishing basic services and critical infrastructure in a time efficient manner. the attainment of fully functioning facilities and productive capacity through rehabilitation and reconstruction will be measured through a detailed technical analysis with the objective of reaching disaster resistance, and fulfilling technical standards across the board and performance criteria required by the bank. . ii. socioeconomic analysis. for emergency response, the socioeconomic analysis will be limited to the evaluation of the cost-effectiveness of restoring the basic services and critical infrastructure. if information is scarce, the analysis may be done based on comparable data from similar operations elsewhere. any delays in the analysis and processing of the emergency financing may limit the bank to have a meaningful contribution to resolve critical needs that are affecting the population, urgent re-establishment of basic services and critical activities. the analysis for rehabilitation and reconstruction investments will follow standard bank practices. if future project benefits cannot be estimated, cost-effectiveness analysis will be carried out. . iii. evaluation of institutional capacity and coordination. in order to gain sustainability, existing agencies are preferred to the establishment of new, ad hoc entities. a rapid analysis will be carried out of the institutional capacity, procurement management capability, and financial track record of the existing agencies. based on its results, it will be determined if the resources will be disbursed on an ex post or on a concurrent basis. the administrative and technical responsibilities of all the participating institutions in different sectors and means of coordination need to be clearly defined to facilitate successful execution in a limited time frame. planned strategies and activities need to be coordinated with other international agencies participating in the post-disaster financing. . i. procurement procedures. the applicable bank policy and rules will be followed for the procurement of goods and services. as an exception, for emergency situations, specific procurement procedures are available, in view of the special nature of these operations and the urgency involved. . ii. transparency in financing. the financial management and evaluation of procurements, expenses, and the utilization of goods and services to be funded with bank resources for emergency situations will be audited on a concurrent basis, following current bank practices. for rehabilitation and reconstruction investments the review may be on a concurrent or ex post basis depending on risk of lack of transparency estimated by the project teams. loan resources can be used to contract the services of independent public accountants to audit the operation's financial statements as required by the bank. . iii. monitoring and evaluation. bank resources will be subject to review on a concurrent basis for emergency investments. for rehabilitation and reconstruction, an audit will be required on a concurrent or ex post basis, depending on the risk of lack of transparency as estimated by the project team. data collection will be planned for monitoring and evaluation. only direct project impacts will need to be evaluated. . . vulnerability should not be replicated when designing disaster response financing. in the preparation of reformulations for rehabilitation and reconstruction, a proportion of the resources of the operation should be allocated to prevention and mitigation activities. the percentage of the total cost that will be dedicated to prevention and mitigation should be defined and the viability of these investments assessed by the project team. the project team should also justify any potential deviations from international practices in these allocations for disaster prevention and mitigation. purpose and scope . . the purpose of this section is to provide assistance to project teams on the implementation of directive b- : reconstruction. specifically guidance is provided on the precautions that country programming process and project teams should take to promote revitalization of development efforts in the aftermath of disasters, while ensuring that rehabilitation and reconstruction projects do not lead to a rebuilding of or an increase in vulnerability. as indicated in directive b- of the policy: . . avoiding rebuilding vulnerability. operations that finance rehabilitation and reconstruction after a disaster require special precautions to avoid rebuilding or increasing vulnerability. these include the precautions mentioned in a- , as well as correcting deficiencies in risk management policies and institutional capacity as reflected in a- . a significant share of the new investment will be earmarked to reduce vulnerability to future disasters and improve the country's capacity for comprehensive disaster risk management. particular attention must be given to lessons learned from recent hazard events. the bank will not assume that pre-disaster conditions persist in whole or in part in the affected area. disaster risk assessment of the reconstruction project should be carried out taking into account the specifics of the area, the sector, and the infrastructure concerned, as well as the current environmental, social, and economic situation and any changes in the affected area as a result of the disaster. . . reconstruction may follow as a response to the impacts of natural hazard events, and physical damage (such as structural collapse and explosions) resulting from technological accidents or other types of disasters resulting from human activity. . . the guidelines for directive a- : risk and project viability, as described in section of these guidelines, also apply to rehabilitation and reconstruction projects. for projects identified as high risk, the disaster risk assessment, and design and implementation of risk reduction measures, will incorporate the lessons learned from the disaster event, including the performance of the physical works, the relevant sectors, institutions, and other project components. risk reduction measures will include enhancements in national, regional, and sectoral risk management policies and strengthening of institutional capacity. . . in order to avoid the rebuilding of or an increase in vulnerability, a proportion of the resources of the operation will be allocated to prevention, mitigation, and risk transfer. the percentage of the total cost is at the discretion of the project team, but will be guided by international practices. used with permission from iadb, . reconstruction facility or immediate response facility for emergencies caused by natural and unexpected disasters. the emergency reconstruction facility can use up to $ million of the iadb's ordinary capital or up to $ million of the fund for special operations to assist an impacted country the ifis described in this chapter bstdb) • caribbean development bank (cdb) • council of europe development bank (coeb) • development bank of southern africa (dbsa) • european bank for reconstruction and development (ebrd) • islamic development bank (idb) • north american development bank (nadb) proposed loan: peoples republic of china emergency assistance for wenchuan earthquake reconstruction project european commission fao's mandate. fao website facing the challenge of natural disasters in latin america and the caribbean service sector severely affected by typhoon haiyan nato (north atlantic treaty organization), . pakistan earthquake relief operation ocha- fm c/$file/ocha_ar _hi% res.pdf?openelement japan diverts rice to tsunami survivors. world food programme (wfp) roadmap towards a strategy for disaster and climate resilient development in the pacific (srdp) by : executive summary un-habitat. secretary-general's envoy for youth overview of global humanitarian response johannesburg plan of implementation. united nations website disaster profiles: third un conference on least developed countries united nations educational, scientific, and cultural organization) unhcr in dubai: first line responder in emergencies. unhcr supply office, dubai. unicef (united nations children's fund) about the unjlc. unjlc website connect and convince to reduce disaster impacts lives saved in viet nam by involving women in disaster planning. press release economics, health, and development: some ethical dilemmas facing the world bank and the international community fast food: wfp's emergency response food aid information system: quantity reporting emergency response framework world bank group to support flood recovery in bosnia and herzegovina through their efforts to mitigate, prepare for, respond to, and recover from natural disasters, multilateral organizations have a major role in international disaster management. all nations are at risk from disasters and, likewise, all nations face the prospect of one day finding themselves requiring help from one or more of these organizations. multilateral organizations direct the collective experience and tools of their member states to benefit all nations in need of assistance-even the wealthiest ones. the progress witnessed by the international disaster management community in recent years can be traced directly to the work of these multilateral organizations, especially focused initiatives such as the international strategy for disaster reduction. provide effective and efficient support to borrowing members in reducing disaster risks and (ii) to facilitate rapid and appropriate assistance by the bank to its borrowers after a disaster. the guidelines are part of the bank's framework for the management of development risk at the country and project levels. there are four possible strategies to manage risks: (i) acceptance, when risks remain below levels deemed tolerable by the parties involved; (ii) prevention and mitigation; (iii) sharing, when risks can be effectively transferred to a third party, for example through insurance; and (iv) rejection ("avoidance"), when the level of risk exceeds the risk level deemed acceptable but cannot be lowered at a reasonable cost. . . the policy directives outline the actions that are to be used both by the iadb staff and by teams of the borrowers, who are responsible for a. country programming-policy directive a- b. preparation and execution of new projects-directive a- c. loan reformulations for financing disaster response-directive b- d. preparation and execution of reconstruction projects-directive b- . . the guidelines will contribute to the mainstreaming of disaster risk management (drm) into the bank's programming exercises with the borrowers, particularly in high-risk countries.to determine which of the idb's borrowing member countries will require a country risk assessment, a provisional classification of all countries has been prepared. . . the guidelines will be used for the design and implementation of lending programs, technical cooperations, small projects, cofinancing, and preinvestment activities consistent with the identified risk level. they will address ways to manage risk in public and private sector activities within the same project or to another existing project, in order to finance unplanned disaster response. reformulations may thus involve just a single loan or several operations. . . loan reformulation allows for the reallocation of resources from existing loans to other projects under certain circumstances, in the aftermath of disasters, as stipulated in directive b- of the policy: . . the bank may approve the reformulation of existing loans in execution in response to disasters if: (i) a state of emergency or disaster has been officially declared by the government; (ii) the impact of the loan reformulation has been estimated taking into account the intended uses and project objectives of the loan or loans to be reformulated relative to the new proposed use of the funds, thereby creating the conditions for more informed decisions on the part of the approving authorities; (iii) adequate transparency and sufficient mechanisms for monitoring, auditing, and reporting the use of the redirected funds are in place, while taking into account the need of a timely response given the nature of the situation; and (iv) a significant share of the redirected funds will be earmarked to reduce the borrower's vulnerability to future disasters and improve the country's capacity for comprehensive disaster risk management. in order to be considered for loan reformulation funding in response to a disaster, the government must have declared a state of emergency or its equivalent, for a region or the country as a whole, according to the laws and regulations of the country. . . the country office should prepare an originating document after the formal declaration of state of emergency by the government, recommending the decisions that should be taken in relation to the projects/programs potentially affected by the disaster. . . the bank may offer technical support to the government in preparing an official request for financing through loan reformulation, on the basis of the originating report. . . once a financing request is received, a project team is appointed, and the approval process of the reformulation operation(s) will follow the established bank procedures on delegation of authority, according to regular bank procedures. once the bank has received an official request from the borrowing country for financing disaster response, the possibility of using fresh idb resources, such as through the immediate response facility (gn- - and gn- - ), is analyzed. if their use is not considered feasible, the impact of the loan reformulation will be estimated by vpc, with support from vps, taking into account the intended uses and project objectives of the loan(s) to be reformulated either: (i) as a provider of funding or (ii) as a recipient of resources. the analysis for operations receiving funding in response to a natural hazard or physical damage from technological activities or other types of disasters resulting from human activity will reflect the nature of the projects, available information, and use of the reallocated resources for an emergency, rehabilitation, and reconstruction. . . the revision of the portfolio in emergency situations should be done jointly with the borrower. those projects whose development objective is unlikely to be achieved should be considered first as candidates for reformulation. the team responsible for the portfolio key: cord- -hrgtaunt authors: rabelo, luiza a.; nunes-souza, valéria; bader, michael title: animal models with a genetic alteration of the ace /ang-( - )/mas axis date: - - journal: the protective arm of the renin angiotensin system (ras) doi: . /b - - - - . - sha: doc_id: cord_uid: hrgtaunt the aim of this chapter is to describe the animal models generated by transgenic technology for the functional analysis of the protective axis of the renin–angiotensin system, consisting of angiotensin-converting enzyme (ace ), angiotensin (ang)-( - ), and mas. transgenic overexpression of the components of this axis in general led to an ameliorated cardiac and vascular damage in disease states and to an improved metabolic profile. knockout models for ace and mas, however, show aggravated cardiovascular pathologies and a metabolic syndrome-like state. in particular, the local production of ang-( - ) in the vascular wall, in the heart, and in the brain was found to be of high physiological relevance by the use of transgenic animals overexpressing ace or ang-( - ) in these tissues. the study of hormone systems involved in cardiovascular and metabolic diseases, such as the renin-angiotensin system (ras), can only be performed in whole organisms due to the complex interplay of different organs, which determines cardiovascular physiology. in contrast to pharmacological interventions in these systems, which often lack specificity, the targeted genetic alteration of the expression of single-hormone system components is the most straightforward method to analyze their functions in cardiovascular and metabolic homeostasis and disorders. accordingly, the generation of transgenic and knockout (ko) animals was widely used to study the role of ras components in cardiovascular control and in the pathogenesis of diseases. , the aim of this chapter is to describe the animal models generated by transgenic technology for the functional analysis of the protective axis of the ras, consisting of angiotensin-converting enzyme (ace ), ang-( - ), and mas. in biomedical research, the use of rats and mice has become a major tool, considering the easiness of breeding, growth, and maintenance and the similarity with human organisms in most cardiovascular and metabolic systems. currently, the use of transgenic technologies to access animal physiology is routine, but the pioneering work was performed in the s. the first successful genetic modifications of a mouse were achieved by gordon and colleagues. this group demonstrated that foreign genes can be integrated into the mouse genome by transfer of dna constructs into the pronuclei of zygotes. by the use of specific promoters, the investigator can direct the expression of the transgene into specific tissues. about years later, the same technology was also established for the rat, interestingly first targeting the ras component renin. since , the elimination of gene expression is also possible by homologous recombination-mediated targeted gene ko in embryonic stem (es) cells. [ ] [ ] [ ] to this purpose, firstly, a targeting vector has to be designed and constructed that contains parts of dna sequences homologous to the gene of interest and the intended mutation. following the transfection of this vector into es cells, a few cells will incorporate it into the endogenous gene via homologous recombination and can be selected by appropriate methods. these successfully targeted es cells are injected into host blastocysts, and a chimeric animal is obtained. this animal carries the mutation in part of its cells and will eventually pass it on to its offspring, which will be heterozygous or homozygous (ko) for the mutant gene. the ko animals will present the physiological phenotype caused by the absence of the gene product. in general, this method is still the most commonly used for the targeted alteration of the mouse genome. it took more than years for this technology to also became available for the rat by the discovery of a method to establish germ-line-competent es cells from this species in , and a few ko rat models have been developed since using es cells. , however, recently, novel methods for the targeted alteration of genes in the mouse and rat genome have become available, which will probably replace the relatively complicated es cell method in the near future. they are based on nucleases that are targeted to a certain site in the genome by different methods. zinc finger (zfn) and tale nucleases use protein domains that specifically recognize a dna sequence of choice, while the crispr/cas system uses a guide rna that binds dna by specific base pairing. since only one animal model for the ras has been described yet produced by zfn technology, the renin-ko rat, and since the mas-ko rat has obviously been generated using zfns (http://rgd.mcw.edu/rgdweb/report/strain/main.html?id= ; access . . ) but is not yet published, we will not go into more detail on these novel technologies. our group and others have developed several transgenic and ko rat and mouse models with genetic deletion and/or overexpression of components of the ace /ang-( - )/mas axis. some of these models produced pleiotropic phenotypes depending on the genetic background of the strain they were generated in. in the following, we will list these models and summarize the insights into the physiology of the protective ras axis gained by their analysis (see also table ). the ace gene is located on the x chromosome, and thus, heterozygous deletion (ace −/y ) already results in the complete absence of the enzyme in male animals. deletion of ace in mouse leads to several cardiac abnormalities. in an elegant study, crackower and colleagues provided the first in vivo evidence supporting the hypothesis that the loss of ace promotes heart dysfunction. however, later studies on the function of ace in the heart resulted in contradictory observations. two independent groups showed that the baseline cardiac function and morphology appeared normal in their ace -deficient mouse lines. , nevertheless, ace −/y and even heterozygous female ace +/− mice are more susceptible to pressure overload or diabetes-induced cardiac injury. [ ] [ ] [ ] moreover, the lack of ace also exacerbated diabetic and shock-induced kidney injury. [ ] [ ] [ ] there is also still a debate whether the deletion of ace changes basic blood pressure in mice. most likely, this effect depends strongly on the genetic background of the mice analyzed: mice show hardly any effect, while c bl/ or fvb/n ace −/y mice are clearly hypertensive (our unpublished results). nevertheless, c bl/ ace −/y mice displayed high blood pressure during pregnancy and reduced weight gain and gave birth to smaller pups. besides an upregulation of oxidative stress in the brain and consequently of the sympathetic nervous system, the cause for the hypertensive effect of ace deletion may be an endothelial dysfunction as evidenced by an impaired acetylcholine-induced aortic vasodilatation. ace deletion in apolipoprotein e (apoe) ko mice, a classical model for atherosclerosis, worsened plaque formation and vascular inflammation. , in low-density lipoprotein receptor ko mice, fed a high-fat diet, ace deletion also aggravated atherosclerosis. moreover, loss of ace led to increased arterial neointima formation in response to endovascular injury in the femoral artery accompanied by an overexpression of inflammation-related genes. several studies have shown an important role of ace in metabolism. c bl/ ace −/y mice show impaired glucose homeostasis at different ages. , also, ace gene deletion aggravated liver fibrosis in models of chronic hepatic injury. ace −/y mice displayed aggravated pathologies in the acute respiratory distress syndrome and in bleomycin-induced lung injury rendering ace an important target for inflammatory lung diseases. ace −/y animals were also instrumental for the surprising finding that the protein is not only an enzyme but also a trafficking molecule in the gut being responsible for the functional expression of the amino acid transporter slc a . , ace −/y mice, therefore, show reduced levels of large amino acids, such as tryptophan, in the circulation, altered gut microbiota, and intestinal inflammation. besides being an enzyme and trafficking molecule, ace is also the receptor for the human severe acute respiratory syndrome (sars) coronavirus. in order to study this function, transgenic mouse models have been generated by several groups that overexpress human ace using the mouse ace promoter, the cytomegalovirus promoter, , or the cytokeratin promoter specific for the airway and other epithelia. , as expected, human ace expression led to an increased susceptibility of the transgenic mice to sars virus infection. in the kidney, ace overexpression protected the mice from shock-induced injury. the transgenic mouse overexpressing human ace in the brain using the synapsin promoter has confirmed the important actions of central ang ii in the pathogenesis of cardiovascular diseases and the protective role of ang-( - ) . the animals are protected from hypertension induced by low peripheral infusions of ang ii and doca-salt treatment. moreover, they show an amelioration of cardiac hypertrophy induced by angii, of chronic heart failure induced by coronary ligation, and of stroke induced by middle cerebral artery occlusion. , in most cases, an increase in ang-( - ) and a decrease of ang ii in the brain both influencing the levels of local no and the autonomic nervous system were shown to be instrumental. increased blood pressure during pregnancy increased susceptibility to cardiac damage [ ] [ ] [ ] aggravated atherosclerosis , disturbed glucose homeostasis , aggravated kidney [ ] [ ] [ ] and lung injury , amino acid uptake deficiency in the gut transgenic mice overexpressing human ace in the heart surprisingly showed an increase in ventricular tachycardia and sudden death, which was due to a dysregulation of connexins. the underlying mechanism and the involved peptides could not be elucidated. when human ace was overexpressed in podocytes of mice using the nephrin promoter, the nephropathy induced by diabetes was ameliorated. the authors suggest that this is due to reduced renal ang ii levels leading to a reduced expression of tgf-beta, but they could also not exclude a protective effect of ang- ( - ) . ace is highly expressed in the endothelium and smooth muscle cells (smc), and its expression is reduced in the spontaneously hypertensive stroke-prone rat (shrsp). when human ace was overexpressed in vascular smc of transgenic shrsp, endothelial dysfunction and hypertension were ameliorated, which was accompanied by a reduction in oxidative stress linked to a decrease in ang ii and/or an increase in ang-( - ). in , we generated mas-deficient (mas −/− ) mice on the mixed ×c bl/ background and showed that these animals were healthy in appearance and grew normally and exhibited normal ang ii plasma levels. however, male (but not female ) mas −/− mice displayed increased anxiety on the elevated-plus maze. we also showed not only that long-term potentiation was markedly increased in the hippocampal ca region of mas −/− mice but also that object recognition memory is impaired. collectively, these data support a role of mas in behavior. c bl/ mas −/− mice show a marked cardiac dysfunction both in vitro and in vivo accompanied by an increase in extracellular matrix proteins, such as collagen i, collagen iii, and fibronectin. furthermore, mas −/− animals exhibit vascular oxidative stress, endothelial dysfunction, and high blood pressure at least on the fvb/n genetic background. , consistently, endothelial function is also impaired in isolated mas −/− vessels. consequently, mas −/− mice exhibit a pronounced decrease in blood flow and a marked increase in resistance in different vascular beds. these functional changes in both regional and systemic hemodynamics in mas −/− mice suggest that the ang-( - )/mas axis plays an important role in vascular regulation. a dysregulation of the vascular function in the corpus cavernosum is probably also the reason for the erectile dysfunction observed in mas −/− mice. pinheiro et al. showed an imbalance in renal function in mas −/− mice: reduced urine volume and fractional sodium excretion and increased glomerular filtration rate and proteinuria. surprisingly, a proinflammatory role for ang-( - ) and mas was reported in a model of unilateral ureteral obstruction in mice in contrast to the anti-inflammatory effects of ang-( - ) and mas in other models of kidney nephropathy. certainly, additional studies are needed to clarify the role of ang-( - ) and mas in the kidney. the anti-inflammatory actions of mas were also recently confirmed in a lipopolysaccharide (lps)-induced endotoxic shock model. santos and colleagues have revealed the effects of mas deficiency on lipid and glucose metabolism. they used mas −/− mice on the fvb/n background and demonstrated that loss of mas increases the risk of metabolic complications by causing several features of the metabolic syndrome, such as type diabetes mellitus, hypertension, dyslipidemia, and nonalcoholic fatty liver disease. mas deletion decreased the responsiveness of adipocytes to insulin accompanied by a decreased expression of pparγ in adipose tissue. moreover, silva et al. recently showed that mas deletion in apoe −/− mice leads to dyslipidemia and liver steatosis. when rat mas is overexpressed in the retina of transgenic mice using the opsin promoter, degeneration of photoreceptors is the consequence, which is probably induced by proliferative signaling pathways activated in these cells due to the constitutively active mas protein. prolylcarboxypeptidase (prcp, ec . . . ) is another enzyme that can generate ang-( - ) from ang ii, but it is also not specific for angiotensin peptides. prcp-ko mice show elevated levels of α-melanocyte-stimulating hormone (α-msh), an anorexigenic neuropeptide, in the hypothalamus. the phenotype of these animals is characterized by a decrease in body weight and body length under normal diet, accompanied by decreased white adipose tissue. the lean phenotype is also observed after high-fat diet-induced obesity (schadock et al., unpublished results). moreover, prcp-ko mice display hypertension and vascular dysfunction probably due to an increase in reactive oxygen species and uncoupled enos. in addition, prcp also regulates angiogenesis and vascular repair. how much of these cardiovascular actions are due to alterations in ang ii or ang-( - ) levels remains to be elucidated. based on an elegant system to generate ras peptides by release from an artificial protein, which is processed by furin during secretion, , several transgenic animal models with altered ang-( - ) levels were generated. the transgenic rat tgr(a - ) expresses the ang-( - )-producing protein mainly in the testis. in this model, ang-( - ) levels are chronically elevated in plasma and testis ~ . -fold and ~ . -fold, respectively. surprisingly, this chronic increase in ang-( - ) did not alter basal blood pressure levels measured by telemetry. however, tgr(a - ) rats displayed a marked reduction in isoproterenol-induced heart hypertrophy and an improvement of postischemic systolic function. botelho-santos and colleagues showed changes in systemic and regional hemodynamic parameters in these rats, resulting in an increase of vascular conductance in several tissues and a decreased total peripheral resistance. furthermore, the transgenic rats showed a significant increase in stroke volume and cardiac index and a reduction in basal urinary flow, leading to increased urinary osmolality and osmolal clearance. furthermore, chronic elevation of circulating ang-( - ) levels considerably improved the lipid and glycolytic profile and lowered the fat mass accompanied by a decrease in triglycerides and cholesterol in plasma and an improved glucose tolerance and insulin sensitivity and reduced gluconeogenesis in the liver. , transgenic mice and rats overexpressing ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the heart transgenic mice carrying the ang-( - )-releasing construct under the control of the alpha-cardiac myosin heavy-chain promoter exhibit about an eightfold increase of the peptide in the heart and show a normal basic cardiac function but are protected from hypertensive cardiac hypertrophy induced by ang ii infusion but not from myocardial infarction. transgenic rats generated with the same construct showed a slightly improved resting cardiac function and were also protected from hypertrophy in this case induced by isoproterenol. transgenic and ko rodent models were pivotal for our understanding of the protective functions of the novel ras axis, ace /ang-( - )/mas. transgenic overexpression of the components of this axis in general led to ameliorated cardiac and vascular damage in disease states and to an improved metabolic profile. ko models for ace and mas, however, show aggravated cardiovascular pathologies and a metabolic-syndrome-like state. in particular, the local production of ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the vascular wall, in the heart, and in the brain was found to be of high physiological relevance by the use of transgenic animals overexpressing ace or ang-( - ) in these tissues. inducible and cell-type-specific ko models for mas and ace will be helpful in the future to deepen our understanding of the ace /ang-( - )/mas axis. mouse knockout models of hypertension genetically altered animal models for mas and angiotensin animal models for metabolic, neuromuscular and ophthalmological rare diseases genetic transformation of mouse embryos by microinjection of purified dna fulminant hypertension in transgenic rats harbouring the mouse ren- gene advances in the use of embryonic stem cell technology introduction of homologous dna sequences into mammalian cells induces mutations in the cognate gene targetted correction of a mutant hprt gene in mouse embryonic stem cells capture of authentic embryonic stem cells from rat blastocysts production of p gene knockout rats by homologous recombination in embryonic stem cells efficient gene targeting by homologous recombination in rat embryonic stem cells barbas rd cf. zfn, talen, and crispr/cas-based methods for genome engineering creation and characterization of a renin knockout rat angiotensin-converting enzyme is an essential regulator of heart function angiotensin converting enzyme- confers endothelial protection and attenuates atherosclerosis altered blood pressure responses and normal cardiac phenotype in ace -null mice angiotensin-converting enzyme deficiency is associated with impaired gestational weight gain and fetal growth restriction deletion of angiotensin-converting enzyme accelerates pressure overloadinduced cardiac dysfunction by increasing local angiotensin ii cardioprotective effects mediated by angiotensin ii type receptor blockade and enhancing angiotensin - in experimental heart failure in angiotensin-converting enzyme -null mice heterozygote loss of ace is sufficient to increase the susceptibility to heart disease genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse deletion of angiotensin-converting enzyme promotes the development of atherosclerosis and arterial neointima formation loss of angiotensin-converting enzyme leads to impaired glucose homeostasis in mice loss of ace exaggerates high-calorie diet-induced insulin resistance by reduction of glut in mice loss of angiotensin-converting enzyme- (ace ) accelerates diabetic kidney injury role of angiotensin-converting enzyme (ace and ace ) imbalance on tourniquetinduced remote kidney injury in a mouse hindlimb ischemia-reperfusion model loss of ace accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice angiotensin-converting enzyme protects from severe acute lung failure angiotensin converting enzyme abrogates bleomycin-induced lung injury defective intestinal amino acid absorption in ace null mice tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations ace links amino acid malnutrition to microbial ecology and intestinal inflammation mice transgenic for human angiotensin-converting enzyme provide a model for sars coronavirus infection lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme virus receptor differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins podocyte-specific overexpression of human angiotensin-converting enzyme attenuates diabetic nephropathy in mice brain-selective overexpression of human angiotensin-converting enzyme type attenuates neurogenic hypertension ace -mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function angiotensin converting enzyme /ang-( - )/mas axis protects brain from ischemic injury with a tendency of age-dependence transgenic angiotensin-converting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function sustained long term potentiation and anxiety in mice lacking the mas protooncogene sex specific behavioural alterations in mas-deficient mice impairment of in vitro and in vivo heart function in angiotensin-( - ) receptor mas knockout mice endothelial dysfunction and elevated blood pressure in mas gene-deleted mice ablation of angiotensin ( - ) receptor mas in c bl/ mice causes endothelial dysfunction evidence that the vasodilator angiotensin-( - )-mas axis plays an important role in erectile function genetic deletion of the angiotensin-( - ) receptor mas leads to glomerular hyperfiltration and microalbuminuria mas deficiency in fvb/n mice produces marked changes in lipid and glycemic metabolism murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis prolylcarboxypeptidase regulates food intake by inactivating alpha-msh in rodents expression of an angiotensin-( - )-producing fusion protein produces cardioprotective effects in rats improved lipid and glucose metabolism in transgenic rats with increased circulating angiotensin-( - ) decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin expression of an angiotensin-( - )-producing fusion protein in rats induced marked changes in regional vascular resistance - ) blunts hypertensive cardiac remodeling by a direct effect on the heart attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-( - )-producing fusion protein in the heart angiotensin ii-mediated oxidative stress and inflammation mediate the agedependent cardiomyopathy in ace null mice angiotensin-converting enzyme gene targeting studies in mice: mixed messages angiotensin-converting enzyme deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor−/− mice angiotensin-converting-enzyme inhibits liver fibrosis in mice brain angiotensin-converting enzyme type shedding contributes to the development of neurogenic hypertension angiotensin-converting enzyme over-expression in the central nervous system reduces angiotensin-ii-mediated cardiac hypertrophy brain-selective overexpression of angiotensin-converting enzyme attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure neuronal over-expression of ace protects brain from ischemia-induced damage angiotensin-( - )/mas axis integrity is required for the expression of object recognition memory effects of genetic deletion of angiotensin-( - ) receptor mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart endothelial dysfunction through genetic deletion or inhibition of the g protein-coupled receptor mas: a new target to improve endothelial function altered regional blood flow distribution in mas-deficient mice angiotensin-( - ) and the g protein-coupled receptor mas are key players in renal inflammation beneficial effects of the activation of the angiotensin-( - ) mas receptor in a murine model of adriamycin-induced nephropathy receptor mas protects mice against hypothermia and mortality induced by endotoxemia angiotensin-( - ) mas-receptor deficiency decreases peroxisome proliferatoractivated receptor gamma expression in adipocytes mas receptor deficiency is associated with worsening of lipid profile and severe hepatic steatosis in apoe-knockout mice degeneration of cone photoreceptors induced by expression of the mas protooncogene prolylcarboxypeptidase promotes angiogenesis and vascular repair tissue targeting of angiotensin peptides renal function in transgenic rats expressing an angiotensin-( - )-producing fusion protein circulating rather than cardiac angiotensin-( - ) stimulates cardioprotection after myocardial infarction key: cord- -andje wu authors: dorobantu, cristina m.; albulescu, lucian; harak, christian; feng, qian; van kampen, mirjam; strating, jeroen r. p. m.; gorbalenya, alexander e.; lohmann, volker; van der schaar, hilde m.; van kuppeveld, frank j. m. title: modulation of the host lipid landscape to promote rna virus replication: the picornavirus encephalomyocarditis virus converges on the pathway used by hepatitis c virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: andje wu cardioviruses, including encephalomyocarditis virus (emcv) and the human saffold virus, are small non-enveloped viruses belonging to the picornaviridae, a large family of positive-sense rna [(+)rna] viruses. all (+)rna viruses remodel intracellular membranes into unique structures for viral genome replication. accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ros). for instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the golgi-localized phosphatidylinositol -kinase iii beta (pi kb), while cardioviruses replicate independently of the kinase. by which mechanisms cardioviruses develop their ros is currently unknown. here we show that cardioviruses manipulate another pi k, namely the er-localized phosphatidylinositol -kinase iii alpha (pi ka), to generate pi p-enriched ros. by sirna-mediated knockdown and pharmacological inhibition, we demonstrate that pi ka is an essential host factor for emcv genome replication. we reveal that the emcv nonstructural protein a interacts with and is responsible for pi ka recruitment to viral ros. the ensuing phosphatidylinositol -phosphate (pi p) proved important for the recruitment of oxysterol-binding protein (osbp), which delivers cholesterol to emcv ros in a pi p-dependent manner. pi p lipids and cholesterol are shown to be required for the global organization of the ros and for viral genome replication. consistently, inhibition of osbp expression or function efficiently blocked emcv rna replication. in conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ros. remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis c virus, a member of the flaviviridae family, but not other picornaviruses or flaviviruses. thus, our results highlight the convergent recruitment by distantly related (+)rna viruses of a host lipid-modifying pathway underlying formation of viral replication sites. picornaviridae is a large family of positive-sense rna viruses [(+)rna viruses] comprising many clinically relevant human and animal pathogens. members of the genus enterovirus include important human viruses like poliovirus (pv), the causative agents of poliomyelitis, coxsackieviruses (cv), causing meningitis and myocarditis, and rhinoviruses (rv), responsible for the common cold and exacerbations of asthma and chronic obstructive pulmonary disease. perhaps the best-known non-human picornavirus is foot-and-mouth-disease virus (fmdv, genus aphtovirus), which can cause devastating outbreaks in cattle leading to severe economic loss. closely related to the apthovirus genus is the genus cardiovirus, composed of three species: theilovirus (tv), encephalomyocarditis virus (emcv) and the more recently discovered boone cardiovirus. the species theilovirus includes, among others, theiler's murine encephalomyocarditis virus (tmev) and saffold virus (safv), a human cardiovirus. while tmev is known to cause enteric infections and sometimes more severe encephalitis or chronic infection of the central nervous system [ ] , as yet, safv has not been firmly associated with a clinical disease [ ] . emcv can infect a wide range of animals, of which rodents are considered the natural reservoir. of all domesticated animals, pigs are most prone to emcv infection, which can lead to fatal myocarditis [ ] , reproductive failure in sows or sudden death of piglets [ ] [ ] [ ] . like other (+)rna viruses-such as hepatitis c virus (hcv), dengue virus (denv), chikungunya virus (chikv) and coronavirus (cov)-picornaviruses replicate their genomic rna on specialized, virus-modified intracellular membranes. these remodeled membranes termed replication organelles (ros) arise from the concerted actions of both viral nonstructural virus-host interactions, indicating that diverse rna viruses might have a limited choice of pathways in the remodeling of host membrane network for virus replication. unlike enteroviruses, cardioviruses do not require pi kb for replication [ ] . to investigate whether other pi ks might be involved in cardiovirus replication, we depleted each of the four distinct cellular pi ks by sirna-mediated gene knockdown using a set of sirna sequences (ambion) which we previously tested for efficiency and toxicity [ ] , and monitored the subsequent effects on replication of emcv. we observed inhibitory effects on emcv replication when silencing pi ka, but not upon silencing of the other pi ks (fig a) . to confirm the importance of pi ka for emcv replication, we performed another series of knockdown experiments using another set of sirna sequences (qiagen). depletion of pi ka, but not pi kb, significantly reduced emcv infection, measured by end-point titration of progeny virus production ( fig b) . we next wondered which step in the virus life cycle is dependent on pi ka. to omit the step of virus attachment and cell entry, emcv rna was in vitro transcribed and subsequently transfected in cells depleted of pi ka by sirnas. virus replication was strongly inhibited upon pi ka silencing, as measured by end-point titration of progeny virions ( fig c) . this indicated that pi ka is involved in a post-entry step in the virus life cycle. to elucidate whether emcv requires pi ka for viral genome amplification, we infected cells with a renilla luciferase-encoding emcv (rluc-emcv) and quantified the luciferase activity as a measure of viral rna replication. emcv rna replication was severely impaired in cells lacking pi ka, but not pi kb (fig d) . we excluded that inhibition of emcv replication by pi ka silencing was due to cytotoxic effects by a cell viability assay ( fig e) and verified the knockdown efficiency by western blot analysis ( fig f) . altogether, these results showed that pi ka plays a key role in emcv genome rna replication. next, we investigated whether emcv required the enzymatic activity of pi ka using al- , a pi k inhibitor that also blocks pi kb, but at -fold higher concentration [ ] . cells were infected with emcv or rluc-emcv at moi . and treated with increasing concentrations of al- for h. coxsackievirus b (cvb ), as well as all other enteroviruses, has been previously shown to hijack the golgi-localized pi kb for replication [ , ] and was included as a control. as measured by end-point titration and analysis of the luciferase activity ( fig g and h ), emcv replication was efficiently inhibited by al- in a dose-dependent manner with complete inhibition detected at μm, while cvb replication was hampered only at μm ( fig g) , which is in line with the -fold preference of al- for pi ka over pi kb. dipyridamole, a well-established inhibitor of emcv rna replication, was included here as positive control. importantly, al- inhibited emcv replication also when infection was performed at high moi (s a fig, moi ). to corroborate that pi ka activity is required for the step of viral genome replication, we performed a time-of-addition experiment in which al- was added to the cells at different time points after infection with rluc-emcv. similar to dipyridamole, al- strongly inhibited replication when added up to h after infection (s b fig), indicating that not entry but rather a step during genome replication was blocked by al- . next, we tested whether other members of the cardiovirus genus also depended on pi ka for replication. similar to emcv, replication of the human cardiovirus saffold virus (safv ) (species theilovirus) was also sensitive to al- treatment (fig i) . the cell viability assay demonstrated that al- treatment only exerted slight cytotoxic effects at the highest concentration tested (fig j) . these results indicated that different cardiovirus species required the enzymatic activity of pi ka for genome replication. soon after infection, the cytoplasm of emcv-infected cells accumulates an impressive amount of vesicular membranous structures [ , ] . as yet, there is little information available regarding which viral proteins and host factors are associated with these new virus-induced organelles [ , ] . we set out to investigate whether pi ka was present at emcv ros. despite repeated efforts, we were unable to detect the endogenous kinase by immunofluorescence staining in any of the cell lines tested. as an alternative, we chose to analyze possible changes in the subcellular distribution of ectopically expressed pi ka upon emcv infection. in mock-infected cells (fig a, upper panel) , gfp-pi ka was distributed diffusely throughout the entire cytoplasm, as previously reported by others [ , ] . in infected cells visualized by dsrna staining, we instead observed a clear difference in the localization of the kinase, which was redistributed to discrete cytoplasmic punctae in a perinuclear region (fig a, lower panel) . we next aimed to elucidate whether these pi ka punctae coincided with the viral ros. the small picornaviral protein a and its precursor ab are membrane-associated and play key roles in viral rna replication and recruitment of essential host factors [ , [ ] [ ] [ ] [ ] . hence, we considered ab as a suitable marker for emcv ros and compared the staining of pi ka to that of ab in infected cells. we observed a striking overlap of gfp-pi ka with ab-positive structures ( fig b, lower panels) and could confirm this phenotype when analyzing the localization of ectopically expressed pi ka bearing an ha-tag (s fig). by contrast, the signal for gfp-pi kb, which was mainly localized at the golgi in non-infected cells (fig c, top panel) , failed to overlap with ab ( fig c, lower panels) . interestingly, although in close proximity to dsrna signals ( fig a, lower panel) , pi ka did not clearly overlap with dsrna (fig a, insets) . taken together, these data demonstrated that pi ka is selectively recruited to emcv ros. interestingly, we noticed a loss of the typical golgi localization of pi kb in emcv-infected cells ( fig c, lower panel) , suggesting that golgi integrity might be affected upon emcv infection. prompted by this and our finding that emcv utilizes the er-localized pi ka for replication, we set out to elucidate whether other er or golgi components are present at emcv ros. in order to be able to use more antibody combinations in immunofluorescence, we constructed a recombinant emcv bearing an ha-tag in the nonstructural protein c. the tag was introduced after the second amino acid, leaving the b- c cleavage site intact (s a fig), and did not impair virus replication (s b fig). first, we checked whether c-ha and ab are present on the same membranes by immunofluorescence microscopy. indeed, c and ab signals greatly overlapped (s c fig), supporting the idea that these proteins occupy the same membranes of the ros. using this tagged emcv, we noticed that the golgi structure was indeed altered in infected cells, from h p.i. onwards, as revealed by the dispersed pattern of both cisand trans-golgi markers gm ( fig a) and tgn , respectively ( fig b) . however, neither tgn nor gm were present at c-ha-positive structures, suggesting that emcv ros are not golgi-derived. ergic , a marker of the er-golgi intermediate compartment also , cells were infected with emcv (a-c) or rluc-emcv (d) at an moi of . or transfected with full-length infectious emcv in vitro-transcribed rna (c). after h, cells were freeze-thawed to release intracellular virus particles and the total virus titers were determined by endpoint titration (a-c). alternatively, cells were lysed and renilla luciferase activity was determined as a measure of viral rna replication (d). in parallel, a cell viability assay was performed to evaluate the cytotoxicity of the sirna treatment (e). (f) western blot analysis showing efficient knockdown of pi ka and pi kb. actin was used as loading control. (g-i) al- treatment inhibits cardiovirus replication. hela r cells were infected with virus at an moi of . (g and h) or (i), followed by al- treatment for h, after which cells were lysed and virus replication was measured by endpoint titration (g and i) or by determining the renilla luciferase activity (h). cytotoxicity of al- was determined in a cell viability assay run in parallel (j). bars represent mean values of triplicates ± standard error of the means (sem). means were statistically compared using unpaired t tests. *p < . , **p< . ; ***p< . . appeared scattered throughout the cytoplasm in infected cells, but without overlapping c-ha ( fig c) . we next compared the localization of ab with sec (copii-coatomer complex component), an er exit site (eres) marker, and the er marker calreticulin. while in noninfected cells, sec displayed mainly a typical perinuclear localization, in emcv-infected cells it appeared dispersed, but without significantly colocalizing with ab ( fig d, mander's colocalization coefficient m = . ± . , fraction of sec overlapping ab). we observed a greater degree of overlap between ab and calreticulin ( fig e, m = . ± . , fraction of calreticulin overlapping ab). images acquired with higher magnification revealed that most of ab was in close contact with er tubules (fig f) . taken together, these data suggested that emcv possibly replicates on er-derived membranes. based on the extensive overlap between pi ka and ab and the drastic change in pi ka pattern in infected cells, we hypothesized that pi ka might be recruited to replication sites by interacting (directly or indirectly) with one or more of the viral nonstructural proteins. to investigate this, we used the stable cell line huh -lunet/t that allows ectopic protein expression under the control of a t promoter and has been previously optimized and validated as a reliable and reproducible cellular system to study pi ka-protein interactions by radioactive co-ip assays [ , ] . myc-tagged emcv nonstructural proteins a, b, c, a, ab, c and d were individually co-expressed together with ha-pi ka in huh -lunet/t cells, radioactively labeled, and affinity purified from cell lysates using anti-myc specific antibodies. autoradiography analysis showed that ha-pi ka was specifically co-purified by a and ab, but not by the other viral proteins (fig a) . to confirm this interaction by co-immunoprecipitation (co-ip) coupled with western blot analysis, myc-tagged emcv a was co-expressed with ha-pi ka and subjected to affinity purification using either monoclonal or polyclonal antimyc antibodies. as shown in fig b, ha-pi ka only interacted with emcv a, but not with cvb a, which interacts with pi kb [ , , ] and was included here as a negative control. these data implied that emcv nonstructural protein a is responsible for pi ka recruitment to ros. interestingly, a diffuse band just below kda appears to co-purify with emcv c when ha-pi ka is co-expressed ( fig a, indicated by à ). we reasoned this could be indicative of a temporal regulation of the pi ka activity during infection via c-dependent degradation. to explore this possibility, we performed western blot analysis of endogenous pi ka during the time course of infection, but did not detect any bands indicative of degradation, neither in huh -lunet/t or hela r cells (s fig). to test if a alone can recruit pi ka, we examined by immunofluorescence the subcellular localization of ha-pi ka when co-expressed with a, ab or b, which we considered as a negative control. when expressed alone, ha-pi ka localized throughout the cell in a diffuse pattern (fig c, top panel) , as previously described [ ] . emcv a-and ab-myc were both localized throughout the cytoplasm and at discrete punctate structures, of which a subset was also positive for pi ka ( fig c) . b-myc was also distributed in punctae throughout the cytoplasm, but failed to recruit pi ka (fig c) . collectively, these results indicated that emcv a is the viral protein responsible for engaging pi ka in replication. while pi kb produces pi p at golgi membranes, pi ka is responsible for the synthesis of the pi p pool at the plasma membrane, where it dynamically localizes [ , [ ] [ ] [ ] . our finding that pi ka activity was critical for emcv rna replication prompted us to investigate whether pi p metabolism is altered during virus replication. given that emcv replicates on the picornavirus emcv converges on the host lipid pathway used by hcv intracellular membranes, we monitored potential changes in the subcellular distribution of both plasma membrane (pm) and intracellular (ic) pools of pi p in huh lunet/t cells following emcv infection. the two pools of pi p can be selectively visualized using two different immunocytochemistry protocols previously established by hammond et al [ ] . while the plasma membrane pool of pi p appeared unaffected in emcv-infected cells (fig a) , the intracellular pi p distribution changed from a perinuclear, golgi-like pattern in mock-infected cells to dispersed throughout the cytoplasm in emcv-infected cells ( fig a) . we observed similar pi p phenotypes in hela cells (s fig), indicating that the observed effects were not cell line-specific. notably, quantitative analysis of the fluorescent pi p signals revealed a marked increase in the level of intracellular pi p in infected cells ( fig b) . to rule out a possible involvement of pi kb in establishing the elevated pi p levels observed in emcv infected cells, we treated cells with the pi kb inhibitor bf (compound ) [ ] . for simultaneous detection of pi p and viral ros by immunofluorescence, we infected cells with emcv- c-ha. short treatment with bf severely depleted the golgi pi p pool in non-infected cells (fig c) , thus reflecting an effective inhibition of pi kb activity. however, the pi p phenotype remained unaltered in infected cells (fig c) , demonstrating that the emcv-induced accumulation of intracellular pi p was not mediated by pi kb. most pi p localized in the vicinity of c-ha, with at least a small subset of pi p overlapping with c-ha ( fig c) . these data together with the finding that emcv requires pi ka activity suggested that pi ka-derived pi p lipids play a central role in emcv genome replication. various cellular proteins carrying a pi p-binding domain called the pleckstrin-homology domain (ph), such as the ceramide-transfer protein (cert), four-phosphate-adaptor protein (fapp ), or the oxysterol-binding protein (osbp) can sense and specifically bind pi p lipids [ , [ ] [ ] [ ] . recently, we and others showed that enteroviruses generate pi p-enriched membranes to recruit osbp, which in turn exchanges pi p for cholesterol at ros [ , , ] . moreover, we showed that emcv is sensitive to itraconazole, which we identified to be an osbp inhibitor [ ] , and that cholesterol shuttling is important for emcv replication [ ] . we therefore reasoned that in emcv-infected cells one purpose of pi p lipids might be to recruit osbp to replication membranes to support viral rna replication. to test if osbp is required for emcv replication, we efficiently reduced osbp expression in hela cells by sirna gene silencing ( fig a) and evaluated the subsequent effects on emcv replication by end-point titration analysis. replication of emcv was significantly reduced in cells in which osbp was depleted compared to control-treated cells (fig a) , indicating that osbp is required for efficient replication. we further used osw- , an osbp ligand that interferes with normal osbp functioning [ ] , to pharmacologically inhibit osbp and analyze whether its lipid transfer function is linked to emcv infection. using luciferase-encoding emcv, we observed a complete inhibition of genome rna replication after h of treatment with osw- at nanomolar concentrations, with no cytotoxicity present (fig b) . a similar inhibition by osw- was observed when infection was performed at high moi (s fig, moi ) . furthermore, by performing osw- time-of-addition experiments, we excluded the possibility that osbp was involved in early steps in the virus life cycle (fig c) . similar results were obtained when using -hydroxycholesterol ( -hc, fig c) , another established osbp ligand [ , ] . next, we wondered whether endogenous osbp was present at emcv ros and if so, whether this localization was dependent on the pi p pool generated by pi ka. to this end, cells were infected with emcv for . h and then treated with dmso or al- for min to acutely deplete pi p, prior to immunofluorescence analysis. while in non-infected cells osbp huh -lunet/t cells were mockinfected or infected with gfp-emcv at an moi of . at h p.i., cells were fixed and stained with antibodies against pi p using specific protocols for detection of plasma membrane and intracellular pi p pools, as described previously [ ] . nuclei were stained with dapi (blue). (b) quantification of pi p levels by immunofluorescence analysis. huh -lunet/t cells were mock-infected or infected with emcv at an moi of . at h p.i., cells were fixed and stained for intracellular pi p and viral dsrna. the intensities of fluorescent pi p signals from whole-cell z-stacks were quantified using imagej. shown are mean values ± sem of cells per condition. means of mock and infected cells were statistically analyzed using the mann-whitney test. ***p< . . (c) emcv ros contain pi p. huh -lunet/t cells were mock-infected or infected with emcv- c-ha at an moi of . at . h p.i., cells were treated with dmso or μm bf (pi kb inhibitor) for min, then fixed and stained for intracellular pi p and ha using specific antibodies. nuclei were stained with dapi (blue). the crop panels at the bottom depict an enlargement of the boxed areas. scale bars represent μm. the picornavirus emcv converges on the host lipid pathway used by hcv localized throughout the cytoplasm and at the golgi, osbp was mainly found at ros in infected cells, where it largely colocalized with ab ( fig d, pearson's correlation coefficient = . ). since other golgi proteins were not present at the ros (fig a and b) , these results suggested that osbp is specifically recruited by emcv. following inhibition of pi ka by short treatment with al- , we observed a strong and significant reduction of osbp and ab colocalization (fig d, pearson's coefficient = . ). importantly, the subcellular localization of osbp in non-infected cells was not affected by al- treatment (fig d) , demonstrating that the presence of osbp at emcv replication structures is conditioned by pi ka-produced pi p. given the colocalization of osbp with ab, we sought to verify whether emcv a was responsible for osbp recruitment. to this end, myc-tagged emcv a-, ab-or b-myc were ectopically expressed in huh -lunet/t cells and recruitment of endogenous osbp was analyzed by immunofluorescence analysis. in cells expressing a and ab, osbp was redistributed in punctate structures throughout the cytoplasm, with some of these punctae colocalizing with a/ ab (fig e) . by contrast, osbp remained localized at the golgi and did not localize at c-positive punctae (fig e) . these data indicated that during emcv infection, osbp is recruited to ros via a. to test whether osbp is involved in transferring cholesterol to ros in a pi p-dependent manner, cells were infected with emcv for h, treated with al- or osw- for h to block pi ka activity or osbp function respectively, and subjected to immunofluorescence analysis. in non-infected cells, cholesterol mainly localizes at endosomes in the perinuclear area and at the plasma membrane, as visualized by filipin staining [ ] . in infected cells treated with dmso, we detected cholesterol primarily colocalizing with ab-positive structures (pearson's coefficient = . ), while in drug-treated cells this colocalization was markedly reduced (fig , pearson's coefficient = . for al- and . for osw- ). this result confirmed that emcv ros acquire cholesterol through the actions of both pi ka and osbp. (+)rna viruses display immense genetic diversity, yet they all rely on remodeled membranes for viral genome replication. a diverse array of cellular organelles can be remodeled into viral replication structures. for instance, picornaviruses from the genera enterovirus and kobuvirus are thought to replicate at modified golgi membranes [ , , ] , while the flavivirus hcv replicates on a membranous web originated from the er [ ] . to do so, viruses rewire host pathways involved in lipid synthesis and transport to generate replication membranes with unique lipid signatures [ ] [ ] [ ] [ ] . how picornaviruses from the genus cardiovirus build their ros is currently unknown. here, we identified pi ka and osbp as essential host factors for genome replication of the cardiovirus emcv. our data suggest that emcv ros may be derived from the er and that pi ka was recruited to ros by interacting with the viral protein a(b). pi ka recruitment led to a significant increase of intracellular pi p levels in infected cells, which proved important for the downstream recruitment of osbp. finally, data are presented suggesting that the osbp-mediated exchange of pi p and cholesterol at ro-mcss is critical for emcv genome replication and the global organization of ros. membrane alterations in the cytoplasm of cardiovirus-infected cells were already observed decades ago by electron microscopy [ , , ] . as also described for enteroviruses, cardiovirus-induced membranes consist of perinuclear clusters of heterogeneous single-and doublemembrane vesicles (dmvs). while recent studies greatly contributed to our understanding of the origin and biogenesis of enterovirus ros [ , , , , , ] , for cardioviruses these details have remained scarce. in a report by zhang et al it was proposed that emcv subverts the autophagy pathway to promote virus replication and ro formation [ ] . the authors observed induction of autophagy and accumulation of cytoplasmic double-membrane vesicles (dmvs), a hallmark of autophagosomes, upon emcv infection. however, inhibition of autophagy had stronger effects on extracellular than intracellular virus yields, which pointed towards a role of autophagy in virus release. indeed, recent studies using enteroviruses and flaviviruses support the hypothesis that autophagy-derived membranes rather serve as means of non-lytic virus release and spread than as a membrane source for the viral ros [ ] [ ] [ ] [ ] . as opposed to enteroviruses, members of the cardiovirus genus are insensitive to gbf depletion by sirna [ ] or treatment with bfa [ , , ] , a compound that targets gbf and subsequently blocks activation of arf , a key regulator of membrane trafficking in the secretory the picornavirus emcv converges on the host lipid pathway used by hcv pathway. furthermore, cardiovirus replication does not require the golgi-localized pi kb, which is essential for enterovirus replication [ ] . collectively, these data suggested that cardioviruses do not rely on golgi components for replication. in line with these previous findings, we here present data suggesting that emcv may derive its ros from er membranes. confocal microscopy analysis revealed that emcv nonstructural proteins partially overlapped with the er marker calreticulin, but not with markers of eres, ergic, cis-or trans-golgi network, which appeared dispersed in infected cells, even at the earliest stages of infection. furthermore, pi ka, which normally resides at the er network, was redistributed in emcv-infected cells to discrete cytoplasmic structures that also contained the viral protein ab. interestingly, the majority of pi ka-positive punctae were detected in close proximity to viral dsrna, but did not completely overlap with dsrna, suggesting a spatial segregation of dsrna from the viral replication membranes, previously also shown for coronaviruses [ , ] . using a pharmacological inhibitor of pi ka, we prove that emcv and safv, which belong to distinct cardiovirus species, both require the lipid kinase activity for replication. in agreement with this result, we observed elevated pi p levels at intracellular membranes in infected cells, suggesting an important role of pi p lipids in cardiovirus replication. in non-infected cells, osbp plays a critical role in lipid homeostasis by exchanging cholesterol for pi p at the interface of er and golgi membranes, to which it localizes under normal conditions [ ] . in this process, pi p lipids also serve as a membrane anchor for osbp. we hypothesized that in cardiovirus infection, pi p may serve to recruit osbp and cholesterol to viral replication sites. indeed, osbp was present at emcv ros, where it colocalized with the viral protein ab. this colocalization was markedly reduced upon al- treatment, demonstrating that osbp is recruited through pi ka-produced pi p. osbp is an essential cardiovirus host factor, since both genetic depletion by sirna treatment and pharmacological inhibition by osw- and -hc blocked viral genome replication. cholesterol was redistributed to emcv ros upon infection, and treatment with al- or osw- resulted in a significantly reduced colocalization of cholesterol with ab, arguing that accumulation of cholesterol at ros is mediated by both pi ka and osbp. these data are in agreement with our recent findings that cholesterol shuttling is important for cardiovirus genome replication [ ] and that cardioviruses are sensitive to itraconazole, which we recently discovered to be an. inhibitor of osbp [ ] . our results indicate that pi p and cholesterol are vital for the global organization of emcv ros. however, as these lipids fulfill multiple functions in various cellular processes [ , [ ] [ ] [ ] , other roles in virus replication should be envisaged. a potential task of pi p in virus replication may be linked to the pi( , )p synthesis pathway, since pi p is the major precursor of pi( , ) p lipids, which were recently attributed an important role in hcv replication [ ] . cholesterol homeostasis was recently shown to play an important role in efficient pv polyprotein processing [ ] . whether cholesterol also ensures a proper microenvironment that supports cardiovirus polyprotein processing remains to be determined. interestingly and in apparent parallel with the distantly related enteroviruses, exploitation of the pi k-osbp pathway by hcv correlates with the induction of membranes of positive curvature [ ] . by contrast, the flavivirus denv, although closely related to hcv, does not require pi k or osbp [ ] and generates membranes of negative curvature [ ] . hence, the interplay between pi p and cholesterol may dictate the positive curvature of the membranes at which diverse rna viruses replicate their genomes. through co-ip assays, we identified pi ka as a novel interaction partner of emcv proteins a and its precursor ab. emcv a is a small protein ( amino acids) of unknown structure, containing a predicted hydrophobic domain in the c-terminus half. expression of a alone was sufficient for pi ka recruitment in intact cells, arguing that in infection, pi ka is recruited to ros by this viral protein. enteroviruses and kobuviruses recruit pi kb to ros also via their a protein [ , , , ] , raising the possibility that diverse picornaviruses might use an evolutionary conserved and a-mediated mechanism to generate pi p-enriched membranes. however, the a proteins of entero-, kobu-and cardioviruses do not share any apparent sequence similarity, their name simply reflecting the occupancy of the same locus ( a) in the respective viral genomes. with the exception of their catalytic domain, also the pi ka and pi kb isoforms do not share any sequence similarity [ ] . furthermore, unlike a of most enteroviruses, cardiovirus a does not interact with gbf nor blocks protein transport in the secretory pathway when expressed alone [ ] , highlighting the functional diversification associated with these small viral proteins. emcv and hcv converged to recruit a common lipid-modifying pathway in building replication sites several lines of evidence suggest that the picornavirus emcv and the distantly related flavivirus hcv have evolved to exploit common host components in assisting virus rna replication. first, hcv genome replication occurs at the "membranous web" (mw), a network of single and dmvs that, like the emcv ro, also mainly originates from the er [ ] . second, both emcv and hcv express a viral protein dedicated to recruitment of pi ka, in order to induce a pi p-rich environment at the replication sites [ , ] . third, in hcv infection, pi p lipids were also shown to be important for the recruitment of osbp, which mediates cholesterol transfer to the mw [ ] . fourth, inhibition of either pi ka or osbp induced clear alterations in the global distribution of emcv ros, which appeared more "clustered" upon treatment with al- or osw- . a similar clustering effect was also observed for replication structures of the hcv mw upon pi ka or osbp inhibition [ , ] , whereas no obvious disruption of the enterovirus ros was observed upon pi kb or osbp inhibition [ , , ] . together, these observations indicate that emcv and hcv replication structures share critical host components, and possibly also a similar architecture, although the latter still remains to be determined. based on at least two lines of emerging evidence in the context of phylogeny of flavi-and picornaviruses, emcv and hcv have likely converged on rather than retained their functional similarities upon divergence from the common ancestor (fig ) . first, the observed commonalities between emcv and hcv are not shared by other characterized viruses in their respective families, indicating that they are not a manifestation of the properties conserved among the two families. for instance, picornaviruses from different genera exhibit different host factor requirements. members of the cardiovirus genus hijack the er-localized pi ka (this study), whereas members of the enterovirus and kobuvirus genera depend on the golgi-localized pi kb [ , , ] . in contrast, equine rhinitis a virus (erav, member of aphthovirus genus, which is prototyped by fmdv) and hepatitis a virus (hepatovirus genus) seem to replicate independent of both pi kb and pi ka (s fig and [ , ] ). likewise, the flaviviruses denv and wnv, representing a sister genus to that of hcv, do not rely on either pi ka or pi kb [ , ] . while denv was also shown not to require osbp [ ] , for wnv this is not known yet. importantly, cardioviruses targeting pi ka occupy a lineage that is farther from the root compared to those of entero-and kobuviruses targeting pi kb (fig ) . this phylogenetic pattern is indicative of the relatively recent emergence of the emcv-specific target properties. second, emcv and hcv employ apparently unrelated proteins to mediate the interaction with pi ka, namely a and ns a (although hcv ns b may contribute as well [ , ] ). both proteins are membrane-bound, albeit through a hydrophobic domain located at either n-terminus (ns a) or in the c-terminus-half ( a), and each includes another region which is among the least conserved in the nonstructural proteins of the respective families [ , ] . our study contributes to the hypothesis that viruses may be confronted with powerful constraints that limit the diversity of host pathways recruited for efficient replication. thus, a common pathway is used by different rna viruses that either only moderately diverged (e.g. different species of same genus) or converged on a host target while diverging profoundly (different families-e.g. emcv and hcv). to date, only a small number of (+)rna viruses have been studied in terms of host lipid requirements. identification of the lipid pathways used by other viruses will hopefully provide a deeper insight on the constraints that viruses are confronted with during the endeavor to replicate their genome. buffalo green monkey (bgm) kidney cells, baby hamster kidney (bhk- ) and hela r cells were grown at °c and % co in dulbecco's modified eagle's medium (dmem, lonza) supplemented with % fetal bovine serum (fbs). huh lunet/t cells (provided by r. bartenschlager, department of molecular virology, university of heidelberg, heidelberg, germany) [ ] were grown in dmem (lonza) supplemented with % fbs and μg/ml blasticidin (paa) . bgm cells were purchased from ecacc and bhk- cells were purchased from atcc. hela r cells were obtained from g. belov (university of maryland and virginia-maryland regional college of veterinary medicine, us) [ ] . al- and osw- were kind gifts from j. neyts (rega institute for medical research, university of leuven, leuven, belgium) and m.d. shair (department of chemistry and chemical biology, harvard university, cambridge, usa) respectively. -hc was purchased from santa cruz. bf [ ] was provided by galapagos nv. filipin iii and dipyridamole were from sigma. [ ] , and the entire tree is rooted according to gibrat et al., [ ] . *in addition to ns a, ns b has also been suggested to be involved in the interaction with pi ka [ , ] the picornavirus emcv converges on the host lipid pathway used by hcv plasmids constructs ptm-ha-pi ka [ ] , pegfp-pi ka (provided by g. hammond, nichd, national institutes of health, bethesda, usa) [ , ] and p a(cvb )-myc [ ] were described previously. pgfp-pi kb was a kind gift from n. altan-bonnet (laboratory of host-pathogen dynamics, national institutes of health, bethesda, usa). to generate c-terminal myc-tagged emcv proteins, genes encoding emcv nonstructural proteins a, b, c, a, ab, c and d were amplified by pcr using the plasmid pm . [ ] and primers introducing restriction sites bamhi and hindiii. pm . contains the full-length infectious cdna sequence of emcv, strain mengovirus. the pcr products were then cloned into the p a (cvb )-myc backbone from which the cvb - a gene was removed using the same restriction enzymes. to allow ectopic expression of pi ka under a cmv promoter, the gene encoding ha-pi ka was amplified by pcr using ptm-ha-pi ka as template and introduced in the pegfp-n backbone using restriction enzymes sali and noti. emcv- c-ha infectious clone was generated by introducing the ha coding sequence (ypydvpdya) in-frame after codon in c of pm . using mutagenesis primers and the q site-directed mutagenesis kit (new england biolabs). emcv, emcv- c-ha and rluc-emcv, which contains the renilla luciferase gene upstream of the capsid coding region [ ] , were obtained by transfecting bhk- cells with rna transcripts derived from full length infectious clones pm . , pm . - c-ha and prluc-qg-m . , respectively, linearized with bamhi. gfp-emcv, which contains the egfp gene upstream the capsid region, was generated similar as rlucemcv [ ] . cvb (strain nancy) was obtained by transfecting bgm cells with rna transcripts of the full length infectious clone p cb /t [ ] linearized with sali. saffold virus (type ) was described previously [ ] . erav (nm / ) was kindly provided by david rowlands and toby tuthill (university of leeds, united kingdom). virus infections were performed by incubating subconfluent cell monolayers for min at °c with virus, after which the virus-containing medium was removed and fresh (compound-containing) medium was added to the cells (t = ). in the time-of-addition experiments, medium without compound was added at t = and replaced by medium with compound at the indicated time points. at the given time points post infection, cells were either fixed for immunolabeling, freeze-thawed to determine virus titers or, in the case of rluc-emcv, lysed to determine replication by measuring the intracellular renilla luciferase activity using the renilla luciferase assay system (promega). virus titers were determined by endpoint titration according to the method of reed and muench and expressed as % tissue culture infective doses (tcid ). hela r or huh lunet/t cells were grown to subconfluency on coverslips in -well plates. where indicated, cells were transfected with ng of plasmids using lipofectamine according to the manufacturer's protocol and/or infected with emcv at the specified multiplicity of infection (moi), followed by compound treatment where specified. at the indicated time points, cells were fixed with % paraformaldehyde (pfa) for min at room temperature (rt). permeabilization was done with pbs- . % triton x- for min or pbs/ . % saponin/ % bsa for min, in the case of filipin staining. cells were incubated sequentially with primary and secondary antibodies diluted in pbs containing % normal goat serum (ngs). the following primary antibodies were used for detection: mouse monoclonal anti-gm (bd biosciences), rabbit polyclonal anti-tgn (novus biologicals), mouse monoclonal anti-ergic (enzo life sciences), rabbit polyclonal anti-sec (kindly provided by b.l tang, department of biochemistry, the national university of singapore, singapore), rabbit polyclonal anti-calreticulin (sigma), rabbit polyclonal anti-ha (santa cruz), mouse monoclonal anti-ha (abcam), mouse monoclonal anti-c-myc (sigma), rabbit polyclonal anti-myc (thermo scientific), mouse anti-pi p igm (echelon biosciences), mouse monoclonal anti-dsrna (j , english & scientific consulting), mouse monoclonal anti-emcv ab (kind gift from a.g. aminev) [ ] and rabbit polyclonal anti-osbp (kindly provided by m.a. de matteis, telethon institute of genetics and medicine, naples, italy) [ ] . alexa fluor -, -conjugated igg and alexa fluor -or -conjugated igm (invitrogen, molecular probes) were used as secondary antibodies. cholesterol was stained with μg/ml filipin iii (sigma) for h at room temperature, included during the incubation with the secondary antibody. nuclei were counterstained with dapi. staining of plasma membrane or intracellular pi p was performed as described elsewhere [ ] . briefly, for pm staining, cells were fixed at rt in % pfa and . % glutaraldehyde. all subsequent steps were performed on ice. cells were blocked and permeabilized for min in buffer a ( mm pipes, ph . , mm nacl, . mm kcl) containing % ngs, mm nh cl and . % saponin. slides were incubated with primary and secondary antibodies in buffer a containing % ngs and . % saponin for h. finally, slides were post-fixed in % pfa in pbs for min. the intracellular pi p staining was performed at rt as follows: cells were fixed with % pfa, then permeabilized for min in μm digitonin in buffer a, blocked for min in buffer a with % ngs and mm nh cl and then incubated sequentially with primary and secondary antibodies in buffer a with % ngs, before post fixation in % pfa. all coverslips were mounted with fluorsave (calbiochem). images were acquired with a leica spe-ii dmi- confocal laser scanning microscope or a nikon ti eclipse microscope equipped with an andor du- emccd-camera. pi p quantification was performed for at least cells for each condition, using the imagej software as described elsewhere [ ] . to determine colocalization of sec or calreticulin with ab, images were first deconvoluted using nis advanced research . software (nikon) ( iterations) and further processed using image j as follows. individual infected cells were outlined and a mask was created, and all signal outside the mask was cropped to exclude it from the calculations. manders' colocalization coefficient was calculated for at least cells for each condition using the jacop plugin [ ] with a manually set threshold. colocalization of osbp with a in infected cells was analyzed using imagej by determining pearson's coefficient for at least cells per condition using the coloc plugin with default settings. to quantify colocalization of filipin with ab, images were first deconvoluted using nis software ( iterations), then imagej was used to select infected cells and the pearson's coefficient of colocalization for at least cells per condition was calculated using the coloc plugin with default settings. hela r cells were reverse-transfected with pmoles of sirna per well of a -well plate ( cells/well) using lipofectamine (invitrogen) according to the manufacturer's indications. scrambled sirna (allstars neg. control, qiagen) was used as a control. sirna against hpi ka (cat. no. s ) and hpi kb (target sequence: '-uguuggggc uucccugccctt- ') were from qiagen. sirna against hosbp (two sirnas mixed at : ratio, cell viability was determined in parallel with virus infection as follows. one day after seeding cells in a -well plate, the compounds were added to the cells and incubated for h. alternatively, cells were transfected with sirnas and incubated for h. subsequently, the medium was replaced with celltiter aqueous one solution reagent (promega) and optical densities were measured at nm. the obtained raw values were converted to percentage of untreated samples or samples transfected with scrambled sirnas, following correction for background absorbance. metabolic labeling of myc-tagged emcv proteins and ha-pi ka was performed as described elsewhere [ ] . briefly, huh -lunet/t cells seeded in -well plates were co-transfected with μg of plasmid encoding emcv nonstructural proteins and μg of either ptm ha-pi kiiia or an empty ptm vector (mock) using lipofectamine (invitrogen) according to the manufacturer's instructions. h later, cells were starved in methionine/cysteine-free medium for h. radiolabeling of cells was done by overnight incubation in methionine/cysteine-free medium, supplemented with mm glutamine, mm hepes, and μci/ml of express protein labeling mix (perkin elmer, boston). cells were then harvested and lysed in lysis buffer ( mm tris-cl [ph . ], mm nacl, % nonidet p- and protease inhibitors) for h on ice, followed by centrifugation at , g for min at °c. supernatants were further subjected to immunoprecipitation by a h incubation at °c with anti-c-myc rabbit polyclonal antibody (santa cruz). immunocomplexes were then captured with protein g-sepharose beads (sigma) by an additional h incubation at °c. beads were washed three times in lysis buffer, followed by elution of immunocomplexes by boiling in sample buffer, separation by polyacrylamide-sds gel electrophoresis and detection by autoradiography. for co-ip followed by western blot, cells were seeded in cm dishes and transfected with . μg of each plasmid using polyethylenimine (pei) (polysciences). immunoprecipitation was carried out as described above, but using protein a-sepharose beads (ge healthcare) and mouse monoclonal anti-c-myc (sigma) or rabbit polyclonal anti-myc (thermo scientific) antibodies. samples separated by sds-page were transferred to nitrocellulose membranes (bio-rad). membranes were incubated with the following primary antibodies: rabbit polyclonal anti-pi ka (cell signaling), rabbit polyclonal anti-pi kb (upstate), rabbit polyclonal anti-osbp (proteintech), rabbit polyclonal anti-emcv capsid (kind gift from ann palmenberg) and mouse monoclonal anti-β-actin (sigma). secondary antibodies included irdye -conjugated goat anti-mouse or irdye -conjugated goat anti-rabbit (li-cor). images of blots were acquired with an odyssey fc imaging system (li-cor). where indicated, unpaired one-tailed student's t-test or two-tailed mann-whitney test were applied as statistical analyses using the graphpad prism software. supporting information s fig. al- inhibits emcv at the step of genome replication. (a) al- is effective against emcv also at high moi. hela r cells were infected with rluc-emcv at an moi of followed by al- treatment for h, after which cells were lysed and virus replication was measured by determining the renilla luciferase activity. (b) al- blocks emcv genome replication. hela r cells were infected with rluc-emcv at an moi of . at the indicated time points, dmso, μm al- or μm dipyridamole was added to the cells and virus replication was measured by determining the renilla luciferase activity at h p.i. bars represent mean values of triplicates ± standard error of the means (sem). means were statistically compared using unpaired t tests. hela r cells were mockinfected or infected with gfp-emcv at moi . at h p.i., cells were fixed and stained with antibodies against pi p using specific protocols for detection of plasma membrane and intracellular pi p pools [ ] . nuclei were stained with dapi (blue). scale bars represent μm. the genetics of the persistent infection and demyelinating disease caused by theiler's virus saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life molecular analysis of encephalomyocarditis viruses isolated from pigs and rodents in italy persistence of encephalomyocarditis virus (emcv) infection in piglets reproductive failure in pigs caused by encephalomyocarditis virus reproductive failure in sows following experimental infection with a belgian emcv isolate +)rna viruses rewire cellular pathways to build replication organelles rewiring of cellular membrane homeostasis by picornaviruses requirements for assembly of poliovirus replication complexes and negative-strand rna synthesis induction of membrane proliferation by poliovirus proteins c and bc membrane rearrangement and vesicle induction by recombinant poliovirus c and bc in human cells a viral protein that blocks arf -mediated cop-i assembly by inhibiting the guanine nucleotide exchange factor gbf molecular determinants of the interaction between coxsackievirus protein a and guanine nucleotide exchange factor gbf hijacking components of the cellular secretory pathway for replication of poliovirus rna viral reorganization of the secretory pathway generates distinct organelles for rna replication recruitment of pi kiiiβ to coxsackievirus b replication organelles is independent of acbd , gbf , and arf rhinovirus uses a phosphatidylinositol -phosphate/cholesterol counter-current for the formation of replication compartments at the er-golgi interface phosphatidylinositol- kinase iii beta and oxysterol-binding protein accumulate unesterified cholesterol on poliovirus-induced membrane structure a four-step cycle driven by pi( )p hydrolysis directs sterol/pi( )p exchange by the er-golgi tether osbp complex dynamic development of poliovirus membranous replication complexes the transformation of enterovirus replication structures: a three-dimensional study of single-and doublemembrane compartments oxysterol-binding protein is a phosphatidylinositol -kinase effector required for hcv replication membrane integrity and cholesterol trafficking recruitment and activation of a lipid kinase by hepatitis c virus ns a is essential for integrity of the membranous replication compartment morphological and biochemical characterization of the membranous hepatitis c virus replication compartment hepatitis c virus stimulates the phosphatidylinositol -kinase iii alpha-dependent phosphatidylinositol -phosphate production that is essential for its replication autophagy promotes the replication of encephalomyocarditis virus in host cells inhibition of cellular autophagy deranges dengue virion maturation coxsackievirus b exits the host cell in shed microvesicles displaying autophagosomal markers nonlytic viral spread enhanced by autophagy components phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses gbf , a guanine nucleotide exchange factor for arf, is crucial for coxsackievirus b rna replication differential effects of the putative gbf inhibitors golgicide a and ag on enterovirus replication a novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol -kinase iiiβ coxsackievirus mutants that can bypass host factor pi kiiiβ and the need for high levels of pi p lipids for replication metabolism of phosphatidylinositol -kinase iiiα-dependent pi p is subverted by hcv and is targeted by a -anilino quinazoline with antiviral activity cytopathology of mengovirus infection ii. proliferation of membranous cisternae effect of mengovirus replication on choline metabolism and membrane formation in novikoff hepatoma cells differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae mapping of functional domains of the lipid kinase phosphatidylinositol -kinase type iii alpha involved in enzymatic activity and hepatitis c virus replication ptdins p synthesis by pi kiiiα at the plasma membrane and its impact on plasma membrane identity determinants of membrane association for poliovirus protein ab properties of purified recombinant poliovirus protein ab as substrate for viral proteinases and as co-factor for rna polymerase dpol interaction between the '-terminal cloverleaf and ab/ cdpro of poliovirus is essential for rna replication the a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd to recruit pi kiiiβ the lipid kinase phosphatidylinositol- kinase iii alpha regulates the phosphorylation status of hepatitis c virus ns a a plasma membrane pool of phosphatidylinositol -phosphate is generated by phosphatidylinositol -kinase type-iii alpha: studies with the ph domains of the oxysterol binding protein and fapp maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol -kinase iiialpha pharmacological and genetic targeting of the pi ka enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol -phosphate and phosphatidylinositol , -bisphosphate levels immunocytochemical techniques reveal multiple, distinct cellular pools of ptdins p and ptdins( , )p( ) phosphatidylinositol -kinase iiibeta regulates the transport of ceramide between the endoplasmic reticulum and golgi targeting of golgi-specific pleckstrin homology domains involves both ptdins -kinase-dependent and-independent components the multiple roles of ptdins( )p-not just the precursor of ptdins( , )p cholesterol shuttling is important for rna replication of coxsackievirus b and encephalomyocarditis virus natural products reveal cancer cell dependence on oxysterol-binding proteins translocation of oxysterol protein to golgi apparatus triggered by ligand binding acbd -mediated recruitment of pi kb to picornavirus rna replication sites three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication modulation of lipid synthesis and trafficking pathways by picornaviruses membranous replication factories induced by plus-strand rna viruses ultrastructure of the replication sites of positive-strand rna viruses architecture and biogenesis of plus-strand rna virus replication factories replication of theiler's murine encephalomyelitis viruses in bhk cells: an electron microscopic study increased long chain acyl-coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin a visualizing coronavirus rna synthesis in time by using click chemistry sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum the lipid kinase pi kiiiβ preserves lysosomal identity gabaraps regulate pi p-dependent autophagosome:lysosome fusion cinderella story: pi p goes from precursor to key signaling molecule phosphatidylinositol , -bisphosphate is an hcv ns a ligand and mediates replication of the viral genome enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication composition and three-dimensional architecture of the dengue virus replication and assembly sites phosphatidylinositol -kinases: old enzymes with emerging functions effects of picornavirus a proteins on protein transport and gbf -dependent cop-i recruitment novel perspectives for hepatitis a virus therapy revealed by comparative analysis of hepatitis c virus and hepatitis a virus rna replication west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol- -phosphate lipids the ns a protein of hepatitis c virus is a zinc metalloprotein analyses of the radiation of birnaviruses from diverse host phyla and of their evolutionary affinities with other double-stranded rna and positive strand rna viruses using robust structure-based multiple sequence alignments and advanced phylogenetic metho role of annexin a in the production of infectious hepatitis c virus particles poliovirus proteins induce membrane association of gtpase adpribosylation factor identification of a series of compounds with potent antiviral activity for the treatment of enterovirus infections a novel probe for phosphatidylinositol -phosphate reveals multiple pools beyond the golgi a proline-rich region in the coxsackievirus a protein is required for the protein to inhibit endoplasmic reticulum-to-golgi transport cloning and synthesis of infectious cardiovirus rnas containing short, discrete poly(c) tracts encephalomyocarditis viral protein a localizes to nucleoli and inhibits cap-dependent mrna translation a guided tour into subcellular colocalization analysis in light microscopy we are grateful to raffaele defrancesco, francesco peri, petra neddermann and johan neyts for providing al- , matthew shair for osw- , gerrald hammond for the plasmid pegfp-pi ka, nihal altan-bonnet for the plasmid pgfp-pi kb, maria antonietta de matteis for the rabbit polyclonal antibody against osbp, aleksey aminev for the mouse monoclonal antibody against ab (emcv), ann palmenberg for the rabbit polyclonal antibody against emcv capsid, bor luen tang for the rabbit polyclonal antibody against sec and the center for cell imaging (faculty of veterinary medicine, utrecht university) for support with microscopy experiments. key: cord- -vy yirek authors: baró, j.; segalés, j.; martínez, j. title: porcine circovirus type (pcv ) enteric disease: an independent condition or part of the systemic disease? date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: vy yirek intestinal disorders in growing and finishing pigs have been associated with several infectious agents, including porcine circovirus type (pcv ). this virus has been mainly related with pcv -systemic disease (pcv -sd); nevertheless, some authors have suggested a possible restricted intestinal infection of this virus associated with enteric clinical signs. this condition has been referred as pcv -enteric disease (pcv -ed). the present study analysed retrospectively, from a pathological point of view, the relation between intestinal disorders and pcv infection in nursery and growing-finishing pigs. among the selected pigs suffering from enteric disease and submitted for necropsy between and , the most prevalent enteric lesions were catarrhal enteritis/colitis ( . %), followed by fibrinous lesions ( . %), granulomatous inflammation ( . %) and other lesions such as haemorrhages or ulceration ( . %). seventy-two pigs ( %) were positive for pcv by in situ hybridization (ish). among positive pigs for pcv ish, animals suffered from pcv -sd and had no lymphoid lesions but low amount of viral nucleic acid in several lymphoid tissues, therefore, these animals did not qualify for pcvd-ed. in conclusion, all animals with enteric disorders that were positive to pcv by ish had evidence of viral systemic infection. these results suggest that pcv -ed is probably a negligible condition and pcv mainly contributes to enteric clinical disorders in relation to pcv -sd occurrence. intestinal disorders in growing-finishing pigs are a common problem in most commercial farms and significantly limit the efficiency and profitability of swine production globally. clinical presentation consists of diarrhoea, poor growth and variable mortality. intestinal disorders in pigs older than four weeks of age can be produced by several infectious agents such as rotaviruses, some coronaviruses, porcine circovirus type (pcv ), escherichia coli, brachyspira spp., salmonella spp., yersinia spp., lawsonia intracellularis, oesophagostomum dentatum and trichuris suis, among others. however, most cases featuring postweaning diarrhoea and colitis are due to concurrent aetiologies (thomson and friendship, ) . pcv is recognized as one of the most important viruses causing severe economic impact in the swine industry worldwide and it has been described to cause different conditions depending on the virus, host immunity, veterinary microbiology ( ) intestinal disorders in growing and finishing pigs have been associated with several infectious agents, including porcine circovirus type (pcv ). this virus has been mainly related with pcv -systemic disease (pcv -sd); nevertheless, some authors have suggested a possible restricted intestinal infection of this virus associated with enteric clinical signs. this condition has been referred as pcv -enteric disease (pcv -ed). the present study analysed retrospectively, from a pathological point of view, the relation between intestinal disorders and pcv infection in nursery and growing-finishing pigs. among the selected pigs suffering from enteric disease and submitted for necropsy between and , the most prevalent enteric lesions were catarrhal enteritis/colitis ( . %), followed by fibrinous lesions ( . %), granulomatous inflammation ( . %) and other lesions such as haemorrhages or ulceration ( . %). seventy-two pigs ( %) were positive for pcv by in situ hybridization (ish). among positive pigs for pcv ish, animals suffered from pcv -sd and had no lymphoid lesions but low amount of viral nucleic acid in several lymphoid tissues, therefore, these animals did not qualify for pcvd-ed. in conclusion, all animals with enteric disorders that were positive to pcv by ish had evidence of viral systemic infection. these results suggest that pcv -ed is probably a negligible condition and pcv mainly contributes to enteric clinical disorders in relation to pcv -sd occurrence. ß elsevier b.v. all rights reserved. co-infections and other environment characteristics (segalé s et al., ; madec et al., ) . these diseases are grouped under the name porcine circovirus diseases (pcvds). pcvds include pcv -systemic disease (pcv -sd, formerly known as postweaning multisystemic wasting syndrome, pmws), porcine dermatitis and nephropathy syndrome (pdns), pcv reproductive disease (pcv -rd), pcv -lung disease (pcv -ld) and pcv -enteric disease (pcv -ed) (segalé s, ). diarrhoea is a feature of some pcvds and has been described in cases of pcv-sd and pcv-ed (kim et al., ; segalé s et al., ; opriessnig et al., ) . pcv -sd occurs in pigs between one and six months of age with the highest number of cases occurring between two and three months (segalé s and cortey, ). it is a multifactorial disease, in which infection by pcv is essential but other factors are usually required for its complete expression (segalé s et al., ; madec et al., ) . from a clinical point of view, there may be a potential diagnostic overlapping between pcv -sd and pcv -ed since diarrhoea can be easily present in cases of the systemic disease (opriessnig et al., ) . however, some authors have suggested that pcv -ed could be a distinct clinical manifestation of pcv- (kim et al., ) . to differentiate between these two conditions, histopathological findings and examination not only of gut but also lymphoid tissues is crucial. pcv-sd is diagnosed when the following are present: (a) clinical signs consisting of weight loss and paleness of skin (respiratory and/or digestive clinical signs may be present as well), (b) moderate to severe lymphocyte depletion with granulomatous inflammation of lymphoid tissues or other organs, and (c) moderate to high amount of pcv in damaged tissues. on the other hand, pcv-ed is diagnosed when (a) pigs suffer from diarrhoea; (b) a granulomatous enteritis and lymphocyte depletion is observed microscopically in peyer's patches, but not in other lymphoid tissues, and (c) a moderate to high amount of pcv is detected in intestinal mucosa or peyer's patches without detection in other lymphoid tissues (segalé s, ) . from a case definition point of view, pcv -ed and pcv -ld share a common characteristic, which is the presence of lesions and pcv antigen/genome in intestines and lung, respectively, in absence of lesions and viral detection in systemic locations (segalé s, ). however, the specific study of pcv -ld in a set of pigs suffering from respiratory disease, indicated that this condition is probably negligible and pcv mainly contributes to respiratory disease in relation to pcv -sd occurrence (ticó et al., ) . this type of assessment has not been yet conducted for pcv -ed. therefore, in order to clarify some points of pcv -ed, the present study aimed to investigate the prevalence of pcv infection in growing-to-finishing pigs with diarrhoea in spain and assess the potential overlap between pcv-ed and pcv -sd. in this study, pigs ( . %) were selected from a total of pigs that were submitted for necropsy between and to the veterinary pathology diagnostic service at the veterinary school of barcelona (spain). animal selection criteria included the following four features: (a) older than four weeks of age (nursery, growing and finishing pigs), (b) clinical digestive signs, (c) availability of intestinal and lymphoid tissues to assess histopathological lesions and to detect pcv dna by in situ hybridization (ish) and (d) presence of microscopic lesions in the intestine. formalin-fixed, paraffin-embedded samples of intestines (portions of jejunum, ileum and colon) and lymphoid organs (mesenteric and inguinal superficial lymph nodes, and tonsil) corresponding to the selected pigs were included in this study. all samples were analysed by the same pathologist. classification of lesions was performed in regards of gross and/or microscopic findings; hence, diagnostic criteria were as follows. catarrhal enteritis or colitis (ce/cc) was consistent with serous or mucous intestinal content in the lumen and moderate to severe lymphoplasmacytic infiltration was observed microscopically within the intestinal mucosa. granulomatous enteritis or colitis (ge/gc) was assessed when multifocal aggregates of macrophages (granulomas), with or without multinucleated giant cells or intracytoplasmic inclusion bodies, and lymphoid depletion were observed in the mucosa and/or peyer's patches. haemorrhagic enteritis (he) was classified when haemorrhagic content was observed in the small intestine. fibrinous enteritis or colitis (fe/fc) consisted of accumulation of fibrin on the mucosa of caecum or colon. ulcerative colitis (uc) was characterized by multifocal to coalescing ulceration of the large intestinal mucosa. mucous-haemorrhagic colitis (mhc) consisted of mucous and haemorrhagic content in the caecal or colonic lumen. lesions were characterized as ''single'' or ''combination'' when one or more lesion types were observed, respectively. diagnosis of pcv -sd was established following internationally accepted criteria previously described (segalé s, ) . regarding the pcv -ed, criteria proposed in the same article were used: ( ) lympho-histiocytic to granulomatous enteritis and lymphocyte depletion with granulomatous inflammation in peyer's patches, ( ) positive ish result for pcv in the intestines, and ( ) absence of pcv genome and histopathologic pcv -sd lesions in other lymphoid tissues (segalé s, ). the presence of pcv in tissues was determined by an ish technique (rosell et al., ) , which was performed on lymph node, tonsil and intestines, using a bp digoxigenin labelled dna probe corresponding to orf of pcv (dig- -cct tcc tca tta ccc tcc tcg cca aca ata aaa taa tca aa- ). quantification of pcv nucleic acid detection was made according to a subjective evaluation of the percentage of positive cells from the totality of the tissue submitted in the slide: -(negative), + (less than %), ++ (between % and %) and +++ (more than %). these samples were analysed in a blinded fashion manner by the same pathologist. all pigs came from different spanish farms, each one with its own particular management, system production and size. from pigs studied, animals ( . %) were necropsied between and (pre-pcv vaccination) and only pigs ( . %) were submitted from to . forty-four animals were among four and eight weeks old (nursery pigs) and fifty-two were older than eight weeks (growing-finishing pigs). a summary of enteric lesions and pcv detection is shown in table , where numbers of pigs with single or combined lesions are given in detail. the majority of pigs displayed a ''single'' lesion ( . %), while only two pigs had a combination of two types of lesions ( . %). the most common lesion was colitis, observed in pigs ( . %), followed by enterocolitis in animals ( . %) and enteritis in pigs ( . %). catarrhal lesions were the most prevalent with pigs affected ( . %), followed by fibrinous lesions observed in animals ( . %), granulomatous inflammation in four ( . %) and other lesions, such as haemorrhages, ulceration or mucohaemorrhagic exudate, in seven pigs ( . %). no lesions compatible with proliferative ileitis were observed. focusing on the age of animals, catarrhal lesions were slightly more frequent in nursery pigs ( . %) compared to growing-finishing animals ( . %). in contrast, fibrinous lesions were more prevalent in the growing-finishing ( . %) than in the nursery period ( , %). only nine animals ( . %) were submitted between - (post-pcv vaccination). in regards of ish positivity for pcv among the intestinal samples, enterocolitis was positive in . % of the animals ( / ) and . % of these cases ( / ) had a diagnosis of pcv -sd. colitis were positive in . % ( / ) and . % ( / ) corresponded to a pcv -sd. enteritis cases were positive to pcv in . % of cases ( / ) and . % of animals ( / ) where diagnosed as a pcv -sd. besides, there was a group of animals where pcv was not detected in the intestine, but yielded positive in lymph nodes and tonsils; this occurred in . % of pigs ( / ); from them, corresponded with a pcv-sd diagnosis. taking only into account the positivity for pcv in the intestinal lesions, from the pigs with intestinal lesions, ( %) were positive for pcv ish and pigs were negative for this virus in intestine and lymphoid tissues (fig. ) . thirty-nine pigs out of the pcv ish positive ( . %) suffered from pcv -sd. the remaining pigs with pcv ish positivity in the intestine had also low or moderate quantity of viral genome in lymph nodes and tonsil. therefore, none of the studied animals qualified for a pcv -ed diagnosis. intestinal disorders are a common health problem in postweaning pigs. however, in the present work, considering the selection criteria, a small percentage of animals were investigated compared to other similar studies performed in pigs with respiratory disorders (ticó et al., ) . several factors could be involved in this low casuistic. first, the necessary presence of gross or microscopic intestinal lesions to be included in the study, as some intestinal disorders such as osmotic or dietary diarrhoeas do not produce lesions. second, most intestinal pcv -sd: porcine circovirus type systemic disease. + ish-pcv was negative in all the tissues analysed. * ish-pcv was negative in the intestine but positive in lymph nodes and tonsil. disorders are usually treated with antibiotics or diet restriction and no animals are submitted for necropsy unless there is no recovery of clinical signs. and third, casuistic of digestive problems in nursery and growingfattening pigs were of lower prevalence than respiratory disorders in the study period. different agents have been involved, including infectious and non-infectious factors, in intestinal disorders in growing-fattening pigs (thomson and friendship, ) . although bacteriological analyses were not performed in the studied cases, from a pathological point of view, intestinal lesions are usually related with specific infectious agents (segalé s and martínez, ). the most prevalent intestinal lesion was catarrhal inflammation of different parts of the intestine. ce is usually associated with colibacillosis, or less frequently with yersiniosis; on the other hand, cc is generally related with the infection of some species of brachyspira. however, other noninfectious factors, such as the composition of diet, could also be involved. another less prevalent lesion was fe/fc which is generally associated with salmonella spp. infections. other type of lesion, such as he and uc or mhc, usually associated with clostridiasis or swine dysentery, appeared in a very small number of cases. surprisingly, only four pigs had granulomatous enteritis (highly suggestive of pcv infection) compared to the high rate of detection of this virus in the intestine or other organs of these animals. these findings suggest that intestinal disorders exclusively attributed to pcv infection in the intestine could be low, but this virus may predispose to opportunistic bacterial overgrowth or dysbacteriosis. pcv has been included in the list of agents involved in the intestinal disorders of these animals (kim et al., ; segalé s et al., ; opriessnig et al., ) . evidence of immunosupression in pcv infected pigs has extensively been reported in regards of several features; for example, the lymphoid depletion observed in infected pigs and the association of pcv -sd with opportunistic pathogens (carrasco et al., ; segalé s and mateu, ; zlotowski et al., ) . another virus associated with immunosuppression is porcine reproductive and respiratory syndrome virus (prrsv) . in the present study, the presence of this virus in lung and lymphoid tissues was analysed by immunohistochemistry in from the pigs, but only were positive (data not shown). in contrast, the present results showed a high prevalence of pcv infection in postweaning pigs with digestive disorders, reinforcing the hypothesis of altered immune response in animals infected by this virus. in this way, only a small percentage of animals corresponded to post-pcv vaccination period. the diagnostic overlapping between pcv -sd and pcv -ed has been previously discussed (opriessnig et al., ; segalé s, ) . kim et al. ( ) proposed that pcv -associated enteritis (equivalent to pcv -ed) should be differentiated from pcv -sd based on clinical and histopathological criteria. however, in the present study, most of pigs showing clinical intestinal disorders with pcv involvement were histopathologically diagnosed as pcv -sd or suffered from subclinical systemic infection of the virus. moreover, the scenario in which pcv was present in intestine but not in lymphoid tissues, as requested to diagnose pcv -ed (segalé s, ), did not occur in any case; so that, pcv -ed as specific clinicopathological entity was absent in the current study. these results coincide with those published by ticó et al. ( ) ; these authors studied the role of pcv associated lung disease (pcv -ld) in cases of respiratory disease of nursery and growing-finishing pigs, but pcv infection exclusively restricted to the lung was not found in any case. all these results support the idea of pcv as a systemic pathogen, rather than virus able to cause a facultative local infection. although, concomitant viral or bacterial agents could be present in the intestinal studied cases (worsening clinical signs), the amount of pcv detected in intestines was never higher than the one detected in lymphoid lesions of the same animal. moreover, in those cases in which pcv -ed was potentially suspected (not diagnosed as pcv -sd), the viral load in intestinal and lymphoid tissues was lower than the amount of virus detected in pcv-sd cases. in fact, obtained findings in pigs which were non-pcv -sd, but pcv -infected, suggest a pcv systemic infection which can be interpreted as: ( ) subclinical pcv- systemic infection, being other pathogens the major contributors of diarrhoea, or ( ) recovery phase from an old pcv -sd infection. the results obtained in this study suggest that pcv is frequent in cases of intestinal disorders in nursery and growing-finishing pigs. from a diagnostic point of view, the present retrospective study shows that the casuistic of enteric disorders cases that could be categorized in the group of pcv -ed was non-existent in contrast to other published data (kim et al., ; opriessnig et al., ) , and pcv mainly contributes to enteric clinical disorders in relation to pcv -sd occurrence. intestinal chlamydial infection concurrent with postweaning multisystemic wasting syndrome in pigs enteritis associated with porcine circovirus in pigs post-weaning multisystemic wasting syndrome and other pcv -related problems in pigs: a -year experience porcine circovirus type associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (pmws) in pigs porcine circovirus type (pcv ) infections: clinical signs, pathology and laboratory diagnosis changes in age at diagnosis of pmws in pigs in spain laboratory diagnosis of enteric disorders immunosuppression as a feature of postweaning multisystemic wasting syndrome porcine circovirus diseases pathological findings associated with naturally acquired porcine circovirus type associated disease digestive system the blurred border between porcine circovirus type -systemic disease and porcine respiratory disease complex porcine reproductive and respiratory syndrome virus (porcine arterivirus) muco-cutaneous candidiasis in two pigs with postweaning multisystemic wasting syndrome the epidemiological data and the paraffin blocks for this study were kindly donated by the servei de diagnòstic de patologia veterinària (sdpv) of the universitat autònoma de barcelona. authors are grateful to the veterinary undergraduate student marta martínez ló pez for her work with the database, and aida neira and blanca pé rez of the sdpv and mó nica pé rez from the centre de recerca en sanitat animal (cresa) for their technical assistance. key: cord- -py awuav authors: willi, barbara; spiri, andrea m.; meli, marina l.; grimm, felix; beatrice, laura; riond, barbara; bley, tim; jordi, rolf; dennler, matthias; hofmann-lehmann, regina title: clinical and molecular investigation of a canine distemper outbreak and vector-borne infections in a group of rescue dogs imported from hungary to switzerland date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: py awuav background: canine distemper virus (cdv) is a major pathogen of dogs and wild carnivores worldwide. in switzerland, distemper in domestic dogs is rarely reported. in recent years, the import of dogs from eastern europe to switzerland has steadily increased. in the present study, we describe a distemper outbreak in rescue dogs that were imported from hungary to switzerland by an animal welfare organisation. the data on vaccination and medical history were recorded ( dogs), and the samples were collected to investigate cdv and vector-borne infections ( dogs) and canine parvovirus infection ( dogs). the dogs were monitored for six months. results: one dog was euthanised directly after import. thirteen dogs showed clinical signs after arrival, i.e., diarrhoea ( %), coughing ( %) and nasal and/or ocular discharge ( %); radiographic findings that were compatible with bronchopneumonia were present in four dogs. cdv infection was diagnosed in dogs ( %); dogs ( %) tested pcr-positive in conjunctival swabs. vector-borne infections (babesia spp., leishmania infantum, dirofilaria immitis) were found in dogs ( %). three dogs were hospitalized, and six dogs received ambulatory therapy for up to two months until recovery. none of the dogs developed neurological disease. cdv shedding was detected for a period of up to four months. because dogs were put under strict quarantine until cdv shedding ceased, cdv did not spread to any other dogs. the cdv isolates showed % sequence identity in the ha gene among each other and belonged to the arctic-like lineage of cdv. conclusions: the present study highlights the imminent risks of spreading contagious viral and vector-borne infections through the non-selective import of sick dogs and dogs with incomplete vaccination from eastern europe. cdv shedding was detected for several months after the cessation of clinical signs, which emphasised the roles of asymptomatic carriers in cdv epidemiology. a long-term follow-up using sensitive pcr and strict quarantine measures is of upmost importance in preventing the spread of infection. dog owners and animal welfare organisations should be educated regarding the importance of complete vaccinations and the impact of dog imports on the spread of viral and vector-borne pathogens. canine distemper virus (cdv) is one of the most important viral pathogens in domestic dogs and causes high morbidity and mortality worldwide, particularly in unvaccinated dogs or dogs with incomplete vaccination [ ] . cdv is a small, enveloped rna virus that belongs to the family paramyxoviridae and the genus morbillivirus [ ] . cdv has a wide natural host range that includes a variety of terrestrial carnivores [ ] . dogs are thought to be the major reservoir host for cdv [ , ] . infection occurs by direct contact with oronasal secretions of infected animals [ ] ; indirect transmission plays only a minor role in cdv epidemics because the virus is quickly inactivated in the environment [ ] . the course of the cdv infection is strongly dependent on the immune response in infected animals [ ] . in this context, vaccination is critically important. dogs that develop an adequate immune response can clear the virus from most tissues, whereas in dogs that show an intermediate immune response, cdv infects the epithelial tissues and induces clinical signs. in dogs that have a weak immune response, cdv disseminates to various tissues, and the clinical signs are usually severe with the persistence of the virus until death [ ] . invasion of the central nervous system occurs when viraemia is sufficiently high [ , ] . more than % of all cdv infections are perceived to be subclinical [ ] . in clinically affected dogs, the disease usually starts with fever and a serous-to-mucopurulent conjunctivitis, followed by a dry to productive cough, depression, anorexia, vomiting and diarrhoea [ ] . neurological signs usually develop within one to three weeks after recovery from systemic illness, but can occur weeks to months later [ ] . since the introduction of highly protective cdv modified live virus (mlv) vaccines more than years ago [ ] , the incidence of cdv infection in completely vaccinated dogs has decreased [ ] . however, in regions with a low proportion of vaccinated dogs, in stray dogs and in shelter environments, the incidence of cdv epidemics is high. in switzerland, the last cdv epidemic in domestic dogs occurred in - ; this outbreak was suspected to be attributed to an inadequate vaccination rate in the swiss dog population at that time [ ] . furthermore, a cdv epidemic associated with high morbidity and mortality commenced in the spring of in wild carnivores in switzerland [ ] . the latter was perceived to be part of a large transnational outbreak that spread from eastern to western europe. only one domestic dog was affected in switzerland during this outbreak [ ] . remarkably, the -year-old mixed breed dog died of a cdv-associated neurological disease, although it had received the standard anti-cdv vaccination protocol. according to the animal identity service (anis) in switzerland, the import of dogs increased by % within one year ( - ) [ ] . the imported dogs comprised primarily stray dogs that were adopted by animal welfare organizations or pure breed dogs to meet the increasing demand of miniature breeds in western europe. in the present study, we report on a distemper outbreak in rescue dogs that had been imported from hungary to switzerland. the study provides data on vaccination, medical history, clinical examinations and diagnostic imaging of the dogs and cdv testing, testing for canine parvovirus (cpv) and vector-borne infections. additionally, the study gives prospectively collected follow-up data on the treatment, clinical course and outcome of the infections and the period of cdv shedding. finally, a molecular characterization of the cdv isolates was performed. a group of rescue dogs that derived from a shelter in kecskemét, hungary, was imported to switzerland in october . one dog was euthanised within several days of import because of clinical deterioration; no data regarding this dog were available. the other dogs comprised nine female ( spayed) and five male ( castrated) mixed breed dogs, aged months to years old, and weighing kg to kg ( table ). all of the dogs had received rabies vaccination (rabisin®, biokema sa, crissier, switzerland) six to weeks before import and deworming (containing praziquantel, pyrantel and fenbendazol, uniwerm®, provet, beograd, serbia) seven to nine days before their arrival in switzerland. additionally, the dogs had been vaccinated with one shot of a combined mlv vaccine containing cdv, canine adenovirus- , cpv, leptospira spp. and canine parainfluenzavirus (biocan® dhppi & l, table ), either seven to eight days (dogs to and to ) or one month prior to arrival in switzerland (dog ); dog had been revaccinated in switzerland one week prior to sample collection for cdv pcr (table ) . after arrival on october , , the rescue dogs were directly distributed to private households throughout switzerland (table , fig. ). seven dogs (dogs , , , , , and ) were placed in multidog households. after arrival, the new owners observed clinical signs in of the dogs (table ) , i.e., diarrhoea ( %), coughing ( %), nasal and/or ocular discharge ( %), vomiting ( %), gagging ( %), lameness ( %), apathy and sneezing (each %). because of these symptoms, five dogs (dogs , , , and ) were presented to private veterinarians within one week of arrival. three of these dogs (dogs , and ) were subsequently referred to small animal clinics for additional investigations. haematology and blood biochemistry results were available for dogs (tables and ), of which were cdv-pcr positive (dogs to , see below). at initial presentation, anaemia ( / , %), leucocytosis ( / , %), eosinophilia ( / , %), neutrophilia ( / , %) and monocytosis ( / , %) were common ( table ) . dogs and showed severe pancytopenia and moderate bicytopenia, respectively; both were cdv pcr-positive, co-infected with babesia spp. (dog ) or positive for anti-leishmania infantum antibodies (dog , see below), and they exhibited fever, increased inspiratory lung sounds, purulent ocular and nasal discharge and radiographic signs that were compatible with bronchopneumonia (tables to ). dog showed slight anaemia and leucopenia ( table ) ; this animal was co-infected with cdv and babesia spp. (tables and ) . dog showed a pronounced eosinophilia ( table ) ; this animal was cdv-pcr negative but dirofilaria immitis positive (tables and ). blood biochemistry results revealed only unspecific changes in the dogs (table ) . the radiographic examinations of the thorax revealed moderate-to-severe interstitial lung changes with variable bronchial thickening in four dogs (dogs - ). the changes were generalised and most pronounced in the dorsal (dog ), perihilar (dogs and ) and caudal lung areas (dog , fig. ). the radiographic findings were compatible with bronchopneumonia in all four dogs, and all of the dogs tested cdv pcr-positive. the thoracic radiographs of dog revealed mild right-sided cardiomegaly and mild generalised bronchointerstitial lung changes; echocardiography showed mild tricuspid and aortic regurgitation but no signs of pulmonary hypertension or right ventricular pressure overload. dog tested d. immitis-positive but was negative for cdv. eleven of the dogs tested cdv pcr-positive during the initial examination ( table ). the positive pcr results were most commonly obtained from conjunctival swabs ( of the cdv-positive dogs, table ). the vaccine-specific real-time reverse transcription (rt)quantitative (q)pcr was negative for all ten dogs that were tested, which supports the finding of infection with a wild-type cdv strain. all three vaccines that were tested (biocan® dhppi & l, bioveta, ivanovice na hané, czech republic; nobivac® dhhpi, msd animal health, luzern, switzerland; canigen® sha ppi, vibac, glattbrugg, switzerland) exhibited a positive pcr result. in gel electrophoresis of the pcr products, a appropriate-sized band was detected for all ten dogs, as were the three vaccines, as expected [ ] . the sequence of the amplification product of the biocan® dhppi & l vaccine used in the dogs of the present study was clearly distinct from the sequence alb albumin; ap alkaline phosphatase; alat alanine aminotransferase; na sodium; k potassium; p phosphorus of the cdv isolates of the ten rescue dogs (fig. ) and most closely related to the cdv vaccine strain onderstepoort ( % nucleotide identity to ab ). sequencing of the ha gene of cdv isolates of five of the infected dogs revealed that the dogs were infected with a similar cdv strain ( . - % nucleotide identity among the ha gene of cdv isolates of dogs , , and ); the ha gene of the cdv isolate of dog differed in only one nucleotide position from the ha gene sequences of the other four cdv isolates. the phylogenetic analysis revealed that the isolates from the five import dogs belonged to the arctic-like lineage of cdv (fig. ) . the ha gene sequences of the cdv isolates were most similar to a published ha gene sequence of a cdv strain from a domestic dog from italy (kf , % nucleotide identity, fig. ) [ ] . they were only distantly related to cdv strains isolated during a cdv epidemic in wild carnivores in switzerland (jf and jf , . % nucleotide identity, fig. ) overall, two dogs (dogs and ) exhibited cdv pcrnegative results one month after the initial examination (table ). two months after the initial examination, another three dogs were found to be cdv pcr-negative (dogs to ); three months after the initial examination, dog was cdv-negative and the remaining three dogs tested cdv-negative four months (dog ) and five months (dogs and ) after the initial examination (table ) . all twelve dogs that were tested for cpv at the initial presentation were pcr-negative (table ) . vector-borne infections were detected in dogs ( %, table ): infection with babesia spp. was detected in dogs and ; infection with l. infantum was diagnosed in dog and infection with dirofilaria immitis was found in dog . dog , which tested positive in d. immitis antigen and knott tests, had received a certificate from a laboratory in budapest, hungary, that stated a negative result in the knott test in august . none of the rescue dogs tested positive for ehrlichia canis (table ) . the cdv-infected dogs , and were hospitalized for two to three days and received intravenous infusions of crystalloids, intravenous antibiotic therapy and inhalation (table ) . dog , which was co-infected with babesia spp., was treated with two imidocarb injections two weeks apart. dog , which was co-infected with l. infantum was treated using allopurinol. all three dogs showed rapid clinical improvement with treatment and were discharged with oral antibiotic therapy. repeated haematological examination in dog two weeks later revealed that the pancytopenia had resolved and had returned to moderate neutrophilia, eosinophilia and slight monocytosis. mild anaemia was still present in dog at that time (data not shown). repeated haematology in dog two weeks after the initial presentation showed normal platelets counts and nearly normal pcv values (data not shown). all three dogs were clinically asymptomatic in the -month follow-up period. dog was ambulatory and was treated with oral antibiotics and antibiotic eye drops ( table ). the dog showed several relapses with purulent nasal and ocular discharge after antibiotic therapy ceased and was repeatedly treated with antibiotics for two months. thereafter, there was no relapse, and the dog was clinically asymptomatic in the remaining -month follow-up period. five cdv pcr-positive dogs (dogs to and ) received oral antibiotic therapy (amoxicillin clavulanic acid or doxycycline) for seven to ten days after their arrival in switzerland. dog developed watery diarrhoea two weeks after arrival and was additionally treated with metronidazole, deworming and a highly digestible diet. dog , which was co-infected with babesia spp., received two injections of imidocarb diproprionate two weeks apart and antibiotic ear drops because of otitis externa. at the end of the -month follow-up period, all of the cdv pcr-positive dogs had recovered and none had developed neurological signs. all of the owners of the cdv pcr-positive dogs were instructed by the first author (bw) to quarantine the dogs until they tested cdv pcr-negative. the owners of the cdv-positive dogs in multidog households (dogs , , , and ) were instructed to separate the infected dog from the other dogs in the household. however, several dogs (dogs , , and ) had already had contact with adult dogs within the household at the time when the cdv diagnosis was made. all of the contact dogs had been vaccinated against cdv, although several dogs had only received the initial vaccination series as puppies and had received no booster vaccinations (data not shown). two dogs that were in close contact with dogs and were tested for the cdv infection with pcr one month and two months after the initial cdv diagnosis in dogs and . one contact dog exhibited a single, very weak cdv-positive result in the conjunctival swab in the first sampling but was negative in all of the swabs collected one month later (data not shown). the other contact dog tested pcr-negative in all of the collected samples (data not shown). in all of the other multidog households, no samples were collected for cdv pcr, but no clinical signs of the disease were noted in the month follow-up period. the present study describes a distemper outbreak in rescue dogs in switzerland that had been imported by an animal welfare organization. one dog had to be and of ten wild-type cdv isolates of the rescue dogs (dogs to and to ) is shown. grey-shaded letters: identical nucleotides between vaccine strains and wild-type isolates. black letters: nucleotides that differ between vaccine strains and wild-type isolates. grey bars: schematic drawing depicting the positions of the primers and the probe used in the cdv vaccine specific real-time rt-qpcr assay [ ] . consensus: consensus sequence of % identical bases matching all of the sequences (most of the ambiguities) euthanized directly after import for humane reasons, whereas nine dogs required therapy for up to two months; three of these dogs were hospitalized at veterinary clinics. the imported animals shed cdv for up to four months, which necessitated long-term quarantine measures. moreover, four of the dogs were infected with vector-borne pathogens. the present study underscores the risk of introducing contagious or vector-borne pathogens to central european countries by importing rescue dogs with incomplete vaccination. based on the data of the anis in switzerland, . % of the newly registered dogs in were imported [ ] . the imported dogs primarily comprised small and miniature purebred dogs to meet the increasing demand for these types of breeds in switzerland, and mixed breed dogs that are rescued by animal welfare organisations [ ] . the dogs of both groups are at risk for carrying infectious diseases because of the inadequate vaccination policies and quarantine measures in many breeding kennels and animal shelters in eastern europe. in the animal shelter in hungary where the dogs originate, no quarantine measures or reliable vaccination policies had been implemented. the dogs had only received a single cdv vaccination either one week or one month before importation to switzerland. at this time point, several dogs had likely been infected with cdv. after the outbreak, the rescue organisation was instructed to introduce vaccination policies and quarantine measures within the animal shelter. in switzerland, the vaccination rate in dogs is insufficiently high to provide population immunity against the cdv infection. the proportion of vaccinated dogs in a population must be > % to provide protection for the dog population, in contrast to protection of only a single vaccinated dog [ ] . a vaccination rate of - % has been estimated for swiss dogs based on the numbers of sold vaccine doses in [ ] . mandatory courses for new dog owners have been introduced in switzerland in which freshly adopted dogs come in close contact with each other. together with the decreasing vaccination rate and the increasing number of imported dogs, this situation has clearly increased the risk for canine distemper outbreaks. dog owners and animal welfare organisations should be informed concerning the critical importance of complete vaccination schedules in domestic dogs. in the present outbreak, the infection could be prevented from spreading to other dogs by extensive followup examinations of the dogs and a thorough education of the dog owners concerning the critical importance of strict quarantine measures. luckily, no cdv-positive dogs of this study had already had contact with young unvaccinated dogs at the time of diagnosis. the cdv vaccines are known to induce a strong and long-lasting immunity when no interference with maternally derived antibodies occurs [ , ] . cdv was reported to be shed by infected animals for up to three months [ , ] . we demonstrated cdv shedding in some dogs in the present study even for up to four months. shedding was detected for several months after the cessation of clinical disease. our results underscore the role of asymptomatic carriers in cdv epidemiology and the importance of a long-term followup of cdv-positive dogs for preventing the spread of infection. several studies have reported the excretion of vaccine strains after cdv vaccination [ , ] . this was not observed in the present study: the dogs that tested cdv pcr-positive were shown to be infected with wildtype cdv, although the majority of the dogs had received mlv cdv vaccination within one to seven weeks before pcr testing. consistent with the published data [ ] , pcr from conjunctival swabs was found to be most reliable for detecting cdv in acutely infected animals and during follow-up examinations. however, in one animal, only the nasal but not the conjunctival swab tested positive for cdv. nine of the dogs in the present study were hospitalized or required ambulatory therapy for up to two months. another animal was euthanised directly after import because of clinical deterioration. in all of the affected dogs, the respiratory and gastrointestinal symptoms predominated. in four dogs, signs of bronchopneumonia were evident. remarkably, none of the dogs developed a neurological disease. whether the latter was due to the intrinsic properties of the cdv strain or to the age and immune status of the dogs is unknown. the sequence analyses of the cdv strains detected in the rescue dogs indicated that all of the dogs were infected with the same wild-type cdv strain, which belongs to the arctic-like lineage of cdv [ , ] . the initial description of arctic-like lineage of cdv dates back to the late s, when epizootics were observed in seals in northern europe and siberia [ ] [ ] [ ] . (see figure on previous page.) fig. phylogenetic relationship between selected cdv strains based on the complete haemagglutinin (ha) gene sequence. the cdv isolates analysed in this study appear in bold. nine cdv lineages are shown: asia- , europe, america- , europe-wildlife, africa, arctic-like, asia- , asia- and america- . phocine distemper virus (pdv- ) was used as the outgroup. genbank accession numbers, host species and geographical origin are indicated, if known. the numbers at the nodes were generated from bootstrap resamplings; only values > are shown. the bar represents the mean number of differences per sites. strain af (giant panda isolate) is the resultant of a genetic recombination between "asia- " and a "europe-wildlife" strain [ ] the strains of the arctic-like lineage are closely related to the cdv strains in north america, china and greenland and were recently isolated from domestic dogs in hungary [ ] and domestic dogs and wolves in southern italy [ , ] . the present study shows how fast these strains can spread to central european countries by import of infected domestic dogs. the present study indicates that rescue dogs may also play an important role in the spread of vector-borne infections to central european countries. babesia spp., l. infantum or d. immitis infections were detected in four of the rescue dogs. several dogs were reported to be free of these pathogens based on the laboratory certificates provided by the animal welfare organisation. in the case of dog , d. immitis infection was diagnosed in this study using the antigen enzyme immunoassay and the knott test in november . the dog was found to be negative in the knott test in august . the d. immitis antigen and knott tests are known to turn positive not before five to eight months after infection [ ] ; therefore, the infection could have been missed in the first testing. l. infantum serology specimen, which was positive in dog , has similar limitations in that negative serological results cannot exclude infection and seroconversion can occur many months after infection [ ] . future dog owners should be properly informed concerning the limitations of these tests and the costs of treatment for vector-borne infections. the present study highlights the risks of spreading contagious viral and vector-borne infections by a nonselective import of sick and unvaccinated dogs or dogs with incomplete vaccination from eastern european countries. the animal welfare organisations should be thoroughly informed concerning the critical importance of complete vaccination schedules and quarantine measures in animal shelters to combat the outbreaks and the spread of viral pathogens to other countries. dog owners in switzerland should be educated regarding the risk of and the potential costs of adopting sick dogs or dogs with incomplete vaccination and the critical importance of sufficiently high vaccination rates in domestic dogs in switzerland. the present study describes a distemper outbreak in fifteen dogs originating from an animal shelter in kecskemét, hungary, and the prospective follow-up of the infected animals. the dogs were imported to switzerland by an animal welfare organisation on october , . one dog had to be euthanised within a several days of arrival. no data on that dog were available. of the remaining fourteen dogs, the signalment, vaccination and medical history and clinical signs were recorded (table ) and dogs were monitored for six months. sample and data collection and sample processing nasal and/or conjunctival swabs were collected from dogs for cdv-specific real-time rt-qpcr and rectal swabs from dogs for cpv real-time qpcr as indicated in tables and . edta blood and serum samples were collected to obtain haematology and blood biochemistry results, and for cdv real-time rt-qpcr and testing for vector-borne infections (babesia spp., e. canis, l. infantum and d. immitis) as indicated in tables through . all of the samples were processed within h of collection. in of the cdv-infected dogs (dogs to and to ), monthly follow-up examinations for cdv were performed from conjunctival, nasal and oropharyngeal swabs ( table ). the swabs were collected by the owner based on written and image instructions regarding how to collect and ship the samples. all of the swabs were sent by priority mail to the clinical laboratory, vetsuisse faculty, university of zurich, within one day of collection. follow-up was continued in each dog until the dog tested cdv pcrnegative, except for dog , in which the owner declined further testing despite a pcr-positive result at three months of follow-up. haematology and blood biochemistry tests were performed at the clinical laboratory, vetsuisse faculty, university of zurich (dogs and , to and ), at the clinical diagnostic laboratory, vetsuisse faculty, university of bern (dog ), at a private laboratory (dog : alomed, radolfzell-böhringen, germany) or by a private veterinarian (dog , tables and ). the laboratory's own device-specific reference intervals were applied; for dogs aged to months (dogs , to ), published reference intervals for phosphorous, alkaline phosphatase, urea and creatinine were used [ ] . at the time of the initial examinations, conjunctival, nasal or rectal swabs were incubated for min in μl of phosphate buffered saline (pbs) at °c; the swabs were subsequently turned upside down and centrifuged for min at × g and the supernatant was used for tna extraction (see below). for the follow-up examinations, the two conjunctival and the two nasal swabs, respectively, were pooled in a total of μl of pbs before incubation at °c for min and tna extraction. the tna extraction was performed from μl of edta blood, μl of swab supernatant or vaccine material that was resuspended in μl of pbs (see below) using the magna pure lc (roche diagnostics ag, rotkreuz, switzerland) and the magna pure lc tna isolation kit (roche diagnostics) following the manufacturer's instructions. with each batch of extraction, a negative control consisting of μl of pbs was used to monitor for cross-contamination. the tna was stored at − °c until pcr analysis was performed. for cdv testing, a published real-time rt-qpcr assay was used as previously described [ ] . for the detection of cpv, a published real-time qpcr assay developed for the detection of feline parvovirus (fpv) was applied [ ] . the assay amplifies a bp sequence of the highly conserved vp /vp gene region using the following primers and probe: pv f: ′-actgcatcattgat ggttgca- ′; pv r: ′-ggtatggttggtttc catgga- ′ pv p: ′-fam-cccaatgtctcaga tctcatagctgctgg- -tamra- ′. sequence comparison revealed that the primer and probe-binding sites in the vp /vp gene region of fpv and cpv showed > % sequence identity (genbank accession numbers m , m , m , m , m , m , m , km , jq ). during cdv follow-up, a published canine (c)gapdh pcr assay was applied to ensure that the quality of the swabs collected by the dog owners was adequate [ ] . during follow-up, dogs were stated cdv-negative if they tested pcr-negative for cdv in conjunctival, nasal and oropharyngeal swabs and the swabs showed a cgapdh threshold cycle below . all of the real-time pcr reactions were run using an abi fast real-time pcr system (applied biosystems, rotkreuz, switzerland). negative and positive controls were included in each pcr run. to ensure that the cdv real-time rt-qpcr signal was due to infection and was not due to a recent vaccination, a published real-time rt-qpcr system based on the cdv m gene and m-f intergenic region was used [ ] . the primers of the pcr assay amplify the mlv vaccine and wild type cdv strains, whereas the probe of the assay only binds to the mlv vaccine strains (fig. ) . because no sequence data have been published on the m gene and the m-f intergenic region of cdv u strain contained in the vaccine used in the rescue dogs (biocan® dhppi & l, bioveta), the latter vaccine, together with two other cdv vaccines that are commercially available in switzerland (nobivac® dhhpi, msd animal health; canigen® sha ppi, vibac) were tested to confirm that the cdv strains contained in these vaccines are detected by the assay. the tna from the three vaccines and from the conjunctival or nasal swabs from of cdv pcr-positive dogs (dogs to and to ) were subjected to real-time rt-qpcr. the pcr products were separated using . % agarose gel and appropriate-sized bands ( bp) cut out, extracted using the minelute® gel extraction kit (qiagen, hombrechtikon, switzerland) and sequenced (microsynth, balgach, switzerland). the complete haemagglutinin (ha) genes of the cdv isolates from five dogs were sequenced (dogs , , , and ). the tna extracted from the conjunctival swabs was used as a template. for amplification, five previously published primer pairs were used [ ] ; single nucleotides in one primer pair ( f and r) had to be changed because of mismatches within the primer binding sites for the five cdv isolates ( f_new: '-ctgtacat caccaagtcata- ' and r_new: '-tagaatac catcttgtgaat- '). reverse transcription was performed using the high capacity cdna reverse transcription kit (applied biosystems) according to the manufacturer's instructions. the pcr amplification was conducted using μl of x hf pcr buffer (finnzymes, bioconcept, allschwil, switzerland), nm each primer, μm each dntp (sigma-aldrich, buchs, switzerland), u phusion high-fidelity dna polymerase (finnzymes), and . μl template cdna made up to μl with water. the thermal cycling conditions comprised °c for min, cycles at °c for s, °c for s, °c for min, and a final elongation at °c for min. after the pcr run, the amplification products were separated using % agarose gel; appropriately sized products were excised and purified using the minelute gel extraction kit (qiagen). direct sequencing of the purified amplicons was performed using the amplification primers in a commercial laboratory (microsynth, balgach, switzerland) under standard conditions. vector-borne infections were tested in dogs, as shown in table . the analyses were performed at the clinical laboratory (for e. canis) and the institute of parasitology (for babesia spp., l. infantum and d. immitis) of the vetsuisse faculty, university of zurich, and at private laboratories (dog : labor am zugersee, hünenberg, switzerland; dog : alomed; dog : idexx diavet ag, bäch, switzerland) ( table ). the laboratories' own reference values were used for defining the positive, negative and intermediate results. the microscopic blood smear evaluation for the presence of babesia spp. organisms was conducted in dog by the private veterinarian and in dog by a commercial laboratory (alomed) ( table ) . the obtained sequences were edited and aligned with a consensus sequence using geneious version . . [ ] . only the nucleotides available for all of the included sequences ( nucleotides of the ha gene) were used to calculate the percent nucleotide identities and perform the phylogenetic analyses. for the phylogenetic analyses, the sequences were aligned with known distemper sequences from genbank (see fig. ) using geneious version . . . a bootstrap phylogenetic tree demonstrating the relationship between the isolates was created using the maximum-likelihood method [ ] and a distance matrix corrected for nucleotide substitutions based on the kimura -parameter model [ ] . the dataset was resampled times to generate bootstrap values. the phylogenetic and molecular evolutionary analyses were conducted using the mega version [ ] . nucleotide sequences obtained in this study have been submitted to genbank under accession numbers kr to kr . canine distemper spillover in domestic dogs from urban wildlife canine distemper in terrestrial carnivores: a review canine distemper virus-a 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emergence of canine distemper in bavarian wildlife associated with a specific amino acid exchange in the haemagglutinin protein geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data prospects for inferring very large phylogenies by using the neighbor-joining method a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences molecular evolutionary genetics analysis version . cross-species recombination in the haemagglutinin gene of canine distemper virus evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic leishmania infections in dogs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors thank the animal rescue organisation and the owners of the dogs for providing the data and supporting diagnostic work-up. we thank t. meili, e. goenczi, s. childers and the technicians of the clinical laboratory for their excellent laboratory assistance. the laboratory work was performed using the logistics of the center for clinical studies, vetsuisse faculty, university of zurich. as was supported by a research grant (forschungskredit, fk- - - ) from the university of zurich. the authors declare that they have no potential conflicts of interest to disclose.authors' contributions rhl and bw conceived the study. bw and ams were responsible for the study coordination and the data and sample collections. lb, tb and rj were responsible for the clinical work-up and medical care of the dogs. mlm was responsible for the molecular laboratory aspects, fg was responsible for the analyses of vector-borne infections, br was responsible for the haematology and blood biochemistry tests and md was responsible for all aspects of diagnostic imaging. rhl and bw drafted the manuscript. all of the authors read and approved the final manuscript. key: cord- -acr xkz authors: long, charles tyler; williams, morika; savage, mason; fogle, jonathan; meeker, rick; hudson, lola title: probable primary polydipsia in a domestic shorthair cat date: - - journal: jfms open rep doi: . / sha: doc_id: cord_uid: acr xkz case summary: a -month-old neutered male domestic shorthair cat presented with a month history of polyuria and polydipsia. after a thorough diagnostic work-up the only abnormal findings were hyposthenuria and an elevated random plasma osmolality level. trial therapy with the oral and ophthalmic forms of desmopressin failed to concentrate urine. a modified water deprivation test confirmed the ability to concentrate urine above a urine specific gravity (usg) of . . after transitioning the cat to a higher sodium diet and instituting several enrichment changes to the cat’s environment, average water consumption and urine output levels decreased to almost normal levels and usg increased from . to . . these findings provide strong evidence that primary polydipsia was the underlying etiology of the cat’s condition. relevance and novel information: this case report exemplifies the challenges faced when a cat presents for polyuria and polydipsia without an obvious cause identified on routine diagnostics. to our knowledge, this is the first report of primary polydipsia in a cat. polyuria and polydipsia (pu/pd) are apparent in a cat if urine specific gravities (usgs) are consistently < . and water consumption is > ml/kg/day. , once pu/pd is confirmed to exist, the diagnostic approach should include a thorough history, physical examination and minimum database, including a complete blood count (cbc), serum chemistry profile, thyroxine (t ) concentration, urinalysis (ua) and urine culture to rule out more common causes of pu/pd such as diabetes mellitus, renal disease, hepatic insufficiency, hyperthyroidism and pyometra. [ ] [ ] [ ] [ ] when a low usg remains the only abnormal finding, central and nephrogenic diabetes insipidus, as well as primary polydipsia, should be ruled out, as treatment varies with each condition. this case report summarizes a cat with persistent pu/pd and hyposthenuria that was ultimately diagnosed with primary polydipsia after completion of a modified water deprivation test. a specific pathogen-free (spf), . kg, -month-old neutered male domestic shorthair cat housed in a research animal vivarium was reported for drinking and urinating approximately twice as much as conspecifics housed in the same facility. after questioning husbandry staff it was discovered that the cat drank more water and soiled its litterbox to a greater degree than other research cats since arrival from a commercial vendor months earlier. prior to arrival, the cat was housed in a closed barrier facility and tested to be spf for the following agents: feline herpesvirus, calicivirus, feline panleukopenia, feline immunodeficiency virus (fiv), feline infectious peritonitis, feline leukemia virus, feline coronavirus, feline chlamydia, toxoplasmosis and rabies. the cat was up to date on vaccinations (rabvac , fel-o-vax pct; boehringer ingerheim) and was fed a commercial feline diet (adult light dry; hill's science diet) once daily. research manipulations were not yet performed on the cat, which was housed in a climate-controlled room with five other research cats, each with their own cm × cm × cm enclosure. all enclosures were equipped with an elevated resting board, two hide boxes, various enrichment toys and a litterbox with corncob litter (bed-o'cobs; andersons). cats could visualize each other at all times and received at least mins of play time per day outside of their enclosures to allow for cleaning and litter removal. physical examination yielded no abnormal findings and the cat was subsequently anesthetized with isoflurane (isothesia; henry schein) the next day for collection of cerebrospinal fluid (csf), blood and urine as part of baseline data for a fiv neuropathogenesis study the cat was enrolled in at the time. reverse transcriptase pcr analysis of plasma and csf samples confirmed the cat was negative for fiv. cbc and serum biochemistry values for this collection and a subsequent collection days later were within normal limits. ua performed at both time points was normal except for persistent hyposthenuria; usg . and . , respectively (reference interval [ri] < . ). additional testing included a t level, which was within normal limits ( . μg/dl [ri . - . μg/dl]), and an elevated random plasma osmolality ( mosm/kg [ri - mosm/kg]). a urine sample obtained by cystocentesis was submitted for aerobic culture but revealed no growth after days. a urine cortisol/creatinine ratio was also within normal limits ( [ri . ± . ]). abdominal ultrasound was performed by a veterinary radiologist ( mhz curved probe, mylab xvg; bio sound esaote) and both kidneys were normal in size and architecture, with no indication of underlying disease such as pyelonephritis. examination of the rest of the abdomen revealed no abnormal findings and normal thyroid and parathyroid glands were visualized on ultrasound of the ventral cervical region. the cat remained pu/pd when allowed free access to water, with an average consumption of ml/kg/day (ri < ml/kg/day) and urine output of approximately ml/kg/day (ri < ml/kg/day). random usg levels measured over the next months on a refractometer (model - ; schuco) were never > . . based on diagnostic results thus far, the three main differentials for pu/pd in this cat were complete or partial central diabetes insipidus (cdi), nephrogenic diabetes insipidus (ndi) and primary polydipsia (pp). because of possible complications and reported difficulties in the interpretation of test results, - the modified water deprivation test (mwdt) was delayed and the cat was treated with desmopressin, a synthetic analog of the natural antidiuretic hormone arginine vasopressin. first, oral tablets ( . mg desmopressin acetate; watson pharma) were administered at . mg q h. owing to continual polydipsia and hyposthenuria, therapy was stopped after days and a week later . mg/ml compounded desmopressin eye drops ( . % desmopressin acetate; diamondback drugs) were administered at a dose of - drops into the conjunctival sac twice daily for days. both forms of desmopressin failed to decrease water consumption or increase the usg above untreated levels (table ) ; therefore, therapy was discontinued. as both diagnostic trials of desmopressin failed it was decided to move forward with a gradual water restriction phase before the mwdt in order to minimize the reported negative effects that renal medullary washout can have on test results. , , water allotment was incrementally reduced over a day period down to ml/kg/day on the day prior to testing. this allowed close monitoring of the cat for changes in mentation, neurologic status, body weight, usg and serum osmolality levels ( table ) . a cbc and chemistry profile were repeated and determined to be within normal limits. on day , after withholding food overnight, the cat was sedated with µg/kg dexmedetomidine hydrochloride (dexdomitor; zoetis) intramuscularly to allow for bladder catheterization and emptying. every hour thereafter the cat's mentation and hydration status were assessed and body weight recorded. to measure usg levels, urine samples were obtained by manual bladder compression. twenty-one hours after initiating the mwdt, the cat lost % body weight and a urine sample was collected with a usg of . (figure ), thus ending the test. table average water consumption and urine specific gravity (usg) levels over a day period with and without synthetic desmopressin treatment in a -month-old domestic shorthair cat with polyuria/polydipsia average water consumption (ml/day)* average usg † over the next days water was gradually reintroduced, to prevent overconsumption and secondary cerebral edema. after testing was completed ml of water was provided every h to the cat for the first h and the entire amount was noted to be consumed within a few hours; usg was . approximately h after reintroducing water. over the next h ml water was offered twice, which the cat consumed entirely and usg was . . on day after testing ml water was offered twice and the consumption rate was ml/ h. thereafter, the cat was provided water ad libitum and remained normal on daily physical examinations, regaining % of original body weight by day . nine days after completing the mwdt the cat was noted by animal care staff to be polydipsic again and had a usg of . . two months after completing the mwdt all research cats were transitioned over to a hairball control diet (adult hairball control light dry; hill's science diet) because some hairballs were noted in the enclosures of a few cats. the new diet contained a higher sodium level of mg/ kcal metabolizable energy (me) vs the previous diet, which contained a sodium level of mg/ kcal me. in an attempt to redirect the chronic polydipsic behavior additional enrichment was added to the cat's daily routine; for example, food pellets were placed in a treat dispenser apparatus (egg-cersizer cat toy; petsafe) and grooming by animal caretakers was increased to twice weekly from once weekly. a cat wheel (the cat wheel) was also placed in the room for exercise during pen cleaning. two weeks after instituting the once pu/pd is confirmed to exist the diagnostic challenge of identifying the underlying mechanism begins. in this case initial testing and advanced diagnostics, such as abdominal ultrasound, urine cortisol/creatinine ratio and random plasma osmolality testing, did not provide a definitive diagnosis for our cat's chronic pu/pd and hyposthenuria, although it did eliminate several differential diagnoses. when a low usg and elevated plasma osmolality were the only abnormal findings, the differential diagnosis list remained cdi (complete and partial), ndi and pp. cdi causes polyuria and secondary polydipsia via insufficient hypothalamic-pituitary secretion of arginine vasopressin (avp), also known as antidiuretic hormone, which interacts with distal renal tubular and collecting duct cells to resorb water and concentrate urine. this can be a result of a deficiency in the synthesis, , transport, or release of the hormone. , cdi in cats has been reported to occur as a result of congenital pituitary malformation, head trauma and neoplasia; , , - ; however, most cases are idiopathic. , in complete cdi there is a total absence of avp, while in partial cdi some avp is present. animals with complete cdi will not be able to concentrate urine above . , despite % dehydration, but when given desmopressin their urine concentration should increase by at least % and, concurrently, there should be a % decrease in water intake and polyuria. , with partial cdi, a mwdt should cause urine to concentrate above . but less than . at a % dehydrated state, and there should be a similar response to desmopressin administration as with complete cdi. although ophthalmic desmopressin raised the usg above . , water consumption and polyuria still continued during the treatment period with both forms of the drug, necessitating a mwdt to prove definitively that the cat could concentrate urine without the administration of exogenous avp. ndi is also a primary polyuric disorder that results from an inability of the nephron to respond to avp. in contrast to cdi, avp is present in normal or increased amounts. ndi can be primary (familial) or secondary (acquired) in dogs; however, no cases of primary ndi have been reported in cats. , secondary ndi is caused by renal or metabolic disorders that interrupt the normal interaction of avp with its tubular receptors in the kidney, disrupt renal tubular function or result in loss of the hypertonic renal medullary interstitial gradient. several diseases, such as hyperadrenocorticism, pyometra, pyelonephritis, hyperthyroidism and hyperaldosteronism can lead to secondary ndi in the cat. with ndi there should be minimal-to-no improvement in pu/pd signs and usg when desmopressin is administered or when a mwdt is performed. by performing diagnostic bloodwork and imaging many causes of secondary ndi were ruled out, although the lack of response in the desmopressin trial indicated ndi was still a possible diagnosis. however, the mwdt ultimately ruled out primary and secondary ndi as the cat was able to concentrate urine above . , proving adequate renal responsiveness to avp. pp is defined as a marked increase in water intake that cannot be explained as a compensatory mechanism for excessive fluid loss. in humans this can be further subdivided into dipsogenic diabetes insipidus and psychogenic polydipsia. the underlying physiological disorder with dipsogenic diabetes insipidus appears to be an osmotic threshold for thirst that is abnormally low. yet not only is the thirst threshold low in affected patients, but also for unknown reasons the osmotic threshold for avp release tends to be in a high normal range, causing plasma osmolality to remain at a level above that necessary to eliminate thirst but below that required to stimulate avp release. , consequently, patients with this disease tend to drink themselves into a water diuresis. the pathogenesis of dipsogenic diabetes insipidus is unknown but is hypothesized to be due to a disruption of one or more afferent pathways that regulate the thirst and avp osmostats. lesions of the hypothalamic thirst center leading to compulsive water drinking have yet to be reported in dogs or cats. , because advanced intracranial imaging was not performed in the cat presented here, a lesion affecting this area of the brain cannot be ruled out. however, there was never an indication of underlying neurologic disease on repeated examinations and behavioral monitoring. although some authors do not distinguish between primary and dipsogenic polydipsia, as it is not universally accepted as a distinct pathophysiologic state, a study by robertson found about the same incidence of dispogenic diabetes insipidus as for ndi and psychogenic polydipsia and half of that for partial cdi in human patients. psychogenic polydipsia describes a patient in which an underlying psychiatric disorder has been diagnosed along with abnormal water intake. the terms 'primary' and 'psychogenic' polydipsia are often used interchangeably, especially in veterinary medicine. , , , although uncommon, pp has been briefly described in dogs as a psychological disorder and behavioral problem but no reports yet exist in cats. , , in dogs this syndrome may be a result of a concurrent disease or a learned behavior due to a change in environment. treatment for dogs diagnosed with psychogenic polydipsia centers around gradual water restriction with or without the addition of salt to the diet and changes in the environment to provide additional enrichment to the dog's daily routine. , when the cat presented here remained on ad libitum water and a new diet with a slightly higher sodium content was started, the first concern was that the polydipsia would continue at the same rate or even worsen. in order to prevent this from happening, additional enrichment activities were added to the cat's daily routine, as mentioned previously. although our cat remained pu/pd with water consumption > ml/kg/day and urine output > ml/kg/day, both conditions improved to almost normal levels weeks after making the enrichment modifications, and the usg increased from an average of . to an average of . . although these findings provide further evidence for the diagnosis of pp in this cat, we realize that without advanced imaging of intracranial structures and constant monitoring of plasma avp levels to determine a threshold for release a distinction between dipsogenic diabetes insipidus and psychogenic polydipsia cannot be made in the case presented here. variability exists in criteria used to diagnose pp. patients with pp should have lower than normal plasma osmolality (< mosm/kg) owing to increased blood volume, while those with cdi or ndi have plasma osmolality levels > mosm/kg owing to the loss of free water through the kidneys and subsequent decreased blood volume. however, there can be considerable overlap of plasma osmolality values in animals with these three disorders. when allowed free access to water, a plasma osmolality level > mosm/ kg is consistent with cdi, ndi or pp, whereas levels < mosm/kg would suggest pp alone. , , plasma and urine osmolality have also been shown to vary throughout the day in dogs with pp, and both hyponatremia and normal serum sodium levels have been reported in humans and dogs with this disease. , with the cat featured in this case study, serum sodium levels were always normal (ri - mmol/l) and plasma osmolality levels were either in the normal range or elevated. as mentioned previously, human patients diagnosed with dipsogenic diabetes insipidus have an osmotic thirst threshold that is set lower than the threshold for avp release. , this could also be the case for the cat presented here. to compensate for an abnormally low thirst threshold it is thought that the kidneys of affected patients adapt a blunted response to avp in order to protect the body against dangerous levels of hyponatremia. in this scenario serum sodium and plasma osmolality levels would remain normal despite compulsive drinking, but the cat could properly concentrate urine when fluid deprived during the mwdt. robertson noted three human patients diagnosed with dipsogenic diabetes insipidus that had basal plasma osmolality and sodium levels in the upper normal range as opposed to being suppressed; these patients were found to have normal avp secretion but a higher threshold for release. initially it was thought that avp secretion was normal in patients with pp, but that may not always be the case as this remains a topic of debate in human and veterinary medicine. a study by van vonderen et al. showed that four dogs previously diagnosed with pp had decreased avp responses during a mwdt, and that two of four dogs had plasma osmolality and serum sodium levels at the upper limits of normal, indicating that a non-linear relationship between avp and plasma osmolality levels may exist in some dogs with pp. plasma osmolality and serum sodium levels that are not decreased in a pu/pd animal should not necessarily rule out pp and emphasizes the fact that multiple diagnostic tests may be needed before reaching a final diagnosis. although helpful in diagnosing cdi in one feline case, there are no published avp reference intervals in cats. , avp levels were not measured in our cat to determine if there was abnormal synthesis and release during the mwdt, but this is a topic that warrants further research in cats with pu/pd. this case exemplifies the challenges faced when initial diagnostics fail to determine an obvious cause of chronic pu/pd in a cat. when both the oral and ophthalmic forms of desmopressin failed to concentrate urine and decrease water intake, gradual water restriction along with a mwdt proved that the cat could concentrate urine above a usg of . . these findings, along with improvement in clinical signs after increasing environmental enrichment, provide strong evidence that primary polydipsia was the underlying cause of the cat's chronic pu/pd and hyposthenuria. to our knowledge, this is the first reported case of feline primary polydipsia. polyuria and polydipsia: diagnostic approach and problems associated with patient evaluation water metabolism and diabetes insipidus canine and feline endocrinology and reproduction clinical use of the vasopression analog desmopressin for the diagnosis and treatment of diabetes insipidus disorders of the hypothalamus and pituitary gland bsava manual of canine and feline clinical pathology normal laboratory values. in: norsworthy gd urine cortisol:creatinine ratio in healthy and sick cats bsava manual of canine and feline endocrinology traumatic partial hypopituitarism in a cat diabetes insipidus: diagnosis and treatment of a complex disease head trauma as a possible cause of central diabetes insipidus in a cat congenital diabetes insipidus in a kitten trauma-induced central diabetes insipidus in a cat central diabetes insipidus in five cats: clinical presentation, diagnosis and oral desmopressin therapy central diabetes insipidus in a cat with central nervous system b cell lymphoma multiple endocrine diseases in cats: cases ( - ) hyperadrenocorticism in cats: seven cases ( - ) uterine torsion and metabolic abnormalities in a cat with a pyometra changes in clinical and laboratory findings in cats with hyperthyroidism from to primary hyperaldosteronism in the cat: a series of cases disturbed vasopressin release in dogs with so-called primary polydipsia dipsogenic diabetes insipidus: report of a novel treatment strategy and literature review dipsogenic diabetes insipidus: a newly recognized syndrome caused by a selective defect in the osmoregulation of thirst psychogenic polydipsia review: etiology, differential, and treatment evaluation of the plasma vasopressin, plasma sodium, and urine osmolality response to water restriction in normal cats and a cat with diabetes insipidus we would like to thank andrea thomson and the laboratory animal research husbandry staff for their training and care of the research cats. institutes of health (grant number r ns - a ). the authors declared no potential conflicts of interest with respect to the research, authorship, and/ or publication of this article. key: cord- - h authors: späth, peter j.; granata, guido; la marra, fabiola; kuijpers, taco w.; quinti, isabella title: on the dark side of therapies with immunoglobulin concentrates: the adverse events date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h therapy by human immunoglobulin g (igg) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. the success of igg concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of frontiers in immunology. a part of the success is the adverse event (ae) profile of igg concentrates which is, even at life-long need for therapy, excellent. transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. the cornerstone of the regulatory network is current good manufacturing practice. non-infectious aes occur rarely and mainly are mild to moderate. however, in recent times, the increase in frequency of hemolytic and thrombotic aes raised worrying questions on the possible background for these aes. below, we review elements of non-infectious aes, and particularly focus on hemolysis and thrombosis. we discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence ae profiles and efficacy of igg concentrates. i.e., cold-ethanol or ion-exchange chromatography, contaminants, route of application, i.e., intra muscular (imig), intravenous (ivig), or subcutaneous (scig), the rate of increase of the exogenous igg in the circulation of the recipient over time and, last but not least an eventually existing risk factor from patients' side ( figure ) as well as incorrect handling of the concentrate are factors having a role in inducing non-infectious aes related to administration of igg concentrates ( table ) . igg concentrates represent a defined part of the adaptive immune system, are isolated from pooled human plasma of at least donors, which contribute to the repertoire diversity in the final product. therapies with igg concentrates manufactured according to regulators requirements are acknowledged to be safe in general. this does not exclude the occurrence of aes which in their majority are rare and clinically mild to moderate. below, we like to give a few insights into various aspects and possible mechanisms of aes. table . contemplating on the active ingredient of the concentrates, the administration of the igg molecules inevitably results in the interactions of the exogenous igg with the various parts of the immune system of the recipient and vice versa. this interaction in its principle generates an inflammatory condition. a key parameter deciding on the intensity of such a condition is the rate of increase over time of exogenous igg in the circulation of the recipient. insert to figure (shadowed): ∆igg in the circulation over ∆time is dependent on the combination of infusion rate (ir) and the strength of the solution applied. main figure: the mode of application, i.e., intravenous or subcutaneous, are additional factors being decisive for kinetics and the area under the curve of generation of pro-inflammatory mediators and for passing the individual threshold (short-dashed line) for a clinically noticeable ae. the threshold in turn depends on eventually exiting risk factors form the patient side. although the area under the curve might be the same for low (long-dashed line) and high (solid line) irs, at high ir, the system might not be able to cope with the extent of reactions. at low ir (long-dashed line), all the events might remain below the threshold of clinically observable ae. subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent than with ivig. in contrast, local mild to moderate aes are more frequent with scig. virus inactivation and virus elimination methods by validating an already performed step of the fractionation process or by introduction of dedicated steps (figure ) . a hallmark of virus elimination introduced in the late s in berne by the team of christoph kempf is the large-scale virus filtration technique (formerly also termed "nanofiltration") ( ) . meanwhile, virus filtration became a versatile tool to eliminate a variety of pathogens, the suspected agent of variant creutzfeldt-jakob disease included. thanks to the tightly implemented regulatory framework, pathogen safety of plasma products is at a level never reached before. this is well supported by the fact of reports missing in the last decade of transmission by igg concentrates of emerging viruses (sars coronavirus, west nile virus, mers coronavirus, and others), zoonotic pathogens, or the agent of variant creutzfeldt-jakob disease (vcjd). furthermore, the development of specific mass screening techniques might help to eradicate in any blood product the transmission of vcjd in the future ( ) . the human immune system is in charge of controlling invading organisms and mediates homeostasis. the immunoglobulin pools in mammals (igm, igg, and iga) to its smaller part provide defense and to the larger part homeostasis. human igg has a role in both. efficient host defense is supported by "immune antibodies." these have undergone somatic hypermutations and have in their vast majority narrow specificities and high affinities. antibodies, generated in absence of external stimuli are termed "natural antibodies" (nabs). they occasionally recognize self structures. in general, the v(d)j genes of nabs are in germ-line configuration or have undergone a few somatic hypermutations only. furthermore, they have broad specificity, are of low affinity (with exceptions) and high avidity ( ) . these nabs can participate in primary host defense, i.e., at a time point when an immunologic reaction has not provided the specific antibodies, react, e.g., with repetitive structures which can be found on bacteria or viruses ( ) . the self-reactive nabs, which we like to term physiologic autoantibodies, comprises various populations of antibodies such as (i) those able to interact through their complementary variable regions (v-regions) with the v-regions of circulating and membrane-bound (bcr) immunoglobulins and the t cell receptor (tcr) β-chain variable region, providing a peripheral immune network (v-connected network) ( ); (ii) the nabs reacting in a non-idiotypic manner with the hinge region of immunoglobulins ( , ) ; (iii) populations of nabs showing a wide variety of specificities toward growth factors, cytokines, or anaphylatoxin ( ); (iii) and the population reacting with the soluble or membrane-bound forms of cell surface molecules having immunological importance, the last described being the fc receptors cd and cd ( ) . antibodies reacting with docking structures for viruses or bacteria can have additional first-line defense potential ( ) . these populations of nabs were described having a peripheral immune network homeostatic and anti-inflammatory function ( , ) . although the primordial humoral proteins comprising the complement and lectin-like proteins in the plasma play a definite role, another population of self-reactive nabs reacting with, e.g., epitopes conserved over the evolution apparently has tissue homeostatic function and might support the efficient removal of roughly altered/senescent cells of the body per day (for references see below). the signal for research on nabs in ivig was the description of igg autoantibody-mediated immune thrombocytopenia (itp) being corrected by infusion of a polyclonal, polyspecific igg concentrate ( , ) . this research has expanded ever since. the populations of immune antibodies and nabs in igg concentrates upon infusion/injection inevitably react with occasional pathogens, toxins, or superantigens and concomitantly infusion/injection also results in recognition of a wide array of tissue antigens and v-regions of the recipient's immune system. reactions with tissue antigens and v-regions are conveyed by the self-reactive antibodies of the many donors in the igg concentrate. vice versa, the recipient's immune system reacts with the infused igg. a bewildering wide range of possible reactions can occur which primarily are dependent on the immune status of the recipient at the time of therapy and to a smaller part on the igg concentrate(s). the therapeutic effect achieved depends on the disease treated, and can depend on the concentration reached locally, i.e., can have agonistic or antagonistic effects ( , ( ) ( ) ( ) . in summary, it is our opinion that igg concentrates always provide more or less the same "bouquet" of igg specificities (similarity); however, it is the recipient's actual immune condition which decides from which igg specificities the patient's derailed immune system is profiting (diversity). parameters of igg-mediated aes are: (i) the content in the product of biologically highly active likely beneficial ingredients that have to be kept under control (e.g., content of "dimers" devoid of remarkable complement activation in vivo; see below), and the content in unwanted active ingredients that have to be discarded during manufacturing (alloantibodies); (ii) impurities such as iga (anaphylactoid reaction); (iii) activated coagulation and contact activation factors (thromboembolic events) and; (iv) excipients such as sucrose (osmotic nephrosis). below, we like to add and contemplate on how fully native igg molecules not harmed by the manufacturing process might add to aes. the above mentioned inevitable interaction of the exogenous igg with the immune system of the recipient and vice versa in its principle might evoke an inflammatory condition. the sum of the potentially beneficial reactions might overshoot and lead to aes (figure ) . the principle of induction of mild inflammatory www.frontiersin.org figure | staying within a regulatory network is mandatory for remaining at the sunny side of the moon. handling of blood/plasma products to obtain therapeutic goods has to be performed within a regulatory framework. manufacturing of stable blood product has to adhere strictly to current good manufacturing practice (cgmp). application of cgmp starts from the moment of collecting whole blood and isolation of plasma thereof (r = recovered plasma) or the machine-supported collection of plasma (s = source plasma, apheresis plasma). application of cgmp ends at delivery of blood/plasma products to health care professionals. not following cgmp can lead to withdrawal of a plasma product from the market from one to the other day. conditions upon each infusion of a well-tolerated ivig was confirmed when several dozen normogammaglobulinemic volunteers in all cases except one, showed a more or less moderate inflammatory reaction as indicated by the increase of tumor necrosis factor alpha (tnfα) at . h post initiation of infusion. the only person in the cohort not showing a measurable tnfα increase was a woman caring at home for her brother with full blown aids ( ) . subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent compared to ivig but they are not absent ( - ) (a case of unintentional i.v. application of scig is not considered). in contrast, local mild to moderate aes are more frequent with scig ( ) . in summary, the intensity of the resulting aes is depending on the immune status of the recipient, the infusion rate (ir), e.g., how rapidly the active ingredients (the various igg specificities), the impurities, and the excipients reach the circulation of the recipient. thus, the i.v. application has the highest chance for the occurrence of aes. in the early days of ivig therapy, complement-mediated "anaphylactoid" (i.e., immediate) and "phlogistic" (i.e., inflammatory) aes were distinguished ( , ) . the complement-mediated aes were considered to be caused by aggregates in the product ("spontaneous complement activation" or anti-complementary activity or aca) or by in vivo formation of immune complexes (ics, patient's condition related; e.g., subclinical infections or the unnoticed presence of anti-iga antibodies) and therefore only igg concentrates with low or absent aca is accepted by authorities for human use. below, we present one instructive case of each type of reaction. the first reports of rapid onset aes concerned either the application of complement-activating fractions in an igg concentrate or the in vivo formation of complement-activating ics ( ) ( ) ( ) . a very rare but potentially fatal condition is the formation of iga/anti-iga complexes in patients being initiated on replacement therapy and having serum igg antibodies against infused iga not recognized before the start of the ivig infusion ( ) . prerequisite for the presence of anti-iga antibodies is the most common primary immunoglobulin defect, i.e., selective iga deficiency (sigad) or igad associated with diminution of other immunoglobulin classes. igad is defined by serum levels of < . or < . g/l (depending on laboratories). a marked diminution of serum iga consistent with igad in various ethnic groups is estimated being : to : , ( ) . the mean frequency in caucasians is approximately : ( ) . up to % of patients with igad have been reported having anti-iga antibodies in the serum with titers ranging between : and : , . in approximately % of patients with common variable immunodeficiency (cvid), and occasionally in patients with other primary immunodeficiency diseases, measurable anti-iga can be detected ( , ). these antibodies are predominantly of the igg class, but anti-iga antibodies of other immunoglobulin classes have been described as well ( , ) . the reason for their emergence remains unknown. taken the above numbers, the infusion of human-derived products containing iga resulting in severe anaphylactoid type aes should be considerable. this is not the case ( ) . questions about the clinical relevance of above numbers emerge as soon as blood banks (i) estimate the theoretical risk of iga anaphylactic reactions ( ); (ii) assess the relation of severe igad with the presence or absence of anti-iga antibodies ( ) ; (iii) screen donors for very low iga levels in order to become able to provide blood and plasma-derived products free of iga and find a considerably lower frequency than expected ( ) . alternatively, the test systems may not reliably detect anti-iga antibodies being as yet insensitive and inaccurate or -at least -do not correspond to the clinically relevant fraction of antibodies. this comes to mind when a more close look to "anti-iga" gives "unexpected" results, including "anti-iga" in blood donors with normal serum iga level or "anti-iga" that cannot be neutralized with purified iga ( ); or when blood products containing proven anti-iga do not elicit severe aes ( ) . among patients on replacement therapy, those with cvid may rarely develop severe immediate aes ( ) . the discrepancy between anti-iga positive patients and frequency of aes raises the question about the nature of the many reported anti-iga antibodies and also raises the question about the immunologic condition which allows the formation of anaphylactoid anti-iga antibodies. there might be some logic in supposing that anaphylactoid anti-iga cannot evolve at iga levels otherwise fulfilling the definition of igad. such a condition would constantly generate ics which in turn could activate complement, react with immune cells, and be deposited in lung and kidney. indeed, horn et al. found anti-iga frontiers in immunology | primary immunodeficiencies antibodies in cvid patients missing iga + b cells and presenting with iga levels < . g/l, a level which is more than -to fold lower than the threshold for igad ( ) . however, a possibility for an iga-mediated anaphylactoid reaction at measurable iga serum levels might exist. serum iga contains approximately % subclass of iga (iga ) and only % subclass of iga (iga ). selective deficiency of iga and -although evidence is lackingthe presence of a highly specific anti-iga antibody theoretically could elicit a severe ae. the kinetics of anti-iga after infusion of blood products have been studied in a few cases. in these patients, a fall in anti-iga titers has been noticed followed by an increase during subsequent weeks or months. this suggests that at appropriate proportions, iga of the infused material and anti-iga present in the patients' serum combine with each other to form ics. in turn, ics activate complement that are bound and eliminated by macrophages most likely leading to cytokine release. the increase in anti-iga titers over time indicates that the infused iga-containing product has a booster effect ( , , ) . such boosting effect together with the presence of anti-iga before the application of an igg concentrate can be taken as the ultimate confirmation of a supposed iga/anti-iga reaction. figure depicts a well-documented case of iga/anti-iga reaction in a patient who progressed from sigad to cvid. the events during the first h at occasion of the first infusion of ivig were as follows (shadowed area in figure ): min after the start of the infusion, having received eight drops of an igg solution (iga < . g/l; % solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped. after approximately h, the reaction has weaned, and h later the patient felt well again, and the infusion of total g igg could be continued without further complications. although the patient fairly assured having never received any blood or plasma product in the past, the follow-up of her anti-iga titers from before infusion to year later confirmed a true anaphylactoid reaction mediated by anti-iga, as the anti-iga became undetectable immediately after the infusion and showed a boosting phenomenon during the following months. true anaphylactoid reaction was further confirmed by follow-up of total complement hemolytic activity (ch ) on the day of infusion. interestingly enough, the ch value reached its nadir at the end of the infusion when the patient had no complains. although a single case only, the events during the first infusion call for the following remarks: (i) severe aes most likely occur at concomitant complement and cell activation with cytokine release; (ii) infusion of minute to low amounts of ivig hours before the main infusion can "anergize" cells and stop release of pro-inflammatory www.frontiersin.org figure | an individual's slithering into the dark. a female patient has been suffering from recurrent airway infections since adolescence, occasionally complicated by pneumonia. at age , selective iga deficiency was diagnosed. ten years later, she was hospitalized with pneumonia. within these years, her serum igg had dropped from to . g/l (long-dashed line) and iga was undetectable. the diagnosis was corrected into cvid, and ivig replacement therapy was initiated (shadowed area). two minutes after the start of the infusion of a % igg solution (iga < . g/l; % solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped and later continued without further complications (see text). the confirmation of a true anaphylactoid reaction due to anti-iga in the serum of the patient was achieved by follow-up of anti-iga (solid line) and ch (short-dashed line). cytokines; (iii) "anergized" cells loose reactivity toward ongoing formation of ics and complement activation products. a non-complement-mediated anaphylactoid reaction was ascribed to the unforeseen release of elastase and other proinflammatory substances from neutrophils activated by the formation of in vivo iga/anti-iga complexes. complement activation or mast cell-dependent release of vasoactive substances was excluded as pathogenic mechanisms. although the iga/anti-iga complexes usually do not cause clinically relevant neutrophil degranulation within the circulation, the presence of a rare genotype encoding a novel gain-of-function igg receptor on neutrophils may provoke premature degranulation by these complexes. this phenomenon was only relevant in hypogammaglobulinemic patients in the presence of in vivo iga/anti-iga complexes ( ). the low prevalence of this genotype combined with an igad or cvid may add how to explain the rarity of serious anaphylactoid reactions in newly ivig-treated patients. authors share the opinion of janne björkander who at occasion of a discussion panel "dilemmas in diagnosis and management of antibody deficiencies: ask the experts" held at occasion of the th annual meeting of the american academy of allergy, asthma & immunology (aaaai), new york city, march - , came to the following conclusion: a clinician has to be aware of the risk, particularly at occasion of first infusions, but otherwise iga is not a major concern (from tape record). in the early days of ig-therapy, the nature of the "phlogistic" aes was obscure. however, it was already known that an ae can be prevented or its evolution halted when the patient receives a low dose of ivig first or the infusion is stopped early and is continued several hours later. hours later the infusion can be (re)started at high rates without further problems ( figure ) . one of the authors had a particular opportunity to get an insight into what a "phlogistic reaction" might be. at the occasion of a voluntary infusion of an investigational liquid ivig, he encountered a severe flu-like ae of more than h duration. before injection, the investigational liquid preparation had passed all release criteria for human use, including spontaneous complement activation assessed by aca and was free of prekallikrein activator (pka). in those days, assays for cytokines in biological samples just began to become available and were included into the parameters assessed in the study. infusion was stopped after h because of a drop of pulse rate and heavy discomfort provoking the laconic comment by the proband's technician who was taking samples:"you look green." the infusion was continued after another min when the heart rate had almost normalized. the infusion could be completed within an additional . h (a total of . g/kg b.w.) without further aggravation of malaise. the leukocyte count transiently had dropped to a nadir of % at h followed by a leukocytosis peak at h. complement activation, as assessed by generation of c a/c a[desarg] and the formation of the terminal complement complex c b- , apparently did not occur: the c a/c a[desarg] value reached a maximum of ng/ml (norm: < ng/ml) at h while the c b- value never moved outside the normal range. instead, a sequence of rapid transient massive increases of pro-inflammatory cytokines was observed: (i) tnfα started to increase min post initiation of infusion from a value of pg/ml to a peak value at h which was above the calibration range of the test kit of pg/ml; (ii) interleukin (il- ) increase started after h from pg/ml and peaked at . h with pg/ml post initiation of infusion; (iii) interleukin (il- ) secretion started after h with an undetectable level and peaked at h with pg/ml. all pro-inflammatory cytokines fell sharply while the second part of the infusion was still ongoing. the day after infusion, the pro-inflammatory cytokine profiles were back to normal and the flu-like syndrome was gone. in contrast, interleukin receptor antagonist (il- ra) values started increasing at . h ( pg/ml), peaked at h (> , pg/ml), and decreased slowly to reach a value of , pg/ml h after initiation of the infusion. soluble tnf receptor p level started at . ng/ml, reached a peak with . ng/ml at the same time as il- ra, and h after initiation of infusion was still at . ng/ml. thus, in this normogammaglobulinemic subject similar cytokine profiles and leukocyte number changes were observed as reported for hypogammagobulinemia under replacement therapy ( , ) . a series of further experiments with investigational and marketed ivigs was performed. all igg concentrates were analyzed for their molecular weight (mw) distribution. the most remarkable differences emerged in the mw range of dimers while the presence of minute amounts of higher oligomers could not be excluded with certainty. below, we will use the term "dimers" for that fraction of igg with higher mw. subsequent findings indicated that levels of "dimers" > % were responsible for complement-independent cell activation and cytokine release. the tnfα peaks assessed at . h post initiation of infusions correlated with "dimer" content of the ivigs and mirrored a clinical score of aes ( ) ( ) ( ) . frontiers in immunology | primary immunodeficiencies a few years before a complement-independent induction of a hypotensive factor by igg di-and oligomers was reported in animal experiments ( ) . a key role for macrophages in the generation of the hypotensive lipid factor was identified as plateletactivating factor, being induced by dimers and polymers ( , ) . several years later, the dimer-mediated aes in animal experiments were confirmed ( , ) . yet, at the same time, the dimer content of ivig apparently correlated with the clinical efficacy in a murine itp model ( , ) . variables such as "ir," "genetic background," "endogenous immunoglobulin levels," or "proportional fraction of polymers versus dimers" may impact on the balance between the phlogiston (being cytokines, active lipid substances, or a combination of factors) and the therapeutic efficacy (blocking igg receptors on liver/spleen macrophages to prevent clearance of "opsonized" material such as platelets in itp). as of today, reports on release of cytokines in humans in association with aes or tolerability toward dimers remain scarce and to the best of our knowledge studies in humans of causative factors/fraction in an igg concentrate has not been adequately addressed ( , , ( ) ( ) ( ) ( ) . in igg preparations, various forms of dimers might be present: formed through covalent binding ( ) by denaturation, hydrophobic interactions of the fc-parts, and by idiotype/anti-idiotype interactions, as part of the v-connected network of peripheral immune homeostasis ( ) . for a commercially viable fractionation process, pooling of donated plasma is mandatory in order to obtain a volume of starting material large enough to cover ever increasing costs for documentation, in-process, and batchrelease testing as it is required by cgmp. pooling also intends smoothing the batch-to-batch differences in antibody titers, a goal apparently difficult to achieve to levels as theory might imply ( ) . consequences of pooling are on the one hand the enrichment of public/common immune antibodies while diluting out individual specificities; on the other hand, the antibodies of the immune network of an individual donor are exposed to those of many other donors. the more individuals contribute to the pool, the more complex the possible "immune-network" interactions among iga, igg, and igm will become. the subsequent fractionation process has far-reaching effects on immunoglobulins from a given pool: only trace amounts of iga and igm are retained in the final product, i.e., igg is deprived of its counterparts of the v-connected immune network. the igg molecules of the homeostatic network "naked" at their v-regions can interact with each other at random combining site-interactions of single donor-derived monomeric igg ( ), otherwise not existing in vivo. this interaction is largely reversible. with increasing numbers of donors included into the pool, the immune network recognition among the "naked" igg molecules of the v-connected network becomes more and more complex, and the dimer and lower oligomer content in the resulting igg concentrate increases ( ) ( ) ( ) . in lyophilized igg concentrates, the dimer formation is "frozen" at a low level while in liquid preparations an equilibrium between monomers and dimers is achieved over time reaching a dimer content of % or more if not hampered by stabilizers. specificities, as far as they have been addressed, in the dimer fraction considerably differ from the monomeric fraction ( ) ( ) ( ) ( ) ( ) . in conclusion, the immunomodulatory efficacy of igg concentrates in part depends on the capacity and extent to form "dimer" fractions devoid of remarkable complement activation in vivo. the "art" of manufacturing a liquid igg concentrate is not to eliminate the monomeric igg having potential for "dimer" formation but to inhibit extensive"dimerization."in summary,aes might be associated with the induction of pro-inflammatory cytokines in absence of measurable complement activation in vivo where all regulatory mechanisms and removal processes of a body are at disposition. at reasonable irs in the open system of the human body, clinically relevant systemic complement activation apparently needs oligomers formed of three or more igg molecules. there are multiple reports of ig-induced hemolytic anemia (ha) in patients receiving high doses of ivig ( , - ) (table ; figure ; www.adrreports.eu). by spontaneous reporting, risk factors recognized for ig-induced hemolysis include beside high doses (more than g ivig over - days), female gender and histo-blood group type a, b, or ab of recipients. www.frontiersin.org a significant proportion of patients receiving ivig develop a positive direct antiglobulin test (dat) detectable after h for up to days after the ivig infusion ( , ) . however, it should be underlined that the dat positivity due to the factors mentioned above ( , ) is not sufficient per se to diagnose hemolysis and dat positivity does not necessarily imply the presence of active hemolysis. dat-positive mild hemolytic reactions can be easily missed and the true incidence of such reactions is difficult to document without careful clinical and laboratory follow-up. in the majority of reports on ha, intravascular red blood cell (rbc) destruction via complement activation or extravascular rbc sequestration and removal by the reticulo-endothelial system was proposed to result from igg alloantibodies with specificity for rbc antigens a, b, d, or c. hemolytic anemia induced by high-dose ivig has an average incidence of . % ( ) . low-dose igg replacement therapy is considered universally as safe, and only few cases of hemolysis following low-dose ivig or scig administration have been described ( , , ) . a baseline wbc and rbc count prior to ivig initiation and a close clinical and laboratory follow-up was suggested as a useful tool for early diagnosis and treatment. a possible work up might be to check hemoglobin (hb) level prior and - h after ig infusion. in case of a drop of hb, the presence of dat, an increase in unconjugated bilirubin, lactate dehydrogenase (ldh), and reduced haptoglobin level, followed by a rise in reticulocyte count should be assessed (figure ) . we systematically reviewed case reports related to ivig-induced hemolysis from to and identified articles containing reports of patients. baseline characteristics of the patients are shown in table . when available, blood group, dat, hb drop, and outcome are indicated. all reports showed positive dat, except for a case of yin et al. ( ) ; in this case, dat was performed days after ivig administration and the dat negativity might have been due to a rapid removal of sensitized rbcs. in the majority of patients, the outcome was positive: out of patients recovered with or without packed rbc transfusions; three patients died after ha, with the hemolytic episode representing a precipitating factor of a severe underlying condition. elution experiments were performed and the search for blood group antibodies revealed anti-a and anti-b specificity in the majority of cases; anti-d specificity was assessed in four reports, often associated with other specificities ( , , ) . a search for other specificities such as anti-band or anti-gal was not performed. only one report detected anti-c specificity in three patients; in one of them associated with anti-d irregular antibodies ( ) . although studies were restricted to blood group antibodies, this finding demonstrated that polyvalent igg preparations might contain clinically significant non-blood group antibodies, which are not part of the lot-release criteria in that their titration is not yet required by the european pharmacopeia. antibodies in ha, such as anti-c, may have unexpected hemolytic consequences ( ) ( ) ( ) ( ) ( ) . beside passive transfer of alloantibodies, igg administration also has been demonstrated to lead to unspecific enhanced erythrocyte sequestration, in particular, in patients with underlying inflammatory disorders ( , ) . in , the canadian ivig hemolysis pharmacovigilance group elaborated criteria to define an "ivig-induced hemolysis" ( ) . they included a reduction of hb levels ≥ g within days after ig administration, with frontiers in immunology | primary immunodeficiencies appearance of a positive dat and, at least, two of the following criteria: increase in the reticulocyte count, elevation of ldh and unconjugated bilirubin serum levels, low haptoglobin, hemoglobinuria, hemoglobinemia, presence of significant spherocytosis, in the absence of alternative causes of anemia. the passive transfer of igg alloantibodies through igg concentrates is difficult to explain as polyvalent igg is prepared from plasma of thousands of donors. since immunization to rbc alloantigens can occur because of past transfusions or pregnancy, the hypothetical numbers of alloimmunized plasma donors should be rather low. recently, other mechanisms underlying alloimmunization related to molecular mimicry have been demonstrated ( ) . the mechanism of high-dose ivig-induced ha is complex and it might vary from patient to patient. ivig cause hemolysis due to: (i) diseaseassociated pre-coating of rbcs; (ii) igg with hemolysis triggered by passive transfer of igg binding to blood group antigens; (iii) transfer of high levels of alloantibodies to rbc pre-coated at a low level only; or (iv) transfer of clinically tolerable levels of isoagglutinins plus transfer of additional rbc-reacting physiological autoantibodies. indeed, hemolytic reactions could not be related exclusively to transfer of alloantibodies. hence, antibodies other than histo-blood group alloantibodies (pre-)coated to rbcs might contribute to hemolysis in igg recipients need to be identified. in addition, hemolytic episodes may possibly be precipitated by some sort of complexed/denatured igg that co-purify with other igg in the product ( , , , ) . recently, a two hit mechanism for ivig-induced hemolysis has been proposed: the passive transfer of alloantibodies through ivig representing the first hit and the underlying inflammatory state representing the second hit ( ) . nowadays all commercial ig products have to undergo anti-a and anti-b testing and regulatory requirement ask for respective igg antibody titers of ≤ : at % solution strength (w/v) ( , ) . nevertheless, hemolysis might occur even in recipients of igg products that meet these specifications ( ) . consequently, it has been suggested that igg recipients should be monitored for clinical signs and symptoms of hemolysis ( ) . with the detection of the immunomodulatory potential of igg concentrates, their clinical use has continuously increased ( ) . to cover the need, at a first glance, an increase of the volume of plasma fractionated seems to be the most convenient option. however, this might economically not be viable because fractionation of plasma products is interconnected ( ) and before increasing output of one product (e.g., ivig), the market absorbance of the other products as well (e.g., albumin) must be ascertained. on a longer-run, a more viable option is to improve recovery. considering recovery, the cold-ethanol fractionation apparently has reached its limits. as of today, four manufactures have invested into a"modern"fractioning technique on the basis of ion-exchange chromatography. ion-exchange chromatography allows elevated recovery at high purity. as of today, five ivigs, one scig, and one anti-d concentrate are fractionated by ion-exchange chromatography. pharmacovigilance has shown that all chromatographically fractionated ivig and scig, more or less prominently, show a tendency for elevated frequencies of hemolytic aes. anti-a and anti-b alloantibody titers are now lot-release criteria (see above) as they constitute the major risk parameter for hemolytic reactions mediated by igg concentrates. to overcome the threat of end up on the dark side of the moon, two manufacturers have taken measures to reduce anti-a and anti-b titers in their igg products. one measure chosen was adsorption of the alloantibodies by affinity chromatography ( ) . reported reduction in both alloantibodies was significant and levels were similar to those in cold-ethanol fractionated immunoglobulins ( ) . the other measure chosen was reduction in anti-a using an automated indirect agglutination test for donor screening and exclusion of high-titer donations (approximately . %) from plasma pooling and fractionation ( ) . this measure reduced anti-a in the igg concentrate by one titer step. to ensure staying on the safe and sunny side, the manufacturer has announced the introduction of an alloanti-a and alloanti-b immune-affinity chromatography step into the manufacturing process ( ) . preliminary results indicate depletion in anti-a and anti-b by > % in investigational lots. subsequently, we want to discuss possible consequences of (extensive) removal of antibodies reacting with histo-blood group antigens a and b. three facts have initiated our thinking about possible consequences of removal of histo-blood group a and b reacting www.frontiersin.org antibodies from igg concentrates. (i) in collaboration with hans u. lutz, formerly biochemistry eth zurich, we have observed the non-intended removal of natural anti-c autoantibodies regulating complement activation by large-scale immune-affinity adsorption of iga from an igg concentrate ( ) . anti-c antibodies belong to the family of "nabs" and have a particular role in homeostasis: they control activation of complement, among others, in the frame of nab-mediated opsono-phagocytosis of altered or senescent cells, including rbcs ( ) ( ) ( ) . thus, the intention to target one particular antibody by affinity chromatography might reduce that antibody specificity but at the same time affect other specificities as well. (ii) it should be kept in mind that the blood groups a and b are in fact "histo-blood group" antigens, i.e., they are also found on white blood cells, t lymphocytes, and proteins and also can be found in soluble form ( ) . alloantibodies reacting with histo-blood group antigens a and b thus have much broader tissue recognition than rbcs only. in addition, alloanti-a and alloanti-b belong to the population of nabs recognizing non-self and most likely participate in primary host defense ( ) . (iii) in contrast to cold-ethanol fractionation, where low titers of alloanti-a and alloanti-b are achieved on basis of their isoelectric points (ieps), the (extensive) immune-affinity removal might affect a much wider iep range, thereby removing broadly reacting antibodies and impairing some desirable functions of the igg concentrate. thus, the struggle for staying on the sunny side of the moon might have consequences for the antibody repertoire in an igg concentrate. antibodies reacting with terminal di-, tri-, and tetrasaccharides belong to the large family of human anti-glycan nabs. histo-blood group a and b epitopes in terminal position are tetrasaccharides. alloantibodies to these tetra-saccharides are found in the plasma of healthy individuals depending on the blood group they have. a considerable body of research into the nature of these nabs has been performed so far, all using for isolation the corresponding terminal di-or tri-saccharides ( ) ( ) ( ) . recently, the repertoire and epitope specificity of such immunoglobulins was addressed in depth by including the tetra-saccharide as well ( , ) . it proved that serum of healthy individuals contain respectable amounts of di-or tri-saccharide-reacting nabs. these nabs proved to be pseudo-anti-a and pseudo-anti-b nabs as they are not reacting with the tetra-saccharide of histo-blood groups a and b. in contrast, alloanti-a and -b antibodies able to react with tetra-saccharides are reacting with the corresponding terminal di-and tri-saccharides. reasoning about the biological role of these "high-titer and population conservative" anti-di-and anti-tri-saccharide nabs and the consequence of their potential removal by immunoaffinity is outlined below. a population of the anti-glycan nabs are the anti-αgal nabs which recognize galα - gal and galα - (fucα - )gal epitopes. anti-αgal nabs have been described being xenoreactive, recognizing bacterial galα - gal ( ) and having tissue homeostatic function. the daily removal of altered/senescent cells of the body is~ . removal is mainly mediated by apoptosis (no inflammation, no necrosis). rbcs, when they do not encounter a pathological condition, over their life span of - days remain intact although they shrink, do not undergo apoptosis but progressively become senescent, mainly due to cumulative oxidative stress. removal of intact rbcs with a daily turnover of~ × , corresponding to~ g cell mass, is effectuated by increased exposure of otherwise cryptic structures such as spectrin, band , or αgal epitopes. these exposed structures are recognized by low affinity, high avidity, c -bearing nabs, which promote the efficient removal of intact senescent rbcs ( , , ) . immunoaffinity adsorption by tri-saccharides columns of di-and tri-saccharide reacting nabs from igg concentrates can eliminate anti-histoblood a and b alloantibodies while it also eliminates αgal and this might have a janus effect. the face directed to the sun tells that adsorbing αgal nabs reacting with altered and senescent self on rbc might prevent an increase in the igg load of rbcs over the threshold level of relevant hemolysis in individuals at risk. the face directed to the dark indicate that adsorption of tissue homeostatic antibodies might deprive an igg concentrate of potentially beneficial antibodies. although they are nabs, tri-saccharide reacting antibodies can be induced by feeding bacteria bearing the corresponding carbohydrate epitopes ( ) . these inducible nabs are considered to participate in primary host defense. other antibodies possibly involved in primary host defense are the anti-αgal nabs. they show a broad specificity and can react with a number of related αgal-terminated oligosaccharides, including those on bacteria ( ) . thus, the immunoadsorption of di-and trisaccharide reacting nabs might diminish the potential of an igg concentrate to mediate primary host defense. therefore, when choosing affinity resins for immunoadsorption, there might be some aspects worth to consider. in summary, the principles of avoiding co-fractionation through cold-ethanol fractionation ( ) versus immune-affinity removal of histo-blood group alloantibodies can have an impact on the presence of homeostatic and first-line defense antibodies. according to present knowledge, only resins coated with the corresponding tetra-saccharides can ascertain the selective removal of histo-blood group alloantibodies presumably involved in ha. resins coated with the corresponding di-and tri-saccharides also remove blood group alloantibodies, however not selectively. such resins in addition might remove a broad range of nabs present in igg concentrates at relative high amounts. in the literature, the use of tri-saccharide-coated resin was reported ( ) ( ) ( ) . we have found no information available in the public domain indicating which type of resin is/will be used for reduction of the histo-blood group alloantibodies in large-scale fractionation of igg. furthermore, we suggest that the effect of reduction of anti-a and ant-b reacting antibodies by immune-affinity on the antibody repertoire of igg concentrates can only be assessed by, e.g., using pathogens/commensals, which share the saccharide epitopes, that have been used to coat the affinity resins or alternatively by exposing senescent rbcs stripped off the iggs coated in vivo. finally, techniques are required, which allow detection of low affinity, high avidity nabs. ivig administration-related aes, including thrombosis, have been extensively described ( ) . thrombotic aes are severe aes and patients with risk factors require a special care. reported average incidence of ivig-induced thrombosis ranges from to % ( ) . recognized risk factors for ivig-induced thrombosis frontiers in immunology | primary immunodeficiencies include male gender; age > ; diabetes; renal insufficiency, dyslipidemia; hypertension; immobility; coronary disease; pre-existing vascular disease, family history of early thromboembolic disease; atrial fibrillation, high-dose and high-speed ivig infusions. ivig-induced thrombosis is reported both as venous events such as thrombosis stroke, pulmonary embolism (pe), deep venous thrombosis (dvt), and arterial ischemia events such as myocardial infarct and stroke. the mechanisms leading to ivigassociated thrombosis are still not completely clear; three main mechanisms have been proposed, emphasizing the role of an increased blood viscosity causing a hypercoagulable state ( ) , the role of anticardiolipin antibodies passively transferred through ivig ( ) , and the role of factor xia or other biologically highly active factors passively transferred via igg concentrates, such as pka. avoiding activated coagulation factors in igg concentrates starts with appropriate anticoagulation of donated blood/plasma, i.e., careful mixing of anticoagulant and sample over the whole donation process. alterations in an established manufacturing process neglecting appropriate controls can also lead to increased risk of transmission of activated coagulation factors. high mw proteins passively transferred by ivig are probably contributing to this phenomenon ( ) . in patients with other risk factors, such as vascular disease, the increase in blood viscosity can precipitate thromboembolic events. as elderly individuals are prone for such aes, we like to point to the possibility of elevated altered/senescent self-reacting with infused homeostatic nabs being a possible factor facilitating thrombotic events as well. a relationship between ivig administration and cerebral vasospasm has also been suggested by sztajzel et al. ( ) ; blood viscosity is a determinant for oxygen delivery to the tissues, and changes in viscosity can lead to a reduction in cerebral or myocardial perfusion. we systematically reviewed case reports related to iviginduced thrombosis from to (figure ) . literature search identified articles containing reports concerning patients ( , , , . when data were available, diagnosis, risk factors, the number of ivig infusion prior to thrombosis event, and outcome have been indicated. baseline characteristics of the patients are shown in table . high-dose ivig induced thromboembolic events in patients at low to medium ivig doses. marie et al. ( ) observed that the frequency and type of arterial events was inversely related to the time elapsed from ivig infusion; almost % ( versus reports) of arterial ischemic events occurred within h following ivig, while about % of venous thrombosis occurred after more than h. no correlation between number of infusions and occurrence of ae was observed. the main risk factors observed in this review were hypertension ( cases, % of prevalence), previous vascular disease ( %), and dyslipidemia ( %). average mortality for thrombotic events was % (arterial ischemia % versus venous thrombosis %, pe representing the main venous fatal event). predicting iviginduced thrombosis is difficult. risk factors should be assessed for each patient including instrumental exams when needed. doppler ultrasound can be useful as early diagnostic tool for thrombosis or to detect the presence of abnormal blood flow especially after prolonged immobility. ivig should be administered at low ir to reduce the risk. the administration of antiplatelet or anticoagulant prophylaxis was suggested in patients with several risk factors ( ) . however, thrombotic events have been reported even after several previous uncomplicated courses of treatment. www.frontiersin.org ( ), arrhythmia ( ) first ( ), several ( ) arterial ( ), venous ( ) days recovery ( ), death ( ) al-riyami et al. ( ) second ( ), third ( ), several ( ) arterial ( ), venous ( ) h ( ), days ( ), weeks ( ) recovery ( ) stamboulis et al. ( ) several ( ) arterial ( ) h ( ), days ( ) death ( ) clinical and immunological characteristics of patients described in case reports. numbers in parenthesis indicate the number of patients with the given condition. in such cases, patients should be examined for signs and symptoms of thrombosis during each courses of ivig. immunoglobulin g concentrates are widely acknowledged to offer a safe, high-dose, long-term therapy option for a variety of diseases. aes occur rarely and mainly are mild to moderate. deviations from this rule of thumb are addressed by authorities and the plasma fractionation industry to achieve corrections. above, we have reviewed two types of ae which have shown elevated frequency in the near past. we tried to give some insights which might help in reducing frequencies of aes bed side. authors are deeply grateful to hans-hartmut peter, freiburg, germany, for his careful reading of the manuscript, the valuable comments, and the correction of english. epidemic hepatitis b caused by commercial human immunoglobulin intravenous administration of human γ-globulin adverse reactions following administration of human gamma-globulin igg antibodies to iga in two patients with hypogammaglobulinemia treated with commercial gammaglobulin contact-activated factors: contaminants of immunoglobulin preparations with coagulant and vasoactive properties fatal thrombotic events during treatment of autoimmune thrombocytopenia with intravenous immunoglobulin in elderly patients effect of high-dose intravenous immunoglobulin therapy on blood rheology pathogen safety of immunoglobulin preparations population screening for variant creutzfeldt-jakob disease using a novel blood test: diagnostic accuracy and feasibility study intravenous immunoglobulin: exploiting the potential of natural antibodies inherent specificities in natural antibodies: a key to immune defense against pathogen invasion the natural human igg anti-f(ab') antibody recognizes a conformational igg hinge epitope stimulation of complement amplification by f(ab') -containing immune complexes and naturally occurring anti-hinge antibodies -possible role in systemic inflammation natural autoantibodies to fcγ receptors in intravenous immunoglobulins multi-faceted role of naturally occurring autoantibodies in fighting pathogens immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin intravenous immunoglobulin: an update on the clinical use and mechanisms of action demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura high-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood concurrent presence of agonistic and antagonistic anti-cd autoantibodies in intravenous ig preparations a differential concentration-dependent effect of ivig on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms verträglichkeit und virussicherheit von intravenösen immunglobulinen occurrence of hemolytic reactions (hrs) on the same day as immune globulin (ig) product administrations during cerebral venous and sinus thrombosis associated with subcutaneous immunoglobulin injection and oral contraceptive use multiple immunoglobulin intolerance without antibody's anti-immunoglobulin a: a case report common variable immunodeficiency: a patient with anaphylaxis to intravenous and subcutaneous immunoglobulin prospective study on cvid patients with adverse reactions to intravenous or subcutaneous igg administration severe adverse reaction to subcutaneous immunoglobulin therapy in a patient with common variable immunodeficiency evaluation of the relationship between injection site reaction rate and scig doses in patients with primary immunodeficiencies immunthemotherapy: a guide to immunoglobulin prophylaxis and therapy prophylactic infusions with an unmodified intravenous immunoglobullin product causing few side-effects in patients with antibody deficiency syndromes the role of anti-iga antibodies in causing adverse reactions to gamma globulin infusion in immunodeficient patients: a comprehensive review of the literature prevalence of immunoglobulin a deficiency (igad) in shanghai blood donors and efforts to establish a rare blood bank of igad in shanghai hammarström l, persson maa, smith cie. anti-iga in selective iga deficiency -in vitro effects and ig subclass pattern of human anti-iga immunoglobulin prophylaxis in patients with antibody deficiency syndromes and anti-iga antibodies follow-up of anti-iga antibodies in primary immunodeficient patients treated with gamma-globulin anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia: detection of ige antibodies to iga management of immunoglobulin a deficiency: lessons from haemovigilance screening of canadian blood services donors for severe immunoglobulin a deficiency large scale detection of iga deficient blood donors unexpected reactions of the anti-iga antibody particle gel immunoassay allergic transfusion reactions from blood components donated by iga-deficient donors with and without anti-iga: a comparative retrospective study anti-iga antibodies in common variable immunodeficiency (cvid): diagnostic workup and therapeutic strategy induction of unresponsiveness against iga in iga-deficient patients on subcutaneous immunoglobulin infusion therapy release of cytokines, soluble cytokine receptors, and interleukin- receptor antagonist after intravenous immunoglobulin administration in vivo neutropenia as a complication of intravenous immunoglobulin (ivig) therapy in children with immune thrombocytopenic purpura: common and non-alarming igg dimers in liquid intravenous immunoglobulin preparations laboratory parameters measured during infusion of immunoglobulin preparations for intravenous use and related tolerability welltolerated liquid intravenous immunoglobulin g preparations (ivgg) have a low immunoglobulin g dimer (igg-dimer) content an animal model for the detection of hypotensive side effects of immunoglobulin preparations key role of macrophages in hypotensive side effects of immunoglobulin preparations. studies in an animal model vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages -studies in a rat model hypotension with intravenous immunoglobulin therapy: importance of ph and dimer formation therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin g dimers: studies in experimental immune thrombocytopenia tumor necrosis factor and intravenous gammaglobulins in common variable immunodeficiency ivig adverse reactions: potential role of cytokines and vasoactive substances acute hemolysis after intravenous immunoglobulin amid host factors of abo-mismatched bone marrow transplantation, inflammation, and activated mononuclear phagocytes intravenous immunoglobulin induces interferon-γ and interleukin- in vivo human igg can form covalent dimers variable regionconnected, dimeric fraction of intravenous immunoglobulin enriched in natural autoantibodies ivig -mechanisms of action igg dimers in multidonor-derived immunoglobulins: aspects of generation and function immunoglobulin g dimer: an idiotype-anti-idiotype complex a view of the human idiotypic repertoire -electron microscopic and immunologic analyses of spontaneous idiotype-anti-idiotype dimers in pooled human igg polyreactive antibodies in multidonorderived immunoglobulin g: theory and conclusions drawn from experiments comparative analysis of antigen specificities in the monomeric and dimeric fractions of intravenous immunoglobulin monomerization of dimeric igg of intravenous immunoglobulin (ivig) increases the antibody reactivity against intracellular antigens antibodies in the dimer fraction of ivig have the capacity to bind beta amyloid an analysis of anti-fas and anti-siglec- autoantibodies in monomeric and dimeric fractions of ivig dimeric ivig contains natural anti-siglec- autoantibodies and their antiidiotypes intravenous immunoglobulininduced hemolytic anemia in a patient with juvenile dermatomyositis intravenous immunoglobulin-induced haemolysis: a case report and review of the literature massive intravascular haemolysis after high dose intravenous immunoglobulin therapy maternal hemolysis after intravenous immunoglobulin treatment in fetal and neonatal alloimmune thrombocytopenia hemolytic anemia following intravenous immunoglobulin therapy in patients treated for kawasaki disease: a report of cases ivig -a hemolytic culprit haemolysis after treatment with intravenous immunoglobulin due to anti-a hemoglobinuria and acute kidney injury requiring hemodialysis following intravenous immunoglobulin infusion a pediatric case series of acute hemolysis after administration of intravenous immunoglobulin acute hemolysis after high-dose intravenous immunoglobulin therapy in highly hla sensitized patients hemolytic transfusion reactions after administration of intravenous immune (gamma) globulin: a case series analysis hemolytic anemia following intravenous immunoglobulin administration acute hemolysis in a patient with cytomegalovirus pneumonitis treated with intravenous immunoglobulin (ivig) a prospective study of the immediate and delayed adverse events following intravenous immunoglobulin infusions adverse effect of polyvalent immunoglobulin in the treatment of guillain-barré syndrome hemolysis after administration of high-dose immunoglobulin in a patient with myocarditis haemolytic anaemia associated with high dose intravenous immunoglobulin therapy in a child with guillain-barré syndrome insisting on intravenous polyvalent immunoglobulin therapy in polymyositis in spite of the occurrence of sever hemolytic anemia -poursuite du traitement d'une polymyosite par les immunoglobulines intraveineuses polyvalentes malgré la survenue d'une anémie hémolytique sévère severe hemolytic anemia following high-dose intravenous immunoglobulin administration in a patient with kawasaki disease hemolytic anemia associated with intravenous immunoglobulin hemolysis during immunoglobulin therapy -hemolisis durante el tratamiento con inmunoglobulinas hemolytic anemia following highdose intravenous immunoglobulin administration acute hemolysis due to passively transfused high-titer anti-b causing spontaneous in vitro agglutination hemolysis after high-dose intravenous ig immune hemolysis, disseminated intravascular coagulation, and serum sickness after large doses of ivig for kawasaki disease acute intravascular haemolysis associated with high dose immunoglobulin after bone marrow transplantation for acute myelogenous leukemia hemolysis after intravenous immune globulin therapy: relation to igg subclasses of red cell antibody autoimmune hemolytic anemia in kawasaki disease: a case report haemolysis induced by intravenously-administered immunoglobulin massive intravascular hemolysis associated with intravenous immunoglobulin in bone marrow transplant recipients hemolytic anemia following intravenous gamma globulin administration hemolysis following intravenous immune globulin therapy transient haemoglobin drop following high dose intravenous immunoglobulin in vivo administration of intravenous immunoglobulin (ivig) can lead to enhanced erythrocyte sequestration hemolysis in patients with antibody deficiencies on immunoglobulin replacement treatment batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-rh d activity hematologic toxicities associated with intravenous immunoglobulin therapy anti-a and anti-b activity in batches of different intravenous immunoglobulin products determined using a direct haemagglutination method international collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti-a and anti-b in normal intravenous immunoglobulin products european directorate for the quality of medicines and healthcare. anti-a and anti-b haemagglutinins european directorate for the quality of medicines and healthcare. test for anti-d antibodies in human immunoglobulin immune complex-like moieties in immunoglobulin for intravenous use (i.v.ig) bind complement and enhance phagocytosis of human erythrocytes regulation of primary alloantibody response through antecedent exposure to a microbial tcell epitope a follow-up study of adult patients with idiopathic thrombocytopenic purpura treated with high-dose immunoglobulins and anti-d immunoglobulins hemolysis upon intravenous immunoglobulin transfusion report of the fda meeting on strategies to address hemolytic complications of immune globulin infusions kreuth ig working group. european consensus proposal for immunoglobulin therapies the art of balanced production in vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations patient safety through an ivig mastered manufacturing process. posters isoagglutinin reduction in human immunoglobulin products by donor screening igg product development isoagglutinin reduction measures. oral presentation naturally occurring antibodies/autoantibodies in polyclonal immunoglobulin concentrates. lutz, h. u. naturally occurring antibodies (nabs) naturally occurring anti-band- antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes high doses of immunoglobulin g attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of c b in c bn-igg complexes intravenously applied igg stimulates complement attenuation in a complementdependent autoimmune disease at the amplifying c convertase level histoblood group antigens as allo-and autoantigens blood group isoantibody stimulation in man by feeding blood group-active bacteria normal human serum contains natural antibodies reactive with autologous ab blood group antigens a unique natural human igg antibody with anti-alpha-galactosyl specificity normal human serum contains high levels of anti-galα - glcnac antibodies natural anti-a and anti-b of the ab system: allo-and autoantibodies have different epitope specificity repertoire of human natural anti-glycan immunoglobulins. do we have autoantibodies? interaction between human natural anti-alpha-galactosyl immunoglobulin g and bacteria of the human flora innate immune and non-immune mediators of erythrocyte clearance comparison of serum anti-band and anti-gal antibody binding to density-separated human red blood cells specificity of human antibodies against galα - gal carbohydrate epitope and distinction from natural antibodies reacting with galα - gal or galα - gal anti-a and anti-b haemagglutinin trend analysis during manufacturing process of ivig. presentation at workshop "strategies to address hemolytic complications of immune globulin infusions in vitro evaluation of the efficacy and biocompatibility of new, synthetic ab immunoabsorbents high molecular weight blood group a trisaccharide-polyacrylamide glycoconjugates as synthetic blood group a antigens for anti-a antibody removal devices specific antibody filter (saf) binding capacity enhancement to remove anti-a antibodies intravenous immunoglobulin: adverse effects and safe administration thromboembolic complications of intravenous immunoglobulin therapy in patients with neuropathy: a two-year study high-dose intravenous immunoglobulin and serum viscosity: risk of precipitating thromboembolic events antired blood cell antibodies, free light chains, and antiphospholipid antibodies in intravenous immunoglobulin preparations soluble hla class i and class ii concentrations in commercial immunoglobulin preparations high-dose intravenous immunoglobulin treatment and cerebral vasospasm: a possible mechanism of ischemic encephalopathy? high dose intravenous immunoglobulin may be complicated by myocardial infarction fatal case of bilateral internal jugular vein thrombosis following ivig infusion in an adolescent girl treated for itp cerebral sinus thrombosis following iv immunoglobulin therapy of immune thrombocytopenia purpura graft rupture after high-dose intravenous immunoglobulin therapy in a renal transplant patient cerebral sinus thrombosis in a patient with humoral immunodeficiency on intravenous immunoglobulin therapy: a case report a case of deep vein thrombosis and pulmonary thromboembolism after intravenous immunoglobulin therapy acute stroke with high-dose intravenous immune globulin venous and arterial thrombosis following administration of intravenous immunoglobulins intravenous immunoglobulin-associated vena cava thrombosis intravenous immunoglobulin-associated arterial and venous thrombosis; report of a series and review of the literature thromboembolic events as an emerging adverse effect during high-dose intravenous immunoglobulin therapy in elderly patients: a case report and discussion of the relevant literature diffuse venous thromboemboli associated with ivig therapy in the treatment of streptococcal toxic shock syndrome: case report and review deep vein thrombosis after intravenous immunoglobulins associated with methylprednisolone deep venous thrombosis after high-dose intravenous immunoglobulin in the treatment of pemphigus vulgaris thromboembolic complications of intravenous immunoglobulin treatment acute myocardial infarction following intravenous immunoglobulin therapy for chronic inflammatory demyelinating polyneuropathy in association with a monoclonal immunoglobulin g paraprotein stroke and deep venous thrombosis complicating intravenous immunoglobulin infusions thrombosis complicating high dose intravenous immunoglobulin: report of three cases and review of the literature acute thromboembolic events associated with intravenous immunoglobulin infusion in antibody-deficient patients transverse sinus thrombosis and ivig treatment: a case report and discussion of risk-benefit assessment for immunoglobulin treatment thrombotic complications after intravenous immunoglobulin therapy in two patients pulmonary embolism after intravenous immunoglobulin adverse effects of intravenous immunoglobulin therapy in patients with autoimmune diseases acute myocardial infarction associated with high dose intravenous immunoglobulin infusion for autoimmune disorders. a study of four cases deep venous thrombosis of the arm after intravenous immunoglobulin infusion: case report and literature review of intravenous immunoglobulin-related thrombotic complications cerebral infarction complicating intravenous immunoglobulin therapy in a patient with miller fisher syndrome central retinal vein occlusion complicating treatment with intravenous immunoglobulin acute myocardial infarction during treatment with intravenous immunoglobulin for idiopathic thrombocytopenic purpura (itp) myocardial infarction as a complication of immunoglobulin therapy iatrogenic central retinal vein occlusion and hyperviscosity associated with high-dose intravenous immunoglobulin administration the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - mbr authors: curran, e. t. title: standard precautions: what is meant and what is not date: - - journal: journal of hospital infection doi: . /j.jhin. . . sha: doc_id: cord_uid: mbr nan in , the cdc introduced the term 'universal precautions' with the aim of preventing occupational exposure of hcws to bloodborne viruses (bbvs). universal precautions applied to blood and some, but not all, body fluids; the precautions were to be used for all patients regardless of their known infection status. in , the cdc replaced the term 'universal precautions' with 'standard precautions', which aimed to prevent nosocomial infection in patients as well as hcws, and concerned other micro-organisms as well as bbvs. the cdc updated the definition of standard precautions in to include new elements of respiratory hygiene as a consequence of lessons learned in the outbreak of severe acute respiratory syndrome, and safe injection practices as a result of the multiple outbreaks involving bbvs and other organisms that occurred principally from the re-use of needles and contaminated multi-dose vials. multiple healthcare agencies have now modified the cdc's standard precautions. the standard precautions from who contain a more limited number of actions compared with the cdc's standard precautions. standard infection control precautions published by health protection scotland include both a policy and independent supplementary literature reviews to provide evidence for their required actions, similar to, but not overlapping with, the cdc model. standard principles within epic for england have been updated recently. the epic account lacks some of the basics of the cdc's standard precautions, but includes critical information on several high-infection-risk deviceassociated procedures. the european centre for disease control has also recommended and promoted the use of standard precautions, but has not specified what is included in the term. some of the content variation of these documents can be explained by different national organizations having different jurisdictions, and by variation in mandates given to the authors; this has to be a problem that can be overcome. to the present author's knowledge, no standard precautions include actions that should be omitted; they all need to be done, all of the time. transmission-based precautions are used in addition to standard precautions when patients are at risk of having, or confirmed to have, any of a specified list of infections or microorganisms. to decide if a person requires transmission-based precautions, there has to be an infection risk assessment at every patient admission. this infection risk assessment is, therefore, a critical standard precautions action. this assessment is where patients with diarrhoea, vomiting, infected wounds, symptoms suggestive of tuberculosis or at high-risk of carrying a multi-drug-resistant organism are identified as presenting an infection risk to others; as a consequence, transmission-based precautions commence in addition to standard precautions. when new infection control challenges arise, such as that presented by carbapenemase-producing enterobacteriaceae, guideline writers need to determine whether anything in standard precautions needs to change (e.g. hand hygiene materials or methods). if there is no evidence to change standard precautions, this should be stated explicitly in the new guidance. emphasizing the importance of standard precautions with adjectives such as 'strict', 'effective', 'good', 'excellent' or 'robust' is unhelpful because it implies that one only needs to practice safely if an infection risk is recognized, or it is acceptable not to undertake standard precautions all the time. this is not the case: standard precautions are the standard and they need to be undertaken for and by everyone in the care environment. the concept is simple: standard precautions represent what needs to be done every time, and what needs to be present in all care environments all of the time, to minimize the risk of people acquiring infection. however, actually putting this down in clear, concise and caring language is difficult. the guidance released after the pseudomonas spp. outbreaks in neonatal intensive care units involved actions such as not tipping body fluids into wash hand basins. this action is relevant in all care settings all of the time; ergo it should be incorporated within standard precautions. over time, standard precautions have advanced from protecting hcws from acquiring bbvs, to protecting hcws and patients from exogenous organisms, to what is now evolving into protecting people in the care environment from infections of both exogenous and endogenous origin. the following definition is proposed in an attempt to present an easy-tounderstand summary of standard precautions: standard precautions are designed to prevent cross-transmission and infection (including from bbv infection) when receiving care, delivering care or being present in the care environment. they are the minimum set of actions that are to be undertaken in every care environment and to be used for every care procedure, every time. there are three action categories. basics to ensure a safe environment: actions performed to inanimate objects such as equipment, environmental surfaces and linen. for example, a clean environment, decontaminated equipment ready for use by the next patient, safe disposal of waste and safe disposal of blood and body fluids (bbf). basics for the safe care of people (i.e that which is done by and with people). for example, hand hygiene, use of personal protective equipment, respiratory hygiene, assessment pre-patient placement and effective bbf exposure response. basics for the safe care of people who require high-infectionrisk procedures. any procedure that involves an invasive device or access to a sterile body area presents a high risk of infection and should be avoided wherever possible. where the procedures cannot be avoided, they should be practised in such a way as to minimize risks. such procedures include safe invasive device procedures (including endoscopy) and safe injection practices (including intravenous drug preparation and lumbar puncture). this is a novel summary but it presents a simple division of what needs to be done to inanimate objects in the healthcare setting and people to prevent infection. it also allows new actions to be slotted in on the basis of meeting the criteria for needing to be done for everyone, every time. by separating the invasive procedures into the third category, they can be named as what they present to patients: a high risk of infection. the first and second categories are uncluttered with instructions for procedures which may never be performed in some care settings. this brief paper highlights that standard precautions are, at present, anything but standard. furthermore, what is required to be part of standard precautions continues to evolve as understanding of infection risks in care settings changes. in order to prevent carbapenemase-producing enterobacteriaceae from becoming endemic, agreed standard precautions must become standard. at present, a common language is not spoken with regard to standard precautions. national and international guidelines should not extol standard precautions unless and until they make explicit what they mean by the term. a case is made for gathering relevant experts, including clinicians and human factors experts, to agree what is and what is not meant by standard precautions. what it is and what it is not e a familiar expression. standard precautions in healthcare: aidememoire. epidemic and pandemic alert and response guideline for isolation precautions: preventing transmission of infectious agents in health care settings infection control team, health protection scotland. national infection prevention and control manual epic : national evidencebased guidelines for preventing healthcare-associated infections in nhs hospitals in england perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis b virus, and other bloodborne pathogens in health-care settings guideline for isolation precautions in hospitals. hospital infection control practices advisory committee ecdc technical report: risk assessment on the spread of carbapenemaseproducing enterobacteriaceae (cpe) through patient transfer between healthcare facilities, with special emphasis on cross-border transfer interim guidance on pseudomonas and neonatal units hss(md) / . belfast: department of health, social services and public safety notes on nursing: what it is and what it is not. london: harrison and sons none declared. none. key: cord- -d htyfcl authors: gaglia, marta maria; rycroft, chris h.; glaunsinger, britt a. title: transcriptome-wide cleavage site mapping on cellular mrnas reveals features underlying sequence-specific cleavage by the viral ribonuclease sox date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d htyfcl many viruses express factors that reduce host gene expression through widespread degradation of cellular mrna. an example of this class of proteins is the mrna-targeting endoribonuclease sox from the gamma-herpesvirus kaposi’s sarcoma-associated herpesvirus (kshv). previous studies indicated that cleavage of messenger rnas (mrna) by sox occurs at specific locations defined by the sequence of the target rna, which is at odds with the down-regulation of a large portion of cellular transcripts. in this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify sox cleavage sites across the mrna transcriptome. these data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. this degenerate element is well represented in both human and kshv mrna, and its presence correlates with rna destabilization by sox. this represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables rna cleavage at specific locations without restricting the range of targets. furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. triggering wide-spread rna degradation is a common strategy that viruses use to decrease host gene expression, also known as host shutoff [ , ] . viral factors from many different families including herpesviruses, coronaviruses and orthomyxoviruses either directly cut rnas or indirectly stimulate rna cleavages in an endonucleolytic fashion [ , ] . cellular rna exonucleases are then recruited to degrade the fragments, resulting in a reduction in rna and consequently protein levels [ ] . despite the fact that the proposed role of most of these host shutoff ribonucleases (rnases) is to modulate immune responses, they are generally thought to have little or no specificity and to affect host messenger rnas (mrnas) indiscriminately. however, increasing evidence suggests that this view may be overly simplistic, and that some of the rnases display selectivity for or against specific targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this type of specificity may provide an additional level of regulation in viral control of the host transcriptome. how this selectivity is achieved and how it is balanced with the widespread shutoff phenotype remain open questions. the sox family of proteins from gamma-herpesviruses is an example of a viral rnase that displays both broad targeting of rnas and a poorly understood level of selectivity. gammaherpesviruses include the human pathogens kaposi's sarcoma-associated herpesvirus (kshv), which causes kaposi's sarcoma as well as lymphomas in immunocompromised individuals and remains a leading cause of cancer-linked death in sub-saharan africa. the sox (orf ) protein is expressed early during the lytic cycle of kshv infection and its expression triggers rna degradation, which is recapitulated by expression of the protein alone [ ] . homologs of sox in the other human gamma-herpesvirus, epstein barr virus (ebv bglf ), and in the model murine pathogen murine herpesvirus (mhv musox) also degrade rna in cells [ , ] . studies in mhv suggest that host shutoff by the sox family of proteins is crucial for viral replication in specific cell types and for systemic spread of the virus and establishment of a latent infection [ ] . transcriptomic studies of mrna levels during kshv or mhv infection and in cells overexpressing sox demonstrate that this family of proteins triggers the degradation of a majority of both host and viral transcripts [ , , ] . however, in-depth mechanistic studies of sox reveal a more complex picture. sox targets mrnas, as opposed to non-coding rna species, a specificity that is related to the association of sox with polyribosomes [ ] . moreover, selected transcripts, like the cytokine interleukin (il- ) [ ] and apoptosis enhancing nuclease (aen) [ ] , are spared from sox-mediated decay. in the case of il- , protection is conferred by the presence of a protective sequence in the ' untranslated region (utr) [ ] , but aen appears to be intrinsically resistant to sox mediated degradation [ ] , without a clear protective element in its sequence. the most unexpected observation, however, is that kshv sox and ebv bglf cut rnas at specific locations that appear to be determined by an unknown targeting element [ , ] . these specific cleavages become apparent upon knockdown of the human rnase xrn , the major '- '-directed rnase in eukaryotic cells, which is responsible for clearing the ' rna fragments generated by these rnases [ ] . this ability of sox to cut at specific locations within mrna yet target the majority of transcripts argues for a degenerate targeting motif. in general, the principles guiding the positioning of rna cleavages by cellular and viral mrna endonucleases are not well understood. in the case of endonucleases, the term "sequence specificity" is sometimes used to refer to preferential cutting at specific dimers, often inferred from in vitro studies (for example in datta et al. [ ] ). however, this specificity cannot explain cutting of rnas at single locations in mrnas. additional specificity can be conferred by localization of the target mrna to a specific site in the cells [ ] or proximity to a "landmark" feature on the rna, such as the ' of the transcript [ , ] or the stop codon location [ ] . the sox targeting system is unprecedented because the sequence of the targeting element alone appears to direct cleavages in mrna, and because the targeting element is longer than nt [ ] . to address how sox specificity is mediated, we applied a degradome sequencing technique called parallel analysis of rna ends (pare) [ ] to map cleavage sites of the sox protein across the human transcriptome. development of a stringent python-based analysis algorithm, which we term pydegradome, allowed identification of sox-dependent cuts at specific locations across the mrna transcriptome. the sequences surrounding these sites contained no strong consensus sequence, but rather a degenerate sequence pattern that nonetheless conferred specificity when analyzed experimentally in endogenous mrna targets. the presence of a more complex targeting motif explains how sox achieves cleavage specificity without sacrificing target breadth, and offers a framework for understanding how additional viral and cellular endonucleases may operate. development of a novel bioinformatics pipeline to detect highconfidence sox cleavage sites across the transcriptome following pare prior analyses of individual mrnas indicated that the kshv rnase sox cuts at specific locations within the rna, in a manner dependent on the sequence surrounding the cleavage site [ ] . by performing ' rapid amplification of cdna ends ( ' race) on the gfp reporter mrna, we found that the gfp mrna was cleaved in the same location regardless of whether sox was transiently expressed in t cells or expressed from the kshv genome in lyticallyreactivated islk. cells (s fig) . this is in agreement with the fact that sox activity does not rely on any additional viral proteins, and that its cleavage activity can be studied in transfected cells [ , , ] . to dissect the specificity of sox cleavage across the mrna transcriptome, we designed an approach to map sox cleavage sites in endogenous mrnas using pare [ , ] . pare is an rna-seq based methodology that allows mapping of the ' ends of uncapped, phosphorylated rna species, such as mrna degradation fragments ( fig a) . as we previously found that the sox degradation intermediates are cleared by the host '- ' exonuclease xrn [ ] , we stabilized degradation intermediates in t cells expressing a gfp-sox fusion [ ] by knocking down xrn (s a fig) . cells expressing gfp alone were used as controls to filter out rna fragments generated by cellular rnases or other processing events. this was important because multiple studies have shown that pare and similar techniques detect a large number of rna fragments in human cells, many of which are of unknown origin [ ] [ ] [ ] . we prepared and sequenced pare experimental procedure and peak finding analysis pipeline. a) diagram of the pare procedure. b) schematic of pydegradome analysis approach, which uses read counts in a control sample to generate a table of thresholds to compare test sample counts to. the table lists thresholds for a particular user-defined confidence level and for a range of ratios between control and test samples. the applicable ratio for each position is computed by multiplying a user-defined multiplicative factor by the ratio of total read counts for the exon in test vs. control samples, thus accounting for variation in rna levels and total mapped reads. read counts in test sample at each position must exceed the threshold to be identified as part of a peak. c) example of plot of read counts ( ' end only) for nt surrounding a sox cut site identified by pydegradome within the '-most exon of the limd nm_ transcript in the four samples. note that y-axis has a logarithmic scale. this example shows the expected distribution for a cut site followed by exonucleolytic degradation due pare libraries from two replicates of sox-expressing or gfp control cells and extracted the ' end of each mapped read, which represents the cleavage site (s table) . conventional analysis of pare and similar degradome datasets relies on detecting cut sites in each condition and then comparing conditions to each other a posteriori, but initial attempts at detecting and validating cut sites indicated that this approach did not provide sufficient discriminatory power to identify sox-specific cleavage sites. in previous studies using pare or similar approaches [ , ] , additional information about the pathways, such as the mirna sequences for mirna cut site studies or the proximity of the site to a stop codon for studies of smg and nonsense mediated decay, was used to further select "true" cut sites. however, such contextual information does not exist for sox cleavage specificity. therefore, we devised a python-based analysis approach that would directly use our control dataset as a baseline, which we termed pydegradome ( fig b) . the analysis uses a bayesian probability framework to determine whether the read counts at a given location differ significantly between control and test samples, taking into account random variations in the number of reads. using bayes' theorem, we determine for each location whether the underlying rate of fragment production in the test sample is a multiplicative factor larger than the control rate at a user-defined confidence level. the user also chooses the multiplicative factor. for a given control read count, we thus compute a threshold that the read counts in the test samples have to exceed to be considered part of a "peak" (fig b) . to improve efficiency when testing thousands of locations, the software builds a reference table of the thresholds for the entire dataset. this approach allowed the identification of locations within the transcriptome where the read counts were statistically higher in the samples from sox-expressing cells than in control samples (peaks). in order to correct for up-or down-regulation of the rnas and for the total number of reads obtained for each sample, the ratio used to determine thresholds was computed from the user-defined multiplicative factor and the ratio of the total number of reads mapping to each exon in test vs. control samples (fig b) . to prevent isolated high read counts from skewing our analysis, we integrated read counts within small windows ( nt) rather than single nucleotides. adjacent windows that passed the cutoff were then combined into a single peak. we optimized the userdefined confidence level and ratio by determining how many peaks were detected when comparing each sox replicate sample to its gfp control to detect sox-specific peaks, or performing the opposite comparison to detect gfp-specific peaks. in addition, we also ran the program to detect peaks specific to only one biological repeat, by comparing the two sox or gfp replicates to each other, as these peaks may represent experimental noise. although we consistently detected more sox-specific peaks than gfp-specific peaks, varying the parameters improved discrimination of sox-specific peaks (s b fig) and reduced detection of "noise" peaks specific to one repeat. based on this optimization, we empirically set the final iteration of the program to detect nt windows with read counts in the test samples that are four fold higher than read counts in the control samples within a confidence level of . %. because these parameters are conservative, we expect that the sox cut sites we detected do not represent a comprehensive list of all sox cut sites, but rather only the highest confidence sites. within each peak, we also determined the position where the read count was highest, and we considered this position the location of putative cut site (with the cleavage occurring ' of this position) (fig b and c ). with similar optimization, this program could be used to identify the ends of degradation fragments in other degradome datasets that contain matching test and control samples. sox cuts sites are abundant and not positioned relative to landmark features of mrna using the approach detailed above, we detected a higher number of peaks specific to sox-containing samples relative to control samples, consistent with broad mrna targeting by sox (fig a; s table) . even when varying the allowed distance between peaks from - nt in sample replicates, the sox samples contained~ - times the number of reproducible ("shared") sox-specific peaks (s c fig). up to % of the sox-specific peaks but fewer than % of the control gfp-specific peaks were shared between the replicates, indicating that many of the sox cleavages reproducibly occur at a given site (fig b) . the read counts at the putative cut site in the two sox replicates were highly correlated ( fig c, spearman's ρ = . , p value < . ), further demonstrating that these peaks correspond to specific sox-mediated cleavages. for downstream analyses, we focused on cut sites that were detected in both the replicates using the . % confidence level and were - nt apart (s table) . example plots of the read counts around identified cut sites are shown in figs c and s a-s f. several virally encoded host shutoff factors that trigger rna degradation, including herpes simplex virus vhs and sars coronavirus nsp , are thought to induce sequence-independent cuts near the ' end of the message [ , ] . to examine whether sox cleavage sites in endogenous mrnas are position-specific, we compared the location of the sox-specific cut sites to those found only in control samples using the human transcript annotation from ensembl grch . in both sox and gfp control samples, more cut sites occur towards the ends of the transcripts, most frequently corresponding to the ' and ' untranslated regions (utrs) of the mrna (fig d and e ). it remains unclear whether this non-specific end bias is due to a general preference for cleavage in non-translated regions or a consequence of the pare approach. we also computed the position of the cuts relative to landmarks such as the transcript start site, start codon, stop codon or annotated ' end. only a fraction of the peaks was located within nt of any of these landmarks in either case (fig f) . although a greater percentage of the sox cut sites occurred within nt of start codons, this still only accounted for % of the cut sites. furthermore, the - % of both sox and gfp peaks near annotated transcription start sites may represent the beginning of full-length decapped mrnas rather than endonuclease cleavage fragments. collectively, these analyses indicate that sox cut sites are not restricted to a particular region of the mrna, although cleavage sites in both sox and control gfp samples may be enriched in areas of the transcripts that are not covered by ribosomes. these findings are consistent with our previous reporter mrna data [ ] . we next selected seven sox cut sites for independent experimental validation ( fig a) . the selection was based on three criteria: ) position more than nt from the annotated ' end of the transcript in order to eliminate potential transcription start sites, ) high number of mapped reads, and ) clear sox-specific peaks in a visual inspections of the read plots (figs c and s a-s f). we used two approaches to validate the cut sites, targeted ' race and insertion in reporter constructs, and found that all sites validated in at least one of the two assays. we detected a ' race fragment that appeared specifically in sox-expressing cells (s g fig) and whose size corresponded to the predicted sox cleavage location for four of the transcripts. (we were unable to detect the rnas for bloc s and srsf using control primers). our second validation approach was designed to test the hypothesis that specific rna sequences or structures flanking endogenous cut sites direct cleavage by sox even when removed from their native context, as we had seen for reporter mrnas [ ] . we inserted nt pare analysis identifies sox-specific cut sites in endogenous rnas. a) number of peaks/cut sites identified specifically in sox or gfp samples. b) fraction of the peaks identified in sox or gfp samples that were detected in both biological replicates ("shared peaks"), relative to the maximum distance allowed between the peaks. c) correlation plot of the heights of peaks found in both sox+ samples. peak height is defined as the highest read count within the peak window, at the position defined as the putative cut site. d) position of the cut sites found in both replicates ("shared cut sites") within the mrnas relative to the length of the transcript. e) position of the shared cut sites relative to the coding region of the mrna (in all samples > % of surrounding the cleavage sites from the mrna targets identified by pydegradome into a gfp reporter ( fig b) . we then co-expressed these constructs with sox in xrn -depleted cells and tested whether the inserted sequence caused a sox-specific cut in the mrna. the gfp reporter we used is normally cut by sox at~nt of the coding region [ ] , generating a degradation intermediate that is~ nt shorter than the full length mrna. we found that the insertion of the sequences from six out of seven of the candidate sox cleavage sites resulted in the appearance of a second rna fragment in sox-expressing cells (fig c and d ). interestingly, the intensity of the additional cleavage products varied between the insertions, suggesting some sequences are better sox targets than others. in particular, in the gfp reporters with insertions from the limd mrna (fig d) , we found that the original cleavage site in the gfp coding region was almost completely abolished in favor of the new cleavage site, as evidenced by the disappearance of the longer degradation intermediate. moreover, insertion of the limd nt sequence in a different position in the gfp mrna also elicited sox cleavage ( fig e) , further demonstrating functionality of the targeting sequence regardless of its broader context. taken together, these data indicate that we have identified bona fide sox cleavage sites in endogenous mrnas, and that these sites contain specific elements that lead to sox targeting. the sox cleavage site likely occurs in an unstructured region of the mrna and is characterized by an a-rich sequence just upstream of the cleavage to identify features that define a sox cleavage site, we searched the sequences surrounding the sox cut sites detected in both biological repeats for structural or sequence similarities, using the cut sites shared by the two gfp samples as a comparison set. first, localfold [ ] was used to determine the likelihood that the nucleotides around the cut are located in unpaired regions (i.e. accessibility). this program is a variation on the vienna algorithm rnafold and is based on the assumption that potential structures are formed locally, which is consistent with the success of our insertion experiments. we found that the nucleotides ' of the sox cut were more accessible (thus presumably unstructured) than surrounding sequences ( fig a) . this pattern was different from the sequences surrounding the gfp sites, suggesting that it is feature specific to sox cleavage sequences. we also computed the log likelihood for different nucleotides at positions around the sox cleavage ( fig b and c ). although no strong consensus sequence emerged, two features stood out from this analysis. first, the position right after the cut site (position ) was preferentially c or t. when we computed the frequency of different nucleotides at the cut site for both soxspecific and gfp-specific cleavages, we found that the pyrimidines c or t were found at % of the sox cut sites, whereas c or a were the most frequent bases at position in cuts specific to gfp control samples ( fig d) . this distribution is not due to a bias in library preparation, as a was the most frequent base at the beginning of both aligned (s a we also found that there were more as and fewer cs in the nt ' to the cleavage site. we computed the fraction of putative sox cut sites that had a dimers or trimers in the nt preceding the cut and found that a stretches were found before~ % of our mapped cut sites (fig the peaks fall within coding transcripts). nd = not determined, because multiple transcript isoforms are present in the annotation and the cut site position would differ between isoforms. na = no coding sequence annotated. f) position of shared cut sites relative to annotated landmarks on transcripts. for all panels in this figure, a scanning window of nt, a multiplicative factor of , a confidence level of . %, were used to predict cut sites. all cuts sites nt apart in the two replicates were used for the analyses in panel c-f (sox-specific peaks: n = , gfp-specific peaks: n = ). doi: . /journal.ppat. .g . the gfp-based reporters were then co-expressed with sox ("+" or "+sox") or an empty vector control ("-") in shxrn -treated e). this was not a general feature of the sequences that produce rna fragments, as only five of the cut sites found solely in the control samples were preceded by an a dimer and none by longer a stretches. these analyses suggest that although sox cut sites are defined by a degenerate sequence pattern, this sequence is enriched for pyrimidines at a cut site adjacent to an unstructured stretch of a residues. experimental analysis of an endogenous sox cleavage sequence confirms role of oligo a sequence and potential structural element to probe the sox targeting element further we examined more in detail the sox targeting element in the validated endogenous mrnas (fig a) . the structure prediction program rnafold [ ] predicted that the - nt surrounding the cut sites in six out of seven of the rnas fold in hairpin structures with oligo-a stretches and the cleavage sites in unpaired loops (figs a and s a). the only exception was mapk ip , which also lacked the oligo-a sequence. these structure predictions mirror the accessibility results from localfold analysis (fig a) , which indicated that the positions from - to relative to sox cut sites are more likely to be unpaired. similarly, we predicted the structures of all nt sequences surrounding the soxspecific cut sites in our dataset and determined how many of the sequences presented either the cut site, an a dimer or both the cut site and an a dimer in an unstructured region. for % of the sox cut sites, at least one of these two features was predicted to be in an unpaired region, with over % of the sequences predicted to have both ( fig b) . we reasoned that if these structural features are important for cleavage, the efficiency of the cleavage could vary depending on the length of the inserted endogenous sequence. fragments of different sizes may not be able to fold into the native structure equally well and the stability of the resulting structures may vary. consistent with this idea, we found that changing the length of the inserted fragments for two different rnas (limd and srsf ) changed the efficiency of sox cleavage, measured by the intensity of the degradation intermediate (figs c, d, s b and s c), although sequences of nt were sufficient to elicit sox cleavage. in particular, when we shortened the limd inserted sequence from nt to nt, nt and nt, the efficiency of cleavage progressively diminished (fig c and d ), consistent with limd sequences adopting a stem-loop structure that becomes destabilized upon sequential deletions of the putative stem region. surprisingly, shortening the inserted sequence for srsf had the opposite effect and increased the efficiency of the cleavage (s b and s c fig). because the same sequences are present in the nt and the nt srsf insertion, this observation cannot be explained by the presence of a targeting sequence alone. instead, we hypothesize that the shorter insertion folds more stably into an autonomous element that is required for targeting by sox. we previously found that mutating a tgaagt sequence nt before the gfp cut site to tgagtg could abolish the cleavage site in gfp (s a fig). the limd site is also preceded by a similar tgaaag sequence predicted to be in an unpaired loop. to test directly whether the a trimer in the limd sequence was required for the positioning of the cleavage, we mutated the tgaaag sequence in our insertion reporter to tgcccg, tggggg or tgtttg. as predicted by our data analysis, we found that mutation of the a trimer prevented the limd sequence from eliciting sox cleavage, indicating that the aaa is an integral part of the sox recognition site (fig e) . moreover, deletion of one of the three as in the nt insertion cells and the gfp mrna from these cells was detected using northern blotting. the arrowheads point to the additional cleavage fragments resulting from the insertions. images are representative of results from at least three experimental replicates. [ ] representation of the frequency of each base in the nt surrounding the cut sites found in both sox samples (n = ). the position of the cut site is reduced the efficiency of cleavage dramatically (fig c and d) , while rnafold structure prediction suggested that this deletion is unlikely to substantially alter the structure of the rna (s b fig). similarly, mutating an a dimer just upstream of the cut site in a srsf insertion construct reduces sox-mediated cleavage (fig g) . while the upstream a dimer likely contributes to sox targeting, we note that it is not always required, as a similar mutation in a pgam insertion construct did not abolish cleavage (s c fig). overall, these data are consistent with the idea that the a-stretch is an important feature of the sox cleavage specificity. lastly, our analysis indicated that the nucleotide g is under-represented at the position immediately following the sox cut site (position , fig b- d ). we found that mutating the nucleotide at position from an a to a g prevented sox-mediated cleavage in two out of three of the rna we tested (limd and srsf , but not the pgam ) (figs f, g and s d). these data suggest that sox activity is inhibited by the presence of a g nucleotide as the residue ' of the cleavage. collectively these data experimentally validate our computational finding and strongly suggest that sox cut sites are defined by a combination of sequence and structural features. a conserved sequence pattern is specific to sox-dependent cut sites although sequences flanking the sox cleavage sites lacked a strong consensus motif, our analysis showed that the frequency of the bases around the cut site diverged from the expected distribution for human rna sequences (fig b and c ). this suggested to us that there is a conserved sequence pattern among sox target sequences. in order to be able to search rna sequences for this variable motif, we derived a position weight matrix (pwm) for the positions - to + from the sox cleavage site ( being the nucleotide ' of the cut site, fig c) . the pwm is a matrix that lists the probabilities (transformed into log likelihood to correct for the background frequencies of the nucleotides) for each of the four bases at each of the positions of the putative motif. the log likelihoods for our pwm were derived from very high confidence sox cut sites identified in both of our experimental repeats using a very stringent confidence level of . % and located at least nt away from a transcriptional start site. the logo in s a fig is a pictorial representation of the pwm. we then used the pwm to confirm that sox cut sites specifically matched the motif by scoring several subsets of potential sox target or control sequences using the pwm. the presence of preferred nucleotides in positions - to + from the cut site results in higher (positive) log likelihood scores, whereas a poorer match to the motif produces a lower (negative) log likelihood score. indeed, the sequences flanking the set of reproducible sox cut sites (identified with a confidence level of . %) were a closer match to the motif compared to those surrounding gfp-specific fragment ends, as shown by the distribution of the log likelihood scores (fig a) . a similar difference was seen when analyzing the sequences surrounding all potential sox and control (gfp) cut sites from each of the two biological repeats. sequences around sox cut sites were a good match (positive log likelihood score) to the motif more frequently than control sequences (fig b) . in both analyses (fig a and b) , we removed the sequences of the high confidence cut sites used to derive the pwm from the analyzed sets. the results of these analyses indicate that although the indicated. d) percentage of sequences with each of the four nucleotides at the cut site (position ) among the gfp or sox specific peaks. e) percentage of sequences surrounding putative cut sites that contain at least one oligo-a stretch within the nt upstream of the cut (for d and e, sox: n = , gfp: n = ). for all panels in this figure, the shared cut sites were determined based on a scanning window of nt, a multiplicative factor of and a confidence level of . %. all cut sites with the same exact position in both sox or both gfp samples that were > nt away from an annotated transcription start site and had sufficient surrounding sequences within the same annotated exon ( nt in a, nt in b and nt in c and d) were included in these analyses. the varying number of sequences used for the analyses in the different panels is a result of the requirement for sufficient flanking sequences in the same annotated exon, but as many sequences as possible were analyzed in each case. doi: . /journal.ppat. .g fig . the sequence features at the sox cut site in limd and srsf , as well as a structural element around the site, are required for sox cleavage. a) predicted structure of the nt surrounding the sox cut site in limd , highlighting the a trimer (asterisk) and the cut site (arrow), as well as the ends of the nt, and nt insertions used in d. b) rnafold was used to predict the structure of all -nt sequences surrounding sox-specific and gfpspecific cut sites (based on a scanning window of nt, a multiplicative factor of and a confidence level of . %). the proportion of cut site where the location of the cut was predicted to be unpaired, that had an unpaired a dimer within nt of the cut or both is plotted. c-g) gfp reporters were co-expressed with sox ("+") or an empty vector control ("-") in shxrn -treated cells. the gfp mrna was detected using northern blotting. the empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowheads point to the normal gfp cleavage fragment. c-d) - nt surrounding the sox cleavage site in the limd mrna were inserted into the gfp reporter at nt . in the nt Δa construct, one of the three as found at positions - to - from the cut site was deleted. a representative blot is shown (c), as well as the quantified intensity of the signal from the different rna species (d), plotted relative to the intensity of the bands from the nt insertion construct. error bars = standard deviation, ** p < . , *p < . (student's t-test). e) the a trimer preceding the sox cut site in limd was mutated to a c, g or t trimer in the limd nt insertion construct. images are representative of results from at least three experimental replicates. f) the a immediately ' of the sox cut site in limd was mutated to a g in the nt insertion construct. g) the a immediately ' of the sox cut site in srsf was mutated to a g (a ! g) and the a dimer preceding the sox cut site was mutated to a c dimer (aa ! cc) in the nt insertion construct. fig . a degenerate motif defines sox cut sites. a position weight matrix (pwm) for nucleotide likelihood in the nt surrounding the sox cut sites was generated from the sequences that contained sox-specific sites with a confidence level of . %. sequences were scored using this matrix, after removing the "parent" sequences where applicable. a) frequency distribution histogram of log likelihood scores for the nt surrounding the gfp-or sox-specific cut sites. gfp: n = ; sox: n = . b) frequency distribution histogram of log likelihood scores for the nt surrounding all cut sites found in the two gfp and sox samples. gfp rep : n = ; gfp rep : n = ; sox rep : n = ; sox rep : n = . c) all human and kshv annotated precise sequence composition may vary, there is a specific element marking sox cut sites that is not observed in control samples. furthermore, our stringent analysis parameters have likely resulted in an underestimation of the number of true sox targeting sites, because even sites detected in only one of our two replicates were generally a good match to the sox targeting motif. we next used the pwm to test whether the prevalence of the targeting element differed between the human and viral mrna transcriptomes. we analyzed annotated transcripts with a nt scanning window, computed a log likelihood score for every possible nt sequence, determined the highest possible motif score for each transcript and plotted the distribution of the scores. in agreement with the widespread mrna cleavage by sox, most of the annotated human transcripts have at least one sequence that is a good match to the motif (log likelihood score > , fig c) . the prevalence of high scoring sequences may explain how sox is able to degrade most transcripts. moreover, we found that kshv transcripts also contained sequences that matched the motif (fig c) , which suggests that sequence specificity is not used by sox to discriminate between host and viral mrnas. this results is consistent with findings from the related gamma-herpesvirus mhv [ ] that show degradation of viral transcripts by proteins of the sox family. we have listed examples of human and kshv rnas with high log likelihood scores in s table. interestingly, within human transcripts, coding rnas tended to contain sequences that were slightly better matches to the motif than non-coding rnas (ncrnas, fig d) , as did spliced rna in comparison to non-spliced rnas (s b fig). the meaning of these small but statistically significant differences is unclear, particularly since the log likelihood scores for all groups were generally high, indicating the presence of good sequence matches to the motif. we also wanted to test whether the sequences around the experimentally determined sox cleavage sites were more likely to match the motif than other locations on the same transcripts. we analyzed the transcripts containing sox cuts sites identified in the pare data using a -nt scanning window as described above. we then ranked the log likelihood scores for all possible -mers in the transcript, and asked how highly ranked the score for the actual cut site was. % of the experimentally observed cut sites ranked within the top % of scores for their rna, indicating that the surrounding sequences were a very good match to the motif compared to other sites in the same mrna ( fig e) . finally, we tested whether the relative degradation of different human transcripts by sox was correlated with how well their sequences matched our degenerate motif. human transcripts were classified as down-regulated by sox or sox escapees based on the data from an rnaseq experiment comparing human mrna levels in cells overexpressing gfp-sox or gfp alone [ ] the best motif score for each rna detected in the rnaseq experiment was then computed using a -nt scanning window as described above. we found that the down-transcripts were scored using the pwm. the frequency distribution histogram for the top scores for each transcript is plotted. d) human transcripts were divided into coding and non-coding based on the annotation in ensembl. the frequency distribution histogram for the top scores for each transcript in the two sets of human rnas is shown. p value (kolgorov-smirnoff test) < . . e) all possible scores for mrnas carrying an observed sox cut site far from the transcription start site (n = , confidence level = . %) were computed and ranked in comparison to all the possible scores for the transcript containing the cut. out of ( %) of the cuts were found at the site with the best score. the cumulative frequency distribution for the percentile of the score at the cut was plotted. f) human rnas were classified into sox targets ("down-regulated rnas") or sox escapees ("escapees rna") based on the results from clyde and glaunsinger [ ] . the frequency distribution histogram for the top scores for each transcript in the two sets is shown. p value (kolgorov-smirnoff test) < . . regulated rnas (fold change < . ) had better motif scores than the escapee rnas (fig f) . similarly, when we plotted the fold change for each gene against the best motif score for that gene, we found that there was a modest but significant inverse correlation between the fold change in mrna levels and the motif scores (spearman's ρ = - . . p value < . , s c fig) . these analyses suggest that the level of down-regulation of mrnas by sox is in part determined by the degree to which their sequence is a good match for the sox targeting motif. we have identified key sequence features of the targeting element that directs the rna endonuclease sox to cleave a significant fraction of the mrna transcriptome. as we had hypothesized from the analysis of individual reporter mrnas [ ] , sox cleavages in endogenous mrnas occur in a sequence-specific manner. although surprisingly large, this element is not defined by strong sequence consensus, but instead contains a small number of conserved residues. structural features may also contribute to motif-driven sox targeting. our data resolve how a sequence-specific nuclease can target such a breadth of targets. sox presents a model of rna targeting in which cleavages are at the same time sequence specific and highly promiscuous. this is achieved through the use of a degenerate sequence/structure pattern that is anchored by key residues to define specific rnase targeting locations. good matches for a loosely defined sequence pattern can be found in all viral and host rnas, enabling cleavage to be simultaneously specific and widespread. although this approach may be less efficient than the location-driven targeting of the cap-proximal region reported for other host shutoff factors that promote mrna cleavage [ , ] , such a mechanism may provide more regulatory opportunities. also, it may explain why sox has less dramatic effects on rna than other viral rnases [ ] . many cellular endonucleases have few described targets and transcriptome-wide targeting analyses of other cellular and viral rnases are limited. it will thus be of interest to apply high-throughput sequencing approaches to isolate degradation fragments in other systems and investigate whether any other viral or cellular rnases use principles similar to those employed by sox to achieve target specificity. notably, recent studies of specificity of rnase l, a host rnase that is activated in response to viral infection and cleaves viral rnas and host rrnas, have also suggested that a combination of sequence and structure is important for targeting [ , ] . however, the requirements for rnase l targeting appear less stringent than those for sox, as the preferred cleavages sites occur at uu/ua dinucleotides in unpaired regions of structured rnas [ , ] . the rna motif underlying sox cleavage specificity could be used for target selectivity, enabling a subset of viral and cellular mrnas to escape cleavage. our observation that the majority of both viral and cellular transcripts contain the sox targeting motif is in agreement with the fact that in mhv , viral mrnas are broadly susceptible to degradation by the sox homolog musox [ ] . however, subsets of viral and cellular transcripts are not susceptible to host shutoff [ , , , ] , and it is likely that at least some of these escape due to the absence of a robust targeting element. indeed, the correlation between the scores of matches to the motif and the level of degradation of host mrnas suggest that sequences within the motif can influence rates of degradation of different rnas. this may result in a more nuanced effect of soxmediated degradation. we also note that the level of degradation seen by steady-state level measurements is likely influenced by additional variables that are unrelated to the efficiency of sox cleavage, including the efficiency of removal of different sequences by xrn and a reduction of transcriptional rate due to a feedback loop triggered by the rna degradation [ ] . therefore the relationship we see may be an underestimation of the contribution of targeting preference. an additional level of regulation for transcript selectivity is also provided by the presence of dominant protective elements like the sox-resistance element we have identified in the ' utr of the il- gene [ , ] , which prevents cleavage of the gfp rna despite the presence of a strong targeting sequence. we expect that both dominant and passive mechanisms of escape from sox-mediated targeting ultimately shape the landscape of host gene expression during sox-mediated host shutoff. a limitation of our analyses is that we are unable to readily explore, both computationally and experimentally, the contribution of structural elements to the sox cleavage site. computational analysis of shared structures is difficult when the sequences involved are not evolutionarily related. moreover, even in well-characterized examples of the same protein binding different rnas, for example in the case of bacterial ribosomal proteins binding both ribosomal rnas and their own ' utrs, the features that are recognized are highly variable [ ] , and the remainder of the structure may serve as a scaffold. nonetheless, our identification of endogenous cut sites makes experimental analysis of a putative sox target structure possible in the future. a major outstanding question is how sox recognizes the targeting motif. in vitro studies indicate that the binding affinity of sox for rna is much lower than its affinity for dna [ ] , which is also processed by sox during viral genome replication in the nucleus. this suggests that sox may not recognize rna targets directly but may instead be recruited by a protein partner. this model is supported by the observation that point mutations that abolish sox host shutoff activity in cells do not affect its rnase activity in vitro [ , ] , pointing at a likely protein-protein interaction. a sox partner protein would have to be a fundamental factor in rna metabolism and/or a rna binding protein with promiscuous specificity, as it must bind a large portion of the cellular rnas. another possibility is that sox directly recognizes its target sequence, and that the apparent low affinity for rna in vitro is due to the fact that a noncognate sequence was used for the binding assay. however, the fact that sox cleaves the gfp rna sequence when gfp is expressed from an rna polymerase ii promoter, but not when it is expressed from an rna polymerase i or iii promoter [ ] argues against this scenario. how this motif potentiates sox targeting, as well as whether it is used as a protein-binding scaffold for other purposes in the cell related to mrna fate remain important questions for the future. cells and xrn knockdown hek t, hek t shxrn and kshv-infected islk- [ ] and islk- shxrn cells were maintained in high-glucose dmem (gibco) supplemented with % fetal bovine serum (hyclone). shxrn cells were generated using ptripz-shxrn (thermoscientific, clone v ths_ /rhs - , targeting sequence: tatggtgagatatactatg). to induce expression of the shxrn , cells were treated with μg/ml doxycycline (fisher) for - days prior to harvesting. lytic induction of kshv was induced in islk- cells [ ] by treating cells with μg/ml doxycycline and mm sodium butyrate for - days prior to harvesting. the same induction also led to anti-xrn shrna expression in the islk- shxrn cells. for the second biological repeat of the pare experiment, cells were transfected twice with the sirna against xrn as previously described [ ] . plasmids pd egfp-n was purchased from clontech. pcdna . -gfp-sox was previously described [ ] . pcdef -sox was previously described [ ] . pd egfp-Δtgaag was previously described [ ] . to test sox-mediated cleavage of endogenous mrna sequences, - nt surrounding putative cleavage sites in the human mrnas to be tested were cloned from hek t cdna using vent polymerase (neb). they were then inserted into the bsrgi site at nt of the gfp coding region in pd egfp-n , as previously done to test gfp sequences [ ] , either by restriction enzyme digest or through a modified version of quikchange mutagenesis [ ] . an ecorv site was also generated at nt of the gfp coding region using quikchange mutagenesis (agilent) and the nt surrounding the cleavage site in limd were inserted at this location. quikchange mutagenesis (agilent) was used to insert out the following mutations: ) mutate the aaa sequence preceding the limd cleavage site to ccc, ggg and ttt in the pd egfp construct containing the nt limd fragment; ) mutate the aa sequence preceding the cleavage site to cc in the pd egfp constructs containing either the nt pgam or the nt srsf fragments; ) mutate the cleavage site from a to g in the pd egfp constructs containing either the nt limd fragment, the nt pgam fragment or the nt srsf fragment; ) mutate the gfp tgaagt to tgagtg. all primers used for cloning are listed in s table. pare library preparation and sequencing hek t cells treated with sirnas against xrn (repeat ) or expressing shrnas against xrn (repeat ) were transfected either with pcdna . -gfp-sox or pd egfp-n . in both cases > % transfection efficiency was observed. one day after transfection total rna was harvested and purified using rnabee (teltest). rna was then treated as described in zhai et al. [ ] to generate pare libraries. briefly, poly(a)+ rna was purified, and rna adapters were ligated to free ' phosphate-bearing rna ends. a second poly(a) purification was used to remove unligated adapter. cdna was synthesized using oligodt directed primers, and the cdna was then amplified times. as the adapter includes an mmei restriction endonuclease site, mmei was used to cut the double stranded amplicons bp downstream of the adapters. ' dsdna adapters were then ligated to the ' end of the amplicons. this created libraries of bp tags corresponding to the ' end of rna fragments flanked by adapters, similar to small rna libraries. libraries were checked on an agilent bioanalyzer and sequenced at the vincent j. coates genomics sequencing laboratory at uc berkeley using a hiseq illumina sequencer. raw data are available on the ncbi gene expression omnibus database as study gse . reads flagged by the casava . program were eliminated and cutadapt [ ] was used to trim away the adapter sequence at the read ' end (sequence: tggaattctcgggtgc-caaggaactccagt). because the pare protocol should produce - nt sequence tags from the ' ends of phosphorylated rna fragments, trimmed reads that were longer than nt or shorter than nt were discarded. the resulting sequences were aligned using tophat . . [ ] using bowtie as recommended for short sequences. no mismatches were allowed (-n option), and only alignments that uniquely mapped to the annotated portion of the genome (-t -x options) were retained, to simplify downstream analysis. for the alignment and subsequent analyses, grch and the ensembl annotation for this genome build were used. these and other analysis were carried out on an imac computer (mid model, . ghz intel core i , gb ram). a bayesian probability framework was used to find peaks that were specific to test samples compared to control samples, which takes into account random variations in the observed number of reads. at a given location and a given experiment, we assume that there is an underlying rate at which reads are produced, and the observed count follows a poisson distribution with mean equal to this rate. in both the control and test data sets, we find that the frequency of reads per location follows a power-law distribution, as is typical for gene expression and deep sequencing data [ , ] , and we therefore assume that the prior distributions for the underlying rates follow this powerlaw distribution, where the power is fitted from the data. at a given location, we then use bayes' theorem to construct posterior distributions for the rates, given the observations of the read counts. we then deem that there is a significant difference between the control and test at that location, if the posterior probability of the test rate being a multiplicative factor larger than the control rate exceeds some confidence level. the multiplicative factor (ratio) and confidence level are chosen by the user. the observed counts vary over a large range, from single digits up to values in the millions, and a key feature of the method is that is can effectively deal with these variations within a unified theoretical framework. in practice, for a given control read count, we can compute a threshold for the test read count, beyond which the difference in underlying rates is significant. the software builds a table of the thresholds using bicubic splines so that many locations can be tested efficiently. the peak finding python scripts are attached as s files. parameters were empirically optimized for the analysis so that a scanning window of nt, a multiplicative factor between test/control read counts of and a confidence level of . % or . % were used to output specific peaks. parameters used in the different analyses are specified in the figure legends. after identifying peaks in single test/control comparisons, the peaks found in the biological repeats were compared. for subsequent bioinformatics analysis of sequences only peaks that were found in both biological repeats were used (figs d, b, s c ). sequences surrounding cleavage sites as defined by chromosomal positions were extracted, using the human genome assembly grch build as a reference. as many sequences as possible were used for each analysis. however, because the short reads do not provide information about mrna isoform and splicing, for all sequence analyses only cut sites that had sufficient flanking sequences within the same annotated exon were used. for motif analysis (figs b, c and s a) and rnafold structure prediction (fig b) nt or nt on either side of the cut site were used. for the accessibility computation (fig a) , localfold.pl [ ] , a modification of the rnaplfold algorithm within the vienna rna package (v . . ) [ ] was used using default settings (window = nt and maxspan = nt) and sequences of nt on each side of the cut site were analyzed. the log likelihood of each base at each position was calculated using background frequencies of nucleotides derived from the human cdna list from the ensembl grch build. weblogo [ ] was used to generate a graphical representation of the sequence motif from aligned sequences. to score matches to the motif, a position weight matrix was generated using log likelihoods for positions - to + relative to the cut site using cut sites that were deemed high confidence in our analysis (i.e. identified using a . % confidence level and position at least nt away from an annotated transcription start site). the log likelihood score was then calculated for all sequences surrounding cut sites that were identified in different subsets of the data. the cut sites used to generate the matrix were always eliminated from the sets that were analyzed. to compute the score matches in human and kshv mrna, the log likelihood score for each nt sequence was calculated in all sequences longer than nt listed in the human cdna fasta repository associated with the ensembl grch build or in a kshv mrna fasta-formatted list (compiled using data from arias et al. [ ] ). the highest score was recorded for each mrna. to separate the human rnas into coding and non-coding their ensembl annotation was used. rnas annotated as "protein coding", "nonsense mediated decay" and "non stop decay" were considered coding, whereas rnas annotated as "antisense", "lincrna", "mirna", "snorna", "processed transcript", "unprocessed pseudogene", "pseudogene", "transcribed unprocessed pseudogene", "transcribed processed pseudogene", "processed pseudogene", and "unitary_ pseudogene" were considered non-coding. the ensembl annotation was also used to determine whether the transcripts were spliced. the motif scores for human mrnas detected in clyde and glaunsinger [ ] were also compared to the level of degradation, that is the fold change in steady-state mrna levels between gfpexpressing and gfp-sox-expressing samples in the cited study. for the analysis in fig f, the transcripts were categorized into "down-regulated" (fold change in sox vs. gfp < . ) and "escapees" (fold change in sox vs. gfp > . ). the structure of the sequences surrounding the validated cut sites was predicted using the rnafold webserver (vienna rna package [ ] ). rnafold v. . . [ ] was used to predict structures around all candidate cut sites, and the results were analyzed to determine whether either of the nucleotides at position - and was predicted to be unpaired. they were also analyzed to determine whether they had an a dimer within nucleotides ' of the cut site that was also predicted to be unpaired. custom python . scripts (s files) were used unless otherwise noted. where noted in the figure legends, the kolgorov-smirnoff test was used to determine whether the distribution of scores were significantly different. total cellular rna was isolated for northern blotting using trizol (life technologies). rna was separated on formaldehyde gels ( x mops buffer, . % agarose, . m formaldehyde) in mops buffer ( mm mops (sigma), mm sodium acetate, mm edta, ph . ) and transferred by capillary blotting onto nitrocellulose membrane (bio-rad) using x ssc buffer ( . m nacl, . m sodium citrate, ph . ). northern blots were probed with p-labeled dna probes made using decaprime ii (ambion), against the ' utr of the gfp reporters. blots were imaged using a fujifilm scanner fla- . quantification of the blots was carried out using imagej [ ] . ' rapid amplification of cdna ends (race) was carried out on μg of total rna using the first choice rlm-race kit following manufacturer's protocol (life technologies). ' race primers are listed in s table. protein harvesting and western blotting protein was isolated for western blots in protein lysis buffer ( mm tris ph . , mm nacl, % triton x- ) containing complete edta-free protease inhibitors (roche), separated on sds-page gels run in tris-glycine buffer and transferred onto pvdf membranes (emd millipore). western blots were performed with mouse anti-xrn antibodies (bethyl laboratories or santa cruz biotechnology, : ) or mouse anti-tubulin antibodies ( : , sigma aldrich). secondary antibodies were used at : dilution and purchased from southern biotech. a) the tgaagt sequence at positions - to - relative to the cut site in the gfp rna (see s b fig) was either partially deleted (Δtgaag) or mutated to tgagtg. the wild-type gfp and the two mutated reporters were co-expressed with sox ("+") or an empty vector control ("-") and the gfp mrna was detected using northern blotting. the arrowhead points at the position of the normal gfp cleavage fragment. b) predicted structures (with rnafold) of the nt surrounding the sox cut site in limd , showing the wild-type sequence on the left (" nt") and the mutated sequence lacking one of the as on the right (" nt Δa"). c-d) gfp reporters were co-expressed with sox ("+") or an empty vector control ("-") in shxrn -treated cells. the gfp mrna was detected using northern blotting. the empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowhead in e points to the normal gfp cleavage fragment. the a dimer preceding the sox cut site in pgam was mutated to a c dimer (c) or the a at position was mutated to g (d) in the pgam nt insertion construct. (eps) s fig. additional analyses of prevalence of the degenerate sox targeting motif. a) weblogo [ ] representation of the frequency of each base in the nt surrounding the cut sites found in both sox samples using confidence level setting of . % and excluding sites near an annotated transcription start site (n = ). b) human transcripts were divided into "spliced" and "not spliced" based on the annotation in ensembl. the frequency distribution histogram for the top scores for each transcript in the two sets of human rnas is shown. p value (kolgorov-smirnoff test) < . . c) the fold change in mrna levels in sox-expressing vs. control cells from clyde and glaunsinger [ ] is plotted against the best motif score for that gene. spearman's ρ = - . , p < . . (eps) s table. number of reads obtained from pare. % mapping (no restrictions) indicates the percentage of reads that map to the human genome if the requirement for unique mapping to a previously annotated region of the genome is removed. (docx) s table. number of peaks detected. the number of peaks detected by using each of the samples as test or control in the pydegradome program (and plotted in fig a) is listed. parameters used for this analysis were a scanning window of nt, a multiplicative factor of , a confidence level of . %. (docx) s table. sox cut sites identified by our analysis. this table lists sox cut sites identified in both replicates with confidence level of . % or in one with confidence level . % and in the second with confidence level . %, and that were nt apart in the two replicates. the table includes the chromosomal position of the cut site, the read count at the cut site in each replicate, the gene name and the confidence level setting used for the identification. it also indicates whether the cut site could be a transcriptional start site (tss) and whether the cut site was used for the analyses in figs and or to generate the pwm for analyses in fig . only cut sites identified in both replicates with confidence level . % were used for the analyses shown in figs and . (xlsx) s table. list of human and kshv transcripts with highest log-likelihood scores of a match to sox targeting motif. (docx) s table. primers used for cloning and ' race analysis. (docx) s files. compressed archive of scripts required for pydegradome analysis. "readme.txt" file with instructions on how to run the analysis, as well as the two scripts required for the analysis are included in the archive. (zip) s files. compressed archive of scripts used for analyses in the paper. "readme.txt" file with instructions on how to run the analyses, and several scripts used to analyze the data. (zip) emerging roles for rna degradation in viral replication and antiviral defense kshv and the pathogenesis of kaposi sarcoma: listening to human biology and medicine a common strategy for host rna degradation by divergent viruses an overlapping protein-coding region in influenza a virus segment modulates the host response coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn . renne r, editor highly selective escape from kshv-mediated host mrna shutoff and its implications for viral pathogenesis deep sequencing reveals direct targets of gammaherpesvirus-induced mrna decay and suggests that multiple mechanisms govern cellular transcript escape an rna element in human interleukin confers escape from degradation by the gammaherpesvirus sox a ribonucleoprotein complex protects the interleukin- mrna from degradation by distinct herpesviral endonucleases sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage the ul protein of herpes simplex virus mediates selective stabilization or degradation of cellular mrnas the herpes simplex virus ul gene-dependent destabilization of cellular rnas is selective and may be sequence-specific lytic kshv infection inhibits host gene expression by accelerating global mrna turnover host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm host shutoff during productive epstein-barr virus infection is mediated by bglf and may contribute to immune evasion gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mrna degradation host transcript accumulation during lytic kshv infection reveals several classes of host responses characterization of pa-n terminal domain of influenza a polymerase reveals sequence specific rna cleavage decay of endoplasmic reticulum-localized mrnas during the unfolded protein response the herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target rnas in cell extracts a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein smg promotes endonucleolytic cleavage of nonsense mrna in human cells construction of parallel analysis of rna ends (pare) libraries for the study of cleaved mirna targets and the rna degradome rapid construction of parallel analysis of rna end (pare) libraries for illumina sequencing diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon micrornas, drosha, and additional nucleases human nonsense-mediated rna decay initiates widely by endonucleolysis and targets snorna host genes identification of smg cleavage sites and a preferred rna cleavage motif by global analysis of endogenous nmd targets in human cells global or local? predicting secondary structure and accessibility in mrnas viennarna package . ribonuclease l and metal-ion-independent endoribonuclease cleavage sites in host and viral rnas rnase l targets distinct sites in influenza a virus rnas viral nucleases induce an mrna degradation-transcription feedback loop in mammalian cells bacterial rna motif in the ' utr of rpsf interacts with an s :s complex crystal structure of a kshv-sox-dna complex: insights into the molecular mechanisms underlying dnase activity and host shutoff the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable generation of a doxycycline-inducible kshv producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction integration of pcr fragments at any specific site within cloning vectors without the use of restriction enzymes and dna ligase cutadapt removes adapter sequences from high-throughput sequencing reads tophat : accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions zipf's law in gene expression methods for analyzing deep sequencing expression data: constructing the human and mouse promoterome with deepcage data weblogo: a sequence logo generator kshv . : a comprehensive annotation of the kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features nih image to imagej: years of image analysis we thank albert tai at the tufts genomics core facility for discussion on the analysis, rachel brem and members of the brem lab (buck institute of aging research) and glaunsinger lab for helpful discussions. we thank alicia bicknell and members of the glaunsinger lab and gaglia lab for critical reading of the manuscript. conceived and designed the experiments: mmg. performed the experiments: mmg. analyzed the data: mmg chr bag. contributed reagents/materials/analysis tools: chr. wrote the paper: mmg bag. key: cord- - bxx u authors: lee, sunhee; lee, changhee title: functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: bxx u porcine deltacoronavirus (pdcov) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. like other coronaviruses, pdcov commonly contains major structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. among these, the n protein is known to be the most abundant and multifunctional viral component. therefore, as the first step toward understanding the biology of pdcov, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the pdcov n protein. similar to n proteins of other coronaviruses, the pdcov n protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. we then assessed alterations in production levels of proteins in the n-expressing pk (pk-pdcov-n) cells at different time points by means of proteomic analysis. according to the results of high-resolution two-dimensional gel electrophoresis, a total of protein spots were initially found to be differentially expressed in pk-pdcov-n cells in comparison with control pk cells. of these spots, protein spots showed a statistically significant alteration, including up-regulated and down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. the affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. notably, two members of the heat shock protein family were found to be up-regulated in pk-pdcov-n cells. these proteomic data will provide insights into the specific cellular response to the n protein during pdcov infection. porcine deltacoronavirus (pdcov) is an emerging viral pathogen that was first reported in in hong kong, china (woo et al., ) . in february , the detection of pdcov was first announced in ohio, united states, in conjunction with outbreaks of diarrhea without other etiologic agents. since its emergence, this novel coronavirus has been detected in us states, and almost % of the tested samples corresponded to cases of coinfection of pdcov with other enteric viral pathogens such as a rotavirus or porcine epidemic diarrhea virus (li et al., ; utr. orf a/b encompasses two-thirds of the genome encoding overlapping viral replicase polyproteins, a and ab, which are then proteolytically processed into mature nonstructural proteins. as in other coronaviruses, production of polyproteins a and ab requires a − ribosomal frameshift during translation of the genomic rna. the last third of the genome encodes the structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as two accessory genes, nonstructural gene (ns ) and ns gene, between m and n, and within n, respectively (lai et al., ; lee and lee, ; li et al., ; marthaler et al., a; woo et al., ) . among the structural proteins of coronaviruses, the n protein is abundantly produced in infected cells and has multiple functions in viral replication and pathogenesis (mcbride et al., ) . as the sole structural component of the viral capsid, the n protein of coronaviruses interacts with the nucleic acid and itself for selfassociation to protect the viral genome from extracellular agents, serving as the critical basis for ribonucleoprotein (rnp) complexes during virus assembly (mcbride et al., ) . the entire life cycle of coronaviruses takes place in the cytoplasm of infected cells, and accordingly, the n protein is distributed mainly in the cytoplasmic compartments. in addition to their cytoplasmic localization, coronaviral n proteins are commonly localized to the nucleolus, suggesting their non-structural functions in ensuring successful virus infection mcbride et al., ) . although a variety of studies have confirmed that n possesses multifunctional significance in coronavirology (mcbride et al., ) , detailed characteristics of this protein and its role in the replication of pdcov remain unknown. in the present study, alterations in cellular gene expression that are caused by the n protein were evaluated as a first step toward understanding the biological role of the n protein in pdcov replication. to accomplish this task, stable porcine-origin cell lines constitutively expressing the pdcov n protein were generated and characterized in this study. changes in expression patterns of various cellular proteins in the n protein-expressing porcine cells in comparison with control cells were examined by proteomic analysis at different time points. our proteomic data are expected to provide novel information for better knowledge of the properties and functions of the n protein during pdcov infection. hek- t cells (crl- ) were purchased from the american type culture collection (atcc, manassas, va) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca) with % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions ( ×; invitrogen). pk- cells were grown in rpmi medium (invitrogen) supplemented with % fbs and antibiotic-antimycotic solutions ( ×). the cells were maintained at • c in an atmosphere of humidified air containing % co . pam-pcd -n cells that stably express the n protein of porcine reproductive and respiratory syndrome virus (prrsv; sagong and lee, ) were cultured in rpmi medium (invitrogen) supplemented with % fbs, antibiotic-antimycotic solutions ( ×), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ×; invitrogen) in the presence of g/ml zeocin (invitrogen) and g/ml g (invitrogen). the anti-prrsv nucleocapsid (n) monoclonal antibody (mab) and anti-myc mab were purchased from median diagnostics (chuncheon, south korea) and invitrogen, respectively. antibodies to the × histidine tag, glucose-regulated protein (grp ), heat shock cognate -kda protein (hsc ), ␤-actin, ␣-tubulin, and sp were purchased from santa cruz biotechnology (santa cruz, ca). dna manipulation and cloning were performed according to standard procedures (sambrook and russell, ) . the escherichia coli strain dh ␣ (rbc bioscience, taiwan) was used as the host for general cloning. the full-length n gene was amplified from the pdcov knu - strain (lee and lee, ) with the following primer pair: knu - -n-fwd ( -gccggtcgacatggctgcaccagtag- ) and knu - -n-rev ( -cggctctagacgctgctgattcctgc- ), where underlines indicate the sali and xbai restriction enzyme sites, respectively. the pcr amplicon was initially inserted into the pbudce . vector (invitrogen) that contains a myc epitope and repetitive histidine codons, and the resulting plasmid pbud-pdcov-n was verified by nucleotide sequencing. because the pdcov genome contains one accessory ns gene within the n gene in a different reading frame, the presence of ns could affect our functional studies of the pdcov n protein. to prevent any such side effects, the translation initiation codon and the fifth codon of the ns orf were modified to disrupt its expression by pbud-pdcov-n. to accomplish this, overlapping pcr was conducted to simultaneously change the atg start codon and the fifth codon of the ns gene to tta and taa at genomic nucleotide positions , - , and , - , , respectively, using pbud-pdcov-n as a template with the following primers for the t c/t a mutation: ns -ko-fwd ( -ggcaacggagttccgctaaactccgccatc- ) and ns -ko-rev ( -gatggcggagtttagcggaactccgttgcc- ), where lowercase letters indicate the mutated nucleotides. both mutations were translationally silent with respect to the orf encoding the n protein, and the resulting plasmid pbud-pdcov-n w/ons was verified by nucleotide sequencing. a fragment of pdcov n w/ons cdna that was prepared from pbud-pdcov-n was then subcloned into the pfb-neo retroviral vector (stratagene, la jolla, ca) using the sali and ecori restriction sites to construct the pdcov n gene expression plasmid pfb-neo-pdcov-n-myc/his that produces recombinant n protein. the retrovirus gene transfer system (stratagene) was used to generate cell lines constitutively expressing the recombinant pdcov n gene or an empty vector only as described elsewhere nam and lee, ; oh and lee, ) . antibioticresistant continuous cell clones were analyzed by rt-pcr to determine the presence of the full-length n gene, and the positive clones (pk-pdcov-n and pk-neo) were amplified for subsequent experiments. pk-pdcov-n cells were grown on microscope coverslips placed in -well tissue culture plates. at h post-seeding, the cells were fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked using % bovine serum albumin (bsa) in pbs for min at rt and then incubated with an anti-his tag or anti-myc antibody for h. after washing times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca). the cells were finally counterstained with , -diamidino- phenylindole (dapi; sigma, st. louis, mo), and the cell staining was visualized by the fluorescence leica dm il led microscope (leica, wetzlar, germany) or a confocal laser scanning microscope (carl zeiss, gattingen, germany). the n expression in pk-pdcov-n cells was analyzed by flow cytometry. the cells were trypsinized at h post-seeding and centrifuged at × g (hanil centrifuge fleta ) for min. the cell pellet was washed with cold washing buffer ( % bsa and . % sodium azide in pbs), and cells were resuspended in % formaldehyde solution in cold wash buffer for fixation at • c in the dark for min followed by centrifugation and incubation of the pellet in . % triton x- in pbs at • c for min for permeabilization. after centrifugation, the cell pellet was resuspended in a solution of the primary anti-his tag antibody or normal mouse igg (santa cruz biotechnology) and the mixture was incubated at • c for min. the cells were washed and allowed to react with an alexa fluor -conjugated anti-mouse igg secondary antibody at • c for min in the dark. the stained cells were washed again and analyzed on the bd facsaria iii flow cytometer (bd biosciences, belford, ma). the growth properties of cells expressing pdcov n protein and control cells were determined by a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability as described previously (kim and lee, ) . all mtt assays were performed in triplicate. whole cell lysates were prepared from pk-pdcov-n cells grown at × cells/well in -well tissue culture plates at indicated time points using lysis buffer as described previously . for cell fractionation, pk-pdcov-n cells were fractionated using the nuclear/cytosol fractionation kit (biovision, mountain view, ca) according to the manufacturer's manuals. the protein concentrations of the cell lysates were determined using the bca protein assay (pierce, rockford, il). the cell lysates were mixed with × nupage sample buffer (invitrogen) and boiled at • c for min. equal amounts of total protein were separated in a nupage - % gradient bis-tris gel (invitrogen) under non-reducing or reducing conditions, and electrotransferred onto an immobilon-p membrane (millipore, billerica, ma). the membranes were blocked with % powdered skim milk (bd biosciences) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at • c for h and reacted with the primary antibody against the × his-tag, grp , hsc , or ␤-actin at • c overnight. the blots were then incubated with a horseradish peroxidase (hrp)-labeled goat anti-mouse igg or goat anti-rabbit igg antibody (santa cruz biotechnology) at the dilution of : for h at • c. finally, the proteins were visualized by enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the instructions of the manufacturer. to analyze the expression kinetics of cellular proteins in pk-pdcov-n cells, the band density of each protein was quantified relative to ␤-actin using densitometry with the wright cell imaging facility (wcif) version of the imagej software package (http://www. uhnresearch.ca/facilities/wcif/imagej/). a chemical cross-linking assay was performed as described previously . briefly, pk-pdcov-n cells and pam-pcd -n cells were grown in a -well tissue culture plate for h. for cross-linking studies, the cells were washed twice with cold pbs and then incubated at rt for min with a mm solution of a membrane permeable and thiol-cleavable cross-linker, , dithiobis(succinimidyl propionate) (dsp; pierce), dissolved in % dimethyl sulfoxide (v/v in pbs). the reaction was quenched with mm tris-hcl [ph . ] and incubated for an additional min. the cells were lysed with lysis buffer, and the resultant cell lysates were then subjected to a western blot assay as described above. pellets of cultured cells were washed twice with ice-cold pbs, placed in sample lysis solution consisting of m urea, m thiourea, % -[( -cholamidopropyl) dimethyammonio]- propanesulfonate (chaps), % dithiothreitol (dtt), % pharmalyte (ph . - , amersham biosciences), and mm benzamidine, and were then sonicated for s using a sonoplus device (bandelin electronic, germany). the samples were subsequently incubated for h at • c. after centrifugation at , × g for h at • c, insoluble cellular debris were discarded, and the supernatant was collected and stored at − • c until use. the protein concentrations were measured by the bradford assay as described previously (bradford, ) . protein samples were analyzed by de using commercial ipg dry strips (ph - , cm; genomine inc., pohang, south korea) for the first-dimensional separation (isoelectric focusing; ief) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) for the second dimension. the ipg dry strips were loaded independently with g of each protein sample and rehydrated overnight in rehydration buffer ( m urea, m thiourea, % chaps, % dtt, and % pharmalyte). ief was performed at • c using a multiphor ii electrophoresis unit and an eps xl power supply (amersham biosciences) with the following parameters: gradient increase from to v for h and v for a total of , v h. following ief separation, the ipg strips were incubated for min at rt with gentle shaking in equilibration buffer ( mm tris-cl ph . , containing m urea, % sds, and % glycerol) containing % dtt and were subsequently incubated in the equilibration buffer containing . % iodoacetamide under the same conditions. each equilibrated strip was then inserted into - % polyacrylamide gels ( cm × cm) and sds-page was run using a hoefer dalt d system (amersham biosciences) at • c for v h according to the manufacturer's instructions. the d gels were stained by the silver staining method as described previously with some modifications (oakley et al., ) . to compensate for the variability of de, three independent experiments were conducted for further statistical analysis. the stained gels were imaged using a high-resolution d gel ccd image analyzer, dyversity (syngene, frederick, md), and quantitative analysis of the digitized gel images was carried out using the pdquest software (version . , bio-rad, hercules, ca) according to the protocols provided by the manufacturer. data representing independent de experiments for each sample were statistically analyzed using student's t-test, and p-values of less than . were considered to be statistically significant. the quantitative data from each protein spot were normalized based on the total valid spot intensity for each gel. only protein spots that showed significant differential expression (p < . ) with a ± -fold consistent change in the expression level in comparison with the control sample were selected for mass spectrometry (ms) analysis. each selected protein spot was excised manually from the silverstained gels and was enzymatically digested in-gel as previously described (shevchenko et al., ) and using modified porcine trypsin (sequencing grade; promega, madison, wi). the gel pieces were washed with % acetonitrile (acn), air-dried thoroughly at rt, and then rehydrated for - h at • c with modified sequencing grade porcine trypsin ( - ng/l). the proteolytic reaction was terminated by addition of l of . % trifluoroacetic acid (tfa). the tryptic peptides were recovered by combining the aqueous phase from several extractions of gel pieces with % aqueous acn. after concentration, the peptide mixture was desalted using c ziptips (millipore), and the peptides were eluted in - l of % acn. an aliquot of this solution was mixed with an equal volume of a saturated solution of ␣-cyano- -hydroxycinnamic acid (chca; sigma) in % acn/ . % tfa, and l of this mixture was immediately spotted onto the target plate. maldi-tof analysis was performed on a microflex lrf instrument (bruker daltonics, billerica, ma) as described by fernandez et al. ( ) . the spectra were collected from shots per spectrum over the m/z range - and calibrated by two point internal calibration using trypsin auto-digestion peaks (m/z . , . ). the peak list was generated using flex analysis . . we used the following threshold for peak-picking: for minimum resolution of monoisotopic mass, for s/n. the search program mascot from matrix science (http://www.matrixscience.com/) was used for protein identification by pmf. the following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (cys) as a complete modification, oxidation (met) as a partial modification, monoisotopic masses, and a mass tolerance of ± . da. the pmf acceptance criterion was probability scoring. total rna was extracted from the lysates of pk-pdcov-n cells at and h post-seeding by the trizol reagent (invitrogen) and was treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was performed using a thermal cycler dice real time system (takara) with genespecific primer sets as described previously (sagong and lee, ) . the primer sequences are available upon request. the rna levels of cellular genes were normalized to porcine ␤-actin mrna, and relative quantities (rq) of mrna accumulation were calculated using the − ct method (livak and schmittgen, ) . to detect alterations of cellular mrna levels in the presence of the pdcov n protein, the relative fold change of each cellular gene was calculated in accordance with the comparison of pk-pdcov-n and control pk-neo cells. we used the student's t-test for all statistical analyses, and differences with p-values < . were considered statistically significant. a set of seven novel mammalian and avian coronaviruses was recently discovered, including one porcine coronavirus, pdcov (woo et al., ) . pdcov is the fifth coronavirus infecting pigs in addition to transmissible gastroenteritis virus, porcine respiratory virus, porcine hemagglutinating encephalomyelitis virus, and porcine epidemic diarrhea virus (pedv). to date, interactions between this novel virus and the host have not been studied yet. furthermore, alterations of cellular protein expression in response to pdcov or each viral protein upon infection currently remain undetermined. in the current study, the pdcov n protein was chosen in the current study for the proteomic analysis to evaluate specific host cellular responses, which is not only the most abundant viral component but also performs several biological functions in completing viral replication. for this purpose, sublines of pk- cells were established to stably express recombinant pdcov n under the control of a retroviral longterminal-repeat (ltr) promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blotting to confirm the n gene expression at mrna and protein levels, respectively (data not shown). according to the results of the western blot analysis, one pk-pdcov-n cell clone that constitutively expressed the highest levels of n was selected for subsequent studies. to characterize pk-pdcov-n cells, we examined intracellular expression levels of n by ifa, facs analysis and western blotting. as shown in fig. a , the specific cell staining was clearly evident when pk-pdcov-n cells were reacted with the anti-his tag antibody, confirming the constant high expression level of the n protein. furthermore, the majority of the cells consistently exhibited specific fluorescent signals, indicating a homogenous population of cells in terms of n expression (fig. b) . time-course western blot analysis revealed that the pk-pdcov-n cells stably express and accumulate robust levels of a ∼ kda recombinant n protein, larger than its predicted molecular weight of approximately kda possibly due to post-translational modifications and the presence of c-terminal myc and histidine tags (fig. c ). in addition, the overall growth kinetics of pdcov n gene-expressing pk cells was found to be similar to that of the parental pk-neo cells, indicating that the pdcov n expression has no effect on cell proliferation (fig. d) . self-association of the n protein has been observed in many viruses, including coronaviruses, and is essential for assembly of the viral core constructing the basic architecture of viruses. sequence analysis of the pdcov n protein indicated that it is composed of amino acids residues and contains no cysteine residues. as expected, no band corresponding to a disulfide-linked n dimer was detected by sds-page under non-reducing conditions ( fig. a, lane ) , indicating that the pdcov n protein does not undergo cysteine-linked homodimerization. as a positive control, the prrsv n protein in the n-expressing stable pam cells was clearly demonstrated to form -kda n-n dimers under the same conditions (sagong and lee, ; fig. a, lane ) . the ability of n to form non-covalent dimers was further investigated in a chemical crosslinking experiment. as shown in fig. b , the prrsv n protein in pam-pcd -n cells formed a number of higher-order oligomers (lane ) as reported previously . when the n protein in pk-pdcov cells was subjected to cross-linking, numerous multimeric forms of the n protein were identified (lane ), indicating that the n protein of pdcov exists in the form of noncovalently linked oligomers that are used for assembly the viral capsid. we next determined whether the recombinant n protein expressed in pk-pdcov-n cells is subject to nucleolar localization that is known to be a common feature of coronaviral n proteins. the staining pattern in pk-pdcov cells was found to be predominantly cytoplasmic and nucleolic; this pattern persisted for up to h after seeding (fig. a) . nearly all cells expressing pdcov n showed distinct fluorescent signals in the nucleolus at h post-seeding, and thereafter, the pdcov n protein was localized mainly to the cytoplasm at h post-seeding. the nucleolar localization of pdcov n was confirmed by transient transfection of bhk- or st cells with the plasmid pbud-pdcov-n (fig. b) . the cell fractionation assay also revealed the presence of the n protein in both the cytoplasmic and nucleic fractions (fig. c) . these observations demonstrated that, like n proteins of other coronaviruses, pdcov n protein is mostly distributed in the nucleolus along with the cytoplasm and has a conserved subcellular localization property. cellular proteins in the parental pk-neo cells and pk-pdcov-n cells were extracted and subjected to de analysis to compare the host protein expression profiles. to reduce the variability of gel electrophoresis, three independent de analyses of cellular extracts from control or n gene-expressing pk cells were performed, and spot intensity data from triplicate gels were selected for statistical analysis. on average, ± protein spots were resolved by de within a ph gradient - and were visualized using silver staining; the molecular weights of the spots ranged from to kda. these spots were used for the comparative analysis. fig. a shows representative images of de gels from control pk cells (upper panel) and n gene-expressing pk cells (middle and lower panels). a total of protein spots were initially found to be differentially expressed in pk-pdcov-n cells when compared with control pk-neo cells. on the basis of the statistical comparison, only those spots that consistently showed alteration in expression levels between pk-pdcov-n cells and the control cells were chosen for further protein identification. to identify the differentially expressed cellular protein spots in pk-pdcov-n cells at different time points, protein spots with a statistically significant alteration, including up-regulated and down-regulated protein spots (fig. b) , were selected and manually excised from the stained gels. subsequently, the trypsin-digested spots were subjected to maldi-tof analysis. with combined pmf and database searching, identity of all proteins was successfully determined. information on all these proteins in pk-pdcov-n cells, with their protein scores and sequence coverage, is summarized in table . to better understand the implications of the cellular responses to the pdcov n protein, we further (c) nuclear and cytoplasmic fractionation of pk cells expressing pdcov n. each nuclear and cytosolic fraction was prepared from pk-pdcov-n cells at indicated time points post-seeding and subjected to western blot analysis with the antibody specific for his-tag (top panel), sp as a nuclear protein marker (second panel), or ␣-tubulin as a cytosolic protein marker (third panel). all blots were also reacted with ␤-actin antibody to verify equal protein loading (bottom panel). categorized the identified proteins by biological processes according to the gene ontology database as described previously (zhang et al., ) . these proteins showing altered expression were associated with various cellular functions including intracellular transport, metabolic processes, gene regulation, the stress response, protein synthesis, cytoskeleton networks, and cell division. since changes in cellular protein expression may be attributed to alterations in the corresponding mrna levels, transcriptional changes for all the identified proteins were tested by real-time rt-pcr to confirm the results of the proteomic analysis (fig. ) . we were able to detect mrnas corresponding to nearly all proteins identified by proteomics, except for histone h . although the altered expression levels of the remaining proteins were consistent with the real-time rt-pcr results, we observed only modest increase or reduction in mrna levels. these results could be explained the case the correlation between mrna and protein abundance could be insufficient to predict protein expression levels from quantitative mrna data (gygi et al., ) , suggesting that the differences in protein levels might be due to post-translational modifications or protein stability rather than mrna levels. the quantity of each spot was normalized based on the total valid spot intensity for each gel and the relative fold change of each spot was then calculated between control pk-neo and pk-pdcov-n cells. data representing three independent de experiments for each sample were statistically analyzed and error bars represent standard deviations. *p = . - . . two members of the hsp family, glucose-regulated protein (grp ) and heat shock cognate -kda protein (hsc ), were found to be up-regulated in the pdcov n-expressing cells by the proteomic analysis. to further verify the dynamic alterations in fig. . transcriptional alteration of identified cellular genes in pk-pdcov-n cells. the mrna level of each gene was assessed by quantitative real-time rt-pcr and normalized to that of porcine ␤-actin. relative quantities (rq) of mrna accumulation were evaluated by − ct method and the relative fold change of each gene was then calculated between control pk-neo and pk-pdcov-n cells. results are expressed as the mean values from three independent experiments in duplicate and error bars represent standard deviations. *p = . - . ; † p < . . expression of these stress-response proteins under the influence of pdcov n, we performed time-course western blot analysis. total cellular lysates were prepared from pk-pdcov-n cells at different time points, and the expression kinetics of the grp and hsc was compared between control and n protein-containing extracts. higher expression of both proteins was first evident in pk-pdcov-n cells as early as at h post-seeding in comparison with control pk-neo cells, and the production of both proteins was persistently high during the later time points, suggesting that their enhanced expression is dependent on the n protein (fig. ) . these results were consistent with the proteomic analysis of de gels and realtime rt-pcr, demonstrating that the synthesis of grp and hsc is indeed up-regulated in response to expression of the n protein of pdcov. since viruses are obligate intracellular parasites, they must utilize the cellular machinery and biosynthetic components of the host cell for their own replication. after viral infection, the infected cells launch a number of host antiviral defensive responses, which are turned on to eliminate the invading viruses. on the other hand, viruses employ their own evasion strategies to complete their replication and to successfully spread to neighboring cells. this series of interactions between the virus and host results in compensatory modifications in cellular gene production. accordingly, numerous studies have been extensively conducted by means of various molecular and genomic methods to understand how the altered host gene expression affects viral infection and the associated pathogenic processes. the proteomic analysis coupling high-resolution de and maldi-tof/ms is a tool that can generate ample data on cellular protein profiles modified by viral replication. with the proteomic techniques, it is now possible to identify relative changes in protein abundance for evaluation of host cellular fig. . differential expression of the hsp family in pk-pdcov-n cells. cell lysates were prepared from pdcov n gene-expressing pk cells at indicated time points and immunoblotted to determine the expression profile of each protein with antibodies specific for grp and hsc (top to second panels). the blot was also reacted with anti-his tag (third panel) and anti-␤-actin (bottom panel) antibodies to confirm the status of n expression and equal protein loading, respectively. each cellular protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the ␤-actin gene and pk-pdcov-n sample results were compared to pk-control results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = . - . ; † p < . . responses to viral infection or viral protein expression and to gain specific insights into the cellular mechanisms involved in viral pathogenesis (alfonso et al., ; brasier et al., ; neuman et al., ; oh and lee, ; ringrose et al., ; sagong and lee, ; zhang et al., ) . in the present work, a proteomic approach was applied for profiling the global protein expression changes in host cells in response to the n protein of pdcov, which is a major constituent of the virion and infected cells. therefore, we initially established a porcine cell line stably expressing the pdcov n protein and then characterized the n protein produced in those cells. due to the absence of cysteine residues, a disulfide-linked homodimeric n protein of pdcov was not detected in pk-pdcov-n cells. rather, the pdcov n protein was shown to be self-assembled via non-covalent oligomerization as a structural component for formation of the viral capsid. interestingly, the n protein of pdcov was found to be predominantly present in both the cytoplasm and nucleolus of pk-pdcov-n cells. the localization of nidovirus n proteins to the nucleolus may be necessary for control of rna synthesis or ribosome biogenesis via association with ribosomal subunits and interaction with nucleolar proteins (chen et al., ; wurm et al., ; yoo et al., ; ) . similarly, the pdcov n protein may participate in such a viral strategy to favor viral replication and pathogenesis. in addition, the nucleolar localization sequence detector program (http://www.compbio.dundee.ac. uk/www-nod/) predicted that pdcov n harbors a putative nucleolar localization signal (nols) consisting of a stretch of basic amino acids. therefore, further research is needed to identify a functional nols to confirm the intracellular localization of the pdcov n protein. these biological characteristics of n gene-expressing cells have yet to be confirmed using an authentic n protein in pdcovinfected cells, but according to our results, pdcov n appears to act as a multifunctional protein playing structural and non-structural roles contributing to completion of viral replication. our proteomic data revealed that differentially expressed cellular proteins are identifiable in pdcov n gene-expressing porcine cells. the proteins that we identified in this study are involved in diverse cellular processes: metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication, and cell division. the significance of the functional roles of the selected host proteins affected by the interaction of the pdcov n protein with the host cell is discussed below. the most interesting finding in the present study is that two cellular chaperone proteins belonging to the hsp family are up-regulated concomitantly by the n protein of pdcov. the first up-regulated protein is grp in cells expressing the pdcov n protein, which is associated with endoplasmic reticulum (er) stress. in eukaryotic cells, the er is the major site for synthesis and folding of transmembrane and secreted proteins, and accordingly, animal viruses also use the er as a site of synthesis and processing of their own proteins. the amount of protein entering the er can differ under physiological and environmental conditions. if protein synthesis exceeds the folding capacity of the er, unfolded proteins accumulate there, resulting in er stress. to maintain the er homeostasis, cells have developed a signaling pathway known as the unfolded protein response (upr) that transmits signals across the er membrane to the cytosol and the nucleus and ultimately reduces protein translation and enhances the er folding capacity by up-regulating chaperone proteins (ron and walter, ) . coronavirus infection of cultured cells is known to cause er stress and to induce the upr, which then crosstalks with various cellular signaling pathways, including mitogen-activated protein kinase cascades, autophagy, apoptosis, and innate immune responses, indicating the involvement of upr activation in virus-host interactions and viral pathogenesis . more interestingly, global proteomic and microarray analyses have shown that the expression of chaperon proteins, such as grp and grp , is up-regulated in cells infected with a human coronavirus or in cells expressing the s subunit (jiang et al., ; yeung et al., ) . furthermore, the n protein of pedv, another porcine coronavirus, overexpression was shown to trigger er stress (xu et al., ) . thus, it appears that pdcov infection may induce er stress and the upr, leading to the up-regulation of grp to counteract the er stress; hence, the n protein may be responsible for this stress response pathway. the second identified hsp family protein is hsc , also known as heat shock -kda protein (hspa ). hsc is a molecular chaperone with multiple functions in protein folding and trafficking in all eukaryotic cells and in protecting cells from apoptosis or a wide array of stressors such as heat and infection (morano, ; powers et al., ; takayama et al., ) . accumulating evidence has shown that hsc plays important roles in certain processes involved in viral infection by modulating cell entry, virion disassembly and assembly, and the cellular antiviral response and apoptosis (chuang et al., ; gutiérrez et al., ; ivanovic et al., ; liu et al., ; radhakrishnan et al., ; yan et al., ) . apoptosis is considered an innate defense mechanism that limits propagation of a virus by eliminating infected cells (everett and mcfadden, ) . therefore, many viruses have evolved to employ various strategies that inhibit apoptosis in order to prevent premature cell death, thereby securing sufficient time for progeny production. thus, a conceivable explanation is that pdcov may take the advantage of suppressing apoptosis by up-regulating hsc in the early phase of the infection, and the n protein seems to be involved in this mechanism. in addition, hsc mediates the nuclear export of the influenza virus ribonucleoprotein complex via interaction with viral proteins m and ns (watanabe et al., (watanabe et al., , . in the present study, we found that pdcov n can be localized to the nucleolus in nexpressing cells. on the other hand, n proteins must be trafficked from the nucleolus to the cytoplasm to accomplish nucleocapsid assembly as well as viral rna synthesis and virus-host interactions. according to our results and existing data, hsc may facilitate the export of pdcov n from the nucleolus to complete the viral life cycle. like many other viruses, coronaviruses turn off host protein translation, while continuing to the synthesis of their own gene products to finish viral replication. one of the mechanisms behind this translational suppression is through interaction of the n protein with elongation factor ␣ (ef ␣), a major translation factor in mammalian cells . in the present study, the amount of another translational factor, ef , was affected by the host response to the pdcov n protein. although we do not know whether the increased expression of ef is related to its binding to pdcov n, such differential expression of a translation factor may be associated with regulation of both host and viral protein synthesis. we also found that expression of the cytoskeletal protein ezrin is significantly enhanced by the n protein. ezrin is a member of the ezrin-moesin-radixin (emr) family of host cytoskeletal proteins that organize the cortical cytoskeleton by mediating interactions between actin and the plasma membrane proteins and function as signal transducers in numerous signaling pathways (neisch and fehon, ) . the emr also modulate rna virus infection by regulating stable and dynamic microtubule formation (bukong et al., ; haedicke et al., ; naghavi et al., ) . given the biological features of the emr proteins, the up-regulation of ezrin in our study suggests that the pdcov n protein may manipulate the host cytoskeletal network and cell signaling, possibly to facilitate the processes of viral infection and replication. in contrast, the expression of translocon-associated protein subunit delta (trapd) is suppressed in cells expressing the pdcov n protein, according to our results. trapd is a part of the trap complex that is involved in translocating proteins across the er membrane and in regulating the retention of er resident proteins (fons et al., ) . coronaviruses acquire their lipid envelop via budding of the nucleocapsid through the er-golgi intermediate compartment (mcbride et al., ) . the down-regulation of trapd may lead to the release of cellular proteins from the er during pdcov replication, which in turn, promotes translocation of envelope-associated viral structural proteins to the er membrane -the site of budding -to facilitate virus assembly. eukaryotic cells reprogram their metabolism to adapt to stress-induced damage in response to environmental stressors. among our differentially expressed proteins, some are either directly or indirectly involved in metabolism. one hypothesis that can explain this result is that production of the n protein affects cellular biosynthesis by modulating the expression of metabolism-related proteins, which in turn remodels the intracellular environment for optimal pdcov replication. in conclusion, to our knowledge, this is the first report of a proteomic analysis of cellular responses to the pdcov n protein. although definite functions of the proteins that we identified here were not determined, it is likely that alterations in their expression are involved in virus-host interactions. to resolve these questions as well as performing various pdcov research, obtaining a korean pdcov isolate that can grow in cell culture is necessary; we are currently working on this task. in future studies, we are planning to assess cellular responses to pdcov by proteomic analysis to 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macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor alpha key: cord- -wy jhsb authors: nansseu, jobert richie n.; alima yanda, anastasie nicole; chelo, david; tatah, sandra a.; mbassi awa, hubert d.; seungue, judith; koki, paul olivier n. title: the acute chest syndrome in cameroonian children living with sickle cell disease date: - - journal: bmc pediatr doi: . /s - - - sha: doc_id: cord_uid: wy jhsb background: although sub-saharan africa (ssa) is particularly affected by sickle cell disease (scd), there is dearth of research on this topic in the region, specifically targeting the magnitude of scd-related complications. we therefore conducted this study to determine the burden of acute chest syndrome (acs) and describe its clinical and therapeutic aspects among scd children in cameroon, a ssa country. methods: this was a retrospective study carried-out from september to june at the scd unit of the mother and child centre of the chantal biya foundation, a pediatric reference centre in yaoundé, cameroon. we enrolled all scd children with confirmed diagnosis of acs, and recorded their clinical presentation at admission along with their evolution during hospitalization. results: twenty one cases of acs were identified during the study period, from hospitalizations of children with scd. ages ranged from months to years with a mean (standard deviation) of . ( . ) years, and a male/female sex ratio of . / . we noticed relatively low levels of hbf, from . to . % with a mean of . % ( . %). the three main symptoms at admission were fever ( . %), cough ( %) and chest pains ( . %). two patients ( . %) developed acs days after admission. the mean values of leukocytes, neutrophils, serum crp, serum ldh and hemoglobin were respectively . ( . )/mm( ), ( . )/mm( ), . ( . ) mg/l, . ( . ) iu/l and . ( . ) g/dl. the main localizations of radiological alveolar consolidations were the lower lobes ( . %). treatment associated broad-spectrum antibiotics ( %), hydration ( %), analgesics ( . %), whole blood transfusion ( . %), and oxygen supplementation ( . %). blood transfusion significantly improved hemoglobin level (p = . ). the duration of hospitalization, the mean of which was . ( . ) days, was influenced by none of the tested variables (all p values > . ). conclusion: acs is frequent among scd children in our milieu. its etiologies seem to be multifactorial. patients’ parents should be educated to recognize early signs and symptoms of the disease, and consult rapidly. additionally, clinicians must be trained to diagnose acs, and manage it promptly and efficiently to avoid its related catastrophic consequences. sickle cell disease (scd), described for the first time by herrick in , is an autosomal recessively inherited genetic disorder caused by a single point mutation in the gene encoding the β-globin chain of hemoglobin [ , ] . it is the most widespread and severe monogenetic disorder in the world [ ] , especially in sub-saharan africa (ssa) where it represents a real public health hazard with to % of babies born affected [ ] . scd is associated with multiple acute and chronic complications such as painful vasoocclusive events, cerebral vasculopathy, priapism, chronic kidney disease, acute chest syndrome (acs) and pulmonary hypertension among others [ , ] . acs is an acute lung injury syndrome that occurs frequently in patients with scd. indeed, it is the second most common cause of hospitalization, and the leading cause of death, contributing to almost % of scd-related mortality [ , ] . moreover, nearly half of deaths due to acs occur in scd patients less than years of age [ ] . repeated episodes of acs negatively impact long-term lung function, resulting thereby in chronic lung diseases [ ] . acs is currently defined as a new pulmonary infiltrate on chest x-ray consistent with alveolar consolidation but not atelectasis, in conjunction with at least one of the following clinical findings: fever (> . °c), reduced oxygen saturation or pao (< mmhg), tachypnea, intercostal retractions, nasal flaring or accessory muscle use, chest pain, cough, wheeze or rales [ , ] . in , charache et al. [ ] first suggested using the term "acute chest syndrome" for this complication, acknowledging the difficulties in determining its pathogenesis. till nowadays, the pathophysiology of acs remains not fully elucidated, yet multiple risk factors have been identified including: male sex, younger age, higher steady-state of leukocyte counts and hemoglobin level, lower hemoglobin f (hbf) concentrations, previous history of acs, history of asthma, active smoking or environmental smoke exposure. furthermore, acs may be more severe in individuals with hbss as compared with hbsc disease [ , , [ ] [ ] [ ] [ ] . the etiology of acs is multifactorial. the three primary studied mechanisms include pneumonia or systemic infection, fat embolism, and direct pulmonary infarction from hbs-containing erythrocytes [ , , ] . the management of this condition is still largely determined by the experience of individual practitioners, and currently there are no conclusive randomized controlled clinical trials to guide therapy [ ] . the most common therapy includes nonspecific supportive care strategies aimed at hastening recovery to baseline, including: hospitalization, hydration, analgesics, broad-spectrum antibiotics, bronchodilators, incentive spirometry, supplemental oxygen, and blood transfusions [ , , , ] . although ssa is particularly affected by scd [ ] , there is paucity of data on this topic in the region, specifically targeting the magnitude of scd-related complications. this study was thus undertaken, aiming at determining the burden of acs and describing its clinical and therapeutic aspects among scd children in a ssa tertiary pediatric health care facility. this was a retrospective study conducted from september to june at the scd unit of the mother and child centre of the chantal biya foundation. this is a pediatric reference centre located in yaoundé, the capital city of cameroon, a ssa country. this tertiary health care facility receives almost , consultations with almost , hospitalizations annually, with patients coming from all over the country. its scd unit was opened in december , and is used to dealing with routine out-patient consultations as well as daily follow-up of hospitalized scd children. we consecutively and exhaustively included all the patients aged below years and hospitalized in the scd unit during the study period. these were known scd children and adolescents with a confirmed diagnosis of acs (at entry or during hospitalization). the diagnosis of acs was based on a new radiological alveolar consolidation at the chest x ray, alongside clinical signs and/or symptoms, in keeping with ballas et al. [ ] , namely: fever (> . °c), reduced oxygen saturation, tachypnea, intercostal retractions, nasal flaring or accessory muscle use, chest pain, cough, wheeze or rales. patients who did not perform a chest x ray or whose chest x ray did not show any radiological alveolar consolidation were not included in the study. data were recorded on socio-demographic characteristics, past medical history and events, main complaints and clinical examination at entry, biological and radiological exams (full blood count, thick blood smear, hemocultures, serum crp (normal < mg/l), serum ldh (normal range - iu/l)) as well as the administered treatment and evolution during hospitalization. statistical analyses used spss, version . (spss inc, chicago, illinois, usa). results are expressed as mean (standard deviation) or count (proportion) as appropriate. qualitative variable comparisons used the χ test or equivalents, and that of quantitative ones, the student t test for paired samples or non-parametric equivalents. odds ratios (or) with % confidence intervals (ci) were used to appreciate the impact of different variables on the duration of hospital stay, and were calculated by logistic regression analyses. a p value < . was used to characterize statistically significant results. all the procedures used in this survey were in accordance with the current revision of the helsinki declaration. this study received approval from the authorities of the mother and child centre of the chantal biya foundation, and was delivered an ethical clearance by the ethical review board of the faculty of medicine and biomedical sciences, university of yaoundé i, cameroon. as the study was retrospective, we could not obtain participants' parents/guardians' consent. nonetheless, the need for consent was waived by the just-cited institutional review board. we recorded on the whole cases of acs during the study period, on hospitalizations of children with scd, hence an in-patient prevalence of . %. table displays the study population's general profile. ages ranged from months to years, with a mean of . ( . ) years, and males were more represented than females with a sex ratio of . / (tables and ). only seven patients had a hemoglobin electrophoresis dating back to less than one year, hbs ranging from % to % with a mean of . ( . ) %, and hbf, from . % to . % with a mean of . ( . ) % (table ). only . % of children were regularly followed-up, i.e. were used to visiting the unit for a check-up at least every months. besides, not more than . % of our patients had been adequately vaccinated in accordance with their ages, and only children aged - years ( . %) were currently taking an antibiotic prophylaxis (table ) . according to our patients' parents, duration of evolution of symptoms before consultation ranged from less than h to almost days, with a mean of . ( . ) days. the presenting complaints are reported in table . the three main symptoms were fever ( . %), cough ( %), and chest pains ( . %). the main clinical signs we observed were pulmonary consolidation ( %), an altered general appearance ( . %), clinical signs of anemia ( . %), a systemic inflammatory response syndrome ( . %), and signs of respiratory distress ( . %). two patients ( . %) who were initially admitted for vaso-occlusive crises (voc) developed acs after days of hospitalization. results of biological tests showed very high levels of leukocytes, neutrophils, serum crp and serum ldh. indeed, their mean values were . ( . )/mm , ( . )/mm , . ( . ) mg/l, and . ( . ) iu/l respectively. contrariwise, we observed relatively low levels of hemoglobin, with a mean of . ( . ) g/dl ( table ). the main localizations of radiological alveolar consolidations were the lower lobes ( . %). none of the hemocultures performed was positive, and only one of the thick blood smears performed to seek for malaria parasites was positive to plasmodium species, specifically plasmodium falciparum ( . %; table ). patients were placed on two to three antibiotics: a betalactam (either ampicillin or ceftriaxone) + a macrolide (oral azithromycin) ± an aminoglycoside (gentamicin) ( table ). two days after the fever had dropped, patients were switched to oral cephalosporin (cefixim) or amoxicillin accordingly, for a total duration of to days (both intravenously and orally). two patients ( . %) initially placed on ceftriaxone were still presenting fever on the fourth day of antibiotherapy; they were subsequently switched to vancomycin for seven days with a good improvement. adjuvant therapy included hyperhydration ( %), paracetamol ( %) for fever or pains, tramadol ( . %) and ibuprofen ( . %). fourteen patients ( . %) were transfused with whole blood, and all of them concurrently received an anti-malarial treatment to prevent transfusion-transmitted malaria. all the seven patients ( . %) who presented signs of respiratory distress were placed on oxygen, its duration ranging from to days with a mean of . ( . ) days (tables , and ) . a control full blood count was performed only by seven of our patients h after blood transfusion (table ) . we did not find any statistical difference between the first and second leukocyte counts (p = . ), this being the same for neutrophil counts (p = . ). by contrast, we did notice a significant increase in hemoglobinemia (p = . ). the duration of hospitalization ranged from to days with a mean of . ( . ) days. one patient of the female sex died on the fourth day of hospitalization, hence a mortality rate of . % (tables and ). while investigating factors that could have impacted the duration of hospitalization, we found that age, sex, regular follow-up, immunization status, duration of evolution of symptoms prior to consultation, transfusion, oxygen, and the antibiotic used were not implicated in the determination of duration of hospitalization (all p values > . as depicted by table ). this study showed that acs accounted for . % of scd children hospital admissions with almost . scd patients presenting acs per month. this proportion is comparable to the . acs episodes/month among hospitalized scd children in bruxelles [ ] , and higher than the . , . and . acs episodes/month respectively reported in brazzaville, antananarivo and french guiana [ ] [ ] [ ] . however, our prevalence of acs is lower than the - % rate of hospital admissions reported by miller and gladwin in their review [ ] . our prevalence may be an underestimate of the real burden of acs among our children suffering from scd for some reasons. first, hemoglobin electrophoresis is not systematically performed in our milieu, and many parents neither know their status nor that of their kids. as we considered only known scd patients, some unknown scd patients may have presented this condition and escaped from enrolment. second, due to parents' financial constraints, we are used to performing just one chest x-ray during hospitalization. as a result, we could have missed all those whose first chest x-ray was normal but who developed acs later on, given that radiographic findings in case of acs may progress over time [ , , ] . if it is true that a positive chest x-ray is the key element to define the disease [ ] , there have been some claims that a single negative chest x-ray cannot exclude the disease [ ] [ ] [ ] , taking into account that clinical assessment may appear inadequate to identify acs [ ] . it has been also suggested that every scd patient presenting with fever must undergo a chest x-ray [ , ] . consequently, close clinical monitoring alongside serial radiographic evaluation (without ignoring the risk of radiation) is necessary. in fact, it has been shown that acs may develop to days after admission for voc as there may exist a close relationship between acs and voc [ , , , ] . this is truer for adults than children, as the latter usually present with acs at admission [ ] . in our study for instance, only children ( . %) developed acs days after admission, and pains were concomitantly associated in . % of cases. some risk factors involved in the development of acs during hospital stay have been identified such as the male sex, past medical history of acs, thoracic pains at entry and use of morphine during hospitalization [ , ] . although we did not assess risk factors of developing acs, we observed a male predominance and relatively low levels of hbf, in line with the literature [ , , ] . we also found that fever ( . %), cough ( %) and thoracic pains ( . %) were the main symptoms in keeping with previous reports [ , , ] , these symptoms being mainly in favor of pulmonary infections. despite the fact that we [ , , ] . besides, we observed that radiological abnormalities of the lower lobes ( . %) were the prevailing ones, mirroring previous findings [ , ] . contrariwise, other studies showed that the main radiological localizations of acs were the upper lobes in children, and the lower ones in adults [ , ] . while the upper lobes predominance has been associated with an infectious etiology especially in children, the lower or multi-lobes predominance has been linked to pulmonary thrombosis and fat embolism [ , , ] or rib infarction [ ] . our results are therefore suggestive that the pathogenesis of acs in our setting is also multifactorial. further well-designed studies with large sample sizes are warranted to better elucidate the etiology of acs in our setting. in line with the literature [ , , ] , our management of acs included broad-spectrum antibiotherapy, hydration, analgesics, supplemental oxygen and transfusion, but none of these supportive care impacted the duration of hospital stay. actually, only patients presenting respiratory distress were placed on oxygen, mainly due to limitation of resources. likewise, we transfused only scd patients who had a hemoglobin level ≤ g/dl given that blood safety remains an issue of major concern in the milieu [ ] . although transfusion did not reduce the hospital stay, it significantly increased the hemoglobin level (p = . ), perhaps improving therefore the clinical state of our patients. early transfusion of scd patients presenting with acs should be encouraged in ssa settings, especially transfusion of packed red blood cells instead of whole blood to reduce transfusion-related adverse reactions, despite recurrent blood shortages that occur in the region [ ] . nonetheless, more studies are needed to underpin this suggestion with robust scientific evidence and indicate which threshold should be considered to transfuse scd children suffering from acs. the mean duration of hospitalization we found ( . days) is comparable to the days-duration reported by bertholdt et al. [ ] , but a bit higher than findings from vichinsky et al. ( . days) [ ] and lower than what has been reported by vichinsky et al. in another study [ ] and hunald et al. [ ] : more than days and days respectively. introduction of other supportive care in our practice like bronchodilators and incentive spirometry as elsewhere [ , ] , along with systematic oxygen supplementation and early blood transfusion could substantially reduce the duration of hospital stay. furthermore, the use of dexamethasone deserves some close considerations. in fact, there is body of evidence bolstering that low dose dexamethasone ( . mg/kg/ × doses) significantly diminished the duration of hospital stay, the need for blood transfusion, oxygen and analgesics use, and duration of fever, without a rebound effect [ ] which was observed with high dose methylprednisolone in cases of voc [ ] . one of our patients died, hence a mortality rate of . % which concurs the % percentage obtained by bertholdt et al. in belgium [ ] . this was an inadequatelyvaccinated and unregularly-followed female scd patient aged months, with a hbf level of . %. she was brought to consult for fever, cough and respiratory distress, and the chest x-ray revealed medium and lower right lobes alveolar consolidations. titers of ldh and crp were respectively iu/l and mg/l. she was placed on a triple antibiotherapy (ceftriaxone, gentamycin and azithromycin), oxygen supplementation, and was transfused as well. but she died at day of hospitalization in a context of severe sepsis. unfortunately, the retrospective design and small sample size constitute some weaknesses of the present study. besides, generalization of our results may be impeded by the use of data collected from only one hospital centre. another limitation is that biological data were incomplete due to parents' financial limitations, as the study was not sponsored. nonetheless, in this particularly difficult context, we carried-out serious and reliable data collection and analyses, and have shown that despite the difficulties in managing our patients, the outcome is relatively similar when compared with resource-rich settings in terms of duration of hospital stay and mortality rate. eventually, and to the best of our knowledge, this is the first study conducted in our milieu targeting acs burden and presentations. acs is frequent among scd children in our milieu. its etiologies seem to be multifactorial including infection, pulmonary thrombosis and fat embolism. after an initial normal chest x-ray, especially in a patient presenting with voc, repeated clinical evaluation must be conducted and possible changes in the clinical status should indicate the necessity of a new radiographic examination. patients' parents should be educated to recognize early signs and symptoms of the disease, and consult rapidly. additionally, clinicians must be trained to correctly diagnose acs, and manage it promptly and efficiently to avoid its related catastrophic consequences. additive treatment such as incentive spirometry, bronchodilators and even low dose dexamethasone could perhaps be introduced in our settings to reduce the hospital stay and hasten recovery. on recovery, treatment with hydroxyurea should be discussed to reduce the likelihood of recurrent episodes. abbreviations acs: acute chest syndrome; hb: hemoglobin; scd: sickle cell disease; ssa: sub-saharan africa; voc: vaso-occlusive crisis. deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes sickle-cell disease a case for developing north-south partnerships for research in sickle cell disease prevalence and risk factors for pulmonary artery systolic hypertension among sickle cell disease patients in nigeria health-related quality of life in children with sickle cell disease: child and parent perception pulmonary complications of sickle cell disease mortality in sickle cell disease. life expectancy and risk factors for early death respiratory distress and drepanocytosis].[article in french acute chest syndrome in sickle cell disease: clinical presentation and course. cooperative study of sickle cell disease impact of acute chest syndrome on lung function of children with sickle cell disease acute chest syndrome of sickle cell disease definitions of the phenotypic manifestations of sickle cell disease acute chest syndrome" in adults with sickle cell anemia asthma in children with sickle cell disease and its association with acute chest syndrome respiratory complications of sickle cell anemia in children: the acute chest syndrome]. [article in french the acute chest syndrome in sickle cell disease: incidence and risk factors. the cooperative study of sickle cell disease hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease pulmonary complications of sickle cell disease how i treat acute chest syndrome in children with sickle cell disease sickle-cell crisis in the child and teenager in brazzaville, congo. a retrospective study of cases acute thoracic syndrome in child sickle-cell disease]. [article in french associated factors of acute chest syndrome in children with sickle cell disease in french guiana causes and outcomes of the acute chest syndrome in sickle cell disease. national acute chest syndrome study group painful vasoocclusive crisis as a prodromal phase of acute chest syndrome. is only one chest x-ray enough? a case report the role of rib infarcts in the acute chest syndrome of sickle cell diseases complications aiguës de la drépanocytose morphine is associated with acute chest syndrome in children hospitalized with sickle cell disease acute chest syndrome in children with sickle cell disease. a retrospective analysis of hospitalized cases sero-epidemiology of human immunodeficiency virus, hepatitis b and c viruses, and syphilis infections among first-time blood donors in edéa, cameroon what is the best strategy for the prevention of transfusion-transmitted malaria in sub-saharan african countries where malaria is endemic? beneficial effect of intravenous dexamethasone in children with mild to moderately severe acute chest syndrome complicating sickle cell disease submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors gratefully acknowledge the medical staff of the scd unit of the mother and child centre, as well as all the nurses, especially mrs. essomba evarine and tanjon rita for their constant devotion to care for these vulnerable scd children. they also want to thank chavely gwladys monamele who helped them to revise the manuscript. the authors declare that they have no competing interests.authors' contributions jrnn conceived and designed the study, collected and analyzed the data, and drafted the manuscript. anay participated in study conception and design, in data collection, and in critical revision of the manuscript. dc, sat, hdma, js and ponk participated in critical revision of the manuscript. all authors read and approved the final manuscript. key: cord- -f pyn rx authors: roman, gheorghe title: mannich bases in medicinal chemistry and drug design date: - - journal: eur j med chem doi: . /j.ejmech. . . sha: doc_id: cord_uid: f pyn rx the biological activity of mannich bases, a structurally heterogeneous class of chemical compounds that are generated from various substrates through the introduction of an aminomethyl function by means of the mannich reaction, is surveyed, with emphasis on the relationship between structure and biological activity. the review covers extensively the literature reports that have disclosed mannich bases as anticancer and cytotoxic agents, or compounds with potential antibacterial and antifungal activity in the last decade. the most relevant studies on the activity of mannich bases as antimycobacterial agents, antimalarials, or antiviral candidates have been included as well. the review contains also a thorough coverage of anticonvulsant, anti-inflammatory, analgesic and antioxidant activities of mannich bases. in addition, several minor biological activities of mannich bases, such as their ability to regulate blood pressure or inhibit platelet aggregation, their antiparasitic and anti-ulcer effects, as well as their use as agents for the treatment of mental disorders have been presented. the review gives in the end a brief overview of the potential of mannich bases as inhibitors of various enzymes or ligands for several receptors. the classical mannich reaction, a three-component condensation between structurally diverse substrates (xeh) containing at least one active hydrogen atom, an aldehyde component (generally r -cho) and an amine reagent leads to a class of compounds generally known as mannich bases (scheme ). because mannich bases may be regarded as derivatives of the substrate obtained through substitution by an aminoalkyl moiety, mannich reactions are also known as aminoalkylation reactions. in the particular instance when formaldehyde is employed as aldehyde component, the substrate is converted into the corresponding mannich base through an aminomethylation process. although primary amines and even ammonia (in the form of an ammonium salt) may be employed as amine reagents in aminomethylations or aminoalkylations, secondary aliphatic amines (r nh) are the most commonly encountered as amine reagents in the mannich reaction. as formaldehyde is used to a great extent as aldehyde component in the mannich reaction, the structural diversity of mannich bases stems primarily from the miscellaneous types of the substrates that can be subjected to aminomethylation, and secondarily from the variety of amine reagents that can be potentially employed in the mannich reaction. regardless of their structural diversity, the substrates should all have an activating functional group as a crucial structural feature that is required to render the substrate active in the mannich reaction. the carbonyl function in ketones, the phenolic hydroxyl in phenols, the terminal carbonecarbon triple bond in alkynes, the heteroatom in heterocycles, or electronwithdrawing groups that substitute the carbon atom a to the carboxylate group in esters of aliphatic carboxylic acids are common examples of pairs of activating groups and corresponding substrates, but the list is far from being exhaustive. a general classification of the most common types of mannich bases with respect of the substrates from which they derive and the nature of the atom substituted by the aminomethyl function is given in fig. . under normal reaction conditions, substitution of a substrate with a single aminomethyl function results in mono-mannich bases, but two aminomethyl groups may also be grafted onto a substrate containing more than one active hydrogen atom, leading to double mannich bases such as e derived from dialkyl ketones, alkyl aryl ketones, -substituted phenols and pyrrole, respectively (fig. ) . also, the aminomethylation of substrate xeh with amine reagents other than secondary amines (such as ammonia, having three reactive hydrogen atoms at nitrogen, or primary amines renh , having two reactive hydrogen atoms at nitrogen) may lead to tris-mannich bases and bis-mannich bases , respectively (fig. ). in addition, the capability of some polyfunctional substrates to aminomethylate chemoselectively at a single potential reaction site under the appropriate reaction condition, or aminomethylate indiscriminately at multiple reaction sites, or even undergo aminomethylation simultaneously with ring closure, contributes considerably to the structural variety of the resulting mannich bases. two excellent, albeit rather old reviews provide more details on the synthesis and reactions of mannich bases to the interested reader [ , ] . mannich bases have found numerous practical applications in the treatment of natural macromolecular materials such as leather, paper and textiles, the production of synthetic polymers, as additives used by the petroleum industry, as products used in water treatment, analytical reagents, cosmetics, dyes, etc. [ ] . nonetheless, the most important application of the mannich reaction lies in the field of medicinal chemistry, and this claim is supported by the substantial number of papers published on this topic every year. first of all, mannich bases could present interesting biological activities, many of these having yet to be discovered through a diligent screening process. second, aminomethylation of drugs could be used to improve their delivery into the human body. aminomethylation may increase the hydrophilic properties of drugs through the introduction of a polar function in their structure, the long-known rolicycline being one of the most common examples [ ] . the solubility in water of a drug could be further enhanced through the quaternization of the nitrogen atom in its aminomethyl derivative and conversion into an ammonium salt. alternatively, the lipophilic properties of a drug could be tailored through a mannich reaction if the appropriate amine reagent is employed [ ] . in addition, the aminomethylated drugs could act as prodrugs, releasing the active substance under controlled hydrolytic conditions via deaminomethylation [ ] or deamination [ ] . in spite of the tremendous potential of mannich bases in medicinal chemistry, the wealth of information from studies concerning the structureeactivity relationship (sar) involving mannich bases or the use of aminomethylated drugs as prodrugs does not appear to have inspired many recent literature reviews of consequence, to the best of our knowledge. the present review fills this void by providing a comprehensive coverage of the most relevant developments in the medicinal chemistry of mannich bases generated exclusively through aminomethylation, the information being ordered according to the reported biological activity. due the large number of articles published on this topic, the coverage of this review is limited to the last decade. anticancer properties and cytotoxicity of ketonic mannich bases (with an emphasis on mannich bases of type derived from acetophenones [ ] ) and of structurally related a,b-unsaturated ketones [ ] were reviewed years ago. these two groups of compounds were shown to exert their cytotoxic action through the alkylation of cellular thiols such as glutathione or cysteine, and may be useful in sensitizing tumor cells to antineoplastic agents, and even reverse drug resistance [ ] . it is therefore no surprise that compounds having both a ketonic mannich base moiety and an activated unsaturated carbonecarbon double bond in their structure (for example, mannich bases of chalcones such as ) have been considered as candidates for the evaluation of the sequential cytotoxicity theory [ ] . this theory hypothesizes that the successive release of two or more cytotoxic agents will result in increased toxicity to malignant tissue rather than to normal cells [ ] . in addition, mannich bases of enones, which are easily accessible from alkyl aryl ketones in one synthetic step, also demonstrated marked toxicity towards numerous cancer cell lines [ ] . furthermore, as ortho-phenolic mannich bases undergo deamination easily to yield ortho-quinone methides, mannich bases of chalcones derived from either phenolic aldehydes or ketones, a class of compounds for which structure is prototypical, have been examined also as cytotoxic agents [ ] . in the last decade, the quest for more potent anticancer agents amongst these four general types of cytotoxic mannich bases e (fig. ) has steadily continued. cytotoxicity of ketonic mannich bases of type with various substitution patterns in the aromatic ring and of a few types of their derivatives has been studied in detail. gul et al. have shown that the structural modification of single mannich bases (r ¼ r ¼ h) derived from acetophenone and secondary aliphatic amines into double mannich bases (fig. ) generally results in increased cytotoxicity against mouse renal carcinoma (renca) and transformed human t-lymphocyte (jurkat) cell lines [ ] to the extent that double mannich bases were more cytotoxic than reference drugs -fluorouracil or melphalan. the cytotoxicity of these single and double mannich bases and , respectively, was reversed when the compounds were used in a brine shrimp bioassay, presumably due to the fast deamination of the double mannich bases before they could reach their target [ ] . also, ketonic mannich bases with dimethylamino, -piperidinyl, -morpholinyl as amine moiety and featuring either an unsubstituted, variously monosubstituted phenyl rings, or a thiophene ring were evaluated with respect of their cytotoxicity towards jurkat cells [ , ] or androgen-independent prostate cancer (pc- ) cells [ ] , and the cytotoxicity for some of these compounds was . -to . -fold higher than that of the standard -fluorouracil. in addition, mannich bases of type derived from -aryloxyacetophenones were shown to display moderate cytotoxic properties towards murine l cells as well as human molt /c and cem t-lymphocytes, and a number of these compounds possessed remarkable potencies towards seven human colon cancer cell lines [ ] . the use of primary aliphatic amines in the mannich reaction leads to reaction products with diverse structures (fig. ) . aminomethylation of acetophenones using methylamine as amine reagent afforded both bis-mannich bases (r ¼ ch ) and piperidinols (r ¼ ch ) , and the evaluation of their cytotoxicity towards jurkat cells showed that bis-mannich bases were generally more potent than the corresponding piperidinols or mono-mannich bases [ ] . besides their ability to alkylate cellular glutathione, compounds (r ¼ ch ) may exert their cytotoxic action through the inhibition of dna topoisomerase i; as the corresponding piperidinols were generally devoid of dna topoisomerase i inhibitory action, the authors tentatively attribute the activity of bis-mannich bases to their linear structure and the possibility of formation of hydrogen bonds with dna nucleotides [ ] . on the other hand, aminomethylation of acetophenones using isopropylamine [ ] and n-butylamine [ ] as amine reagent yielded only secondary mono-mannich bases (r ¼ ch(ch ) , (ch ) ch ), whose cytotoxicity was evaluated against huh- hepatoma cells, human jurkat and rat skeletal muscle derived myoblasts (l ) cells. compared to reference drug -fluorouracil, these compounds were . -to . -fold more cytotoxic towards huh- hepatoma cells, . -to . -fold more cytotoxic towards human jurkat cells, and . -to . -fold more cytotoxic towards l cells. aminomethylation of acetophenones using phenethylamine as amine reagent could led under carefully controlled reaction conditions either to mono-mannich bases (r ¼ ch ch c h ) [ ] or to the corresponding piperidinols (r ¼ ch ch c h ) [ ] ; the cytotoxicity of these compounds towards androgen-independent prostate cancer (pc- ) cells ranged from . to . mm, whereas the best compounds from each series had an average value for dna topoisomerase i interference of approximately %. anticancer activity of ketonic mannich bases has been compared with that of derivatives of the carbonyl function (fig. ) . a series of azines of ketonic mannich bases were designed as bifunctional cytotoxic agents, but their activity towards jurkat cells [ ] or pc- cells [ ] was less potent or, in the best of cases, equipotent to the parent ketonic mannich bases. on the other hand, hydrazones were consistently more cytotoxic than the corresponding ketonic mannich bases [ ] . noteworthy is the contribution of gul et al. to the understanding of the mechanism of the cytotoxic action of these compounds. his group has provided evidence that connects the anticancer activity of ketonic mannich bases of various structures or piperidinols with their ability to alkylate glutathione [ , e ] , whereas a few of the same compounds had mixed effects on thioredoxin, glutaredoxin, or heat shock proteins hsc and grp [ ] . only a limited number of examples of mannich bases of a,bunsaturated ketones of type with cytotoxic action are available in recent publications. a small series of mannich bases of arylidene- -tetralones ( fig. ) were evaluated as cytotoxic agents using human molt /c and cem t-lymphocytes, as well as murine p and l leukemic cells [ ] . compared to the parent a,bunsaturated ketones, mannich bases were consistently more potent, with half maximal inhibitory concentration (ic ) values in the . e mm range. furthermore, mannich bases derived from aromatic aldehydes substituted with chlorine, carboxyl, methoxy or cinnamoyloxy groups exhibited significant potencies towards human tumor cell lines, with an emphasis on their antileukemic effect. in most instances, the compounds prepared in this study demonstrated selective toxicity to different cells, which further enhances their potential utility. in addition to compounds , simpler mannich bases derived from benzylidenecyclohexanones were synthesized, and their evaluation against the same cell lines proved once more that mannich bases were more cytotoxic than the corresponding arylidenecyclohexanones, some of them showing growthinhibiting properties (ic of approximately mm) more potent than reference drug melphalan [ ] . because n-myristoyltransferase is expressed in larger quantities in tumors than it is in normal cells, this enzyme has been under consideration as a molecular target for cancer [ , ] . however, the substantially high ic value of mm towards n-myristoyltransferase for a representative compound of the series of candidates suggests that the inhibition of this enzyme does not play an important role in the mechanism of the cytotoxic activity of these compounds. several compounds were also tested against murine cancer cells mac (sensitive to most cytotoxic agents) and mac (resistant to most cytotoxic agents), and they demonstrated high cytotoxicity against the latter, but also against normal murine cells c c and t [ ] . on the other hand, mannich bases showed no activity against mac , which points to the importance of the double bond conjugated to the carbonyl function. the exploration of possible mechanisms of cytotoxic action of these compounds revealed that compounds may interfere with a number of essential cellular mechanisms by alkylation of thiols on enzyme or proteins, by disrupting mitochondrial electron transport, or by creating holes in the cell membrane, and thus promoting atp leakage [ ] . finally, mannich bases had high cytotoxic activity against two human breast cancer cell lines (mcf- and mcf- /adr) cells and human leukemia hl- cells, showed glutathione binding ability, and exhibited inhibitory action on glutathione-s-transferase p, whereas their analogues obtained through the hydrogenation of the double carbonecarbon bond were slightly less active [ ] . the nature of the dialkylamino group did not seem to affect the cytotoxic activity of these compounds, while the substitution of the aromatic rings with a methyl selectively increased the cytotoxic effect on breast cancer cells, but not on immortalized mammary epithelial ( b ) cells. the literature reporting the anticancer activity of mannich bases of type is even scarcer. given the significant antineoplastic properties of compounds [ ] , a novel series having substituents r in the phenyl ring that were carefully selected with a view to impart a variety of physicochemical properties has been designed and synthesized [ ] . since a gradual release of mannich base from niosomes had improved its bioactivity in vivo, amino alcohols (fig. ) , which may slowly undergo dehydration to yield the desired mannich base , were also examined as cytotoxic agents. finally, in order to explore the hypothesis that cytotoxicity would be retained even when a thiol is liberated, the synthesis of adducts was carried out. compounds , and ( fig. ) were evaluated against both human widr colon cancer cells and human crl- foreskin fibroblasts. ic values lesser than mm were obtained when compounds were evaluated towards human widr colon cancer cells, and the corresponding candidates also had ic values in the low micromolar range. on the other hand, conversion of mannich bases into the corresponding adducts led to a -fold reduction in potency. in addition, compounds and demonstrated a preferential cytotoxicity to cancer cells compared to normal fibroblasts [ ] . further studies showed that compounds and are cytotoxic towards a large number of human tumor cell lines, two important features of many of these compounds being their lethal effects toward promyelocytic leukemic hl- cells and their selective toxicity for the aforementioned cancer cell line (selectivity index of or more) [ ] . because divergence in the mechanism of action is required for drug candidates that are developed to be tumor-specific and spare normal tissues, it is noteworthy that a representative mannich base caused apoptosis and activated caspase- , caspase- , and caspase- in hl- cells, but not in hsc- cells. cytotoxic phenolic mannich bases of chalcone analogues (type in fig. ) are well represented in the recent literature. one of the strategies that are available for the synthesis of this type of mannich bases consists in the aminomethylation of chalcone analogues derived from at least either a phenolic aldehyde or a phenolic ketone. in line with this strategy, a series of five mono-mannich bases (fig. ) were obtained through a mannich reaction of chalcone analogues derived from -hydroxyacetophenone, employing piperidine as amine reagent [ ] . despite the use of an excess of both paraformaldehyde and piperidine, no double mannich bases analogous to were isolated. the evaluation of compounds against androgene-independent prostate cancer (pc- ) cell line showed that although three of them were more potent than the parent chalcone analogues, the most cytotoxic mannich base was . -fold less potent than the reference drug fluorouracil. based on correlations between cytotoxicity and hammet constant on one hand and cytotoxicity and partition coefficient on the other hand, the authors hypothesized that an increase in the general hydrophobicity of the molecule would result in enhanced cytotoxicity. therefore, a novel series of mannich bases was designed to incorporate a dibenzylaminomethyl residue as a replacement for the piperidinomethyl group [ ] . again, every attempt to obtain double mannich bases by varying the reaction conditions failed, and only mono-mannich bases could be isolated. when evaluated against androgene-independent prostate cancer (pc- ) cell line, these compounds consistently displayed lower cytotoxicity than that of the parent chalcone analogues. unexpectedly, cytotoxicity in the series of mannich bases with a dibenzylaminomethyl residue was also much lower than that of the corresponding mannich bases in the series containing a piperidinomethyl motif, thus invalidating the hypothesis put forth by the authors in the previous study. furthermore, no adduct between ethanethiol and a representative mannich base could be detected after h, whereas the incubation of ethanethiol with the corresponding parent chalcone analogue under the same conditions resulted in formation of small amounts of adduct. the authors concluded that no active cyclohexadienone species are formed following a potential deamination of mannich base , and that the introduction of the dibenzylaminomethyl group further reduces the ability of the chalcone moiety to undergo thiol addition. with a view to explore the effect of variation of dialkylamino moiety on the cytotoxicity of phenolic mannich bases of chalcone analogues, candidates were synthesized through aminomethylation of three chalcone analogues derived from hydroxyacetophenone and diverse secondary aliphatic amines [ ] . equimolar ratio of reactants afforded mono-mannich bases of type , which were evaluated against hepatocellular carcinoma (hepg ), human lung carcinoma (sk-lu- ), and human breast cancer (mcf- ) cell lines. mannich bases with -phenylpiperazine residue exhibited reduced cytotoxicity towards all three lines of cancer cells, whereas the candidates with -methylpiperazine or ethylpiperazine residues were the most active in each series, but less cytotoxic than reference drug ellipticine. with respect to the substitution pattern in the b phenyl ring, mannich bases ( fig. ) derived from -chlorobenzaldehyde (r ¼ h, r ¼ cl) or methoxybenzaldehyde (r ¼ och , r ¼ h) were consistently more active than those derived from -methoxybenzaldehyde (r ¼ h, r ¼ och ). the screening identified five compounds whose ic values against mcf- cell line were lower than mg/ml, whereas the most cytotoxic compound (r ¼ h, r ¼ cl, nr ¼ ethylpiperazinyl) had ic values lower than mg/ml against all three cancer cell lines used in this study. a larger library of phenolic mannich bases of chalcone analogues featuring the dialkylaminomethyl moiety either in ring a or ring b of the chalcone system was synthesized through the clai-seneschmidt condensation of the appropriately substituted mannich bases of phenolic aldehydes or ketones with heterocyclic ketones or aldehydes, respectively [ ] . the use of -alkoxy- hydroxyacetophenones as substrates in the mannich reaction yielded a mixture of -aminomethylated derivative with the isomeric -aminomethylated derivative, the former being the major reaction product in all cases. the adept tailoring of the ratio between the substrate, formaldehyde and morpholine in the mannich reaction of -hydroxyacetophenone resulted in the selective preparation of either single or double mannich base from this substrate. on the other hand, -hydroxyacetophenone afforded a mixture of -morpholinylmethyl derivative with morpholinylmethyl derivative and , -bis(morpholinylmethyl) derivative, even when an equimolar ratio between the reagents was used. isovanillin, which underwent aminomethylation only at position with morpholine and piperidine as amine reagents, was employed as an example of a phenolic aldehyde substrate in the mannich reaction. with the help of these intermediates, several small series of mannich bases of heterocyclic chalcone analogues e (fig. ) were synthesized and evaluated for cytotoxic activity against four human cancer cell lines, namely pc- , mcf- , nasopharyngeal carcinoma (kb), and resistant nasopharyngeal carcinoma (kb-vin). the rich diversity within this library comprised of structurally related entities allowed interesting insight on the cytotoxicityestructure relationship. first, the presence of two phenolic groups in ring a seems to enhance the cytotoxic activity, as proven by a candidate of type (r ¼ h, het ¼ -pyridinyl), which was the most potent in the entire library against all four types of cancer cell lines. then, the presence of a methoxy group in series (r ¼ ch ) appears to be generally preferable to ethoxy and isopropoxy, whereas a comparison of the cytotoxicity of similar compounds of type and type usually favors the candidates in the latter series, which have the aminomethyl group para to the phenolic hydroxyl. an analysis of the cytotoxicity within series of candidates of type demonstrated that six-membered heterocycles (particularly a -pyridinyl residue) are preferred to five membered heterocycles as ring b moieties, and this observation was validated by the inspection of anticancer activity of compounds in series . however, the presence of a second morpholinylmethyl group appears to be detrimental to the cytotoxic activity of candidates . shuffling of hydroxy and morpholinylmethyl groups in mannich bases and led to compounds in series e , which are generally less cytotoxic than their counterparts derived from chalcone analogues having a -hydroxy substituent in ring a. selective high cytotoxicity against mcf- cell line was displayed by the compounds in series featuring the morpholinylmethyl moiety in ring b of the chalcone system. overall, more than % of the mannich bases in this library of mannich bases of heterocyclic chalcone analogues e are cytotoxic (ic < mg/ml), while four members of the library are highly cytotoxic (ic < mg/ml) against all four cell lines, and other four against at least three cell lines, with mcf- and pc- being usually more sensitive than kb and kb-vin cell lines. later, novel mannich bases of type (ar ¼ pyridinyl) were evaluated against several cancer cell lines, and the candidates showed cytotoxicity in the low micromolar range only towards promyelocytic leukemic cells (hl- ) and oral squamosa cell carcinomas (hsc- , hsc- and hsc- ), whereas the ic values against non-malignant gingival fibroblasts, pulp cells and periodontal ligament fibroblasts were higher [ ] . the tumor selectivity of these mannich bases may be the result of their proven ability to cleave poly[adp-ribose]polymerase- in hsc- cells, but not in gingival fibroblasts cells. in addition, the cytotoxic activity of mannich bases of chalcone analogues with an aminomethyl moiety in b ring structurally similar to against a panel of breast cancer (mcf ), melanoma (uacc ) and renal cancer (tk ) cell lines was described in a patent [ ] . most compounds were active, and some were potent mostly towards the first two cancer cell lines. a series of mannich bases of bichalcone analogues (fig. ) , in which the two chalcone units are linked through a bis(aminomethyl) function generated by the use of a bifunctional amine reagent such as piperazine, has also been synthesized and evaluated against cancer cell lines [ ] . aminomethylation of acetovanillone with piperazine afforded a bis-mannich base, which subsequently led to compounds through a claiseneschmidt condensation with various aldehydes. surprisingly, compound (ar ¼ -pyridinyl) was selectively cytotoxic to human tongue squamous carcinoma (cal- ) and human pharyngeal squamous carcinoma (fadu) cell lines, whereas -pyridinyl and phenyl analogues were the most cytotoxic compounds towards all cell lines. substitution of the phenyl ring (ar ¼ c h ) with methoxy groups stripped mannich bases of their cytotoxicity towards most cell lines, whereas the decrease in cytotoxic activity induced by the presence of chlorine as substituent was not so drastic. replacement of phenyl with -furanyl or -thiophenyl led to compounds that are selectively cytotoxic to one or more cell lines, but further substitution with methyl of these five-membered heterocycle renders them devoid of cytotoxicity against all lines. despite a few notable examples of selectivity, the results obtained for this collection of compounds are not very encouraging, and they suggest that the incorporation of a second chalcone unit does not enhance the cytotoxicity of mannich bases derived from chalcone analogues. cytotoxic activity of mannich bases of bichalcone analogues was further explored using candidates with a modified design. the synthetic strategy comprised the synthesis of mono-phenolic mannich bases starting from -hydroxyacetophenone or acetovanillone as substrates and employing -( -(piperazin- -yl)phenyl) ethanone as amine reagent, then the bichalcone unit was generated through a claiseneschmidt condensation of both acetyl functions with aromatic aldehydes [ ] . only mannich bases from bichalcone analogues featuring a pyridinyl moiety (such as candidate , fig. ) were active towards prostate cancer (du ), non-small cell lung cancer (a ), ileocecal (hct) and nasopharyngeal carcinoma (kb) cell lines with ic values between . and mm; all other compounds had ic values greater than mm. compared to compound , mannich base having a single chalcone unit was less active (ic between and mm). an exploration of the mechanism of action for these compounds suggested that compound most likely acted via the fas/cd apoptosis signaling pathway. ferulic acid and its derivatives provide another example of a type of substrate containing an a,b-unsaturated system activated by an electron-withdrawing group, similar to that in phenolic chalcone analogues, from which phenolic mannich bases can be synthesized. the growing body of evidence suggesting that -azido- -deoxythymidine (azt), a known antiviral, also possesses anticancer activity [ e ] has sparked a study aiming at cytotoxic evaluation of a series of conjugates of azt and ferulic acid derivatives [ ] . thus, propargyl ester and n-propargylamide of ferulic acid chemoselectively underwent aminomethylation ortho to the phenolic hydroxyl with various secondary aliphatic amines, and the resulting phenolic mannich bases (x ¼ o, nh) reacted through the terminal alkyne moiety with azt via a cu(i)-catalyzed click chemistry process to afford the corresponding , , -triazoles. evaluation of cytotoxicity for both mannich bases (fig. ) and the related , , -triazoles against human breast adenocarcinoma (mda-mb- ), lung adenocarcinoma (sk-lu- ) and colon adenocarcinoma (sw ) cell lines showed that only some of compounds were cytotoxic. despite the presence in their structure of an identical scaffold comprising a phenolic mannich bases moiety and an activated carbonecarbon double bond motif that has been deemed responsible for the cytotoxic effect of mannich bases , all of the corresponding , , -triazoles were inactive. out of eight mannich bases of propargyl ester of ferulic acid reported in this study, three were inactive against all three lines, while the rest had weak to moderate cytotoxicity, and the most active candidate (ic ¼ e mg/ml) was mannich base (x ¼ o) having a pyrrolidinylmethyl moiety. with the exception of mannich base (x ¼ nh) with a piperidinylmethyl moiety, all others candidates derived from n-propargylamide of ferulic acid were inactive. other aminomethylated phenols with cytotoxic activity have been reported besides phenolic mannich bases of chalcone analogues. unfortunately, the wide structural diversity of phenolic substrates from which these phenolic mannich bases originate and the lack of a systematic and comprehensive search for structureecytotoxic activity relationships within a particular type of phenolic substrate undermine any efforts to discover good lead compounds for further development as drugs. phenolic substrates subjected to the mannich reaction with a view to obtain novel cytotoxic agents include both simple and very complex structures. examples that illustrate structurally simple phenolic substrates are -naphthol and -hydroquinoline, whose mannich bases with piperidine and -arylsulfonylpiperazines exhibited growthinhibitory effects towards a panel of carcinoma cell lines, including hela (cervical epithelioid carcinoma cell), bt (mammary gland adenocarcinoma cell), skhep (hepatocellular carcinoma cell), and ce t (esophageal carcinoma cell) [ ] . although the cytotoxic effect of aminomethylated naphthols and hydroxyquinoline derivatives has been known for some time [ , ] , a mechanistic study presented in the aforementioned report showed that these phenolic mannich bases induce apoptosis by activation of caspase-dependent pathways. furthermore, upon addition of copper ions, these compounds dramatically stimulate production of reactive oxygen species and activate various kinases, a group of enzymes that are known to be important in the oxidative stress-mediated cell death. candidate (fig. ) was the most potent in this small series, with a concentration for % cell growth inhibition of . mm; this value decreased to . mm in the presence of mm copper. a more detailed study [ ] of the structureecytotoxic activity relationship within this class of compounds showed that either replacement of sulfonyl function in with a methylene group, or replacement of piperazine ring with an ethylenediamino moiety (as in compound , fig. ) led to a significant increase in cytotoxic activity. the cell lines used in this study exhibited selective sensitivity towards different mannich bases, which appeared to be modulated by the nature of the arylsulfonyl group. as for the -hydroxyquinoline part of the molecule, substitution at position of the quinoline motif (especially with a nitro group) had a beneficial effect, whereas its replacement with phenol, -hydroxypyridine or -naphthol led to a decrease of cytotoxic activity [ ] . cytotoxicity of enantiomerically-enriched mannich bases (fig. ) derived from -naphthol, aromatic aldehydes and either -piperidinol (r ¼ h) or its acetylated counterpart (r ¼ coch ) against murine leukemic l and human lymphoblast molt /c and cem cell lines has been examined by another study [ ] . all of the compounds were only moderately cytotoxic, with ic values in the middle micromolar range, and were also e -fold less potent than that reference drug melphalan. substitution of the aryl group in with r ¼ dialkylaminoethoxy resulted in a series of compounds whose cytotoxicity potential against estrogen-responsive human mcf- breast cancer cells was found to be comparable to that of tamoxifen. however, removal of the -piperidinol moiety from mannich bases led to benzylnaphthols with enhanced cytotoxicity against mcf- cells, which is most likely due to their binding and antagonistic effects against human estrogen receptor alpha [ ] . flavones represent another type of phenolic substrate from which cytotoxic mannich bases have been synthesized. because flavones such as chrysin bear structural resemblances to androgens, mannich bases fig. ) have been designed as inhibitors of human aromatase, an enzyme which converts androgens to estrogens, and therefore represents a key target in the treatment of hormone-dependent tumors, including breast cancer [ ] . several single and double mannich bases of chrysin were found to inhibit human aromatase more effectively than reference drug aminoglutethimide [ ] . in addition, mannich bases (r ¼ oh, r ¼ h, r ¼ aminomethyl) of apigenin ( fig. ) have been prepared from aliphatic primary and secondary amines via chemoselective aminomethylation at c- in the benzopyran ring system [ ] . antiproliferative activity of these mannich bases against four human cancer cell lines, namely human cervical (hela), human liver (hepg ), human lung (a ), and human breast (mcf- ) cancer cells, was determined using the standard -( , -dimethylthiazol- -diphenyl-tetrazolium) bromide (mtt) assay. pyrrolidine mannich base of apigenin was the most promising compound in this series, its inhibition of cell proliferation being greater than % against all four cell lines at a concentration of mg/ml. many natural or synthetic carbazoles, either simple or condensed with other heterocycles (such as pyridocarbazoles, indolocarbazoles, pyranocarbazoles, pyrrolocarbazoles, etc), have been reported as anticancer agents. a recent study examines the cytotoxicity of a series of oxazinocarbazoles, which were the major products arising from the mannich reaction of n-substituted -or -hydroxycarbazoles with primary amines (fig. ) [ ] . under the appropriate reaction condition, -hydroxycarbazoles yielded , , , -tetrahydro [ , ] oxazino [ , - c]carbazoles , whereas hydroxy- -methylcarbazole led to a mixture of regioisomeric , , , -tetrahydro [ , ] oxazino [ , -b] carbazoles and , , , tetrahydro [ , ] oxazino[ , -a]carbazoles . use of allylamine, , -dimethylallylamine or benzylamine as amine reagents afforded as the major product, while isomer was the major component of the mixture when -pyridinylmethylamine was employed. in addition, small to moderate yields of bis-mannich bases or were isolated from n-substituted -hydroxycarbazoles and hydroxy- -methylcarbazole, respectively, but only when pyridinylmethylamine was employed as amine reagent. evaluation of the antiproliferative action of these compounds against cem (t cell leukemia), jurkat (acute t cell leukemia), raji (burkitt's lymphoma), mcf- (breast cancer cells) and caco- (colorectal cancer) using the wst- colorimetric assay showed, after the primary screening at mm, that bis-mannich bases and were less active than the oxazinocarbazoles. in the series of compounds , the best antiproliferative effect was observed for candidates having either an allyl or a prenyl group at the carbazole nitrogen atom and an allyl group at the oxazine nitrogen atom. thus, jurkat and raji cell lines. the majority of candidates and exhibited significant antiproliferative action at mm, and compound (r ¼ -pyridinylmethyl) was the most active towards jurkat and raji cell lines (ic ¼ mm) [ ] . aminomethylated derivatives of hydroxycarbazoles have been also mentioned in a different study [ ] , which described the synthesis of a small series of phenolic mannich bases (fig. ) obtained from -substituted -hydroxy- h-benzo[b]carbazole- , -diones along with their in vitro anticancer evaluation at national cancer institute (nci) using an in-house developed screening panel of approximately cell lines derived from nine different types of cancer. only one mannich base (r ¼ -h coc h ) was more active than the parent benzocarbazoledione, and a compare analysis [ ] revealed that its mechanism of action is novel and does not resemble the known mechanisms of action for standard anticancer drugs, but this candidate was not selected for further studies concerning its interaction with dna. in a related report, phenolic mannich bases of -hydroxy- h-naphtho [ , -g] indoles were found to be inactive in a similar screening [ ] . quinones are a class of compounds that have been widely investigated as anticancer agents [ ] . besides anthracycline antibiotics, other natural hydroxyquinones such as plumbagin [ , ] , juglone [ , ] or lapachol [ ] and their derivatives have been reported to exhibit significant cytotoxicity. the mannich bases of another natural phenolic quinone, namely lawsone, and their pt(ii) complexes ( fig. ) have been synthesized and shown to be highly cytotoxic towards six cancer cell lines: mda-mb- (melanoma), hl- (promyelocytic leukemia), hct- (colon), sf- (brain), ovcar- (ovary) and pc- (prostate) [ ] . the ligands and the complexes that have long alkyl chains (r ¼ n-heptyl or ndecyl) were the most active (the complexes were actually more cytotoxic than cisplatin), whereas the neutral complexes (x ¼ cl) were generally more cytotoxic than the corresponding charged complexes (x ¼ h o or nh ). examination of the mechanism of action for some of these complexes has shown that aqua complexes were more efficient inhibitors of ethidium bromide intercalation into dna than amino complexes, and candidate (r ¼ (ch ) ch , x ¼ h o) was more efficient than cisplatin [ ] . the same aqua complex also induced dna strand breaks, while the corresponding amino complex was ineffective. the ability of these complexes to inhibit topoisomerase i was also examined. most chlorido and amino complexes were as active as reference drug camptothecin in the dna relaxation assay, and did not cause major unwinding of dna, with the exception of complex (r ¼ n-decyl, x ¼ cl). in addition, cellular platinum accumulation was shown to increase with the increase of the length of the alkyl chain of the amino moiety in the mannich base ligand. the chlorido pt(ii) complexes were oxidized to the corresponding chlorido pt(iv) complexes, but the cytotoxicity of these new complexes was comparable to the cytotoxicity of the parent pt(ii) complexes, presumably owing to the rapid reduction of pt(iv) complexes before entering the cancer cells [ ] . furthermore, miscellaneous aminomethylated phenols from structurally diverse substrates have been reported as anticancer agents (fig. ) . thus, a small series of seven phenolic mannich bases were synthesized through aminomethylation of naturally occurring antibiotic lasalocid, with simultaneous elimination of the carboxyl group neighboring the phenolic hydroxyl [ ] . antiproliferative effect of mannich bases was evaluated against mcf- (human breast adenocarcinoma), a (human lung adenocarcinoma), ht- (human colon carcinoma) and p (murine leukemia) using either mtt or sulforhodamine b (srb) assay, and four candidates were more cytotoxic towards a , ht- and mcf- cell lines that anticancer drug cisplatin. these candidates also presented higher selectivity towards cancer cells than towards balb/ t (normal murine embryonic fibroblast) or hlmec (human lung microvascular endothelial) cell lines. the lack of activity of the other three mannich bases was attributed to the aralkyl or long alkyl chains in the amine moiety of these compounds [ ] . camptothecin, another naturally occurring phenolic substrate and lead compound for a plethora of cytotoxic substances, was converted into the oxazino derivatives (fig. ) by means of the mannich reaction using primary aliphatic and aromatic amines [ ] . evaluation of these novel hexacyclic camptothecin derivatives towards nine human cancer cell lines (bxpc- , nci- , mcf- , hepg- , a , a , bel , ht- , and kb), using mtt assay and camptothecin and topotecan as reference compounds, showed that most of them exhibit cytotoxicity towards several cell lines that is superior or comparable to topotecan, while only a few of the candidates presented cytotoxicity comparable to camptothecin. because candidates (r ¼ c h or n-c h ) were the most potent antiproliferative agents in this series, the presence of a small alkyl group at the nitrogen in the oxazine moiety seems to be preferable for a high cytotoxicity [ ] . a series of oxazino derivatives (r ¼ ch ) (fig. ) were also prepared from g-tocotrienol and primary amines via the mannich reaction, whereas d-tocotrienol afforded under the same conditions a mixture of oxazino derivatives (r ¼ h) and , in which the former is the major component [ ] . no reaction occurred when secondary amines were used instead, but two phenolic mannich bases were obtained indirectly from the corresponding oxazino derivatives . out of candidates in this library, thirty compounds had greater antiproliferative activity against the highly metastatic þ sa mouse mammary epithelial cancer cells than that of the parent tocotrienols (ic ¼ mm), and seven candidates had ic values in the nanomolar range. mannich bases were less active than the corresponding oxazino derivatives against nci's standard panel of cell lines, which suggests that the oxazine ring is an essential pharmacophore for the cytotoxic activity of these tocotrienol derivatives. generally, the oxazino derivatives of dtocotrienol were more active than the corresponding isomers derived from g-tocotrienol, and a long alkyl chain at the nitrogen atom (preferably with a terminal hydroxyl group) proved beneficial for the antiproliferative activity. the same conclusions were drawn after the evaluation of structureeantimigratory activity relationship using the highly metastatic mdamb- breast cancer cell line [ ] . novel g-quadruplex ligand/alkylating hybrid structures ( fig. ) were obtained by tethering a naphthalene diimide core having g-quadruplex recognizing properties to phenolic mannich bases using flexible spacer [ ] . the assessment of cytotoxic effects of these compounds (n ¼ , , or ) and their corresponding methiodides against human embryonic kidney t cell line by mtt assay suggests that the length of the spacer between the core and the phenolic mannich bases moiety modulates the cytotoxicity: candidates having two-carbon atoms and one-carbon atom spacers were the most active (ic . and . mm, respectively), whereas the mannich base with a three-carbon atoms spacer was less active. cytotoxicity of these compounds parallels their ability to alkylate dna, and the grafting of the alkylating mannich base moiety to the central core contributes to the enhancement of the g-quadruplex folding induction and stabilization. a similar g-quadruplex ligand/alkylating hybrid structure was shown to significantly slow the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression [ ] , which suggests that these hybrids could be possible candidates for the development of novel targeted anticancer therapies. in connection to this, the methiodide of double mannich base (fig. ) with a quinazoline core was also shown to cross-link linear dna at concentrations as low as mm, and to inhibit dna transcription almost completely at mm [ ] . phenolic mannich bases of norvisnagin ( fig. ) were prepared through direct aminomethylation, and their ability to interact with dna was evaluated using both a qualitative binding assay and a colorimetric microassay based on the displacement of methyl green from dna [ ] . three of the candidates (r ¼ pyridinyl- -amino, diethylamino, and methylamino) showed moderate dna binding affinity, and could be potentially cytotoxic. also, mannich bases (r ¼ h) of , -dihydroxyxanthone ( fig. ) displayed moderate to good cytotoxicity against lung cancer (nci-h ), tongue squamosa cell carcinoma (tca- ), liver cancer (bel- ), hepatocarcinoma (hepg ), gastric carcinoma (sgc- ) and urinary bladder carcinoma (t ) in an mtt assay [ ] . several reports of mannich bases of indoles as cytotoxic agents are also available in recent literature. cytotoxicity of a few indole mannich bases derived from -substituted piperazines (fig. ) was evaluated against liver (huh ), breast (mcf ) and colon (hct ) cancer cell lines using srb assay, and some of these compounds had ic values in the lower micromolar range. compound (r ¼ , -dichlorobenzyl) was cytotoxic towards all three cancer cell lines, and fared better than reference drug -fluorouracil ( -fu) [ ] . on the other hand, n-mannich bases of methylindole did not inhibit the growth of cancer cells or had high ic values. in spite of this fact, a subsequent study [ ] was dedicated exclusively to the investigation of cytotoxicity of a novel series of n-mannich bases of type against the same cancer cell lines. the broadening of the nature of substituent at position of piperazine proved favorable, as several compounds reported in this later study presented cytotoxic activity comparable to reference drug -fu. a comparison between the morphological features of cancer cells for which apoptosis was induced either by a selected metylindole n-mannich base or by paclitaxel suggests that compounds (r ¼ ch ) and paclitaxel share the same mechanism of action. design of novel cytotoxic mannich bases in which indole itself was the substrate for aminomethylation was also revisited using an extended panel of -substituted piperazines as amine reagents in aminomethylation [ ] . the novel candidates proved to be cytotoxic towards the same cancer cell lines (huh , mcf , and hct ); however, no cytotoxicity was observed for the candidates with an electron-withdrawing group (such as nitrophenyl, benzoyl or acetyl) as substituent at position of piperazine. other c-mannich bases of indole derivatives have been claimed as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (imct), an enzyme that plays an important role in the posttranslational modification of proteins that are involved in the regulation of cell growth, and therefore represents a potential therapeutic target in oncogenesis. among the few small molecules inhibitors of imct that were discovered so far, the most promising appears to be cysmethynil, an indol- -ylacetamide derivative which impairs growth factor signaling and induces cell cycle arrest and autophagy. because cysmethynil suffers from poor water solubility and strong binding to plasma proteins, rational modification of this hit compound has been expected to yield more potent imct inhibitors with improved bioavailability. replacement of the acetamide function in cysmethynil with various aminomethyl moieties led to compounds (r ¼ dialkylamino) with an inhibitory effect on icmt that was generally e -fold more potent than that of the parent inhibitor [ ] . evaluation of the effects of these candidates on viability of mda-mb- human breast cancer cells using the colorimetric tetrazolium assay confirmed the results for icmt inhibition. thus, compounds ( fig. ) were found to be more cytotoxic (ic e mm) than cysmethynil (ic mm). other modifications of the lead compound (r ¼ diethylamino) either preserved the potency of the candidates (e.g., shuffling of the methyl group on the phenyl ring), or resulted in a potency decrease (e.g., replacement of n-octyl with prenyl). however, the replacement of m-tolyl moiety in with more polar heteroaromatic rings led to submicromolar ic values in the icmt inhibition assay, and to ic values in the antiproliferative assay on breast mda-mb- and prostate pc cell lines that are e -fold lower than that of fig. . cytotoxic indole and azaindole mannich bases. the lead (r ¼ diethylamino) [ ] . compound (fig. ) was the most potent compound in this series, and presented a series of improvements of the drug-like profile over cysmethynil, such as good solubility in water, acceptable permeability through an artificial membrane, and limited tendency to form light scattering aggregates. using naphtho[ , -f]indole- , -dione as scaffold, a series of aminomethylated derivatives was synthesized, and four candidates were evaluated as antiproliferative agents against the standard panel of human cancer cell lines at nci [ ] . compounds (r ¼ primary or secondary aliphatic amine residue, r ¼ oh) (fig. ) were less potent than doxorubicin against any of the cell lines, but multidrug resistant breast cancer cells were found to be more sensitive to than to doxorubicin. in addition, mannich bases showed potency for cancer cell lines that are otherwise resistant to anticancer drugs, such as the p-glycoprotein-positive subline of k leukemia cells or the p -null subline of hct colon carcinoma cell line. replacement of phenolic hydroxyl groups r in (r ¼ dimethylamino) by -aminoethyleneamino moieties led to mixed results, as the potency improved for some of the cell lines and declined for others, but the sensitivity of multidrug resistant breast cancer cells to these modified candidates was completely lost [ ] . furthermore, candidate (r ¼ quinuclidin- ylamino, r ¼ oh) was shown to inhibit topoisomerase i-mediated relaxation of dna, but the suppression of the topoisomerase i activity is presumably the leading although probably not the only factor contributing to cytotoxicity of mannich bases of naphtho [ , -f]indole- , -diones [ ] . preobrazhenskaya et al. have also shown that a series of single and double mannich bases (r ¼ h or dialkylaminomethyl) of , -bis(indol- -yl)maleimides (fig. ) , structurally related to rebeccamycin or staurosporine, were highly cytotoxic towards К and hcТ cell lines, but their cytotoxicity does not correlate well with their ability to either inhibit protein kinase c-a or constrain activation of multiple drug resistance [ ] . mannich bases of an indole isostere, namely h-pyrrolo[ , -d] pyrimidine, have been designed as inhibitors of phosphatidylinositol- -kinase a (pi ka), a lipid kinase that modulates activity of the pi k downstream effectors akt and mtor. since the consequences of biological activation of akt include tumor progression, proliferation, survival, growth, invasion, angiogenesis, and metastasis, pi ka represents an attractive target for development of anticancer drugs. although aminomethylated pyrrolopyrimidine (fig. ) was an efficient inhibitors of pi ka (ic ¼ nm) and showed good selectivity for pi ka over mtor ( -fold) , this compound exhibited low cytotoxicity towards pc cancer cells [ ] . in addition, all the other analogues of mannich base were found to be even weaker inhibitors of pi ka than the lead compound. isatin is nowadays a well recognized and privileged scaffold in the design of cytotoxic and anticancer compounds [ ] . several isatin-containing substrates, namely isatin and its -halogenated analogues, the corresponding imine derivatives obtained from sulfadiazine, sulfadoxine and trimethoprim, and a hydrazone derived from isoniazid, were aminomethylated using gatifloxacin as amine reagent [ ] . the resulting mannich bases were tested against nci's standard panel of cell lines using srb assay, and compound (fig. ) emerged as an efficient anticancer agent that was generally more potent than reference drug etoposide against most cell lines in the panel. another library of mannich bases derived from isatin, -halogenated isatins and their schiff bases with -amino- -methylbenzothiazole was evaluated for cytotoxic effects on three breast cancer cell lines (mda-mb , mdamb and mcf ) using srb assay [ ] . the introduction of a halogen as substituent at position of isatin mannich bases led to an increase in cytotoxicity, and modification of isatins into schiff bases followed by aminomethylation resulted in mannich bases further enhanced the cytotoxicity of these candidates. compounds and ( fig. ) were the most potent in each series (ic < mm), and more potent than reference drug cisplatin, while their cytotoxicity against normal cells was low. as far as their mechanism of action is concerned, compound induced cell cycle arrest in g /m phase at concentrations similar to those observed for cell growth inhibition, whereas compound did not [ ] . furthermore, another collection of mannich bases of isatin imines generated either from aminobenzimidazole or -amino- , -dihydrothiazole was evaluated against mcf- human breast adenocarcinoma cell line using srb assay, but the cytotoxicity of these compounds was moderate (ic > mm) and inferior to that of doxorubicin [ ] . generally, aminomethylated schiff bases of isatin derived from aminobenzimidazole were more cytotoxic than their counterparts derived from -amino- , -dihydrothiazole, compound ( fig. ) being the most potent in this collection (ic ¼ . mm). cytotoxic mannich bases of isatins were also designed employing hybridization of isatin with a -aminoquinoline scaffold to generate the substrate subjected to aminomethylation [ ] . the cytotoxicity of these compounds towards breast cancer cell lines mda-mb and mcf was moderate (ic values between and mm), but the activity improved slightly in the series of the corresponding thiosemicarbazones (ic in the range of e mm). mannich bases and (fig. ) were the most potent candidates in every series ( e -fold more cytotoxic than cisplatin), and they preferentially inhibited the growth of cancer cell over normal cells. studies using flow cytometry also suggest that these compounds induce cancer cell death by apoptosis. beside isatin derivatives, several other classes of nh-azoles have been aminomethylated with a view to synthesize cytotoxic mannich bases. , -dihydro- , , -oxadiazole- -thiones appear to be the preferred substrate within this category, most likely owing to their straightforward preparation. starting from methyl salicylate, good yields of the corresponding oxadiazolethione mannich bases ( fig. ) were obtained in three steps [ ] . most compounds in this collection arise from primary aromatic amines diversely substituted in the aromatic ring, whereas mannich bases derived from secondary aliphatic amines are poorly represented. selected candidates from this series have been initially evaluated by nci against a panel consisting of nci-h (lung), mcf (breast), and sf- (glioblastoma) cancer cell lines using srb assay. seven of these thirteen mannich bases , most of them having either chlorine or carboxy group as substituent in the aromatic ring of the amine moiety, reduced the growth of nci-h cell line to % or less, and they were further selected for the standard -cell lines panel assay. compounds (r ¼ h, r ¼ -clc h or -clc h ) presented higher cytotoxicity than reference drugs -fu or cyclophosphamide against most cancer cell lines in this panel [ ] . the ability of several oxadiazolethione mannich bases featuring variously substituted aromatic ring at position of the oxadiazolethione ring (r ¼ h and r ¼ no , oh, ch , or r ¼ r ¼ cl) to inhibit the growth of tumors in vivo has been also investigated [ ] . tumor volume and tumor weight in mice injected with ehrlich ascites carcinoma cells were reduced by e % at a dose of mg candidates (fig. ) per kg body weight, whereas similar dose of reference drug -fu inhibited tumor formation by %. mannich bases , especially those having hydroxyl, methyl or chloro substituents on the phenyl ring at position , were the most potent. also, the counts of red blood cells and leukocytes, as well as hemoglobin levels, have been restored almost to the normal values in mice treated with mannich bases [ ] . aminomethylated oxadiazolethiones (fig. ) were generally more cytotoxic against colon carcinoma (ht ) and less cytotoxic against breast cancer (mcf ) cells using srb assay, but the most potent candidate (nr ¼ nhc h ch - ) was -fold less cytotoxic than reference drug doxycycline [ ] . cytotoxicity of a series of mannich bases (fig. ) of a norharmaneoxadiazolethione structural hybrids was evaluated against a panel comprising melanoma (uacc- ), breast (mcf ), ovarian resistant (nci/adr), renal ( - ), lung (nci- ), prostate (pco- ), ovarian (ovcar) and colon (ht- ) cell lines using srb assay [ ] . several of candidates (r ¼ h or n(ch ) , r ¼ isopropylamino or benzylamino) exhibited a broad spectrum cytotoxic activity, and aminomethylation of the parent oxadiazolethiones significantly enhanced the cytotoxicity of each resulting mannich bases compared to that of the corresponding substrate. furthermore, mannich bases of a fluoroquinoloneeoxadiazolethione hybrid were prepared using either secondary aliphatic amines or substituted arylamines as amine reagents [ ] . the in vitro evaluation of cytotoxicity against hep b cancer cells using mtt assay showed that compounds ( fig. ) were more potent than the parent fluoroquinolone pefloxacin. also, mannich bases in this series derived from aliphatic amines were generally more cytotoxic than those derived from arylamines, and candidate (r ¼ n(ch ) ) was even more potent than reference drug bisantrene. , -dihydro- , , -triazole- -thiones could also act as substrates for the preparation of cytotoxic n-mannich bases. schiff bases obtained from -aryloxymethyl- -amino- -mercapto- , , -triazoles and ( )-substituted pyrazole- -carboxaldehydes were aminomethylated using either morpholine or diphenylamine to afford mannich bases (fig. ) , whose cytotoxicity against hepg cell line was evaluated using mtt assay [ ] . out of five tested candidates, mannich bases having a morpholine moiety were more potent than those with a diphenylamine moiety, but they were still e -fold less cytotoxic than reference drug doxorubicin. eleven dimethylamine mannich bases , that were obtained through aminomethylation of schiff bases derived from a -amino- , , triazole- -thione having at position a moiety originating from fluoroquinolone ofloxacin, were screened for cytotoxicity against murine leukemia cell line (l ) and human leukocytoma cell line (hl ) [ ] . mannich bases (fig. ) were generally more cytotoxic than the corresponding parent schiff bases, and candidates with a hydroxyl group in the aromatic ring of the azomethine function (r ¼ oh) were the most cytotoxic compounds in the series (ic values in the range of . e . mm). mannich bases of thiazolidinone derivatives have also been investigated as cytotoxic agents. c-aminomethylation of two thiazolidinones using secondary aliphatic amines afforded mannich bases (x ¼ o, ch ) (fig. ) , which were evaluated against colon (hct ) and breast (t d) cancer cell lines by srb assay, but their cytotoxicity was generally moderate to low (ic values between and mm) [ ] . in addition, n-aminomethylation of two thiazolidine- , -diones (r ¼ cl, och ) with morpholine, piperidine and variously -substituted piperazines yielded mannich bases (fig. ) , which were investigated at nci against the standard -cell lines panel using srb assay, and proved to be virtually inactive (growth inhibition for the most sensitive cell line between and %) [ ] . several examples of p-mannich bases derived from organic esters of phosphorous acid that were disclosed as cytotoxic agents are available in the literature. thus, a-aminophosphonates were synthesized from alkyl phosphites (r ¼ ch , c h , n-c h , i-c h , n-c h ), fluorine-substituted benzaldehydes and aminobenzothiazoles, and their cytotoxicity was evaluated against pc (prostate), a (melanoma), a (epidermoid carcinoma), and bcap- (breast) cancer cells using mtt assay [ ] . most mannich bases ( fig. ) exhibited low to moderate growth inhibition of a and bcap- cells, but their cytotoxicity towards pc and a cells was generally greater. the nature of the fluorine substituent appears to influence cytotoxicity, as candidates having fluorine directly attached to the aromatic ring are more potent than those with a trifluoromethyl substituent. an improvement of cytotoxicity with the increase of the length of the alkyl residue r from the initial phosphite was also noted. p-mannich bases (r ¼ ch , c h , i-c h ) (fig. ) were prepared using thieno [ , -c] pyridine- -carboxaldehyde as aldehyde component in the mannich reaction of dialkyl phosphites with variously substituted arylamines [ ] . the majority of candidates showed good cytotoxicity against esophageal cancer cells (ec ), but they were generally more potent against hepatocellular liver carcinoma cells (hepg ) at a concentration of mg/ml. a few aaminophosphonates (fig. ) were obtained through a mannichtype process from diphenyl phosphite, -acetylpyridine and variously substituted anilines, and proved to be cytotoxic to hepg liver carcinoma cell line (ic ~ mm) and mcf breast adenocarcinoma cell line (ic ~ mm) using mtt assay [ ] . although these candidates were approximately -fold less cytotoxic than reference drug doxorubicin, their ld values were greater than the corresponding ic values, making them safe to use. aminomethylation of diethyl phosphite with either aromatic aldehydes and aromatic diamines or terephthaldehyde and various amines led to bis(aaminophosphonates) or (fig. ), respectively, whose cytotoxicity against jurkat (t-cell lymphoma), raji (burkit's lymphoma) and mcf- (breast cancer) cell lines was determined using mtt assay [ ] . in this structurally diverse series of compounds, a few were devoid of cytotoxicity while most of them were moderately cytotoxic. compound , derived from tryptamine as amine reagent in the mannich reaction, emerged as the most potent in this series, its cytotoxicity being comparable to that of doxorubicin. furan belongs to the category of electron-rich heterocycles known to undergo electrophilic substitutions such as the mannich reaction with great ease. for example, aminomethylation of synthetic lactone of natural furanditerpene a, b-dihydroxyvouacapan- b-oic acid as substrate and using various secondary aliphatic amines led to the furan mannich bases (fig. ) [ ] . antiproliferative effect of these compounds was more potent than that of the parent lactone against a panel of nine cancer cell lines (melanoma (uacc- ), breast (mcf ), ovarian expressing the resistance phenotype for adryamycin (nci-adr/res), kidney ( - ), lung, non-small cells (nci-h ), prostate (pc ), ovarian (ovcar- ), colon (ht- ), and k erythromyeloblastoid leukemia), as determined by srb assay. mannich bases were also equipotent to reference drug doxorubicin against at least one, if not many, of these cancer cell lines, often with ic values as low as mg/ml. in addition, , , -triazolo[ , -a]pyrimidine- -amines having at position a side chain capped with a furan ring were aminomethylated with various secondary aliphatic amines to yield a large series of furan mannich bases (fig. ) [ , ] . cytotoxicity of compounds against liver cancer (bel- ) and fibro sarcoma (ht- ) cells was established using mtt assay, and the results suggest that both the substitution of the arylamine moiety and the nature of the aliphatic amino residue in the aminomethyl function have a significant influence on the potency of these candidates. in particular, the presence of a -trifluoromethyl group or a -fluoro- -trifluoromethyl substitution pattern in the arylamine moiety led to high cytotoxicity against both cell lines at levels comparable to that of reference drug cisplatin. also, the presence of dimethylamino, -piperidinyl or -pyrrolidinyl moieties as aliphatic amino residues in the aminomethyl group of mannich bases appears to result in significant antiproliferative effects, while -morpholinyl or -methylpiperazinyl moieties drastically decrease or even abolish cytotoxicity. titanium-based chemical entities enjoy the reputation of having a tremendous potential against solid tumors. recently, a number of studies presented the synthesis and the cytotoxicity for a series of titanocenes, some of them featuring various aminomethylated fivemembered heterocycles as substituent of either one or both cyclopentadiene moieties [ e ]. thus, titanocenes and ( fig. ) containing mono-and bis-aminomethylated pyrroles, respectively, or titanocenes and (fig. ) having a dimethylaminomethyl group at position and of indole, respectively, as well as titanocene (fig. ) presenting an aminomethylated imidazole ring, have been screened against pig kidney epithelial cells (llc-pk ) or human renal cancer cells (caki- ) and found to have ic values quite similar to that of cisplatin (in the range of e mm). the presence of at least one aminomethyl function in their structure is claimed to be crucial for the high cytotoxicity of these titanocenes. the aminomethyl groups are able to coordinate the titanium center, and could therefore stabilize the mono-or dication formed through hydrolysis of either one or both chlorine atoms inside the cell. this results in enhanced interactions between titanocene and dna, leading to cell death at low concentrations. aminomethylation of terminal alkynes has also been employed for the generation of mannich bases with potential cytotoxic effect. -(prop- -ynil)phenothiazines underwent aminomethylation with secondary aliphatic amines to give propargylamines (r ¼ h, cl, cf ) (fig. ), which were first evaluated for cytotoxic activity using two hematological tumor cell lines, namely hl (promyelocytic leukemia) and the ccrf/cem (lymphocytic leukemia), and were afterwards tested, in combination with doxorubicin, for ability to revert activity in the corresponding multidrug resistant variants, hl r and cem/vbl [ ] . although most compounds were devoid of significant antiproliferative effect on the sensitive cell lines, a few of them were highly cytotoxic for the resistant cell lines, and appear to arrest cells in g phase of the cell cycle, unlike classic anticancer agents. furthermore, several mannich bases were able to restore sensitivity to doxorubicin of the resistant cell lines, an effect that was concentration-dependent and reached maximum at mm. propargylamines seem to induce apoptosis by activating the caspase cascade, although neither the extrinsic nor the intrinsic pathways appear to be involved in apoptosis [ ] . betulin derivatives bearing an ethynyl function have also served as starting materials for acetylenic mannich bases with cytotoxic potential. for example, propargylamines ( fig. ) have been obtained from alkynes prepared through addition of an organometallic derivative of acetylene to the carbonyl function in betulonic acid esters [ ] , and alkynes synthesized from betulin through a sequence comprising the oxidation of the primary alcohol function to aldehyde, followed by addition of an organometallic derivative of acetylene to aldehyde carbonyl, afforded propargylamines (fig. ) [ ] . cytotoxicity of these mannich bases was evaluated on a panel of nine human cancer cell lines using srb assay, and the results prove that some of these compounds show considerable toxicity (ic values as low as mm). introduction of the aminomethyl group significantly improved the cytotoxicity of propargylamines and compared to that of the parent alkynes, presumably by enhancing their solubility and bioavailability. highly hydrophobic and sterically hindered amino moieties, such as dicyclohexylamino or dibenzylamino, led to a decrease in cytotoxicity of the corresponding aminomethylated alkynes. mannich bases of this type were shown to act by triggering apoptosis, although a complementary process of autophagy could also be involved. in an attempt to circumvent the resistance mechanism developed by cancer cells after prolonged administration of doxorubicin and address the issues of poor solubility, short lifetime and high toxicity of prodrug doxoform [ ] , a second-generation, watersoluble prodrug of doxorubicin was developed by conjugation of the active drug with salicylamide by means of a mannich reaction [ ] . doxorubicinesalicylamide conjugate doxaliform (fig. ) has a half-life of approximately one hour, and was more cytotoxic than doxorubicin against mcf- sensitive ( -fold) and mcf- /adr resistant ( -fold) breast cancer cells. furthermore, doxaliform is amenable to functionalization with a view to provide a site for attachment of a releasable targeting group that might direct the conjugate to a specific receptor that is overexpressed by cancer cells. because many breast cancer cells overexpress estrogen receptor a, this receptor was chosen for targeting by doxasaliform having tethered a hydroxytamoxifen moiety, as in prototype ( fig. ) [ ] . cytotoxicity of these candidates as a function of the length of the tether showed that a triethylene glycol unit provides a lead compound whose growth inhibition of four selected breast cancer lines (mcf- , mcf- /adr, mda-mb- and mda-mb- ) was enhanced up to -fold relative to doxorubicin. later work confirmed that uptake of hydroxytamoxifen-targeted doxorubicinesalicylamide conjugate is mediated by both the antiestrogen binding site and estrogen receptor [ ] . also, several doxorubicineformaldehyde conjugates tethered to the nonsteroidal antiandrogen cyanonilutamide were designed, synthesized and evaluated as androgen receptor-targeted ligands for specific delivery of the conjugate to prostate cancer cells [ ] . such a construct was later used in studies intended to evidence binding to androgen receptor in live pc prostate cancer cells and the subsequent translocation of the construct bound to the receptor to the nucleus, but the results were not very promising [ ] . other efforts [ ] were directed towards the conjugates of doxasaliform with the cyclic peptide neme-vrgdf (known as cilengitide), which is a potent antagonist of a v b integrin involved in many cell-matrix recognition and cell adhesion phenomena, and plays an important role in angiogenesis and tumor metastasis. although the complete construct maintained a high affinity for a v b integrin, the ic for growth inhibition of mda-mb- cells was -fold greater than that of doxasaliform; the poor results have been tentatively blamed on limitation of drug delivery caused by the specific reduced abundance of receptors in this type of cell. eventually, because doxasaliform is not as active as prodrugs doxoform or doxazolidine, this line of research was terminated. however, the topic was later revisited by other authors, who reported the synthesis of constructs derived from either doxorubicinesalicylamide, daunorubicinesalicylamide or their -acyloxymethyl derivative, and amino-terminated poly(ethylene glycol), and their use for in vitro and in vivo studies [ ] . these constructs presented cytotoxicities comparable to those of the parent drugs, and the lifetime of one of these constructs was determined to be longer than that of doxorubicin. also, the construct was more efficient than doxorubicin at reducing the weight of s- xenografted tumors [ ] . cytotoxic mannich bases derived from miscellaneous, structurally unrelated substrates are grouped in the last paragraph of this section. -aminomethylated -alkyl- , , , -tetrahydrocarbazole- -ones (fig. ) show moderate to potent cytotoxicity towards a (human lung adenocarcinoma), sgc (human gastric cancer), k (human myelogenous leukemia), hct (human colorectal carcinoma), and kb-vcr (human oral cancer) cells using mtt assay [ ] . one of candidates (r ¼ c h , r ¼ ch ) was more cytotoxic than reference drug taxol against a cell line (ic ¼ nm), and at least one of its mechanism of action appears to be the inhibition of tubulin polymerization. a series of aminomethyl imidazo[ , -b]benzothiazoles (fig. ) were evaluated for antiproliferative activity against hepatocellular carcinoma (hepg ), human breast (mcf- ) and human cervical (hela) cancer cell lines using mtt assay [ ] . mannich bases inhibited the proliferation of cancer cells at concentrations lower than mm, arrested the cell cycle at g /m phase while downregulating cyclin b and upregulating chk protein, and appeared to induce apoptosis based on the elevated levels of caspase- . -cinnamoyl-benzoxazol- -ones (r ¼ h, ch o) were the substrates used for the synthesis of mannich bases (fig. ) , which had high to moderate cytotoxicity (ic between and mm) towards human pre-b-cell leukemia cell line bv- and chronic myeloid leukemia k- , and appear to exert their cytotoxic action at least in part through induction of apoptosis [ ] . replacement of the methoxy substituent in compounds with chlorine led to a marginal improvement of ic values [ ] . a series of mannich bases (r ¼ h, ch ; x ¼ ch , o, n-r ) (fig. ) of fused pyridazinone derivatives were synthesized and tested in vitro using srb assay to determine their growth inhibitory properties at a single mm dose against sixty different human tumor cell lines. most of them showed no activity of all, but two candidates moderately inhibited the growth of non-small cell lung cancer (ekvx and hop- ) and glioblastoma (snb- ) cell lines [ ] . an impressive number of articles dealing with the antibacterial activity of mannich bases have been published in the last decade. from a structural point of view, mannich bases reported in these studies are derived from nearly all of the major types of substrates capable of undergoing aminomethylation. these mannich bases have been evaluated against both gram-positive and gramnegative bacteria belonging to various families. the evaluation of the antibacterial activity was performed using different screening methods, and not always the standardized versions, and this complicates the interpretation and the comparison of results obtained in different studies. owing to the particular relevance of tuberculosis, the reports concerning the screening of mannich bases against mycobacterium species are presented in a separate section. ketonic mannich bases with antibacterial potential are at the heart of several studies within the period of time covered by this review. aminomethylation of a,b-unsaturated cyclic ketones with variable ring sizes afforded a library of compounds (n ¼ e ) (fig. ) derived from secondary cyclic aliphatic amines and having on the aromatic ring either no substituent at all, or a methyl group, or a variable number of methoxy groups [ ] . evaluation of the antibacterial activity of these mannich bases against a panel of both gram-positive and gram-negative bacteria using the serial dilution method showed that the candidates derived from seven-or eightmembered a,b-unsaturated cyclic ketones were the least active of all, and that the nature of the amine moiety, or the number and the position of the methoxy groups on the aromatic ring, did not influence the antibacterial activity. none of the compounds were active against pseudomonas aeruginosa, and only a few were moderately active against escherichia coli. mannich bases affected gram-positive bacteria (minimum inhibitory concentration (mic) < . mg/ml in most cases), but the candidates were less potent than reference antibiotics. another set of mannich bases ( fig. ) obtained from benzo-fused cyclic ketones (indanone, tetralone, benzosuberone) were tested under the same conditions against an extended panel of gram-positive and gram-negative bacteria [ ] . in this collection, both the nature of the amine moiety and the alkoxy substituent r influence the antibacterial activity, to a greater extent for gram-positive bacteria and to a lesser extent for gram-negative bacteria. indanone derivatives were active against both classes of bacteria (mic between . and mg/ml), whereas tetralone and benzosuberone derivatives were mostly active against gram-positive bacteria (presumably due to the higher outer membrane permeability of these bacteria). also, a statistical comparison between the antibacterial activity of mannich bases and mannich bases showed that the latter were more active than the former [ , ] . based on oxazolidinone antibacterials linezolid and eperezolid, a small series of mannich bases (r ¼ nhcoch or nhcsch ) (fig. ) derived from cyclohexanone or benzo-fused cyclic ketones was synthesized and evaluated against three selected gram-positive microorganisms using the standard serial dilution method [ ] . all of the compounds having an acetamido group had low antibacterial activity, but the replacement of this group with thioacetamido restored the activity up to the level exhibited by the parent oxazolidinone antibacterial. further modification (r ¼ nhcsnh ) produced a candidate whose antibacterial activity was superior to that of linezolid. , -dimethoxybenzofuran- ( h)-one double mannich bases (fig. ) had moderate to high antibacterial activity, as determined using disc diffusion method at a concentration of . % in dmso [ ] . the majority of compounds were active against e. coli, and those derived from pyrrolidine, diethylamine and di-nbutylamine as amine reagents showed significant antibacterial activity (zone of inhibition e mm) towards most bacteria, comparable to that of reference drug gentamycin (zone of inhibition e mm). on the other hand, compound derived from n-ethylmethylamine was inactive (no inhibition at concentrations higher than mg/ml). also, structurally diverse types of mannich bases derived from acetophenones or -acetylthiophene, such as mono-mannich bases of type (r ¼ ch ) (fig. ) , bis-mannich bases of type (r ¼ ch ) (fig. ) , and piperidinols of type (r ¼ ch ) (fig. ) , as well as their azine derivatives (fig. ) , are devoid of antibacterial activity against a wide range of human-and plant-pathogenic bacteria [ ] . methiodide (fig. ) of dimethylamine mannich base of acetophenone was the sole active compound in this series, and only against staphylococcus aureus, but the diameter of the inhibition zone measured mm (compared to cm in the case of reference drug ofloxacin), and its mic was mg/ml. the antibacterial activity of ketonic mannich bases generated from arylamines and aromatic aldehydes was also investigated by several groups. starting from cyclohexanone, a set of eleven compounds was obtained, and screening against four common bacteria using serial dilution method showed that many of these candidates have excellent antibacterial activity, comparable to that of ceftriaxone [ ] . however, the lack of rational design and systematic use of the aldehyde and amine components used to generate mannich bases ( fig. ) preclude any logical conclusions regarding sar in this collection. use of four differently substituted acetophenones, -fluorobenzaldehyde and several para-substituted anilines led to mannich bases (fig. ) , which were screened against four common bacteria [ ] . most of these compounds were inactive, but candidate (r ¼ -br, r ¼ f) was more potent than reference drug ampicillin against all four microorganisms, whereas another candidate (r ¼ -br, r ¼ h) was more potent than ampicillin against e. coli and s. aureus. arylamine mannich bases and ( fig. ) were obtained from -acetylcoumarine and -acetylbenzofuran, respectively, and evaluated against e. coli, s. aureus and p. aeruginosa using disc diffusion method [ ] . since most compounds in this series are derivatives of -aminobenzoic acid, it has been expected that at least a few of these compounds have good antibacterial activity. three candidates (r ¼ h, cl or f) had antibacterial activity comparable to that of reference drug streptomycin, but the rest were almost inactive. the series of candidates provided only one remarkably active mannich base (r ¼ n(ch ) ), but the rest of the compounds with a benzofuran moiety are generally more active than coumarine-derived candidates . -(arylamino)- -ferrocenyl- -propanones ( fig. ) showed broad spectrum activity against both gram-positive and gram-negative bacteria, the highest degree of growth inhibition being obtained for s. aureus [ ] . mic values varied between . and . mg/ml, making these compounds approximately -fold less active than reference drug tetracycline. because compounds ( fig. ) with a similar structure but with a phenyl ring instead of ferrocene have been reported to possess moderate to high antibacterial activity [ ] , it appears that the replacement of phenyl in with ferrocene could be responsible for the substantial decrease in activity recorded in the case of compounds . aminomethylated phenols have also been reported to have antibacterial activity. mannich bases ( fig. ) obtained from t-butylcatechol and secondary aliphatic amines have been screened for antibacterial activity against seven types of bacteria, but their potency was rather low to moderate [ ] ; their corresponding copper(ii) complexes fared better, as two of them had mics comparable to that of chloramphenicol ( . mg/ml). the mannich reaction of a derivative of another dihydroxylic phenol with either secondary aliphatic amines or primary arylamines afforded compounds (fig. ), whose zones of inhibition for three common bacteria, determined at two concentrations ( and mg/ml) using disc diffusion method, were smaller than those observed for reference drug ofloxacin at mg/ml [ ] . because chemical modification of natural antibiotic novobiocin by means of aminomethylation was hypothesized to increase permeability through the outer membrane of gram-negative bacteria, phenolic mannich bases (fig. ) were designed, synthesized and evaluated against s. aureus (one of novobiocin's usual targets), francisella tularensis and e. coli [ ] . candidates were significantly less potent than novobiocin against these three microorganisms, the antibacterial activity of the most potent of these compounds being almost times weaker than that of novobiocin. thirteen novel phenolic mannich bases (fig. ) derived from lawsone, benzaldehydes and primary aliphatic amines, together with their corresponding copper(ii) complexes, were tested for antibacterial activity against seven types of bacteria [ ] . with a few exceptions, mannich bases were generally more potent than the corresponding complexes, presumably due to the greater solubility of the pro-ligands. two mannich bases had antibacterial activity comparable or higher to that of chloramphenicol against bacillus subtilis, e. coli and s. aureus, whereas the other compounds inhibited bacterial growth at concentrations greater than mmol/l. antibacterial activity of fused oxazines obtained through the mannich reaction of phenols and naphthols with primary amines has also been examined. -acetyl- , -benzoxazines ( fig. ) were screened at mg/ml against three common microorganisms using disc diffusion method, and they showed good growth inhibitory activity towards s. aureus, but only moderate potency against e. coli and b. subtilis [ ] . the antibacterial activity was evaluated for , -benzoxazines ( fig. ) substituted with chloro and methyl groups in the aromatic ring, bis( , -benzoxazines) ( fig. ) , naphtho[ , -e]- , -oxazines (fig. ) , as well as naphtho[ , -e]- , -oxazines (fig. ) using serial dilution method [ ] . regardless of the aliphatic or aromatic nature of the moiety at the nitrogen atom, most compounds, and especially naphthoxazines and , had mic values greater than mg/ ml, but two benzoxazines derived from -chlorophenol and aromatic amines were more potent than ampicillin against e. coli, whereas compound derived from , -dichlorophenol was equipotent to gentamycin against s. aureus. in addition, benzoxazines and naphthoxazines and having benzazole moieties at the nitrogen atom were tested against four common types of bacteria [ ] . although the majority of the compounds were inactive, the antibacterial activity of few candidates was -folde -fold greater than that of reference drug tetracycline; the enhancement of activity for these candidates has been associated with the presence of a benzimidazole system at the nitrogen atom and at least one halogen in the phenolic starting material. in contradiction to the previously mentioned results, a number of naphtho[ , -e]- , -oxazines obtained from variously substituted arylamines were found to be quite active as antibacterial agents towards e. coli and b. subtilis [ ] . these compounds were generally more active against the latter pathogen, and the candidates derived from -fluoroaniline, -ethoxyaniline and , , tribromoaniline were even more potent than reference drug streptomycin. the mannich reaction of phenols fused with heterocycles has also been investigated for the preparation of antibacterials. mannich bases of -hydroxyquinoline have been quaternized at the heterocyclic nitrogen atom with bromoacetic acid esters of superior alcohols (r ¼ n-c h , n-c h , n-c h ) to give compounds (x ¼ o, ch ) (fig. ) [ ] . the antibacterial activity of candidates , determined for three concentrations ( , . and mg/ml) using disc diffusion method, showed that the compounds were active towards both gram-positive and gram-negative bacteria, but the presence in their structure of long alkyl chains rather than the aminomethyl function is most likely responsible for their activity. phenolic mannich bases (r ¼ h, br) ( fig. ) were evaluated against e. coli and bacillus cirroflagellosus at mg/ml using disc diffusion method, but their antibacterial activity was generally weak, with the exception of candidates derived from morpholine as amine reagent [ ] . aminomethylated derivatives (r ¼ oh, fig. ) also manifested antibacterial activity in various degrees; the most active candidates were the mannich base derived from cyclohexylamine, which was as potent as tetracycline against b. subtilis (mic . mg/ml), and the mannich base derived from morpholine, which had the same antibacterial activity as ampicillin against s. aureus (mic mg/ml) [ ] . phenolic mannich bases derived from -hydroxy- h-pyran- ones received special attention as potential antibacterials. allomaltol ( -hydroxy- -methyl- h-pyran- -one) was converted into mannich bases (fig. ) , whose antibacterial activity against four different gram-positive bacteria was only moderate (mic > mg/ml) [ ] . on the other hand, the antibacterial activity of mannich bases ( fig. ) derived from chlorokojic acid was superior to that of analogous , and the candidates were shown to possess a broad spectrum, while being more active against standard strains (mics between and mg/ml) than towards clinical, drug-resistant isolates [ ] . there is no striking difference between the antibacterial activity of aminomethylated derivatives having -benzylpiperazines and those having -substituted piperidines as amine moiety. the growth inhibition of grampositive bacteria (with the exception of enterococcus faecalis) occurred at mic values that were lower than those required for the growth inhibition of gram-negative bacteria. mannich base with -( -chlorobenzhydryl)piperazine as amine moiety seems to be best compound in this series, but its activity was consistently lower than that of any reference antibacterial drug. subsequently, a novel series of mannich bases was synthesized, this time using various -(substituted aryl)piperazines as amine reagent, but the compounds in this collection were weaker antibacterials than analogous derived from -benzylpiperazines [ ] . screening of another novel set of compounds with either benzylpiperazines of -arylpiperazines did not identify any compounds with improved or remarkable antibacterial activity, as the best candidates in this study had mic values between and mg/ ml [ ] . however, when piperazinyl-substituted fluoroquinolones were employed as amine reagents in the mannich reaction of hydroxy- h-pyran- -ones as substrates, the resulting hybrids (r ¼ cl) (fig. ) had a broad spectrum and were highly active against a panel comprising both gram-negative and gram-positive bacteria [ ] . generally, aminomethylated derivatives of chlorokojic acid ( , r ¼ cl) were more potent antibacterials than aminomethylated derivatives of kojic acid ( , r ¼ oh). mannich base (r ¼ cl) derived from fluoroquinolone ciprofloxacin was the most potent candidate in the series. replacement of the quinolone scaffold in candidates derived from norfloxacin with a naphthyridone moiety (as in mannich bases derived from enoxacin) resulted in decreased antibacterial activity. among the bacteria used in this study, e. coli and klebsiella pneumoniae were the most sensitive microorganisms to hybrids . docking of the most active compound into topoisomerase ii dna-gyrase active site (which is the traditional target of quinolones) showed that mannich base derived from ciprofloxacin binds in a manner similar to ciprofloxacin to the enzyme's active site, and that the hydroxypyran- -one moiety is anchored with additional hydrogen bonding within the binding pocket, thus increasing the stability of the inhibitoreenzyme complex. mannich bases of isatin derivatives have also been investigated as antibacterial agents. aminomethylation of isatin semicarbazone with (hetero)arylamines afforded mannich bases (fig. ), whose antibacterial activity was moderate towards e. coli and good towards s. aureus [ ] . in addition, a series of mannich bases (r ¼ secondary aliphatic amines) (fig. ) derived from a hydrazone of isatin incorporating the biologically relevant moieties of , , triazine, sulfa drugs and azacarbazole had moderate to good antibacterial activity against the previously mentioned microorganisms [ ] . hydrazidones obtained from isatin and hydrazides of variously substituted acetic acids have also been aminomethylated with a view to synthesize antibacterial mannich bases. for example, mannich bases ) derived from substituted isatins and morpholine, piperidine or -methylpiperazine generally showed poor antibacterial activity against the two aforementioned types of bacteria, with the exception of compound (r ¼ -methylpiperazinyl, r ¼ -br), whose inhibition determined by disc diffusion method was approximately half of that for reference drug gentamycin [ ] . another collection of mannich bases (r ¼ -or , -substituted -ethylphenyloxy) generated from substituted isatins as substrates and morpholine or piperidine as amine reagents exhibited weak to moderate antibacterial activity towards e. coli, s. aureus and shigella flemeri (zone of inhibition e mm) compared to norfloxacin (zone of inhibition e mm) [ ] . schiff bases generated from isatin and various (hetero)aromatic amines represent another type of isatin-derived substrate that was subjected to aminomethylation to obtain potential antibacterial agents. the use of isatin schiff bases in the mannich reaction with -(( , -dichlorophenyl)amino]) phenylacetic acid as the amine reagent yielded compounds (r ¼ substituted phenyl) (fig. ) , which proved to be e -fold less potent than reference drug ciprofloxacin against an array of bacteria [ ] . when -chloro- -fluoroaniline was used to generate schiff bases from isatin and -chloroisatin, aminomethylation with secondary aliphatic amines gave mannich bases (fig. ) ; these compounds had moderate to good activity against e. coli, s. aureus, p. aeruginosa and salmonella typhi in disc diffusion assay, and showed also moderate to good b-lactamase inhibitory activity [ ] . schiff base of chloroisatin and trimethoprim was aminomethylated to give compounds (r ¼ -amino- -( , , -trimethoxybenzyl)pyrimidin- -yl), but only mannich bases derived from ciprofloxacin, lomefloxacin or gatifloxacin as amine reagents in the mannich reaction were at least as potent, if not more potent, than the corresponding parent fluoroquinolones against a large panel of bacteria [ ] . antibacterial activity of mannich bases (r ¼ benzyl- -thioxo- h- , , -triazol- -yl) derived from schiff bases of isatin or -halogen-substituted isatins and an aminotriazolethione was generally good against four common microorganisms, as the size of the inhibition zones for some of the candidates (especially those derived from halogenated isatins and having piperidine or morpholine residues in the aminomethyl group) was comparable to the size of the inhibition zone for reference drug ciprofloxacin [ ] . recently, mannich bases ( fig. ) obtained using schiff bases derived from -fluoroisatin and -arylideneaminoanilines as substrates and ciprofloxacin as amine reagent were shown to be generally less potent antibacterials than reference drug ciprofloxacin, although some candidates had mic values comparable to those of ciprofloxacin against the investigated bacteria [ ] . a large number of papers published in the last decade report the antibacterial activity of mannich bases of , -dihydro- , , triazole- -thiones. from a structural point of view, mannich bases from triazolethiones that were synthesized through aminomethylation in these reports fall into two major categories: substituted -aminomethyl- h- , , -triazole- -thiones and -substituted -aminomethyl- -arylideneamino- h- , , triazole- -thiones . the relevant information from these studies (i.e., substituents at position and of the triazole ring, amine reagents used in aminomethylation, microorganisms employed in the antibacterial evaluation) is presented in a condensed manner in table . in the series of compounds a, the nature of the amine moiety in the aminomethyl group appears to be critical for the antibacterial activity, as mannich bases from piperazines were consistently more potent than mannich bases from arylamines, and some of them were even more potent than reference drug ampicillin [ ] . a comparison between antibacterial activities of a and structurally related b suggests that the replacement of substituent at position of the triazole ring in a with hydroxybenzylideneamino in b further enhances the antibacterial activity. although it was shown that substitution with chlorine of the phenyl at position of the triazole ring improves the antibacterial activity in the series of compounds b and c, and that aminomethylation also increases the bacterial growth of mannich bases c compared to the parent triazolethione, no candidates with substantial antibacterial activity could be singled out from these two collections [ e ]. however, use of ciprofloxacin as amine reagent in the synthesis of mannich bases c and d led to a substantial increased antibacterial activity of these compounds, and some of them were as potent as reference drugs ciprofloxacin and vancomycin against several types of bacteria and methicillin-resistant s. aureus (mrsa), respectively [ ] . mannich bases e or f having a diphenylsulfone moiety at position of the triazole ring had only weak antibacterial activity [ ] . a small number of mannich bases f, especially those having a halogen as substituent of the phenyl at n- of the triazole ring, inhibited the growth of gram-positive bacteria [ ] . mannich bases g, especially candidates with morpholine, benzylpiperazine, n-methylpiperidine and trifluoromethylphenylpiperazine in the aminomethyl group, had very good antibacterial activity (mic . e mg/ml) [ ] , whereas analogous h having -furyl instead of -thiophenyl were less potent (inhibition zones of e mm, compared to e mm for ampicillin) [ ] . the three mannich i bases reported in a study gave interesting results, as the candidates derived from methylpiperazine (inhibition zones of e mm for all bacteria) and -( -morpholinyl)ethylamine (inhibition zones of e mm for some of the microorganisms under evaluation) were more potent than reference drug ampicillin, whereas the bis-mannich base i derived from piperazine was inactive [ ] . however, further elaboration of i into compound p abolished the antibacterial activity [ ] . mannich bases j, which feature a pyridinyl moiety at position of the triazole ring, were generally poorer antibacterials than reference drug ampicillin, although there were isolated examples amongst these candidates that show more potency than ampicillin against certain bacteria [ ] . furthermore, the replacement of -pyridinyl with -quinolinyl as substituent at position of the triazole ring, and modification of phenyl into benzyl as substituent at position of the triazole ring led to inactive mannich bases k [ ] . both types of mannich bases l and l, featuring a , , -triazole moiety at position of the triazolethione scaffold, showed good antibacterial activity, but compounds l were generally more potent than l, and two of candidates l actually had mic values comparable to those of reference drug ciprofloxacin [ ] . mannich bases m and n (continued on next page) [ ] were devoid of effective antibacterial activity [ , ] , as was structurally related candidate o (a singular and notable exception is this compound's activity against bacillus cereus) [ ] . mannich bases q were also inactive as antibacterials, except for a few candidates that were more potent against p. aeruginosa than reference drug ampicillin [ ] . every member of the series of [ ] h primary arylamines diphenylamine piperazines s. aureus s. pyogenes [ ] mannich bases a was active against the tested bacteria, but not as potent as reference drug nitrofurazone [ ] . in addition, the nature of substituent at position of the triazole ring in the series of compounds a had no noticeable influence on their antibacterial activity, but the data (based on a single example) suggest that presence of chlorine in the aromatic ring of substituent x of the pyrazole moiety could improve the activity. mannich bases c were mostly active against s. aureus and b. subtilis, and candidates derived from ethyl piperidine- -carboxylate were generally more potent than those derived from piperazines [ ] . it should be noted that the presence of halogen in the structure of c (r ¼ , -difluorophenyl or , -dichlorophenyl) did not enhance the antibacterial activity of these mannich bases. in contrast, substitution with halogen in compounds d (e.g., r ¼ , dichlorophenyl) led to the most potent candidates in the series, and their antibacterial activity was comparable to that of reference [ ] drug ciprofloxacin [ ] . use of , -dichloro- -fluorophenyl moiety at position of the triazole ring in mannich bases e also produced candidates with good to excellent antibacterial activity [ ] . compounds e having secondary aliphatic amines in the aminomethyl group were generally more potent than those having arylamines, even if the anilines used in aminomethylation were substituted with additional halogen atoms [ ] . although mannich base g had no antibacterial activity, a close analogue of g such as compound (fig. ) was . e -fold more potent than ampicillin against most bacteria in the panel, with the exception of s. aureus [ ] . replacement of -methylpiperazin- yl with -morpholinyl in the aminomethyl group of mannich base g, and use of substituted phenyl other than -fluorophenyl as r afforded structurally related candidates h with moderate antibacterial activity [ ] . antibacterial activity of mannich bases i was moderate to good, but even the most potent compounds in this series (all of them having a -methylpiperazin- -yl moiety in the aminomethyl group) were at least -fold less active than reference drug ciprofloxacin [ ] . mannich bases j also had moderate to good antibacterial activity (inhibition zones of e mm), but were less potent than tetracycline (inhibition zones of mm) [ ] . overall antibacterial activity was reasonable in the series of mannich bases k, while a few compounds had mic values against various bacteria close to those determined for reference drugs [ ] . a few mannich bases m were more potent against p. aeruginosa than reference drugs levofloxacin and amikacin, while most candidates exhibited broad, moderate to good antibacterial activity [ ] . evaluation of mannich bases of , -dihydro- , , -triazole- ones as antibacterial agents received little attention in recent literature. a small series of six mannich bases (fig. ) derived from -(substituted (hetero)arylidendeamino)- -methyl- , , triazole- -ones was synthesized and tested against seven types of bacteria, and the antibacterial activity of the candidates ranged from moderate to excellent [ ] . all compounds were more potent than reference drug ampicillin against e. coli and p. aeruginosa, and candidate (r ¼ -hydroxyphenyl, nr ¼ -( -morpholinyl) ethylamino) was at least as active as ampicillin against all types of bacteria used in the study, with the exception of s. aureus. out of mannich bases of type (fig. ) , only those derived from morpholine or piperidine were excellent, broad spectrum antibacterials, whereas other analogues were either inactive, or weak antibacterials, or moderately active against the tested bacteria [ ] . a single mannich base (fig. ) of , , -triazole- -ones having a -quinolinyl moiety as substituent at position of the triazole ring was synthesized and evaluated for antibacterial activity, and its effect on e. coli and p. aeruginosa was comparable to that of ampicillin [ ] . three mannich bases having a -pyridinyl moiety at position of , , -triazole- -one scaffold, analogous to compounds j derived from , , -triazole- -thione, had moderate to good activity against most bacteria in the panel, but were not superior to reference drug ampicillin [ ] . antibacterial activity of mannich bases of , -dihydro- , , oxadiazole- -thiones has been investigated extensively. the structural features of the compounds reported in these studies have been summarized in table , along with the microorganisms used for screening. mannich bases a had moderate to good antibacterial activity (mic values between . and mg/ml), but none was as potent as reference drug ciprofloxacin, and no definite sar could be inferred from the biological results [ ] . the growth inhibition of mannich bases b appears to be modulated by the nature of the amine in the aminomethyl residue; thus, candidates derived from -trifluoromethylaniline were the most potent in this series and had antibacterial activity comparable by that of reference drug ciprofloxacin, followed in order by mannich bases b derived from morpholine, -chloro- -methylaniline and -chloro- -trifluoroacetylanline [ ] . in the case of mannich bases c, candidates derived from secondary aromatic amines were inactive, whereas the remaining compounds were less active against gram negative bacteria, and more active against gram positive bacteria (mic values e -fold lesser than those of reference drug ampicillin) [ ] . both mannich bases d reported in this study [ ] had moderate to good antibacterial activity, and both were more potent than ampicillin against e. coli and e. faecalis, but the candidate derived from -methylpiperazine was generally more potent than mannich base derived from -( -morpholinyl)ethylamine. in the case of thiazole-containing mannich bases, antibacterial activity of compounds e was moderate (mic values between and . mg/ml) [ ] , whereas compounds two of compounds f emerged as effective antibacterials, especially against gramnegative bacteria, with mic values e -fold lesser than those for reference drug chloramphenicol [ ] . several mannich bases g (nr ¼ nhc h f- , nhc h cl- , nhc h cf - , nhc h f - , ) had antibacterial activity comparable to that of reference drug penicillin, and, for some bacteria, even comparable to reference drug streptomycin [ ] . mannich base h (nr ¼ -piperazinyl) was equipotent to reference drug ciprofloxacin against s. aureus, but the antibacterial activity of the rest of candidates in this series was weaker than that of ciprofloxacin against the tested bacteria [ ] . the compounds reported in a study, including mannich bases i, are claimed to exhibit moderate antibacterial activity, but lack of access to the original information (which is only provided in the online edition as supplemental material, and is missing) makes data assessment impossible [ ] . phenylpiperazine-containing mannich base j was generally more potent that candidate j derived from ethyl isonipecotate, but both had moderate antibacterial activity compared to reference drug ampicillin [ ] . three out of eight synthesized mannich bases k were screened for antibacterial activity, but their growth inhibition was only weak to moderate (inhibition zones of e mm) compared to that of reference drug chloramphenicol (inhibition zones of e mm) [ ] . none of the synthesized mannich bases l presented any antibacterial activity at the maximum concentration tested ( mg/ml) [ ] . mannich base m was the only compound with antibacterial activity in the structural diverse library evaluated in the study, but its effects were moderate at best [ ] . screening results for mannich bases n were also disappointing, as they only barely inhibited the growth of e. coli at a concentration of mg/ml [ ] . out of two synthesized mannich bases o, the candidate having a morpholine residue in the aminomethyl group displayed excellent antimicrobial activity against all the tested microorganisms, while the candidate having a methylpiperazine residue had good or moderate activities against most bacteria in the panel, with the exception of e. coli and k. pneumoniae [ ] . the growth inhibition exerted by mannich bases p was moderate compared to reference drugs, and it was uninfluenced by the nature of the amine moiety in the aminomethyl group [ ] . nalidixic acid-based oxadiazolethione mannich bases q exhibited very weak antibacterial activity (mic values > mg/ml) compared to reference drug streptomycin (mic ¼ mg/ml), and only towards b. subtilis, but some of the structurally related mannich bases of , -dihydro- , , -thiadiazole- -thione ( fig. ) were quite active (mic values as high as . mg/ml) against the microorganisms used in the study [ ] . finally, mannich bases having a , dichlorophenyl moiety at position of , -dihydro- , , oxadiazole- -one ring (fig. ) were inactive against s. aureus, e. coli, p. aeruginosa and bacillus megaterium [ ] . two studies presented the synthesis and antibacterial evaluation of c-mannich bases of thiazolidinones (fig. ) having a thiazolyl- -imino substituent at position [ , ] . while candidates having alkyl or aryl groups as r substituent at c- of thiazolidinone ring showed no antibacterial activity against a panel of eight bacteria, some of their analogues unsubstituted at c- (r ¼ h) had weak antibacterial activity against s. aureus (mic values of e mg/ml). two of mannich bases (r ¼ -hoc h ) of thiazolidinones featuring a , -dibromo- oxoquinazolin- -yl moiety (fig. ) were -fold less potent against s. typhimurium (when nr ¼ diethylamino) and -fold less potent against b. cereus (when nr ¼ -piperidinyl) than reference drug ciprofloxacin [ ] . antibacterial activity of mannich bases (fig. ) was found to be more potent (mic values between . and mg/ml) than that of parent thiazolidinones, and the sar suggests that the nature of substituent r influences significantly the ability of these compounds to inhibit the growth of various bacteria [ ] . aminomethylation of pyrimidine- -thiones and pyrimidine- ones, substrates that can be easily obtained in a considerable structural variety through a simple biginelli reaction, has also been investigated in relation with the antibacterial activity of the resulting mannich bases. thus, pyrimidine- -thione mannich base (fig. ) was obtained using natural alkaloid cytosine as amine reagent in aminomethylation, and its evaluation as antibacterial agent showed that it is a potent growth inhibitor of s. aureus and b. subtilis, and it has weak activity against p. aeruginosa and e. coli [ ] . double mannich bases (fig. ) of pyrimidine- -thiones (x ¼ s) were good antibacterial agents (zones of inhibition of to % of the zone of inhibition for reference drug streptomycin) against e. coli and b. subtilis [ ] , and analogous double mannich bases of pyrimidine- -ones (x ¼ o) were even more potent, with the exception of the candidate having a -methoxyphenyl moiety at position of the pyrimidine ring (r ¼ -och , r ¼ h) [ ] . taking advantage of the lability of the proton located at the nitrogen atom in the amide function, several n-mannich bases of -chloro- -methoxybenzamide ( fig. ) obtained from either sulfonamides or common secondary amines as amine reagents have been synthesized and evaluated for antibacterial activity against a panel of bacteria [ ] . candidates derived from sulfonamides were more potent than the parent compounds, whereas mannich base derived from morpholine was an antibacterial agent with broad spectrum and good activity. n-mannich bases of glutarimides (r ¼ h, c-c h , c-c h , -clc h ) (fig. ) have been screened for antibacterial activity, and claimed to be potent very potent, although no comparison to currently-in-use antibacterials was available [ , ] . compounds derived from sulfonamides showed improved growth inhibition activity of the tested microorganisms over the corresponding parent amines, whereas analogues generated from secondary aliphatic amines were generally equipotent to those obtained from sulfonamides [ ] . also, n-mannich bases of succinimide (r ¼ h or c h , nr ¼ nhc h , -pyridinyl) (fig. ) and an n-mannich base from phthalimide were weak to moderate antibacterials against e. coli, s. typhi and b. subtilis [ ] . well-known antibiotics have also been subjected to aminomethylation with a hope of increasing their antibacterial activity or improving their bioavailability. hybrids of tetracycline and sulfonamides ( fig. ) have been generated using the mannich reaction, and their screening against salmonella enteritidis and pasturella multocida showed that their antibacterial activity is higher than that of the parent sulfonamides; unfortunately, no comparison with the activity of tetracycline or a reference drug is provided in the study [ ] . aminomethylation is one of the modifications that have also been explored in the case of vancomycin. after an n-decylaminoethyl moiety was introduced into the structure of vancomycin with a view to restore antibacterial activity against vancomycin-resistant enterococci while retaining potency against mrsa, grafting a hydrophilic group has been considered as a way to reduce the overall lipophilicity of the n-decylaminoethylmodified vancomycin and to restore the initial favorable distribution properties of vancomycin. several types of amine reagents (amino acids, amino sugars, phosphonic acids) have been considered for the synthesis of mannich bases (fig. ) , and some of these modifications led to candidates active against vancomycinresistant bacteria, and ultimately to the discovery of telavancin (td- ) as a lead compound with improved absorption, distribution, metabolism and excretion (adme) properties over ndecylaminoethylvancomycin [ ] . (fig. ) was subsequently screened against a large number of predominantly gram-positive isolates with developed antibacterial resistance, and showed to be highly active against methicillin-resistant staphylococci (mic . e mg/ml), streptococci (mic values . mg/ml), and vanb-type enterococci (mic values mg/ml) [ ] . with respect to its mechanism of action, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion, bound to cell wall with high affinity, and perturbed bacterial cell membrane potential and permeability [ ] . aminomethylation of vancomycin has also been used as the first step in the construction of glycopeptide/b-lactam heterodimers via linkage of aminomethylated vancomycin and various cephalosporin cores through an adipic acid moiety [ ] . these heterodimers were all found to exhibit excellent potency against a range of important gram-positive bacteria, and a subset of these candidates also demonstrated rapid bactericidal activity against mrsa, whereas two of the most attractive compounds have been shown to exhibit in vivo efficacy fold greater than that of vancomycin. finally, a patent claims a series of mannich bases of vancomycin as compounds suitable for oral delivery and having enhanced antibacterial potency [ ] . antibacterial activity of mannich bases derived from miscellaneous substrates is covered in the last paragraph of this section. mannich bases (r ¼ ch , oc h ) (fig. ) derived from either ethyl acetoacetate or diethyl malonate as substrates, variously substituted benzaldehydes as aldehyde components and benzyl carbamate as amine reagent had poor to moderate activity against five types of common bacteria [ ] . hydrazidones derived from isoniazid and -alkoxybenzaldehydes have been aminomethylated at position ortho to the alkoxy group to give mannich bases (fig. ) . a few members of this collection having an ethoxy group demonstrated at concentration of mg/ml antibacterial activity superior to that of reference drug amoxicillin at mg/ml [ ] , whereas candidates having a propoxy group were less potent [ ] . mannich bases derived from -methyl- -(substituted aryl) imidazo[ , -a]pyridines (fig. ) and having two halogens as substituents in the phenyl ring at position of the fused heterocyclic system were the most potent in this series against e. coli, p. aeruginosa, s. aureus and streptococcus pyogenes [ ] . mannich bases derived from an imidazo[ , -b]- , , -thiadiazole ring system (fig. ) showed weak activity against gram-positive bacteria and moderate activity against vibrio cholera, but their growth inhibition activity of e. coli was comparable to reference drug norfloxacin [ ] . evaluation of the antibacterial activity of mannich bases derived from -alkyl- -hydroxy-pyridine- ( h)-ones showed that only one candidate (fig. ) had moderate activity against s. enteritidis ( mg/ml) and s. aureus ( mg/ml) compared to reference drug ciprofloxacin ( mg/ml and . mg/ml, respectively, for the mentioned microorganisms) [ ] . mono-mannich bases (r ¼ ch ) and double mannich bases generated from sydnones ( fig. ) were both found to inhibit moderately the growth of four standard microorganisms, regardless of the nature of the amine reagent used in aminomethylation, but no useful conclusions regarding sar in this series could be drawn for further development [ ] . also, most mannich bases (r ¼ och ) had weak to moderate activity against both gram-positive and gramnegative bacteria, with the exception of candidate (r ¼ och , nr ¼ -nitrobenzothiazole- -ylamino), which was as potent as reference drugs ciprofloxacin and ampicillin against gram-positive bacteria [ ] . in contrast, aminomethylation of another sydnone derivative using various primary and secondary aliphatic amines was reported to occur in the at n- in the pyrazoline ring to give mannich bases instead (fig. ) , which had good antibacterial activity compared to reference drug ampicillin against four common bacteria, and particularly against b. subtilis [ ] . the copper(ii) complex of pyrazole c-mannich base (fig. ) inhibited the growth of b. subtilis at concentrations as low as . mm, most likely by promoting mutagenesis and inducing cell death [ ] . screening of a few of pyrazolone n-mannich bases (fig. ) against various bacteria resulted in inhibition zones that were comparable to those of reference drug ciprofloxacin, but the mic values for these candidates were ultimately far greater from those of ciprofloxacin [ ] . some of the acetylenic mannich bases (r ¼ c h , -clc h ; r ¼ c h , -c h ) (fig. ) had antibacterial activity comparable to reference drug ofloxacin against s. aureus, fewer showed good activity against e. coli, and only one candidate was equipotent to ofloxacin against p. aeruginosa [ ] . various substrates (e.g., benzimidazole, methylpyrazole- ( h)-one, succinimide, phthalimide or naphthalimide) were aminomethylated using norfloxacin as amine reagent, and some of the corresponding mannich bases were more potent than the parent fluoroquinolone, especially against e. coli and p. aeruginosa [ ] . sulfonamides and primary or secondary aliphatic amines were employed as amine reagents in aminomethylation of isoniazid to give mannich bases (fig. ) , and some of the candidates had antibacterial activity that was superior to that of the parent sulfonamides, whereas mannich base (nr ¼ nhch ) was the most potent compound in the series against s. aureus and b. anthracis [ ] . use of hydantoins as substrates in the mannich reaction led to -aminomethylated derivatives or to -aminomethylated derivatives (fig. ) , depending on the substituent on n- of imidazole ring, and some of these mannich bases had moderate to good antibacterial activity against four common bacteria [ ] . aminomethylation and aminoalkylation of saccharin using various types of amine reagents led to mannich bases ( fig. ) with moderate to excellent antibacterial activity against s. aureus (zones of inhibition between and mm, compared to mm for reference drug chloramphenicol) [ ] . a note reports in a concise manner the synthesis and antibacterial activity of mannich bases (fig. ) derived from a phenothiazine and primary aromatic amines, and some of the compounds in this study were potent and had a broad spectrum against the tested bacteria [ ] . mono-mannich base of quinoxaline- , ( h, h)-dione (fig. ) was screened at concentration of mg/disc, and was found to be more potent than reference drug nalidixic acid at concentration of mg/disc against four common bacteria, with the exception of proteus vulgaris [ ] . moderate to good antibacterial activity was determined for mannich bases of -aryl- -thioxo- , -dihydroquinazolin- ( h)one (fig. ), but none of the candidates was as potent as reference drug amikacin against the four common bacteria used in this study [ ] . in addition to their anticancer activity evaluation, p-mannich bases (fig. ) were also screened for antibacterial activity, and the mic values were generally approximately mg/ml, regardless of candidate's structure and type of bacteria, but no comparison with reference drugs was provided in the study [ ] . a paper claiming the synthesis of s-mannich bases of , -diaryl- mercaptopyrimidine and secondary aliphatic amines (fig. ) also reports their antibacterial activity weak to moderate, as the most potent candidates are -to -fold less potent than reference drug ciprofloxacin against four types of bacteria [ ] . tuberculosis is a chronic disease whose magnitude and gravity can be gleaned from a study by world health organization stating that in alone, . million new cases of tuberculosis were reported, and . million deaths were caused by tuberculosis, out of which . million people were infected with both human immunodeficiency virus and mycobacterium [ ] . continuous rise of the number of reported cases of drug-resistant and latent tuberculosis is also alarming and calls for urgent discovery of novel classes of compounds with antimycobacterial activity capable of overcoming the resistance of mycobacterium strains to current drugs. mannich bases are among these novel classes of antimycobacterial compounds that have recently caught the attention of researchers in the field. several examples of papers dealing with antitubercular ketonic mannich bases are available. thus, dimethylamine mannich bases of -benzylidenecyclohexanones ( fig. ) were reported to have mic values between . mg/ml and more than . mg/ml against mycobacterium tuberculosis h rv (for candidate (n ¼ , r ¼ r ¼ h) and candidate (n ¼ , r ¼ f, r ¼ h), respectively), which is approximately e % of the efficiency of reference drug rifampin against this microorganism [ ] . in addition, the ability of the same candidates to inhibit the growth of mycobacterium avium at concentration of . mg/ml ranged from to %. however, no significant correlations between the structural patterns of mannich bases (e.g., presence or absence of the cinnamoyl double bond, substitution pattern in the phenyl ring, electronic, hydrophobic or steric properties of the substituents in the aromatic ring) and their antimycobacterial activity could be established. mannich base (n ¼ , r ¼ och , r ¼ h) emerged from this series as a viable hit compound, having a mic value of . mg/ml and an ic value for vero cells of . mg/ml. this compound presents therefore a greater toxicity towards mycobacterium over normal mammalian cells, which has been tentatively explained by its ability to affect respiration in isolated rat liver mitochondria, and had mic values against several drug-resistant strains of mycobacterium that were similar or identical to the values determined for the h rv strain [ ] . isocitrate lyase is an enzyme in the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate, and, together with malate synthase, bypasses the two decarboxylation steps of the tricarboxylic acid cycle [ ] . glyoxylate cycle is essential for the survival of pathogens, including m. tuberculosis, inside the host [ ] , and isocitrate lyase has been identified only in bacteria, fungi, and plants, but no analogous enzyme has been documented in humans. therefore, isocitrate lyase has been pursued as a promising target for the development of novel antimycobacterial agents [ ] . a high-throughput screening of a large library of ketonic mannich bases against this enzyme led to the identification of candidate (fig. ) with a potent inhibitory activity (ic ¼ . mg/ml), but this compound's activity against m. tuberculosis has not been actually evaluated [ ] . phenolic mannich bases ( fig. ) derived from the hydrazone generated from isoniazid and -hydroxyacetophenone were screened against m. tuberculosis h rv, and found to inhibit its growth with mic values ranging from . to . mm [ ] . six compounds in this series were more potent than isoniazid (mic ¼ . mm), and the most potent candidate (nr ¼ piperazinyl) was shown to be equipotent to isoniazid in decreasing bacterial load in lungs of infected mice at a dose of mg/kg. anti-mycobacterium activity of novobiocin mannich bases (fig. ) was evaluated, and two of these candidates (nr ¼ n-acetoxyacetyl-n-methyl and n-acetaminoacetyl-nmethyl) showed an increased potency over novobiocin, although no comparison to established antimycobacterial drugs was provided [ ] . phenolic mannich bases derived from allomaltol and piperazines (fig. ) had good activity against mycobacterium smegmatis, with mic values between and mg/ml [ ] . although the growth inhibition of mycobacterium by the candidates in the library was moderate to good (mic values between and mg/ml), mannich bases derived from -phenylpiperazine and its analogues substituted with halogens in the aromatic ring were among the most potent compounds in the series. departure from substitution of piperazine in candidates with bulky, aromatic rings directly linked at n- generally led to decreased potency against mycobacterium, although some substituents (such as tbutoxycarbonyl, ethoxycarbonyl, -hydroxyethyl, cyclohexyl, benzoyl, furfuryl or -(dimethylamino)ethyl) appear to be tolerated better than others (e.g., isopropyl, allyl, -methoxyethyl, ethoxyethyl, phenethyl or benzyl). an extension of this study, aimed at evaluating against m. tuberculosis h rv the library of mannich bases of type (fig. ) obtained by replacement of piperazines in compounds with piperidines, led to valuable insight on the sar for this particular type of compounds [ ] . inspection of the sar in the subset of mannich bases suggested that the position of the substituents and, to a lesser extent, the nature of these substituents plays an important role in their antimycobacterial activity, as the most potent candidates in this subset are generally those substituted at position of piperidine ring [ ] . replacement of allomaltol with chlorokojic acid as substrate afforded piperazine mannich bases (r ¼ -substituted piperazin- -yl) (fig. ) , analogous to both and and having comparable antimycobacterial activity (mic values between and many papers reporting the antimycobacterial activity of mannich bases of pyrrole derivatives have been published in the last decade. the intensive research in this field stems from the discovery by italian researchers at universit a "la sapienza" in rome of bm (fig. ) as a potent agent, not only against several collection strains and clinical isolates of m. tuberculosis, but also against non-tuberculosis mycobacterial strains and drug-resistant mycobacteria, with mic values that are comparable to those of reference drugs isoniazid and streptomycin [ ] . although the quest for a more potent antimycobacterial agent through systematic modification of lead compound bm was not successful at first, it provided experimental proofs for the importance of the presence and nature of substituents at positions and of pyrrole ring [ ] . subsequently, novel pyrrole mannich bases analogous to bm (fig. ) , derived from -methylpiperazine but also from thiomorpholine as amine reagents, and having either chlorine [ ] or chlorine and fluorine [ ] as substituents of the phenyl rings in positions and of pyrrole, have been synthesized. two candidates (r ¼ f, r ¼ h or r ¼ h, r ¼ f) showed an improved activity (mic values of . and . mg/ml) against m. tuberculosis over mannich base (mic ¼ . mg/ml) [ ] . investigation of structureeantimycobacterial activity relationship in analogues of and was further pursued with a view to optimize the structure of these candidates. thus, synthesis of novel mannich bases of pyrrole using iterative introduction of more lipophilic -naphthyl or -fluoro-or -chlorophenyl moieties at either one of positions and in pyrrole ring of these lead compounds [ ] , or a recombination through shuffling of any of the substituents employed so far on the phenyl rings at these two positions of the pyrrole ring [ ] was undertaken. these studies confirmed the superior antimycobacterial activity of mannich bases containing a thiomorpholinyl moiety compared to the activity of the corresponding analogues with a -methylpiperazinyl moiety, evidenced the importance of fluorine substitution (especially at position of the phenyl ring) for antimycobacterial activity, and proved the decrease of activity with the introduction of -naphthyl moieties in the structure of lead compounds and . unfortunately, no novel candidates, more potent than mannich bases and , have been identified during these investigations. however, replacement of one halogen in the structure of lead compound with more lipophilic substituents (methyl, ethyl n-propyl, i-propyl) afforded candidates with improved antimycobacterial activity (mic values between . and . mg/ml) over and even over reference drug streptomycin [ , ] . in order to further increase the lipophilicity, replacement of the methyl group at position of the pyrrole ring with ethyl was also examined, but these novel mannich bases (x ¼ s or nch ) (fig. ) were generally less active against several m. tuberculosis strains than their methylsubstituted analogues, although their cytotoxicity towards normal cells was lower than that of the methyl-substituted counterparts [ ] . further exploration of the relationship between the nature of substituents on phenyl rings at positions and of pyrrole ring and the antimycobacterial activity of pyrrole mannich bases led to the identification of a novel lead compound (fig. ) with a very high activity toward both m. tuberculosis and h rv strains (mic ¼ . mg/ml) [ ] . antimycobacterial activity of mannich base was comparable to that of reference drugs streptomycin or rifampin, while the candidate demonstrated low cytotoxicity towards normal cells (selectivity index ic /mic > ) [ ] . subsequently a series of morpholine derivatives were designed and synthesized with a view to lower the clearance rate in mouse microsomal fractions associated with the presence of thiomorpholine [ ] . although the replacement of thiomorpholine with morpholine generally resulted in a decrease in potency, candidate (fig. ) was found to be equipotent to any of the lead compounds, showed improved microsomal clearance and lower cytotoxicity to normal cells, and was ultimately selected for in vivo pharmacokinetic and efficacy studies in an acute murine tuberculosis infection model. mannich base had an efficacious dose that results in a % colony-forming unit reduction in the lung of mg/kg, which is within the range of commonly employed tuberculosis drugs. a recent study showed that mutations in the mmpl gene in mycobacterium strains are responsible for resistance to bm , and suggested that products of this gene are cellular targets for bm , which makes mmpl , a member of the mmpl (mycobacterial membrane protein, large) family, a new potential druggable target for the treatment of tuberculosis [ ] . point mutations in mmpl gene that confer resistance to bm and other analogous pyrrole mannich bases have been later identified in several mycobacterium mutants [ ] . a large number of antimycobacterial pyrrole mannich bases were also employed to obtain a final multiprobe -d qsar model, which was shown to offer good predictions for antimycobacterial activity of an external, unrelated test set of compounds [ ] . accounts at various stages of the endeavors concerning the synthesis of pyrrole mannich bases as antimycobacterial agents, along with the related biological results obtained by the italian researchers, have also made the object of several author's reviews along the years [ e ]. a patent claiming the antimycobacterial activity of pyrrole mannich bases derived from -arylpiperazines is also worth mentioning [ ] . on the other hand, attempts to make a more drastic departure from these already established structural features of antimycobacterial pyrrole mannich bases (such as the introduction of an oxazolidinone moiety reminding of antibacterial linezolid) led to a series of compounds (x ¼ acetamido of -(hetero)aryl- , , -triazol- yl) (fig. ) that were at best e -fold less potent than mannich base [ ] . in addition, pyrrole mannich bases (fig. ) were shown to act as inhibitors (ic values ranging from . to mm) of mycobacterium protein tyrosine phosphatase b, an enzyme that is essential for the survival of bacteria in the host, but no in vitro evaluation using mycobacterium strains was performed [ ] . mannich bases of isatin and its derivatives have been evaluated as antimycobacterial agents as well. several isatin mannich bases (fig. ) containing a semicarbazide moiety were equipotent (mic ¼ . mg/ml) to isoniazid against m. tuberculosis, and two of the candidates were more potent in vitro than either isoniazid or rifampicin against a multidrug-resistant mycobacterium strain (mic ¼ . mg/ml) [ ] . schiff bases obtained from -substituted isatins (r ¼ f, cl, f) and lamivudine were aminomethylated using fluoroquinolones (r ¼ ethyl, cyclopropyl; r ¼ h, ch ) as amine reagents, and the resulting mannich bases (fig. ) showed e % growth inhibition of m. tuberculosis h rv at a concentration of . mg/ml; inhibition for one selected schiff base was %, and lamivudine alone did not inhibit the growth of this mycobacterium strain at the given concentration [ ] . several morpholine mannich bases of -substituted thiosemicarbazones of -trifluoromethoxyisatin (r ¼ c-c h , -fc h , -clc h , -brc h ) (fig. ) were potent antimycobacterial agents (mic values between and mg/ml) [ ] . replacement of the trifluoromethoxy group in with nitro led to a slight decrease in potency, which became more drastic when fluorine replaced the trifluoromethoxy group, whereas the substitution of morpholine with piperidine generally decreased the antimycobacterial activity of the candidates [ ] . using fluoroquinolone gatifloxacin as amine reagent, sixteen mannich bases derived from isatins, isatin semicarbazones, isatin thiosemicarbazones, isatin hydrazones with isoniazid, and isatin schiff bases with sulfadiazine were prepared and tested for antimycobacterial activity [ ] . mic values recorded for % growth inhibition of mycobacterium by these compounds ranged from . mg/ml to . ng/ml, whereas mic values between . and . mg/ml were determined for multidrug-resistant strains. promising results were obtained when the most potent compound in this series was tested in a murine model at a dose of mg/kg, and the analysis of data for inhibition of m. tuberculosis dna gyrase suggested that this enzyme could be the target of these isatinefluoroquinolones hybrids. good results were obtained from the evaluation as antimycobacterial agents of mannich bases obtained from -substituted isatins and their semicarbazones and thiosemicarbazones as substrates and ciprofloxacin as amine reagent (mic values between . and . nm) [ ] , and mannich bases generated from -substituted isatins, the corresponding semicarbazones, thiosemicarbazones and oxime ethers using methoxyciprofloxacin as amine reagent had comparable potencies [ ] . analogous mannich bases derived from moxifloxacin as amine reagent were also potent (mic value of mg/ml for m. tuberculosis, and between and mg/ml for multidrugresistant mycobacterium strains), but all of these isatinemoxifloxacin conjugates were generally less potent antimycobacterials than parent fluoroquinolone [ ] . evaluation as antimycobacterial agents of aminomethylated , dihydro- , , -oxadiazole- -ones, , -dihydro- , , -oxadiazole- thiones and , -dihydro- , , -triazole- -thiones has led to interesting results. a direct comparison between mannich bases (x ¼ o) of , -dihydro- , , -oxadiazole- -one and mannich bases (x ¼ s) of , -dihydro- , , -oxadiazole- -thione (fig. ) , having both a -pyridinyl substituent at position of the azole ring and derived from cyclic secondary aliphatic amines, showed that the former were significantly more potent against an isoniazidsensitive m. tuberculosis h rv strain than the latter, thus suggesting that the presence of oxygen is crucial for the antimycobacterial activity of this type of compounds [ ] . in a continuation of the initial study, the search for better antimycobacterial agents was restricted to mannich bases of , dihydro- , , -oxadiazole- -ones, while the variety of amine reagents employed in aminomethylation was expanded to include n-(substituted benzyl)methylamines, a structural modification that preserved the good activity of the candidates (mic values of e mg/ml) [ ] . however, replacement of the -pyridinyl substituent at position of mannich bases (x ¼ o) with phenyl, pyridinyl of pyrazinyl resulted in a significantly decreased antimycobacterial activity, whereas replacement with -pyridinyl did not generally affect the activity of the candidates. bis-mannich bases (fig. ) derived from dapsone as amine reagent, benzaldehyde and furan- -carboxaldehyde as aldehyde reagents and variously -substituted , , -oxadiazole- -thiones as substrates had excellent antimycobacterial activities against both isoniazidsensitive and isoniazid-resistant strains of m. tuberculosis, as they were -fold more potent than isoniazid against the sensitive strain and -fold more potent than isoniazid against the resistant strain [ ] . a study allowed another direct comparison, this time between mannich bases e of , -dihydro- , , -oxadiazole- thiones ( table ) and mannich bases i (table ) of , dihydro- , , -triazole- -thiones having the same isopropylthiazole- -yl substituent at position of the aforementioned azoles. some of candidates i were -to -fold more potent than the most potent mannich base e against m. tuberculosis h rv strain, but they were still -fold less potent than isoniazid [ ] . on the other hand, mannich bases (r ¼ h, oh; r ¼ c h , ch ch]ch , c h , -brc h ; r ¼ ch c h , c h , substituted phenyl, -pyrimidinyl) derived from , , -triazole- thiones (fig. ) had low antimycobacterial activity (mic values between and mg/ml) [ ] . the majority of mannich bases derived from either , -dihydro- , , -triazole- -thione or , dihydro- , , -triazole- -one having a -pyridinyl moiety at position of the triazole ring were more potent antimycobacterials (mic between and mg/ml) than reference drug streptomycin against m. smegmatis [ ] . in addition, , -dihydro- , , -triazole- -one mannich base (nr ¼ -piperidinyl) (fig. ) was equipotent to reference drug streptomycin (mic ¼ mg/ml) against m. smegmatis [ ] . mannich bases of imidazo[ , -b]- , , -thiadiazole derivatives ( fig. ) have been screened for antimycobacterial activity. evaluation of a library of mannich bases (r ¼ c-c h , -furyl, -thienyl; r ¼ h, br; nr ¼ -pyrrolidinyl, -piperidinyl, morpholinyl) against m. tuberculosis h rv showed that all of the candidates had mic > . mg/ml [ ] , but a second collection of aminomethylated imidazo[ , -b]- , , -thiadiazole derivatives (r ¼ n-c h , c-c h , -thienyl; r ¼ cl, ch , och ; nr ¼ -pyrrolidinyl, -piperidinyl, -morpholinyl) consisted of mannich bases with good antimycobacterial activity ( e % growth inhibition) at a concentration of . mg/ml [ ] . amongst mannich bases (r ¼ -ch oc h ch ), pyrrolidine-containing derivatives were consistently the best agents against m. tuberculosis h rv [ ] . also, compounds (nr ¼ -pyrrolidinyl, -piperidinyl, morpholinyl) (fig. ) had moderate antimycobacterial activity (mic ¼ mg/ml) [ ] . in addition to the types of substrates presented so far, miscellaneous other substrates have been subjected to the mannich reaction with a view to synthesizing compounds with antimycobacterial activity. aminomethylation of the amide function in tetracyclines using fluoroquinolones as amine reagents afforded mannich bases (fig. ) with excellent activity against m. tuberculosis (mic values between . and . mg/ml) [ ] . pyrazinamide was also aminomethylated at the amide function using piperazines (including four fluoroquinolones) as amine reagents; the resulting mannich bases (fig. ) were at least as potent as reference drug pyrazinamide against m. tuberculosis, and showed a significantly improved antimycobacterial activity over pyrazinamide against multidrug-resistant mycobacterium strain [ ] . anti-hiv drug efavirenz was aminomethylated using either common piperazines or piperazine-containing fluoroquinolones as amine reagents to afford hybrids (fig. ) , but only the mannich bases derived from fluoroquinolones had good activity against m. tuberculosis (no comparison with standard antimycobaterial drugs was available though) [ ] . investigation of antimycobacterial activity of a small number of indole mannich bases obtained from -arylpiperazines (fig. ) uncovered a few candidates that inhibited the growth of m. tuberculosis h rv almost completely at a concentration of . mg/ml [ ] . (fig. ) had good antimycobacterial activity (mic < mg/ml) [ ] , while mannich base (fig. ) was moderately active against m. tuberculosis h rv [ ] . finally, pyrazolone mannich base (nr ¼ nhcoc h ) (fig. ) was more potent than reference drugs ethambutol and ciprofloxacin, but inferior to isoniazid, against m. tuberculosis h rv [ ] . the incidence of fungal infections of any variety has been steadily increasing over the last decades, and it has become one of the main causes of morbidity and mortality, especially in patient with compromised immune systems [ ] . development of resistance to currently-in-use antifungal drugs also represents a major concern, and the discovery of novel antifungal agents, preferably outside any of the six major classes that are presently available for treatment, is one of the highest priorities for pharmaceutical industry. the latest advances in the evaluation of mannich bases with various structures as potential antifungals are presented in this section. first reports on the antifungal activity of ketonic mannich bases are relatively recent. a few mono-mannich bases of type (fig. ) and double mannich bases (fig. ) of acetophenone (r ¼ r ¼ h; nr ¼ dimethylamino, -piperidinyl, -morpholinyl) have been shown to have weak antifungal activity, but the corresponding methiodides were more potent than reference drug amphotericin b against yeasts saccharomyces cerevisiae and candida krusei, and against dermatophytes trichophyton mentagrophytes, trichophyton rubrum and microsporum canis [ ] . thus, quaternization of the nitrogen atom in mannich bases of type appears to be a chemical modification leading to efficient antifungals [ ] . various substitution patterns in the phenyl ring in ketonic mannich bases also did not improve significantly the antifungal activity of these compounds [ ] . bis-mannich bases (r ¼ c h ) derived from various acetophenones (fig. ) were only active against aspergillus fumigatus, but the antifungal activity of the related piperidinols (r ¼ c h ) (fig. ) was broader and slightly more potent compared to that of corresponding mono-mannich bases and bis-mannich bases , especially against dermatophytes t. rubrum and m. canis [ ] . however, a later study showed that a minor modification such as replacement of ethyl with methyl as substituent at nitrogen increased the antifungal activity of bis-mannich bases (r ¼ ch ), while it decreased the potency of piperidinols (r ¼ ch ) against t. rubrum and m. canis [ ] . use of phenethylamine as amine reagent in the mannich reaction with various acetophenones afforded secondary ketonic mono-mannich bases (r ¼ ch ch c h ) (fig. ) along with piperidinols (r ¼ ch ch c h ), but these compounds had relevant activity (most mic values were either . or mg/ml) only against m. canis, a dermatophyte whose growth was not inhibited by reference drug nystatin in the concentration range used in this study [ ] . most members of a library of mannich bases of enones had antifungal effects e -fold more potent than amphotericin b against at least one (if not many) selected plant fungi [ ] . ketonic mannich bases derived from arylamines as amine reagents have also been reported to have antifungal effects. thus, mannich bases (r ¼ -br, r ¼ h or f) (fig. ) were almost twofold more potent than reference drug ampicillin against candida albicans [ ] , whereas candidates (r ¼ f or cl) derived from -acetylcoumarin and (r ¼ cl or n(ch ) ) derived from -acetylbenzofuran (fig. ) had mic values comparable to those of reference drug fluconazole against aspergillus flavus, chrysosporium keratinophilum, and c. albicans [ ] . evaluation of mannich bases (fig. ) derived from cyclic ketones (n ¼ e ) showed that these compounds had good anti-candida and anti-saccharomyces activity, but they were less potent against aspergillus strains, and none of the compounds were as potent as reference drug amphotericin b [ ] . the anti-candida potency of candidates generally diminished with the increasing size of the ring, while the substitution pattern of the aromatic ring or the nature of the amine in the aminomethyl moiety did not influence the antifungal activity considerably. in addition, the a,bunsaturated ketone system is a structural requirement for the antifungal activity of mannich bases , as the amino alcohols obtained through the reduction of the carbonyl group in were mostly inactive towards all the fungi used in this study. inhibition of ergosterol synthesis does not appear to be the mechanism by which mannich bases exert their anti-candida activity, although they are able to influence the development of pseudohyphae and induce noticeable changes in the protein composition in c. albicans [ ] . inspection of sar disclosed in the previous study also proved valid for mannich bases derived from cyclic ketones fused with an aromatic ring (fig. ) , except that the mic values for compounds were consistently better than those for mannich bases [ ] . transcript profiling of c. albicans cells treated with candidate (n ¼ , nr ¼ -morpholinyl) showed that the transcriptional response is typical for oxidative stress and similar to that of a c. albicans cap transcriptional activator, which suggests that the ability of mannich bases to directly trigger oxidative stress may be at least in part the reason for their antifungal activity [ ] . mannich bases of thiocroman- -ones (fig. ) obtained either from secondary aliphatic amines or from primary arylamines had good activity against several types of fungi, and two candidates (nr r ¼ n(ch ) , r ¼ -cl, r ¼ -f or -cl) were more potent than reference drug ketoconazole against the fungi investigated in the study [ ] . antifungal activity of double mannich bases (fig. ) against several fungi was also good, although the potency of compounds against c. albicans was only moderate [ ] . several studies reporting the antifungal activity of phenolic mannich bases are available. the hydrazone obtained from , dinitrophenylhydrazine and salicylaldehyde was aminomethylated using various secondary amines to give mannich bases (fig. ) [ ] the most potent anti-candida candidates (nr ¼ diphenylamino, -morpholinyl, -piperazinyl) were e fold weaker than reference drug clotrimazole, whereas the growth of aspergillus niger was inhibited by the most potent compounds at mic values in the range of . e . mg/ml [ ] . most aminomethylated -t-butylcatechols (fig. ) , along with their copper(ii) complexes, have significant antifungal activity (radial inhibition of mycelial growth ! %) against several plant pathogenic fungi, which is comparable, or even higher in a several cases, to that of reference drugs nystatin and terbinafine [ ] . even at a concentration -fold greater than that of reference drug voriconazole, only three of mannich bases (nr r ¼ methoxyphenylamino, -piperidinyl, -methylpiperazinyl) (fig. ) barely showed anti-candida activity that was comparable to that of voriconazole [ ] . aminomethylation of mulundocandin, an antifungal lipopeptide belonging to echinocandin antifungals, was undertaken with a view to improve its solubility in water; the resulting mixture of mannich bases (r ¼ ch nr , r ¼ h) and double mannich bases (r ¼ r ¼ ch nr ) (fig. ) was separated before the antifungal activity of the compounds was evaluated in vitro, but only one candidate showed good inhibition against c. albicans and a. fumigatus [ ] . nonetheless, in vivo testing of the collection was promising, as many compounds showed anti-candida activity comparable to parent mulundocandin, while a significant improvement in anti-candida activity compared to mulundocandin was observed for mono-mannich base (r ¼ -phenylpiperazinylmethyl, r ¼ h), which was equipotent to fluconazole. three members of a collection of acetyl- , -benzoxazines (r ¼ ch , r ¼ -br; r ¼ h, r ¼ -och or -f) (fig. ) had inhibition zones against a. niger comparable to that of reference drug fluconazole [ ] . benzoxazines and , as well as naphthoxazines and (fig. ) were either inactive or had weak activity against candida spp., a. fumigatus and cryptococcus neoformans, but a few compounds in this library had moderate activity against sporothrix schenckii and t. mentagrophytes [ ] . in contrast, naphtho[ , -e]- , -oxazine (r ¼ -c h oc h ) was more potent than reference drug griseofulvin against c. albicans, whereas naphtho[ , -e]- , oxazine (r ¼ -ch oc h ) was equipotent to griseofulvin against a. niger [ ] . examples of mannich bases from phenols fused with heterocyclic rings that were investigated for antifungal activity include compounds (fig. ) , which had only moderate to weak activity against a. niger and penicillium spp. compared to reference drug griseofulvin [ ] , and compounds (fig. ) , which were efficient against aspergillus spp. and c. albicans at concentrations as high as mg/ml [ ] . mannich bases of chlorokojic acid (fig. ) had good anti-candida activity (mic values between and mg/ml) [ e ], but the antifungal activity against dermatophytes was slightly poorer [ ] . in contrast, mannich bases (fig. ) derived from allomaltol and substituted piperazines had poor antifungal activity against c. albicans and candida parapsilosis compared to that of reference drug fluconazole, but some members of the library were equipotent to fluconazole against c. krusei [ ] . antifungal activity of various mannich bases of isatin derivatives has been examined jointly with their antibacterial activity. mannich bases (fig. ) obtained from isatin semicarbazone as substrate and (hetero)arylamines as amine reagents had good anti-candida activity ( e % of the activity of reference drug fluconazole) [ ] , whereas mannich bases (r ¼ secondary aliphatic amines) (fig. ) had moderate antifungal activity against aspergillus spp., and consistently lower than that of reference drug fluconazole [ ] . for the most active mannich bases (r ¼ -or , -substituted -ethylphenyloxy, r ¼ -piperidinyl or morpholinyl), the antifungal potency against c. albicans and a. niger was / of the potency of reference drug griseofulvin [ ] . a couple of mannich bases (r ¼ substituted phenyl) (fig. ) were moderately active against a. niger, but the activity against c. albicans and penicillium notatum was generally poor [ ] . mannich bases (r ¼ -benzyl- -thioxo- h- , , -triazol- -yl) (fig. ) were moderately potent against c. albicans and a. niger compared to reference drug fluconazole [ ] . finally, in their evaluation against a. niger, mannich base (ar ¼ c h oh- ) (fig. ) was equipotent to reference drug ketoconazole against a, fumigatus, whereas mannich base (ar ¼ c h n(ch ) - ) was twofold more potent than ketoconazole, and three other analogues were equipotent to ketoconazole [ ] . evaluation of mannich bases of , -dihydro- , , -triazole- thiones (table ) as antifungal agents has been reported in conjunction with their antibacterial activity. in the series of mannich bases a, with the exception of candidate a (r ¼ c h cl- , nr ¼ nhc h f - , ) whose anti-candida activity was / of that of reference drug clotrimazole, all other compounds were inactive, and so were mannich bases b investigated in the same study [ ] . compared to reference drug fluconazole, most active mannich bases h were -fold less potent against c. albicans and s. cerevisiae [ ] , mannich bases j were either inactive or showed low antifungal activity [ ] , whereas mannich bases k were inactive against c. albicans and s. cerevisiae [ ] . mannich bases i lacked any anti-candida activity [ ] , as did mannich bases n and o [ ] , and most mannich bases q [ ] . the majority of mannich bases a were equipotent to reference drug fluconazole against c. albicans [ ] , and a small number of candidates c were moderately active against the same fungus, whereas the rest were inactive [ ] . while most mannich bases d were moderately active against aspergillus spp., c. albicans and penicillium marneffei, two candidates d (r ¼ -clc h or , -cl c h ) derived from -methylpiperazine as amine reagent were equipotent to reference drug ciclopiroxolamine [ ] . several mannich bases e were also equipotent to reference drug fluconazole against aspergillus spp., t. mentagrophytes and p. marneffei [ ] . neither compound g, nor candidate (fig. ) exhibited any anti-candida activity [ ] , whereas mannich bases i had weak to moderate activity against candida tropicalis, s. cerevisiae and a. niger [ ] . the antifungal activity of mannich bases j against aspergillus spp. and penicillium spp. was moderate to good, and mannich base j (x ¼ y ¼ ch , r ¼ -no c h ) was more potent than reference drug fluconazole against c. albicans [ ] . a few mannich bases k also showed good antifungal activity against c. albicans and fusarium solani [ ] . the best anti-candida activity in the series of mannich bases m was recorded for the candidate with r ¼ c h och - ( % of the inhibition zone of reference drug fluconazole) [ ] . in addition, , , -triazolo[ , -b]- , , -thiadiazines (fig. ) have been synthesized through a double mannich reaction starting from -benzyl- h- , , -triazole- ( h)-thione and primary aliphatic and aromatic amines, and these compounds had good antifungal activity against chrysosponium tropicum and t. rubrum, but were completely inactive against a. fumigatus, a. flavus and microsporum nanum [ ] . mixed results were obtained for other fungi, as a few candidates were more potent than reference drug clotrimazole against a. niger, but were moderately potent against c. albicans or fusarium oxysporum, whereas the rest of the compounds in the series were totally inactive against these three fungi. as far as the aminomethyl derivatives of , -dihydro- , , -triazole- -ones are concerned, candidates and (fig. ) had no activity against c. albicans and s. cerevisiae [ , ] , whereas in the series of mannich bases featuring an imidazole moiety (fig. ) , candidate (nr ¼ -piperidinyl) was twofold more potent against s. cerevisiae and twofold less potent against c. albicans than reference drug fluconazole [ ] . antifungal activity of aminomethylated , -dihydro- , , oxadiazole- -thiones ( table ) has been reported in the same studies dealing with these compounds' antibacterial activity. several mannich bases a were equipotent to reference drug ciclopiroxolamine against t. mentagrophytes, a. flavus and a. fumigatus (mic . mg/ml), but not against p. marneffei [ ] . mannich bases b derived from either morpholine or trifluoromethylaniline showed high activity against p. marneffei, t. mentagrophytes, a. flavus and a. fumigatus compared to reference drug ciclopiroxolamine, whereas those derived from other primary aromatic amines had only weak antifungal activity [ ] . only a few of mannich bases c derived from piperazines had moderate anti-candida activity, and the rest of the collection was inactive [ ] . oxadiazolethione mannich bases d [ ] and j [ ] , having at position of the oxadiazole ring either a pyridine or a quinoline moiety, had no noticeable anti-candida activity, and the activity of mannich bases e against s. cerevisiae, c. tropicalis and a. niger was moderate at best [ ] . most mannich bases f had weak antifungal activity against c. albicans, a. niger and aspergillus clavatus, but one candidate f (nr ¼ nhc h och - ) was -fold more potent than reference drug ketoconazole against the first two fungi, and almost equipotent to ketoconazole against the last fungus [ ] . several mannich bases g had similar mic values as reference drug amphotericin b against c. albicans, a. fumigatus, t. rubrum and t. mentagrophytes [ ] . most imidazole-containing mannich bases h exhibited better antifungal activity against c. albicans, t. rubrum and t. mentagrophytes than reference drug fluconazole [ ] . the compounds reported in a study, including mannich bases i, are claimed to exhibit significant antifungal activity against c. albicans, but lack of access to the original information (which is only provided in the online edition as supplemental material, and is missing) makes data assessment impossible [ ] . three mannich bases k were moderately active against aspergillus spp. and curvularia lunata [ ] , while mannich bases l [ ] and o [ ] had no anti-candida activity, and mannich base m showed no antifungal activity against c. albicans and s. cerevisiae [ ] . in addition, several aminomethylated , dihydro- , , -oxadiazole- -ones were more potent than reference drug nystatin against fungal plant pathogens aspergillus spp. ( [ ] . anti-candida activity of c-mannich bases of thiazolidinone (fig. ) was found to be -fold weaker than that of reference drug terbinafine [ ] . while three mannich bases were moderately active against c. albicans and a. flavus, one candidate (r ¼ c h , nr ¼ n(c h ) ) (fig. ) showed against the latter fungus a growth inhibition potency (mic ¼ . mg/ml) which was half of that determined for reference drug fluconazole [ ] . also, aminomethylated thiazolidinones (fig. ) had moderate activity against aspergillus spp. and c. albicans compared to reference drug ketoconazole [ ] . pyrimidine derivatives that have been aminomethylated with a view to evaluate the antifungal activity of the resulting mannich bases include pyrimidin- -ones, pyrimidin- -thiones and (thio) barbituric acid. double n-mannich bases (x ¼ o or s) (fig. ) had e % of the anti-candida activity of the clotrimazolecontaining commercial drug imidil [ , ] . c-mannich bases of (thio)barbituric acid (fig. ) , obtained using furan- carboxaldehyde or indole- -carboxaldehyde as aldehyde components and -aminopyridine or -aminoantipyrine as amine reagents in the mannich reaction, showed good activity against aspergillus spp. compared to reference drug salicylic acid [ ] . antifungal evaluation of mannich bases from miscellaneous substrates that do not belong to any of the previously mentioned classes is the topic of several studies. p-mannich bases (fig. ) showed good activity against c. albicans and s. cerevisiae (mic ¼ mg/ml) compared to reference drug amphotericin b (mic ¼ mg/ml) [ ] . various mannich bases of type (fig. ) derived from imides presented anti-aspergillus activity that ranged from weak to good, but no correlations between structure and activity for the small number of compounds in this series could be established [ ] . anti-candida activity of the most potent mannich bases (fig. ) was only half of the activity of reference drug fluconazole [ ] . aminomethylated hydrazidones (r ¼ c h ) derived from isonicotinic acid hydrazide (fig. ) had good anti-candida activity [ ] , but their analogues (r ¼ n-c h ) were less active, and their activity against a. niger was even poorer [ ] . several dimethylamine mannich bases derived from -methyl- -(substituted aryl)imidazo[ , -a]pyridines (fig. ) were -folde -fold more active against aspergillus spp. and c. albicans than reference drug griseofulvin [ ] . when evaluated against the same fungi, mannich bases (r ¼ ch ) and of sydnones (fig. ) were consistently less active than both reference drugs griseofulvin and nystatin [ ] , and the majority of mannich bases (r ¼ och ) behaved similarly, with the exception of candidate (r ¼ och ; nr ¼ -nitrobenzothiazole- -ylamino), which was more potent than reference drugs fluconazole and nystatin against c. albicans [ ] . all of the mannich bases derived from -alkyl- -hydroxy-pyridine- ( h)-ones (fig. ) were inactive against aspergillus spp. and c. albicans [ ] . a few aminomethylated pyrazolines (fig. ) had anti-candida activity (mics between . and mg/ml) comparable or even better than that of reference drug clotrimazole (mic ¼ mg/ml) [ ] , while several acetylenic mannich bases (fig. ) were equipotent to reference drug ketoconazole against c. albicans [ ] . mannich bases and derived from hydantoins ( fig. ) were generally moderate growth inhibitors for c. albicans and a. niger [ ] , and phenothiazine mannich bases (fig. ) were all active against a. niger (sizes of inhibition zone between and mm) [ ] . antifungal activity of mannich base (fig. ) of quinoxaline- , ( h, h)-dione ( mg/disc) against c. albicans and a. niger was superior to that of reference drug clotrimazole ( mg/disc) [ ] , while antifungal activity of mannich bases of -aryl- -thioxo- , -dihydroquinazolin- ( h)-one (fig. ) equipotent to reference drug fluconazole against c. albicans (mic . mg/ml) [ ] . as much as % of the world population live in areas with high risk of contracting malaria, and millions of people suffer from acute malaria each year, while approximately half million died from the disease in [ ] . malaria has been largely eradicated in many parts of the world, but the total number of recorded cases is still on the rise. one of the causes for this grave situation is the proliferation of malaria parasites that are rapidly becoming resistant to antimalarial drugs, especially those that are most frequently used, such as chloroquine. the development of novel antimalarials has stagnated since the s owing to lack of interest in developed countries for this topic, and limited market potential for these drugs. initiation of numerous programs supported by public funding led to a surge of interest in drugs for neglected diseases, and antimalarials are certainly amongst these drugs. use of the mannich reaction for the synthesis of antimalarials has a long and rich history, the quest for aminomethylated substrates for treatment of malaria starting with amodiaquine (fig. ) , the first mannich base that was used to treat malaria. many of these early studies focused on derivatives of aminoquinolines, and the sar within this class of antimalarials suggested that the chlorine atom at position of the quinoline scaffold and the phenolic mannich bases moiety are structural features that are essential for the antimalarial activity of these compounds. nowadays, the use of amodiaquine has declined owing to its considerable toxicity as a result of its in vivo conversion into a reactive quinone-imine metabolite by cytochrome p . it was therefore hypothesized that swapping the phenolic hydroxyl and the aminomethyl side chain in amodiaquine should prevent the formation of toxic metabolites, and help circumvent the adverse side effects associated with the use of amodiaquine. this hypothesis led to the design and synthesis of several amodiaquine analogues that retain the antimalarial activity, while lacking the potential to form a quinone-imine derivative [ ] . out of these analogues, mannich base (the isomer of amodiaquine henceforth named isoquine, fig. ) expressed in vitro activity against both chloroquine sensitive hb strain and highly chloroquine-resistant k strain of plasmodium falciparum below nm, and was times more potent that chloroquine. isoquine (as its diphosphate salt) was subsequently tested in vitro against the murine plasmodium yoelii ns strain, and found to be -fold more potent than amodiaquine when administered orally. moreover, no toxic metabolites or any excessive accumulation of isoquine have been noticed in animal models, but unacceptably high first pass metabolism of isoquine to n-dealkylated metabolites in four animal species compromised isoquine's activity against parasites, and complicated the development process of isoquine into a marketable drug. a novel candidate, n-tert-butyl isoquine (gsk ) (fig. ) , was identified using isoquine as lead, and shown to exhibit low nm activity against chloroquine-resistant and sensitive parasites, as well as in vivo oral activity equivalent to amodiaquine, but with a significantly improved antimalarial exposure profile [ ] . in addition, n-tert-butyl isoquine had a better overall preclinical safety profile than chloroquine or amodiaquine, and can be synthesized in a scalable and cost-effective way. the study of disposition of candidates and showed that isoquine undergoes in vivo oxidative n-dealkylation to desethyl-isoquine, a metabolite that had an improved blood clearance over isoquine; unfortunately, desethyl-isoquine also had a more potent inhibitory action of cyp a than isoquine [ ] . on the other hand, n-tertbutyl isoquine had human plasma protein binding ability and inhibition of cyp d similar to those of isoquine and desethylisoquine, but a reduced rate of n-dealkylation and a higher oral bioavailability compared to isoquine and desethyl-isoquine, and these properties made n-tert-butyl isoquine a better candidate than isoquine for further development. a study of the in vitro activity of isoquine against p. falciparum clinical isolates collected in kenya in relation to amino acid changes in pfcrt and pfmdr , the two genes associated with -aminoquinoline resistance, showed that isoquine possesses high activity against field isolates (ic was nm, compared with nm for chloroquine, and nm for amodiaquine), and that isoquine's activity could be correlated with polymorphisms in pfcrt, but not in pfmdr [ ] . starting from n-tert-butyl isoquine as a template, three benzoxazines (r ¼ c h , n-c h , ch c h ) (fig. ) were designed and generated through a double mannich reaction, but despite the good anti-plasmodium activity against both chloroquine-sensitive (ic between and nm) and chloroquine-resistant (ic between and nm) strains, the rapid ring opening of benzoxazine at low ph makes them unsuitable for further development [ ] . novel analogues (nr ¼ aliphatic primary and secondary amines, aromatic primary and secondary amines) of amodiaquine have been tested in vitro against chloroquine-sensitive strains of p. falciparum, and while all of the compounds were active, only mannich base (nr ¼ morpholinyl) (fig. ) showed good activity (mic ¼ ng/ml) compared to chloroquine (mic ¼ ng/ml) [ ] . antimalarial activity of analogues of isoquine (fig. ) has also been evaluated, but regardless of the nature of the amine in the aminomethyl moiety, their activity was considerably lower than that of reference drug chloroquine [ , ] . use of primary amines derived from bicyclic scaffolds for the synthesis of novel isoquine analogues led to the discovery of (fig. ) , which was approximately twofold more potent that either chloroquine or isoquine against chloroquine-sensitive and resistant p. falciparum strains [ ] . a series of isotebuquine analogues (r ¼ cl or cf , r ¼ h), the corresponding double mannich bases (r ¼ ch nhc(ch ) ), as well as n-oxides derived from a mono-mannich base and a double mannich base of type (fig. ) were also evaluated against chloroquine-sensitive and resistant p. falciparum strains [ ] . while only a few of candidates were more potent than chloroquine against the chloroquine-sensitive strain, the majority of these mannich bases were more potent than chloroquine against the chloroquine-resistant strain, and mannich bases having one aminomethyl function were generally more potent than double mannich bases. pyronaridine (fig. ) is another example of antimalarial drug that feature a phenolic mannich bases moiety in its structure, but the aminoquinoline scaffold that was typical for amodiaquine and its congeners has been replaced in pyronaridine by a naphthyridine ring system. pyronaridine was first synthesized in at the chinese institute of parasitic disease, and has been used solely in china as treatment for malaria with good results for over years [ ] . beside the difficultly accessible literature in chinese, there are several recent studies that provide information on its physicochemical properties [ ] and adme properties in rats [ ] , interaction with other antimalarials [ ] , potential use in combination therapy with artesunate [ ] , or its mechanism of action based on inhibition of b-hematin formation and subsequent glutathione-dependent hematin degradation process [ ] . based on the observation that substitution of the quinoline scaffold in amodiaquine with a napthyridine ring system in pyronaridine preserved the antimalarial action, g€ orlitzer initiated an ambitious synthetic program aiming at exploring the effect on the antimalarial properties of replacement of quinoline in quinoline-based antimalarials with other fused heterocyclic systems containing one pyridine ring. thus, single (r ¼ h) and double mannich bases (r ¼ ch nr ) structurally related to amodiaquine and pyronaridine, respectively, and featuring pyrido[ , -b]indol- -yl (compounds , x ¼ nh) [ ] , benzofuro [ , - [ , ] naphthyridin- -yl (compound ) [ ] moieties were synthesized and evaluated against chloroquine-sensitive and resistant p. falciparum strains (fig. ) . as expected, all of the mannich bases presented in these studies exhibited good antimalarial activity, and double mannich bases were generally more potent than single mannich bases. however, in spite of the variety of fused heterocyclic systems that were investigated, no compound with antimalarial activity at least comparable to that of chloroquine emerged, with the exception of one pyronaridine analogue ( , x ¼ nh, r ¼ ch nr , nr ¼ -pyrrolidinyl). this candidate had ic ¼ nm against chloroquine-sensitive strain d , ic ¼ nm against chloroquine-resistant strain dd , and its evaluation against plasmodium winckei in a murine malaria model (intraperitoneal dosage of mg/kg) showed that no intact parasite could be noticed after three days of treatment [ ] . the phenolic mannich base moiety in the previously mentioned antimalarial agents can be also found in candidates that were designed through its incorporation into hybrids with dual mode of action. thus, the combination of a tetraoxane and a phenolic mannich base moiety afforded hybrids called mannoxanes (fig. ) , which are active at low nanomolar concentrations and surpass the ability of artesunate, tetraoxane rka and a peroxide/amodiaquine combination to cure malaria in mice at mg/kg [ ] . hybridization of artemisin with a phenolic mannich base moiety led to candidates (fig. ) , which are up to times more potent than artemisin against p. yoelii nigeriensis, could be easily converted into water soluble forms, can be administered orally, are presumed to have good bioavailability, and lack the capability to form neurotoxic metabolite dihydroartemisinin [ ] . hybrids (fig. ) are structurally very similar to , and have been shown to be more active than sodium artesunate against chloroquine-sensitive nf and chloroquine-resistant k p. falciparum strains [ ] . screening of a collection of compounds containing a -hydroxyethylamino motif in their structures for inhibition of parasite growth in red blood cells infected with chloroquine-sensitive nf p. falciparum strain identified a hit compound, whose further elaboration led to several hybrids featuring several types of phenolic mannich moieties as the second pharmacophore [ ] . their evaluation against nf strain, k strain, and the rodent parasite plasmodium berghei showed that the introduction of the phenolic mannich bases moiety resulted in candidates that were very potent against the parasites in the h assay. for example, compound (fig. ) the design of hybrids with dual mode of action has also been broadened to include pyrrole mannich bases. thus, hybrids from -amino- -chloroquinoline and pyrrole mannich bases (fig. ) were evaluated against chloroquine-sensitive d and chloroquine-resistant w p. falciparum strains, but they were all less potent than isoquine against both strains [ ] . however, candidate (r ¼ -clc h , nr ¼ n(c h ) ) was equipotent to chloroquine against d- strain, and four of the candidates were also more potent than chloroquine against w strain [ ] . several candidates from a small library of mannich bases (r ¼ h or ch ) of c- pyrrole analogues of artemisin (fig. ) had antimalarial activity that was superior to both artemisin and chloroquine against chloroquine-sensitive d p. falciparum strain, and selected mannich bases from this library were more potent than the aforementioned reference drugs against chloroquine-resistant k p. falciparum strain [ ] . all of the compounds tested displayed good activity in peter's day suppressive test using mg/kg over days e post-infection, and candidates ( showed complete elimination of parasites. these three mannich bases were further investigated in vivo, and had effective doses between . and . mg/kg that eliminate % of p. berghei in mice, whereas candidate (r ¼ ch , nr ¼ methyl- -piperazinyl) reached % clearance of parasitemia h after the last treatment and increased mouse survival to days, a biological profile that render it superior to clinically used sodium artesunate [ ] . phenolic mannich bases of chalcone analogues (such as , fig. ) have been claimed to possess antimalarial activity against chloroquine-sensitive d p. falciparum strain at concentrations ranging from . to mg/ml [ ] . taking into account the structural similarity between phenolic mannich bases and ketonic mannich bases of a,b-unsaturated ketones, a possible mechanism action for compounds could be the inhibition of thioredoxin fig. . antimalarial phenolic mannich bases derived from miscellaneous substrates. reductase in p. falciparum [ ] . phenolic mannich bases of , dihydroxybenzaldehyde, its thiosemicarbazone and semicarbazone (fig. ) , as well as related quinoline-containing derivatives (fig. ) have been screened against chloroquine-resistant w p. falciparum strain [ ] . only candidates derived from -( chloroquinolin- -yl)piperazine had antimalarial activity, while the remaining mannich bases were inactive. all of the candidates were potent antimalarial agents, with mannich base (nr ¼ -( -chloroquinolin- -yl)piperazin- -yl) featuring two quinoline motifs in its structure being the most potent (ic ¼ nm) [ ] . imines of ( h)-pyridones (r and r ¼ h or ch ) (fig. ) having a phenolic mannich base moiety were active against chloroquine-resistant w p. falciparum strain (ic values between and mm) and atovaquone-resistant fcr p. falciparum strain (ic values as low as mm), but their potency was lower than that of reference drugs chloroquine or atovaquone [ ] . in addition, aminoalkylation of lawsone using various aldehydes and n-alkyl-ferrocenylmethylamines afforded mannich bases (r ¼ alkyl) (fig. ) structurally resembling antimalarial drug atovaquone, but evaluation of three candidates (r ¼ n-c h , n-c h , n-c h ) against p. falciparum showed that they were less potent than atovaquone itself [ ] . because of their self-limiting nature, most viral diseases (with the exception of those caused by human immunodeficiency virus, hiv) do not require specific therapy, although treatments are used to bring the condition or its symptoms to an end more quickly. currently available drugs are designed to target four main groups of viruses, namely herpes viruses, hepatitis viruses, influenza viruses, and hiv. recent investigations in the antiviral activity of mannich bases deal mostly with aminomethylated phenols and aminomethylated isatins, although examples of mannich bases with antiviral properties derived from other types of substrates are available in literature. phenolic mannich base arbidol (fig. ) [ ] is a broadspectrum antiviral agent commonly used in russia to treat acute respiratory viral infections, which acts by inhibiting virus-mediated fusion with target membrane and subsequent blockage of virus entry into target cells [ , ] . using arbidol as lead compound, a group at shenyang pharmaceutical university led by gong initiated a program aiming at investigating structureeanti-hepatitis b relationship for a series of phenolic mannich bases derived from various heterocyclic ring systems. in their first report on this topic, the chinese researchers presented a series of analogues of arbidol (x ¼ so, r ¼ ch , c-c h , r ¼ br) (fig. ) having various substituents r in the aromatic sulfoxide moiety, and presenting amino residues different than the original dimethylamino group in arbidol (e.g., secondary aliphatic amines, nh-azoles belonging to imidazoles and , , -triazoles) in the aminomethyl function in position [ ] . sar investigation was based on the ability of these compounds to inhibit replication of hepatitis b virus (hbv) and the production of surface antigen of the hepatitis b virus (hbsag) and extracellular form of hepatitis b core antigen (hbeag) in hepg . . cells infected with hbv, and showed that half of these mannich bases exhibited inhibitory effects on hbv that were superior to reference drug lamivudine. replacement of the original methyl group at n- in with cyclopropyl led to an increase in antiviral activity, but enhanced the cytotoxicity of these candidates as well, whereas the presence of fluorine or chlorine as substituents in the aromatic sulfoxide moiety resulted in an improvement of the anti-hbv activity. departure from the original phenylmercapto function in arbidol reduced the cytotoxicity of mannich bases (x ¼ so), leaving in the same time the anti-hbv activity practically unchanged. also, the nature of the amino group in the aminomethyl function appears to have little effect on the anti-hbv activity of the mannich bases reported in this study [ ] . the relationship between the presence of a linker (containing two or three atoms between the sulfur atom in the side chain and the phenyl moiety) and the anti-hbv activity of mannich bases (x ¼ soch ch y, y ¼ ch , o or null, r ¼ ch , r ¼ br) was also explored, and candidates having a phenethyl residue proved to be inactive, whereas candidates with a -phenylpropyl residue presented remarkable anti-hbv activity [ ] . a subsequent study [ ] of the anti-hbv activity of arbidol analogues (x ¼ so or so , r ¼ ch , c-c h , r ¼ h) unsubstituted at position of the indole scaffold claimed that the nature of the amino function plays an important role, and that pyrrolidine-and imidazole-containing candidates exhibited better anti-hbv activity than candidates having different amino residues. for another set of mannich bases (x ¼ so or so , r ¼ ch , r ¼ h), the authors suggested that the presence of -methylpiperazin- -yl residue as amino moiety, rather than -imidazolyl, -methyl- -imidazolyl or -piperidinyl, afforded more potent anti-hvb agents [ ] . replacement of the sulfinyl group in the side chain with sulfonyl appeared to enhance the antiviral activity while reducing cellular toxicity of these mannich bases [ ] , although the results in the later study [ ] supported the opposite conclusion. substitution of the indole scaffold with quinoline in mannich bases (x ¼ s or so) (fig. ) resulted in potent inhibition of hbv dna replication ( -to -fold compared to reference drug lamivudine) [ ] . analysis of sar for compounds revealed that the presence of -fluorophenyl moiety linked to sulfur in the side chain at position of the quinoline scaffolds, in conjunction with amine moieties such as pyrrolidinyl, -piperidinyl, -imidazolyl (but not - fig. . phenolic mannich bases inspired by arbidol and useful as anti-hepatitis b agents. methylpiperazinyl or morpholinyl) in the aminomethyl function are required for good anti-hbv activity. evaluation of mannich bases (fig. ) derived from a tetracyclic ring system comprising the quinoline scaffold fused with a benzothiopyrane ring system that has various substitution patterns in the aromatic ring identified a significant number of candidates with good inhibition of hbv replication [ , ] . the search for anti-hbv agents with similar structures continued with the evaluation of a series of phenolic mannich bases (r ¼ ch , c h , ch(ch ) , r ¼ h or f, x ¼ s or so) of -hydroxybenzimidazoles substituted at position with -alkylmercaptophenyl residues (fig. ) [ ] . these compounds exhibited inhibitory effect on the secretion of hbsag that was superior to reference drug lamivudine, but lacked the ability to inhibit the replication of hbv dna in hepg . . cells at concentrations that were inferior to those corresponding to % of their cytotoxicity. the presence of fluorine at position of benzimidazole scaffold and the presence of sulfinyl function appears to be critical for the inhibition of hbsag secretion of mannich bases . evaluation of another series of mannich bases (x ¼ s, so, so , r ¼ h, -f, -och , -ch ) of -hydroxybenzimidazoles (fig. ) showed that the thioethers in this series did not inhibit the replication of hbv dna, but the candidates with -methylpiperazin- -yl as amino moiety were good inhibitors of secretion of hbsag, and the inhibitory effect significantly decreased upon oxidation of sulfur in thioethers to sulfinyl and sulfonyl [ ] . phenolic mannich bases (r ¼ h, -f, -cl, -br, -ch , -och ) derived from bromo- -hydroxyimidazo[ , -a]pyridine- -carboxylates (fig. ) , especially those having -morpholinyl or -methylpiperazin- -yl moieties in the aminomethyl function, inhibited the replication of hbv dna, but had no inhibitory effect on secretion of hbsag or hbeag [ ] . the nature of substituents in the arylmercapto residue modulates the anti-hbv activity, as the decrease of bulkiness of the substituents (from bromine to fluorine, for example) and the presence of lipophilic substituents (e.g., a methyl group) enhance the inhibition of replication of hbv dna, whereas the presence of a -methoxy group further increases the activity. phenolic mannich bases derived from chlorokojic acid (fig. ) have been evaluated against herpes simplex virus type- (hsv- ) and parainfluenza- virus (pi- ). amongst the candidates reported in one study, only two mannich bases had inhibitory concentration in the range of . e . mg/ml against hsv- , whereas all of the compounds were active in various degrees against pi- [ ] . all of the mannich bases derived from arylpiperazines as amine reagents inhibited both hsv- and pi- , and one candidate (r ¼ -( -methoxyphenyl)piperazin- -yl) was as potent as reference drug acyclovir against hsv- [ ] . in addition, candidate (r ¼ -( -chlorophenyl)piperazin- -yl) presented remarkable activity ( . e . mg/ml) against pi- . other aminomethyl derivatives of various phenolic substrates have been tested against different viruses. only one phenolic mannich base (r ¼ nhch ) of norvisnagin (fig. ) was moderately active against hsv- [ ] . evaluation of aminomethylated -hydroxycoumarin derivatives (fig. ) against flaviviridae and other viruses led to mixed results [ ] . phenolic mannich bases (r ¼ h, r ¼ h or ch ) were generally inactive against bovine viral diarrhea virus (bvdv), yellow fever virus (yfv) or respiratory syncytial virus (rsv), and a few other candidates that were active against bvdv or yfw were also cytotoxic. o-alkylated phenolic mannich bases (r ¼ n-c h , r ¼ h or ch ), and especially candidates having a methyl group at position of the coumarin ring system, were active against bvdv, but they were inactive against yfv or rsv. o-acylated phenolic mannich bases (r ¼ coc h , r ¼ h or ch ) were generally inactive against all three viruses, but one candidate (r ¼ coc h , r ¼ h, nr ¼ , , , -tetrahydroisoquinolin- -yl) had a remarkable activity against rsv, comparable to that of reference drug -azauridine, and had also very low toxicity. compounds were not active against a panel of viruses comprising hiv- , coxsackievirus b , sb- strain of marek's disease virus, vesicular stomatitis virus and a reovirus. in addition, screening of an extensive library of compounds identified mannich bases of -chloro- -hydroxyquinoline (fig. ) as reactivators of latent hiv- , which could prove helpful in eradicating the latent reservoir of hiv- in resting memory cd þ t cells, either alone or in combination with other treatments [ ] . antiviral activity of mannich bases of isatin and its derivatives is the topic of several investigations. screening of twelve mannich bases derived from semithiocarbazones of -nitroisatin (fig. ) against a panel of other viruses afforded no compound with antiviral properties, with the exception of candidate (x ¼ o, r ¼ allyl), which had weak activity against yfv (strain d) at subtoxic concentrations. this candidate was more potent than reference drug ribavirin, but was also more cytotoxic [ ] . based on results of molecular studies aiming at designing inhibitors of hiv reverse transcriptase, a series of mannich bases (fig. ) have been synthesized [ ] . candidates having secondary aliphatic amino moieties in the aminomethyl function showed to % inhibition of the enzyme, whereas mannich bases (nr ¼ nhc h ) showed no inhibition. aminomethylation of schiff bases obtained from -substituted isatins (r ¼ f, cl, f) and lamivudine using fluoroquinolones (r ¼ ethyl, cyclopropyl; r ¼ h, ch ) as amine reagents gave mannich bases (fig. ) , which were less potent against hiv than the parent schiff bases, and the most potent candidates (r ¼ f) were also -fold less potent than lamivudine itself against hiv [ ] . starting from schiff bases of trimethoprim, mannich bases (fig. ) were obtained using fluoroquinolones as amine reagents. their evaluation against hiv and hepatitis c virus (hcv) showed that mannich bases (r ¼ cl) inhibited replication of hiv in mt- cells at effective concentrations (ec ) ranging from . to mg/ml, and most compounds were active against hcv rna replication ( % inhibition at mg/ml) [ ] . mannich base (r ¼ ch , nr ¼ -( chlorophenyl)piperazin- -yl) and three mannich bases (r ¼ ch ) derived from fluoroquinolones as amine reagents showed inhibition against replication of hiv in mt- cells at ec ranging from . to . mg/ml [ ] . in addition, all of the compounds (r ¼ ch ) were active against hcv rna replication ( % inhibition at mg/ml) [ ] . furthermore, a study reports candidate (r ¼ h, nr ¼ -( -nitrophenyl)piperazin- -yl) as inhibitor of japanese encephalitis virus and west nile virus in vitro, and a remarkable inhibitor of japanese encephalitis virus in a murine model [ ] . investigations into the antiviral activity of mannich bases derived from substrates other than phenols or isatins are available in literature as well. the majority of mannich bases (r ¼ cl, och ) (fig. ) , obtained through n-aminomethylation of thiazolidine- , -diones with morpholine, piperidine and variously substituted piperazines, showed no activity against severe acute respiratory syndrome (sars) coronavirus [ ] . also, all of these mannich bases had antiviral activity against types a and b of influenza virus, but virus inhibition occurred almost at cytotoxic concentration. aminomethylation of the amide function in tetracyclines using fluoroquinolones as amine reagents afforded mannich bases (fig. ) , and four candidates derived from tetracycline and one derived from minocycline were found to inhibit hiv replication with ec values below mm, while their toxicity against mock infected cem cell line was greater than mm [ ] . in addition, all mannich bases showed moderate inhibition of both -processing and strand transfer steps of hiv- integrase. evaluation of efavirenz mannich bases (fig. ) led to the discovery of three candidates that were at least as potent as the parent compound against hiv [ ] . a large number of indole mannich bases (r ¼ aminomethyl, -amino- -propyn- -yl) (fig. ) derived from a pentacyclic core, along several acetylenic mannich bases with the same core, have been claimed to be active against hcv and members of the flaviviridae family of viruses [ ] . none of the mannich bases (fig. ) derived from indophenazine as substrate and sulfonamides, anthranilic acid or aminopyridine as amine reagents either showed any activity against hiv above their cytotoxic concentrations, but some presented weak activity against hsv, vsv, or vaccinia virus [ ] . anticonvulsants are drugs used in the treatment of seizures in epilepsy, a common chronic neurological disorder that affects around million people of all ages worldwide. the discovery of novel antiepileptic drugs relies either on rational design based on the use of well-known pharmacophores (e.g., imides), or on random screening of libraries of compounds. ketonic mannich bases (r ¼ -substituted aryloxy, substituted arylthio, r ¼ h, nr ¼ n(c h ) ; r ¼ fluorophenyloxy, r ¼ h, nr ¼ dimethylamino, -piperidinyl, morpholinyl, -pyrrolidinyl) (fig. ) and the related piperidinols (r ¼ -substituted aryloxy, r ¼ c h ) (fig. ) were examined for anticonvulsant activity in the maximal electroshock (mes) and subcutaneous pentylenetetrazole (scptz) screens [ ] . none of the compounds provided protection in the scptz screen, but several candidates and demonstrated anticonvulsant activity in the mes screen below their neurotoxic levels ( mg/kg). four ketonic mannich bases of type derived from acetophenone or hydroxyacetophenone as substrates and common secondary aliphatic amines as amine reagents, as well as the corresponding azines (fig. ) , were assessed as anticonvulsants using the same two tests. the results showed that anticonvulsant activity of candidates was superior to that of azines , and compounds derived from -hydroxyacetophenone were active at a dose of mg/kg in the mes test, but no candidate showed anticonvulsant activity in the cptz test [ ] . bis-mannich bases (r ¼ h, -cl, -ch , thienyl, r ¼ c h ) and the corresponding piperidinols were protective in the mes test at mg/kg and/or above, while candidate (r ¼ -cl, r ¼ c h ) was protective in the scmet test at mg/kg after h [ ] . the presence of a -chlorophenyl moiety appears to be important for the anticonvulsant activity of these compounds, and analogues having the same moiety were identified as good anticonvulsant agents in a previous study [ ] . anticonvulsant activity of phenolic mannich bases of hydroxy- -pyranones has been investigated extensively by aytemir's group. screening of derivatives of allomaltol having a substituted piperazin- -ylmethyl group (fig. ) using mes and scptz tests showed that candidate (r ¼ -cf c h ) provided excellent protection against pentylenetetrazole-induced seizures, but was neurotoxic at high dose ( mg/kg), while candidate (r ¼ -clc h ) had high anticonvulsant activity in mes test at all doses after min, without being neurotoxic [ ] . evaluation of another series of mannich bases of allomaltol revealed that candidate (r ¼ , -(ch ) c h ) was active in scptz test at mg/kg after h, along with candidate (r ¼ -clc h ), which was active in mes test at mg/kg after h [ ] . although mannich bases of allomaltol obtained using morpholine or -( -piperidinyl)piperidine as amine reagents had no anticonvulsant activity [ ] , a subsequent study dealing with novel mannich bases generated from piperidines as amine reagents reported that candidates (r ¼ -ch , r ¼ -ch ; r ¼ -hoch ch , r ¼ h; r ¼ -c h ch , r ¼ h) (fig. ) were protective in scptz test, while only candidate (r ¼ -hoch ch , r ¼ h) provided protection in mes test at mg/kg [ ] . use of other piperidines as amine reagent in the mannich reaction with allomaltol as substrate led to candidates (r ¼ -(un)substituted phenyl, r ¼ oh, cn, coch ), and two of these mannich bases (r ¼ -brc h , r ¼ oh; r ¼ -clc h , r ¼ oh) were active in scptz test at mg/kg after h, while all of them were active in mes test either after . h or after h [ ] . in contrast, when allomaltol was replaced with kojic acid as substrate in the mannich reaction, but the same piperidines were used as mine reagents, the resulting candidates (nr ¼ , -disubstituted piperidines) (fig. ) were all active in scptz test either after . h or after h, but only two of them (r ¼ -brc h , r ¼ oh; r ¼ -clc h , r ¼ oh) were active in the mes test at mg/kg after . h, and one candidate (r ¼ c h , r ¼ coch ) was active at any dose after h [ ] . mannich bases (nr ¼ -substituted piperazines) of kojic acid as substrate and piperazines as amine reagents were generally better anticonvulsants than analogous mannich bases of allomaltol, or than mannich bases derived from kojic acid and , disubstituted piperidines [ ] . the authors tentatively explain the enhanced anticonvulsant activity of mannich bases of kojic acid compared to the activity of mannich bases of allomaltol through the possible formation of an extra hydrogen bond in the former compounds. however, mannich bases derived from chlorokojic acid were also good anticonvulsants, and they present, like mannich bases of allomaltol, only one hydrogen bond in their structure [ ] . hydantoin represents the core structure of the old generation of antiepileptic drugs, such as phenytoin, and substitution with an aminomethyl moiety was shown to improve activity against mes seizures in mice [ ] . evaluation of a library of n-mannich bases (fig. ) derived from -cyclopropyl- -arylhydantoins having -substituted piperazines in the aminomethyl function showed that the majority of candidates were effective in the mes or/and scptz screens, and quantitative studies in rats after oral administration showed that three mannich bases (r ¼ h, r ¼ c h , c h ch , -ch c h ch ) were more potent than phenytoin in mes test [ ] . results from another study [ ] showed that candidates were generally more active in mes test than in scptz screen, and chlorine-substituted mannich bases (r ¼ cl) were generally less active than those unsubstituted in the aromatic ring (r ¼ h), whereas candidates derived from , , , -tetrahydroisoquinoline as amine reagent in the mannich reaction were less potent than those derived from morpholine or piperazines. compared to candidates obtained from arylpiperazines as amine reagents, mannich bases having alkylene, alkenylene, carbonyl or ester linkers between the piperazine moiety and phenyl ring presented enhanced anticonvulsant protection to pentylenetetrazole-induced seizures, which was noticeable not only after . h, but after h as well [ ] . taking into account the established anticonvulsant properties of many spirohydantoins [ e ], mannich bases (x ¼ nh) of spirohydantoins (fig. ) have been synthesized and evaluated as anticonvulsants, and while some of them were effective in mes or/ and scptz screens, and were more potent than reference drug phenytoin, their high neurotoxicity precluded further testing [ ] . besides hydantoin, succinimide presents the structural requirements for the core structure of good anticonvulsant agents (namely, a nitrogen-containing heteroatomic system with a least one carbonyl group), and the well-established drug ethosuximide is an example for this class of antiepileptic drugs. the group of obniska has been developing novel anticonvulsants for a long time, and mannich bases derived from variously substituted succinimides has been one of the classes of compounds that provided some of the most interesting results reported by these researchers. based on the significant number of candidates that have been synthesized, aminomethylated derivatives of phenylsuccinimides (fig. ) [ ] , generally showed protection in mes screen, but some of them were also effective in scptz screen. moreover, a few candidates presented activity not only . h after administration, but also after or h, which is indicative of quick onset and long duration of anticonvulsant activity. mannich bases of -arylsuccinimides derived from other secondary aliphatic amines, such as morpholine, benzylpiperidine, -cyclohexylpiperazine, were generally effective in both screens [ , ] . despite the large number of compounds evaluated in these studies, no consistent sars could be established. compounds with good anticonvulsant properties emerged from almost all of these studies, but none of these anticonvulsant mannich bases was deemed sufficiently promising to advance to clinical studies. aminomethylated spirosuccinimides have also been investigated as anticonvulsants. candidates (x ¼ ch ) derived from -( -trifluoromethylphenyl)piperazine and -( chlorophenyl)piperazine as amine reagents in the mannich reaction were the most potent anticonvulsants in this series in mes test, and replacement of substituted aryl with -hydroxyethyl rendered the candidates active in both tests [ ] . the activity of mannich bases of simpler spirosuccinimides (fig. ) appears to depend [ ] and candidates (n ¼ or ) derived from -( methylphenyl)piperazine [ ] were devoid of anticonvulsant activity. on the other hand, mannich bases (n ¼ or ) obtained from -( -trifluoromethylphenyl)piperazine as amine reagent were efficient in mes screen, but not in scptz screen [ ] . evaluation as anticonvulsant agents of mannich bases of succinimides , disubstituted with identical ( (fig. ) has also been the topic of several recent papers [ e ]. generally, mannich bases obtained from , diphenylsuccinimide were more potent than those obtained from -alkyl- -phenylsuccinimides, which, in turn, were more potent than mannich bases derived from , -dialkylsuccinimides, which were actually inactive in most cases. candidates generated from arylpiperazines as amine reagents in the mannich reaction were efficient only in mes test, and the nature and position of the substituent in the aromatic ring attached to piperazine modulate the anticonvulsant activity of these compounds. however, mannich bases derived from -( -hydroxyethyl)piperazine or benzylpiperidine were efficient in both screens [ ] . the effect of mannich bases with potent anticonvulsant activity on na v . sodium channel currents was also investigated as a potential mechanism of action for these compounds, and the results showed that the anticonvulsant activity of these candidates correlates nicely with their effectiveness as sodium channel blockers [ , ] . in addition, there was no direct correlation between anticonvulsant properties and -ht a , -ht a , -ht a and/or -ht serotonin receptor affinity [ , ] . besides hydantoins and succinimides, other substrates featuring the ureido motif in their structure have been aminomethylated with a view to obtain anticonvulsant agents. barbituric acid and its thio analogue have been subjected to the mannich reaction with two amine reagents having a quinazolinone moiety, and the resulting candidates and (x ¼ o or s, r ¼ -br, -i, , -br ) (fig. ) provided good protection ( e %, and e %, respectively) in both mes and scptz screens at a dose of mg/kg, while lacking neurotoxicity, or sedative and hypnotic effects [ ] . mannich bases of -aryl- -thioxo- , -dihydroquinazolin- ( h)-one (fig. ) showed significant anticonvulsant activity in mes test, as some of these candidates afforded results comparable to those obtain for reference drug phenytoin [ ] . several studies concerning the anticonvulsant activity of fused seven-membered ring systems containing two heteroatoms are available in literature. aminomethylated , -dihydro- , benzoxazepines (fig. ) provided protection in the mes test in the range of e % at a dose of mg/kg; candidate (r ¼ -och - -oh, r ¼ -och ) was equipotent to reference drugs phenytoin and lamotrigine in mes screen, and was also equipotent to reference drug valproate in scptz screen [ ] . another study allowed a direct comparison between the anticonvulsant activities of analogous aminomethylated , -dihydro- , -benzoxazepines (x ¼ o) and , -dihydro- , benzothiazepines (x ¼ s) (fig. ) [ ] . at a dose of mg/ kg, the latter provided more protection ( e %) in mes test than the former, with mannich base (x ¼ s, r ¼ -cl, r ¼ och ) being the most active compound in this series. ketonic mannich bases (r ¼ h, cl, no ; r er ¼ r er ¼ (ch ) or r ¼ r ¼ r ¼ ch , r ¼ h) having a , -dihydro- , benzodiazepine scaffold as the amino moiety (fig. ) were evaluated using a model in which the seizures were induced chemically, and some of these compounds provided protection after . h, whereas only candidates derived from acetophenone (r ¼ h) provided protection up to h [ ] . inflammation is part of the complex, nonspecific immune response of vascular tissue that occurs in reaction to any type of injury, such as pathogens, irritants, damaged cells, etc. the initial response of the body to harmful stimuli is initiated by cells already present in all tissues, and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from blood into the injured tissue, where they release a series of cell-derived mediators, triggering afterwards a cascade of biochemical events that propagate the inflammatory response. nonsteroidal antiinflammatory drugs (nsaids) are commonly used for treatment of inflammation, along with its symptoms (fever, pain, swelling), but they present significant side effects after long-term usage, such as gastrointestinal lesions, kidney injury, and cardiovascular risk. along with many other classes of compounds, mannich bases of various structures have been investigated as part of the ongoing search for novel anti-inflammatory drugs. ketonic mannich bases (r ¼ h, ch , so ch ) derived from arylidenecyclohexanones and aromatic amines (fig. ) have been evaluated using xylene-induced ear swelling test and carrageenan-induced paw edema, and candidate (r ¼ h) was superior to reference drug ibuprofen in both tests, whereas several candidates (r ¼ ch ) were less efficient, but still more effective than ibuprofen, in the same tests [ ] . administration of ketonic mannich base (fig. ) to rats with a chronic inflammatory process induced by insertion of cotton pellets led to increased phagocytic capacity of peripheric neutrophils, enhanced activity of the serum complement system, and higher catalase activity, while a slightly decrease in the activity of superoxide dismutase was observed [ ] . a few double mannich bases of , dimethoxybenzofuran- ( h)-one (fig. ) inhibited the production of pro-inflammatory cytokines tnf-a and il- by e % at a concentration of mm, but they were more cytotoxic than reference drug dexamethasone, which had comparable effects with candidates on the production of the same cytokines at only mm [ ] . investigation of the anti-inflammatory activity of mannich bases (r ¼ h, f, cl, br, or ; r ¼ och and oc h -n) having amino acids isoleucine and methionine as amine moieties (fig. ) was performed using both carrageenan-induced paw edema model and pellet-induced granuloma model, and resulted in the discovery of two anti-inflammatory agents (r ¼ och and oc h -n; r ¼ ch ch sch ) derived from methionine [ ] . at a dose of mg/kg, these two mannich bases had % and %, respectively, of the efficiency of the reference drug diclofenac (dose of mg/kg) on the reduction of paw swelling. at the same dose, these two candidates also showed % and %, respectively, of the activity of reference drug indomethacin (dose of mg/kg) on chronic inflammation. a continuation of this study was undertaken with a view to broaden the nature of amino acids (cysteine, threonine, methionine, isoleucine, asparagine, and glutamine) as amine moieties in mannich bases , and also the nature of the alkoxy substituents in the aromatic ring, but these novel candidates had no anti-inflammatory activity at mg/kg, with the exception of two candidates (r ¼ oc h ; r ¼ ch sh, ch conh ), which displayed mild activity in the acute inflammation model [ ] . only one example of mannich bases obtained from simple phenols as potential anti-inflammatory agents is available in recent literature. thus, evaluation of four phenolic mannich bases (fig. ) using formalin-induced acute inflammation model in mice revealed that one candidate had reliable activity upon parenteral administration ( mg/kg), whereas reference drug diclofenac sodium exhibited only two-thirds of its activity, albeit at mg/kg [ ] . phenolic mannich bases of resveratrol analogues containing a pyridinyl moiety and either one, two or three aminomethyl residues (fig. ) were tested using xylene-induced ear edema in mice at mg/kg, and two of them had % of the efficiency of reference drug ibuprofen, whereas the remaining candidates were less potent [ ] . the anti-inflammatory activity of mannich bases of polyhydroxylic phenols was also investigated. at a dose of mg/kg, two candidates (nr r ¼ -piperidinyl and -morpholinyl) derived from , -diacetylresorcinol (fig. ) had a slightly lower activity than that of reference drug indomethacin in carrageenan-induced paw edema [ ] . three candidates (r ¼ -cl, nr ¼ -methylpiperazin- -yl; r ¼ -br, nr ¼ piperidinyl or -pyrrolidinyl) (fig. ) were more efficient at inhibiting the production of tnf-a at a concentration of mm than reference drug dexamethasone at mm [ ] ; under the same experimental conditions, most mannich bases in the study were also more efficient at inhibiting the production of il- than dexamethasone. the same researchers also investigated the action on enzymes that are involved in inflammation (such as cyclooxygenases (cox), trypsin and b-glucuronidase) of phenolic mannich bases (r ¼ -f, -cl, -br, -f, -cl, -br) of chalcone analogues (fig. ) having the same substitution pattern in ring a as candidates [ ] . with one exception, none of the candidates inhibited the activity of trypsin, whereas most mannich bases in this study inhibited the activity of b-glucuronidase. candidates derived from chalcone analogues having a halogen at position of the b ring were generally more potent than those derived from chalcone analogues having a halogen at position of the b ring. in addition, a few mannich bases were poor inhibitors of cox- , but excellent inhibitors of cox- , and the majority of the candidates were more efficient at inhibiting cox- than reference drug aspirin. in a different study [ ] , two out of four phenolic mannich bases of other chalcone analogues were found to reduce rat paw edema induced by carrageenan, and although these compounds were found to be good inhibitors of trypsin this time, no satisfactory correlation with their antioxidant, free radical scavenging, or lipooxygenase inhibition could be established. because inducible nitric oxide synthase (inos) generates high levels of nitric oxide that modulates inflammations through multiple pathways, the inhibition of this enzyme by phenolic mannich bases of heterocyclic analogues of chalcones with various structures was also investigated. this type of phenolic mannich bases was found to strongly inhibit no production, with ic values ranging between . and . mm [ ] . benzoxazines (fig. ) , easily obtained from hydroxyacetophenones through a double mannich reaction, inhibited the swelling of rat paw in various degrees when administered orally in doses equimolar to mg/kg of indomethacin [ ] ; compared to indomethacin, one candidate (r ¼ -f) was more efficient, while another candidate (r ¼ -och ) was equipotent. chalcone analogues (r ¼ -och or -ch ) derived from these benzoxazines (fig. ) had a more pronounced antiinflammatory activity compared to candidates , and all of them displayed e % of the efficiency of reference drug indomethacin [ ] . phenolic mannich bases of naturally-occurring benzopyranones have also been tested as anti-inflammatory agents. most mannich bases derived from hydroxycoumarin (fig. ) were superior to reference drug indomethacin at reducing rat paw edema induced by carrageenan, and two candidates (nr ¼ -morpholinyl, -piperazinyl) were . times more efficient than indomethacin at mm without significant inhibition of cox- [ ] . irisolidone is an isoflavone which was isolated from pueraria spp., and was found to exert its antiinflammatory action through suppression of inos gene expression and pro-inflammatory cytokines in activated microglia [ ] . chemical modification of irisolidone by means of the mannich reaction using primary aliphatic and aromatic amines as amine reagents led to candidates ( fig. ) with enhanced ability to inhibit nitric oxide production compared to parent irisolidone [ ] . anti-inflammatory activity of mannich bases of , -dihydro- , , -triazole- -thiones has also been investigated. several candidates f (table ) have been evaluated using carrageenan- induced rat paw edema model, and the activity of one of them ( f, r ¼ -ch c h , nr ¼ -morpholinyl) was comparable to that of reference drug ibuprofen [ ] . mannich bases i with secondary aliphatic amino moieties (dimethylamino, diethylamino, -pyrrolidinyl) in the aminomethyl function were as efficient as reference drug celecoxib in reducing rat paw edema both after h and after h, and they also had lower ic values (around nm) for the inhibition of cox- compared to celecoxib ( . nm) [ ] . as far as -substituted -aminomethyl- -arylideneamino- h- , dihydro- , , -triazole- -thiones (table ) are concerned, the majority of mannich bases c had, upon administration of either mg/kg or mg/kg, a lower anti-inflammatory activity in carrageenan-induced rat paw edema model than reference drug indomethacin ( mg/kg), although one candidate ( c, r ¼ , -cl c h , nr ¼ -phenylpiperazin- -yl) inhibited edema formation at the higher dose almost as efficiently as indomethacin [ ] . starting from ibuprofen, two separate studies reported the synthesis and anti-inflammatory activity of mannich bases having different arylideneamino moieties at position of the , -dihydro- , , -triazole- -thione scaffold (fig. ) . the most potent compounds in this series were those having either a -morpholinyl or a -methylpiperazin- -yl residue in the aminomethyl function. when common -substituted benzaldehydes (r ¼ -clc h , -ch c h , -brc h , -no c h ) were used to generate the azomethine moiety, several candidates had anti-inflammatory activity comparable with that of reference drug diclofenac, and were generally more efficient than ibuprofen at every time interval up to h [ ] . also, the most potent compounds in this series were those having either a -morpholinyl or a -methylpiperazin- -yl residue in the aminomethyl function. on the other hand, when aryl- -formylsydnones were used to generate the azomethine moiety, the resulting mannich bases were consistently more potent than the analogues in the previously mentioned series of compounds, but all of these candidates were less efficient than reference drug indomethacin in reducing rat paw edema [ ] . several mannich bases h showed a peak in their antiinflammatory activity at h post-injection (from % to % edema inhibition compared to indomethacin), whereas the antiinflammatory activity of other candidates h peaked at h post-injection (from % to % edema inhibition compared to indomethacin) [ ] . all of the mannich bases (fig. ) had only moderate anti-inflammatory activity, with the most potent compound in the series showing approximately % of the efficiency of reference drug indomethacin [ ] . a few mannich bases and other structurally related aminomethylated , -dihydro- , , -triazole- -thiones (fig. ) reduced significantly the inflammatory response (maximum inhibition between and %) compared to reference drug diclofenac (inhibition between and %), and some of them presented fast onset of their antiinflammatory action, while others had a long-lasting anti-inflammatory effect [ ] . the anti-inflammatory activity of mannich bases of , -dihydro- , , -oxadiazole- -thiones has been scarcely examined. several mannich bases a (table ) , especially those derived from ibuprofen as starting material for the , , -oxadiazole- -thione substrate and generated from -arylpiperazines as amine reagents in the aminomethylation step, were as efficient as reference drug diclofenac at reducing rat paw edema [ ] . also, mannich base (fig. ) had anti-inflammatory activity comparable to that of reference drug indomethacin in carrageenan-induced rat paw edema test [ ] . several papers published by g€ okhan's group have reported the anti-inflammatory activity of mannich bases of benzoxazolinones. one of their studies showed that candidates (r ¼ ch , r ¼ -or -clc h co) having an acyl moiety at position of the benzoxazolidinone scaffold were more potent than their analogues (r ¼ -or -clc h co, r ¼ h) with the same acyl group at position (fig. ); in addition, the antiinflammatory activity of two of these candidates was equipotent to that of reference drug indomethacin [ ] . substitution with fluorine in the phenyl ring of the acyl moiety appears to be less favorable for the anti-inflammatory activity of candidates (r ¼ h, r ¼ difluorobenzoyl) [ ] . mannich bases (r ¼ ch , r ¼ h) having -arylpiperazine residues in the aminomethyl function were all less potent than reference drug indomethacin in carrageenan-induced rat paw edema test, but the results suggest that the nature of the substituent in the aryl group of piperazines plays an important role in the anti-inflammatory activity of these compounds [ ] . a few mannich bases (r ¼ no , r ¼ h) had an anti-inflammatory effect comparable to that of reference drug indomethacin h after administration, but their efficiency in reducing the swelling reached a plateau afterwards, whereas that of indomethacin continued to increase in time [ ] . two studies have reported the anti-inflammatory activity of mannich bases of isatins. aminomethylation of derivatives of methylisatin thiosemicarbazone afforded mannich bases (r ¼ r ¼ h or ch , nr ¼ secondary aliphatic amines) (fig. ) , which showed moderate anti-inflammatory activity ( e % inhibition of edema at a dose of mg/kg) compared to reference drug diclofenac ( % inhibition of edema at a dose of mg/kg) [ ] . several mannich bases (fig. ) , which were obtained from a derivative of isatin hydrazone, had anti-inflammatory activity comparable to that of reference drug diclofenac, and the highest level of activity was observed after h [ ] . a correlation in this series between anti-inflammatory activity and the nature of the amino moiety in the aminomethyl function was noticed, as the activity decreased with the increasing lipophilicity of the amino group. a series of n-mannich bases of benzimidazole derivatives (fig. ) were evaluated as anti-inflammatory agents using formalin-induced paw edema method. compared with reference drug diclofenac ( mg/kg), they all caused significant reduction of paw edema, albeit at different doses ( mg/kg for mannich base (r ¼ h or ch ) and mg/kg for mannich bases of styrylbenzimidazole) [ ] . also, several mannich bases (r ¼ c h ), derived from both secondary aliphatic amines and primary arylamines, showed moderate anti-inflammatory activity h after administration ( e % of the activity of reference drug aspirin at the same dose of mg/kg) [ ] . mannich bases obtained from substrates other than those mentioned so far have been examined as anti-inflammatory agents as well. thus, n-mannich bases (nr ¼ -pyrrolidinyl, piperidinyl, -morpholinyl) of pyrimido[ , -a]azepine derivatives (fig. ) had moderate activity ( e % reduction of edema compared to that recorded for reference drug diclofenac) [ ] , whereas n-mannich bases of a tricyclic system derived from pyrimido[ , -a]azepine (fig. ) were less potent (anti-inflammatory activity of approximately % of that of diclofenac) [ ] . two aminomethylated pyridazinones bearing a thiophene ring, namely compounds and (fig. ) , showed % and % reduction of rat paw edema, respectively, compared to reference drug indomethacin [ ] . even when administered in a higher doses relative to that of reference drug, none of the mannich bases (r ¼ h, ch , och , cl, x ¼ o or ch ) derived from isoxazolines (fig. ) was as efficient as indomethacin in reducing rat paw edema [ ] . out of the two acetylenic mannich bases (x ¼ o or ch ) with a betulonic acid scaffold (fig. ) that were investigated as antiinflammatory agents, only the piperidine derivative was almost as efficient as reference drug indomethacin in reducing rat paw edema; the morpholine analogue had only half of the activity of the piperidine mannich base [ ] . as pain is a symptom of inflammation, the anti-inflammatory and analgesic activities of novel candidates are usually evaluated at the same time. therefore, it is not surprising that many of the studies that report the anti-inflammatory activity of mannich bases also offer information on their analgesic potential. several ketonic mannich bases (r ¼ h, ch , so ch ) derived from -arylidenecyclohexanones and aromatic amines (fig. ) were equipotent to reference drug ibuprofen in both acetic acid-induced writhing test and hot plate test [ ] . one of the most potent candidate was compound (r ¼ r ¼ h) derived from ptoluidine, but a few mannich bases derived from -( methylsulfonylbenzylidene)cyclohexanone were also efficient as pain relievers. phenolic mannich bases of -and -naphthols substituted in either rings of naphthalene system with various functions, which were synthesized using preformed aminomethylation reagents (e.g., imonium salts obtained from aromatic aldehydes and secondary aliphatic amines), were claimed to have analgesic activity [ ] . the claim is difficult to assess, because only two candidates have been evaluated, and no comparison with established analgesics was provided. in addition, while candidate (x ¼ ch ) (fig. ) was efficient ( % inhibition of the writhing reaction), the second candidate (x ¼ o) offered only modest protection against pain ( % inhibition of the writhing reaction). mannich bases of , -dihydro- , , -triazole- -thiones with analgesic activity have been reported in several studies. candidates f (r ¼ -ch c h , nr ¼ -morpholinyl; r ¼ -ch oc h , nr ¼ -methylpiperazinyl) ( table ) , which had good antiinflammatory activity, were also tested for analgesic activity; their efficiency, determined using tail flick method in albino rats, was comparable to that of reference drug ibuprofen [ ] . several mannich bases (fig. ) showed analgesic activity (tail flick latency between . and . s) that was comparable with that of reference drug pentazocine (tail flick latency of . s), while the rest of the compounds were moderately active [ ] . mannich bases (fig. ) were less efficient in the tail flick test, as the reaction time for the most potent compound in the series was approximately % of the latency provided by reference drug pentazocine [ ] . two candidates a (table ) , both derived from ibuprofen as starting material for the , -dihydro- , , oxadiazole- -thione substrate and generated using either ethyl piperidine- -carboxylate or -( -fluorophenyl)piperazine as amine reagents in the aminomethylation step, were more efficient analgesics than reference drug diclofenac in hot plate test [ ] , while mannich base (fig. ) was equipotent to reference drug diclofenac in acetic acid-induced writhing test [ ] . analgesic activity of mannich bases of -benzoxazolinones was also determined for the same candidates that were investigated for anti-inflammatory activity. in the library of mannich bases derived from -benzoxazolinones (fig. ) carrying a benzoyl moiety in the aromatic ring (r or r ¼ substituted benzoyl), no significant difference in analgesic activity between the benzoylated and the -benzoylated analogues could be observed [ ] . although most candidates provided moderate to low protection in either tests used to determine their analgesic activity (namely acetic acid-induced writhing test and p-benzoquinoneinduced abdominal constriction test), two mannich bases derived from -(substituted benzoyl)- -benzoxazolinones were found to be as efficient as reference drug aspirin. thus, analgesic activity of mannich base (r ¼ h, r ¼ , -f c h co, nr ¼ -( acetylphenyl)piperazin- -yl) was equipotent to that of aspirin [ ] , while analgesic activity of mannich base (r ¼ ch , r ¼ -clc h co, nr ¼ -( -fluorophenyl)piperazin- -yl) was slightly poorer than that of aspirin [ ] . in the series of mannich bases derived from -methyl- -benzoxazolinone, several compounds showed better analgesic activity compared to reference drug aspirin, and the analgesic activity for all the compounds was consistently higher than their anti-inflammatory activity, suggesting that these mannich bases might exert their analgesic activity centrally [ ] . two mannich bases derived from -nitro- benzoxazolinone were also found to possess analgesic activity comparable to that of aspirin [ ] . mannich bases of derivatives of -methylisatin thiosemicarbazone (fig. ) were moderately efficient ( e % protection) in preventing acetic acid-induced writhing in mice at a dose of mg/kg, while reference drug diclofenac provided % protection in the same test at a dose of only mg/kg [ ] . mannich bases of isatin hydrazone (fig. ) carrying a quinazoline moiety and derived from acyclic secondary aliphatic amines (dimethylamine, diethylamine) were the most potent; their analgesic effect was superior or comparable to that of reference drug diclofenac h after administration, but it decreased rapidly afterwards [ ] . n-mannich bases (fig. ) obtained from benzimidazole using dimethylamine or diethylamine as amine reagents were found to be moderate analgesics at a dose of mg/kg relative to paracetamol ( mg/kg), while mannich base derived from methylbenzimidazole as substrate and diphenylamine as amine reagent was a poor analgesic candidate [ ] . owing to their toxicity, mannich bases derived from -styrylbenzimidazole were administered at a lower dose ( and mg/kg), and their analgesic activity ranged from moderate (nr ¼ -morpholinyl) to very good (nr ¼ diethylamino or -piperidinyl) when compared to paracetamol [ ] . n-mannich bases of -ethylbenzimidazole had analgesic activity comparable to that of reference drug pentazocine only when administered in doses that were times greater than that of pentazocine [ ] . other mannich bases derived from -substituted benzimidazoles (r ¼ ch nhnhc h or -hoc h ) also showed moderate to good analgesic activity in acetic-acidinduced writhing test ( e % of the analgesic activity of diclofenac at the same dose) [ ] . evaluation of a first series of n-mannich bases derived from h- , -dimethyl- -oxo- , -dihydroisothiazolo[ , -b]pyridine ( fig. ) led to identification of weak to moderate analgesic agents [ ] . amongst them, candidates derived from -aryl-or benzylpiperidine and those having a -( -substituted phenyl) piperazine as the amine moiety were the most efficient analgesics in writhing and hot plate tests. a second series of mannich bases was subsequently synthesized, and some of the candidates displayed significant activity in the writhing test, with analgesic activity to times more potent than that of aspirin and . to times weaker than that of morphine, used as reference drugs in the study [ ] . excess of reactive oxygen species produced in living organisms can cause oxidative stress and damage cells by initiating chain reactions that lead to lipid peroxidation, dna damage or protein oxidation. besides the complex system of antioxidant metabolites and enzymes that naturally prevent cell damage, exogenous antioxidants may sometimes be required to keep reactive oxygen species at an optimum level. use of the mannich reaction to generate novel chemical entities capable of acting as antioxidants is presented in this section. ketonic mannich bases and (fig. ) , which were obtained from arylamines as amine reagents and -acetylcoumarine and -acetylbenzofuran as substrates, respectively, were tested for antioxidant activity by evaluating their ability to scavenge , diphenyl-picrylhydrazyl (dpph) radical [ ] . the most efficient candidates in each series, namely (r ¼ h) and (r ¼ n(ch ) ), showed moderate potency in scavenging dpph radical (approximately %) compared to standard butylated hydroxytoluene ( %). substitution of the aromatic ring with electron-withdrawing groups appears to reduce the antioxidant ability of these mannich bases. a large number of phenolic mannich bases have been reported in the literature as potential antioxidants. antioxidant activity of two phenolic mannich bases derived from thymol (fig. ) has been assessed by means of xanthine oxidase inhibition test for the cell-free system, and by inhibition of lipid peroxidation using ratliver homogenate [ ] . both candidates, and especially mannich base having morpholine as amine moiety, presented enhanced antioxidant activity in both tests compared to parent thymol. three phenolic mannich bases (nr r ¼ -ch oc h nh, piperidinyl and -morpholinyl) derived from , diacetylresorcinol (fig. ) , designed primarily as antiinflammatory agents, were also evaluated for their ability to inhibit lipid peroxidation; results showed that these candidates were more efficient than indomethacin at preventing lipid peroxidation, and that the antioxidant activity may be correlated with their ulcerogenic activity [ ] . only three mannich bases (fig. ) , all of them having piperidine as the amine moiety (nr ¼ -piperidinyl), showed moderate to high ability in scavenging dpph radical ( e % of dpph scavenging ability of standard gallic acid), while the rest of the candidates were either poor scavengers of dpph radical, or failed to react with dpph radical at all [ ] . although all of the phenolic mannich bases showed efficiency as antioxidants in various degrees, candidates (nr ¼ diphenylamino, -morpholinyl, -piperazinyl) (fig. ) were the most potent scavengers of hydrogen peroxide, their activity being comparable to that of standard ascorbic acid, but inferior to that of standard butylated hydroxyanisole [ ] . selected tacrinee -hydroxyquinoline hybrids (fig. ) , that were developed primarily as agents for the treatment of alzheimer's disease, exhibited also potent peroxyl radical absorbance capacities ( . e . trolox equivalents/mmol of mannich base), as determined by means of orac-fl method (oxygen radical absorbance capacity by fluorescence) [ ] . phenolic mannich bases of natural flavanones have been designed and synthesized with the view to improve antioxidant efficiency, bioavailability and water solubility of parent flavanones. only three mannich bases (r ¼ oh, r ¼ h, r ¼ -pyrrolidinyl, diethylamino or diisopropylamino) of apigenin (fig. ) exhibited antioxidant activity greater than that of apigenin, while the rest of candidates presented antioxidant activity comparable to that of apigenin [ ] . antioxidant activity of these apigenin derivatives was concentration-dependent, and dpph radical scavenging ability of the most potent mannich bases was almost the same as that of the standard ascorbic acid at a concentration of . mg/ml. scutellarein was also aminomethylated to give mannich bases (r ¼ r ¼ oh, r ¼ aminomethyl), which had the ability to scavenge % of dpph radical in the sample at concentrations ranging from to mm, but no comparison with a well-established antioxidant was provided in the study [ ] . mono-and bis-mannich bases fig. ) were synthesized and evaluated as inhibitors of photooxidation of a e (a pigment of the lipofuscin of retinal pigment cells, thought to play a role in macular degeneration) [ ] . these candidates showed sufficient antioxidant ability to inhibit noncellular and intracellular photooxidation of a e, and were superior as antioxidants to quercetin itself. on the other hand, phenolic mannich bases obtained from other known antioxidants such as sesamol or -( -hydroxy- -methoxyphenyl)- phenylprop- -en- -one were ineffective inhibitors of a e photooxidation at mm [ ] . phenolic mannich bases of chalcone analogues such as candidates (r ¼ -f, -cl, -br, -f, -cl, -br) (fig. ) were modest to poor scavengers of dpph radical ( e % reduction of dpph absorption) compared to standard quercetin ( % reduction of dpph absorption) [ ] . results for dpph scavenging ability of phenolic mannich bases derived from other chalcone analogues obtained in a different study are also in line with the antioxidant data recorded for mannich bases , as the greatest reduction of dpph absorption was only % at concentrations as high as mm of mannich base [ ] . however, one candidate in this series scavenged superoxide radical efficiently, while another candidate was a potent inhibitor of heme dependent lipid peroxidation [ ] . several mannich bases g derived from -( -thienyl)- , dihydro- , , -triazol- -thiones (table ) were as efficient antioxidants as standard ascorbic acid (over % reduction of dpph radical), and showed enhanced antioxidant activity over the parent , -dihydro- , , -triazole- -thiones [ ] . a series of mannich bases [ , ] dioxin- -yl, nr ¼ substituted primary arylamines) ( table ) were designed as antioxidant agents, and were evaluated using scavenging dpph radical assay, scavenging , -azino-bis( -ethylbenzothiazoline- sulfonic acid) radical cation (abts) assay, and ferric reducing antioxidant power assay [ ] . more than half of these mannich bases showed greater dpph radical scavenging ability than butylated hydroxytoluene (ic ¼ mm), and seven of them exhibited greater antioxidant activity than trolox (ic ¼ mm) in the same assay. a few candidates with high dpph scavenging activity, along with other mannich bases , were also very good scavengers of abts radical cation (their activity was greater than that of trolox). in ferric reducing antioxidant power assay, three mannich bases showed better activity than trolox, and other seven were more potent than butylated hydroxytoluene. mannich bases nr ¼ , , -f c h ) were the only compounds to exhibit high potency in all three assays, and they were also more efficient than trolox in inhibiting lipid peroxidation in mice liver microsomes homogenate [ ] . n-mannich bases of succinimide (r ¼ h or phenyl, nr ¼ nhc h , -pyridinyl) (fig. ) and an n-mannich base of phthalimide showed moderate dpph scavenging activity compared to standards vitamin c and vitamin e, while their ability to inhibit peroxidation of linoleic acid was moderate to weak [ ] . also, several n-mannich bases of saccharin (fig. ) were antioxidants as potent as standard ascorbic acid in the abts assay, and more potent antioxidants than saccharin itself [ ] . n-mannich base derived from a , -disubstituted pyrazoline (fig. ) had dpph scavenging and nitric oxide scavenging activities comparable to standard antioxidants ascorbic acid and rutin; the presence of the phenolic hydroxyl group could be responsible for the antioxidant activity of this compound [ ] . finally, antioxidant activity of two c-mannich bases of uracil (fig. ) was determined by means of measuring the rate of oxidation of -propanol [ ] ; addition of compounds to the reaction mixture reduces the rate of oxidation to levels comparable to the rate of oxidation in the presence of butylated hydroxytoluene. a small number of studies deal with the investigation of mannich bases as antihypertensive agents. phenolic mannich bases appear to be particularly remarkable as blood pressure-lowering substances. several single and double mannich bases of various phenols and having thiomorpholine as amine moiety showed a gradual effect on systolic, diastolic and mean arterial pressure, starting at a dose . mg/kg; in the case of reference drugs captopril, losartan and omapatrilat, the same effect was noticeable at . mg/kg [ ] . in a manner similar to the aforementioned reference drugs, these phenolic mannich bases also induced a gradual (but significant) reduction of heart rate in anesthetized mice. double mannich base (fig. ) emerged as a valuable candidate, with one of the lowest effective dosage in this collection, while exhibiting the highest antihypertensive activity in conscious spontaneous hypertensive rat model [ ] . the same researchers examined the antihypertensive effect of double mannich base with a , -dihydropyridine moiety in its structure (fig. ) , but the results showed that the activity of this candidate was inferior to that of double mannich base , presumably due to the change in bulkiness of the substituent para to the phenolic hydroxyl [ ] . the presence of two aminomethyl functions in the structure of may have been essential in preserving the antihypertensive activity of this candidate to a reasonable level, while replacement of thiomorpholine in with morpholine in could be also the cause for the poorer antihypertensive properties of candidate relative to those of mannich base . in an attempt to synthesize aminomethylated -(naphthyloxy) butanoic acids (e.g., compound , were obtained when -(naphthalen- -yloxy)butanoic acid was subjected to aminomethylation [ ] . at a concentration of mg/ kg, candidate (nr ¼ -piperidinyl) lowered blood pressure in anesthetized cats similarly to propranolol, while compound (nr ¼ -phenylpiperazin- -yl) was superior to propanolol at the same dose (fall of blood pressure of mm hg, duration of the effect of min). it is also worth mentioning that the antihypertensive effect of a c-mannich base of -naphthol, namely - fig. . mannich bases as agents for blood pressure regulation. pyrrolidinylmethyl- -naphthol (fig. ) , was reported in an earlier publication [ ] . a large collection of mannich bases (r ¼ h, ch , c h , ch ch(ch ) , c h , och , cl) (fig. ) was evaluated for antihypertensive activity by the non-invasive tail cuff method, and several candidates were found to significantly reduce mean arterial blood pressure, albeit at higher doses than reference drug hydralazine [ ] . the presence of alkyl groups as substituent r seems to be favorable for the antihypertensive activity, whereas the nature of the amine moiety does not appear to influence the activity. several mannich bases (r ¼ ch , c h , n-c h , ch c h ; r ¼ ch , c h ) of imidazo[ , -a]benzimidazoles (fig. ) were found to reduce arterial blood pressure in anesthetized rats with % at doses that were lower than the dose at which reference drug dibazole produces the same effect ( mg/kg) [ ] . analogous aminomethylated imidazo[ , -a]benzimidazoles (r ¼ ch , n-c h , ch c h ) (fig. ) were more efficient antihypertensive agents than mannich bases , as they reduce arterial blood pressure with % at doses lower than those recorded for candidates . several octahydroquinazoline derivatives (r ¼ h, -f, -f, -cl, -br, -ch , -oh) (fig. ) were obtained through a double mannich reaction starting from -( -chlorophenylamino)- , dimethyl- -cyclohexenone and arylamines, and were shown to produce insignificant changes in both arterial blood pressure and heart rate at a dose of mg/kg [ ] . however, derivatives (r ¼ och conhn]chc h r , r ¼ h, -ch , -no ) of a candidate in the initial series afforded significant decreases in both systolic and diastolic arterial blood pressure, with rapid onset of action ( min) and minor decrease of heart rate in anesthetized male adult albino rats [ ] . on the other hand, candidate (r ¼ -cl) produced a time-dependent significant increase in both systolic and diastolic arterial blood pressure, without causing tachycardia for min, which makes this compound useful for treatment of hypotension. a small number of studies report the activity of mannich bases against parasites other than plasmodium spp. among these parasites, which are the cause of parasitic infections especially in developing countries, different species from the trypanosomatidae family are pathogenic to humans and cause african trypanosomiasis (sleeping sickness, trypanosoma brucei), american trypanosomiasis (chagas' disease, trypanosoma cruzi) or leishmaniasis (leishmania spp.). in addition, one study investigated the activity of mannich bases against entamoeba histolytica, and one study reported the anti-schistosoma activity of mannich bases derived from praziquantel. all parasitic protozoa belonging to trypanosoma spp. have a unique thiol metabolism based on the flavoenzyme trypanothione reductase. this enzyme could be therefore considered a promising target for rational drug design against african sleeping sickness, chagas' disease, and different forms of leishmaniasis, owing to its absence of in the mammalian host, the structural differences to related host enzymes, and its essential role for parasite survival. because unsaturated ketonic mannich bases react readily with thiols, and because several such compounds were shown to be efficient mechanism-based inhibitors of p. falciparum thioredoxin reductase [ ] , a study concerning the ability of these compounds to interact with both trypanothione reductase and free trypanothione was undertaken [ ] . candidates (nr ¼ dimethylamino, -piperidinyl, -morpholinyl) (fig. ) inactivated trypanothione reductase, but only in the presence of nadph, suggesting that reduction of this enzyme prior to its interaction with the mannich base is essential. the divinyl ketone arising from the deamination of candidates is the actual inhibitor, and its activity against trypanothione reductase was higher than that of parent mannich bases; unfortunately, this divinyl ketone was also too reactive to be considered a drug candidate. mannich bases displayed only modest activity against all strains of intracellular parasites, which may be explained by reaction with glutathione present in millimolar concentrations in the cytosol of the mammalian host cells. nonetheless, they showed a significant effect against extracellular t. b. rhodesiense, which might (at least partially) be due to their high reactivity toward trypanothione reductase and trypanothione. with trypanothione reductase validated as a drug target in trypanosomiasis, design and synthesis of novel unsaturated mannich bases based on melaminophenyl arsenical drug melarsoprol was subsequently pursued [ ] . candidates (r ¼ cl, h, ch ) (fig. ) were efficient in vitro trypanothione reductase inhibitors, and while they did not display any significant activity in cell-based assays against t. cruzi, they were active towards t. brucei. the presence of the melamine residue para to the enone motif did not result in any improvement in the trypanocidal potency of (r ¼ cl) compared to hit compounds . however, the presence of the melamine residue meta to the enone motif significantly lowered the ic against the human cell line used in the study, which is indicative of these compounds' high cytotoxicity. on the other hand, phenolic mannich base (fig. ) was basically inactive towards all parasites. mannich bases were taken up effectively into cells despite the absence of p and hapt carriers, which suggests that the main route of entry for these compounds was not through aminopurine transporters. stemming from the observation that heterocyclic pyrazolopyridine ring system can be considered analogous to quinoline, and because aminoquinoline derivatives are efficient antimalarials, several derivatives featuring the pyrazolopyridine scaffold were designed and tested against leishmania amazonensis, in the evolutive form of promastigotes [ ] . thus, both mannich bases (r ¼ c h , r ¼ ch , c h ) (fig. ) are active antileishmanial agents with ic values of and nm, whereas aminoquinoline derivative amodiaquine had ic ¼ nm. from the structureeactivity point of view, the results suggest that pyrazolopyridine ring system is a bioisostere of quinoline, and that the presence of the carbethoxy group in the structure of candidates does not influence significantly the biological activity. amebiasis is a contagious disease of the human gastrointestinal tract that is caused by parasitic protozoa e. histolytica. all the candidates in a small library of piperazine mannich bases d of -( pyridinyl)- , -dihydro- , , -oxadiazole- -thione ( table ) were active against hm :imss strain of e. histolytica, but only three of them were more potent than reference drug metronidazole (ic ¼ . mm) [ ] . the amine residues in the aminomethyl function of these active mannich bases were -ethylpiperazin- -yl (ic ¼ nm), -( -fluorophenyl)piperazin- -yl (ic ¼ nm), and -( -methoxyphenyl)piperazin- -yl (ic ¼ . mm). in addition, cytotoxicity of these antiamoebic candidates was low (in the concentration range of . e mm). out of four newly synthesized mannich bases of praziquantel (fig. ) , two candidates (nr ¼ n(c h -n) and nhch ch oh) exhibited significant in vitro anti-schistosoma activity ( % worm killing at mm and mm, respectively), but they were not as efficient as praziquantel itself ( % worm killing at mm) [ ] . platelets are crucial for hemostasis, as they connect to one another through receptor bridges, form aggregates, and finally plug the tear in the interrupted endothelium. however, the aggregation of platelets leading to formation of clots can also be triggered by irregularities on the vessel wall, resulting in abnormal clot formation, which is the primary factor in the development of thrombotic disorders such as unstable angina, myocardial infarction, stroke and peripheral vascular diseases, especially when it occurs in the coronary artery. platelet aggregation is also initiated by endogenous substances, such as collagen, thrombin, prostaglandin endoperoxides, thromboxanes, arachidonic acid, adenosine diphosphate (adp), etc. inhibition of platelet aggregation represents a promising strategy for the treatment of thrombotic diseases, and several studies have reported the activity of mannich bases as inhibitors of platelet aggregation. several types of phenolic mannich bases represented by structures e (fig. ) were also evaluated as inhibitors of platelet aggregation induced by adp or collagen at concentrations of mm or mg/ml, respectively [ ] . numerous candidates having various structures showed good inhibitory effects and were more potent than reference drug clopidogrel, whereas nine compounds in this collection exhibited inhibitory activity in the range of e % in both models. good platelet aggregation inhibitory activity was associated with the presence of a pyridyl moiety as ring b in the structure of chalcone analogues, and also by the presence in ring a of a hydroxy group meta to the carbonyl function. phenolic mannich bases (r ¼ r ¼ oh, r ¼ aminomethyl) of scutellarein (fig. ) were investigated as thrombin inhibitors, and their influence on several parameters such as prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen was determined [ ] . all of these candidates showed greater thrombin inhibitory activity compared to parent scutellarein. mannich base derived from scutellarein and containing a morpholinyl residue in the aminomethyl function was the most potent of all, and was subsequently selected for molecular docking experiments with thrombin. these experiments revealed that the morpholinylmethyl group occupies deep pocket s of the thrombin binding site, whereas the scutellarein part of the inhibitor's molecule is anchored by three hydrogen bonds within the active site. the effect of mannich bases (r ¼ ch , c h , n-c h , ch c h ; r ¼ ch , c h ) and (r ¼ ch , n-c h , ch c h ; (fig. ) on adp-induced aggregation of rabbit thrombocytes was studied in vitro [ ] . several candidates and most candidates were found to have lower effective concentrations (ec ) at which they decrease the degree of aggregation by half than reference drug acetylsalicylic acid. cellular effects of thrombin, including platelet aggregation, are mediated through the activation of thrombin receptor par- belonging to the family of four g-protein-coupled receptors called protease-activated receptors. par- has become an attractive drug discovery target, and peptide-mimetics or small organic molecules with par- antagonist properties have already been designed and synthesized. an indole mannich base motif has been incorporated in the structure of a series of novel peptide-mimetics (r ¼ -ch oc h ch , , -f c h ch , naphthalen- -ylmethyl, naphthalen- -ylmethyl) ( fig. ) capable to bind to par- [ ] . the ability of these candidates to inhibit par- -induced platelet aggregation has been tested by measuring the degree of aggregation of human platelets, and also by establishing the degree of inhibition of contraction of aortic rings. compounds (r ¼ -ch oc h ch , , -f c h ch ) belonging to the series of -aminoindole derivatives were the most potent candidates, with values of inhibitory concentration that were about times lower than reference compound rwj for % inhibition of platelet aggregation. in addition, these results were confirmed by the enhanced inhibitory activity of these two candidates on the contraction of aorta rings when compared to the activity of rwj , an effect that became more evident with the increase of par- dose. candidates in the series of -aminoindole derivatives were found inactive or weak inhibitors in both assays. anti-ulcer agents are a class of drugs used to treat ulcers in the stomach and the upper part of the small intestine. acid peptic disease is a chronic pathology that affects millions of people worldwide, and it has been estimated that % of the world population will develop this condition in their lifetime [ ] . an imbalance between aggressive factors (such as gastric acid, helicobacter pylori infection, excessive intake of anti-inflammatory drugs, immoderate consumption of alcohol, high concentrations of reactive oxygen species) on one hand, and protective factors (e.g., mucus, bicarbonate anion, prostaglandins, good blood flow, efficient cellular repair, endogenous and exogenous antioxidants) on the other hand is considered to be the cause of ulcers. treatment of ulcers and their symptoms relies on pain relievers, antiacids and cytoprotective agents to allow the healing of ulcers, and agents to prevent the recurrence of ulcers, such as proton pump inhibitors, muscarinic antagonist pirenzepine or h receptor antagonist cimetidine. the ability of mannich bases to act as anti-ulcer agents has been reported in a small number of studies. anti-ulcer activity of saminomethyl derivatives generated from , -diaryl- mercaptopyrimidines (fig. ) as substrates and secondary aliphatic amines as amine reagents in the mannich reaction was evaluated in vivo using aspirin-induced ulcer model in albino rats [ ] . based on the ulcer score, the mean ulcer index and the degree of protection were calculated and compared to those of reference drug omeprazole. five candidates (r ¼ -clc h , r ¼ -no c h or -ch oc h ) offered more than % protection, while other five mannich bases (r ¼ -clc h , r ¼ -clc h , -no c h or -(h c) nc h ) had only moderate anti-ulcer activity (approximately % protection) compared to omeprazole ( % protection). aminomethylation of , -dihydropyrimidines with sulfanilamide as amine reagent afforded mannich bases (fig. ) , whose ability to reduce the volume of acid secretion was determined by pyloric ligation method [ ] . three candidates (r ¼ -ch oc h , -ch o- -hoc h , -furyl) presented good antiulcer activity (ulcer indices in the range of . e . at a dose of mg/kg), while reference drug omeprazole had an ulcer index of . at a dose of mg/kg. a small number of furo [ , -g] flavones of type (r ¼ c h , -clc h , -pyridinyl; r ¼ h or ch ) and (r ¼ c h , -pyridinyl) (fig. ) , aminomethylated either at position or , have been evaluated as gastroprotective agents using the ethanol-induced gastric ulcer model in rats [ ] . the mean values of the protection index for these mannich bases were in the range of e %, and no comparison with an anti-ulcer reference drug was provided in the study. the best three candidates (r ¼ -pyridinyl, r ¼ ch , nr ¼ -methylpiperazin- -yl), (r ¼ -pyridinyl, r ¼ och , nr ¼ -methylpiperazin- -yl) and (r ¼ c h , r ¼ h, nr ¼ -morpholinyl) have a methoxy group at position as a common structural feature; the corresponding mannich bases having a hydroxyl group instead of methoxy were significantly less active. mental disorders comprise a broad range of problems, with different symptoms, generally characterized by some combination of abnormal thoughts, emotions, behavior and relationships with others. common neurological conditions labeled as mental disorders include depression and anxiety, while schizophrenia and bipolar disorder stand out as mental disorders that are severe and disabling. untreated mental, neurological and substance use disorders exact a high toll, accounting for % of the total global burden of disease, while unipolar depressive disorder is the third leading cause of disease burden, accounting for . % of the global burden of disease. current predictions indicate that depression will be the leading cause of disease burden globally by [ ] . a range of different types of treatment for mental disorders are available, and the most suitable treatment depends both on the disorder and on the individual. a major option for many mental disorders is psychotherapy, while another option is psychiatric medication. amongst several groups of drugs that are currently employed in psychiatric medication, antidepressants treat clinical depression as well as anxiety and a range of other disorders, anxiolytics (including sedatives) are used for anxiety disorders and related problems such as insomnia, mood stabilizers are used primarily in bipolar disorder, and antipsychotics are used for psychotic disorders, notably for positive symptoms in schizophrenia. selective serotonin reuptake inhibitors (ssris) play an important role in pharmacotherapeutic treatment of depression. in an attempt to modify the general ssri structural motif of g-phenoxypropylamine, two ketonic mannich bases (r ¼ cl, br) (fig. ) derived from -chloro-and -bromoacetophenone as substrates and -benzylpiperidine as amine reagent were synthesized, the carbonyl function in these amino ketones was subsequently reduced, and the resulting secondary hydroxyl was then converted into an ether group through reaction with -chloro- trifluoromethylbenzene [ ] . although the designed ssri analogues targeted in this study showed no antidepressant activity, the intermediate ketone mannich bases were as effective as reference drugs fluoxetine, sertraline or imipramine at similar dosage in a validated experimental model of depression in mice such as the forced swimming test. an innovative computer-assisted approach based on the prediction of activity spectra for substances (pass) has been applied for the discovery of new anxiolytics [ ] . an initial database comprising structures was generated by virtual combinatorial design of highly diverse chemical compounds, including different types of heterocycles such as thiazoles, pyrazoles, isatins, fused imidazoles, with the view to increase the probability of finding new chemical entities as anxiolytics. out of the eight hits obtained from this database, four candidates were mannich bases. ketonic mannich base , mannich base derived from an imidazo[ , -b] benzo[d]thiazole and two mannich bases (r ¼ f, r ¼ ch ; r ¼ no , r ¼ c h ) derived from imidazo[ , -a]pyridines (fig. ) were synthesized and tested as potential anxiolytics using the conflict situation test. all of the candidates showed an anxiolytic effect that was comparable or greater than that of reference drug medazepam. mannich base (r ¼ no , r ¼ c h ) was the most potent anxiolytic in this study, being two times more potent than medazepam. a collection of compounds sharing a benzoxepin scaffold as common structural feature was evaluated for their sedativeehypnotic effect using phenobarbital-induced sleep test [ ] . among these compounds, three ketonic mannich bases (nr ¼ -piperidinyl, -methylpiperazin- -yl, -morpholinyl) (fig. ) decreased the onset of phenobarbital-induced sleep and prolonged the duration of hypnosis when administered in a dose equimolar to that of reference drug phenobarbital. the hypnotic activity of candidates (nr ¼ -piperidinyl, -methylpiperazin- -yl) was comparable to that of phenobarbital, while mannich base (nr ¼ dimethylamino) exhibited only moderate activity, and mannich base (nr ¼ -( -chlorophenyl)piperazin- -yl) was virtually inactive. therefore, the nature of the amino residue in these mannich bases seems to have a significant impact on their hypnotic activity. in addition to biological activities of mannich bases presented so far, isolated studies were found to report various other biological activities for mannich bases. these singular results are covered in this section, without any attempt to arrange them in a systematic order. two phenolic mannich bases derived from , -and , -di-tbutylphenol as substrates and dimethylamine as amine reagent were evaluated as hepatoprotective agents against experimental toxic hepatitis induced by tetrachloromethane. the degree of liver damage was evaluated in terms of alanine aminotransferase activity in blood serum and malonic dialdehyde content in liver homogenates. even at a dose of % of the corresponding ld , the ability of these two candidates to diminish the hepatotoxic action of tetrachloromethane was superior to that of reference compound emoxypine [ ] . in addition, in an assay using the same tetrachloromethane-induced hepatitis model, two acetylenic mannich bases (x ¼ o or ch ) with a betulonic acid scaffold (fig. ) were shown to decrease alkaline phosphatase activity and lower alanine aminotransferase and aspartate aminotransferase activities in blood serum compared to control [ ] . acetylenic mannich base (fig. ) was tested on guinea pig spontaneously beating atria with a view to evaluate its negative chronotropic activity, but the potency of this candidate was approximately four times lower than that of bradycardic agent zatebradine, and was therefore excluded from subsequent testing as blocker of hyperpolarization-activated current [ ] . ondansetron analogues (r ¼ ch ) having a piperazine moiety instead of imidazole (fig. ) were synthesized and evaluated as anti-emetic agents using a retching model [ ] . all the candidates were effective to some extent when administered at a dose of mg/kg, but only mannich base (r ¼ -pyrimidinyl) had anti-emetic activity comparable to that of reference drug ondansetron at low dosage ( mg/kg). several -aminomethylated b-thujaplicin analogues ( fig. ) were tested against oxidative stress-induced death of ht cells following exposure to glutamate [ ] . piperazine-containing mannich bases were more potent than parent b-thujaplicin in protecting glutamate-challenged ht cells, with ec values ranging from . to . mm. because the most potent candidates were those having a chroman moiety as substituent at n in the piperazine residue, the high in vitro neuroprotective activity of these mannich bases may be due to the potent antioxidant effect imparted by the chroman moiety. analogous morpholine mannich base was also active in protecting ht cells from oxytosis, but it was less active than the piperazine-containing b-thujaplicin derivatives. the presence of the isopropyl group also seems to be important for the neuroprotective activity of these compounds, since an analogous mannich base derived from tropolone was not active. estrogen deficiency after menopause is one of the most common causes of osteoporosis. hormone replacement therapy is widely used to prevent bone loss, although it presents potential drawbacks such as increased risk of uterine bleeding and/or hyperplasia, increased risk of endometrial, breast or ovarian cancer, higher occurrence of myocardial infarction, cardiovascular disease, etc. conjugate of b-estradiol and iminodiacetic acid (fig. ) was designed as an estrogen-containing, bone-seeking agent that could prevent bone loss with lesser side effects, and was subsequently synthesized by means of the mannich reaction [ ] . mannich base showed significant affinity for bone, but lower affinity for ovary and uterus than b-estradiol, while it maintained % of the affinity of b-estradiol for osteoblast estrogen receptors. candidate did not induce uterine hypertrophy, and lower levels of biochemical markers of bone turnover (e.g., osteocalcin, alkaline phosphatase, c-terminal telopeptide fragment of type i collagen cterminus) were found in the group of rats treated with than in ovariectomized rats, which suggests decreased bone turnover. administration of candidate improved bone mineral density and trabecular architecture after ovariectomy, but did not suppress body weight increase. these results suggest that compound is effective in preventing ovariectomy-induced bone loss while exhibiting exhibited fewer adverse side effects than b-estradiol, making mannich base a better choice for the prevention of postmenopausal osteoporosis. three bis-p-mannich bases (r ¼ r ¼ ch ; r er ¼ (ch ) ; r er ¼ (ch ) ) (fig. ) , derived from diethyl phosphite as substrate and amino acids as amine reagents in the mannich reaction, have been used to investigate the induction of adipogenic or osteogenic differentiation of mesenchymal stem cells isolated from human adipose tissue (hmads cells) [ ] . candidates did not affect the adipocyte differentiation, but induced the inhibition of osteoblast formation without any detectable cytotoxic effect, whereas reference drug sodium alendronate elicited a cytotoxic effect even at the lowest concentration used in the study ( À m). therapeutic efficacy and toxicity profiles of ketonic double mannich base (fig. ) , a putative immunosuppressant which preferentially inhibited jak as opposed to several other kinases, were examined [ ] . candidate blocked il- -induced activation of jak and its downstream substrates stat a/b, while it failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, src family members, and serine/threonine protein kinases. mannich base alone prolonged kidney allograft survival and induced transplantation tolerance, and its combination with cyclosporin a presented therapeutic synergism. candidate showed no nephrotoxicity, did not affect hematopoiesis or lipid metabolism, and was not metabolized by the cytochrome p a isoform. therefore, because mannich base prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs, it may provide significant clinical benefits for transplant patients. an important number of studies report the activity of mannich bases as enzyme inhibitors. although the studies covered in this section usually validate the importance of the topic through a rational and evidence-supported connection between the enzyme under scrutiny and a certain medical condition, they only examine the action of mannich bases as enzyme inhibitors, and do not provide any experimental evidence that these mannich bases could be actually useful agents for the treatment of specific medical conditions. starting from tacrine, the first drug approved for the treatment of alzheimer's disease and a potent inhibitor of both acetylcholinesterase (ache) and butyrylcholinesterase (buche), multifunctional compounds (fig. ) that combine neuroprotective, antioxidant, metal-binding properties, and dual inhibition of ache and buche in a single small molecule have been designed and synthesized through the mannich reaction [ ] . tacrinee hydroxyquinoline hybrids were potent inhibitors of both ache and buche of bovine origin with ic ranging from submicromolar to nanomolar concentrations. candidates containing an unsubstituted -hydroxyquinoline moiety and a methylene tether of e carbon atoms had the most potent inhibitory activities. selected mannich bases were evaluated as inhibitors of human cholinesterases, and they exhibited ic values in the range of . e . nm against ache, and in the range of . e nm against buche. mannich base (r ¼ r ¼ h, n ¼ ) was the most potent dual inhibitor of human ache and buche in this library [ ] . other mannich base hybrids (fig. ) were designed as dual binding site inhibitors of ache by combining tacrine (a recognized inhibitor of catalytic binding site of ache) with the indanone moiety of donepezil (known to be responsible for the binding of this drug to the peripheral site of ache) [ ] . their evaluation as inhibitors of ache (bovine erythrocyte) and buche (human serum) using ellman's method showed that the activity of two of these candidates was modest, while the third candidate (z ¼ h, r ¼ och , x ¼ (ch ) ) exhibited moderate activity against ache ( nm) and good activity against buche ( . nm). apparently, the difference in activity between these candidates is due to the lengthening by one methylene group of the alkyl chain linking the tacrine and indanone moieties. -benzoxazolone mono-mannich bases (fig. ) derived from secondary aliphatic amines or primary arylamines, and bis-mannich bases derived either from primary aliphatic amines (x ¼ nr, r ¼ c h , r c h ch ch , r ¼ h, -cl, , -(ch o) ) or piperazine have been evaluated for ache inhibitory activity using ellman's method. the degree of inhibition was in the range of e % at a concentration of mm, but decreased to e % at . mm, whereas the degree of ache inhibition by tacrine was virtually the same at both concentrations (greater than %) [ ] . the most potent inhibitor in this series at both concentration was bis-mannich base derived from piperazine. inhibitory activity against ache and buche of phenolic mannich bases (r ¼ h, ch , allyl, prenyl; nr ¼ n(ch ) , n(c h ) , pyrrolidinyl, -piperidinyl, -morpholinyl) of xanthone derivatives ( fig. ) was found to be moderate to good compared to that of reference drug galantamine [ ] . candidates derived from diethylamine as amine reagent in the mannich reaction were generally the most potent inhibitors of both enzymes. the nature of alkyl moiety r modulates the selectivity of inhibitors towards one of these enzymes; thus, the most potent inhibitors of ache feature a methyl group as r , whereas the increasing bulkiness of this substituent appears to favor the inhibition of buche. mannich base (r ¼ prenyl, nr ¼ n(c h ) ) was the most potent dual inhibitor in this series, and molecular docking studies were performed with the view to garner information on the binding mode of this inhibitor with both enzymes. several phenolic mannich bases of naturally occurring flavanone oroxylin a (fig. ) were more potent inhibitors of intestinal a-glucosidase than parent oroxylin a, but the same candidates were less potent than oroxylin a against yeast a-glucosidase [ ] . nonetheless, the most potent inhibitor of intestinal aglucosidase in this series was still . less potent than reference drug acarbose. because all of the candidates which failed to inhibit both enzymes were derived from acyclic secondary amines as amine reagents in the mannich reaction, the alicyclic nature of secondary amines may responsible for the good inhibitory activity against a-glucosidases in this series. ketonic mannich bases derived from nabumetone (fig. ) possess modest inhibitory activity against intestinal a-glucosidase (approximately mm), in addition to modest inhibitory activity against another therapeutic target for type diabetes mellitus, obesity and related states of insulin resistance, namely proteintyrosine phosphatase b [ ] . however, two mannich bases (r ¼ h or -oh) were good agonists of peroxisome proliferatoractivated receptors, which are major regulators of lipid and glucose metabolism. a novel series of inhibitors was designed by retaining the nabumetone moiety, while sulfanilamide and amino- -methylisoxazole served as amine reagents in the synthesis of ketonic mannich bases of type and , respectively (fig. ) [ ] . candidates were generally more potent inhibitors of a-glucosidase than analogous , suggesting that sulfonamide moiety is a more potent pharmacophore than aminoisoxazole. mannich base (r ¼ -br) inhibited % of a-glucosidase activity at a dose of mg/ml. replacement of the sulfonamide moiety with -aminobenzoic acid, with a view to mimic the acidic, hydrophilic part of molecules with known antidiabetic activity, led to a novel series of mannich bases (r ¼ h). a second series of candidates (r ¼ c h ) (fig. ) was also evaluated as a-glucosidase inhibitors, but their activity was generally poorer than that of mannich bases (r ¼ h) derived from -aminobenzoic acid [ ] . for example, the most potent a-glucosidase inhibitor ( % inhibition) in the series derived from -aminobenzoic acid was candidate purine nucleoside phosphorylase (pnp) catalyzes the phosphorolytic cleavage of inosine, guanosine and their -deoxy analogues to (deoxy)ribose -phosphate and hypoxanthine or guanine. genetical deficiency in pnp leads to complete t-cell deficiency early in life and eventual death of infants from virus infections, and pnp has been identified as a target for the treatment of t-cell lymphoma, rheumatoid arthritis, psoriasis, multiple sclerosis, and other t-cell mediated disorders. a transition state analogue is a compound that occupies the catalytic site of an enzyme and induces the slow conformational changes that would normally occur to place the catalytic site in the appropriate geometry for the search that would locate the transition state with the normal substrate of the enzyme. schramm et al. have developed a series of mannich bases of -deazahypoxanthine as transition state analogues for purine nucleoside phosphorylase inhibition, generally called immucilins. although aminomethylated purines and analogues have been present within the firstand second-generation of pnp inhibitors [ , ] , they have been obtained through reductive amination, and not through direct mannich reaction. because a more expeditious synthesis of second-generation pnp inhibitors by means of the mannich reaction has been later developed [ ] , the third generation of these inhibitors included several examples of mannich bases (fig. ) obtained through direct aminomethylation of substrate -deazahypoxanthine with amino alcohols such as ethanolamine, -amino-propan- -ol, -amino-butan- -ol, diethanolamine and -( -hydroxyethylamino)propan- -ol as amine reagents [ ] . unfortunately, all these candidates obtained through direct aminomethylation proved to be modest or poor inhibitors of pnp (dissociation constants k i in the range of to , pm) compared to other mannich bases previously investigated, such as (dadme-immucilinh, k i ¼ . pm) or fig. . mannich bases as inhibitors of a-glucosidase. (dadme-immuciling, k i ¼ . pm) (fig. ) . nonetheless, this study identified two acyclic, simplified alternatives of immucilin analogues, namely (k i ¼ . pm) having an open-chain amino alcohol with two asymmetric carbon atoms, and (k i ¼ . pm) derived from achiral dihydroxyaminoalcohol seramide (fig. ) , as potent inhibitors of pnp. because the enantiomers of both immu-cilinh and dadme-immucilinh have been shown to have pnp binding properties that differ considerably [ ] , and since access to enantiomerically pure starting azasugar employed for the generation of requires a suitable enzyme-based route, the discovery of the two simpler mannich bases and represents a significant step forward in the development of clinically useful pnp inhibitors. sulfur-containing transition state analogues have been developed as well, originally as specific inhibitor of pnp from p. falciparum, but it was later shown that sulfur-containing candidates analogous to dadme-immucilinh or mannich base also bind to human native pnp [ ] . although human pnp tolerates substitution of -hydroxyl in transition state analogues and with alkylthio and arylthio groups, the slow-onset nature of inhibition is lost, and inhibitor dissociation constants increase by two to three orders of magnitude. fluorine-containing analogue (fig. ) of dadme-immucilinh has also been evaluated as human pnp inhibitor, and its enantiomers exhibit slow-onset binding constants of pm for [( s, s)- ] and . nm for [( r, r)- ] [ ] . in addition, direct mannich reaction has been used to generate in a similar fashion potent transition state inhibitors for other enzymes, such as methylthioadenosine phosphorylase methylthioadenosine nucleosidase [ , ] or purine specific nucleoside hydrolase [ ] . a large library of a-, band g-amino ketones was screened with a view to discover inhibitors of transglutaminase, but only b-amino ketones (ketonic mannich bases) strongly inhibited this enzyme [ ] . mannich bases derived from aliphatic ketones as substrates, or those derived from alkyl aryl ketones as substrates and primary amines as amine reagents were weak inhibitors (ic > mm) of transglutaminase. potent transglutaminase inhibitory activity resided with ketonic mannich bases derived from alkyl aryl ketones as substrates and secondary aliphatic amines as amine reagents. both the nature of the aryl moiety in the substrate and the nature of the alkyl groups attached to the nitrogen atom influenced transglutaminase inhibitory activity of these candidates. generally, heteroaryl-substituted substrates ( -thienyl, -benzothienyl, furyl, -furyl, pyridinyls, pyrazinyl) fared better than substrates having aryl moieties (phenyl, naphthyls), and t-butyl, isopropyl, benzyl or -hydroxyethyl groups (but not phenyl) as alkyl substituent at nitrogen afforded potent transglutaminase inhibitors, such as candidate (ic ¼ nm) (fig. ) . because disulfides are well-known transglutaminase inhibitors, a novel series of b-amino ketones (r ¼ h, n ¼ ; r ¼ cooch , n ¼ ; r ¼ cooch , n ¼ ) (fig. ) derived from cystamine, dimethyl cystine ester, and dimethyl homocystine ester as amine reagents in the mannich reaction were synthesized and found to be approximately times more active than the starting disulfides [ ] . mannich bases derived from -acetylthiophenes were once more the most potent inhibitors in this series, while the nature of the disulfide-containing amine moiety did not significantly influence the activity of candidates having the same heterocyclic or carbocyclic moiety r . mannich bases derived from , -dimethylphenol ( fig. ) have been evaluated in vitro as inhibitors of two human carbonic anhydrase (hca, ec . . . ) isozymes i and ii using the hydratase and esterase assays, respectively [ ] . only two candidates exhibit weak hca ii inhibitory effects on esterase activity, whereas other two mannich bases could be used as carbonic anhydrase activators. phenolic mannich bases derived from quercetin (fig. ), derived from baicalein and derived from -( -hydroxy- methoxyphenyl)- -phenylprop- -en- -one ( fig. ) were screened for inhibition of cyclin-dependent kinases using a biochemical assay method based on fluorescence resonance energy transfer [ ] . candidates and are essentially devoid of inhibitory activity of cyclin-dependent kinases; on the other hand, baicalein mannich bases were e times more potent than the parent flavone, suggesting that the presence of a nitrogen atom at c- is crucial for the inhibition of cyclin-dependent kinases, while the presence of a second heteroatom in the amine moiety (e.g., oxygen in morpholine, sulfur in thiomorpholine, nitrogen in n-methylpiperazine) further increases the potency of these candidates. virtual screening of a commercial library containing drugs and drug-like molecules against a d model of heparanase led to the identification of several candidates with high scores for binding affinity; they were subsequently tested in nmr competitive inhibition experiments using suramine as a known heparanase inhibitor [ ] . one of these candidates is amodiaquine (fig. ) , and a subset of fourteen representative compounds that retained the characteristic -phenylamino-chloroquinoline scaffold of amodiaquine, but presented different functional groups in the phenylamino moiety, was further selected for nmr competitive inhibition experiments. although no candidate with improved heparanase inhibitory activity over that of amodiaquine emerged from this subset, several structural requirements for the binding of an inhibitor to the active site of the enzyme were obtained. four mannich bases of nitroxoline (fig. ) were found to inhibit efficiently the activity of methionine aminopeptidase- from burkholderia pseudomallei with ic values ranging from low micromolar to nm, and a few candidates in this series also showed in vitro growth inhibition of burkholderia thailandensis [ ] . hexacyclic indole mannich bases (r ¼ h or ch , nr ¼ n(c h ) , -pyrrolidinyl) and (r ¼ ch , nr ¼ morpholinyl) derived from annelated maleimidoindolodiazepines (fig. ) were tested against a panel of human protein kinases, but they were inactive against all of the tested enzymes at micromolar concentrations [ ] . out of four synthesized acetylenic mannich bases (nr ¼ n(c h ) , -pyrrolidinyl, n-allyl-n-methyl, n-( dimethylaminoethyl)-n-methyl) derived from -(prop- -ynyloxy) chromen- -one (fig. ) , three were inhibitors of squalene-hopene cyclase [ ] . candidate (nr ¼ n-allyl-n-methyl) was half as potent as the hoffmanela roche's anticholesteremic drug ro - , which is an effective inhibitor of both squalene-and oxidosqualene cyclases that features the same secondary amino group in its structure. thiazolidinone c-mannich bases (r ¼ -ch c h , -no c h , -furyl, , , -(ch ) c h ; nr ¼ n(c h ) , morpholinyl, -methylpiperazinyl) (fig. ) were evaluated as inhibitors of schistosoma mansoni cercarial elastase, but they were all inactive [ ] . oxadiazolethione n-mannich bases having a naphthalen- ylmethyl residue at c- ( fig. ) were all active against mushroom tyrosinase, and to times more potent than reference compound kojic acid [ ] . the presence of a thione group able to chelate with copper, and the ability of the cyclic secondary amine to form extensive hydrophobic contacts within the binding site of tyrosinase appear to be responsible for the activity. another oxadiazolethione n-mannich base, namely m (table ) , presented moderate inhibitory activity against urease within the series of compounds investigated, but no comparison with the activity of a well-established urease inhibitor was provided [ ] . . . ligands of -hydroxytryptamine receptors -hydroxytryptamine receptors ( -ht receptors), also known as serotonin receptors, belong to the group of g protein-coupled receptors (with the exception of -ht , which is a ligand-gated ion channel). they are found in the central and peripheral nervous systems, and their function is to mediate both excitatory and inhibitory neurotransmission. -ht receptors are activated by their natural ligand, the neurotransmitter serotonin. mannich bases (n ¼ or ) (fig. ) were evaluated as ligands for the types of -ht receptors that could be involved in the mediation of the anticonvulsant effect of these compounds, but the candidates exhibited only moderate to low affinity for both -ht a (k i ¼ e nm) and -ht a receptors (k i ¼ e nm) [ ] . the nature of the spiranic cycloalkyl group did not influence serotonin receptor affinity significantly, although cyclohexylsubstituted candidates were slightly more active than cyclopentyl-substituted analogues. the nature of the substituent in the aryl moiety at n- of piperazine ring modulates the selectivity towards one of these -ht receptors; thus, mannich bases (r ¼ -och ) showed the highest affinity for ht a receptors and the lowest affinity for -ht a receptors, while the highest -ht a receptor affinity was shown by mannich bases (r ¼ -cf ). in addition, computer-aided design of novel mannich bases of imidazoline- , -diones led to the identification of several compounds with potential dual affinity for ht a receptors and serotonin transporter, whose synthesis and evaluation finally led to candidates (r ¼ h or f) (fig. ) having the desired pharmacological profile [ ] . a series of quinoxaline- -carboxamides n-substituted with phenolic mannich bases moieties having either one or two aminomethyl functions have been synthesized and tested for -ht receptor antagonistic activity in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against -ht agonist, -methyl- -ht [ , ] . candidates (r ¼ cl) (fig. ) exhibited mild to moderate antagonist activity, and double mannich bases were generally less potent than their mono-mannich bases counterparts, but mannich base (r ¼ cl, r ¼ h, nr ¼ -methylpiperazin- -yl) had -ht receptor antagonistic activity comparable to that of reference compound ondansetron [ ] . replacement of chlorine with methoxy led to less potent candidates (r ¼ och ), whereas candidates with an ethoxy group at position of quinoxaline ring were even less potent [ ] . the presence of piperazines in the aminomethyl moiety of candidates seemed to be more favorable for -ht receptor antagonistic activity than the presence of other cyclic secondary amines. dopamine receptors are g protein-coupled receptors that are widely distributed in the brain, and whose primary endogenous ligand is the neurotransmitter dopamine. there are at least five subtypes of dopamine receptors, but d - receptor subtypes usually predominate, as they are e times more numerous than d [ ] [ ] [ ] subtypes. discovery of l- , [ ] and fauc [ ] (fig. ) as potent and selective d receptor ligands has fueled the search for novel chemical entities having the ability to bind to dopamine receptors. based on the observation that these two ligands and many other dopamine receptor ligands feature a methylene bridge between the piperazine ring and the (hetero)aromatic part of the molecule, various analogues have been designed and synthesized. however, the majority of these analogues were obtained either through nucleophilic displacement by an appropriately substituted piperazine of a halogen atom from a halomethyl derivative of a selected (hetero)aromatic substrate, or through reductive amination of a formyl-substituted (hetero)aromatic substrate in the presence of a piperazine. literature search revealed a few examples when the mannich reaction was employed to synthesize the ligands to be evaluated for dopamine receptor binding ability. in addition to pyrrolo [ , -b] pyridine and pyrazolo [ , -a]pyridine ring systems used to in the generation of ligands and , imidazo[ , -a]pyridine is another example of an azaindole ring system that was aminomethylated to yield mannich bases (fig. ) [ ] . receptor binding profiles of candidates and several mono-mannich bases derived from pyrrole ( fig. ) were determined in vitro by measuring their ability to compete with [ h]spiperone for the cloned human dopamine receptor subtypes d long , d short , d and d . , while d receptor affinities were measured employing an assay that used d selective ligand [ h]sch and porcine striatal membranes. all of the mannich bases and were selective ligands for the d receptor subtype, and the candidates with an imidazo[ , -a]pyridine scaffold were more potent than their counterparts having a pyrrole ring. the most potent mannich bases (r ¼ -c h o, , -cl , -cf ) showed an affinity for the dopamine d receptor in the low nanomolar range, and their selectivity for this receptor subtype exceeded that of reference drug clozapine [ ] . several small series of candidates comprising mannich bases , and (r ¼ h or cl) having a pyrrolo[ , -d]pyrimidine scaffold (fig. ) were designed and evaluated as ligands for dopamine receptor subtypes d , d long , d short , d and d . , but they had no appreciable affinity for any of the dopamine receptors tested [ ] . the strongest binding in these series was recorded for candidates (r ¼ -ch o) and (r ¼ h, r ¼ -ch o) at dopamine d . receptor, with k i values of . and . nm, respectively. novel heteroaromatic scaffolds have been employed in the synthesis of mannich bases , and (fig. ) as potential d receptor ligands [ ] . candidate derived from pyrrolo[ , c]pyridine ring system bound selectively at d . receptor (k i ¼ . nm). evaluation of mannich bases generated from imidazo [ , -c] pyrimidines and having various substituents at positions , and led to different results, depending on the nature and number of these substituents. thus, mannich base (r ¼ r ¼ r ¼ h) derived from the unsubstituted scaffold and all of the mannich bases (r ¼ ch , r ¼ r ¼ h) obtained from methylimidazo[ , -c]pyrimidine had no effect on dopamine d receptor. the results for the binding assay for mannich bases derived from dimethyl-substituted imidazo [ , -c] pyrimidines showed that candidates (r ¼ r ¼ ch , r ¼ h) have binding affinities to dopamine d receptor in the range of reference drug clozapine, while candidate (r ¼ r ¼ ch , r ¼ h) is a weak ligand. further modifications, such as introduction of a carbethoxy group in the structure of candidates (r ¼ r ¼ ch , r ¼ cooc h ), or two methoxy groups in mannich bases (r ¼ r ¼ och , r ¼ h), led to compounds without affinity for dopamine d receptor. in the series of mannich bases derived from , , -trimethylimidazo[ , -c]pyrimidine, only candidate (r ¼ r ¼ r ¼ ch , r ¼ -och ) had good binding properties for the dopamine d receptor, whereas another candidate (r ¼ r ¼ r ¼ ch , r ¼ -och ) exhibited a slight selectivity for dopamine d receptor. use of pyrrolo [ , -d] pyrimidine as scaffold led to some of the most potent ligands of dopamine d receptor in this study (k i values between . and . nm). the nature of the amino group at position of pyrrolo [ , -d] pyrimidine scaffold modulates the activity; thus, mannich bases (nr ¼ pyrrolidinyl) had good affinities for dopamine d receptor, but replacement of pyrrolidine with morpholine resulted in significant loss of dopamine d receptor binding affinity. two novel indole mannich bases (r ¼ -c h oc h , , -cl c h ) (fig. ) were synthesized through direct aminomethylation and evaluated as ligands for several dopamine receptor subtypes, along with other heteroarylmethylpiperazines [ ] . both candidates were potent and selective dopamine d receptor ligands, and mannich base (r ¼ -c h oc h ) was the most potent compound in this library (k i ¼ . nm). in addition, mannich base (r ¼ , -cl c h ) was the most potent candidate having a -( , -dichlorophenyl)piperazine moiety, suggesting the importance of indole as (hetero)aromatic moiety in this type of dopamine d receptor ligands. the effect on the affinity to d , d and d dopamine receptor subtypes of replacement of -arylpiperazinyl in mannich bases with other monocyclic or bicyclic amine residues was also investigated [ ] . mannich bases (x ¼ ch or n) (fig. ) derived from -( -chlorophenyl)- -azabicyclo[ . . ]octan- -ol as amine reagent in the mannich reaction had modest affinity and moderate selectivity for dopamine d receptor subtype compared to d and d receptor subtypes. pyrrolidinol analogues (x ¼ ch or n) (fig. ) had poor binding affinities to all subtypes of dopamine receptors, while homopiperazine analogue (fig. ) had moderate binding affinity to dopamine d receptor subtype (k i ¼ . nm). candidate derived from , -diazabicyclo[ . . ] nonane ring system as amine reagent in the mannich reaction ( fig. ) bound poorly to all types of dopamine receptor investigated in this study. although candidate (fig. ) derived from , -diazabicyclo[ . . ]heptane ring system as amine reagent in the mannich reaction had the highest d receptor affinity of all the compounds tested in this paper (k i ¼ nm), it also had poor selectivity towards d (k i d ¼ nm, k i d ¼ nm). mannich base derived from -( -pyrimidinyl)piperazine (fig. ) had modest binding affinity to dopamine d receptor subtype (k i ¼ nm), but good selectivity over d and d subtypes. thyroid receptors are nuclear receptors belonging to a superfamily whose members function as hormone activated transcription factors. the regulation of transcription for thyroid receptors is controlled by their natural ligand, the thyroid hormone. both unliganded and liganded thyroid receptors can bind to and regulate genes under the control of thyroid response elements, but the liganded thyroid receptor complex can also recruit a particular coactivator protein out of many available, with a view to steer the course of transcriptional regulation. high-throughput screening of a library of compounds identified a number of hits for inhibitors of the interaction between thyroid receptor and its coactivator src (steroid receptor coactivator ), and two of these hit compounds were ketonic mannich bases and (fig. ) [ ] . these candidates represent a new class of thyroid receptor antagonists that have exceptional selectivity towards b isoform and are active in the presence of thyroid hormone, while being able to silence its signaling. mannich bases and are most likely covalently bound to thyroid receptor as a result of an alkylation process, thus inhibiting its hormone-induced gene transcription. subsequent preliminary sar showed that a hydrophobic moiety para to the ketone function in other ketonic mannich bases analogous to and is an important structural feature of potent inhibitors of the thyroid receptorecoactivator interaction [ ] . a second series of candidates explored the effect that the nature of the amino group in ketonic mannich bases analogous to and derived from -nhexylacetophenone has on the interaction between coregulatory protein src and both isoforms of thyroid receptors. all the synthesized candidates inhibited the interaction in the low micromolar range, with a roughly -fold selectivity towards isoform b. these ketonic mannich bases most likely function as prodrugs for the true active species, namely the a,b-unsaturated ketones resulted through deamination. therefore, a collection of enones having various substituents at the carbonecarbon double bond was also tested with a view to provide an insight on the mechanism of action of ketonic mannich bases as inhibitors of this interaction. the results showed that the inhibition of the interaction between thyroid receptor and coactivator protein src depends strongly on the substitution pattern at the carbonecarbon double bond in these a,b-unsaturated ketones. the most potent was the unsubstituted enone (fig. ) , which formally arises from through deamination, whereas all other substituted enones were weaker inhibitors than . subsequent investigations confirmed that enone is the actual inhibitor, and that compound is generated within the binding site, where it remains bound until it reacts with one of the local cysteine residues [ ] . in addition, an attempt to examine a complex between thyroid receptor b and mannich base by x-ray crystallography led to the detection in the activation function- cleft of thyroid receptor b of enone instead of mannich base . although enone did not react rapidly with thyroid hormone, it positioned close to several cysteine residues. several other experiments showed that, once formed, enone slowly alkylates nearby cys residue to occlude activation function- pocket of thyroid receptor b. significant improvement of pharmacological properties (especially potency and therapeutic index) of inhibitors of thyroid hormone receptor coactivator binding based on ketonic mannich bases was achieved with a series of novel candidates having in the aromatic ring electron withdrawing substituents associated with high efficacy, and a sulfonyl group to reduce cytotoxicity. also, piperazinone and -acetylpiperazine were used as preferred amine moieties with a view to minimize ion channel activity of these inhibitors [ ] . the most potent compounds and (r ¼ , -cl or , -cl ) (fig. ) showed outstanding thyroid hormone receptor coactivator interaction inhibitory potency, good therapeutic index, while no inhibition of kcl-stimulated depolarization of hek -herg cells was observed for these mannich bases. androgen receptor is a ligand-regulated transcription factor in the nuclear receptor superfamily. the receptor, which represents an important molecular target for the treatment of prostate cancer, is activated through the binding of its two natural endogenous ligands, testosterone or a-dihydrotestosterone. subsequent activation of androgen-responsive genes can be blocked by androgen receptor antagonists through competitive inhibition. highthroughput screening of a structurally diverse chemical library comprising , synthetic and natural compounds led to the identification of hit compounds that exhibited more than % competitive inhibition of a-dihydrotestosterone binding to androgen receptor [ ] . mannich base (r ¼ r ¼ ch , r ¼ c h ) (fig. ) was selected for further development owing to its consistent androgen receptor antagonist activities in a cellbased assay using monkey kidney fibroblast cv- cells, and fortynine analogues were designed based on its b-amino ketone core structure. nine of these analogues showed higher binding affinity to androgen receptor than a-dihydrotestosterone, and evaluation of the enantiomers of candidate (r ¼ no , r ¼ cl, r ¼ furyl) showed that the androgen receptor-modulating activity and binding resided with the dextrorotatory isomer. sar within this library of ketonic mannich bases revealed that the presence of chlorine as substituent r , preferably para to the amino group, is important for androgen receptor antagonistic effects, while the presence of an electron-withdrawing group as substituent r is crucial for high-affinity binding to the receptor. good androgen fig. . mannich bases as inhibitors of the interaction between thyroid receptors and their coregulators. receptor binding activity was found among analogues having phenyl, -furyl and -thienyl as substituent r , and mannich bases with heteroaryl moiety exhibited less cytotoxicity towards hela cells than mannich bases with r ¼ (substituted)phenyl. candidate (r ¼ no , r ¼ cl, r ¼ -furyl) inhibited the growth of sc cells (a mouse mammary cell line that responds well to androgens), while its effects on the proliferation of pc cells (a human prostate cancer cell line that lacks measurable androgen receptors) were very weak. mannich bases of type were subsequently disclosed as selective non-steroidal antagonists of another member of the nuclear receptor superfamily, namely progesterone receptor, and sar within a collection of mannich bases showed that coordinating effects of substituents r and r can modulate the potency and selectivity for progesterone receptor over androgen receptor [ ] . mannich bases that modulate the activity of acetylcholine receptors have been the topic of two recent studies. two quaternary salts (r ¼ ch , allyl) derived from bis-aminomethylated pyrazinodiindole (fig. ) were synthesized in order to probe their binding to the allosteric binding domain of muscarinic receptor m [ ] . these quaternary salts showed only slightly higher binding affinity to muscarinic m receptor subtype than the corresponding parent double mannich bases, which in turn exhibited relatively poor allosteric potency. due to the structural similarity of candidates and neuromuscular-blocking agents, their binding affinity to the muscle-type of the nicotinic acetylcholine receptors was also determined. thus, compound (r ¼ ch ) had a -fold higher affinity for the muscle type nicotinic acetylcholine receptor than for the allosteric site of m receptors. double mannich bases (nr ¼ -piperidinyl, -methyl- -piperidinyl, -azepanyl) derived from phthalamide (fig. ) had a competitive antagonistic effect to acetylcholine on isolated guinea pig ileum that was poorer than that of reference compound atropine, and behaved as noncompetitive antagonists at higher concentration [ ] . in addition, candidates (nr ¼ -piperidinyl, -methyl- -piperidinyl, -azepanyl) antagonize the motor effect of oxotremorine in mice, while mannich base (nr ¼ , -dimethyl- -piperidinyl) was inactive in both tests. indole mannich base (r ¼ phenethyl) (fig. ) had moderate binding affinity to both sigma- (k i ¼ . nm) and sigma- (k i ¼ . nm) receptors, and can be considered as a potent, but non-selective ligand for sigma receptors [ ] . ketonic mannich bases (fig. ) derived from cyclic ketones such as a-tetralone (x ¼ ch ch ), chromanone (x ¼ och ), indanone (x ¼ ch ) or benzosuberone (x ¼ (ch ) ) as substrates and either benzylpiperidine (z ¼ ch ) or -benzylpiperazine (z ¼ n) as amine reagents in the mannich reaction were also evaluated for their affinity towards sigma- and sigma- receptors [ ] . generally, -benzylpiperazine-containing mannich bases were higher-affinity sigma receptor ligands than the corresponding benzylpiperidine-containing analogues. compared to other alkylpiperidines or alkylpiperazines that were evaluated in this study, ketonic mannich bases had only moderate affinity for sigma receptors. the presence of the carbonyl group in their structure appears to be detrimental, especially for the affinity to sigma- receptors, and the binding affinity seems to be influenced by the size of the saturated ring fused to the aromatic ring. thus, sixmembered candidates are more potent than mannich bases derived from indanone or benzosuberone. however, candidate (x ¼ ch ch , z ¼ ch ) was one of the most selective compounds in this study, with a selectivity towards sigma- receptors of approximately . (k i-s ¼ nm, k i-s ¼ . nm). in contrast, mannich bases derived from -benzylpiperazine were consistently better ligands for sigma- than for sigma- receptors. mannich bases of indoles substituted in the carbocyclic ring with a carboxamide group were evaluated as human histamine h receptor antagonists [ ] . preliminary analysis of the binding affinity of candidates (r ¼ i-c h , c-c h , x ¼ ch , o, nc h -i, nc h -c) (fig. ) to human h receptor with respect to different positional isomers within this series established a correlation between the , -substitution pattern in and good human h receptor affinity, while the , -substitution pattern was the least favorable for h receptor binding affinity. the presence of piperidine and morpholine in the aminomethyl group led to compounds with excellent histamine h receptor affinity, whereas the presence of -substituted piperazines in the aminomethyl group was not well tolerated. several mannich bases of indoles with a , substitution pattern also presented reasonable potency as human h antagonists; in this case, the presence of a cyclobutyl residue in the piperazine ring in the carboxamide group seems to be more favorable for human histamine h binding affinity than substitution with an isopropyl residue. in the search for new ligands able to discriminate among the three a -adrenergic receptor subtypes, a series of mannich bases (r ¼ ch , n-c h , ch c h , ch c h -c, c h ) derived from , , , -tetrahydro- h-carbazol- -ones (fig. ) , that are structurally related to the potent a -adrenergic receptor antagonist heat, were synthesized and evaluated [ ] . as a general trend, all candidates showed lower affinities than heat for all three a -adrenergic receptor subtypes, and replacement of tyramine with -( substituted phenyl)piperazines did not enhance the affinity towards any subtype in particular. in addition, mannich bases derived from -acetylindole and the same amines ( fig. ) were designed as "open chain" analogues of candidates , and their evaluation showed a slight improvement in the affinity for all three a -adrenergic receptor subtypes. starting from a hit obtained by screening a large library of compounds, and employing subsequent stepwise refinement of structural features, a series of mannich bases (r ¼ (substituted) phenyl) of pyrazolones (fig. ) was designed as ligands of cc chemokine receptor (ccr ), a potential molecular target for treatment of allergic asthma [ ] . the evaluation of binding to human ccr receptor of candidates showed that most compounds display ic values around nm, and that only two of these mannich bases (r ¼ -fc h and -pyridinyl) bound more tightly to ccr than the initial hit compound (ic ¼ nm). because the lead compound (r ¼ -fc h , ic ¼ nm) suffered from poor water solubility and metabolic stability, less lipophilic analogues were prepared by the introduction of ionizable groups in different parts of the molecule. replacement in the structure of (r ¼ -fc h ) of the -chlorophenyl moiety with -fluorophenyl, -nitrophenyl, -sulfonamidophenyl, aminophenyl or pyridinyl moieties, as well as substitution of the , -diflurophenyl moiety with pyridinyl or pyridinyl-n-oxide led to a second series of pyrazolone mannich bases ( fig. ) with improved solubility in water and enhanced metabolic stability. although structural modulations in the r moiety generally decreased the binding affinity of candidates to ccr , modifications in the r part afforded potent compounds (ic ¼ nm). two n-mannich bases (r ¼ h or f) of a pyrazole derivative (fig. ) were reported in a study that examined the interaction of a series of -heteroaryl- -phenylquinolines with neurokinin nk- and nk- receptors, but they were inactive (k i > mm) [ ] . throughout the last decade, a large number of novel mannich bases have been synthesized and evaluated as potential treatments for a multitude of diseases and medical conditions, as prodrugs, or as molecules eliciting a response from biological targets. the vast amount of data concerning the structureeactivity relationship for mannich bases derived from structurally diverse substrates has added to previous knowledge, thus allowing better insight into the design of more effective drug candidates based on the aminomethylation reaction in the future. tremendous progress has been made in the field of anticancer agents and antimicrobials, and the last decade has witnessed the chemical modification of many biologically-active, naturally-occurring substrates or well established drugs by means of the mannich reaction with a view to improve their biological activity. as such, the mannich reaction has earned its rightful place as a powerful tool in medicinal chemistry, both for the synthesis of novel chemical entities endowed with various and interesting biological properties, and for the modification of physico-chemical properties of a candidate, that ultimately influence the candidate's biodisponibility, performance and pharmacological activity as a drug. advances in the chemistry of mannich bases further advances in the chemistry of mannich bases mannich bases: chemistry and uses effect of aminomethyl (n-mannich base) derivatization on the ability of s -acetyloxymethyl- -mercaptopurine prodrug to deliver -mercaptopurine through hairless mouse skin prodrugs e an efficient way to breach delivery and targeting barriers gilmer, b-aminoketones as prodrugs with phcontrolled activation anticancer and cytotoxic properties of mannich bases bioactivities of chalcones cytotoxic thiol alkylators sequential cytotoxicity: a theory evaluated using novel -[ -( -aryl- -propenoyloxy)phenylmethylene]cyclohexanones and related compounds evaluation of some mannich bases of cycloalkanones and related compounds for cytotoxic activity cytotoxic and anticancer activities of some -aryl- -dimethylaminomethyl- -propen- -one hydrochlorides cytotoxic activities of mannich bases of chalcones and related compounds cytotoxic activities of mono and bis mannich bases derived from acetophenone against renca and jurkat cells cytotoxic activities of some mono and bis mannich bases derived from acetophenone in brine shrimp bioassay cytotoxicity of some azines of acetophenone-derived mannich bases against jurkat cells synthesis of some mannich bases with dimethylamine and their hydrazones and evaluation of their cytotoxicity against jurkat cells evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells cytotoxic and anticonvulsant aryloxyaryl mannich bases and related compounds biological evaluation and structureactivity relationships of bis-( -aryl- -oxo-propyl)-methylamine hydrochlorides and -aryl- -arylcarbonyl- -methyl- -piperidinol hydrochlorides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic t cells effect of some bis mannich bases and corresponding piperidinols on dna topoisomerase i the design and cytotoxic evaluation of some -aryl- -isopropylamino- -propanone hydrochlorides towards human huh- hepatoma cells cytotoxicity of -aryl- -butylamino- -propanone hydrochlorides against jurkat and l cells synthesis of -aryl- -phenethylamino- -propanone hydrochlorides as possible potent cytotoxic agents biological activity of -aryl- -phenethylamino- -propanone hydrochlorides and -aroyl- -aryl- -phenethyl- -piperidinols on pc- cells and dna topoisomerase i enzyme effect of acetophenone derived mannich bases on cellular glutathione level in jurkat cells. a possible mechanism of action syntheses and stability studies of some mannich bases of acetophenones and evaluation of their cytotoxicity against jurkat cells effects of mannich bases on cellular glutathione and related enzymes of jurkat cells in culture conditions the effects of some mannich bases on heat shock proteins hsc and grp , and thioredoxin and glutaredoxin levels in jurkat cells cytotoxic mannich bases of -arylidene- -tetralones cytotoxic and topographical properties of -arylidene- -dimethylaminomethylcyclohexanone hydrochlorides and related compounds potential role of nmyristoyltransferase in cancer inhibition of protein n-myristoylation: a therapeutic protocol in developing anticancer agents synthesis and cytotoxic and mechanistic studies of a-arylidenecyclohex(pent)anone or aarylcyclohexanone a'-mannich bases and their deoxo bisaryl cyclohex(pent) ene analogs synthesis and anticancer activity of -alkylaminomethyl- -diaryl-methylenecyclopentanone hydrochlorides and related compounds dimmock, a-substituted -aryl- -dimethylaminopropanone hydrochlorides: potent cytotoxins towards human widr colon cancer cells -aryl- -dimethylaminomethyl- -propen- -one hydrochlorides and related adducts: a quest for selective cytotoxicity for malignant cells synthesis of -hydroxy- -piperidinomethylchalcone derivatives and their cytotoxicity against pc- cell lines synthesis and cytotoxicity of novel -aryl- -( -dibenzylaminomethyl- -hydroxyphenyl)-propenones and related compounds design, synthesis and in vitro cytotoxic activity evaluation of new mannich bases design, synthesis, and biological evaluation of mannich bases of heterocyclic chalcone analogs as cytotoxic agents -( -aminomethyl- -hydroxyphenyl)- -pyridinyl- -propen- -ones: a novel group of tumour-selective cytotoxins preparation of aminoalkyl substituted chalcones for use in anticancer and antimalarial pharmaceutical compositions preparation of a series of novel bichalcones linked with a , -dimethylenepiperazine moiety and examination of their cytotoxicity new bichalcone analogs as nf-kb inhibitors and as cytotoxic agents inducing fas/cd -dependent apoptosis azidothymidine is effective against human multiple myeloma: a new use for an old drug hłado n, synthesis and anticancer activity of -chloromethylphosphonates of -azido- -deoxythymidine (azt) antileukemic activity and cellular metabolism of the aryl phosphate derivative of bromo-methoxy zidovudine (compound whi- ) synthesis and in vitro cytotoxic activity evaluation of novel mannich bases and modified azt derivatives possessing mannich base moieties via click chemistry synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and mapk pathway activation cytotoxicity and antimicrobial activity of some naphthol derivatives synthesis and cytotoxicity evaluation of some -hydroxyquinoline derivatives synthesis and structure-activity relationship study of -hydroxyquinoline-derived mannich bases as anticancer agents design, synthesis and bioevaluation of novel candidate selective estrogen receptor modulators recent advancement in nonsteroidal aromatase inhibitors for treatment of estrogen-dependent breast cancer facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors nitrogen-containing apigenin analogs: preparation and biological activity synthesis and antiproliferative activity of oxazinocarbazole and n,n-bis(carbazolylmethyl)amine derivatives synthesis, antitumour activity and structureeactivity relationships of h-benzo[b]carbazoles display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and compare algorithm -anthradiquinone as precursor for antitumor compounds the natural anticancer agent plumbagin induces potent cytotoxicity in mcf- human breast cancer cells by inhibiting a pi- kinase for ros generation anti-tumor effects of novel -o-acyl plumbagins based on the inhibition of mammalian dna replicative polymerase activity inhibitory effect of novel -o-acyl juglones on mammalian dna polymerase activity, cancer cell growth and inflammatory response cytotoxic activity of naphthoquinones with special emphasis on juglone and its -o-methyl derivative antiproliferative activity of synthetic naphthoquinones related to lapachol. first synthesis of -hydroxylapachol novel platinum(ii) complexes of -(aminomethyl)naphthoquinone mannich bases: synthesis, crystal structure and cytotoxic activities exploring the dna binding/cleavage, cellular accumulation and topoisomerase inhibition of -hydroxy- -(aminomethyl)- , -naphthoquinone mannich bases and their platinum(ii) complexes novel -(aminomethyl)naphthoquinone mannich base-platinum(iv) complexes: synthesis, characterization, electrochemical and cytotoxic studies one-pot synthesis and cytotoxicity studies of new mannich base derivatives of polyether antibiotic e lasalocid acid novel hexacyclic camptothecin derivatives. part : synthesis and cytotoxicity of camptothecins with an a-ring fused , -oxazine ring optimization of tocotrienols as antiproliferative and antimigratory leads quinone methides tethered to naphthalene diimides as selective g-quadruplex alkylating agents hybrid ligandalkylating agents targeting telomeric g-quadruplex structures synthesis and biological activities of quinazoline derivatives with ortho-phenol-quaternary ammonium salt groups dna binding, antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives synthesis and biological evaluation of , -dihydroxyxanthone mannich base derivatives as potential antitumor agents synthesis and in vitro evaluation of novel indole-based sigma receptors ligands synthesis and cytotoxic activity of novel -methyl- -[( -substituted- -piperazinyl)methyl]- h-indole derivatives design, synthesis, and biological evaluation of indole-based , -disubstituted piperazines as cytotoxic agents amino derivatives of indole as potent inhibitors of isoprenylcysteine carboxyl methyltransferase functionalized indoleamines as potent, drug-like inhibitors of isoprenylcysteine carboxyl methyltransferase (icmt) -aminomethyl derivatives of , -dihydroxynaphtho[ , -f]indole- , -dione for circumvention of anticancer drug resistance synthesis and structureeactivity relationship studies of naphtho[ , -f]indole- , -dione aminoalkyl derivatives: a new class of topoisomerase i inhibitors synthesis of -substituted -[ -(dialkylaminomethyl)indol- -yl]maleimides and study of their ability to inhibit protein kinase c-a, prevent development of multiple drug resistance of tumor cells and cytotoxicity synthesis and sar of novel -morpholinopyrrolopyrimidine derivatives as potent phosphatidylinositol -kinase inhibitors cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from synthesis and in-vitro cytotoxicity evaluation of gatifloxacin mannich bases hybrid pharmacophore design and synthesis of isatinebenzothiazole analogs for their anti-breast cancer activity synthesis of novel isatin-thiazoline and isatin-benzimidazole conjugates as anti-breast cancer agents design and synthesis of anti-breast cancer agents from -piperazinylquinoline: a hybrid pharmacophore approach hydroxyphenyl)- -substituted- , -dihydro- , , -oxadiazole- -thione derivatives: promising anticancer agents synthesis of some novel , -disubstituted , , -oxadiazole derivatives and anticancer activity on eac animal model synthesis, quantitative structure-activity relationship and biological evaluation of , , -oxadiazole derivatives possessing diphenylamine moiety as potential anticancer agents synthesis, antitumor and antimicrobial activity of novel -substituted phenyl- -[ -alkylamino(methyl)- -thioxo- , , -oxadiazol- -yl]-b-carboline derivatives synthesis and antitumor activity of fluoroquinolone c -isostere derivatives: oxadiazole mannich base derivatives synthesis, characterization and in vitro cytotoxic properties of some new schiff and mannich bases in hep g cells design, synthesis and antitumor activities of fluoroquinolone c- heterocycles (iv): s-triazole schiffemannich bases derived from ofloxacin facile synthesis and quantitative structureeactivity relationship study of antitumor active -( -oxo-thiazolidin- -ylidene)- -oxo-propionitriles synthesis and biological activity evaluation of -pyrazoline substituted -thiazolidinones synthesis, xray crystallographic analysis, and antitumor activity of n-(benzothiazole- -yl)- -(fluorophenyl)-o,o-dialkyl-a-aminophosphonates synthesis and biological activities of aaminophosphonates derivatives containing thieno[ , -c]pyridine (in chinese) synthesis, antimicrobial and anticancer activities of a novel series of diphenyl -(pyridin- -yl) ethylphosphonates design and one-pot synthesis of a-aminophosphonates and bis(aaminophosphonates) by iron(iii) chloride and cytotoxic activity synthesis, antiproliferative activity in cancer cells and theoretical studies of novel a, b-dihydroxyvouacapan- b-oic acid mannich base derivatives synthesis and anti-tumor activities of novel synthesis and cytotoxicity studies of novel dimethylamino-functionalised and n-heteroaryl-substituted titanocene anticancer drugs: synthesis and cytotoxicity studies synthesis and cytotoxicity studies of new dimethylamino-functionalised and indolylsubstituted titanocene anti-cancer drugs synthesis and cytotoxicity studies of new dimethylamino-functionalized and azolesubstituted titanocene anticancer drugs synthesis and preliminary cytotoxicity studies of achiral indolyl-substituted titanocenes multidrug resistance reverting activity and antitumor profile of new phenothiazine derivatives synthesis of antitumoractive betulinic acid-derived hydroxypropargylamines by copper-catalyzed mannich reactions cytotoxic betulin-derived hydroxypropargylamines trigger apoptosis doxoform and daunoform: anthracyclineformaldehyde conjugates toxic to resistant tumor cells doxsaliform: a novel n-mannich base prodrug of a doxorubicin formaldehyde conjugate design, synthesis, and biological evaluation of doxorubicin-formaldehyde conjugates targeted to breast cancer cells antiestrogen binding site and estrogen receptor mediate uptake and distribution of -hydroxytamoxifen-targeted doxorubicin-formaldehyde conjugate in breast cancer cells rational design and synthesis of androgen receptortargeted nonsteroidal anti-androgen ligands for the tumor-specific delivery of a doxorubicin-formaldehyde conjugate studies of targeting and intracellular trafficking of an anti-androgen doxorubicin-formaldehyde conjugate in pc- prostate cancer cells bearing androgen receptor-gfp chimera doxorubicin-formaldehyde conjugates targeting a v b integrin poly (ethylene glycol) prodrug for anthracyclines via n-mannich base linker: design, synthesis and biological evaluation synthesis and invitro antitumor activities of some mannich bases of -alkyl- , , , -tetrahydrocarbazole- -ones synthesis and biological evaluation of novel mannich bases of -arylimidazo [ , -b]benzothiazoles as potential anti-cancer agents cytotoxic mannich bases of -( -aryl- -propenoyl)- ( h)-benzoxazolones new synthetic chalcones: cytotoxic mannich bases of -( -chlorocinnamoyl)- ( h)-benzoxazolone synthesis and cytotoxicity of novel hexahydrothieno-cycloheptapyridazinone derivatives synthesis and antibacterial study of unsaturated mannich ketones synthesis and antibacterial activity of fused mannich ketones novel mannich ketones of oxazolidinones as antibacterial agents synthesis and biological evaluation of a novel series of , -bisaminomethylated aurone analogues as anti-inflammatory and antimicrobial agents evaluation of antimicrobial activities of several mannich bases and their derivatives synthesis and biological evaluation of aminoketones highly efficient one-pot synthesis, antimicrobial and docking studies of newer b-amino carbonyl derivatives catalyzed by silica sulfuric acid synthesis of b-amino carbonyl derivatives of coumarin and benzofuran and evaluation of their biological activity vuki cevi c, antibacterial -(arylamino)- -ferrocenylpropan- -ones: synthesis, spectral, electrochemical and structural characterization synthesis and characterization of some mannich bases as potential antimicrobial agents synthesis, characterization and antimicrobial evaluation of copper(ii) complexes of -tert-butyl-pyrocatechin-derived mannich bases synthesis and microbiological evaluation of mannich bases derived from , -diacetylresorcinol mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities novel aminonaphthoquinone mannich bases derived from lawsone and their copper(ii) complexes: synthesis, characterization and antibacterial activity synthesis, antiinflammatory and antimicrobial activity of some new -( -phenyl- , -dihydro- h- , -benzoxazin- -yl)ethanone derivatives an eco-friendly synthesis and antimicrobial activities of dihydro- h-benzo-and naphtho- , -oxazine derivatives synthesis and antibacterial evaluation of benzazoles tethered dihydro[ , ]oxazines ]oxazine derivatives catalyzed by zirconyl chloride and evaluation of its biological activities biocidal activity of some mannich base cationic derivatives some electrophilic substitution reactions on -substituted- -acetyl/carbethoxy- -hydroxy- -methylindole and the antimicrobial activity of these new indole derivatives mannich base derivatives of -hydroxy- -methyl- h-pyran- -one with antimicrobial activity a study of cytotoxicity of novel chlorokojic acid derivatives with their antimicrobial and antiviral activities synthesis and biological activities of new mannich bases of chlorokojic acid derivatives design, synthesis and in vivo/ in vitro screening of novel chlorokojic acid derivatives mannich bases of -piperazinylquinolones and kojic acid derivatives: synthesis, in vitro antibacterial activity and in silico study synthesis and antimicrobial screening of novel mannich bases of isatin derivative synthesis and antimicrobial evaluation of some novel trisubstituted s-triazine derivatives based on isatinimino, sulphonamido and azacarbazole synthesis and antimicrobial screening of certain novel mannich bases bearing , -naphthyridine moiety synthesis and antimicrobial activities of oxadiazoles, phthalazines and indolinones synthesis and antimicrobial activity of mannich bases of isatin and its derivatives with -[( , -dichlorophenyl) amino]phenylacetic acid synthesis, antimicrobial screening and beta lactamase inhibitory activity of -( -chloro- -fluorophenylimino)indolin- -one and -chloroindolin- -one derivatives, turk synthesis, antiviral and antibacterial activities of isatin mannich bases synthesis and bioevaluation of schiff and mannich bases of isatin derivatives with -amino- -benzyl- , -dihydro- h- , , -triazole- -thione synthesis, characterization and in vitro antimicrobial activity of some novel -substituted schiff and mannich base of isatin derivatives synthesis and antimicrobial activity of novel -( -adamantyl)- -aminomethyl- -substituted- , , -triazoline- -thiones microbiologically active mannich bases derived from , , -triazoles. the effect of c- substituent on antibacterial activity synthesis and antimicrobial activity of thiosemicarbazides, s-triazoles and their mannich bases bearing -chlorophenyl moiety synthesis and antibacterial activity of some novel n -hydroxymethyl and n -aminomethyl derivatives of -aryl- -( -chlorophenyl)- , -dihydro- h- , , -triazole- -thione synthesis and in vitro activity of , , -triazole-ciprofloxacin hybrids against drug-susceptible and drug-resistant bacteria synthesis, characterization and antibacterial activity of some triazole mannich bases carrying diphenylsulfone moieties synthesis of some new s-alkylated , , -triazoles, their mannich bases and their biological activities synthesis and biological activities of some novel aminomethyl derivatives of -substituted- -( -thienyl)- , -dihydro- h- , , -triazole- -thiones design, synthesis and antimicrobial activities of some azole derivatives synthesis of some new , , -triazoles, their mannich and schiff bases and evaluation of their antimicrobial activities syntheses and biological activities of new hybrid molecules containing different heterocyclic moieties alpay karao glu, preparation and antimicrobial activity evaluation of some quinoline derivatives containing an azole nucleus convenient one pot synthesis and antibacterial evaluation of some new mannich bases carrying , , -triazolyl moiety synthesis and antimicrobial activities of some new biheterocyclic compounds containing , , -triazol- -one and , , -thiadiazole moieties synthesis of some new , , -thiadiazol- -ylmethyl- , , -triazole derivatives and investigation of their antimicrobial activities synthesis and biological activities of methylenebis- h- , , -triazole derivatives regioselective reaction: synthesis, characterization and pharmacological studies of some new mannich bases derived from , , -triazoles el-emam, synthesis, antimicrobial and anti-inflammatory activities of novel -( -adamantyl)- -[(arylidene)amino]- -mercapto- , , -triazoles and related derivatives convenient one pot synthesis and antimicrobial evaluation of some new mannich bases carrying -methylthiobenzyl moiety synthesis and biological activity of schiff and mannich bases bearing , -dichloro- -fluorophenyl moiety synthesis of some new , , -triazoles starting from isonicotinic acid hydrazide and evaluation of their antimicrobial activities synthesis, antiinflammatory, analgesic, and antibacterial activities of some triazole, triazolothiadiazole, and triazolothiadiazine derivatives synthesis and pharmacological evaluation of clubbed isopropylthiazole derived triazolothiadiazoles, triazolothiadiazines and mannich bases as potential antimicrobial and antitubercular agents regioselective reaction: synthesis of novel mannich bases derived from -( , -disubstituted- -thiomethylpyrimidyl)- -amino- -mercapto- , , -triazoles and their antimicrobial properties new class of triazole derivatives and their antimicrobial activity synthesis, characterization, and biological activity of novel schiff and mannich bases of -amino- -(n-phthalimidomethyl)- , , -triazole- -thione alpay karao glu, reduction, mannich reaction and antimicrobial activity evaluation of some new , , -triazol- -one derivatives preparation and antimicrobial activity evaluation of some new bi-and triheterocyclic azoles synthesis and biological evaluation of some , , -oxadiazole derivatives synthesis, characterization and antimicrobial activity of some disubstituted , , -oxadiazoles carrying -(aryloxymethyl)phenyl moiety synthesis and antimicrobial activity of new -( -thienyl)- , , -triazoles and -( -thienyl)- , , -oxadiazoles and related derivatives synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed , , -oxadiazoles synthesis and antimicrobial activity of linked heterocyclics containing pyrazole and oxadiazoles synthesis and antimicrobial studies of some mannich bases carrying imidazole moiety synthesis and antimicrobial activity of some new -( -chloro- -benzothiophen- -yl)- , , -oxadiazole- -thiol and their derivatives synthesis, antimicrobial and antiinflammatory activities of , , -oxadiazoles linked to naphtho[ , -b]furan antimicrobial and antiurease activities of newly synthesized morpholine derivatives containing an azole nucleus synthesis and antimicrobial activities of some new , , -triazole derivatives synthesis of novel nalidixic acid-based , , -thiadiazole and , , -oxadiazole derivatives as potent antibacterial agents synthesis, characterization and antimicrobial activity of novel , -disubstituted- , , -oxadiazole- -ones synthesis of mannich bases of some , -disubstituted -thiazolidinones and evaluation of their antimicrobial activities synthesis, characterization and evaluation of antimicrobial activity of mannich bases of some -[( -carbethoxymethylthiazol- -yl)imino]- -thiazolidinones novel , -dibromo- ( h)quinazolinone derivatives of antibacterial and anti-fungal activities synthesis of -{ -[ -dimethylamino- -( -methyl- -oxo- h-chromen- -yloxy)-[ , , ]triazin- -ylamino]-phenyl}- -phenyl- -( -pyridin- -yl-piperazin- -ylmethyl)-thiazolidin- -one and their biological evaluation synthesis and biological activity of , -dihydropyrimidine- -thione aminomethylene derivatives based on the alkaloid cytosine synthesis and in vitro study of biological activity of heterocyclic n-mannich bases of , -dihydropyrimidine- ( h)-thiones synthesis and in vitro study of biological activity of heterocyclic n-mannich bases synthesis and in vitro study of novel mannich bases as antibacterial agents synthetic, spectral, antimicrobial and qsar studies on novel mannich bases of glutarimides evaluation of antimicrobial activity and qsar study of a molecule library of the mannich bases of glutarimides syntheses, spectral, crystallographic, antimicrobial, and antioxidant studies of few mannich bases synthesis and biological study of medicinally important mannich bases derived from -(dimethylamino)- , , a, , a, , , a-octahydro- , , , , a-pentahydroxynaphthacenecarboxamide hydrophobic vancomycin derivatives with improved adme properties: discovery of telavancin in vitro activity of telavancin against resistant gram-positive bacteria telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillinresistant staphylococcus aureus exploring the positional attachment of glycopeptide/b-lactam heterodimers vancomycin derivatives iodine catalyzed three-component synthesis of b-amino-b-keto-esters and their antimicrobial activity deep, synthesis, characterization and antimicrobial evaluation of novel derivatives of isoniazid synthesis and evaluation of some novel derivatives of -propoxybenzylideneisonicotinohydrazide for their potential antimicrobial activity synthesis and antimicrobial screening of novel series of imidazo[ , -a]pyridine derivatives synthesis and evaluation of antibacterial and antitubercular activities of some novel imidazo synthesis, antimicrobial evaluation and qsar study of some -hydroxypyridine- -one and -hydroxypyran- -one derivatives synthesis of novel biologically active methylene derivatives of sydnones synthesis, characterization, and antimicrobial screening of some mannich base sydnone derivatives facile syntheses of mannich bases of -[p-( -arylpyrazolin- -yl)phenyl]sydnones, as anti-tubercular and anti-microbial agents, under ionic liquid/tetrabutylammonium bromide catalytic conditions copper(ii) complex with the tridentate ligand n,n-bis( -ethyl- -methylimidazol- -ylmethyl)phenylethylamine (biaq). x-ray crystal structure and biological activity on bacillus subtilis of synthesis, sar study and evaluation of mannich and schiff bases of pyrazol- ( h)-one moiety containing -(hydrazinyl)- -phenylquinazolin- ( h)-one design, synthesis, antibacterial activity and physicochemical parameters of novel n- -piperazinyl derivatives of norfloxacin in vitro study of some medicinally important mannich bases derived from antitubercular agent microwave-assisted synthesis of some novel and potent antibacterial and antifungal compounds with biological screening synthesis and antimicrobial and antioxidant activities of simple saccharin derivatives with nbasic side chains synthesis and antimicrobial activity of -arylaminomethyl- -methoxyphenothiazine- -carboxylic acid in vitro antitubercular and antimicrobial activities of -substituted quinoxaline- , ( h, h)-diones synthesis of some new thioxoquinazolinone derivatives and a study on their anticonvulsant and antimicrobial activities synthesis and anti bacterial and anti-ulcer evaluation of new s-mannich bases of , -diaryl- , -dihydropyrimidin- ( h)-thiones world health organization antimycobacterial arylidenecyclohexanones and related mannich bases methoxyphenylcarbonyloxy)benzylidene]- -dimethylaminomethyl cyclohexanone hydrochloride: a mannich base which inhibits the growth of some drug-resistant strains of mycobacterium tuberculosis major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis mycobacterium tuberculosis isocitrate lyases and are jointly required for in vivo growth and virulence isocitrate lyase: a potential target for anti-tubercular drugs identification of mannich base as a novel inhibitor of mycobacterium tuberculosis isocitrate by high-throughput screening synthesis and in vitro and in vivo antimycobacterial activity of isonicotinoyl hydrazones h-pyran- -one derivatives: leading molecule for preparation of compounds with antimycobacterial potential molecular modeling and antimycobacterial studies of mannich bases: -hydroxy- -methyl- h-pyran- -ones bactericidal activities of the pyrrole derivative bm against multidrug-resistant and intramacrophagic mycobacterium tuberculosis strains synthesis and microbiological activities of pyrrole analogs of bm , a potent antitubercular agent new pyrrole derivatives as antimycobacterial agents analogs of bm importance of the thiomorpholine introduction in new pyrrole derivatives as antimycobacterial agents analogues of bm antimycobacterial compounds. new pyrrole derivatives of bm antimycobacterial compounds. optimization of the bm structure, the lead compound for a new pyrrole derivative class antimycobacterial agents. novel diarylpyrrole derivatives of bm endowed with high activity toward mycobacterium tuberculosis and low cytotoxicity botta, , -diphenylpyrrole derivatives as antimycobacterial agents. probing the influence on antimycobacterial activity of lipophilic substituents at the phenyl rings -diaryl- -ethyl pyrrole derivatives as antimycobacterial agents: design, synthesis, and microbiological evaluation identification of a novel pyrrole derivative endowed with antimycobacterial activity and protection index comparable to that of the current antitubercular drugs streptomycin and rifampin improved bm mmpl inhibitor analogue shows efficacy in acute murine model of tuberculosis infection mmpl is the cellular target of the antitubercular pyrrole derivative bm pharmacophore assessment through -d qsar: evaluation of the predictive ability on new derivatives by the application on a series of antitubercular agents bm and its derivatives as a new class of antimycobacterial active agents new pyrroles with potential antimycobacterial, antifungal and selective cox- inhibiting activities. synthetic methodologies new derivatives of bm : a class of antimycobacterial compounds based on the pyrrole ring as a scaffold developing pyrrole-derived antimycobacterial agents: a rational lead optimization approach pyrrole derivatives as antimycobacterial compounds, us patent antitubercular agents. part : a new class of diarylpyrroleeoxazolidinone conjugates as antimycobacterial agents organocatalytic multicomponent reaction for the acquisition of a selective inhibitor of mptpb, a virulence factor of tuberculosis novel -( -substituted benzylidene)- -( -(substituted methyl)- , -dioxoindolin- -yl)semicarbazide derivatives for use against mycobacterium tuberculosis h rv (atcc ) and mdr-tb strain synthesis, anti-hiv and antitubercular activities of lamivudine prodrugs synthesis and antituberculosis activity of -methyl/trifluoromethoxy- h-indole- , -dione -thiosemicarbazone derivatives synthesis and structureeantituberculosis activity relationship of h-indole- , -dione derivatives gatifloxacin derivatives: synthesis, antimycobacterial activities, and inhibition of mycobacterium tuberculosis dna gyrase synthesis and antimycobacterial evaluation of various -substituted ciprofloxacin derivatives synthesis and in vitro antimycobacterial activity of -och ciprofloxacin methylene and ethylene isatin derivatives synthesis and in vitro antimycobacterial activity of moxifloxacin methylene and ethylene isatin derivatives antimycobacterial activity of new -substituted -(pyridin- -yl)- h- , , -oxadiazol- -one and -thione derivatives. preliminary molecular modeling investigations antimycobacterial activity of new , -disubstituted , , -oxadiazol- ( h)-one derivatives. molecular modeling investigations oxadiazole mannich bases: synthesis and antimycobacterial activity augustynowicz-kope c, synthesis and tuberculostatic activity of some -piperazinmethylene derivatives , , -triazole- -thiones synthesis and evaluation of antitubercular activity of imidazo mannich bases and novel benzothiazole derivatives of imidazo[ , -b]- , , -thiadiazoles and their biological evaluation synthesis & evaluation of antitubercular activity of novel mannich bases of imidazo newer tetracycline derivatives: synthesis, anti-hiv, antimycobacterial activities and inhibition of hiv- integrase synthesis of pyrazinamide mannich bases and their antitubercular properties efavirenz mannich bases: synthesis, anti-hiv and antitubercular activities indole mannich bases and their antimycobacterial effect synthesis of novel isothiazolopyridines and their in vitro evaluation against mycobacterium and propionibacterium acnes studies on pyrazine derivatives. . synthesis, reactions, and tuberculostatic activity of epidemiology of invasive mycoses in north america antifungal activity of some mono, bis and quaternary mannich bases derived from acetophenone antimicrobial evaluation of some mannich bases of acetophenones and representative quaternary derivatives antifungal evaluation of bis mannich base derived from acetophenones and their corresponding piperidinols and stability studies synthesis and antifungal activity of -aryl- -phenethylamino- -propanone hydrochlorides and -aroyl- -aryl- -phenethyl- -piperidinols synthesis and antifungal evaluation of -aryl- -[(dimethylamino)methyl]- -propen- -one hydrochlorides antifungal unsaturated cyclic mannich ketones and amino alcohols: study of mechanism of action antifungal activity of fused mannich ketones triggers an oxidative stress response and is cap -dependent in candida albicans synthesis and antifungal activity of -substituted thiochromanones deep, synthesis, characterization and evaluation of mannich bases as potent antifungal and hydrogen peroxide scavenging agents mannich reaction: an approach for the synthesis of water soluble mulundocandin analogues evaluation of bioactivities of chlorokojic acid derivatives against dermatophytes couplet with cytotoxicity synthesis and evaluation of anticonvulsant and antimicrobial activities of -hydroxy- -methyl- -substituted h-pyran- -one derivatives synthesis and studies of triazolothiadiazines. an approach toward new biologically active heterocyclic compounds, phosphorus ecofriendly synthesis of novel antifungal (thio)barbituric acid derivatives world health organization malaria isoquine and related amodiaquine analogues: a new generation of improved -aminoquinoline antimalarials candidate selection and preclinical evaluation of ntert-butyl isoquine (gsk ), an affordable and effective -aminoquinoline antimalarial for the st century comparative preclinical drug metabolism and pharmacokinetic evaluation of novel -aminoquinoline anti-malarials antimalarial activity of isoquine against kenyan plasmodium falciparum clinical isolates and association with polymorphisms in pfcrt and pfmdr genes mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents synthesis and in vitro and in vivo antimalarial activity of novel -anilinoquinoline mannich base derivatives synthesis and antimalarial activity evaluation of some isoquine analogues synthesis and antimalarial activity study of some new mannich bases of -chloro- -aminoquinoline novel amodiaquine congeners as potent antimalarial agents synthesis and antimalarial activity of new isotebuquine analogues review of pyronaridine anti-malarial properties and product characteristics determination of the physicochemical properties of pyronaridine e a new antimalarial drug absorption, distribution, excretion, and pharmacokinetics of c-pyronaridine tetraphosphate in male and female spra-gueedawley rats in vitro interactions between piperaquine, dihydroartemisinin, and other conventional and novel antimalarial drugs, antimicrob anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo targeting of hematin by the antimalarial pyronaridine pyrido [ , -b]indol- -yl-amine e synthese und prüfung auf wirksamkeit gegen malaria benzofuro[ , -b]pyridin- -ylamines e synthese und prüfung auf wirksamkeit gegen malaria benzothieno[ , -b]pyridin- -yl-amine e synthese und prüfung auf wirksamkeit gegen malaria thieno[ , -c]chinolin- -ylamine e synthese und prüfung auf wirksamkeit gegen malaria thieno[ , -c]chinolin- -ylamine e synthese und prüfung auf wirksamkeit gegen malaria naphthyridin- -yl-arylamine e phenol-mannich-basen vom amodiaquin-, cycloquin-und pyronaridin-typ antimalarial mannoxanes: hybrid antimalarial drugs with outstanding oral activity profiles and a potential dual mechanism of action aromatic amino analogues of artemisinin: synthesis and in vivo antimalarial activity artemisinin derivatives bearing mannich base group: synthesis and antimalarial activity novel in vivo active anti-malarials based on a hydroxy-ethyl-amine scaffold modular synthesis and in vitro and in vivo antimalarial assessment of c- pyrrole mannich base derivatives of artemisinin mechanism-based inactivation of thioredoxin reductase from plasmodium falciparum by mannich bases. implication for cytotoxicity synthesis and biological evaluation of phenolic mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain- and a chloroquine resistant strain of plasmodium falciparum design, synthesis and structureeactivity relationships of ( h-pyridin- -ylidene)amines as potential antimalarials synthesis and in vitro activities of ferrocenic aminohydroxynaphthoquinones against toxoplasma gondii and plasmodium falciparum pershina, indole derivative having antiviral arbidol: a broad-spectrum antiviral compound that blocks viral fusion mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol synthesis and in vitro anti-hepatitis b virus activities of some ethyl -bromo- -hydroxy- h-indole- -carboxylates synthesis and in vitro anti-hepatitis b virus activity of some ethyl -hydroxy- -substituted aminomethyl- -sulfinylmethyl- h-indole- -carboxylates synthesis and in vitro anti-hepatitis b virus activities of some ethyl -hydroxy- h-indole- -carboxylates synthesis and in vitro-anti-hepatitis b virus activities of several ethyl -hydroxy- h-indole- -carboxylates synthesis and anti-hepatitis b virus evaluation of novel ethyl -hydroxyquinoline- -carboxylates in vitro synthesis and in-vitro antihepatitis-b virus activity of h synthesis and in vitro anti-hepatitis b virus activity of h-[ ]benzothiopyrano[ , -b]quinolin- -ols synthesis and in vitro anti-hepatitis b virus activity of h-benzimidazol- -ol derivatives synthesis and biological evaluation of h-benzimidazol- -ols as potent hbv inhibitors synthesis and in-vitro anti-hepatitis b virus activity of ethyl -bromo- -hydroxyimidazo[ , -a] pyridine- -carboxylates activity of mannich bases of -hydroxycoumarin against flaviviridae novel structurally related compounds reactivate latent hiv- in a bcl- -transduced primary cd þ t cell model without inducing global t cell activation synthesis and primary antiviral activity evaluation of -hydrazono- -nitro- -indolinone derivatives design of potential reverse transcriptase inhibitor containing isatin nucleus using molecular modeling studies newer aminopyrimidinimino isatin analogues as non-nucleoside hiv- reverse transcriptase inhibitors for hiv and other opportunistic infections of aids: design, synthesis and biological evaluation n-methylisatin-beta-thiosemicarbazone derivative (sch ) is an inhibitor of japanese encephalitis virus infection in vitro and in vivo condensed pentacyclic derivatives for use in the treatment of flaviviridae infections synthesis, antiviral and cytotoxicity studies of novel n-substituted indophenazine derivatives, indian synthesis of some mono-mannich bases and corresponding azine derivatives and evaluation of their anticonvulsant activity evaluation of anticonvulsant activities of bis( -aryl- -oxo-propyl)ethylamine hydrochlorides and -aryl- -arylcarbonyl- -ethyl- -piperidinol hydrochlorides synthesis and evaluation of anticonvulsant activities of some bis mannich bases and corresponding piperidinols synthesis of some new hydroxypyranone derivatives and evaluation of their anticonvulsant activities synthesis of some novel mannich bases derived from allomaltol and evaluation of their anticonvulsant activities anticonvulsant and neurotoxicity evaluation of some novel kojic acids and allomaltol derivatives synthesis and anticonvulsant activity of new kojic acid derivatives synthesis of aminomethyl-substituted cyclic imide derivatives for evaluation as anticonvulsants synthesis and anticonvulsant activity of new n-mannich bases derived from -cyclopropyl- -phenylhydantoins synthesis and anticonvulsant activity of new n-mannich bases derived from -cyclopropyl- -phenyl-and -cyclopropyl- -( -chlorophenyl)-imidazolidine- , -diones synthesis and anticonvulsant activity of new n- synthesis and potential anticonvulsant activity of new n- -substituted , -cyclopropanespirohydantoins synthesis and pharmacological evaluation of novel karolak-wojciechowska, design, synthesis, and anticonvulsant activity of new n-mannich bases derived from spirosuccinimides and spirohydantoins synthesis and anticonvulsant properties of new n-[( -arylpiperazin- -yl)-methyl] derivatives of -aryl pyrrolidine- , -dione and -aza-spiro synthesis and anticonvulsant activity of new n-[( -arylpiperazin- -yl)-alkyl] derivatives of -phenylpyrrolidine- , -dione synthesis, physicochemical and anticonvulsant properties of new n-[( -aryl- -piperazinyl) alkyl]- -phenyl- , -pyrrolidinedione and n-[( -aryl- -piperazinyl)alkyl]- -( -methylphenyl)- , -pyrrolidinedione derivatives synthesis and anticonvulsant properties of new mannich bases derived from -aryl-pyrrolidine- , -diones synthesis and anticonvulsant activity of new n-mannich bases derived from -( -fluorophenyl)-and -( -bromophenyl)-pyrrolidine- , -diones. part ii design, synthesis and anticonvulsant properties of new n-mannich bases derived from -phenylpyrrolidine- , -diones duszy nska, synthesis, anticonvulsant activity and -ht a , -ht a receptor affinity of new n-[( -arylpiperazin- -yl)-alkyl] derivatives of -azaspiro synthesis and anticonvulsant properties of new mannich bases derived from , -disubstituted pyrrolidine- , -diones. part iv synthesis and anticonvulsant properties of new n-mannich bases derived from , -diphenyl-and -ethyl- -methyl-pyrrolidine- , -diones. part iii synthesis and biological properties of new n-mannich bases derived from -methyl- -phenyl-and , -dimethyl-succinimides. part v anticonvulsant activity and -ht a / -ht receptors affinity of piperazine derivatives of synthesis of some newer derivatives of substituted quinazolinonyl- -oxo/thiobarbituric acid as potent anticonvulsant agents synthesis and pharmacological evaluation of newer substituted benzoxazepine derivatives as potent anticonvulsant agents synthesis and evaluation of some new substituted benzothiazepine and benzoxazepine derivatives as anticonvulsant agents synthesis and anticonvulsant activity of various mannich and schiff bases of , -benzodiazepines novel -(e)-substituted benzylidene- -(n-substituted aminomethyl)cyclohexanones and cyclohexanols as analgesic and anti-inflammatory agents biochemical effects of some new mannich base from ortho-hydroxyaryl alkyl ketones on rats with chronic inflammation synthesis and pharmacological activity of n-[b-(para-substituted benzoyl)ethyl]isoleucine and -methionine synthesis and anti-inflammatory, analgesic, and antipyretic activities of n-[b-(p-substituted benzoyl)ethyl]amino acids anti-inflammatory activity of isobornylphenol derivatives synthesis and antiinflammatory activity of resveratrol analogs new , -diacetylresorcinol mannich bases: synthesis and biological evaluation synthesis and biological evaluation of nitrogencontaining benzophenone analogues as tnf-a and il- inhibitors with antioxidant activity synthesis and biological evaluation of nitrogen-containing chalcones as possible anti-inflammatory and antioxidant agents synthesis and antiinflammatory activity of chalcones and related mannich bases inhibitory effects of mannich bases of heterocyclic chalcones on no production by activated raw . macrophages and superoxide anion generation and elastase release by activated human neutrophils synthesis of some new , -dihydro- h- , -benzoxazines under microwave irradiation in solvent-free conditions and their biological activity synthesis and antiinflammatory activity of coumarin derivatives anti-inflammatory mechanisms of isoflavone metabolites in lipopolysaccharide-stimulated microglial cells synthesis and no production inhibition of novel mannich base derivatives of irisolidone synthesis and in-silico studies of some diaryltriazole derivatives as potential cyclooxygenase inhibitors regioselective reaction: synthesis and pharmacological study of mannich bases containing ibuprofen moiety regioselective reaction: synthesis, characterization and pharmacological activity of some new mannich and schiff bases containing sydnone synthesis, characterization and pharmacological activity of -{[ -substituted aminomethyl- -arylideneamino- -sulfanyl- , -dihydro- h- , , -triazol- -yl] methyl}- h- , -benzothiazin- ( h)-ones synthesis and anti-inflammatory activity of new polyheterocyclic schiff bases and mannich bases docking, synthesis, and pharmacological investigation of novel substituted thiazole derivatives as non-carboxylic, anti-inflammatory, and analgesic agents synthesis, analgesic and antiinflammatory properties of certain -/ -acyl- -( -substituted- -piperazinylmethyl)- -benzoxazolinones derivatives synthesis and evaluation of analgesic, anti-inflammatory and antimicrobial activities of -acyl- -piperazinomethyl- -benzoxazolinones some new mannich bases of -methyl- -benzoxazolinones with analgesic and antiinflammatory activities analgesic and antiinflammatory activities of some new mannich bases of -nitro- -benzoxazolinones synthesis and pharmacological evaluation of -( -((substituted)methyl)- -methyl- -oxoindolin- -ylidene)- -(substituted pyridin- -yl)thiosemicarbazide synthesis, analgesic, antiinflammatory and ulcerogenic properties of some novel n -(( -(substitutedamino)methyl)- -oxoindolin- -ylidene)- -( -(methyl/phenyl)- -oxoquinazolin- ( h)-yl)benzohydrazide derivatives synthesis, pharmacological screening, quantum chemical and in vitro permeability studies of n-mannich bases of benzimidazoles through bovine cornea synthesis and biological evaluation of mannich bases of benzimidazole derivatives synthesis, antiinflammatory and ulcerogenicity studies of some substituted pyrimido[ , -a]azepine derivatives potential antiinflammatory activity and ulcerogenicity study of some novel pyrimido synthesis and anti-inflammatory activity of certain piperazinylthienylpyridazine derivatives design and synthesis of -[ -(substituted phenyl)- -piperidin- -ylmethyl/- -morpholin- -ylmethyl- , -dihydro-isoxazol- -yl]- h-indoles as potent anti-inflammatory agents efficient synthesis of the first betulonic acideacetylene hybrids and their hepatoprotective and anti-inflammatory activity substituted and -naphthol mannich bases synthesis and analgesic activity of novel derivatives of , -substituted benzimidazoles synthesis and in vivo pharmacology of new derivatives of isothiazolo[ , -b] pyridine of mannich base type synthesis, analgesic activity and computational study of new isothiazolopyridines of mannich base type thymol analogues with antioxidant and l-type calcium current inhibitory activity alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and copper-complexing properties mannich bases of scutellarein as thrombin-inhibitors: design, synthesis, biological activity and solubility synthesis of antioxidants for prevention of age-related macular degeneration synthesis and antioxidant activity of novel mannich base of , , -oxadiazole derivatives possessing , -benzodioxan design, synthesis, and in vitro antioxidant activity of , , -trisubstituted- -pyrazolines derivatives synthesis and antioxidant activity of aminomethylated -methyluracil derivatives synthesis and antihypertensive effects of new methylthiomorpholinphenol derivatives antihypertensive and antiarrhythmic properties of a para-hydroxy[bis(ortho-morpholinylmethyl)] phenyl- , -dhp compound: comparison with other compounds of the same kind and relationship with logp values -and -substituted naphthalenes: a new class of potential hypotensive agents studies on the cardiovascular action of tpyb: an antihypertensive agent with antiplatelet activity synthesis, characterization and antihypertensive activity of pyridazinone derivatives synthesis and pharmacological activity of -and -(aminomethyl)imidazo [ , -a]benzimidazoles new octahydroquinazoline derivatives: synthesis and hypotensive activity irreversible inactivation of trypanothione reductase by unsaturated mannich bases: a divinyl ketone as key intermediate unsaturated mannich bases active against multidrugresistant trypanosoma brucei brucei strains antileishmanial pyrazolopyridine derivatives: synthesis and structureeactivity relationship analysis mannich base derivatives of , , -oxadiazole: synthesis and screening against entamoeba histolytica in vitro antischistosomal evaluation of some newly synthesized praziquantel derivatives structureeactivity relationships of chalcone analogs as potential inhibitors of adpand collagen-induced platelet aggregation synthesis and pharmacological evaluation of peptide-mimetic protease-activated receptor- antagonists containing novel heterocyclic scaffolds the association of helicobacter pylori infection and nonsteroidal anti-inflammatory drugs in peptic ulcer disease synthesis and antiulcer activity study of , -dihydropyridines and their mannich bases with sulfanilamide synthesis of -and -alkylaminomethyl furoflavones as gastroprotective agents submitted to the th world health assembly synthesis and antidepressant-like profile of novel -aryl- -[( -benzyl)piperidine- -yl]propane derivatives design, synthesis, computational and biological evaluation of new anxiolytics benzoxepin derivatives: design, synthesis, and pharmacological evaluation with sedativeehypnotic effect synthesis and hepatoprotector activity of water-soluble derivatives of aminoalkylphenols design, synthesis and preliminary biological evaluation of zatebradine analogues as potential blockers of the hyperpolarization-activated current synthesis and antiemetic activity of , , , -tetrahydro- -methyl- -( -substituted-piperazin- -ylmethyl)- h-carbazol- -one derivatives synthesis of tropolone derivatives and evaluation of their in vitro neuroprotective activity bone selective protective effect of a novel bone-seeking estrogen on trabecular bone in ovariectomized rats a one step/one pot synthesis of n,nbis(phosphonomethyl)amino acids and their effects on adipogenic and osteogenic differentiation of human mesenchymal stem cells the mannich base nc promotes long-term allograft survival and spares the recipient from multiple toxicities donepeziletacrine hybrid related derivatives as new dual binding site inhibitors of ache synthesis and acetylcholinesterase (ache) inhibitory activity of some n-substituted- -chloro- ( h)-benzoxazolone derivatives synthesis and biological evaluation of , -dihydroxyxanthone mannich base derivatives as anticholinesterase agents synthesis and biological evaluation of novel -aminomethylated oroxylin a analogues as a-glucosidase inhibitors synthesis and evaluation of -( -methoxynaphthalen- -yl)- -aryl- -( -(trifluoromethyl) phenylamino)pentan- -ones as potential antidiabetic agents synthesis and antidiabetic performance of b-amino ketone containing nabumetone moiety synthesis of novel b-amino ketones containing a p-aminobenzoic acid moiety and evaluation of their antidiabetic activities exploring structureeactivity relationships of transition state analogues of human purine nucleoside phosphorylase synthesis of second-generation transition state analogues of human purine nucleoside phosphorylase synthesis of a transition state analogue inhibitor of purine nucleoside phosphorylase via the mannich reaction third-generation immucillins: syntheses and bioactivities of acyclic immucillin inhibitors of human purine nucleoside phosphorylase syntheses and bio-activities of the l-enantiomers of two potent transition state analogue inhibitors of purine nucleoside phosphorylases immucillins in custom catalytic-site cavities a bfluoroamine inhibitor of purine nucleoside phosphorylase azetidine based transition state analogue inhibitors of n-ribosyl hydrolases and phosphorylases design and synthesis of potent "sulfur-free" transition state analogue inhibitors of -methylthioadenosine nucleosidase and -methylthioadenosine phosphorylase augustyns, narylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase potent transglutaminase inhibitors, aryl b-aminoethyl ketones potent transglutaminase inhibitors, dithio b-aminoethyl ketones synthesis and characterization of phenolic mannich bases and effects of these compounds on human carbonic anhydrase isozymes i and ii nitrogen-containing flavonoid analogues as cdk /cyclin b inhibitors: synthesis, sar analysis, and biological activity hit identification of novel heparanase inhibitors by structure and ligand-based approaches discovery of inhibitors of burkholderia pseudomallei methionine aminopeptidase with antibacterial activity search for inhibitors of bacterial and human protein kinases among derivatives of diazepines[ , ] annelated with maleimide and indole cycles umbelliferone aminoalkyl derivatives, a new class of squalene-hopene cyclase inhibitors synthesis of new -oxo- -thioxo- , , , -tetrahydropyrimidine derivatives with an incorporated thiazolidinone moiety and testing their possible serine protease and cercarial elastase inhibitory effects with a possible prospective to block penetration of schistosoma mansoni cercariae into the mice skin synthesis and biological activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors novel mannich bases, -arylimidazolidine- , -dione derivatives with dual -ht a receptor and serotonin transporter affinity pharmacophore based synthesis of -chloroquinoxaline- -carboxamides as serotonin ( -ht ) receptor antagonist synthesis and biological evaluation of a novel structural type of serotonin -ht receptor antagonists ]pyridine: an antagonist with high affinity and selectivity for the human dopamine d receptor azaindole derivatives with high affinity for the dopamine d receptor: synthesis, ligand binding studies and comparison of molecular electrostatic potential maps phenylpiperazinylmethylheterocycle derivatives: synthesis and dopamine receptor binding profiles synthesis of -[( -phenylpiperazin- -yl)methyl]pyrrolo [ , -d]pyrimidine derivatives as potential dopamine d receptor ligands design, synthesis and dopamine d receptor binding activities of new n-heteroaromatic / -ring mannich bases discovery of highly potent and selective d ligands by interactive sar study synthesis and evaluation of ligands for d -like receptors: the role of common pharmacophoric groups discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators inhibitors of the interaction of a thyroid hormone receptor and coactivators: preliminary structureeactivity relationships structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor improvement of pharmacological properties of irreversible thyroid receptor coactivator binding inhibitors discovery and biological characterization of a novel series of androgen receptor modulators aromatic b-amino-ketone derivatives as novel selective non-steroidal progesterone receptor antagonists synthesis and biological evaluation of n ,n -bis-[ -(t-amino)- -butynyl]phthalamides as oxotremorine and acetylcholine antagonists synthesis and structureactivity relationships of -aralkyl- -benzylpiperidine and -aralkyl- -benzylpiperazine derivatives as potent s ligands indole-and benzothiophene-based histamine h antagonists new , , , -tetrahydro- h-carbazol- -one derivatives: analogues of heat as ligands for the a -adrenergic receptor subtypes pyrazolone methylamino piperidine derivatives as novel ccr antagonists synthesis of new -heteroaryl- -phenylquinolines and their pharmacological activity as nk- /nk- receptor ligands the author declares that there is no conflict of interest. key: cord- - n aq k authors: qiu, sangsang; pan, hongqiu; zhang, simin; peng, xianzhen; zheng, xianzhi; xu, guisheng; wang, min; wang, jianming; lu, hui title: is tuberculosis treatment really free in china? a study comparing two areas with different management models date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: n aq k objective: china has implemented a free-service policy for tuberculosis. however, patients still have to pay a substantial proportion of their annual income for treatment of this disease. this study describes the economic burden on patients with tuberculosis; identifies related factors by comparing two areas with different management models; and provides policy recommendation for tuberculosis control reform in china. methods: there are three tuberculosis management models in china: the tuberculosis dispensary model, specialist model and integrated model. we selected zhangjiagang (zjg) and taixing (tx) as the study sites, which correspond to areas implementing the integrated model and dispensary model, respectively. patients diagnosed and treated for tuberculosis since january were recruited as study subjects. a total of patients ( patients from zjg and patients from tx) were interviewed with a response rate of %. the economic burden attributed to tuberculosis, including direct costs and indirect costs, was estimated and compared between the two study sites. the mann-whitney u test was used to compare the cost differences between the two groups. potential factors related to the total out-of-pocket costs were analyzed based on a step-by-step multivariate linear regression model after the logarithmic transformation of the costs. results: the average (median, interquartile range) total cost was . ( , - ) cny for patients in zjg, which was significantly higher than for patients in tx (mean: . , median: , interquartile range: – ) (z = . , p < . ). after excluding expenses covered by health insurance, the average out-of-pocket costs were . cny in zjg and . cny in tx. based on the multivariable linear regression analysis, factors related to the total out-of-pocket costs were study site, age, number of clinical visits, residence, diagnosis delay, hospitalization, intake of liver protective drugs and use of the second-line drugs. conclusion: under the current “free of diagnosis and treatment” policy, the financial burden remains heavy on tuberculosis patients. policy makers need to consider appropriate steps to lessen the burden of out-of-pocket costs for tuberculosis patients in china and how best to improve service delivery for poor patients. tuberculosis (tb) is a global health problem and remains a major cause of morbidity and mortality in developing countries [ ] . china has the world's second largest tuberculosis epidemic, accounting for % of the total number of cases. in , there were million new cases and tuberculosis-related deaths in china [ ] . the rising multidrug-resistant (mdr) tuberculosis is increasing an already heavy burden on china's health system [ , ] . tuberculosis has been regarded as a "poverty-related disease" due to the association with poverty and malnutrition, which are more prevalent in developing countries. for example, in south africa, tuberculosis is referred to as a "barometer of poverty" [ ] . tuberculosis-affected patients and their family members face many economic and social problems, such as high medical costs, loss of productivity, stigmatization and social isolation [ , ] . in , china initiated its modern national tuberculosis control program (ntp) with directly observed treatment, short-course (dots) [ ] . recently, especially after the outbreak of severe acute respiratory syndrome in , the chinese government has taken a series of measures to strengthen its public health system with great efforts towards tuberculosis control. by , china achieved the global targets for tuberculosis control with % dots coverage and over % treatment success [ ] . each year in china, more than million tuberculosis patients receive dots therapy [ ] . to reduce the financial barriers to and burdens on patients seeking essential healthcare, a "free-tb service policy" has been implemented gradually throughout the country [ , ] . under this policy, tuberculosis suspects are provided a free diagnosis and anti-tuberculosis treatment, including a free chest x-ray examination, sputum smear test and designated firstline anti-tuberculosis drugs [ ] . initially, the free-service policy was only performed for sputum smear-positive patients. now it has expanded to sputum smear-negative patients. moreover, the government has taken more measures to reduce the patient burden, including the establishment of universal health coverage and increasing the reimbursement rate for patients with tuberculosis [ ] . the central government's spending on tuberculosis control increased from million chinese yuan (cny) in to million cny in [ ] . china has established universal health coverage for million rural residents through the rapid expansion of the new cooperative medical scheme (ncms). moreover, a free-service policy has been gradually adopted in order to lighten the economic burden of patients with tuberculosis. despite these policy changes, previous studies have revealed that patients still bear a high financial burden, which could affect their health care-seeking behaviors and treatment outcomes [ , [ ] [ ] [ ] [ ] . the revenue-driven practices in some health facilities, such as over-prescription of medication, poor referral and high hospitalization rates not only influence a patient's economic burden but also affects the entire tuberculosis control program [ , ] . there are three common types of tuberculosis management models in china: the tuberculosis dispensary model, specialist model and integrated model [ ] . for the dispensary model, patients are diagnosed and treated in tuberculosis dispensaries, which are usually affiliated with local cdc (center for disease control and prevention). the specialist model is similar to the dispensary model, but a specialized hospital is also responsible for treating the patients. for the integrated model, tuberculosis diagnosis and treatment is integrated into a general hospital which is referred to as a "designated hospital" [ ] . this study describes the economic burden on patients with tuberculosis, identifies related factors by comparing two areas with different management models, and provides a policy recommendation for the tuberculosis control system in china. the institutional review board (irb) of nanjing medical university approved the study. written informed consent was obtained from all of the participants. ethical practices were used throughout the study period. a descriptive study was performed in two counties of jiangsu province, china. one county, zhangjiagang (zjg), is located in the southern part of jiangsu, which is a relatively rich area and ranked as the third most developed county of china in . another county, taixing (tx), is located in the middle of jiangsu, which is a moderately developed county. in , the per capita gdp was chinese yuan (cny) in zjg and cny in tx (http://www. jssb.gov.cn/). in the same year, the per capita gdp was cny in china (http://www.stats. gov.cn/tjsj/ndsj/). zjg adopted the integrated model, and the diagnosis and treatment of tuberculosis are provided by the designated county-level general hospital. tx implemented the dispensary model, and tuberculosis patients are diagnosed and treated at the local cdc (tuberculosis dispensary). in zjg, the free service covered the first-line anti-tuberculosis drugs, four liver function tests, three chest x-ray examinations and all sputum smear microscopy tests. moreover, each patient could receive a reimbursement of cny for liver protection drugs and each migrant patient with tuberculosis could receive an additional subsidy of cny. in tx, the free service policy covered the first-line anti-tuberculosis drugs, two liver function tests, one chest x-ray examination and all sputum smear microscopy tests. hospitalization and second-line anti-tuberculosis drugs were not free of charge in either area. baseline characteristics of the two study settings were listed in table . in zjg, villages were selected as study sites based on a random cluster sampling method. three hundred and forty patients who were diagnosed with pulmonary tuberculosis since january were recruited for the investigation. the patients had already completed the standard anti-tuberculosis treatment prior to march . in tx, we randomly selected villages as study sites. three hundred and ninety patients who were diagnosed with pulmonary tuberculosis since january were enrolled. patients with mdr-tb were excluded from the study. due to the gradually evolving tuberculosis management models in tx since , the recruited patients were limited to those who had finished anti-tuberculosis treatment prior to . a total of patients ( patients from zjg and patients from tx) were successfully interviewed and involved in the analysis with a response rate of %. with the patients' informed consent, trained investigators interviewed them at their homes using a structured questionnaire to gather information, including demographic characteristics, socioeconomic status, health insurance, health care-seeking history and costs related to tuberculosis diagnosis and treatment. the economic burden of the patients with tuberculosis consisted of direct and indirect costs. the direct costs were medical costs related to tuberculosis diagnosis and treatment (including outpatient and inpatient expenses), transportation, accommodation and food [ , ] . the indirect costs were calculated as a loss of income due to an inability to work due to the disease. we examined the loss of income (or decreased earning ability) for both patients of the labor force and their caregivers [ ] . the total out-of-pocket costs were calculated by excluding reimbursement through medical insurance. the out-of-pocket direct costs were direct costs calculated by excluding reimbursement through medical insurance. the costs are expressed in the chinese yuan (cny). in , cny was equal to . usd. in order to calculate the costs for outpatient visits to the doctor, the number and costs of the visits made for tuberculosis diagnosis and treatment were obtained from the patients and their family members. if the patients could not remember the costs of the doctor's visits, medical records and invoices were checked. the hospitalization cost of the patients was computed based on the cost that they paid when being discharged from the hospital. non-medical, direct costs included transportation; commuting and food costs (of patients and their families) during their visit to health facilities; the cost of purchasing extra health products that were required due to the tuberculosis; the cost of residing in other cities for treatment and nursing patients at home. indirect costs depended on daily income; the number of sick leaves; the average daily income of each patient's companion; and the duration of absence from work resulting from nursing and caring for the patient. the individuals' wages were used to calculate lost income. for patients who were not willing to declare their income and also for housekeepers, the local average daily wage was used. we used epidata . (http://epidata.dk) software to input data with double entry for consistency. statistical analyses was performed using stata . software (college station, texas, usa). considering the distribution of costs, we used the mean and median (interquartile range, iqr) to describe a patient's economic burden. the mann-whitney u test was used to compare the cost difference between the two groups. the cost was logarithmically transformed as the dependent variable, and a multivariate linear regression model was performed to analyze potential factors related to the out-of-pocket costs of patients with tuberculosis. we included product terms in our model to individually account for each possible two-way interaction, considering as statistically significant those interactions with a p-value < . . this study recruited tuberculosis patients, including men ( . %) and women ( . %). among them, ( . %) resided in zjg, and ( . %) resided in tx. in zjg, there were many young, migrant patients with tuberculosis, and fewer patients had a treatment history. the proportion of hospitalized patients and patients taking liver protective drugs were also higher in zjg than tx (table ). as shown in table , the average (median, iqr) total costs was . ( , - ) cny for patients in zjg, which was significantly higher than for patients in tx (mean: . , median: , iqr: - ) (z = . , p < . ). after excluding the expenses covered by health insurance, the total out-of-pocket costs was . cny in zjg and . cny in tx. the median (iqr) ratio of total out-of-pocket cost to the annual family income was . % ( . %- . %) in zjg and . % ( . %- . %) in tx (table ). in zjg, the average direct costs was . cny with a median (iqr) of ( - ). in tx, the average direct costs was . cny with a median (iqr) of ( - ). when we excluded the expenses covered by health insurance, the average out-of-pocket direct costs was . table ) . the common factors contributing to the total out-of-pocket costs in these two areas were diagnosis delay, hospitalization, and intake of liver protective drugs and second-line drugs. a univariate analysis also identified several peculiar features related to the total out-of-pocket costs in tx, including the patients' age, treatment history, adverse drug reactions and the number of clinical visits (table ) . we further performed a step-by-step linear regression analysis by including multiple variables in the model. significant factors related to the total out-of-pocket costs were study setting (t = - . , p = . ), age (t = - . , p < . ), number of clinical visits (t = . , p < . ), residence (t = . , p = . ), diagnosis delay (t = . , p = . ), hospitalization (t = . , p < . ), intake of liver protective drugs (t = . , p = . ) and intake of second-line drugs (t = . , p = . ) ( table ) . a significant interaction was found between the number of clinical visits and study setting (p interaction = . ). we then performed a multivariate linear regression analysis stratified by study setting. as shown in table , age, residence, diagnosis delay and hospitalization entered into the model in zjg. the number of clinical visits, age, diagnosis delay, hospitalization, intake of liver protective drugs and intake of second-line drugs entered into the model in tx. tuberculosis has historically been endemic in china, primarily due to limited health resources and government neglect [ ] . in recent years, the political commitment to public health has significantly increased [ ] . china is moving towards primary health care based on community services [ ] . for example, the government pays sustainable funds per person for the prevention of disease, and some funds are allotted to tuberculosis control. financial concern is the most important factor guiding health care seeking behavior among the chinese population [ ] [ ] [ ] . however, as demonstrated in the current study and previous reports [ , , , ] , most of the patients have complained about paying a major part of the treatment cost through out of pocket payments. to modify the current tuberculosis control strategies, policy makers must identify the factors related to patient economic burden. in this study, we selected two counties from the jiangsu province using different tuberculosis management models. zjg adopted the integrated model. one advantage of this model is that it can shorten the delay time of diagnosis and treatment [ ] . previous studies have demonstrated that multiple clinical visits and a delay of diagnosis can increase the financial burden on patients and influence the successful control of tuberculosis [ ] . patient delay also occurs due to a lack of knowledge or negligence in seeking health care services until their symptoms deteriorate [ ] [ ] [ ] . the inability of doctors to diagnose tuberculosis during the patient's early visits leads to an institutional delay [ ] . suspected patients are usually provided symptomatic treatment resulting in increased expenses. the who recommends outpatient treatment for non-complicated tuberculosis patients. however, unnecessary hospitalization has been reported, which not only leads to higher inpatient expense, but also results in extra costs related to accommodation and transportation. the integrated tuberculosis management model may increase the hospitalization rate of suspected patients in the designated hospital. a study demonstrated that in tuberculosis patients without concomitant disorders, the initiation of treatment at a hospital adversely influenced the outcome of treatment, as reflected by the percentage of completers [ ] . however, for patients with complicated or severe symptoms, hospitalization can be advantageous. the intake of liver protective drugs and second-line anti-tuberculosis drugs were two common factors influencing a patient's economic burden. one of the most adverse reactions caused by anti-tuberculosis drugs is drug-induced liver damage. to avoid this reaction, clinical doctors in china usually prescribe protective drugs. these drugs include herbals, manufactured herbal products, and combinations of vitamins and other non-herbal substances (although their preventive effects have not been proven) [ , ] . liver-protecting drugs are not free. over-prescription of these medications also increases the patients' out-of-pocket costs [ ] . currently, in many settings in china, the prescription of second-line drugs primarily depends on the treatment history of the patients rather than the drug susceptibility test. as reported in one cross-sectional study conducted in five provinces within china, approximately . % of the patients had a history of taking second-line drugs [ ] . even worse, there was no significant association between the prescription of second-line drugs and the drug resistant statuses of the patients. abuse of second-line drugs merely based on clinical judgments not only increases the risk of mdr-tb and xdr-tb but also results in extra economic burden on the patients [ ] . in addition to the medical costs due to the disease, the loss of working time experienced by patients and their family members should not be ignored. a dutch study demonstrated that patients lose (on average) . months ( days) of productive days due to tuberculosis [ ] . in developed areas, the indirect costs would be much higher. for example, in our study, patients in zjg had nearly four times higher indirect costs compared with patients in tx, which might be attributed to a higher level of economic development and per capita income in zjg. besides the common factors related to the total out-of-pocket costs in zjg and tx, the univariate analysis revealed that age, treatment history, adverse drug reactions and number of clinical visits were significant factors in tx but not in zgj. these particular factors may play different roles in a patient's economic burden in different areas. for example, a significant interaction was found between the clinical visits and study setting. the number of clinical visits was more important in tx as compared with zjg. also, the relatively small sample size in zjg may reduce the statistical power, resulting in nonsignificant findings. some limitations existed in this study. first, we selected zjg and tx as the study sites representing areas using the integrated model and tb dispensary model. however, the socio- economic status and local health system may also influence a patient's direct and indirect costs. the cost differences between these two areas could not be completely attributed to the disparity of tuberculosis management models. second, although we estimated the expenses by interviewing patients and their family members and checking medical records, recall bias should not be neglected. third, the mean costs may not be reflective of the median. considering the non-normal distribution of the costs, we used the mean and median (interquartile range, %- %) to describe a patient's economic burden. we also applied a non-parametric test to compare the cost differences between groups. a logarithmic transformation was performed to normalize the distribution when we performed a step-by-step multivariate linear regression analysis. in this study, we revealed several common and specific factors affecting the economic burden of patients' with tuberculosis. health policy makers should consider these factors by enhancing financial support, strengthening health facilities and involving human resources to achieve success in tuberculosis control. tuberculosis control in china is a long-term, public health challenge and needs the support of affordable and sustainable health resources. community health resources within a strengthened health system might be the best approach [ ] . evidence-based measures to improve healthcare-seeking behavior, reduce patient and detection delays, address financial and system barriers, improve the quality of direct observed therapy and increase the access to health promotion are urgently needed [ ] . policy makers need to further document their challenges when implementing tuberculosis management models [ ] . under the current "free diagnosis and treatment" policy, the financial burden remains heavy on tuberculosis patients. policy makers need to consider appropriate steps to lessen the burden of out-of-pocket costs for tuberculosis patients in china and how best to improve service delivery for poor patients. global tuberculosis report : world health organization multidrug-resistant tuberculosis around the world: what progress has been made? epidemiology of anti-tuberculosis drug resistance in a chinese population: current situation and challenges ahead economic support to improve tuberculosis treatment outcomes in south africa: a pragmatic cluster-randomized controlled trial economic impact of pulmonary tuberculosis on patients and their families of dharan municipality, nepal perception and social consequences of tuberculosis: a focus group study of tuberculosis patients in sialkot dots in china-removing barriers or moving barriers? progress in tuberculosis control and the evolving public-health system in china adverse reactions due to directly observed treatment strategy therapy in chinese tuberculosis patients: a prospective study analysis of the economic burden of diagnosis and treatment of tuberculosis patients in rural china barriers to tb care for rural-to-urban migrant tb patients in shanghai: a qualitative study tuberculosis patient expenditure on drugs and tests in subsidised, public services in china: a descriptive study effective reimbursement rates of the rural health insurance among uncomplicated tuberculosis patients in china tuberculosis control in china: striving for sustainability how affordable are tuberculosis diagnosis and treatment in rural china? an analysis from community and tuberculosis patient perspectives perceptions and experiences of health care seeking and access to tb care-a qualitative study in rural jiangsu province adherence to anti-tuberculosis treatment among pulmonary tuberculosis patients: a qualitative and quantitative study why india should become a global leader in high-quality, affordable tb diagnostics revenue-driven in tb control-three cases in china patient medical costs for tuberculosis treatment and impact on adherence in china: a systematic review china tuberculosis policy at crucial crossroads: comparing the practice of different hospital and tuberculosis control collaboration models using survey data comparing patient care seeking pathways in three models of hospital and tb programme collaboration in china tuberculosis and poverty: the contribution of patient costs in sub-saharan africa-a systematic review patients are paying too much for tuberculosis: a direct cost-burden evaluation in burkina faso early appraisal of china's huge and complex health-care reforms perceptions of tuberculosis and health seeking behaviour in rural inner mongolia equity in health and health care: the chinese experience persistent problems of access to appropriate, affordable tb services in rural china: experiences of different socio-economic groups factors affecting patient delay of diagnosis and completion of direct observation therapy, short-course (dots) among the migrant population in shandong delays in diagnosis and treatment of pulmonary tuberculosis in india: a systematic review factors associated with patient, and diagnostic delays in chinese tb patients: a systematic review and meta-analysis can hospitalization provide better compliance in smear positive tuberculosis patients? perspectives on tuberculosis among traditional chinese medical practitioners in new york city's chinatown drugs and herbs given to prevent hepatotoxicity of tuberculosis therapy: systematic review of ingredients and evaluation studies experiences in anti-tuberculosis treatment in patients with multiple previous treatments and its impact on drug resistant tuberculosis epidemics direct and indirect costs of tuberculosis among immigrant patients in the netherlands a sustainable agenda for tuberculosis control and research are we doing enough to stem the tide of acquired mdr-tb in countries with high tb burden? results of a mixed method study in chongqing i would like to thank dr. stefanie röhrs (institute of tropical medicine & international health, charité -universitätsmedizin berlin) for her editorial support on this manuscript. key: cord- -mai eggf authors: bai, lu; gu, li; cao, bin; zhai, xiao-li; lu, min; lu, yong; liang, li-rong; zhang, lei; gao, zi-fen; huang, ke-wu; liu, ying-mei; song, shu-fan; wu, lin; yin, yu-dong; wang, chen title: clinical features of pneumonia caused by influenza a(h n ) virus in beijing, china date: - - journal: chest doi: . /chest. - sha: doc_id: cord_uid: mai eggf background: data on symptoms and radiographic changes in patients with pandemic influenza a(h n ) (a[h n ]) pneumonia during convalescence have not been reported. methods: during october , , and january , , adult patients with pneumonia with laboratory-confirmed or clinically suspected a(h n ) infections were observed for clinical characteristics, high-resolution chest ct scan, and lung function test changes during acute and -month convalescent phases. results: of the case subjects, the median age was (interquartile range [iqr], - ) years, . % were men, and . % had at least one underlying medical condition. sixty-two patients started oseltamivir therapy within a median of (iqr, - ) days from the onset of illness, and received iv corticosteroids. ards developed in patients, and were treated initially with noninvasive positive pressure ventilation (nppv). in this group, nppv was successful in patients ( . %). nine patients died at a median of (iqr, - ) days after onset of illness. multivariate cox regression identified two independent risk factors for death: progressive dyspnea after resolution of fever (relative risk, . ; % ci, . - . ; p = . ) and a higher apache (acute physiology and chronic health evaluation) ii score on presentation (relative risk for each point, . ; % ci, . - . ; p < . ). at -month follow-up of survivors with a(h n ), ground-glass opacities were still present, although diminished, in . %, and diffusing capacity for carbon monoxide was mildly reduced in . %. conclusions: ground-glass opacities and decreased diffusing capacity were the main abnormalities observed at -month follow-up of survivors of a(h n ). the clinical spectrum of this disease has ranged from self-limited illness to respiratory failure and death. in our initial report of the a(h n ) virus infection in china, the majority of patients had mild illness. , since the fi rst report of pneumonia caused by the a(h n ) virus in mexico, severe cases have been documented throughout the world. as of march , , Ն , laboratoryconfi rmed cases of death have been reported by the six world regions. in mainland china, there were . , confi rmed cases up to february , , including deaths. many studies have been published on the clinical manifestations of a(h n ) pneumonia during the acute phase of illness, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] but no information has been reported on symptoms and radiographic and lung function changes in convalescence. we studied clinical manifestations during the acute phase, antiviral and corticosteroid therapy, noninvasive positive pressure ventilation (nppv), and the histopathologic changes of a fatal case. survivors were followed up after discharge for a period of months. we believe patient follow-up and further investigation at follow-up were carried out according to clinical need, so written informed consent was not sought. high-resolution ct (hrct) scanning was ordered only in those with persisting symptoms, chest signs, or radiologic fi ndings on discharge or on last visit. no contrast was given with ct scan, and the possible radiation harm was also explained to patients. lung function tests (lfts) were ordered in those patients still attending at months. the ethics board committee at beijing chao-yang hospital approved the study design. the severity of lung changes was evaluated initially and on follow-up examinations. each lung was divided into three zones. the number of abnormal zones and the changes in ground-glass opacities (ggos), consolidation, reticular-nodules, and interlobular septal thickening were evaluated by hrct scanning. all evaluations were performed by two radiologists who were blinded to the clinical information. lfts, including lung volume, spirometry, and diffusing capacity for carbon monoxide (d lco ) were performed months after the onset of symptoms. all lfts were performed in accordance with recommended standards. d lco was measured with a singlebreath technique, adjusted for hemoglobin and alveolar volume. lft measurements were considered abnormal if they were , % of the predicted value. continuous variables were summarized as means ( sd) or medians (interquartile range [iqr] ). differences between groups were assessed using the x test or fisher exact test for categorical variables and the mann-whitney u test for continuous variables. we used univariate and multivariate cox regression to identify independent predictors of mortality. all analyses were performed by spss software,version . (spss inc; chicago, illinois). a p value Յ . was considered statistically signifi cant. from october , , to january , , a total of , cases of infl uenza-like illness were reported in our hospital, of which were laboratory-confi rmed a(h n ) cases. during the epidemic, a total of patients were eligible for this study, including patients with laboratory-confi rmed a(h n ) and three patients with high clinical suspicion for a(h n ) infection. among the patients, were hospitalized, and were treated as outpatients. median age was years, . % were men, and . % had at least one underlying medical condition ( table ) . dyspnea persisted in . % of patients after resolution of fever. smokers were more common in the ards group ( p . ), and moist rales and wheezing were signifi cantly more frequent in this group. although leukocyte counts were similar in the two our work can help optimize treatment, and also lead to a better understanding of the symptomatic, radiologic, and lung functional changes during the convalescent period. data were collected retrospectively and prospectively on all patients with confi rmed a(h n )-related pneumonia treated at beijing chao-yang hospital between october , , and january , . the diagnosis of pneumonia was based on respiratory symptoms combined with a new infi ltrate on chest radiograph. real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay was used to confi rm the diagnosis of a(h n ) infection. patients presenting with pneumonia with high clinical suspicion of a(h n ) infection but negative rt-pcr test results for a(h n ) were also included in this study. children younger than years of age were excluded. most patients were hospitalized for treatment, whereas those who presented with less serious illness and did not need oxygen supplementation were treated as outpatients under home quarantine. treatment decisions for all patients were made by their attending physicians. hospitalized patients were discharged when their temperatures had returned to normal for at least days, most infl uenza-like symptoms had disappeared, and they were clinically stable. information recorded included demographic data, underlying medical conditions, symptoms, signs, laboratory and chest radiograph fi ndings before therapy and during follow-up, and the clinical course, treatment, and adverse events during hospital stay. apache (acute physiology and chronic health evaluation) ii scores were determined in all patients to assess the severity of illness. during hospitalization, clinical data were collected retrospectively from medical records. zones on chest radiograph ( p . ) than did those who were outpatients. sixty-two of patients started oseltamivir therapy within a median of (iqr, - ) days from the onset of illness. dosages and duration of antiviral therapy are listed in table . thirty-one patients received iv corticosteroids for a median duration of (iqr, - ) days, with a dose of methylprednisolone, - mg/kg/d. adverse effects involving hallucinations and disorientation occurred in three male hospitalized patients to h after beginning corticosteroids or oseltamivir. two of the three patients received both drugs, and the other one received only oseltamivir. symptoms groups, lymphocyte counts were signifi cantly lower and serum potassium levels signifi cantly higher in the ards group. patients with ards also required more frequent use of higher doses of oseltamivir, longer duration of oseltamivir treatment, and more frequent use of corticosteroids and vasopressors, and more frequently had positive bacterial and fungal cultures. the most common initial radiologic fi ndings on hrct scan were bilateral ggos involving several zones with or without associated multifocal areas of consolidation. centrilobular nodules were also common, and small pleural effusions were present in . % of patients ( fig ) . in patients without ards, those who were hospitalized more frequently had diarrhea ( p . ), moist rales ( p . ), a lower serum albumin level ( p . ), and more involved lung ; without ards group (n ); with ards group (n ). g chest radiograph was performed within wk after onset of symptoms. total (n ); without ards group (n ); with ards group (n ). h high-resolution chest ct scanning was performed at a median (interquartile range, - ) days after onset of symptoms. total (n ); without ards group (n ); with ards group (n ). disappeared to days after stopping corticosteroids and oseltamivir or lowering the dose of oseltamivir. among patients with ards, required ventilation support, all of whom were initially treated with nppv. in this group, nppv succeeded in ( . %) (duration . . days) and ( . %) failed and were intubated at a median of (iqr, - ) h after admission; the last one refused intubation and died. among the patients who were intubated, eight died. patients who failed nppv treatment had higher apache ii scores on presentation (median [iqr, [ ] [ ] [ ] [ ] ) compared with those who succeeded table ) , who was treated successfully with oseltamivir and noninvasive positive pressure ventilation. a, initial high-resolution ct (hrct) scan obtained days after onset of illness shows bilateral extensive ground-glass opacities (ggos) and multifocal consolidation that had a predominant subpleural distribution. b, hrct scan obtained days after onset of illness shows ggos, interlobular septal thickening, and reticular nodules pattern (arrows). c, on day , only ggos are seen. d, at a -month visit, ggos are still present but are much improved. e and f, the same scan as a shows centrilobular nodules in the left upper lobe (arrows in e) and a very small amount of right pleural effusion (arrow in f). (median [iqr, [ ] [ ] [ ] ; p . ). barotrauma occurred in two patients, one during extracorporeal membrane oxygenation therapy. sputum or transtracheal aspirate specimens obtained for bacterial culture were positive in patients ( table ) , including acinetobacter baumannii, four; klebsiella pneumoniae, four; pseudomonas aeruginosa, two; enterobacter aerogenes , one; escherichia coli, one; staphylococcus aureus, one; and a spergillus spp, six . only one patient had a positive sputum culture within the fi rst h of hospitalization ( klebsiella pneumoniae ). all other positive bacterial or fungal cultures were obtained Ն h after hospitalization. an autopsy was performed on a -year-old previously healthy man who was admitted days after onset of symptoms and died of severe ards on day of hospitalization ( fig ) . gross examination of lung tissue revealed prominent congestion and consolidation, with increased weight (left, g; right, , g). an abscess was seen in the right lower lobe that among the patients, nine died, of whom eight had hemorrhagic respiratory secretions. one -yearold man died of severe hemoptysis within h of admission. the death rate among patients with ards was . % ( / ). the main cause of death was refractory hypoxemia. two factors were found to be independently associated with death: progressive dyspnea after resolution of fever (relative risk, . ; % ci, . - . ; p . ) and a higher apache ii score on presentation (relative risk for each point, . ; % ci, . - . ; p , . ) ( table ) . of the survivors, had one or more follow-up visits. among who completed the -month visits, symptoms reported at the last visit included exertional dyspnea (four), hair loss (two), and cough (one). the duration of symptoms was as follows: sputum . . days, bloody sputum . . days, fatigue . . days. a -year-old female patient who was previously healthy still had a low platelet count of , per mm at days after the onset of illness. changes in lung abnormalities from initial to follow-up hrct scan examinations are shown in table . among the patients who completed their -month visit, still showed lesser degrees of ggos ( fig ) . in those who had ards (n ), "involved zones" were signifi cantly ( p . ) more frequent than in those without ards (n ). lfts were performed at visit for patients ( table ). all had been hospitalized, and there was no statistical difference in clinical and laboratory characteristics between these patients and those in whom lfts were not obtained. impairment of d lco was the most common ( / [ . %]) abnormality detected. data are presented as median (interquartile range) unless otherwise indicated. a(h n ) infl uenza a(h n ). see table our series of cases of a(h n ) identifi ed two independent risk factors associated with fatal pneumonia: progressive dyspnea after resolution of fever and a higher apache ii score on presentation. three months later, ggos of less severity were still present on chest radiographs in . % of patients ( / ) . lfts revealed decreased d lco ( , % predicted) in eight ( . %) of the patients tested. the clinical characteristics of a(h n ) pneumonia we described during the acute phase were similar to those reported by others. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this report, most patients complained of dyspnea, which usually occurred within week after illness onset. dyspnea continued to progress after resolution of fever in . % of the patients, a fi nding that has not been reported by others. in this report, the success rate for nppv was . %, which is much higher than that reported by others ( . %- . %). , [ ] [ ] [ ] although the death rate ( / [ %]) in patients who received invasive ventilation in our study was higher than that reported in another study, among patients with ards, the death rate was . %, similar to other reports. , , [ ] [ ] [ ] [ ] [ ] moreover, although nppv was used widely in the ward specifi cally set aside for patients infected with a(h n ), none of the doctors and nurses who were in direct contact with these patients developed respiratory symptoms or infl uenza-like illnesses. therefore, we believe that with proper infection-control procedures, nppv can be used successfully and safely for treating patients with a(h n ) pneumonia complicated by ards. it has been reported that % to % of autopsied patients with a(h n ) had evidence of bacterial coinfection. , streptococcus pneumoniae, streptococcus pyogenes , and s aureus were the most predominant pathogens. however, in our study, community-acquired bacterial infection (defi ned as sputum collected within h of hospitalization) was detected in only one of patients ( k pneumoniae ). the low yield of gram-positive bacteria before or within h of hospitalization may be due to the widespread use of prophylactic antibiotic therapy. in contrast, nosocomial infection was common in the patients ( / [ . %]), and gram-negative bacilli were the predominant causative pathogens. aspergillus spp was also seen. progressive a(h n ) infection, intubation, a prolonged hospital stay, iv antibiotic use, and use of oral or iv corticosteroids may be risk factors for nosocomial infection caused by gram-negative bacilli and aspergillus spp. we showed that symptoms and laboratory abnormalities in survivors of a(h n ) virus infection returned to normal within month of the onset of illness. nonetheless, ggos were still found at months, although no fi brotic changes were seen. in survivors of a(h n ) virus infection, persistent radiologic abnormalities including ggos, often with a reticular pattern, have been seen as long as year after illness onset. in survivors of severe acute respiratory syndrome (sars) followed for year, marked improvements in pulmonary fi brosis have been seen, but some patients still had residual changes. because this kind of fi brosis was reversible, it has been suggested that these fi ndings were partially caused by postinflammatory atelectasis rather than by genuine fi brosis alone. the resolution of lung abnormalities in patients with a(h n ) viral pneumonia seemed better than that seen in patients with sars and infl uenza a(h n ) infection. impairment of d lco was the most common ( / [ . %]) abnormality in lung function testing months after the onset of illness, followed by restrictive defects ( / [ . %] ). the d lco fi ndings were similar to the fi ndings of one study of patients with sars at -month follow-up visits. the impairment of d lco in survivors of sars persisted for year in . % of patients reported by other investigators. although the number of cases with lfts in our series is limited (only cases, of whom eight had a reduction of d lco ), it seemed that patients who had bilateral ggos on hrct scan were more likely to have an impaired d lco . during the convalescent period of ards, ggos may consist of intralobular fi brosis that is below the limits of resolution of hrct scanning. a longer follow-up study is needed to determine whether lung function abnormalities in patients infected with a(h n ) are irreversible and radiologic changes persist over time. to our knowledge, this is the fi rst report of symptoms and radiographic changes in patients with a(h n ) pneumonia during the convalescent period. there were several limitations. first, it is a singlecenter study with a limited number of patients. second, monthly follow-up visits were offered to all patients when they were discharged but some of the patients felt that was inconvenient and did not come back. as a result, out of survivors had one or more follow-up visits. third, most patients had underlying medical conditions that could have contributed to the lung function abnormalities. in conclusion, we found that progressive dyspnea after resolution of fever and a higher apache ii score on presentation were independent risk factors associated with death in patients with a(h n ) viral pneumonia. at the -month follow-up visit of survivors of a(h n ) pneumonia, some degree of ggos persisted in most patients and decreased d lco was common. clinical management of human infection with pandemic (h n ) : revised guidance. world health organization web site national infl uenza a pandemic (h n ) clinical investigation group . clinical and epidemiologic characteristics of early cases of infl uenza a pandemic (h n ) virus infection, people's republic of china national infl uenza a pandemic (h n ) clinical investigation group of china . clinical features of the initial cases of pandemic infl uenza a (h n ) virus infection in china iner working group on infl uenza . pneumonia and respiratory failure from swine-origin infl uenza a (h n ) in mexico utah due to novel influenza a(h n ) infection anzic influenza investigators . critical care services and h n infl uenza in australia and new zealand severe acute respiratory syndrome: temporal lung changes at thin-section ct in patients bacterial coinfections in lung tissue specimens from fatal cases of pandemic infl uenza a (h n )-united states pulmonary pathologic fi ndings of fatal pandemic infl uenza a/h n viral infections radiological features of lung changes caused by avian infl uenza subtype a h n virus: report of two severe adult cases with regular follow-up dynamic changes of serum sarscoronavirus igg, pulmonary function and radiography in patients recovering from sars after hospital discharge severe acute respiratory syndrome: thin-section computed tomography features, temporal changes, and clinicoradiologic correlation during the convalescent period pulmonary function and exercise capacity in survivors of severe acute respiratory syndrome the -year impact of severe acute respiratory syndrome on pulmonary function, exercise capacity, and quality of life in a cohort of survivors acute respiratory distress syndrome: imaging of the injured lung pandemic (h n ) . world health organization web site ministry of health of the people's republic of china . pandemic h n in china. ministry of health web site h n semicyuc working group . intensive care adult patients with severe respiratory failure caused by infl uenza a (h n )v in spain pandemic influenza a (h n ) virus hospitalizations investigation team . hospitalized patients with h n infl uenza in the united states hospitalised adult patients with pandemic (h n ) infl uenza in melbourne, australia california pandemic (h n ) working group . factors associated with death or hospitalization due to pandemic infl uenza a(h n ) infection in california predictors and outcomes of respiratory failure among hospitalized pneumonia patients with h n infl uenza in taiwan canadian critical care trials group h n collaborative . critically ill patients with infl uenza a(h n ) infection in canada critically ill patients with infl uenza a(h n ) in mexico clinical fi ndings and demographic factors associated with icu admission author contributions: dr bai: contributed to data collection and analysis and the fi rst draft of the manuscript. dr gu: contributed to data collection and analysis. dr cao: contributed to data collection and analysis, the fi rst draft of the manuscript, and revision of the manuscript . dr zhai: contributed to radiologic evaluation and review and revision of the manuscript. dr m. lu : contributed to postmortem examination and review and revision of the manuscript. dr y. lu: contributed to lung function testing and review and revision of the manuscript. dr liang: contributed to statistical analysis and review and revision of the manuscript. dr zhang: contributed to radiologic evaluation and review and revision of the manuscript. dr gao: contributed to postmortem examination and review and revision of the manuscript. dr huang: contributed to lung function testing and review and revision of the manuscript. dr liu: contributed to infl uenza virus testing and review and revision of the manuscript. dr song: contributed to data collection and review and revision of the manuscript. dr wu: contributed to data collection and review and revision of the manuscript. dr yin: contributed to data collection and review and revision of the manuscript. dr wang: contributed to data analysis and careful revision of the manuscript. financial/nonfi nancial disclosures: the authors have reported to chest that no potential confl icts of interest exist with any companies/organizations whose products or services may be discussed in this article . other contributions: all work was performed at the beijing chao-yang hospital, capital medical university and the school of basic medical sciences, peking university, bejing, china. we key: cord- -lzybfwc authors: savarino, andrea; shytaj, iart luca title: chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids date: - - journal: retrovirology doi: . /s - - - sha: doc_id: cord_uid: lzybfwc the restoration of the immune system prompted by antiretroviral therapy (art) has allowed drastically reducing the mortality and morbidity of hiv infection. however, one main source of clinical concern is the persistence of immune hyperactivation in individuals under art. chronically enhanced levels of t-cell activation are associated with several deleterious effects which lead to faster disease progression and slower cd (+) t-cell recovery during art. in this article, we discuss the rationale, and review the results, of the use of antimalarial quinolines, such as chloroquine and its derivative hydroxychloroquine, to counteract immune activation in hiv infection. despite the promising results of several pilot trials, the most recent clinical data indicate that antimalarial quinolines are unlikely to exert a marked beneficial effect on immune activation. alternative approaches will likely be required to reproducibly decrease immune activation in the setting of hiv infection. if the quinoline-based strategies should nevertheless be pursued in future studies, particular care must be devoted to the dosage selection, in order to maximize the chances to obtain effective in vivo drug concentrations. the quest for clinical candidates to counteract immune activation has become a "hot topic" in aids research, because hiv infection is characterized by malignant immune hyperactivation which correlates with disease progression and poor response to antiretroviral therapy (art) [ ] [ ] [ ] [ ] [ ] . moreover, immune hyperactivation is also regarded as a major obstacle to a cure for aids [ ] . in the beginning of the millennium, an article authored by one of us launched chloroquine as a tool to inhibit viral replication and the related malignant immune activation associated with some viral diseases [ ] . this article sparked a new wave of studies, in that it extended a theory, previously designed for hiv/aids [ ] , to other viral diseases characterized by excessive immune activation. as will be discussed below, by accumulating in the acidic organelles, chloroquine exerts both direct antiviral effects on enveloped viruses and decreases activation of several cell types involved in the immune response. chloroquine has since shown promise in preclinical studies (both in vitro and in vivo), as a therapeutic agent against emerging viruses such as mers cov [ ] . of note, chloroquine has been indicated as a promising candidate for filovirus treatment [ ] , especially during the latest ebola epidemic [ , ] . in two studies out of three, chloroquine showed antiviral activity in mice at the maximum tolerated dose [ , , ] , thus rendering this drug an interesting agent for further testing of combination anti-ebola therapies. however, the effects of chloroquine and its hydroxyl analogue hydroxychloroquine, on hiv infection, i.e. the initial target for the repurposing of these drugs, have remained controversial. on the one hand, based on the results of some earlier clinical trials, chloroquine/hydroxychloroquine has been recently resuggested as a promising candidate to restrict the hivrelated immune activation [ , ] . on the other hand, the results from the latest clinical trials indicate that chloroquine/hydroxychloroquine has no beneficial effect on immune activation [ , ] . we here provide a state of the art of the studies investigating the use of chloroquine/hydroxychloroquine as a therapeutic tool for hiv/aids and suggest the possible biological grounds for the clinical results obtained. moreover, we describe the reasons why our group decided to proceed further with strategies based on another drug, i.e. auranofin, which shares with chloroquine an antirheumatic effect [ ] . several reviews have recently been published on immune activation in hiv infection [ , , , ] . briefly, immune hyperactivation, commonly measured as the expression levels on peripheral blood lymphocytes of markers such as hla-dr, cd , or cd correlates with, and also predicts, disease progression (reviewed in [ , ] ). immune activation gradually decreases following therapy initiation [ ] and is maintained high in immunological non-responders, who are individuals maintaining low cd counts despite prolonged exposure to art [ , ] . while the initial studies were focused on the relationship between disease progression and activation of cd + t-cells [ ] , later studies better concluded that there is a broader relationship between disease progression and immune hyperactivation, involving also cd + t-cells [ , ] and innate immunity [ ] . immune activation and viral replication are believed to be mutually enhanced in a vicious circle. the virus, recognized by the immune system as non-self, induces immune activation, which, in turn, fuels viral replication by furnishing to the virus material to synthesize the different viral components. for example, lymphocyte activation increases the cytoplasmic levels of deoxyribonucleotides necessary for viral dna synthesis by reverse transcriptase [ ] . this vicious circle may still persist in anatomical compartments incompletely penetrated by art. hiv-induced immune activation is not limited to specific immunity, but exerts its effects on innate immunity as well. hiv- was shown to activate plasmacytoid dendritic cells (pdcs), which, differently from myeloid dendritic cells (the most potent antigen-presenting cells in the body), induce innate antimicrobial immunity by producing type i interferons ( figure ) [ ] . pdcs internalize hiv- through viral envelope/cd interactions, and the internalized virus activates these cells mainly through toll-like receptor (tlr- ) signaling ( figure ). comparative pathology corroborates the hypothesis that over-stimulation of this pathway may be associated with deleterious effects. sooty mangabeys (cercocebus atys), which can be infected by a simian homolog of hiv (i.e. simian immunodeficiency virus, siv) but do not develop aids, display weak ifn-α production upon stimulation with tlr- antagonists [ ] . on the contrary, rhesus macaques (macaca mulatta), which do progress to aids, produce high amounts of ifn-α when their pdcs are subjected to the same stimuli [ ] . moreover, another species displaying nonpathogenic siv infection, i.e. the african green monkey (chlorocebus aethiops), is characterized by an efficient control of ifn-α production following acute infection [ ] . pdcs decrease in peripheral blood during progression to aids, because, upon activation, they migrate to the lymphoid tissue [ ] . as a huge number of cells reside in the gut-associated lymphoid tissue (galt), according to the microbial translocation theory, the intestinal mucosa damaged by the consequent inflammation may become permeable to products of the gut microbiome which further enhance hiv-related immune hyperactivation [ , ] . finally, immune activation is one primary driver of both generation and maintenance of the viral reservoir, which is mainly constituted by latently infected, central and transitional memory cd + t-cells (henceforth t cm and t tm , respectively) [ ] . also in this case, comparative pathology has provided clues for understanding this phenomenon. it was shown that cd + t cm cells from sooty mangabeys express, upon activation, low levels of ccr , the main coreceptor for virus entry into cells, thus limiting infection of this important cellular compartment [ ] . instead, activated t cm cells from aids-developing species, such as humans and rhesus macaques, up-regulate the levels of ccr to a higher extent than cells from sooty mangabeys, thus facilitating the generation of a consistent viral reservoir [ ] . after these cells become quiescent, viral replication switches off, and latently infected, hiv-reservoir cells proliferate through low-level antigenic stimulation (t cm ) or il- -driven homeostatic proliferation (t tm ) [ ] . both processes are enhanced by generalized immune activation. multiple in vitro effects of chloroquine could support its possible use as a modulator of immune activation in hiv/aids: . chloroquine and its hydroxyl analogue hydroxychloroquine were shown in several studies to inhibit hiv- replication (reviewed in: [ ] ). the effects of these quinolines, mainly due to the induction of a defect in the maturation of the viral envelope glycoprotein gp [ , ] , might mimic the effects of broadly neutralizing antibodies directed against the viral envelope, although the effects of these antibodies are weaker than those directed against the cd binding site [ ] . these effects are additive to those of non-nucleosidic reverse transcriptase inhibitors (nnrtis) and synergistic to those of protease inhibitors (pis) [ ] . as quinoline drugs accumulate in lymphoid tissues [ ] , they might decrease ongoing viral replication during art in anatomical sanctuaries and, consequently switch off one of the main drivers of immune activation. chloroquine is also an inhibitor of p-glycoprotein (p-gp) and multidrug resistance proteins (mrps) [ , ] , cell surface glycoproteins which extrude several antiretroviral drugs to the extracellular medium. in line with this evidence, chloroquine was shown to increase the intracellular levels of pis [ ] . the effects of chloroquine in com-bination with nrtis are instead controversial: some reported an additive effect [ ] , while others did not detect it [ ] . the combined effects of chloroquine and integrase inhibitors are as yet unknown. . chloroquine accumulates in phagosomes of pdcs and inhibits their hiv-induced activation [ ] . it might therefore impact on innate immunity-induced immune hyperactivation. . a recent study showed that hydroxychloroquine selectively induces apoptosis in the memory t-cell compartment (cd ra − cd ro + ) [ ] . as, upon activation, naïve t-cells (cd ra + cd ro − ) acquire a cd ra − cd ro + phenotype, the "antimemory" effect should limit immune activation (figure ) [ ] . there is growing consensus that induction of apoptosis in the memory t-cell compartment might have a detrimental effect on the viral reservoir [ ] [ ] [ ] . in this light, chloroquine/hydroxychloroquine should have an anti-reservoir potential. this view is supported by another recent study which shows that chloroquine sensitizes to apoptosis the latently infected cells upon viral reactivation, likely by removing the anti-apoptotic effect of the virus structural gag gene products [ ] . these effects are potentially interesting, since it has been well demonstrated that viral reactivation from latency does not necessarily result in cell death [ ] . the macaque aids model is an important tool for preclinical assessment of strategies aimed at treating hiv/ aids [ ] . to our knowledge, chloroquine has been tested in this model on two occasions. in a first study, chloroquine ( mg every other day for days, i.e. a cumulative dosage comparable to that administered to humans with rheumatioid arthritis) was administered to three chinese rhesus macaques infected with the simian hiv-homologue, sivmac [ ] . although a decrease in activated pdcs was shown, no effects were seen on viral load and cd + and cd + t-cell activation (measured as cd expression) [ ] . as the immune activation set point is established during acute infection [ ] , vaccari et al. [ ] treated with chloroquine ( . mg/day for consecutive days) seven sivmac -infected rhesus macaques during the viral load peak that characterizes acute infection. apart from an unexpected, although transient, increase in the comparison of the susceptibility to chloroquine/hydroxychloroquine and auranofin of the cellular subsets involved in hiv production and persistence. shown in the figure is a schematic depiction of a activation and b differentiation stages of cd + t-lymphocytes and their correlation with viral production, latency and viral reactivation. both chloroquine/hydroxychloroquine and auranofin can influence these transitions by exerting a pro-apoptotic effect, the efficacy of which is graphically exemplified by the intensity of the blue color in the corresponding rectangles. efficacy gradients are based on data derived from refs. [ , , ] . expression of interferon-regulated genes (perhaps not population-relevant as possibly driven by only one animal), no significant differences were reported in viral load and t-cell activation and proliferation (measured as expression of cd and ki , respectively) [ ] . a trend was however noticed for maintenance of decreased levels of ki , cd and ccr in the gut of the chloroquinetreated animals, although the differences with values from the control group did not reach statistical significance. the effect of chloroquine in this simian model in the presence of art is still unknown. chloroquine and hydroxychloroquine have so far been tested in several hiv clinical trials. the results summarized in figure support the hypothesis that the chloroquine/hydroxychloroquine dosage may be an important driver of at least partial clinical success. suppressive effects on immune activation by chloroquine were shown in the trial conducted by murray et al. [ ] . however, in this trial, the dosage administered was not the same for all individuals, some of them receiving mg/die instead of mg/die. it is thus possible that the statistical significance of the effects reported in this study was driven by the higher dosage of the drug. this view is supported by a later study which tested chloroquine at mg/die and failed to show any effect of the drug [ ] . in two clinical trials conducted in the s, sperber et al. reported suppressive effects on immune activation (measured at that time as il- production) and viral load in individuals treated with mg of hydroxychloroquine/day (bioequivalent to mg/day of chloroquine) [ , ] . the other clinical trials testing hydroxychloroquine at a lower dosage (i.e. mg/day) led to conflicting results. earlier studies [ , ] and the more recent study of piconi et al. [ ] reported significant effects on viral load [ ] , cd counts [ ] , and immune activation. [ ] . instead, a more recent clinical trial, randomized and double blind, showed disappointing results, even hinting at possibly deleterious effects of hydroxychloroquine on viral load and cd counts [ ] . this trial was conducted in the absence of art, and this might explain differences between this study and the study of piconi et al., which was conducted on individuals under art [ ] . another trial in art-treated patients is currently ongoing and will provide more information on the effects of hydroxychloroquine (clinicaltrials.gov identifier: nct ). the hydroxychloroquine levels show high inter-subject variability and, although individuals receiving the higher hydroxychloroquine dosages ( and , mg/day) also showed significantly higher blood levels of the drug than those receiving mg/die, the range of the blood concentrations was in part overlapping in the different dosage groups [ ] . chloroquine has similar pharmacokinetics [ ] ; therefore, not only the dosage but also individual differences in drug metabolism and distribution may explain the different conclusions of the aforementioned studies. a large clinical trial has recently been completed (clinicaltrials.gov identifier: nct ) and its results can help to better represent the response of a population, thus abolishing the bias due to limited sample size. in this trial, however, chloroquine has been tested at mg/day in the absence of art; thus, in light of the results of the aforementioned clinical trials and considerations derived from basic science (see next paragraph), it is not surprising that the preliminary results released so far for this trial (https://clinicaltrials.gov/ct / show/nct ) do not show any significant effect of chloroquine on immune activation, viral load and cd counts. chloroquine/hydroxychloroquine-treated individuals display blood concentrations that are highly variable and only rarely exceed or µm, respectively [ , ] . therefore, at the steady state levels, these blood concentrations only in part overlap those at which a therapeutic effect is expected. for example, the ec of chloroquine on pbmc proliferation upon activation is, in general, ≥ µm [ ] , and this value can explain the varying results obtained in the different clinical trials, with clearer effects associated with the higher drug dosages. similarly, the pro-apoptotic effect of hydroxychloroquine on the memory t-cells is only moderate at the concentrations reachable in blood, especially in the lower range [ , ] . the pro-apoptotic effect of chloroquine described by li et al. on latently infected cells upon viral reactivation is instead more marked, although still partial, at the upper range of clinically achievable blood concentrations ( - µm) [ ] . this effect could therefore be visible in vivo in terms of viral reservoir reduction, but only treating with high chloroquine dosages in the presence of suppressive art. moreover, to maximize the chances to obtain viral reservoir reduction in vivo, chloroquine treatment should be prolonged, as the events of virus reactivation from latency are rather rare (estimated as one event of transition from latency to productive infection every ml of blood each day) [ ] . the effect of chloroquine on pdc activation (see figure published clinical studies evaluating the effects of chloroquine/hydroxychloroquine administration, alone or in combination with other drugs, in hiv infected subjects. highlighted in blue, red or white are the studies that have reported a positive, negative, or neutral outcome of the therapy respectively. cq chloroquine, hcq hydroxychloroquine. [ ] . in this case, the in vitro effect is in line with the results of two in vivo studies [ , ] . the use of chloroquine-related compounds with increased potency is yielding promising results in vitro [ ] , and it will be interesting to test the best-performing candidates in the simian aids model. the effects of chloroquine/hydroxychloroquine on viral replication have been repeatedly shown in vitro at lower drug levels than those inducing the cellular effects [ , , , ] . the blood concentration/ec ratio is however much narrower than those shown by antiretroviral drugs [ ] . the antiretroviral effects of chloroquine/hydroxychloroquine may though become visible in anatomical sanctuaries of those individuals treated with pi-containing antiretroviral regimens. in any case, we recommend that chloroquine/hydroxychloroquine be tested at the highest recommended dosages in future hiv clinical trials. alternative/complementary interpretations of the results so far obtained are possible. for example, the effectiveness of the art regimen employed may play a role in determining the magnitude of the effects (if any) observed following chloroquine/hydroxichloroquine addition. the study of piconi et al. [ ] , showing some benefit in immunological non responders, may indicate that the effects of chloroquine may be visible only in some subsets of individuals with peculiar immunological characteristics, and that these effects can be hindered when immunologically non homogeneous cohorts are studied. in this regard, larger studies, with cohorts stratified according to immunological responsiveness to art, could provide further information on the effects of chloroquine/hydroxychloroquine. another open question remains the influence of the duration of drug exposure, as it has been shown that chloroquine/hydroxychloroquine has cumulative effects [ ] . as a proportion of hiv-infected patients in africa may already be on chloroquine medication to prevent malaria, it might be worth examining the long-term effects of this treatment. in this regard, an ongoing phase iii clinical trial will assess the long-term effects of chloroquine and trimethoprim-sulfamethoxazole phrophylaxis on survival and disease control in hiv-infected individuals with suppressed viral load and good clinical response to art [ ] . given the aforementioned problems in the pharmacokinetics of chloroquine/hydroxychloroquine, our group chose to follow a different, yet partly similar, approach to corroborate treatment of hiv/aids. based on the feedback received from basic science studies and clinical trials that have been published throughout the years, we decided to use drugs the desired effects of which be striking in vitro at concentrations lower than the trough plasma concentrations in vivo. we also decided to redirect our research on the basis of the plasma concentrations rather than on whole-blood concentrations (widely used for chloroquine/hydroxychloroquine), because we thought that the former might better mimic the tissue culture concentrations. the drug that we selected is the gold-based compound auranofin, the pharmacodynamics and pharmacokinetics of which are well known, due to its decade-long employment for treatment of rheumatoid arthritis [ ] . the main rationale for the use of auranofin in our studies was its ability to target the central/transitional memory cd + t-cell compartment ( figure ) [ , ] , which is known to harbor the main viral reservoir in patients receiving art [ ] . auranofin is drastically active at submicromolar (i.e. ≤ nm) concentrations, which are below those readily achievable in human plasma [ ] . the administration of auranofin ultimately led to a reduction of the viral reservoir in art-treated sivmac -infected macaques [ ] . a review on our preclinical studies has recently been published [ ] and the reader is addressed to it for further detail. not surprisingly for a drug effective against an autoimmune disease such as rheumatoid arthritis, auranofin may as well be beneficial in terms of reduction of cell activation. in particular, the downregulation of the cd molecule induced by auranofin can disrupt the co-stimulatory signal often crucial for lymphocyte activation [ ] . moreover, apart from memory cd + t-cells, auranofin also targets the memory cd + t-cell compartment [ ] , i.e. a cellular subset known to be hyperactivated during hiv infection [ ] . interestingly, as described for hydroxychloroquine [ ] , auranofin was shown to disrupt in various cell lines the tlr- signaling [ ] , which is activated by bacterial lipopolysaccharides and likely constitutes another source of immune hyperactivation. in vitro data indicate that the impact of auranofin on lymphocyte activation may be mediated, at least in part, by modulation of oxidative stress [ ] . of note, the addition of a potent pro-oxidant drug, such as buthionine sulfoximine (bso), increases the potency of auranofin, decreasing phytohemagglutinin-induced activation and expression of the α-chain of the il- receptor [ ] . this is in line with our preliminary data in sivmac -infected macaques, in which a combined regimen of art, auranofin and bso induced a functional cure-like condition following suspension of all therapies [ ] . these observations provide proof of concept that drastically decreasing immune hyperactivation arrests siv disease progression and turns the virus/immune system balance in favor of the latter. clinical trials will be required to assess the potential of auranofin to decrease immune activation in art-treated subjects. finally, other drugs used or proposed for treatment of rheumatoid arthritis might find a place in the treatment of hiv/aids. for example, the janus kinase inhibitors tofacitinib and ruxolitinib have shown a promising in vitro activity against hiv replication [ ] . the ongoing in vivo studies on these compounds could provide an opportunity to analyze the effects of this treatment on viral replication and immune activation. as and ils contributed to the ideas and to the interpretation of the data presented in the manuscript. as and ils contributed to manuscript drafting and preparation of the figures. both 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auranofin and sodium aurothiomalate auranofin, as an anti-rheumatic gold compound, suppresses lps-induced homodimerization of tlr the interaction of auranofin and buthionine sulfoximine blocks activation of human peripheral t lymphocytes investigational treatment suspension and enhanced cell-mediated immunity at rebound followed by drug-free remission of simian aids ruxolitinib and tofacitinib are potent and selective inhibitors of hiv- replication and virus reactivation in vitro submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www ils is supported by a fellowship from the "sapienza" university (rome, italy). the istituto superiore di sanità holds a us patent on the use of the auranofin for treatment of hiv/aids. as is the leading inventor of the patent. key: cord- -wy ac y authors: khan, saima; pandotra, pankaj; manzoor, malik muzafar; kushwaha, manoj; sharma, rajni; jain, shreyansh; ahuja, ashok; amancha, vishal; bhushan, sashi; guru, santosh kumar; gupta, ajai prakash; vishwakarma, ram; gupta, suphla title: terpenoid and flavonoid spectrum of in vitro cultures of glycyrrhiza glabra revealed high chemical heterogeneity: platform to understand biosynthesis date: - - journal: plant cell tissue organ cult doi: . /s - - - sha: doc_id: cord_uid: wy ac y simultaneous qualitative and quantitative assessment of eight flavonoids and two terpenoids were performed in fourteen in vitro raised morphogenic cultures of glycyrrhiza glabra. our study revealed that the spectrum and production of ten compounds, under investigation, were higher in organized tissue than the undifferentiated mass, however, aerial portions of the in vitro raised plants (leaf and stem) were found to be devoid of therapeutically relevant triterpenoid, glycyrrhizin. a correlation was observed between cell maturation, morphological differentiation and glycyrrhizin accumulation. mature stolons ( months) were characterized by the maximum accumulation of glycyrrhizin ( . µg/mg) in in vitro plantlets. the cytotoxic effect of the extracts evaluated against a panel of human cancer cell lines (in vitro) indicated that the pancreatic cell line (mia-paca- ) were sensitive to all the fourteen extracts investigated. to the best of our knowledge this is the first comprehensive report relating plant growth regulators to metabolite spectrum and cytotoxic assessment in in vitro raised g. glabra cultures. overall, our findings demonstrated that the metabolite spectrum of in vitro raised morphogenetic lines, under different stages of maturation, might offer a platform to understand the regulatory aspects of the concerned metabolite pathway and their consequent role in differentiation. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. terpenoids and flavonoids are low-molecular-weight natural products ubiquitous in vascular plants encompassing a diverse range of structure-dependent biological functions. they have been accredited with a substantial role in the biochemistry, physiology and ecology of the plants. these secondary metabolites are acknowledged to provide a rich electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. pool for exploring novel compounds that may be exploited commercially in various industries (manach et al. ). many of these compounds found in licorice roots (glycyrrhiza glabra/glycyrrhiza uralensis) have received attention in the recent years owing to their multitude of biological activities (asl and hosseinzadeh ) . the root extract has shown to possess anti-inflammatory, hepatoprotective, anti-ulcer, anti-allergy (park et al. ) , and antiviral activity against various dna and rna viruses (fiore et al. ) , including human immunodeficiency virus (ito et al. ) and coronavirus (cinatl et al. ) . recently focus on the plant has escalated due to a triterpenoid saponin, glycyrrhizin, exhibiting potent anticancer activity in human (zhang et al. ) and mice (khan et al. ) . since almost all the licorice root used in mercantile, is collected from wild resources, therefore, its high demand and low supply has invited establishing alternative systems for production in renewable resources. however, selection of highly productive lines and optimization of chemical and physical culture milieu of the cells for copious production is a prerequisite for the commercial application of these systems. it has been demonstrated that the biosynthetic activity of cultured cells can be enhanced by regulating various factors like environment (ayabe et al. ), selecting hyper-producing lines (jiao et al. ; mardamshin et al. ) precursor feeding (yang et al. a (yang et al. , and transformation method . the expanding commercial importance of the plant based anti-cancer compound has kindled the researchers to look for their production in tissue culture system. exploitation of the biosynthetic capabilities of different cell types under a defined set of parameters, would not only give higher yields of the desired compound but will also provide deeper insight into regulatory processes thereon. various in vitro systems including callus, organ (gupta et al. ) , cell suspension (yang et al. (yang et al. , b root (du et al. ) and shoot cultures (kojoma et al. ) , have been exploited and subjected to terpenoid and flavonoid investigations in the past. in-vitro culture based metabolite analyses have shown the presence of flavonoids, chalcones and isoflavonoids (kobayashi et al. ) . flavonoids like formononetin, isoliquiritigenin, echinatin, glabridine (man et al. ; tamura and oda ) , liquiritigenin, p-hydroxy benzoic acid, -hydroxy formononetin have been reported from the in vitro culture lines of glycyrrhiza species. a recent study on adventitious roots of g. uralensis revealed production of flavonoids at par with the intact plant . similar results on flavonoid hyper-producing cultures were reported by (man et al. ) in suspended cells of glycyrrhiza. however, reports on oleanane-type triterpenoids (glycyrrhizin and glycyrritinic acid) are few and recent (gupta et al. ; shirazi et al. ; yang et al. ). on the contrary, lupane type triterpenoids (betulinic acid) and soya saponins, have been found in most of the earlier studies pertaining to tissue culture raised plants (hayashi et al. ) , exhibiting metabolite distribution pattern in various organs of the in vitro plant (hayashi et al. ) . literature survey revealed most of the studies on terpenoids and flavonoids detection in in vitro plant cultures remain restricted to few compounds (hayashi et al. ; yang et al. b) , restraining a comprehensive picture of the metabolite composition. until now, only a single report on the chemical analysis of six flavonoids and two terpenoids in suspension cells of g. uralensis has been accounted (man et al. ). here we are reporting the simultaneous assessment of ten compounds-eight flavonoids (butin, quercetin, isoliquiritigenin, formononetin, glabridin, -o-methylglabridine, hispaglabridin a and hispaglabridin b) and two terpenoids (glycyrrhizin and a-glycyrritinic acid) in the licorice root and in vitro raised callus, organ culture and plantlets of g. glabra. also, the in vitro cytotoxicity, against a panel of four human cancer cell lines, were evaluated for these samples. the present study broadens the concept to investigate the biosynthetic potential of in vitro raised g. glabra cultures. glycyrrhiza glabra rhizome, collected from selected clones of g. glabra obtained from hissar, was cultivated in the experimental farm of iiim jammu in ( . °n and . °e). plant raised under field conditions, following good agricultural practices, was exploited as a source material. in the present study, three-year-old field grown plant was utilized for in vitro experiments. for chemical analysis, field grown intact plant was harvested and divided into aerial and underground parts and extracted for analyzing using lc-esi-ms and hrms techniques. for in vitro studies, explants (leaf and nodal segment) from the same plant were employed. the explants were rinsed in detergent ( . % tween ) for min and washed in running water for h. after that, they were surface sterilized with sodium hypochlorite ( %, v/v) for min and dried on filter paper followed by thorough washing with autoclaved distilled water. finally, the explants were cultured aseptically. for culture establishment, three basal media (murashige and skoog, white and gamborg) with different pgr combinations were tried using leaf, stem and nodal segments as explants. the explants were dissected to the size of - mm each, and inoculated in their respective basal media (ms, gamborg and white) . all the cultures were maintained at ± °c under continuous cool white fluorescent light (phillips, india) having a light intensity of lmol/m /s. axenic root cultures and root based explants were incubated under dark conditions. the observations were taken after weeks of inoculation. the cultures were sub-cultured after every weeks. in each experiment, at least ten explants were used in triplicates in a petri dish. the best response was observed in the nodal segments in ms medium. subsequent culture development was done using nodal segment as explant. nodal explant (* mm) was used for callus induction using ms medium supplemented with % sucrose and , -dichloroacetic acid ( m) and -benzylaminopurine ( m). shoot induction from the nodal explants was observed in ms media having % sucrose supplemented with ba ( m) and naa ( . m). multiple shoots could be obtained after three sub-culturing in the same medium. three months old shoots (in pairs) were transferred to rooting ms medium supplemented with % sucrose and naa ( . lm). stolon regeneration from the root was performed according to gupta et al. ( ) . growth index (fresh weight) was determined at the time of harvest of the callus, and chemical profiling of the callus was employed in -week cultures. for the chemical analysis of morphogenetic lines, weeks old green stolons and weeks old brown stolons were harvested, weighed and analyzed for the flavonoid and terpenoids. twelveweek-old shoots and plantlet were harvested, divided into aerial and underground parts and analyzed for related flavonoids and terpenoids contents. the cyanocobalamine ( . ml/l) and calcium pantothenate ( mg/l) were added to vitamins in r medium. roots from in vitro grown plantlets were subjected to axenic root culture establishment. different pgr's with varying concentrations and combinations (naa, iaa, and iba) and vitamin composition (r ) in three basal media (ms, white's and gamborg's) were tested for faster, healthier root initiation and development in g. glabra. after weeks, the explants responded with different morphology and coloration. after two sub-culturing ( weeks), three different morphologies could be observed (roots, root ? callus, and callus). best axenic root growth was seen in ms medium supplemented with indol- -butyric acid ( m). roots, root with callus and callus were harvested and subjected to chemical analyses after weeks. human pancreatic cancer cell line miapaca- , human lung cancer line a , human breast cancer cell line mcf- and human colon cancer cell line hct- was purchased from sigma-aldrich, india (ecacc, type). cells were grown in mem medium containing % fcs, u/ml penicillin and mg/ml of streptomycin medium, maintained in a co incubator (thermocon electron corporation, houston, tx) at c with % humidity and % co gas environment. cells treated with tested materials were dissolved in dmso while the untreated control cultures received only the vehicle (dmso \ . %). in-vitro cytotoxicity of the cancer cells were plated in well plates at a density of . in ll of medium per well as published in (bhushan et al. ) . cultures were incubated with lg/ml concentration of test material for h. the medium was replaced with fresh medium containing lg/ml of -( , -dimethylthiazol- yl)- , -diphenyltetrazolium bromide (mtt) for h. the supernatant was aspirated, and mtt-formazan crystals were dissolved in ll dmso and the od of the resulting solution was measured at k nm (reference wavelength, k nm) on elisa reader (thermo labs, usa). the percentage of cell viability and growth inhibition were calculated according to the following equation the % of cell viability ¼ absorbance of treated cells À absorbance of blank  absorbance of control cells À absorbance of blank % growth inhibition ¼ À % cell viability since the assay was a quantitative colorimetric method for determination of cell survival and proliferation, the metabolic activity of viable cancer cells was assessed. metabolically active cells reduced pale yellow tetrazolium salt (mtt) to dark blue dmso soluble formazan crystals, which were quantified calorimetrically. the absorbance of the formazan directly correlates with the number of viable cells. chemicals and reagents: lcms-grade solvents (merck, germany) and milli-q-plus filter systems (millipore, plant cell tiss organ cult ( ) : - bedford, ma, usa) were used for the study. isolated, identified and authenticated investigated pure flavonoids(butin, quercetin, isoliquiritigenin, formononetin, glabridin, -omethylglabridine, hispaglabridin a, hispaglabridin b) and tri terpenoids( -a glycyrrhetinic acid, glycyrrhizin) were analyzed after ascertaining the purity (c . %) by hplc. stock solutions of the ten compounds under investigation namely, butin ( . mg/ ml), buercetin ( . mg/ ml),glycyrrhizin ( . mg/ ml), isoliquiritegenin ( . mg/ ml), formononetin ( . mg/ ml), glabridin ( . mg/ ml), -omethylglabridine ( . mg/ ml), -a glycyrrhetinic acid ( . mg/ ml), hispaglabridin a ( . mg/ ml) and hispaglabridin b ( . mg/ ml) were prepared in volumetric flasks individually in mobile phase. standard working solutions were then obtained by mixing and making appropriate dilutions of stock solutions using mobile phase ( : v/v). all the standard solutions were filtered through a . lm membrane filter (millipore) and injected directly. the stock and working solution were stored at ? °c. before making calibration curve, one blank injection was run to check the noise level of the system. lyophilized samples of field grown intact plant and in vitro raised callus cultures, organ cultures and plantlets were extracted following protocol published in (hayashi et al. ). for the simultaneous detection, identification and quantification of ten compounds under investigation lc-ms/ms analysis was performed on an agilent lc/ms-ms (agilent technologies, usa) triple-stage quadrupole mass spectrometer equipped with the electrospray ionization (esi) interface. liquid chromatography was performed on an agilent infinity (agilent, usa) quaternary pump equipped with an auto sampler, column heater, and online degasser. analytical chromatographic separations of g. glabra extract was carried out on a chromolith performance rp- e column ( . mm, merck, germany) protected by a chromolith guard column of the same company. the column temperature was maintained at °c; flow rate was optimized to . ml/ min and the sample injection volume was ll. the solvent system optimized was a linear gradient of acetonitrile/water and formic acid. the mobile phase was programmed at acetonitrile % for min, % for min, b; % for min, % for min, % for min, and from to % in min. esi positive mode with single ion monitoring was chosen for the quantification of investigated compounds. sample solutions of the extract of g. glabra were injected directly without pre-treatment under the optimum condition developed, for the simultaneous detection, identification and quantification of flavonoids and terpenoids from g. glabra. full-scan spectra obtained for the samples and pseudomolecular ions were selected for the selected ion monitoring (sim). compound identities were confirmed by comparison of molecular ion (sim) with that of standard and formula generated through high-resolution mass spectroscopy (hrms) analysis (supplementary figure ) fig. that the retention time of ten ( - ) investigated compounds were . , . , . , . , . , . , . , . , . and . min., respectively. the procedure could be completed in a short analysis time (within min), simultaneously separating all the ten investigated compounds. further, it was also found that analysis of the protonated molecular ion [m ? h]? and [m ? na]?, of the compounds ( - ) yielded different retention time ( . , . , . , . , . , . , . , . , . and . min) indicating their presence. all the quantification were performed in triplicate (fig. ) . the separated and identified ten peaks were subjected to hrms analysis for confirmation. the biosynthetic potential of plantlets and morphogenetically distinct culture lines were analyzed for the two triterpenoids and eight flavonoids in different stages of maturation and at various time intervals (table ) in the in vitro raised cultures of g. glabra. the metabolite spectrum of triterpenoids and flavonoids in the aerial and underground parts of plantlets were found to deviate significantly. the biologically active triterpenoid saponins (glycyrrhizin and -a glycyrrhetinic acid) were not detected in the aerial part of the months old in vitro raised plantlet (tc ). however, underground rhizome showed presence of ( . lg/mg) glycyrrhizin. among the organ culture analyzed, roots originating from in vitro plantlets, axenic root cultures (tr & tr ), yellow stolons (tc ), brown stolons (tc ) and brown root callus (tr & h ), produced glycyrrhizin under in vitro conditions. the glycyrrhizin content in these morphogenetic lines varied widely between . and . lg/mg. analysis of the organ cultures revealed that the mature brown stolons ( weeks) had a better capacity to form glycyrrhizin ( . lg/mg) than the yellow/green immature ( weeks) stolons ( . lg/mg). similarly, older roots showed more glycyrrhizin content ( . lg/mg) than the younger cultures ( . lg/mg). further observations exhibited root cultures grown in solid media, in isolation, produced least glycyrrhizin. it seems certain amount of maturation along with differentiation is required for glycyrrhizin biosynthesis. hayashi et al. ( ) had concluded similarly observing thickening of the roots and xylem tissues being tightly associated with glycyrrhizin accumulation (hayashi et al. ). literature has limited reports on glycyrrhizin production in cell cultures. nevertheless, the results of the present study corroborated well with earlier studies on in vitro raised stolons of g. glabra (gupta et al. ) , g. uralensis (kojoma et al. ) , callus tissues (wongwicha et al. ) and suspension cultures in glycyrrhiza species. the present study also detected glycyrrhitinic acid, aglycon of glycyrrhizin, in appreciable amount ( . lg/mg) in one of the axenic root culture (h ). among the eight flavonoids analyzed, four of them namely, hispaglabridine b ( . lg/mg), quercetin ( . lg/mg), butin ( . lg/mg) and formononetin ( . lg/ mg) could be detected in aerial parts of the plant. the underground parts of the plantlet, however, had an appreciable amount of all the eight investigated flavonoids. the roots analyzed from mature plantlet ( months old) had a moderate amount of all the investigated compounds, except quercetin and -a glycyrrhetinic acid which were not detected in the younger culture ( months) also. the underground parts of the plantlet showed an appreciable amount of glabridine ( . lg/mg) and -o-methylglabridine ( . lg/mg), other investigated flavonoids ranged between ( . lg/mg, quercetin to . lg/mg). formononetin. hispaglabridine b and glabridine showed interesting pattern of accumulation. hispaglabridine b, which was present in the highest amount ( . lg/mg) in the aerial part, was appreciably lesser in quantity in the underground rhizome. while glabridine that was undetected in the aerial part was present in the maximum amount ( . lg/mg) in the underground rhizome of the table biochemical constituents and yield (average of three in ng/g) along with relative standard deviation (rsd %) of the ten compounds identified in the cultured tissues at different stages of morphogenetic differentiation in glycyrrhiza glabra morphology gly. acid rsd acid rsd in vitro raised cultures under investigation. the stolon cultures and the axenic root tissues detected good amounts of formononetin ( . - . lg/mg) and isoliquiritigenin ( . - . lg/mg), and moderate amounts of other flavonoids, except quercetin, which was not detected in any of the stages under investigation. contrary to observation in glycyrrhizin, no correlation could be deduced between flavonoid accumulation and maturation of the axenic root cultures. the part of tissue present between stem and root had a moderate amount of all the compounds except -a glycyrrhetinic acid. it showed maximum presence of glabridine ( . lg/mg) followed by -o-methylglabridine ( . lg/mg) and isoliquiritigenin ( . lg/mg) and hispaglabridine b ( . lg/mg). literature survey on flavonoid contents of in vitro raised plantlets/organ culture in glycyrrhiza species gave scattered information. most of the published studies have reported various flavonoids in cell suspension cultures of glycyrrhiza inflate , glycyrrhiza echinata, g. glabra (furuya et al. (furuya et al. , and g. uralensis ). many of the researchers are of the view that in vitro cultures of glycyrrhiza has a better capability of production of flavonoids than the triterpenoids . however, reports about preferential accumulation and age-dependent synthesis of secondary metabolite are available in plants (piatczak et al. ) . basal medium and plant growth regulators (pgrs) play a significant role in tissue morphogenesis, differentiation and secondary metabolite production (raj et al. ) . as most of the terpenoids and flavonoids were found in the licorice roots, the role of basal media composition and pgr effect on root tissue differentiation and secondary metabolite production was investigated in in vitro raised cultures of g. glabra. five basal media namely, full strength murashige and skoog (fms), half strength ms (hms), r medium (ms with different vitamin composition), white's (w) and gamborg's (b) media supplemented with seven ( - ) pgr combinations were used to assess the response of in vitro raised root explants. simultaneous quantification of all the lines in various combinations demonstrated that irrespective of the basal medium, the root explants evoked three types of morphogenetic response, in % of the cultures ( / ). five out of sixteen cultures showed a very weak response, either due to the browning of the callus or very slow growth and hence were not studied further. among the cultures responded, root ? callus mixed morphology was observed in cultures ( %) while pure callus and pure root morphology were seen in and pgr combinations, respectively. maximum response ( out of combinations) was observed in half strength ms medium (hms), followed by r medium ( / ) and full ms and gamborg's media ( / ). while white's medium responded only in one pgr combination ( / ), recorded after weeks of culturing. a better response ( ) was observed in ms medium when vitamin composition of full ms was changed to r vitamin combination. in the present study, the response of root explants in gamborg's medium was noteworthy, as it responded in only out of combinations, and all the combinations produced only root type morphology, unlike the other basal media, where pure callus and root ? callus morphology were dominant. in accordance with earlier observations of gopitha et al. ( ) , the present study also concludes that iba augmented media is better in root induction than iaa. browning was observed in fm & cultures, parsaeimehr and mousavi ( ) also reported browning of callus when bap and naa growth hormones were used (parsaeimehr and mousavi ) . wongwicha et al. ( ) reported ms basal medium supplemented with , d or naa ? kinetin growth regulators, to be a better medium for calli induction in g. uralensis (wongwicha et al. ) . the present study also observed better root multiplication in ms medium supplemented with high concentrations of auxin (iaa/ iba) as was reported by thengane et al. ( ) . findings of the study suggested growth hormone naa in combination with iaa or iba evoked callusing while naa alone resulted in root initiation. similar observations were recorded by (elias et al. ) in shoot induction in ecinocereus cinerascens under in vitro conditions. the biochemical spectrum of the root explants, grown in different cultures in various pgr combinations, varied widely (table ) . among the flavonoids, maximum flavonoids ( ) were detected in root ? callus cultures having the combination of naa ? bap growth regulators (f ). as can be seen from the table , butin ( . - . lg/mg) and hispaglabridine a ( . - . lg/mg) were found in all the cultures in varied quantities. hispaglabridine b and glabridine were not detected in any of the cultures, while formononetin ( . - . lg/mg), isoliquiritigenin ( . - . lg/mg) and quercitin ( . - . lg/mg) were found in few cultures. -a glycyrritinic acid could not be detected in most of the samples except in axenic roots (h ) supplemented with ms media containing ( mg/ml) iba. the varied chemical spectrum is probably due to pgr effect as the root explants were of same age and origin and were grown under similar in vitro conditions. recent works of on anacyclus pyrethrum (singh et al. ) and securinega suffruticose (raj et al. ) have demonstrated the effect of pgr combinations on plant differentiation and secondary metabolite accumulation. the maximum number of compounds was reported from the mature stolons/roots of the plantlets (table ). all the investigated compounds, except the -a glycyrrhetinic acid, was present in the mature root of the in vitro culture, and their quantity was found to increase with an increase in the maturity or age. further, it was observed that hispaglabridine a was not found in and -week old axenic root and stolon cultures, the compound could only be detected in the brown stolons and underground plant part ( months), indicating a certain level of cellular organization or maturity of the tissue is a prerequisite for its biosynthesis. it was further noted that ms medium supplemented with auxins (naa/iba) had a positive effect on glycyrrhizin production. this is also supported by the observation that auxin predominately induced rooting, while its combination with kinetin or bap resulted in callusing, in the present study. these findings are also in agreement with the culture medium composition recorded in earlier studies where glycyrrhizin was detected in glycyrrhiza species (gupta et al. ; kojoma et al. ) . careful analysis of the quantification data (table ) showed mature stolon to be a good source of many flavonoids including hispaglabridine b, glabridine, -o-methylglabridine, butin, and isoliquiritigenin, while aerial plant part was predominantly rich in hispaglabridine b ( . ng/ mg) and quercitin ( . ng/mg). secondary metabolites are synthesized in specific tissues at a certain developmental stage of the plant. their production is significantly influenced by delicate physiological changes. with a better understanding of the cell culture behavior, their higher production through media modulation can be achieved. literature reports on flavonoid detection in glycyrrhiza species are dispersed. most of the reports refer to total flavonoid contents under in vitro conditions (ayabe ; jiao et al. ) . however, few reports support formononetin production in suspension cultures of g. glabra (arias-castro et al. ) , g. echinata (ayabe et al. ) and g. paddiflora (li et al. ) are available. similarly, glabridine has been detected in the in vitro plantlet (kinoshita et al. ) and suspension cells (man et al. ; tamura and oda ) . hispaglabridine a/b (chin et al. ; denisova et al. ) and isoliquiritigenin, (shirazi et al. ) were shown to be primarily distributed in the underground rhizomes of the naturally grown field plants. isoliquiritigenin was similarly detected in the underground parts of the plantlets and organ cultures, in the present study. its quantity, though, was found to be more in organized tissue than the undifferentiated mass. the isoliquiritigenin content varied between . lg/mg (mature stolon) to . lg/mg (the part between root and stolon), confirming the earlier report (man et al. ) on isoliquiritigenin production in tissue culture raised plantlet of g. glabra ( . - . lg/mg). our literature search revealed no reports on hispaglabridine a/b, butin, a flavanone, and quercetin, flavonoid detection from the tissue cultures of glycyrrhiza species. in the present study hispaglabridine a/b were found in most of the organ culture analyzed (table ) including underground plant parts and organ cultures. however, the compound was found to be absent in the aerial part and unorganized cell mass analyzed in the present study. the present lcms-based analyses detected butin in all the in vitro raised samples analyzed, ranging between . lg/mg (f ) callus to . lg/mg (h ) in mixed morphology. the observation carries significance as flavonoids such as hispaglabridinea/b, glabridine and butin are being reported for the first time in g. glabra under tissue cultures conditions. flavonoids and terpenoids have been known to possess anticancer properties (fukai et al. ; zhang et al. ). preliminary in vitro cytotoxicity of all the tested compounds ( - ) was evaluated by performing a comprehensive screening at a single dose of lg/ml, employing mtt assay, against various human cancer cell lines encompassing human breast cancer (mcf- ), pancreatic cancer cell lines (mia paca- ), lung cancer cell line (a ) colon cancer cell line . the results are summarized in table and expressed as a percentage (%) of growth inhibition. the results demonstrated that extracts induced cell growth inhibition ranged between nil to % in breast cancer, % in lung cancer and % in colon cancer cell lines. pancreatic cancer cell lines showed inhibition ranging between - %. out of the fourteen samples tested, pancreatic cancer cell lines responded to all the extracts examined, while the minimum response was observed in colon cancer cell lines ( ). field root, spb shoot, and pm extracts exhibited more than % cell growth inhibitory effect in a and mcf- cells. root extract prepared from field grown plant was found to be the most potent amongst the series that exhibited around % cell growth inhibition against breast cancer mcf- cells (supplementary figure ) . further, extracts having a higher amount of glycyrrhizin notably showed a higher percentage of growth inhibition in breast cancer cell lines, while relatively more content of flavonoid displayed higher inhibitory activity in lung cancer cell lines in the present study. the pattern and distribution of secondary metabolites in cell cultures are combinations of many interacting factors, including physiological status of culture, the origin of explants, age and degree of differentiation/developmental stage. present results points towards a close association between cell differentiation and glycyrrhizin metabolism and also indicated the influence of the state of differentiation (under the influence of pgrs and basal media) on flavonoid spectrum. metabolite spectrum of tissues at different stages of differentiation observed during the present study, offers a platform for understanding the inter-play between gene function and cellular regulatory processes involved in triterpenoid and flavonoid metabolism in g. glabra. it is a well-established fact that the functions of genes belonging to multi-gene families are widely divergent regarding substrate specificity and reaction sensitivity. in this context, medium modulation to regulate tissue differentiation could help in understanding the regulatory mechanisms involved in the production of the studied eight flavonoids and two terpenoids in the plant under investigation. additionally, the study has further extended the biochemical spectrum of the in vitro raised morphogenetic lines, their metabolite flux and role in distribution pattern inside the tissue and cell differentiation. the effect of cultural conditions on the accumulation of formononetin by suspension cultures of glycyrrhiza glabra review of pharmacological effects of glycyrrhiza sp. and its bioactive compounds regulation of flavonoidal biosynthesis in cultured glycyrrhiza echinata cells studies on plant tissue cultures. part . flavonoids from the cultured cells of glycyrrhiza echinata triterpenoid biosynthesis in tissue cultures of glycyrrhiza glabra var a novel lignan composition from cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia molt - and hl- cells anti-oxidant constituents of the roots and stolons of licorice (glycyrrhiza glabra) glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus isolation and gc-ms determination of flavonoids from glycyrrhiza glabra root culture system establishment and chemical constituent analysis of hairy root of glycyrrhiza uralensis the effects of plant growth regulators on shoot formation, regeneration and coloured callus production in echinocereus cinerascens in vitro antiviral effects of glycyrrhiza species cytotoxic activity of low molecular weight polyphenols against human oral tumor cell lines plant tissue cultures. xi. echinatin, a new chalcone from tissue culture of glycyrrhiza echinata studies on plant tissue cultures. . licodione, a new dibenzoylmethane derivative from cultured cells of glycyrrhiza echinata effect of the different auxins and cytokinins in callus induction, shoot, root regeneration in sugarcane direct rhizogenesis, in vitro stolon proliferation and high-throughput regeneration of plantlets in glycyrrhiza glabra examination of triterpenoids produced by callus and cell suspension cultures of glycyrrhiza glabra mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (hiv) research advances on production of flavonoids by licorice tissue culture glycyrrhizic acid suppresses the development of precancerous lesions via regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of wistar rats isoflavan derivatives from glycyrrhiza glabra (licorice) formation of chalcones and isoflavones by callus culture of glycyrrhiza uralensis with different production patterns in vitro proliferation and triterpenoid characteristics of licorice (glycyrrhiza uralensis fischer, leguminosae) stolons flavonoids from glycyrrhiza pallidiflora hairy root cultures studies on the hplc fingerprint of flavonoids in suspension culture cell of glycyrrhiza inflata bat stable transformation of suspension-cultured glycyrrhiza inflata batalin cells with agrobacterium tumefaciens chemical analysis and anti-inflammatory comparison of the cell culture of glycyrrhiza with its field cultivated variety chemical analysis and anti-inflammatory comparison of the cell culture of glycyrrhiza with its field cultivated variety the complex links between dietary phytochemicals and human health deciphered by metabolomics callus tissue of licorice (glycyrrhiza glabra l.) is a superproducer of flavonoids antiallergic activity of beta-glycyrrhetinic acid- -o-beta-d-glucuronide producing friable callus for suspension culture in glycyrrhiza glabra iridoid and phenylethanoid glycoside production and phenotypical changes in plants regenerated from hairy roots of rehmannia glutinosa libosch effect of plant growth regulators on the accumulation of indolizidine alkaloids in securinega suffruticosa callus cultures glycyrrhizin and isoliquiritigenin production by hairy root culture of glycyrrhiza glabra micropropogation of anacyclus pyrethrium and chemical profiling of the regenerated plants for pellitorine, the active principle glabridine manufacture by tissue culture of glycyrrhiza micropropa gation of licorice (glycyrrhiza glabra l.) through shoot tip and nodal cultures establishment and quality assessment of tissue cultures in glycyrrhiza uralensis fisch production of glycyrrhizin in callus cultures of licorice studies on callus tissue of glycyrrhiza inflata and glycyrrhizic acid content. hebei shifan daxue xuebao ziran kexueban the effect of precursor feeding on flavonoids biosynthesis in cell suspension cultures of glycyrrhiza inflata bat production of flavonoids in cell suspension culture of glycyrrhiza inflate (leguminosae) differentiation of roots of glycyrrhiza species by h nuclear magnetic resonance spectroscopy and multivariate statistical analysis inhibition of b-hydroxysteroid dehydrogenase type ii selectively blocks the tumor cox- pathway and suppresses colon carcinogenesis in mice and humans key: cord- -hcf gsv authors: lin, k.h.; chen, l.f.o.; li, s.d.; lo, h.f. title: comparative proteomic analysis of cauliflower under high temperature and flooding stresses date: - - journal: sci hortic (amsterdam) doi: . /j.scienta. . . sha: doc_id: cord_uid: hcf gsv high-temperature and waterlogging are major abiotic stresses that affect the yield and quality of cauliflower. cauliflower cultivars ‘h ’ and ‘h ’ are tolerant to high temperature and flooding, respectively; however, ‘h ’ is sensitive to both stresses. the objectives of this study were to identify the proteins that were differentially regulated and the physiological changes that occurred during different time periods in ‘h ’, ‘h ’, and ‘h ’ when responding to treatments of flooding, °c, and both stresses combined. changes in the leaf proteome were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof-ms) and identified by mascot peptide mass fingerprint (pmf) and database searching. stress treatments caused significant reductions in electrolyte leakage, chlorophyll fluorescence fv/fm, chlorophyll content, and water potential as stress times were prolonged. by the comparative proteomic analysis, protein peaks that were differentially expressed in response to combination treatments at , , and h, ( in ‘h ’, in ‘h ’, and in ‘h ’) were identified, of which were cultivar specific. differentially regulated proteins predominantly functioned in photosynthesis and to a lesser extent in energy metabolism, cellular homeostasis, transcription and translation, signal transduction, and protein biosynthesis. this is the first report that utilizes proteomics to discover changes in the protein expression profile of cauliflower in response to heat and flooding. cauliflower (brassica oleracea var. botrytis), a member of the brassicaceae family, is an economically and nutritionally important cole crop. the optimum mean temperature range for growing cauliflower is - • c. high temperatures cause cauliflower to form uneven and loose heads, puffy buds, yellow eyes and leaves in the head, narrow leaves, reduced leaf growth, delayed initiation of heading, and increased petiole-to-blade ratio, all of which lower the quality of or even make the heads unmarketable (wurr et al., ; nowbuth and pearson, ) . however, heat-tolerant cultivars are able to form heads at mean temperatures higher than • c. to expand the area of food production, crops are grown under stressful environments that likely lead to lower yields. the main contributing factors in the reduction of yield and quality in these areas are variations in climatic conditions such as flooding caused by rains (lin et al., ) . heavy rainstorms and standing water can leave soils saturated for days before draining, making waterlogging a problem in many parts of the world. air pockets in the soil become filled with water during saturation, thus creating hypoxic conditions followed by anoxia. flooding causes oxygen starvation, which is a consequence of the relatively slow diffusion of gases in water and from oxygen consumption by plant roots (takeshi and julia, ) . rapid leaf chlorosis is seen when cauliflower is subjected to waterlogging (shih et al., ) . flooding from large rainfall events is a major risk to fresh-market cauliflower production in taiwan, and most cauliflower cultivars are unable to tolerate flooding during typhoon-caused heavy summer rains. waterlogging and increasing temperatures associated with global warming are a growing concern, as they limit plant growth and productivity, especially in temperate species. over the past decade in the tropics and subtropics, there have been considerable increases in production because of the availability of new, tropically adapted cultivars, resulting in increased farmer incomes. in the field, the co-occurrence of several abiotic stresses rather than individual stresses are most damaging to crop production (mittler, ) . many physiological changes occur during high temperature and flood stressing that result in increased heat and flood http://dx.doi.org/ . /j.scienta. . . - /© elsevier b.v. all rights reserved. (hf) tolerance. the physiological mechanisms of hf tolerance are extensively studied in various plant species; however, our current understanding of the molecular biology of acquired tolerances to high temperature and waterlogging are still limited, and relatively little is known about proteins that are critical for controlling these dual mechanisms (wahid et al., ) . understanding these processes requires the identification and analysis of major proteins that underlie stress-regulatory networks. plant adaptations to environmental stresses depend on the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of stress-related proteins. knowledge of hf-responsive proteins is critical for further understanding of the molecular mechanisms of stress tolerance. plants respond to stress in part by modulating protein regulation, which eventually leads to restoration of cellular homeostasis, neutralization of toxins, and recovery of growth. however, there are no reports on the effect of hf stresses on the functioning of cauliflower. proteins are the direct effectors of the response of plants to stress. they not only include enzymes that mediate changes in the levels of metabolites but also serve as components of the transcription and translation machinery. in recent years, methods for analyzing the proteome have advanced considerably, and together with emerging sequence information in crops, plant proteomics has become increasingly useful for understanding gene functioning and networks in response to environmental stimuli. proteomics is a powerful tool for the quantitative analyses of different biochemical pathways including plant stress-related responses. comparative proteomic analyses of plants subjected to specific stress conditions allow the exploration of various defense-related mechanisms. for instance, proteomics approaches for the comparative analysis of protein abundance between untreated and stress-treated or tolerant and intolerant rice plants have greatly facilitated the study of plant cellular stress responses (komatsu et al., ) . therefore, identifying novel proteins and studying their differential display patterns in response to hf stresses will provide the molecular and physiological bases for improving tolerance to hf by cauliflowers. understanding the basis of hf stress signaling and tolerance mechanisms in cauliflowers is required for engineering local cauliflower genotypes that are more tolerant to heat and flood stressing. this goal can be achieved by deciphering the physiological and proteomic responses of cauliflower genotypes, heat-tolerant 'h ', flood-tolerant 'h ', and hf-sensitive 'h ' to flood stress at • c. the objective of this study was to identify the leaf proteins that are differentially regulated in response to hf stress using a pro-teomelab pf- d aided by improved databases. our hypothesis was that both hf stresses trigger plant responses that result in quantifiable changes in the proteins of 'h ', 'h ', and 'h '. proteomic characterization and functional analysis should facilitate a better understanding of the hf response mechanisms in brassica so that effective strategies for the genetic improvement of hf-tolerant plant cultivars can be established. to the best of our knowledge, there are no published reports addressing the identification of hfresponsive proteins in brassica using a proteomic approach. . . plant materials, culturing, and heat-and flood-stress treatments seeds of cauliflower (b. oleracea var. botrytis) 'h ', 'h ', and 'h ' were obtained from chin-long seed co. (tainan, taiwan). 'h ' is a heat-tolerant cultivar used especially in warm-subtropical regions such as southern taiwan, where average day temperatures reach as high as • c during the summer (june-august). 'h ' is a popular cultivar grown in taiwan and is flood-tolerant during the rainy season, particularly in rain-fed lowlands. 'h ' is a heat-and flood-sensitive cultivar, and mostly grown during winter in taiwan due to its cold tolerance. seeds were sterilized with . % (v/v) sodium hypochlorite, rinsed with distilled-deionized (dd) h o, and sown in a commercial potting soil mixture, and germinated seedlings were transplanted into . -cm diameter plastic pots and raised in a growth chamber under mol m − s − light with a h photoperiod and / • c day/night temperatures at a relative humidity (rh) of %. plants were watered three times a week and fertilizer ( : : , n:p:k) applied once a week to maintain optimal growth for days before the imposition of stress treatments. pots containing 'h ' and 'h ' plants were subjected to four treatments: non-flooding at • c (nfc, as control), flooding at • c (fc, control temperature), non-flooding at • c (nfh, high temperature), and flooding at • c (fh), for periods of , , , , , , and h in four growth chambers having a h photoperiod at mol m − s − radiation and % rh (lin et al., ) . in the case of flooding treatments, pots were randomly placed in cm × cm × cm plastic buckets and subjected to flooding by filling the buckets with tap water to cm above the soil surface. pots were removed from the buckets at different times following flooding, and plants were removed and their leaves from each plant clipped, frozen in liquid nitrogen, and stored at − • c in an ultrafreezer until used. three replicates of each time period for the four treatments were randomly placed in a growth chamber. the experiment was performed twice independently in a randomized design for growth environment, sampling day, and physiological analyses. . . determination of electrolyte leakage (el), chlorophyll fluorescence (cf), chlorophyll content (cc), and water potential (wp) cell membrane stability was estimated by measuring leaf ion leakage according to the method of huang and guo ( ) . leaves were excised and immersed in ml of distilled water in test tubes overnight at room temperature. the initial conductivity of the water was determined using a conductivity meter (model cdm , radiometer, cedex, france). tubes were placed in boiling water for min and then cooled to room temperature, and conductivity was again determined. the relative el (%) was calculated as the ratio of conductivity before boiling to that after boiling. cf components were quantified with a portable modulated fluorometer (mini-pam photosynthesis yield analyzer, walz, effeltrich, germany). the measurement of variable fluorescence to (fv)/maximum fluorescence level in light-adapted leaves (fv/fm) was previously described (lin et al., ) . relative cc per unit leaf area was determined using a spad (soil plant analysis development) analyzer (spad- chlorophyll meter, konica minolta, tokyo, japan). wp (in bars) was measured on the third leaf from the top of each plant using a pressure chamber (plant water system, skye skpm , tokyo, japan) (sairam et al., ) . the data shown in tables - represent the means of at least two independent sets of experiments with similar results. measurements of physiological parameters were analyzed by a three-factor completely randomized anova that compared cultivars, treatments, and time periods. for significant values, means were separated by a least significant difference (lsd) test at p ≤ . using pc sas . (sas institute, cary, nc, usa). because the negative effects of flood stressing on hf-sensitive 'h ' at • c were observed after h of stress treatments (see section ), 'h ', 'h ', and 'h ' plant leaves subjected to hf conditions for , , and h were used for subsequent proteomics analysis. proteins were extracted according to a previously published method (yan et al., ) with some modifications. briefly, two grams of plant leaves were ground in liquid nitrogen and crude protein extracts solubilized in ml of extraction buffer containing . % sds (sodium dodecyl sulfate), % ␤-mercaptoethanol, % glycerol, . % polyvinylpyrrolidone, and mm tris-hcl, ph . after min of incubation at • c, samples were centrifuged for min at , × g at • c; these two steps were done twice. the extraction buffer was further replaced by ml of start buffer ( mm tris-hcl, ph ) with a pd- column (ge healthcare) according to manufacturer (proteomelab pf- d kit, beckman coulter) protocols. protein concentrations in the samples were determined using the bradford assay (bio-rad, hercules, ca, usa) and bovine serum albumin (bsa) was used to generate a standard curve. a g aliquot of the total protein sample was passed through a . m filter before injection into the chromatography column. the high performance chromatofocusing (hpcf) column was treated according to manufacturer instructions (proteomelab pf- d protein fractionation system, beckman coulter, fullerton, ca, usa). briefly, the column was washed with volumes of water at a flow rate of . ml/min for min and then equilibrated with volumes of start buffer for min at . ml/min. after equilibration, each sample was introduced with a manual injector into the column and absorbance of the column effluent was monitored at nm. in the first dimension, proteins were bound to a strong anion exchanger and eluted with a continuously decreasing ph from . to . . fractions were collected at ph intervals of . in a deepwell plate. proteins eluted in the gradient were then separated in the second dimension using high performance reversed phase (hprp) chromatography. fractions were separated from the second dimension with an rp-hplc column using two solvents: . % trifluroracetic acid (tfa) in hplc water (solvent a) and . % tfa in acetonitrile (acn) (solvent b) (lee et al., ) . separation was performed at • c with a flow rate of . ml/min and protein fractions were detected by uv absorbance at nm. equilibration was achieved with solvent a for min followed by solvent b for min prior to each injection. from the selected first dimension fractions, . ml were injected, run for min, and the column eluted with a linear gradient of - % solvent b for min. thereafter, solvent b was continued for min, followed by re-equilibration with % solvent a for min (irar et al., ) . fractions from the second dimension were analyzed with karat tm version . (beckman coulter). protein extraction and two-dimensional gel electrophoresis ( -de) were carried out on three independent technical replicas of the bulk samples. proteins were in-gel digested with trypsin in the automatic investigator progest robot of genomic solutions. briefly, excised gel bands were washed sequentially with mm ammonium bicarbonate (nh hco ) buffer and acn. proteins were reduced and alkylated, respectively, by treatment with mm dithiothreitol (dtt) solution for h at • c and treatment with a mm solution of iodine acetamide. after sequential washings with buffer and acn, proteins were digested overnight at • c with g/ml of trypsin. tryptic peptides were extracted from the gel matrix with % formic acid and acn, and extracts were then pooled and dried in a vacuum centrifuge. digested peptide solutions were desalted with zip-tip pipet tips (nikkyo technos, tokyo, japan) and redissolved in l of . % tfa in % acn according to manufacturer instructions. proteins excised from two-dimensional gels were analyzed in matrix-assisted laser desorption ionization time-of-flight (maldi-tof, proteomics analyzer, applied biosystems) mass spectrometers. a l aliquot was mixed with the same volume of a matrix solution ( mg/ml ␣-cyano- -hydroxycinnamic acid in . % tfa in % acn) and spotted on a maldi plate. ms spectra were acquired in the positive reflector mode, with shots per spectrum being accumulated. three major peaks were selected for further characterization by ms/ms analysis. ms spectra were acquired using collision-induced dissociation (cid) with atmospheric air as the collision gas, the ms kv positive mode being used. the peak lists for ms/ms spectra from same-replicate samples were merged into a single file prior to database searching. the pkl and mgf files were searched against the swiss-prot database using mascot (http://www.matrixscience.com; london, uk). search parameters were set as follows: missed cleavage, fixed modification, peptide charge ± , and variable modifications were carbamidomethyl of cysteine and oxidation of methionine. trypsin was specified as the proteolytic enzyme. peptide tolerance and ms/ms mass tolerance were ppm and . da, respectively. peptide mass fingerprinting (pmf) match confidence was based on the mowse score and confirmed by accurate overlapping of matched peptides with mass spectrum major peaks. scores greater than (p < . ) were considered positive. peaks with multiple proteins detected by ms were not considered. only significant hits, as defined by mascot probability analysis (p < . ), were accepted. characteristic physiological responses of plants to different temperature and waterlogging treatments were evaluated. table illustrates the comparison of fv/fm values under four treatments at seven different times in leaves of three genotypes of cauliflower. levels of fv/fm in all plants progressively decreased as flood stress and heat stress (nfh and fh) durations were extended. fv/fm in 'h ' plants showed significantly lower values after h with table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( table lists the different points in time that waterlogging and high temperature treatments were monitored by measuring the changes in wp. when different treatments across time were compared, wp with stress treatments was significantly lower (more negative) than those with nfc treatment from to h for all genotypes, indicating that high temperature and/or flooding induced a decrease in leaf water level, subsequently affecting leaf wp. the trends in wp differed in all stressing treatments from to h in all plants. thus, the wp of the different genotypes responded differently to specific stresses. the lowest value in wp (− . mpa) was detected at h with exposure to nfh treatment in 'h '. proteome alterations in the three genotypes in response to short-term exposures to flood and high-temperature conditions were compared relative to treatments and controls. the proteome-lab system uses two-dimensional liquid chromatography based on high-performance chromatofocusing in the first dimension followed by high-resolution reversed-phase chromatography in the second dimension, and has become available for sample fractionation and more resolution at extreme ph values (soldi et al., ; pirondini et al., ) . protein peaks were identified and their expression patterns analyzed. protein fractions were separated according to isoelectric point (pi) and hydrophobicity and detected in a ph range of . - . by proteomelab pf- d. proteins were analyzed by maldi-tof-ms and identified via pmf and database searching by their calculated molecular weight, pi score, and percent coverage, and their database accession numbers and alteration values among stress treatments are represented in tables - . changes in the protein levels of 'h ' exposed to fh stresses at and h and their differentially expressed protein peaks are listed in table . compared to nfc control treatment, two protein peaks ( of phosphoglucosamine mutase and of rotidine -phosphate decarboxylase) under fc treatment for h were up-regulated (supplementary fig. s a) . furthermore, the expression level of s-adenosylmethionine synthetase (peak ) was increased during high-temperature stressing over h (supplementary fig. s b ). two identified peaks, of kda calcium-binding protein and of acetyl-coenzyme a carboxylase carboxyl transferase subunit beta, were also up-regulated when 'h ' plants were treated with fc for h in comparison to nfc treatment at h (supplementary fig. s c) . nine protein expression patterns were detected in 'h ' plants under high-temperature and flood stress at h, and the dynamic changes in the level of each protein are displayed in table . compared to nfc treatment at h, nfh treatment for h showed that the abundances of peaks (phosphoserine aminotransferase), (imidazole glycerol phosphate synthase subunit hisf), table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( (phosphoribosyl formyl glycin amidine synthase ), and (probably hydrogenase nickel incorporation protein hypa) were up-regulated (supplementary fig. s a) . moreover, the abundance of peak (putative holliday junction resolvase) in 'h ' plants was also up-regulated during h of fc treatment as compared to nfc treatment at h (supplementary fig. s b ). the last comparison includes four proteins (peaks , , , and , namely trna-methyltransferase, arginyl-trna synthetase, elongation factor g, and methionyl-trna formyltransferase, respectively) whose abundances were consistently up-regulated during flood stressing for h at • c. interestingly, all peaks were up-regulated after all treatments for h and h in both 'h ' (table ) and 'h ' (table ) plants. the identified proteins showed altered expressions by upregulation or down-regulation in 'h ' plants at the h and h time points when combination treatments were compared (table ) . only six protein peaks ( , , , , , and ; supplementary fig. s ) from comparative proteomic analyses between fh and nfc for h were down-regulated, all others being up-regulated. there were peaks found in fh vs nfc for h, with the remaining peaks being among other comparisons, including fh vs nfh, fc vs nfc, nfh vs nfc, nfh vs fc, and fh at h vs nfc at h. the carbohydrate metabolism related protein, fructose- , -bisphosphatase (peak ), was down-regulated under fh treatment compared to nfc treatment. nevertheless, the photosynthetic-related proteins ribulose bisphosphate carboxylase (rubisco) small chain (peak ) and large chain (peak ) were increased in abundance in 'h ' under flooding for h. additionally, dna mismatch repair protein muts was found in peak with down-regulation (fh vs nfc) and peak with up-regulation (fc vs nfc) in protein abundance through a homologue search. differentially expressed proteins identified in 'h ' plants at h and h under stressing are shown in table . the protein abundances of eight peaks ( , , , , , , , and of ribose import atp-binding protein, phenylalanyl-trna synthetase, rubisco large subunit, gmp synthase, atp synthase subunit, and phosphoribosylformyl glycinamidine synthase) were consistently down-regulated during the fh condition when compared to nfc treatment for h, but the other peaks were up-regulated in protein abundance. mitochondrial inner membrane protease (peak ) and rubisco small subunit (peaks and ) were up-regulated in 'h ' upon fc treatment compared to nfc after h ( supplementary fig. s a ). peaks and , respectively identified as uncharacterized . kda and sec cytosolic factor, were increased under heat stress at h ( supplementary fig. s b) . meanwhile, rubisco small subunit (peak ) was also increased in 'h ' under fh at h as compared to nfc at h ( supplementary fig. s c) . notably, the majority of peaks found in 'h ' at h represented rubisco down-regulation (peaks , , and ) and up-regulation (peaks , , , , , , , , , and ) in protein abundance. because flood and heat often co-occur in stress-prone environments, this study compared the combined effects of flood and heat with those of the single stresses on plant physiology. in general, the changes in plant physiology were greater under the combined treatment than under heating or flooding alone. the differences in responses to flood and heat in fv/fm, cc, el, and wp seen between cultivars suggested that the three genotypes have unique mechanisms for coping with environmental stresses. the heat-tolerant 'h ' genotype showed significantly higher fv/fm, cc, and wp values (tables , and ) and lower el% (table ) than the sensitive 'h ' genotype when heated for h under nfh treatment, which reflects its tolerant nature. the exposure to flooding for at least h under fc treatment provoked greater reductions in fv/fm and cc values in 'h ' plants than 'h ' plants (tables and ). in addition, flooding also significantly decreased the el in 'h ' plants relative to 'h ' plants after - h ( table ), indicating that 'h ' is flood-tolerant. similar losses of fv/fm, cc, and wp and an increase in el were observed in 'h ' under flooding and high temperature stresses (fh), but responded at different levels over time (tables - ) . table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( a soil water potential of approximately − mpa was used in greenhouse experiments to simulate field situations under severe stress conditions (ashoub et al., ) . the leaf wp of 'h ' plants under high-temperature treatment was significantly lower than in 'h ' and 'h ' plants after h, indicating that 'h ' plants suffered from heat exposure. a lower osmotic potential is responsible for maintaining turgor when water loss occurs due to high temperature. thus, high electrical conductivity due to a high concentration of electrolytes in the leaf sap of 'h ' plants should lead to alterations in membrane permeability and a reduced ability to retain solutes and water during high temperature and waterlogging. however, the ability of 'h ' and 'h ' plants to tolerate heat and flood stress, respectively, strongly depends on adjustments in water balance and ionic leakage. a decline in wp and an increase in el during heat and flood stress were associated with leaf water deficits as well as increases in ionic leakage. at the physiological level, the many effects of flooding and heat stresses indicate the importance of protecting plants from oxidative damage caused by the overproduction of ros that is elicited by increased ion leakage (kangasjarvi et al., ) . the chlorophyll fluorescence emission parameter, fv/fm, which is widely used as a proxy for the maximum quantum efficiency of ps ii photochemistry, was correlated with flooding and high temperature tolerance. in healthy leaves, the fv/fm value is close to . , which is a typical value for uninhibited plants. a lower table identification of differentially expressed proteins found in cauliflower 'h ' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for and h. peak ( value indicates that some proportion of the psii reaction centers are damaged, which is often observed in plants under stress (camejo et al., ) . flooding and high temperatures inhibit photosynthetic co fixation and damages photosynthetic electron transport at the site of psii where there exists a very sensitive photosynthesis apparatus. this reduction in the co assimilation rate observed in 'h ' plants was generated by effects on the calvin cycle and also on psii functioning. exposure of 'h ' plants to flooding and high temperature may lead to reductions in net photosynthetic rate, stomatal closure, and cell activities that damage photosynthetic membranes (jiang and huang, ) . these results suggested that the chlorophyll fluorescence parameters were stress specific and were not expressed solely in response to an increasing excess of photon energy. chloroplast development in 'h ' plants may be particularly sensitive to high temperature and flooding. alternatively, the pigments of the sensitive cultivar might have been destroyed because of a high sensitivity to oxidative stress. 'h ' plants suffered greater cc losses than 'h ' and 'h ' plants after - h of exposure. flooding and heat may induce stomatal closure and consequently reduce cc levels and wp as well. typically, the amount of cc is reduced by stress. this is consistent with our observations that chlorophyll loss was more pronounced in waterlog-and heat-sensitive 'h ' plants in accordance with the more pronounced and increased visible symptoms of leaf injury. leaf curling or folding were the initial and most obvious changes observed. most 'h ' leaves progressively became necrotic, epinastic, or wilted over the course of time; however, most 'h ' and 'h ' leaves visually appeared to be green and healthy after h of flooding and high temperature (photos not shown). along with visual symptoms, reduced cc could be used to monitor flooding and heat damage in green or senescent leaves. attempts have been made to breed for increased flooding tolerance and modify brassica cultivation or management practices to avoid injury from flooding and heat. environmental stresses represent the most limiting conditions for horticultural productivity and plant exploitation worldwide. important factors among them are water and temperature. flooding and high temperature are major abiotic stresses resulting in serious problems for the growth and yield of flood-and heat-sensitive plants. it is necessary to identify the physiological characteristics that reflect their complex underlying genetic make-up. these easily measured physiological biomarkers could be used as generic tools to develop a reliable method for selecting cauliflower cultivars having flood-and heat-stress resistance. tables - demonstrate that the chlorophyll losses in 'h ' plants were in concert with increasing el accumulations after h of flooding and heat treatments, which is an index of oxidative damages to cell constituents in general. this suggests that fv/fm and cc can be effectively used as reporter signals in screening for flooding and high temperature tolerances. these easily measured physiological biomarkers could be used as generic tools for developing a reliable method to select 'h ' and 'h ' cultivars having flooding and heat-stress resistances. these findings are important for farming in high-temperature areas and wetlands or other areas subject to short and intense rainfall events. proteomics techniques are becoming indispensable for understanding how plants adapt to abiotic stresses, and have promoted the identification of numerous stress-related proteins and the pathways in which they function (kosová et al., ) . changes in protein accumulation under stress are directly related to the physiological phenotypic responses of plants to stress. the proteomic responses to high-temperature or flood stresses are known for several plants, including rice (lin et al., ) , wheat (majoul et al., ) , barley (süle et al., ) , soybean (komatsu et al., ) , radish (zhang et al., ) , arabidopsis thaliana (palmblad et al., ) , agave americana (shakeel et al., ) , and populus euphratica (ferreira et al., ) . in the present study, we analyzed physiological and proteomics data in different genotypes of cauliflower under conditions of high-temperature and waterlogging stresses over different time periods. approximately peaks were detected at the tested time points, whereas peaks were successfully observed to be differentially regulated ( down-regulated, up-regulated) and expressed at a minimum of one time point. among them, , , and proteins were identified in 'h ', 'h ', and 'h ' plants, respectively, indicating that they were regulated in a genotype-specific manner ( supplementary fig. s ). rubisco small and large chains, kda calcium-binding protein, molybdenum cofactor biosynthesis protein a, and phosphoserine aminotransferase were observed in both 'h ' and 'h ' plants. phosphoribosylformyl glycinamidine synthase and arginyl-trna synthetase were both found in 'h ' and 'h ' plants ( supplementary fig. s ). moreover, in 'h ' plants, peaks and were both identified as dna mismatch repair protein muts, while and were identified as phosphoglucosamine mutase (tables and ). these sister peaks may represent close homologues, but this could not be resolved based on mass spectrometric data and might instead be isoforms resulting from differential post-translational modifications (rollins et al., ) . as stress times increased, additional responsive proteins continued to be identified. for instance, (table ) and (table ) peaks were observed in 'h ' under stress treatments for h and h, respectively. in this study, some of the proteins identified were well characterized in terms of response to stressing, while others were not. the biological functions of differentially regulated proteins include roles in photosynthesis, energy metabolism, cellular homeostasis, response to stimuli, transcription and translation, protein biosynthesis, mediation of signal transduction, and other functions. for survival, plants must respond to flood and heat stresses in a different manner from regulating protein expressions for biochemical and physiological adaptations. photosynthesis is one of the systems that is most sensitive to high-temperature stress. changes in environmental temperatures are primarily reflected in photosynthesis, triggering a response that is aimed toward the best possible performance under new conditions. for this, a balance is sought between the energy of absorbed light, carbon assimilation, and consumption by metabolic sinks. several studies have shown that high-temperature stress can significantly inhibit the rate of photosynthesis (yan et al., ; salvucci and crafts-brandner, ) . in our study, rubisco units (small and large chain) related to photosynthesis were differentially expressed and regulated by combination treatments and among genotypes. for example, the up-regulation of rubisco was detected in 'h ' (peaks and , table ) and 'h ' (peaks (peaks , (peaks , (peaks , , , (peaks , . however, the level of expression of the rubisco large subunit was down-regulated in 'h ' during fh treatment for h (peaks , , and , table ). rubisco proteins resist high-temperature and flood stresses by inhibiting photosynthesis and other nonessential metabolic processes. in contrast, to increase energy metabolism, the expression of proteins related to redox homeostasis and response to stimuli were up-regulated, thereby maintaining physiological balance during stress. rubisco catalyzes the first major step of carbon fixation in photosynthesis. affinity of rubisco for co decreases with increasing temperature (yan et al., ) . the diminished capacity of rubisco and its low affinity for co suggest that carbon fixation or assimilation was highly inhibited when 'h ' plants were exposed to stress, especially under the high temperature and flooding stresses ( table ) that lead to reduced rates of photosynthetic co assimilation. photorespiration serves as an energy sink, preventing the over-reduction of the photosynthetic electron transport chain and photoinhibition. lee et al. ( ) reported that high temperature over h significantly inhibits the activity and quantity of rubisco subunits in rice seedlings. the effects of drought stress on rubisco represent reductions in rubisco activity (parry et al., ) . exposure to salt stress brings about a reduction in the overall growth and productivity of plants by disturbing the function of vital components of photosynthesis, such as psii and rubisco (ghaffaria et al., ) . consequently, a strong reduction in fv/fm value (table ) and cc content ( table ) while maintaining photosynthesis under stress after h was seen in all plants. some researchers report its up-regulation (ge et al., ; budak et al., ) , whereas others find downregulation (gaoa et al., ) or even both (guoa et al., ) in response to abiotic stresses. as a result, an up-regulation in rubisco levels may also indicate an increase in the photorespiration rate. molecular chaperones, including heat shock proteins (hsps), are key components of innate immunity in plants. they have major roles in protein folding and signal-transduction networks, cellcycle control, protein degradation, and protein trafficking (wang et al., ) . hsp chaperone pathways require energy in the form of atp hydrolysis in order to function. the atp-dependent clp proteases (hsp ) represent a class of hsp. in addition to their function as molecular chaperones, they function in protein disaggregation and protein degradation when removal of potentially harmful polypeptides arising from misfolding, denaturation, or aggregation are important for the maintenance of cellular homeostasis. the mechanism for rescuing proteins from aggregation is proposed to include the cooperation hsp (kregel, ; sarkar et al., ) . hsps are typically induced when cells are exposed to various types of environmental stresses. the synthesis of hsps under high temperatures is reported to be related to thermotolerance in plants, and plants with a decreased expression of hsps show compromised tolerance to acquired thermo-tolerance (charng et al., ) . hsps are closely related to the acquisition of heat resistance in plants. choi et al. ( ) identify several high molecular weight hsps whose expression was up-regulated significantly in pyropia tenera under high-temperature stress for h and h. furthermore, lee et al. ( ) report that several hsps are expressed in rice leaves during heat stress. high temperature might increase the potential risk of protein misfolding, and an active protein quality control system inside cells plays an important role in plant tolerance to heat stress. hsps are also increased by drought stress in the sugar beet (hajheidari et al., ) , wheat (demirevska et al., ) , and sugarcane (jangpromma et al., ) . in our study, peak of atp-dependent clp protease proteolytic subunit was up-regulated in response to heat stress for h in 'h ' plants (table ) . this suggests that the increased level of hsp under high-temperature stress allowed the heat-tolerant 'h ' to produce more energy through atp hydrolysis and the degradation or reactivation of damaged proteins and prevented protein misfolding, resulting in reestablishing normal protein conformations and cellular homeostasis. s-adenosylmethionine (sam) synthetase catalyzes the synthesis of sam that serves as a methyl group donor in transmethylation of proteins, nucleic acids, and a precursor in the biosynthesis of polyamines, biotin, and nicotianamine in plants (roeder et al., ) . sam in shoots underwent increased protein synthesis in a heat-tolerant barley cultivar and showed a reduced trend in an abiotic stress-susceptible cultivar (süle et al., ) . in our study, the expression of sam synthetase was significantly up-regulated (peak , table ) in heat stressed 'h ' over h. therefore, it is most likely that an increased amount of sam synthetase (as a result of the increased formation of sam methyl donors) in 'h ' contributes to its stronger heat tolerance compared to controls. in addition, peak of adenylate kinase (adk) was observed in 'h ' under heat stress for h (table ) . adk catalyzes the salvage synthesis of adenine monophosphate from adenosine and atp (wang et al., ) . heat and flood treatments markedly increased the abundance of two elongation factors (efs): ( ) elongation factor g was increased in 'h ' (peak ) under flooding at h ( table ) and ( ) transcription elongation factor grea was increased in 'h ' (peak ) under fh at h ( table ). the elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukaryotes. efs are the critical regulators of protein synthesis, which is influenced by high temperature and drought stress in arabidopsis (rizhsky et al., ) and rapeseed (mohammadi et al., ) . regulation of the transcriptional and translational machinery is considered to be an important component of cellular stress response. the increased expression of ef grea in 'h ' and ef g in 'h ' likely led to increased protein synthesis, which served to reduce the effects of treatment stress. plants under stress can respond by sensing and transferring stress signals through signal transduction networks. the response to stress is likely mediated through signaling pathways that regulate the expression levels of a range of proteins (hirayama and shinozaki, ) . in our study, gtp-binding protein lepa (peak , table ) involved in signal transduction was identified. the up-regulation of the lepa protein in response to flooding at • c might reflect the role of this protein in cell signaling under stress. ca + -binding protein is mainly resident in the endoplasmic reticulum where it serves as a calcium modulator and chaperone of newly synthesized glycoproteins (nam et al., ) . the latter have functions in plant growth and development as well as biotic and abiotic stress responses. aghaei et al. ( ) indicated that ca + -binding protein was up-regulated by salt stress in potato leaves. salinity induces ca + accumulation in the cytosol that starts the signaling pathway. the modulation of intracellular ca + levels is regulated by calcium-binding proteins, which, after activation, induce specific kinases (capriotti et al., ) . a kda calcium-binding protein was identified as an up-regulated peak ( ) under flooding (table ) , suggesting the involvement of intracellular calcium homeostasis and signal transduction in 'h ' during waterlogging. atp synthase is a large multi-subunit transmembrane enzyme complex. the catalytic site for atp synthesis is localized primarily on the beta subunit. atp synthase is known to be involved in energy metabolism and was found in 'h ' plants upon fh stressing for h (table ) . down-regulation of the catalytic subunit of atp synthase subunit beta (peak ) suggested that the energy production systems of 'h ' were highly affected by heat and flood stresses. huseynova et al. ( ) also found that the atp synthase complex was less accumulated in drought-sensitive wheat 'giymatli- / ' than in drought-tolerant azamatli- under water stress. in addition, impaired atp synthesis under drought is regarded as a major constraint to photosynthesis (flexas and medrano, ) . fructose- , -bisphosphatase (fbpase) is involved in carbohydrate metabolism during gluconeogenesis, and also an important enzyme in the calvin cycle that plays a crucial role in plant responses to some abiotic stimuli (fan et al., ) . the downregulation of fbpase in 'h ' under fh at h (peak , table ) indicated that gluconeogenesis was inhibited under stressing. an increase in ribosomal protein is a candidate for initiating a translation site, and the phosphorylation of ribosomal proteins is important in the regulation of protein synthesis (fatehi et al., ) . the s ribosomal subunit catalyzes the peptidyl transfer reaction of mrna-directed protein biosynthesis (sobhanian et al., ) . up-regulation of the s ribosomal protein l p (# ) was observed under high temperature stress at h in 'h ' (table ) , indicating the resistance of 'h ' plants to the inhibitory effect of high temperature on protein biosynthesis. in addition, pentatricopeptide repeat (ppr) proteins are characterized by tandem arrays of degenerate amino acid motifs (o'toole et al., ) and are thought to be rna binding proteins involved in posttranscriptional processes in mitochondria and chloroplasts (schmitz-linneweber et al., ) . meanwhile, ppr proteins are also essential for rna editing in chloroplasts (emi et al., ) . peak (pentatricopeptide repeat containing protein) of 'h ' was up-regulated under fh within h ( table ), suggesting that 'h ' was able to increase rna maturation and protein function in chloroplasts under fh. taken together, genetic differences are supported by the high number of proteins differentially regulated between genotypes. our data suggest that the early response of cauliflower to flooding and high temperature might be an important stress adaptation for survival following not only hypoxia and heat, but also direct damage to cells by flooding and high temperature. all of the identified proteins likely work cooperatively to reestablish cellular homeostasis under stress. the long term goal of our work is to help breed a competitively higher flood-and heat-tolerant cauliflower to be grown in lowlands during summer. the identification of the unique stress-responsive proteins will allow further dissection of the genetic basis of this transgressive performance in offspring. our results not only provide information for selecting lines having better tolerance to waterlogging and heat stresses, but also provide a basis for understanding cauliflower metabolic pathways and their cross-talk under stress. further verification of the correlative stress-responsive protein by gene expression analyses of these stressed-responsive genes, such as rubisco, efs, hsp , fbpase, adk, atp synthase, sam synthetase, ppr protein, gtp-binding protein lepa, ca + -binding protein, and ribosomal protein l p, would facilitate our understanding of the heat-and flood-response mechanism in cauliflowers. this study provides information on mechanisms underlying the contrasting responses to hf stresses. different genotypes showed differing physiological responses to combinations of stress treatments. all dynamics of the peaks were analyzed across all treatments, and a comparative analysis identified stressresponsive proteins in cauliflower under short-term stressing. the majority of these stressed-responsive proteins were genotype specific. these identified proteins emerged as key participants in stress tolerance. most of the changes in the expression levels of these proteins in response to stressing were involved in photosynthesis. 'h ' plants resisted high-temperature stressing by inhibiting photosynthesis and other unnecessary metabolic processes. energy metabolism was increased and up-regulation of the expression of proteins related to cellular homeostasis, as well as those that respond to stimuli, served to maintain physiological balance during stress. concurrently, the expression levels of proteins related to transcription, translation, and signal transduction were also up-regulated to promote survival under hf stressing. the genetic variations identified in the proteomes, plant growth, and photosynthetic performance in response to flood and heat represent stress adaption mechanisms to be exploited in future cauliflower breeding efforts. proteome analysis of potato under salt stress comparative analysis of barley leaf proteome as affected by drought stress proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanolc-esi-ms/ms changes in photosynthetic 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limitations revisited proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis ( d-dige) comparative proteomic analysis of grain development in two spring wheat varieties under drought stress physiology and proteome responses of two contrasting rice mutants and their wild type parent under salt stress conditions at the vegetative stage comparative proteomic analysis of salt response proteins in seedling roots of two wheat varieties proteome analysis of sugar beet leaves under drought stress research on plant abiotic stress responses in the post-genome era: past, present and future responses of antioxidative system to chilling stress in two rice cultivars differing in sensitivity structural-functional state of thylakoid membranes of wheat genotypes under water stress proteomic analysis of wheat embryos with -de and liquid-phase chromatography (proteomelab pf- d) -a wider perspective of the proteome a proteomics analysis of drought 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temperature on expression chilling stress and chilling tolerance of sweet potato as sensed by chlorophyll fluorescence identification of flooding-response genes in eggplant roots by suppression subtractive hybridization proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heatresponsive proteins from nonprolamins fraction abiotic stress, the field environment and stress combination comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid f line under drought stress comparative proteomic analysis of early salt stress-responsive proteins in roots of snrk transgenic rice the effect of temperature and shade on curd initiation in temperature and tropical cauliflower on the expansion of the pentatricopeptide repeat gene family in plants heat-shock response in arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling rubisco activity: effects of drought stress a -d liquid-phase chromatography for proteomic analysis in plant tissues when defense pathways collide: the response of arabidopsis to a combination of drought and heat stress sam levels, gene expression of sam synthetase, methionine synthase and acc oxidase, and ethylene emission from n. suaveolens flowers leaf proteome alterations in the context of physiological and morphological responses to drought and heat stress in barley (hordeum vulgare l.) role of antioxidant systems in wheat genotypes tolerance to water stress inhibition of photosynthesis by heat stress: the activation state of rubisco as a limiting factor in photosynthesis heat shock proteins: molecules with assorted functions a pentatricopeptide pepeat protein facilitates the trans-splicing of the maize chloroplast rps pre-mrna proteomic and transcriptomic analyses of agave americana in response to heat stress physiological index for tolerance to high temperature and waterlogging in cauliflower proteome analysis of soybean leaves, hypocotyls and roots under salt stress proteome profile of human urine with two-dimensional liquid phase fractionation proteomic analysis of small heat shock protein isoforms in barley shoots plant responses to hypoxia -is survival a balancing act? heat tolerance in plants: an overview role of plant heat-shock proteins and molecular chaperones in the abiotic stress response proteomic alternations of brassica napus root in response to boron deficiency investigating trends in vegetable crop response to increasing temperature associated with climate change proteomic profile analysis of pyropia haitanensis in response to high-temperature stress proteomic analysis of heat stress response in leaves of radish (raphanus sativus l.) this research was supported by grants (nsc , - -b- - -my ) from national science council, taiwan, roc. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.scienta. . . . key: cord- -ogu f o authors: abdelnabi, rana; neyts, johan; delang, leen title: antiviral strategies against chikungunya virus date: - - journal: chikungunya virus doi: . / - - - - _ sha: doc_id: cord_uid: ogu f o in the last few decades the chikungunya virus (chikv) has evolved from a geographically isolated pathogen to a virus that is widespread in many parts of africa, asia and recently also in central- and south-america. although chikv infections are rarely fatal, the disease can evolve into a chronic stage, which is characterized by persisting polyarthralgia and joint stiffness. this chronic chikv infection can severely incapacitate patients for weeks up to several years after the initial infection. despite the burden of chikv infections, no vaccine or antivirals are available yet. the current therapy is therefore only symptomatic and consists of the administration of analgesics, antipyretics, and anti-inflammatory agents. recently several molecules with various viral or host targets have been identified as chikv inhibitors. in this chapter, we summarize the current status of the development of antiviral strategies against chikv infections. chikungunya virus (chikv), belonging to the alphavirus genus of the togaviridae family is an arthropod-borne virus transmitted by female mosquitoes of the aedes species [ ] . chikv infections cause chikungunya fever which is characterized by abrupt fever, rash and bilateral symmetric arthralgia. in most of the chikvinfected patients the acute phase is followed by persistent disabling polyarthritis that can severely incapacitate the patient for weeks up to several months [ ] . despite the widespread emergence of chikv and the high morbidity rate associated with it, there is no approved vaccine or antiviral treatment available at the moment. the current therapy is therefore purely based on the relief of the patient's symptoms and consists of the administration of analgesics, antipyretics, anti-infl ammatory agents such as paracetamol, and nonsteroidal anti-infl ammatory drugs, and of bed rest and fl uids intake [ ] . the use of aspirin during chikv infection is to be avoided because of the risk of bleeding and the potential risk of developing reye's syndrome [ ] . in addition, the use of systemic corticosteroids is not recommended due to the strong rebound effect after cessation of treatment [ ] . in severe cases, where patients have limited response to nonsteroidal anti-infl ammatory drugs, disease-modifying antirheumatic drugs (dmards) such as methotrexate, hydroxychloroquine, or sulphasalazine can be administered to relieve the symptoms [ , ] . the development of novel, potent antiviral drugs against chikv is thus urgently needed. antiviral agents targeting the entry of enveloped viruses are of major interest since they inhibit an early step in the viral life cycle which minimizes the cell damage caused by intracellular viral replication. in addition, viral entry inhibitors may target extracellular components, which are more accessible; therefore, they could be effective in lower dosages with limited toxicity [ ] . chloroquine is an antimalarial drug that has also been shown to inhibit the in vitro replication of several viruses, including hiv, severe acute respiratory syndrome (sars) coronavirus, and alphaviruses [ ] . chloroquine was reported to inhibit chikv entry into cells, possibly by raising the endosomal ph and thus preventing the fusion of the chikv e protein with the endosomal membrane [ , ] . the potential effect of chloroquine treatment was assessed in two clinical trials. in a fi rst clinical trial improvement of symptoms in the chronic phase of chikv infection were reported following chloroquine treatment [ ] , whereas another study failed to prove the effi cacy of chloroquine as a treatment for the acute phase of chikv infection [ ] . therefore, the use of chloroquine as anti-chikv antiviral requires further study to prove its effectiveness and to determine the appropriate dosage and length of treatment. arbidol is a broad-spectrum antiviral that has been licensed in russia and china for the treatment and prophylaxis of infl uenza and other respiratory infections [ ] . arbidol was also reported as an inhibitor of chikv infection in mrc- cells [ ] . the mechanism of its anti-chikv activity has not been totally elucidated. an arbidol-resistant chikv strain could be selected and was shown to have acquired a mutation at amino acid (g r) in the chikv e glycoprotein, which may be involved in binding to host receptors [ ] . recently, a series of arbidol analogues have been synthesized and evaluated for their anti-chikv activity [ ] . two analogues in this series (iiie and iiif) inhibited the chikvinduced cytopathic effect (cpe) with selectivity indices higher than that of the parent compound arbidol [ ] . phenothiazines are clinically approved antipsychotics. in a screening assay using semliki forest virus (sfv) as a bio-safe surrogate for chikv, six compounds containing a h-phenothiazine core, including chlorpromazine, perphenazine, ethopropazine, thiethylperazine, thioridazine, and methdilazine were identifi ed as possible sfv entry inhibitors. the antiviral activity of these molecules was confi rmed using a recombinant chikv strain carrying a luciferase reporter gene (chikv-rluc). when compared to the sfv-based screening results, the molecules showed similar potencies against the chikv-rluc; however, the ec values determined using the chikv-rluc were higher. the antiviral target of these inhibitors still needs to be identifi ed [ ] . epigallocatechin gallate (egcg) is the major constituent of green tea. recently, egcg has been reported to have a modest but signifi cant antiviral activity against chikv [ ] . the inhibition of chikv entry and attachment to the target cells by egcg was confi rmed using pseudo-particles carrying the chikv envelope proteins [ ] . rna interference is induced by small interfering rnas (sirna) that are homologous in sequence to the gene that needs to be silenced. small interfering rnas are - nucleotides long dsrna molecules having ′-overhangs of two nucleotides. treatment of cells with exogenous sirnas results in the assembly of a rna-induced silencing complex (risc) which degrades specifi c complementary mrna molecules. consequently, protein expression of the targeted gene is markedly reduced. small interfering rna (sirna) sequences targeting chikv nsp and e genes were reported to signifi cantly reduce chikv titers at h post-infection in transfected vero cells [ ] . however, the inhibitory effect of these sirnas was transient and diminished after days of infection. these sirnas could thus be used in combination with other antivirals for more effective treatment. in a more recent study, nsp and e sirnas were generated and their potential activity was evaluated in cell culture and in animal models [ ] . sirnas directed against nsp and e as well as their combinations, reduced in vitro chikv replication in vero cells with more than %. interestingly, when chikv-infected mice were injected days post-infection with these sirnas, chikv replication was completely inhibited at the highest dose of sirna tested ( mg/kg body weight; [ ] ). plasmid-based small hairpin rnas (shrnas) were also designed and evaluated as strategy to inhibit chikv replication. shrnas produced from the shrna-plasmid construct resulted in their intracellular processing to sirnas causing specifi c knockdown of viral rna and subsequent inhibition of viral protein expression. stable cell clones expressing shrna against chikv e and nsp showed signifi cant and sustained inhibition of chikv infection [ ] . in addition, mice pretreated with e targeting shrna were completely protected against chikv induced disease and their survival was observed up to days post-infection (untreated animals died between and days post-infection; [ ] ). harringtonine, a cephalotaxine alkaloid derived from cephalotaxus harringtonia , has been reported asa potent inhibitor of chikv with minimal cytotoxicity [ ] . in addition, homoharringtonine, a more stable analogue of harringtonine, also showed anti-chikv activity. homoharringtonine has been recently approved by the fda for the treatment of chronic myeloid leukemia in . harringtonine and homoharringtonine were found to suppress the production of viral nsp and e proteins, most likely through the inhibition of the host cell protein translation machinery [ ] . in addition, the decrease in nsp production resulted in a reduction of viral replicase complexes formation. consequently, the level of negative-sense rnas was decreased leading to the reduced synthesis of the viral positive-sense rna genome [ ] . ribavirin, a structural analogue of guanosine, is a broad-spectrum antiviral drug that has been approved by the fda for the treatment of respiratory syncytial virus in infants [ ] , and in combination with pegylated interferon alpha (ifn-α) for treatment of chronic hepatitis c virus infection [ ] . ribavirin was shown to inhibit the replication of chikv in vitro [ ] . in addition, the combination of ribavirin and ifn-α b was reported to result in asynergistic inhibitory effect against chikv replication in vero cells [ ] . the mechanism of the antiviral action of ribavirin is likely different for different viruses. the ′-monophosphate metabolite of ribavirin acts a competitive inhibitor of inosine monophosphatedehydrogenase (impdh) resulting in a depletion of intracellular gtp (and dgtp) pools [ ] . the predominant mechanism by which ribavirin inhibits the replication of other rna viruses such as fl aviviruses and paramyxoviruses has been shown to be mediated by depletion of gtp pools [ ] . other suggested mechanisms by which ribavirin inhibits rna virus replication include the inhibition of viral rna capping, inhibition of the viral polymerase, and lethal mutagenesis of the rna genome [ ] . -azauridine is a broad-spectrum anti-metabolite that inhibits the replication of both dna and rna viruses [ ] . it is a uridine analogue that competitively inhibits the orotidine monophosphate decarboxylase enzyme, which is involved in the de novo synthesis of pyrimidines [ ] . -azauridine showed strong inhibitory effect against chikv replication in vero cells with an ec value of . μm [ ] . mycophenolic acid (mpa) is a non-competitive inhibitor of impdh which has been used clinically as an immunosuppressant to prevent the rejection of transplant organs. mpa inhibits in vitro chikv replication [ ] . the inhibition of chikv replication appears to be due to the depletion of intracellular gtp pools [ ] . favipiravir (t- ) is a broad-spectrum antiviral agent that was originally discovered as an inhibitor of infl uenza a virus replication. in the cell, t- is metabolized to its ribofuranosyl ′-triphosphate form, which was shown to be a competitive inhibitor for the incorporation of atp and gtp by the viral rnadependent rna polymerase (rdrp; [ ] ). however, the exact mechanism of action of t- has not been totally clarifi ed yet. recently, it has been reported that t- and its defl uorinated analogue, t- inhibit the in vitro replication of chikv [ ] . in addition, the oral treatment of chikv-infected ag mice with t- protected these mice from severe neurological disease and reduced the mortality rate by more than %. low-level t- -resistant chikv variants have been selected. these variants carried a k r mutation in the f motif of the rdrp that was shown to be responsible for the observed resistance to t- . this position is highly conserved in the polymerase of + ssrna viruses [ ] . the chikv nsp protein exhibits rna triphosphatase/nucleoside triphosphatase activity, as well as helicase activity within the n-terminal half while the c-terminal half encodes the viral cysteine protease required for processing of the non-structural viral polyprotein [ , ] . in addition, nsp plays an important role in shutting down host cell mrna transcription via degradation of a subunit of the dna-directed rna polymerase ii. it also inhibits the host antiviral response by suppressing transcription and type i/ ii interferon-stimulated jak/stat signaling [ ] . in a highthroughput screening for chikv nsp inhibitors that target the nsp -mediated transcriptional shut off, a natural compound derivative (id - ) was shown to partially block the nsp activity resulting in inhibition of chikv replication in cell culture [ ] . in another study, a number of nsp inhibitors were identifi ed using a computer-aided screening procedure of which one lead compound (compound ) showed a signifi cant antiviral activity against chikv [ ] . this compound was predicted to bind to the central portion of the nsp protease active site. recently, a number of arylalkylidene derivatives of , -thiazolidin- -one have been shown to inhibit the in vitro replication of chikv. the inhibition of the chikv protease was suggested to be the mechanism of action of these compounds [ ] . cellular furins and furin-like proteases are involved in the cleavage of viral pe into mature e and e proteins. the inhibition of cellular furin may therefore be expected to inhibit the formation of mature viral particles. decanoyl-rvkr-chloromethyl ketone (dec-rvkr-cmk) is an irreversible furin inhibitor that was shown to inhibit chikv infection in vitro via inhibition of viral glycoprotein maturation [ ] . the combination of dec-rvkr-cmk and chloroquine resulted in an additive inhibitory effect on chikv replication. surprisingly, pretreatment of cells with dec-rvkrcmk revealed a signifi cant inhibition of viral entry, indicating that dec-rvkr-cmk treatment could alter the cleavage of proteins involved in chikv endocytosis or early replication steps or that this molecule could even inhibit chikv receptor maturation [ ] . prostratin and -o -tetradecanoylphorbol -acetate (tpa) are well-known tigliane diterpenoids with a basic phorbol carbon skeleton esterifi ed at position [ ] . due to their chemical structure, they act as natural analogues of diacylglycerol that induce the activation of protein kinases c. previously, prostratin and tpa were reported to have antiviral activity against hiv [ ] . prostratin and tpa were also identifi ed as potent and selective chikv inhibitors in vitro [ ] . further studies are required to determine their mode of action against chikv. in a cell-based screening of a kinase inhibitor library, six kinase inhibitors were found to inhibit chikv-associated cell death in a dose-dependent manner [ ] . of these molecules, four compounds have a benzofuran core scaffold, one a pyrrolopyridine and one a thiazol-carboxamide scaffold. using image analysis, it was shown that chikv-infected cells treated with these molecules had less prominent apoptotic blebs, which are typical for the chikv cytopathic effect. moreover, these compounds reduced viral titers up to -fold. it was suggested that the inhibition of the virusinduced cpe by these compounds was the result of inhibition of kinases involved in apoptosis [ ] . hsp- is a family of highly conserved molecular chaperones which includes two cytoplasmic isoforms: stress-induced hsp- α and constitutively expressed hsp- β. in general, hsp- is involved in maturation, localization, and turnover of its client proteins in a cell. hsp- has been reported to play an important role in the replication of many dna and rna viruses such as hepatitis b virus, hepatitis c virus, human cytomegalovirus and infl uenza virus. consequently, hsp- inhibitors may have a role as broad(er)-spectrum antiviral agents. two hsp- inhibitors, hs- and snx- , were reported as chikv replication inhibitors. the treatment of chikv-infected mice (sva ) with hs- and snx- signifi cantly reduced the serum viral load at protein kinase c(pkc) activators h post-infection and protected against the chikv-induced infl ammation in the limb of infected mice [ ] . in coimmunoprecipitation studies chikv nsp and nsp were shown to interact with hsp- . interestingly, the knockdown of the hsp- α subunit resulted in a more pronounced inhibition of viral replication than targeting the hsp- β subunit. hsp- α is thought to be involved in the stabilization of chikv nsp and the formation of the chikv replication complex [ ] . further mechanistic studies are required to unravel the role of hsp- in the replication cycle of chikv. the innate immune system plays an important role in the acute phase of chikv infection. detection of chikv rna by toll-like receptors (tlrs) , , and , as well as rig-i like receptors during the acute phase of infection is believed to trigger the production of type i ifns. consequently, type i ifns activate the transcription of interferon-stimulated genes (isgs), which encode proteins involved in the host antiviral defense leading to clearance of the infection [ ] . therefore, activation of the innate immune response could be interesting for the treatment of chikv infections. treatment with ifn-α a and ifn-α b inhibited chikv replication in vero cells in a dose-dependent manner [ ] . the combination of ifn-α b and ribavirin resulted in a synergistic antiviral effect on in vitro chikv replication. a chikv strain carrying the e a v mutation was reported to be more sensitive to the antiviral activity of recombinant ifn-α than wild-type virus [ ] . the role of oas in the innate immunity towards chikv was investigated using a stable hela cell line expressing oas [ ] . the expression of oas by this cell line effi ciently inhibited chikv infection by blocking the early stages of virus replication. a chikv variant with a glutamine-to-lysine mutation at position of the envelope e glycoprotein proved resistance to the antiviral activity of oas [ ] . rig-i (retinoic acid-inducible gene i) is a member of the rig-i like receptor family which recognizes viral dsrna leading to activation of multiple antiviral factors that block viral infection at different stages. interestingly, chemically or enzymatically synthesized dsrna molecules with an exposed ′-triphosphate end ( ′ ppp) polyinosinic acid-polycytidylic acid rig-i agonists were reported to induce rig-i [ , ] . it has been recently shown that pretreatment of mrc- cells with an optimized ′ triphosphorylated rna molecule triggered rig-i stimulation resulting in protection against chikv infection [ ] . moreover, the protective response against chikv induced by this ′ ppp rna was largely independent of the type i ifn response. these results suggest the potential effi cacy of rig-i agonists as an antiviral treatment for chikv infection. trigocherrierin a is a new daphnane diterpenoid orthoester isolated from the leaves of trigonostemon cherrieri [ ] . trigocherrierin a inhibits chikv in cell culture but the mechanism of its action remains elusive. debromoaplysiatoxin and -methoxydebromoaplysiatoxin are marine toxins isolated from the marine cyanobacterium, trichodesmium erythraeum [ ] . both compounds had signifi cant antiviral activity against chikv at non-toxic concentrations. the compound was reported to block a post-entry step in the chikv lifecycle. a number of , -dihydroxyfl avones (apigenin, chrysin, naringenin, and silybin) were identifi ed as inhibitors of the chikv subgenomic replicon [ ] . the molecular target of these compounds is still unknown. the global re-emergence of chikv and the high morbidity rate associated with its infection emphasizes the need to develop potent antiviral agents against chikv. so far, a number of classes of compounds that inhibit viral replication by targeting either a viral or a host factor have been reported. most of the compounds have relatively modest activity and for most of them, activity in infection models (in mice) was not assessed. some of these classes may serve as a starting point for the design of more specifi c and selective inhibitors of chikv replication. also, to the best of our knowledge, no information is available yet on the effect of antivirals on the chronic stage of chikv infection. recently, mouse models for chikv-induced arthritis and chronic joint disease have been developed which will help the evaluation of chikv antiviral agents in different stages of chikv infection [ , ] . several other viruses belonging to the alphavirus genus, in particular the equine encephalitis viruses are considered to be a potential bioterroristic threat. when designing/developing antivirals against the chikungunya virus it may be important to develop classes of compounds that have pan-alphavirus activity and that could thus also be used for the treatment of alphaviruses other than chikv. among the reported chikv inhibitors, favipravir, ribavirin, arbidol, and ifn-α have been approved previously for the treatment of other viral infections. this could markedly facilitate their evaluation for clinical use in chikv-infected patients. favipravir, a drug with a broad-spectrum antiviral activity, has been approved in japan for the treatment of infl uenza virus infections. it is currently also being evaluated in western africa for the treatment of ebola virus infection. if its activity is demonstrated against this infection, this compound may be considered for treatment of other infections such as those caused by chikv. however, given the growing number of patients suffering from chikv infections, it may be justifi ed to develop specifi c chikv/alphavirus drugs. highly potent drugs are today available for the treatment of infections with herpes viruses, hiv, the hepatitis b and c virus. without a doubt, it should also be possible, when investing suffi cient effort, to develop highly effective and safe drugs for the treatment (and perhaps even prophylaxis) of infections with alphaviruses. chikungunya virus infection chikungunya: an emerging and spreading arthropod-borne viral disease cebula-byrska i ( ) chikungunya fever arthritis after infection with chikungunya virus mechanism of inhibition of 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shrna against the e and nsp genes effectively silenced chikungunya virus replication inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression respiratory syncytial virus: current and emerging treatment options new hepatitis c therapies: the toolbox, strategies, and challenges in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-α and ribavirin combination cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells the anti-yellow fever virus activity of ribavirin is independent of error-prone replication ribavirin for the treatment of chronic hepatitis c virus infection: a review of the proposed mechanisms of action antiviral action and selectivity of -azauridine favipiravir (t- ), a novel viral rna polymerase inhibitor mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (t- ), a broad-spectrum antiviral replication cycle of chikungunya: a reemerging arbovirus the c-terminal domain of chikungunya virus nsp independently governs viral rna replication, cytopathicity, and inhibition of interferon signaling a phenotypic assay to identify chikungunya virus inhibitors targeting the nonstructural protein nsp computer-aided identifi cation, design and synthesis of a novel series of compounds with selective antiviral activity against chikungunya virus thiazolidone derivatives as inhibitors of chikungunya virus inhibition of chikungunya virus infection in cultured human muscle cells by furin inhibitors: impairment of the maturation of the e surface glycoprotein prostratin and -o-tetradecanoylphorbol -acetate are potent and selective inhibitors of chikungunya virus replication protein kinase c: one pathway towards the eradication of latent hiv- reservoirs identifi cation of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library chikungunya virus nsp & nsp interacts with hsp- to promote virus replication: hsp- inhibitors reduce chikv infection and infl ammation in vivo biology and pathogenesis of chikungunya virus chikungunya virus isolates with/without a v mutation show different sensitivity to ifn-alpha, but similar replication kinetics in non human primate cells the large form of human ′, ′-oligoadenylate synthetase (oas ) exerts antiviral effect against chikungunya virus the e -e k substitution restores chikungunya virus growth in oas expressing cells by acting on viral entry poly (i:c), an agonist of toll-like receptor- , inhibits replication of the chikungunya virus in beas- b cells ′-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to singlestranded rna bearing ′-phosphates inhibition of dengue and chikungunya virus infection by rig-i-mediated type i ifnindependent stimulation of the innate antiviral response trigocherrierin a, a potent inhibitor of chikungunya virus replication anti-chikungunya viral activities of aplysiatoxinrelated compounds from the marine cyanobacterium trichodesmium erythraeum chronic joint disease caused by persistent chikungunya virus infection is controlled by the adaptive immune response a mouse model of chikungunya virusinduced musculoskeletal infl ammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence the original work of the authors is supported by eu fp silver (contract no. health-f - - ) and the belspo iuap consortium belvir (belgium). leen delang is funded by the scientifi c fund for research of flanders (fwo). key: cord- -npug c p authors: liu, yang; liu, jianying; pang, xiaojing; liu, tao; ning, zhijie; cheng, gong title: the roles of direct recognition by animal lectins in antiviral immunity and viral pathogenesis date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: npug c p lectins are a group of proteins with carbohydrate recognition activity. lectins are categorized into many families based on their different cellular locations as well as their specificities for a variety of carbohydrate structures due to the features of their carbohydrate recognition domain (crd) modules. many studies have indicated that the direct recognition of particular oligosaccharides on viral components by lectins is important for interactions between hosts and viruses. herein, we aim to globally review the roles of this recognition by animal lectins in antiviral immune responses and viral pathogenesis. the different classes of mammalian lectins can either recognize carbohydrates to activate host immunity for viral elimination or can exploit those carbohydrates as susceptibility factors to facilitate viral entry, replication or assembly. additionally, some arthropod c-type lectins were recently identified as key susceptibility factors that directly interact with multiple viruses and then facilitate infection. summarization of the pleiotropic roles of direct viral recognition by animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. keywords: lectin; virus; direct interaction; antiviral immunity; viral pathogenesis lectins, a highly diverse group of proteins that recognize carbohydrates, have been demonstrated to play a vital role in numerous life processes and to be critical for several viral infections and pathogeneses in a variety of organisms [ , ] . based on their conserved structure of sequence motifs for sugar binding and carbohydrate specificities, lectins have been categorized into many families conventionally designated as calnexin, c-type, l-type, p-type/mannose- -phosphate receptors (mprs), i-type/siglecs, m-type, f-type (absent in mammals), r-type, f-box, chitinase-like lectins, galectins and intelectins (table ) [ ] . the features of carbohydrate recognition domains (crds), such as structure peculiarity, carbohydrate binding selectivity and geometrical arrangement of multiple crds, determine the different properties of lectins [ ] [ ] [ ] . furthermore, the diversity of locations and functions indicates the importance of lectins in the basic life processes of organisms. lectins are exploited as susceptibility factors to facilitate viral entry, replication or assembly ( figure ). insight into direct lectin-based viral recognition will provide a deep understanding of host-virus interactions. mammalian lectins have been categorized into multiple classes according to the features of their crds, as well as their sugar recognition specificity. some lectins generally play a role outside the cell, whereas others are predominantly intracellular and located on cytoplasmic organelles. extracellular lectins, including c-type, r-type, i-type/siglecs lectins and galectins, are secreted into the extracellular milieu or are localized to the plasma membrane and are capable of mediating cell adhesion, immune signaling and pattern recognition activities for host-pathogen interactions. however, intracellular lectins, such as the calnexin family, m-type, l-type and p-type lectins, are located in luminal compartments of the secretory pathway and function in the trafficking, sorting and maturation of glycoproteins [ ] [ ] [ ] . as lectins play diverse roles in mammalian physiological processes, we only focus herein on a portion of lectins that directly interact with viral components and describe their functions in viral propagation and pathogen-host immune responses. c-type lectins (ctls) are a large group of proteins in metazoans that were originally named according to their property of ca + (c-type)-dependent carbohydrate binding. sequence and structural comparisons of c-type lectins have suggested that their carbohydrate-binding activity is mediated by a specific crd that is conserved in a variety of organisms. although some c-type lectins do not possess carbohydrate-binding activity, all of them show distinct sequence similarity and are believed to descend from a common ancestor during evolution [ ] [ ] [ ] . to date, c-type lectins have been divided into subgroups according to their domain architecture and the phylogenetic relationship between their crd sequences [ ] . in general, c-type lectins can be separated into two groups, mannose-binding and galactose-binding c-type lectins, based on the specificity of their carbohydrate-binding activity. the binding specificity of these two groups is mediated by diverse residues flanking the conserved cis-proline in the long loop region, in which the sequence of the core motif is e-p-n for mannose-binding and q-p-d for galactose-binding specificity [ , ] . previous studies have demonstrated that interchange of the e-p-n and q-p-d sequences is sufficient to switch the mannose-and galactose-binding specificity ( figure ) [ ] . however, several lectins are exceptions to this rule. for example, surfactant protein a, possessing an e-p-k but not an e-p-n motif, binds to mannose sugars [ , ] . although human tetranectin contains a galactose-type q-p-d motif, it is not responsible to the lectin-carbohydrate interaction [ ] . therefore, other determinants, including modifications around the binding sites and stereochemical factors, should be taken into consideration when examining binding specificity [ ] [ ] [ ] . robust investigations have shown that multiple mannose-/galactose-binding c-type lectins play important roles in viral infections in mammals. mbl, one of the most intensively studied lectins, is a member of the collectin family, a subgroup of c-type lectins ( figure a ). the mbl molecule contains four domains that are standard for collectin family proteins: a cysteine-rich region, a collagen-like domain, a neck region and a carbohydrate recognition domain. the native functional form of mbl is a hexamer; however, although mbl can form several oligomeric forms, the dimers and trimers do not have biological activity, and at least a tetramer form is needed to activate the complement cascade [ ] . mbl functions as a soluble pattern recognition receptor in the host complement system and is involved in resistance to many viral infections [ ] . the crds of mbl multimers recognize carbohydrate patterns on the virus surface, and consequently, the binding of mbl and viral particles results in activation of the lectin pathway of the complement system. masps (mannose-binding lectin-associated serine proteases), which are protease zymogens (an inactive form of an enzyme) similar to the c r and c s molecules of the classical complement pathway, are activated to cleave complement components c and c into c a/c b and c a/c b, respectively. interactions between c b and c b produce the c convertase, which continuously activates c further downstream in the cascade to eliminate viruses ( figure a ) [ ] . current investigations have reported a resistance role of mbl in infections of multiple human viruses, including human immunodeficiency virus (hiv) [ ] , hepatitis b virus (hbv) [ , ] , hepatitis c virus (hcv) [ ] , west nile virus (wnv) [ ] and dengue virus (denv) [ ] . specific recognition of these viral particles by mbl is a central event for activation of the lectin-based complement cascade. the mechanism of viral elimination by the mbl-based complement cascade is still unclear. unlike bacterial elimination by the complement system, mbl-based complement components do not appear to form a membrane-attacking complex on the viral surface; therefore, viral eradication may not be mediated by known complement mechanisms. three possible mechanisms have been proposed for mbl-mediated viral elimination. ( ) mbl-mediated complement c /c deposition onto the viral surface. viral neutralization can be processed in cell-free serum and is completely dependent on c and c activation, but not on c q and c , suggesting that neither opsonization nor the classical/alternative complement pathway is sufficient for viral neutralization [ ] . ( ) mbl can directly neutralize viruses. pre-incubation of serial concentrations of recombinant mbl with hiv cell-derived living particles was found to dramatically neutralize hiv infection [ , ] . however, other independent investigations have suggested that some primary hiv isolates resist direct neutralization by mbl [ ] , indicating that the possible neutralizing activity depends highly on the different carbohydrate structures on the surfaces of various viral strains. ( ) mbl can block the recognition of viruses and receptors. dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign), which is present on the surface of dendritic cells, functions as a key attachment factor used for the recognition and uptake of multiple viruses [ ] [ ] [ ] [ ] [ ] . a study reported that mbl can prevent interaction between hiv and dc-sign, thereby inhibiting the hiv infection of t cells, which is mediated by dc-sign [ ] . furthermore, mbl interacts with the viral envelope glycoproteins of ebola and marburg viruses (marv), resulting in the impairment of viral internalization by blocking virus-dc-sign interaction ( figure b ) [ ] . two soluble collectins, designated sp-a and sp-d ( figure b ,c), have been found to be involved in the recognition of viral particles for limiting infection in humans. sp-a is produced within the respiratory tract, gastrointestinal tract, and possibly other sites; conversely, sp-d is primarily synthesized in the respiratory tract [ ] . these factors are constitutively secreted into the lungs by alveolar type ii cells, unciliated bronchial epithelial cells and other mucosal tissue cells [ , ] . the specific location of surfactant proteins suggests a defensive role against viral invasion of the respiratory system. both sp-a and sp-d interact with different strains of influenza a virus (iav) via glycosylated hemagglutinin (ha) and neuraminidase (na) on the viral surfaces. the binding of iav to sp-a leads to agglutination of the virions, inhibition of iav infectivity and dissemination and also facilitates clearance by macrophages and neutrophils ( figure a ,b) [ ] . sp-d binds to iavs and thereby inhibits virus attachment and entry by viral aggregation ( figure b ) [ ] [ ] [ ] [ ] [ ] and also controls iav infection in human by activating neutrophil chemoattraction ( figure a ) [ , ] . respiratory syncytial virus (rsv) infects humans via the respiratory tract. sp-a has been reported to bind fusion (f) and adherence (g) glycoproteins on the surfaces of rsv virions, resulting in opsonization to reduce infection by enhancing viral uptake by peripheral blood mononuclear cells (pbmcs) and alveolar macrophages ( figure a ) [ , ] . sp-d also directly interacts with rsv surface g protein to modulate host immune responses to control rsv infection [ ] . dc-sign (cd ) and its homolog l-sign (also called dc-signr, cd l) are one of the most investigated c-type lectins involved in viral infection ( figure f ). unlike the main role of collectins in host defense, dc-sign and l-sign have been widely reported to be susceptibility factors that facilitate viral entry into host immune cells [ , , , ] . both dc-sign and l-sign are trans-membrane proteins that are composed of a short cytoplasmic tail, which is responsible for signaling and internalization, a transmembrane region, a neck domain, which consists of eight repeat regions of amino acids, and a carbohydrate recognition domain [ ] . the roles of dc-sign/l-sign in viral infections have been summarized and reviewed previously [ , ] . studies have shown that dc-sign/l-sign are capable of binding to the surface proteins of hiv [ ] , cytomegalovirus (cmv) [ ] , denv [ ] , wnv [ , ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] , hcv [ , ] , ebola virus [ ] and marv [ ] and consequently facilitating viral entry ( figure c ). differential glycosylation patterns of viral surface proteins strongly influence the efficiency of viral recognition by dc-sign/l-sign [ , ] . for example, only mannosylated envelope (e) glycoproteins on denv, but not e proteins with complex glycosylation, have been shown to interact with dc-sign-expressing cells [ ] . the mannose receptor (mr, cd ) is another c-type lectin that functions as a viral recognition receptor on the cell membrane ( figure g ). mr is mainly expressed in multiple immune cells, including macrophages and dendritic cells, and is a key susceptibility factor for denv infection of human macrophages. binding of mr to denv e glycoproteins enhances viral attachment, thus facilitating denv internalization into macrophages, and deglycosylation of the denv e glycoprotein enables the abrogation of this binding, and denv infection of primary human macrophages can be blocked by anti-mr antibodies [ ] . moreover, the interaction between mr and hbv surface antigen (hbsag) enhances viral uptake by dendritic cells (dcs), resulting in the impairment of dc function and the ineffective antiviral response of chronic hbv [ ] . the recognition of viral surface glycoproteins by mr is also beneficial to influenza virus [ , ] and hiv [ ] invasion into host cells ( figure c ). overall, dc-sign/l-sign and mr function as receptors/attachment factors for viral entry into particular cell types. clec a/mdl- (myeloid dap -associating lectin) is a c-type lectin associated with dap ( -kda dnax-activating protein) on myeloid cells such as monocytes, macrophages and neutrophils ( figure h ) [ ] . a recent study has found that clec a binds to dengue glycoproteins. however, in contrast to other c-type lectin receptors, the association between clec a and denv does not result in viral entry, but rather induces dap -mediated immune signaling to stimulate the release of pro-inflammatory cytokines that potentially contribute to the pathogenesis of dengue hemorrhagic fever [ , ] . clec a also directly interacts with japanese encephalitis virus (jev) to induce dap phosphorylation in macrophages and therefore plays a role in jev-induced neuro-inflammation and lethality ( figure c ) [ ] . the blocking of clec a in mice can significantly reduce the infiltration of jev-harboring leukocytes into the central nervous system, thus attenuating neuro-inflammation and protecting the animals from jev-induced lethality [ ] . the discovery of a role of clec a in flaviviral pathogenesis suggests that the extracellular crd modules are generally responsible for the recognition of viral glycoproteins; nonetheless, the intracellular modules determine the role of c-type lectins in viral infection. langerin (also known as cd ), containing a single ca + -dependent crd domain, is a type ii transmembrane c-type lectin that is specifically expressed on langerhans cells ( figure i ). the physiological function of langerin is to trigger the cellular membrane superimposition and zippering that benefit birbeck granule (bg) formation [ ] . langerin is capable of directly binding to hiv- envelope protein gp and thus serves as a potential receptor for hiv- infection in langerhans cells ( figure c ) [ , ] . however, a recent study reported that langerin is a natural barrier for hiv- transmission among langerhans cells. langerin is capable of directly capturing hiv- and sequentially degrading it in bgs to promote t cell elimination of hiv- infection [ ] , suggesting that langerin plays a pleiotropic role in hiv infection. furthermore, langerin functions as an attachment factor to facilitate measles virus (mv) infection in langerhans cells [ ] . a large number of host proteins are abundantly glycosylated. therefore, microbial recognition by c-type lectins relies on the mechanism for distinguishing carbohydrate structures between self and non-self, and the c-type lectin structure largely influences binding avidity and selectivity in the recognition of self and non-self carbohydrate structures [ , ] . based on the x-ray crystal structures of mannose-binding lectin (mbl), the mbl crd sites in the trimer form are too far apart to spatially interact efficiently with common mammalian high-mannose oligosaccharides. however, the dense and repeated arrays of carbohydrates present on the microbial surface can span the distance between the binding sites in mbl, resulting in highly avid multivalent interaction [ , ] . moreover, the number of crds is another determinant for the avidity and strength of differential binding by c-type lectins. the eight different c-type crds of mr contribute to its high binding affinity for single sugars, even though each individual crd motif only displays weak affinity. the crd domain organization also confers mr with the ability to recognize the wide range of different carbohydrates found on the pathogen surface and to distinguish between self and foreign glycoproteins [ ] . galectins are a group of secreted proteins that associate with specific cell surface glycans containing beta-galactosides ( figure d ,e) [ ] . although mammalian galectins lack conventional signal sequences, they reach the cell surface via a particular mechanism. galectins accumulate directly beneath the plasma membrane and are subsequently involved in the establishment of membrane-bound vesicles that pinch off before release outside the cell; galectins then bind to glycoconjugates on the plasma membrane or remain in the extracellular matrix [ ] [ ] [ ] . fifteen galectins have been identified in mammals and are categorized into three structural forms: dimeric, tandem or chimeric. dimeric galectins, also called prototypical galectins, are homodimers and include galectin- , - , - , - , - , - , - , - and - . tandem galectins contain at least two distinct crds within one polypeptide and include galectin subtype- , - , - , - and - . galectin- is specific to mammals, has one crd and a long non-lectin domain, and exists in either a monomeric form or a multivalent complex associated via the non-lectin domains of monomers [ ] ; this property allows galectin- to effectively bridge different ligands to form adhesive networks. current investigations have indicated that direct recognition between galectin- /- and viral-surface glycoproteins is important for host-virus interaction. [ ] . because of its particular binding specificity for galactosides, galectin- recognizes the surface envelope proteins of many human viruses and therefore is involved in viral infection. galectin- binds to niv-f, a viral envelope glycoprotein of nipah virus (niv), to reduce the niv-f-mediated fusion of endothelial cells and thereby inhibit niv-induced syncytium formation [ ] . galectin- directly interacts with the envelope glycoproteins of influenza a/wsn/ virus and inhibits its hemagglutination activity, resulting in the reduction of influenza virus infectivity ( figure b) [ ] . however, galectin- has also been reported to be a susceptibility factor for viral entry. hiv- exploits galectin- to enhance gp -cd interaction, leading to faster viral entry and more robust viral replication ( figure c ) [ ] [ ] [ ] [ ] . in addition to galectin- , the role of galectin- in viral infection has been elucidated by several studies. galectin- has been shown to interact with herpes simplex virus- (hsv- ). rnai-mediated knockdown of galectin- in human corneal keratinocytes significantly impaired hsv- infection, suggesting that hsv- exploits galectin- to enhance its attachment to host cells [ ] . recently, proteomic-based studies have identified galectin- as a host-binding partner of parvovirus minute virus of mice (mvm). the authors proposed that galectin- binding facilitates the access of mvm to its receptor(s) at the plasma membrane and thus promotes mvm endocytosis ( figure c ) [ ] . the above-mentioned evidence indicates a pleiotropic role of galectins during viral infections. the endoplasmic reticulum (er) of mammalian cells contains molecular chaperones and foldases, which are required for forming the active structures of newly synthesized peptides and thus serve as components of the er quality control system. the er-resident chaperones include bip, calnexin ( figure l ) and calreticulin (a calnexin-like soluble form without the transmembrane region) [ , ] . both calnexin and calreticulin are lectin-like, membrane-bound molecular chaperones that associate with newly synthesized proteins in the er. in addition, several studies have indicated that calnexin and calreticulin preferentially interact with glycoproteins that carry monoglucosylated n-linked oligosaccharides [ ] [ ] [ ] [ ] . the maturation of virus-encoded proteins occurs in the er, and calnexin family proteins have been shown to transiently interact with multiple viral proteins that consequently undergo rapid maturation ( figures d and a) . both calnexin and calreticulin can transiently interface with envelope glycoproteins f and hn of sendai virus (sev) [ ] and glycoproteins g /g of uukuniemi virus (uukv) (bunyaviridae family) [ ] to facilitate the rapid maturation of these proteins. during sars-cov infection, maturation of the viral s protein due to its interaction with calnexin is essential for the formation of infective virions [ ] . calnexin/calreticulin also plays a role in the assembly and secretion of hbv middle (m) envelope protein [ ] , hiv- envelope protein gp [ ] and gp [ ] . furthermore, during the rotavirus life cycle, calnexin binds to the er-associated viral transmembrane protein nsp , a nonstructural glycoprotein that acts as a toxin capable of inducing diarrhea in animals [ ] [ ] [ ] . calnexin/calreticulin is also associated with the glycosylation of hantaan virus (htnv, also known as hantavirus) envelope proteins gn and gc and plays a crucial role in the folding of htnv glycoproteins with a high content of high-mannose oligosaccharides [ ] . accumulated evidence suggests that the lectin-like calnexin proteins interact with viral components to largely facilitate viral assembly and protein maturation. the p-type lectin/mannose -phosphate receptors (mprs) are transmembrane glycoproteins that target lysosomal enzymes located in either intracellular organelles or the plasma membrane ( figure j ,k). mprs can bind newly synthesized lysosomal hydrolases in the trans-golgi network (tgn) and deliver them to pre-lysosomal compartments. the mpr crd was originally identified in two types of proteins, cation-independent and cation-dependent mannose -phosphate receptors (ci-mpr and cd-mpr, respectively), both of which recognize mannose- -phosphate (m- -p) to identify and route lysosomal enzymes to the lysosomal compartment [ ] . a previous study demonstrated that human herpes simplex virus (hsv) glycoprotein d (gd) binds to both ci-mpr and cd-mpr. these mprs sort glycoproteins modified with m- -p to lysosomes in the trans-golgi compartment and divert them to the endosomal pathway ( figures e and b) [ , ] . mprs were also found on the surfaces of mammalian cells as serving as putative cellular receptors for hsv entry and cell-cell viral spread; furthermore, chemo-or immuno-blocking mprs was shown to inhibit hsv entry and the production of hsv plaques in monkey cells ( figure c ). mouse cells lacking both ci-mpr and cd-mpr remain sensitive to hsv infection [ ] , suggesting that the expression of mprs is not essential for hsv invasion. varicella zoster virus (vzv) is known as a highly infectious human pathogen, and multiple vzv envelope glycoproteins are modified by m- -p; therefore, ci-mprs appear to be important for vzv infection. intracellular ci-mpr contributes to the transport of enveloped vzv to late endosomes, and the plasmalemmal form is necessary for cellular entry through cell-free vzv particles [ ] . l-type lectins are widely distributed in plants and animals. animal l-type lectins are intracellular luminal proteins that are involved in protein sorting in the luminal er-golgi compartments of animal cells. there are four l-type lectins in mammals: ergic- ( figure m) , ergl, vip , and vipl [ , ] . a recent study has shown that intracellular cargo receptor ergic- interacts with the glycoproteins of arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus particles. ergic- is also essential for the propagation of arenavirus, coronavirus, and filovirus; in the absence of ergic- , viral particles can be formed but are noninfectious (figures e and c ) [ ] . in addition to the above-mentioned mammalian lectins, there are some other lectin classes in animals, e.g., m-type, r-type, i-type, chitinase-like, f-box lectins and intelectins. as we have not found reports on direct interactions between these lectins and viral components, we cannot determine the role of these lectins in viral infection based on the current knowledge. lectin crds are conserved throughout evolution, and many lectin homologues have been identified and reported in invertebrates. a homolog of galectin plays a role in opsonization for bacterial clearance in marsupenaeus japonicus [ ] . similarly, a galectin-like factor is expressed on the surface of oyster hemocytes and plays a role in oyster physiology through the recognition of oligosaccharides [ , ] . several proteins identified in crassostrea hongkongensis [ ] , venerupis philippinarum [ ] and trypanosoma cruzi [ ] have been categorized as homologs of mammalian i-type lectins/siglecs with high sialic acid-binding activity. in arthropods, multiple lectins identified in shrimp, such as l-type, p-type/mprs, m-type, and calnexin family factors, have been proposed to be important in shrimp innate immunity [ ] . many c-type lectin homologues in aedes and anopheles mosquitoes have been found to be involved in insect immune responses and pathogenesis [ ] [ ] [ ] . the current investigations of the immune roles of arthropod lectins mainly focus on their anti-bacterial or anti-parasite functions, including microorganism-induced lectin up-regulation, lectin-mediated microorganism recognition and opsonization [ , ] . however, little is known about the molecular details of lectins in arthropod immunity and pathogenesis, especially with regard to the function in arthropod-virus interactions. a recent study on c-type lectins in aedes aegypti initially assessed lectin functions in viral infections of arthropods. tens of c-type lectins were identified in aedes [ , ] and anopheles [ ] mosquitoes, and most are soluble forms [ ] . previous studies have shown that an aedes aegypti c-type lectin, mosquito galactose-specific c-type lectin- (mosgctl- ), interacts with wnv in a calcium-dependent manner to form a mosgctl-virus complex. this complex consequently interacts with mosquito protein tyrosine phosphatase- (mosptp- ), a mosquito homolog of human cd in a. aegypti, to enable viral attachment to the plasma membrane and enhance viral entry. in vivo experiments showed that mosgctl- and mosptp- function as part of the same pathway and are critical for wnv infection of mosquitoes [ ] . further investigations identified that another mosgctl paralogs facilitate dengue infection of mosquitoes. these mosgctls interact with denv- surface e protein and virions, functioning as susceptibility factors for dengue viral entry into mosquito cells. however, mosptp- did not influence dengue infection in mosquitoes, suggesting that other membrane receptors may recruit the denv-mosgctl complex onto the cell membrane for viral entry [ ] . in agreement with the findings in mosquitoes, a recent study has identified a c-type lectin in the shrimp marsupenaeus japonicus that interacts with an envelope protein of white spot syndrome virus (wssv) and consequently associates with a cell-surface calreticulin, which serves as a membrane receptor that facilitates viral entry in a cholesterol-dependent manner [ ] . the study therefore suggested that c-type lectins might play a broad role in expediting many viral infections of arthropods. the role might not be limited to wnv/denv in mosquito and wssv in shrimp but might extend to other virus infections in arthropods. lectins are potential targets for the development of antiviral drugs and vaccines. such lectin-based antiviral strategies are divided into two parts: ( ) lectin-based immune activation and ( ) blockade of lectin receptors against viral entry [ ] . many envelope viruses are protected by their dense carbohydrate shield against efficient recognition and persistent neutralization by the host immune system. various natural and synthetic carbohydrate-binding agents have been screened to refine candidates that can reinforce the recognition of specific pathogens, enhance the cascade amplification of the innate immune response and interrupt virus attachment to receptors. in fact, lectins have been considered as drug targets for many years. several heterologous lectins derived from various organisms have been already selected and introduced into pre-clinic trials for hiv therapy, including svn (scytovirin), a . -kd lectin isolated from aqueous extracts of the cyanobacterium scytonema varium [ ] , and uda (stinging nettle lectin), a . -kd plant lectin isolated from urtica dioica [ , ] . furthermore, the combined usage of uda with hha (amaryllis lectin, from a hippeastrum hybrid) and gna (snowdrop lectin from galanthus nivalis), another two carbohydrate-binding agents, showed broad anti-viral activity against four serotypes of denv in monocyte-derived dendritic cells by preventing virus attachment [ ] . additionally, an interesting monoclonal antibody, g , which interacts with specific, highly conserved glycosylation sites on hiv envelop protein gp shows a broad anti-hiv neutralizing activity. the mechanism of this antibody specifically targeting n-linked glycans is very similar to that of lectins [ ] [ ] [ ] . with regard to arthropod-borne viruses, vector ligands that interact with pathogens are ideal targets for interfering with the successful acquisition of the virus from the vertebrate host. due to the importance of c-type lectins in dengue infection of mosquitoes, these lectin factors may be proposed as targets for the development of vaccines or antiviral drugs. studies show that treatment with mosgctls antisera dramatically interrupted denv- infection of mosquitoes through blood feeding. therefore, the humoral response against mosgctls in mammals could feasibly impair dengue infection of mosquitoes. the approach to blocking mosquito c-type lectins may direct a future avenue for the development of a transmission-blocking vaccine that interrupts the mosquito-borne viral life cycle and reduces disease burden [ ] . lectins comprise highly diverse proteins with different carbohydrate recognition activities and play pleiotropic roles in the immune responses and pathogenesis of many viral infections (summarized in table ). the interaction between lectins and viral glycoproteins may lead to the three following consequences: ( ) lectins, such as mbl and sps, function as pattern recognition molecules that bind a repertoire of viruses and activate antiviral immune responses; ( ) lectins are employed as attachment factors that recruit viral particles to the cell membrane to enhance viral entry, e.g., some mammalian lectins (dc-sign, l-sign, mr and mprs) or their homologs in arthropods (mosgctls); and ( ) some intracellular lectins, such as calnexin and ergic- , function as susceptibility factors associated with virus-encoded proteins to facilitate viral replication or assembly (please refer to figures and ) . interestingly, the same lectin may show opposing roles in different virus infections. for example, galectin- binds to niv to inhibit syncytium formation and recognizes iav to reduce its infectivity [ , ] ; however, galectin- was also reported to be a susceptibility factor that enhanced gp -cd interactions, thus facilitating hiv entry [ ] [ ] [ ] [ ] . the current accumulated knowledge indicates that lectins are crucial host factors with complex and profound roles in the process of viral infection. lectins for investigation of proteins and carbohydrates molecular structure animal lectins animal lectins: a historical introduction and overview demonstration of carbohydrate recognition activity in diverse proteins which share a common primary structure motif two distinct classes of carbohydrate-recognition domains in animal lectins mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. complete primary structures and homology with pulmonary surfactant apoprotein the c-type lectin-like domain superfamily engineering galactose-binding activity into a c-type mannose-binding protein binding of sugar ligands to ca( +)-dependent animal lectins. ii. generation of high-affinity galactose binding by site-directed mutagenesis studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein a and comparison with two other collectins: mannan-binding protein and conglutinin the major lung surfactant protein, sp - , is a calciumdependent, carbohydrate-binding protein interaction of tetranectin with sulphated polysaccharides and trypan blue. scand lectin-carbohydrate interactions: different folds, common recognition principles mechanism of n-acetylgalactosamine binding to a c-type animal lectin carbohydrate-recognition domain mechanism of ph-dependent n-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil collections and ficolins: humoral lectins of the innate immune defense two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases interaction of mannose-binding lectin with hiv- is sufficient for virus opsonization but not neutralization high frequency of variant alleles of the mannose-binding lectin (mbl ) gene are associated with patients infected by hepatitis b virus mannose-binding lectin in chronic hepatitis b virus infection mannose-binding lectin mbl gene polymorphisms and outcome of hepatitis c virus-infected patients direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type binding and neutralization by mannose-binding lectin dc-sign, a dendritic cell-specific hiv- -binding protein that enhances transinfection of t cells c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses dc-sign (cd ) mediates dengue virus infection of human dendritic cells inhibition of dc-sign-mediated trans infection of t cells by mannose-binding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization localization of lung surfactant protein d on mucosal surfaces in human tissues immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung expression sites of the collectin sp-d suggest its importance in first line host defence: power of combining in situ hybridisation, rt-pcr and immunohistochemistry mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins pulmonary surfactant protein d in first-line innate defence against influenza a virusinfections site-directed mutagenesis of cys- and cys- of pulmonary surfactant protein d. expression of a trimeric protein with altered anti-viral properties mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro inhibition of influenza viral neuraminidase activity by collectins evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses recombinant sp-d carbohydrate recognition domain is a chemoattractant for human neutrophils surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity surfactant protein a enhances uptake of respiratory syncytial virus by monocytes and u macrophages respiratory syncytial virus and pulmonary surfactant c-type lectin receptors on dendritic cells and langerhans cells dc-sign: escape mechanism for pathogens the c type lectins dc-sign and l-sign: receptors for viral glycoproteins nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of ifn-alpha and tnf-alpha dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign -sign) is a receptor for severe acute respiratory syndrome coronavirus l-sign (cd l) is a liver-specific capture receptor for hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dc-signr dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals the mannose receptor mediates dengue virus infection of macrophages the mannose receptor acts as hepatitis b virus surface antigen receptor mediating interaction with intrahepaticdendritic cells involvement of themannose receptor in infection of macrophages by influenza virus reading pc macrophage receptors for influenza a virus: role of the macrophage galactose-type lectin and mannose receptorin viral entry oligomerization of the macrophage mannose receptor enhances gp -mediated binding of hiv- immunoreceptor dap bearing a tyrosine-based activation motif is involved in activating nk cells clec a is critical for dengue-virus-induced lethal disease clec a is critical for dengue virus-induced inflammasome activation in human macrophages clec a regulates japanese encephalitis virus-induced neuroinflammation and lethality langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules the role of dendritic cell c-type lectin receptors in hiv pathogenesis diversity of receptors binding hiv on dendritic cell subsets langerin is a natural barrier to hiv- transmission by langerhans cells human langerhans cells capture measles virus through langerin and present viral antigens to cd + t cells but are incapable of cross-presentation the c-type lectin superfamily in the immune system self-and nonself-recognition by c-type lectins on dendritic cells trimeric structure of a c-type mannose-binding protein galectins: a family of animal beta-galactosidebinding lectins secretion of the galectin family of mammalian carbohydrate-binding proteins secretion of the baby hamster kidney -kda galactose-binding lectin from polarized and nonpolarized cells: a pathway independent of the endoplasmic reticulum-golgi complex plasma membrane targetting, vesicular budding and release of galectin from the cytoplasm of mammalian cells during secretion galectins: regulators of acute and chronic inflammation galectins: structure, function and therapeutic potential endothelial galectin- binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation galectin- binds to influenza virus and ameliorates influenza virus pathogenesis galectin- promotes hiv- infectivity in macrophages through stabilization of viral adsorption galectin- acts as a soluble host factor that promotes hiv- infectivity through stabilization of virus attachment to host cells galectin- and hiv- infection host-soluble galectin- promotes hiv- replication through a direct interaction with glycans of viral gp andhost cd binding of transmembrane mucins to galectin- limits herpesvirus infection of human corneal keratinocytes proteomic analysis identifies a novel function for galectin- in the cell entry of parvovirus role of n-oligosaccharides endoplasmic reticulum processing reactions in glycoprotein folding and degradation lectins as chaperones in glycoprotein folding association of folding intermediates of glycoproteins with calnexin during protein maturation role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins glycan-dependent and -independent association of vesicular stomatitis virus g protein with calnexin kinetics of interactions of sendai virus envelope glycoproteins, f and hn, with endoplasmic reticulum-residentmolecular chaperones, bip, calnexin, and calreticulin transient association of calnexin and calreticulin with newly synthesized g and g glycoproteins of uukuniemivirus (family bunyaviridae) monitoring of s protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus role for calnexin and n-linked glycosylation in the assembly and secretion of hepatitis b virus middle envelopeprotein particles calreticulin interacts with newly synthesized human immunodeficiency virus type envelope glycoprotein, suggesting a chaperone function similar to that of calnexin effects of inefficient cleavage of the signal sequence of hiv- gp on its association with calnexin, folding, and intracellular transport the rotavirus nonstructural glycoprotein nsp possesses membrane destabilization activity rotavirus nonstructural glycoprotein nsp alters plasma membrane permeability in mammalian cells the molecular chaperone calnexin interacts with the nsp enterotoxin of rotavirus in vivo and in vitro analysis of n-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking lysosomal enzyme binding to mouse p d macrophage membranes lacking the -kda mannose -phosphate receptor: evidence for the existence of a second mannose -phosphate receptor herpes simplex virus glycoprotein d acquires mannose -phosphate residues and binds to mannose -phosphate receptors role of mannose- -phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission mannose -phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster a putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins structures of the carbohydrate recognition domain of ca + -independent cargo receptors emp p and emp p the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles a galectin from the kuruma shrimp (marsupenaeus japonicus) functions as an opsonin and promotes bacterial clearance from hemolymph hemocytes and plasma of the eastern oyster (crassostrea virginica) display a diverse repertoire of sulfated and blood group a-modified n-glycans the galectin cvgal from the eastern oyster (crassostrea virginica) binds to blood group a oligosaccharides on the hemocyte surface a novel sialic acid binding lectin with anti-bacterial activity from the hong kong oyster (crassostrea hongkongensis) cloning and characterization of a sialic acid binding lectins (sabl) from manila clam molecular interaction of siglecs (sialic acid-binding ig-like lectins) with sialylated ligands on trypanosoma cruzi diversity and multiple functions of lectins in shrimp immunity transmission-blocking antibodies against mosquito c-type lectins for dengue prevention cloning and characterization of a mannose binding c-type lectin gene from salivary gland of aedes albopictus the genome sequence of the malaria mosquito anopheles gambiae purification, characterization and cdna cloning of a novel lipopolysaccharide-binding lectin from the shrimp penaeus monodon a novel c-type lectin (fclec ) facilitates the clearance of vibrio anguillarumin in vivo in chinese white shrimp a c-type lectin collaborates with a cd phosphatase homolog to facilitate west nile virus infection of mosquitoes collaboration between a soluble c-type lectin and calreticulin facilitates white spot syndrome virus infection in shrimp targeting the c-type lectins-mediated host-pathogen interactions with dextran overexpression and purification of scytovirin, a potent, novel anti-hiv protein from the cultured cyanobacterium scytonema varium the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborineand the (n-acetylglucosamine)n-specific plant lectin from urtica dioicaare potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp a new therapeutic concept to hit the achilles heel of hiv broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of alpha → mannose residues on the outer face of gp antibody domain exchange is an immunological solution to carbohydrate cluster recognition dissection of the carbohydrate specificity of the broadly neutralizing anti-hiv- antibody g key: cord- -usl z ep authors: zheng, wen-zhi; wei, tian-li; ma, fen-lian; yuan, wu-mei; zhang, qian; zhang, ya-xin; cui, hong; zheng, li-shu title: human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in beijing, china date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: usl z ep background: hpyv is a novel human polyomavirus (hpyv), and neither its natural history nor its prevalence in human disease is well known. therefore, the epidemiology and phylogenetic status of hpyv must be systematically characterized. methods: the vp gene of hpyv was detected with an established taqman real-time pcr from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. the hpyv -positive specimens were screened for other common respiratory viruses with real-time pcr assays. results: the prevalence of hpyv was . % ( / ), and children ≤ years of age accounted for % ( / ) of cases. all hpyv -positive patients were coinfected with other respiratory viruses, of which influenza virus a (ifva) ( / , . %) and respiratory syncytial virus ( / , . %) were most common. all hpyv -positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from . to . copies/μl nasopharyngeal aspirate specimen. the most common symptoms were cough ( %) and fever ( . %). the complete -bp genome (bj strain, genbank accession number km ) was amplified and showed % identity to hpyv strain a. conclusions: the prevalence of hpyv was . % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time pcr. because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between hpyv and respiratory diseases. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the family polyomaviridae contains viruses that are among the smallest known to infect humans, in terms of both their particle sizes and genome lengths. the - nm nonenveloped icosahedral particles carry a circular doublestranded dna genome of approximately bp, which is divided into three functional regions: the noncoding control region, the early gene region encoding the large t antigen and the small t antigen, and the late coding region, encoding capsid proteins vp , vp , and vp [ ] . human polyomaviruses (hpyvs) have not been associated with any severe acute disease in healthy humans. however, the viral proteins expressed by polyomaviruses (pyvs) can initiate the transformation and immortalization of cultured cells and cause cancer in experimental animals [ ] . the hpyv family currently consists of members, including jcpyv [ ] , bkpyv [ ] , wupyv [ ] , kipyv [ ] , mcpyv [ ] , hpyv [ ] , hpyv [ ] , tspyv [ ] , hpyv [ ] , hpyv [ ] [ ] [ ] , stlpyv [ ] , hpyv [ ] and njpyv- [ ] . previous studies have indicated that a number of hpyvs are associated with human diseases, such as progressive multifocal leukoencephalopathy (jcpyv), hemorrhagic cystitis (bkpyv), merkel cell carcinoma (mcpyv), and trichodysplasia spinulosa (tspyv) [ , , , [ ] [ ] [ ] . a serological study demonstrated that the seroprevalence of these viruses in plasma samples from healthy adult blood donors was bkpyv ( %), jcpyv ( %), kipyv ( %), wupyv ( %), mcpyv isolate ( %); and mcpyv isolate ( %). the seroprevalence of all polyomaviruses in children under years of age (n = ) was similar to that in the adult population, suggesting that primary exposure to these viruses occurs in early childhood and seems to result in lifelong persistence [ ] . nevertheless, the natural histories of most hpyvs and their prevalence in human diseases are not yet well known. in , mcpyv was discovered by feng et al. [ ] . hpyv and hpyv were discovered with mcpyv in skin swabs from the foreheads of healthy volunteers [ ] . however, later research could not demonstrate a relationship between hpyv and merkel cell carcinoma or other skin diseases. a phylogenetic analysis of the complete hpyv genome indicated that hpyv shared a branch with kipyv and wupyv. previous reports have shown that genomic fragments of kipyv and wupyv have been regularly detected in nasopharyngeal aspirates of children with respiratory tract infections (rtis) and are suspected of a causal relationship with respiratory disease. however, the link between these pyvs and respiratory diseases remains speculative [ , , , ] . a real-time pcr assay was established here to determine the prevalence of hpyv throughout a time period of months (from october to september ). the prevalence of hpyv was . % ( / ). a complete hpyv genome was amplified, sequenced and found to be identical with a hpyv isolate from usa. with the standard curve derived from serial dna dilutions, the dynamic range of the real-time pcr assay was - copies/μl and the limit of detection was one copy. the coefficient of determination (r = . ) showed a good linear correlation. the taqman-based real-time pcr assay to detect hpyv did not amplify any other viral pathogen, showing the excellent specificity of this assay. four different dna concentrations ( - copies per reaction) were repeated five times in each run. the maximum coefficient of variation was . %, which indicates good precision (data not shown). a total of npa samples were obtained from children with rti. the sex ratio (male:female) was : ( . : ) and the median age was months (ranging from days to years). the prevalence of hpyv was . % ( / ) in the npa specimens tested. of the hpyv -positive patients, eight were male ( / , . %) and seven were female ( / , . %), so the prevalence was similar in both sexes (p > . ). the ages of the infected patients ranged from days to years, and children ≤ years of age accounted for % ( / ) of the total hpyv -positive children. the age distribution of the hpyv -infected children indicated that those aged - months had the highest infection rate table ) . all hpyv -positive patients were diagnosed with lower rti, including bronchopneumonia in nine ( %), acute bronchitis in three ( %), bronchitis in two ( . %), and pneumonia in one ( . %). the most common symptom was cough, which occurred in all patients ( %). other clinical presentations included fever (n = , . %), gasping (n = , %), vomiting (n = , %), and diarrhea (n = , . %). among the hpyv -positive specimens, the hpyv genome copies ranged from . to . copies/μl npa on a realtime pcr assay ( table ). the hpyv genome copy number was . copies/μl npa in children suffering lower rtis only, and was slightly higher than . copies/μl npa in those infected with lower rtis and other diseases, but these did not differ significantly (p > . , mann-whitney u test). to determine the complete hpyv genomic sequence, overlapping genomic fragments were amplified with nested pcr using pairs of virus-specific primers (listed in additional file : table s ), and the complete -bp genome was compiled with bioedit . software. the genome sequence of hpyv was deposited in genbank under accession number km . blast analysis with the complete hpyv genome showed a high level of nucleic acid identity to the six full hpyv genomes available in genbank. relationsship to the hpyv strain a was %. hpyv , thought to be a skin-tropic polyomavirus, was initially described in . since then, subgenomic fragments of hpyv dna have been detected in a variety of specimen types, including skin, respiratory secretion samples, and various tumor samples (for instance, the answers to these questions will require more information on the biology and epidemiology of the hpyvs. the genomes and proteins of hpyv , one of the novel hpyvs, show little sequence homology with previously reported hpyvs (bkpyv and jcpyv). although hpyv encodes a conserved, potentially carcinogenic ltag, previous studies have shown no association between hpyv and tumors [ ] . furthermore, a phylogenetic analysis indicated that ltag of hpyv (km ) is only distantly related to its homologues in other cancerassociated hpyvs. the hpyv , wupyv, and kipyv strains formed a clade in the complete genome and vp amino acid phylogenies, whether hpyv also associate with respiratory infection which need more clinical and experimental evidences to support. hpyv has been detected in specimens from the human respiratory tract, but there are as yet insufficient epidemiological data to demonstrate a correlation between hpyv and respiratory disease. because initial infections with most hpyvs occur in infancy, the prevalence of hpyv in npas from children was detected with real-time pcr. hpyv displayed an overall prevalence of . % in npa samples collected from children in a hospital in china, which is similar to its prevalence reported previously ( . - %) [ , ] . it has not been confirmed that hpyv infects humans via the respiratory tract, but the respiratory tract may be a possible route of transmission. in this study, hpyv was mainly detected in children less than years of age, and the peak incidence occurred in spring. all hpyv -positive patients were coinfected with other respiratory viruses, of which ifva and rsv were the most common. the hpyv -positive patients were diagnosed with lower rtis, % had bronchopneumonia, and the most common symptoms were cough and fever. although known hpyvs cause disease in patients with immune-system imbalances, they do not seem to cause obvious illnesses in the great majority of infected individuals. however, bkpyv can induce nephropathy in kidney transplantation recipients and jcpyv causes progressive multifocal leukoencephalopathy. progressively increasing or high viral loads are also associated with high-level viral replication and disease. for instance, progressive multifocal leukoencephalopathy and hemorrhagic cystitis are related to high viral loads of jcpyv and bkpyv, respectively. the present study cannot confirm that hpyv is the cause of rtis in hospitalized children, because the viral loads of hpyv were low ( . - . copies/μl) and the coinfection rate with other respiratory viruses was high. in previous reports, hpyvs were detected in the respiratory tract and skin but, to date, there has been insufficient evidence that hpyv is associated with any respiratory tract disease or skin disease [ ] . whether hpyv induce any disease requires the analysis of further data. the detection rate for hpyv by real-time pcr assay was . % in npa samples collected from hospitalized children with rti. an association between hpyv and respiratory diseases could not been revealed due to the high coinfection rate and the low hpyv viral load. from october, , to september, , nasopharyngeal aspirate (npa) specimens were collected continuously from hospitalized children with rti, who ranged in age from days to years, at beijing friendship hospital, beijing, china. the patients' parents or guardians gave their written informed consent for specimen collection and testing, and the project was approved by the ethical committee of the beijing friendship hospital. the npa specimens were collected and transported immediately to the national institute for viral disease control and prevention, china cdc, and stored at − °c until further processing. all demographic data and clinical findings were recorded on a standard form. total nucleic acids were extracted from each npa specimen using the qiaamp minelute virus spin kit (qiagen, beijing, china). a taqman-based real-time pcr assay for the detection of vp gene of hpyv was designed with primer express . software. the primer sequences used were hpyv -f '-ttaacacccttctttgtgctgcta- ' and hpyv -r '-gcccaattattcaaagcagctaa- ' , and the probe sequence was hpyv -p fam-ctgtcacag gcctgctgagcaatagatttc-tamra. the specificities of the primers and probe were evaluated in genbank with blast. the primers and probe were synthesized by invitrogen (beijing, china). a common reaction mix was prepared for the real-time pcr assays. briefly, the final μl reaction mix contained μl of taqman gene expression master mix, . μl of each primer ( pmol/μl), . μl of probe ( pmol/μl), μl of pmd -t/vp plasmid template (plasmid pmd -t linked to the vp gene of hpyv ), and . μl of h o. the amplification conditions included an initial incubation at °c for min and °c for min, followed by cycles of °c for s and °c for min, using the mx p qpcr system (agilent stratagene). ten-fold serial dilutions of the pmd -t/vp plasmid (from to copies/μl) were added to the real-time pcr reactions in duplicate. the results were used to generate a standard curve for hpyv . specificity was assessed by testing mixed samples of other common hpyvs, including wupyv, kipyv, jcpyv, bkpyv, and hpyv . to test the reproducibility of the assay, we added - copies/μl of pmd -t/vp plasmid to each reaction and each concentration of dna was repeated five times. in addition, housekeeping gene glyceraldehyd- -phosphate dehydrogenase (gapdh) was used as internal control. μl of nucleic acid of each npa specimen was added to each reaction. only samples that were positive according to both pcr and dna sequencing were considered "positive". fifteen overlapping fragments of the complete genome of hpyv strain bj were pcr amplified (table ) with the takara ex taq kit. the overlapping fragments were then cloned into pmd -t and sequenced (invitrogen). the nucleotide sequence of the full-length hpyv genome was then compiled using bioedit . software. the full-length hpyv sequence was aligned with the sequences of other hpyvs and other hpyv strains available in genbank with dnastar software. a neighbor-joining tree was constructed with mega . . the significance of differences between the prevalence rates and viral loads of various groups was tested with fisher's exact test and the mann-whitney u test. all analyses were performed with spss . software. genome analysis of the new human polyomaviruses human polyomaviruses in disease and cancer cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy new human papovavirus (b.k.) isolated from urine after renal transplantation identification of a novel polyomavirus from patients with acute respiratory tract infections identification of a third human polyomavirus clonal integration of a polyomavirus in human merkel cell carcinoma merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient a novel human polyomavirus closely related to the african green monkeyderived lymphotropic polyomavirus complete genome sequence of a tenth human polyomavirus discovery of a novel polyomavirus in acute diarrheal samples from children identification of mw polyomavirus, a novel polyomavirus in human stool discovery of stl polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique t antigen by alternative splicing identification of a novel human polyomavirus in organs of the gastrointestinal tract identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy association between a high bk virus load in urine samples of patients with graftversus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation progressive multifocal leukoencephalopathy and promyelocytic leukemia nuclear bodies: a review of clinical, neuropathological, and virological aspects of jc virus-induced demyelinating disease sv and human cancer: a review of recent data seroepidemiology of human polyomaviruses presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection molecular epidemiology of ki and wu polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus:assessment of assay sensitivity and specificity of real-time screening and typing methods wu and ki polyomaviruses in respiratory samples from allogeneic hematopoietic cell transplant recipients development and evaluation of real-time pcr assays for the detection of the newly identified ki and wu polyomaviruses human bocavirus in patients with respiratory tract infection pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement impact of human coronavirus infections in otherwise healthy children who attended an emergency department exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer prevalence of human polyomaviruses in common and rare types of non-merkel cell carcinoma skin cancer detection of novel polyomaviruses, tspyv, hpyv , hpyv , hpyv and mwpyv in feces, urine, blood, respiratory swabs and cerebrospinal fluid human polyomaviruses in children undergoing transplantation no evidence for association of hpyv or hpyv with different skin cancers submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we thank the beijing friendship hospital, capital medical university, for providing the samples, and the laboratory staff who contributed to this study. no compensation was given to subjects for participation in this study. additional file : table s . primers and probes used to detect respiratory viruses. table s . primers used to amplify hpyv with nested pcr (docx kb) the authors declare that they have no competing interests. key: cord- - hqqheho authors: ng, kim tien; oong, xiang yong; pang, yong kek; hanafi, nik sherina; kamarulzaman, adeeba; tee, kok keng title: outbreaks of enterovirus d in malaysia: genetic relatedness to the recent us outbreak strains date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: hqqheho nan temperature ( c), relative humidity (%), number of rain days, and the amount of rainfall (mm) collected from the nearest meteorological station (latitude: n, longitude: e) was analyzed using pearson's correlation. specimens positive for enteroviruses were further confirmed using standard molecular approaches that involved amplification and sequencing of the human enterovirus vp /vp gene using primers described previously. pcr amplification of the capsid (p ) region ( bp) of ev-d was performed when vp /vp gene was identified as ev-d . a combination of previously published primers, namely -forward, -reverse, fwd , rev , fwd , rev , and newly designed primers were used to amplify the p region by forming several overlapping fragments. these newly designed primers are ev p . f ( -tca aaa tty act gaa cca gt- ), ev p . r ( -gtt gcc atg aag ctv cca ca- ), ev p . r ( -gat atg ttt cct act ara gt- ), ev p . f ( -cca ggg car gtc cgy aac atg- ), ev p . r ( -cca ytt gwa aaa gtt ytt gtc- ), ev p . f ( -gtg gar tca atg gag at- ), and ev p . r ( -gct gat tta tca cyg tgc gag- ). a total of vp /vp , vp , and p of ev-d retrieved from genbank (accessed on february ) were analyzed using neighbor-joining method implemented in mega version to deduce the viral phylogenies. the statistical robustness of the branching orders was evaluated by bootstrap analysis of replicates. in this molecular epidemiological surveillance, approximately % ( / ) of patients had at least one viral pathogen detected by the multiplex assay, of whom . % ( / ) were infected with ev-d . these ev-d cases were detected in the second half of (june to december) and between december and january , of which september and january were the peak months of infection. no ev-d cases were detected in other months, suggesting transient outbreaks of ev-d . the patients (five males and seven females) from whom ev-d was detected ranged in age from to years old. during recruitment, most of the patients experienced mild sneezing and moderate-to-severe cough. correlation of ev-d infections with meteorological factors was not observed (correlation coefficient , . ). based on previously described ev-d classification, the newly sequenced strains from malaysia were found within clade a (my-cluster- ) and clade b (my-cluster- ). phylogenetic analysis of the p region indicated that . % ( / ) of the malaysian ev-d formed clusters, suggesting the transient ev-d outbreaks were most likely caused by at least two viral lineages ( figure ) . a sporadic ev-d strain ( mykl ) that did not cluster with the major lineages was also observed. of note, my-cluster- contains ev-d mostly sampled in , while my-cluster- contains ev-d sampled in / . such observation suggests an ongoing ''clade shift'' or lineage replacement of circulating ev-d in causing new outbreak, as observed in other enterovirus-associated outbreaks. sequence homology comparison based on the p region indicated that the malaysian ev-d strains shared . %- . % (nucleotide) and . %- . % (amino acid) sequence identities with the fermon strain (ay ), a prototype strain isolated in the usa in . phylogenetic tree also showed that the representative ev-d sequences sampled from recent outbreaks in the usa were grouped into three distinct lineages. these lineages are designated as outbreak lineage (consists of sequences isolated from missouri, mo), lineage (illinois, il and kentucky, ky), and lineage (ky) (figure ) . interestingly, malaysian ev-d sequences (my-cluster- ) was related to outbreak lineage while outbreak lineage (us/ky/ - ) was clustered with the my-cluster- , supported by strong statistical evidence (bootstrap values of % and %, respectively) at the internal tree nodes and genetic distance of f . . likewise, outbreak lineage was closely related to the ev-d sequences (cu and cu ) reported recently in thailand. all members of my-cluster- and outbreak lineage exhibited unique amino acid substitution at two residue positions (s a and t a), while members of my-cluster- and outbreak lineage sequences displayed unique amino acid substitutions at four residue positions (n e, g e, f y, and i v) relative to the fermon strain (figure ). these ''clade-defining'' substitutions differentiate outbreak lineage from other outbreak and non-outbreak lineages. a unique substitution t a discriminated outbreak lineage from outbreak lineage , further suggesting that the former is indeed closely related to the thai strains. contribution of these amino acid substitutions on virus pathogenesis however remains unclear. although the amino acid substitutions are located in the putative immunogenic bc and de loops, experiments in susceptible cell systems are needed to prove the importance of such substitutions. the divergence dates of the malaysian outbreak and other major lineages were estimated by the bayesian coalescent-based relaxed molecular clock model with constant population size, implemented in the beast software. . our data suggest that the recent ev-d strains associated with unprecedented severe respiratory outbreaks in the usa in were probably descended from the recent ev-d lineages circulating in thailand and malaysia. however such observation remains presumptuous largely because limited ev-d sequence data originating from the usa (due to inadequate surveillance) are available for genealogical analysis. given the close relationship of the southeast asian isolates with the us strains from recent outbreak, a more active enterovirus surveillance should be in place to monitor risks of impending outbreak. centers for disease control and prevention. enterovirus surveillance -united states clusters of acute respiratory illness associated with human enterovirus -asia severe respiratory illness associated with enterovirus d -missouri and illinois genome sequence of enterovirus d from genome sequence of enterovirus d and clinical disease seven strains of enterovirus d detected in the united states during the severe respiratory disease outbreak transmission of the common cold to volunteers under controlled conditions. i. the common cold as a clinical entity evaluation of nuclisens easymag for automated nucleic acid extraction from various clinical specimens comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus worldwide emergence of multiple clades of enterovirus mega : molecular evolutionary genetics analysis version . evolutionary genetics of human enterovirus : origin, population dynamics, natural selection, and seasonal periodicity of the vp gene can sequence data predict enterovirus d infection outcome? beast: bayesian evolutionary analysis by sampling trees key: cord- -uoszpnst authors: watanabe, yohei; ito, tetsuo; ibrahim, madiha s.; arai, yasuha; hotta, kozue; phuong, hoang vu mai; hang, nguyen le khanh; mai, le quynh; soda, kosuke; yamaoka, masaoki; poetranto, emmanuel djoko; wulandari, laksmi; hiramatsu, hiroaki; daidoji, tomo; kubota-koketsu, ritsuko; sriwilaijaroen, nongluk; nakaya, takaaki; okuno, yoshinobu; takahashi, tadanobu; suzuki, takashi; ito, toshihiro; hotta, hak; yamashiro, tetsu; hayashi, tsukasa; morita, kouichi; ikuta, kazuyoshi; suzuki, yasuo title: a novel immunochromatographic system for easy-to-use detection of group avian influenza viruses with acquired human-type receptor binding specificity date: - - journal: biosens bioelectron doi: . /j.bios. . . sha: doc_id: cord_uid: uoszpnst a switch of viral hemagglutinin receptor binding specificity from bird-type α , - to human-type α , -linked sialic acid is necessary for an avian influenza virus to become a pandemic virus. in this study, an easy-to-use strip test to detect receptor binding specificity of influenza virus was developed. a biotinylated anti-hemagglutinin antibody that bound a broad range of group influenza a viruses and latex-conjugated α , (blue) and α , (red) sialylglycopolymers were used in an immunochromatographic strip test, with avidin and lectin immobilized on a nitrocellulose membrane at test and control lines, respectively. accumulation of a sialylglycopolymer–virus–antibody complex at the test line was visualized by eye. the strip test could be completed in min and did not require special equipment or skills, thereby avoiding some disadvantages of current methods for analyzing receptor binding specificity of influenza virus. the strip test could detect the receptor binding specificity of a wide range of influenza viruses, as well as small increases in the binding affinity of variant h n viruses to α , sialylglycans at viral titers > hemagglutination units. the strip test results were in agreement with those of elisa virus binding assays, with correlations > . . in conclusion, the immunochromatographic strip test developed in this study should be useful for monitoring potential changes in the receptor binding specificity of group influenza a viruses in the field. emerging infectious diseases, such as severe acute respiratory syndrome (sars) and avian influenza, have been of increasing public concern in the past few decades. these diseases involve animal-to-human transmission of zoonotic pathogens (pang and guindon, ) . in particular, an influenza pandemic would be devastating and a serious threat to human health and the global economy. avian influenza (ai) viruses were the origin of the influenza a viruses and also have been involved in the emergence of all past influenza pandemics (webster et al., ) . therefore, surveillance of ai viruses to assess their evolution in the field is crucial for preparing for an influenza pandemic (watanabe et al., b) . influenza viruses are classified into subtypes based on the antigenic properties of their two surface glycoproteins: hemagglutinin (ha) and neuraminidase (na) (webster et al., ) . to date, ha subtypes and na subtypes have been identified. almost all possible combinations of ha and na subtypes have been detected in influenza viruses isolated from aquatic birds, poultry and other bird species. the ha subtypes are phylogenetically categorized into group (h , h , h , h , h , h , h , h , h , h , h and h ) and group (h , h , h , h , h and h ) has, with has in the same group antigenically related, although they can be distinguished by subtype-specific antibodies (watanabe et al., a) . ha is the main determinant of viral infectivity and consists of a head region and a stalk region (imai and kawaoka, ; sriwilaijaroen and suzuki, ) . influenza viruses attach to host cells by specific binding between the ha head region and sialylglycan that is expressed on the host cell surface. influenza viruses also recognize terminal sialic acid (sia) and galactose linkage patterns on sialylglycans (imai and kawaoka, ) . human influenza viruses preferentially bind to α , -linked sia (α , sia), whereas most avian viruses preferentially bind to a sugar chain ending in α , -linked sia (α , sia). this plays a key role of the interspecies barrier that prevents ai viruses from easily infecting humans. therefore, it is believed that a switch of ha receptor specificity from α , sia to α , sia is essential for the emergence of a pandemic influenza virus (watanabe et al., b) . the highly pathogenic ai virus subtype h n (h n virus) that emerged in china around has become endemic in birds in some areas, including china, viet nam, indonesia and egypt (oie, ) . h n virus can be directly transmitted from birds to humans and cause a severe respiratory disease with high morbidity ( %) (who, ) . fortunately, all human h n infections have been restricted to people with close contact with infected poultry and there has been no sustained human-to-human transmission. however, repeated bird-to-human transmission may allow h n viruses to acquire ha mutations that change their receptor specificity from α , sia (bird-type) to α , sia (human-type), thereby generating a pandemic virus. h n virus has now diverged genetically to form phylogenetically and phenotypically distinct clades (designated clades - ) in different geographic areas (watanabe et al., ) . such complex ecology and diversification in the field increase the pandemic potential of h n virus (peiris et al., ) . in addition, other subtype ai viruses, such as h n and h n , have also been directly transmitted to humans (garcia-sastre and schmolke, ). thus far, human-adaptive changes in ai viruses had been thought to occur during ai virus infections in humans and/or pigs. however, recent studies showed that ai virus could acquire increased human-type receptor binding affinity during viral transmission and infection in birds (watanabe et al., ; sriwilaijaroen and suzuki, ) . these results highlight the importance of monitoring possible changes in ai receptor binding specificity in the field to enable rapid response to the emergence of a pandemic influenza virus. several assay techniques have been developed to analyze receptor binding affinity of influenza virus (smith and cummings, ; stevens et al., ; watanabe et al., ; yamada et al., ) . the solid-phase virus binding assay is a quantitative system that can detect small changes in receptor binding affinity of influenza virus (watanabe et al., ; yamada et al., ) . glycan microarrays enable multiplex analysis using a panel of glycans with different topologies (smith and cummings, ; stevens et al., ) . a new assay system has been developed combining a virus binding assay and real-time rt-pcr (takahashi et al., ) . however, these very sensitive assay systems require a high level of technical expertise, expensive reagents and specialized equipment. these limitations indicate the need for a system that is easier to use for detecting changes in influenza virus binding specificity. immunochromatographic assays based on specific antigen-antibody reactions are very useful diagnostic tools and do not require specialized equipment or complicated handling procedures (gopinath et al., ; sakurai and shibasaki, ) . despite their relatively moderate sensitivity, immunochromatographic assays have been used in various rapid diagnostic kits to detect virus infections for clinical diagnosis and surveillance, since they are fairly simple and rapid. in this study, we developed a new easy-to-use immunochoromatographic strip test to detect the emergence of ai viruses with increased human-type receptor specificity and confirmed the applicability of this test using ai viruses isolated in several different geographic areas. avian and human influenza viruses were grown in -day-old embryonated chicken eggs and mdck cells, respectively. the allantoic fluids and culture supernatants were then harvested, precleared by centrifugation at rpm for min, passed through . mm filters, and stored as seed viruses at À °c. viral titers were assayed as hemagglutination units (hau) by hemagglutination assays as described below. all experiments with live h n viruses were performed in biosafety level (bsl ) conditions at osaka university (japan), tottori university (japan), national institute of hygiene and epidemiology (viet nam) and airlangga university (indonesia). recombinant viruses were generated with a plasmid-based reverse genetics system in the genetic background of influenza virus a/duck/egypt/d br/ (eg/d ), which is one of the parental egyptian h n strains, as previously described (watanabe et al., ) . mutant ha genes were generated by pcr-based sitedirected mutagenesis. recombinant eg/d virus and an eg/d variant carrying an ha mutation were denoted here as reg/d and reg/d mutation , respectively. recombinant viruses were propagated by single passage in eggs. the ha genes of the virus stocks were sequenced to detect the possible emergence of revertants during amplification. all studies with recombinant dnas were conducted at osaka university under the applicable laws and approved by the biological safety committee of the research institute for microbial diseases, osaka university (approval number ). stocks of avian and human influenza viruses were serially diluted with phosphate-buffered saline (pbs) and mixed with . % turkey red blood cells (nippon biotest, japan) and . % guinea pig red blood cells (nippon biotest, japan), respectively. hemagglutination by avian and human influenza viruses was observed after incubation at room temperature for min and h, respectively, to determine their hau titers. receptor binding specificity was analyzed by a solid-phase direct binding assay (watanabe et al., ) with sialylglycopolymers (totani et al., ; suzuki et al., ) containing sia linked to galactose through siaα , -lacnacβ-pap or siaα , lacnacβ-pap. a mg/ml solution of each sialylglycopolymer (non-conjugated with colored latex) was prepared in pbs, and ml of this solution was added to each well of -well microtiter plates (polystyrene universal-bind microplates, corning, usa). the plates were then irradiated with nm ultraviolet light for min, and each well was washed three times with ml pbs. each well was blocked with ml superblock blocking buffer (thermo scientific, usa) at room temperature for h. after washing with ice-cold pbs containing . % tween (pbst), a serial dilution of hau of an influenza virus in pbst was added to the wells, and the plates were incubated at °c for h. after five washes with ice-cold pbst, monoclonal antibody against influenza virus nucleoprotein, which is a structural protein of the viral particle, was added to each well, and the plates were incubated at °c for h. the wells were then virus biotin -ha antibody the mixture was then applied on the sample pad. during migration though the sialylglycopolymer pad, virus-c antibody complexes formed a colored sialylglycopolymer-virus-biotinylated antibody complex, which was captured by avidin at the test line. accumulation of the trimetric complex produced a visible test line with the color of the bound sialylglycopolymer (α , sia, blue; α , sia, red). excess complexes and free sialylglycopolymers were captured by lectin at the control line. the strip test was completed min after the virus-antibody complex was applied on the sample pad. a photograph of the strips is also shown. (b) schematic illustration of the viral receptor binding specificity patterns on the test strip. based on the patterns of visible bands on the α , sia and α , sia strips, viral receptor binding specificity was identified as indicated. washed five times with ice-cold pbst and incubated with peroxidase-conjugated goat anti-immunoglobulin (histofine simple stain max-po, nichirei, japan) at °c for h. after washing five times with ice-cold pbst, ml sureblue reserve peroxidase substrate (kpl, usa) was added to each well and, after incubation at room temperature for min, absorbance at nm (developed blue color) was measured. the test system in this study included an expansion solution, an antibody solution, and two test strips. the expansion solution was a mixture of surfactant agents: . % triton x- , % tween , and μg dana (n-acetyl- . -didehydro- -deoxyneuraminic acid)/ml in tris-hcl buffer (ph . ). these surfactant concentrations were sufficient to inactivate influenza virus infectivity and sialidase activity. the antibody solution contained . mg biotinylated anti-ha antibody c /ml. c is a monoclonal antibody against the antigenically conserved ha stem region (okuno et al., ; sakabe et al., ) , and has been reported to bind to a broad range of group influenza has (h , h , h , h and h ) (pan et al., ; smirnov iu et al., ) . a preliminary study showed that c reacted with all of the group influenza has in this study, while monoclonal antibodies against the antigenically variable ha head region only reacted with some of these has (table s ). c binding to the ha stem region has been reported to not interfere with the interaction between sialylglycans and the ha head region (okuno et al., ) , which enabled its use in the strip test to detect sialylglycan-ha complexes. each test strip consisted of four components assembled on a plastic backing: a sample pad, a sialylglycopolymer pad, a nitrocellulose (nc) membrane and an absorption pad (fig. a) . the three pads were as follows: the sample pads were rayon nonwoven fabric (advantec), each mm wide, cm long and . mm thick; the sialylglycopolymer pads were glass fiber (millipore corporation), each mm wide, mm long and . mm thick; and the absorption pads were filter paper (advantec), each mm wide, . cm long and . mm thick. the nc membranes (millipore corporation) were mm wide and cm long, and the plastic backings were adhesive backing cards (lohmann technologies corp.), each mm wide and . cm long. a sialylglycopolymer pad was prepared by spraying it with either blue latex-conjugated α , sialylglycopolymer or red latex-conjugated α , sialylglycopolymer (denoted as an α , sia strip and an α , sia strip, respectively) and drying at room temperature. our previous study showed that the latex-conjugated sialylglycopolymers are remarkably stable without any aggregation even in boiling water (totani et al., ). an . μl sample of a mg avidin/ml solution and a . μl sample of a mg lectin/ml solution were dispensed on each nc membrane to form test and control lines, respectively, and the membrane was air-dried. the distance between test and control lines was about mm. to assemble a test strip, an nc membrane was pasted onto the center of the plastic backing (using acrylic adhesive) and covered with the sialylglycopolymer pad at one end and the absorption pad at the other end. a sample pad was pasted onto the plastic backing at the top of the sialylglycopolymer pad. the nc membranes with attached pads were then cut into mm wide test strips and stored desiccated at °c. virus samples ( ml) were mixed with ml of expansion solution and ml of antibody solution, and then incubated for min to form virus-c complexes (fig. a) . each mixture was then pipetted onto a test strip sample pad or, alternatively, a sample pad was immersed in the virus mixture. this allowed the mixture to migrate along the strip by capillary action. during migration through the sialylglycopolymer pad, the virus-c complex was able to form a complex with α , sia or α , sia depending on the virus ha receptor binding affinity. principle of the subsequent reaction is mainly based on two types of interaction: avidin-biotin interaction and lectin-glycan interaction. as the virus-c -sialylglycopolymer complex migrated along the test strip, it was captured at the test line by avidin-biotinylated c interaction. accumulation of the trimeric complex resulted in visual blue and red test lines on the α , sia strips and α , sia strips, respectively. if a viral ha did not bind either sialyglycopolymer, there was no visible test line because the captured virus-c dimeric complex was colorless. excess complexes and free sialylglycopolymers were captured at the control line by lectinglycan interactions, resulting in visible blue and red control lines on the α , sia strips and α , sia strips, respectively. the results of this assay were observed by eye min after deposition of the virus mixture onto the strips. the presence of double blue lines on the α , sia strip and double red lines on the α , sia strip indicated virus binding affinity to α , sia and α , sia, respectively. only a blue control line on the α , sia strip and only a red control line on the α , sia strip indicated no binding affinity to α , sia and α , sia respectively, or that the amount of virus was below the limit of detection of this assay. if a control line did not appear within min, the test was considered invalid. thus, the complete assay took only min. in past influenza pandemics, mutant viruses with dual tropism for both α , sia and α , sia have emerged in the immediate early phase of an outbreak (matrosovich et al. ) . these prototype viruses subsequently switched their receptor binding specificity from bird-type to human-type sialyglycans to become established pandemic viruses. therefore, based on the patterns of the test lines on the α , sia and α , sia strips (fig. b) , viral receptor binding specificity was identified as follows: (i) only a blue test line indicated typical bird-type receptor specificity; (ii) both blue and red test lines indicated increased human-type receptor affinity with residual bird-type receptor affinity, denoting an interphyletic mutant virus; and (iii) only a red test line alone indicated a switch of viral receptor affinity from bird-type to human-type receptors, denoting either a novel virus with a high pandemic potential or a pandemic virus. band intensities on test strips were quantified by imagej software, and the test line intensity was calculated by subtracting the background intensity from the test line intensity. for this study, the receptor binding specificity of several virus strains that were used as reference viruses were confirmed by virus binding assays. the reference viruses were as follows: a/suita/ / (suita/ ), a human h n strain; a/japan/ / (japan/ ), a human h n strain; eg/d and recombinant reg/d virus, bird h n strains; and reg/d q h and reg/d Δ/i t , mutant reg/d viruses with enhanced human-type receptor specificity. previous studies reported that ha q h and Δ/i t mutations increased viral binding affinity for α , sia (watanabe et al., ) . therefore, reg/d q h and reg/d Δ/i t were generated by introducing the q h and Δ/i t mutations into eg/d ha to produce representative interphyletic viruses for this study. the virus binding assays showed that suita/ and japan/ had binding specificity for α , sia ( fig. a and b) . in contrast, eg/d and reg/d had binding specificity for α , sia ( fig. c and d) . interphyletic viruses reg/d q h and reg/d Δ/i t had significantly increased binding to α , sia, with binding to both α , sia and α , sia ( fig. e and f) . the binding curves of these viruses were typical sia binding specificity profiles for human viruses, bird viruses, and interphyletic viruses, respectively (boltz et al., ; watanabe et al., ; yamada et al., ) . we evaluated the sensitivity and specificity of the strip test by investigating the reactivity of - hau of the reference viruses to α , sia and α , sia. suita/ gave clear red test lines on α , sia strips for virus titers of - hau (fig. a) . however, even at the highest titer, hau, suita/ produced no visible blue test line on the α , sia strip, indicating human-type receptor specificity. japan/ , which is classified as a group influenza a virus, produced no visible test lines on either α , sia or α , sia strips (fig. b) . these results showed that the visible test lines produced on the test strips were the result of specific c binding to group influenza has. in contrast, eg/d gave clear blue test lines on α , sia test strips when the virus titer was - hau (fig. c) . however, even the highest eg/d titer, hau, produced no visible red test line on α , sia strips, showing bird-type receptor specificity. to further assess the receptor specificity of h n viruses that were circulating in geographical areas other than egypt, we tested a/ chicken/soc trang/ / and a/tree sparrow/indonesia/d / , which were typical bird-type h n viruses isolated in viet nam and indonesia, respectively. these two viruses produced blue test lines only on α , sia strips, with the same sensitivity ( fig. d and e). these results showed that the test strips consistently detected the receptor binding specificity of group influenza viruses. the reactivity of the test strips to the interphyletic viruses with titers of - hau was then evaluated. both reg/d q h and reg/d Δ/i t produced clear blue test lines on α , sia strips at all virus titers tested ( fig. g and h) . in addition, - hau of these viruses produced visible red test lines on α , sia strips, in contrast to the results with the parental reg/d virus (fig. f) . these results showed that the test strips were able to detect human-type receptor binding by interphyletic viruses at higher titers ( hau). based on these results, we used virus isolates with titers of - hau for further studies. band intensities on the test strips were quantified by densitometry and plotted as a function of virus titer. the results showe d that the intensity profiles ( fig. a -f) were similar to the virus binding assay results ( fig. a-f) . scatter plots showed that an increase in band intensity was strongly correlated with binding affinity on both α , sia (r ¼ . , fig. g ) and α , sia strips (r ¼ . , fig. h ), indicating that the results obtained with the test strips and by binding assays were highly correlated. therefore, the test strips produced a semi-quantitative measure of viral receptor binding affinity. a long-term storage test was then performed to evaluate the stability and reproducibility of the strip test. investigations using the same batch of test strips showed that the test strips were stable for at least months at °c without any changes in sensitivity or specificity (table s ) . to verify the potential applicability of the test strip, the ha receptor specificity of influenza virus strains that had been isolated in the several geographical areas were examined. the results showed that strains that were classified as egyptian clade . . sublineage a/b viruses had dual binding affinity to both α , sia and α , sia, indicating that they were interphyletic viruses ( fig. and table ). this was in agreement with the previous report that sublineage a/b viruses have generally acquired an increased human-type receptor binding affinity (watanabe et al., ) . in contrast, all the h n strains that were isolated in viet nam and indonesia showed typical bird-type receptor specificity. several variant-type strains with increased α , sia binding affinity have been reported in some asian countries (auewarakul et al., ; boltz et al., ) . however, acquisition of human-type receptor affinity by asian h n viruses seemed to be strain-specific, which may explain why the asian h n viruses tested in this study did not show dual receptor specificity. further evaluation of the test strips using larger numbers of asian h n strains may detect interphyletic viruses in these areas. in this study, two h n virus strains produced no visible test lines on either α , sia or α , sia test strips (table ) , even though the titers of these viruses were hau. epidemiological studies have reported a few exceptional virus strains that recognized distinct sialylglycan topologies (xiong et al., ) . thus, these two viruses may have such an exceptional receptor binding specificity. however, these viruses may not have bound the c antibody due to amino acid change(s) at the antigen epitope in the ha stem region. further genetic analyses and characterization of these two viruses are needed to elucidate the reason(s) why these viruses did not produce bands on the test strips. the results described above demonstrated the utility of the test strip system developed in this study to analyze the receptor specificity of influenza viruses. compared with conventional methods to analyze receptor binding affinity of influenza virus, the test strip system was simple and easy to use: only a drop of virus and the test strips were required, without any additional methods or equipment. the test strips were shown to be applicable to a broad range of group influenza a vruses and allowed semi-quantitative detection of their receptor binding affinities. these results indicate that the test strips could be applied as a screening device to monitor possible evolutionary changes in ai receptor binding specificity in the field. the sensitivity of the test strips was not adequate for rapid diagnosis of clinical samples (e.g., nasopharyngeal swabs). however, traditional methods for determining viral genome sequences, pathogenicity and drug resistance using isolated viruses are still routinely used in public health and academic laboratories worldwide (kumar and henrickson, ) . since isolated virus samples contain significantly more virus than clinical samples, application of the strip test to influenza virus analysis should enable us to more easily and comprehensively monitor ai evolution and pandemic potential in the field. the strip test system described here could then be applied for rapid diagnosis in the field when its sensitivity is improved. spec no) united states patent application publication microbiol the authors would like to thank dr. tadahiro sasaki and dr. kazuo takahashi for assistance with this project; dr. masato tashiro and dr. takato odagiri for valuable discussions. this work was supported by a grant-in-aid for scientific research from the ministry of education, culture, sports, science and technology, japan (jsps kakenhi grant numbers and ) and by japan initiative for global research network on infectious diseases (j-grid). key: cord- -ex qb zj authors: elena, santiago f.; pybus, oliver g. title: editorial: a home for virology, ecology, epidemiology, and evolutionary biology date: - - journal: virus evol doi: . /ve/vev sha: doc_id: cord_uid: ex qb zj nan santa fe, new mexico, usa and department of zoology, university of oxford, uk *corresponding author. e-mail: sfelena@ibmcp.upv.es; oliver.pybus@zoo.ox.ac.uk. viruses make headline news on an almost daily basis. sometimes the news is positive, a report on the development of new anti-viral drugs or a reduction in transmission, perhaps. however, often the story will relate to a gloomier theme, for example, the appearance of new viral epidemics, the evolution of drug resistance, or falling vaccine coverage. the appearance of ebola virus in west africa since represents just the latest of a long series of devastating viruses that have emerged or expanded in humans in recent years, including middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) coronaviruses, chikungunya virus, highly pathogenic avian influenza viruses, west nile virus, and various human enteroviruses, and bunyaviruses. this list is both selective and anthropocentric and excludes numerous new epidemics in livestock (e.g., schmallenberg virus), crop (e.g., tomato torrado virus), and wild animal populations (e.g., phocine distemper virus). the impacts of viral epidemics may extend beyond death and illness to cause substantial economic losses and social instability. such effects are not limited to new or exotic viruses, as established and well-characterized viral diseases persist despite tremendous efforts to control and eradicate them. important pathogens in this category include hiv/ aids, human influenza viruses, dengue viruses, hepatitis viruses, human papillomaviruses, and rabies virus. one reason that viruses are such potent adversaries is their great potential for genetic diversity and evolvability, a characteristic that they owe to a combination of short generation times, very large population sizes, and (in some but not all instances) error-prone replication mechanisms. strains that escape host immune responses, are resistant to antiviral drugs, or, in the case of plant viruses, that break transgenic or natural genetic resistance, may arise in a viral population soon after it is challenged by the corresponding antiviral measure. together, the outlook is mixed: the perspectives for future eradication or control are balanced by the emergence or re-emergence of new foes. although genetic diversity is an essential part of virus biology, classical approaches to virus control often ignore evolutionary processes and focus on understanding in great detail the molecular bases of pathogenesis, virus-host interaction, and drug-virus interference. this is despite the fact that evolutionary concepts are key to the correct interpretation of molecular variation among virus strains. some experimental biologists, on the other hand, have taken advantage of the rapid evolution of many viruses and chosen to use them as model organisms for the investigation of fundamental questions in evolutionary biology (e.g., elena and sanjuá n ) . moreover, in recent years, virus epidemiologists have begun to exploit the increasing availability of virus genome sequences and incorporated evolutionary thinking in their approach, leading to new insights into the ecological origins and transmission of viruses (e.g., holmes ; pybus and rambaut ). lastly, computational and theoretical biologists are developing increasingly complex models of virus behavior across all biological scales, from the cellular to the global, many of which attempt to explicitly represent the generation and dynamics of viral genetic diversity under different conditions (e.g., nowak and may ; wilke ; luksza and lä ssig ) . unfortunately, it is rare for all these diverse approaches to be taken into account when new strategies for virus control are designed. why? perhaps, at least until recently, there was insufficient exchange of ideas and concepts among these disciplines. the study of virus evolution in its own right has flourished in the last years and the subject undoubtedly gained greater recognition within the biological sciences after the discovery that evolution is an essential component of hiv infection. since then, the number of published articles that use the term 'virus evolution' in the article title or summary has grown exponentially and doubled every - years (fig. a ). an equally striking pattern is seen if we plot occurrence of the same term in books written in english (fig. b) . the rate of growth of scientific papers that include 'virus evolution' is nearly twice that of those that mention only 'virus' (fig. a) and approximately three times faster than the growth rate of pubmed as a whole (lu ) . where do all these manuscripts get published? despite an explosion in virus evolution research activity, publications on the topic are scattered among a large number of journals that belong to a variety of categories from the institute of scientific information (isi). although many studies appear in evolutionary biology journals, particularly those on viral experimental evolution, mathematical modeling, molecular evolution, and phylogenetics, a large proportion are submitted to journals that focus on virology and pathogenesis. in these disciplines, some editors express a preference for 'mechanistic' studies within a clear hypothetico-deductive framework and may not appreciate the importance of inferential and observational work within population and evolutionary biology. further, several virology journals focus on either animal and plant viruses, so that relevant articles may not come to the attention of researchers from the other field. viruses of bacteria, archaea, fungi, and protists are served comparatively poorly by the current literature, yet these groups are very likely to comprise the majority of viral genetic diversity on earth. to add further fragmentation, some important theoretical work on virus variability and evolution is published in specialized mathematical journals that will not be well known to laboratory and field researchers. we believe that the study of virus evolution would benefit from a common forum in which findings and ideas can be shared. we have established the journal virus evolution with this aim in mind, and we hope that it will grow into a successful and dynamic inter-disciplinary community of researchers interested in understanding why and how viruses have and continue to evolve. we aim to cover all aspects of virus evolution, ecology, and diversity with no restriction on host range or research methodology. to achieve this goal, we have assembled an editorial board whose members have made many important contributions to the field. the board has expertise in animal, plant, and bacterial viruses and in a wide range of techniques, including experimental evolutionary biology, molecular epidemiology, metagenomics, structural biology, population genetics, ecology, and molecular virology. one benefit of a focused journal, such as virus evolution, is that the editorial board shares with the authorship a passion for the subject. our editorial philosophy is that virus evolution exists first and foremost to serve its authors and readers. to make publication in the journal a more pleasant experience, we impose no specific formatting requirements at submission: the manuscript can be provided in any style so long as it is readable by reviewers. standard formatting will be requested only 'after' a paper has been accepted, at which point it should seem less of a chore. we will operate a traditional peer-review process but one that will emphasize the quality of reviews as well as their speed. published papers will be available to read by all under an open access model that is compliant with all major funding bodies including the usa national institutes of health and uk wellcome trust. lastly, accepted manuscripts will be visible online in the shortest possible time after acceptance. we very much look forward to receiving your submissions and to begin working with the community to make virus evolution a vibrant and successful home for your research. virus evolution: insights from an experimental approach the evolution and emergence of rna viruses pubmed and beyond: a survey of web tools for searching biomedical literature a predictive fitness model for influenza evolutionary analysis of the dynamics of viral infectious disease probability of fixation of an advantageous mutant in a viral quasispecies key: cord- -pge authors: kotton, c.n.; kuehnert, m.j.; fishman, j.a. title: organ transplantation, risks date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: pge viruses are among the most common causes of opportunistic infection after solid organ and hematopoietic stem cell transplantation (sot and hsct). viral infection is associated with both direct (invasive disease) and indirect (immune modulation) effects affecting susceptibility to other infections and promoting allograft rejection. the transplantation recipient is susceptible to a broad array of viral pathogens. some may be transmitted with the allograft as donor-derived infections, while others result from exposure, either soon after the transplant or from more distant exposures when infection is latent and reactivates in the setting of immune suppression. simultaneous infections with multiple viral or viral and nonviral pathogens are common. the risk for viral infection is a function of the intensity of exposure and virulence of the specific virus, the intensity of immune suppression used to prevent graft rejection or graft-versus-host disease, underlying immune deficits, and factors affecting host susceptibility. studies of viral latency, reactivation, and of the cellular effects of viral infection will provide clues for future strategies in prevention and treatment of viral infections. this article covers specific issues relating to viral infection in sot and hsct; additional details regarding these viral infections are also found elsewhere in this text. transaminases, coagulopathy, graft dysfunction, and either fever or leukocytosis within several weeks after transplantation. given the rarity of such infection diagnosed in normal hosts, there may be a predilection for this in transplant recipients. transplant-transmitted infection is rare and might be difficult to recognize, but physicians should consider the possibility, particularly when unexplained neurologic complications occur. these investigations underscore the challenge in detecting and diagnosing infections that occur in recipients of organs or tissues from a common donor. the potential for disease transmission from donor source may not be considered in recipient evaluation. in these investigations, the ability to connect illnesses to a common organ donor was facilitated by the fact that multiple recipients were hospitalized at the same facility. as organ and tissue transplantation becomes more common, the potential risks of disease transmission may also increase. because of improved diagnostic assays, donor-transmitted infections are increasingly recognized, although often times, the impact of infection is not well understood. the advent of polymerase chain reaction (pcr) and other genetic material-based tests have allowed for detection of active viremia, in contrast to serologic tests, which reflect past acquisition of infection. this is important not only for recipient diagnosis, but also for recognition of donor infection retrospectively; donor diagnosis is only possible if appropriate specimens are stored postmortem. recognition of viral infection transmitted through transplantation is increasing, likely both due to increased recognition of unexpected symptoms consistent with transmission, and due to increasingly sophisticated diagnostic testing in both the recipient and the donor. investigation of potential donor-transmitted infection requires rapid communication among physicians in transplant centers, organ procurement organizations, and public health authorities. an immediate system for tracking and disseminating pertinent patient data to evaluate donor-derived infection and associated adverse event outcomes is needed. until such a system can be established, clinicians should report unexpected outcomes or unexplained illness in transplant recipients to their local organ and tissue procurement organizations. given the frequency of latent viral infection, notably among herpesviruses, reactivation of latent infection provides a major source of infection after transplantation. the specific virus, the tissue infected, stimuli for activation, and the nature of the host immune response impact the nature of viral latency. some viruses are metabolically inactive when latent, while others continue to replicate at low levels that may be determined by the effectiveness of the host's immune response. multiple factors contribute to viral reactivation after transplantation, including graft rejection and therapy, immune suppression (especially reduction of t-cell mediated, cytotoxic immunity), inflammation, and tissue injury. numerous cellular pathways are involved in the control of viral replication and are activated after transplantation, such as nuclear factor kb, ikb, and jak-stat (the janus family of protein tyrosine kinases (jaks) and signal transducers and activators of transcription (stat) proteins). antirejection therapy can also result in a significant release of pro-inflammatory cytokines which may increase viral replication. in general, reactivation of viruses, especially late after transplantation, should suggest new immune defects (e.g., cancer) or relative over-immunosuppression. latency and reactivation has been best studied in the herpesviruses, which establish lifelong, latent infection after primary infection. in general, latency is considered to be the absence of viral replication, with viral genomes present in the cell without replication or spread. studies of other viruses, such as friend virus in mice, suggest that protective antiviral immunity is an active process mediated by 'leaky' (low-level) viral replication. the existence of true latency, as opposed to low-level replication, remains controversial. herpesviruses make 'latency' proteins that both control viral persistence within the target cell and influence other cellular processes. the latent state is characterized by low levels or the absence of detectable viral antigens, minimal transcription of productive or lytic cycle genes, and expression of the latency-associated viral transcripts. viral latency may be occasionally interrupted, leading to reactivation and spread of infectious virus with or without recurrent disease. ebv establishes latency in b lymphocytes in association with expression of a limited set of viral genes. immune control of hsv infection and replication occurs at the level of skin or mucosa during initial or recurrent infection and in the dorsal root ganglion, where latency and reactivation are controlled by immune mechanisms mediated by interferons, myeloid and plasmacytoid dendritic cells, cd (+) and cd (+) t cells, and other cytokines. despite similarities, the molecular details and mechanisms of latency and reactivation vary considerably among the herpesviruses. mechanisms responsible for maintenance of latency are unclear. reactivation of cmv has been extensively studied. cmv viral genomes can be found in cd + monocytes and cd + progenitor cells, although the primary reservoir for latent cmv and the mechanisms by which latency is maintained are unknown. allogeneic immune responses and fever (via tumor necrosis factor-a (tnf-a)) have been shown in vitro to increase both cmv promoter activity and viral replication. immune suppression is not essential for the reactivation of latent cmv, but serves to perpetuate such infections once activated. subclinical activation of cmv is common and increasing diagnosed by sensitive molecular assays. for other viruses such as bk polyomavirus, specific types of tissue damage such as warm ischemia and reperfusion injury may precipitate viral activation; they have been linked to an inflammatory state in grafts (via activation of tnf-a, nuclear factor kappa b (nf-kb), neutrophil infiltration, and nitric oxide synthesis), tubular-cell injury, and enhanced expression of cell-surface molecules, all of which may contribute to viral activation. thus, immune injury, inflammatory cytokines, and ischemia-reperfusion injury stimulate viral replication and change expression of virus-specific cell-surface receptors. the hosts' direct pathway antiviral cellular immune response within allografts is less effective due to mismatched major histocompatibility antigens between the organ donor and host with dependence on indirect pathways of antigen presentation. these factors may render the allograft more susceptible to viral infection. common reactivation infections after transplantation include cmv, hbv, hcv, hiv, hsv- and hsv- , hpv, and vzv (as zoster). other less clinically common viral infections related to reactivation include the polyoma viruses bk and jc, human herpes virus (hhv- ), human herpes virus (hhv- ), and hhv- . reactivation of one virus may lead to reactivation of others; multiple studies have shown that infection with hhv- and/or hhv- are risk factors for cmv disease and cmv infection may trigger hhv- and hhv- reactivation. while some reactivation infections routinely cause significant clinical disease, such as cmv, hsv, and vzv, others may cause more variable illness. hhv- , for example, reactivates with immunosuppression, with clinically significant infection after hsct. by contrast, the role of both hhv- and hhv- in sot recipients is less well defined; while reactivation is common, clinical disease is generally not evident. based on epidemiologic exposures, new infections from the environment are commonly acquired after transplantation. the respiratory viruses are the most common new infections after transplantation, including rsv, influenza, parainfluenza, and adenovirus. newer respiratory pathogens (metapneumovirus and sars coronavirus) also cause major infections in immunocompromised hosts. gastrointestinal viruses such as rotavirus or norwalk virus are common and can cause significant diarrhea and dehydration; diarrheal syndromes may alter absorbance and metabolism of calcineurin inhibitors (e.g., cyclosporine and tacrolimus), with unexpectedly elevated levels of tacrolimus. nonimmune patients can acquire primary ebv, cmv, vzv, parvovirus b , and other infections in the post-transplantation period. in the absence of previous immunity and with the attenuation of immunity due to the immunosuppressive regimen, new infections are often more severe and prolonged than in the general population. for example, parvovirus b infection is often more persistent and relapsing in transplantation patients, occasionally complicated by the unusual findings of hepatitis, myocarditis, pneumonitis, glomerulopathy, arthritis, or transplantation graft dysfunction. the effects of viral infection are conceptualized as 'direct' and 'indirect' (see table ). this classification serves to separate the tissueinvasive viral infection (cellular and tissue injury) from effects mediated by inflammatory responses (e.g., cytokines) or by alterations in host immune responses. syndromes such as fever and neutropenia (e.g., with cmv infection) or invasive disease resulting in pneumonia, enteritis, meningitis, and encephalitis are considered direct effects. indirect effects of viral infections are generally thought to be immunomodulatory responses to viral infections mediated by cytokines, chemokines, and/or growth factors. the impact of these effects is diverse and includes systemic immune suppression predisposing to other opportunistic infections (notably with cmv or hcv infections). in addition, viral infection may alter the expression of cell-surface antigens (e.g., major histocompatibility antigens) provoking graft rejection and/or cause disregulated cellular proliferation (contributing to atherogenesis in cardiac allografts, obliterative bronchiolitis in lung transplantation, or to oncogenesis). increased viral replication and persistence may contribute to allograft injury (fibrosis) or chronic rejection. infection with one virus may stimulate replication of other viruses in a form of viral 'cross talk'. as was noted above, infection with hhv- and/or hhv- serve as risk factors for cmv disease and vice versa. the direct and indirect effects of hhv- reactivation can be significant: hhv- infection is associated with high levels of il- and tnf-a, and in pediatric renal or bone marrow transplantation, hhv- reactivation is strongly associated with acute rejection. co-infection with hcv and cmv predicts an accelerated course for hepatitis. co-infection with cmv and ebv increases the risk for post-transplantation lymphoproliferative disorders (ptld) by - -fold. a more theoretical concern is that t-cell responses against viral infections are thought to produce cross-reacting immune responses against graft antigens, possibly via 'alternative recognition' within the t-cell receptor. this cross-reactivity is termed 'heterologous immunity' and may provoke abrogation of graft tolerance. the hhv family has eight members affecting humans, all of which can cause significant disease in transplantation recipients. the risk of many of these infections is reduced by the use of valganciclovir or acyclovir after transplantation. hsv- and À (hhv- and À ) usually cause oral and genital ulceration, although it may occur in more unexpected areas as well. recurrent disease can be problematic for some patients and warrants consideration of secondary prophylaxis with antiviral therapy. vzv (hhv- ) is common with an incidence of herpes zoster among patients after sot of . % (liver . %, renal . %, lung . %, and heart . . ptld constitutes a spectrum of disease, which is often responsive to reduced immunosuppression in previously immune hosts. ebv-seronegative individuals with primary infection after transplantation are at increased risk for ebv-mediated ptld. the clinical presentation of cmv (hhv- ) can range from a 'cmv syndrome' including fever, malaise, leukopenia, to a 'flu-like' illness with myalgias and fatigue, to a more significant end-organ disease with pneumonitis, colitis, encephalitis, hepatitis, or chorioretinitis. cmv is the single most important pathogen in transplantation recipients due to direct and indirect effects (see above). hhv- commonly reactivates after transplantation, especially after hsct where it is associated with hepatitis, pneumonitis, cmv reactivation, bone marrow suppression, and encephalitis. hhv causes less symptomatic clinical infection after sot, although the indirect effects of reactivation have not been studied. hhv- commonly reactivates after transplantation. the clinical symptoms caused by hhv- are uncertain, although neurological symptoms seem to be significant, especially in children. hhv- causes kaposi's sarcoma and is seen in sot recipients at a rate - higher than the general population, with a prevalence of . - % depending on the patient's (and donor's) country of origin. hepatitis b and c are among the most common indications for liver transplantation, and can complicate other transplantations as well. hepatitis c is currently the most common indication for liver transplantation, accounting for - % of cases in recent times. recurrent post-transplantation hepatitis c infection poses a conundrum between treating the hepatitis c and reducing immunosuppression without precipitating rejection. given the risk of precipitating graft dysfunction, hepatitis c treatment with interferon and ribavirin was often deferred in extra-hepatic transplant recipients. novel therapies for hepatitis c should reduce the risk of treatment complications after transplant. for hepatitis b, the goal is complete viral suppression before and after transplantation, using antiviral agents and sometimes hepatitis b immunoglobulin. respiratory viruses are the most common community-acquired infections in transplantation recipients. given the increased rates of pneumonia and bacterial and fungal superinfection, prevention (vaccination, avoidance of sick individuals) is essential. diagnosis of respiratory viruses within a few hours via enzyme-linked immunosorbent assay (elisa) or immunofluorescent staining is available in many medical centers. viral cultures are time consuming and expensive. respiratory syncytial virus and parainfluenza are the most common community-acquired respiratory viruses, followed by influenza and adenovirus. antiviral medications (rimantidine, amantidine, or oseltamivir) may prevent or reduce the severity of illness. the use of ribavirin or rsv table direct and indirect effects of cmv infection fever and neutropenia syndrome (leukopenia, fever, myalgia, fatigue, thrombocytopenia, hepatitis, nephritis) increases risk of ptld gastrointestinal invasion with colitis, gastritis, ulcers, bleeding, or perforation increases risk of cardiovascular events hepatitis increases risk of immunosenescence, mortality pancreatitis chorioretinitis increases risk of new-onset diabetes mellitus after transplantation immune globulin in adults to prevent rsv infection is not well studied. ribavirin is sometimes used for documented rsv infections of the lower respiratory tract in transplant recipients. metapneumovirus and severe acute respiratory syndrome-associated coronavirus (sars-cov) are emerging pathogens in transplantation patients. the clinical spectrum of disease from metapneumovirus ranges from symptomatic (even fatal) to asymptomatic cases. some groups have suggested a possible correlation with graft rejection in lung transplant recipients. diagnosis is often made using molecular assays; the full impact of this infection in transplantation is yet to be realized. sars, caused by a zoonotic coronaviruses, is a highly contagious and rapidly progressive form of viral pneumonia, which spread from asia to many parts of the world in early . a number of transplantation patients were infected, some of whom died. the impact of sars and resulting infection control issues was significant for both active organ transplantation (i.e., concerns about transmitting donorderived infections) as well as routine follow-up care for transplantation patients, some of who deferred healthcare visits. gastrointestinal viruses such as rotavirus or norwalk virus may cause significant diarrhea and dehydration; prolonged infections for months have been seen in transplant recipients. enteroviral infections in the summer months in the northern hemispheres are common and can have a more complicated and prolonged course in renal transplantation recipients. bk virus is associated with a range of clinical syndromes in immunocompromised hosts: viruria and viremia, ureteral ulceration and stenosis, and hemorrhagic cystitis. the majority of patients with bk virus infections are asymptomatic. infection by jc polyomavirus has been observed in renal allograft recipients as both nephropathy (in association with bk virus or alone) and/or progressive multifocal encephalopathy (pml). jcv establishes renal latency but receptors are present in multiple tissues including the brain. infection of the central nervous system generally presents with focal neurologic deficits or seizures and may progress to death following extensive demyelination. human papilloma virus (hpv) infections can cause significant disease in renal transplantation recipients, including oral, skin, genital, and rectal lesions ranging from warts and dysplasia to malignancy (especially squamous cell carcinoma). the recent arrival of a vaccine for genital hpv infections may help reduce these infections. in transplantation recipients, parvovirus b infection can cause erythropoietin-resistant anemia, pancytopenia, myocarditis, or pneumonitis. direct renal involvement with glomerulopathy and allograft dysfunction has been reported in renal transplantation recipients. clinical and virologic responses to treatment with intravenous immunoglobulin are usually excellent. several zoonotic viruses have caused major illness and death in the transplantation setting, including wnv, rabies, and lymphocytic choriomeningitis virus (lcmv). all have been recently reported as donor-derived infections related to sot, with clinically subtle infections in the donor and often deadly infections in the recipients. wnv is more morbid after sot; the risk of meningoencephalitis in a sot patient infected with wnv has been estimated to be %, compared with < % in normal hosts. donors in endemic areas should be screened for wnv, as the prevalence can be high. aside from donor-derived infections, rabies and lcmv have not been reported. although hiv-infected patients are living longer and dying less often from complications related to acquired immunodeficiency syndrome (aids), they are experiencing significant morbidity and mortality related to end-stage liver and renal disease. preliminary studies suggest that both patient and graft survival are similar in hiv-negative and hiv-positive kidney and liver transplantation recipients. however, hcv infection appears to be accelerated even in controlled hiv infection. ongoing multicenter trials of transplantation in hiv infections are continuing. drug interactions between the immunosuppressive regimen and antiretroviral drugs necessitate careful monitoring. rapid and sensitive molecular biology-based assays for many of the common viruses after transplantation have replaced, for the most part, serologic testing and in vitro cultures for the diagnosis of infection. serologic assays are generally less sensitive in transplant patients, as humoral immune responses may be delayed or absent. in one series, % of patients with parvovirus b infection as shown by pcr assay had a negative igm assay. quantitative molecular tests allow the optimization and individualization of antiviral therapies for prevention and treatment of infection. this advance is most significant in the management of cmv, ebv, hepatitis b, and hepatitis c viruses, where quantitative assays (such as viral loads or antigenemia tests) guide antiviral therapy. nonquantitative (i.e., qualitative) assays are less useful in management as they do not assess responses to therapy and cannot differentiate primary infection, from reactivation or reinfection. for example, chromosomal integration of latent hhv- dna (which happens in - % of immunocompetent subjects) leads to high levels of viral dna, whether or not the infection is active; one group concludes that any diagnosis of hhv- encephalitis should not be made without first excluding chromosomal hhv- integration by measuring dna load in csf, serum and/or whole blood. latent infections due to ebv and cmv may be qualitatively positive by pcr, confirming the need for quantitative assays. blood tests may not always accurately reflect the level of end-organ diseases; thus, it may be useful to test specific affected tissues as well the blood. in this regard, histologic evaluation of tissues using pathogen-specific immunohistochemistry may augment systemic assays. patients with cmv colitis, for example, may have negative molecular or antigenemia blood assays for cmv. in addition, patients may shed cmv in secretions without true infection, limiting the diagnostic capacity of a positive culture. bk polyomavirus may be detectable in the urine (either by cytology, looking for the classic decoy cells, or by pcr) before it is detectable in the blood, providing a window of opportunity for reducing immunosuppression possibly in advance of invasive disease. adenovirus may be detectable in local infections, such as cystitis, with negative blood assays. the treatment of viral infections in the renal transplantation recipient includes: the reduction of immunosuppression, antiviral therapy, diagnosis and treatment of co-infections (such as cmv, ebv, hhv- , or À ), and use of adjunctive therapies such as immunoglobulins or colony stimulating factors. the overall level of immunosuppression has a major impact on both the risk of reactivation of latent infection and the ability to clear such an infection. reducing the immunosuppressive regimen during active viral infection can be a major contribution toward clearing infection, although it presents a risk of graft rejection. as protective cytotoxic immunity to viruses is generally t-cell (cd +) mediated, an initial reduction of antimetabolites (if neutropenic) and calcineurin inhibitors merits consideration. in contrast, a reduction of the steroid dose during the acute phase of a febrile illness may cause acute adrenal insufficiency. when available, antiviral therapies (such as acyclovir, ganciclovir, ribavirin, lamivudine, and oseltamivir) are often essential. the toxicity of some agents (such as cidofovir, foscarnet, and ribavirin) may complicate management, notably in the face of reduced renal function in patients receiving calcineurin inhibitors as part of the immunosuppressive regimen. the duration of therapy is often longer in transplant patients than in normal hosts, and often reflects the ability to reduce the overall level of immunosuppression, that is. less immunosuppression may result in a shorter treatment time. the increased information provided by molecular diagnostics may allow for more directed treatment regimens. antiviral therapy is often used for prophylaxis in the post-transplantation period, especially for individuals at risk for primary infection. in general, acyclovir and related agents are used to prevent hsv and vzv and ganciclovir or valganciclovir to prevent cmv as well as hsv and vzv. the relative advantages of universal prophylaxis (i.e., the use of antiviral medication in all susceptible patients for a period after transplantation) or monitoring with preemptive (early) therapy remain to be established. meta-analyses suggest that universal prophylaxis with antiviral medications in sot recipients reduces cmv disease and cmv-associated opportunistic infections, graft rejection, and mortality; their use is recommended for high risk individuals (cmv-positive recipients and in cmv-negative recipients of cmv-positive organs). some studies indicate that universal prophylaxis and preemptive therapy are effective in reducing the incidence of cmv disease. in the hsct setting, where the risks of bone marrow toxicity from valganciclovir are higher, many programs choose to use preemptive monitoring and therapy for cmv. various adjunctive therapies have been helpful in treating and preventing viral infections. immunoglobulins (i.e., intravenous immune globulins (ivig), cmv, and hbv hyper-immune globulins (both prepared from plasma preselected for high titer antibodies to cmv and hbv, respectively), as well as monoclonal antibodies such as those for rsv) have been helpful in preventing and treating viral infections, likely due to both direct and indirect immunomodulatory properties. a significant percent of patients have post-transplantation hypogammaglobulinemia, which has been linked to increased mortality and may benefit from globulin repletion. this may be most apparent in the setting of active infections, as well as prophylaxis. immunostimulatory agents, such as interferon-alpha used to treat hepatitis c, can be helpful at treating the viral infection but may perturb the relationship between the graft and the host, precipitating rejection. reversal of neutropenia can be done using colony stimulating factors such as g-csf, which is generally well tolerated in sold organ transplantation patients. vaccines should be given to patients as early as possible in the course of organ failure and well in advance of transplantation to optimize immune responses. in general, a response to vaccination is more likely to occur when given pre-transplantation rather than after transplantation. nonlive viral vaccines such as hepatitis b, hepatitis a, injectable influenza, rabies, hpv, injectable polio may be given both pre-and post-transplantation (see table for classifications). live viral vaccines such as attenuated influenza for protection against varicella in nonimmune subjects, as well as zostavax, for protection against zoster), yellow fever, oral polio, and vaccinia (smallpox) should generally be avoided after sot and in patients who are chronically immunosuppressed (such as those with gvhd); some may be given after hsct. consideration of future travel or trips home should also be considered in the pre-transplantation period a prospective survey of human herpesvirus- primary infection in solid organ transplant recipients ast infectious diseases community of practice ( ) vaccination in solid organ transplantation parvovirus b infection after transplantation: a review of cases transmission of lymphocytic choriomeningitis virus by organ transplantation infection in solid-organ transplant recipients prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients zoonoses in solid-organ and hematopoietic stem cell transplant recipients cmv: prevention, diagnosis and therapy viral infection in the renal transplant recipient prevention of infection in adult travelers after solid organ transplantation updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation a seroprevalence study of west nile virus infection in solid organ transplant recipients idsa clinical practice guideline for vaccination of the immunocompromised host notes from the field: a cluster of lymphocytic choriomeningitis virus infections transmitted through organ transplantation -iowa hhv- dna level in csf due to primary infection differs from that in chromosomal viral integration and has implications for the diagnosis of encephalitis key: cord- -wlhmcdcn authors: rutschke, nils; zimmermann, jan; möller, ronny; klöck, gerd; winterhalter, mathias; leune, annika title: hot start reverse transcriptase: an approach for improved real-time rt-pcr performance date: - - journal: j anal sci technol doi: . /s - - - sha: doc_id: cord_uid: wlhmcdcn background: reverse transcriptase is an indispensable enzyme for real-time reverse transcriptase (rt)-pcr, a standard method in molecular diagnostics for detection and quantification of defined rna molecules. the prevention of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature is one of the biggest challenges in real-time rt-pcr. in the present study, an aptamer directed against the reverse transcriptase was analyzed for its potential to attain a temperature-dependent reverse transcriptase (“hot start” rt). findings: the hot start effect was investigated in a one-step real-time rt-pcr assay for the detection of middle east respiratory syndrome coronavirus (mers-cov). results with aptamer revealed a reduced rt activity at low temperatures while achieving full activity at the specific annealing temperature of °c. sensitivity (limit of detection (lod) %) of the mers-cov assay was increased by about two times in the presence of aptamer. conclusions: the study demonstrates the potential of aptamer-dependent hot start rt for the improvement of diagnostic real-time rt-pcr assays. real-time rt-pcr is the method of choice in molecular diagnostics for detection and quantification of defined rna molecules (mackay ) . this technique utilizes reverse transcriptase (rt) to convert rna into complementary dna (cdna), a thermostable dna-dependent dna polymerase for the amplification of specific target sequences and target specific probes (oligonucleotides) labelled with fluorophores for the detection of amplified dna (gibson et al. ) . real-time rt-pcr is regarded as a method with high sensitivity and specificity (martel et al. ) . however, this is challenged by non-specific products generated by elongation of misprimed primer that competes with the synthesis of specific amplification products in each cycle (chou et al. ; li et al. ). the probability of nonspecific product formation increases with the complexity of the real-time rt-pcr system and the background nucleic acid in the specimen (brownie et al. , handschur et al. ). ultimately, non-specific products can severely decrease sensitivity as well as specificity of real-time rt-pcr assays (sharkey et al. ; birch et al. ; jayasena ) . assays for the detection of rna viruses are often highly complex (high quantity of different oligonucleotides) due to low sequence conservation of the rna genome (gardner et al. ; brownie et al. ) . in general, mispriming occurs at temperatures below the specific annealing temperature of the oligonucleotides (jayasena ) . thus, the formation of non-specific products can be reduced by using hot start variants of the enzymes, which are inactive at low temperatures and activated at higher temperatures, appropriate for specific primer annealing to the target nucleic acid (birch et al. ) . several biological or chemical hot start concepts exist for taq polymerase, a thermostable dna-dependent dna polymerases. the taq polymerase can be inactivated by binding of specific antibodies or aptamers, by incubation with chemicals or by altered molecular kinetics (sharkey et al. ; birch et al. , hermann and patel ; gening et al. ; kermekchiev et al. ) . the activation is obtained by heating up to ≥ °c during the initial denaturation step of the pcr. in contrast to taq polymerase, which is heat-stable up to temperatures of ≥ °c, the rt is only stable at temperatures ranging from to °c (pfaffl ; gallup ) . therefore, other hot start concepts for the reversible inactivation of reverse transcriptase need to be developed. a high-affinity rna ligand (aptamer), which targets moloney murine leukemia virus (m-mlv) rt was described by chen and gold ( ) . the aptamer is assumed to inactivate the rt by blocking the nucleic acid binding site of the rt (chen and gold ; chen et al. ) . in the present study, the aptamer was analyzed in a one-step real-time rt-pcr assay for the detection of middle east respiratory syndrome coronavirus (mers-cov) to investigate the potential of a hot start rt for improved real-time rt-pcr performance. mers-cov was first identified in ). since the discovery, the world health organization noted cases of mers-cov, and of these, led to a lethal outcome (who ) . therefore, a more sensitive detection system, especially in the presence of low viral loads in patients, would be favorable for an early diagnosis and treatment. the one-step real-time rt-pcr was performed in a -μl reaction mix containing μl of rna template, x pcr reaction buffer (altona diagnostics gmbh), . mm mgcl (sigma-aldrich), μg/μl bsa (roche), u of platinum® taq dna polymerase high fidelity (invitrogen), u of superscript® iii reverse transcriptase (invitrogen). the mers-cov specific primer and probe, targeting the genomic region upstream of the envelope gene (upe), were synthesized as published (corman et al. a; corman et al. b ). the rt-pcr reaction included . μm of primer upe-fwd (gcaacgcgcgattcagtt), . μm of primer upe-rev (gcctctacacgggacccata), and . μm of probe (fam-ctcttcacataatcgccccga gctcg-tamra). thermal cycling conditions were °c for min (rt-step), min at °c (taq polymerase activation and rt inactivation), followed by cycles of s at °c (denaturation), s at °c (annealing and acquisition) and s at °c (extension). in case of thermal gradient real-time rt-pcr, the rt-step was performed in parallel at °c for one part and °c for the other part of the -well plate. all real-time rt-pcrs were performed on a cfx touch™ real-time pcr detection system (biorad). the aptamer ( ′-cuuaccacgcgcucuuaacugcua gcgccauggccaaaacu- ′) published by chen and gold ( ) was synthesized at altona diagnostics gmbh. a ′ phosphorylation was added to the aptamer to eliminate any possible elongation of the aptamer. the reverse transcriptase was incubated with increasing concentrations ( , . , , , , or μm) of aptamer for min at room temperature ( °c) before adding to the reaction mix. to demonstrate an aptamer-dependent hot start rt effect, the rt-step was carried out in parallel at °c, the specific annealing temperature of the primer and probe, and at °c, at which the rt shows a significant activity but which is below the specific annealing temperature of the primer and probe. each aptamer concentration was analyzed in three replicates. an in vitro transcribed rna (ivt) based on a sequence of mers-cov strain emc/ was used as real-time rt-pcr template. the concentration of the ivt was determined by spectrophotometry. the analytic sensitivity (limit of detection (lod)) is defined as the concentration (copies/reaction) of mers-cov specific rna (ivt) molecules that can be detected with a positive rate of ≥ %. the analytic sensitivity was determined by analyzing a half-logarithmic serial dilution table hit rate of μm aptamer and without aptamer in real-time rt-pcr mers-cov assay. half-logarithmic serial dilutions of mers-cov rna, ranging from − to copies per reaction were analyzed. the ratio of positive signals to all signals, followed by the data in percentage is given below copies (mers-cov ivt)/reaction . . − . − aptamer ( fig. probit regression analyses of a real-time rt-pcr with μm aptamer and without aptamer. probit regression analysis was carried out with target rna loads from − to copies per reaction. each dilution was analyzed in four independent runs with six replicates. the target rna concentration is plotted on the x-axis, and the y-axis displays the hit rate. circles are experimental data points; the inner lines represent the corresponding probit curve, outer lines the % confidence intervals. a without aptamer; b μm aptamer of the ivt ranging from to . copies/reaction. each concentration was analyzed four times in six replicates (n = ). hit rates were subjected to probit regression and correlation analysis in statsdirect software (version , , ; statsdirect statistical software). the rt was either incubated with μm aptamer or without aptamer for min at room temperature ( °c), before adding to the reaction mix. the reverse transcriptase was incubated with different concentrations of the aptamer ( , . , , , , and μm per reaction) before adding to the reaction mix. the rt-step was performed in parallel at and °c. incubation of rt with aptamer and rt-step at °c resulted in a mean delayed cycle threshold of up to Δc t . ( μm aptamer, fig. a ) compared to the reference without aptamer, indicating reduced rt activity (fig. a, b) . the results can be summarized as, the higher the aptamer concentration the later the amplification signal. two hundred micromolar of aptamer revealed no amplification signal at all. the rt-step at °c, with . and μm aptamer showed slightly earlier cycle thresholds compared to the reference without aptamer (Δc t . and . ), while concentrations of μm and higher resulted in delayed amplification signals, compared to the reference without aptamer (fig. a, b) . since the aptamer concentration of μm allowed full rt activity at °c but significantly reduced rt activity at °c, this concentration was chosen to survey the influence of the aptamer on the analytic sensitivity of the mers-cov assay. in order to investigate, if the hot start rt lead to an increase in analytical sensitivity of the mers-cov assay, a half-logarithmic serial dilution of the mers-cov ivt ranging from to . copies per reaction was analyzed with a reaction mix containing hot start rt ( μm aptamer) and standard rt (without aptamer). real-time rt-pcr was carried out to determine concentration-dependent hit rates (table ) , which were analyzed in a probit regression (fig. ) . the mers-cov assay with the hot start rt has a lod of . copies/reaction ( % confidence interval (ci): . - . copies/reaction), whereas the assay without aptamer has a lod of . copies/reaction ( % confidence interval (ci): . - . copies/reaction) (fig. a, b) . hot start taq polymerases have proven to be valuable tools to improve analytical sensitivity and specificity in real-time pcr assays, by reducing non-specific products. based on this experience, the idea arose to improve the performance of real-time rt-pcr assays by developing a hot start concept for the reverse transcriptase. in this study, we demonstrated that a hot start rt can be generated by using an aptamer directed against m-mlv rt. the use of this hot start rt in a mers-cov assay led to an increase of the analytical sensitivity of about twofold compared to the analytical sensitivity of the same assay with standard rt. in summary, we could demonstrate that hot start rt has the potential to improve the sensitivity of real-time rt-pcr assays. this finding will be of special interest for more complex assays or for assays which are used with specimen with a high load of non-target nucleic acids in routine molecular diagnostics. simplified hot start pcr the elimination of primer-dimer accumulation in pcr selection of high-affinity rna ligands to reverse transcriptase: inhibition of cdna synthesis and rnase h activity inhibitory rna ligand to reverse transcriptase from feline immunodeficiency virus prevention of pre-pcr mis-priming and primer dimerization improves low-copy-number amplifications detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections qpcr inhibition and amplification of difficult templates sequencing needs for viral diagnostics rna aptamers selected against dna polymerase ß inhibit the polymerase activities of dna polymerases ß and k a novel method for real time quantitative rt-pcr preanalytic removal of human dna eliminates false signals in general s rdna pcr monitoring of bacterial pathogens in blood adaptive recognition by nucleic acid aptamers aptamers: an emerging class of molecules that rival antibodies in diagnostics cold-sensitive mutants of taq dna polymerase provide a hot start for pcr direct electrophoretic detection of the allelic state of single dna molecules in human sperm by using the polymerase chain reaction real-time pcr in the microbiology laboratory a simple method for elimination of false positive results in rt-pcr the road from qualitative to quantitative assay: what is next? in: bustin sa (ed) the pcr revolution: basic technologies and applications antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment -as of isolation of a novel coronavirus from a man with pneumonia in saudi arabia authors nr, al, rm, and jz are employees of altona diagnostics gmbh. the authors declare that they have no competing interests.authors' contributions nr has performed the experimental and analytical work and prepared the draft of the manuscript. jz provided oligos and aptamers. rm involved in experiment design and data analysis. the guidelines and supervision of this work was provided by al and gk. al and mw read and modified the manuscript. all authors read and approved the final manuscript. key: cord- -rkadl r authors: macfall, janet; lelekacs, joanna massey; levasseur, todd; moore, steve; walker, jennifer title: toward resilient food systems through increased agricultural diversity and local sourcing in the carolinas date: - - journal: j environ stud sci doi: . /s - - - sha: doc_id: cord_uid: rkadl r biological and agricultural diversity are connected to food security through strengthened resilience to both anthropogenic and natural perturbations. increased resilience to stress via increased biodiversity has been described in a number of natural systems. diversity in food production can be considered on the following three levels: (a) genetic diversity as reflected in the range of cultivars which can be selected for production; (b) species diversity, captured through production of a wide range of crops on each farm; and (c) broad ecosystem diversity, described by the diversity of production between farms and within the broader food system. a network of locally based food producers and entrepreneurs provides opportunity for high diversity at each network stage, with increased adaptive capacity and the ability for rapid response to disturbance. we argue that production techniques that use carefully planned diverse plantings, such as biointensive cultivation, increase resilience by increased water use efficiency, yield and nutrient retention while reducing pressure from pests and pathogens. we present a model for a diverse, distributed food system in the north carolina piedmont and analyze an existing distributed network by a food hub in south carolina. through these models, we argue that a shift in the food network has the potential to increase local food security by having food more reliably available where it is needed and by contributing to local resilience through community economic development. the shift in food production and distribution systems serves multiple goals: when crop loss occurs, other crops still contribute to overall harvest, reducing net loss. diverse on-farm production can support a more distributed network of food aggregators, processors, and markets than the current approach of large-scale consolidation. finally, a distributed food supply network supported with diverse agricultural products can increase resilience by providing access to diversified markets for producers and improved food access to consumers with more food choices, while expanding the need for skilled jobs supporting the regionally based food industry. the term bresilience^has been used in many social, economic, and ecological contexts with varied meanings and interpretations. in its broadest form, the term resilience refers to the ability to recover from or to adjust to change (gunderson ) . uncertainties in an era of rapid change in agriculture have led to uncertainty in the capacity to meet growing food needs. concerns about chronic malnutrition, environmental degradation, livelihood security, food safety and hygiene, and equitable access raise important questions about how and where food is grown and eaten. in this paper, resilience is defined as the capacity for a system to absorb disturbance and to reorganize, while retaining system function and self-organizing processes (gunderson ; folke et al. ; folke et al. ) . resilience can be measured by the magnitude of disturbance that can be absorbed before the system is redefined into a different state (gunderson ) . in the case of agriculture and food security, resilience can be considered from the perspective of food availability with a return to a predictable and adequate supply following a disturbance. in this case, disturbances may occur in production (as with the loss of a crop following a plant disease or pest outbreak, severe weather, or climate change), transportation, processing, distribution, and access/supply. locally based and controlled food production systems with high diversity can provide opportunities for adaptive capacity through the ability to rapidly respond to disturbance and to changing conditions in production and market conditions (hendrickson and heffernen ) . the prevailing perception is that food production is predictable with a constancy of relationships. as anyone who has been involved with food production, processing, and distribution has experienced, uncertainty in food production and post-harvest handling is common-and risks are high. adaptive capacity and adaptive management acknowledge that the system being managed will always change, so humans can respond by adjusting the system quickly (gunderson ) . a growing body of evidence highlights the importance of biodiversity for ecosystem functionality (peterson et al. ; fischer et al. ; kerkhoff and enquist ) . although food and agricultural systems are highly managed, they are still guided by ecological principles through provision of essential natural resources, ecological function, and ecosystem services. just as with natural systems, biodiversity within food systems can be considered across a range of spatial scales from single farms to larger landscapes. food system diversity also includes social dimensions such as food distribution and access. sustaining ecosystem function in agricultural activities is essential for sustaining farm livelihoods and the food supply (tomimatsu et al. ) . functional redundancy within an ecosystem may increase resilience to environmental fluctuations, facilitating successful reorganization of ecological systems. high response diversity may ensure ecosystem functionality, providing a range of ways to respond to environmental change and uncertainty (folke et al. ) . biodiversity in agricultural systems can improve crop protection and soil fertility through these expanded functionalities (altieri ) . below, we discuss two examples of risks from biodiversity loss, the southern corn leaf blight epidemic of and porcine epidemic diarrhea, which demonstrate the negative possible outcomes of genetic uniformity in food systems. building on this, the insurance hypothesis for ecosystem resilience through conservation of functional diversity and responsive scale may be transferable to food systems. in the case of food systems, diversity in farm scale and number, diversity of crops, type of market opportunities, and higher numbers of farms equate to high numbers of species that respond differently to external pressures in ecosystems where diversity provides binsurance^if one component of the system declines or is lost. to overcome such risks, species with similar traits may be functionally redundant; if a crop is lost because of high disturbance sensitivity (drought or disease, for example), other crops will still provide agricultural product (mori et al. ) . some techniques such as biointensive agriculture which emphasize high biological diversity are most easily adaptable to small acreage. a concern can be about yield loss under such production techniques. however, intensively managed biointensive farms can produce yields which are comparable and often greater than conventional methods, while enhancing ecosystem structure and functions such as biogeochemical cycles and pest population controls. the contemporary food system is built on a complex network of related activities, ranging from on-farm production to harvest and sale to distribution, processing, and marketing, ending with consumer access, purchase, consumption and resource, and waste recovery. many related factors including environmental, social, and economic disruptions have the potential to contribute both chronic and acute disturbance to all points in this complex system. it is important to consider resilience at all points in the food system, minimizing vulnerability in each individual sector. therefore, it is crucial to link the shift in food production systems with a shift in the food distribution mechanisms. indeed, greater crop diversity offers opportunities for more diverse markets, and a locally embedded network of markets for food sale and access may provide greater resilience by insuring multiple points of entry for sale and access if the system is disrupted. as the two models below, north carolina central piedmont network and the south carolina food hub demonstrate, decentralized models that link producers to consumers provide opportunities for farmers that utilize high-yield, low input techniques such as biointensive and other agroecological techniques a convenient and reasonable access to markets. since the techniques can be developed both in urban and rural areas on smaller acreage farms, they can reduce the upfront capitalization costs for a start-up farm and also provide access to food related entrepreneurial opportunities and to food for low-income, ethnically and racially diverse consumers. this paper explores the link between diversity and resilience in our food networks-from production to distribution. we first illustrate risks from loss of biological diversity in food production systems and discuss how biointensive techniques can help overcome such risks. we then discuss two models of food value chains in the carolinas and how a shift in food distribution mechanisms built on principles of biological diversity can help build food security and community resilience. we also discuss the challenges of establishing such models on the ground and thus offer guidelines for practitioners on how to establish decentralized/local/ networked food production, distribution, and access systems. conditions of extreme genetic uniformity for production efficiency. the outcome was the epidemic of southern corn leaf blight, causing a crop loss of over $ billion (priced at dollars) (american phytopathological society ). corn leaf blight is caused by a fungus, an ascomycete, cochliobolus heterostrophus (also known as bipolaris maydis, or its name at the time of the epidemic, helminthosporium maydis). the fungus was common across the southern corn production areas, with annual losses of around . % under normal production. cultural practices to reduce leaf wetness and maintain general resistance throughout the corn population were the most affordable and consistently effective means of control. stubble left following harvest was plowed under to enhance decay, with corn production moved to other fields the following year (levings iii ; schumann ) . early in the twentieth century, corn breeders found that greatest yields were produced by progeny from the crossing of two genetically different inbred parental lines (buckner ). production of seed from defined crosses, however, required carefully controlled pollination and removal of the corn tassels. the detasseling process is very labor intensive and must be done by hand before pollen shedding. a genetic factor was discovered that caused plants to become male sterile, the texas male-sterile (tms) cytoplasm (levings iii ) . this meant that plants used for corn production no longer had to be de-tasseled prior to pollination-a huge economic benefit to the corn industry. by , nearly % of the corn produced in the usa contained tms cytoplasm. genetically, two changes occurred simultaneously in the host (the corn) and the pathogen populations. the corn crop now had the new tms gene throughout the population, and a genetic change occurred in the population of the fungal pathogen. a new race, named race t, evolved which had genes to produce a toxin that only affected the tms plants. the fungus could now complete its life cycle more quickly and was able to infect not only the leaves and husks but also the developing ears. for disease to occur, three conditions must be met-a susceptible host, a virulent pathogen contacting the host, and an appropriate environment. the genetic uniformity of the corn with the tms genes introduced the susceptible host. the genetic change in the pathogen, evolved in response to changes in the host genetics, greatly increased the virulence. the weather during the summer of was warm and wet, ideal conditions for disease to develop. the infection started in early summer in the southeastern united states. by early fall, corn across the entire east coast and westward past the mississippi river had become infected, with - % crop loss in many areas. the result was near complete loss of the corn crop that year, with a huge economic impact. fortunately, producers returned to still available non-tms lines of corn, again requiring manual detasseling. with the return to production of corn varieties lacking the tms genes, epidemics of this disease have not returned. however, as current corn production becomes less diverse, similar risks may again become important. most pork producers raise hogs for specific markets, resulting in limited genetic variability. breeds and hybrid lines are often selected for uniformity and product predictability, such as size, feed conversion, time to market, and/or meeting the requirements of the integrator or marketing company (martinez and zering ) . the disease porcine epidemic diarrhea has had widespread and damaging impacts throughout the pork industry. the disease was first confirmed in the usa in april . it is caused by a highly transmissible coronavirus genetically related to strains found in china (stevenson et al. ). this disease can be contracted by pigs of all ages; % mortality often occurs with suckling pigs. the incubation period is only - h. symptoms include vomiting and severe diarrhea, frequently followed by dehydration and death. there are no effective vaccines or pharmaceutical therapies for the disease at this time. the disease is highly contagious and environmentally stable. a tiny amount of material taken from intestines of an infected animal can be highly diluted (diluted - ) and still remain infective. air-borne transmission is under investigation. trucks and trailers used for dead haul, transport of animals to processing plants, feed deliveries, trash removal and other activities on infected farms have likely been associated with disease spread (national pork board ). the toll on the swine industry has been high. of the million hogs in the usa, about million have been lost to this disease (us department of agriculture ). some individual swine farms have lost as many as , pigs. in response to the rapid disease spread, quarantine measures were put into place in fall . farm visits were not allowed, and movement of people and materials between farms and processors was restricted and monitored. biosecurity measures, isolating one farm from another and preventing movement of infectious material between farms, have been the only effective method for control (national pork board ). separation of farms across north carolina and across the country is comparable to high landscape level diversity with species separation in natural systems, similar to the space between farms and sanitation providing a physical barrier to pathogen movement. most hogs are grown by independent farmers, who are producing on contract for sale of the animals to a larger integrator (animal processor). for example, one such integrator has contracts with over independent farmers for swine production across the usa. the integrators are requiring quarantine between farms as part of the contractual agreement. many of these independent farmers are small to mid-sized concentrated animal feeding operations (us environmental protection agency ) . when compared to production on only a few very large swine farms (> , animals), this distributed national network of independent, smaller farms provides opportunities for implementation of the needed biosecurity measures and physical separation. farms can be widely spaced across the landscape, reducing potential for aerial transmission, and strict sanitation measures have been effectively implemented with farm isolation. in addition, risk is distributed across the farmer network, rather than being concentrated in only a few centralized production farms. this suggests that resilience in the hog industry may be dependent on small farms producing animals independently of each other, rather than depending on large, centralized concentrated animal feeding operations. it also argues that genetic diversity between growers and by each independent grower should be increased to add potential disease resistance and resilience within the industry. in agricultural systems focused on local market channels, resilience can be enhanced by a diversification of on-farm products as well as by distribution of more smaller farms across the landscape, as suggested by the above two examples (thrupp ) . on farms, production risk is distributed across the products being grown for harvest and sale. each product represents an ecologically functional group, with varied ranges of tolerance for conditions within the production environment. for example, some crop plants may have a wider or shifted range of tolerance for temperatures or available moisture. based on observations from natural systems, biological and agricultural diversity can be considered from several perspectives: & diversity of markets based on diverse crops and production some crop production techniques mimic and enhance ecological processes and function within agricultural settings. biointensive production is an example of one approach which is focused on application of principles of agroecology (grow biointensive ). biointensive production emphasizes high crop spatial and temporal diversity via crop selection and multi-cropping practices. soil is highly managed for fertility and biological activity through an initial deep tillage and frequent carbon and nitrogen additions through green manures and compost. these techniques are often most suitable for small-scale farming and are able to provide product to the local food supply network for sale and distribution. there is also efficient land use and minimal reliance on mechanized equipment. these options are severely limited in conventional agriculture which is dependent on high acreage, uniform mechanized production (jeavons ) . a number of studies in natural systems have shown a correlation between high biodiversity and pressures from pests and pathogens (janzen ; connell ; wright ; peterman et al. ; terborgh ; bagchi et al. ). in some ecosystems, there is a feedback between pest and pathogen populations and biological diversity, with pests/ pathogen pressures reduced as individuals of each species become more widely separated with increasing biological diversity, reducing the potential for disease and insect spread. production methods which can enhance agricultural diversity such as organic production have been compared to conventional production in a number of studies. two large-scale meta-analysis studies have suggested that high acreage crop yield with organic production is generally lower when conventional methods are used. however, the yield differences between the two methods varied widely and were highly contextual, differing between crop groups, regions, site characteristics, and cropping techniques (di ponti et al. ; seufert et al. ). in addition, organic production can be done in large acreage monoculture, minimizing biodiversity (mission ). in contrast, some techniques that are most easily adaptable to small acreage, intensively managed farms can produce yields which are comparable and often greater than conventional methods, while enhancing ecosystem structure and functions such as biogeochemical cycles and pest population controls. biointensive techniques with high density diverse plantings have shown a - % increase in production with improved water use efficiency, as for tomato, basil, and brussel sprouts (jeavons ; jeavons ; bomford ; grow biointensive ) . in another study evaluating yield of nine onion cultivars, kg/ m were produced with conventional practice compared to kg/ m with biointensive techniques (moore ). in addition, energy efficiency was improved from an energy efficiency ratio of . : with conventional tillage compared to : with biointensive production-meaning . cal of food were produced for cal used for production. with biointensive production, most of the energy input was from direct human input (renewable) rather than from fossil fuels (moore ) . high biological diversity is a characteristic of biointensive production, with at least different types of plants grown together, including food for local consumption, food for sale, and compost crops. one farm in california, woodleaf farm, grows over different crops on ha of land with ha in woodland and meadow, using similar cultivation techniques. this farm notes anecdotal evidence that populations of beneficial insects have increased while pests and pathogens are minimized (woodleaf ). enhanced productivity, water use efficiency, creation of unique microclimates, and pest/pathogen management are also attributed to high crop diversity (woodleaf ). soil management in biointensive systems is directed toward enhancing ecosystem processes (e.g., nitrogen fixation, mineral immobilization) which are associated with soil fertility (perrings et al. ; d'haene ) . common practices include double-digging soils (loosening two layers of soil) to a depth of . m with annual amendments of cured compost and green manures. communities of bacteria acting synergistically can suppress plant pathogens, promoting plant growth and crop production (kinkel et al. ; mendes ; hadar and papadopoulou ; gaiero ) and regulating carbon flux (fitter et al. ) . biologically active soil amended with organic materials will also provide ecosystem services by retaining water in soil, regulating biogeochemical processes, increasing cation exchange capacity to enhance nutrient retention and plant availability and reducing material loss from the production beds (cooperband ) . environmental benefits with biointensive and similar types of agricultural production include (jeavons from to , average farm size in the usa increased from to acres, while the number of farms declined by about , . significantly, the number of small farms, those most suitable for biointensive and similar types of production ( - acres) declined nationally from , to , . the number of large farms (≥ acres) increased from , to , during the same period of time (us department of agriculture ). these trends suggest that agricultural production in the usa has the potential for decreased resilience, as farming becomes more consolidated into large acreage operations growing a small number of crops on each farm. biointensive and similar practices have the potential to greatly enhance the adaptive capacity of the food production network, increasing crop/product diversity, while shortening the supply chain from production to consumption. they also have the potential to increase productivity per unit area, while protecting and enhancing ecosystem health. through more diversified agricultural production, as described above, greater resilience can also be attained within locally based food systems and associated economies. a distributed and regional network of small-scale aggregators, processors, distributors, and markets has the capacity to make venues for sale of farm products more accessible to small farmers, especially as they diversify their crops. greater crop diversity offers opportunities for more diverse markets, especially in specialty and ethnic foods. a distributed network can foster regional job growth within the food system and enhance food access to underserved communities. a distributed network of producers, buyers, processors, and consumers that share adjacency also have more opportunity for sharing information and knowledge such as technical advice, information about buyers and markets, new regulations, market opportunities, and new innovative practices. the distributed network can also support current models of centralized markets by consolidating products from small growers into larger quantities for sale/purchase. currently, large aggregators leave little room for small producers who cannot meet minimum quantities required for sale. they also usually require a substantial supply of single, uniform crops, creating a disincentive for on-farm crop diversification. in contrast, small producers have found much of their success by selling directly to consumers through farmers markets and community supported agricultural (csa) models (low and vogel ) . among us farmers, in , nearly % of farms sold primarily through direct-to-consumer (dtc) markets, including farmer markets and csas while another % used dtc marketing in addition to other venues. the number of american farms with dtc sales increased by % and sales increased by % between and ; however, between and , the number of farms with dtc sales increased . %, with no change in total dtc sales. that dtc sales did not increase may be a reflection that consumer demand has been met (low and vogel ) . effectiveness of these direct market outlets for small farmers can be place-based and the cost of marketing can be high. additionally, most farmers benefit by having a combination of sale approaches and types of crops to market. as in other industries, market diversification in the produce business is a means to minimize risks-a diversity of market connections supports a financially sustainable farm enterprise (izumi et al. ; vogt and kaiser ; us department of agriculture ). additionally, the food system benefits by having a diversity of local and global channels for the distribution, storage, and value-added processing of food through an increased base of skilled labor. in the event of a disruption (natural disaster, economic collapse, contamination of a supply chain), there are multiple alternative channels that can supply a population's food needs, including supplies closer to communities needing food. disaster notwithstanding, the resilience of the local economy may be increased because of the geographically-linked, skilled jobs that are created: farming, logistics, marketing, storage and distribution, value-added processing, equipment maintenance, input supplier, and many other jobs that cannot be outsourced to non-local entities. in the current national food system, where food is grown and harvested several states away and aggregated by large, centralized corporations, these jobs and skills are not available locally. an increase in non-transferable, geographically dependent, skilled jobs increases the resilience of the local economy immediately. according to the the north carolina commission on workforce development, state of the north carolina workforce report ( ), rural areas of north carolina continue to lose employment opportunities, and bmiddle jobs^that supplied a family-sustaining wage for workers with little formal education are disappearing rapidly. the median family income in north carolina is $ , , substantially below the national median of $ , , with % of the population living in poverty. median incomes in rural counties are often % or more below the state median (u.s. census bureau ). central north carolina is also challenged by many communities having limited access to food. north carolina is ranked th in the nation for degree of food hardships, and greensboro, north carolina in the north carolina piedmont, has been identified as the most food-limited metropolitan area in the nation (food research and action center ) . for many small farmers in the piedmont region of north carolina, farm income is a supplemental, yet crucial, part of their household economy. in , about half of the people identified as farmers ( , individuals) in north carolina by the us department of agriculture did not farm full time. of those, , worked more than days off farm (us department of agriculture ). for many of these families, small farm operations provide much needed household income to supplement off-farm, low-wage job earnings (us department of agriculture ). small farming operations also present economic opportunities for partially-or fullyretired growers, as well as opportunities for beginning producers interested in scaling up to full-time farm businesses. when farmers utilize high-yield, low input techniques such as biointensive and other agroecological methods, farming businesses can be developed in both urban and rural areas, as smaller acreage farms (typically . - acres) are proving their viability. reduced land and equipment requirements also reduce the upfront capitalization costs for a start-up farm. one of the barriers that small farmers experience when developing a farm enterprise is identifying convenient and reasonable access to markets for the sale of their products. generally, individual small growers do not have the physical infrastructure (e.g., cold storage), product volume or market access to sell to institutions or buyers that can pay a fair wholesale price and provide a consistent market for their products. there is room for growth and diversification in the local food sector. according to the us department of agriculture, in north carolinians spend $ billion per year on food (us department of agriculture , center for environmental farming systems , a, b). although spending on local foods is difficult to track, cefs is encouraging north carolina consumers to spend at least % of their food dollars on locally produced food. the north carolina % campaign is in the third year of its promotion to bring local foods spending closer to this benchmark. at %, $ . billion would be generated for the local economy, increasing opportunities for entrepreneurial jobs, skilled employment, and healthy foods. at the same time, there is a renewed interest in local foods to foster healthier north carolinians and protect and preserve the rural landscape (curtis et al. ). there is a tremendous untapped opportunity for selling locally grown produce to indirect markets such as stores, restaurants, institutions, and distributors, particularly for small growers that cannot market their produce directly to consumers. developing a marketing network geared toward these small and micro-farmers simultaneously builds the resilience of local economies through job creation, the resilience of the food system through a diversified, decentralized network of small suppliers, and resilience in production through cultivation of diversified crops. many of the jobs that have been created since the end of the recession have been low-wage service jobs. rural areas of north carolina continue to lose jobs, while metropolitan areas slowly begin adding new jobs (forter et al. ) . this slow economic recovery is a reflection of a lack of resilience in the local economy-largely in response to changes in the manufacturing foundation of the region. fostering locally embedded sustainable, high-yield/low-input agriculture and markets for product sale has the potential to replace the former manufacturing economic foundation. potential for distribution, sale and access to locally produced foods in the north carolina piedmont region to assist local governments in fostering sustainable neighborhoods through food, a model was designed to describe a decentralized, produce aggregation network serving smalland micro-producers within the north carolina piedmont (piedmont together ; walker ). questions of economic and food system resilience were addressed by asking: what opportunities exist to build the supply and demand for local produce while engaging and involving rural and urban, low-resource, diverse communities in ways that generate individual and community wealth and security? more specifically, what sectors are not currently engaged in local food system efforts that hold potential for growing their businesses while contributing to a more robust local food system? (walker ) . the economy of the piedmont region of north carolina during the latter half of the th century-dependent largely on low-skilled manufacturing jobs in textiles, furniture, and tobacco in urban areas and tobacco production in rural areasis a prime example of low resilience, as attested by the lingering effects of the recent economic downturn. similarly, while the prevalence and growth of farmers markets across the piedmont is certainly a welcomed addition, a food system dependent on a small number of relatively high-priced, geographically distant markets with limited hours lacks the resilience possible in a food system that includes markets characterized by a high diversity of product, price, location, and methods of sale. the goal of the model designed to address the participation gaps in the local food system described above was to enhance resilient economic development through creative and sustainable diversification of employment opportunities and market types within the local foodshed through opportunities for aggregation, storage, and sale of local food products, exploiting the growing diversity of locally grown agricultural products. figure shows the model developed. dashed boxes, focusing on micro-, small-, and medium-scale aggregators, are assets that could provide opportunities for sale and distribution of local foods from small-and micro-scale producers that currently lack many opportunities for indirect sales of their products. these small-scale aggregators would fill a niche that links a diverse set of producers (part-and full-time, rural and urban) and diverse agricultural products to retail outlets that cater to populations that currently lack access to local food, such as small rural grocers, urban convenience stores, locally owned mid-priced restaurants, and city-and county-managed institutions. diversifying crops also provides opportunities for consumer access to more diverse produce and other foods, such as culturally important or desired foods. this model demonstrates one method by which resilience is increased through supplementing and integrating with the existing, larger-scale food system, not creating a parallel, stand-alone blocal^system. resilience, defined earlier as the capacity for a system to absorb disturbance and to reorganize, while retaining system function, structure, and self-organizing processes, can best be achieved within the food system by more food produced, distributed, and consumed within local geographies, while keeping the larger-even national-structures that fill gaps which cannot be filled locally or where those structures inform and support local efforts. another important concept model is illustrated in fig. , where the inner ring of the diagram shows a direct-toconsumer relationship within a typical local food system. while this smaller model is more highly favored by local food system activists who place a priority on bknowing one's farmer,^it can exclude opportunities for generating community wealth, resilience, and new jobs through the multiplier effect: increasing the number of times a dollar cycles through a community increases the economic impact of that dollar on the local economy (morgan ) . a study by the iowa state university concluded that buying local food has a multiplier effect of . - . throughout the wider local economy, depending on the rural or urban context and commodities and scale of the community economy (swenson ) . additionally, current research suggests that adding one skilled job in the tradable sector generates . jobs in local goods and services, with high potential for the non-tradable sector (moretti ) . the larger circle in fig. illustrates the inclusion of these bvalue-added^industries, including aggregators, institutions and wide variety of retail outlets. the larger circle also illustrates multiple points of entry for community members into a food production/aggregation/processing/distribution network. programs such as farm incubators can provide training and technical assistance in sustainable production techniques (e.g., biointensive, agroecology), access to on-farm resources and infrastructure, initial entry into post-harvest processing/marketing, and land access opportunities to facilitate the start of new businesses. additionally, underutilized facilities such as convenience stores, restaurants, grocery stores, and local institutions can provide the capacity for storage, post-harvest processing, and/or distribution that can serve as points of entry for people interested in starting non-farm food system enterprises. retail outlets such as restaurants, small grocers, institutions, and innovative outlets, for example mobile or pop-up markets, can widen the distribution of the local food network increasing (walker ) access for community members who would not normally be a part of a regional foodshed based only around direct farm-toconsumer marketing. currently, there are a number of projects, programs, and initiatives geared toward assisting mid-scale farmers who wish to enter mainstream markets. for example, started in , the north carolina farm to school program now facilitates sale of locally grown food to of the counties in north carolina. during the - school year, $ , , was spent purchasing fruits and vegetables grown in north carolina, which were then served in schools (north carolina department of agriculture and consumer services ). both approaches are necessary to build a resilient local food system that can absorb disturbances such as natural disasters and disruptions to the national economy and transportation network. the models illustrated in figs. and highlight the opportunities for new, small-scale, locally-based markets that can be the portal for sale of products by locallyconnected producers, thus increasing the resilience of the food system through dynamic, adaptive, and responsive enterprises. to achieve this increase in locally sourced food through decentralized, networked distribution channels, three main issues must be addressed. ) many of the resources for the promotion of local foods development over the past decade have gone to supporting mid-and larger-scale farms within north carolina. large foundations and universities have focused on helping farmers who were affected by the tobacco buyout program transition to other viable crops, as well as the renewed public health focus on obesity, diabetes, and stroke prevention. this led to much public support for getting fresh, local foods into a variety of markets and food access programs. working with larger farmers and distributors has made a difference in the prevalence of local foods available to consumers. however, smallscale farmers are often not able to sell to these markets. for example, during the - school year, the north carolina farm to school program purchased strawberries from only farms and sweet potatoes from farms. for comparison, there are north carolina farms of - acres identified in the us department of agriculture agricultural census, who are also potential suppliers for this program (north carolina department of agriculture and consumer services ; us department of agriculture ). in order to engender greater resilience of the local food system, more attention needs to be paid to the contribution potential for smalland micro-scale farms. ) nationally, the local food movement has often been hegemonic in its lack of inclusion of minority participantsbe they underrepresented racial, ethnic, class, or genders (hinrichs ; jarosz ) . greater attention paid to the barriers for participation by individuals, businesses, and communities that are not currently part of the local food movement will help in creating greater equity of food access, economic opportunity, and environmental health (holley ) . it will also expand the range of products available for production, processing, and purchase, increasing both social and economic resilience within the local network. in the same way that a diversity of scale and market type will strengthen the ecosystem of the local food system in the piedmont region, greater involvement from a diversity of stakeholders throughout the system will engender more dynamism and resilience (page ; holley ) . expanding and diversifying the community of growers and non-farm entrepreneurs will also expand markets and market access to parts of the community now underserved by a centralized food marketing network. ) for many small farmers, resources and equipment that would facilitate sale to a network are not available. for example, the cost of an × ′ cold storage unit is typically $ -$ , . for many farmers who have substantial capital, off-farm income, or access to credit, this is attainable. however, need for capital is a barrier for most low-resource farmers. the local food models outlined above for central north carolina's small and mid-sized farmers and aggregators can be described in a set of five bdesign guidelines^that increase economic, social, and agricultural resilience. these guidelines are intended as a beginning resource for people interested in starting or adding to some aspect of the decentralized aggregation and cold storage local food network. they are suggestions that can be utilized by decision makers, designers, and planners that may be working on other community-wide issues such as transportation, housing, and economic development. having design guidelines that summarize a model of diversification and resilience based on food system localization allows for the inclusion of ideas generated through academic research into practical, applied community practice. guideline # : promote networks and nodes intent: develop a regional food system that utilizes complexity (a system of networks and nodes) as a mechanism for achieving greater economic and food system resiliency. a. look for opportunities to situate aggregation and storage facilities throughout the region and close to production farms, while encouraging strong interconnectedness throughout. b. cluster small aggregation and storage facilities near one another, forming bnodes^of the local food supply chain. fifty miles has been defined as a maximum distance between aggregation facilities in rural north carolina (bruno and hossfeld ) . facilities may be closer in the more urban piedmont. guideline # : engender equity and inclusivity intent: build on the strengths of the piedmont triad region by integrating cross-barrier collaborations into the aggregation or cold storage business initiative. a. understand the political geography of your community. while doing business in a new area may present challenges, new business opportunities, collaborators, and markets also await. b. build new lines of community rapport across racial, economic, class, and language boundaries. c. work to promote access to financing and entrepreneurial assistance for diverse communities that are specific to current trends in food system development. guideline # : plan for appropriate transportation options intent: ensure that the cold storage chain of all produce is maintained from farm to fork and that handling is appropriately documented. each segment of the supply chain should work in tandem with other segments. a. ensure that there is access to a refrigerated truck or trailer to maintain the cold chain. b. know the acceptable upper and lower temperature and humidity limits of the produce being aggregated. c. manage traceability of products. keep all appropriate records regarding the place of origin, place of sale, and storage notes for all produce that moves through the aggregation facility. guideline # : design an effective management plan intent: design a management structure and plan that addresses the goals and limitations of your aggregation or cold storage business, while adhering to all regulatory requirements. a. choose a management structure that fits with your business. a simple cold storage unit leased to several farmers who are marketing their own produce to aggregators will still require facility management, rent collection, and marketing to new farmers. b. ensure that your facility is compliant with all regulatory requirements, including appropriate recordkeeping. track and document temperature and humidity through the duration of the entire supply chain. c. develop procedures that allow access to storage space by clients when needed and ensure that everyone who enters keeps appropriate logs for food safety requirements. guideline # : build appropriately-sized cold storage facilities intent: invest in infrastructure wisely. cold storage units can be re-configured, and their temperature and humidity changed with relative ease if planned for up front. however, there is an economy of scale, even when working exclusively with small-scale farmers and buyers. develop a business plan that helps to determine what and when your enterprise should build. a. estimate how much cold storage space you need based on the amount of produce your suppliers will need to store. b. approximate energy costs ahead of time and investigate alternative energy as a way to lower the utility bill. c. know what the optimal temperature and humidity settings are for each produce type you will store and aggregate. d. determine what storage system you will use within the cold storage units based on the farmers and buyers you are working with. some smaller growers who are storing their own produce to sell at a local farmers' market may prefer industrial shelves where they can store produce boxes, while larger growers may prefer palettes and use palette jacks to move their product around. also, there is a need to consider the storage of certified organic and gap-certified produce, which need to remain segregated from non-certified foods in order to maintain their certification. the distributed network of aggregation and distribution centers proposed for the piedmont of north carolina provides an integrated, regional model, reducing transport needs for farmers and expanding distribution, sale, and access. a south carolina food hub has been successful in making local foods more available, providing an aggregation/distribution/service network to benefit farmers and consumers. growfood carolina is presented below as a case study of a food hub that is helping the charleston food system move toward a fledgling level of resilience. growfood carolina provides a model for one of the nodes in fig. -an aggregation/distribution hub that connects small-scale growers to consumers in the same region. it also has the potential to serve as a central hub networked to smaller aggregation and value-added processing centers throughout the region, like the spokes of a wheel. charleston, south carolina is the center of one of the southeast's real estate development booms, aided by an influx of retirees moving to the area due to its climate and the availability of still-relatively affordable land. charleston county has grown from , in to almost , in , a . % increase (us census ). a corollary to the above growth and international recognition for charleston's reputation is the recent development of a vibrant, local food culture. green grocer farms on wadmalaw island, located outside of charleston is representative of this growing local food culture. in the s, the farm grew a variety of row crops under regional organic certification, which were sold in savannah and atlanta. now, years later, pastured eggs and pastured raw milk are produced, and the farm is unable to keep up with local demand for their products. the growth in demand for local foods is also seen at the charleston farmers market. ten years ago there were approximately five farmers; now there are over twenty-six farms selling a variety of local and regional products, including meat, eggs, milk and cheese, vegetables, flowers, shrimp, rice and fruit. one other area of growth is in the local restaurant industry, where almost every local restaurant advertises that they support local farmers and purchase local food, with one nationally recognized restaurant, husk, advertising a strictly regional menu. growfood carolina-a regional food hub growfood carolina is a working example of how a food hub contributes to resilience in the lowcountry of south carolina. the us department of agriculture defines a food hub as ba centrally located facility with a business management structure facilitating the aggregation, storage, processing, distribution, and/or marketing of locally/regionally produced food products^(us department of agriculture ). food hubs have operational services which can range from aggregation to distribution and sale, including branding, promotion, packaging, and product storage. they can also assist with producer services, ranging from transportation, training, business guidance, value-added product development, and providing liability insurance, to an often key service, linking buyers with producers. food hubs can also help with the promotion of community/social missions, such as distribution of products to food deserts, raising awareness about benefits of supporting local agricultural businesses, providing opportunities to community youth, offering composting and other forms of regionally-appropriate farming/agricultural workshops. growfood carolina is a subsidiary of south carolina's leading environmental advocacy group, coastal league (ccl), started in with the mission to bprotect the natural environment of the south carolina coastal plain and to enhance the quality of life in our communities by working with individuals, businesses, and government to ensure balanced solutions( coastal conservation league ). to realize this mission, in , ccl started two bin-house^policy and advocacy groups to help protect the coastal corridor of south carolina. one group focuses on climate change and energy, while the other group focuses on sustainable agriculture. the ccl sustainable agriculture committee helps ccl's overall strategy of land conservation: if farming is not economically viable, then small and mid-sized farmers will be squeezed out of the market with subsequent changes in land use. thus, ccl actively supports local, sustainable agriculture. to support their mission, growfood was created to provide infrastructural help to regional small-and mid-sized farmers. in , a -ft warehouse in downtown charleston was donated to ccl. after renovating it and hiring a general manager, growfood officially opened for business in . the warehouse contains ft of refrigerated space. although not climate controlled, the building meets leadership in energy and environmental design (leed) standards. growfood also owns a -ft refrigerated truck purchased with funds from a us department of agriculture grant and has an urban garden demonstration plot on site where they host various workshops and school groups. the warehouse is located directly off interstate i- leading inland to columbia, sc, and is close to hwy , a north/south artery. this location is strategic, allowing growfood to meet its mission as a food hub, consolidating production and distribution within a -mile radius. this radius allows growfood to work with farmers growing produce from georgia to north carolina, and ccl has plans to open a second hub in greenville, south carolina that will service the upstate, mountain region of south carolina. farms within the -mile radius contact the growfood general manager, who visits the farm for bfull transparency. restaurants and retail stores, and growfood either delivers the produce, or retail customers come to purchase it from the warehouse. after a sale, % of the purchase price returns to the grower, and % remains with growfood to help cover operating costs. growfood does all sales, marketing, and distribution via a two-person marketing team, and they work with over local businesses, distributing days per week. this requires five fulltime staff and two part-time drivers. in , they had five growers and customers, and as of , they have growers with regular restaurant and retail customers. currently, demand far outstrips supply, demonstrating demand for additional local foods and food hub services in the south carolina foodshed. with farms from to acres in size in south carolina, clearly, there is additional production and distribution capacity (us department of agriculure ). the goal of the ccl staff is to expand both growfood as well as the local farming community, helping them to diversify, increase production and transition to organic production. therefore, besides retail distribution, growfood is an advocate for small-scale, sustainable farming. south carolina currently has certified organic growers, compared to over in north carolina. to expand the number of sc organic growers, ccl does outreach and education about the benefits of sustainable agriculture, focusing especially on the next generation of farmers. the average age of farmers in south carolina is . there are currently not enough young farmers to supply current demand, let alone create new farms that can help meet growing regional food needs. but this also presents an entrepreneurial opportunity for increasing the resilience of the regional food system through creation of new, small, sustainable farms if land, training, and resources for start-up business are made available. dirt works incubator farm on johns island outside of charleston is working to support development of new farmers starting their new farm businesses in the area. growfood as a food hub is helping the community in three important areas. & bthe environment.^use of sustainable production techniques that improves the soil with each cropping cycle and use of the food near the production farms, reducing transportation impacts & bdiversify local agriculture.^smaller farms have the adaptive capacity for increased diversity of crops in production both on-farm and between farms, also increasing nutritional and preference opportunities for consumers. & brural economic development.^as the manager of growfood stated, bcash flow is the most important thing for a grower.^this cash flow allows farmers to be economically solvent, allowing them to continue farming. median income in south carolina is $ , , well below the national average, with % living in poverty. despite the ready support of local restaurants, it is recognized the client base needs to expand, so further goals are to sell to local schools, hospitals, and retail supermarkets, and to advocate for a year-round farmers' market. lastly, their larger goal is to have % of charleston's food come from within the -mile radius of the food hub by . if successful, charleston and the lowcountry will demonstrate a powerful move toward food resilience as a disturbance response. thus, ccl's goal is to develop an aggregated food hub network in the state, as is proposed for the north carolina piedmont in the previous section, while working on education and training, advocating for policy shifts at the state level, and creating food network market resilience. food hubs can potentially add redundancy and resilience at regional scales and across industries (production, distribution, consumption) and thus act as catalysts fostering reorganization of regional food networks. as anyone who has farmed knows, if a farm cannot make money, then there will be no farm. food hubs can become key nodes in food networks, and because they interact with farmers and retailers on a daily basis, they can serve as a platform to quickly disseminate information, creating synergies between and among the diverse members of a food system. rapid information transfer throughout the food network strengthens adaptive capacity for growers, processors, and distributors, allowing rapid response to changes in market and/or environmental conditions. however, a note of caution: food hubs may not be appropriate in all places, and local production may not fill all food needs. even growfood is years away, at best, from helping create a critical mass of local farms that can provide even a small percentage of the lowcountry's daily food needs. the food hub model in south carolina and the proposed model for north carolina may also be appropriate for other regions, extending the capacity of the regional food networks with strengthened resilience. it is an open question as to whether our global food system contains the capacity to respond to a variety of disturbances, which may threaten the integrity of system function and structure and the ability to provide needed food to all people. by definition, if such structure and function were to collapse and be unable to reorganize, such a food system lacks resilience, with potentially severe human consequences. however, adaptive capacity and adaptive management acknowledge that a system being managed will always change, so humans can respond by adjusting the system in response to disturbance and changing conditions (gunderson (gunderson , (gunderson , . in the case of agriculture and food security, resilience can be considered from the perspective of food availability with a return to a predictable, dependable, safe supply following disturbance. this issue of the journal of environmental studies and science grapples with diverse perspectives of this challenge (marten and atalan-helicke ) . while appearing to make agricultural production more consistent and predictable, genetic uniformity also has the potential to increase risk from pests, pathogens, and other disturbances, as highlighted earlier in this paper. in contrast, high biological diversity has the capacity to decrease risk with genetic diversity within a single crop and through distribution of risk between a variety of products being grown for harvest and sale. these insights should help us think more clearly about what are long-term goals for a truly resilient food system. small-scale, locally-managed farm operations using agroecological techniques such as biointensive agriculture have the adaptive capacity for increasing biological and ecological diversity both on and between farms, increasing resilience through the ability to rapidly respond to changing environmental and market conditions. highly managed, low acreage production systems such as the biointensive technique have shown greater yields per unit area than conventional production systems reliant on larger acreage. these highly managed systems also result in more efficient water use, reduced pest/pathogen pressures, and protection of adjacent environments. interspersing small farms with large farms and returning some lands to a non-farmed condition to provide localized ecosystem services has the potential to engender social, economic, and environmental resilience through regionally-enhanced, responsive, and more highly-integrated food networks. development of small, diverse farming operations can help communities respond to chronic economic stress by providing supplemental income for some growers and opportunities to build larger, full-time farm businesses for others. high-yield, low acreage techniques can also make farming accessible to a diverse community of people who had not been involved with agriculture, such as urban residents, minorities, and women. however, one barrier to small farmers is often inconvenient access to markets for sale of their products. to have a resilient food system, you have to make it economically viable to farm. therefore, it is crucial to link the shift in food production systems with the shift in food distribution networks. there is a growing emphasis for the need for new, locally based markets that can be the portal for sale of products by small-scale, locally connected producers. indeed, several regional food hubs and networks have emerged in the usa that aim to close the gap between producers and consumers. regional food network and hub models can increase opportunities for direct consumer purchase, or they can serve as aggregation centers for food to be passed into a centralized distribution network, connecting with large volume sellers such as grocery stores, processing facilities, and wholesale distributors. a food system that is predicated upon supporting and cultivating farmers of all scales, who utilize various sustainable production methods for a diversity of crops, and have a variety of market outlets will inevitably be stronger, more resilient, and more adaptable to change than a system that only responds to one scale or type of farmer or one type of market. using biological and agricultural diversity to expand locally based, sustainable farming systems, foster new farmers and food entrepreneurs, and build distributed aggregation, processing and marketing networks that focus on triple bottom line benefits-environmental, social, and economic-have the potential to strengthen our food security and our 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agriculture, agricultural marketing service, north carolina: population profile, transportation and marketing getting to scale with regional food hubs regulatory definitions of large cafos, medium cafos and small cafos. access may still a time to act: a review of institutional marketing of regionally-grown food planning for a networked produce storage and aggregation system for the piedmont region plant diversity in tropical forests: a review of mechanisms of species coexistence key: cord- - zy unz authors: long, jason; wright, edward; molesti, eleonora; temperton, nigel; barclay, wendy title: antiviral therapies against ebola and other emerging viral diseases using existing medicines that block virus entry date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: zy unz emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. the former was the only available intervention when the current unprecedented ebolavirus (ebov) outbreak in west africa began. prior to this, the development of ebov vaccines and anti-viral therapies required time and resources that were not available. therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of ebov and could be used immediately. here we test three such medicines and measure their ability to inhibit pseudotype viruses (pvs) of two ebov species, marburg virus (marv) and avian influenza h (flu-h ). we confirm the ability of chloroquine (cq) to inhibit viral entry in a ph specific manner. the commonly used proton pump inhibitors, omeprazole and esomeprazole were also able to inhibit entry of all pvs tested but at higher drug concentrations than may be achieved in vivo. we propose cq as a priority candidate to consider for treatment of ebov. past outbreaks have been of limited size affecting a local population, however a strain of ebov-z is the causative agent of the current outbreak that began in late and has since become an unprecedented and devastating epidemic , , resulting in over , suspected cases, of which those confirmed had a case fatality rate of around % in the afflicted west african countries (http://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary- ?lang=en and http://www.who.int/csr/disease/ebola/ situation-reports/en/). towards the end of the trend in case numbers reversed in liberia and the epidemic slowed in sierra leone and guinea, but the virus continues to transit in new geographical areas . this epidemic has triggered a significant global health response relying on primary hygiene and other containment measures that have proved successful in limiting the spread of the virus in previous outbreaks. given the scale of this outbreak and the fear that traditional containment measures may fail to prevent global spread, several vaccines have been fast-tracked into phase i clinical trials - although even if proved efficacious, the limited supply of sufficient quantities of vaccine will hinder their use in the current situation. for disease treatment, patients suffering a haemorrhagic fever have relied on the clinical management of symptoms (http://www.cdc.gov/vhf/ebola/treatment/). with a handful of patients in this outbreak receiving experimental therapies such as zmapp, tkm-ebola, brincidofovir and favipiravir (http://www.nature.com/news/ebola-trials-to-start-in-december- . ) - . alternatively antibody treatment by transfusion therapy using blood or plasma from ebola virus survivors has been approved , - ; although issues with safety and lack of resources for this method limit its suitability in west africa today. having no approved or widely available therapeutics for ebov, as with many other emerging viral diseases, focus has turned to possible re-purposing of drugs already licensed for other uses by the ema and fda. several clinically approved drugs have been identified by researchers - , including amiodarone, one of the several cationic amphiphiles found to inhibit filovirus entry which is currently being trialled in sierra leone . however reservations have been expressed about the complications that could be caused by side effects of the drug in ebov patients. the anti-malarial drug chloroquine (cq) has also been shown to inhibit ebov entry and protected mice from ebov infection , and has been previously highlighted as a possible drug to treat ebov infection . the possible difficulties that may arise with use of re-purposed drugs include unforeseen interactions between virus/drug and host causing exacerbation of disease. therefore it is important to try and understand the mechanism of virus inhibition by such drugs. to this end we re-examined the anti-viral properties of cq, and show here that it inhibited the ph-dependent endosomal entry of a pseudotyped virus (pv) bearing ebov glycoproteins, in the same way as did the potent and specific vacuolar-atpase (vatpase) inhibitor bafilyomycin a (bafa ) (a non-medical laboratory compound). we also show that licensed and widely used proton pump inhibitors (ppis) for treatment of gastric acid reflux, omeprazole (om) and esomeprazole (esom), inhibited pv ebov entry, likely by their off-target inhibitory activity on endosomal vatpase. human embryonic kidney ( t/ ) (atcc) and human lung adenocarcinoma epithelial cells (a ) (attc) were maintained in dulbecco's modified eagle's medium (dmem; invitrogen) supplemented with % fetal calf serum (fcs) (biosera) and % penicillinstreptomycin (ps) (invitrogen). the cell lines were maintained at °c in a % co atmosphere. chloroquine diphosphate salt (cq), bafilomycin a from streptomyces griseus (bafa ), omeprazole (om) and esomeprazole magnesium hydrate (esom) (sigma) were resuspended as per manufacturer's instructions and aliquots stored at - °c: cq was prepared in sterile dh o; bafa , om and esom were prepared in sterile dmso (sigma). the bundibugyo ebolavirus (ebov-b) envelope glycoprotein (gp) (fj ) coding sequence was synthesised (bio basic inc.) and the ha glycoprotein of avian influenza a/turkey/england/ - / (h n ) (flu-h ) was amplified from the ha plasmid of the h n reverse genetics system . both were sub-cloned into the pcaggs expression vector. expression vectors containing the envelope glycoproteins of zaire ebolavirus (mayinga) (ebov_z), marburg virus (lake victoria isolate; marv) and gibbon ape leukemia virus (galv) (modified to contain the trans-membrane domain of amphotropic murine leukemia virus (a-mlv) envelope glycoprotein) are described previously , . the renilla luciferase gene was sub-cloned into pcaggs expressing vector from a minigenome reporter described previously . the generation of all lentiviral pseudotype viruses was based on the methods detailed previously [ ] [ ] [ ] . briefly, t/ cells were seeded into cm tissue culture plates (nunc™ thermo scientific). the hiv gag-pol plasmid, pcmv-Δ . and the firefly luciferase reporter construct, pcsflw, were transfected together with either influenza ha, galv, ebov or marburg gp expression constructs at a ratio of : . : (core:reporter:envelope) using fugene transfection reagent (promega). at h post-transfection, cells were washed and fresh media applied. for the generation of h the units for chloroquine in table have been corrected from nm to μm. pvs, u exogenous recombinant neuraminidase from clostridium perfringens (sigma-aldrich) was also added h after transfection to effect egress from the producer cells. pv supernatants were harvested at and h post-transfection and passed through a . m pore filter. ebov pvs were aliquoted and stored at °c; the remaining pvs were stored at - °c. entry inhibition assay t cells in cm plates were transfected with ug of renilla luciferase expressing plasmid using lipofectamine according to manufacturer's instructions (life technologies™). cq, bafa , om and esom were serially diluted in -well white-bottomed plates (nunc™ thermo scientific) to give the final described concentrations. after h the transfected cells were trypsinised and × cells were added to each well. after min cells were transduced with no more than × rlu of pv per well (estimated from raw rlu values of previously infected t cells), and to an equal volume per well. h later supernatant was removed and cells were lysed with µl of passive lysis buffer (promega), and firefly/renilla luciferase activity measured using a fluostar omega plate reader (bmg labtech) and the dual luciferase assay system (promega). a cells were pre-treated with drug h before nm of the ph sensitive lysotracker ® red dnd- (life technologies™) was added to the media of each well . after minutes in growth conditions, cells were analyzed for fluorescence using an axiovert confocal laser (cfl) microscope and an axiocam mrc camera (carl zeiss). statistical analysis pv transduction rlus were normalised to the renilla value in the corresponding wells. percent infection of each drug dilution was calculated compared to untreated cells. two-way anova with bonferroni's multiple comparisons test between untreated and treated mean values (α- . ) was performed to measure statistically significant differences. ic values were calculated using nonlinear regression analysis (log[inhibitor] vs normalised response). all manipulation of data was performed on graphpad prism (graphpad software). the envelope glycoproteins of several emerging viruses with high pathogenicity and pandemic potential were used to create lentiviral based pseudotype particles as previously described . pvs were generated bearing the envelope glycoproteins from zaire ebolavirus (mayinga strain) (ebov-z), bundibugyo ebolavirus (ebov-b), marburg (lake victoria isolate) virus (marv), h ha from a highly pathogenic avian influenza virus a/turkey/ england/ - / (h n ) (flu-h ), and gibbon ape leukaemia virus (galv). galv pvs were included because galv is a virus that does not require acidification of endosomes for its entry into cells. all the pvs generated were shown to transduce t cells and firefly luciferase expression from the packaged reporter gene was measured above mock infected cells (non-transduced cells) (dataset ). in order to assess the ability of cq, bafa , om and esom to inhibit pv entry, drugs were serially diluted in triplicate in white bottomed -well plates. next, t cells transfected hours previously with a renilla luciferase expression plasmid to allow monitoring of cell viability, were added to each well. appropriately diluted pvs were then added to each dilution, including a no-drug control. after hours incubation, the supernatant was removed and firefly and renilla luciferase rlus were recorded using the dual luciferase assay system (promega). pv rlus were normalised to the corresponding renilla values, which reduced the edge effect observed in the -well plates, and controlled for toxicity of the drugs. only bafa appeared to reduce expression of renilla at the highest concentrations, suggesting cellular toxicity, (dataset ) and visible cytopathic effect was not observed in cells treated by cq, om and esom at the concentrations used in figure . both bafa and cq reduced ebov-z, ebov-b, marv and flu-h entry in a dose dependent manner ( figure a and b). the ic value of bafa was in the nm range for ebov-z, ebov-b, flu-h and marv and inhibition of entry was statistically significant at the nm concentration compared to the untreated control (table ) . cq inhibited ebov-z, ebov-b, marv and flu-h with ic of . , . , . and . µm respectively, and inhibition was statistically significant (table ). in contrast, galv entry was augmented by both bafa and cq above that of the untreated cells to a maximum of . % ( . nm) and . % ( . µm) respectively. both om and esom reduced entry of all pvs tested at µm but galv pv was the least affected ( figure c and d). inhibition of entry for ebov-z, ebov-b, marv and flu-h pvs by esom was significant at µm, and galv pv was not significantly inhibited at this dose ( figure d and table ). increasing endosome ph as a mechanism of inhibiting virus ph-dependent entry bafa and cq are known endosomal acidification inhibitors (bafa being a potent and specific vatpase inhibitor and cq a licensed lysotropic agent) . the effects of om and esom on endosomal acidification have also been previously reported , . to confirm that endosomal ph was being affected at doses used here, a cells were treated with drug for hour before applying lysotracker ® red dnd- (lifetechnologies). a cells were chosen here because t cells are poorly imaged due to their morphology. the lysotracker probe specifically fluoresces in acidic organelles. fluorescence was decreased in cells treated with bafa and cq in a dose dependant manner, but was unaffected in cells treated with vehicle alone (figure ). om and esom appeared to decrease fluorescence, and therefore increase endosomal ph, only at a concentration of µm, higher than that required to inhibit pv entry. moreover cellular toxicity was observed at this concentration after hours. table . after attachment to cells, viruses require a mechanism of fusion to deliver the viral genome. preventing this action by fusion inhibitors has been successful approach for hiv antiviral therapy . unlike hiv, ebov and many other viruses are dependent on the naturally low ph of acidic endosomes to activate and trigger fusion by their envelope glycoproteins. in this instance, a 'fusion inhibitor' could target the host cell machinery preventing acidification of the endosome, working to inhibit virus entry of several different viruses. here we have reiterated that cell entry by pvs representing ebov, flu-h and marv can be inhibited by increasing the endosome ph using bafa and cq (figure ) , and this correlates with their ability to prevent the acidification of intracellular organelles (figure ). cq has shown antiviral activity against several viruses in vitro, including ebov, influenza, nipah, hendra, dengue and chikv - . disappointingly, this antiviral activity has not always translated into efficacy in vivo models or clinical trials, although cq was effective in a mouse model against ebov , , [ ] [ ] [ ] [ ] [ ] . the variability in in vivo results may depend on study design and strains of virus used. in one study bafa treated mice were not protected from influenza infection but treatment with a related compound, saliphe, was protective, even though both drugs were potent in vitro . inhibition of endosome acidification as a target for inhibiting ebov can be justified by the knowledge that the filoviruses depend on the low ph for two separate steps of their entry pathway. not only is the fusion by g protein triggered by low ph, but its cleavage into a fusogenic form is carried out by endosomal enzymes cathepsins b and l whose activation is also ph dependent . some have argued that g protein cleavage by cathepsin is less essential than previously thought , and that ebov species other than zaire together with closely related marv do not require cathepsin cleavage for entry , . nonetheless, entry of marv pvs was still inhibited in our assays suggesting that inhibiting fusion alone is sufficient. recently, using computational modelling, ekins et al. suggested the anti-ebov mechanism of cq may be by binding the vp protein of ebov . if this drug had activity on several steps of the replication cycle it may not only be more effective in vivo but it may be even less likely that the virus could mutate to escape inhibition. at first we were surprised that cq actually increased entry of galv pv (figure ). however this effect has been noted before for other retroviruses, including a-mlv and hiv- , and is accounted for by the inhibitory effect of cq on the autophagy pathway. cq prevents degradation of phagosomes that contain virus particles and prevents them from otherwise being degraded [ ] [ ] [ ] . cq has been used for many years as an anti-malarial drug, although it is now only effective in parts of central america and the caribbean due to accumulation of drug resistance by the plasmodium parasite . interestingly, compounds belonging to the omeprazole family have also been described as having anti-malarial properties in vitro, possibly via their reported ability to target vatpase in the plasma membrane of plasmodium parasite . soon after its discovery om was found to also inhibit intracellular vatpase at µm concentrations as opposed to its licensed target of gastric h+/ k+-atpase against which it is effective at much lower concentrations , . indeed there are a plethora of publications indicating use of om and esom in cancer therapy, as a means to inhibit the characteristic acidic intracellular environment, and thus permit sensitivity to cytotoxic therapies [ ] [ ] [ ] [ ] [ ] . a role of om and esom has also been noted in the suppression of bone resorption, another physiological process dependent on ph [ ] [ ] [ ] . given the volume of research suggesting these off target effects depend on an ability to affect intracellular ph, we hypothesised that these drugs would, like cq and bafa , inhibit ebov, marv and influenza virus ph dependent entry. we used galv as a control again since its entry is reportedly independent of ph. indeed, ebov, flu-h and marv were inhibited by lower doses of om or esom than galv ( figure and table ). galv entry was also inhibited at the highest concentration, but we cannot exclude that this was due to a toxic effect that was not measured by the renilla control we employed here. we did not observe as close a correlation between drug doses that mediated the inhibition of ebov or influenza pv entry and increase in ph of intracellular vesicles for om and esom as for cq and bafa, (figure and figure ). more recently, it has been reported that om and esom altered the localisation of vatpase in the cell as well as the ph of intracellular vesicles and this may explain their ability to inhibit pv entry more potently than the ph changes we observed would suggest. inhibition of influenza virus entry to cells by means of inhibiting acidification of endosomes has been known for decades , although no current antivirals for influenza have been licensed on this basis. some epidemiological evidence from population studies suggests that om could exert a protective effect against influenza-likeillness , but our studies suggest that doses required for potent inhibition might be difficult to achieve without significant toxicity. despite these drugs being readily available, even without prescription in some countries, the licensed dosing would generate a plasma concentration reportedly . - . µm for esom that falls short of the ic calculated in this study, although higher doses have been used clinically . therefore it seems unlikely that om and esom would be a suitable therapy for ebolavirus infection, but more specifically designed vatpase inhibitors may have potential as broad acting antivirals against several emerging viruses in the future. with regard to cq, the evidence suggests a more promising position for use against ebolavirus. standard adult dosing ( mg/kg) achieves plasma concentration of µm, close to our ic value against ebov pv entry. protection in the mouse model was previously shown with a mg/kg dosage , . using re-purposed drugs to treat outbreaks of emerging diseases must surely be approached with caution. in ebola patients with severe life-threatening disease it would be important to ensure that any side effects of a therapy did not enhance disease progression, particularly if higher doses of re-purposed drugs, as suggested here, were considered. on the other hand, cq has been taken prophylactically in a tropical setting for many years to prevent malaria and we suggest that, with little additional need for scale up of production of a new agent, this might represent a useful adjunct to the current antiviral strategies being trialled in west africa. we envisage that in contacts of ebov cases, cq might decrease the viral load that establishes in the early days after virus transmission. further work in in vivo models including guinea pig and primates should inform about doses and administration regimens. author contributions dr jason long, dr edward wright and dr eleonora molesti generated the pvs. jason long performed the drug entry assay and ph assay. this work was planned by prof wendy barclay, dr nigel temperton and dr jason long. all authors were involved in preparing and revising the manuscript. evolutionary history of ebola virus entry mediated by the ebola virus glycoprotein pubmed abstract | publisher full text | free full text cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change pubmed abstract | publisher full text | free full text cathepsin b & l are not required for ebola virus replication pubmed abstract | publisher full text | free full text cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss expression a common feature pharmacophore for fdaapproved drugs inhibiting the ebola virus toll-like receptor binds single-stranded cpg-dna in a sequence- and ph-dependent manner tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production autophagy in health and disease: a double-edged sword pubmed abstract | publisher full text | free full text ph regulation in the intracellular malaria parasite, plasmodium falciparum. h( + ) extrusion via a v-type h( + )-atpase effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs proton pump inhibitors may reduce tumour resistance v-atpase as an effective therapeutic target for sarcomas v-atpase is a candidate therapeutic target for ewing sarcoma proton pump inhibitors induce apoptosis of human b-cell tumors through a caspase-independent mechanism involving reactive oxygen species effect of omeprazole, an inhibitor of h + ,k( + )-atpase, on bone resorption in humans omeprazole, a specific inhibitor of h + -k + -atpase, inhibits bone resorption in vitro weiss division of infection and immunity the authors wish to thank caroline goujon for the kind provision of the galv construct and olivier moncorgé for generating the renilla expression plasmid. no competing interests were disclosed. this work was supported by the grants flupig eu fp and bbsrc bb/k / . this is an elegant study employing envelope pseudotypes of highly pathogenic viruses which demonstrates that certain inhibitors of low endosomal ph can inhibit viral entry. because some of these molecules such as chloroquine have been in clinical use for decades, and are inexpensive, they might tip the balance between survival and death during human infection.i have no criticism of the experimental work. however, i have been told by a reliable physician who has recently cared for patients with ebola infection that treatment with chloroquine offered no clinical benefit. thus it is possible that an observation may not translate into a useful treatment . so one in vitro in vivo should be wary about the conclusions. no competing interests were disclosed. the work was of particular interest especially in light of several viruses shown to be taken up in this rather non specific way. i would have perhaps also like some discussion of receptor and channel mediated virus uptake -there are several publications in this space. the interplay between such different mechanisms may point to multiple targets or need for combined approaches to block them.the authors describe amiodarone, but there are many molecules that have been found as ebola replication or pseudoviral entry inhibitors, had they looked at more molecules to see if the ph mechanism was common across them? i would likely suggest adding repurposing in the title of the article.the conclusion might benefit from comparison of the chloroquine data with that previously published the conclusion might benefit from comparison of the chloroquine data with that previously published (higher ec ), potential ocular toxicity etc.some discussion as to whether the ph effect is an in vitro specific effect or something of in vivo relevance -would also be worth mention.this study confirms the previous work on chloroquine and suggestions by others as to its potential utility. this begs the question why it is not used clinically. what other data would be needed to show that chloroquine could be clinically useful?the study is well designed and reported and adds to the growing literature on chloroquine and its potential as an antiviral.i have read this submission. i believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.no competing interests were disclosed. competing interests: key: cord- -do m authors: gyawali, rabin; minor, radiah c.; donovan, barry; ibrahim, salam a. title: inclusion of oat in feeding can increase the potential probiotic bifidobacteria in sow milk date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: do m simple summary: in this study we isolated and characterized potential probiotic bifidobacteria from sow milk. the bifidobacterial population in milk has been attributed to the existence of prebiotic oat in feeding systems. since breast feeding protects the newborns against several infectious diseases, milk from sows fed with oat could improve the health of piglets. abstract: the objectives of this study were to (i) investigate the impact of feeding oat on the population of bifidobacteria and (ii) evaluate their probiotic potential. in this study, we investigated the effects of supplementing sows’ gestation and lactation feed with % oat (prebiotic source) on the levels of probiotic population in milk. we found that dietary inclusion of oat during lactation and gestation resulted in increased levels of bifidobacteria compared to lactobacilli in sow milk. furthermore bifidobacteria within the sow milk samples were further evaluated for probiotic potential based on aggregating properties, and acid- and bile-tolerance after exposure to hydrochloric acid (ph . ) and bile salts ( %, . %, . %, . % and . %). all isolates survived under the condition of low ph and bile . %. autoaggregation ability ranged from . % to %. these isolates also showed antimicrobial activity against e. coli o :h . together our results suggest that inclusion of oat in feeding systems could have the potential to improve the intestinal health of piglets by increasing the population of bifidobacteria. maintaining healthy hogs and increasing production is important to the pork industry, consumers, and the economy. many strategies are employed by the pork industry to increase production. one common strategy involves weaning piglets at approximately days of age in order to allow for more piglet litters per sow each year [ ] . while weaning piglets at early ages shortens the time-period when the sows are out of production, performance of the piglets can be negatively affected. weaning piglets early is challenging because their digestive and immune systems are not yet fully mature [ ] . consequently, piglets are more susceptible to infections and gastrointestinal (gi) issues. one gi disease that piglets are prone to develop, particularly when they are weaned before four weeks of age, is post weaning diarrhea (pwd). pwd leads to anorexia, growth inhibition, and death of piglets and therefore is an important issue with economic consequences to the swine industry. to avoid post-weaning diarrhea, antibiotic growth promoters have been included in piglet diets; however, antibiotic growth promoters are potentially linked to the emergence of antibiotic resistant bacteria [ ] . therefore, alternatives to antibiotic growth promoters that maintain piglet health and boost immunity are being sought [ ] . in swine, nutrients consumed during gestation and lactation has been shown to be key to the viability and health of offspring. alexopoulos et al. [ ] reported that supplementation of sow diets with probiotics during gestation and lactation has been shown to lead to increased body weights and reduced incidences of diarrhea in their offspring. to promote the health of weaned pigs and protect against pwd, it is important that the gut be colonized by beneficial bacteria and that strong intestinal/mucosal immunity develops. beneficial bacteria, like bifidobacteria and lactobacilli can stimulate the immune system, inhibit the growth of pathogens, and reduce the incidence of diarrhea and constipation in pigs [ , ] . research has shown that a mother's diet has the potential to positively affect the overall health of her offspring and contribute to development of their offspring's immune systems. for example, breastfed infants of mothers who consumed probiotics had fewer gastrointestinal issues such as diarrhea [ ] . in this study, we sought to evaluate whether supplementation of feeding system with oat would lead to changes in the levels of probiotics in milk. the objectives of this study were to investigate the impact of feeding oat on the population of bifidobacteria and to evaluate their probiotic potential. the study consisted of two trials with sows, each with sows of first to third parity. sows were of duroc, landrace, yorkshire, and berkshire genetic backgrounds. the feeding treatment groups were: ( ) control, and ( ) ground whole oat ( %) ( table ). the experimental feed was distributed during the last month of gestation and the first month post-partum. the sows had free access to feed and water. the diets were formulated to meet the protein ( %) and energy ( . mcal de/kg) requirements of the sow, and vitamins and minerals were supplemented according to the nutrient requirements of swine published by the u.s. national research council (nrc). to facilitate lactation prior to milk sample collection, ml of oxytocin was given via im injection to each sow each weeks for three weeks starting on day of farrowing. a total of milk samples were collected from each sows. after the milk was collected, it was placed on ice and transported to the lab within h of collection. for each weekly evaluation, samples were pooled so the end result was one ml tube per diet condition. all for the enumeration of lactobacillus and bifidobacteria, each milk sample was plated onto man-rogosa-sharpe (mrs) and bifidobacterium iodoacetate (bim- ) agar media respectively. bim- is a selective medium for isolation and enumeration of bifidobacterium spp. [ ] . plates were incubated anaerobically at ˝c for - h before colonies were counted. since our interest was to isolate and characterize bifidobacteria, colonies from the bim- agar media were inoculated and incubated in trypticase phytone yeast (tpy) broth for h. among these, isolates were determined to grow well; isolates that showed aggregation (determined by visual observation) were selected for the next phase of the study. in addition, two nonaggregating isolates were selected for evaluation of their other probiotic potential. initially, selected colonies grown in bim- agar were picked out, gram's stained and examined under the microscope for the cell morphology. an agar spot test described by awaisheh and ibrahim [ ] , with slight modification, was used to determine the antimicrobial activity. ten microliters of active culture from isolates was spotted on the surface of the mrs agar plate. a µl of an overnight culture of indicator bacteria e. coli o :h ( ) was mixed with ml of soft bhi agar ( . %) and poured over the plate. the plates were incubated at ˝c for h and zones of inhibition (mm) were measured. mrs broth supplemented with , . , . , . , and . (w/v) ox-gall (sigma chemical co., st. louis, mo, usa.) were freshly prepared and sterilized by autoclaving at ˝c for min. a . ml of appropriately diluted overnight suspensions of each isolates was inoculated into the tubes containing different bile salt concentrations. samples were incubated at ˝c for h, the bacterial growth was determined by measuring the optical density at nm. a . ml of overnight suspensions of each isolates was inoculated into mrs broth adjusted to ph . using hcl. bacterial populations were enumerated by plating the samples onto bim- agar plates after incubation at ˝c for min. survival of bacterial population was compared to the control sample (unacidified mrs broth) of ph . . to determine whether these acid challenged isolates could be recovered, the isolates were further incubated at ˝c for h using -well microplate reader (biotek institute, winooski, vt, usa). each bacterial isolate was inoculated ( %, v/v) in mrs broth supplemented with different concentrations of antibiotics (ampicillin at and mg/l, chloramphenicol at and mg/l, erythromycin at and mg/l, and gentamicin at and mg/l) and growth was examined after h incubation at ˝c using a -well microplate reader. antibiotic concentrations were selected based on microbiological breakpoints values [ ] . graph prism . was used to graph results and perform statistical analysis for bacterial population. one-way anova with a tukey's multiple comparison post-test was used. data used for bacterial characterization were analyzed using sas version . (sas inst., cary, nc, usa.). comparison between groups was performed using a paired t-test or a one-way analysis of variance with post hoc analysis by duncan multiple test. p < . was considered statistically significant. plate counts on milk samples collected from sows that were fed oat had significantly higher levels of bifidobacteria compared to ( ) control group (p < . ) ( figure ). in contrast, we did not observe significant changes in lactobacillus levels between the diet groups. animals , each bacterial isolate was inoculated ( %, v/v) in mrs broth supplemented with different concentrations of antibiotics (ampicillin at and mg/l, chloramphenicol at and mg/l, erythromycin at and mg/l, and gentamicin at and mg/l) and growth was examined after h incubation at °c using a -well microplate reader. antibiotic concentrations were selected based on microbiological breakpoints values [ ] . graph prism . was used to graph results and perform statistical analysis for bacterial population. one-way anova with a tukey's multiple comparison post-test was used. data used for bacterial characterization were analyzed using sas version . (sas inst., cary, nc, usa.). comparison between groups was performed using a paired t-test or a one-way analysis of variance with post hoc analysis by duncan multiple test. p < . was considered statistically significant. plate counts on milk samples collected from sows that were fed oat had significantly higher levels of bifidobacteria compared to ( ) control group (p < . ) ( figure ). in contrast, we did not observe significant changes in lactobacillus levels between the diet groups. there are several studies on isolation and evaluation of probiotic potential especially lactobacilli from sow milk. to our knowledge this is the first study to isolate bifidobacteria from sow milk. in addition, our study showed significantly higher population of bifidobacteria compared to the control diet. therefore, we sought to further characterize the probiotic potential of the bifidobacteria present in the sow milk. bifidobacterial strains were isolated from sow milk using bim- agar as a selective medium. colonies from bim- agar were examined under the microscope for morphological characterization. gram-positive straight rod (branching and bifurcations) were selected as being the characteristics of bifidobacteria. they were thus identified as members of the genus bifidobacteria prior to further phenotypic characterization based on the visual appearance of the cell suspensions, samples were selected for the autoaggregation assay. as shown in figure a , autoaggregating isolates grown in mrs broth adhered around the surface of the tubes giving a clear supernatant ( . % to % autoaggregation, table ). the non-aggregating strains showed turbid suspension. based on our calculation, isolates were classified in different groups: high autoaggregation (> %), medium autoaggregation ( %- %), and low autoaggregation (< %) [ ] . table also shows the antimicrobial activity of isolates against indicator bacteria (e. coli o :h strain ). all isolates were able to inhibit indicator bacteria producing a clear zone of inhibition in the range of - mm in diameter ( figure b ). there are several studies on isolation and evaluation of probiotic potential especially lactobacilli from sow milk. to our knowledge this is the first study to isolate bifidobacteria from sow milk. in addition, our study showed significantly higher population of bifidobacteria compared to the control diet. therefore, we sought to further characterize the probiotic potential of the bifidobacteria present in the sow milk. bifidobacterial strains were isolated from sow milk using bim- agar as a selective medium. colonies from bim- agar were examined under the microscope for morphological characterization. gram-positive straight rod (branching and bifurcations) were selected as being the characteristics of bifidobacteria. they were thus identified as members of the genus bifidobacteria prior to further phenotypic characterization based on the visual appearance of the cell suspensions, samples were selected for the autoaggregation assay. as shown in figure a , autoaggregating isolates grown in mrs broth adhered around the surface of the tubes giving a clear supernatant ( . % to % autoaggregation, table ). the non-aggregating strains showed turbid suspension. based on our calculation, isolates were classified in different groups: high autoaggregation (> %), medium autoaggregation ( %- %), and low autoaggregation (< %) [ ] . table also shows the antimicrobial activity of isolates against indicator bacteria (e. coli o :h strain ). all isolates were able to inhibit indicator bacteria producing a clear zone of inhibition in the range of - mm in diameter ( figure b ). table shows the growth (absorbance readings at nm) of tested isolates in mrs broth containing bile concentration from %- . %. during h of incubation at °c, all the isolates survived well at a concentration level of bile of . %. these results showed that all the isolates tolerated . % bile with an absorbance readings ranging from . - . indicating resistance to . % bile. table shows the growth (absorbance readings at nm) of tested isolates in mrs broth containing bile concentration from %- . %. during h of incubation at ˝c, all the isolates survived well at a concentration level of bile of . %. these results showed that all the isolates tolerated . % bile with an absorbance readings ranging from . - . indicating resistance to . % bile. table shows the population of isolates before and after min of incubation at ph . and . at ˝c. all the isolates were able to survive after exposure to ph . for min. the initial population ranged from . - . log cfu/ml. in the control sample (ph . ), the population reached to . - . log cfu ml/ml after min of incubation. significant differences (p < . ) were observed when mean populations were compared using a paired t-test at ph . and . at and min. however, all the isolates were able to survive conditions mimicking the gastro intestinal environment. the isolate population ranged from . to . log cfu/ml. since stress recovery is an important criteria to determine the effectiveness of probiotic, we further determined whether these acid-stressed isolates could recover (grow). figure shows the recovery and growth of all the tested isolates at ˝c for h. after - h, all the isolates showed full recovery and continued to grow ( figure b ). these isolates continued growing further and reached the maximum absorbance of . to . (o.d. nm) similar to the reference isolates ( figure a ) that were grown at ph . under the same condition. the values are the averages of duplicate analysis˘s.d. all the isolates were able to survive conditions mimicking the gastro intestinal environment. the isolate population ranged from . to . log cfu/ml. since stress recovery is an important criteria to determine the effectiveness of probiotic, we further determined whether these acid-stressed isolates could recover (grow). figure shows the recovery and growth of all the tested isolates at °c for h. after - h, all the isolates showed full recovery and continued to grow ( figure b ). these isolates continued growing further and reached the maximum absorbance of . to . (o.d. nm) similar to the reference isolates ( figure a ) that were grown at ph . under the same condition. table shows the antibiotic susceptibility test of isolates to four different antibiotics at different concentrations. bacterial strains were considered resistant when they showed mic values higher than the mic breakpoints established by the european food safety authority [ ] . all the tested isolates were sensitive to ampicillin and chloramphenicol and resistant to gentamicin at tested concentration. however, with antibiotic erythromycin, isolates , , , and showed sensitivity while the rest of the isolates were resistant, according to the breakpoints set by efsa, indicating that these isolates could be strain-dependent. antibiotic concentrations were selected based on microbiological breakpoints values (mg/l) for bifidobacterium spp. [ ] . s-sensitive, r-resistant. hog production is an important industry to the us and worldwide market. post weaning diarrhea is responsible for major economic losses due to mortality, morbidity, decreased growth rate, and cost of medication [ ] . pwd is a multifactorial disease that can be caused by many different agents. several pathogens that cause diarrhea include lawsonia spp., clostridium spp., brachyspira spp., campylobacter spp., salmonella ssp., coronaviruses, and transmissive gastroenteritis viruses [ , ] ; however, in the swine industry, the most common cause of post-weaning diarrhea is enterotoxigenic escherichia coli [ ] . to avoid post-weaning diarrhea, antibiotic growth promoters have been included in piglet diets. however, antibiotic growth promoters are known to suppress beneficial organisms in the gut and, because of a possible link to the emergence of antibiotic resistant bacteria, there is a push to phase out or eliminate their use in swine production [ ] . intestinal microflora is a key contributor to intestinal health. among probiotic genera, bifidobacteria are one of the predominant bacterial groups existing in the animal and human intestine [ ] and are believed to play a beneficial role in maintaining the health of the host. our study showed that the inclusion of oat diet increased the bifidobacterial population more than lactobacilli. the possible explanation for this could be that oat contains oligosaccharides that function as prebiotic for the bacterial growth including bifidobacteria [ ] . tuohy et al. [ ] reported that bifidobacteria as specialized oligosaccharides-degrading bacteria. these bacteria have a broad range of glycolytic enzymes and carbohydrate uptake channels with high affinity for prebiotic oligosaccharides and their composite sugars. these prebiotics could have served as a growth-promoting factor for bifidobacteria [ ] . this is further confirmed by hinz et al. [ ] , who also reported that bifidobacteria play an important role in degradation of oligosaccharides. consistent with our findings, a study conducted by connolly et al. [ ] showed that oat flakes increase bifidobacteria populations in vitro, which also suggest that oats have prebiotic properties. additionally, using culture, isolation, sequencing, and fingerprinting methods, jost et al. [ ] demonstrated that the same probiotic bacteria are found in the feces of mothers and offspring as well as in mother's milk, suggesting that the probiotic bacteria travel from mother's gut into milk. therefore, in our study, one can speculate that the diet containing oat led to increased bifidobacteria in the intestine of the sows and that these bacteria travelled to the milk. the principle criterion for selection of a good probiotic is to overcome physiological barriers through the stomach (acid ph) and small intestine (bile salt) in order to arrive in the large intestine in a viable physiological state [ ] . in addition, these probiotics should adhere to the intestinal mucosa, possess antimicrobial properties, and should not have transmissible antibiotic resistance genes [ , ] . acid and bile tolerance are considered to be an important characteristic of probiotic strains. this tolerance supports survival and growth of probiotics and thus allows probiotics to exert their beneficial effects in the gastrointestinal tract [ ] . yun et al. [ ] reported the survivability of lactobacillus strains isolated from pig feces at % (w/v) bile, which is the condition found in the stomach and intestine. all the isolates tested in our study were able to survive at . % (w/v) bile concentration. however, bile salt tolerance usually varied among the strains of bifidobacteria [ ] . it has been reported that probiotic strains, and particularly bifidobacterial strains, have lower acid tolerance system, with the exception of b. animalis subsp. lactic [ ] . however, probiotic strains may have adaptive capabilities to survive in acidic conditions, allowing the strains to improve recovery and to continue growth. despite having a slightly low bacterial count (log cfu/ml), growth of isolates were not affected following min exposure to ph . when further grown in ph . mrs broth. the basic requirement for probiotics to exert expected positive effects is to be viable. similarly, all the isolates were capable of surviving and growing at ph . and % (w/v) bile salt, which are the conditions, found in the stomach and intestine. all the tested isolates survived well in low ph ( . ) and showed a similar growth trend. acid tolerance response (atr) may have developed in these isolates after the exposure of cells to low acid (ph . ), which might account for the isolates' good growth after the recovery period [ ] . aggregation properties of probiotic bacteria indicate that they can adhere to mucosal surfaces that will have prolonged survival in the body of the host. this property of probiotics bodes well for the use of probiotics and could be promising candidates for use in animal feed or to develop functional foods [ ] . ibrahim et al. [ ] studied several bifidobacterial strains and classified them into autoaggregation sensitive (ě %), moderate ( %- %), and resistant groups (< %) similar to our classification of high, medium and low autoaggregation behavior. in the present study, all the isolates also showed a clear zone of inhibition against e. coli o :h . from these observed zones, it is apparent that these isolates possess antimicrobial properties. the determination of antibiotic susceptibility of intestinal microorganisms is another important criterion for selecting an organism as a probiotic. agaliya and jeevaratnam [ ] reported the resistivity of l. plantarum isolated from fermented olives towards gentamicin ( µg/disc) whereas strains were sensitive to ampicillin ( µg), chloramphenicol ( µg), and erythromycin ( µg). argyri et al. [ ] also reported the resistivity of various lactic acid bacteria strains to ampicillin, chloramphenicol, erythromycin, and gentamicin when concentrations were used above mic values set by efsa. high resistance rates were observed with low-level kanamycin ( µg) and gentamicin ( µg) for % of the tested strains of bifidobacterium spp., similar to our results. however, tested strains were sensitive to high-level of kanamycin ( µg) or gentamicin ( µg,) using the disc diffusion method [ ] . similarly in another study, bifidobacterium spp. isolated from dairy and pharmaceutical products were resistant to gentamicin (mic = . µg/ml), and susceptible to chloramphenicol (mic = µg/ml), erythromycin (mic = . µg/ml), and ampicillin, (mic = , µg/ml) [ ] . in cases of co-administration with antibiotics to prevent and treat intestinal disorders, probiotics should be resistant to certain antibiotics in order to survive in the gastrointestinal tract [ ] . however, the use of antibiotic-resistant probiotic strains is still a controversial subject as antibiotic-resistant genes could transfer to the other bacteria present in the host gastrointestinal tract [ , ] . in addition, if such probiotic bacteria are added to different feed products, they could contribute to a potential source for the spread of antibiotic-resistance genes [ ] . therefore, it is still a debatable subject whether one should select antibiotic-resistant or sensitive strains. thus, more work is needed to address the potential impact of antibiotic-resistant bacteria to the host. in this study, we isolated potential probiotic bifidobacteria from sow milk. our study suggests that inclusion of oat in feeding could have the potential to improve the intestinal health of piglets by increasing the population of bifidobacteria. however, further research on long term supplementation of oats in feeding systems is needed before any recommendation about oats' beneficial effects on pig health can be made. it is equally important to identify these isolates up to species and strain level before being used in any products. further studies on the antimicrobial activity of these isolates against other pathogens, adhesion to intestinal epithelial cells, characterization of bacterial cell wall composition, and cell surface hydrophobicity is also required. national institute of food and agriculture. a part of this study was completed by barry donovan as partial fulfillment of the requirements for a master of science degree in animal health, north carolina a &t state university. the authors thank the north carolina state feed mill for preparing the sow diets, the personnel of the north carolina agricultural & technical state university's swine unit for animal care and nabin gyawali at virginia polytechnic institute and state university for his assistance with the statistical analysis. rabin gyawali and barry donovan collected samples and conducted the research experiments. rabin gyawali, radiah c. minor, and salam a. ibrahim designed, interpreted the results and wrote the manuscript. all authors read and approved the final manuscript. attempts to induce conception in lactating sows weaning pigs at an early age decreases cellular immunity farm factors associated with the use of antibiotics in pig production alternatives to antibiotic growth promoters in prevention of diarrhoea in weaned piglets: a review field evaluation of the efficacy of a probiotic containing bacillus licheniformis and bacillus subtilis spores, on the health status and performance of sows and their litters probiotics as a dietary additive for pigs: a review antimicrobial activity of bifidobacterium longum (ncfb ) as influenced by spices gut microbiota and probiotics in maternal and infant health selective medium for isolation and enumeration of bifidobacterium spp effect of media composition and incubation temperatures on autoaggregation behavior of bifidobacteria screening of antibacterial activity of lactic acid 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of dairy propionibacteria to intestinal epithelial tissue in vitro and in vivo screening of lactobacillus plantarum isolated from fermented idli batter for probiotic properties antimicrobial susceptibility of bifidobacteria antibiotic resistance of lactic acid bacteria and bifidobacterium spp. isolated from dairy and pharmaceutical products antibiotic susceptibility of potentially probiotic lactobacillus species high levels of spontaneous drug resistance in lactobacillus current status of antibiotic resistance in lactic acid bacteria. antibiotic resistance: causes and risk factors, mechanisms and alternatives this work was supported by the agricultural research program at north carolina agricultural and technical state university with funding from the usda nifa, hatch project number nc.x- - - - - (salam a. ibrahim), and ncx- - - - - (radiah c. minor). its contents are solely the responsibility of the authors and do not necessarily represent the official views of the the authors declare no conflict of interest. key: cord- -fbilhauu authors: savarin, carine; bergmann, cornelia c.; gaignage, melanie; stohlman, stephen a. title: self-reactive cd (+) t cells activated during viral-induced demyelination do not prevent clinical recovery date: - - journal: j neuroinflammation doi: . /s - - - sha: doc_id: cord_uid: fbilhauu background: microbial infections have been implicated in initiating and enhancing severity of autoimmune diseases including the demyelinating disease multiple sclerosis (ms). nevertheless, the incidence of both acute and persisting viral infections without evidence of autoimmune sequelae suggests that this process is well controlled. the conditions promoting or stemming self-reactive (sr) t cells following viral-induced tissue damage thus need to be better defined. using a non-fatal viral mouse model of encephalomyelitis associated with demyelination and disability, yet ultimate clinical improvement, this study set out to monitor uptake and presentation of endogenous myelin antigens, as well as induction and fate of sr t cells. methods: activation and central nervous system (cns) recruitment of myelin-specific cd t cells was analyzed by flow cytometry during encephalomyelitis induced by a glia tropic murine coronavirus. potential antigen-presenting cells (apc) ingesting myelin were characterized by flow cytometry and their ability to activate sr t cells tested by co-culture with carboxyfluorescein succinimidyl ester (cfse)-labeled myelin-specific cd t cells. endogenous sr t cell kinetics was analyzed within both cervical lymph nodes and cns by enzyme-linked immunospot (elispot) following viral infection. results: the data demonstrate the presence of apc capable of activating sr t cells in both draining lymph nodes and the cns temporally correlating with overt demyelination. while both the cns-infiltrating myeloid population and microglia ingested myelin, only cns-infiltrating apc were capable of presenting endogenous myelin antigen to sr t cells ex vivo. finally, sr t cell activation from the endogenous t cell repertoire was most notable when infectious virus was controlled and paralleled myelin damage. although sr t cell accumulation peaked in the persistently infected cns during maximal demyelination, they were not preferentially retained. their gradual decline, despite ongoing demyelination, suggested minimal re-stimulation and pathogenic function in vivo consistent with the lack of autoimmune symptoms. conclusions: the results demonstrate the potential for cns tissue destruction to induce and recruit sr t cells to the injury site and support a host suppressive mechanism limiting development of autoimmunity. multiple sclerosis (ms) is an autoimmune disease of the central nervous system (cns) characterized by demyelination, axonal loss, and increasing disability [ , ] . while the etiology of ms remains unknown, various genetic and environmental factors have been associated with ms pathogenesis [ ] [ ] [ ] . among the environmental cues, viral infections, most prominently epstein-barr virus and human herpes virus type , have been linked to initiation or progression of disease [ , [ ] [ ] [ ] [ ] ; however, their causal nature remains unclear. anti-viral antibodies and viral antigens have been detected in brain and cerebrospinal fluid of ms patients [ ] [ ] [ ] . nevertheless, active virus replication has yet to be demonstrated in the cns of ms patients. by contrast, infections have even been proposed to limit autoimmunity [ ] . indeed, the decreased incidence of infectious diseases in the past decades inversely correlates with an increased incidence of autoimmune diseases, including ms [ ] . for example, viral infection in an animal model of type i diabetes prevents disease development [ , ] . similarly, mice bred under conventional conditions are less susceptible to diabetes than mice bred in a pathogen-free environment [ ] , supporting the concept that microbiome burden may reduce the incidence of autoimmunity [ ] . therefore, the role of viral infections in autoimmune diseases is complex and remains largely elusive. activation of self-reactive (sr) immune cells by epitope spreading, molecular mimicry, cryptic antigen, and bystander activation have all been proposed to explain how viral infections may ultimately result in autoimmunity [ , ] . this is exemplified by the biphasic disease induced by theiler's murine encephalomyelitis virus (tmev) [ ] . minimal clinical deficits appear during the acute phase of tmev infection, characterized by neuronal infection. the protective anti-viral immune response fails to completely eliminate infectious virus, resulting in chronic infection of microglia and macrophages [ ] , ascending paralysis and progressive demyelination [ ] . whereas initial tissue destruction occurs as a bystander effect of the anti-viral response, paralysis and demyelination are associated with autoimmunity mediated by sr t cells activated via epitope spreading during chronic infection [ , ] . tmev infection thus provides a paradigm for autoimmunity associated with chronic infection and sustained inflammation. by contrast, demyelination induced by infections with the neurotropic mouse hepatitis virus (mhv) strain jhm (jhmv) or the related dual hepato-and neurotropic strain mhv-a suggests that persistent cns infections, even those associated with myelin destruction, do not necessarily result in clinically apparent cns autoimmune disease. although a vigorous cd + t cell-and th -dominated immune anti-viral response eliminates infectious virus [ ] , viral antigen and rna persist within the cns without evidence of active virus replication [ , ] . demyelination requires both infection of oligodendrocytes and adaptive immunity and is initiated by t cell-mediated virus control. similar to chronic tmev infection, persistent mhv infections are associated with ongoing demyelination. however, following acute viral-induced tissue injury, sustained demyelination is balanced by myelin repair, associated with clinical improvement [ , , ] . the absence of disease progression during chronic mhv infection suggests that autoimmune responses are either not initiated or suppressed. nevertheless, during acute mhv-a infection, sufficient myelin-derived self-antigen (ag) is presented in cervical lymph nodes (cln) to support proliferation of exogenously added sr t cells. however, little or no sr t cell proliferation occurred within the cns, suggesting that t cells activated in cln during acute infection migrate to the cns but are unable to promote a local autoimmune response. the present study set out to determine whether ag-presenting cells (apc) in the cns support sr t cell proliferation and whether endogenous sr t cells are induced during jhmv-induced demyelination. the data demonstrate that sr t cell activation is agdriven and mediated by a population of cd b + apc present within both the cln-and the cns-infiltrating cells. despite myelin uptake and expression of major histocompatibility complex (mhc) class ii molecules, microglia do not support sr t cell activation. importantly, the kinetics of cd b + cell-mediated sr t cell activation parallels demyelination and correlated with myelin-specific t cells derived from the endogenous host t cell repertoire. nevertheless, despite sustained demyelination and cns inflammation, the sr t cell response declined during viral persistence, suggesting that chronic jhmv infection establishes an environment which supports ongoing clinical improvement and regulates autoimmune responses. wild-type (wt) c bl/ mice were purchased from the national cancer institute (frederick, ms, usa). myelin oligodendrocyte glycoprotein (mog)-specific t cell receptor (tcr) transgenic ( d ) mice expressing the congenic marker cd . [ ] were obtained from dr. v.j. kuchroo (harvard university, boston, ma). ovalbumine (ova)-specific tcr transgenic (ot-ii) mice expressing the congenic marker ly . were provided by dr. b. min (cleveland clinic, cleveland, oh). mice expressing the green fluorescent protein (gfp) under the myelin proteolipid protein (plp) promoter (plp-gfp) [ ] were obtained from dr. w.b. macklin (university of colorado, denver, usa). all transgenic mice were bred and maintained at the biological resources unit of the cleveland clinic lerner research institute under sterile conditions. all procedures were performed in compliance with the cleveland clinic institutional animal care and use committee approved protocol number - . peptides were obtained from bio-synthesis (lewisville, tx, usa) and included m - (gtvyvrpiiedyht), mog (mevgwyrspfsrvvhlyrngk), and mbp - (shhaartthygslpqksqr). mice were infected between and weeks of age in the left hemisphere with pfu of the glia tropic jhmv neutralizing monoclonal antibody (mab)-derived . v- variant [ ] . mice were assessed daily for clinical disease severity according to the following scale: , healthy; , hunched back and ruffled fur; , partial hind limb paralysis or inability to maintain the upright position; , complete hind limb paralysis; and , moribund or dead. anesthetized mice were perfused with phosphate-buffered saline (pbs). single-cell suspension was prepared from the cln by mechanical disruption through a -μm cell strainer in rpmi medium containing mm hepes (ph . ) supplemented with % fetal calf serum (fcs). brain and spinal cord were homogenized using a ten-broeck tissue grinder. tissue homogenates were adjusted to % percoll (pharmacia, uppsala, sweden). a -ml % percoll underlay was added prior to centrifugation at g for min at °c. cns-derived cells were recovered from the - % interface, washed, and resuspended in roswell park memorial institute (rpmi) medium % fcs. splenocytes were isolated from naïve mice by mechanical disruption and red blood cells eliminated using gey's solution. ninety-six-well filtration plates (millipore, billerica, ma, usa) were coated overnight at °c with anti-interferon-γ (ifn-γ) capture ab ( μg/ml; bd biosciences, san diego, ca, usa). serial dilutions of clnand cns-derived mononuclear cells pooled from a minimum of eight mice per time point were cultured in triplicate in rpmi complete (rpmi medium containing mm l-glutamine, non-essential amino acid, mm sodium pyruvate, μg/ml gentamicin, and × − m -mercaptoethanol) supplement with % fcs in the presence of irradiated splenocytes ( . × cells/well) pre-incubated with or without μm peptide for h at °c. after h at °c, spots were visualized by sequential incubation with biotinylated anti-ifn-γ mab ( μg/ml; bd biosciences) overnight at °c, horseradish peroxidaseconjugated streptavidin (bd biosciences) for h at room temperature, and , ′-diaminobenzidine substrate (sigma aldrich, st. louis, mo, usa). spots were counted using a ctl immunospot analyzer (cellular technology ltd., shaker heights, oh, usa). spots detected in wells with no stimulation (no peptide) were subtracted and results presented as the number of ifn-γ-secreting cells normalized to input cells. non-specific binding was blocked with mouse serum and anti-mouse fcγiii/ii mab for min on ice. cells were stained for surface markers for min on ice using fluorescein isothiocyanate (fitc), phycoerythrin (pe), peridinin chlorophyll protein complex (percp), or allophycocyanin (apc)-conjugated mab (all from bd biosciences) specific for cd (clone ly- ), cd (clone gk . ), cd (clone - . ), cd (clone pc ), cd (clone im ), cd . (clone a ), cd . (clone ox. ), cd b (clone m / ), cd (clone - a ), and cd (clone gl- ). cells were washed twice in facs buffer (pbs, % bovine serum albumin (bsa)) prior analysis. for proliferation study, bromodeoxyuridine (brdu, mg/mouse) (bd biosciences) was injected i.p. h prior to sacrifice. mononuclear cells from cln and cns were prepared as described above, stained for surface markers followed by intracellular brdu according to the manufacturer's instructions using the fitc brdu flow kit (bd biosciences). data were analyzed using a facscalibur flow cytometer (bd biosciences) and flowjo software (tree star inc., ashland, or, usa). cd + t cells were enriched from naïve d (tcr transgenic mice specific for mog or ot-ii (tcr transgenic mice specific for ova) mice by negative selection using the cd + t cell isolation kit ii (miltenyi biotec inc., auburn, ca, usa), according to the manufacturer instructions. naïve (cd − cd lo ) cd + t cells were then purified by cell sorting (facsaria, bd biosciences). sub-lethally irradiated ( rad) wt recipients received equal numbers ( cells) of purified naïve ot-ii and d cd + t cells by intravenous injections and were challenged with virus weeks after adoptive transfer. cln, brains, and spinal cords were isolated at various times post-infection (p.i.) from pbs-perfused mice. cln were mechanically disrupted through a -μm cell strainer as described above, whereas brains and spinal cords were finely minced with a razor blade. suspensions were digested in rpmi medium containing % fcs, . % collagenase ( mg/ml) (roche, basel, switzerland), and % dnase i ( mg/ml) (sigma aldrich) for min at °c. collagenase was then inactivated by addition of % . m edta for min at °c. following centrifugation at g for min at °c, cln-derived cells were directly resuspended in facs buffer for staining, whereas cns-derived cells were isolated using percoll gradients prior to staining as described above. cln-derived cd b − and cd b + cells, cnsinfiltrating cd hi cd b + , microglia (cd lo cd b + ), and cd hi cd b − cells were purified using a cell sorter (facsaria) and resuspended in rpmi complete % fcs. naïve cd + t cells were purified from splenocytes of naïve d mice using the cd + cd l + t cell isolation kit ii (miltenyi biotec) according to the manufacturer instructions. naïve d cd + t cells were then stained with carboxyfluorescein succinimidyl ester (cfse, . μm) (molecular probes, carlsbad, ca, usa) and cultured in a -well plate in the presence of cln-derived cd b − or cd b + cells, cns-derived cd b − or cd b + cells, or microglia (t cells/apc ratio : ) in rpmi complete % fcs for days at °c. rat anti-mhc class ii blocking mab (clone m / ) (abcam, cambridge, ma) or mog - peptide ( μm) were added at the initiation of the cultures for some experiments. t cell proliferation was assessed by measuring the percentage of cfse dilution by flow cytometry. mice were perfused with ice-cold pbs followed by % paraformaldehyde (pfa). spinal cords were dissected, fixed for h in % pfa at °c, and then incubated with sucrose gradients as follows: min with % sucrose at room temperature, min with % sucrose at °c, and, finally, overnight with % sucrose at °c. tissues were stored in cryoprotection solution until preparation of μm microtome sections. after antigen retrieval with . m sodium citrate buffer ph . , sections were treated with % triton x- for min, treated with blocking solution (pbs, % bsa, % normal goat serum) for min, and stained with rabbit anti-mouse iba (wako, richmond, va) and mouse anti-mouse plp (millipore) primary mabs overnight at °c. alexa fluor goat anti-rabbit (invitrogen, carlsbad, ca) and alexa fluor goat anti-mouse (invitrogen) were added for h at room temperature. sections were mounted with vectashield mounting medium with ′- -diamidino- -phenylindole (dapi) (vector laboratories) and analyzed using a leica tcs confocal microscope. results represent the mean ± sem and are plotted using graphpad prism software. statistics were calculated using a two-tailed unpaired student's t test, anova with bonferroni post-test, and dunn's multiple comparison test, and p values < . were considered statistically significant. infection with the mhv-a strain suggested that acute encephalomyelitis provides a milieu capable of supporting proliferation of transferred mog-specific t cell receptor (tcr) transgenic t cells within the cln [ ] . however, neither their reactivation within the cns, prolonged survival, or potential to induce autoimmunity have been explored. to determine whether sr cd + t cells are retained during chronic infection, mogspecific d cd + t cells were transferred to sublethally irradiated wt mice prior to jhmv infection. by enhancing engraftment of donor t cells, this approach increased sr t cells to numbers amenable to flow cytometric analysis, while maintaining a host anti-viral immune response. bone marrow-derived inflammatory (cd hi ) cells were minimal within the cns of recipients prior to infection (fig. a) , indicating non-specific activation and that cns recruitment was prevented by intact blood brain barrier. at day p.i., maximal antiviral t cell responses [ , ] coincided with a decreased percentage of transferred sr t cells in cln (fig. b, c) . grafted sr t cells were undetectable within the cns at day p.i. following jhmv infection (fig. b, c) in contrast to their early migration into the cns during acute mhv-a infection [ ] . nevertheless, transferred sr t cells were present in the cns of jhmv-infected mice by day p.i. (fig. b, c) ; furthermore, similar proliferation of grafted sr t cells and host cd + t cells suggested identical activation (fig. d) . although the kinetics differed, these data are consistent with cns recruitment of sr t cells during mhv-mediated demyelination, independent of the virus strain and tropism [ ] . importantly, retention of transferred sr t cells at slightly declining frequencies within the total cns cd population out to day p.i. (fig. b, c) negated preferential expansion/survival during chronic viral infection. the absolute numbers of grafted sr cd + t cells gradually declined (fig. c) concomitant with contraction of the overall cd + t cell population, supporting a lack of ongoing self-ag-driven survival. furthermore, retention of sr t cells within the cns did not alter disease severity out to days p.i. (fig. e) . within the cln, transferred sr t cells comprised~ % of activated cd hi cells (data not shown) and their absolute numbers remained stable during ongoing chronic jhmv infection (fig. c) . limited proliferation of sr t cells within the cns during acute mhv-a infection [ ] suggested the possibility of non-specific t cell recruitment. to determine the specificity of recruitment into the cns, heterologous ova-specific t cells were co-transferred with sr t cells to sub-lethally irradiated mice. prior to infection, the frequencies of both the sr-and ova-specific t cells were similar in cln (fig. f ) and blood (data not shown), demonstrating equivalent survival. following jhmv infection, the relative percentages of both the sr-and ova-specific t cells were reduced in the cln coincident with the expansion of virus-specific t cells. however, the decline in transferred sr t cells was less dramatic, suggesting enhanced survival cues via agspecific activation (fig. f ) . consistent with an absence of non-specific peripheral activation, ova-specific t cells were not detected in the inflamed cns at day p.i. by contrast, transferred sr t cells constituted a discrete population (fig. f ) , confirming migration of peripherally activated cells and supporting agdependent cns retention of sr cd + t cells. cln constitute the major site of peripheral lymphoid drainage from the cns [ , ] . this is supported by activation of jhmv-specific t cells within the cln [ ] and detection of myelin antigens in ms patients and rodents with experimental autoimmune encephalomyelitis (eae) [ ] [ ] [ ] , as well as following demyelination induced by oligodendrocyte death [ ] . myelin, either in a soluble form or associated with apc, drains to cln, where it can potentially activate naïve sr t cells [ , ] via presentation by cd b + cells, comprising both dendritic cells (dc) and macrophages [ ] . during eae, the most efficient population-presenting myelin antigen in the cns is cd b + cd c + [ , ] . cd b + cells represent a small subset (between . and %) of the cln population, which only varies slightly throughout the course of jhmv infection (fig. a) . importantly, cd c + cells constitute the vast majority of cd b + cells (~ %) and their frequencies within the cd b + population remain constant between days and p.i. (fig. a) . we therefore tested unfractionated clnderived cd b + cells for the presentation of self-ag following jhmv infection. cd b + and control cd b − cells were purified from the cln at different times p.i. and were co-cultured with cfse-labeled mog-specific cd + t cells as a source of sr t cells. neither cd b + nor cd b − cells isolated at day p.i. supported sr t cell activation (fig. c) . however, by day p.i., when myelin damage becomes apparent, both cd b + and cd b − cells initiated minimal t cell proliferation, which reached statistical significance relative to unstimulated conditions (no apc) for cd b + cells (fig. c) . activation of sr t cells by the cd b + population increased at days and p.i. concomitant with enhanced demyelination [ , ] , while the cd b − population retained only a minimal ability to support sr t cell proliferation (fig. b, c) . the ability of clnderived cd b + cells to support sr t cell proliferation declined, but was sustained, by day p.i., while the minimal ability of the cd b − population dropped to below detection by days p.i. (fig. c) . the presence of myelin ag within the cln by day p.i. supports the possibility that endogenous sr t cells could be activated following jhmv-induced demyelination. the absence of clinically apparent autoimmunity, coincident with a decline rather than progressive increase in sr t cells in the cns during chronic jhmv infection (fig. c) , suggested that sr t cells fail to be continuously reactivated within the cns. in the inflamed cns, resident microglia and myeloid cells constitute heterogeneous populations of apc potentially capable of presenting self-ag [ ] . to determine whether cnsinfiltrating myeloid cells, comprising macrophages and dendritic cells, and/or microglia are capable of processing and presenting self-ag within the cns following infection, both potential apc populations were purified based on differential cd expression [ , ] at distinct times p.i. and tested for the ability to support sr t cell activation in the absence of exogenously added ag. infiltrating myeloid cells at day p.i. (fig. a) were comprised of~ % cd c + cells, a proportion that gradually increased during the course of infection to reach~ % at day p.i. (fig. a) . at the peak of acute inflammation (day p.i.), neither the cd hi cd b + , cd hi cdllb − , nor microglia populations supported t cell proliferation (fig. c) . however, sr t cell proliferation was modestly increased by the cd b + apc subset at day p.i. (fig. c) and prominently by day p.i. (fig. b, c) , similar to the cln (fig. ) . on a per cell basis, the ability of infiltrating cd b + cells to support proliferation remained relatively stable during chronic jhmv infection (fig. c) . although cd b − cells appeared to promote minimal proliferation, their activity remained near threshold levels throughout the infection. similar to the cd b − population, microglia were unable to support sr t cell proliferation at any time p.i. (fig. b, c) . the kinetics of sr cd + t cell activation by cns-infiltrating cd b + cells correlate with viralinduced myelin destruction as a source of self-ag [ ] . in addition, anti-mhc class ii ab blocked proliferation, supporting mhc class ii-dependent t cell activation (fig. d) rather than non-specific proliferation signals derived from the inflamed cns environment (fig. d) . mhc class ii expression was compared on microglia and infiltrating cd b + cells by flow cytometry to examine whether the inability of microglia to support t cell proliferation correlated with differential mhc class ii expression. while no differences were noted in the percentage of class ii-expressing cells within the populations (fig. a) , microglia were inferior to infiltrating cd b + cells in their level of class ii expression based on mhc class ii mean fluorescence intensity (mfi) (fig. a) , even at the peak of ifn-γ release at day p.i. [ ] . similarly, the mfi of the co-stimulatory molecule cd was slightly lower on microglia compared to i. c percentages of proliferating d cells co-cultured with media only (no apc), cd b − , and cd b + cells isolated from the cln of jhmv-infected mice at days , , , , and p.i. data represent the mean ± sem with n = - pooled mice per time point from three separate experiments. #p < . and ###p < . depict significant differences between no apc and cd b + cells, whereas § § §p < . depicts significant differences between cd b − and cd b + cells determined by a two-way anova with bonferroni post-test. significant differences between time points are indicated by **p < . and ***p < . using dunn's multiple comparison test infiltrating cd b + cells, while no differences were observed for cd expression levels (fig. b) . moreover, while mhc class ii expression was sustained at similar levels on microglia, it was further upregulated between day and p.i. on infiltrating cd b + cells (fig. a) . to assess whether myelin ag added to the cell culture could overcome the inability of microglia to activate sr t cells, cd b + apc populations were pre-incubated with mog peptide. under these conditions, microglia induced proliferation of sr cd + t cells as well as infiltrating cd b + cells (fig. c) . these data demonstrate that while class ii levels on microglia suffice to present the inability of microglia to support sr t cell proliferation may reflect a defect in myelin uptake or ag processing. myelin engulfment by iba + cells supports myelin uptake by microglia and/or infiltrating myeloid cells during jhmv-induced demyelination (fig. a) . however, the amoeboid morphology of microglia during inflammation prevents histological distinction between myelin in macrophages versus microglia (fig. a) . mice expressing gfp under the plp promoter were therefore infected to quantify gfp as a surrogate marker for myelin ingestion by infiltrating cd b + cells versus microglia. flow cytometry revealed gfp within both infiltrating myeloid cells (cd hi cd b + ) and microglia (cd low cd b + ) (fig. b, c) . percentages of infiltrating myeloid cells and microglia containing gfp were relatively stable between days and p.i. (fig. b) , with a higher proportion of microglia containing gfp, supporting their primary role in demyelination [ ] . in contrast to percentages, mfi analysis showed that the amount of gfp differed throughout the course of infection (fig. c) . infiltrating cd b + cells exhibited peak uptake at days p.i., which slowly decreased at later time points (fig. c) , but remained higher than gfp mfi in the control cd hi cd b − population, as well as cd hi cd b + cells isolated from naïve animals (fig. c) . by contrast, a gradual increase in gfp was observed within microglia throughout the course of jhmv infection independent of auto-fluorescence (fig. c) . these data imply that both the infiltrating myeloid and microglial populations phagocytoze myelin during viral-induced demyelination. therefore, the inability of microglia to support sr t cell activation is less likely to reside in fig. myelin debris within cns cells. a plp (red) and iba (green) stainings on spinal cord tissue isolated from wt-infected mice at day p.i. and analyzed by confocal microscopy. scale bar, μm. (i) d reconstruction image view, yz (ii), and xz (iii) projections show plp + myelin in close contact and engulfed (arrow head) by iba + cells. b percentage of cd hi cd b + and microglia (cd low cd b + ) containing gfp between days and p.i. during infection of plp gfp mice. c mean gfp fluorescent intensity (mfi) analyzed by flow cytometry in cns-infiltrating cd hi cd b − and cd hi cd b + cells and microglia (cd low cd b + ) between days to p.i. hashed bars represent microglia autofluorescence detected within infected wt mice between days and p.i. data represent the mean ± sem from two separate experiments with n = per time point per experiment a defect in myelin uptake, rather than inefficient ag processing and/or presentation. myelin damage, uptake and subsequent presentation of self-ag by potential apc, and sustained cns inflammation without overt clinical evidence of autoimmune disease following jhmv infection pose a dilemma. plausible explanations may be that sr t cells are either not, or only minimally, induced or that they are not reactivated within the cns. indeed, demyelination triggered by oligodendrocyte death fails to initiate autoimmunity, despite myelin ag drainage to cln [ ] . to determine if jhmv-induced demyelination results in the activation of endogenous sr t cells, cd + t cell responses to the h- b restricted encephalitogenic myelin epitopes mog and mbp were assessed by enzyme-linked immunospot (elispot) to detect lowfrequency responder cells. responses to the h- b -restricted immuno-dominant viral m epitope [ ] were used as positive controls. analysis focused on ifn-γproducing cd + t cells, as jhmv infection induces a vigorous th response with negligible il- or il- components (data not shown), which is supported by no affects on pathogenesis in the absence of il- [ ] . virus-as well as myelin-specific t cell frequencies were lower than and positive cells per , respectively, within the cln and spleen of naïve animals (fig. a) . within cln, the frequency of virus-specific cd + t cells peaked at day p.i., declined by day p.i., and remained relatively stable up to day p.i. (fig. a) . within the cns, frequencies of virus-specific t cells were~ higher than cln at day p.i., continued to increase at day p.i., and declined as the overall inflammatory response resolved due to virus control (fig. a) . sr t cells specific for both the immuno-dominant mog and myelin basic protein (mbp) epitopes within the cln were above baseline throughout acute and chronic jhmv infection, albeit at very low frequencies (fig. a) . maximum sr t cell frequencies were observed at day p.i. (fig. a) with responses near detection limits in a large proportion of infected mice throughout infection (fig. b) . a very limited number of infected mice harbored sr t cells within the cns during acute infection at days and p.i. however, by day p.i., the number of responder mice exhibiting sr t cells within the cns reached % (fig. b) , coincident with peak frequencies of both mog-and mbp-specific t cells between days and p.i. (fig. a) . these data are consistent with maximal clinical disease throughout days - p.i. in all infected mice (fig. c) . although the sr t cell frequencies in the cns were variable among individual mice, and always reduced relative to virus-specific t cells, they were increased relative to the cln at day p.i. and thereafter (fig. a) . importantly, cns sr t cells were sustained above baseline during resolution of clinical symptoms, with preferential stability of mog-specific t cells (fig. a, b) . however, compared to peak sr t cell frequencies at days / p.i., frequencies, as well as the number of mice harboring cns sr t cells, declined as clinical disease resolved (fig. c) . these data demonstrate that endogenous sr t cells are induced in the vast majority of jhmv-infected mice. however, they are only sustained within the cns of~ % of mice during chronic demyelination, when lesion formation is counterbalanced by repair (fig. a, b) . importantly, segregation of mice harboring sr t cells within the cns after day versus mice in which sr t cells were undetectable showed no differences in overall clinical recovery (fig. d) . these data further supported our previous observations that cns recruitment and retention of sr t cells did not alter disease severity (fig. e) . animal models have demonstrated that viral infections can promote the initiation as well as increase the severity of autoimmune diseases [ ] . however, evidence for a viral etiology of human autoimmune diseases, including ms, remains elusive. moreover, given the extent of both acute and persisting viral infections without evidence of autoimmune sequelae, it has also been proposed that infections may even provide protection from autoimmunity. specifically, the conditions promoting or stemming activation of auto-ag-specific t cells following viralinduced tissue damage have not been extensively explored. chronic cns infection by the naturally occurring mouse pathogen tmev promotes activation of sr t cells, which mediates increasing tissue destruction accompanied by an ascending paralysis [ ] . by contrast, infection with jhmv also leads to immune-mediated demyelination; however, persistent infection correlates with clinical recovery [ ] . while clinical improvement throughout persistence despite ongoing demyelination supported the absence of an autoimmune component following neurotropic mhv infection, previous studies clearly demonstrate sufficient auto-ag presentation to induce sr t cell responses [ , , ] . in an effort to reconcile peripheral activation of sr t cells, but no disease exacerbation, this study set out to assess kinetics of auto-ag presentation as well as the fate of sr t cells following jhmv infection. the data demonstrate the presence of apc capable of activating sr t cells in both the cln and the cns with maximal stimulating activity during the time of most overt demyelination. while both the cns-infiltrating myeloid population and microglia ingest gfp as a surrogate for myelin, only the cnsinfiltrating apc were capable of presenting endogenous , and mbp - detected in naïve animals. red bars represent the mean ± sem. significant differences between time points are indicated *p < . , **p < . , and ***p < . . significant differences compared to baseline levels are indicated #p < . , ##p < . , and ###p < . using unpaired t test. b percentage of mice exhibiting ifn-γ-secreting mog and mbp-specific cd + t cells above maximal baseline levels in the cln and cns between days and p.i. c clinical symptoms following jhmv infection. data represent the mean ± sem of mice. d correlation between frequencies of sr t cells at days and p.i. with average clinical scores, coefficient of determination r = . myelin ag to sr t cells ex vivo. finally, the results demonstrate that sr t cells are activated from the endogenous t cell repertoire when myelin destruction is evident, but infectious virus is already controlled. although sr t cells migrate into and are most prominent in the persistently infected cns during maximal demyelination, no evidence was found to suggest preferential expansion despite ongoing demyelination, consistent with minimal re-stimulation in vivo. mechanisms underlying sr t cell activation following cns infections include molecular mimicry between pathogen and self-epitopes, presentation of self-peptides from tissue damage, and dysregulation of regulatory tolerance mechanisms [ , ] . myelin-specific t cells isolated from ms patients have been shown to cross-react with human coronavirus [ , ] . similarly, homology was found between mhv and myelin peptides, potentially triggering autoimmune disease [ ] . nevertheless, sr t cells were near detection levels within the cns during acute jhmv infection in the vast majority of mice and their frequency only increased as myelin damage increased. moreover, their numbers declined as clinical symptoms dropped due to increased repair. self-ag-specific activation of sr t cells is further supported by the lack of t cell activation with irrelevant specificity and blockade of activation by anti class ii ab, ruling out bystander events. overall, our data support the hypothesis that release of myelin debris due to jhmv-induced tissue damage results in cell-free or cell-associated myelin drainage to cln, where a population of cd b + apc can support the activation of sr t cells. activated sr t cells access the cns at a time when the vast majority of infectious virus is already controlled, but persistence drives ongoing expression of proinflammatory molecules capable of recruiting activated cells from circulation [ ] . local encounter with myelin-loaded cd hi cd b + apc, but not microglia, is then poised to drive sr t cell reactivation. the apparent inability of microglia to activate sr cd + t cells ex vivo remains unclear as their uptake of myelin is consistent with demyelination independent of inflammatory monocyte recruitment following jhmv infection [ ] . nevertheless, microglia also failed to support cd + t cell responses following tmev infection and induction of eae [ , ] . in fact, several studies question the role of microglia as apc in vivo [ ] despite their implication as apc based on expression of mhc class ii and costimulatory molecules in both ms patients [ ] and eae [ ] , as well as their ability to activate cd + t cells in vitro [ ] . detection of myelin within iba + cells increased gfp mfi in microglia, as well as detection of myelin in microglia in other models [ , ] supports self-ag uptake during jhmv infection. however, these data suggest that microglia may be poor at processing myelin ag for loading onto mhc class ii. this notion is also supported by effective priming of sr t cells using exogenous peptide, despite reduced mhc class ii expression relative to infiltrating cd b + cells. in addition, as activated microglia can adapt to their surroundings to mediate both pro-and anti-inflammatory functions [ ] , it cannot be excluded that microglia may negatively regulate sr t cell responses during chronic jhmv infection by limiting t cell proliferation or by inducing apoptosis [ ] . the results also indicate a discrepancy between apc presentation capacity ex vivo, yet apparently limited sr t cell activation in vivo. although apc in cln sustain their ability to activate sr t cells throughout days to p.i., endogenous sr t cells are only detected at low frequencies and exhibit a brief peak at day p.i. maximal accumulation of sr t cells in the cns between days and p.i. in all mice supports efficient egress from cln. whether frequencies in the cns are supported by ongoing recruitment or local expansion remains unclear. the capacity of cns-infiltrating cd b + cells to activate sr t cells ex vivo in the absence of exogenous peptide indicates that apc are competent to reactivate sr t cells throughout chronic infection. however, no evidence for preferential expansion or retention of sr t within the cns relative to virus-specific cd + t cells suggests that the absence of sustained activation may contribute to the lack of increasing clinical symptoms during chronic infection. these results clearly differ from the tmev model, in which sr t cells aggravate pathology and clinical disease. one possible scenario is that persistent jhmv infection may establish an environment that suppresses the sr t cell response. jhmv infection induces both agspecific and foxp + regulatory t cells [ , ] which are efficient at controlling autoimmunity in other settings [ , ] . treg depletion [ ] , elimination of agspecific treg [ ] , and adoptive transfer of foxp + treg [ ] during acute jhmv infection all demonstrated that, in contrast to tmev [ ] , tregs have a limited effect on virus clearance. nevertheless, foxp + tregs play a critical role in limiting tissue destruction [ ] , suggesting that they regulate the activation or effector function of sr t cells following jhmvinduced demyelination [ ] . although treg 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t lymphocyte final effector function and death shifting hierarchies of interleukin- -producing t cell populations in the central nervous system during acute and persistent viral encephalomyelitis role of cd (+) cd (+) t cells in acute and persistent coronavirus infection of the central nervous system foxp + cd + cd + natural regulatory t cells in dominant self-tolerance and autoimmune disease curing cns autoimmune disease with myelin-reactive foxp + treg regulatory t cells inhibit t cell proliferation and decrease demyelination in mice chronically infected with a coronavirus virus expanded regulatory t cells control disease severity in the theiler's virus mouse model of ms virus-specific regulatory t cells ameliorate encephalitis by repressing effector t cell functions from priming to effector stages the authors would like to thank natasha towne, kate stenson, and megan mcconnell for their technical assistance and jennifer powers for the facs purification. this work was supported by the national institutes of health grant ns . the authors declare no financial conflicts of interest.authors' contributions cs designed and performed the experiments, collected and analyzed the data, and wrote the manuscript. ccb interpreted the data and wrote the manuscript. mg assisted with the experiments and edited the manuscript. sas designed the research, provided the materials, interpreted the data, and wrote the manuscript. all authors read and approved the final manuscript. key: cord- -cob hf q authors: otter, j. a. title: the inaugural healthcare infection society middle east summit: ‘no action today. no cure tomorrow.’ date: - - journal: journal of hospital infection doi: . /j.jhin. . . sha: doc_id: cord_uid: cob hf q nan the healthcare infection society (his) decided to run its spring meeting in dubai this year as the inaugural his middle east summit. the conference was well attended, with delegates from all over the world. most of the presentations can be viewed on the his website. the conference opened with professor tawfik khoja outlining the challenges to infection prevention and control in the middle east. professor khoja focused his thoughts on the impressive joint gulf plan for infection prevention ( e ). this strategy document aims to raise the standards of infection prevention and control in the region, and is already yielding success. dr tim boswell then followed with a presentation describing the challenges in europe. among the challenges he covered were public reporting and external scrutiny, hand hygiene, antibiotic resistance, the healthcare environment, surveillance and outbreaks, an increasingly elderly population, new threats [such as ebola and middle east respiratory syndrome coronavirus (mers-cov)], meticillinresistant staphylococcus aureus (mrsa), c. difficile, and invasive devices and new complex equipment. to these i would add the increasingly cost-constrained financial environment that we face in europe. how can we invest in infection control when some hospitals can't afford to buy new pens? the next session covered viruses with pandemic potential: mers-cov, influenza and ebola (although only the first two really have pandemic potential!). dr ali omrani gave an extremely current overview of mers-cov, tracking the outbreak in south korea with up-to-the-minute slides. this illustrated how quickly the picture can change with a pandemic virus such as mers-cov. one striking aspect of dr omrani's talk was findings from saudi arabia that . % of the population are likely to have encountered mers-cov, since this is the proportion of a large community sample who were seropositive for mers-cov antibodies. this suggests a sizeable and previously unrecognized burden of asymptomatic exposure e and possibly shedding. furthermore, animal handlers are more likely to have mers-cov seropositivity, reinforcing the link between animals and mers-cov. dr omrani then discussed sharp spikes of mers-cov in jeddah, saudi arabia in , and the recent outbreak in south korea, attributing both to breakdowns in simple infection control. dr nick phin from public health england (phe) then discussed influenza, and carole fry preparedness for ebola. dr phin highlighted a useful cdc toolkit providing advice on respiratory protection for healthcare workers, and also a recent bmj review concluding that facemasks may help to prevent the spread of respiratory viruses in the community. , carole fry added some cultural perspective to ebola considerations: there are still some 'ebola deniers' in west africa! in terms of containing ebola, the british approach of using trexler isolators is safe for staff, but pretty miserable for patients. is this an appropriate trade-off? also, our careful use of personal protective equipment (ppe) for ebola has highlighted our careless use of ppe for other organisms! there are some parallels in preparing for all three of these unlikely but potentially very serious threats (mers-cov, ebola, and influenza). one of the most important challenges is dealing with the paranoia that always seems to engulf these preparations! valerie harmon delivered a useful lecture on achieving hand hygiene compliance. self-reported hand hygiene compliance rates are usually reported to be > %, but who would believe this with such a huge conflict of interest? the problem with using human beings to monitor hand hygiene compliance is that the moment another person is there, compliance improves! so, automation of hand hygiene compliance monitoring seems the best way forward. valerie's colleagues demonstrated a stateof-the-art automated hand hygiene compliance monitoring approach based on google glass. although the technology was rather prototype, the principle is there, and it seems likely that automated hand hygiene monitoring systems will come into play over the next few years. but this will not alter a fundamental problem: self-protection is a large and rather unhelpful driver for hand hygiene compliance. accurate monitoring of hand hygiene compliance using technology is important, but it must be combined with effective education to help staff to overcome themselves to comply. tim boswell's talk on the environment summarized the evidence that contaminated surfaces make an important contribution to the transmission of key pathogens, including: mrsa, c. difficile, vancomycin-resistant enterococci, norovirus, and acinetobacter baumannii. this is demonstrated most convincingly by the 'prior room occupancy' studies, showing that admission to a room previously occupied by a patient with these pathogens is a risk factor for acquisition for the incoming occupant. underpinning this are the poor levels of conventional cleaning and disinfection, illuminated by techniques such as fluorescent marking of surfaces to evaluate the cleaning process. a failure to eliminate key pathogens from hospital surfaces by cleaning and disinfection processes designed to do just this at the time of patient discharge has been demonstrated for multiple pathogens. automated room disinfection systems (principally hydrogen peroxide and ultraviolet systems) offer the potential to reduce or remove reliance on the operator to assure adequate distribution and contact time of a disinfectant e and can help to reduce the transmission of hospital pathogens. professor tibor pal provided an overview of the concerning epidemiology of multidrug-resistant gram-negative rods (mdr-gnr) in the arabian peninsula: lots of overseas healthcare and travel, and huge antibiotic usage is a toxic mix in this regard. professor pal's view is that all carbapenemases are not equal: oxa- is weedy and klebsiella pneumoniae carbapenemase (kpc)/new delhi metallo-beta-lactamase (ndm) are scary! e not least due to the emergence of resistance to last-line agents such as colistin. it does seem from the limited available data that the rate of carbapenem-resistant enterobacteriaceae (cre) in the middle east is considerably higher than in europe, and increasing fast! switching to the non-fermenters, professor pal highlighted the remarkable environmental survival properties of acinetobacter, combined with a propensity towards antibiotic resistance. although data are limited, it seems that around % of acinetobacter in the arabian peninsula are carbapenem resistant. i was next to speak, and i outlined approaches to mdr-gnr control in europe based on current guidelines. cre is a big deal in europe, especially in the uk, and has prompted unprecedented action on a national level. one key question is: do we go universal or targeted? there has been much discussion recently about abandoning traditional targeted (also known as vertical) approaches in favour of universal (horizontal). interestingly, all guidelines that i reviewed favoured a targeted approach for mdr-gnr, centred around screening and isolation of carriers. we are hamstrung by the lack of high quality studies telling us with any certainty what works to control mdr-gnr. also, how do you go about producing good guidelines? plus, importantly, how do good guidelines translate through a good policy into good practice? as to which interventions we should use for each organism, this depends on organism and setting, although screening, isolation, stewardship, hand hygiene, and cleaning/ disinfection are the pillars of infection control. but what do we do about the more controversial areas: decolonization, screening of staff, cohorting staff and patients, environment screening, and education? dr muhammad halwani then gave an overview of infection control in the middle east, focusing on acinetobacter and pseudomonas. his comprehensive review set out prevalence and resistance rates across the region, highlighting limited surveillance data e but high rates where data are available. dr halwani then reviewed the guidelines available in the region, most importantly the guidelines for isolation precautions, and the gcc strategic plan for combatting antimicrobial resistance (which has a most apt tagline: 'no action today. no cure tomorrow.'). professor david leaper gave an entertaining critique of the evidence base for infection prevention. he began by highlighting the drying of the antibiotic pipeline: the 'variations on a theme' are losing efficacy due to resistance. there is strong evidence for some interventions to prevent infection, for example, the timing of antibiotic prophylaxis prior to surgery, but other areas are more controversial. professor leaper cited the example of nasal decolonization for meticillin-susceptible s. aureus, based on this study being less convincing than you may think. but can we expect a randomized controlled trial for everything? even for parachutes when jumping out of aeroplanes? finally, carole fry and martin kiernan gave an engaging overview with uk perspectives on reductions in mrsa and clostridium difficile infection. carole described the political situation in the early s, with daily stories in the papers about the mrsa 'scandal'. this led to huge scrutiny and political drive for improvement, which has been successful contrary to the predictions of many (most?) experts! but what has caused the rate of mrsa to fall so dramatically? with many interventions during the same period, it is impossible to tell. but the high degree of scrutiny almost certainly played a key role. martin gave some useful guidance about performing an effective root cause analysis (to get to the root of the problem!): make sure the right stakeholders are around the table (junior doctors won't do); be a toddler (ask why? why? why?), document and evaluate improvement. on a personal note, the conference was very enjoyable. it had a good mixture of delegates from the middle east and elsewhere, and the talks prompted much interesting discussion! presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study hospital respiratory protection program toolkit facemasks for the prevention of infection in healthcare and community settings self-protection as a driver for hand hygiene among healthcare workers preventing surgicalsite infections in nasal carriers of staphylococcus aureus parachute use to prevent death and major trauma related to gravitational challenge: systematic review of randomised controlled trials author is a consultant to gama. key: cord- -knd avhu authors: mulpuru, sunita; aaron, shawn d.; ronksley, paul e.; lawrence, nadine; forster, alan j. title: hospital resource utilization and patient outcomes associated with respiratory viral testing in hospitalized patients date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: knd avhu testing patients for respiratory viruses should guide isolation precautions and provide a rationale for antimicrobial drug therapies, but few studies have evaluated these assumptions. to determine the association between viral testing, patient outcomes, and care processes, we identified adults hospitalized with respiratory symptoms from through at a large, academic, tertiary hospital in canada. viral testing was performed in % ( , / , ) of hospital admissions and was not associated with reduced odds for death (odds ratio . , % ci . – . ) or longer length of stay (+ day for those tested). viral testing resulted in more resource utilization, including intensive care unit admission, but positive test results were not associated with less antibiotic use or shorter duration of isolation. results suggest that health care providers do not use viral test results in making management decisions at this hospital. further research is needed to evaluate the effectiveness of respiratory infection control policies. i n , the coronavirus responsible for the severe acute respiratory syndrome (sars) outbreak infected and killed , persons worldwide ( ) . it was quickly recognized that this virus spread between close contacts, because % of infected case-patients were health care workers caring for patients infected with the sars coronavirus ( , ) . during the outbreak, respiratory infection control policies were developed by clinical infectious disease and public health experts, and their use was mandated in all canadian hospitals. these measures were attributed to the eventual control of the outbreak ( ) ( ) ( ) ( ) . as a result, infection control practices, including strict hand hygiene, viral testing of patient samples, and use of isolation precautions, quarantine rooms, and personal protective equipment, were mandated for routine use with all patients who sought treatment at emergency departments (eds) with respiratory symptoms and fever ( , ) . national guidelines suggest that patients admitted to acute care hospitals with infectious respiratory symptoms should receive screening for viral infections by answering symptom-based questionnaires, and they should be placed under droplet isolation precautions until definitive evidence rules out a transmissible respiratory illness ( , ) . viral testing in this setting is carried out with a nasopharyngeal (np) swab sample, which is processed by direct fluorescent antibody (dfa), pcr, or both to identify a viral pathogen. viral testing in these patients should improve diagnostic clarity, reduce the number of subsequent diagnostic tests and procedures required, and prevent infection transmission to other patients and health care workers by guiding the use of isolation precautions. however, these outcomes can only occur if physicians and infection control practitioners assess the results of the viral test and feel confident ruling out viral disease on the basis of the results. to date, whether respiratory viral testing in patients improves outcomes or care processes has not been proven in large studies. two small studies demonstrated that knowledge of the viral test results did not affect length of stay and subsequent antibiotic use ( , ) . however, previous study demonstrated reduced length of stay, mortality, and cost when using viral testing ( ) . these studies were limited by the following: relatively small sample sizes; only single winter seasons being evaluated; and utilization of hospital resources, including isolation precautions, not being assessed ( ) ( ) ( ) . to address this gap in evidence, we set main objectives for this study. first, we aimed to determine the association between the use of viral testing and subsequent hospital resource utilization (antibiotic/antiviral drugs prescribed; radiology studies conducted; cultures and bronchoscopies performed), including the duration of isolation precautions. second, we aimed to determine whether viral testing was associated with in-hospital deaths, admission to intensive care, and length of stay in the hospital. we conducted a large retrospective observational cohort analysis based at the ottawa hospital (toh), an adult academic hospital located in ottawa, ontario, canada, with ≈ , inpatient beds. toh is a tertiary care referral center that provides care for . million patients in the eastern ontario region. we created the study cohort using hospital administrative and clinical data from the ottawa hospital data warehouse, a relational database containing information from toh's patient registration system, the clinical data repository (containing laboratory, pharmacy, radiology, and clinical notes), and the discharge abstract database. data from these operational systems are loaded into the database on a daily basis and linked by patient unique identifiers. extensive assessments of data quality were performed during the development of the database. we identified hospitalizations of adult patients from january , , through december , . hospitalizations were included if the patient was admitted through the ed with any combination of cough, fever, or shortness of breath. we excluded hospitalizations resulting from a transfer from another health institution (such as a long-term care facility or another regional hospital). if a patient had been seen in the ed with respiratory symptoms but was not subsequently admitted, the patient was also excluded from the study. the np swab sample, processed by dfa or pcr, was the exposure of interest for each hospitalization. the standard of practice at our center during the study period was to process np swab samples with dfa. however, during the influenza a(h n ) pandemic season, multiplex pcr was used to detect viruses. the multiplex pcr can detect influenza a or b, respiratory syncytial virus, parainfluenza virus, enterovirus, adenovirus, human metapneumovirus, rhinovius, and coronavirus. we developed and validated an algorithm within our dataset to determine whether np swab samples were analyzed, and categorized them as positive or negative on the basis of the dfa or pcr result (positive test refers to identification of a respiratory virus). the primary outcomes considered in this study were number of inpatient deaths, and length of hospital stay. secondary outcomes included admission to the intensive care unit (icu) and measures of resource utilization, including antibiotic and antiviral prescriptions, chest radiograph and computed tomography imaging, blood and sputum cultures, bronchoscopy, and use and duration of isolation precautions in the hospital. the unit of analysis in this study was the patient's hospitalization. patient characteristics were compared across groups (with and without viral testing performed) and were described by using proportions, means with sds, and medians with interquartile range when appropriate. using similar methods, we then compared groups with positive and negative np swab sample results among the hospitalizations in which an np swab sample was analyzed. we assessed the difference of means and sd for continuous variables using a -way analysis of variance test (anova) and for differences between proportions using a χ test. for all statistical tests, p< . was considered statistically significant. we measured patient coexisting conditions using the elixhauser score ( , ) , a validated scoring system which summarizes comorbid illness and can predict the patient's risk of death in the hospital ( ) . it was derived and validated by using hospitalization data from toh, and the score was based on the original comorbidity diagnosis groups in the elixhauser comorbidity classification system ( , ) . the elixhauser summary score ranges from a minimum of - to + , which are associated, respectively, with a . % and . % risk of in-hospital death ( ) . baseline risk of death at the time of hospitalization was calculated for each hospitalization by using a regression model previously validated by data from toh's patient population ( , ) . we defined influenza seasons on the basis of dates recorded in the public health agency of canada's national surveillance system for influenza, flu-watch ( ). we used this information to categorize hospital admissions according to whether or not they occurred during an influenza season. we created multivariate logistic regression models to investigate whether having an np swab sample obtained and tested was associated with probability of death and icu admission. for each outcome in which the patient died or was admitted to the icu, univariate odds ratios (ors) were calculated for patient sociodemographic factors, clinical factors related to the hospitalization, patient comorbidity, and whether the patient was admitted to the hospital during influenza season. a multivariate model was created on the basis of significant predictors of death and icu admission and was reduced by using stepwise variable selection. we used unadjusted and adjusted linear regression models to determine the change in length of stay in hospital when an np swab sample was tested during the admission process. we used a natural logarithm transformation of the length of stay variable to adjust for the left skewed distribution of this variable. in a secondary analysis, the same methods were used to develop multivariate logistic regression models to investigate the association between the np swab sample testing (positive or negative test result) and odds of death and icu admission. multivariate linear regression was used to evaluate length of stay. we conducted a sensitivity analysis of a subgroup of hospitalizations in which the most responsible discharge diagnosis was a pulmonary infection or exacerbation. this was done to account for the fact that patients seeking treatment for respiratory symptoms could have received a diagnosis of a noninfectious condition (such as heart failure or pulmonary embolism). we limited the discharge diagnosis to diagnoses of respiratory infections or exacerbations to determine whether there was any effect on the study outcomes. in this group, we assessed the potential association between having an np swab sample tested and clinical outcomes (online technical appendix table , http://wwwnc. cdc.gov/eid/article/ / / - .pdf). all analyses were conducted by using sas . statistical software (sas institute inc., cary, nc, usa). this study was approved by the ottawa health sciences network research ethics board, and a waiver of patient consent was granted. during the -year study period, we identified , hospital admissions in which the patient sought treatment at the ed reporting chief symptoms of fever and/or cough and/or shortness of breath. these admissions represented , unique patients. baseline characteristics of the study cohort are described in table . an np swab sample was tested in % ( , / , ) of admissions. overall, patients who had an np swab sample tested were younger, more likely to be admitted during influenza season, and more likely to be female. table describes likelihood of deaths, icu admission, length of stay, and use of isolation precautions in the study cohort and among hospitalizations in which the patient had a positive or negative np swab sample. during hospitalizations in which an np swab sample was tested, length of stay in hospital was longer ( . days vs. . days, p< . ) and mean duration of isolation precautions was longer ( . days vs. . days, p< . ) than in hospitalizations in which an np swab sample was not tested. there was no significant difference in the mean number of days spent in isolation between hospitalizations in which the patient had positive or negative np swab samples ( . ± . vs. . ± . days, p = . ). table describes the use of hospital resources (antibiotic drugs, antiviral drugs, chest imaging studies, cultures, and bronchoscopy) among hospitalized patients with positive and negative np swab samples. among hospitalizations in which the sample was positive ( / , ) and hospitalizations in which it was negative ( , / , ), no significant differences were found in process of care variables, with exception of more antiviral drug use and less use of computed tomography chest scans in the group with positive swab samples. hospitalizations in which an np swab sample was analyzed used statistically more resources than those in which no swab sample was tested (p< . , for all hospital resources measured). after adjustment for confounding variables, there was no association between having an np swab sample tested in the hospital and odds of death (or . , % ci . - . ). we identified a significant increase in icu admission when a patient's np swab sample had been tested (or . , % ci . - . ). finally, linear regression analysis demonstrated a nonsignificant -day increase in length of stay among hospitalized patients for whom a sample was tested (p = . ; ors with % cis are shown in online technical appendix table ). among the hospitalizations in which an np swab sample was tested (n = , ), no significant associations were found between a positive swab sample and odds of death, icu admission, or length of stay (online technical appendix table ). in a restricted cohort of hospitalized patients in which an infectious respiratory diagnosis was made (n = , ), the fact that an np swab specimen was tested was not associated with reduction in chance of death but was significantly associated with increased icu admission. length of stay was also significantly increased by day ( % ci . - . days, p = . ), which was not the case in the primary analysis (online technical appendix table ). in this study, viral testing of respiratory samples during hospitalization was not associated with a significant reduction in odds of patient deaths or length of hospital stay after adjustment for critical clinical confounding factors. viral testing, however, was associated with increased likelihood of admission to the icu. our study also did not find that respiratory viral testing was associated with significant reductions in antibiotic use, chest imaging studies, bronchoscopy, or microbiologic cultures among patients with infectious respiratory symptoms. most notably, a positive viral test result did not lead to significant reductions in antibiotic use, number of chest radiographs obtained, and number of blood cultures requested. it is plausible that lack of any observable beneficial effect on these outcomes is a result of health care providers neglecting to adjust care processes on the basis of the testing results. although more isolation precautions were used with patients with positive viral test results than with those with negative test results ( % vs. %, p< . ), the test result did not influence the duration of isolation precautions. no statistical difference was found in the mean number of isolation-days between hospitalizations in which positive and negative viral test results were obtained ( . days vs. . days, p = . ). this finding could have several potential causes, however. first, health care providers may not be translating negative test results into the action of removing isolation precautions because of lack of infection control directives for front-line staff (nurses and physicians) to guide the safe removal of isolation precautions. as a result, patients may remain under isolation precautions for a standard fixed duration, regardless of the viral test result. second, perhaps front-line staff fear the possibility of infection transmission (even when the np swab sample is negative) and continue the precautions as a conservative measure. we found that hospitalized patients for whom np swab samples were tested had a greater chance of icu admission, after adjustment for confounders, including admission during influenza season, isolation status, and baseline risk for death. this observation remains unexplained. it may be due to residual confounding, but it is also conceivable that obtaining the np swab sample and subsequent isolation precautions may put some patients at risk for adverse events that require icu admission. abad et al. conducted a systematic review and found that isolation precautions are associated with greater adverse drug events, less physician and nurse care, and increased patient scores for anxiety and depression ( ) . in a prospective study, stelfox et al. found that patients in isolation experienced more preventable adverse events in the hospital, made more formal complaints to the hospital about their care, were more likely to have had no vital signs done when ordered, and had more days with no physician progress notes, when compared with nonisolated controls ( ) . relatively few studies have evaluated the effects of respiratory viral testing on processes of care and clinical ( ) . their overall conclusion was that early knowledge of the viral test result did not significantly reduce the duration of antibiotics, or costs, when compared with those of a group in which the viral test results were not made available ( ) . the results of these small prospective studies are generally congruent with our results. however, we also found that a positive viral test result did not affect other processes of care, including whether blood and sputum cultures, bronchoscopy, and chest radiographs were obtained and, most notably, duration of isolation precautions. our study also examined the outcomes of patient death and icu admission, which were not addressed in the previous studies. our study has several strengths. it is the largest study conducted to evaluate the effects of respiratory viral testing on clinical outcomes in adult hospitalized patients. also, our data spanned years, including seasons of viral infections. given our sample size, all adjusted regression models had adequate power to evaluate the chance of death and icu admission outcomes ( ) . our study also has several limitations. the retrospective nature of this study makes the results vulnerable to unmeasured confounding. we accounted for temporal confounding due to influenza seasonality and for confounding by indication using validated measures of baseline mortality risk and comorbidity in the adjusted regression models. however, we did not capture acute vital signs and other nonlaboratory clinical data at the time patients sought treatment, which may have influenced the outcomes we studied. finally, the study was conducted by using data from a single academic center, which has implications for the generalization of these results to other medical institutions. however, the tertiary care hospital in this study follows national and international recommendations for infection control practices, which reduces the likelihood that practices would be significantly different from other major medical centers. our results suggest that in this academic center during the study period, respiratory viral testing did not achieve the goals of reducing antibiotic prescriptions and other diagnostic tests, nor did it result in timely discontinuation of isolation precautions. because the duration of isolation is not guided by the viral test result, one questions whether the process of viral testing is helping reduce viral infection transmission in the hospital. we could not assess infection transmission in this study, but future work is required in this area. our findings should encourage hospital administrators and infection control practitioners to reevaluate current practices, so that viral test results are used appropriately to modify subsequent treatments and guide provision of isolation precautions. this study sets the foundation for further research to ensure that current policies and practices result in efficient resource utilization and prevent infection transmission in hospitals. world health organization global alert and response. summary of probable sars cases with onset of illness from epidemic viral pneumonia preventing respiratory illnesses: protecting patients and staff. recommended infection control and surveillance standards for febrile respiratory illness (fri) in non-outbreak conditions identification and containment of an outbreak of sars in a community hospital the sars outbreak and its impact on infection control practices transmission of influenza: implications for control in health care settings routine practices and additional precautions for preventing the transmission of infection in healthcare settings best practices for infection prevention and control programs in ontario in all health care settings no impact of early real-time pcr screening for respiratory viruses on length of stay and use of antibiotics in elderly patients hospitalized with symptoms of a respiratory tract infection in a single center in norway impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study comorbidity measures for use with administrative data a modification of the elixhauser comorbidity measures into a point system for hospital death using administrative data the kaiser permanente inpatient risk adjustment methodology was valid in an external patient population risk-adjusting hospital inpatient mortality using automated inpatient, outpatient, and laboratory databases adverse effects of isolation in hospitalised patients: a systematic review safety of patients isolated for infection control impact of the rapid diagnosis of influenza on physician decisionmaking and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial effect of point-of-care influenza testing on management of febrile children clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial the effect of rapid diagnostic testing for influenza on the reduction of antibiotic use in paediatric emergency department a simulation study of the number of events per variable in logistic regression analysis key: cord- -dmb x authors: lee, sujin; nguyen, minh trang title: recent advances of vaccine adjuvants for infectious diseases date: - - journal: immune netw doi: . /in. . . . sha: doc_id: cord_uid: dmb x vaccines are the most effective and cost-efficient method for preventing diseases caused by infectious pathogens. despite the great success of vaccines, development of safe and strong vaccines is still required for emerging new pathogens, re-emerging old pathogens, and in order to improve the inadequate protection conferred by existing vaccines. one of the most important strategies for the development of effective new vaccines is the selection and usage of a suitable adjuvant. immunologic adjuvants are essential for enhancing vaccine potency by improvement of the humoral and/or cell-mediated immune response to vaccine antigens. thus, formulation of vaccines with appropriate adjuvants is an attractive approach towards eliciting protective and long-lasting immunity in humans. however, only a limited number of adjuvants is licensed for human vaccines due to concerns about safety and toxicity. we summarize current knowledge about the potential benefits of adjuvants, the characteristics of adjuvants and the mechanisms of adjuvants in human vaccines. adjuvants have diverse modes of action and should be selected for use on the basis of the type of immune response that is desired for a particular vaccine. better understanding of current adjuvants will help exploring new adjuvant formulations and facilitate rational design of vaccines against infectious diseases. vaccines are the most effective and cost-efficient method for preventing diseases caused by infectious pathogens. despite the great success of vaccines, development of safe and strong vaccines is still required for emerging new pathogens, re-emerging old pathogens, and in order to improve the inadequate protection conferred by existing vaccines. one of the most important strategies for the development of effective new vaccines is the selection and usage of a suitable adjuvant. immunologic adjuvants are essential for enhancing vaccine potency by improvement of the humoral and/or cell-mediated immune response to vaccine antigens. thus, formulation of vaccines with appropriate adjuvants is an attractive approach towards eliciting protective and long-lasting immunity in humans. however, only a limited number of adjuvants is licensed for human vaccines due to concerns about safety and toxicity. we summarize current knowledge about the potential benefits of adjuvants, the characteristics of adjuvants and the mechanisms of adjuvants in human vaccines. adjuvants have diverse modes of action and should be selected for use on the basis of the type of immune response that is desired for a particular vaccine. better understanding of current adjuvants will help exploring new adjuvant formulations and facilitate rational design of vaccines against infectious diseases. [immune network ; ( ): [ ] [ ] [ ] [ ] [ ] [ ] [ ] keywords: vaccine, adjuvant, infectious disease, innate immunity, adaptive immunity vaccines infectious diseases remain the second leading cause of death worldwide after cardiovascular disease, but the leading cause of death in infants and children ( ) . vaccination is the most efficient tool for preventing a variety of infectious diseases. the ultimate goal of vaccination is to generate a pathogen-specific immune response providing long-lasting protection against infection ( ). despite the significant success of vaccines, development of safe and strong vaccines is still required due to the emergence of new pathogens, re-emergence of old pathogens and suboptimal protection conferred by existing vaccines. recent important emerging or re-emerging diseases were severe acute respiratory syndrome (sars) in , the h n influenza pandemic, and ebola virus in ( ) . last year, the most widespread epidemic of ebola virus caused significant mortality in several west african countries ( ) . as a result, we are aware of pursuing a new approach towards the rapid development of vaccines against emerging diseases. three different types of vaccine are currently used in humans: live-attenuated vaccines, inactivated vaccines and subunit vaccines ( ) . many of the most effective vaccines in use are live-attenuated vaccines. as an attenuated vaccine is composed of a virus or bacterium that can replicate within the host, this type of vaccine elicits robust humoral and cell-mediated immunity (cmi). examples of live-attenuated vaccine include mmr (measles, mumps, rubella), chicken pox, oral polio (sabin), influenza (the seasonal flu nasal spray and the table i . the benefits of adjuvants . decrease the dose of antigen needed (dose sparing) . decrease the number of vaccine doses needed . enhance vaccine efficacy in infants, the elderly and immunocompromised people . increase functional antibody titer . induce more rapid and long-lasting immune responses . induce robust cell-mediated immunity . provide broad protection (cross-reactivity) . facilitate mucosal immunity . overcome antigen competition in combination vaccines h n nasal spray), rotavirus and yellow fever vaccine. inactivated (killed) vaccines (e.g. inactivated polio -salk, hepatitis a) are either heat-inactivated or chemically inactivated particles of the pathogen. although these vaccines are safe and non-infectious, they stimulate only weak, short-lived and often insufficient immunity. thus, large and multiple doses of inactivated vaccine are required to confer protective immunity ( ) . in contrast to live-attenuated vaccines, inactivated vaccines elicit mainly humoral immunity, with little to no induction of cmi. purified or recombinant subunit vaccines derived from non-living vaccine antigens are poorly immunogenic and require the addition of some components to help stimulate protective immunity. in some cases, these vaccines utilize epitopes recognized and bound by antibodies or t-cells. because subunit vaccines contain only an essential part of the antigen instead of the entire microbe, the chances of adverse reactions to the vaccine are relatively low ( ) . subunit vaccines have been made for hepatitis b virus (hbv), influenza virus (injection) and pertussis (part of dtap combined immunization). recently developed subunit vaccines are less immunogenic and reactogenic than traditional vaccines such as live-attenuated, and inactivated vaccines. thus, repeated boost immunizations or the addition of adjuvant are necessary to enhance the efficacy of subunit vaccines. the word "adjuvant" is derived from the latin adjuvare, meaning "to help" or "to aid". adjuvants have been defined as agents added to vaccine formulations that enhance the immunogenicity of antigens and induce protection against infection. vaccines made from live-attenuated or inactivated pathogens can elicit robust protective immune responses because those vaccines contain naturally occurring adjuvants. in contrast, protein-based vaccines in most cases have limited immunogenicity although they have some advantages in terms of safety and cost-effectiveness. thus, adjuvants are necessary to help these proteins become effective vaccines by inducing strong and long-lasting protective immune responses. indeed, some protein-based vaccines have been successfully developed in use for human vaccines by mixing with aluminium salts (alum). however, new vaccine targets will require not only strong antibody responses but also robust cmi including t helper (th) cells and cytotoxic t lymphocytes (ctl). alum alone will be insufficient for such cases because it is a poor inducer of t cell responses. the use of appropriate adjuvants will allow for vaccine formulations that selectively trigger innate immunity and/or adaptive immunity to obtain a desired type of antigen-specific immune responses. we also describe the practical and functional reasons for why adjuvants are needed as a component in vaccines in table i . licensed adjuvants in use for human vaccines are listed in table ii . in , glenny et al. reported the adjuvant activity of aluminium compounds utilizing a suspension of alum-precipitated diphtheria toxoid (dt) ( ) . aluminium salts are the most widely used adjuvants in human vaccines. these adjuvants have been used in practical vaccination for more than years and are generally considered stimulators of th immunity ( , ) . until aluminium salt (referred to as "alum") adjuvants were the only ones contained in vaccines licensed for human use in the united states. alum is a component of licensed human vaccines such as hepatitis a virus (hav), hepatitis b virus (hbv), human papilloma virus (hpv), diphtheria, tetanus, haemophilus influenzae type b (hib) and meningococcal. although there are a number of adjuvants more potent than alum, they have not been used for human vaccine formulations due to high levels of toxicity. surprisingly, despite the wide use of alum adjuvants in licensed human vaccines, the mechanisms of action are not well characterized. the most well-known mechanism of action of alum is the "depot effect", first proposed by glenny in , whereby depot formation was cited to facilitate con- tinuous antigen release from the injection site ( ) . even though depot formation still remains somewhat controversial, recent studies have clearly demonstrated that depot formation is not required for alum adjuvanticity ( ) ( ) ( ) . alum has been shown to facilitate humoral immunity via th type immune responses (igg , ige, il- , il- and eosinophil) ( , ) . the advantages of alum are high safety record, antigen stabilization and augmentation of high and long-lasting antibody titer. however, alum does not have the ability to elicit th type immunity or cytotoxic t cell responses and vaccines containing alum adjuvant cannot be sterilized by filtration, frozen or lyophilized ( ) oil-in-water (o/w) emulsions: mf and adjuvant system (as ) emulsions are unstable two-phase systems consisting of at least two immiscible liquids, combined with a surfactant for stabilization. the major benefits of using emulsions are antigen dose sparing and enhancement of antibody titer. both mf (novartis) and as (glaxosmithkline) are squalene based oil-in-water emulsions ( , ) . mf has been approved for the h n pandemic influenza vaccine (fluad) and also for the h n influenza vaccine (focetria and celtura) in europe ( , ) . it recruits monocytes and macrophages into injection sites by the induction of local chemokine secretion ( ) . mf can also augment antigen uptake by dendritic cells (dcs) and activate cd t cells ( ) . as a result, mf generates high antibody titers with balanced igg :igg a responses. mf has been evaluated in conjunction with herpes simplex virus (hsv), human immunodeficiency virus (hiv), hbv and cytomegalovirus (cmv) vaccine trials ( ) . as is included in licensed h n and h n pandemic influenza vaccines. although both mf and as contain squalene oil, they have different compositions. as contains α-tocopherol. moral et al. demonstrated that as induced a non-specific activation of the immune system in mice in the presence of α-tocopherol ( ) . unfortunately, recent studies have reported a possible association between narcolepsy and the use of as adjuvanted h n influenza vaccine ( , ) . although oil-in-water emulsions seem to be very effective and promising adjuvants, further detailed characterization and analysis of components used in emulsion preparations need to be examined. a virosome is a reconstituted viral envelope possessing membrane lipids and viral glycoproteins, but devoid of viral genetic information ( ) . the virosome vaccine for influenza virus (inflexal v) is approved in europe and hepatitis a virus (epaxal) vaccine is approved in asia, europe and south america ( ). both vaccines utilize virosomes derived from influenza virus represented by immunopotentiating reconstituted influenza virosomes (iriv) harboring the influenza hemagglutinin (ha) protein ( ) . inflexal v is the only viroso- combination enhances antibody titer, th and th type immunity and cd t cell-mediated immunity. combined with saponin and phospholipid. phase mal adjuvanted influenza vaccine licensed for all age groups including children, adults and the elderly. as virosomal adjuvants present antigen via both major histocompability complex (mhc) i and mhc ii, virosomes are able to induce both humoral immunity and cmi ( , ) . major advantages of using virosomes in vaccines are: ) high quality and long-lasting antibody responses, ) conformational stabilization of antigen, ) protection of antigen from degradation, ) excellent safety profile, ) suitability to specific populations such as infants, immunocompromised patients, and the elderly ( ). toll-like receptors (tlr) are transmembrane signaling proteins, comprising a family of pattern recognition receptors (prr) ( ) . tlr agonists, the natural ligands which activate tlrs, are immunostimulatory adjuvants. advances in the design of efficient adjuvants based on the use of tlr agonists have been promising and some of these adjuvants have already been licensed for human vaccines. mpl, a tlr agonist, is a chemically detoxified derivative of the parent lipopolysaccharide (lps) from salmonella minnesota r strain ( ) . mpl increases the production of pro-inflammatory cytokines such as il- and ifn-γ, resulting in the generation of th immune responses ( ) . as is composed of mpl adsorbed to aluminium salts ( ) . two as -adjuvanted vaccines are licensed for human use: the hpv vaccine (cervarix) and hbv vaccine (fendrix) for haemodialised patients ( , ) . since mpl still retains the ability to activate innate immunity by interaction with tlr , it leads to activation of nf-κb signaling and production of pro-inflammatory cyto-kines and chemokines. subsequently, chemokines such as ccl and ccl recruit monocytes and macrophages, and activate dendritic cells (dcs) at the injection site ( ) . mature dcs that have migrated to the draining lymph node can interact with t-cells to stimulate cmi. a benefit of using as adjuvant in human vaccines is the effective induction of robust th -type immune responses by promoting il- and ifn-γ production, which cannot be achieved by using alum alone. a recent study showed that the antigen and as should be co-localized in lymph nodes in order to elicit an adjuvant effect on antigen presenting cells ( ) . table iii summarizes a subset of the adjuvants that have been tested in human clinical trials. all adjuvants listed in table iii are known as "immunostimulators" or "immune potentiators". tlrs provide a bridge between innate and adaptive immunity. a new class of effective vaccine adjuvant is based on the tlr pathway. here, we will focus on tlr , and which are in clinical trials of vaccines against infectious pathogens. tlr is one of the more advanced adjuvant candidates among tlr agonists ( ) . unmethylated cpg oligodeoxynucleotides (odn), a type of tlr agonist, enhance antigen-specific immune responses and induce proinflammatory cytokines such as tnf-α, il- , il- and ifn-γ. cpg odn are an example of immunostimmulatory sequences (iss) currently being evaluated for hbv vaccine (heplisav-b, dynavax) ( ) . polyriboinosinic acid-polyribocytidylic acid (poly i:c) mimics viral dsrna and is a promising candidate for a vaccine adjuvant against intracellular pathogens. poly i:c binds to tlr and enhances robust cmi and potent type i interferon response. however, the major draw-back of stability and toxicity issues need to be addressed before proceeding to clinical application of dsrnas. recently, a clinically safe dsrna, polyi:c analogue (ampligen), was investigated as an adjuvant for intranasal h n influenza virus vaccines ( ) . bacterial flagellin, a tlr agonist, is a known immunostimulator that induces high antibody titer, and mixed th and th type immune responses. the d portion of flagellin binds to tlr and can be expressed in a fusion protein with selected vaccine antigens. due to this characteristic of flagellin, a major advantage of the tlr -dependent adjuvant is that a fusion protein can co-deliver antigen and tlr agonist to the apc ( ) . thus, flagellin fusion proteins are suitable adjuvants for the development of vaccines to induce robust antigen-specific immune responses. indeed, a flagellin/ hemagglutinin-based vaccine (vax ) and a flagellin/matrix protein ectodomain (m e) vaccine (vax ) are in clinical trials of vaccines against influenza ( , ) . although further studies in humans are required, it appears that tlr agonists may be attractive candidates for use in human vaccines. iscoms are another promising lipid-based adjuvant formation. iscoms are spherical and ring-like structures spontaneously formed upon mixing antigens with saponin, cholesterol and phospholipid ( ) . the compound qs- , a potent immunostimulatory saponin, was extensively studied as an adjuvant in various vaccines, though it has not yet been approved for human vaccine use due to the toxicity of qs- . since iscom allows for the reduction in qs- dose, it is being considered as a new approach to overcome the issue of toxicity. the second type of iscom is called iscommatrix, which doesn't contain antigen. the major advantage of iscom and iscommatrix is their exceptional stability owing to the high affinity between saponin and cholesterol, therefore allowing them to be effective adjuvants for mucosal vaccines ( ) . main benefits of these adjuvants are induction of high and long-lasting antibody titer, induction of balanced th and th type immunity, and induction of cmi including cytotoxic t cell response ( ) . the adjuvant properties of iscom and iscommatrix are currently being evaluated in clinical trials of influenza, hcv and hpv. collectively, usage of iscom and iscommatrix as adjuvants could be an alternative approach in vaccine development against infectious pathogens. adjuvant systems (gsk) refer to various combinations of classical adjuvants such as aluminium salts, o/w emulsions, liposomes and immunostimulators designed to adjust the adaptive immune responses against pathogens ( ) . the challenge for this strategy is to define the best combination for an effective and safe formulation in which individual components can synergize with one another to elicit a more robust immune response. as described in table ii , as and as have been approved as adjuvants in several human vaccines. here, we will discuss as and as , which are in recent development. as is identical to as (α-tocopherol＋squalene) with the addition of mpl and qs . while as induces biased th type immune responses, as induces high antibody titer and dominant th type immune responses owing to the addition of mpl. although some local and systemic reactogenicity has been reported, as is in clinical trials for various vaccine applications, including malaria, hbv, hpv, tuberculosis, and hiv ( ) . as combines three components such as liposomes, mpl and qs ( ) . unlike as , as was designed to improve cd t cell responses. as induces robust th type immune responses, enhances antigen presentation to apc, and induces high antibody titer. recent studies demonstrated that an as malaria vaccine induces increased igg titers and polyfunctional cd t cells expressing il- , ifn-γ, tnf-α, or cd l ( ) . several clinical trials are in progress with as -containing vaccine candidates against infectious pathogens, including hiv, tuberculosis and malaria. the ultimate goal of vaccination is to generate potent and long-term protection against diseases. such protective immunity can be elicited by using vaccine formulations containing appropriate antigens and adjuvants. adjuvants are important components of vaccines and can affect the outcomes of vaccination. past approaches of vaccine formulation with adjuvants were focused on single-type adjuvants such as alum or emulsions. however, new vaccine targets require the induction of well-defined cmi in addition to high titer of antibody. consequently, new immunostimulant adjuvants in vaccine formulations are needed in order to stimulate robust immune responses including humoral immunity and cmi. as great progress has been made in the field of adjuvant research over last two decades, vaccinologists are now able to select an appropriate adjuvant from classical adjuvants, immunostimulants or combinations thereof to enhance vaccine efficacy. taken together, recent successful clinical studies conducted with new adjuvants suggest that a panel of novel immunostimulant adjuvants will be utilized for human vaccine formulations in a near future. the availability of these adjuvants in various combinations will facilitate the rational design of vaccines against infectious diseases. infectious diseases: considerations for the st century unmet needs in modern vaccinology: adjuvants to improve the immune response global capacity for emerging infectious disease detection ebola virus disease in west africa--the first months of the epidemic and forward projections vaccine adjuvants: key tools for innovative vaccine design active and passive immunity, vaccine types, excipients and licensing key roles of adjuvants in modern vaccines immunological notes. xvii-xxiv (how) do aluminium adjuvants work? aluminium adjuvants--in retrospect and prospect the antigenic effect of intravenous injection of diphtheria toxin antigen depot is not required for alum adjuvanticity mechanisms of action of adjuvants towards an understanding of the adjuvant action of aluminium new horizons in adjuvants for vaccine development aluminum compounds as vaccine adjuvants the mechanism of action of mf -an innately attractive adjuvant formulation the adjuvanted influenza vaccines with novel adjuvants: experience with the mf -adjuvanted vaccine impact of prior or concomitant seasonal influenza vaccination on mf -adjuvanted h n v vaccine (focetria) in adult and elderly subjects vaccine adjuvants alum and mf induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes the history of mf (®) adjuvant: a phoenix that arose from the ashes adjuvant system as containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity designing and building the next generation of improved vaccine adjuvants formation of virosomes from influenza subunits and liposomes influenza virosomes as a combined vaccine carrier and adjuvant system for prophylactic and therapeutic immunizations applications of influenza virosomes as a delivery system toll-like receptors and innate immunity vaccine adjuvants: putting innate immunity to work bacterial cell wall products as adjuvants: early interferon gamma as a marker for adjuvants that enhance protective immunity glaxosmithkline adjuvant systems in vaccines: concepts, achievements and perspectives safety of human papillomavirus (hpv)- / as -adjuvanted vaccine for cervical cancer prevention: a pooled analysis of clinical trials safety and immunogenicity of a new hepatitis b vaccine for the protection of patients with renal insufficiency including pre-haemodialysis and haemodialysis patients as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity therapeutic potential of toll-like receptor activation the potential of iss adjuvant in hepatitis b vaccines: heplisav review development of mucosal adjuvants for intranasal vaccine for h n influenza viruses use of defined tlr ligands as adjuvants within human vaccines development of vax , a recombinant hemagglutinin (ha) influenza-flagellin fusion vaccine with improved safety and immune response safety and immunogenicity of a recombinant m e-flagellin influenza vaccine (stf . xm e) in healthy adults iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses an overview of adjuvant formulations and delivery systems iscom technology-based matrix m tm adjuvant: success in future vaccines relies on formulation recombinant liver stage antigen- (lsa- ) formulated with as or as is safe, elicits high titer antibody and induces ifn-gamma immunomodulatory properties of the vaccine adjuvant alum alum adjuvanticity: unraveling a century old mystery recent advances in vaccine adjuvants: the development of mf emulsion and polymeric microparticles development and evaluation of as , an adjuvant system containing alpha-tocopherol and squalene in an oil-in-water emulsion adjuvants for pandemic influenza vaccines liposomes as vaccine delivery systems: a review of the recent advances influenza virosomes as vaccine adjuvant and carrier system role of as in human papillomavirus vaccine: mode of action and clinical profile the authors declare no conflict of interest. key: cord- -konm x authors: decaro, nicola; mari, viviana; elia, gabriella; lanave, gianvito; dowgier, giulia; colaianni, maria loredana; martella, vito; buonavoglia, canio title: full-length genome analysis of canine coronavirus type i date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: konm x canine coronavirus types i (ccov-i) and ii (ccov-ii) are usually responsible for mild enteritis in dogs. while the ccov-ii genome has been completely sequenced, to date there are no complete genomic sequence data available publicly for ccov-i. thus, the aim of the present study was to analyze the full-length genome of a ccov-i prototype strain that had been recovered from a dog with diarrhea in italy. ccov-i strain / has a genome of , nucleotides, excluding the ′ poly(a) tail, displaying the typical alphacoronavirus- organization and the highest genetic relatedness to ccov-ii. however, two distinct features were observed in the ccov-i genome: (i) the presence of an additional orf between the spike (s) protein gene and orf a; (ii) the diversity of the s protein, which is more closely related to that of feline coronavirus type i and presents a furin cleavage site. the present study may contribute to a better understanding of the alphacoronavirus- evolutionary pattern and may be paradigmatic of how coronaviruses evolve through gene losses, acquisition and exchanges among different members. coronaviruses (covs) are large, single-stranded, positive-sense rna viruses, which are responsible for enteric and/or respiratory disease in mammals and birds. canine coronavirus (ccov) is usually responsible for mild enteritis in young dogs buonavoglia, , ) , although fatal disease has been associated to a pantropic variant of the virus (decaro et al., , a marinaro et al., ; zicola et al., ; ntafis et al., ) . based on the genetic distance encountered in the spike (s) protein gene (pratelli et al., ) , two ccov genotypes are known, ccov type i (ccov-i) and type ii (ccov-ii), which are variously distributed worldwide (decaro et al., mcelligott et al., ; ntafis et al., ; licitra et al., ; cavalli et al., ; costa et al., ) . ccov-ii has been found to exist in two different subtypes, ccov-iia and ccov-iib, the latter being the result of homologous recombination with transmissible gastroenteritis virus of swine (tgev) (decaro et al., (decaro et al., , b . intermediate viruses between ccov-i and ccov-ii have been also detected . ccov-i and ccov-ii form a unique viral species, alphacoronavirus- (family coronaviridae, genus alphacoronavirus), along with feline coronavirus types i (fcov-i) and ii (fcov-ii), tgev and porcine respiratory coronavirus (prcov) ). an additional orf, named orf , was found in the ccov-i genome, whereas only its remnants were evident in the genomes of ccov-ii and tgev, revealing an intriguing evolutionary history within the alphacoronavirus- species . while the full-length genomes of several strains of ccov-ii have been determined (decaro et al., ) , to date there are no complete genomic sequence data available publicly for ccov-i. thus, the aim of the present study was to analyze the full-length genome of a ccov-i prototype strain that had been recovered from a dog with diarrhea in italy. strain / was detected during an epidemiological survey for ccov in italian dogs with diarrhea . the ill dog, a male german shepherd of weeks of age, belonged to a kennel located in the apulia region, southern italy. the feces were collected by a vet directly from the rectal ampulla into a sterile container during the clinical examination of the dog. ccov-i rna detection in the specimen was obtained by means of genotype-specific pcr and real-time rt-pcr (decaro et al., ) . virus isolation attempts using different cell lines of canine and feline origin were unsuccessful, since ccov-i has not been adapted to the in vitro growth buonavoglia, , ) . the original fecal sample was aliquoted and stored at − • c until rna extraction. an aliquot of the original fecal specimen was clarified by centrifuging at × g for min. one-hundred-forty microliters of the supernatant were then used for rna extraction by means of qiaamp ® viral rna mini kit (qiagen s.p.a., milan, italy), following the manufacturer's protocol and the rna template was stored at − • c until its use. the rna extract was subjected to a previously-established taqman-based real-time rt-pcr assay for rapid detection and quantification of ccov rna , with minor modifications. briefly, a one-step method was adopted using superscript ® iii platinum ® one-step qrt-pcr kit (life technologies srl, milan, italy) and the following -l mixture: l of master mix, l of superscript ® iii rt/platinum taq mix, nm of primers ccov-for and ccov-rev, nm of probe ccov-pb and l of template rna. duplicates of log dilutions of standard rna were analyzed simultaneously in order to obtain a standard curve for absolute quantification. the thermal profile consisted of reverse transcription at • c for min and activation of platinum taq dna polymerase at • c for min, followed by cycles of denaturation at • c for s, annealing at • c for s and extension at • c for s. ccov genotyping was achieved by means of two distinct genotype-specific assays (decaro et al., ) performed by using superscript ® iii platinum ® one-step qrt-pcr kit (life technologies srl) and the following oligonucleotide sets (final concentrations were and nm for primers and probes, respectively): primer pair ccovi-f/ccovi-r and probe ccovi-pb for ccov-i and ccovii-f/ccovii-r and probe ccovii-pb (decaro et al., ) for ccov-ii. the thermal protocol was as described for ccov detection except for different annealing temperatures ( • c and • c for ccov-i and ccov-ii, respectively). overlapping fragments of the genome of ccov-i strain / were obtained through rt-pcr reaction carried out using primer sets designed based on the genome sequence of other alphacoronaviruses and the kit superscript tm one-step rt-pcr for long templates (life technologies srl). additional rt-pcr assays and subsequent sequencing attempts were performed to close gaps between assembled contigs and to sequence unresolved genomic regions using primers designed on the alignment of the reference alphacoronavirus strains. the very and ends were amplified using and race system for rapid amplification of cdna ends (invitrogen), respectively, following the manufacturer's instructions. the pcr products were detected by electrophoresis through a . % agarose gel and visualization under uv light after ethidium bromide staining. rt-pcr products were subjected to direct sequencing at the baseclear b.v. (leiden, the netherlands). the sequences were manually edited and analyzed using the geneious platform (http:// www.geneious.com) and the ncbi's (htttp://www.ncbi.nlm.nih. gov) and embl's (http://www.ebi.ac.uk) analysis tools. nucleotide (nt) sequences of the different orfs were converted into amino acid (aa) sequences and comparative sequence analysis with reference coronavirus sequences was carried out in the full-length genome and encoded structural and nonstructural proteins. phylogenetic and molecular evolutionary analyses were conducted using mega . beta (tamura et al., ) . in order to include in the analysis ccov-iib, whose genome has not been completely sequenced, pylogenetic trees were elaborated on a , genomic sequence (encompassing from the end of orf a to the utr) and on the amino acid (aa) sequences of s, membrane (m), and nucleocapsid (n) proteins using both parsimony and neighbor-joining methods, supplying a statistical support with bootstrapping over replicates. the following alphacoronavirus reference strains were used for phylogeny ( the full-length genome of ccov-i strain / was deposited in genbank under accession number kp . by the real-time rt-pcr panels, the fecal sample was confirmed to contain a ccov-i strain, whose titer was calculated as . × rna copies/l of template. the specimen had no traces of ccov-ii rna. the genome of ccov-i strain / has a size of , nt, excluding the poly(a) tail, and shows typical alphacoronavirus- organization (table and fig. ). the utr consists of nt including the leader sequence (l, nt - ) and the conserved core -cuaaac- (nt - ) of the transcription regulatory sequence (trs), which controls the mrna synthesis through interaction with the viral polymerase during the discontinuous transcription of the negative strand subgenomic rna of the nidovirales members (enjuanes et al., ) . similar trs signals precede each of the putative mrna encoding for the structural and nonstructural proteins ( table ) . the end of the viral genome consists of a -nt utr that is followed by the poly(a) tail. sequence analysis showed intact structural and non-structural proteins with respect to reference ccov-ii, fcov-i and fcov-ii genomes. about two-thirds of the viral genome is occupied by the replicase gene encoding for two large polyproteins (pp), pp a and pp ab, the latter being synthesized through ribosomal slippage at position , . the polyproteins of the replicase complex are processed by viral proteinases, resulting in several products with different size and function. sequence comparison with other alphacoronavirus- genomes led to the detection of three putative papain-like proteinase cleavage sites and putative c-like proteinase cleavage sites, producing nonstructural proteins (table ) . four structural proteins were detected downstream of the replicase gene, namely the spike (s), small envelope (e), membrane (m) and nucleocapsid (n) proteins. the s protein has a size of aa, thus being longer than the analogous protein of glycosylated asn residues. the n protein of strain ccov-i / is -aa long product with three potential n-glycosylation sites. analogously to ccov-ii and fcov-i/ii, some accessory genes were detected between orfs (s-protein gene) and (e-protein gene) and downstream of orf (n-protein gene). the s-e intergenic region contains the canonical three orfs a, b and c, encoding for products with sizes of , and aa, respectively, plus an additional accessory protein gene, orf , encoding for a putative aa protein, which has been found to be unique to the ccov-i genome . the end accessory genes were orfs a and b that encodes for -aa and -aa long proteins, respectively. alignment of complete genome sequences of ccov-i strain / and reference alphacoronaviruses showed the closest genetic relatedness with ccov-iia isolates ( . - . % nt identity), followed by tgev ( . %) and . no comparison was possible with ccov-iib since there are no full-length genomes available in the genbank database for this virus. when the spike protein was analyzed, ccov-i displayed a higher aa identity to fcov-i ( . %) than to ccov-iia/iib ( . - . %), fcov-ii ( . %) and tgev ( . %). among extant ccov strains, the closest identity was observed with isolate a , which has been proven to have a ccov-i/ii recombinant s protein (regan et al., ) . the e, m and n proteins of strain / were all more closely related to the analogous products of ccov-ii and porcine cov reference strains (table ) . in order to include in the analysis the nt sequences available for ccov-iib, phylogeny was first constructed on a , -nt fragment spanning from the end of orf a to the very end of the viral genomes. in the neighbor-joining tree elaborated using these sequences, strain ccov-i / clustered separately from extant ccov/fcov isolates leading to the formation of an outlier branch ( fig. a) . the prototype ccov-i strain formed a separate branch from other analyzed alphacoronaviruses also in the trees elaborated using the m (fig. c) and n (fig. d) proteins, whereas the s protein revealed a clustering with fcov-i (fig. d) . the same tree topologies were obtained through phylogenetic analysis using the maximum-parsimony method (data not shown). ccov has progressively emerged as being responsible for moderate to severe enteritis in dogs, with different genotypes and subgenotypes being detected in recent years buonavoglia, , ) . ccov-ii is the oldest genotype, which has been known since (binn et al., ) , whereas ccov-i was discovered only years later thanks to molecular methods, since this virus has not been adapted to grow in vitro (pratelli et al., ) . more recently, two ccov-ii subtypes have been recognized, namely ccov-iia and ccov-iib, on the basis on the relatedness of the spike protein of the latter virus to that of tgev (decaro et al., (decaro et al., , b . in addition, a virulent ccov-iia biotype, strain cb/ , has been proven to cause fatal infections and long-lasting lymphopenia in naturally (buonavoglia et al., ; decaro et al., ; ntafis et al., ; zicola et al., ; pinto et al., ) and experimentally marinaro et al., ) infected dogs. to date, while the ccov-ii genome has been fully determined (decaro et al., ) , only fragments of the genomic end are available for ccov-i. therefore, in the present study we have carried out the complete genome sequence analysis for this canine pathogen. the results showed that the ccov-i genome displays some distinct features with respect to ccov-ii and other alphacoronaviruses. the first finding is the presence of an additional accessory protein gene, orf , which was located between the s protein gene and orf a. this additional gene, which has been recently characterized, was found to be unique to the ccov-i genome, whereas ccov-ii and tgev exhibit only and end remnants . however, fcov strains harboring different forms of table percent (%) identities of ccov-i / to alphacoronavirus- reference strains in the complete genomic sequence (nucleotide, nt) and structural proteins (amino acid, aa). orf and a ccov-i n protein gene have been detected, thus proving the circulation of fcov-i/ccov-i recombinant viruses . another finding of the ccov-i genome is that the s protein gene is closely related to that of fcov-i, whereas the rest of the genome displays a higher relatedness to ccov-ii. in addition, a furin cleavage site leads to the potential generation of two subunits, s and s . a similar basic motif is present, approximately in the same position, in most beta-and gammacoronaviruses. cleavage of the cov s protein has been correlated to cell-fusion activity in vitro but the potential implications in viral pathobiology have not been fully determined (hingley et al., ) . in addition to ccov-i, furin cleavage motifs have been detected in fcov-i, but even in this case the biological consequences were not completely understood (de haan et al., ) . unlike ccov-ii and fcov-ii that display a similar s protein as a consequence of homologous recombination, ccov-i does not grow in cell cultures and only few fcov-i strains have been adapted to grow in vitro. the cov s protein interacts with cell receptors, thus being responsible for binding of virions to the cell surface (enjuanes et al., ) . thus, the different s proteins between ccov-i/fcov-i and ccov-ii/fcov-ii are likely to be responsible for the diverse biological behaviors in cell cultures. the cell receptor for most alphacoronavirus- isolates is the cell surface glycoprotein aminopeptidase n (apn), but there is no evidence for this receptor being used by ccov-i/fcov-i (de haan et al., ) . on the basis of the most recent findings, the evolutionary history of alphacoronavirus- members has been tentatively reconstructed, suggesting that ccov-i and fcov-i are the ancestral viruses from which tgev, ccov-ii and fcov-ii have originated through gene losses and recombination events . our findings corroborate the hypothesis that ccov-i is the ancestor for ccov-ii, 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brazil genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea characterization of a recombinant canine coronavirus with a distinct receptor-binding (s ) domain molecular characterization of a virulent canine coronavirus bgf strain mega : molecular evolutionary genetics analysis (mega) software version . characterization of a recombinant canine coronavirus with a distinct receptor-binding (s ) domain fatal outbreaks in dogs associated with pantropic canine coronavirus in france and belgium the authors are grateful to carlo armenise, arturo gentile and donato narcisi for their technical assistance. key: cord- -hqrd e p authors: rozell, daniel j. title: assessing and managing the risks of potential pandemic pathogen research date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: hqrd e p nan h n virus experiments occur in special facilities (bsl- ϩ) and, using the erasmus mc facility as an example, he estimated that the risks are much lower due to extra physical barrier biosafety measures, lab personnel vaccinations, and available antiviral therapeutics. thus, he estimated the risk of a laboratory-acquired infection (lai) to be less than ϫ Ϫ per person-year. taking into account that any infected lab worker would have already been vaccinated against a homologous h n virus, would be taking antiviral medication, and would be quarantined, fouchier estimated that a lab-induced pandemic would occur every billion years-more than twice the known age of the universe. he concluded with the observation that there have been no confirmed influenza virus lais or releases in decades, which suggests current measures are sufficient. a reply by lipsitch and inglesby ( ) questioned fouchier's claim that virology labs are safer than other bsl- labs. they also noted that fouchier's calculations incorrectly accounted for the uncertainty associated with observed events ( ) . furthermore, the assumption of events was claimed to be unreasonable, because viral lais have occurred in non-u.s. facilities ( ) . in separate comments, klotz ( ) argued that fouchier's calculations were based on the wrong method of calculating the elapsed time of escape for an lai and that the estimate for an lai was too low. a reply by fouchier ( ) argued that klotz did not provide "scientific justification" for higher estimates. within this debate among competing risk estimates, there appears to be disagreement as to not only what constitutes the appropriate methodology, but also what constitutes evidence. for example, fouchier ( ) does not believe that recent laboratory errors (most notably at the cdc) constitute relevant data, because either the errors did not result in lais, the pathogen was not an engineered avian influenza virus, or the work was not conducted specifically in a bsl- ϩ laboratory. however, critics contend that these errors demonstrate the general failure of laboratory safety procedures upon which fouchier's calculations depend. adding to this concern is a study ( ) that estimated a % to % probability that a laboratory escape event would go undetected. likewise, investigative reporting on u.s. labs ( ) suggested that laboratory accident records are poorly tracked, generally underreported, and difficult for the public to access. a review of these various assessments suggests that the most useful contribution of a single independent quantitative risk as-sessment may be to standardize the language of the debate. it is difficult enough to assess the quality of the data and validity of assumptions of each risk assessment. further comparisons are made nearly impossible because of use of inconsistent units (e.g., escape probability, risk per lab-year, and risk per worker-year) and different treatments of uncertainty (e.g., point estimates versus % confidence intervals). by using a single rba as a starting point, hopefully the various stakeholders will at least be able to argue using the same mathematical framework. despite the nih request for a comprehensive quantitative riskbenefit analysis, there is acknowledgment that this may not be possible. during the nrc symposium ( ), both baruch fischhoff and ronald atlas discussed the difficulty of estimating benefits from the gof research or, more generally, any basic research, due to its unpredictable and serendipitous nature. likewise, the public health benefits of gof research are difficult to estimate because they are conditioned on factors outside the laboratory ( ) . that is, while the risk of accidental release is largely controlled by laboratory conditions, the beneficial use of any discovered knowledge depends on the existing public health system, which varies widely among communities, regions, and nations. for example, year into the influenza pandemic, there was still only enough vaccine for one-quarter of the world's population ( ) . further complicating a benefits analysis are the multiple ways in which evidence can be interpreted. for example, during the nrc symposium, it was widely acknowledged that genetic analysis of ppps currently could not predict the resulting phenotype ( ) . critics of gof research argued that this lack of predictive ability severely limits the benefits of this line of research for any practical therapeutic purposes (e.g., vaccine design). however, proponents argued that this lack of knowledge was the very reason that gof research should continue. thus, an argument against the current practical value of the research is being interpreted by others as a supportive argument from the perspective of basic science. in this case, interpretation of a benefit is a subjective value judgment. the gof controversy includes many other value-laden debates regarding risks, benefits, and assessment methodologies. for example, proponents argue that gof research has a unique scientific value ( ) , while critics argue that the scientific value may be no greater than that of safer alternatives, which should be considered an opportunity cost in an rba ( ) . the debate also extends to disagreements regarding: the practical value of gof experiments to policy makers ( , ) , how we should count and compare the various ways of valuing research (e.g., intrinsic value versus instrumental value) ( , ) , and how publication criteria should compare public health risk(s) to scientific merit ( , ) . considerable disagreement even exists regarding ancillary effects, such as the impact of the various moratoria and regulations on the decisions of young scientists to work in virology ( ) ( ) ( ) . the gof controversy even includes debates over definitions. as discussed at the nrc symposium ( ), gof research is already widely used for multiple beneficial and largely benign purposes, including increasing vaccine yields ( ) , expanding genomic sequence surveillance databases ( ) , and creating animal models of human viral infections to aid further research. furthermore, naturally arising gof mutations are common in research labs that work with rna viruses. because the current state of science is unable to predict what genomic changes will increase danger, we cannot be sure what experiments will result in new undesirable traits. proponents of the moratorium argue that the wording was specific enough that only federally funded projects were affected and that public health surveillance and vaccine development activities were exempt ( ) . rather, proponents accuse critics of the moratorium of attempting to widen the definition of what might be banned in hopes of weakening support for any restrictions. this is not the only debate over terminology. it has been argued that the use of the term "pandemic" itself is an "apocalyptic rhetorical device" ( , ) that preempts any reasonable discussion of risks and benefits by appealing to our innate fear of rare but catastrophic events. however, this assumes that the risk of a pandemic is actually rare, despite considerable disagreement among informed scientists regarding the likelihood of such an event. ironically, labeling the use of "pandemic" as rhetorical sophistry may itself be a rhetorical trick if it is used to dismiss a category of serious claims without due consideration of merit. ultimately, the purpose of summarizing and critiquing some of the arguments within the gof/ppp debate is to emphasize the many epistemic and ethical value judgments inherent to rba and to provide evidence for prior claims that a consensus-building quantitative assessment is unlikely ( ). this naturally leads us to wonder if there is a better alternative. fischhoff suggests that, rather than use rba to only inform the eventual policy decision, it should instead be used to improve research design ( ). lipsitch and galvani ( ) made the same argument for improving gof/ppp research design, but in the context of responsible research principles. they argued that most gof/ppp experiments are not ethically justifiable because they do not meet the criterion of yielding humanitarian benefits not attainable by safer alternatives. one approach to improving research design is to use the design principle of inherent safety ( ) ( ) ( ) , which focuses on attempting to eliminate material hazards in research and manufacturing. in contrast, conventional risk management generally focuses on reducing the likelihood of an accident through safety procedures and equipment. the formal inherent safety concept is frequently used in the chemical and nuclear engineering communities but it has not been widely adopted by scientists and engineers in other fields ( ) . while this idea seems to be common sense, it is a departure from most previous work on biosafety and biosecurity ( , ) , which was focused on improving risk management through formalized processes and training. the continued emphasis on these methods is unfortunate, given the generally poor record of implementation ( , ) . an additional benefit of the inherent safety concept is its ability to address security concerns ( ) . for example, a traditional safety measure, such as removing all ignition sources near an explosive material, is of little security value; malevolent actors will bring their own ignition source. likewise, terrorists are attracted to hazards that already instill public dread. inherently safe design makes terrorism more difficult by removing the exploitable hazard. because safety has traditionally been the concern of engineers at the production level, the r&d community often fails to consider these principles in the early stages of research when the most impact can be made ( ) . however, inherent safety in research is sometimes recognized in hindsight. a cdc report ( ) that summarized an internal review of the june exposure of laboratory workers to potentially viable bacillus anthracis at a cdc bio-letter to the editor terrorism response lab noted that an avirulent strain could have been used as a substitute in the experiment. it is also interesting that in its list of responses, the report focused primarily on revised biosafety protocols and procedures. a reference to reducing the hazard (i.e., inherent safety) was made only within the fifth of eight recommendations. the calls for inherently safe design appear to have yielded some consensus from the opposing camps in the gof/ppp controversy. one sign during the nrc symposium was provided by yoshihiro kawaoka, a principal investigator of one of the two original studies that started the debate ( ) , who endorsed the idea that some research could be conducted with alternative techniques, such as loss-of-function studies, use of less-pathogenic viruses, and phenotypic analyses ( ) . proponents of inherently safe ppp research have also been buoyed by recent successes. for example, langlois et al. ( ) showed that species-specific microrna targeting can be used to conduct relevant animal model ppp research that still poses low risks to humans. as michael imperiale stated, "you can develop safer approaches to do these types of experiments; it just needs a little bit of imagination on the part of researchers" ( ) . as summarized here, many of the disagreements within the gof/ppp debate involve epistemic and ethical value judgments that suggest that definitive quantitative risk-benefit analysis is not possible. this does not devalue rba; it is still useful as a tool for engaging experts and the public in a conversation about riskbenefit tradeoffs. however, if calls for rba become knee-jerk responses to what are essentially quantitatively intractable technological risk problems, everyone will be disappointed. rba works best when expectations are realistic. when data are plentiful and there are no moral or cultural differences among the stakeholders, rba can generate "answers" for policy formulation. however, for emerging technologies and controversial research where data are sparse and uncertainty is high, putting a number on a subjective quantity only engenders suspicion. the question of whether the benefits of gof/ppp outweigh the risks is unlikely to be resolved by an independent formal rba. however, this question may become less relevant if safer approaches can achieve the same goals. that is, inherently safe design may be the best compromise solution for the gof/ppp controversy. because the inherent safety principle will not be invoked unless a risk is perceived, the appropriate next step is to regard the eventual results of the rba as a tool for risk exploration, which then inspires more inherently safe research. over the long term, changing the biosafety/biosecurity culture in the life sciences to emphasize inherent safety principles will help avoid similar heated controversies in the future. risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion mbio addresses the pause in gain-offunction (gof) experiments involving pathogens with pandemic potential (ppp) the h n moratorium controversy and debate conducting risk and benefit analysis on gain-of-function research involving pathogens with pandemic potential moratorium on research intended to create novel potential pandemic pathogens the unacceptable risks of a man-made pandemic the consequences of a lab escape of a potential pandemic pathogen. front public health : monitoring select agent theft, loss and release reports in the united states- - airborne transmission of influenza a/h n virus between ferrets potential risks and benefits of gain-of-function research: summary of a workshop studies on influenza virus transmission between ferrets: the public health risks revisited studies on influenza virus transmission between ferrets: the public health risks revisited probability of adverse events that have not yet occurred: a statistical reminder strengthening risk governance in bioscience laboratories comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory comments on fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory containing the accidental laboratory escape of potential pandemic influenza viruses inside america's secretive biolabs great expectations-ethics, avian flu and the value of progress perspective: ill prepared for a pandemic improving pandemic influenza risk assessment an epistemological perspective on the value of gain-of-function experiments involving pathogens with pandemic potential can limited scientific value of potential pandemic pathogen experiments justify the risks? mbio reply to "can limited scientific value of potential pandemic pathogen experiments justify the risks valuing knowledge: a reply to the epistemological perspective on the value of gain-of-function experiments valuing knowledge: a reply to the epistemological perspective on the value of gainof-function experiments an avian h n gain-of-function experiment of great concern the decision to publish an avian h n influenza virus gain-offunction experiment vagueness and costs of the pause on gain-of-function (gof) experiments on pathogens with pandemic potential, including influenza virus a brain drain due to increased regulation of influenza virus research is highly speculative reply to "a brain drain due to increased regulation of influenza virus research is highly speculative influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness use of highly pathogenic avian influenza a (h n ) gain-of-function studies for molecularbased surveillance and pandemic preparedness the apocalypse as a rhetorical device in the influenza virus gain-of-function debate ethical alternatives to experiments with novel potential pandemic pathogens what you don't have, can't leak inherently safer plants how to make inherent safety practice a reality developments in inherent safety: a review of the progress during - and opportunities ahead biosafety risk assessment methodology biosafety controls come under fire can biosecurity be embedded into the culture of the life sciences promoting inherent safety are we too risk-averse for inherent safety? report on the potential exposure to anthrax experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets microrna-based strategy to mitigate the risk of gain-of-function influenza studies key: cord- -by albr authors: van ginkel, frederik w.; padgett, justin; martinez-romero, gisela; miller, matthew s.; joiner, kellye s.; gulley, stephen l. title: age-dependent immune responses and immune protection after avian coronavirus vaccination date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: by albr infectious bronchitis virus (ibv) is an endemic disease of chickens and a major contributor to economic losses for the poultry industry despite vaccination. recent observations indicated that chicks may have an immature immune system immediately after hatching when vaccinated for ibv. therefore we hypothesized that early ibv vaccination will generate an immature, poorly protective ibv-specific immune response contributing to immune escape and persistence of ibv. to test this hypothesis the ibv-specific immune response and immune protection were measured in chicks vaccinated at different ages. this demonstrated a delayed production of igg and iga plasma antibodies in the , and -day-old vaccination groups and also lower iga antibody levels were observed in plasma of the -day-old group. similar observations were made for antibodies in tears. in addition, igg antibodies from the -day-old group had lower avidity indices than day vaccinated birds. the delayed and/or lower antibody response combined with lower igg avidity indices coincided with increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon ibv field strain challenge. the lack of vaccine-mediated protection was most pronounced in the -day-old vaccination group and to a lesser extent the -day-old group, while the -day-old and older chickens were protected. these data strongly support ibv vaccination after day post hatch. ibv is endemic and currently one of the most important causes of economic losses for the poultry industry and represents a continuous threat for this industry. in the past, it was estimated that with the best possible management of flocks ibv infection will reduce income by approximately % when compared to an ibv-free flock [ ] . there is approximately a % vaccine failure for arkansas (ark) serotype of ibv [ ] , the most prevalent vaccine serotype used in the usa. symptoms of ibv infection include, but are not limited to, wet eyes, swollen face, tracheal and kidney lesions, respiratory disease, reduced weight gain in broilers, decreasing and poor egg quality in layers [ , ] . the existence of various ibv serotypes as abbreviations: ark dpi, arkansas delmarva poultry industry; calt, conjunctivaassociated lymphoid tissue; elispot, enzyme-linked immunospot; hg, harderian gland; halt, head-associated lymphoid tissues; hrp, horseradish peroxidase; ibv, infectious bronchitis virus; eid , median embryo infectious dose; tlr, toll-like receptor. * corresponding author. well as antigenic variants [ ] complicates vaccination programs. since immunity induced by vaccination against a single serotype generally provides insufficient protection against other serotypes [ , ] . mucosal immunity plays a role in the control of ibv in chickens as was demonstrated using ibv-resistant and ibv-susceptible inbred chicken lines [ ] . this combined with the finding of gelb et al. [ ] , in which ocular immunization with the massachusetts connaught strain of ibv only on day or on day plus day followed by challenge with massachusetts provided protection of % and % of the chickens, respectively, while the same spf white leghorns only ocularly immunized on day were % protected [ ] . bsa immunization of , and day old broiler chickens obtained very similar results [ ] . this raises questions pertaining the maturity of the immune system and in particular the mucosal immune system, and the ability of chicks to generate a protective immune response when vaccinated at a very young age. conjunctiva-associated lymphoid tissue (calt) and harderian glands do not fully mature as a lymphoid organ until weeks after hatching [ , , [ ] [ ] [ ] . this combined with the practice of immunizing and boosting for ibv early after hatching may set up the immune http://dx.doi.org/ . /j.vaccine. . . - x/© elsevier ltd. all rights reserved. response for failure to protect. the second ibv immunization on day of age, which by itself is fully protective, does not completely compensate for the premature priming on day [ ] . field studies by de wit et al. [ ] demonstrated a lack of protective immunity when birds were boosted between day through day of age. the percentage of birds in a commercial flock positive for ibvspecific igm antibodies was correlated with vaccine protection and increased with the age of boosting. this data supports the notion that early vaccination and boosting of the ibv immune response may limit induction of protective immune responses to ibv. unlike the study by gelb et al. [ ] , the de wit et al. [ ] study can also be interpreted that maternal antibodies interfere with vaccine delivery during the first weeks of life [ ] . further evidence that the immune response may be limited during the first weeks of life comes from the observation that iga levels are undetectable in plasma the first week of life and igm levels are low [ ] . this indicates that immunoglobulin class switching and production of antibodies is very limited during the first week post hatch and therefore chicks are highly dependent on maternal igy antibodies for protection against ibv, which drops ∼ % during the first week of life [ ] . besides diminished b cell response after vaccination, splenic t cells from one week old chickens are also less responsive to polyclonal activation than that of older chickens. the splenic t cells from day old chicks even produce inhibitory factors for proliferation of mature t cells in vitro [ ] . furthermore, splenic lymphocytes of day old chicken displayed better antigen specific proliferation after oral salmonella exposure than day old chicken [ ] . when measuring gene expression in lung and trachea in and week old birds after avian influenza exposure a reduced expression of immune-related genes was shown and included innate immune response genes in the younger birds [ ] . additional evidence that innate immune mechanisms are diminished in young chickens was demonstrated by a lower salmonella phagocytic index of heterophils during the first few days of life [ ] . thus, early exposure to pathogens or vaccines may induce suboptimal innate and adaptive immune responses. based on these observations we hypothesized that early ibv vaccination, i.e., within the first week after hatching, will generate an immature, poorly protective ibv-specific immune response contributing to ibv immune escape and persistence. therefore, the ability of spf chickens of different age to induce an ibv-specific antibody response and protect against challenge with an ibv field strain was measured. our data indicate that early vaccination is suboptimal for induction of ibv-specific immune responses and immune protection. chickens: specific-pathogen-free (spf) white leghorn eggs were obtained from sunrise farms, inc., catskill, ny, hatched and used in all experiments. all hatched chickens were used for the below outlined experiments regardless of sex. chickens were housed in cages in bsl facilities for the duration of the experiment. food and water were provided ad libitum. all experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal use guidelines. auburn university college of veterinary medicine is an association for assessment and accreditation of laboratory animal care (aaalac)-accredited institution. ibv-vaccination and challenge: spf chickens were ocularly vaccinated with × % embryo infectious doses (eid ) of a live attenuated arkdpi ibv vaccine strain (zoetis, new york, ny) in l pbs, which was expanded in our laboratory. chickens were vaccinated day of age and , , or weeks of age. all groups were challenged ocularly with . × eid of the al/ / ibv field strain days after vaccination sample collection: tears were collected as previously described [ ] . blood samples were obtained by puncturing the brachial vein with a sterile g needle into kendall monoject, edta containing, blood collection tubes (tyco healthcare group lp, mansfield, ma) and incubated on ice. blood samples were centrifuged at × g for min. plasma was collected and stored at − • c until tested. ibv propagation and purification for elisa: ibv was propagated in spf white leghorn embryonated chicken eggs (sunrise farms, inc., catskills, ny) by inoculation on day of embryonation as previously reported [ ] . supernatants were titrated for the ibv virus using the reed and muench method [ ] . ibv was treated with . % ␤-propriolactone for min at • c [ ] . inactivation of the virus was confirmed by injection into embryonated eggs. the inactivated ibv was purified based on a previously published protocol [ ] . the virus was then stored at − • c until used. in order to measure igg (igy), iga and igm antibody levels in plasma and tears of chicken, an ibv-specific enzyme-linked immunosorbent assay (elisa) was developed as previously described [ ] . in brief, elisa plates were coated with ␤propriolactone killed, purified ibv at g/ml in carbonate buffer. the plates were blocked with pbs-bsa ( %) after which the samples were loaded at two-fold dilutions. binding of chicken antibodies was detected using biotinylated anti-chicken-igg, -iga and -igm monoclonal antibodies (southern biotechnology associates, inc., birmingham, al) followed by streptavidin-horseradish peroxidase. the plates were developed using tmb ( , , , -tetramethylbensidine; invitrogen corp., frederick, md) substrate. the highest sample dilution with at least an optical density of . above background level at nm was defined as the endpointtiter. ten to thirteen chickens were analyzed per group. the control group consisted out chicken from each age group for iga and igg, which were pooled in one group of since no differences were observed between the controls. for igm levels in plasma the controls (each group containing chickens except day which had ) were displayed separate for each age group. this was done because significant differences were observed between control igm levels to ibv in different age groups. the avidity index was determined as previously described [ , ] using the above described ibv-specific elisa. plasma and tears were diluted : in elisa buffer and were loaded on ␤propiolactone killed ibv coated elisa plates ( g/ml) [ ] . after overnight incubation of these samples at • c, l of increasing concentrations of potassium thiocyanate ( . , . , . , . , . , . , . m kscn) were loaded into the wells and incubated for min at room temperature. after washing the plates, the detection of ibv-specific antibodies was accomplished as previously reported [ ] . the data were normalized to percent inhibition relative to samples not exposed to kscn. the concentration of kscn to inhibit % of the reactivity of the elisa was defined as the avidity index [ , ] . the od read-out for the kscn inhibition data was curve-fitted using -order polynomial regression analyses in microsoft office excell program. the excell provided formula for the inhibition curve was used to determine the x values of y = . , which are the concentrations of kscn inhibiting % of the elisa reactivity, representing the avidity indices of those samples. all samples were analyzed in triplicates and - samples were analyzed per group. the cranial / of tracheae was collected days after ibv challenge with . × eid of al/ / ibv field strain. the tracheae were formalin-fixed and embedded in paraffin. longitudinal m sections were made and were hematoxylin and eosin (h&e) stained and analyzed for mucosal thickness using aperio scan scope and the image j morphometry program (rsb.info.nih.gov/ij/download.html). to measure the mucosal thickness measurements were made at regular intervals on one tracheal ring (see supplemental fig. ). as stated above, histopathology was analyzed in h&e stained tracheal slides days after ibv challenge. besides mucosal thickness (see supplemental fig. ), deciliation (see supplemental fig. ), goblet cells (see supplemental fig. ) and lymphocytes scores (see supplemental fig. ) of the tracheal mucosa were evaluated blindly and scored through based on severity (i.e., normal, mild, moderate, marked, severe). five chickens were used as positive and negative controls, i.e., one of each age group, and - chickens were analyzed for each age group. a visual depiction of the scoring of these parameters is provided in the supplemental data (supplemental figs. - ) . statistical analysis: data were analyzed using a one-way anova test with newman-keuls multi-comparison test or the t-test using graphpad prism software. groups were considered significantly different when p < . . to determine whether age of ibv vaccination affected the humoral immune response, plasma samples were collected and days and tears days after vaccination with × eid of a live-attenuated arkdpi ibv vaccine strain on , , or days of age. the ibv-specific igg endpoint titers in plasma days after vaccination are significantly lower for day vaccinated birds with a mean of . ± . when compared to day , and vaccinated birds, which means vary between . - . . the -day-old group does not differ significantly from day or later vaccinated bird (fig. a) . the igg ibv-specific plasma levels days after vaccination demonstrate, that the day and old vaccination groups chickens were vaccinated at day , , , or of age. unvaccinated chickens of the different age groups served as negative control. the data was analyzed by one way anova with the newman-keuls post-test. the control group contains data points for each age group, which were pooled in one group (n = ). all vaccinated age groups contained between and chickens for igg and iga. a significant difference is observed at p < . and is indicated by different letters. igm levels in ibv vaccinated birds are depicted by squares (n = ) and the controls by circles (n = , d group n = ). for the igm controls the different age groups are shown separately since significant differences were observed between them. a significant increase (p < . ) of igm levels in ibv vaccinated birds over their control group is indicated by a (*). stayed the same, while igg antibody titers in groups vaccinated on day , and still increased (fig. c) . this shows that early vaccination causes a delay in the ibv-specific igg antibody kinetics. the ibv-specific iga plasma antibody titers are not significantly different between groups, although the day vaccinated group mean antibody titer is the lowest of all groups (fig. b) and at least -fold lower than the next lowest group. unlike the igg antibody titers only the day vaccination group increases in mean iga plasma titer on day post vaccination, while day group stays the same and the day , and groups decline in mean iga titer. thus, only the day group increases antibody titers on day when compared to older birds. thus, the day group displays a delayed response in antibody production compared to older birds. the day and day groups have plasma iga titers to ibv that are comparable to, or higher than, the day and groups on day of the response. unlike the day and day vaccination groups the day and groups are declining on day of the response compared to the immune response on day . this indicates that they are past their peak response on day and possibly even on day based on previous observations [ ] . these data are consistent with a delay in the iga plasma response to ibv in birds vaccinated at a younger age and a non-significant decline in mean iga titers in the -day-old group. ibv-specific igm antibody titers were measured in plasma. the plasma samples analyzed were collected on day post vaccination. this time point was selected based on the literature in which the peak igm response was observed between - days after virus challenge or live virus vaccination [ ] [ ] [ ] . due to the variability of ibv-reactive igm in the controls between different age groups, independent controls were included for each age group. the igm levels in the controls decreased considerably by ∼ week of age after ( days after the day old chick vaccination) which increased one week later and stabilized in older age control groups (fig. e) . the day through day control igm levels were significantly higher than in the day and day age group controls. and the day control was significantly lower than the day control igm levels. all igm titers from the ibv vaccinated age groups were significantly higher when compared to their controls (p < . ) with exception of the day age groups (p = . ). this was due to higher control values, which were ∼ . fold higher than in the age or days old groups. this may reflect an initial peak of natural antibodies induced to ibv before stabilizing. the ibv vaccinated age groups did not differ significantly in igm antibody levels to ibv with exception of the day ibv vaccinated group, which mean ± se was . ± . had significantly lower ibv-specific igm levels in plasma than the day ( . ± . ) day ( . ± . ) and day ( . ± . ) vaccinated birds but did not differ significantly from the day ( . ± . ) vaccinated birds (fig. e) . the day igm antibody titers were also significantly lower than those in the day vaccination group but not compared to the other age groups. in tears the ibv-specific igg response is significantly higher in the day and vaccinated groups than in the day and vaccinated chickens for day of the immune response ( fig. a) . the igg endpoint titer in the day immunized group is even significantly lower than the day immunized group, while the day immunized group is intermediate between the day group and chickens vaccinated at an older age. thus, a correlation between age of vaccination and the magnitude of the ibv-specific igg response in tears is observed on day of the ibv-specific immune response. the day and groups have significantly higher iga anti-ibv responses in tears on day after vaccination compared to the day group. the day group is not significantly different from the day and vaccination groups. the day group is also significantly lower than the day and groups (fig. b) . this is likely due to the day group displaying faster kinetics for iga antibody levels in tears after vaccination, rather than a lower response [ ] . the day group iga response is significantly higher than the day and day groups consistent with a delay or deficiency in the mucosal antibody response when vaccinated at an earlier age. our data indicate there is a delay in antibody production when vaccinated at a younger age. the day vaccination group not only displays a delay but also lower levels of antibody production. to determine whether there are not only quantitative differences between antibodies produced when vaccinated on day but also qualitative differences we compared avidity indices for igg and iga antibodies from plasma and tears generated in day old versus fully matured day old birds days after ibv vaccination. as is illustrated in fig. a ,b a significantly higher avidity index is observed for igg plasma antibodies for the day vaccination group when compared to the day vaccinated birds, while no significant difference is observed for iga plasma antibodies. the same observations are also made for tear igg and iga antibodies (fig. c,d) . ciliated cells and goblet cells are the primary target of ibv in the respiratory tract [ ] . fig. displays the deciliation (fig. a ) and goblet cell (fig. b ) scores days after ibv challenge. ibv challenge decreased ciliated epithelial cells and goblet cell score. ciliated cells were fully protected when vaccinated on day of age or later but not when vaccinated on day of age (fig. a) . the protection of goblet cells increases with the age of vaccination and were fully protected when vaccinated on day of age or older (fig. b) . the lymphocytes score and mucosal thickness were also measured in tracheal samples on day post al/ / ibv field strain challenge as indicators of inflammation. as is illustrated in fig. a , a significant decrease in lymphocyte score was observed in the day and vaccination groups when compared to the day group. the day vaccination group did not significantly differ from the day fig. . the ibv-specific igg and iga response in tears. endpoint titers of ibv-specific igg (a) and iga (b) on day of the immune response were measured by elisa. chickens were vaccinated on day , , , or of age. unvaccinated chickens of the different age groups served as negative control. depicted are the means and standard error of each age group and the control group, and different age groups are as described in fig. (n = - per group) . the data was analyzed by one way anova with the newman-keuls post-test. differences were considered significant at p < . and are indicated by different letters. fig. . tracheal deciliation and goblet cell depletion after ibv challenge. to measure the degree of protection against ibv challenge after vaccination tracheal deciliation and goblet cell depletion were measured. chickens were vaccinated at day , , , or of age and challenged days later. unvaccinated/unchallenged chickens of all age groups served as negative control and unvaccinated/ibv challenge chickens as positive control. trachea were collected days post challenge. depicted are the mean and one standard error. for the negative and positive controls n = for the different age groups n = - . the data were analyzed by one way anova with the newman-keuls post-test. significant difference was observed at p < . and are indicated by different letters. and vaccination groups due to a small increase in lymphocyte score in the latter two groups (fig. a) . the day group displayed the highest lymphocytes score from all vaccination groups, which is consistent with the highest inflammatory response to ibv challenge. this was also supported by a significant increase in mucosal thickness in the day vaccinated group when compared with the day , and groups but not day group, which was intermediate between the day and groups vaccinated at an older age (fig. b ). based on our data, the hypothesis that early ibv vaccination will generate an immature, poorly protective ibv-specific immune response, is confirmed. ibv vaccination on day of age, which is routinely performed in the poultry industry, will not be fully protective and as a consequence the chicks remain vulnerable to ibv exposure. thus, early vaccination perpetuates the ibv problems and is a factor in the estimated $ million or more annual loss to the poultry industry due to ibv infection [ ] . our measurements of mucosal and systemic antibody levels demonstrates a delayed production of igg and iga plasma antibodies in the day , day and of age vaccination groups. iga antibody levels in the day group, unlike igg antibodies, do not recover later in the response. besides delayed igg kinetics, the day group displays also a lower avidity index than the day vaccinated group. lower avidity index is not observed in iga antibodies. the delayed and/or lower antibody response and lower igg avidity index translated in increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon ibv challenge when compared to chicks vaccinated later in life. a lack of vaccine-mediated protection is most noticeable in the day of age vaccination group and to a lesser extend the day vaccination group, while the day and older vaccinated chickens are protected. the igm antibodies specific for ibv were significantly elevated above controls on days of the ibv immune response in all age groups except the day group. the day group was not quite significant (p = . ) because of higher igm levels in the control group. this could be due to an initial surge of natural igm antibodies to ibv in this age group. a significant decline in the day old group is observed when comparing the igm antibody levels to ibv in the control day group. this would be consistent with a drop of presumably natural maternal ibv-specific igm antibodies in these spf chickens in the day control age group. these igm antibodies rapidly increases in the day group after which they stabilize in the older age groups. this indicates that a considerable portion of igm antibodies in plasma from the older vaccination groups reacts with ibv without seeing the virus, indicating these are natural antibodies to ibv, which only increase after day of age. bacterial colonization of the intestinal tract of chickens is established during the first two weeks post-hatch [ ] . this, combined with the observation that probiotics enhance natural antibodies in chicken [ ] , indicates that ibv-specific natural igm antibodies to ibv are possibly generated following intestinal colonization presumably by stimulating b cells, which are the main producers of natural igm antibodies in sera of mammals [ ] . in the ibv vaccinated groups we see a steadily incline of igm ibv-specific antibodies in plasma with age as has been reported by de wit et al. [ ] . only the day age group displays significantly lower igm antibody levels when compared with the day - age groups but not with the day age group. this is consistent with an early in life deficiency or delay in the igm response. the lower avidity index for the igg antibodies in -day-old chicks is an important factor contributing to decreased protection to ibv challenge. increased antibody affinity maturation to virus vaccines strongly correlated with better protection [ , ] . a lack of antibody affinity maturation observed following vaccination against respiratory syncytial virus was due to a lack of tlr stimulation [ ] . this indicates that -day-old birds may be deficient in tlr expression. evidence that this is the case comes from a recent publication [ ] demonstrating, that significantly lower levels of tlr expression in spleen and small intestines were observed when comparing -day-old chicks with -to -week-old chickens. inclusion of tlr activating adjuvants could alleviate the problems of early vaccination by boosting antibody production and affinity maturation in -day-old chicks. neither monomeric plasma nor dimeric tears-derived iga [ ] displays this drop in avidity index for the day vaccinated group (fig. b,d) . although there is a lower level of iga antibodies produced by the day vaccinated birds compared to the older groups, which is consistent with a delay in class-switching in the day old group, it is not clear why the lack of a mature mucosal immune system in the -day-old group [ , , [ ] [ ] [ ] did not result in lower affinity maturation of mucosal iga antibodies compared to the -week-old group. another factor influencing early vaccination is the level of maternal antibodies, an issue not addressed in this study. there exists a linear relationship with the hens' plasma antibody levels and transfer of igy to the chicks' circulation [ ] . chicks were over % protected against ibv challenge on day if they had high levels of maternal antibodies but less than % protected when challenged on day . this protection correlated with local respiratory antibodies and not serum antibodies [ ] . ibv-specific maternal antibodies decreased the induction of neutralizing antibodies following boosting [ ] . despite this inhibition by maternal antibodies of the memory response, low or erratic maternal antibody titers to ibv in broiler flocks are associated with ibv-induced economic losses [ ] . this further supports that protection by maternal antibodies, which are predominantly of the igg isotype, is important to prevent activation of an immature immune system that is not capable generating a fully protective immune response early in life. in several studies ibv vaccination was effective against ibv challenge in both spf chickens and commercial broilers when the initial vaccination was performed on day [ ] [ ] [ ] . however, in these studies day vaccination was followed with a second vaccination two weeks later for optimal protection against challenge, which would have masked the relative poor ibv-specific immune responses after the day immunization. extensive immunization on the day of hatch containing three different live attenuated viruses, which caused severe vaccine symptoms, provided similar protection as two single live attenuated ibv virus vaccines on day of hatch and day of age when challenged with a heterologous virus [ ] . this seems to indicate that induction of cross-protective immunity may be less impaired when vaccinated early in life. however, no direct comparison of the same vaccination protocol was analyzed between these two challenge groups, which makes this data harder to interpret in the context of age-dependent immune responses. a decreased humoral immune response to vaccines early in life as seen in chickens is also observed in humans. neonates are highly dependent upon passively acquired maternal antibodies, since their humoral immune system remains underdeveloped [ ] . these passively obtained antibodies in infants can alter humoral and antibody-dependent immune responses to vaccines [ , ] . in broiler chicks maternal antibodies to pathogens persisted only for ∼ days [ ] . in our study interference of maternal antibodies was excluded. the observation that -day-old and -day-old birds are not fully protected when vaccinated are confirming the importance of pathogen-specific maternal antibodies during this period. in summary we can say that ibv vaccination of chickens on day of age contribute to the ibv problem in the poultry industry by inducing lower levels and/or slower kinetics of antibody production as well as lower avidity igg antibodies. this results in a poorly protective immune response as is demonstrated by subsequent ibv field strain challenge. therefore, it is advisable for the poultry industry based on our data to change their practice from vaccinating chicks on day of age to vaccinating after day of age. infectious bronchitis infectious bronchitis virus field 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oligomerization of h -ha that induces better antibody affinity maturation and enhanced protection against h n and h n viruses compared to inactivated influenza vaccine lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease chicken tlr acts as a functional homologue to mammalian tlr in the recognition of cpg oligonucleotides induction of mucosal immunity in the avian harderian gland with a replication deficient ad- vector expressing avian influenza h hemagglutinin maternal antibody to infectious bronchitis virus: its role in protection against infection and development of active immunity to vaccine infectious bronchitis serology in broilers and broiler breeders: correlations between antibody titers and performance in vaccinated flocks breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by vaccination programme based on the ma and / serotypes induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d (genotype qx) by maternally derived antibodies and by early vaccination the maturing immune system: implications for development and testing hiv- vaccines for children and adolescents determinants of infant responses to vaccines in presence of maternal antibodies decay of maternal antibodies in broiler chickens we thank the alabama agricultural experiment station for funding this research ala - - . supplementary material related to this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . key: cord- - pcb enl authors: siedner, mark j.; gostin, lawrence o.; cranmer, hilarie h.; kraemer, john d. title: strengthening the detection of and early response to public health emergencies: lessons from the west african ebola epidemic date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: pcb enl mark siedner and colleagues reflect on the early response to the ebola epidemic and lessons that can be learned for future epidemics. • strategies to consider include development of a more precise system to risk stratify geographic settings susceptible to disease outbreaks, reconsideration of the international health regulations criteria to allow for earlier responses to localized epidemics before they reach epidemic proportions, increasing the flexibility of the world health organization director general to characterize epidemics with more granularity, development of guidelines for best practices to promote partnership with local stakeholders and identify locally acceptable response strategies, and, most importantly, making good on international commitments to establish a fund for public health emergency preparedness and response. • the recent success of the global action to stem the ebola virus disease epidemic is laudable but should not encourage complacency in our efforts to improve the global public health infrastructure. in march , guinea identified cases of ebola virus disease (evd) and reported them to international health agencies [ ] . nearly twelve months later, the epidemic, having exploded into neighboring sierra leone and liberia, has reached over , cases and nearly , deaths. the toll on human life, impact on health infrastructure, diversion of funding from routine-but critical-priorities, and concentrated mortality among health care workers places the epidemic among the worst disease outbreaks in recent history. after a delayed response [ ] , the world health organization (who) laid out a programmatic roadmap to mobilize financial support and human resources [ ] . in addition, for the first time in its history, the united nations (un) security council has authorized an emergency health mission, the un mission for ebola emergency response, with assets typically committed to peacekeeping. the massive response that followed, involving multiple foreign governments, multinational partners, and regional ministries of health, has brought unprecedented resources to the west african region. towards the end of , the epidemic showed signs of coming under control, particularly in guinea and liberia. compared to worst-case scenario estimates from earlier in the epidemic, this global response has likely saved many thousands of lives [ ] . but while the international response has become an example of the great potential of the global public health community, it also revealed critical weaknesses. had these same partners responded earlier and more effectively after the first signs of an uncharacteristic outbreak, it is likely that the number of lives lost, the impact on health infrastructure, and the magnitude of the eventual response could have been drastically diminished. it is incumbent upon the global public health community to identify gaps revealed during the early stages of the epidemic so that we improve our collective ability to detect and respond early to the inevitable next emerging disease. we offer lessons from the west african ebola epidemic and propose solutions for future international health emergencies. experts have observed that large-scale threats from evd are limited primarily to countries with weak public health systems [ ] . the current epidemic has supported, if not confirmed, this observation. previous evd epidemics, almost all of which occurred in low-and-middle-income countries (lmics) and predominantly in rural areas, have been controlled within weeks, with the largest prior outbreak claiming less than lives. in contrast, the current west african outbreak has now killed more people than all previous evd outbreaks combined. whereas who generally considers the health infrastructure of involved countries when assessing the risk of a potential public health emergency, this outbreak has revealed that a more granular consideration of risk will be of value. guinea, sierra leone, and liberia are all recovering from prolonged periods of civic unrest and suffering from decimated health systems with limited human resource capacity and thus demonstrate that all lmics should not be considered the same. for example, nigeria, another country broadly characterized as a lmic, provides a clear illustration of how a functional, albeit limited, public health infrastructure can successfully bring an evd outbreak under control [ ] . the country responded rapidly through efforts in public education, isolation, quarantine, contact tracing, and case identification to control an epidemic after only cases and deaths in a little over a month. consequently, when a disease of epidemic potential emerges, the international community should pay increased attention to the capacity of the local health system. for example, who could create and maintain a curated scoring system of lmics to include standard measures of health infrastructure, including the availability and sufficiency of the health care work force, surveillance and laboratory capacity, and personal protection equipment availability and supply chains. moreover, a careful analysis of factors that contributed to variations in epidemic severity might lead to identification of additional characteristics to include in a ranking system. for example, local burial practices, having had prior local experience with a similar outbreak, and urban versus rural environments appear to have contributed to variations in the severity of recent west african evd epidemics. with a more precise risk stratification system, who and international partners could give expedited and focused attention to countries on the list that have a particularly weak health infrastructure. this would facilitate faster and stronger responses to both routine and extraordinary health threats, as well as help to target routine support for health systems strengthening. who primarily relies upon the revised international health regulations (ihr) to define a public health emergency of international concern (pheic), and to determine when such announcements should be made to alert the global community. to improve their predictive accuracy and effectiveness, who should reexamine the ihr's criteria for declaring a pheic following each major public health emergency. this strategy will enable who to incorporate the lessons learned from each event to guide future responses. multiple characteristics of the west african evd epidemic have revealed areas for potential improvement. first, this outbreak exemplified the importance of an often neglected criterion in the ihri.e., paying particular attention to cases in which "external assistance [is] needed to detect, investigate, respond, and control" the incident [ ] . although this criterion is included in the ihr as a consideration, it is subordinated to others, such as the present number of cases, which risks missing detection of threats before they reach epidemic proportions. this epidemic, in which the regional health infrastructure was quickly overwhelmed, taught us that a need for external assistance ought to become a primary condition for declaring a pheic. doing so would assist struggling member states with weak health infrastructures in the crucial early stages of response. second, the ihr partially define a pheic as a disease outbreak that "constitutes a public health risk to other states through the international spread of disease," or poses a "significant risk of international travel or trade restrictions" [ ] . by this definition, outbreaks must transcend a national border before they legitimately trigger an international response. from the standpoint of national sovereignty, this requirement is understandable. states retain authority to handle health threats purely within their territories. however, the current epidemic began in december , months before it crossed borders into both sierra leone and liberia. this realization should challenge us to reconsider whether national borders should have such clout in considerations of potential global health emergencies. at the very least, the risk of international spread-particularly to other countries with weak public health infrastructures-should be carefully examined to prevent epidemics from growing out of control. a more modern approach to global public health should de-emphasize the a priori criterion of international spread. modeling studies have demonstrated that, for pathogens with relatively short generation times, early cross-border coordination between nations is crucial when epidemic diseases arise in border regions [ ] . but even for diseases with relatively long generation times, as with the current evd epidemic, waiting for cases to cross borders can result in disastrous delays. as such, removing the requirement that an outbreak be an "international threat"-or at minimum interpreting that criterion liberally-will decrease delays between reporting and response and allow much needed support to at-risk countries before epidemics have reached a tipping point. lastly, in hindsight this outbreak might have been defined as a public health emergency long before the who director general (dg) declared it a pheic, and the delay probably exacerbated the slow global response to the outbreak. there are reasons not to prematurely declare a pheic. doing so could damage already vulnerable political systems, lead to misallocation of scarce global health funding, and diminish the influence of the ihr. additionally, a pheic declaration may inadvertently trigger harmful measures, such as border closings and travel bans, which can hinder the response to an epidemic. however, the current epidemic has taught us that delaying an announcement imperils lives and health systems and, in the long run, may do more economic harm [ ] , cost more response dollars, and undermine domestic political legitimacy much more than an early, errant pheic declaration. therefore, for high-consequence diseases of epidemic potential, a precautionary principle should take precedence. the who dg should be empowered to declare pheics early if criteria are met. in fact, the dg has broad discretion to determine when an outbreak constitutes a pheic [ ] . who should interpret this discretion to declare graduated pheics, ranging from high-consequence localized events (e.g., the current ebola outbreak as of june ) to clear global threats (e.g., severe acute respiratory syndrome in ). it can then tailor its recommendations, as well as funding needs, as the scenario unfolds. in doing so, we could expect pheic announcements for outbreaks that never reach devastating proportions. a similar approach has been implemented in other domains-such as genocide prevention-in which a concerted international response is required to prevent urgent problems before they become catastrophic [ ] . in fact, the emergency response framework, also published by who, which aims to grade and motivate responses to international emergencies, public health or otherwise, specifically includes a "no regrets" policy, stating that "it is better to err on the side of over-resourcing the critical functions rather than risk failure by under-resourcing" [ ] . although it garnered relatively less attention than the pheic announcement, who declared the west africa evd epidemic a grade international emergency (the highest classification) on july , two weeks before the pheic was declared [ ] . similar discretion for declaring a pheic might help prevent the next large epidemic from gaining such momentum before the international community responds appropriately. overwhelmed and under-resourced, the affected countries have implemented drastic measures to control the epidemic, including hospital and school closures, local and national quarantines, and border closures [ ] . not surprisingly, these measures have engendered widespread public distrust of health authorities [ ] . in contrast, prior successful responses to ebola [ , ] and other epidemics [ ] have prioritized early partnerships with local authorities, anthropologists, and civil society to establish buy-in from multiple stakeholders. this strategy helps ensure that the design and implementation of control measures are culturally appropriate. in hindsight, some of the negative fallout from decisions to use extraordinary measures might have been avoided had who, in partnership with local community leaders and public health experts, more assertively used their legitimacy to caution against the use of coercive measures without an evidence base. when acting under emergency powers-and especially when using extraordinary control measures such as regional quarantines-governments should prioritize formation of community advisory bodies in the affected region. a rich institutional knowledge about best practices for community advisory boards exists from the research community [ , ] and, in combination with recent experience gained through collaborations with community leaders during the current epidemic, can serve as the basis for much-needed guidelines for public health activities. members should represent divergent interests and include religious leaders, community representatives, nongovernmental organizations (ngos), and other stakeholders. the body should be briefed on the status of the threat and called on to offer recommendations on community desensitization, capacity building, and control measures. while national and local governments hold primary responsibility for creating community advisory bodies, international actors should provide support, including technical assistance for overstretched ministries as they weigh evidence about appropriate interventions. declaring a pheic is a critical early step to marshal an effective response. a pheic announcement alerts partners and should initiate a concerted, coordinated response. but a pheic is of little value without corresponding ammunition. for example, although the pheic was announced on august , six weeks passed between the declaration and the united states government's commitment of us$ million and the planned deployment of , military personnel and public health service commissioned corps officers [ ] . in the interim, total cases of evd increased from approximately , to over , , and confirmed deaths more than tripled, from under , to approximately , [ ] . the steady march of the epidemic, not only before the pheic declaration but also long after it, reinforces the fact that global health preparedness is contingent on the immediate availability of funding and human resources to respond. this need can be fulfilled through the establishment of a global emergency fund and the formation of a corps of trained health workers that can be deployed rapidly to curb an outbreak, with expertise ranging from epidemic surveillance to supply chain management. an international health systems fund, through a sustained investment by global partners, would provide much needed preparedness in future cases of outbreaks in lmics, where local resources are not capable of controlling epidemics [ ] . in , a who committee proposed the establishment of a us$ million contingency fund for rapid response in a declared pheic [ ] . however, the commitment remains unfulfilled. instead, the organization has been forced to rely on mobilizing funding from member states and other partners [ ] , which inevitably delays a robust international response. spurred by the current evd epidemic, in january who's secretariat repeated calls for the creation of a rapid response fund, to be financed by member states [ ] , and who's executive board agreed to establish the fund [ ] . yet, the onus remains on who and the international community to follow through with this request even after the present emergency has faded and ensure this call to action does not meet a similar fate as previous attempts. arguably the greatest lesson to emerge from the ebola virus epidemic is that both national ministries and the global public health community were caught off guard and unprepared [ ] . in addition to the many thousands of lives taken from both ebola infections and interrupted access to routine health services, billions of dollars that could have otherwise been used for development and health systems strengthening were allocated to direct the ebola response. the deleterious impact on local economies is equally staggering. the world bank predicts that the three most affected countries will lose us$ . billion in economic growth in , corresponding to an average gross domestic product (gdp) loss of % across the three countries. in sierra leone, a loss of nearly % of the gdp is projected [ ] . all this resulted from a disease that is relatively easy to control in settings with established health systems. had a disease like sars, with airborne transmission and a high case-fatality rate emerged in a similar location, the fallout could have been far worse. what can be done today to prepare for the unavoidable public health threats of tomorrow? in the long term, it will be essential to build more robust health systems. beyond public health emergencies, strong health systems will improve the health and wellbeing of the population in lmics by delivering a range of essential services [ ] [ ] [ ] . the ihr currently mandate that technical support-surveillance, epidemiology, and laboratory and other core capacities-be provided by high-income countries to low-income countries to build capacity to survey and respond to outbreaks and that a detailed international framework exist for defining and assessing these capacities [ ] . however, although this is a central component of the obligations borne by countries for collective health security, capacity building support to low-income countries has long been underfunded. as a result, most countries in sub-saharan africa continue to suffer from weak systems for disaster preparedness and emergency response [ , ] . to ensure a truly robust response to global health hazards, states must abide by their ihr obligations to build core public health capabilities in regions of need. human resources capacity building, laboratory infrastructure, and epidemiologic surveillance expertise are all urgently needed in west africa and beyond. these inputs should be routinely assessed through a demonstrated capability to deploy resources successfully and in a timely fashion to respond to emergencies [ ] . moreover, such investments clearly will have secondary benefits for routine health services in the areas they are employed. while successful implementation of these elements of the ihr will require substantial resource expenditures by high-resource countries, it is a legal and moral duty to which wealthy countries bound themselves when joining the ihr. the evd epidemic has brought broad realization that health systems strengthening will be crucial to realize the benefits of a global community protected against international infectious disease threats. a positive legacy of the otherwise disastrous evd outbreak should be a global community with renewed commitment to the establishment of a capable emergency response infrastructure. while current efforts to bring the evd epidemic under control should be widely applauded, the delayed response during the early stages of the evd epidemic in west africa exemplifies not only the danger posed by disease outbreaks in states with weak health systems but also their widespread impact in an increasingly globalized world. the international public health community-who, states, and stakeholders-can learn from missteps during the first stages of the epidemic. if instead we accept the status quo by relying on overwhelmed and undersupported domestic health systems and international charity to respond to threats after they have become emergencies, history will repeat itself. at stake are the values of the ihr and the legitimacy of who. the power of global health law and global health institutions will remain seriously unrealized and deeply compromised if the ebola epidemic does not spur fundamental reform. emergence of zaire ebola virus disease in guinea-preliminary report the global response to the ebola fever epidemic: what took so long? speaking of medicine: blogsplosorg/speakingofmedicine: plos medicine ebola response roadmap estimating the future number of cases in the ebola epidemic-liberia and sierra leone what's missing in the ebola fight in west africa ebola virus disease outbreak-nigeria world health organization ( ) international health regulations strategies for containing an emerging influenza pandemic in southeast asia the economic impact of the ebola epidemic: short and medium term estimates for liberia developing a strategy, methods, and tools for genocide early warning emergency response framework (erf) ebola virus disease outbreak response plan in west africa the ebola epidemic: a global health emergency ebola in west africa: gaining community trust and confidence ebola then and now ebola virus disease in the democratic republic of congo weapons for battling viruses: effective handling of nipah outbreak in south asia offers lessons for controlling ebola in west africa building community partnerships: case studies of community advisory boards at research sites in peru ethics in public health research: protecting human subjects: the role of community advisory boards global ebola response kicks into gear at last ebola virus disease timeline of ebola: towards an international health systems fund report of the review committee on the functioning of the international health regulations ( ) in relation to pandemic (h n ) reforming the world health organization ensuring who's capacity to prepare for and respond to future large-scale and sustained outbreaks and emergencies ebola: ending the current outbreak, strengthening global preparedness and ensuring who's capacity to prepare for and respond to future large-scale outbreaks and emergencies with health consequences assessment of ebola virus disease, health care infrastructure, and preparedness-four counties, southeastern liberia the economic impact of ebola on sub-saharan africa: updated estimates for reshaping global health the joint action and learning initiative: towards a global agreement on national and global responsibilities for health do we need a world health insurance to realise the right to health summary of states parties report on ihr core capacity implementation expert opinion on implementation strategies for the international health regulations biosurveillance capability requirements for the global health security agenda: lessons from the h n pandemic key: cord- -dohglrm authors: scully, c; samaranayake, lp title: emerging and changing viral diseases in the new millennium date: - - journal: oral dis doi: . /odi. sha: doc_id: cord_uid: dohglrm most viral infections encountered in resource‐rich countries are relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestations (enanthema), including oral in some. however, the apparent benignity may be illusory as some viral infections have unexpected consequences – such as the oncogenicity of some herpesviruses and human papillomaviruses. infections are transmitted from various human or animal vectors, especially by close proximity, and the increasing movements of peoples across the globe, mean that infections hitherto confined largely to the tropics now appear worldwide. global warming also increases the range of movement of vectors such as mosquitoes. thus recent decades have seen a most dramatic change with the emergence globally also of new viral infections – notably human immunodeficiency viruses (hiv) – and the appearance of some other dangerous and sometimes lethal infections formerly seen mainly in, and reported from, resource‐poor areas especially in parts of asia, latin america and africa. this study offers a brief update of the most salient new aspects of the important viral infections, especially those with known orofacial manifestations or other implications for oral health care. professor jens pindborg, in a lecture in the s, spoke of the very few viral infections then known, most of which were regarded as relatively trivial, transient and with few or no serious sequelae and which were largely clinical phenomena with few therapies available (scully, ) . indeed, most viral infections encountered in resource-rich countries were and still are, usually relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestationsan enanthema, and these have been well documented (scully and samaranayake, ) . gradually, however, the unexpected consequences of some oral viral infections have emerged and been recognised, not without some surprise (scully, ) especially the oncogenicity of some herpesviruses (eglin et al, ) and human papillomaviruses (hpvs) which we (eglin et al, ; maitland et al, ; cox et al, ) and many others (e.g. lind et al, ) have explored, culminating in the appreciation of unanticipated transmission routes for some cancers, such as sexual (scully, ) . viruses are increasingly appreciated to cause a wide range of human diseases, ranging from acute self-resolving conditions to acute or chronic fatal diseases. effects that arise long after the primary infection can increase the propensity for chronic conditions (e.g. erythema multiforme) or in some, lead to the development of cancers other than oral (herrington et al, ) . in addition, other viruses such as hepatitis c virus (hcv) have been implicated in common but diverse oral lesions such as lichen planus/lichenoid lesions and sicca syndrome (baccaglini et al, ) , the latter also sometimes arising after some other viral infections (youinou et al, ) although the full aetiopathogenesis of sjogren syndrome remains unclear (kivity et al, ) . the possible viral associations in many other oral conditions including periodontitis (vincent-bugnas et al, ; ambili et al, ; contreras et al, ; ly et al, ) and several conditions of unknown aetiology, however, remain enigmatic, and associations are not necessarily causal. the recent several decades have also seen a most dramatic change with the emergence globally of new viral infectionsnotably human immunodeficiency viruses (hiv)and the appearance also in resource-rich countries, of some other dangerous and sometimes lethal infections hitherto latent, unrecognised or unappreciated in resource-poor areas. rare lethal and other dangerous viral infections were formerly seen mainly in, and reported from, resourcepoor areas of asia, latin america and africa. pandemics of severe acute respiratory syndrome (sars) and influenza viruses (both h n and h n ), heralded the surge of zoonotic virus outbreaks. laboratory diagnosis of viral infections is traditionally based on the isolation of the virus, detection of viral nucleic acid or antigens, or serological confirmation (generally presence of igm antibodies), and it is clear that the rate of discovery of new viruses is accelerating, due to a combination of true emergence of new pathogens, increasing awareness and the advance of new mainly molecular biology technologies making rapid detection and characterisation possible (wang, ) . various respiratory viruses and ebola virus disease (evd) in particular have served to awaken humanity to the related social inequalities and challenges. this study discusses the importance of emergent new infections and recent findings in relation mainly to those viral infections that may manifest orally or may affect oral health care. fuller descriptions of ebola virus, marburg virus, coronaviruses, nipah virus, noroviruses, enteroviruses, hiv, measles, mumps, respiratory syncytial virus (rsv), influenza, cytomegalovirus (cmv), varicella zoster virus (vzv), epstein-barr virus (ebv), hpvs and kaposi's sarcoma-associated herpesvirus (kshv) are available elsewhere (e.g. herrington et al, ) . it is increasingly evident that there is now an amazing range of viral agents, many unidentified, which may be new or newly recognised or old, re-emerging. risk factors for infection include more risk • from greater exposure, • if defences are down, • if agents evade defences, • generally in warm, moist places. emerging viral diseases have arisen mainly because of increasing and changing air travel habits, but other factors that have also increased exposure to the vectors of these infections include conflicts, displacement and migration, as well as changing climate and vector distribution, changing farming/manufacturing, changing lifestyles such as promiscuity and changing clinical practices (mathis et al, ) . transmission of respiratory and enteric viruses is highwhile sexually shared infections (ssi) are of low infectivitygenerally requiring the close apposition of infected mucosae, increased when there is epithelial discontinuity. however, with the existence of transnational sexual networks in many countries including usa (hughes, ) and europe, with high rates of migration and travel between, for example, amsterdam, barcelona, berlin, london and paris, there have been ssi outbreaks among sexually exploited people of both genders but especially in hiv-positive men. such close encounters, as well as large gatherings of humans, and/or prolonged periods in confined spaces, can enhance transmission of many agents; this has led to the development of a new area of medicine -'mass gathering medicine (mgm)' (memish et al, ) . mass gatherings consist of large numbers of people attending an event at a specific site for a finite time. some of the largest mass gatherings are spiritual in nature but other examples include olympics, rock concerts and political rallies. the public health issues associated with the hajj (the annual muslim pilgrimage to mecca, saudi arabia) is the best reported and has international or even intercontinental implications in terms of infection spread. hajj routinely attracts . million muslims (memish et al, ) , and the unavoidable overcrowding raises the risk of respiratory infections. 'hajj cough' is the most frequently reported but influenza (shafi et al, ; haworth et al, ) and bacterial meningococcal w strains are more seriousas are severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronavirusesnew viruses that cause severe acute lower respiratory infections (ari), with % and % mortality rates, respectively, and > % mortality rates seen in older and immunosuppressed people (gralinski and baric, ) . the past decade has also seen the emergence of several other novel viruses that have appeared in resource-rich countriesoften in travelling peopleincluding an h n influenza a virus, a swine-like influenza h n variant virus and a human adenovirus p (gautret et al, ) . a number of zoonotic and vector borne viral diseases have emerged in south-east asia and the western pacific including japanese encephalitis, barmah forest and ross river viruses. australia sees ross river and barmah viruses now appearing regularly. nipah virus causes limited but deadly epidemics in south-east asia. finally, infections by lyssa viruses, kunjin, murray valley and zika viruses are also emerging (bhutta et al, ; carson et al, ) . in usa, the centers for disease control and prevention (cdc) recognise such threats and have a mission cri-tical (frieden et al, ) which focuses on antibiotic resistance and viral infections as shown in box . coronaviruses cause illnesses ranging from the common cold to sars. mers-cov has an especially high mortality and has recently spread widely outside the middle east to asia. dr frieden, director of cdc, had stated 'in this interconnected world we live in, we expected mers-cov to make its way to the united states. we have been preparing since for this possibility (frieden et al, ) '. avian influenza a (h n ) and a (h n ) viruses have continued to circulate widely in some poultry populations and infect humans sporadically. sporadic human cases of emerging and changing viral diseases c scully and lp samaranayake avian a(h n ), a(h n ) and a(h n ) have also emerged. closure of live poultry markets in china has reduced the risk of a(h n ) infection (hui and zumla, ) . standard infection control precautions are mandatory to prevent cross-transmission from recognised and unrecognised sources of infection. these sources of (potential) infection include blood and other body fluid secretions or excretions (excluding sweat, non-intact skin or mucous membranes) and any equipment or items in the care environment which are likely to become contaminated (booty et al, ) . rna viruses, with their high potential for mutation and epidemic spread, are the most common new viral causes of human disease. most emerging viruses are zoonoses; they have jumped from mammals or birds to humans. the annual rate at which novel viruses have been found remains at - a small number which is an artefact of inadequate surveillance in resource-poor countries, where even established endemic pathogens are often misdiagnosed. indeed, many of the emerging viruses of the future are already infecting humans (rosenberg, ) . more awareness, internet communications, advances in diagnostic technology [high-throughput sequencing and specific polymerase chain reaction (pcr) assays] now help assists recognition. promed-mail is one robust and sensitive mechanism for the discovery of emerging disease outbreaks and for rapid dissemination of information to the public health community (madoff and woodall, ) . for example, severe fever with thrombocytopenia syndrome (sfts) was recognised as associated with a novel sfts bunyavirus (sftsv) (bao et al, ; jiang et al, ) using real-time reverse transcription pcr (rt-pcr), viral culture, genetic sequencing, microneutralisation assay (mna) and indirect immunofluorescence assay (ifa). another example is a new arenavirus related to lymphocytic choriomeningitis viruses that was recognised in a cluster of fatal transplant-associated diseases (palacios et al, ) . specific pcr assays showed the virus in the kidneys, liver, blood and cerebrospinal fluid of the transplant recipients. awareness of the infections possible and likely, their epidemiology, and a history of travel, contact and relevant vaccinations is crucial to diagnose an infection, but the history is often overlooked. virus infections typically manifest with a fever, but this is neither always present nor measured by the clinician. clinical features are often non-specific and may include malaise, myalgia, rashes and mucosal lesions such as ulceration or bleeding. these are not always major features, not always checked by a clinician, or may often be seen by non-orally trained healthcare workers. examination conditions in the field may be less than ideal; thus, underdiagnoses or misdiagnoses are likely to be quite common. although evd has been recently discussed fully elsewhere (samaranayake et al, (samaranayake et al, , scully et al, ) , we provide a brief overview account of its oral manifestations below. the three cardinal oral signs and symptoms of evd are gingival bleeding, mucosal lesions (see below) and pain (odynophagia). other important features typical of early and mild forms of disease, which may help oral health care providers suspect evd, include epistaxis, bleeding from injection sites, conjunctivitis and rash. bleeding is very frequent in advanced forms of evd, but it is only relatively frequent in mild or early evd forms; thus, it is likely that ebola-infected patients seeking care may not show such a sign. typically, gingival bleeding is concomitant with other forms of bleeding, particularly epistaxis and bleeding from injection sites. concurrent bleeding at disparate sites is a discriminatory sign of evd. mucosal lesions such as white or red patches, aphthouslike ulceration and greyish exudative lesions may be seen in evd, but these remain to be more accurately defined. odynophagia (discomfort), the consequence of oedema and mucosal lesions, may range from sore throat to severe dysphagia, when mucosal lesions are ulcerated. ebola virus disease is readily transmitted by body fluids, so universal infection control is essential. there is no reliably effective antiviral against this agent yet available. dengue viruses (denvs) cause denguethe most common arthropod-borne viral disease in humansnow seen in more than tropical countries. the word dengue is obtained from swahili phrase ka-dinga pepo meaning 'cramp-like seizure'. it is also called break-bone fever. dengue virus infection can be asymptomatic or a selflimited, acute febrile disease ranging in severity. the classical form is characterised by high fever, headache, abdominal pain, morbilliform rash, myalgia and arthralgia. in a small proportion of cases, the disease progresses to life-threatening dengue haemorrhagic fever, which results in non-infectious bleeding, thrombocytopenia and leakage of plasma, or dengue shock syndrome. there is no reliably effective antiviral against this agent yet available. in the absence of a vaccine and antiviral drugs, the sole control measure is limiting the mosquito vectors. the main mosquitoes, aedes aegypti and aedes albopictus, are potential vectors of numerous arthropod-borne viruses (arboviruses), and have now expanded from the tropics and subtropics, although a. albopictus still plays a relatively minor role compared to a. aegypti in denv transmission. oral manifestations in dengue are rarely reported; gingival bleeding is the most common (lambrechts et al, ; mithra et al, ; roopashri et al, ) . postoperative haemorrhage (dubey et al, ) , gingival and lip swelling (pontes et al, ) or mucous membrane involvement with no further details (desruelles et al, ) have also been noted. neurological sequelae include hypoglossal palsy (jaganathan and raman, ) and taste emerging and changing viral diseases c scully and lp samaranayake changes (scully, ) . of interest in this context is that saliva has been attempted to be used as a diagnostic fluid for dengue fever (ravi banavar and vidya, ) . chikungunya virus (chikv), a togaviridae family alphavirus has emerged as a worldwide threat, causing fever and devitalising arthritis (the name reflects the condition of many of the stricken, 'bent down or become contorted', in the tanzanian makonda language) in a range of countries including many holiday destinations (hasan et al, ) . chikungunya virus is a mosquito-borne virus, like dengue transmitted by aedes mosquitoes and has features of fever, headache, rash, nausea, vomiting, myalgia and arthralgia. the presentation differs in adults and childrenthe latter may present with fever, a rash on the face and arms and intra-oral lesions reported as 'koplik spots' (centers for disease c & prevention, ). oral manifestations reported also include ulceration (macdonald-ottevanger et al, ) and candidiasis (bandyopadhyay and ghosh, ) . thus, chikungunya mimics dengue and like dengue, there is no reliably effective antiviral against this agent yet available. indigenous to tropical africa, recent large chikungunya outbreaks have been reported in south-east asia, the indian ocean islands, the caribbean, and the united states and europe (balkans, france, greece, ireland, italy, madeira, the netherlands and spain), mainly in travellers (kumar et al, ) . the rapid spread of a. albopictus into europe and the americas coupled with high viraemia in infected travellers returning from endemic areas increases the risk that chikv could establish itself in new endemic regions (thiboutot et al, ) . a mutation in chikv (e -a v) appears to improve virus survival in a. albopictus and increase its virulence (burt et al, ). the majority of virus infections of the oral mucosa are due to the herpes group, which are dna viruses. the classical oral manifestations of these virus infections ranging from herpes simplex, herpes zoster to kaposi`s sarcoma, to infections caused by ebv are adequately described elsewhere (balasubramaniam et al, ) . the description below refers to recent developments in this regard. herpes simplex virus infections. herpes simplex virus (hsv) infections are increasingly seen in adults, are sometimes caused by hsv- and can be an ssi with stomatitis or pharyngitis (looker and garnett, ) . herpes labialis recurrences are now recognised to be significantly common in immune defects and to occur where the innate antiviral immune response involving the interferon (ifn-k) promoter is lacking due to polymorphisms within the ifn-k gene (griffiths et al, ) . therapy, despite many studies, still involves antivirals such as aciclovir or penciclovir, but hydrocolloid patches may have a place (karlsmark et al, ; stoopler and balasubramaniam, ) . herpes simplex virus has long been associated with cranial neuropathies and now has also been associated with alzheimer disease (l€ ovheim et al, ) . human papillomavirus infections. one of most common ssi in world, hpv can cause cutaneous or mucosal papillomas, common warts (verruca vulgaris), genital warts (condyloma acuminatum) and multifocal epithelial hyperplasia (heck disease). some hpv types (oncogenic or 'high-risk' hpv types such as hpv ) are now implicated in some mouth cancer, especially oropharyngeal cancer (opc). opc incidence has significantly increased, predominantly in economically developed countries (scully et al, ; scully, ; chaturvedi et al, ; brewer and calo, ) . hpv is a strong and independent prognostic factor for better survival of opc (ang, ; lowy and munger, ; o'rorke et al, ) , human papillomaviruses infection does not necessarily lead to opc. a study of males in brazil, mexico, usa; year showed that . % acquired incident oral hpv, . % acquired oral oncogenic hpv infection and . % acquired oral hpv infection. new oral oncogenic hpv infections are rare and most are cleared spontaneously within year (kreimer et al, ) . the united states food and drug administration (fda) approved the quadrivalent hpv vaccine for girls in and for boys in aimed at genital, hpv-related lesions. vaccination has proved to be successful at preventing hpv and associated cervical and other anogenital tumours. hpv vaccines are effective and also against the hpv strains that are most commonly found in the oropharynx (wierzbicka et al, ) and have reduced infections (herrero et al, ) , but vaccination is yet to be universally recommended for all adolescents. there are two vaccines; both regarded as safe and usually given as a three dose series. • cervarix: recommended for females from to years of age and protects against hpv and hpv . • gardasil: recommended for -and year-old girls and also females to -year-old. gardasil is also recommended for -to -year-old males to protect against some genital warts. this vaccine protects against hpv , hpv , hpv and hpv . human parvoviruses. the only known parvovirus to infect humans is b , which is transmitted by droplets, touch and occasionally in blood, and infects rapidly dividing tissues, most commonly the foetus, intestinal epithelium or haematopoietic system. b infection in pregnancy is associated with early foetal loss, although the probability of this is low (< %). b also acutely depresses erythrocyte production, which is of little clinical significance, except in patients with other haematological diseases, particularly sickle cell disease, when haemolytic crises may be precipitated. emerging and changing viral diseases parvovirus commonly causes 'fifth disease' (erythema infectiosum; slapped cheek syndrome), a mild illness with a lace-like rash on the face, trunk and extremities, usually in children. papular-purpuric glove-and-sock syndrome is pruritus, oedema and symmetrical erythema, with a 'gloves-and-socks' distribution and oral blisters, erosions and ulcers; % of published cases are related to parvovirus b infection (segura saint-gerons et al, ) . cranial neuropathies have also been reported (soares-fernandes and mar e, ) . in many patients ( %) infected with b , there is also arthropathy, particularly in adults. as the vaccination induced disappearance of rubella, parvovirus is the commonest cause of infection-related transitory arthritis, particularly if it affects the hands. there is no specific therapy available for parvovirus infections. the new century has seen the emergence of several novel viruses that cause respiratory tract infections in humans including sars coronavirus infection mainly in china, mers-cov in saudi arabia and in asia, an h n influenza a virus in eastern china, a swine-like influenza h n variant virus in the usa and a human adenovirus p also in the usa. mers-cov and h n viruses are still a major worldwide public health concern, but the pathogenesis and mode of transmission of mers-cov and h n influenza a virus are poorly understood and their oral manifestations, if any, ill-defined (gautret et al, ) . there are no reliably effective antiviral agents available for these agents. hand, foot and mouth disease. hand, foot and mouth disease (hfmd) is a common childhood illness characterised by fever and vesicular eruptions on hands and feet and in the mouth. complications are rare, but pneumonia, meningitis or encephalitis may occur. hand, foot and mouth disease is not associated with one single agent; it is caused by members of the family picornaviridae in the genus enterovirus; there are over serotypes of enterovirus species a-d, which are the common cause of hfmd and illnesses in infants, such as meningitis and encephalitis . outbreaks of hfmd have been mainly caused by two types of enterovirus a species, coxsackievirus (cv) a (cva ), or enterovirus (ev ), and only sporadic cases involve other members of the enterovirus a species have been reported (osterback et al, ). most cva associated infections cause only mild symptoms; however, some cva infections can lead to severe complications and even death (chen et al, a) . a chinese study of hfmd showed many patients were infected with cva , fewer with ev , some were co-infected with cva and ev , and a few were infected with other enteroviruses. upper respiratory tract infection was significantly higher in cva -associated patients, while neurological complications and hyperglycaemia were significantly higher in ev -infected patients (liu et al, ) . enterovirus infections remain an important public health problem, and other enteroviruses and other agents are emerging as major causative agents of hfmd in some epidemics. serotypes causing severe symptoms such as hfmd including ca and ev are decreasing, while the proportion of unidentified ev serotypes causing herpangina and viral encephalitis are on the rise . there may be an upward trend in cocirculation of the two pathogens globally and a new role that recombinants play in the emergence of new enterovirus variants. in , a large, hfmd outbreak in fuyang city of anhui province in south-eastern china resulted in a large number of fatalities. phylogenetic analyses of the entire vp capsid protein sequence showed isolates belonging to the c a cluster of the c subgenotype, and additionally, genetic recombinations were found between the fuyang hev strain and cv-a , resulting in a recombination virus. in , another nationwide outbreak of hfmd was reported in day care centres and schools in finland (osterback et al, ) . from vesicle fluid specimens of hospitalised children, the authors identified the aetiological agent as coxsackievirus a . enterovirus d (ev-d ) appears to cause severe respiratory illness in childrenaffecting most severely those with asthma in the usa epidemic, and may cause paralyses, including affecting cranial nerves (chen et al, b; foster et al, ; maloney et al, ) . there is no reliably effective antiviral against these agents yet available. an assay using multilocus pcr and reverse transcription pcr coupled with electrospray ionisation mass spectrometry (rt-pcr/esi-ms), which simultaneously detects and identifies human enterovirus a-d, adenovirus a-f, human herpesvirus - , parvovirus b and polyomavirus, detected not only enteroviruses in hfmd, but also herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses in % hfmd specimens (chen et al, a) . virus and the orofacial implications of infection have been extensively scrutinised and reported (greenspan, ; nokta, ; scully, ) . hiv infection appeared in the s in the congo from simian origins but was not recognised until the s. the pan epidemic continues to expand with worldwide latest figures of new reported infections ( ) at . m, a total of over m, and infection in sexually active people now at in (http://www.who.int/hiv/data/en/). worldwide, hiv is mainly heterosexually transmitted and, in the over s, there is a threefold increase. africa has a known infected rate > %, and in eastern europe and central asia, there has been a known increase of % in years. uk has among highest rates of new hiv in europe, outstripped by portugal, ukraine, estonia and russia. most hiv infections are in heterosexuals, on vacation but many men having sex with men have hiv. hiv infection is undiagnosed in one in three affected. multidrug-resistant hiv has appeared. the two main viruses, hiv- (most common) and hiv- , infect cells with cd surface receptors, mainly t-helper lymphocytes and brain glial cells, and replicate within and damage them, causing hiv disease which may be emerging and changing viral diseases c scully and lp samaranayake symptomless but, over time, ultimately damages cd + cells crucial to host defences against fungi, viruses, mycobacteria and parasites, thus causing hiv disease and ultimately producing symptoms (mainly infections and tumours) and the acquired immune deficiency syndrome (aids) and a range of lesions. hiv/aids common manifestations are infections, neoplasms, neurological and autoimmune disorders. infections with viruses, mycobacteria, fungi and parasites, particularly pneumocystis carinii (jiroveci) pneumonia and mucosal candidiasis, are common. loss of weight and wasting ('slim disease') is common. aids is a lethal infection, defined as hiv infection plus or more aids-defining illnesses and a cd t lymphocyte count < /l. without antiretroviral treatment (art), all eventually develop aids within - years. candidiasis is universal especially in hiv subtype b strain crf infection, but other infections in hiv/ aids depend also upon their environmental exposure; thus, tb is particularly common in people from africa and in urban iv drug users in usa; leishmaniasis is common in persons from around the mediterranean; mycoses such as penicillosis are seen mainly in northern thailand. neoplasms may include virally related kaposi sarcoma, lymphomas or carcinomas (jose et al, ; mthethwa et al, ; meless et al, ; nair et al, ) . body fluids such as semen may contain hiv as may saliva, breast milk and blood. hiv transmission is sexual mainly: most new cases are via heterosexual intercourse. hiv can also be transmitted by infected blood or blood products, including plasma or tissues. hiv transmission by contaminated needles and syringes is an important route in injecting drug users. cross-placental transfer is not uncommon. transmission by needlestick ('sharps') injury is an occasional risk for healthcare workers. there is no reliable evidence for hiv transmission by normal social contact or by biting insects. pre-exposure prophylaxis using safe sex practices plus tenofovir and emtricitabine is highly effective to prevent hiv infection. as for treatment, art is typically effective although drugs are costly and often associated with resistance and/ or adverse reactions. immune reconstitution inflammatory syndrome (iris) may follow art and can include exacerbation of some lesions such as tuberculosis, mycobacterium avium complex infections, zoster, hpv, cmv, cryptococcosis and kaposi sarcoma (tsang and samaranayake, ) . every effort must be made to avoid needlestick (sharps) injuries, as these could transmit hiv, hepatitis viruses or other infections. in the event of such an injury, speed is of the essence, and where appropriate, counselling and postexposure prophylaxis (pep). current pep for hiv (and other blood-borne agents such as hepatitis b and c) viruses in uk consists of (samaranayake and scully, ); • hiv: tenovir, emtricitabine, lopinavir, ritonavir, • hbv: hepatitis immunoglobulin plus hbv vaccine or booster, • hcv: interferon plus ribavirin. mumps. mumps is a common childhood infection caused by the mumps virus (muv), a member of the paramyxoviridae family of enveloped, non-segmented, negative-sense rna viruses. the defining feature of classical mumps is swelling of the parotid gland, but this is not present in all cases and it can also occur in various other disorders. only mumps causes epidemic parotitis. other causes of parotitis include infection by parainfluenza virus types and , influenza a virus, coxsackie a virus, echovirus, lymphocytic choriomeningitis virus, human immunodeficiency virus, human t lymphotropic virus and non-infectious causes (e.g. drugs, tumours, immunologic diseases and salivary duct obstruction). mumps virus not only affects salivary glands but also other glands including reproductive glands and pancreas, leading to orchitis or oophoritis (hviid et al, ; ternavasio-de la vega et al, ) . acute hormonal disturbances are common including decreased testosterone and inhibin b levels with low or normal levels of gonadotropins in % and a high incidence of sperm disturbance. importantly, mumps is highly neurotropic, and central nervous system infection ensues in approximately half of cases and may lead to aseptic meningitis, viral encephalitis, and rarely deafness and pancreatitis (rubin et al, ) . mumps is vaccine preventable, and one dose of mumps vaccine is about % effective against the disease. routine vaccination has proven highly effective in reducing the incidence of mumps and is presently used by most developed countries; however, there have been outbreaks of disease in vaccinated populations (hviid et al, ) .since the introduction of the mumps vaccine, the age of appearance of mumps infection has shifted from children to adolescents and young adults, groups with a higher incidence of disease complications and sequelae. in , a large epidemic peaked in the uk, and, in , the american mid-west had several outbreaks. in both countries, the largest proportion of cases was in young adults. in the uk, susceptible cohorts too old to have been vaccinated and too young to have been exposed to natural infections were the primary cause of the mumps epidemic. in the usa, effectiveness and uptake in combination appear not to have been sufficient to obtain herd immunity for mumps in populations such as college students. severe fever with thrombocytopenia syndrome virus (sftsv) infection. an emerging infectious disease, sfts, was identified to be associated with a novel sfts rna bunyavirus (sftsv). transmission of the disease among humans has been described, but clinical impact factors and transmission mechanisms still need further study (bao et al, ; li, ) . risk factors assessment of the person-to-person transmission revealed that the major exposure factor was blood contact without personal protection equipment. information from this study provided solid references of sfts incubation time, clinical and laboratory parameters related to sfts severity and outcome, and biosafety issues for preventing personto-person transmission or nosocomial infection of sftsv. emerging and changing viral diseases there is no reliably effective antiviral against this agent yet available. viruses are ubiquitous, and they cause a wide range of human diseases, ranging from acute self-resolving conditions to fatal diseases. we continue to witness the emergence of new viruses and infections and there appear to be no signs of abeyance in the march of these elusive enemies. apart from the acute 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fevers with emphasis on ebola virus disease and orodental healthcare fungal and viral infections, skeletal disorders and malignancies. hospital update viruses and cancer: herpesviruses and tumors in the head and neck. a review oral squamous cell carcinoma; from an hypothesis about a virus, to concern about possible sexual transmission persistent metallic taste churchill livingstone: london, uk. oral diseases emerging and changing viral diseases c scully and lp samaranayake clinical virology in oral medicine and dentistry papillomaviruses: the current status in relation to oral disease infection control: ebola aware; ebola beware; ebola healthcare papular purpuric gloves and socks syndrome. presentation of a clinical case hajj: health lessons for mass gatherings isolated velopalatine paralysis associated with parvovirus b infection topical and systemic therapies for oral and perioral herpes simplex virus infections mumps orchitis in the post-vaccine era ( - ): a singlecenter series of patients and review of clinical outcome and trends chikungunya: a potentially emerging epidemic? immune reconstitution inflammatory syndrome after highly active antiretroviral therapy: a review ebv infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis discovering novel zoonotic viruses hpv vaccination in head and neck hpv-related pathologies viruses contribute to the development of sj€ ogren's syndrome an epidemic analysis of hand, foot, and mouth disease in zunyi we are grateful to dr nihal bandara for the editorial assistance rendered and to the royal college of surgeons of edinburgh for support of professorships (king james iv) for both authors. both authors have equally contributed to the review. key: cord- -fgdyzzty authors: tan, joshua; pieper, kathrin; piccoli, luca; abdi, abdirahman; perez, mathilde foglierini; geiger, roger; tully, claire maria; jarrossay, david; maina ndungu, francis; wambua, juliana; bejon, philip; fregni, chiara silacci; fernandez-rodriguez, blanca; barbieri, sonia; bianchi, siro; marsh, kevin; thathy, vandana; corti, davide; sallusto, federica; bull, peter; lanzavecchia, antonio title: a lair- insertion generates broadly reactive antibodies against malaria variant antigens date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: fgdyzzty plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies( – ). although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. here, we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different p. falciparum isolates and opsonize these cells by binding to members of the rifin family. these antibodies acquired broad reactivity through a novel mechanism of insertion of a large dna fragment between the v and dj segments. the insert, which is both necessary and sufficient for binding to rifins, encodes the entire amino acid collagen-binding domain of lair- , an ig superfamily inhibitory receptor encoded on chromosome . in each of the two donors studied, the antibodies are produced by a single expanded b cell clone and carry distinct somatic mutations in the lair- domain that abolish binding to collagen and increase binding to infected erythrocytes. these findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal dna transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine. expanded b cell clone and carry distinct somatic mutations in the lair- domain that abolish binding to collagen and increase binding to infected erythrocytes. these findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal dna transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine. to identify individuals that may produce antibodies that broadly react with p. falciparuminfected erythrocytes (ie), we developed an improved mixed agglutination assay (fig. a) . plasma from adults (n = ) living in a malaria-endemic region in kilifi, kenya, were initially tested in pools of five (fig. b) and then individually for their capacity to agglutinate mixtures of erythrocytes infected with three culture-adapted kenyan parasite isolates, each stained with a different dna dye. most plasma samples formed single-color agglutinates, but three were able to form mixed-color agglutinates with at least six isolates (fig. c) . from two selected donors (c and d) whose plasma formed mixed agglutinates with eight parasite isolates, we immortalized igg + memory b cells and screened the culture supernatants for the capacity to stain erythrocytes infected with the eight isolates. surprisingly, most antibodies isolated from these donors stained multiple isolates, with the best antibodies, such as mgc , mgd and mgd , recognizing all eight isolates tested (fig. d) . conversely, a few antibodies, such as mgd , were specific for a single isolate. in all cases, only a fraction of ie was stained (fig. e ) and this fraction varied with different antibodies, possibly reflecting different clonal expression of the relevant antigen. overall, these findings show that broadly reactive antibodies against ie can be generated in response to malaria infection. we investigated the molecular basis of the broad antibody reactivity by comparing the sequences of the antibodies isolated from the two donors. while the antibodies with narrow reactivity showed classical vdj organization of the heavy (h) chain gene, all the broadly reactive antibodies ( from donor c, from donor d) carried a large insert of more than amino acids between their v and dj segments ( fig. a and extended data fig. - ) . in both donors, the core of the inserts encoded an amino acid sequence that was - % homologous to the extracellular domain of lair- , a collagen-binding inhibitory receptor encoded in the leukocyte receptor locus on chromosome (chr ) . however, in each donor, the broadly reactive antibodies used a distinct vh/jh combination (vh - /jh in donor c and vh - /jh in donor d) and had junctions of distinct length between the v, lair- and j segments. in addition, the broadly reactive antibodies from donor d shared a single light (l) chain (vk - /jk ), while the antibodies from donor c had one of three different l chains (vk - /jk , vk - /jk , vl - /jl ) (extended data fig. ). all the broadly reactive antibodies carried a high load of somatic mutations spanning the whole v-lair- -dj region. the mutations in the vh were used to reconstruct genealogy trees showing a developmental pathway with progressive acquisition of somatic mutations (fig. b-c) . notably, the trees were consistent with those generated using only the lair- insert or the vl sequence (extended data fig. ). these findings indicate that, within each europe pmc funders author manuscripts individual, a single b cell clone carrying a lair- insert expanded following stimulation by malaria antigens and progressively accrued mutations in the lair- , vh and vl regions. to explore the mechanism that led to the generation of the lair- -containing antibodies, we compared cdna and genomic dna sequences obtained from the antibody-producing b cell clones (fig. d) . in both donors, the genomic dna contained a lair- insert that was larger than that found in the corresponding cdna. in particular, in donor c, the insert comprised not only the bp exon encoding the extracellular lair- domain, but also a bp ' intronic region of the lair- gene that was partially spliced out in the mrna, and a shorter bp ' intronic region that was maintained in the mrna (extended data fig. a) . donor d had a somewhat different genomic insertion, with larger ' ( bp) and ' ( bp) lair- intronic sequences, and, ' of the lair- insertion, an additional sequence of bp corresponding to an intergenic sequence of chr (extended data fig. b-c) . in this donor, the entire lair- ' intronic sequence and much of the ' chr sequence were spliced out in the mrna (extended data fig. d ). the spliced intronic lair- region contained a duplicated bp element with a very high load of somatic mutations (extended data fig. e ). the finding that the inserts were located exactly between v and d/j segments and were joined to these segments by n-nucleotides would suggest that rag might be involved in the insertion process. indeed, cryptic recombination signal sequences (crsss) that followed the / rule were found flanking both the lair- and the chr inserts, although their rss prediction scores were low and they were not positioned precisely at the ends of the inserts (extended data fig. ). as rag acts by excising a target dna sequence, we investigated whether, in b cells making lair- -containing antibodies, one of the two lair- alleles on chr would be deleted. by sequencing genomic dna from t cells of donor c, we identified a heterozygous nucleotide site in the chr lair- exon sequence. surprisingly, both alleles were also present in the b cells producing lair- -containing antibodies (extended data fig. ), a finding that is inconsistent with the "cut-and-paste" function of rag. to determine the contribution of the mutated vh, vl and lair- domains to the antibody specificity, we generated a panel of constructs and fusion proteins based on the broadly reactive antibody mgd (fig. a) . substitution of the v, j or l chain of mgd with that of an unrelated antibody did not affect binding to ie (fig. b) , suggesting that these elements are dispensable for binding. in contrast, deletion of the lair- insert, or its reversion to the unmutated genomic sequence, led to a complete loss of binding. furthermore, fusion proteins displaying only the mutated lair- exon bound to ie, although with lower affinity. to dissect the contribution of the somatic mutations of the lair- insert to antigen binding, we created a set of lair- -fc fusion proteins carrying, in various combinations, the mutations shared by mgd with other antibodies of the same clonal family. we tested the mutants for binding to ie and to collagen, which is the natural ligand of lair- . interestingly, two distinct kinds of mutations were identified: those that reduced collagen binding (p s and p r) and those that increased binding to ie (t l, n s and a t) (fig. c) . collectively, these findings indicate that the binding of the broadly reactive antibodies to ie relies mainly on the mutated lair- domain, which evolves under selective pressure to lose collagen binding and gain binding to ie. to identify the antigen(s) recognized by the lair- -containing antibodies, we generated stable p. falciparum d lines that were enriched ( d -mgd + ) or depleted ( d -mgd -) of mgd reactivity (extended data fig. a ). western blot analysis showed two specific mgd -reactive bands of - kda in erythrocyte ghosts and in mgd immunoprecipitates (ip) prepared from d -mgd + ie (fig. a) . analysis of the mgd ip by liquid chromatography-coupled mass spectrometry (lc-ms) revealed that a member of the a-type rifin family (pf d _ ) was significantly enriched in d -mgd + ip as compared to d -mgd -ip (log fold change > ; p < . ) (fig. b ). pf d _ and a second a-type rifin (pf d _ ) were also identified in d -mgd + but not in d -mgd ghosts in the absence of immunoprecipitation (extended data fig. b ). in contrast, four other rifins, including one recently characterized for its capacity to induce rosetting (pf d _ ) , were detected in similar amounts in both d -mgd + and d -mgd ghosts. we found that enrichment for d -mgd + ie greatly increased recognition by all the other broadly reactive antibodies from donor d tested and, notably, by two broadly reactive antibodies from donor c (extended data fig. c ), suggesting that these antibodies recognize the same antigens. similar results were obtained with the kenyan isolate (extended data fig. d -e). the binding of the lair- -containing antibodies to specific rifins was confirmed by the finding that mgd stained cho cells transfected with the candidate antigens (pf d _ and pf d _ ), but not with irrelevant rifins that were similarly expressed (pf d _ and pf d _ ) or not detected (pf d _ ) in d -mgd + and d -mgd ghosts (fig. c ). furthermore, mgd and an fc fusion protein containing the mgd lair- exon stained cho cells transfected with a rifin chimera containing the constant region of pf d _ and the variable region of pf d _ , but not cells transfected with the inverse chimera (extended data fig. f g), indicating that mgd binds to the variable region. collectively, these results indicate that the lair- -containing antibodies recognize specific members of the rifin family in different p. falciparum isolates. addition of mgd to d culture did not interfere with parasite growth and did not result in decreased expression of the antigen(s) (extended data fig. h -i). in addition, when tested in a rosette inhibition assay with o + or a + erythrocytes, mgd did not show a consistent inhibitory effect (p > . for both blood groups) (extended data fig. j ). in contrast, mgd , as well as mgc , could agglutinate erythrocytes infected with d or the kenyan isolate (extended data fig. k ). furthermore, mgd showed a strong capacity to opsonize d ie for phagocytosis by human monocytes (fig. d ). opsonization was dependent on an intact fc, as a mutant lacking fc receptor binding (mgd lala) did not induce phagocytosis. similar results were obtained with other broadly reactive antibodies isolated from both donors and with a different parasite isolate ( ) (extended data fig. l ), suggesting that these broadly reactive antibodies could be effective in promoting phagocytosis and destruction of ie in vivo. our study opens several questions as to the potential use of rifins as targets for passive and active vaccination. rifins represent the largest family (~ genes) of variant antigens expressed on ie, some of which have been implicated in severe malaria . the lair- containing antibodies have potent agglutinating and opsonizing activity, which would be consistent with their role in decreasing the burden of ie in vivo by enhancing parasite clearance. however, the staining of only a fraction of ie by the lair- -containing antibodies is consistent with the clonal expression of rifins and suggests that these antibodies may not be sufficient to take full control of the infection. it will be interesting to determine whether the lair- -containing antibodies recognize rifins that are expressed at other stages of the parasite life cycle, such as sporozoites, merozoites and gametocytes , , which may open interesting opportunities for vaccine design. the unusual architecture of the lair- -containing antibodies illustrates a novel mechanism of inter-chromosomal dna transposition that can contribute to antibody diversification (extended data fig. ). the precise location of the lair- and chr inserts between the v and d/j segments, as well as the presence of n-nucleotides and cryptic / rsss at the ends of the inserts, would be compatible with a role for the rag enzyme. rag has been implicated in inter-chromosomal genomic rearrangements at cryptic rsss outside the ig and tcr loci , , and in the formation of chromosomal translocations found in human lymphomas , . however, rsss are frequently found in the genome and are generally inactive, according to recent data , . furthermore, the conservation of the two lair- alleles in b cells producing lair- -containing antibodies is inconsistent with a ragmediated "cut-and-paste" mechanism and suggests a new mechanism by which lair- dna is duplicated. it is possible that gene conversion or aid-dependent genomic instability caused by chronic plasmodium infection may contribute to the production of lair- -containing antibodies. aid can lead to insertions and deletions of multiple codons in the v-genes, which contribute to the specificity of the antibody in the context of the whole v-gene , . nevertheless, to the best of our knowledge, these insertions are distributed over the whole v-gene sequence, are of smaller size and cannot be traced back to a particular genomic sequence as in the case of lair- . the transposition of lair- (and chr ) sequences into v-dj genes is the first example of an insertion that gives rise to a functional antibody where the insert represents the fundamental binding element. it remains to be established how often this novel mechanism may give rise to functional antibodies and whether sequences other than lair- are transposed into ig genes. we anticipate that lair- -containing antibodies will be frequently found in malaria-endemic regions and speculate that the transposed lair- domain may serve to bind other foreign antigens and possibly also collagen in patients with rheumatic diseases. the plasmodium falciparum clone d and nine laboratory-adapted parasite isolates from severe and non-severe malaria patients in kilifi, kenya (sampled between and ), were cultured in vitro according to standard procedures and cryopreserved at the late . adults in this area are clinically immune from febrile malaria, having acquired immunity during childhood. the two donors were p. falciparum-negative during sample collection. following informed consent, plasma samples were taken from to from adults living in a malaria-endemic region within kilifi county on the coast of kenya. the study was approved by the kenya medical research institute ethics review committee and the oxford tropical research ethics committee. ie from three parasite isolates were separately stained with μg/ml dapi, μg/ml ethidium bromide or . × sybr green i for one hour at room temperature. the stained parasites were washed five times, mixed in equal proportions, and diluted to a % haematocrit in incomplete rpmi medium. ten μl of the parasite mixture was rotated with . μl of adult plasma for . h at room temperature, and agglutinates formed were examined by fluorescence microscopy. in the primary screen, pools of five adult plasma were tested against six kenyan isolates (in two separate reactions). pools that formed mixed-color agglutinates were identified and individual plasma within these pools were tested against nine isolates using the same assay. a single isolate ( ) was not detected in mixed agglutinates formed by any of the plasma and was therefore excluded from the study. two adults (donors c and d) with plasma that formed mixed agglutinates with eight parasite isolates were selected for monoclonal antibody isolation and, following further informed consent, an additional blood sample was taken from each individual in february . igg + memory b cells were isolated from cryopreserved pbmcs by magnetic cell sorting with mouse anti-cd -pecy antibodies (bd pharmingen, cat. no ) and mouse anti-pe microbeads (miltenyi biotec, cat. no - - ), followed by flow cytometry sorting for igg + igm -igdcells. the b cells were immortalized with epstein-barr virus (ebv) in the presence of cpg-dna ( . μg/ml) and irradiated feeder cells as described previously . two weeks post-immortalization, culture supernatants were tested for the ability to stain ie from eight parasite isolates by flow cytometry. cryopreserved ie were thawed, stained with × sybr green i, and incubated with the b cell supernatants for h at ºc. antibody binding was detected using . μg/ml of goat alexa fluor -conjugated anti-human igg (jackson immunoresearch, cat. no - - ). reactivity was calculated based on the percentage of late-stage parasites (high sybr green) recognized by each antibody. cdna was synthesized from selected b cell cultures and both heavy chain and light chain variable regions (vh and vl) were sequenced as previously described . the usage of vh and vl genes and the number of somatic mutations were determined by analyzing the homology of vh and vl sequences of mabs to known human v, d and j genes in the imgt database .genomic dna was isolated from two b cell lines of donor c and one b cell line of donor d with a commercial kit (qiagen), and antibody-encoding sequences were amplified and sequenced with primers specific for the v and j regions of the given antibody. sequences were aligned with clustalw . potential cryptic rss sites were identified using the rsssite web server . to determine the heterozygosity of lair- on chr , the following primers were used to perform pcrs on genomic dna (lair _intr_fw ggcggtgggcactcaggttc; lair _intr_rev cacaggcagtcaccgggtctagg; lair _intr_fw ggatgcaccatgtcacccagtcctgg). genomic dna isolated from pha-and il- stimulated t cells from donor c was used as a control for sequence analysis. unmutated common ancestor (uca) sequences of the vl were inferred with antigen receptor probabilistic parser (arpp) ua inference software, as previously described . uca sequences of the vh were constructed using imgt/v-quest and the genomic insert sequences. nucleotide sequences of the mutated antibodies and the uca were aligned using clustalw , and phylogenetic trees were generated with the dna maximum likelihood program (dnaml) of the phylip package, version . , . antibody heavy and light chains were cloned into human igg , igκ and igλ expression vectors and expressed by transient transfection of expi f cells (thermofisher scientific) using polyethylenimine (pei). cell lines were routinely tested for mycoplasma contamination. the antibodies were affinity purified by protein a chromatography (ge healthcare). variants of the mgd antibody were produced by i) exchanging v h , d h , j h elements or the light chain with the corresponding sequences of an irrelevant antibody (fi , reactive to influenza virus ), ii) deleting selected segments, or iii) reverting somatic mutations to the germline configuration with reference to the imgt database and the original lair- genomic sequence (ncbi reference sequence: nc_ . ). in addition, lair- -fc fusion proteins were produced recombinantly by cloning the mutated or unmutated lair- fragment into a plasmid designed for expression of human igg fusion proteins (pinfuse-higg -fc , invivogen). based on an alignment of the most potent lair- -containing antibodies with the unmutated lair- sequence, five key residues that could contribute to gain of binding to ie and loss of binding to collagen were identified and added alone or in various combinations to the unmutated lair- -fc fusion protein. ghosts and immunoprecipitates were dissolved in × sds sample buffer (bio-rad) and run on a % polyacrylamide gel under non-reducing conditions. the proteins on the gel were transferred onto a pvdf membrane, which was blocked with % milk in tbs with . % tween (tbst) for h at room temperature. the membrane was incubated with μg/ml mgd overnight at ºc, washed with tbst, and developed with hrp-conjugated sheep anti-human igg (ge healthcare, cat. no na ) used in combination with a chemiluminescent substrate. genes encoding the a-rifins pf d _ , pf d _ , pf d _ , pf d _ , and pf d _ were produced by gene synthesis (genscript) and cloned into the pdisplay vector (invitrogen), which contains an ha tag, as previously europe pmc funders author manuscripts described . rifin chimeras containing the constant region of pf d _ (residues - ) and the variable region of pf d _ (residues - ) [pf d _ c_ v], or containing the constant region of pf d _ (residues - ) and the variable region of pf d _ (residues - ) [pf d _ c_ v], were generated. the pdisplay constructs were transiently transfected into chok -sv cells (gs-system, lonza) using pei. cell lines were routinely tested for mycoplasma contamination. briefly, one day before transfection, chok -sv cells were seeded at . × cells/ml in ml cd-cho medium (invitrogen) supplemented with mm l-glutamine in ml erlenmeyer flasks (corning). on the day of transfection, μg dna was diluted in opti-pro sfm medium (invitrogen) and mixed with μg pei for min at room temperature. the dna-pei complexes were added to the cells, which were cultured in a co shaker incubator at °c, rpm. after hours, the expression of rifins and their recognition by the lair- -containing antibodies were tested by flow cytometry. briefly, μg/ml of rabbit anti-ha tag and μg/ml of mgc or mgd antibodies were added to the rifin-transfected cells. antibody binding was detected by μg/ml of alexa fluor -conjugated goat anti-rabbit igg (life technologies, cat. no a ) and . μg/ml of alexa fluor -conjugated goat anti-human igg (jackson immunoresearch, cat. no - - ). dead cells were excluded by staining with -aad (bd biosciences). d -mgd + ( % parasitemia, ring stage) was cultured with various concentrations of mgd or bkc for d. after d, × sybr green i was added to aliquots of each culture and parasitemia was quantified by flow cytometry. the remaining parasites in each culture were washed to remove the antibodies and incubated for d to allow the parasites to reach the late trophozoite/schizont stage. mgd recognition of these cultures was detected using . μg/ml of alexa fluor -conjugated goat anti-human igg (jackson immunoresearch, cat. no - - ). -mgd + ie at the late trophozoite/schizont stage were purified from uninfected erythrocytes and ring-stage parasites using a magnetic column (miltenyi biotec) and were resuspended in culture medium with % human serum. the purified ie were incubated with μg/ml of mgd or bkc for h at ºc, mixed with o + erythrocytes or a + erythrocytes in a : ratio, and incubated for min at room temperature to allow rosetting to occur. the ie were stained with × sybr green i, and the number of rosettes formed by at least ie was counted by fluorescence microscopy to calculate the rosetting rate. d -mgd + and -mgd + ie ( - % parasitemia) were diluted to a % haematocrit in a × sybr green i solution containing μg/ml of the test monoclonal antibody. each sample was rotated for h at room temperature and subsequently examined by fluorescence microscopy. ie were stained with μg/ml dapi for min at room temperature, washed four times and run on a magnetic column (miltenyi biotec) to purify late stage parasites. the purified parasites were opsonized with serially diluted antibodies for h at ºc. monocytes were isolated from fresh pbmcs of healthy donors using mouse anti-cd microbeads (miltenyi, cat. no - - ) and mixed with the opsonized parasites in a : ratio for h at ºc. extracellularly bound, non-internalized ie were lysed by treatment with red blood cell lysis solution (miltenyi biotec) for min at room temperature. the cells were stained with mouse anti-cd -pecy (beckman coulter, cat. no a ) and analyzed by flow cytometry. the mean fluorescence intensity (mfi) of dapi in cd + cells was used as a measure of phagocytosis of ie by monocytes. the wilcoxon signed-rank test was used for statistical comparisons of pairs of data groups in rosetting experiments. refer to web version on pubmed central for supplementary material. heterozygosity of the chr lair- exon in cells from donor c showing that both lair- alleles are intact in b cells producing lair- -containing antibodies. displayed are the chromatograms obtained for b cell clones with or without a lair- insertion (lair- + or lair- -b cell) and for polyclonal t cells. surface antigens of plasmodium falciparum-infected erythrocytes as immune targets and malaria vaccine candidates antigenic variation in plasmodium falciparum rifins are adhesins implicated in severe plasmodium falciparum malaria parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus the inhibitory collagen receptor lair- (cd ) discovery of gene function by expression profiling of the malaria parasite life cycle a proteomic view of the plasmodium falciparum life cycle in vivo transposition mediated by v(d)j recombinase in human t lymphocytes v(d)j recombinase-mediated transposition of the bcl gene to the igh locus in follicular lymphoma mechanisms of chromosomal translocations in b cell lymphomas cellular origin of human b-cell lymphomas rag represents a widespread threat to the lymphocyte genome chromosomal loop domains direct the recombination of antigen receptor genes what role for aid: mutator, or assembler of the immunoglobulin mutasome? plasmodium infection promotes genomic instability and aid-dependent b cell lymphoma somatic hypermutation introduces insertions and deletions into immunoglobulin v genes immunoglobulin gene insertions and deletions in the affinity maturation of hiv- broadly reactive neutralizing antibodies crystal structure and collagen-binding site of immune inhibitory receptor lair- : unexpected implications for collagen binding by platelet receptor gpvi human malaria parasites in continuous culture wind direction and proximity to larval sites determines malaria risk in kilifi district in kenya efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning imgt, the international immunogenetics information system clustal w and clustal x version . rsssite: a reference database and prediction tool for the identification of cryptic recombination signal sequences in human and murine genomes reconstructing a b-cell clonal lineage. i. statistical inference of unobserved ancestors co-evolution of a broadly neutralizing hiv- antibody and founder virus rapid development of broadly influenza neutralizing antibodies through redundant mutations maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification global quantification of mammalian gene expression control we thank m. nussenzweig for providing reagents for antibody cloning and expression. this work was supported by shown is the insertion of a fragment of lair- into the ig heavy chain locus through a mechanism still to be molecularly defined, followed by the acquisition of somatic mutations that increase binding to ie and abolish binding to collagen. refer to web version on pubmed central for supplementary material. key: cord- -h su authors: guan, lili; liu, jin; wu, xia min; chen, dafang; wang, xun; ma, ning; wang, yan; good, byron; ma, hong; yu, xin; good, mary-jo title: unlocking patients with mental disorders who were in restraints at home: a national follow-up study of china’s new public mental health initiatives date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: h su background: in , china implemented a demonstration program known as “ ” to scale-up nation-wide basic mental health services designed to improve access to evidence-based care and to promote human rights for people with severe mental disorders. as part of the program, teams “unlocked” and provided continuous mental health care to people with severe mental disorders who were found in restraints and largely untreated in their family homes. we implemented a nation-wide two-stage follow-up study to measure the effectiveness and sustainability of the “unlocking and treatment” intervention and its impact on the well-being of patients’ families. methods: patients unlocked from in “ ” demonstration sites across china were recruited in stage one of the study in . in , of the cases were re-interviewed (the stage two study). outcome measures included the patient medication adherence and social functioning, family burden ratings, and relocking rate. we utilized pre-post tests to analyze the changes over time following the unlocking efforts. results: % of patients were diagnosed with schizophrenia. prior to unlocking, their total time locked ranged from two weeks to years, with % having been locked multiple times. the number of persons regularly taking medicines increased from one person at the time of unlocking to % in and % in . pre-post tests showed sustained improvement in patient social functioning and significant reductions in family burden. over % of patients remained free of restraints in . conclusion: practice-based evidence from our study suggests an important model for protecting the human rights of people with mental disorders and keeping them free of restraints can be achieved by providing accessible, community based mental health services with continuity of care. china’s “ ” program can inform similar efforts in low-resource settings where community locking of patients is practiced. % of patients were diagnosed with schizophrenia. prior to unlocking, their total time locked ranged from two weeks to years, with % having been locked multiple times. the number of persons regularly taking medicines increased from one person at the time of unlocking to % in and % in . pre-post tests showed sustained improvement in patient social functioning and significant reductions in family burden. over % of patients remained free of restraints in . mental disorders constitute a huge global burden of disease, and there is a large treatment gap, particularly in low and middle-income countries (lmics). in china, mental and behavioral disorders contribute to . % of years lived with disability (ylds) [ ] . recognizing the gap between the population's needs for mental health care and the level of resources and services available [ ] [ ] [ ] , the chinese government invested in an innovative reform of mental health services [ ] and mental health legislation [ ] [ ] . in , as part of an effort to rebuild the public health infrastructure following the severe acute respiratory syndrome (sars) outbreak, china officially placed mental health services within the public health system. in , the government supported the launching of the national continuing management and intervention program for psychosis, known as " " after initial funding of cny . million [ , [ ] [ ] [ ] . this program has contributed to improving care for patients with severe mental disorders, including schizophrenia, psychosis, bipolar disorder and schizoaffective disorder, through increasing access to treatment and integrating hospital and community services designed to provide continuity of evidence-based care and to address patients' rights. the program prioritized equal access to a basic package of community based mental health care for patients with severe mental illness, especially for those living in poverty. the program focused on promoting rehabilitation and recovery, family education and support, and making medications continuously available [ ] . according to liu and colleagues [ ] , implementing the program has led to significant progress in scaling up mental health services in china. by , demonstration sites had been established, two in each of china's provinces, covering million people in " " catchment areas. by the end of , . million people from the general population in cities were covered by this program, a total of , patients ( . % of the general population) were registered, and , patients received regular follow-up [ ] . as the program was being initiated, teams found seriously mentally ill individuals who were physically restrained or "locked" by family members accounted for approximately . % of the patients registered in the " " demonstration sites. although these were rare events in the community, the patients indeed lived miserable lives, without care from medical personnel. many were secluded in separate rooms or outbuildings, such as huts; some were also restrained by ropes, belts or chains; a few were locked in iron cages. program staff used "free-medication" funds, which were developed by program for providing a subsidy (cny per case per year) for the expenses of essential antipsychotic medication to the patients who were socio-economically disadvantaged, to unlock and treat the patients in restraints between and . from , the program provided additional funds (cny , per case per year) for the "unlocking and treatment" intervention to "free" the locked patients and to provide immediate medical care and enrollment in " " follow-up services. widespread reports by journalists, human rights activists, and mental health specialists make it clear that in societies with limited mental health resources in asia and africa, many persons with severe mental illness are locked by family members and live in appalling conditions [ ] [ ] [ ] [ ] [ ] . others are locked or chained in temples or compounds of traditional healers [ ] . despite such reports, we find no report of empirical studies of the long-term effectiveness of programs designed to "unlock" the patients and provide mental health services in such settings. only one report published in english demonstrated that restrained patients with schizophrenia-spectrum disorder exhibited clinical improvement within months of psychiatric treatment after unlocking in bali, indonesia [ ] . however, no specific outcome measures were clearly indicated. the present study aims to measure the long-term effects of an "unlocking and treatment" intervention undertaken from in the context of a larger mental health services reform program in china. patients with severe mental disorders were followed-up about their medication adherence, mental health status, social functioning and family burden in and to investigate the changes over time following the unlocking efforts. we hypothesized that the patients and families would benefit from the intervention and the improvements achieved could be largely sustained through . this report concludes by suggesting the implications of this intervention for other settings with limited mental health resources. we designed a nation-wide two-stage follow-up study to investigate the initial phases of the program "unlocking and treatment" intervention. individuals who fulfilled the following criteria were included in the study: ) met diagnostic criteria for schizophrenia, schizoaffective disorder, bipolar disorder, delusional disorder, mental retardation with psychotic symptoms, or schizophrenia-like psychosis in epilepsy; ) received the "unlocking and treatment" intervention provided by the program between january , and july , ; and ) were not inpatients at the time of investigation. by july , the program had enrolled and unlocked a total of patients in the demonstration sites across provinces of china. (no locked cases were reported by the demonstration sites in beijing, shanghai, jiangsu and zhejiang at that time.) these represented a complete sample of the locked individuals among the patients with severe mental disorders registered in " " demonstration sites at that moment. among them, were included in stage one of the study between august , and september , . information about patients and families were gathered for both the time when patients were unlocked (t ) and the time of stage one interviews (t ) to allow pre-post comparison. in june , of these patients and families were followed-up in stage two of the study (t ) (fig ) . case report forms designed by the " " implementation and evaluation research team from peking university institute of mental health (pkuimh) were utilized at both stage one data collection (concerning t and t ) and stage two data collection (t ) to collect the same data at each time point. t was defined as one month prior to the " " unlocking action; t was defined as the month prior to participation in the stage one of the study in ; and t was defined as the month prior to the date of participation in the stage two study in . one or two key investigators from each province were trained to collect patient clinical data concerning t , t and t from existing medical records routinely recorded by clinical staff for each unlocked patient, dated from the time patients were first unlocked by the program (i.e., t ). hospital psychiatrists and community case managers reviewed the accuracy of the case records from hospitalizations as well as records from the community clinics. in addition, the key investigators were trained to interview family members in their homes during stage one (concerning t and t ) and stage two (t ) studies to collect data about the locking and relocking history as well as family members' subjective experiences of family burden. the study was approved by the ethics review board at peking university institute of mental health (pkuimh). patients and/or their family members/guardians consented to participate in a written form. neighborhood/village committee staff, who were trained by program to be mainly responsible for helping find the patients and leading community advocacy, provided information about the seriously mentally ill individuals who were locked in their family homes and assisted the unlocking team reach the patients. the unlocking process began with requesting permission from families of locked patients to allow a small group of trained professionals to free the patients. among the patients registered by program, were in restraints at home, and all of the families agreed to participate in the "unlocking and treatment" intervention program. the unlocking team consisted of three or more staff, including psychiatric health professionals (psychiatrists and psychiatric nurses), community persons who were often health workers, and neighborhood committee members. following unlocking, patients received physical exams and psychiatric diagnoses. patients assessed to be in need of hospitalization were admitted immediately to the provincial psychiatric hospitals. costs of medical and psychiatric treatment, including medications, were covered by the program. upon discharge, patients were enrolled in " " community services and followed monthly in their community by specially trained " " case managers, who are mainly primary health care workers. persons unlocked who were assessed as not needing hospitalization were enrolled in " " community services immediately after being freed; costs of antipsychotic and other prescribed medications, regular follow-ups, and community-based rehabilitation were covered by the program. as an important component of the package of care provided by the program, health education and psychosocial support were also provided to patients and their family members during follow-up visits at community health centers or village clinics. psychiatrists, psychiatric nurses, lay health care providers and non-health workers made up multidisciplinary teams to provide mental health services and non-professional services. changes in patients were assessed and recorded on a regular basis on follow-up by staff who were trained by the program to conduct standardized clinical assessments. these procedures continue into the present. in case that there is failure of preventing "relocking", the " " teams would take immediate action to restart the "unlocking and treatment" intervention as far as possible. mental health status, diagnoses, and violent behaviors. following unlocking, a psychiatrist conducted interviews with the patient and a family member who best knew the patient. overall psychiatric symptoms were assessed with the brief psychiatric rating scale (bprs) and global severity was assessed with clinical global impression (cgi) scales. a final diagnosis, based on icd- criteria, was made by a senior psychiatrist who reviewed the medical history, illness symptoms and associated impairments, and other available information. violent behaviors related to initiating restraint were evaluated on level to level by using the scale established by the national working group of program [ ] , defined as follows: level : threats other people verbally, or shouts at others, but doesn't damage any physical issue; level : damages only domestic property at home, and can be persuaded to stop; level : damages property at home or out of home, but cannot be persuaded to stop; level : repetitively damages property or hurt other people at home or out of home, but cannot be persuaded to stop; level : hurts other people with any kind of weapon, or sets fire, or explodes a bomb, etc. in the program, level and above is regarded as cases with high violence. medication adherence. adherence with antipsychotic medication over the previous month was rated by the clinician in closest contact with the patient and recorded on a three point measure as good adherence, uses antipsychotics intermittently, and does not use medications. "good adherence" was defined as patients actively take medications as prescribed, which includes following the prescribed dosing schedule, taking the full dose and refilling prescriptions. patients were rated as "uses antipsychotics intermittently" if they followed the prescription about half of the time during last month. "does not use medications" was defined as patients who did not receive antipsychotic medications for longer than two weeks. the rating was based on knowledge of the patient from routine clinical contact. social functioning. assessments of social functioning include five categories: daily life activities, housework, productive labor or work, learning ability, and interpersonal relationships. each category of social functioning was evaluated on a three point measure as good, average or poor [ ] . the assessments were based on clinician's observation of the patient's performance as well as reports from patient and family members. family burden ratings. we explored the impact of patient mental illness on the well being of families, taking changes in caregivers' ratings of their family burden experiences as a subjective measure. family members were asked to rate their subjective experiences on analogue scales from "no impact at all" to "extremely negative impact" for the five categories of family burden-stigma, psychological pressures, economic burden, loss of personal energy, and interpersonal relationships. ratings concerning t were obtained by families' retrospective reflections at stage one study. relocking rate. patients remaining free of restraints after receiving " " unlocking and treatment intervention was considered a sign of success of the program. the relocking rate was defined as the proportion of patients who were relocked after the initial "unlocking and treatment" intervention. the comparison of variables at t (before unlocking), t (after unlocking ) and t (after unlocking ) was performed using wilcoxon signed rank tests, as the data about medication adherence and social functioning were of ordinal categorical variables, and the data about family burden ratings were not normally distributed and did not meet the criteria of compound symmetry. chi-square tests were used to measure male/female and rural/urban differences in terms of psychiatric diagnosis and duration of illness. the level of statistical significance was defined a priori as a two-sided p-value of . or less. all calculations were performed with spss version . . as ( %) cases of the total sample dropped out of the study at t , data from ( %) cases were used in the statistical analysis and related comparisons at t . based on our analysis of demographic features, restraint history, and measures of mental health status, violent behaviors, and social functioning at both t and t , there was no evidence of systematic bias introduced by loss to follow-up of patients. a total of subjects aged - (mean age . [sd . ]) were included in the study at stage one. as presented in table , the "locked" population suffered from grave mental illnesses of long duration, with % afflicted with schizophrenia. there were no male/female and rural/ urban differences in terms of psychiatric diagnosis or duration of illness (p> . ). table describes the restraint history of these patients before being unlocked (t ). total time of restraint varied from two weeks to years, with % being locked multiple times. the vast majority of families reported that the top two reasons for "locking" a mentally ill family member were financial problems associated with caring for the patient ( %) and serious difficulties in finding capable caretakers ( %). families also reported that patient violence triggered the most recent restraint; % of patients exhibited serious violent behaviors, at level or above on the program's violent behavior scale [ ] . after being unlocked by the program, patients ( %) were admitted to a psychiatric hospital for professional treatment; patients ( %) were immediately enrolled in " " community outpatient programs. upon discharge from psychiatric hospital, % of inpatients were regarded by medical staff as improved and % as significantly improved; two (< %) showed no improvement. changes in patient medication adherence and social functioning table illustrates changes in patients as measured in stage one (t and t ) and stage two (t ) studies. prior to unlocking, % of the patients were not receiving antipsychotic medication; % of the patients were receiving medications and used them intermittently; only one patient showed good adherence to medication. all patients were assessed to require antipsychotic medications at the time of unlocking. significant changes in medication adherence occurred after patients were unlocked and entered into the " " treatment program. three quarters of patients were assessed as having good adherence to medication treatment both at t and at t . social functioning in each of the five categories also improved significantly, showing major changes from t to t (p< . ). although the proportion of patients with poor functioning increased slightly from t to t (p< . ) except in "productive labor or work" category (p = . ), most changes were sustained through , as indicated by the t -t comparisons (p< . ). table . changes in patient medication adherence and social functioning (t , t and t comparison). t t t t -t test t -t test t -t as illustrated in table , when comparing family members' evaluations on how much they were affected by the patients' mental illness, post-unlocking scores illustrated significant improvement in caregivers' experience with a reduction in rating scores on each of the measures of family burden from t to t (p< . ). although measures of family burden except stigma (p = . ) increased slightly from t to t (p< . ), the improvement in five categories of family burden ratings were largely sustained as shown in the t -t comparisons (p< . ). by the time of the stage two study, ( %) of the initial individuals had an episode of "relocking" following the " " intervening efforts. poor adherence to treatment, including "uses antipsychotics intermittently" and "does not use medications", was found at t in ( %) cases who were relocked. families noted the top two reasons for relocking to be similar to reasons for initial locking: no capable care-giver was available and financial difficulties. this study reports findings of an implementation research project designed to measure the effectiveness of an "unlocking and treatment" intervention that was developed in the context of china's program [ ] . our analysis of cases of patients who had been unlocked, entered into treatment, and returned to their homes- at baseline in , and in -provides the first practice-based evidence for long-term benefits of an intervention approach that is linked to a major mental health services reform. the persons locked by family members in rural and urban china were primarily patients who had lived with severe mental illness for many years, as a result of a functional failure of the traditional hospital-based mental health model. less than % of the total sample were in regular treatment. unlocking patients and providing access to free hospital treatment and continuing mental health care in the community were shown to have powerful and sustained benefits for these patients. it is encouraging that the number of persons regularly taking medicines increased from one person at the time of unlocking to three quarters in and in , table . changes in family burden ratings (t , t and t comparison). t a t t t -t test t -t test t -t data are mean (sd). family members were asked to rate their subjective experiences on analogue scales from "no impact at all" to "extremely negative impact". the higher the score the higher the family members experience family burden that was associated with patients' mental illness. especially given that an epidemiological study suggests only % of persons with psychotic illness in china have ever sought specialized mental health care [ ] . besides the improved clinical outcome achieved, improved social functioning was shown to hold not only through (t ) but through (t ), compared with the time the patients were freed by the program (t ). improved access to care and social functioning of patients had direct benefits for the lives of families as well. family caregivers described significant reductions in feelings of stigma, psychological pressures, economic burden, loss of personal energy, and negative personal relationships at both t and t . we observed some decline in patient social functioning and increased family burden from t to t . this might be due to the habituation effect. over the years after the "unlocking", families became accustomed to the initial impact of the initiative on patients' clinical condition, and thereby their ratings were biased toward minimizing the initial improvement at t . other possible explanations might be: ) patients received more intensive services during t -t , especially immediately after being enrolled in the "unlocking and treatment" intervention, than during t -t ; such services included inpatient care, assistance from the disabled federation and the civil affairs association, the " " rehabilitation programs, etc. ) those were the most vulnerable population in the community with the least societal resources. therefore the families had very limited ability to take care of the patients and cope with family burdens. it is suggested that further efforts are needed to continue to support the long-term recovery of patients from severe mental illness and to improve the well being of the families. our study suggests that successful eradication of restraint practices can be achieved and sustained by scaling up services to provide effective, accessible and affordable care for people living with mental disorders [ ] [ ] . the finding that more than % of those unlocked and entered into continuous treatment by the program remained free of restraints by demonstrates the feasibility of improving the human rights of persons with severe mental illness by increasing access to mental health care in the community [ ] , even with limited societal resources. nevertheless, the failure to prevent relocking for individuals suggests that considerable room for improvement of our mental health care practice still exists. continuous and efficient treatment and strong social supports are warranted to prevent the recurrence of restraints. persons with severe mental illness worldwide live in "zones of abandonment" [ ] . in north america, these include the streets, halfway houses, homeless shelters, and prisons. in countries with very limited mental health resources, these are often homes of family members, where a combination of lack of medical care, outbursts of violence inside the household or a tendency to wander outside the house, stigma and embarrassment, and lack of ability of families to provide care lead to seclusion and restraints [ ] . this phenomenon, which occurs throughout the world [ ] [ ] [ ] [ ] [ ] [ ] [ ] , represents human rights violations of individuals with mental and psychosocial disabilities [ ] . to prevent patients with severe mental disorders from being pushed to the margins of society, continuous development of accessible services in the community should be guaranteed for the long run. policies and laws should be well formulated to stimulate advocacy and education campaigns, as well as establish and develop legal and oversight mechanisms to prevent human rights violations [ , ] . limitations of this study grow out of the fact that it was designed after the program was underway; it is not a prospective study with an experimental design. family burden ratings at t were obtained by family care-givers' retrospective reflections, which may introduce bias; these findings should therefore be interpreted with caution given their subjective nature. however, family care-givers, usually parents and spouse of the patients, had very clear memories concerning the unique and painful experience of locking a mentally ill family member. we believe that with limitations, the rating of family burden based on retrospective recall thus has important validity. the sample of the study was % schizophrenia; this might make it difficult to generalize findings to locked individuals with other serious mental illnesses. at the time of this study, it was possible that one or more patients with severe mental disorders remained locked and were not reached by the program during implementation in the demonstration sites. the program was implemented according to the annual budget and tasks allocated by the central government, and patients who were the most mentally ill and lived in poverty were prioritized. the patients registered at the initial phase of the program were . % of the general population, and . % of all those registered were found to be in restraints and therefore freed and treated by the program. in a few cases, although the exact number was not available, patients or families refused to be enrolled by program for some concerns. further efforts are warranted for the program to cover the whole population of patients with severe mental disorders and provide effective mental health care for people in need. with such efforts, it is hoped that those individuals who remain in restraints at home can be fully identified, and "locking" of patients can be ultimately eliminated. there is obviously much to be done in china to scale up the program for the whole nation [ ] [ ] and to improve quality of care. this will require substantially increased investment in mental health services. however, the success of the program in maintaining the severely ill individuals in treatment over three to seven years, and the benefits of the intervention for those who have lived for years with untreated psychosis and for their families, attests to the feasibility and social value of the " " model. the chinese " " model can serve to inform similar efforts in other lmics where restraint of the patients continues [ ] [ ] [ ] [ ] [ ] [ ] [ ] . extending protection of human rights of persons with severe mental illness and providing patients equitable access to effective care in the community remain enormous challenges in the least resourced countries [ ] [ ] . findings of this study suggest that the chinese model may be one of the important models for low-resource settings. the program demonstrates the feasibility of building model mental health programs to scale up services [ , ] , by using limited investment of central government to mobilize administrative and societal resources, allocating funds from local government and lay organizations, as well as by involving lay health care providers and non-health workers in the multidisciplinary mental health service team [ , ] . rapid health transition in china, - : findings from the global burden of disease study prime: a programme to reduce the treatment gap for mental disorders in five low-and middle-income countries packages of care for mental, neurological, and substance use disorders in low-and middle-income countries prevalence, treatment, and associated disability of mental disorders in four provinces in china during - : an epidemiological survey mental health system in china: history, recent service reform and future challenges mental health law of the people's republic of china (english translation with annotations) china's national mental health law: a -year work in progress integrating mental health into primary care: the policy maker's perspective and experience in china integration of hospital and community services-the ' project'-is a crucial component in the reform of china's mental health services significance of the program for china and for global mental health packages of care for schizophrenia in lowand middle-income countries mentally iil man caged for decade in china lost in paradise: the chained-up mentally ill of bali another side of african psychiatry in the st century-chaining as containment breaking the chains local suffering and the global discourse of mental health and human rights: an ethnographic study of responses to mental illness in rural ghana treating the untreated: applying a community-based, culturally sensitive psychiatric intervention to confined and physically restrained mentally ill individuals in bali, indonesia. eur arch psychiatry china ( ) management and treatment guidelines for psychosis human rights violations of people with mental and psychosocial disabilities: an unresolved global crisis scale up services for mental disorders: a call for action the wpa action plan vita-life in a zone of social abandonment the zone of social abandonment in cultural geography: on the street in the united states, inside the family in india aceh free pasung: releasing the mentally ill from physical restraint global mental health: a failure of humanity a qualitative study of religious practices by chronic mentally ill and their caregivers in south india wpa guidance on steps, obstacles and mistakes to avoid in the implementation of community mental health care scale up of services for mental health in low-income and middle-income countries resources for mental health: scarcity, inequity, and inefficiency barriers to improvement of mental health services in low-income and middle-income countries treatment and prevention of mental disorders in low-income and middle-income countries we thank the patients and the family members who participated in the study. we acknowledge all " " staff in china who contribute to implementing "unlocking and treatment" intervention and providing mental health care to the patients. key: cord- -uej d zf authors: witkowski, peter t.; heinemann, patrick; klempa, boris; krüger, detlev h. title: molekulare identifikation von hantaviren in neuen wirten date: - - journal: biospektrum (heidelb.) doi: . /s - - - sha: doc_id: cord_uid: uej d zf in addition to classical virus isolation in cell culture, the molecular detection of new virus variants by pcr techniques allows broader epidemiological insights into the world of viral pathogens. for the detection of hantaviruses–zoonotic viruses leading to fever and organ failure in humans–we developed a genus-wide nested rt-pcr format, which enables the discovery of new members within this virus genus. the methodological approach allowed the demonstration of first hantaviruses from africa and revealed new hantavirus reservoir hosts, as shrews, moles, and bats. ó das auffinden neuer viren als krankheitserreger geschieht klassischerweise über die virusisolierung in der zellkultur. in der aufsuchenden virusdiagnostik nutzt man dazu eine reihe von zelllinien in der hoffnung, dass sich wenigstens eine davon für die vermehrung des noch unbekannten virus als geeignet erweist. im zellrasen lässt sich der zytopathische effekt nachweisen, eine auflösung der lichtmikroskopisch sichtbaren struktur des zellverbandes. nicht alle viren sind jedoch in der lage, sich in den angebotenen zellkulturen zu vermehren. selbst wenn die virusanzucht gelingt, ist die prozedur langwierig und zunächst unspezifisch, zudem erfordert der test frisches probenmaterial, da intakte viruspartikel zur vermehrung benötigt werden. die molekulare detektion neuer viren mittels polymerasekettenreaktion (polymerase chain reaction, pcr) bietet demgegenüber etliche vorteile. die pcr wurde zum ersten mal in den er-jahren erwähnt [ hantaviren bilden ein genus innerhalb der familie der bunyaviren. sie sind umhüllte viren mit einem segmentierten rna-genom negativer polarität. man bezeichnet sie als emerging viruses, weil unsere kenntnisse zu ihrer bedeutung als krankheitserreger beim menschen sowie zu ihrer geografischen ausbreitung schnell zunehmen. sie werden von kleinen säugetieren (die virusreservoire sind) auf den menschen übertragen und verursachen hämorrhagische fieber mit renaler und kardiopulmonaler manifestation. bei infektion mit bestimmten hantaviren kann die sterblichkeit bis zu prozent betragen [ ] . in deutschland treten das puumala-virus und das dobrava-belgrad-virus als krankheitserreger auf [ , ] . bisher waren allein nagetiere (ratten, mäuse, wühlmäuse) als natürliche reservoire für die einzelnen hantavirus-spezies angesehen worden. bis war lediglich ein hantavirus bekannt, welches nicht mit einem nagetier-wirt assoziiert war (thottapalayam-virus) und das über lange zeit nicht als hantavirus klassifiziert wurde [ ] . nach ihrer ersten beschreibung in den er-jahren sind mitglieder des genus hantavirus zunächst in asien und europa als erreger zoonotischer erkrankungen identifiziert worden. erst im jahr wurden zum ersten mal "neuwelt-hantaviren" in den usa und später auch in südamerika als pathogene gefunden [ ] . australien und afrika verblieben seitdem als terra incognita in bezug auf das vorkommen von hantaviren. überprüft werden müssten. um diesen nachteil auszugleichen und gleichzeitig einen möglichst universellen pcr-assay zu erhalten, wurde von uns ein weiteres degeneriertes primerpaar verwendet, welches innerhalb des abschnitts lag, der durch die erste pcr amplifiziert wurde. es entstand eine nested pcr (npcr) mit zwei zeitlich hintereinander geschachtelten kettenreaktionen, von denen nur die zweite elektrophoretisch ausgewertet wird. das produkt der ersten reaktion dient in kleiner menge als edukt für die zweite und unterdrückt auf diese weise die entstehung unspezifischer produkte. mit der so entstandenen "pan-hanta-pcr" wurden die ersten afrikanischen proben getestet und zwei neuartige, natürlich vorkommende hantaviren im westafrikanischen guinea gefunden [ , ] . trotz der genannten vorteile ist der einsatz der npcr sehr aufwendig. probensammlungen, die nach einem primären screening mittels npcr einen positiven nachweis liefern, können potenziell einen noch höheren anteil positiver befunde liefern, wenn sie anschließend mit der spezifischen qpcr erneut getestet werden. spezifische untersuchungen (z. b. einer neuen reservoirspezies) dienen der bestimmung des aufkommens des virus im natürlichen wirt und ermöglichen erste aussagen zur virusökologie. mittels phylogenetischer analysen werden evolutionäre zusammenhänge innerhalb der untersuchten virusfamilien verdeutlicht. die spezifische qpcr kann ebenfalls zur bestimmung der viruslast in patienten verwendet werden, wenn der erreger bekannt ist [ ] . biospektrum | . | . jahrgang seit wurden mittels neuer testmethoden viele neuartige hantaviren identifiziert. neben dem ersten afrikanischen hantavirus aus einer maus (sangassou-virus) wurde auch das erste insektivoren-assoziierte hantavirus (tanganya-virus) ebenfalls in afrika gefunden [ , ] . durch die nutzung des von klempa und kollegen [ ] veröffentlichten pan-hanta-assays war es auch anderen forschern weltweit möglich, das spektrum bekannter hantaviren enorm zu erweitern [ ] . seit wurden mehr als neue hantaviren molekular nachgewiesen, viele davon in vorher unerwarteten reservoirwirten, wie spitzmäuse und maulwürfe aus der ordnung der insektenfresser. einen weiteren meilenstein in der erforschung der molekularen epidemiologie dieser viren stellt der erste fund von fledermausassoziierten hantaviren dar [ ] . fledermäuse und flughunde werden als träger vieler zoonotischer viren, wie ebolavirus oder sars-coronavirus, angesehen. sie sind in der lage, durch die größe ihrer populationen pathogene effektiv zu erhalten und durch ihre mobilität ebenso zu verbreiten. es sind mittlerweile weitere hantaviren aus fledermäusen beschrieben, deren pathogenes potenzial für den menschen jedoch insgesamt noch unbekannt ist. zurzeit wird darüber diskutiert, ob hantaviren, die als nicht von insekten übertragene viren eine ausnahme innerhalb der familie der bunyaviren bilden, im laufe ihrer evolution einen wirtswechsel von insekten in insektenfressende fledermäuse vollzogen haben [ ] . abbildung zeigt einen molekularphylogenetischen stammbaum, der die verwandtschaftsgrade der hantaviren untereinander verdeutlicht und fünf gruppen umfasst. zu den klassischen phylogruppen der viren, die von echten mäusen (gruppe i), wühlmäusen (gruppe iia) und neuweltmäusen (gruppe iib) beherbergt werden, sind nun zwei neue gruppen gekommen, die eine große zahl neuer, bisher meist nur molekular nachgewiesener viren umfassen, die von spitzmäusen, fledermäusen und maulwürfen getragen werden. obwohl in den letzten jahren die entwicklung des next generation sequencing schnell voranschreitet, haben unsere erfahrungen gezeigt, dass die anwendung dieser technologie zur detektion von rna-viren mit einem segmentierten genom direkt aus biologischem probenmaterial noch nicht zufriedenstellend etabliert ist. solange die optimierung dieser methodologie nicht ausreichend fortgeschritten ist, wird die pcr als klassische methode für die aufsuchende virusdiagnostik weiterhin ihren bestand haben. afrika gilt als schmelztiegel bei der entstehung neuer viren, auch der aids-erreger vollzog hier den wirtswechsel vom tier zum menschen. während malaria, aids und tuberkulose die großen herausforderungen bei der bekämpfung von infektionskrankheiten in afrika darstellen, dürften hantavirus-erkrankungen zu den vernachlässigten krankheiten (neglected diseases) gehören. die durchführung von probensammlungen im feld (abb. ), der ordnungsgemäße transport unter strenger einhaltung der kühlkette sowie die korrekte probenaufarbeitung sind vorbedingungen für die durchführung der pcr, die vor ort oft nur mit großen anstrengungen zu realisieren sind. das pcr-gestützte auffinden der verschiedenen viren in den natürlichen reservoiren ist wiederum die voraussetzung dafür, ihre rolle als mögliche krankheitserreger des menschen zu untersuchen. wir danken frau brita auste für ihre exzellente technische unterstützung der arbeiten zur molekularen epidemiologie von hantaviren. außerdem möchten wir die sehr gute kooperation mit der gruppe von dr. fabian leendertz am robert koch-institut und mit einer vielzahl afrikanischer und deutscher partner hervorheben, ohne die unser projekt nicht möglich wäre. wir bedanken uns bei der deutschen forschungsgemeinschaft für die finanzierung -insbesondere im rahmen der "deutsch-afrikanischen kooperation in der infektiologie". ó studies on polynucleotides. xcvi. repair replications of short synthetic dna's as catalyzed by dna polymerases hantaviruses -globally emerging pathogens multiple synchronous outbreaks of puumala virus hantavirus disease in germany due to infection with dobrava-belgrade virus genotype kurkino thottapalayam virus: a presumptive arbovirus isolated from a shrew in india genetic identification of a hantavirus associated with an outbreak of acute respiratory illness hantavirus in african wood mouse novel hantavirus sequences in shrew detection and typing of human pathogenic hantaviruses by real-time reverse transcription-pcr and pyrosequencing hantaviruses: rediscovery and new beginnings hantavirus in bat the evolution and emergence of hantaviruses am institut für medizinische virologie der berliner charité wird in der arbeitsgruppe von detlev h. krüger an themen wie der molekularen epidemiologie und pathogenesemechanismen von hantaviren gearbeitet. das hier angesiedelte nationale konsiliarlaboratorium für hantaviren bietet spezielle testverfahren und fachliche beratung zu diesen pathogenen der biologischen risikostufe an. key: cord- - xiqz w authors: song, daesub; moon, hyoungjoon; kang, bokyu title: porcine epidemic diarrhea: a review of current epidemiology and available vaccines date: - - journal: clin exp vaccine res doi: . /cevr. . . . sha: doc_id: cord_uid: xiqz w porcine epidemic diarrhea virus (pedv), an alphacoronavirus in the family coronaviridae, causes acute diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. pedv can also cause diarrhea, agalactia, and abnormal reproductive cycles in pregnant sows. although pedv was first identified in europe, it has resulted in significant economic losses in many asian swine-raising countries, including korea, china, japan, vietnam, and the philippines. however, from april to the present, major outbreaks of pedv have been reported in the united states, canada, and mexico. moreover, intercontinental transmission of pedv has increased mortality rates in seronegative neonatal piglets, resulting in % loss of the us pig population. the emergence and re-emergence of pedv indicates that the virus is able to evade current vaccine strategies. continuous emergence of multiple mutant strains from several regions has aggravated porcine epidemic diarrhea endemic conditions and highlighted the need for new vaccines based on the current circulating pedv. epidemic pedv strains tend to be more pathogenic and cause increased death in pigs, thereby causing substantial financial losses for swine producers. in this review, we described the epidemiology of pedv in several countries and present molecular characterization of current strains. we also discuss pedv vaccines and related issues. throughout europe. however, during the s and s, the number of ped outbreaks decreased markedly in the re gion. only a few severe outbreaks have been reported since the s in europe. however, ped has become an endemic disease in asian pig farming countries, such as korea, china, vietnam, japan, the philippines, taiwan, and thailand [ ] . until , ped was thought to have been restricted to asian countries. however, an outbreak of pedv infection occurred in the united states in iowa in april , and within year, pedv had spread to canada and mexico [ ] , which share borders with the united states. additionally, ped outbreaks occurred in korea and japan, across the pacific ocean, also within year of the us outbreak [ , ] . the pedv strain iso lated in the united states was genetically related to the chi nese pedv strain reported in [ ] . interestingly, the ko rean and taiwanese pedv strains isolated after the us out break were genetically related to the us pedv strain [ ] . after spring , ped was no longer found only in asia. us scientists who had not researched pedv began to study this disease, which had previously not been a problem in ame ri can. from this, a ped vaccine reflecting the genetic charac teristics of the pedv strain isolated during the us outbreak was commercialized [ ] . moreover, many veterinary scien tists have focused on the development of more effective ped vaccines because of the major economic losses associated with ped outbreaks. after the us outbreak, sporadic ped out south korean, piglets < week of age died from severe watery diarrhoea after showing signs of dehydration. after the acute outbreak, piglets were anorectic, depressed, vomiting, and producing water faeces that did not contain any signs of blood. (c) necropsies of deceased piglets from the gimpo outbreak uncovered gross lesions in the small intestines, which were typically fluidic, distended, and yellow, containing a mass of curdled, undigested milk. atrophy of the villi caused the walls of the small intestines to become thin and almost transparent. (d) yellowish watery diarrhea in sucling piglets after acute infection of pedv. breaks occurred in germany. therefore, european countries were not isolated from the spread of the us pedv [ ] . thus, the us outbreak was important turning point in ped research, and ped research can be said to be divided into two eras: be fore and after . this review covers the vaccine, epidemiology, genetic struc ture, and characteristics of ped/pedv after . this report may improve our understanding of this disease, which is cur rently the most fatal disease in pigs and one of the most costly health issues in animals. furthermore, this review may pro vide insight into important topics for investigation in ped re search. the genome of pedv is positivesense, singlestranded rna. the size of pedv genomic rna is about kb. the organiza tion of the pedv genome is presented in table . from the ′ cap to the ′ poly a tail, ped genomic rna contains seven open reading frames (orfs) encoding viral proteins. orf a and orf b encode the viral polymerase. orf encodes a non structural protein with an unknown function; this protein is thought to be related to viral pathogenicity [ ] . additionally, the other orfs have specific names according to the proteins encoded in these regions, i.e., spike (s), envelope (e), matrix (m), and nucleocapsid (n) proteins [ , ] . table presents the characteristics of the pedv proteins as described in pre vious articles. of the pedv proteins, the s protein is considered the most antigenic. as summarized in table , the s protein is respon sible for the interaction with host cellular receptor molecules [ , ] . this interaction is crucial for the entry of the virus and is related to induction of neutralizing antibodies against the virus [ , , ] . additionally, these critical characteristics of the s protein are used for analysis of the molecular epidemi ology of pedv. thus, pedv researchers have started to analyze the geno types of pedv using the s gene [ ] . while other genes, such as the gene encoding the m protein and the gene encoded in orf [ ] have been used for phylogeny or molecular epi demiological studies, genetic diversity of the s gene is the fo cus of this review. one of the most interesting characteristics of the s gene is the diversity in this gene that has occurred from the end of s to the present. genotypes in the s gene of pedv could be important because this gene may affect the pathogenicity of novel ped outbreaks based on variations in the s gene [ , , , ] . moreover, the chinese ped out break in involved the presence of new variants based on the nucleotide similarities in the s gene. this variant pedv exhibited % similarity with the cv prototype pedv strain in the s gene [ ] . moreover, the pedv isolated after the us outbreak also exhibited % % similarity with the s protein of cv [ , ] . phylogenetic analyses of pedv rna have been reported in many previous studies [ , , , , ] . whether the % nucleotide difference often observed in the s gene can affect the pathogenesis of pedv has not been clarified. however, the similarities in the s gene, which is re sponsible for the host interaction and normalization, the s region in particular could be important in vaccine efficacy and development strategies [ , ] . neutralizing epitopes of the pedv s protein have been investigated [ ] . in particular, the co k equivalent (coe) is a neutralizing epitope within the co k collagenase fragment in transmissible gastroen teritis virus (tgev) [ , ] . in the prototype pedv (cv ) and vaccine strains, certain coes are different from field iso lates that were isolated after . the different coes are t and g in the prototype and vaccine strains, respectively (table ) ; however, other coes that differ from that of cv have been shown to be identical to the vaccine strains p th or attenuated dr [ ] . in pedv isolates acquired af ter in china, these amino acids were changed to serine; similar observations were made for the pedv strain from the us outbreak [ ] . the new variant pedv strains have similar mutational patterns for the neutralizing epitope. in contrast, pedv strains isolated from to exhibit different pat terns in t and g compared to strains isolated after . interestingly, at amino acid position , some strains exhibit no changes; however, in most strains, amino acid is chang ed to arginine (r). additionally, most strains exhibit a change from g to s at amino acid position , similar to current stra ins [ ] . in order to elucidate the positive correlations between chan ges in the s protein and virulence or vaccine efficacy, further studies involving animal models are needed. in addition to the changes in the s protein described above, more variations have been discovered. for example, park et al. [ ] reported large deletion in the s protein from amino acid to amino acid , within the s and s domains. this virus was detected in , and other regions of the s protein share . % . % amino acid identity in the partial s domain with other strains isolated in korean in and . in the united states, a deletion in the s gene was also reported after the united states outbreak. in december , a strain with deletion of the s gene was detected and isolated in ohio [ ] . this strain exhibited patterns that were different from those of the korean strain with the s gene deletion. the us deletion mutant contains a large deletion ( amino ac ids in length, from amino acid to amino acid ) in the s domain [ ] . the effects of deletion in the s protein should be further investigated and are reminiscent of porcine respirato ry coronavirus arising from s protein deletion in the genome of tgev [ , ] . through pedv research, unique genetic characteristics other than those in the s gene have been reported. fulllength analysis of the orf gene of pedv revealed a bp deletion in the cellattenuated pedv strain dr [ ] . this deletion in the orf gene has not been identified in wildtype pedv; however, in addition to the attenuated pedv dr strain, live pedv vaccine strains available in korea exhibit similar dele tion patterns by reverse transcription polymerase chain reac tion (rtpcr) comparisons [ ] . this deletion pattern is clin ically useful for differentiation of live vaccine strains from wild type pedv casing diarrhea. the final genetic mutation we will discuss is deletion of the e gene in the cellattenuated pedv strain dr . genetic anal ysis has focused on the s, n, and m genes. from analysis of the e gene, a unique deletion was found. moreover, compar ed to other pedv strains, including the wildtype and vaccine strains, only the attenuated pedv strain dr has been shown to have a bp deletion in this gene [ ] . the e protein of the coronavirus is responsible for the assembly of the virus and cell stress responses [ ] . the effects of the e gene deletion are under investigation. in the czech republic, rodak et al. [ ] reported that out of fecal samples from diarrheic piglets (< days old) were positive for pedv. this outbreak occurred between may and june in an area densely populated with pigs in the po valley in northern italy [ ] . some pedvpositive farms ( out of ) were detected between mid and the end of ; however, the disease progressively disappeared [ ] . during the period from to , mild clinical signs were report in pigs of all ages, and mortality was observed in pig lets only in pedvpositive farms [ ] . [ ] , and the southern prov inces of vietnam [ ] . in october , a largescale outbreak of pedv was reported in several provinces in southern china. pedv also spread to other regions of the country, particularly in northwest [ ] . pedv is now circulating in at least chi nese provinces [ ] . in october , japan reported a pedv outbreak to the world organization for animal health (oie) [ ] after a period of years without an outbreak. according to the information provided by japan's national institute of animal health, pedv isolates from this outbreak are geneti cally related to the pedv isolates recovered from china and the united states in . in addition, in late , pedv out breaks were reported in south korea and taiwan [ , , ] . the us pedv strains identified during the us outbreak were genetically related to the chinese strains (china/ /ah ) reported in [ , ] . pedv was first identified with in the united states in iowa in may , although testing of historical samples showed that pedv occurred the month before in ohio. pedv rapidly spread throughout the country and was confirmed on farms from states, including ohio, indiana, iowa, minnesota, oklahoma, illinois, and north car olina, by the end september [ ] . pedv was detected in mexico for the first time in july [ ] . in october , pedv was identified for the first time in peru [ ] . in novem ber , pedv was also identified as the cause of outbreaks of diarrhea in farms in the espaillat province, dominican re public. by september , ped outbreaks were reported in seven of the provinces in the dominican republic [ ] . in april , canada reported outbreaks of pedv to the oie; these outbreaks started in january and affected herds in four provinces [ ] . an acute outbreak of diarrhea and death in lactating piglets was observed in columbia in march . by september , samples from six departments were confirmed via laboratory testing [ ] . several reports have described the development of rtpcr as a diagnostic technique for detection of both laboratory and field isolates [ ] . primers derived from the m gene can be used in an rtpcr system to obtain pedvspecific fragments [ ] , and duplex rtpcr has been used to differ entiate between tgev and pedv [ ] . within the past few years, several useful modifications of the basic rtpcr meth od have been reported. for example, it is possible to estimate the potential transmission of pedv by comparing viral shed ding load with a standard internal control dna curve [ ] and by multiplex rtpcr to detect pedv in the presence of various viruses [ ] -a technique that is particularly useful for rapid, sensitive, and costeffective diagnosis of acute viral gastroenteritis in swine. the commercial dual priming oligo nucleotide system (seegene, seoul, korea) (fig. ) has been developed for the rapid differential detection of pedv. this system employs a single tube onestep multiplex rtpcr with two separate primer segments to block nonspecific priming [ ] . recently, a proteinbased enzymelinked immunosor bent assay (elisa) system was developed to detect pedv. using this technique, a polyclonal antibody is produced by immunizing rabbits with purified pedv m gene after its ex pression in escherichia coli. immunofluorescence analysis can then be carried out with the antipedvm antibody in order to detect pedvinfected cells among other enteric vi ruses [ ] . another useful reverse transcriptionbased diagnostic tool is rt loopmediated isothermal amplification. this assay, which uses primers that recognize regions of the tar get dna, is more sensitive than gelbased rtpcr and eli sa, largely because this method produces a greater quantity of dna [ ] . immunochromatographic assay kits can be used at farms in order to detect the n (nucleocapsid) protein of pedv with % sensitivity and % specificity. moreover, a rapid technique for differential detection of pedv and por cine rotavirus (rv) has recently been commercialized and is now widely used in the field (fig. ) . this technique is less sensitive than rtpcr, but allows for diagnosis within min utes. thus, it is particularly effective for quickly determining quarantine or slaughter policies in the field. interestingly, some reports have commented on the detec tion of pedv genomic dna in sera. genomic detection in gno tobiotic piglets has been reported for serum viral rna con centrations ranging from . to . log genomic equivalents (ge)/ml after inoculation of the us pedv strain. similar de tection of the pedv genome has been observed in diarrheic pigs at age weeks ( . . log ge/ml) [ ] . however, no infectious pedv has been recovered from genomeposi tive sera samples. unfortunately, after intensive screening and trials to isolate the pedv genome from serum samples, we have not succeeded in this endeavor (data not shown). enteropathogenic viruses can be divided into two types (type i and ii) according to their infection site in the intestine [ ] . viruses infecting villous enterocytes, including tgev, pedv, and rv, are type i viruses and can be suppressed by local gut associated immunity. diseases caused by type ii viruses, which infect crypt enterocytes basolaterally (e.g., canine parvovirus), can be prevented by inducing systemic or mucosal immuni ty. in this review, we discuss control strategies for reducing viral shedding, mortality, and the transmission of pedv in swine herds, such as transmission occurring from artificial oral exposure (i.e., the feedback method) and vaccines. in naïve swine herds, ped is characterized by vomiting and acute diarrhea and results in high mortality rates in piglets less than weeks of age. neonatal pigs are born without ma ternal antibodies if they are not infected in utero, and they should receive passive lactogenic immunity (igg and iga) through intake of colostrum and milk to promote survival af ter birth. therefore, maternalderived immunity at an early age is critical for passive protection of neonatal pigs; for this purpose, immunization of the dam preparturition has been used successfully [ ] . igg is the major immunoglobulin com ponent in pig colostrum, consisting of more than % of all immunoglobulins, but is not found in milk. iga accounts for a substantially reduced percentage of colostrum immunoglob ulin content; however, iga is more effective than igg or igm at protecting animals from orally infected agents because it is more resistant to the activity of proteolytic enzymes in the in testine and has a higher neutralizing ability than igg and igm [ ] . bohl et al. [ ] and saif et al. [ ] demonstrated that oral inoculation of seronegative sows with live virulent tgev re sults in high rates of protection in suckling piglets. in these sows, passive protection is associated with high titers of se cretory iga (siga) in colostrum and milk. this investigation suggested the presence of a gutmammary glandsiga axis; that is, iga plasmablasts stimulated in the gut by virulent path ogens migrate to the gut lamina propria and mammary glands. several highly attenuated oral tgev vaccines, which repli cate lower in the gut, induce poor milk siga titers compared with virulent tgev in sows and result in lower protective effi cacy in piglets [ , ] . this research could be employed to maternal immunization strategies for pedv. in areas where no effective ped vaccines are available, some veterinarians have recommended artificial infection of sows (i.e., the feedback method) during pregnancy to supply lacto genic immunity to their piglets [ ] . the recommended feed back material is pooled feces collected from infected piglets during the first hours of infection. every sow on the farm should be simultaneously administered feces containing high titers of pedv, allowing all sows and gilts to recover at approx imately the same time and stop shedding the virus. one of advantage of this type of feedback method is strong stimulation of mucosal immunity in the gut and a quick re sponse after immunization. after successful feedback, the piglets will be protected during the first few days after birth by passive antibodies through colostrum and milk. however, there is a potential risk of transmission of the contaminated viral or bacterial agents in the inoculum (e.g., porcine circovi rus type [pcv ] infection, porcine reproductive and respi ratory syndrome virus [prrsv] , and salmonellosis) [ , ] . additionally, it is possible that pedv may spread rapidly in pigs of all ages in the index farms. the severity of the disease may vary with unknown factors, such as stress, nutrition, or coinfection. in addition, there is a risk that the virulent virus used for feedback materials may spread to and produce dis ease in other herds. irregular immune responses in sows after feedback may also be a major concern for optimal induction of herd immunity for protection. all of these possibilities em phasize the need for a safe and effective pedv vaccine to pro tect both sows and piglets. for the prevention of pedv infection, several vaccines have been reported in asian countries; the predominance of vac cines in asian countries, but not in europe or america, is thou ght to be related to the occurrence of severe ped outbreaks and major economic losses in asia [ ] . commercial pedv vaccines include live attenuated vaccines and binary ethyl enimine (bei) inactivated vaccines. some of these vaccines have been combined with vaccines for tgev (a bivalent vac cine) and porcine rv (a trivalent vaccine) and used in china and south korea [ , ] . moreover, an attenuated virus vac cine using cell cultureadapted pedv has been administered to sows in japan since . oral vaccination with a cellat tenuated vaccine has been used in south korea since and in the philippines since [ ] . although these commer cial vaccines are considered effective and have been widely used, not all animals develop solid lactogenic immunity. sev eral factors are thought to be associated with the poor lacto genic immunogenicity of the commercial vaccine, including the immunizing route of the vaccine. song et al. [ ] demon strated that oral inoculation of pedvseronegative pregnant sows with live attenuated pedv reduces the mortality of suck ling piglets more effectively rather than injection after chal lenge, and this protection is associated with elevated iga con centrations in colostrum and milk. despite the reduction in mortality rates in piglets delivered from orally vaccinated sows, there was no shortening of the duration of virus shedding and no reduced severity of diarrhea after challenge between vac cinated and control pigs. thus, some researchers may con clude that passive immunity by vaccination with the highly attenuated pedv strain dr does not prevent virus shedding after challenge. protection against virus challenge in conven tional pigs is related to the inoculation dose of the virus in the vaccine and the challenge dose of the virulent virus. at low doses of the attenuated pedv, % of pigs are protected against pedv challenge; however, this proportion increases to % when pigs are inoculated with a dose times higher [ ] . moreover, loss of body weight and the content of viral shed ding decrease in orally vaccinated pigs compared with those in intramuscularly vaccinated and unvaccinated pigs follow ing challenge with a low dose of virulent virus ( ld , . tcid /dose). additionally, the lethal dose of pedv changes depending on the body weights of the infected piglets and in fection of sows with the challenge virus (data not shown). however, several publications have questioned the efficacy and/or safety of pedv vaccines used in asia [ , , , , ] . in particular, after the us outbreak, the efficacy of commer cial ped vaccines in korea became controversial, and simul taneously, there was an urgent need for a new vaccine in or der to establish solid immunity in sow herds prior to farrow ing to protect piglets. furthermore, many groups have debat ed the appropriate standards for evaluation of the efficacy of vaccines after official challenge tests using currently available pedv vaccines in south korea in . information on pedv mucosal immunity is limited. moreover, due to the complex characteristics of mucosal viral diseases, simple criteria, such as serum neutralizing antibodies, severity of diarrhea, and mortality after virulent challenge, are insufficient for the ac curate and optimal evaluation of pedv vaccine efficacy. for detailed identification of standards for the evaluation of pedv vaccines after virulent challenge, the following criteria, which may not be controllable, should be taken into consideration: characteristics of piglets used in the challenge test: con ventional or specific pathogenfree piglets, weight of pig characteristics of sows: parity number, existence of aga lactia or coinfection with other viral and bacterial patho gens, number of delivered piglets quantity of challenged virus: amount of viral load for the challenge virus may result in discrepancies in the observ ed mortality rates of piglets cohabitation with sows: the conditions of piglets challeng ed with virulent virus could be affected by the occurrence of viral shedding by sows duration of the challenge test the vaccine strains commonly used in korea display a max imum difference of % at the amino acid level compared with field viruses. additionally, using sn assays, the sm vaccine strain was shown to exhibit variable crossreactivity with several antisera against other vaccines and field viruses, implying that these vaccines would confer protection against the vaccine prepared using wildtype pedvs from the field [ ] . however, the crossprotection between vaccine strains and field viruses should be elucidated through animal exper iments, which can show protection based on lactogenic im munity after vaccination. for the ideal development of pedv vaccines, new vaccine strains that are genetically related to field viruses are critical. furthermore, several criteria, includ ing factors related to the reduction in virus shedding and loss of body weight in piglets as well as the details of mucosal im munity and the relationships between protection and immu nity, should be considered and identified during the develop ment of nextgeneration ped vaccines. pedv research has become a hot topic in veterinary virology since the us outbreak in . ped had been a regional dis ease primarily found in asian countries. however, ped was transmitted to the united states and subsequently to neigh boring countries, including mexico and canada. european countries have also encountered pedv. accordingly, with the spread of the ped outbreak area, research on pedv has in creased rapidly. veterinarians and veterinary scientists are striving to breakthroughs to combat current ped outbreaks worldwide, and remarkable advances have been made in the relatively short time after the outbreak. therefore, reasonable methods for ped vaccine evaluation, along with the development of new vaccines, are urgently need ed, and this complicated process could provide valuable in formation on pedv, immune responses to pedv infection, and pedv pathobiology. furthermore, rational evaluation procedures can help swine farmers understand and control ped more efficiently. a new coronaviruslike particle associated with diarrhea in swine isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of differ ent ages outbreak of porcine epidemic diarrhea in suckling pig lets letter to the editor cloning and sequence analysis of the m gene of porcine epidemic diarrhea virus ljb/ isolation and se rial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate porcine epidemic diarrhoea virus: a com prehensive review of molecular epidemiology, diagnosis, and vaccines isolation of porcine epidemic diarrhea virus (pedv) in korea an outbreak of swine diarrhea of a newtype associated with coronaviruslike particles in japan distinct charac teristics and complex evolution of pedv strains complete genome sequence of k jb , a novel variant strain of por cine epidemic diarrhea virus in south korea uslike strain of por cine epidemic diarrhea virus outbreaks in taiwan origin, evolu tion, and genotyping of emergent porcine epidemic diar rhea virus strains in the united states ped vaccine gains conditional approval sci entific opinion on porcine epidemic diarrhoea and emerg ing porcine deltacoronavirus genetic variabil ity and phylogeny of current chinese porcine epidemic diarrhea virus strains based on spike, orf , and mem brane genes porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunopro phylaxis the coro navirus spike protein is a class i virus fusion protein: struc tural and functional characterization of the fusion core complex the gprlqpy motif located at the carboxyterminal of the spike protein induces antibo dies that neutralize porcine epidemic diarrhea virus identification of the epit ope region capable of inducing 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pigs multiplex reverse transcrip tionpcr for rapid differential detection of porcine epide mic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus onestep multiplex rt pcr for detection and subtyping of swine influenza h , h , n , n viruses in clinical samples using a dual prim ing oligonucleotide (dpo) system development of a porcine epidemic diarrhea virus m proteinbased elisa for virus detection development of reverse transcription loopme diated isothermal amplification for rapid detection of por cine epidemic diarrhea virus enteric viral infections of pigs and strategies for in duction of mucosal immunity protection against rotavirusinduced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies antibody respons es in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus isolation of porcine immuno globulins and determination of the immunoglobulin class es of transmissible gastroenteritis viral antibodies exposing sows to pedv to build herd immunity national hog farmer detec tion of porcine circovirus in mammary and other tissues from experimentally infected sows colostral transmission of porcine circovirus (pcv ): reproduction of postweaning multi systemic wasting syndrome in pigs fed milk from pcv in fected sows with postnatal porcine parvovirus infection or immunostimulation oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr strain mucosal and systemic isotypespecific antibody responses and protec tion in conventional pigs exposed to virulent or attenuat ed porcine epidemic diarrhoea virus isolation and characterization of a variant porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu genetic characterization of porcine epidemic diarrhea virus in korea from key: cord- - bccxgbd authors: saxena, latika; khanna, madhu title: production and characterization of human monoclonal antibodies from the cells of a(h n )pdm influenza virus infected indian donors date: - - journal: procedia in vaccinology doi: . /j.provac. . . sha: doc_id: cord_uid: bccxgbd abstract analysis of human monoclonal antibodies (mabs) developed from influenza infected donors have enormously contributed to the identification of neutralization sensitive epitopes of influenza virus. the ha protein is a crucial target of neutralizing antibodies and at monoclonal level only abs binding to ha have been able to neutralize the virus. in this study, eight a (h n )pdm seropositive patients within the age range of - years (median= years) were recruited. two anti-ha mabs secreting stable clones, d and f were established under optimized conditions from the peripheral blood mononuclear cells (pbmcs) of the volunteers. these antibodies efficiently neutralized the homologous laboratory isolated strain of the pandemic virus as well as the reference strain. our study suggests that the anti-ha antibodies derived from infected indian patients display neutralization potential against the a(h n )pdm virus. this is the first ever study of generation of mabs against the pandemic influenza virus involving the immune repertoire if indian patients. molecular characterization of the target regions will help in identifying potential immunogens in the indian pandemic isolates and confer protective immunity against this virus. influenza a virus is a member of the orthomyxoviridae family and it is one of the main causes of prevalent infection of the respiratory tract in humans. the susceptibility to influenza illness is most common in immunologically naive infants, immunocompromised individuals and elderly . the virulence of influenza a virus is because of its easy spread by aerosol; the frequent changes in the viral antigens by antigenic drift and antigenic shift that enables it to escape from protective immunity. influenza infection in humans is self-limiting, but the virus is known to cause substantial mortality and morbidity worldwide . during the most devastating influenza pandemic of the global mortality reached to million individuals in one year. in a stark reminder to the pandemic, the population that is considered fittest was the most vulnerable during the recent h n pandemic . despite intensive efforts, the threat to influenza persists as there are many limitations to the use of existing vaccines and antiviral therapies. the protection provided by vaccines that contain killed or recombinant viral glycoprotein is weak and may last as little as months. moreover, the vaccine efficacy in immunocompromised and elderly individuals is only % . above all the vaccines have to be formulated annually as the existing year's vaccine may not provide protection to the newly emerging strains. currently four antiviral drugs are approved for use against influenza virus but, their use is restricted due to possible side effects and rapid emergence of resistant strains in the recent years. hence, the development of new therapeutic targets and strategies to control pandemic and seasonal influenza virus infection in humans is urgently needed. passive immunotherapy against influenza has been reported since the influenza pandemic, where convalescent sera was used for treating patients . however, use of convalescent plasma is being largely replaced with monoclonal antibody preparations owing to the recent advances in monoclonal antibody engineering. there are several reports where antibody therapy using polyclonal and monoclonal antibodies has been used effectively as prophylaxis against varicella, hepatitis a, hepatitis b, rabies, and respiratory syncytial virus infections . the use of human mab or humanized mab to key epitopes of infectious pathogens may help in defining the humoral responses with significant therapeutic potential. the use of human mabs may also lead to more effective post exposure prophylaxis including their use intranasally in viral diseases . human monoclonal antibody technology has seen various advances recently, like the use of epstein barr virus (ebv) to transform human b cells, development of several new heteromyeloma cell lines and cpg oligonucleotides that further enhance the efficiency of b cell transformation . in this study, we generated strongly neutralizing novel human monoclonal antibodies that were selected from the immune repertoire of influenza infected seropositive patients. we generated two stable monoclonal antibody secreting fusion clones that produced antibodies specific against a(h n )pdm virus. we further investigated the in vivo prophylactic and therapeutic efficacy of these monoclonal antibodies against influenza a virus infection in the in vivo model. this study supports the fact that fully human mabs with neutralization activity can be successfully generated from the peripheral blood of convalescent patients under optimized conditions. madine darby canine kidney (mdck) cells were procured from the national centre for cell science (nccs), pune, india. ebv transformed marmoset leukocyte cell line (b - ) was a kind gift, from dr. rahul pal, nii, delhi. the hmma . human mouse heteromyeloma was provided by dr. lisa cavacini, beth israel deaconess medical centre, boston, usa. pandemic virus reference strain a/cal/ / (h n ) was procured from victoria infectious disease reference laboratory (vidrl), australia. eight a(h n )pdm seropositive patients ( males and females) within the age range of - years (median = years) were recruited in this study. rna was isolated from the nasal and throat swab samples and influenza virus infection was detected by real time rt pcr. all the volunteers had low ct (cycle threshold) values (below ) for influenza approximately - weeks after onset of the disease. ml blood samples were collected in heparanized vacutainers from these donors. - ml plasma samples were collected from each sample and stored at - °c for serological examination. the remaining blood was used for pbmc isolation. plasma samples were treated with receptor destroying enzyme (rde) for the destruction of non-specific inhibitors. serial two fold dilution of the plasma samples was prepared in pbs in a microtiter plate.the plasma samples (diluted : ) were added in equal volume ( μl) of ha units of a/cal/ / (h n ). plates were covered and incubated at room temperature (rt) for min. the contents of the plate were mixed by gently agitating the plates manually. μl of the . % guinea pig rbcs were added to each well. plates were covered and incubated at rt for min. the hai titre was the highest dilution of the serum that showed hemagglutination activity. the monoclonal antibodies were generated from the pbmcs of infected patients by the method as per gorny m, , with minor modifications. mononuclear cells were isolated from the blood of the seropositive patients by density gradient centrifugation and were resuspended in the ebv transformation medium (b - culture supernatant that was diluted : with complete medium (rpmi+ % fcs) to a concentration of x cells/ml in the presence of cpg odn- ( μg/ml) and were cultured overnight at °c. after three weeks of culture, proliferating transformed colonies of b lymphocytes were observed using an inverted microscope . the positive clones were screened by microneutralization and expanded to t- tissue culture flasks. the positive clones were fused with hmma . (at a ratio of : ) cell lines by adding ml of warm peg/dmso solution dropwise for min. the cell culture supernatant of the stable clones was collected and the monoclonal antibodies were subsequently purified by affinity chromatography by protein g columns (sigma aldrich, usa). madin darby canine kidney (mdck) cells were grown in wells tissue culture plates, at a density of . x cells per well for h.cells were infected with tcid units of a (h n ) pdm virus. h after infection, cells were fixed with acetone: methanol ( : ) for min, followed by permeabilization with triton x . fixed cells were blocked with % bsa in pbs for h at °c and washed twice with pbs. monoclonal antibodies were added at a concentration of μg/ml and the plates were incubated for h at °c. plates were washed thrice with pbs. bound antibodies were detected with fitc labeled goat anti-human igg and observed under an immunofluorescent microscope . hemagglutination inhibition assay of the purified monoclonal antibodies was performed by the end point dilution method . two fold serial dilutions of the purified monoclonal antibodies were taken, with the maximum concentration of . μg/ml. the hai titers were expressed as the lowest concentration of the antibody that completely inhibited ha units of the pandemic virus. microneutralization assay of the purified antibodies was performed by the end point dilution method . briefly, two fold serial dilutions of the antibodies were prepared in pbs starting with the highest dilution of . μg/ml in well tissue culture plates. tcid of the pandemic isolate as well as reference strain were incubated with equal volume of the antibody for h at °c. μl mdck cells ( . x cells/ml) were added to each well. the plates were incubated for - days at °c in % co and examined for cpe. the residual infectivity was tested in four wells per dilution. the neutralizing titer was determined as the lowest concentration of the antibody at which the infectivity of tcid of the respective virus for mdck cells was completely neutralized in % of the wells. infectivity was identified by the presence of cytopathic effect on day and the titer was calculated by the reed-muench method . the infected cell lysates were first resolved on % sds-page and then transferred to pvdf membrane using trans-blot sd semi-dry transfer cell (bio-rad laboratories, ca, usa). the membrane was blocked with % non-fat milk in tbst for h at room temperature and was washed thrice with tbst. the blot was then cut into strips and denatured antigens were probed by incubating each strip with a single purified monoclonal ab at a concentration of μg/ml. anti-human igg was taken as the negative control. the strips were then washed thoroughly with x tbst for three times, min each, followed by incubation with : dilution of horse-radish peroxidase (hrp) conjugated goat anti-human igg as secondary antibody (diluted in % milk-tbst) for h at room temperature. after washing, the immuno-reactive bands were visualized by the addition of pbs containing μl/ml of % h o and . mg/ml of dab (sigma aldrich, usa). to evaluate the degree of protection of monoclonal antibodies, - weeks old female balb/c mice (n= per group) were intraperitoneally injected with purified monoclonal antibodies in three different concentrations ( μg, μg, μg), h before (pre), h after ( h post) and h after ( h post) challenge with x pfu a/cal/ / (h n ) virus. mice were monitored daily for sickness, weight loss and death for days. significance of the differences between means was determined by student t test using graph pad prism software version . (san diego, ca, usa). the results were presented as mean + s.d. plasma samples of all the donors showed serologic reactivity with the ha of the reference pandemic strain as was evident with the hemagglutination inhibition assay and showed a titer of < : .the individual titers of the plasma samples are mentioned in table i. pbmcs from all the eight seropositive volunteers were transformed with ebv and the supernatants of the transformed colonies were screened by microneutralization assay for the influenza virus specific antibodies (table ii) . the positive clones were fused with the hmma . heteromyeloma cells. after fusion only two stable fusion clones secreting neutralizing monoclonal antibodies against the pandemic virus were established. pbmcs of all patients were successfully transformed with ebv. the mean of pbmcs isolated from patients was . million per individual (range . - . ). pbmcs were plated in well tissue culture plates at , - , cells per well. approximately . % wells ( / ) were positive for neutralizing antibodies against their autologus virus. both the clones d and f clones were derived from separate donors (donor and donor respectively). most of the transformed colonies lost reactivity in subsequent rounds of screening due to possible out-growth of nonsecretors and instability in antibody generation. the mabs were purified by affinity chromatography on protein g columns. the monoclonal antibodies were analyzed in vitro by a series of assays. d and f bound to the pandemic virus a/cal/ / on elisa in a dose dependent manner. f showed lesser binding compared to the d antibody ( figure ). similar results were obtained when the two monoclonal antibodies were tested for their binding to the ha of a panel of viruses selected for the hai assay. the mabs showed binding to the reference pandemic strain as well as the laboratory isolate but did not show binding to any other h n or h n virus they were tested against. >indicates that hai activity was not detected at any concentration that was tested up to . μg/ml. the immunofluorescence data further supported the above findings that d bound more strongly to influenza virus infected mdck cells compared to the f mab ( figure ). the gel was run in the denaturing condition, to allow ha protein to denature to ha and ha . antibody d reacted with the denatured viral ha protein as was evident by a band at kda (figure ). in contrast antibody f showed no binding activity to the denatured ha .it may be hypothesised that f that did not show binding to any region in the denatured ha protein, as it might be recognizing a conformational epitope. mice were treated with graded doses ( μg, μg, and μg) of the two mabs ( d and f ), h pre, h post and h post infection with the respective strains of influenza viruses as mentioned in the methods section. the antibodies were evaluated for their prophylactic or therapeutic efficacy by the assessment of morbidity (loss of weight), mortality, viral load and histopathologic examination of the lungs. the survival and weight loss experiments suggested that the mab d achieved considerable protection at the doses μg and μg when administered prophylactically and therapeutically (table iv) . the f mab was more effective at the dose of μg. viral load analysis was performed in the subgroups h pre and h post challenge with μg mab. the lung virus titers of the treated mice confirm that upon treatment with the mabs, the virus titer considerably reduces in the lungs (table v) . the production of human monoclonal antibodies is a much more complex procedure than the production of murine monoclonal antibodies. scarcity of antibody producing cells in the peripheral blood and the difficulty to obtain the blood from naturally infected patients at the right time is one of the major concerns. moreover, ebv transformed b cells are hard to grow and secrete very low amount of antibodies . the instability of antibody production by epstein-barr virus transformed cells or fused cells is another concern. these factors result in the decreased fusion efficiency and the chances of obtaining a human hybridoma against an antigen of interest is only of the order of - to - . the human hybridoma technology is based on two main methods: fusion of ab producing b cells to myeloma cells and transformation of b lymphocytes with ebv . combination of both the methods has proved to be more beneficial than either of them alone . in this study, we report the generation of two stable fusion clones, secreting monoclonal antibodies against a(h n )pdm influenza viruses. the conditions of ebv transformation and fusion with the heteromyeloma cell line were optimised by overcoming the limitations of the current technologies. the convalescent phase plasma of the eight influenza positive patients was screened for influenza virus specific antibodies, as the serum/plasma antibody titre is a critical factor for the generation of monoclonal antibodies from pbmcs. the pbmcs were ebv transformed in the presence of cpg oligonucleotides as it has been reported to increase the rate of ebv transformation . an overall . % transformation efficiency was achieved and the cells that were positive for antibodies against the pandemic h n virus were fused with hmma . cells. the best antibody secreting hybrids were cloned at one cell /well to ensure monoclonal antibody generation and stable fusion clones were obtained- d and f that secreted mabs against a(h n )pdm and showed neutralization in the in vitro as well as in vivo conditions. monoclonal antibody d showed the maximum binding in the in vitro assays and neutralized the human isolate of the pandemic strain as well as the reference strain at lowest concentrations when compared to the f antibody. mab f showed the lesser binding in elisa and immunofluorescence assay and although it showed hai and neutralizing activity, but at a higher concentration compared to the d antibody. the western blot analysis of the monoclonal antibodies revealed that d bound to the ha region in the denatured ha protein. f did not show binding to any of the region in the denatured ha protein. hence, it is hypothesised that f may be binding a conformational epitope in the ha region. however, the binding properties of f need to be investigated further to confirm the hypothesis. the monoclonal antibodies did not show binding to the seasonal h n or any other h n or h n virus against which its hai activity was determined. the reference strains available in the laboratory were picked randomly for the hai studies. further studies regarding the mapping of the epitope of these antibodies can reveal more information on whether or not they exhibit cross reactivity to other influenza virus strains. the antibodies however, showed comparative neutralization and hai activity between the laboratory isolates of the pandemic virus and the reference strain a/cal/ / (h n ). this data reflects the fact that these indian pandemic isolates have not shown much deviation from the prototype pandemic strain in terms of antibody binding sites. the prophylactic and therapeutic efficacy of these antibodies was determined in mice. the antibody treatment was given h before, h after and h after challenge with the pandemic strain. the in vivo results were consistent with the in vitro study and all the antibodies were able to considerably protect mice from lethal influenza challenge. both the antibodies were protective prophylactically but, f could achieve only % protection in terms of survival assay at μg, but achieved % survival rate at μg. the antibodies were less protective h post infection as compared to h, the fact that is supported by the survival assays and weight loss percentage. the histopathologic examination of the lungs was done in the group of mice that were treated therapeutically at h the hisptopathology data further supported the survival and morbidity results, as the lungs from the mice that were given therapeutic doses of the antibodies, showed considerably less signs of alveolar damage and neutrophilic infiltrates resulting from virus infection. the antibodies d and f may be employed as therapeutic agents for at least h after influenza infection as is indicated by the studies in mice. to, the best of our knowledge, these antibodies are the first fully human monoclonal antibodies generated from the immune repertoire of indian patients infected with a(h n )pdm virus. molecular characterization of their epitopes with indian pandemic isolates will reveal more information of their cross-protectivity and binding characteristics. the strength of our approach for antibody generation lies in the fact that it has used human immune repertoire rather than animals and the antibodies have been generated in response to a natural infection of influenza virus. since, the antibodies have originated from humans, self -reactivity against self-antigens is minimised in comparison to antibodies that have been generated in mice or through the technique of phage display. new class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription broadly protective monoclonal antibodies against h influenza viruses following sequential immunization with different hemagglutinins human serum in influenza antibodies for the prevention and treatment of viral diseases intranasal antibody prophylaxis for protection against viral disease production of human monoclonal antibodies via fusion of epstein-barr virus-transformed lymphocytes with heteromyeloma generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune b cells an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus detection of antibody to avian influenza a (h n ) virus in human serum by using a combination of serologic assays a simple method of estimating fifty percent endpoints human monoclonals from antigen specific selection of b lymphocytes and transformation by ebv continuous cultures of fused cells secreting antibodies of pre defined specificity eb virus induced b lymphocyte cells lines producing specific antibody an optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies the author saxena l, acknowledges the senior research fellowship granted to her by the council of scientific and industrial research, government of india, without which this work would not have been possible. key: cord- -u bqca authors: tekelioglu, b. k.; berriatua, e.; turan, n.; helps, c. r.; kocak, m.; yilmaz, h. title: a retrospective clinical and epidemiological study on feline coronavirus (fcov) in cats in istanbul, turkey date: - - journal: preventive veterinary medicine doi: . /j.prevetmed. . . sha: doc_id: cord_uid: u bqca abstract the presence of antibodies to feline coronavirus (fcov) and feline immunodeficiency virus (fiv), together with feline leukemia virus (felv) antigen was investigated in ill household and stray cats attending a veterinary surgery in istanbul in – . the estimated fcov and fiv seroprevalence ( % confidence intervals) were % ( – %) and % ( – %), respectively and felv prevalence was % ( – %). fcov seroprevalence increased until years of age, was highest in and among household cats living with other cats and with outdoor access, and was lower in fiv seropositive compared to seronegative cats. symptoms typically associated with wet feline infectious peritonitis (fip) including ascites, abdominal distention or pleural effusion, coupled in many cases with non-antibiotic responsive fever, were observed in % ( / ) of cats, and % ( / ) of these cats were fcov seropositive. fcov seropositivity was also associated with a high white blood cell count, high plasma globulin, low plasma albumin and low blood urea nitrogen. the percentage of fcov seropositive and seronegative cats that died in spite of supportive veterinary treatment was % ( / ) and % ( / ), respectively. these results indicate that fcov is widespread and has a severe clinical impact in cats from istanbul. moreover, the incidence of fcov infections could be rising, and in the absence of effective vaccination cat owners need to be made aware of ways to minimize the spread of this virus. feline coronaviruses (fcovs) are enveloped, positivesense, single-stranded rna viruses classified as "subgroup a" in the family coronaviridae within the order nidovirales (vijaykrishna et al., ) . fcovs consist of two biotypes designated as feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv), which are both divided into two serotypes, i and ii. serotype i is of feline origin and difficult to grow in cell culture. serotype ii appears to have arisen from the recombination of fcov serotype i with canine coronavirus and grows rapidly in cell culture causing a lytic cytopathic effect (benetka et al., ; hartmann, ; pedersen, ) . it is thought that the fipv biotype may arise from fecvs in individual cats by internal mutation, often in immune suppressed cats (poland et al., ; vennema, ) . an alternative hypothesis is that fecvs and fipvs form distinct viral populations with infection by fipv causing fip (brown et al., ). fcovs are transmitted by the fecal-oral route and the virus can persist on fomites for - weeks where they pose a risk of transmission (hartmann, ; pedersen, ; http://dx.doi.org/ . /j.prevetmed. . . - /© elsevier b.v. all rights reserved. kipar et al., ) . fcovs primarily infect enterocytes and spread from the intestine by monocyte-associated viremia (gunn-moore et al., ; kipar et al., ) . they have also been shown to replicate in monocytes/macrophages of healthy cats (can-sahna et al., ; dye et al., ) . vertical transmission has not been demonstrated (foley et al., ) . persistently infected, asymptomatic carriers spread fcov since most of these cats shed the virus for a period of months or years, either continuously or transiently (foley et al., ; cave et al., ; dye et al., ; kipar et al., ; sabshin et al., ) . the symptoms of fcov infection are highly variable. most fcov-infected cats look healthy with the exception of a mild enteritis (pedersen, ) . up to % of fcov infected cats develop feline infectious peritonitis (fip), which is a fatal form of the infection (addie et al., ) . development of fip is strongly associated with stress, immunity, multicat households and mainly occurs in young cats between and months of age (cave et al., ; hartmann, ; bell et al., ; addie et al., ; vogel et al., ) . clinically, two forms of fip are well documented: a 'wet' or effusive form (polyserositis and vasculitis) and a 'dry' or non-effusive form (pyogranulomatous lesions in organs) (kipar et al., ) . ascites is the most prominent manifestation of 'wet form' fip while lethargy, anorexia, weight loss and fever refractory to antibiotics are common and non-specific signs of fip (kipar et al., ; addie et al., ) . diagnosis of fip is complicated and the cat's clinical history together with results from several analyses including serology, pcr and postmortem analyses are often required before a definite diagnosis can be reached (shelly et al., ; hartmann et al., ; addie et al., addie et al., , pratelli, ; sharif et al., ; taylor et al., ) . hematological and biochemical changes in fip cases are not very specific, but ascites, increase in serum protein level, increase in bilirubin, decrease in hematocrit and decrease in a:g ratio are prominent (addie et al., ) . serological tests may fail to detect recent infections and cross-reactions occur between fipv and low pathogenic fecv strains (hartmann, , sharif et al., . molecular detection systems like standard and real time reverse transcription polymerase chain reaction (pcr) have certain advantages as they are rapid and sensitive, particularly when using abdominal or pleural fluidor tissue biopsy or aspirates (pedersen, ; sharif et al., ) . a recent pcr test that is commercially available (fip virus realpcr tm test, idexx) allows differentiating fipv and low pathogenic fecv biotypes, and according to the manufacturers, the test was . % accurate in samples from % infected cats with a positive pcr result. pcr results should be evaluated together with clinical findings and postmortem samples should be analyzed by molecular methods (sparkes et al., ; hartmann et al., ; pratelli, ; addie et al., ; pedersen, ; sharif et al., ) . worldwide the prevalence of fcov infections may be up to % in multi-cat environments and - % in household cats (herrewegh et al., ; pedersen et al., ; bell et al., ; addie et al., ; sharif et al., ; taharaguchi et al., ) . detection of fcov antibodies in the early stage of infection can be useful to minimize the spread of fcovs in a breeding cattery, multi-cat household and fcov-free household (cave et al., ; dye et al., ; drechsler et al., ) . therefore, it is important to monitor cats living in multi-cat environments in order to reduce and control fcov infection. the aim of this study was to investigate fcov seroprevalence and its relationship with the animal's signalment, habitat, hematological and biochemical parameters and symptoms in cats from istanbul. during years, from january to april , a total of cats with symptoms compatible with feline viral infections were included in the study population. they included individuals with fever, depression, dullness and/or weight loss. they were examined by two different veterinarians working at a private veterinary clinic in istanbul. the animals' gender, breed, age and hábitat whether household, shelter or street (stray cats) was recorded. other data from household cats included if they were adopted or home raised from birth, they cohabitated with other cats and had outdoor access. cats were clinically examined to detect fever, skin lesions, behavioral changes (insidious onset, depression) and symptoms related to organ systems were recorded; specifically, cardiorespiratory (dyspnea, abnormal heart and lung sounds), gastrointestinal (anorexia, weight loss, stomatitis, enteritis, abdominal distension, vomication, ascites), urinary, circulatory (lymphoadenopathy, anemia, icterus), ocular lesions (keratic precipitates, uveitis, hyphema, iridocyclitis, chorioretinitis) and central nervous system (epileptic seizures, ataxia) symptoms. blood samples were taken from the cephalic vein by the veterinarians examining the cats for hematological and biochemical analyses and to detect antibodies against fcov and feline immunodeficiency virus (fiv) together with feline leukemia virus (felv) antigen as described below. all analysis except fcov ifat antibodies were carried out at the veterinary clinic within an hour of taking the blood sample. ifat tests and protein electrophoresis were carried out at an external private veterinary laboratory. disease progression of the study cats was evaluated during repeat visits to the clinic and mortality was considered to be associated to the current infection when the cat did not respond to standard treatments which included fluid and antibiotic therapy. all serum samples (n = ) were analyzed by rapid tests for the presence of antibodies to fcov (bionote, anigen, fcov) and fiv (bionote, anigen fiv ab), and felv antigen (bionote, felv ag) following kits' instructions. according to the manufacturers, the sensitivity (se) and specificity (sp) of the fcov test compared to the reference immunofluorescence antibody test (ifa) were . % and . %, respectively, se and sp of the felv test versus virus isolation were . % and . %, respectively, and that of the fiv tests versus western blot were . % and . %, respectively. sera found to be positive for antibodies to fcov by the rapid test were analyzed by ifa in an external private laboratory to confirm the result. serum giving fluorescence at a dilution above : was considered positive for antibodies against this virus. all blood samples were analyzed for a complete blood hemogram-histogram ( parameters) using a veterinary specific mindray blood analyzing kit and the hemogram instrument (mindray). samples were also analyzed for comprehensive blood biochemistry ( parameters); samples were analyzed using the vet-scan (abaxis) kit and the remaining samples were analyzed using reflotron (roche) kit. serum protein electrophoresis was performed for serum samples positive for antibodies to fcov. ascitic fluid from cats was analyzed for albumin/globulin (a:g) ratio. hematological and biochemical tests included total white blood cell count (wbc), lymphocyte and red blood cell counts, hematocrit, hemoglobin, total protein, albumin (alb.), globulin (glob.), alkaline phosphatase (alp), alanine transaminase (alt), amylase, total bilirubin, blood urea nitrogen (bun), creatinine, glucose, calcium, phosphorus, sodium and potassium. data were analyzed using r (http://cran.r-project.org/) software. approximately unbiased estimates of prevalence were calculated assuming known values of test se and sp using the rogan-gladen statistic (greiner and gardner, ) . yates-corrected chi squared test, or when appropriate fisher's exact test (kirwood and sterne, ) , was used to compare the proportion of fcov seropositive cats according to cat demographic and habitat explanatory variables and the proportion of signs in fcov seropositive and seronegative cats. biochemical and hematological results were categorized as being within (normal), above (high) or below (low) reference values (villiers and blackwood, ) . the independent relationship between fcov serological status, and a cat's demographic, habitat and fiv serological status was further investigated using logistic regression analysis (kleinbaum and klein, ) . fcov was the binary outcome variable (seropositive or seronegative). the explanatory variables included those associated at p < . with fcov serological status in the univariable analysis; they were fiv serological status, age, examination year and the variable reflecting living place, outdoor access and contact with other cats (tables a and b). all variables were included in the model as categorical variables as shown in tables a and b, except age, which included seven levels after combining data from to year old cats into a single level. model parameters were estimated using the maximum likelihood estimation method and significance was taken for alpha less than % for a double sided test. antibodies to fcov and fiv were detected in / and / cats. two out of cats were positive for felv antigen. the estimated fcov and fiv seroprevalence and felv prevalence ( % ci) adjusted for tests se and sp, were % ( - %), % ( - %) and % ( - %), respectively. all cats testing fcov antibody positive to the rapid test were also ifat antibody positive. fcov seroprevalence varied significantly by study year, origin and habitat variables (p < . ) (tables a and b) . furthermore, fcov seroprevalence was % ( / ) and % ( / ) among fiv seropositive and seronegative cats, respectively (p < . ). logistic regression analysis confirmed the independent relationship of fcov serological status with examination year, age, fiv status, habitat and contact with other cats (table ) . clinical examination revealed depression or dullness, fever and low body weight in % ( / ), % ( / ) and % ( / ) of study cats, respectively, and % ( / ) of cats had all three signs. the percentage of cats with ascites, abdominal distension and pleural effusion was % ( / ), % ( / ) and % ( / ), respectively. all three symptoms were present in only one cat, no cats had pleural effusion and ascites or abdominal enlargement alone; in contrast, ascites and abdominal enlargement without pleural effusion were observed in % ( / ) of cats and % ( / ) of cats presented one of these three conditions. the cat with all three symptoms was fcov seronegative instead; fcov seroprevalence was % ( / ) in cats with ascites and abdominal enlargement and % ( / ) in cats with at least one of the three symptoms, and % ( / ) of these were dull or depressed, had fever and/or low body weight. the percentage of some clinical signs differed according to the cat's fcov serological status (table a) . other symptoms found included dyspnea ( / ), stomatitis ( / ), ocular signs ( / ), urinary tract signs ( / ) and epilepsy ( / ) (not shown in table format). the prevalence of these symptoms was not significantly different between fcov seropositive and seronegative cats. thirty-three percent of fcov seropositive cats ( / ) and % ( / ) of seronegative cats died from the condition for which they were admitted in spite of receiving treatment (p < . ). results of the hematology and clinical chemistry are shown in table b . abnormalities were particularly frequent in cats with ascites and pleural effusion and % ( / ) and % ( / ) of cats with these signs had high wbc and low a:g ratio, respectively (not tabulated). this study shows that fcov infections are widespread in cats from istanbul and this is in agreement with other studies elsewhere (sparkes et al., ; pedersen et al., ; pesteanu-somogyi et al., ; sharif et al., ; taharaguchi et al., ; paris et al., ) . moreover, fcov seroprevalence increased in compared to previous years and this may suggest that fcov infections are an increasing health problem in cats in istanbul. high fcov seroprevalence (up to %) has been reported in many countries (sparkes et al., ; holst et al., ; pratelli, ; pratelli et al., ; sabshin et al., ; taharaguchi et al., ) . in contrast, fcov seroprevalence was comparatively low in chronically ill ( . %) and even lower in healthy cats ( . %) in korea (dong-jun et al., ) . prevalence may vary depending on the inclusion criteria used (normal versus ill cats) and estimates may be affected by selection bias, analytical errors and imperfect diagnostic tests. several risk factors have been reported to be associated with fcov infection and with fip development, including age, breed, gender, multi-cat environment and stress (bell et al., ; pesteanu-somogyi et al., ; addie et al., ; sharif et al., ; worthing et al., ) . in the present study, fcov serological status was significantly associated with year, age, fip serological status and habitat variables. the risk of infection would be expected to rise during the first months or years of life due to increasing cat-to-cat contact. however, it is possible that infection prevalence among . - . year-olds may have been underestimated as several weeks would be needed for anti-fcov antibodies to develop following infection. other studies have reported greater fcov prevalence in cats - months of age (bell et al., ; pedersen, ; taharaguchi et al., ) . instead, a study in australia and malaysia found no association between age and fcov infection in cats (bell et al., ; sharif et al., ) . household cats living alone had the lowest risk of being fcov seropositive, as reported in other studies (addie et al., ; drechsler et al., ) . in contrast, this study found that household cats that cohabitated with other cats had a high risk of being fcov seropositive, as has been previously shown (foley et al., ; herrewegh et al., ; pedersen et al., ; pesteanu-somogyi et al., ; sharif et al., ; sabshin et al., ) . moreover, in the present study, fcov seroprevalence was lower in stray cats ( %) compared to cats living at home ( %). it is possible that stray cats have poorer health and increased risk of dying from fcov infections compared to household cats. alternatively, stray cats could be exposed to less fcov compared to household cats, who commonly share the same litter box and eat from the same food bowl as other cats in the household. furthermore, immunological differences could exist between stray and household cats, with the latter being naturally selected for a protective th- mediated rather than a th- antibody mediated response. this, however, has not been investigated and remains speculative. interestingly, fiv seroprevalence was negatively associated with fcov infection in this study. the reason for this is unclear. it could be because household cats are vaccinated for fiv in istanbul. it is also possible that coinfected cats are at greater risk of dying as a result of fiv immunosuppression compared to cats that are only fcov seropositive. in the present study, no difference in fcov seroprevalence was found between females and males. similar results have been found by others (cave et al., ; bell et al., ; holst et al., ; sharif et al., ; taharaguchi et al., ) . instead, in australia and the usa, male cats were found to be more frequently infected with fcov (pesteanu-somogyi et al., ; worthing et al., ) . there is no known biological reason supporting gender-associated susceptibility and resistance to fcov, and differences between studies could be related to males and females having different lifestyles and fcov exposure. breed was not associated with fcov seropositivity in the present study. in contrast, higher fcov seroprevalence has been reported in purebred cats compared to non-pedigree cats in several studies (bell et al., ; pesteanu-somogyi et al., ; holst et al., ; taharaguchi et al., ) . in japan, seroprevalence was higher among pedigree cats including american curl, maine coon, norwegian forest cat, ragdoll and scottish fold compared to american shorthair, himalayan, oriental, persian, and siamese (taharaguchi et al., ) . in australia, siamese, persian, domestic shorthairs and bengal cats had significantly lower prevalence than british shorthairs, cornish rex and burmese cats (bell et al., ) . australian studies reported fcov to be prevalent among british shorthair, devon rex and abyssinian breeds (worthing et al., ) . in malaysia, fcov seroprevalence was higher in persian ( %) than in mix-breed cats ( %) (sharif et al., ). in the present study there were too few cats of specific breeds to allow robust statistical comparisons. moreover, pure and mixed breed cats did not differ significantly in terms of street access and contact with other cats. critical evaluation is necessary for a cat to be diagnosed with fip. diagnosis is based on evaluation of history, symptoms, hematological and biochemical parameters, diagnostic tests, radiology and tissue biopsy results (shelly et al., ; sparkes et al., ; hartmann et al., ; addie et al., ; pedersen, ; sharif et al., ; tsai et al., ) . it has been reported that up to % of fcov infected cats develop fip (hartmann, ; addie et al., ; pedersen, ) . although in the present study no definitive diagnosis of fip was attempted, % of cats had typical signs of wet fip including abdominal distension, ascites and pleural effusion, and increased ␥-globulin and decreased albumin-to-globulin (a:g) ratio. differences in the percentage of fcov cats developing fip between studies could be associated with the cat populations examined; cats investigated in this study were clinically ill and suspected of having a viral infection. this study indicates that fcov infection in cats from istanbul is high and possibly increasing. preventive actions are necessary in multi-cat environments (shelters, catteries and pet shops) and single household cats with outdoor access. cats presenting with general malaise, including fever not responding to antibiotics, depression, ascites, abdominal distension, diarrhea, pleural effusion, postsurgical complications and a low a:g ratio should be suspected of suffering from fip. feline infectious peritonitis. abcd guidelines on prevention and management evaluation of an in-practice test for feline coronavirus antibodies the relationship between the feline coronavirus antibody titre and the age, breed, gender and health status of australian cats prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis genetics and pathogenesis of feline infectious peritonitis virus the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr risk factors for feline coronavirus seropositivity in cats relinquished to a uk rescue charity prevalence of korean cats with natural feline coronavirus infections feline coronavirus in multicat environments evaluation of real-time rt-pcr for the quantification of fcov shedding in the faeces of domestic cats patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments application of diagnostic tests in veterinary epidemiologic studies detection of feline coronaviruses by culture and reverse transcriptase polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis feline infectious peritonitis comparison of different tests to diagnose feline infectious peritonitis persistence and evolution of feline coronavirus in a closed cat-breeding colony prevalence of antibodies against feline coronavirus and chlamydophila felis in swedish cats morphological features and development of granulomatous vasculitis in feline infectious peritonitis sites of feline coronavirus persistence in healthy cats logistic regression. a self-learning test enteropathogen co-infection in uk cats with diarrhoea common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus a review of feline infectious peritonitis virus infection: - prevalence of feline infectious peritonitis in specific cat breeds two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus prevalence of feline coronavirus antibodies in cats in bursa province, turkey, by an enzyme-linked immunosorbent assay comparison of serologic techniques for the detection of antibodies against feline coronaviruses enteropathogens identified in cats entering a florida animal shelter with normal feces or diarrhea prevalence of feline coronavirus in two cat populations in malaysia diagnostic methods for feline coronavirus: a review protein electrophoresis on effusions from cats as a diagnostic test for feline infectious peritonitis an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis feline coronavirus antibodies in uk cats prevalence of feline coronavirus antibodies in japanese domestic cats during the past decade serum protein electrophoresis in cats clinicopathological findings and disease staging of feline infectious peritonitis: cases from to in taiwan genetic shift and drift during feline coronavirus evolution evolutionary insights into the ecology of coronaviruses bsava manual of canine and feline clinical pathology pathogenic characteristics of persistent feline enteric coronavirus infection in cats risk factors for feline infectious peritonitis in australian cats the authors declare that they have no conflict of interest. key: cord- -jr mbh s authors: calore, elisabetta; marson, piero; pillon, marta; tumino, manuela; tison, tiziana; mainardi, chiara; de silvestro, giustina; rossin, sara; franceschetto, genny; carraro, elisa; pescarin, matilde; varotto, stefania; destro, roberta; gazzola, maria vittoria; basso, giuseppe; messina, chiara title: treatment of acute graft-versus-host disease in childhood with extracorporeal photochemotherapy/photopheresis: the padova experience date: - - journal: biol blood marrow transplant doi: . /j.bbmt. . . sha: doc_id: cord_uid: jr mbh s acute graft-versus-host disease (agvhd) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. systemic steroid treatment represents the first-line therapy for agvhd and is associated with a response rate of % to %. steroid-resistant patients have a poor prognosis with high transplantation-related mortality (trm). several second-line therapies have been proposed for the management of unresponsive agvhd, without proven beneficial effects on patients' outcome or overall long-term survival. for these reasons, extracorporeal photochemotherapy/photopheresis (ecp), a cell-based approach to control gvhd that spares generalized immunosuppression, seems to be promising. in this study, we report the outcome of consecutive pediatric patients treated with ecp between and for agvhd. among them, patients had steroid-resistant agvhd, had steroid-dependent agvhd, and did not receive steroid as first-line therapy because of clinical contraindications. a complete response was obtained in % of patients, a partial response was observed in %, and there was no response in % of patients. at day + , trm was % in the whole cohort; trm was % and % among responders and nonresponders to ecp, respectively (p < . ). the -year overall survival was %, showing a difference between responders and nonresponders of % and %, respectively (p = . ). the -year time to progression of primary disease was %, without any significant difference between the groups. moreover, the -year progression-free survival of primary disease was %, with a significant difference (p = . ) between responders ( %) and nonresponders ( %) to ecp. in conclusion, this study demonstrates that ecp is highly effective in agvhd without a negative impact on primary disease. allogeneic (allo) hematopoietic stem cell transplantation (hsct) is increasingly used as a therapeutic approach for hematological and nonhematological diseases. in the last decade, improvements in infection monitoring and prophylaxis, immunosuppressive (is) strategies, high-resolution hla typing, and supportive care measures led to better outcomes after this procedure. despite these advancements, acute graft-versus-host disease (agvhd) remains the major cause of morbidity and mortality after allo-hsct [ , ] . to standardize diagnosis and management of agvhd, a british guideline was published by a joint working group of the british committee for standards in haematology and the british society for bone marrow transplantation. this document included recommendations for diagnosis and management of agvhd as well as primary treatment options for patients with steroid-refractory (sr) disease [ ] . standard management of agvhd included steroids at different doses depending on agvhd grade. if no improvement of agvhd after days was noted or progression within hours were observed, then the addition of secondline agents should be considered. second-line options are mycophenolate mofetil (mmf), anti-tnf antibodies, mammalian target of rapamycin inhibitors, il- receptor antibodies, and extracorporeal photochemotherapy/photopheresis (ecp) [ ] . unfortunately, despite multiple clinical trials, no single agent improving overall survival (os) for patients who failed steroid treatment has been identified [ , ] . moreover, the current survival at years in patients who respond to steroids is about % versus % in nonresponders (nr) [ ] and it has been shown that transplantation-related mortality (trm) is higher in steroidresistant patients [ , ] . in our study, we focused on ecp, one of the most promising treatments for agvhd. briefly, ecp consists of procedures: collection of peripheral leukocytes cells, irradiation of cells by ultraviolet a light in presence of methoxypsoralen , and reinfusion of treated cells in the patient. the underlying mechanism of action of ecp in gvhd is not completely understood [ , ] . within hours, this process induces apoptosis of all treated cells (including t cells) and subsequent phagocytosis by antigen-presenting cells; as a result, this might regulate immune homoeostasis, modulate the cytokine production, and induce tolerance of antigen-presenting cells [ ] [ ] [ ] [ ] . ecp has been demonstrated to be effective in treating both steroid-resistant and steroid-dependent patients with agvhd [ ] [ ] [ ] [ ] . in the pediatric setting in particular, the reported response rate ranges from % to %, according to the organs involved. in agvhd steroid-resistant patients, - year os is significantly increased in complete responders to ecp, % compared with % for nr [ ] . in , the italian society of haemapheresis and cell manipulation and the italian group of bone marrow transplantation elaborated best practice recommendations for ecp, which reflected the common clinical practice in most italian transplantation centers where ecp is performed with a total of procedures per year [ ] . despite this large use of ecp, most of the published reports deal with retrospective data that are difficult to compare, as patients' selection criteria, treatment schedules, patients' monitoring, and patients' assessment protocols differ among institutions. moreover, no randomized studies have been conducted in patients with agvhd. here, we report our single-center experience on ecp treatment in pediatric patients with agvhd. the response to ecp, trm, os, progression-free survival (pfs) of primary disease, and time to progression (ttp) of primary disease of patients treated with ecp were analyzed. from january to june , consecutive pediatric patients ( males, females) affected by agvhd were treated with ecp at the hsct unit of university hospital of padova. fifteen of these patients have been previously reported [ ] . the clinical characteristics of patients are shown in table . the median age at ecp was . years (range, . to . years) and the median body weight was kg (range, to kg). fifteen children weighted less than kg. the last follow-up was fixed on june . in detail, patients were treated with ecp for agvhd refractory to steroids, which was defined as a progression or no improvement in agvhd after at least days or days on methylprednisolone (mp) ! mg/kg body weight, respectively (sr group); patients for steroid-dependant agvhd, defined as a flare-up of agvhd during the tapering of mp (sd group); and children with agvhd who required a reduction of pharmacological is or contraindications to is therapy for viral reactivations, systemic mycoses, or intolerable side effects (group with infectious complications [ic]). in particular, of patients in the ic group (ic-a group) underwent ecp without steroids as a first-line therapy because of contraindications: for tcr ab and cd edepleted haploidentical transplantation with probable invasive pulmonary aspergillosis (ipa) and adenovirus (adv), for proven ipa, for concomitant proved ipa and cytomegalovirus (cmv) reactivation, for probable ipa and cmv and bk virus (bkv) reactivations, for proven ipa and multiple viral reactivations, including adv, cmv, and epstein-barr virus (ebv), for cmv reactivation, for cmv and ebv reactivations, and for cmv, ebv, adv, and bkv reactivations. the other patients (ic-b group) had sd agvhd and cyclosporin a (csa)erelated renal insufficiency in only patient; sd agvhd and concomitant infections in the remaining patients: cmv reactivations, ; cmv and ebv reactivations, ; cmv and bkv reactivations, ; cmv, adv, and bkv reactivations, ; ebv reactivations, ; ebv and adv reactivations, ; ebv reactivations and probable aspergillosis, ; hepatitis b virus, ; hepatitis b virus and ebv reactivations, ; cmv, ebv, and bkv reactivations, ; adv, human herpesvirus- , bkv, and coronavirus, ; hepatitis c virus, cmv, and proven ipa, n ¼ . in our practice, surveillance for viral and fungal infections in blood is routinely performed during the first days after hsct in all patients and includes ebv-dna, cmv-dna, adv-dna, human herpesvirus- dna, bkv-dna, jc-dna, and galactomannan antigen search. this schedule is performed once each week in allo-hsct recipients from hla-identical sibling and twice each week in allo-hsct recipients from unrelated or haploidentical donors. monitoring viral infections in urine comprises cmv-dna, bkv-dna, jcv-dna in a weekly search. blood, urine, and stool cultures; nasal and throat swabs; and nasopharyngeal aspirates were weekly performed. search for other viruses or microbiological agents was performed upon clinical symptoms. viral reactivations were detected by pcr positivity for ebv-dna (cut-off: copies/ml), cmv-dna (cut-off: copies/ ml), and adv-dna in qualitative test. clinical systemic fungal infections were defined proved or probable according to european organization for research and treatment of cancer criteria [ ] . the cut-off for the galactomannan index was set at . (enzyme immuno assay (e.i.a.) method). the algorithm for agvhd treatment used in our center is shown in figure . csa was administered for and months in children who received hsct from an hla-identical sibling or unrelated donor, respectively. in unrelated hsct, short-term methotrexate and rabbit antithymocyte globulin (atg) were given. in unrelated cord blood hsct, prophylaxis included csa and atg. in haploidentical setting, ex vivo elimination of ab t cells and cd b cells was done and atg was administered before the cells were infused; no other is therapy was given after hsct. informed consent was obtained from patients' parents, as well as from the patients themselves when possible, and the use of ecp was approved by the ethical committee of the hospital of padova. the clinical organ involvement was graded and then combined to obtain an overall grade, according to glucksberg criteria for agvhd [ ] . histological confirmation was obtained whenever clinically indicated to confirm gvhd diagnosis. eligibility criteria for ecp treatment were as follows: children with sr agvhd (n ¼ ); children with sd agvhd (n ¼ ); patients with agvhd in whom is therapy was contraindicated or who required a rapid decrease of is therapy for increasing ebv viral load, cmv reactivation in subsequent samples, systemic fungal infections, intolerable side effects (n ¼ ). all children must present in complete hematological remission and full donor chimerism; white blood cell (wbc) count >  /l, and no concomitant treatment with either atg or monoclonal antibodies. ecp was performed using different techniques: "in-line" treatment (uvar photopheresis instrument, therakos, exton, pa) was used in of patients and the "off-line" technique (cobe spectra, bct terumo, lakewood, co) was used in of children. technical descriptions have already been published [ ] . the "off-line" technique was introduced in to treat lowweight children. for patients weighing less than kg, priming of the leukapheresis circuit with irradiated and leuko-reduced red blood cells (regardless of baseline hemoglobin level) was performed, as recommended in the italian society of haemapheresis and cell manipulation-italian group of bone marrow transplantation indications [ , ] . pre-ecp hemoglobin levels were maintained between g/dl and g/dl. the cell product was treated with -mop and diluted to a final concentration of mg/ ml to mg/ ml, according to specific procedures (in-line technique, mg/ ml; off-line technique, mg/ ml). in all patients, a to french hickman double-lumen central catheter was systematically used for the procedure. to provide adequate flow rates, ie, to ml/kg/minute, anticoagulation with urokinase , u for hours as lock-therapy was performed on the day of the procedure. the leukapheresis product contained a median of wbc of .  /ul (range, . to .  ), a median of mononuclear cells (mnc) of . % (range, % to %). the median number of wbc reinfused to the patients was  (range, to ,  ), whereas the median number of mnc reinfused to the patients was  (range, . to .  ). patients were treated with ecp twice each week for the first month, every weeks during the second and third months, and then monthly for at least more months, for a total of procedures. progressive tapering and discontinuation of ecp were decided upon evaluation of individual response. any concomitant is therapy was initially maintained, then modified or discontinued according to the clinical response. criteria for defining response to ecp were previously reported [ ] . all patients enrolled for ecp before day þ were included in this group and response to ecp was evaluated at day þ , day þ , and at the end of ecp treatment. complete response (cr) was defined as the resolution of all signs of agvhd and partial response (pr) as at least a % improvement in the clinical signs. in the latter case, given the complexity of assessing response, we defined pr for each organ as follows: for the skin, at least a % reduction in the body surface area affected; for the gi tract and liver a % reduction in the volume of diarrhea or value of bilirubin. any worsening of organ involvement, as well as the appearance of new signs or symptoms of gvhd, was defined as progressive disease (pd). patients with stable or pd were considered nr. seventy-two consecutive patients with agvhd received ecp at a median time of days (range, to ) after hsct and days (range, to ) from the diagnosis of agvhd. sixty-four patients had skin involvement (grade iv, n ¼ ; grade iii, n ¼ ; grade ii, n ¼ ; grade i, n ¼ ). fifty-five patients had gastrointestinal (gi) agvhd (grade iv, n ¼ ; grade iii, n ¼ ; grade ii, n ¼ ; grade i, n ¼ ). twelve patients had liver involvement (grade iii, n ¼ ; grade ii, n ¼ ; grade i, n ¼ ). regarding the number of organs involved: in patients skin was affected and presented gi involvement, whereas patients had combined skin and gi agvhd, patient had combined gi and liver agvhd, and patients had combined skin, gi, and liver agvhd. ic-a group is those with infectious complications and no steroid before ecp; the ic-b group is those with infectious complications and steroid before ecp. * hla match considered / . the overall clinical grading of agvhd was as follows: grade i, n ¼ ; grade ii, n ¼ ; grade iii, n ¼ ; and grade iv, n ¼ ; details of different grades in the patients' subgroups can be found in table clinical evaluation of the patients was conducted at every ecp procedure. sixty-three of patients with agvhd grades i to iv received mg/kg/ day of mp as first-line therapy. the median dose of steroid at the beginning of ecp was mg/kg/day. in detail, the is therapies before ecp were csa, n ¼ ; csa plus steroid ( mg/kg), n ¼ ; tacrolimus plus steroid ( mg/kg), n ¼ ; and csa or tacrolimus plus mmf plus steroid, n ¼ . ecp was used as first-line therapy in of patients, as second line therapy in of patients (among them, haploidentical hsct was treated only with csa), as third-line in of patients, and in of patients as fourth-line therapy. patients' characteristics were compared using the chi-squared or fisher's exact test (as appropriate) in case of discrete variables, or the mann-whitney test in case of continuous variables. trm was calculated from the date of hsct to day þ and to the last follow-up, considering as event any nonrelapse cause of death. os was calculated from the date of hsct to the date of death from any cause, or to the last follow-up. pfs was calculated from the date of hsct to the date of relapse of underlying primary disease or death for any cause or to the last follow-up. ttp was calculated from the date of hsct to the date of relapse of primary disease or to the last follow up. cumulative incidences (ci) of relapse of underlying disease were estimated in the competing risk model, considering death from any cause or cgvhd as the competing events. survival analysis was performed using kaplan-meier method with % confidence interval. standard error (se) for each survival and incidence rate is given. differences between groups were compared using the log-rank test and the gray's test. all reported p values were -sided, and statistical significance was set at a ¼ . (sas institute, cary, nc; release . ) [ ] . response to ecp treatment, evaluated according to the overall grading of agvhd and to the organ involvement at day þ , day þ , and at the end of ecp, is summarized in table . at the end of treatment with ecp, of ( %) patients had a cr, of ( %) had a pr, and of ( %) were nr. among the patients showing a cr, patients had agvhd grade i, patients had grade ii, had grade iii, and had grade iv. in particular, the cr rate for patients with agvhd grades i and ii and grades iii and iv were % and %, respectively (p ¼ . ), whereas the pr rate for patients with agvhd grades i and ii and grades ii to iv were % and %, respectively (p ¼ . ). no significant statistical difference in cr rate was observed according to the subgroups analyzed (sr, %; sd, %; ic groups, %) (p ¼ . ). at ecp discontinuation, cr of agvhd manifestations of skin, gut, and liver was observed in %, %, and % of patients, respectively. maximal response to ecp was observed after weeks of treatment ( procedures). as a result of ecp, at the end of treatment, it was possible to discontinue is therapy in patients ( %) and reduce it in patients ( %), of them who received allo-hsct from an unrelated donor. regarding the steroid tapering, in patients treated with mg/kg/day before ecp, the steroid dose was reduced by % after month of ecp treatment, % after months, and % after months of ecp treatment. the median lansky/karnofsky performance score improved from % before ecp to % after completing the treatment. no association was found between responders and nr to ecp and the major clinical risk factors affecting agvhd (table ) . cgvhd twenty-three of patients ( %) presented clinic manifestations of cgvhd (table ). in detail, patients ( %) had progressive cgvhd ( nr and pr to ecp) and patients ( %) had quiescent cgvhd onset after a median of months (range, days to months) from the end of ecp. overall grading of cgvhd, based on the national institutes of health consensus [ ] , was mild for patients, moderate for patients, and severe for patients. among the patients with progressive cgvhd, ecp was used with other is therapies in of patients, obtaining cr in only of them. overall, of patients were alive at the last follow-up: of had no cgvhd and discontinued is therapy, whereas only patient presenting with cgvhd was still in treatment. all the patients with quiescent cgvhd were alive at the last follow-up: patients were free from cgvhd and without is therapy and the other patients had cgvhd and were still on treatment with is therapy plus ecp. no association was found between responders and nr to ecp and the onset of quiescent cgvhd. at day þ , the overall trm was % (se, %). trm was % (se, %) and % (se, %) for responders and nr to ecp, respectively (p < . ). at last follow-up, the overall trm was % (se, %), whereas trm stratified between responders and nr was % (se, %) and % (se, %), respectively (p < . ) (figure a,b) . the -year os was % (se, %) with a statistically significant difference between responders and nr ( %; se, % versus %; se, %, respectively; p ¼ . ) ( figure a,b) . the -year pfs of primary disease for all the group was % (se, %), with a significant difference (p ¼ . ) between responders ( %; se, %) and nr ( %; se, %) ( figure a,b) . overall, the -year ttp of primary disease was % (se, %), without any significant difference between the groups (responders: %; se % versus nr: %; se, %; p ¼ . ) ( figure a,b) . we compared patients' survival rates on ecp treatment used as first, second, or third/fourth-line therapy. no difference was observed at -years between responders and nr in term of os (p ¼ . ), pfs (p ¼ . ), and ttp (p ¼ . ). the overall -year ci of relapse of the underlying disease was % (se, %); in particular, it was % (se, %) and % (se, %) for responders and nr to ecp, respectively ( figure a,b) . overall, at the last follow-up (median time from hsct of years; range, . to . years), patients were alive ( %); of them ( %) were without gvhd and without any is therapy. twenty-one patients ( %) died: from relapse of primary disease and from nrm. among this last group, patient with agvhd died at day þ from hsct because of sepsis; patients with cgvhd died from cmv pneumonia ( case), acute hepatitis from hcv infection ( case), encephalopathy ( case), and multiorgan failure ( cases); and patient died from cmv pneumonia at day þ from hsct, without evidence of cgvhd. side effects observed during ecp were generally mild and more frequent in low-weight children. ecp caused mild hypotension in patients associated with abdominal pain in all cases ( episodes out of apheresis sessions). these adverse effects did warrant suspending the procedure. a transient reduction in hemoglobin, platelet, and/or wbc count during the ecp treatment, likely independent from the post-transplantation course and putatively ecp-related, was observed in , , and patients, respectively. one patient with grade iv agvhd on high-dose steroid therapy ( mg/kg/ day) experienced acute gi bleeding after the second course of ecp: gi endoscopy showed multiple ulcers in the stomach. a girl with pre-existing cardiac impairment showed acute heart failure for fluid overload after the procedure that quickly responded to adequate therapy. one girl, after ecp procedures, had anaphylaxis (cough, vomiting, abdominal pain, hypotension, and palpebral edema) a few minutes after the end of -mop irradiated bag infusion. she responded to antihistamine and steroid therapy, but ecp treatment was then stopped. the aim of this retrospective study was to analyze the role of ecp for the treatment of agvhd. the efficacy of ecp is well established for treatment of cgvhd [ , ] , whereas in agvhd, no prospective randomized studies have been published. however, the use of ecp is suggested as second-line therapy, together with mammalian target of rapamycin inhibitors, mmf, il- receptor antibodies, and anti-tnf antibodies [ ] . the largest phase prospective study exploring feasibility and efficacy of ecp in treatment of agvhd in adults, involving patients, was performed by greinix et al. [ ] and reported a cr rates of % for skin and % for liver and gi agvhd. so far, data on pediatric patients treated with ecp for agvhd have been reported, showing an overall cr rate ranging from % to % and a survival rate ranging from % to % [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to date, our is the largest pediatric case series treated in a single center. in our sample size, we found a higher overall response rate to ecp compared with a multicenter retrospective study of the italian association for pediatric hematology/oncology (aieop) ( % versus %) [ ] . we attempted to determine the factors that may have influenced our observed higher response. in the last years, many changes have been introduced in hsct, such as highresolution hla typing, new agents in the conditioning regimen, more use of atg, monoclonal antibodies, and new antifungal drugs. it is difficult to determine which modification may have influenced the outcome. we could also hypothesize that specific expertise in pediatric hsct and earlier treatment with ecp ( days in our series versus days in aeiop study) may have improved the overall outcome. further studies are needed to address this topic. no association was found between responders and nr setting to ecp and major risk factors for agvhd. in addition, in our series, no difference was found according to the grade of gvhd (grade i and ii, %; versus grade iii and iv, %; p ¼ . ) and to the subgroups of patients analyzed (sr, %; sd, %; ic, %; p ¼ . ). our results showed better response rate than those reported in literature for advanced stages of disease, where higher grades and poorer response to is therapy correlate with a worst outcome [ ] . nevertheless, higher cr rates were observed in grade ii gvhd, suggesting that an early start of ecp sessions may be beneficial, even if in our study the timing to start ecp (< versus > days) did not influence the response. in our group, ecp seemed to be effective in all the involved organs. as previously reported, our results support that ecp is a steroid-sparing treatment; in fact, the steroid dose was reduced by %, %, and % after , , and months, respectively from the onset of ecp. we performed ecp as front-line therapy in patients with fungal infection and viral reactivation and agvhd, with complete response in of them. to our knowledge, this is the first report of ecp as first-line treatment. is therapy was either discontinued or reduced in % of responding patients. it is well known from the literature that is therapy increases the risk of infectious complications and relapse of underlying disease after allo-hsct [ , , , , , ] . in children, who may be particularly vulnerable to the consequence of gvhd itself and prolonged treatment with is agents, the use of ecp is particularly appealing. the efficacy of ecp in controlling gvhd did not affect the preservation of graft-versus-leukemia effect; in fact, the low incidence of relapse of underlying disease was recorded by us and others [ , , , ] . many concerns has been raised related to the technical aspects of apheresis in the pediatric setting. in children with low body weight, the caregivers should carefully monitor patients for signs and symptoms of hypovolemia. in our experience, ecp was well tolerated, with few and mild adverse effects, the most frequent of which were mild hypotension, abdominal pain, and headache. curiously, these symptoms were recorded more often during ecp compared with other apheretic procedures [ , ] . the majority of side effects were observed in the earliest period in which ecp was performed in our center. all these observations support the idea that there has been a learning process for the management of technical elements and side effects. in our experience, ecp was feasible even in very young children with low body weight (< kg). technically, we performed priming of the circuit with irradiated and leukodepleted red blood cells (regardless of baseline hemoglobin level). some authors recently reported that saline infusion or albumin boluses may be an alternative priming approach in patients with body weight ranging from to kg [ ] . however, it should be proven that this approach could be safely transferred to population weighting less than kg. currently used ecp techniques include the "off-line" and the "in-line" devices [ ] . in our center, both techniques were used in different time periods with no difference in response rate observed. the number of wbc collected and mnc reinfused did not affect clinical outcome. notably, all patients underwent the procedure with the bilumen central venous line already in place (hickman-broviac bard access systems, salt lake city, ut, usa), which is different from the majority of reports, in which a larger central venous line (for instance, quinton) is placed. it would be interesting to extend our experience to determine if urokinase anticoagulation allows the proper flow rate of pre-existing central venous line. further, because of the experience of the staff in completing the procedure, no patient required sedation. the ci of cgvhd in pediatric population ranged from % to % according to the source of stem cells (hla-identical sibling cord blood versus matched unrelated donor peripheral blood) [ , ] , whereas in the aieop experience, the ci of cgvhd was reported to be % [ ] . in our small series, the incidence of cgvhd was %. the majority of our children presented progressive cgvhd ( %) and few had quiescent cgvhd ( %). for this reason, it is hard to determine if patients previously treated with ecp for agvhd could benefit from retreatment. our data are consistent with literature and the results encourage us in exploiting this promising approach for agvhd. in conclusion, a standardized approach to ecp treatment is needed for pediatric patients. from this perspective, sharing single-center experience is of great value in building experience; however, it is time to propose randomized prospective trials. graft-versus-host disease risk factors for acute gvhd and survival after hematopoietic cell transplantation diagnosis and management of acute graft-versus-host disease how i treat refractory acute gvhd etanercept, mycophenolate, denileukin, or pentostatin plus corticosteroids for acute graft-versushost disease: a randomized phase trial from the blood and marrow transplant clinical trials network steroid-refractory acute gvhd: predictors and outcomes treatment of moderate/severe acute graft-versus-host disease after allogeneic bone marrow transplantation: an analysis of clinical risk features and outcome a retrospective analysis of therapy for acute graft versus host disease: initial treatment technology insight: ecp for the treatment of gvhdecan we offer selective immune control without generalized immunosuppression? potential mechanisms of photopheresis in hematopoietic stem cell transplantation extracorporeal photopheresis: a focus on apoptosis and cytokines update on the mechanism of action and on clinical efficacy of extracorporeal photopheresis in the treatment of acute and chronic graft versus host disease in children extracorporeal photopheresis (photochemotherapy) in the treatment of acute and chronic graft versus host disease: immunological mechanisms and the results from clinical studies the effect of intensified extracorporeal photochemotherapy on long-term survival in patients with severe acute graft-versus-host disease extracorporeal photopheresis for the treatment of steroid refractory acute gvhd role of extracorporeal photopheresis (ecp) in treatment of steroid-refractory acute graft-versus-host disease extracorporeal photopheresis in acute and chronic graft-versus-host disease extracorporeal photochemotherapy for paediatric patients with graft-versus-host disease after haematopoietic stem cell transplantation extracorporeal photopheresis for the treatment of acute and chronic graft-versus-host disease in adults and children: best practice recommendations from an italian society of hemapheresis and cell manipulation (sidem) and italian group for bone marrow transplantation (gitmo) consensus process extracorporeal photochemotherapy may improve outcome in children with acute gvhd defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus consensus conference on acute gvhd grading best practice recommendations in: ( ) peripheral blood stem cell mobilization and collection and ( ) acute and chronic gvhd treatment using extracorporeal photopheresis. a joint effort from sidem (società italiana di emaferesi e manipolazione cellulare) and gitmo (gruppo italiano trapianto di midollo osseo) analyzing survival data from clinical trials and observational studies national institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: i. diagnosis and staging working group report a multicenter prospective phase randomized study of extracorporeal photopheresis for treatment of chronic graft-versus-host disease progressive improvement in cutaneous and extracutaneous chronic graft-versus-host disease after a -week course of extracorporeal photopheresisdresults of a crossover randomized study extracorporeal photopheresis for paediatric patients experiencing graft-versus-host disease (gvhd) update on extracorporeal photochemotherapy for graft-versus-host disease treatment photopheresis in pediatric graftversus-host disease after allogeneic marrow transplantation: clinical practice guidelines based on field experience and review of the literature extracorporeal photopheresis for steroid resistant graft versus host disease in pediatric patients: a pilot single institution report extracorporeal photochemotherapy in graft-versus-host disease: a longitudinal study on factors influencing the response and survival in pediatric patients extracorporeal photochemotherapy as second-or first-line therapy of acute gvhd? the italian sidem registry for apheresis: an overview of the statistics management of acute graft-versus-host disease the use of fluid boluses to safely perform extracorporeal photopheresis (ecp) in low-weight children: a novel procedure graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an hla-identical sibling. eurocord and international bone marrow transplant registry working committee on alternative donor and stem cell sources comparable long-term survival after bone marrow versus peripheral blood progenitor cell transplantation from matched unrelated donors in children with hematologic malignancies chronic graft-versus-host disease in children: incidence, risk factors, and impact on outcome financial disclosure: the authors have nothing to disclose. conflict of interest statement: there are no conflicts of interest to report. key: cord- - wzyfb j authors: zeng, zheng; huang, xiang-rong; lu, pu-xuan; le, xiao-hua; li, jing-jing; chen, de-ming; yuan, jing; li, guo-bao; liu, ying-xia; zhou, bo-ping title: imaging manifestations and pathological analysis of severe pneumonia caused by human infected avian influenza (h n )() date: - - journal: radiol infect dis doi: . /j.jrid. . . sha: doc_id: cord_uid: wzyfb j objective: to investigate the imaging and pathological findings of severe pneumonia caused by human infected avian influenza (h n ), and therefore to further understand and improve diagnostic accuracy of severe pneumonia caused by human infected avian influenza (h n ). methods: the relevant clinical and imaging data of cases, including males and females, with pneumonia caused by human infected avian influenza (h n ) was retrospectively analyzed. one of the cases had received percutaneous lung biopsy, with the clinical, imaging and pathological changes possible to be analyzed. results: the lesions were mainly located at lower lobes and dorsal of lungs, involving multiple lobes and segments. ground-glass opacities and/or pulmonary opacities were the more often imaging manifestations of severe pneumonia caused by human infected avian influenza (h n ) in early and evolving phases ( / , %). by biopsy following percutaneous lung puncture, exudation of slurry, cellulose, rbc and neutrophils, formation of hyaline membrane, squamous metaplasia and organizing exudates were observable at the alveolar space. some of alveoli collapsed, and some responded to show compensatory emphysema. conclusion: the imaging features of severe pneumonia caused by human infected avian influenza (h n ) include obvious ground-glass opacity and pulmonary consolidation, mainly at lower lobes and dorsal of lungs, with rapid changes. the cross-analysis of imaging and pathology preliminary can elucidate the pathological mechanisms of ground-glass opacities and pulmonary consolidation of severe pneumonia. such an intensive study is beneficial to prompt clinicians to observe and evaluate the progress of the disease. in addition, it is also in favor of managing the symptoms and reducing the mortality rate. human infected avian influenza (h n ) is an acute respiratory infection caused by h n subtype of avian influenza a virus [ ] . the disease was firstly reported in the spring of in yangtze river delta, china [ ] . a total of cases of human infected avian influenza (h n ) was admitted to our hospital from december, to april, , with cases suffering from severe pneumonia. in this study, we retrospectively analyzed imaging and pathological manifestations of the disease to shed light on its clinical diagnosis, therapeutic efficacy evaluation and prognosis. the clinical, radiological and pathological data of cases with severe pneumonia caused by human infected avian influenza (h n ) from december , to april , in shenzhen third people's hospital, china, were collected. all the patients were tested positive to nucleic acid of h n subtype of avian influenza virus by cdc of guangdong province and shenzhen city, china, in line with the diagnostic criteria of severe human infected avian influenza. the cases included males and females, aged e years with a median of years. two patients had preexisting hypertension; had hypertension and diabetes; had tuberculosis; and had right pulmonary embolism. fever was the most common symptom, found in all cases ( %), and ardent fever ( c or above) showed up in patients ( . %). cough was also the most common symptom, occurring in all cases ( %), with expectoration in cases ( . %) and case coughing up dark red bloody sputum. anhelation occurred in cases ( . %). all patients were admitted to our hospital at d e after onset, averagely . days, and received antiretroviral and respiratory supporting therapies. six patients assured of a history of contact to live poultry, by another patients denied a history of contact to live poultry. the epidemiologic data of the other patients was not available. wbc count was detected to have a decrease in cases, being from .  ^ /l to .  ^ /l, with a mean of .  ^ /l. neutrophil percentage increased in cases, but remained normal level in cases, being from . % to . %, with a mean of . %. according to the diagnostic and treatment protocol for human infected avian influenza a (h n ) established by national council on health and family planning commission, p. r. china (edition, ), the cases with any one of the following criteria can be diagnosed as severe. ( ) chest x-ray demonstrates lesions with multiple lobes involved or lesions progress more than % within h; ( ) difficulty breathing, with more than breaths per minute; ( ) severe hypoxemia, with oxygen flow at e l per minute and spo %; ( ) shock, ards or mods (multiple organ dysfunction syndrome). biopsy following percutaneous lung tissue puncture was performed in one case to observe the pathological changes. the pathogenic bacteria was observed after pas and masson staining. acid-fast bacteria staining was performed to detect possible infection of mycobacterium tuberculosis. conventional chest x-ray was performed using philips didi th/vr, with the tube voltage kv and automatic tube current. bedside chest x-ray was performed using hitachi sirisu hp mobile dr, with a tube voltage kv and automatic tube current. ct scanning was performed using toshiba tsx- a -slice spiral ct, with a tube voltage kv, automatic tube current, a pitch of . , a matrix of  , an fov of mm  mm, a thickness of . e . mm, and an interval of mm. ct scanning was from the apex to the bottom of lungs continuously. lung window was reconstructed using conventional mm and high resolution of mm after the scanning. all the images were independent analyzed by two radiologists with a title above associate chief-physician, and consensus was reached after consultation and discussion. the images were analyzed in terms of distribution and range of lesions, morphology of the lesions as well as changes of mediastinum and pleura. ( ) ct manifestations at the early phase (d e after onset) the lesions more often onset from a lower lung lobe ( / ), only cases had their lesions onset from an upper lung lobe. poorly-defined patches of shadows and fragmental ground-glass opacities were demonstrated by ct scanning. changes of pulmonary interstitium were observed, including interlobular septal thickening, acinar nodules, and other changes. chest ct scanning demonstrated rapid progress of the lesions within a short period of e days, with rapid expansion, fusion, formation of large patchy opacities, and the lesions were demonstrated to involve multiple lobes (fig. ). ( ) ct manifestations at the evolving phase (d e after onset) the lesions of cases involved both lungs ( / , . %), and only case showed lesions with unilateral lung involved ( / , . %). the lesions of cases involved median lobe (lingual lobe) or lower lobe ( / , %). the lesions of cases involved to lung segments ( / , . %). ground-glass opacity was demonstrated by ct scanning in all cases ( %), which were poorly defined in fragments and large patches. pulmonary consolidation was also demonstrated in all cases ( / , %), which was more commonly found at the lower lung lobes to cross segments or lobes with higher density. air bronchogram was observed in the area of consolidation. air sacs in lungs were demonstrated in cases ( / , . %), which were round with smooth lining, different sizes, various shapes and could be absorbed. lymphadenectasis was demonstrated in case ( / , . %), with several enlarged lymph nodes in the mediastinum, the larger one in front of the tracheal eminence, and the smaller one having a diameter of about mm.pleural effusion was demonstrated in cases ( / , . %), mostly in a small quantity, cases of unilateral and cases of bilateral ( fig. ) . ( ) ct manifestations at the absorbing phase (d -after onset) all the patients were admitted to our hospital at d e after onset (mean, . days), and by chest ct scanning, the lesions began to be absorbed at d e after onset, averagely at d . after onset. interval between onset and beginning of absorption ranged from day to days, with an average of . days. the group of patients received antiretroviral and symptomatic therapies. in patient, the lesions began to be absorbed at d after admission to hospital, and in the remaining patients, the lung lesions began to be absorbed at d after hospitalization. during absorption, the range of fig. . a female patient aged years was definitively diagnosed with severe pneumonia caused by human infected avian influenza a (h n ). a) chest ct scanning at d after onset demonstrates large patchy consolidation at the right lower lung lobe, with observable air bronchogram; b) chest ct scanning at d after onset demonstrates substantial progress of the lesions, with large patchy consolidation and poorly defined patchy ground-glass opacities at lower lobes of both lungs and the right middle lung lobe. consolidation gradually reduced with decreased density, consolidation and recruitment of lung. the lesions at the upper and median lung lobes were absorbed earlier than those at the dorsal part of lower lung lobe and the subpleural lesions. the earlier emerging lesions were absorbed later, and vise versa. finally, the residual lesions were more often located at dorsal part of the lower lung lobe and under the pleura. in this group of patients, were cured after hospitalization for e days (averagely . days). by ct scanning, consolidation disappeared in cases ( / , . %); multiple patches of shadows were shown in cases ( / , . %); lamellar ground-glass opacities were shown in cases ( / , . %); linear shadows in cases ( / , . %); latticed shadows and subpleural line in cases ( / , . %); air sacs or pseudocavity, paraseptal emphysema, scar emphysema, subpleural bulla in cases ( / , . %) (fig. ) . death occurred in case due to respiratory and renal failure (fig. ). one case of this group received biopsy following percutaneous lung tissue puncture at the advanced period of the condition. histopathology mainly showed fibrotic changes. necrosis and abscission of some alveolar epithelia as well as reactive hyperplasia of alveolar epithelium were observed (fig. ced) , but with no viral inclusions within epithelial cells. slurry, cellulose, rbc, effused neutrophils, formation of hyaline membranes, squamous metaplasia and organizing exudates were observed in alveolar space (fig. e ). some pulmonary alveoli were shown with atrophy or compensating emphysema. pulmonary interstitial fibrosis, sometimes leukomonocyte, phlogocyte and reactive hematophagocyte were observable. formation of hyaline thrombus and dic were shown in capillary of pulmonary interstitium, sometimes with vascular occlusion (fig. f) . empsyxis and secondary infection was sometimes found (fig. ). human infected avian influenza a (h n ) is a newly emerging infectious disease caused by h n subtype of avian influenza a virus. the disease was firstly reported in china in , with main manifestations of flu-like symptoms such as fever, cough, and so on [ ] . severe pneumonia prompts critical condition, often in combination with ards, infectious shock, and so on. chest ct scanning and diagnostic imaging are the important means for its clinical diagnosis and therapeutic evaluation [ ] . this group of cases with severe pneumonia caused by human infected avian influenza a (h n ) had received more than three times of chest ct scanning. after comprehensive analysis, the ct manifestations of these patients include: ( ) the lesions are more often found at the lower lung lobes, with cases of this group showing lesions at or lower lung lobes. with progress of the disease, the lesions rapidly progress to involve multiple segments and lobes of both lungs, often or more lung lobes up to all segments and lobes of both lungs. pulmonary opacities are mainly located at the dorsal part of lungs. ( ) ground-glass opacities and pulmonary opacities are the cardinal imaging infestations to severe pneumonia caused by human infected avian influenza a (h n ). when the condition is mild or at its early stage, the lesions are mainly ground-glass opacities. at the evolving phase, the percentage of pulmonary opacities increases, with air bronchogram in the pulmonary opacities. ground-glass opacities mainly distribute at the anterior border of pulmonary opacities, showing as sporadic patchy opacities, even as "white lung" when in severe condition. the patient may develop significant hypoxemia, respiratory distress syndrome and respiratory failure. ( ) pleural effusion is also one of the imaging features of human infected avian influenza a (h n ), with cases of this group showing pleural effusion, and its incidence rate is higher than the previous report [ ] . it may be related to the fact that all patients of this group suffered from severe pneumonia. the pleural effusion may be caused by systemic inflammatory response triggered by direct involvement of pleura by virus and/or virus evoked cytokine storm. ( ) pulmonary interstitial fibrosis is the main findings at the recovery phase, with all cases of this group showing as small patchy shadow under the pleura and/or at dorsal part of lower lobes. fibriform cords, latticed shadows, fragmented ground-glass opacities and other pulmonary fibrosis are also ct changes. at the same time, in this group, subpleural paraseptal emphysema and ulotic emphysema, subpleural bullas and localized bronchiectasis are observable. pathological findings in the cases of severe pneumonia caused by human infected avian influenza a (h n ). influenza virus is categorized into orthomyxovirus, and it is a single minus strand, segmental rna virus that is enveloped [ ] . the hemagglutinin in the tunica external of influenza virus a plays a key role in the pathomechanism of human infected avian influenza a (h n ). inflammatory factors may be mediate systemic inflammatory response syndrome and ards. therefore, severe pneumonia is demonstrated as extensive ground-glass opacities and obvious consolidation by ct scan whose underlying mechanism is damaged pulmonary alveoli and pulmonary capillaries caused by virus, extensive effusion of pulmonary interstitium and pulmonary parenchyma [ ] . diffuse alveolar damage, interstitial fibrosis and air cavity extension, infiltration of leukomonocyte and plasma cells were demonstrated by histopathology. therefore, the ct manifestations of severe pneumonia caused by human infected avian influenza a (h n ) can be explained by the histopathologic findings as its pathomechanism. and, the main pathological changes of body organs are the result of phagocytosis of red blood cells, white blood cells and platelets by macrophages, known as reactive hemophagocytic syndrome. it can also explain different degrees of reduction of peripheral blood counts by clinical laboratory tests at the early and evolving phases of severe pneumonia cause by human infected avian influenza a (h n ). and the varying degrees damage of cd t lymphocytes indicates immune responses. . the differential diagnosis of severe pneumonia caused by human infected avian influenza a (h n ) severe pneumonia caused by human infected avian influenza a (h n ) should be distinguished from influenza a (h n or h n ) and severe acute respiratory syndromes, and other conditions. pneumonia caused by human infected avian influenza a (h n , h n and h n ) is all caused by influenza a virus, all with flu-like symptoms. the conditions are demonstrated as multiple ground-glass opacities in different sizes and consolidation by chest ct scan [ e ]. compared to pneumonia caused by influenza a (h n ), the lesions caused by h n and h n occupy a larger range and develop more rapidly, and air bronchogram is more common. the disease progressions of influenza h n is rapid, following by h n and h n . by ct scans, pneumonia caused by influenza a (h n ) is mainly displayed as large patchy ground-glass opacities and consolidation at both lungs. the lesions distribute widely and progress rapidly [ , ] . in some cases, the lesions are erratic and absorbed slowly, with obvious pulmonary interstitial fibrosis and a mortality rate of about %. onset from middle lobe and lower lobe of lungs, the lesions in the cases of human infected avian influenza a (h n ) are mainly displayed as ground-glass opacities and lung consolidation, which change rapidly and are absorbed slowly, too [ , , ] , with a mortality rate of about %. the lesions are mainly displayed as multiple patchy ground-glass opacities fig. . a male patient aged years was definitively diagnosed with severe pneumonia caused by human infected avian influenza a (h n ), and this is the case that received biopsy following percutaneous lung tissue puncture. aeb) chest ct scanning at d after onset shows large patchy consolidation and ground-glass opacities in all lobes of both lungs, with poorly defined boundary. air bronchogram is observable in the lesions, and mediastinal emphysema is demonstrated; c) the lung tissue is demonstrated to be degenerated and necrotic, with a few lymphocytes infiltrated; d)reactive hyperplasia of the alveolar epithelium and pulmonary interstitial fibrosis are demonstrated; e)the alveolar epithelium is demonstrated to be dropped, with light red liquid, cellulose and a few lymphocytes in alveolar space; f) alveolar epithelial cellsare demonstrated with hyperplasia, with some alveolar epithelium dropped and vascular occlusion observable. and patchy or large patchy high-density consolidation in the cases with influenza a (h n ), with segmental atelectasis and pleural effusion [ , ] as well as a mortality rate of about %. only based on radiological findings, the identification of infections by these different influenza viruses is challenging. and the differential diagnosis should be made in combination to epidemiological and etiological findings. chest ct scans also display sars as ground-glass opacities and lung consolidation, which progress rapidly with involvement of both lungs in about % of the patients, more often at middle and lower lung lobes. the lesions mainly distribute in the peripheral pulmonary tissue, with detectable interlobular septal thickening by high-resolution ct as broken paving-stones. accompanying bronchiolectasis and a small quantity pleural effusion are also detectable [ ] . these findings resemble to those of human infected avian influenza a (h n ), but the interstitial changes in the cases of human infected avian influenza a (h n ) are not obvious. the lesions of human infected avian influenza a (h n ) show up in a more extensive range, with no obvious coverage at the peripheral pulmonary tissue. epidemiological history and laboratory tests for its pathogen are the key to identify the two conditions. generally, severe pneumonia caused by human infected avian influenza a (h n ) is demonstrated with ground-glass opacities and lung consolidation by chest ct scan. these lesions onset from middle and lower lobes of both lungs, and the condition may develop into pulmonary cavity or aerothorax, hydropneumothorax. the differential diagnosis for identification of influenza a virus (including h n , h n and h n ) is challenging based only on radiological findings. and their identification should be made in combination to epidemiological and etiological findings. cross analysis of radiology and pathology is a way to further understand severe pneumonia caused by human infected avian influenza a (h n ). and such a study is beneficial to clinical observation and evaluation of the condition, and is of great significance for disease control and mortality reduction. national health and family planning commission. diagnostic and treatment protocol for human infections with avian influenza a (h n ). edition characteristics of the imaging manifestations and dynamic changes in patients with severe pneumonia caused by h n avian influenza virus epidemiology and imaging manifestations of avian influenza clinical and imaging diagnosis of emerging infectious disease. beijing: people's medical publishing house preliminary imaging findings of novel reassortant avian-origin influenza a (h n ) pneumonia the correlation of ct manifestation, viral load and cd þt lymphocyts in patients with h n avian influenza pneumonia ct manifestation and dynamic changes of grave pneumonia in adults caused by h n subtype of human avian influenza virus imaging features of pneumonia caused by highly pathogenic h n subtype human avian influenza virus human avian influenza types and manifestations in ct images of pneumonia caused by influenza a (h n ) imaging manifestations of the severe cases of pneumonia caused by h n subtype human avian influenza virus correlative study of semi-quantitative score of chest ct findings and viral load in novel influenza a (h n ) virus infection imaging diagnosis of sars key: cord- -dcui lw authors: broadbent, andrew j.; boonnak, kobporn; subbarao, kanta title: respiratory virus vaccines date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: dcui lw this chapter reviews the main viral pathogens of the respiratory tract, the immune responses they induce, currently available vaccines, and vaccines that are in development to control them. the main viruses responsible for acute respiratory infection in people include respiratory syncytial, influenza, human parainfluenza, human metapneumo-, human rhino-, corona-, and adenoviruses. licensed vaccines are available only for influenza virus, with vaccines against the other pathogens either in clinical trials or in preclinical stages of development. the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies are known to protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. as we breathe, we sample an estimated l of air per minute (kohlmeier and woodland, ). the mucosa of our respiratory system is, therefore, in direct and continual contact with the environment and, as such, is highly exposed to microorganisms, some of which may be pathogenic. respiratory infections are among the leading causes of acute illness and mortality worldwide, being responsible for nearly million deaths annually, the majority of which occur in infants and children in developing countries (girard et al., ) . the main viruses responsible for acute respiratory infection include respiratory syncytial virus (rsv), influenza virus, human parainfluenza virus (hpiv), human metapneumovirus (hmpv), human rhinovirus (hrv), coronaviruses, and adenoviruses. however, despite the public health importance of these infections, licensed vaccines are currently available only for influenza viruses. protective immunity against respiratory virus infection is a complex interplay between systemic and mucosal responses. however, immune responses generated during a natural infection may not provide complete protection from reinfection and may actually contribute to the pathogenesis of disease (reviewed in sections pathogenesis and immune responses to respiratory virus infection). vaccine-induced immune responses must, therefore, aim to be more protective and less pathogenic than those induced naturally. in addition, our understanding of the relative contribution of mucosal and systemic immunity to protection remains incomplete. for example, it is well known that inactivated vaccines against influenza given intramuscularly (i.m.) are protective owing to the induction of systemic humoral immunity in the absence of a robust mucosal immune response, and current guidelines for vaccine licensure require influenza vaccines to induce systemic immune responses. however, it is also evident that some intranasal vaccines are protective owing to the induction of mucosal immunity, despite less impressive systemic immune responses (reviewed in section respiratory virus vaccines). unfortunately, standardized methods of measuring mucosal immune responses are lacking, and reliable correlates of protection for vaccines that protect through mucosal immunity have not been identified. in this chapter, we review the main viral pathogens of the respiratory tract, the immune responses they induce, current vaccines, and vaccines that are in development to control them. the viruses that infect the respiratory tract belong to various families and vary in their genome composition, the presence or absence of an envelope, and their replicative cycles. the majority of respiratory viruses that are responsible for acute respiratory infection belong to the paramyxoviridae family and include rsv, hpiv, and hmpv. these viruses infect cells lining the respiratory tract by first attaching to the cell through the interaction of viral envelope glycoproteins, with one or more cellular receptors in the host cell plasma membrane. for example, the hpiv envelope protein hn binds sialic acid residues extending from host cells (schomacker et al., ) , and the g protein of rsv and hmpv binds to glycosaminoglycans (gags) that comprise long chains of disaccharides that form part of the cellular glycocalyx (feldman et al., ) . the rsv and hmpv f protein are also known to bind gags, and findings indicate that the f protein of these viruses is involved in attachment by interacting with the cellular proteins nucleolin and integrin αvβ , respectively (tayyari et al., ; cseke et al., ) . upon binding to the host cell, the f protein undergoes a conformational change that exposes a hydrophobic fusion peptide, which is responsible for the fusion of the paramyxovirus envelope and the host cell plasma membrane. after viral fusion, the genome is released into the cytoplasm, viral genes are transcribed, and viral genomes are replicated (collins and crowe, ; collins and melero, ) . the paramyxovirus genome comprises single-stranded, negative-sense, nonsegmented rna. the viral rna (vrna) must first be transcribed into positive-sense messenger rna (mrna) before viral proteins can be translated by the host cell machinery. this is achieved by a viral rna-dependent rna polymerase (the large, l, protein) that is packaged into the virion and enters the host cell upon infection. the l protein is also responsible for genome replication, in which positive-sense complementary rna (crna) serves as an intermediate template for the production of vrna. an essential cofactor for the l protein is the phospho (p) protein, which tethers the polymerase so it can reach the bases in the vrna and also binds the n protein, which encapsidates the vrna and crna. there is also evidence that transcription is enhanced by the m - protein and that the switch from transcription to replication is mediated by the m - protein (collins and crowe, ; collins and melero, ) . once transcribed, viral structural proteins assemble and newly synthesized viral genomes are packaged into virions that bud from the plasma membrane. the matrix, or m protein, lines the inner surface of the viral envelope and may play a role in budding (henderson et al., ; teng and collins, ) . in addition, the hn protein of hpiv is also involved in budding and in clearing sialic acid residues from the plasma membrane (karron and collins, ) . to complete the replication cycle, paramyxoviruses have evolved multiple mechanisms to prevent the activation of cellular defenses in response to infection, such as the nonstructural (ns) proteins and of rsv (collins and crowe, ) and the c or v proteins of hpiv (karron and collins, ) . one additional protein found in paramyxoviruses is the short transmembrane glycoprotein (sh) that is anchored to the envelope and shares structural features with viroporins, a group of hydrophobic molecules that insert into the membrane of infected cells and increase their permeability to small molecules and ions (gonzalez and carrasco, ) . the orthomyxoviridae family includes influenza viruses, which bind to terminal sialic acid-galactose linkages by the hemagglutinin (ha) envelope glycoprotein. orthomyxovirus attachment to the host cell initiates receptor-mediated endocytosis and endosome acidification. protons are permitted to enter the influenza virion via the m ion channel, and acidification results in a conformational change in the ha protein, revealing the fusion peptide that initiates membrane fusion between the viral envelope and the endosome membrane (reviewed by palese and shaw ( ) ). the ha is synthesized as a precursor (ha ) that is cleaved into its active form (ha and ha ) by cellular proteases, and the amino acid sequence at the cleavage site determines the type of protease that is able to activate the ha. if trypsin-like proteases are required for cleavage, the virus is limited in its tissue tropism to the respiratory tract of humans and the gastrointestinal tract of birds; however, the presence of multiple basic residues at the cleavage site extends the range of proteases that can cleave the ha, resulting in a disseminated, often lethal, infection in poultry (wright et al., b) . once the virus envelope has fused with the endosome, the influenza genome enters the cytoplasm. the orthomyxovirus genome comprises seven or eight segments of singlestranded, negative-sense rna, and each segment encodes one or more proteins. each segment is encapsidated in nucleoprotein (np) and forms a panhandle comprising the ′ and ′ ends, to which a polymerase complex is attached. together, these are known as the viral ribonucleoprotein complex. in the nucleus, orthomyxovirus vrna is either transcribed into mrna or replicated by means of a positive-sense crna intermediate. viral mrna molecules exit the nucleus and are translated in the cytoplasm by the host cell machinery. structural proteins assemble at the plasma membrane, where newly synthesized viral genomes are packaged and virions bud (palese and shaw, ) . how the individual segments traffic to the plasma membrane and are packaged remain active areas of research. the matrix (m ) protein lines the virion beneath the envelope and may be important for morphology and viral assembly at the plasma membrane. in addition, the neuraminidase (na) protein permits budding by cleaving sialic acid residues from the host cell plasma membrane (palese and shaw, ) . to complete the replication cycle, influenza viruses inhibit interferon (ifn) production and signaling. this is achieved by the ns protein (palese and shaw, ) (section adenoviruses). the genomes of coronaviruses and rhinoviruses comprise positive-sense, single-stranded rna that can be translated by the host cell machinery in the cytoplasm (kennedy et al., ) . coronaviruses, which belong to the coronaviridae family, are enveloped and attach to host epithelial cells by the spike (s) envelope proteins (blau and holmes, ) . fusion occurs at the plasma membrane, or after endocytosis, and the genome is translated into a polyprotein, which is then posttranslationally processed into structural proteins that form viral particles and nonstructural proteins that aid in viral genome replication (lai et al., ) . rhinoviruses, which belong to the picornaviridae family, are not enveloped and instead have a capsid of icosahedral symmetry comprising four proteins, vp - (reviewed by greenberg ( ) , kennedy et al. ( ) ). the majority of rhinoviruses bind to intercellular adhesion molecule- (icam- ) (greve et al., ) , and binding leads to a conformational change in the capsid that creates a pore, through which the genome enters the cytoplasm to be translated and replicated (bella and rossmann, ) . adenoviruses are nonenveloped and possess a capsid of icosahedral symmetry. at each of the corners, a fiber protrudes from the capsid that makes contact with the host cell receptor to initiate receptor-mediated endocytosis. acidification of the endosome results in conformational changes in the capsid that lead to viral uncoating and the release of the double-stranded dna genome into the cell. the genome is transported into the nucleus, where it is transcribed into rna, which is alternatively spliced into monocistronic mrnas that are translated by the host cell machinery into early gene products. early gene products remodel the intracellular environment to favor viral replication and are responsible for viral replication. the late phase of the viral life cycle is concerned with the production of structural proteins in sufficient quantities to ensure adequate packaging of the newly synthesized genomes and maximizing the production of progeny virions, which are released by cell lysis (berk, ) . respiratory viruses can infect various parts of the respiratory tract and cause a range of illness. mild upper respiratory tract (urt) infection (urti) can be complicated by sinusitis or otitis media, and a lower respiratory tract (lrt) infection (lrti) can lead to bronchiolitis or pneumonia and possible postinfectious respiratory complications such as sensitization to asthma. the major public health impact is from lrtis, and rsv is responsible for the majority of cases in infants. hmpv, hpiv, and influenza can also lead to lrti, with hpiv and hmpv affecting infants almost as young as those afflicted with rsv, whereas hpiv , hpiv , and influenza are often diagnosed in children months of age or older. rsv and influenza are also recognized as an important cause of lrtis in the elderly and in those with cardiopulmonary disease or immunosuppression (schmidt, ) . moreover, influenza pandemics occur at irregular and unpredictable intervals with widespread morbidity and mortality and economic consequences. in addition, although there have been no cases of severe acute respiratory syndrome (sars) since , several novel coronaviruses have been identified, including the virus responsible for middle east respiratory syndrome (mers) (zaki et al., ) . given the clinical significance of these infections, and the fact that licensed vaccines are available only for influenza viruses, there is an unmet need for vaccines. humans are the only natural host for rsv, with infections occurring in annual epidemics during winter and spring months in temperate climates and the rainy season in the tropics (girard et al., ) . the virus is highly contagious, with most children being infected in the first year of life. the peak of severe disease usually occurs before months of age, with the peak incidence of hospitalization in -to -month-old infants (collins and melero, ) . reinfection is also common. in one study, among children who had been infected in their first year of life, % were reinfected in their second year, and % in their third year of life (glezen et al., ) . moreover, reinfection is independent of antigenic changes in the virus, implying that the protective immunity mounted during an infection does not protect against subsequent reinfection (collins and melero, ) . this is of note when attempting to induce protective immune responses by vaccination. globally, there were an estimated million cases of lrti caused by rsv in children under years of age in , with % requiring hospitalization (martinez et al., ) . in the united states, one study estimated that . million children under years of age require medical attention each year owing to rsv (botosso et al., ) , and another study estimated that rsv was responsible for , - , hospitalizations per year (girard et al., ) . in the united kingdom, the total annual incidence of hospitalization attributed to rsv was . per children under year of age and . per children between and years of age (waris, ) . more than half of the hospitalizations for rsv occur in previously healthy, fullterm infants, and children who experienced severe lrti caused by rsv were at increased risk of wheezing and asthma later in life (girard et al., ) . pediatric mortality from rsv in the united states was estimated to be between . per , per year in infants under year of age and . per , per year in children to years of age in one study (cooney et al., ) , and in another u.s. study, rsv was estimated to be responsible for - deaths per year (girard et al., ) . however, an estimated % of rsv-associated deaths occur in developing countries, possibly because of limited health-care resources (martinez et al., ) . in healthy adults, reinfection rates are approximately - % per year, though hospitalizations are rare. morbidity and mortality are more pronounced in the elderly and it has been estimated that rsv causes an average of , deaths annually in the united states, with % occurring in individuals over years of age (cooney et al., ) . in addition, the cost of caring for patients with severe lrti from rsv and its sequelae are substantial (girard et al., ) . rsv infection induces antibodies against the two main antigens, the f and g envelope glycoproteins. the g protein is the most variable protein in rsv and is the basis for the separation of strains into two antigenic groups (a and b). moreover, sites of positive selection that partially coincide with epitopes recognized by anti-gprotein monoclonal antibodies (mabs) suggest immunedriven rsv evolution (botosso et al., ) . however, most anti-g mabs do not neutralize infectivity (martinez et al., ) and the selection pressure is, therefore, weak. this favors a slow coevolution of several rsv lineages, and multiple genotypes within each group can cocirculate within the same season, with shifts in the predominance of groups a and b occurring in -to -year cycles (waris, ) . in contrast, the sequence of the f gene is highly conserved among rsv isolates, despite the identification of a number of neutralizing mabs against the protein that should impart a selection pressure for mutation (collins and melero, ) . this implies that the function of the f protein confers structural restrictions that may limit antigenic diversity. human parainfluenza viruses are also a common cause of acute respiratory infection, with % of children seropositive by years of age (cooney et al., ) . as with rsv, reinfection is common (schomacker et al., ) . there are four serotypes of hpiv (hpiv - ), with each serotype associated with a broad spectrum of upper and lower respiratory symptoms. hpiv and are, however, more frequently associated with croup (laryngotracheobronchitis), and hpiv is more likely to cause bronchiolitis, pneumonia, and lrti resembling disease caused by rsv (schomacker et al., ) . hpiv is a less frequent cause of clinically significant disease, though a study found hpiv in % of hpiv-positive samples in a day-care setting (fairchok et al., ) . hpiv lrti is a major cause of hospitalization in children under years of age, second only to rsv, though infection is usually self-limiting and rarely fatal, unless an individual is immunosuppressed. severe infection may have long-term effects on lung function, but this remains unclear (schomacker et al., ) . hmpv belongs to the same subfamily as rsv, and two major groups (a and b) and four minor subgroups (a , a , b , and b ) have been identified, based on sequence variability in the g and f glycoproteins (kroll and weinberg, ) . as with rsv, and hpiv, by years of age, most children will have been infected with hmpv, and reinfections are common (kroll and weinberg, ) . the virus also has a seasonal distribution, with the main occurrence in winter and spring (kahn, ) . hmpv typically leads to flu-like symptoms in otherwise healthy adults, but is responsible for - % of hospitalizations for lrti in children and can lead to severe disease in the elderly or immunocompromised hosts (papenburg and boivin, ; boivin et al., ) . influenza viruses are divided into types a, b, and c based on antigenic differences in the np and m genes. influenza a viruses are the most clinically significant and are divided into subtypes based on antigenic differences in the ha and na genes. to date, ha and na subtypes have been identified from waterfowl (palese and shaw, ) and th and th subtypes of ha have been identified from bats in guatemala and peru (tong et al., (tong et al., , . influenza viruses cause a spectrum of clinical illness associated with infection of the upper and lower respiratory tract, with more severe disease associated with lrti. the viruses are spread by respiratory droplets or direct contact. annual influenza epidemics have a seasonal distribution, with the main occurrence in winter months (seasonal influenza) in temperate climates (girard et al., ) . however, unlike rsv, hpiv, and hmpv, influenza a viruses have a broad host range that includes birds, pigs, dogs, horses, marine mammals, and humans, with the main reservoir for infection being aquatic birds (palese and shaw, ; wright et al., b) . this broad host range, together with the segmented nature of the influenza virus genome, makes the epidemiology of influenza complex and gives rise to zoonotic infections and pandemics. pandemic influenza can arise if a novel virus emerges that readily transmits from person to person and if the majority of the population is susceptible to infection. if an avian or animal virus crosses the species barrier to circulate in humans, the population will probably be immunologically naïve and, therefore, susceptible to infection. however, the virus must be able to transmit efficiently from person to person for a pandemic to occur. as the influenza virus genome is segmented, if a host is infected with two or more influenza viruses, the potential exists for the gene segments to reassort, such that a progeny virus containing genes from each parent virus can be produced (wright et al., b) . if a virus that circulates within the human population reassorts with one that is novel for humans, the resultant virus may possess genes that allow it to replicate efficiently in humans, but with glycoproteins to which the population is immunologically naïve, and a pandemic could occur. the introduction of a virus with a novel ha subtype into the human population is known as "antigenic shift" (wright et al., b) . three global influenza pandemics were recorded in the twentieth century from viruses of the subtypes h n , h n , and h n , respectively. in , another pandemic h n (ph n ) influenza virus emerged in mexico (girard et al., ) . this h n virus was antigenically unrelated to previously circulating seasonal h n viruses, and molecular studies revealed that it was a reassortant with genes derived from viruses that had been circulating in pigs: the north american h n triple-reassortant, the classical swine h n , and the eurasian "avian-like" swine h n viruses. in most countries, the median age of infection during the pandemic was estimated to be - years, and in most individuals, infection led to a mild, self-limiting urti. however, - % of confirmed cases in the united states and canada required hospitalization, and the case-fatality rate was . - . %. moreover, nearly one-third of the fatalities among hospitalized patients occurred in previously healthy individuals (girard et al., ) . after each pandemic, the newly emerged subtype became established and caused annual seasonal influenza epidemics. in the united states, it has been estimated that - million cases of influenza occur annually, with approximately , requiring hospitalization (lambert and fauci, ) . current vaccines are aimed at the circulating h n and h n subtypes of influenza a and the predominant circulating strain of influenza b and are therefore trivalent vaccines. however, two antigenically distinct lineages of influenza b viruses (victoria and yamagata) cocirculate, and the world health organization recommended that influenza vaccines should contain both of these lineages. clinical trials of quadrivalent vaccines containing the h n and h n influenza a viruses and the victoria and yamagata influenza b viruses have been conducted, and their use in the united states received an interim recommendation of the advisory committee on immunization practices for the - influenza season (dolin, ) . owing to the low fidelity of the rna-dependent rna polymerase of influenza, and immune selection pressure on the ha protein, viral replication can yield a quasi-species that may differ antigenically from the parent virus. therefore, each season, the predominant circulating strain may be antigenically distinct from the previous year. this phenomenon is known as "antigenic drift" and leads to a need to update the influenza vaccine annually (wright et al., b) . sporadic infections by h n , h n , h n , and h n subtypes of influenza have been reported in humans who were in close direct contact with infected animals. additionally, h n variant viruses have infected humans, the majority of whom were in close contact with pigs (epperson et al., ) . so far, there has been limited transmission of these viruses between people, though there is concern that they may acquire mutations or gene segments that allow efficient spread from person to person. coronaviruses are frequent causes of the common cold, causing urtis throughout the world, in all age groups, leading to millions of days of work and school absence, physician visits, and frequent inappropriate antibiotic use (greenberg, ) . coronaviruses are transmitted by respiratory droplets and are reported to cause - % of common colds, with a peak prevalence in late fall, winter, and early spring. the first human coronaviruses (hcov) to be recognized as significant respiratory pathogens, hcov- e and oc , were identified in the s (greenberg, ) . whereas infection with the majority of coronaviruses is associated with self-limiting urt symptoms in otherwise healthy individuals, a coronavirus was identified as the agent responsible for sars in (drosten et al., ; ksiazek et al., ) . the sars coronavirus (sars-cov) emerged in the guangdong province of china in november and spread to countries, leading to cases and deaths worldwide by the time the outbreak was brought under control in june (who, ) . subsequently, heightened international surveillance for coronaviruses led to the identification of the strains hcov-nl , nh, and hku in (greenberg, previously, hrvs were classified into two species, hrv-a (containing serotypes) and hrv-b (containing serotypes). in , a novel species, hrv-c, was identified, which contains at least serotypes (jacobs et al., ) . hrvs are spread by direct contact, hand-to-hand contact or aerosols. traditionally, they have been associated with a urti, causing between and % of common colds (makela et al., ) . however, they are increasingly recognized as a cause of lrti, particularly in patients with asthma and in infants, the elderly, and immunocompromised individuals (jacobs et al., ) . bronchiolitis is a common clinical manifestation in hospitalized children infected with hrv, and hrv is also a common pathogen in viral community-acquired pneumonia. hrv has also been associated with exacerbations of asthma and chronic obstructive pulmonary disease (jacobs et al., ) . as of this writing, serotypes and seven species of adenovirus have been identified. tissue tropism and clinical manifestations vary between the serotypes, and the viruses are responsible for both febrile respiratory disease and gastrointestinal illness (reviewed by lynch et al. ( ) ). adenoviruses are estimated to be responsible for - % of pediatric and - % of adult respiratory tract infections (ison, ; lee et al., ) . they are spread primarily via respiratory droplets, direct contact, or fomites, and more than % of cases are in children under years of age (lynch et al., ) . however, epidemics have also been described in children and adults, especially in military recruits in closed or crowded settings. most individuals develop a self-limiting urt infection that may be asymptomatic, but conjunctivitis, tonsillitis, otitis media, or croup can occur. infection can also spread to cause bronchiolitis or pneumonia or disseminate to cause viral meningitis or encephalitis that can be fatal (lynch et al., ) . there are many factors that determine the pathogenesis of disease and clinical outcome, including factors pertaining to the virus, host genetics, host immune responses, and the environment. the site of viral replication may influence the pathogenesis of disease and outcome of infection. for example, seasonal influenza viruses usually infect the epithelium of the urt, which is consistent with the most common clinical manifestations of seasonal influenza, whereas highly pathogenic avian influenza viruses of the h n subtype show a stronger tropism for the lrt than for the urt (kuiken et al., ) . h n viruses attach abundantly to "clara" or club cells lining the bronchioles, type ii pneumocytes lining the alveoli, and alveolar macrophages in the alveoli, consistent with the clinical manifestation of diffuse alveolar damage (kuiken et al., ) . rsv also targets both type i alveolar and nonbasilar epithelial cells and possibly alveolar macrophages, which may contribute to lrti (van drunen littelvan den hurk and watkiss, ). tissue tropism is determined, in part, by the receptor preference of the virus. for example, cells lining the urt of humans predominantly possess sialic acid residues with a terminal α , linkage to galactose, whereas cells lining the human lrt have both α , and α , linkages (shinya et al., ) . it is believed that the ability of an influenza virus ha to preferentially bind α , -or α , -linked sialosaccharides therefore partly determines the tissue tropism and hence the clinical outcome. when host cells are infected, type i ifn and proinflammatory cytokines are expressed, cellular translation is suppressed, and an antiviral state is induced (discussed in section immune responses to respiratory virus infection). however, most viruses that infect the respiratory tract modulate the host response to infection by blocking ifn activation and/or signaling and inhibiting apoptosis. this prevents the host from effectively clearing virally infected cells and inducing an antiviral state in neighboring cells, thereby promoting viral replication in infected tissues that may contribute to the observed pathology. rsv is the most effective paramyxovirus at subverting host cell responses, inhibiting apoptosis and type i ifn production and signaling by means of the ns and proteins, inhibiting nuclear factor-κb (nf-κb) activation through the binding and sequestration of cellular protein kinase r (pkr) by the viral n protein, and inhibiting the production of stress granules that can restrict viral replication (collins and melero, ) . influenza a viruses encode the ns protein that downregulates ifn production (palese and shaw, ) , and hpiv encodes either a c protein or a v protein that suppresses ifn induction and signaling (karron and collins, ) , whereas sars-cov encodes eight proteins that antagonize the ifn response by a variety of mechanisms (totura and baric, ) . in addition to blocking ifn activation and signaling, viruses employ other means of ensuring their replication in the face of host immunity. the rsv g protein is highly glycosylated, a feature that may inhibit the binding of antibodies, and the protein is highly variable, enabling a substantial population of immune-escape mutants to be produced during infection. additionally, a truncated, soluble form of the g protein is produced during infection, which acts as a decoy antigen that can bind rsv-specific antibodies, thus reducing their availability to neutralize virus. rsv also infects antigen-presenting cells (apcs), such as dendritic cells (dcs), and can affect their maturation and antigen-presentation functions and can lead to dysregulation of adaptive immune responses (van drunen littel-van den hurk and watkiss, ). a number of virulence determinants have also been identified in influenza viruses, including the ha and the polymerase complex. for example, the presence of a multibasic site in the ha gene renders viruses highly pathogenic in poultry, as they are able to replicate systemically (bosch et al., ; kawaoka and webster, ) . in addition, substitution of a glutamic acid (e) residue for a lysine (k) residue at amino acid position in the pb protein (e k) of the polymerase is associated with altered host range (subbarao et al., ) and virulence in humans and in mice that are experimentally infected with avian h n and h n viruses (hatta et al., ; munster et al., ; subbarao et al., ) . in the absence of the e k mutation, a pb d n mutation is also correlated with increased virulence (li et al., ) , and in the absence of both the pb e k and the d n mutations, pb s and r were found to contribute to ph n influenza virus replication and virulence (mehle and doudna, ). in addition, an n s mutation in the pb f protein has been shown to contribute to the virulence of h n and pandemic h n viruses (conenello et al., ) , and several virulence determinants have been identified in the ns protein, including d e, p s, l f, and i m, reviewed by kuiken et al. ( ) . there are several host factors that are known to contribute to the severity of respiratory virus disease and the outcome of infection. for example, it is known that the more severe rsv lrti in infants is associated with prematurity, chronic lung disease, congenital heart disease, and t cell immunodeficiency. other risk factors include low birth weight, multiple births, male gender, and low titers of maternally derived anti-rsv antibodies (groothuis et al., ; van drunen littel-van den hurk and watkiss, ) . additionally, low levels of vitamin d in cord-blood of healthy neonates is correlated with an increased risk of rsv lrti in the first year of life (belderbos et al., ) . infants are also at a greater risk of lrt disease from hpiv infection than older children; this has been attributed to smaller airways that are more susceptible to obstruction, immature immune responses, and the presence of anti-hpiv maternal antibodies that can suppress primary antibody responses (crowe and williams, ; karron and collins, ) . in adults, immunodeficiency, immunosuppression, or old age may lead to more severe illness (collins and melero, ) . in addition, a number of genetic polymorphisms have also been described in host genes that may affect the outcome of respiratory virus infection and disease severity. single-nucleotide polymorphisms have been identified in genes that encode surfactant proteins, such as surfactant proteins a, b, c, and d; pattern recognition receptors (prrs), such as toll-like receptors (tlrs); chemokines and cytokines, such as rantes, il- , - , - , - , - , - , - , and - , tnf-α, tgf-β, and ifn-γ; chemokine and cytokine receptors, such as ccr , il- ra, and il- ra; adhesion molecules, such as icam- , vcam- , and e-selectin; and hla molecules such as hla-a and -b, among others (miyairi and devincenzo, ; poland et al., ) . however, few consistent results have been obtained between studies, probably a result of differences in study design, sample size, etc., in addition to true variability. clearly the contribution of genetic polymorphisms to disease outcome is complex and remains an active area of research. as well as being responsible for viral clearance and protection against reinfection, the host innate and adaptive immune responses to respiratory viruses can lead to pathology and enhanced disease. this is especially important to bear in mind in vaccine development, as vaccine-induced immune responses must protect against infection without leading to immunopathology. excessive inflammation is an important component in the pathogenesis of respiratory virus infections. upregulation of il- leads to the recruitment of neutrophils to the site of infection, and although these cells may participate in virus elimination, in high numbers they can also cause pathology. upregulation of il- is known to correlate with rsv disease severity (goetghebuer et al., ) and, during an influenza infection, overproduction of cytokines such as tnf-α, il- , il- , and type i and ii ifns and chemokines can also result in the recruitment of immune cells to the site of infection and result in damage to lung tissue (cheung et al., ; de jong et al., ) . elevated il- /cxcl , mip α and β/ccl and , rantes/ccl , and cxcl have also been described in children with hpiv disease, with an association of more severe hpiv disease with high concentrations of il- and ip- (reviewed by schomacker et al. ( ) ). additionally, the rsv soluble g protein can lead to leukocyte recruitment by mimicking the chemokine fractalkine (tripp et al., ) and this can further exacerbate inflammation. pathogenesis can also be enhanced by an insufficiency of anti-inflammatory immune responses in the lung, such as the cytokines il- and tgf-β (carlson et al., ; lebouder et al., ; sun et al., ) , or insufficient numbers of immunosuppressive resident alveolar macrophages (rygiel et al., ; snelgrove et al., ) . dysregulation of adaptive immune responses can also lead to increased pathology, and a th -biased cellular immune response has been implicated in the immunopathogenesis of rsv disease (van drunen littel-van den hurk et al., ) . various defense mechanisms have evolved in the respiratory tract to prevent and control infection. currently, there is considerable effort to develop or improve vaccines against respiratory viruses. however, achieving this goal has been complicated by an incomplete knowledge of how the immune system recognizes, contains, and eliminates respiratory viruses. this section discusses the immune responses against respiratory virus infections, from the initiation of innate and adaptive responses following primary virus infection to the recall of immune responses during a secondary infection. in addition, advances in our understanding of respiratory mucosal immunity are discussed. a common feature of respiratory virus infections is that the initial infection is established in epithelial cells lining the respiratory tract. epithelial cells, as well as alveolar macrophages and dcs, are exposed to the contents of the airway lumen and detect the presence of an invading virus through prrs (holt et al., ) . the recognition of pathogen-associated molecular patterns (pamps) by these receptors initiates a cascade of signals that results in the production of cytokines and chemokines. the release of these inflammatory mediators into the surrounding environment establishes a local antiviral state. in addition, chemokines provide the necessary signals for the recruitment of leukocytes to the site of infection. finally, the combination of inflammatory cytokines and prrs initiates the process of dc maturation and trafficking that is required for the induction of adaptive immune responses (holt et al., ) . the best described of the prrs are those of the tlr family. with respect to respiratory viruses, tlr , , and recognize various products of viral replication (doublestranded rna, single-stranded rna, and unmethylated cpg dna, respectively) (alexopoulou et al., ; diebold et al., ; hagglund et al., ; lund et al., ) , and tlr recognizes the f protein of rsv (kurt- jones et al., ) . tlrs that recognize nucleic acids are located in late endosomes. this location optimizes the ability of tlrs to interact with viral nucleic acids while limiting their access to host-derived nucleic acids (heil et al., ; matsumoto et al., ) . although tlrs expressed on the cell surface or within the cell utilize different signaling pathways, each of these receptors can activate the transcription of ifninducing genes . viral rna is also recognized by cytoplasmic sensors such as rna helicases. the retinoic acid-inducible gene i protein interacts with ′-triphosphate rna and is important for early cytokine production in response to numerous rna viruses (hornung et al., ; pichlmair et al., ; yoneyama et al., ; kato et al., ; pothlichet et al., ; graham et al., ) . the melanoma differentiationassociated gene protein is a related helicase that recognizes polyinosinic polycytidylic acid and is crucial for innate recognition for picornaviruses and human metapneumovirus infection (banos-lara mdel et al., ) . similar to signaling through tlrs, the pathways utilized by rna helicases ultimately trigger ifn-regulatory factor and nf-κb activation (le goffic et al., ) . the key difference between these molecules and tlrs is that the rna helicases are localized throughout the cytosol, rather than being restricted to intracellular compartments. thus, pathogens such as paramyxoviruses that do not enter endosomes can trigger innate immune responses via rna helicases. the innate recognition of viral components through prrs described above leads to a program of gene expression that promotes a localized antiviral state and elicits the recruitment of inflammatory cells to the site of infection. type i interferons, including ifn-α and ifn-β, are most commonly associated with early antiviral responses in the lung, and numerous studies in mice and humans have shown that plasmacytoid dcs (pdc's) are the primary source of these cytokines after infection with viruses (asselin-paturel et al., ; mcgill et al., ) . however, there is a level of redundancy with respect to ifn production, with alveolar macrophages or pdcs predominating depending on the type of viral infection (pribul et al., ) . the precise contribution of pdcs to lung antiviral immunity is also controversial; several in vitro studies show that respiratory viruses, including influenza viruses, can infect pdcs and pdcs can activate virus-specific cd + t cells (wikstrom and stumbles, ; geurtsvankessel and lambrecht, ) , but evidence for a major role of pdcs in controlling influenza virus in vivo is absent. type i ifns produced after respiratory virus infection act in concert with prr signaling to form a feedback loop, by signaling through the ifn-α/β receptor to promote sustained production of proinflammatory cytokines such as tnf-α, il- , and il- by lung-resident innate immune cells (chan et al., ; nakajima et al., ; almansa et al., ) . these proinflammatory cytokines and prrmediated signals also prompt alveolar macrophages, dcs, and epithelial cells to initiate a coordinated program of chemokine production after viral infection. for example, dcs secrete successive waves of chemokines after influenza virus infection, beginning with those capable of recruiting inflammatory cells such as neutrophils and natural killer (nk) cells to the lung, followed by chemokines associated with the recruitment of monocytes and t cells (piqueras et al., ; marois et al., ; teijaro et al., ) . the cytokines il- β, il- , and il- activate monocytes, macrophages, and neutrophils and drive the development of cd + t cell adaptive responses in both mice and humans. cd + t cell differentiation in the presence of il- β, il- , or il- results in th , th , or th effector cells, respectively (chung et al., ; dinarello, ; lasiglie et al., ; ohno et al., ). these cytokines are processed as a result of caspase- activation, and activation of caspase- is regulated by the inflammasome, a large mutimeric structure (martinon et al., ) . a subgroup of the nucleotide-binding domain, leucine-rich repeat-containing proteins (nlrps) are key mediators of the inflammasome. activation of the inflammasome can be divided into two categories: activation driven by host-and environmentderived molecules and activation driven by pathogen-associated activators, including pamps derived from bacteria, virus, fungus, and protozoa. paramyxoviruses such as rsv and orthomyxoviruses such as influenza viruses can activate the nlrp inflammasome (kanneganti et al., ; segovia et al., ; komune et al., ) , which has been shown to play a critical antiviral role in influenza virus-infected mice (thomas et al., ; ichinohe et al., ; allen et al., ). after infection of the lrt, antigen-bearing mature dcs enter the lymph nodes draining the lung, where they form stable interactions with naïve t cells specific for that antigen through t cell receptors (tcrs). signals delivered by antigen recognition, in addition to accessory signals delivered through costimulatory molecules, result in t cell priming and the clonal expansion of antigen-specific effector t cells (moon et al., ; obar et al., ) . the instructions delivered by dcs during the initial expansion phase can have a dramatic impact on the survival and function of the responding t cells. for instance, expression of fasl on dcs after influenza infection has been shown to regulate the magnitude of the cd + t cell response (legge and braciale, ) . resident cd α + conventional dcs in the mediastinal lymph node also mediate the induction of protective immunity to influenza virus, and these cells have been found to cross-present viral antigens to cd + t cells without being directly infected by the virus (belz et al., ) . after activation and clonal expansion of antigen-specific effector t cells in the draining lymph nodes, the cells lose their preference for the lymphoid tissue and migrate via the bloodstream to the site of infection, where the antiviral mechanisms described below are exerted. analysis of chemokine expression in the lung during the adaptive phase of the immune response has shown elevated expression of numerous molecules associated with effector t cell trafficking (monick et al., ) . for instance, the trafficking of effector t cells during rsv infection is partially dependent on cxcr , and this chemokine receptor may play a role in other paramyxovirus infections (harcourt et al., ) . the appearance of antigen-specific effector t cells at the site of virus infection is first observed around - days after infection with influenza and parainfluenza viruses in mice (pommerenke et al., ; kohlmeier and woodland, ; lawrence and braciale, ; roman et al., ) . the continual migration of effector t cells from lymphoid tissues during acute infection results in a massive increase in the number of antigen-specific cells in the lung airways and parenchyma from to days after influenza virus infection (flynn et al., ) , and the arrival of effector t cells has an immediate and dramatic impact on the viral load (kohlmeier et al., ) . the adaptive immune responses induced after a respiratory virus infection are shown in figures - . upon arrival at the effector site (figure ), antigen-specific t cells first interact with apcs such as dcs (shen et al., ) . moreover, a subset of dcs, classed as cd c hi , present a crucial t cell survival factor (il- ) to antiviral cd + t cells in trans (mcgill et al., ) . immune responses are determined by the cytokine milieu in the respiratory tract, as well as the type and level of costimulatory molecules expressed by apcs. for instance, lung dcs are biased to promote th responses, most likely via the production of il- in the absence of the th -prone il p cytokine or through the production of leukotriene ltc (dodge et al., ; barrett et al., ) . effector t cells employ one of the following three antiviral mechanisms ( figure ). first, t cells can promote the lysis of infected cells by exocytosis of granules containing perforin and granzyme (trapani and smyth, ; hou and doherty, ) . second, t cells can induce apoptosis of infected cells by expressing cd (fas) ligand (fasl) hou and doherty, ) or tnf-related apoptosis-inducing ligand (trail) (brincks et al., ) . third, t cells can produce proinflammatory and regulatory mediators, such as ifn-γ, after an encounter with virally infected cells (hamada et al., ) . several studies suggest a crucial role for the cytolytic functions of cd effector t cells in influenza virus infection. the direct lysis of infected cells requires tcr-mediated recognition of processed viral antigens on the infected target cell (brincks et al., ; topham and doherty, ) . in contrast, the release of proinflammatory mediators such as ifn-γ by cd + t cells has only a modest impact on virus clearance and recovery. there is also evidence from models of influenza infection that infected alveolar epithelial cells may be eliminated by the host response through the action of macrophages capable of triggering apoptosis through a trail-dependent mechanism (herold et al., ) . effector cd + t cells have also been found to exhibit cytotoxic activity in vitro, but the contribution of this mechanism to virus clearance in vivo is modest (agrewala et al., ; graham et al., ) and is restricted to the cytolysis of cells that bear viral antigens presented by major histocompatibility complex (mhc) class ii molecules. such cells include cd + mononuclear phagocytic cells, and a few cd − lung parenchymal cells, such as type ii alveolar epithelial cells, that express mhc class ii molecules in either a constitutive or an inducible manner (debbabi et al., ) . the primary role of antiviral cd + t cells is to support the activation and differentiation of b cells, which leads to antibody production (topham et al., ; topham and doherty, ) as discussed below. another aspect of effector t cells is their potential to exert cytotoxic activity and simultaneously produce cytokines such as ifn-γ. for example, during an influenza infection, the interaction of primed cd + t cells with lung dcs elicits both cytokine production and cytotoxic phenotypes (hufford et al., ) . to summarize the cellular immune responses during a primary influenza infection, specific cd + and cd + effector t cells in the lung predominantly produce ifn-γ and figure upon virus infection, viral antigen from dying respiratory epithelial cells is transported from the lung to the lymph node by migratory antigen-presenting cells such as mouse cd + dendritic cells (dcs) or human monocyte-derived dcs that can stimulate both cd + and cd + t cell responses. viral antigen can also be transferred to lymph node-resident cd α + dcs and presented to naïve cd + t cells. in addition, plasmacytoid dendritic cells (pdcs) are an early source of antiviral type i interferon via recognition of viral rna. tnf-α, and cd + effector t cells also produce il- and il- (carding et al., ; pipeling et al., ; mayer et al., ) . cd + effector t cells localize to the respiratory epithelium and induce apoptosis of infected epithelial cells through fas-fasl interactions or the exocytosis of cytolytic granules containing perforin and granzyme tripp et al., ) . b cells are found interspersed in the lung interstitium and in the cervical and mediastinal/bronchial lymph nodes that drain the upper and lower respiratory tract, respectively. during a respiratory virus infection, a tertiary lymphoid structure also forms along the branching point of the bronchial tree, called the bronchus-associated lymphoid tissue (balt) (brandtzaeg, ) . the balt contains organized b cell areas, germinal centers, and antibody-forming cells (randall, ) . b cell responses can be classified into three categories: innate-like b cell responses, t-dependent b cell responses, and t-independent b cell responses. innate-like b cell responses consist of antibodies produced almost exclusively from b- cells, a small subset of b cells characterized by a unique developmental origin, phenotype, tissue distribution, and regulation, compared with conventional b cells (baumgarth et al., ; baumgarth, ) . t-dependent b cell responses are b cell responses that are facilitated by cd t cells, whereas t-independent b cell responses are not. it has been shown that cd t cell deficiency results in a drastically reduced humoral response to influenza virus infection in mice (mozdzanowska et al., ) . however, mice lacking cd and cd t cells are still protected from lethal infection mozdzanowska et al., ) , highlighting the importance of t-independent b cell responses. b cell responses are critical for viral clearance in primary respiratory virus infections, such as influenza (gerhard, ) . although control of early infection ( - days postinfection) is not impaired in b-cell-deficient mice, the mice fail to clear the virus and ultimately succumb to infection (graham and braciale, ; lee et al., ) . studies in influenza virusinfected mice also show that serum antibody titers are first detected around - days postinfection, at least days later than responses are detected in the respiratory tract. they steadily increase for about a month, after which relatively high figure during the effector phase of the response, influenza-specific cd + t cells elicit a cytotoxic response. tnf-α and nitric oxide (no) produced from a subset of dcs and neutrophils contribute to both viral clearance and immunopathology. lung cd c hi dcs present the crucial t cell survival factor il- to antiviral cd + t cells in trans and promote the production of chemokines. in draining lymph nodes, cd b + dcs contribute to the expansion of specific cd + t cells, and conventional dcs (cdcs) induce the th response. plasmablasts initially produce igm, and effector cd + t cells provide help for virus-specific b cell/plasmablast differentiation and proliferation. pdcs also play a detrimental role by eliminating virus-specific cd t cells in a process involving fasl. antibody titers are maintained for life. virus-specific antibodyforming cells reside transiently in the spleen, from around or days postinfection, and persist for the long term in the bone marrow (jones and ada, ; hyland et al., ) . after recovery from an infection (figure ), a state of immunological "memory" ensues, in which the individual is better able to control a subsequent infection with the same pathogen (ahmed and gray, ) . immunological memory is maintained by both t and b cell subsets, and there are profound differences in the generation, trafficking, and maintenance of t and b cell memory. antigen-specific memory t cells persist at increased frequencies, have a reduced requirement for costimulatory signals in comparison to naïve t cells, and respond quickly to antigenic restimulation (woodland et al., ) . in the case of influenza and parainfluenza (piv) virus infections, it has been clearly established that both cd + and cd + memory t cell subsets respond to and control secondary infection (woodland and dutton, ) . b cell memory is characterized by two distinct populations: long-lived plasma cells that continually secrete antibody and memory b cells that persist in a quiescent state (bachmann et al., ; slifka and ahmed, ) . the generation of long-lived plasma cells is dependent on cognate t-b cell interaction and cd signaling that occurs in the germinal center (noelle et al., ; lee et al., ) . antigen-specific igg and iga antibodies are maintained long after infection and may be protective against heterologous strains of virus. for example, up to % of people born between and in finland had preexisting antibodies to the ph n influenza virus, probably because of its relationship to the h n pandemic influenza virus that circulated in the first part of the twentieth century (ikonen et al., ; yu et al., ) . the presence of crossreactive antibodies contributed to the unexpectedly low numbers of the elderly with severe illness during the pandemic, compared with seasonal influenza virus strains (monsalvo et al., ; o'donnell et al., ) . although neutralizing antibodies directed against the ha globular head are highly efficient at preventing and clearing influenza virus infection, they can also figure in the memory phase, migratory lung dcs capture viral antigen retained on follicular dcs (fdcs) in tertiary lymphoid organs and present it to specific t cells in the respiratory draining lymph nodes. these stimulated t cells upregulate the expression of cd , causing them to be retained in the lymph nodes that drain the site of primary infection. resident dcs are able to reactivate memory responses in the lymph nodes. lung dcs can promote the production of iga in a process that depends on tgf-β. the generation of b cell memory, particularly the generation of long-lived plasma cells, is dependent on cognate t-b cell interaction and cd signaling that occurs in the germinal center. provide a selective pressure for viral immune evasion. crossprotection against various influenza a subtypes, termed heterosubtypic immunity, requires the immune system to recognize epitopes that are conserved between subtypes. such epitopes can be found in the membrane-proximal stalk region of ha (han and marasco, ) or in internal proteins such as nucleoprotein or the m protein. anti-stalk antibodies do not inhibit virion binding to mammalian host cells, but inhibit fusion between the viral envelope and the endosomal membrane. they have broadly neutralizing activity and passively protect mice from lethal challenge in vivo (okuno et al., ; throsby et al., ; sui et al., ). interestingly, cross-reactive anti-ha stalk monoclonal antibodies have been generated from the acute response to h n pandemic virus and also from healthy subjects vaccinated with inactivated virus (corti et al., ; sui et al., ; wrammert et al., ) . how to induce high-titers of anti-ha stalk antibodies in humans remains an active area of universal influenza vaccine research. cellular immune responses to cross-reactive epitopes (often expressed on internal viral proteins) also provide a substantial degree of protection against serologically distinct viruses (yewdell et al., ; rimmelzwaan and osterhaus, ) , and although these heterosubtypic cellular responses are not able to prevent reinfection, they can ameliorate disease by reducing the maximal viral load, mediating faster viral clearance, and providing a substantial degree of protection against challenge with a lethal dose of virus in animal models (hillaire et al., ) . there is also some epidemiological evidence that heterosubtypic cellular immunity plays a role in the response to infection with novel influenza viruses in humans; however, the protective effect appears to be weak and may wane over time (epstein, ; epstein and price, ) . it has, therefore, been suggested that protective cellular immunity could be sustained by reinfection or annual immunization. the effector mechanisms of heterosubtypic immunity remain ambiguous . in murine models of influenza a virus infection, heterosubtypic immunity is observed in the absence of antibodies that recognize influenza envelope glycoproteins and is thought to be mediated primarily by cd + t cells, with a relatively small contribution by cd + t cells mckinstry et al., ) . heterotypic influenza-specific cd + t cells have also been shown to lyse influenza virus-infected cells (nguyen et al., ) . however, heterosubtypic immunity has been observed in cd + t-cell-deficient mice, but not in mice lacking b cells , indicating that there is redundancy in the system. the same investigators found that the heterosubtypic immunity does not require ifn-γ (nguyen et al., ) , but does require a properly diversified antibody repertoire (nguyen et al., ) . the mucosal immune system has been described in detail elsewhere in this book; however, there are some features unique to the respiratory tract that are worth noting. like the immune system in general, the mucosal immune system of the respiratory tract uses innate and specific mechanisms to prevent and limit infection. innate defenses include physical and chemical factors such as the secretion of mucus, which traps microorganisms and antigens and facilitates their transport out of the body by mucociliary motion. mucosal secretions also contain chemically active substances, including acids, lactoferrin, and lysozyme, which inhibit the growth of microbes. in addition, the luminal side of the respiratory tract is physically protected by layers of epithelial cells that adhere to each other at tight junctions, using occludin and various members of the claudin family, and at adherent junctions using e-cadherin (tsukita et al., ) . in humans, the respiratory tract can be divided anatomically into the urt, which comprises the nose, mouth, and pharynx, and the lrt, which includes the trachea, bronchi, and lungs, with the lymphoid tissue of waldeyer's ring representing the line of demarcation. the unpaired nasopharyngeal tonsils (also called the adenoids) and the palatine and lingual tonsils constitute most of waldeyer's ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components (dolen et al., ) . this lymphoid tissue is functionally comparable to the nasal or nose-associated lymphoid tissue (nalt) in rodents, which is composed of two paired lymphoepithelial structures beside the nasopharyngeal duct, dorsal to the cartilaginous soft palate (kuper et al., ; fukuyama et al., ) . however, rodents do not have tonsils. waldeyer's ring is more strategically situated than the nalt to generate mucosal immunity, because its elements are exposed to both airborne and alimentary antigens. in addition, human tonsils have deep antigen-retaining crypts, and tonsils express germinal centers shortly after birth, whereas the rodent nalt has a plain surface and requires an external stimulus to induce the expression of germinal centers (brandtzaeg, ) . tissue equivalent to waldeyer's ring has also been found in nonhuman primates (loo and chin, ; harkema et al., ) and horses (mair et al., (mair et al., , , but functional studies have not been performed in these species. the mucosa-associated lymphoid tissues (malt), including the nalt and balt, consist of follicle-associated epithelium and t-cell-and b-cell-enriched areas. the initiation of antigen-specific immune responses occurs at special gateways, which comprise microfold (m) cells located in the epithelium overlying the malt follicles. the cilia of the apical side of the m cells are shorter than those of conventional epithelial cells, and on the basal side, there is a large pocket-like structure that can hold immunocompetent cells required for the generation of immune responses such as t cells, b cells, and apcs. as lysosome development in m cells is poor, in most cases antigens pass through the cells unmodified and are taken up by dcs in the pocket (sato and kiyono, ) . upon encountering antigen, dcs migrate to the t cell region of the malt and present peptide antigen via mhc molecules to naïve t cells. antigen-specific t cells become primed, clonally expand, and leave the follicle to enter the circulation. they then home to effector sites to elicit mechanisms involved in viral clearance as described above. in the b cell region, a germinal center forms and antibody class switching occurs (cerutti, ) . a class switch to iga predominantly occurs in the malt owing to the action of the iga-associated cytokine family of tgf-β, il- , il- , il- , il- , and il- (mcghee et al., ; mestecky and mcghee, ; cerutti, ) . postswitched iga + b cells leave the malt through efferent lymph vessels under the control of the lipid mediator, sphingosine- -phosphate, and the cells then enter the circulatory system (gohda et al., ; lazarus et al., ) and home to effector sites found in unorganized lymphatic tissue spread over the lamina propria that underlies the mucosal epithelium. in the lamina propria, b cells differentiate into plasma cells and secrete iga, igd, igm, and igg antibodies, although iga is the major mucosal antibody isotype (shikina et al., ; shimoda et al., ) . surgical removal of the murine nalt or cervical lymph nodes does not, however, abrogate cellular or antibody immune responses to experimental influenza infection, suggesting that there may be additional inductive sites other than the nalt and demonstrating that dissecting the relative contributions of anti-influenza immunity is difficult. in the urt, iga is the major mediator of immunity to influenza. in mice that had recovered from an influenza infection, immunity to reinfection was abrogated by the intranasal instillation of anti-iga antibodies, but not anti-igg or igm (renegar and small, a) , and intravenous passive transfer of iga resulted in iga in nasal secretions that protected mice from intranasal challenge with influenza (renegar and small, b) . after passive transfer, nasal iga titers that conferred protection were at a concentration equivalent to that seen in convalescent mice, whereas igg transudation into the urt could be detected only after . times the normal convalescent serum titer had been passively transferred (renegar et al., ) . moreover, recombinant iga is sufficient to prevent influenza transmission in a guinea pig model (seibert et al., ) . in humans, iga is present in monomeric and dimeric forms. during transcytosis through mucosal epithelial cells, an extra polypeptide secretory component is added to dimeric iga and the resulting molecule is known as secretory iga (s-iga). dimeric and s-iga are - times more efficient than monomeric iga at neutralizing influenza viruses (renegar et al., ) . dimeric and s-iga are represented by two subclasses, iga and iga , with covalently or noncovalently joined dimers, respectively. both subclasses are detected in nasal secretions after an influenza infection; however, ha preferentially stimulates an iga response (brown et al., ) . s-iga is not considered to be inflammatory because the fc region is not available to activate immune cells or bind complement. s-iga is also resistant to proteolysis and can neutralize viruses inside epithelial cells and transport viruses that have passed the epithelial barrier to the lamina propria back to the lumen (sato and kiyono, ) . s-iga may therefore be useful in preventing viruses from breaching the mucosal barrier, while avoiding immunopathology by not activating inflammatory responses directly and by limiting the number of antigen-antibody complexes in the lamina propria that can trigger inflammation. it is currently thought that plasma igg serves as a backup for s-iga in the urt, whereas in the lrt, igg is the dominant antibody involved in protection (renegar et al., ) . the fc receptor for igg mediates transport of igg across epithelial barriers by transcytosis, permitting the transudation of igg from the serum into the lung where it is able to neutralize viruses (spiekermann et al., ) . this explains why passively transferred igg is effective at preventing severe disease from respiratory infections in experimental animals and why serum igg antibodies are the main correlate of protection for parentally administered inactivated influenza vaccines in humans (section respiratory virus vaccines). viruses that can cause repeated infection are typically characterized either by a failure to induce robust immunity or by significant antigenic diversity in the face of protective immune responses. influenza viruses can reinfect hosts because the antigenic sites evolve and drift to avoid neutralization by prior immunity. infection induces a strong homosubtypic neutralizing antibody response in healthy individuals that contributes to recovery and protection from repeat influenza virus infection with homologous virus or an antigenically similar virus (wrammert et al., ) . moreover, natural infection can lead to long-lasting immunity to the infecting virus. for example, when the influenza a h n subtype reemerged in , the most susceptible members of the population were those born after the time when similar h n viruses had previously circulated, the s (shortridge et al., ) . however, because influenza viruses undergo antigenic drift and shift, the effective period of protection may last only until an antigenic variant emerges. in contrast to the robust strain-specific protection after influenza virus infection, primary infection with rsv, hpiv, and hmpv provides only partial protection from reinfection. rsv commonly reinfects the host even though genetic diversity in the virus is not extreme. in healthy adults challenged every few months with the same strain of rsv, about % were infected each time and about half of those became symptomatic (hall et al., ) . these studies are now being reproduced with experimental human challenge infection (devincenzo et al., ) , and access to modern immunological techniques may provide some insight into the mechanism of immune evasion. most reinfections are limited to the upper respiratory tract, unless subjects are immunocompromised. reinfections may be the consequence of a highly prevalent and contagious virus, effective evasion of local and innate immunity, or a steep gradient for transudation of antibody from the serum to the nasal epithelium (graham, ) . alternatively, rsv infection may alter the characteristics of the adaptive immune effectors and memory. although rsv infection provides a sufficient antigenic stimulus to induce both antibody (shinoff et al., ) and t cell responses (heidema et al., ) , the durability of the antibody is poor (handforth et al., ; dakhama et al., ; collarini et al., ). the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. it seems logical to consider live attenuated vaccines delivered intranasally for protection against respiratory viruses, as they would induce a mucosal immune response. however, a systemic immune response can be protective if it is sufficiently robust, such as that induced by inactivated influenza vaccines administered by the i.m. route. moreover, achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of maternal antibody, is a challenge. this section describes licensed vaccines as well as vaccines that are currently in development. vaccination remains the primary strategy for the prevention and control of influenza (lambert and fauci, ) . as described in section immune responses to respiratory virus infection, after an influenza infection, both cellmediated immunity and systemic and mucosal neutralizing antibodies are produced. whereas cell-mediated immunity contributes significantly to the clearance of a primary influenza virus infection, and can ameliorate disease caused by reinfection, neutralizing antibodies play an important role in preventing reinfection. the goal of vaccination is to prime the immune response to limit viral replication upon subsequent infection, and inactivated, recombinant hemagglutinin and live attenuated influenza vaccines (laivs) are licensed for use, with novel vaccines in varying stages of development. this section focuses on licensed vaccines and the immune responses they elicit, as determined from clinical trial data. owing to antigenic drift in circulating viruses (discussed in section virology), an influenza vaccine from one season may not be effective in subsequent seasons. each year, the strains that are to be included in the vaccine for the next influenza season are chosen and vaccine seed viruses are generated (lambert and fauci, ) . for inactivated vaccines, the influenza a seed viruses are reassortant viruses, with the ha and na gene segments derived from the circulating virus and the internal protein genes derived from a vaccine donor strain that is adapted for high yield in eggs (a/puerto rico/ / ; pr ) (kilbourne, ) . licensed laivs also contain the ha and na from the circulating virus, combined with the internal protein genes from temperature-sensitive, cold-adapted, attenuated master donor viruses that limit replication of the vaccine viruses to the cooler upper respiratory tract (maassab, ) . if the yield of the vaccine virus in eggs is poor, they may be "egg-adapted" through serial passage. vaccine viruses are then amplified in hundreds of millions of eggs and purified. the inactivated vaccine viruses are treated with formalin or β-propriolactone and "split" with detergents before being formulated for clinical use, with or without thimerosal as a preservative (fiore et al., ) . until , seasonal influenza vaccines were trivalent, containing two subtypes of influenza a viruses (h n and h n ) and one influenza b virus. however, from the winter season in the northern hemisphere, quadrivalent vaccines, containing two subtypes of influenza a viruses and two strains of influenza b viruses, have become available. it takes several months from the generation of a seed virus to the manufacture and distribution of a vaccine. typically, for seasonal influenza in the northern hemisphere, manufacturers amplify vaccine viruses and inactivate or purify them between february and late summer and formulate and distribute them for administration in the fall before the anticipated peak of the influenza season (lambert and fauci, ) . in , the h n pandemic virus emerged in april, when the manufacture of seasonal trivalent vaccines was already under way. a monovalent h n vaccine was produced as quickly as possible in addition to the seasonal vaccines, but the monovalent vaccine was not available for widespread use until after the pandemic had peaked in the northern hemisphere and was not available at all during the winter season in the southern hemisphere (broadbent and subbarao, ; skowronski et al., ) . in addition, current global vaccine production capacity does not cover the high-risk population around the world (girard et al., ) . one means of increasing capacity is to move toward using cell culture instead of eggs for vaccine production, and several companies are investigating this. additionally, the amount of ha in each vaccine dose could be reduced and used with an adjuvant such as mf or as . however, adjuvanted seasonal influenza vaccines are not yet licensed in the united states. trivalent inactivated vaccines (tivs) against seasonal influenza are administered i.m. and have an efficacy ranging from to % in preventing influenza morbidity and mortality in healthy adolescents and adults (osterholm et al., ) . a trivalent laiv consisting of an a or b ann arbor cold-adapted backbone together with the ha and na of the target viruses (flumist ® , medimmune), administered by a nasal spray, is licensed in several countries, including north america and europe. in the united states, the seasonal laiv is licensed for healthy individuals between and years of age, but it is currently not approved for use in children under years of age, and it is not approved for use in the elderly or in immunocompromised individuals (ambrose et al., ) . there is a substantial body of evidence from largescale randomized clinical trials demonstrating the effectiveness of laivs, and a number of clinical studies have also shown that the efficacy of laivs is equivalent or superior to that induced by i.m. vaccination with tivs in children (reviewed by luke et al. ( ) ). in adults, however, the majority of double-blind, randomized, placebo-controlled trials have shown tivs to have a greater efficacy than laivs monto et al., ; ohmit et al., ohmit et al., , . in the military, vaccine effectiveness was also found to be higher for tivs than for laivs, except in new recruits (eick-cost et al., ; wang et al., ). tivs primarily induce serum antibodies against the influenza ha glycoprotein, which are typically measured by hemagglutinin inhibition (hi) assays, which serve as a surrogate for virus-neutralization assays. in healthy individuals who are immunologically primed by previous infection or vaccination, influenza-specific antibodysecreting cells in the peripheral blood peak week after vaccination and serum antibody levels peak to weeks postvaccination. however, in unprimed individuals, for example, children, it may take weeks or longer for serum antibody levels to peak after vaccination (brokstad et al., ; cox et al., ; el-madhun et al., ) . the rise in serum antibody titer after tiv administration has been documented for multiple isotypes, including igm, iga, and igg, with a more pronounced rise in igg titers (moldoveanu et al., ) . in contrast, vaccination with an laiv leads to seroconversion more frequently in immunologically naïve individuals than in those who are immunologically primed. for example, in children, after a single dose of trivalent laiv, seroconversion rates of - %, - %, and - % have been reported against influenza a h n , h n , and b, respectively, which increased to % and % for h n after a second dose at day or , respectively (belshe et al., ; lee et al., ) . however, in healthy adults, serum antibody titers are lower after laiv than tiv vaccination (moldoveanu et al., ) , and in one study only % of laiv recipients had an increase in serum igg titer compared to % of tiv recipients . protection mediated by inactivated vaccines therefore correlates with serum neutralizing antibody titers, whereas other immune mechanisms contribute to protection mediated by laiv. a higher percentage of laiv recipients have mucosal antibodies and antibody-secreting cells than those receiving tiv. one study recorded that % of laiv recipients had increased influenza-specific iga in the mucosa, compared to only % of tiv recipients . moreover, levels of iga in nasal wash specimens correlated with protection against challenge with wild-type influenza viruses . mucosal iga in adults vaccinated with laiv declined months after vaccination . in addition to iga, igg has also been found in nasal secretions after laiv (moldoveanu et al., ) . as described (section immune responses to respiratory virus infection), iga is the major mediator of immunity to influenza infection in the urt, with igg serving as a backup. this also appears to be the case after vaccination with laiv. the majority of antibodies induced by influenza vaccines that are associated with protection are directed against the globular head of the ha. recently, neutralizing antibodies have also been identified that bind to a conserved epitope in the ha stem. the ha stem antibodies have been found to be broadly neutralizing, and there is much effort to generate vaccines that elicit these antibodies to produce a more broadly cross-protective vaccine (corti et al., ; ekiert et al., ; kashyap et al., ; okuno et al., ; sui et al., ). in addition, antibodies directed against the na protein are also generated after vaccination with both tiv and laiv (murphy et al., ) . anti-na antibodies restrict virus release from infected cells and reduce the severity of disease by limiting spread (murphy et al., ) . however, currently licensed inactivated vaccines are standardized to ha, but not na protein content. the amount of na protein varies from vaccine to vaccine, and the contribution of vaccine-induced anti-na antibodies to protection against influenza is not well understood (hassantoufighi et al., ) . in addition to humoral immunity, influenza-specific cd + cytotoxic t lymphocytes (ctls) are associated with accelerated clearance of virus and recovery from infection. however, the extent to which the cellular immune response is protective against infection is unknown because the recall response is likely to occur after the peak of viral replication (subbarao et al., ) , and cell-mediated immunity induced after vaccination has been less well studied than the humoral response, and the results are variable. studies have shown that immunization of healthy adults with whole-virus inactivated vaccine resulted in enhanced ctl responses in peripheral blood, whereas immunization with a subunit vaccine resulted in poor ctl responses, the duration of which varied from several months to years (ennis et al., ; mcmichael et al., ; powers and belshe, ) . in addition, an increase in ifn-γ-producing t cells was seen in children ages months to years of age who were vaccinated with inactivated vaccine; however, similar responses were not induced in adults (he et al., ) . h n -specific cd + t cells were also detected after a single dose of as -adjuvanted inactivated vaccine and was amplified by a second dose of vaccine (moris et al., ) . in addition, numerous studies have found a significant increase in ifn-γ-producing cd + and cd + t cells after vaccination with laiv in both adults and children (basha et al., ; forrest et al., ; he et al., a; hoft et al., ; lanthier et al., ) ; however, the role of cellular immune responses in laiv-mediated protection needs further investigation. more than % of annual influenza-related deaths in the united states occur in individuals years of age or older (thompson et al., ) . vaccinating elderly individuals is therefore a public health priority. however, a randomized placebo-controlled trial estimated the efficacy of the tiv to be % for the prevention of influenza in older adults (govaert et al., ) . elderly individuals often have a significantly reduced antibody response to influenza vaccination (goodwin et al., ) . in a quantitative review of elderly subjects, %, %, and % seroconverted to h n , h n , and influenza b vaccination, respectively, compared to %, %, and % in younger subjects (goodwin et al., ) . the impaired ability of the elderly to mount an adequate immune response to influenza vaccines has been attributed to immunosenescence. immunosenescence is the decline in the body's ability to fight infection, mount novel immune responses, and recall responses (targonski et al., ) , and both innate and adaptive responses are implicated. impaired function of costimulatory molecules, altered secretion of inflammatory cytokines, and diminished function of natural killer cells, macrophages, and neutrophils have been observed in the elderly, as well as a decreased proliferative capacity of b cells and impaired t cell memory recall (sullivan et al., ) . in addition, thymic involution and a decline in t cell output are features of advancing age. this, together with a lifetime of exposure to a variety of pathogens, leads to a reduction in the naïve t cell pool and a relative increase in the proportion of memory t cells in the elderly compared with young adults. the most pronounced functional changes are in the cd + t cell subset, in which progressive exhaustion occurs (pawelec et al., ) , whereas the cd + t cell subset is less affected by replicative senescence (czesnikiewicz-guzik et al., ) . the tiv is standardized on the basis of the amount of ha protein, with one dose of μg per strain being recommended in healthy, previously primed individuals. increasing the dose increases serum antibody response to the vaccine, and in an attempt to enhance immune responses to influenza vaccines in the elderly, a high-dose tiv ( μg ha protein per strain) was licensed in for use in persons ages years and older. postlicensure studies have shown enhanced immune responses in this age group, compared to the standard dose (sullivan et al., ) , and vaccine effectiveness studies are under way. in addition, an as -adjuvanted tiv (discussed below) containing μg ha protein per strain showed a % higher efficacy than a nonadjuvanted tiv in a phase randomized clinical trial in the elderly, but the difference was not statistically significant (mcelhaney et al., ) . laivs are not licensed for use in the elderly at present; however, they have been evaluated in clinical studies in persons years of age and older and are safe and well tolerated. in clinical trials, laivs were administered in addition to tivs, and coadministration was reported to enhance local ha-specific iga antibody responses. however, the efficacy of the combination was not greater than that of tiv alone (gorse et al., ; powers et al., powers et al., , treanor et al., ) . in individuals with chronic or immunocompromising conditions, serological responses to tiv vaccination are typically lower than in healthy adults. antibody responses against influenza were adequate in hiv-seropositive individuals who had no or minimal immunodeficiency or had responded well to antiretroviral therapy (chadwick et al., ; huang et al., ; madhi et al., ; staprans et al., ) . however, in individuals with advanced hiv disease and low cd t cell counts, tivs may not induce protective titers even after two doses (kroon et al., ; miotti et al., ) . laivs are not licensed for use in immunocompromised individuals. although there is a large amount of data available on the immune responses to seasonal influenza vaccines in humans, improved seasonal and pandemic influenza vaccines are evaluated first in animal models. the most commonly used models are mice and ferrets. mice immunized with inactivated influenza vaccines develop serum hi and neutralizing antibodies, the titers of which correlate with protection from subsequent challenge (luke and subbarao, ) . although cellular immune responses are mounted during a secondary influenza infection (woodland et al., ) , passively transferred antibodies protected immunosuppressed mice, suggesting that cell-mediated immunity is not essential for protection if sufficient antibody is present (virelizier, ) . the goal of parenteral immunization with inactivated influenza vaccines is to induce sufficient serum antibody titers to limit influenza disease. this protection is mediated by serum igg that transudes into the lower respiratory tract, neutralizing virus. passive transfer of immune serum to naïve mice reduced the replication of influenza virus in the lungs and protected recipient mice from lethal influenza pneumonitis, but did not prevent tracheitis or replication of the influenza virus in the urt (ramphal et al., ) . during a natural influenza infection of the urt, mucosal immune responses, including secretory iga antibodies, play an important role in controlling disease (section immune responses to respiratory virus infection). several studies have documented that higher levels of serum antibodies are required to provide protection against respiratory viruses in the urt compared to the lrt (prince et al., ; ramphal et al., ; takiguchi et al., ) . additionally, influenza in ferrets is primarily a urt infection and vaccination with killed or inactivated influenza viruses does not protect against influenza infection unless administered with an adjuvant (potter et al., a,b) . adjuvants are probably required for parenterally administered inactivated vaccines to elicit the higher levels of serum igg antibody that are needed to restrict viral replication in the urt, in the absence of robust mucosal immune responses. in mice, laivs induce a range of systemic and pulmonary immune effectors and protect animals against challenge virus replication (chen et al., ; lau et al., ) . the magnitude of the pulmonary iga and memory cd + t lymphocyte responses depends on the replication efficiency of the vaccine virus in the respiratory tract, but systemic immunity, such as serum antibody titers and memory cd + t lymphocytes in the spleen, does not . after one dose of an h n laiv that replicated in the lungs of mice and induced local immunity, influenza-specific lymphocytes in the lung contributed to the clearance of challenge virus from the lungs, whereas the contribution of serum antibody and splenic cd + t cells was negligible. after two doses, complete protection was achieved and was associated with maturation of the antibody response (lau et al., ) . taken together, the data suggest that laiv protects animals by inducing multiple arms of the immune response, including mucosal immunity and pulmonary and systemic antibody and cellular immune responses, in a manner similar to natural infection. however, inactivated vaccines aim to induce serum antibody alone. although this does not mimic a natural infection, if antibody titers are sufficiently high, the inactivated vaccine will protect against disease caused by influenza viruses. for inactivated influenza vaccines, serum anti-ha antibody titers correlate well with resistance to influenza infection in people as well as in animal models. lower antibody titers are associated with an increased risk of illness, though a specific antibody titer that can guarantee protection from infection has not been identified. an hi titer of : represents the level at which it is anticipated that approximately % of persons will be protected (hobson et al., ) , and this benchmark forms the basis of the licensing criteria for inactivated vaccines (cber, ) . although several papers refer to "seroprotection," there is insufficient evidence to support the use of this term for vaccines. the benchmark of an hi titer of : was defined in healthy adults who were experimentally challenged with an influenza virus (hobson et al., ) . however, antibody titers that correlate with protection in healthy adults may not translate to clinical improvements in influenza outcomes in the elderly (gorse et al., ) . in addition, laivs are effective despite inducing variable serum hi antibody titers. therefore, alternative correlates of protection are needed. as iga and cellular immune responses are generated in the lungs of mice vaccinated with laiv, in addition to systemic antibody and cellular responses, the extent to which the different arms of the immune response contribute to laiv-induced protection are beginning to be evaluated (chen et al., ; lau et al., lau et al., , . laiv-induced nasal wash igg and iga correlated with protection from virus replication, and in human challenge studies, either serum antibody or nasal wash iga was a predictor of protection (belshe et al., b) . cellular immune responses have also been investigated as a correlate of laiv-induced protection, and one study found a correlation between ifnγ-producing t cells (measured by elispot) and protection from culture-confirmed influenza illness in young children (forrest et al., ) . in addition, it has been shown that laivs alter the expression of ifn-related genes, whereas tivs do not, indicating that the innate immune response plays an important role in protection mediated by laivs (nakaya et al., ) . adjuvants are added to vaccine formulations to enhance immune responses to the antigen in the vaccine. aluminum salts (alum) are the most commonly and historically used adjuvants worldwide. they act by capturing antigens at the injection site, so the antigen is slowly processed and presented by the immune system (the so-called depot effect), and they cause mild cell damage and inflammation that promotes a th immune enhancement (tetsutani and ishii, ) . moreover, alum particles enter host cells and bind dna, introducing it into the cytoplasm of antigen-bearing dcs, where it engages receptors that promote both mhc class ii presentation and dc-t cell interactions (mckee et al., ) . oil-in-water adjuvants, such as mf (novartis) and as (glaxosmithkline), are more effective at inducing high-titer antigen-specific serum antibody responses than alum and have been used with inactivated split-virion influenza vaccines in europe. these adjuvants induce broad, cross-clade humoral responses and permit dose sparing, in which comparable immune responses are induced with a reduced amount of ha in the vaccine (galli et al., ; leroux-roels et al., ) . our understanding of the mechanism of action of mf and as remains incomplete. neither appears to act via a depot effect; instead they induce a local and transient proinflammatory cytokine and chemokine response at the injection site and draining lymph nodes that recruit immune cells from the circulation. in mice, as induced the cytokine il- and chemokine cxcl , which peaked locally by h postvaccination. the neutrophil-mobilizing cytokine csf and lymphocyte-mobilizing cytokines ccl , , and were induced by h postvaccination. in addition, the eosinophil-recruiting chemokine ccl and the cytokine il- β were induced at low levels, and ifn-γ, csf (gm-csf), and tnf-α were induced at levels that were only marginally above background. ifn-α and -β were not induced. the local cytokine response was paralleled by an enhanced recruitment of monocytes and granulocytes in the draining lymph node (morel et al., ) . mf also induces local upregulation of cytokines, chemokines, and other innate immunity genes, promoting the recruitment of immune cells such as monocytes, dendritic cells, and granulocytes. however, the mechanism of action of mf was found to be independent of the nlrp inflammasome, but required myd for a tlr-independent signaling pathway (seubert et al., ) . whereas the inactivated vaccine viruses are typically disrupted with detergents to make split-virion (subunit) vaccines, inactivated whole influenza virion (wiv) vaccines have also been developed, in which the virions are left intact. these are less widely used because of increased reactogenicity and adverse events (fiore et al., ) . however, mice immunized with wiv vaccines consistently showed higher hi titers and virus-neutralizing antibody titers than subunit vaccines, as well as an increased production of proinflammatory cytokines by dendritic cells and ifn-α by plasmacytoid cells, resulting in a desired th response (geeraedts et al., ; koyama et al., ) . the approach of using inactivated whole influenza vaccines is being revisited with pandemic influenza vaccines. whole-virion inactivated h n and h n vaccines administered with alum are immunogenic in humans (kulkarni et al., ; lagler et al., ; lin et al., ; nakayama et al., ) . dna vaccines encoding one or several proteins of influenza viruses induce an immune response targeting the encoded proteins (fynan et al., ; ulmer et al., ; wolff et al., ) . dna vaccines can be produced rapidly and at low cost; however, designing dna vaccines is complex. over the years, it has been shown that numerous factors play roles in the efficiency of expression, such as the promoter, the g/c content, supercoiling, polyadenylation, and codon optimization (laddy and weiner, ) . in addition, safety remains a concern, as there might be a risk of integration into the host genome (klinman et al., ) . numerous studies have evaluated dna vaccines expressing np, m , or ha proteins in animal models (fu et al., ; saha et al., ; tao et al., ; ulmer et al., ulmer et al., , a . in mice, the administration of dna vaccines encoding the np protein of influenza induces a strong ctl response, which correlates with protection against challenge with homologous or heterologous viruses (ulmer et al., ) . in addition, one study showed that delivering the vaccine by in vivo electroporation instead of the classical epidermal route also induces protective humoral and cellular immune responses in mice, ferrets, and nonhuman primates (laddy et al., ) . recently, a phase clinical trial with an adjuvanted plasmid dna vaccine encoding influenza h , ha, np, and m elicited t cell responses against ha in the majority of the subjects and against np and m in some (smith et al., ) . licensed influenza vaccines primarily elicit an immune response to the globular "head" region of the ha glycoprotein. however, immune selection pressure leads to antigenic drift (discussed in section clinical features and epidermology), so new influenza vaccines need to be selected for each season as well as when a pandemic emerges. it remains difficult to predict which strains will circulate in the upcoming influenza season, and rates of morbidity and mortality are greater in influenza epidemics when the virus and vaccine are "mismatched" (pica and palese, ) . the expectation is that vaccines capable of protecting against a broad(er) spectrum of influenza viruses would result in less frequent updating of seasonal influenza vaccines and would provide a degree of preexisting immunity if a novel strain emerges. vaccines that aim to provide broad cross-protection against multiple subtypes of influenza are known as universal vaccines, and several platforms are in development, including those that target the ha, m , np, m , and na proteins (subbarao and matsuoka, ) . unlike the head region of ha, the "stalk" domain has a high degree of sequence conservation between influenza viruses, and broadly neutralizing stalk-reactive antibodies have been identified. these antibodies may inhibit phinduced conformational changes in the ha required for cellular entry, prevent the cleavage of ha into ha and ha (discussed in section virology), or act via antibodydependent cell-mediated cytotoxicity (adcc) or through the activation of complement . however, the ha stalk is less immunogenic than the head, and several techniques have been employed to elicit stalk-reactive antibodies, including removing the head, novel prime-boost strategies, and sequential vaccination with chimeric ha molecules that contain the same stalk region but "exotic" head domains that aim to boost antibodies to the conserved epitopes within the stalk . these strategies have been tested in mice with varying degrees of success in generating protective immune responses against heterologous influenza viruses (subbarao and matsuoka, ) . taken together, ha stalkbased strategies show promise as universal influenza vaccine candidates. the influenza m protein extends beyond the viral envelope, and antibodies against the m extracellular domain (m e) may act via adcc, nk cell activity, or complementmediated lysis. in addition, the sequence of the m e is conserved among human influenza a viruses, making it an attractive target for universal vaccines. however, the peptide is poorly immunogenic, and techniques to improve immunogenicity are under investigation in mice, including fusing the peptide to an immunogenic carrier protein or delivering the antigen in a virus-like particle (vlp) (reviewed by subbarao and matsuoka ( ) ). the influenza np and m proteins are also conserved among influenza a viruses and have potential for use in a universal vaccine, but as they are not exposed on the surface of virions or infected cells, they mainly induce cellular immune responses. phase and clinical trials with modified vaccinia virus ankara (mva) expressing np and m proteins report a % reduction in laboratory-confirmed influenza infection after experimental challenge (lillie et al., ) ; however, larger studies are required to confirm this. immune responses directed against the influenza na protein do not protect against infection, but rather limit the spread of infection and reduce the severity of disease. recently, baculovirus-expressed vlps containing n na proteins were found to induce heterosubtypic na antibodies in mice that protected them against homologous and heterologous challenge (quan et al., ) . a universal vaccine may therefore benefit from anti-na immune responses. it may be useful to combine several components into a universal vaccine, for example, np and m to induce cellular responses and ha and m e to induce humoral responses. however, there are several challenges to the development of a universal vaccine. as outlined above, most of the viral proteins that potentially induce broad heterosubtypic immunity are poorly immunogenic and require a large dose of antigen, multiple doses, addition of adjuvants, fusion to immunogenic carriers, or the use of vectors or vlps. additionally, regulatory challenges include how to determine and define the potency of the vaccine and the need to identify immune correlates of protection and develop validated assays to measure them. in addition, clinical trials will be challenging because it is likely that efficacy will be measured in terms of amelioration of disease rather than preventing infection (subbarao and matsuoka, ) . given these hurdles, achieving a truly universal vaccine that protects against all types or subtypes of influenza will probably proceed in a stepwise manner, with the first step being a vaccine that is more broadly cross-protective than currently licensed vaccines. the primary target populations for rsv vaccination are young infants and the elderly, because hospitalization rates are the highest in these age groups and % of rsv-related deaths occur in individuals over years of age (thompson et al., ) . there is a large body of evidence that protection against infection is conferred mainly by neutralizing antibodies (collins and melero, ) ; however, multiple doses of vaccine might be necessary in young infants, because of the immature immune system and the presence of maternal antibodies. in addition, protective immunity mounted during infection does not protect against subsequent reinfection. reinfections are common and are independent of antigenic changes in the virus (collins and melero, ) . these factors present challenges to rsv vaccination and, as yet, vaccines have not been licensed. this section discusses clinical trial data from various approaches to rsv vaccination. the first approach to vaccination was the development of a formalin-inactivated rsv vaccine (f -rsv) that was evaluated in the s in infants and young children. a concentrated f -rsv vaccine, given i.m. with alum, was well tolerated and moderately immunogenic, but was poorly protective against infection. in fact, vaccinees who were exposed naturally to infection during the subsequent rsv season had immune-mediated enhancement of disease, with % of individuals requiring hospitalization and two fatalities (fulginiti et al., ; kapikian et al., ; kim et al., ) . subsequent studies revealed that vaccine-induced immune responses were different from those elicited after natural infection, with poor induction of neutralizing antibodies (murphy and walsh, ) and an exaggerated cd + t-cell response (kim et al., ) . vaccine-mediated enhancement of disease also occurred in murine models, with poor induction of neutralizing antibodies probably due to denaturation of antigen in the vaccine or a lack of antibody affinity maturation after poor tlr stimulation (delgado et al., ). in addition, an exaggerated th response and a loss of regulatory t cells contributed to disease (connors et al., (connors et al., , de swart et al., ; loebbermann et al., ; waris et al., ) . therefore, it is clear that in addition to a neutralizing antibody response, a balanced cd and cd t cell response is also desirable. after this experience, inactivated rsv vaccines were considered unsuitable for pediatric use, and alternative approaches were sought. in the s, a series of live attenuated vaccines (lavs) was developed by serial passage of rsv at suboptimal temperatures (cold passage, cp) or by growth in the presence of mutagens to produce temperature-sensitive (ts) mutants. the lav did not cause disease enhancement in animal models or in clinical trials (wright et al., a) . the most promising vaccine generated by this method, cpts / , was well tolerated and immunogenic in seronegative infants and children greater than months of age, but caused mild congestion in younger infants and was deemed to be insufficiently attenuated (wright et al., ) . several lavs have also been developed using reverse genetics techniques and were evaluated in clinical trials. the most promising, ra cp / / δsh (medi- ), was strongly ts and was well tolerated in infants . the first dose provided substantial reduction in replication of a second dose of vaccine given months later. however, the majority of individuals did not have increases in serum antibody titers, indicating that other immune mechanisms might play a role in the protection from replication of the second dose of vaccine. as with live attenuated influenza vaccines, establishing correlates of protection for live rsv vaccines is an active area of research, made more challenging by the weak immune responses in infants and limitations on sampling. as of this writing, clinical trials are under way to further monitor tolerability and immunogenicity and the ability of candidate vaccines to induce protection against natural rsv exposure in the community (collins and melero, ) . genetic changes have been employed to generate candidate lavs, including deletion of the m - coding sequence, which increases transcription and antigen synthesis at the expense of viral replication (bermingham and collins, ) , and deletion of the ns and ns genes, which increase ifn production and signaling and might limit viral replication and pathology, but increase immunogenicity (teng et al., ) . the use of attenuated parainfluenza virus as a vector to express rsv f and/or g protein has also been considered as a pediatric vaccine against both hpiv and rsv, as piv has the advantage of improved in vitro growth and stability compared with rsv. bovine piv is attenuated in humans and has been used as a vector into which the f and hn genes from human piv and the f and/or g protein of rsv are incorporated to make a bivalent vaccine against both hpiv and rsv (schmidt et al., ; tang et al., ) . one example of this approach is in clinical trials in children older than months of age and who are seronegative to rsv and hpiv (collins and melero, ) . the vaccine in this study comprises a bovine piv vector into which the human piv f and hn genes and the rsv f gene have been added (tang et al., ) . as an alternative to bovine piv , murine piv (sendai virus) is also being evaluated as a vaccine backbone into which rsv antigens are inserted (jones et al., ) . there is substantial antigenic crossreactivity between sendai virus and hpiv , and the virus may be attenuated in humans. other approaches to live vaccines for rsv include the use of alphaviruses (elliott et al., ) or replicationdefective adenoviruses (shao et al., ) as vectors for rsv antigens. in addition to an rsv vaccine in infants, there is also a need for rsv vaccines in older children, adults, and the elderly. however, these individuals are likely to have been previously infected with rsv, and preexisting immunity may restrict the replication of lavs. in addition, vectored vaccine approaches should not be based on common human pathogens, as preexisting immunity to the vector may limit vaccine virus replication and interfere with the development of immunity. the use of protein-based vaccines in these populations is currently being evaluated. disease enhancement has not been documented in adults who were previously exposed to rsv, and candidate rsv f protein-based vaccines are well tolerated in healthy adults, children over months of age, pregnant women, and the elderly (girard et al., ) . in a phase clinical trial involving children - years of age with cystic fibrosis, a purified f protein- (pfp- ) vaccine induced a fourfold rise in serum antibody titer, but was not associated with significant protection against lrti episodes compared to placebo . in another approach, a subunit vaccine of copurified f, g, and m proteins from rsv-a was tested in adults and found to induce neutralizing antibodies in - % of vaccinees, but titers waned after year, suggesting that annual immunization might be necessary with this vaccine (girard et al., ) . interestingly, in a phase clinical study of pregnant women vaccinated with pfp- , anti-f antibodies were persistently elevated in newborns of the vaccinated mothers, without enhancement of disease. maternal immunization could, therefore, be a strategy to protect infants under months of age, for whom the development of an rsv vaccine has been such a challenge (durbin and karron, ) . a recombinant postfusion form of the f protein with a deletion of the major hydrophobic regions was also produced. this subunit vaccine forms stable trimers that are recognized by neutralizing mabs. high levels of neutralizing antibodies were induced in the sera of vaccinated rodents, and the animals were protected from challenge. this may represent an improved subunit vaccine (mclellan et al., ; swanson et al., ) . another protein-based vaccine, bbg na, has been evaluated in clinical trials. bbg na consists of a fragment of the rsv g protein that contains a central conserved domain fused to the albumin-binding domain of streptococcal g protein, expressed in bacteria. however, this vaccine was not very immunogenic in clinical trials and was associated with hypersensitivity reactions (purpura) in some individuals (power et al., ) . vlps have also been evaluated for use as an rsv vaccine, and one such formulation (rsv-f particle vaccine) is currently in clinical trials (collins and melero, ) . it is the hope that improved methods of producing rsv antigens or using vlps will result in an effective vaccine that could be given to adults periodically, possibly with the annual influenza vaccine. however, in children, it remains to be seen whether a vaccine can be developed that is sufficiently attenuated, yet immunogenic enough to provide protection. however, even if complete protection is unrealistic, immunity that results in substantial reduction of virus replication may be sufficient to prevent severe disease. as % of children are seropositive for hpiv by years of age, and hpiv lrti is a major cause of hospitalization in this age group (schomacker et al., ) , young infants and children are the target population for hpiv vaccination. protective immunity against infection is mediated primarily by mucosal and serum neutralizing antibodies; however, reinfection is common owing to a difficulty in maintaining protective titers of s-iga and igg in the respiratory lumen, thus representing a challenge to vaccine development (glezen et al., ) . this section reviews data from clinical trials of inactivated, live, and vectored hpiv vaccines. the first approach taken to hpiv vaccination was the use of formalin-inactivated piv (fi-piv ) in an intramuscular vaccine. although vaccine-induced disease enhancement seen with formalin-inactivated rsv was not observed with fi-piv , there was no evidence of protection either (kim et al., ) . subsequent vaccine efforts, therefore, focused on the generation of live attenuated vaccines, and two approaches were investigated: the use of a ts hpiv strain and the use of a bovine piv strain. successive passage at lower temperatures has been shown to attenuate hpiv. the most promising vaccine candidate using this approach, cp , is designated by the number of times the virus was passaged at low temperature in african green monkey kidney cells (belshe and hissom, ; ray et al., ) . the vaccine was safe and immunogenic in adults, seropositive and seronegative children, and infants month of age or older (belshe et al., b) . in a phase clinical trial in -to -month-old children, the vaccine was well tolerated and % of previously seronegative vaccinees had a fourfold or greater rise in serum geometric mean antibody titer (belshe et al., b) . moreover, coadministration of an rsv and cp piv vaccine was successful, with little evidence of interference (belshe et al., a) . however, the cp piv vaccine was overattenuated in seropositive children and adults. bovine piv makes an attractive lav as it is known to be antigenically related to human piv, is attenuated in humans, and protects monkeys against challenge with hpiv . clinical trials were conducted in adults, seropositive children, seronegative infants, and children - months of age with residual maternal antibodies. in seropositive individuals, the vaccine was overattenuated, but in seronegative children and infants, the vaccine virus was highly infectious. despite replication of the vaccine virus, serum antibody responses to hpiv were low, consistent with an incomplete antigenic relatedness between the human and the bovine piv hn genes (greenberg et al., ; karron et al., ) . a phase clinical trial of children showed that seroconversion to bovine piv occurred in - % of vaccinated children after three doses, but that seroconversion to hpiv occurred in only - % of vaccinees (greenberg et al., ) . additionally, during the study, % of the placebo group experienced piv infection, highlighting the ubiquitous nature of the pathogen. after these studies, it was concluded that bovine piv might better serve as a backbone for the insertion of hpiv genes. as described above, at this writing, one such vaccine, with hpiv hn and f genes and the rsv f gene inserted into a bovine piv backbone, is being evaluated in clinical trials in seronegative children. this vaccine candidate was highly attenuated in seropositive children and adults. additional vaccine candidates include those generated by reverse genetics. one such vaccine contains mutations in the p/c gene, which reduces the virus's ability to inhibit type i ifn induction and signaling, and another contains ts and attenuating mutations from other paramyxoviruses (sato and wright, ) . by the age of years, most children will have been infected with hmpv. reinfections are common, and hmpv is responsible for - % of childhood hospitalizations for lrti. in addition, severe disease can also be seen in the elderly or immunocompromised individuals (feuillet et al., ) . despite this clinical need, hmpv vaccines have not yet entered clinical trials, and the data reviewed in this section are from studies in animal models. as with rsv, vaccination of macaques and mice with inactivated hmpv led to disease enhancement after challenge and a strong th -type immune response associated with a lack of neutralizing antibodies (herfst et al., a; hamelin et al., ) . therefore, alternative approaches have been evaluated, including soluble protein-based subunit vaccines, live attenuated vaccines, or dna vaccines encoding viral proteins. soluble f-protein subunit vaccines have been shown to generate high titers of neutralizing antibody in serum in golden syrian hamsters and cotton rats, associated with protection against subsequent infection (cseke et al., ; herfst et al., ) . however, stable and long-term immunity was not induced in monkeys (herfst et al., b) . live attenuated vaccines have been generated either by cold passage or by reverse genetics. temperature-sensitive viruses conferred complete protection against heterologous hmpv challenge in golden syrian hamsters (herfst et al., a) , and a recombinant hmpv lacking a region of the sh (Δ − sh), the g, or the m - protein induced high titers of neutralizing antibody and protected hamsters and african green monkeys against piv and hmpv challenge (biacchesi et al., ; buchholz et al., buchholz et al., , . a chimeric vaccine has also been developed in which the f subunit of the hmpv fusion protein is inserted into a backbone of bovine piv that also contains the hpiv f and hn genes (tang et al., ) . this b/hpiv /hmpv f vaccine induced neutralizing antibodies and protected hamsters and african green monkeys against piv and hmpv challenge (tang et al., . coronaviruses such as hcov- e and oc are frequent causes of respiratory illness throughout the world, and sars-cov and mers-cov represent significant public health threats. however, at present there are no licensed vaccines for human coronavirus infections, though a number of vaccines are licensed against animal coronaviruses, and several vaccine platforms have been developed for sars-cov that show great promise in preclinical studies. whole sars-cov particles inactivated with β-propiolactone, formalin, uv light, or a combination of two techniques have been evaluated in mice, ferrets, rabbits, and nonhuman primates. such vaccines have been administered by a variety of routes and tested in the presence or absence of various adjuvants. in general, vaccination elicited a serum igg response in animal models, with higher doses of vaccine eliciting higher igg antibody titers ( subbarao, ) . moreover, some studies demonstrated that inactivated sars-cov vaccines were efficacious in protecting mice (see et al., ; spruth et al., ; stadler et al., ) and nonhuman primates (qin et al., ; zhou et al., ) , although many studies did not investigate protective efficacy. in addition, an inactivated sars vaccine that was evaluated in clinical trials was safe and well tolerated and induced neutralizing antibodies (lin et al., ) . many subunit vaccines comprise purified, recombinant sars-cov s protein, as this protein is the target for protective neutralizing antibodies elicited by sars-cov infection. a truncated soluble form of the s protein that lacks the transmembrane domain also neutralized infectivity of sars-cov (bisht et al., ; he et al., b; zhou et al., ) and substantially reduced the titer of challenge virus replication in the respiratory tract of mice (bisht et al., ) . a trimeric spike protein vaccine (trispike) was also immunogenic in mice and hamsters and provided protection against challenge in hamsters, with reduced occurrence and severity of pneumonitis and no evidence of pulmonary consolidation or sars-cov-associated hepatic cellular necrosis (kam et al., ) . however, sera from animals immunized with trispike were associated with a -to -fold increase in virus entry into fcγrii-positive (ace -negative) human b cells, which has led to concerns about antibody-dependent enhancement of disease (kam et al., ) . vectored vaccines have been developed against sars-cov. in this approach, sars-cov proteins are expressed by venezuelan equine encephalitis virus replicon particles (vrps), chimeric bovine/human piv vectors, recombinant replication-defective human adenovirus- (ad- ), poxvirus mva, attenuated rabies viruses, or attenuated vesicular stomatitis virus (subbarao, ) . when animals were immunized with vectored vaccines expressing the s protein of sars-cov, neutralizing antibodies were induced, and vaccinated animals were protected from challenge. however, if other sars-cov proteins were expressed, animals were not protected from challenge. for example vrps expressing the sars-cov s protein provided protection from challenge in mice; however, vrp expressing the sars-cov n protein (vrp-n) did not (deming et al., ) . moreover, vrp-n vaccination resulted in a marked bronchiolitis, alveolitis, and interstitial accumulation of eosinophils and mononuclear leukocytes (deming et al., ) . in addition, chimeric bovine/ human (b/h) piv vectors expressing the sars-cov s protein or the s, m, and e proteins together also generated neutralizing antibodies in hamsters and protected animals from challenge, whereas b/h parainfluenza viruses expressing m, n, or e proteins were not protective . the route of administration is also important in determining protective efficacy of a vaccine. an ad- vector expressing the sars-cov s and n genes given i.m. had a limited effect in reducing pulmonary replication of challenge virus, despite eliciting higher serum igg titers and a greater cellular immunity than when the vaccine was given intranasally (i.n.). however, the vaccine administered i.n. induced higher mucosal iga responses than when given i.m. and provided protection from pulmonary replication, suggesting that mucosal immunity is important in mediating protection (see et al., ) . mice, rabbits, and monkeys immunized with a modified vaccinia virus mva-s vaccine were protected from replication of challenge virus (bisht et al., ; chen et al., ) . however, in ferrets, mva-s antibodies declined rapidly after vaccination, and the vaccine did not protect the animals from replication or spread of the challenge virus (weingartl et al., ) . a recombinant live attenuated sars-cov vaccine virus in which expression of the e protein was abrogated by point mutations and a deletion in the nucleotide sequence was restricted in vitro and attenuated in hamsters (dediego et al., ) . in addition, inactivation of the ′- ′ exonuclease attenuated sars-cov, and the resulting virus was protective in an aged immunocompromised mouse model of sars infection (graham et al., ) . dna vaccines expressing sars-cov s protein (either without the cytoplasmic domain or without the cytoplasmic and transmembrane domains) also induced humoral and cellular immunity in mice and protected animals from replication of challenge virus . in addition, priming with a dna vaccine and boosting with an inactivated vaccine resulted in an increase in cd + t cells and a stronger antibody response (zakhartchouk et al., ) . the dna vaccine was well tolerated and produced cellular immune responses and neutralizing antibody in a phase clinical trial (martin et al., ) . despite these successes in preclinical vaccine development, as the sars outbreak was declared over in , the impetus was not sustained long enough for many of these products to be evaluated in clinical trials. a cautionary note about sars-cov vaccines is the association of a variety of vaccine platforms with pulmonary immunopathology in mice after challenge with the virus, despite immunogenicity and protective efficacy (tseng et al., ) . the relevance of the rodent model to vaccines in humans remains uncertain. adenovirus epidemics have been described in adults, especially in military recruits in closed or crowded settings (lynch et al., ) . live attenuated, orally administered adenovirus vaccines against serotypes and , which cause respiratory disease, were used by the u.s. military starting in (top, ) . during this time the incidence of disease fell substantially. however, when the manufacturer ceased vaccine production in , epidemics reemerged in military facilities (lynch et al., ) . in , a new live attenuated adenovirus vaccine was issued to military recruits during basic training, and there has since been a reduction in the rate of febrile respiratory illness (armed forces health surveillance, ). the vaccine is not available to children or the civilian adult population at present, though adenoviruses are estimated to account for - % of pediatric and - % of adult respiratory tract infections (lynch et al., ) . in summary, the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because a large body of evidence indicates that, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. because respiratory pathogens infect the host at mucosal surfaces, the induction of mucosal immunity by vaccination is desirable. vaccines that are administered by intramuscular or subcutaneous injection induce protective immunity in the systemic immune compartments, but are generally poor at inducing mucosal immune responses. mucosally delivered vaccines, in contrast, induce both mucosal and systemic immunity and are also more readily accepted because they do not require needles or syringes (levine and dougan, ; yuki and kiyono, ) . ongoing research into the molecular and cellular mechanisms of surface immunological barrier systems has provided practical strategies for the development of a new mucosal vaccine for the control of respiratory viruses. m cells, located in the follicle-associated epithelium of the malt, play a pivotal role in the uptake of luminal antigens in the respiratory tract and the induction of antigen-specific immune responses in both the systemic and the mucosal compartments . it is, therefore, logical and attractive to develop m-cell-targeted mucosal vaccines. several molecules have been found to bind preferentially to m cells, such as ulex europaeus agglutinin- (uea- ), which has specificity for α( , )-fructose (sharma et al., ) , and rho-protein derived from reovirus, which binds to a carbohydrate structure containing α , -linked sialic acids on the plasma membranes of m cells (helander et al., ) . moreover, intranasal vaccines conjugated to either uea- (manocha et al., ) or rho-protein induced not only strong antigen-specific plasma igg and mucosal iga responses, but also ctl immunity (wu et al., ) . in addition, a novel m-cell-specific monoclonal antibody has recently been identified that selectively recognizes m cells, but not goblet cells or epithelial cells. this has been used as a carrier for an m-cell-targeted mucosal vaccine (wu et al., ; nochi et al., ) . concerns have been raised about the potential induction of unwanted mucosal inflammation associated with m cell targeting (kuolee and chen, ) , and additional research is needed to better understand how m cells sample antigens and transcytose them to the basolateral membrane. however, most microparticles administered orally become trapped in the mucus, and only a small fraction of them enter mucosal inductive sites. new approaches that are being developed to transitionally or conditionally enhance the number and function of m cells (neutra and kozlowski, ) are likely to reduce these concerns. there is substantial interest in the exploitation of nanoparticle technology for drug and vaccine delivery. nanoparticles are solid particles ranging in size from to nm in diameter that are often made from biodegradable materials. an antigen payload can be dissolved, entrapped, adsorbed, attached, or encapsulated into the matrix of the particle and released as the particle degrades over a period of time, which may vary from days to weeks, depending on the formulation (adair, ) . several types of nanoparticles have been investigated for vaccine delivery and have proven to be safe, nontoxic, and suitable for loading with antigens. these include the polyesters (poly(lactic acid), poly(glycolic acid), and their copolymers), polyorthoesters, polyanhydrides, and polycarbonates (reddy et al., ) . these materials can protect antigens from degradation, and the particles can be prepared in a chemically reproducible manner within a narrow size limit. in addition, some biopolymers exhibit a natural adjuvant behavior, for example, poly(lactide co-glycolide) (plga) appears to activate the maturation of dcs, possibly by providing a necessary stimulatory signal, though the exact mechanism is not fully elucidated (bennewitz and babensee, ; yoshida and babensee, ) . surface modifications of nanoparticles that change their overall charge and hydrophobicity are aimed at improving mucoadherence properties (des rieux et al., ) . this can be achieved by coating with stabilizers, other polymers, or surfactants. polyethylene glycol has been used for its stabilizing properties and because it can enhance the affinity of the nanoparticles for mucosal surfaces. molecules such as lectins and chitosan can also increase interaction with and transport across the mucosal surface. interestingly, chitosan is reported to be able to open tight junctions between epithelial cells, facilitating the transport of encapsulated macromolecules across the epithelial layer . nanoparticles coated with mannose (mannan) have also been produced with the aim of targeting mannose receptors on apcs, thus improving cell adhesion and uptake (cui and mumper, ) . a plga-coated nanoparticle vaccine encapsulating influenza virus proteins has been developed. the influenza ha protein retained its antigenicity after encapsulation, and mice vaccinated with plga particles of . - . μm in diameter mounted both systemic and mucosal responses, which were protective against intranasal challenge with an h n influenza virus (amidi et al., ; moldoveanu et al., ) . chitosan-coated nanoparticles containing purified influenza virus have also been tested in human volunteers, in which a fourfold or greater increase in anti-ha antibodies was observed in > % of the volunteers . moreover, a single dose of the intranasal vaccine resulted in high titers of nasal iga antibody and strong systemic antigen-specific responses, which were greater than those induced after intramuscular inoculation with soluble influenza antigen (amidi et al., ) . nanoparticleconjugated rsv proteins have also been shown to generate strong systemic th immunity in mice, associated with protection against wild-type rsv challenge (kalkanidis et al., ; xiang et al., ) . in addition, plgacoated nanoparticles incorporating hpiv hn and f glycoproteins have been shown to induce virus-neutralizing antibodies, which were protective against challenge infection in hamsters (ray et al., ) . studies in animal models and humans have shown that the choice of adjuvant can dramatically affect the immediate immune response and long-term protective effect of a vaccine (ogra et al., ; galli et al., ) . in addition, the quality of the immune response, especially the development of high-affinity b cells, long-lived memory b cells, and plasma cells can be influenced by the choice of adjuvant (galli et al., ) . although the mechanisms have not yet been fully elucidated, mucosal adjuvants can be broadly classified into two categories: those that act as immunostimulatory molecules and those that facilitate vaccine delivery (o'hagan, ) . the former include adjuvants that are toxin-based or cytokine-based and molecules associated with innate immunity, for example, pamps. the latter contain immune-stimulating complexes, liposomes, live attenuated vectors, and chitosan (discussed above). from a mechanistic point of view, mucosal adjuvants modulate innate immune responses in the same way as parenterally administered vaccines (lambrecht et al., ) , and tlr agonists constitute a major category of mucosal adjuvants. these adjuvants are based on pamps and are often formulated as oil-inwater emulsions. virosomes are virus-like particles that have been investigated for their potential as vaccines bungener et al., ) . they closely resemble native virus; however, they are nonreplicating and consist of reconstituted viral envelopes, generated by treatment with a detergent. they have a diameter of - nm and, as such, fall into the size range of small particles. virosomal influenza vaccines are available commercially and have been shown to induce ha-specific antibodies after i.m. administration. phase clinical trials revealed the tolerability and immunogenicity of a matrix tm -adjuvanted virosomal h n dna vaccine (cox et al., ) . influenza virosomes that incorporate the rsv-fusion protein have also been constructed and have been shown to generate virus-specific iga in the urt and lrt after i.n. administration with adjuvant in mice (cusi et al., ) . immune stimulating complexes (iscoms) are nonreplicating particles of ∼ nm diameter, comprising viral glycoproteins complexed with saponin derived from the bark of the tree quillaia saponaria (gregory et al., ) . iscom antigens from a number of microorganisms, including viruses, bacteria, and parasites, have been shown to induce humoral and cell-mediated immunity; th immune responses appear to predominate . extensive studies have been carried out with influenza virus iscoms in several species, including mice and monkeys, and an equine influenza iscom vaccine is available commercially (mumford et al., ). an influenza iscom vaccine (sundquist et al., ) stimulated high levels of virus-specific igm and igg serum antibodies and proliferative t cell responses in macaques, and the animals were completely protected from intratracheal challenge with the virus (rimmelzwaan et al., ) . intranasal immunization of mice with rsv iscoms induced very high levels of long-lasting rsv-specific iga in both the urt and the lrt (hu et al., ) . bovine rsv iscoms inoculated subcutaneously into calves were completely protective against challenge with virulent bovine rsv, whereas calves immunized with a conventional bovine rsv vaccine developed moderate to severe respiratory disease after challenge (hagglund et al., ) . the iscom-vaccinated animals developed high titers of nasal and serum virus-specific igg as well as serum iga, which correlated with protection. detoxified derivatives of cholera toxin (ct) and heat-labile enterotoxin (lt) produced by enterotoxigenic escherichia coli are effective mucosal adjuvants that promote mucosal and systemic immunity to coadministered protein antigens via oral or nasal routes (freytag and clements, ; mestecky et al., ) . the results of murine studies demonstrated that both ct and lt induced the expression of b - and/or b - on apcs that deliver costimulatory signals to cd + t cells (freytag and clements, ; mestecky et al., ) , but ct acts in the presence of il- to induce predominantly th responses, whereas lt supports th responses with ifn-γ production. recent vaccine development efforts have focused on the nasal administration of antigen with ct and lt derivatives for the induction of mucosal iga (byun et al., ; fujihashi et al., ) . both ct and lt are ab -type molecules, consisting of one a subunit and five b subunits. chimeric molecules, made by the spontaneous association of the a subunit of ct with the b subunit of the lt, or vice versa, are effective mucosal adjuvants for protein vaccines. the type of t helper responses induced is dictated by the origin of the b subunit (takahashi et al., ) . one such example is the nasal administration of an influenza ha subunit vaccine together with a chimeric mutant (m) ct-a/lt-b adjuvant. in murine studies, this adjuvanted vaccine protected animals from influenza virus challenge (kweon et al., ) . however, there are some concerns about the safety profile of toxin-derived adjuvants. nasal administration of mct-a/lt-b targeted neuronal tissues, though it did not affect trafficking of coadministered vaccine antigens into the neuronal tissues (kweon et al., ) . taken together, these findings suggest that although recombinant chimeric toxin-based molecules show promise as a new generation of mucosal adjuvants, the safety of the ct adjuvant may need to be improved. the inactivated lt-adjuvanted intranasal influenza vaccine (nasalflu) was withdrawn because of concomitant facial nerve paralysis (bell's palsy) that was noted as a potential adverse event caused by the ct adjuvant kunzi et al., ; garner-spitzer et al., ). several cytokines associated with innate immunity and inflammation support the generation of antigen-specific s-iga and serum igg/iga and may be of use as mucosal adjuvants. mice nasally immunized with soluble antigens, including ovalbumin or tetanus toxoid (tt) plus il- α and il- β developed antigen-specific antibody responses that were similar to those induced by coadministered ct adjuvant. the cytokines il- α and il- β promoted antigen-specific igg antibody responses initially, followed by igg b, with minimal igg a antibody responses, a pattern associated with a predominantly th -type response (staats and ennis, ; thompson and staats, ) . furthermore, levels of antigen-specific s-iga antibody similar to those induced by ct were found in mucosal secretions of mice that received nasal il- . these results indicate that il- could be a useful mucosal adjuvant. apcs are known to contribute to the creation of an appropriate cytokine environment for the growth and development of th or th responses. two well-known cytokines secreted by apcs, il- and il- , can influence the outcome of th and th cell subset-mediated immune responses (vajdy et al., ; rincon et al., ) . when tt was used as an antigen, nasal coadministration of il- or il- induced antigen-specific serum igg antibody responses and promoted protective immunity against lethal challenge with tetanus toxin. interestingly, whereas nasal treatment with il- promoted mixed th /th -type responses, il- supported predominantly th -type responses. in addition, il- but not il- can induce antigen-specific mucosal iga antibody (boyaka et al., ) . these findings suggest that il- could be a potent mucosal cytokine for the upregulation of antigen-specific mucosal immune responses. type i ifn affects the differentiation and function of immune cells, including t cells and dcs, and efficiently enhances a primary antibody response (marrack et al., ; luft et al., ) . it was reported that type i ifn was effective as both a systemic and a mucosal adjuvant, promoting th -type immune responses (proietti et al., ) . nasal administration of influenza vaccine with type i ifn was effective at inducing serum antigen-specific igg a and mucosal iga antibody responses and at providing full protection against influenza virus challenge (proietti et al., ) . it is well known that i.n. vaccination stimulates immune responses in the nalt and is effective at inducing mucosal immunity in the respiratory tract (brandtzaeg, ; jabbal-gill, ) . intranasal administration of the live attenuated influenza vaccine flumist (medimmune) has proven effective in protection against seasonal influenza virus infection and protects children against drifted influenza virus strains (belshe et al., a; mendelman et al., ) . furthermore, owing to the development of novel technologies, both aerosol spray and droplet delivery of vaccines are attractive possibilities (jabbal-gill, ) . in addition to the i.n. route of vaccine inoculation, the sublingual route has been explored (shim et al., ; czerkinsky et al., ) . the sublingual epithelium harbors a dense lattice of dcs, and vaccine delivered via this route stimulates broad and disseminated mucosal and systemic immune responses, similar to intranasal inoculation . sublingual vaccination with soluble or particulate antigens promotes strong mucosal iga and systemic igg antibody responses as well as cd + t cell responses. overall, sublingual immunization was comparable to nasal immunization in the magnitude, breadth, and anatomic dissemination of the induced immune responses. importantly, sublingual administration did not redirect antigens and/or adjuvants to the brain . sublingual vaccination against influenza has been shown to protect mice against lung infection (song et al., ) . in addition, sublingual administration of inactivated influenza a/pr (h n ) vaccine virus together with a mucosal adjuvant such as ct (cuburu et al., ) or a nontoxic mct-a/lt-b adjuvant induced both systemic and mucosal virus-specific antibody responses as well as ctl responses with protective immunity after respiratory challenge with the a/pr virus (kweon et al., ; song et al., ) . these studies demonstrated that sublingual administration of an inactivated influenza virus with a toxin adjuvant such as ct did not migrate to or replicate in the central nervous system. moreover, using mucosal adjuvant such as ct mobilizes dcs within the sublingual epithelium. these observations suggest that sublingual immunization may be another attractive and safe mucosal route for the administration of respiratory virus vaccines. given the global burden of respiratory tract infection, there remains an unmet need for effective methods of intervention. the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. systemic immune responses can be protective in the absence of mucosal immunity if they are sufficiently robust, such as that induced by i.m. inactivated influenza vaccines; however, many vaccines in development for respiratory viruses are live attenuated viruses that are mucosally administered. although live vaccines show promise, our understanding of the mechanism of protection at mucosal surfaces is incomplete, and there is currently a lack of adequate immune correlates of protection for these vaccines. in addition, immune responses mounted to a natural infection are often not sufficient to prevent reinfection and may also be involved in the pathogenesis of disease. achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of an immature immune system 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isolation of a novel coronavirus from a man with pneumonia in saudi arabia immunogenicity, safety, and protective efficacy of an inactivated sarsassociated coronavirus vaccine in rhesus monkeys a recombinant baculovirus-expressed s glycoprotein vaccine elicits high titers of sars-associated coronavirus (sars-cov) neutralizing antibodies in mice key: cord- -fzijbney authors: nemoto, manabu; oue, yasuhiro; higuchi, tohru; kinoshita, yuta; bannai, hiroshi; tsujimura, koji; yamanaka, takashi; kondo, takashi title: low prevalence of equine coronavirus in foals in the largest thoroughbred horse breeding region of japan, – date: - - journal: acta vet scand doi: . /s - - - sha: doc_id: cord_uid: fzijbney background: equine coronavirus (ecov) is considered to be a diarrheic pathogen in foals. in central kentucky in the united states, it has been shown that approximately % of thoroughbred foals are infected with ecov and thus it is considered widely prevalent. in contrast, the epidemiology of ecov and its relationship to diarrhea in foals are poorly understood in japan. we investigated ecov in rectal swabs collected from thoroughbred foals in japan. results: we collected rectal swabs from diarrheic foals in the hidaka district of hokkaido, the largest thoroughbred horse breeding region in japan, between and . in addition, rectal swabs were collected from healthy foals in . these samples were tested by reverse transcription loop-mediated isothermal amplification and a real-time reverse transcription-polymerase chain reaction. all samples collected from diarrheic foals were negative, and only three samples ( . %) collected from healthy foals were positive for ecov. compared with central kentucky, ecov is not prevalent among thoroughbred foals in the hidaka district of hokkaido. conclusion: ecov is not prevalent and was not related to diarrhea in thoroughbred foals in the hidaka district of hokkaido between and . certain pathogens are common causes of diarrhea in foals, equine rotavirus being the most common [ ] . equine coronavirus (ecov) is also a diarrheic pathogen in foals [ ] . recently, several ecov outbreaks in adult horses occurred in the united states [ ] and japan [ ] [ ] [ ] . fever, anorexia, lethargy, leukopenia and digestive disorders were observed, and these clinical signs were reproduced in an experimental challenge study [ ] . equine coronavirus was also detected in fecal samples of diarrheic foals in the united states [ , ] , but there have been no reports of an ecov outbreak in foals. slovis et al. [ ] reported that approximately % of healthy and diarrheic thoroughbred foals in central kentucky in the united states were infected with ecov, using a real-time reverse transcription-polymerase chain reaction (rt-pcr) assay. these results indicate that ecov is prevalent among thoroughbred foals in central kentucky. in japan, ecov outbreaks had previously occurred only in the draft racehorse population. draft racehorses include french percheron, breton and belgian horses, with body weights around kg. outbreaks of ecov had not been reported in the japanese thoroughbred horse population, and its epidemiology is poorly understood. it is also unclear whether ecov is related to diarrhea in japanese foals. therefore, we investigated ecov using molecular diagnostic methods on rectal swabs collected from thoroughbred foals in the hidaka district open access *correspondence: nemoto_manabu@equinst.go.jp epizootic research center, equine research institute, japan racing association, - shiba, shimotsuke, tochigi - , japan full list of author information is available at the end of the article of hokkaido, which is the largest thoroughbred horse breeding region in japan. between and , we collected rectal swabs from diarrheic foals aged days to months in the hidaka district of hokkaido by using bd bbl cul-tureswab ez (becton, dickinson and company, fukushima, japan). rectal swabs of twenty-two and four diarrheic foals were collected twice and three times, respectively. multiple samples were collected with - days intervals (average . days). we collected samples from farms in , samples from farms in and samples from farms in . rectal swabs from diarrheic foals were stored at − °c in the veterinary clinic until transport. they were transported to the diagnostic laboratory at around − °c, and after arrival they were kept at − °c until use. additionally, rectal swabs were collected from healthy foals on farms in the same region in by using bd bbl cultureswab plus (becton, dickinson and company). healthy foals were between and days of age. rectal swabs from healthy foals were stored and transported at °c for several days. after arrival, they were kept at − °c until use. these samples were immersed in maintenance medium [ ] or phosphate buffered saline. viral rna was extracted from samples with magna pure lc total nucleic acid isolation kit (roche diagnostics, mannheim, germany). reverse transcription loop-mediated isothermal amplification (rt-lamp) and real-time rt-pcr assays were selected because these molecular methods have high sensitivity for ecov [ ] . the rt-lamp reaction was performed using a primer set described previously [ ] and a loopamp rna amplification kit (eiken chemical, tokyo, japan) according to the manufacturer's instructions. calcein, a fluorescent detection reagent (eiken chemical, tokyo, japan), was added to the reaction mixture for visual detection. the mixtures were incubated at °c for min and then heated at °c for min to terminate the reaction. real-time rt-pcr assay was conducted using a primer set described previously (ecov- f, ecov- r and ecov- p) [ ] and taqman fast virus -step master mix (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. thermal cycling was performed as [ ] : °c for min and °c for s, followed by cycles at °c for s and °c for s. for sequence and phylogenetic analysis of the nucleocapsid (n) gene, rt-pcr was performed using the primer set ecov-nf ( ′-tcaggcatggacaccgcattgtt- ′) and ecov-nr ( ′-ccaggtgccgacataaggttcat- ′) [ ] using pri-mescript ii high fidelity one step rt-pcr kit (takara bio, otsu, japan). rt-pcr products were directly sequenced commercially by fasmac (atsugi, japan). sequence analysis was performed using the blast and clustalw programs, and vector nti advance software (invitrogen, carlsbad, ca, usa). phylogenetic analysis of nucleotide sequences was conducted with mega software version . [ ] . a phylogenetic tree was constructed based on nucleotide sequences using the neighbor-joining method. statistical analysis of the tree was performed with the bootstrap test ( replicates) for multiple alignments. [ , ] , obi-hiro [ ] , tokachi [ ] and obihiro - [ ] phylogenetic analysis was performed for the nucleotide sequences of the n gene (fig. ) . phylogenetic analysis showed that hidaka-no. / and hidaka-no. / are closely related to the obihiro - and tokachi strains, respectively. in this study, all diarrheic samples were negative for ecov. this indicates that ecov is not a causative agent of diarrhea in thoroughbred foals in hidaka district of hokkaido. using rectal swabs collected from healthy foals, only three samples ( . %) were positive for ecov. rectal swabs from healthy foals preserved at worse condition than that of diarrheic foals as described above. however, ecov was only detected in rectal swabs from healthy foals, and therefore it is unclear whether storage condition influenced the result in this study. compared with central kentucky, ecov is not prevalent among thoroughbred foals in hidaka district of hokkaido, but some outbreaks have occurred in draft racehorses. one reason for the lack of ecov positive samples in thoroughbred foals may be that they do not usually have contact with draft horses. these results suggest that the prevalence of ecov varies greatly depending on region. to further show this, it is necessary to investigate ecov in other countries as well. in sequence and phylogenetic analyses of the n gene, there is little difference between hidaka-no. / and obihiro - . obihiro - was isolated in the ecov outbreak in march [ ] , and therefore the hidaka in conclusion, this study shows that ecov is not prevalent and was not related to diarrhea in thoroughbred foals in the hidaka district of hokkaido between and . infectious agents detected in the feces of diarrheic foals: a retrospective study of cases viral diarrhea emerging outbreaks associated with equine coronavirus in adult horses prevalence of disease with inference of equine coronavirus infection among horses stabled in a draft-horse racecourse isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc strain epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in experimental inoculation of equine coronavirus into japanese draft horses neonatal enterocolitis associated with coronavirus infection in a foal: a case report characterization of a coronavirus isolated from a diarrheic foal infectious agents associated with diarrhoea in neonatal foals in central kentucky: a comprehensive molecular study virucidal effect of commercially available disinfectants on equine group a rotavirus rapid detection of equine coronavirus by reverse transcription loopmediated isothermal amplification mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods balasuriya ub. genomic characterization of equine coronavirus submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we are grateful to all of the equine practitioners of the hidaka agriculture mutual aid association for collecting rectal swabs and to mr. akira kokubun, ms. kazue arakawa, ms. akiko suganuma and ms. kaoru makabe for invaluable technical assistance. mn outlined the design of the study, performed the experiments and drafted the manuscript. yo participated in the design of the study and interpretation of the data. th and yk carried out the clinical observations of horses and the sample collections. hb, kt, ty and tk participated in interpretation of the data and helped to draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -o s nw authors: furuse, yuki; okamoto, michiko; oshitani, hitoshi title: conservation of nucleotide sequences for molecular diagnosis of middle east respiratory syndrome coronavirus, date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: o s nw infection due to the middle east respiratory syndrome coronavirus (mers-cov) is widespread. the present study was performed to assess the protocols used for the molecular diagnosis of mers-cov by analyzing the nucleotide sequences of viruses detected between and , including sequences from the large outbreak in eastern asia in . although the diagnostic protocols were established only years ago, mismatches between the sequences of primers/probes and viruses were found for several of the assays. such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. a slight modification in the primer design is suggested. protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as mers-cov. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus with a positive-sense rna genome. infection with the virus causes severe respiratory symptoms in humans, with a case fatality rate as high as %. camels may be a source of infection to humans. human-to-human transmission is also possible, but this requires close contact, such as health care-related contact without proper measures for infection control and prevention. the earliest case of mers was reported in jordan, and mers-cov was subsequently isolated from cases in saudi arabia only a short time later. since then, infections have been endemic mainly in the middle east. however, mers-cov has spread sporadically to other areas, including europe, north america, africa, and southeast and east asia, by travelers from the middle east. the laboratory diagnosis of mers-cov infection is mainly performed using real-time reverse transcription pcr (rt-pcr) to detect viral rna in specimens. interim recommendations from the world health organization (who) in for the laboratory testing of mers-cov included protocols for rt-pcr that were developed by the university hospital bonn and the us centers for disease control and prevention. [ ] [ ] [ ] [ ] this document included seven assays: ( ) the upe assay, which is considered highly sensitive and is recommended for screening, ( ) the orf a assay, which is considered equally as sensitive as the upe assay, ( ) the orf b assay, which is considered less sensitive than the orf a assay, , and the ( ) n and ( ) n assays, which can complement upe and orf a assays for screening and confirmation. , to date, these assays have shown no cross-reactivity with other human coronaviruses. [ ] [ ] [ ] sequencing protocols for further confirmation, namely the ( ) rdrpseq and ( ) nseq assays, were also developed. because mers-cov is an rna virus that can evolve rapidly, there remains concern that these protocols may not be suitable for the detection of current mers-cov because of a mismatch among sequences in the primer/probe regions. this study was performed to analyze recent viral genomic nucleic acid sequences and to discuss the efficacy of the rt-pcr protocols for the molecular diagnosis of mers-cov infections. data for these sequences, including complete as well as partial genome sequences, were obtained and analyzed. sequence data were aligned with clustalw to assess genetic changes in the nucleotide sequences of the primer and probe regions of the assays described above. the numbers of viral sequences that matched the primer/ probe sequences perfectly were counted. as mentioned in the introduction above, the upe, orf a, n , and n assays can be used for screening because of their high sensitivity. [ ] [ ] [ ] [ ] among these, only the primer and probe designs of the orf a assay showed % conservation of all sequence data available today (table ) . minor mismatches were found for the upe assay (one nucleotide substitution in two sequences) and n assay (one nucleotide substitution in one sequence), and significant mismatches were found for the n assay. the primer/probe regions were found to be well conserved, except for the n assay. in addition, mismatches were not found in the end region of primers for the upe and n assays ( table ). the sensitivity of the assays may not be greatly affected. no mismatches were found for the orf b assay. with regard to the sequencing assays, no sequence data that matched the sequence of the reverse primer for the rdrpseq assay was found. however, a single common mismatch in all sequence data was found. when the mismatched nucleotide was corrected, the rdrpseq assay matched all the sequence data perfectly ('corrected reverse primer', table ). in addition, viral sequences of the reverse primer region for the nseq assay were not highly conserved; the sequence matched only % of strains. based on these results, the use of a modified reverse primer for the assay is suggested, in order to reduce the possibility of a mismatch ('modified reverse primer', table ). several mismatches among viral sequences in the primer/probe regions for molecular diagnosis were identified in this study. such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. the mismatched sequence data could have been generated by errors in pcr or sequencing during viral nucleotide sequence analysis because of the incorporation of the wrong nucleotide. however, it is more likely that the rna virus has evolved and that this has accidentally resulted in the induction of mutation/s in the region targeted by the primer/probe for rt-pcr, only years after the establishment of the protocols. fortunately, no or few mismatches were found for most of the mers-cov screening assays. nevertheless, protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as mers-cov. middle east respiratory syndrome coronavirus (mers-cov) evidence for camel-to-human transmission of mers coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): situation update and cases reported in the netherlands. who assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus world health organization. laboratory testing for middle east respiratory syndrome coronavirus-interim guidance (revised). who fidelity of dna polymerases in dna amplification this research was supported by the japan initiative for global research network on infectious diseases (j-grid) from the japan agency for medical research and development, amed. the funding source had no involvement in the study design, in the collection, analysis, and interpretation of the data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.conflict of interest: all authors declare no conflicts of interest. table conservation of the primer and probe region sequences of the who-recommended assays for the molecular diagnosis of mers-cov key: cord- -rg gcc authors: aoyagi, yumiko; beck, charles r; dingwall, robert; nguyen-van-tam, jonathan s title: healthcare workers' willingness to work during an influenza pandemic: a systematic review and meta-analysis date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: rg gcc to estimate the proportion of healthcare workers (hcws) willing to work during an influenza pandemic and identify associated risk factors, we undertook a systematic review and meta-analysis compliant with prisma guidance. databases and grey literature were searched to april , and records were screened against protocol eligibility criteria. data extraction and risk of bias assessments were undertaken using a piloted form. random-effects meta-analyses estimated (i) pooled proportion of hcws willing to work and (ii) pooled odds ratios of risk factors associated with willingness to work. heterogeneity was quantified using the i( ) statistic, and publication bias was assessed using funnel plots and egger's test. data were synthesized narratively where meta-analyses were not possible. forty-three studies met our inclusion criteria. meta-analysis of the proportion of hcws willing to work was abandoned due to excessive heterogeneity (i( ) = · %). narrative synthesis showed study estimates ranged from · % to · % willingness to work, depending on context. meta-analyses of specific factors showed that male hcws, physicians and nurses, full-time employment, perceived personal safety, awareness of pandemic risk and clinical knowledge of influenza pandemics, role-specific knowledge, pandemic response training, and confidence in personal skills were statistically significantly associated with increased willingness. childcare obligations were significantly associated with decreased willingness. hcws' willingness to work during an influenza pandemic was moderately high, albeit highly variable. numerous risk factors showed a statistically significant association with willingness to work despite significant heterogeneity between studies. none of the included studies were based on appropriate theoretical constructs of population behaviour. although variable in severity, , one consistent feature of pandemic influenza is a surge in demand for health care. , hospitalization due to influenza a(h n )pdm in the usa was estimated at approximately cases between april and april contrasting with annual influenza-associated primary hospitalizations from to . in - , the availability of intensive care unit beds came under pressure in most national health systems. , healthcare workers (hcws) play key roles during an influenza pandemic, but a serious shortage of personnel may occur at peak times or in severe pandemics because of absenteeism due to illness, caring for family members who are ill, or refusal to work. effective preparation for the next pandemic requires estimates of hcws' willingness to work and an understanding of influencing factors. the available data are highly variable. one nigerian study found only one quarter of hcws stating they would be willing to work in a unit treating patients with influenza a(h n )pdm , whilst an australian qualitative study of family physicians found % of participants willing to work. chaffee first reviewed willingness to work during disasters and reported that the following factors would be influential: type of disaster, concern for close family, friends and pets, responsibility for dependants, the perceived value of one's response, belief in a duty of care, access to personal protective equipment (ppe), provision of basic needs (water, food, rest, shelter and communication tools) and prolonged working hours. three published reviews reported that similar factors would be associated with willingness to work during an influenza pandemic, [ ] [ ] [ ] but the data were not summarized quantitatively. we addressed this evidence gap by conducting a systematic review and meta-analysis in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) statement. the review questions sought to elucidate the proportion of hcws willing to work during an influenza pandemic, and to identify risk factors associated with willingness to work. our findings are interpreted with reference to sociological understandings of population behaviour, which have to date largely been absent from the peer-reviewed literature, but are highly relevant to the development of appropriate interventions to minimize refusal to work. the study protocol was registered with the national institute for health research international prospective register of scientific reviews (prospero; #crd ) prior to executing the literature search strategy. the prisma checklist is available as supporting information. we sought to analyse data collected exclusively from hcws including doctors, nurses, hospital workers, emergency healthcare service workers, public health workers, medical and nursing students, non-clinical support staff and retirees. the outcome measures of interest were the proportion of hcws reporting willingness to work during an influenza pandemic, and odds ratios or case counts allowing the derivation of odds ratios pertaining to factors associated with willingness to work. we included study manuscripts written in english reporting original quantitative research derived from a cross-sectional design, studies pertaining to a prior or hypothetical influenza pandemic, and studies reporting data pertaining to the aforementioned outcome measures, with no limitations on the time and place of publication. the following databases were searched from their inception to april : medline, embase, web of knowledge, scopus, amed, assia, bioethicsweb, cinahl, cochrane library and psycinfo. google scholar and opengrey were also searched. search terms were 'pandemic + influenza + willingness to work/report to work' to avoid including studies on willingness to accept vaccination. these terms were used in both keyword and mesh searches as appropriate for each database as follows: # . pandemics (mesh); # . influenza, human (mesh); # . 'attitude of health personnel' (mesh) or willingness (keyword); # . hospital administration (mesh) or report to work (keyword); # . willing* adj work (keyword); # . respon* adj work (keyword); # . would come (keyword); # . # or # or # or # or # ; # . # and # and # (see also table s ). reference lists in eligible articles were also searched. all identified records were imported to endnote software x (thomson reuters, toronto, ca, usa) and duplicate entries removed. the remaining records were screened by a single researcher (ya) against the protocol eligibility criteria following a sequential assessment of the study title, abstract and full-text article. where this was unclear, agreement on eligibility of each study was achieved through discussion with a second researcher (rd or jsn-v-t). data extraction was performed by a single researcher (ya) using a piloted form collecting details of study characteristics {title, author, publication year, place, study period, study design, participants, subject [pandemic of avian influenza origin/influenza a(h n )pdm /non-specified, hypothetical influenza pandemic]}; definition of outcome measures; questionnaire type; validation; statistical analysis and any stated limitations; percentage of willingness to work; and risk factors association with willingness. odds ratios (ors) of factors both unadjusted and adjusted were extracted to estimate the association with willingness to work. crude case counts and the percentage of people in each risk factor stratum were extracted where available. risk of bias was assessed for each study using a newcastle-ottawa assessment scale modified for crosssectional studies by herzog et al. descriptive statistics were calculated using microsoft â office excel â (microsoft corporation, richmond, va, usa). random-effects meta-analysis estimated the proportion of hcws (including % confidence intervals [cis]) who reported willingness to work during an influenza pandemic. random-effect meta-analysis of pooled odds ratios (including % cis) estimated the association of factors with willingness to work. heterogeneity between studies was assessed using the i statistic. we considered it statistically inappropriate to perform meta-analysis where i exceeded %. to explore sources of heterogeneity, we planned to conduct subgroup analyses according to the type of influenza pandemic; geographical region; survey time period; type of questionnaire; type of participants; sex of participants; and newcastle-ottawa assessment scale score. we used galbraith plots to detect those studies that contributed substantial heterogeneity and conducted sensitivity analyses excluding them from our pooled estimates. for each meta-analysis, publication bias was assessed graphically using a funnel plot of effect size versus standard error and statistically using egger's regression test. meta-analysis of pooled proportions was conducted using statsdirect version . . (statsdirect ltd., cheshire, uk), and meta-analysis of pooled odds ratios was conducted using we identified a total of unique records of which studies met protocol eligibility criteria (see figure ). two the included studies comprised entirely of cross-sectional surveys including two pre-/post-intervention studies and are summarized in table . the participant population sizes ranged from to with a median of (interquartile range [iqr] - ). the earliest publication was in , and the majority of articles were published in ( ; Á %) and ( ; Á %). of ( Á %) studies used a hypothetical influenza pandemic as the subject, ( Á %) were conducted in the usa, and ( Á %) investigated both clinical and non-clinical staff within hospital settings. assessments using the modified newcastle-ottawa scale showed that of studies were at moderate risk of bias ( - of five stars) for the selection domain, whilst studies were at low risk ( - stars) and ten studies were at high risk ( - stars); many studies used convenience sampling and few justified the study sample size, appropriately considered nonresponders and used a validated measurement tool. for the comparability domain, were at high risk ( of two stars), eight at moderate risk (one star) and at low risk of bias (two stars). many studies did not clarify how statistical adjustment for confounding variables was carried out, or reported unadjusted estimates only. for the outcome domain, studies were at moderate risk of bias (two of three stars) and four were at high risk (one star). willingness to work was self-reported in all studies although the statistical test used was clearly described in only studies (see figure s ). the percentage of participants who expressed a willingness to work ranged from Á % (community nurses during the influenza a(h n )pdm pandemic in hong kong in ) to Á % (a study of us medical students targeting a hypothetical influenza pandemic). we abandoned metaanalysis to estimate a pooled mean proportion of hcws willing to work due to very high statistical heterogeneity between studies (i = Á %). our planned subgroup analyses were unable to adequately explain the sources of heterogeneity between studies as this remained above our threshold of % in each analysis. the percentage of willingness to work seemed to depend on the particular context of the study. studies of hypothetical influenza pandemics, which did not include detailed conditions such as virulence of the strain and availability of protective equipment, tended to show a high level of willingness to work. however, studies of precise scenarios or those which investigated willingness during the relatively mild influenza a (h n )pdm pandemic tended to present relatively low levels of willingness. this finding may correspond with earlier work by syrett et al. which showed that willingness factors associated with willingness to work data were extracted from studies. pooled estimates from meta-analyses of individual factors associated with willingness to work are summarized in table . overall, females were one-third less likely to be willing to work compared with males. by occupational group, physicians were most likely to be willing to work, followed by nurses, then other health workers. urban or metropolitan area workers were less likely to be willing to work than rural area workers. full-time workers were more likely to be willing to work than parttime employees. respondents living with children or having childcare obligations were one-third less likely to be willing to work compared with those without these obligations. one study identified that pregnancy in a family member reduced willingness to work. marital status (not meta-analysed) did not influence willingness to work. perceived personal safety at work and perception of pandemic risk (aware that a pandemic was likely) were both associated with increased willingness to work. likewise, the provision of protective measures (mainly personal protective equipment) increased willingness to work, although metaanalysis was abandoned due to high heterogeneity (i = Á %). training in pandemic preparedness, general and specific role knowledge, confidence in personal skills, good communication skills and perception of role importance all had positive effects on willingness to work. confidence in employers as judged by 'belief that the employer can provide timely information' also positively influenced willingness to work, although meta-analysis was abandoned due to high heterogeneity. the funnel plot of the percentage of hcws willing to work did not present a clear funnel shape, appeared to scatter widely without any detectable association with the standard error and overflowed the false % ci range. egger's regression test reached statistical significance and showed that studies reporting a lower percentage were more likely to be published (p = Á ). funnel plots and egger's regressions tests pertaining to meta-analyses of factors associated with willingness to work revealed no evidence of publication bias except for previous training and comparison of physicians and nurses (see table ), which suggested possible underreporting of studies with an adverse result. this study advances knowledge from previous reviews on willingness to work during influenza pandemics by adding further new studies and subjecting the findings to statistical evaluation where possible. the search was conducted comprehensively and yielded studies from countries. however, quality of the included studies was not uniformly high and excessive statistical heterogeneity prevented metaanalysis of the primary outcome measure. whilst it was not possible to identify a single clear source of the heterogeneity encountered, almost certainly the wide variation in settings, scenarios and respondents contributed significantly. metaanalysis suggested that sex and job category would affect willingness to work although studies varied greatly in the composition of their samples. hypothetical scenarios varied in virulence, stage and the amount of information provided to respondents. studies of influenza a(h n )pdm were conducted at different junctures during the evolution of the - pandemic. there was no consistency in terms of how respondents were asked about their willingness to work, and the design of questionnaires used to collect outcome data from respondents varied between studies. remarkably, despite such high heterogeneity, some factors emerged showing a consistent association with willingness to work. whilst previous reviews suggested these from a narrative approach, this study has confirmed them statistically. being male, a physician or nurse (especially the former), and a full-time worker were all positively associated with willingness to work. these factors are essentially nonmodifiable; without access to the raw data, we could not disentangle any potential confounding between being male and the likelihood of being a physician or full-time worker in studies providing only unadjusted ors. nevertheless these were consistent findings across most studies and firm knowledge that these are reliable and statistically proven influencers of willingness to work is important information for both policy makers and healthcare service managers, even though they are difficult factors to influence. childcare obligation was a consistent barrier to hcws' willingness to work. the importance of this factor may be an artefact of the high participation of women in the hcw workforce in most countries, combined with traditional cultural expectations that they will take primary responsibility for childcare. it is, nevertheless, an important finding for managers. it is not clear whether this is driven mainly by practicality, that is the need to provide childcare at home, or by concerns about whether the safety of children might be compromised by infection brought in from the parental workplace. paradoxically, the evidence that hcws are at increased risk of influenza infection is rather mixed and somewhat inconsistent, whereas the evidence that children (rather than adults) are usually the introducers of influenza infection into households is firmly established. this question should be further investigated because it has implications for appropriate organizational responses. if it is simply a practical matter, then managers need to consider what help could be given in emergencies through the expansion of onsite or community childcare provision. if it is a concern about cross-infection, then appropriate education and information programmes may resolve the problem. in either case, it is unlikely that simple disciplinary sanctions will be effective, because of the social force of parental obligations. indeed, these may well be counterproductive, if other workers perceive them to have been unreasonably applied by managers unsympathetic to real personal dilemmas. confidence in safety, risk perception, prior training, general and role knowledge and confidence in skills were statistically proven facilitators for willingness to work. these are all addressable by detailed pandemic preparedness educational activities at healthcare unit level. importantly, one message arising from assessments of pandemic planning activities prior to the - pandemic was that whilst national level pandemic planning was generally successful, the level of planning at local level was insufficient, including training on pandemic influenza for hcws. a particular feature of pandemics is the level of anxiety provoked by the disruption of 'business as usual' and the destabilization of usually stable organizational environments. whilst it is not necessary to retrain hcws frequently, this is a topic that should be addressed in their basic education and managers should ensure that updating materials are readily available, and regularly revised, so that programmes can rapidly be rolled out when a pandemic is identified. evidence of organizational preparedness will contribute to the confidence of hcws that they will not be placed at undue risk by being asked to work in different ways or in different environments from those that they are accustomed to. a number of limitations with the present study warrant discussion. our literature search was limited to records published in english. therefore, we cannot exclude the possibility of having omitted outcome data published in other languages. many of the included studies were at moderate or high risk of bias. moreover, only a small number were available for analysis in relation to some risk factors; these results should be interpreted cautiously. the possibility of publication bias might also be a limitation. however, considering that the percentage of willingness was relatively high in most studies, this suggests that unpublished data may not have found statistically significantly higher percentages of willingness to work. whilst some studies used questionnaires based on recognized psychological theories, these were commonly 'fear-appeal' theories. unfortunately, this may not be appropriate as the preferable behaviour (working during an influenza pandemic) would not result in release from personal fear. we did not identify any studies that investigated the interaction between individual and organizational responses, which biased the findings towards individual fears rather than the social conditions that might provoke or alleviate these. as important as our specific results themselves, is the fact that we identified a multiplicity of approaches to studying the issue of hcw willingness to work during a pandemic; mainly small, ad hoc enquiries, not based on any consistent scenarios or theoretical approaches. to solve this, a consistent methodological framework is needed before any further studies are undertaken. the outbreaks of ebola virus disease in west africa and mers-cov in the middle east offer two very different settings in which to improve study designs and understanding of hcws' willingness to work where infectious disease creates appreciable personal risk. in the meantime, policy makers should recognize that hcw willingness to work during an influenza pandemic is likely to be improved by practical measures to support childcare responsibilities and by the timely provision of relevant and high-quality training and information as a pandemic develops. whilst the above would hold true for influenza, the actual risks and perceptions are not consistent across all novel respiratory viruses. for example, % of nurses in ontario refused to work during the sars crisis when the risk to hcws was almost exclusively nosocomial (compared with pandemic influenza where the risk is community-wide). similarly, in the ongoing mers-cov epidemic, the risk of nosocomial infection is presently greater than in wider community settings. , conclusions hcws' willingness to work during an influenza pandemic is moderately high although highly variable, and substantial statistical heterogeneity precluded formal meta-analysis. numerous risk factors are associated with willingness of hcws to work during an influenza pandemic, revealing potential points of intervention to increase willingness to work. we identified a wide variety of approaches to the study of willingness to work. for improved future understanding, we advocate a coordinated global approach with standardized protocols and based on appropriate theoretical constructs; and the evaluation of packages of intervention through controlled studies. additional supporting information may be found in the online version of this article: table s . full electronic search strategy (medline). figure s . summary of risk of bias of included studies using the modified newcastle-ottawa scale (n = ). data s . prisma checklist. pandemic influenza preliminary estimates of mortality and years of life lost associated with the a/h n pandemic in the us and comparison with past influenza seasons world health organization. pandemic influenza preparedness and response: a who guidance document. geneva: world health organization america's forgotten pandemic estimating the burden of pandemic influenza a (h n ) in the united states influenza-associated hospitalizations in the united states complications among adults hospitalized with influenza: a comparison of seasonal influenza and the h n pandemic pandemic influenza and hospital resources pandemic ): how prepared are healthcare providers in calabar, nigeria? the gp's response to pandemic influenza: a qualitative study willingness of health care personnel to work in a disaster: an integrative review of the literature healthcare workers willingness to work during a pandemic factors associated with the willingness of health care personnel to work during an influenza public health emergency: an integrative review healthcare workers' willingness to report to work during an influenza pandemic: a systematic literature review an international registry of systematic-review protocols are healthcare workers' intentions to vaccinate related to their knowledge, beliefs and attitudes? a systematic review meta-analysis in clinical trials measuring inconsistency in meta-analyses influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective graphical display of estimates having differing standard errors bias in meta-analysis detected by a simple, graphical test will the community nurse continue to function during h n influenza pandemic: a cross-sectional study of hong kong community nurses? perspectives of future physicians on disaster medicine and public health preparedness: challenges of building a capable and sustainable auxiliary medical workforce will emergency health care providers respond to mass casualty incidents? how would australian hospital staff react to an avian influenza admission, or an influenza pandemic incidence of influenza in healthy adults and healthcare workers: a systematic review and metaanalysis estimating household and community transmission parameters for influenza who regional office for europe. recommendations for good practice in pandemic preparedness: identified through evaluation of the response to pandemic (h n ) . copenhagen: who regional office for europe introduction: why a sociology of pandemics? putting the fear back into fear appeals: the extended parallel process model spring of fear, chapter : the nurses' survey hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus gauging us emergency medical services workers' willingness to respond to pandemic influenza using a threat-and efficacy-based assessment framework assessment of local public health workers' willingness to respond to pandemic influenza through application of the extended parallel process model assessment of medical reserve corps volunteers' emergency response willingness using a threat-and efficacy-based model determinants of emergency response willingness in the local public health workforce by jurisdictional and scenario patterns: a cross-sectional survey nurses' ability and willingness to work during pandemic flu health care workers' ability and willingness to report to work during public health emergencies hospital health care workers' understanding of and attitudes toward pandemic influenza in beijing who will show up? estimating ability and willingness of essential hospital personnel to report to work in response to a disaster will they just pack up and leave?"-attitudes and intended behaviour of hospital health care workers during an influenza pandemic characterizing hospital workers' willingness to report to duty in an influenza pandemic through threat-and efficacy-based assessment ethical planning for an influenza pandemic survey study of the knowledge, attitudes, and expected behaviors of critical care clinicians regarding an influenza pandemic hospital disaster staffing: if you call, will they come? survey of alberta family physicians' reactions to the spring and summer phase of ph n (pandemic swine flu) outbreak willingness of university nursing students to volunteer during a pandemic preparing for an influenza pandemic: healthcare workers' opinions on working during a pandemic willingness of frontline health care workers to work during a public health emergency factors associated with the ability and willingness of essential workers to report to duty during a pandemic will the nhs continue to function in an influenza pandemic? a survey of healthcare workers in the west midlands healthcare workers' perceptions of the duty to work during an influenza pandemic evaluation of a pandemic preparedness training intervention of emergency medical services personnel pre-pandemic planning survey of healthcare workers at a tertiary care children's hospital: ethical and workforce issues anticipated behaviors of emergency prehospital medical care providers during an influenza pandemic assessing public health department employees' willingness to report to work during an influenza pandemic local public health workers' perceptions toward responding to an influenza pandemic senior clinical nurses effectively contribute to the pandemic influenza public health response pandemic-related ability and willingness in home healthcare workers mitigating absenteeism in hospital workers during a pandemic knowledge and anticipated behavior of health care workers in response to an outbreak of pandemic influenza in georgia a national survey of emergency nurses and avian influenza threat are belgian senior medical students ready to deliver basic medical care in case of a h n pandemic ensuring adequate human medical resources during an avian influenza a/h n pandemic nurses' fears and professional obligations concerning possible human-to-human avian flu survey of hospital healthcare personnel response during a potential avian influenza pandemic: will they come to work knowledge and attitudes of healthcare workers in chinese intensive care units regarding h n influenza pandemic perception, attitudes and knowledge regarding the swine-origin influenza a (h n ) virus pandemic among health-care workers in australia influenza vaccination and intention to receive the pandemic h n influenza vaccine among healthcare workers of british columbia, canada: a cross-sectional study nurses' perspectives and concerns towards an infectious disease epidemic in egypt influenza a h ni (pandemic ): how prepared are healthcare providers in calabar, nigeria? factors associated with motivation and hesitation to work among health professionals during a public crisis: a cross sectional study of hospital workers in japan during the pandemic (h n ) we thank the authors of the articles cited in this paper. we also thank nicola darlington (university of nottingham) for assistance with developing the search terms and john mair jenkins (health education east midlands) and roshni joshi (university of nottingham) for help with manuscript preparation. this research was supported by the university of nottingham as a master of public health dissertation project. jsn-v-t and crb are respectively editor-in-chief and associate editor for influenza and other respiratory viruses; however they played no role whatsoever in the editorial process for this paper, including decisions to send the manuscript for independent peer-review or about final acceptance of a revised version. all of the above functions were handled alone by dr john wood, senior editor (reviews). key: cord- - u otbf authors: vainionpää, r.; waris, m.; leinikki, p. title: diagnostic techniques: serological and molecular approaches date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: u otbf virus laboratory diagnostics has an increasingly important role in modern patient care. virological methods are needed to investigate the etiology of acute viral infection or the reactivation of a latent infection, as well as to follow virus load in antiviral treatments. serological assays are also used for screening of blood products for the risk of certain chronic infections, evaluation of the immune status, and need for prophylactic treatments in connection with organ transplantations. for diagnostic purposes the following approaches can be used: demonstration of presence of infectious virus or its structural components directly from a patient's specimens or investigation of specific antibody response in serum specimens. amplification techniques, most commonly polymerase chain reaction (pcr) is currently the workhorse of nucleic acid testing for the detection and quantitation of virus genomes. virus isolation is used to demonstrate infectious virus in a patient's specimens, whereas virus antigens are investigated by antigen detection assays. serological diagnosis is based on either the demonstration of the presence of virus-specific igm antibodies or a significant increase in the levels and/or avidity of specific igg antibodies. immunoassays are the most commonly used serological assays. point-of-care tests (poc tests), for antigens, antibodies, and also nucleic acids are also becoming more and more common in diagnostic use. in order to reach the best diagnostic efficiency for each patient it is important to select the most suitable method using the right sample collected at the right time. glossary eia enzyme immunoassays are methods used to estimate virus-specific igg and igm antibodies or virus antigens by enzyme-labeled conjugates. pcr by the polymerase chain reaction (pcr) and with specific primers, dna sequences can be multiplied. rt-pcr for rna, nucleic acid has to be transcribed with reverse transcriptase (rt) enzyme to complementary dna prior to pcr. specific virus diagnostics can be used to determine the etiology of acute viral infection or the reactivation of a latent infection. two approaches can be used: demonstration of a specific antibody response or of the presence of the virus itself. nucleic acid testing has become the main approach for the demonstration of the presence of virus while cultivation is used by fewer specialized laboratories and antigen detection methods have moved to the point of care. serological methods are used for measuring the antibody response caused by an active infection. in this article, we briefly describe the principles of the most important serological methods and molecular applications that are used to provide information about the viral etiology of the clinical condition presumed to be caused by a viral infection. the diagram of the course of acute virus infection (figure ) indicates the optimal methods for viral diagnosis. following transmission, the virus starts to multiply and after an incubation period clinical symptoms appear with simultaneous shedding of infectious virus. the presence of infectious virus or viral structural components can be investigated directly from various clinical specimens either by nucleic acid detection assays, virus isolation, or antigen detection assays. irrespective of the method of direct virus detection, specimens collected at the site of symptoms give the most conclusive diagnosis. virus-specific antibodies appear somewhat later (from some days to weeks, called a window period). when the virus-specific antibody production reaches the level of detection, at first immunoglobulin m(igm) antibodies and some days later immunoglobulin g(igg) antibodies appear, and the amount of infectious virus starts to decrease. if this is the first encounter with this particular virus, that is, a primary immune response, igg antibody levels can stay at a relatively low level, whereas in a later contact with the same antigen, that is, in secondary response, igg levels increase rapidly and reach high levels while igm response may not be detectable at all. antibodies are usually investigated from serum samples taken at acute and convalescent phase of the infection. in selected cases other materials such as cerebrospinal fluid and other body fluids can also be analyzed. in order to reach the best diagnosis for each patient, it is important to select the most suitable method using the right sample collected at the right time. during most primary infections igm antibody levels peak at - days after the onset of illness and then start to decline, disappearing after some weeks or months. an igm response is usually not detected in reactivated infections or reinfections. the production of igg antibodies starts a few days after igm response and these antibodies often persist throughout life. serological diagnosis is usually based on either the demonstration of the presence of specific igm antibodies or a significant increase in the levels of specific igg antibodies between two consecutive samples taken - weeks apart. the antigen for the test can be either viable or inactivated virus or some of its components prepared by virological or molecular methods. isotype-specific markers or physical separation are used to demonstrate the isotype of the reacting antibody. in some cases, even igg subclass specificities are determined although they have limited value in diagnostic work. during the early phase of primary infection the specific avidity of igg antibodies is usually low but it increases during the maturation of the response. diagnostic applications of the measurement of the avidity of igg antibodies against specific antigens have been developed to help distinguish serological responses due to acute infections from those of chronic or past infections. serological assays are useful for many purposes. in primary infections they often provide information about the etiology even after the acute stage when infectious virus or its components can no longer be demonstrated in the samples. they are widely used for screening of blood products for the risk of certain chronic infections, evaluation of the immune status, and need for prophylactic treatments in connection with certain organ transplantations. serology may also be used to confirm an acute infection in cases when the virus can be asymptomatically present. they are also widely used for epidemiological studies, determination of vaccine-induced immunity, and other similar public health purposes. serological assays have their limitations. in some infections the antibody response is not strong enough or the limited specificity of the antigens used in the assay does not allow unambiguous interpretation of the results. in infections of newborns the presence of maternal antibodies may render the demonstration of the response in the baby impossible. in immunocompromised patients the serological response is often too weak to allow the demonstration of specific responses. in these cases other virological methods should be considered. other clinical specimens than sera can be used for antibody assays. igm and igg antibody determinations from cerebrospinal fluid are used for diagnosis of virus infections in the central nervous system although new molecular methods are increasingly replacing them. recently, increasing attention has been given to the use of noninvasive sample materials such as saliva or urine. they are becoming important for public health purposes but their value for diagnosing individual patients is still limited. antibodies that decrease the infectious capacity of the virus are called neutralizing antibodies. they are produced during acute infection and often persist during the entire lifetime. they are also useful as an indication of immunity. both igm and igg antibodies participate in the neutralization. in the assay, known amounts of infectious virus are mixed with the serum sample and incubated for a short period after which the residual infectivity is measured using cell cultures or test animals. this infectivity is then compared with the infectivity of the original virus and the neutralizing capacity is calculated from this result. today, neutralizing antibody assays are often done by plaque reduction assays with better accuracy but with somewhat more complex technical requirements. neutralizing antibody assay is specific and sensitive, but time-consuming and laborious, and therefore it is not widely used in routine diagnostic services. many viruses bind to hemagglutinin molecules found at the surface of red blood cells of various animal species and this can cause aggregation of red cells in suitable conditions. prevention of this aggregation, called hemagglutination inhibition, by specific antiviral antibodies in the patient's serum has been widely used for diagnostic purposes. the test, known as hemagglutination inhibition test, has important diagnostic and public health applications in certain infections, most notably in influenza where antibodies measured by this test show additional specificity compared to other tests and therefore provide more detailed information about the immunity and past infections of individuals. however, for the diagnosis of individual patients, the assay is no longer widely used and is replaced by more modern immunoassays. in the test, a virus preparation with a predetermined hemagglutinating capacity is mixed with the serum sample and after proper incubation the residual hemagglutination capacity is measured. both igm and igg antibodies are able to inhibit hemagglutination. the complement fixation test (cft) is a classical laboratory diagnostic test, which is still used for determination of virus antibodies in patient sera or cerebrospinal fluid samples during an acute infection. the test mainly measures igg antibodies. the test is based on the capacity of complement, a group of heat-labile proteins present in the plasma of most warm-blooded animals to bind to antigen-antibody complexes. when the complexes are present on the surface of red blood cells, complement causes their lysis which can be visualized by a suitable experimental setup. in the actual test, the complement in the patient's serum is first destroyed by heating; the serum is then mixed with appropriate viral antigen and after incubation; when the antigen-antibody complexes are formed, exogenous complement (usually from fresh guinea pig serum) is added. this complement then binds to the complexes and having been 'fixed,' it is then no longer able to cause lysis of added indicator red cells. usually, sheep red cells coated with antisheep red cell antibodies are used as indicator to measure the presence of any residual complement. the effect is measured by a suitable test protocol. serial dilutions of the patient serum are used and the highest dilution where the serum can still prevent complement activity in the indicator system is taken as the cft titer of the sample. the tests are usually carried out on microtiter plates and the results are observed by eye. cft is still used for diagnosis of acute virus infection. it measures certain types of antibodies which occur only during the acute phase of the infection. therefore, cft is not suitable for investigation of immune status. the assay procedure is quite complex, because the test is dependent on several biological variables, which have to be standardized by pretesting. the method is less sensitive than many other immunoassays. in addition, the method is very labor intensive and is not amenable to automation. the use of cft in virus diagnostics is increasingly replaced by modern immunoassays. in immunoassays, antibodies binding to specific immobilized antigens can directly be observed using bound antigens and proper indicators such as labeled anti-immunoglobulin antibodies. the antigens can be immobilized to plastic microtiter plates, glass slides, filter papers or any similar material. different immunoassays are nowadays widely used to measure virus-specific igm and igg antibodies. the most recent formats of immunoassays make it possible to detect simultaneously both antigens and antibodies decreasing significantly the window period between infection and immune response. numerous commercial kits with high specificity and sensitivity are available. automation has made immunoassay techniques more rapid, accurate, and easier to perform. in the basic format of solid-phase immunoassays, virus-infected cells, cell lysates, purified or semipurified, recombinant viral antigens or synthetic peptides are immobilized to a solid phase, usually plastic microtiter wells or glass slides. patient's serum is incubated with the antigen and the bound antibody, after washing steps, is visualized using labeled anti-immunoglobulin antibodies ('conjugate') ( figure (a) ). if the label used is an enzyme, the test is called enzyme immunoassay (eia) or enzymelinked immunosorbent assay (elisa) and the bound antibody is detected by an enzyme-dependent color reaction. if a fluorescent label is used, the method is called immunofluorescent test (ift). the enzyme labels most commonly used are horseradish peroxidase (hrp) and alkaline phosphatase (ap). in hrp-eia the color-forming system consists of ortho-phenyldiamine (opd) as a chromogen and hydrogen peroxidase (h o ) as a substrate. if the hrp-conjugate is bound to antibody-antigen complexes, the colorless chromogen becomes yellow and color intensity is measured with a photometer at a wavelength of - nm. the intensity of the color is proportional to the amount of bound conjugate and to the amount of specific antibodies in a patient serum sample. if the serum contains no specific antibodies, the conjugate is not bound and no color reaction occurs. by using either anti-igg or anti-igm conjugates it is possible to determine separately immunoglobulin subclasses. the specificity and sensitivity of these immunoassays are generally high. the most common source of potential misinterpretation is false positive igm reaction due to the presence of rheumatoid factor, itself igm molecule, reacting with virus-specific igg. the specificity can be improved by using an additional incubation step where igm antibodies are first enriched ('captured') in the sample by using anti-igm immunoglobulin. alternatively igg can be adsorbed or blocked by serum pretreatment. immunofluorescent tests were used in the past for measuring virus-specific antibodies, but are now replaced by eia techniques. the principle of the method is similar to eias. in ift, infected cells are placed on a glass slide and bound antibodies are detected by fluorescein-labeled anti-immunoglobulin antibodies. the glass slides are examined under a fluorescence microscope. the method is specific and sensitive, but quite labor intensive, and reading the test demands considerable experience. in some infections (e.g., that caused by human immunodeficiency virus (hiv)), antibodies against certain components of the virus are more informative than other less-specific antibodies and they are detected by immunoblotting assays. different virus antigens, prepared by gel diffusion or other techniques, are absorbed as discrete bands on a solid strip of cellulose or similar material and the strip is incubated with the patient's serum. antibodies present in the serum bind to specific antigens and are detected using an hrp-conjugate and nitroblue tetrazolium as the precipitating color chromogen. the color reaction is observed and compared to positive and negative control samples assayed on separate strips. immunoblotting assays are commonly used to confirm highly sensitive eia screening assay results for antibodies against hiv, hepatitis c virus (hcv) and human t-cell lymphoma virus (htlv). a technique known as lateral-flow technology has also been used to identify antibodies or antigens. these tests involve application of serum or other samples directly on a strip of suitable material such as cellulose, where the antibodies are diffused laterally and eventually reach a site in the strip where appropriate antigen has been applied and chemically fixed. specific antibodies become bound to the site while nonreacting antibodies diffuse out from the area. the presence of antibodies is visualized using labeled conjugates. although such tests are not quantitative, they are valuable for infections where the presence of specific antibodies is indicative, such as hiv infection. performance of the test is often very simple and the result is available in a few minutes or a few hours, making such tests suitable for bed-side screening. in more advanced tests, several different antibodies can be detected by a single assay and the test conditions can be modified further so that antigens can also be detected. many such tests have become commercially available in recent years. for some applications, coated latex particles have replaced strips with fixed antigen as the solid phase. binding of specific antibodies can be visualized with chromogenic or otherwise labeled indicator antibodies or a positive reaction can be detected by agglutination of the latex particles. the presence of viral antigens in clinical specimens, such as nasopharyngeal aspirates, fecal specimens, vesicle fluids, tissue specimens, as well as serum samples can be demonstrated by antigen detection assays. in immunofluorescence tests, cells from a clinical specimen are fixed on a glass slide and viral antigens present in the cells are detected by fluorescein-labeled virus-specific antibodies. less reader-dependent results can be obtained using enzyme or other immunoassays. solubilized antigens in clinical specimens are first captured using specific monoclonal antibodies bound to a solid phase, and are then detected with virus-specific detector antibodies (figure (b) ). the functionality of monoclonal antibodies (mabs) with high specific binding affinity is better preserved when labeled with a small molecule as compared to a bulky enzyme molecule. in eia, biotinylated mabs are used with streptavidin-enzyme conjugate. in time-resolved fluoroimmunoassay (tr-fia), detector mabs are labeled with an europium chelate. antigen detection methods are especially recommended in the case of virus reactivation, for example, for herpes simplex and varicella zoster virus diagnosis where the serological response can be very weak. antigen detection assays are also widely used in respiratory tract infections like influenza and respiratory syncytial virus infections. a simple test for the demonstration of rotavirus and adenovirus antigens in children with gastroenteritis is also available. direct demonstration of viral nucleic acids in clinical samples has become the technique with the widest repertoire of diagnostic virus targets. using the polymerase chain reaction (pcr) with specific primers, viral sequences can be rapidly multiplied and identified. these techniques have largely replaced classical virus isolation. they are rapid to perform and in many cases more sensitive than virus isolation or antigen detection methods making earlier diagnosis possible. they have proved particularly valuable for the diagnosis of emerging viruses and viruses that cannot be cultivated such as papillomaviruses, parvoviruses, hepatitis viruses, and rhinovirus species c. semiquantitative and quantitative applications have been developed allowing monitoring of viral load during antiviral treatment. these tests cannot distinguish between viable and replication-incompetent virus, warranting caution in the interpretation of the results in certain cases. while sensitivity to cross-over contamination throughout the diagnostic pathway cannot be undermined, use of real-time pcr technology has diminished these problems in clinical laboratory settings. the specificity of these tests is based on the extent of pair-matching sequences between the viral nucleic acids and the primers. extremely high sensitivity is typical for pcr methods; - copies of viral nucleic acid can be detected in about one to few hours. pcr methods are available for both rna and dna viruses. for rna viruses viral nucleic acid has to be transcribed with reverse transcriptase (rt) enzyme to complementary dna in a combined assay called rt-pcr. viral nucleic acid is extracted from the sample material and amplified in three successive steps. the double-stranded dna is first heat-denaturated and separated into single strands. the specific target fragment of dna strand is then amplified (figure ) by pairs of target-specific oligonucleotide primers, each of which anneal to one strand of unfold double-stranded dna. each annealed primer acts as an origin for heat-stable polymerase enzyme and a complementary strand is synthesized via sequential addition of deoxyribonucleotides. in real-time pcr, annealing and extension steps are often combined to a single step at c. these cycles are repeated about times, each cycle resulting in an exponentially increasing numbers of copies. after the amplification is completed, the products can be detected by several methods. agarose gel electrophoresis combined with ethidium bromide staining of the products is a classical method (figure ) . the size of the amplified product is compared to control amplicons and other standards in the same gel. various hybridization assays, based on labeled complementary oligonucleotides (probes), are also used to improve the sensitivity and specificity of the detection. for applications with a large number of targets, such as respiratory virus detection or papillomavirus typing, conventional pcr with post-pcr hybridization is still a popular solution. products of highly multiplexed pcr assays can be identified by probe hybridization on microarrays using scanning fluorometer or on fluorescent microbeads using flow-cytometric bead counting. the amplified fragments can also be sequenced giving additional information about the virus. comparison of the sequences with known virus sequences allows identification of species, strains, or subtypes that may be important for public health or medical purposes. sequencing after rt-pcr is also the current method-of-choice for investigating the emergence of antiviral drug resistance among hiv-infected patients. real-time pcr instruments monitor accumulation of amplicons by measuring the fluorescence continuously in each cycle of the reaction. fluorescence is generated by a dsdna dye or a fluorescent probe system included into the reaction mix. the earlier the amplification product becomes detectable over the background, the higher is the amount of virus in the sample ( figure ) . a dsdna dye (e.g. sybr green i) reacts with any dsdna formed during amplification, but amplicon specificity can be confirmed by melting curve analysis. it also allows testing for more than one virus from the same sample ( figure ). there are many alternative probe chemistries for real-time pcr, the most common including dual label probes. since probes are usually more selective for their target sequences than primers, probe based real-time pcr methods offer highest specificities with fastest reaction times. multichannel instruments allow the use of probes with different exitation and emission spectra for the simultaneous detection of several different analytes in a single tube. the pcr assays are extremely sensitive and can therefore be influenced by inhibitors of the polymerase enzyme that are sometimes present in clinical samples. internal controls can be included into reaction mixtures. nucleases present in samples or in figure rt-pcr with real-time detection (a) and with melting curve analysis (b) for the detection of respiratory syncytial virus (rsv), rhinovirus, and enterovirus in respiratory secretions (c). c t is a threshold cycle number, t m is a melting temperature, and ntc is a nontemplate control. unpublished results by waris m, tevaluoto t, and Ö sterback r. reagents can also cause false negative results by degrading viral nucleic acids. furthermore, amplicons may also cause product carryover and false positive results. extremely high care has to be applied in handling the clinical specimens, the reagents, as well as the reaction products. the key to a succesful pcr method is the careful design of the primers and probes. they have to be specific for the target and follow certain structural rules to produce efficient amplification. the high specificity of the pcr, especially probe-based real-time pcr, makes the technique sensitive for mutations, sense or non-sense, in the target sequences of highly variable viruses. that must be taken into the consideration not only in design of the assays, but also in continuous evaluation of them for changes in sensitivity caused by new mutations affecting critical primer or probe binding sites. one of the great advantages of the pcr technology is its potential to detect new emerging viruses. by using primers from related viruses or so-called generic primers important information regarding the new virus can be obtained for further development of more specific tests. a recent example is the middle-east respiratory syndrome (mers) coronavirus, for which specific diagnostic tests became available soon after the taxonomic position of the virus became known. the technology also allows safe handling and transport of virus samples, since extraction buffers added to the samples inactivate virus infectivity. poc tests are becoming increasingly common in clinical practice. in proper use, they offer a cost-efficient diagnostic guidance for quick clinical decision making. their performance is rarely as good as that of corresponding laboratory tests; use of them should be quality-controlled, and a back-up should be arranged with a virology lab. most of them are based on easy-to-use lateral-flow or latex particle technology and are able to give the result in a few minutes. poc tests are nowadays available for antibody screening of an increasing number of virus infections (hiv, hepatitis c virus (hcv), varicella-zoster virus (vzv), cytomegalovirus (cmv), epstein-barr virus (ebv)). with the breakthrough of nucleic acid testing, antigen detection methods are becoming obsolete in high-end virus laboratories, but their development have continued for poc testing. most commercially available tests for antigen detection are lateral flow immunochromatographic assays, and they are typically targeting influenza viruses, respiratory syncytial virus, adenoviruses, rotavirus, norovirus and hepatitis b virus. multianalyte testing for common respiratory virus antigens is possible with a commersial multianalyte respiratory infection poc test platform including a small bench-top instrument suitable for polyclinical use. also compact all-in-one pcr systems for the detection of respiratory pathogens are now available. driven by public health, scientific and commercial interests, new diagnostic tests for the laboratory diagnosis of viral infections are continuously being developed. the development of new molecular detection methods continues on two lines. on the one hand, it includes compact, fully integrated automates requiring minimal training to run and suitable for poc use. on the other hand, separate instruments for specimen handling, nucleic acid extraction, pcr set-up, and amplification are integrated into high throughput flow systems, which allow efficient use of both commercial and laboratory designed assays. use of multianalyte methods is becoming a practical reality and they might significantly change diagnostics of infectious diseases in future. multiplex pcr in a single tube or miniature format still have the challenge of reaching the sensitivity of simpler pcr targeting only one or few analytes. microarray technology for pcr product detection competes with sequencing, but still provide a rapid alternative for identification of a large number of virus types. next generation sequencing technology will be the primary way of identification of new or emerging viruses. simple microarrays, readable without bioinformatics, would reduce the cost of serological screening and allow the use of control measures to improved quality of the results. outside the specific virus diagnostics, measures of virus induced interferon response is an area of potential development. next-generation sequencing technologies in diagnostic virology avidity of igg in serodiagnosis of infectious diseases principle and practice of clinical virology clinical and economical impact of multiplex respiratory virus assays microarray-based detection and genotyping of viral pathogens detection and monitoring of virus infections by real-time pcr point-of-care diagnostics for global health key: cord- -nvzfpntu authors: nan title: research communications of the th ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: nvzfpntu nan cobalamin concentrations were previously investigated in cats, but little information is available concerning the follow up of hypocobalaminemic cats. we aimed to assess the frequency of hypocobalaminemia within a large cohort of cats with gastrointestinal signs and describe the epidemiological, clinical, biological and follow-up characteristics of hypocobalaminemic cats. cats with gastrointestinal signs and for which a cobalamin assay (simultrac-snb radioassay kit vitaminb Ò , mpbiomedical) was performed between and at the ldhvet laboratory were retrospectively included in the study. ( . %) cats presented for gastrointestinal signs had an hypocobalaminemia: the majority were domestic short hair, % were males ( % castrated) and % females ( % castrated), aged from months to years. the main clinical signs included chronic diarrhea ( %), weight loss ( %), polyuropolydypsia ( %), vomiting ( %), polyphagia ( %), fatigability ( %) and dysorexia ( %) with a median duration of months before diagnosis. cobalamin values ranged from to ng/l (median: ng/l). % of the hypocobalaminemic cats had also a hyperfolatemia (folate > ng/l) at diagnosis. ft was measured in the older cats (> years) and revealed an hyperthyroidism (ft > pmol/l) in % of the cases. / hypocobalaminemic cats had a known clinical and biological follow-up (median time follow-up = days): cobalamin significantly improved month after treatment ( lg/kg im cyanocobalamin in a single dose) for % of the cats, even if % remained hypocobalaminemic. % of the followed cats were clinically improved, of which % with an associated higher cobalamin value. clinical and biological improvement after cobalamin supplementation was significantly associated with an increase in folate concentration (p-value = . ). however, % of the cats with an improved cobalamin value did not show any clinical improvement. hypocobalaminemia is frequently observed in cats as a consequence of gastrointestinal signs. cobalamin concentrations could be used as an indicator of the severity of various gut diseases more than a primary cause, because one third of the cats did not show any clinical improvement despite an improved cobalamin value. a hyperfolatemia appearing after treatment of hypocobalaminemia seems to be a good indicator of a clinical improvement associated with a return to a normal intestinal integrity. disclosures: no disclosures to report. cobalamin malabsorption is common in old cats with weight loss, macronutrient malabsorption and enteric protein loss due to idiopathic chronic enteropathy, ice (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, - , , williams and czarnecki-maulden, proc rd ecvim-ca congress, ) . high dose oral cobalamin supplementation reverses subnormal serum concentration within week but serum cobalamin can become undetectable within as little as month following cessation of supplementation (williams and czarnecki-maulden, proc rd ecvim-ca congress, ). the objectives of this study were to determine if serum cobalamin concentrations in cats with ice and the response to oral supplementation and withdrawal are associated with differences in the intestinal microbiome. the study evaluated cats older than years of age that were being fed nutritionally complete and balanced diets that included a fortification of vitamin b . thirty-one of these cats had ice demonstrated by increased fecal fat (> %), subnormal fat digestibility (< %), subnormal serum cobalamin or increased serum methylmalonic acid, but without exocrine pancreatic insufficiency as assessed by assay of serum trypsin-like immunoreactivity. serum cobalamin was determined by competitive binding assay and the fecal microbiome by roche sequencing and analysis by qiime (quantitative insights into microbial ecology), pca (principal component analysis) and opls (orthogonal projections to latent structures). of these ice cats were supplemented with oral cobalamin for months. serum cobalamin concentrations were determined monthly during supplementation and for months after cessation of supplementation. in the cats there was a significant (p ≤ . ) association between serum cobalamin and the fecal microbiome, with species being positively correlated with serum cobalamin concentration and species being negatively correlated. serum cobalamin was subnormal (< ng/l) in of the ice cats at the start of the supplementation study and subsequently became normal or supranormal. within to months after cessation of supplementation serum cobalamin was subnormal in the original cats and additional cat. it is concluded that serum cobalamin concentration and the responses to oral supplementation and subsequent cessation of supplementation are significantly associated with the composition of the intestinal microflora as reflected in the fecal microbiome. disclosures: the study described in the abstract was performed at nestle-purina facilities and funded entirely by nestle-purina. david williams is a consultant and adviser for nestle-purina, idexx laboratories, and the gastrointestinal laboratory at texas a&m university. he receives royalties from idexx laboratories and has given continuing education lectures sponsored by nestle-purina. inflammatory bowel disease (ibd) is a common cause of chronic gastrointestinal signs in cats. typically, lymphoplasmacytic inflammation is found in biopsies, but a subset of cats with ibd has neutrophilic inflammation. the clinical significance of neutrophilic infiltration is unclear. the aim of this retrospective study was to use fluorescence in situ hybridisation (fish) to look for the presence of any microorganisms within the intestinal epithelium of cats diagnosed with ibd and then to identity those micro-organisms. our hypothesis was that neutrophilic enteritis in cats would be associated with intestinal mucosal invasion by microorganisms, and specifically by campylobacter spp. the study included cats presented to the small animal hospital, langford veterinary services for investigation of gastrointestinal disease which had duodenal biopsies collected endoscopically. thirteen cats were diagnosed with neutrophilic inflammation (study group) and cats with lymphoplasmacytic inflammation (control group). fluorescence in situ hybridisation (fish) targeting either all eubacteria or campylobacter jejuni, coli and upsaliensis was used to identify and count intra-mural bacteria in the intestinal biopsy samples. neutrophils were detected simultaneously using a fish probe to neutrophil elastase. the pixel distance between different bacterial species and neutrophils was measured. all animals in both groups showed the presence of intra-epithelial bacteria and the number of bacteria present did not differ between the control and study groups. similarly, campylobacter jejuni and upsaliensis were present in some animals in each group but numbers did not differ between the groups. in contrast, campylobacter coli was present in significantly more study cats than control cats (p = . ; chi-squared test) and the study group showed significantly higher numbers of c. coli in the tissue than the control group (p = . ; mann-whitney u test). co-localisation of neutrophils and c. coli was demonstrated with c. coli closer than any of the other bacteria to the neutrophils. this association was statistically significant (p < . ; mann-whitney u test). the role of the intestinal virome in health and disease is gaining increased attention in human medicine. the use of next generation sequencing (ngs) technologies has allowed identification of diversity and distribution of the virome. these approaches can equally be applied to dogs.this study aimed to identify and characterise the virome present in faeces of dogs with chronic enteropathy (ce) compared to the virome of healthy dogs (hd). faecal samples were evaluated from hd and dogs with ce ( food, antibiotic and steroid responsive) using a ngs approach. a viral enrichment protocol, using a series of centrifugation, endonuclease treatments and bacterial filtration were performed. the enriched viral dna and rna were extracted and amplified using sequence-independent single-primer amplification (sispa) protocol, and subsequently sequenced by ngs using the illumina miseq platform at the agrf. two bioinformatic pipelines were used to analyse the viral population. after selecting high quality reads and removing dog and bacterial sequences, sequence information was compared against reference databases. we identified a total of , viral contigs, with , dna viral sequences and , rna sequences across all dog samples. the majority of viral hits from both groups of faecal samples were bacteriophage ( . % hd and . % ce), from several families mainly from the caudovirales order. after all analyses, only viral eukaryotic families were identified across all samples. two groups of sequences similar to known virus families, reoviridae and papillomaviridae, were identified in both groups (hd / and / and ce / and / , respectively). sequences similar to picornaviridae were identified only in one dog with ce and sequences similar to adenoviridae, parvoviridae and coronaviridae were identified only in healthy dogs ( / each).further genomic characterisation and phylogenetic analysis was undertaken on viruses. the genome segments of a rotavirus (reoviridae) isolate were determined. similarly, the sequence of the entire coding region of a kobuvirus (picornaviridae) isolate was determined. preliminary analyses indicated that all rotavirus gene segments exhibited between % - % nt homology to previously reported canine rotaviruses. the kobuvirus sequence exhibited moderate nt homology ( %) to previously described genomes and clustered with other canine kobuvirus sequences available in genbank. in conclusion, viral sequences from a range of different virus families, including both rna and dna families, and known pathogens were identified and characterised, and the largest proportion of viral contigs identified belonged to bacteriophages. disclosures: no disclosures to report. endoscopy is widely used to perform targeted and minimally invasive biopsies for histopathology of the gastrointestinal tract of dogs and cats. only a few studies have focused on the diagnostic contribution of cytological samples of the alimentary tract. the aims of this study were to compare 'imprint' and 'squash' techniques to obtain valuable cytological samples from endoscopic biopsies, and to evaluate the potential interest of cytology compared to histology in reaching the definitive diagnosis. eighteen dogs and cats presenting gastrointestinal symptoms that underwent an endoscopy of their alimentary tract were prospectively included. five biopsies of each area of interest were collected for regular histopathological analysis. an additional biopsy from each area was used to obtain cytological specimens by imprint and squash techniques. cytology samples were all reviewed blindly by the same pathologist. cytology samples of insufficient quality were considered as 'non diagnostic' and were excluded from further analysis. diagnostic conclusions of both cytological and histological analyses were classified into defined categories (inflammation or neoplasia with subcategories, fibrosis, epithelial hyperplasia, and normal) to allow comparison between the techniques. agreement between cytology and histology was determined by cohen's kappa coefficient. from the cases, biopsy specimens were collected from different localizations for histology and cytology slides were obtained. final diagnosis was neoplasia in cases and inflammatory disease in . considering imprint technique, / were considered 'non diagnostic'. for squash technique, only / of cytological samples were considered as 'non diagnostic'. squash cytology and histology gave the same results in . % of the cases (n = ) and agreement between the techniques was considered 'moderate' (k = . ( % confidence interval [ci] . ; . )). agreement was 'fair' between imprint cytology and histology (n = ) (k = . [ % ci . ; . ]). gastric spiral organisms (gso) were observed in cases. in cases they were identified only on cytology. amongst these cases, mast cells were identified on cytology in cases, and not on histology in any cases. mast cells were not found in any other cases. this prospective pilot study demonstrated that cytological examination of gastrointestinal biopsy squash samples obtained during endoscopy of the alimentary tract may give relevant information, which can help the clinician to initiate treatment while histopathological analysis is pending. furthermore, it can give additional information (presence of potential pathogens, mast cells) that may not be identified on histopathology. disclosures: no disclosures to report. intramucosal escherichia coli are implicated in the pathogenesis of granulomatous colitis of boxer dogs. clinical remission hinges upon its eradication, most commonly achieved with fluoroquinolones. antimicrobial resistance is not uncommon among e. coli isolated from dogs with gc and impairs successful treatment. published data is lacking on efficacious therapies for gc dogs with fluoroquinolone-resistant e. coli. the aim of this study was to characterize the antimicrobial resistance patterns and molecular characteristics of e. coli isolated from dogs with gc. additionally, to evaluate the clinical outcome of dogs treated with antimicrobials guided by culture and susceptibility results. the study population was ( boxers and french bulldogs) client-owned dogs with gc. gc biopsies with fish-confirmed intramucosal e. coli were submitted for bacterial culture. antimicrobial susceptibility was determined by broth microdilution. most strains were further characterized by phylogroup and overall genotype using triplex and random amplified polymorphic dna polymerase chain reaction, respectively. treatment and clinical outcomes data were obtained. culture yielded e. coli strains ( - per dog, med ) from / dogs. resistance to fluoroquinolones was identified in / dogs; this was correlated with resistance to other macrophage-penetrating antimicrobials (p < . ). phylogroup a was over-represented among enrofloxacin-resistant strains. in dogs with e. coli isolated at multiple time points, phylogroup changed over time. clinical remission was achieved in / dogs with fluoroquinolone-sensitive e. coli. dogs with fluoroquinolone-resistance had a more variable response; treatment with meropenem (median mg/kg sq q hours for weeks) resolved clinical signs in / . we conclude that antimicrobial resistance is a growing concern. gc-associated e. coli appear genetically diverse. clinical remission can be achieved in the face of fluoroquinolone-resistance though in vitro antimicrobial susceptibility does not consistently predict a positive response. disclosures: no disclosures to report. canine s a has potential as a biomarker of inflammation in dogs. fecal s a concentrations were increased in dogs with chronic gastroenteropathy (ce), and correlated with the severity of clinical and endoscopic disease. a negative outcome was associated with higher fecal s a concentrations in ce dogs, but the response to different forms of treatment and fecal s a has not been reported, and this information will be important to further evaluate the utility of fecal s a as a biomarker for gastrointestinal disease. aim of this study was to evaluate the association between responses to various treatments (i.e., elimination diet, antimicrobial drugs, or corticosteroids/other immunosuppressants) and fecal s a in dogs with ce. fecal samples were collected from dogs diagnosed with ce, and fecal s a was measured in all specimens using an established in-house elisa. based on the response to treatment, dogs were classified as having antibiotic-responsive diarrhea (ard), food-responsive diarrhea (frd), or steroid-responsive/therapy-resistant idiopathic inflammatory bowel disease (ibd). statistical analysis was performed using non-parametric -or multiple-group comparisons, the likelihood ratio to evaluate the association between groups of dogs and response to treatment, and a receiver operating characteristic curve to calculate sensitivity and specificity at the optimum cut-off concentration. a total of dogs with ce (median age: . years; males/ females) were included in the study, the final diagnosis of which were ard (n = ), frd (n = ), or ibd (n = ). response to treatment was complete remission (n = ), partial response (n = ), or no response (n = ). fecal s a concentrations ranged from to , ng/g, and higher s a levels were seen in dogs with ibd than in dogs with frd (p = . ) or ard (p = . ). dogs that did not respond to treatment had significantly higher s a levels than dogs with partial (p = . ) or complete (p = . ) remission, but response to treatment was associated with disease classification (p = . ). despite a small number of patients, fecal s a levels of > , ng/g at the time of diagnosis distinguished dogs that failed responding to treatment from those with at least partial remission with a sensitivity of % and specificity of %. we conclude that, in line with our previous finding that fecal s a may be a useful biomarker of disease severity in dogs with ibd, fecal s a may also have utility in predicting the lack of response to treatment in dogs with ce. the utility of serial fecal s a concentrations to monitor treatment response in dogs with ce warrants further research. disclosures: dr. heilmann and dr. steiner have filed a patent application that includes the s a elisa used for this study. constipation is a common presenting complaint in dogs and cats. differential diagnosis for this clinical sign is well-known but strictures resulting from gastro-intestinal inflammation are not commonly included and have been rarely reported in the literature. acute diarrhea and bone ingestion can lead to anal or rectal stricture which is responsible for the constipation. the aim of this retrospective study was to describe the prevalence of inflammatory rectal and anal stricture in small animals and to describe a simple and effective treatment. medical records of dogs and cats presented for constipation, dyschezia or tenesmus and diagnosed with an inflammatory stricture were obtained from the database of the gastro-intestinal diseases consultation of referral centers in gastroenterology between and ; and were reviewed. signalment, presenting complaint, clinical findings, treatment protocol and outcome were recorded. five dogs and cats were included in the study. of the cats, were purebred kitten between . and months, and among them were persians. the fifth cat was a -year-old female domestic shorthair. three out of cats had history of acute diarrhea and cats had constipation since adoption with unknown history. digital rectal examination under anesthesia revealed stricture in all cats which was treated by bougienage every days and high-fiber diet. of the dogs, age ranged from . to years; dogs had history of acute diarrhea and had ingested bones in prior days. colonoscopy and biopsies were performed in all dogs and showed a lymphoplasmocytic infiltration in all of them. dogs were treated with digital bougienage every other day until disappearance of the stricture, metronidazole, lubricant laxatives, corticosteroids and highfiber diet. the prevalence of inflammatory stricture in dogs was . % based on dogs presented with the same complaints between and . for all dogs and cats, clinical signs related to the stricture resolved for the duration of their follow-up. benign strictures secondary to gastro-intestinal inflammation should be systematically included in the differential diagnosis of constipation. strictures are easily palpated on digital rectal examination, which should always be performed during clinical examination. histology should be a routine part of the diagnosis workup to exclude neoplasia. endoscopy-assisted balloon dilatation with concurrent intralesional injection of triamcinolone has been used in dogs and reported in the human literature. the treatment described here is simpler and effective. in dogs, it can be done at home by the owner. disclosures: no disclosures to report. acute pancreatitis is a diagnostic challenge because of anatomic inaccessibility of the pancreas, vague clinical signs and physical examination findings, and inconsistent laboratory results. common, yet non-specific, clinical signs include abdominal pain, anorexia, vomiting, and diarrhea. ultrasonography is the imaging modality of choice to evaluate the pancreas. the purpose of this study was to compare clinical signs with ultrasonographic findings in dogs with acute pancreatitis to account for differences in clinical presentation depending on the region of the pancreas affected as determined by ultrasonography. the hypothesis was that there would be differences in clinical presentation depending on the pancreatic region involved. records of client-owned dogs diagnosed with acute pancreatitis based on history, clinical signs, laboratory testing, and abdominal ultrasonography were retrospectively evaluated. based on ultrasonography, dogs were divided into groups: group - dogs with changes within the left limb of the pancreas exclusively; group - dogs with changes within the right limb of the pancreas exclusively; and group - dogs with diffuse pancreatic involvement. presence of abdominal pain, anorexia, vomiting, and diarrhea was correlated between groups using chi-square and fischer's exact test. no significant differences regarding age, breed and sex were noted between groups. in group pain was noted in %, anorexia in %, vomiting in %, and diarrhea in % of dogs. in group pain was present in %, anorexia in %, vomiting in %, and diarrhea in % of dogs. in group pain was noted in %, anorexia in %, vomiting in %, and diarrhea in % of dogs. pain was noted with a significantly higher frequency in diffuse pancreatic disease as compared to disease restricted to the left or right limb of the pancreas. anorexia was significantly more common with right limb involvement. both vomiting and diarrhea were significantly more common with disease restricted to the left limb as compared to diffuse parenchymal or right limb involvement. despite overlap between groups, these findings indicate that pain response is expected to occur with a higher frequency in diffuse pancreatitis but overall is not a very common clinical sign. anorexia is more prevalent in dogs with pancreatitis of the right limb whereas vomiting and diarrhea both are more evident in dogs left limb pancreatitis. differences between the groups can possibly be ascribed to gastric involvement when the left side of the pancreas is affected. disclosures: no disclosures to report. increased physical exercise has been reported to improve the clinical symptoms of chronic enteropathies, such as inflammatory bowel disease, in human patients. the aim of this investigation was to evaluate the impact of an intervention to increase physical exercise in dogs with chronic enteropathies. twenty-two dogs ( each in the exercise and control groups) with chronic enteropathies and no response to an elimination diet were included. routine diagnostic work-up (haematology, plasma biochemistry profile, urinalysis, faecal parasitology, abdominal radiographs, and ultrasound) was conducted in all dogs to eliminate underlying causes. all dogs were given oral prednisolone ( mg/kg/day) for days, followed by a tapering dosage over weeks. after weeks of prednisolone treatment, a certified canine rehabilitation therapist instructed the owners of dogs in the exercise group on how to increase their dogs' physical exercise. the exercise protocol combined aerobic and resistance exercises in low-to moderate-intensity interval training. owners of dogs in the control group were asked to maintain the dogs' routine lifestyles. modified canine inflammatory bowel disease activity scores (cib-dais), based on the parameters of activity level, appetite, vomiting, stool consistency, stool frequency, bloating, and weight loss, were calculated pre-treatment and and weeks post-treatment for all dogs. cibdai scores were compared among timepoints (pre-treatment and post-treatment assessments) and between groups (exercise and control) using multivariate repeated-measures models for multiple comparisons. all dogs showed improvement after weeks of prednisolone treatment. modified cibdais decreased in the exercise (from . ae . to . ae . ) and control (from . ae . to . ae . ) groups. after weeks of the increased physical exercise intervention, the modified cibdai in the exercise group decreased significantly ( . ae . ) relative to the first post-treatment assessment (p = . ), whereas this index remained similar ( . ae . ) in the control group. modified cibdais differed significantly between groups after weeks of treatment (p = . ). all parameters of the modified cibdai were significantly affected by the intervention of increased physical exercise; the largest difference was found for body weight (p < . , adjusted r = . ) and faecal frequency (p < . , adjusted r = . ) and activity level (p < . , adjusted r = . ). an increased physical activity intervention had positive effects on clinical symptoms in dogs with chronic enteropathies. disclosures: no disclosures to report. corticosteroid therapy is commonly required in veterinary patients for treatment of inflammatory, immune-mediated, neurological and neoplastic diseases. some of these patients also require assisted enteral nutrition via percutaneous endoscopic gastrostomy (peg) tubes. this retrospective case-control study evaluated the complications associated with peg tube use in veterinary patients receiving corticosteroids in a referral teaching hospital. medical records of dogs and cats in which a peg tube was placed in the qmha between january and march were reviewed. patients were included if the peg tube was in use for at least hours and if complete medical records, including clinical notes from referring veterinarians, kennel sheets, communication records with patients' owners and notes from tube removal, were available. to be included in the steroid group, patients must have received corticosteroid therapy (> mg/kg/day) for at least % of the length of time the peg tube was in use. control patients were not treated with corticosteroids. forty-two cases were included ( dogs and cats). fourteen patients ( dogs and cats) were included in the steroid group and patients ( dogs and cats) were included in the control group. complications were scored in terms of severity as minor ( ), moderate ( ) and major ( ) and compared between groups using the mann-whitney u-test. values of p < . were considered significant. complications included: serous discharge (n = ), sanguineous discharge ( ), purulent discharge ( ), stoma site inflammation ( ), peg tube dislodgement ( ) , pain around the stoma ( ), peg tube blockage ( ) and peg tube chewed by the patient on its tip ( ) or at the stoma ( ). median (interquartile range) of maximum complication scores for control and steroid groups were respectively ( ) and ( ). the maximum complication scores were not significantly different between groups (u = . , p = . ), though patients receiving corticosteroids showed a trend towards higher maximum complications scores than those in the control group. in conclusion, owners of dogs and cats receiving corticosteroids in which a peg tube is planned should be appraised of the possibility of complications beyond those normally associated with tube placement alone. disclosures: no disclosures to report. canine obesity is usually treated with dietary energy restriction, but data are limited regarding nutritional adequacy. the aim of the current study was to compare intake of essential nutrients with national research council recommendations in obese dogs during weight management with a purpose-formulated diet. twenty-seven dogs were included in this non-randomized retrospective observational cohort study. all were determined to be systemically well, and without significant abnormalities based upon physical examination and clinicopathological assessments. the dogs underwent a controlled weight loss protocol of at least weeks, to achieve ideal condition and using a high protein high fiber weight loss diet. median, maximum, and minimum daily intakes of all essential nutrients were compared against nrc recommended allowances (ra) for adult dogs. median weight loss was % ( - %), median daily energy intake was kcal/kg . ( - kcal/kg . ), and no signs of nutrient deficiency were observed in any dog. based upon the average nutrient content of the diet, daily intake of the majority of essential nutrients was greater than their nrc ra (per kg body weight . ), except for selenium, choline, choline ( / dogs) and methionine+cysteine ( / dogs), all essential nutrients remained above nrc minimum requirements (mr) throughout the trial. daily intakes of most essential nutrients meet both their nrc ra and mr in obese dogs throughout a period of weight loss. in light of absence of signs of nutrient deficiency, the significance of the borderline intakes for some nutrients (especially selenium and choline) is not known, and further studies are recommended. disclosures: the following conflicts of interest apply: ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational mate-rial, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin; the diet used in this study is manufactured by royal canin; ss and vb are employed by royal canin. esvcn-o- endocrine profile of obese dogs. d. j. rochel, c. amato, p. nguyen, l. jaillardon, b. siliart. oniris, nantes atlantic college of veterinary medicine, food science, engineering, nantes cedex , france obesity is a frequent condition of the dog, associated with many endocrine and metabolic disturbances leading to major organ dysfunctions. we therefore aimed to assess biochemical and hormonal profiles of a large cohort of obese dogs. obese dogs were retrospectively included in the study, based on an overweight over % the ideal body weight (ibw). endocrine profiles consisted in assessing prolactin, leptin, insulin like growth factor type (igf ), cortisol after an acth stimulation test, insulin, free thyroxine (ft ) and ctsh serum concentrations. obese dogs ( % females of which % spayed and % males of which % castrated) were from different breeds and ranged from to years [median years, . % between and years]. % of the dogs suffered from generalized (versus abdominal) obesity and long-term obesity (> year) was described in % of the cases. the main observed clinical signs were abdominal distension ( %), fatigability ( %), polyphagia ( %), decline of interest for usual activities, ( %) and polyuropolydipsia ( %). biochemical profile was unremarkable except that % of the dogs had hypercholesterolemia (cholesterol > mmol/l). a high prolactin value (> ng/ml) was observed in % of the dogs, a high leptin value (> lg/l) in %, a high igf value [igf > lg/l (ibw < kg), > ( < ibw < kg) and > (ibw> kg)] in % and a high insulin value in % (> lui/ ml), without significant correlation with glucose concentration in % of the cases. % of the dogs had a high cortisol value (> nmole/l after acth stimulation) and % had alow ft (ft < pmol/l) with % having a high tsh value (> , ng/ml). canine obesity is associated with many endocrine disorders including hyperprolactinemia, hyperleptinemia, high igf value, hypercortisolemia and/or a hypothyroxinemia associated with a high ctsh value. the endocrine profile could be very interesting for the diagnosis and prognosis of canine obesity and could allow the veterinarian to choose a better treatment, particularly when the diet is unsuccessful. further investigations could be done to assess the prognostic value of the endocrine profile at the diagnosis of canine obesity to control the treatment efficiency. disclosures: no disclosures to report. a better understanding of how dogs undergo healthy ageing would benefit owners and veterinarians alike. in july a longitudinal study began to evaluate health and longevity in labrador retrievers ( males and females, all neutered, mean age . years), continuously fed a fixed plane of nutrition with identical housing, standardised husbandry and veterinary care. body condition score was maintained between and on a -point scale. standard veterinary protocols were used for any medical conditions; cancer and severe or life threatening conditions were managed individually based on quality of life assessments. the 'average' lifespan of labrador retrievers was estimated to be years. dogs were classified according to lifespan as 'typical' if they died between and ≤ . years of age, 'long' ≥ to . years and 'exceptional' ≥ . years (corresponding to % longer than the average lifespan). data were analysed using linear mixed models with random effects for slopes and intercepts and a fixed effect for lifespan grouping variable. on st july , dogs ( %) were classified as exceptional with still alive, typical (n = ) and long (n = ). gender and age at neutering were not associated with survival time or risk of death (p≥ . ). body weight change showed a quadratic trend: up to age , body weights increased for all lifespan groups but the changes were not significantly different. there was a significant change in body weight from to years as exceptional dogs increased body weight while the long-lifespan dogs lost weight (+ . versus À . kg/dog/year, p = . ). after age the exceptional and long groups both had similar losses. dual-energy x-ray absorptiometry scans revealed that wholebody fat (g) increased in all lifespan groups to age but the change was significantly slower for the long lifespan dogs when compared with typical dogs which accumulated fat at > times the rate. all groups lost a similar amount of whole-body lean tissue (g) through age (p > . ). up to age the mean % gain in whole body fat, and % loss of whole-body lean tissue, was slower and the mean change in fat to lean ratio was lower in the exceptional and long-lived dogs compared to the typical dogs (p £ . ). typically aged labradors showed a greater gain of fat tissue, and greater loss of lean tissue, up to years of age than exceptional dogs. disclosures: the eukanuba diet used in this study is manufactured by spectrum brands whilst dmm is employed by spectrum brands. vja is an independent epidemiologist who helped analyze the data and was financially supported by spectrum brands for this work. pjw has participated in veterinary seminars organised by spectrum brands and has received an honorarium for this work. introduction: in healthy animals, the phosphate (p) in combination with calcium homeostasis is regulated in comparatively narrow limits: excessively ingested p is excreted via urine. common knowledge is, however, that p is a progressive factor in chronic renal failure (crf) wherefore typically a p restricted diet is prescribed for affected patients. in , pastoor demonstrated that a p excess (significantly at~ mg p/mj me; ca/p . / for days) impairs renal function even in healthy cats, diagnosed mainly by reduced endogenous creatinine clearance. dietary p originates from meat and other protein sources, bones and cartilages, mineral supplements and technical additives (water binding, palatability a/o texture enhancer etc.). the daily amount ingested with complete diets from the european market often exceeds the recommended daily allowance (rda; nrc , fediaf considerably (up to times, anonymus ). our own studies were done with the aim ( ) to survey the reproducibility of the results of pastoor ( ) and ( ) to test effects of different ca/p ratios and p sources on parameters of renal function in healthy adult cats. animals, materials and methods: up to adult, healthy cats were appointed to groups in every trial period. firstly, a balanced diet was fed for days including a days balance trial. one group was then switched to a high p diet whereas the other remained on the balanced diet, again completed by a balance trial. after days of wash-out (balanced diet) both groups were switched in a cross-over design repeating the days trial period. this design was carried out repeatedly with high p diets differently composed concerning ca/p ratios and p sources. endogenous creatinine clearance, glucosuria, microalbuminuria, water and mineral balance were determined at each period. the study was approved by the proper authority for animal welfare. results and discussion: the studies confirmed the results of pastoor ( ): a p content of approximately mg/mj me in a diet consumed by healthy cats at maintenance may lead to a decrease of the creatinine clearance. markers of acute tubular damage, i.e. glucose and microproteins in the urine, showed positive results in several trials. the p concentration of a diet alone is no sufficient marker of its tolerance since ca/p ratio and p origin influence the effects. therefore, high p diets cannot be considered safe and should be avoided also in healthy cats. literature the study aim was to investigate serum global proteomes in dogs with overt dilated cardiomyopathy (dcm) and to correlate protein expression in serum with that in ascitic fluid. eight dogs diagnosed with dcm based on echocardiographic evidence including increased left ventricular dimension at diastole and systole, increased e point to septal separation, and decreased fractional shortening were included in the study. serum and ascitic fluid samples were analyzed for proteomes using a label-free lc-ms/ms method. eight dogs from different breed, sex and age served as controls. proteome analyses revealed significantly different expressions of eight proteins in all samples. expressions in serum of apolipoprotein a , ig heavy chain v, superoxide dismutase and plasminogen were higher (p < . ), while expressions of clusterin, hemoglobin subunit ß, apolipoprotein c ii, b glycoprotein i (ß gpi) were lower (p < . ) in dogs with dcm than in control dogs. in addition, apolipoprotein a , clusterin, hemoglobin subunit ß, ig heavy chain v, plasminogen and ß gpi were down-regulated whereas apolipoprotein c ii and superoxide dismutase were upregulated in ascitic fluid compared with serum in dogs with dcm. data obtained in the present study suggest that serum and/or ascitic fluid proteomes may help explain some of the pathophysiological mechanisms involved in the progression of dcm. tick paralysis is an important disease of dogs and cats in australia, induced by toxins of the paralysis tick ixodes holocyclus, very commonly occurring from spring to autumn on the eastern seaboard. respiratory failure is one of the major clinical derangements occurring in severe cases of tick paralysis, although its pathogenesis is poorly characterised. there is some suggestion that the respiratory failure is secondary to toxin-induced myocardial dysfunction with the subsequent development of cardiogenic pulmonary oedema. the purpose of this study was to determine cardiac involvement in dogs infested with ixodes holocyclus, through measurement of cardiac biomarkers. a cross-sectional study of client-owned dogs was undertaken. dogs enrolled in the study belonged to one of groups: dogs with tick paralysis and no-mild respiratory compromise (group a), dogs with tick paralysis and moderate-severe respiratory compromise (group b) and a control group of dogs with neither tick paralysis nor respiratory compromise. respiratory compromise was scored using a commonly employed grading system. each animal had the following parameters determined: serum cardiac troponin i (ctni) concentration, plasma n-terminal pro-btype natriuretic peptide (nt-probnp) concentration and serum creatinine concentration. for most dogs, but not all, spo was also determined. mean nt-probnp concentrations were significantly lower in dogs with tick paralysis than those in the control group, with no statistical difference detected between dogs with and without respiratory compromise. there was no significant difference in mean ctni concentrations between groups, however there were some high outliers of ctni concentration. creatinine concentrations differed significantly between each group, with the control group having the highest mean creatinine and those in group b having the lowest mean creatinine. there was no significant difference in spo between groups. this study showed no compelling evidence of cardiac insult as measured through cardiac biomarkers in our cohort of dogs with tick paralysis; however there was evidence supporting reduced preload in these dogs. in addition, the results of this study suggested that a small subset of patients with systemic hypoxaemia might have some loss of cardiomyocyte integrity. disclosures: employee/salary: gp nicolson, r malik and nj beijerink are employees of sydney university; alh mcgrath is a student at sydney university; ra webster is an employee of the animal emergency service; carrara; s kaye is an employee of queensland veterinary specialists, stafford heights; j li is an employee of northside emergency veterinary service, forestville. grants/research: this study was funded by bequest grants provided by the faculty of veterinary science at the university of sydney. idexx laboratories provided some funding for the laboratory tests. no other disclosures. speaking & consultancies: none related to this presentation. investments/commercial interests: none related to this presentation. gifts, hospitality, travel support: none related to this presentation. other: none related to this presentation. the use of ntprobnp, troponin i (high-sensitivity, ctni) and pdk pre-screening for occult dilated cardiomyopathy(odcm) in the doberman pinscher(dp) has been previously reported. the aim of this prospective collaborative study was to identify robust pre-screening recommendations for dp utilizing the current generation of commercially available diagnostic tests. a cohort of asymptomatic dp were evaluated at the american doberman national specialty show in , , and (n = , median age years, range - ). evaluations consisted of auscultation, echocardiography (echo), -minute ecg (ecg), ntprobnp (cardiopet plus r ), ctni, and pdk . dp were classified as affected (odcm) if their lvids was > the protect entry criteria with or without vpcs (n = ). dp were classified as normal (nl) if their lvidd and lvids < protect entry criteria and they had no vpcs (ntprobnp:n = , ctni:n = , pdk ;n = ). roc analysis comparing odcm and nl was done for ntprobnp, ctni, and pdk . overall accuracy (percent correctly classified) was considered for individual tests as well as a variety of combinations. the goal of combining tests was to eliminate false negatives while minimizing false positives. the auc for ntprobnp, ctni and pdk was . , . and . respectively with the percentage correctly classified equal to . , . and . (including false negatives for pdk ) when a cut-off of pmol/l, . ng/ml or a positive pdk (hetero-or homozygous) were used respectively. when the cutoffs for ntprobnp and ctni are used in combination the auc was . and . % were correctly classified ( false negatives, false positives). disclosures: research and programatic support from idexx the lab that runs ntprobnp. the study was sponsored by boehringer ingelheim, idexx and the doberman pinscher society of america. with renal disease. r. langhorn , a.s. kloster , l.r. jessen , a. jensen , j. koch . university of copenhagen, frederiksberg c, denmark, copenhagen small animal hospital, valby, denmark cardiac troponins are sensitive and specific markers of myocardial injury. however, their reliability in renal disease has been questioned due to possible renal involvement in troponin elimination. the purpose of this study was to examine whether cardiac troponin i (ctni) is elevated in cats with renal disease and no concurrent cardiac disease, and whether ctni is measurable in urine of cats with normal and compromised renal function. cats presenting with renal disease or primary structural cardiac disease were enrolled in a renal and a cardiac group, respectively. a healthy control group was similarly included. clinical and echocardiographical examination was performed and blood and urine samples obtained for each cat. the mann-whitney u test was applied to evaluate differences between groups. seven cats with renal disease, cats with cardiac disease, and healthy cats were included. serum ctni concentrations were (median [range]) . [ . - . ] ng/ml for the renal group, . [ . - . ] ng/ml for the cardiac group, and . [ . - . ] ng/ml for the control group. the renal group had significantly higher serum ctni concentrations than the control group (p = . ), but was not significantly different from the cardiac group (p = . ). urine ctni was measurable in . % ( / ) of cats in the renal group ( . [ . - . ] ng/ml), % in the cardiac group, and . % ( / ) of controls ( . ng/ml). it was concluded that elevated serum ctni in cats with renal disease may occur without concurrent cardiac disease. moreover, compromised renal function was associated with presence of ctni in urine. disclosures: no disclosures to report. sudden death (sd) commonly occurs in dog breeds with a high predisposition to vpds and vt, occuring in about % of asymptomatic doberman pinschers (dp) and % of dp with chf, and reported in % of boxers with arvc. in human patients with atrial fibrillation (af) on anticoagulant therapy for stroke prevention (n = ), cardiac death (sd and progressive heart failure) has been reported to account for . % of all deaths. the objective of this study was to evaluate the incidence of sd in irish wolfhounds (iw) with dcm and/or af. iw from western europe (n = ) were examined by physical examination, standard echocardiography and electrocardiography between / - / (av). dogs were longitudinally followed, and owners instructed to report date and circumstances of death. dcm and/or af were diagnosed in %. long-term follow-up until death was possible in ( m, f) dogs with dcm and ( m, f) dogs with lone af. based on the initial diagnosis, disease groups were established. results: sd occurred in to % of all groups with dcm or af: ( ) out of dogs with dcm +af, sd occurred in % after median ( - ) days, median age . ae . years. ( ) out of dogs with dcm +sinus rhythm, . % died from sd after median ( - ) days, median age . ae . years. ( ) out of iw with dcm, af +chf, . % died from sd after median ( - ) days, median age . ae . years. ( ) out of iw with lone af, sd occurred in . % after median ( - ) days, median age . ae . years, of these, dogs had developed dcm prior to death. sudden cardiac death (sd) from cardiac arrest is the most common cause of death in people worldwide, accounting for > % of all deaths from cardiovascular disease. ventricular tachycardia (vt)/ fibrillation (vf) is the most common cause of sd, other causes include pulseless electrical activity. the fatal arrhythmia has not recorded in iw. in this study, vpds were recorded at one or more occasions in / iw with af, and in / with dcm, while in iws without heart disease vpds were seen in . % of males and in . % of females. in conclusion, sd occurs in . % of iw with lone af before or after development of dcm and chf, and in . % of dogs with dcm. disclosures: no disclosures to report. tachycardia may induce dilated cardiomyopathy (dcm). irish wolfhounds (iw) are commonly affected with dcm and atrial fibrillation (af). the objective of this study was to compare heart rates (hr) of iw with lone af with hr of an age and gender matched control iw cohort that had neither af nor dcm until death and to iw with dcm with either congestive heart failure (chf), af or sinus rhythm (sr). all disease groups had hr recorded before and after - months of medical therapy. out of iw with cardiovascular examinations including standard echocardiography and electrocardiography long-term follow-up until death was possible in ( m, f) dogs with dcm and ( m, f) dogs with lone af. based on the initial diagnosis, disease groups were established. dogs received single or combination treatment of metildigoxine, aceis, pimobendan, diltiazem, furosemide, spironolactone, atenolol and sotalol. mean hr during minutes ecg monitor recordings with print-outs were evaluated. the differences in hr in the disease groups before and after treatment versus controls were examined by analysis of variance with post hoc multiple comparisons (dunnett t ). mean hr in ( m, f) control dogs was . ae . bpm. mean hr in iw with lone af was . ae . bpm before, and . ae . bpm with therapy. mean hr of dogs with dcm +af was . ae . bpm before, and . ae . bpm with therapy. mean hr of dogs with dcm +sinus rhythm (sr), was . ae bpm before, and . ae sd bpm with therapy. mean hr of iw with dcm, af +chf, was . ae . bpm before, and median . ae . bpm with therapy. in conclusion, compared to control dogs, untreated iw with chf, with dcm+af, and iw with lone af had statistically significant (p = . ) increased hr, but not dogs with dcm and sr, while under medical therapy elevation of hr was only significant (p = . ) in iw with chf and dcm. disclosures: no disclosures to report. echocardiography, as a noninvasive method, is being increasingly used as a complementary means of diagnosis in small animal clinical practice. the need for standardization of techniques by ultrasound operators in the measurement of the different echocardiographic parameters is essential for a proper examination. the aim of this work was to check a potential correlation between the values obtained in right parasternal long-axis and short-axis views in -dimensional mode and m-mode. twenty persian cats were submitted to a complete physical examination, clinicopathologic tests (hematocrit, total solids and t hormone), systolic blood pressure measurement using doppler and echocardiography. seventeen cats fulfilled the criteria inclusion and were included in the study. two-dimensional mode and m-mode echocardiograms were recorded, in systole and diastole, from both short-axis and long-axis views for evaluation of left ventricular internal diameter (lvd), interventricular septum thickness (ivs), left ventricular free wall thickness (lvpw), aorta diameter, left atrium diameter (la), pulmonary artery diameter, shortening fraction (fs) and ejection fraction (fe). statistical analysis included paired t-test (wilcoxon test) and a linear regression analysis with graphical analysis to assess agreement between the methods of data acquisition. there was a highly significant correlation (p < . ) between the values obtained in short-axis and long-axis views for the parameter la diameter (longitudinal: . ae . cm; transversal: . ae . cm), a very significant correlation (p < . ) for the parameter lvds (longitudinal: . ae . cm; transversal: . ae . cm), and significant correlation (p < . ) for the parameters ivss (longitudinal: . ae . cm; transversal: . ae . cm), lvpws (longitudinal: . ae . cm; transversal: . ae . cm) and fs (longitudinal: . ae . %; transversal: . ae . %), with no significant correlation (p > . ) between the methods for the remaining parameters. in conclusion, the data obtained from right parasternal short-axis and long-axis recordings cannot be used interchangeably in the evaluation of diastolic parameters in normal adult cats. disclosures: no disclosures to report. real time three-dimensional transesophageal echocardiography (rt dtee) is an established imaging modality for interventional cardiac procedures in humans. it has been shown to yield comprehensive views of the cardiac valves and congenital heart defects. it potentially provides a more accurate echocardiographic means of evaluating cardiac chamber volumes and a more precise pre and postoperative tool. rt dtee was used in combination with conventional -dimensional transesophageal ( -d tee) and transthoracic echocardiography (tte) standard imaging protocols. the pulmonic valve anatomy and function was evaluated in client-owned dogs with severe valvular pulmonic stenosis prior to and post-balloon valvuloplasty. the -d images were obtained with the phillips ie and cx cardiac ultrasound systems using a -d transesophageal - mhz xmatrix probe. standard cardiac - mhz, - mhz and - mhz sector array probes were used to acquire d tte images. diagnostic images were obtained in all examined patients. rt dtee did not change the balloon size decision when compared with -d tee, but provided additional views, detailed anatomy of the pulmonic valve cusps and commissures, as well as thickness and mobility of the pulmonic valve cusps, when compared to those obtained with -d tee or tte. successful balloon valvuloplasty was achieved in of the patients. repeatable artifacts occurred with respiratory excursions and insufficient probe contact. no complications related to rt dtee were observed. rt dtee provided enhanced views of the pulmonic valve while aiding in the procedure guidance and evaluation of the results post-balloon valvuloplasty. a better understanding of the anatomy of the pulmonic valve may improve procedure success. immediate visualization of the results post-balloon valvuloplasty may reduce patient risk and fluoroscopy time. a larger sample and further research will be needed to establish guidelines and predict success based on particular valve anatomy. we can conclude that rt dtee provided additional anatomical and intraprocedural information and was well tolerated in this group of dogs. disclosures: no disclosures to report. pimobendan is an inodilator utilised extensively in the treatment of canine congestive heart failure. several retrospective studies evaluating clinical records have suggested that it is well tolerated in cats; however its efficacy in this species remains ill-defined. moreover, a recent pharmacokinetic study found peak plasma concentrations of the drug to be around ten times greater than those reported in the dog, thus highlighting inter-species differences in the pharmacokinetics and, potentially, pharmacodynamics of this drug. this study was conducted to evaluate the cardiovascular effects following oral doses of pimobendan in healthy cats. a placebo-controlled, randomised, operator-blinded crossover study was conducted in healthy cats (weight range . - . kg) to evaluate the effect of doses of pimobendan (high dose [hd]: . mg vetmedin chewable tablet po; low dose [ld]: . mg vetmedin chewable tablet po) and placebo ([pl]: water po) on cardiovascular parameters over time. standard echocardiography ( d, m-mode, and spectral doppler) and oscillometric blood pressure measurements (vethdo) were performed repeatedly for hours following dosing. each measured parameter was evaluated for between-and within-treatment effects over time using linear mixed modeling with reml estimation to account for intercat variability. heart rate was used as a proxy for the level of anxiety experienced by the cats, and adjustment for this was performed through inclusion of heart rate as a fixed effect in the final model. the effect of treatment with pimobendan was most evident in the left ventricular internal diameter in systole (lvids). maximal effects occurred hours following treatment with hd and ld. the predicted mean reduction from baseline following heart rate adjustment at this time for lvids was . mm ( % reduction) and . mm ( % reduction) for hd and ld, respectively. although there were no significant differences between hd and ld in the magnitude of effect at any given time point, lvids remained significantly reduced from baseline and the pl group for longer in the hd ( minutes to hours following dosing) than in the ld group ( to hours following dosing). significant treatment effects on aortic velocity and fractional shortening were also present, but to a lesser degree. these results demonstrate that treatment with pimobendan results in measurable changes to systolic indices in cats. a dose-dependent increase in duration of effect was also observed. further studies are required to characterise the optimal dose of pimobendan in cats and to evaluate its efficacy in clinical patients. disclosures: this study was funded by grants provided by the faculty of veterinary science at the university of sydney. m. yata received financial support from luoda pharma, the australian postgraduate awards scholarship, and the eric horatio maclean scholarship whilst undertaking this project. none of the authors involved in this study have current affiliations with the drug company that manufactured the product used in this study (boehringer ingelheim pimobendan has positive inotropic, positive lusitropic and vasodilator effects and is licensed for use in dogs with cardiac disease in many countries. numerous studies have shown benefit with the use of pimobendan in canine dilated cardiomyopathy and chronic degenerative mitral valve disease, and whilst not licensed for use in cats, recent studies have reported benefits with the use of oral pimobendan in a variety of cardiac diseases including dilated and hypertrophic cardiomyopathies. an intravenous formulation has been available in the uk since january . the use of intravenous pimobendan in cats in the clinical setting has not previously been described. the aim of this study was to describe the use of intravenous pimobendan in cats with naturally occurring heart failure and report tolerability and side effects/adverse reactions. the hospital data base was searched for the use of intravenous pimobendan in feline patients. signalment, presenting signs, investigations, diagnosis, dose and time of pimobendan administration, concurrent medications, short-term outcome and adverse reactions were recorded. a boarded-certified cardiologist retrospectively reviewed all the cases in order to confirm the diagnosis. all owners had signed consent forms to permit use of off licensed drugs. eight cats were included in the study. median age was . years (range, . - . ) . six ( %) were male and ( %) were domestic short-haired. weight ranged from . to . kg. all presented with dyspnoea. three out of cats ( %) had a heart murmur and out of ( %) had a gallop rhythm. different heart conditions were diagnosed including / cats with cardiomyopathy and / with suspected endocarditis. median dose of intravenous pimobendan was . mg/kg (range, . - . ). concurrent drugs administered included frusemide, dalteparin, terbutaline, dexamethasone, amoxicillin-clavulanate, maropitant, midazolam, butorphanol, methadone, glyceryl trinitrate, clopidogrel, aspirin and potassium gluconate. no immediate adverse reactions/side effects were observed in any of the cats. five of the cats were discharged from the hospital between and hours post pimobendan administration. one cat was euthanatized, one died during thoracocentesis and one had a thromboembolic episode between and hours post pimobendan administration. intravenous pimobendan was well tolerated by this clinical population of cats with heart failure. no immediate adverse reactions/ side effects were observed. the intravenous route may be considered as an alternative method of administration of pimobendan in cats with heart failure. disclosures: no disclosures to report. spironolactone (sp) is an aldosterone receptor antagonist, registered in europe for the treatment of congestive heart failure (chf) caused by valvular regurgitation in dogs, in combination with standard therapy. in cats, cardiomyopathy (cm) is the predominant cause of heart failure. to evaluate the safety and efficacy of sp in cats with cm, a double blind, randomized placebocontrolled study has been conducted with cats receiving either sp ( . to . mg/kg po once daily) or placebo for up to months in addition to benazepril and furosemide (dose at clinician's discretion). cats ( dsh, ragdoll, siamese and burmese) with cm of various types ( hypertrophic, dilated, unclassified and arrhythmogenic right ventricular) were enrolled. the cats were randomized to either group a or b according to the presence of hcm or not and whether the cat required hospitalization due to clinical need or not. cats were recruited to group a (sp) and cats recruited to group b (placebo). the only significant difference between the groups at baseline were aortic diameter (p = . ) larger in the sp group, and la:ao ratio (p = . ) larger in the placebo group. the survival analysis showed a survival rate at months respectively of % and % in the intention to treat (itt) and per protocol (pp) populations in the sp group and % and % in the placebo group. the difference between the groups was significant (log rank test: itt population p = . ; pp population p = . ). the hazard ratio indicates an % (itt) and % (pp) reduction in risk of an event occurrence in the sp group. the effect of covariates (age, weight, bcs, systolic blood pressure, ratio la/ao) was not significant. although this is a pilot study with small numbers of cats, this data would suggest that spironolactone is likely to be beneficial in the treatment of cats with congestive heart failure secondary to a cardiomyopathy. disclosures: the study was joint funded by ceva and the university of nottingham. the authors have the right to publish the results. of the study irrespective of outcome. the objectives of this study were to describe pulmonary transit time and myocardial perfusion normalized to heart rate (nptt and nmp, respectively), evaluated by means of contrast echocardiography, in dogs with stable stage c acvim myxomatous mitral valve disease (mmvd), and to assess short-term effects of pimobendan on these parameters. we hypothesized that nptt and nmp are increased in dogs with mmvd compared to normal dogs. additionally, we hypothesized that treatment with pimobendan will decrease both variables in dogs with mmvd. we prospectively enrolled normal dogs and dogs with stable stage c acvim mmvd. all dogs had a standard and contrast echocardiographic examination at the beginning of the study. at this time, mmvd dogs were randomly assigned to receive either pimobendan ( . - . mg/kg) or not. all dogs with mmvd were re-evaluated by means of standard and contrast echocardiography after week (t ), by operators blinded to the dog's treatment. our results show that nptt was significantly increased in dogs with mmvd (p = . ), compared to normal dogs. nptt was significantly decreased at t in dogs receiving pimobendan (p = . ). nmp was not significantly different in dogs with mmvd, compared to healthy dogs (p = . ), and it was not significantly different at t in the treatment group (p = . ). in conclusion, contrast echocardiography is a valid, complementary tool for echocardiographic analysis of dogs with mmvd. pimobendan decreases nptt in dogs affected by mmvd. myocardial perfusion is not different in dogs with mmvd and is not changed by pimobendan treatment. disclosures: michele borgarelli has received research funding by boheringher inghelheim for this study. in human beings, assessment of atrial function using -dimensional speckle tracking echocardiography (ste) is useful in several cardiovascular diseases. to date information on the use of ste for the evaluation of canine atrial function is lacking. we assessed the feasibility and reproducibility of ste in the assessment of left atrial (la) function in healthy dogs and dogs with myxomatous mitral valve disease (mmvd) and we compared ste derived indices with other parameters of left atrial and ventricular function and morphology. privately owned dogs including clinically healthy dogs (control, h) and dogs with mmvd subdivided according to heart failure class (b , b , c+d) were enrolled. standard echocardiographic examination was carried out in all dogs. furthermore, video clips were acquired from a -chamber apical view and ste analysis was done using dedicated software. for the ste analysis a region of interest was drawn including the entire left atrial wall. the software provided a strain/time curve that represents the degree of deformation of the la wall over the entire cardiac cycle. similarly, la areas are provided. the following variables were evaluated: peak atrial longitudinal strain (pals, %), as the point of maximal systolic strain; peak atrial contraction strain (pacs, %) just before atrial contracting phase; contraction strain index (csi, %) calculated from these variables. la areas were recorded during ventricular systole (la maximum area, laamax, cm ) and atrial contraction (la minimum area, laamin, cm ), and the la fractional area change (fac, %) was then calculated. the intra-and inter-observer variability was assessed using the coefficient of variation (cv, %). variability was low for all variables (cvs < left atrial measurements are commonly obtained by cardiologists to assess severity of left heart disease. traditionally, measurements were obtained from m-mode images, however several studies have examined measurements of left atrial dimensions and areas from -dimensional ( d) images. studies have demonstrated the interobserver variability of aortic valve measurements, and the effects of timing of the measurements throughout the cardiac cycle. however, few studies have examined interobserver variability of left atrial measurements from d images, factors affecting variability, or the consequences of this variability on ascribing degree of disease severity to a patient. images of the right parasternal short-axis view of the left atrium and aorta were provided to cardiologists or cardiology residents. the images depicted left atria of varying size, from both dogs and cats, ranging from normal to markedly enlarged. each participant placed arrows on each image to denote the start and finish points of their left atrial measurements without prior instructions -the first being near the interface with the aorta and the second being along the caudolateral border of the left atrium. thus, sets of images were analyzed. d distributions were mapped and analyzed to determine dispersion of the start and finish points. these were compared between images to look for association with severity (estimated as the median la:ao for each image) and image complexity. results: variability of measurements around the origin of the la measurement (interface with aorta) was small, and scaled with increasing heart size. variability at the distal measurement point was complex. in only / images was interobserver variability < . la:ao, and ranged up to la:ao ( % to % variability). a systematic observer effect was noted. variability did not appear to scale with severity of disease or image complexity, although the cases with the greatest variability had severe enlargement and indistinct margins. conclusion: this study demonstrates that highly trained individuals vary considerably in their measurement of left atria from the right parasternal short-axis view. the variability did not increase with increasing disease severity or image complexity. in some instances, the same patient could be classified differently by different observers if relying on la:ao thresholds. the study suggests that standardized methods of measurement should be developed to minimize this variability. disclosures: the study was supported by veterinary information network (salary, imaging software). there is growing evidence that fibrosis plays an important role in the development of remodeling and heart failure during cardiac diseases. at cellular level, fibrotic processes are prior to clinical manifestation of symptoms. fibrosis and extracellular matrix remodeling influences cardiac function in a negative manner. to our knowledge there is no biomarker, which is able to properly detect heart-specific fibrotic processes and remodeling in the peripheral blood. such a biomarker would be of great importance in cardiac diagnostics, risk stratification and therapy monitoring. in a preliminary study, using microarray method and pathway analysis in pooled samples, we were able to identify heart specific gene-expression profile representing fibrotic and inflammatory processes in the peripheral blood of tachypacing-induced cardiomyopathy model dogs. our results were validated by histopathology and quantitative real time rt-pcr (qrt-pcr). based on our microarray results, in this current study we aimed to select and investigate a panel of possible cardiac fibrosis and remodeling specific genes in blood. whole blood and left ventricular samples of tachypaced dogs (n = ), healthy controls (n = ) and blood samples of canine clinical patients (n = ) with different cardiomyopathies were collected in rna-stabilizing solution. rna integrity was confirmed by capillary electrophoresis (rin> ). exon-spanning primers were designed. expression of selected genes were measured by sybr-green based qrt-pcr and normalized to different housekeeping genes (hprt , rps ) by ddct method. quality controls were made by melting curve analysis and size determination of the pcr products by agarosegel electrophoresis. for data evaluation descriptive statistics, student's t-test and mann whitney u-test were used. the selected gene-expression panel consisted of different mrnas which represent main biological processes related to fibrosis, remodeling and impaired contractility. col a , mmp , timp , vcan, spp are directly related to collagen turnover and remodeling. il , ccl are inflammatory markers. stc , hsp , s a are early stress response genes. tgfb has central role in fibrosis while myh and myh ensure cardiac specificity. we found significant up regulation (p < . ) of col a , timp , vcan, spp , il , ccl , stc , hsp , s a , tgfb and down regulation of myh , myh genes in the heart tissue samples. all targets also showed similar changes in the blood samples except mmp , however not all alterations were significant. based on this selected panel clinical cases could be clearly differentiated from healthy dogs using their gene-expression pattern in blood samples. our findings suggest that the peripheral blood may have a potential to reveal cardiac specific fibrosis in dogs. disclosures: no disclosures to report. within the distal nephron, the enzyme -beta hydroxysteroid dehydrogenase ( bhsd ) protects the mineralocorticoid receptor (mr) from activation by cortisol, allowing it to interact with aldosterone. in humans, mutations of bhsd cause apparent mineralocorticoid excess, characterised by sodium and water retention with resultant hypertension. sodium and water retention is also a hallmark of canine congestive heart failure (chf). this could partly be explained by dysregulation of renal bhsd activity, exposing mr to activation by cortisol. the aim of this study was to investigate the activity of renal bhsd in canine chf by measuring the concentration of cortisol and its metabolites in the urine from affected dogs. owners collected urine in a home environment from healthy adult dogs (n = ), and from dogs prior to presentation with non-cardiac chronic disease (n = ), and dogs with cardiac disease (isachc ib, n = ; isachcii or iii, n = ). levels of cortisol (f) and cortisone (e) excreted in urine were measured by mass spectrometry. urinary cortisol was normalised to creatinine to account for variations in glomerular filtration rate. cortisol was also measured in plasma obtained from all unhealthy dogs. plasma cortisol levels (p = . )and urinary cortisol:creatinine ratio (p = . ) did not differ between groups. however, the f/e ratio, was increased in dogs with class ii-iii heart failure (p = . ). an increased f/e ratio, in the presence of unchanged plasma cortisol, implies decreased renal bhsd activity and enhanced mr activation by cortisol in canine chf. this data suggests that changes in renal cortisol metabolism in canine chf cannot be explained by chronicity of disease, that the urinary f/e ratio has potential as a biomarker for canine chf and that renal bhsd could offer a therapeutic target in its management. further studies investigating bhsd expression and bioactivity in canine chf are ongoing. disclosures: supported by the fiona and ian russell seed corn grant through the university of edinburgh. in humans endomyocardial biopsy (emb) is highly recommended in case of unexplained left ventricular dysfunction associated to ventricular arrhythmias (va) or high-grade atrioventricular block (avb). despite the frequency of these conditions in dogs, histopathology data are lacking. the aims of this study were to describe the feasibility of emb in dogs and to investigate a possible role of viral myocarditis in case of unexplained dilated cardiomyopathy (dcm) phenotypes, high-grade avbs, supraventricular arrhythmias (sva) and va. twenty-five dogs of different breeds, m/f , , mean age . + . years, mean body weight . + . kg, presented for third degree avb / , dcm / , va / , sva / , and va+sva / , underwent percutaneous right emb under general anesthesia throughout the jugular vein. for each dog clinical records were analyzed. in all dogs at least one right ventricular sample (range - ) was collected for histopathology and immunohistochemistry; in / dogs at least one sample (range - ) for viral pcr was collected. all histopathologic samples were stained with haematoxylin and eosin, masson's trichrome and red elastic picrocirius. in selected cases stains with monoclonal anti-cd and anti-cd were performed. nucleic acids were obtained after sample storage in rna later solution, disruption with tissue lyser and extraction with trizol; and tested for canine viruses (enteric and respiratory coronavirus, herpes virus, distemper virus, adenovirus and , and parvovirus) and for west nile virus and bartonella spp. seven out of dogs had aspecific signs of cardiomyopathy and / suggestive of arrhythmogenic right ventricular cardiomyopathy (arvc). emb gave normal samples in / dogs and not diagnostic in / dog. nine out of samples were suggestive of myocarditis at different stages ( third degree avb, dcm and sva). two of these dogs resulted positive for virus ( enteric coronavirus, herpes virus). none of the dogs had positive immunoistochemical stains. two dogs with cardiomyopathy were positive for herpes virus and for herpes virus and parvovirus, respectively. both of these dogs came from a kennel. no complication was noted in / dogs, one dog had self-limiting pericardial effusion. this study showed, similarly to human cardiology, that emb is a safe and useful technique that allows recognition and classification of unexplained myocardial and rhythm disorders, % of which possibly associated with viral myocarditis. further studies are needed to prove the relationship between viruses and myocarditis in a larger cohort of dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right ventricle (rv) is challenging. studies lack quantitative assessment of the rv in dogs. the goal of this study therefore was to evaluate rv morphology and systolic function using different transthoracic echocardiographic (te) views and to compare the results with magnetic resonance imaging (mri) measurements in adult healthy anesthetized beagles. te variables were rv wall thickness (wt) in short axis and from a subcostal view, fractional shortening (fs), rv fractional area change (fac) from different apical views (an optimized view for the rv and a standard chambers view), tricuspid annular plane systolic excursion (tapse) from different apical views, right ventricular outflow tract diameter at proximal (rvot ) and valvular (rvot ) levels both obtained in long and short axis, tissue doppler imaging (tdi) derived tricuspid annulus systolic wave (s¢), isovolumic contraction velocity (ivcvel), and isovolumic contraction acceleration time (ivcat).mri variables were rv wt, rvot in short and long axis and rvot , ejection fraction (ef), and stroke volume (sv) based on flow quantification. there was no difference between rv wt measured with te in both short axis ( . ae . mm) and subcostal views ( . ae . mm) and mri ( . ae . mm). no difference was found between rvot or rvot when measured in long ( . ae . mm for the former, . ae . mm for the latter) and short axis ( . ae . mm for the former, . ae . mm for the latter) with te; however rvot in both short and long axis was overestimated by te compared to mri ( . ae . mm in long axis, and . ae . mm in short axis). both rvot in short and long axis obtained by te were lower than mri values ( . ae . mm). te fs was . ae %. values of tapse varied significantly when using different apical views, optimized ( . ae . mm) and standard chambers ( ae . mm); the same was true for fac (optimized view, . ae . %; standard chambers, . ae . mm). tdi s¢ was . ae . m/s, ivcvel was . ae . m/s, and ivcat was . ae . msec. the only te correlations found were tapse with fac and s¢. the only echocardiographic variables correlating with the mri based sv ( . ae . ml) were fac and s¢. mri based ef ( . ae . %) did not correlate with any echocardiographic variable. this new approach to assess rv function revealed problems similar to earlier attempts. without a reliable standard for comparison of quantitative results, the value of any te parameter is questionable. furthermore, te parameters obtained from different views produced different results, indicating that standardization maybe difficult, respectively increasing the risk of variability. disclosures: no disclosures to report. spontaneous echo contrast (sec) or 'smoke' is caused by low blood velocity and appears on ultrasound as a swirling blood flow pattern. it is associated with increased risk of thromboembolism in small animals. detection of sec is entirely subjective and there is limited consensus in veterinary medicine regarding the echocardiographic technique that is best for detection of sec. the main hypothesis of this study was that dimensional ( d) colour tissue doppler imaging (tdi) would significantly outperform d colour and grey scale as the best echocardiographic technique to view sec. a further hypothesis was that colour blindness would have no influence on results. echocardiographic data was obtained retrospectively from small animal cases that presented to the university of glasgow small animal hospital. all cases had evidence of sec. using a ge vivid echocardiography machine each of the cases had one video loop recorded with d colour tdi. colour tdi was then replaced by different d colours (gold, sepia and aqua) and different grey scale colours (machine presets and ) and video loops recorded. for each case the order of the recorded video loops was randomised. the video loops from the cases were viewed in standardised conditions by observers (veterinary cardiologists, residents, interns and students); observers had full colour spectrum vision (fcsv) and were red-green colour blind (deuteranopia). each observer ranked their ability to see sec with each colour, with being the least able to visualise sec and being the best able to visualise sec. binary logistic regression using minitab (version . ) was used to identify factors associated with whether tdi was ranked best or not. potential explanatory variables that were examined were view and colour blindness. observer and case were also fitted as fixed and random effects to account for clustering within these variables. tdi was chosen by the observers / ( %) occasions as the best technique to diagnose sec, which was significantly more frequently than all other d colour views together (p < . ). there was no significant difference between colour blind observers and those with fcsv (p = . ) and there was no influence of observer or case on the final model. in conclusion, d colour tdi may assist in easier diagnosis of spontaneous echo contrast in veterinary medicine regardless of colour visual spectrum of the observer. easier detection of sec would allow earlier implementation of preventative measures. disclosures: no disclosures to report. the foramen ovale (fo) is a slit-like passageway between septum secundum and primum that typically closes after birth by fusion of these septa. in - % of humans, a patent foramen ovale (pfo) persists into adulthood. the prevalence of pfo in small animals is unknown. this interatrial channel may serve as a bypass to the pulmonary circulation and is an important cause of paradoxical embolism (pe) and stroke in people. the primary aim of the study was to evaluate the prevalence of pfo in a large population of dogs and cats. secondary aims were to gather data on the prevalence of atrial septal defects (asd) and on the potential association between pfo and a) clinical/pathological signs of stroke or thromboembolism and b) the presence of right-sided heart disease. hearts of all dogs and cats that underwent a full diagnostic post mortem examination were prospectively evaluated for a pfo in a blinded fashion (to clinical history and cause of death). in selected cases with patent and closed fo respectively, a histological examination of the interatrial septum was undertaken. clinical information and the results of the post mortem examination were only evaluated after all hearts had been examined. a total of hearts ( cats, dogs) were examined, of which cats ( %) and dogs ( %) with a median age of [ day- years] and years [ day- years] respectively, exhibited a probe-patent pfo. in adult animals with presumed normal right atrial pressure the prevalence of pfo was % (cats) and % (dogs). none of the animals had an asd. one dog with a pfo also exhibited an aortic thrombus; otherwise there was no evidence of pe in this population. in % (dogs) and % (cats) with closed fo the left side of the interatrial septum (septum primum) was still partially probe-patent through a channel extending from the left atrial crescentic ridge (ostium secundum) to the limbus, but closed at this level by a thin, easily rupturable membrane. in conclusion, pfos are common in dogs and cats, but less prevalent than in humans. in contrast, asds appear to be rare in either. despite the high prevalence of pfo, clinical complications seem to be very rare. the majority of dogs with a closed fo have a fossa ovalis that is weakly fused to the limbus, which may facilitate blunt trans-septal atrial catheterisation and provide an easier access to the left atrium. disclosures: jose novo matos and tony glaus have performed consultancy work for boehringer ingelheim and vetoquinol. in human medicine diabetes mellitus (dm) is known to lead to cardiovascular dysfunction and heart failure, characterized by early diastolic and late systolic dysfunction. diabetic cardiomyopathy has been defined as the existence of left ventricular dysfunction in diabetics without coronary artery disease, hypertension or other potential etiological conditions. the prevalence of a diabetic cardiomyopathy in cats has not been previously studied. we sought to prospectively identify if cardiac diastolic dysfunction was present or would develop in a population of cats with newly diagnosed dm. cats were recruited based on a diagnosis of primary dm. patients received physical examination, biochemical and hematologic profiles including thyroxin and insulin-like growth factor , urinalysis, blood pressure measurement, thoracic and abdominal radiographs and abdominal ultrasound. echocardiography was performed at both diagnosis and months post diagnosis. echocardiographic assessment included conventional d, m-mode, spectral and tissue doppler measurements. patients with relevant concomitant systemic illness or secondary dm were excluded from the study. healthy age matched control cats were retrospectively enrolled. thirty-two diabetics (d ) were enrolled in the study. eighteen were females and were males. mean age was . years. on march , cats had received a month echocardiographic control (d ). ten control cats were enrolled (c). eight were males and females. mean age was . years. results ( fluoroscopically guided pacemaker implantation imparts a risk of radiation exposure. ideally exposure risk should be minimized or avoided. we previously reported using transthoracic (tte) and transesophageal echocardiography to guide pda occlusion and balloon valvuloplasty. therefore, we hypothesized that tte could be used to minimize fluoroscopy time during pacemaker implantation in dogs. we implanted single bipolar lead pacemakers (vvir) in dogs, using either active or passive fixation, as determined by tte assessment of the right ventricular apical myocardium thickness: in dogs with an apical rv thickness < . mm we implanted passive fixation leads. dogs were anesthetized, positioned in right lateral recumbency on a standard echocardiography table and a left jugular vein exteriorization and venotomy were performed. in all dogs, a permanent pacing lead was advanced through the left jugular venotomy and was directed from cranial vena cava through the right atrium into the rv with tte guidance. echocardiographic right parasternal views optimized to visualize the pacing lead were used, starting with a short axis image of the right atrium and ending with a long axis view of the rv optimized to image the ventricular apex. after placing the pacing lead in the rv apex with tte guidance, and after acceptable measures of the capture threshold and impedance had been obtained, fluoroscopy was used to confirm lead placement. the pulse generator was connected to the pacing lead and secured within a right dorsal cervical pocket. incisions were closed routinely and post-implantation thoracic radiographs were performed. the pacing lead appeared hyperechoic on tte images and tte guidance provided images of a quality sufficient to clearly monitor implantation in real-time. real-time monitoring allowed for immediate corrections to pacing lead malpositioning or excessive looping. with active-fixation leads, tte allowed visualization of the fixation helix being implanted into the myocardium in some cases. the right parasternal echocardiographic window allowed imaging of the different positions of the lead in the cardiac chambers during the entire procedure. the implantations were successful in all dogs but in the first cases we required fluoroscopic guidance to follow the intraventricular progression of the lead. in the last dogs we only used the fluoroscope as an intraoperative x-ray machine (no cine mode). we have demonstrated that tte monitoring can guide pacemaker lead implantation and that fluoroscopy is only required to confirm the correct placement of the lead just before the end of the procedure. disclosures: no disclosures to report. deciding whether a cardiac murmur is innocent or the result of a congenital cardiac anomaly could be challenging. the gold standard method to differentiate innocent murmurs from congenital cardiac anomalies is echocardiogram, performed by a skilled operator. the present study investigated whether objective auscultation-criteria can differentiate innocent from pathologic murmurs in asymptomatic puppies between . - months of age. the null-hypothesis was that a systolic murmur with an intensity of - out of with a musical character is innocent. a total of puppies were included between july and january . these puppies originated either from breeders who brought their puppies to the clinic for screening for congenital porto-systemic shunts, or were referred to the cardiology service for evaluation of a murmur. of the puppies that were brought for shunt-screening puppies (age - days) had a murmur that was audible on every beat. all these dogs were small terrier breeds. dogs with intermittently audible murmurs were excluded. the remaining puppies (age - days) were referrals to the cardiology service. based on the above described auscultation criteria the murmur was classified as innocent in dogs and pathologic in dogs. on each of the dogs an echocardiogram was performed. no abnormalities were seen in of the dogs whose murmur was classified as innocent. echocardiography revealed one or more congenital cardiac anomaly in of the dogs whose murmur was classified as pathologic. the congenital anomalies were patent ductus arteriosus, pulmonic stenosis ( severe, moderate and mild; in combination with a small ventricular septal defect), aortic stenosis ( severe and moderate), ventricular septal defects, mild mitral valve dysplasia and double-chambered right ventricle (both moderate, isolated and combined with a mild aortic stenosis, small ventricular septal defect and mild mitral valve dysplasia). the puppy with the isolated mild mitral dysplasia had a soft systolic murmur with a musical character. on every puppy with a murmur of the shunt-screening group also a phonocardiogram was performed. phonocardiograms of the innocent murmurs revealed early systolic murmurs with low amplitude. auscultation turned out to be a sensitive and specific method to differentiate innocent murmurs from pathologic ones. phonocardiogram did not have an additional value. limitation: the above described findings may not be used in breeds predisposed to aortic stenosis, such as boxer. disclosures: no disclosures to report. since the first description of feline hyperthyroidism (feht ) several epidemiological studies have suggested diet as a causal factor of feht . the aim was to critically assess the evidence presented in epidemiological studies suggesting food-associated factors in etiology of feht . scientific literature was screened for peer reviewed publications investigating food-associated factors in feht since it was first described in . study designs were checked against classical epidemiology biases. food-associated factors showing an increased risk for feht were assessed for compatibility with the bradford-hill (b-h) criteria, which are used to evaluate whether an association involves a causal component. evidence for a causal component is higher when more b-h criteria are met. a total of publications investigating food-associated factors in feht were identified, all retrospective. three publications investigated qualitative factors only (e.g. preferred food flavors) but not quantitative. feeding canned food was not found to be a significant risk factor for feht in one study, while it was found to be a significant and quantitative risk factor in publications. however there were important limitations in their design: controls included sick cats ( studies), cases outnumbered controls ( studies), cases and controls differed in age ( studies), or diet information did not reflect diet fed at time feht developed ( studies). three out of b-h criteria were met when considering canned diet feeding as an increased risk for feht development. from the available literature there is insufficient evidence to conclude that canned diet is a food-associated factor in the etiology of feht . retrospective studies only describe an association and not a cause to effect relationship, are sensitive to bias from host and environment, and are less likely to identify etiologic factors when prevalence is below % as is the case in feht . hypotheses linking food-associated factors to feht (such as bpa, pbdes, flavonoids and iodine content) have never been proven to date, and lifelong prospective studies are required to investigate food-associated factors in etiology of feht . an iodine-restricted food has been recently introduced as a potential therapeutic option for feline hyperthyroidism. no controlled studies have been published on this treatment. the aim of this non-randomised controlled trial was to evaluate the effects of the iodine-restricted food on serum tt concentrations, clinical and clinicopathological parameters in client-owned cats with hyperthyroidism. indoor cats with newly diagnosed hyperthyroidism (consistent clinical signs and serum tt concentration > nmol/l), with or without mild to moderate azotemia (iris stage≤ ), were included. cats with severe concurrent disorders (i.e. lymphoma, neoplasia, malabsorption or severe azotemia [iris> ]) were excluded. cats were allocated on owner preference into groups: group a received an iodine-restricted food as a single therapy; the control group (group b) received transdermal methimazole in pluronic Ò lecithin organogel (plo, mg/ml) at the starting dose of . mg/cat q h. in both groups clinical parameters, biochemistry and serum tt were evaluated at baseline (t ), (t ), (t ), (t ), and (t ) days after treatment began. twenty-five cats were enrolled in the study, were included in group a, and in group b. no statistical differences were present between group at t for signalment, clinical and laboratory findings, including tt concentrations. in group a, only / cats ( . %) completed the study. the causes of interruption were: food refusal in / ( . %), loss to follow-up in / ( . %), death of unrelated cause in / ( . %), and poor owner compliance in / ( . %). in group b, / cats ( . %) completed the study. the causes of interruption were: suspected methimazole induced hepatotoxicity in / ( . %) and loss to follow-up in / ( . %). median serum tt concentration at t was nmol/l (range - nmol/l) in group a, and nmol/l (range - nmol/l) in group b. no significant differences were found in serum tt concentrations between group a and group b in any of the evaluated timing. bodyweight and serum creatinine significantly increased only in group b between t and t (p = . and p = . , respectively); nevertheless, urea did not significantly change in both groups. ast, alt, and alp significantly decreased only in group b between t and t (p = . , p = . and p = . , respectively). these results suggest that iodine-restricted food is effective in reducing serum tt concentration in hyperthyroid cats. compared to transdermal methimazole, food does not produce an increase in serum creatinine, but apparently is less effective in improving bodyweight and liver parameters. disclosures: no disclosures to report. the objective of this study was to identify whether pre-treatment plasma ionized calcium (ica) concentrations are predictive of hypocalcemia following parathyroid removal or heat ablation in dogs with primary hyperparathyroidism. fifty-five dogs seen between january , and february , met the inclusion criteria of having persistent hypercalcemia (defined as ica > . mmol/l) due to primary hyperparathyroidism, absence of pre-emptive calcitriol therapy, and ica monitoring post-operatively. each dog was treated with surgery (n = ) or ultrasound-guided percutaneous heat ablation (n = ). hypercalcemia resolved for all dogs within hours of the procedure. dogs were split into pretreatment ica groups of . - . mmol/l, . - . mmol/l and > . mmol/l. there was a significant association between higher pretreatment hypercalcemia and lower post treatment hypocalcemia. in addition, there was a significant dose-response relationship between pretreatment calcium concentration and the absolute decline in calcium following treatment. sixteen dogs became notably hypocalcemic (ica < . mmol/l). four out of dogs with pretreatment ica ≤ . mmol/l, out of dogs with ica between . - . mmol/l and out of dogs with pretreatment ica ≥ . mmol/l became hypocalcemic (ica < . mmol/l). adverse effects of hypocalcemia were observed in dogs, of which had pretreatment ica > . mmol/l. given the risk of significant hypocalcemia following parathyroid removal in dogs with pretreatment ica > . mmol/l, these patients should be treated to prevent rapid decline and development of clinical hypocalcemia. disclosures: no disclosures to report. diabetes mellitus is estimated to affect . % of pet dogs in the uk. most cases are thought to result from insulin deficiency resulting from loss of pancreatic beta cells, similar to human type diabetes. juvenile-onset diabetes is rare (catchpole, et al., ) . seven labrador retriever puppies were diagnosed with diabetes mellitus at between and weeks of age on the basis of the presence of hyperglycaemia, glucosuria and compatible clinical signs including polyuria, polydipsia and polyphagia. two of the dogs were known to be related (full siblings); of the remaining cases, other family members (full or half siblings or one sire) were also reported to be affected, though clinical details were not available. pancreatic histopathology at post mortem in cases showed islet hypoplasia. in the only puppy tested, hypoinsulinaemia was identified. two puppies were euthanased soon after diagnosis, were lost to follow up and survived to > years of age with well controlled diabetes. due to the early age of onset and occurrence within a single breed, it was hypothesised that diabetes in these cases might be due to mutation(s) in a single gene. three candidate genes were selected on the basis that they are the more common genetic causes of monogenic diabetes in human neonates or young children (kcnj , hnf a, hnf a). the coding regions of the canine orthologues of those genes (q bmm _canfa, hnf a, hnf a, respectively) were amplified and sequenced from genomic dna from all affected puppies and unaffected siblings and control labradors. no variants were identified in the coding sequences of hnf a or hnf a. in q bmm _canfa, novel, synonymous coding, single nucleotide base substitution variants were identified in of the affected dogs. however, these variants were considered unlikely to be the cause of the clinical syndrome, as their presence did not co-segregate with disease within families or across the cohort and did not change the protein coding sequence. in conclusion, we report a case series of labrador puppies suspected to be affected with monogenic diabetes mellitus, and results of candidate gene screening which did not identify a causa-tive mutation. further investigation of a possible genetic cause would be required to elucidate the cause of early onset diabetes in this breed. disclosures: er's salary and laboratory costs were paid using a grant from the wellcome trust. samples were submitted to the canine diabetes register at the rvc which has received funding from masterfoods, kennel club charitable trust, msd animal health and eu grant fp 'lupa'. meh is a trustee of the kennel club charitable trust. he was not part of discussions to award funding to the canine diabetes register. some samples were submitted as part of ljd's phd which was funded by the rvc (clinical research fellowship) with additional funding from intervet pharma r&d (currently msd animal health). diabetes mellitus (dm) is a common feline endocrinopathy and pathophysiologically similar to human type diabetes (t dm). t dm occurs due to a combination of insulin resistance and b-cell dysfunction. several studies have identified environmental and genetic susceptibility factors for t dm. in cats, environmental factors such as obesity and physical inactivity have been linked with dm; however, identification of genetic factors has been challenging. to date, mc r is the only gene shown to be associated with increased susceptibility to dm in overweight domestic short hair (dsh) cats. the aim of the present study was to perform a genome-wide association study (gwas) to identify loci associated with dm in lean dsh cats. illumina infinium k iselect dna arrays were used to interrogate genomic dna samples from lean diabetic dsh cats from the royal veterinary college feline dm archive and control dsh cats. the data was analysed using plink whole genome data analysis toolset. significance was established at p < À . snps with a minor allele frequency below . and a call rate below % and individuals with a genotyping rate < % were excluded from analysis. a total of , snps were available for analysis. after excluding cats with low genotypic rate, control dsh and lean diabetic dsh cats were evaluated. diabetic cats had a mean (sd) age of . ( . ) years; ( %) were male, ( %) female. non-diabetic cats had a mean (sd) age of . ( . ) years; ( %) were female, ( %) male. control cats were significantly older than diabetic cats (p < . ; t-test). five significant snps were identified: chra . (p = . À ); chrun . (p = À ); chrun . (p = À ); chrun . (p = À ) and chrun . (p = À ). the first snp is located within chromosome a ; the others are located within a . mb region towards the end of chromosome a . the snp in chromosome a is located kb upstream of dipeptidyl-peptidase- (dpp ), a peptidase similar to dpp- , involved in incretin inactivation. within the identified region of chromosome a , genes of interest include tmem and acp ; both have been associated with t dm in humans, most likely causing insulin resistance. this is the first gwas of dm in cats. a number of significant snps have been identified; some of which are located in proximity to genes that have been associated with t dm in humans others could be involved in pathophysiology related to dm. further investigation of these candidate genes is warranted. disclosures: snp chips for the gwas were provided by the morris animal foundation. diabetes mellitus (dm) and its treatment have been documented to exert a negative psychosocial impact on cats and their owners. common owner concerns include hypoglycaemia worry, socialand work-life impact and a desire for more control over their cat's treatment. home blood glucose monitoring (hbgm) has been suggested to enable superior glycaemic control and could address some of the above-mentioned quality-of-life (qol) issues. conversely, hbgm could also exert negative psychosocial effects such as disturbance of the cat-owner bond. this prospective -month trial aimed to document the acceptance rate of hbgm among owners of diabetic cats, reasons for declining hbgm, possible impact on glycaemic control and whether acceptance altered pet and owner qol measured through the use of a validated psychometric diabetic pet qol-tool (diaqol-pet). at baseline (m ) all owners of recently diagnosed cats received a veterinary glucometer (alphatrak Ò , zoetis) and a standardised demonstration of its use on their cat. diabetic management was standardised and included a low carbohydrate diet, twice-daily insulin following a standardised dose-adjustment-protocol according to, initially, weekly blood glucose curves (bgcs) carried out through hbgm (if adopted; hbgm-group) or in-hospital (if not adopted; non-hbgm-group). mann-whitney u-tests assessed for significant differences in fructosamine, average bgc-value, insulin dose and diaqol-pet-scores at m and month (m ) between the hbgmand the non-hbgm-group. fisher's exact test was used to compare remission rates; average values are given as median (range). hbgm was introduced to owners and was successfully adopted by ( . %). reasons for failure were patient aggression (n = ), owner concerns about patient distress (n = ) and lack of available assistance (n = ). at m , there was no significant difference in fructosamine (hbgm: ( - )lmol/l, non-hbgm: ( - )lmol/l; p = . ), insulin dose (hbgm: . ( - . )u/kg/dose, non-hbgm: . ( . - . )u/ kg/dose; p = . ), average bgc-value (hbgm: . ( . - . ) mmol/l, non-hbgm: . ( . - . )mmol/l; p = . ) or overall diaqol-pet-score (hbgm: À . ( . to À . ), non-hbgm: À . (À . to À . ). on examination of individual diaqolpet-categories hbgm-cat owners reported no significant difference in the bond they felt with their cat (p = . ), degree of worry about hypoglycaemia (p = . ) or restriction to their work-(p = . ) or social-life (p = . ) compared to the non-hbgmgroup. four hbgm-cats ( . %) achieved diabetic remission; none of the non-hbgm-group did (p = . ). hbgm can be successfully adopted in a majority of cat-owner combinations without a demonstrable extra burden on cat and owner's qol. hbgm also did not appear to compromise owners' relationships with their cat. a larger sample size is likely needed to assess whether hbgm promotes superior glycaemic control and remission. disclosures: the research presented in this abstract was supported by zoetis. the clinic at which this research was conducted is also supported by boehringer ingelheim and nestle purina pet-care. ruth gostelow and christopher scudder both receive phd funding from the evetts-luff animal welfare trust. stijn neissen acts as a consultant for the veterinary information network (vin). a major difficulty in the management of diabetes mellitus (dm) is our inability to predict blood glucose values in response to an insulin dose. this is linked to the existence of numerous factors impacting on blood glucose in the diabetic patient (e.g. caloric intake, type of food, exercise, insulin type and dose, stress). artificial neural networks (anns), which are statistical learning algorithms, have shown potential to aid in this prediction process in human dm. their development has been hugely aided by the introduction of continuous glucose monitoring systems (cgms), enabling generation of sufficient data-points. the goal of the current study was to develop an ann for feline dm. algorithms were developed with matlab Ò (mathworks, uk) using data on exogenous insulin dose, serial blood glucoses (obtained through traditional blood glucose curves and cgms) and caloric intake over hours of diabetic cats. all cats were maintained in an environment that enabled normal activities, limiting stress. the neural network toolbox tm (mathworks, uk) was used to construct and test types of anns: multi-layer perceptron (mlp) and radial basis function (rbf). both anns were trained using the diabetic cat data, followed by so-called detrending, elimination of outliers and configuration of external delays. in order to increase accuracy, neural architecture was set as a single hidden layer with a maximum of neurons; inclusion of saturated neurons was forbidden. the accuracy of the resulting mlp and rbf models was assessed by calculating the mean squared normalised error (threshold: . ), generated through comparison of predicted data with actually registered data in recruited diabetic cats. in total, diabetic cats were recruited for this study ( males, females, . ae . [sd]kg, age . ae . ; burmese, dsh, other breeds). all had a -hour blood glucose curve performed (n = with cgms) and response to insulin was predicted by mlp and rbf. calculation of the mean squared normalized error revealed that in / diabetic cats ( %; mlp) and / ( %; rbf), the dynamics of the blood glucose curve were correctly predicted by the ann. in conclusion, our study is the first to describe the successful development of an ann to predict blood glucose dynamics in insulin-treated diabetic cats. further evaluation is indicated, though ann-model-based prediction of glucose concentration may allow clinicians in future to optimise insulin management protocols or may allow the development of an artificial pancreas for the diabetic cat. disclosures: no disclosures to report. glucagon-like peptide (glp) - analogues induce significant weight loss in humans; presumably by slowing gastric emptying and increasing satiety. in lean purpose-bred cats, short-term glp- analogue treatment also induced weight loss. we evaluated the effect of weeks exenatide, a glp- analogue approved for treatment of human type diabetes, or placebo treatment on body composition and adipokines in obese, client-owned cats. cats were randomized to subcutaneous saline (n = ) or exenatide (n = ) injections; . lg/kg q h during the initial weeks and . lg/ kg q h during the following weeks. body weight, body composition using dual-energy x-ray absorptiometry and adipokine levels were measured before and after treatment. all cats were obese (body condition score ≥ out of ). mean body weight was . kg (range . - . kg) and mean % body fat was . % ( . - . %). median percent loss of baseline body weight was . % (range . - . %) for exenatide and . % (range À . to . %) for placebo. only the exenatide group had a significant absolute weight loss; however the difference in median percent loss between groups was not significant. change in total amount or % body fat were not different between groups. correspondingly, plasma leptin and total adiponectin were unaltered by treatment. complications were limited to a single, mild hypoglycemic episode and cases of self-limiting gastrointestinal signs. we conclude that the appointed dose of exenatide was well tolerated and safe in obese healthy cats. a larger study population may be required to fully elucidate the effect of exenatide in obese cats. disclosures: no disclosures to report. feline diabetes mellitus (dm) is recommended to be treated through addressing underlying diseases, bid insulin injections and low carbohydrate diets. good glycaemic control is suggested to promote diabetic remission. a recent systematic review of feline dm literature identified studies on glargine and lente insulin to be proportionately overrepresented compared to other insulin types. additionally, most insulin studies suffered from lack of screening for concurrent disease, homogeneity in management and assessment of quality of life (qol). until recently, only porcine lente insulin was available as a veterinary-licensed product. this prospective trial evaluated the impact of newly veterinary-licensed human-recombinant protamine zinc insulin (prozinc tm , boehringer ingelheim) on clinical signs, glycaemic control and qol in diabetic cats. recently (< months) diagnosed diabetic cats, treated with caninsulin Ò (msd) bid for at least weeks and receiving a specific low carbohydrate diet were recruited. a full history, physical examination, diabetic clinical score (dcs; range [no diabetic signs] - [many diabetic signs]), fructosamine concentration, hour blood-glucose-curve (bgc) and qol-assessment (diaqolpet-score) were performed before and months after transition to prozinc tm (start dose: . - . unit/kg bid), following a set protocol of weekly bgcs and dose adjustments ( . - unit change/injection/week guided by nadir). cats were excluded if screening (biochemistry, urinalysis, fpli, tt , igf- , abdominal ultrasound) identified: ketoacidosis, clinical pancreatitis, glucocorticoid/ progestogen administration, hyperthyroidism, acromegaly, other conditions impairing treatment response. data were assessed for normality and reported as meanae-standard deviation; changes were assessed using paired t-tests (p < . ; without multiple comparisons correction following latest statistical guidelines). sixteen cats were recruited ( male neutered, female neutered; age ae months); all completed the trial. at time of entry cats received . ae . unit/kg caninsulin bid, had a dcs of . ae . ; diaqol-pet-score of - . ae . ; bgc-value of . ae . mmol/l; and fructosamine of ae lmol/l. three months after transitioning to prozinc tm , cats were receiving . ae . unit/kg bid; had a significantly lower mean dcs ( . ae . , p = . ) and diaqol-pet-score (À . ae . , p = . ); bgc-value ( . ae . mmol/l) and fructosamine ( ae lmol/l) were also lower, though not significantly (p = . and p = . , respectively). three cats entered diabetic remission ( %). these results show that transitioning cats from caninsulin to prozinc tm produced a significant improvement in clinical signs and qol. more cases are likely needed to document any additional significant glycaemic impact after transition. finally, in veterinary dm research, this represents the first clinical trial to include validated quantitative qol-assessment as an outcome parameter. disclosures: the study described in this abstract received financial support from boehringer ingelheim. the clinic in which this research was produced also receives support from nestle purina petcare and zoetis. ruth gostelow and christopher scudder both receive phd funding from the evetts-luff animal welfare trust. stijn niessen is a consultant for the veterinary information network (vin). a. hope, s. spence, i.k. ramsey. university of glasgow small animal hospital, bearsden, uk serial blood glucose measurements are currently used as an accepted method to assess diabetic control, however extended curves are expensive and can be technically challenging. a new day continuous glucose monitoring system (cgms) called the dexcom g platinum Ò that incorporates a glucose oxidase-based sensor to measure interstitial blood glucose was evaluated by comparing the results to the blood glucose measured contemporaneously on a glucometer (alphatrak Ò ). these measurements were made at least twice daily at the time of calibration of the cgms, which was a variable period (but not more than hours) after the previous calibration. a total of measurements from eight dogs' glucose curves (blood glucose range - . mmol/l, with a median of . mmol/l) were compared to a paired measurement of interstitial glucose by calculation of the pearson correlation coefficient (r). a minimum of measurements were obtained from each dog. the device only provides a specific measurement of interstitial glucose in the range . to . mmol/l. values above and below this range were not included in the study. subjectively, the device was easy to use with an intuitive user interface that provided wireless real time measurements and was much smaller than older cgms systems. overall, there was excellent correlation between the glucometer and the cgms readings (r = . ), which was statistically significant (p = < . ). the range of differences between the blood and interstitial glucose concentrations was - . mmol/l with a median of . mmol/l. problems encountered with the system included detachment of the system from the dog's skin, as well as variably correlated glucometer and cgms readings in individual dogs (r range . - . , median . ). in conclusion, the dexcom Ò g cgms can be used to assess interstitial glucose concentrations in dogs, and these are generally well correlated with blood glucose concentrations. more work is needed (with larger numbers of patients) to determine the relationship of the correlation to the timing of calibration, and to determine why some dogs seem to have poorer correlation than others. disclosures: no disclosures to report. several continuous glucose monitoring systems that measure interstitial glucose (ig) are currently available. however, they require multiple calibrations and therefore multiple blood sampling; moreover, the sensors are quite expensive and can be used only for a few days. a new human flash glucose monitoring system (fgm) (freestyle libre, abbott, uk) measures ig, does not require calibration, is rather inexpensive and the sensor can be used as many as days. it is composed by a small sensor applied subcutaneously that has to be "scanned"with a reader to obtain real time glucose values. the aim of this study was to assess the accuracy of this fgm in diabetic dogs. in all dogs the sensor was placed on the neck area and fixed with an adhesive patch. during the st - nd, th - th, th - th days from the application, the ig measurements were compared with the peripheral blood (edta plasma) glucose (pg) concentrations analysed by a reference hexochinase based method (olympus/beckman coulter au ). linear regression, bland altman plots and the clarke error grid analysis were used to assess the accuracy. ten client-owned diabetic dogs on insulin treatment were included. median age was . years (range - ), were female (spayed), were male ( neu-tered). median body weight was . kg (range . - . ). four hundred and sixty four simultaneous measurements were taken with fgm (ig) and with the reference method (pg): samples were in the hypoglycemic range (< mg/dl), in the euglycemic range ( - mg/dl) and in the hyperglycemic range (> mg/dl). considering all the measurements together a positive significant correlation between ig and pg concentrations (r = . ) was found. meanaestandard deviation difference from the reference method was À . ae mg/dl in the hypoglycemic range, À . ae mg/dl in the euglycemic range, À . ae mg/dl in the hyperglycemic range. ig values differed > mg/dl from the reference method in %, % and % and > mg/dl in %, % and % in the hypoglycemic, euglycemic and hyperglycemic range, respectively. underestimation-overestimation of ig compared to pg was observed in - %, - % and - % of hypoglycemic, euglycemic and hyperglycemic measurements, respectively; . % and . % of glucose values measured by fgm fall in zone a and zones a+b of the error grid analysis, respectively. the application of the sensor was easy and apparently painless; a mild local erythema after sensor removal was found in / dogs. fgm is a simple and promising glucose monitoring system that seems accurate for the clinical use in diabetic dogs. disclosures: no disclosures to report. the cornerstone of treatment in diabetic cats is insulin. among other issues, insufficient duration of insulin action may lead to poor metabolic control and persistence of clinical signs. with the aim to improve current therapeutic options, the present study evaluated pharmacodynamics parameters, such as onset of action, time to glucose nadir and duration of action, of protamine zinc insulin (prozinc Ò , boehringer ingelheim) and insulin degludec (tresiba Ò , novo nordisk) in healthy cats. additionally, the accuracy of different sensors, sof-and enlite-sensor, of the continuous glucose monitoring system (cgms) ipro Ò (medtronic) was determined with particular attention to the low glycemic range, since reliability of cgms in case of hypoglycemia is crucial. three different doses ( . , . and . iu/kg) of each insulin and both ipro Ò sensors were tested in healthy purpose bred cats in a randomized crossover trial. the sensors were placed in the neck area for days. paired glucose measurements were obtained every - hours with a validated portable blood glucose meter (alphatrak Ò , abbot) set as standard and accuracy was assessed by using iso criteria. additionally, to determine onset of insulin action, time to glucose nadir and duration of action, glucose concentrations were measured and minutes before and , , , , , , and minutes after each insulin administration, then every hours for hours. median (range) onset of action was . ( . - ) hours for pro-zinc Ò and . ( . - ) hours for tresiba Ò . median (range) time to glucose nadir and duration of action were ( . - ) hours and ( - ) hours for prozinc Ò and . ( ) ( ) ( ) ( ) ( ) ( ) hours and ( -> ) hours for tresiba Ò , respectively. with regard to ipro Ò , % of paired glucose measurements with both sensor types were in zone a and b of the consensus error grid. at glucose concentrations < . mmol/l % ( / ) of sof-sensor measurements and % ( / ) of enlite-sensor measurements were within ae . mmol/l of the standard; at glucose concentrations > . mmol/l % ( / ) of sof-sensor measurements and % ( / ) of enlite-sensor measurements were within ae % of the standard. in conclusion, healthy cats injected with prozinc Ò and tresiba Ò showed similar onset of action. later glucose nadir and longer duration of action was seen in cats treated with tresiba Ò compared to those treated with prozinc Ò . both ipro Ò sensors revealed good clinical accuracy and performed similarly in the low glycemic range. disclosures: no disclosures to report. the canine adrenal cortex consists of concentric zones: the zona glomerulosa (zg), the zona fasciculata (zf) and the zona reticularis (zr), which produce mineralocorticoids, glucocorticoids and androgens, respectively. in humans, critical step for the production of either aldosterone or cortisol are the zg-specific aldosterone synthase (cyp b ) and the zf-specific b-hydroxylase cytochrome p (cyp b ). the fact that humans and dogs have the same adrenocortical end products, i.e. aldosterone and cortisol, has led to the assumption that canine steroidogenesis is identical to that of humans. however, in dogs, the zonal expression of steroidogenic enzymes has not been studied previously. moreover, in dogs the expression of cyp b / is unclear, as only one coding gene sequence (cyp b ) has been published in the ncbi database and, adjacent to this, a large non-sequenced gap is present. we hypothesized that canine adrenals possess only one cyp b gene, similar to sheep and pigs. zg and zf tissue was isolated separately by use of laser-guided microdissection of adrenals of healthy dogs. the zone-specificity of the tissues was confirmed by specific markers, with mrna relative expression of wnt , angiotensin ii receptor and disabled- being significantly higher (p = . , p = . , p = . , respectively) in the zg compared to the zf. rt-qpcr analysis of mrna relative expression of steroidogenic enzymes demonstrated a significantly higher fold change of steroidogenic acute regulatory protein (star), cytochrome p side chain cleavage (cyp a ) and a-hydroxylase/ , -lyase (cyp ) (p = . , p = . , p = . , respectively) in the zf compared to the zg. the zfspecific presence of cyp was also demonstrated by immunohistochemistry. no significant difference (p = . ) in the mrna relative expression of cyp b mentioned in the database was found, and southern blot analysis showed that the non-sequenced gap does not contain another cyp b gene. we conclude that there is only one functional cyp b enzyme in canine adrenals. the zone-specific production of aldosterone and cortisol is probably due to zone-specific cyp expression. its presence in the zf is crucial for cortisol synthesis, while lack of cyp in the zg conducts steroidogenesis to mineralocorticoid production. this is the first report providing insights in one of the most important physiological mechanisms of the canine adrenal cortex, its zone-dependent steroidogenesis. disclosures: no disclosures to report. the pathogenesis of cortisol-secreting adrenocortical tumors (ats) has become more clear recently. mutations of the gnas gene provide an explanation for acth-independent hormonal activity of ats, but the autonomous growth remains greatly undisclosed. an approach to elucidate the proliferative capacity of ats is to learn from the current understanding of adrenal growth biology. the sonic hedgehog (shh) signaling pathway plays an essential role in the development of the adrenal gland and in regulating adrenocortical cell proliferation. the members of the shh signaling pathway are present in progenitors of the steroidogenic cells of the normal adrenal gland and dysregulation of shh signaling has been implicated in adrenal cancer in humans. we hypothesized that shh signaling is also enhanced in canine ats, predominantly in carcinomas. we examined the relative expression of shh pathway components (shh, ptch , smo, gli , gli and gli ) by rt-qpcr analysis in cortisol-secreting adenomas (n = ) and carcinomas (n = ) and normal canine adrenals (n = ). the relative expression of members of the shh pathway was detected in both ats and normal adrenal tissue. a significant (p < . ) lower mrna expression of gli was detected in carci-nomas when compared to normal tissue. amongst the other genes, no significant differences were found. since gli is mainly a repressor of genes activated by the shh pathway, a down regulation of gli in carcinomas could point to enhanced shh signaling in adrenocortical carcinomas and could theoretically be responsible for their expansive growth. in conclusion, dysregulation of shh pathway might be involved in the pathogenesis of canine cortisol-secreting carcinomas. modulating shh expression might provide a new target therapy for adrenocortical carcinomas and will need to be explored in the future. disclosures: no disclosures to report. feline hypersomatotropism (acromegaly) has been suggested to be an underdiagnosed endocrinopathy among diabetic cats. treatment options include management of the secondary diabetes mellitus alone, medical pituitary inhibition, radiotherapy (rt) and hypophysectomy (hpx). tools to diagnose the disease and monitor treatment effect are limited, with insulin-like growth factor- (igf- ) currently being the only easily accessible blood test. serum igf- has previously been shown to be insensitive when assessing rt effect. development of additional serological diagnostic tools that can be measured alongside serum igf- is therefore desirable. a pilot study previously validated an n-terminal type iii pro-collagen propeptide (piiinp) elisa for use in cats and found this peripheral biomarker of collagen turnover to be significantly elevated in a small number of hypersomatotropic diabetic (hsdm) cats. this study therefore aimed to: . further evaluate the use of serum piiinp to differentiate hsdm from dm; and . to evaluate piiinp as a marker for treatment success. piiinp concentrations were measured in cats with uncomplicated diabetes mellitus (dm) (igf- < ng/ml [radioimmunoassay], < . iu/kg/injection exogenous insulin requirement) and with confirmed hsdm (igf- > ng/ml, pituitary mass on computed tomography) using the previously validated elisa. additionally, piiinp and igf- were measured in preand post-treatment ( - months) samples of hsdm cats that responded favourably (decreased insulin requirement) to radiotherapy (rt; n = ) or hypophysectomy (hp; n = , of which had achieved diabetic remission at time of sampling). serum piiinp concentrations were significantly higher in hsdm cats (median . ng/ml; range: . - . ) compared to dm cats (median . ng/ml; . - . ; p < . , mann whitney u-test). receiver-operator-curve-analysis revealed a . ng/ml cut-off to differentiate between dm and hsdm cats with % sensitivity and % specificity (auc: . ; % confidence interval: . - . ). after rt, piiinp increased significantly (median pre-rt . ng/ml, . - . ; post-rt . ng/ml, . - . ; p = . , wilcoxon signed rank-test) despite absence of significant change in igf- concentrations (median pre-rt ng/ml, - ; post-rt ng/ml, - ; p = . , wilcoxon signed rank-test). following hpx, serum piiinp concentrations did not change significantly (median pre-hpx . ng/ml, . - . ; post-hpx . ng/ml, . - . ; p = . , wilcoxon signed rank-test) despite significant serum igf- decreases (median pre-hpx ng/ml, - ; post-hpx ng/ml, - ; p = . , wilcoxon signed rank-test). in conclusion, serum piiinp concentration was confirmed to be a useful additional parameter when differentiating hsdm from dm in cats. however, the current data do not suggest piiinp to be a reliable marker of treatment success following rt or hpx. disclosures: no disclosures to report. decreased frequency and function of peripheral regulatory t cells (tregs) have been documented in people with immune-mediated haemolytic anaemia (imha), suggesting that defects in peripheral tolerance may play a role in the pathogenesis of this disease. the aim of the current study was to test the hypothesis that the frequency of peripheral tregs is decreased in dogs with primary imha, accompanied by increases in t helper (th) cells, cytotoxic t (tc) cells and b cells. residual edta-anticoagulated blood samples were obtained from dogs with primary imha (n = ), dogs with inflammatory diseases (n = ) and healthy dogs (n = ). primary imha was diagnosed in dogs with regenerative anaemia (packed cell volume [pcv] < %) and either persistent agglutination of erythrocytes after saline dilution or detection of spherocytosis on a fresh blood smear. the study was approved by an institutional ethics and welfare committee. after erythrocyte lysis, peripheral blood mononuclear cells were stained with fluorophore-conjugated antibodies specific for extracellular (cd , cd , cd ) and intracellular (cd b, foxp ) antigens. multicolour flow cytometry was undertaken to determine the proportion of lymphocytes that were b cells (cd b hi ), th cells (cd hi cd + ), tc cells (cd hi cd + ) and tregs (cd hi cd +-foxp + ); the kruskal-wallis test was used to compare proportions between groups. correlations between the proportions of tregs and pcv and serum total bilirubin concentration (tbil) in dogs with imha at presentation were assessed using spearman's rho coefficients. the median proportion of cd + t cells that expressed foxp was . % (inter-quartile range [iqr]: . - . ) in dogs with imha, . % (iqr: . - . ) in dogs with inflammatory diseases and . % (iqr: . - . ) in healthy dogs, with no difference between groups (p = . ). there was no difference in proportions of t cells that were cd + (p = . ) or cd + (p = . ) between groups, nor in the proportion of b cells (p = . ). the proportion of cd hi cd + foxp + tregs was positively correlated with tbil in dogs with imha (spearman's rho . , p = . ), but not pcv (rho À . , p = . ). though limited by its size, the results of this pilot study suggest that the frequency of tregs is not decreased in dogs with imha; proportions of th, tc and b cells were also comparable to those in control dogs. further work is required to determine whether the function of tregs is altered, or whether other defects in peripheral tolerance contribute to development of this disease. disclosures: no disclosures to report. xenotransfusion of canine blood to cats may be a life-saving procedure when treating an acute anaemic syndrome and compatible feline blood cannot be obtained. published evidence in a limited number of cases dating from the s indicates that cats do not appear to have naturally-occurring antibodies against canine red blood cell antigens. in fact compatibility tests before the first transfusion did not demonstrate evidence of agglutination or haemolysis of canine erythrocytes in feline serum and no severe acute adverse reactions have been reported in cats receiving a single transfusion with canine blood. severe acute reactions not reported so far cannot however be excluded and we decided to perform a pilot study to evaluate the presence of naturally occurring antibodies against canine red blood cell antigens in cats and viceversa. surplus material from diagnostic samples (blood edta and blood serum samples) of cats and dogs was used to perform test-tube major and minor cross-match tests (at °c, °c and room temperature (rt)) and blood typing, after obtaining the informed consent from owners. hemolysis, macro-and micro-ag-glutination were investigated in each test tube and were considered markers of a positive matching. blood from each cat was tested with blood from to different dogs for a total of major and minor cross-matchings each one performed at the different temperatures of incubation. thirty-eight overall major cross-matchings proved positive at °c, at rt and at °c respectively. the minor cross-matching was positive in all but tests performed at °c. no cat tested totally negative ( °c, °c, rt) at both major and minor cross-matching procedures performed towards any single dog. ten cats experienced positive major and minor cross-matching at °c, rt and °c towards - different dogs. five cats were positive in the major cross-match, at least at °c, towards - different different dogs. seven cats obtained a positive major cross-match at rt and/or at °c towards - dogs. only cats tested completely negative at °c, rt and °c, in one out of the different major cross-matchings performed. in conclusion, naturally occurring antibodies against canine red blood cell antigens appear to be frequently detected in cats as well as those against feline red blood cell antigens in dogs. xenotrasfusion of canine blood to cats should only be performed after the selection of a compatible donor by means of at least a negative major cross-match test result. disclosures: no disclosures to report. anti-thymocyte serum (ats), a potent immunosuppressive agent, is commonly used perioperatively in human patients to increase graft survivaland decrease rejection of transplanted tissue. ats has been reported as part of an immunosuppressive protocol to treat immune-mediated diseases including aplastic anemiaand myelodysplastic syndromes (mds). rabbit anti-dog thymocyte serum (radts) has been used in veterinary medicine for perioperative immunosuppression in canine renal transplants. however, there are no reports regarding the use of radts in the treatment of dogs with immune-mediated disorders. the medical records of dogs diagnosed with imha, dogs with itp and dog with mds were reviewed. median age was . years ( . months to years). all dogs failed to respond to traditional immunosuppressive therapy and received radts. none of the dogs experienced any adverse reaction. lymphocyte counts were used to monitor the response to therapy. all dogs, except , had a significant decrease in their lymphocyte count; / had a decrease to < % of the initial lymphocyte count, which was the aim in previous studies on radts. all dogs were discharged, however, the same dog with no changes in his lymphocyte count experienced a relapse of his imha after week and was euthanized. all other cases achieved clinical remission with immunosuppressants being tapered or discontinued. radts appeared to be a safe immunosuppressant agent of interest in refractory immune mediated diseases. disclosures: no disclosures to report. in cats with upper respiratory tract aspergillosis (urta), invasive disease is common. in other species, invasive mycoses are associated with immunodeficiency. characterisation of the humoral immune response in feline urta serves to identify whether selective immunodeficiency underlies susceptibility and to determine the utility of class-specific antibody detection for early diagnosis. we have shown that serum anti-aspergillus igg has high sensitivity and specificity for diagnosis. the aims of the study were to ( ) determine whether serum anti-aspergillus iga can be detected in cats with urta, and ( ) evaluate the sensitivity and specificity of iga detection for diagnosis. sera were collected from groups of cats; group -confirmed urta (n = ), group -upper respiratory disease without aspergillosis (n = ), group -healthy cats (n = ) and cats with non-fungal, non-respiratory illness (n = ). an indirect elisa to detect anti-aspergillus iga was developed. inter-assay and intraassay coefficients of variation were . % and . %, respectively. serum iga was detected in . %, % and % of group , , and cats, respectively. using an optimal elisa cut-off value for diagnosis ( . elisa units/ml), determined by receiver-operating curve analysis, assay sensitivity for group cats was . %. specificity was highest ( . %) when group was used as the control, compared to group ( . %) or group and combined ( . %). we found no evidence of a role for primary iga deficiency in the pathogenesis of feline urta. serum anti-aspergillus iga detection has moderate sensitivity and moderate specificity for diagnosis of urta. disclosures: there is no conflict of interest. the study was partially funded by a australian companion animal health foundation grant . non-invasive topical infusion therapies are widely used in canine sinonasal aspergillosis (sna) but are time-consuming and associated with prolonged recovery and increased costs. therefore, the main goal of the present study was to compare the efficacy of a simplified infusion protocol (d e) with a -hour infusion protocol (d eb). d eb consisted in endoscopic debridement followed by minutes % enilconazole infusion and % bifonazole cream depot into the affected frontal sinus through endoscopically placed catheter. for d e protocol, after debridement, enilconazole infusion was shortened to minutes, with the dog remaining in dorsal recumbency, head flexed at °, during the whole procedure. adjunctive oral itraconazole therapy was prescribed in both protocols. effective debridement of fungal plaques is considered as an essential therapeutic step. unfortunately, it is not always possible to achieve perfect debridement of the sinonasal cavities, due to incomplete accessibility of the whole sinusal area with the endoscope; however, its effect has never been assessed as such. therefore, the second aim of this study was to evaluate the effectiveness of debridement on success rate after the first treatment. fisher's exact test was used to assess the difference in success rate between both protocols and in function of full debridement. twenty-eight dogs with sna were treated with d e and dogs with d eb. the median (range) duration of d e was only minutes ( - ) compared to minutes ( - ) for d eb. first treatment success rate did not differ between both protocols and were % for d e and % for d eb. both protocols had an overall success rate of % after procedures. in contrast to the majority of dogs with d eb, all dogs receiving d e recovered quickly and were discharged the same day. completeness of debridement was assessed endoscopically in dogs ( treated with d e and with d eb). debridement was judged complete in / dogs and had a significant effect on first treatment success rate (p = . ). when debridement was complete, % of the dogs (d e: / dogs; d eb: / dogs) were cured after the first procedure, compared to % (d e: / dogs; d eb: / dogs) of the dogs with incomplete debridement. we concluded that ( ) the simplified infusion protocol is quick, safe, easy and effective, and offers a favourable alternative to hour infusion protocols for treatment of canine sna; ( ) completeness of the debridement undoubtfully is an important step for treatment success of infusion protocols. disclosures: no disclosures to report. gastroesophageal (ge) symptoms are commonly reported in dogs with brachycephalic upper airway obstructive syndrome (bs). since ge symptoms frequently occur during situations of increased inspiratory effort (excitement, respiratory distress), dynamic disorders of the ge junction (gej) are probably involved, due to transient increased negative intrathoracic pressure. however, according to a previous study, only few dynamic abnormalities of the gej are observed during gastroscopy. we hypothesized that both anaesthesia and endotracheal intubation during gastroscopy lead to underestimation of gej abnormalities. the aim of this study was to improve detection of dynamic gej abnormalities during gastroscopy using obstructive manoeuvers mimicking and reproducing upper airway obstruction of variable severity. twenty-six dogs presented with bs were prospectively included. respiratory and digestive symptoms as well as endoscopic abnormalities were scored at initial diagnosis and at control month after surgery. during each endoscopy, gej was assessed and scored (based on esophagitis, gej atony, ge reflux, cranial displacement of the gej) in the consecutive situations: ( ) absence of obstruction with the dog intubated (ob- ), ( ) presence of natural obstruction with the dog extubated (ob-nat) and ( ) during complete manual obstruction of the endotracheal tube during up to spontaneous breathings (ob-compl). spearman's rank test was used to assess correlations between the different clinical and endoscopic scores. taking all endoscopic procedures together, the severity of respiratory symptoms correlated significantly with the severity of respiratory endoscopic abnormalities (p < . , r = . ) and the severity of digestive symptoms (p = . , r = . ). at diagnosis, dogs ( %) presented digestive symptoms while endoscopic gej abnormalities were observed in ( %), ( %) and ( %) dogs during ob- , ob-nat and ob-compl respectively. gej atony, ge reflux, cranial displacement of the gej and sliding hiatal hernia were present in ( . %), ( . %), ( . %) and dogs during ob- , in ( . %), ( . %), ( . %) and ( . %) dogs during ob-nat and in ( . %), ( . %), ( %) and ( . %) dogs during ob-compl, respectively. a significant correlation was found between digestive and endoscopic gej scores during ob-compl (r = . , p = . ) as well as during ob-nat (r = . , p = . ) but not during ob- . it can be concluded that in dogs with bs ( ) gej abnormalities are dynamic and related to the degree of upper airway obstruction; ( ) the use of obstructive manoeuvres during gastroscopy improves the detection of gej abnormalities. disclosures: no disclosures to report. tracheobronchial foreign bodies are common causes of respiratory disease in children but they are rare in veterinary medicine. particularly in cats, reports of tracheobronchial foreign bodies are scarce. this study aimed to describe clinical presentation, diagnostic findings and treatment modalities in confirmed cases of tracheobronchial foreign bodies in cats. we hypothesize that bronchoscopy is highly effective in their extraction in cats. cases of confirmed tracheobronchial foreign bodies in cats admitted to referral centers in france, from may to november , were included. files were retrospectively analyzed for age, sex, breed, clinical signs, delay between onset of signs and presentation, diagnostic procedure, method of extraction, location and nature of foreign bodies. twelve cats were included ( males, females). mean age at presentation was years old ( . years ae . ). cough was the main chief-complaint, being present in / ( %) cats. while / ( %) cats presented to consultation in the first week after the beginning of respiratory signs, / ( %) cats exhibited clinical signs for more than week. chest radiographs were done in / cats. bronchoscopy was performed in / cats, confirming the presence of foreign body material and allowing their extraction in / animals ( %). in / cats ( %), bronchoscopic extraction was unsuccessful and a pulmonary lobectomy was required. the foreign body was located in the trachea in / cats ( %) and in the bronchial tree in the remaining cats ( / in the right caudal bronchus, / in the left caudal bronchus and / in the main left bronchus). / ( %) were vegetal foreign bodies (grain seeds and foxtail awns), / ( %) were mineral (a bone fragment, a teeth and a small stone) and / ( %) of undetermined origin. all the mineral foreign bodies were extracted from the trachea, whilst the majority of the vegetal ones ( / - %) were removed from the bronchial tree. in this case series, bronchial foreign bodies were as frequent as tracheal foreign bodies in cats. this finding differs from previous data reporting that trachea is the preferential location for feline respiratory foreign bodies. vegetal foreign bodies are more common. due to their nature and shape, they may be more prone to lodge in the bronchial tree, while mineral foreign bodies remain in the trachea. according to our results, bronchoscopy is highly effective for identification and extraction of tracheobronchial foreign bodies in cats. disclosures: no disclosures to report. mycophenolate mofetil is the prodrug of mycophenolic acid (mpa). it is a selective non-competitive inhibitor of inosine- monophosphate dehydrogenase, which is expressed in many cell types. mpa's ability to induce lymphocyte cytotoxicity, reduce monocyte recruitment, and suppress dendritic cell maturation, are useful targets in treating immune mediated and inflammatory diseases. it has been used extensively in human medicine for transplant recipients and more recently in veterinary medicine in dogs with various immune mediated diseases. however, its use in cats is limited in part because mpa is primarily metabolized by glucuronidation and cats inherently have a decreased ability to glucuronidate many drugs. we proposed that cats may glucuronidate mpa more slowly than humans and dogs and conducted a series of in vitro studies to explore this hypothesis. we used liver microsomes from cats ( individual and pooled), dogs (pooled), and humans (pooled). these liver samples were incubated at °c in a water bath with mpa and udp-glucuronic acid or udp-glucose. udp-glucose was studied since mpa glucoside is a minor metabolite in humans but may be a major metabolite in other species. hplc-ms was used to determine concentrations of mpa-glucuronide (phenol and acyl) and mpa-glucoside (phenol) formed by incubation. cats formed much less mpa phenol glucuronide ( . nmoles/ min/mg) than dogs ( . nmoles/min/mg), or humans ( . nmoles/ min/mg). cats formed similar amounts of mpa acyl glucuronide ( . nmoles/min/mg) than humans ( . nmoles/min/mg), but less than dogs ( . nmoles/min/mg). in contrast, cats ( . nmoles/min/ mg) formed more mpa phenol glucoside than humans ( . nmoles/min/mg) but less than dogs ( . nmoles/min/mg). when the pathways of metabolism were summed for each species, cats ( nmoles/min/mg) metabolized mpa much less efficiently than dogs ( nmoles/min/mg) or humans ( nmoles/min/mg). variability in metabolite formation between the individual cats was high ranging from fold for mpa glucoside, to fold for mpa phenol glucuronide, to fold for mpa acyl glucuronide. our preliminary results confirm that cats glucuronidate mpa less rapidly than dogs and humans, however cats and dogs were found to glucosidate mpa more efficiently than humans. in addition, individual cats are variable in their ability to glucuronidate and glucosidate mpa. this preliminary in vitro data will be compared to in vivo studies of mpa pharmacokinetics to elucidate proper dosing of mpa in cats. disclosures: no disclosures to report. cystocentesis is the gold standard sampling method for urine microbiology in dogs, as voided samples are associated with a higher risk of contamination. however, the accuracy of the veterinary cut-off values currently recommended for detection of clinically significant bacteriuria in voided urine has been poorly investigated. the aim of this study was to evaluate the accuracy of veterinary and human criteria for diagnosis of urinary tract infection (uti) in dogs using voided urine samples. dogs with suspected uti were prospectively enrolled. paired urine samples collected by cystocentesis and voiding, respectively, were stored at • c and cultured within hours. bacterial counts in voided urine were interpreted using both the veterinary (≥ . colony forming units (cfu) per ml) and the human (≥ . cfu/ml plus presence of clinical signs) criteria for diagnosing uti, and compared to those obtained in urine collected by cystocentesis (gold standard). significant bacteriuria in cystocentesis samples was defined as ≥ . cfu/ml. sixty-five dogs were included in the study. when applying the veterinary criteria for diagnosing uti in voided samples, the diagnostic accuracy was % (sensitivity %, specificity %, positive predictive value (ppv) % and negative predictive value (npv) %). when applying the human criteria the accuracy fell to % (sensitivity %, specificity %, ppv % and npv %). the results indicate that, in most dogs with suspected uti, an accurate diagnosis can be obtained using voided urine, if applying the current veterinary cut-off values to samples stored at refrigeration temperature and cultured shortly after collection. disclosures: the study was supported financially by the uc-care research centre, university of copenhagen, and minor external foundations. antimicrobial resistance (amr) is a major public health concern and will likely be the first cause of mortality in human medicine in . canine bacterial urinary infection is a frequent condition and might be implicated in interspecies transmission of resistance mechanisms. this study aimed to retrospectively describe the evolution of the amr of uropathogens over a -year period in a veterinary teaching hospital. positive urinary cultures obtained by cystocentesis (> cfu/ ml) from dogs treated at the national veterinary school of toulouse (envt) between and , were reviewed. annual prevalence of amr for several enterobacteriaceae, staphylococcus spp, enterococcus spp and streptococcus spp were recorded for various veterinary and human antimicrobials. frequency of extended spectrum beta-lactamase-producing enterobacteriaceae (esbl) and multidrug resistant (mdr) bacteria were noted. logistic regression was performed to analyze the evolution of amrs. a p-value < . was considered significant. over the study period, isolates with stable annual distribution were identified. considering enterobacteriaceae, amr for several antimicrobials significantly evolved over time: despite a possible increase in for some antimicrobials, a general decrease of amr was observed. prevalence of esbl and mdr bacteria remained stable with mean prevalence of % and %, respectively. trends of amr of enterobacteriaceae over the study period in envt are not in accordance with the worrisome general tendency and could be consistent with a rationalized antimicrobials use. however, the persistently elevated prevalence of esbl and mdr bacteria, and the possible increase of amr during the last studied year warrant further investigation and surveillance. disclosures: no disclosures to report. klebsiella pneumoniae are important pathogens that cause urinary tract infections (uti). antibiotic-resistant and virulent bacterial clones with high interhost transmission may play an important role in the spread of antimicrobial resistance. this study aimed to characterize the antimicrobial resistance and virulence of clinical klebsiella isolated from animals and humans with uti and to determine the lineages of companion animal klebsiella pneumoniae resistant to third-generation cephalosporins ( gc). thirty-five companion animal clinical klebsiella spp., obtained between and january , and human clinical strains isolated in were included. antimicrobial susceptibility testing was performed using disk diffusion and clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) and plasmid-mediated ampc genes were detected by pcr whenever resistance to gc was detected. furthermore, gc-resistant klebsiella were characterized by multi-locus sequence typing. regarding virulence, pcr for detection of fimh (adhesin type- fimbriae), mrkd (adhesin type- fimbriae), entb (enterobactin), ybts (yersiniabactin) and rpma (regulator of mucoid phenotype-a) genes was conducted on and companion animal and human strains, respectively. k. pneumoniae was the main species isolated in companion animals ( . % n = / ) and in humans ( . %, n = / ) but klebsiella oxytoca was also identified. resistance to gc (cefotaxime or ceftazidime) was present in . % (n = / ) and . % (n = / ) of companion animal and human strains, respectively. overall, cg-resistant k. pneumoniae were found to be ctx-m group- ( . %, n = / ), cmy ( . %, n = / ) and dha ( . %, n = / ) producers. both companion animal k. oxytoca were dha producers. companion animal cg resistant klebsiella were frequently ( . %, n = / ) co-resistant to fluoroquinoles and trimethoprim/sulphamethoxazole rendering them as multidrug resistant. moreover most companion animal cg resistant strains were also resistant/intermediate to amoxicillin/clavulanate ( . %, n = / ). overall, companion animal klebsiella resistance to amoxicillin/clavulanate ( . %, n = / ), fluoroquinolones ( . %, n = / ) and trimethoprim/sulphamethoxazole ( . %, n = / ) was high. companion animal k. pneumoniae resistant to gc belonged to st (n = ), st (n = ), st (n = ) and st (n = ) lineages. concerning virulence, all k. pneumoniae were positive for fimh, mrkd and entb. yersiniabactin was present in . % (n = / ) and . % (n = / ) of companion animal and human k. pneumoniae, respectively. k. oxytoca (n = ) were positive for entb and ybts. all tested strains were negative for rpma. the detection of k. pneumoniae lineages highly important for humans in companion animals with uti raises great concerns regarding their role as reservoirs. moreover, the fact they were also found to share gc resistance genes and common virulence factors with humans further extends the risk of transfer. disclosures: conflicts of interest: the first author currently receives a phd grant funded by the portuguese foundation for science and technology. feline lower urinary tract disease (flutd) refers to a heterogeneous group of disorders with similar clinical signs. some diets are designed to manage flutd by promoting struvite stone dissolution and addressing key risk factors (overweight, low water intake, stress. . .). the goal of this study is to compare the struvite dissolution potential of diets marketed for flutd, in standardized in vitro conditions. six adult healthy cats were fed successively dry diets (a = royal canin s/o-biopeptide; b = hill's c/d-urinary-stress) for days, with urine collection on the last days. urines collected for each diet were pooled and distributed in bottles containing the mean urine volume produced daily per cat. urine ph and struvite relative supersaturation (rss) were measured for each pool. two feline struvite uroliths homogenous in shape and weight were immersed separately in a urine bottle of each diet, and put in a stove at °c. twenty-four hours later, the urine was filtered to collect the stones, which were dried and weighed. every day, the stones were placed in new bottles of the corresponding urine until the first complete stone dissolution. diet effect on urine ph, volume and rss was analysed non parametrically (wilcoxon paired rank test). diet effect was combined with a period effect (period = days dissolution), in a complete factorial design to analyse struvite stones weight evolution at each period for each diet (mixed model with diet, period, diet x period as fixed effects). fdr method was applied to compare diets at each period. diet a induced a higher mean urine volume ( . versus . ml/kg/day), and a lower rss than diet b ( . versus . ) (p < . ). the urine ph of the diets were not significantly different ( . and . ) (p > . ). after days, the struvite stone immersed in urine from diet a was totally dissolved, versus % dissolution for the stone in urine from diet b. when considering periods of days, the struvite weight diminution was significantly higher when struvite stone was immersed in urine from diet a than from diet b (diet effect: p < . ). the interaction between diet and period effect revealed that the difference in dissolution rate between the diets was significant as soon as the first day period (p < . ), and increased over the other periods (p < . ). a diet inducing a lower struvite rss and a greater urine dilution allows faster struvite stone dissolution. disclosures: the author and co-authors work for royal canin, the company commercializing one of the diets evaluated. serum cystatin c (scysc) and urinary cystatin c (ucysc) are potential markers for detection of feline chronic kidney disease (ckd). our aims were twofold. firstly, we evaluated cysc as marker for ckd. we compared scysc and ucysc between ckd and healthy cats, correlated scysc and scr with glomerular filtration rate (gfr) and calculated sensitivity, specificity for detecting decreased gfr. secondly, we compared assay performance of the turbidimetric assay (petia) with the previously validated nephelometric assay (penia). forty-nine ckd (iris stage - ) and healthy cats were included. gfr was measured with plasma exogenous creatinine (pect), endo-(penict) and exo-iohexol (pexict) clearance test in ckd and healthy cats. based on pexict, scysc was evaluated to distinguish normal, borderline and low gfr. sensi-tivity and specificity to detect pexict< . ml/min/kg were calculated. validation of petia was performed and scysc results of penia and petia were correlated with gfr. statistical analysis was performed using general linear modelling. serum cysc and ucysc were significantly higher (p < . ) in ckd cats. however, ucysc was detected only in / ckd cats. r values between gfr and scr or scysc were . and . respectively. sensitivity and specificity were % and % for scysc and % and % for scr. serum cysc could not distinguish healthy from ckd cats, nor normal from borderline or low gfr, in contrast to scr. penia appeared superior to petia. in conclusion, scysc is not a reliable marker for gfr in cats and ucysc could not be detected in all ckd cats. disclosures: for this work support was received from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l.f.e. ghys. soblechero, f.j. duque, p. ruiz, r. barrera. university of extremadura, c aceres, spain serum cystatin c (scys c) is a marker of glomerular filtration rate with advantages over serum creatinine. in human medicine, some authors observed that scys c is influenced by methylprednisolone or prednisone administration. with the aim to follow the course of this maker of renal function in acutely diseased patients with a receiving glucocorticoid medication, we followed at dog's whit steroid responsive meningitis. the study was carried out on patients that where divided in groups: control group ( clinical healthy dogs), group b, dogs treated with prednisone due to steroid responsive meningitis (treated with mg/kg prednisone during days, followed to mg/kg prednisone for another days). dogs had no known preexisting renal disease, and have no previous glucocorticoid medication. group c was established to test the effects of endogenous steroids: dogs with hyperadrenocorticism were evaluated. serum cys -c was measured by turbidimetric latex and creatinine by jaffe reaction (spinreac Ò ) and were determined at time of diagnosis for group b, and on days , and in the meningitis dogs. a statistically significant increase of scys-c was observed in group b, with doses of mg/kg of prednisone ( . ae . mg/l; p < . ), and doses of mg/kg of prednisone ( . ae . mg/l; p < . ) respect to these same dogs before treatment ( . ae . mg/l) and compared to the control group ( . ae . mg/l). however, the serum concentration observed in hyperadrenocorticism ( . ae . mg/l) was similar to the one find in the healthy animals group. the creatinine concentration was not increased either, during the prednisone treatment, or in the case of hiperadrenocorticism. in conclusion, the present study agrees with that is described in human medicine, and confirms the effects of glucocorticoids on scys c concentrations in dogs. the administration of high doses of prednisone is associated with a scys c increase. on the other hand endogenous cortisol increase (hyperadrenocorticism) in dogs is not seen to modificate the scys c. disclosures: no disclosures to report. chronic kidney disease (ckd) produces progressive reduction in the number of functional nephrons and directly affects the homeostasis of the solutes excreted in the urine, including phosphorus. hyperphosphatemia is considered a factor directly related to the increased mortality in humans, cats and dogs. in order to provide data from controlled clinical studies to examine the effects of hyperphosphatemia on the progression and survival of naturally occurring canine ckd, the following study was conducted. for the present study dogs, which were followed up by the veterinary teaching hospital of the university of extremadura (spain), were used for the study. distributed in the following groups: group i ( healthy adult dogs) and group ii ( adult dogs with ckd). this second group had a subclasification attending to different factors: phosphatemia: iia ( dogs with phosphatemia < . mg/dl) and iib ( dogs with phosphatemia > . mg/dl). leishmaniasis: iic ( dogs with ckd due to leishmaniasis) and iid ( dogs with ckd not due to leishmaniasis). iris classification: iris ( dogs), iris ( dogs), iris ( dogs) and iris ( dogs). the results of survival were as followed: statistically lower survival was found between the groups iia and iib (p < . ) also between the iris grades , and and the iris grade (p < . ), iris and with iris (p < . ) and iris with iris (p < . ). no significant differences between positive and negative leishmaniasis. in conclusion, plasma phosphate concentration in dogs increases as chronic kidney disease develops. and an inverse relationship to survival in dogs with phosphorus concentrations above mg/dl, and as it progresses the iris scale was observed. disclosures: no disclosures to report. studies analyzing to which extent upc in dogs is influenced by pyuria have yielded conflicting results. moreover, there is no data on the effect of proteinuria on plasma acute phase proteins in dogs. in dogs upc was prospectively measured. upc and if available, results of plasma biochemistry including measurement of c-reactive protein (crp) from the same day were analyzed using the mann-whitney-u-test. samples without sediment analysis (n = ) were excluded resulting in urine samples for analysis. hematuria (> erythrocytes/hpf), pyuria (> leucocytes/hpf), and bacteriuria were present in , and samples, respectively. upc in samples with hematuria was significantly (p = . ) higher (median . ; th - th percentile . - . ) compared to samples without hematuria ( . ; . - . ). in dogs with pyuria upc was significantly (p < . ) higher ( . ; . - . ) compared to samples without pyuria ( . ; . - . ). % of the samples with pyuria had an upc > . this is in contrast to data reported previously where only % of pyuric urine samples had an upc > (vet clin path ; : ). bacteriuria did not influence upc (p = . ). samples of dogs with negative protein dipstick results had an upc > . ( . , . , and . , respectively). all samples had low urine specific gravity ( . - . ) and alkaline ph. for a total of samples corresponding plasma data on albumin (reference interval ri: . - . g/dl) and crp (ri: - . mg/l) were available. crp was significantly (p = . ) higher in dogs with upc > . ( . mg/l; . - . ) compared to dogs with upc ≤ . ( . mg/l; . - . ). albumin was significantly (p < . ) lower ( . g/dl; . - . ) in dogs with upc > . compared to dogs with upc ≤ . ( . g/dl; . - . ). naturally occurring pyuria has a more profound effect on upc results than previously reported. proteinuria is associated with changes of acute phase proteins such as hypoalbuminemia and increased crp. whether this is consequence or cause of the proteinuria needs further investigation. furthermore, animals with low urine specific gravity may have clinically relevant proteinuria even in the light of a negative dipstick result. therefore measurement of upc is recommended to exclude renal protein loss in hypo-and isosthenuric dogs. disclosures: no disclosures to report. high blood pressure causes an increase in vascular endothelial growth factor (vegf) secretion. feline hypertension is commonly associated with chronic kidney disease (ckd) amlodipine is the first choice antihypertensive treatment in cats but could have a negative effect on the kidney by increasing glomerular pressure through afferent arteriolar dilatation. the aims of this study were to: ( ) validate a method for the quantification of vegf in feline serum samples; ( ) assess the association between urinary vegf, serum vegf (svegf) and biochemical and clinical variables in hypertensive cats and ( ) investigate changes in urinary vegf with amlodipine treatment. a randomised, double blinded, placebo controlled parallel group study (n = ) was conducted in phases to determine the efficacy and safety of amlodipine in cats with naturally occurring hypertension. the placebo group was crossed-over to amlodipine after day . a canine vegf elisa (previously validated for feline urine) was used to measure urine and serum vegf. urine vegf concentration was normalised to urinary creatinine (urinary vegf to creatinine ratio [uvc]). univariable linear regression models, followed by a backwards multivariable linear regression model, were performed to identify independent predictors of svegf and uvc. a linear mixed measures model was used to compare the effect of placebo and amlodipine on uvc ( days) and to investigate potential changes in uvc with long-term amlodipine treatment ( days). intra-assay and inter-assay cv of svegf measurements were . - . (n = ) and . - . (n = ) respectively. dilutional parallelism indicated a mean recovery of . % ae . % (n = ). urea and urine protein:creatinine (upc) were independent negative and positive predictors of svegf respectively. plasma creatinine was an independent negative predictor of uvc, upc and sodium were independent positive predictors. no association was found between svegf and uvc. no significant changes in uvc or differences between groups were found with days of amlodipine or placebo treatment. mean uvc at screening was . and . lg/g after days of amlodipine treatment (p = . ), both within the healthy cat reference range ( . to . lg/g). the lack of correlation between urinary and serum vegf suggests that uvc reflects renal vegf production, and is possibly a biomarker of renal stress. uvc does not significantly change with amlodipine treatment suggesting that amlodipine may not cause renal stress when used in cats with hypertension and concurrent ckd. disclosures: this study was sponsored by orion inc. and ceva animal health and used residual samples collected from animals involved in a clinical trial run by orion inc. jonathan elliott provides consultancy advice to the following companies: bayer animal health, ceva animal health, orion inc., elanco animal health, zoetis ltd, boehringer ingelheim, vetoquinol ltd., waltham centre for pet nutrition, idexx ltd. the group is in receipt of research funding from the following companies: novartis animal health, royal canin ltd, zoetis, ceva animal health / orion inc. jonathan elliott serves on the following advisory boards: international renal interest society, european emesis council. diffuse large b-cell lymphoma (dlbcl) is the most frequent subtype of non-hodgkin lymphomas in dogs. in humans, common morphological variants have been recognized by the world health organization (who) classification: centroblastic, immunoblastic and anaplastic. the who classification was recently adapted to canine lymphomas. however, no study clearly correlated prognosis to each morphological variant of canine dlbcl. the objective of this retrospective study was to correlate morphological variants of dlbcl to prognosis, in dogs treated with a standardized chemotherapy protocol. medical records from dogs with a cytological diagnosis of dlbcl between and were retrospectively reviewed by a single boarded clinical pathologist. the centroblastic (dlbcl-cp) and immunoblastic morphotypes (dlbcl-ib) were defined as previously described. anaplastic variant is very rare in dogs, and no case meeting all inclusion criteria were diagnosed during the study period. a fourth borderlines morphological variant was identified and distinguished in this study for clinical considerations (immunoblasts rich centroblastics (dlbcl-irc)). it was characterized by the presence of a higher number of immunoblasts compared to dlbcl-cp. complete initial and follow-up clinical information and application of a standardized chemotherapy protocol were part of the main inclusion criteria. statistical analysis was performed using kaplan-meier analysis. fourty-nine dogs were included. thirty-four ( . %) were dlbcl-cp, ( . %) were dlbcl-ib and ( . %) were dlbcl-irc. median first remission duration for dlbcl-cp, dlbcl-ib, dlbcl-irc were respectively and . and days (p < . ). median overall survival time for dlbcl-cp, dlbcl-ib, dlbcl-irc were respectively , and . days (p = . ). a significant shorter time to obtain complete remission (p = . ) and a significant longer duration of first remission (p < . ) in dogs with dlbcl-cp in comparison to dlbcl-ib were observed when dlbcl-irc were included in the dlbcl-ib group. interestingly for cases, dlbcl-irc variant was observed in peripheral lymph nodes whereas dlbcl-ib variant was observed in the spleen. moreover, / recurrent dlbcl-cp and / of dlbcl-irc displayed progression towards dlbcl-ib variant. in conclusion, this study showed, for the first time, significant prognostic differences between the morphological variants of canine dlbcl, suggesting the prognostic impact of immunoblastic features as it is discussed in humans. disclosures: the residency program of david sayag is supported in part by zoetis. canine lymphoma is a heterogenous group of diseases and evidence exists to describe different behaviours between b-cell and tcell phenotypes of disease. this study aims to describe the response to treatment and survival of canine b-cell multicentric lymphoma (cbcml) cases treated at the royal veterinary college. signalment, clinical findings, staging, treatment, response and survival times were recorded retrospectively. sixty-three cases of cbcml were identified. forty-nine percent presented as stage , % stage , and % stage . sixty-two percent were substage b and % were substage a. forty-four percent received "chop" induction protocols, % "cop," % "coap," and the rest received various induction protocols. ninety-five percent of dogs responded to induction treatment. median first remission duration (frd) was . days. thirty-seven dogs ( %) received rescue protocols with a response rate of %. median overall survival time (os) was days. follow-up was days. this study showed that cop protocols followed by doxorubicin rescue therapy gave no significant difference in os compared with both chop induction alone and chop followed by a rescue protocol (p = . ). os was significantly increased by increased frd (p = . ), absence of an aberrant immunophenotype (p = . ), complete response to therapy (p = . ), and use of rescue protocol (p = . ). frd was significantly increased by use of a chop induction protocol compared with a cop protocol (p = . ), and complete response to therapy (p = . ). age, bodyweight, sex/neuter status, stage, substage, and cell size had no effect on frd or os. seven ( %) of the dogs had a prolonged os in excess of years, and of these dogs remain alive. dogs in the prolonged os group were more likely to be anaemic on presentation (pcv< %, p = . ), experienced a greater frd (p = . ) and were more likely to be treated with a rescue protocol (p = . ) than other dogs. no other significant differences in signalment, clinical presentation, stage, substage, or treatments received were found between this group of dogs and others. in this group of dogs, chop induction therapy gave no survival benefit over the cheaper, less intense cop protocol, providing doxorubicin rescue therapy was later employed. the proportion of dogs receiving chop induction versus cop did not significantly differ between dogs with prolonged survival and those without. the use of rescue protocol, complete response to treatment, aberrant immunophenotype and first remission duration were shown to have prognostic relevance. disclosures: no disclosures to report. lymphoma is the most common malignant haemopoietic tumour in the dog. gene expression profiling (gep) of canine lymphoma has highlighted the important signalling pathways including b-cell activation, b-cell receptor and nf-kb signalling. next-generation sequencing offers benefits over microarray technology for gep in identification of novel transcripts and sequence variants. the aim of this study was to examine gene expression and variant calling in canine lymphoma using rna-seq. lymph node samples were collected from canine multicentric lymphoma patients as part of their clinical investigations. diagnosis was confirmed cytologically or histologically and cell lineage established by pcr for antigen receptor rearrangements (parr) and flow cytometry. cdna was prepared from extracted rna and sequencing performed on an illumina nextseq sequencer generating bp paired-end reads. samples were from b-cell tumours ( stage v, stage iv, stage iii and stage ii) and t cell tumours ( stage iii, each stage ii, iv, v). million reads (mean) per sample were obtained with % mapping to the canine genome. b-and t-cell samples clustered separately on principal component analysis indicating distinct gene expression patterns. genes were upregulated (log fc > , q value < . ) in b-cell lymphomas, many involved in bcr signalling, primary immunodeficiency and haematopoietic cell lineage pathways, innate immune and inflammatory responses. genes were upregulated (log fc > , q value < . ) in t cell lymphomas, most affecting tcr signalling, but also natural killer mediated cytotoxicity, jak-stat signalling, haemopoietic cell lineage and cancer pathways. compared to the reference genome, . million sequence variants were detected across the samples; % not previously described. using the sift (sorting intolerant from tolerant) algorithm, % were predicted to be deleterious for protein function. functional analysis of the affected genes indicated many were involved in bcr signalling and cancer-related pathways. some such as bcl and map k were affected in almost all cases, although proportionally more frequent in b cell lymphoma. others such as traf were exclusive to b cell cases. genes affecting a large number of cases such as bcl tended to have a common variant present in or more cases whereas other genes had variants unique to each single case. although it remains to be confirmed if the detected variants represent true mutations rather than polymorphisms, rna-seq of canine lymphoma samples has generated interesting pilot data that need to be expanded with more samples to validate the results. disclosures: manikhandan mudaliar works for glasgow polyomics which is a commercial company within glasgow university and carries out genomic and polyomic assays. the next gen sequencing was done at glasgow polyomics, partly using an ecvim grant. feline large granular lymphocyte (lgl) lymphoma is uncommonly described in the literature and it is caused mainly by t-cell lymphocyte. to date a standard protocol has not yet been established and long term prognosis is poor. a recent study (krick et al. ) described a median survival time of days (range: - ) in cats with lgl lymphoma receiving mainly a cop-based protocol and in few cases adjuvant surgery or orthovoltage radiation therapy. surprisingly, in that study the longest survival time was achieved from a cat in the non treated group (median survival time days; range: - ) that received only prednisone and single agent cyclophosphamide. considering these data, and the advantages in treating with more than one alchylating agents t-cell lymphoma in dogs (brodsky et al ), the aim of this study was to assess if the sequential use of different alchylating agents was of any benefit in cats with lgl lymphoma. to all owners of cats with a cytological or hystopatological diagnosis of lgl lymphoma that presented to the san marco veterinary clinic from july till december were offered a treatment with sequential alchylating agents and prednisone (saa&p) protocol or palliative care with only prednisone. the saa&p protocol consisted of prednisone at mg/kg q h and chlorambucil at mg/cat (for cats > kg) to q h (for cats < kg). when despite treatment progressive disease or stable disease plus clinical signs referable to the lgl lymphoma were present chlorambucil was substituted with cyclophosphamide at mg/cat q days. finally, when cats stop responding to cyclophosphamide, this drug was substituted with lomustine at - mg/m q - week. during the study period cats were diagnosed with a lgl lymphoma. on owner request cats were euthanased at the time of diagnosis, cats were sent home with no treatment and lost to follow-up, cats received prednisone alone, and cats received the saa&p protocol. median survival time for cats treated with prednisone alone was days (range: - days, % ci - days) and for cats treated with the saa&p protocol was days (range: - days, % ci - ). survival kaplan-meier curves of the treatment group were significantly different (log rank test = . ; p = . ). survival time in cats with lgl lymphoma treated with the saa&p protocol is significantly longer than in cats receiving only prednisone and seems to be longer than historical reported data of cats receiving cop-based protocol. disclosures: no disclosures to report. mast cell tumors are often accompanied by eosinophilic inflammation as they are known to produce eosinophil chemotactic factors. however, little is known about frequency or eventual prognostic significance of blood eosinophilia in mct bearing dogs. thus, the aim of this study was to determine frequency of absolute and relative peripheral blood eosinophilia as well as eosinopenia and to evaluate potential influence on progression free interval (pfi), overall survival time (ost) and tumor specific survival (tss). dogs with mast cell tumors diagnosed between and were included into this retrospective study. data were collected from medical records and follow up phone conversations with patient owners or referring veterinarians. medical records were reviewed to rule out underlying clinical conditions other than mct that could cause eosinophilia. tumor diagnosis was made either by fine needle aspirate and/or tumor biopsy. a patient was allocated to the eosinophilic group, when the eosinophil concentration was > . * /ll or the relative percentage was > %, respectively. when the eosinophil concentration was < . * /ll patients were categorized as eosinopenic. groups were compared by the pearson chi-square test. the pfi, ost and tss curves were generated by the kaplan-meier product limit method. a log rank test was used to compare the curves. one-hundred dogs were included into this study. absolute eosinophilia was detected in / patients and in / a relative eosinophilia was present. median concentration of eosinophils was . * /ll (range, - ) and median relative percentage was . % (range, - %). eosinopenia was found in % of all dogs. a positive association between relative eosinophilia and low grade tumors was detected with both patnaik (p = . ) and kiupel (p = . ) grading system. a positive linear correlation was further noticed between absolute eosinophilia and ost (p = . ). positive correlation was confirmed between relative eosinophilia and pfs (p = . ), ost (p = . ), and tss (p = . ). accordingly a negative linear fit was found between eosinopenia and pfi (p = . ), ost (p = . ) and tss (p = . ). data indicate that peripheral blood eosinophilia might serve as an easily available additional prognostic tool for mast cell tumor bearing dogs. disclosures: no disclosures to report. limited literature is available about prognostic factors for canine renal carcinomas. in humans, histologic differentiation and tumor type are strongly associated with outcome. in dogs only one publication so far has reported this association. cox- expression is documented in several canine neoplasias with prognostic value in canine mammary carcinomas. in renal carcinomas cox- expression has been demonstrated but its significance is not known. the aim of this study was to evaluate clinical and histopathological features of canine renal carcinomas, including cox- expression, and to correlate them with outcome. our hypothesis was that advanced disease, higher histological grade and increased cox- expression would be associated with shorter survivals. this retrospective multi-institutional study within veterinary institutions, included histologically confirmed cases of canine renal carcinoma undergoing nephrectomy between - , with available follow up. histologic features and cox- immunostaining scoring were reviewed by independent pathologists where available. signalment, clinical presentation, stage, adjuvant therapy and survival times were recorded and statistical analysis performed. sixty-two cases were included. male to female ratio was : , median age was . years. cross-breed dogs (n = ) and labrador retrievers (n = ) were over-represented. on presentation dogs had metastasis. overall median survival time (mst) was days ( - days) . dogs without metastasis lived longer (mst versus days, p = . ). twenty-seven dogs received adjuvant therapy post-nephrectomy, without impact in mst (treatment days, no treatment days, p = . ). fifty samples were available for histopathological review, and for cox- immunostaining. shorter survival times were seen in solid histological type compared to others (solid days, papillary days, tubular days, p = . ). histologic degree of differentiation was associated with mst (well days, moderate days, undifferentiated days; p = . ). vascular invasion was associated with shorter survival (mst days versus days if absent; p = . ). marked invasiveness was associated with shorter mst ( days versus mild = days, moderate days; p = . ). patients with low cox- expression had longer mst ( days) than those with high ( days, p = . ). mitotic index, clear cell type, nuclear morphology, fuhrman nuclear grading, presence of pseudocapsule, necrosis, haemorrhage and type of inflammation were not significantly associated with mst. histopathological findings (degree of differentiation, invasiveness, vascular invasion, solid histologic type), cox- expression and metastasis present at diagnosis are strongly associated with survival in canine renal carcinomas and can be used as prognostic factors. disclosures: no disclosures to report. the detection of leishmania infantum-specific antibodies has been extensively exploited for specific diagnosis and monitoring of treatment in canine leishmaniosis. high levels of l. infantum-specific antibodies are commonly observed in dogs with moderate to severe disease. controversial results have been described regarding the use of kinetics of l. infantum specific antibodies during treatment monitoring. the majority of the studies reported that antibodies often decreased slowly but remained detectable over a long period of time while older studies considered that serology was not useful for treatment monitoring. a consensus statement is that measurement of antibody levels is meaningless before months of treatment. the aim of this study was to evaluate l. infantumspecific antibodies at the time of diagnosis and during treatment follow-up visits and to correlate these with the dog's clinical status. nineteen dogs were diagnosed (day ) and followed-up during treatment (days , and ). the treatment protocol was a combination of meglumine antimoniate ( mg/kg/ h sc for month) and allopurinol ( mg/kg/ h po for year). physical examination and baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) were performed during all visits. leishmania infantumspecific antibodies were assessed by an end point sera dilution elisa method. the majority of dogs (n = ) were classified as leishvet stage ii (moderate disease) at the time of diagnosis. three dogs were classified as leishvet stage iii (severe disease, n = ) or iv (very severe disease, n = ). results showed high and variable levels of specific antibodies at the time of diagnosis (meanaesd: ae elisa units (eu)). interestingly, a rapid significant reduction (p < . ) was observed at day during treatment (mean aesd: ae eu). a continuing significant decrease of specific antibodies was also determined at days (mean ae sd: ae eu) and (mean aesd: ae eu). all dogs improved clinically with treatment with the exception of one dog that clinically relapsed at months post-treatment and its specific antibodies level slightly increased. in conclusion, this study reports, for the first time, a rapid reduction of l. infantum specific antibodies after days of treatment by an end point sera dilution elisa method associated with clinical improvement. it is important also to highlight that a marked decrease of antibody levels was also noted after months and year of treatment. disclosures: no disclosures to report. a broad range of clinical manifestations and immune responses have been described in canine leishmaniosis. canine l. infantum infection can manifest as a chronic subclinical infection, self-limiting disease, or non-self-limiting illness. a protective cd + t-cellmediated immune response characterized by production of inter-feron-gamma, il- and tnf-alpha is believed to be present in resistant subclinical dogs while this response seems to be diminished or absent in sick dogs. however, there are few and poorly standardized assays to evaluate this response in the dog. in addition, cellular mediated immunity has been mainly investigated in subclinical or vaccinated dogs but limited information is available in sick dogs with different degrees of disease severity. the aim of this study was to investigate l. infantum-specific cellular immunity in dogs with clinical leishmaniosis at the time of diagnosis. twenty-six dogs were diagnosed based on physical examination, routine laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) and l. infantum-specific antibody levels measured by quantitative elisa. heparin whole blood was stimulated with l. infantum soluble antigen (lsa) and the mitogen concavalin a (cona) and incubated during days. unstimulated whole blood from each dog was used as control. supernatants were collected and ifngamma concentration was measured with a commercial sandwich elisa. the majority of dogs (n = ) were classified as leishvet stage ii (moderate disease). the rest of dogs were classified as stage i (n = ), stage iii (n = ) and stage iv (n = ). twelve dogs (stage i, n = ; stage iia: n = , stage iii: n = ) produced ifngamma after stimulation with lsa (mean ae sd: ae pg/ ml) and cona (meanaesd: ae pg/ml). in contrast, dogs (stage iia: n = ; stage iib: n = ; stage iii = n = ; stage iv, n = ) did not produce detectable ifn-gamma after stimulation with lsa but dogs produced ifn-gamma after stimulation with cona (mean ae sd: ae pg/ml) while one dog was unresponsiveness to cona. no differences in ifn-gamma concentration were found between ifn-gamma producer and nonproducer dogs when blood was stimulated with cona (p = . ). interestingly, ifn-gamma producer sick dogs presented with a lower antibody levels (meanaesd: ae elisa units (eu)) when compared with ifn-gamma non-producer sick dogs (mean ae sd: ae eu) but differences were not statistically significant (p = . ). the results of this study suggest that sick dogs with a more exaggerated humoral response and a more severe disease lack l. infantum specific ifn-gamma production in stimulated blood. disclosures: no disclosures to report. canine anaplasma platys infection (capi) results in thrombocytopenia, but is considered as a subclinical disease in the united states where the disease was first identified. in europe, where the disease seems to be more severe, it has been suggested that circulating strains are more pathogenic, although co-infection with other vector-borne pathogens (vbp) may also contribute to the expression of clinical signs. however, the availability of pcrbased investigation of the impact of co-infection in naturally infected dogs is limited, compromising the assessment of their clinical significance under field conditions. the aim of the present study was to describe epidemiological and clinical features of capi under field conditions in areas endemic for several canine vbp. a study was conducted in veterinary clinics across italy, spain and portugal. sick animals were included when fitting at least clinical and/or biological criteria compatible with ehrlichial disease. serological tests (snap Ò dx, snap Ò leish tests) and diagnostic pcr for ehrlichia canis, anaplasma platys, anaplasma phagocytophilum, babesia/theileria spp, hepatozoon canis and leishmania infantum detection were performed to identify the etiological agents. capi was considered on the basis of suggestive signs associated with positive pcr-based assay for anaplasma platys. among the dogs included, were pcr positive for a. platys. the annual incidence risk of capi was . % and the probability to diagnose capi when facing clinical and/or biological signs suggestive of ehrlichial disease was evaluated at . %. nine dogs were mono-infected, and dogs were co-infected with e.canis ( ), l.infantum ( ), babesia sp. ( ) and h.canis ( ). for dogs, all tests were not performed. anorexia ( %) and weight loss ( %) were common reasons for visit. lymphadenomegaly ( %), hyperthermia and cutaneous signs ( %) were frequent findings whereas musculoskeletal disorders ( %), petechiae/ecchymosis ( %), splenomegaly ( %), dehydration and ocular lesions ( %) then epistaxis ( %) were less common. haematological abnormalities included thrombocytopenia and anaemia ( %), leucopoenia ( %) and leucocytosis ( %). a risk-analysis conducted between mono-and co-infected dogs didn't highlight significant differences except for anorexia that was significantly more frequent in mono-infected dogs. this study illustrates the magnitude of capi in the mediterranean basin and supports the existence of virulent strains in this area. co-infections were common but had a weak impact on clinical expression. these results emphasize also the importance of testing dogs for multiple vbp due to the difficulty in assigning a specific symptom or haematological abnormality to a specific vector-borne infection in endemic areas. disclosures: no disclosures to report. leptospirosis is a worldwide zoonotic disease with high mortality rate in humans and dogs. clinicopathologic changes may reflect renal disease, hepatic disease, or both causing often vomiting, polyuria/polydipsia and diarrhea. therefore, severe electrolytes and anion gap (ag) abnormalities could be expected. the aim of this cohort study was to investigate serum electrolytes and ag in dogs with natural occurring leptospirosis and to assess their prognostic values. the electronic data-base of the san marco veterinary clinic p.o.a system-plus . Ò was searched between october- and april- for dogs with diagnosis of leptospirosis (group ; n = ). inclusion criteria for group were consistent clinicopathologic signs and a positive microscopic agglutination test (titer≥ : in vaccinated dogs, titer≥ : in nonvaccinated dogs or≥ -fold increase in convalescent titer) and/or a positive pcr (urine and/or blood) for leptospirosis. parameters studied were: serum electolytes (sodium, chloride, potassium), ag, and ag albumin-adjusted (ag alb-adjusted = ag + . x ( . -[alb] ).two control populations of randomly healthy dogs (group ; n = ) and sick dogs without leptospirosis (group ; n = ) dogs were created and matched to group for age (ae months), sex (including sexual status) and breed. statistical differences between groups were evaluated by kruskal-wallis test and post-test analysis were performed by wilcoxon-mann-whitney. mortality relative risk (mrr) at days post-admission between group and group was evaluated. roc curves were used to identify the best prognostic analyte. significance level for all statistical test was set at p < . . serum sodium and chloride concentrations were significantly decreased in group compared to group and (p < . for both comparisons). serum potassium concentration was significantly decrease in group compared to group (p = . ), while no difference was present between group and (p = . ). serum ag and ag alb-adjusted were significantly increased in group compared to group and (p < . for all comparisons). there was a significantly increased in mortality rate in group (n = , . %) compared to group (n = , . %) (mrr = . ; % ci = . - . ). between the variables studied serum potassium (auc = %; p = . ) and chloride (auc = %; p = . ), ag (auc = %; p = . ) and ag alb-adjusted (auc = %; p < . ) were prognostic. serum electrolytes, ag, and ag alb-adjusted were significantly different in dogs with leptospirosis compared to healthy and sick dogs without leptospirosis with the exception of serum potassium that was similar between sick dogs with and without leptospirosis. between the variables studied the ag alb-adjusted resulted the best parameter in predicting death in dogs with leptospirosis. disclosures: no disclosures to report. enterococci causes urinary tract infection (uti) in companion animals and may carry important resistance genes such as for the bifunctional enzyme. furthermore, their virulence factors are seldomly reported in veterinary medicine. thus, this study aims to characterize the uropathogenic enterococci antimicrobial resistance, virulence genes and the clonallity of high-level gentamicin resistance (hlgr) enterococcus faecalis. antimicrobial susceptibility testing of clinical uropathogenic enterococci isolated from dogs and cats with uti, isolated between - , was performed by the disc diffusion method. clsi clinical breakpoints were applied. strains showing hlgr were screened for aac( )-ieaph( ')-ia and aph( ')- d genes by pcr. e. faecalis harbouring hlgr genes were typed by multi-locus-sequencing. fifty-nine strains were further characterized by pcr for the presence of gel e (gelatinase), ace (collagen binding antigen), asa- (aggregation substance), and efa a (endocarditis) virulence genes. e. faecalis was the most frequently isolated ( . %, n = / ) followed by enterococcus faecium ( . %, n = / ). overall, antimicrobial susceptibility results were: . % (n = / ) resistance to penicillin/ampicillin; . % (n = / ) resistance to fluoroquinolones (enrofloxacin or ciprofloxacin); . % (n = / ) resistance to nitrofurantoin; . % (n = / ) resistance to chloramphenicol and . % (n = / ) resistance to tetracycline. hlgr was detected in . % (n = / ) enterococci, namely e. faecalis and two e. faecium. all hlgr e. faecalis were aac ( )-ieaph( ')-ia carriers. one e. faecium was positive for aac ( )-ieaph( ')-ia whereas the other was positive for aph( ')- d. interestingly, ampicillin-resistance was only detected in e. faecium ( out of isolates). furthermore, all hlgr e. faecium were also ampicillin-resistant. hlgr e. faecalis were found to belong to st , st , st , and st major lineages circulating in both hospital and community settings in portugal. considering all enterococci, . % (n = / ), . % (n = / ), . % (n = / ) and . % (n = / ) were positive for gel e, ace, asa- and efa a virulence genes, respectively. uropathogenic e. faecium were only positive for ace gene ( out of ), thus e. faecalis had higher virulence genes frequencies. in this study we detected important human hlgr e. faecalis belonging to the clonal complex , such as e. faecalis st , among uropathogens in companion animals. the presence of major clonal lineages in companion animals highlights their role as communityassociated hosts and possible reservoirs of putative human pathogenic enterococci. disclosures: conflicts of interest: c atia marques currently receives a phd grant funded by the portuguese foundation for science and technology. haemotropic mycoplasmas (haemoplasmas) can cause haemolytic anaemias in many species, including people. three feline haemoplasmas have been identified: mycoplasma haemofelis (mhf), 'candidatus mycoplasma haemominutum' (cmhm), 'candidatus mycoplasma turicensis' (cmt). mhf is considered the most pathogenic, whilst cmhm and cmt usually only cause anaemia in cats with concurrent disease or immunosuppression. the aim of this study was to estimate the prevalence of feline haemoplasmas in serbia and identify potential risk factors for infection. surplus edta blood samples from cats in the belgrade region were used. for each cat, the following variables were recorded: age, health status, gender, outdoor access, presence of ectoparasites and haematological results. samples were stored at À °c and transported to the university of bristol for haemoplasma quantitative pcr testing. serology (petchek, idexx) was performed for felv (n = ) and fiv (n = ) infection. statistical analysis was performed using spss; univariable associations between variables and haemoplasma status were first evaluated (v for categorical variables, t-test/mann-whitney u test for continuous variables) followed by multivariable analysis. two samples were negative for internal control s rdna and excluded from the study. of the remaining cats, ( . %) were infected with one or more haemoplasma species; were singly infected ( mhf, cmhm, cmt) , dually infected ( mhf/cmhm, mhf/cmt, cmhm/cmt) and triple infected. the overall prevalences of mhf, cmhm and cmt were . %, . % and . %, respectively. / ( . %) cats were felv infected whilst / ( . %) were fiv infected. multivariable analysis identified significant associations between haemoplasma infection and anaemia (anaemic/non-anaemic, odds ratio (or) . , ci . - . , p = . ), male gender (male/female, or . , ci . - . , p < . ), outdoor access (yes/no, or . , ci . - . , p < . ), non-pedigree breed (non-pedigree/pedigree, or . , ci . - . , p = . ) and fiv positive status (positive/ negative, or . , ci . - . , p = . ). the overall prevalence of feline haemoplasmas in serbia ( . %) is similar to that reported in other european countries ( . - . %). cmhm was the most prevalent haemoplasma species ( . %) in the current study, similar to other european studies (range: . - . %). most previous studies reported that cmt infection is the least prevalent feline haemoplasma species, but in the current study the prevalence of cmt was greater than that of mhf. similarly to previous studies, the presence of anaemia, male gender, outdoor access, non-pedigree status and fiv infection were significantly associated with haemoplasma infection. disclosures: no disclosures to report. canine parvovirus type (cpv- ) is a frequent digestive pathogen in dogs, responsible for high mortality rates in puppies. the control of the infection by disinfection and isolation of patients is of limited efficiency, raising questions about the contagion sources. the aim of our study was to evaluate the epidemiological role of dams in viral circulation during the reproductive period. a total of bitches (mean ae standard deviation: . ae . years old) from one kennel were enrolled in the study. all were annually vaccinated (nobivac dhppi-lepto vaccine; msd, beaucouz e, france). dams were followed from mating to whelping and dams were followed from whelping until weaning. all puppies from the lactating dams (n = ) were followed since until weeks of age. cpv- fecal excretion was evaluated by real time pcr on rectal swabs [ ] every days during gestation (dams) and every days during lactation (dams and puppies). data were analyzed through logistic regression and mixed linear models. a total of samples were collected. during pregnancy, % of the bitches excreted cpv at least once, but only one sample was above the quantification threshold ( . copies/g feces). dur-ing lactation, all bitches were found positive at least once (and times in mean) and % went above the quantification threshold at least once. during lactation, excreted viral loads were significantly higher at d ( . /g feces; p = . ), d ( . /g feces; p < . ) and d ( /g feces; p < . ) compared to the early lactation (< copies/g feces; d to d ). despite threshold for a clinical parvovirosis is . /g feces, none of the bitches expressed any symptom. in % of the cases, the dam excreted before her puppies. viral loads excreted by puppies were not correlated with those excreted by dams. the proportion of puppies excreting viral loads above the clinical threshold increased from d to d (from to % per litter), with overall mortality of only % ( / ). this study demonstrates that appropriately vaccinated adult female dogs may excrete cpv during gestation and lactation. due to the high quantity of cpv- excreted, females probably represent a major source of contamination for their puppies. viral excretion by bitches after lactation until the next breeding period and by males would be interesting to follow to better understand the role of adults in cpv circulation. [ feline panleukopenia virus (fpv) is responsible for one of the most severe infectious diseases in cats, but only few studies have addressed factors of prognostic importance. in an earlier investigation spanning over years, leukopenia, thrombocytopenia, hypoalbuminemia and hypokalemia were found associated with poor outcome. here, we aimed at identifying outcome predictors during shelter outbreaks of panleukopenia between and ; we limited our analysis to fresh cases treated and followed until recovery or death at the same institution. clinical records of the affected cats were reviewed and information was collected at diagnosis and during hospitalization. the data included anamnestic history, physical examination, complete blood count, biochemical profile, blood gas analysis and treatments, including types of antibiotic, antiviral, antiemetic, analgesic, crystalloid, colloid and hemoderivative administered. outcome predictors were analyzed using logistic regression and mixed-design analysis of variance. the study included cats diagnosed with panleukopenia based on clinical findings and positive fecal elisa, of which . % were < months old, and . % females. clinical signs at diagnosis included lethargy ( . %), vomiting ( . %) and diarrhea ( . %). at admission, median (range) leukocyte counts were , /ll ( - , ) and platelets , /ll ( - , ); . % had hypoalbuminemia and . % hypokalemia. treatments included administrations of amoxicillin-clavulanate ( . %), interferon-x ( . %) and intravenous glucose solution ( %). overall, . % of the cats did not survive. lethargic cats were more likely to die (or: . , ci %: . - . , p < . ). leukocyte counts at diagnosis were not associated with outcome, but were after days of hospitalization; in particular, cats alive at days, which succumbed later, had leukocyte counts of /ll ( - , ) whereas survivors had , /ll ( - , ) (p < . ). survivors were more likely to have received amoxicillin-clavulanate (or: . , ci %: . - . , p < . ) and less likely intravenous glucose solutions (or: . , ci %: . - . , p < . ). thrombocytopenia, hypoalbuminemia, hypokalemia and administration of interferon-x were not associated with the outcome. our results suggest that infected cats with lower leukocyte counts later during hospitalization are more likely to die despite treatment. in this study, and different from previous data, lower leukocyte counts at admission did not predict outcome, possibly due to inclusion of cats with early fpv diagnosis. administration of intravenous glucose was associated with poor outcome, perhaps because of an increased risk of sepsis; also, cats in critical conditions were more likely to receive intravenous glucose. the beneficial role of amoxicillin-clavulanate in sick cats might be due to its broad-spectrum bactericidal activity; interferon-x did not show any conspicuous effect. disclosures: no disclosures to report. according to prior studies up to % of cats in southern germany do not have protective antibodies against feline panleukopenia virus and thus, are likely susceptible for feline panleukopenia infection. until now, it is unknown how healthy adult cats with different antibody titers react to feline panleukopenia vaccination in the field. therefore, the aim of the study was to measure antibody titers in healthy adult cats within days after feline panleukopenia vaccination. one hundred and twelve healthy adult cats were vaccinated with a rcp vaccine. before vaccination (day ) and on days and antibodies against panleukopenia virus were determined by hemagglutination inhibition. in . % ( / ) of the cats, no antibodies prior to vaccination were detected; of these cats were vaccinated regularly. nearly one third of the cats ( . %; / ) showed no antibody increase after vaccination and in . % ( / ) of the cats, antibody titer decreased despite vaccination within the days. however, all of these cats were likely protected by their preexisting antibody titer. in cats no antibodies were detected neither prior to nor after vaccination. a large number of adult cats has no protective antibodies and is therefore at risk for feline panleukopenia virus infection. on the other hand, many other cats show high antibody titers and do not develop antibodies due to vaccination. therefore, evaluation of individual antibody status in cats and vaccination only in those cats, that have no antibodies or low titers, should be recommended. disclosures: independent study financed by merial. there was no influence on the results of the study by merial and there is no co-authorship planned with merial. the feline coronaviruses (fcov) occur as pathotypes with an enigmatic, even controversial, relationship: the low virulence or nonvirulent feline enteric coronavirus (fecv) and the highly lethal feline infectious peritonitis virus (fipv). recently, sequence differences within the spike gene region encoding the putative fusion peptide were described and proposed to correlate with the mutated form of fecv (i.e. fipv) leading to the clinical presentation of feline infectious peritonitis (fip). in this presentation, the development and validation of an allelic discrimination real-time pcr typing test which can identify each mutation separately will be described. the diagnostic sensitivity and specificity will be reported from a set of european clinical samples acquired from either fip confirmed cats or from healthy cats that previously tested fcov positive. of these archived samples, fcov positive samples were included into the validation. from these, samples did not pass quality control and had virus levels that were below the limit of detection of the pcr assay. of the remaining samples, were typed correctly with an accuracy of . %. one fip characterized sample was typed fecv (diagnostic sensitivity . %) while all of the healthy cats were typed fecv ( % diagnostic specificity). to confirm that these spike gene mutations are not unique to european cats with fip, additional validation studies from us and japanese samples were conducted. the us clinical study included cases, from fip suspicious cases and with non-fip compatible disease. the fipv realpcr biotyping assay was able to accurately differentiate between the fip or non-fip (fecv) etiologies (p < . ) and did not biotype cats with confirmed non-fip disease as fipv, confirming the high diagnostic specificity of the molecular test. disclosures the aim of this study was to compare the diagnostic accuracy for fip of conventional clinico-pathological tests (routine hematology, serum protein electrophoresis, a -acid glycoprotein -agpmeasurement and analysis of the effusions) with that of molecular tests such as routine pcr and pcr followed by the sequencing of the spike (s) gene. blood, effusion and tissues specimens were collected from cats with symptoms imputable to fip. the in vivo examination consisted of clinico-pathological tests such as complete blood count, serum protein electrophoresis, agp measurement, cytological and biochemical examination as well as the evaluation of the sysmex dtnc of effusions, when present, and of molecular tests such as a screening pcr (directed towards the utr region) and the pcr directed towards the s gene followed by sequencing of the amplification products in order to detect the aminoacidic substitution considered diagnostic for fip. the same molecular techniques were applied to the tissues samples collected during necropsy, which also allowed to divide the cats in a fip group ( cats) and in a non fip group ( cats) based on histology and immunohistochemistry. the diagnostic accuracy (sensitivity, specificity, negative and positive predictive values) of each test was calculated. the best test on tissues was immunohistochemistry (sens: . %; spec: %), while the screening pcr suffered of low sensitivity and very low specificity (sens: . %; spec: . %). the s gene sequencing, positive when revealing the mutated nucleotide, showed very low sensitivity (sens: . ; spec: %). on effusions, the best tests resulted the screening pcr and cytology (sens and spec: %) in comparison with the dtnc measurement (sens: . %; spec: %) and the s gene sequencing (sens: . %; spec: %). in blood samples, agp measurement demonstrated the best diagnostic accuracy (sens: . %; spec: %), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: . %; spec: %). screening pcr (sens: . %; spec: %) and s gene sequencing (sens: . %; spec: %) proved again low accuracy, demonstrating that a negative result with these molecular tests does not allow to exclude fip. . chang hw, egberink hf, halpin r, spiro dj, rottier pjm "spike protein fusion peptide and feline coronavirus virulence"emerg infect dis ( ) diagnosis in feline infectious peritonitis (fip) is still challenging, especially in cats without body cavity effusion. uveitis in cats with fip commonly presents without effusion, which makes a definitive confirmation of fip difficult. the aim of this study was to evaluate the diagnostic utility of an immunocytochemical (icc) assay using aqueous humor in cats suspected of having fip. samples of cats with immunohistochemically confirmed fip and cats that were suspected of having fip due to similar clinical or laboratory changes, but that were definitively diagnosed with another disease were examined. aqueous humor was collected post-mortem after the cats were euthanized due to their diagnosed diseases. icc analysis was carried out using an anti-feline coronavirus mouse monoclonal igg a and an avidin-biotin complex method. sensitivity, specificity, negative and positive predictive values were determined and % confidence intervals ( % ci) calculated. of the aqueous humor samples, ( with fip, controls) revealed positive icc results. false positive icc results were obtained in cats suffering from lymphoma and pulmonary adenocarcinoma. diagnostic sensitivity of the icc assay in aqueous humor was . % ( % ci . - . ); diagnostic specificity was . % ( % ci . - . ); the negative predictive value was . % ( % ci . - . ); the positive predictive value was . % ( % ci . - . ) . unfortunately, false positive results occurred, and specificity, which is considered the most important parameter in a fatal disease like fip, was only . %. positive icc results in aqueous humor should be interpreted cautiously and cannot confirm a suspicion of fip. disclosures: no disclosures to report. alanine aminotransferase (alt) level in plasma is the most commonly used indicator for hepatocellular injury in dogs. however, dogs with advanced liver disease can present with alt levels within reference range. recent studies have shown the potential of circulating micrornas as a biomarker for liver injury. hepatocyte-derived microrna- (mir- ) was identified to be liver specific with superior sensitivity over alt levels in mice and humans. the aim of the present study was to investigate the potential for circulating mir- to serve as a diagnostic serum biomarker of hepatocellular injury in dogs. hereto, liver biopsies of labrador retrievers were collected. liver histology, including grade of hepatitis and stage of fibrosis, was reviewed by a boardcertified veterinary pathologist (tsgamvdi). concurrently, serum samples were collected and analyzed for alt levels and mir- levels. dogs were included into one of the following groups: normal liver and normal alt levels (control group), liver pathology and normal alt levels, or liver pathology and high alt levels. comparative statistics between groups were performed using the mann-whitney u test and associations between mir- and alt levels, grade, stage and hepatic copper concentrations were analyzed using the spearman's rank correlation coefficient. logistic regression models were used to assess the accuracy of mir- and alt to detect the presence of hepatocellular injury. in total, dogs had normal liver histology and normal alt levels. thirtyeight dogs had liver pathology whereof only dogs had increased alt levels. in the high alt group the median level of mir- was (range, - ) times higher compared to the control group (p < . ). even in dogs with liver pathology and normal alt levels, the median mir- level was (range, . - ) times higher compared to the control group (p < . ). univariate logistic regression showed that only mir- and not alt level was a significant predictor for abnormal liver histology (p < . ). serum levels of mir- were positively correlated with alt levels, histological grade and stage of fibrosis (r = . , r = . , r = . , respectively, p < . for all). this study highlights the potential of mir- as an early and sensitive biomarker for liver injury in dogs and is more sensitive than alt levels. early diagnosis of hepatocellular injury opens the opportunity to institute treatment in a subclinical stage of disease with a possibly more favorable outcome. disclosures: this study was financially supported by the ecvim clinical studies fund. the authors declare no further conflict of interest. the evaluation of liver fibrosis is of major importance for the management of chronic liver disease and the prediction of prognosis. although liver biopsy is the gold standard for evaluation of fibrosis, non-invasive tests enable the clinician to stage and monitor a variety of liver diseases in human medicine. as such transforming growth factor b (tgf-b) and hyaluronic acid (ha) are biomarkers of hepatic fibrogenesis, that reflect the activity of the fibrogenic and fibrinolytic process, their use has not been validated in dogs. the aim of this study was to evaluate the measurement of tgfb and ha and assess their sensibility and specificity for the monitoring of dogs with different level of hepatic fibrosis. eighty three adult dogs were prospectively enrolled based on a persistent elevation of alt, with the exclusion of those with focal hepatic lesions on ultrasound examination. all dogs underwent liver biopsy and serum blood collection. lf was staged according to histopathological wsava criteria and the amount of collagen was measured through morphometric analysis. quantitative variables were expressed as meanaesd. bean plots described the relationships between variables. bivariate analysis between ha, alt and pal with lf were performed by spearman correlations. a parametric test (student test) was carried out to assess the relationship between tgfb and lf. diagnostic cut-offs were determined according to the maximum youden index [sensitivity (se) + specifity (spe) - ]. preliminary results in dogs showed that . % of the individuals with ha below ng/ml had a density of < . % collagen. the serum concentration of ha was significantly (p-value = . ) higher in the group whose fibrosis density was ≥ . %. tgf-b was the most sensitive marker but its specificity to diagnose dogs with more than . % of collagen was quite low. the preliminary results show a potential interest of ha as a biomarker to detect liver fibrosis in dogs but the interest of the tgfb could not be demonstrated. ha combines a good sensitivity with a fair specificity. the complete results of the study will help to refine the cut-off value and to improve the diagnostic performance of ah and to evaluate the interest of tgfb. a combination of several markers would be helpful to elaborate a sensitive and specific diagnostic test for liver fibrosis. disclosures: the speaker declares a potential conflict of interest with the company echosens . echosens covers a part of the biological measurements expenses in the clinical trial associated to this abstract. primary hepatitis is a common disorder in dogs. treatments for primary hepatitis are typically symptomatic and importantly, predicting prognosis at point of diagnosis remains challenging. in contrast to human medicine, where the type of hepatitis is defined by the inciting cause, few causes of chronic hepatitis have been identified in the dog, and the majority of cases are idiopathic. systemic inflammation is well recognised in humans with liver disease. systemic inflammatory response syndrome (sirs) is the clinical expression of the action of complex intrinsic mediators of the acute phase reaction. the presence of sirs has been linked to a poor outcome in various liver diseases. the prevalence and predictive value of a sirs in dogs with primary hepatitis has not been examined in dogs with liver disease. this is surprising given the accumulating evidence that sirs is linked to the development of hepatic encephalopathy (he) in dogs with liver disease. although the pathogenesis of he is not completely understood, it has been indicated that ammonia and inflammatory cytokines play a crucial role in the development of he. he is an important cause of morbidity and mortality in patients with liver disease. the hypothesis of this study was to examine the prevalence and severity of sirs n dogs with histological confirmed primary hepatopathies. eighty dogs with primary hepatopathies (confirmed with histopathology) were included in this study. a sirs score was calculated for each (respiration rate > breaths per minute; heart rate > beats per minute; total white blood cell count < or > ^ /l and rectal temperature < . or < . °c). sirs scores presented as a value from to . patient's date of arrival in hospital and date of death were all recorded; therefore survival time (days) could be determined. sirs scoring was applied and survival time was recorded. the median survival for sirs ( - ) was days, while sirs ( - ) had a median survival of days (p value < . , log rank test). this study demonstrates that sirs is a common feature of dogs with primary hepatitis and is valuable in predicting clinical outcome. disclosures: no disclosures to report. the aim of this study was to determine the prevalence of congenital portosystemic shunts (cpss) in deerhounds, focussing on the uk and the usa, and to determine how many deerhound breeders routinely test their puppies for cpss. congenital portosystemic shunts (cpss) are over-represented in certain breeds such as irish wolfhounds, maltese and yorkshire terriers. anecdotal evidence suggests that there is also an increased prevalence in deerhounds. this has not been confirmed, however. the study was questionnaire-based, distributed online to deerhound breeders worldwide (particularly the usa and uk). in addition it was distributed by post in the uk. the questionnaire passed ethical review at the department of veterinary medicine, university of cambridge. fifty-six breeders worldwide returned questionnaires (uk , usa , other countries ). the uk response rate was . % (including postal and online responses). the prevalence of shunts was found to be . % of puppies with prevalences in the uk and the usa of . % and . %, respectively. worldwide, % of breeders were found to test routinely for cpss in their puppies, while the proportions in the uk and the usa were % and %, respectively. the prevalence of cpss in uk and usa deerhounds found in this study was higher than was found for mixed-breed dogs ( . %) in a separate study. this suggests a genetic component to the disease in deerhounds. a lower proportion of breeders rou-tinely tested for cpss in the usa compared with the uk. the prevalence of cpss was also lower in the usa. these findings may be related. it would be advisable for all breeders to routinely test their puppies for cpss before sale, and to avoid breeding from affected animals. disclosures: st catharine's college, cambridge, contributed to the cost of this study. the deerhound club (uk) and the scottish deerhound club of america gave their support, helping to distribute and advertise the study. measurement of neuroendocrine markers can offer diagnostic, prognostic, and therapeutic information that cannot be obtained by clinical examination. nt-probnp is a potential biomarker for hypertensive target organ damage (tod). circulating concentrations of this biomarker are increased in human hypertensive patients with tod and with poor response to antihypertensive treatment. cats with hypertension and tod have significantly higher nt-probnp concentrations than non-hypertensive cats. the aim of this study was to investigate the utility of nt-probnp as a biomarker of hypertension, tod and efficacy of antihypertensive treatment. plasma samples from hypertensive cats seen at first opinion practices were retrospectively identified. hypertension was diagnosed based on systolic blood pressure (sbp) ≥ mmhg with evidence of hypertensive retinopathy (tod-group; n = ) or sbp≥ mmhg on consecutive visits - weeks apart without evidence of retinal pathology (notod-group; n = ). all cats achieved sbp control (defined as < mmhg) on . - . mg amlodipine once daily and had samples available on both a hypertensive visit and the first visit target sbp was achieved. additionally, healthy cats ≥ years old (n = ) and normotensive cats diagnosed with ckd (plasma creatinine ≥ lmol/l in conjunction with usg< . ; n = ) were identified. nt-probnp concentration was measured at an external laboratory. if necessary, variables were log-transformed to meet normality of distribution. binary logistic regression was used to investigate nt-probnp as a predictor of hypertension (using the healthy and ckd cats as comparator group) and tod (using notod as comparator group). comparisons between groups and of response to treatment were performed using mann-whitney u and wilcoxon rank sum tests respectively. higher nt-probnp concentration significantly increased the probability for a cat to be hypertensive (odds ratio log(nt-probnp) = . , [ % confidence interval . , . ], p < . ), but could not reliably predict tod (p = . ). nt-probnp concentration was however significantly higher in cats with tod than in cats with no tod ( . [ . , . ] these data suggest that increased plasma nt-probnp concentration predicts hypertensive status in cats. cats without tod have significantly lower nt-probnp concentrations at diagnosis of hypertension than cats with tod. nt-probnp concentration decreases with effective antihypertensive treatment. further studies are required to determine whether nt-probnp remains elevated in cats with poorly controlled blood pressure. disclosures: esther bijsmans's phd is funded by zoetis. hypovitaminosis d has previously been shown to be prevalent amongst dogs with protein losing enteropathy (ple). outcome is generally poor in canine ple, and there is a lack of studies identifying underlying risk factors. the hypothesis of this study was that low vitamin d serum concentrations could be a risk factor for bad outcome in such patients. medical records for dogs seen at the royal veterinary college between and were reviewed to identify dogs with a diagnosis of ple confirmed by histopathology. dogs were included in the study if they had serum samples frozen within minutes after sampling, had been kept at - degrees c until analysis, and if clinical activity scoring (cce-cai) had been recorded at the time of diagnosis. forty-three dogs were included in the study. follow-up with referring veterinarians was made to determine outcome of patients. patients were divided into groups: patients deceased due to ple (poor outcome group, n = ) and patients alive or deceased due to another disease (good outcome group, n = ). treatments for patients were allocated to groups: one group consisted of patients who were prescribed diet only and the other group received diet and immunosuppressive agents. samples were sent on dry ice to michigan state university's diagnostic center for population and animal health. ionised calcium (ica) was measured using an ion specific electrode and (oh)d was measured using a commercially available radio-immunoassay that has been validated for use in veterinary medicine. comparisons of outcome groups for age, ccecai, treatment, serum (oh)d and ica were performed using a mann-whitney u test or chi . logistic regression analysis was performed to determine possible risk factors for poor outcome. results: ccecai scores, age, and ica concentrations between the groups were not significantly different. there was a significantly greater number of dogs treated with food alone in the group with good outcome ( / ) than in the poor outcome group ( / , p = . ). furthermore, median serum (oh)d concentration was significantly lower in patients with poor outcomes ( . nmol/l, range - nmol/l) compared to patients with good outcomes ( nmol/l, range - nmol/l, p = . ). using logistical regression, (oh)d serum concentration was a statistically significant factor for poor outcome (p = . ), with an increase of (oh)d serum concentration reducing the odds of having a poor outcome (odds ratio = . , % ci: . - . ). further studies are required to investigate vitamin d as a potential adjuvant therapeutic agent in ple patients. disclosures: no disclosures to report. campylobacter jejuni (cj), c. upsaliensis (cu) and c. helveticus (ch) are commonly isolated from dog and cat faeces but association with clinical signs is discordant or lacking. cj is a recognized human pathogen, cu is considered an 'emerging' pathogen and ch is not considered pathogenic despite a high level of genetic similarity. recently, the greater wax moth, galleria mellonella, was described as an animal model of disease; these invertebrates have a high degree of functional and structural homology with the mammalian innate immune system. this study aimed to evaluate the pathogenic potential of cj, cu and ch using the galleria mellonella larvae model. twelve isolates of cj, of cu and of ch from dogs and cats were used for the inoculation of larvae. inocula were prepared by suspending isolates in phosphate-buffered saline (pbs) from which -fold dilutions were made. each dilution was tested in duplicate sets of larvae. each larva was injected with - ll into the haemocoel via the last left pro-leg using g insulin syringes. controls consisted of pbs inoculated larvae and un-inoculated larvae. survival of larvae at °c in a h enriched microaerobic atmosphere was monitored for days postinjection. one subset of isolates was grown in mueller-hinton broth and used for the preparation of secretory products, and another grown on blood-agar and suspended in pbs for heat inactivation of minutes at °c for testing of whole-cell lysates and heat-stable insoluble and soluble components. the overall median survival of larvae was % with cj [iqr - ], % with cu [iqr - ], % with ch [iqr - ], % with pbs [iqr - ] and % for un-inoculated larvae [iqr - ]. a dose-dependent association was evident for each species with larval survival being similar between a low bacterial dose and pbs. larval survival presented a consistent pattern between species for medium and high bacterial loads; cj had a higher and faster larval death rate than cu and ch (p < . ), but no difference was observed between cu and ch (p = . ). there were no significant differences between species in any of the assays with secretory products, inactivated cells and soluble/insoluble cellular components. the observations within this invertebrate disease model support a varying pathogenic potential between the species studied that appears related to the (patho)biology of the species rather than their cellular components or metabolic products. the invertebrate animal model is promising in comparative pathogenicity studies. disclosures: no disclosures to report. acute stress from medium or high duration high-intensity exercise has been reported to be associated with an increase in serum c-reactive protein (crp) concentrations, an important acute-phase reactant in dogs. however, the effect of exercise on fecal s a concentration, a biomarker of intestinal inflammation has not previously been evaluated in dogs. the goal of this study was to determine if moderate intensity short duration exercise causes an increase in crp and/or s a concentrations in dogs, potentially leading to misinterpretation of their results. adult military working dogs (german and belgian shepherd dogs; males; mean age = years [ . - . ]) were included in the study. fecal quality, fecal s a , and serum crp concentrations were evaluated just before and after standardized exercise ( minutes of bikejoring at a speed of km/h). fecal quality was evaluated based on a -point scale (from : liquid to : dry and hard feces). fecal s a and crp concentrations were assayed with previously validated elisa tests. data were analyzed with an anova test for repeated measurements (sas software). results are presented as medians and ranges. serum crp concentrations increased significantly after exercise (median before and after excercise mg/l [ - ] and mg/l [ - ] (p = . ). also, fecal s a concentrations were significantly higher after exercise compared with baseline concentrations ( ng/g [ - ] versus ng/g [ - ], p = . ). no significant effect of exercise on fecal score was observed ( [ . - . ] before and after the exercise; p = . ). our study demonstrates that a moderate-intensity, short-duration effort performed by healthy army dogs causes significant increases in fecal s a and serum crp concentrations, as compared with baseline values, but within the respective reference intervals. therefore, a moderate exercise does not present a confounding variable in the interpretation of fecal s a or serum crp concentrations in healthy dogs. disclosures: this study was performed thanks the financial support of royal canin. imaging is an integral part of the work-up of canine gastrointestinal (gi) disease. radiography and ultrasonography are noninvasive modalities that can evaluate the bowel, but many findings lack desirable sensitivity or specificity. endoscopy directly visualizes gi mucosa, but is limited by the length of the endoscope and the need for general anesthesia, advanced training and expensive equipment. ambulatory light-based imaging (ali) is a new imaging modality that utilizes high-resolution cameras, a microprocessor, and led illumination to non-invasively visualize the gastrointestinal mucosa. ali is performed by oral administration of a fully automated device the size of a pill that is propelled by peristalsis. the aim of this study was to analyze image quality and gi transit times in a series of client owned dogs undergoing ali. dogs were food-restricted for hours before and hours after capsule administration. capsules were retrieved and images were downloaded and analyzed. video clips of frames duration were obtained from the stomach; proximal, middle and distal small intestine; and proximal colon for assessment of image quality. internists rated the images on a scale of - ( = poor, = excellent) based on clarity and resolution of images, and obscuration of the mucosa by fluid, bubbles or debris. scores for each region were compared using general estimating equation analysis. gastric and small intestine transit time were calculated based on visualization of passage of the capsule from the stomach to duodenum, and ileum to colon. clinical analysis of the entire video was performed by one of the authors. ali was successfully performed in / patients, with no adverse effects. average study duration was . ae . hours and mean image acquisition count was , ae , . gastric and small intestinal transit times were . ae . minutes and . ae . minutes, respectively. median (range) image quality scores were ( - ), ( - ) and ( - ), for the stomach, si and colon, respectively. image quality scores were significantly higher in the stomach and si than in the colon (p < . ). visualized lesions were consistent with gi ulcers ( dogs), inflammatory bowel disease ( dog), and bilious vomiting syndrome ( dog). one dog receiving chronic nsaids had a normal study. ambulatory light-based imaging resulted in good to excellent image quality throughout most of the gi tract. bowel preparation should be considered to enhance visualization of the colon. ali was safe and easy to perform in ambulatory dogs, and should therefore be considered in the work-up of canine gi disease. disclosures canine pancreatitis is the most common exocrine pancreatic disorder. the prognosis of canine pancreatitis is variably and no logistic regression constructed severity scoring systems are available. four hundred and thirty nine dogs diagnosed as pancreatitis with acute onset of compatible clinical signs, a positive snap Ò cpl tm test, and/or associated abdominal ultrasonographic abnormalities between january and december were presented at national taiwan university veterinary hospital (ntuvh). one hundred and three dogs hospitalized with complete medical therapy and outcomes were selected for further analysis. the dogs were divided into survival (n = ) and non-survival (n = ) groups. forty-seven parameters including signalment, clinical signs, physical examinations, clinicopathological examination, complications and concurrent diseases were analyzed and compared between the groups. logistic regression analyses were performed in this study. variables with p ≤ . were considered for further analyses. the mortality in this study was . %. age, heart rate, respiratory rate, white blood cell count, albumin, bun, creatinine, potassium, presence of systemic inflammatory response syndrome (sirs) and presence of oliguria or anuria were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each dog by summing all weighting factors. the total scores of each dog ranged from to . the severity scores in this study achieved an area under the receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with a sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was a significant difference (p < . ) between the groups seperated by the cut-off point. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in dogs with pancreatitis. disclosures: no disclosures to report. glucocorticoids (gcs) are known for their anti-inflammatory and immunmodulatory properties and are therefore often used in the therapy of canine inflammatory bowel disease (ibd). it was recently shown that endogenous gcs are also produced in the intestinal epithelium of men and mice and influence the gastrointestinal immune system in case of inflammatory or neoplastic conditions. thus, the aim of this project was to prove that gcs can be produced or metabolized in the canine intestinal epithelium. five healthy beagle dogs were included into this prospective study. all dogs were clinically examined, given a clinical score using the canine ibd activity index (cibdai) scoring system, also gastrointestinal endoscopy was performed. mucosal biopsy specimens from duodenum were examined histologically from a board certified pathologist using the wsava grading. biopsy incubation of - endoscopical mucosal biopsies in tissue culture medium with h-labeled progesterone in the absence of any stimulation was performed. the mean age of the included dogs was . + . years, the mean weight was . + . kg. all beagle dogs had a mean clinical score of + . the mean wsava scoring was + . . after hours, supernatant was harvested and radioactive progesterone metabolites formed were detected using high performance liquid chromatography plus liquid scintillation counting. in all dogs the h-progesterone was metabolized into various steroid species, nevertheless a local production of cortisol could not be proven. in summary, it could be shown that precursors of gcs can be metabolized by healthy canine intestinal mucosal tissue. disclosures: no disclosures to report. we studied the relationship between pancreatitis and cardiac injury in dogs and cats. previously, we validated a cardiac troponin i (ctni; vet j : - , ) assay for sensitive and specific detection of cardiac injury in domestic animals. we found various non-cardiac diseases of dogs and cats were associated with cardiac injury detected by serum cardiac troponin i, including some cases of pancreatitis. also, we validated the dggr-lipase assay for cost-effective, sensitive and specific detection of pancreatitis in dogs and cats (vet clin path : e - , ; :e - , ). herein, we tested the hypothesis that pancreatitis was associated with cardiac injury. ctni was measured by advia centaur tni-ultra assay; dggr-lipase by the randox colourimetric assay. we retrospectively analysed data from dogs and cats admitted to ucd veterinary hospital in which both ctn and lipase had been measured. upper limit of reference range for lipase in dogs is u/l; we consider - indicative of mild pancreatitis, - moderate, and > as marked. upper limit of reference range for lipase in cats is . reference range for ctni is < . ug/l for dogs and cats. we consider . - . indicative of mild cardiac injury, . - as moderate, and > . as marked. dogs and cats had both lipase and ctni measured. dogs had normal troponin; had normal lipase and had normal lipase and normal ctni. dogs ( %) had pancreatitis as indicated by increased lipase. in ( %), pancreatitis was mild, in ( %) it was moderate, and in ( %) it was marked. of dogs had increased ctni: mild in ( %), moderate in ( %), and marked in ( %). cardiac injury in dogs with pancreatitis was absent in %, mild in %, moderate in %, and marked in %. of cats had normal ctn; had normal lipase. of cats had pancreatitis, severely in . lipase and ctni was correlated (r = . ) for dogs and cats. we conclude that both pancreatitis and cardiac injury, as indicated by high-sensitivity and high-specificity assays randox-dggr-lipase and centaur-ctni, respectively, are not uncommon in veterinary hospital cases. we confirm and extend our previous work. pancreatitis in dogs and cats is typically associated with cardiac injury. severities of pancreatitis and cardiac injury are correlated. for~ % of dogs and cats with pancreatitis, cardiac injury is moderate to marked. disclosures: no disclosures to report. intracellular colonization may serve as a protected niche where helicobacter spporganisms evade effective treatment, contributing to recolonization. confocal endomicroscopy (cem) is an endoscopic modality allowing in vivo gastrointestinal imaging at high resolution; and has aided real-time identification of helicobacter pylori and intracellular and mucosally associated bacterial. in dogs, non-helicobacter pylori-helicobacter (nhph) are described intracellularly. the objective of this study was to determine the utility of cem to identify nhph in dogs compared with other diagnostic modalities; and to assess its ability to identify intracellular organisms. fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by cem using topical acriflavine. images were obtained using cem at a minimum of sites within the stomach. endoscopic pinch biopsies were obtained for histopathology, polymerase chain reaction (pcr) and fluorescence in situ hybridisation (fish). methodologies were compared for their sensitivity in detecting the presence and distribution of nhph and their ability to identify intracellular organisms. cem provided high quality images allowing in vivo identification ofnhph in dogs, as did fish post-procedure analysis. standard histopathology identified nhph in only . nhph were identified within the superficial gastric mucus, and gastric pits. distribution throughout the stomach was diffuse and multi-focal. cem findings correlated with fish and pcr, however only fish enabled identification of intracellular nhph which were present in of dogs. cem provides in vivo histology images and is capable of identifying nhph during gastroscopy, but is unable to identify intracellular organisms using the current fluorophore protocol. nhph in the canine stomach are commonly identified intracellularly. disclosures: dr sharman has shares in optiscan imaging pty ltd. chronic enteropathies (ce) and exocrine pancreatic insufficiency (epi) can both cause hypocobalaminemia in cats. current supplementation protocols for cobalamin in cats call for repeated parenteral injections. in humans, several studies have reported equal efficacy of oral administration of cobalamin. there is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. the purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies. a computerized database search for cats treated at evidensia specialist animal hospital, helsingborg, sweden during - was performed. inclusion criteria were cats with symptoms of ce, an initial serum cobalamin concentration below pmol/l (reference interval: - pmol/l) and daily oral treatment with cyanocobalamin ( mg/tablet; ⅛-¼ tablet/cat daily). follow-up serum cobalamin concentration was measured to days after initiation of daily oral cobalamin supplementation. thirteen cats aged - years (median ) of different breeds met the inclusion criteria. presenting complaints included vomiting ( / ), anorexia ( / ), diarrhea ( / ), weight loss ( / ), and lethargy ( / ). increased pancreas specific lipase (spec fpl Ò ) serum concentrations were reported in / cats and / had increased serum alanine transaminase activity. feline serum trypsin like immunoreactivity (ftli) was determined in / cats revealing results within the reference interval. all cats had an abdominal ultrasound, / had changes related to the gastrointestinal tract such as mild-moderate thickening of the small intestinal wall, thickening of the muscularis layer, poor definition of intestinal wall layers, and/or enlargement of the mesenterial lymph nodes, histopathology was performed in / cats, revealing small intestinal inflammation in cats and small intestinal lymphoma in one. serum cobalamin increased in all cats with treatment. the concentration difference ranged from to pmol/l (mean: pmol/l). mean (ae standard deviation) serum cobalamin concentrations were (ae ) pmol/l before and (ae ) pmol/l after supplementation. this difference was statistically significant (p < . , paired t-test). our results suggest that oral cobalamin supplementation is effective in normalizing serum cobalamin concentrations in cats with various enteropathies. prospective studies are warranted comparing cellular cobalamin status in cats being treated with parenteral or oral cobalamin supplementation. disclosures: no disclosures to report. pulmonary thromboembolism (pte) is observed in dogs with idiopathic-inflammatory-bowel disease (ibd) and particularly with protein-losing enteropathy (ple). hypercoagulability has been attributed to antithrombin (at) loss although the pathogenesis is likely to be more complex. in humans, where venous thromboembolism (te) is a wellrecognised complication of crohn's disease and ulcerative colitis, the pathogenesis of te is still not completely understood. derangements in procoagulant and anticoagulant factors have been demonstrated, including increased circulating procoagulant microparticles (mps). the aim of this pilot study was to evaluate mp-procoagulant activity in the plasma of dogs with ibd and ple using a functional elisa assay (zymuphen-mp-activity, aniara). we hypothesised that all dogs with ple and a subset of dogs with ibd but without ple would have increased levels of circulating mps. the study group consisted of dogs with ibd, including with ple. diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. ple was defined as ibd plus hypoproteinaemia (serum total protein < g/l) and hypoalbuminaemia (serum albumin < g/l). pte was diagnosed in one dog with ple, and suspected in a second. a control group comprised healthy dogs undergoing blood sampling for reasons unrelated to the study including blood donor screening (n = ) and health assessment (n = ). dogs were considered healthy based on owner evaluation, physical examination, haematology and serum biochemistry median mp procoagulant activity in dogs with ibd was . nm (range . - . ) compared with . nm (range . - . ) in the control group. median mp activity in ple dogs was . nm (range . - . ) compared with . nm (range . - . ) in non-ple ibd dogs. using kruskal-wallis test for nonparametric data and dunn's multiple comparisons test the groups were not statistically different. interestingly, mp-procoagulant activity value in the dog with documented pte was . nm; in the dog with high clinical suspicion for pte, mp-procoagulant activity was . nm. the highest mp-procoagulant activity was detected in a healthy control dog, raising concerns for pre-analytical or sampling error. removing this measurement had no impact on statistical analysis, which remained nonsignificant. mp-procoagulant activity > nm is considered clinically relevant in humans. employing a similar cut-off, / of controls, / of ibd and / of ple group would be defined as having increased levels of circulating mps. further studies are required to fully evaluate the clinical relevance and diagnostic potential of mp evaluation. disclosures: no disclosures to report. the intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (ce) in dogs. while imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with ce involving the ileum and colon. the aim of the present study was to use fluorescence in situ hybridization (fish) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with ce and controls. tissue sections from the ileum and colon from dogs with inflammatory bowel disease (ibd), dogs with granulomatous colitis (gc), dogs with intestinal neoplasia, and controls were studied by fish targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. the numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (cib-dai score, histopathology) indices. the ileal and colonic mucosa of healthy dogs and dogs with ce was predominantly colonized by bacteria localized to free and adherent mucus compartments. dogs with ce harbored more (p < . ) mucosal bacteria belonging to the clostridium-coccoides/eubacterium rectale group, bacteroides, enterobacteriaceae, and escherichia coli versus controls. within the ce group, ibd dogs had increased (p < . ) enterobacteriaceae and e. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. bacterial invasion with e. coli was present in the ileal and colonic mucosa of dogs with gc (p < . ). dogs with intestinal neoplasia had increased (p < . ) adherent (total bacteria, enterobacteriaceae, e. coli) and invasive (enterobacteriaceae, e. coli, and bacteroides) bacteria in biopsy specimens versus all other groups. increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (cibdai score) in ibd dogs (p < . ). these results indicate that histopathologic lesions of canine ce are associated with different populations in ileal and colonic mucosal microbiota. these spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine ce. disclosures: no disclosures to report. abdominal girth is used as an indicator of human adiposity, with such measurements being made by tape measure. given concerns in precision and accuracy of repeat measurements, some tape measure designs have inbuilt mechanisms to improve consistency. although body condition scoring is the most common method of assessing adiposity in dogs, zoometric systems have also been developed requiring the use of a tape measure. however, the precision and accuracy of such zoometric measurements are not known. the aim of this study was to determine the precision and accuracy of different types of tape measure for a variety of dimensional measurements. a variety of length (head, forelimb, hindlimb) and circumferential (neck, thorax, and abdomen) were made using different tape measures, of which were designed to improve precision (standard tape; myotape tm and gulick ii tm ). to assess intra-operator variability, measurements were taken for consecutive days from healthy dogs; to assess inter-operator variability, operators independently took measurements from a group of dogs of various breeds and sizes. for intra-operator comparisons, precision was good overall (coefficient of variation [cv] ≤ % for all measurements). for inter-operator comparisons, precision was more variable and, although reasonable on average (mean cv - %), it varied depending upon tape measure type (p = . ; greatest for standard tape measure, least for gulich ii tm ), and could be highly variable for some measurements in individuals dogs (maximum cv % for head measurements with standard tape measure). significant differences also existed in the absolute results of circumferential measurements taken by the different tape measure types (neck p = . ; thorax p < . ; abdomen p < . ). finally, significant operator differences were also evident for some measurements (head p = . ; hindlimb p = . ), but not for others (forelimb p = . ; neck p = . ; thorax p = . ; abdomen p = . ). in summary, although precision for individual operators making zoometric measurements is good, significant inter-operator and tape type differences exist. these results have implications for systems using a range of zoometric measures to assess adiposity. in order to ensure precision and accuracy, it is recommended that the same operator take all measurements with the same type of tape. disclosures: the study conducted was not supported by a research grant. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. however, this method is subjective due to its sensory evaluation. therefore, the improvement of the precision of the bcs diagnosis is expected. our previous study has shown that the bcs model that we created improved the precision of the bcs diagnosis ( ). however, a palpation site was not identified. a palpation site must be the site where thickness of subcutaneous fat is able to capture for measuring animal's obesity status. therefore the objective of this study was to find a remarkable body site of the changes with obesity status using ultrasonic diagnostic equipment. nine dogs which varied in the percent of body fat were used in this study. the percent of body fat was measured by a body fat analyzer for dog (kao). the image analysis of a palpation site was evaluated using echo, xario ssa- a (toshiba) which attached to a linear probe. the measurement points were , and o'clock positions on the ribs of t , t and t . the distance (d) from skin surface to the rib was measured in the echogram. the distance (l) from scapula to ilium was measured to offset the difference in physique by dog breeds. the d/l was used to compare relative value of the quantity of fat at each measurement point. bcs of dogs which used in this study were from bcs of to bcs of . there were no dogs in bcs of and bcs of . a statistically significant correlation was found between bcs and d/l value. the d/l value increased in order of t , t and t in bcs of and . this suggests that the thickness of subcutaneous fat in the chest is thicker at the head side than the tail side. also, as for the d/ l value from back to abdomen, the highest value was found at the position of : and : . this tendency was the most remarkable in bcs of but no difference in the d/l value was recognized in the dogs in bcs of . in conclusion, the position of : or : on the t is the suitable palpation point at the chest. ( body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. bcs has been recognized as one of the screen method of nutrition diagnosis by american animal hospital association in . however, this method is subjective due to its sensory evaluation. therefore we made a bcs model to increase the precision of the bcs diagnosis and have shown the efficacy of the bcs model ( ). however, the prototype model which we have reported before tended to have higher bcs than a target bcs. therefore, we improved the bcs model in this study. sixty seven dogs which varied in the bcs were used in this study. body fat percentage was measured by using a body fat analyzer for dogs (kao healthlab bif- ). the bcs model was improved by using several rubber sheets. relative hardness of stacking rubber sheets in each bcs was measured by durometer mj-dua-c (satotec tokyo, japan). bcs diagnosis of dogs was performed by pet owner by using the bcs model. bcs of represents the most hard in the bcs model and the hardness decreased linearly and it was the lowest in of bcs. these values were as expected. high correlation was recognized between bcs and body fat percentage. these results suggested the efficacy of bcs model. however, the body fat percentage in the dogs diagnosed as bcs of was higher than body fat percentage which has been reported in the previous paper. there were no dogs with the body fat percentage < % which were diagnosed as bcs of . we need more study in future to make clear the difference of body fat percentage between our data and date of the previous research. the completion of this bcs model will help provide the precision of nutritional diagnosis in dogs. ( in humans the metabolic syndrome (ms) is a well-recognised and extensively studied entity that comprises obesity, hypertension, dyslipidaemia, and glucose intolerance. it is associated with an increased risk of cardiovascular diseases and diabetes. recently, human ms criteria were adapted for dogs to define the condition of obesity-related metabolic dysfunction (ormd). it was observed that ormd was associated with increased circulating insulin and decreased adiponectin concentrations, suggesting that in dogs, as in humans, there are links between obesity, ormd, and associated diseases, although pathogenetic mechanisms and health significance for dogs remain unknown. the main aim of the present study was to compare plasma proteomes of obese dogs with and without ormd, so as to investigate the mechanisms associated with canine ormd and their possible significance in the health status. eight obese dogs referred for weight management at the royal canin weight management clinic, university of liverpool participated in the study. clinical assessments included physical examination, body condition scoring, blood pressure measurement and routine clinicopathological analysis. surplus plasma was used in proteomic analysis. samples were first treated with proteominer for thedepletion of high-abundance proteins and subsequently analysed by using -de dige methodology. of the dogs in the study, dogs had ormd and dogs did not. image analysis and further statistical analysis allowed identification of spots with differential expression concentration between dogs with and without ormd. among the spots, were over-expressed and were down-expressed in dogs with ormd than in dogs that did not presented ormd. although the results of the present study are preliminary and still the identification of the spots is up to be performed, the observed datareveal that dogs with ormd present alterations in their plasma proteomes that could be responsible for the development of ormd-related pathologies. disclosures: the study was funded by waltham. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. vb is an employee of royal canin and pjm is an employee of wal-tham. the aim of the study was to assess the diagnostic value and the discrimination potential between the normal heart size and microcardia or cardiomegaly of a method which calculates the cardiothoracic ratio (ctr) using area measurement, compared to the vertebral heart scale method (vhs) used as reference for the cardiac size, in dogs. one hundred-nine dog x-rays were accepted into study. the patients belonged to small and medium size breeds, were males and females with age between and years. the analogic xrays were scanned and transferred to a computer where the vhs and ctr was calculated for each patient with a commercial software and the data was collected and processed in a statystical analysis software. the patients were distributed into groups by respiratory phase and heart size. there was a low correlation between the vhs and ctr (r = . ), but statistically significant (p? . ). a good correlation was obtained between vhs and ctr in microcardia, normal heart size and cardiomegaly groups (p < . ). furthermore, between the ctr in dogs with microcardia and those with normal cardiac size, as well as between ctr in dogs with normal cardiac size and those with cardiomegaly, a significantly statistic difference (p < . ), respectively (p < . ), was obtained. among the groups distributed by respiratory phase and vhs, a statistically significant difference was obtained only between normal cardiac size and cardiomegaly during inspiratory phase groups (p > . ). for the x-rays taken in inspiratory phase, a cutoff of . had a sensitivity of % and a specificity of % for diagnosing cardiomegaly. the ctr can be considered a valid method being able to discriminate between the patients with microcardia and cardiomegaly from those with normal heart size. moreover, it was found that a ctr over the cutoff of . , mesured during inspiratory phase is a good predictor for cardiomegaly. key words: cardiac, cardio-thoracic ratio, dog, x-ray. the canine cardiac conduction system is modified by anatomical and functional adaptations of the maternal heart during gestation. however, it is not clear if these changes persist or are modified after parturition. therefore, the aim of this study was to describe canine electrocardiographic features during the course of normal puerperium. twenty healthy pure-bred, - ( . ae . ) year-old, weighing . - kg ( . ae . ) bitches were included in this study. all the animals whelped healthy puppies at term which were weaned on day after parturition (day ). all the dogs were electrochardiographically evaluated on days À , , , , , , and . mean electrical axis (mea; degrees), p wave amplitude (pa; mv) and duration (pd; ms), p-r interval (pr; ms), qrs complex amplitude (qrsa; mv) and duration (qrsd; ms), q-t interval (qt; ms), and s-t segment (st; mv) were calculated at mm/s of velocity. the rr interval immediately preceding each complex was recorded and qt interval was corrected (qtc) by van de water formula [qtc = qt- . (rr- )]. later, lead ii was recorded at mm/sec to analyze heart rate (hr; bpm) and cardiac rhythm (cr; normal sinus rhythm or sinus arrythmia). values of hr, mea, pa, pd, pr, qrsa, qrsd, qt, rr and qtc were analyzed by anova for repeated measures followed by tukey test. cardiac rhythm was analyzed by chi square test (spss . , spss inc. chicago, il, usa). p < . was considered significant. during the study period, hr (p < . ) and qtc (p < . ) progressively decreased, while rr (p < . ) and pa increased (p < . ). qrs complex amplitude diminished in the second week after parturition and then increased during the following weeks (p < . ). mean electrical axis shifted to the right during this period (p < . ). on day À , most of the bitches presented normal sinus rhythm in contrast with day , in which most of the bitches presented sinus arrhythmia (p < . ). from day onward, all the bitches showed sinus arrhythmia. p wave duration, pr, qrsd, qt and st remained unchanged during puerperium. it is concluded that most electrophysiological adaptive changes of canine gestation reverted during normal puerperium. the pre-sent study contributes to the understanding of canine cardiac physiology during this reproductive stage. disclosures: no disclosures to report. esvc-p- echocardiographic assessment of pregnant queens. p.g. blanco, r. rodr ıguez, a. carranza, a. rube, r. vercellini, p.r. batista, m. t ortora, c. gobello. national university of la plata, la plata, argentina cardiovascular adaptation during gestation guarantees an appropriate development of the fetuses and maternal cardiovascular maladaptation is highly correlated with adverse pregnancy outcome. while, the hemodynamic changes occurring during canine pregnancy have been described there is scarce information concerning maternal cardiac variations during feline gestation. thus, the aim of this study was to describe cardiac morphology and systolic function variations during normal feline pregnancy. eighteen pregnant queens were echocardiographically evaluated (toshiba nemio xg, japan, mhz transducer) every days from day (defined as day of mating) to parturition. left ventricular dimensions were measured in the short axis view, during mmode tracing. shortening fraction was calculated as (lvdd -lvds)/lvdd x to assess systolic function. stroke volume (ml) was calculated as the product of the velocity time integral (measured by pulsed-wave doppler) and the cross-sectional area of the aorta. cardiac output (l/min) was calculated as the product of stroke volume and heart rate (bpm) derived from electrocardiographic monitoring. uterine artery resistance index (ri) was obtained by doppler ultrasound. all the parameters were analyzed by repeated measures anova. all the queens delivered healthy kittens at term. throughout the study period, interventricular septum in diastole (p < . ) and systole (p < . ) and left ventricular diameter in diastole (p < . ) augmented during gestation. shortening fraction (p < . ), cardiac output (p < . ) and maternal heart rate (p < . ) also increased up to parturition. conversely, uterine artery resistance index decreased in the same period (p < . ). it is concluded that cardiac structure and function varied during normal pregnancy in these queens. cardiac eccentric hypertrophy, systolic function and cardiac output increases appear to be the consequences of the hemodynamic modifications occurring during pregnancy. the assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline reproduction. disclosures: no disclosures to report. tricuspid annular plane systolic excursion (tapse) is an echocardiographic measure that allows to assess right ventricular systolic function. it has been described reference values for tapse in normal adult dogs, but there is no reference to influence of age in tapse in dogs. this influence has been reported in humans. thus, the goal of this study is to determine the reproducibility of the measure tapse in normal dogs and to determine the relationship between tapse and age in healthy beagle dogs. tapse was measured from an m-mode recording of the lateral aspect of the tricuspid valve annulus obtained through a left parasternal apical -chamber view. tapse values were averaged from measurements on consecutive beats during sinus rhythm. the measurements were recorded by different persons (c-v, a.; m-m f.) with different grade of experience in canine echocardiography studies.. all patients had a complete -dimensional and doppler study using an envisor chd (philips Ò ) ultrasound system. twenty-three healthy beagles were used. the study was approved by the ethical committee of veterinary medicine service of las palmas de gran canaria university (spain) and it was carried out in accordance with the current european legislation on animal protection. these dogs were divided in different groups according to age: group included dogs under years, group included dogs between and years, group included eight dogs older than years. we analyzed differences between groups using non-parametric (kruskal wallis and wilcoxon scores (rank sums)) test. there were no differences with respect to sex. dogs in group presented higher tapse values than group or ( . ae . cm versus . ae . cm versus . ae . cm; p < . ). statistic intra-observer and inter-observer agreement using the intraclass correlation coefficient was . (p < . ). this study showed that tapse measurement is easily obtainable with a standard echocardiography system, and has adequate interobserver agreement. this study showed higher values of tapse in normal young dogs with respect to older dogs. these results are similar to the results obtained in humans, and could reflect a less effective right ventricle with age. the values presented should be taken with caution due to the relatively small number of patients included. it may also be necessary to validate results in future studies with a second independent sample of dogs of other races. disclosures: no disclosures to report. tetralogy of fallot (tof) is a congenital heart disease characterized by abnormalities, i.e., pulmonic stenosis, ventricular septal defect (vsd), aortic overriding and secondary right ventricular hypertrophy, caused by anterior deviation and abnormal septation of the conal septum during the embryonic period. few studies have reported the hemodynamic consequences and clinical outcome of tof in small animals. the objective of this retrospective study was therefore to document the epidemiological, clinical, echo-doppler findings, and survival, in a canine and feline population with tof. the case records of animals diagnosed with tof by combined use of echocardiography and doppler examination were reviewed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tetralogy of fallot was identified in animals ( dogs, cats). the most commonly represented breeds were terriers for dogs ( / , . %), and domestic shorthair for cats ( / , . %). most included animals ( / , . %) were clinically affected at the time of diagnosis. pulmonic stenosis was characterized by a variable systolic doppler-derived pressure gradient both in dogs (median [range] mmhg ) and cats ( mmhg ), and associated with hypoplasia of the pulmonary trunk in one third of the cases ( . %). most vsd were large, with a median vsd:aorta ratio of . [ . - . ] in dogs and . [ . - . ] in cats. median age at death from cardiac cause was . months [ . - . ] without significant difference between dogs and cats (p = . ). these results suggest that in both cats and dogs tof-related death occurs predominantly in young adult animals with major hemodynamic consequences at the time of diagnosis. disclosures: no disclosures to report. the aim of this study was to assess whether and how radiographic and echocardiographic cardiovascular variables differ across age bands of healthy cats. a cohort of clinically healthy cats were categorized into groups: adolescent-adult ( . - years; n = ), middle-aged ( - years; n = ), and geriatric ( - years; n = ). all cats underwent a full physical examination, a complete blood count, routine biochemical profile, a baseline serum total thyroxine concentration, auscultation, non-invasive blood pressure measurements, thoracic radiography, electrocardiography, and echocardiography. cats with hypertension, hyperthyroidism, cardiac, or renal disease were excluded from the study. body weight, body condition score, systolic blood pressure, heart rate, and all echocardiographic indices were similar across the groups. the mean (ae standard deviation [sd]) vertebral heart scale (vhs) value obtained for the geriatric group ( . ae . ) was significantly greater than that obtained for the adolescent-adult group ( . ae . ; p = . ). the mean ratio of the distance between the cardiac base and dorsal sternum to thoracic cavity height at the point of the cardiac base was significantly less in the middle-aged ( . ae . ) and geriatric ( . ae . ) groups than in the adolescent-adult group ( . ae . ; both p < . ). the mean angle between the cardiac long axis and the body axis was significantly smaller in middle-aged ( . ae . °) and geriatric cats ( . ae . °) than in adolescent-adult cats ( . ae . °; both p < . ). the mean angle between the cardiac long axis and the sternum of middle-aged ( . ae . °) and geriatric cats ( . ae . °) was significantly smaller than that in adolescentadult cats ( . ae . °; p = . and p = . , respectively). additionally, the degree of undulation of the thoracic aorta correlated positively with age (r = . , p = . ). these findings suggest that differences in the horizontal alignment of the heart, thoracic-aorta undulation, and vhs in healthy geriatric cats, relative to observations in younger cats, can be considered to be agerelated. disclosures: no disclosures to report. the aim of this study was to investigate the presence of pulmonary hypertension (ph) in young cats affected by single or mixed lungworm infections. twenty-three cats infected with lungworms were examined at the veterinary teaching hospital of teramo, italy, in - . animals underwent to a complete physical examination and to -or -views radiographic analysis of the thorax. a minimum database (i.e. cbc, serum biochemistry, serology for fiv antibody and felv antigen) was obtained for each patient. nine cats were excluded for concomitant diseases, while cats were included in the study. microscopic identification of parasites was confirmed by molecular tests and all cats received an anthelmintic treatment. a single infection by aelurostrongylus abstrusus was diagnosed in cats, while cats had a troglostrongylus brevior infection either alone or in combination with a. abstrusus. transthoracic echocardiography was performed using an ultrasound unit with a mhz phased array transducer. no structural abnormalities of the tricuspid valve and sign of pulmonary stenosis were detected. the -dimensional and m-mode echocardiography showed a cardiac involvement in cats. one cat, infected by a. abstrusus and t. brevior showed a mild systolic tricuspid regurgitant jet with color doppler of . m/sec, while another a. abstrusus-infected cat, had mild tr of . m/sec with a mean paps of mmhg which resolved within weeks after therapy. one cat diagnosed with troglostrongylosis, showed a marked right-sided cardiac enlargement of mm, and a large systolic tricuspid regurgitant jet with a tr peak velocity of . m/s recorded at continuous-wave doppler via a color doppler echocardiography. the minimum pressure difference between the right ventricle and the right atrium was estimated mmhg and the paps was at least mmhg. the echocardiographic and doppler evidence of mild ph persisted at further examination performed until months after diagnosis. ph is rare in cats, despite cases of reversible ph are known in cat aelurostrongylosis. in this study the first case of irreversible ph infection in a cat affected by t. brevior is presented and this finding further supports the high pathogenicity of troglostrongylosis, especially in young patients. in cats with lungworm infection, possible cardiovascular complications must be taken into account and these infections should be always considered in the differential diagnosis in cats with cardiorespiratory signs. disclosures: no disclosures to report. although uncommonly assessed in veterinary cardiology, a right ventricular (rv) function has been shown to be an important prognostic determinant of many congenital and acquired heart diseases in human patients. our group has already demonstrated that -dimensional ( d) color tissue doppler imaging provides a noninvasive evaluation of systolic and diastolic rv function in the awake dog with adequate repeatability and reproducibility. b however, other noninvasive ultrasound imaging variables reflecting rv function need to be further investigated, particularly in correlation with pulmonary arterial pressure (pap) values and left ventricular (lv) function. the aim of this prospective study was therefore to assess several indices of systolic and diastolic rv function using conventional echocardiography and speckle tracking echocardiography (ste) in healthy awake dogs of different breeds with documented systolic pap (spap) and lv function (lv ejection fraction and global lv systolic strain assessed using the simpson's derived method of disks and ste, respectively). imaging rv tested variables included tricuspid annular plane systolic excursion (tapse), right fractional area change (rfac, %), ste longitudinal systolic strain of the rv free wall (rvfw, %) and of the whole rv (i.e., global rv strain, %), ste longitudinal systolic strain rate (sr, s À ) and diastolic early:late sr ratio. additionally, d-guided m-mode ventricular measurements included the end-diastolic rv:lv diameter ratio (rvdd: lvdd) and the end-systolic rvfw:lvfw ratio. correlations between imaging variables were calculated by using spearman's correlation coefficients. means of age and body weight (ae sd; range) of the study population were . years (ae . ; . - . ) and . kg (ae . ; . - . ), respectively. no correlations were found between rv morphological variables (i.e., rvdd:lvdd and rvfw:lvfw ratios) and all indices of systolic and diastolic rv function. global rv strain (meanaesd = . ae . %) and rvfw strain ( . ae . %) were positively correlated (p < . ) with rfac ( . ae . %, r = . and r = . , respectively), and negatively correlated (p < . ) with spap ( . ae . mmhg [ . - . ], r = À . and r = À . , respectively). spap was also negatively correlated with the tapse:body weight ratio and systolic sr (r = À . and À . respectively, p < . ). there was no correlation between indices of lv function and ste indices of rv function, and no correlation either between ste rv indices of systolic function and the diastolic early:late sr ratio. in conclusion, ste provides a rapid and non-invasive evaluation of rv function that may be used for clinical investigations in canine cardiology. doppler-derived +dp/dt and -dp/dt from mitral regurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phase indexes. this study aimed to determine correlation between doppler-deriveddp/dt and other systolic and diastolic echocardiographic indexes, and if they can be used to identify dogs with and without remodeling, with or without congestive heart failure (chf) and for evaluation of chronic mitral valve disease (cmvd) severity. fifty-seven dogs with cmvd (stages b , b , c+d) were included prospectively in an observational cross-sectional clinical study and distributed in groups regarding the presence of remodeling and chf, to evaluate+dp/dt and -dp/dt, and distributed according to tdi-diastolic pattern tocompare -dp/dt. group c+d ( mmhg/s, p -p = - )had +dp/dt significantly lower compared to b ( mmhg/s, p -p = - )and b ( mmhg/s, p -p = - ) (p = . ). groupc+d also had lower -dp/dt, compared to b ( . mmhg/sae . and mmhg/sae . ; p = . ). dogs with chf compared to those without chf, presented lower +dp/dt( mmhg/s, p -p = - ; mmhg/s, p -p = - ; p = . ) and -dp/dt ( . mmhg/sae . ; mmhg/sae . ; p = . ). regardingdiastolic function, -dp/dt was lower for the restrictive pattern group ( . mmhg/sae . )compared to those without diastolic dysfunction, ( mmhgae . ), delayedrelaxation ( mmhgae . ) and pseudonormal patterns ( mmhgae . ) (p < . ).when +dp/dt< mmhg/s, the post-test chance for the dog with cmvd to havechf is twice the chance than not having it. for -dp/dt< mmhg/s theposttest chance of having chf is times higher than not having it. in conclusion, doppler-derived +dp/dt and-dp/dt may contribute respectively, for systolic and diastolic assessment ofdogs with cmvd. disclosures: no disclosures to report. pulmonic stenosis (ps) is one of the most common congenital heart defects seen in veterinarycardiology practice. pulmonary balloon valvuloplasty (pbv) is considered to be the treatment of choice for dogs withsevere stenosis. whether dogs with moderate stenosis benefit from pbv remains unclear, and variables such as degree of hypertrophy, valve morphology, amount of tricuspid insufficiency and presence or absence of clinical signs aregenerally used when recommendations are made to pet owners. in this study we report the effect of valve type on pbv outcome in dogs treated at different academic speciality cardiology practices. baseline echocardiographic images were evaluated at each institution and valve morphology was classified as either type a (n = , . mmhg, range - ) or type b (n = , . mmhg, range - ) and 'no' (n = , mmhg, range - ) or 'yes' (n = , mmhg, range - ) for presence of pulmonary annular hypoplasia when diameter was compared to aortic annulus. twenty four hours following pbv both type a ( mmhg, range - ) and type b ( mmhg, range - ) valves had significant reduction in gradient compared to baseline (p < . ). this reduction remained significant at days (a: mmhg, range - ; b: mmhg, range - ; p < . for both). dogs with annular hypoplasia ( mmhg, range - ) and without annular hypoplasia ( mmhg range - ) had a significant reduction in gradient hours post pbv. it remained significant at days (with annular hypoplasia: mmhg, range - ; without annular hypoplasia: mmhg, range - ; p < . for both). when comparing to baseline, considering valve type, there was no significant difference in percent reduction in gradient for type a versus type b valves at both the -hour (a: %, range - ; b: %, range - ; p = . ) and -day (a: %, range - ; b: %, range - ; p = . ) recheck evaluation time points. additionally, there was no significant difference in gradient reduction when looking only at whether or not there was annular hypoplasia at hours (yes: %, range - ; no: %, range - ; p = . ) and days (yes: %, range - ; no: %, range - ; p = . ). in conclusion, classification of dogs with ps according to valve type and annulus morphology did not help predict the -day response to pbv. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is the most common feline inherited cardiac disease and it is a major cause of morbidity and mortality. the osservatorio italiano hcm felina was formed in by a network of clinicians, geneticists and breeders, to monitor and study hcm in italian cats. since april , adult cats, belonging to various breeds, including maine coon, siberian, norwegian forest cats, ragdoll, sphynx, british sh, birmans and others have been prospectively enrolled. recheck evaluations were performed in cats. each cat underwent a clinical examination, echocardiography, and blood collection for genetic testing (when appropriate) and storage in the italian feline bio-bank. the disease status was defined by echocardiography according to established guidelines (left ventricular diastolic wall thickness < . mm = hcm negative, = . but < mm = hcm equivocal; = mm = hcm positive). the prevalence of hcm in the population was % ( cats); equivocal diagnoses were conferred on % ( cats). these prevalences did not differ between breeds. the prevalence of hcm in the italian feline population was lower compared to those reported by other investigators. evaluation of data from the entire population demonstrated that left ventricular end-diastolic wall thicknesses and aortic diameter showed a weak positive correlation with body weight (p < . , r < . for all variables), suggesting that weight-dependent limits on wall thickness should be considered in cats as is currently practiced in dogs. the lower prevalence of hcm in italian cat breeds compared with those examined elsewhere might be explained by different criteria for determining presence or absence of disease, differences in ages at which the subjects were examined, or a selection bias by breeders in presenting cats they consider 'normal'. disclosures: no disclosures to report. chronic mitral valve disease is by far the most common cardiovascular disease in dogs. the disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately % of cases, it's accompanied by degeneration of the tricuspid valve. it is also described in previous studies that approximately % of affected dogs also have evidence of associated pulmonary arterial hypertension. the prevalence of the disease is higher in small breed dogs (under kg), although large breeds can also be affected and it occurs more frequently in males than in females. the present study aims to characterize the disease in a population of dogs in portugal. we retrospectively reviewed the medical records of dogs presented to hospital veterin ario do porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of years. from this records, cases were identified, from which ( . %) were males and ( . %) were females. most of the dogs were mixed breed ( ) and different breeds of dogs were represented. the poodle was by far the most represented breed (n = ; . %), followed by english cocker spaniel ( . %), yorkshire terrier ( . %), boxer ( . %), epagneul breton ( . %), dalmatian ( . %), pekingese ( . %), labrador retriever ( %) and portuguese podengo ( . %). all other breeds represented . % of the population. regarding weight, . % of the dogs (n = ) weighted < kg, with a mean body weight of . kg (range . - kg). the mean age at diagnosis was . years old. we also observed that . % of the dogs (n = ) had concomitant degeneration of the tricuspid valve and . % (n = ) pulmonary arterial hypertension (ph). we categorized these dogs according to the severity of ph, in mild ph if they had a doppler echocardiography derived systolic pulmonary arterial pressure (spap) of - mm/hg, moderate ph (spap - mm/hg) and severe ph (spap > mm/hg). we found that . % (n = ) of dogs had mild ph; . % (n = ) moderate ph and . % (n = ) severe ph. as described in previous studies, the disease affects mainly males and small breed dogs, with a breed distribution that reflects the canine population in the country, including very including very popular large breed dogs in portugal, as the boxer and labrador. both the presence of concomitant tricuspid valve disease and ph had a higher prevalence in our study than previously described. disclosures: no disclosures to report. in people anemia is frequent in patients with heart failure (hf) and it is associated with poor outcomes. the most likely pathogenic factors include iron deficiency, chronic kidney disease (ckd), and cytokine production, although other factors may contribute. little is known about the prevalence of anemia in dog with cardiovascular disease. the aim of this retrospective study was to define the prevalence of anemia (hct ≤ %) in dogs with mitral valve disease (mvd) and to investigate associated risk factors (age, weight, azotemia, hf, iris/acvim class). medical records of dogs presented at the cardiology service, divet, university of milan (january -march ) were retrospectively evaluated. dogs with mvd with complete physical, thoracic and echocardiographic examinations, and serum biochemical panel, including serum creatinine (scr), were included in the study. dogs with other heart or systemic diseases, except ckd, or neoplasm were excluded. statistical analysis was performed using jmp . (sas institute). a p value < . was considered significant. two hundred and ninety dogs ( males/ females), . ae . years of age, . ae . kg of body weight fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( %), york shire terrier ( %), and cavalier king charles ( %). dogs were % b , % b , % c and % d acvim class. while the % of the dogs were normoazotemic (scr < . mg/dl), . % were staged in iris , % in iris and . % in iris . the prevalence of anemia in dogs with mvd was % ( / ): showed mild ( ≤ hct ≤ %) and moderate ( ≤ hct ≤ %) anemia. sixteen dogs were in b , in b , in c and in d acvim class; were normoazotemic ( %). anemic dogs showed a significant higher scr. normoazotemic dog showed significant higher hb, hct and rbc both in the overall population and in the anemic group. in the overall population dogs in different iris class showed statistically different hb, hct and rbc and hb was significantly lower in decompensated hf dogs. in conclusion although a relationship between anemia and azotemia/ckd was documented in our study, it is important to emphasize that most of the anemic dog were normoazotemic: anemia is not an exclusive finding of cardiorenal syndrome and should be considered as possible complication in dogs with mvd alone. disclosures: no disclosures to report. the objective of this study was to evaluate left atrial (la) function by left atrial total fractional area change (la-factotal) and left atrial ejection fraction (laef) in dogs affected with chronic mitral valve disease (cmvd) naturally acquired with and without congestive heart failure (chf). our hypothesis was that la-factotal and laef decrease with severity of cmvd. eighty dogs were included in a prospective observational cross-section clinical study, grouped according to cmvd severity based on echocardiographic evaluation and clinical signs. the dogs were equally distributed in each group: a, b , b and c, according to american college of veterinary internal medicine staging system. indicators of la function were calculated with the following equations: la-factotal = (lamaximum area -laminimum area)/lamaximum area, measured by apical view; and laef = x (lamaximum volume -laminimum volume)/lamaximum volume, by biplane area-length method from the left apical and chamber views. la-factotal showed lower values (p < . ) in group c ( . %, p - % = . - . ) compared with groups a ( . %, p - % = . - . ), b ( . %, p - % = . - . ) and b ( . %, p - % = . - ). group c had lower laef ( . %, p - % = . - . ) than groups a ( . %, p - % = . - . ), b ( . %, p - % = . - . ) and b ( . %, p - % = . - ) (p < . ). left atrial function, assessed by la-factotal and laef, was reduced in dogs with cmvd and chf compared with healthy and asymptomatic cmvd groups. disclosures: no disclosures to report. recurrent episodes of heart and/or kidney failure are considered one of the causes leading to worsening heart/renal functions in human patients. the aim of this prospective study was to assess the influence of heart/kidney worsening on elected parameters of heart/kidney function in dogs affected by mitral valve disease (mvd). between july and may , dogs affected by mvd in acvim class b and without comorbidities were included in the study group. the control group was constituted by healthy dogs, matched with the cases for age (older than years) and gender. all the dogs underwent physical examination, thorax radiography, ecg, echocardiography, systemic blood pressure assessment, a complete blood count, serum biochemical analysis, including assessment of serum creatinine (scr), serum urea nitrogen (urea) and glycaemia (gly) and urine analysis with urine protein/creatinine ratio (upc). dogs were re-evaluated every -month until october . statistical analysis was performed using ibm spss statistics (p value significant if < . ). twenty-one dogs affected by mvd (cases) were included and healthy dogs (controls) were randomly selected among the eligible population. the % of cases experienced at least one episode of congestive heart failure (chf), but none of these patients developed chronic kidney disease (ckd). the % of cases developed ckd while remaining in acvim class b . no dogs in the control group developed ckd or mvd. correlations between worsening renal function (wrf -scr elevation ≥ . mg/dl or % from baseline), furosemide administration, upc levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. only a statistically significant difference in iris class between the groups according to wrf and in the echocardiographic parameter left atrium to aortic root (la/ao) according to furosemide amount were observed. both these results were expected. none of the cases included experienced renal damage (wrf or iris class change or upc change) concomitant to episodes of chf. the persistence of normal renal condition regardless of chf events and therapy administration was unexpected. in conclusion, experiencing chf seems not to directly affect renal function. to authors' opinion, the use of wrf, better than single scr and urea levels, may be useful in the long term management of aged patients affected by mvd. however, the small number of cases included in this study represents a great limit. we consider this work a pilot study. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is a primary myocardial disease characterized by inappropriate thickening of the myocardium in absence of other causes of hypertrophy including hypertension, hyperthyroidism, aortic stenosis and acromegaly. it is also the most common heart disease in cats. hcm presents a wide variety of clinical sings depending on the severity and location of the hypertrophy. cats affected with hcm have a mean age of . - . years old at the time of the diagnosis however this disease can affect cats as young as months although this later age is unusual hcm is a heterogeneous disease both in terms of phenotypic degree of hypertrophy and clinical outcome. hallmark histopathological hallmarks lesions of hcm are myocyte disarray, small coronary arteriosclerosis and interstitial fibrosis replacement in order to confirm hcm echocardiography has to be made. primary hypertrophy diagnosis is made based on the presence of ventricular hypertrophy, symmetric or asymmetric, in the absence of systemic disorders. the purpose of this study was to assess the prevalence of hcm in a feline population. in order to achieve this goal echocardiograms were made in all cats older of years clinically asymptomatic with or without cardiac murmur. all echocardiograms were made according to the guidelines of the acvim published in . diagnosis of ventricular hypertrophy was made from the right parasternal window using the b mode to measure the diameter of the lvfw and the ivs in diastole. cats with more than mm of wall thickness measured t , bun, crea, blood pressure. only cats within the normal limits of the later parameters were considered hcm positive. total number of cats in this study was cats male and female. from this population had no defined breed, were persian, maine coon, norwegian woods, siamese, chartreaux. no murmour was detected in ( . %) cats, s or s was detected in ( %) cats and differente degree of murmour was detected in ( . %) cats. hypertrophy was detected in cats. from this cats ( . %) were diagnosed as hcm, ( . %) cats were excluded either because of lack of values of t and or because they had high values of blood pressure, t levels or crea. in this study . % of the population had hcm. the epidemiological and phenotype distribution is highly variable. the average age at diagnosis of hcm in this study was . years. disclosures: no disclosures to report. mildly increased concentrations of crp are associated with cardiovascular disease in humans and dogs. it is not known whether increased concentrations of crp are associated with myxomatous mitral valve disease (mmvd) in dogs, or rather its sequel, congestive heart failure (chf). the aim of this study was to investigate whether serum concentrations of crp, determined using a novel automated canine-specific high-sensitivity crp assay (gentian hscrp), were associated with severity of mmvd and certain clinical variables in dogs. the study included client-owned dogs with different severities of mmvd. disease severity was determined by medical history, physical examination, echocardiography and response to diuretic therapy. dogs were allocated into groups based on acvim consensus statement guidelines (group a (n = ), group b (n = ), group b (n = ) and group c (n = )). data were analysed using descriptive statistics and multiple regression analysis. dogs with chf (group c) had significantly higher serum crp concentrations ( . mg/l, [ . ; . ]) (median, [quartile ; quartile ]) compared to dogs in groups a ( . mg/l, [< . ; . ]) (p = . ), b ( . mg/l, [< . ; . ]) (p < . ) and b ( . mg/l, [< . ; . ]) (p < . ). other measures of disease severity including left atrial to aortic root ratio and left ventricular end-diastolic diameter normalized for body weight were positively correlated with serum crp concentration. in conclusion, slightly higher serum crp concentrations were found in dogs with chf whereas the severity of asymptomatic mmvd showed limited association with serum crp concentrations. disclosures: no disclosures to report. medetomidine is a a -agonist widely employed for sedation in dogs but its use is discouraged in cardiac patients even those suffering from myxomatous mitral valve disease (mmvd). however, only one investigation was previously conducted in a wide rangeregarding the class of the disease -of mmvd patients, reporting a general safety of that protocol. the present study was focused just on class b of mmvd, with the aim to provide more detailed information on the cardiovascular effects of medetomidine in such patients, by the analysis of clinical and instrumental parameters suggestive of disease severity or congestive heart failure. dogs weighing < kg, needing a soft clinical procedure and showing a systolic apical heart murmur were screened and selected for the study if la/ao < . . the sedative protocol consisted in an iv injection of lg/kg medetomidine antagonized, after the clinical procedure, by an im injection of the recommended dose of atipamezole. clinical parameters, echocardiographic variables, thoracic radiographs and oscillometric blood pressure measurements were collected at baseline (t ), minutes after medetomidine administration (t ) and hours after atipamezole injection (t ). of dogs screened, were definitively enrolled. at t a significative decrease in the right parasternal regurgitant jet area (rp-arj/laa), peak velocity of mitral regurgitation and shortening fraction was observed along with an increase in lvids (p < . ). left parasternal arj/laa decreased without reaching statistical significance but showing a high correlation with rp-arj/laa (r = . ). interestingly, la/ao changed only mildly and never reached a value > . . the other echocardiographic variables did not show a particular trend. systolic blood pressure showed values at the upper physiologic limit at t , lower values than t at t , and an increase above the initial value at t but without significance. thoracic radiographs were evocative of heart enlargement without pulmonary venous congestion or pulmonary oedema both at t and t . respiratory rate did not change between t and t . the degree of sedation was optimal during the clinical procedure in all cases. sedation with lg/kg medetomidine is safe in dogs suffering from mmvd in stage b (la/ao < . ). the decrease observed in peak velocity and color-doppler appearance of mitral regurgitation at t could be due to a reduction of both myocardial contractility and systolic blood pressure, by a lowering of sympathetic activity via baroreceptors stimulation. disclosures: no disclosures to report. in this retrospective study were included a total of clientowned dogs, undergoing transarterial occlusion of pda with mreye Ò flipper detachable embolization coil (n = ), amplatz â canine duct occluder (acdo; n = ) and amplatzer â vascular plug (n = ). device size selection was based on pda dimensions assessed by transesophageal echocardiography (tee) in cases and transthoracic echocardiography (tte) in cases. angiography was performed during the procedure to assess the success of the occlusion, and it confirmed complete occlusion in dogs and a trivial residual flow in dogs. the following day, transthoracic color-doppler echocardiography revealed that complete ductal closure was achieved in all dogs. the procedure was hemodynamically successful, as evidenced, by a reduction in indexed left ventricular internal diameter in diastole (lvidd; p < . ), fractional shortening (fs; p < . ) and left atrial to aortic ratio (la: ao; p < . ) within hours after procedure. four months after surgery, indexed lvidd was significantly reduced (p = . ) and la:ao remained constant. secondary complications included pulmonary arterial embolization of an acdo and a late rotation of an amplatzer â vascular plug resulting in an increased flow through the pda. the dog with the rotated device required subsequent surgical ligation of the pda. at this time, dogs were reported to be alive and the other dogs were lost to follow up. only one dog remained on congestive heart failure therapy after the pda occlusion. we can conclude that pda occlusion using an acdo for dogs with more than kg and a transarterial coil embolization for dogs with < kg had a high rate of immediate complete occlusion. pda occlusion using those devices proved to be a safe and effective therapeutic method for pda in dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right pulmonary artery distensibility index (rpad index) was recently described as a valuable method for early detection and severity evaluation of pulmonary arterial hypertension in dogs. rpad index is calculated as the percentage change in diameter of the right pulmonary artery (rpa) between systole and diastole, obtained by m-mode echocardiography from the right parasternal long axis view. the aim of this study was to compare the rpad index obtained by different echocardiographic views in dogs. the study design was a prospective, multicenter, observational study. forty-five clientowned dogs from different breeds were included: dogs with heart disease and healthy dogs. two different right parasternal views, long axis (rpla) and short axis (rpsa), were used to measure the rpad index. from the rpla view (method ) and rpsa view (method ) a short axis and a long axis image were respectively optimized for the right pulmonary artery. the rpad index was calculated by m-mode as the percentage change in diameter of the right pulmonary artery: [(systolic diameter -diastolic diameter)/ systolic diameter]* . measurements were done off-line as an average of consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. a pearson and a bland-altman test were used to assess correlation and agreement between the methods, respectively. intra-and inter-observer measurement variability was quantified by average coefficient of variation (cv). level of significance was set at p < . . m-mode evaluation of the rpad index was satisfactorily obtained by both methods in all dogs. pearson test showed a strong positive linear correlation between the values of rpad index obtained from both methods (r = . , p < . ). bland-altman test showed a good agreement between the methods in estimating rpad index (bias = . %, sd = . %, % limits of agreement = À . , . %). the mean difference between the methods was . % ( % confidence interval = À . ; . ). intra-and inter-observer measurement variability was clinically acceptable (cv< %).the study showed a good agreement between short axis and long axis m-mode evaluation of rpa. both methods can be used interchangeably to evaluate rpad index. further studies are needed to evaluate the rpad index in a larger population of healthy dogs and the diagnostic and prognostic role of this echocardiographic parameter in dogs with different types of pulmonary hypertension. disclosures: no disclosures to report. this retrospective study (march to october ) includes client-owned great pyrenees diagnosed with hypoadrenocorticism. the medical records of dogs with a diagnosis of naturally occurring hypoadrenocorticism were reviewed, with an emphasis on great pyrenees' record. the prevalence of hypoadrenocorticism in the studied population, as well as the prevalence per breed, was calculated. data collected included breed, clinical signs, laboratory findings, age at diagnosis, treatment, and cause of the death. one hundred dogs were diagnosed with naturally occurring hypoadrenocorticism, representing . % of the overall canine population studied. thirty-five breeds were represented, with a prevalence per breed varying between . % and . %. a high prevalence was observed in west highland white terriers ( . %), great danes ( . %), standard poodles ( . %), saint-bernards ( . %) and jack russell terriers ( . %). out of gp presented during that period of time, . % (n = ) were diagnosed with hypoadrenocorticism. median age at diagnosis was . years (range: . to . ) in dogs with hypoadrenocorticism, and . years ( . to . ) in gp. the main reason for presentation of the addisonian gp was lethargy (n = ) and anorexia (n = ). clinical findings included hypotension (n = ), poor body condition (n = ), and heart murmur (n = ). the majority (n = ) had serum electrolytes abnormalities, with a na:k ratio ranging from . to . . other major laboratory findings included azotemia (n = ), anemia (n = ) and the absence of a stress leukogram (n = ). the majority (n = ) received fludrocortisone, with prednisone as needed. one gp was euthanized at time of diagnosis. great pyrenees diagnosed with hypoadrenocortism were overrepresented in the studied population, with a prevalence of hypoadrenocorticism in our gp population of . %. therefore, an inherited susceptibility can be suspected. reason for presentation and clinical signs were nonspecific, and similar to what is reported in other breed. in human medicine, haemoglobin a c (hba c), a form of glycosylated haemoglobin, is used as the standard measure of average glycaemic control over to months. the measurement of hba c in dogs has been previously demonstrated however high pressure liquid chromatography techniques are too technically difficult for routine use and other methods are no longer available. the objective of this study was to assess the use of latex immunoagglutination inhibition using a monoclonal antibody for the measurement of hba c in dogs, using the siemens dca vantage Ò . repeatability was assessed by measuring samples times within minutes. the effect of storage on edta anticoagulated samples was examined by measuring samples stored at °c every day for up to days. storage was further assessed by freezing samples and measuring them at , and weeks. the machine was then used to compare the hba c values in groups of dogs with diabetes mellitus (group , n = ), hyperadrenocorticism (group , n = ) or non-diabetic/cushingoid hospitalised patients (group , n = ). differences in the groups were examined for significance using a kruskal-wallis analysis of variance. the reference range of hba c has been previously calculated to be . - . % ( - mmol/mol) and values of . -> % ( -> mmol/mol) are seen in diabetic animals using high pressure liquid chromatography. the median coefficient of variation for the repeatability study was found to be % (range % to %). it was possible to store samples at °c for up to days (median cv% = %, range % to %) and at À °c for at least weeks (median cv% = %, range % to %). the median hba c concentrations were group ; . % ( mmol/mol), group ; . % ( . mmol/mol) and group ; . % ( mmol/mol). group was significantly different from the other groups using kruskal-wallis analysis of variance. in conclusion, the latex immunoagglutination method was repeatable for the measurement of hba c in dogs. in addition, hba c in canine edta anticoagulated samples were stable at °c for up to days and, if frozen, could be stored for at least weeks without significant sample deterioration. the assay provides the expected results in dogs with and without abnormalities of glycaemic control. disclosures: the siemens dca vantage was provided on loan from siemens, as well as the cartridges used on this machine to run all samples in the study. a. wehner , r. anderson , s. reese , k. hartmann . clinic of small animal medicine, munich, germany, institute of veterinary anatomy, histology and embryology, munich, germany measurement of total thyroxine (t ) is often the first diagnostic step in the work up of thyroid disease in dogs and cats. blood samples are routinely sent to a reference laboratory causing a delay in testing which might impact the results. the aim of this study was to validate an enzyme fluorescence assay (elfa) as an inhouse method to measure t in dogs and cats. t was measured in sera of dogs and cats by methods, an enzyme immunoassay (eia) and an enzyme fluorescence assay (elfa). the eia served as the standard method to which the elfa results were compared. the elfa was performed with the minividas automated analyser (biom erieux, craponne, france) according to the manufactors instructions. coefficient of variation (cv) of the elfa in dogs sera was . % and of the eia - . %, respectively. cv of the elfa in cats sera was . - . % and of the eia . - . %, respectively. overall bias of the elfa in dogs was . %; however up to À . % in lower t ranges. maximal bias of the elfa in cats was . %. correlation of both methods was linear only in cats. using bland altman plots limits of agreement were - - % in dogs and - - % in cats. cohen s kappa revealed only slight agreement between both methods in dogs, but a good to very good agreement in cats. the elfa is a fast method with a high precision and can be recommended to measure t in cats, but cannot be recommended for dogs. disclosures: no disclosures to report. dysfunctions of the central nervous system (cns) are the most frequent causes of neurological disorders in dogs. our study aimed to find ( ) if some cns diseases could be associated with a selected group of common microbial or viral pathogens in dogs and ( ) if cns diseases have any characteristic profile with regard to parameters, c-reactive protein (crp) and iga, that are reported to be potentially useful but unspecific markers of cns diseases. we analysed cerebrospinal fluid samples obtained from dogs with varying neurological signs between june and november . real-time pcr was employed with probes for toxoplasma gondii, neospora caninum, anaplasma phagocytophilum and canine distemper virus. igg-antibodies to tickborne encephalitis virus (tbe) were assayed and iga titres were measured using elisa, while crp concentrations were determined by immunoturbidimetric assay. the dogs had a median age of years (range: . - ) and comprised breeds most frequently involving chihuahuas, labrador retrievers, bernese mountain dogs and boxers. gender distribution was . % female, . % spayed bitches, . % male, . % neutered male, and . % non-identified. none of the cases were pcr-positive for toxoplasma gondii or canine distemper virus. one dog was positive for anaplasma phagocytophilum and another for neospora caninum. antibodies to tbe virus were within the borderline range in / dogs. the dogs could be divided into age groups: (= . %) for young dogs (< years, median year) and (= . %) for older dogs (≥ years, median years). igahigh (> . mg/dl) cases represented % and % for young and older dogs, respectively. crp-high (> . mg/l) cases were almost half and equal: . % and . % in young and older dogs, respectively. compared with older dogs, young dogs had higher levels of crp (p = . ) and iga (p = . ). within the iga-high cohorts, crp-high and crp-low cases distributed almost equally ( . % versus . %) in young dogs but disproportionate ( . % versus . %) in older dogs. there were no [iga-low/crp-low] cases in young dogs but . % in older dogs. our present data suggest that ( ) canine cns disorders were largely characterized by high iga and particularly in young dogs ( ) inflammatory types (crp-high) were almost equal in both groups and ( ) although the significance remains yet to be determined, pathogens like anaplasma phagocytophilum and neospora caninum could be detected in a few cases of canine cns disorders. disclosures: the authors breu d and guthardt j are employed at laboklin gmbh & co kg, germany. m€ uller, e is owner/manager of the laboklin gmbh & co kg. internet is a potential source of medical information for pet owners. therefore, it could play an indirect but important role in the veterinary practice. this survey assesses the online search behaviour of french pet owners for their pets' health and its influence on a veterinary consultation. in april , french pet owners coming in a veterinary teaching hospital for a medical or a surgical consultation were surveyed. ( . %) owners fulfilled the questionnaire on a voluntary basis. the survey contained questions dealing with topics: the online search behaviour of owners for their pets' health, their perception of the information found online and the internet's influence on a consultation and on the veterinarian/client relationship. . % of owners use the internet to obtain information on their pets' health. among them, . % use it rarely and . % occasionally. they mainly look for information on a disease ( . %), a symptom ( . %), a breed ( %) or a nutritional advice ( . %). . % of owners try to verify the accuracy of the information found, most often by questioning their veterinarian ( . %). few owners ( . %) think that online information is always trustworthy. most of the research ( . %) is randomly made, websites being found through search engines. the majority of pet owners ( . %) aren't aware of any health certification label for websites. internet enables certain pet owners to feel more at ease with their pets' health care: they ask more questions to their veterinarian ( . %) and feel more involved in medical choices ( . %). . % of owners consider that the internet can positively impact their relationship with the veterinarian. relief is the most common ( . %) emotional response to online research for medical information. however, . % of owners feel overwhelmed by the amount of information found, % are confused and . % frightened by the serious or graphic nature of the information found online. this study emphasizes the frequent but measured use of the internet by french pet owners for their pets' health. they seem to consider information found on the net with a critical mind. unexpectedly, it appears that the internet could be an ally for veterinarians by promoting exchanges between the clients and the veterinarian and by improving compliance with the care project. disclosures: no disclosures to report. sinonasal aspergillosis is a well-known and described fungal infection of the sinonasal cavities in dogs. topical treatment either with enilconazole or itraconazole infusion administered surgically or endoscopically are effective. the use of % clotrimazole cream have been described in a surgical setting after itraconazole infusion by sissener et al. in . the aim of the work was to report the effectiveness of the use of topical % clotrimazole cream as the only treatment for sinonasal aspergillosis in dogs. inclusion criteria were a full medical record with radiological and endoscopical imaging, record of clotrimazole discharge after instillation and endoscopic control between and days after procedure. the % clotrimazole cream was applied through catheters placed under direct endoscopic vision after fungal plaques removal. nine dogs were included. three dogs were mixed breed dogs, dogs golden retriever, one dog a german shepherd and one old english sheepdog, one bull terrier and one cane corso. six dogs were male (one neutered) and female (one intact). mean age was . years. main clinical signs were muco-purulent discharge ( ), pain at sinonasal palpation ( ), nasal planum alterations ( ), epistaxis ( ) . nasal discharge was bilateral in dogs. mean duration of clinical signs was . months. main radiological findings were turbinates lysis ( ), frontal sinus empyema ( ), frontal bone thickening ( ) and frontal bone lysis ( ). rhinoscopy disclosed lysis and remodelling of the nasal turbinates ( ), easy access to the frontal sinus ( ), septum lysis ( ), bilateral sinonasal aspergillosis ( ), monolateral nasal aspergillosis ( ), monolateral sinonasal aspergillosis and contralateral nasal aspergillosis ( ), monolateral frontal aspergillosis ( ) . main duration of nasal cream discharge was . days. all dogs underwent endoscopic control between and days after the procedure. seven dogs were disease free; dogs had persistent fungal plaques and underwent a second treatment. success rate was . %. success rate of this study is comparable to other studies with larger and smaller case series. endoscopic removal of the fungal plaques can be time consuming and topical administration of either enilconazole or itraconazole require an additional hour. the catheter placement and the % clotrimazole cream application lasted minutes for each cavity in the dogs here reported. the use of % clotrimazole cream as the only treatment for sinonasal aspergillosis needs further evaluation on a larger case series. disclosures: no disclosures to report. few studies exist on euthanasia in small animal practice. however, such an act belongs to veterinary procedures, more or less frequently depending of the kind of practice, and may deeply impact both owners and veterinarians. we intended to study practical, ethical and psychological aspects of euthanasia of dogs and cats among french veterinary practitioners. from october to february , an on-line -item questionnaire on small animals' euthanasia, addressing practical aspects of euthanasia, communication with the owners, ethical problems, owners' and veterinarians' perceptions, was emailed via professional associations and networks. results were analyzed using commercial software (sphinx iq Ò and excel Ò ). french veterinarians practicing small animal medicine completed the questionnaire, representing > % of this population. euthanasia occurs rarely at home. over % of veterinarians propose the owners some time alone with their pet, and to stay during euthanasia, performed most commonly by intravenous injection ( . %) mainly after sedation/anesthesia ( . %). ninety nine percent of veterinarians consider communication, including description of events' sequence, and disposal of the body, as important. estimated minimum communication time required varies from - to - minutes. following euthanasia, . % are often thanked by the owners. most veterinarians (> %) have refused a euthanasia, considered unjustified, or had their own suggestion of euthanasia rejected. reasons for such a suggestion include intractable pain ( . %), non-acceptable complications ( . %), financial considerations ( . %) or animal considered dangerous ( . %). veterinarians think most owners ( . %) experience some sense of guilt during euthanasia. themselves perceive euthanasia most commonly as relief of animal's suffering ( . %) and part of veterinary practice ( %), less frequently as a defeat ( . %). almost all veterinarians have experienced emotionally challenging euthanasia, and . % estimate that practicing euthanasia influences their perception of death. practical ( %) and psychological ( . %) aspects of euthanasia have been discussed in most teams. veterinarians' gender influences euthanasia management, mostly concerning some communicational and practical aspects. euthanasia is definitely not an ordinary veterinary act, neither for the owner nor for the veterinarian. therefore, this act must be performed with as much care and communication as possible. disclosures: no disclosures to report. canine pancytopenia is associated with a range of intra-marrow or extra-marrow causes, including though not limited to, infectious agents, drugs, toxins and neoplasms. there is currently limited information regarding the clinicopathological features of the underlying causes or the prognosis in pancytopenic dogs. the objective of this retrospective study was to better define the spectrum of diseases associated with canine pancytopenia, to establish possible clinicopathological discriminators for the common causes and to investigate if the severity of pancytopenia or the underlying disease were associated with the clinical outcome (death or survival). medical records of dogs with a comprehensive diagnostic investigation admitted in a veterinary teaching hospital were retrospectively reviewed. pancytopenia was defined by a hematocrit (hct) < % (< % for dogs < months of age), white blood cell counts (wbc) < , /ll and platelet counts (plt) < , /ll. a control group of dogs without any evidence of blood cytopenia(s) was also established. in total, pancytopenic dogs were studied. bone marrow aspiration cytology was examined in cases and aplasia of all hematopoietic lineages was observed in ( . %) dogs. the most common diagnoses included monocytic ehrlichiosis (cme) (n = ), leishmaniosis (canl) (n = ), parvoviral enteritis (pe) (n = ), and concurrent cme and canl (n = ). mixed breed dogs were more likely to develop pancytopenia as compared to purebreds and pancytopenic dogs tended to be younger than the controls (conditional dependent logistic regression model, p = . and p = . , respectively). among the most common diseases associated with pancytopenia, the mean wbc counts were significantly lower in dogs with cme and pe compared to dogs with canl (one way anova with bonferroni test for multiple comparisons, p = . and p = . , respectively), while plt counts were significantly lower in cme compared to canl (p< . ) or pe (p < . ). total protein concentrations were significantly lower in dogs with pe compared to cme (p < . ) and canl (p < . ). using a univariable logistic regression analysis model, no association was established between the underlying disease and the final outcome. however, higher hct (by at least one percentage unit), wbc (by at least , /ll) and plt (by at least , /ll) values tended to significantly increase the odds of survival (p = . , p < . and p = . , respectively). in the present study, cme, canl and pe were the major causes of canine pancytopenia. potentially useful diagnostic indicators included severe leucopenia (cme, pe), thrombocytopenia (cme) and hypoproteinemia (pe). disclosures: no disclosures to report. laryngeal masses (lm) usually produce air flow limitation during inspiration, expiration or transiently during subsegments of both breathing phases. feline bronchial diseases (fbd) have predominantly expiratory flow restrictions. barometric whole-body plethysmography (bwbp) is a non-invasive pulmonary function test (pft) that allows a dynamic study of breathing patterns widely used to evaluate lower airway responsiveness. the objective of this preliminary study was to evaluate if there were significant differences in respiratory rate [rr ( bwbp detects both upper and lower airways diseases because any site of airway obstruction will result in increased pressure changes associated with breathing. nevertheless our results suggest that there are significant differences in tv(p = . ), ti (p = . ), pef(p = . ), eep(p = . ), penh(p = . ) and pau(p = . ) between lm and fbd cats. no other significant difference in bwbp parameters was found. upper airway obstructions have been previously evaluated in cats by using pft (mckieman, , lin, but in authors' knowledgement this is the first study designed to compare upper and lower airway obstructions by using bwbp. attending our results, there is the evidence that bwbp can help characterize mechanical dysfunction of the airways in cats with lm obstruction. however we must keep in mind some limits of this study as the low number of animals, individual variability in breathing pattern and to have the chance of doing bronchoreactivity tests. disclosures: no disclosures to report. the median lifespan of domestic dogs has been estimated to - years, but little is known about risk factors for mortality in aged dogs: most mortality studies in dogs have been carried out among diseased dogs (renal or heart diseases, cancers, post-operative death). to determine which characteristics are associated with mortality in a priori healthy aged dogs, a prospective cohort study has been conducted in guide dogs, followed from a systematic geriatric examination (ge) to either (all-cause) death or cut-off date (july th , ) . survival analyses (kaplan-meier estimators, log-rank tests, and multivariate cox proportional hazards models) were used to assess the associations with time to death. median age at ge was . years, all dogs were neutered, and % were female. the majority of dogs were golden retriever (n = ) and labrador retriever (n = ). among these dogs, % were obese, % presented skin nodules and % used bus as transportation. a total of dogs died during follow-up, leading to a median survival time from ge of . years. after adjustment for demographic and biochemical variables (age, sex, total proteins, cholesterol and alp), an increased alanine amionotransferase level (≥ ui/l; adjusted hazard ratio [ahr], . ), presenting skin nodules (ahr, . ), and not being a labrador (ahr, . ) were independently associated with a shorter time to death (p < . ). public transportation tended to be associated with mortality (ahr, . ; p = . ), highlighting the importance of environment in mortality. neither sex nor other biochemical parameters were significantly associated with mortality. the alanine amionotransferase level and the presence of skin nodules seem predictors of mortality in senior guide dogs, mostly labrador, golden, or mixed breed of labrador/golden. the impact of environment, in particular urban environment, on mortality needs further investigation. studies in other breeds and pets are also necessary to generalize these results. disclosures: sara hoummady received a grant from mp labo for his phd work about dog aging and marc blondot work at the paris guide dog school. laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. laryngoscopic and bronchoscopic findings in dogs examined by the author between and were evaluated for the presence or absence of laryngeal abnormalities. dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. detection rates for laryngeal abnormalities were compared among disease groups using chi square analysis and fisher's exact test, with significance set at p < . . a total of dogs were evaluated and varied in age between months to . years (median years). weight ranged from . - . kg (median kg), with dogs < kg, dogs from . - . kg, dogs from - kg, dogs from . - kg, and dogs > kg. laryngeal hyperemia or swelling was found in / dogs ( %), and detection rate did not differ among disease processes. laryngeal function was considered suspect in / cases, prompting administration of doxapram, which normalized function in / dogs. laryngeal paresis or paralysis was reported in a total of / dogs ( %). a substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough. disclosures: member of the feline advisory board, speaker honoraria for international, national, and regional continuing education meetings. bronchiectasis is a poorly characterized disease in dogs identified by airway dilatation on radiographs, computed tomography, bronchoscopy, or histopathology. little is known about underlying disease processes associated with bronchiectasis in dogs. medical records from dogs presented to uc davis were searched for identification of bronchiectasis. underlying disease processes and clinical diagnoses were obtained through review of the history, physical examination, respiratory endoscopy and bronchoalveolar lavage fluid analysis and microbiology. historical reports, results of imaging, bronchoscopy and fluid analysis, and scrutiny of pathologic and clinical diagnoses were comprehensively evaluated to identify the most likely underlying disease process associated with bronchiectasis. between and , bronchiectasis was diagnosed in / dogs ( %) that had bronchoscopy performed. dogs ranged in age from . to years (median years) with / dogs < months, / dogs ( %) - years, / dogs ( %) . - years of age and / dogs ( %) over years of age. dog breeds affected more than once included labrador retrievers, cocker spaniels, golden retrievers and standard poodles. duration of cough ranged from days to years (median months). underlying disease processes included pneumonia in / ( %) dogs, inflammatory airway disease in / ( %) dogs, and eosinophilic bronchopneumopathy in / ( %) dogs. twenty-three of dogs ( %) had positive bacterial cultures, with isolation of streptococcus (n = ) and enteric species (n = ) most commonly. this study found that bronchiectasis often occurs in older, large breed dogs with infectious or inflammatory pneumonia. disclosures: johnson: feline advisory board, speaker honoraria. chronic respiratory disease, often characterized by a chronic cough, is common in dogs. the purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing. a retrospective study of client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (etw) or broncho-alveolar lavage (bal), was performed. all dogs were evaluated by means of full clinical examination, hematology and serum biochemistry analyses, survey thoracic radiographs, echocardiography and ecg (if indicated), and bronchoscopy with cytological analysis and aerobic culture of etw or bal fluid. an etw was performed in / ( %) dogs while a bal was performed in / ( %) dogs. a positive aerobic bacterial culture was identified in / ( %) of submitted etw/bal fluid samples. most commonly isolated bacteria included mycoplasma sp. ( %), bordetella bronchiseptica ( %) and pseudomonas aeruginosa ( %). a definitive diagnosis was made in / cases ( . %). chronic bronchitis was the most common diagnosis ( . %), median age years; followed by airway tracheal collapse or bronchomalacia ( %), median age years,; and primary bacterial infections ( . %), median age years. less common etiologies identified included neoplasia ( . %), median age years; parasitic infections ( . %), median age years; and eosinophilic bronchopneumopathy ( . %), median age years. rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. myxodematous mitral valve disease was found concurrently in / ( . %) dogs. this study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and etw or bal with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease. disclosures: no disclosures to report. canine sterile steroid-responsive lymphadenitis (cssrl) is an uncommon cause of lymphadenomegaly. diagnosis is one of exclusion after extensive investigations exclude infectious, inflammatory or neoplastic aetiologies. resolution of clinical signs occurs with corticosteroids. this retrospective study aimed to further define characteristics, progression and treatment regimens. cases were recruited from uk referral centres between - . diagnostic investigations in each case excluded other potential causes of lymphadenomegaly. thirty-six dogs were diagnosed with cssrl from lymph node cytology and/or histopathology. eighteen breeds were represented, of which were spaniels. english springer spaniels (ess) accounted for cases along with cocker spaniels ( ), cavalier king charles spaniels ( ) and border collies ( ) clinical presentation varied between dogs. clinical signs of pyrexia ( %), lethargy ( %) and anorexia ( . %) were the most common. other signs included cough, tachypnoea, dyspnoea, dysphagia, vomiting, diarrhoea, neck or spinal pain, abdominal pain, joint effusion or dermatologic signs. median referral time was days (ess , cockers and border collies days). twenty-two animals were pyrexic at presentation (mean . °c, range . - . °c). thirty-one animals presented with peripheral lymphadenomegaly, but animals displayed only internal lymph node enlargement. cytology was performed in / cases; neutrophilic lymphadenitis ( ), followed by granulomatous ( ), pyogranulomatous ( ) and reactive hyperplasia ( ). histopathology was performed in / cases documenting neutrophilic ( ), pyogranulomatous ( ) or granulomatous ( ) lymphadenitis. lymph node culture or staining (gram, pas, zn) was performed in and animals respectively, all of which were negative. prednisolone was administered in all cases (dose range . - mg/kg daily). animals were initiated therapy at mg/kg q hours. mean treatment length was weeks. dogs relapsed throughout the study period ( ess, cockers, ckcs, border collie, lurcher and beagle). ess relapsed within months of diagnosis. median relapse time was weeks. this study documents dogs with cssrl in the uk suggesting an over-representation in spaniel breeds (particularly ess), with females and young dogs typically affected. cytologic and histopathologic examination confirmed sterile lymphadenitis with animals showing marked and rapid clinical improvement with corticosteroids. disclosures: no disclosures to report. brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (osas). oxidative stress in the body represents the imbalance between the production of reactive oxidative species and the ability of antioxidant defense mechanisms to detoxify the reactive intermediates. oxidative stress is involved in the pathogenesis of many diseases, including hemolytic anemia, atherosclerosis, tissue reperfusion injury and has also carcinogenic potential. several studies have clearly shown an association between obstructive sleep apnea syndrome in humans and oxidative stress, but detailed underlying pathomechanism remains unclear. due to the consideration of brachycephalic dogs as an animal model of human osas, this study was designed to evaluate oxidative stress (paraoxonase type- activity; pon and total antioxidant status; tac) in brachycephalic dogs with brachycephalic airways obstructive syndrome (baos) before and after surgery compared to control dogs. this study was conducted on dogs with baos and control dogs. twenty out of baos dogs were evaluated - months after surgical correction. mean values of tac and pon in different studied groups were as follows: control dogs (tac: . ; pon : . ), baos dogs (tac: . ; pon : . ), baos dogs post-surgery (tac: . ; pon : . ) a statistically significant difference on tac levels is observed between dogs with baos and control dogs (p < . ). no statistically significant differences were observed in pon and tac levels before and after surgery. on the other hand, no differences have been observed between pon and tac levels in baos dogs according type of brachycephalic breed, grade of respiratory and digestive signs or presence of everted ventricular laryngeals. the results of our study showed a statistically significant difference in tac values between control and dogs with baos, confirming the oxidative stress previously described in humans. even that human patients with osas can partially reverse their increase in oxidative stress by using a nasal continuous positive airway pressure treatment, in dogs with baos no differences were observed before and month after surgical treatment. baos surgical treatment is not useful to reduce pon- or tac levels, probably because baos does not induce such an evident inflammatory process in dogs as in human patients with osas. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). the cipf course varies greatly among dogs from rapid deterioration to slowly progressive forms and the survival time from onset of clinical signs ranges from a few months to several years. in human ipf, increased chemokine (cc-motif) ligand (ccl ) concentrations in bronchoalveolar lavage fluid (balf) are indicative of a poor outcome and serum concentrations are correlated with clinical parameters of lung function. in dogs, serum and balf ccl concentrations were shown to be elevated in whwts with cipf compared with healthy whwts. the aim of the present study was to investigate whether serum ccl concentrations measured in whwts with cipf at diagnosis ( ) can be used as an indicator of prognosis and ( ) correlate with lung function parameters. ccl concentrations were determined by elisa (canine ccl quantikine elisa kit, r&d systems) in the serum of whwts suspected of cipf (median age . years, range . - . ), for which a follow-up was available (median follow-up time . months, range - . ). serum sampling extended from may to january . cipf diagnosis was confirmed by thoracic high resolution computed tomography, lung histopathology, or both examinations in , and whwts respectively. kaplan-meier analysis was conducted to investigate differences in survival times according to serum ccl concentrations at diagnosis. spearman analysis was used to assess correlations between serum ccl concentrations and lung function parameters, namely the distance walked in the -minute walking test ( mwd) and the arterial partial pressure of oxygen (po ). among the cipf whwts included, died or were euthanized for cipf-related reason, died or were euthanized for non-cipf-related reason and were still alive at the end of the study. the median survival of whwts with cipf-related death or euthanasia was . (range . - . ) months from diagnosis. serum ccl concentrations above pg/ml were significantly associated with a shorter survival time in whwts affected with cipf (p = . ). a weak negative correlation was found between serum ccl concentrations and the mwd (r = À . , p = . , n = ), while no correlation was observed with arterial po values (n = ). in conclusion, serum ccl concentration provides prognostic information in whwts suffering from cipf, while this marker is weakly correlated with the clinically lung function parameters available in the present study. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). this study was intended to ( ) describe thoracic high-resolution computed tomography (t-hrct) findings obtained in cipf dogs under general anesthesia (ga) using the glossary of the fleischner society and ( ) compare images obtained under ga (t-hrct ga ) with those obtained under sedation (t-hrct s ). t-hrct images from whwts with cipf and control whwts were retrospectively reviewed by observers in consensus. specific t-hrct features were assessed and graded for each lung lobe ( = absence, = mild, = moderate and = severe). a global score was then calculated. the khi² test with the threshold % was used for the statistical analysis. ground glass opacity (ggo) was observed in all cipf whwts and in / of controls (p = . ). in controls, ggo was mild and localised mainly in cranial lobes. in cipf whwts, ggo was mild, moderate or severe in , and dogs respectively, without lobe predilection. consolidation was observed in / cipf whwts but not in controls (p = . ) and was mild ( / ) to moderate ( / ). a mosaic pattern, suggestive of air trapping, was noticed in / cipf whwts but not in controls (p = . ) and was mild, moderate or severe in , and whwts respectively, without lobe predilection. nodules were present in / cipf whwts but not in controls. reticulation, subpleural bands and parenchymal bands were noticed in , , and / cipf whwts respectively. honeycombing, emphysema, pleural effusion and pleural thickening were never observed. bronchial wall thickening and mild bronchiectasis were present in / and / cipf whwts respectively but not in controls (p = . and p = . ). the overall t-hrct s quality was good in / examinations compared with / for t-hrct ga (p = . ). the presence of motion artefacts was higher for t-hrct s (p < . ), but were most frequently graded as mild (p < . ). t-hrct s allowed identification of a mosaic pattern in additional cipf whwts, while consolidation could not be identified in others. there was no difference in identification or gradation for the other features between t-hrct ga and t-hrct s . in conclusion, ggo, consolidation, mosaic pattern and bronchial wall thickening are the main t-hrct features of cipf in whwts. honeycombing, the major feature of ipf in humans, was never observed, which suggests a different pathophysiology between the entities. t-hrct s images are in accordance with t-hrct ga and can be used for cipf diagnosis. disclosures: no disclosures to report. literature on mesenterial lymphadenitis (lad) or mesenterial lymph node abscesses (lab) in small animals is scarce. case files from to were searched for dogs with the diagnosis of mesenterial lad/lab based on cytology and/or histopathology. of cases identified, had to be excluded due to incomplete data. the remaining dogs were of mixed breed (n = ), small munsterlander (n = ) and one each of other breeds. nine dogs were male and female. median age was months (range . diagnosis was based on fine needle aspiration (fna; n = ), histopathology (n = ) or both (n = ). significant findings in the dogs' history included gastrointestinal signs (n = ), puppy whose mother had mastitis, bite wounds/abscesses of the skin (n = ), pulmonic stenosis (n = ) and orthopaedic diseases (n = ). most common presenting complaints were lethargy (n = ), hyperthermia (n = ), diarrhoea (n = ), vomiting/nausea (n = ), inappetence/anorexia (n = ), back/abdominal pain (n = ) and lameness (n = ). diagnostic tests performed included haematology/serum biochemistry (n = ), thoracic (n = ) and abdominal (n = ) radiographs, abdominal ultrasound (n = ), ct/mri (n = ), fnas of other organs (n = ), urinalysis (n = ) with culture (n = ), coagulation profiles (n = ), orthopaedic radiographs (n = ), cpl (n = ), blood cultures (n = ), and csf/joint taps (n = ). dogs were retrospectively divided into group a (n = ): dogs with no other disease than lad/lab ("idiopathic") and group b (n = ): dogs with other diseases diagnosed simultaneously. these included neoplasia (carcinoma n = , lymphoma n = , leiomysarcoma n = , histiocytic neoplasia n = , prostate carcinoma n = ), gastroenteritis (n = ), presumed pancreatitis (n = ), purulent monoarthritis (n = ), purulent hepatitis/ splenitis (n = ), fungal infection at a distant site (n = ), and mycobacteriosis (n = ). eleven dogs received surgical treatment and antibiotics, and dogs conservative medical management consisting of supportive treatment and antibiotics. all dogs were discharged alive. dogs in group a were hospitalized longer (mean days, sd . ) than dogs in group b (mean days, sd . ) (p = . ). the median follow-up time was days ( - days). there was no difference in pretreatment with antibiotics or anti-inflammatories between groups. t-tests or kruskall-wallis tests showed that dogs in group a were borderline significantly younger (p = . ), had significantly higher respiratory rate (p = . ), rectal temperature (p = . ), monocyte count (p = . ) and crp concentration (p = . ) than dogs in group b. the small munsterlander had an odds ratio of over other breeds to suffer from lad/lab. in conclusion, idiopathic mesenterial lad/lab was seen in young dogs with hyperthermia and gastrointestinal signs. diagnosis of purulent lad/lab on fna does not exclude the presence of another underlying pathogenesis. disclosures: no disclosures to report. dogs were included, if they showed respiratory signs (< days) consistent with cird and if non-infectious respiratory conditions could be excluded. nasal and pharyngeal swabs were taken from dogs with cird and tested for respiratory pathogens, including canine parainfluenza virus (cpiv), canine adenovirus (cav), canines distemper virus (cdv), canine herpes virus (chv), canine respiratory coronavirus (crcov), canines influenza virus (civ), and bordetella bronchiseptica (b. bronchiseptica) by polymerase chain reaction. results of clinical and laboratory data were correlated with the underlying pathogen using fisher's exact test and chi-square test (p≤ . ). cpiv was detected in , crcov in , and b. bronchiseptica in dogs; patients showed infections with more than one pathogen. there was no significant difference for age and gender distribution between the groups; however, dogs infected with cpiv more likely originated from a shelter (p = . ). when clinical data were compared, there was no significant difference for the parameters depression, fever, cough, nasal discharge, dyspnoea, and abnormal lung sounds. furthermore, there was no significant difference regarding abnormalities of erythrocytes, platelets, leukocytes, and differential count between groups. the study shows that in dogs with cird clinical and laboratory parameters cannot indicate the underlying pathogen. furthermore, diseases severity does not seem to depend on the infectious organism involved. disclosures: no disclosures to report. breed related predisposition to bacterial bronchopneumonia (bp) has been reported in irish wolfhounds (iwhs). underlying factors are unknown, however immune deficit, ciliary dysfunction and aspiration have been suggested as predisposing factors. the purpose of this prospective study was to evaluate lymphocyte subpopulations in iwhs with one or more previous bp and compare results to elderly iwhs without any previous bacterial respiratory infections. additionally, healthy dogs of other breeds were included as controls. previous bp was confirmed in iwhs (median age . years, interquartile range . - . years). healthy iwhs (n = , . , . - . years) or dogs of other breeds (n = , . , . - . years) had no history or findings suggestive of previous bp. edta blood samples, collected from all dogs when they were healthy, were stained with fluorescent mouse anti dog cd , cd , cd , cd and mhc class ii antibodies (abd serotec Ò ) and flow cytometry analysis was performed with bd facsaria Ò ii cell sorter and facsdiva Ò software. statistical comparison between groups and the effect of age was studied using analysis of covariance (ancova) models. the number of leucocytes did not differ significantly between groups. the total numbers of lymphocytes and numbers of major lymphocyte subpopulations (b-cells, cd + and cd + t-cells) were significantly lower in healthy iwhs and iwhs with previous bp compared to dogs of other breeds (lymphocytes p < . and p < . ; b-cells p = . and p = . ; cd + t-cells p = . and p = . ; cd + t-cells p < . and p = . respectively). percentage and number of mhc class ii+ non-b lymphocytes was significantly higher in both iwh groups than in dogs of other breeds (p < . in all comparisons). lymphocyte numbers and subpopulations did not differ significantly in healthy iwhs compared to iwhs with previous bp. an age-related decline in the total number of lymphocytes (p = . ), t-cells (p = . ), cd + t-cells (p = . ) and mhc class ii+ non bcells (p < . ) was noticed only in the group of iwhs with previous bp. these preliminary results indicate that iwhs may have significantly lower numbers of lymphocytes, b-cells as well as cd + and cd + t-cells than dogs of other breeds. further studies are needed to determine whether these alterations represent a breed related phenomenon or are connected to the predisposition to bp. an age-related decline in lymphocyte, total t-cell and cd + t-cell numbers was detected in iwhs with previous bp. in humans, age related changes in cd + t-cells have been associated with increased susceptibility to infections. disclosures: no disclosures to report. feline chronic kidney disease (ckd) is a common feature of ageing cats. angiotensin-converting enzyme inhibitors (acei) are recommended to treat hypertension associated with ckd to limit target-organ damage and especially glomerular hypertension. in addition, the international renal interest society (iris) guidelines recommends the prescription of an acei in patients with ckd and proteinuria. to our knowledge no study has demonstrated the effects of long term administration of acei in a client owned population of cats suffering from ckd on glomerular filtration rate (gfr). the aim of the study was to evaluate the effect of an acei (imidapril, prilium Ò , v etoquinol sa) on the gfr of cats suffering from naturally occurring chronic kidney disease (ckd) over months. sixty-six cats presenting with clinical and biological signs of ckd were enrolled by european investigators and followed up till months in this randomized blinded study. thirty-two cats provided suitable data for gfr analysis; animals received imidapril at the dosage of . mg/kg/day and received placebo. animals with no available gfr value on day or with no data after day were excluded as well as animals for which the iohexol clearance could not be determined. follow up was censored after months due to small sample sizes for statistical comparisons. on day , month , month and month , cats were sampled , and minutes after intravenous administration of mg of iohexol. gfr was based on determination of the iohexol clearance which was calculated with the phoenix Ò winnonlin . software. statistical analyses were performed with sas/stat . software. as gfr were not available on each time point for a given animal, the treatment groups were compared on each gfr determination point using an ancova (analysis of covariance) with the gfr determined on day as the covariate. on d , gfr were . ae . and . ae . ml/kg/min in the imidapril and placebo group, respectively. a significant statistical difference ( . ae . versus . ae . ml/kg/min in the imidapril and placebo group respectively, p = . ) was observed in favor of a higher gfr in the imidapril treated animals on month . higher gfr were also observed in the imidapril group on mon-th and month but not significantly different from the placebo group. daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease. disclosures: authors are employees of vetoquinol. there is a concern over the potential of cytotoxic drugs which could harm exposed workers. the speciality of oncology of the ecvim-ca published guidelines in order to prevent that risk. however few data exist for evaluation of the real risk of occupationnal exposure. this concern was the aim of our study. biomarkers used were vincristine and cyclophosphamide. surface samples were collected in the consultation room and ward dedicated for chemotherapy. samples were collected with wet filter paper on cm surfaces, or objects (computers for instance). samples were analysed by liquid chromatography and mass spectrometric method.the limit of quantification was . ng/ cm (or . g/object) for vincristine and . ng/ cm (or . g/object) for cyclophosphamide. samples in consultation room were collected after treating dogs with vincristine and after cleaning. samples in dedicated ward were collected after cleaning and after -days stay of treated dogs. aeras/objects were tested in consultation room; in dedicated ward for vincristine; in ward for cyclophosphamide. after treatment of dogs in consultation room, traces of vincristine were detected on the floor and the laboratory bench top ( . to . ng/ cm ). moreover, the surface of auscultation table and extern side of gloves were contaminated after preparation and administration of vincristine (respectively . , and ng/objet). after cleaning, % of samples in consultation room were positive. traces of vincristine were detected on the floor or objects (wall otoscope. . .: . to . ng/ cm ). after cleaning, % of samples in ward were positive. traces of vincristine were detected on the floor and objects (boxes, wastebin, bowls. . .: . to . ng/objet) after cleaning and animals treatment. cyclophosphamide were detected on all areas tested ( . a . ng/objet). despite protective guidelines to avoid spread of cytotoxic drug, environemental exposure was demonstrated. however contaminations were limited and show that handling procedures of cytotoxic drugs are well controlled. most-elevated contamination level for vincristin were noticed on extern side of gloves depsite of using appropriate material. workers should pay a particular attention for gloves withdrawal to limit exposure. small amounts of vincristine were found in inapropriate places: computeur mouse,. . . even if there is few of those residues, we thought about people working on a regular basis in this room for other activities than chemotherapy., and we decided to adapt our clinical practices. evaluation of occupational exposure to cytotoxic drugs is an important step to prevent incidents and heigt awarness of nursing staff to apply those good clinical practices. disclosures: no disclosures to report. the tumor-associated inflammatory response had the effect of enhancing mammary tumorigenesis, helping incipient neoplasias to acquire hallmark capabilities, both in human and dogs. t-cells migration to the tumor site and the following activation may be the essential requirement for their promoting effect on tumors. in human breast cancer the signaling pathways of c-kit have been described as possibly being involved in differentiation, migration, survival, and maturation of t-cells and other inflammatory cells into tumor sites. in order to clarify this subject in canine mammary tumors (cmt), malignant neoplasms were studied by using immunohistochemistry comparing the intratumoral cd + tlymphocytes and c-kit expression together with vegf, microvessel density (mvd) and clinicopathological characteristics of tumor aggressiveness. cd + t cells and high c-kit immunoexpression revealed a positive and statistically significant correlation with vegf (r = . , p < . ; r = . , p = . for cd and c-kit respectively) and cd (r = . , p < . ; r = . , p = . for cd and c-kit respectively). a statistically significant association (p = . ) and a positive correlation (r = . , p = . ) between cd + t-lymphocytes and c-kit was also observed. tumors with high c-kit expression showed higher counts of cd + t-cells. the mvd of high cd /vegf tumors was significantly more elevated (p < . ). a similar association was observed for high c-kit/vegf tumors (p < . ). in this study high cd /vegf, high c-kit/vegf and high cd /c-kit tumors were statistically significantly associated with elevated grade of malignancy (p < . for cd /vegf, c-kit/vegf and cd /ckit), presence of neoplastic intravascular emboli (p < . for cd /vegf and cd /c-kit; p = . for c-kit/vegf) and presence of lymph node metastasis (p < . for cd /vegf, c-kit/ vegf and cd /c-kit). tumors with high cd /vegf (p = . ), high c-kit/vegf (p < . ) and high cd /c-kit (p = . ) expression were associated with shorter os time. inter-estingly the group of tumors with high c-kit/vegf retained their significance by multivariate analysis arising as independent predictor of os. results of this study suggest that t-lymphocytes may share common signaling pathways with c-kit and vegf in cmt progression and may contribute to increased angiogenesis, aggression and shorter os in these tumors. disclosures: no disclosures to report. lgl lymphoma, but no comparisons were made with cats with other diseases or other forms of feline lymphoma. therefore, the objective of this study was to assess differences in prevalence, signalment (breed, sex, and age), physical exam findings (body weight, body condition score, body temperature, heart rate, respiratory rate, and systolic blood pressure), and felv/ fiv status between cats with lgl lymphoma (group ) and all other type of feline lymphoma (group ). the electronic data-base of the san marco veterinary clinic was searched between january- and december- for cats with a cytological or histopathological diagnosis of lymphoma. differences between groups were assessed by t-test, mann whitney, pearson-chi square, pearson chi square yates corrected, and fisher's exact test. during the study period out of sick cats seen at the clinic were diagnosed with lymphoma (group : n = ; group : n = ). the prevalence of all type of feline lymphoma between and compared to sick cats did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). the lymphoma lgl prevalence between and compared to all types of lymphoma did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). among the variables studied, only sex (group : [ . %] females, [ . %] males; group : [ . %] females, [ . %] males; p = . ) and age (group : ae months; group : ae months; p = . ) were significantly different between groups. sixteen out of cats with lgl lymphoma were tested for their felv/fiv status resulting all felv-and one ( . %) fiv+. seventy-four out of cats with all other types of lymphoma were tested for their felv/fiv status resulting ( . %) felv+ and ( . %) fiv+. five cats ( . %) were both felv+/fiv+. prevalence of felv infection was significantly lower (p = . ) in group compared to group . there was no difference in prevalence of fiv infection between groups. lymphoma lgl affects more females and older cats compared to all other type of feline lymphoma. opposite to all other type of lymphoma, and in accordance to previous litterature information, felv+ status does not play a role in the pathogenesis of feline lgl lymphoma. disclosures: no disclosures to report. the activity of t regulatory cells (tregs) is known to be closely associated with the expression of foxp transcription factor. foxp regulatory t cells (foxp treg) are a distinct group of t lymphocytes that have immunosuppressive properties. normally this cells work for prevention of harmful autoimmune responses, however can also interfere with beneficial immune responses such as anti-tumor immunity. in human breast cancer these cells play a crucial role in tumor progression. in canine mammary tumors (cmt) there are only a few studies and this topic are not welldocumented. in this study we included malignant cmt and studied, by immunohistochemistry, the intratumoral foxp expression together with vascular endothelial growth factor (vegf), microvessel density (mvd, by cd antibody) and several clinicopathological characteristics. abundant foxp treg cells was statistically associated with presence of tumor necrosis (p = . ), nuclear grade (p = . ), poor differentiation of tumors (p < . ), high mitotic grade (p < . ), high histological grade of malignancy (p < . ), presence of neoplastic intravascular emboli (p < . ) and presence of lymph node metastasis (p < . ). intratumoral foxp levels were correlated with the levels of vegf (r = . ; p < . ) and intratumoral mvd (r = . ; p < . ). additionally tumors with abundant foxp treg cells were associated with shorter overall survival (os) time (p = . ). results suggest that treg cells play a role in cmt progression and may contribute to increased angiogenesis and aggression in these tumors. the association of intratumoral foxp expression with shorter os of animals suggests a utility of treg cells activity as a prognostic marker. disclosures: no disclosures to report. cyprus is an island state in the eastern mediterranean basin. no epidemiological studies have yet been performed on infectious agents in cats from cyprus. the aim of this study was to determine the prevalence of several infectious agents, including some vector-borne infections, in cats from cyprus. surplus edta-blood and serum samples were recruited from cypriot cats, from which signalment and lifestyle characteristics were recorded. dna was extracted and real-time quantitative polymerase chain reaction (qpcr) assays were used to detect haemplasmas(mycoplasma haemofelis, 'candidatus mycoplasma haemominutum' and 'candidatus mycoplasma turicensis'), leishmania spp.and bartonella henselae. conventional pcr assays were used to detect ehrlichia/anaplasma spp. samples yielding positive results for leishmania spp. or ehrlichia/anaplasma spp. underwent further characterisation (sequencing). elisas were performed for the detection of l. infantum antibodies, feline leukaemia virus (felv) antigen and feline immunodeficiency virus (fiv) antibodies. statistical analysis was performed using spss for the assessment of any associations between variables and infectious agents. of the samples extracted, were excluded due to failure of ≥ one internal control pcr. of the remaining samples, ( . %) were positive by pcr for haemoplasma including ( . %) for m. haemofelis, ( . %) for 'ca. m. haemominutum' and ( . %) for 'ca. m. turicensis'. nineteen ( . %) were positive for b. henselae. one cat ( . %) was pcr positive for ehrlichia/anaplasma spp. and sequencing revealed identity with anaplasma platys. leishmania spp. dna was detected in of the ( . %) cats; sequencing revealed l. infantum in of these cases. l. infantum serology was positive in of the cats tested ( . %). only one cat was positive for both leishmania pcr and serology. of the cats that underwent retroviral serology, ( . %) were felv and ( . %) were fiv positive. statistical analysis identified several significant associations (p < . ) including the following; haemoplasma infection and both outdoor access and feral-shelter cat origin, felv or fiv infection and both anaemia and feral-shelter cat origin. this study documents, for the first time, the presence of haemoplasmas, l. infantum, b. henselae, a. platys, felv and fiv in the feline population of cyprus. the prevalence of haemoplasma, fiv and b. henselae infections were among the highest reported in cats from mediterranean countries, while that of leishmania spp. was similar. this is the second report of a. platys infection in a cat from a mediterranean country. disclosures: no disclosures to report. bartonella species (spp.) are zoonotic pathogens, and infections in cats are common. prevalence in cats from southern germany is still unknown. the aim of this study was to determine the prevalence of bartonella spp. dna in blood of cats in southern germany and to evaluate associations between bartonella bacteremia, housing conditions, feline immunodeficiency virus (fiv), and feline leukemia virus (felv) status, including progressive, regressive, and abortive felv infection. blood samples of cats that were presented to different veterinary clinics in southern germany for various reasons were tested for bartonella spp. dna using a previously published conventional polymerase chain reaction (pcr) targeting a fragment of the s- s rrna intergenic spacer region. for statistical analysis, fisher's exact test was used. prevalence rate of bartonella spp. bacteremia was . % ( / ; ci: . % - . %). b. henselae was amplified in of these cats. one cat was positive for b. clarridgeiae dna. most of the infected cats were clinically healthy, but half of the cats had thrombocytopenia, potentially caused by their bartonella spp. infection. there was no significantly higher risk to be infected with bartonella spp. when living mainly outdoors or being fiv-or felv-infected. prevalence of bartonella spp. bacteremia is low in southern german cats, but there is still a risk of human bartonella infection associated with cat ownership. most clinical changes of the bartonella spp.-infected cats were related to other diseases. however, thrombocytopenia was common and further studies are required to define its potential clinical relevance. disclosures: no disclosures to report. ante-mortem diagnosis of feline infectious peritonitis (fip) is still challenging. the aim of this study was to evaluate sensitivity and specificity of a 'combined reverse transcription nested polymerase chain reaction (rt-npcr) and sequencing approach', detecting mutations at different nucleotide positions within the spike gene, that previously were shown to correlate with the fip phenotype. the study population consisted of cats with confirmed fip and a defined control group of cats for which fip was considered an important differential diagnosis, but that were definitively diagnosed with other diseases. blood and/or effusion samples were examined for feline coronavirus (fcov) rna by rt-npcr and, if positive, nucleotide positions and were sequenced for nucleotide transitions. sensitivity, specificity, negative and posi-tive predictive values were determined and % confidence intervals ( % ci) calculated. rt-npcr detected fcov in cats in blood (n = ) and/or effusion (n = ); all of them had fip. one of the mutations of interest was found in / of the pcr-positive blood samples and in / of the pcr-positive effusion samples. diagnostic specificity of the 'combined rt-npcr and sequencing approach' was % in blood ( % ci . - . ) and effusion ( % ci . - . ). diagnostic sensitivity was . % ( % ci . - . ) in blood and . % ( % ci . - . ) in effusion. a positive test result therefore confirms a suspicion of fip. a negative result, however, cannot be used to rule out fip, especially if only blood is available. therefore, if no effusion is present, diagnosis of fip still remains challenging. disclosures: dr. elisabeth mueller is the managing director of laboklin gmbh & co.kg. dr. karola weider is employed at laboklin gmbh & co.kg. this laboratory offered the 'combined rt-npcr and sequencing approach' on a commercial basis and performed the testing in this study. feline infectious peritonitis (fip) is a viral disease caused by the virulent strain of feline coronavirus (fipv). the disease can appear under clinical forms, dry or effusive, both leading to a fatal outcome. diagnosis was based on histopathologic lesions on necropsy until the recent discovery of mutations associated with the fipv strain in the c and spike (s) genes. our main goal was to detail the distribution of c or s gene mutations in different biological samples of cats suffering from fip. this was a retrospective, observational study of cats showing clinical signs compatible with fip. ten out of cats were of pure breed. . % were males and . % females. median age was . months at presentation. the clinical presentation, pathologic findings and virologic data were reviewed. according to clinical signs, cats were classified with a dry form and with a wet form of fip. the main clinical signs included dehydration, hyperthermia, icterus, abnormal abdominal palpation, neurological and ocular disorders. when possible blood, fecal material, effusion, fine needle aspiration (fna) from relevant organs or a combination of these, was recovered from each cat. feline coronavirus (fcov) was first researched by rt-pcr, then the c and part of the s genes were sequenced to determine the eventual presence of mutations. among the dry cases, fcov was detected in / blood samples, / fecal samples and fna ( / ). among the wet cases, / blood samples, / fecal samples and all effusion samples ( / ) were positive for the presence of fcov. c mutations were never found in fecal samples but were found in / effusion samples and in / fna. s mutations were detected in / fecal samples, / fna and / effusion samples. for cats, no mutation, neither in c or s genes was identified despite the confirmation of fip by necropsy. s gene mutation is more frequently observed than c gene despite in cases where only c mutations were identified. moreover the presence of strains harbouring s mutation in feces has never been described before and could suggest the possible diffusion of fipv among feline population. in conclusion, viral diagnosis of fip based on rt-pcr sequencing in effusion and fna samples is essential. rt-pcr resulting from blood samples should be carefully interpreted because of high risk of missing fipv. finally, searching for mutations in both s and c genes is recommended. disclosures: no disclosures to report. methicillin resistant staphylococcus aureus (mrsa) has recently become a great concern for pet animals' disease and zoonotic infection. mrsa strains transfer between pet animals and humans could occur. the aim of the present study was to determine the occurrence of mrsa in household dogs. from january to june , clinical samples were collected from dogs, patients of the veterinary teaching hospital of the department of veterinary sciences of messina (italy), affected by several diseases of various origins. all samples were processed by bacteriological conventional methods for isolation and identification. all strains were tested for phenotypic susceptibility to oxacillin and were subjected to a pcr protocol for the detection of meca gene. strains carrying the gene were considered methicillin resistant (mrs). lastly, on both mrs and methicillin sensitive (mss) strains, kirby-bauer disk diffusion susceptibility testing were performed to highlight resistance profiles using molecules belonging to the main classes of antimicrobials used in veterinary practice. strains resistant to at least one molecule of or more classes of antibiotics were considered multidrug-resistant (mdr). the statistical analysis of the results was made using the z-test by a primer Ò software. forty staphylococcus spp. strains were isolated, belonging to species. the most frequently isolated microorganisms were staphylococcus aureus with isolations ( %) and staphylococcus pseudintermedius with isolations ( . %), followed by staphylococcus epidermidis with isolations ( %) and staphylococcus cohnii and staphylococcus warneri, both with isolations ( %). a single isolation ( . %) was obtained for each of the species staphylococcus chromogenes, staphylococcus haemolyticus, staphylococcus lentus, staphylococcus lugdunensis, staphylococcus saprophyticus, staphylococcus simulans and staphylococcus sciuri. thirteen ( . %) strains of staphylococcus spp. were phenotypically resistant to oxacillin and staphylococcus aureus ( . %; n. from pyoderma, n. from exudative pleural effusion) were positive for the meca gene. all strains of staphylococcus spp. were mdr. our results showed the presence of mrsa and multidrug-resistant staphylococcal strains in household dogs. a lack of correspondence between antimicrobial susceptibility tests and molecular methods was found in the present study. disclosures: no disclosures to report. a fourth haemoplasma called 'candidatus m. haematoparvun-like' (cmhp) was latter identified in cats. the only published study in chile was carried out in cats, with prevalences by pcr of . % to mhf, and % to cmhm.the aim of this study was to perform molecular detection of haemoplasmas in cats from valdivia, southern chile. blood samples were taken from cats and used for haemoplasmas dna detection by quantitative real time pcr (qpcr) at universidad austral de chile. qpcr protocol was based on detection of feline dna targeting s housekeeping gene and mycoplasma spp. s rrna gene (universal primers, my sf forward, my sr y my sr , both reverse) by sybr green method. the melting temperature (tm) analysis allowed identifying the infecting mycoplasma species (mhf, cmhm, cmt). it was not posible to identify haemoplasmas species on co infected cats, so a second qpcr specie specific protocol was applied on these samples. second qpcr protocol was based on s rrna gene, with specific primers to detect mhf, cmhm, cmt and cmhp. all samples ( / ) were positive to s gene, proving presence of cat dna. from the cats, . % ( / ) were positive to haemoplasmas, where . % ( / ) corresponded to cmhm (tm . - . °c), . % ( / ) to mhf (tm . - . °c), % ( / ) to cmt (tm . - . °c) and . % ( / ) to co infections. associations between cmhm+mhf, cmhm+cmt, cmhm+cmhp and cmhm+mhf+ cmhp were detected on co infected animals. these results agree with those found in previous reports from chile, europe, eua and brazil, where cmhm is the most prevalent species and co infections are less frequent. valdivia cats are infected by different haemoplasma species and cmt and cmhp are reported for the first time in chile.founding: did uach, project s- - disclosures: no disclosures to report. clinical manifestations of canine visceral leishmaniasis (canl) are non-specific and include progressive weight loss, anemia, lymphadenomegaly, hepatosplenomegaly, dermatological, renal and ocular alterations. cardiac lesions resulting in clinical signs has been scarcely described in dogs with vl, and the presence of the parasite in the cardiac tissue has been involved in few reports. accordingly, the present study aimed to evaluate histopathological abnormalities in cardiac tissue from dogs naturally infected by leishmania infantum chagasi. a total of dogs were evaluated. all dogs were symptomatic but no one presented clinical signs of cardiac involvement. in compliance with a federal law and under the owners' signed consent, all dogs were submitted to euthanasia and comprehensive post-mortem evaluation. samples from right atrium free wall (ra), right ventricle free wall (rv), interventricular septum (ivs) and left ventricle free wall (lv) were collected and evaluated. tissue samples were fixed in formalin, embedded in paraffin, sectioned at mm, and stained with hematoxylin and eosin (he) and anti-leishmania immunohistochemistry was also performed. the study was approved by the ethics committee in animal experimentation and animal welfare (protocol number / ). histopathological changes were observed in at least one of the evaluated cardiac regions in % ( / ) of the dogs. the most frequent cardiac injury was an inflammatory reaction, characterized by the presence of mononuclear cell infiltrate in different degrees. of the evaluated regions, ra was the one with the highest incidence of histopathological changes, observed in % ( / ) of the animals, followed by rv, lv and siv, affected in . % ( / ), . % ( / ) and . % ( / ) of the dogs, respectively. immunohistochemistry revealed amastigotes in the cardiac tissue in % ( / ) of the dogs. a positive correlation was found between cardiac lesions and the presence of amastigotes in the myocardium (p < . ). disclosures: no disclosures to report. canine leishmaniosis is a life threatening zoonotic disease. the combination of meglumine antimoniate and allopurinol is consid-ered the most effective therapy for canine leishmaniosis and constitutes the first line protocol against this disease. allopurinol is a parasitostatic drug used in long-term to maintain low parasite loads and to avoid clinical relapses. traditionally, allopurinol is considered a very safe drug in the dog. however, some reports indicate that xanthinuria and xanthine urolithiasis is produced after prolonged therapy with allopurinol in the dog. the aim of this prospective study was to evaluate the prevalence of urinary adverse effects of allopurinol treatment ( mg/kg/bid/po) in dogs with leishmaniosis. diagnosis was made by compatible clinicopathological abnormalitites with leishmaniosis and high leishmania infantum-specific antibody levels assessed by quantitative elisa. once leishmaniosis was diagnosed, a close follow-up (day , , , and during treatment) including physical examination, baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis, urinary protein/creatinine ratio) and abdominal ultrasound was performed. in our preliminary results, dogs were included. dogs did not present any urinary abnormalities based on biochemistry profile, urinalysis and abdominal ultrasound at the time of diagnosis. four out of presented xanthinuria (day- (n = ), day- (n = ), and day- (n = )). two out of dogs presented renal mineralization at day- of treatment. two out of dogs presented bladder urolithiasis since day- of treatment. xanthinuria was presented initially in all dogs that developed renal mineralization or bladder urolithiasis. dogs with renal mineralization and urolithiasis were treated with a restricted protein diet and, so far, they did not develop renal disease. the present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. a thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects. disclosures: no disclosures to report. giardia duodenalis is one of the most important gastrointestinal parasites in dogs and cats with a zoonotic potential. in germany the prevalence in dogs and cats reaches up to % and %, respectively. genotypes of genetic assemblages of the parasites infect humans (assemblages a and b) and other mammals including small animals. in contrast, parasites of the assemblages c and d are specific for dogs, assemblage f for cats. objectives of the study were to analyse the prevalence, potential epidemiological risk factors and symptoms of g. duodenalis infections in dogs and cats. to detect g. duodenalis, feces from dogs and cats was analysed with an elisa technique. after dna extraction real time pcr as well as multi-locus sequence typing was performed for the following gene loci: triosephosphate isomerase-, glutamate dehydrogenase-, beta-giardin-gene, ssu rrna. with a questionnaire possible epidemiological risk factors were evaluated. statistical analyses were performed using spss (odds ratio, kolmogorow-smirnow test, spearman correlation). fecal samples of dogs and cats were collected over a time period of months. the elisa test was positive in / dogs and / cats. of giardia positive dogs and of positive cats had gastrointestinal signs. genotyping was successful in of dog samples and were assigned to assemblages as follows: assemblage a (n = ), a/c (n = ), a/d (n = ), b (n = ), b/d (n = ), c (n = ), c/d (n = ), d (n = ). only one of positive cat samples could be genotyped and was atypically identified as assemblage d. significant correlations between giardia infection and age, clinical signs, deworming status and staying abroad were found. in this monocenter study a prevalence rate of . % in dogs and . % in cats was detected, which is in good accordance with previous studies. the study further highlights a high rate ( %) of asymptomatically g. duodenalis infected animals. as potential zoonotic assemblages were detected, transmission of giardia from small animals to humans (and vice versa) cannot be excluded. especially young and not dewormed animals had a higher prevalence. disclosures: no disclosures to report. canine parvoviral enteritis remains a common cause of morbidity and mortality in young dogs. the goal of this study was to document a large cohort of affected dogs and analyze several factors as possible predictors of fatal outcome. medical records were retrospectively searched for dogs with parvoviral enteritis diagnosed with a positive fecal antigenic test or a fecal pcr. dogs were included only if the medical records were complete. the population was compared to the reference population of the hospital on the same time period with chi square tests and several factors were analyzed as possible predictors of death with a logistic regression. one hundred and fourty seven cases were included. seventy percent of the dogs were non vaccinated puppies under the age of months. intact females and rottweiler, american staffordshire terrier and french beauce shepherd dogs were over-represented. clinical signs such as vomiting, diarrhea and dehydration were present in . %, . % and . % of the dogs respectively. hyperthermia, anemia and leucopenia were observed in . %, . % and . % of dogs respectively. the majority of the affected dogs were hospitalized for to days and the mortality rate was . % ( / dogs). hypoglycemia at admission was observed in / ( . %) dogs in which blood glucose was measured and was the only risk factor associated with death (p < . ). in this study, a predisposition of rottweiler, american staffordshire terrier and french beauce shepherd dogs was observed and hypoglycemia at admission was the only predictor of fatal outcome. disclosures: no disclosures to report. canine parvovirus (cpv) infections in dogs remain widespread around the world and still represent a major health threat in puppies. all vaccine manufacturers include this component in their core vaccination package, recommending injections at - weeks interval from to weeks of age. despite broad vaccination coverage, number of reports suggesting lack of efficacy in vaccinated dogs have been reported, which implicate vaccines belonging to all major manufacturers. these cases are usually considered as being linked to the interference with maternal antibodies (matab), able to persist beyond weeks of age, which has led most expert groups to recommend a third vaccination around weeks of age. persistence of matab actually represents a major issue when immunizing puppies against parvovirosis. indeed, matab titres higher than / in the haemaglutination inhibition (hi) test can still inhibit vaccine uptake whereas such titres do not prevent field virus infection. in contrast, hi titres higher than / to / are usually considered as protective against disease and virus excretion. this "immunity gap"is therefore a critical period for the puppy and the outcome of the vaccination. in order to evaluate the impact of residual mda on the efficacy of a standard primary vaccination protocol, we performed a vaccination field trial with serological follow-up. puppies from to weeks of age presented at veterinary practice received injections at weeks interval. serology was performed by elisa before (at d /v ), during (at d /v ) and after (d ) vaccination. average maternal antibodies titres were strongly correlated with the age of the puppy at primary vaccination, remaining at vaccine inhibiting level until~ week of age. average titres increased significantly after st injection of primary vaccination in most groups and in all groups after the nd injection of primary vaccination. individual variability remained significant: vaccine uptake was inversely and strongly correlated to the pre-vaccinal matab titre at vaccination. out of puppies ( %) didn't seroconvert, despite vaccination complying to the recommended schedule. vaccination was started in such dogs between . and . weeks and completed between . and . weeks and average initial matab titre was . log compared to . for the general population. in conclusion, this trial supports the recommendation of an additional injection of primary vaccination at weeks, especially in areas of high parvovirus prevalence / pressure, where high levels of matab are likely to be transferred to puppies. disclosures: all authors are employees of merial. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . . % of the dogs were pcr positive. the aim of our study was to evaluate the usefulness of determining serum level of c-reactive protein (crp) in dogs naturally infected with bacterium a. phagocytophilum as a possible indicator of the clinical phase of the disease. pcr and/or ifa positive dogs with clinical presentation and/or thrombocytopenia were included in the study. based on the results, the dogs were divided into groups: pcr positive dogs; ifa positive (subdivided according to titer level from : -> : ) and pcr negative dogs; positive control group -pcr and ifa negative dogs with clinical signs and/or thrombocytopenia; negative control group -clinically healthy, pcr and ifa negative dogs. serum level of crp was determined using lifeassays Ò canine crp (lifeassays, lund, sveden) . an elevated concentration of crp (> mg/l) was determined in pcr positive and ifa positive dogs with an ifa titer ≥ : and coincides with the presence of clinical signs (most commonly general clinical signs, elevated body temperature, gastrointestinal problems) and/or mild ( . %) or severe ( %) thrombocytopenia. the assessment of crp concentration, in correlation with certain clinical alterations and thrombocytopenia, suggests that crp concentration is elevated in the acute phase of the disease and is in correlation with the aforementioned changes therefore can serve as an additional diagnostic parameter. the crp concentration in ifa positive dogs, regardless of ifa titer levels, and with present clinical signs and thrombocytopenia is higher (above detectable level, > mg/l) than in dogs without clinical signs or laboratory alterations, which may speak in favor of reinfection or reactivation of a persistent infection at least in cases when no other cause of inflammation can be found. specific treatment would therefore be reasonable in such cases, especially in cases of rising crp concentration. disclosures: no disclosures to report. gallbladder agenesis is a very rare cause of elevated liver enzymes in dogs. in this study, we evaluated the features of dogs [ males ( castrated) and females ( spayed)] with suspected gallbladder agenesis on ultrasonography. five different breeds were included: chihuahua (n = ), toy poodle ( ), german shepherd ( ), jack russell terrier ( ), and shiba dog ( ). the median age was . ( . - . ) years. ten dogs were asymptomatic, while the other dogs showed decreased appetite ( ), vomiting ( ), ascites ( ), seizure ( ), and diarrhea ( ). all dogs showed elevated liver enzymes, with high alanine aminotransferase levels (median, u/l; - u/l) in dogs and high gamma-glutamyl transpeptidase levels (median, u/l; - u/l) in . gallbladder agenesis was confirmed using laparoscopy in dogs and laparotomy in . liver biopsy samples were obtained from all dogs. additional computed tomography cholangiography was performed for dogs using a -slice multidetector computed tomography (mdct) scanner following the intravenous administration of contrast medium (meglumine iotroxate). the obtained images were analyzed on a workstation, and they revealed an absent gallbladder in dogs and a vestigial gallbladder in . the common bile duct was dilated in dogs. for all dogs, laparotomy or laparoscopy was used to visualize the gallbladder and liver abnormalities, including malformed lobes and surface irregularities. acquired portal systemic collaterals were visually confirmed in dogs, who also exhibited hypoplasia of the portal vein on histological examination. in conclusion, most animals with gallbladder agenesis were asymptomatic in our study, indicating a good long-term prognosis. however, symptoms associated with portal hypertension must be monitored in animals with primary portal vein hypoplasia. disclosures: no disclosures to report. assessment of systolic arterial blood pressure (sap) is an important tool in small animal internal medicine practice, especially with diseases or clinical conditions that can cause hypertension or hypotension. the doppler method is noninvasive and has several advantages compared to oscillometric method. there are few studies about the effect of body position on sap in conscious dogs. the hypothesis was that animal positioning during measurement alters sap values. the study design was prospective and randomized regarding order of positioning measurements. one hundred and twenty client-owned, conscious, healthy or sick adult dogs, weighing up to kg were included. sap was recorded by doppler ultrasonography following american college of veterinary internal medicine consensus statement with animals positioned in sternal recumbency, right lateral recumbency and with the dog laying down on owner s lap. the order of body position was raffled at the time of measurement. five consecutive measurements on each body position were performed always on left forelimb and the average was calculated. sap values were higher in sternal recumbency ( mmhg, p %- % = - . ; p < . ) compared to those obtained on the owner s lap ( mmhg, p %- % = . - . ), and both were similar to right lateral recumbency ( mmhg, p %- % = - ). these results suggest that sap measurement obtained on owner s lap or right lateral recumbency can be used on clinic routine, but sap measurement obtained on sternal recumbency should be avoided, because such measures may be overestimated. disclosures: no disclosures to report. canine patients may be presented for blood pressure (bp) assessment when clinical diseases associated with systemic hypertension (ht) are suspected but not confirmed; this population may encompass patients that have normal bp, true ht or situational ht. the clinician's aim is to identify animals with ht reliably, while minimizing false positives. this prospective study investigated the repeatability of duplicate within-visit systolic bp assessments (sbp and sbp ) in consecutive canine patients presented for bp assessment in the small animal clinic ( duplicate sbp recorded from dogs) and in a control group of healthy dogs ( duplicate sbp obtained from control dogs), resulting in duplicate measurements for analysis. doppler methods were used for duplicate assessments and oscillometric methods were used for duplicate assessments; cuff size/location were consistent within any dog. sbp ≤ mmhg was considered normal (nml); sbp > mmhg was considered abnormal (abn). median (range) elapsed time between duplicate readings was ( - ) minutes; % of sbp were obtained within minutes of sbp . there was no correlation between elapsed time and change in sbp (p = . ). % of sbp were equal to or lower than sbp ; median decrease was ( - no dog with sbp > mmhg (n = ) had nml sbp . more dogs with abn sbp were panting ( / scored, %) compared to the group with dogs with nml sbp ( / scored, %, p = . ). sbp of dogs that stopped panting ( / , %) tended to decrease (p = . ). within-visit repeatability of bp diagnosis was good in dogs with nml sbp , but apparent false positive diagnoses of ht occurred in % of dogs with abn sbp . sbp > mmhg was repeatable in all dogs. panting may be associated with increased measured sbp by these methods. duplicate within-day measurements may help identify false positive ht diagnoses in dogs with initial sbp measurements > mmhg. disclosures: no disclosures to report. hypovitaminosis d has previously been shown to be prevalent amongst dogs with protein losing enteropathy (ple). outcome is generally poor in canine ple, and there is a lack of studies identifying underlying risk factors. the hypothesis of this study was that low vitamin d serum concentrations could be a risk factor for bad outcome in such patients. medical records for dogs seen at the royal veterinary college between and were reviewed to identify dogs with a diagnosis of ple confirmed by histopathology. dogs were included in the study if they had serum samples frozen within minute after sampling, had been kept at À degrees c until analysis, and if clinical activity scoring (cce-cai) had been recorded at the time of diagnosis. forty-three dogs were included in the study. follow-up with referring veterinarians was made to determine outcome of patients. patients were divided into two groups: patients deceased due to ple (poor outcome group, n = ) and patients alive or deceased due to another disease (good outcome group, n = ). treatments for patients were allocated to two groups: one group consisted of patients who were prescribed diet only and the other group received diet and immunosuppressive agents. samples were sent on dry ice to michigan state university's diagnostic center for population and animal health. ionised calcium (ica) was measured using an ion specific electrode and (oh)d was measured using a commercially available radio-immunoassay that has been validated for use in veterinary medicine. comparisons of outcome groups for age, ccecai, treatment, serum (oh)d and ica were performed using a mann-whitney u test or chi . logistic regression analysis was performed to determine possible risk factors for poor outcome. results: ccecai scores, age, and ica concentrations between the two groups were not significantly different. there was a significantly greater number of dogs treated with food alone in the group with good outcome ( / ) than in the poor outcome group ( / , p = . ). furthermore, median serum (oh)d concentration was significantly lower in patients with poor outcomes ( . nmol/l, range - nmol/l) compared to patients with good outcomes ( nmol/l, range - nmol/l, p = . ). using logistical regression, (oh)d serum concentration was a statistically significant factor for poor outcome (p = . ), with an increase of (oh)d serum concentration reducing the odds of having a poor outcome (odds ratio = . , % ci: . - . ). further studies are required to investigate vitamin d as a potential adjuvant therapeutic agent in ple patients. disclosures: no disclosures to report. campylobacter jejuni (cj), c. upsaliensis (cu) and c. helveticus (ch) are commonly isolated from dog and cat faeces but association with clinical signs is discordant or lacking. cj is a recognized human pathogen, cu is considered an "emerging" pathogen and ch is not considered pathogenic despite a high level of genetic similarity. recently, the greater wax moth, galleria mellonella, was described as an animal model of disease; these invertebrates have a high degree of functional and structural homology with the mammalian innate immune system. this study aimed to evaluate the pathogenic potential of cj, cu and ch using the galleria mellonella larvae model. twelve isolates of cj, of cu and of ch from dogs and cats were used for the inoculation of larvae. inocula were prepared by suspending isolates in phosphate-buffered saline (pbs) from which three -fold dilutions were made. each dilution was tested in duplicate sets of larvae. each larva was injected with - ll into the haemocoel via the last left pro-leg using g insulin syringes. controls consisted of pbs inoculated larvae and un-inoculated larvae. survival of larvae at °c in a h enriched microaerobic atmosphere was monitored for days postinjection. one subset of isolates was grown in mueller-hinton broth and used for the preparation of secretory products, and another grown on blood-agar and suspended in pbs for heat inactivation of minutes at °c for testing of whole-cell lysates and heat-stable insoluble and soluble components. the overall median survival of larvae was % with cj [iqr - ], % with cu [iqr - ], % with ch [iqr - ], % with pbs [iqr - ] and % for un-inoculated larvae [iqr - ]. a dose-dependent association was evident for each species with larval survival being similar between a low bacterial dose and pbs. larval survival presented a consistent pattern between species for medium and high bacterial loads; cj had a higher and faster larval death rate than cu and ch (p < . ), but no difference was observed between cu and ch (p = . ). there were no significant differences between species in any of the assays with secretory products, inactivated cells and soluble/insoluble cellular components. the observations within this invertebrate disease model support a varying pathogenic potential between the species studied that appears related to the (patho)biology of the species rather than their cellular components or metabolic products. the invertebrate animal model is promising in comparative pathogenicity studies. disclosures: no disclosures to report. a. grellet , s. dubois , a. feugier , c. girardet , s. magnan , v. andr eo , g. trombini , c.a. boehringer , j. suchodolski , j. steiner . royal canin, aimargues, france, veterinary department of the french army health service, suippes, france, gastrointestinal laboratory, texas a&m university, college station, tx, usa acute stress from medium or high duration high-intensity exercise has been reported to be associated with an increase in serum c-reactive protein (crp) concentrations, an important acute-phase reactant in dogs. however, the effect of exercise on fecal s a concentration, a biomarker of intestinal inflammation has not previously been evaluated in dogs. the goal of this study was to determine if moderate intensity short duration exercise causes an increase in crp and/or s a concentrations in dogs, potentially leading to misinterpretation of their results. thirty-seven adult military working dogs (german and belgian shepherd dogs; males; mean age = years [ . - . ]) were included in the study. fecal quality, fecal s a , and serum crp concentrations were evaluated just before and after standardized exercise ( minutes of bikejoring at a speed of km/h). fecal quality was evaluated based on a -point scale (from : liquid to : dry and hard feces). fecal s a and crp concentrations were assayed with previously validated elisa tests. data were analyzed with an anova test for repeated measurements (sas software). results are presented as medians and ranges. serum crp concentrations increased significantly after exercise (median before and after excercise mg/l [ - ] and mg/l [ - ] (p = . ). also, fecal s a concentrations were significantly higher after exercise compared with baseline concentrations ( ng/g [ - ] vs. ng/g [ - ], p = . ). no significant effect of exercise on fecal score was observed ( [ . - . ] before and after the exercise; p = . ). our study demonstrates that a moderate-intensity, short-duration effort performed by healthy army dogs causes significant increases in fecal s a and serum crp concentrations, as compared with baseline values, but within the respective reference intervals. therefore, a moderate exercise does not present a confounding variable in the interpretation of fecal s a or serum crp concentrations in healthy dogs. disclosures: this study was performed thanks the financial support of royal canin. imaging is an integral part of the work-up of canine gastrointestinal (gi) disease. radiography and ultrasonography are noninvasive modalities that can evaluate the bowel, but many findings lack desirable sensitivity or specificity. endoscopy directly visualizes gi mucosa, but is limited by the length of the endoscope and the need for general anesthesia, advanced training and expensive equipment. ambulatory light-based imaging (ali) is a new imaging modality that utilizes high-resolution cameras, a microprocessor, and led illumination to non-invasively visualize the gastrointestinal mucosa. ali is performed by oral administration of a fully automated device the size of a pill that is propelled by peristalsis. the aim of this study was to analyze image quality and gi transit times in a series of five client owned dogs undergoing ali. dogs were food-restricted for hour before and hour after capsule administration. capsules were retrieved and images were downloaded and analyzed. video clips of frames duration were obtained from the stomach; proximal, middle and distal small intestine; and proximal colon for assessment of image quality. internists rated the images on a scale of - ( = poor, = ex-cellent) based on clarity and resolution of images, and obscuration of the mucosa by fluid, bubbles or debris. scores for each region were compared using general estimating equation analysis. gastric and small intestine transit time were calculated based on visualization of passage of the capsule from the stomach to duodenum, and ileum to colon. clinical analysis of the entire video was performed by one of the authors. ali was successfully performed in / patients, with no adverse effects. average study duration was . ae . hour and mean image acquisition count was . ae . . gastric and small intestinal transit times were . ae . minute and . ae . minute, respectively. median (range) image quality scores were ( - ), ( - ) and ( - ), for the stomach, si and colon, respectively. image quality scores were significantly higher in the stomach and si than in the colon (p < . ). visualized lesions were consistent with gi ulcers ( dogs), inflammatory bowel disease ( dog), and bilious vomiting syndrome ( dog). one dog receiving chronic nsaids had a normal study. ambulatory light-based imaging resulted in good to excellent image quality throughout most of the gi tract. bowel preparation should be considered to enhance visualization of the colon. ali was safe and easy to perform in ambulatory dogs, and should therefore be considered in the work-up of canine gi disease. disclosures: drs. hardy and solomon are employed by infiniti medical. escg-p- establishment of a severity scoring system for outcome prediction in dogs with pancre-atitis. p.c. liu , f.r. wu , y.j. lee , b.l. su . graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, national taiwan university veterinary hospital, national taiwan university, taipei, taiwan, institute of veterinary clinical sciences, national taiwan university, taipei, taiwan canine pancreatitis is the most common exocrine pancreatic disorder. the prognosis of canine pancreatitis is variably and no logistic regression constructed severity scoring systems are available. four hundred and thirty nine dogs diagnosed as pancreatitis with acute onset of compatible clinical signs, a positive snap Ò cpl[trademark] test, and/or associated abdominal ultrasonographic abnormalities between january and december were presented at national taiwan university veterinary hospital (ntuvh). one hundred and three dogs hospitalized with complete medical therapy and outcomes were selected for further analysis. the dogs were divided into survival (n = ) and non-survival (n = ) groups. forty-seven parameters including signalment, clinical signs, physical examinations, clinicopathological examination, complications and concurrent diseases were analyzed and compared between the two groups. logistic regression analyses were performed in this study. variables with p ≤ . were considered for further analyses. the mortality in this study was . %. age, heart rate, respiratory rate, white blood cell count, albumin, bun, creatinine, potassium, presence of systemic inflammatory response syndrome (sirs) and presence of oliguria or anuria were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each dog by summing all weighting factors. the total scores of each dog ranged from to . the severity scores in this study achieved an area under the receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with a sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was a significant difference (p < . ) between the two groups seperated by the cut-off point. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in dogs with pancreatitis. disclosures: no disclosures to report. glucocorticoids (gcs) are known for their anti-inflammatory and immunmodulatory properties and are therefore often used in the therapy of canine inflammatory bowel disease (ibd). it was recently shown that endogenous gcs are also produced in the intestinal epithelium of men and mice and influence the gastrointestinal immune system in case of inflammatory or neoplastic conditions. thus, the aim of this project was to prove that gcs can be produced or metabolized in the canine intestinal epithelium. five healthy beagle dogs were included into this prospective study. all dogs were clinically examined, given a clinical score using the canine ibd activity index (cibdai) scoring system, also gastrointestinal endoscopy was performed. mucosal biopsy specimens from duodenum were examined histologically from a board certified pathologist using the wsava grading. biopsy incubation of - endoscopical mucosal biopsies in tissue culture medium with h-labeled progesterone in the absence of any stimulation was performed. the mean age of the included dogs was . + . years, the mean weight was . + . kg. all beagle dogs had a mean clinical score of + . the mean wsava scoring was + . . after hours, supernatant was harvested and radioactive progesterone metabolites formed were detected using high performance liquid chromatography plus liquid scintillation counting. in all dogs the h-progesterone was metabolized into various steroid species, nevertheless a local production of cortisol could not be proven. in summary, it could be shown that precursors of gcs can be metabolized by healthy canine intestinal mucosal tissue. disclosures: no disclosures to report. we studied the relationship between pancreatitis and cardiac injury in dogs and cats. previously, we validated a cardiac troponin i (ctni; vet j : - , ) assay for sensitive and specific detection of cardiac injury in domestic animals. we found various non-cardiac diseases of dogs and cats were associated with cardiac injury detected by serum cardiac troponin i, including some cases of pancreatitis. also, we validated the dggr-lipase assay for cost-effective, sensitive and specific detection of pancreatitis in dogs and cats (vet clin path :e - , ; :e - , ). herein, we tested the hypothesis that pancreatitis was associated with cardiac injury. ctni was measured by advia centaur tni-ultra assay; dggr-lipase by the randox colourimetric assay. we retrospectively analysed data from dogs and cats admitted to ucd veterinary hospital in which both ctn and lipase had been measured. upper limit of reference range for lipase in dogs is u/l; we consider - indicative of mild pancreatitis, - moderate, and > as marked. upper limit of reference range for lipase in cats is . reference range for ctni is < . ug/l for dogs and cats. we consider . - . indicative of mild cardiac injury, . - as moderate, and > . as marked. dogs and cats had both lipase and ctni measured. seventy-eight dogs had normal troponin; had normal lipase and had normal lipase and normal ctni. dogs ( %) had pancreatitis as indicated by increased lipase. in ( %), pancreatitis was mild, in ( %) it was moderate, and in ( %) it was marked. sixty-seven of dogs had increased ctni: mild in ( %), moderate in ( %), and marked in ( %). cardiac injury in dogs with pancreatitis was absent in %, mild in %, moderate in %, and marked in %. of cats had normal ctn; had normal lipase. of cats had pancreatitis, severely in . lipase and ctni was correlated (r = . ) for dogs and cats. we conclude that both pancreatitis and cardiac injury, as indicated by high-sensitivity and high-specificity assays randox-dggr-lipase and centaur-ctni, respectively, are not uncommon in veterinary hospital cases. we confirm and extend our previous work. pancreatitis in dogs and cats is typically associated with cardiac injury. severities of pancreatitis and cardiac injury are correlated. for~ % of dogs and cats with pancreatitis, cardiac injury is moderate to marked. disclosures: no disclosures to report. intracellular colonization may serve as a protected niche where helicobacter spporganisms evade effective treatment, contributing to recolonization. confocal endomicroscopy (cem) is an endoscopic modality allowing in vivo gastrointestinal imaging at high resolution; and has aided real-time identification of helicobacter pylori and intracellular and mucosally associated bacterial. in dogs, non-helicobacter pylori-helicobacter (nhph) are described intracellularly. the objective of this study was to determine the utility of cem to identify nhph in dogs compared with other diagnostic modalities; and to assess its ability to identify intracellular organisms. fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by cem using topical acriflavine. images were obtained using cem at a minimum of five sites within the stomach. endoscopic pinch biopsies were obtained for histopathology, polymerase chain reaction (pcr) and fluorescence in situ hybridisation (fish). methodologies were compared for their sensitivity in detecting the presence and distribution of nhph and their ability to identify intracellular organisms. cem provided high quality images allowing in vivo identification ofnhph in dogs, as did fish post-procedure analysis. standard histopathology identified nhph in only . nhph were identified within the superficial gastric mucus, and gastric pits. distribution throughout the stomach was diffuse and multi-focal. cem findings correlated with fish and pcr, however only fish enabled identification of intracellular nhph which were present in of dogs. cem provides in vivo histology images and is capable of identifying nhph during gastroscopy, but is unable to identify intracellular organisms using the current fluorophore protocol. nhph in the canine stomach are commonly identified intracellularly. disclosures: dr sharman has shares in optiscan imaging pty ltd. chronic enteropathies (ce) and exocrine pancreatic insufficiency (epi) can both cause hypocobalaminemia in cats. current supplementation protocols for cobalamin in cats call for repeated parenteral injections. in humans, several studies have reported equal efficacy of oral administration of cobalamin. there is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. the purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies. a computerized database search for cats treated at evidensia specialist animal hospital, helsingborg, sweden during - was performed. inclusion criteria were cats with symptoms of ce, an initial serum cobalamin concentration below pmol/l (reference interval: - pmol/l) and daily oral treatment with cyanocobalamin ( mg/tablet; ⅛-¼ tablet/cat daily). follow-up serum cobalamin concentration was measured - days after initiation of daily oral cobalamin supplementation. thirteen cats aged - years (median ) of different breeds met the inclusion criteria. presenting complaints included vomiting ( / ), anorexia ( / ), diarrhea ( / ), weight loss ( / ), and lethargy ( / ). increased pancreas specific lipase (spec fpl Ò ) serum concentrations were reported in / cats and / had increased serum alanine transaminase activity. feline serum trypsin like immunoreactivity (ftli) was determined in / cats revealing results within the reference interval. all cats had an abdominal ultrasound, / had changes related to the gastrointestinal tract such as mild-moderate thickening of the small intestinal wall, thickening of the muscularis layer, poor definition of intestinal wall layers, and/or enlargement of the mesenterial lymph nodes, histopathology was performed in / cats, revealing small intestinal inflammation in five cats and small intestinal lymphoma in one. serum cobalamin increased in all cats with treatment. the concentration difference ranged from to pmol/l (mean: pmol/l). mean (aestandard deviation) serum cobalamin concentrations were (ae ) pmol/l before and (ae ) pmol/l after supplementation. this difference was statistically significant (p < . , paired t-test). our results suggest that oral cobalamin supplementation is effective in normalizing serum cobalamin concentrations in cats with various enteropathies. prospective studies are warranted comparing cellular cobalamin status in cats being treated with parenteral or oral cobalamin supplementation. disclosures: no disclosures to report. pulmonary thromboembolism (pte) is observed in dogs with idiopathic-inflammatory-bowel disease (ibd) and particularly with protein-losing enteropathy (ple). hypercoagulability has been attributed to antithrombin (at) loss although the pathogenesis is likely to be more complex. in humans, where venous thromboembolism (te) is a wellrecognised complication of crohn's disease and ulcerative colitis, the pathogenesis of te is still not completely understood. derangements in procoagulant and anticoagulant factors have been demonstrated, including increased circulating procoagulant microparticles (mps). the aim of this pilot study was to evaluate mp-procoagulant activity in the plasma of dogs with ibd and ple using a functional elisa assay (zymuphen-mp-activity, aniara). we hypothesised that all dogs with ple and a subset of dogs with ibd but without ple would have increased levels of circulating mps. the study group consisted of dogs with ibd, including with ple. diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. ple was defined as ibd plus hypoproteinaemia (serum total protein < g/l) and hypoalbuminaemia (serum albumin < g/l). pte was diagnosed in one dog with ple, and suspected in a second. a control group comprised healthy dogs undergoing blood sampling for reasons unrelated to the study including blood donor screening (n = ) and health assessment (n = ). dogs were considered healthy based on owner evaluation, physical examination, haematology and serum biochemistry. median mp procoagulant activity in dogs with ibd was . nm (range . - . ) compared with . nm (range . - . ) in the control group. median mp activity in ple dogs was . nm (range . - . ) compared with . nm (range . - . ) in non-ple ibd dogs. using kruskal-wallis test for nonparametric data and dunn's multiple comparisons test the groups were not statistically different. interestingly, mp-procoagulant activity value in the dog with documented pte was . nm; in the dog with high clinical suspicion for pte, mp-procoagulant activity was . nm. the highest mp-procoagulant activity was detected in a healthy control dog, raising concerns for pre-analytical or sampling error. removing this measurement had no impact on statistical analysis, which remained nonsignificant. mp-procoagulant activity > nm is considered clinically relevant in humans. employing a similar cut-off, / of controls, / of ibd and / of ple group would be defined as having increased levels of circulating mps. further studies are required to fully evaluate the clinical relevance and diagnostic potential of mp evaluation. disclosures: no disclosures to report. the intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (ce) in dogs. while imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with ce involving the ileum and colon. the aim of the present study was to use fluorescence in situ hybridization (fish) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with ce and controls. tissue sections from the ileum and colon from dogs with inflammatory bowel disease (ibd), dogs with granulomatous colitis (gc), dogs with intestinal neoplasia, and controls were studied by fish targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. the numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (cib-dai score, histopathology) indices. the ileal and colonic mucosa of healthy dogs and dogs with ce was predominantly colonized by bacteria localized to free and adherent mucus compartments. dogs with ce harbored more (p < . ) mucosal bacteria belonging to the clostridium-coccoides/eubacterium rectale group, bacteroides, enterobacteriaceae, and escherichia coli versus controls. within the ce group, ibd dogs had increased (p < . ) enterobacteriaceae and e. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. bacterial invasion with e. coli was present in the ileal and colonic mucosa of dogs with gc (p < . ). dogs with intestinal neoplasia had increased (p < . ) adherent (total bacteria, enterobacteriaceae, e. coli) and invasive (enterobacteriaceae, e. coli, and bacteroides) bacteria in biopsy specimens versus all other groups. increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (cibdai score) in ibd dogs (p < . ). these results indicate that histopathologic lesions of canine ce are associated with different populations in ileal and colonic mucosal microbiota. these spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine ce. disclosures: no disclosures to report. abdominal girth is used as an indicator of human adiposity, with such measurements being made by tape measure. given concerns in precision and accuracy of repeat measurements, some tape measure designs have inbuilt mechanisms to improve consistency. although body condition scoring is the most common method of assessing adiposity in dogs, zoometric systems have also been developed requiring the use of a tape measure. however, the precision and accuracy of such zoometric measurements are not known. the aim of this study was to determine the precision and accuracy of different types of tape measure for a variety of dimensional measurements. a variety of length (head, forelimb, hindlimb) and circumferential (neck, thorax, and abdomen) were made using three different tape measures, two of which were designed to improve precision (standard tape; myotape tm and gulick ii tm ). to assess intra-operator variability, measurements were taken for consecutive days from healthy dogs; to assess inter-operator variability, operators independently took measurements from a group of dogs of various breeds and sizes. for intra-operator comparisons, precision was good overall (coefficient of variation [cv] ≤ % for all measurements). for interoperator comparisons, precision was more variable and, although reasonable on average (mean cv - %), it varied depending upon tape measure type (p = . ; greatest for standard tape measure, least for gulich ii tm ), and could be highly variable for some measurements in individuals dogs (maximum cv % for head measurements with standard tape measure). significant differences also existed in the absolute results of circumferential measurements taken by the different tape measure types (neck p = . ; thorax p < . ; abdomen p < . ). finally, significant operator differences were also evident for some measurements (head p = . ; hindlimb p = . ), but not for others (forelimb p = . ; neck p = . ; thorax p = . ; abdomen p = . ). in summary, although precision for individual operators making zoometric measurements is good, significant inter-operator and tape type differences exist. these results have implications for systems using a range of zoometric measures to assess adiposity. in order to ensure precision and accuracy, it is recommended that the same operator take all measurements with the same type of tape. disclosures: the study conducted was not supported by a research grant. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. koizumi , m. noda , c. shimokawa , a. kusumi , t. kobayashi , t. watari , k. otsuji . teikyo university of science, tokyo, japan, grace animal hospital, tokyo, japan, nihon university, fujisawa, japan body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. however, this method is subjective due to its sensory evaluation. therefore, the improvement of the precision of the bcs diagnosis is expected. our previous study has shown that the bcs model that we created improved the precision of the bcs diagnosis ( ). however, a palpation site was not identified. a palpation site must be the site where thickness of subcutaneous fat is able to capture for measuring animal's obesity status. therefore the objective of this study was to find a remarkable body site of the changes with obesity status using ultrasonic diagnostic equipment. nine dogs which varied in the percent of body fat were used in this study. the percent of body fat was measured by a body fat analyzer for dog (kao). the image analysis of a palpation site was evaluated using echo, xario ssa- a (toshiba) which attached to a linear probe. the measurement points were , and o'clock positions on the ribs of t , t and t . the distance (d) from skin surface to the rib was measured in the echogram. the distance (l) from scapula to ilium was measured to offset the difference in physique by dog breeds. the d/l was used to compare relative value of the quantity of fat at each measurement point. bcs of dogs which used in this study were from bcs of to bcs of . there were no dogs in bcs of and bcs of . a statistically significant correlation was found between bcs and d/l value. the d/l value increased in order of t , t and t in bcs of and . this suggests that the thickness of subcutaneous fat in the chest is thicker at the head side than the tail side. also, as for the d/ l value from back to abdomen, the highest value was found at the position of : and : . this tendency was the most remarkable in bcs of but no difference in the d/l value was recognized in the dogs in bcs of . in conclusion, the position of : or : on the t is the suitable palpation point at the chest. ( ) k. otsuji, m. suzuki, n. furukawa, n. kobayashi, a. koizumi, a. kusumi, t. kobayashi efficacy of the body condition score (bcs) model in the bcs diagnosis wsava proceeding p , disclosures: no disclosures to report. body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. bcs has been recognized as one of the screen method of nutrition diagnosis by american animal hospital association in . however, this method is subjective due to its sensory evaluation. therefore we made a bcs model to increase the precision of the bcs diagnosis and have shown the efficacy of the bcs model ( ). however, the prototype model which we have reported before tended to have higher bcs than a target bcs. therefore, we improved the bcs model in this study. sixty seven dogs which varied in the bcs were used in this study. body fat percentage was measured by using a body fat analyzer for dogs (kao healthlab bif- ). the bcs model was improved by using several rubber sheets. relative hardness of stacking rubber sheets in each bcs was measured by durometer mj-dua-c (satotec tokyo, japan). bcs diagnosis of dogs was performed by pet owner by using the bcs model. bcs of represents the most hard in the bcs model and the hardness decreased linearly and it was the lowest in of bcs. these values were as expected. high correlation was recognized between bcs and body fat percentage. these results suggested the efficacy of bcs model. however, the body fat percentage in the dogs diagnosed as bcs of was higher than body fat percentage which has been reported in the previous paper. there were no dogs with the body fat percentage < % which were diagnosed as bcs of . we need more study in future to make clear the difference of body fat percentage between our data and date of the previous research. the completion of this bcs model will help provide the precision of nutritional diagnosis in dogs. ( in humans the metabolic syndrome (ms) is a well-recognised and extensively studied entity that comprises obesity, hypertension, dyslipidaemia, and glucose intolerance. it is associated with an increased risk of cardiovascular diseases and diabetes. recently, human ms criteria were adapted for dogs to define the condition of obesity-related metabolic dysfunction (ormd). it was observed that ormd was associated with increased circulating insulin and decreased adiponectin concentrations, suggesting that in dogs, as in humans, there are links between obesity, ormd, and associated diseases, although pathogenetic mechanisms and health significance for dogs remain unknown. the main aim of the present study was to compare plasma proteomes of obese dogs with and without ormd, so as to investigate the mechanisms associated with canine ormd and their possible significance in the health status. eight obese dogs referred for weight management at the royal canin weight management clinic, university of liverpool participated in the study. clinical assessments included physical examination, body condition scoring, blood pressure measurement and routine clinicopathological analysis. surplus plasma was used in proteomic analysis. samples were first treated with proteominer for thedepletion of high-abundance proteins and subsequently analysed by using -de dige methodology. of the eight dogs in the study, dogs had ormd and dogs did not. image analysis and further statistical analysis allowed identification of spots with differential expression concentration between dogs with and without ormd. among the spots, were over-expressed and were down-expressed in dogs with ormd than in dogs that did not presented ormd. although the results of the present study are preliminary and still the identification of the spots is up to be performed, the observed datareveal that dogs with ormd present alterations in their plasma proteomes that could be responsible for the development of ormd-related pathologies. disclosures: the study was funded by waltham. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. vb is an employee of royal canin and pjm is an employee of wal-tham. the aim of the study was to assess the diagnostic value and the discrimination potential between the normal heart size and microcardia or cardiomegaly of a method which calculates the cardiothoracic ratio (ctr) using area measurement, compared to the vertebral heart scale method (vhs) used as reference for the cardiac size, in dogs. one hundred-nine dog x-rays were accepted into study. the patients belonged to small and medium size breeds, fourty-seven were males and sixty-two females with age between and years. the analogic x-rays were scanned and transferred to a computer where the vhs and ctr was calculated for each patient with a commercial software and the data was collected and processed in a statystical analysis software. the patients were distributed into groups by respiratory phase and heart size. there was a low correlation between the vhs and ctr (r = . ), but statistically significant (p? . ). a good correlation was obtained between vhs and ctr in microcardia, normal heart size and cardiomegaly groups (p < . ). furthermore, between the ctr in dogs with microcardia and those with normal cardiac size, as well as between ctr in dogs with normal cardiac size and those with cardiomegaly, a significantly statistic difference (p < . ), respectively (p < . ), was obtained. among the groups distributed by respiratory phase and vhs, a statistically significant difference was obtained only between normal cardiac size and cardiomegaly during inspiratory phase groups (p > . ). for the x-rays taken in inspiratory phase, a cutoff of . had a sensitivity of % and a specificity of % for diagnosing cardiomegaly. the ctr can be considered a valid method being able to discriminate between the patients with microcardia and cardiomegaly from those with normal heart size. moreover, it was found that a ctr over the cutoff of . , mesured during inspiratory phase is a good predictor for cardiomegaly. key words: cardiac, cardio-thoracic ratio, dog, x-ray. disclosures: no disclosures to report. the canine cardiac conduction system is modified by anatomical and functional adaptations of the maternal heart during gestation. however, it is not clear if these changes persist or are modified after parturition. therefore, the aim of this study was to describe canine electrocardiographic features during the course of normal puerperium. twenty healthy pure-bred, - ( . ae . ) year-old, weighing . - kg ( . ae . ) bitches were included in this study. all the animals whelped healthy puppies at term which were weaned on day after parturition (day ). all the dogs were electrochardiographically evaluated on days À , , , , , , and . mean electrical axis (mea; degrees), p wave amplitude (pa; mv) and duration (pd; ms), p-r interval (pr; ms), qrs complex amplitude (qrsa; mv) and duration (qrsd; ms), q-t interval (qt; ms), and s-t segment (st; mv) were calculated at mm/s of velocity. the rr interval immediately preceding each complex was recorded and qt interval was corrected (qtc) by van de water formula [qtc = qt- . (rr- )]. later, lead ii was recorded at mm/s to analyze heart rate (hr; bpm) and cardiac rhythm (cr; normal sinus rhythm or sinus arrythmia). values of hr, mea, pa, pd, pr, qrsa, qrsd, qt, rr and qtc were analyzed by anova for repeated measures followed by tukey test. cardiac rhythm was analyzed by chi square test (spss . , spss inc. chicago, il, usa). p < . was considered significant. during the study period, hr (p < . ) and qtc (p < . ) progressively decreased, while rr (p < . ) and pa increased (p < . ). qrs complex amplitude diminished in the second week after parturition and then increased during the following weeks (p < . ). mean electrical axis shifted to the right during this period (p < . ). on day À , most of the bitches presented normal sinus rhythm in contrast with day , in which most of the bitches presented sinus arrhythmia (p < . ). from day onward, all the bitches showed sinus arrhythmia. p wave duration, pr, qrsd, qt and st remained unchanged during puerperium. it is concluded that most electrophysiological adaptive changes of canine gestation reverted during normal puerperium. the present study contributes to the understanding of canine cardiac physiology during this reproductive stage. disclosures: no disclosures to report. esvc-p- echocardiographic assessment of pregnant queens. p.g. blanco, r. rodr ıguez, a. carranza, a. rube, r. vercellini, p.r. batista, m. t ortora, c. gobello. national university of la plata, la plata, argentina cardiovascular adaptation during gestation guarantees an appropriate development of the fetuses and maternal cardiovascular maladaptation is highly correlated with adverse pregnancy outcome. while, the hemodynamic changes occurring during canine pregnancy have been described there is scarce information concerning maternal cardiac variations during feline gestation. thus, the aim of this study was to describe cardiac morphology and systolic function variations during normal feline pregnancy. eighteen pregnant queens were echocardiographically evaluated (toshiba nemio xg, japan, mhz transducer) every days from day (defined as day of mating) to parturition. left ventricular dimensions were measured in the short axis view, during mmode tracing. shortening fraction was calculated as (lvdd À lvds)/lvdd to assess systolic function. stroke volume (ml) was calculated as the product of the velocity time integral (measured by pulsed-wave doppler) and the cross-sectional area of the aorta. cardiac output (l/minute) was calculated as the product of stroke volume and heart rate (bpm) derived from electrocardiographic monitoring. uterine artery resistance index (ri) was obtained by doppler ultrasound. all the parameters were analyzed by repeated measures anova. all the queens delivered healthy kittens at term. throughout the study period, interventricular septum in diastole (p < . ) and systole (p < . ) and left ventricular diameter in diastole (p < . ) augmented during gestation. shortening fraction (p < . ), cardiac output (p < . ) and maternal heart rate (p < . ) also increased up to parturition. conversely, uterine artery resistance index decreased in the same period (p < . ). it is concluded that cardiac structure and function varied during normal pregnancy in these queens. cardiac eccentric hypertrophy, systolic function and cardiac output increases appear to be the consequences of the hemodynamic modifications occurring during pregnancy. the assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline reproduction. disclosures: no disclosures to report. tricuspid annular plane systolic excursion (tapse) is an echocardiographic measure that allows to assess right ventricular systolic function. it has been described reference values for tapse in normal adult dogs, but there is no reference to influence of age in tapse in dogs. this influence has been reported in humans. thus, the goal of this study is to determine the reproducibility of the measure tapse in normal dogs and to determine the relationship between tapse and age in healthy beagle dogs. tapse was measured from an m-mode recording of the lateral aspect of the tricuspid valve annulus obtained through a left parasternal apical -chamber view. tapse values were averaged from measurements on consecutive beats during sinus rhythm. the measurements were recorded by two different persons (c-v, a.; m-m f.) with different grade of experience in canine echocardiography studies. all patients had a complete two-dimensional and doppler study using an envisor chd (philips Ò ) ultrasound system. twenty-three healthy beagles were used. the study was approved by the ethical committee of veterinary medicine service of las palmas de gran canaria university (spain) and it was carried out in accordance with the current european legislation on animal protection. these dogs were divided in three different groups according to age: group included twelve dogs under years, group included three dogs between and years, group included eight dogs older than years. we analyzed differences between groups using non-parametric (kruskal wallis and wilcoxon scores [rank sums]) test. there were no differences with respect to sex. dogs in group presented higher tapse values than group or ( . ae . cm vs . ae . cm vs . ae . cm; p < . ). statistic intra-observer and inter-observer agreement using the intraclass correlation coefficient was . (p < . ). this study showed that tapse measurement is easily obtainable with a standard echocardiography system, and has adequate interobserver agreement. this study showed higher values of tapse in normal young dogs with respect to older dogs. these results are similar to the results obtained in humans, and could reflect a less effective right ventricle with age. the values presented should be taken with caution due to the relatively small number of patients included. it may also be necessary to validate results in future studies with a second independent sample of dogs of other races. disclosures: no disclosures to report. tetralogy of fallot (tof) is a congenital heart disease characterized by abnormalities, i.e., pulmonic stenosis, ventricular septal defect (vsd), aortic overriding and secondary right ventricular hypertrophy, caused by anterior deviation and abnormal septation of the conal septum during the embryonic period. few studies have reported the hemodynamic consequences and clinical outcome of tof in small animals. the objective of this retrospective study was therefore to document the epidemiological, clinical, echo-doppler findings, and survival, in a canine and feline population with tof. the case records of animals diagnosed with tof by combined use of echocardiography and doppler examination were reviewed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tetralogy of fallot was identified in animals ( dogs, cats). the most commonly represented breeds were terriers for dogs ( / , . %), and domestic shorthair for cats ( / , . %). most included animals ( / , . %) were clinically affected at the time of diagnosis. pulmonic stenosis was characterized by a variable systolic doppler-derived pressure gradient both in dogs (median [range] mmhg ) and cats ( mmhg ), and associated with hypoplasia of the pulmonary trunk in one third of the cases ( . %). most vsd were large, with a median vsd: aorta ratio of . [ . - . ] in dogs and . [ . - . ] in cats. median age at death from cardiac cause was . months [ . - . ] without significant difference between dogs and cats (p = . ). these results suggest that in both cats and dogs tof-related death occurs predominantly in young adult animals with major hemodynamic consequences at the time of diagnosis. disclosures: no disclosures to report. the aim of this study was to assess whether and how radiographic and echocardiographic cardiovascular variables differ across age bands of healthy cats. a cohort of clinically healthy cats were categorized into three groups: adolescent-adult ( . - years; n = ), middle-aged ( - years; n = ), and geriatric ( - years; n = ). all cats underwent a full physical examination, a complete blood count, routine biochemical profile, a baseline serum total thyroxine concentration, auscultation, noninvasive blood pressure measurements, thoracic radiography, electrocardiography, and echocardiography. cats with hypertension, hyperthyroidism, cardiac, or renal disease were excluded from the study. body weight, body condition score, systolic blood pressure, heart rate, and all echocardiographic indices were similar across the three groups. the mean (aestandard deviation [sd] ) vertebral heart scale (vhs) value obtained for the geriatric group ( . ae . ) was significantly greater than that obtained for the adolescent-adult group ( . ae . ; p = . ). the mean ratio of the distance between the cardiac base and dorsal sternum to thoracic cavity height at the point of the cardiac base was significantly less in the middle-aged ( . ae . ) and geriatric ( . ae . ) groups than in the adolescent-adult group ( . ae . ; both p < . ). the mean angle between the cardiac long axis and the body axis was significantly smaller in middle-aged ( . ae . °) and geriatric cats ( . ae . °) than in adolescent-adult cats ( . ae . °; both p < . ). the mean angle between the cardiac long axis and the sternum of middle-aged ( . ae . °) and geriatric cats ( . ae . °) was significantly smaller than that in adolescent-adult cats ( . ae . °; p = . and p = . , respectively). additionally, the degree of undulation of the thoracic aorta correlated positively with age (r = . , p = . ). these findings suggest that differences in the horizontal alignment of the heart, thoracic-aorta undulation, and vhs in healthy geriatric cats, relative to observations in younger cats, can be considered to be age-related. disclosures: no disclosures to report. the aim of this study was to investigate the presence of pulmonary hypertension (ph) in young cats affected by single or mixed lungworm infections. twenty-three cats infected with lungworms were examined at the veterinary teaching hospital of teramo, italy, in - . animals underwent to a complete physical examination and to two-or three-views radiographic analysis of the thorax. a minimum database (i.e. cbc, serum biochemistry, serology for fiv antibody and felv antigen) was obtained for each patient. nine cats were excluded for concomitant diseases, while cats were included in the study. microscopic identification of parasites was confirmed by molecular tests and all cats received an anthelmintic treatment. a single infection by aelurostrongylus abstrusus was diagnosed in eleven cats, while three cats had a troglostrongylus brevior infection either alone or in combination with a. abstrusus. transthoracic echocardiography was performed using an ultrasound unit with a mhz phased array transducer. no structural abnormalities of the tricuspid valve and sign of pulmonary stenosis were detected. the two-dimensional and m-mode echocardiography showed a cardiac involvement in three cats. one cat, infected by a. abstrusus and t. brevior showed a mild systolic tricuspid regurgitant jet with color doppler of . m/s, while another a. abstrusus-infected cat, had mild tr of . m/s with a mean paps of mmhg which resolved within weeks after therapy. one cat diagnosed with troglostrongylosis, showed a marked right-sided cardiac enlargement of mm, and a large systolic tricuspid regurgitant jet with a tr peak velocity of . m/s recorded at continuous-wave doppler via a color doppler echocardiography. the minimum pressure difference between the right ventricle and the right atrium was estimated mmhg and the paps was at least mmhg. the echocardiographic and doppler evidence of mild ph persisted at further examination performed until months after diagnosis. ph is rare in cats, despite cases of reversible ph are known in cat aelurostrongylosis. in this study the first case of irreversible ph infection in a cat affected by t. brevior is presented and this finding further supports the high pathogenicity of troglostrongylosis, especially in young patients. in cats with lungworm infection, possible cardiovascular complications must be taken into account and these infections should be always considered in the differential diagnosis in cats with cardiorespiratory signs. disclosures: no disclosures to report. ventricular function by conventional echocardiography and speckle tracking imag-ing although uncommonly assessed in veterinary cardiology, a right ventricular (rv) function has been shown to be an important prognostic determinant of many congenital and acquired heart diseases in human patients. our group has already demonstrated that two-dimensional ( d) color tissue doppler imaging provides a non-invasive evaluation of systolic and diastolic rv function in the awake dog with adequate repeatability and reproducibility. b however, other noninvasive ultrasound imaging variables reflecting rv function need to be further investigated, particularly in correlation with pulmonary arterial pressure (pap) values and left ventricular (lv) function. the aim of this prospective study was therefore to assess several indices of systolic and diastolic rv function using conventional echocardiography and speckle tracking echocardiography (ste) in healthy awake dogs of different breeds with documented systolic pap (spap) and lv function (lv ejection fraction and global lv systolic strain assessed using the simpson's derived method of disks and ste, respectively). imaging rv tested variables included tricuspid annular plane systolic excursion (tapse), right fractional area change (rfac, %), ste longitudinal systolic strain of the rv free wall (rvfw, %) and of the whole rv (i.e., global rv strain, %), ste longitudinal systolic strain rate (sr, s À ) and diastolic early:late sr ratio. additionally, d-guided m-mode ventricular measurements included the end-diastolic rv:lv diameter ratio (rvdd: lvdd) and the end-systolic rvfw:lvfw ratio. correlations between imaging variables were calculated by using spearman's correlation coefficients. means of age and body weight (aesd; range) of the study population were . years (ae . ; . - . ) and . kg (ae . ; . - . ), respectively. no correlations were found between rv morphological variables (i.e., rvdd:lvdd and rvfw:lvfw ratios) and all indices of systolic and diastolic rv function. global rv strain (mean ae sd = . ae . %) and rvfw strain ( . ae . %) were positively correlated (p < . ) with rfac ( . ae . %, r = . and r = . , respectively), and negatively correlated (p < . ) with spap ( . ae . mmhg [ . - . ], r = À . and r = À . , respectively). spap was also negatively correlated with the tapse:body weight ratio and systolic sr (r = À . and À . respectively, p < . ). there was no correlation between indices of lv function and ste indices of rv function, and no correlation either between ste rv indices of systolic function and the diastolic early:late sr ratio. in conclusion, ste provides a rapid and non-invasive evaluation of rv function that may be used for clinical investigations in canine cardiology. doppler-derived +dp/dt and -dp/dt from mitralregurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phaseindexes. this study aimed to determine correlation between doppler-deriveddp/dt and other systolic and diastolic echocardiographic indexes, and if theycan be used to identify dogs with and without remodeling, with or withoutcongestive heart failure (chf) and for evaluation of chronic mitral valvedisease (cmvd) severity. fifty-seven dogs with cmvd (stages b , b , c+d) wereincluded prospectively in an observational cross-sectional clinical study anddistributed in groups regarding the presence of remodeling and chf, to evaluate+dp/dt and -dp/dt, and distributed according to tdi-diastolic pattern tocompare Àdp/dt. group c+d ( mmhg/s, p -p = - ) had +dp/dt significantly lower compared to b ( mmhg/s, p -p = - ) and b ( mmhg/s, p -p = - ) (p = . ). groupc+d also had lower Àdp/dt, compared to b ( . mmhg/s ae . and mmhg/s ae . ; p = . ). dogs with chf compared to those without chf, presented lower +dp/dt ( mmhg/s, p -p = - ; mmhg/s, p -p = - ; p = . ) and Àdp/dt ( . mmhg/ s ae . ; mmhg/s ae . ; p = . ). regardingdiastolic function, Àdp/dt was lower for the restrictive pattern group ( . mmhg/s ae . ) compared to those without diastolic dysfunction, ( mmhg ae . ), delayedrelaxation ( mmhg ae . ) and pseudonormal patterns ( mmhg ae . ) (p < . ).when +dp/dt< mmhg/s, the post-test chance for the dog with cmvd to havechf is twice the chance than not having it. for Àdp/dt< mmhg/s theposttest chance of having chf is eight times higher than not having it. in conclusion, doppler-derived +dp/dt and Àdp/dt may contribute respectively, for systolic and diastolic assessment ofdogs with cmvd. disclosures: no disclosures to report. pulmonic stenosis (ps) is one of the most common congenital heart defects seen in veterinarycardiology practice. pulmonary balloon valvuloplasty (pbv) is considered to be the treatment of choice for dogs withsevere stenosis. whether dogs with moderate stenosis benefit from pbv remains unclear, and variables such as degree of hypertrophy, valve morphology, amount of tricuspid insufficiency and presence or absence of clinical signs aregenerally used when recommendations are made to pet owners. in this study we report the effect of valve type on pbv outcome in dogs treated at three different academic speciality cardiology practices. baseline echocardiographic images were evaluated at each institution and valve morphology was classified as either type a (n = , . mmhg, range - ) or type b (n = , . mmhg, range - ) and "no" (n = , mmhg, range - ) or "yes" (n = , mmhg, range - ) for presence of pulmonary annular hypoplasia when diameter was compared to aortic annulus. twenty-four hours following pbv both type a ( mmhg, range - ) and type b ( mmhg, range - ) valves had significant reduction in gradient compared to baseline (p < . ). this reduction remained significant at days (a: mmhg, range - ; b: mmhg, range - ; p < . for both). dogs with annular hypoplasia ( mmhg, and without annular hypoplasia ( mmhg range - ) had a significant reduction in gradient hours post pbv. it remained significant at days (with annular hypoplasia: mmhg, range - ; without annular hypoplasia: mmhg, range - ; p < . for both). when comparing to baseline, considering valve type, there was no significant difference in percent reduction in gradient for type a versus type b valves at both the -hour (a: %, range - ; b: %, range - ; p = . ) and -day (a: %, range - ; b: %, range - ; p = . ) recheck evaluation time points. additionally, there was no significant difference in gradient reduction when looking only at whether or not there was annular hypoplasia at hours (yes: %, range - ; no: %, range - ; p = . ) and days (yes: %, range - ; no: %, range - ; p = . ). in conclusion, classification of dogs with ps according to valve type and annulus morphology did not help predict the -day response to pbv. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is the most common feline inherited cardiac disease and it is a major cause of morbidity and mortality. the osservatorio italiano hcm felina was formed in by a network of clinicians, geneticists and breeders, to monitor and study hcm in italian cats. since april , adult cats, belonging to various breeds, including maine coon, siberian, norwegian forest cats, ragdoll, sphynx, british sh, birmans and others have been prospectively enrolled. recheck evaluations were performed in cats. each cat underwent a clinical examination, echocardiography, and blood collection for genetic testing (when appropriate) and storage in the italian feline bio-bank. the disease status was defined by echocardiography according to established guidelines (left ventricular diastolic wall thickness < . mm = hcm negative, = . but < mm = hcm equivocal; = mm = hcm positive). the prevalence of hcm in the population was % ( cats); equivocal diagnoses were conferred on % ( cats). these prevalences did not differ between breeds. the prevalence of hcm in the italian feline population was lower compared to those reported by other investigators. evaluation of data from the entire population demonstrated that left ventricular end-diastolic wall thicknesses and aortic diameter showed a weak positive correlation with body weight (p < . , r < . for all variables), suggesting that weight-dependent limits on wall thickness should be considered in cats as is currently practiced in dogs. the lower prevalence of hcm in italian cat breeds compared with those examined elsewhere might be explained by different criteria for determining presence or absence of disease, differences in ages at which the subjects were examined, or a selection bias by breeders in presenting cats they consider "normal". disclosures: no disclosures to report. chronic mitral valve disease is by far the most common cardiovascular disease in dogs. the disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately % of cases, it's accompanied by degeneration of the tricuspid valve. it is also described in previous studies that approximately % of affected dogs also have evidence of associated pulmonary arterial hypertension. the prevalence of the disease is higher in small breed dogs (under kg), although large breeds can also be affected and it occurs more frequently in males than in females. the present study aims to characterize the disease in a population of dogs in portugal. we retrospectively reviewed the medical records of dogs presented to hospital veterin ario do porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of years. from this records, cases were identified, from which ( . %) were males and ( . %) were females. most of the dogs were mixed breed ( ) and different breeds of dogs were represented. the poodle was by far the most represented breed (n = ; . %), followed by english cocker spaniel ( . %), yorkshire terrier ( . %), boxer ( . %), epagneul breton ( . %), dalmatian ( . %), pekingese ( . %), labrador retriever ( %) and portuguese podengo ( . %). all other breeds represented . % of the population. regarding weight, . % of the dogs (n = ) weighted < kg, with a mean body weight of . kg (range . - kg). the mean age at diagnosis was . years old. we also observed that . % of the dogs (n = ) had concomitant degeneration of the tricuspid valve and . % (n = ) pulmonary arterial hypertension (ph). we categorized these dogs according to the severity of ph, in mild ph if they had a doppler echocardiography derived systolic pulmonary arterial pressure (spap) of - mm/hg, moderate ph (spap - mm/hg) and severe ph (spap > mm/hg). we found that . % (n = ) of dogs had mild ph; . % (n = ) moderate ph and . % (n = ) severe ph. as described in previous studies, the disease affects mainly males and small breed dogs, with a breed distribution that reflects the canine population in the country, including very including very popular large breed dogs in portugal, as the boxer and labrador. both the presence of concomitant tricuspid valve disease and ph had a higher prevalence in our study than previously described. disclosures: no disclosures to report. in people anemia is frequent in patients with heart failure (hf) and it is associated with poor outcomes. the most likely pathogenic factors include iron deficiency, chronic kidney disease (ckd), and cytokine production, although other factors may contribute. little is known about the prevalence of anemia in dog with cardiovascular disease. the aim of this retrospective study was to define the prevalence of anemia (hct ≤ %) in dogs with mitral valve disease (mvd) and to investigate associated risk factors (age, weight, azotemia, hf, iris/acvim class). medical records of dogs presented at the cardiology service, divet, university of milan (january -march ) were retrospectively evaluated. dogs with mvd with complete physical, thoracic and echocardiographic examinations, and serum biochemical panel, including serum creatinine (scr), were included in the study. dogs with other heart or systemic diseases, except ckd, or neoplasm were excluded. statistical analysis was performed using jmp . (sas institute). a p value < . was considered significant. two hundred and ninety dogs ( males/ females), . ae . years of age, . ae . kg of body weight fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( %), york shire terrier ( %), and cavalier king charles ( %). dogs were % b , % b , % c and % d acvim class. while the % of the dogs were normoazotemic (scr < . mg/dl), . % were staged in iris , % in iris and . % in iris . the prevalence of anemia in dogs with mvd was % ( / ): showed mild ( ≤ hct ≤ %) and moderate ( ≤ hct ≤ %) anemia. sixteen dogs were in b , in b , in c and in d acvim class; thirty-four were normoazotemic ( %). anemic dogs showed a significant higher scr. normoazotemic dog showed significant higher hb, hct and rbc both in the overall population and in the anemic group. in the overall population dogs in different iris class showed statistically different hb, hct and rbc and hb was significantly lower in decompensated hf dogs. in conclusion although a relationship between anemia and azotemia/ckd was documented in our study, it is important to emphasize that most of the anemic dog were normoazotemic: anemia is not an exclusive finding of cardiorenal syndrome and should be considered as possible complication in dogs with mvd alone. disclosures: no disclosures to report. the objective of this study was to evaluate left atrial (la) function by left atrial total fractional area change (la-factotal) and left atrial ejection fraction (laef) in dogs affected with chronic mitral valve disease (cmvd) naturally acquired with and without congestive heart failure (chf). our hypothesis was that la-factotal and laef decrease with severity of cmvd. eighty dogs were included in a prospective observational cross-section clinical study, grouped according to cmvd severity based on echocardiographic evaluation and clinical signs. the dogs were equally distributed in each group: a, b , b and c, according to american college of veterinary internal medicine staging system. indicators of la function were calculated with the following equations: la-factotal = (lamaximum area Àlaminimum area)/lamaximum area, measured by apical four view; and laef = (lamaximum volume À laminimum volume)/ lamaximum volume, by biplane area-length method from the left apical four and two-chamber views. la-factotal showed lower values (p < . ) in group c ( . %, p %- % = . - . ) compared with groups a ( . %, p %- % = . - . ), b ( . %, p %- % = . - . ) and b ( . %, p %- % = . - ). group c had lower laef ( . %, p %- % = . - . ) than groups a ( . %, p %- % = . - . ), b ( . %, p %- % = . - . ) and b ( . %, p %- % = . - ) (p < . ). left atrial function, assessed by la-factotal and laef, was reduced in dogs with cmvd and chf compared with healthy and asymptomatic cmvd groups. disclosures: no disclosures to report. recurrent episodes of heart and/or kidney failure are considered one of the causes leading to worsening heart/renal functions in human patients. the aim of this prospective study was to assess the influence of heart/kidney worsening on elected parameters of heart/kidney function in dogs affected by mitral valve disease (mvd). between july and may , dogs affected by mvd in acvim class b and without comorbidities were included in the study group. the control group was constituted by healthy dogs, matched with the cases for age (older than years) and gender. all the dogs underwent physical examination, thorax radiography, ecg, echocardiography, systemic blood pressure assessment, a complete blood count, serum biochemical analysis, including assessment of serum creatinine (scr), serum urea nitrogen (urea) and glycaemia (gly) and urine analysis with urine protein/creatinine ratio (upc). dogs were re-evaluated every -month until october . statistical analysis was performed using ibm spss statistics (p value significant if < . ). twenty-one dogs affected by mvd (cases) were included and healthy dogs (controls) were randomly selected among the eligible population. the % of cases experienced at least one episode of congestive heart failure (chf), but none of these patients developed chronic kidney disease (ckd). the % of cases developed ckd while remaining in acvim class b . no dogs in the control group developed ckd or mvd. correlations between worsening renal function (wrf -scr elevation ≥ . mg/dl or % from baseline), furosemide administration, upc levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. only a statistically significant difference in iris class between the groups according to wrf and in the echocardiographic parameter left atrium to aortic root (la/ao) according to furosemide amount were observed. both these results were expected. none of the cases included experienced renal damage (wrf or iris class change or upc change) concomitant to episodes of chf. the persistence of normal renal condition regardless of chf events and therapy administration was unexpected. in conclusion, experiencing chf seems not to directly affect renal function. to authors' opinion, the use of wrf, better than single scr and urea levels, may be useful in the long term management of aged patients affected by mvd. however, the small number of cases included in this study represents a great limit. we consider this work a pilot study. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is a primary myocardial disease characterized by inappropriate thickening of the myocardium in absence of other causes of hypertrophy including hypertension, hyperthyroidism, aortic stenosis and acromegaly. it is also the most common heart disease in cats. hcm presents a wide variety of clinical sings depending on the severity and location of the hypertrophy. cats affected with hcm have a mean age of . - . years old at the time of the diagnosis however this disease can affect cats as young as months although this later age is unusual hcm is a heterogeneous disease both in terms of phenotypic degree of hypertrophy and clinical outcome. hallmark histopathological hallmarks lesions of hcm are myocyte disarray, small coronary arteriosclerosis and interstitial fibrosis replacement in order to confirm hcm echocardiography has to be made. primary hypertrophy diagnosis is made based on the presence of ventricular hypertrophy, symmetric or asymmetric, in the absence of systemic disorders. the purpose of this study was to assess the prevalence of hcm in a feline population. in order to achieve this goal echocardiograms were made in all cats older of years clinically asymptomatic with or without cardiac murmur. all echocardiograms were made according to the guidelines of the acvim published in . diagnosis of ventricular hypertrophy was made from the right parasternal window using the b mode to measure the diameter of the lvfw and the ivs in diastole. cats with more than mm of wall thickness measured t , bun, crea, blood pressure. only cats within the normal limits of the later parameters were considered hcm positive. total number of cats in this study was cats male and female. from this population had no defined breed, were persian, maine coon, norwegian woods, siamese, chartreaux. no murmour was detected in ( . %) cats, s or s was detected in ( %) cats and differente degree of murmour was detected in ( . %) cats. hypertrophy was detected in cats. from this cats ( . %) were diagnosed as hcm, ( . %) cats were excluded either because of lack of values of t and or because they had high values of blood pressure, t levels or crea. in this study . % of the population had hcm. the epidemiological and phenotype distribution is highly variable. the average age at diagnosis of hcm in this study was . years. disclosures: no disclosures to report. mildly increased concentrations of crp are associated with cardiovascular disease in humans and dogs. it is not known whether increased concentrations of crp are associated with myxomatous mitral valve disease (mmvd) in dogs, or rather its sequel, congestive heart failure (chf). the aim of this study was to investigate whether serum concentrations of crp, determined using a novel automated canine-specific high-sensitivity crp assay (gentian hscrp), were associated with severity of mmvd and certain clinical variables in dogs. the study included client-owned dogs with different severities of mmvd. disease severity was determined by medical history, physical examination, echocardiography and response to diuretic therapy. dogs were allocated into groups based on acvim consensus statement guidelines ( in conclusion, slightly higher serum crp concentrations were found in dogs with chf whereas the severity of asymptomatic mmvd showed limited association with serum crp concentrations. disclosures: no disclosures to report. medetomidine is a a -agonist widely employed for sedation in dogs but its use is discouraged in cardiac patients even those suffering from myxomatous mitral valve disease (mmvd). however, only one investigation was previously conducted in a wide rangeregarding the class of the diseaseof mmvd patients, reporting a general safety of that protocol. the present study was focused just on class b of mmvd, with the aim to provide more detailed information on the cardiovascular effects of medetomidine in such patients, by the analysis of clinical and instrumental parameters suggestive of disease severity or congestive heart failure. dogs weighing < kg, needing a soft clinical procedure and showing a systolic apical heart murmur were screened and selected for the study if la/ao < . . the sedative protocol consisted in an iv injection of lg/kg medetomidine antagonized, after the clinical procedure, by an im injection of the recommended dose of atipamezole. clinical parameters, echocardiographic variables, thoracic radiographs and oscillometric blood pressure measurements were collected at baseline (t ), minutes after medeto-midine administration (t ) and hours after atipamezole injection (t ). of dogs screened, were definitively enrolled. at t a significative decrease in the right parasternal regurgitant jet area (rp-arj/laa), peak velocity of mitral regurgitation and shortening fraction was observed along with an increase in lvids (p < . ). left parasternal arj/laa decreased without reaching statistical significance but showing a high correlation with rp-arj/laa (r = . ). interestingly, la/ao changed only mildly and never reached a value > . . the other echocardiographic variables did not show a particular trend. systolic blood pressure showed values at the upper physiologic limit at t , lower values than t at t , and an increase above the initial value at t but without significance. thoracic radiographs were evocative of heart enlargement without pulmonary venous congestion or pulmonary oedema both at t and t . respiratory rate did not change between t and t . the degree of sedation was optimal during the clinical procedure in all cases. sedation with lg/kg medetomidine is safe in dogs suffering from mmvd in stage b (la/ao < . ). the decrease observed in peak velocity and color-doppler appearance of mitral regurgitation at t could be due to a reduction of both myocardial contractility and systolic blood pressure, by a lowering of sympathetic activity via baroreceptors stimulation. disclosures: no disclosures to report. in this retrospective study were included a total of clientowned dogs, undergoing transarterial occlusion of pda with mreye Ò flipper detachable embolization coil (n = ), amplatz â canine duct occluder (acdo; n = ) and amplatzer â vascular plug (n = ). device size selection was based on pda dimensions assessed by transesophageal echocardiography (tee) in cases and transthoracic echocardiography (tte) in cases. angiography was performed during the procedure to assess the success of the occlusion, and it confirmed complete occlusion in dogs and a trivial residual flow in dogs. the following day, transthoracic color-doppler echocardiography revealed that complete ductal closure was achieved in all dogs. the procedure was hemodynamically successful, as evidenced, by a reduction in indexed left ventricular internal diameter in diastole (lvidd; p < . ), fractional shortening (fs; p < . ) and left atrial to aortic ratio (la: ao; p < . ) within hours after procedure. four months after surgery, indexed lvidd was significantly reduced (p = . ) and la:ao remained constant. secondary complications included pulmonary arterial embolization of an acdo and a late rotation of an amplatzer â vascular plug resulting in an increased flow through the pda. the dog with the rotated device required subsequent surgical ligation of the pda. at this time, dogs were reported to be alive and the other dogs were lost to follow up. only one dog remained on congestive heart failure therapy after the pda occlusion. we can conclude that pda occlusion using an acdo for dogs with more than kg and a transarterial coil embolization for dogs with < kg had a high rate of immediate complete occlusion. pda occlusion using those devices proved to be a safe and effective therapeutic method for pda in dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right pulmonary artery distensibility index (rpad index) was recently described as a valuable method for early detection and severity evaluation of pulmonary arterial hypertension in dogs. rpad index is calculated as the percentage change in diameter of the right pulmonary artery (rpa) between systole and diastole, obtained by m-mode echocardiography from the right parasternal long axis view. the aim of this study was to compare the rpad index obtained by two different echocardiographic views in dogs. the study design was a prospective, multicenter, observational study. forty-five client-owned dogs from different breeds were included: dogs with heart disease and healthy dogs. two different right parasternal views, long axis (rpla) and short axis (rpsa), were used to measure the rpad index. from the rpla view (method ) and rpsa view (method ) a short axis and a long axis image were respectively optimized for the right pulmonary artery. the rpad index was calculated by m-mode as the percentage change in diameter of the right pulmonary artery: [(systolic diameter À diastolic diameter)/systolic diameter]* . measurements were done off-line as an average of consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. a pearson and a bland-altman test were used to assess correlation and agreement between the methods, respectively. intra-and inter-observer measurement variability was quantified by average coefficient of variation (cv). level of significance was set at p < . . m-mode evaluation of the rpad index was satisfactorily obtained by both methods in all dogs. pearson test showed a strong positive linear correlation between the values of rpad index obtained from both methods (r = . , p < . ). bland-altman test showed a good agreement between the methods in estimating rpad index (bias = . %, sd = . %, % limits of agreement = À . , . %). the mean difference between the two methods was . % ( % confidence interval = À . ; . ). intra-and inter-observer measurement variability was clinically acceptable (cv< %).the study showed a good agreement between short axis and long axis m-mode evaluation of rpa. both methods can be used interchangeably to evaluate rpad index. further studies are needed to evaluate the rpad index in a larger population of healthy dogs and the diagnostic and prognostic role of this echocardiographic parameter in dogs with different types of pulmonary hypertension. disclosures: no disclosures to report. naturally occurring hypoadrenocorticism (addison's disease) is an uncommon illness. its prevalence in the general canine population is estimated between . and . %. certain breeds appear to have an increased risk for developing hypoadrenocorticism, including bearded collie, standard poodle, portuguese water dog and nova scotia duck tolling retriever, with reported prevalence of . , . , . and . %, respectively. the objective is to evaluate the prevalence and clinical features of naturally occurring hypoadrenocorticism in great pyrenees (gp) presented at the centre hospitalier universitaire v et erinaire (chuv) of the university of montreal. this retrospective study (march to october ) includes client-owned great pyrenees diagnosed with hypoadrenocorticism. the medical records of dogs with a diagnosis of naturally occurring hypoadrenocorticism were reviewed, with an emphasis on great pyrenees' record. the prevalence of hypoad-renocorticism in the studied population, as well as the prevalence per breed, was calculated. data collected included breed, clinical signs, laboratory findings, age at diagnosis, treatment, and cause of the death. one hundred dogs were diagnosed with naturally occurring hypoadrenocorticism, representing . % of the overall canine population studied. thirty-five breeds were represented, with a prevalence per breed varying between . % and . %. a high prevalence was observed in west highland white terriers ( . %), great danes ( . %), standard poodles ( . %), saint-bernards ( . %) and jack russell terriers ( . %). out of gp presented during that period of time, . % (n = ) were diagnosed with hypoadrenocorticism. median age at diagnosis was . years (range: . to . ) in dogs with hypoadrenocorticism, and . years ( . - . ) in gp. the main reason for presentation of the addisonian gp was lethargy (n = ) and anorexia (n = ). clinical findings included hypotension (n = ), poor body condition (n = ), and heart murmur (n = ). the majority (n = ) had serum electrolytes abnormalities, with a na:k ratio ranging from . to . . other major laboratory findings included azotemia (n = ), anemia (n = ) and the absence of a stress leukogram (n = ). the majority (n = ) received fludrocortisone, with prednisone as needed. one gp was euthanized at time of diagnosis. great pyrenees diagnosed with hypoadrenocortism were overrepresented in the studied population, with a prevalence of hypoadrenocorticism in our gp population of . %. therefore, an inherited susceptibility can be suspected. reason for presentation and clinical signs were nonspecific, and similar to what is reported in other breed. in human medicine, haemoglobin a c (hba c), a form of glycosylated haemoglobin, is used as the standard measure of average glycaemic control over - months. the measurement of hba c in dogs has been previously demonstrated however high pressure liquid chromatography techniques are too technically difficult for routine use and other methods are no longer available. the objective of this study was to assess the use of latex immunoagglutination inhibition using a monoclonal antibody for the measurement of hba c in dogs, using the siemens dca vantage Ò . repeatability was assessed by measuring samples times within minutes. the effect of storage on edta anticoagulated samples was examined by measuring samples stored at °c every day for up to days. storage was further assessed by freezing samples and measuring them at , and weeks. the machine was then used to compare the hba c values in groups of dogs with diabetes mellitus (group , n = ), hyperadrenocorticism (group , n = ) or non-diabetic/cushingoid hospitalised patients (group , n = ). differences in the groups were examined for significance using a kruskal-wallis analysis of variance. the reference range of hba c has been previously calculated to be . - . % ( - mmol/mol) and values of . to > % ( to > mmol/mol) are seen in diabetic animals using high pressure liquid chromatography. the median coefficient of variation for the repeatability study was found to be % (range - %). it was possible to store samples at °c for up to days (median cv% = %, range - %) and at À °c for at least weeks (median cv% = %, range - %). the median hba c concentrations were group ; . % ( mmol/mol), group ; . % ( . mmol/mol) and group ; . % ( mmol/mol). group was significantly different from the other two groups using kruskal-wallis analysis of variance. in conclusion, the latex immunoagglutination method was repeatable for the measurement of hba c in dogs. in addition, hba c in canine edta anticoagulated samples were stable at °c for up to days and, if frozen, could be stored for at least weeks without significant sample deterioration. the assay provides the expected results in dogs with and without abnormalities of glycaemic control. disclosures: the siemens dca vantage was provided on loan from siemens, as well as the cartridges used on this machine to run all samples in the study. a. wehner , r. anderson , s. reese , k. hartmann . clinic of small animal medicine, munich, germany, institute of veterinary anatomy, histology and embryology, munich, germany measurement of total thyroxine (t ) is often the first diagnostic step in the work up of thyroid disease in dogs and cats. blood samples are routinely sent to a reference laboratory causing a delay in testing which might impact the results. the aim of this study was to validate an enzyme fluorescence assay (elfa) as an inhouse method to measure t in dogs and cats. t was measured in sera of dogs and cats by two methods, an enzyme immunoassay (eia) and an enzyme fluorescence assay (elfa). the eia served as the standard method to which the elfa results were compared. the elfa was performed with the minividas automated analyser (biom erieux, craponne, france) according to the manufactors instructions. coefficient of variation (cv) of the elfa in dogs sera was . % and of the eia - . %, respectively. cv of the elfa in cats sera was . - . % and of the eia . - . %, respectively. overall bias of the elfa in dogs was . %; however up to À . % in lower t ranges. maximal bias of the elfa in cats was . %. correlation of both methods was linear only in cats. using bland altman plots limits of agreement were À to % in dogs and À to % in cats. cohen s kappa revealed only slight agreement between both methods in dogs, but a good to very good agreement in cats. the elfa is a fast method with a high precision and can be recommended to measure t in cats, but cannot be recommended for dogs. disclosures: no disclosures to report. dysfunctions of the central nervous system (cns) are the most frequent causes of neurological disorders in dogs. our study aimed to find ( ) if some cns diseases could be associated with a selected group of common microbial or viral pathogens in dogs and ( ) if cns diseases have any characteristic profile with regard to two parameters, c-reactive protein (crp) and iga, that are reported to be potentially useful but unspecific markers of cns diseases. we analysed cerebrospinal fluid samples obtained from dogs with varying neurological signs between june and november . real-time pcr was employed with probes for toxoplasma gondii, neospora caninum, anaplasma phagocytophilum and canine distemper virus. igg-antibodies to tickborne encephalitis virus (tbe) were assayed and iga titres were measured using elisa, while crp concentrations were determined by immunoturbidimetric assay. the dogs had a median age of years (range: . - ) and comprised breeds most frequently involving chihuahuas, labrador retrievers, bernese mountain dogs and boxers. gender distribution was . % female, . % spayed bitches, . % male, . % neutered male, and . % non-identified. none of the cases were pcr-positive for toxoplasma gondii or canine distemper virus. one dog was positive for anaplasma phagocytophilum and another for neospora caninum. antibodies to tbe virus were within the borderline range in / dogs. the dogs could be divided into two age groups: (= . %) for young dogs (< years, median year) and (= . %) for older dogs (≥ years, median years). igahigh (> . mg/dl) cases represented % and % for young and older dogs, respectively. crp-high (> . mg/l) cases were almost half and equal: . % and . % in young and older dogs, respectively. compared with older dogs, young dogs had higher levels of crp (p = . ) and iga (p = . ). within the iga-high cohorts, crp-high and crp-low cases distributed almost equally ( . % versus . %) in young dogs but disproportionate ( . % versus . %) in older dogs. there were no [iga-low/crp-low] cases in young dogs but . % in older dogs. our present data sug-gest that ( ) canine cns disorders were largely characterized by high iga and particularly in young dogs ( ) inflammatory types (crp-high) were almost equal in both groups and ( ) internet is a potential source of medical information for pet owners. therefore, it could play an indirect but important role in the veterinary practice. this survey assesses the online search behaviour of french pet owners for their pets' health and its influence on a veterinary consultation. in april , french pet owners coming in a veterinary teaching hospital for a medical or a surgical consultation were surveyed. ( . %) owners fulfilled the questionnaire on a voluntary basis. the survey contained questions dealing with three topics: the online search behaviour of owners for their pets' health, their perception of the information found online and the internet's influence on a consultation and on the veterinarian/client relationship. . % of owners use the internet to obtain information on their pets' health. among them, . % use it rarely and . % occasionally. they mainly look for information on a disease ( . %), a symptom ( . %), a breed ( %) or a nutritional advice ( . %). . % of owners try to verify the accuracy of the information found, most often by questioning their veterinarian ( . %). few owners ( . %) think that online information is always trustworthy. most of the research ( . %) is randomly made, websites being found through search engines. the majority of pet owners ( . %) aren't aware of any health certification label for websites. internet enables certain pet owners to feel more at ease with their pets' health care: they ask more questions to their veterinarian ( . %) and feel more involved in medical choices ( . %). . % of owners consider that the internet can positively impact their relationship with the veterinarian. relief is the most common ( . %) emotional response to online research for medical information. however, . % of owners feel overwhelmed by the amount of information found, % are confused and . % frightened by the serious or graphic nature of the information found online. this study emphasizes the frequent but measured use of the internet by french pet owners for their pets' health. they seem to consider information found on the net with a critical mind. unexpectedly, it appears that the internet could be an ally for veterinarians by promoting exchanges between the clients and the veterinarian and by improving compliance with the care project. disclosures: no disclosures to report. sinonasal aspergillosis is a well-known and described fungal infection of the sinonasal cavities in dogs. topical treatment either with enilconazole or itraconazole infusion administered surgically or endoscopically are effective. the use of % clotrimazole cream have been described in a surgical setting after itraconazole infusion by sissener et al. in . the aim of the work was to report the effectiveness of the use of topical % clotrimazole cream as the only treatment for sinonasal aspergillosis in dogs. inclusion criteria were a full medical record with radiological and endoscopical imaging, record of clotrimazole discharge after instil-lation and endoscopic control between and days after procedure. the % clotrimazole cream was applied through catheters placed under direct endoscopic vision after fungal plaques removal. nine dogs were included. three dogs were mixed breed dogs, two dogs golden retriever, one dog a german shepherd and one old english sheepdog, one bull terrier and one cane corso. six dogs were male (one neutered) and three female (one intact). mean age was . years. main clinical signs were muco-purulent discharge ( ), pain at sinonasal palpation ( ), nasal planum alterations ( ), epistaxis ( ) . nasal discharge was bilateral in dogs. mean duration of clinical signs was . months. main radiological findings were turbinates lysis ( ), frontal sinus empyema ( ), frontal bone thickening ( ) and frontal bone lysis ( ). rhinoscopy disclosed lysis and remodelling of the nasal turbinates ( ), easy access to the frontal sinus ( ), septum lysis ( ), bilateral sinonasal aspergillosis ( ), monolateral nasal aspergillosis ( ), monolateral sinonasal aspergillosis and contralateral nasal aspergillosis ( ), monolateral frontal aspergillosis ( ). main duration of nasal cream discharge was . days. all dogs underwent endoscopic control between and days after the procedure. seven dogs were disease free; two dogs had persistent fungal plaques and underwent a second treatment. success rate was . %. success rate of this study is comparable to other studies with larger and smaller case series. endoscopic removal of the fungal plaques can be time consuming and topical administration of either enilconazole or itraconazole require an additional hour. the catheter placement and the % clotrimazole cream application lasted minutes for each cavity in the dogs here reported. the use of % clotrimazole cream as the only treatment for sinonasal aspergillosis needs further evaluation on a larger case series. disclosures: no disclosures to report. few studies exist on euthanasia in small animal practice. however, such an act belongs to veterinary procedures, more or less frequently depending of the kind of practice, and may deeply impact both owners and veterinarians. we intended to study practical, ethical and psychological aspects of euthanasia of dogs and cats among french veterinary practitioners. from october to february , an on-line -item questionnaire on small animals' euthanasia, addressing practical aspects of euthanasia, communication with the owners, ethical problems, owners' and veterinarians' perceptions, was emailed via professional associations and networks. results were analyzed using commercial software (sphinx iq Ò and excel Ò ). french veterinarians practicing small animal medicine completed the questionnaire, representing > % of this population. euthanasia occurs rarely at home. over % of veterinarians propose the owners some time alone with their pet, and to stay during euthanasia, performed most commonly by intravenous injection ( . %) mainly after sedation/anesthesia ( . %). ninety nine percent of veterinarians consider communication, including description of events' sequence, and disposal of the body, as important. estimated minimum communication time required varies from - to - minute. following euthanasia, . % are often thanked by the owners. most veterinarians (> %) have refused a euthanasia, considered unjustified, or had their own suggestion of euthanasia rejected. reasons for such a suggestion include intractable pain ( . %), non-acceptable complications ( . %), financial considerations ( . %) or animal considered dangerous ( . %). veterinarians think most owners ( . %) experience some sense of guilt during euthanasia. themselves perceive euthanasia most commonly as relief of animal's suffering ( . %) and part of veterinary practice ( %), less frequently as a defeat ( . %). almost all veterinarians have experienced emotionally challenging euthanasia, and . % estimate that practicing euthanasia influences their perception of death. practical ( %) and psychological ( . %) aspects of euthanasia have been discussed in most teams. veterinarians' gender influences euthanasia management, mostly concerning some communicational and practical aspects. euthanasia is definitely not an ordinary veterinary act, neither for the owner nor for the veterinarian. therefore, this act must be performed with as much care and communication as possible. disclosures: no disclosures to report. canine pancytopenia is associated with a range of intra-marrow or extra-marrow causes, including though not limited to, infectious agents, drugs, toxins and neoplasms. there is currently limited information regarding the clinicopathological features of the underlying causes or the prognosis in pancytopenic dogs. the objective of this retrospective study was to better define the spectrum of diseases associated with canine pancytopenia, to establish possible clinicopathological discriminators for the common causes and to investigate if the severity of pancytopenia or the underlying disease were associated with the clinical outcome (death or survival). medical records of dogs with a comprehensive diagnostic investigation admitted in a veterinary teaching hospital were retrospectively reviewed. pancytopenia was defined by a hematocrit (hct) < % (< % for dogs < months of age), white blood cell counts (wbc) < . /ll and platelet counts (plt) < , /ll. a control group of dogs without any evidence of blood cytopenia(s) was also established. in total, pancytopenic dogs were studied. bone marrow aspiration cytology was examined in cases and aplasia of all hematopoietic lineages was observed in ( . %) dogs. the most common diagnoses included monocytic ehrlichiosis (cme) (n = ), leishmaniosis (canl) (n = ), parvoviral enteritis (pe) (n = ), and concurrent cme and canl (n = ). mixed breed dogs were more likely to develop pancytopenia as compared to purebreds and pancytopenic dogs tended to be younger than the controls (conditional dependent logistic regression model, p = . and p = . , respectively). among the most common diseases associated with pancytopenia, the mean wbc counts were significantly lower in dogs with cme and pe compared to dogs with canl (one way anova with bonferroni test for multiple comparisons, p = . and p = . , respectively), while plt counts were significantly lower in cme compared to canl (p < . ) or pe (p < . ). total protein concentrations were significantly lower in dogs with pe compared to cme (p < . ) and canl (p < . ). using a univariable logistic regression analysis model, no association was established between the underlying disease and the final outcome. however, higher hct (by at least one percentage unit), wbc (by at least /ll) and plt (by at least , /ll) values tended to significantly increase the odds of survival (p = . , p < . and p = . , respectively). in the present study, cme, canl and pe were the major causes of canine pancytopenia. potentially useful diagnostic indicators included severe leucopenia (cme, pe), thrombocytopenia (cme) and hypoproteinemia (pe). disclosures: no disclosures to report. bwbp detects both upper and lower airways diseases because any site of airway obstruction will result in increased pressure changes associated with breathing. nevertheless our results suggest that there are significant differences in tv (p = . ), ti (p = . ), pef (p = . ), eep (p = . ), penh (p = . ) and pau (p = . ) between lm and fbd cats. no other significant difference in bwbp parameters was found. upper airway obstructions have been previously evaluated in cats by using pft (mckieman, , lin, but in authors' knowledgement this is the first study designed to compare upper and lower airway obstructions by using bwbp. attending our results, there is the evidence that bwbp can help characterize mechanical dysfunction of the airways in cats with lm obstruction. however we must keep in mind some limits of this study as the low number of animals, individual variability in breathing pattern and to have the chance of doing bronchoreactivity tests. disclosures: no disclosures to report. the median lifespan of domestic dogs has been estimated to - years, but little is known about risk factors for mortality in aged dogs: most mortality studies in dogs have been carried out among diseased dogs (renal or heart diseases, cancers, post-operative death). to determine which characteristics are associated with mortality in a priori healthy aged dogs, a prospective cohort study has been conducted in guide dogs, followed from a systematic geriatric examination (ge) to either (all-cause) death or cut-off date (july th, ). survival analyses (kaplan-meier estimators, log-rank tests, and multivariate cox proportional hazards models) were used to assess the associations with time to death. median age at ge was . years, all dogs were neutered, and % were female. the majority of dogs were golden retriever (n = ) and labrador retriever (n = ). among these dogs, % were obese, % presented skin nodules and % used bus as transportation. a total of dogs died during follow-up, leading to a median survival time from ge of . years. after adjustment for demographic and biochemical variables (age, sex, total proteins, cholesterol and alp), an increased alanine amionotransferase level (≥ ui/l; adjusted hazard ratio [ahr], . ), presenting skin nodules (ahr, . ), and not being a labrador (ahr, . ) were independently associated with a shorter time to death (p < . ). public transportation tended to be associated with mortality (ahr, . ; p = . ), highlighting the importance of environment in mortality. neither sex nor other biochemical parameters were significantly associated with mortality. the alanine amionotransferase level and the presence of skin nodules seem predictors of mortality in senior guide dogs, mostly labrador, golden, or mixed breed of labrador/golden. the impact of environment, in particular urban environment, on mortality needs further investigation. studies in other breeds and pets are also necessary to generalize these results. disclosures: sara hoummady received a grant from mp labo for his phd work about dog aging and marc blondot work at the paris guide dog school. laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. laryngoscopic and bronchoscopic findings in dogs examined by the author between and were evaluated for the presence or absence of laryngeal abnormalities. dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. detection rates for laryngeal abnormalities were compared among disease groups using chi square analysis and fisher's exact test, with significance set at p < . . a total of dogs were evaluated and varied in age between months to . years (median years). weight ranged from . to . kg (median kg), with dogs < kg, dogs from . - . kg, dogs from - kg, dogs from . - kg, and dogs > kg. laryngeal hyperemia or swelling was found in / dogs ( %), and detection rate did not differ among disease processes. laryngeal function was considered suspect in / cases, prompting administration of doxapram, which normalized function in / dogs. laryngeal paresis or paralysis was reported in a total of / dogs ( %). a substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough. disclosures: member of the feline advisory board, speaker honoraria for international, national, and regional continuing education meetings. bronchiectasis is a poorly characterized disease in dogs identified by airway dilatation on radiographs, computed tomography, bronchoscopy, or histopathology. little is known about underlying disease processes associated with bronchiectasis in dogs. medical records from dogs presented to uc davis were searched for identification of bronchiectasis. underlying disease processes and clinical diagnoses were obtained through review of the history, physical examination, respiratory endoscopy and bronchoalveolar lavage fluid analysis and microbiology. historical reports, results of imaging, bronchoscopy and fluid analysis, and scrutiny of pathologic and clinical diagnoses were comprehensively evaluated to identify the most likely underlying disease process associated with bronchiectasis. between and , bronchiectasis was diagnosed in / dogs ( %) that had bronchoscopy performed. dogs ranged in age from . to years (median years) with / dogs < months, / dogs ( %) - years, / dogs ( %) . - years of age and / dogs ( %) over years of age. dog breeds affected more than once included labrador retrievers, cocker spaniels, golden retrievers and standard poodles. duration of cough ranged from days to years (median months). underlying disease processes included pneumonia in / ( %) dogs, inflammatory airway disease in / ( %) dogs, and eosinophilic bronchopneumopathy in / ( %) dogs. twenty-three of dogs ( %) had positive bacterial cultures, with isolation of streptococcus (n = ) and enteric species (n = ) most commonly. this study found that bronchiectasis often occurs in older, large breed dogs with infectious or inflammatory pneumonia. disclosures: johnson: feline advisory board, speaker honoraria. chronic respiratory disease, often characterized by a chronic cough, is common in dogs. the purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing. a retrospective study of client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (etw) or broncho-alveolar lavage (bal), was performed. all dogs were evaluated by means of full clinical examination, hematology and serum biochemistry analyses, survey thoracic radiographs, echocardiography and ecg (if indicated), and bronchoscopy with cytological analysis and aerobic culture of etw or bal fluid. an etw was performed in / ( %) dogs while a bal was performed in / ( %) dogs. a positive aerobic bacterial culture was identified in / ( %) of submitted etw/bal fluid samples. most commonly isolated bacteria included mycoplasma sp. ( %), bordetella bronchiseptica ( %) and pseudomonas aeruginosa ( %). a definitive diagnosis was made in / cases ( . %). chronic bronchitis was the most common diagnosis ( . %), median age years; followed by airway tracheal collapse or bronchomalacia ( %), median age years,; and primary bacterial infections ( . %), median age years. less common etiologies identified included neoplasia ( . %), median age years; parasitic infections ( . %), median age years; and eosinophilic bronchopneumopathy ( . %), median age years. rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. myxodematous mitral valve disease was found concurrently in / ( . %) dogs. this study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and etw or bal with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease. disclosures: no disclosures to report. canine sterile steroid-responsive lymphadenitis (cssrl) is an uncommon cause of lymphadenomegaly. diagnosis is one of exclu-brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (osas). oxidative stress in the body represents the imbalance between the production of reactive oxidative species and the ability of antioxidant defense mechanisms to detoxify the reactive intermediates. oxidative stress is involved in the pathogenesis of many diseases, including hemolytic anemia, atherosclerosis, tissue reperfusion injury and has also carcinogenic potential. several studies have clearly shown an association between obstructive sleep apnea syndrome in humans and oxidative stress, but detailed underlying pathomechanism remains unclear. due to the consideration of brachycephalic dogs as an animal model of human osas, this study was designed to evaluate oxidative stress (paraoxonase type- activity; pon and total antioxidant status; tac) in brachycephalic dogs with brachycephalic airways obstructive syndrome (baos) before and after surgery compared to control dogs. this study was conducted on dogs with baos and control dogs. twenty out of baos dogs were evaluated - months after surgical correction. mean values of tac and pon in different studied groups were as follows: control dogs (tac: . ; pon : . ), baos dogs (tac: . ; pon : . ), baos dogs post-surgery (tac: . ; pon : . ) a statistically significant difference on tac levels is observed between dogs with baos and control dogs (p < . ). no statistically significant differences were observed in pon and tac levels before and after surgery. on the other hand, no differences have been observed between pon and tac levels in baos dogs according type of brachycephalic breed, grade of respiratory and digestive signs or presence of everted ventricular laryngeals. the results of our study showed a statistically significant difference in tac values between control and dogs with baos, confirming the oxidative stress previously described in humans. even that human patients with osas can partially reverse their increase in oxidative stress by using a nasal continuous positive airway pressure treatment, in dogs with baos no differences were observed before and month after surgical treatment. baos surgical treatment is not useful to reduce pon- or tac levels, probably because baos does not induce such an evident inflammatory process in dogs as in human patients with osas. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). the cipf course varies greatly among dogs from rapid deterioration to slowly progressive forms and the survival time from onset of clinical signs ranges from a few months to several years. in human ipf, increased chemokine (cc-motif) ligand (ccl ) concentrations in bronchoalveolar lavage fluid (balf) are indicative of a poor outcome and serum concentrations are correlated with clinical parameters of lung function. in dogs, serum and balf ccl concentrations were shown to be elevated in whwts with cipf compared with healthy whwts. the aim of the present study was to investigate whether serum ccl concentrations measured in whwts with cipf at diagnosis ( ) can be used as an indicator of prognosis and ( ) correlate with lung function parameters. ccl concentrations were determined by elisa (canine ccl quantikine elisa kit, r&d systems) in the serum of whwts suspected of cipf (median age . years, range . - . ), for which a follow-up was available (median follow-up time . months, range - . ). serum sampling extended from may to january . cipf diagnosis was confirmed by thoracic high resolution computed tomography, lung histopathology, or both examinations in , and whwts respectively. kaplan-meier analysis was conducted to investigate differences in survival times according to serum ccl concentrations at diagnosis. spearman analysis was used to assess correlations between serum ccl concentrations and lung function parameters, namely the distance walked in the -minute walking test ( mwd) and the arterial partial pressure of oxygen (po ). among the cipf whwts included, died or were euthanized for cipf-related reason, died or were euthanized for non-cipf-related reason and were still alive at the end of the study. the median survival of whwts with cipf-related death or euthanasia was . (range . - . ) months from diagnosis. serum ccl concentrations above pg/ml were significantly associated with a shorter survival time in whwts affected with cipf (p = . ). a weak negative correlation was found between serum ccl concentrations and the mwd (r = À . , p = . , n = ), while no correlation was observed with arterial po values (n = ). in conclusion, serum ccl concentration provides prognostic information in whwts suffering from cipf, while this marker is weakly correlated with the clinically lung function parameters available in the present study. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). this study was intended to ( ) describe thoracic high-resolution computed tomography (t-hrct) findings obtained in cipf dogs under general anesthesia (ga) using the glossary of the fleischner society and ( ) compare images obtained under ga (t-hrct ga ) with those obtained under sedation (t-hrct s ). t-hrct images from whwts with cipf and control whwts were retrospectively reviewed by three observers in consensus. specific t-hrct features were assessed and graded for each lung lobe ( = absence, = mild, = moderate and = severe). a global score was then calculated. the khi² test with the threshold % was used for the statistical analysis. ground glass opacity (ggo) was observed in all cipf whwts and in / of controls (p = . ). in controls, ggo was mild and localised mainly in cranial lobes. in cipf whwts, ggo was mild, moderate or severe in , and dogs respectively, without lobe predilection. consolidation was observed in / cipf whwts but not in controls (p = . ) and was mild ( / ) to moderate ( / ). a mosaic pattern, suggestive of air trapping, was noticed in / cipf whwts but not in controls (p = . ) and was mild, moderate or severe in , and whwts respectively, without lobe predilection. nodules were present in / cipf whwts but not in controls. reticulation, subpleural bands and parenchymal bands were noticed in , , and / cipf whwts respectively. honeycombing, emphysema, pleural effusion and pleural thickening were never observed. bronchial wall thickening and mild bronchiectasis were present in / and / cipf whwts respectively but not in controls (p = . and p = . ). the overall t-hrct s quality was good in / examinations compared with / for t-hrct ga (p = . ). the presence of motion artefacts was higher for t-hrct s (p < . ), but were most frequently graded as mild (p < . ). t-hrct s allowed identification of a mosaic pattern in additional cipf whwts, while consolidation could not be identified in others. there was no difference in identification or gradation for the other features between t-hrct ga and t-hrct s . in conclusion, ggo, consolidation, mosaic pattern and bronchial wall thickening are the main t-hrct features of cipf in whwts. honeycombing, the major feature of ipf in humans, was never observed, which suggests a different pathophysiology between the two entities. t-hrct s images are in accordance with t-hrct ga and can be used for cipf diagnosis. disclosures: no disclosures to report. literature on mesenterial lymphadenitis (lad) or mesenterial lymph node abscesses (lab) in small animals is scarce. case files from to were searched for dogs with the diagnosis of mesenterial lad/lab based on cytology and/or histopathology. of cases identified, had to be excluded due to incomplete data. the remaining dogs were of mixed breed (n = ), small munsterlander (n = ) and one each of other breeds. nine dogs were male and female. median age was months (range . diagnosis was based on fine needle aspiration (fna; n = ), histopathology (n = ) or both (n = ). significant findings in the dogs' history included gastrointestinal signs (n = ), puppy whose mother had mastitis, bite wounds/abscesses of the skin (n = ), pulmonic stenosis (n = ) and orthopaedic diseases (n = ). most common presenting complaints were lethargy (n = ), hyperthermia (n = ), diarrhoea (n = ), vomiting/nausea (n = ), inappetence/anorexia (n = ), back/abdominal pain (n = ) and lameness (n = ). diagnostic tests performed included haematology/serum biochemistry (n = ), thoracic (n = ) and abdominal (n = ) radiographs, abdominal ultrasound (n = ), ct/mri (n = ), fnas of other organs (n = ), urinalysis (n = ) with culture (n = ), coagulation profiles (n = ), orthopaedic radiographs (n = ), cpl (n = ), blood cultures (n = ), and csf/joint taps (n = ). dogs were retrospectively divided into group a (n = ): dogs with no other disease than lad/lab ("idiopathic") and group b (n = ): dogs with other diseases diagnosed simultaneously. these included neoplasia (carcinoma n = , lymphoma n = , leiomysarcoma n = , histiocytic neoplasia n = , prostate carcinoma n = ), gastroenteritis (n = ), presumed pancreatitis (n = ), purulent monoarthritis (n = ), purulent hepatitis/ splenitis (n = ), fungal infection at a distant site (n = ), and mycobacteriosis (n = ). eleven dogs received surgical treatment and antibiotics, and dogs conservative medical management consisting of supportive treatment and antibiotics. all dogs were discharged alive. dogs in group a were hospitalized longer (mean days, sd . ) than dogs in group b (mean days, sd . ) (p = . ). the median follow-up time was days ( - days). there was no difference in pretreatment with antibiotics or anti-inflammatories between groups. t-tests or kruskall-wallis tests showed that dogs in group a were borderline significantly younger (p = . ), had significantly higher respiratory rate (p = . ), rectal temperature (p = . ), monocyte count (p = . ) and crp concentration (p = . ) than dogs in group b. the small munsterlander had an odds ratio of over other breeds to suffer from lad/lab. in conclusion, idiopathic mesenterial lad/lab was seen in young dogs with hyperthermia and gastrointestinal signs. diagnosis of purulent lad/lab on fna does not exclude the presence of another underlying pathogenesis. disclosures: no disclosures to report. canine infectious respiratory disease (cird) is a disease complex caused by different viral and bacterial pathogens. aim of the study was to evaluate clinical and laboratory factors associated with different infectious agents. dogs were included, if they showed respiratory signs (< days) consistent with cird and if non-infectious respiratory conditions could be excluded. nasal and pharyngeal swabs were taken from dogs with cird and tested for respiratory pathogens, including canine parainfluenza virus (cpiv), canine adenovirus (cav), canines distemper virus (cdv), canine herpes virus (chv), canine respiratory coronavirus (crcov), canines influenza virus (civ), and bordetella bronchiseptica (b. bronchiseptica) by polymerase chain reaction. results of clinical and laboratory data were correlated with the underlying pathogen using fisher's exact test and chi-square test (p ≤ . ). cpiv was detected in , crcov in six, and b. bronchiseptica in dogs; patients showed infections with more than one pathogen. there was no significant difference for age and gender distribution between the three groups; however, dogs infected with cpiv more likely originated from a shelter (p = . ). when clinical data were compared, there was no significant difference for the parameters depression, fever, cough, nasal discharge, dyspnoea, and abnormal lung sounds. furthermore, there was no sig-nificant difference regarding abnormalities of erythrocytes, platelets, leukocytes, and differential count between groups. the study shows that in dogs with cird clinical and laboratory parameters cannot indicate the underlying pathogen. furthermore, diseases severity does not seem to depend on the infectious organism involved. disclosures: no disclosures to report. breed related predisposition to bacterial bronchopneumonia (bp) has been reported in irish wolfhounds (iwhs). underlying factors are unknown, however immune deficit, ciliary dysfunction and aspiration have been suggested as predisposing factors. the purpose of this prospective study was to evaluate lymphocyte subpopulations in iwhs with one or more previous bp and compare results to elderly iwhs without any previous bacterial respiratory infections. additionally, healthy dogs of other breeds were included as controls. previous bp was confirmed in iwhs (median age . years, interquartile range . - . years). healthy iwhs (n = , . , . - . years) or dogs of other breeds (n = , . , . - . years) had no history or findings suggestive of previous bp. edta blood samples, collected from all dogs when they were healthy, were stained with fluorescent mouse anti dog cd , cd , cd , cd and mhc class ii antibodies (abd serotec Ò ) and flow cytometry analysis was performed with bd facsaria Ò ii cell sorter and facsdiva Ò software. statistical comparison between groups and the effect of age was studied using analysis of covariance (ancova) models. the number of leucocytes did not differ significantly between groups. the total numbers of lymphocytes and numbers of major lymphocyte subpopulations (b-cells, cd + and cd + t-cells) were significantly lower in healthy iwhs and iwhs with previous bp compared to dogs of other breeds (lymphocytes p < . and p < . ; b-cells p = . and p = . ; cd + t-cells p = . and p = . ; cd + t-cells p < . and p = . respectively). percentage and number of mhc class ii+ non-b lymphocytes was significantly higher in both iwh groups than in dogs of other breeds (p < . in all comparisons). lymphocyte numbers and subpopulations did not differ significantly in healthy iwhs compared to iwhs with previous bp. an age-related decline in the total number of lymphocytes (p = . ), t-cells (p = . ), cd + t-cells (p = . ) and mhc class ii+ non bcells (p < . ) was noticed only in the group of iwhs with previous bp. these preliminary results indicate that iwhs may have significantly lower numbers of lymphocytes, b-cells as well as cd + and cd + t-cells than dogs of other breeds. further studies are needed to determine whether these alterations represent a breed related phenomenon or are connected to the predisposition to bp. an age-related decline in lymphocyte, total t-cell and cd + t-cell numbers was detected in iwhs with previous bp. in humans, age related changes in cd + t-cells have been associated with increased susceptibility to infections. disclosures: no disclosures to report. feline chronic kidney disease (ckd) is a common feature of ageing cats. angiotensin-converting enzyme inhibitors (acei) are recommended to treat hypertension associated with ckd to limit target-organ damage and especially glomerular hypertension. in addition, the international renal interest society (iris) guidelines recommends the prescription of an acei in patients with ckd and proteinuria. to our knowledge no study has demonstrated the effects of long term administration of acei in a client owned population of cats suffering from ckd on glomerular filtration rate (gfr). the aim of the study was to evaluate the effect of an acei (imidapril, prilium Ò , v etoquinol sa) on the gfr of cats suffering from naturally occurring chronic kidney disease (ckd) over months. sixty-six cats presenting with clinical and biological signs of ckd were enrolled by european investigators and followed up till months in this randomized blinded study. thirty-two cats provided suitable data for gfr analysis; animals received imidapril at the dosage of . mg/kg/d and received placebo. animals with no available gfr value on day or with no data after day were excluded as well as animals for which the iohexol clearance could not be determined. follow up was censored after months due to small sample sizes for statistical comparisons. on day , month , month and month , cats were sampled , and minutes after intravenous administration of mg of iohexol. gfr was based on determination of the iohexol clearance which was calculated with the phoenix Ò winnonlin . software. statistical analyses were performed with sas/stat . software. as gfr were not available on each time point for a given animal, the two treatment groups were compared on each gfr determination point using an ancova (analysis of covariance) with the gfr determined on day as the covariate. on d , gfr were . ae . and . ae . ml/kg/min in the imidapril and placebo group, respectively. a significant statistical difference ( . ae . versus . ae . ml/kg/min in the imidapril and placebo group respectively, p = . ) was observed in favor of a higher gfr in the imidapril treated animals on month . higher gfr were also observed in the imidapril group on month and month but not significantly different from the placebo group. daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease. disclosures: authors are employees of vetoquinol. there is a concern over the potential of cytotoxic drugs which could harm exposed workers. the speciality of oncology of the ecvim-ca published guidelines in order to prevent that risk. however few data exist for evaluation of the real risk of occupationnal exposure. this concern was the aim of our study. biomarkers used were vincristine and cyclophosphamide. surface samples were collected in the consultation room and ward dedicated for chemotherapy. samples were collected with wet filter paper on cm surfaces, or objects (computers for instance). samples were analysed by liquid chromatography and mass spectrometric method.the limit of quantification was . ng/ cm (or . g/object) for vincristine and . ng/ cm (or . g/object) for cyclophosphamide. samples in consultation room were collected after treating dogs with vincristine and after cleaning. samples in dedicated ward were collected after cleaning and after -days stay of treated dogs. aeras/objects were tested in consultation room; in dedicated ward for vincristine; in ward for cyclophosphamide. after treatment of dogs in consultation room, traces of vincristine were detected on the floor and the laboratory bench top ( . - . ng/ cm ). moreover, the surface of auscultation table and extern side of gloves were contaminated after preparation and administration of vincristine (respectively . , and ng/objet). after cleaning, % of samples in consultation room were positive. traces of vincristine were detected on the floor or objects (wall otoscope: . - . ng/ cm ). after cleaning, % of samples in ward were positive. traces of vincristine were detected on the floor and objects (boxes, wastebin, bowls: . - . ng/objet) after cleaning and animals treatment. cyclophosphamide were detected on all areas tested ( . a . ng/objet). despite protective guidelines to avoid spread of cytotoxic drug, environemental exposure was demonstrated. however contaminations were limited and show that handling procedures of cytotoxic drugs are well controlled. most-elevated contamination level for vincristin were noticed on extern side of gloves depsite of using appropriate material. workers should pay a particular attention for gloves withdrawal to limit exposure. small amounts of vincristine were found in inapropriate places: computeur mouse. even if there is few of those residues, we thought about people working on a regular basis in this room for other activities than chemotherapy, and we decided to adapt our clinical practices. evaluation of occupational exposure to cytotoxic drugs is an important step to prevent incidents and heigt awarness of nursing staff to apply those good clinical practices. disclosures: no disclosures to report. the tumor-associated inflammatory response had the effect of enhancing mammary tumorigenesis, helping incipient neoplasias to acquire hallmark capabilities, both in human and dogs. t-cells migration to the tumor site and the following activation may be the essential requirement for their promoting effect on tumors. in human breast cancer the signaling pathways of c-kit have been described as possibly being involved in differentiation, migration, survival, and maturation of t-cells and other inflammatory cells into tumor sites. in order to clarify this subject in canine mammary tumors (cmt), malignant neoplasms were studied by using immunohistochemistry comparing the intratumoral cd + tlymphocytes and c-kit expression together with vegf, microvessel density (mvd) and clinicopathological characteristics of tumor aggressiveness. cd + t cells and high c-kit immunoexpression revealed a positive and statistically significant correlation with vegf (r = . , p < . ; r = . , p = . for cd and c-kit respectively) and cd (r = . , p < . ; r = . , p = . for cd and c-kit respectively). a statistically significant association (p = . ) and a positive correlation (r = . , p = . ) between cd + t-lymphocytes and c-kit was also observed. tumors with high c-kit expression showed higher counts of cd + t-cells. the mvd of high cd /vegf tumors was significantly more elevated (p < . ). a similar association was observed for high c-kit/vegf tumors (p < . ). in this study high cd /vegf, high c-kit/vegf and high cd /c-kit tumors were statistically significantly associated with elevated grade of malignancy (p < . for cd /vegf, c-kit/vegf and cd /ckit), presence of neoplastic intravascular emboli (p < . for cd /vegf and cd /c-kit; p = . for c-kit/vegf) and presence of lymph node metastasis (p < . for cd /vegf, c-kit/ vegf and cd /c-kit). tumors with high cd /vegf (p = . ), high c-kit/vegf (p < . ) and high cd /c-kit (p = . ) expression were associated with shorter os time. interestingly the group of tumors with high c-kit/vegf retained their significance by multivariate analysis arising as independent predictor of os. results of this study suggest that t-lymphocytes may share common signaling pathways with c-kit and vegf in cmt progression and may contribute to increased angiogenesis, aggression and shorter os in these tumors. disclosures: no disclosures to report. few epidemiological data have been published on feline large granular lymphocyte (lgl) lymphoma. a recent study (krick et al. ) described clinicopathologic features in cats with lgl lymphoma, but no comparisons were made with cats with other diseases or other forms of feline lymphoma. therefore, the objective of this study was to assess differences in prevalence, signalment (breed, sex, and age), physical exam findings (body weight, body condition score, body temperature, heart rate, respiratory rate, and systolic blood pressure), and felv/ fiv status between cats with lgl lymphoma (group ) and all other type of feline lymphoma (group ). the electronic data-base of the san marco veterinary clinic was searched between january- and december- for cats with a cytological or histopathological diagnosis of lymphoma. differences between groups were assessed by t-test, mann whitney, pearson-chi square, pearson chi square yates corrected, and fisher's exact test. during the study period out of sick cats seen at the clinic were diagnosed with lymphoma (group : n = ; group : n = ). the prevalence of all type of feline lymphoma between and compared to sick cats did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). the lymphoma lgl prevalence between and compared to all types of lymphoma did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). among the variables studied, only sex (group : [ . %] females, [ . %] males; group : [ . %] females, [ . %] males; p = . ) and age (group : ae months; group : ae months; p = . ) were significantly different between groups. sixteen out of cats with lgl lymphoma were tested for their felv/fiv status resulting all felv-and one ( . %) fiv+. seventy-four out of cats with all other types of lymphoma were tested for their felv/fiv status resulting ( . %) felv+ and ( . %) fiv+. five cats ( . %) were both felv+/fiv+. prevalence of felv infection was significantly lower (p = . ) in group compared to group . there was no difference in prevalence of fiv infection between groups. lymphoma lgl affects more females and older cats compared to all other type of feline lymphoma. opposite to all other type of lymphoma, and in accordance to previous litterature information, felv+ status does not play a role in the pathogenesis of feline lgl lymphoma. disclosures: no disclosures to report. the activity of t regulatory cells (tregs) is known to be closely associated with the expression of foxp transcription factor. foxp regulatory t cells (foxp treg) are a distinct group of t lymphocytes that have immunosuppressive properties. normally this cells work for prevention of harmful autoimmune responses, however can also interfere with beneficial immune responses such as anti-tumor immunity. in human breast cancer these cells play a crucial role in tumor progression. in canine mammary tumors (cmt) there are only a few studies and this topic are not welldocumented. in this study we included malignant cmt and studied, by immunohistochemistry, the intratumoral foxp expression together with vascular endothelial growth factor (vegf), microvessel density (mvd, by cd antibody) and several clinicopathological characteristics. abundant foxp treg cells was statistically associated with presence of tumor necrosis (p = . ), nuclear grade (p = . ), poor differentiation of tumors (p < . ), high mitotic grade (p < . ), high histological grade of malignancy (p < . ), presence of neoplastic intravascular emboli (p < . ) and presence of lymph node metastasis (p < . ). intratumoral foxp levels were correlated with the levels of vegf (r = . ; p < . ) and intratumoral mvd (r = . ; p < . ). additionally tumors with abundant foxp treg cells were associated with shorter overall survival (os) time (p = . ). results suggest that treg cells play a role in cmt progression and may contribute to increased angiogenesis and aggression in these tumors. the association of intratumoral foxp expression with shorter os of animals suggests a utility of treg cells activity as a prognostic marker. disclosures: no disclosures to report. clinical manifestations of canine visceral leishmaniasis (canl) are non-specific and include progressive weight loss, anemia, lymphadenomegaly, hepatosplenomegaly, dermatological, renal and ocular alterations. cardiac lesions resulting in clinical signs has been scarcely described in dogs with vl, and the presence of the parasite in the cardiac tissue has been involved in few reports. accordingly, the present study aimed to evaluate histopathological abnormalities in cardiac tissue from dogs naturally infected by leishmania infantum chagasi. a total of dogs were evaluated. all dogs were symptomatic but no one presented clinical signs of cardiac involvement. in compliance with a federal law and under the owners' signed consent, all dogs were submitted to euthanasia and comprehensive post-mortem evaluation. samples from right atrium free wall (ra), right ventricle free wall (rv), interventricular septum (ivs) and left ventricle free wall (lv) were collected and evaluated. tissue samples were fixed in formalin, embedded in paraffin, sectioned at mm, and stained with hematoxylin and eosin (he) and anti-leishmania immunohistochemistry was also performed. the study was approved by the ethics committee in animal experimentation and animal welfare (protocol number / ). histopathological changes were observed in at least one of the four evaluated cardiac regions in % ( / ) of the dogs. the most frequent cardiac injury was an inflammatory reaction, characterized by the presence of mononuclear cell infiltrate in different degrees. of the evaluated regions, ra was the one with the highest incidence of histopathological changes, observed in % ( / ) of the animals, followed by rv, lv and siv, affected in . % ( / ), . % ( / ) and . % ( / ) of the dogs, respectively. immunohistochemistry revealed amastigotes in the cardiac tissue in % ( / ) of the dogs. a positive correlation was found between cardiac lesions and the presence of amastigotes in the myocardium (p < . ). disclosures: no disclosures to report. canine leishmaniosis is a life threatening zoonotic disease. the combination of meglumine antimoniate and allopurinol is considered the most effective therapy for canine leishmaniosis and constitutes the first line protocol against this disease. allopurinol is a parasitostatic drug used in long-term to maintain low parasite loads and to avoid clinical relapses. traditionally, allopurinol is considered a very safe drug in the dog. however, some reports indicate that xanthinuria and xanthine urolithiasis is produced after prolonged therapy with allopurinol in the dog. the aim of this prospective study was to evaluate the prevalence of urinary adverse effects of allopurinol treatment ( mg/kg/bid/po) in dogs with leishmaniosis. diagnosis was made by compatible clini-copathological abnormalitites with leishmaniosis and high leishmania infantum-specific antibody levels assessed by quantitative elisa. once leishmaniosis was diagnosed, a close follow-up (day , , , and during treatment) including physical examination, baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis, urinary protein/creatinine ratio) and abdominal ultrasound was performed. in our preliminary results, dogs were included. dogs did not present any urinary abnormalities based on biochemistry profile, urinalysis and abdominal ultrasound at the time of diagnosis. four out of presented xanthinuria (day- [n = ], day- [n = ], and day- [n = ]). two out of dogs presented renal mineralization at day- of treatment. two out of dogs presented bladder urolithiasis since day- of treatment. xanthinuria was presented initially in all dogs that developed renal mineralization or bladder urolithiasis. dogs with renal mineralization and urolithiasis were treated with a restricted protein diet and, so far, they did not develop renal disease. the present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. a thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects. disclosures: no disclosures to report. giardia duodenalis is one of the most important gastrointestinal parasites in dogs and cats with a zoonotic potential. in germany the prevalence in dogs and cats reaches up to % and %, respectively. genotypes of two genetic assemblages of the parasites infect humans (assemblages a and b) and other mammals including small animals. in contrast, parasites of the assemblages c and d are specific for dogs, assemblage f for cats. objectives of the study were to analyse the prevalence, potential epidemiological risk factors and symptoms of g. duodenalis infections in dogs and cats. to detect g. duodenalis, feces from dogs and cats was analysed with an elisa technique. after dna extraction real time pcr as well as multi-locus sequence typing was performed for the following gene loci: triosephosphate isomerase-, glutamate dehydrogenase-, beta-giardin-gene, ssu rrna. with a questionnaire possible epidemiological risk factors were evaluated. statistical analyses were performed using spss (odds ratio, kolmogorow-smirnow test, spearman correlation). fecal samples of dogs and cats were collected over a time period of months. the elisa test was positive in / dogs and / cats. sixty-seven of giardia positive dogs and of positive cats had gastrointestinal signs. genotyping was successful in of dog samples and were assigned to assemblages as follows: assemblage a (n = ), a/c (n = ), a/d (n = ), b (n = ), b/d (n = ), c (n = ), c/d (n = ), d (n = ). only one of positive cat samples could be genotyped and was atypically identified as assemblage d. significant correlations between giardia infection and age, clinical signs, deworming status and staying abroad were found. in this monocenter study a prevalence rate of . % in dogs and . % in cats was detected, which is in good accordance with previous studies. the study further highlights a high rate ( %) of asymptomatically g. duodenalis infected animals. as potential zoonotic assemblages were detected, transmission of giardia from small animals to humans (and vice versa) cannot be excluded. especially young and not dewormed animals had a higher prevalence. disclosures: no disclosures to report. canine parvoviral enteritis remains a common cause of morbidity and mortality in young dogs. the goal of this study was to document a large cohort of affected dogs and analyze several factors as possible predictors of fatal outcome. medical records were retrospectively searched for dogs with parvoviral enteritis diagnosed with a positive fecal antigenic test or a fecal pcr. dogs were included only if the medical records were complete. the population was compared to the reference population of the hospital on the same time period with chi square tests and several factors were analyzed as possible predictors of death with a logistic regression. one hundred and forty seven cases were included. seventy percent of the dogs were non vaccinated puppies under the age of months. intact females and rottweiler, american staffordshire terrier and french beauce shepherd dogs were over-represented. clinical signs such as vomiting, diarrhea and dehydration were present in . %, . % and . % of the dogs respectively. hyperthermia, anemia and leucopenia were observed in . %, . % and . % of dogs respectively. the majority of the affected dogs were hospitalized for - days and the mortality rate was . % ( / dogs). hypoglycemia at admission was observed in / ( . %) dogs in which blood glucose was measured and was the only risk factor associated with death (p < . ). in this study, a predisposition of rottweiler, american staffordshire terrier and french beauce shepherd dogs was observed and hypoglycemia at admission was the only predictor of fatal outcome. disclosures: no disclosures to report. canine parvovirus (cpv) infections in dogs remain widespread around the world and still represent a major health threat in puppies. all vaccine manufacturers include this component in their core vaccination package, recommending two injections at - weeks interval from to weeks of age. despite broad vaccination coverage, number of reports suggesting lack of efficacy in vaccinated dogs have been reported, which implicate vaccines belonging to all major manufacturers. these cases are usually considered as being linked to the interference with maternal antibodies (matab), able to persist beyond weeks of age, which has led most expert groups to recommend a third vaccination around weeks of age. persistence of matab actually represents a major issue when immunizing puppies against parvovirosis. indeed, matab titres higher than / in the haemaglutination inhibition (hi) test can still inhibit vaccine uptake whereas such titres do not prevent field virus infection. in contrast, hi titres higher than / to / are usually considered as protective against disease and virus excretion. this "immunity gap"is therefore a critical period for the puppy and the outcome of the vaccination. in order to evaluate the impact of residual mda on the efficacy of a standard primary vaccination protocol, we performed a vaccination field trial with serological follow-up. eighty-eight puppies from to weeks of age presented at veterinary practice received injections at weeks interval. serology was performed by elisa before (at d /v ), during (at d /v ) and after (d ) vaccination. average maternal antibodies titres were strongly correlated with the age of the puppy at primary vaccination, remaining at vaccine inhibiting level until~ week of age. average titres increased significantly after st injection of primary vaccination in most groups and in all groups after the nd injection of primary vaccination. individual variability remained significant: vaccine uptake was inversely and strongly correlated to the pre-vaccinal matab titre at vaccination. seven out of puppies ( %) didn't seroconvert, despite vaccination complying to the recommended schedule. vaccination was started in such dogs between . and . weeks and completed between . and . weeks and average initial matab titre was . log compared to . for the general population. in conclusion, this trial supports the recommendation of an additional injection of primary vaccination at weeks, especially in areas of high parvovirus prevalence / pressure, where high levels of matab are likely to be transferred to puppies. disclosures: all authors are employees of merial. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . . % of the dogs were pcr positive. the aim of our study was to evaluate the usefulness of determining serum level of c-reactive protein (crp) in dogs naturally infected with bacterium a. phagocytophilum as a possible indicator of the clinical phase of the disease. pcr and/or ifa positive dogs with clinical presentation and/or thrombocytopenia were included in the study. based on the results, the dogs were divided into groups: pcr positive dogs; ifa positive (subdivided according to titer level from : to > : ) and pcr negative dogs; positive control group -pcr and ifa negative dogs with clinical signs and/or thrombocytopenia; negative control groupclinically healthy, pcr and ifa negative dogs. serum level of crp was determined using lifeassays Ò canine crp (lifeassays, lund, sweden). an elevated concentration of crp (> mg/l) was determined in pcr positive and ifa positive dogs with an ifa titer ≥ : and coincides with the presence of clinical signs (most commonly general clinical signs, elevated body temperature, gastrointestinal problems) and/or mild ( . %) or severe ( %) thrombocytopenia. the assessment of crp concentration, in correlation with certain clinical alterations and thrombocytopenia, suggests that crp concentration is elevated in the acute phase of the disease and is in correlation with the aforementioned changes therefore can serve as an additional diagnostic parameter. the crp concentration in ifa positive dogs, regardless of ifa titer levels, and with present clinical signs and thrombocytopenia is higher (above detectable level, > mg/l) than in dogs without clinical signs or laboratory alterations, which may speak in favor of reinfection or reactivation of a persistent infection at least in cases when no other cause of inflammation can be found. specific treatment would therefore be reasonable in such cases, especially in cases of rising crp concentration. disclosures: no disclosures to report. gallbladder agenesis is a very rare cause of elevated liver enzymes in dogs. in this study, we evaluated the features of dogs [six males (three castrated) and nine females (two spayed)] with suspected gallbladder agenesis on ultrasonography. five different breeds were included: chihuahua (n = ), toy poodle ( ), german shepherd ( ), jack russell terrier ( ), and shiba dog ( ). the median age was . ( . - . ) years. ten dogs were asymptomatic, while the other five dogs showed decreased appetite ( ), vomiting ( ), ascites ( ), seizure ( ), and diarrhea ( ). all dogs showed elevated liver enzymes, with high alanine aminotransferase levels (median, u/l; - u/l) in dogs and high gamma-glutamyl transpeptidase levels (median, u/l; - u/l) in . gallbladder agenesis was confirmed using laparoscopy in dogs and laparotomy in three. liver biopsy samples were obtained from all dogs. additional computed tomography cholangiography was performed for dogs using a -slice multidetector computed tomography (mdct) scanner following the intravenous administration of contrast medium (meglumine iotroxate). the obtained images were analyzed on a workstation, and they revealed an absent gallbladder in nine dogs and a vestigial gallbladder in three. the common bile duct was dilated in five dogs. for all dogs, laparotomy or laparoscopy was used to visualize the gallbladder and liver abnormalities, including malformed lobes and surface irregularities. acquired portal systemic collaterals were visually confirmed in five dogs, who also exhibited hypoplasia of the portal vein on histological examination. in conclusion, most animals with gallbladder agenesis were asymptomatic in our study, indicating a good long-term prognosis. however, symptoms associated with portal hypertension must be monitored in animals with primary portal vein hypoplasia. disclosures: no disclosures to report. assessment of systolic arterial blood pressure (sap) is an important tool in small animal internal medicine practice, especially with diseases or clinical conditions that can cause hypertension or hypotension. the doppler method is noninvasive and has several advantages compared to oscillometric method. there are few studies about the effect of body position on sap in conscious dogs. the hypothesis was that animal positioning during measurement alters sap values. the study design was prospective and randomized regarding order of positioning measurements. one hundred and twenty client-owned, conscious, healthy or sick adult dogs, weighing up to kg were included. sap was recorded by doppler ultrasonography following american college of veterinary internal medicine consensus statement with animals positioned in sternal recumbency, right lateral recumbency and with the dog laying down on owner s lap. the order of body position was raffled at the time of measurement. five consecutive measurements on each body position were performed always on left forelimb and the average was calculated. sap values were higher in sternal recumbency ( mmhg, p %- % = - . ; p < . ) compared to those obtained on the owner s lap ( mmhg, p %- % = . - . ), and both were similar to right lateral recumbency ( mmhg, p %- % = - ). these results suggest that sap measurement obtained on owner s lap or right lateral recumbency can be used on clinic routine, but sap measurement obtained on sternal recumbency should be avoided, because such measures may be overestimated. disclosures: no disclosures to report. canine patients may be presented for blood pressure (bp) assessment when clinical diseases associated with systemic hypertension (ht) are suspected but not confirmed; this population may encompass patients that have normal bp, true ht or situational ht. the clinician's aim is to identify animals with ht reliably, while minimizing false positives. this prospective study investigated the repeatability of duplicate within-visit systolic bp assessments (sbp and sbp ) in consecutive canine patients presented for bp assessment in the small animal clinic ( duplicate sbp recorded from dogs) and in a control group of healthy dogs ( duplicate sbp obtained from control dogs), resulting in duplicate measurements for analysis. doppler methods were used for duplicate assessments and oscillometric methods were used for duplicate assessments; cuff size/location were consistent within any dog. sbp ≤ mmhg was considered normal (nml); sbp > mmhg was considered abnormal (abn). median (range) elapsed time between duplicate readings was ( - ) minutes; % of sbp were obtained within minutes of sbp . there was no correlation between elapsed time and change in sbp (p = . ). % of sbp were equal to or lower than sbp ; median decrease was ( - no dog with sbp > mmhg (n = ) had nml sbp . more dogs with abn sbp were panting ( / scored, %) compared to the group with dogs with nml sbp ( / scored, %, p = . ). sbp of dogs that stopped panting ( / , %) tended to decrease (p = . ). within-visit repeatability of bp diagnosis was good in dogs with nml sbp , but apparent false positive diagnoses of ht occurred in % of dogs with abn sbp . sbp > mmhg was repeatable in all dogs. panting may be associated with increased measured sbp by these methods. duplicate within-day measurements may help identify false positive ht diagnoses in dogs with initial sbp measurements > mmhg. disclosures: no disclosures to report. median age at presentation was years. ess, cocker spaniels, ckcs and border collies presented at . , . , . and . years respectively. females were over-represented with / females ( / fn and / fe) and males ( / mn and / me). % of the ess cases presented were fn dogs. clinical presentation varied between dogs. clinical signs of pyrexia ( %), lethargy ( %) and anorexia ( . %) were the most common. other signs included cough, tachypnoea, dyspnoea, dysphagia, vomiting, diarrhoea, neck or spinal pain, abdominal pain, joint effusion or dermatologic signs thirty-one animals presented with peripheral lymphadenomegaly, but five animals displayed only internal lymph node enlargement. cytology was performed in / cases pyogranulomatous ( ) or granulomatous ( ) lymphadenitis. lymph node culture or staining (gram, pas, zn) was performed in and animals respectively, all of which were negative twenty-four animals were initiated therapy at mg/kg q hours. mean treatment length was weeks median relapse time was weeks. this study documents dogs with cssrl in the uk suggesting an over-representation in spaniel breeds (particularly ess), with females and young dogs typically affected. cytologic and histopathologic examination confirmed sterile lymphadenitis with animals showing marked and rapid clinical improvement pastor . hospital clinic veterinari animal medicine and surgery department, faculty of veterinary medicine of murcia, murcia, spain if only blood is available. therefore, if no effusion is present, diagnosis of fip still remains challenging. disclosures: dr. elisabeth mueller is the managing director of laboklin gmbh & co.kg. dr. karola weider is employed at laboklin gmbh & co.kg. this laboratory offered the "combined rt-npcr and sequencing approach which samples are the most relevant for analysis? a retrospective study of cases from maisons alfort, france, umr virologie inra-enva-anses cyprus is an island state in the eastern mediterranean basin. no epidemiological studies have yet been performed on infectious agents in cats from cyprus. the aim of this study was to determine the prevalence of several infectious agents, including some vector-borne infections, in cats from cyprus.surplus edta-blood and serum samples were recruited from cypriot cats, from which signalment and lifestyle characteristics were recorded. dna was extracted and real-time quantitative polymerase chain reaction (qpcr) assays were used to detect haemplasmas (mycoplasma haemofelis, "candidatus mycoplasma haemominutum" and "candidatus mycoplasma turicensis"), leishmania spp.and bartonella henselae. conventional pcr assays were used to detect ehrlichia/anaplasma spp. samples yielding positive results for leishmania spp. or ehrlichia/anaplasma spp. underwent further characterisation (sequencing). elisas were performed for the detection of l. infantum antibodies, feline leukaemia virus (felv) antigen and feline immunodeficiency virus (fiv) antibodies. statistical analysis was performed using spss for the assessment of any associations between variables and infectious agents.of the samples extracted, were excluded due to failure of ≥one internal control pcr. of the remaining samples, ( . %) were positive by pcr for haemoplasma including ( . %) for m. haemofelis, ( . %) for "ca. m. haemominutum" and ( . %) for "ca. m. turicensis". nineteen ( . %) were positive for b. henselae. one cat ( . %) was pcr positive for ehrlichia/anaplasma spp. and sequencing revealed identity with anaplasma platys. leishmania spp. dna was detected in of the ( . %) cats; sequencing revealed l. infantum in of these cases. l. infantum serology was positive in of the cats tested ( . %). only one cat was positive for both leishmania pcr and serology. of the cats that underwent retroviral serology, ( . %) were felv and ( . %) were fiv positive.statistical analysis identified several significant associations (p < . ) including the following; haemoplasma infection and both outdoor access and feral-shelter cat origin, felv or fiv infection and both anaemia and feral-shelter cat origin.this study documents, for the first time, the presence of haemoplasmas, l. infantum, b. henselae, a. platys, felv and fiv in the feline population of cyprus. the prevalence of haemoplasma, fiv and b. henselae infections were among the highest reported in cats from mediterranean countries, while that of leishmania spp. was similar. this is the second report of a. platys infection in a cat from a mediterranean country.disclosures: no disclosures to report. bartonella species (spp.) are zoonotic pathogens, and infections in cats are common. prevalence in cats from southern germany is still unknown. the aim of this study was to determine the prevalence of bartonella spp. dna in blood of cats in southern germany and to evaluate associations between bartonella bacteremia, housing conditions, feline immunodeficiency virus (fiv), and feline leukemia virus (felv) status, including progressive, regressive, and abortive felv infection.blood samples of cats that were presented to different veterinary clinics in southern germany for various reasons were tested for bartonella spp. dna using a previously published conventional polymerase chain reaction (pcr) targeting a fragment of the s- s rrna intergenic spacer region. for statistical analysis, fisher's exact test was used.prevalence rate of bartonella spp. bacteremia was . % ( / ; ci: . %- . %). b. henselae was amplified in eleven of these cats. one cat was positive for b. clarridgeiae dna. most of the infected cats were clinically healthy, but half of the cats had thrombocytopenia, potentially caused by their bartonella spp. infection. there was no significantly higher risk to be infected with bartonella spp. when living mainly outdoors or being fiv-or felv-infected.prevalence of bartonella spp. bacteremia is low in southern german cats, but there is still a risk of human bartonella infection associated with cat ownership. most clinical changes of the bartonella spp.-infected cats were related to other diseases. however, thrombocytopenia was common and further studies are required to define its potential clinical relevance.disclosures: no disclosures to report. ante-mortem diagnosis of feline infectious peritonitis (fip) is still challenging. the aim of this study was to evaluate sensitivity and specificity of a "combined reverse transcription nested polymerase chain reaction (rt-npcr) and sequencing approach", detecting mutations at two different nucleotide positions within the spike gene, that previously were shown to correlate with the fip phenotype. the study population consisted of cats with confirmed fip and a defined control group of cats for which fip was considered an important differential diagnosis, but that were definitively diagnosed with other diseases. blood and/or effusion samples were examined for feline coronavirus (fcov) rna by rt-npcr and, if positive, nucleotide positions and were sequenced for nucleotide transitions. sensitivity, specificity, negative and positive predictive values were determined and % confidence intervals ( % ci) calculated.rt-npcr detected fcov in cats in blood (n = ) and/or effusion (n = ); all of them had fip. one of the mutations of interest was found in / of the pcr-positive blood samples and in / of the pcr-positive effusion samples. diagnostic specificity of the "combined rt-npcr and sequencing approach" was % in blood ( % ci . - . ) and effusion ( % ci . - . ). diagnostic sensitivity was . % ( % ci . - . ) in blood and . % ( % ci . - . ) in effusion.a positive test result therefore confirms a suspicion of fip. a negative result, however, cannot be used to rule out fip, especially feline infectious peritonitis (fip) is a viral disease caused by the virulent strain of feline coronavirus (fipv). the disease can appear under two clinical forms, dry or effusive, both leading to a fatal outcome. diagnosis was based on histopathologic lesions on necropsy until the recent discovery of mutations associated with the fipv strain in the c and spike (s) genes. our main goal was to detail the distribution of c or s gene mutations in different biological samples of cats suffering from fip.this was a retrospective, observational study of cats showing clinical signs compatible with fip. ten out of cats were of pure breed. . % were males and . % females. median age was . months at presentation. the clinical presentation, pathologic findings and virologic data were reviewed. according to clinical signs, cats were classified with a dry form and with a wet form of fip. the main clinical signs included dehydration, hyperthermia, icterus, abnormal abdominal palpation, neurological and ocular disorders.when possible blood, fecal material, effusion, fine needle aspiration (fna) from relevant organs or a combination of these, was recovered from each cat. feline coronavirus (fcov) was first researched by rt-pcr, then the c and part of the s genes were sequenced to determine the eventual presence of mutations.among the dry cases, fcov was detected in / blood samples, / fecal samples and fna ( / ). among the wet cases, / blood samples, / fecal samples and all effusion samples ( / ) were positive for the presence of fcov. c mutations were never found in fecal samples but were found in / effusion samples and in / fna. s mutations were detected in / fecal samples, / fna and / effusion samples. for three cats, no mutation, neither in c or s genes was identified despite the confirmation of fip by necropsy.s gene mutation is more frequently observed than c gene despite in two cases where only c mutations were identified. moreover the presence of strains harbouring s mutation in feces has never been described before and could suggest the possible diffusion of fipv among feline population.in conclusion, viral diagnosis of fip based on rt-pcr sequencing in effusion and fna samples is essential. rt-pcr resulting from blood samples should be carefully interpreted because of high risk of missing fipv.finally, searching for mutations in both s and c genes is recommended.disclosures: no disclosures to report. methicillin resistant staphylococcus aureus (mrsa) has recently become a great concern for pet animals' disease and zoonotic infection. mrsa strains transfer between pet animals and humans could occur. the aim of the present study was to determine the occurrence of mrsa in household dogs.from january to june , clinical samples were collected from dogs, patients of the veterinary teaching hospital of the department of veterinary sciences of messina (italy), affected by several diseases of various origins. all samples were processed by bacteriological conventional methods for isolation and identification. all strains were tested for phenotypic susceptibility to oxacillin and were subjected to a pcr protocol for the detection of meca gene. strains carrying the gene were considered methicillin resistant (mrs). lastly, on both mrs and methicillin sensitive (mss) strains, kirby-bauer disk diffusion susceptibility testing were performed to highlight resistance profiles using molecules belonging to the main classes of antimicrobials used in veterinary practice. strains resistant to at least one molecule of three or more classes of antibiotics were considered multidrug-resistant (mdr). the statistical analysis of the results was made using the z-test by a primer Ò software.forty staphylococcus spp. strains were isolated, belonging to species. the most frequently isolated microorganisms were staphylococcus aureus with isolations ( %) and staphylococcus pseudintermedius with isolations ( . %), followed by staphylococcus epidermidis with isolations ( %) and staphylococcus cohnii and staphylococcus warneri, both with isolations ( %). a single isolation ( . %) was obtained for each of the species staphylococcus chromogenes, staphylococcus haemolyticus, staphylococcus lentus, staphylococcus lugdunensis, staphylococcus saprophyticus, staphylococcus simulans and staphylococcus sciuri. thirteen ( . %) strains of staphylococcus spp. were phenotypically resistant to oxacillin and three staphylococcus aureus ( . %; n. from pyoderma, n. from exudative pleural effusion) were positive for the meca gene. all strains of staphylococcus spp. were mdr. our results showed the presence of mrsa and multidrug-resistant staphylococcal strains in household dogs. a lack of correspondence between antimicrobial susceptibility tests and molecular methods was found in the present study.disclosures: no disclosures to report. haemotropic mycoplasmas (haemoplasmas) are bacteria that infect domestic cats. approximately % of ill cats have haemoplasma infection. three main feline haemoplasma species have been detected worldwide; mycoplasma haemofelis (mhf), "candidatus m. haemominutum" (cmhm) and "candidatus m. turicensis" (cmt). a fourth haemoplasma called "candidatus m. haematoparvun-like" (cmhp) was latter identified in cats. the only published study in chile was carried out in cats, with prevalences by pcr of . % to mhf, and % to cmhm.the aim of this study was to perform molecular detection of haemoplasmas in cats from valdivia, southern chile. blood samples were taken from cats and used for haemoplasmas dna detection by quantitative real time pcr (qpcr) at universidad austral de chile. qpcr protocol was based on detection of feline dna targeting s housekeeping gene and mycoplasma spp. s rrna gene (universal primers, my sf forward, my sr y my sr , both reverse) by sybr green method. the melting temperature (tm) analysis allowed identifying the infecting mycoplasma species (mhf, cmhm, cmt). it was not posible to identify haemoplasmas species on co infected cats, so a second qpcr specie specific protocol was applied on these samples. second qpcr protocol was based on s rrna gene, with specific primers to detect mhf, cmhm, cmt and cmhp. all samples ( / ) were positive to s gene, proving presence of cat dna. from the cats, . % ( / ) were positive to haemoplasmas, where . % ( / ) corresponded to cmhm (tm . - . °c), . % ( / ) to mhf (tm . - . °c), % ( / ) to cmt (tm . - . °c) and . % ( / ) to co infections. associations between cmhm+mhf, cmhm+cmt, cmhm+cmhp and cmhm+mhf+cmhp were detected on co infected animals. these results agree with those found in previous key: cord- -a qr nl authors: pires, sara m.; fischer-walker, christa l.; lanata, claudio f.; devleesschauwer, brecht; hall, aron j.; kirk, martyn d.; duarte, ana s. r.; black, robert e.; angulo, frederick j. title: aetiology-specific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: a qr nl background: diarrhoeal diseases are major contributors to the global burden of disease, particularly in children. however, comprehensive estimates of the incidence and mortality due to specific aetiologies of diarrhoeal diseases are not available. the objective of this study is to provide estimates of the global and regional incidence and mortality of diarrhoeal diseases caused by nine pathogens that are commonly transmitted through foods. methods and findings: we abstracted data from systematic reviews and, depending on the overall mortality rates of the country, applied either a national incidence estimate approach or a modified child health epidemiology reference group (cherg) approach to estimate the aetiology-specific incidence and mortality of diarrhoeal diseases, by age and region. the nine diarrhoeal diseases assessed caused an estimated . billion ( % uncertainty interval [ui] . – . billion) cases and , ( % ui , – , ) deaths worldwide in . the largest number of cases were caused by norovirus ( million; % ui – , million), enterotoxigenic escherichia coli (etec) ( million; % ui – million), shigella spp. ( million; % ui – million) and giardia lamblia ( million; % ui – ); the largest number of deaths were caused by norovirus ( , ; % ui , – , ), enteropathogenic e. coli ( , ; % ui , – , ), etec ( , ; % ui , – , ) and shigella ( , ; % ui , – , ). there were marked regional differences in incidence and mortality for these nine diseases. nearly % of cases and % of deaths caused by these nine diarrhoeal diseases occurred in children under five years of age. conclusions: diarrhoeal diseases caused by these nine pathogens are responsible for a large disease burden, particularly in children. these aetiology-specific burden estimates can inform efforts to reduce diarrhoeal diseases caused by these nine pathogens commonly transmitted through foods. diarrhoeal diseases are a major cause of disease burden worldwide [ ] . the global burden of disease study (gbd ) ranked diarrhoeal diseases as the fourth largest disease burden, accounting for . % of the total disease burden globally. diarrhoeal diseases accounted for an even higher proportion ( %) of the total disease burden in children < years of age [ ] . diarrhoeal diseases have a substantially higher impact in low-income countries and regions with poor water quality, sanitation and food safety. diarrhoeal diseases are caused by a variety of bacteria, viruses, and parasites, many of which are commonly transmitted through food [ ] . despite the large disease burden caused by these pathogens, the global contribution of specific aetiological agents of diarrhoeal diseases is largely unknown. for example, recent studies have estimated the worldwide incidence of diarrhoeal diseases in children < years of age [ ] , and in older children and adults [ ] , but did not provide aetiology-specific estimates. lanata et al. [ ] and fisher walker et al. [ ] provided aetiology-specific estimates, but only for diarrhoeal deaths among children < years of age, and diarrhoeal cases in persons years of age, respectively. another large-scale study estimated diarrhoeal aetiologies in children < years of age in specific study sites, particularly in sub-saharan africa and south asia [ ] . other studies have estimated the incidence of specific foodborne diseases, many of which cause diarrhoea, but each focused on a single developed country [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to identify and prioritize targeted interventions to reduce the public health impact of foodborne diseases, public health policy makers and other stakeholders need aetiology-specific regional and global estimates of the incidence and mortality of diarrhoeal diseases caused by pathogens that are commonly transmitted through foods. these estimates, combined with knowledge on the proportion of this burden that is derived from foods, will form the basis for the estimation of the global and regional burden of foodborne diseases. as part of the effort by the world health organization (who) foodborne disease burden epidemiology reference group (ferg; http://www.who.int/foodsafety/areas_work/ foodborne-diseases/ferg/en/) to estimate the disease burden of foodborne diseases, we estimated the global and regional incidence and mortality of diarrhoeal diseases which are commonly transmitted through foods. after reviewing the epidemiology of diseases caused by bacteria, viruses, and protozoa that are commonly transmitted to humans through food, and which are included in ferg, we selected nine pathogens which commonly cause diarrhoea for inclusion in our study: campylobacter global spp., cryptospordium spp., entamoeba histolytica, enterotoxigenic escherichia coli (etec), enteropathogenic e. coli (epec), giardia lamblia, norovirus, salmonella spp., and shigella spp. we did not include shiga-toxin producing e. coli (stec), and vibrio cholerae, diarrhoeal pathogens included in ferg, because the incidence and mortality of diseases caused by these agents have been estimated using different approaches and published elsewhere [ , ] . although some countries have published national incidence and mortality estimates for diseases caused by most of these nine pathogens, such estimates of foodborne diseases, which are considered to be the highest quality burden estimates for these diseases [ ] , are only available from a limited number of countries. we therefore used two approaches to estimate the incidence and mortality of the diseases caused by the nine selected diarrhoeal pathogens, and applied one or the other approach in each country in the six regions. approach was applied to all countries in the european region (euro) and all other countries with overall low mortality as defined by who (http://www.who.int/choice/demography/mortality_strata/en/); approach was applied to all remaining countries. using these two approaches allowed us to utilize the highest quality available data in countries with overall low mortality which have similar sanitary and public health infrastructure and presumed incidence of foodborne diseases. the final results of the two approaches were merged in a last step to determine global estimates and estimates for the regions. the first approach utilized available national incidence and mortality estimates of diseases caused by the nine selected pathogens and was applied to countries in euro (sub-regions a, b, and c), and low-mortality countries in american region (amro; sub-region a) and western pacific region (wpro; sub-region a). we conducted a literature review and consulted with the international collaboration of foodborne disease burden of illness studies (http://www.cdc.gov/ncezid/dfwed/international/enteric-burden-collaboration.html) to identify all published national estimates (with associated uncertainty intervals) of foodborne diseases. such estimates were derived through studies that corrected national surveillance data to account for under-diagnosis and underreporting [ ] . in cases where national estimates were expressed as number of cases and deaths, and not as incidence and mortality rates (per , population), we used united nations population figures for the national study year to create such rates. if a national estimate only included domestically-acquired infections, we used the proportion of infections acquired during international travel in a neighbouring country to derive revised estimates that included infections acquired abroad. we applied the national incidence and mortality estimates, with the associated uncertainty intervals, to each country with such national estimates, and the median incidence and mortality of all studies, with uncertainty intervals associated with those median estimates, to each country in euro, amro sub-region a, and wpro sub-region a that did not have national incidence or mortality estimates. this approach resulted in incidence and mortality estimates (per , population), with uncertainty intervals, for each of the countries for each of the nine diseases. overall estimates were partitioned to the age groups < years of age and years of age on the basis of the age distribution of the population in each region. further details of this approach are available in s appendix. regional incidence and mortality of diarrhoea. first, we identified the total number of diarrhoeal cases in the countries for by combining estimates based on systematic reviews for children < years of age and persons years of age [ , ] . for the estimate of the total number of diarrhoeal deaths, we obtained data on the total number of deaths in the countries in attributed to diarrhoeal diseases from who (http://www.who.int/gho/en/; accessed june ); we used the range of diarrhoeal deaths estimated in the global burden of disease study (gbd ) to derive an uncertainty interval for the death envelope. we derived the final number of diarrhoeal cases ("diarrhoeal envelope") and diarrhoeal deaths ("diarrhoeal death envelope") by subtracting published estimates of the number of diarrhoeal cases and deaths caused by stec [ ] and v. cholerae [ ] . aetiology proportions. our next step was to estimate the proportion of diarrhoeal illnesses and deaths due to the nine pathogens by extracting the aetiological proportions of diarrhoeal cases and deaths for each pathogen by region from systematic reviews of studies reporting stool sample isolation and detection from inpatient, outpatient, and communitybased studies of persons with diarrhoea. we assumed that the distribution of pathogens observed among outpatient and community studies represented the pathogen prevalence among diarrhoeal cases, and that the distribution of pathogens among inpatients hospitalized for severe diarrhoea represented the pathogen prevalence among diarrhoeal deaths for all age groups. due to data scarcity, we also included inpatient studies to estimate the aetiological proportions of diarrhoeal cases in persons years of age (i.e. all available studies). we assumed that g. lamblia infection was unlikely to result in death based upon the data available from the national foodborne disease mortality estimates, and therefore excluded it from mortality estimates. to determine aetiological proportions, we used systematic reviews to identify studies that reported isolation or detection of pathogens from stool specimens or rectal swabs collected from persons with diarrhoea. for norovirus, we used a previously published systematic review including studies published between and and then updated through [ ] . the norovirus-specific review was conducted because of the increased recent use of molecular diagnostics globally and as part of a parallel effort to estimate the global burden of norovirus disease [ ] . for all other pathogens we included two previously published systematic reviews: ) the aetiology of diarrhoeal disease studies for children years of age published between and [ ] and ) the aetiology among children < years of age published between and [ ] . we updated each of these reviews to include studies published through . s appendix details the systematic review methodologies and results of the updated reviews, including search terms and inclusion/exclusion criteria. studies focusing only on norovirus collected in the general reviews were excluded to avoid duplicates with the norovirus-specific review. the general systematic reviews collected data on isolation or detection of the nine pathogens included in our study and on pathogens not commonly transmitted by food (e.g. rotavirus, sapovirus, astrovirus, and coronavirus); the latter were grouped together as "other pathogens". we initially calculated study-specific aetiology-proportions by dividing the number of samples positive for the pathogen by the total number of samples tested in that study (studyproportions); we then estimated regional proportions by calculating the median aetiology-proportion of all study-estimates within each region (regional-proportions). for example, if studies conducted in the western pacific region (wpro) provided data on the number of salmonella isolates among diarrhoeal cases, this region's salmonella-proportion was estimated as the median of the estimated study-specific aetiology proportions. since several of the studies among children < years of age used narrower age ranges, in analyses a and b we calculated an age-adjusted proportion for this age group by calculating a conversion factor as the ratio of the median proportion in the age group - months to the median proportion in age group x ((median (prev - )/median (prevx)). we applied this approach when three or more studies for each pathogen contributed to each of the two medians. if this condition was not met, we borrowed the conversion factor for the age group from a similar age group within the same pathogen (for example, used the conversion factor calculated for studies including infants - months of age for studies that included infants - months of age). for persons years of age, we have assumed that differences between narrower age categories would be diluted in this very broad population group and have chosen not to age-adjust medians. to estimate the proportion of diarrhoeal stools due to unknown aetiology, we included studies that sought pathogens and reported patients with an unknown cause of disease. if only one study tested for a given pathogen in one region, we applied criteria to identify outliers and prevent potentially non-representative studies from misrepresenting the final regional aetiology-proportions. an outlier was defined as a regional estimate ± times the global median. if a regional estimate derived from studies was identified as an outlier, the individual studies' prevalence and sample size were evaluated, and a particular study was excluded if it represented an outlier when compared to remaining studies within the region. outliers were excluded from the results and taken as a missing value. if a regional median aetiological proportion estimate was missing (due to either missing data or exclusion of an outlier), the missing regional estimate was replaced by the global median of the aetiological proportions for that pathogen. all global medians were estimated after the exclusion of potential outliers. statistical analysis. to account for uncertainty in these calculations, we used monte carlo simulation in all steps of the analysis. we applied a bootstrapping analysis to derive % uncertainty intervals (ui) around aetiology-proportions. 'pseudo-data sets' were created by sampling each study with replacement from the real dataset. in each of these , pseudo-datasets, a different random number of positive samples for each study was generated from a binomial distribution defined on the basis of the number of samples and the expected proportion in that study. all pseudo-datasets were used in the estimation procedure described above to generate corresponding , study-specific aetiology proportions, and regional aetiology proportions. the . th and . th percentile of these proportions gave the % ui. data management and analyses were conducted in sas enterprise guide . . we then constrained the aetiological proportions for the nine diarrhoeal pathogens, for other pathogens not commonly transmitted by food, and unknown aetiology to ensure that they did not sum to more than % in each region. for this purpose, we first fitted univariate beta(α, β) distributions to the median proportions and simulated quantiles (i.e., the . th and . th percentiles). the distributions' optimized parameters were estimated via one dimensional optimization, minimizing the squared distance between the estimated and fitted quantiles, and ensuring the median of the fitted distribution to be similar to the estimated median. next, , random deviates were sampled from the fitted beta distributions. if needed, an "unknown aetiology" category was created as minus the sum of simulated proportions across aetiologies, per iteration. finally, the random deviates were normalized iteration-wise by dividing them by the sum of simulated aetiological fractions. once estimates of aetiological proportions for cases and deaths for the nine pathogens were derived, the regional aetiological proportions for each disease were multiplied by the respective estimates for total diarrhoeal illnesses and deaths in those regions, accounting for uncertainty using a stochastic model with , iterations. we then derived age-stratified incidence and mortality estimates, with associated uncertainty, for each disease for each region using country-level population data for from the revision of the united nations world population prospects (esa.un.org/wpp, accessed june , ); among the countries, all countries in the same region were assumed to have the same incidence and mortality, and associated uncertainty, for each pathogen. in order to aggregate results with estimates derived for low-mortality countries (approach ), age-specific estimates were then grouped into overall estimates. the stochastic model was implemented in r version . . (r core team, ). global estimates (combining approach and approach ). the incidence and mortality estimates, with associated uncertainty, for each disease for each region obtained through approach and approach were combined in a final step to produce final global estimates and estimates for each of the six regions. we identified national incidence and mortality estimates of the nine diarrhoeal diseases for seven countries: australia, canada, france, netherlands, new zealand, united states of america, and the united kingdom [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the national estimates from australia and canada included only domestically-acquired infections; revised national estimates were derived using data on international travel-acquired infections from the new zealand and the united states, respectively. estimates of incidence and mortality for each of countries are available in s appendix. the systematic reviews conducted to collect data to estimate the proportion of diarrhoeal illnesses and deaths in the other countries yielded outpatient and community studies (fig ) and inpatient studies (fig ) among children < years of age, and inpatient, outpatient, and community studies (fig ) among persons years of age. table lists the number of studies for each of the nine pathogens by region. the final number of studies providing data for individual pathogens in different regions varied substantially. the data available for persons years of age were scarcer and the number of studies for each region was low, particularly for the afro and amro regions. table a in s file presents the estimates of the regional envelopes of diarrhoeal cases and deaths, and tables b to b in s file present the contribution of each pathogen for these envelopes. we estimated that the nine pathogens included in our study resulted in a total of . billion cases ( % ui . - . ) and . million deaths ( % ui . - . ) in worldwide (table ). in the countries in euro and amro and wpro a regions, we estimated that these nine pathogens resulted in . million cases ( % ui . - , . ) and , deaths ( % ui , - , . in the remaining countries, we estimated these pathogens resulted in a total of . billion cases ( % ui . - . billion) and , , deaths in ( % ui , , - , , ). global and regional estimates of incidence and mortality rates caused by these pathogens are presented in s there were marked regional differences in incidence and mortality caused by the nine diseases (table ; figs and ). among the nine pathogens, norovirus was the most common cause of cases in all regions, particularly among persons years of age (fig ) . among these nine pathogens, norovirus and g. lamblia were the cause of the highest incidence in children < years of age; in afro, amro, emro and wpro campylobacter caused the third highest incidence in afro and wpro, and cryptosporidium spp. made a substantial contribution in emro. the second highest incidence of cases among persons years of age was caused by salmonella spp. in emro, searo and wpro. for deaths caused by the nine pathogens, epec and norovirus were the most frequent causes in children < years of age in afro, amro, searo and wpro (fig ) . etec was also estimated to be an important cause of mortality, particularly in afro and amro, and shigella spp. was the leading cause in emro and third leading in afro. salmonella spp. caused the largest proportion of diarrhoeal deaths in this age group in euro. in persons years of age, norovirus was the leading cause of mortality in all regions. these are the first global and regional estimates of incidence and mortality caused by nine pathogens that cause diarrhoea and are commonly transmitted through foods. of these agents, we showed that norovirus was responsible for the largest number of cases followed by etec, g. lamblia and shigella spp. norovirus caused the most deaths among these nine pathogens, followed by epec and shigella spp. nearly % of all cases and % of deaths caused by these nine pathogens occurred in children < years of age, who represent only % of the global population. we also demonstrated regional differences in both the incidence and mortality of diseases caused by these nine pathogens. these differences are important to consider when defining interventions to reduce the burden of diseases commonly transmitted through foods. as an example, campylobacter and salmonella, which are pathogens that have mainly domestic food-producing animals as reservoirs and usually infect humans due to contamination in the food production chain, play a major role in the incidence of foodborne enteric disease in the euro region but are less frequently observed in who-defined high mortality countries. in high mortality countries where poor sanitary conditions and food and water contamination are more important, pathogens such as g. lamblia and etec play more important roles. the predominance and relative ubiquity of norovirus across all regions suggests that targeted interventions, such as vaccines, may be necessary to reduce the burden of this human reservoir pathogen. our study focused only on diarrhoeal pathogens that are commonly transmitted through foods. it is important to recognize that the estimates we present include disease from all modes of transmission, including contaminated food, water, and environments, along with infected persons and animals. our inclusion of only pathogens that are commonly transmitted through food also meant that we did not present estimates for important diarrhoeal agents, such as rotavirus. by including them into other pathogens, we have assured that we did not overestimate the incidence and mortality of remaining pathogens when distributing the diarrhoeal total cases and deaths to the nine diseases of interest. we used two approaches to estimate the relative contribution of different aetiologies for disease because the quality and availability of data varied substantially between countries and regions. national incidence and mortality estimates were available only from seven low mortality countries, but given the high quality of these estimates, we gave priority to these data by using them as the basis for estimating the incidence and mortality of the diseases caused by the nine pathogens for all euro and other low mortality countries (sub-region a in amro and wpro). for all other countries, we adapted the cherg approach for estimating the burden of diarrhoeal diseases in these countries [ ] . this approach was facilitated by the availability of estimates of the envelope of diarrhoeal deaths, along with recent advances in diarrhoeal disease diagnosis, such as widespread application of polymerase chain reaction (pcr) for norovirus detection. we have divided countries for the two approaches on the basis of their overall mortality status, as defined by who, which we assume can be used as a proxy for differences in the public health status of the countries. the two approaches differed in terms of methodology, assumptions and type of data used. while approach analysed national incidence and mortality of disease by pathogens commonly transmitted through foods estimated primarily by correcting surveillance data to account for underreporting and under-diagnosis, approach relied on systematic reviews of studies identifying causative agents in patients with diarrhoea. applying approach for all regions was not possible due to the scarcity of community, inpatient and outpatient studies from low mortality countries for most pathogens. because of these differences, we acknowledge that comparisons between regions should be made with care. we relied on studies that estimated the incidence of potentially foodborne diseases in a specific country by correcting public health surveillance data for underreporting and under-diagnosis for approach . such studies have their own limitations, such as relying on a variety of data with variable quality and representativeness, multiple modelling approaches and a wide range of assumptions. as an example, most use population surveys on care-seeking behaviours of patients with diarrhoea, which are subject to recall bias that can influence the results [ ] . furthermore, in approach we have assumed that the median aetiology-proportion of the seven collected studies (along with its uncertainty interval) represented the relative *not all studies provide data for pathogens included in our analysis. remaining studies were used to estimate the aetiology-proportion of "others". †studies conducted in countries in the euro region, amro sub-region a, or wpro sub-region a were not included in the analyses. doi: . /journal.pone. .t contribution of each agent for the diarrhoeal morbidity and mortality estimates, and have taken a simplified approach to estimate uncertainty. approach to estimate the contribution of aetiologies for diarrhoeal cases and deaths used studies that tested for varying numbers of pathogens. studies that focused on a single pathogen may tend to overestimate the importance of that pathogen because they are potentially more likely to be conducted in a study site with a high prevalence of that pathogen [ ] or have selective inclusion criteria e.g. acute watery diarrhoea. however, we needed to include them due to estimates exclude the proportion of the incidence envelope attributable to "other pathogens", because these were not considered in approach (applied to countries within euro, amro sub-region a, and wpro sub-region a). doi: . /journal.pone. .g the low number of studies focusing on multiple pathogens, particularly in the older age group. due to scarcity of data, some regional aetiological proportion estimates were informed by a single study. to avoid that studies with potentially over or underestimated aetiology-proportion estimates misrepresented the estimate for that region, we have defined criteria to identify outliers, and have excluded these estimates from the results. these overestimations or underestimations can have various causes, such as study locations with a particularly high or low prevalence of that pathogen, a small sample size, or a country not representative of the whole region. excluded outliers were treated as missing values, and the aetiology-global median used to represent the regional estimate. in this approach, we also assumed that the distribution of pathogens among inpatients hospitalised with severe diarrhoea represented the relative importance of these pathogens for diarrhoeal deaths. this meant that we used severity of disease as a proxy for mortality, which may lead to an over-or under-estimation of the number of deaths caused by some pathogens. in the analyses for persons over five years of age, due to sparseness of data we decided to use data from inpatient studies to estimate aetiology-proportion of cases, which may also have led to an erroneous estimation of the contribution of pathogens to diarrhoeal incidence. additionally, this approach relied on stool samples, but some patients that tested positive for a pathogen may have had asymptomatic infections. this is more likely to happen for pathogens that have long excretion periods after illness (e.g. norovirus). carefully conducted longitudinal studies would be needed to distinguish clinical cases from asymptomatic infections [ ] . in approach , we estimated the proportion of the diarrhoeal cases and deaths that was caused by unknown aetiology. the defined strategy was to base our estimates on studies that had collected data for eight or more pathogens and had reported "aetiology unknown". undiagnosed cases could be caused by truly unrecognized agents, but also could be due to e.g. the use of incorrect or insensitive testing methods, to antimicrobial-therapy prior to stool sampling, or to non-infectious diarrhoeal causes. this strategy could only be adopted for the analyses for children < years of age, and still there were few studies available for the calculations. in remaining analyses, unknown was calculated as the difference between % and the sum of all remaining aetiologies (including others). in both approaches, we assumed that studies conducted in a given country would be representative of the region or sub-region of this country. when several studies providing data for a given pathogen were available for that region, calculating a median estimate ensured that the influence of extreme values was restricted. on the contrary, when only one or few studies were available, this study's/studies' estimate was assumed to represent all countries within a region. in addition, the six regions' classification was based on the geographic distribution of countries, and countries within each are very diverse. we tried to avoid inappropriate extrapolations by applying distinct approaches to low mortality and medium and high mortality countries, assuming that a country's mortality status may also reflect the nation's public health and food safety situation. in the euro region, we made an extra extrapolation and assumed that all countries were similar to sub-region a countries in terms of foodborne diseases' incidence and mortality. this region includes countries with other mortality status (euro b and euro c), and some of these countries may have a different distribution of aetiologies. due to lack of data, we were not able to explore the extent of these differences. a recent large prospective matched case-control study in children < years of age conducted over three years at selected sites in africa and asia (the gems study) estimated the burden of aetiology-specific diarrhoea in these sites, and concluded that rotavirus, cryptosporidium spp., etec and shigella spp. were the most important causes [ ] . this study was fundamentally different from ours: among other differences, it calculated attributable cases of diarrhoea to each aetiology, comparing patients (moderate to severe diarrhoeal cases seen in health facilities) and controls (un-matched healthy children in a community sample). the gems study excluded mild diarrhoeal cases, which along with their moderate diarrhoeal cases would constitute our non-fatal incident cases. by including moderate and severe cases instead of only severe hospitalized cases, the gems study does not have a good proxy for fatal cases as we defined it. still, the study's findings are generally consistent with ours; epec, etec, shigella spp. and cryptosporidium spp. were among the most important causes of diarrhoea. one important difference was on the relative contribution of norovirus for diarrhoea incidence and mortality: our results suggest that norovirus is amongst the most important causes of diarrhoeal cases and deaths, whereas the gems study estimated a lower contribution of this pathogen. this discrepancy may indicate that our approach may lead to an overestimation of the proportion of diarrhoea attributable to norovirus or other pathogens that are likely to be carried asymptomatically after the first exposure (like certain types of etec, e.g. lt-etec) because it does not account for the potential detection of norovirus in the stool of healthy individuals [ ] . however, the gems study required that control individuals did not have a diarrhoeal episode in the days prior to sampling; because norovirus patients can excrete the virus for to weeks after infection, it is likely that some controls included in this study corresponded to asymptomatic carriers of norovirus [ ] . these findings may suggest limitations in ascribing pathogenicity based on case-control studies, particularly in developing country settings in which frequent exposures and a high degree of transmission results in common reinfections [ ] . a recent community based prospective cohort study (the "etiology, risk factors, and interactions of enteric infections and malnutrition and the consequences for child health and development project", the mal-ed study), supported our results and estimated that norovirus was the most important cause of diarrhoea in young children [ ] . our study provides information on the incidence and mortality of nine pathogens that cause diarrhoeal and are commonly transmitted through food. most of these agents can also infect humans through other sources (e.g. environmental, contact with animals and person-toperson transmission), and the relative contribution of these sources for disease varies between aetiologies and regions [ ] . to estimate the number of foodborne aetiology-specific diarrhoeal cases and deaths, our results need to be combined with regional source attribution-proportions and other aetiological agents [ ] . these results, as well as the foodborne disease burden of each disease in terms of disability adjusted life years (dalys) are presented elsewhere [ ] . our results provide public health policy makers, including risk managers, and other stakeholders with information for advocacy for improved regulation and control of diseases commonly transmitted through foods. we highlight the most important diarrhoeal diseases in different regions and age groups, which will allow policy makers to define and improve control strategies targeted at different pathogens, settings and countries. supporting information s appendix. estimating the aetiology-specific incidence and mortality of diarrhoea in countries in the region of the americas (amro) sub-region a, western pacific region (wpro) sub-region a, and european region (euro; sub-regions a, b and c). (docx) s appendix. detailed description of the methods of the systematic reviews used to identify studies that provided data to derive aetiology-proportion estimates for all included pathogens except norovirus. (docx) table b : regional and global etiology-proportions of diarrhea cases in children < , (median % and % ci). table b : regional and global etiology-proportions of diarrhea cases in persons > , (median % and % ci). table b : regional and global etiology-proportions of diarrhea deaths in children < , (median % and % ci). table b : regional and global etiology-proportions of diarrhea deaths in persons > total global incidence and mortality rate (per , ) of foodborne diarrhoeal pathogens (median and % uncertainty intervals) disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study evolving public health approaches to the global challenge of foodborne infections diarrhoea morbidity and mortality in older children, adolescents, and adults. epidemiology and infection global burden of childhood pneumonia and diarrhoea global causes of diarrhoeal disease mortality in children < years of age: a systematic review etiology of diarrhea among older children, adolescents, and adults: a systematic review burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study foodborne illness acquired in the united states-unspecified agents disease burden of foodborne pathogens in the netherlands longitudinal study of infectious intestinal disease in the uk (iid study): incidence in the community and presenting to general practice estimates of the burden of foodborne illness in canada for specified pathogens and unspecified agents, circa foodborne illness, australia, circa and circa foodborne infections in france risk ranking for foodborne microbial hazards in new zealand: burden of disease estimates global incidence of human shiga toxin-producing escherichia coli infections and deaths: a systematic review and knowledge synthesis. foodborne pathogens and disease the global burden of cholera. bull world health organ the global prevalence of norovirus among cases of gastroenteritis estimating the burden of acute gastroenteritis, foodborne disease, and pathogens commonly transmitted by food: an international review epidemiologic implications of asymptomatic reinfection: a mathematical modeling study of norovirus pathogen-specific c burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed) assessing the applicability of currently available methods for attributing foodborne disease to sources, including food and food commodities who foodborne disease burden epidemiology reference group's (ferg) expert elicitation for estimating the relative contribution of food to the global burden of diseases commonly but not only acquired through the consumption of food who foodborne disease burden epidemiology reference group (ferg) estimates of the global and regional disease burden of foodborne bacterial, protozoal and viral diseases the world health organization funded this study under the foodborne disease burden epidemiology reference group through contributions from member states and international agencies. we would also like to acknowledge the support from the bill and melinda gates analyzed the data: smp. wrote the paper: smp clfw cfl bd ajh asrd reb mdk fja. key: cord- -f qeytxc authors: zhou, yanchen; vedantham, punitha; lu, kai; agudelo, juliet; carrion, ricardo; nunneley, jerritt w.; barnard, dale; pöhlmann, stefan; mckerrow, james h.; renslo, adam r.; simmons, graham title: protease inhibitors targeting coronavirus and filovirus entry date: - - journal: antiviral research doi: . /j.antiviral. . . sha: doc_id: cord_uid: f qeytxc abstract in order to gain entry into cells, diverse viruses, including ebola virus, sars-coronavirus and the emerging mers-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. the respective enzymes are thus excellent targets for antiviral intervention. in cell culture, activation of ebola virus, as well as sars- and mers-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin l (ctsl) and cathepsin b (ctsb). in addition, sars- and mers-coronavirus can use serine proteases localized at the cell surface, for their activation. however, it is currently unclear which protease(s) facilitate viral spread in the infected host. we report here that the cysteine protease inhibitor k , (( s)-n-[( e, s)- -(benzenesulfonyl)- -phenylpent- -en- -yl]- -{[(e)- -methylpiperazine- -carbonyl]amino}- -phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. k is already in advanced stages of development for a number of parasitic diseases, such as chagas disease, and has proven to be safe and effective in a range of animal models. k inhibition of sars-cov and ebola virus entry was observed in the sub-nanomolar range. in order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized k -derivative with that of camostat, an inhibitor of tmprss and related serine proteases. employing a pathogenic animal model of sars-cov infection, we demonstrated that viral spread and pathogenesis of sars-cov is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of sars and potentially mers, while vinyl sulfone-based inhibitors are excellent lead candidates for ebola virus therapeutics. in order to gain entry into cells, diverse viruses, including ebola virus, sars-coronavirus and the emerging mers-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. the respective enzymes are thus excellent targets for antiviral intervention. in cell culture, activation of ebola virus, as well as sars-and mers-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin l (ctsl) and cathepsin b (ctsb). in addition, sars-and mers-coronavirus can use serine proteases localized at the cell surface, for their activation. however, it is currently unclear which protease(s) facilitate viral spread in the infected host. we report here that the cysteine protease inhibitor k , (( s)-n-[( e, s)- -(benzenesulfonyl)- -phenylpent- -en- -yl]- -{[(e)- -methylpiperazine- -carbonyl] amino}- -phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. k is already in advanced stages of development for a number of parasitic diseases, such as chagas disease, and has proven to be safe and effective in a range of animal models. k inhibition of sars-cov and ebola virus entry was observed in the sub-nanomolar range. in order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized k -derivative with that of camostat, an inhibitor of tmprss and related serine proteases. employing a pathogenic animal model of sars-cov infection, we demonstrated that viral spread and pathogenesis of sars-cov is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of sars and potentially mers, while vinyl sulfone-based inhibitors are excellent lead candidates for ebola virus therapeutics. Ó published by elsevier b.v. emerging viral diseases pose a unique risk to public health. ebola virus, severe acute respiratory syndrome coronavirus (sars-cov) and members of the henipavirus genus of paramyxoviruses are all highly pathogenic viruses that have arisen in the past years and caused, or threaten to cause, major outbreaks. new viral threats continue to emerge, most recently demonstrated by a novel beta-coronavirus, middle east respiratory syndrome coronavirus (mers-cov), which was identified in (zaki et al., ; memish et al., ; de groot et al., ) . there are currently no approved vaccines or therapeutics for many of the highly pathogenic viruses potentially dependent on cathepsins, including ebola virus, nipah virus (niv), mers-cov and sars-cov. broadspectrum antiviral drugs, with overlapping therapeutic indications, would facilitate rapid responses to new or changing pandemic threats, potentially even without precise identification of the agent. targeting host factors involved in viral entry provides an excellent avenue for such drug development, due to the limited number of pathways involved the glycoproteins of corona-, filo-and paramyxoviruses facilitate viral entry into target cells by binding to receptors and by driving fusion of viral and host cell membranes. however, the glycoproteins are synthesized as inactive precursors and depend on activation by host cell proteases to acquire a fusion active form. as a consequence, the respective enzymes are potential targets for broad-spectrum antiviral intervention. cell culture studies demonstrated that endosomal cysteine proteases, in particular cathepsin b (ctsb) and/or l (ctsl), can activate the glycoproteins of filoviruses, sars-cov, other coronaviruses, and niv and hendra (hev) viruses to facilitate entry into certain cell lines. in addition, activation of coronaviruses can also be accomplished by tmprss , or other serine proteases located at the cell surface, or secreted into the extracellular space (simmons et al., ) . however, the respective roles of endosomal and cell surface proteases in viral spread in the infected host is unknown. the development of protease inhibitors able to inhibit ctsl, ctsb and related proteases would be an excellent starting point for development of broad-spectrum antiviral therapies . we describe here the discovery of k and its related compounds, as broad-spectrum antivirals targeting endosomal proteases involved in viral entry. k , a cysteine protease inhibitor, blocked infection when viral entry did not require activating serine proteases, as is the case with ebolavirus (ebov). k also fully inhibited coronavirus infection, but only when target cell lines lacking activating serine proteases were used. if cells expressed cell-surface serine proteases known to activate coronaviruses, both k and a serine protease inhibitor, such as camostat were required for full inhibition. thus, both compounds were deployed to examine which activation pathway is predominant in vivo. camostat displayed antiviral activity in a pathogenic animal model for sars-cov infection, indicating that serine protease inhibitors are suitable for treatment of sars and potentially mers. the predicted effect of k and related cysteine protease inhibitors versus ebola virus in vivo must await studies in approved biocontainment facilities. the cysteine protease inhibitor library screened in this work has been described elsewhere (ang et al., ) . briefly, the library includes $ electrophilic cysteine protease inhibitors of various chemotype (glycine nitriles, ketobenzoxazoles, ketooxadiazoles, vinylsulfones, etc.), which were synthesized during the course of industrial drug discovery programs targeting human cathepsins (palmer et al., (palmer et al., , (palmer et al., , rydzewski et al., ) . camostat mesylate, leupeptin, bafilomycin a , ammonium chloride, and chloroquine were purchased from sigma-aldrich. k and novel p derivatives were synthesized according to the general approach described previously (jaishankar et al., ) and as illustrated here (scheme ). the n-substituted piperazines were obtained from commercial sources or (for r = cyclopentyl and cyclopropylmethyl) were prepared by reductive amination of boc-protected piperazine followed by treatment with hcl in dioxane ( - % over two steps). we find that the final coupling of p /p carboxylic acid to vinylsulfone amine is best accomplished via the mixed anhydride, as this minimized epimerization of the phenylalanine side chain. final vinylsulfone analogs were > % pure as judged by lc/ms analysis. characterization data for final analogs is provided below. . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . human embryonic kidney cells, t cells, clone ( t/ ), and vero cells were obtained from american type culture collection (atcc), while the huh . cell line was a gift from apath llc. all cells were grown in dulbecco's modified eagle medium (dmem; invitrogen) supplemented with % fbs and penicillin and streptomycin ( u/ml). t/ stably expressing ace ( t/ace ) were established by transfecting t/ cells with pcdna (invitrogen) encoding the ace gene and selecting for stable transformants using blasticidin s ( . lg/ml). stably expressing human cd [also called aminopeptidase n (apn)] ( /cd ) were established by transfecting cells with pcdna (invitrogen) encoding the cd gene and selecting for stable transformants using geneticin ( . mg/ml). expression of cd was measured with flow cytometric analysis. lentiviral pseudotypes were generated from two plasmids, one encoding the envelope and the second an envelope-deficient hiv reporter construct -either pnl - luc-r -e -(pnl-luc) or pnl - .ren.r-e- connor et al., ) . plasmids encoding spike (s) proteins from human coronaviruses sars-cov, nl and e, mers-cov, glycoproteins (gp) from filoviruses ebov (formerly known as ebolavirus zaire), sudan ebolavirus (sudv), tai forest ebolavirus (tafv), reston ebolavirus (restv), and marburg (marv), as well as niv f and g, lassa virus gp, vesicular stomatitis virus (vsv) g protein, chikungunya virus (chikv) e /e , and mlv envelope, have been described simmons et al., simmons et al., , simmons et al., , salvador et al., salvador et al., , ). bundibugyo ebolavirus (bebov) gp was a gift from edward wright (university of westminster). hcv e e was synthesized (genscript, ca) while junin virus g protein was a gift from sean amberg (siga technologies, or). plasmids encoding cellular type ii transmembrane serine proteases (ttsp) tmprss were previously described . lentiviral pseudovirions were produced essentially as previously described (zhou et al., ) . briefly, t/ cells were transfected with up to lg of viral envelope encoding plasmid and lg of pnl - reporter backbone per -cm dish by calcium phosphate transfection. the next day, expression was induced with sodium butyrate ( mm) for h before washing once. forty hours after transfection, supernatant was filtered through a . lmpore-size filter and frozen at À °c. virus was titrated essentially as it would be used in the screening assay. if required, virions were purified and concentrated by ultracentrifugation ( , rpm in a sw rotor, beckman) over a % sucrose cushion, resuspended in hank's balanced salt solution (hbss) and stored at À °c as aliquots. pseudoviruses were normalized for equal infectivity by transduction of target cells with serially diluted stock followed h later by determination of luciferase activity in cell lysates according to the manufacturer's instruction (promega). vsv-based pseudotypes bearing junin virus g were produced essentially as described (steffen et al., ) by transfecting t cells with lg of junin g plasmid and then infecting the cells with recombinant vsvdg-gfp(vsv-g). progeny vsvdg-gfp(junin-g) virus was then collected, titrated and used for inhibition studies. in the case of niv, vsvdg-gfp(niv f/g) viruses were produced via calcium phosphate transfection of t cells with ug of niv f and lg of niv g. transfected cells were left for h before an initial medium change; then infected with recombinant vsvdg-gfp(vsv-g) (moi . - . ) after five additional hours. media alone or compound of interest were then added at the desired concentration and cells were incubated overnight before supernatant was harvested and filtered. to assay for inhibition, production of entry-competent virus was examined. target cells were pre-plated at , cells/ ll in well plates and allowed to attach overnight. ll of undiluted vsvdg-gfp niv f/g made in the presence or absence of inhibitor was added. cells were incubated at °c with % co for two days, and then washed and fixed with % paraformaldehyde and gfp fluorescence determined by flow cytometry using a becton dickinson lsrii cytometer and flowjo software. % infection was determined with samples infected with pseudovirons produced from cells with no compound exposure. high-throughput screens for viral entry inhibitors were performed in -well plates using the dual-envelope pseudotype (dep) assay . briefly, compounds and controls were diluted in dmem with % fbs to lm ( % dmso) and ll were transferred to -well white tissue culture plates (nunc) using a biomek fx-p (beckman-coulter). a mixture of the target virus [e.g., (hiv-luc(sars-cov s)) and the control virus [hiv-ren(lassa gp) or hiv-ren(mlv env)] was made, with the concentration and ratio derived empirically to give similar robust levels of reporter expression. ll of reporter virus mix was added to each well using a matrix well-mate (thermo scientific). ll of cells ( , cells per milliliter) were then added to all wells. plates were incubated for two days at °c/ % co and firefly and renilla luciferase reporter expression was determined using the dual-glo luciferase assay substrate (promega). assays for dose response curves were performed in -well white tissue culture plates (nunc). target cells were pretreated with test compounds or inhibitors serially diluted in medium, followed by either a single virus or a two reporter virus mixture, depending on the purpose of the assay. the env/reporter combinations were reversed in order to demonstrate inhibition was not directed at the backbone or reporter enzyme rather than entry. plates were incubated for two days at °c/ % co and firefly and renilla luciferase reporter expression was determined using the dual-glo luciferase substrate (promega), or detection of firefly luciferase reporter expression using the bright-glo™ luciferase substrate (promega). the infectivity for pseudotyped vsvs with niv f/g was analyzed by measuring the number of gfp expressing cells by flow cytometric analysis. either caco or -cd cells transiently expressing tmprss were pretreated with serially diluted k , a combination of serially diluted k and camostat mesylate at or lm or a combination of serially diluted camostat mesylate and k at . lm for min at °c and then incubated with infectivity-normalized pseudoviruses in the presence of the inhibitors. the cells were then cultured at °c/ % co for two days and luciferase expression was measured. antiviral replication with urbani and toronto- strains of live sars-cov, as well as cytotoxicity of selected compounds was investigated using three in vitro assays, cytopathic effect (cpe) inhibition assay, neutral red (nr) uptake assay, and virus yield reduction assay as described in kumaki et al. ( ) . for cell viability assays, cells were seeded in -well black tissue culture plates (costar) and treated with compounds with final concentration of % dmso. the quantity of the atp present in metabolically active cells was determined with celltiter-glo Ò luminescent cell viability assay kits (promega, madison, wi). smdc ( mg/kg), camostat ( mg/kg) alone, smdc ( mg/kg) combined with camostat ( mg/kg), or negative control (water) were administrated into - week old female balb/c mice by oral gavage twice a day for days beginning h prior to virus exposure. ten mice were assigned to each group. the texas biomedical research institute's institutional (texas biomed) animal care and use committee approved all animal protocols. live virus assays were performed at the absl- facility at texas biomed using a mouse adapted strain of sars-cov (ma ) kindly provided by ralph baric (university of north carolina). mice were infected by administering , pfu of virus by intranasal instillation. statistical calculations were performed in excel (microsoft, seattle, wa). compounds from the primary screens were considered inhibitory when the luciferase readings of sars-cov, but not the internal control pseudotyped viruses, fell below the predefined cut-off, mean-  sd (m- sd). ic ( % inhibitory concentration) and cc ( % cell cytotoxic concentration) values were calculated using non-linear regression analysis based on the sigmoidal dose response equation using prism (graphpad software inc) (applied to the percent inhibition and concentration data. a selectivity index (si) was calculated using the formula si = cc /ic . we recently developed an internally-controlled dual virus hts assay for identification of inhibitors of viral entry . using sars-cov entry assays, we screened a library of $ cysteine protease inhibitors with confirmed activity against human cathepsins. unsurprisingly, a large number of hits were identified. upon validation of the hits, the most robust activity was observed for k (( s)-n-[( e, s)- -(benzenesulfonyl)- -phenylpent- -en- -yl]- -{[(e)- -methylpiperazine- -carbonyl] amino}- -phenylpropanamide) (fig. a) , a compound known to inhibit cruzain, a cathepsin-l like protease from the protozoan parasite trypanosoma cruzi (engel et al., ) . in addition, k inhibits a variety of cysteine proteases, including human cathepsins (choy et al., ) and cathepsin-like proteases from several other parasites (ndao et al., ; abdulla et al., ) . to determine whether k can inhibit entry driven by other viral envelope proteins, hiv-based pseudotypes bearing spikes from coronaviruses (sars-cov, hcov- e, nl , mers-cov) or glycoproteins from filoviruses (ebov, sudv, tafv, restv, bebov and marv) were examined together with control pseudotypes. we also tested the ability of k to prevent activation and hence infectivity during production of vsv-based pseudotypes (salvador et al., ) bearing niv f and g. k was active against all the major enveloped viruses previously known to require cathepsin-mediated proteolysis, including a variety of coronaviruses and filoviruses, especially ebov ( fig. b; table ). k inhibited sars-cov pseudovirus entry with an ic of . nm (fig. b , table ) while no toxicity was observed, cc > lm (data not shown). mers-cov and nl envelope required higher concentrations of k for inhibition, likely due to less reliance on ctsl hofmann et al., ) . nevertheless, the ic s were very low: nm for mers-cov and < nm for nl . in contrast, nm k did not inhibit infection mediated by envelope glycoproteins from an alphavirus (chikv), a rhabdovirus (vsv), a flavivirus (hcv), the retroviruses mlv-a and xmrv or two arenaviruses, lassa and junin virus. coronaviruses including sars-cov, human coronavirus e (hcov- e) and mers-cov use two distinct pathways for cell entry: (i) the endosomal pathway, in which spike activation is facilitated by the ph-dependent endosomal protease ctsl; or (ii) entry at the plasma membrane, which relies on spike activation by secreted or surface proteases -such as trypsin and type ii transmembrane serine proteases hat (human airway trypsin-like protease) or tmprss bertram et al., bertram et al., , . the serine protease inhibitor camostat mesylate (camostat) inhibits the enzymatic activity of tmprss and other cell-surface proteases involved in coronavirus activation (kawase et al., ) . we therefore assessed whether k displays antiviral activity in tmprss expressing cells. for this, we incubated target cells with camostat, k , or a combination of k and camostat and then infected with pseudoviruses bearing e-s. k alone demonstrated up to $ % inhibition of e-s-mediated transduction. simultaneous treatment with camostat and k increased inhibition to $ % (fig. a, left panel) . similar inhibition patterns were obtained using the human intestinal epithelial cell line caco- , which express endogenous tmprss and cathepsins (fig. b) . in contrast, k alone fully blocked ebola pseudovirus infection, while camostat had no impact on viral infection (fig. a, middle panel) . finally, treatment of cells with k , camostat or both, had no impact on vsv-g driven viral entry (fig. a, right panel) , which is known to be independent of cysteine and serine protease activity. these results indicate that both serine and cysteine proteases can activate e-s for viral entry, as expected, while ebov-gp exclusively relies on cysteine proteases for activation. we next synthesized a series of k analogs to further explore the antiviral activity of vinylsulfone-class protease inhibitors ( table ). given that the piperazine ring in k is basic (pka $ . for the conjugate acid) we considered that the compound might accumulate in the acidic (lysosomal and endosomal) compartments where target proteases such as ctsl and ctsb are abundant. to explore this notion and to more generally evaluate structure-activity trends, we synthesized new vinylsulfone analogs in which the substituent on the piperazine ring nitrogen atom was modified systematically. while the majority of these analogs (table ) retain a basic piperazine ring, the n-phenyl analog smdc- is only weakly basic (pka $ . for the conjugate acid) and thus will be neutral at physiological ph and would not be expected to exhibit lysosomotropic behavior. nearly all of the new analogs possessed potency comparable or superior to k against sars-cov and ebov (table ) , the most potent analogs being smdc (sars-cov ic = . nm; ebov ic = . nm), smdc (sars-cov ic = . nm; ebov ic = . nm) and smdc (sars-cov ic = . nm; ebov ic = . nm). flaviviridae ssrna(+) huh . > a ic (inhibitory concentration) values are the concentrations required to inhibit the infectivity of the pseudotyped viruses on cells by %, which were determined from dose response curves. all envelopes apart from nipah and junin were used to make hiv-based pseudotypes. target cells ( t, t expressing ace or cd , or vero cells) were then pretreated with serial dilutions of k and exposed to virus. vsv-based pseudotypes were made by transfecting cells with nipah f and g plasmids, or junin envelope, and transducing with vsvdg(gfp)-g. progeny virus was then collected and titered on target cells. a non-linear regression analysis based on the sigmoidal dose response equation was applied to the percent inhibition and concentration data. data is shown as means of triplicate measurements ± standard deviation. values are representative of at least two independent experiments. of particular note from the structure-activity data is that the weakly basic analog smdc- was - -fold less potent than the other basic and protonatable vinylsulfone analogs ( table ). the reduced potency of smdc- is likely not related to the size of the phenyl substituent, since even larger, biaryl p substituents are known to be well tolerated in cathepsin-l like proteases such as cruzain (beaulieu et al., ) . also consistent with this interpretation, we find that other bulky tertbutyl and cyclopentyl groups are tolerated in analogs like smdc- and smdc- . therefore, the most likely explanation is that as a weak base and the only analog expected not to be protonated at physiological ph, smdc- does not accumulate in the lysosome to the same extent that the other, more basic, analogs do. conversely, k and the other basic analogs accumulate in acidic endosomal compartments where target cysteine proteases such as ctsl and ctsb are located. to further verify the antiviral effects of three of the most efficient drug candidates, inhibition assays were carried out with two strains (urbani and toronto- ) of replication competent sars-cov, and using two separate readouts of replication (summarized (si, cc /ic ) ranged from . (smdc inhibition against the toronto- strain) to over . thus, these compounds were identified as ideal tools to determine whether cysteine or serine proteases promote sars-cov spread in the host. the pharmacokinetics and bioavailability of smdc and smdc in male and female sprague-dawley rats were determined following a single i.v. or p.o. dose administration (data not shown) and demonstrated similar profiles to k (jacobsen et al., ) . in initial experiments, the antiviral efficacy of low-dose ( - mg/kg) smdc was examined in a lethal sars-cov mouse model (day et al., ) . while there was a trend toward protection, there was no statistically significant reduction in mortality or disease severity (data not shown). experiments were therefore repeated at higher doses of cysteine protease inhibitor ( mg/kg), either alone or in combination with the serine protease inhibitor, camostat ( mg/kg) (fig. ) . smdc- alone was no more effective than vehicle treated controls (fig. ) . in contrast, camostat was effective in protecting mice against death due to a lethal infection by sars-cov, with a survival rate of $ %. combining both classes of inhibitors did not significantly improve survival versus camostat alone. thus, sars-cov depends on serine protease activity for viral spread in vivo. viral entry is a multi-step process and an attractive target for antivirals (zhou and simmons, ) . the fact that disparate pathogenic viruses such as sars-cov, ebov and niv all utilize a common host factor for entry -ctsl -suggested that inhibitors of cathepsins might have broad applicability. cysteine proteases have proved to be druggable targets and their inhibitors are generally of low toxicity. we screened a library of drug-like compounds with established activity against ctsl and ctsb for activity against sars-cov and filoviruses, including ebov. we describe here the confirmation that protease inhibitors, such as k and related compounds, are broad-spectrum antiviral drug candidates targeting viral entry. a number of additional vinylsulfone analogs were synthesized, some of which exhibited enhanced potency compared to k . most notably, potent antiviral activity was correlated with the presence of a basic piperazine ring at the p position, a finding that is consistent with accumulation in endosomal (acidic) compartments where the target cysteine proteases required for viral entry are located. the vinylsulfones described herein were broadly active against viral entry for three viral families: the corona-, filo-and paramyxoviruses, and are very well tolerated in vivo (barr et al., ) . the notion that coronaviruses, including sars-cov, use both a cathepsin-dependent endosomal pathway and a direct cell-surface serine protease-mediated pathway for entry (simmons et al., ) is supported by our finding that the combination of k and camostat was superior to either compound alone. in contrast, ebov infection was effectively inhibited by k , but not by camostat. while unidentified additional proteases have been reported to mediate infection by other filoviruses, such as marv (gnirss fig. . effects of per os administered smdc and/or camostat on survival of balb/c mice infected with a lethal sars-cov. ten mice per group were dosed twice a day by oral gavage with smdc and/or camostat or diluent alone (sterile water) for days beginning h prior to infection with , pfu of mouseadapted sars-cov. et al., ) and sudv (misasi et al., ) , efficient inhibition by the vinylsulfone analogs suggests that the unidentified proteases are cysteine proteases related to ctsb and l. activation of niv and hev appears to be fully dependent on ctsl and/or ctsb (pager et al., ; diederich et al., diederich et al., , . thus, vinylsulfones are promising antiviral lead compounds for further optimization as potent inhibitors of these two important groups of pathogenic emerging viruses, including ebov. previous reports showed that compound k and analogs have satisfactory safety and pharmacokinetic profiles in rodents, dogs and primates (abdulla et al., ) . the fact that k , as a vinylsulfone, is an irreversible and not highly selective cysteine protease inhibitor does not appear to be a liability, at least if it is used as a short course antiviral. indeed, in the case of filoviruses, the lack of target selectivity is likely a boon -increasing effectiveness by also inhibiting secondary proteases (gnirss et al., ; misasi et al., ) . the availability of a novel, highly potent and largely non-toxic cysteine protease inhibitor, smdc , afforded the opportunity to assess whether the activity of cysteine or serine proteases is required for viral spread in vivo. for this, a mouse model for lethal sars-cov infection was employed. notably, only inhibition of serine proteases mitigated sars-cov pathogenesis in vivo. thus, future development of anti-coronavirus therapeutics should focus on inhibiting serine rather than cysteine proteases, with camostat being an excellent starting candidate. indeed, in japan camostat is used clinically, particularly to treat chronic pancreatitis (ikeda et al., ; sai et al., ) , with a reasonable safety profile (fiopan Ò tablets, ). our results showed that targeting viral entry, and more specifically, the endosomal proteolysis step of entry, is an attractive strategy to discover new antiviral agents -particularly for filoviruses, like ebov, and some paramyxoviruses. although endosomal and cell-surface proteases can facilitate coronavirus entry in cultured cells, only the activity of serine proteases is required for viral spread in the infected host. nevertheless, the highly potent cysteine protease inhibitors identified here might be excellent starting points for the development of highly effective inhibitors of ebola virus and paramyxovirus entry, and constitute excellent research tools for dissecting the molecular mechanisms of viral entry. schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor 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clinical trial with camostat mesylate in vitro evaluation of the disposition of a novel cysteine protease inhibitor potency and selectivity of p /p -modified inhibitors of cysteine proteases from trypanosomes simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry inhibition of severe acute respiratory syndrome coronavirus replication in a lethal sars-cov balb/c mouse model by stinging nettle lectin, urtica dioica agglutinin family cluster of middle east respiratory syndrome coronavirus infections filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences a cysteine protease inhibitor rescues mice from a lethal cryptosporidium parvum infection a mature and fusogenic form of the nipah virus fusion protein requires proteolytic processing by cathepsin l vinyl sulfones as mechanismbased cysteine protease inhibitors design and synthesis of tri-ring p benzamide-containing aminonitriles as potent, selective, orally effective inhibitors of cathepsin k keto- , , -oxadiazoles as cathepsin k inhibitors peptidic -cyanopyrrolidines: synthesis and sar of a series of potent, selective cathepsin inhibitors efficacy of camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease characterization of chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in e glycoprotein filoviruses utilize glycosaminoglycans for their attachment to target cells ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research characterization of the bas-congo virus glycoprotein and its function in pseudotyped viruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia development of novel entry inhibitors targeting emerging viruses a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms inhibitors of sars-cov entry -identification using an internally-controlled dual envelope pseudovirion assay development and application of a high-throughput microneutralization assay: lack of xenotropic murine leukemia virus-related virus and/or murine leukemia virus detection in blood donors this work was supported by grants r ai and r ai from the national institute of allergy and infectious diseases (to g.s.) and funding from the sandler foundation (to a.r.r.). preliminary animal studies were supported by niaid contracts hhsn c (sri international) and hhsn i, pi. john morrey, phd (to d.b.). key: cord- -d bxe c authors: yuan, xiaomin; lin, huixing; fan, hongjie title: efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: d bxe c to explore the possibility of developing a vaccine against transmissible gastroenteritis virus (tgev) infection, a recombinant swinepox virus (rspv-sa) expressing a tgev protective antigen has been constructed. immune responses and protection efficacy of the vaccination vector were assessed in both mice and pig models. an indirect elisa assay suggested that when mice were vaccinated with rspv-sa, the level of igg against tgev was enhanced dramatically. the cytokine assays were employed and the results indicated that both the th -type and th -type cytokine levels raised after vaccination with rspv-sa in mice models. results from the passive immunity protection test of new born piglets demonstrated that the recombinant live-vector vaccine, rspv-sa, could % protect piglets from the spv infection, and there was no significant clinical symptom in the rspv-sa treatment group during this experiment. the data suggest that the novel recombinant swinepox virus is a potential vaccine against tgev infection. transmissible gastroenteritis virus (tgev) is a member of coronaviridae, which is the etiological agent of transmissible gastroenteritis. although the virus is capable of infecting swine of all ages, suckling piglets are the most susceptible and have a mortality rate up to % [ ] . tgev is a pleomorphic enveloped virus containing a positive-stranded rna genome and four structural proteins: the spike (s) protein, the integral membrane (m) protein, the minor envelope (e) protein, and the nucleocapsid (n) protein. among which, the spike (s) protein, one of the key structural membrane proteins of coronaviruses, is an attractive target for generating neutralizing antibodies against the virus due to the critical role it plays in the host cell invasion [ , ] . precisely, the s protein mediates the attachment of virus particles to targets via binding of itself to the specific receptors. at the n terminus of the s protein, there are four antigenic sites, a, b, c, and d, which have been shown to be involved in the stimulation of neutralizing antibodies (fig. ) [ ] . previous studies have determined that the a site (which is fully dependent on glycosylation for proper folding) is predominantly responsible for stimulating neutralizing host antibodies [ ] [ ] [ ] [ ] [ ] [ ] . spv is a natural mild attenuated virus and has been widely applied as a vaccine. given that poxvirus-vectors can prevent a great deal of important diseases in both humans and animals it is not surprising that many of these vectors been licensed and used extensively [ ] [ ] [ ] . additionally, spv is a safe vaccine vector as there is no risk of cross-species infection [ ] . therefore, both for biological and clinical practicality, spv is regarded as an appropriate and promising veterinary vaccine for swine, owing to its ability to effectively express foreign genes, its large packaging capacity for recombinant dna, its low cost of delivery and its specific host restriction [ ] . the potential value of spv as a live vector vaccine is being studied extensively. because spv is able to pack large amounts of recombinant dna and to induce appropriate immune responses in vivo, it is a promising candidate for the development of a recombinant vaccine [ , ] . as of yet, pigs are the only known hosts of swinepox virus and therefore may be useful in developing a safe vaccine for clinical application [ , ] . in this study, we constructed a recombinant swinepox virus expressing s-a (a epitope of the s protein) of tgev and characterized recombinant virus replication and expression of the s protein in pk- cells. we further investigated the potential of this approach for use in the vaccination of pigs against tge. type culture collection. swine transmissible gastroenteritis virus (tgev, china strain, shxb) was purchased from the jiangsu academy of agricultural sciences, and the titer was determined as × pfu/ml st cell. tgev convalescent positive serum was purchased from the jiangsu academy of agricultural sciences, and the neutralizing antibodies were used at a dilution of : , . the pusz swinepox virus vector was generated previously [ ] . two primers, sa ( -gcgtcgacatgggtcttggtatga-agcgtag- ) and sa ( -cgggatcctta tagcgtcctgt-tagtttgtc- ) were used to amplify the s-a gene ( bp, km ) from the tgev genome, which was inserted into the pusz plasmid to construct pusz -sa subsequently. the recombinant swinepox virus, rspv-sa, was generated by homologous recombination of wtspv with pusz -s-a as previously described [ ] . pcr and indirect immunofluorescence were employed to analyze the s-a gene expression and the expression of s protein. the replication capacity and genetic stability of rspv-sa were also evaluated by. the generation and screening of the recombinant swinepox virus assays were performed as described previously [ ] . a subconfluent culture of pk- cells was infected with wtspv ( . moi) for h, and subsequently transfected with g of the pusz -sa plasmid using exfect tm transfection reagent (vazyme biotech co., ltd). after h, pk- cells were harvested and lysed by five rounds of freezing and thawing. subsequently, the lysate was used to infect pk- cells grown in a -well plate for further purification of recombinant viruses. . ml of medium with % lmp agarose (dingguo, beijing, china) was added to each well and incubation was continued for five days until plaques became visible under a light microscope. after - days, a second overlay medium containing x-gal was added. the plaques were resuspended in . ml of medium with % fbs. plaque isolation was repeated for - rounds until all plaques in a given well were stained blue. the recombinant spv bearing s-a of tgev was designated as rspv-sa. the rspv-sa genomic dna from the pk- cells infected with rspv-sa was extracted by sds-protease k-phenol. we utilized wtspv genomic dna from pk cells infected with wtspv as a negative control. pcr was performed for min at • c; followed by cycles of min at • c, min at • c, and min at • c. amplifications were performed with dna polymerase (promega, shanghai, china) using primers sa ( -gcgtcgacatgggtcttggtatgaagcgtag- ) and sa ( -cgggatccttatagcgtcctgttagtttgtc- ). indirect immunofluorescence assays (ifa) were performed as described previously [ ] . pk- cells were grown on a -well plate and infected with the wtspv and rspv-sa at × pfu/ml per well. pbs-treated cells were used as a negative control. at h post-infection, cells were washed three times in pbst and fixed with cold methanol for min at − • c. cells were then washed three times with pbst and blocked by pbst with % bsa. preparations were incubated for h at • c with tgev convalescent positive serum ( : in dilution buffer, pbst with % bsa). after three washes with pbst, cells were treated with the rhodamineconjugated secondary antibody (staphylococcal protein a-rhod, boshide, wuhan, china) at a : dilution (diluted in pbs) for min at • c. after a final wash with pbs, all wells were examined by fluorescence microscopy (zeiss, germany). nine six-week-old balb/c mice were randomly divided into three groups ( mice per group), and immunized three times at , , and days with rspv-sa ( × pfu/ml in . ml of pbs) or wtspv ( × pfu/ml in . ml of pbs), the control group injected with pbs. eight one-month-old swine (large white) were randomly divided into four groups ( pigs per group) and were immunized twice at and days with infectious rspv-sa ( × pfu/ml in ml of pbs), inactivated-tgev ( × pfu/ml in ml of pbs), wtspv ( × pfu/ml in ml of pbs) or pbs, each time via three routes: oral, nasal, and intraperitoneal. serum was collected days after the last immunization. twelve one-day-old pigs were randomly divided into four groups for passive immunization experiments ( pigs per group). high titers of antibodies were collected from piglets following the first immunization. mice and swine serum were incubated at • c min to complement inactivated. all experimental protocols involving mice or swine were approved by the laboratory animal monitoring committee of jiangsu province. pk- cell monolayers were infected with wtspv and rspv-sa (moi of ) and incubated for h at • c. extracts, representing approximately × cells, were electrophoresed through an sds- % polyacrylamide gel and the separated proteins were transferred onto a pvdf membrane. after a h transfer, the membrane was blocked with % skim milk in phosphate buffered saline with . % tween- (pbst) overnight at • c. the membrane was incubated with swine convalescent serum ( : dilution) containing tgev for h at • c and washed three times with pbst. immunodetection was performed with staphy-lococcal protein a-hrp at • c. following the secondary antibody probing, the membrane was washed four times with pbst. the membrane was then developed with , -diaminoben-zidine substrate until optimal color development was observed. serum was collected from mice and pigs, and detected the tgev-specific antibodies by indirect elisa. the purified tgev was resuspended in l pbs (ph . ), and used the best titer of virus for coating -well plates, which was determined by titration. samples were then incubated overnight at • c. this incubation was followed by three pbst washes, and blocking with % skim milk (in pbst) at • c for h. serum samples were serially diluted and incubated at • c for h. the samples were set up at the same time and divided into three groups: the tgev positive serum, the negative control serum (spv positive serum) and the blank control (without serum). after three pbst washes, horseradish peroxidase (hrp)-conjugated goat anti-spa igg ( : , diluted in pbst, signalway antibody) was added to each test well. the plates were then incubated at room temperature in the dark for min and then washed three times with pbst. the tmb microwell peroxidase substrate system (tiangen) was used to develop the reaction. samples were developed for min and the reaction terminated with . m sulphuric acid. all assays were performed in duplicate. a microplate reader (bio-rad) was used to measure the reaction product at an absorbance of nm [ ] . evaluation of cellular immunity was performed by detecting levels of ifn-␥ and il- . three mice were sacrificed at days after the first inoculation with wtspv ( × pfu/ml in . ml), pbs or rspv-sa ( × pfu/ml in . ml). the mouse spleen was removed aseptically. splenocytes were isolated, counted, and diluted to a density of × cells/ l. the evenly separated cells were aliquot into -well plates ( l/well). then, l/well of dmem with tcid / l tgev was added to each well. after a h incubation, the supernatants were collected and the mrna of ifn-␥ and il- were probed by rt-qpcr relative to ␤-actin as described previously [ , ] . an assay for neutralizing antibodies was performed as described previously [ , ] . to explore whether mice or swine generated tgev neutralizing antibodies, serum from the pbs, wtspv, inactivated-tgev and rspv-sa treated mice and pig were collected at , , , , days post-primary immunization ( : - : , dilution in a l volume). and sera were mixed with equal volume of tcid /ml tgev and incubated at • c. after . h incubation, we utilized sera treated viruses to infect st cells in -well plates and overlaid cells with agar at • c in a % co atmosphere. cells were monitored daily for three days to detect tgev-specific cpe. the virulent tgev strain shxb ( × pfu/ml in ml) was mixed with ml of the porcine antiserum induced by recombinants rspv-sa, inactivated tgev or pbs, incubated at • c for min, and administered using a gastric tube to -day-old swine born from tgev-seronegative sow. inoculated animals were fed three times per day with formula for newborns that contained ml of the antiserum. at three days following viral infection, small intestine tissue was collected from newborn pigs sectioned for histology. microstructure characteristics were analyzed under the microscope (olympus, cx fs , philippines) [ ] . all data were analyzed using one-way anova and values of p < . were considered significant. to analyse the recombinant virus and confirm the presence of the sa gene, two specific primers were designed to amplify the inserted sa gene. the gene encoding the neutralizing antigen epitopes of tgev is a . kb fragment and the specific fragments was detected in spv-sa as shown in fig. a . the recombinant spv-sa was also confirmed by observing blue foci in plaque assays. rspv-sa and wtspv were determined to be approximately × pfu/ml in ml for both. as shown in fig. b , the ifa demonstrated that the expression of the a epitope of the s protein was present in infected cells. western blot analysis showed a specific band of target protein with a size of kda in the cell lysates infected with rspv-sa (fig. e) . the kda molecular weight is consistent with the predicted size of the sa protein of tgev. according to these data, we suggested that the a epitope of the s protein was expressed efficiently by the rspv-sa virus (fig. ) . to monitor the s-specific antibody titers of mice vaccinated with rspv-sa, an elisa was used. after an initial boost days postvaccination, the s-a-specific antibody titers increased gradually in mice (fig. a) . the antibody titers in rspv-sa-immunized mice were higher at all-time points post-vaccination (p < . , n = ) compared to wtspv or pbs-treated mice. the p/n value of the positive group was greater than . . persistent high levels of neutralizing antibodies (fig. a) were detected in the rspv-sa group with a mean titer of : at days post-inoculation. in mice vaccinated with wtspv and rspv-sa at days postinoculation (fig. b) , the qpcr results showed a distinct variation of il- and ifn-␥ between wtspv and rspv-sa treatment groups. the relative quantity normalized by beta-actin of il- mrna in rspv-sa was . times higher compared to wtspv. the ifn-␥ mrna levels increased . fold in rspv-sa when compared to wtspv. the concentrations of il- and ifn-␥ in rspv-sa-vaccinated mice were significantly higher than the control groups. these results indicate that rspv-sa induces th -type and th -type cytokine responses during cellular immunity. during the vaccination procedure, several poxes of mmϕ could be observed around the injection position at five days post-inoculation in both rspv-sa and wtspv treatment groups, respectively, which was not observed in the inactivated-tgev and pbs treatment groups. this symptom could be disappeared spontaneously in the following days. no pigs developed further symptoms such as fever or severe inflammation, and the spiritual condition and appetite of both treatment groups was considered as good. these two group pigs recovered in days. all group pigs maintained rectal temperatures of . - . • c. from this experiment, we reveal that vaccination with rspv-sa and wtspv is well tolerated by pigs. rspv-sa induced a moderate level of tgev-specific igg as shown in fig. . persistent high levels of tgev-specific neutralizing antibodies are shown in fig. b . the second boost led to of the levels of tgev-specific neutralizing antibodies. persistent high levels of neutralizing antibodies were detected in the rspv-sa group at a mean titer of : in swine (p < . , n = ). all newborn piglets in different groups were fed with the mixture of tgev and the corresponding sera from pigs vaccinated with pbs, wtspv, inactivated-tgev, or rspv-sa. both pbs and wtspv treatment groups developed a very severe diarrhea symptom, significantly losing of weight and appetite, and the mortality rate was up to % in days. meanwhile, in the inactivated-tgev and rspv-sa treatment groups, there was no obvious clinical symptoms and % morality rate observed (fig. . and table ). after the sacrificing of all the piglets, the small intestine tissue from different groups were used for the pathological examination. histological samples from pbs and wtspv treatment groups showed prominent histopathological changes in the small intestine characterized by serious fracture of the small intestine mucosa, epithelial expansion, vacuolar degeneration, necrosis, shedding, and lamina propria congestive edema and hemorrhage. and such pathological changes were not observed during the examination of the samples from inactivated-tgev and rspv-sa treatment groups. all the results indicate that rspv-sa inoculation provides complete protection passive immunity against tgev challenge in pigs (fig. ) . in the present study, we have engineered the swinepox virus to express the a epitope of the tgev s protein. our data showed that this recombinant virus was not only able to induce a strong immune response against the a antigen of tgev in mice and pigs, but also had safety degree and protection efficacy against the virulent homologous tgev infection in pigs. the traditional way to protect swine from tgev infection is to immunize pregnant sows with inactivated or attenuated vaccine, resulting in the production of secretory iga in the colostrum. despite the protection that secretory iga offers piglets, this method does not provide protection after the cessation of breastfeeding. furthermore, inactivated or attenuated viral vaccines still maintain the ability of developing the viral toxicity [ ] . currently, there are various potential vaccines against tgev but many present certain challenges, such as difficulties in the vaccination process, high production cost, low immunogenicity, biological risk and carcinogenicity. the results from our experiments indicate that the rspv-sa vaccine is a very promising candidate in the safety and immunogenicity respect. however, it should be noticed that, as an alive viral vector vaccine, it is possible that the spv per se would interfere the vaccination. to evaluate this possibility, randomly sampled pig sera were tested via the agarose gel diffusion assay, and the results showed the spv positive rate was around %. moreover, there was no report of spv epidemic in these recent years. thus, together with gel diffusion assay, the interfering from spv per se could be considered as minimum. for the first time, we generated a recombinant spv that expresses the neutralizing epitopes (a epitope in protein s) of tgev, and we verified that the s-a was expressed efficiently in our system. the antigen induced neutralizing antibodies against tgev in st cells, and potentiated strong th -type and th -type cytokine responses in our mouse model. these results suggest that rspv-sa induces humoral and cellular immune responses effectively and efficiently. given the th cell secretion of il- , ifn-␥, ifn-␣, and tfn-␤, it is apparent that th cells play an important role in antiintracellular pathogenic infection. because th cells in our study secreted il- , il- , il- and il- , it is likely that these cells were able to effectively stimulate b cell proliferation and hence, igg and ige antibody production (relevant to humoral immunity). we defined ifn-␥ and il- to be representative of the th -type and th -type cytokine secretory capacity, respectively. our results indicate that in our hands, both th -type and th -type cytokine levels increased dramatically. due to the immature immune system, the maternal antibodies from breast milk account for the immune defensive ability of piglets. based on the feasibility and relevant published papers [ ] , we decided to mimic the breast milk from female pig by mixing the anti-serum generated by vaccinating the days old piglets with spv-sa and the normal cow milk, and use it for passive immunity protection test on the days old piglets. in the study, the recombinant spv vectors were capable of protecting neonatal piglets against mortality and severe disease after a challenge with virus. the rspv-sa vector induced high titers of antibodies in swine. however it is worth noting that rspv-sa may enhance the anti-tgev antibody titer in swine as well as the anti-spv antibody, which will likely affect immune efficiency. with respect to clinical value, it is imperative to study this multivalent vaccine. to summarize, we first report that spv can be used as a live vector vaccine when it expresses the s-a protein of tgev. we determined that not only b-cells, but also t-cells were induced successfully. thus, rspv-sa provides thorough protection against virulent tgev challenge in swine. lastly, our data indicate that rspv-sa is a promising vaccine to prevent tgev infection. transmissible gastroenteritis virus infection: a vanishing specter coronaviruses: structure and genome expression antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein bacterial expression 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recombinant lactococcus lactis expressing porcine transmissible gastroenteritis virus spike glycoprotein the authors of this paper have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- -oe yv y authors: dutta, ritaban title: replacement management in cattle: health management date: - - journal: reference module in food science doi: . /b - - - - . - sha: doc_id: cord_uid: oe yv y replacements are the future of the dairy industry. focusing on improving health management of replacements will yield tremendous returns through decreased losses of animals with the greatest genetic potential on the dairy, decreased costs of medication, improved growth rates, improved feed efficiency and earlier entry into the milking herd. health management begins before replacements are born with attention to the nutrition of lactating and dry cows, the vaccination of lactating and dry cows, control of length of the dry period and both control of the disease status of the dams and the cleanliness of the calving environment. greater attention must be paid to animal and environmental biosecurity to prevent introduction of diseases into the herd and to digestive disorders such as diarrhea, internal parasites and appropriate vaccination programs for the calves. health management of replacements is often overlooked because producers do not see the immediate returns for their efforts. common sense management in cattle, historical facts, experience based practice cultural and social aspects, combined with research, would depict that having adequately optimised balanced diets for the replacements, without producing excessive body conditions, could achieve a production of healthy replacements with superior levels of milk production. continual video monitoring of the herd, modern thermal infrared imaging of the dry cows and calves body parts to identify early symptoms, and overall animal health and biosecurity risk analysis could achieve a sustainable and efficient replacement management practice in cattle industry. replacements are the future of the dairy industry. focusing on improving health management of replacements will yield tremendous returns through decreased losses of animals with the greatest genetic potential on the dairy, decreased costs of medication, improved growth rates, improved feed efficiency and earlier entry into the milking herd. health management of dairy replacements begins before the replacements are born. several factors, such as nutrition of lactating and dry cows, vaccinations of lactating and dry cows, length of dry periods, cleanliness of the calving environment and disease status of the dams, will ultimately affect disease resistance and health of replacements. it is important to note, however, that cows that are overfed tend to have difficulty calving because of being excessively over conditioned. cows that are underfed, which results in mineral or vitamin deficiencies or lack of body condition, may produce inferior and low-volume colostrum. they also may experience difficulty calving. protein deficiency in cows during the dry period may lead to low birth weights, low metabolic rates and poor vigor of calves, resulting in poor survivability. some research also indicates that inadequate protein and energy nutrition of the dam results in poor absorption of immunoglobulins from colostrum by the calf. cows that lose condition during the dry period are also at greater risk of experiencing calving difficulty. calves that experience difficult births require more time before being able to stand, experience an increase in the time to voluntary suckling and have a decreased ability to absorb immunoglobulins. all these problems result in decreased transfer of passive immunity from the dam to the calf and increased risk of disease in calves. as the degree of calving difficulty increases, the risk of mortality for calves increases. proper nutrition of dairy cows during lactation and the dry period will help decrease disease risks for replacements. in recent times researcher are employing electronic cattle tags to remotely monitor cows' general health conditions correlated with the movements of the head and other body parts. some farming communities apply more direct video surveillance mechanism to monitor milking herd to prevent any possible disruption in milk production by taking care of early animal health symptoms and unexpected animal behaviours. intelligent processing of video stream, combined with in-situ visual observations by a farmer could provide a better management culture in replacement management. low cost thermal imaging cameras could also be deployed to monitor the cows and calves body temperature, environmental conditions, and any unusual spots in a herd that has significantly higher temperature than normally expected. early prevention of any bacterial and viral disease spread is extremely important for precalving and calving management. this issue has become ever more important in today's world, where market accessibility for a local producer of milk and dairy products, could be global. bigger market access and business proposition comes with a significantly higher level of responsibility. zero tolerance attitudes towards animal and food biosecurity are key to greater market access and business sustainability. greater awareness is needed to achieve a standard biosecurity management in cattle industries. informed decision on planned vaccinations of the dams will also impact disease resistance of dairy replacements and a better preventive management. proper vaccination of the dairy herd will increase the concentration of antibodies (immunoglobulins specific for diseases) in colostrum. dams may be vaccinated during the dry period against pathogens that are common causes of diarrhea in calves, such as escherichia coli, rotavirus and coronavirus. vaccination of the dams increases the concentration of antibodies against these pathogens in colostrum, thus providing increased protection for calves, resulting in decreased incidence or duration of diarrhea. vaccination of the dams during the dry period is more effective for prevention of disease in calves than vaccination of calves at an early age. the immune system of neonatal calves is unable to respond quickly to a vaccination or an infection because the immune system of the newborn is immature at birth. both numbers and effectiveness of antibody-producing cells are lower in calves at birth than in adult cattle. therefore, it is important for calves to obtain antibodies against common diseases of calves by consumption of colostrum rather than from an attempt to vaccinate calves at an early age. vaccination of the dams against pneumonia may also help to decrease the incidence or severity of this disease in replacements. another important factor that may affect the health of replacements is the length of the dry period of the dam. a dry period that is too short, i.e. less than weeks, may not provide enough time for involution of the mammary gland and preparation for the next lactation. cows with shortened dry periods produce small quantities of colostrum that may also have low concentrations of immunoglobulins. it is important for health of replacements, therefore, that cows have at least a -week dry period for production of high-quality colostrum. management of the calving environment has a tremendous impact on the health of replacements. it is important for calves to be born in a clean, dry environment. wet, sloppy stalls provide a perfect environment for growth of bacteria. calving on a grass lot may be the best alternative when the climate is dry and mild. if a maternity barn is used, it is important to clean stalls thoroughly between calvings to prevent transfer of disease. maternity stalls should only be used for calving and never for housing sick cows. maternity pens and sick pens should be kept in separate facilities in order to prevent transfer of disease to highly vulnerable neonates and periparturient dairy cows. it is also important for the cows to be as clean as possible at calving in order to prevent calves from contracting disease organisms when suckling or attempting to suckle their dams. preferably, calves should be separated from dams prior to suckling in order to prevent the calf from ingesting pathogens present on the legs, belly, flanks or udder of the cow as the calf attempts to nurse. separating the calf from the dam and feeding colostrum by bottle also ensures adequate intake of colostrum for transfer of passive immunity from the dam. finally, it is important to know the disease status of cows prior to calving. diseases such as johne's disease, milk fever (hypocalcaemia) close to calving, bovine viral diarrhea (bvd), and bovine leucosis virus (blv) may be passed in utero or through colostrum. calves should only be fed colostrum from cows known to be free of these diseases. it is important, therefore, to maintain a supply of frozen, high-quality colostrum from cows free of such diseases. today's cost could be tomorrow's gain, so conservativeness towards deploying new technologies in replacement management could be addressed by community based farming, to try new practice, to tackle ever growing biosecurity related threats in dairy industries. the importance for baby calves of adequate consumption of immunoglobulins from colostrum has been reviewed elsewhere. mortality resulting from lack of consumption of adequate amounts of immunoglobulins is commonly greater than % and has been reported to be as high as %. others have indicated a -fold increased risk of mortality when calves do not consume colostrum. % of the hypocalcaemia cases occur within day of calving, because extremely high rate of drain calcium drainage (and other substances) from the blood during the milk and colostrum production, which cattle bodies are unable to replace quickly enough, bring severe health risk and subsequent economic loses. along with economic losses from high mortality rates as a result of lack of colostrum consumption, there are also increased costs associated with increased medication and poor feed efficiency. transfer of passive immunity (absorption of immunoglobulins from colostrum) can be determined using commercial kits that measure immunoglobulins in the blood. for adequate protection of calves, blood immunoglobulin concentrations should be at least mg ml À . serum protein concentrations in calves are also highly correlated with the concentration of immunoglobulins in blood and can be used to determine adequate transfer of passive immunity. a hand-held refractometer can be used to measure serum protein; levels greater than . g ml À by h of age indicate adequate consumption of colostrum. the similar technology could be used to measure 'specific gravity of urine' and 'refractive index of serum', as researchers are aiming to combine these factors for better diagnosis. the use of colostrum substitutes and replacers may help improve disease resistance in calves when high-quality colostrum is not available. it's also important to have a better feeding management plan for the dry cows weeks before the calving, as this effects the amount of calcium available to replace blood calcium, and how efficiently body can utilize the available calcium at a very critical period. the most prevalent health problem of calves on most farms in the united states is diarrhea. organisms such as cryptosporidium parvum, rotavirus and coronavirus that cause diarrhea will not respond to antibiotic treatment. for cryptosporidiosis, the only means of prevention is sanitation, which includes controlling flies. for rotavirus and coronavirus, the most effective prevention is vaccination of the dam to increase antibodies in the colostrum against these organisms. other organisms, such as e. coli and salmonella sp., may be resistant to many of the commonly used antibiotics. producers often give antibiotics to calves during episodes of diarrhea in order to prevent secondary infections; however, this practice often does more damage than good, killing beneficial gut microflora and damaging the gut lining. the first step in caring for calves with diarrhea is to provide fluids for hydration and electrolytes for mineral loss, while continuing to provide milk for protein and energy. an electrolyte solution can be fed from min to h after each feeding of milk or milk replacer until feces return to normal. secondly, the organism causing diarrhea should be identified to determine whether antibiotic treatment is needed. pneumonia is the second most prevalent health problem of replacements, especially for replacements raised indoors. research has shown that calves raised in individual hutches (plastic, fiberglass or wooden structures providing individual housing) perform very well and have fewer health problems, especially pneumonia, than calves raised in closed buildings. open-front housing for older heifers should also help prevent pneumonia. adequate, draught-free ventilation is important for prevention of pneumonia. hutches, pastures and open-front housing for replacements provide optimal ventilation. in addition, hutches can be moved from location to location, giving producers the opportunity easily to remove old bedding and to break disease cycles. no matter what type of housing is used for replacements, cleanliness, dry bedding and adequate ventilation are essential to decrease incidence of disease. another important factor for controlling disease in replacements is grouping of heifers. most producers in the united states house young calves individually. in other areas, housing calves in groups and using mob-feeders is an efficient method of rearing calves during the liquid feeding phase. after weaning, calves should be housed in small groups of or fewer until they have successfully made the transition from liquid feed to dry feed and the transition from individual housing to competing for food. the grouping could be done better by multi-sensory historical recording of individual cow behavioural patterns, body temperature, provenance of any previous disease records, long term eating patterns from the young age, and any obvious known symptoms at the time of the grouping. additionally, by housing in small groups (rather than mixing large groups of animals at one time), producers can limit the exposure of calves to disease organisms and match calves more closely by size. as calves age, they can be housed in increasingly larger groups; however, animals should be grouped so there is not more than kg difference in size of animals up to months and not more than kg difference in size for older animals. biosecurity is an outcome of a human systemso people are at its core. all dairy producers must actively institute biosecurity measures to prevent introduction of disease into the herd and to minimize spread of disease within the herd. for replacements, it is extremely important to prevent exposure of younger animals to older animals that may have johne's disease. exposure is not limited merely to animal-to-animal contact, but also includes articles of transmission, such as manure on hands, clothing and boots of workers, manure from older animals on equipment for feeding and handling replacements, or water that has been contaminated by older animals. in addition, flies can transfer diseases from older to younger animals. producers must determine whether to have a closed herd or to allow introduction of new animals to the farm. if new animals are brought to a farm, the producer should work closely with a veterinary surgeon to determine which vaccinations animals should receive prior to coming to the farm. once new animals arrive on the farm, or animals return to the farm from contract-growers or exhibitions, they should be quarantined for at least days. this will allow time to determine if the new animals are likely to become ill and to allow the new animals to be exposed more slowly to any disease organisms currently on the farm. airborne disease spread in cattle farming is rear but when it happens, severity of damage is significantly higher. prions, the proteins that cause mad cow disease and creutzfeldt-jakob disorder could be spread through the air, rather than just through contaminated food, as recent studies have reported. foot-and-mouth disease (fmd) is a severe, highly contagious viral disease of cattle and swine, primarily get spread under favourable weather conditions. the disease may be spread considerable distances by over a route, within a short time period, directed by wind speed, air pressure and temperature. given the fact that airborne spread is probably the one of the most serious issue in animal biosecurity management, a predictive meteorological service for national level biosecurity risk pathway analysis is essential, for prevention of large-scale livestock disease control. in recent time researchers have reported interesting results and applications of a data driven predictive model to track a probable route of an airborne disease, an alerting system to provide better biosecurity decision support for efficient management of cattle health. other potential sources of disease entry into replacements are visitors, vehicles removing dead animals, feed-delivery vehicles, wild and domestic animals, and birds. within the herd of replacements, diseases can be transferred by using needles on more than one animal or using the same glove to palpate more than one animal. producers must identify potential sources for transfer of disease-causing organisms within the herd and from outside the herd and institute a management plan to control them. digestive disorders can occur in dairy replacements, resulting in problems such as acidosis and overeating diarrhea. overeating diarrhea is found in replacements during the liquid feeding phase and may be prevalent in systems using accelerated feeding programs. this form of diarrhea can be treated by decreasing the amount of dry matter offered to calves in the liquid diet until the consistency of the feces returns to normal. care should be taken to determine whether increased fluidity of the feces is caused by overeating or by disease organisms. if caused by disease organisms, treatment should include administration of an electrolyte solution and may require use of antibiotics. acidosis can occur in replacements if they consume large amounts of grains. forages comprise the basis for diets for replacements after months of age. animals that gain access to fields of maize or bags of feed by accident will often suffer acidosis leading to laminitis (founder) or even death. animals that are affected will generally have severe diarrhea. they can be treated by withholding grain until feces return to normal, followed by gradual reintroduction of grain into the diet. several types of internal parasites are found in dairy replacements. perhaps the most common problem is coccidiosis. coccidiosis causes diarrhea, which may be severe, resulting in weight loss, dehydration and anemia. animals can be treated with a coccidiostat, such as amprolium, for severe coccidiosis. coccidiostats such as decoquinate or lasalocid may be included in grain rations or even in milk replacers to help control coccidiosis. another common internal parasite of calves is cryptosporidium parvum. this organism causes diarrhea in young calves at - days of age that lasts approximately a week. there are no cures for cryptosporidiosis and no means of prevention other than sanitation to decrease the pathogen load. treatment involves electrolyte solutions along with continued milk feeding. replacement animals are very vulnerable to internal parasites (especially worms) during their first grazing season. deworming of heifers yields economic returns in growth rates and feed efficiency. producers should consult their veterinary surgeon to determine the most effective method of treating internal parasites both to decrease the parasite load in the animals and to prevent shedding of eggs onto pastures. depending on geographical location, different deworming strategies are needed to control internal parasite populations. producers should be aware that cold temperatures cause larvae to undergo arrest, even when ingested into the host. during this arrested stage, the larvae are resistant to most deworming agents. many external parasites, including various species of flies, affect health and growth of replacements. several species of blood-sucking flies affect replacements. horn-flies, face-flies, stable-flies, ticks, lice and mite can be a major problem for cattle. they can cause substantial blood loss, transmit diseases including mastitis to replacements and decrease growth rates. use of forced back rubs is probably the most effective method of decreasing populations of horn-flies. additionally, removal of manure, which is the major breeding habitat for horn-flies, helps decrease populations. another type of fly, the stable-fly, breeds in wet feed. severe infestations of stable-flies can cause up to a % decrease in milk production. counts of flies per animal cause economically important losses in milk production and growth. removal of waste feed from under feed troughs and other areas to decrease breeding areas is the most important mechanism for control. horse-flies and deer-flies are also blood-sucking flies and may be responsible for spread of several diseases but are impractical to control. common house-flies are not blood-sucking insects but feed on muzzles, eyes and open wounds. they can be contaminated with more than viruses and bacteria, as well as other disease-causing organisms. the main form of control for common houseflies is sanitation and removal of breeding material because many house-flies are resistant to insecticide sprays. cattle grubs are another parasite common in north america. the main damage to cattle is caused by the migration of the grubs through host tissues and production of cysts on the animals' backs. growth rates can be adversely affected with infestations of cattle grubs. appropriate insecticide treatment will kill grubs; however, care must be taken not to administer insecticides when large numbers of grubs may have accumulated in the spinal canal. killing of large numbers of grubs at once can lead to anaphylaxis in cattle. other external parasites that may affect dairy cattle include fleas, lice, ticks and mites. itchiness and formation of scabs should be examined by a veterinary surgeon who can prescribe appropriate forms of treatment. many disease occurrences can be prevented or at least minimized by appropriate vaccination programs. the program that is appropriate, however, will vary from region to region, and even farm to farm. establishment of a vaccination program requires knowledge of diseases prevalent in the area, history of diseases on the farm, history of diseases in the herd, vaccinations used previously in the herd and an assessment of the risk of contracting economically important diseases based on management of the herd (open or closed). producers should, therefore, consult their veterinary surgeon to develop a vaccination program appropriate for their animals, management and location. timing of vaccinations is important for replacement animals. if the colostrum consumed by the calf contained antibodies against the disease organism present in the vaccine, the vaccine will not generate a sufficient immune response in the replacement animal. maternal antibodies obtained from colostrum may be present up to months of age, preventing an adequate response to vaccinations. it may be beneficial to wait until months of age or greater for many initial vaccinations in calves in order to avoid interference from maternal antibodies. additionally, many vaccines are not effective in neonatal calves because their immune system is not sufficiently developed to generate a protective response. common mistakes made in vaccination programs are lack of booster vaccinations at the appropriate time and lack of frequent vaccinations. if the vaccination protocol calls for an initial vaccination followed by a booster vaccination within - weeks, maximum protection will not be achieved without the booster vaccination. essentially, the money spent for the first injection is wasted. the second problem, lack of frequent vaccinations, is seen especially with leptospiral vaccines. leptospiral vaccines should be administered every months to achieve adequate protection. it is also important for heifers to start receiving leptospiral vaccinations at months of age so that they have received two vaccinations by the time they are used for breeding. health management of replacements requires attention to many different areas, ranging from nutrition and management of late lactation and dry cows to vaccinations of replacements. a better-informed management plans for biosecurity risk and analysis is essential for the next generation replacement cattle management. recent development of low cost sensory technology and an introduction to infectious disease control on farms: biosecurity. american feed ingredients association arlington. bovine alliance on management and nutrition large dairy herd management internal parasites of dairy cattle integrated manure management to reduce environmental impact: ii. environmental impact assessment of strategies dynamic cattle behavioural classification using supervised ensemble classifiers vaccines and vaccination programs application of an integrated outbreak management plan for the control of leptospirosis in dairy cattle herds nutrient and immunity transfer from cow to calf pre-and postcalving optimal management of replacement heifers in a beef herd: a model for simultaneous optimization of rearing and breeding decisions replacement management in cattle: health management. encyclopedia of dairy sciences key: cord- -t z hbox authors: ogawa, hirohito; koizumi, nobuo; ohnuma, aiko; mutemwa, alisheke; hang’ombe, bernard m.; mweene, aaron s.; takada, ayato; sugimoto, chihiro; suzuki, yasuhiko; kida, hiroshi; sawa, hirofumi title: molecular epidemiology of pathogenic leptospira spp. in the straw-colored fruit bat (eidolon helvum) migrating to zambia from the democratic republic of congo date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: t z hbox the role played by bats as a potential source of transmission of leptospira spp. to humans is poorly understood, despite various pathogenic leptospira spp. being identified in these mammals. here, we investigated the prevalence and diversity of pathogenic leptospira spp. that infect the straw-colored fruit bat (eidolon helvum). we captured this bat species, which is widely distributed in africa, in zambia during – . we detected the flagellin b gene (flab) from pathogenic leptospira spp. in kidney samples from of e. helvum ( . %) bats. phylogenetic analysis of flab fragments amplified from e. helvum samples and previously reported sequences, revealed that of the fragments grouped with leptospira borgpetersenii and leptospira kirschneri; however, the remaining flab fragments appeared not to be associated with any reported species. additionally, the s ribosomal rna gene (rrs) amplified from randomly chosen flab-positive samples was compared with previously reported sequences, including bat-derived leptospira spp. all rrs fragments clustered into a pathogenic group. eight fragments were located in unique branches, the other fragments were closely related to leptospira spp. detected in bats. these results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. our study indicates that pathogenic leptospira spp. in e. helvum in zambia have unique genotypes. leptospirosis is an important reemerging zoonotic disease caused by pathogenic spirochetes of the genus leptospira. the disease is found worldwide, especially in tropical regions. human leptospirosis presents with a variety of signs and symptoms, including general febrile disease an influenza-like illness, and results in liver or kidney failure. as a result, this disease is often confused with other diseases, such as dengue fever, hemorrhagic fever and malaria, all of which are common in tropical and subtropical regions of the world (world health organization, ) . environments (e.g., soil and water) (adler and de la peña moctezuma, ) . humans become infected mainly through leptospira-contaminated water or soil, or from contact with urine from animals infected with this bacterium (adler and de la peña moctezuma, ) . rodents are the most important reservoir of leptospira among a variety of wildlife reservoirs. over the past decade, there have been many reports of bats being an important reservoir and vector of emerging infectious diseases, such as ebola and marburg viral diseases, severe acute respiratory syndrome (known as sars), nipah and hendra viral infections, and rabies (calisher et al., ) . bats (order chiroptera) are the second largest order in mammals after rodents (order rodentia) and are geographically widespread. loss of habitat for bats, caused by recent anthropogenic activities, may increase contact between bats and humans, resulting in transmission of various pathogens from peridomestic bats to humans (de jong et al., ) . transmission of viral pathogens from bats to humans has been the main focus of studies in this area; however, there have not been many studies on pathogenic bacteria in bats (mühldorfer, ) . a variety of pathogenic leptospira spp. have been identified in bats worldwide (bessa et al., ; bunnell et al., ; cox et al., ; fennestad and borg-petersen, ; harkin et al., ; lagadec et al., ; matthias et al., ; tulsiani et al., ) ; however, little is known about the role of bats in the transmission of leptospirosis. in this study, we performed a molecular epidemiological investigation of leptospira spp. in straw-colored fruit bats (eidolon helvum) captured from to , which were migrating from the democratic republic of congo to zambia (richter and cumming, ) . a total of kidney samples were collected from captured e. helvum that were roosting in trees (muleya et al., ; ogawa et al., ) in kasanka national park in central province and in ndola in copperbelt province of zambia (table ). this research was performed under the research project ''molecular epidemiology of bacterial zoonoses in zambia'' approved by the zambia wildlife authority, in the republic of zambia. the kidney samples collected from e. helvum were placed directly in korthof or ellinghausen-mccullough-johnson-harris (emjh) media (world health organization, ) and homogenized for dna extraction and leptospira isolation by crushing with beads. dna was extracted from % (w/v) kidney homogenates using a dna isolation kit for mammalian blood (roche diagnostics, indianapolis, in, usa) according to the manufacturer's instructions with minor modifications. a nested pcr based on the flagellin b gene (flab) sequence was used to amplify the extracted dna samples (n = ) to detect the flab gene of pathogenic leptospira spp. (koizumi et al., ) . some of the flab-nested pcr-positive samples (n = ) were examined further. to identify leptospira species, we also performed a nested pcr based on the s ribosomal rna gene (rrs) and the preprotein translocase gene (secy) using the primer sets shown in supplementary tables and . the pcr products from the flab-nested pcr ( bp including the bp primer sequence), the rrs-nested pcr ( bp including the bp primer sequence) and the secy-nested pcr ( bp including the primer sequence) were purified and subjected to direct sequencing using a bigdye terminator v . cycle sequencing a kit (life technologies, waltham, ma, usa) according to the manufacturer's instructions, and a xl genetic analyzer (life technologies). the sequence data were aligned using the clustal w software, and a maximum-likelihood phylogenetic tree was generated with , bootstrap replications using mega . . software (tamura et al., ) . the ddbj accession numbers for the flab and rrs sequences from the uncultured leptospira spp. detected in e. helvum comprised lc to lc and lc to lc , respectively (supplementary table ). a bp fragment of the leptospira flab gene was detected in out of e. helvum kidney samples ( . %, table ). among the flab-nested pcr-positive samples, were used for direct sequencing and nine samples were not able to be sequenced because of insufficient dna. phylogenetic analysis (fig. ) revealed that the flab sequences fell into seven clusters (fc -fc ). six flab fragments (zfb - , zfb - , zfb - , zfb - , zfb - and zfb - ) in the fc cluster were related to the corresponding gene sequences, all of which were identical to leptospira borgpetersenii strains including jules, de , arborea, poi, and veldrat batavia . the six fragments shared sequence identities ranging from . % to . % with the l. borgpetersenii strains described above. the nucleotide identity of the flab fragment for zfb - in the fc cluster with the leptospira kirschneri strains moskva v and c was . % and . %, respectively. the nucleotide sequence identities of five flab fragments (zfb - , zfb - , zfb - , zfb - and zfb - ) in the fc cluster with moskva v and c l. kirschneri strains were . % and . %, respectively. the nucleotide sequence identities of the remaining flab fragments belonging to fc to fc with that of the closest species, l. borgpetersenii, were from . % to . %. in a previous report, l. borgpetersenii, l. kirschneri, and leptospira interrogans were isolated predominantly from rodents in africa (ahmed et al., ; nalam et al., ) . a novel species, leptospira mayottensis, for which strains were isolated in mayotte located in the comoros archipelago, was reported to be genetically similar to l. borgpetersenii (bourhy et al., (bourhy et al., , . this new species was included in phylogenetic analysis; however, the flab fragments belonging to the fc -fc clusters were also distantly related to l. mayottensis. the flab fragments belonging to the three clusters fc -fc appear to group with those of l. borgpetersenii and l. kirschneri; however, the remaining flab fragments belonging to the fc -fc clusters appear not to be associated with those of any reported species. accordingly, the leptospira flab sequence data from kidney samples of captured e. helvum bats indicates that leptospires from e. helvum in zambia have genotypes distinct from those previously reported. the results of phylogenetic analysis of the secy gene, which has been used as a valuable tool for discriminating between leptospira spp. (gravekamp et al., ; rahelinirina et al., ; victoria et al., ) , were in accordance with the flab-based phylogenetic tree ( supplementary fig. ) , also supporting the hypothesis that leptospires from e. helvum in zambia have unique genotypes. subsequently, we examined another gene from leptospira, rrs, which has been used before to identify leptospira spp. (matthias et al., ; postic et al., ) . fragments of the rrs gene (each bp) were amplified and sequenced from samples randomly selected from the seven clusters (fc -fc ) (fig. ) . phylogenetic analysis showed that all rrs fragments from e. helvum kidney samples grouped into a pathogenic group. zfb - , zfb - and zfb - , belonging to the fc cluster, were associated with the l. kirschneri strain, kambale (fj ), and uncultured leptospira sp. (jq ) from triaenops menamena bats captured in madagascar (lagadec et al., ) (fig. ) . zfb - (fc ) and zfb - (fc ) were closely related to l. borgpetersenii and uncultured leptospira sp. (ay ) from bats captured in peru, respectively (matthias et al., ) (fig. ) . the rrs fragments of zfb - fig. . maximum-likelihood phylogenetic tree based on the nucleotide sequences of leptospira spp. flab in e. helvum bats. the dendrogram was constructed with the jc model, and with , replications using mega . . software (tamura et al., ) . numbers at nodes indicate bootstrap supports > %. the sequences determined in this study are shown in bold. the samples colored red were also used in the phylogenetic analysis of rrs (fig. ) . genbank accession numbers are indicated in parentheses. scale bar indicates the number of nucleotide substitutions per site. (fc ), zfb - (fc ), zfb - (fc ) and zfb - (fc ), all of which were identical, as well as those of zfb - (fc ), zfb - (fc ), zfb - (fc ) and zfb - (fc ), were located on unique branches (fig. ) . the other rrs fragments were closely related to an uncultured leptospira sp. (jq ) from rousettus obliviosus bats captured in comoros; the latter sequence was closely related to l. borgpetersenii (lagadec et al., ) (fig. ) . non-significant coevolutionary congruence was reported between the rrs sequence from leptospira spp. and that of bats at the bat species level (lei and olival, ) . however, the rrs sequences from bats are genetically related to each other and show no regional variations in phylogenetic analysis of the rrs sequences from various kinds of hosts (fig. ) , suggesting that leptospira have evolved uniquely in this bat population. dietrich et al. reported that the host is an important factor in leptospira diversification (dietrich et al., ) , also supporting our findings. zambia is bordered by eight countries. epidemiological studies in these countries have been reported; however, almost all of these reports were serological surveys using l. interrogans as the antigen, and most data originated from tanzania and zimbabwe (de vries et al., ) . in zambia, data regarding circulating leptospira spp. are limited. serosurveys of leptospira spp. in rodents and leptospira weilii in pigs have been reported (de vries et al., ) . although e. helvum examined in this study were migrating to zambia from the democratic republic of congo (richter and cumming, ) , data in this country are also lacking and there are no previous reports on l. borgpetersenii that may be related to the leptospira spp. detected in this study. e. helvum captured in kasanka national park were more frequently infected than those captured in nodla (x = . , df = , p < . ). the roosting environment and colony size may influence this difference. no significant difference in the prevalence of the leptospira flab gene was found between males and females. the phylogenetic analyses of flab and rrs infer that genes from potentially pathogenic leptospira spp. were present in the kidney samples of e. helvum in zambia. to the best of our knowledge, this is the first report of pcr detection of leptospira spp. in fruit bats from the african continent. in addition, the nested pcr-positive rate for leptospira ( . %) in e. helvum in zambia was relatively higher than that of previous reports (mühldorfer, ) . although isolation of leptospira directly from bat kidney samples using korthof and emjh media was not successful, the relatively high infection rate in the kidneys of e. helvum is likely to result in excretion of leptospira via the urine. contaminated urine has therefore been proposed as the potential transmission pathway of leptospira spp. from fruit bats to rodents (tulsiani et al., ) . it is suggested, therefore, that e. helvum might be a candidate natural reservoir for leptospira in zambia. continued surveillance in e. helvum, as well as in humans and rodents, is required to gain a better understanding of how leptospira is maintained in, and transmitted by, e. helvum bats in zambia. fig. . maximum-likelihood phylogenetic tree based on the nucleotide 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in the transmission of pathogenic leptospires in australia conservation of the s -spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus leptospira human leptospirosis: guidelines for diagnosis, surveillance and control. world health organization we thank ms. yuka thomas, drs. emiko nakagawa, akihiro ishii, reiko yoshida, yasuko orba, ichiro nakamura, kimihito ito, and many phd students and postdoctoral fellows at the research center for zoonosis control, hokkaido university for technical assistance. we also thank drs. katendi chabgula, edgar simulundu and musso munyeme of the university of zambia, dr. frank willems of the kasanka trust, the ministry agriculture and livestock, the zambia wildlife authority, and mr. bwalya chisha. this work was supported by the japan initiative for global research network on infectious diseases (j-grid) and the science and technology research partnership for sustainable development (satreps) by the japan science and technology agency (jst) and japan international cooperation agency (jica). supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.meegid. . . . key: cord- -pd lt n authors: mathieu, cyrille; dhondt, kévin p.; châlons, marie; mély, stéphane; raoul, hervé; negre, didier; cosset, françois-loïc; gerlier, denis; vivès, romain r.; horvat, branka title: heparan sulfate-dependent enhancement of henipavirus infection date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: pd lt n nipah virus and hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genus henipavirus. they infect humans as well as numerous mammalian species. both viruses use ephrin-b and -b as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. we have previously reported that nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. here, this attachment molecule was identified as heparan sulfate for both nipah virus and hendra virus. cells devoid of heparan sulfate were not able to mediate henipavirus trans-infection and showed reduced permissivity to infection. virus pseudotyped with nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediated trans-infection of henipaviruses. moreover, heparin was shown to bind to ephrin-b and to restrain infection of permissive cells in vitro. consequently, treatment with heparin devoid of anticoagulant activity improved the survival of nipah virus-infected hamsters. altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections. required for cell-mediated trans-infection. as proteoglycans are used by several viruses for binding to target cells ( , ) , we investigated their potential role as a henipavirus attachment receptor. heparan sulfate (hs) is a complex and sulfated polysaccharide of the glycosaminoglycan (gag) family that is linked to ubiquitous core proteins of the cell surface and extracellular matrix of all eukaryotes. hs is composed of a repetition of a d-glucuronic acid/ n-acetyl-d-glucosamine disaccharide motif that can be further modified by addition of sulfate groups. hs has the ability to bind to a vast repertoire of proteins and is involved in many physiological as well as pathological processes ( ) . in addition, the long carbohydrate chains of hs provide easily accessible binding sites for a wide range of pathogens, including viruses, bacteria, and parasites ( , ) . commercial heparin (hp), which is widely used for its anticoagulant properties, is a gag that is chemically related to hs ( , ) and displays protein-binding properties similar to hs. in this study, we show that both niv and hev can use hs as an attachment receptor to mediate trans-infection. in addition, hs facilitates henipavirus infection in cis, and heparin can compete with hs for binding to the virus, thereby limiting both transinfection and infection itself. finally, heparin significantly reduced niv infection in hamsters, suggesting its potential use to treat henipavirus infections. we first asked whether leukocytes could capture hev and transmit it to susceptible cells in trans without becoming infected themselves (fig. a) . as we previously found for niv ( ) , peripheral blood lymphocytes (pbls) also transmit cell-attached hev to susceptible cells, indicating that trans-infection is a mechanism shared by both members of the henipavirus genus. the trans-infection by leukocytes can also be mimicked in cho cells ( ) , which are resistant to henipavirus infection due to the lack of entry receptors efn-b and -b ( ) . the treatment of cho-k cells with heparinase inhibited their trans-infection properties by Ͼ %. accordingly, cho-derived cell lines that lacked expression of hs because of their deficiency in gag biosynthesis enzymes ( ) were also highly deficient in their ability to mediate trans-infection of niv (fig. b) . to further characterize the niv-gag interaction, we compared the ability of virus particles pseudotyped with niv g and f glycoproteins or vesicular stomatitis virus g-protein (vsv-g) ( ) to bind surfaces coated with the hs analog heparin by using surface plasmon resonance (spr). the strong hyperbolic association shape observed during the injection phase of the sensorgram for niv, but not for vsv pseudoparticules, clearly indicated a much stronger binding affinity of niv-pseudotyped viral particles (fig. c) , underlining the importance of niv glycoproteins in the analyzed interaction. to assess the specificity of the niv-gag interaction toward hs, by using spr we compared the binding of niv-pseudotyped virus to surfaces displaying hs, hp, or dermatan sulfate (ds), another sulfated gag (fig. d) . although niv-pseudotyped virus efficiently bound to hp and hs, we did not detect any interaction either niv or hev, washed, cultured for h, and then transferred to vero cell monolayers, which were used for the determination of the viral titers after days of coculture, using infectious center assays. (b) cho-k cells, treated or not with heparinase , and three hs-deficient cho lines, pgsa- , pgsb- , and pgsd- , were incubated with niv and analyzed for their capacities to transmit infection to susceptible vero cells in trans. results are expressed as a percentage of inhibition compared to results with untreated cells Ϯ sd. *, p Ͻ . ; ***, p Ͻ . (mann-whitney u test). (c) spr analysis of the binding of mlv pseudotyped either with vsv-g (green) or with niv glycoproteins g and f (red) to hp-activated sensor chip surfaces. (d) spr analysis of the binding of niv g and f pseudoparticles to surfaces activated by either hs (blue), ds (green), or hp (red). the binding response, in ru, was recorded as a function of time; results from of experiments are presented. with the ds surface, suggesting an implication of specific carbohydrate features. altogether, these results suggested that niv could specifically use hs as an attachment receptor to promote efficient trans-infection. heparin is a competitive inhibitor of henipavirus transinfection. as niv-pseudotyped virus binds heparin in addition to hs (fig. d) , we next analyzed the effect of heparin on the ability of pbls and cho-k to mediate niv trans-infection. heparin reduced the trans-infection property of pbls and cho-k cells by % and %, respectively ( fig. a) . strikingly, heparin was also active when applied after contact with the virus, and both pretreatment and posttreatment with heparin were effective in inhibiting human pbl-mediated trans-infection of either niv or hev (fig. b ). this inhibition reflects the known capacity of heparin to bind multiple cell surface proteins due to its high negative charge ( ) . furthermore, in a pretreatment regimen, heparin also . trans-infection of vero cells was than determined as described for fig. . results are expressed as a percentage of inhibition compared to results in untreated cells, Ϯ the sd. *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . (mann-whitney u test). (c) analysis of niv binding to cho-k and hs-deficient cho-pgsa- cells, pretreated or not with heparin ( . mg/ml). cells were put into contact with niv for h, and the number of viral rna (n gene) copies was determined by rt-qpcr. (d) spr analysis of niv pseudoparticle binding to an hp-activated surface in the absence or presence of soluble heparin at g/ml or g/ml. (e) spr analysis of the binding of niv pseudoparticles to hp-activated sensor chips, after preincubation with either pbs or g/ml of soluble heparin, cs-a, cs-c, or ds. (f) spr analysis of the binding of niv pseudoparticles, expressing either niv-g or niv-f protein, to hp-activated sensor chips. the binding response, in ru, was recorded as a function of time after removal of the background provided by nonpseudotyped particles. results are presented as the averages of separate experiments (Ϯ standard errors of the means) and reflect statistically significant binding of niv-g to hp. **, p Ͻ . (one-sample t test). prevented the binding of niv to cho-k cells by %, as measured by cell-associated viral rna, while the residual binding to the hs-defective pgsa- cho cell line remained unaffected (fig. c ). these results indicated the ability of heparin to elute viral particles from cell surface hs by competition. accordingly, soluble heparin was able to compete and inhibit niv-pseudotyped binding to immobilized hp or hs by spr ( fig. d and data not shown) with % and~ % reductions observed for g/ml and g/ml heparin, respectively. notably, other gags found on cell surface proteoglycans, such as chondroitin sulfate a (cs-a), cs-c, and ds, were devoid of inhibition properties (fig. e) , revealing the specificity of the inhibition of niv binding to hs by heparin. finally, to determine which of two niv membrane glycoproteins, f or g, was responsible for binding to heparin, we analyzed hp binding of pseudotyped niv expressing either niv-f or -g by spr (fig. f) . significantly more binding was obtained with niv-g than with niv-f (fig. f) , and results were further confirmed using hela cells transfected with either niv-g or -f (data not shown). we concluded from these results that niv trans-infection requires binding to cell surface hs via niv-g, a process that can be inhibited by competition with heparin. hs facilitates henipavirus infection in cis. as gags may play multiple roles in viral infection ( , ) , we next investigated whether the hs could be directly involved in henipavirus infection in cis, in addition to its role in trans-infection. permissive vero cells were treated with heparinase , to remove cell surface hs, prior to the infection with niv. this resulted in a modest but significant reduction in the number of infected cells at days postinfection (fig. a) . furthermore, the inhibition of hs sulfation in vero cells after treatment with sodium chlorate significantly reduced infection with both niv (fig. b) and hev (fig. c ), the latter being much more sensitive to sodium chlorate pretreatment than the former. the obtained results therefore suggest that hs sulfation status is determinant for virus attachment to cells and subsequent infectivity. we then tested whether soluble gags could interfere with henipavirus infection in cis. heparin pretreatment of vero cells reproducibly reduced the number of cells infected by either niv or hev (fig. a ). furthermore, pretreatment of either the cells or the virus was efficient, even with very low concentrations ( g/ml) (fig. b ). to distinguish the contribution of heparin-mediated inhibition of niv-g binding to cell surface hs from binding to an efn-b / entry receptor, hs-negative cho-pgsa- cells stably expressing either efn-b or -b ( ) were pretreated with . g/ml of heparin and infected with niv (fig. c ). heparin treatment modestly, but significantly, reduced the percentage of infected cells from both cell lines, indicating that this molecule may also directly inhibit or delay the binding of niv to its entry receptors efn-b and -b . this effect may be the consequence of direct heparin binding to efn-b and/or -b , as recently suggested ( ) . indeed, spr analysis suggested that both cho-pgsa- -efn-b and -b cells bound to heparin, in contrast to nontransfected cho-pgsa- cells (fig. d ), and proportionally to the level of expressed efn-b and -b transcripts (fig. e) . accordingly, soluble recombinant efn-b bound to heparin in the spr assay as well (fig. f) , fitting a : langmuir binding model and yielding a k d of Ϯ nm (mean Ϯ standard deviation [sd]) for the interaction. additional spr analysis showed interactions of efn-b with both hs and heparin, but not with cs (data not shown). thus, by its ability to compete with hs for binding to niv-g and to interfere with efn-b /b receptors, heparin can restrict both transand cis-infection by henipavirus. heparin treatment restricts nipah virus infection in animals. well known for its anticoagulant properties, heparin binds and activates anti-thrombin iii (at-iii) through a specific pentasaccharide sequence ( , ) . to test the antiviral effect of heparin during niv infection in vivo and to avoid potential hemorrhagic complications, we produced heparin lacking anticoagulant activity by using periodate oxidation (po-heparin), which alters the integrity of the at-iii-binding pentasaccharide motif ( ) . since po-heparin inhibited lymphocyte-mediated niv transinfection in vitro similarly to heparin (fig. a) , we tested its antiviral properties in the golden hamster model of niv infection, which closely reproduces the niv pathogenesis seen in humans ( ) . while all nontreated animals succumbed to infection in less than days, survival in the po-heparin-treated group increased moderately (p ϭ . ) (fig. b ), thus suggesting a biological relevance for niv-hs interaction and revealing potential antiviral properties of heparin-like molecules in vivo. the rapid dissemination of nipah virus and hendra virus in an infected host may play a critical role in the high pathogenicity of henipaviruses. our previous work demonstrated that human peripheral blood lymphocytes are not permissive to niv infection but that they could capture the virus and transmit it to susceptible target cells in trans ( ) . in contrast to human lymphocytes, specific subsets of porcine lymphocytes could be infected with niv and thus participate in the transmission of the virus in the swine host, also in cis ( ) . low levels of viral replication were detected in human dendritic cells, suggesting that this cell population could contribute to transmission of niv both in cis and in trans ( ) . recently, a cd -dependent trans-infection pathway in dendritic cells was demonstrated for henipaviruses ( ) ; however, the molecular mechanism of niv trans-infection mediated by lymphocytes, which do not express cd , has remained obscure. this report reveals hs to be a cell surface attachment receptor for both niv and hev that is implicated in the trans-infection properties of human leukocytes as well as in henipavirus infection in cis. hs has been shown to bind many different viruses by interacting with either one or more viral glycoproteins. interactions between hs and the hiv- envelope glycoprotein gp ( , ) and with vaccinia virus a l protein ( ) have been demonstrated. furthermore, hs can bind measles virus hemagglutinin (h) ( ) and the fusion (f) proteins of respiratory syncytial virus (rsv) ( ) and human metapneumovirus ( ) . in the cases of bovine rsv and canine distemper virus, both h and f seem to interact with hs ( , ) . our results suggest that niv-g interacts with hs. although vsv-g protein has been shown to interact with hs ( ), our spr analysis indicated that binding of niv-g to hs had a higher affinity. further studies using recombinant soluble viral g protein should give better insights into the biochemical characteristics of this interaction and identify binding sites on both ligands. hs has been previously suggested to capture, protect, and transmit hiv- ( ) and human t-cell lymphotropic virus type (htlv- ) ( ) in trans. similarly to what was shown for hiv- ( ), the other sulfated gags, such as cs or ds, were inactive, implying that structural features beyond charge density on the polysaccharide backbone could be important for the activity. the implication is that different hs-binding sites may be involved, as shown recently for papillomavirus, for which sequential engagement of three different hs-binding sites leads to virus attachment, interaction with the receptor, and entry into the cell ( ) . likewise, hs stabilizes varicella-zoster virus, making it more accessible for binding to its entry receptor ( ) . thus, similar to its role in other viral infections, hs could improve the contact of henipavirus with host cells so as to help the virus reach the cellular receptors efn-b and -b (summarized schematically in fig. ). we observed that heparin inhibits henipavirus trans-infection in vitro. owing to its capacity to bind multiple cell surface proteins via negatively charged sulfated groups ( ) , heparin could prevent henipavirus attachment to cell surface hs. as it is more sulfated than hs, it could also bind viruses with higher affinity than hs and thus act as an efficient competitive inhibitor for virus binding. furthermore, heparin significantly reduces direct virus infection in cis, in addition to affecting infection in trans. this effect may also result from the interaction between heparin and henipavirus receptors, as evidenced by spr for efn-b and in agreement with a recent report ( ) . these results allowed us to propose a model for the role of hs in henipavirus binding and entry and possible interference of heparin with viral infection (fig. ). in addition to the inhibition of henipavirus infection by binding to either viral g glycoprotein or viral entry receptors, heparin may significantly limit virus spread within the organism by blocking binding to the attachment receptor hs and consecutive trans-infection of permissive cells. the mechanism by which a heparin molecule devoid of anticoagulation properties ( ) restrains virus infectivity in vivo most likely depends on the combination of its different biological activities. in addition to affecting henipavirus infection in cis and in trans, heparin could constrain inflammation and consequent tissue damage by binding various inflammatory molecules. indeed, both heparin and hs bind to important immunoregulatory molecules, such as the chemokine cxcl ( ), which is strongly upregulated during niv infection ( ) . moreover, as heparin is a heavily sulfated electronegative molecule, it might enhance the antiadhesive properties of the endothelium against leukocytes ( ) . this effect may rely on the ability of heparin to bind directly to several adhesion molecules that are expressed during inflammation, including l-selectin ( ), cd b/mac ( ) , and p-selectin ( ) . finally, heparin-like derivatives can stabilize the endothelium ( ) and help in blocking the passage of both humoral and cellular immune factors, as well as viruses, through the blood-brain barrier ( , ) . this process may also contribute to the antiviral effect observed during in vivo experiments, together providing a "proof of concept" for further development of this antiviral approach. the heparin-mediated inhibition of henipavirus infection both in vitro and in vivo highlights the antiviral potential of this gag, which is well tolerated and has already been used in the clinical environment as an anticoagulant for more than years. indeed, heparin treatment reduces niv infection in a hamster animal model, thus opening interesting therapeutic perspectives to complement treatment of this highly lethal infection. additionally, the acute nature of henipavirus infection makes it more prone to the regulatory action of heparin, compared to some chronic infections, including hiv or htlv, where heparin showed in vitro antiviral activity ( , ) . the hs mimetic pi- has already been shown to have significant beneficial effect in vivo in the outcome of dengue virus and encephalitic flavivirus infections ( ) . the use of derivatives that mimic the heparin/hs structure ( ) , synthetic antilipopolysaccharide peptides that bind hs moieties on cell surfaces ( ) , or polyanionic compounds with longer halflives in vivo ( ) , devoid of anticoagulant activity and with potentially higher affinity to henipavirus g-protein, may further improve therapeutic effects. altogether, this study demonstrates a previously unrecognized hs-henipavirus interaction involved in both niv and hev infection and dissemination within the host. it may allow henipaviruses to bind to different circulating cells, to use them for transport for efficient spreading within the host organism, and for target cells, to allow accumulation on their surface, enhancing the efficacy of infection in cis. these results reveal heparan sulfate as a potential therapeutic target for the development of novel approaches against these highly lethal infections. in this context, heparin or its derivatives may be used in a metaphylaxis approach of virus-exposed and potentially infected animals, before the appearance of clinical symptoms, during regular hendra equine epizootics in australia ( ). ethics statement. venous blood from anonymous healthy human volunteers was obtained from the blood transfusion centre (etablissement francais du sang, lyon, france) in accordance with its guidelines, with informed written consent from each volunteer. all animals were handled in strict accordance with good animal practices as defined by the french national charter on the ethics of animals experiments, and all efforts were made to minimize suffering. animal work was approved by the regional ethical committee ceccapp (p _ _ ), and experiments were performed in the inserm jean mérieux bsl laboratory in lyon, france (french animal regulation commitee number a ). cell culture. the cho cell line k and cho pgsa- cells stably transfected with human ephrin-b and -b , (pgsa-efnb and pgsa-efnb ; all generously provided by b. lee [ucla, united states] [ ] ) were maintained in f- medium (invitrogen) supplemented with % fetal calf serum (fcs), u/ml penicillin, . mg streptomycin, mm hepes, and mm l-glutamine at °c in % co . vero e , hs-deficient cho-k cells cho pgsa- , cho pgsb- , and cho pgsd- cells ( ) , hela cells, and stable transfected hela cells with phcmv-nipah-g-neo and phcmv-nipah-f-neo, expressing niv-g and -f proteins, respectively (hela-g and hela-f), were maintained in dulbecco's modified eagle's medium (dmem; invitrogen) supplemented as described above. human peripheral blood was obtained from different healthy donors from the blood transfusion centre (lyon, france). peripheral blood mononuclear cells were isolated by density ficoll/hypaque gradient centrifugation and then centrifuged through a % percoll gradient (pharmacia fine chemicals) for min at ϫ g. pbls were recovered from the high-density fraction. for spr analysis, cells were detached by versene treatment and washed twice with hbs-p buffer ( mm hepes, mm nacl, . % surfactant p , ph . ). virus infection and titration. nipah virus (isolate ummc ; gen-bank accession number ay ) ( ), recombinant niv expressing enhanced green fluorescent protein ( ) , and hendra virus (australia/ horse/ ) obtained from porton down laboratory, united kingdom, were prepared on vero-e cells as described previously ( ) , and infection virus was used in the inserm jean mérieux bsl laboratory in lyon, france. all cells were infected at a multiplicity of infection (moi) of , for h at °c, washed twice, and observed by inverted and/or fluorescence microscopy daily or harvested for rna isolation or for use in transinfection assays. at the indicated times postinfection, l of cell culture supernatant was collected and frozen prior to viral titration. viral titration was performed by plaque assay as detailed elsewhere ( ) . the viral infection level in cocultures of leukocytes and cho cells with vero cells was determined using a previously described infectious center assay in -well plates ( ) , after min of formaldehyde fixation and crystal violet staining. alternatively, in heparin-mediated inhibition assays, titrations were directly performed by plaque assay, using vero cells or cho-pgsa -efn-b or -b cell monolayers. pseudotyped viral particles, containing either niv-g and/or -f or control vsv-g were produced using friend's murine leukemia virus (mlv) particles and t cells, as described previously ( ) . treatment of cells with sodium chlorate. vero cell monolayers were grown to confluence and maintained in dmem supplemented with % fcs and sodium chlorate (naclo ; , and mm) for h to inhibit hs sulfation in cells, as rapid turnover of proteoglycans in the cells ( ) required prolonged incubation with naclo . cells were then detached by using trypsin- . % edta, distributed into new -well tissue culture plates, and grown to confluence in the presence of the same range of concentrations of naclo and infected with either niv or hev ( or pfu/well). following h of incubation in cmc/dmem supplemented with % fcs and naclo , virus was titrated by using crystal violet staining. trans-infection assay. cells were put in contact with virus (moi, ) for h at °c, % co . after two washes with phosphate-buffered saline (pbs), cells were cultured at °c for h and then collected and washed, and -fold serial dilutions were added to cell monolayers of vero cells for determination of cell-associated infectious niv in a infectious center assay, as described previously ( ) . in some experiments, cells were incubated for h at °c with u/ml of heparinase (sigma) and washed times before contact with niv. treatment with heparin (porcine intestinal mucosa; sigma) of either cells or virus and pseudotyped viral particles was performed for min at °c. in heparin pretreatment experiments, after incubation with heparin, cells were thoroughly washed before adding the virus. in heparin posttreatment experiments, niv-resistant cells were initially put in contact with either niv or hev for h and incubated with heparin afterward, for min at °c, washed, and analyzed in a transinfection assay as described above. spr-based binding assays. spr experiments were performed on a biacore x apparatus (for analysis of cells and pseudotyped viral particles) or a biacore apparatus (for pseudotyped viral particles and recombinant ephrin-b ), using cm (for pseudotyped virus and recombinant efn-b ) and cm (for cells) sensor chips and hbs-p buffer. hs (celsus, cincinnati, oh, united states), hp (sigma), and ds (sigma) were biotinylated and immobilized on biacore sensor chips, as described before ( ) . briefly, two flow cells were activated with a mix of . m n-ethyl-n=-(diethylaminopropyl)-carbodiimide (edc) and . m n-hydroxysuccinimide (nhs). then, streptavidin ( g/ml in mm acetate buffer [ph . ]) was injected over the activated flow cells, to obtain an immobilization level of~ , response units (ru). one of these flow cells served as a negative control, while biotinylated hp, hs, and ds were injected on the other flow cells, to obtain suitable immobilization levels ( to ru for recombinant human ephrin-b fc and pseudotyped viral particles analysis; ru for cells). interaction assays were performed at a flow rate of l/min and involved -to -min injections of sample over the hp and negative-control surfaces, followed by a -min washing step with hbs-p buffer to allow dissociation of the complexes formed. at the end of each cycle, gag surfaces were regenerated by sequential injections of . % sds ( min) and m nacl ( . min). typical sample concentrations used were g/ml for recombinant human ephrin-b fc (r&d systems), ϫ to . ϫ particles/ml for pseudotyped viral particles and . ϫ to . ϫ cells/ml for cho-pgsa- and hela cell transfectants. the sensorgrams shown correspond to on-line subtraction of the negative-control signal from the gag surface signal. competition assays were performed by preincubating niv pseudoparticles with gags ( to g/ml, final concentration) for min prior to injection. rna isolation and rt-qpcr. rna was isolated from cells and plasma by using an rneasy minikit (qiagen) in rlt buffer, according to the manufacturer's instructions. reverse transcription (rt) was performed on . g of total rna by using oligo(dt) and random hexamer oligonucleotide primers (iscript cdna synthesis kit; bio-rad) and run in a biometra t-gradient pcr device, and cdnas were diluted / . quantitative pcr (qpcr) was performed with cdna samples by using platinum sybr green qpcr supermix-udg with a rox kit (invitrogen). qpcr was run on the stepone plus pcr system (applied biosystems) as follows: °c for min and cycles of °c for s and °c for min, followed by a melting curve of up to °c at . °c intervals. all samples were run in duplicate, and the results were analyzed using stepone software v . . the glyceraldehyde -phosphate dehydrogenase (gapdh) gene was used as a housekeeping gene to normalize the samples. gapdh and standard references for the corresponding genes were included in each run to check for rna integrity, rna load, and inter-pcr variation. after normalization, the results were expressed as the number of mrna copies of the gene of interest per microgram of analyzed rna. all calculations were done using the ⌬⌬ct model ( ) , and experiments were performed according to the miqe guideline ( ) . primer used included the following: niv n forward, ggcaggattcttcgcaaccatc, and reverse, ggctcttg ggccaatttctctg; murine gapdh forward, gcatggccttccgt gtcc, and reverse, tgtcatcatacttggcaggtttct; efn-b forward, caagttctgctggatcaa, and reverse, gatgttgttccccg aatg, efn-b forward, atggaaagagaccgaggg, and reverse, ga ggttgcattgctggtg. production of heparin devoid of anticoagulant activity (poheparin). to eliminate its anticoagulant properties, heparin was treated with periodate, as previously described ( ) . briefly, mg of heparin from porcine intestine ( . usp units/mg; sigma) was dissolved in l of . m naio in . m sodium acetate buffer (ph ) and stirred at °c for days. the unreacted naio was then neutralized by addition of glycerol ( l), dialyzed against h o, and lyophilized. the sample was resuspended in l of . m nabh in . m ammonium bicarbonate and incubated for a further h at °c. after acidification with glacial acetic acid (to eliminate any remaining nabh ), the reaction mixture was neutralized by addition of naoh ( m), dialyzed against water, and lyophilized. infection of hamsters. eight-week-old golden hamsters (mesocricetus auratus; janvier, france) were anesthetized and infected intraperitoneally with . ml containing niv ( % lethal doses [ld s] preincubated with heparin [ . mg/ml; min at °c]). groups of animals were treated daily subcutaneously with the po-heparin ( mg/kg of body weight) for days, starting from the day of infection. animals were followed daily for weeks. statistical analysis. data are expressed as means Ϯ sd or as the percentage of survival. statistical analyses were performed using mann-whitney u test, one-sample t test, and mantel-cox test within graph-pad's prism software. a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus hendra and nipah viruses: why are they so deadly? nipah virus-a potential agent of bioterrorism? ephrin-b ligand is a functional receptor for hendra virus and nipah virus ephrinb is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb are critical for its use as an alternative receptor for nipah virus nipah virus uses leukocytes for efficient dissemination within a host syndecan captures, protects, and transmits hiv to t lymphocytes human t-cell leukemia virus type envelope glycoprotein gp interacts with cell surface heparan sulfate 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are required for the infectious entry of human papillomavirus type infection of cells by varicella zoster virus: inhibition of viral entry by mannose -phosphate and heparin heparin displaces interferon-gammainducible chemokines (ip- , i-tac, and mig) sequestered in the vasculature and inhibits the transendothelial migration and arterial recruitment of t cells lethal nipah virus infection induces rapid overexpression of cxcl heparan sulfate proteoglycans modulate monocyte migration across cerebral endothelium differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. implications for the use of unfractionated and low molecular weight heparins as therapeutic agents heparin attenuates tnf-alpha induced inflammatory response through a cd b dependent mechanism gmp- binding to neutrophils is inhibited by sulfated glycans polyanionic drugs and viral oncogenesis: a novel approach to control infection, tumor-associated inflammation and angiogenesis contribution of proteoglycans to human immunodeficiency virus type brain invasion antiviral effect of the heparan sulfate mimetic, pi- , against dengue and encephalitic flaviviruses highly sulfated k escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures a new class of synthetic peptide inhibitors blocks attachment and entry of human pathogenic viruses establishment of a nipah virus rescue system acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model specific detection of nipah virus using realtime rt-pcr (taqman) transgenic mice expressing human measles virus (mv) receptor cd provide cells exhibiting different permissivities to mv infections a kinetics and modeling study of rantes( - ) binding to heparin reveals a mechanism of cooperative oligomerization a new mathematical model for relative quantification in real-time rt-pcr the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments periodatetreated, non-anticoagulant heparin-carrying polystyrene (nac-hcps) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung this work was supported by inserm, anr- -mien- - , and anr-astrid- . c.m. was supported by cluster of infectiology rhône-alpes, and k.d. was supported by a dga/inserm research fellowship.we thank i. bally and n. thielens, from the ibs platform of the partnership for structural biology and the institut de biologie structurale in grenoble for assistance and access to the biacore facility and members of the group "immunobiology of viral infections" for their help during the realization of the study. in addition, we thank the labex ecofect (anr- -labx- ) of université de lyon, france, as well as i. grosjean from the cellulonet facility (sfr bioscience gerland-lyon-sud, ums /us ) for their help. we are also grateful to b. lee (ucla, usa) for providing us efn-b -and -b -expressing cho cells and to p. lawrence (ciri lyon) for english editing. we also thank d. cornec, f. jacquot, a. duthey, a. valve, l. barrot, and other biosafety team members from inserm bsl "jean mérieux" for their assistance. key: cord- -af lsj g authors: svobodova, tamara; mejstrikova, ester; salzer, ulrich; sukova, martina; hubacek, petr; matej, radoslav; vasakova, martina; hornofova, ludmila; dvorakova, marcela; fronkova, eva; votava, felix; freiberger, tomas; pohunek, petr; stary, jan; janda, ales title: diffuse parenchymal lung disease as first clinical manifestation of gata- deficiency in childhood date: - - journal: bmc pulm med doi: . /s - - - sha: doc_id: cord_uid: af lsj g background: gata- transcription factor deficiency has recently been described in patients with a propensity towards myeloid malignancy associated with other highly variable phenotypic features: chronic leukocytopenias (dendritic cell-, monocyto-, granulocyto-, lymphocytopenia), increased susceptibility to infections, lymphatic vasculature abnormalities, and sensorineural deafness. patients often suffer from opportunistic respiratory infections; chronic pulmonary changes have been found in advanced disease. case presentation: we present a case of a -year-old previously healthy caucasian male who was admitted to the hospital with fever, malaise, headache, cough and dyspnea. a chest x-ray revealed bilateral interstitial infiltrates and pneumonia was diagnosed. despite prompt clinical improvement under antibiotic therapy, interstitial changes remained stable. a high resolution computer tomography showed severe diffuse parenchymal lung disease, while the patient’s pulmonary function tests were normal and he was asymptomatic. lung tissue biopsy revealed chronic reparative and resorptive reaction with organizing vasculitis. at the time of the initial presentation to the hospital, serological signs of acute infection with epstein-barr virus (ebv) were present; ebv viremia with atypical serological response persisted during two-year follow up. no other infectious agents were found. marked monocytopenia combined with b-cell lymphopenia led to a suspicion of gata- deficiency. diagnosis was confirmed by detection of the previously published heterozygous mutation in gata (c. c > t, p.r c). the patient’s brother and father were both carriers of the same genetic defect. the brother had no clinically relevant ailments despite leukocyte changes similar to the index patient. the father suffered from spondylarthritis, and apart from b-cell lymphopenia, no other changes within the leukocyte pool were seen. conclusion: we conclude that a diagnosis of gata- deficiency should be considered in all patients with diffuse parenchymal lung disease presenting together with leukocytopenia, namely monocyto-, dendritic cell- and b-lymphopenia, irrespective of severity of the clinical phenotype. genetic counseling and screening for gata mutations within the patient’s family should be provided as the phenotype is highly variable and carriers without apparent immunodeficiency are still in danger of developing myeloid malignancy. a prompt recognition of this rare condition helps to direct clinical treatment strategies and follow-up procedures. defects of transcription factor gata- have recently been identified in a few overlapping phenotypes associated with myeloid malignancies: dendritic cell, monocyte, b-and nk-cell deficiency; monomac syndrome (monocytopenia with mycobacterium avium complex infections); emberger syndrome (early onset primary lymphedema, multiple warts, sensorineural deafness, dysmorphism); and familial mds/aml with no additional known phenotype. these syndromes share autosomal-dominant inheritance with variable manifestation of immunodeficiency [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the respiratory tract is frequently affected by viral, fungal or mycobacterial infections. chronic lung tissue changes and pulmonary alveolar proteinosis (pap), as well as pulmonary arterial hypertension, have been described in adult patients [ , [ ] [ ] [ ] . we present an adolescent male with gata- deficiency and early manifestation of diffuse parenchymal lung disease (dpld) as well as an atypical course of epstein-barr virus (ebv) infection. a -year-old caucasian male presented to the hospital with acute fever, malaise, headache, cough and dyspnea. a bilateral pneumonia with signs of systemic inflammation corresponding to bacterial infection (c-reactive protein mg/l) was diagnosed and antibiotic treatment initiated. no causative microorganism was identified. despite rapid clinical improvement, chest x-ray showed persistent interstitial changes ( figure a) . a subsequent high-resolution computer tomography (hrct) revealed marked lung damage suggestive of bronchiectasis with peribronchitis, fibrotisation, subpleural cystic remodeling (honey-combing) and emphysema ( figure b) . interestingly, pulmonary function tests showed normal vital capacity, total lung capacity as well as diffusing capacity (table ) . thus, we detected chronic lung figure pulmonary changes in the index patient. a: diffuse bilateral linear and reticular opacities, compatible with interstitial pulmonary involvement (chest x-ray). b: diffuse subpleural fibrotic changeshoneycomb (black asterisk), areas of subpleural consolidations (blue asterisk) and bronchectasis (red asterisk) in the upper lobes (high-resolution computer tomography scan). c: chronic reparative and resorptive reaction: fibrosis and cystic rearrangement (green arrows) and cholesterol clefts (blue arrows) in the upper left lobe (hematoxylin and eosin tissue stain; original magnification x). d: thickened arterial wall, destruction of the elastic layer, thrombosis showing organizing vasculitis (red arrows) in the upper left lobe (elastin tissue stain; original magnification x). changes with no functional correlate during the first episode of pneumonia in a previously healthy boy. further investigations to unfold the cause for the diffuse parenchymal lung disease were initiated. a complete blood count showed leukocytopenia with marked monocytopenia (table ) . immunological assays detected b-cell lymphopenia with predominance of memory b cells. despite the very low numbers of b cells, normal serum immunoglobulin levels of igm and iga and increased levels of igg were present ( . g/l). antibody response to routine vaccination was normal. no serum autoantibodies were found. functional testing of granulocytes (respiratory burst test: analysis of the ability of granulocytes to release reactive oxygen species after in vitro stimulation) and of t cells (evaluation of proliferative response of t cells to various in vitro stimuli) excluded chronic granulomatous disease and t-cell proliferation defects. serology corresponded with primary ebv infection (table ) . however, the ebv viral load in peripheral blood was low. bronchoalveolar lavage (bal) showed ebv presence in the bronchial fluid. immunological analysis of the bal fluid showed lymphocytosis with predominance of cd pos with increased hla-dr expression (especially on cd pos pos ); alveolar macrophages were present, cd a pos cells were not detected. no pas (periodic acid-schiff) positive material was evident in the alveolar macrophages (table ) . neither bacterial, fungal, mycobacterial (including nontuberculous mycobacteria) nor viral (cytomegalovirus, human herpes virus , varicella zoster virus, human herpes virus, respiratory syntitial virus, influenza, adenovirus, enterovirus, coronavirus, parainfluenza, human rhinovirus, human metapneumovirus, bokavirus and papillomavirus tested) infection was revealed via culture, serology or molecular genetic testing in peripheral blood and bronchoalveolar fluid. hence, the extensive microbiological analysis revealed only the presence of ebv in peripheral blood and lungs. histopathological investigation of the lung parenchyma was prompted. thoracoscopic lung biopsy from a severely affected region of the right upper lobe showed fibrosis, cystic rearrangement and cholesterol clefts with signs of organizing pneumonia and vasculitis (figure c, d) . inflammatory infiltration was predominantly lymphoplasmocytic with presence of activated macrophages. no changes compatible with pulmonary alveolar proteinosis or other alveolar filling disorder were seen. despite an ebv presence ( copies/ . genomic equivalents, g.e.) in the lung tissue found with polymerase chain reaction, hybridization probes for ebv-encoded small rna (eber) were negative in the histology slides. thus, no clear relationship between ebv and the histopathological parenchymal changes could be stated. given the severe affliction of the lung parenchyma with fibrotic remodeling, ongoing inflammation with activated cd pos t cells in the bronchoalveolar fluid and lack of clear evidence for an infectious cause, a treatment with an oral steroid was initiated to suppress further tissue destruction. a prophylactic antibiotic (azithromycin) was added and the patient was closely monitored. immunoglobulin levels normalized and signs of systemic inflammation regressed. after months a stable finding was documented via hrct and no clinical symptoms were present. the ebv viral load remained low in peripheral blood. minimal presence of the virus was seen in repeated bal. however, the serological signs of active ebv infection persisted and no ebna antibodies were detected at the follow up. no lymphoproliferation was present and the patient remained asymptomatic. the condition was classified as persistent ebv viremia accompanied by an atypical serological response. molecular genetic testing of sh d a was carried out. a normal result excluded x-linked lymphoproliferative disease, the most common inborn cause of abnormal immunological reaction to ebv infection. details on other possible genetic causes, not yet tested in our patient, are in the discussion. the patient has a history of occasional uncomplicated respiratory infections; at the age of years he suffered from acute bronchitis, a chest x-ray was performed and retrospective analysis of the image showed some interstitial changes present already at that time. monocytopenia was documented as early as at the age of years. the persistent profound monocytopenia and b-lymphocytopenia at follow up prompted gata sequencing. the diagnosis of gata- deficiency was confirmed by the finding of a known heterozygous pathogenic variation c. c > t (p.r c) [ ] . myeloid malignancy was excluded by morphological, flow cytometric and cytogenetic analysis of the bone marrow aspirate. detailed immunophenotypic analysis of the bone marrow showed suppression of cd pos and cd pos precursors; impairment of b-cell lineage (only . % b cells were present, out of those plasma cells constituted % and mature cd pos cd neg cells %, the precursors cd pos cd pos were scarce) and lower percentage of monocytes as well as their progenitors (cd high cd pos ssc med ). additional testing showed lack of myeloid and plasmacytoid dendritic cells. the immunosuppressive treatment was stopped and the patient was further treated with prophylactic antibiotics and antimycotics. vaccination against human papillomavirus (hpv) was performed as recommended [ ] . he has been monitored closely, including regular checks of bone marrow aspirate for early detection of clonal myeloid proliferation. in case of myelodysplasia, transplantation of hematopoietic stem cells would be initiated. after two years of follow-up the patient did not develop any clinical symptoms. he was treated once for pseudomonas aeruginosa found in the bronchoalveolar fluid detected in the second bal analysis performed months after the first one. otherwise, there were no clinical signs of increased susceptibility to infection. pulmonary function tests remained normal, no progression of the pulmonary parenchyma affliction have been detected so far (tables , and ) . the same heterozygous mutation in gata was found in the patient's -year-old brother and year-old father, whereas his mother was healthy. the brother had been without any clinical symptoms so far, blood tests revealed leukocytopenia and marked monocytopenia. hrct scan showed normal parenchyma, no ebv activity was documented. the father suffered from bilateral ankylosing spondylitis (hla-b positive). apart from low b-cell numbers ( . % cd pos cells of lymphocytes, with prevailing memory phenotype: % cd pos cells out of b cells; norm < %) no leukocyte count abnormalities were detected. the lymphocyte changes in the three family members carrying the gata mutation stimulated investigation of bone marrow output. newly emerging t and b cells can be assessed via t-cell recombination circle (trec) and kappadeleting element recombination circle (krec) analysis in the peripheral blood [ , ] . as expected, both siblings had no detectable krec copies in the peripheral blood, indicating severe impairment of b cell development. trec analysis showed normal results. bone marrow examination of the younger brother also showed complete negativity of krec with normal trec copies. krec copies were absent in the peripheral blood of the father as well. the krec/trec copies were normal in the unaffected mother. interestingly, dna obtained from the newborn guthrie card of the younger brother was analyzed showing a normal amount of krec/trec copies. this indicates that the impairment in b lymphocyte development occurred postnatally. gata- deficiency is a protean disease with a broad spectrum of symptoms. most of the patients present with hematological abnormalities (cytopenias, early-onset myeloid malignancies) and an increased susceptibility to opportunistic infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the recently published cohort of patients treated at the national institute of health (nih, bethesda, usa) [ ] % of the patients had severe viral infections, particularly infection with hpv ( %) presenting with recalcitrant warts, condylomata, and/or dysplasia. severe herpesvirus infections were present in % of patients: recurrent herpes stomatitis, esophagitis, genital infection, severe varicella in % of cases, and cytomegalovirus pneumonia or disseminated disease. interestingly, in % of patients persistent ebv viremia similar to our patient was documented; in patients ebv-positive skin tumors occurred. infection with non-tuberculous mycobacteria was seen in %, severe bacterial infection was observed in % and severe invasive fungal infection in % of the patients. eighteen percent of patients showed no increased susceptibility to infection. additionally, vascular/lymphatic defects (venous thrombosis, lymphedema), sensorineural hearing loss, miscarriages and hypothyroidism were found [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pulmonary involvement in gata- deficiency is frequent, involving infections and pap [ , , , ] , particularly in more advanced stages of the disease. in the nih cohort, % and % of the patients had diffusion and ventilatory defects, respectively. pap was found in % and pulmonary arterial hypertension in % of patients. structural abnormalities included nodules, reticular and ground glass opacities, subpleural blebbing, "crazy paving" , and paraseptal emphysema [ ] . similar picture could be seen in our patient. the surprisingly normal pulmonary function test results in our patient could possibly be explained by localized affliction of the pulmonary tissue. the infiltrated and fibrotic tissue decreased the elasticity of the lung parenchyma, however, there was still enough normal tissue that kept the static volumes and transfer factor normal (table ) . unfortunately, it is not possible to compare our findings with other pediatric patients as the data on pulmonary infliction in children are scarce. there were children in the nih cohort. data on the pulmonary function tests were presented for only of them (median age at testing years, range - years; median time from disease manifestation . years, range - years). all those children suffered from myeloid malignancy, in four of them a chronic infection with herpesviruses or mycobacteria species was documented. mild to severe diffusion defects were found in all tested patients, in two children a bronchial obstruction was seen. no information on structural lung changes in the affected children was provided [ ] . the median age at initial presentation in the nih cohort was years but was highly variable (range months - years). of note, four individuals ( %) had no apparent clinical manifestations as of the last follow-up (range - years). the proportion of patients without symptoms was % by age , % by age , and % by age irrespective of the type of genetic change in gata . the phenotype varied within families significantly [ ] . this fact strongly argues for a substantial impact of epigenetic, infectious and environmental factors on disease manifestation. effects of germline or somatic mutations in other genes may play a role as well. this may explain the variability of symptoms in the three individuals carrying the same gata mutation within our index family. an intriguing issue is the etiology of the chronic diffuse parenchymal lung tissue changes in our patient in the absence of respiratory symptoms. the extensive investigations revealed only ebv presence in peripheral blood as well as in pulmonary tissue without specific tissue changes or clinically apparent ebv infection (e.g. mononucleosis-like symptoms, lymphoproliferation). as the patient presented with serological signs of acute ebv infection whereas the pulmonary changes were chronic, a decisive role of ebv in the pathogenesis of the pulmonary tissue changes in our patient was improbable. possibly repeated mild infections in an environment of impaired regulation of the endothelial nitric oxide synthetase expression [ ] , defective phagocytosis and impaired gm-csf signaling in pulmonary macrophages [ , ] played a role in the pathogenesis of the chronic pulmonary inflammatory changes. poor control of ebv replication resulting in persistent ebv viremia irrespective of lung involvement is a known phenomenon in gata- deficient patients [ , [ ] [ ] [ ] . it has been shown that the inability to confine viral infections in patients with gata- deficiency correlates well with the extent of cytopenias, namely with the lack of dc-, nk-and cd pos t-cells. similarly, the defective antibody response at more advanced stages is associated with b cell lymphopenia [ , ] . however, with regards to the possible oligogenic etiology of immunodeficiency in gata- deficiency, impact of other genes implicated in ebv control should be considered. we have excluded only the most common syndrome -xlinked lymphoproliferative disease type caused by a defect in an adapter protein sap, involved in signalling of cell-cell interactions. other molecules implicated in ebv control encompass for example: the ubiquitously expressed xiap with both antiapoptotic function and multiple signalling pathway connections; the surface molecule cd , important for intercellular communication; the nk cell activating receptor for antibody-dependent cell cytotoxicity (cd ), and minichromosome maintenance (mcm ) crucial for nk-cell function; or il- -inducible t-cell kinase (itk), coronin a, serine-threonine kinase (stk) and magnesium transporter, magt , indispensable for t-cell receptor signalling and tcell homeostasis [ ] . the search for a gata- defect in our patient was prompted by the abnormalities in the leukocyte and lymphocyte counts. another serum marker useful in diagnostics as well as in monitoring of the disease progression (correlating with cytopenia) is the stem cell growth fmsrelated tyrosine kinase ligand (flt ligand) [ ] . we have shown that the newborn screening using krec/trec analysis [ ] cannot be used to screen for gata- deficiency. the prognosis of individuals with gata mutations is difficult to establish due to high clinical variability, incomplete penetrance and lack of close phenotype-genotype correlation data [ , , ] . antibiotics (e.g. azithromycin) and hpv vaccination are the recommended prophylactic measures [ ] . use of steroids or other immunosuppressive therapy is not indicated and exclusion of immunodeficiency in dpld prior to use is warranted. a large proportion of patients will develop myeloid malignancy later in life [ , [ ] [ ] [ ] . the only curative therapy is allogeneic hematopoietic stem cell transplantation. two patients with gata- deficiency with pulmonary involvement transplanted for advanced mds were reported to have profited significantly from this procedure [ ] . diffuse parenchymal lung diseases are a heterogeneous group of disorders with an often insidious onset of symptoms [ ] . the underlying immunodeficiency may not be apparent and an immunological and genetic work-up is required, in particular if abnormalities in peripheral leukocyte counts are revealed. as demonstrated in our patient, an aberrant immune response to common respiratory infections may result in diffuse lung disease with bronchial and bronchiolar damage, significant chronic changes of pulmonary parenchyma and fibrotic remodeling. the structural changes might be present prior to any severe infection. diffuse parenchymal lung disease may become the first manifestation of the gata- deficiency. early genetic diagnosis is critical to direct clinical management, prophylaxis, transplantation, and family screening. autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses, and myelodysplasia mutations in gata are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (monomac) syndrome the human syndrome of dendritic cell, monocyte, b and nk lymphoid deficiency mutations in gata cause primary lymphedema associated with a predisposition to acute myeloid leukemia (emberger syndrome) high frequency of gata mutations in patients with mild chronic neutropenia evolving to monomac syndrome, myelodysplasia, and acute myeloid leukemia gata haploinsufficiency caused by mutations in a conserved intronic element leads to monomac syndrome successful allogeneic hematopoietic stem cell transplantation for gata deficiency the evolution of cellular deficiency in gata mutation gata deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity gata haploinsufficiency caused by mutations in a conserved intronic element leads to monomac syndrome factors affecting thymic function after allogeneic hematopoietic stem cell transplantation replication history of b lymphocytes reveals homeostatic proliferation and extensive antigen-induced b cell expansion molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium a modular enhancer is differentially regulated by gata and nfat elements that direct different tissue-specific patterns of nucleosome positioning and inducible chromatin remodeling effect of transcription factor gata- on phagocytic activity of alveolar macrophages from pneumocystis carinii-infected hosts cellular immune controls over epstein-barr virus infection: new lessons from the clinic and the laboratory neonatal screening for severe primary immunodeficiency diseases using highthroughput triplex real-time pcr highly variable clinical manifestations in a large family with a novel gata mutation interstitial lung diseases in children written informed consent was obtained from the patient and the family for publication of this case report and any accompanying images. a copy of written consent is available for review by the editor of this journal. the authors declare that they have no competing interests.authors' contributions ts identified the patient and drafted the manuscript. em acquired and analysed the flow cytometry data and revised the manuscript. us and tf acquired and analysed the genetic data and revised the manuscript. ms analysed and interpreted the clinical and laboratory data and revised the manuscript. ph acquired and analysed the virology data and revised the manuscript. rm, mv and lh acquired and analysed data on pulmonary tissue pathology and revised the manuscript. md acquired and analysed the radiology data and revised the manuscript. ef and fv analysed the krec/trec data and revised the manuscript. pp acquired and analysed the pulmonary function tests and revised the manuscript. js provided supervision, analysed the clinical data and revised the manuscript. aj is the corresponding author, he initiated the study and wrote the manuscript. all authors read and approved the manuscript. key: cord- -be qely authors: lyoo, hey rhyoung; park, soo young; kim, ji young; jeong, yong seok title: constant up-regulation of bip/grp expression prevents virus-induced apoptosis in bhk- cells with japanese encephalitis virus persistent infection date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: be qely background: persistent infection of the japanese encephalitis virus (jev) has been reported in clinical cases, experimental animals, and various cell culture systems. we previously reported the establishment of spontaneous jev persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in bhk- cells devoid of virus-induced apoptosis, cbs - and cbs - . however, cell-specific factors may play important roles in controlling jev replication and have never been assessed for this specific phenomenon. recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (upr). results: to identify the host cell factors that affect jev persistence, we investigated the expression of essential upr factors in cbs - and cbs - cells. of the selected upr factors tested, the most noticeable deviations from those of the normal bhk- cells with jev acute infection were as follows: the suppression of c/ebp homologous binding protein (chop) and the constant up-regulation of immunoglobulin binding protein (bip) expression in cbs - and cbs - cells. in jev acute infection on normal bhk- cells, silencing chop expression through specific sirna blocked cell death almost completely. meanwhile, depletion of bip by specific sirna unlocked chop expression in cbs - and cbs - cells, resulting in massive cell death. fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the jev persistently infected cells still contained functional arms for cell fate decisions. conclusions: bhk- cells with jev persistent infection strive against virus-induced apoptosis through constant up-regulation of bip expression, resulting in the complete depletion of chop even with apparent virus amplification in the cells. accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of jev persistent infection in mammalian cells. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. viruses have evolved a wide range of strategies to persist in their hosts. it remains a challenge to understand the mechanisms whereby viral persistence is established and maintained, especially viral persistence within a cell or group of cells. mechanisms by which rna virus persistence is initiated and maintained usually involve two virus-specific factors: the generation of defective interfering (di) particles or temperature-sensitive mutation of wild-type virus [ , ] . research suggests that host factors involved in the control of persistent infection relate to elements of innate immunity in morbillivirus [ ] and cellular protein synthesis in reovirus [ ] . protein synthesis and folding occurs in the endoplasmic reticulum (er). mammalian cells have evolved many sophisticated signaling pathways to monitor any abnormality, including the accumulation of misfolded proteins; these pathways are known as the unfolded protein response (upr) [ ] . these signaling pathways monitor the er's capacity to refold and/or remove abnormally folded proteins and to make cell-fate decisions according to the homeostatic balance [ , ] . in all known animal cells, the following are known to be activated to initiate the upr: three er-localized transmembrane upr transducers, inositol requiring kinase (ire ), double-stranded rnaactivated protein kinase-like kinase (perk), and activating transcription factor (atf ) [ ] . under basal conditions, these three sensors are associated with immunoglobulin binding protein (bip), also known as grp , which is a chaperone of the heat shock protein family. each branch operates parallel with a particular target downstream and contributes to both cell-protective and celldeath pathways [ , ] . under severe or chronic er stress, the upr switches its mode of action toward apoptosis. c/ebp homologous binding protein (chop), also known as growth arrest and dna damage-inducible protein (gadd ), is the pro-apoptotic transcription factor that plays an important role in regulating cell death after er stress [ , ] . several molecular mechanisms of chopinduced apoptosis have been cited, such as compromised alteration of bcl- family proteins [ , ] . a variety of viruses induce er stress and the upr, having evolved various mechanisms to cope with the upr [ ] . west nile virus modulates all three arms of the upr and induces numerous apoptotic responses, including induction of chop expression [ ] . modulation of the upr by the west nile virus is regulated differentially along with its replication cycle [ ] . similar to other flaviviruses, the dengue virus also induces the three arms of the upr and chop expression. however, activated chop does not induce its downstream apoptotic markers, such as suppression of anti-apoptotic protein bcl- and activation of caspase- or caspase- [ , ] . in addition, studies of the hepatitis c virus have shown that both viral structural (envelope) and non-structural (ns ) proteins can induce er stress and the upr activation with up-regulation of bip and chop [ , ] . japanese encephalitis virus (jev), a member of the flaviviridae, is the causative agent of encephalitis in humans and can be transmitted by persistently jevinfected mosquitoes [ ] . viral persistence in the nervous systems of jev-infected patients has been shown in approximately % of jev cases, suggesting that jev persistence may contribute to neural sequelae after acute infection [ ] . though jev is usually cytolytic for susceptible cells, persistent infection of jev has been established in various cell cultures, including baby hamster kidney (bhk)- [ ] [ ] [ ] [ ] [ ] [ ] [ ] as well as in a mouse model [ ] . the underlying mechanisms for jev persistence in cultured cells are not clearly described. we have previously demonstrated spontaneous establishment of persistent jev infection in bhk- cells via serial undiluted passages without any supplemental treatment [ ] . examples of supplemental treatment include bcl- overexpression [ ] or indirect infection with supernatants from persistently jev-infected mosquito cells [ ] . our previous study suggested that di particle accumulation and helper virus attenuation are possible mechanisms for the development and maintenance of jev persistence in bhk- cells. nonetheless, labile cellular factors that may play a role in jev persistence have never been identified in this system. jev also induces er stress and the upr, and studies suggest that the activation of the upr is a major cause of jev-induced apoptosis [ ] . the upr has never been assessed in jev persistent infection, however. in this study, we utilized two persistently jevinfected bhk- cell clones (previously reported in [ ] ) to identify the cell-specific factors of the upr involved in jev persistence in mammalian cells. these cell clones seldom, if ever, undergo apoptosis, while jev replicates actively within. we observed that there was no chop expression at any time, but a significant amount of bip expression was constant in these cells. knockdown of bip expression resulted in chop induction and subsequent cell death. because the level of jev amplification in these cells was not low enough to hold an apoptotic process, we suggest that the readjustment of bip expression in host cells could be one of key factors involved in cell fate decision under viral persistence. many viruses that are originally cytopathic have been found to lose their cytopathicity when the persistent infection is established [ ] . jev infection induces severe cytopathic effects in various cell culture systems, including bhk- cells, and researchers have documented the er stress response and subsequent apoptosis in the jevinfected cells [ , ] . in order to assess how much of the jev persistently infected bhk- cell population is destined to apoptosis while continuously producing infectious virus particles, the two bhk- cell clones with jev persistent infection-cbs - and cbs - -were subjected to flow cytometry analysis after annexin v/propidium iodide staining. the number of apoptotic cells and the late apoptotic or necrotic cells increased significantly upon wild-type jev infection in normal bhk- cells ( figure a ). on the contrary, the number of cbs - or cbs - cells with jev persistent infection undergoing apoptosis seemed to remain below the basal level shown in the naïve bhk- cells. the amount of intracellular infectious jev particles produced in the persistently infected cells was -to -fold less than that of an acute infection ( figure b ). all cells subjected to the flow cytometry were plated and analyzed at the same time points. the cells were grown under the same culture conditions, and no notable difference in the cell numbers of each population was observed. constant expression of elevated amounts of bip and complete depletion of chop were associated with the survival of the persistently infected bhk- cells jev infection is known to induce the upr in bhk- cells through bip-perk and/or bip-ire arms, which is followed by chop-mediated apoptosis [ , , ] . based on these reports, this study primarily assessed the expression profile of the upr factors in the bip-perk arm of the bhk- cells with jev persistent infection. normal bhk- cells were infected with jev and harvested at , , , , , , and hr post-infection (p.i.) in order to obtain cell lysates. two jev persistently infected bhk- cell clones, cbs - and cbs - , were freshly seeded and harvested at their confluence on the culture flask; their cell lysates were subjected to western blotting. phosphorylation of perk was gradually increased, peaked at hr p.i., and decreased thereafter in acutely jev-infected cells ( figure a ). in persistently jev-infected cells, however, there was only a minute amount of both perk and p-perk. unlike perk, the expression of eif α in the persistently infected cells seemed enhanced, as shown in normal bhk- cells with jev acute infection at - hr p.i.; however, the amount of p-eif α was barely detectable (figure a) . phosphorylation of eif α often leads to inhibition of protein translation in general, but the translation of atf is promoted by p-eif α [ ] . this phenomenon was reconfirmed in this experiment as the expression of atf increased gradually hr p.i. in jev acutely infected cells (figure a) . chop, the key mediator of er stressinduced apoptosis, was also induced and accumulated gradually along with the atf activation. in contrast, for the jev persistently infected cell clones, the expression of chop was not detected at all, even in the presence of atf for its transcription (figure a ). unlike cbs - , impaired expression of atf in cbs - was repeatedly noticed in several independent experiments. it was noteworthy that a much higher level of bip was maintained in both jev persistently infected cells throughout the culture period. to further investigate whether the jev persistently infected cell clones kept the upr pathway intact, naïve bhk- cells and the two cell clones with jev persistent infection were treated with . μg tunicamycin ml − for hr. compared to the dmso-treated control, both tunicamycin-treated normal bhk- cells and cbs - cells showed perk-eif α-atf pathway activation followed by chop induction ( figure b ). most of the cells treated with tunicamycin, including cbs - and cbs - cells, succumbed to apoptotic cell death within hr (data not shown). although chop was also clearly induced, expression of p-perk or atf in cbs - cells was not comparable to the normal bhk- cells or cbs - cells on tunicamycin treatment for unknown reasons. this observation suggests that cbs - cell clones may utilize another pathway to induce chop expression, perhaps involving ire activation. these differences in the activation process of the bip-perk arm between cbs - and cbs - imply that individual cells comprising a cell batch with jev persistent infection could have their own unique modification in cellular physiology to avoid the virus-induced apoptosis. taken together, these results suggest that the jev persistently infected cells avoid fulminant apoptosis by maintaining a constant, highly-elevated level of bip, which results in the complete suppression of chop induction. based on the observations, there was no detectable chop expression during continued virus replication in the jev persistently infected cells ( figures b, a , and additional file : figure s ). therefore, we attempted to clarify the effects of chop on jev-induced apoptosis and on virus replication efficiency. naïve bhk- cells transfected with specific sirna for chop were infected with jev at hr post-transfection and harvested at hr p.i. the cell lysates were examined by western blotting with antibodies against chop, bcl- , caspase- , and jev ns . the expression of chop was efficiently silenced, and cleavage of caspase- was not detected in sichop-transfected cells ( figure a ). we also found that, even in the reduced expression of bcl- , sichoptransfected cells were highly resistant to the jev-induced cytopathic effect, as measured by trypan blue exclusion ( figure a and b) . the level of jev ns protein in the cells transfected with sichop and then infected with jev was slightly lower than that of the other cells transfected with scramble sirna ( figure a ). this observation aligns with the result that the virus titer obtained from the chopsilenced cells was slightly compromised compared to those from the control cells at hr p.i. (figure c ). these results are also consistent with a report noting that up-regulation of chop during jev infection plays a key role in virus-induced apoptosis [ ] . as infectious bronchitis virus-induced apoptosis was suppressed and virus replication inhibited in the chop-knockdown cells [ ] , the complete blockage of chop expression in cbs - or cbs - cells might intervene in jev replication and therefore assist in the development of jev persistent infection. however, a clear explanation for this observation is presently beyond the scope of this study. in this study of jev persistently infected cells compared to an acute infection in normal bhk- cells, the most distinguishable aspects regarding the upr factors were the complete depletion of chop and the constant upregulation of bip expression (figure a and additional file : figure s ). some previous studies also showed that bip overexpression attenuates er stress signaling and is protective against apoptosis [ ] . therefore, we decided to assess the implications of the constant expression of bip for maintaining cell viability against virus-induced apoptosis in the jev persistently infected cells. the cbs - and cbs - cells were transfected with specific sirna for bip and harvested hr later. the effect of bip silencing was examined by western blotting with antibodies against bip, chop, caspase- , and jev ns . the expression of bip was suppressed almost completely by sibip in both cbs - and cbs - cells, while the chop expression was clearly induced ( figure a ). in addition, the numbers of viable cells decreased significantly in both cell lines according to cleavage of caspase- ( figure a and b) . the results suggest that the constant overexpression of bip in the jev persistently infected cells somehow holds back chop expression, resulting in the prevention of virus-induced apoptosis. this observation is consistent with reports that the inhibition of chop guarantees a higher survival rate both in vivo and in vitro even though chop is not the sole factor promoting cell death undergoing er stress [ , ] . furthermore, these results revealed that resistance against virus-induced apoptosis of the cbs - and cbs - cells did not result from the lower level of virus replication efficiency; rather it was ascribable primarily to the active participation of cellular factors in the upr. in conclusion, bhk- cells with jev persistent infection strive against virus-induced apoptosis through constant up-regulation of bip expression, a key chaperone involved in er stress. as demonstrated in tunicamycin treatment, these cells maintained their capacity to decide their own cell death fate by inducing chop, although some of the upr factors relaying the bip-perk-atf arm appeared to be impaired. in a previous study, we successfully established jev persistent infection in several mammalian cells in the presence of di particle generation [ ] . certain modifications in the genetic makeup of helper jev could also play a role in the development and maintenance of the viral persistent infection [ ] . therefore, the observations made in this experiment demonstrate that both the compromised virus replication capacity and certain cellular factors, such as the upr factors, also participate in establishing viral persistent infection in mammalian cells. our study highlights the importance of certain host cell factors in er stress-signaling pathways for jev persistence by utilizing an in vitro model for the first time. this work provides new insight into the complex mechanism of viral persistence and potentially contributes to developing useful agents and tools for therapeutic intervention in the clinical sequelae of japanese encephalitis. baby hamster kidney- (bhk- ; korea cell line bank) cells were maintained in a minimum essential medium (mem; gibco) containing % fbs (gibco) and units of penicillin-streptomycin (gibco) ml − . the persistently jev-infected bhk- cells have been described previously [ ] . two cell clones, cbs - and cbs - , were chosen for use. all cells were grown at °c in a % co incubator. jev k p strain (provided by the korean national institute of health) was employed throughout this study. propagation of the virus was carried out in bhk- at °c in mem supplemented with % fbs for hr. after infection, the virus-containing supernatant was collected and centrifuged to remove cell debris, and then stored at − °c. the virus was inoculated on a monolayer of bhk- cells in mm plates; an overlay medium was applied containing % fbs, % penicillin-streptomycin ( , u), % × mem, % × mem, and % agarose. after to days incubation, the cells were fixed with . % formaldehyde in phosphate buffered saline (pbs) for hr and stained with . % crystal violet. to titrate the intracellular virus particles, the infected cells were washed with pbs, trypsinized, and resuspended in ml of mem. after three times of freeze and thaw cycles, the cell debris was pelleted before collecting the virus-containing supernatant. the virus titer was determined by a plaque-forming assay. to analyze apoptosis, an annexin v-fluorescein isothiocyanate (fitc) and pi double-staining method (meb-cyto apoptosis kit; mbl) was used according to the manufacturer's protocol. after the adherent cells were harvested, they were re-suspended in binding buffer, and μl annexin v-fitc and . μl pi were added to the cell samples. the mixtures were incubated for min in the dark at room temperature and then analyzed by flow cytometry (bd facscalibur). tunicamycin (sigma-aldrich) was dissolved in dmso. rabbit anti-ns antibody was kindly provided by professor radhakrishnan padmanabhan (georgetown university, usa). antibodies against p-perk, total perk, p-eif α, total eif α, bip, and caspase- were purchased from cell signaling technology. antibodies against chop and bcl- were purchased from santa cruz biotechnology. anti-atf antibody and anti-β-actin antibody were sourced from abcam and neomarkers, respectively. hrpconjugated goat anti-mouse antibody and hrp-conjugated goat anti-rabbit antibody were obtained from molecular probes and invitrogen, respectively. the bhk- cells, cbs - and cbs - , were seeded in -well plates and grown to % confluence. sichop, sibip, and scramble sirna were purchased from santa cruz. transfection of sirna was conducted using jet-prime™ (polyplus-transfection) according to the manufacturer's instructions. the bhk- cells were infected with jev at hr post-transfection. the cells and the supernatant were harvested at hr post-infection, and the pi cell clones were harvested at hr posttransfection for further analysis. the collected supernatant was used for the quantification of the viral production, and viable cells were counted using trypan blue exclusion. for total protein extraction, virus-or mock-infected cells in monolayers were washed with cold pbs and then lysed in ice-cold m-per buffer (pierce) with a cocktail of protease inhibitors (roche). proteins were separated with . % or % gradient page using the gradi-gel™ gradient analysis kit (elpis biotech); they were subsequently transferred to pvdf membrane (millipore). the membrane was blocked with % skim milk in tbst. primary antibodies were incubated at °c with membrane overnight in % skim milk in tbst or % bsa in tbst. after primary incubation, the membrane was washed in tbst once for min and three times for min; it then was incubated with secondary antibodies in % skim milk in tbst at room temperature for hr. the membrane was washed again three times in tbst for min, and the proteins were detected with an enhanced luminol-based chemiluminescent detection kit (abfrontier) according to the manufacturer's instructions. the intensities of bands from western blot analysis were quantified using the imagej program (national institutes of health) according to the developer's instructions. the data were presented as mean ± standard deviations (sd) of three independent experiments. the differences between groups were assessed by student's t test. a p value < . was considered statistically significant. all statistical analyses were performed using spss. additional file : figure s . modulation of bip and chop expression in the persistently jev-infected cell clones. protein samples were collected from bhk- cells infected with jev at an moi of at to hr p.i., and from persistently jev-infected (pi) cell clones cbs - and cbs - . cell lysates were analyzed by western blotting for chop, bip, jev ns , and the internal control β-actin. band intensities for bip and ns were determined by densitometry and normalized to those for β-actin. submit your next manuscript to biomed central and take full advantage of: persistent infection of tissue culture cells by rna viruses strategies of virus persistence molecular mechanisms of measles virus persistence molecular mechanisms of persistent infection by reovirus endoplasmic reticulum stress in disease pathogenesis the unfolded protein response: from stress pathway to homeostatic regulation modulating stress responses by the uprosome: a matter of life and death the endoplasmic reticulum and the unfolded protein response identification of novel stress-induced genes downstream of chop roles of chop/gadd in endoplasmic reticulum stress mechanisms of er stress-induced apoptosis in atherosclerosis integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress viruses, endoplasmic reticulum stress, and interferon responses west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion dengue virus modulates the unfolded protein response in a time-dependent manner dengue virus serotype infection specifies the activation of the unfolded protein response hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response hepatitis c virus ns protein triggers endoplasmic reticulum stress and suppresses its own viral replication overview: japanese encephalitis persistence of japanese encephalitis virus in the human nervous system persistent infection of cultured mammalian cells by japanese encephalitis virus continuous mouse brain cell lines chronically infected with japanese encephalitis virus clonal analysis of mammalian cell cultures persistently infected with japanese encephalitis virus persistent infection of porcine kidney cells with japanese encephalitis virus antiapoptotic but not antiviral function of human bcl- assists establishment of japanese encephalitis virus persistence in cultured cells integrative effect of defective interfering rna accumulation and helper virus attenuation is responsible for the persistent infection of japanese encephalitis virus in bhk- cells characterization of homologous defective interfering rna during persistent infection of vero cells with japanese encephalitis virus persistence, latency and reactivation of japanese encephalitis virus infection in mice defective interfering rnas of japanese encephalitis virus found in mosquito cells and correlation with persistent infection japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response effect of enforced expression of human bcl- on japanese encephalitis virus-induced apoptosis in cultured cells stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy er stress, autophagy, and rna viruses translational control in the endoplasmic reticulum stress response upregulation of chop/gadd during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway bip overexpression, but not chop inhibition, attenuates fatty-acid-induced endoplasmic reticulum stress and apoptosis in hepg liver cells chop is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum chop induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum we would like to thank professor radhakrishnan padmanabhan (georgetown university, usa) for providing flavivirus ns -specific antibody. the authors declare that they have no competing interests. key: cord- -ffulkqrf authors: versluys, anne birgitta; van der ent, korstiaan; boelens, jaap j.; wolfs, tom; de jong, pim; bierings, marc b. title: high diagnostic yield of dedicated pulmonary screening before hematopoietic cell transplantation in children date: - - journal: biol blood marrow transplant doi: . /j.bbmt. . . sha: doc_id: cord_uid: ffulkqrf pulmonary complications are an important cause for treatment-related morbidity and mortality in hematopoietic cell transplantation (hct) in children. the aim of this study was to investigate the yield of our pre-hct pulmonary screening program. we also describe our management guidelines based on these findings and correlate them with symptomatic lung injury after hct. since , all patients undergo a dedicated pulmonary screening consisting of pulmonary function test (pft), chest high-resolution computed tomography (hrct), and bronchial alveolar lavage (bal) before hct. we systematically evaluated the yield during the first years of our screening program. we included consecutive children. in % of patients, abnormalities were found. in % of patients, or more pft results were < % of normal. chest hrct showed abnormalities in %; % of these abnormalities were considered “clinically significant.” bal was abnormal in % of patients; respiratory viruses (pcr) were found in patients, fungi (antigen or culture) in , and bacteria (culture) in . all screening tests contributed separately to clinically relevant information regarding pulmonary status in these pre-hct children. in patients ( %), screening results had diagnostic and/or therapeutic implications. we found an association between pre-sct hrct findings and lung injury after transplantation. pre-hct screening with the combination of modalities, reflecting different domains of respiratory status (function, structure, and microbial colonization), reveals important abnormalities in a substantial number of patients. whether this improves patient outcome requires further investigation. hematopoietic cell transplantation (hct) is a curative treatment for various diseases. pulmonary complications, both infectious and noninfectious, are frequently seen in patients undergoing hct. in children, the incidence of pulmonary complications varies from % to % and is associated with a significantly increased risk for mortality [ ] [ ] [ ] . because of the risk of life-threatening complications of the procedure, patients are routinely screened for hct eligibility. lung screening can potentially impact selection of hct patients as well as affect preemptive treatment and prognosis. invasive fungal infections (ifi) are an important cause of morbidity and mortality during hct. diagnostic imaging, culturing pathogens, and antigen detection can be helpful to identify patients at high risk for ifi, which may guide therapy [ ] . also, respiratory viruses (rv) may have impact on the overall survival of hct, either directly as a cause of pneumonitis in the severe immune-compromised patient or indirectly by triggering allo-immunity in the setting of allogeneic transplantation [ ] . in , we implemented extensive pre-hct lung screening, which includes pulmonary function test (pft), chest high-resolution computed tomography (hrct), and bronchial alveolar lavage (bal) in all patients. here, we evaluate the yield of such an extensive pulmonary screening biology of blood and marrow transplantation j o u r n a l h o m e p a g e : w w w . b b m t . o r g program and describe our treatment guidelines according to these findings as well as the outcome of patients. all consecutive pediatric patients undergoing a first allogeneic hct in our center between january and august were included. patients were enrolled in the hct research protocol after providing written informed consent for data collection and analysis, according to national ethical regulations (ethical commission number / and / k). patient characteristics (age, gender, underlying disease), clinical symptoms, results of pulmonary screening tests, and occurrence of symptomatic lung disease after hct was registered. standard pre-hct pulmonary screening is performed in the week before transplantation and consists of a pft, hrct scan, and bal. pft includes spirometry, whole body plethysmography, and measurement of carbon monoxide diffusion capacity. measurements are performed in children aged years and older, according to american thoracic society/ european respiratory society criteria, using calibrated pneumotachometer systems (jaeger, hochberg, germany). values are expressed as percentage of predicted values for age, race, sex, and height-matched controls (the utrecht data set, koopman [ ] ). forced expiratory volume in second, forced vital capacity, total lung capacity, and lung diffusion capacity for co, corrected for hemoglobin and alveolar volume < % of predicted values are considered to be abnormal. residual volume of > % of total lung capacity is considered to be abnormal and suggestive for trapped air. hrct scans are acquired using a -detector row scanner (philips medical systems, best, netherlands). for infants and young children, scans are obtained at -cm h o pressure (inspiration) and -cm h o pressure (expiration). for older children, who were able to cooperate with breath hold instruction, scans were obtained at full inspiration and at end of exhalation. inspiration images are obtained using fixed kvp and to mas (depending on bodyweight). for expiration images, we used kvp and mas. acquisition was volumetric thin-slice for both inspiratory and expiratory computed tomography. all hrct scans were assessed by a pediatric radiologist. fleischner society terms for thoracic imaging were used [ ] . all abnormalities, as stated in the radiology report, were registered. those abnormalities with clinical implications, such as antimicrobial treatment, guided lung biopsy or diuretics, were defined as clinically significant. bal was performed under general anesthesia. bal fluid was cultured and processed in accordance with standard microbiological procedures. galactomannan (gm) tests are performed using biorad platelia aspergillus eia. any positive culture or gm levels > . was considered to be abnormal. nucleic acids are extracted using the total nucleic acid protocol with the magna pure lc nucleic acid isolation system (roche diagnostics, basel, switzerland). for detection of rna-viruses cdna is synthesized by using multiscribe reverse transcriptase and random hexamers (applied biosystems, foster city, ca). detection of viral and atypical pathogens was performed in parallel, using real-time pcr assays specific for the following viruses: bocavirus, human herpesviruse , respiratory syncytial virus, influenzavirus a and b, parainfluenzavirus to , rhinoviruses, adenoviruses, human coronavirus oc , nl and e, human metapneumovirus, and mycoplasma pneumoniae. real-time pcr procedures were performed as described previously [ ] . any positive pcr is considered to be abnormal. the total costs for the pulmonary screening were approximately euro. chest hrct costs euro, rv panel pcr , bacterial cultures euro, gm euro, pft (complete) cost euro. for further analysis, patients were classified according to their risk for pre-hct pulmonary problems, based on underlying disease, immune competence, infection risk, and pretreatment with potentially lung-toxic therapy. we distinguished groups of patients: those with an inherited immune deficiency, those with a malignant disease and chemotherapy before transplantation, and those with inborn errors of metabolism, mild bone marrow failure, and malignancies without chemotherapeutic treatment. antibiotic prophylaxis involved daily ciprofloxacin and fluconazole, from the start of conditioning until the resolution of neutropenia. additional prophylaxis against streptococcus viridans was given with cefazoline in the mucositis phase. empiric antibiotic treatment for febrile neutropenia included vancomycin and ceftazidime. pneumocystis jeroveci pneumonia prophylaxis was started from month after transplantation as cotrimoxazole times a week. in case of positive serology for herpes simplex virus in all patients, and in case of positive serology for varicella zoster virus in cord blood transplantation recipients, prophylaxis with aciclovir was given. no other antiviral prophylaxis was given. in patients at high risk for ifi, according to our protocol, based on pretreatment, duration of neutropenia, and history of fungal infection, aspergillus prophylaxis was given with daily voriconazole or twice weekly amphotericin b. patients with severely impaired pft (< % of normal) were considered to have an unacceptable high risk for treatment-related mortality and were excluded for hct. patients with rv from bal were considered to have a high risk for alloimmune-mediated lung syndromes. in elective hct procedures, hct was postponed until the rv was cleared. in other casesdwhen the underlying disease did not allow treatment delaydtapering of immune suppression after hct was adjusted to prevent alloimmune-mediated lung syndromes. in cases with probable fungal disease (positive cultures or gm from bal), antifungal treatment was considered. patients with positive bacterial cultures from bal were not treated, unless pulmonary symptoms developed. bacterial culture results guide the choice of empirical antibiotic treatment for neutropenic fever after hct. in patients with nodular lesions on hrct, lung biopsy was considered to identify the possible infectious cause and antimicrobial resistance pattern. in patients with possible or proven ifi based on bal findings, biopsy results, or hrct findings, antifungal treatment was started and granulocyte transfusions or haploidentical stem cell support (combined with cord blood grafts) were considered. calculation of mean values and standard deviation was done for pfts. comparing the results with predicted values for age, race, sex, and heightmatched controls was done using t-test (test value %). comparison of the means between the different disease groups was done using anova. the chi-square test was used for comparison of proportions between or more groups. differences with a p value of < . were considered statistically significant. associations between pre-hct pulmonary screening findings and clinically manifested lung injury after hct were analyzed using cox proportional hazard models. dichotomous outcomes were used as dependent variables. univariate predictors with a p value of < . were used for multivariate analysis. all statistics were done using spss . we included consecutive children receiving a first allogeneic hct. apart from mild upper respiratory tract symptoms in some, all patients were asymptomatic for lung disease at the time of pre-hct screening. patient characteristics are shown in table . in patients, no pre-sct lung screening was performed at all; of them were under months of age. in patients ( %), all planned screening modalities could be performed. in children, only some of the tests were done either for logistic reasons (n ¼ ) or because screening bal was the only reason for general anesthesia, which was then considered a disproportional invasive procedure (n ¼ ). in children above the age of , pft was not feasible because of developmental delay. in patient, hrct was omitted because of the risk of irradiation damage related to underlying disease (fanconi anemia). pft were performed in patients. this was % of all patients in whom we were able to perform these tests, according to age and developmental level. results are shown in table . we found pft patterns of restrictive and obstructive lung disease as well as diffusion abnormalities with forced expiratory volume in second, mean . % (sd . ); forced vital capacity, mean . % (sd . ); total lung capacity, % (sd . ); and lung diffusion capacity for co, corrected for hemoglobin and alveolar volume, . % (sd . ). compared with the reference population with values of % (sd ), all differences were statistically significant; with p values of < . . there was no difference in abnormalities in pft between the different disease categories, see table . chest hrct was performed in ( %) patients. in patients ( %), abnormalities were seen; in of patients ( %) these findings were "new" findings. in the group of patients without pretreatment with chemotherapy or immune deficiency, the incidence of hrct abnormalities before hct was significantly lower than in the other patients ( figure ). in patients ( %), clinically significant abnormalities were found. four ( %) had lesions suspect for fungus. fourteen patients ( %) showed other abnormalities, including bronchiectasis, pleural effusion, consolidations, and aspecific nodules > cm. these were new findings in of patients ( %). most clinically significant abnormalities were found in the subgroup of patients with immune deficiencies, but this did not reach statistical significance ( figure ). bal was performed in ( %) patients. unfortunately, for logistic reasons, it was not always possible to do all microbial tests. overall, in % of tested patients, or more of the microbial tests were positive. positive pcr for rv was found in ( %) of tested patients. rhinovirus was the most frequently detected virus (table ). in ( %) patients, we found microbial evidence of fungal colonization either with positive cultures or gm. in only patients, a positive gm corresponded with a positive culture for aspergillus; in patient with a positive aspergillus culture, gm from bal was negative. the positive findings for the whole cohort are shown in table . in patients under years of age, the incidences of bal abnormalities in general, rv positivity, and bacteria positivity were significant higher than in older patients (p values of . , . , and . respectively). figure shows the yield of all screening tests. abnormalities were found in tests of % of patients. we found abnormalities in radiological tests among patients with normal and abnormal pfts, as well as positive microbial test results in patients with normal pfts and normal hrct. in patients, the screening outcome had implications, such as guided bal/lung biopsy (n ¼ ), change in antifungal treatment/prophylaxis (n ¼ ), granulocyte transfusions (n ¼ ), addition of haploidentical stem cells (n ¼ ), postponement of hct (n ¼ ), or guided tapering of immune suppressive agents (n ¼ ). these interventions overlapped in some patients. in patients, the pre-hct hrct showed new or progressive signs of infiltrative fungal infection. antifungal therapy was intensified in based on resistance pattern of the cultured pathogen. in patients, we postponed hct. in cord blood transplant recipients, we added haploidentical cd þ cells from a family donor for early myeloid support, and in patients we gave granulocyte transfusions during the period of neutropenia. one patient had graft rejection and showed fatal progression of aspergillus infection in prolonged neutropenia. the others did not show progression of infection, and therapy could be stopped safely after engraftment. no patient with rv or bacteria isolated showed progression of pulmonary infection during hct. none of the patients with isolated positive findings for fungus from bal, of whom the majority received intensification of fungal prophylaxis/ treatment, developed pulmonary fungal disease. cox regression analysis did not show a relation between pre-hct screening findings in bal or pft and symptomatic lung injury after hct. a clinically significant abnormality on chest hrct before hct, however, was a predictor for the development of immune-mediated lung injury after hct (hazard ratio, . ; % confidence interval, . to . ; p ¼ . ). our study in pediatric patients shows that pulmonary screening before hct with pft, hrct, and bal is feasible. we could perform all the tests in the majority of patients ( %). abnormalities were found in % of patients. in % of * galactomannan in bal > . ¼ positive. y there was overlap between fungal culture results and galactomannan findings in bal ( of aspergillus-positive patients were also galactomannan positive, patient with candida and with penicillium also had positive gm) so patients were considered positive for fungus. patients, these abnormalities led to supportive/preemptive treatment according to guidelines. only in patients with clinically significant chest hrct, abnormalities a higher incidence of lung-injury was noted after hct. although not negligible, the costs seem justified in relation to the findings. it is well known that pulmonary function declines early after hct [ , ] , and some studies have shown a continuous decline without reaching a plateau during prolonged followup [ ] . several studies have demonstrated that impaired pft before transplantation increases the risk for post-transplantation lung complications and mortality [ , , [ ] [ ] [ ] [ ] . possible explanations for these observations are that patients can have such marginal lung reserve capacity, endangering a period of critical illness and/or further lung toxic events. also, in patients with pre-existing lung injury, this organ may be at increased risk for allo-immune phenomena, such as graft-versus-host disease. we evaluated the yield of hrct scanning. omitting hrct from our screening in this cohort would have missed ( %) children with abnormalities, including of the with infiltrative lesions suspect for fungus. on the other hand, hrct leads to radiation exposure and may require general anesthesia in children and, therefore, deserves critical appraisal. the relevance of abnormal findings on hrct are a matter of debate. in the radiological reports in this study, abnormalities were described in % of patients. because the severity of the reported abnormalities varied considerably, we chose to take into account those hrct findings which had "significant clinical meaning" at time of transplantation, such as consolidations requiring antibiotic or antifungal therapy, bronchiectasis as a risk factor for infections warranting change in prophylaxis, or pleural effusions requiring diuretics. in most patients, a plain chest x-ray was available but showed abnormalities in only %, and, of note, did not show any abnormalities in the patients with signs of invasive fungal infection on hrct (data not shown). the yield of bal procedures was high in our study. omitting bal would have missed ( %) patients with fungal colonization and ( %) with rv. all these patients had normal hrct scans and no significant pulmonary symptoms. figure illustrates that all tests contribute separately to information regarding pulmonary status in pre-hct children. this argues that all these screening modalities, reflecting different domains of respiratory status (function, structure, and microbial colonization), should be done in all pediatric pre-hct patients, if a sensitive pre-hct screening for pulmonary pathology is desired. the impact of the finding and the invasiveness of the test should guide clinicians in decision-making on whether or not to perform all tests. as far as we know, this is the first report on such comprehensive pulmonary screening with pft, hrct, and bal in a large cohort of children before hct. we have shown considerable decrease in pulmonary function, a significant amount of clinical important hrct findings, and high prevalence of infectious agents. this is most likely because of underlying disease, pretreatment with chemotherapy, and the age distribution of our patients. in this cohort of patients, there is a significant association between clinically significant hrct findings before hct and lung injury after hct. the findings in bal and pft were not related with outcome. this might be due to small numbers. we might also conclude that with current treatment strategies for this group of pulmonary compromised patients, we manage to have comparable outcome. we conclude that this screening protocol is feasible and provides important information for risk classification with therapeutic consequences. we would advocate all screening methods, as they all contribute separately. prospective studies are needed to further identify the importance of baseline abnormalities in the risk for pulmonary complications and treatment-related mortality and whether outcome is improved by using intensive screening. financial disclosure statement: there are no items to disclose. conflict of interest statement: there are no conflicts of interest to report. authorship statement: a.b.v. had full access to all data in the study and takes responsibility for integrity of data and analysis. c.k.e., j.j.b., t.w., p.d.j., and m.b. contributed equally and substantially to study design, interpretation of data, and writing of the manuscript. lung function, pulmonary complications and mortality after allogeneic blood and marrow transplantation in children lung function and late pulmonary complications among survivors of hematopoietic stem cell transplantation during childhood natural history of pulmonary complications in children after bone marrow transplantation diagnosis of invasive fungal infections in hematology and oncology-guidelines from the infectious disease working party in haematology and oncology of the german society for haematology and oncology (agiho) strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes reference values for paediatric pulmonary function testing: the utrecht dataset fleischner society: glossary of terms for thoracic imaging diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection pulmonary dysfunction in survivors of childhood hematological malignancies after allogeneic hematopoietic stem cell transplantation lung function after allogeneic hematopoietic stem cell transplantation in children: a longitudinal study in a population based cohort pulmonary function testing prior to hematopoietic stem cell transplantation influence of pretransplantation restrictive lung disease on allogeneic hematopoietic cell transplantation outcomes lung function and long-term complications after hematopoietic cell transplant pretransplant lung function is predictive of survival following pediatric bone marrow transplantation key: cord- - kiahjyy authors: nandy, ashesh title: chapter the granch techniques for analysis of dna, rna and protein sequences date: - - journal: advances in mathematical chemistry and applications doi: . /b - - - - . - sha: doc_id: cord_uid: kiahjyy abstract: the very rapid growth in molecular sequence data from the daily accretion of large gene and protein sequencing projects have led to issues regarding viewing and analyzing the massive amounts of data. graphical representation and numerical characterization of dna, rna and protein sequences have exhibited great potential to address these concerns. we review here in brief several different formulations of these representations and examples of applications to diverse problems based on what this author had presented at the second mathematical chemistry workshop of the americas in bogota, colombia in . in particular, we note several insights that were gained from such representations, and the applications to the bio-medicinal field. my first brush with a dna sequence, in around , left me totally puzzled: i could not "see" nor get a "feel" of anything noteworthy in the apparent jumble of characters that symbolized a dna, not the least because i had never studied biology myself. my background was physics, and i began a search for, to me, a more meaningful exposition of the sequence of characters that represented the dna sequence. my studies led me to appreciate and anticipate the immense potential opening up with the sequencing of genome length sequences and the concomitant need for rapidly scanning and analyzing dna sequences for matters of interest [ ] , and to get excited at the new insights being gained from a global perspective of the dna sequences: jeffreys [ ] had shown through his chaos generator representation that such sequences had an inherent fractal nature; peng et al. [ ] speculated that dna sequences had long-range correlations, an observation that raised a storm of papers in very short order; and voss [ ] showed that long range fractal correlations existed in dna sequences with the degree of correlation varying with evolutionary divergence. but a close up look at a dna sequence and how the bases were distributed along it still lacked an appealing representation. experimenting with various formats i determined that a d graphical representation, as explained later in this chapter, was what i could relate to on a purely personal basis. after many graphs of various sequences and discussions with some eminent persons to ensure that such a simple stratagem was not already familiar to cutting edge biologists, i published a paper on it in current science (bangalore) in [ ] . imagine my consternation when i was informed soon after that gates had already anticipated such a device, albeit with different axes assignments, way back in [ ] , but which seemed to have been in limbo since! a short note had to be published soon after informing of this oversight and explaining the differences although both used cartesian co-ordinate system to plot the graphs [ ] . however, a physics background demands some quantitative appraisal of whatever nature has to offer. i had observed certain similarities and changes in plots of conserved gene sequences of various species, but coming up with some way to measure the changes posed difficulties with these plots of discrete numbers. i had done some number crunching with individual gene segments like introns and exons [ , ] , but now the need was for whole sequences for which we came up with a geometrical interpretation to describe in general a macro-molecular sequence and measure sequence differences. we presented our scheme at the first indo-us workshop on mathematical chemistry in shantiniketan, west bengal, india in [ ] where we reported, as stated in the abstract, that "geometrisation of macromolecular sequences in the form of a graphical representation provides one … technique where the nucleotides in a gene sequence can be viewed as objects in a -dimensional space; the method can be extended, in principle, to include, say proteins, in a -dimensional space. we have found a reduced -dimensional representation of dna sequences very useful in studies of nucleotide distribution and composition. …. we here propose a new measure of the dispersion of dna graphs that can be used to quantify the differences between two or more graphs of genes of various organisms …. lt also appears that once standardized the proposed scheme may help study molecular phylogeny in evolutionary time scale." although the participants in the shantiniketan workshop included stalwarts in the field like prof. milan randić, prof. haruo hosoya, prof. paul mezey and others, our scheme did not seem to evoke any response, not surprising since they apparently did not know about dna issues. but prof. subhash basak of the university of minnesota, usa and co-organiser of the workshop was intrigued enough by our work and its potential to describe dna sequences through graph invariants to meet me in kolkata after the workshop to discuss the possibility of using invariants for dna sequences as descriptors. subsequently prof. basak invited me the following summer to duluth to carry out further research on dna mathematical descriptors in his group funded by the natural resources research institute (nrri). prof. milan randić and some other distinguished scientists were also invited there to begin to work on dna descriptors in a project funded by nrri. it began with a talk i gave at the university of minnesota, duluth about my work on mathematical descriptors of dnas arising from my graphical representation method. among the attendees was prof. milan randić who, with prof. basak, immediately saw the potential for converting a dna sequence graph to a matrix and thereby extract numerical invariants which could be a more meaningful way to characterize dna sequences. we collaborated then on a proposal for a d graphical representation and a matrix method for extracting graph invariants for the first exons of beta globin sequences of several species. this was published in in the journal of chemical information and computer science [ ] and led very soon to a whole host of papers on the dna graphical representations and numerical characterisations and applications of them that continues still as more and more areas keep opening up and a new field of research seems to have begun. this review is a brief introduction to the readers of this new and exciting field of research on graphical representation and numerical characterization (granch) of bio-molecular sequences, based on the talk i presented at the second mathematical chemistry workshop of the americas in bogota, colombia, in july [ ] . some of the various applications made to date using these techniques are also covered briefly, with special emphasis on our recent work that provides a possible approach to anti-viral vaccine design that could be expected to be less susceptible to invalidation through mutational changes in the viral proteins. more details can be found in the several reviews [ ] [ ] [ ] [ ] [ ] and book chapters [ ] [ ] [ ] that have appeared on the subject, and of course there are always the original papers. (note added in proof: see also bibliography in ref [ ] .) as sequence data on long stretches of dna began to become available in the late 's, there arose a problem on how to view them and a curiosity to know whether any systematics lay hidden in the apparently random arrangement of characters representing the bases in the sequence. h j jeffrey [ ] came up with the idea of plotting them in a square grid where the four corners were identified with the four bases a, c, g, t. the algorithm was to start from the center of the square and for the first base plot a point midway between the origin and the home corner of the base. for the second base he started from the point representing the first base and plotted a point midway between it and the home corner of the second base. continuing in this way filled up the square with a series of points until the entire sequence was plotted. this diagram he called the chaos generator representation (cgr) of the dna sequence. he noticed that different animal kingdoms showed different patterns -double scoop depletion regions for vertebrates, striped patterns for plant sequences, an apparently random distribution for bacterial genomes. overall, each sub-square section of the cgr pattern seemed a replica of the whole, i.e. dna sequences had properties of selfsimilarity or fractal nature. the cgr diagrams of various sequences were investigated by several researchers to find different properties of biological interest. burma et al. [ ] showed the structures observed in cgr diagrams arises from skews in base composition and presence of repetitive sequences or specific motifs. dutta and das [ ] reported that a cgr plot can be reproduced by suitable algorithms by manipulating different combinations of strings of bases with appropriate frequencies. thus, the double scoop depletion patterns seen in vertebrate cgrs arises from scarcity of cg dinucleotides, and so on. baranidharan et al. [ ] developed quantitative methods to generate similarity/dissimilarity maps of genomic sequences and showed that for certain mitochondrial genomes species wise characteristic features could be seen when nucleotide stretches of or more bases at a time were analysed. in a slightly different vein, peng et al. [ ] considered the structure of the bases in a dna sequence and analysed them on the basis of their being pyrimidines (c,t) or purines (a,g) only. on an x-y graph where the x-axis counted the nucleotide number, they plotted the appearance of the bases in a sequence by taking a step diagonally upwards if it was a purine or downwards if it was a pyrimidine to the next nucleotide number. plotting the whole sequence step by step in this manner they generated a graph with an irregular up-down structure which they called a "dna landscape". by taking subsections of the graph they found that the subsections also looked similar to the up-down structure of the whole, and the same was true of sub-sub-sections and so on showing that the purine-pyrimidine structure of a dna sequence had self-similarity, which was what jeffrey had remarked two years ago. peng et al. then searched for possible correlations by estimating frequencies of different lengths of nucleotide stretches and found that all gene sequences with the mosaic structure of introns and exons had long-range correlations whereas intronless genes did not show this feature. the implications of such an observation, on the face of it, are huge: the beginning of the dna sequence should, theoretically, be knowing what the end would be like! such an observation quite naturally led to a storm of papers on the subject until it quitened down after the observation that since dna sequences are known to elongate by duplicating long stretches of subsequences, it was possible that such sequences showed apparent long range correlations. around the same time voss [ ] conducted a rigorous analysis of dna sequences with over million bases covering organisms of all classes to search for long range correlations. using a spectral density function analysis he concluded that "(a) long range fractal correlations exist in dna sequences, (b) the degree of correlation as measured by a spectral exponent varies systematically with evolutionary category and (c) short range periodicities of period are prominent while other periods, e.g. , are also present. the fractal correlations have been seen to extend over long ranges of nucleotide positions, with the smallest for phage and bacteria and extending to over , bases for the higher classes" [ ] . to get a feel for the actual distribution of bases along a dna sequences you need a more direct graphical depiction than what the abstract representations of jeffrey [ ] or peng et al., [ ] can offer. this problem was addressed many years ago by hamori and ruskin [ ] with their proposal for a -dimensional graphical representation of a dna sequence. they proposed a hypothetical square on the xy plane with four corners (nw, ne, se, sw) identified with the four bases a, c, g, t and the nucleotide number to be counted along the z-axis. thus for a dna sequence like acggt, one would plot a point on the a-corner at z-coordinate , then draw a line to the next base, c in this case, in its corner with z now equal to , and so on. for a sequence like acgtacgtacgt this would generate a spiral around the z-axis; in case there was a preponderance of one base or the other the curve would flow along those corners. these curves the authors called h-curves. visualizing such a d image on the d plane of the paper is admittedly difficult. however, the authors suggested that drawing two such curves at slightly different angles would allow stereoscopic vision so that the dna could be seen in d. taking the bacteriophage m as an example they showed that in their representation they could easily identify regions of sharp changes in base composition through visualization that would be difficult to determine from the normal character representation. this author's search for a meaningful display of dna sequence information led him to propose a -dimensional graphical representation where the four cardinal directions are associated with the four bases [ ] . the method is to take a walk in the negative x-direction if there is an adenosine in the sequence, in the positive ydirection for a cytosine, positive x-direction for a guanine and the negative ydirection for thymine. proceeding to walk in succession in the appropriate direction in the order of the bases making up the particular dna sequence generates a path that visually depicts the arrangement of bases in the sequence. these dna plots were found to be characteristic of the types of gene sequences and that the same genes from different species showed almost the same pattern. since we know that specific genes from different species have significant homology, and in fact that is how often new genes are recognized, it is not surprising that their graphical plots will show basically the same shape. it was found later that gates [ ] had already proposed a similar scheme to depicting gene sequences, although his assignment of bases were different from nandy's scheme. a year later leong and morgenthaler [ ] independently proposed another d scheme, where the base assignments were again different from the two just mentioned. on a d cartesian co-ordinate system the assignments of the bases with the cardinal directions in the three schemes are, starting from the negative x-direction and going clockwise, gtca (gates), acgt (nandy) and ctag (leong and morgenthaler). it is interesting to note that these three axes representations exhaust all possible d schemes of this type, and these can be seen to be like d projections of the hamori-ruskin h-curves. the d plots can be scaled to accommodate from the largest to the smallest dna sequences depending on the level of detail one wishes to observe. in reference (the illustration ( )) depicts the thousand base long human beta globin sequence that contains the beta, eta, delta and the gamma globins of less than bases each, which can individually be plotted on a smaller scale. plots such as these provide a quick estimation of base composition and distribution along a dna sequence. an inspection of the human beta globin sequence graph shows that it has two sections that are mainly a-t rich with one part in between that is t-dominated. a plot of a sequence like the chicken myosin heavy chain gene is represented in illustration (loc. cit.,) shows that it also is at-rich; from the angle it makes with the axes, it is evident that the sequence is dominated by larger percentage of t's than a's, and likewise one can determine preponderance of structures like amtn from inspections of such plots. further applications of these graphs are taken up later in this chapter. the d representations, however, suffer from degeneracy in that nucleotide pairs, like ag or ct in the nandy scheme, will result in only one step instead of two. bielińska-wąż et al. [ ] have shown that this can be accounted for by a mathematical method of using a weight parameter for each visit to the same location, but a number of researchers has been to propose different ways to represent dna sequences graphically that reduces or removes this degeneracy. an extensive coverage of these methods can be found in nandy, harle and basak [ ] , but we may mention here that one of the first proposals to reduce the degeneracy was the scheme of guo, randić and basak [ ] where the unit vectors for the four bases were aligned at a small angle to the cardinal directions. yau et al. [ ] used a two-quadrant representation in d space where a,g were inclined to the x-axis in the th quadrant and t,c were inclined to the x-axis but in the first quadrant, and the nucleotide count was recorded along the x-axis; this generated a dna graph extending in the positive x-direction and had no degeneracy. he et al., [ ] proposed to characterize a dna sequence by their chemical (amino, keto), structural (purine, pyrimidine) and bond strengths (weak, strong) and plotted this set of three reduced sequences as characteristic curves that extended along the x-axis with nucleotide number thus avoiding degeneracy altogether. randić proposed several constructs, among them a horizontal line scheme [ ] where the four bases were plotted in order of the sequence along four lines parallel to the x-axis and placed unit distance apart wile the nucleotide number was again counted along the x-axis, a compact "worm curve" representation [ ] , four-color maps [ ] , "spectrum-like" curves [ ] among others which reduced or eliminated the degeneracy inherent in the classical d approach. d and higher dimensional representations have been proposed to more faithfully reproduce the features of a dna sequence or enable more accurate calculations. hamori and ruskin [ ] had originally proposed a d model where the four bases were plotted in four dimensions and the fifth was for nucleotide count, but since this was difficult to visualize he had moved to the d h-curve representation. d representations and variations were also proposed by randić, vracko, nandy and basak [ ] , and li and wang [ ] to name a few. a d method was proposed by chi and ding [ ] , a d method by liao and wang [ ] and an d method by liu and wang [ ] . the interested reader can refer to the reviews [e.g. ref ] and the literature for details of these interesting developments. thus, the study of dna sequences is facilitated in many ways by graphical representation, but making intra-and inter-sequence comparison becomes meaningful when the similarities and differences can be quantified in some manner. the difficulty is that since the graphical plots are composed of a set of discrete points, one has to apply either novel geometrical methods or use graph theory where the points are considered as nodes and the connections between the nodes as edges. we describe below first the geometrical methods and then the graph theoretic methods. two techniques were devised, one for intra-sequence comparison and another for inter-sequence comparison. for the variations within a sequence arising from the base distribution, we had observed [ ] that coding regions of mammalian gene sequences appeared as a dense cluster of points in the d graphical representations implying high degree of mixing of the four bases in almost equal proportions, whereas the non-coding regions that were a-t or g-c rich usually appeared as long filaments. we therefore devised a cluster density measurement by enclosing such regions in a square grid and dividing the number of points in the grid by the area of the square. this was complemented by an inverse displacement method and a fractal coefficient method to numerically assess the differences between these two types of regions. analysis of introns (noncoding regions of a gene) and exons (coding regions) of genes from various species by these measures showed [ ] that (a) cluster density of non-coding regions are very small and fall off exponentially rapidly, (b) the cluster density of coding regions grows to about . per unit area and falls off gradually, (c) exons of evolutionarily later genes have higher cluster densities, (d) cluster densities of intronless genes like the phage m genome or the bacteriophage lambda are very low, closely paralleling intron densities and (e) more recent genes show greater fragmentation and smaller lengths of the exons. the cluster density measure also enabled us to propose a way of predicting protein coding regions in new dna sequences [ ] and was used to analyse the human chromosome contig and predict existence of several genes [ ] . gates [ ] had proposed a manhattan distance computation to compare two or more sequences, but this method is suitable for equal length sequences, whereas gene sequences are not generally of equal lengths. to study similarities and dissimilarities of genes from various species we devised a new and different methodology, which was reported for the first time in the first indo-us workshop [ ] and published the following year [ ] , as mentioned earlier. since we have in the d graphical representation a set of discrete points comprising each gene sequence, we defined a function to describe the sequence as where s is the zeroth-order term representing the coordinates x f , y f of the end points, s is the linear term representing the first-order moments about the two axes, s the second-order term representing the variance about the mean, s the third order term representing the skewness, etc., all of which taken together became a descriptor for the sequence. for the initial presentation we computed the first order moments as weighted center of mass only and defined a graph radius, g r , the distance of the weighted center of mass from the origin, for each sequence and a g r to estimate the difference between two sequences plotted on the same scale; this scheme gave a reasonable fit to the dispersion of the beta globin genes from various species [ ] . because of the cumulative nature of the sequence plots, differences in base distributions will lead to progressively increasing differences in the plots. closely related sequences with less mutational changes between them will have smaller g r while unrelated sequences can be expected to lead to larger values of the g r . as remarked by the authors, this method could clearly be generalized to apply to the case of protein and other sequences where one may represent the sequences in a multidimensional hyperspace with a view to eventually develop phylogenetic trees. these techniques have been used by several authors (e.g., [ , ] ). bielińska-wąż et al. [ ] have computed the moments to various higher orders in a d dynamic graph with statistical moments of mass-density distributions as new descriptors. computing the moments for a set of histone genes, they showed that the larger number of descriptors improved the characterization of the object and different aspects of the dna could be compared separately while retaining the simplicity of the d graphs. nandy and nandy [ ] showed that the g r s were quite sensitive measures where base composition or base arrangement differences caused the g r to change and that two or more sequences will not have the same g r value except in some pathological cases. the graph theoretic method arose out of deliberations after the first presentation of the d graphical representations in duluth in . the method described in the paper by randić, vracko nandy and basak [ ] was to first represent a dna sequence graphically in a d cartesian grid and then convert the points to elements of a matrix by computing the ratio of euclidean distance to graph theoretic distance between all possible pairs of points taken systematically. matrix methods are well studied and have well recognized properties. the d/d matrix generated by the distance measures was analysed to yield a set of eigenvalues with the leading eigenvalue being taken as invariant of the matrix and therefore of the sequence. differences between the leading eigenvalues of various gene sequences could then be taken as indicative of their evolutionary distances, although this seminal paper limited itself to computation on the basis of the first exons of beta globin genes only. the interesting point to note is that this paper led to generation of intense interest among researchers and many different ways of representing dna sequences and computation of evolutionary distances subsequently ensued (see review nandy et al. [ ] ). authors such as randić et al. [ ] , randić [ ] , he and wang [ ] , song and tan [ ] and many others proposed different ways to graphically represent dna sequences and convert the plots to mathematical objects, and derive leading eigenvalues as invariants of the sequences. for example, he and wang [ ] reduced the dna sequences to a set of three sequences based on their structural, chemical and bonding nature and devised a vector of the three leading eigenvalues of the matrices associated with each of the reduced sequences which they proposed as being characteristic sequences of the original dna sequence. distances between two sequences then were computed by determining the distances between the end points of the two vectors. song and tan [ ] similarly devised a -component vector characterizing a sequence, others came up with other ways of computing the intersequence distances based on vectors devised out of the matrix eigenvalues. such matrix invariants from their own representations were used by liao et al. [ ] , wang et al. [ ] , liao et al. [ ] and others to draw phylogenetic trees for mitochondrial genes, sars coronavirus genomes, etc. the graph theoretic method, however, does not seem to have been applied so far to determine specific features within a sequence. developments in the graphical representation and numerical characterization of dna sequences raised the possibilities of using similar analysis of protein sequences, albeit with difficulty arising from the fact that now we have to contend with amino acids making up a protein chain whereas dna sequences were made up of only four nucleotides. although meeta rani [ ] had shown as early as the presence of statistical self-affinity, a kind of self-similarity, in protein sequences that implies a fractal nature, graphical representation methods for proteins drew attention with the paper of randić [ ] . the basic idea here was to start with the cgr method of jeffrey to plot a rna sequence drawing triangles for every triplets of bases, i.e., the genetic codes, and taking the centers of each such triangle as corresponding to the residue the triplet would code for. thus starting with the mrna, this method generates a cgr-equivalent d graphical representation for the protein sequence. randić et al. [ ] carried the method further to construct a zigzag curve for the a-chain of human insulin which allows a direct conversion of a protein sequence into a numerical sequence of (x,y) coordinates that can be used subsequently for construction of the graph-theoretic matrices and sequence invariants. the technique was refined to remove some arbitrariness that were inherent in the d scheme by converting the d graph to a d graphical representation where the triplets were assigned to the corners of a tetrahedron structure; although visual inspection of the graphical patterns had to be discarded in this scheme, the authors claimed that construction of graph invariants in this manner was more accurate and unique. randić et al. [ ] proposed a magic circle representation where the protein sequence graph starts from the centre following the sequence by moving half way towards the corresponding amino acids which are positioned equally spaced on the circumference of a unit circle. the result of the complete execution of the protein sequence within the circle produces a typical graph for a particular protein, except for large protein sequences which are often found to have lesser visual benefits. bai and wang [ ] considered the triplet codon concept and using a complex coordinate scheme constructed a purine-pyrimidine graph on the left half of the complex plane, with purines (a and g) in the first quadrant and pyrimidines (t and c) in the fourth quadrant. a protein sequence can then be drawn from the triplet codons extending along the x-axis allowing visual inspection of the trends and also from the co-ordinates generate graph-theoretic matrices and their leading eigenvalues as descriptors of the sequences. bai and wang [ ] next proposed a d graphical representation for protein sequences where the amino acids are represented as end points in a dodecahedron embedded in the d space, i.e. each amino acid is represented at one of the vertices of the dodecahedron. this allows construction of a sequence graph following the amino acids in the sequence where each point in the plot can be considered as a node of the graph, from which one can again generate matrices and sequence invariants. liao et al. [ ] used a d graphical representation method to compare coronavirus sequences where the four cardinal directions were associated with particular properties of the amino acids. they classified the amino acids of a protein sequence into four separate groups according to the chemistry of their r groups: amino acids a,v,f,p,m,i,l to the hydrophobic chemical group; amino acids d,e,k,r to charged chemical group; amino acids s,t,y,h,c,n,q,w to polar chemical group; and the g amino acid to glycine chemical group. starting with the nucleotide sequence, this enabled them to construct three d graphs (one for each reading frame) for each gene sequence and compute a distance matrix. in a similar construction, aguero-chapin et al. [ ] grouped the amino-acids into four categories: acidic, basic, polar and non-polar and assigned the four groups to the four cardinal directions of a cartesian frame to compute numeric descriptors of sequences of polygalacturonases. in recent years the field has progressed rapidly to numerically characterize protein sequences for application to different issues. gonzález-díaz and collaborators have extended these representations to the study of protein sequences [ ] and applied to mass spectral data of proteins and protein serum profiles in parasites [ ] . gonzalez-diaz has found that using different type of numerical indices derived from the protein d molecular graphics to perform qsar studies is simpler than having to work with the protein d structures [ ] . integrated qsars [ ] developed using chemodescriptors for ligands and biodescriptors of a molecular entity connect structural information of drug molecules, dna and rna sequences or rna secondary and protein tertiary structures. basak et al. [ ] using a new differential qsar approach for study of dihydrofolate reductases (dhfr) from multiple strains of plasmodium falciparum showed that dhfr from the wild strain is substantially different from four mutant strains of their study; this indicated that the protocols indicated in the paper can be used for the development of drugs to combat drug-resistant pathogens arising continuously in nature due to mutations. nandy et al. [ ] showed that their d graphical representation of protein sequences (explained later) was useful in generating phylogenetic relationships between sequences without necessity of multiple alignments and for determining conserved surface exposed stretches on viral proteins that could be useful in drug and vaccine designs [ ] . we mention in passing that randić [ ] , basak and gute [ ] had developed mathematical techniques for analysis of proteomics data drawing parallels with dna granch techniques, but we do not go into any details about this topic in this review. a detailed review of graphical representations of proteins including of proteomics has been made by randić et al. [ ] . any new technique needs to be tested through applications to real problems and these methods of graphical representation and numerical characterization of biomolecular sequences are no exception. the intense interest which these granch techniques have evoked amongst researchers have led to many and varied applications which shows the wide applicability and great potential of the methods. we cover some of these applications in brief here, with a novel application to anti-virus drug targeting in slightly more detail. as a natural application of the graphical representation of dna sequences, consider the visualization of patterns in base arrangements that are otherwise difficult to see in the normal character representation. as already mentioned, gates [ ] had noticed large scale repeats that were revealed by his d graphical plots and nandy [ ] showed that conserved genes have shapes on the d maps that are similar across species. from a detailed analysis of the graphical plots of families of conserved gene sequences that these altered with evolution such that the constituent bases appear to tend to greater homogeneity in base composition and higher complexity in base composition in the protein coding sequences [ ] . also, visual inspection of the graphical plots can enable new insights into similarities of different stretches of dna sequences. larionov et al. [ ] had thus found long range palindromes in the mouse and human chromosomes. nandy, gute and basak [ ] reported on a stretch of the h n avian flu neuraminidase gene that appeared to be well conserved among the various strains of the avian flu and reported on the possibility of using this site as drug or vaccine target so that these can be effective over many mutational changes (see below). further observations and numerical computations on over h n neuraminidase sequences showed the wide dispersion and mutations of the gene sequences and especially the possible exchange of structural parts of the genes, which was a new observation for this type of virus [ ] . based on the observations of the plots of several conserved gene sequences, nandy [ ] showed that the base arrangements of these sequences could be conceived as bound by a characteristic function of the instantaneous population of the four bases as one moves along the sequence. based on spot mutations, nandy proposed an equation connecting the instantaneous values of the purine and pyrimidine population asymmetries. it was hypothesized that this may have important consequences for genetic engineering since it implied that stability of engineered gene sequences required these constraints to be followed. an important issue in molecular biology is identification of protein coding regions in dna sequences. nandy showed from the d graphical representations that exon and intron regions of mammalian genes showed distinctly different patterns and how these could be used to discriminate between the exons and introns [ ] . this method was used by ghosh et al. [ ] to analyse a newly sequenced human chromosome iii contig dna to identify coding regions and predict, using webbased tools, possible genes in the sequence. he, li and wang [ ] used the numerical characterization of characteristic sequence representation of he and wang [ ] to suggest a protein coding gene finding algorithm specific for the yeast genome and found that the total number of protein coding genes in the yeast genome was , which matches very well with estimates from other methods of - . discrimination between protein coding and non-coding regions was also proposed on an entropy-based approach [ ] differentiating the dna sequence into three subsequences and using shannon's formula. wiesner and wiesnerova [ ] did an interesting application of granch techniques to study plant germplasm identificators. for their study of multiallelic marker loci from begonia × tuberhybridas, they used a d random walk digitization of the dna sequences by three transform classes according to the prescription of bai et al. [ ] and derived invariants from the respective matrices to compute sequence similarities and dissimilarities. principal component analysis done to compare the marker loci to the dna invariants found statistical correlations between the genetic diversity of the marker loci and the random walk invariants. based on their results, the authors concluded that "dna walk representation may function as an efficient pre-scanning procedure, which can predict allele-rich genomic loci as highly informative dna markers solely using the information from their primary sequence." one of the early observations was that these graphical and numerical techniques allowed comparison of dna and protein sequences without having to do multiple sequence alignment since here we are dealing with numbers derived from the method rather than having to compare base by base or residue by residue. almost all proposals of schemes for graphical representations have computed distances between dna sequences to determine similarities and dissimilarities without multiple sequence alignment and obtained fairly good, though not uniform, results. for example, liao et al. [ ] used a d graphical representation proposed by liao [ ] to derive a phylogenetic tree from the elements of a similarity matrix for eleven mitochondrial gene sequences without having to go through any multiple alignment procedure. they constructed a x covariance matrix of the weighted centers of masses from the co-ordinates of each base of a sequence and computed the euclidean distance between pairs of sequences to obtain their similarity/dissimilarity matrix. liao et al. [ ] also investigated the phylogeny of sars coronavirus genomes by their d graphical representations for protein sequences where they could draw three plots for each sequence by considering the three reading frames. these generate three eigenvalues for each sequence which are then used to compute a distance matrix from which they could diagrammatically show the relationships of various strains of the virus. in another exercise, bai and wang [ ] compared nine different neurocan nerve protein sequences in their d dodecahedron representation scheme. a direct comparison of these protein sequences through alignments is difficult since these protein sequences have different lengths. using -and -component vectors from their model, they compared the distances between end-points of the vectors corresponding to each of the nine genes and built phylogenetic trees. nandy et al. [ ] used their d representation of protein sequences to compute distances between sequences of the families of globin, the rat and human voltage gated sodium channel alpha subunit and their phylogenetic relationships. it is to be noted that deriving phylogenetic trees from protein sequences is usually a difficult matter when the sequences are of different lengths; but with the granch techniques where d/d and other matrices can be computed for any length sequence and only the eigenvalues compared, the sequence length differences become irrelevant. jayalakshmi et al. [ ] generalized these methods to compute alignment free sequence comparison using n-dimensional similarity space. h gonzalez-diaz and his group have used d graphical methods for extensive work in the bio-medicine field. based on pseudo-folding lattice network (ln) and star-graphs (sg) topological indices they proposed two dna promoter qsar models to predict promoter sequences in the function regulation of several mycobacterial pathogens [ ] . aguero-chapin et al. [ ] using their reduced four groups of amino acids on a d cartesian co-ordinate framework computed numerical descriptors for polygalacturonases through a markov model and were able to discriminate between these and other proteins and predict polygalacturonase activity of a new protein. comparison of rna secondary structures are important to understand their catalytic properties. bai et al. [ ] considered a d graphical representation of rna characteristic sequences taken bases at a time to compare similarities and dissimilarities in viral rnas of nine species. they computed three modular lengths and three phases for each sequence from which they constructed a component vector characteristic for each viral sequence. two sequences were considered to be similar if their vectors pointed in the same direction and difference between sequences could be quantified by computing the euclidean distance between the end points of the two vectors: the bigger the distance the less similar the sequence. the resultant difference table showed how methods such as these could be used to do cluster analysis without having to use alignment tools which are time consuming and requires several assumptions. in another instance, gonzalez-diaz et al. [ ] has computed d-rna coupling numbers by adapting the d graphical representation method for dna sequences. a novel application of granch techniques was proposed by ghosh et al. [ ] to determine targets on viral proteins for drug and vaccine design. viruses are known to mutate very fast and therefore become resistant to drugs and vaccine sin short time scales; the virulence of the avian flu led to an apprehension that it might mutate to a form that would enable human to human transmission of the disease and thus cause widespread infection and possible death as had happened in the case of the spanish flu outbreak in when millions died. new drugs and vaccines, especially ones that could be readily moved from table to dispensaries were badly needed. we had already noticed in early that certain parts of the neuraminidase gene appeared to be fairly well conserved [ ] . the neuraminidase, along with hemagglutinin, are surface proteins that enable the viral particles to enter and leave the human cells where they proliferate, and of these the neruaminidase is the preferred target of the currently available drug, tamiflu. we therefore determined to search the neuraminidase protein for surface residues that were well conserved. our procedure was to scan a small stretch of the neuraminidase protein sequences of +strains of the h n virus and then slide the window by one base and scan again to calculate the protein graph radius in our d representation system. we know that these radii are very sensitive to any changes in the sequence, so equal values of the radii in one stretch over all the strains implied that this stretch was conserved. by covering the entire sequence for all strains we could get a good profile of regions of least variability. the next step was to determine which parts of the sequences were surface exposed. there are several on-line engines available to scan a sequence and assign parameters to predict the degree of probability that certain portions were surface exposed. matching these predictions with the hard facts we had on low variability we were able to identify six regions in the neuraminidase protein that were surface exposed and largely stable to mutational changes. these included the peptide we had identified earlier as being exceptionally stable. however, in a recent report on influenza virus rna structure [ ] , it has been noted that the structures seen in the crystalline form may be one of several structural forms in vivo and confirmation will need to be experimentally determined. the results of the analysis on the h n neuraminidase protein sequence was published in [ ] . subsequently we have done a similar study on the vp protein of the rotavirus, a mainly tropical disease responsible for causing deaths to over half a million children every year. we identified four regions on the vp which appeared to be surface situated and quite stable. our findings were reported at the nd mathematical chemistry workshop at bogota, colombia in [ ] and the indian biophysics conference, delhi [ ] . while a number of applications have shown the usefulness of the granch approach to analyzing dna, rna and protein sequences, this remains as yet a nascent field where many issues need to be looked into and problems resolved for the potential to be well realized. an early indication of some of these areas was outlined some years ago [ ] , but they are worth recapitulating along with some more issues that may bear scrutiny. the intense interest in this field of graphical representation and numerical characterization of bio-molecular sequences have led to proposals for a vast array of models for depicting the sequences, some real and some virtual, more for dna sequences, less for protein and rna sequences. this has almost become an intellectual sport, with new ideas being propounded on regular basis, generally without a proper rationale for yet another method or critical comparison with earlier proposals. what appears to be lost in the process is the target: how useful are these representations to the practicing biologist? critical to this issue is the problem of determining the domains of applicability of the various representations if different, i.e., which model is best suited to address which classes of problems. as of now, the vast majority of proposals have addressed themselves to comparisons of similarity and dissimilarity, but as we have seen in the previous section, the issues that we can address and which biologists need answers for are more varied. from the applications made to date, the d graphical representations where the sequence data are easily viewed have generated the most interest. even aside for the global characteristics revealed by the investigations of jeffreys [ ] and peng et al. [ ] , the particular patterns of intron and exon segments [ ] or characteristic curves of he et al. [ ] have led to models to predict protein coding regions, determination of long-range palindromes [ ] , identification of target segments for vaccine development for viral proteins [ ] and determination of allele-rich genomic loci for plants [ ] among other applications have been based on d representation schemes. hamori had identified regions of sharp changes in base compositions from his d h-curves [ ] , but for almost all other d, d and higher dimensionality representations applications have been restricted to sequence similarities and generation of phylogenetic trees. the mathematical technique involved in generating the descriptors and characterizers for dna sequences are still at a preliminary level. while the first moments in the geometrical method for generating descriptors have generally yielded reasonable results in comparing intra-and inter-genic sequences, attempts to calculate higher moments to increase the accuracy and effectiveness of these descriptors have only lately begun [ , ] . the leading eigenvalues from the euclidean and graph theoretic distance ratios matrix have so far been used mainly to compute inter-sequence distances; given the rigorous mathematics of matrix mechanics, it may be worthwhile to try and extend the applications to other areas. for the benefit of users of these methods, it would be useful to have a comparison of the geometrical and graph theoretic models to determine at what level the two could give comparable results. in the case of d graphical representations using cartesian co-ordinates, we had seen that gene sequences take characteristic shapes [ ] . this raises the possibility that some day we could create an atlas of gene sequences where samples of each gene would be depicted and the descriptor parameters listed for easy reference and rapid visual identification. we have described quantitatively the gross features of the graphical plots in the d representations by using the first moments in a geometrical method [ , ] ; better descriptors can be determined through higher order moments [ ] to quantify the curvature, skewness and other properties. these, and the leading eigenvalues from the graph theoretic approach, could be considered as a list of parameters describing the sequence, akin to the quantum numbers that are used to describe elementary particles. such a scheme then provides a method to electronically store, retrieve and compare data between various sequences more efficiently, especially with a view to quickly scan newly sequenced dna, rna and proteins to determine the genes and functions. we have considered the moments calculated from the geometrical approach to d graphical representations as numerical "descriptors" of the dna sequence and taken tentative steps to enhance the number of descriptors of a sequence by computing higher order moments to more completely describe the sequences. in the matrix method applied to different varieties of graphical representation, leading eigenvalues arising from the matrices have been taken as "invariants" of the sequence in the strict mathematical sense. however, the concept of invariants derived from these matrix methods of numerically characterizing dna sequences may require some modification to account for the fact that dna sequences constantly change due to mutations in the bases. the vast majority of these changes do not affect the functioning of the protein or the enzyme coded by the gene due to synonymous mutations in the coding segments or in the non-coding part; e.g. in the case of intronless gene like the neuraminidase of the avian flu h n we had found [ ] out of total sequences prevalent over the period to had undergone mutations in one or more bases in the gene, but even then, all of these variants coded for a functioning flu neuraminidase protein. for a beta globin gene, the common standard example of most graphical representation schemes, a sample from one person may differ by a base or more from the next person due to mutational changes. determining an "invariant" from one sample sequence of these genes, while being mathematically precise, may not adequately express or characterize a gene sequence from a practical point of view. perhaps a biologically more relevant measure would be a sampling of several such sequences and from them to compute an average eigenvalue with a standard deviation and derive a numerical to characterize the gene. in fact, in the absence of a sensitivity analysis or a standard deviation, it would be difficult to accept that the computations through leading eigenvalues of distances between several sequences that are only a few percentage points apart could be statistically meaningful. the descriptors are no exception either. once these basic issues are attended to, the granch techniques can become a useful tool in the medicare field. since the computations of the numerical descriptors/characteristics are quite simply done, they can be incorporated into the dna sequencing schemes so that there will be automatic computations of, e.g., g r and p r values which would enable the physician to immediately ascertain the presence of any harmful genetic disorders; huntington's potential to degenerate into a disease for the patient, or some similar genetic problem areas could be easily read out as the genome is sequenced provided we know the characteristic locus and have a standard genome, for example the readout for a normal person from the family, available for comparison. the viral application already discussed in detail in the previous section could be automated and extended to other viruses and bacterial genomes to promote new generation of drugs and vaccines. the researches of gonzalez-diaz [ , , ] and basak [ , ] are already pointers to new directions. many potential application areas remain to be explored. since the numerical descriptors mentioned previously are seen to be quite sensitive to changes in base composition and distribution, the potential exists to devise schemes to index various aspects related to the bio-molecular sequences. initial attempts have been made to index toxic chemicals that have damaging effects on dna sequences [ ] , and to index snp gene sequences measured against some standard sequences [ ] . however, these need to be refined and made more useful for the confidence to be generated for their use in laboratory situations. one area that requires in depth study is how to address non-contiguous sequence segments. for example, in the case of epitopes, it is found that there could be continuous epitopes and discontinuous epitopes; in the latter case the folded protein brings residues from different parts of the amino acid sequence close together, which then become sites for the antibodies to act upon. the methods delineated so far for g r and p r or leading eigenvalue evaluation require contiguous span of the bases or residues for the numbers to be calculated. one way to circumvent this difficulty is to work on small segments of the sequence at a time as had been done in ref. [ ] . however, this is time consuming and inefficient, and more improved methods to be able to focus on regions of interest and calculate a minimum number of the parameters could offer better rewards. in summary it is apparent that graphical representation and numerical characterization of molecular sequences hold far-reaching potential of rapidly analyzing the sequences to extract numerous information. it opens up new ways to look at these sequences, and to gain new insights such as long range palindromes, fractal properties and intra-purine intra-pyrimidine relationships not seen by any other means. it allows one to compute many aspects of biological and medicinal interest and provide novel methods of tackling old problems; we have seen examples of gene identification, analysis of evolutionary trends and generation of phylogenetic trees, identification of conserved sites on viral proteins for drug and vaccine targeting, predict promoter sequences and new properties of polygalacturose proteins, among others and many possibilities remain unexplored, or barely scratched. still, from plants to viruses, from mammalian genes to mitochondrial genomes, a varied series of applications have been formulated. although many issues doubtless remain yet such as handling non-contiguous stretches of bases and residues like discontinuous epitopes, it is apparent that the granch techniques hold a lot of promise for a new direction in molecular analysis. recent investigations into characteristics of long dna sequences. ind chaos game representation of gene structure long range correlation in nucleotide sequences evolution of long-range fractal correlations and /f noise in dna base sequences a new graphical representation and analysis of dna sequence structure: i. methodology and application to globin genes simple way to look a dna graphical representation of long dna sequences graphical analysis of dna sequence structure: iii. indications of evolutionary distinctions and characteristics of introns and exons two dimensional graphical representation of dna sequences and intronl-exon discrimination in intron-rich sequences indexation schemes and similarity measures for macromolecular sequences. paper presented at the indo-us workshop on mathematical chemistry indexing scheme and similarity measures for macromolecular sequences on -d representation of dna primary sequences novel analysis of dna and protein sequences through graphical representation and numerical characterization techniques novel techniques of graphical representation and analysis of dna sequences -a review visualization and analysis of dna sequences using dna walks mathematical descriptors of dna sequences: development and applications new approaches to drug-dna interactions based on graphical representation and numerical characterization of dna sequences graphical representation and mathematical characterization of protein sequences and applications to viral proteins dna sequence 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targets with the march-inside approach generalized lattice graphs for d-visualization of biological information predicting pharmacological and toxicological activity of heterocyclic compounds using qsar and molecular modeling characterization of dihydrofolate reductases from multiple strains of plasmodium falciparum using mathematical descriptors of their inhibitors numerical characterization of protein sequences and application to voltage-gated sodium channel alpha subunit phylogeny computational analysis and determination of a highly conserved surface exposed segment in h n avian flu and h n swine flu neuraminidase on graphical and numerical characterization of proteomics maps mathematical biodescriptors of proteomics maps: background and applications graphical representation of proteins investigations on evolutionary changes in base distributions in gene sequences chromosome evolution with naked eye: palindromic context of the life origin graphical representation and numerical characterization of h n avian flu neuraminidase gene sequence computational study of dispersion and extent of mutated and duplicated sequences of the h n influenza neuraminidase over the period - empirical relationship between intra-purine and intra-pyrimidine differences in conserved gene sequences relative entropy of dna and its application d random walk representation of begonia × tuberhybrida multiallelic loci used for germplasm identification a representation of dna primary sequences by random walk a d graphical representation of dna sequence on graphical and numerical representation of protein sequences alignment-free sequence comparison using n-dimensional similarity space analysis of similarity between rna secondary structures d-rna-coupling numbers: a new computational chemistry approach to link secondary structure topology with biological function influenza virus rna structure: unique and common features characterization of conserved regions in rotaviral vp proteins: a graphical representation approach towards epitope prediction theory and computation: old problems and new challenges, g. maroulis and t. simos graphical and numerical representations of dna sequences: statistical aspects of similarity simple numerical descriptor for quantifying effect of toxic substances on dna sequences quantitative descriptor for snp related gene sequences the author confirms that this chapter contents have no conflict of interest. key: cord- -jlrievxl authors: abd el wahed, ahmed; weidmann, manfred; hufert, frank t. title: diagnostics-in-a-suitcase: development of a portable and rapid assay for the detection of the emerging avian influenza a (h n ) virus date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: jlrievxl background: in developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. objectives: the development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. study design: a suitcase laboratory “diagnostics-in-a-suitcase” ( cm × . cm × . cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (rt-rpa) assay was developed. as an example, two rt-rpa assays were established for the detection of hemagglutinin (h) and neuraminidase (n) genes of the novel avian influenza (h n ) virus. results: the sensitivities of the h and the n rt-rpa assays were and rna molecules, respectively. the assays were performed at a single temperature ( °c). the results were obtained within – min. the h n rt-rpa assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. all reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. in addition, the diagnostics-in-a-suitcase was operated by a solar-powered battery. conclusions: the developed assay protocol and mobile setup performed well. moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection. reliable rapid diagnostic tools can generally help confirm infection in suspected cases during disease outbreaks, and therefore they can help improve hygiene management and prevent the further spread of the disease. the most extensively used tools are rapid antigen detection tests (rdts) [ , ] . rdts are simple, fast ( - min) , and suitable for point-of-care screening. nevertheless, many reports stress the limited sensitivity and specificity of rdts [ ] [ ] [ ] [ ] , and rdt results are yet to be confirmed by real-time polymerase chain reaction (pcr). currently, real-time pcr is the standard of molecular diagnosis [ ] [ ] [ ] . samples are sent to regional or central laboratories for testing. alternatively, a mobile real-time pcr system can be used at an outbreak site for the detection of pathogen nucleic acid. however, mobile real-time pcr devices are large, expensive, and complex. in addition, the test run time varies between and min [ ] . for rapid wide-scale testing, robust easy-to-use molecular test systems that can be applied in the field provide a big advantage, as they allow rapid on-site nucleic acid detection at the point of need. smart mobile assays should be able to discover silent or clinical relevant cases even in rural areas with low infrastructure. recombinase polymerase amplification (rpa) [ ] represents an alternative technology to the real-time pcr. rpa is an isothermal dna amplification and detection technology. this assay is rapid ( - min) and operated via a compact portable device (i.e., a tube scanner, cm × . cm) [ ] [ ] [ ] [ ] . in this study, reverse transcription rpa (rt-rpa) assays were developed for the detection of avian influenza a (h n ) virus. moreover, "diagnostics-in-a-suitcase" (dias) was established in order to facilitate its use at the site of an outbreak. the quality control for molecular diagnostics, glasgow, scotland, uk; the landesgesundheitsamt niedersachsen, hanover, germany; and robert koch institute, berlin, germany, provided the human viruses and viral nucleic acids (table ) used in this study. the avian respiratory viral nucleic acid panel (table ) was provided by the friedrich-loeffler-institute, greifswald-insel riems, germany. two rt-rpa assays were developed. as no program or strict rules for designing rpa primers and probe were available, three forward primers (fps), three reverse primers (rps), and one exo-probes (exo-ps) were tested for each assay to select the combination producing the highest rt-rpa assay analytical sensitivity ( table ). oligonucleotides were synthesized by tib molbiol (berlin, germany). to simplify the use of h n rt-rpa assays at the site of an outbreak, dias ( fig. ) was created. dias contains all reagents and equipment necessary for performing h n rt-rpa assays. a table list of viral nucleic acids. h n rt-rpa assays detected only h and n rna but not other nucleic acids of viruses causing respiratory manifestations. h rt-rpa assay n rt-rpa assay coronavirus e, nl , and oc case of size cm × . cm × . cm (zarges gmbh, weilheim, germany) is lined with foam at its base to act as a shock absorber during transport. a pvc layer is attached to the foam, into which indents are cut and sealed to render them watertight (fig. ) . these indents house the equipment needed to perform the sample processing and amplification reactions: a tube scanner (twista, twistdx, cambridge, uk), disinfectant wipes (pursept ® -a xpress, merz consumer hygiene gmbh, luetjenburg, germany), a waste container (sarstedt, nuembrecht, germany), a vortex (lab-dancer, ika ® , staufen, germany), a sprout ® minicentrifuge (omnilab, bremen, germany), and two easily refillable pipette tip boxes (brand, wertheim, germany), in addition to a micro-tube rack, scissors, and a magnetic stand (beckman coulter, krefeld, germany). the lid of the case contains a box for gloves, an ffp mask, protection table sequence of primers and exo-probes for h n rt-rpa assays. name sequence - amplicon size (nt) h -rpa-fp and h -rpa-rp are forward and reverse rpa primers for the h rt-rpa assay; n -rpa-fp and n -rpa-rp are forward and reverse rpa primers for the n rt-rpa assay; h -rpa-p and n -rpa-p are exo-probes; bhq -dt, thymidine nucleotide carrying black hole quencher- ; thf, tetrahydrofuran spacer; fam-dt, thymidine nucleotide-carrying fluorophore; nt, nucleotides. goggles, - -and - -l automatic micropipettes (eppendorf ag, hamburg, germany), marker and boxes for storage buffers, and rpa kits. a solar panel and a power pack (yeti set, goalzero, south bluffdale, ut, usa) provide the energy needed. the workflow consisted of total viral nucleic acid extraction using magnetic beads (dynabeads ® silane viral na, life h and (b) the n rt-rpa assays. fluorescence development via real-time detection using a dilution range of - rna molecules/l of the h and n rna molecular standards (graph generated by esequant tube scanner studio software). the sensitivities were and rna molecules for the h (a) and the n (b) rt-rpa assays, respectively. the reverse transcription took place in the first minute. to increase the sensitivity, a mixing step was performed after min (no fluorescence signal was detected). represented by black line; , gray; , red; , blue; , green; , cyan; , dark khaki; and negative control, orange. (for interpretation of the references to color in this text, the reader is referred to the web version of the article.) technologies, darmstadt, germany) according to the manufacturer's instructions with few modifications. in summary, l of the sample was incubated with l of proteinase k and l of lysis buffer for min at room temperature. thereafter, l of isopropanol and l of dynabeads were added and incubated for min followed by two washing steps. nucleic acid bound to the dynabeads was left to dry for min, followed by elution. the total time needed for the extraction was min. for the rt-rpa assay, the ready-to-use twistamp tm rt exo (twistdx, cambridge, uk) was used as described [ ] . briefly, rpa primers ( nm), the probe ( nm), l of rehydration buffer containing mm mg acetate, and l of extracted rna were added. the tube was closed and mixed well, and then placed in the tube scanner for min at • c. the total time for the rt-rpa test was min. all pipetting steps were performed in the suitcase area. surfaces outside of the suitcase were not used. in vitro transcribed ha and na rnas of the h n a/anhui/ / virus (accession numbers: epi , epi ) were provided by the friedrich-loeffler-institute, greifswald-insel riems, germany [ ] . to determine the analytical sensitivity of rt-rpa assays, rna molecular standard quantities ranging from to rna molecules/l were used. the rt-rpa assay was performed using the ready-to-use twistamp tm rt exo (twistdx, cambridge, uk) as described before [ ] . using prism (graphpad software inc., san diego, ca, usa), the threshold time was plotted against molecules detected and a semilog regression was calculated. to determine the limit of detection at % probability, a probit regression was performed using statistica (statsoft, hamburg, germany). the specificity of rt-rpa assays was determined by testing six h n -negative chicken samples and a reference human genome. in addition, the cross-reactivity was tested using the viral nucleic acids listed in table . for comparison, h n real-time rt-pcr was performed as previously described [ ] . two rt-rpa assays were designed for the detection of avian influenza a (h n ) virus. two molecular in vitro transcribed rna standards (h and n ) were implemented to evaluate both rt-rpa assays. a dilution range of - molecules/l of the rna h and n molecular standards was used to select the rt-rpa primer and probe combination to produce the highest assay analytical sensitivity. rpa primers and exo-ps listed in table yielded an analytical sensitivity between and rna molecules for the h and n rt-rpa assays, respectively (fig. ) . using the data of eight rt-rpa runs of rna molecular standard serial dilutions, a semilog regression (fig. ) and probit regression analysis (fig. ) were carried out. the run times for both assays were between and min (fig. ) . the limits of detection in % of cases were calculated to be (fig. a) and (fig. b ) rna molecules detected for the h and the n rt-rpa assays, respectively. both the h and n rt-rpa assays were specific. they detected neither viral nucleic acids of other respiratory viruses (table ) nor the human or the avian genome. unfortunately, no clinical samples were available to test the clinical sensitivity of the assay. the avian influenza a (h n ) virus, which mainly infects birds, also occasionally infects humans [ ] . as of december , deaths of laboratory-confirmed human cases were recorded by the world health organization (who) in restricted parts of china semilogarithmic regression of the data collected from eight h n rt-rpa test runs on the rna molecular standard using prism software. both assays yielded results between and min. in the h rt-rpa assay, - rna molecules were detected in eight out of eight, and in six of eight rt-rpa runs. in the n assay, - rna molecules were detected in all rt-rpa runs, in seven out of eight, and in two out of eight. probit regression analysis using statistica software on the data set of the eight rt-rpa assay runs. the limit of detection at % probability ( and rna molecules for the h and n rt-rpa assays, respectively) is represented by a triangle. including hong kong. by november , the number of confirmed human cases increased to , of which were fatalities. in addition, infected cases were reported in taiwan and malaysia [ ] , suggesting the spread of the virus to other countries as well. thus, it is absolutely necessary to detect an h n outbreak as early as possible to initiate appropriate control measures and prevent further spread among birds or transmission to humans. several real-time rt-pcrs were developed for sensitive detection of avian influenza (h n ) virus [ , , ] . they produced results in - min. to avoid contamination of reagents or the workplace with pcr amplicons, the preparation of the master mix, addition of the sample rna to the mix, and the pcr reaction must be performed in separate biological safety cabinets or pipetting hoods. moreover, real-time rt-pcr reagents must be stored in a freezer at − • c. therefore, real-time rt-pcr is not easy to use at the point of need or in the field. the detection of avian influenza (h n ) virus was also performed by reverse transcription-loop-mediated isothermal amplification (rt-lamp) [ ] [ ] [ ] [ ] [ ] . rt-lamp amplification was achieved at • c in > min, and readout was done with the naked eye or sybr green-based detection. however, rt-lamp needs six to primers for the amplification of the h and n genes, which are not easy to design especially for highly variable rna viruses. by contrast, the h n rt-rpa assays developed in this study were very fast ( - min). they were operated at a single temperature ( • c), and only four primers and two exo-ps were needed for the amplification and detection of two genes (h and n ). moreover, the analytical sensitivity and specificity of the rt-rpa assays were as good as that of the published real-time-rt-pcr [ ] . to allow for the use of the rt-rpa assay at quarantine stations, ports, or the site of outbreak, the developed dias contains the complete equipment and reagents needed to perform on-site rpa-based diagnostic test. dias has the same size as a standard suitcase, which is easy to carry, transport, and ship. the dias currently costs around euro. the system can be used with a solar panel and/or power pack as a source of energy for easy use in lowresource settings. all rt-rpa reagents and oligonucleotides can be stored at ambient temperature for up to months. rna extraction is achieved by a magnetic-bead-based method, which reduces the risk of environmental contamination because no centrifugation step is needed. six samples can be tested at the same time, in addition to negative and positive controls. kits for up to samples can be stored in the dias. the dias storage box can be refilled whenever needed. results are displayed as positive or negative on the screen of the tube scanner without the need of a laptop. overall, nine pipetting steps are needed. further improvements to reduce the pipetting steps could be to include lyophilized rpa primers and probe into the rpa pellet, to employ a multiplex rt-rpa assay, and to apply simple non-purification lysis. in addition, generic influenza rt-rpa assays for detection of influenza type a and b as well as h n are being developed, but they have not yet been included [ ] . in conclusion, the developed dias represents a portable, rapid, and simple platform for the detection of avian influenza a (h n ) virus at the point of need and in low-resource settings. dias depends on the rpa technique for identifying the rna of the h n virus, which can be optimized for the detection of the genome of other infectious agents as well. the development of avian influenza a (h n ) assays was funded by the federal ministry of education and research (bmbf), germany (project name: rescheck, grant no: fkn: sv ). the funders had no role in design of the study, data collection and analysis, 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amplification assay for rapid detection of foot-and-mouth disease virus reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay recombinase polymerase amplification assay for rapid detection of rift valley fever virus nucleic acid-based detection of influenza a virus subtypes h and n with a special emphasis on the avian h n virus specific detection by real-time reverse-transcription pcr assays of a novel avian influenza a(h n ) strain associated with human spillover infections in china. euro surveillance: bulletin europeen sur les maladies transmissibles h n : preparing for the unexpected in influenza who. avian influenza a (h n ) virus detection of a novel avian influenza a (h n ) virus in humans by multiplex one-step real-time rt-pcr assay development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of subtype h n avian influenza virus rapid and sensitive detection of novel avian-origin influenza a (h n ) virus by reverse transcription loopmediated isothermal amplification combined with a lateral-flow device development of reversetranscription loop-mediated isothermal amplification assay for rapid detection of novel avian influenza a (h n ) virus development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza a (h n ) virus infection rapid and sensitive detection of h n avian influenza virus by use of reverse transcription-loop-mediated isothermal amplification development of a panel of recombinase polymerase amplification assays for detection of respiratory viruses the authors thank dr. bernd hoffmann, friedrich-loeffler-institute, greifswald-insel riems, germany, for providing the h and n in vitro transcribed rna, avian respiratory viruses, and h n negative chicken samples; the global initiative on sharing all influenza data (gisaid) for the avian influenza a (h n ) sequence; and quality control for molecular diagnostics (qcmd), glasgow, scotland, uk, landesgesundheitsamt niedersachsen, hanover, germany, and robert koch institute, berlin, germany, for human viruses and viral nucleic acid. we thank the university medical centre, goettingen, germany, for providing support and workspace until september . mr. manfred lawendel helped us fix the pvc layer to the bottom of the case. we thank doris heidenreich for technical help, and dr. christiane stahl-hennig and shereen petersen for english proofreading. the authors have declared that no competing interests exist. this is non-applicable as no materials from humans or animals were used. key: cord- -homxj uz authors: rodhain, f. title: bats and viruses: complex relationships date: - - journal: bull soc pathol exot doi: . /s - - -z sha: doc_id: cord_uid: homxj uz with more than species, bats and flying foxes (order chiroptera) constitute the most important and diverse order of mammals after rodents. many species of bats are insectivorous while others are frugivorous and few of them are hematophagous. some of these animals fly during the night, others are crepuscular or diurnal. some fly long distances during seasonal migrations. many species are colonial cave-dwelling, living in a rather small home range while others are relatively solitary. however, in spite of the importance of bats for terrestrial biotic communities and ecosystem ecology, the diversity in their biology and lifestyles remain poorly known and underappreciated. more than sixty viruses have been detected or isolated in bats; these animals are therefore involved in the natural cycles of many of them. this is the case, for instance, of rabies virus and other lyssavirus (family rhabdoviridae), nipah and hendra viruses (paramyxoviridae), ebola and marburg viruses (filoviridae), sars-cov and mers-cov (coronaviridae). for these zoonotic viruses, a number of bat species are considered as important reservoir hosts, efficient disseminators or even directly responsible of the transmission. some of these bat-borne viruses cause highly pathogenic diseases while others are of potential significance for humans and domestic or wild animals; so, bats are an important risk in human and animal public health. moreover, some groups of viruses developed through different phylogenetic mechanisms of coevolution between viruses and bats. the fact that most of these viral infections are asymptomatic in bats has been observed since a long time but the mechanisms of the viral persistence are not clearly understood. the various bioecology of the different bat populations allows exchange of virus between migrating and non-migrating conspecific species. for a better understanding of the role of bats in the circulation of these viral zoonoses, epidemiologists must pay attention to some of their biologic properties which are not fully documented, like their extreme longevity, their diet, the population size and the particular densities observed in species with crowded roosting behavior, the population structure and migrations, the hibernation permitting overwintering of viruses, their particular innate and acquired immune response, probably related at least partially to their ability to fly, allowing persistent virus infections and preventing immunopathological consequences, etc. it is also necessary to get a better knowledge of the interactions between bats and ecologic changes induced by man and to attentively follow bat populations and their viruses through surveillance networks involving human and veterinary physicians, specialists of wild fauna, ecologists, etc. in order to understand the mechanisms of disease emergence, to try to foresee and, perhaps, to prevent viral emergences beforehand. finally, a more fundamental research about immune mechanisms developed in viral infections is essential to reveal the reasons why chiroptera are so efficient reservoir hosts. clearly, a great deal of additional work is needed to document the roles of bats in the natural history of viruses. résumé avec plus de espèces répertoriées, l'ordre des chiroptères (les chauves-souris) constitue, parmi les mammifères, le second en importance après celui des rongeurs. beaucoup d'espèces sont insectivores, d'autres sont frugivores et quelques espèces sont hématophages. certains de ces animaux sont nocturnes ou crépusculaires alors que d'autres sont actifs de jour ; les uns entreprennent d'importantes migrations saisonnières, d'autres adoptent un domaine vital restreint. cependant, malgré l'importance du rôle des chauves-souris dans le fonctionnement de la biosphère, leur bio-écologie, au demeurant très variée, demeure mal connue. plus d'une soixantaine de virus a été isolée ou détectée chez des chauves-souris qui, selon différentes modalités, se trouvent ainsi impliquées dans la circulation de beaucoup d'entre eux ; c'est le cas, notamment, de rhabdoviridae du genre lyssavirus, de paramyxoviridae comme les virus nipah et hendra, de filoviridae (virus ebola et marburg) ou de coronaviridae comme les agents du syndrome respiratoire aigu sévère (sras) et du syndrome respiratoire du moyen-orient (mers). certaines espèces de chauves-souris semblent constituer, pour ces agents infectieux, des réservoirs ou d'efficaces disséminateurs, voire des agents transmetteurs. des groupes entiers de virus se sont développés chez ces mammifères par des mécanismes de coévolution entre virus et chauves-souris. le caractère généralement asymptomatique de ces infections virales chez les chiroptères est remarqué depuis longtemps mais les mécanismes de cette tolérance ne sont pas encore bien compris. certains des virus en question étant hautement pathogènes pour l'homme ou les animaux domestiques ou sauvages, les chauves-souris représentent un risque important en santé publique humaine et animale. pour mieux comprendre leur rôle dans la circulation de ces zoonoses virales, les épidémiologistes doivent se pencher sur certains de leurs traits de vie comme leur longévité, leurs régimes alimentaires, leur comportement grégaire, l'hibernation, les migrations ou le fonctionnement de leur système immunitaire. il convient également d'analyser les interactions entre les chauves-souris et les modifications écologiques induites par l'homme et d'en suivre attentivement les populations et les virus qu'elles hébergent en mettant sur pied des réseaux d'épidémio-surveillance impliquant médecins, vétérinaires, spécialistes de la faune sauvage, écologistes, etc. dans l'espoir de comprendre les mécanismes d'émergence, de tenter de les prévoir et, peut-être, de les prévenir. enfin, une recherche plus fondamentale portant sur les mécanismes immunitaires mis en jeu lors des infections virales s'avère indispensable pour saisir les raisons qui font des chiroptères des réservoirs aussi efficaces. mots clés chiroptères · chauves-souris · virus · zoonoses virales · homme · Épidémiologie · tolérance immunitaire · Épidémies émergentes abstract with more than species, bats and flying foxes (order chiroptera) constitute the most important and diverse order of mammals after rodents. many species of bats are insectivorous while others are frugivorous and few of them are hematophagous. some of these animals fly during the night, others are crepuscular or diurnal. some fly long distances during seasonal migrations. many species are colonial cave-dwelling, living in a rather small home range while others are relatively solitary. however, in spite of the importance of bats for terrestrial biotic communities and ecosystem ecology, the diversity in their biology and lifestyles remain poorly known and underappreciated. more than sixty viruses have been detected or isolated in bats; these animals are therefore involved in the natural cycles of many of them. this is the case, for instance, of rabies virus and other lyssavirus (family rhabdoviridae), nipah and hendra viruses (paramyxoviridae), ebola and marburg viruses (filoviridae), sars-cov and mers-cov (coronaviridae). for these zoonotic viruses, a number of bat species are considered as important reservoir hosts, efficient disseminators or even directly responsible of the transmission. some of these bat-borne viruses cause highly pathogenic diseases while others are of potential significance for humans and domestic or wild animals; so, bats are an important risk in human and animal public health. moreover, some groups of viruses developed through different phylogenetic mechanisms of coevolution between viruses and bats. the fact that most of these viral infections are asymptomatic in bats has been observed since a long time but the mechanisms of the viral persistence are not clearly understood. the various bioecology of the different bat populations allows exchange of virus between migrating and non-migrating conspecific species. for a better understanding of the role of bats in the circulation of these viral zoonoses, epidemiologists must pay attention to some of their biologic properties which are not fully documented, like their extreme longevity, their diet, les chauves-souris sont des animaux mal connus et généralement mal aimés (fig. ) . un peu partout dans le monde, ces mammifères nocturnes sont accusés de porter malheur. ils sont souvent considérés comme mystérieux, inquiétants, malfaisants ; ils suscitent la crainte, mais, comme nous le verrons, pas pour de bonnes raisons. ces êtres, il est vrai, apparaissent quelque peu inhabituels : des mammifères d'aspect étrange, aux doigts démesurés, qui, la nuit tombée, volètent silencieusement et apparemment sans but, qui, le jour, dorment la tête en bas, réfugiés dans des grottes ou des greniers, qui, même en europe, sont réputés boire le sang des animaux et des hommes (alors que cette habitude n'est le propre que de trois espèces américaines), etc. les chauves-souris ont donné lieu à d'innombrables légendes et superstitions. le temps n'est pas si loin où, en france, ces créatures démoniaques étaient clouées aux portes des granges. ailleurs toutefois, les mayas et les aztèques les considéraient comme des êtres protecteurs et, en chine, elles sont symboles de longévité et de bonheur. de plus, dans de nombreux pays tropicaux, les chauves-souris frugivores sont consommées avec délectation. les écologistes nous apprennent que les chauves-souris occupent une place importante dans le fonctionnement de la biosphère, notamment en détruisant d'innombrables insectes dont elles contribuent à réguler les populations, en assurant la pollinisation de nombreuses plantes à fleurs ou en disséminant des graines. mais ce qui nous intéresse ici chez les chauves-souris, et qui, pour le coup, serait une bonne raison de les redouter, est le rôle que, partout dans le monde, ces animaux jouent dans la circulation de très nombreux agents infectieux, notamment des virus. quelques données générales sur les chauves-souris ne sont pas inutiles pour comprendre leur importance épidémiologique. l'ordre des chiroptères constitué par les seuls mammifères volants est répandu sur tous les continents sauf les régions polaires et certains archipels isolés ; il regroupe un grand nombre d'espèces : avec plus de nous reviendrons plus bas sur ces caractéristiques de la biologie des chauves-souris pour en examiner l'importance épidémiologique. malheureusement, nos connaissances sur la biologie des chiroptères demeurent très superficielles, y compris pour les espèces connues pour héberger des virus pathogènes. alors que, jusque-là, la question n'intéressait guère que les spécialistes de la rage, les épidémiologistes se trouvent confrontés, depuis une vingtaine d'années, à un nombre croissant de virus souvent très pathogènes pour l'homme, dans la circulation desquels sont impliqués des chiroptères. au sein de la faune sauvage, rares sont en effet les groupes d'animaux susceptibles d'être infectés d'autant d'agents viraux ; plus d'une soixantaine de virus ont été détectés ou isolés du sang, des excrétas ou des organes des chiroptères ( , , , ) . ceci conduit les épidémiologistes à se pencher sur les raisons de cette situation, sur les risques qu'elle présente pour les hommes et les animaux domestiques et sur ce qu'il est possible de faire pour en limiter l'importance. il n'est pas question de dresser ici une liste exhaustive de tous les virus hébergés par les chauves-souris (bat-borne viruses : babovirus). nous ne ferons qu'en citer les principaux groupes à l'origine de zoonoses virales (tableaux , ). toutefois, la prévalence réelle de ces infections dans les populations de chiroptères demeure très mal connue ; les incidences enregistrées ne reflètent que l'intensité de la surveillance, elle-même très variable d'un pays à l'autre et d'une année à l'autre (environ cas en europe dans les dernières années). en france, sur une période de ans ( - ) , cas d'infection de chauves-souris ( par eblv- et par le virus bokeloh) ont été détectés ( , ) . apparemment non pathogènes pour les chauves-souris qui les hébergent, les deux virus eblv- et eblv- sont capables d'entraîner chez l'homme, un syndrome tout-à-fait comparable à la rage mais les observations en sont très rares. par deux fois, le virus eblv- a été transmis à un chat domestique en france ( ) . en australie, le virus australian bat (ablv), d'abord isolé de la chauve-souris frugivore pteropus alecto en , fut ensuite observé chez trois autres espèces de pteropodidae mais aussi chez une espèce insectivore (saccolaimus flaviventris). on en distingue deux lignées différentes. contrairement aux autres agents viraux présents chez les chauves-souris, ce virus semble capable de causer chez elles des encéphalites. sur le continent américain, où le seul lyssavirus présent est le virus rabique classique (rabv), une lignée particulière (lignée dite « indigène américaine ») circule à la fois chez les carnivores et de nombreux chiroptères insectivores ou hématophages ( ) . bien tolérée par les chauves-souris, l'infection pourrait se transmettre d'un animal à un autre par morsure, grooming, léchage ou par inhalation d'aérosol. l'existence d'espèces hématophages pose bien entendu des problèmes bien spécifiques. des trois espèces de vampires (famille des phyllostomidae), la principale est desmondus rotundus dont la densité et la répartition géographique sont en augmentation. la lignée de rabv associée aux vampires serait la plus ancienne. ici encore, les cas de contamination humaine restent très rares (une vingtaine par décennie) mais il n'en est pas de même pour les animaux domestiques qui ont avec les vampires des contacts fréquents puisqu'ils constituent pour eux une source de sang habituelle. les pertes ainsi enregistrées par mortalité du bétail domestique seraient de l'ordre de millions de us$ par an. en afrique, le virus lagos bat (sérotype ), dont on distingue quatre lignées de répartition et de pathogénicité probablement différentes, est connu pour circuler chez plusieurs espèces de chauves-souris frugivores mais il fut également isolé chez une espèce insectivore (nycteris gambiensis) ainsi que chez une mangouste (atilax paludinosus) ; il n'a pas été observé chez l'homme. d'autre part, trois cas humains, dont un mortel après morsure de chauve-souris, dûs au virus duvenhage (sérotype ) sont connus. cet agent circule chez des chauves-souris insectivores. quant au virus mokola, dont le réservoir est inconnu, il a provoqué deux cas humains au nigeria dont un mortel. nous ne savons pratiquement rien des virus shimoni bat et ikoma. en asie, la mise en évidence de lyssavirus chez les chauves-souris reste exceptionnelle mais la présence du virus rabique a été signalée en inde chez un pteropus et celle de virus du sérotype (ablv) aurait été détectée aux philippines ; d'autres cas semblables sont connus en asie du sud-est ( ) . le fait que, dans une région donnée, un même variant viral peut être retrouvé à la fois chez différents mammifères et chez des espèces différentes de chiroptères sympatriques est en faveur de l'existence d'une transmission interspécifique aussi bien de chauve-souris à chauve-souris que de chauve-souris à carnivore comme chats, renards, moufettes, etc. ( ) . cependant, si la plupart des mammifères s'avèrent réceptifs aux différents lyssavirus, chacun de ces virus semble préférentiellement associé à un hôte réservoir ; les franchissements des barrières d'espèces ne doivent pas être fréquents et ils ne permettent généralement pas la maintenance chez un hôte inhabituel. ce sont le plus souvent des culs-de-sac évolutifs. ainsi, steicker et al ( ) estiment que la fréquence des transmissions inter-espèces du virus rabique aux États-unis serait seulement de pour transmissions intra-spécifiques. le plus souvent, l'infection des chauves-souris par un lyssavirus demeure sans conséquence pathologique. toutefois, une maladie parfois mortelle peut s'observer, notamment chez les vampires, et on sait qu'une chauve-souris infectée par un lyssavirus peut parfois changer de comportement et se montrer agressive vis-à-vis d'autres espèces. les lyssaviroses humaines transmises par chiroptères entraînent une maladie comparable dans son incubation, sa présentation et sa durée d'évolution à l'encéphalite provoquée par le virus de la rage classique. cependant, même si les contacts entre les hommes et les chauves-souris sont faibles, la rareté des infections humaines par des lyssavirus n'appartenant pas au génotype n'est pas clairement expliquée car certains au moins de ces virus sont très largement répandus depuis longtemps ; sans doute leur véritable pathogénicité pour l'homme est-elle faible (l'observation de cas non mortels et de sujets en bonne santé apparente porteurs d'anticorps semble en témoigner), mais les déterminants de cette pathogénicité demeurent inconnus. il faut néanmoins noter qu'aux États-unis, des variants viraux apparemment nouveaux circulant chez deux espèces particulières de chauves-souris présentent une forte infectiosité pour l'homme, sans pathogénicité établie ( ) . la diversité des lyssavirus pose évidemment la question de l'efficacité des vaccins, tous dirigés contre le virus rabique classique ; pour ce qui concerne les virus des génotypes et notamment, éloignés antigéniquement des virus du génotype , ces vaccins ne semblent pas -ou pas assezprotecteurs. cette famille de virus comprend notamment le genre henipavirus qu'il convient d'examiner tout d'abord en raison de l'importance des deux virus zoonotiques qui le composent : les virus nipah (niv) et hendra (hev). le virus nipah est connu depuis son émergence en septembre sous la forme d'une épidémie/épizootie en malaisie ( cas humains, décès) qui a entrainé l'abattage de plus d'un million de porcs. ce virus cause chez les porcs des syndromes respiratoires (bronchopneumonie avec toux sévère, détresse respiratoire, parfois signes méningés), chez l'homme des syndromes encéphalitiques (fièvre, céphalées, convulsions, troubles de la conscience, séquelles neurologiques chez % des sujets « guéris ») ou respiratoires (pneumonies atypiques), avec une létalité élevée ( ) . la contamination à partir des porcs avait eu probablement lieu par aérosols. des personnels des abattoirs de singapour ont aussi été infectés à cette occasion. rapidement, le réservoir naturel de niv fut identifié comme étant des chauvessouris frugivores du genre pteropus (en particulier pteropus hypomelanus et p. vampyrus en malaisie) et les conditions de transmission ont été éclaircies : le facteur majeur était la proximité des porcheries avec des arbres fruitiers fréquentés par les pteropus, la transmission d'un hôte à l'autre étant assurée par aérosol, par la salive ou les urines. pour tenter d'expliquer les raisons de l'émergence à ce moment, on a suspecté le rôle qu'auraient pu jouer des déplacements de populations de chauves-souris à la suite d'une sécheresse marquée et des énormes incendies de forêt survenus peu auparavant dans certaines îles indonésiennes ; il convient également de considérer le rôle de l'accroissement de l'élevage porcin dans la région. À partir de , le virus nipah, dont il existe au moins quatre génotypes dénommés nipah-bangladesh, nipah-india, nipah-malaysia et nipah-cambodia, a été impliqué au bangladesh dans de petites épidémies saisonnières (décembreavril) localisées mais avec une létalité atteignant % ; toutefois, les porcs, peu nombreux dans ce pays, n'ont pas été impliqués et la transmission d'homme à homme était la règle. le virus nipah est également présent en inde (épidémies en et - ), au cambodge, en thaïlande (notamment chez p. lylei), à sumatra (chez p. vampyrus) et un virus, dont l'identification comme nipah n'est pas certaine à ce jour, semble à l'origine d'une petite épidémie survenue en aux philippines avec, semblet-il, des cas équins ( ) . enfin, au vu d'enquêtes sérologiques, des virus identiques ou proches de nipah circuleraient également chez des chauves-souris en chine, au vietnam, en indonésie (java, sumatra, sulawesi, sumba), au timor oriental, en papouasie-nouvelle guinée, mais aussi en afrique (ghana, guinée équatoriale, cameroun, république du congo) et à madagascar. il paraît clair aujourd'hui que la principale voie de contamination de l'homme est la consommation de jus de palmier non cuit contaminé par l'urine ou la salive des chauvessouris, notamment pteropus giganteus, mais des infections inter-humaines ont aussi été observées. la survenue de ces petites épidémies localisées est probablement liée à la densité élevée de la population humaine, à la fragmentation des massifs forestiers qui est globalement favorable aux pteropus et à la présence, dans les villages, de certains arbres (albizia spp., swietenia mahogani) constituant des dortoirs pour ces pteropus ( ) . dans certains pays comme le cambodge, ces chauves-souris fréquentent les arbres plantés dans les vergers, les jardins et notamment dans l'enceinte des pagodes. plus généralement, les émergences du virus nipah résultent certainement des multiples changements écologiques liés aux activités humaines (déforestation, intensification de l'agriculture et de l'élevage, structure des villages, démographie, etc.) et de leurs conséquences en termes de contacts entre les réservoirs naturels de virus (chiroptères pteropodidae), les animaux domestiques (porcins) et les populations humaines ( ) . quant au virus hendra, il fut découvert en à l'occasion d'une épizootie de pneumopathies survenue chez des chevaux au queensland ( chevaux atteints, décès) avec cas humains concomitants (détresse respiratoire, dont mortel). d'autres épisodes comparables eurent lieu dans les années suivantes sous forme de cas sporadiques chez des chevaux et parfois des hommes, généralement sans diffusion aux zones adjacentes. la contamination des chevaux a certainement lieu par l'intermédiaire de l'urine des chauves-souris. ce virus circule, lui aussi, chez différentes espèces de pteropus en australie (p. conspicillatus, p. alecto, p. scapulatus, p. poliocephalus). ces mégachiroptères sont actuellement en expansion au queensland et en nouvelles galles du sud et il en est de même du virus hendra. des anticorps anti-hev montrent aussi la présence de ce virus en papouasie-nouvelle guinée chez p. neohibernicus. par ailleurs, on sait que, chez les pteropus, hendra peut être expérimentalement transmis de la mère au jeune. néanmoins, les infections humaines diagnostiquées ont toutes été contractées à partir de chevaux malades, probablement par les sécrétions nasales. ces deux virus admettent donc pour réservoirs différentes espèces de chauves-souris frugivores du genre pteropus qui, une fois infectées, tolèrent bien ces virus (sauf peut-être s'ils causent des avortements) et les éliminent dans la salive et l'urine. on connait près de espèces de pteropus, un genre répandu dans le sous-continent indien, dans les iles de l'océan indien, en asie du sud-est, en australie et dans les iles du sud-ouest du pacifique ; certaines de ces espèces présentent une vaste répartition et sont partiellement sympatriques avec d'autres dont la distribution est plus restreinte. les pteropus se déplacent facilement selon les saisons, en fonction de la disponibilité des fruits de certains arbres, et par conséquent des fluctuations climatiques ; ceci peut affecter la circulation des henipavirus ( ). toutefois, une bonne compréhension des facteurs en cause nécessiterait que l'on dispose de meilleures connaissances sur l'écologie des pteropodidae ( ) . les porcs et les chevaux servent probablement d'hôtes-relais entre les chauves-souris et l'homme, ce dernier étant un cul-de-sac épidémiologique. sur le plan biogéographique, on a suggéré que la séparation des aires de distribution de ces deux virus coïncide avec la ligne de wallace ; toutefois, le virus nipah existe à timor et d'autres henipavirus encore non caractérisés semblent présents dans cette région ( ) lors d'une épidémie d'ebola, tous les individus paraissent également exposés, quels que soient l'âge, le sexe, le type d'activité, ce qui est en faveur d'une source de virus située dans le village-même. la transmission inter-humaine est alors habituelle mais le cas initial résulte généralement d'un contact avec un singe malade ou mort, ou peut-être avec une chauve-souris infectée, ou encore par ingestion de fruits contaminés par la salive de chiroptères. dans le cours d'une épidémie, en revanche, les contacts avec les fluides corporels contaminants concernent essentiellement l'entourage des malades et surtout les professionnels médicosociaux. sans que cela ait pu être formellement prouvé par des isolements de virus, on estime que des pteropodidae, chauves-souris frugivores très abondantes dans les zones forestières d'afrique centrale et occidentale, y compris dans les villages, et chez lesquelles l'infection est vraisemblablement asymptomatique, constituent le réservoir naturel des virus ebola. plusieurs espèces semblent impliquées, comme epomops franqueti, hypsignathus monstrosus, ou myonycteris torquata. beaucoup de ces chauves-souris sont trouvées porteuses d'anticorps spécifiques ; leur salive, leurs déjections et leur consommation seraient contaminantes pour les animaux sauvages (singes) ou domestiques (porcs) comme pour les humains. de plus, les grandes chauvessouris frugivores sont couramment chassées et consommées en afrique ; le contact avec les fluides biologiques (sang, urine, …) lors de la manipulation et du dépeçage de ces animaux constitue aussi une source de contamination possible (le virus est tué lors de la cuisson). il ne faudrait pas négliger, toutefois, une éventuelle implication de chauves-souris insectivores. aux philippines, le virus reston a, lui aussi, été mis en évidence chez plusieurs espèces de chiroptères. il en est de même pour le virus marburg, dont le génome a été détecté chez des roussettes de forêt, rousettus aegyptiacus, au gabon ( ) et en ouganda, avant que des anticorps spécifiques ne soient trouvés en ouganda et au congo chez le même animal (chez cette chauve-souris, les séroprévalences semblent varier selon la saison et les périodes de reproduction). il est intéressant de noter à cet égard que des cas humains sont survenus chez des personnes ayant visité des grottes hébergeant des chauves-souris dans le massif du mont elgon (kenya). actuellement, les mécanismes d'émergence de ces virus zoonotiques ne sont pas connus. on ignore pourquoi certaines régions demeurent apparemment épargnées. on suspecte que les cycles épidémiques des virus ebola (épidémies apparaissant tous les ou ans) pourraient correspondre à des épisodes de fructification de certains arbres attirant en nombre les chiroptères ; la raréfaction des ressources alimentaires en saison sèche pourrait aussi être à l'origine de contacts plus étroits entre singes et chauves-souris fréquentant alors les mêmes arbres à la recherche de fruits ; les singes pourraient encore s'infecter en mangeant des fruits contaminés par la salive des chauves-souris et laissés au sol ; ce ne sont là que des hypothèses et beaucoup d'autres facteurs écologiques, naturels et anthropiques, interviennent probablement dans ces phénomènes. outre les contacts avec les sujets infectés ou décédés lors d'une épidémie, il convient donc d'éviter également, dans les pays concernés, tout contact direct avec des primates ou des porcs malades ou morts et surtout avec les chauves-souris. jusqu'en , les virus à arn de la famille des coronaviridae étaient connus pour infecter des rongeurs, des bovins, des porcs ou des chiens et, bien sûr, des humains. mais, en - , l'émergence en chine (province du guangdong) du betacoronavirus responsable du syndrome respiratoire aigu sévère (sras) sous la forme d'une épidémie importante (plus de cas et de décès) a amené les épidémiologistes à revoir les modalités de circulation et la classification de ces virus. rapidement, plusieurs espèces de mammifères sauvages ont été impliquées dans la transmission à l'homme de ce virus, en particulier la civette palmiste masquée (paguna larvata), le chien viverrin (nyctereutes procyonoides) et un mustélidé, le « blaireau-furet » (melogale moschata) ; qu'ils soient capturés dans la nature ou élevés dans des fermes, ces animaux sont très communément proposés sur les marchés chinois dans un état de stress susceptible de compromettre le bon fonctionnement de leur système immunitaire ( ) . mais il ne s'agissait vraisemblablement que d'hôtes accidentels agissant comme amplificateurs et hôtes-relais entre les réservoirs sauvages et les humains, et ce sont finalement des chiroptères, principalement du genre rhinolophus, qui ont été reconnus comme constituant les réservoirs du virus en cause, sars-cov, ainsi que de nombreux autres virus « sars-cov-like » en chine ( , ) . chez certaines de ces chauves-souris, la prévalence des anticorps peut atteindre % sans qu'aucune maladie ne soit observée chez elles. de plus, des sérologies positives détectées chez des roussettes (rousettus leschenaulti) ont orienté la recherche vers des mégachiroptères frugivores. la transmission inter-humaine ultérieure, facile (nombreux cas nosocomiaux) est la conséquence de mutations adaptatives survenant dans le génome viral. par la suite, beaucoup d'autres coronavirus (appartenant aux genres alphacoronavirus et il parait vraisemblable que les dromadaires ne sont, pour le virus mers-cov, qu'un hôte-relais entre un réservoir naturel sauvage et l'homme. forts de l'expérience acquise lors de l'épidémie de sras, les virologistes recherchant la nature de ce réservoir se sont rapidement intéressés aux chauves-souris, d'autant plus qu'au sein des betacoronavirus, le virus mers-cov appartient au lignage c, aux côtés de deux autres virus (hku et hku ) isolés de chiroptères. cependant, jusqu'à présent, les résultats des recherches demeurent médiocres. une séquence de quelques nucléotides identique au segment homologue du mers-cov a été retrouvée chez une chauve-souris insectivore (taphozous perforatus) capturée en arabie dans la maison d'une personne contaminée ( ) . ici encore, on peut penser que la nourriture ou l'eau proposée aux animaux d'élevage peuvent se trouver contaminées par l'urine, la salive ou le guano des chauves-souris. il convient d'autre part de noter que d'autres betacoronavirus ont été isolés de chiroptères en asie, en afrique ( ) et même en europe : allemagne, pays-bas, roumanie, ukraine ( ). ainsi, en chine, un nouveau betacoronavirus du lignage c et proche du mers-cov a été identifié chez vespertilio superans ( ) ; un autre en thaïlande. plus récemment, au brésil, un alphacoronavirus a été mis en évidence chez plusieurs espèces de chiroptères urbains ( ) et, en afrique du sud, a été détecté chez la chauve-souris neoromicia capensis le génome d'un autre coronavirus qui, au vu de critères taxinomiques, pourrait représenter l'ancêtre du mers-cov ( ) . malgré l'absence de preuve permettant de considérer les chiroptères comme réservoirs naturels du virus mers-cov, cette hypothèse reste vraisemblable (on connait espèces de chiroptères dans la région considérée du moyen-orient). au total, il existe donc, partout dans le monde, une grande diversité de coronaviridae chez les chiroptères qui en constituent souvent les réservoirs et les agents disséminateurs. les quelques essais de transmission expérimentale de coronavirus à des chauves-souris frugivores maintenues en captivité n'ont pas révélé de pathogénicité chez ces animaux. néanmoins, rien n'indique aujourd'hui que les chauvessouris jouent un rôle de quelque importance dans l'épidémiologie de ces virus hormis peut-être une longue conservation lors de l'hibernation. il est bien certain que nombre d'autres virus sont encore hébergés par les chiroptères à notre insu, dont certains pourront peut-être dans l'avenir s'avérer pathogènes pour l'homme ou les animaux domestiques ou sauvages. il n'est peut-être pas sans intérêt de remarquer que les paramyxoviridae, les filoviridae, et les rhabdoviridae, souvent associés à des chauves-souris, sont des familles relativement proches, appartenant à l'ordre des mononégavirales (virus à génome arn négatif et non segmenté, chez lesquels les fréquences élevées de mutation des virus à arn peuvent faciliter le franchissement des barrières d'espèce). les paléontologistes nous disent que les plus anciens fossiles de chiroptères remontent à millions d'années (eocène) au moins, ce qui correspondrait à l'émergence des premiers ( ) . quant aux paramyxovirus, certains chercheurs ont estimé que tous proviendraient des chauves-souris, y compris des virus que l'on a l'habitude de considérer comme spécifiques de l'homme comme ceux de la rougeole et des oreillons. nous sommes donc en présence de phénomènes de co-évolution entre des familles entières de virus et les chauves-souris. plusieurs traits du comportement des chiroptères (en particulier : diversité des espèces, grande longévité, hibernation, migrations, contacts inter-spécifiques, infections virales prolongées) font de ces mammifères des hôtes privilégiés pour l'évolution des virus. ils peuvent en effet se trouver infectés par plusieurs virus différents et ces infections mixtes sont susceptibles de conduire, avec les virus à arn, à l'apparition de nouveaux mutants, recombinants ou réassortants. on sait aussi que, d'une manière générale, la diversification et l'émergence de lignées évolutives chez les virus à arn sont grandement favorisées à l'occasion du passage sur un nouvel hôte ( ) . la spécificité des virus pour les différentes espèces de chauves-souris apparait très variable : certains virus ne sont identifiés que chez une unique espèce, ce qui suggère une maintenance intra-spécifique, voire intra-populationnelle, alors que d'autres sont isolés de nombreuses espèces (le virus issyk-koul a été isolé de espèces différentes, le virus de la rage est présent chez espèces de chauvessouris insectivores au brésil). quoi qu'il en soit, les chiroptères sont donc capables d'héberger de manière persistante un grand nombre de virus, apparemment sans en souffrir alors que certains au moins de ces agents s'avèrent très pathogènes pour l'homme et les autres mammifères. ainsi, les infections à lyssavirus sont très rarement létales pour les chauves-souris, alors qu'elles le sont fréquemment chez les autres mammifères, notamment chez les carnivores. nous avons cependant un certain nombre d'observations de présence d'anticorps antirabiques chez des carnivores ou chez des humains non vaccinés qui n'ont présenté aucun symptôme ou seulement des signes très frustes comme des céphalées à répétition. quelles sont les raisons de cette tolérance ? celle-ci est-elle en rapport avec ces millions d'années de coexistence des chiroptères et des virus ? est-ce en relation avec un fonctionnement particulier du système immunitaire ? comment celui-ci peut-il contrôler le niveau de la réplication virale et éviter ainsi des réponses immuno-pathologiques sans pour autant éliminer les particules virales infectieuses ? pour les lyssavirus, on peut concevoir que leur migration vers le système nerveux central les met à l'abri de l'immunité et donc d'une pression de sélection immunologique ( ). l'immunobiologie des chauves-souris est toujours mal comprise, de sorte que nous ne sommes pas en mesure d'apporter de réponses aux nombreuses questions que pose cet état de fait. en réalité, les mécanismes du système immunitaire des chauves-souris ne paraissent pas fondamentalement différents de ceux que l'on connait chez les autres mammifères mais le fonctionnement du système parait particulier. il semble que leur immunité innée (notamment les récepteurs « toll-like » impliqués dans les processus de reconnaissance, les interférons et autres cytokines, etc.) et les caractères de leur réponse en anticorps présentent des mécanismes de contrôle de la réplication virale et de la réponse antivirale particulièrement efficaces qui, en évitant des réactions immunitaires extrêmes, conduiraient au caractère asymptomatique des infections et à la persistance des virus qui échapperaient à la réponse immune stérilisante. nous ne pouvons ici entrer dans le détail de ces hypothèses qui sont évoquées dans de récentes publications ( ) . selon certains auteurs, la réponse immune serait, chez ces animaux, étroitement liée aux variations de la température corporelle. le vol, qui augmente la température quelques heures chaque jour, pourrait accroître l'efficacité de la réponse immunitaire ( ) . l'aptitude au vol serait alors un élément clé de l'adaptation des virus aux chiroptères. À l'appui de cette thèse, une équipe de chercheurs a remarqué, en séquençant le génome entier de deux espèces de chiroptères asiatiques (l'une insectivore : myotis davidii, l'autre frugivore : pteropus alecto), des gènes particuliers, impliqués dans la détection et la réparation des dégâts provoqués sur l'adn par les dérivés réactifs de l'oxygène (radicaux libres) produits par l'élévation considérable de l'activité métabolique durant le vol ; supprimant ou atténuant les effets nocifs de ces radicaux libres, ces gènes sont liés à la capacité de vol et sans doute aussi au vieillissement, deux caractéristiques majeures des chauves-souris. autrement dit, selon cette hypothèse, le vol pourrait avoir prédisposé les chiroptères à être les réservoirs de nombreux virus en augmentant l'efficacité de leurs défenses immunitaires ( ) . il y aurait donc un lien entre le vol, la longévité et le contrôle des infections virales. il est encore trop tôt pour l'affirmer mais la piste est des plus intéressantes. par ailleurs, nous ne connaissons pas la dynamique de l'immunité d'origine maternelle chez les jeunes. le rôle exact des chauves-souris dans la maintenance, la transmission et l'évolution de ces virus est complexe et demeure très mal compris. le fait que ces animaux soient, dans de nombreux pays dont ceux de l'europe, intégralement protégés rend très difficiles les recherches expérimentales. les études de terrain sur l'écologie des chauves-souris ne sont pas simples non plus à mettre en oeuvre. il en résulte que nos connaissances quant à la bio-écologie des chiroptères sont encore très préliminaires. plusieurs traits de la bioécologie des chauves-souris sont importants en épidémiologie en favorisant l'acquisition, la maintenance et le transport de virus. c'est le cas notamment de leur vie en colonies mono-ou plurispécifiques parfois considérables (jusqu'à plusieurs millions d'individus), de leur longévité et de leurs capacités d'hibernation qui sont en faveur d'un rôle de réservoir, de leurs réponses aux modifications de l'environnement ou encore de leur mobilité qui peuvent les amener à jouer un rôle de disséminateurs d'agents infectieux. ce rôle repose principalement sur le caractère habituellement asymptomatique (ou paucisymptomatique) des infections enzootiques persistantes chez les chiroptères, sur leurs capacités d'hibernation, sur la biomasse des chauves-souris, principalement en zone inter-tropicale, et sur leur longévité. les infections abortives semblent fréquentes, pour ne pas dire la règle, avec réplication contrôlée dans les organes profonds et présence d'anticorps neutralisants dans le sang. les durées d'incubation sont parfois très prolongées chez les chauves-souris ; on a observé, avec des lyssavirus, des maladies se déclarant après neuf mois de captivité ( ). cependant, nous l'avons vu, les mécanismes permettant à ces agents infectieux de persister dans les populations de chauves-souris ne sont pas encore bien compris. la voie de contamination (par exemple l'infection par aérosol chez les espèces vivant en colonies par opposition à la transmission par morsure) ainsi que la dose de virus reçue jouent peut-être un rôle dans le déroulement de l'infection. quoi qu'il en soit, on conçoit qu'une longue durée d'infectivité, résultant d'une infection persistante associée à une grande longévité augmente fortement le rôle d'hôtes de maintenance de ces animaux. enfin, on a pu démontrer, dans certains cas, une transmission trans-placentaire du virus rabique (chez tadarida brasiliensis). l'hibernation, qui ralentit le métabolisme et crée une hypothermie, serait favorable à la maintenance d'agents viraux, notamment de lyssavirus, dans les populations de chauves-souris des pays tempérés. il parait vraisemblable en tout cas que l'hibernation entraîne des incubations très prolongées, sans doute plusieurs mois. de nombreux virus infectent d'ailleurs de manière persistante les chiroptères en hibernation qui, dès lors, peuvent en assurer la maintenance durant plusieurs mois sans présenter de maladie. À la remontée de la température, des virémies transitoires ont pu être observées avant l'apparition d'anticorps circulants, ce qui suggère la possibilité d'une remise en circulation des virus ; de telles observations ont été réalisées expérimentalement avec, entre autres, le virus de l'encéphalite japonaise. des situations plus ou moins comparables ont déjà été observées chez d'autres mammifères hibernants comme les écureuils terrestres américains. des titres élevés de virus ont aussi été obtenus à partir de la graisse brune de chauves-souris inoculées avec du virus rabique puis gardées à basse température. pour les coronavirus au moins, l'amplification virale semble avoir lieu principalement dans les nurseries lors de la parturition. il faut aussi préciser que plusieurs espèces peuvent hiberner dans les mêmes sites, favorisant ainsi les transmissions inter-espèces. même si les infections asymptomatiques sont fréquentes chez les chiroptères, il arrive que certains virus manifestent une certaine pathogénicité. le caractère grégaire de beaucoup d'espèces entraîne le regroupement en un même site d'un grand nombre de chauves-souris, parfois jusqu'à plusieurs millions, avec d'étroites interactions entre les individus ; la taille des colonies est toutefois variable avec la saison (par exemple en fonction de la disponibilité des fruits). les densités parfois très fortes observées au sein des colonies peuvent sans doute permettre l'existence permanente d'infections aiguës quelquefois susceptibles d'entraîner la mort des individus infectés. il est également possible qu'au sein d'une même espèce, des épizooties frappent tour à tour différentes sous-populations lorsqu'on a affaire à des populations à structure spatialement hétérogène (ce pourrait être le cas, par exemple, avec le virus rabique chez des vampires, ou avec le virus hendra chez les pteropus australiens). le taux de reproduction de base d'une maladie (r ), c'est-à-dire le nombre de nouvelles infections se produisant dans une population réceptive à partir d'un individu est fonction de la durée d'infectivité, du taux de contact entre individus infectants et individus réceptifs et de la probabilité de transmission lors d'un tel contact. chez les chauvessouris, une longue durée d'infectivité, résultant d'une forte longévité associée à une infection persistante, et une forte densité liée au comportement grégaire sont favorables à une circulation active des virus dans la population, voire au risque de survenue d'infections secondaires. on connait très mal la répartition géographique des différentes espèces de chiroptères. certaines, exigeant des conditions écologiques précises, paraissent localisées ; d'autres ont au contraire une très vaste distribution. ainsi, les vampires hématophages (famille des phyllostomidae) n'existent que dans certaines régions d'amérique centrale et du sud. le genre pteropus (famille des pteropodidae), quant à lui, est présent depuis certaines îles de l'océan indien jusque dans le sud du continent asiatique, en nouvelle guinée et en australie et même dans certains archipels du sud-ouest du pacifique (îles cook) et, si quelques espèces de ce genre présentent des aires géographiques restreintes (certaines sont propres à une île ou à un archipel), beaucoup d'autres présentent une très large distribution ( ) ; il en est de même sur le continent africain avec les eidolon comme e. helvum. il en résulte que certaines sont souvent sympatriques. ainsi, par le jeu des sympatries partielles entre espèces, une liaison entre le pakistan et l'australie peut être établie par l'intermédiaire de trois espèces seulement de pteropus ( ). À l'intérieur de leur domaine vital, ces animaux circulent de manière saisonnière, en fonction notamment des périodes de floraison ou de fructification des différents arbres. l'existence d'importantes migrations (parfois plus de kilomètres) chez de nombreuses espèces de chauves-souris est évidemment un facteur favorable à une dissémination des virus qu'elles hébergent. le phénomène demeure cependant très difficile à mettre en évidence ( ), même si des études portant soit sur les flux géniques, soit sur le suivi d'individus par radiotélémétrie ou, plus récemment, par télémétrie satellitaire, ont permis de préciser les déplacements de quelques espèces, par exemple entre les îles de la sonde, la nouvelle guinée et l'australie ( ) . il est également possible que, globalement, les espèces migratrices, qui sont amenées à fréquenter des écosystèmes variés et à avoir davantage de contacts inter-spécifiques, hébergent plus de virus que les espèces non migratrices ( ) . en raison du partage d'une même cavité naturelle par différentes espèces, on peut penser que des échanges de virus ont lieu entre espèces migrantes et non-migrantes, par exemple dans le cas d'infection par un lyssavirus qui, en outre, peut parfois rendre l'hôte agressif pour les individus d'autres espèces. certains épidémiologistes ont émis l'hypothèse que, chez les vampires d'amérique latine, les déplacements d'individus d'une colonie à une autre entraîneraient une asynchronie spatiale des infections rabiques qui pourrait s'avérer importante pour expliquer la persistance du virus dans une région. dès lors, l'étude de la dynamique temporo-spatiale de ces animaux s'avère indispensable pour la compréhension du phénomène. dans ce cas, malgré les taux très faibles de reproduction des vampires, les tentatives d'extermination parfois entreprises dans le cadre de la prévention de la rage seraient non seulement inefficaces mais sans doute contreproductives car favorisant les déplacements des chauvessouris cherchant à occuper des sites devenus temporairement libres ( ) . ceci est probablement vrai pour toutes les espèces de chiroptères. on a d'ailleurs observé, en ouganda, que les destructions de roussettes nichant dans les mines, effectuées dans le cadre de la prévention des infections à virus marburg, se sont soldées par une réinvasion de ces sites par des chauves-souris réceptives et des réintroductions multiples du virus. les nombreuses modifications introduites par l'homme dans les écosystèmes naturels ont mis en relation des espèces animales qui, jusque-là, n'avaient entre elles que peu de contact, voire aucun. il parait évident que les changements de l'environnement, qu'ils soient naturels (changements climatiques) ou anthropiques (déforestation, conséquences des activités agro-pastorales, plantations de palmiers à huile ou de manguiers, urbanisation…) sont de nature à rapprocher les populations de chauves-souris des habitats humains ou des élevages d'animaux domestiques. nous avons mentionné plus haut les déplacements de populations de chiroptères à la suite d'incendies de massifs forestiers en asie tropicale. les déforestations massives entreprises pour la mise en culture des terres (plantation de palmiers à huile en indonésie par exemple) ou pour l'exploitation minière, ou encore, plus simplement, pour l'installation de réfugiés chassés de leur pays par l'insécurité, la sécheresse, la famine, etc. entraînent de profonds bouleversements écologiques. l'intrusion des humains dans certains écosystèmes les met ainsi en contact avec des éléments de la faune sauvage habituellement éloignés des populations humaines. ailleurs, des contacts ponctuels peuvent survenir, comme c'est le cas avec les chauves-souris qui rencontrent des spéléologues. mais n'oublions pas que certaines espèces, se regroupant dans des clochers, des caves ou des greniers, ont développé un certain degré d'anthropophilie et vivent parfois dans les centres urbains. ces comportements et les contacts écologiques qu'ils ont pu entraîner depuis le néolithique sont évidemment favorables au franchissement de barrières d'espèces et à l'émergence de zoonoses. en théorie, la transmission directe d'agents infectieux d'un vertébré à un autre peut avoir lieu par contact, léchage, griffure ou morsure, voire par consommation. en réalité, en raison de leur biologie, ces mammifères volants et le plus souvent nocturnes que sont les chiroptères n'ont, en règle générale, qu'assez peu de contacts directs avec l'homme, hormis bien sûr le cas des biologistes qui les étudient et sont amenés à les manipuler, comme les virologistes, les chiroptérologistes (soit environ personnes en france) ou encore celui des chasseurs qui les capturent ou les tuent, les manipulent et les préparent pour les consommer. d'autres, comme les récolteurs de guano, ont des contacts réguliers avec les déjections des chauves-souris. il ne faut pas oublier d'autre part que certaines espèces se sont bien adaptées au milieu urbain. or, les fluides biologiques des chauves-souris infectées contiennent généralement des particules virales. nous avons vu que, dans le cas des henipavirus, la contamination des animaux domestiques et de l'homme avait sans doute lieu par ce moyen ; la consommation des jus de palmiers souillés par les déjections ou l'urine des pteropus constitue probablement le mode habituel de la contamination humaine. il en est sans doute de même avec les coronaviridae dont la circulation doit être assurée par l'intermédiaire des fluides oropharyngés, des déjections ou de l'urine. les filovirus seraient aussi transmis par les déjections de pteropus ou par leur salive lors de la consommation de fruits par les singes ou par les humains. on a signalé des transmissions directes de lyssavirus par aérosol de sécrétions diverses ou de poussières de guano. certains ont aussi émis l'hypothèse que la production des ultra-sons lors du processus d'écholocation pouvait s'accompagner d'émission de gouttelettes de fluides oropharyngés susceptibles de contenir des particules virales (des virus rabiques ont été isolés de mucus provenant de chauves-souris infectées). la transmission par griffure ou morsure est, elle, bien réelle, soit entre chauves-souris (on sait que l'infection par un lyssavirus peut rendre une chauve-souris agressive pour les individus d'autres espèces), soit de chauve-souris à un autre animal ou à l'homme. la manipulation de chauvessouris conduit souvent à de tels micro-traumatismes accidentels. en europe, les cas de lyssaviroses contractés à partir de chauves-souris sont rares : huit seulement ont été recensés entre et ( en ukraine, en russie, en finlande, en lettonie, en ecosse). il convient toutefois de prêter attention aux risques liés aux animaux exotiques importés le plus souvent frauduleusement : une roussette d'egypte porteuse du virus lagos bat fut introduite dans le gard en et personnes ont dû être traitées. reste le problème posé par les morsures délivrées par les espèces hématophages du continent américain, dont la salive aux propriétés anticoa-gulantes peut notamment contenir des lyssavirus. en fait, les morsures délivrées à l'homme par les espèces hématophages américaines (phyllostomidae) demeurent peu nombreuses ; les animaux domestiques, en revanche, en sont beaucoup plus fréquemment victimes. par ailleurs, les arthropodes hématophages associés aux chiroptères sont le plus souvent spécifiques et ne s'aventurent guère sur d'autres hôtes. de nombreuses espèces d'arthropodes hématophages, parfois même des familles entières de punaises, de pupipares, de tiques, de puces, sont spécifiquement associées aux chiroptères et servent probablement de vecteurs pour beaucoup d'agents infectieux, en particulier de virus, propres aux chauves-souris mais elles ne semblent guère impliquées dans la transmission à l'homme ou à d'autres animaux. c'est donc surtout par une voie indirecte que les chauvessouris peuvent être impliquées dans des infections virales humaines ou animales. un hôte-relais amplificateur est alors nécessaire pour qu'ait lieu le passage d'un virus des chauves-souris à l'homme. c'est ce qui est observé avec le porc pour le virus nipah, le cheval pour le virus hendra, les singes pour les virus ebola, le dromadaire pour le mers-cov, les civettes et d'autres mammifères pour le virus du sras, etc. ces épidémies peuvent ensuite se propager sans retour au réservoir animal. la menace constituée par l'émergence de maladies virales est permanente, en santé publique tant humaine que vétérinaire. on sait que l'origine de la plupart des virus émergents est à rechercher dans la faune sauvage. bien que cela n'ait été reconnu que récemment, les chauves-souris constituent à l'évidence des réservoirs pour de nombreux agents infectieux émergents ou potentiellement émergents. cependant, on prête relativement peu d'attention aux chauves-souris. elles représentent d'importants risques sanitaires potentiels et on ne s'en méfie pas assez. ce manque d'intérêt reflète bien sûr un manque chronique de moyens, sans doute aussi une ignorance profonde de la biologie de ces animaux mais aussi des modalités de circulation des agents infectieux et du fonctionnement des cycles épidémiologiques. actuellement, ce n'est que lorsqu'une émergence survient que l'on peut mettre la main sur le virus responsable qui, le plus souvent, nous était jusqu'alors inconnu. attendre la survenue d'un tel événement n'est évidemment pas une attitude satisfaisante. c'est la négation d'une politique de prévention. dès lors, que faire ? prévention individuelle À l'échelle individuelle, on ne peut que conseiller d'éviter tout contact direct volontaire avec des chauves-souris, qu'elles soient malades, blessées ou apparemment saines et de renoncer à en garder en captivité. il convient, bien entendu, de fermement déconseiller la chasse et la consommation des chauves-souris (tout comme pour ce qui est des singes et de la viande de brousse en général, même si nous savons bien que celle-ci représente parfois une ressource importante pour les populations forestières). on peut recommander de rendre « bat-proof » différentes constructions comme les habitations ou les porcheries, de limiter l'accès de grottes à chauves-souris aux visites touristiques, de protéger les récipients destinés à recueillir les jus de palme ( , ) . cependant, il n'est pas rare qu'une personne exposée à la suite d'un bref et discret contact avec une chauve-souris n'y ait pas prêté attention et, de ce fait, n'ait pas fait l'objet d'une prise en charge adéquate. il convient, en premier lieu, de rappeler quelques données relatives à la situation des populations de chiroptères. beaucoup d'espèces sont en forte régression en raison de l'usage des pesticides et des changements écologiques liés aux activités humaines (déforestation, développement des zones cultivées, pollution lumineuse, etc.). par ailleurs, il faut aussi prendre en considération le rôle des chauves-souris dans la biosphère : régulation des populations d'insectes (on a même cherché autrefois à les utiliser pour lutter contre les moustiques en construisant des tours destinées à attirer les chauvessouris et à en favoriser la reproduction ; ce ne fut pas un succès !), pollinisation de plantes à fleurs et dissémination de graines, propriétés fertilisantes du guano, etc. pour l'ensemble de ces raisons, ces animaux sont intégralement protégés dans de nombreux pays, notamment en france. par conséquent, les épidémiologistes doivent recourir, pour les captures et les prélèvements (prises de sang, écouvillonnages rectaux et oro-pharyngés, etc.), à des techniques ne nécessitant pas de tuer ou de traumatiser les animaux. les chauvessouris sont bien entendu exposées à un certain nombre d'agents infectieux pathogènes pour elles, en particulier le champignon kératinophile pseudogymnoascus destructans (anciennement geomyces destructans), responsable en amérique du nord d'une maladie émergente, le syndrome du nez blanc (white nose syndrome), qui a détruit , millions de chauves-souris depuis , et jusqu'à % de certaines colonies de chauves-souris. cette maladie, qui se manifeste par divers troubles métaboliques et une forte augmentation de la consommation des réserves énergétiques durant l'hibernation, aurait fait perdre , milliards de dollars par an aux agriculteurs américains par prolifération d'insectes nuisibles aux cultures, mais nous n'avons pas d'information quant aux conséquences éventuelles sur les viroses associées aux chauves-souris. ce champignon semble également présent en europe (notamment en france) mais, curieusement, sans entraîner de mortalité chez ses hôtes. pour ces différentes raisons, il n'est pas envisageable de chercher à diminuer les populations de chiroptères, et encore moins à les supprimer totalement, tâche qui serait évidemment impossible à mettre en oeuvre en pratique. dès lors, que peut-on proposer pour protéger les populations humaines et animales contre les virus hébergés par les chiroptères ? plusieurs types d'actions peuvent être envisagés, organisés selon trois axes majeurs : renforcement de la surveillance épidémiologique, renforcement de notre effort de recherche en épidémiologie, développement d'une recherche fondamentale sur le fonctionnement du système immunitaire de ces animaux. une meilleure surveillance des populations de chauvessouriset plus généralement de la faune sauvagenotamment sur le plan de l'écologie et de la génétique des populations est cruciale, parallèlement à une détection plus rapide et plus fiable des virus qu'elles hébergent. pour ce faire, un système de surveillance standardisé, impliquant vétérinaires, médecins, spécialistes de la faune sauvage et les populations elles-mêmes, avec échange des informations recueillies, est nécessaire ( ) . en france, il existe, au sein du réseau d'épidémio-surveillance de la rage assuré par l'anses, un réseau spécial de surveillance des lyssavirus des chiroptères ayant pour principal objectif de mieux cerner les risques pour l'homme (depuis , chauvessouris, dont sérotines, ont été découvertes porteuses de lyssavirus ebl ) ( , ) . cf. page : cas d'ebl de à . même si cela est difficile et parait même quelque peu illusoire, nous devons nous efforcer de recueillir partout les données sur les virus en circulation chez les chiroptères et diffuser rapidement ces informations ( ) . dans cette optique, il nous faut améliorer, généraliser et standardiser les méthodes de détection de virus zoonotiques susceptibles d'émerger. autrement dit, surveiller partout et tout le temps, plus efficacement que nous le faisons aujourd'hui. là résident nos seules chances d'isoler des agents infectieux « nouveaux » ou non, de comprendre les mécanismes de leur circulation et tenter de prévoir leur émergence afin d'intervenir à temps, c'est à dire avant l'émergence. en second lieu, de nombreuses questions tenant à l'épidémiologie et à l'évolution des virus demeurent non résolues. l'émergence de virus associés, ou paraissant préférentiellement associés aux chauves-souris, nous amène à nous poser les mêmes questions qu'à propos des autres agents infectieux : s'il s'agit d'un simple franchissement de barrière d'espèce, pourquoi le passage chauve-sourishomme ou animal n'a-t-il pas eu lieu plus tôt ? ces passages sont-ils rares, accidentels ? sont-ils liés à un contact écologique fortuit, à des bouleversements écologiques liés aux activités humaines, à un changement du climat ? existe-t-il des saisons ou des écosystèmes plus propices que d'autres ? pourquoi certaines régions semblent épargnées, par exemple par les virus ebola ? la détection des émergences est-elle simplement liée à une amélioration des activités de surveillance et de dépistage des virus, à l'action conjointe de plusieurs de ces facteurs ? certains au moins des virus propagés par les chauves-souris frugivores sont-ils, à l'origine, des virus de végétaux ? les chauves-souris insectivores sont-elles, pour certains virus, de simples hôtes-relais entre les insectes et les mammifères ? pourquoi les infections par certains virus, comme hendra, menangle et d'autres, demeurent-elles rares et sporadiques, du moins jusqu'à présent, alors que d'autres virus donnent lieu à des épidémies/épizooties massives ? comment mieux comprendre la dynamique spatiale de ces infections chez les chiroptères et délimiter les zones à risque d'émergence ? d'un point de vue très général, il conviendra certainement dans l'avenir de limiter les modifications écologiques induites par l'homme et de mieux « gérer » les interactions qu'elles créent entre les écosystèmes et les populations animales et humaines, ainsi que de tenter de prévoir les conséquences éventuelles des changements climatiques. ainsi, à propos des henipavirus ou des filovirus, on soupçonne que la déforestation et la fragmentation des massifs forestiers ou encore la présence, dans les villages et les vergers, de certains arbres-dortoirs, pourraient jouer, dans la survenue des épidémies, un rôle décisif. les déforestations pourraient aussi être à l'origine de la prolifération des vampires hématophages, et donc des cas de rage, observés au brésil mais d'autres auteurs incriminent plutôt l'accroissement du bétail domestique, les deux explications n'étant pas incompatibles. on remarquera aussi que la différentiation des coronavirus de chauves-souris date du néolithique, c'est-à-dire justement de l'époque où a commencé à se manifester la pression exercée par l'homme sur l'environnement par son intrusion dans les milieux naturels, les déboisements pour les besoins de l'agriculture et de l'élevage, par le développement des déplacements commerciaux, le tout aboutissant à des contacts jusque-là tout à fait inhabituels et à la dissémination d'agents infectieux. dans ce contexte où, de plus, interviennent des changements immunogénétiques entrainés chez l'homme par l'accroissement démographique et les croisements inter-populationnels, ces changements sont autant de facteurs de spéciation et d'évolution pour les virus à arn chez lesquels la fréquence des mutations est élevée. quoi qu'il en soit, la mise en évidence chez les chiroptères de virus très proches des agents responsables de maladies telles que la rougeole ou les oreillons pourrait, si ces résultats se trouvaient confirmés, nous amener à reconsidérer nos stratégies de prévention et même les perspectives d'éradication de ces endémies. nous sommes bien dans le concept « one health/une seule santé » prôné par diverses organisations internationales. enfin, il nous faut comprendre le fonctionnement du système immunitaire de ces animaux. nous devons arriver à saisir les raisons qui font des chiroptères des réservoirs aussi efficaces. des pistes de recherche ont été proposées pour ce faire ( ) . les virus doivent échapper à la réponse immunitaire de leur hôte réservoir pour que soit possible une transmission à un autre hôte ou pour que s'établisse une infection persistante sans phénomène immunopathologique grave. sur ce point, la fréquence des infections persistantes à virus à arn pourrait traduire un fonctionnement particulier des mécanismes immunitaires (immunité innée ?) qui, chez les autres mammifères, permettent l'élimination de ces virus ? des modèles d'infection doivent être trouvés pour ces agents de zoonoses, en particulier les mononégavirales qui semblent nouer de manière privilégiée des associations avec les chauves-souris, mais, en l'absence d'élevages en continu de chauves-souris (en raison notamment de leurs exigences écologiques et nutritionnelles, de leur faible taux de reproduction et de leur longévité), des cultures cellulaires et des observations physiopathologiques sur des colonies de chiroptères en captivité devraient être développées en laboratoire de sécurité, logistique évidemment beaucoup plus difficile à mettre en oeuvre qu'avec des rongeurs. au-delà de la compréhension de l'immunobiologie propre aux chiroptères et du rôle joué par ces mammifères dans la biologie de tous ces virus, les biologistes mettent beaucoup d'espoir dans cette recherche qui, à terme, pourrait déboucher sur des immuno-thérapies ou des vaccins intéressants. liens d'intérêts : l'auteur déclare ne pas avoir de liens d'intérêts. rapport sur la rage des chiroptères en france métropolitaine. afssa human betacoronavirus c emc/ -related viruses in bats, ghana and europe host switching in lyssavirus history from the chiroptera to the carnivora orders bats and lyssavirus resolving the roles of immunity, pathogenesis and immigration for rabies persistence in vampire bats bats without borders: long-distance movements and implications for disease risk management the distribution of henipaviruses in southeast asia and australasia: is wallace's line a barrier to nipah virus? les migrations de la pipistrelle de nathusius, pipistrellus nathusii, en france. ses incidences possibles sur la propagation de la rage bats: important reservoir hosts of emerging viruses detection and phylogenetic analysis of group coronaviruses in south american bats interspecies transmission and emergence of novel viruses: lessons from bats and birds outbreak of henipavirus infection fatal encephalitis due to nipah virus among pig-farmers in malaysia rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat interdisciplinary approaches to understanding disease emergence: the past, present, and future drivers of nipah virus emergence bats host major mammalian paramyxoviruses the natural history of hendra and nipah viruses investigating the role of bats in emerging zoonoses: balancing ecology, conservation and public health interests the role of landscape composition and configuration on pteropus giganteus roosting ecology and nipah virus spillover risk in bangladesh new alphacoronavirus in mystacina tuberculata bats henipaviruses: gaps in the knowledge of emergence use of infrared camera to understand bats' access to date palm sap: implications for preventing nipah virus transmission severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus in bats, saudi arabia emerging epidemiology of bat-associated cryptic cases of rabies in humans in the united states la vengeance de la civette masquée coronavirus antibodies in african bat species molecular phylogenetics of the lyssaviruses -insights from a coalescent approach date palm sap collection: exploring opportunities to prevent nipah transmission bat flight and zoonotic viruses distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus e in bats bat rabies in france: a -year retrospective epidemiological study genetic divergence of rabies viruses from bat species of colorado bat coronavirus in brazil related to appalachian ridge and porcine epidemic diarrhea viruses host phylogeny constrains cross-species emergence and establishment of rabies virus in bats detection of novel sars-like and other coronaviruses in bats from kenya marburg virus infection detected in a common african bat correlates of viral richness in bats public health awareness of emerging zoonotic viruses of bats: a european perspective bat coronaviruses and experimental infection of bats, the philippines bats as a continuing source of emerging infections in humans mers-related betacoronavirus in vespertilio superans bats comparative analysis of bat genomes provides insight into the evolution of flight and immunity key: cord- -ssnvr nj authors: berry, michael; gamieldien, junaid; fielding, burtram c. title: identification of new respiratory viruses in the new millennium date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ssnvr nj the rapid advancement of molecular tools in the past years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. the inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. in , this led to the discovery of human metapneumovirus (hmpv) and soon following that the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (hcov) nl and hcov-hku . human bocavirus, with its four separate lineages, discovered in , has been linked to acute respiratory tract infections and gastrointestinal complications. middle east respiratory syndrome coronavirus (mers-cov) represents the most recent outbreak of a completely novel respiratory virus, which occurred in saudi arabia in and presents a significant threat to human health. this review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since . viral infections of the upper and lower respiratory tract are among the most common illness in humans. children and infants bear the major burden of infection, typically presenting with to episodes annually [ ] . these infections are often associated with significant patient morbidity and related mortality. for this reason, urtis and lrtis represents the leading cause of death in children younger than five years of age worldwide [ , ] ; this accounts for approximately million deaths annually [ ] . acute respiratory tract disease is the leading cause of hospitalization in children and febrile episodes in infants younger than three months of age [ , ] . bacteria only represent approximately % of all upper respiratory tract infections with the subsequent % of infections caused by respiratory viruses [ ] . despite the viral aetiological origin of most respiratory infections, antibiotics are often prescribed in the treatment of such diseases [ ] , exacerbating antibiotic abuse. the morbidity and fiscal implications associated with respiratory infections are significant, with approximately million cases reported in the united states alone each year with subsequent direct and indirect costs to the us economy estimated at $ billion annually [ ] . the burden of respiratory tract infections is increased in patients with chronic comorbidities or clinical risk factors including asthma [ ] , chronic obstructive pulmonary disease (copd) [ ] , young, elderly [ ] and immunocompromised [ , ] . the viruses primarily associated with upper respiratory tract infections commonly include rhinoviruses, enteroviruses, adenoviruses, parainfluenza viruses (piv), influenza viruses, respiratory syncytial viruses (rsv) and coronaviruses [ , , , ] . in recent years six new human respiratory viruses have been reported including human metapneumovirus (hmpv) [ ] , bocavirus and four new human coronaviruses including severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus nl (hcov-nl ), hcov-hku and middle east respiratory syndrome coronavirus (mers-cov). this review will detail these newly discovered and emerging respiratory viruses. coronaviruses affect a diverse group of animal hosts, and cause a plethora of diseases in animals including progressive peritonitis, acute and chronic hepatitis, gastroenteritis, nephritis, and encephalitis [ ] . in humans coronavirus infection results in respiratory tract complications with varying degree of severity and have been associated with gastroenteritis. four human coronaviruses (hcov- e, hcov-oc , hcov-nl and hcov-hku ) are endemic in the human population and are mainly associated with mild, self-limiting respiratory illnesses. another two human coronaviruses, namely sars-cov and mers-cov cause severe respiratory syndromes and present a significant threat with their high fatality rates. the first human coronaviruses were identified in the s by tyrell and bynoe who passaged a virus, named b , in human embryonic tracheal organ cultures. when this virus was inoculated intranasally into human volunteers, common cold-like symptoms were produced [ , ] . hamre and procknow ( ) later isolated a virus, which they were able to grow in tissue culture, from subjects presenting with symptoms of the common cold. the virus was later named human coronavirus e (hcov- e) [ ] . seroepidemiological studies have shown that % of wild animal traders and % of people responsible for the slaughtering of animals in the region where human sars was thought to originate, were seropositive for sars-cov, although all cases were asymptomatic. this indicates that these people were previously exposed to a sars-like-cov, which resulted in asymptomatic infection [ ] . sars presents as an atypical pneumonia [ , ] , with pneumocytes being the primary target of infection. infection results in haemorrhagic inflammation in most pulmonary alveoli with alveolar thickening, diffuse alveolar damage, desquamation of pneumocytes, formation of hyaline membranes and multinucleated pneumocytes with capillary engorgement and microthrombosis [ ] . approximately % of patients deteriorated in the second week of infection, presenting with persistent fever, dyspnoea and oxygen desaturation [ ] . approximately %- % of patients were subsequently admitted to intensive care, where mechanical ventilation was necessary [ ] . a surprising finding with the sars outbreak was that it was not as great a threat to infants and children [ , ] . clinical presentation was less severe in infants and no children aged between and required intensive care or mechanical ventilation [ , ] . this is in sharp contrast to the age related burden of other respiratory infections and the underlying biological mechanism remains unclear [ ] . a family of viruses that were previously understood to cause mild, self-limiting upper respiratory tract infections was showcased by the sars-cov outbreak to be a significant threat to global public health. the economic losses brought on by the sars pandemic was estimated to be in the region billion dollars [ ] with hong kong bearing a significant proportion of the losses. tourism, entertainment and restaurant industries in the area recorded up to % loss in business. the pressure on healthcare facilities in affected areas was substantially exacerbated by the spread to healthcare workers. several hospitals were forced to close to new admissions as large numbers of staff became infected with sars-cov. to view this as an isolated incidence would be naï ve and the potential for the emergence and re-emergence of new and existing infectious agents poses a probable risk. understanding the sars-cov outbreak has provided immense knowledge and an excellent model to replicate in the event of further outbreaks of communicable diseases. in a month old child presenting with bronchiolitis and conjunctivitis was screened for several respiratory viruses to identify the causative agent, with all diagnostics yielding negative results. the group led by lia van der hoek then used a modified cdna amplified restriction fragment-length polymorphism (cdna-aflp) technique (virus-discovery-cdna-aflp or vidisca), to identify the causative agent. briefly, the technique utilizes reverse transcription-pcr of viral rna with subsequent restriction digest of the cdna using frequently cutting restriction enzymes. since the restriction sites are selected and therefore known, the resultant "sticky ends" can be ligated into anchors for amplification and sequencing with specific primers. the results showed highest sequence similarities with known coronaviruses, but with significant sequence divergence indicating the discovery of a new coronavirus species, later named human coronavirus nl [ ] . at about the same time, two other independent groups identified essentially the same virus [ , ] . shortly after the van der hoek paper [ ] , a novel coronavirus that replicated efficiently in tertiary monkey kidney and vero cells, was retrospectively isolated from a nose swab sample collected in from an month-old boy presenting with pneumonia. this virus was reported to be similar to hcov-nl and named hcov-nl [ ] . in , also reported the identification of a novel coronavirus isolated in new haven, connecticut, which was named hcov-nh. this novel virus was identified by pcr which was adapted to amplify a conserved region within the replicase a or pol gene [ ] . subsequent sequence analysis showed that these three viruses were essentially the same virus or variants thereof [ , ] . it is obvious that hcov-nl has been circulating in the human population since before [ ] . in fact, molecular clock analyses have shown that hcov-nl and hcov- e diverged from a most recent common ancestor, in a zoonotic event, approximately to years ago [ ] . hcov-nl later diverged into two lineages with subsequent recombination of the two lineages during co-infection. this frequent recombination has given hcov-nl a mosaic structured genome with multiple recombination sites [ ] . a year old male patient from china was admitted to hospital with pneumonia in january . viral cultures, rt-pcr and direct antigen detection from nasopharyngeal aspirate were all negative for respiratory viruses. a pan-coronavirus rt-pcr targeting a conserved region of the pol gene confirmed the presence of a coronavirus however attempts to culture the causative agent were all unsuccessful. sequencing the gene segment amplified by the pan-coronavirus assay indicated a high homology to other viruses of the βcov genus including hcov-oc but of novel origin. the human coronavirus, termed hcov-hku , was later isolated from another female patient. efforts to culture the virus had posed complicated and the complete genome was isolated, amplified and sequenced directly from rna extracted from a nasopharyngeal aspirate [ ] . successful propagation of the virus was achieved recently in human ciliated airway epithelial cells [ ] but culturing of hcov-hku still remains a daunting task. the presence of the he gene further characterizes hcov-hku as belonging to the βcov genus. since the discovery of hcov-hku , its prevalence has shown a global distribution and retrospective analysis on stored nasopharyngeal aspirates have confirmed its existence since [ ] ; however, phylogenetic analysis suggests a much earlier divergence. june saw the most recent emergence of a completely novel strain of human coronavirus. a sputum sample was collected from a year old male patient presenting with severe respiratory disease in the dr. soliman fakeeh hospital in jeddah, saudi arabia. viral assays frequently used could not identify any aetiological agent responsible for the disease. the sputum sample was sent to dr. ron fouchier at the erasmus medical college in rotterdam, netherlands, where the virus was identified as a novel coronavirus, provisionally termed hcov-emc (human coronavirus erasmus medical college). the patient later succumbed to the disease with acute pneumonia and subsequent renal failure [ ] . a retrospective study further traced the virus back to april where an outbreak of pneumonia, resulting in two fatalities, occurred in health care workers in an intensive care unit in zarqa, jordan [ ] . since the initial discovery, several new isolates were identified and described in scientific literature, databases and media under various names. this provoked the convening of the coronavirus study group to introduce a naming convention and avoid confusion within the research field, health care authorities, governments and general public. the name middle east respiratory syndrome coronavirus (mers-cov) was coined and has been widely accepted by the discoverers of the virus and pioneers of the field, the who and saudi ministry of health [ ] . primary infections, to which all cases were linked directly or indirectly, occurred in middle eastern countries including saudi arabia, qatar, jordan, oman and united arab emirates and subsequently spread to united kingdom, tunisia, france, italy and germany with egypt and the united states recently reporting their first laboratory confirmed cases [ , ] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [ ] and only reported in close, sustained contact [ ] such as within families [ ] , healthcare workers [ ] and as nosocomial transmission [ ] , especially if the patient represents with comorbidities. there has been no evidence to suggest sustained community transmission [ ] . the ever increasing case numbers and countries affected by the disease however suggests mers-cov does represent potential for widespread, global outbreak [ ] . from the first reported case in june , until february , a total of cases had been reported with fatalities. according to the latest figures available from the who, as of june , the virus had resulted in a total of laboratory confirmed cases with a total of fatalities [ ] . these statistics indicate that within a month period case numbers had more than trebled suggesting the virus is far from controlled. the fatality rate of % is also substantially increased in patients with comorbidities, with a large number of reported cases being identified in immunocompromised patients or those with underlying disease [ , ] and a severe threat of nosocomial transmission has been demonstrated [ ] . clinical presentation is similar to sars and includes a spectrum of respiratory diseases with the most common symptoms including cough, fever and gastrointestinal symptoms [ ] before progressing to pneumonia [ ] . acute respiratory distress syndrome (ards), renal failure, pericarditis and disseminated intravascular coagulation have also been reported [ ] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [ ] and only reported in close, sustained contact [ ] such as within families [ ] , healthcare workers [ ] and as nosocomial transmission [ ] , especially if the patient presents with comorbidities. there has been no evidence to suggest sustained community transmission [ ] . the ever increasing case numbers and countries affected by the disease however suggests that mers-cov does represent potential for widespread, global outbreak [ ] . the inefficient spread between infected patients strongly suggests zoonotic transmission, however over two years on from the initial discovery of mers-cov it is still unclear where it originated from and how it infects humans. during the outbreak of sars-cov the identification of the wet markets of china as the probable source greatly contributed to the control of the disease [ ] . this highlights the importance in identifying the origins of disease outbreak. phylogenetic analysis places it in the same genus as sars-cov but within a new lineage, c. the closest detectable relatives suggests origins from bat coronavirus species with relative high sequence homology between bat-cov hku and hku in china [ ] , vm in the netherlands [ ] and a recently discovered isolate from south africa [ ] . assuming that bats also form the immediate source is improbable and as with sars-cov an intermediate animal host species for transmission to humans is of greater likelihood [ ] . high levels of neutralizing antibodies, viral rna and infectious viruses have been discovered in dromedary camels suggesting a potential for camels to form the source for human transmission [ ] [ ] [ ] [ ] . a recent study identified identical single nucleotide polymorphisms in a sequence of mers-cov isolated from an infected patient and camel, which was cared for by the patient. phylogenetic analysis of these two sequences supported the conclusion that transmission occurred between patient and camel, however the direction of transmission could not be confirmed [ ] . it has since been proven that a patient who succumbed to mers-cov infection obtained the virus directly from his camel herd. the direction of transmission was confirmed as camel-to-human by serological analysis [ ] . these facts strongly contribute to an increasingly popular hypothesis that mers-cov infections in humans are directly acquired from camels, however very few patients have had reported contact with camels. many residents of the arabian peninsula frequently consume unpasteurized camel milk and this has been suggested as a potential source as the virus has been detected and can remain viable for prolonged periods in milk [ , ] . the who, saudi arabia and qatar have recently issued warnings and recommended against consuming unpasteurized camel milk and to be cautious when interacting with dromedary camels [ ] . the outbreak of sars-cov in and mers-cov less than years later highlights the significant threat of coronaviruses to humans and confirms that the sars outbreak was not an isolated incident. with the ever increasing diversity of animal coronavirus species, especially within bats, the likelihood of recombination leading to future outbreaks is high and the threat of potential pandemics is real as highly pathogenic coronaviruses continue to spill over from zoonotic sources into the human population. misdiagnosis of these outbreaks pose a further substantial threat to healthcare workers with nosocomial spread to other patients putting further pressure on an already strained healthcare system. understanding the dynamics and molecular characteristics of human coronaviruses currently in circulation and how they emerge, infect and cause disease, we can be better prepared for future pandemics allowing for improved response, management and treatment of related conditions. the clinical presentation of human coronaviruses clearly follows two distinct lines of progression. sars-cov and mers-cov present with severe respiratory complications and often multisystem involvement with renal failure and enteric symptoms. the high fatality rates associated with these infections is not reflected in the remaining four human coronaviruses, hcov- e, hcov-oc , hcov-nl and hcov-hku . the clinical importance of sars-cov and mers-cov are evident and discussed in detail in previous sections allowing for the presentation of the involvement of the remaining non-severe human coronaviruses and there implications in human health. the clinical presentation of these four non-severe human coronaviruses is largely identical and indistinguishable symptomatically, commonly presenting with rhinorrhoea, sore throat, cough and fever [ , ] . majority of infections are associated with self-limiting upper respiratory tract disease or "the common cold" but can also present with high morbidity outcomes of the lower respiratory tract including bronchiolitis, pneumonia, [ ] [ ] [ ] , asthmatic exacerbations [ ] acute exacerbations of chronic obstructive pulmonary disease (copd) [ ] and croup in hcov-nl infected patients [ ] . a report by esper et al. found a correlation between hcov-nl infections and kawasaki disease [ ] , although other studies could not replicate the association [ ] [ ] [ ] . febrile seizures have been reported for most human coronaviruses but the significance of hcov-hku is alarming with one study indicating that % of patients infected with hcov-hku experience febrile seizures [ ] . human coronaviruses affect all age groups [ , ] but elicit more serious disease in young, elderly and immunecompromized [ , , ] , frequently resulting in hospitalization. reports on the prevalence of human coronaviruses and their association with upper and lower respiratory tract disease vary but range between . % and % [ , , , ] . over % of the general public has seroconverted towards all four non-severe human coronaviruses with primary infection shown to occur in childhood [ ] and reinfection occurring throughout life [ ] . given the high prevalence of respiratory infections, human coronaviruses represent a substantial disease burden which is exacerbated by the high implications of healthcare workers in coronavirus outbreaks [ ] . high rates of co-infection with other respiratory viruses are commonly reported [ , , , ] . viruses frequently associated with co-infection include enterovirus, rhinovirus and piv [ ] however reports of co-infection with two human coronaviruses are limited. dijkman et al. recently demonstrated that hcov-oc and hcov-nl may elicit immunity that protects against hcov-hku and hcov- e, respectively [ ] . clinical progression and outcomes of disease in patients presenting with co-infection are however similar to patients presenting with mono-infection [ , ] . there is also no substantial difference in coronaviral load between co-infected and mono-infected patients. no substantial difference in disease progression in co-infected versus mono-infected patients has been reported and therefore understood to have little impact; however, the role in facilitation of infection of one respiratory virus by another is still speculative [ ] . all four human coronaviruses are endemic worldwide but the prevalence, regional distribution and pathogenicity of individual human coronaviruses is unclear and highly subjective on study conducted with parameters including population sampled, respiratory sample collected, sensitivity of diagnostic assay used, region where study was conducted and duration of the study playing a substantial role in epidemiological findings. a common finding in majority of studies conducted is the increased prevalence of human coronaviruses during winter months in temperate climates [ , ] . however even this has shown to be geographically dependent with a spring/summer predominance in subtropical and tropical climates [ , ] . biennial outbreaks are frequently reported [ , , , ] for all strains of non-severe human coronaviruses. in a previously undiscovered virus was identified in epidemiologically distinct patients in the netherlands. patient symptoms were similar to those infected with rsv and, several patients required hospitalization and mechanical ventilation. viral isolates were cultured in tertiary monkey kidney (tmk) cells and cytopathic effects caused by the virus were largely identical to those caused by rsv. electron microscopy of infected cell supernatants revealed paramyxovirus-like particles; however, rt-pcr assays to detect known paramyxoviruses were all negative. the low stringency of the assays used indicated a currently unknown, genetically distinct virus. a rap-pcr assay was then utilized to obtain sequence information of the unknown virus and fragments amplified by the rap-pcr allowed for further sequencing of the '-end of the genome. based on the sequence homology and gene organization, the unidentified virus displayed closest homology with avian pneumovirus, but to be a tentative new member of the metapneumovirus genus and the first virus in the genus to infect humans, provisionally termed human metapneumovirus (hmpv) [ ] . symptomatic differentiation between hmpv and other respiratory viruses cannot be made as there is a significant overlap in clinical presentation [ , ] . the most common presentation of hmpv in children includes complications of the upper respiratory tract with rhinorrhoea, cough and fever [ ] . acute otitis media is also frequently reported [ , ] and conjunctivitis, rash, diarrhea and vomiting are reported but infrequently [ ] . bronchiolitis, pneumonia, croup and asthmatic exacerbations are the most frequently associated lower respiratory tract complications [ ] and viral load is directly associated with disease severity [ ] . hmpv infection in the young and elderly frequently requires hospitalization and fatalities have been reported in the elderly [ , ] . an increased morbidity in elderly patients with a delayed clearance of symptoms has been reported and is likely related to the age related impairment of the innate and adaptive immunity [ ] or an over stimulated immune response leading to inflammation [ ] . elderly patients requiring hospitalization most frequently present with acute bronchitis, copd exacerbations, pneumonia and congestive heart failure [ ] . in healthy adults asymptomatic infections or cold-and flu-like symptoms are the most prevalent presentation [ ] . the pathogenesis of hmpv infection is strongly affected by bacterial coinfections with pneumococcus. one study has shown that administration of a conjugate pneumococcal vaccine is sufficient to reduce the incidence of hmpv infection of the lower respiratory tract and the incidence of clinical pneumonia in both hiv positive and negative patients [ ] . these finding suggest that the incidence of hospitalizations in hmpv infections may be decreased by vaccination with a conjugate pneumococcal vaccine. another case report of severe respiratory failure was found to be caused by coinfection with hmpv and streptococcus pneumonia in a year old patient [ ] . both in vitro and in vivo studies have shown that infection with hmpv facilitates adhesion of pneumococcal bacteria, which may provide an explanation for the coinfection with pneumococcal strains and hmpv [ ] . viral coinfections between hmpv and rsv have been reported, but remain a contentious issue. the typical seasonal overlap of the two viruses has been suggested to promote viral coinfection. one study reported a -fold increase in risk of admission to an intensive care unit in pediatric patients coinfected with rsv and hmpv and associated the dual infection as capable of augmenting severe bronchiolitis [ ] . other studies do not support this finding and further report a decreased correlation between hmpv-rsv coinfections and hospitalization and additionally lists dual infection, along with breastfeeding, as having protective effects [ ] . although hmpv was only discovered in , it has been shown by phylogenetic analysis to have been in existence for approximately years [ , ] . soon after the discovery of hmpv, it was evident that two lineages, a and b, existed. these two lineages were further subdivided into two sublineages per lineage, a -a and b -b [ ] . a recent report analyzing sequence divergence of the attachment and fusion surface glycoproteins indicates the presence of five sublineages, namely a , a a, a b, b and b [ ] . from long term retrospective studies it was evident that these lineages are not restricted to certain locations or times and that multiple lineages can exist in the same location and period [ , ] . it has also become evident that old sublineages may be replaced by new variants [ ] . disease progression or varying clinical outcomes related to different lineages of hmpv has become a contentious issue. several studies have reported that lineage a presents with more severe clinical outcomes [ ] [ ] [ ] where the same is reported for lineage b by other groups [ , ] . it has been further reported that there is no difference in disease outcomes related to the two lineages [ , , ] . between % and % of all cases of respiratory infections in children are caused by hmpv, in both hospitalized and outpatients [ , , ] and has been reported to be the second most frequently identified virus in respiratory tract infections [ ] . extrapolation of consensus data suggests a total of hospital and one million clinic visits annually in the us among children younger than [ ] . children hospitalized with hmpv infections are also more likely to present with pneumonia or asthma and required longer stay in intensive care units with supplemental oxygen, when compared to other respiratory viruses [ ] . seroprevalence studies indicates that % of young adults are seropositive for hmpv with stable neutralizing titres, which further suggests that reinfection occurs throughout life [ ] , with a potential for genetic variation between clades promoting reinfection [ ] . hmpv has a worldwide distribution and affects all age groups but predominantly affects young, elderly and immunocompromised patients [ ] , with children younger than five years of age being most susceptible to infection [ ] . children and adults with underlying or chronic conditions such as asthma, chronic lung disease, congenital heart disease, cancer or copd are more likely to be hospitalized with hmpv infection [ ] . infection with hmpv occurs throughout the year but seasonal prevalence in late winter and spring has been observed and coincides with the peak of rsv infection [ , [ ] [ ] [ ] . the first human bocavirus (hbov) was discovered in from nasopharyngeal aspirates of patients with unresolved lower respiratory tract infections in sweden. researchers utilized a novel technique which included steps of dnase treatment to exclude contaminating, or non-viral, nucleic acids followed by pcr amplification by nonspecific primers. the pcr-products were subsequently cloned with large-scale sequencing of the clones. bioinformatic analysis of generated sequence data yielded the discovery of a new parvovirus with a high homology to bovine and canine minute parvoviruses. the genus name bocavirus was in fact derived from the species infected by the known virus strains, namely bovine and canine. the new virus was named hbov and was the first virus to be discovered by molecular virus screening [ ] . three additional species of hbov were later discovered in and added to the genus; these were named hbov , hbov and hbov [ ] [ ] [ ] . hbov is a respiratory pathogen affecting all regions of the globe and is associated with approximately %- % of all upper and lower respiratory tract conditions [ ] [ ] [ ] . hbov productively infects human airway epithelium cell cultures and leads to damage of airway epithelial cells [ ] [ ] [ ] , which supports clinical observations that infection does result in respiratory disease. in contrast, hbov - , are found in the gastrointestinal tract and hbov , and possibly hbov , are associated with gastroenteritis [ , [ ] [ ] [ ] . interestingly, hbov is the only enteric bocavirus to be isolated from nasopharygeal aspirates and may, therefore, also be associated with respiratory disease [ , ] . hbov is detected in all age groups, but predominantly in young children between the ages of - months [ , , ] and is rarely detected in adults [ ] [ ] [ ] [ ] [ ] . transmission and infection occurs throughout the year, but predominantly during winter and spring months [ , [ ] [ ] [ ] . seroprevalence studies suggest that maternal antibodies, which provide protection, are present in infants younger than months of age [ , ] , after which seropositivity decreases with low levels of detection until - months. virtually % of children aged are seroconverted for hbov and as reinfection occurs throughout life this remains into adulthood [ , [ ] [ ] [ ] [ ] . the presence of the three enteric bocaviruses does however complicate the findings of seroconversion as cross-reactivity does exist [ ] . as with many respiratory viruses, clinical differentiation with hbov infection is not possible by symptomatic presentation [ ] . common features of infection of the upper respiratory tract include common cold-like symptoms with cough, rhinorrhoea and acute otitis media [ ] . infection of the lower respiratory tract in children is associated with pneumonia, acute wheezing, asthmatic exacerbations and bronchiolitis [ , , [ ] [ ] [ ] , but life-threatening complications are rare with hbov infection [ ] . although hbov has been isolated from stool samples, there is no statistical evidence to associate hbov with gastrointestinal disease [ ] . hbov has not only been found in the upper and lower respiratory tract and gastrointestinal specimens, but also in urine samples, serum, saliva, and tonsils [ ] . rather than having a role in disease pathogenesis, this viraemia and systemic spread may be a feature common to all parvoviruses as they require proliferating host cells for replication [ ] . interestingly, hbov appears to be more than just a respiratory or gastrointestinal virus. in a recent study, hbov was identified in . % of lung (n = / ) and . % of colorectal (n = / ) tumors screened. unfortunately, the study did not investigate whether the hbov genomes were in fact incorporated into the host genome as reported for other known parvoviruses. therefore, based on their observations as well as previous studies on other parvoviruses, the authors speculate that hbov could contribute to the development of some lung and colorectal tumors. however, they do also acknowledge that these tumors could simply be providing an optimal environment for hbov replication and more conclusive studies are required to resolve this issue [ ] . hbov has been associated with a prolonged period of persistence in the mucosa of the respiratory tract. this prolonged presence has possibly led to a high frequency of coinfection found with hbov infections of the both the upper and lower respiratory tract [ , , ] . the high rate of detection of multiple respiratory viruses within up to % of respiratory specimens, and the presence of asymptomatic hbov infections, does complicate the determination of the actual pathogenic role of hbov [ ] . high viral load is statistically associated with symptoms [ ] and this may therefore be a better indication of coinfections which are related to disease severity or symptomatic presentation. it has been further suggested that patients presenting with viraemia are better candidates to assess the symptoms of disease when compared to investigations of respiratory secretions [ ] . the effects and mechanisms of latency, persistence, reactivation and reinfection are however poorly understood and therefore its effects on coinfection and its contribution to active disease cannot be accurately stated [ ] . the etiological agents of %- % of lower respiratory tract infections still remain to be identified [ , ] . these results may vary significantly depending on the sensitivity of the diagnostic assay used, respiratory site sampled and even geographical location of the study but it does suggest that many uncharacterized respiratory pathogens could still remain elusive awaiting discovery. the vast improvements in molecular techniques within the past decade have however led to the discovery of four previously circulating respiratory viruses and also the rapid characterization of two completely novel viruses, namely sars-cov and mers-cov. all these viruses have varying but significant impact on human health and the potential for outbreak of completely novel, emergent respiratory viruses, seen with sars and mers, poses their own unique threats. lessons learned from these viruses, and others currently in circulation, provide health care authorities and scientists with suitable expertise and knowledge to rapidly identify and combat novel respiratory viruses and as our techniques 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public health legal preparedness (phlp) for emergencies is a core component of the health system response. however, the implementation of health legal preparedness differs between low- and middle-income countries (lmic) and developed countries. objective: this paper examines recent trends regarding public health legal preparedness for emergencies and discusses its role in the recent ebola outbreak. design: a rigorous literature review was conducted using eight electronic databases as well as google scholar. the results encompassed peer-reviewed english articles, reports, theses, and position papers dating from to . earlier articles concerning regulatory actions were also examined. results: the importance of phlp has grown during the past decade and focuses mainly on infection–disease scenarios. amid lmics, it mostly refers to application of international regulations, whereas in developed states, it focuses on independent legislation and creation of conditions optimal to promoting an effective emergency management. among developed countries, the united states’ utilisation of health legal preparedness is the most advanced, including the creation of a model comprising four elements: law, competencies, information, and coordination. only limited research has been conducted in this field to date. nevertheless, in both developed and developing states, studies that focused on regulations and laws activated in health systems during emergencies, identified inconsistency and incoherence. the ebola outbreak plaguing west africa since has global implications, challenges and paralleling results, that were identified in this review. conclusions: the review has shown the need to broaden international regulations, to deepen reciprocity between countries, and to consider lmics health capacities, in order to strengthen the national health security. adopting elements of the health legal preparedness model is recommended. p lanning for the prevention and mitigation of morbidity, mortality, and environmental damage is fundamental to public health system preparedness for emergencies ( ) . an essential element in this planning process is the creation of a legal infrastructure to be activated during all phases of the emergency ( ) from preevent to recovery. the existence of a legal framework is particularly important in large scale crises ( ) and scenarios requiring humanitarian assistance ( ) . public health legal preparedness (phlp) involves more than legislation. moulton et al. ( ) defined phlp as 'legal bench-marks or standards essential to the preparedness of that system'. accordingly phlp includes ) laws/legal authorities; ) competencies of those who apply the law; ) information relevant for the application of the law; and ) coordination across sectors/jurisdictions. benjamin et al. ( ) state that although these components can be broadened the improvement of legal preparedness must address all global health action ae global health action . # odeya cohen et al. this is an open access article distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. four core elements and not focus on one element (e.g. legislation) only, which will lead to an incomplete solution ( ) . globalisation requires integrated, joint actions aimed to facilitate management of international threats including the use of phlp. the aim of this paper is to review recent theoretical and research trends regarding legal preparedness of the public health system for emergencies. in particular, the phlp during the current ebola crisis will be discussed. two parallel literature reviews were conducted to explore ) theoretical and research trends of phlp in developed countries, as well as in low-and middle-income countries (lmics), and ) the application of phlp during the recent ebola crisis. the reviews were conducted from september to december and encompassed peerreviewed articles published in english as well as reports, theses, and position papers. the study search encompassed eight electronic databases: cochrane, lexisnexis, proquest, pubmed, science direct, scopus, social science research network, and web of science; the google scholar search engine was also employed. the keywords used to extract relevant articles were public health, legal preparedness, and emergency. the year of publication was limited from to . however, if one of the reviewed articles emphasised findings that were based primarily on specific previous models or legislative actions, these earlier articles were also reviewed. due to the very limited findings concerning lmics, and in order to identify elements within the context of phlp that are implemented in those countries, additional keywords Á regulation and legislation Á were introduced to the review procedure. articles were only included in this review if they dealt with legal aspects of emergency situations. an exclusion criterion was a focus on routine issues (e.g. the obesity epidemic). this criterion was used to screen all abstracts included in the study. in the final stage, the articles were grouped by themes. review of application of phlp during the current ebola crisis keywords used to extract relevant articles were ebola, public health, legal preparedness, and emergency. the review was limited to . articles were included if they focused on legal issues regarding the ebola outbreak. this criterion was used to screen all abstracts included in the study. in the final stage, the articles were grouped by themes. the role of phlp in emergency response phlp plays an important role in the overall functioning of the health system during emergencies ( , ) . providing legal assistance in the midst of a disaster is central to any response plan ( , ). adini et al. ( ) found that following standard operating procedures in an emergency is crucial for assuring preparedness. other aspects of legal preparedness relate to the status of volunteers and their ability to provide humanitarian aid after a disaster ( ) . orenstein ( ) asserts that some laws, though appropriate for routine health activities, may hinder operations during emergencies. while declaring a state of emergency may facilitate waiving laws, the implications of such waivers must be carefully evaluated. during emergencies, as maintained by courtney et al. ( ) , healthcare providers operate under challenging conditions that may require deviation from existing treatment protocols, necessitating the development of strategies to protect against legal liabilities. similarly, chan ( ) argues in favour of granting legal immunity to private physicians to protect them against damage claims. conversely, it is important to protect the vulnerable population from uncertified personnel performing beyond their capacity. according to wang ( ) , this issue manifests itself more intensely in situations of cross-national mutual aid that are regarded as reciprocal gestures of goodwill, where programmes cross boundaries and achieve their expected goals quickly. thus, it is important to define obligations and rights and to establish roles, items, and standards. other researchers ( , ) perceived the competency of health workers as one of the core elements of phlp. pandemic outbreaks inflict widespread suffering and may negatively impact international economic stability ( ) . managing pandemics involves coordinating different aspects of the health and social systems. in such situations, the law, which is a small but crucial component of emergency preparedness ( , ) , assumes increased importance ( Á ). the speed with which viruses spread makes it imperative to ensure that a legal framework is in place to delineate mechanisms for effective epidemic management, within the country itself as well as globally ( , , ) . accordingly, efforts and reforms in this direction have been proposed and implemented worldwide, in developed countries as well as lmics ( ) from the united states to china ( ) . according to hodge ( ) , although legal reforms have occurred in the united states in the last years, three core challenges will continue to engage experts during the next decade: the legal implications of multiple emergency declarations, legal triage, and liability protection for practitioners and entities implementing crisis standards of care in response to declared emergencies ( ) . following outbreaks of severe acute respiratory syndrome (sars) and avian flu (h ni) in Á , the world health organization (who) adopted the international health regulations (ihr), whose goal is 'to prevent, protect against, control and provide a public health response to the international spread of disease' ( ). the who also published a checklist designed to help countries prepare effectively for infectious emergencies ( ) . the experiences of the global health system in dealing with the pandemic flu (h n ) had a significant impact on subsequent implementation of the ihr ( , , , ) . the ihr made valuable contributions in various areas, including issuing clear-cut reports from afflicted countries, integrating information from diverse sources, and monitoring unnecessary human rights limitations ( ) . in addition, it has become a useful decision support tool ( ) . however, analysis of how the ihr are applied in lmics reveals the challenges that still lie ahead ( ) . the main weakness is lack of resources and the ensuing inability to meet ihr demands and develop effective public health services ( , , , ) . in addition, the ihr do not make allowances for local and cultural variability ( , Á ). wilson et al. ( ) note several arenas in which ihr application needs to be strengthened, including declaring a health emergency in international scenarios and developing mechanisms to improve compliance with who and ihr recommendations. kool et al. ( ) noted that in order to achieve the ihr goals it is crucial to simplify identification and detection capacities in countries lacking advanced health systems. for example, it is necessary to base disease definitions on clinical signs and symptoms without the need for laboratory confirmation ( ) . epidemic scenarios that involve laws and phlp concerning isolation, quarantine, and social distancing are worthy of broad attention. limiting individual freedom in order to protect the public health has significant implications for managing infectious diseases ( , , , ) . preparing a legal infrastructure to administrate these situations is crucial, including declaration of an emergency situation which authorises public health officials to activate such means ( ) . nonetheless, operationalising them should not be based on legal facets alone, but should rather also consider judicial aspects. coercion may decrease the effectiveness of protection that could be achieved through voluntary compliance. it may also increase the probability of a constitutional crisis ( ). mosher ( ) notes that the law's promise of protection against abuses during an epidemic offers limited space for critics worried that individual liberties will yield to national security and public health concerns. in such cases, it is important to create an alternative framework in order to voice the views of those who are socially marginalised and have been largely silenced ( ) . the balance between individual rights and the common good a central aspect of phlp is balancing individual needs and the common good. the need to protect individual freedom arises in all types of emergency situations, but has significant implications for the management of infectious disease outbreaks ( , ) . turnock ( ) describes two major aims of public health laws: to protect and foster public health and to safeguard the rights of the individual. in emergency situations, imposing limitations on individual rights is unavoidable ( ) . in the united states, in the supreme court case jacobson v. massachusetts the court sustained the right of the authorities to use penalties to pressure people to be vaccinated during a smallpox epidemic. this ruling interpreted the use of the public health authority and the way the court balanced two strong competing values: the public good and individual liberties ( ) . gostin ( ) argues that the resulting ethical conflict is more acute in the period preceding the emergency. however, early legislation enables legal definition of individual rights, thereby facilitating optimal actions during the emergency itself ( ) . according to gerwin ( ) , during pandemics, governance that assures a legal balance between the needs of the public and those of the individual enhances public trust in the authorities' ability to manage the situation. by contrast, in the wake of the terror attack in the united states, the rights of the individual were significantly curtailed by a powerful government supported by legal measures ( ) . limited information was found regarding legal preparedness for emergencies in lmics. most sources focused exclusively on infectious disease management while only few related to legal aspects in the wider context of health system operation. fischer and kats ( ) mention the rural-to-urban migration phenomenon that produces 'mega-cities' of million or more inhabitants. according to united nations estimates, three-quarters of the megacities are located in lmics. this global trend exposes populations to disaster vulnerabilities and is associated with a dearth of risk management infrastructures Á physical, governmental, and legal ( ). nishtar et al. ( ) , analysing the pakistani health system, stated that one of its strengths is its legislative activism and the federal structure of the government, which promote the health system's ability to overcome challenges. according to them ( ) , it is important to establish laws and regulations governing publicÁprivate interactions, insurance, and e-health and thus contribute towards a coordinated, joint preparedness. van niekerk et al. ( ) found in studying south africa that certain laws enacted for disaster risk reduction are not implemented due to lack of funds and that laws need to be updated to ensure coordination between public and private sectors. globally, lmics play a crucial role in promoting prevention measures and immunisation programmes against diseases ( ) . kaddar et al. ( ) contend that the focus should be on middle-rather than low-income countries. regarding immunisation, vaccines are designated by the who according to the population's needs, supported by the global alliance for vaccines and immunization ( ) . an additional aspect of the legislative infrastructure relates to counterfeit medicines. despite the efforts of the who ( ), the regulatory structures prevailing in lmics cannot cope with the problem of counterfeit drugs and their use ( ) . health legal preparedness is more prevalent in developed countries compared to lmics. most publications dealing with phlp focus on the creation of legal conditions (e.g. emergency declaration, legal immunity) which may promote public health preparedness for emergencies, rather than implementation of international regulations. while the increasing importance of phlp is a worldwide trend, it is especially well developed in the united states. the first call to address the issue of health system legislation was published by the institute of medicine in ( ). this call was heeded after different emergency events that occurred during the first decade of the twenty-first century (the world trade center attack in , the mailing of envelopes containing anthrax, the sars epidemic in , and hurricane katrina in ) ( ) . these natural disasters and man-made events significantly impacted us society and government ( ) . in addition, they demonstrated the need for a policy that would include, inter alia, a legal response defining how the health system should manage such scenarios. a health legal preparedness model was developed and adapted by the us legal and health systems. it encompasses four core components ( , ): ) law Á the authoritative infrastructure of public health bodies that activate the public health system, ) competencies Á qualification of experts in areas common to legislation and health preparedness, ) information Á updating and publicising health laws for content experts and healthcare workers and ) coordination between legal systems and within emergency response agencies. current publications dealing with this model discuss researching and expanding its components ( Á ), as well as broadening its application to situations that the health system faces routinely ( ). current us literature reflects the recent improvements in the phlp field. studies dealing with preparedness are designed to reveal and resolve ethical and legal dilemmas in order to promote an optimal response for future situations. in addition to extensive coverage of legal aspects dealing with pandemic scenarios ( , , , , ) , publications also consider the impact of phlp on issues such as legal triage ( ), motivating health workers during emergencies ( , , ) , human rights during crises, legal coordination of relevant emergency bodies ( ) , and the effect of an emergency declaration on health system management ( , ) . hodge ( ) notes that upon declaration of a public health emergency the model state emergency health powers act ( ) authorises public health officials to undertake a set of activities dealing with early detection, as well as to care for and protect public health, treat exposed or infected persons, and seek out-ofstate volunteers. the declaration is defined to inform the population, through the media, using language that is cross-culturally accessible and understandable ( ) . all the items mentioned above reflect the creation of a legal infrastructure targeted for enhancing the us public health system. in the european union, emphasis is placed on routine application of a 'no border' policy, with minor references to emergencies ( Á ). european union law is based on legislation (directives and regulations) and decisions of the court of justice, which intervenes when the meaning or implementation of the legislation is unclear or fails during implementation ( ) . in the context of emergency preparedness, references to legal aspects focus on management of communicable disease outbreaks ( , , ) . greer et al. ( ) claim that the european union's laws play a dominant role in safeguarding the population's health, as their common laws and regulations significantly affect the health system. nonetheless, due to factors stemming from the complexity of the european union and the variability that exists among the european states' health systems, decisions are interpreted very generally with apparently vague provisions in the treaties. as a result, legislative interventions sometimes fail to adequately promote public health. for example, hatzianastasiou et al. ( ) found that greek laws are aligned with accepted practices of international law in the context of communicable diseases in terms of safeguarding individual rights, but exhibit a lack of coherence, clarity, or systematisation and are often perceived as incomprehensible ( ) . in order to understand phlp, it is vital to review studies which cover diverse types of legal management. although this topic could be significantly broadened, the description of current trends in phlp would be incomplete without referring to this issue. studying phlp during emergencies is complex, as there is a need to refer to diverse types of knowledge such as that concerning governance, policy, and perceptions of emergency professionals in order to provide meaningful insights ( ). legal preparedness shapes the health system and its emergency response in different ways. as such, there are various methodologies that prove efficient in studying these mechanisms. the integration of legislation into emergency management still seems to be a relatively neglected area ( ) . according to jacobson et al. ( ) , this is due to the lack of recognition that information about public health laws promotes best practices during emergencies. fox ( ) noted two methodological concerns regarding research on health policy governance and diseases. the first concerns the researchers' definition of governance, which influences what information they obtain and how they assess it. the second is that governance determines how diseases are conceptualised in order to make and implement policy. burris et al. ( ) classify three types of health laws: infrastructural laws, intervention/implementation laws, and secondary legislation. most studies in the field of law and health relate to intervention and secondary legislation and only a few relate to infrastructure and its effects on public health ( ). differences were found in the type of studies conducted in lmics versus developed countries. in the former, the majority of studies focused on the effectiveness of infrastructural laws, international regulations, and the need for more flexible regulations ( , ) . in developed states, studies focused on internal legislation, legal issues that might arise during emergencies, and advanced planning for future challenges ( , , ) . most studies relating to developed countries were conducted in the united states. differences among the individual states regarding the statutory/judicial system and the structure of healthcare agencies make it possible to investigate the effects of public health laws on the population's health. on the other hand, the variance that exists among the states influences knowledge management, which in turn affects health knowledge implementation within the framework of public health laws ( ) . rutkow et al. ( ) analysed differences between individual states in an effort to identify laws that impact the public health workforce and willingness to respond to emergencies. these differences can be well noted concerning emergency management, such as ensuring accessibility of the public to information via media that is influenced by cultural characteristics as defined in the model state emergency health powers act ( ) ( ) . reviewing the studies relating to phlp which were conducted in the last few years indicated that declaration of an emergency situation by the authorities was an essential component of emergency response and provided the health sector with flexibility and guidance concerning response parameters ( ) . other researchers studied perceptions regarding public health laws among organisations involved in managing emergencies. jacobson et al. ( ) found a gap between experts' perceptions of these laws and their basic aims, leading to severe deficiencies in health system preparedness. public health and disaster management professionals may differ in their understanding of the law ( ) , which hampers their ability to cooperate effectively during emergencies. according to kaufman et al. ( ) , staff training is the key to bridging differences in perception between public health workers and legal advisors. other studies dealt with legal means for motivating healthcare workers and offering enhanced legal protection against liability while reducing the incidence of harm claims during disasters and pandemics ( , ) . to deal with the lack of familiarity with legal preparedness, multi-professional panels were created to reach consensus concerning relevant issues ( , ) . in both developed and underdeveloped countries, researchers that investigated regulations and laws that activate the health system during emergencies found inconsistencies and lack of coherence ( , , ) . the ebola crisis: a case study of legal preparedness during a worldwide outbreak the ebola outbreak clearly illustrates the involvement of legal preparedness and response during an international crisis. this section will briefly review legal issues that demonstrate the different facets of the phlp. the re-emergence of ebola in in west africa, followed by the evacuation of stricken western healthcare workers to the united states, captured the world's attention ( , ) . the fatality rate of the ebola outbreak ranged from to % ( ) . affected countries are characterised by limited resources and political instability, with low capacity health systems and a lack of essential equipment and personnel. the probability that the ebola virus will take root in a high-resource country is small ( , , ) . the ebola crisis displayed the importance of legal preparedness for emergency situations from a global perspective, presenting ethical and legal dilemmas of affected and unaffected countries. managing such a crisis necessitated a multi-dimensional response, including effective functioning of health systems; coordination of diverse disciplines; international distribution; use of experimental drugs and medical procedures; safeguarding human rights; and consideration of implications in the health, political, and economic arenas. in addition, the ebola crisis brought to the forefront basic dilemmas concerning responsibility and reciprocity among developed states and lmics. it was essential to integrate the legal dimension into the global response in order to maximise strategies aimed at coordinating joint efforts to contain the ebola epidemic and save lives ( ) . the global response to the ebola outbreak was insufficient ( , ) . gostin and friedman ( ) argue that the outbreak uncovered a failure in global health leadership and that the who should be the global health leader. however, its budget is not commensurate with its responsibilities. as a result, some countries departed from the who directives and responded with excessive severity (e.g. imposing mandatory travel bans). in addition, several contaminated states could not realistically implement who recommendations and thus did not show full compliance with the guidelines ( ). hodge et al. ( ) note four issues that should be considered when creating legal preparedness for the ebola crisis: health workers' willingness to respond, experimental drugs and medical procedures, social-distancing measures in medical settings, and potential liabilities of healthcare workers and entities. addressing these issues could help mitigate fears, improve the public health response, protect the safety of healthcare workers and communities, and promote comprehensive medical and public health services ( ) . effective management of emergencies such as the ebola outbreak depends on the health systems' capacities ( ) . therefore, the most effective way to curb such outbreaks is to strengthen weak health systems and infrastructures ( , , ) . considering the vast differences between developed countries and lmics, immediate and extensive assistance should be provided ( ) . regarding the high mortality rate of healthcare workers in the ebola crisis ( ), hodge et al. ( ) noted that the infrastructures must offset the risks taken by frontline personnel with a commitment to their protection. this issue came to the forefront because western healthcare workers were evacuated, while frontline workers from affected places were not. in addition, the commitment to protect healthcare workers has to be standardised legally in affected countries as well as in ascending countries. in addition to the above, a central legal issue highlighted by this crisis is protection of human rights. the acute nature of the recent outbreak necessitated imposing quarantine, isolation, and other restrictive measures, in addition to monitoring movement of travellers. however, these measures were applied excessively and, rather than proving beneficial, caused under-reporting of diagnosed patients, lowered trust in the government, and sharpened economic, political, and social challenges ( , , , ) . during this crisis, human rights violations were wideranging, including blockage of rural areas in sierra leone by the army, shooting of people who unlawfully entered liberia from sierra leone, and broad-sweeping barricades in liberia that prevented access to food, medicine, and life-sustaining services. limiting travel from affected countries is contrary to who guidelines ( ) . rothstein ( ) noted four ethical principles that should be considered in the process of deciding whether quarantine is needed: necessity, effectiveness, and scientific rationale; proportionality and minimal infringement; humane supportive services; and public justification. regarding experimental treatments, it is important to formulate and adhere to ethical rules ( , , ) in order to mitigate inadvertent damage, which could worsen already strained relationships between health professionals and their patients ( ) . the factors mentioned above significantly affect the public's trust in responding agencies and governance systems activated during crises such as the ebola outbreak. the relationships between international health organisations are thus impacted ( ) in both underdeveloped ( ) and developed states ( , ) . this study reviews current trends regarding phlp for emergencies. over the past decade, in order to improve disaster planning and response ( ) , phlp has steadily grown in importance ( ) . however, while legal preparedness is important in diverse types of emergencies, most of the literature focuses exclusively on pandemic scenarios. furthermore, the status of phlp is influenced by the characteristics of the country involved. ihr represent a potentially revolutionary change in global health governance ( , ) . one criticism is that the regulations fail to make due allowance for local conditions and characteristics ( , Á ). in order for ihr to be appropriately realised, health organisations must create conditions that enhance the capacities of countries in need. regulatory attention paid to health systems' capacities may have a positive economic consequence: the provision of structured support during the pre-crisis period would significantly reduce post-outbreak outlays for assistance ( ) . according to mccloskey et al. ( ) , there is a need to develop trust and nurture effective, meaningful collaborations between countries to facilitate rapid detection of potential pandemics and initiation of public health actions. from this perspective, international regulations need to standardise the implementation of legal activities, motivated by a concern for global public health and well-being. coordination of such activities would promote routine inter-state assistance as well as collaboration during emergencies. thus, allowing the development of responses adapted to different countries' capacities without losing sight of the overall public health goals would help expand the capabilities of poorer countries. the ebola crisis reveals the importance of creating legal preparedness that takes into account the needs and capacities of both affected and unaffected countries ( , , , ) . inadequate capacities create severe stress on a country's ability to deal with the crisis. as a result, first and foremost, medical care is harmed. moreover, it inflicts a severe blow to individual rights. another facet of ethical and legal implications which was evident in the current crisis concerns the protection of health workers. this topic impacts both affected countries and the countries that provide assistance. reciprocity between developed countries and lmics is a global concern targeted to protect the world's health. the current global situation and the health status in developing countries directly affect the health of the world's population. thus, developed countries have an interest, beyond their responsibility, in catering to the health status of countries that lack vital means and resources. international legal preparedness, which considers the needs and capacities of different countries, will improve the assistance provided to countries that need it and regulate cooperation between the various stakeholders. implementation of the phlp model the us phlp model was intended to strengthen health system preparedness for routine scenarios as well as emergencies ( , ) . implementing the four core elements (law, competencies, information, and coordination) ( ) may increase legal preparedness by addressing the response of local legal and health systems for emergencies. the adoption of elements of the us model by developing countries, may promote the capacities of health systems' preparedness, which, in turn, will contribute to the increase of global health security. developed countries have an interest in assisting the local health capacities in lmics. international regulations incorporating these elements would in effect expand the resources available for coping with local and international public health emergencies. at present, resources are provided to underdeveloped states mainly to manage public health threats that affect developed countries Á even though other public health hazards may pose a greater threat to the lmic ( ) . the challenges identified in lmics in the course of the ebola crisis should be studied in order to assist the tailoring of appropriate legal preparedness in these countries. in certain countries, such as the united states, laws regarding infectious diseases provide the legal framework for health system operations in routine situations as well as during emergencies ( ) . it is vital to understand the ways in which less developed countries manage epidemics and translate this into a legislative framework that can promote the effectiveness of local health systems. research in the field of phlp efforts to improve health preparedness must be supported by adequate research. although a relatively new field, public health law provides important contributions to policy making and, by extension, to the health of the population ( ) . the dearth of studies in this area indicates a gap that needs to be filled. systematic research employing advanced techniques and sensitive data analyses can facilitate the study of legal preparedness and help elucidate the causal relationship between legal reforms and emergency preparedness of healthcare systems. the findings will help promote emergency preparedness in every country. this article provides highlights of current trends regarding phlp as reflected in the professional literature. nonetheless, this study did not fully examine legislation in the investigated countries. the keywords used in the literature search process did not include the word disaster. nevertheless, in checking the articles that were found using the search engines, researchers noted only one article that was not included in the findings based on the term emergency. in addition, the literature review only included papers in the english language, which might have excluded publications from non-english speaking countries. this article provides an overview of various issues regarding phlp but does not encompass all the particulars. the role of the legal component in building emergency health preparedness is gaining increasing recognition worldwide, although in many countries this has been expressed only in the context of infectious diseases. the ebola epidemic revealed that despite adoption of international regulations by lmics their health systems still lack the capacity to manage such epidemics. there is a need to boost effective implementation of international regulations by these states, thereby strengthening their ability to deal with routine and emergency situations and fostering global health security. the ihr present a good starting point, although additional work is needed to find a legal framework that will strengthen the willingness of the various stakeholders with different interests to cooperate and coordinate health preparedness programs. it is recommended that the components of the phlp model be widely adopted as a comprehensive basis for promoting legal preparedness in local health systems, backed by sophisticated methods of analysis directed at elucidating the effect of phlp on the capacity 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and the law in the united states: a short guide to public health authority and practical limits. emory legal studies research paper Á progress in disaster planning and preparedness since global health security agenda and the international health regulations: moving forward emerging infectious diseases and pandemic potential: status quo and reducing risk of global spread for the public's health: revitalizing law and policy to meet new challenges ac conducted the literature review and drafted the article. ba was responsible for the overall supervision of the study and critically revised the draft article. pfb and ybd critically revised the article drafts. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. key: cord- -l wrrapv authors: duchêne, david a.; duchêne, sebastian; holmes, edward c.; ho, simon y.w. title: evaluating the adequacy of molecular clock models using posterior predictive simulations date: - - journal: mol biol evol doi: . /molbev/msv sha: doc_id: cord_uid: l wrrapv molecular clock models are commonly used to estimate evolutionary rates and timescales from nucleotide sequences. the goal of these models is to account for rate variation among lineages, such that they are assumed to be adequate descriptions of the processes that generated the data. a common approach for selecting a clock model for a data set of interest is to examine a set of candidates and to select the model that provides the best statistical fit. however, this can lead to unreliable estimates if all the candidate models are actually inadequate. for this reason, a method of evaluating absolute model performance is critical. we describe a method that uses posterior predictive simulations to assess the adequacy of clock models. we test the power of this approach using simulated data and find that the method is sensitive to bias in the estimates of branch lengths, which tends to occur when using underparameterized clock models. we also compare the performance of the multinomial test statistic, originally developed to assess the adequacy of substitution models, but find that it has low power in identifying the adequacy of clock models. we illustrate the performance of our method using empirical data sets from coronaviruses, simian immunodeficiency virus, killer whales, and marine turtles. our results indicate that methods of investigating model adequacy, including the one proposed here, should be routinely used in combination with traditional model selection in evolutionary studies. this will reveal whether a broader range of clock models to be considered in phylogenetic analysis. analyses of nucleotide sequences can provide a range of valuable insights into evolutionary relationships and timescales, allowing various biological questions to be addressed. the problem of inferring phylogenies and evolutionary divergence times is a statistical one, such that inferences are dependent on reliable models of the evolutionary process (felsenstein ) . bayesian methods provide a powerful framework for estimating phylogenetic trees and evolutionary rates and timescales using parameter-rich models (huelsenbeck et al. ; yang and rannala ) . model-based phylogenetic inference in a bayesian framework has several desirable properties: it is possible to include detailed descriptions of molecular evolution (dutheil et al. ; heath et al. ) ; many of the model assumptions are explicit (sullivan and joyce ) ; large parameter spaces can be explored efficiently (nylander et al. ; drummond et al. ) ; and uncertainty is naturally incorporated in the estimates. as a consequence, the number and complexity of evolutionary models for bayesian inference has grown rapidly, prompting considerable interest in methods of model selection (xie et al. ; baele et al. ) . evolutionary models can provide useful insight into biological processes, but they are incomplete representations of molecular evolution (goldman ) . this can be problematic in phylogenetic inference when all the available models are poor descriptions of the process that generated the data (gatesy ) . traditional methods of model selection do not allow the rejection, or falsification, of every model in the set of candidates being considered. gelman and shalizi ( ) recently referred to this as a critical weakness in current practice of bayesian statistics. a different approach to model selection is to evaluate the adequacy, or plausibility (following brown a), of the model. this involves testing whether the data could have been generated by the model in question (gelman et al. ). assessment of model adequacy is a critical step in bayesian inference in general (gelman and shalizi ) , and phylogenetics in particular (brown a) . one method of evaluating the adequacy of a model is to use posterior predictive checks (gelman et al. ) . among the first of such methods in phylogenetics was the use of posterior predictive simulations, proposed by bollback ( ) . the first step in this approach is to conduct a bayesian phylogenetic analysis of the empirical data. the second step is to use simulation to generate data sets with the same size as the empirical data, using the values of model parameters sampled from the posterior distribution obtained in the first step. the data generated via these posterior predictive simulations are considered to represent hypothetical alternative or future data sets, but generated by the model used for inference. if the process that generated the empirical data can be described with the model used for inference, the posterior predictive data sets should resemble the empirical data set (gelman et al. ) . therefore, the third step in assessing model adequacy is to perform a comparison between the posterior predictive data and the empirical data. this comparison must be done using a test statistic that quantifies the discrepancies between the posterior predictive data and the empirical data (gelman and meng ) . the test statistic is calculated for each of the posterior predictive data sets to generate a distribution of values. if the test statistic calculated from the empirical data falls outside this distribution of the posterior predictive values, the model in question is considered to be inadequate. previous studies using posterior predictive checks of nucleotide substitution models have implemented a number of different test statistics. some of these provide descriptions of the sequence alignments, such as the homogeneity of base composition (huelsenbeck et al. ; foster ) , site frequency patterns (bollback ; lewis et al. ) , and unequal synonymous versus nonsynonymous substitution rates (nielsen ; rodrigue et al. ). brown ( b) and reid et al. ( ) introduced test statistics based on phylogenetic inferences from posterior predictive data sets. some of the characteristics of inferred phylogenies that can be used as test statistics include the mean tree length and the median robinson-foulds distance between the sampled topologies in the analysis (brown b) . although several test statistics are available for assessing models of nucleotide substitution (brown and eldabaje ; brown a; lewis et al. ) , there are no methods available to assess the adequacy of molecular clock models. molecular clocks have become an established tool in evolutionary biology, allowing the study of molecular evolutionary rates and divergence times between organisms (kumar ; ho ). molecular clock models describe the pattern of evolutionary rates among lineages, relying on external temporal information (e.g., fossil data) to calibrate estimates of absolute rates and times. the primary differences among the various clock models include the number of distinct substitution rates across the tree and the degree to which rates are treated as a heritable trait (thorne et al. ; drummond et al. ; drummond and suchard ; for a review see ho and duchêne ) . for example, the strict clock assumes that the rate is the same for all branches, whereas some relaxed clock models allow each branch to have a different rate. we refer to models that assume a large number of rates as being more parameter rich than models with a small number of rates . although molecular clock models are used routinely, the methods of assessing their efficacy are restricted to estimating and comparing their statistical fit. for example, a common means of model selection is to compare marginal likelihoods in a bayesian framework (baele et al. ). however, model selection can only evaluate the relative statistical fit of the models, such that it can lead to false confidence in the estimates if all the candidate models are actually inadequate. in this study, we introduce a method for assessing the adequacy of molecular clock models. using simulated and empirical data, we show that our approach is sensitive to underparameterization of the clock model, and that it can be used to identify the branches of the tree that are in conflict with the assumed clock model. in practice, our method is also sensitive to other aspects of the hierarchical model, such as misspecification of the node-age priors. we highlight the importance of methods of evaluating the adequacy of substitution models in molecular clock analyses. to evaluate the adequacy of molecular clock models, we propose a method of generating and analyzing posterior predictive data. in this method, the posterior predictive data sets are generated using phylogenetic trees inferred from branchspecific rates and times from the posterior samples ( fig. ). because this method uses branch-specific estimates, it requires a fixed tree topology. the first step in our method is to conduct a bayesian molecular clock analysis of empirical data. we assume that this analysis obtains samples from the posterior distribution of branch-specific rates and times. these estimates are given in relative time, or in absolute time if calibration priors are used. in the second step, we take a random subset of these samples. for each of these samples, we multiply the branch-specific rates and times to produce phylogenetic trees in which the branch lengths are measured in substitutions per site (subs/ site), known as phylograms. to assess model adequacy, we randomly select samples from the posterior, excluding the burn-in. from these samples, posterior predictive data sets are generated by simulation along the phylograms and using the estimates of the parameters in the nucleotide substitution model. the third step in our approach is to use a clock-free method to estimate a phylogram from each of the posterior predictive data sets and from the empirical data set. for this step, we find that the maximum likelihood approach implemented in phangorn (schliep ) is effective. to compute our adequacy index, we consider the branch lengths estimated from the posterior predictive data sets under a clock-free method, such that there is a distribution of length estimates for each branch. we calculate a posterior predictive p value for each branch using the corresponding distribution obtained with the posterior predictive data sets. this value is important for identifying the length estimates for individual branches that are in conflict with the clock model. our index for overall assessment is the proportion of branches in the phylogram from the empirical data that have lengths falling outside the % quantile range of those estimated from the posterior predictive data sets. we refer to our index as a, or overall plausibility of branch length estimates. we also provide a measure of the extent to which the branch length estimates from the clock-free method differ from those obtained using the posterior predictive simulations. to do this, we calculate for each branch the absolute difference between the empirical branch length estimated using a clock-free method and the mean branch length estimated from the posterior predictive data. we then divide this value by the empirical branch length estimated using a clockfree method. this measure corresponds to the deviation of posterior predictive branch lengths from the branch length estimated from the empirical data. for simulations and analyses of empirical data, we present the median value across branches to avoid the effect of extreme values. we refer to this measure as "branch length deviation," of which low values represent high performance. we also investigated the uncertainty in the estimates of posterior predictive branch lengths. this is useful because it provides insight into the combined uncertainty in estimates of rates and times. the method we used was to take the width of the % quantile range from the posterior predictive data sets, divided by the mean length estimated for each branch. this value, along with the width of the % credible interval of the rate estimate from the original analysis, can then be compared among clock models to investigate the increase in uncertainty that can occur when using complex models. we first evaluated the accuracy and uncertainty of substitution rate estimates from simulated data. to do this, we compared the values used to generate the data with those estimated using each of three clock models: strict clock, random local clocks (drummond and suchard ) , and the uncorrelated lognormal relaxed clock (drummond et al. ) . we regarded the branch-specific rates as accurate when the rate used for the simulation was contained within the % credible interval. we found that rate estimates were frequently inaccurate under five circumstances: clock model underparameterization; rate autocorrelation among branches (kishino et al. ) ; uncorrelated beta-distributed rate variation among lineages; misleading node-age priors (i.e., node calibrations that differ considerably from the true node ages); and when data were generated under a strict clock but analyzed with an underparameterized substitution model ( fig. a ). when analyses were performed using the correct or an overparameterized clock model, more than % of branch rates were accurately estimated, such that the true value was contained within the % credible interval ( fig. a) . in most simulation schemes, the uncorrelated lognormal relaxed clock had high accuracy, at the expense of a small increase in the uncertainty compared with the other models ( fig. b ). these results are broadly similar to those of drummond et al. ( ) , who also found that underparameterization of the clock model resulted in low accuracy in rate estimates, whereas overparameterization had a negligible effect on accuracy. we analyzed data generated by simulation to test our method of assessing the adequacy of molecular clock models. the a index was approximately proportional to the branch length deviation ( fig. a ). we found a to be ! . (indicating high performance) when the model used in the analyses matched that used to generate the data, or when it was overparameterized. when the assumed model was the top right box shows the first step in assessing model adequacy using pps. in our analyses, this step is performed using branch-specific rates and times. the bottom box shows our procedure for testing the clock model, which is based on the clock-free posterior predictive distribution of the length of each branch. the thin arrows indicate that the test statistic is the posterior predictive p value for each branch. pps, posterior predictive simulations. duchêne et al. . doi: . /molbev/msv mbe underparameterized, a was . . the uncertainty obtained using posterior predictive branch lengths was sensitive to the rate variance in the simulations. for this reason, estimates from data generated according to a strict clock or an uncorrelated lognormal relaxed clock had lower uncertainty than estimates from data generated under local clocks, regardless of the model used for analysis ( fig. b ). estimates made using the uncorrelated lognormal relaxed clock had a larger variance in three analysis schemes: when data were generated with autocorrelated rates across branches; when data were generated with beta-distributed rates across branches; and when there was a misleading prior for the node ages. for analyses with substitution model underparameterization, our method incorrectly provided greater support for the more complex clock model, indicating that rate variation among lineages was overestimated ( fig. ) . we used our simulated data and posterior predictive simulations to investigate the performance of the multinomial test statistic for evaluating the adequacy of molecular clock models. this test statistic was originally designed to assess models of nucleotide substitution (goldman ; bollback ) and can perform well compared with some of the other existing test statistics (brown b) . the multinomial test statistic for the empirical alignment can be compared with the distribution of test statistics from posterior predictive data sets to produce a posterior predictive p value. we find that the multinomial test statistic correctly identified when the substitution model was matched or underparameterized ( fig. ) . the multinomial likelihood did not have the power to detect clock model adequacy, but it was sensitive to rate variation among lineages, primarily from the simulation involving autocorrelated rates and when the node-age prior was misleading ( fig. ). we used three clock models, as in our analyses of simulated data, to analyze a broad range of nucleotide sequence data fig. . mean values of (a) accuracy and (b) uncertainty of branch rate estimates from molecular clock analyses of simulated data. each cell shows the results of replicate analyses. accuracy is measured as the proportion of data sets for which the rate used for simulation was contained in the % credible interval of the estimate. darker shades in (a) represent high accuracy. uncertainty is measured as the width of the % credible interval as a proportion of the mean rate. dark shades in (b) represent small ranges in branch length estimates, and therefore low uncertainty. the initials stand for each of the schemes for estimation or simulation. sc, strict clock; loc, local clock; ucl, uncorrelated lognormal relaxed clock; rlc, random local clock; acl, autocorrelated relaxed clock; bim, beta-distributed bimodal clock; pri, misleading node-age prior; gtrg, data simulated under the parameter-rich general time-reversible substitution model with among-site rate heterogeneity. mean values of (a) plausibility, a, and (b) uncertainty as described by the posterior predictive simulations from clock analyses of simulated data. each cell shows the results of replicate analyses. values in parentheses are the branch length deviations, of which lower values indicate good performance. the darker shades represent higher values of a and less uncertainty. high values of a represent good performance. in the case of uncertainty, small values indicate small ranges in posterior predictive branch lengths, and therefore low uncertainty. the initials stand for each of the schemes for estimation or simulation. sc, strict clock; loc, local clock; ucl, uncorrelated lognormal relaxed clock; rlc, random local clock; acl, autocorrelated relaxed clock; bim, beta-distributed bimodal clock; pri, misleading node-age prior; gtrg, data simulated under the parameter-rich general time-reversible substitution model with among-site rate heterogeneity. assessing the adequacy of clock models . doi: . /molbev/msv mbe sets: the m (matrix) gene of a set of coronaviruses; the gag gene of simian immunodeficiency virus (siv; wertheim and worobey ); complete mitochondrial genomes of killer whales orcinus orca (morin et al. ) ; and mitochondrial protein-coding genes of marine turtles (duchene et al. ) . the uncorrelated lognormal relaxed clock was the bestfitting clock model according to the marginal likelihood for the coronaviruses, siv, and the killer whales (table ) . for the marine turtles, the random local clock provided the best fit. in all the analyses of empirical data sets, the uncorrelated lognormal relaxed clock had the best performance according to our a index. the highest a index was . for the siv and the killer whales, and the lowest uncertainty in posterior predictive branch lengths was . for the killer whales. the uncertainty for all other data sets was above , indicating that it was larger than the mean of the posterior predictive branch lengths. we calculated the multinomial test statistic for the empirical data sets using the posterior predictive data from a clock model analysis, as well as under a clock-free method. the multinomial test statistic from both methods suggested that the substitution model was inadequate for the siv and the marine turtles, with posterior predictive p values below . . the substitution model was identified as inadequate for the coronavirus data set by the multinomial test statistic estimated using posterior predictive data sets from a clock analysis (p < . ); however, it was identified as adequate when using a clock-free method (p = . ). the mitochondrial data set from killer whales represented the only case in which the substitution model was adequate according to both multinomial likelihood estimates. for the data sets from coronaviruses and killer whales, the clock models with the highest performance had a indices of . and . , respectively (table ). these indices are substantially lower than those obtained in analyses of simulated data when the clock model used for simulation and estimation was matched. however, we evaluated the posterior predictive p values for all branches in these empirical data sets and found that at least two-thirds of the incorrect estimates correspond to relatively short terminal branches (supplementary information, supplementary material online). the branch length deviation in the empirical data ranged between . for the uncorrelated lognormal relaxed clock in the turtle data and . for the killer whale data analyzed with a strict clock (table ) . low values for this metric indicate small differences between the posterior predictive and the empirical branch lengths. although scores for this metric varied considerably between data sets, they were closely associated with the a indices for the different models for each data set individually. for example, in every empirical data set, the lowest branch length deviation was achieved by the model with the highest a index (indicative of higher performance). importantly, the branch length deviation was not directly comparable with the a index between data sets. mbe this is probably because the posterior predictive branch lengths have different amounts of uncertainty. in particular, the a index will tend to be low if the posterior predictive branch length estimates are similar to the empirical value but have low uncertainty. this would create a scenario with a small branch length deviation but also a low a index. this appears to be the case for the coronaviruses, for which all the clock models appear inadequate according to the a index, but with the uncorrelated lognormal relaxed clock having a small branch length deviation. assessing the adequacy of models in phylogenetics is an important process that can provide information beyond that offered by traditional methods for model selection. although traditional model selection can be used to evaluate the relative statistical fit of a set of candidates, model adequacy provides information about the absolute performance of the model, such that even the best-fitting model can be a poor predictor of the data (gelman et al. ). there have been important developments in model adequacy methods and test statistics in the context of substitution models (ripplinger and sullivan ; brown b; lewis et al. ) and estimates of gene trees (reid et al. ). here we have described a method that can be used for assessment of molecular clock models, and which should be used in combination with approaches for evaluating the adequacy of substitution models. the results of our analyses suggest that our method is able detect whether estimates of branchspecific rates and times are consistent with the expected number of substitutions along each branch. for example, in the coronavirus data set analyzed here, the best-fitting clock model was a poor predictor of the data, as was the substitution model. our index is sensitive to underparameterization of clock models and has the benefit of being computationally efficient. in addition, our metric of uncertainty in posterior predictive branch lengths is sensitive to some cases of misspecification of clock models and node-age priors, but not to substitution model misspecification, as shown for our analyses of the coronavirus data set. analyses based on the random local clock and the data simulated under two local clocks generally produced low accuracy ( fig. a) , with lower a indices than the other models that were matched to the true model ( fig. a) . the substandard performance of the random local clock when it is matched to the true model is surprising. a possible explanation is that our simulations of the local clock represented an extreme scenario in which the rates of the local clocks differed by an order of magnitude. previous studies based on simulations and empirical data demonstrated that this model can be effective when the rate differences are smaller (drummond and suchard ; dornburg et al. ) . in our analyses of empirical data, even the highest values of our index were lower than the minimum value obtained in our analyses of simulated data when the three models matched those used for simulation. this is consistent with the results of previous studies of posterior predictive simulations, which have suggested that the proposed threshold for a test statistic using simulations is conservative for empirical data (bollback ; ripplinger and sullivan ; brown b) . it is difficult to suggest a specific threshold for our index to determine whether a model is inadequate. however, the interpretation is straightforward: a low a index indicates that a large proportion of branch rates and times are inconsistent with the expected number of substitutions along the branches. under ideal conditions, an a index of . or higher means that the clock model accurately describes the true pattern of rate variation. however, our method allows the user to inspect the particular branches with inconsistent estimates, which can be useful for identifying regions of the tree that cause the clock model to be inadequate. measuring the effect size of differences in the branch length estimates of the posterior predictive and empirical data can also be useful for quantifying potential errors in the estimates of node times and branch-specific rates. an important finding of our study is that overparameterized clock models typically have higher accuracy than those that are underparameterized. this is consistent with a statistical phenomenon known as the bias-variance trade-off, with underparameterization leading to high bias, and overparameterization leading to high uncertainty. this was demonstrated for molecular clock models by . although our results show a bias when the model is underparameterized, we did not detect high uncertainty with increasing model complexity. this probably occurs because the models used here are not severely overparameterized. this is consistent with the fact that bayesian analyses are robust to mild overparameterization because estimates are integrated over the uncertainty in additional parameters (huelsenbeck and rannala ; lemmon and moriarty ) . we note that our index is insensitive to the overparameterization in our analyses. this problem is also present in some adequacy statistics for substitution models (bollback ; ripplinger and sullivan ) . identifying an overparameterized model is challenging, but a recent study proposed a method to do this for substitution models (lewis et al. ). an equivalent implementation for clock models would also be valuable. another potential solution is to select a pool of adequate models and to perform model selection using methods that penalize an excess of parameters, such as marginal likelihoods or information criteria. we find that our assessment of clock model adequacy can be influenced by other components of the analysis. for example, multiple calibrations can create a misleading node-age prior that is in conflict with the clock model (warnock et al. ; duchêne et al. ; heled and drummond ) . although our simulations with misleading node calibrations were done using a strict clock, our method identified this scenario as clock model inadequacy when the models for estimation were the strict or random local clocks ( fig. a) . in the case of the uncorrelated lognormal relaxed clock, our method identified a misleading node-age prior as causing an increase in uncertainty ( fig. b ). this highlights the critical importance of selecting and using time calibrations appropriately, and we refer the reader to the comprehensive reviews of assessing the adequacy of clock models . doi: . /molbev/msv mbe this topic (benton and donoghue ; ho and phillips ) . another component of the analysis that can have an impact on the adequacy of the clock model is the tree prior, which can influence the estimates of branch lengths. although one study suggested that the effect of the tree prior is not substantial (lepage et al. ), its influence on divergence-time estimates remains largely unknown. we found that substitution model underparameterization led to a severe reduction in accuracy. overconfidence in incorrect branch lengths in terms of substitutions can cause bias in divergence-time estimates (cutler ) . however, this form of model inadequacy is incorrectly identified by the methods we used for estimation as a form of rate variation among lineages. for our data generated using a strict clock and an underparameterized substitution model, the a index rejected the strict clock and supported the overparameterized uncorrelated lognormal relaxed clock. on the other hand, the multinomial test statistic was sensitive to substitution model underparameterization, and to some forms of rate variation among lineages. the sensitivity of the multinomial likelihood to rate variation among lineages might explain why the substitution model was rejected for the coronavirus data set when using a clock model, but not when using a clock-free method. due to this sensitivity and the substantial impact of substitution model misspecification, we recommend the use of a clock-free method to assess the substitution model before performing analyses using a clock model. our results suggest that it is only advisable to perform a clock model analysis when an adequate substitution model is available. other methods for substitution model assessment that are less conservative than the multinomial likelihood represent an interesting area for further research. we find that the a index is sensitive to patterns of rate variation among lineages that conflict with the clock model used for estimation. this is highlighted in the simulations of rate variation among lineages under autocorrelated and the unusual beta-distributed rates. in these cases, the a index identified the uncorrelated lognormal clock as the only adequate clock model, despite an increase in uncertainty in both cases. although other studies have also suggested that the uncorrelated lognormal relaxed clock can account for rate autocorrelation (drummond et al. ; ho et al. ) , an increase in uncertainty can impair the interpretation of divergence-time estimates. we suggest caution when the uncertainty values are above , which occurs when the widths of the % credible intervals are greater than the mean parameter estimates. in our analyses of the two virus data sets, the multinomial test statistic suggested that the best-fitting substitution model was inadequate. in the analyses of the siv data, our index of clock model adequacy was . , similar to that of killer whales, for which the substitution model appeared adequate. we recommend caution when interpreting estimates of evolutionary rates and timescales when the substitution model is inadequate. this typically suggests that the substitution process is not being modeled correctly, which can affect inferences of branch lengths regardless of whether a clock model is used or not. for this reason, the a index of . for the siv data set might be overconfident compared with the same index obtained for the killer whale data. previous research has also suggested that there are processes in the evolution of siv that are not accounted for by current evolutionary models . we also found that all the clock models were inadequate for the coronavirus sequence data. our results might provide an explanation for the lack of consensus over the evolutionary timescale of these viruses. for example, a study of mammalian and avian coronaviruses estimated that these viruses originated at most , years ago (woo et al. ). this result stands in contrast with a subsequent study that suggested a much deeper origin of these viruses, in the order of millions of years (wertheim et al. ) . our results suggest that estimating the timescale of these viruses might not be feasible with the current clock models. our analysis of mitochondrial genomes of killer whales shows that even if the clock model performance is not as high as that obtained in the simulations that match the models used for estimation, a large proportion of the divergence-time estimates can still be useful. examining the estimates of specific branch lengths can indicate whether many of the node-age estimates are reliable, or whether important branches provide unreliable estimates. we recommend this practice when the substitution model has been deemed adequate and when a substantial proportion of the branch lengths are consistent with the clock model (i.e., when the a index is high). we note that the mitochondrial genomes of killer whales have the lowest a index of any data set when analyzed using a random local clock. this might occur because the model identified an average of - rate changes along the tree ( . rate changes; table ). although rate variation is likely to be higher in this data set, it might not be sufficiently high for the model to detect it. analyses of mitochondrial protein-coding genes from marine turtles identified the substitution model as inadequate using the multinomial test statistic. the clock model with the highest performance had an a index of . , which might be considered sufficient to interpret the divergencetime estimates for at least some portions of the tree. again, the fact that the substitution model is inadequate precludes further interpretation of the estimates of evolutionary rates and timescales. this is a surprising result for a mitochondrial data set with several internal-node calibrations. a potential solution is to assess substitution-model adequacy for individual genes and to conduct the molecular clock analysis using only those genes for which an adequate substitution model is available. we believe that, with the advent of genomic data sets, this will become a feasible strategy in the near future. some of the reasons for the paucity of studies that assess model adequacy in phylogenetics include computational demand and the lack of available methods. in this study, we have presented a method of evaluating clock model adequacy, using a simple test statistic that can be computed efficiently. assessment of clock model adequacy is an important complement to traditional methods of model selection for two primary reasons: it allows the researcher to reject all the available models if they are inadequate; and, as implemented in this study, it can be used to identify the branches with length estimates that are implausible under the assumed model. the results of our analyses of empirical data underscore the importance of evaluating the adequacy of the substitution and clock models. in some cases, several models might be adequate, particularly when they are overparameterized. in this respect, methods for traditional model selection are important tools because they can be used to select a single best-fitting model from a set of adequate models. further research into methods, test statistics, and software for evaluating model adequacy is needed, both to improve the existing models and to identify data sets that will consistently provide unreliable estimates. we generated pure-birth trees with tips and root-node ages of my using beast v . (bouckaert et al. ) . we then simulated branch-specific rates under five clock model treatments using the r package nelsi (ho et al. ) . this program simulates rates under a given model and multiplies rates by time to produce phylogenetic trees in which the branch lengths represent subs/site, known as phylograms. these phylograms were then used to simulate the evolution of dna sequences of , nt in the r package phangorn. the five clock model treatments included the following: ) a strict clock with a rate of  À subs/site/my; ) an uncorrelated lognormal relaxed clock (drummond et al. ) , with a mean rate of  À subs/site/my and a standard deviation of . ; ) a treatment in which a randomly selected clade with at least ten tips experienced an increase in the rate, representing a scenario with two local clocks (yoder and yang ) , with rates of  À and  À subs/ site/my; ) a treatment with rate autocorrelation, with an initial rate of  À subs/site/my and a parameter of . (kishino et al. ) ; and ) a treatment with rate variation that followed a beta distribution with equal shape parameters of . and centered at  À subs/site/my, resulting in a bimodal shape. in every simulation, the mean rate was  À subs/site/my, which is approximately the mean mitochondrial evolutionary rate in mammals, birds, nonavian reptiles, and amphibians (pereira and baker ) . we selected this mean rate instead of sampling from the prior because our estimation methods involved an uninformative rate prior, and random samples from this can produce data sets with high sequence saturation or with low information content. we used the jukes-cantor substitution model for simulation (jukes and cantor ) . this model allows us to avoid making arbitrary parameterizations of more parameter-rich models, which is not the focus of this study. to explore the effect of substitution model underparameterization, we simulated additional data sets under a strict clock and a general time-reversible model with gammadistributed rates among sites, using parameters from empirical data (murphy et al. ) . we analyzed these data sets using the same method as for the rest of the simulated data, including the use of the simpler jukes-cantor substitution model. we also explored the effect of using misleading node-age priors. to do this, we placed two time calibrations with incorrect ages. one calibration was placed in one of the two nodes descending from the root selected at random, with an age prior of . times its true age (i.e., younger than the truth). the other calibration was placed on the most recent node in the other clade descending from the root, with an age of . of the root age (i.e., older than the truth). for this scenario, we only used trees with more than one descendant in each of the two oldest clades. we show an example of the simulated phylogeny compared with this kind of marginal prior on node ages in the supplementary information, supplementary material online. our study had simulated data sets for each simulation treatment, for a total of simulated alignments. we analyzed the simulated alignments using bayesian markov chain monte carlo (mcmc) sampling as implemented in beast. we used three different clock models to analyze each of the simulated alignments: the strict clock, uncorrelated lognormal relaxed clock (drummond et al. ) , and random local clock (drummond and suchard ) . we used the same tree prior and substitution model for estimation as those used for simulation. we fixed the age of the root to my and fixed the tree topology to that used to simulate sequence evolution in every analysis. we analyzed the simulated data with an mcmc chain length of  steps, with samples drawn from the posterior every  steps. we discarded the first % of the samples as burn-in, and assessed satisfactory sampling from the posterior by verifying that effective sample sizes for all parameters were above using the r package coda (plummer et al. ) . we performed analyses using each of the three clock models for each of the simulated data sets, for a total of clock analyses. we assessed the accuracy and uncertainty of the estimates made using each of the analysis schemes ( fig. ) . to do this, we compared the simulated rates with the branch-specific rates in the posterior. next, we tested the power of our method for assessing clock model adequacy using the simulated data under each of the scenarios of simulation and analysis. we provide example code and results in a public repository in github (https://github.com/duchene/modadclocks, last accessed july , ). we also tested the power of the multinomial test statistic to assess clock model adequacy in each of the analyses. this test statistic quantifies the frequency of site patterns in an alignment and is appropriate for testing the adequacy of models of nucleotide substitution (bollback ; brown b ). we used four published data sets to investigate the performance of our method of assessing clock 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and dolphins (mammalia: cetacea) relaxed phylogenetics and dating with confidence bayesian coalescent inference of past population dynamics from molecular sequences bayesian random local clocks, or one rate to rule them all marine turtle mitogenome phylogenetics and evolution the impact of calibration and clock-model choice on molecular estimates of divergence times efficient selection of brach specific models of sequence evolution statistical inference of phylogenies a tenth crucial question regarding model use in phylogenetics bayesian data analysis model checking and model improvement simulating normalizing constants: from importance sampling to bridge sampling to path sampling philosophy and the practice of bayesian statistics statistical tests of models of dna substitution a dirichlet process prior for estimating lineage-specific substitution rates calibrated birth-death phylogenetic timetree priors for bayesian inference the changing face of the molecular evolutionary clock molecular-clock methods for estimating evolutionary rates and timescales simulating and detecting autocorrelation of molecular evolutionary rates among lineages accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times frequentist properties of bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models bayesian inference of phylogeny and its impact on evolutionary biology evolution of protein molecules performance of a divergence time estimation method under a probabilistic model of rate evolution molecular clocks: four decades of evolution computing bayes factors using thermodynamic integration the importance of proper model assumption in bayesian phylogenetics a general comparison of relaxed molecular clock models posterior predictive bayesian phylogenetic model selection complete mitochondrial genome phylogeographic analysis of killer whales (orcinus orca) indicates multiple species resolution of the early placental mammal radiation using bayesian phylogenetics mapping mutations on phylogenies bayesian phylogenetic analysis of combined data a mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock coda: convergence diagnosis and output analysis for poor fit to the multispecies coalescent is widely detectable in empirical data assessment of substitution model adequacy using frequentist and bayesian methods computational methods for evaluating phylogenetic models of coding sequence evolution with dependence between codons mrbayes . : efficient bayesian phylogenetic inference and model choice across a large model space phangorn: phylogenetic analysis in r model selection in phylogenetics estimating the rate of evolution of the rate of molecular evolution exploring uncertainty in the calibration of the molecular clock a case for the ancient origin of coronaviruses relaxed molecular clocks, the bias-variance trade-off, and the quality of phylogenetic inference dating the age of the siv lineages that gave rise to hiv- and hiv- discovery of seven novel mammalian and avian coronaviruses in deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus improving marginal likelihood estimation for bayesian phylogenetic model selection molecular phylogenetics: principles and practice estimation of primate speciation dates using local molecular clocks we thank the editor, tracy heath, and an anonymous reviewer for suggestions and insights that helped improve this article. this research was undertaken with the assistance of resources from the national computational infrastructure, which is supported by the australian government. d. assessing the adequacy of clock models . doi: . /molbev/msv mbe for each analysis of the empirical data sets, we ran the mcmc chain for steps, with samples drawn from the posterior every steps. we discarded the first % of the samples as burn-in and assessed satisfactory sampling from the posterior by verifying that the effective sample sizes for all parameters were above using the r package coda. we used stepping-stone sampling to estimate the marginal likelihood of the clock model (gelman and meng ; lartillot and philippe ; xie et al. ) . for each bayesian analysis, we performed posterior predictive simulations as done for the simulated data sets, and assessed the substitution model using the multinomial test statistic. in addition, to estimate the clock-free multinomial test statistic, we analyzed each of the empirical data sets using mrbayes . (ronquist et al. ) . for these analyses we used the same chain length, sampling frequency, sampling verification method, and substitution model as in the analyses using clock models.our empirical data sets included nucleotide sequences of coronaviruses. this data set contained sequences of nt of a portion of the m (matrix) gene, as used by wertheim et al. ( ) . these sequences were sampled between and . the best-fitting substitution model for this data set was gtr+g. we also used a data set of the gag gene of sivs, which comprised sequences of nt, sampled between and (wertheim and worobey ). the best-fitting substitution model for this data set was gtr+g. we used the bayesian skyline demographic model (drummond et al. ) for the analyses of both of the virus data sets, and used the sampling times for calibration.we analyzed a data set of the killer whale (o. orca), which contained complete mitochondrial genome sequences of , nt (morin et al. ) . we calibrated the age of the root using a normal distribution with mean of . and a standard deviation of % of the mean, as used in the original study. the best-fitting substitution model for this data set was hky+g. finally, we analyzed a data set of several genera of marine turtles, which comprised sequences of the mitochondrial protein-coding genes (duchene et al. ) , and we selected the gtr+g substitution model. following the scheme in the original study, we used calibrations at four internal nodes. the pure-birth process was used to generate the tree prior in the analyses of the killer whales and the marine turtles. supplementary information is available at molecular biology and evolution online (http://www.mbe.oxfordjournals.org/). key: cord- - oes g k authors: botta, giorgia; bizzarri, bruno mattia; garozzo, adriana; timpanaro, rossella; bisignano, benedetta; amatore, donatella; palamara, anna teresa; nencioni, lucia; saladino, raffaele title: carbon nanotubes supported tyrosinase in the synthesis of lipophilic hydroxytyrosol and dihydrocaffeoyl catechols with antiviral activity against dna and rna viruses date: - - journal: bioorg med chem doi: . /j.bmc. . . sha: doc_id: cord_uid: oes g k hydroxytyrosol and dihydrocaffeoyl catechols with lipophilic properties have been synthesized in high yield using tyrosinase immobilized on multi-walled carbon nanotubes by the layer-by-layer technique. all synthesized catechols were evaluated against a large panel of dna and rna viruses, including poliovirus type , echovirus type , herpes simplex virus type (hsv- ), herpes simplex virus type (hsv- ), coxsackievirus type b (cox b ), adenovirus type and type and cytomegalovirus (cmv). a significant antiviral activity was observed in the inhibition of hsv- , hsv- , cox b and cmv. the mechanism of action of the most active dihydrocaffeoyl derivative was investigated against a model of hsv- infection. catechol derivatives show different biological properties, including inhibition of hypoxia inducible factor-prolyl hydroxylase- (hph) in the treatment of colitis, antiepileptogenic, pulmonary fibrosis, anticancer, - antimicrobial, and anti-parkinson activities. moreover, they are active against viruses, as in the case of rhinovirus, hiv- integrase, , hiv- reverse transcriptase, and coronavirus. the biological activity of catechols is associated to their antioxidant property, that is the capacity of transferring single-electron and/or hydrogen-atom to reactive free radicals, [ ] [ ] [ ] as well as, to binding pro-oxidant metal ions. the antioxidant activity can be oriented toward specific cellular compartments by controlling the chemical and physical properties of the substituents on the aromatic ring. for example, the limited accessibility of highly hydrophilic catechols to specific intracellular targets has been improved by the synthesis of lipophilic derivatives possessing long carbon alkyl side chains. [ ] [ ] [ ] in the case of bioactive hydroxytyrosol and dihydrocaffeic acid derivatives, , which are characterized by the concomitant presence of alcoholic and ortho-diphenol groups, , the side-chain functionalization requires expensive and tedious protection/deprotection sequences. as an alternative, we described the synthesis of lipophilic catechols by selective oxidation of side-chain functionalized phenol derivatives, using tyrosinase supported on eupergit c l resin (tyr/ecm), by sequential deposition of alternatively charged poly(allylamine hydrochloride) (pah) and polystyrene sulfonate (pss). tyrosinase is a copper enzyme which catalyzes the hydroxylation of monophenols to ortho-diphenols and ortho-quinones using dioxygen (o ) as primary oxidant. in this latter case, dihydrocaffeoyl catechols showed antiviral activity against influenza a virus, an infection that continue to represent a severe threat wordwide. derivatives characterized by antioxidant activity and longer carbon alkyl side-chains were more effective, suggesting the possibility of novel inhibition mechanisms based on both redox and lipophilic properties. these data were in accordance with previous findings about the activity of lipophilic glutathione (gsh) derivatives, which were able to enter into the cell more easily than gsh, thus inhibiting the replication of dna and rna viruses. the efficacy of tyrosinase in the synthesis of simple catechol derivatives was successively increased by immobilization of the enzyme on multi-walled carbonanotubes (mwcnts), using the layer-by-layer (lbl) procedure. the lbl procedure is based on the consecutive deposition of alternatively charged polyelectrolytes onto the active species, able to protect proteins from high-molecular-weight denaturing agents. mwcnts are characterized by high surface area for enzyme loading, as well as biocompatibility, and favorable electrochemical and mechanical properties. , the best catalyst (indicated in the follow as mwcnt/tyr) was obtained by co-immobilization of tyr and bovine serum albumin (bsa) on oxidized mwcnts, in the presence of polydiallyldimethyl ammonium chloride (pdda). here we report the use of mwcnt/tyr for the improved synthesis of lipophilic hydroxytyrosol and dihydrocaffeoyl catechols, and their antiviral activity against a large panel of dna and rna viruses, including poliovirus type , echovirus type , herpes simplex virus type (hsv- ) and type (hsv- ), coxsackie virus type b (cox b ), adenovirus type and type , and cytomegalovirus (cmv). some of these compounds were endowed with antiviral activity at sub-toxic concentrations. the mechanism of action of the most active dihydrocaffeoyl derivative was investigated in detail against a model of hsv- infection. the mwcnt/tyr was prepared as previously reported. briefly, mushroom tyr from agaricus bisporus and bsa (bsa/tyr ratio : ) were immobilized on oxidized mwcnts, by deposition of a layer of pdda (mwcnts-pdda/tyr ratio of : ) in sodium phosphate buffer (pbs; . m, ph ) at room temperature ( fig. , step a) . the excess pdda was removed by centrifugation/re-dispersion cycles since residual pdda in the solution can precipitate upon mixing with enzyme. the concentration of adsorbed pdda was evaluated by uv-vis analysis (at nm) of the supernatant after treatment with comassie brillant blue (cbb) (experimental details and the calibration curve for pdda are in supplementary information, si # , table a , fig. ). the amount of pdda adsorbed per mg of starting material was found to be . ± . mg. this amount is similar to the 'just enough' amount of pdda ( . mg) required to cover all the surface of mwcnts ( % covering efficacy), as previously reported. the presence of cationic pdda facilitates the loading of tyr, that is negatively charged at the operative ph . (isoelectric point of tyr . - . ). bovine serum albumin (bsa), an inert enzyme characterized by an isoelectric point close to tyr, was used during the co-immobilization procedure to reduce the surface area available to the enzyme, avoiding undesired conformational changes due to enzyme strives for the greatest surface coverage. glutaraldehyde (ga) was then added to increase the reticulation grade and stability of the catalyst (fig. , step b ). crosslinking with glutaraldehyde is a widely applied procedure for the immobilization/reticulation of enzymes on different kind of support. in accordance with data reported in the literature, the effectiveness of the crosslinking procedure was confirmed by ft-ir analysis, observing the appearance of a new absorption band in the range of - cm À attributable to the formation of schiff bases (representative ft-ir spectra of mwcnt/tyr with or without ga are in supplementary information, si # , figs. and ). the activity of native tyr ( . u/mg) was determined by the dopachrome assay following the oxidation of l-tyrosine at nm (the enzyme unit is defined as the increase in absorbance of À unit/min at °c in . m phosphate buffer, ph . ). the activity of mwcnt/tyr ( . u/mg), and the value of activity parameters, namely immobilization yield ( %), and activity yield ( . %), were in accordance with our previous data (for the definition of these parameters see in the experimental part). the scanning electron microscopy (sem) and atomic force microscopy (afm) of mwcnt/tyr were in accordance with data previously reported, and confirmed the polyelectrolyte deposition and enzyme immobilization. in particular, the afm analysis clearly showed functionalized nanotubes characterized by a smooth surface probably due to the presence of bsa in the interstitial sites between tyr molecules. the maximum height and width were higher than that observed for mwcnts alone (experimental procedures, sem analysis, and magnified details of the bi-dimensional high resolution afm images of mwcnt/ty and mwcnts with the corresponding profiles, are in supplementary information si # , figs. and ). kinetic parameters for native tyr and mwcnt/tyr were evaluated in ch cl and pbs, using tyrosol acetate a as a selected substrate to be oxidized (see next). the initial reaction rates were measured at substrate concentrations ranging from . to mm at °c. kinetic constants were evaluated by using different linear regression equations (lineweaver-burk, hanes and eadie-hofstee). the kinetic parameters (k m , v max , and k cat ) for the native enzyme and mwcnt/tyr are reported in table . mwcnt/tyr shows a performance about two times lower than the native enzyme, in agreement with the expected decrease of catalytic efficiency after immobilization. the catalytic efficacy of mwcnt/tyr (defined as v max /k m ) was found to be . - (as the average value of the three tests). the ester derivatives of commercially available ( -hydroxyphenyl)propanoic acid and tyrosol ( -hydroxyphenylethyl alcohol) were used as substrates. in particular, the ester derivatives a-d (scheme ) were prepared by reaction of with an excess of the appropriate alcohol in the presence of trimethylchlorosilane (tmcs) at °c (experimental details are in supporting si # ). the esterification of to yield compounds a-d (scheme ) was carried-out using lipase from candida antarctica to avoid the possible formation of mixture of esters, due to competition between alcoholic and phenol groups (supporting si # ). the oxidation of compounds and a-d ( . mmol) was performed with mwcnt/tyr ( u) in ch cl /buffer (na phosphate . m ph , ch cl /buffer ratio . : . ) at °c under o atmosphere for h (scheme , table ). the use of ch cl as reaction solvent was necessary to increase the solubility of hydrophobic substrates, in accordance with improved stability and selectivity (with limited formation of ortho-benzoquinones and polymeric side-products) reported for tyr in organic solvents. the oxidation of and a (as selected samples) was also performed using previously reported tyr/ecm as reference, in which tyr is covalently immobilized on micro-spheres of the synthetic resin eupergit c l (methacrylamide, n,n -methylenbis-acrylamide polymer). mwcnt/tyr selectively afforded catechol derivatives and a-d in quantitative conversion of substrate and yield of product (table ). in these reactions, mwcnt/tyr showed a reactivity similar to tyr, and higher than tyr/ecm ( table , entry versus entries and , and entry versus entries and ). in this latter case u were required to obtain the quantitative yield of products (table , entries , and ). these data further confirm the benign role of mwcnt as nanosized supports for the immobilization of tyr in synthetic applications. in a similar way, the oxidation of tyrosol esters a-d afforded the corresponding lipophilic catechols a-d (scheme ) in quantitative conversion of substrate and yield of product ( table , entries [ ] [ ] [ ] [ ] [ ] . again, mwcnt/tyr performed better than tyr/ecm ( table , entry vs entry ). recycling experiments proceeded with success in the oxidation of compound under similar experimental conditions. after the first oxidation, mwcnt/tyr was recovered by centrifugation, washed, and reused with fresh substrate. as shown in table , mwcnt/tyr was used for at least six cycles with only a slight decrease of efficiency to give ( table , entry vs entry ). note that mwcnt/tyr was more stable than tyr/ecm, for which a significant decrease in the activity was observed (ca. %; table , entry vs entry ). the development of antiviral agents has progressed rapidly over the last decades. the drugs approved for use generally inhibit specific steps of viral replication usually by targeting viral proteins with an enzymatic function. unfortunately, a large majority of the available antiviral drugs are limited in their efficacy by systemic toxicity and drug resistance. many different circumstances favor the selection of drug resistant strains, for example, high viral loads, intrinsic mutation rates, and prolonged exposure to the drug. for this reason, natural or de-novo designed molecules able to inhibit the replication of dna or rna viruses with different mechanisms have been synthesized. derivatives b, d, a and b were characterized by selective antiviral activity. in particular, compounds b and a demonstrated the highest level of activity against hsv- (dna virus), with id values of and lg/ml, respectively. moreover, the same compounds showed slight activity against hsv- ( and lg/ml, respectively) and cox b (rna virus; and lg/ml, respectively). with regard to b, a modest activity against hsv- was observed, probably due to toxic effect on cell monolayer, as suggested by the low value of cd ( lg/ml). finally, b and d were effective against cmv (dna virus), d being the most active compound with an id value of lg/ml. since b was effective against several rna or dna viruses, especially in the case of herpetic viruses (hsv- , hsv- and cmv), its mechanism of action was investigated in more detail using a model of hsv- infection. in particular, compound b was added at different times on vero cells infected with . moi of hsv- , to determine the inhibition of the virus yield during specific periods in the virus life-cycle. the results clearly demonstrate that b interferes with an early step of the viral replicative cycle. indeed, the viral replication was blocked during the first hour of infection. otherwise, no reduction was observed when b was added after h. moreover, a slight reduction of virus yield was observed during the adsorption period (fig. ) . as b exerted its activity through the inhibition of the early events in hsv- replication, we set up some experiments in order to deepen its mechanism of action. first, the effect of the compound was studied during the viral adsorption period by means of the infective center assay. results obtained from this experiment demonstrated that b did not significantly inhibit adsorption of hsv- at concentrations higher than times the id (fig. ) . furthermore, it was important to establish if any virucidal effect or protective actions for vero cells was produced. our results demonstrated that b was not virucidal for hsv- and did not exerted any protective action for the cells, thus suggesting that the reduction in the virus yield, immediately after the adsorption period, could be related to the interference of the compound with penetration, un-coating and/or another early step of hsv- replication. mwcnts/tyr was an efficient catalyst for the synthesis of tyrosol and dihydrocaffeoyl catechols by oxidation of phenol ester derivatives under mild experimental conditions, in quantitative conversion of substrate and yield. the activity of mwcnt/tyr was comparable to native tyr and greater than previously reported tyr/e- catalyst, confirming the benign role of carbon nanotubes in the enzyme immobilization process. mwcnt/tyr was a stable catalyst for at least six recycle experiments. among lipophilic catechols, compounds b, d, a and b were active against hsv- , hsv- , cox b and cmv. in a previous study, compounds a and b were also active against influenza a/pr /h n virus, showing the highest antioxidant activity as measured by the , -diphenyilpicrylhydrazyl (dpph) radical scavenging assay. in the case of the inhibition of hsv- and hsv- viruses, the highest antiviral activity prevailed in derivatives characterized by a low/medium long alkyl side-chain. the mechanism of action of compound b was studied in detail using a model of hsv- infection. data showed that the inhibition of virus replication was effective in earlier stages of the replication cycle, probably related to penetration, un-coating and/or another early step of hsv- replication. mushroom tyrosinase from agaricus bisporus (tyr), multiwalled carbon nanotubes (mwcnts), l-tyrosine, bovine serum albumin (bsa), glutaraldehyde (ga), polydiallyldimethylammonium chloride (pdda), sodium sulfate anhydrous (na so ), ( -hydroxyphenyl)propanoic acid ,,tyrosol ( -hydroxy phenyl ethyl alcohol) , alcohols and organic solvents were purchased from sigma-aldrich. all spectrophotometric measurements were made with a varian cary uv-vis spectrophotometer equipped with a peltier thermostatted single cell holder. ft-ir measurements were performed using a perkin elmer ft-ir spectrometer in kbr. all samples were dried before the analysis. h nmr and c nmr spectra were recorded on a bruker ( mhz) spectrometer. mass spectra were recorded on a vg / s spectrometer with an electron beam of ev. dichloromethane (ch cl ) was dried on anhydrous sodium sulfate prior to use. all experiments were done in triplicate using native and immobilized tyrosinase in dichloromethane/buffer system and in h o medium. sodium phosphate [(pbs) . m, ph . ] was used as the buffer solution. mwcnt/tyr was prepared as previously reported. briefly, pdda coated mwcnts in pbs were treated with a mixture of tyr ( . mg) and bsa ( . mg) for min. glutaraldehyde (ga, . %) was added to reach a final volume of ll and the mixture was shaken at °c for min and at °c overnight. the excess enzyme and ga were removed by centrifugation ( rpm  min) and the surnatant was used for the calculation of activity parameters. the catalyst was finally treated with . ml tris-hcl . m ph . by shaking for h at °c and centrifuged. mwcnt/tyr was washed several times with pbs in order to ensure the complete removal of unbounded tyr (as evaluated by the bradford method). the activity of native and immobilized tyr was determined by measuring the oxidation of l-tyrosine. the reaction was started by adding l-tyrosine to the solution of tyr or mwcnt/tyr in pbs under magnetic stirring. the initial rates were measured as linear increase in optical density at nm, due to dopachrome formation. one unit of enzyme activity was defined as the increase in absorbance of . per minute at ph , °c in a . ml reaction cuvette containing . mm of l-tyrosine and mm of pbs ph . . the activity was expressed as activity unit per milligram of support: activity ðu=mgÞ ¼ ux=wsupport where: ux is the activity of the immobilized enzyme assayed by dopachrome method. the activity yield represents the % of the ratio of activity of the immobilized enzyme to the total units of native enzyme used: activity yield ð%Þ ¼ ½ux=ðua À urÞ Á where: ua is the total activity of enzyme (unit) added in the solution and ur is the activity of the residual tyr (unit) evaluated by dopachrome method in the washing solutions. the immobilization yield is defined as: immobilization yield ð%Þ ¼ ½ðua À urÞ=ua Á : the catalytic properties of native and immobilized tyrosinase were determined in the organic solvent media by measuring initial rates of the reaction with the substrate at °c. the reactions were carried out by using different concentrations of tyrosol acetate a, ranging from to mm. the appropriate amount of a was dissolved in dcm ( . ml) and free or immobilized tyrosinase in the optimum aqueous amount ( ll of pbs) was added. the reaction mixture was stirred for min. sampling was performed every . min, the absorbance at nm was measured and the sample returned to the flask as rapidly as possible. one unit of enzyme activity was defined as the increase in absorbance of . at nm, °c, ch cl and pbs . m, ph . . k m and v max values were calculated by plotting data in lineweaver-burk, hanes and eadie-hofstee. mwcnt/tyr ( u) was added to a solution of the appropriate substrate ( . mmol) in ch cl ( . ml) in pbs ( ll), and the mixture was stirred at °c under o . after h, the catalyst was recovered by centrifugation and the organic fraction was concentrated and treated with a solution of sodium dithionite in thf and h o [ : (v/v)]. the mixture was stirred at °c for min to allow the complete reduction of benzoquinones to catechols and extracted twice with ethyl acetate (etoac; . ml  ). the collected organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to yield catechol derivatives , a-d and a-d. all experiments were conducted in triplicate. the structure of catechol derivatives was characterized without further purification by comparison with data previously reported in the literature. oil; oil oil; oil; vr- ) was propagated in human foreskin fibroblast cell (hff- : scrc- ). viruses and cells were purchased from the american type culture collection (atcc). cell lines were kept at °c in a humidified atmosphere with % co and grown in dulbecco's modified eagle's minimum essential medium (dmem) supplemented with % heat inactivated fetal calf serum (fcs), mm l-glutamine, . % sodium bicarbonate, lg ml - of streptomycin and units ml - of penicillin g. working stocks of all viruses were prepared as cellular lysates using dmem with % heat inactivated fcs (maintenance medium). the compounds were initially dissolved in dimethyl sulfoxide (dmso) and further diluted in maintenance medium before use to achieve the final concentration needed. the final dilution of test compounds contained a maximum concentration of . % dmso, which was not toxic to our cell lines. acyclovir was used as the reference compounds. the cytotoxicity of the test compounds was evaluated by measuring their effect on cell morphology and growth. cell monolayers were prepared in -well tissue culture plates and exposed to various concentrations (lg/ml) of the compounds. plates were checked by light microscopy after , , and h. cytotoxicity was scored as morphological alterations (e.g., rounding up, shrinking, and detachment). cell growth was determined by the -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide (mtt) method. the cells were seeded at  /ml ( ll/well) in -well tissue culture plates such that cell replication remained logarithmic all along the -day incubation period. the % cytotoxic dose (cd ) was expressed as the highest concentration of the compound that resulted in % inhibition of cell growth. the assay of the antiviral activity against all the viruses tested was carried out by the % plaque reduction assay or by % virus-induced cytopathogenicity, as previously described. [ ] [ ] [ ] the compound concentration required to inhibit virus plaque formation or virus-induced cytopathogenicity by % is expressed as the % effective concentration (id ) and calculated by dose-response curves and linear regression. monolayers of cells were grown to confluence in -well plates and inoculated with viruses at a moi (multiplicity of infection) of . . the plates were incubated for h at °c to ensure synchronous replication of the viruses, with or without compound r for the adsorption period. then, the inoculum was removed and fresh medium, with or without the compound, was added at various times after the adsorption period. the plates were incubated at °c for h, then cultures were frozen and virus yield was determined by plaque assay. infective center assay was used to study the effect of compound b on the virus adsorption step. a vero cell suspension ( cells/ml) was cooled to °c for at least h. hsv- ( pfu/ml), incubated for min at °c with different concentrations of test compound, was cooled to °c, and subsequently added to the cell suspension. cells were incubated with the virusdrug mixtures for min at °c to prevent the virus entering the cells. after the adsorption period, un-adsorbed virus and free compound were removed by washing three times with cold dmem. the cells were then diluted serially and plaque assayed for cell-associated viral activity. pre-treatment of cultures was performed by exposing the cell monolayers to different concentrations of the test compound in maintenance medium for and days at °c. after treatment the cell monolayers were washed thoroughly with pbs and infected with hsv- at a moi of . to allow viral cytopathic activity. the cell monolayers grown in maintenance medium without the test compounds were used as control. virus titration was performed as described above. to test possible virucidal activity, equal volumes ( . ml) of viral suspension (containing pfu/ml) and dmem containing compound b (  the id ) were mixed and incubated for h at °c. infectivity was determined by plaque assay after dilution of the virus below the inhibitory concentration. alarcón-de-la-lastra supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.bmc. . . . key: cord- - wnmdvg authors: nan title: p – p date: - - journal: clin microbiol infect doi: . /j. - . . _ _ .x sha: doc_id: cord_uid: wnmdvg nan resistance rates (rr) over a period of years were generally better than those reported for a total of icus. the mean mrsa rr was . %, for all the sari icus, whereas it was only . % in the study icu. by the end of duration of treatment for pneumonia had been reduced to - days and written guidelines on empiric antibiotic treatment and prophylaxis were revised with respect to the resistance situation of the study icu. the significant decrease between and in total antimicrobial ad from , to in the study icu resulted mainly from the reduced consumption of nd generation cephalosporins, carbapenems and imidazoles. ni did not change significantly over time. compared to the year , the costs for antibiotics were halved from € , to , , which corresponds to € . /pd and € . /pd, respectively. the percentage of antibiotics in the total icu budget for pharmaceuticals decreased from . % to . %. conclusion: surveillance and feedback of antibiotic use and resistance can serve as a valuable quality control instrument and can have an impact on antibiotic treatment. from to , antibiotic use was reduced by % and costs for antibiotics/pd were cut by two third in the icu study without any increase in device associated nosocomial infection rates. the resistance situation was generally better than in all sari icus, but showed heavy fluctuations. similar illness burden but different antibiotic prescription to children: a population-based study k. hedin, m. andre, a. håkansson, n. rodhe, s. mö lstad, c. petersson (växjö, falun, malmö, linköping, se) objectives: respiratory tract infections are the most common reason for antibiotic prescription in sweden as in other countries. the prescription rates vary markedly in different countries, counties and municipalities. the reasons for these variations in prescription rates are not obvious. the aim of the study was to find possible explanations for different antibiotic prescription rates in children. therefore a prospective population based log book study was conducted in four municipalities which, according to official statistics, had high and three municipalities which had low antibiotic prescription rates. methods: during one month, parents recorded all infectious symptoms, physician consultations and antibiotic treatments, from -month-old children in a log book. the children's parents also answered a questionnaire about socioeconomic factors and concern about infectious illness. results: antibiotics were prescribed to . % of the children in the high prescription area and . % in the low prescription area (crude or . ( % ). after multiple logistic regression analyses taking account of socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations, differences in antibiotic prescription rates remained (adjusted or . ( %ci . - . )). the variable that impacted most on antibiotic prescription rates although it was not relevant to the geographical differences was a high level of concern about infectious illness in the family. conclusion: the differences in antibiotic prescription rates could not be explained by socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations. the differences may be attributable to different prescription customs, in which case physicianś prescription patterns are not always rational. decreasing outpatient antibiotic prescribing in germany, germany, - , does not include newer macrolides, fluoroquinolones and extendedspectrum beta-lactams w.v. kern, k. de with, k. nink, h. schrö der (freiburg, bonn, de) objective: the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project has shown that outpatient antibiotic prescribing in germany has been comparatively low among european countries. we assessed trends over time and regional variation of outpatient antibiotic use in germany, and wondered if the observable decreasing trend included all drug classes to a similar extent. methods: prescription data (compulsory health insurance covering > % of the population, sample of . % until the year , all prescriptions thereafter) were analysed using the atc/who methodology and current ddd definitions. we specifically defined the following drug groups: ''basic'' penicillins (bpens, oral penicillin or aminopenicillins), extended-spectrum betalactams (esbls, oral cephalosporins, staphylococcal penicillins, aminopenicillin/betalactamse inhibitor combinations, parenteral cephalosporins and broadspectrum betalactams), newer macrolides (nmls, roxithromycin, clarithromycin, azithromycin) versus older macrolides (omls). quinolones (fqs), folate synthesis inhibitors (t/ss) and tetracyclines (tets) were also assessed. data were expressed in yearly ddd/ persons covered by the insurance (ddd/ ). findings: outpatient prescribing in was ddd/ (corresponding to . did = ddd/ and day) and decreased to ddd/ in the year and to ddd/ in . the decreasing trend over the last years was observed in all regions. the decrease was most significant for omls () %), t/ss () %), tets () %), and bpens () %) while there was no decreasing use of esbls (± %) and increases in the rate of prescribing nmls (+ %) and fqs (+ %). tets and bpens, however remained the most prescribed antibiotics in . regional variations in remained large for bpens (> -fold) with very low prescribing rates in the eastern region, but were small for t/ss, nmls and fqs (< -fold). conclusions: over a decade we observed a % decreasing outpatient antibiotic prescribing that included relevant antibiotic drug classes except esbls, nmls and fqs. the relative increase was most significant for fqs. severe community-acquired pneumonia admitted to the intensive care unit: impact of antibiotic therapy delay on hospital mortality antibiotic therapy were enrolled in the study. pts were divided in groups according to time to treatment (< h gi, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . baseline severity scores (apache ii, sofa, psi, curb- ), microbiological documentation, and hospital outcome were compared for all groups. results: pts were included in the study. microbiological documentation was achieved in % of all pts, positive blood cultures in / ( . %), s. pneumoniae in / ( %). mean age was ± , apache ii . ± . , sofa . ± . , psi ± , curb- . ± , mechanical ventilation in . % and vasopressors use in . %. overall icu and hospital all-cause mortality were . % and . %, respectively. baseline severity scores were comparable in all groups and their respective hospital mortality is provided in table . conclusions: in severe cap, treated with a combination therapy, time to treatment seems to have an impact on hospital all-cause mortality. based on our results, antibiotic treatment should be initiated within the first hours after hospital admission. (period ii) -cefoperazone/ sulbactam ( g a day) as monotherapy were used as the empirical antibacterial therapy of vap. the rotation from cefepime to cefoperazone/sulbactam was performed due to our previous study demonstrated high frequency of esbl producers among enterobacteriaceae. the samples from the lower respiratory tract were obtained by mini-bal. the sensitivity of microorganisms to the antibiotics studied (ceftazidime, cefepime, cefoperazone/sulbactam and carbapenems) was determined by the disk diffusion method. results: the main pathogens of vap were s. aureus ( %), p. aeruginosa ( %), enterobacteriaceae ( %) and this structure did not changed during both periods. the antibiotic sensitivity of p. aeruginosa and enterobacteriaceae (k. pneumoniae and e. coli), was studied separately. a high level of resistance of enterobacteriaceae to cefepime can be explained by the strains prevailing in the given icu, which produced extended spectrum beta-lactamases (ctx-m). the resistance of enterobacteriaceae to cefepime was . % in i period and . % in ii period, to ceftazidime - . and %, meropenem - and %, imipenem - . and . %, cefoperazone/sulbactam - . and . %, respectively. a change of cefepime for cefoperazone/sulbactam was not followed by any decrease of enterobacteriaceae resistance level to cefepime during ii period. the resistance level of p. aeruginosa to cefepime was . % in i period and . % in ii period, to ceftazidime - . and . %, meropenem - . and . %, imipenem - . and . %, cefoperazone/ sulbactam - . and . %, respectively. conclusion: the exclusion of cefepime for months didn't improved the sensitivity of enterobacteriaceae to this medication. the level of resistance of p. aeruginosa and enterobacteriaceae to cefoperazone/sulbactam did not increased despite a wide use of this antibiotic during months. antibiotic consumption in german acute care hospitals m. steib-bauert, k. de with, e. meyer, p. straach, w.v. kern (freiburg, frankfurt, de) objective: outpatient antibiotic use in germany differs substantially between eastern and southern parts of the country (relatively low use) and western part (relatively high use). there is no nationwide estimate of hospital antibiotic use and its geographic variation if any. the aim of the present study was to provide an estimate of recent hospital antibiotic use density in germany and to identify basic unit/hospital characteristics associated with excess use. methods: data on hospital consumption of systemic antibiotics in anatomical therapeutic chemical (atc) class j were obtained from a convenience sample of acute care hospitals in germany that participated in an ims survey in the year and had complete data (dispensed drugs and patient-days per year) for at least one non-paediatric, non-psychiatric department or ward. a total of non-icu surgical departments/wards, non-icu non-surgical (general medicine, oncologyhaematology, neurology/stroke) departments/wards, and icus covering > million patient-days were analysed. data were expressed in ddd (who/atc definition version ) or ''prescribed/recommended daily doses'' (pdd, better reflecting [high] dosages given to hospitalized patients) per patient days (ddd/ and pdd/ ). findings: the weighted mean over all departments/wards incl. icus was . ddd/ ( . pdd/ ). as expected, icu antibiotic use density was much higher than use in non-icu areas, and use in haematology-oncology was higher than in other non-surgical departments/wards. in univariate analyses, bed-size category and university affiliation (icus, surgical wards), region (icu, surgical and non-surgical wards) and haematology-oncology as specialty (non-surgical wards) were associated with use density, but these associations were only partly confirmed in multivariate logistic regression analyses of factors associated with excess ( ‡ %) use density which showed university affiliation and haematology-oncology to be independently associated with high use. conclusions: based on this hospital sample, antibiotic use in german hospitals shows little, non-significant regional variation and appears to be similar to what has been described from other european countries. adjustment of the data at least for university affiliation and haematology-oncology is important in comparative analyses of hospital antibiotic consumption. impact of formulary change in medical intensive care unit on outcome of infection and antimicrobial resistance sought to evaluate a formulary change and impact it has on infection and resistant. methods: prospectively, all patients in a -bed icu were followed for a period of months in phase i ( patients per patient days) and to collect baseline data after a decrease in the use of piperacillin-tazobactam (pt) when substituted by cefepime for a period of months in phase ii ( patients per patient days). results: total infections in phase i vs. phase ii were lower respiratory tract (lrti) patients ( %) vs. patients ( %); urinary tract infection (uti) patients ( %) vs. patients ( %); and sepsis of undetermined aetiology patients ( %) vs. patients ( %), respectively. there were no significant differences in death ( % vs. %) , cure or improvement of infection ( % vs. %), readmission to the unit ( . % vs. . %), hospital risk of death ( . % vs. . %), mean length of icu stay ( . days vs. . days), or rates of nosocomial infection ( . % vs. . % for lrti; . % vs. . % for uti; . % vs. . % for soft tissue infection; . % vs. . % for bacteremia; . vs. . per patient days for intravenous catheter infection) in phase i and ii respectively. the cost of antimicrobial acquisition in phase i and ii were $ and $ per patient respectively (p < . ). the mean antimicrobial treatment costs per patient for pt were $ vs. $ and cefepime were $ vs. $ in phase i and ii respectively (p < . ). the in vitro susceptibility and rate of infection and colonization with escherichia coli were unchanged in both study periods. there were vs. staphylococcus aureus (p < . ); of these percnt vs. percnt were methicillinresistant s. aureus and vs. enterococcus faecium ( % vs. % vancomycin-resistant enterococci) in phase i and ii respectively. there were % vs. % pseudomonas aeruginosa and % vs. % klebsiella pneumoniae extended spectrum beta lactamases in phase i and ii respectively. conclusion: the implementation of formulary substitution of pt to cefepime in the medical icu had resulted in a decrease in the use of pt. in addition, there were decreased costs and less s. aureus infections without adversely affecting the outcome of infection or antimicrobial resistance. intravenous antibiotic use in scottish hospitals; evaluation of the glasgow antimicrobial audit tool r.a. seaton, d. nathwani, p. burton, e. douglas (glasgow, dundee, uk) introduction: there are few data on antibiotic prescribing within scottish hospitals and a coordinated multisite point prevalence survey had not been performed before. there is concern that antimicrobials are overused in hospitals. methods: antibiotic use in acute medical and surgical units in scottish hospitals across trusts, was investigated using a point prevalence survey. data were collected by pharmacists. appropriateness of the iv route of administration was determined by review of data by an infectious diseases physician (idp) and compared with a specifically designed computerised algorithm. the idp also judged the appropriateness of the chosen iv agent against local guidelines. patients from hospitals in regions were surveyed on a single day. ( . %) were receiving an antibiotic, ( . %) intravenously. receiving oral antibiotics had received an iv previously. median duration of iv therapy was days (iqr - days) and time from iv to oral switch was . ( ) ( ) ( ) ( ) ( ) . the idp judged appropriate iv route in % patients compared with . % by the algorithm. the sensitivity of the algorithm was . % and specificity . %. the positive predictive value was . % and the negative predictive value was . %. the idp judged iv agents to have been chosen and administered appropriately in %. most frequently prescribed iv agents were rd generation cephalosporins ( gc) ( . %), co-amoxiclav ( . %), metronidazole ( . %), glycopeptides ( . %). significant regional differences were seen for most antibiotic groups including gcs ( . % (site ) vs . % (sites , , , ) , p < . ) and glycopeptides [ . % (site ) vs . % (site , , , ) , p < . ]. it is possible to coordinate, collect and compare data from scottish hospitals. the gaat gives a good estimate of the appropriateness of iv therapy. significant differences in prescribing patterns between similar patient groups across different hospital sites were demonstrated. such data may usefully inform local and national audit and support prescribing initiatives. associations between continuous variables were tested in univariate analysis with the spearman correlation test (r). multiple linear regression analysis was performed in a backward stepwise approach. results: the median rate of total hospital glycopeptides use was . (range . to . ) ddds per , pd with higher consumption in large public hospitals. consumption was higher in intensive care areas (median . ; range . to ) than in surgery areas (median . ; range . to . ) and in medicine (median . ; range to ). glycopeptides use correlated with number of central line per , pd (r: . ; p: . ) and with size of the various areas in the hospital (for intensive care, r: . ; for medicine areas, r: . and for surgery areas, r: . ; p < . ). median incidence of mrsa was . per , pd. incidence of mrsa explained a small proportion of the variation in hospital glycopeptides consumption (r : . ). in a multivariate linear regression model, incidence of mrsa and number of beds in surgery areas were independent predictors of total glycopeptides use in the hospital (r adjusted: . ). after controlling for these factors, number of central-line per , pd was no more associated with glycopeptides use. conclusion: in our hospitals, total glycopeptides use was not heavily determined by incidence of mrsa. although glycopeptides use in surgery areas was not the highest, the total number of surgery beds in the hospital explained a large variation of the total hospital glycopeptides use. therefore we had to take it into account to interpret these consumption and to decide further evaluation. antibiotic management of acute lower respiratory tract infections among dutch elderly patients in primary care j. bont, c. birkhoff, t. verheij, e. hak on behalf of esprit objectives: acute lower respiratory tract infection (lrti) can cause various complications leading to morbidity as well as mortality notably among elderly patients. antibiotic treatment of lrti is common, despite dutch clinical guidelines recommending antibiotics only in case of pneumonia or high risk of serious complications. we assessed the course of illness and outcome of pneumonia, acute bronchitis and exacerbations of copd or asthma among dutch elderly patients in primary care and assessed whether gps were inclined to prescribe antibiotics more readily to patients with potential risk factors for complications in acute bronchitis or exacerbations of copd/ asthma. methods: we retrospectively analysed medical data from , episodes of lrti among patients ‡ years of age presenting in primary care to describe the course of illness and outcome. the relation between prescriptions of antibiotics and patients with risk factors for a complicated course was assessed by means of multivariate logistic regression. risk factors for a complicated course included heart failure, history of myocardial infarction, angina pectoris, diabetes, history of stroke, dementia, malignancy, and history of pneumonia or hospitalisation in preceding year. results: one or more complications arose in % of episodes of lrti. among these, % suffered from pulmonary complications, % had cardiovascular complications (heart failure, myocardial infarction etc.), % had a protracted course and . % had a diabetes event. in . % of the patients complications led to hospital admission and in . % lrti were fatal. antibiotics were more readily prescribed to patients aged ‡ years, when heart failure was present and in patients with diabetes. no significant association was observed in patients with other co-morbid conditions. patients diagnosed with an exacerbation of copd or acute bronchitis with a history of pneumonia or hospitalisation in the preceding year were not more likely to receive antibiotics. conclusions: a considerable part of elderly patients with a lrti suffers from a severely complicated course in primary care. although gps are inclined to prescribe more readily antibiotics in the very old and those with heart failure or diabetes, other potential risk factors are not taken into account. objectives: in this study it was aimed to analyse the infectious diseases (id) trainees' night/weekend shift consultation process in terms of patient and consultant characteristics, types of recommendations, and compliance with recommendations. methods: all consultations performed by id trainees in night shift and at the weekends between june th-august th were analysed in terms of consultation type [treatment continuation (tc), consultation for surgical antibiotic prophylaxis (pa), and consultation with or without a request of a specific antibiotic (others)]. appropriateness of recommendations was assessed the day after the consultation by infectious diseases specialists (ids). adherence to recommendations was assessed days after the consultation by idss. recommendations including antibiotics were considered appropriate, if they were appropriate according to national and international guidelines. recommendations were considered complied, if they were done in up to hours after the consultation (except the consultations in the emergency medicine and the consultations in which antibiotics were started by the counselling idss). results: of consultations was for tc, was for pa and was for others. the clinic where id consultations were requested mostly was general surgery clinic ( / , . %) . in % of all consultations trainees consulted the specialists. overall consultations ( for sp, for a clinical infectious disease diagnosed clinically, for an infectious disease diagnosed microbiologically) were for requesting spesific antibiotic(s). pa were approved in of consultations. antibiotic was not recommended in of other consultations. in six of consultations for pa antibiotic was changed for a clinically diagnosed infectious disease. in one of consultations for tc antibiotic was changed due to lack of response to the given antibiotic, in others tc was approved. inappropriate antibiotic recommendation rate was . % ( / , inappropriate choice, inappropriate dosage, one antibiotic unnecessary). overall compliance to id recommendations was . % ( / ). rate of compliance to antibiotic recommendations was evaluated in consultations and was found . % ( / ) and was higher than compliance to other (microbiology etc.) recommendations ( . %, / , chi square p < . ). conclusion: methodologies to improve the compliance to nontreatment based recommendations and optimizing antibiotic selection is necessary. study of the influence of online practice guidelines on the appropriateness of antibiotic prescribing in a university-affiliated psychiatric hospital j.f. westphal, c. nonnenmacher, d. gregoire, m. hittinger, c. oulerich, f. jehl (brumath, strasbourg, fr) background: problems with the dissemination of guidelines are frequently cited as a major reason for failure to impact practice. reviews of the effectiveness of various methods of guideline dissemination show that the most predictable impact is achieved when the guideline is made accessible through computer-based reminders that are integrated into the clinician's workflow. we report a time-series prospective investigation aimed at comparing the appropriateness of antibiotic (ab) orders for pneumonia at the treatment initiation level after vs. before having embedded our current ab guidelines for pneumonia in the computerized physician drug-order entry system of our teaching psychiatric hospital comprising adult beds. methods: in total, consecutive ab orders for pneumonia were evaluated by the pharmacy department, including orders just before and orders just after implementation of online ab guidelines. appropriateness of ab orders relative to the guidelines was assessed according to criteria: ( ) the choice of ab with respect to the mode of acquisition (community-or hospital-acquired) of pneumonia or the presence of clinical risk factors for involvement of gramnegative bacilli, ( ) the daily dosage, ( ) the planned duration of treatment. data were extracted from the computerized infection declaration system that recorded all ab-requiring infections in our hospital. results: the number of ab orders with at least criterion of inappropriateness tended to decrease, yet not significantly (p = . ), after vs. before implementation of online guidelines: / ( . %) and / ( . %), respectively. the number of criteria of inappropriateness relative to all ab orders for pneumonia was significantly lower in the post-implementation period: . % vs. . % before implementation (difference . %, % ci . - . , p < . ) , with a trend to a decreased number of orders containing more than criterion of inappropriateness. analyzed separately, the numbers of inappropriate orders for the choice of the ab, or the daily dosage, or the planned duration of treatment decreased, yet not significantly (p > . for each criterion), in the post-vs. preimplementation period : vs. , vs. , vs. , respectively. conclusion: in this study, the moderate impact on ab prescribing practices of online guidelines available at the time of drug order shows that additional types of intervention are needed to improve further the quality of ab prescribing. material: the pilot hospitals had a median capacity of (range, to ) beds; their regional distribution was representative of population size; were general hospitals, teaching hospitals and general hospitals with teaching beds. results: ams were internists ( ), microbiologists ( ) and pharmacists ( ). amts included a mean of members who met every weeks on average. all hospitals irrespective of size or affiliation had undertaken a wide range of antibiotic management interventions in , which increased in ; these included (in and , respectively) : major review of formulary (in and hospitals), development of clinical guidelines ( and topics), restricted access to selected antibiotics (carbapenems, glycopeptides, quinolones, new drugs; in and hospitals). in , antibiotic consumption databases were established in hospitals and antibacterial susceptibility databases in hospitals. in , cross-analysis of these databases was performed in hospitals. in , prescribing assistance, antibiotic stop orders, treatment streamlining and iv/po therapy switch were implemented in , , and hospitals, respectively. in , hospitals reported a better use of target antibiotics, hospitals a decrease in consumption of restricted antibiotics, hospitals a decrease of total antibiotic consumption, hospitals a decrease in high consumer departments. conclusion: all hospitals participating in the amt pilot scheme have developed multiple antibiotic policy interventions and established monitoring and guidance of antibiotic prescription. preliminary data from some hospitals indicated success in meeting self-defined targets of appropriate use and reducing the consumption of selected antimicrobial agents. more systematic evaluation using standard quantitative and qualitative indicators is planned. antibiotic prescribing practices at two linked london teaching hospitals p comparison of different antibiotic consumption measurement methods in large multidisciplinary hospital e. pujate, i. apine, u. dumpis (riga, lv) objectives: antibiotic selection pressure is determined by the total amount of antibiotics, number and density of patients treated with antibiotics in the particular geographical area. several antibiotic consumption detection methods should be combined in the hospital setting. our objective was to evaluate efficacy of different approaches in large multidisciplinary hospital. methods: point prevalence studies were repeated annually at [ ] [ ] [ ] in stradins university hospital ( beds) in latvia. all patients receiving antibiotics on the day of the survey were identified and their medical records were reviewed. data on antibiotics, dose and route of administration were collected. in addition, annual data on antibiotics dispensed to the departments were collected from pharmacy. total used grams for each antibiotic were expressed into defined daily doses (ddd-who). bed days (bd) and admission days (ad) were used as denominators. results: table total use of antibiotics in stradins university hospital hospital - the most commonly used antibiotic groups in the pharmacy study were st generation cephalosporins ( . ddd/ bd in , . in , . in ) and penicillin's with extended spectrum ( . , . , . ) followed by fluoroquinolones ( . , . , . ) and metronidazole ( . , . , . ). there was no significant difference between distribution of different antibiotics from prevalence and pharmacy studies if calculated in ddds. in contrast, distribution of antibiotics calculated per patient in the prevalence study was quite different; st generation cephalosporins ( . %, . %, . % in , , respectively) and fluoroquinolones ( . %, . %, . %) with smaller proportion of extended spectrum penicillins ( . %, . %, . %) and metronidazole ( . %, . %, . %). conclusions: there were no differences in the distribution of antibiotics calculated in ddds per bed days and admissions. distribution of antibiotics in annual pharmacy studies and point prevalence studies if calculated in ddds were also similar. in contrast, the prevalence data expressed as a proportion of patients with selected antibiotics showed quite different distribution. studies using only ddds may overestimate use of certain antibiotic groups in our setting where who ddds are significantly different from actual pdds used. a study of prescribing patterns and errors of antibiotics in a saudi hospital m. al-jamal, m. al-barrak (riyadh, sa) background: the term ''prescribing patterns'' has been used extensively in studies to describe different aspects of the prescribing process. antibiotics as well as other drugs are prescribed for the purpose of achieving definite therapeutic outcomes that improve a patient's quality of life while minimizing risk. in the clinical literature, the incidence of antibiotics prescribing errors ranges between . % and . %. objective: in this study we will address antibiotics prescribing patterns and the incidence of prescribing errors in a tertiary hospital and the potential relationship between them. methods: a prospective study of all prescriptions in a -month period (june to august ) in a tertiary hospital has been analysed. the hospital provides both primary and secondary levels of care. criteria used include frequency of selected prescribed drugs, average number of items per prescription, compliance to the hospital formulary, frequency of prescriptions for antibiotics, generic prescribing and diagnosis. the prescribing patterns and the incidence of prescribing errors were performed. results: total number of prescriptions for the -month study was , . emergency room (er) and primary care have the highest number of prescriptions ( . %). the average number of items per prescription is . . the most prescribed drugs by primary care ( . % errors), emergency are antibiotics ( . %), medicine ( . ), ophthalmology ( . ), gynaecology ( . ), and paediatrics ( . ). the prescription errors were . % in primary care and . % in emergency department. discussions and conclusions: over prescriptions were included in this study. the incidence of prescribing errors was . % the average number of items per prescription was . . total prescription errors are also related to frequency of prescribing antibiotics. there was a relation between prescribing of antibiotics and prescribing of trade names (p < . ), and compliance to the hospital formulary (p < . ). several factors influence prescribing patterns and variations in prescribing rates has been identified. these include general physician behavior, differences in morbidity and mortality patterns, social perception toward illness, and physician clinical skills, experience and qualification, as well as physician continuing education and training. special antibiotic prescribing guidelines and restrictions should target primary care and emergency department physicians. effect of a policy for restriction of selected classes of antibiotics on antimicrobial drug cost and resistance of the non-restricted antibiotics. the logistic regression model we performed showed that the new policy had an independent positive effect on the in vitro antimicrobial susceptibility of pseudomonas aeruginosa (p = . ) but not of acinetobacter baumannii and escherichia coli isolates. conclusion: our data suggest that there are considerable limitations of the programs aiming to reduce the consumption of restricted antibiotics through the approval of their use by specialists, at least in a proportion of settings. education programs that aim to involve the medical staff directly responsible for the care of patients in voluntary decisions regarding the appropriate use of antimicrobial agents may have more profound and sustainable success, and thus, deserve to be studied. estimating hospital versus ambulatory care consumption of antibiotics in southwestern germany k. de with, m. steib-bauert, h. schrö der, k. nink, w.v. kern (freiburg, bonn, de) objective: preliminary data from the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project indicated that the proportion of hospital care (hc) antibiotic use on total antibiotic use in several european countries ranges between and %. only few countries, however, have so far been able to report representative countrywide information on both hc and ambulatory care (hc) antibiotic consumption. we estimated ac versus hc consumption of antibiotics for one of the german federal states located in the southwestern part of the country with a . million population. methods: data on hc consumption (atc class j ) were obtained from a convenience sample of acute care general hospitals (n = ), extrapolated to state-wide consumption (using official statistics for the total state-wide general plus special non-psychiatric/non-paediatric/non-radiotherapy hospitals), expressed in defined daily doses per inhabitants and day (did), and finally compared to ambulatory care antibiotic use density in the same region and period of time (years and ) . findings: the estimated state-wide hc consumption of antibiotics was . did ( % confidence interval, . to . did) in both years. state-wide antibiotic consumption in the ac setting during the same time was did (~ % of total consumption). ac consumption of fluoroquinolones ( . - . did, %) and macrolides/clindamycin ( . did, %) made up a major proportion of total use of that drug classes. conclusions: hospital antibiotic use in the southwestern part of germany can be estimated to contribute~ % to overall antibiotic consumption in the general population. antibiotic use profile and temporal trends during a -year period at a greek university hospital: implications for antibiotic policy changes e.i. kritsotakis, p. assithianakis, p. kanellos, n. tzagarakis, m.c. ioannides, a. gikas (heraklion, gr) objectives: to investigate the profile and temporal trends of inpatient antimicrobial use over a -year period at the university hospital of heraklion crete, greece. further, to examine the way in which frequency of data collection and stratification by different patient-care areas provides guidance to antibiotic policy changes. methods: retrospective monitoring of antimicrobial consumption was carried out according to the who anatomic therapeutic chemical classification (atc) and defined daily dose (ddd) measurement methodology. pharmacy records were used to obtain aggregate data of drug deliveries to individual wards. results were expressed as usage density rates in ddds per bed-days (ddd/ bd). linear regression was used in order to assess the statistical significance of a temporal trend in usage densities. results: during - , hospital-wide antimicrobial use (atc group j ) significantly increased by %, from . to . ddd/ bd. the annual average increase rate was . ddd/ bd. stratification by clinical service demonstrated differences in the intensity and profile of class use, as well as varying temporal trends (figures , ) . pooled usage rates in ddd/ bd, overall percentage increases and annual average increase rates were respectively . , . %, . for medical wards; . , . %, . for icu's; and . , . %, . for haemato-oncology wards. surgical wards had a fairly constant usage rate ( . ). a shift towards the newer broad-spectrum antibiotics to the detriment of the older penicillins and cephalosporins was noted in all hospital areas. conclusion: surveillance of aggregate data on the consumption of antimicrobials using the atc/ddd system provided a clear picture of the profile of hospital usage. monthly data over a sufficient surveillance period allowed the assessment of temporal trends. stratification of usage rates by clinical service allowed areas of concern to be specified. thus, surveillance of monthly antimicrobial consumption rates stratified by patientcare area can provide a simple, rapid and efficient tool for triggering antibiotic policy changes in the hospital and targeting more detailed quality-of-use audits. appropriate use of aminoglycosides: the impact of an antibiotic control team c. rioux, p. lesprit, j.r. zahar, a. hulin, a. bernier-combes, c. brun-buisson, e. girou (créteil, paris, fr) objectives: many factors are involved in the appropriate use of aminoglycosides (ag), such as modalities of administration, serum monitoring and duration of treatment. we assessed prospectively the risk factors and the impact of an antibiotic control team on the appropriateness of ag prescriptions. methods: in a setting of a restricted delivery system of ag in our hospital, we first performed an observational audit (oa) to assess the appropriateness of prescriptions including justification of prescribing, adequacy of drug choice, adequacy of administration modalities, modalities of serum monitoring and duration of treatment. after implementation of specific guidelines hospital wide, we then performed an interventional audit (ia) where an antibiotic control team could interfere when ag prescriptions were considered inappropriate. appropriateness of ag prescriptions between the audits was then compared. results: prescriptions were analysed. during the ia, % of prescriptions were modified by the control team. as compared to the oa, prescriptions in the ia were significantly more appropriate with regard to treatment duration ( vs %, p = . ) and serum monitoring ( vs %, p = . ). median treatment duration was shorter in the ia ( d) than in the oa ( d) (p < . ). a logistic regression model showed that risk factors for appropriate treatment duration were (adjusted or, % ci, p value): hospitalization in intensive care unit ( . , . - . , . ) , polymicrobial infection ( . , . - . , . ) and antibiotic control team intervention ( . , . - . , . ) . table: conclusions: despite a restricted delivery system, ag use was frequently associated with excessive treatment duration and errors in monitoring modalities. reinforcing practice guidelines through direct counselling improved appropriateness of prescriptions. hospital antibiotic consumption in southern and eastern mediterranean countries: preliminary results from the armed project p. zarb, m.a. borg, h. goossens, m. ferech for the armed participants introduction: armed is an international research project investigating antimicrobial resistance and consumption in southern and eastern mediterranean countries through the collection of comparable and validated antimicrobial resistance data as well as information about antibiotic consumption patterns and infection control initiatives. objectives: the consumption part of the study aims to collect data on antimicrobial use within participating hospitals in the region, which information is currently unavailable. methods: data collection is planned over a -month period using anatomical therapeutic chemical (atc) classification, a validated methodology adopted by the european surveillance of antimicrobial consumption (esac -www.ua.ac.be/esac). hospitals are participating: cyprus ( hospitals); egypt ( ); jordan ( ); malta ( ); tunisia ( ); turkey ( ). results are expressed in ddd/ bed-days. results: data from , the first year of data collection, indicates that turkish hospitals seem to show the lowest overall consumption [ - ddd/ bed days], whilst the cypriot hospitals show highest values [ - ddd/ bed days]. the most common antibiotics used are the beta-lactams, especially the penicillins although in jordan and turkey cephalosporin consumption is very close to the penicillins. broad-spectrum penicillins [j ca] are the mostly utilised penicillins in cyprus, jordan and tunisia whereas in malta and turkey the combination penicillins [j cr] are the most widely used. there is more variability where cephalosporin consumption is concerned. cyprus utilises mainly first generation, jordan and malta the second generation. in egypt, tunisia and turkey there is significant variability between hospitals; nevertheless use of third generation cephalosporins appears to be significant. conclusion: a significant variability was evident between countries. this is likely to be multifactorial depending on the antibiotics licensed in a country, the national and/or hospital formulary, the type of hospital as well as any antibiotic donations that are relevant in some of the study hospitals. nevertheless, the preliminary results suggest that trends within hospitals of the same country tend to be similar. furthermore, the region as a whole seems to utilise a considerable quantity of broad-spectrum antimicrobials. this can be a factor in the high prevalence of resistance already documented in the study. russian pharmacoepidemiology study of the antibiotic prescription during pregnancy results: mean age of the patients was . ± . (min - , max - ) years, mean gestational ages at admission to hospital was . ± . ( to ) weeks. most often ( . %) infection was community acquired and . % -nosocomial, in % patients there was not to estimate origin of the infection. the most prevalent infections during pregnancy in russia was urinary tract infections - . %, std - . %, candidiasis - . %, rti - . % therefore the most interest was analysing the antibiotic prescription for uti in pregnancy (table) . in % cases were used topical (intravaginal) antimicrobial administration. most often of topically administrated antimicrobials ( . % of all prescriptions) were prescribed combined drugs included antibacterials and amtimycotics. in . % cases antimicrobials were prescribed systemically. mostly prescribed antimicrobials were beta-lactams ( . % for outpatients and . % for inpatients), ampicillin was prescribed more often ( . % for outpatients and . % for inpatients). amoxicillin + clavulanic acid was prescribed in . % of outpatients and . % inpatients pregnant women with uti. cephalosporins were prescribed in . % and . % for outpatient and inpatient uti (mainly iii-and ist generations). mitroimidazoles - . - . % (in general metronidasole), nitro-furanes - . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . %, aminolglycosides - . - . % were prescribed quite often but unjustified. other antimicrobials (fluoroquinolones, doxicicline, antiviral drugs, antifungals) were prescribed relatively rarely. despite the fact that most prescribed drugs were class b by fda, . % all antimicrobials prescribed to pregnancy were class c, . % class d and . % were unclassified. conclusions: most often prescribed antimicrobials for uti (the most prevalent infections during pregnancy in russia) are betalactams and combined topical antibacterials. in . % cases were prescribed antimicrobials of class c, d or unclassified by fda. in % outpatient and . % inpatient were used antibiotics with low in vitro activity for uropathogens. objectives: to study the dynamics of the antibiotic usage in children from orphanages located in different russian cities as the result of interventions with the increased use of the most active antimicrobials and restrictions on use of the least active. methods: the study was performed in orphanages ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) from cities of european russia (moscow, saint-petersburg, smolensk, karachev, bryansk) . use of antimicrobials during the previous months was analysed upon reviews of medical records of children < years in . appropriate recommendations on predominant use of selected beta-lactams (e.g. amoxicillin/clavulanate -amc) with restriction of antimicrobials of other classes (e.g. co-trimoxazole -sxt) were made where applicable on the basis of the expert analysis of antibiotic usage and pneumococcal nasopharyngeal resistance rates. repeated antibiotic usage analysis was performed months later in upon reviews of medical records of children < years. results: total usage of antimicrobials increased . increase of resistance to pen and aminopenicillins. enhanced use of cephalosporins led to increase of resistance to these drugs. in spite of recommendations to restrict usage of am/ox, aminoglycosides and sxt, the analysis showed that these antimicrobials still accounted for . %, . % and . % of all prescriptions, respectively, thus dictating the need for further enforcement measures. antibiotic consumption in ambulatory care in latvia, s. berzina, m. ferech, g. ozolins, h. goosens (riga, lv; antwerp, be) objectives: to collect data on antibiotic consumption in ambulatory care (ac) in latvia according to the esac data collection protocol. esac (european surveillance of antimicrobial consumption, granted by dg sanco of the ec) is an international network of national surveillance systems, aiming to collect reliable and comparable data on antibiotic consumption in europe. methods: the data on ac antibiotic consumption for have been collected using atc/ddd classification (who, version ) and expressed in defined daily doses per inhabitants per day (did). data were obtained from the state medicinal agencybased on the reports of the wholesalers for ac. results: the overall use of antibiotics in ac was . did in , which positions latvia to countries with comparatively low antibiotic consumption in europe. the mostly used class of antibiotics in ac were penicillins with extended spectrum (mainly amoxicillin) - . did ( . %). other frequently used antibiotics were tetracyclines (mainly doxycycline), representing . did ( . %), combinations of penicillins/with betalactamase inhibitors (essentially co-amoxiclav) - . did ( . %), macrolides (mainly clarithromycin) . did ( . %), fluoroquinolones (essentially ciprofloxacin) - . did ( . %) and combinations of sulphonamides and trimethroprim, incl.derivatives - . did ( . %). the most frequently used antibiotics in ac in latvia, in , were amoxicillin ( . did) , doxycycline ( . did) , and co-amoxiclav ( . did) . conclusions: valid data on outpatient antibiotic use in latvia has been for the first time collected and delivered to european surveillance of antimicrobial consumption. this allows international comparison of the pattern of antibiotic consumption in latvia with other european countries. trends in glycopeptide antibiotics consumption over a -year period in a general hospital, athens, greece introduction: glycopeptide use is under restriction in hellenic hospitals since late ' s. the aim of our study was to record trends in their consumption over the last years in our hospital (''a. fleming'' general hospital - beds) and to correlate these data with the numbers of important gram (+) strains isolated in our hospital during the same time period. methods: we measured glycopeptide use for the period - by using data from the pharmacy computer. consumption was expressed as ddds/ patient days (abc calc . ). furthermore we correlated these data with data from the microbiology department concerning numbers of mrsa, mrse and enterococci isolated during the same period. results: glycopeptide consumption was . , . , . , . , . and ddds/ patient days for the years , , , , , ( % increase) . at the same time the cumulative number of mrsa, mrse and enterococci isolated were , , , , , respectively ( % increase) . when both types of data were put on the same graph, glycopeptide consumption correlated well with the number of important gram(+) strains isolated (figure). furthermore vre percentage among enterococci was , , . , . , . , for the study years respectively. it is worth noting that % of our mrsa strains were sensitive to rifampin, % to clindamycin, % to cotrimoxazole, % to clindamycin + rifampin and % to cotrimoxazole + rifampin. linezolid has not been introduced in our hospital yet. conclusions: (a) despite the restriction policy, a tremendous increase in glycopeptide use was recorded in our hospital during the study period and this correlated to the number of the important gram(+) strains isolated; (b) nevertheless, vre is not a significant problem for our hospital yet; and c. the huge increase in glycopeptide use could be avoided at least in part, since other, older and simpler antibiotics could substitute for glycopeptides in many cases. an audit of linezolid use in a university teaching hospital, galway, ireland objective: to audit linezolid use over a six-month period among the in-patient population of a -bed teaching hospital that includes most medical and surgical specialties with the exception of nephrology, rheumatology and orthopaedics. methods: a prospective audit was carried out of the prescribing of linezolid to in-patients from october to april . the ward pharmacist recorded the details of all patients who were prescribed linezolid. a chart review was performed to assess the profile of patients prescribed linezolid, clinical and microbiological indications for treatment, adherence to treatment guidelines and documented adverse events. results: over the -month period courses of linezolid were prescribed. fifty two percent of the patients for whom linezolid was prescribed were from surgical specialties; half of these patients were under the care of one surgeon. pneumonia was the clinical indication for use in % of cases and soft tissue infection in % of cases. the microbiological indication was clear in % of cases where mrsa or vre had been isolated. in % of cases therapy was either ( ) empiric with no significant organisms isolated prior to prescription of linezolid or ( ) therapy was directed against an organism that could have been treated with an alternative agent. duration of treatment exceeded to days in % of courses. an adverse event was recorded in the case of only one course of linezolid. conclusion: in more than a third of cases linezolid use was prescribed without clear justification. avoidable use of linezolid is associated with increased costs and risks of acquired resistance. participants prior to on an oral interview during which the interviewer filled in the answers. results: a total of cm specialists completed the inquiry. this represents approximately % of the national quorum. mean age was years ( - ). of the interviewed cm specialists worked full-time with more full-time employment in hospital labs (hl). next to routine microbiology, other activities performed by cm specialists are mainly the other domains of clinical biology, hospital hygiene and to a lesser extent quality control and lab management. almost two thirds of the interviewed cm specialists believes that their training hasn't prepared them properly for the tasks they are performing now. most desired changes include more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. cm does not exist as a separate speciality in belgium but is included in the 'clinical biology' speciality training. the majority of the respondents thinks that cm should become a sub-speciality (still part of clinical biology) but with a specific minimal training that needs to be defined. the majority of the cm specialists also believes that cm can share lab infrastructure with other disciplines and that the essential aspect of cm lies predominantly in the medical expertise. conclusion: cm training should put more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. the majority of the respondents feels that cm should become a sub-specialty (still part of clinical biology), with a well defined training curriculum. objectives: since more than years, all infectious disease consultations have been recorded in a computerized database (epi info . d, cdc). here we report on consultations of a fellow, conducted during year, compared with consultations conducted by two veteran board-certified infectious disease consultants during the same period. methods: we analysed computerized consultation records, including demographic details of patients; referring department; initiative for, route and purpose of the consultation; and recommendations; and compared between the different consultants. results: a larger percentage of veterans' compared to the fellow's consultations, were requested by attending physicians ( % vs. %, p < . ), while follow-up ( % vs. %, p < . ), laboratory results ( % vs. %, p < . ) or prescription for a restricted antimicrobial agent ( . % vs. . %, p < . ) were more prevalent in fellow consultations. the fellow had a higher rate of additional consultations (in which the patient was seen more than once) ( % vs. %, p < . ), and performed more bedside consultations ( % vs. %, p < . ) or consultation by curb side discussion ( % vs. . %, p < . ), and less consultations by telephone ( . % vs. . %, p < . ). diagnosis and prophylaxis were more often the purposes of the veterans' consultations ( . % vs. . %, p < . , . % vs. . %, p < . , respectively), and they also offered new diagnoses more frequently (p < . ). the veteran consultants more often conducted consultation for communityacquired infections ( % vs. %, p < . ), and more often started antibiotic treatment ( % vs. . %, p < . ). conclusions: significant differences were detected between consultations conducted during the first year of a fellow compared to those of veteran infectious disease consultants. these differences reflect the changing demands and activities in the consultant's work as experience and knowledge accumulate. periodic analysis of computerized data of consultations facilitates supervision as well as direction of consultants' work, addressing issues such as antibiotic use and patterns of microbial resistance. bridging the gap between health care and public health; capacity building in infectious disease control objectives: in recent years, the european union (eu) has developed and supported many activities in the field of communicable diseases. these activities not only concern surveillance networks of specific infectious diseases (e.g. enter-net for salmonella and escherichia coli infections, ewgli for legionella infections, eiss for influenza infections), but also eu training programmes like epiet (european programme for intervention epidemiology training) and a eu communicable disease bulletin. even more recent are the eu's initiative bichat to improve preparedness and response to bioterrorism, and the development of a eu cdc. moreover, a major part of the new programme of community action in the field of public health (ph) ( ) ( ) ( ) ( ) ( ) ( ) concerns id, with not only a commitment to improve information and information exchange, but particularly to strengthen the international rapid response capacity.all this, to illustrate the importance of idc on the eu agenda. methods: the european public health association (eupha) is an umbrella organisation for ph associations in eu. in eupha has created an eupha section idc bringing together eupha members with expertise in this field and representatives of the various above mentioned eu initiatives in order to: promote and strengthen research in the field of idc; provide a platform for the exchange of information, experience and research in idc; bring together researchers, ph practitioners and policymakers active in idc; encourage joint activities in idc; and improve idc training. results: by now the section has members from different countries. as of the section is represented in the ecdc advisory forum. different section activities: organising workshops, a breakfast meeting and a pre-conference meeting on timely idc topics during eupha conference. objectives: to establish a cross-border dutch-german network (www.mrsa-net.org) providing a user-friendly knowledge centre for hospitals, public health authorities, gps, nursing homes and laboratories. primary purpose is to aid in the reduction of mrsa-rates and limit the cross-border transmission of mrsa. guidelines and their implementation play a significant role in reaching these aims. cross-border (ca-) mrsa guidelines will be redesigned according to international standards and socio-cultural differences between the nations. methods: based on quality standards for safety and healthcare documentation used in high risk chemical organizations, a framework for a systematic content analysis of national mrsaguidelines was developed. national guidelines were analysed on the basis of this framework. results: a content analysis of the current national mrsaguidelines showed five dominating mrsa-perspectives: rule-, expert-, risk-, demand-and community-driven. german guidelines are mainly dominated by the rule-and expertdriven perspectives (guidelines are literally derived from law and follow the infection transmission route), in contrast to the dutch which focus on the demand of the user and the community (addressed to public health and acceptability of guidelines by users). conclusion: the analysis showed that the fact that there are different guideline-perspectives results in an enormous, confusing set of guidelines. the management and use of guidelines becomes uncontrollable and leads to an illusory organisation where healthcare workers don't act in accordance with the guidelines and start applying their own insights. this might lead to cost-increasing and contrasting situations. to implement guidelines successfully in a cross-border situation, a cultural and technical synchronisation alongside an integrated approach of the different perspectives of guidelines is necessary, inline with the current disease management models. further research about the redesign and the evaluation of those guidelines in practice will help achieving this. r. tsiklauri (tbilisi, ge) background: animal bites are a common but under recognized public health problem. it has been estimated that there are - bites each year in georgia, and based on an average visit and post-exposure treatments cost at list $ per year. despite the frequency and expense of these injuries, there is little information about the incidence of animal bites because of a lack systematic reporting and a lack of measurement of the quality and completeness of reported data. objectives: to investigate animal bites and rabies reported cases, revealed unreported cases, analyse and based on study results find more effective epidemiological measures of animal bites and deaths (due to rabies) prevention in georgia. methods: the capture-recapture method was used, along with log-linear modelling. for sources were used to identify victims: policlinic/ambulatory reports, hospital reports, animal control reports and victim reports. results: in - years dog and other animal bites were reported. the capture-recapture method estimated that there were unreported bites. during these period deaths due to rabies was registered in georgia and ( %) cases among them have been registered during the last years. the reasons of fatal cases were untreated ( %), uncompleted treated ( %) and late began post-exposure treated ( %) cases of bites (mostly dog bites). about % of bitted persons did not know about rabies and it's prevention measures. about % had incorrect information about prevention and only % of them knew epidemiological and clinical aspects of disease. about % of physicians who were responsible on quality post-exposure treatment had not an adequate knowledge. conclusion: dog and other animal bites are common but preventable injuries. to improve surveillance and prevention of rabies in georgia, the focus should be on educating the general public about the serious consequences of animal bite injuries and developing the animal's vaccination strategy. pharmacoeconomics and electronic resources p the expected economic burden of methicillinresistant staphylococcus aureus in complicated skin and skin structure infections: a modelling approach a. kuznik, r. mallick, d. weber (collegeville, chapel hill, us) objective: to model the expected rate of clinical failure of initial empiric therapy and economic burden likely to be associated with the increasing prevalence of methicillinresistant staphylococcus aureus (mrsa) in patients hospitalised with complicated skin and skin structure infections (csssi) in the united states. methods: using published data on ( ) the prevalence of mrsa and other bacterial pathogens causing csssi in the us, ( ) the in-vitro susceptibility rates of commonly used regimens in csssi in the us in relation to the most pervasive pathogens identified above, and ( ) estimated costs of failure of initial, empiric treatment from a recent study of a large us multi-hospital database, we developed a model to predict the expected clinical and economic impact of increasing prevalence of mrsa. specifically, clinical failure of of the more commonly used initial regimens in csssi was modeled in terms of their in-vitro susceptibility rates with respect to mrsa, weighted by mrsa prevalence. varying the rate of mrsa further yielded projected clinical failure rates and costs attributable to increasing levels of methicillin resistance over time. results: given current % prevalence of s. aureus pathogens in csssi, half of them methicillin-resistant (base case mrsa = %), the model projected an overall clinical failure rate of . % for of the more commonly used initial regimens, with an expected overall treatment cost (in us dollars) of $ , per patient (range, $ , - , ) . if none of the s. aureus pathogens were resistant (mrsa = %), clinical failure rate was projected to be . % and treatment cost to be $ , per patient. the differences in the two scenarios translated to an expected clinical failure rate of . %, an incremental cost of $ per patient, and for the , patients hospitalised for csssi annually in the us, an expected health care system burden of $ million attributable to mrsa. under a "worst-case" scenario in which mrsa was the only causative pathogen (mrsa = %) in csssi, clinical failure rate was projected to be . %, and treatment cost per patient was expected to be $ , . conclusions: going beyond existing estimates, our model generated a substantial expected clinical failure rate and economic impact attributable to current mrsa levels, as well as simulations of the expected impact of increasing mrsa prevalence over time, varying levels of mrsa across regions and choice of initial empiric regimens. treatment of complicated skin and skin structure infections in the us: expected cost differences between tigecycline and vancomycin/aztreonam r. mallick, a. kuznik, d. weber (collegeville, chapel hill, us) objective: to compare tigecycline and vancomycin/aztreonam in terms of treatment-related costs for patients hospitalised in the united states with complicated skin and skin structure infections (csssi). methods: we conducted a retrospective analysis of pooled data from us centres in two randomized, double-blind clinical studies comparing tigecycline and vancomycin/aztreonam in the treatment of csssi. using regression analysis, we estimated the effect of tigecycline treatment on hospital length of stay (los), controlling for other significant predictors. using published estimates of daily hospitalisation cost of csssi in the us from a multi-hospital audit, we then translated the estimated impact on los into economic terms. this analysis was repeated for the subgroup of patients in which the primary pathogen was methicillin-resistant staphylococcus aureus (mrsa). clinical efficacy (tigecycline %, vancomycin/aztreonam . %; p = . ) was similar across treatments and was not included as a model parameter. results: our retrospective analysis of the pooled clinical data from us centres found that tigecycline was associated with a shorter los [) . days (p = . )] compared with the combination of vancomycin/aztreonam in the treatment of patients with csssi. at a mean daily hospitalisation cost (in us $) of $ , excluding antibiotic costs, this translated into expected medical cost savings of $ , per patient for tigecycline compared with vancomycin/aztreonam. in the mrsa subgroup, comprising % of the clinical study sample, tigecycline was associated with a greater reduction in los [) . days (p = . )] compared with vancomycin/ aztreonam, translating to expected medical cost savings of $ , per patient treated with tigecycline. these expected medical cost savings more than offset the higher average daily drug acquisition costs of tigecycline ($ /day) relative to the vancomycin/aztreonam combination ($ /day). conclusion: in a retrospective analysis of pooled clinical data of patients with csssi treated at us centres, tigecycline was associated with a significantly reduced length of hospital stay relative to vancomycin/aztreonam; this translated into substantial cost savings, especially in the subset of csssi patients with mrsa. the economic impact of linezolid in the treatment of skin and soft tissue mrsa infections in italy m. eandi, p. dale, s. sorensen, t. baker, m. procaccini, s. duttagupta (turin, it; london, uk; bethesda, us; rome, it; new york, us) objective: linezolid has been shown to be highly effective against infections caused by methicillin-resistant staphylococcus aureus (mrsa) in patients with complicated skin and soft tissue infections (cssti). the objective of this study was to evaluate the clinical and economic consequences of using linezolid for the empiric treatment of cssti from the italian hospital perspective. methods: a decision-analytic model was developed to calculate the clinical and cost outcomes of empiric treatment of hospitalized patients with cssti in italy prescribed linezolid, vancomycin or teicoplanin. efficacy data were derived from clinical trials. costs from published sources were applied to tests, adverse events, and days of intravenous and oral (linezolid only) treatment and hospitalization by ward type (general, intensive-care). resource use and utilization patterns were obtained from a combination of clinical trial data and expert opinion. outcomes included total costs per patient, cost per cure and cost per death avoided. uncertainty surrounding the ce ratio was tested using one-way sensitivity analysis. results: starting empiric treatment with linezolid resulted in . % of patients cured from mrsa compared to . % with vancomycin. the average cost per patient treated with linezolid was € , versus € , for patients treated with vancomycin. this resulted in a cost per cure of € , . in a separate analysis more patients were cured using linezolid ( . %) compared to teicoplanin ( . %). the average total cost per episode was € , for linezolid treated patients versus € , for teicoplanin treated patients, resulting in a cost per cure of € , . the most sensitive parameters included hospital los and mrsa resistance rate. conclusions: in the treatment of cssti due to suspected mrsa in italy, the empiric use of linezolid is cost-effective when compared to vancomycin and teicoplanin p outpatient and home parenteral antimicrobial therapy for the treatment of cellulitis: evaluation of efficacy and cost h. ziglam, r. tilley, c. wootton, j. morrison, d. nathwani (dundee, uk) objective: outpatient and home parenteral antibiotic therapy (ohpat) programmes are effective, well tolerated and economically advantageous in carefully selected patient populations. skin and soft tissue infections represent a high burden disease which in amenable to treatment by ophat programmes. we retrospectively analysed our outcomes registry to evaluate the clinical and health economic impact of treating cellulitis in this setting. methods: we have reviewed patients with cellulitis and erysipelas who were treated with ohpat. each patient treatment has a full integrated care pathway (icp). the icp documents the microbiological outcome, drug and vascular access complication rates, impact on drug costs and in-patient bed days on the number of patients treated from april to march are presented here. we also reviewed using the smr o inpatient discharge diagnosis data from the information statistics division scotland (isd) and the dundee infectious diseases units (didu) outcomes registry database. the key diagnosis (icd codes) groups considered were cellulitis (lo , , , , , ) and erysipelas (a x) over eight consecutive years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: the patients received intravenous antibiotic therapy for a mean duration of . days. the two primary agents administered were once-daily ceftriaxone in %of patients and teicoplanin in . % of patients. of the patients, ( . %) were cured or improved; worsened and required surgery. tinea pedis was found in % of patients treated for cellulitis. economic benefits were realized despite use of more expensive agents. data from the dundee outcomes registry revealed a mean reduction in length of hospitalization from . days ( / ) to . in - -a reduction of % compared to scottish data from isd which did not show any changes in length of hospitalization between year / ( . days) and year - ( . days) . conclusions: we have found that ohpat is clinically effective and can be administered safely and successfully in an outpatient setting. the majority of complications were minor, and % of patients were cured. tinea pedis and were found to be significant risk factors for acute cellulitis and indicate that improved awareness and management of toe web intertrigo might reduce the incidence of cellulitis. this analysis also supports the premise that an adult ohpat programme can substantially reduce healthcare resource use in the european healthcare setting. cost-effectiveness analysis of intravenous moxifloxacin compared to levofloxacin in hospitalised elderly patients with communityacquired pneumonia objective: to evaluate the cost-effectiveness of moxifloxacin compared to levofloxacin in hospitalised patients aged ‡ with community acquired pneumonia (cap). methods: a randomised double-blind parallel group study was conducted in us hospitals. patients had radiological evidence of bacterial pneumonia confirmed by at least other signs, were aged ‡ years and were managed as inpatients on initiation of treatment. patients initially received moxifloxacin mg iv o.d. or levofloxacin mg iv o.d., and once stabilised were switched to oral therapy with the same agent. the effectiveness endpoint for the economic analysis was the percentage of patients successfully treated, defined as patients with marked improvement, resolution or clinical cure at test of cure visit after - days of therapy who did not experience a serious cardiac adverse event. total costs were estimated from the perspective of the treating hospital and included antibiotic drugs, hospital stay, hospital re-admission within days and cost of managing treatment failures. results: patients were included in this analysis, randomised to moxifloxacin and to levofloxacin. % ( % ci: - %) of moxifloxacin and % ( - %) of levofloxacin treated patients were successfully treated (resolution, clinical cure at toc and no serious drug related aes). in the moxifloxacin group patients reported a mean of . days of iv antibiotic treatment and . days inpatient stay with . days iv antibiotic treatment and . days inpatient stay in the levofloxacin group. mean per patient drug cost was $ in the moxifloxacin group, $ in the levofloxacin group. mean total cost was $ , in the moxifloxacin group and $ , in the levofloxacin group. findings were consistent across a range of patient subgroups. costs were sensitive to length of hospital stay. conclusions: patients in the moxifloxacin group had higher rates of successful treatment at slightly lower average costs than the levofloxacin group. this confirms the results of the target study where moxifloxacin showed superior clinical efficacy in comparison to co-amoxiclav with or without clarithromycin in hospitalised cap patients. antibiotic costs were slightly higher in the moxifloxacin group than the levofloxacin group but total costs were slightly lower, due to reduced hospital stay. economic impact of invasive fungal infections in icu patients in a tertiary care hospital in switzerland a. imhof, w. zingg, r. laffer, c. ruef (zurich, ch) objectives: invasive fungal infections (ifi) cause significant morbidity and mortality. the management of invasive fungal infections is currently undergoing important changes due to the availability of new therapeutic agents with improved safety profiles but the acquisition costs of these new agents are high. we evaluated the average overall cost of management (microbiological diagnosis and treatment) of invasive fungal infection in critically ill patients at a large university hospital. methods: a retrospective ( ) ( ) , pairwise-matched cohort study was performed on surgical icus and one medical icu at our university hospital. icu patients with documented ifi (n = ) were matched with control subjects (n = ) on the basis of disease severity, sex and age (± years). clinical outcome was principally evaluated by in-hospital mortality. the economic impact of microbiological studies and antibiotic treatment was assessed. calculations were based on the period between admission and diagnosis of ifi in cases and the duration of hospital stay in controls, respectively. results: the median length of hospital and icu stay differed significantly between cases and controls ( vs days, vs days, p < . , respectively). ifi occurred after a median hospital stay of (range - ) days. the mortality rate for patients with ifi and matched control subjects were . % and . %, respectively (p = . ). there was no significant difference between cases and controls for charlson index, mccabe and saps ii score. median number of antibiotic treatment courses was for cases and for controls (p = . ), with a median duration of therapy of days vs day (p < . ), respectively. microbiological studies (mis) were conducted times/ patient-days (pd) in cases and times/ pd in the control group (p = . ). the most frequent samples were bloodcultures in both groups. swabs were ordered significantly more frequently in cases (median (range: - ) vs. ( - ); p = . ). the cost for mis was ' euro/ pd in cases vs. euro/ pd in controls, and the costs for antifungal therapy ' euro/ pd vs. euro/ pd, respectively. conclusions: ifi is associated with excess length of icu and hospital stay, increased use of antibiotics and microbiological diagnostics. the microbiological studies have a significant economic impact on the treatment of ifi. cost-effectiveness of voriconazole to amphotericin b deoxycholate in early and late treatment of invasive aspergillosis r. greene, j. mauskopf, c. roberts, t. zyczynski, h. schlamm (boston, research triangle park, new york, us) objective: we estimate the cost-effectiveness of alternative initial drug treatments of invasive pulmonary aspergillosis (ipa) in suspected earlier and later lung involvement, based on the presence or absence of the halo sign on thoracic computed tomography (ct). methods: we constructed a decision analysis model comparing -week treatment outcomes for a subset of patients enrolled in a clinical trial of initial treatment of ipa with amphotericin b dexoycholate (ambd) vs voriconazole (vor). patients included those with suspected lung involvement who underwent a baseline thoracic ct. the subset was subdivided into two groups based on the presence or absence of a characteristic ct halo sign, a perimeter of ground glass ct opacity surrounding a solid lung nodule ‡ cm diameter, known as an early indicator of ipa. healthcare resource use and survival data were obtained directly from the clinical trial. us unit costs for drugs and health care services were applied from standard data sources. cost and survival at -weeks were estimated for those with and without a halo sign at baseline. incremental cost-effectiveness ratios comparing vor to ambd were calculated for both patient subgroups. sensitivity of results to uncertainty in health care use and cost estimates was tested. results: patients in the halo subgroup had better survival than those in the no-halo subgroup ( . % vs . %), with lower total treatment cost ($ , vs $ , ) . survival was higher for vor than for ambd in both patient subgroups (halo: . % vs . %; no-halo: . % vs . %). in the halo subgroup, total costs were lower for those treated with vor than for those treated with ambd ($ , vs $ , ) . in the no-halo subgroup, total cost per patient was slightly higher for those treated with vor ($ , vs $ , ) . accounting for the difference in survival, the incremental cost-effectiveness ratio for vor compared to ambd was $ , per additional -week survivor in this subgroup. conclusions: earlier identification and treatment of ipa appears to result in better survival and potentially lower costs than later treatment. initial treatment of ipa with vor improves survival in patients with early or late disease compared with ambd, is cost saving in the halo sub-group, and is cost-effective in the no-halo subgroup, within the constraints of our analysis. objective: to describe and compare the nursing labor time required for preparation and administration of liposomal amphotericin b (l-amb), amphotericin b deoxycholate (ambd), and voriconazole (vor). methods: activities associated with nurse preparation and administration of the three study drugs were timed by trained observers at five hospitals (one in italy, three in france, and one in the united kingdom). target tasks were classified as those likely to be affected by the difference between the drugs and excluded those tasks likely to differ because of site-specific factors (e.g., travel time to a patient room in different hospitals). target tasks included: obtain supplies and medications; prepare medications; educate patient; administer medications; monitor for adverse events; and prepare follow-up medications. the mean times for administration of a single day of study drug were summarised and compared, accounting for a single daily dose of l-amb and ambd and daily doses of vor iv or oral. results: sixty-nine patients were observed receiving doses of study medications at the five hospitals. time of administration in minutes per day was , , , and for l-amb, ambd, vor iv, and vor oral, respectively. administration time was significantly lower for vor iv compared with l-amb (p < . ) and for vor oral compared to all iv regimens (p < . ). the task of preparation of medications required the most time for iv formulations, and was longer in the l-amb group than the others (l-amb: mins vs ambd: mins; vor iv: mins). ambd required more time for patient monitoring and administration of followup drugs than other formulations (ambd: mins vs l-amb: mins; vor iv: mins). conclusion: vor iv required significantly less time to prepare and administer on a daily basis compared to l-amb. measurements of iv antifungal versus oral vor administration suggest the opportunity to save - minutes per day by switching to oral therapy when possible. need of cost-effectiveness investigation focused on diagnosis, management and prevention of osteopenia and osteoporosis in the setting of hiv disease treated with haart: when to act, how to act, which patients are the first target of intervention r. manfredi, l. calza, f. chiodo (bologna, it) background: osteopenia/osteoporosis are emerging untoward effects of hiv infection/haart. the pathogenesis is multifactorial, involving all classes of anti-hiv drugs, although protease inhibitor use, overall haart duration, and the male sex, seem related to a greater risk.epidemiologicalclinical data. in an ongoing study at our centre where > hiv-infected patients (p) are followed, bone mineral density was assessed in lumbar spine/femural head by a dual energy xray absorptiometry (dexa) exam to estimate the prevalence of osteopenia/osteoporosis. in a screening of~ p, the frequency of osteopenia and osteoporosis (based on lumbar t-score) was % and~ %, respectively. an increased risk was found in p treated with protease inhibitors versus those receiving nonnucleoside reverse transcriptase inhibitors or triple nucleoside/ nucleotide combinations. discussion and future insights: prospective studies of extensive p samples are needed, to elucidate the epidemiology, pathogenesis, clinical issues and evolution of hiv-associated bone metabolism anomalies. when planning strategies for their early diagnosis, prevention and management also cost-effectiveness issues should be considered, since no pharmacoeconomic data still exist in this setting. although severe consequences (e.g. pathological fractures, prosthetic implants) are expected to be infrequent their consequences in terms of length and intensity of hospitalization, related costs, and especially severe consequences on the p's quality of life, play a notable role. anyway, the most reliable diagnostic procedure (dexa) has affordable costs (around eur . for a total-body scan which also offers a body composition assessment), as well as the first-line drugs for osteopenia, e.g. supplementation with calcium (eur - . /month), and vitamin d (eur /month). these costs cannot be compared with the costs of a standard care of an asymptomatic haart-treated p (eur to /month) and the immunological, virologic, laboratory and clinical controls made at least quarterly. like postmenopausal osteopenia/osteoporosis (burdened by a greater risk of bone mass anomalies) also hiv disease should be investigated from multiple cost-effectiveness points of view to establish which p are the preferred candidates for a dexa screening when this examination is more useful during hiv disease course and therapy, when the exam should be repeated and when and how to intervene pharmacologically to prevent serious and potentially invalidating complications. a comparative study on the cost of new drugs in different therapeutic categories m. falagas, k. fragoulis, g. zouglakis, i. karydis (athens, gr) objectives: drug treatment is becoming more expensive due to the increased cost for the introduction of new drugs and there seems to be an uneven distribution of medication cost for different therapeutic categories. we hypothesized that the cost of new antimicrobial agents may differ from that of other therapeutic categories and this may play a role in the stagnation of development of new antibiotics. methods: we performed a pharmaco-economical comparative analysis of the drug cost of treatment for new agents introduced in the united states drug market in various therapeutic categories. we calculated the drug cost [in us dollars (usd)] of a -day treatment of all new drugs approved by the fda during the period between january and july , according to the red book pharmacy's fundamental reference. results: new anti-neoplastic agents were found to be the most expensive drugs in comparison to all other therapeutic categories with a median -day drug-treatment cost of usd compared to the median -day drug-treatment costs of all other categories ranging from to usd (table) . on the other hand, new antimicrobial drugs were found to be much less expensive with a median -day drug-treatment cost of and usd for all anti-microbial agents and for anti-microbial agents excluding anti-hiv medications, respectively. conclusion: the drug-treatment cost of new medications varies considerably by different therapeutic categories. this fact may influence industry decisions regarding the development of new drugs and may play a role in the shortage of new anti-microbial agents in the fight against the serious problem of anti-microbial resistance. usage and expenditure of f-quinolones in a tertiary hospital in t.a. peppas, k. malengou, n. zachos, d. voutsinas, o. kosmopoulou, n. galanakis (piraeus, athens, gr) objective: our aim was to assess -f-quinolone ( fq) usage, distribution and expenditure over years. objective: to analyse antiobiotic utilization in croatia using anatomical-therapeutic-chemical (atc) drug classification system and number of defined daily doses (ddd). methods: data on the number of packages and purchase price were collected for each individual drug. these data were used to calculate the number of defined daily doses (ddd) and ddd per inhabitants per day (ddd/tid). data obtained from % of pharmacies and % of hospitals were extrapolated to the total number of pharmacies and hospitals in croatia. drug utilization % (du %) segment was used as a prescribing quality indicator. results: in , the overall utilization of antibiotics in croatia amounted to . ddd/tid. according to drug groups, penicillins (j c) showed highest utilization ( . ddd/tid), predominated by the subgroup of penicillin combinations (including beta-lactamase inhibitors, j cr) with . ddd/ tid, within which the combination of amoxicillin + clavulanic acid accounted for . % with . ddd/tid. broad-spectrum penicillins (j ca) accounted for . %( . ddd/tid) of total penicillin utilization, with a . % predominance of amoxicillin ( . ddd/tid). cephalosporins (j d) ranked second with . ddd/tid, followed by macrolides and lincosamides (j f) with . ddd/tid, with an . %predominance of macrolides (j fa) with . ddd/tid. among the latter, azithromycin showed highest utilization with . ddd/tid, accounting for . % of total macrolide utilization. tetracyclines (j a) ranked fourth with . ddd/tid, accounting for . % of overall antibiotic utilization, followed by quinolones with . ddd/tid, other antimicrobials with . ddd/tid, and aminoglycosides with . ddd/tid. sulfonamides (j e) accounted for a negligible proportion of overall utilization. du % segment included of antibiotics registered in croatia, with amoxicillin + clavulanic acid as the leading one, followed by cephalexin with . , cefuroxime with . , azithromycin and norfloxacin with . each, nitrofurantoin with . and clarithromycin with . ddd/tid. hospital utilization accounted for . % of overall antibiotic utilization expressed in ddd/tid and . % of the respective financial cost, predominated by aminoglycosides (j g) with % and . %, and lowest proportion of tetracyclines (j a) with . %, and . %, respectively. conclusion: the utilization of antibiotics in croatia is among the highest in europe, mostly due to overuse of amoxicillin + clavulanic acid, which has no rational ground in professional guidelines. objective: the objective of the research was to analyse antibiotics prescripsion behavior by family doctors and specialists treatments prior to and after the introduction of the health care reform in poland. materials and methods: prescriptions from the first six months of and were compared. the data was collected from two randomly chosen pharmacies in the city of zabrze that supply the citizens of the silesian agglomeration from various social backgrounds. taking into account the value of a single antibiotics package and the price a patient has to pay for it an average price of medications prescribed by family and specialist doctors was calculated. results: a total of prescriptions were analysed out of which dated from and from .in the first half-year of the percentage of prescriptions for antibiotics reached . % on average, and in the year -the average was . %. in the first half-year of family doctors mostly prescribed: penicyllins ( %), makrolids ( %), cephalosporins ( %), tetracyclins ( %), chinolons ( %). in the same period spcialist doctors prescribed: penicyllins ( %), cephalosporins ( %), makrolids ( %), tetracyclins ( %), lincozamids ( %), chinolons ( %). in the first half-year of family doctors most often prescribed penicyllins -( . %), makrolids -( . %), cephalosporins -( . %), tetracyclins -( . %) and lincozamidsbased ( . %) treatments specialist doctors, on the other hand, prescribed penicllins ( . %), makrolids ( . %), cephalosporins ( . %), tetracyclins ( . %), lincozamids ( . %) and chinolons ( %). the average prices of the prescribed medications in the years and were, respectively: for family doctors-eu . and . , for specialist doctors eu . and . . conclusions: there has been a considerable increase in the percentage of prescriptions for antibiotics from . % (in ) to . % (in ). the tendency towards prescribing antibiotics in the specific groups of doctors has not changed significantly. in both years prescriptions for antibiotics were in line with the recommendations. also, prices of medications prescribed by family doctors have risen. internet guide on antimicrobial resistance a. sosa, f. traub, s. valovic, p. chea (boston, us) objectives: ( ) to organize the plethora of information available, providing clinicians the tools to easily access available online resources that include academic institutions, professional societies as well as sites maintained by private individuals; ( ) to inform clinicians of new advances in the epidemiology, diagnosis, treatment, and prevention of most common infections; ( ) to inform on subjects such as clinical trials in antimicrobial resistance, information about specific pathogens and their infections, genomic resources, culture collections, electronic images of pathogens and antimicrobial agents, antimicrobial resistance lecture and teaching materials, environmental health and safety information, and a listing of websites of infectious disease and clinical microbiology professional societies. methods: we defined four inclusion criteria after extensive consulting with apua staff and scientific advisory members: ( ) recognized/reputable source; ( ) high quality of information presented; ( ) potential usefulness to medical professionals and the general public; and ( ) ease of navigation. ideal parameters were determined for the guide's scope and the appropriate sources identified online were subsequently reviewed. a set of broad categories was established to organize the topics and the online resources. sites reviewed included those maintained by the federal government, academic institutions, nonprofit organizations, and commercial entities. some personal websites were included because of their quality and their association with academic institutions. this review is intended as an introduction to amr websites. results: with use of popular search engines, such as google, yahoo!, and altavista, we initially identified a great number of websites. using broad search terms, such as "antibiotic resistance," we identified , , web addresses. the term "antimicrobial resistance" generated , hits, and the term "drug resistance" generated , , hits. conclusions: websites found were classified following a systematic topic structure. each website listed describes: ( ) full citation of the resource: author/editor and title of website; ( ) date of publication or last revision; ( ) methods and results: the portal is free to use but requires registration and has more than registered users as of november . of these, % are in-training positions, % hold a faculty position in infectious diseases (id), microbiology or hemato-oncology, % are specialists in a non-university, but a teaching setting. the remaining are a mixed group of primary physicians, other speciality doctors and pharmaceutical company workers. running costs of the portal are partially covered by educational grants from pharmaceutical sponsors who have no role in organization of the site, but their names are acknowledged. articles chosen by the two faculty members, one id physician and one haematologist, are sent to registered users by daily e-mail postings. these are selected from toc alerts of various core clinical journals and well known educational web sites (e.g. cdc, who, medscape). a short turkish summary is provided and the reader is referred to the abstract and if available, to the free full text of original article with a link. other materials included guidelines, free slide sets, study protocols and updates from the group, cme activities and meeting announcements. registrants may also use the site for expert opinion. during the trial period, the site has been visited times with hits. approximately . gb material was downloaded. the frequency of readings are related with the time (highest between . - . am during weekdays and lowest during weekends), the type of documents (i.e. educational materials and guidelines), popularity of the news (e.g. peaked during an epidemic of avian influenza when related news and articles announced). the pages are most frequently visited by id specialists followed by clinical microbiology, haematology and pharmaceutical company workers ( %, %, % and % respectively). conclusion: timely published medical data have high attraction rates among physicians. our results also indicated that, a web page gets ''old'' after about a month of publishing, emphasizing the importance of well-timed announcements of the portal material. neli and nric survey: information needs of infectious disease professionals p. kostkova, s. d'souza, g. madle, j. mani-saada, s. wiseman, a. roy, j. weinberg (london, uk) healthcare professionals are increasingly facing the problem of information overflow. it is getting impossible to keep up-to-date with the latest research findings, care guidelines and pathways, government strategies and national and local policies. internet enables an instant information dissemination enabling access to the latest results at any time as well as informal knowledge exchange by using chat rooms and discussion forums. however, it is getting increasingly difficult for busy professionals to find reliable quality-assured information on the internet when they need it. national internet libraries in the uk are addressing this problem: the umbrella portal national electronic library of infection neli (http://www.neli.org.uk) providing a single access portal to quality-assured information on treatment, diagnosis, prevention and management of infection diseases, and the national resource for infection control nric (http:// www.nric.org.uk) -a single-stop shop for policies, guidelines and research around infection control, hosted by neli. to better meet the information needs of these internet portals, accessed by unique users per month, we are conducting an information needs study to explore clinical questions, user needs and disease priorities of users seeking answers on neli and nric. a pilot qualitative online questionnaire-based study revealed that our users come from the variety of professionals: clinical scientist, consultant, registrar, psychotherapist, lecturer, gp, medical librarian, information scientist, health protection. these have questions mainly around hiv, tinea, molluscum contagio, meningitis, cold, mrsa, lyme, toxoplasma, chicken pox, influenza, diarrhoea and vomiting, rash, staph. aureus, traveller infections antibiotics resistance, malaria, mmr, meningitis, viral myocarditis, anthrax, smallpox, and tb. this is in line with our quantitative weblog-based evaluation of the most commonly access topics on neli by nhs-based users: antimicrobial resistance and hae ( . %), tb ( . %), meningitis ( . %), hiv ( . %), chlamydia ( . %), e. coli ( . %), staph. aureus ( . %), adenovirus ( . %), blood borne infections ( . %). the results of the ongoing analysis of google search keywords that brought users to neli and nric will be discussed. further results identifying the needs specific to the infection disease professions will be discussed in relation to differences in the national variations in information needs and priorities. training in infection s. d'souza, p. kostkova, f. cooke, a. holmes (london, uk) specialists today require prompt access to quality information in order to work effectively. the diversity of specialist interests in the field of infection has led to the formation of a large number of professional and scientific societies. these play an increasingly important role in ensuring that the trainee is effectively supported, not only during the period of training, but also in longer-term personal development. details of relevant societies, conferences, grants etc are, on most society websites, confined to those for either that society or others in that specialty only, and knowledge of the numerous places in which to look for this information is necessary to find out the latest information. training in infection (tii -www.trainingininfection.org.uk) is an online resource, primarily aimed at infection specialist trainees but useful throughout the career path, which brings together this information into one central access point, so that users from all infection specialties can find the appropriate information for their specialty quickly and easily. it identifies and links to the key relevant resources covering a broad range of infection related disciplines in a dynamic database structure. information on societies, conferences, grants, journals, textbooks and more are available on the site, and have been put together to create a one-stop infection training portal. online discussion forums to be implemented will allow trainees' to share ideas and make the most of their combined expertise, and users will be able to receive alerts on new information in their specialty as well as be reminded of conference deadlines, journal submission deadlines etc. the ability to discuss regional issues online within specialties also aims to promote greater local and international collaboration. training in infection is endorsed by the national electronic library of infection (neli -www.neli.org.uk), an established digital library bringing together the best available online evidence-based resources on the investigation, treatment, prevention and control of infectious diseases. research designs and statistical methods in medical abstracts m. kompoti, m. matsagoura, a. koutsovasilis, a. koutsovasili, s. drimis (athens, gr) statistical methods used in biomedical research articles are being increasingly scrutinized in medical journals. however, no such strict policy is generally applied in abstracts presented in medical congresses. objective: this study aimed at assessing the frequency of research designs and statistical methods reported in abstracts presented in two successive years of the european congress of clinical microbiology and infectious diseases (eccmid). material and methods: we reviewed all abstracts included in the abstract book of the th eccmid (prague ) (pg) and the th eccmid (copenhagen ) (cp). all abstracts of original research studies but no abstracts of lectures were included in our study. two independent investigators read all abstracts and extracted information concerning origin, type (clinical, laboratory, animal model), research design, sample size and statistical methods used in the study. data analysis was performed with logistic regression and pearson's chi-square test for categorical variables and student's t-test for continuous variables. statistical significance level was set at p < . . results: a total of abstracts were included in the analysis according to eligibility criteria ( from pg and from cp). laboratory studies prevailed ( %) followed by clinical studies ( %) and experimental studies with animal models ( %). the majority ( . %) of the studies were observational (retrospective, prospective, cross-sectional) of which . % concerned diagnostic accuracy testing of laboratory methods and . % were pharmacological studies, . % were randomized controlled trials. statistical evaluation was clearly described in . % of abstracts ( . % in pg and . % in cp, p < . ), while the rest of abstracts included only descriptive statistics or no statistics at all. the proportion of statistical methods reporting varied according to the type of the study (animal model studies . %, clinical studies . % and laboratory studies . %, p < . ). multicentre research studies reported statistics more frequently than single-center studies ( . % vs. . %, respectively, p = . ). conclusions: statistical analysis is an inseparable part of original research. research design as well as the implemented statistical methods should always be reported in an adequate manner, thus improving the scientific quality of abstracts. antimicrobial pk/pd p nxl -oral streptogramin: a phase i, doubleblind, single escalating oral dose study to evaluate safety, tolerability and pharmacokinetics in healthy adult male volunteers m. rangaraju, j. rey, j. hodgson (romainville, vitry sur seine, fr) background: nxl (formerly xrp ) is a novel semisynthetic oral streptogramin that consists of a / (w/w ratio) association of a pristinamycin ia (pi) derivative and a pristinamycin iib (pii) derivative. nxl is being developed for the treatment of respiratory tract and skin and skin structure infections. methods: healthy male subjects were enrolled in this study. subjects in each of cohorts ( mg, mg, mg, mg, mg and mg) received either nxl ( ) or placebo ( ) . an additional cohort of subjects received a single dose of mg nxl in fasting and fed conditions. blood and urine samples for pk analysis were collected at multiple time points. safety was assessed via adverse events, physical examination, clinical laboratory data, ecg and cardiac monitoring. results: nxl administered as mg capsules at single doses from mg to mg was well tolerated and safe. there was no serious or severe adverse event, no dosedependency in the number of aes or their severity, no significant variation in blood pressure or heart rate, no abnormality on ecg recording, and no clinically significant changes compared to baseline for laboratory parameters. both components were rapidly absorbed; pi being slightly more rapidly absorbed than pii. the cmax and auc ( -t) increased approximately in proportion with dose. the proportion of pi and pii components estimated on mean exposure values was approximately comparable to that administered ( / ), indicating that the relative bioavailabilities of pi and pii are simila. elimination half-life ranged from between to hours for pi to to hours for pii. food increased the bioavailability of pi and pii by approximately %. conclusions: nxl is safe, well tolerated and exhibits predictable pk properties in healthy volunteers in doses up to mg administered as a single dose. correlation of vancomycin and daptomycin susceptibility in staphylococcus aureus in reference to accessory gene regulator polymorphism and function w. rose, m. rybak, b. tsuji, g. kaatz, g. sakoulas (detroit, new york, us) objective: polymorphism at the accessory gene regulator (agr) locus in s. aureus (sa) defines groups (i-iv). agr group ii sa have been associated with glycopeptide treatment failure in patients. sa with loss of agr function appear to have a higher tendency to become vancomycin (v) resistant. it is unknown whether this association only pertains to glycopeptides. we examined the effect of varying v and daptomycin (d) against agr+ and agr null pairs in an in vitro pharmacodynamic model (ivpm). methods: agr group i and ii wild-type prototype and knockout (tetm::agr) pairs were evaluated. mic values were determined according to clinical laboratory standards institute. ivpm glass and hollow fibre models were used to simulate dosages and auc/mic exposures for v ranging from . mg- g q h fauc/mic range - mg/l*h, and d . mg/kg- mg/ kg/day fauc/mic range . - . the dosage regimen and auc/mic breakpoints that produced resistance was then evaluated in the hollow fibre ivpm. all ivpm simulations were performed in duplicate over h. resistance was evaluated using and x mic screening plates at , , , , and h. results: pre-exposure mic values for agr i± and agr ii± were . / and for v and . lg/ml for d respectively. vintermediate resistance (mic = mg/l) was detected in both agr i and ii null strains at a simulated v dosage of . mg q h (auc/mic ), representing an mic increase of - fold. this breakpoint for resistance was verified in the hollow fibre model. although significant regrowth was noted with suboptimal dosing of d, no resistance was detected on d screening plates for any daptomycin regimen evaluated. conclusions: exposure of sa to v approximating / of optimal serum concentrations resulted in the development of heteroresistance in the agr null group i and ii. loss of agr function did not correlate with the development of d resistance despite suboptimal simulations of d exposures. these results implicate loss of agr function important to the development of glycopeptide resistance but not to loss of susceptibility to d. teicoplanin efficiently penetrates into the rabbit infected vitreous but may enhance expression of virulence factors at sub-inhibitory concentrations e. forestier, f. jehl, c. gallion, r. andres, s. bronner, l. leininger, g. prévost (strasbourg, fr) objectives: fluoroquinolones are the antibiotics that most efficiently penetrate inside vitreous. however, alternative treatments for endophtalmitis may be required in some cases for example resistant bacteria. we used a rabbit experimental model of endophtalmitis to evaluate the penetration of teicoplanin in different conditions. the influence of subinhibitory concentrations of teicoplanin was also evaluated on the expression of s. aureus virulence factors. methods: new zealand rabbits (> kgs) received one or repeated doses of intra-venous (iv) teicoplanin ( mg/kg) every hours for days plus one dose a day for more days. another group of rabbits was infected by cfu of a methicillin resistant s. aureus ibs (cmi = . mg/l) producing enterotoxin a, panton-valentine leucocidin and luke-lukd. they were administrated - hours later with mg/kg teicoplanin, as a single dose or as to reach the steady state. vitreous ( ll) was sampled before new injections of teicoplanin or at indicated time as well as blood before or min after teicoplanin injection. teicoplanin concentrations were measured by hplc. bacterial counts were recorded and expression of virulence factors was semi-quantified by dedicated competitive rt-pcr tests. results: in safe eyes, teicoplanin penetration remains moderate reaching about mg/l within about - h after one iv injection. the half-life of teicoplanin in the rabbit vitreous is about hours. after days of repeated injections, intra-ocular concentration stabilises around mg/l while residual blood concentrations were comprised between - mg/l. in infected eyes, teicoplanin, when repeatedly administrated after the beginning of clinical signs, i.e. h postinfection = cfu/ml, reaches intra-vitreal concentrations of . ± . mg/l h post-infection, and increases to . ± . mg/l h post-infection and h after a fourth injection. however, at sub-inhibitory concentrations (~ mg/l), it may be responsible for a significant increase of agr, gammahemolysin hlga, luked and panton-valentine leucocidin luk-pv expressions with ratio ranging from to folds. conclusion: these preliminary results strongly suggest that teicoplanin iv administration constitutes an interesting alternative therapy for endophtalmitis provided high intraocular concentrations are rapidly obtained. investigations now concern optimisation of teicoplanin dosage regimen. pharmacokinetics of temocillin in intensive care patients and monte carlo simulations to evaluate susceptible breakpoints j.w. mouton, r. dejongh, v. basma, p. tulkens, s. carryn (nijmegen, nl; genk, brussels, be) background: temocillin (tmo) is a narrow spectrum penicillin with good activity against gram negative micro-organisms including esbl and ampc producers. little pharmacokinetic data are available however. we performed a pharmacokinetic study in icu patients receiving tmo g q h. parameter estimates were used to predict concentrations during continuous infusion (coinf) and compared with data obtained from other icu patients receiving coinf to validate the model. the model was then used to perform monte carlo simulations (mcs) to determine probabilities of target attainment (ptas) for pharmacodynamic indices (pdi) in order to evaluate and suggest clinical breakpoints. methods: blood samples were taken from icu patients prior to (t = ) and after (t = , , , , h) a m infusion of g tmo (n = ) or after h during coinf with g/ h (n = ), and then cooled, centrifuged and stored at ) °c until analysis by hplc. protein binding was determined using an ultrafiltration method. results were used to estimate population pharmacokinetic parameters by winnonmix including the covariance matrix. miclab was used to perform simulations for coinf as well as to perform mcs ( cycles) and obtain ptas for the unbound fraction including % confidence intervals (ci) for the target concentrations. ft>mic was chosen as the pdi because of the pharmacodynamic properties of tmo. results: protein binding was %. a one-compartment model best fitted to the data, with estimates (se) of vc = . ( . ) l and k = . ( . ) /h corresponding to a mean half-life of . h. using these estimates, the predicted unbound concentration during coinf was . mg/l, while the mean concentration in the other patients was . mg/l, a bias of less then %. the breakpoint mic for a mean ft>mic of % was mg/l. however, mcs -taking the variation in the population into account -indicated that % ptas of a g q h dose were obtained at , , and mg/l for , and % ft>mic, respectively. the % ci at % ft>mic indicated a clinical breakpoint of mg/l. the % ci was relatively large, as expected from data obtained in patients rather than volunteers. conclusion: the population pharmacokinetic estimates from icu patients were very well in agreement with the validation study, with a bias of < %. the mcs indicate a susceptible breakpoint for temocillin of £ mg/l provided an administration of g q h is used. tissue penetration and pharmacokinetics of moxifloxacin in diabetic foot infections:an interim analysis j. majcher-peszynska, k. karrasch, m. saß, r. mundkowski, a. gussmann, p. kujath, b. ruf, w. schareck, h. koch, b. drewelow (rostock, bad saarow, lubeck, leipzig, beeskow, de) objectives: with its broad spectrum of activity against grampositive, gramnegative and anaerobic organisms moxifloxacin covers the pathogens of the mainly polymicrobial infections associated with the diabetic foot. inflammatory and fibrotic processes in diabetic foot infections (dfi) contribute to impaired tissue penetration of antibiotics. in addition, diabetic patients represent a pharmacological risk population, physiological changes in diabetic patients may alter the pharmacokinetics of antibiotics. the study was designed to investigate the penetration of moxifloxacin into perinecrotic tissue in patients with dfi and the pharmacokinetic properties of moxifloxacin in diabetic patients. methods: the interim analysis of this open, multicentre study included adult, hospitalized male and female patients (mean age: . years) with type diabetes mellitus and dfi. the pharmacokinetic parameters of moxifloxacin and penetration into dfi tissue at steady state (day to ) following once daily administration of mg iv or po were evaluated. correlations between penetration of moxifloxacin and clinical and laboratory parameters were examinated. results: in all patients the moxifloxacin concentrations measured in infected diabetic foot wounds hours after administration exceeded the in vitro mic values of susceptible staphylococci ( . mg/l). the moxifloxacin concentrations achieved in dfi tissue correlated more strongly with the auc - (r = . ; p < . ) than with the corresponding plasma concentrations (r = . , p < . ), but not with the extent of the systemic inflammation and the blood glucose level. taking into account the predictive pk/pd parameters for moxifloxacin (based on an in vitro mic value of . mg/l for staphylococcus aureus) a therapeutic success can be expected (auc /mic: . ; cmax/mic: . ). significant differences between the routes of administration (iv vs po) were only observed for tmax (p < . ) and t / (p < . ), but not for other clinically relevant parameters (auc - , cmax, moxifloxacin tissue concentration). this allows sequential therapy i.v./p.o. in this indication. conclusion: based on adequate plasma concentrations in diabetic patients, the sufficient penetration into dfi tissue and the possibility of a sequential therapy, moxifloxacin representsfrom a pharmacological point of view -a valuable therapeutic option in the treatment of diabetic foot infections caused by susceptible organisms. fluoquinolones effects on patient lymphocytes during prolonged treatments e. bertazzoni minelli, a. benini, d. doria, p. franceschetti, m.e. fracass (verona, it) fluoroquinolones (fq), widely used in clinical practice, are well tolerated. the most common adverse reactions are those affecting gastrointestinal tract, phototoxicity and allergy. the aim of the study is to evaluate the possible cellular damage in lymphocytes of patients treated with different fqs according to pharmacokinetic data. blood samples obtained from thirty-six patients treated with ciprofloxacin (cpx, pts), levofloxacin (lvx, pts.) and moxifloxacin (mfx, pts) at different doses were analysed. patients treated with cpx and lvx were in therapy with other drugs (diuretics, cardiovascular drugs, omeprazole, antiinflammatory drugs, etc.). mxf treated patients were not in therapy with other drugs. samples were collected at time (before fqs administration) and after and days of treatment. serum levels of fqs were determined with microbiological method and hplc. comet test was performed on lymphocytes, to evaluate dna damage. gsh levels were determined as efficiency marker in metabolic process of detoxification. cpx showed good serum concentrations; its levels increases proportionally with administered doses (from to mg). lvx concentrations resulted in good inhibitory levels after treatments ( mg) both per os and i.v. patients orally treated with mg showed similar serum levels (from . to . mg/l). mfx levels were between . and . mg/l after and days. repeated cpx administration induced a dose-dependent increase in all dna damage parameters, with statistical differences after treatments. mfx ( mg) and lvx administration didn't induce dna damage after and days. intracellular levels of gsh were similar in all treated groups, even if cpx treated patients showed the lowest concentrations. no statistical correlations were found between all parameters studied. these data indicate that cpx induce dna damage in lymphocytes in combination with a reduced efficiency in detoxification system. this effect does not seems to depend on high intersubject variability for fqs administered doses, co-administration of other drugs, different ages of patients and low samples numbers. effects of single fqs molecules seems to be structurespecific and selective. objectives: ertapenem is a carbapenem commonly used to treat intra-abdominal infections. the antibacterial spectrum includes the major causative pathogens. clinical trials proved excellent clinical and microbiological efficacy in peritonitis. on the other hand in inflammatory pancreatic diseases sufficient antibiotic concentration in the inflammatory tissue is vital for the outcome of the disease. we therefore investigated ertapenem concentrations in pancreatic tissue and juice in comparison to the plasma levels measured at the same time. methods: in a prospective clinical trial ertapenem was given in a dosage of g i.v. minutes prior to operation in patients ( - years) suffering from chronic pancreatitis or pancreas carcinoma undergoing pancreas resection. blood samples were collected every minutes during the operation. moreover we collected pancreatic tissue and pancreatic juice shortly before resection and shortly before finalisation of the anastomosis. the samples of ertapenem (blood, juice, tissue) were determined by hplc. results: in patients ( female, male, mean age . ± . years) ertapenem blood concentrations were determined and demonstrated intraoperatively high concentration ( ± mg/l) above mic values for major expected pathogens. concomitantly in of these patients ertapenem concentration was determined also in pancreas tissue and pancreas secretion (in further patients in pancreas secretion only). in / patients sufficiently high ertapenem levels were detected in pancreatic tissue. in patients with chronic pancreatitis no accumulation was seen. mean pancreas tissue concentration was . ± . lg/g tissue. of patients with pancreas carcinoma had increased ertapenem levels in pancreas secretion but only of patients with chronic pancreatitis. conclusion: in patients with pancreas carcinoma, ertapenem levels were measured in pancreatic tissue as well as in pancreatic secretion and penetration seems to be similar to imipenem. due to chronic inflammation and possibly altered microcirculation only in one half to one third of chronic pancreatitis patients ertapenem levels were detected. bacterial strain-independent pharmacodynamics of linezolid/doxycycline combinations with staphylococcus aureus: -day simulations using an in vitro dynamic model m. smirnova, i. alferova, i. lubenko, y. portnoy, s. zinner, a. firsov (moscow, ru; cambridge, us) objective: to delineate the possible advantages of linezolid (l)/doxycycline (d) combinations over monotherapy, the pharmacodynamics of l, d and l+d were studied with s. aureus. methods: s. aureus atcc and a clinical isolate s. aureus were exposed to twice-daily l (half-life h) and once-daily d (half-life h), alone and in combination ( : ratio based on -h auc/mics), for five consecutive days. to provide simultaneous mono-exponential elimination of l and d with different half-lives, a previously described dynamic model was modified according to blaser and zinner. the mics of l were . and . mg/l and mics of d were . and . mg/l for s. aureus atcc and s. aureus , respectively. nine dosing regimens were simulated with each organism exposed to different auc/mics (in hours): l , l and l ; d , d and d ; l + d , l + d and l + d . the cumulative antimicrobial effect was expressed by its intensity (ie) measured from the start of treatment to the time after the last antibiotic dose when numbers of antibiotic-exposed bacteria reached at least cfu/ml. emergence of resistance was monitored daily by quantitating surviving organisms on agar plates containing x and xmic of l or d. results: with both s. aureus atcc and s. aureus exposed to l or d, ie increased with increasing simulated auc/ mic ratios, although significantly higher ies were produced with l , l and l treatments relative to d , d and d treatments. each of the combined treatments, i.e., l + d , l + d and l + d , produced much greater ies than the sum of l and d ies observed in the respective mono-treatments with both s. aureus strains. based on population data, a pronounced selection of s. aureus resistant to d occurred in all mono-treatments with d. it was also observed with l + d and, to a lesser extent, with l + d but not with l + d . no resistance to l was observed with l mono-or combination treatments. conclusions: these data predict a synergistic interaction of l with d against s. aureus. anti-staphylococcal effects of telavancin in an in vitro dynamic model: impact of different half-lives and initial concentrations that simulates normal (nek) and impaired elimination kinetics (iek). materials and methods: a glycopeptide intermediately susceptible strain of s. aureus (gisa) mu- with a telavancin mic of . mg/l was selected for the study. with both nek and iek simulations at a starting inoculum of log cfu/ml, gisa mu- was exposed to different ratios of the peak concentration (cmax) to the mic of telavancin (as a single dose), i.e., . , . and . based on time-kill data, the intensity of the antimicrobial effect (ie -the area between control growth and time-kill curves) was determined from time zero to the time when the effect no longer could be detected, i.e. the time after the last dosing at which the number of antibiotic-exposed bacteria reached log cfu/ml. results: in each treatment, bacterial regrowth followed gradual reduction in the starting inoculum during the first h (similar in nek and iek simulations) that led to significantly lower minimal numbers of surviving organisms in iek simulations compared to nek simulations. despite similar rates of initial killing, times to regrowth were much longer in iek than nek simulations. at a given cmax/mic ratio, the ies observed in iek were greater than in nek simulations (figure). conclusions: these findings demonstrate pharmacokineticdependent pharmacodynamics of telavancin with staphylococci. pharmacokinetics of amoxicillin in pregnant women with pre-term premature rupture of the membranes objectives: amoxicillin is widely used during pregnancy, in particular to treat group b streptococcus. insufficient knowledge on the pharmacokinetics just before and during delivery, could pose patients with preterm premature rupture of the membranes (pprom) at serious risk for under dosing. we investigated the pharmacokinetics in patients with pprom in this critical situation. methods: seven healthy women at - weeks of gestation were included. they received g (first dose g) amoxicillin for pprom according to local guidelines. from each patient - blood samples were taken. antibiotic serum concentrations were determined by a validated hplc method. pharmacokinetic parameters were estimated by population pk modeling using nonmem. to discriminate between various models the minimum value of the objective function (mvof) was used. a reduction of > in mvof was considered significant. results: a three-compartment pharmacokinetic model best described the time course of amoxicillin. the clearance and volume of distribution of the central compartment (vc) were estimated at respectively ± . l/h and ± . l (mean ± se). estimates of the parameters and model discrimination improved when we assumed the size of the third compartment to be equal to the first compartment. the residual error was found to be proportional to the serum concentrations. most of the inter-individual variability could be explained by variation of clearance. the mean volume of distribution at steady state (vss) and terminal half-life were . l and . h respectively. estimated values of elimination and distribution rate constants were: k = . h- , k = . h- , k = . h- , k = . h- and k = . h- . as was to be expected due to the small population size, no significant relationship was observed between the individual posthoc estimates for clearance and patient characteristics. conclusion: here we describe the pharmacokinetics of amoxicillin in pregnant women with pprom. it was found that the pharmacokinetics clearly differs from that in nonpregnant individuals. clearance and vss were significantly higher and the terminal half-life was shorter. furthermore, a -compartment model was found to describe the data better than a -compartment model. it is an intriguing question whether this rd compartment is a unique feature associated with pregnancy. these data offer a theoretical basis to make proper dose-adjustments in a particular patient group in a critical condition. penetration of piperacillin and tazobactam in severe acute pancreatitis objectives: acute necrotizing pancreatitis is still related to an extremely high mortality rate, based on local infectious complications, particularly in necrotizing areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotizing pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of betalactamases. on that score, the penetration of co-administrated pip and bli into inflamed or necrotic pancreatic tissue has not been investigated yet. methods: adressing the penetration capability of bsp and bli a clinical trial was designed to investigate the penetration of piperacillin (pip) and tazobactam (taz) in patients with severe necrotizing pancreatitis undergoing pancreas surgery. samples (n = ) were taken from plasma (pl), necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of . g pip and . g taz. concentrations of pip/ taz were determined by hplc/ uv. results: mean plasma concentrations at . h after application were . ± . mg/l (pip) and . ± . mg/l (taz). exceeded in pl and bs, nearly reached in pn but not in pft. the concentration of pip in combination with taz exceeded or reached the mic in pl, pn and bs against e. coli, klebs. spp., enterobacter, proteus spp. and clostr. spp., in pl and bs even against pseudomonas and bacteroides. conclusion: given in combination both -pip and taz -have been demonstrated to reach rapidly effective inhibitory concentrations in inflamed and necrotic compartments of pancreatic and peripancreatic tissue. co-administration of piperacillin and tazobactam may have a potential clinical benefit in prevention and treatment of local infectious complications of severe necrotizing pancreatitis. pk/pd challenges of in vitro time-kill curves -a new modelling approach s. schmidt, o. burkhardt, w. treyaprasert, h. derendorf (gainesville, us; bangkok, th) objective: in vitro pk/pd models, based on time-kill curve data, have become a powerful tool to predict the in vivo situation. up to date, several modelling approaches have been undertaken to develop suitable pk/pd models that fit in vitro data sufficiently well. widely used simple sigmoid emax models meet these criteria only partly. a further approach was undertaken to address the weak points of currently used models and applied to model the effects of ceftriaxone against escherichia coli. methods: constant concentration time-kill curves were performed in mueller-hinton broth (mhb, difco) with and without bovine serum albumin (bsa) g/l. using concentrations of ceftriaxone, ranging from . mic to mic, the change in number of bacteria (cfu/ml) versus time was linked to its effect. escherichia coli atcc was employed as the test organism. samples were taken at , . , , . , , , , and hours. the data were modelled simultaneously, using a modified sigmoid emax model and the software scientist Ò for windows tm . results: a differential equation, characterized by growth rate constant (k ) times the starting number (n) of bacteria represent the simplest case. barging from log-growth phase to stationary phase can be described by an additional nmax term. however, bacteria do not necessarily start growing in the loggrowth phase. this delay in onset of growth can be modeled by an exponential term, characterized by a factor beta (b) and time (t). to describe the overall change in number of bacteria not only growth but also concentration (c) dependent kill has to be taken into account. from certain drug specific concentrations on, a maximum effect is reached, described by the maximum kill rate constant kmax. however, it may be necessary to model a delay in the onset of kill with an additional exponential term, characterized by a factor alpha (a) and time (t). finally, a hill factor/shape factor (h) is necessary to smooth the predicting curves out. as shown in figure this new model meets the in vitro time-kill curve data sufficiently well. the final equation including all parameters described above is: conclusion: the proposed model was able to describe the observed data much better than a simple emax-model. incorporating two additional terms into the model, the in vitro situation could be described much better, taking the delay in onset of growth and kill into account. objectives of this study were ( ) to describe the pharmacodynamic (pd) profiles of bpr mg iv q h as a hr infusion & mg iv q h as a -hr infusion; ( ) to determine the overall probability of target attainment (pta) by weighting for the expected distributions (dis) of renal function (rfx) in the populations (pop) of interests; ( ) to determine the organism-specific pta against the pathogens encountered in phase ii trials. methods: subjects (total samples) were studied (phase i/ii subjects). samples were analysed using bignpod. to assess the impact of differing degrees of rfx impairment on pta, crcl (crcl-cockcroft & gault method) was employed as a covariate in the pop pk analysis. monte carlo simulation (mcs) ( subjects) was performed with adapt ii. overall pta was calculated for - % ft>mic. weighting for the expected dis of rfx in the pop of interests was accomplished by using the dis of crcl observed in previous registration studies of the same indications (cssti and np). dis of mics for pathogens was supplied by sponsor. results: in the pop pk analysis, the pop mean (sd) values for volume, clslope, clintercept, kcp and kpc were: . ( . ) l, . ( . ) l/hr, . ( . ), . ( . ) hr- and . ( . ) hr- , respectively. the obs-pred plot was obs = . x pred + . ; r = . after the bayesian step. in the mcs analysis of bpr mg iv q h, the pta of achieving % ft>mic & % ft>mic exceeded % for mics values £ mg/l & £ . mg/l, respectively. for bpr mg iv q h, the pta of achieving % ft>mic exceeded % for mics values £ mg/l. in the organism-specific analysis, the pta of a static effect ( % ft>mic) exceeded % for both mssa & mrsa for bpr mg iv q h. bpr mg iv q h provided a > % pta of a cidal effect ( % ft>mic) for both mssa & mrsa. for gnb, the pta of bpr mg iv q h in achieving a cidal effect ( % ft>mic) exceeded % for non-ampc-bearing gnb. for ampc-bearing gnb, the pta of achieving a cidal effect was . %. conclusions: an extensive evaluation of the pd of bpr was performed to estimate the overall activity of bpr against target pathogens. these findings need to be validated in the clinical trial arena. investigation of different levofloxacin regimens in patients with acute complicated urinary tract infections p. tenke, r. benko (budapest, szeged, hu) objective: in the present study we aimed to find out if a continuous or an intermittent levofloxacin ( · mg) treatment is more advantageous for patients with acute complicated urinary tract infection (uti) caused by urinary obstruction. we investigated if levofloxacin adsorbs to the surface of the foreign body, which was inserted with the aim of temporary resolution of ureteral obstruction. preventive effect of levofloxacin on bacterial biofilm formation and incrustation was also evaluated. methods: we enrolled and randomised patients who had acute uti caused by urinary obstruction. obstruction was resolved with double j stent (djs) insertion or percutaneous nephrostomy (pcn) and meanwhile, antibiotic treatment was started in all patients. patients (group ) were on antibiotics till the day of definitive curative operation when all foreign bodies were removed. in the other % of the patients (group ) the antibiotic therapy was stopped days after the djs or pcn insertion. short term antibiotic course -which is advisable for prevention of uti before invasive endoscopic treatmentwas administered in both groups from the day of the operation (after djs or pcn removal) and it was continued until the removal of all possible urinary foreign bodies used during the operation. in both groups of patients we recorded and evaluated early and late clinical and microbiological recovery. retrieved stents were sectioned for further laboratory examinations. adsorbed levofloxacin in the conditioning film layer and on the stent surface was detected by hplc. rasterelectron microscopy (rem) was used to examine biofilm formation and encrustation. results: we did not find any significant differences between the two groups of patients, neither in clinical (presence of fever, back pain, flank pain, leukocyte count) nor in microbiological recovery. statistical analysis showed that significantly greater amount of levofloxacin adsorbed to the conditioning film than to the stent surface in both groups of patients ( . ± . vs. . ± . in group and . ± . vs. . ± . in group ). no viable, adherent bacteria were recovered by sonication and culture in any of the patients, and no biofilms or encrustation were seen under rem either. conclusion: our data prove the hypotheses that continuous antibiotic treatment does not have any clinical or microbiological advantages in patients with indwelling ureteral stents compared to intermittent therapy. objective: the prophylaxis of bacterial infections during cardiac surgery is widely used in clinical practice. staphylococcus aureus, staphylococcus epidermidis and enterococcus spp are the pathogens most frequently involved in infective complications of cardio-pulmonary bypass (cpb) surgery. it is generally agreed that the success of prophylaxis is dependent on the ability to reach and maintain free antibiotic concentration in tissues higher than the mics for the most common pathogens. so we estimated the tissue concentrations of linezolid into sternal bone of patients undergoing cpb surgery. methods: six patients undergoing routine cpb surgery were given mg linezolid as a min iv infusion along with conventional prophylaxis of . g of cefuroxime immediately before surgery. two hours after the end of infusion blood samples and sternal bone tissues were collected. the local medical research ethics committee approved this study and all patients gave written informed consent. samples were assayed for the presence of linezolid by a high-performance liquid chromatography (hplc) method. results: following a mg infusion of linezolid, mean serum concentration for the six patients were . mg/l (range . - . mg/l) hours after the end of infusion. the concentration of linezolid into sternal bone was . mg/l (range . - . mg/ l) hours after the end of infusion. the penetration of linezolid into sternal bone was . %. conclusion: the penetration of linezolid into bone was . % of the simultaneous blood levels. in all bone samples the concentration of linezolid exceeded the mic for susceptible pathogens (< mg/l). although these data have been obtained from healthy, well-perfused bone the values suggest that linezolid may be a useful agent in the management of multidrug-resistant gram-positive bone infections. the antibacterial effect of daptomycin, teicoplanin and vancomycin against s. aureus studied in an in vitro pharmacokinetic model of infection a. noel, k. bowker, a. macgowan (bristol, uk) objectives: daptomycin (dap) is the first cyclic lipopeptide antibiotic approved for parenteral use in gram-positive infection. as yet, no comparative pharmacodynamic studies have been performed using dap and the two most common iv therapies teicoplanin (tei) and vancomycin (van). we used a pharmacokinetic (pk) model to study the antibacterial effect (abe) of these agents against two typical mrsa strains (ukemrsa & ) and a hetero vancomycin intermediate mrsa (hvisa). methods: an in vitro dilutional model was used to simulate the free drug concentration over h associated with doses of -dap mg/kg hrly (cmax . mg/l, t / h); tei mg hrly (cmax . mg/l, t / h); van g hrly (cmax . mg/l, t / h). an inoculum of cfu/ml was used and the experiments performed in triplicate. abe was assessed by area-under-the-bacterial-kill-curve - h (aubkc ) and logcfu/ml.h objectives: linezolid, the first oxazolidinone, is active against methicillin resistant staphylococcus aureus and has been effective in a variety of acute infections. however long-term administration, although desirable in bone infections caused by resistant gram-positive organisms, is hampered by the occurrence of anaemia and thrombocytopenia. administration of vitamin b has been reported to prevent myelosuppression. methods: patients attending the infectious disease clinic with bone infections caused by resistant gram-positive bacteria and treated with linezolid ( mg b.d. orally), received vitamin b ( mg o.d. orally) for the period of administration of linezolid. full blood counts were followed-up weekly. linezolid treatment was discontinued if haemoglobin declined below mg/dl or platelets below /ll. data from sixteen patients with osteomyelitis and with prosthetic joint infections were evaluated. comparisons were performed with matched historical controls receiving linezolid without b by kaplan-meyer curves with the log-rank test. results: the median follow-up of patients receiving b was . weeks and of controls weeks. in the b group % of the patients discontinued while in the control group % of the patients discontinued treatment because of side effects (p ns). % of patients receiving b discontinued due to thrombocytopenia and % due to anaemia. respective percentages in the control group were % and % (p ns for all comparisons). mean time to the occurrence of thrombocytopenia was weeks in the patients who received b and . weeks in the control patients. respective times to occurrence of anaemia were . and . weeks. all cases of myelosuppression were reversible. conclusions: administration of b failed to prevent or delay both thrombocytopenia and anaemia in patients receiving linezolid. other methods should be investigated to facilitate longer administration of linezolid in this group of patients. therapeutic drug monitoring of colistin -a -year review from a uk clinical antibiotic assay service k. bowker, a. noel, s. tomaselli, a. macgowan (bristol, uk) objectives: over the last years there has been increased use of colistin (col). however, little clinical data is available on the therapeutic levels of colistin. monitoring of col is useful in terms of therapeutic levels and avoidance of toxicity for patients with cystic fibrosis and complicated gram-negative infections. previous in vitro data has shown that col is bactericidal at col concentration is ‡ mg/l. here we assessed col data collected from our antibiotic assay service from the last seven years in order to establish if such levels were obtained. methods: col levels were determined by bioassay. data was retrospectively collected from the hospital information management system. data was assessed collectively and stratified by known cystic fibrosis patients, sex and age. objectives: ßlactams like ceftriaxone (cfx) and quinolones such as moxifloxacin (mox) are widely used to treat pneumococcal infection. we studied the antibacterial effect of (abe) after the first dose exposure to free drug serum concentrations of iv cfx g and po mox mg against s. pneumoniae strains with typical mics at low and high inocula. methods: a hollow fibre in vitro model was used to simulate free drug concentrations over h of cfx ( g h iv, cmax mg/l, auc mg/laeh, t / h) and mox ( mg, po, cmax . mg/l, auc mg/laeh, t / . h). the cfx mic was . mg/l (t>mic cfx %) and mox mic . mg/l (auc/ mic ). initial abe was measured by the slope of the log viable count - h and total abe over the dose interval ( h) by log reduction in viable count at h (d ) and the area-underthe-bacterial-kill-curve (aubkc ). inocula of cfu/ml and cfu/ml were used. results: the initial and total abe at low and high inocula were: given the pk/pd indices modelled both drugs showed a maximal effect. clearance from the model occurred at h ( inoculum) and h ( inoculum). there were no significant differences in speed or extent of abes comparing cfx and mox. conclusion: the abes of iv cfx and po mox against s. pneumoniae is similar in the first hrs of drug exposure. emergence of resistance in e. coli and ent. cloacae after exposure to ceftriaxone or ertapenem in an in vitro model of infection k. bowker, a. noel, a. macgowan (bristol, uk) objectives: emergence of resistance (eor) is an emergent factor in therapeutic choice. we studied eor to ceftriaxone (cfx) and ertapenem (ert) in e. coli (ec) and ent. cloacae (entclo), a more challenging species within inducible blactamases. methods: an in vitro dilutional model was used to simulate free drug concentrations associated with g hrly cfx (cmax mg/l, t½ h) and ert (cmax mg/l, t½ h) over h. two inocula and cfu/ml were used and eor assessed by population-analysis-profiles (pap). the area under the pap (auc-pap) was used to measure eor. ert mics were . mg/l ec and . mg/l entclo, cfx mic . mg/l ec and . mg/l entclo. experiments were performed in triplicate and mean values presented. results: observations at h were similar to those at h, hence data to h is given. at and cfu/ml, ec viable counts were reduced by fi logs, there was no eor. against entclo inoculum and , cfx resulted in an initial - log drop, then regrowth, ert produced a > log reduction. eor as measured by the mean auc-pap (n = ) with entclo is shown below: dosing with cfx resulted in eor to cfx and ert at high and low inoculum. dosing with ert resulted in no eor at inoculum, at resistance emerged to both cfx and ert. conclusions: eor depends on species (entclo > ec); duration of exposure (long > short) and agent (cfx > ert). ert appears to induce less eor both to itself and cfx than cfx does to itself and ert. however, initial use of cfx may reduce the effectiveness of ert. comparative serum activity of telithromycin, azithromycin, and amoxicillin/clavulanate against aerobic and anaerobic respiratory pathogens objectives: the purpose of this investigation was to study the clinical potential of telithromycin, a new ketolide antibiotic, for the treatment of mixed aerobic/anaerobic respiratory infections. in this study, we compared the pharmacodynamics (duration of inhibition/killing) of telithromycin (tel) to azithromycin (azi) and amoxicillin/clavulanate (a/c) against aerobic and anaerobic pathogens associated with mixed respiratory infections. methods: following written informed consent, ten healthy adult subjects (ages, - yrs) received single doses of tel ( mg), azi ( mg), and a/c ( mg) one week apart following a -h fast. venous blood samples were obtained at , , , and h after the dose and stored at ) °c. inhibitory and bactericidal titre s were determined by microdilution (s. pneumoniae & h. influenzae) and agar dilution (peptostrep. magnus, peptostrep. micros, prev. bivia, & prev. melaninogenica) procedures following clinical & laboratory standards institute methodology. bactericidal titres in serum endpoint was determined as the highest dilution of serum yielding . % killing. the median titres at each time point were calculated and the duration of activity was used for comparison of these agents. conclusions: in this ex-vivo study, we found that tel can provide prolonged ( % of the dosing interval) inhibitory activity in serum against macrolide-resistant strains of s. pneumoniae, bl pos. and neg. strains of h. influenzae, and common respiratory anaerobic pathogens. these findings suggest that tel could have clinical utility in the treatment of community-acquired mixed aerobic-anaerobic respiratory tract infections, including sinusitis, bronchitis, and pneumonia. objectives: increasing resistance in isolates of e. coli ( %) and p. aeruginosa ( %) to fluoroquinolones (fq) is a concern since these antibiotics are commonly used in the treatment of complicated urinary tract infections (utis). currently, no interpretive standards exist for ''susceptible'' isolates in urine for the newer fq. the purpose of this investigation was to evaluate the activity of high-dose levofloxacin against fqresistant urinary pathogens. methods: in this study, we determined the serum and urine levels of high dose ( mg) levofloxacin (l) as well as its bactericidal activity in urine (uba) against l-resistant isolates of e. coli (mics = to lg/ml) and p. aeruginosa (mics = to lg/ml). following written informed consent, blood and urine samples were collected from healthy adult (ages, - y/o) fasting subjects ( m and f) prior to and at . , , , , and hours after a single mg dose of l. serum and urine concentrations were measured by a validated hplc assay ( . - . % cv). the testing methodology for uba was similar to the microdilution assay used for serum bactericidal testing (clsi) with the exception that antibiotic-free urine was used to dilute these samples. the median titre ( : - : ) at each time point for the subjects was used to determine uba. results: the mean serum pharmacokinetic parameters were similar to previously published values: cmax = . lg/ml, auc = mgaeh/l, and t / = . h. mean urine concentrations ranged from lg/ml ( h) to lg/ml ( h). uba (titres > : ) was maintained for at least hours in all subjects for e. coli isolates with mics = , , and lg/ ml. for the e. coli strain with a mic = lg/ml, subjects exhibited uba at h but only subjects exhibited uba at h. similar results were observed against the p. aeruginosa isolates. conclusions: the results from this ex-vivo pharmacodynamic study in healthy volunteers found that mg of l provides prolonged (at least half the dosing interval) uba against l-resistant strains of e. coli and p. aeruginosa up to lg/ml. this suggests that a separate urinary susceptibility breakpoint is indicated for urine isolates treated with mg doses of l. objectives: exposure of methicillin-resistant s. aureus (mrsa) to acid ph restores its susceptibility to beta-lactams (sabath and al., aac, ) . in macrophages, s. aureus is mainly confined within phagolysosomes where the ph is acidic. we showed that meropenem (mem) displays similar intracellular activity against mrsa atcc and mssa atcc in macrophages. in the present study, we have investigated the intraphagocytic activity of mem and cloxacillin (clx) against mrsa clinical isolates (including one visa strain), in comparison with the reference mrsa atcc and mssa atcc strains. methods: mic's were determined in mhb (plus nacl %) by micro-dilution method. meca expression was examined at neutral and acidic ph by a semi-quantitative rt-pcr ( s rrna as housekeeping gene). intracellular activity was assessed in human thp- macrophages exposed to extracellular concentrations equivalent to human cmax (total drug; mem: mg/l; clx: mg/l) by examining the decrease in cellassociated cfu after h from the original, post-phagocytosis inoculum (controls [no antibiotic]; approx. log cfu increase). results: the table shows the mics (in broth) at neutral and acid ph and the intracellular activity for the strains studied. in atcc , meca expression was similar for bacteria maintained in broth at ph . conclusions: the intracellular environment markedly enhances the activity of beta-lactams against mrsa, probably through exposure to acid ph, although the latter does not affect meca expression. comparative activity of dalbavancin against european gram-positive isolates i. morrissey, c. dencer, j.w.t. dallow, j. childers, a. brook, j. cowan (london, uk) objectives: dalbavancin (dal) is a new semisynthetic lipoglycopeptide with a half-life of . days, enabling onceweekly dosing. this study compared the activity of dal with other agents against gram-positive isolates from europe. methods: isolates from belgium, the czech republic, denmark, finland, france, germany, hungary, italy, the netherlands, poland, spain, sweden and the uk were included. the clsi broth microdilution method was used to determine mic using dried microtitre plates. the following antimicrobial agents were evaluated: dal, vancomycin (van), teicoplanin (tei), daptomycin (dap), linezolid (lzd), dalfopristin/quinupristin (syn), erythromycin (ery), levofloxacin (lev) and tetracycline (tet). results: selected data are shown in the objectives: dalbavancin (dal) is a next generation lipoglycopeptide antibiotic in development for the treatment of complicated skin and skin structure infections (csssi). a population pharmacokinetic (pk) analysis was performed to estimate patient parameters and to determine significant covariates. incorporating the pk model, pharmacodynamic (pd) parameters were simulated to support the effectiveness of a weekly dosage. methods: the pk analysis included dal concentrations from patients across clinical trials. most patients received mg on day and mg on day . possible covariates examined included demography and concomitant medications, including medications that are considered inhibitors, inducers, and substrates of cytochrome p enzymes. the pk-pd analysis employed monte carlo simulations of time-dependent and concentration-dependent parameters. distributions of mics were obtained directly from clinical studies, and were also simulated to explore the effect of higher mics. results: dal pk fit a -compartment model with interpatient variability (ipv) on all parameters. the typical value and ipv (cv%) of clearance (cl) was . l/h ( . %), influenced by body surface area (bsa) and creatinine clearance (clcr). volume of distribution (v ) was . l ( . %) and influenced by bsa. the inter-compartmental clearance and peripheral volume were . l/h and . l, respectively. free drug concentrations were simulated using a dal protein binding of %. for a weekly -dose regimen, free dal remained above mg/l for the majority (> %) of patients for more than days. using previously described area under the curve (auc)/mic targets for staphylococcus aureus, a proposed mic of at least . mg/l was associated with a greater than % probability of target attainment. conclusions: dal pk were predictable, demonstrating low ipv. bsa and clcr were the only sources of variability, but described less than % of the ipv. pd simulations support the use of dalbavancin in a weekly regimen. objectives: methicillin-resistant staphylococcus joint infection due to peri-operative contamination is a complication after arthroplasty. the objective of this study was to assess the distribution of radioactivity in bone and related structures using quantitative autoradiography after administration of [ c]dalbavancin in rabbits. methods: new zealand white male rabbits were given a single intravenous (iv) bolus dose of mg/kg [ c]-dalbavancin (n = ) or control vehicle (n = ). plasma, cerebrospinal fluid (csf), bone, bone marrow, and nucleus pulposus were collected at , , , , and h post-dose by necropsy, homogenized, combusted, and analysed for total drug-derived radioactivity using liquid scintillation counting (lsc). in addition, the left hindlimb from rabbit/time point was flash-frozen and cryosectioned for quantitative autoradioluminography. results: [ c]-dalbavancin-derived radioactivity was rapidly and widely distributed into bone, bone marrow and, to a lesser extent, in csf and nucleus pulposus. autoradioluminography data indicated that concentration of radioactivity was highest in bone marrow, whole blood, articulate cartilage, ligament, epiphyseal plate, periostium, and meniscus. at h postdose, [ c]-dalbavancin-derived radioactivity was measurable in all tissues, and remained at relatively high concentrations in bone marrow ( . lg equiv/g), epiphyseal plate ( . lg equiv/g), periostium ( . lg equiv/g), and articular cartilage ( . lg equiv/g). in homogenized bone using lsc, mean concentration after hours was . lg equiv/g. conclusion: [ c]-dalbavancin-derived radioactivity rapidly penetrated knee joint tissues and persisted at relatively high concentrations for at least h after a single iv dose in rabbits. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound ( %) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution ( . l/kg) means that it may be removed by cvvh. cvvh is widely used in the management of critically ill patients. the objective of this study was to determine cvvh telavancin transmembrane clearance (cl) with commonly used hemofilters (an , polysulfone) at conventional ultrafiltrate flow rates. methods: tlv cl was assessed in our in vitro cvvh model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run using an (m , gambro) and polysulfone (f nr, fresenius) hemofilters. ultrafiltrate (uf) flows were , , and l/hr with sufficient blood flows [(qb) - ml/min] to maintain uf rates. blood samples were collected from the pre-filter line and uf samples from the post-filter uf port. concentrations of tlv in plasma and uf samples were assayed using validated lc-ms/ms methods. tlv cl was determined using the following formula:cl = (uf flow rate) [tlv]uf/[tlv]arterial. cl differences between the filter types were compared using a two-tailed, unpaired t-test. conclusion: tlv is substantially cleared by cvvh and cl increases significantly with increasing uf rate. cl did not differ by hemofilter type. cvvh cl at higher uf flows exceeds the total cl reported in patients with normal renal function. tlv likely will require dose adjustments in patients receiving cvvh. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound ( %) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution ( . l/kg) means that it may be removed by cvvhd. cvvhd is used in the management of critically ill patients. the objective of this study was to determine cvvhd tlv transmembrane clearance (cl) with commonly used hemodialyzers at conventional cvvhd dialysate flow rates. methods: tlv cl was assessed in our in vitro cvvhd model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run times using an (m , gambro) and polysulfone (f nr, fresenius) hemodialyzers. dialysate flows were , , and l/hr with sufficient blood flows [(qb) - ml/min] to maintain appropriate transmembrane pressures. blood samples were collected from the pre-filter port (a) and post-filter port (v), and spent dialysate samples (d) from the post-filter d port. plasma tlv concentrations (arterial and venous) and dialysate samples were assayed using validated lc-ms/ms methods and tlv cl was determined using the following formula: cl = (d flow rate) [tlv]d/(([tlv]arterial+[tlv]venous)/ ). dialytic cl between filter types was compared using a two-tailed, unpaired t-test. conclusion: tlv is effectively cleared by cvvhd. the higher permeability polysulfone dialyzer was associated with significantly increased cl vs. the an dialyzer as dialysate flow increased. the degree of tlv cl seen with cvvhd suggests that dose adjustments will be necessary in patients receiving cvvhd. objective: it is still a subject of controversy that only free, unbound drug is responsible for antibacterial activity of antibiotics. to provide further proof, that only free drug contributes to antimicrobial efficacy a comparative, doseranging time-kill curve study was performed. to exclude influence factors resulting from different mechanisms of action this was done within the antibiotic class of carbapenems, using compounds with different serum protein binding. methods: constant concentration time-kill curves were performed in % serum for the slightly serum protein bound mer-openem (~ %) and imipenem ( %) as well as for the highly serum protein bound ertapenem ( %) and faro-penem ( - %), ranging from · mic to · mic. the change in number of bacteria (cfu/ml) versus time was linked to their effect. escherichia coli atcc , klebsiella pneumoniae bay , staphylococcus aureus bay and streptococcus pneumoniae atcc were used as the test organisms. samples were taken at , . , , , and hours. the data were modelled simultaneously using the software scientist Ò for windows tm and a modified sigmoid emax model characterized by growth rate constant (k ), maximum kill rate (kmax), and concentration at half maximum effect (ec ). results: for all four bacterial strains investigated, there were dramatic increases ( - %) in ec for the highly se-rum protein bound carbapenems (ertapenem, faropenem) in the presence of serum proteins (fig. ) . for both substances no significant differences in k and kmax were determined. in contrast, imipenem and meropenem showed only minor differences in ec in the presence and the absence of % serum. conclusion: only free, unbound drug is responsible for the antimicrobial activity. analysis of these time-kill curves clearly showed that the antibacterial efficacy was significantly decreased in the presence of % serum for the highly bound ertapenem and faropenem while being unaltered for the slightly bound meropenem and imipenem. objective: numerous in vitro experiments have shown that protein binding (pb) is an important factor for antimicrobial activity, especially for highly bound antibiotics. however, the experimental conditions that simulate the in vivo situation best are still subject of controversy. therefore, an in vitro microdialysis experiment was performed that evaluates various influence factors on the pb of the highly bound betalactams ceftriaxone (pb - %). methods: a comparative, dose-ranging in vitro microdialysis study was conducted to determine free, unbound ceftriaxone concentrations in lactated ringer's solution and todd hewitt broth (thb) both with and without bovine serum albumin (bsa; sigma, st. louis) g/l and human plasma at °c. furthermore, in vitro constant concentration time-kill curves were performed, using escherichia coli atcc , streptococcus pneumoniae atcc and streptococcus pneumoniae atcc as the test organisms. the data was analysed using an appropriate pk/pd model, characterized by growth rate constant (k ), maximum kill rate (kmax), and concentration at half maximum effect (ec ) and correlated to free ceftriaxone thb concentrations determined by hplc-uv. results: there were only minor differences in both unbound drug concentrations and anti-infective activity when bsa g/ l was added to either lactated ringer's (pb % and . ± . %, with and without bsa respectively) or thb (pb % and . ± . %, with and without bsa respectively). no significant changes in k , kmax and ec were observed. however, using human plasma, unbound concentrations (pb %) %) were altered dramati-cally. conclusion: only free, unbound drug is responsible for the antimicrobial activity. however, one cannot rely on that binding to commercially purchased bsa is consistent with reported protein binding values. unbound concentrations should be measured under the respective experimental conditions to be able to correctly interpret the experimental results. in vitro postantibiotic effect of faropenem on penicillin-resistant streptococcus pneumoniae and beta-lactamase-producing haemophilus influenzae c.l. young, i.a. critchley, u.a. ochsner, n. janjic (louisville, us) objectives: faropenem (far) is an oral penem with potent activity against respiratory pathogens such as penicillin (pn)resistant streptococcus pneumoniae (sp) and beta-lactamase (bla)-producing haemophilus infleunzae (hi). the postantibiotic effect (pae) is a pharmacodynamic (pd) parameter that monitors suppression of bacterial growth following short exposure and removal of the drug. paes are clinically important for agents such as far with short half-lives ( h) . the aim of the study was to determine the pae of far on resistant phenotypes of sp and hi. methods: nine clinical isolates of sp, pn-s, pn-i, and pn-r and six clinical isolates of hi, bla-negative and blapositive were tested in pae studies. paes were determined in cation-adjusted mueller-hinton broth with - % lysed horse blood for sp and haemophilus test medium for hi. exponential cultures ( cfu/ml) were exposed to far at , and x mic. far was removed by serial washing ( , -fold dilution) prior to transfer to fresh media. control cultures were treated in the same way. bacteria were incubated with shaking and viable cfus determined at , , , , and h. counts of log cfu were plotted against time and pae defined as the difference is the time required for count in test culture and control (untreated culture) to increase log above the count observed immediately after removal. results: significant paes of > . h were observed for all strains of sp at and x mic. however, the pae was more prolonged on the pn-r strains with mean paes of . h at and x mic. among hi, little or no pae was observed on bla-negative strains but a significant pae was observed on the bla-positive isolates (mean paes of . h and . h at and x mic respectively). conclusions: far demonstrates a prolonged pae on key resistant phenotypes of sp (pn-r) and hi (bla-positive) compared with susceptible strains. the observation of pae in bla-positive hi is unique in the class of beta-lactams. far exhibits in vitro pd properties that may contribute to its clinical efficacy against pn-r sp and bla-positive hi. telavancin is more efficacious than vancomycin in a murine model of bacteraemic peritonitis induced by methicillin-resistant staphylococcus aureus s. hegde, n. reyes, b. benton, r. skinner (south san francisco, us) objective: telavancin (tlv) is a novel lipoglycopeptide that operates through multiple mechanisms to produce potent and rapid bactericidal activity against clinically relevant grampositive bacteria including methicillin-resistant staphylococcus aureus (mrsa). the present studies evaluated the in vivo efficacy of tlv vs vancomycin (van) in a model of mrsa induced peritonitis in neutropenic mice. methods: female nsa immunocompromised mice were inoculated intraperitoneally with atcc mrsa and treated, beginning at h post-infection, with subcutaneous doses (q h) of vehicle (veh) or test compound. mouse pharmacokinetic data were generated and used to choose doses of tlv ( mg/kg) and van ( mg/kg) in order to equate clinical exposures (auc's (free drug) of and lg.hr/ml, respectively). in survival studies, deaths were recorded for days post-infection and survival curves were compared using log-rank test. in bacterial titer determination studies, designated groups of control and drug-treated surviving animals were humanely euthanized at various times post-treatment and their blood and spleen were harvested to determine bacterial titers. results: mics of tlv and van were . and . lg/ml, respectively. mortality was % in animals treated with veh or van. mortality was % in tlv-treated animals (p < . vs veh and van). the pre-treatment bacterial titres were . log cfu/ml and . log cfu/g in the blood and spleen, respectively. analysis of the time kill curves for both blood and spleen revealed that tlv exhibited significantly greater killing activity than van (p < . , two-way anova). at hrs after the first dose, the titers in the blood were reduced to a greater extent by tlv () . log cfu/ml) compared to van () . log cfu/ml). at hrs after the second dose, the splenic titers were reduced to a greater extent by tlv () . log cfu/g) when compared to van () . log cfu/g). conclusions: the data described here demonstrate that tlv's in vivo bactericidal activity is superior to that of van against mrsa and results in successful infection resolution and, consequently, improved survival in the murine peritonitis model. proper use of carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa y. kobayashi (tokyo, jp) methods: regimens of carbapenems were given to healthy adult subjects. changes in their blood concentrations of carbapenems were compared by using pharmacokinetic parameters (two-compartment model analysis) of meropenem (mepm), imipenem (ipm), and panipenem (papm) and by applying the lognormal distribution to the probability distribution of distribution volume and plasma half-life with monte carlo simulation (mcs). based on the data on distributions of the minimal inhibitory concentrations (mic) of various carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa isolated/identified at keio university hospital between october and october (mic : mepm mcg/ml, ipm mcg/ml, papm mcg/ml), the mics in the subjects were obtained with mcs. from the changes in blood concentrations and mics in the subjects, the probability of achieving t>mic was calculated for each carbapenem regimen, using the formula reported by kuti et al.: %t>mic = ln (dose/vd*mic)*(t / / . )*( /di) <<ln: natural logarithm, dose: dose (mg), vd: distribution volume (l), t / : plasma half-life (h), di: dosing interval (h)>>. based on craig's data, the maximum bactericidal effect on gram negative bacilli is attained when %t>mic is approximately %. we focused on this information and analysed our data. results: the probability of achieving t>mic % was . % for mepm mg bid, followed by . % for ipm mg bid and . % for papm mg bid. when the dose was increased from mg to mg, it was . % for mepm mg bid, followed by . % for ipm mg bid and . % for papm mg bid. when the dose remained at mg and the dosing frequency was increased to three times daily, it was . % for mepm mg tid, followed by . % for ipm mg tid and . % for papm mg tid. regarding mepm, it was . % for mg gid? and . % for mg tid showing higher probabilities. discussion: in severe sepsis caused by pseudomonas aeruginosa, remarkably higher t>mic % was achieved with carbapenems at mg tid, although the daily dose ( mg) was lower, compared to mg bid. carbapenems with a low mic distribution, i.e. a superior antibacterial activity, showed higher probability of achieving t>mic. therefore, the optimal treatment for such sepsis is mepm mg tid. mepm mg qid appeared to provide comparable therapeutic effects with those at mg tid, the usual dose in foreign countries. penetration of moxifloxacin into normal and infected subcutaneous tissue in patients with spinal cord injury measured by microdialysis background: skin breakdowns, also termed decubitus ulcers or pressure sores, are a major complication associated with spinal cord injury, resulting in infection and tissue death. moxifloxacin (mfx) is approved for the treatment of sssi. our objective was to construct a population pk model for mfx disposition in plasma, normal and infected subcutaneous tissue in spinal cord injured patients with infected decubitus ulcer. methods: patients receiving mg mfx orally daily were enrolled in this study. blood, saliva and interstitial tissue fluid samples (microdialysis in normal and infected tissue) were collected over a time period of hrs. mfx concentrations were measured by a validated hplc. concentration-time data obtained in the present study were pooled with previously published mfx data (n = ). population pk modelling was performed with nonmem. results: the concentrations of mfx achieved in plasma, saliva, normal subcutaneous tissues and infected decubitus ulcers showed parallel profiles versus time. the pk was best described by a -compartment model with a link to interstitial tissue fluid. the population pk parameters were as follows (given as estimate with percent interindividual variability in parentheses): cl . l/h ( %); central vd . l ( %); intercompartmental cl . l/h ( %); peripheral vd . l ( %); and elimination rate constant for interstitial tissue fluid . h) ( %). with a conservative mic of . mg/l, the peak/mic ratios were higher than and the auc /mic ratios were higher than for plasma, saliva and interstitial tissue fluids. conclusions: this study showed the good diffusion of mfx into subcutaneous tissue in spinal cord injured patients with decubitus ulcers. the interstitial tissue fluids reached bactericidal levels for common bacteria found in infected skin lesions. objective: investigations of pharmacodynamic parameters such as postantibiotic effect and postantibiotic subminimum inhibitory concentration effect have been employed for design of dosing schedules of antimicrobial agents. in this study we compared postantibiotic effect and postantibiotic subminimum inhibitory concentration effect of ciprofloxacin, levofloxacin, and moxifloxacin for clinical isolates of methicillin susceptible staphylococcus aureus, methicillin resistant staphylococcus aureus and pseudomonas aureginosa. methods: the following strains were tested in this study: methicilline-susceptible staphylococcus aureus (n: ), methicilline resistant -staphylococcus aureus (n: ) and pseudomonas aeruginosa (n: ). the pae was determined by viable plate count method using mueller hinton broth. tubes containing ml of broth and the antibiotic to be tested at , , and x the mic were inoculated with approximately · cfu/ml. growth controls with an inoculum but not antibiotic were included with each experiment. result: postantibiotic effects of ciprofloxacin, levofloxacin and moxifloxacin increased with increasing concentration of the drug. the longest postantibiotic effect was observed for moxifloxacin. moxifloxacin showed no postantibiotic effect one p. aureginosa at all concentration and had no post antibiotic effect to another p. aureginosa at x mic and mic. in our study the longest postantibiotic subminimum inhibitory concentration effect against mssa was determined with moxifloxacin. similarly the moxifloxacin induced the longest effect against mrsa. however, this time frame was shorter than that of mssa. conclusions: all three antibiotics, showed for longer postantibiotic subminimum inhibitory concentration effect in all submic concentrations, immeasurable within the study period i.e. hours. lack of horizontal transmission of fluoroquinolone resistance between s. mitis and s. pneumoniae objectives: fluoroquinolone (fq) resistance can arise in s. pneumoniae through acquisition of dna from s. mitis and subsequent homologous recombination. the frequency at which this occurs is unknown, and while likely a rare event, increases in fq resistance among s. mitis may increase the rate at which horizontal transmission occurs. we sought to determine the frequency at which fq resistance could be transferred from s. mitis to s. pneumoniae or from s. pneumoniae to s. mitis. methods: s. mitis (either fq^r,tetracycline[tet^s], fq^r,penicillin[pen^s], or fq^s,pen^r) and s. pneumoniae (either fq^s,tet^r, fq^s,pen^r or fq^r,pen^s) were grown in co-culture using a pharmacodynamic model in the presence of either moxifloxacin (mxf) or levofloxacin (lfx) at salivary drug concentrations. after incubation, aliquots were plated onto either tet or pen containing sba plates to select for the recipient strains. fq susceptibility was performed using microbroth dilution. the entire parc and gyra genes were amplified and sequenced to determine if horizontal transmission occurred. results: in initial experiments tet was used as the selective agent. however tet resistance was transferred and therefore pen^r was used as a selective marker. an increase in the lfx mic in was observed in s. pneumoniae and s. mitis strain. sequencing of the parc gene revealed the selection of ser phe and ser tyr mutations in s. pneumoniae and ser phe in s. mitis consistent with fq resistance. sequencing of the entire gene failed to uncover evidence of horizontal transmission. no mutations were detected in gyra. selection of st step parc clinical microbiology and infection, volume , supplement , mutations occurred only after exposure to lfx. mxf eradicated both s. mitis and s. pneumoniae and failed to either select for resistance or support horizontal transmission. conclusions: although st step parc mutations were selected in strains ( s. pneumoniae, s. mitis), we failed to find evidence of horizontal transmission between s. pneumoniae and s. mitis under our laboratory conditions. the phenomenon of horizontal transfer resulting in fq resistance has been described, however, based on our results, we must speculate that it is an extremely rare event and not likely to be a major driver of fq resistance. of interest, the parc mutations were selected only under the selective pressure of lfx. mxf completely eradicated both s. pneumoniae and s. mitis and did not select for the development of fq^r mutations. objectives: the aim of the present study was to assess the killing activity of ertapenem (ert) and metronidazole (mtr) against four selected bacteroides fragilis strains with different mic values in an in vitro pharmacokinetic/pharmacodynamic (pk/pd) model. since anaerobes are often present in mixed infections, kill kinetics were also established for mixed inocula employing the b. fragilis strains together with four selected escherichia coli strains. the killing activity was analysed for kinetic concentrations of the antimicrobial agents simulating human serum kinetics. methods: a pk/pd in vitro model was established by adding appropriate amounts of broth every half hour. at the same time intervals samples were obtained and plated. after incubation colony forming units were counted. human serum concentrations were simulated with cmax = mg/l and t / of hours for ert and cmax = . mg/l and t / of hours for mtr. mann trend test was used for statistical analysis. results: as to be expected the e. coli strains were not killed by mtr both in pure as well as in mixed cultures whereas the susceptible e. coli strains were effectively killed by ert. in pure cultures the b. fragilis strains were effectively killed by mtr and the growth of the susceptible b. fragilis strains was reduced by ert by about two to four logs. however, in some mixed cultures the killing activity of mtr against the b. fragilis strains was significantly reduced. conclusion: the in part moderate in vitro activity of ert against the b. fragilis strains and the reduced activity of metronidazole in mixed cultures against the b. fragilis strains may explain some of the difficulties in treating mixed aerobic/ anaerobic infections. penetration of ciprofloxacin into human cerebrospinal fluid and brain tissue a. tsona, s. metallidis, e. koumentaki, j. nikolaidis, p. kollaras, g. lazaraki, p. nikolaidis (thessaloniki, gr) objectives: the aim of the present study was to determine the penetration of ciprofloxacin into cerebrospinal fluid (csf) and brain tissue of humans. methods: a total of patients undergoing brain tumor excision were evaluated. the patients received a single intravenous dose of mg ciprofloxacin. samples of blood, cerebrospinal fluid and brain (brain-adjacent tumour tissue) were collected during surgery h after drug administration. ciprofloxacin concentrations in serum, cerebrospinal fluid and brain homogenate were analysed by means of a validated hplc method. results: ciprofloxacin concentrations in plasma (mcg/ml), cerebrospinal fluid (mcg/ml) and tissue homogenate (mcg/g), respectively, after h ranged . - . mcg/ml, . - . mcg/ ml and . - . mcg/g. csf-to-serum ratio ranged between . and . . tissue-to-serum ratio ranged between . and . . mean (±s.d.) csf/serum concentration ratios and brain tissue/serum concentration ratios were respectively . ± . and . ± . . conclusion: these findings suggest that valuable informations on brain tissue penetration can be obtained only from brain material. data from csf penetration cannot be extrapolated to the brain since the blood: sf barrier differs from the blood:brain barrier. concentrations of ciprofloxacin in cerebrospinal fluid were lower than those in serum, in contrast to the brain tissue concentrations that exceeded serum concentrations. the achieved concentrations in brain tissue were generally above the mic of common pathogens in central nervous system infections (h. influenze, n. meningitidis, s. pneumoniae, l. monocytogenes, escherichia coli, aerobic gram-negative bacilli, group b streptococci, mssa). cerebrospinal fluid concentrations exceed the mics of neisseria meningitidis and most gram-negative aerobic bacilli. our findings suggests that ciprofloxacin may be an acceptable alternative for the treatment of meningitis due to susceptible gram-negative aerobic organisms and for the treatment of brain abscesses. objective: to model the performance of imipenem (imi), meropenem (mem), and ertapenem (etm) against esbl producing e. coli and klebsiella spp in order to identify possible pd differences among compounds. methods: minimal inhibitory concentrations (mics) were generated for randomly selected esbl producing isolates of ec (n = ) and kl (n = ) collected during from brazilian hospitals as part of the mystic program. mic testing for imi, mem, etm, ceftazidime (ctz), and cefotaxime (ctx) were done by e-test methodology. esbls were confirmed via ctz/clavulanate and ctx/clavulanate e-test. pd exposure, measured as percent time above the mic for free drug (ft>mic), was modelled via a subject monte carlo simulation for the following -minute infusions:imi gram every hours, mem gram every hours, and etm gram every hours, using pharmacokinetics from healthy volunteers. the bactericidal cumulative fraction of response (cfr) was calculated for each regimen against the populations of ec, kl, and against all esbl isolates together. bactericidal cfr was defined as % ft>mic for all agents. results are reported as cfr ( % confidence interval). results: isolates were % susceptible (s) to imi and mem (mic range . - . and . - mg/l, respectively), and % s to etm (mic range . - mg/l conclusions: these findings support other data that although etm is likely to be an effective empiric agent against most esbl producing ec and kl, its ability to achieve high bactericidal pd exposure will be dependent on the presence of less susceptible organisms in the population. imi and mem should remain first line for esbl infections. objectives: this study analyses eradication and resistance selection in streptococcus pneumoniae with moxifloxacin, levofloxacin and azithromycin, using a parental serotype infecting strain (a) and subsequent resistant step-mutants (isolates b, c and d) selected in vivo in a patient with pneumonia. methods: moxifloxacin, levofloxacin and azithromycin mics were , and . lg/ml for the parental strain, , , and lg/ ml for isolate b, and , and > lg/ml for isolates c and d, respectively. a pharmacokinetic computerized device was used to simulate serum and epithelial lining fluid (elf) concentrations. initial inocula was approx. cfu/ml. population analysis profiles were performed using plates with increasing antimicrobial concentrations on a mic basis. results: in serum, moxifloxacin eradicated the parental isolate (isolate a), with an auc - h/mic value of . . serum auc - h/mic values of . and . for levofloxacin and azithromycin, respectively, were not able to eradicate isolate a. in elf, moxifloxacin showed a bactericidal pattern against all isolates with a minority (approx. cfu/ml) of the survival population (isolates b, c and d) growing in plates with moxifloxacin concentrations higher than those obtained in elf. levofloxacin and azithromycin showed a bactericidal pattern only against isolate a, with the whole population of isolates b, c and d growing in plates with levofloxacin concentrations higher ( - lg/ml) than those obtained in elf, and in plates with azithromycin concentrations as high as lg/ml (for isolates c and d). in elf, moxifloxacin auc - h/mic values were . for isolate a, and . for isolates b, c and d. levofloxacin auc - h/mic values were . for isolate a, and . for isolates b, c and d. azithromycin auc - h/mic values were . for isolate a; . for isolate b; and for isolates c and d. conclusion: if prevention of resistance depends more on the eradication of possible emerging mutants in pulmonary tissues than of the parental susceptible strain, moxifloxacin concentrations in elf may provide advantages over previous quinolones and macrolides in preventing clinical failures. objectives: to explore how antimicrobial pressure influences the evolution of streptococcus pneumoniae populations sharing the same ecological niche. methods: an in vitro computerized pharmacodynamic model simulating physiological concentrations obtained over h after mg o.d levofloxacin, mg b.i.d ciprofloxacin, and mg o.d azithromycin was used to investigate its effect on a mixed culture of five s. pneumoniae serotypes (s) as an approach to ecology of population dynamics. resistance patterns were: s was susceptible to study drugs, s was low-level macrolideresistant (efflux phenotype), s was high-level macrolideresistant (erm genotype), s v was low-level quinoloneresistant, and s was high-level quinolone-resistant. initial mixed inocula (time ) included similar percentages of each serotype. results: mean colony counts in antibiotic-free plates (whole pneumococcal population) increased (from to h) from log . to . in drug-free simulations (control), from log . to . in levofloxacin simulations, from log . to . in ciprofloxacin simulations, and from log . to . in azithromycin simulations. at h of control drug-free experiments, dominant strains were s v ( . %) and s ( . %) with marginal populations of s , s and s . azithromycin selected in a much higher extent the strain with low-level resistance to macrolides (s ) than the strain with high-level resistance (s ) (accounting for . % vs. . % of total population at h). ciprofloxacin selected in a higher extent low-level (s v) than high-level (s ) quinolone resistance ( . % vs. . %). levofloxacin decreased the proportion of the predominant s v in controls to . % (an intermediateresistant strain with mic = lg/ml), and unmasked the highlevel resistant strain (mic = lg/ml) up to . %. conclusion: strain distribution in antibiotic-free environment depends on bacterial fitness in mono-and multi-strain niches. the selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. selective potential only for resistance phenotypes with very low prevalence (as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes. re-evaluation of the role of broad-spectrum cephalosporins against staphylococci applying contemporary in vitro results and pharmacokinetic-pharmacodynamic principals h. sader, s.m. bhavnani, p.g. ambrose, r. jones (north liberty, us) objectives: to re-evaluate the current in vitro activity and to assess the pk-pd target attainment of cefepime (cpm), ceftriaxone (cro) and ceftazidime (caz) against staphylococcus spp. methods: the potency of cpm, cro and caz against staphylococci was accessed through the sentry antimicrobial surveillance program database, worldwide. during the - period , s. aureus (sa; % oxacillin [oxa]-susceptible [s]) and , coagulase-negative staphylococci (cons; % oxa-s) were s tested against cpm, cro, caz and numerous comparators by clsi broth microdilution methods. using volunteer pk data and a linear intermittent intravenous infusion model, and an animal-derived pk-pd target of % time above mic, expected probabilities of target attainment (pta) for cephems were evaluated using monte carlo simulation. pta were determined for the following dosing regimens: cpm gm q and q hours, caz gm q hours and cro gm q hours, each representing the most common dosing patterns applied clinically. cephem susceptibility (%s) was calculated based on the current clsi ( ) breakpoints (bkps) and also on bkps derived from a pta > %. results: against oxa-s sa, mic / values were (in mg/l): / for cpm, / for cro and / for caz, respectively; and against oxa-s cons mic / values were (in mg/l) . / for cpm, / for cro, and / for caz, respectively. the calculated %s of these cephems are summarized in the table: twenty year-old clsi bkps would rank the tested agents cpm ‡ cro > caz and by pk-pd pta cpm ‡ caz > cro. cpm has a potency advantage over caz ( -to -fold) and superiority at the usual dosing over cro ( . - . %) for oxa-s staphylococci. caz pk overcomes by-weight activity disadvantages, while a low proportion (< %) of active freedrug penalizes cro in the pta calculations. pta remained at > % to a bkp of mg/l for cpm ( gm q ) and caz and to a bkp of mg/l for cro. conclusions: regardless of applied bkp (clsi or pk-pd), cpm has the widest and more potent anti-staphylococcal activity among commonly used ''third-or fourth-generation'' cephems. when used at doses ‡ gm/day, cpm assures maximal coverage of oxa-s staphylococci whether using existing (clsi) or modified (pk/pd) bkps. cro should be used with caution. methods: the mic for all strains were determined by serial two-fold macrodilutions. an in vitro kinetic model was used to investigate the antibacterial efficacy of constant drug concentrations during hours. the selection of the doses of azithromycin tested in each bacterial strains was based on their mic values. bacterial counts were determined on appropriate agar plates using an adapted drop-plate method. twelve different pk/pd models were fitted and compared to the time-kill data by using non-linear regression. results: a simple pk-pd model was not sufficient to describe the pharmacodynamic effects for the four bacterial strains. appropriate models that gave good curve fits included a saturation term for the number of bacteria (nmax), delay terms ( -e-zt) for the initial bacterial growth phase and/or the onset of anti-infective activity as well as a hill factor (h) to capture the steepness of the concentration-response relationship. azithromycin had high potency against s. pneumoniae strains and m. catarrhalis while the potency of azithromycin against h. influenzae was poor. conclusions: the developed pk/pd models are suitable for describing the pharmacodynamics of azithromycin. applications of these pk-pd models will eventually provide a tool for rational antibiotic dosing decisions. objectives: optimal antimicrobial dosage regimens aim to achieve successful clinical outcomes without drug toxicity or emergence of bacterial resistance. for concentration dependent antibiotics, such as the fluoroquinolones, in humans a cmax:mic ratio of > is considered more important for efficacy and reduced selection of resistance than prolonged antibiotic concentrations just above the mic. fluoroquinolone resistance in zoonotic bacteria is a matter of public health concern, and fluoroquinolone treatment of poultry can rapidly select for bacteria with reduced fluoroquinolone susceptibility. in this study we compared basic pharmacokinetic parameters for the recommended dose of baytril (enrofloxacin) % oral solution in poultry to . x this dose for birds dosed by continuous water (standard) compared to pulsed water treatments and dosing by gavage.methods. for the pulsed versus continuous water treatments, groups of chickens received baytril % oral solution at (recommended) or ppm continuously in the water or at (recommended) or mg/kg pulsed in the water. for each group, three birds were killed at , , , , , and hours after start of antibiotic treatment and caecal contents, liver, lung and sera were taken and the concentration of fluoroquinolone determined by fluorescence hplc. for gavage treatment, dosing was at and mg/kg by crop intubation and four birds were killed in each group at , and hours after gavage; caecal contents, liver and sera were taken and analysed as above. basic pharmacokinetic parameters were determined using pk solutions software. results: the mean fluoroquinolone cmax in caecal contents (and sera) for gavage, pulsed water and continuous water treatments respectively was . ( . ), . ( . ) and . ( . ) mg/ml after the recommended dose and . ( . ), . ( . ) and . ( . ) mg/ml after . x the recommended dose. cmax of antibiotic in liver and lung was increased by the modified regimens in similar proportions to above. both pulsed water and gavage treatment not only resulted in higher cmax values, but also a faster rate of fluoroquinolone clearance than continuous water treatment ( figure ). dosing by gavage is not practical for thousands of chickens. however, pulsed dosing at . x the recommended dose can increase cmax values about fourfold and so could improve efficacy and reduce selection of resistance, compared to the current recommended treatment regime. objectives: nephrotoxicity is the major concern arising with the use of intravenous colistimethate sodium. methods: a prospective cohort study was performed at ''henry dunant'' hospital, a -bed tertiary care center in athens, greece. patients who received intravenous colistin for at least days for the treatment of multidrug resistant gram-negative bacterial infections were included in the study. the development of nephrotoxicity through evaluations of serum creatinine, blood urea, serum electrolytes, urinalysis, and creatinine and sodium in -hour urine collection during intravenous colistin therapy was the primary end point of the study. results: twenty-six patients were included in the study, of whom received colistimethate sodium (cms) for at least days and were evaluated further. the mean (± sd)/median daily dose, cumulative dose, and duration of treatment of intravenous cms was . (± . )/ million iu, . (± . )/ million iu, and . (± . )/ days (range - days), respectively. three of the evaluable patients ( . %) developed nephrotoxicity during the intravenous treatment with cms. the cumulative dose of the administered cms was statistically correlated with the difference between the end and start of cms treatment values of serum creatinine (r = . , p = . by spearman's test). a statistically but not clinically significant decrease of the mean baseline serum sodium concentration was observed between start and end of treatment [mean . (± . ) to . (± . ) mmol/l, p = . ]. no other toxic events were noted during the intravenous administration of colistimethate sodium. conclusion: although this is an evaluation of a small number of patients, our prospective study shows that nephrotoxicity was not commonly observed in this group of patients who received intravenous colistimethate sodium. however, caution should be taken to avoid the prolonged administration of the antibiotic. objectives: the objective of the present work is quantitative structure-activity relationship (qsar) analysis of antimicrobial activity of the -thiazolidone derivatives and consequent computational design of new antimicrobials. methods: for the achievement of the formulated objectives the qsar investigation has been carried out using computational chemistry approach based on simplex representation of molecular structure (sirms). on the framework of sirms it is possible to develop the molecular design of the new effective antimicrobials. results: systematic researches of relationship between antimicrobial activity (staphylococcus aureus -methicillin-sensitive (mssa) strain, pseudomonas aeruginosa -r and s strain, klebsiella pneumoniae, candida albicans s and Ñ itrobacter freundii) and a structure of about one hundred fifty compounds ( -thiazolidone derivatives and analogs). the elucidation of structure-activity relations allows predicting biological properties of such compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of their biological effect. completely adequate statistical partial least squares models (r = . - . , q = . - . ) have been obtained for all of the studied cultures. on the base of the first ones the molecular fragments both promoting and interfering the given antimicrobial activity have been determined. they give a possibility to realize the computer high throughput screening and molecular design of active compounds. the results of prognosis are verifying by the experimental investigations. also the influence of heterocycle system evolution on antimicrobial activity has been revealed. conclusion: qsar analysis of antimicrobial activity of -thiazolidone derivatives allows us to discover that the presence of naphthalene-substituted fragment (independently on its location in molecule) has distinctly negative influence on antimicrobial action. the requirements to molecular design have been formulated. for example, high active compounds must include -indolyl fragment. computational design of the new antimicrobials based on the substituted crown ethers activity of the row of substituted crown ethers and consequent molecular design of new antimicrobials. methods: the well-known hierarchic system of qsar models based on simplex representation of molecular structure has been used for the solution of the formulated problem, within the framework of which one it is possible to develop the molecular design of the new effective antimicrobial agents. results: we tried to conduct systematic researches of relationship between antimicrobial activity (planococcus citreus, streptococcus lactis, micrococcus lysodeiktious, staphylococcus aureus, streptococcus faecalis, bacillus subtillum) about two hundred fifty crowns ethers including aromatic, cyclic and heterocyclic etc. fragments and a structure of these molecules, in particular -macro cycle size, it dentacy, lipophily, nature of the substituents, and other factors. the elucidation of similar relations allows predicting biological properties of crown compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of biological effect of such kind of compounds. completely adequate qsar models (r = . - . , q = . - . ) have been obtained using partial least squares method for all of the studied cultures. on the base of the first ones the molecular fragments with positive or negative influence on the explored properties have been determined. they give a possibility to realize the virtual screening and molecular design of compounds with the high level of target activity. the results of prognosis are verifying by the experimental investigations. conclusion: qsar analysis of antimicrobial activity of crown ethers allows us to suppose the presence of two different mechanisms of their antimicrobial action. it is discovered that the presence of diphenyloxide and tert-butyl fragments promotes; diphenyl-sulphide and diamino-biphenyl -prevents the antimicrobial action. it is shown that the hexadenthal crown ethers containing aromatic fragments with a tert-butyl group are the most perspective antimicrobials. objectives: methionyl trna synthetase (mrs) catalyses the covalent attachment of methionine to its cognate trna. rep is a synthetic inhibitor of mrs with potent antibacterial activity against staphylococcus aureus including clinically-relevant resistant strains (mic equals . to . lg/ml). we determined the biochemical potency and mechanism of action of rep and related compounds with respect to s. aureus mrs enzymatic activity. we also evaluated the enzyme kinetic properties of mutated forms of s. aureus mrs. methods: the mets gene from s. aureus was expressed in e. coli and mrs was purified to near homogeneity by ammonium sulfate fractionation and anion exchange chromatography. aminoacylation of trnamet was measured using scintillation proximity assays (spa). the kinetics of the atp:ppi exchange were determined using thin layer chromatography (tlc). mutants of s. aureus mrs were selected by serial passage and spontaneous resistance in the presence of rep . results: rep exhibited strong inhibition of s. aureus mrs in the aminoacylation reaction, having an ic limited by the enzyme concentration. in order to estimate the true inhibition constant (ki), we utilized an atp:ppi exchange assay. rep showed potent inhibition of s. aureus mrs, with a ki of pm. related inhibitors were analysed, and a correlation was observed between the ki for mrs and the mic for s. aureus. rep was found to be competitive with methionine binding, but uncompetitive with atp binding (i.e., increasing the atp concentration resulted in tighter binding of rep ). the majority of analogs exhibited comparable mechanism of action; altered mechanism of action was observed with a subset of analogs. mutated s. aureus mrs variants (derived from strains with elevated mics) showed substantially weaker binding by rep . all of the mutated enzymes exhibited impaired trna aminoacylation activity, with defects ranging from reduced turnover rates to weaker affinities for one or more substrates. conclusions: rep is a potent inhibitor of s. aureus mrs. enzymatic potency of this class of inhibitors correlates with microbiological potency. mutations that confer resistance to rep result in functionally impaired mrs, encompassing a wide variety of enzymatic phenotypes. we report here the antibacterial and antifungal activity of newly synthesized and physico-chemically characterised thioureides of -( -chlorophenoxy)-benzoic acid. the new compounds were prepared in three stage. firstly, the -( -chlorophenoxymethyl)-benzoic acid was prepared by treating the phtalide with p-chlorophenol potassium salt in xylene. the second stage was the synthesis of -( -chlorophenoxymethyl)benzoyl chloride by treating the corresponding acid with thionyl chloride using anhydrous , -dichloroethane as solvent, followed in the third stage, by the treatment of the above-mentioned chloride with ammonium thiocyanate. the -( -chlorophenoxymethyl)-benzoyl isothiocyanate resulted after refluxing the reaction mixture in dry acetone. the new compounds were prepared by refluxing the isothiocyanate with primary aromatic amines in dry acetone. the obtained compounds have been characterized by their physical properties and their chemical structures were confirmed using the spectral analysis. the aim of this study was also to evaluate the in vitro antimicrobial activity of the new compounds. the in vitro antimicrobial testing was performed by binary microdilution method, in multi-well plates, in order to establish the minimal inhibitory concentration (mic), against gram-positive (listeria (l.) monocytogenes, staphylococcus (s.) aureus, bacillus (b.) subtilis), gram-negative (psedomonas (p.) aeruginosa, escherichia (e.) coli, salmonella (s.) enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, depending on the nature of the substituents and their position on the benzene ring, both concerning the microbial spectrum and the mic value. the mics values widely ranged between mcg/ml and mcg/ml. the most active proved to be n-[ -( -chlorophenoxymethyl)-benzoyl]-n'-( , -dichloro-phenyl)-thiourea and n-[ -( -chlorophenoxymethyl)-benzoyl]-n'-( -bromo-phenyl)-thiourea, showing a large spectrum of antimicrobial activity against enterobacterial strains (e. coli and s. enteritidis), l. monocytogenes, s. aureus and candida sp. all the tested compounds were highly active against s. aureus (mic = mcg/ml). four of the tested compounds exhibited antifungal activity (mic = - mcg/ml), and p. aeruginosa as well as b. subtilis were resistant to all tested compounds. in vitro antimicrobial activities of novel dianthraquinones produced by a marine streptomyces sp. against clinical staphylococcus aureus and enterococcus faecium isolates k.l. laplante, k. lor, a. socha, d.c. rowley (providence, north kingston, us) objectives: the escalation of antibiotic resistance among grampositive pathogens presents increasing treatment challenges and requires the development of new therapeutic agents. recently we discovered a new class of dianthraquinone antibiotics produced by a marine streptomycete. the inhibitory and bactericidal activity of four dianthraquinone secondary metabolites and four semi-synthetic derivatives were measured against clinical strains of vancomycin resistant e. faecium (vre), methicillin susceptible and methicillin resistant s. aureus (mssa and mrsa, respectively). two compounds, daq a and daq , were tested against an expanded panel of pathogens. methods: thirty-two clinical strains of vre (n = ), mssa (n = ) and mrsa (n = ) were obtained from patients at the veterans affairs medical center in providence, ri. mic's were performed using methodologies described by clsi. control isolates were atcc and atcc . the bactericidal activity of each antimicrobial agent was evaluated with time-kill experiments using randomly selected mssa (n = ), mrsa (n = ), and vre (n = ) isolates tested at times the respective mic. conclusions: the potent activities and unusual structures of the dianthraquinones tested here suggest that these may provide a new molecular scaffold for the development of novel antimicrobial agents. more biological testing is warranted to more fully explore the clinical potential of these antibiotics. efficacy of the novel antimicrobial peptide plectasin to staphylococci objective: the purpose of the investigation was to investigate the in vitro efficacy and kill kinetics of plectasin against staphylococcus aureus. plectasin is a newly discovered defensintype antimicrobial peptide found in the fungus pseudoplectania nigrella which showed activity against several gram-positive bacteria including drug resistant strains (mygind ph. et al. plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. nature ; : - ). methods: all experiments were determined according to clsi/ nccls guidelines. bactericidal activity was characterized by time kill experiments at and times the mic. staphylococcus aureus (s. aureus) atcc were used as the test organism and vancomycin was used for comparison. the kill kinetics and post antibiotic effect (pae) were evaluated by cfu determination. inoculum sizes ranging from e to e cells were used to test the inoculum effect. e cells were employed for determination of mutant prevention concentration (mpc) and the frequency of spontaneous resistance. results: plectasin is bactericidal as evidenced by kill kinetics showing a . log reduction in cfu/ml after hour of incubation and a reduction of . log cfu/ml after hours. this is superior compared to the activity of vancomycin. no inoculum effect was observed in the employed range of cells. the observed pae had a duration of hours and minutes. no spontaneously resistance mutation was observed among e cells of staphylococci and the mpc were determined to be times mic. conclusions: plectasin is a novel antimicrobial peptide that shows potent antimicrobial activity against gram-positive bacteria including drug-resistant organisms. the potent, excellent bactericidal activity in vitro, lack of cross-resistance to clinical used antibiotics, low spontaneously resistance mutation frequency and good pae properties, suggest that plectasin may have potential as a therapeutic agent against staphylococci. in vitro antimicrobial activity of the novel polymeric guanidine akacid plus Ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: cationic antimicrobials are widely used for disinfection within clinical settings. in the present study the bactericidal and fungicidal activity of akacid plus Ò , a novel polymeric compound of the cationic family of disinfectants, was evaluated against quality control strains of staphylococcus aureus, enterococcus hirae, escherichia coli, pseudomonas aeruginosa, candida albicans and aspergillus niger in comparison to chlorhexidine digluconate. methods: the in vitro activity of akacid plus Ò and chlorhexidine was determined by quantitative suspensions tests according to the european committee for standardization at concentrations of . - . % against bacterial strains and c. albicans and at concentrations of . - % against a. niger after exposure for , and min in the presence and absence of . % bovine albumin and dilution in distilled and hard water. results: in the basic quantitative suspension test akacid plus Ò destroyed all bacterial pathogens at a concentration of ‡ . % in £ min contact time. chlorhexidine was also highly active against s. aureus, e. coli and p. aeruginosa, but failed to eliminate e. hirae within min. under high organic burden, the bactericidal activity of both disinfectants was slightly reduced. akacid plus Ò showed fungicidal activity against c. albicans within - min and eliminated a. niger at a concentration of ‡ % in min contact time. chlorhexidine was fungicidal against c. albicans, but did not achieve biocidal activity against a. niger. conclusion: the novel polymeric guanidine akacid plus Ò when compared to chlorhexidine digluconate showed similar bactericidal activity against s. aureus, e. coli and p. aeruginosa and superior biocidal activity against e. hirae and a. niger. investigation of emergence of bacterial resistance to the novel antibacterial photodynamic agent xf- are novel, light activated antibacterial agents ( ) active against gram-positive bacteria, which have greater potency than antibiotics. the emergence of resistance to xf- has been investigated. methods: . mg/l of xf- was added to cells/ml of mrsa. after minutes incubation in the dark the unbound xf- was removed and the culture illuminated with . j/cm of light at nm and cfu analysis undertaken to determine the number of viable cells remaining. surviving clones of the treatment were cultured and subjected to further treatment. cycles were undertaken to determine whether the number of surviving cells increased, suggesting resistance build up to xf- . results: the survival of methicillin-resistant staphylococcus aureus (mrsa) (atcc baa- ) is expressed as log n /n, where n and n are the cfu of untreated and treated suspensions, respectively. the results demonstrate that no detectable resistance build up to the activity of xf- was seen after successive treatments. a low propensity for emergence of resistance is a valuable attribute for new anti-bacterial agents. xf- might be effectively employed in the clinical setting for prophylactic use to decolonise skin and nares and therapeutic use to treat infected wounds/ulcers. objectives: the xf drugs are novel, light activated antibacterial agents ( ) active against gram-positive bacteria which have superior potency to antibiotics but possess a low propensity to induce resistant bacterial strain emergence. a novel ex-vivo porcine skin model has been developed to test the antibacterial activity of xf- on the surface of skin. methods: x cells of methicillin-resistant staphylococcus aureus (mrsa) were inoculated onto a . cm area of ex-vivo porcine skin samples, immobilised in agar. after drying, solutions of xf- were applied and after minutes, the samples were illuminated for minutes with blue light ( nm) with various total light doses using a lumacare tm lc- m lamp. cfu analysis were undertaken to determine the number of viable cells remaining after treatment. controls of drug alone and light alone were included. results: using . mg/l of xf- , cfu analysis demonstrated that at a total light dose of j/cm , there was~ % kill of bacteria. at j/cm , there was . % kill of bacteria, and . % at j/cm and j/cm . at a total light dose of j/ cm , it was found that there was a < % kill by xf- at concentrations of . , . and . mg/l. at a concentration of . mg/l, there was a > . % kill. this kill did not significantly increase at . and . mg/l. conclusions: the results demonstrate that xf- has exceptional activity at low concentrations against mrsa on the surface of porcine skin. xf- and light are non-toxic to skin at therapeutic concentrations. work is in progress to clinically evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objectives: the rise of epidemic methicillin-resistant staphylococcus aureus (emrsa) and the emergence of mupirocin resistance means that it is essential to develop new therapies that cannot be readily overcome by microorganisms. the xf series of novel light activated antibacterial agents ( ) active against gram-positive bacteria addresses this issue and have superior levels of activity to antibiotics but with less likelihood of resistance emergence. the antibacterial activity of the xf drugs against emrsa has been investigated. methods: mic and mbc assays were used to investigate the antibacterial activity of xf- , a novel antimicrobial photodynamic agent against a range of staphylococcus aureus strains. a concentration range of - . mg/l was investigated. minutes of nm light activation ( j/cm ) was applied. light alone had no effect. results: conclusions: the results demonstrate that xf- has exceptionally low mic and mbc values against all of the s. aureus strains tested. the results also demonstrate that xf- is equally effective against mrsa and methicillin-sensitive staphylococcus aureus (mssa) indicating its mode of action is independent of antibiotic resistance. xf- may therefore be useful in prevention and treatment of emrsa. xf- is non-toxic to skin at prophylactic/therapeutic concentrations and has potential for the treatment of skin sepsis and the eradication of nasal and skin mrsa carriage. work is in progress to evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objective: nxl is a novel antibacterial currently in preclinical development. the mechanism of action is directed against topoisomerase, and the spectrum of activity is exclusively against gram positive organisms. the goal of the study was to characterise the activity and time/kill kinetics against common aerobic cocci in comparison to currently marketed molecules: linezolid (lin), vancomycin (van), quinupristin/dalfopristin (q/d) and moxifloxacin (mox). methods: (i) in vitro susceptibility tests: the strains used were from the culture collection of novexel and were of clinical origin. mics were determined by an agar dilution technique. mueller hinton agar medium was used, supplemented with % horse blood for group a streptococci (gas), group b streptococci and s. pneumoniae. overnight cultures were diluted to obtain the final inoculum of cfu/spot. the mic was the lowest concentration which inhibited all visual growth ( or less colonies were ignored). (ii) time/kill kinetics: experiments were performed against strains of s. aureus (n = ) and s. pneumoniae (n = ) in ml volumes of appropriate growth medium with initial inoculum of around cfu/ml of logarithmically growing culture. timed samples over a hour period were enumerated using a spiral plating method. nxl was compared to linezolid and vancomycin and the concentrations tested were , and -fold the mic for both species. results: (i) the mic s of nxl versus comparators are shown in the table. (ii) time kill experiments showed that nxl was bactericidal against s. aureus, including methicillin resistant strains (> log reduction within - hours) compared to a slowly bactericidal effect for vancomycin ( hours). nxl and vancomycin were both bactericidal against s. pneumoniae within - hours. linezolid was bacteriostatic against all strains tested. conclusion: nxl exhibits bactericidal activity against common gram positive cocci, including strains which exhibit resistance to methicillin, vancomycin and fluoroquinolones. nxl warrants further investigation. objectives: the aim of this study was to identify bacterial proteins as targets of the endogenous antiseptic n-chlorotaurine (nct), which is a promising microbicidal agent for topical treatment of infections. in addition, a combination of nct with ammonium chloride which enhances the microbicidal activity significantly was investigated. methods: escherichia coli and staphylococcus aureus were treated with nct and nct plus ammonium chloride for different incubation times between and min -a period where killing takes place. to find out protein changes, d-page of bacterial proteins followed by mass spectrometry was performed. results: incubation in % nct revealed a change of the charge and a separation of numerous proteins into a series of spots with a different isoelectric point. moreover, in e. coli heat shock protein appeared, while ribosome releasing factor, d-ribose periplasmic binding protein, and malonyl-coa transacylase spots decreased. in s. aureus, enolase and a translation elongation factor decreased. these changes appeared more rapidly in the presence of ammonium chloride, which can be explained by formation of the more lipophilic and microbicidal monochloramine. molecular mechanisms of attack comprised mainly oxidation of thio and amino groups as confirmed with model peptides. conclusion: these results fit very well to previous preclinical and clinical findings. they indicate both surface attack and penetration of oxidation capacity into the bacteria and destruction of essential proteins by nct and nct plus ammonium chloride, respectively. objectives: ceftobiprole is a new extended-spectrum cephalosporin with activity against methicillin-susceptible and methicillin-resistant staphylococci, as well as against most enterobacteriaceae. in this study the anti-staphylococcal activity of ceftobiprole is reported from a set of isolates from a recent clinical trial. methods: consecutive clinical isolates of staphylococci from patients enrolled in a multicentre clinical trial involving complicated skin infections were examined for their susceptibility to ceftobiprole and selected anti-gram-positive agents. mics were determined using clsi methodology. results: among these isolates, staphylococcus aureus and coagulase-negative staphylococci (cons) were identified. the percentages of methicillin-resistant strains were % for s. aureus and % for cons. all strains (except one cons with a linezolid mic of mg/l) were susceptible to vancomycin and linezolid, with mics < mg/l.against methicillin-susceptible s. aureus, ceftobiprole mic and mic values were . and . mg/l, respectively, and against methicillin-resistant s. aureus, ceftobiprole mic and mic values were . and mg/l, respectively. ceftobiprole mics ranged from £ . to mg/l against methicillin-susceptible-cons (ms-cons) and methods: consecutive, non-duplicate bacterial isolates ( , strains) acquired from patients with bloodstream, respiratory, and skin and skin structure infections both nosocomial and community acquired were submitted from > medical centres in europe, the americas and the asia-pacific region. all isolates were tested using clsi/nccls broth microdilution methods against grn, the currently marketed fluoroquinolones (fq) including cipro, levofloxacin (levo), gatifloxacin (gati) and representative comparator agents. oxa-and cipro-s and -r subsets were included. a grn-s breakpoint of £ . mg/l was applied for comparative purposes only and was based upon the mic population distributions of strains that included quinoloneresistance determining region (qrdr) mutations. results: potency for grn and comparator fqs tested against sa: (see table) . key resistance patterns (%) among this sa collection included oxa ( . ), cipro ( . ), erythromycin ( . ), clindamycin ( . ), tetracycline ( . ), and trimethoprim/ sulfamethoxazole ( . %); gram-positive-targeted comparator including vancomycin, linezolid, daptomycin and quinupristin/dalfopristin all remained > % s. compared with currently marketed fqs when tested against all sa, grn was -to -fold more active (mic , £ . vs. . or . mg/ l). against both oxa-s and -r sa, grn displayed markedly enhanced potency compared with cipro and levo ( ‡ -fold), and gati ( -to -fold). among cipro-r isolates, grn also maintained ‡ -fold greater potency (mic , vs. ‡ mg/l) although overall s for all fqs was - %. compared to the fq agents tested against sa, grn was the most potent agent and maintained the broadest coverage against oxa-and cipro-r strains even when applying a very conservative epidemiologic breakpoint. when a fq is indicated for staphylococcal coverage, this des-f( ) quinolone may represent a superior alternative among fq class agents, while minimizing selection of resistance. objective: to assess the garenoxacin (grn) potency against a vast number of international respiratory tract infection (rti) pathogens, especially versus phenotypic (high mic) or genotypic (sequence change) qrdr mutants. a total of , isolates from continents were analysed ( ) ( ) ( ) ( ) ( ) ( ) ( ) table) conclusions: grn maintains clinically usable activity (mic, £ mg/l) against important community-acquired rti pathogens having r to presently marketed fluoroquinolones and against those isolates with documented qrdr mutations. continued development of this novel des-f( ) quinolone agent appears desirable. in vitro activity of garenoxacin tested against ciprofloxacin-susceptible and -resistant enterobacteriaceae and acinetobacter spp. strains collected worldwide by the sentry antimicrobial surveillance program ( ) ( ) h. sader, t. fritsche, p. strabala, r. jones (north liberty, us) objective: to evaluate the contemporary activity of garenoxacin (grn) against ciprofloxacin (cipro)-susceptible (s) and cipro-resistant (r) enterobacteriaceae (ent) and acinetobacter spp. (asp). unlike recently marketed fluoroquinolones (fq), grn, a des-f( ) quinolone lacks the c- fluorine. methods: a total of , isolates ( , ent and asp) were consecutively collected from > medical centres from bloodstream, respiratory, urinary and skin and soft tissue infections and tested by reference broth microdilution methods according to clsi/nccls methods and interpretative criteria. a grn s breakpoint of £ mg/l was applied for comparison purposes only. results: the results of the major organism groups tested: (see table) . grn showed excellent activity against this large collection of ent (mic , . mg/l) and . % of isolates were inhibited at £ mg/l. objectives: garenoxacin (grn) is a novel, broad-spectrum des-f( )-quinolone with activity against gram-negative and grampositive aerobes and anaerobes including quinolone-resistant staphylococcus aureus. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in complicated skin and skin structure infections (csssi). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn ( mg iv to po qd) or piperacillin/tazobactam ( . g iv q h) with transition to po amoxicillin/clavulanate ( mg po q h). in the second study, subjects received grn ( mg po qd) or ciprofloxacin/metronidazole ( mg q h/ mg q h). all antimicrobials were administered for to days. subjects were adults ( ‡ y) newly hospitalized or ambulatory outpatients with evidence of csssi who did not have underlying osteomyelitis. microbiologic efficacy was determined to days post-therapy. results: a total of subjects were microbiologically evaluable (grn, n = ; comparators, n = ). the disease diagnosis was similar between grn and comparators and included infected pressure sore ( % vs %), infected diabetic foot ulcer ( % vs %), major abscess ( % vs %), or postsurgical wound infection ( % vs %). the majority of common skin pathogens were eradicated by grn background: acute bacterial sinusitis (abs) is a common infection world-wide, with many patients having an associated an allergic component/history. however the role of antibacterials in these patients (pts) has not been examined. as some fluoroquinolones (fq) have an in-vitro immunomodulatory effect (ie) the clinical efficacy of gem was compared other agents in abs pts with or without allergic rhinitis (ar). methods: phase clinical trials were pooled and pts where allergic rhinitis was identified ( pts) were compared with pts not reporting ar ( pts). clinical response (success or failure) at end of therapy (eot) & at follow-up (fu, approx. - weeks after treatment) was studied. comparators (cmp) were cefuroxime (cef) and trovafloxacin (tro). results: % success based on clinical outcome at eot and fu for ar and non ar pts are shown in the table. for all treatments eot success was high for the non ar pts, but at fu this was reduced, especially with both fqs. in contrast, gem retained a high clinical success rate in pts with ar unlike cef or tro. conclusion: gem has been shown to be very efficacious in a sub group of problematic abs pts. this advantage may be due to the high antibacterial activity of gem vs key abs pathogens and/or a stimulatory ie. both being important with pts having decreased local immune defences. these data also show that not all fluoroquinolones have immuno-stimulatory properties. garenoxacin efficacy against multidrug-resistant streptococcus pneumoniae: retrospective analysis of community-acquired pneumoniae isolates obtained from nine phase ii and iii clinical studies ( ) ( ) ( ) ( ) ( ) t. black, h. waskin, r. hare (kenilworth, us) objective: garenoxacin (grn) is a novel, des-f( )-quinolone with excellent activity against s. pneumoniae, one of the most common pathogens causing community-acquired pneumoniae (cap). the incidence of infections caused by antibiotic-resistant isolates of streptococcus pneumoniae is on the increase, therefore information regarding the activity of new anti-infective drugs against populations of s. pneumoniae that are multi-drug resistant (mdr) is critical. mdr s. pneumoniae (mdrsp) includes isolates previously known as prsp (penicillinresistant s. pneumoniae), as well as strains resistant to two or more of the following antibiotics: second-generation cephalosporins, macrolides, tetracyclines, and trimethoprim/ sulfamethoxazole. methods: pretreatment sputum and blood isolates collected worldwide during grn phase / clinical cap trials ( ) ( ) ( ) ( ) ( ) were retrospectively analysed for the mdrsp phenotype. of the s. pneumoniae isolates originally identified, from subjects were subjected to secondary mdr susceptibility testing by central laboratories. confirmed mdrsp isolates were matched to individual subjects to assess clinical and microbiological outcomes for mdrsp-infections treated with grn. results: expanded susceptibility testing identified / mdrsp isolates from unique subjects. the lowest mic and mic values for mdrsp isolates tested against a panel of representative drugs were observed for grn (table ; . lg/ ml and . lg/ml, respectively). the incidence of resistance to the five classes of drugs was %, %, %, % and % for penicillin, nd generation cep., macrolides, tetracycline and tri/sulf, respectively. no isolates were resistant to grn using a proposed susceptibility breakpoint value of £ lg/ml. thirtyfive percent, %, %, % and % of isolates were resistant to , , , and drug classes, respectively. the worldwide incidence of mdrsp was % with an equivalent geographic distribution of %, % and % among north america, europe and the rest of world. overall, grn provided clinical and bacteriological success for / ( %) cap evaluable subjects with mdr infection, which was similar to clinical success for evaluable subjects with non-mdrsp cap infections / ( %). conclusions: these data demonstrate the ability of grn to successfully eradicate mdrsp associated with cap. . per cent success is shown in the table (ab, antibiotics, copd, chronic bronchitis and obstructive lung disease, hd, heart disease). results: although gemifloxacin showed lower % success than comparator against cap patients with no defined risk factor, gemifloxacin was considerably more successful than comparator against patients associated with risk factors, especially diabetic patients where comparator success was low. this advantage was often more prominent at fu than at eot. patients with other comorbidities such as renal failure or malignancy were not recruited in sufficient number for analysis. conclusions: these data support the use of gemifloxacin in the treatment of cap, especially where the patient has recognised idsa risk factors. microbiologic efficacy of garenoxacin vs. comparators against common pathogens associated with community-acquired pneumonia objectives: garenoxacin (grn) a novel, broad-spectrum des-f( )-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. grn has dual sites of inhibition (dna gyrase and topoisomerase iv) and may be less likely to promote resistance. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in community-acquired pneumonia (cap). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn ( mg po qd for d) or amoxicillin/clavulanate (a/c; mg po q h for - d). in the second study, subjects received grn ( mg po qd for - d) or levofloxacin (lev; mg po qd for - d). adults ( years of age or older) were enrolled with clinical and radiologic evidence of cap [new infiltrate(s) on chest radiograph and fever, leukocytosis, cough, chest pain, auscultatory findings, or sputum production]. the majority of subjects were fine class i/ii in both studies. bacteriologic eradication was assessed to days post therapy. results: a total of treated subjects had pretreatment pathogens (grn, n = ; comparators, n = ) . the overall eradication rate in all treated subjects was % ( / ) for grn and % ( / ) for the comparators. eradication rates for s pneumoniae were % ( / ) for garenoxacin and % ( / ) for the comparators. eradication of s pneumoniae was % and % for a/c and lev, respectively. in strains with reduced susceptibility to penicillin eradication rates were % ( / ) vs % ( / ) in favour of grn. eradication rates for h. influenzae were % ( / ) and % ( / ) for grn and comparators, respectively. lev eradicated % of h. influenzae isolates and a/c eradicated % of the strains isolated. there were very few isolates ( ) of moraxella catarrhalis in the studies. in study grn was % effective against strains of m. catarrhalis and in the other a/c was % effective against the strain isolated. grn eradicated % ( / ) of the staphylococcus aureus isolates vs % ( / ) for the comparators. conclusions: grn was highly active against pathogens commonly associated with cap including drug-resistant strains of s pneumoniae and represents an effective therapeutic option for this patient population. objectives: garenoxacin (grn) a novel, broad-spectrum des-f( )-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. there is a growing problem of resistance in strains of s pneumoniae, with multi-drug-resistant s pneumoniae (mdrsp) becoming increasingly more common. the objective of this study was to evaluate the clinical and microbiologic efficacy of grn in the treatment of communityacquired pneumonia (cap) caused by mdrsp. methods: this was a multinational, open-label, noncomparative study. subjects were adults ( ‡ and < y) with clinical (clinical signs, sputum production), radiologic (new infiltrates on chest radiograph), or microbiologic (predominance of gram-positive cocci in pairs on sputum gram-stain or a positive blood culture for s. pneumoniae) evidence of cap caused by s. pneumoniae. subjects received grn mg po qd or grn mg iv with transition to mg po qd for to days. clinical and microbiologic responses were determined at a test-of-cure visit to days posttherapy. results: a total of subjects were enrolled. of these, ( po only, iv to po) were clinically and microbiologically evaluable. clinical and microbiologic success rates were % ( / ) and % ( / ), respectively. clinical success rates were % ( / ) and % ( / ) for po and iv to po, respectively. documented s. pneumoniae bacteremia was present in % (n = ) of subjects with a clinical success rate of %. among evaluable subjects, resistance rates for s. pneumoniae were penicillin %, second-generation cephalosporin %, macrolides %, tetracyclines %, and trimethoprim/ sulfamethoxazole %. twelve evaluable subjects had pneumonia caused by mdrsp. clinical success rate was % ( / ) in subjects with mdrsp and % ( / ) in non-mdrsp subjects. clinical success of grn for strains resistant to , , , or antimicrobial drug classes, were % ( / ), % ( / ), % ( / ), and % ( / ), respectively. microbiologic success was % ( / ) and % ( / ) for mdrsp and non-mdrsp (susceptible or resistant to class) strains, respectively. grn was generally well tolerated with drug-related adverse events (ae) reported in % ( / ; po) and % ( / ; iv to po) of subjects. conclusions: grn (po or iv to po) is an effective treatment for cap caused by mdrsp and non-mdrsp. grn is well tolerated. in vitro bactericidal activity of daptomycin against staphylococcus aureus and enterococcus spp.: comparison with vancomycin, teicoplanin and linezolid h. drugeon, m. juvin (nantes, fr) objectives: the aim of this study was to evaluate the bactericidal activity (by killing kinetics) of daptomycin (dap) against staphylococcus aureus (sa) clinical isolates with different teicoplanin mics and against enterococcus faecalis (efl) and e. faecium (efm) with different mechanisms of glycopeptide resistance. dap has been compared with teicoplanin (tei), vancomycin (van) and linezolid (lin). methods: sa strains ( mssa and mrsa) with tei mic distributed from . to mg/l, enterococcus ( efl and efm) with glycopeptide phenotypes [s, r-vana, r-vanb] were studied using a killing curve method. antibiotic concentrations were used from mg/l to mg/l in two fold dilutions. surviving bacteria were counted at t , t ', t , t , t , t and t hours using agar plates with inhibitors to prevent antibiotic carry-over. antibiotics tested were daptomycin (dap), teicoplanin (tei), vancomycin (van) and linezolid (lin). results: all the sa isolates were susceptible to dap (mic = . - mg/l), to lin (mic = - mg/l), to van (mic = - mg/l) regardless of susceptibility to methicillin. dap showed the same strong concentration dependent bactericidal activity with mssa and mrsa: at t ' bactericidal activity (ba) (decrease of log cfu/ml) was observed with - mg/l of dap; at t hours, - mg/l of dap was sufficient and at t hours, ba was obtained with mg/l of dap. the other antibiotics showed a time dependent bactericidal activity but ba was observed only with long exposure ( ‡ hours) and with high concentrations. all the enterococcus isolates were susceptible to dap (mic = - mg/ l) and to lin (mic = - mg/l) regardless of the resistance to glycopeptides. ba of dap was also concentration dependent. ba was obtained with - mg/l after hours of contact and with mg/l after hours of contact for efl. ba was observed with - mg/l after hours of contact and with - mg/l after hours of contact for efm. the other antibiotics had a time dependant activity but didn't show bactericidal activity with concentrations mg/l. conclusion: the bactericidal activity of daptomycin was very strong, concentration dependent, and not influenced by the level or mechanism of glycopeptide resistance the bactericidal activity of linezolid was time dependent and observed only with the highest concentration and the bactericidal activity of vancomycin and teicoplanin was time dependent but was influenced by the mechanism of glycopeptide resistance. objectives: telavancin (tlv) is a bactericidal lipoglycopeptide with multiple mechanisms of action that is in phase clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia with a focus on infections due to methicillin-resistant staphylococcus aureus (mrsa). this study evaluated and compared the antibacterial activity of tlv with that of other antibacterial agents against recent gram-positive clinical isolates from germany. methods: a total of aerobic gram-positive bacterial strains recently collected were included. antibiotics tested were tlv, vancomycin (van), teicoplanin, penicillin, oxacillin, ampicillin, cefuroxime, ceftriaxone, daptomycin (dap), linezolid (lzd), quinupristin-dalfopristin, clindamycin, ciprofloxacin, levofloxacin, gentamicin, streptomycin, erythromycin, telithromycin, co-trimoxazole and tetracycline. mics were determined by the broth microdilution procedure according to the guidelines of the clsi. results: tlv exhibited potent activity against all grampositive bacteria including resistant isolates such as mrsa, van-resistant enterococci, pneumococci (including multiple resistant strains with various antibiotic resistance phenotypes) and other streptococcal species. tlv showed excellent in vitro activity against the species irrespective of the antibiotic phenotype tested. for methicillin-susceptible s. aureus (mssa, n = ) and mrsa (n = ) mic of tlv for both phenotypes were . mg/l. for coagulase-negative staphylococci (n = , incl. msse, mrse, mssh, mrsh and others) mic s were . or mg/l. mic s of tlv for enterococcus faecalis (n = ) and e. faecium (n = ) were and mg/l, respectively. for van-resistant strains of e. faecalis (n = ) or e. faecium (n = ) mics for tlv ranged from . to mg/l. against streptococcus pneumoniae (n = ) tlv mics ranged from £ . to . mg/l. all streptococcus pyogenes, streptococcus agalactiae and all viridans group streptococci (n = ) had mics of £ . mg/l. conclusion: based on mic , tlv was more potent than van, dap or lzd against staphylococci, streptococci and e. faecalis. it was superior to dap and lzd against e. faecium and at least as active as dap or lzd against most van-resistant enterococci. tlv appears to be a promising new antimicrobial agent for the treatment of infections caused by gram-positive organisms including multiply resistant isolates. the extent of protein binding (pb) of dap is still under investigation and data available so far indicate pb of either % or %. therefore we tested two fscs: . (corresponding to % pb) and . (corresponding to % pb). the activity of dap was determined in mueller-hinton broth supplemented with mg/l calcium. viability counts were performed at . , . , , , , and h. one methicillin-susceptible staphylococcus aureus (mssa), two methicillin-resistant s. aureus (mrsa), one vancomycin-susceptible (van-s) and one van-resistant (van-r) enterococcus faecalis, one van-s and one van-r enterococcus faecium were tested. bactericidal activity was defined as > . % killing during incubation. results: dap was bactericidal at concentrations of . mg/l and . mg/l in all seven strains. the concentration of . mg/l was bactericidal against the two mrsa and against the van-s e. faecium. in the other four strains the maximum reduction of initial inoculum ranged from . to . log cfu/ml. in six strains a bactericidal effect at . mg/l and . mg/l of dap, respectively, occurred between minutes and h and after h in the van-s e. faecalis. van at . mg/l or . mg/l was bactericidal in only two strains after h ( mssa, mrsa). against the other five strains, van was bacteriostatic with maximum reduction of initial inoculum between . and . log cfu/ml at mg/l after h, respectively. both tpl and lzd were consistently bacteriostatic against the test strains. conclusion: dap at psc of . mg/l as well as at fsc of . mg/l showed a pronounced bactericidal effect within h in / strains. van was bactericidal in only / strains after h. compared to van bacterial killing by dap was very rapid. tpl and lzd were bacteriostatic only. the effect of human serum on the bactericidal activity of daptomycin and comparators against staphylococcus aureus and enterococcus spp. background: daptomycin is a new cyclic lipopeptide antibiotic that shows rapid bactericidal activity and has high protein binding when assessed by standard methodology. this study investigated the bactericidal activity of daptomycin and the effect of protein binding by the addition of % human serum (hs). methods: exponentially-growing methicillin-susceptible andresistant s. aureus (mssa, mrsa) and vancomycin-susceptible enterococcus faecium (vse) and -resistant enterococcus faecium (vre) (ca. cfu/ml) were exposed to daptomycin (dap), vancomycin (van), teicoplanin (tei), piperacillin-tazobactam (ptz) or linezolid (lzd) at peak (p) and trough (t) serum concentrations in mueller hinton broth supplemented with ca + to mg/l with or without hs. viable count was determined at . , . , , & h. plots were made of log reduction in viable count over time and the area-under-thecurve measured to calculate bactericidal indices (bis) from these plots (j antimicrob chemother , : - ). results: daptomycin reduced viable count of mssa & mrsa by approx. logs or more within . h and vse or vre within h at p. other agents either did not achieve this or required h to do so (not shown). bi data are shown below (>represents kill beyond the limit of detection). hs had little effect on dap kill, except against the vre at t. nevertheless, dap at t against vre was more bactericidal than any other antibacterial except dap at p. conclusions: dap was the most bactericidal agent tested as measured either by bi or rate of kill. dap at p reduced mssa and mrsa to below detection within min. the effect of hs was minimal which suggests that protein binding is either weak or highly reversible. these data support the use of dap in the treatment of infections caused by these organisms. daptomycin activity against multi-resistant staphylococcus haemolyticus bloodstream isolates from severe infections objectives: daptomycin, a new cyclic lipopeptide with activity against multidrug-resistant gram-positive pathogens including mrsa, is approved for use in cssst infections (us-fda) and is being reviewed by emea for approval in eu member countries. the rapid bactericidal activity of daptomycin, due to its unique mechanism of action, makes it an attractive antibiotic for serious gram-positive infections. the study was performed: (i) to evaluate the activity of daptomycin and other drugs against multi-resistant clinically relevant staphylococcus haemolyticus (mrsh), isolated from bloodstream infections in various hospitals in italy (ii) to determine epidemiologic and genetic correlation among strains, and (iii) to characterize the sccmec dna of these strains. methods: the mrsh strains were tested against a panel of antimicrobial agents, by broth microdilution method performed according to clsi (clinical laboratory standards institute) guidelines, including supplementation of mg/l calcium for daptomycin. moreover, phenotypic tests and antibiotic susceptibility profiling were carried out and the results compared with molecular typing analysis by using smai-pfge fingerprints and pcr to characterize the mec-complex. results: all isolates were resistant to erythromycin, gentamicin, ciprofloxacin, strains showed reduced susceptibility to vancomycin (mics mg/l), strains were resistant to cotrimoxazole, strains to clindamycin, strains to chloramphenicol and strains to tetracycline. almost all isolates were inhibited by £ mg/l of daptomycin, and only four strains exhibited a mic value of mg/l. pfge analyses showed the existence of at least two multi-resistant s. haemolyticus clones widespread in different hospitals. methicillin-resistance was correlated to the presence of the meca and preliminary results regarding the genetic element carrying the gene, showed an organization of the mec-complex of class a and class c. conclusions: our results suggest that daptomycin has excellent activity against multiresistant mr s. haemolyticus isolates, which represent a serious threat in catheter-related bloodstream infections. furthermore, the emergence of s. haemolyticus exhibiting reduced susceptibility to vancomycin is of particular concern, probably due to the common use of vancomycin as initial therapy for such infections. moreover, the use of additional molecular techniques to fingerprint isolates makes this study of clinically important cons more accurate. objectives: ceftobiprole is a new cephalosporin with a broad spectrum of action including methicillin-resistant staphylococci (mrs) as well as many other gram-positive and gram-negative pathogenic bacteria. this study investigates the structural basis for the good activity against mrs. methods: the primary beta-lactam resistance determinant of mrs, penicillin-binding protein pbp ' (or a) has been cloned and expressed as a soluble form in which the amino-terminal residues forming a membrane-anchor have been deleted. the soluble form has been crystallized and the structure of the complex formed after soaking crystals in a solution containing ceftobiprole has been determined at . angstrom resolution. additional data on the structure of the ceftobiprole-pbp ' complex formed in solution has been obtained using spectroscopic methods such as uv-circular dichroism. results: ceftobiprole reacts rapidly with pbp ' to form a stable acyl-enzyme complex. the ceftobiprole moiety is positioned deep within the active site of the acyl-enzyme complex formed with pbp ', where it forms several hydrogen bonds and hydrophobic interactions. in particular, the -aminothiadiazolylhyroxyiminoacetyl side chain of ceftobiprole sits more deeply within the side-chain binding pocket of pbp ' than does the -acylamino side chain of nitrocefin in the previously determined complex structure. the additional interactions probably add to the enhanced stability of the acyl-enzyme complex formed with ceftobiprole, compared to complexes formed with other betalactams that are inactive against mrs. significant structural rearrangements between apo-enzyme and acyl-enzyme are evident in the crystal structure and in solution. conclusion: ceftobiprole readily forms a stable inhibitory acylenzyme complex with the pbp ', the beta-lactam resistance determinant of mrs. this, together with potent inhibition of the normal complement of beta-lactam sensitive penicillin-binding proteins, accounts for its excellent activity against staphylococci and probably accounts for the low rates of resistance development observed in experimental conditions. incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in a french hospital (november -april c. morate, a. charron, c. bebear, j. maugein (bordeaux, fr) staphylococcus aureus are a major cause of nosocomial infections around the world. glycopeptides remain the drug of choice for severe infections caused by mrsa. however, after the emergence of vancomycin resistance in enterococcus and in the coagulase negative staphylococcus, strains of staphylococcus aureus with reduced susceptibility to glycopeptides (gisa) have been reported in different countries like japan, france, spain, the uk and the united states. the aim of our study was to determine the proportion of vancomycin resistance in clinical s. aureus isolates in a french university hospital, between november and april , then we wanted to define if there was an epidemic clone and study the clinical impact of these gisa strains. the protocol of detection was, first, a screening test on bhi agar containing mg/l of teicoplanin, then, the vancomycin and teicoplanin mics were determined by the method of etest with an inoculum of . mcf on the selected strains. finally, the isolates with mic of the teicoplanin ‡ mg/ l and mic of the vancomycin ‡ mg/l or mic of the teicoplanin ‡ mg/l and mic of the vancomycin £ mg/l were studied on population analysis. after that, pulsed-field gel electrophoresis (pfge) was performed on the different isolates and the pulsotypes were compared. from november to april , s. aureus isolates were collected from patients and screened for glycopeptide resistance on an initial agar screening test containing mg/l of teicoplanin. the teicoplanin mic was > mg/l for isolates ( . %) from patients and these strains were selected for the determination of the mics by ''macromethod'' etest. by this technique, strains were selected and studied by population analysis. all the profiles were compared to the reference strain mu profile. this procedure detected isolates (from patients) with heterogeneous reduced susceptibility to glycopeptides (hgisa). so the incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in our hospital was found to be . %. four strains were resistant to methicillin and were also resistant to gentamicin. the diversity of the strains was confirmed by pfge: there was not an epidemic clone in the hospital. the clinical history showed that patients had received a prior treatment with vancomycin, and that patients had a failure in treatment: of them had cystic fibrosis. objectives: enterococcus faecalis was the most prevalent organism ( . %) involved in enterococcal infections at tehran hospitals followed by e. faecium ( . %). due to widespread expansion of aminoglycoside modifiying enzymes (agmes) genes, the rate of resistance to high level concentration of aminoglycosides has increased in these years. the rate of high level gentamicin resistant isolates of enterococci (hlgr) is high in iran ( %). the aim of this study was to determine the genes encoding resistance to aminoglycosides among enterococci in iran. methods: disks containing lg gentamicin were used to detect hlgr isolates. primers specific for aac ( ') aph ( ") and aph ( ') iiia genes were used in pcr to possibly detect acetyltransferases and phosphotransferas, the common agmes among isolates of enetrococci. theses isolates were resistance to different concentration of gentamicin. results: a bp region of the aac ( ')-aph ( ") gene was amplified by pcr in % hlgr isolates as well as in % of low level getamicin resistant isolates (llgr). moreover the gene aph ( ') iiia was detected in . % and % of isolates of hlgr and llgr respectively. differences between isolates of e. faecalis and e. faecium were found in term of prevalence of aph ( ') iiia gene. conclusion: the bifunctional enzyme aac ( ')-aph ( ") is the main cause of resistance to high concentration of aminoglycosides in our collection of enterococci. this enzyme confers resistance to all clinically useful aminoglycosides with the exception of streptomycin. in the absence of aac ( ')-aph ( "), gentamicin could be used in combination therapy. prevalence and genetic analysis of methicillinresistant staphylococcus aureus expressing highlevel and low-level mupirocin resistance m. kural, t. us, y. akgun (eskisehir, tr) objectives: to investigate the genetic location of mupa gene which encoded mupirocin resistance and characterize mupirocin-resistant methicillin resistant staphylococcus aureus (mrsa) isolated from patients in a turkish university hospital by polymerase chain reaction (pcr) and plasmid analysis. methods: methicillin and mupirocin resistance were detected by disk diffusion (oxoid, uk). the etest (ab biodisk, sweden) was performed to determine mupirocin minimum inhibitory concentrations (mics). the presence of mupa and meca were detected by pcr using specific primers. plasmid analysis were used to study the genetic location of mupa gene. results: a total of ( . %) mrsa strains were identified by disk diffusion in s. aureus. of the clinical isolates ( . %) were from wound, ( . %) from blood, ( . %) from catheter, ( . %) from lower respirator tract (bronchoalveolar lavage, pleural fluid and transtracheal aspirates), ( . %) from sputum, ( . %) from urine and ( . %) from other (serebrospinal fluid, parasynthesis fluid, peritoneal fluid, and bone marrow) clinical samples. among the mrsa isolates, mupirocin resistance was detected in ( . %) strains with disk diffusion and etest. of the mupirosin-resistant isolates ( . %) expressed high-level (muh) and ( %) expressed lowlevel (mul) mupirocin resistance. all isolates were vancomycin, teicoplannin susceptible and chloramphenicol resistant with disk diffusion. isolates with high-level and low level mupirocin resistance due to the mupa gene were also detected with pcr. plasmids were detected in all of the isolates. however only the muh isolates contained a kb plasmid that encoded highlevel resistance. all of the isolates contained a . kb plasmid and resistant to chloramphenicol. conclusion: our results indicated that the mrsa clones detected in the hospital had acquired a high-level mupirocin resistant plasmid. the past observations and recent studies suggested that the numbers of such strians have increased following extensive topical use of mupirocin. the usage of mupirocin in our hospital has not yet been systematically implemented. it is frequently prescribed for the treatment of staphylococcal skin infections and less to eliminate nasal carriage of mrsa. in our hospital we should be aware of the possible emergence and increase of mupirocin highly resistant mrsa strains in the future so that we should be considered when using mupirocin to control the spread of mrsa in hospital. emergence and spread of acquired fusidic acid resistance in staphylococcus aureus objectives: a major route to fusidic acid resistance (fusr) in s. aureus involves acquisition of fusb, a resistance determinant first clinical microbiology and infection, volume , supplement , identified on plasmid pub . here we show that (i) the two currently-circulating major clones of fusr s. aureus identified to date have acquired fusb from pub (or from the same ancestral source as pub ), and (ii) that the pub -encoded fusb is only one of at least three lineages of this protein that appear to have evolved since recruitment of the original, ancestral fusb to the staphylococci. methods: plasmid purification, dna sequencing, pcr amplification, and cloning in s. aureus rn using shuttlevector pcu , were all performed using established methods. antibiotic susceptibility testing was performed by agar dilution. results: the epidemic european fusidic acid-resistant impetigo clone (eefic) and community-acquired mrsa strain st have been shown to carry chromosomal and plasmid-encoded fusb, respectively. dna sequencing of fusb and its surrounding regions in these backgrounds revealed that they are identical to sequences on pub . however, acquired fusr does not always result from acquisition of the prototypical fusb gene. a gene encoding a fusb homologue was recently identified during sequencing of s. aureus strain mssa , and we identified an additional homologue encoded in the genome of s. saprophyticus strain atcc . the products of these genes exhibit~ % homology to fusb and to each other. cloning of pcr amplicons corresponding to these genes and their upstream expression signals into s. aureus established that they both confer resistance to fus. since these functional homologues are more closely related to each other than to those from other gram-positive organisms, it is highly likely that they evolved from an ancestral fusb after its recruitment to the staphylococci. conclusions: the three members of the staphylococcal family of fusb proteins appear to have evolved from the same ancestral protein, which, based on the low level of sequence homology between fusb genes at the nucleotide level, clearly occurred well before the introduction of fus into the clinic. of the three, the fusb protein encoded by pub is by far the most successful, and this gene/plasmid represents the source of (or shares a source with) the major fusr strain lineages. telithromycin activity is reduced by efflux in streptococcus pneumoniae c. benvenuti, r. koncan, g. bahar, a. mazzariol, g. cornaglia (verona, it; ankara, tr) objectives: telithromycin shows an excellent activity against m-type erythromycin-resistant streptococcus pneumoniae, thus is commonly regarded as being capable of overcoming the efflux resistance mechanism. nevertheless, telithromycin mic values in those strains appear to be distinctly higher than in the erythromycin-susceptible ones. the possibility of telithromycin acting as an actual efflux substrate, as it was already demonstrated in streptococcus pyogenes, seemed worth investigating. methods: telithromycin mic distribution was analysed in a collection of italian s. pneumoniae strains originating from multi-centre studies ( ) ( ) ( ) ( ) . the effect of an efflux mechanism was investigated using [ h]-telithromycin. results: telithromycin mic ranges were £ . - . mg/l (mic . mg/l and mic . mg/l) in erythromycinsusceptible strains (lacking both mef and erm genes) and . - mg/l (mic . mg/l and mic . mg/l) in strains endowed with the m phenotype. a distinct telithromycin efflux was detected in the strains expressing the mef gene, but not in those expressing the erm(b) gene, nor in the susceptible strains lacking mef or erm genes. efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. an msr-like sequence was also found in all strains effluxing telithromycin, but not in the others. conclusions: this is the first time that telithromycin has been shown to be effluxed by s. pyogenes isolates. that the efflux is related to the presence of both the mef and the msr-like genes is clearly demonstrated, but -owing to the increasingly evident complexity of s. pneumoniae efflux systems -other genes might also contribute to the efflux. an unusual phenotype of enterococcus faecalis in greece expressing low-level resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin m. maniati, f. kontos, p. liakos, e. petinaki, i. spiliopoulou, a. maniatis (larissa, patras, gr) objectives: to investigate the resistance mechanism of a new described phenotype among enterococcus faecalis expressing lowlevel resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin (q-d). methods: in greece, during , three enterococcus faecalis isolates, expressing this unusual phenotype, were recovered from urine samples. the isolates were studied by pcr for the lsa-gene and by pfge. nucleotide sequencing analysis of lsa and bp of the upstream region was performed. the isolates were also tested by rt-pcr for the expression of the lsa-gene. results: the isolates belonged to three distinct clones and carried the lsa-gene. no stop codons were found in any strain, while some point mutations in the lsa-gene were detected. comparing the lsa mrna production of these unusual strains with that obtained from fully q-d resistant ones no quantitative differences were found. conclusions: the findings of the present study clearly show that the resistance mechanism of quinupristin-dalfopristin is not only correlated with the presence and the expression of the lsagene. some mutations detected in the lsa gene probably are responsible for the production of an lsa protein with decreased activity, resulting to the q-d susceptibility. the presence of erm tr gene is responsible for the macrolide-resistance of streptococcus agalactiae objectives: to investigate the mechanism of resistance to macrolides in strains of streptococcus agalactiae in the area of thessalia, greece during the period - . methods: the subject of this study were strains of s. agalactiae which were collected from clinical specimens ( % vaginal swabs) from pregnant and non pregnant women. the strains were identified by gram stain, the lancefield b antigen, and by api strep system (biomerieux, france). susceptibility to macrolides, lincosamides and streptogrammines b was studied by the disk diffusion method. the mics were also measured by the use of e-test. the differentiation between m and mlsb inducible type was tested by the double disk synergy test (ddst). the detection of the genes mef a, erm tr, and erm b was performed by polymerase chain reaction (pcr). the clonality of the resistant strains was studied by pulse-field gel electrophoresis. results: of the strains, were resistant to erythromycin, lincosamid and streptogrammines b. none was found to be resistant to erythromycin only (m-phenopype). % of the strains were mlsb constitutive phenotype, while % were mlsb inducible. all strains were found to carry the erm tr gene. only one strain was found to carry both erm tr and erm b genes. pfge analysis revealed the emergence of multiple resistant clones. conclusions: the resistance of s. agalactiae to mlsb antibiotics is related with the presence of erm tr gene in central greece. emergence of novel clindamycin resistance phenotype among invasive streptococcus pyogenes isolates in sweden a. jasir, b. luca, c. schalen (lund, se) objectives: in some recent throat group a streptococci (gas) isolates from our diagnostic laboratory total resistance to clindamycin but susceptibility to erythromycin and other -as well as -membered macrolides was found. the isolates were susceptible to -membered macrolides and streptogramin b. these atypical strains thus did not agree with previously known mls resistance phenotypes. the main objective was to characterize theses resistance phenotype and genotypes. method and results: the isolates were examined for resistance genes by pcr. out of strains one harboured an erma gene. the gene was sequenced and showed a mutation in regulatory part and was localized on a transposons. all other strains were negative for any erm genes and were also tested for s rrna mutations with negative outcome. strains were t-and emm typed and showed to belong to different types. conclusions: gas account for common human infections such as acute pharyngotonsillitis and impetigo, which untreated may be followed by the nonsuppurative complications rheumatic fever and acute poststreptococcal glomerulonephritis gas may also give rise to invasive, often life-threatening acute disease, such as scarlatina, erysipelas, endometritis, necrotising fasciitis and sepsis, often accompanied by toxic shock. without known exceptions, gas are fully susceptible to betalactams, which are first-choice drugs for treatment. in cases of allergy or intolerance to penicillins, macrolides are most used, and possibly as a consequence, a significant resistance development to these agents has evolved in many parts of the world. though the role of clindamycin for treatment of streptococcal disease is more limited this drug was shown to be particularly effective in eradicating streptococci after penicillin treatment failure of pharyngotonsillitis. clindamycin, often as a supplement to betalactams, also may have a life-saving effect in the treatment of fulminant streptococcal infections. due to its important role in the treatment of invasive streptococcal disease, resistance development to clindamycin in gas is considered highly undesirable. the alarming finding of a possibly new phenotype of selective clindamycin resistance in gas will motivate a thorough analysis of the phenotype as well as identification of its resistance determinants. a. al-lahham, m. van der linden, r.r. reinert (aachen, de) objectives: telithromycin is a novel ketolide antibiotic with significant in-vitro activity against streptococcus pneumoniae. the aim of this study is to characterize the resistance mechanisms of clinical isolates of s. pneumoniae with reduced susceptibility to telithromycin (> mg/l) and to perform the time-kill kinetics with telithromycin. methods: determination of mics was performed by the microbroth dilution method according to the clsi and the serotyping by the neufeld quellung reaction. multilocus sequence typing, sequencing of the s rrna, sequencing of genes encoding ribosomal proteins (l and l ), and ermb were performed according to standard methods. four isolates were selected for time-kill, two of which with a telithromycin mic mg/l and two strains with a telithromycin mic of mg/l. results: in two nation-wide studies and one european surveillance study (n = ) performed at the national reference center for streptococci (nrcs) in germany, reduced susceptibility to telithromycin (> mg/l) was detected in isolates ( . %). mic /mic (mg/l) of the strains to other antibiotics were as follows: telithromycin / , penicillin g / , cefuroxime / , erythromycin a > /> , clindamycin > /> , tetracycline / , and gatifloxacin . / . . two major serotypes were observed, serotype ( . %) and serotype a ( . %). all isolates possess the cmlsb phenotype (ermb positive). the isolates showed a wide range of combinations of resistance determinants including multiple alterations in the s rrna (a g, c t, a g, a t, and c t), a s n alteration in the ribosomal protein l (n = ), and a n s alteration in the erm(b) gene (n = ). the predominant clone was serotype sequence type ( of isolates), which was seen in france (n = ) and germany (n = ). telithromycin-resistance has also spread to the spain f- clone (st ; n = ) and its serotype a variant. in vitro time-kill showed a minimal kill from - hours and then regrowth. bactericidal activity was achieved only with times the mic in all strains. conclusions: although the incidence of telithromycin resistance remains rare world-wide, the spread of telithromycin resistance to multi-drug resistance clones with world-wide distribution is worrisome. gbs obtained from non-pregnant women. the erythromycin resistant-gbs were identified, phenotypically analysed, screened by pcr for mre(a) gene and for erythromycin resistance genes: erm(b), erm(tr), mef(a) and mef(e), and serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of gbs, ( . %) were erythromycin-resistant: ( . %) erythromycin-resistant gbs were isolated from vaginal swabs of pregnant women and ( . %) from non-pregnant women. the frequency of serotypes in erythromycin-resistant gbs tested, the distribu-tion of their resistance genes and the distribution of serotypes among the different genotypes are illustrated in the table. nt, nontypeable, the mre(a) gene was found in all the gbs strains tested. mics of erythromycin in erythromycin-resistant gbs were: mic and mic , > mg/l; range, to > mg/l for gbs harbouring erm(b) and erm(b)+erm(tr) and mic and mic , mg/l and > mg/l, respectively; range, . to > mg/l for gbs harbouring erm(tr). conclusion: erm(b) was the erythromycin-resistant gene most prevalent among the gbs isolates and these isolates showed the highest mics of erythromycin. the commonest serotypes among erythromycin-resistant gbs isolated were iii, ii and i, and showed genotypic variability harbouring either of the two most prevalent genes, erm(b) or/and erm(tr). methods: we studied the rates of resistance to tetracycline and minocycline among erythromycin-resistant gbs strains isolated at the university hospital lozano blesa of zaragoza, spain. isolates were subsequently phenotypically analysed by means of the disk diffusion method and screened by pcr for erythromycin and tetracycline resistance genes [erm(b), erm(tr), mef(a/e), tet(m) and tet(o)]. the susceptibility to erythromycin, josamycin, tetracycline and minocycline was tested by the agar dilution method according to the nccls. the strains were serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of isolates of macrolide-resistant gbs collected from may to april in our hospital ( . % of the total sgb isolated), ( . %) were tetracyclineresistant. the distribution of tet(m) and tet(o) among the erythromycin-resistant gbs harbouring erm(b) was ( . % and . %, repectively) and harbouring erm(tr) was ( . % and . %). the distribution of tetracycline resistance genes and serotypes among the different genotypes in gbs are illustrated in the table.*nt: non typable isolates carrying tet(m) or tet(m)+tet(o) presented the following mics: tetracycline (mic , mic , range - mg/l); minocycline (mic , mic , range - mg/l). isolates carrying tet(o) presented the following mics: tetracycline (mic , mic , range - mg/l); minocycline (mic , mic , range - mg/l). conclusion: the majority ( . %) of tetracycline-resistant gbs harboured tet(m) alone or in combination with tet(o). the most prevalent serotypes among the total of tetracycline-resistant gbs was the serotype iii ( %) and the serotype ii ( %). serotype iii was more prevalent among the gbs harbouring the tet(m) gene and serotype ii was more prevalent among the gbs harbouring the tet(o) gene. objectives: to know the prevalence of resistance to macrolides in viridans streptococci, its mechanism and the genetic elements which are involved. methods: we studied viridans streptococcus pharyngeal isolates from different patients. mics for macrolides were determined by the agar dilution method. the presence of mef and erma, ermb and ermtr genes, and the presence of mega (macrolide efflux genetic assembly) or tn in resistant, mef + isolates was determined by pcr with specific primers. the similarity to mef genes first described in pneumococci (mefe) and streptococcus pyogenes (mefa) was determined by sequencing. results: viridans streptococci isolates were resistant to macrolides ( . %). out of the resistant isolates harboured mef genes ( . %), one harboured ermb ( . %), and isolates harboured both mef and ermb genes ( . %). no isolates harboured erma or ermtr genes. we studied genetics elements which harbour mef genes in other streptococci, in mef (+) isolates. we found mega insertion element in of isolates ( . %), all of them harbouring mefe. the only isolate in which we found mefa, did not harbour mega, but tn . conclusions: m phenotype is frequent in viridans streptococci, and all of them harbour mef genes. most mlsb phenotype viridans streptococci do not harbour erm genes alone; most of them combine erm and mef genes. most isolates contained the mef sequence corresponding to mefe, and the genetic element (mega) usually described in pneumococci as harbouring this gene. one isolate contained the sequence corresponding to mefa, and the genetic element usually described in s. pyogenes (tn ). the increasing presence of macrolide-resistant pneumococci harbouring mega element might be related with its wide presence in viridans streptococci. the acquisition of mega by pneumococci from viridans streptococci through transformation is being studied. objectives: the principal mechanism of macrolide resistance in streptococcus pneumoniae in italy is target site modification mediated by erm(b). the erm(a) gene is common in streptococcus pyogenes but rare in s. pneumoniae, even if recent studies have demonstrated an increased detection of this resistance determinant. recently, a clinical s. pneumoniae isolate carrying erm(a) has been obtained from a patient with meningitis in italy. the aim of this study is the molecular characterization of this isolate. methods: antimicrobial susceptibility tests were determined by etest. the presence of erythromycin resistance determinants was detected by pcr assays. genotyping was performed by pfge and mlst. the flanking regions of erm(a) were analysed by sequencing a fragment amplified by inverse pcr. transformation and conjugation experiments were carried out. transformants were analysed by pfge and hybridization with an erm(a) probe. results: the isolate belonging to serotype (st) a, was resistant to erythromycin, inducibly resistant to clindamycin and susceptible to penicillin and tetracycline. by pcr, the only macrolide resistance determinant detected was erm(a). pfge analysis revealed the genetic correlation of this strain with other st a s. pneumoniae italian isolates. mlst confirmed this data since the isolate belonged to st , which is a single-allele variant of st , the most common st among italian st a isolates. a bp dna fragment, obtained by inverse pcr and containing the erm(a) gene, was sequenced. this fragment contains an orf upstream erm(a), the gene erm(a) identical to that described in s. pyogenes and orfs, downstream erm(a), one homologous to a hypothetical kinase and the other two to transposases of other gram-positive species. in transformation experiments the gene erm(a) was transferred to an erythromycin susceptible recipient. hybridization analysis of one transformant revealed that the size of the transferred dna fragment was approximately kb. no transconjugant was obtained in mating experiments. conclusions: this is the first italian report of an s. pneumoniae isolate carrying erm(a). erm(a) appears to be contained in a genetic element that includes two transposases, although the gene is not transferable by conjugation. objective: treatment with the first oxazolidinone antibiotic, linezolid, of infections caused by staphylococci has proved effective in most cases. in the present study we present the first three cases of linezolid resistant staphylococci in our hospital. methods: we examined three coagulase negative staphylococcus strains isolated from blood cultures (bactec, becton dickinson). identification and susceptibility testing were performed by the vitek ii automated system (biomerieux) and the results were confirmed by the api system (biomerieux) and e-test (ab biodisk, solna, sweden), according to nccls guidelines. results: three linezolid resistant staphylococci were isolated from blood cultures. identification showed that all three isolates were staphylococcus cohnii subsp. urealitycum and the mic values were lg/ml (n = ) and lg/ml (n = ) which are much higher than the value of lg/ml that characterizes sensitive strains. the isolates derived from three patients in different wards of our hospital. the first two isolates were recovered from two icu patients in april and august and the last staphylococcus cohnii was isolated from a patient in the neurosurgery ward, who is still hospitalized. all patients received prolonged treatment with linezolid. conclusions: although six linezolid resistant clinical isolates of s. aureus were previously reported in the literature, these three isolates are the first coagulase negative staphylococcus isolates resistant to linezolid. it is imperative to screen for resistance to linezolid all staphylococci and take the necessary precausions in order to prevent the spread of a linezolid resistant strain in other wards of our hospital. correlation between mic and number of mutated s rrna genes in oxazolidinone-resistant staphylococcus aureus objectives: to determine the number of mutated s rdna alleles present in clinical and laboratory-generated linezolidresistant staphylococcus aureus isolates. methods: linezolid-resistant isolates were tested, of them clinical isolates (mics - mg/l) and mutants selected in-vitro (mics - mg/l). the mutants were raised by repeated passage on increasing linezolid concentrations and their parentage was verified by pfge. mics were determined by agar dilution. genomic dna was digested with ecori and hybridized with a bp probe corresponding to domain v of the genes encoding s rrna, to determine gene copy number. pyrosequencing was used to quantify the proportions of wildtype and mutated alleles present; assays were designed to detect the presence of mutations conferring oxazolidinone resistance. pyrosequencing and hybridization data were combined to determine the number of mutated alleles present. results: resistance selected in-vitro proved less stable than that in the clinical isolates. pyrosequencing showed that all clinical isolates had the g t mutation, of the in-vitro selected mutants had g t, had t c, had t a, had a g and had g a. the s rdna copy number in the oxazolidinone-resistant clinical isolates varied from - , and from - in the laboratory-generated mutants; / laboratoryselected mutants had changes in copy number, compared with their parent strains, and had changes in fragment size, but not number. the number of mutated copies in lin-resistant isolates ranged from - in laboratory-selected mutants and from - in clinical isolates. an increasing number of mutated genes correlated with increasing linezolid mic. conclusions: in combination, pyrosequencing and hybridization successfully determined the number of mutated s rdna alleles. exposure to linezolid selected changes in s rrna gene copy number as well as sequence in % of in-vitro selected mutants. there was a positive correlation between both the number and proportion of mutated s rdna copies and mic, previously unproven for staphylococci. objectives: linezolid (lzd) is an important antibiotic for the treatment of enterococcal infections, especially when the corresponding strain possesses multiresistance including resistance to vancomycin (van). we report the emergence lzd resistance in clonally related van-susceptible and vanresistant enterococcus faecium isolates originated from an icu patient only days after initiation of linezolid therapy. patient and methods: van-resistant e. faecium was repeatedly isolated from intraabdominal cultures of a -year-old female icu-patient with infected necrotizing pancreatitis after pancreaticoduodenectomy (whipplés procedure). antibiotic susceptibility testing of the bacteria was performed by e-tests; vana gene were detected by pcr. the possible lzd resistance mechanism (mutation in the s rdna of one or more of the six s rrna alleles of e. faecium) was examined by a pcr-based method. molecular typing of the strains was performed by smai macrorestiction analysis. results: van-resistant but lzd-sensitive e. faecium (vrlse) were initially detected in intraabdominal cultures, however, already twelve days after initation of lzd therapy, van-and lzd-resistant e. faecium (vrlre) strains were detected. resistance to lzd was confirmed: mics ranged from to mg/l. all e. faecium isolates showed identical or closely related pfge patterns. throughout the icu period, van-and lzd-susceptible e. faecium (vslse) strains were repeatedly detected in the same specimens from which the vrlse and vrlre were isolated. additionally, van-susceptible e. faecium isolates with resistance to lzd (vslre) were detected. mutations in the s rdna of three out of six alleles led to lzd resistance in the e. faecium isolates examined. two weeks after termination of the lzd therapy, no lzd-resistant strain could be detected in follow-up swabs. conclusions: resistance to lzd in e. faecium can occur already shortly after the initiation of lzd therapy. assessment of antibiotic susceptibilities of all isolates at the start of therapy and regularly during the therapy is advisable, especially during therapy of severe infections. the epidemiological and clinical repercussions of resistance to lzd among enterococci cannot be predicted at this time. attention to proper dosing and prompt removal of infected devices, when feasible, could limit occurrence and spread of lzd-resistant e. faecium. objectives: to investigate the mechanisms of resistance to tetracycline in shigella spp. methods: one hundred and eleven tetracycline-resistant shigella spp strains ( s. sonnei, s. flexneri), were isolated as a cause of enteritis in our geographical area and the remaining recovered from patients with traveller's diarrhoea. antimicrobial susceptibility to tetracycline was determined by the kirby-bauer method. presence of teta, tetb and tetg genes was established by pcr. sequencing of amplified products were used to corroborate the reliability of the pcr results. maentel haenszel test was used to establish the statistical significance. results: the statistical analysis showed that the teta gene was more frequent in s. sonnei (p < . ), while tetb was more usual in s. flexneri (p < . ). although without statistical significance (p: . ), presence of non-determined mechanisms of tetracycline-resistance seems to be more frequent among s. sonnei. conclusions: species-specific differences in the distribuition of the teta and tetb genes has been shown. moreover . % of the analysed strains did not show any of the analysed determinants of tetracycline-resistance. the concomitant presence of more than one of the analysed genes is a rare event. distribution and genetic determinants of tetracycline resistance in laribacter hongkongensis isolates from humans and fish objectives: to study the distribution of tetracycline resistance and to clone and characterize a tetracycline resistance determinant in laribacter hongkongensis, a recently discovered bacterial genus and species associated with communityacquired gastroenteritis. methods: twenty-four l. hongkongensis strains isolated from patients with community-acquired gastroenteritis and l. hongkongensis strains isolated from freshwater fish in hong kong were used in this study. genetic determinants for tetracycline resistance were looked for by screening a genomic dna library of l. hongkongensis. the prevalence of teta gene in other strains of l. hongkongensis was studied by pcr using laboratory-designed primers. the presence of the tetracycline resistance determinants in plasmid was examined by southern blot analysis. results: among human and fish isolates tested, human and fish isolates were tetracycline-resistant. a -bp gene cluster, which consists of putative transposases, a tetr and a teta gene, was cloned by inserting restriction fragments of genomic dna from a resistant strain, hlhk , into pbk-cmv. the -bp teta and -bp tetr genes shared significant nucleotide sequence homology with known teta and tetr genes. while the flanking regions and ' end of the teta were identical to the corresponding regions of a tetc island in chlamydia suis, the teta was almost identical to that of transposon tn and plasmids found in many gram-negative bacteria, suggesting that illegitimate recombination may have occurred to produce the present tetracycline resistant determinant. southern hybridization suggested that the teta gene of hlhk was plasmid-encoded. the tetracycline resistance in l. hongkongensis was associated with teta. pcr amplification of the teta gene in the isolates of l. hongkongensis, including hlhk , showed the presence of teta in all the four tetracycline resistant isolates but none of the tetracycline susceptible ones. in contrast to strain hlhk , the teta of two strains were identical to that of tn , while that of the other strain was more closely related to other gram-negative bacteria plasmids. conclusion: our results indicate that horizontal transfer of genes, especially through tn and related plasmids, between l. hongkongensis and other gram-negative bacteria is probably a frequent event and is an important mechanism for acquisition and dissemination of tetracycline resistance in l. hongkongensis. succesful treatment of infective endocarditis with linezolid t. hryniewiecki, u. lopaciuk, j. stepinska (warsaw, pl) objectives: there is an increasing proportion of resistant strains causing infective endocarditis in recent years. it has changed the approach to choice of antibiotic therapy. linezolid (zyvoxid Ò ) is a new bacteriostatic antibiotic with a wide spectrum of activity against gram-positive organisms and with good efficacy in experimental animal models of endocarditis. unfortunately clinical experience with linezolid in the treatment of endocarditis is limited. the aim of the study was to observe efficacy of linezolid in the treatment of infective endocarditis. methods: the study group consisted of patients hospitalised in institute of cardiology in warsaw ( warsaw ( - due to clinically resistant infective endocarditis. the diagnosis of endocarditis was established according to the duke criteria by clinical examination, echocardiography, laboratory investigations and positive blood cultures with vancomycin mic estimation (in pts). all patients were treated surgically (valve replacement, artificial material removal) in conjunction with different conventional antibiotics and afterwards with mg of linezolid every hours intravenously. results: infective endocarditis was diagnosed as caused by mrcns in pts, mssa in pt, enterococcus faecalis in pt and staphylococcus epidermidis mr in pt. vancomycin mic vary from to mg/l. in pts culture-negative endocarditis was diagnosed. all patients were treated with linezolid intravenously to weeks (average , ). clinical response and eradication of bacteremia were achieved in all patients. leukopenia nad thrombocytopenia as an adverse reaction occurred in patient. conclusions . linezolid is effective in patients with grampositive endocarditis. . linezolid could be also effective in some patients with culture-negative endocarditis. . linezolid may provide an alternative in the treatment of infective endocarditis due to multi-resistant bacteria, in patients with resistant course or with adverse reaction to conventional antibiotics. objectives: to evaluate the safety and efficacy of lzd in a chinese population. methods: this randomized, double-blind, multi-centre study was conducted in china. after obtaining written informed consent, patients from to years of age with pneumonia (pneu) or skin and soft tissue infection (ssti) known or suspected to be caused by a gram-positive pathogen were randomized : to receive either lzd, mg, or vancomycin (van), g, each given iv q h. patients were to be treated for to days, and outcomes were assessed at end-of-treatment (eot) and follow-up (f-u) visits. results: one hundred forty-two patients were enrolled and received study medication, with pneu and with ssti. clinical assessments (effective = ''cured'' plus ''marked improvement'') for patients in the fully evaluable population are summarized in the table. the most frequently isolated pathogen was staphylococcus aureus: all isolates were susceptible to both study drugs. the eradication rates for all pathogens in evaluable patients at the f-u evaluation were / ( . %) in lzd-treated patients and / ( . %) in van-treated patients (p = . ). all patients receiving study drug were evaluated for safety. drug-related adverse events (aes) were reported in ( . %) lzd-treated and ( . %) van-treated patients. the most commonly reported drug-related aes in lzd-treated patients were mild abnormalities in liver function tests and leucopenia ( . % each); rash ( . %) was the most commonly reported ae in van-treated patients. seven ( . %) lzd-treated and ( . %) van-treated patients discontinued study drug because of an ae. conclusions: linezolid is an effective drug for the treatment of infections caused by gram-positive pathogens and is welltolerated. eradication in one patient, by rifamycinlinezolid, of a methicillin-resistant staphylococcus aureus producing panton-valentine leukocidin, responsible for relapses over months, and decolonisation of her family by mupirocin objective: we report the case of the mother who experienced relapses over the period. methods: pvl-mrsa were isolated on routine and mrsa agars (biorad). antibiotypes were studied by disk diffusion method. genetics and pulsotypes were studied by the french centre national de référence des staphylocoques in lyon (cnr). results: mrs kym had her th child on october , in the hospital of orléans. she was healthy and presented no risk factor for delivery. three weeks later she was addressed for surgical treatment of an abscess on buttocks. cultures yielded the special antibiotype: methicillin-r, kanamycin-r and tobramycin-s, of the pvl-mrsa currently spreading across europe and maghreb. the cnr found the luks-pv and lukf-pv-genes.through march , she relapsed times and was treated by pyostacin for a total of weeks. two of her children were addressed for abscesses ( buttocks, thumb) yielding the same bacteria. in march , mrs kym was addressed to the infectious diseases ward because of nasal furonculosis. samples yielded a pvl-mrsa with mls-b phenotype. treatment by rifamycin-linezolid d was initiated. the whole family was screened. the father and the boy, , who had the infected thumb months earlier, were carriers. the girl, , who had an abscess on buttocks months earlier was not. in april the whole family accepted an attempt for decolonisation by % nasal mupirocin or /d for d. cure and decolonisation were confirmed by nares and cutaneous folds samples in may and june. they missed an additional appointment in the beginning of term, but a phone call to the social worker confirmed none of them relapsed. the cnr studied strains ( from mrs kym , , from children abscesses in and , from boy's and father's nares ), and confirmed that they were all identical along the period and across the family. conclusion: short treatment with linezolid-rifamycin in the relapsing case associated with familial decolonisation by nasal mupirocin was an effective strategy to stop a time-prolonged familial outbreak of pvl-mrsa infection. multiple brain abscesses and purulent meningitis by listeria monocytogenes in an otherwise healthy man. favourable linezolid response, hampered by a suspected early drug myelotoxicity introduction: l. monocytogenes cns infection in immunocompetent adults remains rare. meningitis is the most common cns manifestation, with brain abscesses being < % of overall episodes. anecdotal episodes of cns l. monocytogenes infection were reported from immunocompetent patients where both diagnosis and treatment may be hampered by low clinical suspicion and a frequent non-specific presentation. in a -yearlong survey conducted in dallas (us), only cases of nonneonatal l. monocytogenes meningitis were found (estimated incidence rate: . %). case report: a -year-old male with a negligible history and no obvious exposure to l. monocytogenes was hospitalized owing to dizziness. a brain ct scan showed a small, late ischemic lesion. a few days later hyperpyrexia, headache, vomiting and altered mentation occurred. the csf study detected an elevated albumin content ( mg/dl), low glucose ( mg/dl) and a wbc count of cells/ll ( % neutrophils) so that ceftriaxone-chloramphenicol were immediately started. clinical-neurological conditions deteriorated while l. monocytogenes was cultured from the csf so that treatment was changed towards high-dose ampicillin-gentamicin. the persistence of severe clinical-neurological conditions and altered csf assay prompted the introduction of rifampicin-cotrimoxazole after days, but days later other focal neurological deficits appeared and a mri showed small, hyperintense focal lesions involving the medulla oblongata, interpreted as multiple abscesses. the introduction of linezolidmeropenem, despite anemia (requiring rbc transfusions after - days) led to a progressive clinical-csf improvement. our patient recovered completely and a control mri carried out month after discharge confimed the complete disappearance of the multiple brain listeria abscesses. discussion: the l. monocytogenes meningitis and multiple subtentorial abscesses (including rare localizations at cerebellum, bulb, and pons varolii), had an evolving cumbersome presentation. despite the in vitro activity of a broad spectrum of agents, multiple therapeutic changes became necessary, until the last linezolid-meropenem combination, which was proved very effective, although it was affected by relapsing anemia probably attributable to linezolid. linezolid, due to its elevated csf-brain penetration, and its activity against a broad spectrum of cns pathogens (including the intracellular l. monocytogenes), is expected to become a key antimicrobial compound, waiting for rct. discrepancy between favourable in vitro microbiological data and a severe clinical course of a staphylococcal knee and soft tissue infection responsive to oxazolidinone linezolid only after failure of all other therapeutic attempts introduction: to offer therapeutic alternatives for the emerging, multiresistant, serious gram-positive infections, novel molecules (quinupristin/dalfopristin, linezolid, daptomycin) were introduced and are made available when multiresistant gram-positive cocci are documented as no more susceptible to all available drugs including glycopeptides. however, inezolid encompasses unique tissue penetration and diffusion features (regarding soft tissues, lungs, joints and central nervous system) which make this last drug extremely promising in all circumstances where the penetration rate into infectious foci becomes critical. clinical experience: a very intriguing case report of a severe, staphylococcal knee arthtiris associated to an extensive local cellulitis/fasciitis and haematogenous dissemination occurring after a surgical curettage was characterized by a complete lack of response to a prolonged vancomycin/teicoplanin plus rifampicin therapy based on the apparently favourable in vitro sensitivity assays of methicllin-resistant staphylococci, but rapidly responded to i.v. (followed by oral) linezolid administration. the complete lack of clinical activity of a -week glycopeptiderifampicin administration cannot be explained by the in vitro measured mic values of isolated pathogens which showed complete sensitivity of staphylococcus aureus against vancomycina/teicoplanin and rifampicin and susceptibility of a concurrent hematogenous s. epidermidis strain to glycopeptidesrifampicin. since an abscess formation and an underlying osteomyelitis were carefully excluded by adequate instrumental examinations, from a theoretical point of view the active glycopeptide-rifampicin molecules should have been provided appropriate cure. on the other hand, from a strictly clinical issue, only a -week administration of i.v. linezolid followed by one more week of oral linezolid allowed to obtain a complete clinical-bacteriological cure and a complete function recovery without any sequelae after a . -year follow-up. conclusions: when the management of severe, multiresistant gram-positive infections is of concern, the in vitro activity of single drugs and therapeutic classes should be carefully evaluated in relation with the expected penetration and diffusion rates of these drugs into the relevant organs and tissues involved by the ongoing infectious localizations. otherwise, apparently unexplained failures may occur also when in vitro studies point out a complete activity of the tested compounds. epidemiology of resistance to antibiotics -ii p contemporary prevalence of bro betalactamases in m. catarrhalis: report from the sentry antimicrobial surveillance program (usa; l. deshpande, h. sader, r. jones (north liberty, us) objectives: to evaluate the prevalence of bro- and bro- among b-lactamase (bl)-producing m. catarrhalis (mcat) in the usa. although the bl-mediated penicillin (pen) resistance (r) in mcat has been stable at %, the bro- and - occurrence has not been determined in usa isolates since . bro- rates have been reported at < ( s), . ( - ), . ( - ) and . % ( - ) . methods: community-acquired mcat isolates (sentry program were tested by clsi broth microdilution methods including: , worldwide and , in north america (na). bro- and - was detected by pcr methods (levy and walker; ), compared to epidemiologic tests, and mic values. b-lactamase-positive (bl+) mcat samples per year from usa ( sites) and canada (ca; sites) were tested for the odd-numbered years. results: the bro- rate was , , , and % for , , and , respectively; rates in ca ( isolates) > usa ( ). several agents remained active: amoxicillin/clavulanate (mic , £ . mg/l), ceftriaxone (ctri; . ), cefuroxime ( ), erythromycin (£ . - . ), levofloxacin (£ . - . ), tetracyclines ( ) and trimethoprim/sulfamethoxazole (tmp/ smx; £ . / . ). pen mic distribution was tri-modal (£ . , - , > mg/l) and ctri bi-modal ( . , . ), yet bro- and - mic/zone distributions overlap (best discriminated by methicillin (mean zone, . vs. . mm) and pen ( . vs. . ) disks). possible bro- epidemic clusters could not be excluded due to a very common ribotype in centres (ca, sites; usa, ). conclusions: this bro- and - enzymes na prevalence update in mcat isolates ( ) ( ) ( ) ( ) ( ) ( ) ( ) shows stability at - % and - %, respectively. phenotypic tests (zones or mics) cannot easily distinguish between these b-lactamase types, necessitating the use of molecular applications. objective: although resistance to penicillin in beta haemolytic streptococci has not been reported yet, increasing resistance rates for alternative drugs, such as erythromycin, clindamycin or tetracycline is an emerging concern which brings the necessity to carefully monitor penicillin susceptibility. materials and methods: in order to detect any changes in penicillin mics, we performed antimicrobial susceptibility testing for all isolated beta haemolytic streptococci in our hospital between january and november . identification to serogroup level was done using a commercial latex agglutination kit (avipath strep, omega diagnostics ltd., scotland, united kingdom). results: a total of isolates were identified, distribution of groups for serogroup a, b, c and g were . %, . %, . % and . %, correspondingly. penicillin susceptibility was determined using etest (ab biodisk solna, sweden) strips according to manufacturers' instructions. when results are evaluated in year periods, mic increased from . to . mg/ml for group a, from . to . mg/ml for group b, from . to . mg/ml for group c and from . to . mg/ml for group g (table) . conclusions: even though highest mic values were to be found in group b ( . mg/ml), our results indicate the steady increase in penicillin mic for all serogroups. three group a and six group b isolates with penicillin mic of . mg/ml, reaching susceptibility breakpoint concentration according to clsi, and also highly elevated mic concentrations for group b streptococci may be messengers of possible forthcoming resistant strains. objective: to study trends in macrolide resistance rates among s. pneumoniae isolated from children aged to months attending day-care centres in france following implementation of prudent antibiotic use campaigns (alpes maritimes , france and pneumococcal conjugate vaccine (pcv) ( ) . method: nasopharyngeal aspirates were obtained from a random -stage cluster sample of children attending day-care centres in the nord (n) and alpes maritimes (am) areas during consecutive surveys between january and march march , march and . susceptibility to erythromycin and clindamycin and resistance phenotype were analysed by disk diffusion method. serotypes were determined using the quelling reaction. pneumococcal immunization status and antibiotic prescriptions over the previous months were recorded. results: sp was isolated from / , / and / children in , and , respectively (p < ) ). resistance to macrolides declined overall from . % to . % of strains between and (p < ) ). among erythromycin-resistant (e-r) isolates, percentage of erm-b phenotype increased from . % to . % (p = . ). while the proportion of penicillin non-susceptible strains declined from . % to . % of sp isolates (p < ) ), erythromycin resistance remained stable among these strains at . %. overall proportion of treated children fell from . % to . % (p < ) ) between and ; in am this reduction was observed in ( . %; p < ) ), while in n it occurred in ( %; p < ) ) and the percentage of macrolides among prescriptions fell from . % to . % (v for trend: p = . ). serotype distribution showed most e-r isolates were b, , f and f. a % reduction in serotype f was observed in am in and in n in . immunisation with pcv concerned at least . % of children in . conclusion: macrolide resistance has followed a parallel decline with penicillin resistance as a result of antibioticprescription reducing campaigns and pneumococcal immunization against the most prevalent macrolide-resistant serotypes. objective: to evaluate the prevalence of resistance of invasive strains of s. pneumoniae to erythromycin after decline in macrolide consumption. methods: the number of packages of antibiotics was obtained from the institute of public health of slovenia. for the period - the data on outpatient antibiotic consumption were collected using the atc/ddd classification (who version ) and the results were expressed in ddd/ inhabitants per day (did). all invasive strains of s. pneumoniae isolated from sterile body fluids in all slovenian hospitals were included in the study. susceptibility testing was performed using nccls approved disk diffusion test. results: during - the total use of antibacterials in slovenia decreased for . % from . did to . did. the consumption of macrolides which constituted . - . % of total use of antibacterials decreased for . % ( . to . did). short-acting (erythromycin, miocamycin), intermediate-acting (midecamycin, roxithromycin, clarithromycin), and long-acting (azithromycin) decreased for . %, . % and % respectively. in all years the use of intermediate-acting macrolides was the most prescribed subclass of macrolides corresponding for . - . did, followed by long-acting ( . - . did) and short-acting ( . - . did). the resistance of s. pneumoniae strains to erythromycin increased from . % ( / ) to . % ( / ); in children from . % ( / ) to . % ( / ) and in adults from . % ( / ) to . % ( / ) respectively. rates of the isolates resistant to erythromycin and at least of the following agents: penicillin, tetracycline, tmp/smx, chloramphenicol increased from . % ( / ) to . % ( / ); in children from % ( / ) to . % ( / ) and in adults from . % ( / ) to . % ( / ) respectively. conclusion: despite a reduction of macrolide consumption in outpatients the resistance of invasive strains of s. pneumoniae was increasing during the observation period especially in children. multiple drug resistance explains best the changes in s. pneumoniae resistance in a ten-year surveillance study in belgium objective: belgium is located between countries with very high and very low antibiotic resistance rates. modeling how resistance changes over time and place in belgium provides insights into correlates of s. pneumoniae resistance at the population level. methods: surveillance data consists of , s. pneumoniae invasive isolates from - , identified by postal code as well as clinical and demographic information. antimicrobial consumption (ims health services) is expressed in defined daily doses (ddd) per inhabitants per day. changes in resistance by month and postal code were evaluated using mixed effects models for repeated measures, using mathematical models of transmission for the curve shape, and taking into account seasonality. resistance to penicillin, erythromycin, tetracycline, and ofloxacin was considered in the analysis. results: resistance to penicillins, macrolides and tetracyclines peaked in the year , and their levels in were . %, . % and . % respectively. the shape of the curves is similar for most of the antibiotics studied, with a steep rise from to and a plateau thereafter. resistance to two or more antibiotic classes corresponded to % of all resistant isolates and in a multivariate model explains most of the variability through time and place of the antibiotics studied. resistance to only one antibiotic (any) decreased from . % in to . % in , while resistance to two or more increased . times ( % ci . - . , p < . ) from . % in to . % of all isolates years later. more than nine out of ten isolates that were macrolide or tetracycline resistant were also multiply resistant (mr). mr increases . % for each ddd of overall cumulative antimicrobial consumption, and out of all antibiotic classes, macrolides and broad-spectrum penicillins are most associated with resistance. conclusion: resistance to two or more antibiotics is the most important factor in understanding the changes over time for all studied antibiotic classes in belgium. the cumulative impact of antimicrobial exposure of separate antibiotic classes at the population level facilitates the survival and transmission of any isolate that is resistant to two or more antibiotic classes. methods: the isolates were identified by biochemical tests and specific serotyping. antimicrobial susceptibility to ampicilllin (amp), amoxicillin plus clavulanic acid (auc), cloramphenicol (cm), gentamicin (gm), cotrimoxazole (sxt), nalidíxic acid (nal) and tetracycline (tc) were established by the method of kirby bauer. the presence of beta-lactamases encoding genes (tem, carb, oxa -like) as well as the teta, tetb, tetc, tetg, cmla and flor genes, and integrons type was established by pcr, while the presence of plasmid-mediated dhfr was determined by pcr-rflp and the cat activity by a colorimetric assay. results: seven different resistance patterns were identified: i. susceptible ( strains); ii. amp, sxt, gm, a/c ( ); iii. amp, tc, cm ( ); iv. amp, tc, sxt, cm ( ); v. amp, a/c ( ); vi. amp, sxt, a/c ( ); vii ( ). -sxt. no isolate resistant to nalidixic acid was detected. resistance to beta-lactam agents was due to the presence of beta-lactamases type tem-like (pattern v), carb- (iii) and tem-like plus oxa- (ii, v, vi). meanwhile resistance to cloranphenicol and tetracycline was associated to cat activity (iii, iv) and flor (iii), and tetb (iv) and tetg (iii) respectively. no mechanism of cotrimoxazole resistance was detected in the isolates of the patterns ii, vi and vii, while dfra was detected in the isolates of the group iv. resistance to gm was associated to the presence of the gene aadb, detected in the analysis of integrons type . type integrons were detected in isolates belonging toi the pattern ii ( bp -aadb; bp -oxa , aada ), iii ( bp -carb , -aada ), iv ( bp -dfra , aada ), v and vi ( bp -oxa , aada ). conclusions: a great diversity of resistance mechanisms has been detected. those mechanisms might spread among microorganisms resulting in a serious health problem due to the limited number of antibiotic treatments available in the area. small outbreaks of veb- esbl producing acinetobacter baumannii in belgian nursing homes and hospitals through cross-border transfer of patients from northern france methods: from / to / , all belgian acute hospitals were invited to report cases of nosocomial infections/colonisations due to mdr ab isolates presenting a resistance profile similar to the french epidemic strain (resistance to all agents except carbapenems and colistin) and to send such isolates to the reference laboratory for phenotypic confirmation and for genotypic characterization (pcr of veb- and class integron, pfge typing). guidelines for detection of the epidemic strain, screening for carriage in patients transferred from hospitals or nursing homes (nh) close to the french border as well as infection control measures were sent to all hospitals. results: overall ab strains from hospitals were sent to the reference laboratory. only, of these fulfilled the phenotypic resistance patterns and were definitely confirmed as veb- ab and had a pfge pattern identical to the french epidemic clone. two mini-outbreak clusters (each involving cases) were documented in hospitals from two cities (tournai and chimay) closed to the french border. two patients died from their infection. in the first outbreak, all patients were residents who lived in the same nh. two of them were french citizens who had been hospitalised in different acute care hospitals in the north of france within the last year. in the second outbreak, the index case had also been previously hospitalised in a french hospital. secondary transmission to two other hospitalised patients occurred in this outbreak. conclusion: despite the large extension of the veb- ab outbreak in france no similar problem occurred in belgium. however, this national alert allowed to detect two small outbreaks in belgian institutions located close to the french border. in both outbreaks the epidemic strain was imported from france through patient circuits. this study illustrates that transfers between acute care hospitals and nh may explain cross-border spread of multi-resistant epidemic strains. types of extended-spectrum beta-lactamases in salmonella spp. and decreased susceptibility to fluoroquinolones objectives: the aim of this study was to determine the rate of esbl production in clinical isolates of salmonella spp. and to detect decreased susceptibility to fluoroquinolones in esbl positive isolates in turkey. methods: a total of salmonella spp. isolated from clinical samples from thirteen centres between and were included in the study. in vitro susceptibility to ampicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and ciprofloxacin were determined using the agar dilution method on mueller-hinton agar following the clinical and laboratory standards institute (clsi) guidelines. decreased susceptibility to ciprofloxacin was defined as an mic of . - mg/l. salmonella isolates were screened for esbl production by double disk synergy method using amoxicillin/clavulanic acid, cefotaxime and ceftazidime disks. types of esbl enzymes were analysed by pcr for tem, ctx-m, shv and per- genes. results: in salmonella spp. the highest level of resistance was observed against ampicillin ( . %) followed by chloramphenicol ( . %), tetracycline ( . %) amoxicillin/ clavulanic acid ( . %), trimethoprim/sulfamethoxazole ( . %), gentamicin ( . %), and cefotaxime ( . %). ciprofloxacin resistance was observed in one isolate ( . %). among salmonella isolates, ( . %) were shown to produce esbl by double disk synergy testing. these isolates were salmonella typhimurium (n = ), serogroup c (n = ) and salmonella enteritidis (n = ). three isolates were from fecal samples two were from urine and one was from blood. one of the esbl producing isolates were susceptible to cefotaxime in vitro. two isolates showed decreased susceptibility to ciprofloxacin. all the esbl producers were resistant to ampicillin, amoxicillin/ clavulanic acid, chloramphenicol and harbored ctx-m type enzymes. in three isolates a tem-type enzyme was also present. conclusion: albeit being rare, esbl production is an important resistance factor among salmonella spp. in order to prevent treatment failures, decreased susceptibility to fluoroquinolones should be investigated routinely in invasive isolates as well as esbl production. incidence of faecal carriage of esbl-producing enterobacteriaceae in hospital and community patients during two non-outbreak periods of time the identities of the esbl-producing isolates recovered during were: e. coli (n = ), k. pneumoniae (n = ), p. vulgaris (n = ) and e. cloacae (n = ), and isolates recovered during were: e. coli (n = ), k. pneumoniae (n = ), k. oxytoca (n = ), p. vulgaris (n = ), p. mirabilis (n = ), e. cloacae (n = ) and e. aerogenes (n = ). conclusions: a dramatic, significant increase in the frequency of faecal carriage of esbl-producing isolates was demonstrated in among hospitalized ( . %) and ambulatory patients ( . %).the results revealed that the prevalence of faecal carriage among ambulatory patients and hospitalized patients was not significantly different in both periods of time. outpatients came from the community carrying enterobacteria harbouring esbl in the intestinal tract, suggesting that the community could be a reservoir for these microorganisms and enzymes. methods: a total of k. pneumoniae, k. oxytoca, e. coli, c. freundii, s. marcescens, and e. cloacae from university hospitals, isolated from blood, wound, urine, sputum and other clinically significant specimens were proven to produce esbls. antimicrobial susceptibility was determined according to clsi, ; conjugation on a solid medium was performed; isoelectric focusing was followed by bioassay; pcr with beta-lactamase group-specific oligonucleotides was applied, followed by nucleotide sequencing; rapd with eric- a and eric primers was performed. results: mic of ceftazidime varied from to > mg/l, mic of cefotaxime - - mg/l; the addition of sulbactam : reduced mic > -fold. transconjugants exhibited resistance both to extended-spectrum cephalosporins and aminoglycosides in of strains. according to their pi, two clusters of betalactamase producers could be described: first one -esbls focussed at pi . , and the second -pi at . . results from pcr confirmed the presence of two groups esbls: tem and shv. sequencing of representative strains showed the presence of shv- in two participating hospitals and of shv- in only one strain e. cloacae, while tem- like enzyme was found in centres and had a clonal dissemination. objectives: during treatment with selective decontamination of the digestive tract (sdd), four strains of multidrug-resistant (mdr) gram-negative bacteria (three escherichia coli strains and one klebsiella pneumoniae) were isolated at the intensive care unit (icu) in the academic medical center (amc) in amsterdam. these isolates were extended spectrum betalactamase (esbl) positive. we investigated whether this was due to interspecies transfer of resistance genes. methods: the strains were typed by amplified fragment length polymorphism analysis. the plasmids from these strains were characterized by restriction fragment length polymorphism. resistance genes of the mdr-strains were characterized by pcr and sequence analysis. results: aflp analysis confirmed that the three mdr e. coli isolates represented three different strains. the mdr-strains were shown to harbour the same plasmid with identical extended-spectrum â-lactamase (esbl) genes; ctx-m- and shv- . conclusions: identification of the emergence of such mdr gram-negative bacteria and recognition of resistance plasmid transfer during sdd treatment is crucial for optimal application of this regimen in icus. the use of the third generation cephalosporins in sdd may associate with emergence and increase in the prevalence of esbls. therefore, for optimal screening of resistance to cephalosporins in icus, the screening for esbls should be included. objective: carbapenems are the drugs of choice for the treatment of serious infections caused by esbl-producing enterobacteriaceae and the emergence of carbapenem resistance is rarely documented. we investigated pairs of carbapenem-susceptible and resistant k. pneumoniae isolates from three patients, collected before and after therapy with carbapenems. methods: pre-and post-therapy pairs of esbl-producing k. pneumoniae isolates were from three patients with urinary catheter-associated infections who were treated with ertapenem (erp, cases) or meropenem (mem, one case) in a district general hospital with a low incidence of esbl-producing organisms ( . / bed days), and meropenem use of ddd/year. isolates were compared by pfge of xbai-digested genomic dna. mics were determined and interpreted by british society for antimicrobial chemotherapy methodology. blactx-m alleles were sought by multiplex pcr. outer membrane proteins (omps) were extracted, and analysed by sds-page. results: the three patients relapsed following erp or mem therapy, and the post-therapy isolates from repeat urine samples were resistant (table), with mics erp>mem>ipm. all six isolates from the three patients belonged to the same pfge strain, but transmission of the resistant variants is unlikely as the patients were geographically and temporally unrelated and separate selection of resistance in individual patients seems more likely. all isolates had a group ctx-m esbl; the resistant isolate in each pair had lost a major omp, consistent with a porin, compared with its susceptible 'parent'. all three patients were successfully treated with amikacin. the emergence of carbapenem resistance in ctx-m-producing k. pneumoniae following therapy severely limits treatment options. whilst unusual in general, such selection has occurred repeatedly with this strain. wide geographic spread of diverse acquired ampc beta-lactamases in escherichia coli and klebsiella spp. in the uk and ireland objective: to determine the distribution of genes encoding acquired ampc beta-lactamases in cephalosporin-resistant isolates of e. coli and klebsiella spp. submitted to the uk national reference laboratory. methods: mics were determined by agar dilution and interpreted according to breakpoints of the british society for antimicrobial chemotherapy. isolates of e. coli or klebsiella spp. resistant to cefotaxime and ceftazidime, irrespective of addition of clavulanic acid, were inferred to have possible ampcmediated resistance. genes encoding six phylogenetic groups of acquired ampc enzymes were sought with a multiplex pcr assay (perez-perez & hanson. j clin microbiol ; : - ) . selected isolates were compared by pfge, and selected blaampc amplicons were sequenced. results: e. coli isolates and klebsiella spp. from separate patients yielded pcr amplicons indicating the presence of genes encoding acquired ampc enzymes. forty of these e. coli isolates (from hospitals) produced cit-type enzymes, (from irish hospitals) produced acc types, and a dha type. the klebsiella spp. produced acc ( isolates from irish hospitals), fox ( isolates from welsh hospitals) or dha ( irish isolate) enzymes. genes encoding ebc-/ent-and moxtype enzymes were not detected. twelve e. coli isolates from one hospital all produced a cit-type enzyme; these isolates belonged to an epidemic uk strain, designated strain a; isolates also contained blactx-m- linked to an upstream copy of is , as is characteristic of strain a; isolates lacked blactx-m- . sequencing of a representative blaampc amplicon indicated production of a novel cmy- variant in these isolates. conclusions: diverse acquired ampc enzymes are present in e. coli and klebsiella spp. in the uk and ireland, with cit-types the most common, and acc types linked to ireland. the broad resistance profiles of ampc enzymes compromises patient management. hence, the acquisition of a cmy- -like enzyme by epidemic e. coli strain a suggests that acquired ampc enzymes are poised to become an important public health issue in the uk. objective: to characterize the spectrum of activity and potency of dor (formerly s- ) and comparator agents against contemporary wild-type bacterial isolates from medical centres in europe and the middle east in . dor is a novel parenteral -b-methyl carbapenem in late stage clinical development whose molecular structure confers stability to b-lactamases and resistance (r) to renal dehydropeptidases. methods: the collection included non-duplicate, consecutive clinical isolates from patients in medical centres in europe ( ), turkey ( ) and israel ( ) that were submitted to the dor surveillance program ( ) for identification confirmation and susceptibility (s) testing. mic values for > antimicrobials were determined using nccls broth microdilution methods ( ) . a tentative dor susceptible (s) breakpoint of £ mg/l (£ . mg/l for s. pneumoniae) was used for comparative purposes; clsi ( ) criteria were used for other tested agents. results: antimicrobial activities of dor and other carbapenems vs. selected isolates. dor consistently displayed activity against staphylococci and streptococci (mic , . and . mg/l) most similar to that of imipenem, and against e. coli and klebsiella spp. (mic , . and . mg/l, respectively, including . and . % of strains that met esbl screening criteria), most similar to that of meropenem. enterobacter spp. isolates, including . % that were ceftazidime-r (indicative of ampc production), were also highly s to dor and other carbapenems ( . to . % r). dor also provided slightly enhanced coverage against p. aeruginosa ( . % s) and acinetobacter spp. ( . % s) compared to other carbapenems. carbapenem r among these latter strains is, however, a particularly worrisome development. conclusions: dor is a new carbapenem with a competitive profile that incorporates both potent gram-negative and grampositive activity, with enhanced activity against the commonly occurring non-fermentative gram-negative bacilli. carbapenems are assuming a greater therapeutic role in many nations as multi-drug resistance (including emergence of ambler class a, c and d b-lactamases) spreads, necessitating their accelerated development. phenotypic and genetic characterisations of enterococcal isolates in tehran sewage, with emphasis on detection of vana and vanb genes objectives: enterococci are members of the normal gut flora of animals and humans and are thus released into the environment directly or via sewage outlets, where they can survive for long time periods. during the last decade the concern has been focused on enterococci that are resistant to the glycopeptide antibiotic vancomycin [vancomycin-resistant enterococci (vre)]. the aim of the study was to detect and to analyse the biochemical diversity of the entrococci strains in tehran sewage and to determine the genetic characterization of vre. methods: a total of isolates of enterococci were selected on me agar medium. all of the isolates were identified at the species level by the common biochemical tests. drug susceptibility test of isolates was done by disk diffusion method with antibiotics vancomycin, erythromycin, gentamicin, tetracycline, chloramphenicol and ciprofloxacin. the mic was also done by macrobroth dilution assay. analysis of the plasmid profiles and the pcr tests for vana and vanb genes were done. methods: we studied vre isolates collected in the north and center of portugal ( portugal ( - from: (i) clinical isolates from hospitals in different cities, (ii) faecal samples from healthy volunteers, (iii) river water samples, (iv) samples collected downstream of hospital sewage water, (v) samples from urban sewage water, (vi) swine faeces (vii) poultry food samples for human consumption. identification and characterization of vancomycin resistant genes vana, vanb, vanc and vanc were determined by a multiplex pcr. the backbone structure of tn was characterized by a pcr overlapping assay ( overlapping fragments), and further sequencing. conclusion: beta-lactamase production among hi strains has declined significantly since among children attending daycare centres as antibiotic prescriptions fell among this population. results: the mic distribution of am showed % of strains (n = ) with a mic > mg/l and % with a mic of > mg/l, indicating that resistance to am is still relatively rare and does increase as compared to nethmap . the lognormal distribution of both am and amc ( strain r) extended to mg/l but showed tailing to mg/l. this may indicate hidden less susceptible strains but could equally well be explained by testing circumstances, since the left part of the mic distribution showed comparable tailing. all strains were susceptible to moxifloxacin, levofloxacin and cefotaxim. the lognormal distribution of sxt extended to . mg/l with % of strains showing higher values. doxycyclin resistance was less than %. most of the strains were resistant to clarithromycin and azithromycin with a mic > . mg/l for both. conclusions: resistance of hi to common antimicrobials in the netherlands is still low and does not increase. objectives: s. pneumoniae (sp) and h. influenzae (hi) are the two most common pathogens associated with community-acquired pneumonia. changes in the prevalence of resistance or multidrug resistance (mdr) among these pathogens have important therapeutic ramifications. the global surveillance initiative is a longitudinal study that benchmarks antibacterial resistance among respiratory pathogens. methods: during , sp and hi were isolated from patient specimens collected at hospital laboratories in france (fr), germany (ger), italy (it), spain (spa), and the united kingdom (uk). isolates were centrally tested by broth microdilution against lev, penicillin (pen; sp only), azithromycin (azi), erythromycin (ery), clindamycin (cli), ceftriaxone (ctx), cefuroxime (cfx), and trimethoprimsulfamethoxazole (tmp-smx) (nccls, ) . data were analysed according to pen resistance, mdr, and b-lactamase status. mdr was defined concurrent resistance to ‡ of the following agents: ctx, cfx, ery, lev, pen, and tmp-smx. results: for sp, pen r was . % in ger, . % in the uk, . % in it, . % in spa, and . % in fr. azi r was . % in the uk, . % in ger, . % in spa, . % in it, and . % in fr. overall, lev r was rare (£ %) and mic s = mg/l in all countries. . % of isolates were susceptible to all of the drugs tested, the most common phenotype encountered. the prevalence (%) of mdr among sp ranged from . in uk to . in fr. resistance to pen, ery, cfx, and tmp-smx was the most prevalent mdr phenotype found in europe. overall . % of mdr sp were susceptible to lev. for hi, b-lactamase rates varied by country from . % in it to . % in fr. based on mic lev and ctx were the most active agents tested against hi, regardless of b-lactamase status. conclusions: lev showed potent activity against sp and hi. for sp, lev activity was independent of resistance to pen or mdr phenotype. lev maintained consistent activity against sp based on mic , regardless of country studied. antimicrobial surveillance data from studies such as the global offer guidance to physicians for empiric prescribing. sxt was obtained ( sxt was obtained ( - . conclusions: our results suggest that beta-lactamase production does not constitute a threat in hi therapy since values were almost constant. although with an unregulated fluctuation on arnblp percentages, it seems that this mechanism is gaining importance in relation to beta-lactamase production. thus, we conclude the need to be aware of arnblp, as these strains are difficult to detect using the nccls ( ) breakpoints. further molecular studies of the resistance genes responsible of this resistance mechanism are needed. resistance of beta-lactamase producer strains, to other antibiotics decreased during the period of study, due to the diminished use of these antibiotics. this study shows the importance of monitoring antibiotic resistance in hi in order to detect emerging mechanisms. antimicrobial susceptibility of respiratory haemophilus influenzae strains in northern greece k. koraki, p. karapavlidou, d. sofianou (thessaloniki, gr) objectives: to investigate the antimicrobial susceptibility of haemophilus influenzae, one of the most frequent bacterial pathogens of respiratory tract infections. treatment of these infections is most often empirical and considerable geographical resistance variation has been reported. methods: eighty h. influenzae strains were collected from respiratory tract specimens (sputum, bronchoalveolar lavages, endotracheal secretions) in a -year period ( ) ( ) ( ) ( ) ( ) . identification was made by colonial morphology, gram staining characteristics, x-and v-factor requirements and api nh (biomerieux, france). antibiotics were selected to reflect representative current treatment options and susceptibility was determined by kirby-bauer disc diffusion method on haemophilus test medium according to nccls guidelines. results: out of the h. influenzae strains were isolated from children and from adults. % of isolates came from children admitted to the intensive care units and . % from cystic fibrosis patients. a seasonal trend was reported for infections since . % of isolates were collected during springtime and % during autumn months. overall ampicillin resistance was . % and resistant strains were isolated exclusively from children. ampicillin resistance was doubled among cystic fibrosis patients ( . %). all isolates were susceptible to amoxicillin/clavulanate, chloramphenicol, ciprofloxacin and imipenem. the rank order of cephalosporin activity was cefotaxime and ceftriaxone ( %) followed by cefuroxime and cefaclor ( . % and . % respectively). trimethoprim/ sulfomethoxazole was active against . % of isolates while erythromycin was the least potent antimicrobial agent with % of isolates being susceptible to it. no multiresistant phenotypes were detected. conclusion: our results demonstrated that ampicillin resistance among h. influenzae in our area is still relatively low and overall antibacterial susceptibility rates are high. knowledge of antimicrobial resistance among these pathogens is imperative for physicians to choose the most appropriate therapeutic agent. results: nosocomial gram-negative uropathogens were studied. most common uropathogens were p. aeruginosa ( . %), e. coli ( . %), k. pneumoniae ( . %), followed by a. baumannii ( . %), enterobacter spp. ( . %), s. marcescens ( . %), proteus spp. ( . %) and other gram-negative rods ( . %). resistance rates (i+r, %) among p. aeruginosa were: gentamicin - %, levofloxacin - %, ciprofloxacin - %, cefoperazone - %, cefoperazone/sulbactam - %, cefepime - %, piperacillin - %, amikacin - %, ceftazidime - %, imipenem - %, meropenem - %, piperacillin/tazobactam - %, polymyxin b - %. resistance rates (i+r, %) among e. coli were: piperacillin - %, ticarcillin/clavulanic acid - %, amoxicillin/clavulanic acid - %, ciprofloxacin - %, gentamicin - %, moxifloxacin - %, levofloxacin - %, cefoperazone - %, ceftriaxone - %, cefepime - %, ceftazidime - %, cefoperazone/sulbactam - %, piperacillin/tazobactam - %, amikacin - %, all strains were susceptible to ertapenem, imipenem, meropenem. resistance rates (i+r, %) among k. pneumoniae were following: piperacillin - %, cefoperazone - %, ceftriaxone - %, gentamicin - %, amoxicillin/clavulanic acid - %, cefepime - %, ceftazidime - %, ciprofloxacin - %, piperacillin/ tazobactam - %, moxifloxacin - %, cefoperazone/sulbactam - %, levofloxacin - %, amikacin - %, ertapenem - %, imipenem and meropenem were active against all isolates. conclusion: p. aeruginosa, e. coli and k. pneumoniae are the main gram-negative uropathogens in russian icus patients. imipenem, meropenem, ertapenem showed prominent activity against e. coli and k. pneumoniae. cefoperazone/sulbactam, piperacillin/tazobactam, amikacin exhibited considerable activity versus e. coli, while k. pneumoniae were more resistant to them. p. aeruginosa were highly resistant to all tested antimicrobials except polymyxin b, thus leaving virtually no choices for therapy in terms of acceptable patient safety. results: overall gram-negative anaerobic bacteria from patients were studied. isolation sites were represented by intraabdominal - ( . %), soft tissue - ( . %), prostate fluid - ( . %), bone - ( . %), and dental - ( . %) infections. susceptibility of ( . %) prevotella spp., ( . %) bacteroides spp. (predominantly b. fragilis group - strains), ( . %) fusobacterium spp., ( . %) porphyromonas spp., and ( . %) veillonella spp. to ampicillin, clindamycin, metronidazole, imipenem, ertapenem, amoxicillin/clavulanic acid and cefoperazone/sulbactam was determined. all species were susceptible to carbapenems. in prevotella spp. there were % and % strains resistant to ampicillin and clindamycin and % of strains with intermediate resistance to metronidazole. among bacteroides spp. % of strains were resistant to ampicillin and % to clindamycin. no resistance to metronidazole was detected in bacteroides spp. objectives: the objectives of this study were to: analyse our current blood culture practice; describe the frequency of occurrence and antimicrobial susceptibility of bloodstream infections (bsi) isolates; determine the contamination rate. methods: we performed a prospective survey of all positive blood cultures received in the department of microbiology of tartu university hospital ( beds) in . blood culture system used was bactec . duplicates within one week were excluded. isolates were identified using conventional microbiology methods and susceptibility tests were those recommended by nccls. to determine extended spectrum beta-lactamase (esbl) producers an e-test with cefepime and cefepime combined with clavulanic acid was used. nosocomial infections were defined according to cdc criteria. results: during study period blood culture bottles were received, comprising blood culture sets ( . sets per patient-days). these resulted in ( . %) positive blood cultures, ( . %) were considered contaminants and contamination rate was . %. a total of bsi episodes involving patients were identified and ( %) of these were nosocomial. the incidence of nosocomial bsi (n-bsi) and community-acquired bsi (ca-bsi) was . and . per patient-days, respectively. polymicrobial bsi was detected in patients. among n-bsi dominated coagulase-negative staphylococci ( / . %), staphylococcus aureus ( / . %), klebsiella spp. ( / %), and escherichia coli ( / . %). the most frequent pathogens of ca-bsi were e. coli ( / . %), s. aureus ( / . %), haemophilus influenzae ( / . %), and streptococcus pneumoniae ( / . %). susceptibility to oxacillin of s. aureus and cons was % and . %, respectively. all s. pneumoniae isolates were susceptible to penicillin. . % of e. coli strains were susceptible to ciprofloxacin, . % to ampicillin, and % to gentamicin. susceptibility of klebsiella spp. to both ciprofloxacin and gentamicin was . %, and to ampicillin . %. . % of klebsiella spp. and none of e. coli isolates were esbl-producers. the susceptibility patterns of n-bsi and ca-bsi pathogens were similar to each other. conclusion: compared to west and north european countries our number of blood culture sets per patient-days is low. this may explain the relatively low incidence of bsi. the interventions to reduce contamination rate need to be implemented. the susceptibility among bsi isolates was high. recent outbreaks of c. difficile associated diarrhoea (cdad) reported in north america, united kingdom and the netherlands have emphasized the importance for an ongoing surveillance of cdad. the aims of the present study was to determine the epidemiology of cdad over the past years and the rate of nosocomial transmission in our acute care hospital ( -beds). materials and methods: all the cases of cdad diagnosed between january st and december st were retrospectively reviewed. a cdad case was defined as diarrhoea in hospitalised patients with a positive result for c. difficile cytotoxin or with a positive toxigenic culture. cdad was considered as severe if patient fulfilled at least of the following criteria: fever > . c abdominal pain or leukocyte count > , /mm or if the patient had an endoscopically proven pseudomembranous colitis or complications (toxic megacolon, perforation…). cdad was considered as community acquired if the diarrhoea occurred in patients within h after admission and if the patient had no history of hospitalisation in the previous months, otherwise cdad was considered as nosocomial. all the strains were serogrouped and characterized by toxinotyping and pcr-ribotyping. detection of toxin a, toxin b and binary toxin was performed by pcr. results: cases of cdad were diagnosed: clinical charts could be reviewed and strains were studied. global incidence of cdad was . per thousand discharges with higher rates in and . diarrhoea was community acquired in % of patients. for patients with nosocomial cdad, transmission of the strain from patient to patient (i.e. strain with the same serogroup and pcr-ribotype than the strain from another patient hospitalised in the same ward in the previous months) was demonstrated in . % of cases. binary toxin was positive in % of strains. binary toxin was associated to a more severe diarrhoea (p < . ) and to a higher case fatality (p < . ). a specific clone accounted for % of all the strains (serogroup h, pcr-ribotype '' '') but this clone was found both in nosocomial or community cases. three strains belonged to toxinotype iii but further investigations are needed to know whether these strains correspond to the hypervirulent strains involved in recent outbreaks. conclusion: incidence of cdad is low in our hospital and cross infection is limited. these results also suggest that strains with binary toxin might be more virulent. the development and application of a new exact typing method for clostridium difficile: multilocus variable number of tandem repeat analysis objectives: to study the epidemiology of clostridium difficile, a typing method with a higher discriminatory power, typeability and reproducibility than currently available methods is required. multi-locus variable number of tandem repeat analysis (mlva) is a new candidate technique, that has already been tested successfully on a number of bacterial and fungal species. using the whole genomic sequence, we developed mlva for c. difficile and compared the method to standardized pcr-ribotyping. additionally, mlva was tested on a collection of the new emerging hypervirulent pcr-ribotype strains. methods: short tandem repeat loci ( to bp) were identified using tandem repeat finder v . on the genome of c. difficile strain . amplification of the repeats was performed using a single pcr-protocol. pcr-fragments were analysed using multicoloured capillary electrophoresis on an abi , with a rox -marker as internal marker for each sample. the number of repeats per fragment was subsequently determined.the discriminatory power of the mlva was tested on reference strains representing serogroups and toxinotypes. the ability to subtype specific pcr-ribotypes was investigated with subtypes of pcr-ribotype (rep-pcr types - ), tcda-/tcdb+ strains of pcr-ribotype , and strains belonging to pcr-ribotype . of these type strains, were isolated from outbreaks and from endemic cases. results: a total of regions with short tandem repeats were identified. mlva discriminated all reference strains and the known reference strains of pcr-ribotype (rep-pcr - ). two mlva-types were recognized among tcda-/tcdb+ strains; the differences were present in only one of the repeat-regions. of pcr-ribotype strains, outbreak-related strains were identical to each other. interestingly, two endemic type strains differed from the other strains in of the regions. conclusion: mlva is a highly discriminatory genotyping method for c. difficile and is capable to subtype various crribotypes. mlva is also an important new tool to study the epidemiology of the emerging pcr-ribotype strains. comparative study of clostridium difficile diarrhoea in elderly patients treated with moxifloxacin versus amoxycillin for lower respiratory tract infections l. mooney, m. wilcox (leeds, uk) fourth generation fluoroquinolones such as moxifloxacin have improved anti-anaerobic activity. consequently, these new agents could induce c. difficile infection (cdi) by inhibition of 'protective' anaerobic flora. recent reports have suggested such an association. however, further studies are warranted to determine the risk of cdi in elderly in-patients treated with these agents, and notably where exposure to cd is measured/ controlled. methods: we prospectively investigated the propensity of moxifloxacin (mox) or amoxycillin/macrolide (aml/mac) to induce cdi when used to treat lower respiratory tract infections (lrtis) in elderly in-patients, using a -ward, crossover design ( months total). patients prescribed mox or aml/mac were monitored for gastrointestinal symptoms. diarrhoea was assessed as due to cd, viral or other cause. relevant clinical data were collected. concurrent epidemiological surveillance was also performed to determine environmental exposure to cd. results: patients were studied, receiving mox and had aml/mac. univariate analysis indicated that there was no significant difference between mox and aml/mac patients in gender, age ( . vs . mean years, respectively), or duration of hospitalisation (total, prior to and post diarrhoea). duration of antibiotic therapy did not differ significantly between mox and comparator patients (either total days or days before diarrhoea onset). there was a significant association between mox and overall risk of diarrhoea. however, there was no significance between mox treatment and cd, viral or other cause of diarrhoea. risk factor analysis to inform on possible confounders was performed. initial epidemiological survey results indicate that there was no change in environmental exposure levels to cd on each hospital ward. molecular typing of all clinical and environmental isolates of cd is ongoing. conclusions: although recent reports have highlighted a risk of cdi associated with fluoroquinolones (and increased age), none have specifically studied hospitalised elderly populations prospectively and controlled for exposure to cd. diarrhoea occurs relatively frequently after antibiotic therapy in the elderly. mox was associated with an increased rate of diarrhoeal symptoms, but causes other than cdi explained this association. mox treatment was not significantly associated with cdi when compared with amox/mac treatment for lrti in elderly in-patients. prevalence and association of macrolidelincosamide-streptogramin b resistance with resistance to moxifloxacin in clostridium difficile strains isolated from symptomatic adults and children hospitalised in two university hospitals in warsaw h. pituch, d. wultanska, g. nurzynska, p. obuch-woszczatynski, f. meisel-mikolajczyk, m. luczak (warsaw, pl) objectives: clostridium difficile is the main aetiological agent of nosocomial diarhoea. clindamycin, penicillins, and cephalosporins have been associated with cdad. however, several case reports of fluoroquinolone-associated c. difficile diarrhea have been published. c. difficile strains usually exhibits susceptibility to metronidazole, and vancomycin. we describe prevalence and association of macrolide-lincosamide-streptogramin b (mlsb) type resistance with resistance to moxifloxacin of c. difficile strains isolated from adults and children. methods: eighty-three c. difficile strains recovered from adults and children hospitalised in two university hospitals were investigated (hospital : adults n = , and children n = ; hospital : adults n = ). toxin types were determined by commercial test for toxin a and cytotoxicity test for toxin b. tcda, tcdb were detected by pcr. mics of erythromycin, clindamycin, moxifloxacin, vancomycin and metronidazole were determined by e-test (ab biodisk, sweden). the ermb gene was detected by pcr. results: sixty-seven ( %) c. difficile strains were toxigenic. among these, were a+b+, and were a-b+. all strains were susceptible to vancomycin and metronidazole. high level resistance to erythromycin, clindamycin and moxifloxacin was found in %, %, % of the tested strains, respectively. twenty-one c. difficile strains harboured high level resistance to erythromycin, clindamycin and moxifloxacin, simultaneously. among these, all were a-b+ and were isolated from adults, only. twenty-one of the macrolide-lincosamide-streptogramin b (mlsb)-resistant a-b+ strains carried the erythromycin resistance methylase gene (ermb). conclusion: resistance against clindamycin, erythromycin and moxifloxacin among polish a-b+ c. difficile strains was very frequent. fluoroquinolone resistance is associated with resistance to mlsb antimicrobials. we suggest that increasing use of fluoroquinolones is selective pressure for clonal dissemination of a-b+ c. difficile strains. fluoroquinolones use is a strong risk factor for cdad in our hospitals. acknowledgement: this work was supported by the ministry of scientific research and information technology, grant no. p d . national surveillance to the incidence of clostridium difficile-associated diarrhoea in the netherlands s. paltansing, r. guseinova, r. van den berg, c. visser, e. van der vorm, e.j. kuijper (leiden, amsterdam, nl) objectives: the recent outbreaks of clostridium difficileassociated diarrhoea (cdad) due to the new emerging pcrribotype , toxinotype iii strains has renewed the interest of cdad as an important nosocomial infection. to determine the incidence of cdad in the netherlands, we conducted a prospective surveillance study in hospitals in the netherlands. clinical microbiology and infection, volume , supplement , methods: from may st to july st of , participating hospitals registered all patients diagnosed with cdad. a standardized questionnaire was devised to obtain patient information. faeces samples or isolated strains were sent to the reference laboratory at the lumc for culture and the presence of genes for toxins a and b (tcda and tcdb). pcrribotyping was performed according to the method of bidet and toxinotyping as described by rupnik et al. results: routine methods to diagnose cdad in laboratories included combinations of cytotoxicity tests ( %), enzymeimmunoassays ( %) and culture of toxinogenic strains ( %). in total, patients with cdad were reported. the overall incidence (median) of cdad was for , patient admissions and varied from to . of patients with cdad, % was community acquired. the median age of patients with nosocomial acquired cdad was years. of patients with cdad, ( . %) died during the study period. at least different pcr-ribotypes could be recognized among strains. type was identified in patients from hospital. toxinotyping revealed the presence of at least different types. of strains, % were tcda+/tcdb+, % tcda-/tcdb-and % tcda-/tcdb+. conclusions: the incidence of cdad in the netherlands is lower than reported in usa and canada, but varied considerably per hospital. the new emerging type was found in patients from hospital with a high incidence of cdad ( per , admissions). outbreak of clostridium difficile pcr-ribotype toxinotype iii in harderwijk, the netherlands objectives: since , several epidemics of clostridium difficileassociated diarrhoea (cdad) caused by c. difficile pcr-ribotype toxinotype iii have occurred in usa, canada, and the uk. in april , the first outbreak encompassing patients was observed in a medium large hospital of beds in the netherlands. the isolated strain was completely resistant to erythromycin and ciprofloxacin. the patient characteristics, predisposing factors and outcome of cdad were studied. methods: a case-control study was performed in patients and at random selected controls without diarrhoea who stayed at the same department as the patients when the diagnosis of cdad was made. standardized questionnaires were designed to collect data from the patient records and all surviving patients were interviewed months after the diagnosis. faeces samples were cultured for the presence of c. difficile and isolates were typed. results: the incidence of cdad increased from per , patient admissions in to . per , admissions in . between april and september , patients with cdad due to type were identified. of patients, ( %) died of which ( %) as a direct result of cdad. eleven ( %) patients experienced one or more relapses. the average age of the cases was yrs, . % of the patients was male. in a multivariate analysis, antibiotic use (or . , p < . ), duration of hospital stay (cases days, controls days; p < . ) and tube feeding (or . , p = . ) were found to be significantly associated with cdad. in particular, the use of ciprofloxacin (or . , p < . ) and cephalosporins (or . , p < . ) were associated. no association was found between the use of protonpump inhibitors and the risk of cdad. the use of erytromycin was significantly higher in cases ( . %) than in controls ( . %) in a univariate analysis (p < . ), but this relation was not significant in a multivariate analysis. conclusion: antibiotic use (especially ciprofloxacin and cephalosporins), duration of hospital stay and tube feeding were significantly associated with cdad caused by c. difficile type , toxinotype iii in the netherlands. we could not confirm the previously described relation between use of protonpump inhibitors and risk of cdad. clostridium difficile pcr ribotype , toxinotype iii in the netherlands objectives & methods: shortly after the reports in june of clostridium difficile pcr ribotype , toxinotype iii in england, this more virulent type was also detected in the netherlands. in response, the dutch centre for infectious disease control has undertaken measures to monitor and control the outbreak. c. difficile guidelines for infection control and treatment were formulated, separately for hospitals and nursing homes. the leiden university medical centre serves as a reference centre for diagnostics and typing of c. difficile. laboratories are encouraged to send in samples for typing in case of a clear rise in the incidence in c. difficile, rapid spread, or several clinically suspect cases.organisation-based surveillance was set up: questionnaires are sent monthly to institutions with c. difficile associated diarrhoea (cdad) outbreaks to obtain information on incidence, c. difficile testing strategies, antibiotics use and control measures taken.measures taken in hospitals dealing with an outbreak of type include: treatment of cdad with vancomycin in stead of metronidazole, emphasis on frequent and thorough cleaning and disinfection, isolation of all patients with diarrhoea until tested negative for c. difficile toxin, cohort isolation of cdadcases if individual isolation capacity is exceeded and strong restriction of certain antibiotics, including fluorochinolones. results: until november st, , samples from institutions have been sent in for typing, resulting in type positives. epidemic spread of type has been detected in hospitals and one nursing home. furthermore, in retrospective studies in four hospitals isolated cases of type were detected. it became clear that in one region with three hospitals, the cdad incidence had already risen in , and , respectively . unfortunately, no samples from that period were available for typing. in the hospitals with epidemic spread of type , a wide range in the monthly incidence of cdad was observed, from to per , admissions during the outbreaks. the incidence in the pre-epidemic period varied from to (see figure) . conclusions: the outbreaks in hospitals are difficult to control: most hospitals continue to have new cases for a long period, although the incidence is decreasing in several hospitals. fortunately, once a c. difficile outbreak in a hospital is recognised, spread to other hospitals has not been observed. objectives: c. difficile is a major cause of antibiotic associated diarrhoea (aad) and colitis (c). the aim of this study was to determine the incidence of these infections in our hospital ( beds), during a period of months (march-october ) . methods: a number of liquid stools from equal adult patients (mean age y, m: , f: ) receiving broad spectrum antibiotics (especially cephalosporins) were plated in ccfa (oxoid) and anaerobic brucella agar (ba), after alcohol shock procedure. if the culture was positive, an immunochromatographic test was performed for toxin a (colorpactm toxin a, bd, usa). if the last test was negative, a rapid enzyme immunoassay was performed for toxins a+b (immunocard, meridian bioscience inc. cincinatti, ohio). results: c. difficile was isolated in / ( . %) samples. seventeen men (pathological -p, pneumological -pn, surgical -s, urologic -u, outpatients -o, , , , , respectivly) and women (p: , pn: , o: ) harbored c. difficile in their intestin. twelve out of strains ( %) produced toxin a, while the remaining ( %) produced toxin b. eleven patients had severe diarrhoea ( - days). one patient got endoscopic examination, which confirmed colitis findings. the two outpatients received oral cefuroxime in the preceding week of the positive culture. conlusions: ( ) the incidence of c. difficile infections in this study is among these reported in international bibliography ( . %). ( ) since toxigenic b c. difficile strains were demonstrated in half cases, the use of the tests detecting both toxins a and b by clinical laboratories is recommended.( ) molecular technics application (e.g. pfge and ribotyping) will offer a better knowledge of c. difficile spread in our hospital. assay of the cytotoxicity of stool samples to cells in tissue culture is commonly considered the 'gold standard' for detection of c. difficile toxin. however the method is slow and therefore its use can result in delayed patient treatment and implementation of infection control measures. we undertook a comparison of two microtitre plate-based elisa kits (techlab c. difficile tox a/b ii and meridian premier toxins a & b) and three rapid immunoassay card kits (remel xpect clostridium difficile toxin a/b, meridian immunocard toxins a & b and techlab tox a/b quik chek) with an in-house cytotoxin assay. all samples tested had been referred for routine microbiological examination. toxin tests were done on unformed samples from adult hospital in-patients and bone marrow transplant recipients and on samples where c. difficile toxin testing was requested by the referring clinician. all kits were used according to manufacturers' instructions. three hundred and thirty three specimens were tested using all five kits and cytotoxin assay. sensitivities and specificities were calculated both (a) assuming the cytotoxin assay to be the 'gold standard' (universally correct) test and (b) taking a concensus view that any sample with at least two tests positive is truly positive. data are shown in the table below. overall, the microtitre plate-based elisa kits were more sensitive than the rapid immunoassay card kits. the cytotoxin assay was negative for seven samples that were positive by at least two other tests. thus the plate-based elisa kits were also more sensitive (but less specific) than the cytotoxin assay if consensus data was used to judge true positivity. we conclude that some immunoassay kits offer an acceptable alternative to cytotoxin assays for the detection of c. difficile toxin, allowing more rapid diagnosis. location of the enterotoxin gene in strains of clostridium perfringens associated with gastroenteritis objectives: clostridium perfringens type a is a common cause of food poisoning and is also associated with non-food borne gastroenteritis including antibiotic associated, infectious and sporadic diarrhoea. the disease symptoms are due to an enterotoxin produced when the organism sporulates in the human small intestine. the c. perfringens entertoxin gene (cpe) has been shown to be located either on the chromosome or on one of two large plasmids and it is generally accepted that c. perfringens strains associated with food poisoning have a chromosomal cpe gene whilst strains isolated from non-food borne diarrhoea have a plasmid encoded cpe gene. spores from strains possessing a chromosomal cpe gene have been found to be far more heat resistant than spores from strains with a plasmid encoded cpe gene. heat resistant spores are more able to survive the cooking process and go on to cause food poisoning, thus explaining why most food poisoning strains have been found to have chromosomally located cpe genes. the purpose of this study was to determine the location of the cpe gene in a range of c. perfringens strains from the uk, including those from both food borne and non-food borne illness. method: a multiplex pcr assay described by miyamoto et al., ( ) was used to determine the location of the cpe gene in strains of c. perfringens isolates associated with food borne illness and strains associated with non-food borne illness. results: by multiplex pcr assay % of c. perfringens strains associated with food borne outbreaks in the uk were found to have a plasmid encoded cpe gene. these findings have not been described before. all strains associated with non-food borne illness had the cpe gene located on one of two plasmids, as anticipated. conclusions: a significant number of food borne outbreaks of c. perfringens food poisoning were found to be caused by strains of c. perfringens carrying a plasmid encoded cpe gene. since strains of c. perfringens with a chromosomal cpe and plasmid cpe genes have different physiological characteristics this may have a profound impact on their mode of transmission. references miyamoto, k., wen, q. and mcclane, b. a. ( ) multiplex pcr genotyping assay that distinguishes between isolates of clostridium perfringens type a carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an is -like sequence, or a plasmid cpe locus with an is sequence. journal of clinical microbiology , - . novel multiplex-pcr method for simultaneous detection of clostridium difficile toxin a and toxin b and the binary toxin (cdta/cdtb) genes applied on a danish cohort k.e.p. olsen, s. persson (copenhagen, dk) objectives: a new multiplex pcr method was developed for the detection of the clostridium difficile toxin genes: tcda, tcdb, cdta and cdtb. this method was applied on clostridium difficile strains isolated from danish hospitalised patients with diarrhoea in the period from april to october , in order to investigate the present toxin profiles and their correlation to sex and age. method: a -gene multiplex pcr method was developed for the simultaneous amplification of the four clostridium difficile toxin genes tcda, tcdb, cdta, cdtb and s rdna as an internal positive control. template dna was prepared from plate grown bacterial colonies by a simple boiling procedure, and amplicons were visualized by standard gel electrophoresis. results: three different toxin profiles were detected in the danish cohort: tcda+, tcdb+, cdta+/cdtb+; tcda+, tcdb+, cdta-/cdtb-and non-toxigenic tcda-, tcdb-, cdta-/ cdtb-. the prevalence of the binary toxin genes in this study was % of the clinical isolates.more than half of the strains ( %) were isolated from the elderly part of the population (> years), and % of these strains displayed the tcda+, tcdb+, cdta+/cdtb+ profile. of the non-toxigenic strains, % of the patients were females. one fourth of the strains isolated from children under years of age were non-toxigenic. in four patients, two different toxin profiles were obtained from independent faecal samples. conclusion: this method offers a one-step, rapid and specific identification of clostridium difficile toxin genes. this specific toxin profiling allows an evaluation of the pathogenic potential of the isolated clostridium difficile and surveillance of emerging toxin profiles. further studies of the isolated toxigenic clostridium difficile strains will include gene deletion analyses of the tcda and the tcdc (toxin regulating gene) which independently have been observed to cause enhanced pathogenicity. prevalence of clostridium difficile-associated diarrhoea in hospitalised patients with nosocomial diarrhoea in university of medical sciences hospitals, tehran, iran objectives: this study was aimed at determining the prevalence of clostridium difficile associated diarrhoea in hospitalized patients with nosocomial diarrhoea at three university hospitals in tehran from december to august . methods: during the study period, the stool samples of hospitalized patients with nosocomial diarrhoea were cultured and tested by stool cytotoxin assay, toxigenic culture and also of the samples were examined by enzyme immunoassay. results: in ( . %) of samples c. difficile grew and stool samples (prevalence: . %) were toxin positive by stool cytotoxin assay, enzyme immunoassay or toxigenic culture. there were no significant relationships between c. difficileassociated diarrhoea and sex and age of patients. the results of the present study showed that among requested samples the highest percentage of c. difficileassociated diarrhoea was observed from the transplantation department ( . %), followed by icu and paediatric section. objectives: the prevalence of toxigenic clostridium difficile (c. difficile) has been reported about - % in korea. toxin a(-)/ toxin b(+) variant c. difficile strain is also important in nosocomial c. difficile infection. however, characterization of clostridial toxin (toxin a, toxin b) had not been studied. methods: we used pcr for toxin a and toxin b genes in c. difficile isolates from patients admitted in three tertiary hospitals during january to december, . primers for toxin a genes were nk -nk , nk -nk and nk -nk and toxin b gene was nk -nk . results: toxin a and toxin b positive rates using nk -nk , nk -nk and nk -nk were concordant and ranged from . % to . % in hospitals. the proportions of non-toxigenic strains were - %. however, we could differentiate toxin a(-)/toxin b(+) variants using nk -nk primers. the proportion of toxin a(-)/toxin b(+) c. difficile variants were . %, . % and . % in hospitals respectively. objective: administration of antibiotic drugs has long been known to cause alterations in the gut ecosystem. in some patients, these alterations may create a niche that allows the overgrowth of some pathogens such as clostridium difficile, the main causative agent in nosocomial infectious diarrhoea. a predictive tool to assess the risk of development of clostridium difficile, would be of utmost clinical relevance. it remains to be determined whether specific patterns in pre-existing gut microbiota can predict the risk of onset of clostridium difficile, upon initiation of antibiotic treatment. using samples from subjects enrolled in a previously published clinical study on antibiotic-associated diarrhoea (aad), we investigated the potential relationship between their dominant faecal microbiota and the subsequent development of clostridium difficile when subjects received antibiotics. methods: temporal temperature gradient gel electrophoresis (ttge) was used to assess dominant species distribution in gut microbiota. each electrophoregram was digitised from the migration distances and a regression model [partial least square-discriminant analysis (pls)] was built to investigate the correlation between pre-treatment dominant faecal microbiota and the acquisition of clostridium difficile during antimicrobial chemotherapy. results: this pls model could explain % of the subsequent onset of clostridium difficile. this result supports the concept of ''permissive'' flora with preliminary data focusing on clostridium coccoides-phylogenetic group. conclusion: to our knowledge it is the first time that dominant faecal microbiota is found to heighten susceptibility to the subsequent onset of clostridium difficile upon initiation of antibiotic treatment. these findings insinuate that strategies reinforcing the control of dominant faecal microbiota at homeostasis would be of clinical relevance. this study has been partially financed by biocodex laboratories. objectives: selective therapy of c. difficile diarrhea (cdd) requires the reduction of pathogen counts in the colon, but spare the normal flora. to determine if par is selective for cdd, serial stool samples were collected at study entry, at day , and weekly x during the conduct of a phase a study of cdd treatment. methods: patients (n = ) were randomized to receive , or mg twice daily of par for days. no prior therapy was given to patients; receive or doses of standard therapy. as treatment controls, additional patients were treated with vancomycin mg qid for days. five well persons donated stools as normal flora controls. fresh stool samples were cultured - , , , for c. difficile vegetative and spore forms; faecal filtrates were tested for cytotoxin b by cell assay. strains were characterized by tcda/b, ermb, cdta/b pcr and by ribotyping. at study entry and day , aerobic and anaerobic faecal flora cultures, diluted - , , , , were examined for major floral shifts. since bacteroides group organisms are ubiquitously present and cultivable, this genera was selected as a indicator of the integrity of the microbial flora. results: at study entry, mean log cfu + sd vegetative counts of c. difficile (all par patients) were . + . , range - . ; at day , with the exception of one patient receiving mg, all other patients had c. difficile quantitative counts reduced < log /gm faeces. vancomycin was similarly effective. at study entry, bacteroides group counts were < , - , & . - log cfu/gm in~ / each of patients. all normal stools showed complex, multi-genera in high counts, with - bacteroides group species > log cfu/g. mean + sd of log cfu of bacteroides group counts/g feces wet weight at study entry and day for mg/day (n = ) were . + . / . + . (p = . , wilcoxon matched pairs signed-ranks test, tailed); for mg/day (n = ) were . + . / . + . (p = . ); for mg/day (n = ) were . + . / . + . (p = . ); and for vancomycin (n = ) . + . / . + . (p = . ). conclusion: patients with cdd have variably impaired normal flora. par was effective in all dosages in eradicating c. difficile. a dose-dependent reduction in bacteroides counts was not observed. vancomycin significantly reduces bacteroides counts during cdd treatment. par is effective against c. difficile in-vivo, and is relatively sparing of the normal flora. results: the three rt pcr assays were able to detect all enterovirus strains in cell culture supernatants. however the detection limit of the mgb rt pcr was to log more sensitive in out of dilutions assays of vc supernatants compared to the rab and ver rt pcr. all ver and mgb negative csf were vc negative. thirty-two csf specimens from patients suspected of viral meningitis were positive by all rt pcr ( . %), whereas only were found positive by vc ( . %). the rab rt pcr failed to detect csf confirmed positive by vc ( echo and non typable ev). among samples positive by rt pcr, sensitivity of ver, mgb and rab was respectively %, % and . %. conclusion: in our laboratory, mgb rt pcr has a good correlation with ver rt pcr whereas rab rt pcr is less sensitive especially for the detection of echovirus . the mgb rt pcr seems to be the most sensitive of the rt pcr. further studies, including more ev strains should help to precise the sensitivity of this assay. a. dalwai, s. ahmad, e. hussein, a. pacsa, w. al-nakib (kuwait, kw) objectives: enteroviruses generally share tissue tropism and present with overlapping disease spectrum, however certain enteroviruses may be over represented in certain diseases than others. coxsackievirus a though has been reported to cause several diseases such as febrile illness, herpangina, aseptic meningitis and acute flaccid paralysis, the frequency was very low. the study aimed to determine the prevalent enteroviruses causing non-specific febrile illness, aseptic meningitis, encephalitis, neonatal disease and myositis, in kuwait. it also aimed to study the association between a certain enterovirus and a particular disease and its severity. methods: diagnosis of enteroviral infection was based on detection of enteroviral rna by semi-nested rt-pcr of a portion of the 'utr of the enteroviral genome followed by southern hybridization with an enterovirus specific probe to confirm the results. the enterovirus was genotyped by sequencing of the 'utr, the vp and a portion of the vp encoding regions, and the sequence was analysed by blast analysis, clustalw alignment and phylip phylogenetic analysis package. results: enteroviruses were the only etiological agents detected in % ( ) of disease cases investigated. coxsackievirus a was identified to be the second most predominant enterovirus ( %; of cases genotyped) associated with disease, after only echovirus ( %; / ). although identified in all the diseases investigated, coxsackievirus a occurred less frequently in cns disease cases ( %; / ) than in febrile illness cases ( %; / ). in a preliminary study, it was also predominantly detected in % ( / ) of myositis cases. the 'utr of this virus showed % homology with that of coxsackievirus a prototype strain (parker strain) whereas the vp and the adjoining region showed greater homology to human enterovirus b genotype sequence. conclusions: coxsackievirus a was determined to be an emerging enterovirus associated with different diseases in kuwait. it was frequently represented in mild febrile illness and myositis cases than in cns disease suggesting that the isolate might be less neurovirulent. molecular analysis suggests that the isolate might have emerged due to recombination between coding and non-coding segments of coxsackievirus a and human enterovirus b group genomes. acknowledgement: supported by research administration project grants mi / , ym / and college of graduate studies, kuwait university. the new proposed enterovirus type is causing meningitis in spain introduction: several new proposed enteroviruses (ev) have been recently described, including the named ev [ ] . a total of isolates of this serotype were identified from to in america, africa or asia associated mainly with acute flaccid paralysis or unspecified disease. objective: to determine if this new serotype circulates in spain and what type of disease produces. methods: a total of ev isolates coming in to the spanish enterovirus reference laboratory were studied both by micro neutralization assays and by typing pcr [ ] . in the isolates in which ev was suspected by the mentioned methods complete vp gene was amplified and sequenced with specific designed primers. results: four isolates from two different regions of spain were identified as ev (more than % of homology with the published sequences). three of them corresponded to aseptic meningitis in children and were isolated from csf. discussion: the present work demonstrates that this new proposed virus circulates also by europe and is associated to aseptic meningitis. till the moment it seems that is represented in a minor proportion ( / studied), however the possibility of spreading of this viral infection should be considered, as evs may behave in that way, as previously have been demonstrated [ ] . objectives: rotavirus is the most important cause of severe gastroenteritis in infants and young children through the world and is responsible of , deaths annually, mostly in developing countries. therefore, development of rotavirus vaccine is a high priority. rotavirus strains with g types account for the majority of the diarrhoea episodes. recently, a monovalent g attenuated rotavirus vaccine was licensed in mexico. in view of a hypothetical introduction of such vaccine in europe, we investigated the variability over time of vp antigenic genes of g rotavirus strains in our area. methods: fifty strains were selected from a total of g strains obtained from children of less than years of age hospitalised with acute gastroenteritis at the ''g. di cristina'' children's hospital of palermo in the period - . the selected strains were genotyped by rt-pcr and of them were submitted to vp gene sequence analysis. results: all but one of the strains were genotyped as g p( ). the vp sequences of of them were distributed into lineages i and ii. lineage i included strains from different years in the range - . lineage ii included strains from different years in the range - . the degree of similarity among the nucleotide sequences of italian strains in each lineage were comprised between % and %. an alignment of the deduced amino acid sequences showed major lineage specific amino acid changes in the variable antigenic regions with respect to the reference wa strain. conclusions: sequence analysis indicated that in palermo there was co-circulation of g strains belonging to two different lineages. overall, the g strains showed a high degree of similarity inside each lineage and shared specific amino acid modifications. the antigenic differences between circulating strains might permit them to escape neutralization and persist in the infantile population. our results suggest that rotavirus strains belonging to the two g lineages should be both included in a rotavirus vaccine preparation. epidemic spread of recombinant noroviruses with four capsid types in hungary objectives: noroviruses (''winter vomiting diseases'') are the predominant etiological agent in hungary and common pathogen worldwide in outbreaks of gastro-enteritis in humans. noroviruses are genetically diverse group of viruses with multiple genogroups (gg) and genotypes. more recently, naturally occurring recombinant noroviruses were identified. these viruses had a distinct polymerase gene sequence (orf , designated ggiib/hilversum) and were disseminated through waterborne and food-borne transmission in europe. our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in hungary. methods: stool and rna samples -from norovirus outbreaks between january and may -containing ''ggiib/ hilversum polymerase'' (ggiib-pol) were selected for analysis of the viral capsid region (orf ) by reverse transcriptionpolymerase chain reaction (rt-pcr) followed by sequence-and phylogenetic analysis. results: forty ( . %) of confirmed norovirus outbreaks were caused by the new-variant lineage with the ggiib-pol. viral capsid region was successfully characterized in ggiibpol outbreaks. four different recombinants were detected with capsids of hu/nlv/ggii- /mexico/ (n = , . %), hu/ nlv/ggii- /snow mountain/ (n = , . %), hu/nlv- /ggii/hawaii/ (n = , . %) and hu/nlv/ggii- / lordsdale/ (n = , . %). interestingly, outbreaks caused by recombinant ggiib-pol strains mostly associated with outbreaks among children ( . %) and had non-winter seasonality. conclusions: epidemic spread of emerging multiple recombinant norovirus strain ggiib-pol were detected in hungary that became the second most common norovirus variants -next to the endemic ggii- /lordsdale virus -causing epidemics of gastroenteritis in the last . years. the respiratory infections are the most common diseases in the world being the origin of a great morbidity and mortality especially in infants and elderly. ( ) human metapneumovirus (hmpv) was first described in dutch children with acute respiratory tract infections (artis) in june . ( ) very limited studies data are available from tropical and developing countries. we sought to determine the role of hmpv in upper and lower respiratory tract infections in cuban patients and correlated the presence of virus with clinical characteristics of the disease. between october to september clinical samples received from the national surveillance program of artis at the national reference laboratory of respiratory viruses, for virological study, were used to detect hmpv by rt-nested pcr, amplifying a conserved fragment of nucleotides in the polymerase gene. we found rna hmpv in . % of samples from the patients with artis. . % of individuals who tested positive for hmpv were under months of age. patients with evidence of hmpv had symptoms consistent with either upper or lower respiratory tract disease or both. . % of hmpv positive individuals were detected during august-october (table ). the results of this preliminary study shows that hmpv is present among cuban patients with arti. constitute the first report of the frequency of hmpv infection in a non-preselected group of cuban patients with ages ranged from months to years old. it should be noted that this is the first report of hmpv infection in central america and in the caribbean region, further confirming the worldwide distribution of the virus ( ) ( ) ( ) . detection of human metapneumovirus in paediatric nasopharyngeal aspirates by a taqman minor groove binder probe assay: a one-year prospective study in belgium w. verstrepen, p. bruynseels, a. de smet, a. mertens (antwerp, be) objective: human metapneumovirus (hmpv) has a relative high incidence in acute respiratory infections in children but is difficult to isolate in culture. the aim of the study was to decrease the number of undiagnosed viral respiratory infections in our hospital by means of a taqman minor groove binder (mgb) probe assay. methods: from october to september a total of nasopharyngeal aspirates from children presenting at our paediatric facility were analysed. rna extracts from specimens negative for rsv, parainfluenzavirus and influenzavirus with an (in) direct immunofluorescence assay (ifa) were subjected to a taqman mgb probe assay in parallel with a previously published taqman assay. results: of the specimens, ( %) were positive by ifa for either rsv ( ), parainfluenzavirus ( ), influenzavirus a ( ) or influenzavirus b ( ). hmpv was detected in ( . %) of the remaining specimens subjected to the newly developed pcr. of the patients with a positive hmpv assay, / ( . %) presented with respiratory symptoms. % of the positive specimens were from children less than year as compared to only % from children older than years. viral load was highest in children less than year. a prominent seasonal variation was noted since more than half of the positive specimens occurred during the months march and april. there was no significant difference in the proportion nor viral load of positive specimens from ambulatory patients, patients admitted to a general ward or patients requiring intensive care. as compared to the published taqman assay, diagnostic sensitivity and specificity were . % and . % respectively, whereas ppv and npv were . % and . %. method comparison (nccls guideline ep- a) failed to demonstrate a significant difference between both assays when the threshold cycle (ct) was between and . strongly positive specimens (ct < ) were associated with a lower ct using the published taqman assay. however, the new taqman mgb probe assay appeared to be more sensitive for weakly positive specimens (ct > ). conclusion: the number of viral respiratory infections confirmed in our hospital was substantially increased by means of the hmpv taqman mgb probe assay. the new assay is a reliable alternative to the previously published taqman assay for detection of hmpv in nasopharyngeal aspirates. nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of respiratory syncytial virus in paediatric respiratory specimens r. manji, f. zhang, c. ginocchio (lake success, us) background: respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infection in infants and young children, with bronchiolitis and pneumonia being the major clinical manifestations. the rapid diagnosis of rsv infections is of central importance for individual patient management (rational use of antibiotics and antiviral agents), hospital infection control and monitoring epidemiological disease patterns. this study included a technical validation and a retrospective clinical evaluation of a real time nasba assay for the detection of rsv a and rsv b in paediatric respiratory samples. methods: samples tested included: dilution panels of in vitro transcribed rna, local rsv isolates, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from children (age range: d to yr) who were evaluated in the paediatric emergency department for respiratory disease. nucleic acid (na) isolation, amplification and detection were performed using the nuclisens easyq basic kit and nuclisens easyq rsv a+b reagents (biomérieux). specimen nas and a rsv specific internal rna control (ic) were co-extracted using nuclisens magnetic extraction reagents and the nuclisens minimag instrument (biomérieux) and co-amplified using a single rsv specific primer pair. included in the reaction were a rsv specific molecular beacon ( '-fam) and an ic specific molecular beacon ( '-rox). target amplification and continuous monitoring of emitted fluorescence were performed using a nuclisens easyq analyzer (biomérieux). results were compared to direct immunofluorescence (dfa) and/or viral culture using r-mix cells (diagnostic hybrids, oh). results: the limit of detection for rsv was rna copies/rxn and the % detection rate was copies/rxn. the assay was % specific for rsv with no cross reactivity to other respiratory pathogens. the nasba assay detected % more positive specimens than dfa and % more positive samples than vc. the npvs of the assays were: nasba . %, dfa . % and vc . %. the nuclisens easyq rsv assay demonstrated superior sensitivity to both dfa and viral culture for the detection of rsv a and b from respiratory specimens. the assay was easy to use, required minimal hands on time ( hr) and a faster time to results as compared to rapid culture ( hr vs. - hr). respiratory syncytial virus (rsv) is a major cause of acute lower respiratory tract infection in infants and young children. it has previously been shown that hrsv isolates can be divided into two antigens groups a and b. the g protein is the most divergent both between and within the two subgroups and appears to accumulate amino acid changes with time, suggesting evolution under selective pressure. our knowledge of the molecular epidemiology of rsv has so far been based mainly on studies done in the developed world with e temperate climate. very limited epidemiological data are available from tropical and developing countries, where rsv infections may follow a different pattern. in this report we examine the molecular epidemiology and evolutionary pattern of the g protein of both subgroups a and b rsv through consecutive epidemics in cuba. sixthly four nasopharyngeal swabs were collected from children under years of age with respiratory disease to different hospitals in cuba between and , to examine the molecular epidemiology and evolutionary pattern of the g protein of rsv. all samples collected from to were rsv subgroup a; however both subgroups co-circulate during . the cuban isolated from to showed a great homogeneity between them and were resemble to an ancient strain (long) with only five nucleotide differences, this also occur in and with two strain. furthermore was detected different size of g protein ( or for rsv a and for rsv b) due to change in stop codon used he genetic homogeneity of the cuban isolates ( ) ( ) ( ) and their resemble to an ancient strain such as long was an unusual finding in our country. in both subgroups was observed the predominance of strains with almost similar sequences. phylogenetic analysis for subgroup a strains showed that strains were cluster in different genotypes with virus isolated in different geographic regions. both subgroups co-circulated during and clustered whit south african strains that circulate during at the same time. point mutations in respiratory syncytial virus detected by lightcycler pcr and melting curve analysis u. germer, l. nielsen, k. boye, h. westh (copenhagen, dk) objective: the objective was to analyse rsv real-time pcrpositive isolates from clinical samples, which appeared to belong to three different groups according to melting temperature (tm) of the amplicons. the analysis was done according to genotypic and phenotypic difference and related to geographical distribution. materials and methods: nasopharyngeal aspirates were collected from children with respiratory distress in the city of copenhagen. viral rna extracted using the magnapure lc automated extraction system was amplified in a real-time rt-pcr previously described ( ) . five samples from each of the three groups with different tm's were selected for bidirectional dna sequencing using the rsv primers. sequences were analysed using chromas lite version . . results: a total of clinical samples were analysed. ( %) of the samples were positive and ( %) were negative for rsv. three distinctive groups with different tm's could be identified from the melting curve analysis. group (n = ) had a tm with a median of . °c, group (n = ) and (n = ) had lower tm's with a median tm of . °c and . °c respectively. sequence analysis of amplicons showed that the difference in tm was due to differences in genotype between the three groups. genotype and were closely related, differing only in two nucleic acids in position (c to t) and (a to t). both were silent mutations. only position is targeted by the probe. genotype and were both blasted to a complete genome sequence of respiratory syncytial virus subgroup a (genbank rsu ) with the highest identity score for genotype . genotype sequences were blasted to human respiratory syncytial virus mutant cp subgroup b (genbank af ). geographical analysis showed a higher prevalence of the mutant strain (genotype ) in the northern areas of the greater copenhagen area compared to central, southern and western areas (p = . ). conclusion: we found three genotypes of rsv according to the tm of the pcr product. two of the genotypes were closely related with only two point mutations and the same phenotype. genotype was mainly found in clinical isolates from the northern part of copenhagen, suggesting a local epidemic spread. objectives: biomerieux is developing a real-time nasba assay to detect influenza a and b rna in different kind of respiratory clinical samples, by using the nuclisens Ò easyq basic kit in combination with specific primers and molecular beacons. methods: nasal/throat swabs in transport medium from hospitalised children ( - yrs from edouard herriot hospital, lyon, france) were used for this evaluation. influenza rna is isolated using the nuclisens Ò minimag extraction. an internal control is added to the sample prior to nucleic acid extraction. the assay is designed to detect in a single tube, using a three-label approach, the internal control and both influenza a and influenza b rna. amplification reactions were performed in a nuclisens Ò easyq analyser allowing real-time detection. the results of the clinical samples were compared to cell culture results. results: among swabs tested, real-time nasba detected ( . %) samples for influenza a and ( . %) samples for influenza b. comparatively, by cell culture only ( . %) samples were identified as influenza a and non as influenza b. interestingly, influenza a positive sample identified by cell culture was found negative in real-time nasba. conclusions: the data showed that nuclisens Ò easyq influenza a/b assay detected % more influenza a virus than cell culture method. moreover, real-time nasba detected influenza positive samples, which were not detected by cell culture. with this assay a qualitative detection of influenza a and influenza b viruses in a single reaction can be done within hours. it provides a valuable alternative to cell culture method for the clinical management of patients with influenza infections. results: patients have developed mumps meningitis and patients were diagnosed with mumps meningoencephalitis. age limits were from to years and sex ratio m/f was / .clinical manifestations involved fever ( %), stiff neck ( %), nausea and vomiting ( %), headaches ( %), photophobia ( %) and neurological manifestations such as: equilibrium disorders and drowsiness ( %), convulsions ( . %), cerebellum syndrome ( . %). meningeal symptoms have occurred shortly after parotiditis in % of cases and before parotiditis in % of cases; the other cases have evolved without parotid swelling. other localizations of the mumps infection were: parotiditis ( %), pancreatitis ( %), submaxillitis ( %) and orchitis ( %). lumbar puncture yields csf containing between and wbc/mm . the predominating cells were usually lymphocytes, but % of the patients have polymorphonuclear leukocyte predominance at the first puncture. protein levels are normal to mildly elevated in all cases and hypoglycorrachia was founded in % of the patients. therapy for mumps meningitis was symptom-based (analgesics and antipyretics) in % of cases and glucocorticoid therapy in % of cases. conclusions: ( ) neurological involvement in mumps occurred in . % of cases; ( ) men are afflicted two times more often as women, but the age distribution is the same as for uncomplicated mumps; ( ) mumps meningitis was the only localization of the mumps infection in % of cases. mumps is acute generalized infection occurs primarily in schoolaged children and adolescents. most prominent manifestation of mumps is swelling and tenderness of salivary glands especially parotid gland. uveitis is a rare manifestation of mumps. here we present a mumps case complicating with uveitis. years old paediatric nurse was admitted to our emergency department because of headache and malaise. on physical examination bilateral parotid enlargement was noticed. opthalmology consulatation revealed anterior uveitis. local prednisolon and cyclopentholate treatment were prescribed. lumbar puncture revealed lymphocytic pleocytosis without hypoglychorachea and elevated protein levels. mumps igm was found positive. differential diagnosis made with other viral infections and sarcoidosis. her headache diminished day after the hospitalisation. uveitis responded very well to local therapy and patient got well in weeks. clinical and epidemiological aspects of a measles epidemic, bucharest, romania results: there were cases; sex ratio m/f: / . the mainly affected age group is under months ( . %) followed by months- years ( . %), - years ( . %), > years ( . %) and - years ( . %). . % cases were hospitalacquired (mostly in paediatric clinics), . % were communityacquired; in . % cases the source was unknown. the most common clinical features were fever ( %), rash ( %), conjunctival hiperemia ( . %), cough ( . %), micropoliadenopatia ( . %), diarrhoea ( . %). pulmonary complications were described in . % of cases; . % of them were bacterial pneumonia, . % were viral pneumonia. in . % of cases we diagnosed acute stomatitis, in . % bacterial conjunctivitis; in . % of cases -otitis; in . % of cases -pharingitis, and in one case ( . %) -urinary tract infection. . % of the patients were previously diagnosed and treated for pulmonary tb. all cases were confirmed serologically through detection of specific igm antibodies. patients ( . %) had severe clinical forms of measels. the evolution was good in all cases. conclusion: . this year in the south-east part of the country, evolves a measels epidemic with different features comparing to the previous one ( ) ( ) we investigated the recombinant proteins np and hn to develop new antigen with useful properties for applied in elisa test systems. methods: significant antigenic epitopes of nucleoprotein (np) and haemagglutinin (hn) measles virus strain edmonston were generated by computer analysis. using standard geneengineering techniques was evaluated two fusion peptides np and hn consist from only linear t-cell antigenic determinants. the virus-neutralization activity of hyperimmune serum on recombinant proteins was determined by plaque reduction neutralization test (prn). the level of specific igg in serum to genotypes a, d , and d of measles virus was determined by enzyme-linked immunosorbent assay (elisa). we used recombinant proteins np and hn as antigens for elisa. results: hyperimmune serum was collected from mice after immunization by np and hn recombinant proteins. the level of neutralize activity was measured in the prn assay with strain edmonston. the titre reached up to : . and : . for np and hn recombinant proteins, respectively. interestingly that, hyperimmune serum on recombinant protein np in elisa reacted both with np (titre : ) and with hn (titre : ), and in turn serum on recombinant protein hn reacted only with hn (titre : ). the estimation immunological properties of proteins with use of the panel of serum ( samples) collected from patients. the diagnosis of measles infection was confirmed in laboratory (by rt-pcr). the nucleotide sequences of rt-pcr products used for genotyping of mv. selective interaction of antibodies in elisa with recombinant proteins in relation to various genotypes is revealed. the interaction with genotypes a and d was expressed with high level of correlation whereas with genotype d any serum did not react authentically (as the control was used recombinant protein n of sars virus). conclusion: we have shown that neutralize antibodies formed hot only on superficial proteins such as hn, f and sh but also on core proteins such as np. our data demonstrate that the recombinant proteins np and hn could be a cost-effective alternative to current whole virus based elisas for surveillance for immunity to measles and could more efficient in detecting susceptibility to measles in relation to genotypes a and d . episode : a pregnant woman with thirty-eight week of gestation was hospitalized in obstetrics clinic with the complaints of fever, malaise, and severe vaginal bleeding. on admission, white blood cell count was /mm , haemoglobin was . g/l, platelet count was /mm . the level of ast was iu, alt iu, lactate dehydrogenase iu, and creatinin phosphokinase iu. the baby was delivered by cesarean section. in serum cchf igm was positive by elisa, and per oral ribavirin was administered after delivery. at the first day of delivery, the clinical and laboratory of findings of the baby were found to be normal. however, on his th day, he died because of massive bleeding. his cchf igm was found to be negative. episode : a pregnant woman with week of gestation was admitted to the hospital. her complaints were fever, malaise, headache, myalgia, nausea, vomiting, diarrhoea, and subconjuntival bleeding. in her laboratory investigation, white blood cell count was /mm , haemoglobin level was . g/l and platelet count was /mm . the level of ast was iu, alt iu, and ldh iu. in her serological analysis cchf igm and cchf virus -pcr was found to be positive. at the twenty six week of gestation in obstetric ultrasound, fetal intraabdominal fluid was visualized and amniocentesis was performed. in serological analysis of amniotic fluid cchfv-pcr was found to be negative. intraabdominal fluid had increased and scrotal edema was visualized at thirty eighth weeks of the gestation. after her vaginal delivery, baby was severely ill and was operated with the diagnosis of necrotizing enterocolitis. his laboratory findings were normal except high white blood cell count. on his fifth day, thrombocytopenia occurred and he died because of massive bleeding. his cchf igm and pcr were negative. conclusion: to our knowledge, these are the first episodes of intrauterine cchf infection. these episodes show that cchfv can transmit through placenta. obstetricians in endemic countries should consider cchf infection among the patients with massive bleeding and thrombocytopenia. objective: to detect the asymptomatic crimean congo hemorrhagic fever virus (cchfv) infections in an endemic area, and calculate the attack and the infection rate. methods: the study was performed in a cchf endemic region. the household members of the index cases were screened for cchfv igg and igm by elisa. the data related to risk exposure were obtained by a structured form. results: eleven index cases were admitted to the clinic, household members of these cases were screened. all the index patients had positive igm or pcr for cchfv. among the household members, three individuals had igg positivity (%), and only one patient had igm positivity. none of the screened individuals had symptoms. the mean age was (sd ), and % of the subjects were female. tick bite was detected a risk factor (p = . ) for cchv infection, whereas patient care and contact with body fluids of the patients were not (p > . ). eighteen patients had the history of tick bite, and became infected ( %), and five ( %) became ill. among the infected eight individuals, five became ill ( %). conclusion: although we consider that some of the patients do not notice tick bite, we can still suggest that the infection rate of the virus is rather high compared to similar diseases. tick bite is the major risk factor, in comparison to exposure to blood and body fluids of the infected cases. results: children were included in our study. distribution according sex was: . % female and . % male. median of age was years (iqr = ). during the follow-up study we recorded years when the number of cases increased. the distribution of cases among the study was: . % in , . % in , . % in , . % in , . % in and . % in . the proportion of paediatric patients also varied from; . % in , . % in , . % in , . % in , . % in and . % in . in panama city we recorded . % of the infants. we detected an increase in the number of patients in the rain season, from may till november. the mean of days between the onset of symptoms and the first blood sample was . days (ds: . ) a second sample was obtained in . % of our infants with an average time of . days (ds: . ). the frequency of classical symptoms related to dengue virus infection was: fever ( . %), severe headache ( . %), chill ( . %) rash ( . %), myalgia ( . %), retro-orbital pain ( . %), arthralgia ( . %), gastrointestinal symptoms ( . %), inflamed pharynx ( . %), cough ( . %), mild respiratory symptoms ( . %) and diarrhoea ( . %). in our infants the symptoms which were detected first were; fever, severe headache, chill, myalgia, retroorbital pain, arthralgia, mild respiratory symptoms, cough and inflamed pharynx. we did not observed differences on clinical features between girls and boys. however, we detected detected significative differences among symptoms when we compared infants who were £ years old with those who were older (p < . ). four of our patients died because of dengue hemorrhagic fever. conclusion: dengue is endemic in panama as in most tropical countries and is one of the world s major emerging infectious disease. more data about this illness are needed to elaborate sanitary programmes which contribute to control this infection. diagnosis of dengue infection by enzyme-linked immunosorbent assay and reverse transcriptionpolymerase chain reaction from oral specimens dengue. salivary elisa has been shown by various investigators to be useful for dengue diagnosis. we sought to perform a pilot evaluation of diagnostic value of elisa and pcr of oral brushes and saliva for dengue diagnosis in adults. methods: adults with acute fever and suspected of dengue infection admitted to our university hospital were enrolled. dengue diagnosis was made by standard elisa using serum or plasma. patients with negative elisa served as controls. buccal mucosal cells were collected for rt-pcr and saliva for both rt-pcr and elisa at least twice, - days apart. our elisa criteria for saliva were single igm > units or single igg > units or -fold increase in igg titre with the second titre > units for secondary dengue infection. criteria for primary dengue infection were the same as secondary infection plus igm:igg ratio of over . . results: cases and controls were enrolled. our country is endemic for dengue and thus there was no primary dengue adult case in this study. as the study was performed in hospitalized patients, most of the first samples were collected one day before or on the day of defervescence. the specificities of either methods and the sensitivity of elisa method for saliva were %. sensitivities were approximately - % for rt-pcr using buccal cells or saliva specimens. however, a combination of rt-pcr results for both types of oral specimens gave a sensitivity of %. the results are summarised in the table. conclusions: collection of oral specimens is less invasive and may be more acceptable in certain situations. a single, acute specimen is adequate for diagnosis by rt-pcr. our specimens, however, were collected late in the course of illness which affected the sensitivity of rt-pcr's. earlier specimens may give a better yield. a study in paediatric patients is needed to assess the value of these methods for primary dengue infection. objective: the aim of this study was to assess the proportion of seropositives against hantaviruses among healthy blood donors. methods: volunteer donors were recruited by the institute of transfusion medicine, representing the demographic situation in the tyrol regarding gender and residence. sera were tested for igg with a commercially available elisa. positive samples were confirmed by a commercially available dot blot which was also used for identification of the serovar. setting: the study area comprises north tyrol (austria, north of the main ridge of the alps), south tyrol (italy) and east tyrol (austria, both south of the main ridge of the alps). south tyrol belongs to the catchment area of the etsch river, which drains into the adriatic, while north-and east tyrol are part of the catchment area of the danube, which drains to the black sea. results: none of samples from the italian part of the study area yielded a positive result, wherein of donors of the austrian part turned out to be seropositive. two patients were positive for hantaan, patients were positive for puumala, one patient was positive for dobrava and one patient had antibodies against hantaan and dobrava. only one of those patients reported extensive travelling abroad. conclusions: evidence was found for the occurrence of hantaviruses in the austrian part of the region covering the catchment area of the danube, but not in the italian part of the study area covering the catchment area of the etsch river. seropositivity to hantaviruses differs by hydrogeographic areas. objectives: canine coronavirus (ccov) is an enveloped, singlestranded rna virus, belonging to group i coronaviruses within the family coronaviridae. two different ccov genotypes have been recognised, that are designated ccovs type i and type ii on the basis of their genetic relatedness to feline coronaviruses (fcovs) type i and type ii, respectively. ccov is usually responsible for mild, self-limiting infections restricted to the enteric tract. we report the molecular characterisation of a pantropic variant of ccov that caused fatal disease in pups. methods: ccov type ii strain cb/ was isolated from an outbreak of fatal disease affecting seven dogs housed in a pet shop in apulia region, italy and characterised by fever, lethargy, inappetance, vomiting, haemorrhagic diarrhoea, neurological signs, and severe lesions in the parenchymatous organs. in all tissues, ccov antigen was detected by immunoistochemistry and ccov type ii rna was identified by genotype-specific realtime rt-pcr. the ' end of the genome of strain cb/ was determined by amplification of seven partially overlapping fragments. the pcr-amplified products were subjected to direct sequencing and the obtained nucleotide (nt) sequences were assembled and analysed using the bioedit software package and the ncbi's and embl's analysis tools. genbank accession number dq was assigned to the sequenced . -kb fragment. the inferred amino acid sequences (aa) were compared to the analogous proteins available in the online databases. results: the structural proteins s, e, m, n of strain cb/ displayed a high degree of aa identity to the cognate orfs of ccov type ii, although the s protein showed the highest identity to type ii fcovs. while the nonstructural protein (nsp) a had the same length of known ccovs, the nsp b was -aa shorter than expected due to the presence of a -nt deletion at position and to a frame shift in the sequence downstream the deletion that introduced an early stop codon. conclusions: association of strain cb/ to a severe, fatal disease of dogs, together with virus isolation from organs with remarkable lesions, strongly suggests that this virus has changed the tropism, acquiring the ability to spread from the enteric tract to the internal organs. by sequence analysis of the viral genome, the only striking change was the truncated form of nsp b, but the role of the deletion in the orf b in determining the patho-biological change deserves more in-depth investigation. objectives: to perform a surveillance study for sars coronavirus (sars-cov)-like virus in non-caged wild animals from the wild of hong kong special administrative region (hksar). methods: from summer to spring , bats, rodents and monkeys from locations in hksar were captured. nasopharyngeal and anal swabs and blood samples were collected and tested for sars-cov-like virus rna by rt-pcr using conserved primers targeted to a -bp fragment of the rna-dependent rna polymerase (pol) gene. the complete genome of the sars-cov-like virus from bats (bat-sars-cov) was sequenced using rna extracted from three anal swabs of three bats as template. phylogenetic tree construction was performed using neighbor-joining method with growtree using jukes-cantor correction. prediction of signal peptides and cleavage sites was performed using signalp, transmembrane domains using tmpred and tmhmm, potential n-glycosylation sites using scanprosite and protein family analysis using pfam and interproscan. antibodies were detected using a recombinant bat-sars-cov nucleocapsid protein enzyme immunoassay and neutralization assay for human sars-cov. results: we identified a coronavirus closely related to sars-cov (bat-sars-cov) from ( %) of anal swabs of wild chinese horseshoe bats by rt-pcr. sequencing and analysis of three bat-sars-cov genomes from samples collected at different dates showed that bat-sars-cov is closely related to sars-cov from humans and civets. phylogenetic analysis showed that bat-sars-cov formed a distinct cluster with sars-cov as group b coronaviruses, distantly related to known group coronaviruses. most differences between the bat-sars-cov and sars-cov genomes were observed in the spike gene, orf and orf , which are the regions where most variations were also observed between human and civet sars-cov genomes. in addition, the presence of a -bp insertion in orf of bat-sars-cov genome, not in most human sars-cov genomes, suggests that it has a common ancestor with civet sars-cov. antibody against recombinant bat-sars-cov nucleocapsid protein was detected in % of chinese horseshoe bats using an enzyme immunoassay. neutralizing antibody to human sars-cov was also detected in those with lower viral loads. conclusion: our data support the existence of sars-cov-like virus in chinese horseshoe bats in hksar. noroviruses are genetically heterogeneous and form at least genotypes within genogroups, gi, gii, giii, giv, and gv, based on the capsid genes. human novs cause an estimated million cases of illness annually in the united states alone and > % of nonbacterial epidemic gastroenteritis worldwide. porcine calicivirus have been found to be genetically similar to human gii novs or to sapoviruses but calicivirus rna has been detected at low frequency by rt-pcr in adults or fattening pigs. the close genetic relationships between human and porcine novs raise public health concerns regarding their potential for zoonotic transmission and as a potential source of new epidemic human strains. methods: a total of faecal samples of nursing and weaning piglets with enteritis were collected during - in porcine herds in italy. an additional samples were include in the analysis, that had been collected during a rotavirus (rv) surveillance study in - , all which tested positive to rv by electron microscopy and by rt-pcr. viral rna was extracted by the guanidine thiocyanate/glass milk method to eliminate enzyme inhibitors. primer pair con -con , targeted to the vp outer capsid protein, was used for rv detection. a degenerated version of primer pair / was used for nv detection, that targets a conserved region in the rnapolymerase. results: nov rna was detected in / of the screened samples, while rvs were detected in / samples. mixed infections nov+rv were found in samples. screening of the rv positive samples allowed detection of mixed infections with novs. conclusions: in previous investigations novs were detected in of , normal slaughtered pigs in japan, in of pooled pig faecal samples of -to -month-old fattening pigs in the netherlands and in out of healthy adult and finisher pigs in the united states. interestingly, in this study a high rate of positivity to novs ( / ) was found in nursing and weaning piglets with diarrhoea, a finding that may suggest a higher frequency of infection by nov in young pigs or an association between nov infection and occurrence of enteric disease. altogether, these findings demonstrate that novs are common in porcine herds in italy and provide new insights into the ecology of novs. detection of calicivirus genome in calves using ni/e primers m. mahzounieh, t. zahraeisalehi, e. moghtadaei khorasgani (shahrekord, tehran, ir) caliciviruses may cause a wide spectrum of disease in animals and are important etiological agent of viral gastroenteritis in humans. members of the family caliciviridae are small nonenveloped viruses to nm in diameter. they possess a single stranded poly adenylated rna genome. caliciviruses have been isolated from mink, dog, cattle and non-human primates. "norwalk-like viruses" (nlvs) are the most common cause of acute non-bacterial gastroenteritis in humans. cattle may be a reservoir of nlvs although never bovine nlvs have been found in humans. in this study, we try to detect enteric caliciviruses genome from faecal samples of dairy cattle herds in shahrekord area using reverse transcriptase polymerase chain reaction (rt-pcr) assays specific for nlvs found in humans. the primers used for pcr amplification were ni and e , which amplify a -bp product for the detection of both genogroups i and ii srsv rna in fecal material. our results showed that nine specimens ( %) were positive. these findings suggest that calicivirus infection is endemic in dairy herds in shahrekord, iran and may be have an important role in calf diarrhea. objectives: reoviruses are non-enveloped, -segmented dsrna viruses. in humans and mammalians three distinct serotypes exist, whose prototypes are strains lang (t ), jones (t ) and dearing (t ). although reoviruses have been isolated both from the enteric and respiratory tract, no diseases has been clearly associated to reovirus infection in humans. the potential association with extra-hepatic biliary atresia, myocarditis, and, above all, neurological and cutaneous diseases require further investigations. reoviruses are ubiquitous and scarcely speciesspecific. reovirus identification is usually based on electronic microscopy or gel electrophoresis and reovirus incidence seems to be very low in humans and most mammalians. in this study, we investigated the presence of reoviruses in dogs by means of molecular methods. methods: one hundred ninety-two rectal samples from dogs with diarrhoea, ocular swabs, nasal swabs and oropharyngeal swabs from dogs with ocular/nasal discharge were subjected to an rt-pcr assay targeting a conserved region of viral genome segment l (primers l -rv /l -rv ). positive samples were characterised by polyacrylamide gel electrophoresis (page), serotype-specific rt-pcr assays targeting segment s and sequence analysis. to increase the sensitivity, a nested pcr using primers l -rv /l -rv was performed on samples tested rt-pcr negative. results: only faecal swabs ( . %) were found positive (rt-pcr product of bp). by using a serotype-specific rt-pcr assay and/or sequence analysis, two strains was characterised as type and the other ones as type . page of viral dsrna confirmed the genetic characterisation. unexpectedly, in secondround pcr faecal samples ( %), ocular swabs and nasal swabs yielded a bp product, while no oropharyngeal swab was positive. conclusions: these data suggest a wider distribution of reoviruses in dogs than previously thought, even if most reovirus infections were detected only by nested pcr. the ability of reoviruses to induce disease in dogs, alone or in synergism with other pathogens, is still unclear, since attempts to reproduce a specific disease in germ-free dogs have given contradictory results. due to their poor species-specificity, reoviruses may be easily transmitted from animals to humans (and vice-versa). further studies are required to understand reovirus ecology and their potential zoonotic impact. objectives: parvovirus b is a member of a family parvoviridae. on the basis of genetic distances and evolutionary relationships, human parvoviruses are divided into three genotypes: genotype i corresponding to b -related isolates, genotype ii to lali-related isolates, and genotype iii to v -related isolates. parvovirus b causes a common exanthematous disease in childhood or adult age, arthropathy, hydrops fetalis, various haematological disorders and myocarditis. up to now, we have had no data of the prevalence of b virus in slovenian population. consequently, we also lack information on the genotypes of parvovirus b that are involved in the patients who suffer from the infection. methods: to gather information of the genetic variants of b virus present in slovenia, we extracted dna from serum samples that were sent for serologic diagnostic of parvovirus b infection and were positive for specific igm in the period from january to june . nearly half of all patients were children and young adults up to years. the ns region of parvovirus b was amplified by the nested pcr (primers pb f , r and pb f , r ). all pcr products were directly sequenced. the results of our study show that dna of parvovirus b was present in all samples that tested positive for specific igm antibodies. after the first round of pcr reaction, samples were positive, and after the second reaction, all samples were positive. altogether unique genotype variants of parvovirus b were identified and all were clustered in the genotype i group of b -related isolates. most of the distinct genetic variants differed in % to % from the sequences deposited in gene bank. the majority of sequences obtained from the b virus epidemic in represents a single variant of genotype i with the gene bank acc. no. aj . we also found that different genetic variants of parvovirus b were circulating in and were % or % identical to the genotype i variant with the gene bank acc. no. z . in our study, we were not able to identify any variants of other rare genotypes (lali or v ). conclusion: parvovirus b dna was successfully amplified from all igm positive serum samples of the patients. the genotype i of parvovirus b is dominating in infections with parvovirus b in slovenia. objectives: great britain has been free of animal brucellosis since (european commission decision / ). the main source of infection for uk residents is through contact with infected material in foreign countries. the objective of this study is to type human samples received in the uk since using variable number tandem repeats (vntr) molecular typing to confirm results obtained by classical typing and relate these results to the suspected source of infection. methods: classical typing is traditionally used and is based on the phenotypic attributes of each strain and biovar. vntr typing is a recently developed molecular method, which is based on short repeats contained in the dna that can be amplified to give a banding pattern specific to each strain. results: results found using both methods are consistent. the results show geographical differences, consistent with observations of strain genotype distribution found in animal brucellosis. conclusion: patient history has been gathered where possible giving information on recent travels. along with results found by classical typing and confirmed by vntr typing we can draw a picture of the sources of infection. these results illustrate the potential of vntr typing as a tool to aid conventional approaches to epidemiological traceback that, in the presence of a suitably comprehensive database of strain genotypes, could help identify the source of an infection. is -fingerprinting of brucella isolates from humans e.stubberfield (surrey, uk) objective: brucellosis is a zoonotic disease usually associated with cattle, sheep, goats and pigs. human infection has been attributed to b. melitensis, b. abortus, b. suis, b. canis and b. maris. although the uk is officially bucellosis free there are a number of human cases due to travel and occupation that are submitted to our laboratory for diagnosis. definitive diagnosis of brucella is by bacteriological culture and microbial tests (classical typing), however these require skilled personnel and the results can be subjective. there are a number of molecular tests that have been developed to assist with diagnosis more rapidly and in some cases to strain level less subjectively. is -fingerpinting is a molecular technique that has proved useful for the identification of brucella isolates to species and in some cases stain level. is -fingerprinting relies on the variable number and location of the is mobile genetic element found in all brucella isolates. method: brucella isolates from humans have been tested. genomic dna was extracted, digested using restriction endonuclease ecor , and electrophoresed. southern blotting was performed, hybridising with a dig-labelled is probe. results: the number of brucella is copies range from to more than . brucella melitensis remains the most commonly acquired brucella species of travellers, while occupational infections have included b. abortus isolated from cattle farmers and b. suis associated with pig butchers. two marine brucella strains have been isolated originating from an occupational perspective (a laboratory worker) and a natural setting from an unknown source. unusual patterns have been observed, of which are unique. one of the new patterns has been observed only in isolates originating in east african countries. conclusion: although the diagnosis of brucella to species and strain level is not essential for the treatment of human brucellosis, it is useful for epidemiological studies. is fingerprinting is able to identify the three biovars of b. melitensis, many other techniques do not offer this capability, because of this it may be a useful test in epidemiological studies. this method remains an important diagnostic tool for brucella identification. rapid diagnosis of brucellar epididymo-orchitis by real-time pcr assay in urine samples objectives: to study the diagnostic yield of a real-time pcr assay in urine samples for the rapid diagnosis of brucellar epididymo-orchitis, in comparison with conventional microbiological techniques. methods: ten consecutive patients with brucellar epididymoorchitis were included in the study. the diagnosis of brucellosis was established according to one of the following criteria: first, isolation of brucella spp in blood or any other body fluid or tissue sample or, second, the presence of a compatible clinical picture together with the demonstration of specific antibodies at significant titers or seroconversion. epididymo-orchitis was diagnosed in patients with scrotal enlargement, swelling and pain not due to other causes.for dna amplification we used a sybr green i lightcycler-based real-time pcr assay. the assay amplifies a bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein (bcsp ). the pair of nucleotide primers b ( ' tgg ctc ggt tgc caa tat caa ') and b ( ' cgc gct tgc ctt tca ggt ctg ) were used in the amplification process. after dna amplication, we performed melting curve analysis to verify the specificity of the pcr products. in order to study the specificity of the technique, all the samples from the patients with brucellosis were paired with an equal number of samples from controls with urinary tract infection. (e. coli four cases, k. pneumoniae two cases, p mirabilis two cases, and c. freundii and p. aeruginosa one of each). results: the mean age was . years (range - ). the duration of the symptoms prior to diagnosis was . ± . days (range: - ). b. melitensis was isolated from blood cultures in nine cases ( %). wright's seroagglutination was negative or inconclusive in % of cases. brucella was isolated from urine in only one case whereas real-time pcr assay in urine was positive in nine ( %) cases and the results were available in four hours, whereas the mean time to availability of the final blood culture results was . days (range . - days). real-time pcr was negative for all the control samples from patients with urinary tract infections. conclusion: sybr green i lightcycler-based real-time pcr assay in urine samples is highly sensitive and specific, easy to perform and could provide the clinician with the results in under five hours. the technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis. objectives: although the united kingdom remains brucellosisfree, there are more than , new cases of human brucellosis reported each year according to the world health organisation. uk residents returning from worldwide travel may have encountered exposure through contact with infected animals and animal products such as dairy produce and meat. phenotypic characterisation or classical methods remain the definitive diagnosis though require skilled personnel and have their limitations. the increasing range of molecular techniques can aid the rapid detection and characterisation of brucella species and their biovars and may have significance in epidemiological studies. methods: a study of brucella reference and field strains of mainly human isolates from different geographic locations were analysed for diversity of their genes encoding the outer membrane proteins (omps) , a and b. pcr products of the three genes digested with seven restriction enzymes were analysed for polymorphisms. results: a re-occurring unique pattern profile seen only in human isolates was observed originating in some european countries and beyond. a growing database of strain types giving a recent overview of brucella infection of humans of many countries. conclusions: molecular typing methods may have an advantage over classical typing concerning brucella melitensis, the most common brucella infection of humans. the characterisation of human brucella isolates may be useful in epidemiological studies for a variety of purposes. objectives: a study to demonstrate the rapid detection and speciation of campylobacter jejuni and of campylobacter coli isolates directly from enrichment broth using a taqman Ò assay. single nucleotide polymorphism analysis of mapa positive strains was used for rapid identification of c. jejuni clonal complexes. methods: thermotolerant campylobacter species were initially confirmed by culture according to the modified draft iso method, where water samples were filtered through . mm pore size nylon membrane. the filters were transferred to selective enrichment in preston broth to improve their recovery and therefore detection of any campylobacter cells present. dna was extracted directly from the enrichment broth culture for real-time detection of c. jejuni and c. coli using the taqman Ò . samples, which were map a positive were, further characterise by single nucleotide polymorphism profiling for rapid recognition of c. jejuni clonal complexes. results: environmental samples, which were confirmed by culture were also map a positive by taqman Ò . snp profiling of mapa positive isolates identified clonal complexes, which are predominantly contained in isolates of human disease and chicken. conclusions: this study has demonstrated the feasibility of rapid detection and identification of c. jejuni and c. coli following short enrichment incubation using a taqman Ò assay. a rapid turnaround time of between - h per batch of samples was achieved. snp profiling offers important epidemiological grouping at strain level, enabling accurate and phylogenetically valid strain identification for c. jejuni, which may have important host associations for tracing sources of infection and consequently improve public health responses. objectives: campylobacter jejuni and c. coli are recognized as the most common causes of acute bacterial gastroenteritis in humans, c. jejuni being the predominant species in most developed countries. the hippurate hydrolysis test is widely used to differentiate c. jejuni from other campylobacter species. about % of c. jejuni isolates fail to hydrolyze hippurate under laboratory conditions. molecular methods represent an alternative to the phenotype-based methods. we tested two multiplex pcr assays for species identification of human campylobacter strains and compared the results with the hippurate hydrolysis test. methods: campylobacter strains isolated from patients were tested for hippurate hydrolysis with rosco diagnostic tablets. hippurate-negative and hippurate-positive strains were selected for two multiplex pcr assays. one pcr-method was based on distinctive ceue-genes of c. jejuni and c. coli, the other pcr-method detected genes from five major clinically relevant campylobacter species: hipo from c. jejuni, glya from c. coli, c. lari and c. upsaliensis, sapb from c. fetus subsp. fetus, and s rrna gene from campylobacter spp. as an internal validation control. results: the c. jejuni hipo gene was detected in all of the hippurate-positive strains and of the hippurate-negative strains. the c. coli glya was detected in of the hippuratenegative strains. in one hippurate-negative strain, sapb from c. fetus subsp. fetus was detected. species-specific genes were detected in of the strains with the ceue-based pcr assay. c. jejuni ceue was detected in hippurate-positive and hippurate-negative strains. c. coli ceue was detected in hippurate-negative strains. conclusion: all hippurate-positive strains were identified as c. jejuni. of the hippurate-negative strains, % were identified as c. coli, whereas % were identified as c. jejuni and one strain as c. fetus subsp. fetus. the results of the two pcr assays were concordant, although some strains could not be identified with the ceue-based pcr assay. the results suggest that molecular species identification should be performed on hippuratenegative strains after the hippurate hydrolysis test for accurate species identification. multiplex-pcr is quick and easy to perform. using the pcr assay that simultaneously detects five campylobacter species also diminishes the need for further phenotypic testing. phenotypic typing of cryptosporidium species isolated from children in kuwait: a role in unique transmission j. iqbal, p. hira (safat, kw) background: cryptosporidiosis is recognized worldwide as a significant cause of diarrhoeal diseases in both adults and children especially in children less than years of age. objective: cryptosporidium spp. isolated from young children in kuwait were characterized at the molecular level to understand the transmission of infection. the study was approved by the ethical committee, faculty of medicine, kuwait. methodology: over a period of years, faecal specimens from kuwaiti children with persistent diarrhoea found to be positive for cryptosporidium spp. by microscopy were genotyped and sub-typed with a small subunit rrna-based pcr-restriction fragment length polymorphism analysis. informed consent was taken from all individuals included in the study. results: the median age of infected children was . years, and the majority of the infections (> %) occurred during the cooler months january-april, indicating a marked seasonal variation. more than % of the children with cryptosporidiosis had only cryptosporidium infection. socio-demographic information did not reveal any particular mode of transmission of infection. genotyping of the organisms isolated showed that ninety-two ( %) of the children had c. parvum, ( %) had c. hominis, and ( %) had both c. parvum and c. hominis. altogether, subtypes of c. parvum and c. hominis were observed. objectives: the intracellular respiratory pathogen chlamydophila pneumoniae (cp) might be involved in the pathogenesis of atherosclerosis. several studies have demonstrated a serological association between cp and cardiovascular disease and dna from the bacteria has been found in various atheromatous vessels. after infection in the respiratory tract, cp is believed to be disseminated systemically within alveolar macrophages. the prevalence of cp within peripheral blood mononuclear cells (pbmc) has in some studies been shown to be higher in patients suffering from cardiovascular disease than in control patients. we investigated the presence of cp dna in aortic heart valves and pmbc in patients ( men; women; mean age years) undergoing aortic valve replacement because of aortic stenosis. also, the presence of cp mrna was investigated in the sclerotic aortic heart valves as a marker of viable bacteria. methods: dna was extracted from aortic valve biopsies and pbmc using the qiaamp dna mini kit (qiagen). mrna and dna were extracted from another piece of the same biopsy using trizol (invitrogen). real-time pcr directed against the chlamydia momp gene was used to detect cp-specific dna and mrna. patient sera were tested for cp-specific igm, igg and iga antibodies by the microimmunofluorescence technique. results: cp dna was found in aortic heart valves from % ( / ) of the patients and in pbmc from % ( / ) of the patients. in one patient cp dna was found in both pbmc and heart valve. no patient had cp-specific igm antibodies. in patients that were pcr-positive for cp dna in the aortic heart valves, % had igg ‡ : and % had iga ‡ : . in patients that were pcr-negative in the aortic heart valves, % had igg ‡ : and % had iga ‡ : . cp-specific mrna in aortic heart valves will be presented on the poster. conclusion: cp-specific dna was found in sclerotic aortic heart valves from % of patients undergoing aortic valve replacement. this confirms previous investigations supporting a role for cp in the pathogenesis of aortic valve sclerosis. the prevalence of cp in pbmc was % which is comparable to that reported in healthy blood donors and lower than that recorded in patients suffering from other cardiovascular diseases. if the bacteria are involved in the pathogenesis of aortic sclerosis they have likely been spread to the aortic valve long before the patient is in need of surgery because of the stenotic valve. introduction: diarrhoea is one of the most common causes of morbidity and mortality among young children in developing countries. diarrheagenic e. coli strains include several emerging pathogens of worldwide public health. six important categories are entero-aggrigative e. coli (eaec), entropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enterohemorrahgic e. coli (ehec), entroinvasive (eiec) and shigatoxin-producing e. coli (stec). this study investigated the role of different diarrheagenic e. coli in iranian children with acute diarrhoea by molecular methods and the antibiotic susceptibility of isolated strains. methods: from april to january , one thousand eighty five children with acute diarrhoea in tehran hospitals in were enrolled in the study. the fecal samples were cultured on macconkey for conventional bacterial pathogen and sorbitol macconkey agar for non sorbitol fermenting phenotype, than they were incubated in ordm;c. the primary stool cultures were subjected to six different pcr reactions targeting stx and stx gene, heat-labile enterotoxin (lt) producing gene, heatstable enterotoxin (st) producing gene, eae gene and pcvd plasmid. the kirby -bauer disc diffusion method was used for antibiogram of isolated strains from different diarrheagenic e. coli by different antibiotics. results: two hundred seventy one diarrheagenic e. coli strains were detected. stec was the most prevalence with ( . %). the frequency of other strains was . %, . %and . % for etec, eaec and epec, respectively. out of stec isolated strains (% . ) had stx or stx gene, and strains had stx and stx gene. the eae gene was found in ( . ) stec strain. out of tested strains, ( . %) were resistance to ampicllin and cefalotin, and ( %) to streptomycin. conclusion: in this study stec was the most frequent associated with diarrhoea. the strong association between use of antibiotics and colonization with antibiotic resistant e. coli, suggest a major role for selection of resistant strains while using antibiotics. the existence of other unknown intestinal adherence factors has been suggested by the isolation of stec strains that lack the eae gene but are still associated with bloody diarrhoea or hemolytic ureamic syndrome (hus). since there is no specific treatment, there is an urgent need for effective preventive measures based on detailed understanding of the epidemiology of stec infections. identification of shiga toxin-producing escherichia coli in raw beef using dna hybridization with digoxigenin-labelled probes and multiplex pcrs m. weiner, j. osek (pulawy, pl) shiga toxin-producing escherichia coli (stec) is an important cause of bloody diarrhoea, haemorrhagic colitis, haemolytic uremic syndrome and thrombotic thrombocytopenic purpura. transmission of stec occurs through consumption of contaminated food, especially meat, dairy products and water. objectives: to develop a three-steps procedure based on two multiplex pcrs and dna hybridization with digoxigeninlabelled probes for identification of stec in raw beef. methods: beef samples inoculated with different number of e. coli o :h cells were incubated in tsb medium at °c for h. the cultures were then transferred to tsb with mitomycin c and incubated for another h. the resulted cultures were used as a source of dna template. the mpcr- was established to identify shiga toxins genes (conserved sequence). the positive culture samples were subjected to dna hybridization with dig-labelled probes as follows: the culture was diluted and inoculated onto agar plates supplemented with tergitol Ò and incubated at °c for h. then, the nylon membranes were put on agar plates, carefully removed and incubated in denaturation, neutralisation and equilibration solutions following incubation with the stx-specific dig-labelled probes, anti-dig conjugates and finally developed with enzyme substrates (bcip and nbt). dark spots visible on the membranes were compared with the respective bacterial colonies on the original agar plates. the corresponding bacterial colonies were isolated and characterized using the mpcr- test which allows amplification of stx (shiga toxin type ), stx (shiga toxin type ), rfbo (e. coli o ) and flich gene (h antigen). an internal control of amplification (e. coli s rrna gene) was also included in both mpcr tests. results: the first mpcr resulted in two amplification products: bp for stx and bp for s rrna genes. the positive meat samples were further tested with dna probes and positive colonies were then characterized with the second test (mpcr- ), generating the amplicons either of bp (stx ), bp (stx ), bp (rfbo ), bp (flich ) or bp ( s rrna). the specificity of this procedure was confirmed by testing e. coli o :h , o :h-and non-stec bacteria. the sensitivity of the method was estimated as cfu/g of meat. conclusion: the obtained results demonstrated the high specificity of the procedure developed and the possibility of using it for identification of shiga toxin-producing e. coli in raw beef. correlation between virulence pattern, phylogenetic group and extended spectrum betalactamases genes in escherichia coli strains isolated from blood cultures m. damian, c. usein, d. tatu-chitoiu, s. ciontea, d. jardan, a. palade (bucharest, ro) e. coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of human diseases, including extra intestinal and enteric infections.the strains isolated from invasive infections were documented to be carriers of a large number of genetic structures coding for virulence, as well as for resistance to antimicrobial agents. the aim of this study was to evaluate the virulence of strains in comparison with the presence of esbl genes and their distribution among the different phylogenetic groups. a total of e. coli strains, isolated from blood cultures, in hospitalised patients, adults and children, were screened for virulence factors-encoding genes (pap, sfa/foc, afa, hly, cnf, aer and fimh), for genes encoding resistance to extended spectrum betalactam antibiotics (bla shv and bla tem genes) and the appurtenance to one of the main four phylogenetic group based on presence or absence of markers chua, yjaa and tspe .c three strains, negative for all virulence genes, were included in the phylogenetic group a. ten strains, which were positive for five or six virulence genes, were identified as b group. no matter the phylogenetic grouping, the remaining strains possessed at least one virulence gene. no strain was pcr positive for all seven virulence genes targeted. among the strains which were positive in the double disk test, strains exhibited both bla shv and bla tem genes and strains only bla tem gene. restriction with pst i and dde i and sequencing of the amplicons were performed in order to identify the type of esbl gene expression product. taking into account the link between phylogenetic group and virulence, we obtained a good correlation for the bacteremic e. coli strains analysed, but there was no relationship with the production of esbls. isolation of shiga-toxin producing escherichia coli from meat samples, phenotypic and genotypic characterisation of isolated strains f. baghbani-arani, f. jafari, m.r. zali, s. salmanzadeh-ahrabi (tehran, ir) objective: shiga-toxin producing escherichia coli (stec) is an emerging foodborne pathogen of worldwide public health importance. this bacterium has been reported as an etiological agent of many outbreaks and sporadic cases. definition of the diversity and antimicrobial susceptibility of (stec) may be helpful in the management of sporadic cases and outbreaks. studies in different countries show that food items maybe contaminated by this pathogen. the present study was carried out to determine the frequency of contamination of meat samples by stec collected in tehran as well as defining genotypes, serotypes, antibiogram susceptibility patterns and molecular diversity of isolated bacteria. methods: from july to june , beef samples were collected from different part of tehran. a grams of each samples was enriched in ec broth and subculture on mac-conkey agar. dna was extracted from a loop full of bacteria taken from primary first streaking area of mac-conkey agar and was subjected to three different pcr reactions targeting stx , stx and eae genes. as much as colonies required were tested for finding the colony responsible for positive results in the first pcr. antibiogram susceptibility patterns of isolated strains were determined by standard disk diffusing method. the antimicrobial agents were used at this study. all isolates were serotyped by slide agglutination test using standard antisera (mast groups) subtyping of strains was done with rapd-pcr by primer. results: among samples, ( %) samples were positive and their genotypes were as follow: ( . %) stx +, stx -, eae-, ( %) stx -, stx +, eae-, ( . %) stx -, stx + and eae+. ( . %) stx +, stx +, eae-, ( . %) stx +, stx +, eae+. among these positive samples strains were isolated. according to the antibiotic susceptibility tests, all isolates were resistance to erythromycin (e) and oleandomycin (ol), and were sensitive to imipenem (i); gentamicin (g) norofloxazin (nx) enterofloxazin (ex) ciprofloxazin (cf) and ceftazidim (ca). in otyping and htyping the most frequency were o ac and h serotypes. analysis of isolates by rapd-pcr yielded different patterns. conclusion: our results show that contamination of meat samples by stec is a life-threatening health problem. combinational analysis of antibiogram susceptibility patterns and serotypes with rapd-pcr patterns can aid to survey the characteristics of stec strains. factors affecting the conjugative transfer of plasmid pip in enterococcus faecalis a.m. al-qurashi (dammam, sa) objectives: factors which are known to influence plasmid transfer were studies using the conjugative plasmid pi , which encodes erythromycin resistance, in enterococcus faecalis. methods: the donors strains streptococcus a agalactiae v (group b) is resistant to in rifampicin and fusidic acid, non hemolytic and b-lactamase-negative. it contains the broad host range plasmid pi , which confers resistance to erythromycin and chloramphenicol. the recipient is enterococcus faecalis strain jh - group d. results: transfer of pip occured on a agar, on filters and in broth cultures at relatively high densities ( - bacteria/ml). transfer frequency was largely unaffected over a wide range of temperatures ( - °c). the ph of the medium, in the range ph - had little effect on the transfer frequency. log phase cultures and donor: recipient ratios of : - : were required for optimal for plasmid transfer. conclusion: factors which modified the transfer efficiency of the conjugative plasmid pip were mating media, solid or liquid environment, mating time, mating temperature, selection temperature, growth temperature of donor and recipient of prior to mating, ph culture age, and donor/recipient cells ratio, to obtain a better understanding of this plasmid and its transfer process will help understand what role they may have in the dissemination resistance among streptococcal and enterococcal populations. enterococcus faecium blood-culture isolates collected during a five-year period h. billströ m, Å . sullivan, b. lund (stockholm, se) background: enterococcus faecalis and enterococcus faecium have during the last years become a significant nosocomial problem. this could be due to the enterococcus hardy nature combined with intrinsic and acquired antibiotic resistance. since most individuals harbour enterococci in their normal intestinal microflora there has been a discussion regarding the origin of these isolates. during the last ten years the isolation ratio between e. faecalis and e. faecium have shifted from : to : . this could be because of increasing antibiotic resistance among infectious e. faecium isolates compared to infectious e. faecalis ones. it is possible that this increase also depends upon different virulence genes such as enterococcal surface protein (esp), hyaluronidase variant gene (hylefm) and e. faecalis antigen a variant (efafm). objectives: the objectives in this study were to determine the presence and frequencies of seven different enterococcal virulence genes in infectious isolates. further objectives were to see if the number of virulence genes in these isolates vary or increase over time. methods: a total of strains isolated from bacteraemia patients during year - at the karolinska university hospital, huddinge were used. all isolates were screened for seven different virulence genes using a multiplex pcr. these seven virulence genes were aggregation substance (asa), cytolysin (cyt), collagen binding protein (ace), e. faecalis antigen a variant (efafm), enterococcal surface protein (esp), gelatinase (gel) and hyaluronidase (hyl). results: according to the results about half of all isolates were esp-positive. the prevalence of the other virulence genes asa, efafm, gel and hyl were detected, but in low frequencies (< %). conclusion: it seems like the esp gene is the most dominant virulence gene in e. faecium isolates. the occurrence of virulence traits in these isolates further indicates that the potential to cause infection is potentiated among this enterococcal population the data from this investigation supports the hypotheses that enterococci causing infection in hospitalized patients are probably of nosocomial origin rather than endogenous. objectives: the ability of l. monocytogenes to tolerate alkaline stress is of particular importance, as this pathogen is often exposed to such stress in food processing environments cleaned with alkaline detergents or in the mildly alkaline ph values which prevail within engulfing phagolysosomes. this study aims to investigate the alkaline tolerance response (altr) in listeria monocytogenes s using dna microarray technology. knowledge of the alkaline-induced stress response will be useful in understanding how this pathogen tolerates alkaline stress. methods: transcription profiling of l. monocytogenes s was carried out at , and min at high ph in order to capture an early, an intermediate and a prolonged expression response to alkaline stress using oligo arrays from the pathogen functional genomic resource centre. to verify the microarray results the regulation of some ph stress response genes were confirmed by real time quantitative polymerase chain reaction (rt-pcr). results: about genes were upregulated and genes (of open reading frames represented on the arrays) were down regulated at least . fold upon alkaline shock. many of the repressed genes encode enzymes that are involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating a metabolic adjustment of the cells to the high ph. notably, the strongest alkaline-inducible genes were involved in the membrane transport systems. conclusion: the analysis of the data revealed that cells sense and respond to alkaline stress with an extensive program of changes in gene expression. interestingly, there is a strong correlation between the altr and virulence gene expression. comparison to various microarray data already in the literature revealed similarity between the response to alkaline stress and the transcriptional response to stresses such as osmotic shock. engineering improved listerial stress tolerance "with a twist"! r. sleator, c. hill (cork, ie) objectives: to engineer listeria monocytogenes strains with a significantly improved ability to tolerate stresses encountered in the external environment and during gastrointestinal transit, thus, improving listeria's efficacy as a potential vaccine and drug delivery platform. methods and results: using a directed evolution approach, based on a random mutagenesis strategy involving the e. coli xl -red mutator strain, we generated a mutant variant of the listerial betl gene (designated betl*), encoding a secondary betaine uptake system. the mutant betl* promotes a dramatic increase in resistance to a number of biologically relevant stresses when expressed in a variety of different surrogate hosts. using a luciferase (lux) reporter system in combination with the ivis imager system (xenogen corporation, alameda, ca), we tracked betl* expression, in real time, both in vitro under various environmental stresses and in vivo in animal models of infection. in each case strains expressing betl* demonstrated a marked improvement over those expressing wild type betl, both in terms of gene expression and bacterial growth. sequence analysis of the mutated gene revealed a single nucleotide deletion in the spacer region between the - and ) promoter elements upstream of the betl coding region. this deletion presumably introduces a conformational 'twist' in the putative promoter, thereby increasing its transcriptional output. furthermore, the betl* mutation appears to counter the heretofore unreported 'twisted' cell morphology observed using scanning electron microscopy of l. monocytogenes grown at elevated osmolarities. conclusions: it is possible to selectively improve genes required for bacterial stress survival both inside and outside the host. such mutated genes systems may ultimately be used for the construction of more physiologically robust bacterial based vaccine and drug delivery platforms. a.r. samarbaf-zadeh, s. tajbakhsh, s.m. moosavian (ahwaz, ir) introduction: peptic ulceration following infection of stomach with h. pylori is a common disease. accurate and rapid detection of the bacteria can lead to implementation of appropriate treatment and recovery. this research was undertaken to evaluate the sensivity and specificity of fluorescent in-situ hybridization (fish) in the detection of h. pylori in patients who were suffering from dyspepsia. methods: for this purpose, one hundred gastric biopsy samples taken from antrum and corpus of stomach by endoscopy were tested by fish and compared with conventional culture method complemented with biochemical tests. results: fish detected h. pylori in clinical samples while conventional method detected samples. the sensivity and specificity of fish for detection of h. pylori were calculated as % and % respectively. conclusion: the findings of this study suggest that fish is a highly suitable and rapid method for diagnosis of h. pylori, especially when the samples are taken from the antrum and the corpus of the stomach this technique potentially can be applied routinely for detection of this bacterium in clinical samples. objective: numerous studies have demonstrated that h. pylori is ubiquitous; approximately % of the world's population is infected with the organism. gastroduodenal diseases associated with h. pylori infection are manifested principally in adults. however, it's usually during chilhood that the infection is acquired, and it is possibile that mucosal and humoral responses at this time may determine, at least in part, the course of the natural infection. our study will describe the prevalence of the h. pylori oral carriage in children resident in bari, south of italy, using the pcr method. methods: the evaluation was performed in children, with ages ranging from to years, from primary school district of local health unit of bari, italy (ausl ba/ ). the school and the class have been selected using the cluster sampling method. a standardized questionnaire was used to verify socio-economic standard, hygiene and history of previous gastrointestinal disorder. a standard full-mouth examination was made to detect periodontal diseases, then dental plaque and saliva collected from children were placed in pbs and transported in laboratory. h. pylori infection status was checked by pcr method. dna was extracted from oral samples by the boiling method and evaluated for the presence of h. pylori caga and urea genes using commercial kit (ab analitica, padova). results: a total of children ( females and males) partecipated to the study. the presence of gene coding for caga was found in children ( %), but gene urea was detected only in ( %). the bacteria was detected in saliva, supragingival and subgingival plaque, suggested that these sites may be considered reservoirs for h. pylori in ureasi-positive patients. there was statistically significant relationship between who didn't wash their hands frequently and the presence of urea gene (o.r. . ). conclusions: current knowledge implies that acquisition of h. pylori seems to occur predominantly in childhood and that once acquired the infection persists life-long in most infected subjects. it has been reported at a worldwide level that h. pylori infection prevalence in children varies between % and % and increases with low socio-economic and educational levels and age. the results of this study suggest that oral carriage of h. pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission. the role of helicobacter pylori in otitis media with effusion t. yilmaz, m. ceylan, y. akyon, o. ozcakir, b. gursel (ankara, tr) objectives: otitis media with effusion (ome) is such a common disease of childhood and its pathogenesis still remains unsettled. pepsinogen and pepsin has been shown in the middle ear fluid of patients with ome, indicating that gastric juice could reach as far as middle ear. if gastric juice could enter the middle ear, helicobacter pylori, a common inhabitant of gastric juice and mucosa, would also be expected to be found in the middle ear of patients with ome. the objective of this study was to evaluate possible role of helicobacter pylori in pathogenesis of otitis media with effusion. methods: the study group consisted of children who are to undergo bilateral ventilation tube insertion, adenoidectomy, tonsillectomy with a diagnosis of ome, adenoid hypertrophy and chronic tonsillitis. the control group consisted of children who are to undergo adenoidectomy, tonsillectomy with a diagnosis of adenoid hypertrophy and chronic tonsillitis. for the study group, middle ear fluid was aspirated and a small biopsy was taken from the promontorium mucosa. for the control group, myringotomy was done and a small biopsy was taken from the promontorium mucosa. for both groups, mm deep tissue specimens were obtained from tonsil and adenoid. for all the specimens taken from the patients, culture and a nested-pcr were performed to show helicobacter pylori. results: middle ear fluid culture was positive for h. pylori in patients and mucosa culture was positive in patient only. in the control group middle ear mucosa cultures were always negative. when culture and pcr results were combined together; the middle ear was positive for h. pylori in patients in the study group and in patients in the control group. this difference was statistically significant. h. pylori presence in the tonsillar and adenoid tissues by culture and pcr was also significantly more frequent in the study group compared to the control group. conclusion: this study is the first to grow h. pylori in the middle ear in ome. significantly increased colonization by h. pylori of the middle ear, tonsillar and adenoid tissue in patients with ome indicates that the bacteria reaching the middle ear through gastroesophageal reflux might be involved in the pathogenesis of ome. for ome cases resistant to medical treatment it may meaningful to evaluate the patient for gastroesophageal reflux and h. pylori. distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal carriage and invasive staphylococcus aureus strains objectives: this study was designed to examine the distribution of the sdr genes among nasal carriage and invasive staphylococcus aureus strains as well as methicillinsensitive s. aureus (mssa) and methicillin-resistant s aureus (mrsa). methods: the presence or absence of the sdr genes using dna from s. aureus strains was determined by a novel triplex pcr procedure. the two-tailed fisher's exact test was used to analyse the distribution of the sdr genes among s. aureus strains originating from different hosts. p values less than . were considered a statistically significant difference. results: the sdr locus was found in all investigated s. aureus strains although in strains it contained only the sdrc gene (sdrd -sdre-). the sdrc + sdrd -sdre-gene profile was exclusive to mssa strains (fisher's exact test; p = . ) and was not found in the strains collected from bone infections (p = . ). we also found a strong association between the presence of the sdrd gene and mrsa strains (p < . ). conclusion: our findings suggest that mssa strains with the newly uncovered sdrc + sdrd -sdre-gene profile have a substantially decreased potential to establish bone infection. sequencing of luks-pv and lukf-pv in methicillin-sensitive and methicillin-resistant staphylococcus aureus of diverse genetic backgrounds in a swedish county c. berglund, b. sö derquist (Ö rebro, se) objectives: community-aquired methicillin-resistant staphylococcus aureus (ca-mrsa) have been reported to carry the loci for panton-valentine leukocidin (pvl) in high frequency. the aim of this study was to describe variations within the pvl genes (luks-pv and lukf-pv) in methicillinsensitive and methicillin-resistant s. aureus of diverse genetic backgrounds. methods: twelve pvl-positive s. aureus were characterised by multilocus sequence typing (mlst) and mrsa also by staphylococcal cassette chromosome mec (sccmec) typing. ten of these were isolated between - in Ö rebro county, sweden. oligonucleotide primers were designed to yield a product size of~ bp including luks-pv and lukf-pv and flanking regions by pcr amplification. cyclic sequencing was performed with several sets of primers to overlap the sequences on both strands and was separated on abi prism Ò genetic analyzer (applied biosystems). the nucleotide sequences were analysed using abi prism Ò autoassembler tm dna sequence assembly . . software and compared using bioedit . . . results: analysis with mlst differentiated the pvl-positive ca-mrsa into six different sequence types (st , , , , and ) with either sccmec type iv, iv c, v or unknown types. six additional sts (st , , , , and new) were detected among the pvl-positive methicillin-sensitive s. aureus. sequencing luks-pv and lukf-pv revealed eight point mutations among these isolates with twelve different origins. five substitutions had occurred in luks-pv and three in lukf-pv. only one substitution was nonsynonymous (histidine fi arginine). conclusion: the pvl-genes were well conserved despite the different genetic origins of the isolates analysed. the pvl is an extracellular product and the genes are not subject to any selective forces and thereby diversify very slowly. additional nonsynonymous mutations might result in a non-functional toxin. the first case of staphylococcus pseudintermedius in humans isolated from an icd lead l. van hoovels, a. vankeerberghen, k. van vaerenbergh, a. boel, h. de beenhouwer (aalst, be) introduction: staphylococcus pseudintermedius is recently described as a new coagulase-positive species from animals (devriese et al., ) . the pathogenic significance of this novel species remains unclear and to our knowledge no human infection due to s. pseudintermedius has been reported to date. here, we present the first isolation of s. pseudintermedius in humans with important clinical significance. patient and methods: a -year old male patient was referred to our centre for an ischemic cardiomyopathy and ventricle tachycardia for which he recieved an implantable cardioverterdefibrillator (icd) in january . in august he presented with complaints of migration of the icd device. clinical examination revealed perforation of the icd pocket. infection was suspected and confirmed by the presence of pus in the pocket. the infected icd was completely removed and several samples (ventricular lead, pus and a tissue sample from the pocket) were sent for culture.bacteria obtained by routine culture were further characterised by phenotypical identification, pastorex Ò staph-plus (biorad), api staph Ò (biomérieux) and phoenix Ò (bd). for molecular analysis, pcrs were performed targeting the nuclease (nuc) and coagulase (coag) genes of s. aureus. additionally, sequencing of the s rrna gene was performed and further analysed using blast. results: staphylococci with identical phenotypical appearance were isolated from of the icd samples (lead and pus). colonies were beta-hemolytic on sheep blood agar, dnase and coagulase positive but clumping factor, mannitol and pastorex Ò negative. biochemical identification by api staph Ò and phoenix Ò gave a presumptive identification of s. aureus with a confidence value of respectively , % and %.the pcrs for the nuc and coag genes were both negative. s rrna gene sequencing resulted in the identification of s. pseudintermedius based on a % sequence similarity with a previous reported sequence by devriese et al. conclusion: this case report describes the first identification of s. pseudintermedius as a significant pathogen in human. growth characteristics and commercial identification systems misidentify the organism as s. aureus. when confronted with an inconsistent phenotypical identification pattern, clinical labs should consider the use of s rrna gene sequencing for final confirmation. characterisation of staphylococcus aureus isolates recovered from dairy sheep farms (agr group, adherence, slime, resistance to antibiotics) e. vautor, m. sabah, g. mancini, m. pepin, h. carsenti-dellamonica (sophia-antipolis, nice, fr) objectives: the purpose of this study was to investigate staphylococcus aureus natural isolates associated with dairy sheep mastitis for epidemiological key features (agr group, adherence, slime production and antibiotics resistance). methods: the s. aureus isolates (n = ) were recovered from a field study in the southeast of france in - ( from subclinical mastitis, from clinical mastitis, from the environment of the dairy sheep farm). a total of thirteen dairy sheep farms, producing cheeses manufactured with raw ewe's milk, were involved. the agr group were determined by multiplex and real-time pcr. the evaluation of adherence and slime production were assessed with methods previously described by christensen et al. ( ) . the susceptibility patterns to antibiotics were determined using the discdiffusion method on mueller-hinton agar plates. oxacillin susceptibility testing was performed on all the isolates. the others antibiotics susceptibility was only studied on the isolates recovered from subclinical mastitis as they represent the major source of cheese contamination. results: % ( / ) of the isolates belonged to agr group , regardless of clinical findings. % ( / ) were adherent, strongly adherent or with maximal adherence (biofilm producers). % ( / ) were slime producers (moderate or strong producers). all the isolates (n = ), but seven, were susceptible to all the antibiotics tested. two isolates recovered from subclinical mastitis were resistant to oxacillin and partly resistant to ampicillin and penicillin-g. the five other isolates were found: partly resistant to erythromycin (n = ), cefoperazone and penicillin-g (n = ), erythromycin (n = ), neomycin (n = ) or resistant to enrofloxaxin and partly resistant to ampicillin and penicillin (n = ). conclusions: s. aureus isolates recovered from sheep mastitis in the southeast of france are mainly related to agr group suggesting a role for agr-regulated proteins in the persistence of this bacteria in the sheep udders. biofilm and slime production may also be an important aspect for intracellular survival of s. aureus which could promote the development of persistent intramammary infections. finally, ewe's milk does not appear to represent a source of resistant s. aureus and specially methicillin (oxacillin)-resistant s. aureus (mrsa) for human health. detection of virulence genes in staphylococus aureus isolates from dairy sheep, goats and cows mastitis, using single-dye dna microarray e. vautor, v. magnone, g. rios, m. pepin, p. barbry (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy farms animals. although many putative virulence factors have been identified in s. aureus genomes (kuroda et al., ) , the differences in pathogenic potential between naturally occurring isolates remain largely unaddressed. the relative importance of host (tissue) factors versus bacterial virulence determinants in disease pathogenesis is not well known, but it is widely accepted that bacterial factors including toxins, cell wall-associated adhesions, and secreted exoproteins are involved in the process. in this study, we use a single-dye dna microarray assay to investigate the presence or absence of putatives virulence genes in s. aureus isolates recovered from cases of ovine, caprine and bovine mastitis. methods: mastitis s. aureus isolates: sheep (n = ), goats (n= ), cows (n = ).dna microarray: the arrays were spotted with long oligonucleotides ( -mer) representing known virulence genes and new candidates identified in mu genome (a human strain) and other s. aureus genomes. each gene were spotted four time. dna extracted from the strains were labelled with fluorescent cy using the bioprime Ò array cgh (invitrogen). control strains with known genetic and phenotypic characteristics were used to normalize the data. results: (i) the majority of the virulence gene was detected in all the isolates (e.g. coa, ica adbc operon, htra, hysa, nuc, sbi, sdre, ssp, feob, fnb, sib, spa). (ii) genes were not detected in the majority of the isolates (e.g. cna, edin, lukf-pv, sav ,…). (iii) genes were not found in isolates, depending on the herd (e.g. aur or sav absent in isolates from some dairy sheep farm), on the isolates whatever the species (i.g. bsap, caph, entk, eta, fnbb, hsds, lpl , lukd, …) . but we found gene mainly related to species (e.g. agriii, sav ,…) comprehensive results will be given in the poster. conclusions: the present study indicated that the prevalence of virulence genes among s. aureus isolates recovered from dairy farm species depends on the gene. these observations suggest a common occurrence of host-adapted (or tissueadapted) s. aureus strains in which particular virulence genes may play a significant role. when taken with complementary methods such as pcr or/and southern hybridisation, singledye dna microarrays may provide a powerful tool to type s. aureus strains for epidemiological and possibly pathogenesis studies. detection of dna sequences distinguishing two closely related genomes of staphylococcus aureus from subclinical versus gangrenous ewe mastitis strains n. chevalier, c. huard, r. thiery, e. vautor (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy sheep. the severity of mastitis ranges from subclinical to gangrenous forms. subclinical mastitis is an inflammation that is not readily detected clinically whereas gangrenous form is an acute necrotizing mastitis. with the ain to find genetic markers or virulence factors that are only present in gangrenous strains a suppression substractive hybridisation (ssh) method was used in the present study to compare two strains of s. aureus respectively recovered from subclinical or gangrenous mastitis in the same dairy sheep herd. methods: ewes were held in the investigated farm. the subclinical strain was recovered in january from the milk of ewes. the gangrenous strain was recovered in december from an primipare dairy sheep that subsequently died from this acute mastitis. dna extracted from the strains were first compared by pulsed field gel electrophoresis (pfge). then, ssh was performed by using dna from the subclinical strain (driver), as described in a commercial kit (clontech pcr-select bacterial genome substraction kit). results: using pfge, four band differences were found between the two strains. two dna fragments, presumably specific from the gangrenous strain were detected by ssh and sequenced: (i) a bp ( % of homology with the sulfide quinone reductase contained in orf pathogenicity island of the mrsa strain) (ii) bp ( % homology with a gene coding a bacteriophage holine contained in the s. aureus n genome). control pcr tests using primers designed from these specific gene candidates confirmed that they were only present in the s. aureus gangrenous strain. conclusions: according to tenover et al. ( ) , a band difference using pfge indicates that the strains may possibly be related genetically. although genes classically involved in the virulence of s. aureus were not detected in the present study, two putative virulence factors were detected. the sulfide quinone reductase allows s. aureus to growth on sulfide (found in animal manure). the holine protein breaks the internal membrane of s. aureus to release daughter phages suggesting that a mechanism of horizontal gene transfer could have been mediated by bacteriophages and could explain the acquisition of virulence factors. antimicrobial clinical trials p outpatient treatment of acute pyelonephritis in pregnancy after weeks. a randomised controlled trial z. ahmadinejad, s. hantooshzadeh (tehran, ir) objectives: the purpose of this study was to compare the safety and efficacy of outpatient and inpatient treatment of acute pyelonephritis in pregnancy. methods: this was a randomized controlled, clinical trial. one hundred twenty eight gravidas past weeks' gestation admitted in imam khomeini hospital, tehran & sahid dr bahonar hospital, kerman, divided by random blocks to outpatient or inpatient therapy, received two -g doses of intramuscular ceftriaxone at -hour intervals while hospitalized, then were discharged and reevaluated within - hours or remained hospitalized until afebrile for hours. all patients completed a -day course of oral cephalexin. we performed urine cultures on admission and - days after therapy. results: the two groups were similar with respect to age, parity, temperature, estimated gestational age, initial white blood cell count, and incidence of bacteremia. there were not any significant differences between two groups about the clinical improvement after - hours, bacteriuria - days after treatment, relapse of pyelonephritis, requirement to change in antibiotic, date of pregnancy at delivery and preterm labor. the relapse of bacteriuria and preterm labor in inpatients were significantly more than outpatients (pv = . and . respectively). the birth weight of neonate in outpatients were significantly more than inpatients (pv = . ). conclusion: outpatient antibiotic therapy is effective and safe in selected pregnant women with pyelonephritis. however in this study, the neonatal outcomes were better in outpatients and the maternal outcomes in inpatients. experience with daptomycin in patients with renal insufficiency investigators collected demographic, disease state, clinical and microbiological data; outcomes were defined using standard definitions. patients nonevaluable for outcome were excluded. core data were divided and data on cohorts of pts with a creatinine clearance (crcl) ‡ or < ml/min were examined. results: of the pts enrolled, ( %) had evaluable pt outcomes and either crcl ‡ ml/min (nml, n = ) or crcl < ml/min not yet requiring renal replacement therapy (ri, n = ). the distribution of males and females was equal in both groups. ri pts were older ( % ‡ yrs vs %, p < . ). the groups did not differ in the percent coming from the community setting prior to starting dap (nml %, ri %). nml had more frequent history of fractures/orthopaedic procedures ( vs %, p < . ) and haematological cancers ( vs %, p < . ) while ri had higher rates of any renal disease ( vs %, p < . ), chf ( vs %, p < . ) and other immunologic/ inflammatory disease ( vs %, p < . ). ri had higher rates of skin infections ( vs %, p < . ) and endocarditis ( vs %, p < . ). infections that were frequently reported for nml and ri were bacteremia, non-catheter-related ( vs %), bacteremia, catheter-related ( vs %), osteomyelitis ( vs %), and foreign body-orthopaedic ( vs %), all p > . . methicillin-resistant staphylococcus aureus was the most common pathogen; nml %, ri %. ri had higher rates of coagulase-negative staphylococci ( vs %, p < . ) and viridans streptococci ( . vs . %, p < . ). there was no difference in the percentage receiving antibiotics prior to dap; nml %, ri %. the mean dap dose and duration were similar; nml . mg/kg for d, ri . mg/kg for d. the most frequent dose was mg/kg; nml %, ri %. ri initial dap dosing was more frequent than recommended (q h) in %. the mean time to clinical response was similar; nml . d, ri . d. more pts in nml received concomitant antibiotics with dap; vs %, p < . ). the clinical success (cure and improved) rates were; nml %, ri %. conclusion: dap shows favourable clinical success rates in pts regardless of the presence of renal insufficiency. in vitro activity of second line antibiotics against helicobacter pylori infection objective: the aim of our study was to determine the in vitro activity of levofloxacin, ciprofloxacin and rifampicin in clinical strains of h. pylori. material and methods: isolates of h. pylori from biopsies of dyspeptic patients were obtained following standard methodology. in vitro activity of metronidazole, clarithromycin, levofloxacin, ciprofloxacin and rifampicin was determined by e-test using % sheep blood agar and incubated at ordm;c during - days in a co atmosphere. mic was determined as the point of complete inhibition of growth. breakpoint of the nccls for other microorganisms were considered for fluorquinolones: resistant if mic > mg/ l. for rifampicin we considered the strain susceptible if mic < mg/ l, as same studies reported. results: . % of the strains were resistant to metronidazole and % to clarithromycin. mic , mic and range (mg/l) was: . , . and . fi for levofloxacin, . , . and . fi for ciprofloxacin and . , . , and < . - for rifampicin. all the strains were susceptible to rifampicin and only % of them were resistant to fluorquinolones. conclusions: the fluorquinolones tested and rifampicin showed an excellent in vitro activity against h. pylori, despite the high resistance rate to metronidazole and clarithromycin. however, in vitro susceptibility test should be done before the use in clinical practice. vibrio antibodies in serum and breast milk samples of parturient women in calabar, nigeria objectives: serum and breast milk samples from parturient women and serum from non-parturient controls were analysed for prevalence and titres of vibrio antibodies. methods: v. cholerae agglutinins and vibriocidal antibodies in serum samples were analysed by direct agglutination and immune bacteriolysis techniques respectively, using well microtitre plates. the protective value of breast milk was evaluated by haemagglutination inhibition and rabbit intestinal mucosal attachment of v. cholerae cells. results: vibrio agglutinins were detected in serum samples of ( . %) parturient and ( . %) non-parturient subjects (p < . ). high prevalence rates of . % and . % occurred among parturient and control subjects of - years of age respectively. at : cut off titre to evaluate vibrio cholerae specific bacteriocidal antibodies, activity was detected in samples of ( . %) and ( . %) parturients and controls respectively aged - years. breast milk from ( . %) parturients contained vibrio agglutinins with titres ranging between : and : , while milk samples from subjects showed haemagglutination inhibition (hi) activity titres of p : . of the hi positive milk samples ( . %) showed inhibition of v. cholerae adherence to rabbit intestinal mucosa at titres p : , and - % reductions in cell attachment. conclusion: our study confirms that parturient women in calabar may benefit from significant serum titres of v. cholerae antibodies and provide immune protection for their babies through breast milk secretions. moxifloxacin vs clarithromycin for treatment of community-acquired pneumonia associated with common respiratory pathogens: a pooled analysis objectives: streptococcus pneumoniae and haemophilus influenzae are pathogens commonly associated with community-acquired pneumonia (cap). this study compared the clinical and bacteriologic efficacy of moxifloxacin (mxf) to clarithromycin (clar) in cap patients with these pathogens. patients and methods: data were pooled from three doubleblind, multicenter, phase iii trials comparing oral mxf mg qd to clar mg bid for days. all patients included had mild-to-moderate cap. clinical and bacteriologic success rates were identified for s. pneumoniae and h. influenzae isolated from these studies. data for the efficacy-valid population was recorded at the test-of-cure (toc) visit ( - days post-therapy). results: patients were entered, of which were microbiologically evaluable. infection with s. pneumoniae and/or h. influenzae was documented in ( %) of patients ( mxf and clar patients had mixed infection). within this cohort, the two treatment groups were well balanced based on demographic/baseline medical characteristics ( % male, mean age yrs, % smokers, % recent antimicrobial therapy). clinical success and bacteriologic eradication rates (one response per patient) at toc are presented in the table. conclusions: in cap associated with s. pneumoniae and h. influenzae there was a trend towards greater bacterial eradication for mxf vs clar. clinical success rates were significantly higher for mxf monotherapy vs clar. variability of creatinine clearance measurements in inpatients with community-acquired pneumonia r. grossman, s. choudhri, d. haverstock (mississauga, ca; west haven, us) objectives: moxifloxacin, levofloxacin and gatifloxacin have been recommended as empiric therapies for patients with community-acquired pneumonia (cap). levofloxacin and gatifloxacin require dose-adjustment for renal insufficiency while no dose adjustment is required for moxifloxacin. this study was designed to determine the frequency and underlying variability of renal insufficiency in patients with cap. methods: a pooled analysis of data from patients with mild to moderate or severe cap entered into one of six randomized, controlled clinical trials was undertaken. renal function (calculated creatinine clearance; crcl) was assessed in each patient prior to treatment with mxf and then again during and post-treatment. results: baseline crcl levels in this pooled population of patients with cap were: < ml/min in ( . %) of patients, - . ml/min in ( . %) and ‡ ml/min in ( . %) patients. after the pre-treatment crcl measurement patients ( %) were lost to follow-up, so there was no during or post treatment value. in patients with cap the crcl improved from baseline in many patients during or post-treatment, while some patients experienced a worsening of renal function (see table) . conclusions: renal function (crcl) is highly variable in cap patients with baseline evidence of renal insufficiency. renal function should be monitored closely to permit appropriate dose adjustments if levofloxacin or gatifloxacin is used in this patient population. moxifloxacin may be a better empiric choice in this setting as it does not require dose adjustment in patients with renal insufficiency or renal failure. a prospective, controlled, randomised, nonblind, comparative study of the efficacy and safety of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice-daily ceftazidime plus amikacin in the empirical therapy of febrile neutropenic patients objective: empirical antibiotic treatment for febrile neutropenia is well established. the best regimen is still controversial. the purpose of this study was to evaluate the efficacy, safety and cost of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice daily ceftazidime plus amikacin in neutropenic febrile patients. patients and methods: ninety-five patients with febrile neutropenia were included in a prospective, controlled, randomized, non-blind, comparative study. patients were randomly assigned to either treatment group ( in the ceftriaxone/ciprofloxacin group and in the ceftazidime/ amikacin group) and evaluated as successes or failures according to defined criteria. daily assessments were made on all patients all adverse events were record. results: the overall incidence of documented infections was . %: / ( . %) in the ceftriaxone/ciprofloxacin group and / ( %) in the ceftazidime/amikacin group. there was significant difference in clinical efficacy between groups (p = . ) at the end of therapy. ceftriaxone/ciprofloxacin group had an overall incidence of resolution and improvement of , % in comparison to the % of the ceftazidime/amikacin group. thirty-nine organisms were isolated, ( . %) gramnegative and ( , %) gram-positive. there was low incidence of adverse events in both groups. conclusion: the combination of high dose single daily ceftriaxone plus ciprofloxacin was more effective than the standard combination of thrice daily ceftazidime plus amikacn with no significant adverse events in either group. objective: in past studies of azithromycin in children, a posttreatment (pt) benefit was observed at day . in recent phase trials in adults, single-dose zmax was at least as effective as standard comparators for treatment of respiratory tract infections (rtis), including cap. our objective is to demonstrate a pt benefit in this adult population. methods: post-hoc analyses, including respiratory adverse event burden (raeb), were conducted on the all treated population (n = ; az-m, comparators) in the phase studies. the raeb is the sum of duration, in days, of all respiratory adverse events, divided by total number of observation days of all patients, normalized to year. the overall and per study day raeb were calculated for zmax and the pooled comparators for the studies combined. results: raeb, in days/patient year, was . for az-m patients vs . for comparator patients (p = . ). the difference in raeb consistently and progressively favoured zmax, beginning at day and achieving statistical significance between days and , when the upper limits of the % cis around the differences were below zero (figure). faropenem medoxomil (fm) is an oral penem with potent activity against streptococcus pneumoniae and haemophilus influenzae. this integrated analysis was conducted to summarize the efficacy of days of mg bid of fm compared with other beta lactams in the management of community acquired pneumonia (cap). methods: efficacy was determined in three multicenter randomized double-blind controlled trials (rct) and a single uncontrolled study of faropenem medoxomil. comparators were days of cefpodoxime (c), days of amoxicillinclavulanate (ac), or days of amoxicillin (a). the analysis allowed examination of treatment effects by age, race, gender and study site subgroups. results: a total of subjects were studied. studies and were conducted in n. america, studies and in europe, latin america, israel, and s. africa. n. american vs. other studies included subjects at least (vs. at least ) years of age and only out patient (vs. outpatient and hospitalized) subjects. the clinical responses for fm in both per protocol and intention-to-treat populations were non-inferior to comparator for each study and for the three trials combined. no differences were found in treatment effect by age, race, gender, or country. recovery of an etiologic agent from initial respiratory or blood culture varied between . and . % of cases in the studies for a total of microbiologically evaluable subjects. s. pneumoniae was eradicated or presumed eradicated in / ( . %) and / ( . %), h. influenzae in / ( . %) and / ( . %), s. aureus in / ( %) and / ( %), h. parainfluenzae in / ( %) and / ( %), and m. catarrhalis in / ( . %) and / ( %) fm and comparator recipients, respectively. clinical response for s. pneumoniae bacteremic patients was / ( . %) for fm. conclusions: fm efficacy was consistent across studies, within subgroups, and non-inferior to comparators. it is efficacious against the most common bacterial pathogens and in the most severe form (bacteremic) disease. fm is a good option for the treatment of cap. propionibacterium acnes strains isolated from acne vulgaris and severe infections c. oprica, c.e. nord (stockholm, se) propionibacterium acnes is a member of the resident flora of the skin and is an important factor involved in inflammatory reactions in acne patients. during the last years the prevalence of different severe infections due to p. acnes has increased. objectives: ) to detect the prevalence of resistant p. acnes strains isolated from acne patients in stockholm and different severe infections in europe; ) to identify the mechanisms of resistance and the genetic diversity among resistant strains. methods: p. acnes strains isolated from acne vulgaris and severe infections were tested against clindamycin, erythromycin, linezolid and tetracycline and pulsed-field gel electrophoresis was used for further characterization. pcr and sequencing of the genes encoding domain v of s rrna for clindamycin and erythromycin resistant strains and s rrna for tetracycline resistant strains were performed. results: i) antibiotic-resistant strains were more often isolated from antibiotic treated patients with moderate to severe acne area than from non-antibiotic treated acne patients. an individual might harbor different pulsotypes of p. acnes with various degrees of resistance. ii) among the clinical isolates from european countries were found resistant strains to tetracycline, clindamycin, and erythromycin. overall, in the southern europe a higher prevalence of erythromycin-resistant strains was noticed and in southern and eastern europe a higher prevalence of resistance to clindamycin. it was noticed a high genomic diversity and the geographical spread of some clones in related areas but also in geographically distant countries. most clindamycin or erythromycin resistant p. acnes isolates, were found to be members of a single clone that has spread in different geographically countries. iii) p. acnes clindamycin and erythromycin resistant strains carrying one of the described mutations within the s rrna were predominantly isolated from swedish acne patients compared to strains from other infections. forty-four per cent of tetracycline resistant strains were found to carry a mutation in the s rrna. these strains were isolated from swedish acne patients, were highly resistant and were clustered in one pulsotype. conclusion: surveillance of both the prevalence of resistant p. acnes strains and associated resistance mechanisms is important due to the rapid variation in resistance patterns, both in acne patients and other severe infections. antimicrobial activity of unisepta quick and deconex solarsept on the surface contamination and dental instrument in dental clinics in iran f. shahcheraghi (tehran, ir) objectives: quaternary ammonium compounds (qacs) are amphoteric surfactants that are widely used for the control of bacterial growth in clinical and industrial invironment.unisepta quick and deconex solarsept are new generation of qacs is widely used as adjuncts in iran to hygine in dental clinics.the aim of present study was to investigate clinical efficiency of these substances on the surface and instruments in dental clinics. material and methods: the following bacteria and fungi on the base of aoac standard were used.pseudomonas aeruginosa (atcc ), staphylococcus aureus (atcc ) bacillus. subtilis atcc ( ), mycobacterium bovis atcc ( ) and wild types of trichophyton mentagraphit, p. aeruginosa and salmonella typhimorium (a common fungi in iran). a stock solution of deconex solarsept (borer chemie) and unisepta quick (micro unident) was prepared as recommended by the manufacturer. the concentration of bacterial suspention was . macfarland and the results were reported on the base of decreasing in (cfu) colony forming unit from to . results: the results shows that both of these disinfectants have bactericidal and fungicidal activity on the standard p. aeruginosa, s. aureus, s. typhimurium and trichophyton mentagraphit, the number of bacteria decreased significantly (p < . ), but no significant difference was seen with b. subtilis, wild type of p. aeruginosa and m. bovis. conclusion: the results confirm that these qacs are not able to sterilize or disinfect medical and dental instruments, and they can not be used lonely, and it must be used with the other methods for sterilization of surface and dental instruments. macrolide as long-term treatment in patients with bronchiectasis colonised by p. aeruginosa background: a certain efficacy of macrolide against p. aeruginosa has been described in vitro, mainly through mechanisms such disruption of quorum sensing and suppression of inflammation. aim: to evaluate the efficacy of macrolide in patients with bronchiectasis colonised by p. aeruginosa. methods: the study prospectively included patients with bronchiectasis and p. aeruginosa isolated in sputum in stable state. all subjects received either azithromycin mg · days/week or clarithromycin mg daily on long term and completed daily diary cards for symptoms and pef values until the end of therapy. follow-up period was year. results: patients with bronchiectasis and p. aeruginosa evidence in sputum were included ( men, mean age . ± . yrs.). patients received azithromycin and patients clarithromycin, with a mean duration of . ± . months. five ( . %) patients discontinued treatment after less than weeks because of adverse events. at the end of therapy, ( . %) patients showed no evidence of p. aeruginosa in sputum while ( . %) patients still had ps. aeruginosa in sputum. an improvement in the following parameters could be observed in all patients: sputum volume ( . ml/day before therapy versus . ml/day after therapy, p = . ); pef ( . ± . l/min before therapy versus . ± . l/min after therapy, p = . ); number of exacerbations/year ( . in the previous year versus . in the follow-up year, p = . ). conclusion: the study shows that macrolide may be an effective therapy in patients with bronchiectasis colonised by p. aeruginosa. independently of the microbial eradication, an improvement of the clinical symptoms and a reduction of exacerbations were observed in all patients. fungal pathogens from haematoncology patients and their susceptibility to new and old antifungal drugs the expanding population of immunocompromised hosts has been infected with many established and emerging opportunistic fungi. most pathogens can be treated empirically whereas for an increasing number of species proper treatment starts once the mic becomes available. though invasive aspergillosis remains the principle life threatening complication in the haematoncology patients (hop) other pathogens cannot be ignored as selection and resistance during prophylaxis increases the risk of treatment failure.in order to understand the frequency of rare fungal pathogens, selection and emergence of resistance in our trust all fungi from hop were identified using standard mycological techniques and the mics to amphotericin b (amb), flucytosine ( fc), fluconazole (fcz), itraconazole (itz), voriconazole (vcz) and caspofungin (cfg) were determined using the nccls method. specimens were processed, % respiratory, . % blood, . % oral, . % other sterile (bile, csf, drains, lines and tissue biopsies) and . % nonsterile sites. yeasts accounted for % and filamentous fungi (ff) for %, representing candida sp, other types of yeast, aspergillus sp and other types of ff. c. albicans represented . %, c. glabrata . %, c. krusei . %, a. fumigatus % and other aspergillus sp % of all isolates. the mic s for all isolates were amb . , fc , fcz > , itz , vcz and cfg . mg/ l. with the exception of acremonium sp, a. versicolor, a. terreus and scedosporium apiospermum all isolates including the isolates of c. lusitaniae were sensitive to amb. most but not all ff and only one isolate of c. albicans from the yeasts were resistant to fc. all ff, rhodotorula sp, c. albicans %, c. glabrata % and c. krusei % were resistant to fcz. only absidia corymbifera, acremonium sp %, c. albicans %, c glabrata % and saccharomyces cerevisiae % were resistant to itz. for vcz a. corymbifera, acremonium sp %, c. albicans %, c. glabrata %, c. krusei %, c. tropicalis %, rhodotorula sp . % and p. aecilomyces variotii % had an mic ‡ mg/l. with cfg the effective concentration was ‡ . mg/l for a. corymbifera, fusarium solani, geotrichum capitatum, sporobolomyces salmonicolor, acremonium sp % and c. parapsilosis %.the data show that hop are exposed to many different fungal pathogens some of which are resistant to the old and the new antifungals and that amb is still the drug with the broader spectrum and less developed resistance for both yeasts and ff. faropenem medoxomil in the treatment of acute bacterial sinusitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial agent with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute bacterial sinusitis (abs) was evaluated in phase iii trials; , , and . study was conducted in n. america, study was conducted in europe and israel and study was conducted in the us and argentina. and were prospective, randomized, double-blind, active comparator trials and was an open-label ''sinus tap'' trial. the dose of fm was mg bid in all studies. the comparator in and was cefuroxime axetil (cfx) mg bid. the duration of fm treatment in was days and days vs cfx for days. in , fm or cfx were given for days. in , fm was administered for days. the primary efficacy variable in all studies was clinical response at the test-of-cure (toc). microbiologic response at the toc was a secondary efficacy variable in (sinus puncture and endoscopic collection) and (sinus puncture and aspiration). non-inferiority was defined as the difference in cure rates (fm minus comparator) where the lower boundary of the % ci was greater than - %. results: the cure rates at the toc are shown in the table for the valid per protocol (vpp) and the intent-to-treat (itt) populations. the frequency of isolation of key pathogens and the rate of eradication in samples obtained by endoscopicallyguided swab and in samples obtained by tap were consistent across studies. the eradication rates for s. pneumoniae, h. influenzae, and m. catarrhalis were . % vs. . % (fm / d vs. cfx / d), . % vs. . % (fm vs. cfx) and . % vs. . % (fm vs. cfx), respectively. conclusions: fm mg bid x days was shown to be noninferior to cfx in clinical efficacy in two prospective, doubleblind, comparative trials. a third, open-label trial, demonstrated similar efficacy in microbiologically documented abs caused by key pathogens. longer ( d treatment) with fm provided no additional efficacy. faropenem medoxomil in the treatment of acute exacerbation of chronic bronchitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute exacerbation of chronic bronchitis (aecb) was evaluated in phase iii trials. study was conducted in europe, israel, mexico, and south africa. study was conducted in the us and argentina. both were prospective, randomized, double-blind, active comparator trials. the dose of fm was mg bid for days in both studies. the comparators were clarithromycin (clr) mg bid for days and azithromycin (azi) qd for days ( mg on day and mg on . the primary efficacy variable was clinical response at the test-of-cure (toc). microbiologic response at the toc, in subjects with a baseline pathogen was a secondary variable. non-inferiority was defined as the difference in cure rate (fm minus comparator) where the lower boundary of the % ci was greater than ) %. results: the cure rates are shown below for the valid per protocol (vpp), intent-to-treat (itt) and modified itt populations (all itt subjects who met inclusion/exclusion criteria). in both the individual studies and the pooled analyses, for all populations, treatment with fm was not less effective than either comparator. % of treated subjects in and % of subjects in were evaluable for microbiological response. in , the eradication rates for the microbiologically evaluable population was higher in the clr group ( . %) compared with the fm group ( . %) ( % ci ) . , . ) . in contrast, the eradication rate in was similar in the fm ( . %) and azi ( . %) groups ( % ci - . , . ). when the data were pooled across studies, the response rates were similar with fm ( . %) and combined comparator ( . %) groups ( % ci - . , . ). the combined eradication/presumed eradication rates in the pooled fm and comparator groups were . % vs. . %, respectively for s. pneumoniae and . % vs. . %, respectively, for h. influenzae. conclusions: fm was shown to be non-inferior to either azi or clr in clinical efficacy in two adequate and well-controlled trials. pooled analysis further strengthened the clinical noninferiority conclusion. the difference in eradication rates observed in study (clr) was not supported by study (azi). an integrated safety analysis of faropenem medoxomil: results of , subjects from phase ii/iii clinical trials r. echols, r. tosiello (milford, us) objective: to evaluate the safety profile of faropenem medoxomil (fm), a novel oral penem antibiotic. methods: , subjects from phase ii and phase iii clinical trials received fm, mg bid for - days for treatment of acute bacterial infections. randomized controlled trials (rcts) included , fm and comparator treated subjects. analyses were conducted to identify possible disparate adverse event (ae) reporting based on type of infection, subject age ( - , - , , - , > ) and gender, duration of treatment ( / d v. / d), geography (na, eu, row), study design (open label v rct), relationship to treatment. comparisons were made to control treatment based on antibiotic class (b-lactam v. other), and individual antibiotic treatments. results: fm compared favourably to penicillins, cephalosporins and macrolides. fm was better tolerated than tmp/smx and co-amoxiclav. open labeled trials had higher aes reported v. rcts. aes reporting na = row > eu except serious aes and deaths where row = eu>na. aes for fm / d = fm / d. underlying infection did influence ae reporting. female gender had higher ae reporting than male gender. fm was tolerated equally well across age ranges, although deaths and saes were more common in > age group. common aes (> %/related from rcts) were diarrhoea, nausea, fungal vaginosis and headache and were generally less frequent with fm than control rx. no evidence of neuro or cardio toxicity was identified. laboratory tests identified no hepatic, renal or hematopoietic signals. conclusion: faropenem medoxomil, a novel oral penem antibiotic, has the safety profile expected of a b-lactam but is better tolerated than co-amoxiclav with approximately one-third the gi side effects. the efficacy of non-surgical and systemic antibiotic treatment regimens in smoking and non-smoking patients e. pähkla, k. lõ ivukene, p. naaber, m. saag (tartu, ee) periodontitis is a chronic infectious disease, which leads to the destruction of periodontal ligament fibres and alveolar bone until tooth loss. the objective of this study was to compare the longitudinal effect of combination of non-surgical periodontal therapy with systemic antibiotics in smoking (s) and nonsmoking (ns) patients. methods: there were total of patients with severe generalized chronic periodontitis involved in this study ( s, ns), who did not respond well to previous mechanical periodontal treatment. the clinical examination included recordings of visible plaque index (vpi), modified gingival index (mgi), bleeding on probing (bop) and suppuration after probing (sup), probing pocket depths (pd) and clinical attachment levels (cal). the non-surgical periodontal therapy was performed within weeks. clinical parameters were recorded at baseline, - weeks after the first mechanical treatment and months after combined treatment, during a regular check-up visit. as the patients did not respond to the conventional periodontal therapy, the microbiological analyses were taken and a combination of systemic amoxicillin mg · and metronidazole mg · for days, was prescribed. results: the results suggested that the combined systemic antibiotic therapy is effective in case of severe generalized chronic periodontitis, as vpi, bop, sup, cal, and mgi improved significantly after the treatment. in the ns group all parameters, except cal, improved significantly after the treatment. the s showed markedly smaller reduction in sup, mgi, and cal.after instrumentation, no periodontal pathogens were isolated in ( %) patients, while patients ( %) were infected with one to three different pathogens. among the pathogens, prevotella intermedia/nigrescens ( patients) and actinobacillus actinomycetemcomitans ( patients) were dominating. the total level of microbial load (log cfu/ml) as well as the spectrum of pathogens in s and ns patients remained similar. conclusions: despite of positive treatment effect in general, there were insignificant improvements in any clinical parameters in the smoking group. smoking has adverse effect on periodontal therapy; therefore the dentist should cooperate with patients in counselling of smoking cessation to achieve better results in the treatment of periodontitis. objectives: laminin (ln), which is a large multidomain glycoprotein of the extra cellular matrix, has attracted much attention because of its importance in many cellular functions, including induction of cell adhesion, growth promotion and mediation of cell communication. the target of this study was to find out whether there is any relation between the levels of serum ln and the inflammatory activity of a microbial infection. patients/methods: from june to october , immunocompetent adults, with confirmed bacterial infection were admitted to our hospital ( with pneumonia, with pyelonephritis and with cholecystitis) (group ). at the same time hospitalised patients for non-infectious causes (stroke, gastrointestinal bleeding, anaemia) were also studied (group ). the levels of serum ln and crp were measured on the day of admission in both groups. the levels of ln were measured using an enzyme immunoassay kit (takara laminin eia kit) and healthy volunteers were used to determine its normal limits ( - ng/ml). plasma crp concentration was assessed by immunoturbidometric method (using randox, uk kits). normal values were considered those below . results: the mean serum ln levels of patients of group were . ± . (much higher that the normal limits), while the mean crp value was . ± . . the mean corresponding values in group were . ± . for ln (within normal limits) and . ± . for crp. there is a statistically significant difference between the mean ln levels of the two groups (p < . ). additionally, there is a statistically significant correlation between the levels of ln and crp (a well studied serum inflammatory marker) in patients with bacterial infection (group ) (pearson correlation coefficient r = . , p = . ). conclusions: the definition of the ln levels could constitute a new reliable, simple, direct serum marker for the confirmation of an active bacterial infection. additionally, as the crp levels are above normal in group too (patients without infection) while ln lies within normal limits, maybe ln is even more specific than crp. more studies are required in the future, with more patients included, in order to confirm the outcome of this study. performance and clinical significance of a direct tube coagulase test using serum separator tubes for rapid identification of staphylococcus aureus from blood culture broth d. kwa, t. schü lin-casonato, p. sturm (nijmegen, nl) objective: blood cultures are important in the diagnosis of serious infections. early administration of effective antibiotics is associated with improved patient outcome. the performance of the direct tube coagulase (dtc) using serum separator tubes (ssts) for rapid identification of s. aureus from blood culture broth (bcb) was investigated. the clinical significance of rapid identification was assessed. methods: consecutive blood cultures with gram-positive cocci in clusters were tested. bcb was collected in ssts using a subculture-venting unit. after centrifugation, the supernatant was discarded and ml rabbit plasma was added to the remaining pellet of bacteria. coagulation was evaluated after and hours incubation at °c, and after overnight incubation at room temperature. in parallel, a direct tube coagulase test was performed using a : saline dilution of bcb as described previously. isolates were identified by standard microbiology procedures. clinical significance was measured by comparison of antimicrobial prescription based on gram stain results, direct coagulase results, and culture results. results: over a -week period, bcbs from patients were tested. s. aureus was present in bcbs. using the serum separator tube method and the saline dilution method, the sensitivity of the dtc after hours incubation was % and %, and after hours % and %, respectively. the specificity of both methods was %. rapid identification of s. aureus resulted in initiation (n = ) or streamlining (n = ) of antimicrobial therapy in of patients with s. aureus bacteremia. rapid identification of coagulase-negative staphylococci resulted in changes in antimicrobial therapy in of patients. conclusion: the dtc using ssts for bacterial enrichment is a very reliable, rapid, cheap and easy to perform method for identification of s. aureus from bcb. implementation of this test can improve antimicrobial therapy. evaluation of the results of the spanish seimc external quality control program for the diagnosis of enterococcus faecalis and klebsiella pneumoniae infections r. guna, j.l. pérez, n. orta, c. gimeno on behalf of seimc objectives: to evaluate the results obtained from four shipments of two different strains by the participants in the seimc external quality control program (eqcp). these controls were intended to analyse the percentages of correct species identification and the ability of the participants in detecting some special features of the control strains: vanb phenotype in the case of e. faecalis, and the production of extended spectrum betalactamase (esbl) in k. pneumoniae. methods: the same strain of each microorganism was sent in two different shipments. the vanb e. faecalis strain was sent both in a control of year as well as in other of , while the esbl-producing k. pneumoniae was sent in and in to an average of laboratories. the results obtained were compared with those of a reference laboratory that certified both the species identification and the resistance features. results: in the control, . % of participants identified correctly e. faecalis, while . % did it in . as for the glycopeptide resistance pattern of the enterococcal strain, . % and . % of participants detected the vanb phenotype in and , respectively. overall, the k. pneumoniae strain was correctly identified in both separate controls by most of the participants ( . % and . %, respectively). interestingly, the percentage of laboratories that detected the presence of the esbl in the k. pneumoniae strain sharply increased from . % in to . % in . the overall percentages of correct species identification were high for the two microorganisms and for both control points. most important, the ability of the spanish clinical laboratories in detecting the special resistance features of these strains clearly improved along the study period. these data confirm the importance of implement a continuous surveillance of the diagnostic training in the clinical laboratory, as well as the possible positive intervention of the seimc external quality control program in such improvement, since the analysis of results is accompanied of updated reviews on the subject of each control. a. bonnet-pierroz, a. resenterra, o. péter (sion, ch) objective: to evaluate new elisa ridascreen Ò borrelia igg and igm for antibody response in patients with confirmed lyme borreliosis and to compare to the results of vidas lyme (igg-igm) and in-house immunoblots (b. garinii igg and igm for early cases or b. burgdorferi sensu stricto, b. afzelii, b. garinii, b. valaisiana igg for late cases). methods: elisa ridascreen Ò borrelia igg and igm was used to screen sera from patients with clinically confirmed erythema migrans em (n = ). patients with confirmed neuroborreliosis by intrathecal antibody synthesis (n = ) were evaluated for igg antibodies to borrelia. sera from patients with acrodermatitis chronica atrophicans aca (n = ) and sera and synovial fluids from patients with lyme arthritis (n = ) were also evaluated for igg antibodies. patients with syphilis (n = ) and infectious mononucleosis (n = ) were screened for igg and igm antibodies to borrelia in order to estimate the specificity. conclusion: the elisa ridascreen Ò borrelia igg and igm have shown a good sensitivity for the serological diagnosis of lyme borreliosis. the short evaluation for the specificity of the igg test revealed a good assay with few false positive reactions, whereas the igm assay was, as expected more prompt to give false positive results with sera from patients with infectious mononucleosis. so far any equivocal or positive tests should be confirmed by immunoblots. is it necessary to incubate the bact/alert blood culture bottles more than days? objective: to assess the incubation time reduction of the aerobic and anaerobic bact/alert system bottles from to days. methods: from to we processed . blood culture sets and detected . ( %) positive blood cultures with clinical significance. we retrospectively examined the detection time of positive bottles and assessed the clinical significance of the bottles that were positive between the fourth and fifth day. results: out of positive blood cultures with clinical significance, ( . %) were detected within the first days of incubation. out of the positive blood cultures detected between the fourth and fifth incubation days, were recovered in concurrent cultures within the first days. chart reviews were conducted from patients with the remaining isolates. only in patients ( . % positive blood cultures) changes in antimicrobial therapy based upon the positive blood culture results on day to were made, in the other patients the empirical treatment was adequate. the isolated microorganisms in those patients were: gram-positive cocci ( staphylococcus spp. not s. aureus, staphylococcus aureus, streptococcus viridans and streptococcus pyogenes), anaerobes, enterobacteriaceae, pseudomonas aeruginosa, campylobacter spp., candida spp. cryptococcus neoformans, brucella spp. and haemophilus influenzae. conclusions: incubation of bact/alert blood cultures bottles only for days would have represented a detection loss of . % of the clinically significant isolates, which led to antimicrobial therapy changes. although we keep employing a -day incubation for routine blood cultures, we could reduce the incubation time to days depending on current instrument capacity. an enzyme immunoassay for anti-diphtheria antibodies: a practical alternative to the vero cell assay r. budd, e. harley, r. george, a. efstratiou, k. broughton, a. bradwell (birmingham, london, uk) introduction: in this extended study, results from an anti-diphtheria toxoid enzyme immunoassay (eia), specifically designed to detect higher affinity antibodies, were compared with those from a vero cell assay (vca). methods and results: serum samples with antibody concentrations ranging from . - . iu/ml on the vca from the respiratory and systemic infectious laboratory (rsil) were assayed by eia (the binding site ltd, uk). a further samples from rsil selected on the basis of being close to the protective level, were assayed to confirm the performance of the eia. the eia was calibrated, against the nibsc reference material / and the assay measuring range was . - . iu/ml results were compared using the who guidelines of . - . iu/ml as minimum protective level, and > . iu/ml as protective. relative agreement, sensitivity and specificity for the first samples were: . %, . % and . % respectively, for the second set of samples performance was: . %, . % and . %, and for the combined samples results were: . %, . % and . % respectively. roc analysis of the total samples confirmed the highest sensitivity . % and specificity . % occurred at a cut-off of precisely . iu/ml for the elisa assay. conclusion: of the total discrepant samples, had vca and eia values < . iu/ml, therefore we suggest the possibility of establishing an equivocal zone for the interpretation of the eia results. if the test is part of a general immune status assessment a grey zone is not required. if undertaken to determine the requirement for immunization, the use of the equivocal zone is recommended. by applying these criteria in the eia, only one sample would have suggested inappropriate immunization, as indicated by a vca result > . iu/ml. because of the > % agreement between the two assays, significant advantages of cost and speed, ease of use and the potential for automation, the eia could therefore be considered as an alternative to the vca. evaluation of accuracy limits of countable colony-forming units on agar plates j. arbique, a. rendell, k. forward (halifax, ca) objectives: accurate colony counts are an essential component of many microbiology research projects and clinical laboratory processes. the suggested range of accuracy of colony-forming units (cfu) extends from to (standard methods for the examination of water and wastewater). this recommendation dates to , and fails to adequately address the numerous sources of inter-and intra-variability. without more detailed analysis it is difficult to estimate the sample size and number of replicates necessary to ensure accurate results. the purpose of this study was to determine the validity of accuracy limits for quantifying cfus on agar plates. methods: escherichia coli (atcc ) and staphylococcus epidermidis (atcc ) were used to prepare series of four organism densities ranging from approximately - cfu, on three different days. on each day, each of the densities for both organisms was plated on sba and viable organisms were counted following incubation. an average of the margins of error obtained over the days of testing was used to determine the reproducibility of agar plate counts, and to estimate the optimum number of replicate plates (sample size) required for each organism at each concentration. results: margins of error for both organisms were greatest with suspensions yielding approximately cfu, and lowest for suspensions yielding and cfu. nine replicate plates were required for a suspension of s. epidermidis yielding cfu to achieve the same margin of error as obtained with replicate plates at concentrations yielding - cfu. seven replicates plates were required for a suspension of e. coli yielding and cfu to achieve similar margins of error to those obtained with replicate plates at concentrations yielding cfu, and replicate plates at concentrations yielding cfu. conclusion: we found that the greater the concentration ( and cfu), the fewer replicate plates necessary to reliably estimate organism concentrations. the lower the organism density ( cfu), the more plates necessary to reliably estimate cfus. contrary to the recommendations described in standard methods for the examination of water and wastewater, cfu of were reliably reproducible. for greatest accuracy, experiments should be conducted so as to assure that colony counts are in the range of - . direct microscopy: a valuable instrument for diagnosis and prognosis of periodontal disease objective: to appreciate the composition of micro flora from periodontal pockets, using light microscopy and to compare it with clinical status. introduction: it is generally accepted that periodontal disease occurs when anaerobic gram-negative flora increase in number with the subsequent decrease of facultative anaerobe grampositive bacteria. in other words, the switch from gram-positive to gram-negative of sub-gingival flora has a pathologic significance and could be observed using direct microscopy. materials and methods: specimens sampled with sterile paper points from periodontal pockets and samples from clinical healthy persons were included in this study. each sample was diluted in . ml saline solution and, with a calibrated loop, was taken ll aliquots in order to prepare a smear for microscopic examination and for inoculation on solid media (columbia with % sheep blood). the smears were gram stained and the culture plates were incubated in anaerobic conditions ( h, °c) and in air ( h, °c). results: in % of samples from patients with periodontal disease, easily notable, high number of gram-negative bacteria at direct microscopy, associated with abundant growth in anaerobic condition and poor growth in air. in from healthy patients, the gram-negative flora was almost absent and gram-positive bacteria were in high number, correlated with the absence of bacterial growth in anaerobiosis and some growth in air.the presence of treponema spp. at direct microscopy was associated with deep and bleeding periodontal pockets. after few days of proper therapy, the good clinical status was well correlated with an increasing number of gram-positive bacteria. conclusions: ) using a diluted sample for microscopic examination, the value of the method increase, offering important information about the composition of sub-gingival flora. ) the good correlation between the clinical status and microscopic finding recommend it as an easy to use diagnostic method in dentistry. identification of species and glycopeptide resistance among enterococcal isolates by bd phoenix objectives: vancomycin resistant enterococci are emerging in europe necessitating their fast and accurate identification by the laboratory. there was an attempt to evaluate the performance of the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md.) for the correct identification of species and glycopeptide resistance in comparison to the gold standard of diagnosis, pcr, using a large collection of clinical strains. methods: a total of enterococcal isolates were tested by the bd phoenix sytem. these strains were isolated from faecal, urine, pus, blood and samples from other body sites cultures. a multiplex pcr was applied using different pairs of primers, specific for the identification of e. faecium, e. faecalis and the vana, vanb, vanc, vand, vang, vane glycopeptide resistance genotypes. susceptibility to the glycopeptides was also confirmed by the etest (ab biodisk, solna, sweden). results: according to the pcr, there were e. faecium (including vana-positive strains), e. faecalis (including vana-positive and vanb-positive strains) and e. cass/gall isolates. two strains were not identified and were excluded from the analysis. discrepant results between the multiplex pcr and the phoenix system were obtained for / isolates ( %) with similar rates amongst faecal ( / , . %) and the rest of the isolates ( / , . %). the most common discrepancies were the misidentification of e. faecium vana strains and e. faecalis strains as e. cass/gall by phoenix. two e. faecalis strains were incorrectly characterized as vancomycin resistant, two e. faecium strains were misidentified as e. hirae and e.cass/gall, respectively, and one e.cass/gall strain was reported as e. faecium resistant to both glycopeptides. thus, the sensitivity and specificity for the identification of e.cass/gall by phoenix were . % ( / strains) and . % ( / strains), respectively, while . % of vana strains ( / strains) were not recognized by this system. conclusion: this study demonstrates that the new identification system, phoenix, similarly to other automated or manual systems, presents with problems regarding correct identifica-tion of enterococcal species and glycopeptide resistance. specifically, laboratories should be aware that clinically significant isolates identified as e.cass/gall should be confirmed by another method. an audit of sputum requisition practices p. lal, i. balakrishnan (london, uk) objectives: to analyse the indications and rationale for the processing of sputum specimens in a london teaching hospital. methods: sputum samples received from / / - / / were included in this study. data were obtained from the patient requisition forms and the winpath systems and were analysed further as per the objectives. results: a total of specimens were received during this period. ( %) from hospital in-patients and ( %) from general practitioners. out of the total of samples received from hospital-in patients ( . %) had > epithelial cells/ lpf. no clinical details were mentioned in ( . %) and ( . %) were from patients already on antibiotics. repeat specimens within one week were sent in ( . %) cases and ( %) had atypical serology also sent. out of the hospital-in patient samples ( . %) had a significant isolate and ( . %) had normal respiratory tract flora isolated. others were reported as: ''gross oral contamination'' [ ( . %)], ''no growth'' [ ( . %)] and ''no significant growth'' [ ( . %)]. there were a few specimens reported as ''inappropriate specimen -two days old'' [ ( . %)] and ''leaking'' or ''saliva only' ' [ ( . %) ]. out of a total of samples received from gp patients ( . %) of samples had > epithelial cells/lpf, ( . %) had no clinical details provided, ( . %) samples were sent while patients were on antibiotics and ( . %) samples were repeated within one week. only ( . %) had atypical serology also sent. conclusions: -less than one-third of specimens yielded a significant pathogen.-adequate clinical details were lacking in about one-fifth of specimens.-nearly one-third of specimens were repeated within one week, without a clear indication.-about % of specimens were of poor quality.-atypical serology was only performed in . % of outpatients, as compared with % of in-patients.this audit brings forth the fact that the clinical indications for which sputa are being sent for culture need to be clearly defined and an educational campaign instituted amongst relevant healthcare professionals. sputum collection techniques need to be rigorously applied if good-quality specimens are to be obtained. indications for performing atypical serology need to be defined and reinforced, particularly in primary care. a new approach to laboratory diagnostic of infectious gastroenteritis -a follow-up objectives: in order to optimize use of laboratory facilities and ensure flexibility in relation to current epidemiology, a new approach to laboratory diagnosis of infectious gastroenteritis was applied: from an algorithm the decision of which organisms to test for was defined by the demographic, clinical and epidemiological information submitted to the laboratory on paper/electronic request forms. methods: from april , -june , , hospitals and general practitioners submitted a request form with the following information together with the stool sample (s): ( ) acute or persistent diarrhoea (duration > weeks); ( ) bloody stools; ( ) recent history of foreign travel; ( ) > patients within same epidemiological setting; and ( ) nosocomial infection. provision of data is mandatory when submitting electronically. based on these data, analyses were performed according to an algorithm. examination for salmonella, shigella, yersinia, campylobacter, and clostridium difficile was done by culturing. verotoxin producing e. coli (vtec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec) and enteroinvasive e. coli (eiec) were identified by pcr for virulence genes and serotyping. rota and adenovirus were detected by antigen tests and parasites by microscopy. results: in total we examined , samples from , patients. a pathogen was isolated in % of patients. in cases ( %) clinical/epidemiological data were missing. • , patients had diarrhoea < weeks: % with campylobacter, % with salmonella, % with etec, % with giardia, and % each with eiec and vtec • , patients had diarrhoea > weeks: % with campylobacter, % with giardia, % each with epec and vtec • patients had a history of foreign travel: % with campylobacter, % with etec, % with salmonella, and % with giardia • patients had bloody stools: % with campylobacter, % with salmonella, and % with vtec • patients were < years: % with campylobacter, % with epec, % each with giardia, vtec and salmonella, and % with etec conclusions: campylobacter was the most common bacterial pathogens in all groups and rotavirus was the most common pathogen in children < years. the new approach had a number of advantages: more relevant microbiological analysis, collection of data on defined patient groups, and flexibility regarding adaptation to current epidemiological knowledge. increasing use of electronic submission of request forms will optimize the approach used. objectives: small colony variants (scvs) are an emerging infectious disease problem, presenting as a naturally occurring, slow-growing subpopulation of staphylococcus aureus that are characterized by tiny colonies on solid media. studies on scvs recovered from patients with persistent infections are hampered due to their frequent unstable phenotype. in particular, scvs are not easily distinguishable from the normal phenotype in broth media and a reversion of scvs into the normal phenotype is not traceable. methods: a set of isogenic s. aureus isolates comprising the (i) normal and the (ii) scv phenotype (isogenic to the isolate with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding mutants mimicking the scv phenotype (knock-out of hemb), and (iv) their complemented mutants were used to investigate the feasibility of fourier-transform infrared (ftir) spectroscopy to trace the expressed phenotype in broth media. the respective isolates cultured on solid media served as controls. in addition, all isolates were genotyped by pulsed-field gel electrophoresis and spa typing. results: using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information in three different spectral ranges resulted in a dendrogram that showed a clear discrimination between both staphylococcal phenotypes. distinct clusters comprising the clinical and mutant scv phenotype on one hand and the normal phenotype (isolate with normal phenotype and complemented mutant) on the other hand were found. thus, scvs from different clonal lineages gave spectra that were more similar to one another than to their normal growth parent. ftir was also shown to be able to trace the switch of the phenotypes in broth when the medium was supplemented. conclusion: ftir spectroscopy allows a rapid, reproducible and clear discrimination of different phenotypes of s. aureus in fluid media for diagnostic and research purposes. in contrast to genotyping approaches, ftir staphylococcal fingerprinting is only reliable for typing purposes if the isolates exhibit the same phenotype. in future studies, this technique may also provide an approach for tracing the scv phenotype in infected tissues. objectives: triggering receptor expressed on myeloid cells- (trem- ) is a recently discovered cell surface molecule whose expression on phagocytes is up regulated by exposure to bacteria or fungi. a soluble form of trem- (strem- ) can be measured in various body fluids. we studied whether strem- in cerebrospinal fluid (csf) could serve as a biomarker for the presence and outcome in patients with bacterial meningitis. methods: in this retrospective study on diagnostic accuracy we used an elisa to determine levels of strem- in csf from adults with bacterial meningitis, confirmed by csf culture, who participated in the prospective dutch meningitis cohort study; patients with viral meningitis, confirmed by polymerase chain reaction of csf; and healthy control subjects, who underwent lumbar puncture to exclude the diagnosis of subarachnoid haemorrhage. the mann-whitney u test and the chi-square test were used to identify differences between groups. a receiveroperating-characteristic curve (roc) was constructed to illustrate various cut-off csf levels of strem- in differentiating between the presence and absence of bacterial meningitis and diagnostic accuracy was quantified by % confidence intervals ( % ci). results: levels of strem- in csf were higher in patients with bacterial meningitis as compared to those with viral meningitis [median, pg/ml (range, to pg/ml) versus . pg/ml (range, to pg/ml); p = . ] and controls [ pg/ml (range, to pg/ml); p < . ; fig]. patients with viral meningitis and controls had similar csf strem- levels. the area under the roc curve for discriminating between patients with and without bacterial meningitis was . ( % ci, . to . ; p < . ). at a cut-off level of pg/ml, strem- yielded a sensitivity of . ( % ci, . to . ) and a specificity of . ( % ci, . to . ). in patients with bacterial meningitis, csf strem- levels were associated with mortality [survivors versus nonsurvivors: median pg/ml (range, to pg/ml) versus pg/ml (range, to pg/ml); p = . ]. conclusions: measuring strem- in csf may be a valuable new approach to accurately diagnose bacterial meningitis and identify patients at high risk for adverse outcome. therefore, a prospective study on strem- as biomarker in bacterial meningitis is needed. systematic review of rapid diagnostic tests for enterohaemorrhagic e. coli i. abubakar, l. irvine, l. shepstone, s. schelenz, c. aldus, p. hunter (norwich, uk) objective: a variety of rapid tests for the detection of enterohaemorrhagic escherichia coli (ehec) have recently emerged. culture on sorbitol macconkey (smac) agar and biochemical identification, while easy to use and inexpensive, is slow and lacks sensitivity in the detection of non o :h serotypes. this study sought to determine the accuracy of rapid serological or polymerase chain reaction (pcr) assays which have been evaluated for the detection of all ehec serotypes compared to culture. methods: a systematic review and meta-analysis of articles, identified via searches of electronic databases, hand searching of selected journals, and through contact with experts and commercial test manufacturers. the majority of these needed to be excluded due to low quality or lack of accuracy data. sensitivity and specificity of each method was calculated using full biochemical identification as the reference standard. twenty-one studies met the inclusion criteria, of which used pcr methods and used serological assays and were based on culture. a summary receiver operator curve (sroc) was constructed from these data and the area under the curve (auc) calculated (using the trapezium rule). results: serological tests had individual sensitivities ranging from . to . and specificities ranging from . to . . pcr tests had individual sensitivities ranging from . to . and specificities ranging from . to . . additional analysis comparing smac agar culture with toxin detection methods showed poor sensitivity compared to pcr and serological tests (ranging from . to . ) yet the specificity was very good ( . for all studies considered). our results suggest that both molecular and serological tests may have a potential role in detecting ehec infection. whilst there is very little difference in the effectiveness of these techniques, both are faster and have improved sensitivity when compared to traditional culture methods. fast, reliable diagnosis could lead to more informed treatment choices and improved outbreak control measures. however, given the substantial extra cost of these assays, an assessment of economic feasibility is necessary prior to use in everyday practice. antibodies against bordetella pertussis detected by slow agglutination test and elisa in two agerelated groups of vaccinated people suspected of acute pertussis: a comparative study objective: the aim of presented study was to describe differences between results of two tests used for detection of antibodies against bordetella pertussis (slow agglutination and elisa) in two age-related groups of patients suffering from respiratory infection. each of the people has undergone vaccination against b. pertussis. methods: paired sera obtained from two age-related groups of patients [( ). age - years, n = ; ( ). age above years, n = ] suffering from acute respiratory infection were tested. the first group comprised the children who were vaccinated earlier than one year before testing; the second group was determined by longer interval between the vaccination and the testing. the criterion of positivity of the slow agglutination was based on quadruple increase/decrease of the titer of specific antibodies; the criterion of serodiagnosis of the illness was the same. each of the patients was tested by elisa iga,igg,igm (virotech) during the same period, positive results of each of class of immunoglobulins were evaluated as positive elisa. the differences of results obtained by the two tests were assessed inside and between the groups. results: there were found . % (respective . %) concordant positive and . % (respective . %) concordant negative results between the tests in the first (respective second) group. there were found the following discrepancies in the frame of non equal results: agglutination positive/elisa negative sera were present in . % (respective . %) persons and agglutination negative/elisa positive samples were present in . % (respective . %) persons. conclusion: ( ) . the frequency of serologically confirmed infection based on results of slow agglutination is higher in the group of people older six; the interpretation of the results in the younger group is limited by the influence of actual vaccination. ( ) . the elisa evaluated as described above shows extremely high frequency of positivity in both groups, thus, the usefulness for diagnostics of acute infection seems to be low. ( ) . the study will be continued to asses relationships between the positive results detected by slow agglutination and the positive ones detected by elisa in separate classes of specific immunglobulins. accuracy of the microscan walkaway system to identify coagulase-negative staphylococci a. sáez, b. ruiz, l. martínez-martínez (santander, es) objective: to determine the reliability of the identification of coagulase-negative staphylococci (cons) with the microscan walkaway (wa, dade behring) system at species level when a > = % probability is obtained, considering as a reference the results of molecular identification. methods: one hundred and sixty-eight isolates of cons from clinical samples (october -may for which the identification with the wa system was ‡ %, and atcc type strains were evaluated. bacteria were identified with the wa system using pos combo s panels. absence of coagulase was determined with a latex assay (pastorex Ò staph-plus, bio-rad). reference identification was established by sequencing of the s rrna; when identification with wa and s rrna disagree, definitive identification was defined after sequencing of the soda and tuf genes, as previously described (drancourt et al. jcm ; : - and heikens el al. jcm ; : - ) . for identification, the sequences of s rrna, soda and tuf were compared with those in genebank. homologies values above % were considered reliable. results: all type strains were correctly identified by s rrna sequencing as named by the atcc. among the clinical isolates, the molecular method identified the following species (number): s. hominis ( ), s. haemolyticus ( ), s. saprophyticus ( ), s. epidermidis ( ), s. lugdunensis ( ), s. schleiferi ( ), s. capitis ( ), s. simulans ( ), s. pasteuri ( ), s. warneri ( ), s. intermedius ( ) and s. equorum ( ). the wa system correctly identified out of the atcc strains. s. pasteuri is not included in the wa database, and the corresponding atcc strain was misidentified as s. warneri. one hundred and fiftyseven out of the ( . %) clinical isolates were correctly identified by the wa. five s. haemolyticus were identified by wa as s. auricularis ( ), s. simulans ( ) and s. warneri ( ) . other errors corresponded to: two s. pasteuri misidentified as s. warneri, one s. epidermidis as s. hominis, one s. lugdunensis as s. schleiferi, one s. hominis as s. haemolyticus and one s. equorum as s. cohnii. all isolates of s. saprophyticus, s. schleiferi, s. capitis, s. simulans, s. warneri and s. intermedius were correctly identified by the wa system. conclusions: the microscan walkaway is reliable to identify cons at species level when a probability of > = % is obtained. s. pasteuri should be incorporated to the wa database in order to improve its performance. objectives: the aim of this study was to analyse the results of proficiency testing obtained by polish microbiology laboratories participating in polmicro. haemophilus influenzae is an important pathogen causing a variety of community-acquired respiratory tract infections, acute otitis media and purulent meningitis. two mechanisms of ampicillin (amp) resistance in this organism are described. one is mediated by the production of beta-lactamases tem- and rob- ; these amp-resistant strains are termed beta-lactamase-producing, amp-resistant (blpar). the second mechanism involves development of altered penicillin-binding proteins (pbp) with decreased affinity to amp and other beta-lectam agents. strains with resistance mechanisms mediated by pbp alterations are termed beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae. methods: four hundred seventy eight laboratories participated in this part of the scheme. each participating laboratory received haemophilus influenzae (pm- )-beta-lactamase negative, ampicillin-resistant strain (blnar). the laboratories were asked to provide identification to the species level and of the susceptibility results and interpretation. results: correct identification to the species level of this strain was reported by laboratories ( . %) of the labs involved. thirteen laboratories reported the analysed strain as haemophilus parainfluenzae. three hundred ninety eight laboratories ( . %) of correctly detected the mechanism of resistance to beta-lactams. only three laboratories incorrectly reported the organism as beta-lactamase producer. the greatest dispersion of inhibition zone was observed in the susceptibility of h. influenzae to ampicillin, amoxicillin-clavulanic acid and clarithromycin. conclusions: over % of the laboratories correctly identified and interpreted beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae strain. purpose and methods.the architect syphilis tp assay is a chemiluminescent eia that employs three recombinant antigens of treponema pallidum on the solid phase and an anti-human igm and igg conjugate. we evaluated this assay in comparison with a conventional eia (diesse enzywell syphilis screen recombinant) on unselected routine serum samples and on repository specimens for whom the results for specific igg and igm and of the rapid plasma reagin (rpr) assay were already known. in both instances an immunoblot (ib: inno-liatm syphilis score, innogenetics) has been employed on discordant specimens as a confirmatory assay. the precision and robustness of the architect assay were also evaluated.results.on . routine samples ( from volunteer blood donors and from in and outpatients) the concordance between architect and eia was high ( . samples, or . %; positives, , negatives). one of the discordant, positive by architect and negative by eia, was confirmed by ib. the specificity of the architect assay was . % ( % confidence limits: . - . ). the repository samples assayed belonged to three groups: ) biological false positives from subjects: all negative by architect; ) true positives, all positive by architect, with a significantly stronger signal (average s/co: . vs. . ) on the igm positive samples, all of whom were also positive by rpr; ) samples positive by rpr and negative by eia igg: of them were negative by architect as well and for igm, while two specimens were strongly positive by architect and positive also for specific igm and with three specific bands by inno-lia, suggesting a pattern of recent infection. the reproducibility of the architect assay was good, with cvs of . %, . % and . % on replicates over weeks of the assay's negative and positive control and of an internal control; finally, the s/co distribution of negative specimens confirmed the robustness of the assay, with a mean of . , a median of . , standard deviations between the mean and the cut-off value and the th percentile at a s/co value of . .conclusion.the automated assay for anti-treponema pallidum antibodies on the architect system has an excellent sensitivity and a good specificity. the analytical performances, coupled with the elevated throughput and minimal samples handling, make this method a first-choice option for syphilis screening and diagnosis in medium and large volume laboratories. objective: quantitative urine culture is the gold standard for defining the diagnosis of urinary tract infection (uti), because it allows identification of the uropathogenic species. however, this method is time consuming and expensive. approximately, up to % of urine cultures are negative with high cost for unnecessary testing. thus, we have evaluated the usefulness of two automated analysers for uti screening to quickly identify the negative samples that can be prompt reported to the clinicians, improving in the quality of patient care and allowing the laboratory to direct more effort into positive samples. methods: . of midstream urine samples submitted for microbiological examination were analysed by conventional urine culture plates (mcconkey agar + trypticase soy agar + bile esculine azide), sysmex uf- (sysmex, japan) and coral uti screen (coral biotechnology, ca, usa) automated analysers. uti was defined positive as follows: one or two strains of bacteria with at least ufc/ml for the culture plates, more than . bacteria/ll and more than wbc/ll for the uf- and/or an rlu value grater than % of the calibrator value for the coral. when more than two strains of bacteria were found, the culture was classified as contamined. results: the diagnostic performance of sysmex uf- and coral uti screen are shown in table . the sensitivity ( . %) and negative predictive value ( . %) confirm that sysmex uf- and coral uti screen are an excellent screening for uti. after this evaluation, we decide the use of the sysmex uf- and coral uti screen on our routine workflow for uti screening. the results of both the analysers are sent to a software system (labfinity dasit, italy) connected to the lis. if the results are lower than the cut-off values, uti can be excluded and directly reported to the physician. positive results are submitted to microbiological culture and reported within or hours depending on negative or positive bacterial growth. in our experience, evaluated on further . samples, this means that % of samples are immediately reported within very few hours. of the % of positive samples, ( %) were confirmed by culture and reported within hours, ( %) were not confirmed and reported within hours. comparison of the blood and bone marrow culture positivity rates for the diagnosis of brucellosis objectives: brucellosis is a common disease, seen worldwide as well as in our country. the diagnosis of brucellosis is made with certainly when brucellae are recovered from blood, bone marrow. in our study, we aimed to compare the blood and bone marrow culture positivity rates in patient with brucellosis. methods: this study was performed in the infectious diseases and clinical microbiology department of ankara research and training hospital between and . the diagnosis of brucellosis was made on the history, physical findings, serologic findings and the isolation of the organism. the number of patients with brucellosis included to the study was . blood and bone marrow samples were taken from all of the patients on admission and cultured by using the bactec system. results: blood culture positivity for brucellosis was % ( / ), while bone marrow culture positivity was % ( / ). the difference between those positivity rates was found to be statistically significant (p < . ). the isolation ratio from blood cultures among acute cases was % ( / ) while it was % ( / ) among subacute cases. brucella isolation from blood was not detected in chronic cases. the isolation rates of the microorganism from bone marrow of acute, subacute and chronic cases were . %, . %, . % respectively. among our patients, had history of medical therapy for brucellosis before admission and of them was treated inadequately. of those cases, the organism was isolated in ( %) from blood and in ( %) from bone marrow.in the cases with high standard tube agglutination titers, the rate of positivity was also high both in blood and bone marrow cultures. however when compared with low standard tube agglutination titers, that difference was not statistically significant.the mean growing time for the positivity of cultures was . days for bone marrow and was . days for blood cultures. the difference between the mean growing times of two culture types was found statistically significant (t-test. p < . ). conclusion: premedication, subacute and especially chronic phases decrease the possibility of isolation of the microorganism from blood culture. therefore we suggest taking bone marrow culture only for these kinds of patients as it. is a traumatic process. serological findings in blood sera of patients with yersinia-triggered arthritis e. golkocheva, r. stoilov, h. najdenski (sofia, bg) objectives: immunoblot analysis of iga and igg antibody response of blood sera from patient with yersinia triggered reactive arthritis and with undifferentiated arthritis were made. patients and methods: serum samples were obtained from patients admitted to clinic of rheumatology at medical university, sofia, bulgaria with suspicion of yersinia triggered reactive arthritis, based on diagnostic criteria. a total of blood serum samples were analysed by immunoblot analysis with specific antigens-yops (yersinia outermembrane proteins). when y. enterocolitica is cultivated at o c under calcium restriction ( . mm ca + ), large amounts of yops are secreted into medium. these proteins were separated by d-sdselectrophoresis. results: immunoblot analysis of iga and igg antibody response against yops in blood sera from patients with arthralgias and polyarthropathies was carried out. yersinia enterocolitica, serotype o: , was used as source for yop. seven strong bands of the molecular weights kda-yope, kda-yopn, kda-yopd, kda -v-ag, kda-yopb, kda-yopm and kda-yoph were visualized. for immunoblot assay the optimal concentration of antigen was established by analytical electrophoresis. of the blood sera from the patients with yersinia triggered reactive arthritis igg antibodies were detected against yoph, yopm, yopb, yopd, yopn and yope. iga antibodies were established against yopm, yopb, yopd, yopn and yope. all sera from the patients with other rheumatic diseases were negative for the presence of anti-yersinia iga antibodies and two of them were positive for igg against yopd. antibodies from two classes were not detected in sera samples from healthy people. conclusions: yops are borne by the virulence plasmid, which mean that they are clearly associated with virulence properties of pathogenic strains. moreover, yops is not restricted to single serotype and this made them a specific antigen in diagnosis of different yersinia infections. conventional techniques such as culture and demonstration of serum agglutinins prove to be insufficient to demonstrate invasive or chronic yersiniosis in contrast with the determination of specific serum iga and igg antibodies by immunoblot analysis and antigen detection. the detection of anti-yops igg and iga antibodies by immunoblot can be used for diagnosis of yersinia triggered arthritis. acknowledgements: this work was sponsored by natoreintegration grant . objectives: to evaluate the identification and susceptibility results by using suspensions obtained directly from positive blood cultures. methods: during the period between st august and st october we selected all positive cultures grown in bact/ alert Ò sa and sn bottles (biomérieux) from gram-negative bacilli. only the first culture positive from each patient was included. we inoculated ml fluid from a positive bottle into a serum separator tube (bd vacutainer systems, plymouth, united kingdom) and centrifuged at x g for minutes and the supernatant was carefully aspirated. using a cotton swab the bacteria were removed from the top of the separator layer to be suspended in . % saline solution to get . mcfarland. the suspension was processed according to standard inoculation procedure for gn and ast-n vitek Ò cards. positive bact/alert d bottles were also sub cultured and after an overnight incubation several colonies were used to make a . mcfarland suspension in . % saline. the suspension was processed according to standard vitek Ò inoculation procedure for gn and ast-n cards. results: identification: a total gram-negative bacillus from positive blood cultures were investigated. fifty ( . %) strains were correctly identified to the species level, four ( . %) strains were not identified and two ( . %) strains were misidentified. antimicrobial susceptibility testing: in all, mics were determined for isolated by both methods. the unidentified strains ( ) were excluded. the overall mic agreement between direct and standard inoculation was . %. all individual antimicrobial agents scored > %. the overall minor error rate was . % ( of ). the overall major error rate was . % ( of ). the overall very major error rate was . % ( of ). the highest rate of mic agreement was for amikacin, norfloxacin ( %), meropenem ( %), gentamicin and ofloxacin ( . %). conclusion: the direct method from positive bact/ alert&# ; cultures cannot totally replace the approved methods of identification and susceptibility but in some cases provides earlier information which allows a better patient management and also reduce cost in patient care. investigation of listeria monocytogenes "o" antibodies in maternal and cord sera with the agglutination test e. us, a.t. cengiz, o. gelisen (ankara, tr) objectives: listeria monocytogenes is a gram-positive food borne pathogen that is responsible for listeriosis, a human infection with a mortality rate of %, which could cause severe motherto-child infections. this serious pathogen in pregnancy could be treated if diagnosed, but there is no routine screening test for susceptibility to listeriosis during pregnancy. therefore, we investigate different l monocytogenes serotype o antibodies for diagnosis of listeriosis in maternal sera with agglutination test. of them had spontaneous abortion, premature labour or stillbirth (group i), while had no obstetric patology (group ii) in their previous pregnancies. cord bloods were also obtained at the delivery and tested. methods: all sera were being tested against antigens with the o formulation of serotype / c, b, ab, c and d. the antigens were prepared by the method of osebold, and larsen et all. the bacterial suspensions were trypsinized for min at °c to prevent cross-reactions and contaminations. sera were diluted by doubling serially in saline followed by addition of an equal volume of antigen. a positive titre of greater than or equal to : was chosen as positive test result to maximize the sensitivity and specificity. results: . % of cases have ingested raw milk and diary products, . % ready-to-eat foods, and . % developed nonspecific febrile illness (nfi) during their pregnancies. % of group i were found positive ( . % developed nfi) while at group ii % had positive ( . % developed nfi) agglutination titres to one ore more serotypes. all the cord blood sera of group i were found negative, whereas two in group ii (all ab) were positive, with the positive maternal sera of the same serotype. it's evaluated as transmission of the antibody from mother to foetus. at group i the frequent serotypes were / c = ab, at group ii ab, / c, respectively. the newborns showed no symptoms or signs of listerial foeto-maternal infection. conclusion: the women encountered the antigens of l monocytogenes in any period of their life time (most - years of age) and produce antibodies against this pathogen. there is a relationship between nfi and positive titres. if the disease is recognized, it is possible to treat the mother and allow the birth of a healthy infant. we propose the less time consuming and easy to perform agglutination test as a routine screening test for susceptibility to listeriosis during pregnancy to prevent bad pregnancy outcomes. objectives: to evaluate the performance of a real time pcr assay (with a fluorogenic target-specific probe), mrsa-idi (geneohm sciences) for mrsa detection directly from mucocutaneous swabs in hospitalized patients. methods: clinical swabs ( to samples with a median of . samples per patient) from nares (n = ) and skin (n = ) were prospectively collected for mrsa screening from patients admitted to a -bed teaching hospital. swabs were inoculated onto selective mrsa agar (mrsa-id, biomérieux), into the buffer extraction solution for idi-mrsa pcr assay and into enrichment broth (bhi with . % nacl). after h, bhi broths were subcultured onto mrsa-id agar. selective agars were incubated for h at ordm;c and examinated daily. suspected colonies were identified by coagulase testing; oxacillin resistance was tested by cefoxitin disk diffusion according to clsi recommendations. the pcr assay was performed according to the manufacturer's instructions. pcr results were compared with phenotypic identification test results. in case of discordant results, the assay was repeated, but only results from first testing were considered for calculating test performance. results: mrsa was detected by culture in specimen ( . %) from patients. the sensitivity and specificity of the pcr compared with culture was . % and . %, respectively. positive predictive value and negative predictive value were . % and . %, respectively. the sensitivity of pcr ( %) was higher on nasal swabs than on swabs from other sites ( . %, p < . ). the pcr assay detected mrsa in patients ( . %). the pcr assay provided results in to versus to hours for conventional method. conclusion: in our hospital, the id-mrsa pcr assay detected . % mrsa carriers in less than hours when performed on multiple specimen. the assay appeared more sensitive in testing nasal swabs than other clinical specimens. prospective studies are needed to evaluate the impact of this assay for rapid implementation of infection control procedures and its global costs and benefits. the purpose of this study was to establish a rapid and sensitive real-time polymerase chain reaction (pcr) method for detection of methicillin-resistant staphylococcus aureus (mrsa) from blood culture bottle. as a result of over use of broad-spectrum antibiotics after the s in whole the world, an outbreak of mrsa infection has been seen. severe nosocomial infections with mrsa such as bacteraemia and sepsis may lead to multiple organ failure and high mortality in the hospital. although standard method took at least hours to identify mrsa by the blood culture method, the presence of meca and nuc genes which is specific for methicillin resistance and s. aureus was determined by real-time pcr method within only hours after blood culture signal positivity. nineteen s. aureus and coagulase negative staphylococci positive blood culture bottles were studied retrospectively for detection of s. aureus and methicillin resistance. staphylococci were identified with classical methods and mics of oxacillin were determined by etest (ab biodisk) on mueller-hinton agar supplemented with % nacl. real-time pcr was performed to all positive blood culture samples for s. aureus and methicillin resistance determination. nineteen ( %) s. aureus were determined correctly by real-time pcr method. forty-four methicillin resistant and methicillin sensitive staphylococci were detected by etest. using the real-time pcr method, the meca gene was detected in staphylococci except . when compared with etest and realtime pcr method gave sensitivity, specificity, and positive and negative predictive values of %, %, %, % for both positive and negative tests, respectively. agreements between two methods were high ( %); there were discrepant results among the strains were tested. detection of mrsa bacteraemia and methicillin resistance with real-time pcr definitely is useful for reducing mortality and morbidity of this type infection. in conclusion, this method, as many as sensitive and specific for detection of mrsa bacteraemia and clinically should be beneficial for prevention of unnecessary antibiotic use and determination of appropriate antibiotic treatments of mrsa infection. pcr detection of class b, c and d betalactamases in environmental and clinical aeromonas strains t. fosse, c. giraud-morin, f. la louze (nice, fr) objectives: aeromonas spp. strains are waterborne opportunistic pathogens. they are able to produce different types of beta-lactamases (class b, c and d). the determination of beta-lactamase content is not easy by phenotypic methods. we have developed a pcr tool to study diversity and distribution of class b, c and d beta-lactamases in a set of representative clinical and environmental aeromonas species. method: a total of references, environmental and clinical strains were tested. identification was realized by conventional tests and gyrb sequence analysis. beta-lactam antibiotic susceptibility was determined by diffusion agar and micro broth dilution methods. three sets of specific primers were defined for the pcr amplification of the internal region of class b beta-lactamase (mei and mei , bp size), class c beta-lactamase (aercp and aercp , bp) and class d betalactamase (aerd and aerd , bp). all pcr products were sequenced. results: class d pcr was positive with most strains except a. trota, a ticarcillin susceptible species ( strains) . class c pcr was positive with most cephalothin resistant strains (mic > mg/l; / strains, %) including a. hydrophila and a. caviae phenospecies. class b pcr was positive with most strains of a. hydrophila and a. veronii phenospecies ( / ; %) including three imipenem susceptible strains (mic < mg/l). beta-lactamase type distribution was species related and was particularly useful to better characterize environmental species such as a. bestiarum, a. popoffii and a. allosaccharophila. partial beta-lactamase gene sequence analysis allowed phylogenic studies. some cephalosporinase gene from environmental species was probable progenitor of ampc plasmidic beta-lactamase. conclusion: pcr with specific primers was a good method to detect class b, c and d beta-lactamase in aeromonas species. beta-lactamase type distribution and sequence analysis phylogeny were largely species related and could be helpful for molecular diagnostic and taxonomic purpose. objectives: the aim of this study was to develop a convenient dna extraction method and to optimise a pcr reaction in order to detect enterotoxin b producing s. aureus strains directly from milk. methods: we applied a chemical extraction method of bacterial dna from milk samples artificially inoculated with s. aureus. a pcr based method was used for the detection of seb gene (coding for enterotoxin b) and nuc gene (coding for termonuclease). a protocol for the multiplex pcr was developed and optimized. the sensitivity of the reaction was checked by determining the minimum number of organismsaeml - , which can be detected in the multiplex pcr and in each single pcr reaction. amplification specificity of the seb gene was verified by amplicon digestion with restriction endonucleases. results: the specific bands for both genes in the multiplex pcr were detected in samples containing a dna quantity corresponding to organismsaeml ) . in the same reaction, the amplicon for nuc gene was visible for as little as the dna concentration corresponding to organismsaeml ) . the sensitivity of each single pcr reaction was similar with those of multiplex pcr reaction. conclusion: the applied dna extraction method allowed us to obtain a good quality dna and can be used for a direct milk extraction. multiplex pcr reaction is a simple, rapid and reliable method for detecting enterotoxin b producing s. aureus strains from milk. objective: to detect the resistance to fluoroquinolones in acinetobacter baumannii strains by a pcr-rflp assay. methods: thirty a. baumannii clinical isolates were obtained from different specimens (bronchial aspirates, blood-cultures, catheters, etc.) . the mics (minimal inhibitory concentrations) for ofloxacin were determined by agar dilution following standard methodology.a pcr-rflp method using one primer pair for amplification of a bp fragment related to gyra gene (which codifies subunity a of dna-gyrase) and using one restriction enzyme hinf i was developed to study the resistance to ofloxacin in the different a. baumannii strains. when an a. baumannii strain is resistant to fluoroquinolones, a mutation in the position ser of the dna-gyrase has been detected, decreasing the affinity for the antimicrobial. agarosa gel was used to determine the dna pattern: fragments of bp and bp when there is not mutation and fragment of bp when the ser to leu mutation is present. results: the relationship between the pcr-rflp pattern and the mic to ofloxacin is shown in the table . the results of pcr-rflp analysis of most strains were in agreement with the results of mic. one isolate was susceptible to ofloxacin by agar dilution (mic = . mg/l) whereas by pcr-rflp this isolate seems to be resistant because it presents the mutation in gyra gene. two isolates with intermediate mic ( mg/l) showed mutation in gyra. the genotypic study by pcr-rflp proved that ofloxacin resistant a. baumannii strains showed a punctual mutation in gyra gene, in the same position inside the sequence of gene. evaluation of a rapid amplification-detection assay for the identification of vancomycinresistant enterococci j. fuller, l. turnbull, s. shokoples, b. lui, l. rosmus, r. rennie (edmonton, ca) objective: the routine identification of vancomycin-resistant enterococci (vre) in clinical laboratories often yields a lengthy turn-around-time that may impede infection control efforts, particularly in an outbreak situation. in search of an improved vre test, we evaluated the genotype Ò enterococcus assay (hain lifescience, germany), which provides both species and van gene identification for vre, and compared the results to conventional methods. methods: forty clinical enterococcal strains isolated on vrescreen agar media were selected for study. lactococcus and pediococcus were used as negative controls. conventional testing involved basic culture and identification tests, e-test susceptibility testing for vancomycin and teichoplanin, and pcr for vana, b, and c genes. the genotype Ò enterococcus assay involved multiplex dna amplification and reverse hybridization of amplified product on an immobilized dna strip-blot containing probes for e. faecium, e. faecalis, e. casseliflavus, e. gallinarum, vana, vanb, vanc , and vanc / . the genotype Ò enterococcus assay produced correct species and van gene identification for all ( %) vre isolates, including e. faecalis vanb, e. faecium vana, e. faecium vanb, e. gallinarum vanc , e. gallinarum vana-vanc , and e. casseliflavus vanc / . the only minor discrepancy was an e. casseliflavus that hybridized very weakly with the vanc probe in addition to the expected vanc /c probe. the costs per specimen were comparable for each test method. however, the genotype Ò enterococcus assay could be completed within a normal working day in contrast to conventional testing, which required a minimum of two days from the point of isolation on the vancomycin-screen media. conclusion: from this preliminary evaluation, the genotype Ò enterococcus amplification-detection assay provides vre species and van genotype identification in a rapid and costeffective manner, superior to conventional culture methods. although further study is required, this kit may have clinical utility during a vre outbreak. application of minimal sequence quality values prevents misidentification of blashv type in single bacterial isolates carrying different shv extended-spectrum beta-lactamase genes background: detection of extended spectrum beta-lactamase (esbl) genes by pcr and sequence analysis is the gold standard for detection of shv-type beta lactamases. usually, quality values of sequence analyses are not reported. during a study on esbl epidemiology, three strains for which the default sequence assembly showed an shv) or shv- gene, showed low quality values at certain positions in individual sequence traces. we investigated the reason for these lower values. methods: shv genes were amplified by pcr from three isolates (escherichia coli, enterobacter cloacae and pseudomonas aeruginosa). individual sequence traces were analysed with the computer programs phred and codon code. pcr products were ligated in vector pcr . and transformed to e. coli. sequence analysis was performed on eight individual clones from each transformation. results: visual inspection of the low quality positions in the sequence traces showed signals for two different nucleotides at three positions in the shv sequence: a or t at position , a or g at position and a or g at position . the polymorphisms at positions and lead to aminoacid substitutions, the four different combinations would give shv types , a, or . the double signals suggested that two or more blashv alleles were amplified. pcr amplicons were cloned in e. coli, in the sequences of individual clones only two combinations of the three polymorphisms were present: a g a and t a g . these two combinations correspond to shv- and shv- , respectively. conclusions: (i) in isolates of three different species, two different shv genes were present: shv- and shv- . (ii) genotypic detection with default sequence assembly parameters may lead to misidentification of the number and type of shv genes carried by a single strain. (iii) careful interpretation of sequence data of shv genes, including analysis of low quality positions, may further improve our understanding of the epidemiology and evolution of these esbl genes. antimicrobial susceptibilities and epidemiological analysis of salmonella typhimurium human isolates in slovakia by phage typing and pulsed-field gel electrophoresis v. majtán, l. majtánova, m. szabó ová (bratislava, sk) objectives: salmonella typhimurium is a common cause of salmonellosis among humans and animals in many countries. in the last few decades the incidence of multidrug-resistant s. typhimurium infections appears to pose a particular health risk. the objectives of this study were analysis by antibiotic susceptibility, phage typing and pulsed-field gel electrophoresis (pfge) of s. typhimurium human isolates. methods: a total of strains isolated during -september were analysed. the susceptibility of isolates to ten antibiotics was evaluated by a disk diffusion method. the phage types were identified according to anderson et al. ( ) in the national reference center for phage typing of salmonellae. pfge was used to resolve xbai macro restriction fragments from all strains. results: of human isolates ( . %) were resistant to more than two antibiotics. sixty-three of isolates ( . %) showed a classic dt resistance profile to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline (acssut). among this resistance type . % were dt , . % were dt and one strain was dt a. isolates encompassed phage types. the majority of isolates was found to be definitive phage type dt , representing . % of all isolates. other phage types were mainly dt , dt and dt a. nine pulsotypes and subpulsotypes were obtained using xbai restriction enzyme, but pattern x with its subtypes predominated ( . %). a major pulsotype x was represented by . % of dt isolates and was also found among dt isolates. conclusion: results indicated the spread of different clones of the multidrug-resistant s. typhimurium in the slovakia, but with predominance of one clone represented mainly by dt isolates. the phage typing as well as pfge may offer an improved level of discrimination for the epidemiological investigation of s. typhimurium human strains. novel reverse hybridisation assay to identify ctx-m genotype in cephalosporin-resistant isolates from uk and india to validate the assay results by dna sequencing. methods: isolate collection : enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in london and south-east england. these isolates were known to carry phylogenetic group blactx-m, but precise genotypes had not been determined. isolate collection : enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in aligarh, north india. resistance determinants had not been investigated previously. a novel multiplex pcr was used to amplify blactx-m. reverse hybridisation was carried out using biotinylated pcr amplicon and sequence-specific oligonucleotides designed to identify members of ctx-m phylogenetic group . hybridisation results were validated by dna sequencing for representative isolates from each collection. results: / london and se england isolates known to carry group blactx-m gave a consistent profile, corresponding to that for ctx-m- and ctx-m- ; / gave a profile corresponding to ctx-m- and ctx-m- . / indian isolates had blactx-m genes, all of which belonged to group , and all these gave a hybridisation profile corresponding to ctx-m- or ctx-m- . ctx-m- and ctx-m- are rare variants, suggesting that the enzymes present were more likely to be ctx-m- and ctx-m- , and this was confirmed by dna sequencing. conclusions: this is the first reported application of this novel reverse hybridisation assay to the analysis of large numbers of cephalosporin-resistant enterobacteriaceae. results were validated by dna sequencing. the assay is cheap and convenient, enables reasonable throughput, provides results within one day and can be used in place of dna sequencing. we believe it will be valuable for monitoring the prevalence and genotypes of blactx-m genes in enterobacteriaceae. detection of mexa and mexx efflux genes in p. aeruginosa: correlation between qc-rt-pcr and real-time pcr objectives: efflux systems are rarely identified as such in clinical microbiology laboratories. yet, over expression of transporters such as mexab-oprm and mexxy-oprm are likely to cause antibiotic multi-and cross-resistance in pseudomonas aeruginosa, leading to potential clinical treatment failures because of their inducible character. we have previously developed and validated with reference strains a qc-rt-pcr method to quantify mexa and mexx expression levels (eccmid . in the present study, we have developed a real-time-pcr assay and present here the correlation between both methods using control strains and clinical isolates. methods: expression levels of mexa and mexx were measured by both techniques in (i) reference strains expressing only one of these efflux mechanisms [mexa ( ) or mexx ( ) ]; and (ii) clinical isolates, in comparison with the wild-type strain pao (basal mexa and mexx expression levels). results: real-time pcr showed an inter-day reproducibility of ± . % (triplicates of strains). among the clinical strains, over expressed mexa and mexx. the table shows (i) the mean level of overexpression of mexa and mexx in comparison with the wild type strain pao (set at ), as detected by real-time pcr for all strains; (ii) the ratio of these values to those observed by qc-rt-pcr for the corresponding transporters. conclusions: both qc-rt-pcr and real-time-pcr are potentially useful in clinical laboratories as sensitive and rapid diagnostic tools to quantify the expression level of mexa and mexx in p. aeruginosa. combined with phenotypic characterization, this approach may help in a better understanding of the resistance mechanisms and epidemiology of resistance in this difficult-to-treat nosocomial pathogen. molecular detection of penicillin resistance in streptococcus pneumomiae n.g. rizk, n.a. abo khadr, s.m. abdel salam, n.m. gamil, m. hassan (alexandria, eg) objectives: the aim of the study was to detect penicillin resistant streptococcus pneumoniae by using seminested polymerase chain reaction (pcr) and to compare it with minimum inhibitory concentration (mic) of penicillin g. methods: fifty clinical isolates of streptococcus pneumoniae where isolated from patients admitted to alexandria main university hospital in egypt and were recovered from sputum ( strains), throat swabs ( strains), and pleural effusion ( strains) . two species-specific primers a- and a- , which amplified bp region of the pbp a penicillin-binding gene, were used for pneumococcal detection. two resistance primers, a-r and a-r , were used to bind to altered areas of pbp a gene which, together with the down stream primer a- , amplify dna sequences of bp and bp from isolates with penicillin mic > . objective: lipopolysaccharide-binding protein (lbp) is an acute phase protein produced in the liver. the objective of our study was to evaluate lbp as a marker of severity and prognosis in patients with bacteraemia. methods: adult patients with community-acquired bacteraemia were included in a prospective manner. daily blood sampling for lbp and interleukin- (il- ) was performed. the patients were classified according to the systemic inflammatory response syndrome (sirs) criteria. demographic data, co-morbidity, microbiological aaetiology, routine biochemical parameters, focus of infection, severity score and mortality on day were recorded. lbp and il- levels were analysed on plasma samples with a chemiluminescent immunometric assay (immulite- Ò ). results: the median age was yrs. the mortality rate on day was . %. patients had bacteraemia without sirs, patients had sepsis and patients had severe sepsis. lbp concentrations are presented as medians and range: . lg/ml ( . - . ) in patients without sirs, ) in patients with sepsis and . lg/ml ( . - . ) in patients with severe sepsis (p < . ). lbp levels correlated to levels of il- (rs . ), c-reactive protein (rs . ), leukocytes (rs . ) and neutrophils (rs . ) (p < . ). lbp did not predict the outcome of the patients with bacteraemia. conclusion: lbp levels increased with the severity of sepsis in patients with bacteraemia. lbp correlated to il- , c-reactive protein, leukocytes and neutrophils. lbp did not predict the outcome of the patients in this small cohort. pyrosequencing of the gra gene to discriminate type i, ii and iii toxoplasma gondii in clinical samples b. edvinsson, b. evengård on behalf of the esgt objectives: infection with toxoplasma gondii in immunocompromised transplant recipients is rare but often fatal. to increase our knowledge about the significance of the genotype of the parasite during infection, methods suitable for routine use need to be developed. pyrosequencing is a rapid sequencing-bysynthesis method performed in real-time. it is developed for detection of short nucleotide polymorphisms (snps), and is suitable for molecular genotyping of microorganisms. we here present a pyrosequencing assay for rapid and reliable discrimination of toxoplasma gondii type i, ii and iii in clinical samples. methods: twenty-two isolates of t. gondii were used for pyrosequencing analysis of the gra gene. real-time pcr was performed using a lightcycler . instrument to amplify a bp fragment of the gra gene. pyrosequencing analysis of two different snps contained within a bp fragment of the amplified product was preformed to identify t. gondii type i, by detection of nucleotides g and a at these respective positions. type ii was g and g, and type iii was a and a. to test the assay in a clinical context, blood samples and lung tissue from an immunocompromised patient was analysed. results: the detection limit of the assay is parasitic genomes in a sample. reproducibility (r) was calculated as r = nr/n (nr = the number of isolates assigned the same type on repeat testing and n = the number of isolates tested). r was determined using three independent runs, and was , suggesting clearly interpretable results with little variation. typeablility (t) of the assay was calculated as t = nt/n (nt = the number of typeable strains and n = the number of isolates tested). t was determined using three independent runs, each including four atypical isolates. t was . , suggesting that the assay discriminates correctly between the three main genotypes of t. gondii, but does not detect atypical strains. analysis of the clinical samples revealed type ii t. gondii in blood samples and lung tissue. conclusion: when preceded by real-time pcr, pyrosequencing is a rapid process with a high reproducibility and throughput. this makes it a good candidate for routine use. the method does, however, not detect atypical or recombinant strains. more than one gene may have to be analysed for that purpose. acknowledgement: in particular, we want to thank marie-laure dardé and hervé pelloux for provision of the t. gondii isolates. virulence genes in escherichia coli isolates from calves in shahrekord area, iran shiga toxin-producing escherichia coli (stec) strains, also called verotoxin-producing e. coli (vtec) strains, represent the most important recently emerged group of food-borne pathogens around the world. members of this group are a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (hc) or the hemolytic uremic syndrome (hus), which is the main cause of acute renal failure in children. domestic ruminants, mainly cattle, sheep, and goats, have been implicated as the principal reservoir. transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables, or water contaminated by feces of carriers because stec strains are found as part of the normal intestinal floras of the animals.we studied the prevalence of shiga toxinproducing escherichia coli (stec) in stool specimens of calves with diarrhoea or other gastrointestinal alterations from dairy cattle farms of shahrekord city (central of iran). the virulence genes, stx , stx , eae, intimin hly, enterohemolysin, st, lt, were detected by multiplex pcr method. stec strains were detected in ( . %) of e. coli from cases investigated. stec o was isolated in cases ( . %), whereas non-o stec strains were isolated from animals ( %). stec strains were the most frequently recovered enteropathogenic bacteria. pcr showed that ( . %) isolates carried st gene. none of isolates carried an ehxa, eae, and lt (labile toxin) genes. our results suggest that stec strains are a significant cause of calf infections in this area and confirm that, infections caused by stec non-o strains are more common than those caused by o :h isolates. the high prevalence of stec strains (both o and non-o strains) also found in human patients by other investigators, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of stec in spanish clinical microbiology laboratories. objectives: shiga toxins are a-b holotoxin including one enzymatically active a subunit associated non-covalently to five identical receptor binding b subunits. each subunit can cause different signalling pathways in different cells. to assess the effect of each single subunit the specific clones for expressing the single subunit was designed. periplasmic expression yielded native ab holotoxin or b pentamer. methods: o was used as bacterial strain for pcr amplification of shiga toxin gene. each subunit was amplified by specific primers and the amplified genes were cloned in pbad expression vector. the expression of the cloned genes was induced and optimized by different concentration of arabinose. the expressed proteins was assessed on sds-page and detected by elisa and western blotting. the expressed recombinant ab holotoxin and b subunit were purified and assessed for its biological activity on cells. cell cytotoxicity was shown by the expressed (ab ) holotoxin. moreover inhibition was observed by b subunit and antibody against it. results: e. coli clones expressing recombinant shiga toxin a and recombinant shiga toxin b subunits were established to release the toxin to periplasmic space. expressed toxin was examined by sds-page to visualize two subunits. the whole structure of these expressed subunits was checked in native gel. active ab structure expressed in periplasmic space was extracted by polymyxine b. the biological activity of the constructed recombinant shiga toxin showed both vero cell cytotoxicity and inhibition of in vitro protein synthesis. conclusion: in this study it was shown that for b subunit assembly and secretion to periplasmic space as b pentamer homologous leader sequence is not needed. although for biological active holotoxin (ab ) secretion to periplasmic space the presence of homologous leader sequence of gene is essential. these subunits can be used for studying on cell cytotoxicity and also as a vector for antigen presentation in immunotherapeutic approaches. characterisation of gram-positive anaerobic cocci by biochemical tests and partial s rrna sequencing a. bryk, a. kanervo-nordstrom, m. hyvonen, e. kononen (helsinki, fi) objective: gram-positive anaerobic cocci, which are common findings in various infections, are difficult to identify in clinical microbiology laboratories, where identification is based only on few phenotypic tests. in recent years, this group of organisms (traditionally known as peptostreptococci) has encountered several taxonomic changes. the aim of the present study was to compare the characterization made by a selection of key phenotypic tests to that by partial sequencing of the s rrna gene. methods: fifty-nine clinical isolates sent to our laboratory as gram-positive anaerobic cocci were examined for their colony and cell morphologies and biochemically characterized using spot catalase and indole reaction, enzyme reactions by individual diagnostic tablets (rosco), sodium polyanethol sulphate susceptibility, glucose fermentation, and determination of metabolic end products. in addition, commercial identification test kit (rapid id a) patterns were performed. the sequencing of the s rrna gene of the clinical isolates and reference strains comprised of about bp, and the sequences obtained were compared to those in genbank database by using the multisequence advanced blast comparison software from the national center of biotechnology information. results: the biochemical characteristics of the isolates were consistent with those of peptostreptococcus anaerobius (n = ), peptostreptococcus (micromonas) micros (n = ), finegoldia magna (n = ), peptoniphilus asaccharolyticus (n = ), peptoniphilus sp. (n = ) and anaerococcus sp. (n = ), whereas isolates remained as unidentified gram-positive anaerobic cocci. biochemical identification correlated with that obtained by partial s rrna sequencing in / ( %) isolates at genus level and in / ( %) isolates at species level. the agreement of the biochemical and sequence-based identification was % for p. micros and f. magna. of isolates biochemically identified as p. asaccharolyticus, isolates were identified as peptoniphilus harei and remained as peptoniphilus sp. by sequencing. according to the sequence data, the unidentified isolates were peptoniphilus ivorii. conclusion: most isolates from human infections proved to be f. magna. a relatively good agreement of identification was obtained using biochemical testing and partial s rrna sequencing. objectives: molecular methods for identification of infectious agents in patients with clinical infectious disease are increasingly being used. especially in cases where antibiotics have been given prior to sampling or when fastidious bacteria difficult to grow are the aaetiology of the infection. infectious arthritis is a serious disease where identification of the etiological agent is mandatory for optimal antibiotic treatment as well as indication of the primary focus if not the joint it self. methods: in the present prospective study, synovial fluids taken from patients in elucidation of affected joints and sent to a clinical microbiological laboratory in the copenhagen area, denmark, were examined by conventional (culture, phenotypic tests) and molecular methods (pcr/sequencing of s ribosomal genes). conventional methods included gramstaining and microscopy, aerobic and anaerobic culture and identification. pcr/sequencing included dna extraction, pcr assay which produced a bp fragment of s rdna, and sequencing of both dna strands of the amplicons. sequencing data were edited and a blast search in the ncbi database was done. results: overall a microorganism was identified in of the synovial fluids ( . %). in synovial fluids from nine patients bacteria were identified by either methods [staphylococcus aureus (n = ), streptococcus pneumoniae (n = ), streptococcus dysgalactiae (n = ), citrobacter freundii (n = )]. six synovial fluids were only culture positive; in four of those six specimens coagulase negative staphylococci were isolated. in three of the synovial fluids a microorganism was identified by s pcr only. in two synovial fluids s pcr identified only one microorganism, whereas culturing resulted in two isolates. conclusion: the present study indicates a significant contribution by molecular methods (pcr/sequencing of s ribosomal genes) in recognizing and identification of microorganisms from foci normally considered sterile like synovial fluids. continued suspicion of infected arthritis despite of negative cultures should result in use of molecular diagnostics. direct detection of cardiobacterium hominis by broad-range s rrna pcr and sequencing in the serum of a patient with infective endocarditis e. malli, d. klapsa, a. vasdeki, m. morava, m. pitsitaki, e. petinaki, a. maniatis (larissa, gr) objectives: to describe the detection of cardiobacterium hominis directly in the serum of a patient with infective endocarditis, by employment of broad-range s rrna pcr followed by sequencing. methods: a series of blood cultures were taken from the patient before starting empirical treatment. in addition, ml whole blood was collected in rubber sealed pyrogen-free tubes for direct detection of bacterial dna. bacterial dna was detected by a broad range pcr reaction and sequencing process allowed identification of bacteria species. results: cardiobacterium hominis was identified as the causative agent of infective endocarditis, on two days after the serum collection. blood cultures, simultaneously obtained with the serum sample, remained negative after days of routine incubation; however, after a prolonged incubation of twelve days a gram negative bacterium was isolated from the aerobic bottles, that was identified as c. hominis species, by the usual phenotypic studies (catalase, oxidase reaction, indole, nitrate, etc) which are time-consuming. conclusions: to our knowledge this is the first report of direct detection of c. hominis in the serum using molecular methods, emphasizing the need for the establishment of such methods especially for infections caused by fastidious organisms. identification of dangerous bacterial pathogens by s ribosomal rna gene sequence analysis w. ruppitsch, a. stoeger, a. indra, d. schmid, k. grif, c. schabereiter-gurtner, a. hirschl, f. allerberger (vienna, at) to assess the usefulness of partial s rrna sequence analysis for identification of dangerous bacterial pathogens, a total of isolates comprising bacillus anthracis, brucella melitensis, biovars melitensis, suis, abortus and bovis, burkholderia mallei, burkholderia pseudomallei, francisella tularensis, yersinia pestis, and genus-related and unrelated control strains were sequenced and analysed using the genbank database (blast . . , national institute of health, u.s.a), the microseq database (version . . and v . , applied biosystems, foster city, u.s.a.) , the ribosomal database project-ii database (rdp-ii, release , update , michigan state university, u.s.a), and the ribosomal differentiation of medical microorganisms database (ridom, university of wuerzburg, germany). on genus level all isolates were identified using genbank, rdp-ii, and microseq v . . the older microseq . . database identified % of the tested samples correctly on genus level. the ridom database did not include sequence data of the tested species even on genus level, the ridom database none (''there seems to be, at least currently, no close relative available''). genbank and rdp-ii identified all dangerous pathogens correctly. the microseq v . database identified four of the six species of dangerous pathogens. on species level none of the dangerous pathogens was correctly identified using microseq . . or ridom. as previously noted by various other authors, the most important reason for failure of databases in identifying a bacterium is a lack of the s rrna gene sequence of the particular bacterium in the database rather than misidentification because of poor sequence quality. one must also be aware that the following bacterial species or subspecies have the same s rrna gene sequence, which makes differentiation by sequence analysis impossible: b. anthracis and b. cereus, y. pestis and y. pseudotuberculosis, all brucella subspecies, and francisella tularensis ssp. holarctica and mediasiatica. in addition to s rrna gene analysis complementary methods are essential to discriminate between these bacteria on species or subspecies level. identification of nontuberculous mycobacteria by sequence analysis of the s ribosomal rna, the heat-shock protein and the rna polymerase beta-subunit genes s. shin, j.h. yoon, e.c. kim (seoul, kr) objectives: the diagnosis of diseases caused by nontuberculous mycobacteria (ntm) is difficult because ntm are prevalent in the environment such as soil and water and because they have fastidious properties. in this study, we investigated the distribution pattern of ntm clinical isolates and the identification to the species level. methods: among the presumptive ntm clinical isolates, cultured in a third referral hospital from -jan- to -jan- in seoul, south korea, which were negative by probe hybridization method for mycobacterium tuberculosis complex, we selected those of more than colonies or those cultured more than twice in a same patient. a total of isolates were studied for the distribution of ntm including isolates recruited for species identification by direct sequencing of s rrna, hsp and rpob gene segments. ( . %) were also identified in the presumptive ntm isolates. the identification rate by sequencing of s rrna, rpob, and hsp were %, % and %, respectively. hsp or rpob gene was more efficient than s rrna in identification of ntm by sequencing. conclusions: some ntm are considered to be the causative organisms of clinical diseases even in the countries with intermediate burden of tuberculosis, so accurate identification method by direct sequencing can be adapted to clinical laboratories. evaluation of the genotype mtbdr assay for the simultaneous detection of resistance to rifampicin and isoniazid of mycobacterium tuberculosis clinical strains f. brossier, c. truffot-pernot, n. veziris, v. jarlier, w. sougakoff (paris, fr) objectives: the rapid determination of drug resistance in mycobacterium tuberculosis is an important challenge to ensure a rapid effective chemotherapy. the genotype mtbdr test is a commercially available dna strip assay enabling the molecular genetic identification of the m. tuberculosis complex and its resistance to rifampicin (rif-r) and isoniazid (inh-r) by detecting the most commonly found mutations in the genes rpob (asp val, his tyr, his asp, ser leu) and katg (ser thr). here, we report the evaluation of the genotype mtbdr assay from a set of clinical isolates of m. tuberculosis. methods: clinical isolates were collected in france over a years period ( ) ( ) and were included in the study: were rif-r, were inh-r (of which were also rif-r) and were susceptible to both drugs. the susceptibility tests were carried out by the standard proportion method. the mutations involved in rif-r and inh-r in rpob, katg, inha and his promoter region, were characterized by dna sequencing. results: the genotype mtbdr assay identified % of the rif-r strains harbouring mutations in the rpob gene, of which ( %) showed a ser leu mutation and ( %) a his asp or tyr mutation. of the inh-r strains ( %) harboured a ser thr mutation in katg, all identified by the genotype mtbdr assay. of this strains displayed a high level of inh-r. among the other inh-r strains, showed a katg mutation at the level of the regions, which was different from ser thr ( of which showing a low level of inh-r), and one harboured a deletion in katg (with a high level of inh-r). these mutations were also detected by the strip. finally, among the remaining inh-r strains not detected by the mtbdr assay, were characterized by a mutation in position - of the promoter region for the maba-inha regulon ( with a low level of inh-r), by a ser ala mutation in inha (all with a low level of resistance) and by other mutations. conclusions: the mtbdr assay, which can readily be included in a routine laboratory workflow, identified % and % of the strains resistant to rif and inh, respectively. interestingly, of the inh-r strains showing a high level of resistance ( %), but only of the inh-r strains with a low level of resistance ( %), were detected by the mtbdr assay, indicating that complementary tests are necessary for detection of the m. tuberculosis strains having a low level of resistance to inh. variation in the streptococcal s rdna detected by pyrosequencing m. haanperä, p. huovinen, j. jalava (turku, fi) originally the aim of this study was to identify alpha-haemolytic streptococcal isolates to the species level by pyrosequencing the v and v regions of the s rdna and comparing the results to the sequences of type strains that have been determined earlier. however, the isolates could not be unambiguously identified due to sequence variations detected in the alpha-haemolytic isolates. materials and methods: invasive s. pneumoniae isolates (n = ), alpha-haemolytic streptococcal blood culture isolates (n = ) and alpha-haemolytic streptococcal isolates from the normal pharyngeal microbiota (n = ) of six elderly persons were analysed by pyrosequencing the v and v regions. results: varying degree of genetic variation was found in different types of streptococcal isolates. in the pneumococcal isolates, no sequence variation was detected as all the isolates contained the sequence specific for s. pneumoniae in both regions. also the sequences of the alpha-haemolytic blood culture isolates were well in agreement with the sequences of the streptococcal type strains. however, most of these isolates could not be unambiguously identified, as they contained sequences belonging to different species in the v and v region. consequently, only five of the isolates could be unequivocally identified as s. gallolyticus (n = ), s. anginosus (n = ), s. mitis (n = ) and s. sanguinis (n = ). the commensal streptococci contained numerous sequences to which an identical type sequence could not be found. also sequences identical to type strains were found; but similarly to the blood culture isolates, the results enabled the identification of only four isolates: s. mitis (n = ), s. parasanguinis (n = ), and s. salivarius or s. vestibularis (n = ). moreover, the pyrograms of three blood culture isolates and ten pharyngeal isolates indicated heterogeneous s rdna alleles. one such pyrogram of the v region is presented in the figure. interestingly, four of the eight different nonheterogeneous v and v sequence combinations of the blood culture isolates were also present among the pharyngeal isolates. the results of this study indicate that the variation in commensal streptococci is greater than that of the streptococcal type strains and pathogenic isolates. the presence of identical sequence combinations among the blood culture and pharyngeal isolates supports the assumption that potentially pathogenic isolates are present in the normal microbiota. evaluation of partial s rrna gene sequencing for identification of clinical isolates of nocardia species m. marín, m. sánchez, m. del rosal, e. cercenado, p. martín-rabadán, e. bouza (madrid, es) new species of nocardia are being described. conventional identification based on biochemical characteristics and pcrrestriction enzyme analysis is frequently unable to distinguish them. partial sequencing of s rrna gene has proven useful in the identification of bacteria. objective: to evaluate the utility of 'end s rrna gene pcr and sequencing in the identification of clinical isolates of nocardia sp. compared with conventional methods and pcr-rflp of hsp . methods: clinical isolates of nocardia sp. were characterized by biochemical reactions and disk diffusion susceptibility testing. molecular identification was performed by hsp pcr-rflp and pcr of 'end of s rrna gene followed by sequencing. the sequences obtained were compared with those included in genebank. only alignments with similarities higher than % were considered. a comparison of sequences of our nocardia isolates with those deposited in genebank and well characterized phenotypically was performed using clustal x . software. results: distribution of species after pcr-rflp of hsp was n. asteroides vi ( ), n. farcinica ( ), n. nova ( ), n. asteroides i ( ), n. otitidiscaviarum ( ) and n. asteroides iv ( ) . partial sequence analysis of s rrna revealed a great heterogeneity between the isolates of n. asteroides vi, as follows: n. cyriacigeorgica ( isolates), n. abscessus ( isolates) and n. carnea ( isolate). for isolates, no genebank sequence was found with more than % similarity. all n. farcinica isolates had the same sequence and showed % similarity with those deposited in genebank. n. nova, n. asteroides i and n. otitidiscaviarum also showed sequence heterogeneity. three n. nova isolates matched with the recently described n. veterana and with n. nova. n. asteroides i isolates were identified as n. abscessus ( ) and n. beijingensis ( ) . all n. otitidiscaviarum were identified properly. the isolate of n. asteroides iv was identified as n. transvalensis. conclusions: sequencing of 'end s rrna gene is a useful and rapid molecular tool for the identification of nocardia clinical isolates. this method could provide more accurate results than the conventional ones used routinely in our laboratory. sequence analysis of the 'end s rrna has enabled us to recognize great diversity and new species among our nocardia isolates. several species would have gone unnoticed using non-sequencing-based methods. antibacterial susceptibility studies-iii p anaerobic bacteraemia due to fusobacterium necrophorum and clostiridium cadaveris: a case report m. panopoulou, e. alepopoulou, e. chrisafidou, a. tsaroucha, c. simopoulos, s. kartali (alexandroupolis, gr) introduction: anaerobic bacteremia is uncommon accounting . - % of bacteremias and it is associated with a high mortality rate, which is strongly and independently associated with underlying liver disease. case report: a year-old man presented to our hospital with a -day fever and rigor. he had a history of cancer of the extrahepatic biliary tree, which was found incidentally during an operation for the treatment of echinococcal cyst of the liver. physical examination reveals high fever ( c) and tachycardia. blood tests showed the following results: hb: . gr/dl, wbc: . /ul, plt: . /ul, tprot: . each colony type subcultured to blood agar plates and incubated aerobically and anaerobically (aerotolerance test). after hours of incubation the two organisms grew only in anaerobic conditions. they identified by the api a system (bio-merieux-france) as fusobacterium necrophorum and clostiridium cadaveris. the patient's treatment started with metronidazole, amikacin and ceftriaxone and followed by metronidazole and imipenem. he was discharged after weeks in a good condition. conclusions: although anaerobic bacteremia is rare, there is value in performing separate anaerobic blood cultures. the early recognition of anaerobic bacteremia and administration of the appropriate antimicrobial therapy play a major role in preventing mortality especially in patients with underlying disease. fluoroquinolone resistance among enterobacteriaceae strains isolated from urinary tract infections v. skandami-epitropaki, p. fostira, a. tsiringa, a. xanthaki, k. zampitha, m. toutouza (athens, gr) objectives: to study the frequency and antibiotic susceptibility of quinolone resistant bacterial stains isolated from patients with community-aquired bacteriuria and compare it with urinary pathogens from hospitalized patients. methods: during a -month period (october -october a total of bacterial strains were isolated out of urine samples submitted for culture in our hospital laboratory from the community and from hospitalized patients with urinary tract infection symptoms. cultures and bacterial identification were obtained by conventional methods. antibiotic susceptibility testing was done by kirby-bauer disk diffusion method according nccls criteria. results: of the bactrial strains studied (escherichia coli , klebsiella pneumoniae , proteus mirabilis ), . % of them were found to be quinolone resistant. the percentage of quinolone resistance was . % for hospitalized patients (hp) and . % for community patients (cp). the quinolone resistance for e. coli was . % ( . % for hp and . % for cp), for k. pneumoniae . % ( . % for hp and . % for cp) and for p. mirabilis . % ( . % for hp, . % for cp). susceptibility pattern of the quinolone resistant isolates to other antimicrobial agents was for hospitalized patients and community patients respectively as following: for e. coli ampicillin (am) %- . %, amoxicillinclavulanate (amc) . %- . %, piperacillin-tazobactam (tzp) . %- . %, cefuroxime (cxm) . %- . %, trimethoprimsulfamethoxazole (sxt) . %- . %, ceftazidime (caz) . %- . %, cefepime (fep) . %- . %, gentamicin (gm) . %- . %. for k. pneumoniae am %- %, amc %- %, tzp . %- %, cxm . %- %, sxt . %- %, caz . %- %, fep . %- %, gm . %- %. for p. mirabilis am . %- %, amc . %- %, tzp . %- %, cxm %- %, sxt . %- %, caz . %- %, fep %- %, gm . %- %. seven strains of k. pneumoniae ( . %) were carbapenem resistant and metallo-beta lactamase producing. conclusions: high resistance rates to fluoroquinolones were observed in uropathogen bacteria isolated not only from hospitalized patients but also from patients with communityacquired urinary tract infections in greece. increasing resistance rates to the rest antibiotic agents make the treatment of urinary tract infections a very difficult problem. susceptibility of pseudomonas aeruginosa isolated from the mystic programme to the carbapenems: meropenem and imipenem p.j. turner (macclesfield, uk) objectives: the meropenem yearly susceptibility test information collection programme (mystic) was initiated in in order to track the susceptibility of organisms in centres that were prescribing meropenem. this poster seeks to examine the susceptibility of pseudomonas aeruginosa isolates over this period to the carbapenems; meropenem and imipenem and, in particular, records the susceptibility of imipenem-resistant isolates to meropenem and vice versa. methods: pseudomonas aeruginosa isolates were speciated by the methods in current use at the participating centres. minimum inhibitory concentrations of meropenem and imipenem were determined using reference methods described by clsi. results: a total of isolates of pseudomonas aeruginosa have been tested globally, of these . % were susceptible to meropenem at the breakpoint of < mg/l and . % to imipenem. globally, susceptibility to the two carbapenems has remained stable over the period - , however when imipenem-resistant isolates were examined (n = ) . % proved to be susceptible to meropenem, conversely of the meropenem-resistant isolates only . % proved to be susceptible to imipenem. a similar pattern was seen when isolates were separated into global regions:usa imipenem-resistant isolates, . % susceptible to meropenemusa meropenem- results: bacteroides fragilis group (bafg) accounted for % of the isolates, fusobacterium spp. for %, other gram negative bacilli (ognb) for %, clostridia (clos) for %, nonsporeforming gram-positive bacilli (nsfgpb) for % and cocci for %. beta-lactamases (bl) were detected in % of isolates. most bl + strains belonged to bafg ( %) and ognb ( %). at nccls-recommended breakpoints, more than % of isolates were susceptible to tzp, mtz, chl and mem, % to amc but only %, %, % and % to fox, ctt, cli and pen respectively. no nccls-breakpoints for anaerobes are available for mxf, lzd and tig. mic and mic for mxf were and mg/l, for lzd and mg/l and for tig . - mg/l. in comparison with similar surveys conducted in and - susceptibility of bafg to clindamycin decreased from % in , to % in - and % in in bafg % of b. fragilis and % of non-b. fragilis were susceptible to amc in this study; in - susceptibility in these groups was % and % and in - % and % respectively. all isolates, except bafg and clos, were susceptible to mem. % of the isolates were susceptible to chl. susceptibility to mtz remains stable and is high in all groups except nsfgpb where mtz is active on merely % of the isolates. conclusions: tzp, mem and mtz remain very potent antimicrobial agents in the treatment of anaerobic infections. although still rare, resistant organisms were detected to each of them. therefore susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy. further monitoring of background susceptibility is necessary to guide empiric treatment. comparative in vitro activity of levofloxacin against escherichia coli isolated from acute pyelonephritis in france in c.j. soussy, c. lascols, c. dib-smahi and the multicenter group study. objectives: the objective of this study was to evaluate the in vitro activity of levofloxacin (lvx) comparatively to other antibiotics against escherichia coli strains isolated from acute pyelonephritis in women consulting emerging rooms by french hospitals in . methods: mics of lvx, ofloxacin (ofx), ciprofloxacin (cip), nalidixic acid (nal), amoxicillin-clavulanic acid (amc), ceftriaxone (cro), cefixime (cfm), amikacin (an), gentamicin (gm) and cotrimoxazole (sxt) were determined by agar dilution according to the eucast breakpoints approved by recommendations of the comité de l'antibiogramme de la société française de microbiologie. quality control was performed with e. coli strain atcc . results: a total of strains were collected. . % of strains were isolated from urinary samples, . % from blood culture and . % from the two specimens. mics / (mg/l), the range of mics and the percentage of susceptibility (%) are presented in the following table: concerning the fluoroquinolones, mics / of lvx were one/two dilution lower than those of ofx and two/one dilution higher than those of cip. for the other antibiotics, a higher percentage of susceptibility was observed with cro and an, when a lower percentage of susceptibility was observed with amc and sxt. conclusions: levofloxacin exhibited good in vitro activity against e. coli strains isolated from acute pyelonephritis with . % of susceptible strains. in vitro activity of double and triple combinations of colistin, imipenem, rifampicin and linezolid against epidemic strains of multidrug-resistant acinetobacter baumannii producing oxa carbapenamases d.w. wareham, d.c. bean (london, uk) objectives: a. baumannii has emerged as an important cause of nosocomial infection in critically ill patients worldwide. in the uk three strains in particular exhibiting multi-drug resistance and producing oxa carbapenamases have been responsible for ongoing outbreaks. treatment options for infection with these organisms are limited as only colistin and tigecycline retaining significant activity in vitro. animal models and in vitro studies using other multi-resistant strains suggest that drugs in combination with colistin may be effective. we assessed the activity of colistin in combinations including imipenem, rifampicin and linezolid against epidemic strains from a recent uk outbreak. methods: isolates of a. baumannii exhibiting resistance to carbapenems were recovered from patients at barts and the london nhs. isolates were referred to the health protection agency and confirmed as belonging to clones producing oxa carbapenemases. activities of polymyxin, imipenem, rifampicin and linezolid alone and in double and triple combinations were determined using standard chequerboard assays with increasing concentrations of drug on the x axis, drug on the y axis and drug three in multiple replicate plates. after incubation at hours wells were examined for growth and mic's determined for each combination. synergy between agents was defined as a fixed inhibitory concentration index (fici) of < . . results: the isolates tested belonged to the oxa- clone , oxa- clone and the south east clone, as confired by the hpa. colistin was the most active agent alone with mics from - mg/l. imipenem mic's varied from - mg/l. the most active combinations were colistin plus rifampicin (fici = . ) and colistin, rifampicin and imipenem (fici = . ). synergy was not seen with colistin in combination with imipenem alone. linezolid in combination with colistin (fici = . ), or imipenem (fici = . ) was synergistic but at therapeutically unobtainable linezolid concentrations ( mg/l). conclusion: multidrug resistant strains of a. baumannii from the uk producing oxa carbapenemases remain susceptible to polymyxin in vitro. polymyxin exerts its effect on the bacterial cell wall; theoretically assisting other antibiotics to reach their respective targets, and seems a logical choice for inclusion in combination therapy. we have shown that rifampicin is synergistic with polymyxin against these isolates in vitro and may be effective in treating severe a. baumannii infections in man. a comparative in vitro evaluation of resistance development after exposure to teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin in staphylococcus spp. and enterococcus spp. mssa, mrse, msse, e. faecium and e. faecalis strains was determined on agar plates containing each antibiotic at clsi resistance breakpoints and at peak blood concentrations. after incubation at °c for h colonies were counted and compared to the inoculum to calculate frequency of mutation. colonies grown in plates containing antibiotics were sampled for determination of mic values. results: frequency of mutation was less than - for all the tested antibiotics at peak blood concentrations. same results were obtained when breakpoint concentrations for each drug were used. conclusion: this one-step in vitro study demonstrated the ability of teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin to prevent growth of resistant mutants of staphylococci and enterococci, thus suggesting no occurrence of mutational events leading to resistance when bacteria are exposed to blood concentrations of these drugs. in order to establish the development of resistance after in vitro serial exposure to the same antibiotics simulating different in vivo concentrations, further studies are needed and are now in progress (multi step induction of resistance). in vitro activity of antimicrobial agents against legionella obtained from hotel water systems in turkey objectives: the aim of this study was to evaluate the in vitro activity of colistin against endemic pan-resistant acinetobacter baumannii (including resistance to imipenem) isolated during a year period in a university hospital. methods: imipenem-resistant acinetobacter spp. isolates were collected between january and october , from a variety of clinical specimens of different patients attending distinct wards in a university teaching hospital. isolates were identified by api gn and by sequencing the s rrna gene. mics of colistin were determined by agar dilution method, according to nccls susceptible breakpoint (£ mg/l). pfge (apai restriction enzyme) was performed. results: of a. baumannii isolates ( %) were susceptible to colistin. colistin resistance (mic ‡ mg/l) was observed in isolates ( isolates with a mic of ‡ mg/l and isolates with a mic of mg/l) recovered from different patients in distinct wards. among these imipenem-and colistin-resistant isolates, distinct pfge patterns were identified (clones a, b, and c). resistance to almost all beta-lactams (including carbapenems) and variable susceptibility to aztreonam, amikacin and tobramycin was a common feature of clone a. isolates belonging to clone b showed resistance to imipenem, amoxicillin and its association with clavulanic acid (amc), ureidopenicillins and their associations; susceptibility to ceftazidime; and variable behaviour to meropenem, cefepime, cefpirome and aztreonam. the susceptibility profile to aminoglycosides was variable, differing from clone a in its susceptibility to netilmicin and minocycline. clone c was resistant to imipenem, amoxicillin, amc, piperacillin, piperacillin + tazobactam, ticarcillin and ticarcillin + clavulanic acid, but remained susceptible to meropenem, aztreonam, cefpirome, ceftazidime and cefepime. conclusion: only colistin, one of the few effective drugs available against multi-drug-resistant acinetobacter infections, showed in vitro activity against the majority of acinetobacter spp. strains isolated within the sampled hospital. the observed % a. baumannii resistance to the recently re-introduced colistin seems like the first chapter of a novel repeatedly told for several antibiotics. emergence of high-level gentamicin resistance in clinical enterococcal isolates of companion animals in portugal objectives: to characterize in vitro gentamicin susceptibility among enterococci causing infections in cats and dogs, in order to evaluate the impact of high-level gentamicin resistance in small animal therapeutics. methods: the samples were collected at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. from january until november , a total of enterococci were isolated from dogs and cats with urinary tract infection (uti), otitis externa (oe) and pioderma. bbl crystal gram positive id system was used for identification at the species level. minimal inhibitory concentrations (mic) were determined by the microdilution method according to nccls ( ) . the bifuntional enzyme gene that confers high-level gentamicin resistance (hlgr) was detected using pcr ( ) . results: enterococcus faecalis was the predominant isolate (n = ), followed in frequency by enterococcus faecium (n = ). mic cumulative data analysis showed that mic values were lg/ ml and mic lg/ml. six ( %) hlgr clinical enterococcal isolates were detected, with mic ranges between - lg/ml. four of these enterococci were isolated from uti and from oe. four of the phenotypically high-level gentamicin resistant isolates carried the aac( ')-ie-aph( '')-ia gene. conclusions: the importance of enterococcal infection in small animal clinical samples has increased over the last years. mic cumulative data points out low-level gentamicin resistance among clinical enterococci isolates of veterinary origin and the emergence of high-level isolates, as previously detected ( ). this fact compromises cell-wall active agents (such as ampicillin or vancomicin) and aminoglicoside in vivo synergy. the aac ( ')-ieaph( '')-ia gene carriage is of concern because its expression confers resistance also to tobramicin, netilmicin, amikacin and kanamicin. our findings are of critical importance, as they may have a direct impact in therapeutic decision in the management of companion animal's infections by enterococci. furthermore, transfer of resistance genes and resistance strains between animals and owners/caretakers by direct contact is a concerning probability. references: ( ) results: interpretative criteria were used according to nccls .during the study period penicillin resistant strains of s. pneumoniae was noted as follows: % in sputum or ta, % in blood, %in csf,and % in others,against cefuroxim resistant strains: % in sputum or ta, % in blood, % in csf.regarding the susceptibility to ofx,penicillin resistant s. pneumoniae strains from sputum or ta revealed . %.the penicillin resistant strains coming from sputum or ta showed resistance as follows; % to em and % to sxt,against strains isolated from others: % to em and % to sxt.no resistant strain to va was found. conclusion: the percentage of the penicillin resistant s. pneumoniae isolates from the lower respiratory tract, middle ear fluid, eye fluid and sinus was markedly higher than that of the isolates from blood and csf. the most efficient drugs against penicillin resistant pneumococci were cefuroxim and ofloxacin. these results from romania also underline the previous observations regarding the higher emerging rates of resistance in s. pneumoniae worldwide. penicillin resistance in streptococcus agalactiae objectives: streptococcus agalactiae has become recognized as a cause of serious illness in newborns, pregnant women, and adults with chronic medical conditions. heavy colonization of the genital tract with streptococcus agalactiae also increases the risk that a woman will deliver a preterm low-birthweight infant. early-onset infections (occurring at < days of age) are associated with much lower fatality than when they were first described, and their incidence is finally decreasing as the use of preventive antibiotics during childbirth increases among women at risk. penicillin or ampicillin remains the drug of choice for intrapartum antibiotic prophylaxis for streptococcus agalactiae colonization in pregnant women. erythromycin and clindamycin are the drugs of choice for women with serious penicillin allergy who are colonized with streptococcus agalactiae. the objective of this study is to estimate the insorgence of penicillin resistance among streptococcus agalactiae. methods: all streptococcus agalactiae were tested against penicillin by agar dilution method according to clinical and laboratory standards institute (clsi); breakpoints for resistance were those recommended by the clsi. antimicrobial agents were obtained from their manufacture as laboratory grade powder. results and discussion: four hundred seven ( ) clinical isolated were analysed during . streptococcus agalactiae resulted resistant to penicillin in case; and about % resulted borderlines.the present findings indicate a probable evolution in s. agalactiae toward penicillin resistance this finding suggest the need a continuous national and international surveillance programs to provide timely data on the evolution of incidence of penicillin resistance in this pathogen. ciprofloxacin susceptibility of the most common isolates at bacterial conuctivitis conclusion: according to the average numerals we concluded that all the isolated strains are highly susceptible at ciprofloxacin. its application in the conuctivial saccus is especially important in curing the conuctivial infections with resistent strains like pseudomonas aeruginosa. we successfully cure the bacteria chronic conuctivitis with the adequately used therapy according to antibiogram. antimicrobial resistance patterns of acinetobacter baumannii in clinical isolates g.t. tsilika, v.p. pliatsika, m.t. tsivitanidou, d.s. sofianou (thessaloniki, gr) objectives: a. baumannii is a nosocomial pathogen, commonly isolated from critically ill and immunocompromised patients. the aim of the present study was to evaluate the antimicrobial resistance of a. baumannii strains isolated in a tertiary care hospital througout a three-year period. methods: a total of a. baumannii strains were selected from january to december .the specimens were obtained from inpatients hospitalized in intensive care unit (icu) and pediatric intensive care unit (picu) and other departments of our hospital.the identification and the antimicrobial susceptibility testing were performed using the vitek automated system(biomerieux,france conclusions: the emergance and rapid spread of multidrug resistant a. baumannii isolates are of a great concern worldwide.imipenem was one of the most potent agents for treatment of those infections caused by multiresistant strains.the increasing prevalence of imipenem resistance limits therapeutic options and leads to outbreaks of carbapenems resistant strains. tigecycline in vivo studies objectives: antibacterial agents disrupt the ecological balance of the normal human microflora. disturbances may lead to the emergence of antibiotic resistance and/or to infections by potentially pathogenic bacteria. tigecycline, a member of a new class of antibiotics (glycylcyclines), has been shown to have a potent expanded broad-spectrum activity against most grampositive and gram-negative aerobic and anaerobic bacteria. the aim of the study was to investigate the ecological effects of tigecycline on the normal oropharyngeal and intestinal microflora in healthy subjects. methods: thirteen ( ) white subjects ( women, men) aged to years, received mg of tigecycline in the morning on day as a -minute intravenous (iv) infusion, followed by mg doses of tigecycline given every hours as a -minute infusion for days. one ( ) subject was withdrawn on day because of an adverse event. serum, saliva, and faecal samples were collected before, during, and after administration for microbiologic cultivation and for assays of tigecycline. all new colonizing bacteria were tested for susceptibility (resistance > mg/l) during the investigation period. results: the serum concentrations on day , hours after dosing, were . to . mg/l (mean value . mg/l, median value . mg/l, and sd . mg/l). the faecal concentrations on day were . to . mg/kg (mean value . mg/kg, median value . mg/kg, and sd . mg/l). saliva concentrations were generally low, with highest mean value . mg/l, median value . mg/l, on day , hours after dosing. a minor effect on the oropharyngeal microflora was observed. the numbers of enterococci and escherichia coli in the intestinal microflora were reduced at day , while other enterobacteria and yeasts increased. there was a marked reduction of lactobacilli and bifidobacteria but no impact on bacteroides. no clostridium difficile strains were isolated. two ( ) klebsiella strains and enterobacter strains resistant to tigecycline were found. conclusion: tigecycline had a minor effect on the oropharyngeal microflora. tigecycline's effect on the intestinal microflora was due to its spectrum of antibacterial activity and intestinal concentrations. objectives: to examine and report the use of tigecycline (wyeth) in the treatment of multidrug resistant acinetobacter (mdra) culture positive sepsis in patients requiring mutiorgan support. methods: all patients were managed within the liver intensive care unit. physiological data was collected prospectively and entered onto a specialist database. patients received standard intensive care management; antibiotic and antifungal therapy administered as indicated by microbiological cultures. systemic inflammatory response (sirs) features initiated blood cultures (vascular lines and peripheral), drain fluid culture and broncoalveolar lavage (bal). screening swabs were undertaken weekly and samples sent for culture at laparotomy. mdra positive cultures from blood, bal, drain fluid or samples taken at laparotomy in the context of sirs resulted in the initiation of tigecycline treatment. results: patients received tigecycline treatment for mdra infections. the underlying disease states were necrotizing pancreatitis ( ), post hepatectomy ( ), polytrauma ( ), all with postive intra-abdominal cultures. acute and acute on chronic liver failure ( ), mdra +ive broncho-alveolar lavage ± blood cultures and post liver transplant patients (necrotising pancreatitis in one, with recurrent small bowel perforation and with retroperitoneal haemorrhage) all with positive blood cultures and in positive intra-abdominal tissue/clot. mean time from admission to treatment for mdra was days. mean duration of treatment was days (range - ). mean apache ii score at initiation of therapy was (range - ); / patients survived to intensive care discharge and / to hospital discharge. microbiological clearance of mdra was observed in / cases. in those who did not achieve microbiological clearance cause of death was intra-abdominal haemorrhage, recalcitrant organ failure with recurrent small bowel perforation and vasopressor resistant shock. in these patients one remained culture positive for intraabdominal sepsis despite full treatment (small bowel perforation x ). the drug was well tolerated with the only side effect being that of hypercalcaemia observed in / patients, mean corrected calcium . mmol/l, range . - . . in all cases this resolved on drug discontinuation. conclusion: tigecycline appears to be an efficacious agent in the treatment of deep seated mdra infections. objectives: nausea (n) and vomiting (v) have been reported with tigecycline, a new glycylcycline with expanded broad spectrum activity. exposure-response relationships and patient covariates predictive of the first n and v occurrence were evaluated in patients with complicated intra-abdominal infections (ciai). methods: data from patients from ciai trials (one phase and two phase ), receiving mg loading dose and mg every hours, were pooled for analysis. n and v (definitely, possibly, or probably related to tigecycline) reported from the start of infusion until hours after the last dose were included. individual exposure measures [auc - and cmax] were calculated using a previously developed population pk model. logistic regression was used to evaluate predictors of first n and v occurrence. covariates included age, weight, sex, region of treatment, and baseline n and v. results: the dataset included patients ( with pk). mean (sd) age and weight were ( ) years and ( ) kg. % of patients were men and %, %, and % were enrolled in north america, europe, and latin america, respectively. baseline nausea or vomiting was reported in % and %. overall, n and v occurred in % and % of patients receiving tigecycline, however most ( %; %) of first n and v events were mild in nature. women had more n and v ( %; %) than men ( %; %). n and v were lower in europe ( %; %) than in other regions. auc - and cmax were not predictive. the final nausea model included weight, sex, region, baseline nausea, and the interaction of weight/region as predictors of the first nausea occurrence (p = . , . , . , . , & . , respectively objective: because hospitalisation for community-acquired pneumonia (cap) is associated with substantial morbidity and health resource utilisation, we evaluated the predictors of prolonged hospital length of stay (los) and treatment duration. methods: we conducted a retrospective analysis of data from a double-blind, randomised, multicentre clinical study that compared the efficacy and safety of tigecycline with that of levofloxacin in the treatment of patients with cap requiring hospitalisation. patients were stratified by the fine pneumonia severity index and randomly assigned to receive tigecycline or levofloxacin via iv administration for at least days. treatment duration and hospital discharge were based on physician assessment of signs and symptoms of infection and patient condition. we used cox proportional hazards modelling with stepwise selection to identify statistically significant predictors (p < . ) of treatment duration and hospital los. results: among patients with cap in the clinical intent-totreat population with complete hospitalisation data, mean age was . years (range - ) and . % of patients were aged ‡ years. diabetes ( . %), chronic obstructive pulmonary disease ( . %), and congestive heart failure ( . %) were leading co-morbidities. about . % of patients were smokers and . % were characterised by alcohol abuse. median fine pneumonia severity index score was ; . % of patients had a score > . . there were no significant differences between the groups in treatment duration or los. conclusions: tigecycline, a first-in-class glycylcycline, was associated with treatment duration and los similar to that of levofloxacin, adjusting for several identified risk factors. tigecycline effective in treating patients with intra-abdominal or skin/skin structure infections who have bacteraemia e.j. ellis-grosse, r. maroko (collegeville, us) objectives: the treatment of bacteraemia, which is a potentially fatal complication of infections originating at other body sites, is complicated by increasing resistance. tigecycline, a first-in-class glycylcycline, has an expanded spectrum of activity against gram-positive, gram-negative, anaerobic, and atypical bacteria including resistant strains. tigecycline is safe and effective in treating complicated skin and skin structure (csssi) and intraabdominal infections (ciai). this analysis examines tigecycline clinical trial experience in patients with ciai or csssi who had bacteraemia (presence of bacteria in blood) at baseline. objectives: treatment of complicated intra-abdominal infections (ciai) is challenging due to diverse bacteriology and bacterial resistance. the efficacy and safety of tigecycline (tgc), a first-in-class glycylcycline approved in mexico, brazil, peru, colombia and usa for treating ciai and complicated skin and skin structure infections, was compared with imipenem/cilastatin (imi/cis) in adult hospitalised patients with ciai in two double-blind, phase multinational trials. this analysis evaluated tgc efficacy and safety in the european region of the integrated results of these two trials. methods: one study was conducted in centres ( countries) and the other study was conducted in centres ( countries). patients were stratified by disease severity (apache ii score £ vs > but £ ), and randomly assigned to iv tgc ( mg loading, then mg q h) or iv imi/cis ( / mg q h) for - days. clinical response at test-of-cure (toc, - days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) were co-primary efficacy endpoints where cure/failure responses were determined. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, patients were mitt (received ‡ dose), m-mitt ( tgc, imi/cis) and me ( tgc, imi/cis). treatment groups were balanced with respect to demographics. patients were mostly white ( . %) men ( %) with a mean age of years. for me, clinical cure rates at toc were . % ( / ) for tgc vs . % ( / ) for imi/cis ( % ci = ) . , . ; test for non-inferiority p < . ). clinical cure rates for m-mitt were . % ( / ) for tgc vs . % ( / ) for imi/cis ( % ci = ) . , . ; test for non-inferiority p < . ). most commonly reported treatment emergent aes (teaes, mitt) for tgc and imi/cis were nausea ( . % and . %, p = . ) and vomiting ( . % and . %, p = . ). the imi/cis group had significantly higher teaes of fever ( . % imi/cis vs . % tgc, p = . ), hyperglycaemia ( . % imi/cis vs tgc, p = . ) and dyspnoea ( . % imi/cis vs . % tgc, p = . ) where tgc had significantly higher amylase increase ( . % tgc vs . % imi/cis, p = . ) and bun increase ( . % tgc vs imi/cis, p = . ). conclusions: similar to the overall integrated analysis of the two phase trails, in the european analysis, tgc was safe and effective in the treatment of hospitalised patients with ciai in comparison with imi/cis. tigecycline is safe and effective in the treatment of complicated skin and skin structure infections: european experience of two double-blind phase comparison studies with vancomycin/aztreonam r. maroko, n. dartois, d. sarkozy, j. goodrich, e.j. ellis-grosse on behalf of the tigecycline and study groups objectives: tigecycline (tgc) a first-in-class expanded spectrum glycylcycline, has been approved in mexico, brazil, peru, colombia and usa for treating complicated skin and skin structure infections (csssi) and complicated intraabdominal infections. two phase , randomised, double-blind studies were conducted in hospitalised men and women with csssi to determine tgc safety and efficacy compared with vancomycin/aztreonam (v/a). the objective of this analysis was to evaluate the efficacy and safety seen in the european population of the integrated analysis of these phase trials. methods: one study was conducted in centres in countries while the other study was conducted in centres in countries. patients were randomly assigned ( : ) to receive either tgc ( mg, followed by mg iv twice daily) or vancomycin ( g iv twice daily) plus aztreonam ( g iv twice daily) for up to days. clinical response at test-of-cure (toc, - days after therapy) for clinically evaluable (ce) and clinical modified intent-to-treat (c-mitt) populations were coprimary efficacy endpoints in which cure/failure responses were determined. secondary objectives included determination of in vitro susceptibility to tgc of a range of bacteria that cause csssi and microbiological efficacy. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, patients comprise mitt (received ‡ dose of study drug), comprised ce ( tgc, v/a/cis) and comprised c-mitt ( tgc, v/a/ cis). treatment groups were balanced with respect to demographics. patients were mostly white ( . %) men ( . %) with a mean age of years. in the european region, clinical responses to tgc and v/a at test-of-cure were similar: c-mitt, . % ( / ) versus . % ( / ), difference tgc-v/a was - . % ( % ci - . , . ). similar results were noted in the ce population with tgc curing . % ( / ) and v/a curing . % ( / ), difference tgc-v/a was - . % ( % ci - . , . ).most commonly reported treatment emergent aes (teaes, mitt) for tgc and v/a were nausea ( . % and . %, p < . ) and vomiting ( . % and . %, p = . ). the v/a group had significantly higher teaes of sgpt increase ( . % v/a vs . % tgc, p = . ) and rash ( . % v/a vs tgc, p = . ). conclusion: in the european analysis of the integrated phase worldwide clinical studies, tgc monotherapy is as safe and efficacious as the combination of v/a in the treatment of patients with csssi. safety and tolerability of tigecycline r. maroko, n. dartois, g. rose, e.j. ellis-grosse (collegeville, us; paris, fr) objectives: tigecycline (tgc), a glycylcycline, is a first-in-class, extended, broad-spectrum iv antibiotic that has demonstrated clinical activity in patients with complicated intra-abdominal infections (ciai) and complicated skin and skin-structure infections (csssi). the safety of tigecycline was evaluated in four phase iii trials. methods: a total of hospitalized patients from these trials were pooled and evaluable for safety analysis. in the ciai trial, patients received tgc mg q hrs (following a -mg loading dose) or imipenem mg and cilastin mg q hrs. those in the csssi study were treated with either tgc (same dose/schedule) or vancomycin gm with or without aztreonam gm q hrs. results: the most frequently reported adverse events (aes) in both tgc-treated groups were nausea (n) and vomiting (v). the incidence of n was . % while v was approximately . %; these were generally mild to moderate in severity. infection-related serious aes were slightly more frequent with tgc versus comparators ( . % vs . %). discontinuations due to treatment-emergent aes (including n/v) occurred at similar rates with tgc and comparators ( . % vs . %). six patients ( . %) treated with tgc presented with intestinal perforations and developed sepsis/septic shock compared with ( . %) for imipenem/cilastatin, with higher baseline apache ii scores in the tgc group; the relationship to treatment could not be determined. in the overall efficacy analysis, subjects with ''perforation of the intestines'' were balanced between the two groups, and overall efficacy was not statistically different. no clinically significant renal, hepatic, cardiac (qtc), bone marrow, or cns toxicities were noted with tgc. conclusion: tgc appears to be safe and tolerable for patients with ciai and csssi. n/v were generally mild to moderate in severity, self-limiting, and did not result in increased overall drug discontinuation. there did not appear to be clinically significant renal, hepatic, cardiac, bone marrow, or neurological toxicities related to tgc treatment. all-cause mortality rates did not statistically differ between those treated with tgc and the comparators. its demonstrated efficacy and favourable toxicity profile make tgc a good monotherapy option for selected serious infections. tigecycline as effective as imipenem/cilastatin in the treatment of complicated intra-abdominal infections: experience in india objective: due to diverse bacteriology and bacterial resistance, treatment of complicated intra-abdominal infections (ciai) is a challenge. in a double-blind, phase , multinational trial, the efficacy of tigecycline, a first-in-class glycylcycline, was compared with imipenem/cilastatin (imi/cis) in hospitalised patients with ciai. this subanalysis evaluated tigecycline safety and efficacy from investigational sites in india. methods: patients were stratified by disease severity (apache ii score £ vs > but < ), and randomly assigned to iv tigecycline ( mg loading, mg q h) or iv imi/cis adjusted for body weight ( / mg q h for ‡ kg) for - days. clinical response at test-of-cure (toc, - days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) populations were co-primary efficacy endpoints where cure/failure responses were determined. safety evaluations included vital signs, laboratory tests and record of adverse events (aes). results: in india, patients received at least dose (mitt, tigecycline, imi/cis), patients were clinically evaluable (ce), were me, were m-mitt. treatment groups were balanced with respect to demographic/baseline medical characteristics. primary diagnoses (mitt) were complicated appendicitis ( %), gastric/duodenal perforation ( %), perforation of intestine ( %), cholecystitis ( %), peritonitis ( %), and intraabdominal abscess ( %). cure rates at toc in me in india were / ( . %) tigecycline and / ( . %) imi/ cis, which are consistent with overall me results [ . % ( / ) tigecycline vs . % ( / ) imi/cis ( % ci = ) . , . ; non-inferiority p < . )]. in india m-mitt, cure rates at toc were / ( . %) tigecycline and / ( . %) imi/cis, similar to the overall m-mitt results [ . % ( / ) tigecycline vs . % ( / ) imi/cis ( % ci = ) . , . ; non-inferiority p < . )]. noninferiority of tigecycline among india patients could not be statistically demonstrated because of insufficient sample sizes, however, magnitude of response to study drugs in patients treated in india was comparable to that in overall patients. in india, treatment aes were similar with significantly higher incidence of dyspnoea in tigecycline ( . %) vs imi/cis ( . %), p = . . conclusions: efficacy results in india are consistent with findings from the overall study and results at other centres, suggesting tigecycline is noninferior to comparator in treating ciai. nosocomial infection: control of environment, viral infections p bacterial flora contamination of blood pressure cuffs in use on hospital wards n. walker, r. gupta, j. cheesbrough (preston, uk) blood pressure cuffs are a plausbile vehicle for the transmission of nosocomial infection between patients. despite this, few studies have examined the level of bacterial contamination and tested for the presence of common nosocomial pathogens on their surface. we swabbed cuffs currently in use on hospital wards. using sterile gloves, a disposable template measuring · cms was placed onto the cuff and a moistened sterile swab was rubbed onto the defined area for minute and then transported in mls of buffer medium. from each sample, . mls of the buffer was plated onto different media which included a non-selective agar medium for total viable count (tvc) and selective media for s. aureus, mrsa, c. difficile, coliforms and vancomycin resistant enterococci (vre.) bacterial growth was recovered from all cuffs. pathogenic organisms were isolated from cuffs ( %). mssa from , mrsa from and c. difficile from . the remaining three cuffs grew more than one pathogenic organism; mssa + mrsa + c. difficile from one and mssa + c. difficile from cuffs. colifroms and vre were not isolated from any of the cuffs. the range of total viable counts recovered per cm area of the cuff varied from > cfu and the cuffs with the highest counts tended to have more pathogens present. mssa and c. difficile were isolated from % of the cuffs sampled and mrsa from %. while the actual importance of this potential route of transmission for nosocomial pathogens remains unclear, it can not be dismissed. the impracticality of decontaminating blood pressure cuffs between patients suggests that single patient use cuffs or a barrier between cuff and skin would be a more viable option on a busy general ward. needlestick and sharp injuries of health care personnel in a newly founded tertiary hospital: a prospective study m. falagas, i. karydis, g. georgoulias, p. hatzopoulou, d. nikita, i. kostogiannou (athens, gr) objectives: needlestick and sharp injuries of health care workers are a major cause of anxiety and may expose susceptible employees to the risk of infectious diseases. however, the incidence of such injuries has not been examined in a newly founded hospital while preventive programmes are taking place. methods: we prospectively studied the needlestick and sharp injuries of employees in a newly founded tertiary hospital in athens, greece while a vaccination program against hepatitis b virus as well as educational activities for avoidance of injuries were taking place. serologic studies for hepatitis b and c virus as well as human immunodeficiency virus (hiv) were performed in all injured employees and the source patients (when known). results: sixty-eight needlestick, sharp injuries, and splashes were reported during the study period ( / / to / / ) in nurses, housekeepers, technicians, and ambulance workers. the overall incidence (percutaneous injuries and splashes) per full-time employment-years ( fteys) was . % whereas the incidence of percutaneous injuries alone per fteys was . %. a higher incidence of injuries was noted during the first than the second half of the study period ( . % versus . %, p = . ). no source patient was found positive for hepatitis c or hiv. the use of high-titre immunoglobulin after adjustment for the incidence of injuries was higher in the first than the second half of the study period ( . % vs . %, p = . ). conclusion: although we did not adjust for possible confounders, our data show that educational and vaccination preventive programs for needlestick and sharp injuries led to a statistically significant decrease in the incidence of such injuries and use of high-titre immunoglobulin. epidemiology of occupational needlestick and sharps injury among healthcare-workers in turkey s. hosoglu on behalf of the occupational infections study group, turkey background: health care workers (hcws) are frequently exposed to the danger of infectious agents through needle stick and sharps injury (nssi) in their occupational efforts. in turkey, the hepatitis b and c viruses cause an essential threat to the hcws because of their prevalence rate ( %- % and . %- %, respectively). a cross-sectional countrywide survey study was performed on the epidemiology of nssi among hcws at hospitals in cities throughout the country. data relating to the epidemiology of nssis were collected using a standard questionnaire in . results: totally hcws completed the questionnaire forms. nurses are the leading group ( persons) that joined into the study were followed by doctors ( persons) andlaboratory technicians ( ). totally of them ( . %) declared an occupational exposure or nssi in the last months related their job. needle stick injury was reported in of them ( . %), splash into the eye in ( . %), sharp injury in ( . %), and the other injuries in ( . %). the hepatitis positivity was reported in cases ( . %) objectives: to assess the microbiological status of reprocessed single-use devices for interventional cardiology by testing bioburden, sterility and pyrogenic load. methods: a total amount of electrophysiology non-lumen catheters (ep) were collected after the first clinical use on patient. devices were contaminated with bacteria spiked human blood and underwent four different pre-sterilization protocols including chlorine, polyphenol, and enzymatic agents. treated samples were assayed by cultural quantitative methods (cqm) for bactericidal properties and electron microscopy (em) for biologic residuals. ep were tested for sterility. by the repetition of simulated-use (bacteria spiked blood) and regeneration (enzymatic and chlorine treatment, gas plasma sterilization) we obtained , , , , , samples respectively reprocessed , , , , , times. devices were cultured for days in trypticase soy broth. the pyrogenic status of ep was monitored after clinical use, after decontamination-cleaning treatments and after complete reprocessing by lal test. results: high-resolution em and cqm confirmed the superior properties of chlorine releasing agent added to enzymatic detergent for devices treatment before sterilization. hypochlorous acid based protocols were more biocide (> . log cfu reduction) than polyphenolic ( . - . log cfu reduction). sterility tests showed no positive sample to inoculated strain until the fourth cycle of reprocessing. catheters showed the growth of the inoculated strain, bacillus subtilis in / and / samples after five cycles and six cycles respectively. every reprocessed device was non-pyrogenic (< eu/catheter). in addition, tests conducted on in-vitro spiked catheters showed that pyrogenic loads of eu/device were reduced to less than eu/device. conclusions: reprocessing procedures following the adopted regeneration protocol were able to satisfy the fundamental microbiological requirements until five in-vitro reuses. sterility tests showed that devices' sterility was not guaranteed after five reuses. pre-sterilization treatments including enzymatic solutions and chlorine revealed high cleaning properties with effective bioburden reduction. storage intervals among reprocessing steps longer than hours should be avoided in order to limit contamination and pyrogenic load. technical considerations suggest to consider the introduction of reprocessing procedure only in hospitals with a considerable workload. room disinfection in the hospital setting using akacid plus Ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: akacid plus Ò , a novel polymeric guanidine with broad antimicrobial activity also against multi-resistant bacterial strains, was used in the present study as room disinfectant. methods: disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multi-resistant staphylococcus aureus (mrsa), pseudomonas aeruginosa and escherichia coli was performed using akacid plus Ò at concentrations of . %, . % and . % for minutes. bacterial suspensions were distributed on stainless steel plates and placed in a test and control room. recovery of the test bacteria was determinedbefore nebulizing, and minutes after the beginning and hours after the end of room disinfection by a modified simple swab-rinse technique. for the detection of mrsa in isolation units, surface samples were collected by direct swab and enrichment culture. results: the swab-rinse method demonstrated a dose-and time-dependent effectiveness of akacid plus Ò in eradicating s. aureus, e. coli and p. aeruginosa on stainless steel plates. nebulizing of . % akacid plus was successful in eliminating all hospital pathogens in min contact time, while mrsa was still detectable after use of . % akacid plus Ò . . % akacid plus Ò achieved a reduction > cfu of s. aureus and p. aeruginosa, but was only able to eradicate e. coli during the observation time. the results suggest that nebulized akacid plus Ò at a concentration of . % is a potent substance for eradication of pathogenic organisms in the hospital setting. study on the antiviral efficacy of citrofresh Ò , a flavonoid based organic acid complex sanitizer z. nack (north-geelong, au) objective: determine the antiviral efficacy of this organic sanitizer against enveloped and non-enveloped viruses using a carrier based method. seeking registration for citrofresh Ò in australia and in the eu as a hospital grade antiviral sanitizer. methods: the study was performed according to the american society of testing and materials (astm) designation (e - ) recommended by the australian therapeutic goods administration (tga) to determine the efficacy of a disinfectant intended to use on inanimate, environmental surfaces. we tested citrofresh Ò (diluted in standard hard water) in three different concentrations: %, % and % on adherent cell lines (pk- , mrc- , mdck, a , l ) in four replicates against five different viruses including: porcine parvovirus (non-enveloped, high resistant against sanitizer); human rhinovirus- (non-enveloped, high resistant against sanitizer); human adenovirus- (non-enveloped, moderate resistant against sanitizer); human influenza type a (h n ) virus (enveloped, moderate resistant against sanitizer); human herpes simplex virus type (enveloped, low resistant against sanitizers). prior to the viral testings, acute toxicity assay was carried out to determine the adherent cells viability against citrofresh Ò . results: cell lines exhibited > % viability after exposure to all three concentration. herpes simplex type , human influenza type a and human adenovirus- exhibited the most significant viral log reduction of log to at % concentration of citrofresh Ò followed by the human rhinovirus- and porcine parvovirus log reduction at % concentration. the reduction of viable virus load was exhibited after minute exposure time to citrofresh Ò , which means no time-dependant activity. citrofresh Ò clearly exhibited concentration and ph dependent viral load reduction activity against influenza type a and the human adenovirus - and human herpes simplex type virus. the reduction in viral titre for porcine parvovirus and human rhinovirus- is probably ph dependent (the ph of % citrofresh Ò is . , % is . and % is . ). conclusion: our investigation shows that citrofresh Ò is an effective disinfectant on environmental surfaces, eliminating enveloped and non-enveloped viruses and sufficient to achieve the minimum -log reduction with complete viral inactivation which is prerequisite for registration. rapid environmental recontamination of an intensive care unit after decontamination with hydrogen peroxide vapour objectives: to evaluate the effectiveness of hydrogen peroxide vapour (hpv) to reduce the levels of total bacterial and methicillin resistant staphylococcus aureus (mrsa) environmental contamination on an intensive care unit (icu), and to establish the rate of environmental recontamination. methods: the study took place on a bed open plan icu. on each environmental screen sites in each bed space (under the bed, the workstation and the monitor) were examined using broth enrichment for the detection of mrsa. in addition total bacterial counts were determined for under the bed and workstation using rodac plates. environmental screening was carried out monthly for the months preceding the usage of hpv, increasing to weekly for the weeks prior to usage. additional sampling was carried out immediately before patients were discharged from icu, following the subsequent terminal clean and then immediately after hpv use. after readmission of patients sampling was carried out at h, h and then weekly for a period of weeks. patients were screened for mrsa on admission and then weekly. results: sampling of the environment prior to the usage of hpv revealed contamination of the environment with mrsa on / occasions, with mrsa colonised patients being present on only / occasions. after discharge of the patients and terminal cleaning of the environment, mrsa was isolated from ( %) environmental sites. after the use of hpv, mrsa was not isolated from any environmental sites upon immediate sampling, but h after patients were readmitted, including patients known to be colonised with mrsa, mrsa was isolated from sites. these sites were not clustered around the colonised patients but were widespread across the icu. in the weeks post hpv usage mrsa has been isolated every week. the mean total bacterial counts prior to the use of hpv were . / cm underneath the beds and . / cm on the workstations, this was reduced after hpv to . / cm and . / cm respectively. after patients readmission the counts were . / cm underneath the beds and . / cm on the workstations after h and returned to pre-hpv levels of . / cm and . / cm at each site respectively after week. conclusion: hydrogen peroxide vapour is effective in eliminating bacteria from the environment. the rapid rate of recontamination of the environment suggests that the use of hpv is not an effective means of maintaining low levels of environmental contamination on an open plan icu. objectives: the nosocomial infections are more serious and dangerous than community acquired infections since they have high rate of morbidity and mortality as well as they increase the cost of therapy. recently many precautions have been taken to prevent these infections. one of these applications is that covering of the floor of the wards, clinics, intensive care units and operating rooms of the hospitals with vinyl flooring material, which is believed to be cleaned easily and effectively. in this study it was aimed to determine the duration of survive of the staphylococcus aureus, enterococcus feacalis, escherichia coli and pseudomonas aeruginosa, which were most common encountered as nosocomial infection agents, on the surface of flooring materials such as vinyl flooring, ceramic laminated wood and galvanized sheet at room temperature. methods: four kinds of flooring materials were prepared approximately in - cm coupons and sterilized. separate bacterial suspensions equal to mc farland turbidity were swapped to the surface of each flooring materials by sterile cotton swabs. all contaminated test materials were put in sterile petri dishes with cover and kept at room temperature without subjecting to the direct sunlight. on the third day, culture samples were taken from the surface of each material by sterile cotton swaps soaked with sterile saline and streaked on the blood agar surface. culturing procedure was repeated every other day until no growth detected. in case of three consequently, negative culture results obtained culturing was ended. results: overall results of the study were presented on table . conclusions: among the four flooring materials, galvanised sheet seemed to be the most unsuitable one for the bacteria to survive long period. in other words this material should be preferred as to laminated wood for covering benches and laboratory tables. as for the flooring of the floors the vinyl flooring material is better than ceramic. covering the complete cmv ie- and pp proteins.results: cmv seropositive transplant recipients had significantly hightened ie- and pp specific t cell frequencies compared to seronegative individuals. patients withevidence of cmv antigenemia or dnaemia could not be discriminated based on cmv-and donor-reactive t cells or serum creatinine. however, recipients of seropositive grafts with low ie response showed a tendency towards more frequent cmv infection. cmv disease was observed in only / individuals. had no detectable ie or pp -t cell response, the third presented with a dominant pp response. interestingly, ie -specific t cells correlated inversely with early post-tx donor-reactive t cell frequencies during weeks - post-tx. most importantly, ie -specific t cell frequencies correlated inversely with serum creatinine at and months at several times post-tx. in patients without acute rejection, even pre-transplant ie- specific t cells correlated inversely with and months creatinine. conclusion: these data suggest subclinical control of cmv infection by ie- specific t cells and subsequently less graft injury by (cmv-induced) alloimmunity. universal precautions: knowledge, attitude and practice of healthcare workers regarding hiv, hepatitis b and c v. gupta, s. bhoi, a. goel, p. aggarwal (new delhi, in) objectives: increasing incidence of hiv, hepatitis b (hbv) and hepatitis c (hcv) in the patients expose the healthcare professionals of acquiring these infections during occupational exposure. we studied the knowledge, attitudes and practices of healthcare workers regarding hiv, hbv, hcv and the risk of occupational transmission of these diseases. methods: an interview survey was conducted among all the health care workers (hcw) using a standardised questionnaire comprising of items in english and local language, as suitable, by an expert in the emergency ward of a tertiary care teaching hospital of a developing nation. data analysis (bivariate and multivariate analysis) was done using spss version . results: (response rate: %) hcw participated in the study. the mean age was ± years, were females. the study population comprised of % doctors, % nurses, % lab technicians and % support staff. respondents had adequate knowledge about causative ( %) usual transmission ( %), symptoms ( %) of aids but poor knowledge about hbv and hcv ( %, % and % respectively). inadequate knowledge was also revealed about the infectious bodyfluids ( %), disinfection of equipments ( %), pregnancy in hcw as a susceptibility factor ( %), post exposure prophylaxis ( %) and comparative infectivity of hiv and hepatitis ( %). % of hcw became anxious while treating these patients. poor compliance with universal precautions was noticed. high compliance was reported for wearing masks ( %) and wearing gloves ( %). doctors were more likely to suffer needlestick injury (p = . ) occupational exposures was found to be high ( %) with poor declaration rate ( %). guidelines adherence was influenced by profession (p < . ), availability or adequacy of protective equipments but not by work experience as hcw (p = . ). all of the respondents urged for an interactive information session. conclusions: results from this study reveal that there is a fair level of knowledge about hiv/aids but hepatitis b and c have not generated adequate concern among the hcw. incongruity between perceived knowledge and reported practice suggests that there is a need for an interactive awareness course about the universal precautions. the educational programmes need to consider attitudes in conjunction with empirical knowledge. objectives: the sero-prevalence of hepatitis a (hav) antibodies are known to be low in young adults in korea. recently, seventeen cases of hepatitis a have been reported in health-care workers (hcw) of icu in a university hospital from may to july . we performed surveillance, and determined molecular identification of outbreaks. methods: . we checked the hav igm from all the patients of sicu with elevated ast/alt retrospectively and screened ast/alt level from all the nurses and the doctors in contact with suspicious index case. . when we determined the existence of outbreak, the molecular subtypes of hav from a blood of hcw were determined to provide the data for epidemiologic study. we determined the index case, a transmission route and the intervention for control an outbreak were planned. results: . seventeen hcw including nurses and doctors who are to years old, suffered from acute hav over weeks period. . the possible transmission of hav was fecaloral route from the bed-ridden patients with diarrhea to the exposed hcw. . seventeen hcw were identified with a positive anti-hav igm. the eight hcw had a positive hav rna. analysis of the vp -p a region of each isolate showed genotype a in five strains and co-circulation of a and b in others. conclusions: the occurrence of hav outbreak highlights the importance of standard precaution in a hospital. the hav vaccination is considered in young aged-hcw. the genotype identification of blood would be useful for the epidemiologic study of suspicious hav outbreak in a hospital. management of a norovirus-associated gastroenteritis outbreak on two psychiatric wards a. buehling, u. arnold (magdeburg, de) objectives: we report a norovirus-associated outbreak of gastroenteritis on a closed psychiatric and a gerontopsychiatric wards from december to february . during this time patients and healthcare workers (hcws) were affected. introduction and results of hygiene measures based on published guidelines on psychiatric wards are described. methods: effective and adapted measures had to be implemented to stop the outbreak and to prevent the spread of disease to other areas of our hospital. isolation or cohorting of the psychiatric patients was excluded for therapeutic reasons. regular hand disinfection in patient rooms was impossible because of the high risk of abuse. the following measures have been introduced: use of gowns, masks and gloves by hcws during care of infected patients-frequent hand disinfections with alcohol-based disinfectants by hcws using ''pocket bottles''; recommendation for all persons entering the station to use gowns, gloves and masks and to disinfect their hands frequently, distribution of handouts describing the measures; hand disinfection by all patients after using toilet, before and after taking meals (distribution of disinfectants by hcw); increased frequency of routine surface disinfection ( times daily) instead of routine cleaning once daily; routine disinfection of door handles, handrails, wash-basins and -fittings and light switches - times a shift; avoidance of patient transfer via hospital; visitor restriction during outbreak time; daily evaluation of recommended measures and adaptation to the current situation; exclusion of affected staff from the ward until h symptom free. results: the hygienic measures have been explained to the local hcws in daily meetings. they have been fully accepted only after a severe staff shortage in the fifth week of outbreak because of new cases of gastroenteritis during hcws and newly infected patients. because of the restrictive application of the adapted guidelines for these special wards the outbreak has been stopped within further weeks. conclusion: in case of norovirus-based gastroenteritis outbreaks on closed psychiatric wards hygienic measures which are adapted to the concrete situation are necessary. especially in these cases the compliance with guidelines can be increased by daily meetings and daily evaluation of recommendations. staff shortage during the outbreak forced the strict compliance with the recommended measures. regional spread of antibiotic resistance methods: we performed surveillance of patients, healthcare stuff and icu environment and we registered the infections of ab during periods of days each one. the interval between st- nd period was months and nd- rd period was year. rectal, oropharyngeal swabs tracheal aspirates from patients, handswabs from stuff and samples from environment were taken weekly. the identification of ab was performed using vitek ii system the susceptibility was tested by kirby-bauer and mic methods and the <<dna fingerprints>>obtained by pulsed field gel electrophoresis (pfge). results: during the st nd and rd period, patients ( men, women), patients ( men, women) and patients ( men, women) were hospitalized in icu respectively. ab was isolated in from samples ( . %) at the st period, from ( . %) at the nd and from ( %) at the rd period. totally ab was isolated in from specimens ( %) at the st nd and rd period among the patients carrying ab, / ( %), / ( %) and / ( %) were infected respectively. the infections observed during the study period were: sepsis ( ), urinary tract infection ( ), pneumonia ( ), meningitis ( ), thrombophlebitis ( ) . all the isolated ab strains were multiresistant to antimicrobial agents. molecular analysis of isolated strains by pfge distinguished the following types: a ( , subtypes a -a ), b ( ) at the st period a( ), c( ), d( ), e( ), f( ), g( ), h( ), i( ). j( ) at the nd period a( ), b ( ), d( ), h( ), k( ). l( ) at the rd period. infections were caused mainly by a and d types while the same types were isolated from the environment and the hands of the icu stuff. conclusion: there was a high rate of colonization and infection of icu patients by multiresistant clones of ab. the persistence of clone a of a. baumannii and the appearance of b type at the rd period after its disappearance at the nd period despite the application hygiene measures, indicates the need for more strict reinforced infection control in icu. the transmission via the hands of stuff to patients has become the most important contributor factor in patient colonization and/or infection. objectives: the antibiotic resistance and its mechanism of group a streptococci (gas) varies according to nations or study period. we have investigated antibiotic resistance and mechanism of macrolide resistance for the strains isolated from korean children and compared to the previous ( ) results. methods: throat cultures were taken from elementary school children in jinju, korea from october to december, to isolate gas. antibiotic susceptibility test to erythromycin (em), clindamycin (cc), and tetracycline (tc) was performed by disk diffusion method. macrolide resistance phenotype and genotype as well as emm genotype were studied. results: isolation rate of gas was . % ( / ). resistance rates of em, cc, and tc were . %, . %, and . % respectively, which were dramatically decreased from %, %, and % in at the same area. emm / was prevalent ( %), while emm was the most common type ( %) in . cmlsb, m, and imlsb were observed in . %, . %, and . % respectively, compared to %, %, and % in . the strains with cmlsb and imlsb had ermb gene and the ones with m phenotype were positive with mefa gene. conclusion: the resistance rates to em and cc were dramatically decreased compared to the past ( ). education to the public and physicians, decreased consumption of antibiotics, acquisition of immunity to the resistant strains, or change of prevalent emm types could be considered to explain the reason of decrease of antibiotic resistance. although antibiotic resistance rate was decreased, cmlsb type which has high mic was prevalent suggesting treatment failure for those children carrying these resistant strains in jinju, korea. analysis of skin and soft tissue infections in european medical centres: report from the sentry antimicrobial surveillance program ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) g. moet, p. strabala, h. sader, t. fritsche, r. jones (north liberty, us) objective: to analyse the skin and soft tissue infections (ssti) or wound infections in hospitalized patients in the sentry program for pathogen prevalence and resistance (r) variations in european (eu) medical centres for the years to ( years). this program also included north america (na) and latin america (la) for the same years, except . methods: consecutively isolated pathogens/site were collected from each centre per year and varied in number of sites each year in eu: eu: ( ), eu: ( ), eu: ( ), eu: ( ), eu: ( ), eu: ( ), and . susceptibility testing was determined by clsi (formerly the nccls) broth microdilution methods and interpreted by current ( ) breakpoints. results: table of all years total of ssti pathogens. (see table) sa was the predominant pathogen in eu ranging from . % of ssti isolates in to . % in . the top most prevalent organisms accounted for . % of isolates in all years with psa and ec ranking second and third, respectively with . % combined, and enc and ent ranked fourth and fifth with . % of the total isolates. compared to the americas, mrsa and vre isolation was at a lower occurrence rate in the eu; but between the rates of other monitored continents for ctz-r psa, cipro-r ec and ctz-r ent (ampc). vre increased in eu over the year. conclusions: pathogen prevalence in ssti for eu has been consistent over the monitored years although sa (with mrsa) appears to be increasing. eu is not a world leader in any key r marker compared to the americas. however, the r rates are evolving which suggests continued need for surveillance programs at regular intervals to detect mobile genetic r elements. objectives: carbapenems play an important role in the therapy of pseudomonas aeruginosa infections. the aim of our study was to characterize the molecular alteration responsible for changed susceptibility towards carbapenems in multiresistant p. aeruginosa strains from germany. methods: multiresistant p. aeruginosa strains from cystic fibrosis and non cystic fibrosis patients were collected in german hospitals in . the strains showed reduced susceptibility (intermediate or resistant; din guidelines) to imipenem, piperacillin, ciprofloxacin and gentamicin. clonality was tested using pfge. a pcr screening for vim and imp was carried out. effluxpump overexpression was detected using an effluxpump inhibitor (epi) test. oprd and for strains with positive results in the epi test the effluxpump repressorgenes mexr and nfxb were sequenced. results: pfge patterns revealed no clonal relationship among the multiresistant strains. neither vim nor imp was detected. the geno-and phenotypes found are depicted in table . defective oprd genes caused by premature stopcodons or frameshifts were found in strains. among those had no mutations in mexr or nfxb and showed the highest mics found ranging from to > and to mg/l of imipenem and meropenem, respectively. additionally had defective mexr genes, but intact nfxb genes, also had modifications in mexr and nfxb, and showed only in nfxb additional alterations. for strains no alterations in oprd but in mexr were proven. conclusions: the predominating mechanism of carbapenem resistance in multiresistant p. aeruginosa strains from germany was the loss of oprd. accessory overexpression of mexaboprm due to modifications in mexr did not result in significantly elevated mics of meropenem. moreover, the additional overexpression of mexcdoprj did not lower the mic of imipenem. in strains with modifications only in mexr only elevated mics of imipenem indicate a reduced expression of oprd accompanied by overexpression of mexefoprn as conferred by nfxc-type mutants. objective: mart (study for monitoring antimicrobial resistance trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intraabdominal infections (iai). the aim of this sub-analysis was to assess antimicrobial susceptibility patterns among gramnegative bacilli from different regions of the world during . methods: pa total of major medical centres in north america, latin america, europe, middle east/africa, & asia/ pacific tested the in vitro activity of antimicrobial agents commonly used to treat iai against consecutive unique aerobic and facultative gram-negative bacilli from iai using microdilution techniques according to clsi guidelines & breakpoints. results: enterobacteriaceae were recovered from ( %) & non-enterobacteriaceae were recovered from ( %) of the patients in the study worldwide, constituting ( %) & ( %) of the total isolates, respectively. e. coli (n = ; %) and klebsiella spp. (n = ; %) were the most commonly isolated enterobacteriaceae. pseudomonas spp. (n = ; %) and acinetobacter spp. (n = ; %) were the most commonly isolated non-enterobacteriaceae. isolates from asia/pacific and latin america were generally more resistant. ( %) of the enterobacteriaceae & ( %) of the non-enterobacteriaceae were recovered < hours after hospitalization. the % susceptible isolates are reported below: conclusion: in this study, enterobacteriaceae were the predominant intraabdominal isolates recovered both < h and > h after hospitalization. carbapenems were overall the most active agents against enterobacteriaceae worldwide. resistance rates varied among geographic regions, with the asia/pacific and latin america regions generally having the most resistance. characterisation of streptogramin resistance genes among enterococcus faecium isolates from austrian animal husbandry a. eisner, g. gorkiewicz, g. feierl, f. dieber, e. marth, j. kö fer (graz, at) objectives: the streptogramin virginiamycin has been widely used as a growth promoter in animal husbandry in the european union but was banned in because of concerns about evolving cross-resistance to the streptogramin quinupristin-dalfopristin used in human medicine. the aim of the present study was to investigate the prevalence of streptogramin resistance genes of enterococcus faecium recovered from animal faecal specimens collected in southeast austria. methods: we analysed e. faecium isolates of cattle (n = ), pig (n = ), and poultry (n = ) for the presence of streptogramin resistance genes. we used selective enterococcal broth for isolation. species identification was done on basis of gram stain, catalase and pyrrolidonyl arylamidase activity, motility, lancefield group d antigen typing, and by the strep apitest (biomérieux). detection of the resistance genes vat(e), vat(d), and erm(b) was done by pcr. results: the erm(b) gene encoding macrolide, lincosamine, and streptogramin b (mlsb) resistance was found in each e. faecium isolates recovered from pig and cattle, none of the isolates from these animals carried genes coding for streptogramin a resistance. on the contrary, e. faecium isolates from broiler specimens contained the vat(e) gene and one isolate contained the vat(d) gene. all of these isolates also contained the erm(b) gene. conclusion: our data indicate that the use of the meanwhile banned antimicrobial feed additive virginiamycin has created a reservoir of streptogramin-resistant e. faecium in southeast austrian poultry. characterisation of macrolide-resistant streptococcus pneumoniae isolates from russia objectives: streptococcus pneumoniae (spn) resistance to macrolide antibiotics continues to be of major concern. the aim of present study was to analyse phenotypic and genotypic characteristics of macrolide resistant spn isolates. methods: eighty one macrolide resistant spn isolates were collected in moscow, moscow, - . the susceptibility testing was performed according to the clsi guidelines. macrolide resistance phenotypes were characterized by triple-disk diffusion test, using erythromycin, clindamycin and rokitamycin disks. detection of genes, coding resistance to macrolides, was done by rt-pcr. sequencing for qrdr mutations was performed on levofloxacin resistant spn isolates. selected isolates were analysed by mlst. results: by the triple-disk test, isolates were assigned to the m phenotype, of them were carrying mefa gene, and one was negative. twenty eight isolates were cmlsb-phenotype, of them were carrying both ermb and mefa genes, in two isolates only ermb was detected, and one isolate was negative for both genes. imclsb phenotype was demonstrated by isolates, both ermb and mefa genes were detected in of them, and only ermb in . three isolates didn't demonstrated blunting of zone of inhibition around rokitamycin disk. associated resistance to penicillin g, tetracycline, chloramphenicol and co-trimoxazole was observed in . %, . %, . % and . % of spn isolates respectively. nine multidrug resistant isolates, harbouring both mefa and ermb genes, were subjected to mlst. among them one isolate was found to share the allelic profile st (spain f- clone), and four isolates were single-allele variants of st . in four isolates new allelic profiles were detected. three isolates were resistant to levofloxacin (mic ‡ mg/l), in two of them with levofloxacin mic > mg/l (st single-allele variants) e k, s f and i v substitutions were detected in gyra, parc and pare, respectively. d n and i v substitutions were detected in parc and pare of one isolate with new allelic profile. conclusion: high prevalence of macrolide resistant spn, harboring both ermb and mefa genes is observed in moscow, macrolide resistance is associated with resistance to other groups of antibacterials. some multidrug resistant isolates are highly related to internationally disseminated multiresistant clone spain f- . strains with fluoroquinolone resistance in moscow were all single locus variants of the spain f- clone. occurrence of tet(w) gene in a clostridium difficile clinical isolate p. mastrantonio, f. barbanti, p. spigaglia (rome, it) objectives: to investigate the presence of tet(w), a tetracycline resistance gene recently identified in anaerobic commensal bacteria from animals and humans, in c. difficile clinical isolates. methods: several c. difficile clinical isolates from different italian hospitals were analysed for the presence of a tet(w) gene by pcr assays. the primers used were designed on the tet(w) sequences available in genbank. pcr fragments obtained by these amplifications were sequenced. tet(w) dna flanking regions were also examined with a set of pcrs constructed on the sequence of the conjugative transposon tnb of butyrivibrio fibrisolvens . , that is the only element carrying a tet(w) partially characterized so far. tet(w) positive isolates were also examined for the tet(m) gene and for the presence of int and tndx, markers for the tn and the tn like elements, respectively. the tn -like elements were further characterized by pcrs designed on enteroccus faecalis tn sequence. tetracycline mic values were determined by the e-test method. tetracycline resistance gene transfer was evaluated by filter mating experiments, using c. difficile p r strain as recipient. results: a tet(w) gene was found in only one isolate, c. difficile cd , also positive for the tet(m) gene. this isolate was resistant to tetracycline with a mic of mg/l. sequence analysis of the tet(w) pcr fragment (about bp) showed that this gene had an identity of % with the genes found in clostridium spp strain k , mitsuokella multacida and butyrivibrio fibrisolvens. no amplifications were obtained with the primers designed on tnb , indicating the presence of a different genetic support for tet(w) in c. difficile. tet(m) gene of c. difficile cd was carried by a tn -like element that showed nucleotide sequence mutations in the region containing orf - compared to the element of e. faecalis. conjugative transfer of tet(w) was not observed, whereas the tet(m) gene was transferred to the recipient strain. c. difficile transconjugants were resistant to tetracycline with a mic of mg/l. conclusion: the results obtained in this study demonstrate for the first time the presence of a tet(w) gene in a clinical isolate of c. difficile, providing further evidence of the spread of this resistance determinant among gastrointestinal bacteria. macrolide resistance determinants are prevalent and readily selected for in viridans group streptococci among healthy norwegian adults background: norway has a low prevalence of antimicrobialresistant bacteria including macrolide resistant (mr) respiratory tract pathogens. we have observed an increase in macrolide consumption in norway and there is a lack of knowledge on the reservoir of macrolide resistance determinants among viridans group of streptococci (vgs) in the pharyngeal flora. objectives: examine the occurrence, selection and persistence of macrolide resistance determinants in vgs pharyngeal flora in healthy norwegian adults before and after treatment with azithromycin. methods: throat samples were collected before (day ), after treatment (day ) and after months (day ) from healthy volunteers. the samples were plated directly as a lawn on pdmii agar plates with % defibrinated blood with an erythromycin etest strip. photos were used as quantitative comparisons. up to morphological different colonies with erythromycin etest mic ‡ lg/ml from each specimen were collected; day (n = ), day (n = ) and day (n = ). in total representatives mr, vgs-isolates were selected for further studies: (i) mics of erythromycin, tetracycline and penicillin were determined by etest. (ii) pcr's for erm(b), erm(tr), and mef(a/e), and subsequent sequence-typing of mef. species identification was performed by soda sequencing. results: a total of / persons carried a low number (< ) of mr vgs in day specimens, while / had a significant higher number (> ) of mr strains in day specimens. in day specimens, / carried a low number of mr, resembling day . reduced susceptibility to penicillin was observed in / ( %) isolates. tetracycline resistance was found in / ( %), and mainly in erm(b)-positive strains. mef(a/e)-positive dominated day ( %) and erm(b) day specimens %. sequence typing revealed mef(e) (n = ) and mef(a) (n = ). soda sequence; s. mitis (n = ), s. oralis (n = ), s. parasanguinis (n = ), s. salivarius (n = ), and s. sanguinis (n = ). conclusion: there is a pool of vgs carrying macrolide resistance determinants in the normal pharyngeal flora of healthy adults that are readily selected for during azithromycin exposure. the mef(e) and erm(b) were the most prevalent resistance genes and co-resistance to tetracycline was frequently observed, resembling the findings in norwegian clinical isolates of s. pneumoniae. these vgs may provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms. relationships in genotype, phenotype, t type and pfge type among macrolide-resistant streptococcus pyogenes strains isolated in the czech republic v. jakubù , p. urbášková, l. straková (prague, cz) objectives: to determine relationships between phenotypic and genotypic methods among erythromycin-resistant s. pyogenes strains. methods: a total of clinical isolates of s. pyogenes resistant to erythromycin were collected in microbiology laboratories during - . erythromycin susceptibility was tested by the disk diffusion method. strains with an inhibition zone < mm around the erythromycin disk ( lg) were sent to the national reference laboratory for antibiotics (nrl). presences of mlsb resistance genes (ermtr, ermb and mefa) were tested by pcr. t serotypes were determined in randomly selected representatives of each phenotype (n = ). pfge type were determined in strains from year only (n = ). results: the rate of the most prevalent phenotype (constitutive mlsb resistance) was %, % in the year and , respectively. the major prevalent t types among the analysed strains were serotype t ( %), t ( %), t ( %) and t b ( %). gene ermb was the most frequent ( %). the results of pcr method was highly congruent with observed phenotype of resistance. pfge patterns of strains with constitutive mlsb resistance were highly identical. conclusion: m phenotypes, constitutive and inducible resistance to mlsb antibiotics were found and ermtr, ermb and mefa genes were detected among the analysed strains. the t serotype was identified the mainly prevalent in our collection. the majority of strains harbouring t serotype were constitutively resistant to macrolides. the study showed close relationships among genotypes, t types, specific resistotypes (phenotype) and pfge types. objectives: since recognition of transferable clindamycin and tetracycline resistance in bacteroides, we have undertaken a us national survey on the susceptibility of b. fragilis group to analyse emergence of resistance and trends, since these species are not routinely tested for susceptibility in hospital clinical laboratories. methods: agar dilution mics were determined for isolates from - for b. fragilis and related species from geographically diverse centers in the us. antibiotics included carbapenems, b-lactam/b-lactamase inhibitors, quinolones, a tetracycline, clindamycin, metronidazole, chloramphenicol, a glycylcycline and linezolid. isolate identity was confirmed by api a. results: analysis of resistance trends from - showed a decrease in geometric mean mic's (geomic) for imipenem ( . mcg/ml to . mcg/ml, p < . ) and meropenem ( . mcg/ml- . mcg/ml, p = . ) for the bacteroides species. ertapenem geomic remained unchanged ( . mcg/ ml). for the b-lactamase inhibitors, piperacillin-tazobactam geomic declined from . mcg/ml to . mcg/ml (p < . ). ampicillin-sulbactam geomic did not change. few isolates were resistant to any carbapenem or b-lactamase inhibitor combination. clindamycin resistance increased, especially for b. fragilis, b. ovatus and b. thetaiotaomicron (all p < . ). among quinolones, resistance of bacteroides to moxifloxacin increased (geomic went from mcg/ml to . mcg/ml, p < . ). b. fragilis remains the most sensitive bacteroides species to moxifloxacin, although approximately % of stains have mic's ‡to mcg/ml in . tigecycline susceptibility, tested over years, did not change. the first confirmed metronidazole-resistant isolate (mic = mcg/ml) obtained in the us was noted in but none were noted in or . conclusion: improved susceptibility of bacteroides species to some carbapenems and the b-lactamase inhibitor combinations is unexplained but significant. clindamycin resistance continues to increase, especially for b. fragilis. moxifloxacin susceptibility for the non fragilis species shows that the majority of strains are resistant. the first metronidazole resistant isolate has been reported from the us. since resistance trends are associated with species, the differentiation within the species is of extreme importance, since it may impact the choice of antimicrobial agent for the treatment of infections caused by this group of anaerobes. observed duration of nasopharyngeal carriage of penicillin-resistant pneumococci: relations to age and serogroup p. geli, l. hö gberg, h. ringberg, e. melander, m. lipsitch, k. ekdahl (solna, malmö, lund, stockholm, se; boston, us) background and objectives: knowledge of how the duration of pneumococcal carriage varies with age and serogroup is essential to understanding how immunity to carriage arises throughout the course of life, and designing appropriate models for the effects of vaccination or other public health initiatives aiming to reduce the pneumococcal transmission in the community. using data from an ongoing swedish intervention project, the duration of nasopharyngeal carriage of penicillinresistant pneumococci (mic pcg > . mg/l) stratified by both serogroup and age of the carrier were estimated. methods: the mean duration and corresponding % confidence interval was estimated by fitting a gamma distribution to the observed duration of carriage for each serogroup and age stratum. results: the mean duration of carriage for all cases was days ( % ci - ). children below the age of years carried prp for significantly longer periods ( days, % ci - ) compared with older individuals ( days, % ci - ). there were also differences within the group of cases below the age of years, as the duration of carriage became significantly shorter for each year older the cases were. serogroup and were carried for significantly shorter periods compared with serogroup . serogroup also had significantly shorter carriage duration compared with serogroups and for cases - years. for cases years or older, no significant difference in carriage duration for different ages or serogroups could be noted. conclusions: even though the estimate does not cover any correction for the censored carriage duration and therefore not yield an estimate of the total length of carriage, the results highlight the importance to take both serogroup and age of the p exploring the molecular basis for differences in phenotype of salmonella enteritidis typing phage n. delappe, d. morris, m. cormican (galway, ie) objectives: the salmonella enteritidis phage tying scheme of the laboratory of enteric pathogens, health protection agency, uk, is a widely used method for subtyping this important pathogen. the method is rapid and highly discriminatory. interpretation of results can be subjective and the typing phage which are central to the method have not been well characterised. complete sequence data is available for the salmonella typhimurium podovirus phage p . methods: the typing phage were propagated on s. enteritidis pt b (pb ). phage were visualised by electron microscopy. phage dna was extracted and digested with hindiii. consensus pcr primers were designed based on sequences of p and other s. typhimurium phage. additional primers were designed based on the sequence of the s. enterititidis typing phage (a siphovirus). amplification, sequencing and dna probe hybridisation of various phage genes were performed using standard techniques. results: on em the typing phage comprise podoviridae (phage , , , , and ) , siphoviridae (phage , , , , and ) and myoviridae (phage , , and ). digestion with hindiii subdivided each morphotype into groups. the podoviridae contained genes homologous to p while the siphoviridae contained genes homologous to the sequenced s. enteritidis typing phage . some sequence variation was detected in podovirus and siphovirus genes however in some cases phage, which differ in their phenotype had no difference detected in hindiii digestion pattern or partial sequence. conclusions: the s. enteritidis typing phage set comprise distinct phage morphotypes. in some instances distinct phage that contribute to differentiation between s. enteritidis phage types had no dna sequence variation detected. variations in phage typing reactions may in part be due to epigenetic difference in typing phage, e.g. due to methylation of phage dna. salmonella enteritidis typing phage biology could provide a model for developing approaches to phage therapy. tularemia is a zoonotic bacterial disease. the causative agent, francisella tularensis, is spread to humans by direct contact with infected rodents, inhalation, ingestion of contaminated water or by arthropod bites. in some endemic regions, outbreaks occur frequently, whereas nearby rural parts may be completely free. we presented two cases of tularemia in non endemic region of the turkey. case : a year old female patient referred to tertiary hospital due to swollen on the neck for months. before admission beta lactam antibiotics had been prescribed to her for tonsilopharyngitidis. but her complaints had been continued. so she admitted to our hospital. she had been suffered fever sore throat and neck pain. she had a palpable and painfull cervical lymphadenopathy which was not suppurated. leukocytosis and elevated c reactive protein were predominant. at screening there were not any lymphadenopathy detected elsewhere. she had been examined about cytomegalovirus epstein barr virus and brucellosis. they were negative. fine needle aspiration from neck was negative considered as malignancy. cultures were negative for routine bacteriologic examination. microagglutination test for tularemia was / positive. then we decided to treat her with gentamycin for days. after treatment cervical lymphadenopathy became small. leukocyte count and c reactive protein levels were reach normal range. case : a year old female patient referred to university hospital due to cervical lymphadenopathy and fever and sore throat. before admission beta lactam antibiotics were prescribed to her for weeks. but no apparent benefits had been detected. there was a palpable and fistulated cervical lymphdenopathy. drainage was examined microscopically and cultured for bacteria, mycobacteria and fungi. on routine cultures no microorganisms were grown. fine needle aspiration was done. it was reported that suppurative granulamatous lympadenitis. so we were examined for tularemia, cat scratch disease. microaggltunation test for tularemia was / positive. then streptomycin had been given for days and excision of lymphadenopathy had been done. no complications or recurrence occur. results: both patients were applied to us from non endemic and different regions of the turkey. they had no known insect bite history. both of them were diagnosed by serological tests. conclusions: in the differential diagnosis of tonsillopharyngitidis, tularemia also must be considered in the non endemic regions. tularaemia presenting with tonsillopharyngitis and cervical lymphadenitis: two case reports b. kandemir, i. erayman, m. bitirgen, e. turk aribas, a.c. inkaya, s. guler (konya, tr) tularemia is a zoonotic disease caused by francisella tularensis. francisella tularensis is transmitted to humans by direct contact or ingestion of infected animal tissues, through the bite of infected arthropods, by consumption of contaminated food or water, or from inhalation of aerosolized bacteria. in this report we describe two cases of oropharyngeal tularemia who presented with tonsillopharyngitis and cervical lymphadenitis. case i: a years old woman with multiple cervical lymphadenitis has been admitted to our clinic. her complaints started months ago with signs and symptoms of tonsillopharyngitis. she had received non specific treatment (ampicillin+sulbactam) and ten days later cervical lymph nodes appeared. the diagnosis was made serologically. the antimicrobial therapy (streptomycin · g im) was given for fourteen days. the patient recovered completely. case ii: a years old girl with multiple cervical lymphadenitis was admitted to hospital. her complaints started months ago with throat ache after which multiple cervical lymphadenitis appeared. she was admitted to our out patient clinics and diagnosed to have tularemia. anti-microbial therapy (streptomycin · g im+doxycyciline · mg) was given for four weeks but no clinical response was achieved. patient was admitted to the hospital and surgical drainage was performed. treatment against tularemia was prolonged. patient was finally recovered at the end of nine weeks of therapy. it can be concluded that early diagnosis and treatment of tularemia are important. some patients may benefit from surgical drainage and prolonged therapy. a case of nonclostridial crepitant cellulitis which is due to escherichia coli c. ayaz, m. ulug, m.k. celen, m.f. geyik, s. hosoglu (diyarbakir, tr) objectives: this condition is caused by gas forming bacteria that involve the skin, either or as an extension from deeper structures. the origin of infection is an abdominal wound, perianal disease, or operative incisions that have become secondarily infected. tracking of gas-forming organisms from deeper sites of infection may also present as crepitant cellulitis without a break in the skin. diabetics are more likely to acquire such infections, especially in the lower extremites. among the bacteria isolated are anaerobic organisms such as bacteriodes or anaerobic streptococci, or coliform bacteria, especially escherichia coli and klebsiella. because of this reason we reported a case of a nonclostridial crepitant cellulitis which is due to escherichia coli. case: a year old man who was previously healthy, has come with fever, pain, oedema, erythema, crepitant and limitation of movement at the right lower extremity. in his history he had no complaint until weeks ago. perianal abscess has developed at this time and it has drainged spontaneously days later. than his complaints has comprised day duration. on physical examination, the temperature was . °c, pulse rate / minute, respiratory rate /minute and blood pressure was / mmhg. laboratory evaluation showed a haemoglobin . g/dl, leucocyte count of /mm (neutrophils %). serum electrolytes, renal and liver function tests were within normal limits. c reactive protein was elevated up to mg/dl, esr was mm/h. escherichia coli was isolated from wound and blood cultures. he was treated initially with ampicilinsulbactam ( g/day) and required attempt. even with optimal surgical and medical therapy, he dies at the third day of the treatment from septic shock. conclusion: the onset is generally gradual, and there is usually mild local pain and systemic toxicity, allowing clinical differentiation from the more fulminant clostridial myonecrosis. the surgical approach should be aggressive, but tailored specifically to the underlying cause of infection. antibiotic therapy is directed at a mixed aerobic-anaerobic flora, until culture reports are available. a case of iliopsoas abscess which is due to pseudomonas aeruginosa objectives: pyogenic psoas abscess, a rare but life-threatening infection, results from primary suppuration or is secondary to the spread of infection from an adjacent structure. primary iliopsoas abscess occurs probably as a result of hematogenous spread of an infectious process from an occult source in the body. primary iliopsoas abscess can occur in diabetus mellitus, intravenous drug abuse, aids, renal failure and immunosupression. ultrasound is diagnostic in only % of the cases. computed tomography should be done for definitive diagnosis and is considered the gold standard. stapylococcus aureus is the causative organism in patients with primary iliopsoas abscess, but pyogenic psoas abscess caused by pseudomonas aeruginosa is uncommon. because of this reason we reported this case. case: a previously well year old woman presented with a month history of right loin to groin pain, limping or limitation of hip movement, fever and nausea. she was a diabetus mellitus patient for years. on her physical examination, the temperature was . °c, pulse rate /minute, respiratory rate /minute and blood pressure was / mmhg. examination of the respiratory system, cardiovascular system and abdomen were found to be normal. laboratory investigations revealed total leucocyte count of /mm (polymorphs %), c reactive protein was elevated up to mg/dl, esr was mm/h. serum electrolytes, renal and liver function tests were within normal limits, but serum glucose level was elevated to mg/dl. her blood cultures were sterile, but abscess culture yielded pseudomonas aeruginosa which was taken during the surgery. she was treated imipenem ( g/ day) + amicasin ( . g/day) and required surgical drainage. she was treated and followed up days, and discharged at the end of the treatment. conclusion: in these patients treatment involves the use of appropriate antibiotics along with drainage of the abscess. an adequate knowledge of the causative organisms should guide the initial choice of antibiotics. depending on the results of the abscess fluid culture and sensitivity, adjustments should be made. percutaneous drainage or surgical drainage may be done in them. in conclusion early recognition, empiric antimicrobial coverage and aggressive drainage or debriment are indicated in these patients. cervical lymphadenitis in a diabetic woman f. Ç okça, a. azap, s. gö çmen, h. erdi sanli, s. gü l (ankara, kirikkale, tr) objective: rhodococcus equi infections are commonly seen in immunocompromised patients. exposure to domestic animals, such as horses and pigs may play a role in some cases. two thirds of the r. equi infections in immunocompromised were reported in hiv infected patients, and the rest divided between transplant recipients, immunosupressive medications and other kinds of immunosupression. the clinical picture presents with pulmonary infection in % of patients. here, we report a rare case of cervical lymphadenitis in a diabetic women due to r. equi. case: a sixty-year-old diabetic woman was admitted with the complaints of fever, right cervical erythematous swelling with tenderness and warmth. on physical examination; inflammation beginning from the right submandibular region and descending to the upper chest was detected. a tender mass of · · cm. was palpated on the right cervical region. ampicillin/sulbactam g/day was given emprically for a week with no improvement. the ct scan of the neck showed conglomerated lymphadenopathy extending from the submandibular area to the supraclaviculary region with . · cm in size. the mass began to fluctuate and cc abscess material was drained surgically. gram's stain of the purulent material showed polymorphonuclear leukocytes with pleomorphic gram positive coccobacilli. the cultures of the material grew r. equi. therapy was changed to teicoplanin and ciprofloxacin combination and surgical care of the wound with antiseptics was performed. after a month, intrevenous medical therapy was changed to oral route with roxythromycin and ciprofloxacin and was continued to months with complete resolution. conclusion: increased awareness and improved laboratory techniques help for the early diagnosis of rhodococcal infections. timely diagnosis is important because the microorganism is usually resistant to penicillin g, oxacillin, ampicillin, carbenicillin and cefazolin. the use of at least one antibiotic with intracellular activity is necessary in the treatment of r. equi infections. empirical two drug regimens with erythromycin, rifampin and/or ciprofloxacin are recommended. objectives: to analysed the features of spondylodiscitis (sd), their clinical presentation, the commonest diagnostic methods and the kind of treatment applied according to the different groups of the study. methods: a retrospective and descriptive study taking place amongst the patients diagnosed as having sd from till . in each case we studied the presence of underlying disease, primary infectious sources in the prior months, the way symptoms started, location, diagnostic methods, treatment and evolution, comparing between different aetiologies. results: patients with sd were studied. of them had pyogenic sd,( had spontaneous sd and had an sd after spinal surgery) and patients had tuberculous sd. were men ( to years; mean . ). patients with postoperative sd were the youngest (mean . y, p = . ). underlying diseases were found in % of patients, mainly in postoperative sd ( % of cases) (p = . ). an episode of previous bacteremia or infectious source was found in % and % respectively of patients with spontaneous pyogenic sd, significantly higher than in surgical sd ( % had bacteremia and % other infectious source, p < . ). the most common presenting symptoms were back pain ( . %) and neurological deficits ( %). frank fever occurred in % of cases, being more frequent in spontaneous sd ( %) than in postoperative sd ( %) or tuberculous sd ( %), p £ . . leukocytosis was found only in % of patients. postoperative sd presented the lowest levels of esr (p = . ). s. aureus was the most frequent bacteria isolated ( %) in pyogenic spontaneous sd, as coagulase negative staphylococci was in surgical sd. lumbosacral localization was detected in % of spontaneous pyogenic sd and in % of postoperative sd. tuberculous sd predominate in dorsolumbar region. paravertebral abscess formation was observed in % of pyogenic sd and in % of tuberculous sd (p = . ). surgical treatment was required in . % of tuberculous sd and in % of pyogenic sd (p = . ). outcome of patients with spontaneous sd was worse (sequelae in %), than in patients with surgical sd ( . %) or tuberculous sd ( %) (p = . ). conclusions: ) spontaneous sd was the most frequent and it occurred mainly in patients suffering from underlying diseases; ) nearly all patients had pain but only in / of them was accompanied by fever; ) the lumbar zone was the most frequent location; ) the majority of patients had a complete resolution of their symptoms only with medical treatment. background: the ethiopathogenesis of cns abscess includes a broad spectrum of pathogens and predisposing conditions, so that a polymicrobial flora is a quite frequent event. capnocytophaga spp. includes fastidious gram-negative organisms, usually underestimated in the common clinical practice, and poorly tested in vitro for antimicrobial susceptibility. surprisingly, also agents usually active on gram-positive pathogens demonstrated some efficacy against capnocytophaga spp. (i.e. erythromycin, rifampin, tetracyclines, cotrimoxazole, chloramphenicol, and glycopeptides), which is usually responsible of anecdotal episodes of cns infection (meningitis, brain abscess, and subdural empyema). methods and results: the fourth case report of capnocytophaga spp. brain abscess is herewith reported. a probable origin from a recent cat bite and a mandibular granuloma is suspected. due to the lack of clinical and neuroradiological response to neurosurgical debridement and an association therapy including imipenem, amikacin, clindamycin and fluconazole, empiric administration of linezolid ( mg/day) was attempted, and a rapidly favorable clinical, microbiological, and neuroradiological response was achieved. notwithstanding the identification of capnocytophaga spp. as the sole microorganism yielded by purulent drainage of a cns abscess, patients with multiple risk factors and recent surgery are expected to suffer from a polymicrobial cns infection. due to its favourable cns penetration and its dual mode of administration (both i.v. and oral), linezolid may represent an alternative option in the event of cns diseases borne by numerous risk factors and a suspected polymicrobial origin, especially when a lack of response to first therapeutic attempts is of concern. in the management of a cns abscess where the role of microorganisms with an unpredictable sensitivity profile remains of concern, chemotherapy should be directed also against potentially multiresistant organisms. considering also the relevant limitations given by the often poor cns penetration, the activity of glycopeptide agents is limited, compared with that of linezolid. aetiologies and antimicrobial resistance profiles of purulent meningitis study carried out in a hospital of infectious diseases, algiers objectives: bacterial meningitis is a serious clinical and medicolegal consequences if management is incorrect. meningitis protocols have recently been published by the british infection society/meningitis research foundation and are widely disseminated in our institution. local guidelines are also available on the hospital intranet and in the emergency department and acute medical admissions wards. this study investigated the level of understanding about meningitis and knowledge of the guidelines in medical staff of different grades working in the emergency department and the acute medical admissions unit in a large teaching and emergency hospital. methods: medical staff were interviewed faced to face and asked a series of questions on the management of meningitis. results were stored on a database and responses were analysed. results: general knowledge about meningitis was variable. although % knew that bacterial meningitis was a notifiable disease only % knew the procedure for informing the health protection agency and only % would notify viral meningitis. only % of responders were aware that guidelines could be viewed on the hospital intranet. only % correctly identified the indications and cautions for lumbar puncture. although the majority recognised the need for urgent administration of antibiotics % would omit antibiotics until further assessment and lumbar puncture results. only % were aware of the need to consider adding ampicillin to cover listeria in patients over years of age and there was uncertainty about the management of patients with penicillin resistance. conclusions: although protocols and guidelines for meningitis have been produced and are easily accessible the majority of medical staff were uncertain how to access and utilise this information. the level of knowledge and expertise in managing meningitis amongst medical staff working in a and e and the acute medical unit was poor and there is a need for further education to improve patient management. guidelines are of no value if they are not disseminated to front-line medical staff. objectives: the aim of this study was to evaluate the prevalence of penicillin resistant and multi-drug resistant pneumococci isolates in streptococcus pneumoniae meningitis. methods: a retrospective study was carried out on clinical records between january and october . among the csf samples the pneumococcal ethiology was confirmed by % positive cultures and % latex agglutination. antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. isolates of pneumococci with oxacillin zone sides of > mm are susceptible (mic < . microg/ml) to penicillin, while at those of < mm the mic has to be determined (by e -test). results: isolates from patients ( %) were found with penicillin-resistance (prp) -of which % were multi-drug resistant-and ( %) with penicillin susceptibility -of which % were resistant to other drugs. an abrupt onset of disease was found in % prp patients and % from non-prp ones. chest x ray pulmonary determinations were found in % prp patients and % non-prp ones. sixty-six per cent of prp patients and % of non-prp ones had a prior hospitalization. only % of non prp patients had a positive blood culture. antibiotic switch was made in % cases with prp isolates and % cases with non prp ones. the overall rate of mortality was %, with % for prp patients and % for non-prp ones. conclusions: non-prp isolates were the prevalent ethiology of s. pneumoniae meningitis. % of non -prp strains developed other drug resistance, and % prp strains were multi -drug resistant. prp meningites evolved more as a hospital-related pathology, with an abrupt onset, frequently associated with pulmonary determinations and higher mortality rate. background: although vaccination strategies have shifted the age distribution of meningitis to older age groups, few studies have specifically examined bacterial meningitis in the older adult. methods: from october to april , we prospectively included episodes of community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, which occurred in patients aged > years. we dichotomized the cohort with respect to age: patients aged ‡ years were defined as older adults and patients aged - years as younger adults. predictors for an unfavourable outcome (defined as score - on the glasgow outcome scale) were determined by logistic regression. we tested for statistical interaction between age group and potential prognostic factors by adding multiplicative interaction terms to the model. the mann-whitney u test and the chi-square test were used to identify differences between groups. results: of episodes ( %) occurred in older adults and episodes in younger adults ( %). streptococcus pneumoniae was the most common pathogen in older adults ( %). meningitis in younger adults was caused by neisseria meningitidis and s. pneumoniae in % and % of the episodes, respectively. older adults were more likely to present with the classic triad of bacterial meningitis (fever, neck stiffness and altered mental status) than younger adults ( % versus %; p < . ). the prognostic value of independent risk factors for unfavourable outcome was similar in both age groups. older adults had more complications during clinical course, resulting in a higher mortality rate than in younger adults ( % versus %; p < . ). sepsis was the most common cause of death in both age groups ( % in older adults versus % in younger adults; fig) . whereas older adults tended to die more often due to cardiorespiratory failure ( % versus %; p = . ), younger adults more often died due to brain herniation ( % versus %; p = . ). conclusions: bacterial meningitis in older adults is associated with high morbidity and mortality rates. elderly patients often present with classic symptoms and s. pneumoniae is the most common pathogen within this age group. whereas older adults often die due to cardiorespiratory failure, younger adults more often die due to brain herniation. incidence of serogroups and penicillin susceptibility in neisseria meningitidis isolates ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) objective: the aim of this study was to analyse the serogroup incidence and penicillin susceptibility in n. meningitidis before and after spanish epidemic outbreak in . in this year the public health service decided a massive vaccination in our sanitary area (galicia, north-west of spain, . inhabitants) and in autum of the inclusion of vaccine against n. meningitidis serogroup c in vaccination programme. methods: retrospective study of all cases of meningococcal disease confirmed by culture and/or pcr in the health care area of santiago de compostela (galicia) from to . results: in the period - we identified meningococcal disease episodes by microbiologic diagnosis ( . %, . % and . % to b, c and w respectively). in , serogroup c were the % of the isolates. in and the serogroup incidence was almost the same (b = . %, c = . %). from an increase in b serogroup cases were detected, in ( . %), in ( . %) and in ( . %). the most frecuent phenotype has been b p : . in c serogroup. during this period an increase in penicillin susceptibility was observed (in b serogroup % in and % in of the isolates were susceptible and in c serogroup % in and % in ). conclusions: the b serogroup is the most frecuent isolate during this period except in the years and . the strain that cause the epidemic outbreak in (c: b p : . ) was not isolated since . in our health care area, c: a serotype, was isolated for first time in , and since then, is the unique serotype isolated in c serogroup. incidence rate in c serogroup has changed from . / . in to . / . in . this decrease was caused by the drop of incidence rate on the youngest groups (< years and - years). the incidence rate in b serogroup during these years was modified from . / . in to . / . in . a four-year retrospective analysis of infective endocarditis in a belgian university hospital n. de visscher, b. delaere, b. krug, y. glupczynski (yvoir, be) objectives: to establish the epidemiology of infective endocarditis (ie) and determine the prognostic factors for adverse outcome in patients admitted to a university hospital with a cardiovascular surgery department. methods: between / and / , the clinical and laboratory features of all consecutive adult patients with a definite diagnosis of ie (duke criteria) were evaluated retrospectively by two infectious diseases physicians on the basis of clinical data charts and microbiological laboratory. results: patients ( men, women) presented with a definite diagnosis of ie. mean age was , yrs; cases ( %) were native valve endocarditis (nve) and ( %) were prosthetic valve endocarditis (pve); % of patients with nve had underlying valvular abnormalities ( bicuspidies, mitral prolapsus or regurgitation, others). ten out of cases of pve were late-onset episodes (> year after surgery). global mortality was % ( / pts), including patients ( %) still under antibiotic therapy. a higher mortality rate was observed in pve [ / ; ( %)] than in nve [ / ; ( %)]. overall, pts ( %) underwent surgery (mean: days following admission). valvular replacement was contra-indicated in pts because of critical status and/or major co-morbidities. the distribution of isolated pathogens was: streptococci: cases ( %) including cases of s. bovis, s. aureus: cases ( %, including mrsa), enterococci: cases ( %), miscellaneous: cases. the affected valves were: only aortic: ( %), only mitral: ( %), only tricuspidal: , aortic and mitral: , mitral and tricuspidal: , aortic, mitral and tricuspidal: . a high mortality rate was observed in s. aureus ie ( / [ %]), especially in the subgroup of patients with a pve ( / pts [ %] ). the mortality rate in patients with ie episodes caused by streptococci amounted % ( / pts). clinical microbiology and infection, volume , supplement , related, % (n = ) had previous surgery and % (n = ) were related to urinary or digestive tract procedures. only patients had illegal substance abuse. the most frequent predisposing acquired cardiac condition for native valve endocarditis was degenerative valvular disease in % ( / ). twelve percentage (n = ) had prior ie. the most frequent predisposing congenital cardiac condition was a bicuspid aortic valve in % (n = ). in % (n = ), no predisposing heart disease was discernible. causative microorganisms included: staphylococci in % (n = ) with s. aureus in % (n = ), cons in % (n = ), streptococci in % (n = ) with s. viridans in % (n = ), s. bovis in % (n = ), enterococci in % (n = ) and other pathogens in % (n = ). culture negative ie was reported in % (n = ). both in community-acquired and nosocomial ie, s. aureus was the most frequent causative agent. twenty-three percentage ( / ) were methicillin-resistant s. aureus. s. viridans ie was mainly community-acquired while enterococcal ie was nearly equally distributed between community and nosocomial origin. conclusion: compared to older series, we observed a high proportion of nosocomial ie and of prosthetic valve ie. s. aureus and e. faecalis were the most prevalent causative microorganisms. enterococci were nearly equally distributed between community and nosocomial origin, suggesting that nosocomial enterococcemia should be added as a major criterion, as proposed before for s. aureus. the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis: a meta-analysis of comparative trials m. falagas, d. matthaiou, p. papastamataki, i. bliziotis (athens, gr) objectives: the addition of an aminoglycoside to a beta-lactam for the treatment of patients with infective endocarditis has been supported mainly from data from laboratory and animal studies. we sought to review the evidence from the available comparative clinical trials regarding the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis due to gram-positive cocci. methods: the studies for our meta-analysis were retrieved from searches of the pubmed database and from references of relevant articles. included studies were trials that provided comparative data regarding the effectiveness of the treatment and/or mortality in patients receiving monotherapy with a betalactam or beta-lactam/aminoglycoside combination therapy. two independent reviewers performed the literature search, study selection, and extraction of data from relevant studies published in english during the period / - / . results: no clinical trial comparing beta-lactam monotherapy to beta-lactam/aminoglycoside combination therapy for the treatment of enterococcal endocarditis was found. we performed a meta-analysis of available comparative trials ( randomized controlled trials and comparative prospective trial) that included patients with bacterial endocarditis in native valves due to staphylococcus. aureus ( studies) or streptococcus viridans ( study). there was no statistically significant difference between the compared arms regarding mortality (or . , ci % . - . ), treatment success (or = . , ci % . - . ), treatment success without surgery (or = . , , and relapse of endocarditis (or = . , . nephrotoxicity was less common in the beta-lactam monotherapy arm compared to the beta-lactam/aminoglycoside combination therapy (or = . , ci % . - . , p = . ). conclusion: the limited evidence from the available prospective comparative studies does not offer support for the addition of an aminoglycoside to beta-lactam treatment of patients with endocarditis due to gram-positive cocci. a large multicenter randomized controlled trial may be necessary to reach a definitive conclusion on this issue. outpatient antimicrobial therapy for infective endocarditis. single-centre experience objectives: to evaluate the characteristics and outcome of infective endocarditis (ie) patients included in a outpatient antimicrobial therapy (opat) program. methods: from january to may all patients who received opat therapy for an ie were prospectively evaluated. inclusion in opat program require clinical stability and agreement of patients. active drug addiction was contraindicated for inclusion. antibiotic treatment was administered in bolus for once-daily antibiotics regimens. we used cadd-legacy tm plus (deltec, inc. st paul. usa) portable infusion system for either continuous or intermittentprogrammed bolus infusion. results: we included patients, male ( %), mean age years old (sd: . years). the diagnostic of ie was definite in cases ( with pathologic diagnosis), probable and possible. mostly of the cases were community-acquired ie ( %). mitral valve ie was the most frequent anatomical site involved ( %), followed by aortic ( %). native-valve ie represent the majority of cases ( %), but % were prostheticvalve and % were pacemaker lead ie. viridans group streptococci was the most frequent isolate ( patients, %) with cases of s. bovis ie. eleven patients had s. aureus ie ( %). at the time of the diagnosis, patients had valve rupture and patients had periannular abscess. a total of patients required some surgical intervention for the ie [ valvular replacement ( of them associated with aortic graft), pacemaker extraction and aortic graft]. the majority of the patients received outpatient monotherapy ( %). the most frequent antibiotic used was ceftriaxone ( % of the cases), followed by cloxacillin %, gentamycin %, vancomycin %, teicoplanin %, ampicillin % and other antibiotics in %. in % of the patients the vascular access was a perifericallyinserted venous central catheter and in % we used a portable infusion system. twelve patients ( %) had some complication during opat that require hospital readmission, of which could return to opat program. three patients had a fatal outcome (deaths) during admission, not related to ie complications. the mean duration of opat was . days per patient, and globally supposed . days of hospital admission savings. conclusion: opat for ie can be a good therapeutic option for ie stable patients. this procedure can represent a considerable amount of hospital admissions savings, improving also patients' well-being, and must be take into account for the treatment of this disease. objectives: botulism, a neuroparalytic illness, is caused by toxin produced by clostridium botulinum. food born botulism, a potentially lethal neuroparalytic disease, is caused by ingestion of preformed toxin. clinical illness is characterised by cranial nerve paralysis, followed by descending flaccid muscle paralysis. in this article we report a case series including a family group of type e botulism after ingestion of an iranian traditional soup. methods: in january , patients of a family group developed clinical manifestations of botulism - hours following ingestion of a traditional soup. their main clinical presentations were severe weakness ( . %- case) and lethargy ( . %- case). other signs and symptoms were blurred vision, fixed and dilated pupils, diplopia, dry mouth and decreased gag reflex. based on clinical finding, all patients received monovalent antitoxins (a, b, c). stool, gastric fluid and serum samples were sent for toxicological evaluation using the standard mouse bioassay. results: type e toxin was detected in the stool and serum sample of only one patient. all patients recovered and discharged one week after admission. conclusion: this study confirmed that prompt administration of antitoxin can prevent progression of disease based on clinical judgment and also may be life saving. in this case series study, we observed a short incubation period of - hours only in type e botulism. an outbreak of group g streptococcal pharyngitis among hospital personnel considered to be foodborne n. karabiber, a. gurbuz ertas, m. karahan, e. aykut arca, z.c. karahan, a. tekeli (ankara, tr) introduction: food-born outbreaks of streptococcal pharyngitis are relatively rarely reported,and while group a streptococci are the main causative agents, only a few epidemics caused by group g streptococci have been published. we describe here an outbreak of group g streptococcal pharyngitis occurred among the staff of a teaching hospital in ankara.the outbreak: an explosive outbreak of pharyngitis occured mainly among the staff working in certain departments (i.e. intensive care units, operation rooms) of tü rkiye yü ksek ihtisas teaching hospital, in january . methods: a total of ( and ; and from catering firm personel) throat cultures were evaluated in days,and bhs strains were isolated, and on the first and the second days, respectively. presumptive identification by nbacitracin and trimethoprim/sulfametoxazole disk diffusion test showed that strains were non-group a, strains were group a streptococci. in definite grouping by streptococcus grouping kit (avipath-strep,omega), strains were found to be lancefield group g, strains were found to be group a streptococci (gas). one of the gas strains was isolated from a catering staff on the first day, the other two were isolated from two health care personnels on the second day. during the outbreak, of catering firm personel ( %) were found to positive for group g streptococci. all the bhs tested were found sensitive to penicillin g and erythromycin by agar disc diffusion method. conclusions: the configuration of the epidemic curve suggested a common source of exposure. since respiratory spread of streptococci in such a rapid fashion would be highly unlikely and that of positive throat culture were from the staff of the catering firm that provide all the food services for the hospital, and that most of them were working at the departments in which the outbreak occurred, we considered that the outbreak might be food-borne. prompt treatment with penicillin all the ill personnel and -day holiday coming consequently january, terminated the outbreak. all the strains were cryopreserved for further typing studies.we are now typing these strains by pulsed field gel electophoresis (pfge) after digestion with smai restriction endonuclease. our initial results show that these strains are of the same origin. outbreak of acute gastroenteritis in an air force base in western greece e. jelastopulu, t. constantinidis, t. kolokotronis, d. venieri, g. komninou, c. bantias (patras, andravida, gr) objectives: on september , an operative training day at the air force base in western greece, soldiers and staff experienced an outbreak of acute gastroenteritis. the purpose of this study was to determine the causes of the outbreak and develop control measures. methods: following the assessment of descriptive epidemiology, a case-control analytic approach was utilized with randomly selected cases and controls. patients completed a questionnaire pertaining to the presence and severity of gastrointestinal disturbances, date and time of symptoms onset and consumption of food items served in the base on the implied training day. adequate questionnaire was administered to the controls. odds ratios were calculated and statistical significance was determined using x test. samples of food items were collected for bacteriological examination. results: the overall attack rate was at least % among the approximately attendees. the outbreak started abruptly in the late afternoon on september, peaked at midnight and ended about hours later. from the interviews and the analysis it was established that the lunch (beef, macaroni, tomato sauce and grated cheese) consumed several hours prior to onset of symptoms by affected military personnel was the likely source of the outbreak with a strong statistical association. there was only one subject who did not eat lunch. among the symptoms the most prominent were watery diarrhoea ( %) and abdominal pain ( %). relatively few indicated vomiting ( %) and nausea ( %). the mean incubation period was h. in the bacteriological examination, staphylococcus aureus was detected in a sample of raw beef and in two samples of grated cheese (rest-cheese from lunch and an unopened package). conclusion: the short incubation period with abrupt onset, the symptomatology and the short, self-limiting nature of the illness, are suggestive of gastroenteritis caused by an enterotoxin-producing bacterium. s. aureus is considered to be the most likely cause. although mortality and longer-term morbidity are uncommon with food poisoning caused by enterotoxin-producing bacteria, this outbreak highlights its capacity to cause short term, moderately-severe illness in a young and healthy population. it underscores the need for proper food handling practices and reinforces the importance of appropriate microbiological specimen collection from cases, as well as the public health importance of timely notification of such outbreaks. occurrence, characterisation and antimicrobial resistance pattern of staphylococcus aureus strains isolated from dairy products in southern italy g. la salandra, e. goffredo, c. pedarra, m.c. nardella, a. parisi, a. dambrosio, n.c. quaglia, g.v. celano, g. normanno (foggia, valenzano, it) objectives: the ingestion of food contaminated by enterotoxins (ses) synthesized by staphylococcus aureus is responsible of one of the most common foodborne diseases (staphylococcal food poisoning-sfp). since s. aureus is often involved in cases of subclinical mastitis of ruminants, milk may results contaminated. infact, the dairy products are frequently related to cases of sfp, expecially in areas characterized by a high level of consumption of these products. consequently an active microbiological surveillance is needed in order to control the risk of sfp and to allow the improvement of the public health standards. s. aureus also show a large antimicrobial resistance pattern. in this work are reported the results of a survey conducted on the occurrence of s. aureus in dairy products from apulia region (southern italy). furthermore, the isolated strains were characterized in order to determine their ability in synthesizing ses and to evaluate their antimicrobial resistance pattern. methods: samples of dairy products (milk, cheese, mozzarella cheese, ricotta cheeses) were analysed for the detection of s. aureus. the isolated strains were tested for the detection of ses, using the reverse passive latex agglutination test (sea to sed) and submitted to pcr to detect enta, entb, entc, entd and ente genes. furthermore, the strains were tested for susceptibility to ampicillin, tetracycline, gentamicin, eritromycin, enrofloxacin, co-trimoxazole, teicoplanin and vancomycin, by the agar diffusion method. results: out of samples analysed, ( . %) resulted contaminated with s. aureus and, among these, ( . %) have been recognized as enterotoxigenic strains ( samples of milk, samples of mozzarella cheese, samples of cheese from ovine milk and sample of cheese). all the strains tested (one per each positive sample) showed antimicrobial resistance properties but none of these was resistant to teicoplanin and vancomicin. conclusions: the results obtained from this survey show that milk and dairy products from southern italy are frequently contaminated by enterotoxigenic strains of s. aureus and highlighted the need to implement strict hygienic control measures along the food chain in order to decrease the risk of spf. furthermore, the presence of antimicrobial-resistant strains of s. aureus in food may be considered a source of communityacquired infections, with the direct risk of transfer of the antimicrobial-resistance to intestinal human microflora. objectives: infection accounts for about one-third of cases of fever of unknown origin (fuo), which remains a major diagnostic challenge. recently, f- -fluorodeoxyglucose (fdg) positron emission tomography (pet) has entered the field of clinical infectious diseases. fdg accumulates in tissues with a high rate of glycolysis, which is present in malignant cells and in all activated leukocytes. the aim of this prospective multi-centre study was to validate the use of fdg-pet as part of a structured diagnostic protocol in the general patient population with fuo. methods: from december to july , patients with fuo, defined according to the revised petersdorf criteria, were recruited from one university hospital and five community hospitals. a structured diagnostic protocol was used. fdg-pet was performed after certain obligatory laboratory tests, chest xray and abdominal ultrasound. the final clinical diagnosis was used for comparison with the fdg-pet results. results: a final diagnosis was established in patients ( %): infections, malignancies, non-infectious inflammatory disorders and miscellaneous causes. of the total number of fdg-pet-scans, % were helpful. positive predictive value of fdg-pet was % and negative predictive value was %. fdg-pet was helpful in all patients diagnosed with an infection except for one case of pyelonephritis. contribution of fdg-pet to the final diagnosis did not differ significantly between the university hospital and the community hospitals. fdg-pet was not helpful in any of the patients with normal erythrocyte sedimentation rate (esr) and c-reactive protein (crp). conclusion: in addition to the apparent value of fdg-pet in diagnosing different infectious diseases as described in several case series, fdg-pet is a valuable imaging technique as part of a diagnostic protocol in the general patient population with fuo and a raised esr or crp. based on previous studies comparing gallium- -citrate or labelled leukocyte scintigraphy and fdg-pet in patients with fuo and resulting from favourable characteristics of fdg-pet, conventional scintigraphic techniques may be replaced by fdg-pet in institutions where pet is available. emergence of clindamycin-resistant streptococcus pyogenes causing cellulitis epidemiology of viral respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a cause of community-acquired respiratory illness seroprevalence of human metapneumovirus in japan - . carriers into account when studying the dynamics of pneumococcal transmission and modelling the effect of pneumococcal vaccination in young children erythromycin-resistant streptococcus pneumoniae isolated in spain: serotypes, clones and mechanisms of resistance ( %) and f ( %) that accounted for % of eryr strains. among eryr strains ( %) had mlsb phenotype ( % constitutive and % inducible) and ( %) had m phenotype. the genes detected in mlsb isolates were: ermb in isolates, and ermb and mefe genes in isolates. all ( %) mlsb isolates with resistance to tet had tetm gene and of them had int gene (related to tn -like). seven positive ermb strains susceptible to tet had int gene spain f- , spain b- , sweden a- and st - f) accounted for % of these strains. capsular switching was observed in two clones, spain f- (serotypes f and a) and sweden a- (serotypes a, f suggesting the spread of tn -like elements. the ermb positive strains, related to spain f- , spain b- , sweden a- and st - f clones, were more frequently isolated in adults us) objective: to characterize changes in the frequency of occurrence of bacterial pathogens responsible for pneumonia in hospitalized patients in europe for the years - and examine select antimicrobial susceptibilities (s) for predominant pathogens. the emergence of resistance (r) among pathogens responsible for pneumonia has resulted in changes to empiric therapy, with increasing reliance upon third-and fourthgeneration cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, carbapenems and fluoroquinolones. methods: participating european medical centres ( - /year) referred consecutive, non-duplicate pathogens ( isolates) from lower respiratory tract sites determined to be significant by local criteria as the probable cause of pneumonia. all identified isolates were tested for s by the broth microdilution method and . %, respectively), increased significantly in ( . % s), and returned to near- levels in . esblphenotypes (cro or caz or aztreonam mic ‡ mg/l) remained essentially unchanged among ec between and ( . % and . %, respectively), whereas among ksp increases were more substantial ( . % and . %). metallobeta-lactamase-producing pa were identified during the study from italy vim- ) methods: four-hundred consecutive mrsa isolates were collected at centres (max. isolates per centre) as part of a multicentre study conducted throughout germany in . isolates were collected from various sources, including colonization sites as well as infectious foci. only one isolate per patient was included and all isolates were spa-genotyped. cps were determined by slide agglutination with cp-specific antibodies (anti-t -dt, anti-t -conjugate, anti- -repa). the serotypes were confirmed by immunodiffusion using lysostaphin-digested cell lysates. results: in the present study, we serotyped mrsa isolates collected most recently in a german multicentre study. all mrsa isolates evaluated were one of the serotypes tested invasive pneumococcal disease in adults in north-rhine westphalia methods: surveillance for our current study focused on north-rhine westphalia, the largest federal state in germany ( million inhabitants). ( . %) acute care hospitals microbiological laboratories serving these hospitals agreed to participate. we studied hospitalized patients older than years of age. a case of ipd was identified by the isolation of s. pneumoniae from an otherwise normally sterile site. isolates were verified for species diagnosis by optochin testing and bile solubility, and for serotyping by the neufeld quellung reaction. mics of penicillin g, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin us) objectives: carbapenems are the most reliably active betalactam antibiotics against enterobacteriaceae and are often the treatment of choice for infections caused by multi-drug resistant isolates. while carbapenem resistance has occasionally been reported in enterobacteriaceae, there are limited data on its frequency and distribution. methods: two large ongoing surveillance databases were searched for imipenem (imp) and ertapenem (etp) resistance in enterobacteriaceae: smart (study for monitoring antimicrobial resistance trends), a worldwide program focusing on community-and hospital-acquired intra-abdominal pathogens, and iss (icu surveillance survey), a us program focusing on icu isolates from any sterile body site. results: the overall frequencies of carbapenem-resistant enterobacteriaceae remained < % in smart and < % in iss throughout the periods of observation (see table). for % of esbl producing k. pneumoniae and e. coli were resistant to etp or imp and rates varied by geographic region. all isolates studied to date have exhibited multiple resistance mechanisms. conclusion: carbapenem resistance was uncommon among clinical isolates of enterobacteriaceae in these surveillance studies. its observed frequency varied by species and geographic region no significant seasonal variability in the prevalence of emergence of streptococcus b-haemolyticus strains in swabs was observed. conclusions: . seasonal fluctuations of pharyngeal discussion: with regard to high prevalences of giardiasis and enterobiasis it increase the prevalence that intestinal parasitic infections. it is suggested to decrease the rate of these parasitic infections in the region by strict programes that help to increase the knowledge of students, their parents and teachers about hygen. results: the study included patients, with a mean age of . ± . . ( . %) were women. the predisposing factors were: renal lithiasis patients ( . %), prostatic adenoma ( . %), vesical structure disease ( . %), vesical functional disorder ( . %), chronic kidney failure ( . %). the underlying diseases included: diabetes mellitus ( . %), immunosuppression ( . %), previous urinary tract instrumentation ( . %), permanent catheter ( . %). the mean hospital stay was . ± . days. the mean duration of symptoms was . ± . days the absence of leukocyturia or mictional syndrome does not exclude the presence of complicated upper uti. ) the high percentage of bacteriemia necessitates blood cultures, with e. coli being the most common pathogen. ) the associated morbidity and mortality are important in association with sepsis or septic shock gr) objectives: to estimate the incidence of streptococci in community acquired urinary tract infections (uti) and also to carry out the in vitro antibiotic resistance of streptococci in urinary tract infections %) were enterococcus avium, ( . %) were enterococcus gallinarum and ( . %) were streptococci group b. the in vitro antibiotic resistance of enterococcus faecalis was: penicillin g . %, ampicillin . %, gentamicin %, streptomycin . %, nitrofurantoin %, ciprofloxacin . %, tetracyclines . %, vancomycin . %, linezolid %. the in vitro antibiotic resistance of enterococcus faecium was: penicillin g %, ampicillin . % tremolieres for the french aup study group background: short-course therapy for acute uncomplicated pyelonephritis (aup) is the newly suggested standard. talan et al. have demonstrated that oral ciprofloxacin (cip) for days, was associated with greater cure rates than a -day trimethoprim-sulfamethxazole regimen. we assessed efficacy and tolerance of a -d. regimen of cip (study i), then of levofloxacin (lvx) in study ii results: of (i) and (ii) enrolled pts. aged . ± . y., and , aged . ± . y. were retained for itt analysis; . % and . % had positive blood cultures. escherichia coli was the uropathogen in . % and % of cases. finally (i) and (ii) were retained for per protocol (pp) analysis. at v bacterial eradication rates were . % and . %. global cure rates were . and . % at v and . % and . % at v with only less than % of lost to follow-up between v and v in both cases. side effects were observed in . % and . % of pts. who received or more fq doses. conclusions: aup treatment with lvx mg hiv-infected patients and drug addicts were excluded. antimicrobial susceptibilities of all s. pyogenes isolates were studied by microdilution method, and macrolide resistance phenotype by double disc test. macrolide resistance genes were detected by pcr. results: over the -year study period, there were episodes of cellulitis. the infection was microbiologically ). of note, all cases of cellulitis due to clindamycin-resistant strains occurred during the last years of the study. five ( %) patients presented with stss and died ( due to an erythromycin-resistant strain). overall mortality (< days) was % this resistance might become a problem when treating s. pyogenes infections, especially stss cases. p risk factors for community-acquired bacteraemic gram-negative cellulitis an administrative database was used. then we selected the patients with blood cultures obtained at the time of the cellulitis episode using the microbiology laboratory database. nosocomial cellulitis were excluded. a standardized data collection form was used to review the hospital records. in statistical analyses, student's t test was used for the comparison of mean values and chi square test and fisher's exact test for the comparison of categorical data (two tailed). results: of the patients with limb cellulitis identified in the study period, patients had blood cultures and were selected for the analysis. bacteremia was detected in of the patients ( . %), of them due to gram-negative microorganisms hemorrhagic rash was present in . % cases. koplick spot was found in . % cases. measles was associated with streptococcal tonsillitis in . % cases, with oral candidiasis in . % cases and with pulmonary tuberculosis in . % cases. severe forms of evolution were observed in complicated cases with: encephalitis ( . %) or bronchopneumonia ( . %), which required intensive care unit survey. we registered only one deceased, in a case of measles encephalitis in gipsy collectivities even it's very difficult, it's necessary to was performed. respiratory samples were tested routinely for twelve common respiratory pathogens. results: over the study period, of samples processed, -six cases were community-acquired and ( %) patients had significant co-morbidities. cough was the predominant reported symptom. chest x-ray was performed in cases, of which showed abnormalities. bronchiolitis ( / ) was the commonest initial clinical diagnosis. the majority ( %) of patients received antibiotic therapy, but a convincing bacterial pathogen was isolated in less than half of these cases. thirty patients were admitted for management. more than one virus was identified from patients, with rhinovirus being the predominant co-infection. overall, the average length of stay was . days. however, where hmpv was the sole pathogen identified, average length of stay was . days. conclusion: our data suggests that hmpv infections are more common in children with underlying co-morbidities. the rate of radiological imaging was higher than expected and perhaps is a reflection of the patient population or the degree of severity of illness. nosocomial acquisition occurred in cases, which has implications for patient cohorting acknowledgements: the financial support for this study was provided by kuwait university research grant / . evaluation of infection control practices in haematopoietic stem-cell transplant facilities in german-speaking countries: variation of measures reflects lacking evidence s. wenzler-rö ttele, a. conrad, w.v. kern, h. bertz, f.d. daschner, m. dettenkofer (freiburg, de) objective: haematopoietic stem cell transplant (hsct) recipients are highly immunocompromised during pre-and postengraftment. thus, they are cared for in specialised facilities and versatile precautions are practised in order to prevent nosocomial infections. however, there is a lack of evidence whether these interventions are effective. furthermore, most of the measures are cost-intensive and restrict the patients' comfort. for evaluation of precautions, a survey was performed to assess the spectrum of measures commonly practised. methods: a questionnaire was compiled asking in detail for infection control measures differing according to allogeneic and autologous hsct recipients. the questionnaire was sent to hsct facilities in germany, austria and switzerland. results: questionnaires ( %) were filled in and sent back. among the centres, were university hospitals, and teaching hospitals. the overall number of transplantations that were performed by the facilities varied considerably and ranged from to /y for auto hscts and from to /y for allo hscts. % of the institutions performing allo and auto hsct have implemented different precaution standards for each group. some measures regarding allo hsct were routinely adhered to in practically all institutions: accommodation in single rooms ( %), interdiction of plants and opening of windows ( % each) and protection from waterborne bacteria by use of terminal tap water filters ( %). % of hsct facilities perform their allo transplantations in hepa-filtered rooms and % are providing laminar air-flow for this population. there was a broad spectrum of different measures regarding barrier precautions: gowns when entering the room (required in % of centres for allo and % for auto hsct) and face masks ( % allo and % auto hsct). precautions to be followed by the patient varied among centres, e.g. specification of the face mask/respirator to be worn outside the isolation room (for allo hsct: % surgical mask, % ffp , % ffp and % ffp ). conclusion: the broad variety of different preventive measures performed by the different facilities reflects lacking evidence for many infection control precautions that are commonly practised in the care of hsct recipients. this survey provides the basis for further studies within the onko-kiss project (hospital infection surveillance system for patients with haematologic/ oncologic malignancies). objectives: in this study it was our aim to evaluate the microbiological contamination of physiological serum flasks in use in medical day center for wound cleaning and to identify the isolated microorganisms. methods: we have collected saline solutions from health care centres localized in the health sub-region of coimbra. from each centre we have recovered aleatory flasks in current use.the samples were transported at ordm;c and maintained at this temperature until its processing. saline solutions were seeded by the pour-plate technique in plate count agar and plates incubated at and ordm;c for h. the saline solutions were evenly spread over the surface of blood agar and sabouraud chloramphenicol agar (sab chl-d). the transfers of saline solutions flasks were also tested for microbiological contamination with a sterile cotton swab that was rubbed vigorously, over the transfer surface and directly applied on blood agar media. blood agar plates were incubated at °c for h and sab chl-d plates were incubated at °c and °c and examined daily for a period of days before declared as culture negative. microbial identification was firstly accomplished by employing conventional morphological and biochemical tests. when identification was not possible by these methods, s rrna gene sequence determination and phylogenetic analysis were used for bacterial strains and in the case of moulds we performed the amplification and sequencing of internal transcriber spacers region of . s gene. results: from the saline solutions analysed, . % were contaminated. a total of strains were isolated, % could be identified to species level using morphological and biochemical tests, the remaining % were identified by gene amplification and sequencing. about . % of the identified strains were gram-positive cocci, the second dominant type of strain were gram-positive bacilli ( %), and the third dominant type of strains were gram-negative bacilli and moulds, both with . %. the most frequent contaminants belong to human normal flora ( %), supporting the idea that the source of contamination of saline solutions analysed was human, in contrast with % of contamination due to the environment. conclusions: the contamination of the saline solutions is due to inadequate clinical practices. these results claim for more strict hygienic measures and for the replacement of big flasks by single use flasks with an incorporated overture used for wound irrigation. frequencies of cmv-ie specific memory t cells are inversely correlated with alloimmune memory and serum creatinine in kidney transplant patients p. nickel, g. bold, f. presber, c. schö nemann, j. pratschke, d. bitti, f. kern, h.-d. volk, p. reinke (berlin, de) background/aims: cytomegalovirus infection is a significant cause of morbidity in transplant patients and has been associated with allograft rejection. in this study frequencies of ifng-producing t cells following ex-vivo stimulation with protein-spanning peptide pools for cmv proteins pp and ie as well as donor-reactive t cell frequencies were serially determined during the first months after renal transplantation (tx) to analyse the relation of cmv specific t cells, virus control and alloimmunity. patients: kidney transplant recipients were included. immunosuppression generally consisted of anti-il- r mab, calcineurin inhibitor, mmf and steroids. presensitized patients received an induction by x low dose okt- , anti-tnf mab, anti-cd mab and x plasmapheresis. patients received fty- , cyclosporine and steroids. methods: pbmcs from renal transplant recipients were analysed in a computer-assisted elispot assay before and at multiple times (mean ) post-transplantationfor ifn-yproducing t cells following in-vitro stimulation for hrs by irradiated donor cells and pools of overlapping peptides conclusions: although temporary r declines were seen among some european pneumonia pathogens, all showed increasing r to most class agents during the study period. the increase in esbl among enterobacteriaceae, and r among pa to most agents except polymyxin b, are especially worrisome. continued longitudinal comparisons of emerging pathogens and changing susceptibility profiles are critical elements in guiding empiric therapies and epidemiologic interventions. week, all participating hospital inpatients were swabbed on three anatomical sites: throat, nose and groin. we investigated the molecular epidemiology of the mrsa isolates collected from patients in hospitals using the pfge method with the smai restriction enzyme. cluster analysis was carried out using bionumerics software. band-based similarity dice coefficients were used for dendrogram construction, which provides a quantitative assessment of strain similarity. samples were defined to belong to a cluster using a similarity coefficient of % or higher. pfge profiles were compared with the most similar strains from the harmony iums global mrsa database.results: different restriction profiles were observed among the mrsa isolates and patients. isolates from the same patient but from different anatomical sites had similar pfge profiles. clusters of mrsa strains could be identified with the two largest clusters containing ( %) and ( %) patients, respectively. strains from these major clonal clusters occurred in and out of the hospitals, respectively. isolates from the cluster with patients were most similar to the well-known iberian clone: france a ( strains), belgium e ( strains), france b, france c and northern germany i ( strain each). isolates from the next largest cluster of patients correlated with a group of strains previously found in finland and belgium: belgium e ( strains), finland e ( strains) and finland e ( strain). the remaining strains were most closely related to belgium ec ( strains), berlin iv ( strain), southern germany ii ( strains) and uk e ( strains). conclusion: two major clonal clusters of mrsa strains were found to be dominant among hospitals inpatients in luxembourg. the molecular diversity of circulating strains was fairly diverse and profiles were very similar to previously described patterns in neighbouring countries and europe. further sequence-based genotyping is warranted to gain a better understanding of the clonal structure and elucidate transmission patterns. enterococci were identified by basic tests and by pcr amplification of ddl genes. susceptibility testing was performed using the icls broth microdilution method. resistance genes were detected by pcr, selected vana, vanb and vanc amplicons were sequenced. macrorestriction analysis (smai) resolved by pulsed-field gel electrophoresis (pfge) was performed. results: during the study vre isolates with different phenotypes of resistance to glycopeptides were obtained from specimens. the prevalence of vre in the gastrointestinal tract was . %. one e. faecalis (isolated from patient arrived from us) and e. faecium isolates, harbouring vana genes, demonstrated mic's of vancomycin (van) and teicoplanin (tec) - and - mg/l respectively. three e. faecium and four e. gallinarum isolates were vanb-positive, with van and tec mic's > and . - mg/l respectively. all stains were susceptible to linezolide. among e. faecium isolates with vana genes one predominant pegf type was observed, differentiated in nine pegf sub-types. each of three other pegf types detected seemed to be unique. among six vana genes sequenced, four demonstrated similarity to vana gene from e. faecium (genebank af ) and two to -vana gene from e. faecalis (genebank ay ). in two sequenced vanb genes from in e. gallinarum nucleotide substitutions, resulting in seven new amino acid substitutions, were detected. conclusions: heterogeneity of glycopeptide-resistance genes, circulating in haematological centre, leads to the conclusion that their spread is not a local phenomenon. spread of vre is an emerging and, possibly, underestimated problem for russia. study of resistance and clonal relatedness of clinical isolates of stenotrophomonas maltophilia from a hospital in northern spain c. valderrey, e. sevillano, f. calvo, l. gallego (bilbao, es) objectives: the aim of this work was to study the antibiotic resistance and genetic relatedness among clinical isolates of s. maltophilia isolated from patients with tract respiratory infections. methods: the study included s. maltophilia isolates obtained in a hospital from bilbao (northern spain) during (from january to october). susceptibility to antimicrobial agents was determined by the disk diffusion method following the nccls recommendations. the antibiotics tested were imipenem, meropenem, cefotaxime, ceftazidime, cefepime, aztreonam, amikacin, tobramicin, ciprofloxacin, ofloxacin and trimethroprim/ sulfamethoxazole. total dna was used as target for pcrfingerprinting experiments with primers rd , eric , ap , m and rnar and . to detect class integrons, primers cs and cs were used in amplification experiments.results: resistance to antibiotics tested was the following: imipenem ( %), meropenem ( %), cefotaxime ( %), ceftazidime ( %), cefepime ( %), aztreonam ( %), amikacin ( %), tobramicin ( %), ciprofloxacin ( %), ofloxacin ( %) and trimethroprim/sulfamethoxazole ( %). pcr-fingerprinting technique was only useful when eric primer was used identifying distinct genotypes. the other primers were not able to produce reliable band patterns. patients with several isolates maintained the same clone along time, although there are two patients from which two different genotypes have been isolated, and two clones that have been isolated from more than one patient. class integrons were detected in % of isolates ranging in size from of to bp ( isolates bored combinations of two structures). conclusions: trimethroprim/sulfamethoxazole and amikacin showed the best activity against the isolates tested. for pcrfingerprinting experiments the best primer was eric which produced reliable and reproducible band patterns. there was a high clonal diversity since different genotypes were identified among the patients included in the study. many isolates bored class integrons with sizes similar to those detected in other nonfermenters bacilli from the same environment.methods: -identification: the strains were identified by colonial morphology, haemolysis on blood agar plates, biochemistral and antigenic identification; antibiotic susceptibility testing: all the strains were tested by disk diffusion according to the national committee for clinical laboratory standard methods. mics were determinated by screening test or mic evaluation in solid media. results: our study concerns bacterial strains isolated from january to june . among the strains isolated, neisseria meningitidis represented the most number of cases with . %.these were distributed among all different age groups. serogroup a was the most predominant and represented . % of total strains while groups b and c represented . % and . % respectively. streptococcus. pneumoniae represents the second causes of purulent meningitis with . % while haemophilus. influenzae b is the third causative bacterial agent with . %.this last agent is most predominant among infants less than years of age in % of cases. neisseria. meningitis is susceptible to all types of antibiotics tested. however, haemophilus. influenzae b produced an inactivating enzyme (penicillinase) in . % of cases. the resistance was associated to cotrimoxazole in . % of cases. the results of mic done on streptococcus. pneumoniae show that . % of strains has an intermediate resistance to penicillin and high level of resistance in . %. the amoxycillin is active in . % of the strains,in the opposite cefotaxim has an intermediate resistance in . % and a high level of resistance in . % of the strains. the resistance to penicillin was associated with resistance to erythromycin, cotrimoxazole or to both in some cases. conclusion: streptococcus. pneumoniae represents the second causative bacterial agent responsible of purulent meningitis and showed an increasing prevalence of resistance profiles to penicillin and cefotaxim in our hospital. this implicates an effective microbiological and epidemiological control.conclusion: as expected in a referral hospital with a cardiac surgery department, the prevalence of s. aureus ie was elevated as well as the attributable mortality rate. the high global mortality rate may be explained by the high frequency of severe co-morbidities and by the late referral of patients to hospital. our data suggest that there is room for improvement in the diagnosis and management of ie in a multidisciplinary collaborative approach. objective: to determine the clinical, epidemiological, diagnostic, and therapeutic characteristics of a series of cases of prosthetic valve endocarditis. methods: we undertook a retrospective, descriptive study of cases of prosthetic valve endocarditis obtained from a series of definite or probable left sided infectious endocarditis from six second-or third-level andalusian hospitals from to . results: of the cases of prosthetic valve endocarditis, ( . %) were definite and ( . %) possible. the mean age was ± years, and they were more common in men ( %). late infection was more common than early involvement ( vs. cases). the aortic valve was involved in cases ( %) and the mitral valve in cases ( %. most ( %) of the valves were made of metal and prior handling had taken place in cases ( %). clinical characteristics were fever %, constitutional syndrome %, murmur %, vascular events %, and immune phenomena %. complications included left ventricular failure %, kidney failure %, peripheral embolism %, cns embolisms % and heart block %. the etiology was as follows: in early prosthetic valve endocarditis the three most common pathogens were s. coagulase-negative ( %), s. aureus ( %) and enterococcus ( %). late prosthetic valve endocarditis involved s. viridans ( %), s. coagulase-negative ( %) and s. aureus ( %). transesophageal echocardiography alone in cases ( %), and transthoracic followed by transesophageal echocardiography in cases ( %). medical therapy was applied in cases ( . %) and surgery in ( . %). a cure was achieved in cases ( %), the other ( %) dying. of those who underwent surgery, . % died and . % of those who were treated medically died. the death rate from early prosthetic valve endocarditis was greater than that for late prosthetic valve endocarditis ( % vs. %). conclusions: ) prosthetic valve endocarditis is a very serious infection which is still associated with an excessively high mortality, despite advances in diagnosis and treatment. ) early prosthetic valve endocarditis has a worse prognosis than late prosthetic valve endocarditis, due to its distinguishing pathophysiological features. ) the greater mortality seen in patients who underwent surgery is probably associated with the fact that they had more complications, such as perivalvular abscesses or persistent infection. outcome of infective endocarditis e.e. hill, s. vanderschueren, p. herijgers, m-c. herregods, p. claus, w.e. peetermans (leuven, be)objectives: despite progress in diagnosis and therapy, almost half of patients with infective endocarditis (ie) has at least one complication and overall mortality remains high. the aim of the present -year prospective observational study was to define predictors of outcome in patients with ie. methods: from june through december , all first episodes of definite ie by the modified duke criteria, encountered in a single tertiary-care medical center, were registered and followed-up for months. results: overall, patients suffered ie episodes. sixtyone percentage were males. the median age was years (range - ). fifty-five percentage of episodes were referred from another hospital. at least one complication occurred in %. surgical intervention was performed in % and was mainly indicated because of congestive heart failure. the median time from diagnosis to surgery was days (range - ). six-months mortality was % (n = ). in bivariable analyses, factors associated with -months mortality were: age, female gender, causative microorganism, nidus of infection and therapeutic policy. six-months mortality was % for native valve ie and % for prosthetic valve ie; twenty-five% for nosocomial ie and % for community-acquired ie. six-months mortality rates for microorganisms were: staphylococci % (n = ) [s. aureus % (n = ) and cons % (n = )], enterococci % (n = ), streptococci % (n = ) and other microorganisms % (n = ). the -months mortality for patients with a contraindication to surgery was % (n = ), for patients conservatively treated without a contraindication % (n = ) and for combined surgical-medical treatment % (n = ). in multivariable logistic regression predictors of -months mortality were age (or, . ; % ci, . - . ; p = . ), causative microorganism (or, . ; % ci, . - ; p = . ) and a contraindication to surgery (or, . ; % ci, . - ; p < . ). conclusion: in the present prospective single centre study of patients with definite ie, -months mortality rate was . , and was especially high in patients with preestablished contraindications to surgery, in the elderly and in patients with staphylococcal ie. six-months mortality in patients with combined surgical-medical treatment versus exclusively medical therapy in patients without a contraindication to surgery was not statistically significant. staphylococcal and enterococcal ie had a worse prognosis compared to streptococcal ie. epidemiology and aetiology of infective endocarditis e.e. hill, p. claus, m-c. herregods, p. herijgers, s. vanderschueren, w.e. peetermans (leuven, be)objectives: the epidemiological features of infective endocarditis (ie) have changed. we report the results of a -year prospective observational study investigating trends in the epidemiology and etiology of ie. methods: from june through december , we registered definite ie episodes according to the modified duke criteria in patients older than years, hospitalized in a single tertiary-care center. results: sixty-one% of episodes involved males. the median age was years (range - ).fifty-five percentage (n = ) were referred from another hospital. forty-four percentage (n = ) were nosocomial. thirty-four percentage (n = ) involved prosthetic valves and % (n = ) thereof were of early postoperative onset. the mitral valve was most frequently involved. exposure to ie risk factors during the previous months was recorded in % (n = ) of the episodes. twenty-four percentage (n = ) were intravascular catheter- objective: to determine the eco-epidemiology of cryptosporidiosis in the health services executive -western area (formerly the western health board).concerns about the incidence of cryptosporidiosis in the western area prompted the department of public health to undertake further investigation of potential links between cryptosporidiosis and environment by focusing on farming activity and water supplies in the first instance. background: cryptosporidiosis was not notifiable in the republic of ireland prior to , unless cited as a cause of gastroenteritis in a child less than two years old. as a result the incidence of cryptosporidiosis in the republic of ireland at the time was unknown. nationally it was estimated that up to % of cases of gastroenteritis in children less than two years old could be attributed to cryptosporidium. in the western area from to the proportion of cases of gastroenteritis in children less than two years old attributable to cryptosporidium ranged from . % to . %. this was cause for concern.many rural locations in the western area are served by voluntarily-operated water schemes. water quality from these schemes is often microbiologically unsatisfactory. the department of public health methods: initial research involved analysis of notification records for cases of cryptosporidiosis received from to inclusive. crude incidence rates for cryptosporidiosis in the western area were compared with crude incidence rates in england & wales, northern ireland, and scotland for the same time period. cases of cryptosporidiosis from the western area were geo-coded and mapped to visualize the geographic spread of cases, and are being contrasted with geographic data for farming activity, and also with available data on water supplies. the results of the initial phase of this research indicated the incidence of cryptosporidiosis in the western area may be cause for concern. the geographic spread of cases and potential links to farming practices and water supplies will be presented. objective: the evaluation of epidemiology and seasonal fluctactions of bacterial flora in pharyngeal swabs taken from family doctors' patients. material and methods: a total of of positive pharyngeal swabs ordered by primary care physicians from silesia were examined during the - period. the microbiological analysis was performed in silesian analytic laboratories. the intake of material, its transport and final identification complied with laboratory standards. results: the most common pathogens were, in order of prevalence: streptococcus viridans ( . %), moraxella catarrhalis ( . %), staphylococcus aureus along with mrsa ( . %) and mrsa alone ( . %), e. coli ( . %), klebsiella pneumoniae ( . %), streptococcus b. haemoliticus ( . %). candida albicans was identified in . % of positive specimens. considering seasonal fluctuation, the number of positive swabs in each month tended to gradually increase in spring with its culmination in may ( . %). as for the most common pathogens streptococcus viridans and moraxella catarrhalis mirrored the general tendency and dominating in spring season (up to . and . %, respectively) and having less stronger impact in automn (up to . and . %). the frequency of isolation of the other pathogens revealed seasonal fluctuations confined to either spring, as in the case of klebsiella pneumoniae, escherichia coli and staphylococcus aureus strains (up to . , . clinical microbiology and infection, volume , supplement , aim: the aim of this study was to identify the microorganisms isolated from corneal and conjuntival samples, isolated from patients attending the ophtalmology department of a spanish hospital. material and methods: a total of corneal scrapes and conjunctival swabs were obtained since october of to october of in an university hospital of madrid. samples were cultured into blood and chocolate agar plates and incubated at ordm;c in o and co atmospheres, respectively, for two days (conjunctival swabs) and fifteen days (corneal scrapes). identification and susceptibility tests were performed following standard methodology. results: thirty four ( . %) out of corneal samples and ( . %) out of conjunctival swabs yielded positive cultures, respectively. results are summarized in the following table:conclusions : corneal scrapes yielded a higher number of positive cultures than conjunctival swabs. gram-positive microorganisms were more prevalent both from corneal scrapes and conjuntival swabs although the difference was more evident in corneal scrapes. s. aureus was the specie most prevalent in conjunctival samples meanwhile cns were the most prevalent in corneal scrapes. methods: vitreous fluid samples (n = ) were obtained from patients ( male, female) undergoing vitrectomy for endophthalmitis between january and october . specimens of undiluted aqueous and vitreous fluid were cultured for aerobic, anaerobic bacteria and fungi by conventional methods. identification and antibiotic susceptibility were performed by the api system, vitek ii system (biomerieux) and the agar disk diffusion methods according to clsi recommendations. results: ninety one isolates were recovered from the samples. gram stain was positive in / ( . %), while cultures were positive in / ( . %) samples. gram-positive bacteria were the most common isolates ( / , %), followed by gramnegative bacteria ( / , %) and fungi ( / , %). staphylococci coagulase-negative were isolated in / ( %). the next most common species isolated among gram-positive bacteria were s. aureus ( . %), streptococcus spp ( . %), propionibacterium acnes ( . %), bacillus spp ( . %), streptococcus. pneumoniae ( %) and enterococcus faecalis ( %). among gramnegative bacteria eight isolates were enterobacteriaceae, two were non fermenters and one was haemophilus inlfuenzae. two of the fungal isolates were candida albicans, one acremonium spp and six aspergillus fumigatus. polymicrobial growth was observed in six patients with two at least isolates. of staphylococci coagulase-negative / ( %) were resistant to methicillin. only one strain of staphylococcus aureus was methicillin resistant. all gram positive isolates were susceptible to vancomycin. all isolates were sensitive to amikacine and ceftazidime while resistance was observed in / ( %) isolates to fluoroquinolones. conclusion: a variety of microorganisms was isolated from the vitreous fluid of patients. the predominant isolates were grampositive bacteria, especially staphylococci coagulase-negative with low resistance rate to methicillin. so, therapy should be based on the isolation and identification of the infecting agent and the in vitro antibiotic susceptibility to the appropriate antibiotics. the prevalence of intestinal parasitic infection in the students of primary schools in nazloo region in urmia during [ ] [ ] k. hazrati tappeh (urmia, ir)background: intestinal parasitic infections are of the most important hygienic and economical problems of millions of people in all over the world, mostly from developing countries. understanding their epidemiological situation and relation to environmental and social factors is necessary for struggling with them in every society. this investigation was designed to study the prevalence of parasitic intestinal infections among primary school attending students in nazloo region of urmia district in . materials and methods: students were chosen randomly from schools upon their population. having their questionnaires filled, two faecal samples were taken from each student and examined with direct wet mount and formalinether sedimentation technique. scotch tape was also applied in order to detect the enterobiasis and taeniasis. students completed the test. all infected persons by e. vemicularis, h. nana were treated by mebandazole and giardia lamblia were treated by metronidazole. results: overall prevalence of parasitic protozoan infections was . %. giardia lamblia was found in cases ( . %), entamoeba coli in cases ( %) and blastocystis hominis in cases legionella pneumophila as an occupational risk factor for inter-city bus drivers y. polat, Ç . ergin, i. kaleli, a. pinar (denizli, ankara, tr)objectives: legionellaceae are ubiquitous aquatic microorganisms that usually isolated from evaporative condensers. various man-made sources such as cooling towers, whirlpools and spas are sources for legionella pneumophila. in hot climate, bus air-conditioning and aircirculating systems are possible sources for the organism. in this study, serologic status of bus drivers and their assistants for legionella infections as well as bus air-conditoner moisture exit samples for legionella species were investigated.methods: serum samples were collected from bus drivers (n = ) and their assistants (n = ). samples were tested for anti-legionella antibodies by indirect immunofluorescence technique. / dilution was accepted as a positive result for anti-legionella pneumophila antibodies. results were analysed according to risk factors based on hot/cold climate route (aegean and mediterranean parts of the turkey were accepted as hot climate region), immundeficiency, chronic diseases and work hours. according to serologic test results, air-conditioners of buses which has been driven by / dilution seropositive persons, were investigated. air-conditioner moisture exit samples were cultured on bcye-alpha agar supplied with bmpa. same samples were tested by pcr targeting a -bp fragment of the s rrna gene of legionella. results: anti-legionella pneumophila antibodies were positive in ( . %) bus-persons. bus drivers' seropositivity was higher than assistants (p < . ). in hot climate route, seropositivity was higher than cold climate route (p < . ). no positive pcr result was detected. coclusion: in conclusion, higher seropositivity rates in bus drivers were pointed out a newer occupational risk factor for legionellosis. although pcr positivity was not detected for bus air-conditioners, high seropositivity rates show that bus drivers have been somehow exposed to legionella. further legionellosis surveillance studies for bus drivers may help to understand legionella exposure during travel. objective: asymptomatic bacteriuria is an important risk factor contributing to pyelonephritis and renal disfunction in diabetic patients. in this study, the relationship between microalbuminuria and age, body mass index, duration of the disease, the level of glycohemoglobin, glycosuria and glomerular filtration rate is studied prospectively in diabetic patients who have asymptomatic bacteriuria. methods: a hundred and twenty-three type diabetic outpatients who were admitted to baskent university konya medical and research center between january-october were included in the study. ages of the patients were within the range of - years. the diagnosis of asymptomatic bacteriuria was established according to the cdc criteria. concurrent samples for urinary culture, glomerular filtration rate, microalbuminuria and glycohemoglobin were obtained. results: twenty-two of ( . %) patients had significant bacteriuria. of these patients % were female. although age, body mass index, creatinine clearence and presence of microalbuminuria were similar, there was a significant difference in glycohemoglobin levels, duration of diabetes and glycosuria between the two groups (p < . ). e. coli was the most common microorganism obtained from urinary samples. risk factors for asymptomatic bacteriuria were shown in the table. conclusion: the frequency of asymtomatic bacteriuria was found to be similar with the previous studies. high glycohemoglobin levels and long duration of diabetes were found to be the risk factors contributing to asymptomatic bacteriuria in type diabetic patients. descriptive study of complicated pyelonephritis objective: the evaluation of prevalence and contributory factors associated with the development of urinary tract diseases among women with urinary incontinence. material and methods: women aged from to years had their urine culture examination performed. the material was taken from the central stream of first catch urine and transported on uromedium. antibiogram was carried out with the use of becton-dicinson's discs. results: in cases the urine culture tested positively which accounted for . % of subject women. the most common pathogens of urinary tract were, in order of prevalence:e. coli- . %, staphylococcus aureus - . %,citrobacter diversus- . % and klebsiella pneumoniae- . %. candida albicans strains were isolated in one patient. e. coli had the highest sensitivity to norfloxacin - % and cefuroxim - %, amoxicillin with clavulonian acid - . %, ampicillin nitrofurantoin and trimethiprim -sulfamethoxazole - . % in each case, cefalothin - . %, tetracycline - . %, and amikacin - . % but only in . % to amoxacillin. staphylococcus aureus proved sensitivity only to gentamicin ( %) and nitrofurantoin ( %). in the case of citrobacter diversus % sensivity to norfloxacin, nitrofurantoin, tetracycline, trimethoprim / sulfamethoxazole, ceftazidim and cefotaksym was confirmed.klebsiella pneumoniae also proved sensitivity to amoxicillin with clavulonian acid, cefuroksime, nitrofurantoin, norfloxacine, tetracyclin and trimethoprim / sulfamethoxazole. when considering the sensitivity of pathogens to antibiotics in the family practise setting of higher reliabilty are nitrofurantoina, norfloksacyna.after the administration of guided therapy complete release from symptoms was observed in women ( %).conclusions: women with urinary incontinence relatively seldom suffer from urinary tract infections. the most common pathogen among women with urinary incontinence was e. coli sensitive to floxacins and cephalosporins but with impaired reaction to amoxycillin. incidence and in vitro antibiotic resistance of streptococci in community-acquired urinary tract infections uncomplicated community-acquired urinary tract infections (ca-utis) and non-pregnant women in london hospital in kuwait over a period of two years. methods: eighty-six pregnant and non-pregnant women with signs of ca-utis were enrolled in the study. the strains isolated from the patients who had significant bacteriuria were included in the microbiological analyses. the identification of the strains was performed using the api e system (biomerieux), while their susceptibility was determined by disk diffusion method. the interpretation of the results was realized according to nccls guidelines. quality control was performed using reference strain e. coli atcc . oserotyping was carried out with polyvalent and monovalent antisera. hemolysin production was tested on human blood agar plates. possession of k antigen by e. coli was tested with agglutination by murine monoclonal antibodies to the group b meningococcal capsule. results: we found o serogroups o , o , o , o , o , o and o among strains isolated from pregnant and non-pregnant women. hemolysin was presented in % and % respective. k antigen was presented in % of strains in studied groups.there are some statistically significant differences in antimicrobial resistance between both groups. amoxicillinclavulanate (amx-clv) resistance was higher among uti haemolytic isolates of e. coli in pregnant women ( %) then in non-pregnant women ( %). similar distinction in cefuroxime resistance was found - % and . amikacin resistance was higher among uti isolates of e. coli in non-pregnant women ( %) then in pregnant women ( %).conclusions: there are no significant differences in expression of virulence factors of e. coli from pregnant and non-pregnant women with ca-utis in london hospital, kuwait. the resistance rates of e. coli from pregnant women to amx-clv and cefuroxime are significantly higher than in non-pregnant women. the penetration of telithromycin in gynaecological tissues and activity in cervicitis patients h. mikamo (gifu, jp)objectives: chlamydia trachomatis and neisseria gonorrhoeae are major causative organisms for sexual transmitted infections in japan. although several oral antimicrobial agents are active against c. trachomatis, few effective oral antimicrobial agents against n. gonorrhoeae exist in japan. two studies were conducted: a clinical pharmacology study examining penetration of telithromycin (tel), an oral ketolide antibiotic, in female genital organ tissues and a clinical study examining tel mg once daily (qd) in cervicitis patients (pts chronic prostatitis (cp) is believed to be an infectious disease in most cases. both aerobic and anaerobic bacteria are involved in the polymicrobial microbiocenosis found in prostate specific specimens. coryneform bacteria form a remarkable part of this community, yet scarce knowledge exists about their clinical significance, species composition and antibiotic susceptibility.our aim was to compare the corynebacteria of the seminal fluid of cp patients and controls and to evaluate their antibiotic susceptibility.material and methods: semen samples from controls and cp patients (nih iiia or iv category) were analysed. corynebacterium seminale was identified by beta-glucuronidase activity, the rest of coryneforms using api coryne (biomerieux). e-test method was used for susceptibility testing.results: coryneforms were found from % cp patients and % controls (p > . ). twelve species and genera were found among strains identified, the most frequent being c. seminale (in % cp patients and % controls). cp patients harboured significantly more arthrobacter sp. ( % vs %, p = . ) andcorynebacterium group g ( % vs %, p = . ), the latter association was especially eminent in case of patients with serious inflammation (> wbc/ml): % vs %, p = . . all tested strains were susceptible to ampicillin-sulbactam, single strains were resistant to doxycycline ( %) and tmp/smx ( %), however, moderate resistance was common to doxycycline ( %). resistance to clindamycin ( %), benzylpenicillin ( %), nitrofurantoin ( %), erythromycin ( %) and norfloxacin ( %) was observed as well. half of cp-related corynebacterium group g strains showed resistance to nitrofurantoin and benzylpenicillin. in addition, they were often moderately resistant to clindamycin, erythromycin and, finally, norfloxacin frequently used to treat cp. conclusions: most of men have coryneforms in their semen, more than half harbour c. seminale. corynebacterium group g and arthrobacter sp are more frequently found in cp patients than the controls. in the treatment of cp of unknown etiology it is useful to take into consideration the susceptibility profile of corynebacterium group g. objective: to evaluate the role of cmv and listeria monocytogenes in abortion.methods: this descriptive prospective study was done on women, women with spontaneous abortion before th weeks of pregnancy as a case group and healthy woman with full term delivery as a control group. serum samples were taken from all patients. elisa test was done for evaluation of cmv (igg and igm) and listeria antibodies in both groups. prevalence of seropositivity was determined. data were analysed by x and chi-square test.results: seologic tests were done on samples. average age in case group was . ± . and in control group was . ± . years. in cases with abortion ( . %) and in control group ( . %) were seropositive for listeria monocytogenes. difference in seropositivity between groups is statistically significant (p = . ). cmv igg antibodies were positive in ( %) of case group and in ( %) of control group; the difference is significant statistically (p = . ). cmv igm antibody was positive in ( . %) of case group and none in control group. difference is significant (p < . ) there was no correlation number of previous abortion and seropositivity for listeria and cmv. conclusion: the present study showed an important role of listeria monocytogenes and cmv infection in abortion. serum and prostatic tissue concentrations of moxifloxacin ( mg) after a single intravenous infusion in patients with benign prostatic hyperplasia undergoing transurethral resection of the prostate background: the spectrum of bacterial prostatitis comprises gram-negative, gram-positive and atypical pathogens. because of its broad spectrum of activity, moxifloxacin might be a suitable antibiotic for the treatment of bacterial prostatitis. aim: in this study the penetration of moxifloxacin into prostatic tissue after intravenous application of mg as single dose was investigated.methods: in a prospective, multicentric study patients with benign prostatic hyperplasia received a single dose of moxifloxacin mg in an hour lasting infusion ( ml) for perioperative prophylaxis before undergoing transurethral resection of the prostate (tur-p). serum concentrations were determined in all patients before infusion, at the end of infusion (time point ), . , and h after the end of infusion. patients were randomized for tissue sampling either , . , or h after the end of infusion. at the beginning of tur-p approximately g of tissue was sampled for analysis. concentrations of moxifloxacin in serum and tissue were determined by hplc. results: patients were evaluated in the study. the concentrations (mean, sd, median, / % quantile) are shown in the table. the prostatic tissue concentrations of moxifloxacin were approximately twice as high as in serum. at the end of infusion the tissue and serum concentrations were already equilibrated, because the tissue-serum ratios did not differ significantly from the end of infusion until h after the end of infusion. after an intravenous infusion of mg the serum and prostatic tissue concentrations of moxifloxacin were well above the mic values of the most important prostatic pathogens until h after the end of infusion. therefore, moxifloxacin might be a good alternative for the treatment of bacterial prostatitis and/ or perioperative prophylaxis for tur-p. statistical significant differences were detected between patients with and without bgnc in the proportion of patients older than years ( . % vs . %), the antecedent of recent animal bite ( . % vs . %), the presence of immunosuppression ( . % vs . %), the presence of haematological illness ( . % vs . %), and the degree of leukocytosis at admission ( ± vs ± cel/ll). conclusions: bgnc is frequently detected in our patients. age older than years, the existence of immunosuppression, the existence of haematological illness, and the antecedent of animal bite are more frequent among patients with bgnc. patients with bgnc had a lower degree of leukocytosis at admission. these factors should be borne in mind to select empiric therapy for patients with cellulitis. is erysipelas-associated tinea pedis a site of streptococcal colonisation? objectives: tinea pedis is considered the most frequent portal of entry of erysipelas of legs (sel) but whether it is the site of streptococcal colonisation is unknown. methods: from june to october we prospectively searched for clinical tinea pedis in patients hospitalised in our infectious diseases ward for sel (acute and unilateral feature with fever were only retained). all patients had bacteriological samples on inter-digital spaces of both feet (sel side and contra lateral side).results: fifteen patients were included. all but one were treated by intra-venous penicillin-g followed by oral amoxicillin. on sel side: tinea pedis was found in / ( %) and, when present, streptococcal colonisation (c or g streptococcal groups) was found in / ( %), although streptococcal colonisation was never found ( / ) in its absence. on contra lateral sides : no streptococcal colonisation was found without tinea pedis, which was observed in / , with streptococcal colonisation in / . then there is a strength statistical association between streptococcal colonisation and tinea pedis, on sel side (p = . ) as well as on contra lateral side (p = . ). in one patient blood-cultures yielded with the same streptococcus than found in foot samples. discussion: streptococcal colonisation of tinea pedis is a common finding on both feet of patients hospitalised for sel. whether inter-digital colonisation is a primary stage of invasive disease remains unproved. in our experience, a strain of streptococcus that colonised inter-digital space was isolated in patient's blood, suggesting this hypothesis may be true in some cases. if confirmed, this concept could lead to a new strategy for secondary prophylaxis of recurrent sel by decontaminating streptococcal colonisation of tinea pedis. among ggs, different emm types were found; stg , stg and stg predominated. among gas, types were found, emm predominated. one patient had the same ggs isolate in throat and skin. six patients had recurrent infections during the study; two of them with disease episodes. of the culture positive skin samples, were taken from the erysipelas infection focus ( % positive for ggs) and from another site ( % positive for ggs), e.g. wound, intertrigo, between toes or an unknown site.conclusion: a predominance of ggs was seen in the throat of erysipelas patients and their families whereas ggs was not present in control subjects. ggs, instead of gas, also seems to predominate in erysipelas skin lesions. several emm types were present in both groups and there was no clear predominance of a distinct type. the recurrent nature of erysipelas became evident also during this study. the evaluation of fournier's gangrene severity index score in patients m. ulug, m.k. celen, m.f. geyik, c. ayaz, s. girgin (diyarbakir, tr)objectives: fournier's gangrene is synergistic necrotizing fasciitis of the perineum and abdominal wall along with the scrotum and penis in men and the vulva in women. it is rare but life-threatening process. in this study we identify effective factors in the survival of patients with fournier's gangrene and to determine the accuracy of the fournier's gangrene severity index score (fgsis). methods: we evaluate patients with fournier's gangrene who were threated and follewed up from us between january and september in the department of general surgery prospectively.results: the results were evaluated in two groups: those who died (n: ) and those who survived (n: ). no statiscally significant difference was found between the age of the survivors and those who died. the admission and final laboratory parameters that correlated statiscally signinificant with outcome includes leucocyte count, hematocrit, urea, creatinine, lactate dehydrogenase, bicarbonate and albumin. sites of culture were skin/soft tissue ( , and %), respiratory tract ( , , and %) , blood ( , and %), urine ( , and %) , and other ( , and %) . -day mortality was % in this population. % of patients received antibiotic therapy alone, % surgery alone, % antibiotics + surgery, % other therapy, and % no treatment. the most common antimicrobial classes received were vancomycin ( %), beta-lactams ( ), fluoroquinolones ( ), and cotrimoxazole ( ) with % of patients receiving multiple agents. median duration of antibiotic therapy was , , and days, in the ca-mrsa, ha-mrsa and ca-mssa groups respectively. , , and % received adequate antimicrobial therapy (p < . ). hospital admission was required in , , and % of patients (p < . ). clinical success rates of initial therapy were , , and % (p < ), and recurrences were more common in the ca-mrsa group, ( , , and %, p < ). characteristics associated with outcome are listed in table . in multivariate analysis, presence of mrsa and diabetes were predictive of clinical failure.conclusion: in the community setting, mrsa infections are associated with an adverse impact on outcome compared to mssa infections and patients with ca-mrsa are significantly less likely to receive adequate antibiotic therapy. microbiological analysis of root canals associated with periapical abscesses and the antimicrobial susceptibility of isolated bacteria s. ozbek, a. ozbek, m. koseoglu, s. evcil, a. erdogan, a. ayyildiz (erzurum, tr)objective: the periapical abscess is a collection of pus in the pulp or around the root of teeth. many odontogenic infections can be managed without antimicrobial therapy or bacteriologic investigation. however, when an acute bacterial infection has progressed or antimicrobial therapy might be of benefit to patients, antibiotics are prescribed. we aimed to identify microorganisms in root canals with periapical abscess and the antimicrobial susceptibility profile of them and to revise antimicrobial treatment protocols when antimicrobials is used empirically. methods: patients with odontogenic infections included in this study. the microbiologic investigation was performed under strict aseptic conditions. a standardize routine of root canal therapy was instituted, and in each case a single root canal was sampled. in multirooted teeth only the largest canal was sampled to preserve the identity of a single endodontic/ microbiologic ecosystem. for microbial sampling, two sequential paper points were introduced into the full length of the canal, and kept in place for min. one of the paper points was used for aerobic culture and the other one for anaerobic culture. to identify isolated bacteria, whole bacterial fatty acid profiles were evaluated by using microbial identification system. antimicrobial susceptibility results were obtained by disc diffusion test for aerobics, and e-test for anaerobics. results: totally bacterial strains were isolated. of them were aerobic and of them were anaerobic. or % of cultured specimens yielded mixed (aerobic and anaerobic) species. the most prevalent bacteria were staphylococcus spp. as aerobic, peptostreptococcus prevotii and streptococcus morbillorum as anaerobic. conclusion: beta-lactam antibiotics combined with beta-lactam inhibitor (amoxicillin-clavulanic acid) had a quite effect on gram (+) and (-) aerobics. when we take into consideration that beta-lactam antibiotics stimulate production of beta-lactamase, amoxicillin-clavulanic acid combination appears a good first step antimicrobial. clindamycin may be second alternative for that purpose. for anaerobics, cefoxitin and metronidazol had well effect. although imipenem and piperasilin-tazobactam are perfect, they should not be first step of therapy. due to the frequency of mixed infections, a combination of amoxicillinclavulanic acid and metronidazol or a combination of clindamycin and metronidazol considered to have well effect for mixed infections. clinical microbiology and infection, volume , supplement , study is to review the spectrum of p. multocida infections in our centre. methods: we studied the medical records of all patients who had positive cultures for p. multocida between and . demographic, epidemiological, clinical and microbiological data including age, sex, animal exposure, site of infection, underlying diseases, type of therapy and outcome were evaluated. all isolates were identified by standard conventional microbiological methods. antibiotic susceptibility testing was performed by the disk diffusion method onto muller-hinton agar supplemented with % sheep blood and the mics of the antibiotics tested were determined by the e-test method. results: thirteen cases of p. multocida infections were diagnosed during this period. the male to female ratio was : and most patients ( %) were > years of age. respiratory tract infections were most commonly encountered ( . %), followed by soft-tissue infections ( . %) and septicemia ( . %). underlying disease was present in ( . %) patients. among them, presented a kind of malignancy. bullous pemphigoid, mitral valve stenosis, coronary disease, chronic obstructive pulmonary disease, and intracranial haemorrhage served also as predisponding factors. a traumatic animal exposure was reported in only patients and non-traumatic in cases. all isolates were susceptible to beta-lactams (penicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, cefuroxime, ceftriaxone, imipenem, and meropenem), quinolones (ciprofloxacin, norfloxacin, levofloxacin, and sparfloxacin), chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and % were intermediately resistant to aminoglycosides (gentamicin). appropriate antibiotic therapy was administered to all patients and a clinical response was observed in ( %) of them. mortality rate was %. conclusions: pasteurella multocida must be considered as a possible etiology for a variety of infections, even without an obvious animal exposure. although this organism is susceptible to a large spectrum of antibiotics, a failure to treatment may be recorded especially in severe infections and in compromised patients. infections caused by nocardia cyriacigeorgici in zaragoza, spain: identification and antibiotic susceptibility c. villuendas, b. moles, v. rodriguez-nava, a. couble, f. laurent, m. revillo, p. boiron (zaragoza, es; lyon, fr)objectives: nocardia species known to date differ in their clinical presentation, antibiotic resistance patterns and geographic distribution. nocardia cyriacigeorgici is a recently described species.the aim of this study is to analyse the identification results, antimicrobial susceptibility together with the clinical data, of n. cyriacigeorgici clinical isolates, recovered from to in our laboratory. methods: identification of nocardia spp. isolates was achieved in our laboratory on the basis of the following: visualization of the colony, gram stain and parcial acid-fast positivity by modified acid-fast staining, casein, xanthine and tyrosine hydrolysis, opacification of middlebrook h agar, production of arylsulphatase after days incubation and antimicrobial susceptibility pattern.identification at species level was achieved by s rdna gene sequencing (laboratoire de mycologie. faculté de pharmacie. lyon. france)antimicrobial susceptibility tests included commercial broth microdilution (emiza ef sensititre Ò ) and gradient strip agar dilution (e-test ab biodisk Ò ). interpretation of results was done according to nccls standard guidelines. in the six years of study, isolates of nocardia spp. were recovered, of them belonging to n. cyriacigeorgici species ( %). n. cyriacigeorgici represents the third species in frecuency in our serie, after n. abscessus and n. farcinica. the strains were recovered from patients, from respiratory specimens and one from blood-culture.pneumonia was the most frequent clinical manifestation, being copd and previous corticosteroid therapy the most common predisposing conditions. all n. cyriacigeorgici isolates showed susceptibility to: amikacin, tobramycin, cefotaxime, imipenem, trimethoprimsulfamethoxazole and linezolid, and resistance to: amoxicillinclavulanic acid and ciprofloxacine. conclusion: n. cyriacigeorgici is not an infrequent cause of nocardiosis in our geographical area. the uniformity showed in the antimicrobial profile can be useful for its identification. in our hospital, patients with copd and receiving corticoid therapy is the most important group of risk for adquiring n. cyriacigeorgici infection. whit the technics available in our laboratory the isolates were identified as nocardia spp. and identification at species level was only possible by phylogenetic analysis using rdna sequencing. high frequency of single-step resistance mutations in nocardia farcinica exposed to quinolones u.s. jensen, j.d. knudsen, k. schønning (hvidovre, dk)objectives: nocardia farcinica infections often require prolonged antibiotic therapy and perorally administered agents are desirable. isolates commonly display in vitro susceptibility to quinolones when tested by disc diffusion methodology. in the present study, we investigated the activity of three different quinolones (ciprofloxacin, levofloxacin and moxifloxacin) against n. farcinica and assessed the robustness of their activity by determining the frequency of single step resistant mutants when exposed to inhibitory concentrations of quinolones. methods: isolates of n. farcinica were used in the study; correct identification to the species level was verified by s rdna sequencing. mics of ciprofloxacin, levofloxacin and moxifloxacin against n. farcinica as well as s. aureus atcc and e. coli ccug were determined by the agar dilution method using inocula of approximately . cfu and h of incubation. single step mutation frequencies were determined by heavily inoculating selective agar plates containing quinolone at a concentration of x mic and counting resistant colonies after days incubation. inoculum was quantified by seeding a dilution series of the inoculum employed on unselective plates and counting colonies after h of incubation and frequencies were calculated by dividing the number of resistant colonies by the number of cfu present in inoculum. results: when mics were determined by agar dilution method all quinolones displayed roughly the same potency against n. farcinica isolates (mics between . and ). as expected moxifloxacin were the most potent quinolone against s. aureus. however, all three quinolones selected for single step resistant mutants, the frequency of which was higher for ciprofloxacin (~ ) ) than for levofloxacin ( ) - ) ), which again was higher than for moxifloxacin ( ) - ) ). however, even for moxifloxacin the frequency against n. farcinica was comparable to the single step mutation frequency of ciprofloxacin against s. aureus ( - ).conclusions: although quinolones may exhibit activity against n. farcinica, n. farcinica is capable of rapid development of resistance. therefore, quinolones should probably be avoided, at least as single agents, in the treatment of nocardia infections. correlation between clinico-laboratory findings and a positive igm elisa test for leptospira: a retrospective study e. mendrinou, p. goudas, a. regli (patra, gr) objective: to correlate a positive elisa test for igm antibodies against leptospira with the clinical and laboratory findings in patients with suspected leptospirosis. method: we retrospectively analysed the history, clinical course and laboratory findings in a total of patients, with suspected leptospirosis. all patients fulfilled the criteria for clinical diagnosis of leptospirosis. from the patients, had to be transferred to the dialysis unit for haemodialysis and patients had to be admitted to the intensive care unit (icu) due to severe pulmonary haemorrhage. serum samples from all patients were tested for igm antibodies against leptospira. results: from the total of patients death occurred to only four, due to respiratory failure from severe pulmonary haemorrhage. the rest of the patients recovered completely. from the total of patients had a positive elisa igm test for leptospirosis ( . %). however, from the patients that were transferred to the dialysis unit, had a positive leptospirosis test ( %) and from the six patients admitted to the icu, three had a positive test ( %). among other laboratory findings there was a stronger correlation between very low platelet levels (< . mm ) and very high blood bilirubin levels (> mg/dl) with a positive test for leptospirosis. all patients with a positive test had less than . platelets per mm and had blood bilirubin over mg/dl. the differential diagnosis of icterohemorrhagic fevers includes a vast number of pathogens, some of which are untraceable with the common laboratory methods. in our study, from the total of patients, only . % had a positive test for leptospirosis. in of the rest patients, many different pathogens were traced, most of them being several kinds of viruses (cmv, ebv), brucella and coxiella. in of the patients no pathogen was traced. conclusions: taking into consideration the high sensitivity of the elisa test we conclude that: . icterohemorrhagic leptospirosis comprises only a small subtotal of icterohemorrhagic fevers; . there is a correlation between higher levels of bilirubin and/or very low platelet levels with leptospira infection; . there seems to be a correlation between leptospira infection and severity of icterohemorrhagic fevers. evaluation of continuous ambulatory peritoneal dialysis-related peritonitis attacks in ankara s. tekin koruk, m.a. yetkin, i. koruk, f.s. erdinc, s. sahan, n. tulek, m. duranay, a.p. demirö z (ankara, konya, samsun, tr) objectives: peritonitis is a common clinical problem that occurs in patients with end stage renal disease treated by peritoneal dialysis. the aims of this study were to assess demographic aspects, rates of peritonitis, causative organisms, clinical outcomes and treatment approach for continuous ambulatory peritoneal dialysis (capd) -related peritonitis cases. methods: seventy cases of peritonitis occurred in patients treated in infectious diseases and clinical microbiology department between may and april were enrolled into this study. the mean age of the patients was . years (range - years). cloudiness of the peritoneal dialysis fluid and/or abdominal pain were considered suggestive of peritonitis and were confirmed by cell count and culture. baseline cell count, gram stain, and cultures were obtained, and repeated with periodic follow-up. results: the overall incidence of peritonitis was . ± . episodes/patient-year. in . % of patients there were only one peritonitis attack, where as in . % of them had two or more attacks. age, gender, education and profession of the patients have not been found as a risk factor in peritonitis attacks.the most common presenting symptoms of the patients were abdominal pain, cloudiness of the peritoneal dialysis fluid, nausea and vomiting. peritoneal dialysate fluid white blood cell count was ± /mm in episodes. cultures were positive in ( . %) peritonitis episodes; coagulase-negative staphylococci was the most common organism (% . ), followed by staphylococcus aureus (% . ), episodes (% . ) had negative culture results. there was a statistically significant decrease in serum crp and esr levels and at the end of the treatment when compared with the levels on admission.at the end of the study, episodes of peritonitis cases were treated with ip cefazolin and gentamicin protocol. seven of the patients did not respond initiate therapy and the therapy was converted to iv protocol. seven episodes were treated with iv antibiotics on admission for medical reasons (systemic infection and/or concurrent exit-side or tunnel infection). there were two deaths. two catheters were removed and the patients were transferred to haemodialysis programme. conclusion: despite all technical improvements during recent decades peritonitis is still the major complication of capd. for the accurate treatment of complications, causative organisms and their antimicrobial susceptibilities must be known. objective: viruses are a frequent cause of upper respiratory tract infections in children. among the respiratory viruses, influenza viruses are known to cause outbreaks globally. the present study was carried out to identify the influenza virus serotypes causing acute respiratory infection in children attending univesity hospital in konya in turkey. methods: thorat swabs were collected from acute viral upper respiratory infection suspected children attending the out patient clinic of meram medical faculty hospital. two swabs were taken fron each chidren and one of the swabs was used for bacteriological cultures and if these were negative the other one was used for viral diagnosis. totally bacteriological cultures negative swabs were investigated by real-time pcr for the presence of parainfluenza , and , influenza a and b. results: one or more viral pathogens were detected in children, with parainfluenza % being the most commonly identified virus. parainfluenza in % and parainfluenza in %, influenza a were identified in % and influenza b in %. from the specimens of children more than one virus detected. conclusion: the influenza viruses cause morbidity and mortality among children and elderly. this study analysed the occurrence of influenza and paranfluenza respiratory ifections due to influenza and paranfluenza viruses. molecular methods used directly on clinical material have an important role in the rapid diagnosis and surveillance of influenza viruses and can be applied in clinical practice for correct diagnosis and administration of effective treatment. , , , , , and . the demographics, clinical presentations and laboratory findings of the patients with serotype were presented. results: the mean age was y m, ranging from months to y m. seventy percents of children with serotype infection clustered between october and january . the mean duration of a positive culture result was . days. the mean duration of fever was . days, with days before admission. forty ( %) children were treated as outpatients. the mean length of hospital stay was . days. the most common diagnoses were exudative tonsillitis ( %), pneumonia or bronchopneumonia ( %) and pharyngoconjunctival fever ( %). the most common symptoms and signs were fever ( %), cough ( %) and coryza ( %). neurologic complications were noted in children. eighteen children had documented coinfection (including virus, bacteria and mycoplasma pneumoniae). leukopenia (wbc < /microliter) was noted in two of cases while leukocytosis (wbc > /microliter) in ( %). six ( . %) of cases had a normal serum c-reactive protein (crp) level (< mg/l), while % of children had a serum crp greater than mg/l. seventy ( . %) of children ever received antibiotics therapy. the outcomes were excellent in these cases. conclusion: recognizing that children with adenoviral serotype infection may present with prolonged high fever, leukocytosis and elevated crp, which mimics bacterial infection, the clinician may not prescribe unnecessary antibiotics for these children. the infectious mononucleosis like syndrome (im) is an acute febrile disease of older children and young adults, and is characterized by lymphadenopathy, tonsillitis, splenomegaly, liver dysfunction and by the presence of peripheral lymphocytosis with > % atypical lymphocytes. epstein -barr virus (ebv) is responsible for over % of the cases, cytomegalovirus (cmv) for %- % and toxoplasma gondii < %.herpes simplex, rubella and adenovirus are rare. the infection is usually characterized by mild symptoms. however in some cases the clinical manifestations may be rather atypical and severe. objective: to determine the prevalence of im like syndrome among patients in a children's hospital and its possible association with etiologic factors, age, major symptoms and atypical manifestations. material and methods: during a one-year period (january to december ) a total of samples were examined in our laboratory. the study population was children between - years old, which either examined in the outpatient's clinic or hospitalized. all serum specimens were examined by . indirect immunofluorescence for the presence of igg and igm antibodies against the viral capsid antigen (vca) ebv, .immuno chemistry luminescence for the detection of igg and igm antibodies to cmv and .eia for the detection of igg,igm abs of herpes simplex i and ii and toxoplasma gondii. results: of the children examined ( . %) were found positive for igg and igm vca antibodies and ( %) showed positive specific igg and igm antibodies for cmv. these patients had one or more of the primary following symptoms: fever ( %), lymphadenopathy ( %), pharyngalgia ( %), cough ( %), skin eruption ( %). atypical manifestations as meningoencephalitis were found in two children one aged months (caused by ebv) and the other of years old (caused by hsv i) confirmed by pcr. the laboratory data showed positive serology for ebv and cmv infection, the existence of atypical lymphocyte ( %), ldh, asat and alat were moderately elevated ( %) and crp increased ( %). conclusion: the frequency of im like syndrome in greece, though it's relatively low, it's not rare. the above results suggested that ebv, cmv, hsv should be considered in any young patient with im and acute neurological illness of uncertain etiology. objectives: enterovirus, parvovirus b and human herpes virus type (hhv- ) are a common cause of infection in young infants. the objective of this study was to determine what portion of the infants who received a clinical diagnosis of febrile syndrome have a viral etiology by these three genera of viruses. methods: ninety-six patients were included in the study, all of them were admitted to the pediatric casualty of a tertiary care hospital, and all of them presented a febrile syndrome without a clear focus of infection (urinary tract, lung and meningeal infections were discarded). the assay was carried out in blood samples by real-time pcr. dna was isolated from ll of blood by semi-automated system magna pure lc total nucleic acid isolation kit (roche diagnostics, nederland bv). pcr was performed in a lightcycler instrument (roche molecular biochemicals) by a uniform cycling parameters: min at °c for polymerase activation, and cycles of s at °c and s at °c for amplification of the specific target sequence ( utr gene for enterovirus, vp gene for parvovirus b and dna polymerase gene for hhv- ). pcr product formation was detected continuously by the use of taqman probes. results: a viral amplification was detected in ( %) of the patients included in this study. enterovirus was detected in ( . %) of the patients, parvovirus b in ( . %) and hhv- in ( . %). in five cases two viral amplifications were detected at the same time: parvovirus b /hhv- and enterovirus/hhv- . the mean age of the patients was years old (range from days to years). in group of infants < months old (n = ) there were enterovirus and hhv- . in the infants from months to years old (n = ) there were enterovirus, parvovirus and hhv- . in the last group of infants > years old (n = ) there were enterovirus and parvovirus b . conclusions: viral infections are an important cause of sepsis in infants admitted to hospital. enterovirus was the most frequent virus detected in infants < months, parvovirus b the most frequent in children > years old, and the hhv- was detected in all age groups. qualitative real-time pcr in blood is a rapid and sensitive method for diagnosis of enterovirus and parvovirus. however, is not the better method for diagnosis of hhv- , a latent virus, in which this technique is not capable of distinguish between recent and acute infection. objectives: group a rotaviruses are a major cause of acute gastroenteritis in infant and young children worldwide. in this study, the molecular epidemiology and clinical features of rotavirus infection in iranian children was investigated. methods: between february to january , thirty hundred and seventy two diarrhoea stools from children under -years-old with acute diarrhoea that attended the biggest paediatric hospital in tehran (iran), were analysed using elisa, electropherotyping and reverse transcriptionpolymerase chain reaction (rt-pcr). results: ninety-four samples ( . %) were positive for the presence of rotavirus either by page, elisa, or both. according to page, the predominant electrophoretic pattern detected was the long profile of ( . %) followed by the short electropherotype five of ( . %). out of the positive samples, were further characterized by rt-pcr typing assay for identification of g types, resulting in strains of g genotype while samples could not be assigned a g type. all of g genotypes had a long rna electropherotype. among the patients with rotavirus infection, ( . %) required hospitalization. watery diarrhoea ( . %), vomiting ( . %) and fever ( . %) were significantly more frequent in children suffering from rotavirus gastroenteritis. seven out of rotavirus-positive patients had severe dehydration (p < . ). rotavirus infection mostly affected children under years of age with a peak incidence of % in children - years of age and it occurs year round with a seasonal pattern: more frequently during winter ( . %). conclusion: this study revealed that rotavirus is an important etiological agent of acute gastroenteritis in tehran. we found that a major proportion of the specimens were untypeable. improved detection and characterization of incompletely typed strains will help to develop comprehensive strain information that may be required for tailoring effective rotavirus vaccines. serological study of prevalent rotaviruses in tehran e. habibi, s. ghorbani, a. jarollahei, z. habibi (tehran, zanjan, ir)objectives: rotaviruses are icosohedral and non-enveloped viruses that belong to reoviridae family which consist of three layers of protein surrounding segments of dsrna. rotavirus is one of the most important agents of acute gastroenteritis in children. in this survey, the most prevalent serotypes in tehran and seasonal distribution in a year were detected. methods: in this study, a total number of specimens of faecal samples of children and infants with acute gastroenteritis were collected from two children hospitals in tehran. the samples were tested by elisa procedure. serotyping investigation of iranian rotavirus isolates, using serotypes monoclonal antibodies (g -g -g -g -g -g -g ) in elisa tests and immunosorbent electron microscopical studies using trapping and decoration techniques were performed. results: rotavirus type a infection was identified in samples ( %). serotyping investigation in elisa tests proved that serotypes g and g were the most common serotypes circulating among infected children and infants in tehran. by electron microscopic studies the characteristic of rotavirus particles were observed in the faecal samples of infected children. the maximum incidence of infection was determined to occur among the cold months of the year. conclusion: it was approved that g and g serotypes are the main rotavirus serotypes present among children in tehran. it was detected that rotavirus diarrhoea was most prevalent among children of under years of age. results: from patients with varicella presented neurological manifestations (sex ratio m/f: / ). had acut cerebellar ataxia and one had encephalitis. we estabilished the diagnosis on the basis of clinical aspects (including neurological examination), cerebrospinal fluid examination and electroencephalogram. the age interval was between months and years. most cases were diagnosed in children and teenager ( ); one case toddlers, and cases in adults. neurological manifestations appeared in most cases among and days after the onset of rash ( cases). in the order of frequency: gait disorders ( ), cerebellar ataxia ( ), fever ( ), vomiting ( ), nistagmus ( ), seizures and coma ( ) . csf showed limphocytic pleiocytosis and elevated levels of protein ( cases); in cases csf had normal aspects. electroencephalogram had dominant theta wave with totally or partially suppression of alpha activity in all patients. all cases showed clinical and eeg improvement at the end of the treatment. conclusions: the most frequent neurological manifestation was cerebellar. the evolution was good under treatment, with no sequelae at month of follow up. key: cord- - phlylce authors: felberbaum, rachael s. title: the baculovirus expression vector system: a commercial manufacturing platform for viral vaccines and gene therapy vectors date: - - journal: biotechnol j doi: . /biot. sha: doc_id: cord_uid: phlylce the baculovirus expression vector system (bevs) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. nine bevs‐derived products have been approved – four for human use (cervarix®, provenge®, glybera® and flublok®) and five for veterinary use (porcilis® pesti, bayovac csf e ®, circumvent® pcv, ingelvac circoflex® and porcilis® pcv). the bevs platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. this combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize bevs to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. in this review, we explore the bevs platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. to underscore the growth in opportunities for bevs‐derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of glybera® and flublok®, respectively. we anticipate that the utility of the platform will expand even further as new bevs‐derived products attain licensure. finally, we touch on some of the areas where new bevs‐derived products are likely to emerge. the baculovirus expression vector system (bevs) is far from new. for thirty years researchers have been using this platform to express recombinant proteins, and thousands of proteins have been successfully expressed and purified. however, for much of this time, bevs was rele-gated to the ranks of research tool. what we have seen in the last decade is the elevation of bevs from research tool to an established manufacturing platform for production of novel biologic products. ten years ago there were only two commercial products manufactured using the bevs manufacturing platform and both of these were veterinary vaccines to prevent classical swine fever in pigs. since then, seven new products have been licensed, four of which are for humans, including vaccines and therapeutics, and many more products are in development (table ) [ , ] . we have passed a tipping point where bevs-derived products are becoming mainstream, and the bevs platform is being actively utilized by major players in the biotechnology industry to develop new products. although the bevs platform has distinct features that make it an attractive platform for the production of many biologics, it is not ideally suited for all products. factors such as protein complexity, post-translational modification, scale and cost must be considered collectively when selecting a manufacturing platform; these have been extensively reviewed elsewhere [ ] [ ] [ ] . in this review, we explore the bevs platform by looking at how the system works and the advantages and limitations of the platform from a manufacturing and regulatory perspective. the opportunities to develop new products using bevs are abundant, and we review the latest developments in the gene therapy and influenza vaccine fields as examples. finally, we consider some newer areas where bevs-derived products show promise for the future. the bevs platform has been previously described [ , , [ ] [ ] [ ] [ ] . the platform takes advantage of baculoviruses' natural propensity to infect insect cells. in nature, there are more than different types of baculoviruses, all of which have a host range restricted to invertebrates [ ] . in the laboratory and for manufacturing purposes, the most commonly used baculovirus is autographa californica multiple-capsid nuclear polyhedrosis virus (acmnpv), a virus with a double-stranded dna genome of approximately kb [ ] . the large size of its genome gives the baculovirus ample capacity to accommodate large amounts of foreign dna, including multiple genes, an advantage over other expression vectors such as vaccinia and adenovirus [ ] . to begin the bevs process, a recombinant baculovirus is constructed comprising the desired gene(s) of interest (goi) (fig. ) . first, the goi is cloned into a transfer plasmid, typically behind the strong polyhedrin or p promoter that can drive protein expression to high levels in insect cells [ , ] ; notably, these promoters are not very active in e. coli and, therefore, can be stable expression cassettes. the goi is flanked by acmnpv dna, e.g. the polyhedrin promoter on one side and a portion of the essential gene orf on the other. insect cells are then co-transfected with a mixture of the transfer plasmid and parental acmnpv dna that has been linearized such that the parental polyhedrin gene and portion of orf are missing, rendering it non-infectious [ ] . the plasmid and parental dna undergo homologous recombination to generate de novo recombinant baculoviruses. these baculoviruses are plated and individual plaques purified to isolate a single, pure plaque of recombinant baculovirus. this plaque is subsequently passaged through multiple rounds of insect cell infection to generate a high-titer stock and establish a working virus bank (wvb) that can be utilized for protein production. for manufacturing purposes, it is important that wvbs be stable and retain integrity as virus passages are scaled up. laboratory kits such as bac-to-bac ® (life technologies) that employ bacmid technology have been developed that allow researchers to quickly and easily construct recombinant baculoviruses in e. coli rather than [ ] . long-term stability of the baculovirus is an important consideration if large-scale protein production is envisioned. once a high-titer wvb has been established it is used to infect insect cells and stimulate protein production. cells are seeded in culture flasks (for small-scale production) or bioreactors (for large-scale production) and the wvb added to infect the insect cells when they are in their logarithmic growth phase. the baculoviruses reprogram the cellular machinery to produce the recombinant protein(s). following protein expression (typically - hours post-infection), the cells and/or supernatant are harvested, depending on whether the product is intracellular or secreted, respectively, and the proteins are purified according to standard techniques such as ultracentrifugation or column chromatography. many different insect cell lines have been used for bevs but the most common are derived from ovarian cells of the fall army worm, spodoptera frugiperda (e.g., sf- , sf- and expressf+ ® [protein sciences corporation]), and the cabbage looper, trichoplusia ni (high five tm cells, life technologies) [ , , ] . high five cells are used to manufacture the licensed human papillomavirus vaccine, cervarix ® (glaxosmithkline), and sf- cells are used to produce the antigen used in the prostate cancer immunotherapy, provenge ® (dendreon). expressf+ cells are used to manufacture three licensed products: flublok ® influenza vaccine (protein sciences corporation), glybera ® gene therapy for the treatment of familial lipoprotein lipase deficiency (uniqure), and ingelvac circoflex ® veterinary vaccine to protect against porcine circovirus type (boehringer ingelheim vetmedica). large scale manufacturing and commercial production require specific cell line characteristics such as scalability, high yields, the ability to grow in low-cost, serum-free media, and qualification to meet regulatory agency (e.g. fda, ema, etc.) requirements for purity and safety. characteristics of the bevs platform are summarized in fig. . these features as well as other important considerations about the technology are discussed below. the choice of an expression system for manufacturing is a complex decision that must balance many facets, including attainment of specific product features, the demands of the therapeutic indication, and the needs of the manufacturer. comparisons of the bevs platform to other expression systems such as bacteria, yeast, mammalian cells and plants have been made and are useful to consider when selecting a platform [ ] [ ] [ ] . bevs employs recombinant technology, giving researchers and product developers a level of control over the production process that is not possible with other techniques. a good example is found with vaccine manufacturing. traditionally, vaccines are made by cultivating large volumes of the pathogen against which protection is desired to generate the "raw materials" required for the product. this can be accomplished by infecting substrates such as embryonated chicken eggs or mammalian cells, both of which are used to manufacture the majority of the vaccines recommended for routine immunization [ , ] . the pathogen is either weakened and administered live as a live attenuated vaccine, or is killed or inactivated with reagents such as formalin or heat prior to being formulated into vaccine. these methods yield safe and effective vaccines; however, important shortcomings have been observed. the first concerns specificity. for example, traditional influenza vaccine production involves virus propagation in eggs. as influenza viruses are rna viruses, they have a tendency to mutate to optimize their figure . the bevs platform. the bevs platform is an efficient process for producing a wide variety of proteins in a streamlined manner. a gene of interest (goi) is cloned into a transfer plasmid behind a strong promoter (green arrow) and surrounded by dna homologous to the parent baculovirus (yellow and green boxes). a library of recombinant baculoviruses (rbv) can be made using standard cloning techniques and varying the goi. construction of an rbv takes eight days. to generate protein, the appropriate rbv is scaled up (taking on average two to five weeks) and used to infect insect cells, which programs the cells to generate large quantities of recombinant protein that can subsequently be purified to high levels using standard techniques. a single insect cell line can be used to produce all proteins. protein production averages three to five weeks and yields highly pure, biologically active products. growth [ ] . generally, the changes introduced into the virus sequence have little impact on vaccine efficacy. however, since the virus receptors for birds and mammals differ, these changes can be meaningful, and it has been documented that in some cases the changes have rendered egg-based influenza vaccines ineffective [ ] . the bevs process does not involve pathogen growth and, therefore, avoids this complication. rather than cultivate a pathogen to collect "raw materials", a recombinant baculovirus is constructed that codes for the antigen required for protection. since accommodations for growth do not need to be made, the antigen can be an exact sequence match to the human pathogen. this has the potential to solve the ineffectiveness observed for some egg-adapted vaccines as was described by skowronski et al. ( ) [ ] . moreover, specific point mutations can be introduced to enhance features such as stability. for example, it has recently been reported that purified hemagglutinin protein, the protective antigen in influenza vaccines, appears unstable in the srid potency assay due to crosslinking of specific cysteine residues in the protein [ ] . by mutating these cysteine residues to non-thiol residues, it was possible to prevent cross-linking and enhance stability [ ] . a second shortcoming with traditional vaccine manufacturing is process length. again, influenza serves as a good example. influenza epidemics occur every year and annual influenza vaccination is recommended by the centers for disease control and prevention [ ] . in addition, pandemic outbreaks occur occasionally, the most recent of which was the h n swine flu (a/california/ / ) [ , ] . timeliness of vaccine manufacture is critical to ensure that adequate vaccine supply is available. egg-based influenza vaccine manufacturing takes on average about six months, as the process of creating a high-producing, egg-adapted seed virus is slow [ ] . this means that manufacturing of seasonal influenza vaccines must begin the winter prior to an epidemic, before that season's strain prevalence is definitively known, to guarantee adequate supply, and this has resulted in seasons where the available influenza vaccines have not matched the circulating influenza strains [ , ] . moreover, the president's council of advisors on science and technology (pcast) reported that pandemic influenza vaccine in was not readily available until after the pandemic peaked, a major concern for public health and safety (www.whitehouse.gov/sites/default/files/microsites/ostp/ pcast-influenza-vaccinology-report.pdf). pcast identified recombinant technology and freedom from virus growth and egg adaptation as the solution to this problem. bevs-derived influenza vaccines do not require seed viruses and can be produced in as little as days [ ] . in addition to specificity and speed, recombinant technology offers advantages with respect to purity and product design. with respect to purity, recombinant products are free of pathogens, eggs and many chemicals (such as formalin and antibiotics) that can be undesirable or allergenic [ ] . for product design, recombinant techniques make it possible to engineer proteins with desired features, such as fusion proteins that increase immunogenicity or include multiple antigens and truncated proteins with deleted domains to improve yields and ease purification [ ] [ ] [ ] [ ] [ ] . one example of this application is the antigen used in provenge immunotherapy. the antigen is a fusion glycoprotein consisting of prostatic acid phosphatase (pap) linked to granulocyte-macrophage colonystimulating factor (gm-csf) and is used to stimulate autologous antigen presenting cells [ ] . the gm-csf activates the antigen presenting cells and enhances cell viability while the pap serves as the target antigen. another example is the cervarix ® vaccine manufactured by glaxosmithkline. the vaccine is a bivalent virus-like particle (vlp) comprised of the human papillomavirus l proteins (strains and ) . by expressing c-terminally truncated l proteins, the manufacturers are able to prevent intracellular vlp self-assembly, which would complicate purification. instead, the l subunit proteins are purified to a high degree and vlp assembly is achieved in vitro [ ] . the bevs platform has inherent safety measures built in that are attractive from a regulatory perspective. baculoviruses have a narrow host range restricted to specific insects and are considered safe to use as biological pesticides with no negative impact on plants, mammals, birds, fish or non-target insects [ ] . people are exposed to baculoviruses daily by consuming fresh vegetables. for instance, a serving of coleslaw may contain hundreds of millions of baculoviruses [ ] . baculovirus vectors have also been explored as gene therapy vectors. these studies have demonstrated that baculoviruses cannot repli-cate in mammalian cells and cannot express a gene cassette unless it is driven by a mammalian promoter [ , [ ] [ ] [ ] [ ] . a major consideration for regulatory agencies when evaluating a novel cell substrate is the potential to harbor adventitious agents that could threaten patient safety. there are very few adventitious agents that can replicate in both insect and mammalian cells. a notable exception is arboviruses that can be transmitted to humans via insect bites and can cause complications such as encephalitis and hemorrhagic fever [ ] . to mitigate this risk, cells from non-biting insects such as the fall armyworm and cabbage looper have been used with bevs as noted above. nonetheless, adventitious agents have been detected in some insect cell lines. for instance, the trichoplusia ni high five cell line, bti-tn- b - , used to make cervarix ® , was found to be latently infected with an alphanodavirus that was induced by recombinant baculovirus infection [ ] . in addition, other insect cell lines, such as those generated from drosophila melanogaster, have been shown to harbor innate retroelements derived from retroviruses that could potentially be infectious [ ] . more recently, a possible insect-specific virus sf-rhabdovirus was identified in spodoptera frugiperda cells [ ] . although studies showed that this virus could not enter or replicate in human cell lines and, therefore, is unlikely to be a risk, novel cell lines will most likely need to be characterized and monitored for the presence of this virus as they are for nodaviruses, retroviruses and others. in general, because adventitious agents are a potential threat, cell substrates of all origins (including insect and others) must be thoroughly tested for the presence and infectivity of such agents before they are allowed by regulatory agencies for manufacturing use. in addition to the safety considerations just discussed, there are two important regulatory features associated with the bevs platform that should be considered. the first is that bevs is a transient protein expression system; the recombinant baculoviruses used vary based on their foreign gene cassettes but a single cell line can be used for the expression of all proteins (fig. ) . qualifying a cell line is no small feat. it can take years of work to adequately ensure purity and safety in addition to high productivity. stable cell lines have to be independently qualified each time their genetic composition changes [ ] . in contrast, cell lines used with bevs and other transient expression systems remain constant and, consequently, need to be qualified just once. a second feature is the growing number of bevsderived products that have been approved by regulatory agencies worldwide ( table ) . as is the case for all technologies, prior regulatory approvals remove barriers for future product approvals. the technology becomes more mainstream and less novel with each approval, and the safety database of patients that are administered products without complication continues to grow. nine bevsderived products have been licensed, providing regulators confidence with the platform. the following characteristics make the bevs platform appealing for commercial manufacturing: scalability, biosafety, flexibility and existing manufacturing capacity. insect cells are grown in suspension, so if the cells and baculoviruses have been optimized for large scale and multiple passages, the culture size is only limited by the size of the bioreactor. for example, expressf+ cells have been used to produce recombinant proteins at scales ranging from two to l [ ] . the cost of goods for bevs production is largely dependent on capital costs and yields. as discussed by cox ( ), vast global bioreactor capacity (~ l) already exists and presents the opportunity to minimize the investment needed to establish bevs manufacturing facilities [ ] . moreover, opportunities for yield improvements are abundant and include genetic and fermentation-based approaches; these are described elsewhere [ ] . unlike some other production facilities, bevs facilities can be multi-purpose and used to produce a variety of bevs-derived products, especially when disposable or single-use technology is employed [ ] . this is because a single cell line can be used for production of different products. this feature is especially meaningful for regions of the world where limited manufacturing capacity exists. a single bevs facility could, for example, be used to produce vaccines for diseases endemic to a region and quickly be converted to produce pandemic influenza vaccine in an emergency. finally, because the bevs platform does not require the handling of live, potentially dangerous pathogens, requirements for biocontainment that can be very costly are reduced. the bevs platform is a versatile technology useful for the manufacture of many products; however, other platforms may be better suited for the production of certain proteins. for instance, small proteins that do not require post-translational modifications are best made in e. coli that can quickly generate high yields at low cost [ ] . yeasts such as s. cerevisiae can also produce high yields of protein at low cost and are capable of some post-translational modifications [ ] . insect cells are capable of many post-translational modifications but proteins that require complex post-translational modifications and folding may best be made in mammalian expression systems. for www.biotecvisions.com www.biotechnology-journal.com example, the glycosylation patterns produced by insect and mammalian cells are related, and glycoproteins produced in insect cells are often correctly folded, biologically active and immunogenic [ ] . however, insect cells generate less complex n-glycans than mammalian cells and this can negatively impact biological function [ , ] . some developments have been made to address this limitation, including engineering transgenic insect cell lines that stably express mammalian glycosylation enzymes or co-expressing such enzymes with the gene of interest in a single baculovirus [ ] [ ] [ ] [ ] . whether this is required must be assessed on a protein by protein basis. licensure of the first bevs-derived products has paved a regulatory pathway, reducing regulatory uncertainty. in this section we discuss opportunities in the areas of gene therapy and influenza vaccines spawned by the two most recent bevs-derived product approvals, glybera ® (licensed by the ema in ) and flublok ® (licensed by the fda in ), respectively. the bevs approach to gene therapy has largely involved the production of recombinant adeno-associated viruses (raavs) that house therapeutic dna. the use of raavs as a gene delivery vector has gained popularity for several reasons, including long-term gene expression, lack of pathogenicity and ability to transduce a wide variety of cells, both dividing and non-dividing [ , ] . nine different raav serotypes ( - ) are most commonly used for raav-based gene therapy, each serotype with a different propensity for tissue-specific infection and infection kinetics [ ] . recombinant aav-based gene therapies have been in development and shown promise for some time; however, a major limitation to their implementation had been the inability to scale up the manufacturing process to produce sufficient quantities of raavs. the original raav vectors were produced in mammalian tissue culture using adherent cells such as hek cells, which required about -cm flasks to produce enough material for a large animal study or human clinical trial (~ raav particles) [ ] . to overcome this limitation, scientists adopted and optimized the bevs platform for production of large scale, high titer raavs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . by adjusting parameters such as multiplicity of infection, cell density and fermentation mode, raav yields on the order of vector genomes per liter have been reported [ ] . the traditional bevs production strategy for raavs requires the co-infection of insect cells with three differ-ent recombinant baculoviruses: bac-rep that expresses the major aav replication enzymes (rep and rep ); bac-cap that expresses the aav virion coat proteins; and bac-goi that expresses the gene of interest flanked by the aav inverted terminal repeat elements that are required for the rescue, replication and packaging of the gene [ , ] . no adenovirus helper is needed as is required for mammalian cell raav production [ , ] . due to some genetic instability of the bac-rep construct, a streamlined two-baculovirus system has further been developed where rep and cap proteins are expressed from a single baculovirus (bac-repcap) and rep and rep are transcribed from a single mrna species that enhances stability [ ] . alternatively, genetic modifications have been made to the original bac-rep and bac-cap constructs to enhance stability and improve expression [ ] . an important regulatory hurdle was overcome in when glybera received marketing authorization in europe, making it the first gene therapy product approved in the western world and launching the bevs platform into the spotlight as a preferred platform for raav manufacture. glybera (alipogene tiparvovec) is comprised of the human gene lpl s x in a bevs-derived raav serotype vector and is used for the treatment of patients with lipoprotein lipase (lpl) deficiency [ ] . clinical studies have shown glybera to be safe and effective [ , ] . glybera will likely be the beginning when it comes to raav-based gene therapy. the approach is relevant to the estimated thousands of monogenic diseases [ ] . treatments are actively being investigated in a diverse array of therapeutic areas and dozens of product candidates are in clinical development (summarized in table ). hemophilia is one area where progress has been made (reviewed in [ ] ). there are four ongoing human clinical trials involving raav serotypes or , all designed to express factor ix for the treatment of hemophilia b (table ). factor viii raav-based therapy is a target for the treatment of hemophilia a, the most common severe inherited bleeding disorder, and only a modest increase in plasma factor viii levels is expected to be required to be clinically relevant [ ] . raav-based treatments for retinal degeneration, including macular degeneration and leber's congenital amaurosis type , are another area of intense investigation [ , ] . retinal treatments are ideal because of cell accessibility through intravitreal and subretinal injections and the ability to assess structure and function noninvasively. serotype is most commonly used for these therapies but types , , and - are all capable of infecting photoreceptors, the most prominent cell type for retinal degenerations [ ] . diseases of the central nervous system (cns), such as parkinson's and alzheimer's diseases, are also actively being tested with raav-based therapies that are promising. aavs exhibit a strong preference for neuronal transduction, making them a popular gene delivery vehicle for cns therapies but vector improvements are still needed to optimize treatments (discussed in [ ] ). final-ly, duchenne muscular dystrophy (dmd) is a therapeutic target where progress is being made. although the single gene affected in dmd (dystrophin) has long been known, its large size has made gene therapy a challenge. recent treatment approaches to overcome this include exonskipping, trans-splicing and micro-and mini-dystrophin delivery strategies [ , ] . for fifty years influenza vaccine manufacturing technology remained largely stagnant. that changed with the licensure of flublok ® in . flublok ® is a trivalent bevs-derived vaccine for seasonal influenza composed of µg of recombinant hemagglutinin (ha) derived from the two a and one b influenza viruses selected for inclusion in the annual influenza vaccine by the world health organization; the vaccine is licensed by the fda for adults and older [ ] . influenza vaccines are standardized to contain a specific amount of ha, the major surface glycoprotein on the influenza virus [ ] . bevs-derived recombinant ha forms trimers that in turn oligomerize into immunogenic rosettes [ ] . these proteins can be purified to high levels resulting in a vaccine that has been shown to be safe and effective in clinical studies [ , [ ] [ ] [ ] [ ] . the advantages of recombinant bevs vaccines for pandemic influenza are especially important. vaccines for avian influenza viruses such as the h , h and h subtypes are urgently needed because of these viruses' high pathogenicity and mortality rates in humans and the fact that h has previously demonstrated pandemic potential and human-to-human transmissibility [ , ] . a monovalent variation of the flublok ® vaccine called panblok ® has been developed. in a phase ii study of h panblok ® (a/indonesia/ / ), a two-dose schedule of vaccine at doses of . - µg ha formulated with the adjuvant glucopyranosyl lipid a/stable emulsion (gla/se) had an acceptable safety and reactogenicity profile and elicited serologic responses meeting seroconversion criteria in adults - years old [ ] . moreover, an earlier study showed that people administered h panblok ® (a/hong kong/ / ) in were primed for an enhanced immune response following administration of an antigenically variant vaccine strain in [ ] . evaluated a different bevs-derived h subunit vaccine candidate (a/goose/guangdong/ / ) and showed that it protected against a lethal challenge in balb/c mice and in specific pathogen-free and commercial chickens, suggesting it could be useful as both a human and animal vaccine [ ] . multi-component vlp vaccines for pandemic influenza are under development that are composed of recombinant ha, neuraminidase (na) and matrix (m ) proteins produced in sf- cells [ ] [ ] [ ] . evaluation of an h n (a/indonesia/ / ) vlp vaccine in a phase i/ii study of adults - years old showed that two doses of unadjuvanted vaccine at , or µg ha/dose were generally well-tolerated and resulted in seroconversion [ ] . similarly, a phase ii study of an h n (a/california/ / ) vlp vaccine in adults - years old showed it was safe at doses of , or µg ha/dose and elicited high rates of seroprotection ( - %) [ ] . more recently, an h n vlp vaccine was developed that was comprised of ha and na proteins matched to a/anhui/ / (h n ) and m protein matched to a/indonesia/ / (h n ) [ ] . this vaccine candidate was tested with or without saponin-based iscomatrix adjuvant in balb/c mice and was shown to protect against a lethal challenge. antibodies against both ha and na were elicited, with -to -fold higher responses in the iscomatrix groups. although the data are promising, a challenge for the development of multi-component vlp vaccines will be standardizing the vaccines for each of their components (e.g., quantity of ha vs. na vs. m ). an advantage of using recombinant technology for vaccine design is the opportunity to modularly add or subtract antigens to a formulation. inclusion of recombinant na in vlp vaccines has been shown to induce formation of anti-neuraminidase antibodies [ ] . it has been noted that different vaccine compositions (e.g., vlp vs. subunit vs. whole virion) induce different immune profiles in balb/c mice [ ] . while the advantages of these various profiles are not yet clear, the flexibility of the bevs platform enables catering towards different outputs. besides inclusion in vlps, recombinant na can be individually produced via bevs and may serve as a potentially efficacy-enhancing additive to influenza vaccines [ ] . na immunity is infection-permissive but can reduce infection severity and duration [ ] . the potential benefits of including recombinant na in influenza vaccines has recently been reviewed [ ] . other opportunities have emerged with bevs as researchers begin pursuit of a so-called universal influenza vaccine. licensed influenza vaccines offer limited cross protection to heterologous influenza viruses and, thus, there is the need for annual update of seasonal influenza vaccines and concern over pandemic preparedness. a successful universal influenza vaccine would offer long-lasting and broad protection against a range of different influenza virus strains. approaches to universal influenza vaccine design include ha stalk-based constructs and chimeric ha-based vaccines that are composed of conserved stalk domains fused to "exotic" heads, usually of avian origin (these approaches are reviewed elsewhere [ ] ). the bevs platform, being based on recombinant technology, offers the flexibility and genetic control required for the design and manufacture of these universal vaccine candidates. in this review we have taken a close look at the bevs platform, describing how the platform works, outlining the features and limitations of the technology and highlighting the growth opportunities that emerged from the two most recent bevs-derived product approvals. we expect this growth to continue and expand as future bevsderived products attain regulatory approval. the approvals of bevs-derived cervarix ® and flublok ® vaccines have broadened the acceptance of the platform beyond its initial veterinary borders to use in healthy adolescents and adults. human therapeutics is another area of use and gene therapy in particular is a growing area of interest; the approval of glybera drew major attention to bevs-derived raavs. baculoviruses themselves can also be used as gene delivery vectors, and other recombinant protein complexes produced using bevs are being explored for the delivery of various peptides and antigens, such as in the form of the newly characterized vault particles [ ] . the speed at which recombinant proteins can be produced using bevs makes it a particularly attractive platform to design safe and effective vaccines to be available to timely combat new infectious pathogens as they arise. success with this approach has been demonstrated for influenza and can be applied to broader areas. for example, coronaviruses have been plaguing both people and animals especially in the last decade with lethal outbreaks of severe acute respiratory syndrome (sars) in , middle east respiratory syndrome (mers) in late , and porcine epidemic diarrhea virus (pedv) in [ ] [ ] [ ] . all coronaviruses share a similar structure that includes the presence of the spike glycoprotein on the viral envelope that is the dominant immunogen [ ] [ ] [ ] . multiple coronavirus bevs-derived vaccine candidates have been developed that include recombinant spike protein either alone as a subunit vaccine or together with recombinant envelope and membrane proteins as vlps and have demonstrated efficacy in animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the spike protein has been shown to be immunogenic both as full length protein and as a truncated protein containing only the extracellular domain [ , ] , and multiple routes of vaccine administration have been examined [ ] . two recently emerged threats are chikungunya virus and ebola virus, both of which can be addressed with bevs-derived vaccines. chikungunya virus causes a serious disease that involves severe joint pain and can be fatal; a recent outbreak appeared in saint martin in december and has since made its way to more than countries or jurisdictions in the americas, including the continental united states [ ] . chikungunya is an arbovirus, for which there are many opportunities to develop bevs-derived vaccines (reviewed in [ ] ). both subunit and vlp vaccine candidates expressing the chikungunya viral envelope glycoproteins have been developed using bevs, with vlps demonstrating higher immunogenicity in mice [ ] [ ] [ ] . ebola virus is a filovirus that causes lethal hemorrhagic fever in humans and is devastating africa in an ongoing outbreak [ ] . the ebola virus glycoprotein has been shown to be the protective antigen and could be produced similar to a chikungunya virus vaccine [ ] . in addition to glycoprotein-based vaccines, the bevs platform has shown early promise for the production of biotechnology journal toxin-based vaccines. for example, the c-terminal heavy chain domain of clostridial botulinum neurotoxin, a highly toxic protein that causes botulism, has been produced with bevs and has demonstrated immunogenicity and challenge protection in mice [ , ] . recombinant toxin vaccines for other diseases such as clostridium difficile could also be possible [ , ] . in conclusion, bevs is a versatile platform whose potential is just beginning to be realized. the technology offers speed, flexibility, specificity and safety, and the use of a single cell line to manufacture multiple products makes bevs an attractive platform to adopt. bevs can be used to develop a wide variety of products and is especially well suited for combating rapidly 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system rapid immune responses to a botulinum neurotoxin hc subunit vaccine through in vivo targeting to antigen-presenting cells development of a recombinant toxin fragment vaccine for clostridium difficile infection recombinant antigens based on toxins a and b of clostridium difficile that evoke a potent toxin-neutralising immune response reingard grabherr key: cord- -pvo hs x authors: ganguli, ishani; chang, yuchiao; weissman, arlene; armstrong, katrina; metlay, joshua p. title: ebola risk and preparedness: a national survey of internists date: - - journal: journal of general internal medicine doi: . /s - - - sha: doc_id: cord_uid: pvo hs x background: the – ebola virus disease (ebola) epidemic centered in west africa highlighted recurring challenges in the united states regarding risk communication and preparedness during global epidemics. objective: to investigate perceptions, preparedness, and knowledge among u.s. internists with regard to ebola risk. design: cross-sectional web-based national survey distributed by e-mail between december and january . participants: practicing u.s. internists participating in a research panel representative of american college of physicians (acp) membership. main measures: respondents’ perceptions of ebola, reported sources of information, and reported management of possible ebola cases. the primary predictor was the possibility of encountering ebola (based on respondents’ geographic proximity to designated airports or confirmed ebola cases, or on their patients’ travel histories). pre-specified outcomes included reported management intensity in clinical vignettes involving patients at low risk of symptomatic ebola as well as reported ebola preparedness. key results: the survey response rate was . %. among the respondents, . % ( % ci . – . %) reported that they had recently evaluated a patient who had traveled to west africa. seventy percent ( % ci . – . %) reported a practice-level protocol. the centers for disease control and prevention (cdc) was the most popular source for ebola information ( . %, % ci . – . %). most respondents felt very ( . %) or somewhat prepared ( . %) to communicate information about or diagnose ebola, especially those with the possibility of encountering ebola and those who reported medical journals, professional groups, or government as information sources. one-fifth of respondents ( . %, % ci . – . %) reported overly intensive management for low-risk patients. those with the possibility of encountering ebola were less likely to report overly intensive management ( . vs. . %, p = . ). conclusions: internists had wide-ranging views and understanding of ebola risk; those least likely to encounter ebola were most likely to be overly aggressive in managing patients at low risk. our findings underscore the need for better risk communication through various information channels to empower frontline providers in infectious disease outbreaks. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the impact of the - outbreak of ebola virus disease (ebola) was deeply felt in west africa, with more than , cases and , deaths in guinea, liberia, and sierra leone as of the time of publication. in the united states, where there have been four locally diagnosed cases and one fatality to date, the potential domestic impact of the disease received a great deal of public health and media attention, at times to deleterious effect. , following missteps in management of the first locally diagnosed case in dallas, which led to loss of trust in health officials, , several state governors initiated quarantines of even symptom-free international aid workers returning from ebola relief efforts, and there were several high-profile news reports of institutions asking students and employees with spurious connections to the disease to stay home . [ ] [ ] [ ] among health care practitioners, there was evidence of heightened concern as well. transmission of the ebola virus occurs only by direct contact with the bodily fluids of symptomatic individuals, and the typical incubation period ranges from to days-evidence that is the basis for guidelines issued by the cdc. yet of the inquiries to the cdc about suspected ebola cases between july and mid-november , % referenced individuals who had not recently traveled to a country with ebola or been in contact with an ebola patient. furthermore, only % were deemed potential cases based on initial signs, symptoms, and risk factors. in several instances, pursuing ebola pathways may have delayed clinicians' efforts to confirm the true diagnosis. the u.s. experience with the ebola epidemic is reminiscent of prior outbreaks, such as avian flu and severe acute respiratory syndrome (sars), in which public and health professional reactions were poorly matched to communicated risks, , yet factors contributing to these disconnects are not electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. well understood. the west african ebola outbreak offers a unique opportunity to investigate how communication infrastructure and personal factors affect the ability of frontline health care providers to respond appropriately to global health crises. our goal was to measure how the risks of ebola were perceived and acted upon by health professionals, particularly in ambulatory care settings, where preparedness is uniquely challenging. in addition, we wanted to explore the role that different information sources and regional context played in shaping provider attitudes and knowledge. to address these aims, we conducted a national survey of u.s. internists, examining self-reported preparedness among physicians and whether their potential for clinical exposure to ebola was predictive of their management approach in hypothetical clinical scenarios. we conducted a web-based national survey of u.s. internists between december , , and january , . the survey was conducted in collaboration with the american college of physicians (acp) and covered the domains of physician and practice characteristics, risk perception, information sources, preparedness, and knowledge related to ebola. the acp research center surveyed member physicians who agreed to participate in its nationally representative panel established in june . after excluding medical students, affiliate members, honorary fellows, and non-u.s. members, the acp used stratified random sampling to create the internal medicine insider research panel. the panel is regularly adjusted to represent membership across multiple demographic characteristics. panelists agree to participate in an average of two projects per month, and are rewarded for completing each survey with points redeemable for gift cards. within this panel, we selected all self-reported general internists and geriatricians who practiced primary care, and excluded retired physicians, residents, clinical fellows, and clinicians who self-reported less than % of their time in direct patient care. we identified physicians ( % of the panel) who met these pre-established criteria for survey administration. the -item survey was developed based on a literature review of comparable viral outbreaks. , the instrument included five sections. the first section captured demographic and practice characteristics and assessed physicians' exposure to patients at risk for ebola. the second section assessed physicians' perceptions of ebola risk. the third section asked respondents to rate the personal importance of information sources and their level of preparation in order to identify and manage possible cases. the fourth section measured how physicians assessed the risk of ebola versus influenza in a series of clinical scenarios, which were written such that influenza was more likely in each case while the probability of ebola varied. finally, the survey posed clinical vignettes testing knowledge of appropriate management of suspected ebola, transmission methods, and incubation period based on cdc guidelines circulating at the time of survey distribution. the survey was field-tested by primary care physicians at two adult internal medicine clinics affiliated with massachusetts general hospital; insights from the field testers' survey responses and oral comments were incorporated into the final instrument (esurvey). the survey was distributed via e-mail to panel members on december , , and remained in the field for days. five repeat requests were sent via e-mail to non-responders in order to improve the response rate. our pre-specified primary predictor was the possibility of encountering ebola, a binary measure meant to approximate a provider's pre-test probability of diagnosing ebola in his/her patients. possibility of encountering ebola was defined as either self-report of seeing patients who had traveled to west africa or been exposed to the ebola virus, or geographic proximity to a designated u.s. port of entry for flights from ebola-affected west african countries (john f. kennedy international airport, newark liberty international airport, dulles international airport, chicago o'hare international airport, and hartsfield-jackson atlanta international airport) or to a confirmed ebola case (dallas, tx; new york city, ny). geographic proximity was defined by whether a respondent's zip code was included in the combined statistical area of an index city. our primary outcome was level of management intensity, which was defined a priori by responses to two of the clinical vignettes (esurvey - ) evaluating knowledge of transmission methods and incubation period in patients with low likelihood of contagious ebola virus disease. in both vignettes, an overly intense management response based on cdc guidelines was defined as choosing to isolate the patient, test for ebola, or call the local hospital. a moderateintensity response was defined as choosing to ensure health department monitoring (this was guideline-concordant for question a), and a low-intensity response was defined as choosing reassurance/routine care (this was guidelineconcordant for questions b and ). the more intensive of the two vignette responses defined the respondent's intensity level. our secondary outcomes were self-reported preparedness to diagnose cases and communicate risk as well as the presence of a protocol for ebola diagnosis and treatment at the respondent's practice site. we reported respondent demographic and practice characteristics, perceptions of ebola risk, information sources, preparedness for ebola, and approach to managing ebola risk. descriptive statistics included frequency counts and percentages for categorical variables, and ranges and means with standard deviations for continuous variables. we examined associations between outcomes and predictors including provider demographics, practice characteristics, self-reported information sources, and geographic regions. both management intensity and ebola preparedness were considered as ordinal variables, with three response categories (low/moderate/overly intense, and very/somewhat/not at all, respectively). having an ebola protocol was dichotomized into yes vs. no/not sure. in bivariate analyses, we compared groups using mantel-haenszel chi-square tests for ordinal outcomes and chi-square tests for dichotomized outcomes. we conducted a proportional odds ordinal regression model to examine the effect of the possibility of encountering ebola on management intensity while controlling for age, sex, race, and practice setting. there was no evidence of violating the proportional odds assumption based on the score test. all analyses were performed using sas version . statistical software (sas institute inc., cary, nc, usa). all reported p values are two-tailed, and p< . was considered statistically significant. we submitted our proposal to the partners institutional review board and it was granted exemption status. the study had no external funding source. we received completed questionnaires from among potential respondents, for a response rate of . % (efigure). survey responses were excluded for respondents who reported that they spent no time delivering primary care. respondents and non-respondents were similar based on demographic features, including gender and age. among the respondents who were eligible and who completed the survey, age ranged from - years, with a mean of . years ( . ); . % were women (table ) . ten respondents ( . %, % ci . - . %) reported that they had worked in an international medical relief effort, while . % ( % ci . - . %) stated that they had considered engaging in ebola relief efforts in west africa. twenty respondents ( . %, % ci . - . %) reported that they had seen at least one patient within the last months who had traveled to west africa in , and one participant ( . %, % ci . - . %) reported seeing a patient who had been exposed to ebola. thirty-two physicians ( . %, % ci . - . %) met the previously described definition of having the possibility of encountering ebola. nearly two-thirds of respondents either agreed or strongly agreed ( . %, % ci . - . %) that they accepted the risk of contracting ebola as part of their job. nine percent ( . %, % ci . - . %) expressed fear of contracting ebola, . % ( % ci . - . %) stated that their physician colleagues had this fear, and . % ( % ci . - . %) indicated that the fear was present among their nonphysician colleagues (fig. ). the cdc was the most popular self-reported source for ebola information ( . %, % ci . - . %) ( respondents were given a series of clinical scenarios involving a patient with flu-like symptoms presenting in november, , and were asked to choose whether ebola or influenza was the more likely diagnosis. respondents were unlikely to choose ebola when asked about evaluating a patient with flu-like symptoms in clinic, . % ( % ci . - . %) reported they would ask for a travel history first. when asked about managing an asymptomatic international aid worker who had returned from ebola containment efforts, . % ( % ci . - . %) of those who were told she had returned days prior said they would confirm health department monitoring. among those who were told she had returned days prior, . % ( % ci . - . %) said they would proceed with preventive care. more than half of respondents ( . %, % ci . - . %) said they would offer reassurance to a nurse who reported that there was a patient with ebola in her hospital with whom she had no direct contact. based on their responses regarding ebola incubation period and transmission (esurvey - ), . % of physicians ( % ci . - . %) were categorized as reporting overly intense management, while . % ( % ci . - . %) reported moderate-intensity management and . % ( % ci . - . %) low-intensity management, as previously defined. predictors of management intensity. the possibility of encountering ebola was significantly inversely associated with intensity of management. among physicians with a possibility of encountering ebola, . % chose overly intense management, while . % chose moderate-intensity and . % chose low-intensity management strategies. in contrast, . % of physicians without a possibility of encountering ebola chose overly intense management, while . % chose moderate-intensity and . % chose low-intensity management strategies (p= . ). in ordinal logistic regression adjusting for age, sex, race, and practice setting, respondents with a possibility of encountering ebola had lower odds of choosing the more intensive management strategy (or . , % ci . - . , p= . ). there was no statistically significant association between management intensity and age, sex, race, top information source, practice type, or practice setting. the - ebola outbreak centered in west africa left united states public health officials, health systems, and the media struggling at times to mount a measured response. this raised important recurring questions about how we manage information to help frontline clinicians in times of public health crisis. for this reason, we investigated internists' perceptions and knowledge of ebola using a national survey. while few respondents said that they had seen patients who had recently traveled to west africa, most reported consulting cdc guidelines on managing ebola at least once; the cdc was the most frequently cited information source. almost all respondents gave the guideline-concordant answer on obtaining a travel history, but there was considerable variation in responses regarding incubation period and transmission risk. when we looked at management intensity of these responses, we found that the only significantly associated factor was the possibility of encountering ebola: physicians who practiced outside metropolitan areas associated with ebola cases or designated airports, or who did not report patients with recent west african travel, were more likely to endorse overly aggressive management. this may be because these physicians were less motivated to stay abreast of ebola clinical guidelines or due to a lack of institutional or community infrastructure to support the physicians in management-although we found no association between the possibility of encountering ebola and the presence of an institutional protocol. it is not clear whether this is a physician-or institution-level phenomenon, though there was no association with practice setting or type. there was wide variation in the number of respondents reporting that ebola was more likely than influenza in a series of scenarios. notably, recent contact with an asymptomatic ebola patient, the presence of vomiting and diarrhea, and even travel to south africa swayed many respondents toward ebola as the more likely pathogen. reassuringly, a medscape survey of health professionals found that % ranked influenza as a high threat to public health, while only % had this view of ebola. despite variation among respondents in ebola guideline knowledge, we found that almost all physicians felt at least somewhat prepared to communicate the risk of acquiring ebola and to identify possible cases-especially if they used informational sources other than the lay press or had a possibility of encountering ebola. in addition, most reported a there were no missing data protocol in place at their practice site, though only % of solo practitioners reported such a protocol, and many had personally changed their practice accordingly. these results are consistent with those of the medscape survey, in which % of health professionals reported that they were prepared to treat ebola and % reported confidence in their knowledge of ebola. finally, while few respondents reported a fear of contracting ebola, a greater number reported such a fear among their physician colleagues, and nearly half reported the fear among non-physician staff members-which may reflect differences in education level or the effect of physicians choosing more socially desirable opinions for themselves. overall, despite most respondents reporting that they had reviewed cdc guidelines, our results confirm a disconnect between physicians' confidence in ebola preparedness and their knowledge of appropriate diagnosis and management. . this raises important questions about how to engage doctors in providing appropriate care beyond guideline distribution-perhaps through outreach by professional groups, electronic decision support, or government-mandated training. given the popularity of lay press sources ( table ) , it will also be critical to address limitations in the quality of health journalism. , this investigation had several limitations. we achieved a % response rate, suggesting the possibility of response bias, though demographic characteristics were similar between respondents and non-respondents. physicians who self-selected to participate may have had greater knowledge about ebola or were more comfortable with ebola preparedness activities, so our study may overestimate physician confidence, knowledge, and preparedness. while the study population was representative of membership in the nation's largest internal medicine organization, it may not represent all u.s. internists. we do not know whether physicians would act in real life as they reported in hypothetical scenarios. this is a cross-sectional survey, so we cannot show causality. we acknowledge that the constructs used for possibility of encountering ebola and fear were not externally validated, though they had face validity and were based on literature regarding similar epidemics. future studies might investigate the reasons behind individual management decisions and the cost and clinical impact of delays or compromise of routine care due to misplaced concern about ebola or a comparable outbreak. we demonstrated that knowledge, risk perception, and preparedness are influenced by individual practice experiences and location. as global health threats continue to emerge, improved communication of health risks and appropriate management strategies are needed. contributors: we thank stephen calderwood, md (massachusetts general hospital) for his insightful thoughts on our study approach; steven weinberger, md (american college of physicians) for his guidance and support of our study design; and erika shenoy, md, phd (massachusetts general hospital) for her thoughtful review of our manuscript. no financial or material support was provided for the design or conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval of the manuscript. ebola outbreak in west africa-case counts fear, misinformation, and social media complicate ebola fight. time on fear, distrust, and ebola communicating uncertainty-ebola, public health, and the scientific process ebola and quarantine experts offer steps for avoiding public hysteria, a different contagious threat maple shade parents struggle with ebola fears. the philadelphia inquirer kentucky teacher resigns over parents' dumb ebola fears centers for disease control and prevention. ebola (ebola virus disease clinical inquiries regarding ebola virus disease received by cdc-united states better late than never: a reexamination of ethical dilemmas in coping with severe acute respiratory syndrome the potential ebolainfected patient in the ambulatory care setting: preparing for the worst without compromising care sars risk perceptions in healthcare workers a cross-sectional study of primarycare physicians in singapore on their concerns and preparedness for an avian influenza outbreak survey reveals concerns over ebola. webmd health news the ebola virus outbreak and other emerging infectious diseases. preliminary electronic draft what are the roles and responsibilities of the media in disseminating health information conflict of interest: the authors declare that they do not have a conflict of interest.corresponding author: ishani ganguli, md; massachusetts general hospital, fruit street bulfinch , boston, ma , usa (e-mail: iganguli@partners.org). key: cord- -k hlf authors: sun, dongbo; shi, hongyan; guo, donghua; chen, jianfei; shi, da; zhu, qinghe; zhang, xin; feng, li title: analysis of protein expression changes of the vero e cells infected with classic pedv strain cv by using quantitative proteomic technique date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k hlf recent outbreaks of porcine epidemic diarrhea virus (pedv) have caused widespread concern. the identification of proteins associated with pedv infection might provide insight into pedv pathogenesis and facilitate the development of novel antiviral strategies. we analyzed the differential protein profile of pedv-infected vero e cells using mass spectrometry and an isobaric tag for relative and absolute quantification. a total of proteins were identified that were differentially expressed between the pedv-infected and mock-infected groups (p < . , quantitative ratio ≥ . ), among which the expression of proteins was up-regulated and that of proteins was down-regulated in the pedv-infected vero e cells, involving in integrin β /β , cystatin-c. the gene ontology analysis indicated that the molecular function of the differentially expressed proteins (deps) was primarily related to binding and catalytic activity, and that the biological functions in which the deps are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. among the disease-related functions, certain anti-viral pathways and proteins, such as the rig-i-like receptor, rap , autophagy, mitogen-activated protein kinase, pi k-akt and jak-stat signaling pathways, and integrin β /β and cystatin-c proteins, represented potential factors in pedv infection. our findings provide valuable insight into pedv-vero e cell interactions. the porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded positive-sense rna virus that causes porcine epidemic diarrhea (ped), an acute and highly contagious enteric disease in pigs. ped is characterized by severe diarrhea, vomiting, dehydration, and a mortality rate of up to % in suckling piglets (pensaert and debouck, ) . ped was first reported in belgium and the united kingdom in , and frequent outbreaks have occurred in various asian countries . since , acute ped outbreaks have continually occurred in thailand, china, and the usa, which have resulted in substantial economic losses (puranaveja et al., ; li et al., ; chen et al., ; huang et al., ; marthaler et al., ; stevenson et al., ; yang et al., ; chen et al., ) . the continued outbreaks of ped, despite control efforts, have caused widespread concern. the pedv belongs to the genus alphacoronavirus, in the family coronaviridae and order nidovirales (belouzard et al., ) . previous studies have investigated various control measures to protect against pedv infection, such as vaccines, diagnostic tools, and therapeutic drugs (sun et al., ; ren et al., ; sun et al., ; zhu et al., ; guo et al., ; kim and lee, ) . various aspects of pedv infection remain unclear, for example, swine testis (st) cells expressing porcine aminopeptidase n of pedv receptor were not susceptible to pedv infection. african green monkey kidney (vero) cells are highly susceptible to pedv infection, and are widely used for the primary isolation and cultivation of pedv guo et al., ) . therefore, vero lineages are suitable hosts for understanding the mechanisms of pedv infection. proteomics techniques are effective tools for characterizing protein expression profiles, and have been used widely to investigate disease-associated proteins (hondermarck et al., ; boja et al., ; he et al., ; sun et al., ) . among current proteomics methods, quantitative high-throughput proteomics approaches are useful for the analysis of infection-associated proteins of pathogens (linde et al., ; papachristou et al., ; ye et al., ; zeng et al., ) . in our current study, we used a quantitative proteomics approach based on an itraq tandem mass spectrometry (ms/ms) technique to identify proteins differentially expressed between pedv-infected and mock-infected vero e cells. the functions of the differentially expressed proteins (deps) were analyzed to determine whether they might be associated with pedv infection. our findings provide valuable insight into the changes in cellular processes that occur during pedv infection. the cv strain of pedv, kindly provided by maurice pensaert at ghent university (merelbeke, belgium), was used in all of our experiments after being adapted to vero e cells, as previously described (hofmann and wyler, ) . the vero e cell-adapted pedv, the vero e cells, and the monoclonal antibody against the nucleocapsid protein (np) of pedv were stored at the diarrhea-related viruses section, division of swine infectious diseases, national key laboratory of veterinary biotechnology, harbin veterinary research institute of the chinese academy of agricultural sciences. the vero e cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum (fbs) in -cm flasks at • c in a % co atmosphere. when the cells reached - % confluence, they were inoculated with the pedv at a multiplicity of infection of in presence of g/ml trypsin. at h postinoculation, the cells began to exhibit cytopathic effects (cpes) of viral infection, but no cells lysis or shedding had occurred. the cells were washed three times with cold phosphate-buffered saline (pbs, ph . ). a . -ml aliquot of lysis buffer containing % sds, mm dtt, and mm tris-hcl (ph . ) was added to each flask, and the flasks were incubated at • c for min. the cell lysates were collected using a cell scraper, and boiled for min. three cell lysate replicates were prepared for the pedv-infected (v -v ) and mock-infected (c -c ) vero e cells, and stored at − • c. western blotting was performed to confirm pedv infection by detecting the presence of the np of pedv in the vero e cells. aliquots of the cell lysates were subjected to sds-page on a % acrylamide gel, and the protein bands were transferred to a nitrocellulose membrane using a semi-dry transfer device (bio-rad, hercules, ca, usa). the membrane was blocked using % (w/v) nonfat dried milk in pbs at • c for h, before incubation in pbs containing the anti-np monoclonal antibody ( : dilution) at • c for h. after washing three times with % tween in pbs (pbst), the membrane was incubated in pbst containing a horseradish peroxidase-conjugated goat anti-mouse igg ( : dilution) at • c for h. after washing three times with pbs, the membrane was incubated with enhanced chemiluminescence detection reagents (biotopped, beijing, china) at room temperature for min, and the peroxidase-mediated luminescence was digitally captured using the molecular imager chemidoc xrs+ system (bio-rad) and the image lab software (bio-rad). to verify the differential expression of the selected deps, equivalent volumes of the cell lysate replicates from the pedv-infected (v -v ) and mock-infected (c -c ) vero e cells were pooled into the v and c samples, respectively, and western blotting was performed as described above, with the following exceptions: a : dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤ , anti-cystatin-c, anti-protein s -a , anti-apolipoprotein e , and anti-centrin from rabbit (beijing biosynthesis biotechnology, beijing, china) was used as the primary antibody, and a : dilution of the hrp-conjugated goat anti-rabbit igg (sigma-aldrich, st. louis, usa) was used as the secondary antibody. protein digestion of the samples was performed according to the fasp procedure described by wiśniewski et al. ( ) . an aliquot of each cell lysate containing g of protein was combined with l of std buffer containing % sds, mm dtt, and mm tris-hcl (ph . ). the detergent, dtt, and other low-molecular-weight components were removed by dilution in ua buffer containing m urea and mm tris-hcl (ph . ) and repeated ultrafiltration using microcon ( kda) ultrafiltration units. the reduction of cysteine residues was blocked by the addition of l of . m iodoacetamide to the ua buffer. the samples were incubated for min in darkness before ultrafiltration. the microcon filters were washed three times with l of ua buffer, followed by two washes with l ds buffer containing mm triethyl ammonium bicarbonate (ph . ). the final protein suspensions were digested using g of trypsin (promega, madison, wi, usa) in l of ds buffer overnight at • c, and the digested peptides were collected as the filtrate. the peptide content was quantified based on absorbance at nm using an extinction coefficient of . for a . mg/ml solution. the digested peptide mixture was labeled using the -plex itraq reagent (life technologies, carlsbad, ca, usa), according to the manufacturer's instructions. each itraq reagent was dissolved in l of ethanol, and added to the digested peptide mixture. the samples were labeled as c - , c - , c - , v - , v - , or v - . the samples were multiplexed, and vacuum dried. the itraq labeled peptides were fractionated by scxc using the akta purifier system (ge healthcare, waukesha, wi, usa). the dried peptide mixture was reconstituted, and acidified by the addition of ml of buffer a containing mm kh po in % acetonitrile (ph . ). the samples were loaded onto a . mm × mm column packed with polysulfoethyl ( m, Å) chromatography resin (polylc, columbia, maryland, usa). the peptides were eluted at a flow rate of ml/min using a gradient of - % buffer b containing mm kcl and mm kh po in % acetonitrile (ph . ). the gradient elution consisted of - % buffer b for min, - % buffer b for min, and - % buffer b for min. the absorbance of the eluate was monitored at nm, and fractions were collected at -min intervals. thirty fractions were combined into ten pools, and desalted using empore standard density spe c cartridges (sigma-aldrich, st. louis, mo, usa) with a bed diameter of mm and a volume ml. each fraction was concentrated by centrifugation in a vacuum, and reconstituted in l of . % (v/v) trifluoroacetic acid. all samples were stored at − • c until the ms analysis was performed. the lc-ms/ms experiments were performed using a q exactive mass spectrometer coupled to a proxeon biosystem easy nanolc (thermo fisher scientific, waltham, ma, usa). ten microliters of each fraction was injected for nanolc-ms/ms analysis. the peptide mixture ( g) was loaded onto a c -reversed phase column ( cm × m) packed with rp-c ( m) resin in buffer a containing . % formic acid, and eluted with a linear gradient of buffer b ( % acetonitrile and . % formic acid) at a flow rate of . l/min for min using the intelliflow technology. the eluate underwent electrospray ionization for the ms/ms analysis. the ms/ms instrument was run in the peptide recognition mode, and the spectra were acquired using a data-dependent top- method based on the selection of the most abundant precursor ions from the survey scan ( - m/z) for hcd fragmentation. the determination of the target value was based on the predictive automatic gain control, and the dynamic exclusion duration was s. survey scans were acquired at a resolution of , at m/z , and the resolution for the hcd spectra was set to , at m/z . the normalized collision energy was ev, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as . %. the ms/ms spectra were compared to the uniprot cercopithecidae database ( sequences, downloaded november , ) and a decoy database using the mascot search engine, version . (matrix science, london, uk), embedded in the proteome discoverer . software (thermo electron, san jose, ca). the following parameters were used for protein identification: a peptide mass tolerance of ppm; an ms/ms tolerance of . da; trypsin digestion; a missed cleavage value of ; the fixed modifications included carbamidomethyl, itraq plex(k), and itraq plex(n-term); the variable modification was oxidation; and an fdr value ≤ . . protein quantification was performed using the proteome discoverer . software based on the centroided reporter ion peak intensity. the average quantitative value of each protein in samples c , c , and c (mock-infection group) was used as the internal reference. the value of the quantitative ratio for each protein relative to the internal reference was calculated, and averaged to obtain the quantitative ratio (v/c) of the proteins identified in the treatment groups (unwin et al., ) . a protein was considered to be differentially expressed between the pedv-infected and mock-infected groups based on the following criteria: the protein had to be present in three replicates of both groups, the difference in the level of the protein between the two groups had to be statistically significant (p < . ), and the change ratio for the protein had to be ≥ . (yuan et al., ) . the expression of a protein with a v/c > . was considered to be up-regulated, and those with a v/c < . were considered to be down-regulated. the data were analyzed using a two-tailed, paired student's t test. the statistical analysis was performed using the excel software (microsoft, redmond, wa, usa). the deps were annotated using the blast go, version . . , program (ashburner et al., ; quevillon et al., ; götz et al., ) . the deps were blasted against the kegg genes database (human). the gene ontology categories (gocs) were retrieved, and mapped to pathways in the kegg database (kanehisa et al., ) . table the proteins identified from pedv-infected and mock-infected groups. the vero e cells inoculated with pedv displayed distinct cpes at h postinoculation, including cell shrinkage, cell fusion, and a rounded cell morphology, but no cells lysis or shedding was observed (fig. a) . the immunoblotting analysis confirmed that the vero e cells were pedv-infected. the band corresponding to the np of pedv was detected in samples v , v , and v , whereas none was detected in samples c , c , and c (fig. b) . the identified peptides, identified proteins, quantified proteins, known/uncharacterized proteins, and the goc annotations are showed in table . a total of proteins, including peptides, were identified in the pedv-infected and mock-infected groups using the itraq-ms/ms approach, among which ( . %) were quantified, ( . %) were known proteins, and ( . %) were uncharacterized/putative proteins. based on the gocs, ( . %) of the proteins were annotated as biological process, ( . %) were annotated as molecular function, and ( . %) were annotated as cellular components. the quantification and significance of the identified proteins are shown in fig. . the changes in the levels of expression between the two groups were analyzed based on statistical significance. of the proteins identified, ( . %) were not differentially expressed (p > . ), and ( . %) were expressed at statistically different levels between the pedv-infected and mockinfected vero e cells (p < . ), including proteins ( . %) with a p-value between . and . , proteins ( . %) with a p-value between . and . , and proteins ( . %) with a p-value < . . the proteins with a p-value < . were also filtered based on whether the v/c or c/v was ≥ . . based on these criteria, a total of ( . %) of the identified proteins were determined to have been differentially expressed between the pedv-infected and mock-infected groups (table ) . among the deps, . % ( / ) were up-regulated, and . % ( / ) down-regulated. the known proteins and uncharacterized/putative proteins accounted for . % ( / ) and . % ( / ) of the deps, respectively. the dep displaying the greatest increase in expression in the pedv-infected vero e cells was isoform of the ovarian cancer immunoreactive antigen domaincontaining protein ( : . ), and the dep displaying the greatest decrease in expression in the pedv-infected vero e cells was cystatin-c ( : . ). the gene ontology (go) database has been widely used for describing protein function in a standardized format. according to their gocs, the deps were annotated as cellular component, biological process, or molecular function. the go annotations are shown in table , and distributions of the go annotations are shown in fig. . seventy-eight deps were distributed among groups of biological processes (fig. a) . the metabolic process (go: ), cellular process (go: ), single-organism process (go: ), and biological regulation (go: ) groups contained the highest proportions of the biological process deps. there were more up-regulated proteins in the cellular component organization group (go: ) than down-regulated proteins. seventy-four deps were distributed among eight cellular component groups (fig. b) , among which the organelle (go: ) and cell (go: ) groups contained the highest proportion of cellular component deps. there were more down-regulated deps in the membrane group (go: ) than up-regulated deps, and there were more up-regulated deps in the macromolecular complex group (go: ) than downregulated deps. ninety-seven deps were distributed among eight molecular function groups (fig. c) , among which the binding (go: ) and catalytic activity (go: ) groups contained the greatest proportion of molecular function deps. the kyoto encyclopedia of genes and genomes (kegg) pathway is a collection of pathway maps that represent molecular interactions and reaction networks in cells. seventy-five of the deps identified were annotated, and mapped to a total of six kegg pathway categories, which included the metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases pathway categories (fig. ) . the annotations in the metabolism, organismal systems, and diseases pathway categories represented , , and pathway groups, respectively (fig. a, b and f). the annotations in metabolism pathways category included the carbohydrate, energy, lipid, nucleotide, amino acid, glycan biosynthesis, cofactors and vitamins, biosynthesis of other secondary metabolites, and xenobiotics pathway groups (fig. a ). the annotations in the organismal systems category included the tolllike receptor (tlr) signaling (ko ), rig-i-like receptor (rlr) signaling (ko ), and natural killer cell mediated cytotoxicity (ko ) pathway groups (fig. b) , which represent pathways related primarily to the immune response to virus infection. the largest number of deps in the cellular process category were mapped to the lysosome (ko ) pathway group, all ten of which were down-regulated deps (fig. c ). the annotations in the genetic information processing category included pathway groups related to dna replication and repair, transcription, translation, and the folding, sorting, and degradation of proteins (fig. d ). the annotations in the environmental information processing proteins and one up-regulated protein. overall, more disease pathway groups were assigned to a single down-regulated dep than those assigned to up-regulated deps. the integrin (␤ and ␤ subunits) protein was annotated to the largest number of pathway groups ( ), which included the organismal systems, environmental information processing, cellular processes, and diseases categories. the ␤ tubulin as loading control, three down-regulated deps cystatin-c, apolipoprotein e and centrin- , two up-regulated deps integrin-␤ and protein s -a , were selected to verify differential expression between the pedv-infected and mock-infected vero e cells. the immunoblotting analysis showed that the ratios of these proteins between the pedv-infected and mock-infected groups were consistent with those obtained using the quantitative proteomics analysis (fig. ) . in our study, pedv infection significantly alters protein expression in vero e cells. the differentially expressed proteins (deps) annotated to virus infection-associated signaling pathways, autophagy, and virus entry-associated proteins were analyzed further to assess their potential roles in pedv infection. in mammals, the first line of defense against virus infection is the innate immune system. early antiviral responses are initiated upon the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs), resulting in the production of interferons for the innate immune response and the maturation of dendritic cells for establishing acquired immunity (yokota et al., ) . the prrs are grouped into the tlrs, rlrs, and nucleotide binding-oligomerization domain-like receptors. our results showed that pedv infection induced the deps that participated in six signaling pathways involved in viral infection, including the rlr, rap , pi k-akt, mapk, jak-stat, and tlr signaling pathways. the pedv is an enteric virus that infects the intestinal epithelial cells (iec) of swine, causing severe diarrhea. hirata et al. ( ) reported the rig-i signaling pathway plays an important role in antiviral innate immunity mechanisms in iecs. sheikh et al. ( ) reported the rap a signaling pathway was associated with secretory diarrhea. the jak-stat signaling pathway regulates the adaptive and innate mechanisms related to mucosal immunity (heneghan et al., ; wang et al., ) . our results showed that deps induced by pedv infection in vero e cells involved in the rlr, rap , and jak-stat signaling pathways. it has been reported that the tlr, mapk, and pi k-akt signaling pathways play roles in host cell responses to coronaviruses (mizutani et al., ; integrins are cell surface ␣/␤ heterodimeric glycoproteins that contribute to a variety of cellular functions (stewart and nemerow, ) . combinations of the various isotypes of the ␣ and ␤ subunits of integrins generate more than different integrin proteins. previous studies have shown that various integrin molecules are used as receptors for virus attachment (stewart and nemerow, ; sun et al., ) . in our current study, the expression of integrin-␤ and -␤ was down-regulated and up-regulated, respectively, in response to pedv infection. the upregulation of integrin-␤ expression is consistent with that observed in response to dengue virus infection (zhang et al., ) . our pathway analysis revealed that both integrin-␤ and -␤ are involved in pathways that contribute to organismal systems, environmental information processing, cellular processes, and diseases. the integrin ␣v␤ protein has been shown to serve as an entry receptor for various viruses (guerrero et al., ; neff et al., ; chu and ng, ; parry et al., ; wang et al., ) , some of which bind the integrin through an rgd sequence in a viral structural protein to initiate infection (stewart and nemerow, ) . the s protein of pedv is a glycoprotein peplomer on the viral surface that plays an important role in receptor-mediated binding and cell membrane fusion. in our study, the integrin recognized sequences of pedv s protein was analyzed based on ruoslahti's ( ) report. the results indicated that four conserved integrin-recognized amino acid motifs (asp-gly-glu, lys-gly-glu, arg-leu-asp, and leu-asp-val) were found in the s proteins of various pedv strains (data not shown). these data suggest that integrin proteins may act as an infection associated protein for the attachment and entry of pedv. autophagy is an essential component of host defenses against viral infection (dong and levine, ) . maier and britton ( ) reported that ␤-coronaviruses induced autophagy. in our study, more deps were mapped to the autophagy pathway group than any of the other pathway groups. fifteen deps were mapped to the lysosome and phagosome pathways. of the proteins, ( %) were down-regulated deps. although the autophagy pathway plays an antiviral role in virus-infected cells, the autophagy machinery is exploited by certain viruses for viral evasion and propagation. our results showed that pedv infection induced the downregulation of the expression of many autophagy-associated proteins. therefore, pedv infection might inhibit autophagy in vero e cells, thus facilitating virus replication. previous studies have shown that the microtubule-associated protein b is a useful biomarker protein for autophagy (dong and levine, ) . we found that the expression of map b was up-regulated . -fold in the pedv-infected vero e cells. these results suggest that the pedv induces autophagy. cystatin-c has been shown to reduce the replication of certain viruses, including the poliovirus, rhinovirus, and human coronaviruses oc and e (korant et al., ; collins and grubb, ) . the cleavage of s protein has been shown to be essential for the induction of cell-to-cell fusion and coronavirus entry into cells (sturman et al., ) . shirato et al. ( ) reported the transmembrane type ii serine protease enhanced infection of pedv in vero cells by increasing virus release. in our study, the reduced expression of cystatin-c might facilitate pedv replication and release through the activation of cysteine-associated proteases in vero e cells. apolipoprotein e , galectin, clusterin, and transferrin receptor have also been shown to be associated with virus infection (hishiki et al., ; peng et al., ; martin and uprichard, ; tripathi et al., ) , and may therefore function as infectionassociated proteins in pedv-infected vero e cells. additionally, the decreased in vitro expression of the adherens junction protein, such as cadherin, might be associated with a reduced integrity of pedv-infected intestinal epithelial cells in vivo. to the best of our knowledge, our study represents the analysis of the interactions between pedv and vero e cells using a quantitative proteomics technique. pedv infection-associated pathways and proteins are described and discussed based on the bioinformatics analysis of the differentially expressed proteins. our analysis of vero e cell responses to pedv infection identified relevant targets for subsequent in-depth studies of pedv pathogenesis, expand the current knowledge base regarding the interaction between the pedv and the host cell, and provide useful basic information about other coronaviruses. although the vero e cells are highly susceptible to pedv infection and facilitate experimental design and performance for proteomics, the vero e cell line is an interferondeficient cell line and not a pig cell line. so, the detailed functions of these pathways and proteins in pedv infection require further verification in the actual host cells of pedv. gene ontology: tool for the unification of biology mechanisms of coronavirus cell entry mediated by the viral spike protein evolution of clinical proteomics and its role in medicine detection and molecular diversity of spike gene of porcine 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two novel b cell epitopes on porcine epidemic diarrhea virus spike protein influenza a virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein clusterin simultaneous analysis of relative protein expression levels across multiple samples using itraq isobaric tags with d nano lc-ms/ms porcine reproductive and respiratory syndrome virus nsp ␤ inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-␣ degradation integrin alphavbeta is a coreceptor for human cytomegalovirus universal sample preparation method for proteome analysis genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from quantitative proteomics by amino acid labeling in foot-and-mouth disease virus (fmdv)-infected cells the battle between virus and host: modulation of toll-like receptor signaling pathways by virus infection cytotoxicity evaluation of oxidized single-walled carbon nanotubes and graphene oxide on human hepatoma hepg cells: an itraq-coupled d lc-ms/ms proteome analysis proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells up-regulated expression of beta integrin induced by dengue virus serotype infection associated with virus entry into human dermal microvascular endothelial cells expression and purification of the scfv from hybridoma cells secreting a monoclonal antibody against s protein of pedv this work is supported by the national natural science foundation of china (grant no. ), the state national key laboratory of veterinary biotechnology (grant no. sklvbf / ), and the program for new century excellent talents in heilongjiang provincial university (grant no. -ncet- ). key: cord- -honeavwj authors: mair-jenkins, john; saavedra-campos, maria; baillie, j. kenneth; cleary, paul; khaw, fu-meng; lim, wei shen; makki, sophia; rooney, kevin d.; beck, charles r.; nguyen-van-tam, jonathan s. title: the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis date: - - journal: j infect dis doi: . /infdis/jiu sha: doc_id: cord_uid: honeavwj background. administration of convalescent plasma, serum, or hyperimmune immunoglobulin may be of clinical benefit for treatment of severe acute respiratory infections (saris) of viral etiology. we conducted a systematic review and exploratory meta-analysis to assess the overall evidence. methods. healthcare databases and sources of grey literature were searched in july . all records were screened against the protocol eligibility criteria, using a -stage process. data extraction and risk of bias assessments were undertaken. results. we identified studies of sars coronavirus infection and severe influenza. narrative analyses revealed consistent evidence for a reduction in mortality, especially when convalescent plasma is administered early after symptom onset. exploratory post hoc meta-analysis showed a statistically significant reduction in the pooled odds of mortality following treatment, compared with placebo or no therapy (odds ratio, . ; % confidence interval, . –. ; i( ) = %). studies were commonly of low or very low quality, lacked control groups, and at moderate or high risk of bias. sources of clinical and methodological heterogeneity were identified. conclusions. convalescent plasma may reduce mortality and appears safe. this therapy should be studied within the context of a well-designed clinical trial or other formal evaluation, including for treatment of middle east respiratory syndrome coronavirus cov infection. as of may , the world health organization (who) had been informed of persons with laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) infection, of whom ( %) have died [ ] . the current approach to clinical management of mers-cov infection centers on general supportive care, with provision of critical care and organ support when necessary [ ] . it has recently been suggested that administration of convalescent plasma or hyperimmune immunoglobulin will yield a clinical effect for treatment of mers-cov infection [ ] . however, numerous uncertainties remain because the clinical course, viral replication kinetics, and host interactions are yet to be fully established [ ] . furthermore, the underlying evidence is based on studies of varying size and quality that describe clinical experience in treating other viral infections, including those due to sars coronavirus (sars-cov), spanish influenza a(h n ), avian influenza a(h n ), and pandemic influenza a (h n ) (hereafter, "influenza a[h n ]pdm ") [ ] [ ] [ ] [ ] [ ] . we conducted a systematic review and exploratory meta-analysis to evaluate the clinical effectiveness of convalescent plasma, serum, or hyperimmune immunoglobulin for the treatment of severe acute respiratory infections (saris) of viral etiology, to help inform clinical management of mers-cov infection. this systematic review was conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines [ ] . the study protocol was registered with the national institute for health research international prospective register of systematic reviews [ ] . the study eligibility criteria are available elsewhere [ ] . briefly, the study population of interest was human subjects of any age or sex who were hospitalized with saris with a laboratory-confirmed or suspected viral etiology. the intervention of interest was convalescent plasma, serum, or hyperimmune immunoglobulin derived from convalescent plasma. comparator treatments included placebo, sham therapy, or no intervention; studies with no comparator group were also included. outcome measures were derived from the protocol research questions to ascertain the clinical effectiveness of therapy [ ] . two reviewers (j. m.-j. and m. s.-c.) executed the search strategy in july . the sources of information searched and search construct are available elsewhere [ ] . adaptations were made for search interfaces that did not allow use of complex constructs. all search records were imported to endnote x software (thomson reuters, san francisco, ca) or screened manually, using paper records. following the removal of duplicate entries, a -stage screening process was followed to identify eligible records through the sequential examination of each title, abstract, and full text. two reviewers (j. m.-j. and m. s.-c.) screened each record, with provision for arbitration from a third reviewer (c. r. b.). data were collected independently by paired reviewers, using a piloted form. consensus agreement for each extracted data item was reached by discussion, with provision for arbitration from a third reviewer (j. m.-j., m. s.-c., and c. r. b.). the data extraction form is available as an appendix to the study protocol [ ] . risk of bias assessments were performed at the outcome measure level during data collection. the cochrane collaboration tool was used for experimental and prospective cohort studies [ ] , the newcastle-ottawa scale was used for observational studies (excluding prospective cohort studies) [ ] , and a tool published by the us agency for healthcare research and quality was used for systematic reviews [ ] . records limited to abstracts were not assessed, because of the paucity of information contained therein. odds ratios (ors), case-fatality rates (cfrs), absolute differences in cfrs, and difference in means were calculated as summary statistics with % confidence intervals (cis). study characteristics and outcome measures were tabulated. a recognized framework for narrative synthesis was adopted [ ] . because of potential concerns with clinical heterogeneity, analyses were stratified by viral etiology for each research question in accordance with the protocol [ ] . an exploratory, post hoc, random-effects-model metaanalysis was conducted to describe the pooled or of mortality, irrespective of sari etiology, following treatment with convalescent plasma or serum, using the odds after receipt of placebo or no therapy as a reference. results were adjusted by adding . to each cell of the contingency table when no deaths occurred in the exposed group in individual studies [ ] . meta-analysis of crude cfrs, using a random-effects model, was undertaken. statistical heterogeneity was ascertained using the i statistic, and meta-analyses were abandoned when this reached % [ ] . sensitivity analyses were undertaken to investigate the impact of excluding studies with ≤ patients in the exposed group. publication bias was assessed through construction of funnel plots and by use of the egger test. all statistical analyses were conducted using stata software, version . (statacorp, college station, tx), except for meta-analysis of pooled proportions, for which we used statsdirect software, version . . (statsdirect, altrincham, united kingdom). statistical significance was assumed at the % level. the search process yielded records ( figure ). after sifting unique records against the protocol eligibility criteria, we identified studies from reports (supplementary table ). three studies could not be obtained [ ] [ ] [ ] , although results from a study by bass et al [ ] were reported elsewhere [ ] , which enabled their inclusion. french (n = ), german (n = ), and korean (n = ) records were screened by single reviewers because of a lack of multilingual collaborators. the study characteristics are summarized in supplementary table . three systematic reviews met our protocol eligibility criteria [ , , ] . data on patients from case studies [ ] [ ] [ ] [ ] [ ] [ ] , case series [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , case-comparison studies [ , ] , and prospective cohort [ ] were included. we identified observational studies published between and , which studied patients who received a clinical diagnosis of influenza-associated pneumonia or spanish influenza a (h n ) infection [ , , - , - , ] . it is unclear whether some of these studies recruited patients with secondary bacterial pneumonia. sixteen observational studies that met our protocol eligibility criteria were published between and . four studies reported outcomes for patients infected with avian influenza a(h n ) [ , , , ] , studies reported outcomes for patients infected with influenza a (h n )pdm [ , , , ] , and studies reported outcomes for patients with sars [ , , , , , , , ] . the clinical status of patients at the time of treatment administration varied, as did concomitant treatments and comorbidities. convalescent plasma was used in all observational studies of sars-cov, influenza a(h n )pdm , and avian influenza a(h n ) infections (supplementary table ). for spanish influenza a(h n ) infection, observational studies used convalescent plasma, and used convalescent serum (supplementary table ). no studies that used hyperimmune immunoglobulin met our protocol eligibility criteria. the use of sham treatments or placebos was not reported. two systematic reviews were at low risk of bias [ , ] , whereas one was at moderate to low risk of bias across most domains ( table ) [ ] . data extraction was judged to be a moderate source of bias in all systematic reviews. search strategies were also a moderate source of bias in systematic reviews, as grey literature and non-peer-reviewed sources were not considered [ , ] . the risks of bias of outcomes in a single prospective cohort study were considered to be moderate ( table ) [ ] . the lack of randomized treatment allocation may have introduced systematic error, and the viral load outcome was at high risk of bias because of incomplete follow-up of patients. figure summarizes the risk of bias assessments for outcomes from observational studies. studies reported outcomes that were either at moderate risk ( outcomes) or moderate to high risk of selection bias ( outcomes). the majority of studies lacked a comparator group, and studies were at high or very high risk of reporting bias. this suggests that the observational study data included are at moderate to high risk of bias. three studies were not assessed for risk of bias, because they presented insufficient data [ , , ] . table summarizes our narrative synthesis, and supplementary table shows results of the individual studies that included an all-cause mortality outcome. meta-analyses, sensitivity analyses, and assessments of publication bias, by viral etiology, proved unfeasible due to a paucity of suitable data. there were no data available to address study questions relating to organ failure and sepsis or to hospital readmission and recurrence of severe disease. table and supplementary summarize observational studies at moderate to high risk of bias that reported improved mortality after patients received various doses of convalescent plasma [ , , , , , , , ] . a retrospective case-comparison study showed a cfr reduction after plasma treatment that reached statistical significance (absolute reduction in cfr, %; % ci, %- %; p = . ) [ ] . a second study with a comparator group described a cluster of cases of sars-cov infection in which patient received convalescent plasma and survived (absolute reduction in cfr, %; % ci, − % to %; p = . ) [ , ] . three small studies reported treatment of patients with no deaths, and a case series by cheng et al reported a cfr of . % ( of patients) following treatment (supplementary table ) [ , , , , , , ] . within this series, a subgroup analysis of patients found that those treated when pcr-positive but seronegative for sars-cov were more likely to be discharged within days of admission than those who were seropositive at the time of plasma infusion ( % vs %; p = . ). a further subgroup analysis of patients found that receipt of convalescent plasma treatment < days after onset of symptoms improved the likelihood of discharge within days of admission ( % vs %; p < . ); this remained significant after adjustment for age, viral status, time of administration, and lactate dehydrogenase level, suggesting that early treatment with convalescent plasma may be beneficial. however, allocation of treatment was mostly based on the physician's decision and the availability of plasma, and this study was at high risk of bias. four observational studies [ , , , ] and systematic review [ ] reported data on severe cases of influenza a(h n )pdm infection treated with convalescent plasma (table and supplementary table ). hung et al [ ] performed a prospective cohort study in which patients received a single -ml dose of convalescent plasma with a neutralizing antibody titer of > : . univariate analysis showed a significant absolute reduction in cfr of % ( % ci, %- %; p = . ) after treatment. multivariable analysis also showed a significant reduction in the relative risk of mortality (or, . ; % ci, . -. ; p = . ), although the factors adjusted for were not clearly stated. both groups received other treatments, such as neuraminidase inhibitors and steroids (supplementary table ). this nonrandomized study was at moderate risk of bias. a small study by chan et al [ ] at moderate risk of bias reported exclusively on patients who received extracorporeal membrane oxygenation (ecmo) and showed a nonsignificant absolute reduction of % ( % ci, − % to %) in the cfr after convalescent plasma treatment. in a case series at high risk of bias, in which of patients receiving convalescent plasma, a nonsignificant absolute reduction of % ( % ci, %- %; p = . ) in the cfr was observed (supplementary table ) [ ] . three case reports reported recovery among patients who were treated with convalescent plasma [ , , ] . the dose of convalescent plasma varied across each study, and the neutralizing antibody titer was reported for only case ( : ) [ ] . all studies were at high to moderate risk of bias and had patients who were given other therapies concomitantly (including steroids and antivirals), which could have influenced the reported clinical effect. a systematic review and meta-analysis by luke et al [ ] showed that treatment with convalescent plasma, serum, or blood was associated with a significant absolute reduction of % ( % ci, %- %) in the pooled cfr. statistical heterogeneity was low (i = . %), although interventions were clinically heterogeneous. of the studies included in the meta-analysis, reported use of convalescent whole blood; however, these studies only contributed patients ( %) in the treatment group. when timing of treatment was recorded, patients who received early treatment (< days from pneumonia onset) had a cfr of % ( of ), compared with % ( of ) for those treated later [ ] . only studies of convalescent serum reported a comparator group [ , ] . both reported absolute reductions in cfr after treatment, with a reduction of % ( % ci, %- %) in one and % ( % ci, %- %) in the other; the reduction in the latter reached statistical significance (p = . ). the remaining studies observed a cfr ranging from % ( of ) to % ( of ) after treatment (supplementary table ) . a significant absolute reduction in the cfr was observed in a case series of cases, of whom received convalescent plasma (absolute reduction in the cfr, %; % ci, % to %; p = . ) [ ] . a further study of patients treated with convalescent plasma reported a cfr of % ( of ) [ ] . the majority of studies on spanish influenza a(h n ) infection were found to have high risk of bias due to the use of now archaic research methods and a risk of wartime censorship and publication bias [ ] . the post hoc meta-analysis evaluated pooled data from comparative studies: studies of sars-cov infection [ , ] , of influenza a(h n )pdm infection [ , ] , of avian influenza a(h n ) infection [ ] , and of spanish influenza a(h n ) infection [ , , ] . there was a statistically significance lower risk of mortality in the group treated with convalescent plasma or serum ( pooled or, . ; % ci, . to . ; p < . ; i = %; figure ). examination of the funnel plot and findings of the egger test showed no evidence of publication bias. sensitivity analyses that excluded studies with ≤ cases demonstrated little variation in the pooled or or change in statistical heterogeneity (figure ) . meta-analysis of the crude cfr in treated patients was rejected due to excessive statistical heterogeneity (i = %). sensitivity analysis that excluded studies with ≤ cases did not account for this and was similarly abandoned (i = %). convalescent plasma treatment was associated with a significant increase in the proportion of sars-cov-infected patients discharged within days of admission in center (absolute difference, %; % ci, %- %; p = . ) after excluding patients with comorbidities from the analysis (table ) [ ] . a further sars-cov infection case series [ ] reported that % of patients ( of ) were discharged by day , and initiation of therapy was significantly earlier among patients discharged by that time (mean number of days from symptom onset, . vs . ; p < . ). both studies were at moderate to high risk of selection bias and confounding by indication. a case-comparison study at moderate risk of bias [ ] reported no significant difference in length of hospital stay between treatment and control patients with severe pandemic influenza a (h n ) infection who required ecmo ( table ) . a retrospective observational study [ ] reported that convalescent plasma treatment made nonsignificant reductions to the length of time spent in the intensive care unit, days of no data were reported in identified studies. significantly lower viral load after treatment was observed at days , , and after icu admission in subgroup analysis of prospective study, which was at moderate to high risk of bias. one noncomparative study found a reduction in viral load after treatment. no adverse events or complications were reported after treatment. nonsignificant benefits following intervention were reported in study with comparator data. three case reports reported no deaths. no comparative data were reported. the length of hospital stay was d in a case report at high risk of bias. no comparative data were reported. one case report, which had a high risk of bias, cited that treatment allowed discontinuation of mechanical ventilation. specific antibodies were detected between day and day after treatment in a case report at high risk of bias. no comparative data were reported. three studies reported reductions in viral load after treatment. no adverse events or complications were reported after treatment. a pooled absolute reduction of % ( % ci, %- %)in the cfr was reported by a meta-analysis at low risk of bias. this pooled studies, including studies using convalescent blood. subgroup analyses suggested that early treatment was beneficial. the absolute reduction in the risk of mortality ranged from . % ( % ci, . %- . %) to . % ( % ci, . %- . %) in studies at high risk of bias. ten noncomparative studies found that the cfr varied from % ( / ) to % ( / ) . no data were reported in identified studies. no data were reported in identified studies. no data were reported in identified studies. no data were reported in identified studies. three studies reported chills, increased temperature, and sweats after infusion. abbreviations: cfr, case-fatality rate; ci, confidence interval; ecmo, extracorporeal membrane oxygenation; icu, intensive care unit; influenza a(h n )pdm , pandemic influenza a(h n ); sars-cov, severe acute respiratory syndrome coronavirus. a all studies reported use of convalescent plasma, except studies, in which convalescent serum was used to treat spanish influenza a(h n ) infection, and meta-analysis of studies, of which reported use of convalescent blood to treat spanish influenza a(h n ) infection. additional data pertaining to individual studies (including comparator data, where presented) are available in the supplementary materials. mechanical ventilation, or number of days of ecmo for patients with severe pandemic influenza a (h n )pdm infection (table ) . two other case reports of pandemic influenza a (h n )pdm infection [ ] and avian influenza a(h n ) infection [ ] also suggested that convalescent plasma may have aided clinical improvement and reduced the duration of mechanical ventilation. we identified limited evidence relating to levels of viral antibodies after convalescent plasma treatment; studies did not use a comparator and were at high risk of bias. peaks in sars-cov antibody levels occurred - days following receipt of a single dose of convalescent plasma in healthcare workers (table ) [ ] . however, it is likely that other treatments, such as intravenous immunoglobulin, ribavirin and steroids, may have influenced the relationship between plasma and antibody levels. a case report of a patient with avian influenza a(h n ) infection also found that virus-specific antibodies appeared - days following administration of convalescent plasma [ ] . the sars-cov load in the respiratory tract decreased at a higher rate in patients who received convalescent plasma in a subgroup analysis of patients with influenza a(h n )pdm infection in a prospective cohort study (table ) ; [ ] viral loads were significantly lower , , and days after intensive care unit admission. however, there was a high risk of selection bias for this outcome, and concomitant treatments, including oseltamivir, zanamivir, and corticosteroids, may have confounded the results. further studies reported that viral load became rapidly undetectable in the blood of patients with sars-cov infection [ ] and in respiratory tract specimens from a patient infected with influenza a(h n )pdm [ ] after treatment. similar decreases in viral loads in serum and respiratory tract specimens were observed in cases of avian influenza a(h n ) infection, with virus becoming undetectable - days after initiation of convalescent plasma treatment for cases and - days after treatment initiation for the third case (supplementary table ) [ , , ] . no studies reported a serious adverse event, and few studies reported information about treatment complications, although minor complications may be underreported in the literature. two observational studies [ , ] concerned with sars-cov infection reported that treatments did not cause harm when administered to patients. one study involving influenza a(h n ) pdm infection reported that no adverse events were observed in the treatment group [ ] . three studies from - (involving - patients with influenza) reported minor infusion complications, including chills, increased temperature [ , ] , and sweats [ ] . a study of patients did not report chills or any serious complications. the methods and reporting of these studies reflect the period during which they were conducted, and the studies are therefore at high risk of bias. our analyses suggest that convalescent plasma may have a clinically relevant impact in reducing the rate of mortality and viral load in patients with sari of viral etiology. post hoc pooled meta-analysis across all viral etiologies showed a statistically significant % reduction in the odds of mortality among those who were treated with convalescent plasma or serum. we found no evidence of serious adverse events or complications due to therapy and limited evidence of a reduction in the use of critical care resources and the length of hospital stay. of interest is the evidence for a survival benefit after early administration. a recent multicenter, prospective, double-blind, randomized control trial compared the use of hyperimmune immunoglobulin (derived from influenza a(h n )pdm convalescent plasma) to intravenous immunoglobulin manufactured before the pandemic [ ] . for patients from this study who received treatment within days of symptom onset and were excluded per protocol, a multivariate subgroup analysis demonstrated that hyperimmune immunoglobulin had a protective effect (or, . ; % ci, . -. ) [ ] . evidence from studies of sars-cov infection [ ] and spanish influenza a(h n ) infection [ ] showed a survival benefit following convalescent plasma treatment within days and days of symptom onset, respectively. these findings suggest that early initiation of treatment may be of critical importance to reducing mortality in patients with sari of viral etiology. a lack of high-quality studies and a paucity in the volume of relevant literature limited our analyses. observational studies were predominately case reports or series, had no control groups, and had a moderate to high risk of bias. findings were commonly at high risk of confounding by indication. although selection or reporting bias may favor the intervention, recruiting patients who are clinically deteriorating or moribund would bias the result in the opposite direction. adequate methodological or statistical measures were infrequently used to control bias and confounding, and we identified numerous sources of clinical and methodological heterogeneity. we cannot be assured that all spanish influenza a(h n ) infection studies were included since our protocol did not include hand searching of literature from - . although our post hoc metaanalyses were undertaken to help inform clinical decision making, the theoretical rationale for pooling mortality data from different viral etiologies remains to be fully established. the results obtained must be considered experimental and interpreted with an appropriate level of caution. we did not identify any reports of convalescent plasma use for patients with mers-cov infection. the evidence for a reduction in mortality associated with convalescent plasma is strongest for sars and influenza a(h n )pdm infection. although it is clinically rational to consider novel therapies for critically ill patients, there is evidence that maximum benefit from convalescent plasma might be realized through early initiation of therapy. however, many treatment protocols currently mention convalescent plasma as a treatment of last resort. if this treatment is considered for mers-cov-infected patients with sari, it should ideally only be administered in acute centers able to manage potential treatment-related complications, such as transfusionrelated acute lung injury. we consider this a precautionary approach because of the limited clinical experience of administering convalescent plasma to this patient group. improved knowledge regarding the mode of action of convalescent plasma and the virologic and immunologic kinetics of novel respiratory infections that cause sari (such as mers-cov) are needed. this would help clarify the potential benefits and harms of treatment, identify optimal dosage, and ascertain whether repeated treatments are relevant factors for clinical practice. randomized controlled trials or observational studies that adopt a standardized minimum data set are needed to better evaluate convalescent plasma as a therapeutic option for mers-cov infection before it can be fully recommended or before refinements can be made over its current use, other than our current recommendation for early use. the who and the international severe acute respiratory and emerging infection consortium are currently developing a clinical trial protocol to investigate the effectiveness of passive immunotherapy for patients with sari. available evidence suggests that convalescent plasma is likely to reduce mortality during saris of viral etiology, with larger treatment effects if it commenced early after symptom onset. however, this is based on predominately low-quality, uncontrolled studies. our review supports the use of convalescent plasma in critically ill mers-cov-infected patients as part of a well-designed clinical trial or other formal evaluation. we thank the following reviewers from the convalescent plasma study group, who evaluated non-english-language articles on the basis of protocol eligibility criteria and undertook risk of bias assessments and data extraction: dr ana l. p. mateus supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. middle east respiratory syndrome coronavirus (mers-cov)-update clinical management of severe acute respiratory infections when novel coronavirus is suspected: what to do and what not to do state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized 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authors alone are responsible for the views expressed in this article, which do not necessarily represent the views, decisions, or policies of the institutions with which the authors are affiliated. the funder had no role in study design, data collection, analysis, or interpretation of the results; preparation of the manuscript; or decision to publish.financial support. this work was supported by the who pandemic and epidemic diseases department.potential conflicts of interest. w. s. l. has received funding from the national institute for health research to set up a pandemic influenza clinical trial and has received unrestricted funding from pfizer for a study in adult pneumonia. the university of nottingham health protection research group (with which j. s. n.-v.-t. and c. r. b. are affiliated) is an official who collaborating center for pandemic influenza and research and receives limited funding from the who in support for specific activities. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -rttaoiwt authors: hang, jian; li, yuguo; ching, w.h.; wei, jianjian; jin, ruiqiu; liu, li; xie, xiaojian title: potential airborne transmission between two isolation cubicles through a shared anteroom date: - - journal: build environ doi: . /j.buildenv. . . sha: doc_id: cord_uid: rttaoiwt full-scale experiments and cfd simulations were performed to study potential inter-cubicle airborne transmissions through a shared anteroom due to the hinged door opening. when doors are closed, current negative pressure designs are effective for the containment of airborne pathogens in the 'dirty' cubicle with an index patient. when the 'dirty' cubicle door is open, airborne agents can move into the other 'clean' cubicle via the shared anteroom. as the door being opened or closed, the door sweeping effect is the main source of the two-way airflow and contaminant exchange through the doorway. when the dirty cubicle door remains fully open, temperature difference and concentration gradient across the doorway induce the two-way buoyancy-driven flow and transport of airborne agents across the doorway. the longer the dirty cubicle door remains fully open ( s, s or s) or the smaller the air change rate ( – . ach for each cubicle), the more airborne pathogens are being transported into the 'clean' cubicle and the longer time it takes to remove them after the door is closed. keeping the door completely open is potentially responsible for the majority of inter-cubicle transmissions if its duration is much longer than the duration of door motion (only s). our analyses suggest a potential inter-cubicle infection risk if the shared anteroom is used for multiple isolation cubicles. decreasing the duration of door opening, raising air change rate or using a curtain at the doorway are recommended to reduce inter-cubicle exposure hazards. respiratory infectious diseases, such as tuberculosis (tb), influenza, and severe acute respiratory syndrome (sars), threaten lives worldwide. for example, in the early st century, more than cases of sars were reported, resulting in deaths [ ] . infection transmission in hospitals for such highly infectious diseases is an important public health issue. besides direct or indirect contact transmissions [ ] , respiratory infectious diseases can also be spread by airborne transmission of droplet nuclei over large distance. the centers for disease control and prevention (cdc) includes the use of administrative measures and engineering controls among its recommendations for reducing exposure risk in health care facilities [ ] . negative pressure isolation cubicles are usually built to accommodate patients in which a slightly negative pressure relative to the surrounding area may effectively prevent infectious agents from escaping out of isolation cubicles [ , ] . some design guidelines are widely accepted [ , ] . us cdc guidelines and relevant international standards are in agreement with respect to requiring a ventilation rate of air change rates per hour (ach) [ , ] . however if an isolation cubicle door is open, the negative pressure differential across the doorway may decrease to small values and the effectiveness of the containment measure may not be maintained [ e ] . to reduce the risk of infectious droplets and particles escaping into the corridor, an anteroom is commonly added to separate the cubicle and the corridor. with a higher pressure than the isolation cubicle and lower pressure than the corridor, the anteroom acts as an airlock. tung et al. [ ] reported that, if the isolation cubicle door is open and the air velocity through the doorway is less than . m/s, risk of droplet nuclei leakage into the anteroom is high. small-scale experiments and theoretical models [ , ] confirmed that the opening and closing motion of hinged doors can induce transient airflows and turbulence close to the doorway, followed by air exchange and the transport of airborne particles into the anteroom through the doorway. in addition, health care worker (hcw) movement is a significant factor in the spread of airborne pathogens and particles [ e ] . it is difficult to obtain quantitative and high-quality experimental data with meaningful spatial and temporal resolution on how door opening and hcw movement affecting the containment effectiveness of isolation cubicles [ e ]. rydock and eian [ ] carried out tracer gas experiments in which sulfur hexafluoride (sf ) was released at the patient bed position and allowed to mix in the room for min before the release technician exited the room into the anteroom. the technician waited an additional min in the anteroom before entering the adjacent corridor. subsequently, sf was readily detected in both the anteroom and the corridor, indicating tracer gas leakage into the anteroom and corridor during hcw movement. johnson et al. [ ] also reported that containment efficiency was greatly reduced during hcw movement into and out of an expedient isolation enclosure. after fluorescent microspheres were released in the isolation cubicle (particle diameter of mm), adams et al. [ ] measured airborne concentrations in the anteroom and at the corridoreanteroom door with and without hcw movement. they found that containment efficiency rose with greater negative pressure differential, and fell with an increase in human traffic. use of a dynamic mesh technique combined with computational fluid dynamics (cfd) simulations [ e ] has recently allowed simulation of transport of infectious agents induced by door motion and human movement. using large eddy simulations, choi and edward [ ] found that for a non-hospital environment the motion of hinged door and realistic human walking did enhance compartment-to-compartment contaminant transport between two rooms through the doorway and a shared vestibule. poussou et al. [ ] and mazumdar et al. [ ] numerically found that humanmovement-induced wake may carry contaminant to positions far from the source location in an airplane cabin. for hospital isolation rooms with higher air change rates, some published studies verified that the velocity and pressure field were significantly affected by human motion and/or door motion [ e ]. however there was only a small change in airborne transmission of airborne agents, and the risk quickly returned to its original level after the door was closed and hcw motion stoped [ e ]. hang et al. [ ] reported ventilation design and air change rates in a six-bed isolation room affected airborne transmission much more than human motion. goldasteh [ ] and wang and chow [ ] found that human motion can significantly influence particle re-suspension and particle dispersion in the isolation room. most airborne transmissions are via dispersion of droplets/ particles. previous studies have confirmed that droplets/particles of different sizes are released from breathing, coughing, or sneezing and then undergo evaporative water loss in the air. there have been a number of studies on evaporation, dispersion and deposition of respiratory droplets as well as tracer gas dispersion in various indoor environments under different ventilation systems [ e ] . larger particles (diameter > mm) may rapidly deposit onto wall surfaces because the force of gravity is more significant than ventilation [ e , ] ; smaller particles ( . e mm) may remain suspended for a long time and contribute to disease transmission over greater distances, thus their transport process is similar with those of gaseous agents [ e , , ] . both fine particles and gaseous pathogens are significantly influenced by ventilation rates and airflow patterns [ e ] . yin et al. [ ] experimentally studied an one-bed isolation room under the mixing or displacement ventilation system. they found that fine particles (diameter mm and mm) and tracer gas can generate similar contaminant distributions in most region of the ward, except in the areas close to the contaminant source and the exhaust adjacent to the restroom, where the flow may be unstable. two or more isolation cubicles may share the same anteroom to reduce construction costs. however few studies have been carried out to evaluate the risk of inter-cubicle airborne transmission via the shared anteroom if hinged door motion and/or hcw movement occur. our objective was to confirm and quantify the mechanism of inter-cubicle airborne transmission induced by the hinged door motion and the duration of door opening. as a start, this paper first disregards hcw movement and only investigates inter-cubicle transport of tracer gas as a surrogate for very fine droplet nuclei. section and introduce full-scale experiments and cfd setups respectively. section presents results and discussions. finally the conclusions are drawn in section . we carried out a series of full-scale field measurements in the christian medical centre (cmc) hospital, hong kong in . two neighboring isolation cubicles are located in ward b, as shown in fig. aec . in these experiments, tracer gas (sf ) was continuously released from the mouth of a thermal manikin in cubicle a (named as room , 'dirty room'). there was no tracer gas source in cubicle which is named as room ('clean room'). each cubicle has its own toilet (toilet and ) but uses a shared anteroom. exhausts and are located in the ceiling of toilets and . the door in 'dirty' room is referred to as door , and that in 'clean' room as door . each door is . m wide and . m tall (fig. c) . above the two doors there are two air transfer grilles (transfer grilles and , see fig. c ) connecting two cubicles with the shared anteroom. there are downward supply diffusers in each cubicle, i.e. supply in room , and supply in room . in the anteroom, there is also a downward supply diffuser on the ceiling and an air transfer grille connected to the corridor (transfer grille ). the volumetric flow rates were measured by an electronic balometer (model: alnor with apm meter) ( table ). the measured volumetric flow rates through the supply in the anteroom, transfer grille , supply and were . m /s, . m /s, . m /s and . m /s respectively. wind speed at two exhausts in toilet and and transfer grilles and was measured at nine points by using an air velocity meter (model: tsi a-m-gb), and an average air speed was obtained to estimate the air flow rate. because the quality of such calculated flow rates is poor, this paper only uses the measured supply flow rates from the electronic balometer to calculate air change rates, which are ach, ach and ach for the anteroom, room and room respectively. these are much larger than the cdc specified value of ach [ ] , but such larger flow rates are commonly adopted in isolation wards of hong kong. the concentration was continuously monitored by multipoint sampler/doser type & (brüel & kjaer, denmark) at four sampling points (fig. bec) , i.e. p , p , p-anter, one point near and below supply . each sample was collected within about three minutes. as shown in fig. d , nine sets of experiments were performed according to the mode and duration of door opening. in fig. d , the time unit is minute, and is not to scale. prior to each test, all doors were closed for about minutes to approximate conditions of equilibrium. in all cfd simulations, only door is opened for a while ( s, s or s). it is assumed for door to take s to move from the closed state ( ) to fully open ( ). similarly it takes s to close from to . as shown in fig. a , the angular velocity of the sweeping motion varies over time (i.e. modeled by p cos(pt/ )/ as opening and Àp cos (pt/ )/ as closing), implying that door moves the fastest at the beginning and the slowest when it is fully open/closed. note that, here we only tested one speed of door motion (about s) as displayed in fig. . if the speed of door motion was quicker or lower, the sweeping effect of door motion on airborne transmission across door way would possibly differ from present simulation results. in cfd simulations (table ) , it was assumed that supply and have the same flow rate ( . m /s). thus the default air change rates for the anteroom and each isolation cubicle were set as and ach respectively. moreover the impact of smaller air change rates ( . and . ach, % of the operating air change rates) was also investigated in cfd simulations. [ ] was used to simulate indoor turbulent flows. large eddy simulation (les) demands more computer memory and a longer simulation time [ ] . among the reynolds averaged navierestokes (rans) turbulence models, the rng keε model was applied because it was reported to be suitable in terms of accuracy, computing efficiency, and robustness for modeling indoor environments [ ] . the steady flow field and gaseous concentration field when both doors were closed (i.e. t ¼ s) were first solved, then the transient door motion was simulated. the governing equations were discretized using a finite volume method (fvm). the second-order upwind scheme was used for discretizing the convection terms. the simple algorithm was used to couple pressure and velocity. the boussinesq model was adopted for the buoyancy effect. user defined functions (udfs) were dynamically loaded to realize the sweeping motion of door (fig. ). the minimum time step for unsteady cfd simulation in all test cases was . s. to apply a dynamic mesh technique, the integral form of the conservation equation for a general scalar on an arbitrary control volume vol with a moving boundary is defined as [ ] : where r is the fluid density, v ! is the flow velocity vector, v g ! is the grid velocity of the moving mesh, g is the diffusion coefficient, s f is the source term, and vvol represents the boundary of the control volume. all boundary conditions and geometries are summarized in table , and also shown in fig. a . a total of w heat flux was produced by each lying and sleeping manikin, and half was assumed to be transferred through convection (i.e. w) [ ] . following its surface area of . m , the convection heat flux at skin surfaces is w/m . similar to our previous studies [ , ] , the radiation was not simulated, but the radiation heat from thermal manikins was distributed at other wall surfaces. a non-slip wall boundary condition with a standard wall function was used at all wall surfaces. the grids were the finest near manikin's mouth (grid size of . cm), finer near the manikin torso and bed surfaces ( e cm), and the coarsest near wall surfaces (~ e cm). the total number of tetrahedral cells was about . million. this grid arrangements were similar to those described in the literature [ , , ] . to attain default air change rates of / ach, default supply velocities of . m/s, . m/s, and . m/s were defined with the same temperature (t ¼ c) in the supply of each cubicle, the anteroom, and transfer grille . for air change rates of . / . ach, % of the default supply velocities were set. in cfd simulations, tracer gas (carbon dioxide, co) was continuously released into the ward through the source manikin's mouth in room with an emission rate of . mg/s (exhalation velocity . m/s, mass fraction of tracer gas is . , t ¼ c). the height of the source manikin's mouth was . m. the momentum of exhalation has been reported as important for coughing and sneezing but not significant for breathing and talking (gupta et al. [ , ] ). thus, the tracer gas source treatment here was acceptable. the second-order upwind scheme was used for the convection term in the tracer gas transport equation. for the boundary condition, zero normal gradient at exhausts and zero normal flux at wall surfaces were used. a reliable turbulence model, suitable boundary conditions, along with a appropriate numerical scheme and algorithm can improve numerical accuracy. the cfd methodologies adopted are similar to those in the literature [ e , ]. for both full-scale experiments and cfd simulations, t ¼ s is always the time that the door opening motion begins. this paper only displays experimental data in two example tests to verify inter-cubicle transmission. in test a (fig. a) since it is difficult to experimentally capture the evidence of inter-cubicle airborne transmission if the duration of door opening is only s, in test a (fig. b) , we opened and closed both doors together, with both remaining fully open for s. concentration data was sampled at least once when the doors were fully open, therefore, the concentration at p in 'dirty' room was found to decrease by about % at t ¼ s, and tended to recover after door was closed. this fact confirms that the concentration distribution in room completely changed while door remained fully open for s. in addition, the concentration at p-anter in test a increased by % at t ¼ s. moreover the concentration at p in test a increased by % at t ¼ s. finally, the concentration decrease at p-anter after door was closed indicates that contaminants in the anteroom were gradually removed after door was closed. overall, the measurements show that containment failure does occur when door is open and infectious agents indeed spread between two isolation cubicles through the shared anteroom. this suggests that patients with different diseases should not be accommodated in isolation cubicles sharing an anteroom. because the sampling interval was about three minutes at only four sampling points, the temporal and spatial resolution of our full-scale experiments was limited, and it does not permit a good understanding of the inter-cubicle transmission mechanism. cfd simulations with better spatial and temporal resolutions will be more revealing. because the spatial and temporal resolution of our full-scale experiments in subection . was limited, it cannot be used to evaluate the transient cfd simulations of inter-cubicle transmission. the experimental data measured by yin et al. [ ] in an inpatient ward (fig. ) was used to verify the reliability of cfd methodologies in predicting steady-state indoor airflows. in yin et al. [ ] , a ventilation rate of ach ( cfm) was obtained by air supplied from a near floor diffuser. air was exhausted by the bathroom exhaust and the main exhaust, with a ventilation rate of cfm and cfm respectively. vertical profiles of velocity, magnitude, and static temperature at eight poles were measured from the floor to the ceiling. in cfd simulations, medium and fine grid arrangements were utilized with tetrahedral cells of . / . million and a maximum grid size of cm/ cm at wall surfaces. the heat released from patient, caretaker, equipment and tv was w, w, w, and w, which matched the experiment. the other cfd arrangements are introduced in subection . . fig. shows vertical profiles of velocity and temperature at two sample locations (pole and pole ). the height, velocity, and temperature (q ¼ (t À ts)/(te À ts)) were normalized with respect to the height of the inpatient ward (h ¼ . m), supply air velocity (u s ¼ . m/s), temperature at inlet (t s ), and main exhaust (t e ). thermal stratification is clearly shown. by using present cfd set-ups with rng keε model, the medium grid performs as well as the fine grid in simulating steady-state turbulent airflows in such a hospital isolation room. and (fig. a) with total volumetric flow rates of À . m /s. then these airflows reach the vertical wall, go down to the ground (fig. a) , flow around the isolation cubicles, enter the toilets contained in each cubicle and finally leave through the ceiling-level exhausts in these two toilets (fig. b) . in room , the concentration in the recirculation region was much higher than in other regions (fig. b) , and is referred to as the 'highly polluted region'. the concentration in the anteroom and room was approximately zero, showing that there were few infectious agents spreading out of room . case [ , s] is presented as an example. fig. aef shows isosurfaces of contaminant concentration at different times. gaseous contaminants start spreading from room into the anteroom after door begins opening (t ¼ . and . s, fig. aeb ). more agents enter room from s to s while door remains fully open (fig. c at s) and from s to s while door is closing (fig. d at . s) . after door is closed at t ¼ s, the concentration in room first rises (fig. e at s) , then decreases. for example, at s (fig. f) the concentration in room approximates its original level and those in the anteroom and in room become low. to quantify the concentration variation, fig. g shows normalized spatial average concentration <c(t)>/<c ( )> in room and the anteroom for case [ , s], where <c ( )>¼ . ppm is the spatial average concentration in room at t ¼ s in this case. the anteroom first experiences a peak of <c > max ¼ . ppm at t ¼ . s, and afterward <c> decreases. after door is closed at t ¼ s, <c> in the anteroom decreases more quickly, while <c> in room continues increasing until it reaches the maximum concentration of <c> max ¼ . (fig. b) . . the opening motion of door produces two-way airflows and contaminant transport (figs. and ). firstly, we define 'the swept volume of door motion' as the space door passes and moves through. the sweeping motion of door , coupled with the flow from transfer grille produces a negative flow rate leaving the anteroom and entering room through the swept volume (figs. a and ) . secondly, the door motion produces a higher pressure in room than in the anteroom (fig. b) . the pressure is relatively low near the top and bottom of the doorway where considerable positive flow rate (q(in) in fig. a) is produced, and which is the main reason for contaminant flux entering the anteroom from room (q c (þ) in fig. b ). vortexes are induced near the tip of door (fig. a) which are also reported in the literature [ , ] . in this period door is always closed, and contaminant flux through transfer grille (fig. b) is small because little contaminant reaches transfer grille over such a short duration ( s). another type of two-way airflow and contaminant exchange was generated (fig. a) . the temperature difference between the anteroom and room , combined with airflow from transfer grille and the entire ventilation system is the main cause of contaminant leakage. since the net flow rate across the doorway of door is about À . m /s in case [ , s] and its cross area is . m , the average velocity is only À . m/s, which is not sufficient to prevent contaminant leakage out of room when door remains fully open ( . m/ s required by tung et al. [ ] ). contaminants mainly enter the anteroom through the top of door and transfer grille (fig. b) . chen et al. [ ] reported similar findings in a hospital ward consisting of four cubicles with 'positive pressure' towards their shared corridor. if the door is open, the slightly higher temperature in the corridor compared with the cubicles does induce two-way air exchange and contaminant transport across the doorway from one source cubicle into the corridor, and subsequently into the other 'clean' cubicles. a good technique is to install curtains at the doorway [ ] which can reduce the area of the doorway and thus increase this average velocity through it. over this period ( s < t < s), as displayed in and ) . pressure in the anteroom is relatively higher than in room (fig. a) . the closing motion also induces vortexes behind door and a two-way airflow pattern (fig. ). fig. aeb shows that there is considerable positive flow rate and contaminant flux into the anteroom across the doorway of door due to the sweeping effect of the closing motion. meanwhile there is much greater negative flow rate and contaminant flux re-entering room through the bottom and top of the doorway and transfer grille . negative contaminant fluxes entering room through transfer grille continue to rise (fig. b) . as displayed in fig. g , <c(t)> in the anteroom reaches its maximum value at t ¼ . s. as shown in fig. b , total qc of the anteroom becomes negative after t ¼ . s, i.e. the negative contaminant fluxes leaving the anteroom across door doorway and transfer grilles and (qc(À)þqc(grille ) þqc(grille )) start to exceed that entering the anteroom across door doorway (qc(þ)). > s, fig. ). after door is closed, the negative pressure design produces the negative constant flow rates (À . m /s) through transfer grilles and . consequently, the two-way air exchange disappears. contaminants are quickly removed from the anteroom across two transfer grilles (fig. b) . considerable qc across transfer grille entering room can explain why the concentration in room increases until t ¼ s (fig. g) . overall, door remaining fully open for s contributes similarly to inter-cubicle transmission as the opening and closing motion of door (only s). with a scale physical model, fontana and quintino [ ] experimentally confirmed that door operation can produce a dirty air transfer in the clean room, and that transfer entity appears strongly related to air volume displaced in the door opening operation but almost independent from differential pressure and flow rate imbalance. however, our simulation results show that the amount of airborne agents transferred into the clean anteroom depend on not only the transient two-way airflow induced by door motion but also the unsteady concentration gradient across the doorway. thus the transport of airborne agents across the doorway into the clean anteroom and to the neighbor cubicle is a much more complicated and transient process than the scaled model in the literature [ ] . further investigations with various speed of door sweeping motion (for example s, s, s) are still required to check how the speed of door motion influences inter-cubicle transmission in such realistic hospital wards. to investigate the impact of the duration of door opening, fig. a . ppm) . this shows that, the longer door remains fully open, the greater the transport of infectious agents into room through the shared anteroom, but <c> max in the anteroom does not rise, apparently because the net contaminant flux of the anteroom becomes negative at t ¼ . s or . s (fig. a) . to study the effect of air change rate, fig. bec the anteroom increases about e times and that in room increases about e times. more time is also required to reach the peak concentration in the anteroom and room , as well as to remove contaminant leaked into the anteroom and room . thus the amount of inter-cubicle transmission and exposure time in room increases greatly if air change rate decreases. both field experiments and cfd simulations show that, despite the maintenance of negative pressure gradients across the doorway with high air change rates ( ach for the anteroom and ach for each isolation cubicle as in a real ward in a hong kong hospital), infectious agents in one 'dirty' isolation cubicle (room ) with a source manikin can spread into the other 'clean' cubicle (room ) through the shared anteroom if the door is open. throughout the door opening and closing motion, the door sweeping effect in combination with the ventilation system dominates two-way airflow and contaminant exchange. when the dirty cubicle door remains fully open for a while, the average air velocity through the doorway is small and the temperature difference across this doorway is the main cause of two-way contaminant exchange and inter-cubicle transmission. our analysis shows that, if ach is used for each cubicle, the longer the dirty cubicle door remains fully open ( , or s), the more airborne pathogens enter the clean cubicle (room ), but the peak concentration in the anteroom only increases slightly. keeping the door completely open possibly contributes to the major fraction of inter-cubicle transmission when the duration time ( s, s) is much longer than the door sweeping processes (only s). air change rate is another important parameter. if the air change rate for each cubicle decreases from ach to . ach, more infectious agents enter the anteroom and the clean cubicle, meanwhile it requires more time to reach their peak concentration and to remove the leaked airborne pathogens once the dirty cubicle door is closed. overall, the opening of hinged doors does pose a significant risk of containment failure, and the use of shared anterooms for isolation cubicles poses the risk of inducing inter-cubicle infection. considering that the negative pressure in isolation cubicles is sufficient to prevent inter-cubicle airborne transmission if wind speed across the doorway is sufficiently high [ ] , it is recommended that curtains can be installed at doorway locations to reduce the risk of airborne pathogen leakage [ ] . other techniques are also plausible, such as using air cleaners [ ] , using facemasks [ ] , etc. although further investigations are still required with respect to particles and droplets simulations, this paper offers meaningful findings for the inter-cubicle transmission of gaseous agents when a shared anteroom is used. world health organization droplet fate in indoor environments, or can we prevent the spread of infection? guidelines for preventing the transmission of mycobacterium tuberculosis in health care facilities guidelines for design and construction of hospital and health care facilities factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises numerical study on the dispersion of airborne contaminants from an isolation room in the case of door opening door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity 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among general populations this study was financially supported by the national natural science foundation of china (no. ) and (no. ) as well as the fundamental research funds for the central universities of sun yat-sen university (no. ). thanks go to dr wh seto and ms patricia ching, mr lei zhang for help with the field measurement. we also thank the hospital engineers, ward managers, and ward nurses for their assistance during this study. key: cord- -ipaoge authors: sadana, ajit; sadana, neeti title: chapter detection of biomarkers for different diseases on biosensor surfaces part ii date: - - journal: biomarkers and biosensors doi: . /b - - - - . - sha: doc_id: cord_uid: ipaoge abstract in this chapter the authors analyze the binding and dissociation kinetics (if applicable) of ( ) interferon-gamma as a function of aptamer variants and inclusion of spacer, ( ) gst-n protein in pbs and gst-n protein in -fold diluted serum to a localized surface plasmon resonance coupled fluorescence biosensor, ( ) cytochrome c mutant to a superoxide biosensor, ( ) carbonic anhydrase-ii to an -( -aminoethyl)-benzene sulfonamide ligand on an surface plasmon resonance biosensor surface, ( ) glycerol secretion from differentiated (murine t -l ) adipocytes to a microfluidic platform for fluorescence-based assay, and ( ) different concentrations of c-reactive protein in solution to a sandwich-type assay using reflectometric interference spectroscopy (label-free detection method). the previous chapters have described the detection of different biomarkers for different diseases. each chapter analyzed the different biomarker for a particular disease. in this chapter we analyze the binding and dissociation (if applicable) kinetics of other biomarkers on biosensor surfaces. some of the examples analyzed include the following: . binding to and dissociation of different aptamer beacon modifications of interferon (ifn)-gamma in solution using fluorescence resonance energy transfer (fret) and immobilized on an avidin-coated surface (tuleuova et al., ) . . binding and dissociation of glutathione s-transferase fused to the nterminus of a protein (gst-n) protein in phosphate buffered saline (pbs) to a localized surface plasmon resonance coupled fluorescence (lspcf) biosensor (huang et al., ). . binding of a cytochrome c mutant to an amperometric superoxide biosensor (wegerich et al., ). . binding of different concentrations (in micromoles) of carbonic anhydrase-ii (ca-ii) in solution to immobilized -( -aminoethyl)-benzene sulfonamide (abs) using signal-locking surface plasmon resonance (spr) (williams et al., ) . . binding to and dissociation from a microfluidic platform of mm glycerol secreted from differentiated (murine t ) adipocytes (clark et al., ) . . binding of different concentrations of c-reactive protein (crp) to a new sandwich-type assay design using a label-free detection method. some of the other biomarker studies that have appeared in the recent literature or have been presented at conferences include the following: . multichannel mass organic analyzer and microfluidic networks for the automated in situ microchip electrophoretic analysis of organic biomarkers (benhabib et a ., ) . . hybrid magnetic-plasmonic nanoparticles for biomarkers (hirt et al., ) . . engineered knottin peptides: a new class of agents for noninvasive molecular imaging of tumor biomarkers (apte and graves, ) . . identifying secreted biomarkers for murine evasion in cellular models of cancer (kinke, ) . . spr biosensor for parallelized detection of protein biomarkers in diluted blood plasma (pilarik et al., ) . . biomarkers in drug discovery and development: from target identification through drug marketing (colburn and keefe, ) . . validation of analytic methods for biomarkers used in drug development (anonymous, ) . . electrochemical biosensors: toward point-of-care diagnostics (wang, ) . . biosensors for biomarkers in medical diagnostics (mancini and tombelll, ) . . point-of-care biosensor systems for cancer diagnostics/prognostics (sofer et al., ) . . the demonstration of the immunochemical biomarkers in methyl methacrylate-embedded plucked human hair follicles (anonymous, ) . . surface plasmon resonance biosensor based on vroman effect: toward cancer biomarker detection (choi and chase, ). . biogenic nanoporous silica-based sensor for enhanced electrochemical detection of cardiovascular biomarker proteins (lin et al., ) . . nanomonitor: a miniature electronic biosensor for glycan biomarker detection (nagaraj et al., ) . . multifunction dendrimer-template antibody presentation on biosensor surfaces for improved biomarker detection (han et al., ) . . rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow strip (li et al., ) . . a biomarker concept for assessment of insulin resistance, beta-cell function, and chronic system inflammation in type diabetes mellitus (pfutzner et al., ) . . multifunctional au nanoparticle dendrimer-based surface plasmon resonance biosensor and its application for improved insulin detection (frasconi et al., ) . recently published reports are also available that describe in detail the different aspects of biomarkers and their applications in a clinical setting and the collaborative efforts that are required for their successful development (laria, ) . for example, they include the highlights of key technologies that are required for the development of imaging biomarkers. more importantly, case studies are presented of individual imaging biomarkers. finally, the future of imaging biomarkers is also presented. we now use fractal analysis to analyze the binding and dissociation kinetics of some of the different biomarkers available in the open literature. the examples were selected at random, with no particular bias toward analyzing a particular biomarker, or a class of biomarkers. havlin ( ) has reviewed and analyzed the diffusion of reactants toward fractal surfaces. the details of the theory and the equations involved for the binding and the dissociation phases for analyteereceptor binding are available (sadana, ) . the details are not repeated here, except that the equations are given to permit an easier reading. these equations have been applied to other biosensor systems (sadana, ; ramakrishnan and sadana, ; sadana, ) . for most applications, a single-or a dual-fractal analysis is often adequate to describe the binding and the dissociation kinetics. peculiarities in the values of the binding and the dissociation rate coefficients, as well as in the values of the fractal dimensions with regard to the dilute analyte systems being analyzed will be carefully noted, if applicable. in this chapter we analyze the binding and dissociation kinetics (if applicable) of ( ) ifn-gamma as a function of aptamer variants and inclusion of spacer in addition to spacer (tuleuova et al., ) , ( ) gst-n protein in pbs and gst-n protein in -fold diluted serum to an lpscf fiber-optic biosensor (huang et al., ) , ( ) cytochrome c mutant to a superoxide biosensor (wegerich et al., ) , ( ) ca-ii to an abs ligand on an spr biosensor surface (williams et al., ), ( ) glycerol secretion from differentiated (murine t -l ) adipocytes to a microfluidic platform for fluorescence-based assay (clark et al., ) , and ( ) different concentrations of crp in solution to a sandwich-type assay using a label-free detection method, reflectometric interference spectroscopy (albrecht et al., ) . here d f,bind or d f (used later on in the chapter) is the fractal dimension of the surface during the binding step. t c is the crossover value. havlin ( ) indicates that the crossover value may be determined by r c w t c . above the characteristic length, r c , the self-similarity of the surface is lost and the surface may be considered homogeneous. above time t c the surface may be considered homogeneous, since the self-similarity property disappears, and "regular" diffusion is now present. for a homogeneous surface where d f ¼ , and when only diffusional limitations are present, p ¼ ½ as it should be. another way of looking at the p ¼ ½ case (where d f,bind ¼ ) is that the analyte in solution views the fractal object, in our case, the receptor-coated biosensor surface, from a "large distance." in essence, in the association process, the diffusion of the analyte from the solution to the receptor surface creates a depletion layer of width (Ðt) ½ where Ð is the diffusion constant. this gives rise to the fractal power law (analyte.receptor) w t ð Àd f;bind Þ= . for the present analysis, t c is arbitrarily chosen and we assume that the value of the t c is not reached. one may consider the approach as an intermediate "heuristic" approach that may be used in the future to develop an autonomous (and not time-dependent) model for diffusion-controlled kinetics. the diffusion of the dissociated particle (receptor [ab] or analyte [ag]) from the solid surface (e.g., analyte [ag]ereceptor [ab] complex-coated surface) into solution may be given, as a first approximation by ðab$agÞz Àt ð Àdf;dissÞ= ¼ Àt p ðt > t diss Þ ( . ) here d f,diss is the fractal dimension of the surface for the dissociation step. this corresponds to the highest concentration of the analyteereceptor complex on the surface. henceforth, its concentration only decreases. the dissociation kinetics may be analyzed in a manner "similar" to the binding kinetics. sometimes, the binding curve exhibits complexities and two parameters (k, d f ) are not sufficient to adequately describe the binding kinetics. this is further corroborated by low values of r factor (goodness of fit). in that case, one resorts to a dual-fractal analysis (four parameters; k , k , d f , and d f ) to adequately describe the binding kinetics. the single-fractal analysis presented above is thus extended to include two fractal dimensions. at present, the time (t ¼ t ) at which the "first" fractal dimension "changes" to the "second" fractal dimension is arbitrary and empirical. for the most part, it is dictated by the data analyzed and experience gained by handling a single-fractal analysis. a smoother curve is obtained in the "transition" region, if care is taken to select the correct number of points for the two regions. in this case, the product (antibodyeantigen; or analyteereceptor complex, ab.ag or analyte.receptor) is given by in some cases, as mentioned above, a triple-fractal analysis with six parameters (k , k , k , d f , d f , and d f ) may be required to adequately model the binding kinetics. this is when the binding curve exhibits convolutions and complexities in its shape due perhaps to the very dilute nature of the analyte (in some of the cases to be presented) or for some other reasons. also, in some cases, a dual-fractal analysis may be required to describe the dissociation kinetics. tuleuova et al. ( ) have developed an aptamer beacon for the detection of ifn-gamma, which is an important inflammatory cytokine. boehm et al. ( ) indicate that it is secreted by immune cells in response to various pathogens. tuleuova et al. ( ) indicate that the levels of this protein provide important information with regard to infectious diseases and the ability of the body to regulate an immune response. panteleo and koup ( ) indicate that there is vigorous production of ifn-gamma in human immunodeficiency virus-infected patients. tuleuova et al. ( ) indicate that previous antibodybased detection techniques for ifn-gamma were very time consuming. jayasena ( ) indicates that aptamer-based affinity strategies are coming into prominence. ellington and szostak ( ) indicate that aptamers are single-stranded dna or rna oligonucleotides that have been selected to bind to target analytes with high specificity and affinity. aptamers have an advantage over antibodies since they are more robust; thus, aptamer-based biosensors can be regenerated and used over and over again. furthermore, balamurugan et al. ( ) , kirby et al. ( ), nutiu ( , and luzi et al. ( ) indicate that aptamers are amenable to modification due to their simplicity and robustness. tuleuova et al. ( ) indicate that fret may be used to convert aptamers into real-time biosensors (urata et al., ; babendure et al., ) . romangani et al. ( ) and karlsson et al. ( ) indicate that ifngamma is an important immune response marker. thus, tuleuova et al. ( ) have designed a novel immune response marker to detect ifn-gamma. the dna aptamer was biotinylated and immobilized on an spr sensing surface by avidinebiotin interactions. the spr biosensor was used to analyze the influence of biotinylation, fluorophore attachment, and spacer incorporation on the ability of the aptamer to bind to ifn-gamma. ifn-gamma is a type ii cytokine, and is critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control. incorrect ifn-gamma expression is associated with a number of autoinflammatory and autoimmune diseases. it plays an important role in immunostimulatory and immunoregulatory effects. the ifn-gamma monomer consists of six alpha-helices and an extended unfolded chain in the c-terminal region (ealick et al., ; thiel et al., ) . tuleuova et al. ( ) analyzed the influence of aptamer modification on the binding and dissociation of ifn-gamma in solution. figure . (a) shows the binding and dissociation of nm ifn-gamma in solution to the modified aptamer b ( -ggg gtt ggt tgt gtt ggg tgt tgt gt-biotin- ; sequence with modification) beacon. a single-fractal analysis is adequate to describe the binding and the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension for dissociation, d f , for binding and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd for a single-fractal analysis are given in tables . (a) and (b). tuleuova et al. ( ) indicate that the highest level of the cytokine (ifn-gamma) binding was observed for the modified aptamer b. tuleuova et al. ( ) also included a polyethylene glycol spacer between the aptamer and the biotin so that the nucleotides could be more accessible to the target analyte. modified aptamer beacon. in this case, in addition to the biotin (b) a spacer was included (bs). once again, a single-fractal analysis is adequate to describe the binding and the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension, d f , and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , for a single-fractal analysis are given in tables . (a) and (b). it is of interest to note that as one goes from the modified aptamer beacon b to the modified aptamer beacon bs the binding rate coefficient, k, decreases by % from a value of k ¼ . to k ¼ . , and the fractal dimension, d f , increases by a factor of . from a value of d f ¼ . to d f ¼ . . in this case, changes in the fractal dimension (degree of heterogeneity on the biosensor surface) and in the binding rate coefficient are in opposite directions (tables . (a) and (b)). figure . (c) shows the binding of nm ifn-gamma in solution to the bs( -biotin-c -ggg gtt ggt tgt gtt ggg tgt tgt gt- ) modified aptamer beacon. once again, a single-fractal analysis is adequate to describe the binding and the dissociation kinetics. the values of (a) the binding rate coefficient, k, and the fractal dimension, d f , and (b) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , are given in tables . (a) and (b). note that in this case there is an increase in the fractal dimension, d f (the highest when compared with the b and b's aptamer modifications), and a decrease in the binding rate coefficient, k (the lowest when compared with b and b's aptamer modifications). in this case, there is a substantial decrease in the binding rate coefficient, k. the decrease is higher than a factor of four. figure . (a) and tables . (a) and (b) show the increase in the binding rate coefficient, k, for a single-fractal analysis with an increase in the fractal dimension, d f . for the data shown in figure . (a), the binding rate coefficient, k, is given by k the fit is reasonable. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k, exhibits close to a third (equal to . ) order of dependence on the fractal dimension, d f , that exists on the biosensor surface. this indicates that the binding rate coefficient, k, is sensitive to the fractal dimension, d f , or the degree of heterogeneity that exists on the biosensor surface (tables . (a) and (b)). figure . (b) and table . (a) and (b) show the increase in the affinity, k (¼k/k d ), for a single-fractal analysis with an increase in the fractal dimension ratio, d f /d fd . for the data shown in figure . (b), the affinity, k, is given by the fit is very good. only three data points are available. the availability of more data points would lead to a more reliable fit. the affinity, k (k/k d ), is very sensitive to the fractal dimension ratio (d f /d fd ) as it exhibits close to a fifth (equal to . ) order of dependence on the fractal dimension ratio (d f /d fd ). this is a very convenient way of manipulating the affinity, k, by changing the heterogeneity of the biosensor surface. some ingenuity may be required here, since a change in the degree of heterogeneity on the biosensor surface would change both the binding as well as the dissociation rate coefficients. figure . (a) shows the binding of the ifn-gamma to the biotin þ aptamer b variant (tuleuova et al., ) . once again, a dual-fractal analysis is required to describe the binding kinetics. a single-fractal analysis is adequate to describe the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension, d f , for a single-fractal analysis, ( ) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f , for a dual-fractal analysis, and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , for a single-fractal analysis are given in tables . (a) and (b). in this case, the affinity values k (¼k /k d ) and k (¼k /k d ) are . and . , respectively (tables . (a) and (b)). note that for dual-fractal analysis for the binding phase, an increase in the fractal dimension by . % from a value of d f ¼ . to d f ¼ . leads to an increase in the binding rate coefficient by a factor of . from a value of k ¼ . to k ¼ . . tuleuova et al. ( ) also investigated the influence of a fluorophore (f) in addition to the biotin for the modified aptamer b during the binding of ifngamma. this was one of the aptamer variants. figure . (b) shows that a dual-fractal analysis is required to describe the binding kinetics. a singlefractal analysis is adequate to describe the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension, d f , for a singlefractal analysis, ( ) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f , for a dual-fractal analysis, and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , for a singlefractal analysis are given in tables . (a) and (b). the affinity values k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. note that for dual-fractal analysis for the binding phase, an increase in the fractal dimension by . % from a value of d f ¼ . to d f ¼ . leads to an increase in the binding rate coefficient by a factor of . from a value of k ¼ . to k ¼ . (tables . (a) and (b)). also, note that on comparing the affinity values k and k when the fluorophore is used and not used, the k value is slightly lower and the k value is significantly higher (by about %), respectively. huang et al. ( ) have developed a localized lspcf fiber-optic biosensor for the detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in human serum. these authors indicate that sars is a highly infectious disease. drosten et al. ( ) indicate that sars results in death in a large portion of patients. the sars coronavirus (sars-cov) causes sars, and is detectable in the respiratory secretions of patients after infection (foucher et al., ) . wang et al. ( ) emphasize that sars is highly contagious and exhibits the potential of becoming a large-scale future epidemic if effective therapeutic drugs are not discovered. huang et al. ( ) and che et al. ( ) emphasize the need for a rapid, sensitive, specific, and an accurate diagnostic method so that specific patients may be correctly assessed. huang et al. ( ) indicate that there are methods available to detect sars. however, present methods such as reverse transcriptase polymerase chain reaction are not sensitive enough, and also require a specific laboratory with expertise in molecular diagnostics to confirm sars in the acute phase (fujimoto et al., ; drosten et al., ) . huang et al. ( ) indicate that gold nanoparticles (gnps) have been introduced into biosensing (manso et al., ; cui et al., ) . these gnps possess special properties such as localized surface plasmons. huang et al. ( ) have developed a novel fiber-optic biosensor where the property of lspcf has been combined with the sandwich immunoassay. huang et al. ( ) have used their lspcf fiber-optic biosensor to detect sars-cov protein in diluted serum to a limit of pg/ml. this according to these authors exhibits the potential for the early detection of clinical sars-cov infection. figure . shows the binding of l pg/ml gst-n protein in solution to the lspcf biosensor (huang et al., ) . a single-fractal analysis is required to describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis are given in tables . (a) and (b). huang et al. ( ) prepared gst-n protein samples in -fold diluted human serum. this allowed them to test their lspcf biosensor in clinical samples. they measured the temporal fluorescence intensity of the biomolecular interaction between the lspcf probes and the gst-n protein. they did this for the e pg/ml rages gst-n protein in solution. figure . (a) shows the binding of pg/ml gst-n protein in -fold diluted human serum in solution. a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b). for a dualfractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the fractal dimension or the degree of heterogeneity on the lspcf biosensor surface leads to an increase in the binding rate coefficient. figure . (b) shows the binding of pg/ml gst-n protein in -fold diluted human serum in solution. once again, a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b). for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the fractal dimension or the degree of heterogeneity on the lspcf biosensor surface leads to an increase in the binding rate coefficient. figure . (c) shows the binding of pg/ml gst-n protein in -fold diluted human serum in solution. a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b) once again, for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the fractal dimension or the degree of heterogeneity on the lspcf biosensor surface leads to an increase in the binding rate coefficient. tables . (a) and (b) show for a dual-fractal analysis the increase in the fractal dimension, d f with an increase in the gst-n protein concentration in solution in the e pg/ml range. figure not shown. for the data shown in table . the fractal dimension, d f is given by: d f ¼ ð : ae : Þ ½gst À n : ae : ( . ) the fit is reasonable. only three data points are available. the availability of more data points would lead to a more reliable fit. the fractal dimension, d f exhibits less than one-half (equal to . ) order of dependence on the gst-n protein concentration in solution in the e pg/ml range. this indicates that the fractal dimension, d f exhibits a mild dependence on the gst-n protein concentration in solution. the superoxide anion radical is present in several pathophysiological situations, such as sepsis (valko et al., ; vaklko et al., ) . electrochemical biosensors can detect this short-lived species (lisdat, ; prieto-simon et al., ) . wegerich et al. ( ) indicate that the redox protein cytochrome c is used as a recognition element. these authors indicate that superoxide dismutase (sod) biosensors used for the detection of the superoxide anion often lack the reproducibility due to immobilization problems. however, cyt c-based superoxide biosensors are more stable and may be used in in vivo applications (buttemeyer et al., ; scheller et al., ) . in this case, the heme protein is reduced by the superoxide, followed by reduction by an electrode. wegerich et al. ( ) indicate that short-chain modified gold electrodes exhibit a highly efficient communication between cyt c and the electrode (frew and hill, ; hinnen et al., ; nahir et al., ; taniguchi et al., ) . they have been used for cyt c based superoxide sensors. wegerich et al. ( ) analyzed the effect of introducing positive charges (lysines) in human cytochrome c on the redox properties and reaction rates of cyt c with superoxide radicals. these authors claim that the eleven mutants analyzed were modified for structural integrity as well as axial coordination of the heme ion. their results indicate that four mutants exhibited a higher reaction rate with the radical as compared with the wild type. these mutants were then used for the construction of the superoxide biosensors. figure . shows the binding of cyt c in solution to the superoxide biosensor (wegerich et al., ) . a single-fractal analysis is adequate to describe the binding kinetics. the binding rate coefficient, k and the fractal dimension, d f , for a single-fractal analysis are . ae . , and . ae . , respectively. these results are also shown in tables . (a) and (b). a single-fractal analysis is also adequate to describe the dissociation kinetics. the values of the dissociation rate coefficient, k d and the fractal dimension, d fd are . ae . and . and . , respectively. in this case the affinity, k (¼k/k d ) is equal to . . williams et al. ( ) have recently analyzed low noise detection of biomolecular interactions with signal-locking surface plasmon resonance. surface plasmon resonance is a popular technique to analyze biomolecular interactions at a surface, especially since it is label-free. these authors indicate that the spr technique is subject to the influence of noise and drift disturbances since that limits the minimum detectable mass change. the spr technique uses the step response of the biomolecular interactions occurring on the biosensor surface. the technique proposed by williams et al. ( ) measures the biomolecular interactions over a very narrow frequency range. this locks the measured response to a very specific narrow band signal. the authors used their technique to analyze the binding kinetics of carbonic anhydrase-ii (ca-ii) and immobilized -( -aminoethyl)-benzenesulfonamide (abs) to a spr surface. carbonic anhydrases are a family of enzymes that catalyze the rapid conversion of carbon dioxide and water to bicarbonate and protons. these anhydrases are classified as metalloenzymes since the active site of most carbonic anhydrases contains a zinc ion. the primary function of this enzyme in animals is to maintain the acid-base balance in blood and other tissues, and to help transport carbon dioxide out of tissues. carbon anhydrase ii is a novel biomarker for gasterointestinal stomal tumors (parkkila et al., ) . these authors indicate that various carbonic anhydrase (ca) isoenzymes have been identified as potential targets against different cancers. they further indicate that high ca-ii expression is associated with a better disease specific survival rate than low or no expression. figure . (a) shows the binding of . mm ca-ii anhydrase in solution to a -( -amino ethyl)-benzene sulfonamide (abs) ligand on a spr biosensor surface (williams et al., ) . a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dualfractal analysis are given in tables . (a) and (b). it is of interest to note that as the fractal dimension increase by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . figure . (b) shows the binding of . mm ca-ii anhydrase in solution to a -( -amino ethyl)-benzene sulfonamide (abs) ligand on a spr biosensor surface (williams et al., ) . once again, a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysts are given in tables . (a) and (b). it is of interest to note that as the fractal dimension increase by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . figure . (c) shows the binding of . mm ca-ii anhydrase in solution to a -( -amino ethyl)-benzene sulfonamide (abs) ligand on a spr biosensor surface (williams et al., ) . once again, a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b) it is of interest to note that as the fractal dimension increase by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . figure . (a) and table . (a) show the increase in the binding rate coefficient, k with an increase in the ca-ii concentation in solution in the . e mm range. for the data shown in figure . (a), the binding rate coefficient, k , is given by: k ¼ ð : ae : Þ ½ca À ii : ae : ( . a) the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k exhibits less than one-half (equal to . ) order of dependence on the ca-ii concentration in solution in the . e . mm range. this indicates that the binding rate coefficient, k is only mildly sensitive to the ca-ii concentration in solution. figure . (b) and table . (a) show the increase in the fractal dimension d f , p with an increase in the ca-ii concentration in solution in the . e mm range. for the data shown in figure . (b), the fractal dimension, d f is given by: d f ¼ ð : ae : Þ½ca À ii : ae : ( . b) the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the fractal dimension, d f exhibits a very mild dependence (equal to . ; close to zero order) on the ca-ii concentration in solution in the . e . mm range. this indicates that the fractal dimension, d f is only mildly sensitive to the ca-ii concentration in solution. the fractal dimension is based on a log scale, and even small changes in the fractal dimension indicate a reasonable change in the degree of heterogeneity on the biosensor surface. figure . (c) and tables . (a) and (b) show the increase in the binding rate coefficient, k with an increase in the fractal dimension, d f . for the data shown in figure . (b), the binding rate coefficient, k is given by: k ¼ ð : ae : Þd : ae : f ( . c) the fit is good. only three data points are available. the availability of mora data points would lead to a more reliable fit. the binding rate coefficient, k exhibits close to a second (equal to . ) order of dependence on the fractal dimension, d f this indicates that the binding rate coefficient, k is sensitive to the fractal dimension, d f or the degree of heterogeneity on the biosensor surface. (b) show the increase in the binding rate coefficient ratio, k /k with an increase in the fractal dimension ratio d f /d f . for the data in figure . (d) the binding rate coefficient ration k /k is given by: the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient ratio, k /k exhibits an order of dependence between three and three and one half (equal to . ) on the ratio of the fractal dimensions, d f /d f . this indicates that the binding rate coefficient ratio is very sensitive to the fractal dimension ratio figure . (e) shows the increase in the ratio of the binding rate coefficient, d f /d f with an increase in the ca-ii concentration in solution. the ratio, d f /d f is only mildly dependent on the ca-ii concentration in solution. clark et al. ( ) have recently developed a continuous-flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultivated adipocytes. these authors indicate that different studies on using chips to monitor cellular secretion have appeared in the literature (cheng et al., ; lau et al., ; el-all et al., ; kim et al., ; meyvantsso et al., ; urbanskl et al., ) . clark et al. ( ) indicate that physiological studies need to maintain cells or tissues in a controlled environment as one detects their physical, electrical and mechanical properties. these authors indicate micriofluidics facilitates such situations, since they permit creation of highly controlled cell-compatible environments along with measurement and cell maniplation methods. clark et al. ( ) emphasize that the prevalence of obesity-related disorders underscores the need to adipocyte physiology. adipocytes store and release energy. adipocytes store energy as triacylglycerol by lipolysls. ths supplies energy for tissues and organs. getty et al. ( ) and getty-kaushik et al., ( a,b) indicate that the measurement of glycerol is used to determine the function and physiological state of adipocytes. clark et al. ( ) have developed a dual-chip microfluidic system for culturing adipocytes and then monitoring the glycerol using a continuous fluorescent enzyme assay after a perfusion step. the authors used their system to demonstrate transient increases in glycerol secretion during exposure of the cells to isoproterenol, a b-adrenergic agonist. these adrenergic agonists act on receptors. beta receptors are specific molecules found in the body which receive and process signals for the nervous system and various hormones. these beta receptors are located at many places in the body, but are found in high numbers in the heart and blood vessels. here they increase blood pressure when stimulated. thus, they are attractive targets for high blood pressure treatment. clark et al. ( ) analyzed the glycerol secretion data from differentiated adipocytes and response to isoproterenol treatment. figure . (a) shows the binding of glycerol secretion from differentiated (murine t -l ) adipocytes by a continuous-flow enzyme assay on a microfluidic chip. a dual-fractal analysis is required to model the binding and the dissociation kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis, and (d) the dissociation rate coefficients, k d and k d and the fractal dimensions for the dissociation phase, d fd and d fd for a dual-fractal analysis are given in tables . (a) and (b). figure . (b) shows the binding of glycerol secretion from differentiated (murine t -l ) adipocytesin the presence of iso proterenol by a continuous-flow enzyme assay on a microfluidic chip. a dual-fractal analysis is required to model the binding and the dissociation kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b). albrecht et al. ( ) have recently presented a new assay design for clinical diagnostics based on alternative recognition. these indicate that the assay format has an important impact in the practical handling as well in the sensitivity of the testing results. jaras et al. ( ) indicate that for clinical diagnostics the sandwich assay format is frequently used due to (a) its lower limits of detection compared to other formats, (b) and reliable analysis of the different parameters. albrecht et al. ( ) further indicate that a drawback of the sandwich assay format is the need for immobilization of the capture antibody on the surface. this often results in a significant loss in binding activity. also, there is no guarantee that the binding sites on the antibodies immobilized on the surface are oriented in the 'correct' direction. this hinders the biosensor performance parameters such as sensitivity, loss in function, and stability of the sensor surface. in essence, these authors indicate that the recognition element on the sensor surface needs to exhibit a high affinity and specificity towards the antigen (analyte) in solution on being immobilized on the biosensor surface. albrecht et al. ( ) have presented an immunoassay set-up that uses a small and stable peptide sequence as the immobilized recognition element (receptor) (baltzer, ) . albrecht et al. ( ) indicate that their recognition elements are small helix loop-helix motifs. these recognition elements contain natural binders of the target analyte. furthermore, these motifs are easily accessible. also, these authors indicate concerted modifications made for immobilization at the artificial helices do not affect binding properties. albrecht et al. ( ) have presented a new sandwich-type assay for the detection of c-reactive protein (crp). they used a tailored binder as the capture element on the sensor surface, and an antibody as a detection element. c-reactive protein is a protein found in blood. its levels rise in response to inflammation. thompson et al. ( ) indicate that its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells in order to activate the complement system via the c q complex. crp is a general marker for inflammation and infection. it can be used as a very rough proxy for heart disease risk. lloyd-jones et al. ( ) emphasize that since many factors are responsible for crp level elevations, thus it is not a very specific prognostic indicator. also the patients with elevated basal levels of crp are at an increased risk of diabetes (pradhan et al., ) , hypertension and cardiovascular disease. figure . (a) shows the binding and dissociation of .  À m crp in solution to the new sandwich assay design that contains a high affinity polypeptide scaffold as the immobilized capture element and an antibody for detection (albrecht et al., ) . as mentioned above a biosensor based on reflectometric interference spectroscopy (rlfs) was used. a dual-fractal analysis is required to describe the binding kinetics. a single-fractal analysis is adequate to describe the dissociation kinetics. the values of (a) the binding rate coefficient k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dualfractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b). it is of interest to note that for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. in this case, the affinity, k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. figure . (b) shows the binding and dissociation of .  À m crp in solution to the new sandwich assay design (albrecht et al., ) . a dualfractal analysis is once again required to describe the binding kinetics. a single-fractal analysis is adequate to describe the dissociation kinetics. the values of (a) the binding rats coefficient k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b). it is of interest to note that for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. in this case, the affinity, k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. figure . (c) shows the binding and dissociation of .  À m crp in solution to the new sandwich assay design (albrecht et al., ) . a dualfractal analysis is once again required to describe the binding kinetics. a single-fractal analysis is adequate to describe the binding kinetics. the values of (a) the binding rate coefficient k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b) it is of interest to note that for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. in this case, the affinity, k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. figure . (a) and table . (a) show for a dual-fractal analysis the decrease in the binding rate coefficient, k with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (a) the binding rate coefficient, k is given by: k ¼ ð : ae : Þ½crp À : ae : ( . a) the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k decreases with an increase in the crp concentration in solution in the . À .  À m concentration range, and exhibits less than a negative one half (equal to - . ) order of dependence on the crp concentration in solution. figure . (b) and table . (a) show for a dual-fractal analysis the increase in the binding rate coefficient, k with an increase in the fractal dimension, d f . for the data shown in figure . (b) the binding rate coefficient, k is given by: the fit is very good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k exhibits close to a seven and one-half (equal to . ) order of dependence on the fractal dimension, d f on the biosensor surface. this indicates that the binding fate coefficient, k is very sensitive to the fractal dimension or the degree of heterogeneity on the biosensor surface. figue . (c) show for a dual-fractal analysis the decrease in the fractal dimension, d f with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (c) the fractal dimension, d f is given by: the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the fractal dimension, exhibits a very mild dependence on the crp concentration in the . À .  À m concentration range. figure . (d) shows the increase in the affinity k (¼k /k d ) with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (d), the affinity, k is given by: the fit is reasonable. only three data points are available. the availability of more data points would lead to a more reliable fit. the availability of more data points would lead to a more reliable fit. the affinity, k exhibits only a mild, less than one-half (equal to . ) order of dependence on the crp concentration in solution in the . À .  À m range. figure . (e) shows the increase in the affinity k (¼k /k d ) with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (e), the affinity, k is given by: the fit is very good. only three data points are available. the availability of more data points would lead to a more reliable fit. the availability of more data points would lead to a more reliable fit. the affinity, k exhibits only a mild, less than one-half (equal to . ) order of dependence on the crp concentration in solution in the . À .  À m range. figure . (f) shows the decrease in the dissociation rate coefficient, k d with an increase in the crp concentration in solution in the . À . m  À m range. for the data shown in figure : (f), the dissociation rate coefficient k d is given by: k d ¼ ð : ae : Þ ½crp À : À : ( . f) the fit is poor. there is scatter in the data. this is reflected in the error in the estimated value of the order of dependence of k d on the crp concentration in solution. only the negative sign is applicable since the dissociation rate coefficient, k d decreases with an increase in the crp concentration in solution. figure . (g) and table . (a) and (b) show the increase in the dissociation rate coefficient, k d with an increase in the fractal dimension for dissociation, d fd . for the data shown in figure . (g), the dissociation rate coefficient, k d is given by: the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the dissociation rate coefficient, k d exhibits close to a three and one-half (equal to . ) order of dependence on the fractal dimension in the dissociation phase, d fd . this indicates that the dissociation rate coefficient, k d is very sensitive to the degree of heterogeneity that exists on the biosensor surface in the dissociation phase. figure . (h) shows the decrease in the affinity, k (¼k /k d ) with an increase in the ratio of the fractal dimensions, (d f /d fd ). for the data shown in figure . (h) the affinity, k is given by: k ¼ ðk =k d Þ ¼ ð : ae : Þ ðd f =d fd Þ À : À : : ( . h) the fit is poor. only three data points are available. the availability of more data points would lead to a more reliable fit. the poor fit is expressed as the error in the power to which the ration of the fractal dimensions is raised. only the negative power is applicable since the affinity, k decreases with an increase in the fractal dimension, ratio, d f /d fd . tang et al. ( ) have recently developed an integrated automatic electrochemical immunosensor array for the detection of five hepatitis virus antigens: hepatitis a virus (hav), hepatitis b virus (hbv), hepatitis c virus (hcv), hepatitis d virus (hdv), and hepatitis e virus (hev) (alavian and ballantian, ) . tang et al. ( ) further indicate that hepatitis viruses are one of the leading causes of mortality (bilora et al., ) . thus, an early diagnosis for hepatitis b viruses is critical. tang et al. ( ) further emphasize that the simultaneous determination of multiple virus antigens is helpful in clinical diagnosis since the patient usually suffers from multiple virus antigens (cornberg et al., ; gitlin, ) . tang et al. ( ) emphasize that potentiometric assays are highthroughput systems, are label-free, exhibit low assay cost, and their simplicity permits miniaturization as well as signal quantification (wu et al., ; tang et al., ) . thus, tang et al., ( ) have developed their electrochemical immunosensor array for the simultaneous determination of five-type hepatitis virus antigens in five minutes. the binding of ng/ml of hav to the immunosensor array (tang et al., ) may be modeled by a dual-fractal analysis. figure not shown. the values of the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis ar . ae . and . ae . , respectively. for a dual-fractal analysis, (a) the binding rate coefficients, k and k are . ae . , and . ae . , respectively, and (b) the fractal dimensions, d f and d f are . ae . , and . ae . , respectively. note that as the fractal dimension increases by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity or the fractal dimension on the immunosensor array surface leads to an increase in the binding rate coefficient. a fractal analysis is used to analyze the binding and dissociation (if applicable) kinetics of biomarkers to different biosensor surfaces. both single-and a dual-fractal analyses are used to analyze the binding and the dissociation kinetics. the dual-fractal analysis is used only if the single-fractal analysis does not provide an adequate fit. for the binding and dissociation of ifn-gamma in solution to the aptamer modification (tuleuova et al., ) , and for a single-fractal analysis, the ( ) binding rate coefficient, k, exhibits close to a third (equal to . ) order of dependence on the fractal dimension or the degree of heterogeneity that exists on the biosensor surface and ( ) the affinity, k (=k/kd), exhibits close to a fifth (equal to . ) order of dependence on the ratio of fractal dimensions, d f /d fd . this indicates that both the binding rate coefficient, k, and the affinity, k, are very sensitive to the nature or the degree of heterogeneity that exists on the biosensor surface. for the binding of different concentrations of ca-ii anhydrase in solution ( ) the binding rate coefficient, k, exhibits a mild (equal to . ) order of dependence on the ca-ii anhydrase concentration in solution, ( ) the binding rate coefficient, k, exhibits close to a second (equal to . ) order of dependence on the fractal dimension, d f , that exists on the biosensor surface, and ( ) the ratio of the binding rate coefficients, k /k , exhibits higher than a third (equal to . ) order of dependence on the ratio of fractal dimensions, d f /d f . the relationships presented above are typical of the ones presented for the biomarkers for the other diseases analyzed and presented in this chapter. they provide a means by which these rate coefficients or affinities may be manipulated in desired directions in order to improve the different biosensor performance parameters. the more sensitive a biosensor is for a specific biomarker for a particular disease the earlier it may be detected. needless to say the early detection of biomarkers for different diseases should lead to a better prognosis. surely, as expected, there is considerable effort and resources being spent in this direction, and correctly so. alzheimer's: a new theory a new assay design for clinical diagnostics based on alternate recognition elements validation of analytic methods for biomarkers used in drug development transcriptional analysis of an e f gene signature as a biomarker of activity of the cyclin-dependent kinase inhibitor pha- in tumor and skin biopsies from a phase i clinical study engineered knottin peptides: a new class of agents for non-invasive molecular imaging of tumor biomarkers polypeptide conjugate binders for protein recognition multichannel mass 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cellular analyte-receptor binding kinetics using biosensors analysis of the role of interferon-gamma, interleukin and a third factor distinct from interferon-gamma and interleukin in human b cell proliferation. evidence that they act at different times after b cell activation a fractal analysis for the evaluation of hybridization kinetics in biosensors fractal binding and disseriation kinetics for different biosensors applications a dual enzyme electrochemical assay for the detection of organophosphorus compounds using organophosphorus hydrolase and horseradish peroxidase point-of-eare biosensor system for cancer diagnostics/prognostics simultaneous determination of five-type hepatitis virus antigens in min using an integrated automatic electrochemuical immunosensor array observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex the physiological structure of human c-reactive protein and its complex with phosphocholine development of an aptamer beacon for detection of interferon-gamma chemical-biological interactions sars associated coronavirus transmitted from human to pig electrochemical biosensors; towards point-of care cancer diagnostics cytochrome c mutants for superoxide biosensors low noise detection of biomolecular interactions with signal locking surface plasmon resonance low noise detection of biomolecular interactions with signal-locking surface plasmon resonance key: cord- -ktl jw v authors: coccia, eliana m.; battistini, angela title: early ifn type i response: learning from microbial evasion strategies date: - - journal: seminars in immunology doi: . /j.smim. . . sha: doc_id: cord_uid: ktl jw v abstract type i interferon (ifn) comprises a class of cytokines first discovered more than years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. as such, their induction downstream of germ-line encoded pattern recognition receptors (prrs) upon recognition of pathogen-associated molecular patterns (pamps) is a hallmark of the host antiviral response. the acknowledgment that several pamps, not just of viral origin, may induce ifn, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. acting in both autocrine and paracrine manner, ifn interferes with viral replication by inducing hundreds of different ifn-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. on the other hand an inverse interference to escape the ifn system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the ifn pathway, that result in progression of infection or establishment of chronic disease. in this review we discuss the interplay between the ifn system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies. the ability of the host to respond to invading pathogens relies on the activation of the innate immune system that orchestrates adaptive immune responses for pathogen clearance. in recent years, our understanding of the mechanisms involved in the activation of the innate response has evolved significantly with the identification and characterization of the mammalian system of pathogen recognition. the innate immune system detects the presence of a pathogen through a set of germline-encoded membrane-associated or cytoplasmic receptors, termed pattern recognition receptors (prrs) that are engaged by microbial-derived products named pathogenassociated molecular patterns (pamps). major classes of prr include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlr), c-type lectin receptors (clrs), retinoic-acid inducible gene (rig)-i-like receptors (rlrs) and a growing list of cytosolic dna-sensing receptors [ ] [ ] [ ] [ ] [ ] [ ] [ ] . upon engagement, these receptors recruit a number of adaptor proteins to signal downstream and activate three major pathways: the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b), the mitogen-activated protein kinases (mapks) and the ifn regulatory factor (irf) pathway [ , ] . downstream tlrs and rlrs, type i ifn and pro-inflammatory cytokines are mainly produced, while nlrs predominantly activate inflammasomes, resulting in the release of il- and multiple inflammatory cytokines (fig. ) . based on the structure of their receptors, interferons are broadly classified into three groups, type i, ii and iii ifns. type i ifn comprises the largest ifn class that includes ifn-␣, constituted by several partially homologous genes and ifn-␤, ifn-, ifn-, ifnrepresented by a single gene. the ifn-␣ and ifn-␤ are the best characterized as antiviral and the most broadly expressed [ ] [ ] [ ] and will be referred as ifn-i from now on. the ifn-␥, the only type ii ifn, is released by activated t and nk cells; type iii ifns, which include ifn- - , similarly to ifn-i are believed to regulate the antiviral response [ ] . according to the most common view, ifn-i exerts primarily an anti-viral action while ifn-ii acts predominantly on macrophages to induce a microbicidal state against ingested intracellular, non-viral pathogens. anti-microbial ifn-i activity is not intrinsic, but mediated, in both autocrine and paracrine manner, by a unique set of induced genes named ifn-stimulated genes (isgs) [ , , ] . secreted ifn-i, indeed, binds to a common heterodimeric ubiquitously expressed receptor composed of ifnar and ifnar chains, and initiates a signaling cascade that has been characterized in detail and reviewed elsewhere [ ] [ ] [ ] . briefly, canonical ifn-i pathway results in activation of the janus kinase (jak) family (cytoplasmic tyrosine kinases) that, in turn, activate by phosphorylation the signal transducers and activators of transcription (stats) [ ] . activated stats complex with irf to form the heterocomplex ifn-stimulated gene factor (isgf ), that translocates in the nucleus, binds to upstream sequence elements named ifn-stimulated response elements (isre), and activates the transcription of isgs (fig. ) . these genes act to promote viral clearance and establish an antiviral state in uninfected bystander cells, or to induce apoptosis and several anti-microbial mechanisms in infected cells. they also stimulate cells at the interface of innate and adaptive immunity, such as macrophages and dendritic cells (dcs) that trigger the adaptive response [ , ] . the ability to break in innate immunity before the onset of the adaptive response is, thus, crucial for the survival of virtually all mammalian pathogens. on the other side of the coin, an essential part of the early response to pathogens is aimed at limiting their ability to hijack host cellular machinery and evade the ifnmediated antimicrobial mechanisms. nowadays, while it is largely accepted that also bacteria can induce the production of ifn-i, the role of the ifn system in the pathogenesis of bacterial infections can be either detrimental (mainly in intracellular bacterial infections) or protective (mainly in extracellular bacterial infections). the route and tropism of bacterial infection, viral co-infections and last, but not least, the balance between ifn-i and ifn-ii effects are all determinants of the different outcome [ ] . the ratio between ifn-i and ii species produced in response to infection, might differ as consequence of the capacity of the pathogen to stimulate specific cell types in the infected tissues. moreover, taking into consideration that several antibacterial ifn-ii-induced genes are stimulated to a much lesser degree by ifn-i, it is quite difficult in vivo to separate the activities strictly dependent on one of the two ifn families. a more reliable view consists of a crosstalk between the two pathways: if the bacterium is able to stimulate ifn-i production, generally it occurs early after the infection as an immediate innate immune response, while ifn-ii intervenes later on when immune cells (such as t cell subsets and nk cells) are activated. also due to this complex picture, while a number of viral evasion strategies have been mechanistically defined so far, studies aimed at characterizing the bacterial components that inhibit the ifn system are only recently starting to be elucidated in molecular details [ ] [ ] [ ] . main strategies that pathogens have evolved to disarm the ifn-i response include: (i) blocking ifn-i production by modifying, curtailing or limiting production of their pamps to make them inaccessible to prrs and/or hitting the components of prrs signaling pathways; (ii) interfering with the ifn-i signaling by inhibiting signal transducers; (iii) blocking or disturbing the action of isgs; (iv) hijacking host proteins or components of the ifn system. each of these strategies involves a number of different molecular mechanisms and the combination of more strategies may be necessary to overcome the ifn-i response by a single pathogen. depending on the nature of the pathogen, these countermeasures that tip the balance toward the pathogen, may result in increased replication or in the establishment of a persistent infection. leaving out strategies that envision ifn-i repression due to a general inhibition of cellular gene expression, reviewed elsewhere [ , ] , here we summarize recent findings on evasion tricks utilized by pathogens to specifically subvert the ifn system. a special focus is put on few highly pathogenic bacteria and emerging or re-emerging viruses, which represent a major threat to human health due to the lack of effective vaccines and/or therapeutics. for an in-depth coverage of other pathogens and strategies to escape the host immune response, the reader is referred to more comprehensive and specific reviews [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recent years have seen major advances in our understanding of the innate response to infectious pathogens and specifically of how pathogen recognition promotes ifn-i release [ , , ] . cytoplasmic and membrane-associated prrs recognize a variety of pathogen components. viral pamps mainly consist of nucleic acids originating from the uncoating of infecting virions, the transcription of viral genomes and the replication of genomic intermediates, in the form of single-stranded (ss) and double-stranded (ds) dna, and ss and ds rna, as well as viral glycoproteins. bacterial pamps include various molecules, ranging from lipoproteins, lipopolysaccharide (lps), flagellin and peptidoglycan to unique bacterial nucleic acid structures, such as cyclic dinucleotides (cdns). multiple endosome-associated tlrs (tlr , , and ) are specialized in the detection of viral and bacterial nucleic acids. tlr recognizes dsrna, tlr / are bound by ssrna and tlr by cpgcontaining dna. tlr , and , previously considered sensors only for bacterial components, are now been involved also in recognition of viral ligands and in the induction of ifn-i [ ] . all tlrs contain an intracellular domain, the toll-interleukin- receptor (tir), which recruits one or more tir-containing adaptor proteins to transmit signals downstream. tlr signals through the adaptor tir domaincontaining adaptor inducing ifn-␤ (trif) to activate the two related kinases, inhibitor of kb kinase (ikk)-and tank-binding kinase (tbk ) that mediates activation by phosphorylation of irf and irf . tlr / and tlr use, as adaptor, myeloid differentiation primary-response protein (myd ) that then initiates signaling cascades involving il- r-associated kinases (irak - ) and tnfrassociated factor (traf) / proteins, which finally converge at the activation of the ib kinase (ikk) family members ikk-␣, ikk-␤, ikk-and tbk responsible for activation of nf-b and irf / [ ] . in the cytoplasm, two closely related helicases, retinoic acidinducible gene (rig-i) and melanoma differentiation-associated gene- (mda ), recognize dsrna of many replicating viruses in a tlr-independent manner. rig-i preferentially senses short dsrna and ssrna with a -triphosphate ( -ppp rna), while mda recognizes long dsrna and poly i:c [ ] [ ] [ ] . like viral rnas, bacterial rnas can possess -ppp termini and secondary structures that make them rig-i agonists. consistent with this notion, rig-i can act as a sensor of bacterial rna and may help maintain homeostasis to gut microbiota [ , ] . upon recognition of non-self rna, rig-i and mda are recruited to the mitochondrial antiviral signaling protein (mavs; also known as cardif, visa or ips ), which triggers a signaling cascade that leads to the activation of ikk-and tbk and, in turn, irf / phosphorylation [ ] . stimulator of ifn genes (sting), initially identified as a cytosolic dna sensor, also participates in the rig-i signalling [ ] . sting interacts with the adaptor mavs at the mitochondrial associated membranes (mam) facilitating the recruitment of tbk and the activation of irf [ ] . a more general role of sting in innate immune responses, not only limited to its function as adaptor of rna and dna sensors, has been now established [ ] . in addition to these rna sensors, viral rnas may also be recognized by effector molecules that are themselves ifn-induced proteins with antiviral functions. these molecules include dsrnaactivated protein kinase (pkr) that binds and is activated by dsrna from viruses and bacteria [ ] , ifn-induced tetratricopeptide repeat proteins / / (ifit , ifit , and ifit ) that, as rig-i, bind -ppp rna and may recognize viral mrnas that lack -omethylation [ , ] . a growing list of cytosolic dna sensors then recognizes dna from different sources including viruses, bacteria and apoptotic cells [ , , ] . more than ten dna cytosolic receptors have been proposed so far that include dai (dna-dependent activator of irf), rna polymerase-iii, ifn-inducible interferon gamma-interferoninducible protein (ifi ), sting, extrachromosomal histone h b, leucine rich repeat (in flii) interacting protein (lrrfip ), ku , deah box protein (dhx) and dhx , cyclic gmp-amp synthase (c-gas). as other sensors, dna sensor activation results in the production of ifn-i and proinflammatory cytokines and chemokines via the sting-tbk -irf axis. in addition to the activation of irf -and nf-b-dependent signaling cascades, cytosolic dna can also promote an apoptosis-associated speck-like protein containing a card (asc)-dependent inflammasome-mediated response resulting in the secretion of proinflammatory cytokines [ , , , , ] . the intracellular nlrs scaffold large signaling complexes to mediate innate immunity and inflammatory responses. they may trigger the assembly of inflammasomes and modulate the nf-b, mapk and irf signaling pathways. in particular, nod and nod are important for immune detection of intracellular bacterial pathogens and are also involved in a variety of immune homeostatic functions [ ] . upon the recognition of bacterial peptidoglycans by nod and nod , receptor-interacting serine-threonine kinase (rip ) is activated via cellular inhibitors of apoptosis and (ciap and ), subsequently leading to ubiquitination of nf-b essential modulator (nemo) and the activation of the proinflammatory nf-b pathway. in parallel, the recognition of muramyl dipeptide present in all peptidoglycans, can also lead to the activation of mapk pathway via rip , which contributes to cytokine production. for the induction of ifn-i, nod activated by viral rna, signals through the mitochondrial mavs independently of rip . then, some sensors can aggregate with adaptors as apoptosisassociated speck-like protein containing a caspase recruitment domain (asc) and caspase to form multimeric structures named inflammasome [ ] (fig. ) . as first line strategy to face these sensing pathways, pathogens hide detection by modifying their pamps. they may also degrade/inactivate target key signal transduction hubs by counteracting host-induced post-translational modifications required for signaling molecule activity and use concomitant different strategies as better described below and also discussed by thomas kufer and igor brodsky in this issue. we focus here on some members of coronaviruses, flaviviruses, hepaciviruses, filoviruses and retroviruses as well as bacteria whose pamps could in principle activate the ifn system, but that efficiently betray host sensing and effector pathways (fig. ) . coronaviruses (cov) are large enveloped rna viruses, in the family coronaviridae, of both veterinary and clinical importance. two newly emerging viruses in the family, the severe acute respiratory syndrome cov (sars-cov) and the middle east respiratory syndrome cov (mers-cov), have been recently responsible for severe disease in humans (reviewed in [ ] [ ] [ ] [ ] [ ] ). a common trait in their pathogenesis is the lack of induction of a robust ifn-i response in infected cells [ , ] . to do this, cov have developed multiple strategies (recently reviewed in [ , ] ). rig-i, mda and the host isgs ifit and are critically involved in sensing of cov infection. however, as first line of hiding from recognition, cov encode several highly conserved nonstructural proteins (nsps) implicated in viral rna capping activity in order to examples of viral and bacterial antagonists that block subvert or exploit the ifn system. pathogens affect at every step the ifn system by multiple mechanisms. sites of intervention by several antagonists are indicated. ifn antagonists may prevent prr recognition by hiding or modifying pamps, may inhibit prr signaling by directly targeting adaptors and signaling effectors, may interfere with the ifn signaling by impairing signaling transducers and may block or disturb the action of isgs. some antagonists have more than one cellular target while others target common signaling molecules, effectively blocking ifn induction from a variety of pamps. see the text for more details and references. mimick n -and -o-methylated cap structure of cellular mrnas [ ] . these viral proteins include a rna-triphosphatase, a guanine-n -methyltransferase, and a -o-methyltransferase encoded by nsp , and , respectively [ , ] . consistently, human and mouse cov mutants lacking -o-methyltransferase activity induce higher expression of ifn-i and are highly sensitive to ifn-i effects [ , ] . sars-cov nsp also encode a , exoribonuclease that is involved in rna-proofreading, but probably it also functions in degrading viral pamps, further hiding immune detection [ , ] . similarly, nsp encodes a ribonuclease that may degrade viral pamps [ ] . another strategy used to avoid detection is the replication in protected sites as the double membrane vesicles (dmvs) that the virus induces in the host cytoplasm. in the case of sars-cov these membranes contain the replicase complex and the viral genomic rna suggesting that replication occurs in these sites and the generated nucleic acids are soon after shielded [ , ] . in addition, the highly basic nucleocapsid (n) protein of sars-cov has been reported to directly inhibit ifn-i production induced by both poly(i:c) or sendai virus, by a mechanism that involves steps upstream rig-i. thus, it is conceivable that by binding dsrna, n protein prevents rig-i/mda activation [ ] . others sars-covencoded proteins, as orf- b and orf , may also interfere with the rlr recruitment of the adaptor mavs based on their preferential localization at the mitochondrial membrane even if their mechanism of action is not yet elucidated [ , ] . the sars-cov membrane (m) protein blocks transcription of ifn-i when stimulated by dsrna or members of the rig-i signaling pathway including rig-i, mavs, ikk-, and tbk , but it does not influence the transcriptional activity of the ifn promoter when irf or irf are overexpressed. the physical association of sars-cov m with rig-i, tbk , ikk-, and traf suggests that m protein may prevent the formation of the functional complex with tbk thereby inhibiting activation of irf /irf and ifn-i transcription [ ] . similarly, the papain-like protease (plp) domain contained in the sars-cov nsp protein, an essential component of the viral replicase complex, interacts with the adaptor sting blocking the binding to mavs and the recruitment of the tbk /irf complex [ ] [ ] [ ] . finally, through its deubiquitinating activity, the plp protein removes ubiquitin from several components of the rlr pathway blocking their activation (reviewed in [ , , , ] ). recently, two mers-cov accessory proteins, the orf- a and orf- b products, have also been identified as immunosuppressive factors. mers-cov a is a rna-binding protein that interacts in a mrna-dependent manner with pkr-associated activator (pact), a cellular dsrna-binding protein, which potently stimulates rig-iinduced ifn production by binding to the c-terminal repression domain of rig-i [ ] . so, orf- a inhibits rig-i/mda pathway without a direct binding to the sensor, but by perturbing the function of a stimulator of rig-i signaling as pact [ ] . the orf- bencoded accessory protein is also able to inhibit ifn-i induction in vitro, however, the mechanism involved is not yet elucidated [ ] . the genus flavivirus, of the flaviridae family, includes west nile virus (wnv), dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), tick-borne encephalitis virus (tbev) and several other viruses all causing serious medical problems in humans [ ] [ ] [ ] [ ] . currently, vaccines for humans are available only for yfv, jev, and tbev and no clinically approved antiviral therapy is available for the treatment of flavivirus infections [ ] . in particular, denv and wnv are re-emerging as global life-threatening human pathogens [ , ] . both viruses are sensitive to ifn antiviral effects and the severity of the disease is mostly dependent on the ability to avoid and/or attenuate induction of ifn-i and its effector responses through several viral encoded ifn-antagonists [ , ] . interestingly, some of these strategies are shared with cov. the most relevant prrs for the detection of wnv and denv products, described so far, are the tlr , , and the rlrs, rig-i/mda- . suppression of both tlr-and rlr-mediated ifn induction has been shown to be important for viral replication [ , ] . as cov, flaviviruses contain a cap structure that is generated by a methyltransferase mapped to the n-terminal region of the ns protein [ , ] . through -o-methylation of the viral mrna cap wnv, denv and jev evade ifit -dependent and -independent mechanisms of host restriction in vitro and in vivo [ , ] . moreover, to hide their nucleic acids during the replicative cycle, both denv and wnv induce the formation of convoluted membranes in the endoplasmic reticulum (er) and golgi apparatus that envelop the virus replication complex [ , ] protecting viral nucleic acids from both tlr and rlr recognition, as it occurs along the infection with cov. so far, an antagonism at the level of sensing signaling has been clearly defined only for denv that fails to induce an ifn response in myeloid cells where it replicates, despite other pro-inflammatory cytokines and chemokines are produced [ , ] . the ability to inhibit ifn-i production is due to the viral ns b/ protease that binds and cleaves the adaptor/sensor sting [ ] [ ] [ ] . interestingly, first evidences indicate that the ns b/ proteases of other flaviviruses, as jev or yfv, are not able to cleave sting, while the ns b of yfv can do it [ ] . to the same flaviviridae family belongs the hepacivirus genus, distantly related to flaviviruses, that includes hepatitis c virus (hcv) a major cause of chronic liver disease [ , ] . hcv uses several strategies to efficiently evade innate immunity and this escape is considered the main determinant of viral persistence that leads to a chronic infection in - % of infected people [ , ] . hcv rna can be sensed by different prrs, namely rlrs, tlrs, nlrs, and, as recently reported, by protein kinase r (pkr). rig-i is the best described sensor for the poly u/uc region located within the untranslated region (utr) of the viral rna, along with a -ppp. this region is essential for viral replication and, thus, highly conserved among hcv genotypes. the key viral protein involved in the evasion strategies is the ns / a protease, which consists of ns and ns a. the complex is essential for several steps in the viral cycle including viral rna replication, polyprotein processing and viral assembly [ ] . taking advantage of its serine protease activity, ns / a cleaves the adaptor mavs, preventing its dimerization and downstream signaling [ , ] . after cleavage, mavs dissociates from the mitochondrial associated endoplasmic reticulum membranes (mam) where upon hcv-induced rig-i activation it is recruited and colocalizes with ikk- [ ] , thus impairing ifn-i expression [ ] . importantly, the cleavage of mavs by the viral protease has been confirmed in patients [ ] . to antagonize ifn-i production, ns b protein instead targets sting. hcv ns b, indeed, contains a sting homology domain and interacts with sting in the er blocking sting interaction with mavs and tbk [ , ] . even if the exact molecular mechanism involved in ns b inhibition of sting signaling has not yet been defined in the context of viral infection, ns b likely cooperates with ns / a in targeting the rig-i signaling pathway. interestingly, ns b/ and ns b of other members of the flaviviridae family including denv and yfv as mentioned above also block sting signaling and possess the same sting homology domain, indicating a conserved mechanism of sting antagonisms between flaviviruses and hepaciviruses. intracellularly, both tlr and tlr have been shown to sense hcv rna, depending on the infected cell type considered [ ] . sensing by tlr may occur in liver cells, as hepatocytes, and liver resident macrophages kupffer cells, while tlr sensing occurs predominantly in plasmacytoid dcs (pdcs) and macrophages. as reported above for the mavs adaptor, the serine protease ns / a also cleaves the key adaptor of tlr , trif [ ] , thus preventing an ifn response in productively-infected hepatocytes. in these cells, the hcv-induced mir- has been recently reported to be involved in evasion of ifn-i production and stimulation of hcv replication, upon suppression of myd and irak expression, that is required for the tlr -mediated sensing of the virus [ ] . in macrophages, myd signaling is instead targeted by the ns a protein that, upon the direct binding to the adaptor, impairs the recruitment of irak- and cytokine production in response to tlr ligands [ ] . interestingly, tlr and tlr levels are decreased in patients chronically infected with hcv [ , ] and this has been recently correlated with increased levels of mir- [ ] . hcv-infected cells, however, can trigger ifn-i production in non-productively-infected pdcs in a cell-cell contact-and tlr dependent manner depending on the intracellular hcv rna level of cocultured infected cells [ ] . this production of ifn-i by pdcs may thus account for the strong ifn-i response observed in the liver of infected people [ ] . interestingly, a robust expression of isgs correlated with a decreased response to ifn therapy [ ] supporting a pathogenetic role of a high ifn signature in chronically infected individuals where the virus has established a persistent infection. in contrast, in monocytes and macrophages upon clathrinmediated endocytosis and recognition of the virus by tlr , hcv activates the inflammasome and not ifn-i production in an infection-independent process [ ] . an association between tlr-polymorphisms and cytokine production in response to tlr agonist in vitro has also been reported, supporting a pathogenic role of tlr -mediated sensing in immune cells [ ] . interestingly, this cell-type dependent stimulation of ifn-i and inflammasome in response to hcv infection is also observed in human immunodeficiency virus type (hiv- ) infection that as hcv can establish a persistent infection (see discussion below). pkr has been shown to sense hcv rna very early in infection even prior to rig-i sensing [ ] . pkr is a dsrna binding protein that upon activation phosphorylates the ␣ subunit of the eukaryotic translation initiation factor (eif ␣) to inhibit translation of host capped mrna but not of non-capped mrna, as that of hcv. pkr, upon binding hcv rna and independently of its kinase-activity, interacts with mavs to induce the transcription of a number of early isgs including ifn stimulated gene (isg ), but not ifn-i. isg , in turn, deubiquitinates rig-i inhibiting its functions [ ] . by doing so, hcv blocks sensing by pkr and reinforces hcv evasion from rig- signaling. amongst rna viruses that, as hcv, can establish a persistent infection, hiv- , a lentivirus from the retroviridae family, represents a paradigm for its ability to prevent or circumvent the innate immune response mediated by ifn-i. in spite of evidence that a sustained ifn-i response occurs in hiv-infected patients, it fails to clear the infection in the first place and to prevent the early establishment of long-lived hiv- reservoirs [ ] [ ] [ ] . hiv- has, indeed, developed a number of strategies to block the ifn signaling and the activity of ifn-induced host restriction factors. here, we only briefly summarize these strategies some of which have been only recently discovered, leading to the identification of immune pathways, thus far, unrecognized (as recently reviewed elsewhere [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in the past few years, the knowledge on innate immunity against hiv- has evolved enormously with the recent identification and the characterization of the molecular basis of retroviral recognition by prrs [ , , ] . retroviral replication generates several structural and intermediate molecules as ssrna, hybrids rna/dna, ss and ds dna produced upon reverse transcriptase, that are potentially available for recognition by cellular prrs as "non-self". with the exception of pdcs, which produce high levels of ifn-i upon detection of hiv- ssrna by tlr , in all other immune cells ifn-i production is prevented or barely detectable unless viral countermeasures are disabled. in conventional dcs (cdcs), monocytes and resting cd + t cells, indeed, hiv- sensing is prevented by the restriction factor sterile alpha motif (sam) and the hystidine/aspartic acid (hd) domain-containing protein (samhd ) that blocks reverse transcription or directly degrades genomic rna, thus preventing prr recognition [ , ] . this samhd mediated restriction is overcome by the viral protein vpx [ , ] that is a hiv- and siv accessory protein absent in hiv- . this apparent disadvantage is, however, effectively exploited by the virus that maintains a reward by not replicating in myeloid cells and by reducing the impact of ifn-i production in these cells. the block of productive infection in non-cycling cells where samdh is active, particularly in cdcs, results in lack of maturation and thus in impairment in priming of naive, hiv- -specific t cells for optimal anti-hiv- immunity [ ] . furthermore, in the small fraction of cdcs that become infected, the recognition of hiv- genomic rna by tlr paradoxically licenses hiv- transcription [ ] . in this case, productive dc infection allows an increased transmission to t cells while inhibiting ifn-i production. in monocytes instead, recognition of viral rna by tlr , does not trigger ifn-i production but, as in the case of hcv, leads to the formation of the nlrp inflammasome with activation of caspase- and il- ␤ production, favoring the establishment of an inflammatory milieu that fuels hiv- replication [ , ] . in contrast, in cells that are target of a productive infection, such as macrophages and t lymphocytes, ifn-i production is prevented by an escape mechanism mediated by the hiv- protease that drives rig-i to the lysosomes [ ] . the ssdna derived from proviral dna upon rt can, instead, be sensed by the newly identified dna sensor interferon gammainterferon-inducible protein (ifi ) [ ] , however, hiv exploits the host cytosolic nuclease repair exonuclease (trex ) to digest hiv- dna generated during infection that, thus, does not accumulate at levels sufficient to be detected by ifi , unless trex activity is blocked [ ] . finally, resting cd + t cells are not permissive to virus replication due to the expression of an active samhd that, as mentioned before, degrades genomic rna and prevents efficient rt and recognition of ssdna. however, this prevention of sensing may not be complete and partial recognition of rt intermediates by the ifi sensor not only leads to the initiation of ifn-i production, but also to the activation of the inflammasome, triggering cell death mechanisms including pyroptosis and apoptosis [ ] . finally, the viral capsid exploits two cellular proteins, cyclophilin a and cpsf , and binds just a right amount of both to allow opening of the capsid and rt process, while preventing sensing of the viral cdna before integration with the following production of ifn-i [ , ] . overall, viruses as hcv and hiv- have evolved nifty strategies to dampen the host innate response in cells where a productive infection may take place, while they induce infection-independent mechanisms in non-permissive cells to facilitate the viral life cycle and promote a chronic inflammation. the genus filoviruses from the filoviridae family are among the most virulent known human pathogens and comprise one species of marburg virus and five species of ebola viruses, including zaire ebolavirus (ebov) that is the most lethal and responsible of the recent severe outbreak of hemorrhagic fevers causing up to % mortality in untreated humans [ ] . several immunoevasion strategies that result in total impairment of the innate immune system are responsible for most of ebov virulence [ ] . a major target of these strategies that are exerted by few viral proteins, is the ifn-i response that controls in vivo filoviruses infection [ ] . at the level of viral sensing, the ebov vp inhibits rig-i signaling [ , ] . vp binds with high affinity to dsrna and -ppp dsrna in a sequence-independent manner. four crystallographic structures of ebov vp rbd/iid from zaire and reston viruses and of marv vp rbd/iid have elucidated how vp rbd/iid dimers bind to rna strands and how the dimers mimic the rlr shape, hiding the rna recognition site [ , ] . interestingly, the residues involved in both protein-protein interactions at the rbd/iid dimer interface and involved in dsrna binding are highly conserved among all known ebov and marv species. moreover, all residues that are important for dsrna binding are also crucial for ifn-i inhibition. as the knowledge on the importance of ifn-i in controlling the immunity against bacteria increases, studies aimed at characterizing the evasion mechanisms that these pathogens employ to evade, inhibit, or otherwise manipulate the innate immune response are arising. the mechanisms induced by bacteria for ifn-i expression in different cell types are various and reflect the heterogeneity of host-pathogen interactions established along bacterial infections. while the extracellular bacteria activate ifn signaling mainly through the interaction with molecules present on the cell surface, the intracellular bacteria are recognized as they enter a cell by cell-surface or endosome/phagosome bound receptors or by cytoplasmic pathogen sensors once they escape from these compartments [ ] . here, we report only few examples of bacterial evasion mostly related to signaling pathways converging in ifn-i stimulation. a more exhaustive discussion of bacterial evasion strategies from prr-signaling pathways and inflammasome is provided by the contribution of thomas kufer and igor brodsky in this issue. to elude prr recognition many bacterial pathogens, like viruses, have modified the molecular structure of their pamps. lps, an ubiquitous component of gram-negative bacterial cell wall, is a pamp that is recognized by the tlr /md- complex present on the cell surface. some bacterial species have evolved an alternative form of lps resulting in a weak antagonist of tlr /md- signaling. this strategy is utilized by yersinia pestis, the causative agent of pestis infection. the acylation status of lipid a from hexa-to tetra-acylated is reduced when the temperature increases from • c (flea temperature) to • c (human temperature). this alteration renders lipid a less recognizable lps by tlr and, following transmission from fleas to humans, contributes predominantly to the virulence of the bacterium [ ] . another example is shigella that, after internalization and proliferation within epithelial cells, hypoacylates lipid a to become less visible to the immune system once leave the infected epithelial cells [ ] . in addition to lps, other bacterial components that may induce ifn-i, include bacterial nucleic acids and peptidoglycans. these pamps can be recognized outside or inside the host cells, leading to the activation of distinct signaling pathways [ ] . intracellularly bacterial rna and dna nucleic acids are recognized by the several intracellular receptors that also sense viral pamps, while bacterial peptidoglycans are detected by nod and nod system. most of these pathways then converge in the activation of the sting/tbk /irf axis [ ] . as viruses, bacteria also encode proteins with enzymatic activity that interfere with the activation of adaptor molecules involved in prr signaling. this is the case of the yersinia pestis virulence factor yopj that, besides being a potent inhibitor of the nf-b and mapk signaling pathways, also inhibits tlr-mediated ifn response. as a deubiquitinating protease, yopj prevents or removes the k polymerized ubiquitin conjugates, which are required for traf and traf activation in the signal transduction pathway leading to irf activation [ ] . similarly, the type iii effector ospi of shigella flexneri inactivates by deamination the e ubiquitin ligase ubc , a factor important for traf auto-polyubiquitinylation and activation [ ] . another interesting example is represented by the translocated intimin receptor (tir), which is one of the first type iii effector proteins discovered in a/e pathogens including the enteropathogenic e. coli (epec), enterohemorrhagic e. coli o :h (ehec), and citrobacter rodentium. in addition to the role played in the attachment to the host membrane, this factor shares sequence similarities with conserved regions present in the cytoplasmic tails of inhibitory receptors of the host immune system, such as the immunoreceptor tyrosine-based inhibition motifs (itims). tir utilizes these itimlike motifs to mimic an endogenous innate immunoregulatory mechanism. in particular, tir recruits the host src-homologyregion- -domain-containing phosphatase to the adaptor traf and thus prevents polyubiquitinylation and activation of traf in the ifn-stimulated pathway [ ] . adaptor molecules downstream sensors transmit signals to classical ib kinase complex, including nemo/ikk-␥, tank and to the atypical ikk-related kinases ikk-and tbk that trigger activation of nf-b and irfs, respectively [ , ] . ifn-i production downstream of these signaling pathways depends essentially on the presence and activation of irfs and their contribution changes depending on the cell type considered [ ] . the irf family is presently composed of nine mammalian members namely irf to , coded by distinct but related genes that exert a number of functions in the regulation of innate and adaptive immune responses. the irfs with intrinsic antiviral function include irf and irf that are essential for the prr-mediated ifn gene transcription, but also induce some ifn effectors in an ifn-independent manner. irf , as mentioned before, is part of the heterocomplex isgf that drives the expression of most isgs including irf (fig. ) . although irf is itself an isgs, which affects different aspects of the immune response even independently from ifn-i production [ , ] , it also represents a positive regulator of ifn-i gene expression in response to specific stimuli in a cell type specific manner [ ] . moreover, irf plays a crucial role in regulating mavs-dependent signaling from peroxisomes [ ] . in this respect, irf regulates the transcriptional profile of antiviral genes unique to that induced by ifn-i and cooperatively promotes an effective antiviral program against a broad spectrum of viruses [ , ] . irf is instead specifically involved in inflammatory cytokines induction [ ] . given the unique functions exerted by irfs, viruses have evolved strategies aimed at the specific destruction of these transcription factors. with regard to the viruses covered here, most of them inhibit irf activation either indirectly by acting on sensors and elements of the signaling pathway that activate them, as described above, or directly by impairing/hijacking irf activity (fig. ) . as described above, the sars-cov plp affects the activation of both irf and nf-b not directly, as initially suggested [ , ] , but by targeting rig-i, mavs, traf [ ] and, as more recently reported, sting [ , ] . interestingly, this activity is independent from plp protease activity. recently, it has been reported that the plp of mers-cov also suppresses ifn-i transcription by interfering with irf phosphorylation and nuclear translocation [ ] . as sars-cov, mers-cov plp is a viral deubiquitinating enzyme that acts on both k -and k -linked ubiquitination and isg -linked isgylation, two posttranslational modifications that play important roles in regulating the rig-i and sting/irf and nf-b activation [ ] . whether the deubiquitination and deisgylation activity of mers-cov plp are directly responsible for inactivation of irf /nf-b or upstream signaling pathway, it remains unclear. the wnv ns protein inhibits the tlr -induced activation of ifn-i and il- transcription through inhibition of nuclear translocation of irf and nf-b [ ] . recent studies indicate that this effect seems to be dependent on ns domains that control viral replication [ ] . interestingly, in the draining lymph nodes the protein released predominantly from macrophages and dcs can inhibit the innate immune signaling pathways in uninfected cells and impairs cytokine production in response to infection [ ] , thus suggesting that ns could also influence the development of the adaptive immune response directed to wnv. the ns b/ serine protease of denv, instead, blocks the serine phosphorylation and nuclear translocation of irf by directly interacting with ikk-and masking the kinase domain [ ] . two tick-borne flaviviruses, lgtv and tbev, have been recently reported to inhibit irf independently of their ability to antagonize ifn signaling. in particular, a weak expression of irf protein and nuclear localization, without reduction in irf mrna expression, was observed in dcs, an early cellular target of infection [ ] . several hcv proteins interfere with irf activity. the hcv ns protease impairs irf activation by blocking the interaction with tbk and irf [ ] . in addition, the ns protein inhibits, in a dose-dependent manner, ikk--and especially tbk -induced irf phosphorylation [ ] . the basic amino acid region (br ) in the n-terminal region of the core protein is also crucial in inhibiting irf dimerization as well as phosphorylation induced by ndv infection and poly (i:c) [ ] . interestingly, this domain has been identified as the binding region for a dead box protein the ddx , which has been recently found to enhance the tbk /ikk--induced ifn-␤ promoter activity upon binding to the adaptor mavs [ ] . the hcv core also decreased the expression levels of ddx suggesting that the irf inhibition may be mediated by the core effect on ddx . moreover, through binding to ddx , the hcv core protein also promotes hcv replication. thus, the core protein appears to switch ddx from an ifn-inducing mode to an hcv-replication mode [ ] . irf expression is, instead, suppressed by the hcv core at the transcriptional level. this event blocks the expression of several antiviral and immunomodulatory genes of both innate and adaptive immunity and, in doing so, facilitates the establishment of hcv persistent infection [ ] . in line with this hypothesis, accumulating evidence suggests that hcv also targets dcs to control the host antiviral response and trigger persistence. as wnv ns , hcv core is, indeed, a secreted protein found in the peripheral blood of patients with chronic infection that may thus affect directly dc functions. in this context, it has been recently reported that the core protein suppresses ifn-i production in response to tlr agonists and to rig-i stimulated by hcv pamp in a cell culture model of pdcs, through the reduced levels of irf and of phosphorylated stat protein [ ] . the effect on irf is, however, not direct but probably mediated by the reduced levels of ifn-i production by core-stimulated pdcs. whether or not this also occurs along the natural infection and contributes to hcv persistency remains to be determined. to increase virus replication and establish viral persistence and latency, hiv- , besides to dismantle or exploit almost all cell intrinsic innate recognition pathways, as discussed above, also directly hits irfs [ , , ] . in t cells, the virion-associated accessory proteins, vif, vpr and vpu, directly target irf for ubiquitin-associated proteasome degradation [ ] [ ] [ ] . recently, this vpu effect on irf degradation has been, however, challenged and it has been reported that vpu, instead, mediates a partial cleavage of irf in a caspase-dependent manner. interestingly, this cleavage produces a c-terminal fragment that can act as a negative regulator of irf -dependent gene activation [ ] . thus, hiv- , as already reported for several viruses, can also exploit the apoptotic machinery to interfere with irf function [ ] . in myeloid cells, instead, hiv- does not inhibit but, rather, stimulates both irf and irf expression. irf activity is, however, exploited by the virus to induce a distinct subset of isgs that despite displays intrinsic and unique antiviral actions, does not restrict viral infection [ ] . irf is also induced in hiv- -productively infected t cells where it may regulate viral promoter activity even in the absence of the viral transactivator tat driving initial transcription of the viral genome [ , ] . later on, however, when viral replication is mostly accomplished by the viral transactivator, irf is sequestered by tat to accelerate proteasomal-mediated irf degradation (remoli al and battistini a, unpublished) and to quench irf transcriptional activity on target genes [ ] . by so doing, tat disarms the unique antiviral response against viral infections that irf could exert [ ] . a block of ifn transcription in primary cd + t cells may also depend on cd /cd -mediated activation of ikk-. we, indeed, recently reported that ikk-activation results in a peculiar pattern of irf phosphorylation in t cells, including a splicing isoform of irf , which may function as an inhibitor of ifn-␤ expression, and phosphorylation of irf that blocks its activity on ifn-i promoter [ ] . the ebov vp , in addition to prevent rlr recognition, inhibits ifn-i promoter activation mediated by tbk- /ikk-overexpression but not by a constitutively active irf , strongly suggesting a specific inhibition of the kinase activity of tbk- /ikk-. indeed, vp interacts with the tbk- /ikk-kinase domain and functions as a substitute substrate, thus inhibiting both the kinase activity and the binding of the physiological irf / substrate [ , ] . notably, mutations of vp residues, involved in this ifn antagonism, do not alter the function of vp in viral replication and transcription [ ] . in dcs, vp targets irf . by interacting with the small ubiquitinlike modifier sumo e enzyme ubc and the e ligase pias , vp promotes irf sumoylation, a post-translational modification that prevents irf translocation into the nucleus and, in turn, ifn-i gene transcription. a similar effect of vp was also reported for irf [ ] . this vp activity is independent of its ability to recognize dsrna and maps to the n-terminus, which is essential for interactions with both irf and pias . interestingly, sumo modification of irf / is a part of the negative feedback loop of normal ifn-i signaling [ ] that is exploited by ebov to weaken host innate immunity. downstream prrs, bacteria mainly target the mapk pathway and the nemo-ikk-nf-b signaling axis, which primarily induces inflammatory cytokines (reviewed in [ ] and thomas kuper and igor brodsky in this issue). as an example of bacteria that target the irf pathway. listeria monocytogenes suppresses ifn-i gene induction downstream of tlr-triggered myd signaling pathway, acting on irf . indeed, a mapk phosphatase renders irf hypophosphorylated by enhancing the formation of a mapk phosphatase-irf -tbk- ternary complex in response to infection [ ] . in fig. is illustrated the ifn-i signaling pathway downstream the ifn-i receptor (ifnar) (a heterodimer of ifnar and ifnar ) that is activated upon binding of virus-infected cell-secreted ifn-i, and some isgs, including positive and negative feed-back regulators. most of the viruses here covered and some bacteria also target these pathways (fig. ) . upstream the jak/stat pathway, the sars-cov a promotes serine phosphorylation within the ifnar degradation motif and increases ifnar ubiquitination [ ] . the plp from sars-cov has a complex mechanism of interference with the jak/stat pathway. through its de-ubiquitinase activity upregulates the expression of the ubiquitinating enzyme e - k, leading to degradation of the erk kinase that, in turn, interferes with stat phosphorylation [ ] . the orf -encoded protein, instead, antagonizes stat function by interacting and sequestering in the er components of the nuclear import complex, as karyopherin alpha and karyopherin beta . by doing so, orf- competes for the binding of the nuclear import complex to stat , thus inhibiting stat nuclear import [ ] . however, the majority of these evidences have been obtained by overexpression or stably expression of individual viral components in cell culture, which represents an experimental setting that may not accurately reflect the innate immune signaling occurring during sars-cov infection in vivo. wnv interferes with ifnar complex by promoting phosphorylation-dependent ubiquitination and degradation of ifnar . this effect is mediated by the hydrophobic ns a and ns b proteins that potently induce the unfolded protein response (upr). this pathway is physiologically induced by different stimuli, including the accumulation of misfolded proteins in the er [ , ] that, as mentioned before, represents the site of flaviviruses replication. activation of the upr pathway inhibits ifn activation and induces a general er stress response, thus facilitating viral replication. the methyltransferase domain of ns from langat virus and jev, instead, binds directly to ifnar through its methyltransferase domain and inhibits the activation of kinases associated to the receptor [ , ] . several wnv non structural proteins, as ns a, ns b, ns , ns a, ns b and ns , have been reported to prevent the phoshorylation of jak , tyk and, as a consequence, the activation of stat / (recently reviewed in [ ] ). likewise, expression of denv nonstructural protein ns a, ns a, or ns b proteins impairs the jak/stat signaling pathway by reducing the phosphorylation and nuclear translocation of stat [ ] . phosphorylation of stat is also blocked by denv ns through inhibition of jak and tyk activity [ ] . ns also binds to the coiled-coil region in the first half of the human stat protein and acts as a bridge between ubr- , a member of the n-recognin family, and stat leading to stat ubiquitination and proteasomal-mediated degradation [ , ] . interestingly, only proteolytically-processed ns can efficiently mediate stat degradation, though both unprocessed and processed ns proteins are able to bind stat . the jev ns protein greatly reduces tyk and, partially, stat phosphorylation, probably through its phosphatase activity [ ] . the tbev ns , instead, blocks stat phosphorylation by promoting the association with the pdz membrane protein scribble [ ] . by activating the ras/raf/mek pathway, hcv replication has been shown to increase the phosphorylation of a motif contained in the cytoplasmic tail of ifnar , which is involved in controlling the receptor ubiquitin-dependent endocytosis and attenuation of stat / phosphorylation [ ] . several hcv proteins have also been implicated in the regulation of the ifn response pathway interfering directly with the jak/stat signaling. however, contrasting results have been reported that probably stem from the use of different cell lines or different hcv expression/replication systems [ ] [ ] [ ] [ ] [ ] . the core protein has been reported to upregulate the expression of socs , thereby inhibiting tyrosine phosphorylation of stat [ ] , although the decreased stat phoshorylation has not been detected in other studies [ , ] . the hcv core protein, expressed alone, has been reported to directly bind to stat and to prevent its phosphorylation and subsequent expression of downstream isgs [ ] . the ns a, similarly, binds and prevents stat phosphorylation specifically in hepatocyte-derived cell lines [ ] . two strategies are used by ebov vp to limit the jak /tyk mediated activation of stat / and their subsequent nuclear localization. vp binds within the tyrosine-phosphorylated-stat binding region located in the c terminus of members of the npi- subfamily of karyopherin alpha nuclear localization signal receptors preventing their binding and shuttling to the nucleus of tyrosine-phosphorylated-stat [ , ] . the crystal structure of human kpnalpha c terminus in complex with vp has been recently resolved and a unique nonclassical nuclear localization signal binding site on kpna has been identified. this motif is necessary for binding and efficient nuclear import of tyrosinephosphorylated-stat [ ] . ebov vp can also directly bind stat ; whether the binding occurs with the unphosphorylated or phosphorylated stat or both isoforms it is not yet clear [ ] . however, also unphosphorylated stat enters the nucleus to activate and sustain the expression of a number of ifn-induced immune regulatory genes, which are distinct from those activated by the phosphorylated stat . thus, ebov vp binding and inhibition of either forms of stat may be important in the suppression of an antiviral state. notably, in spite of the high similarity of ebov and marv genome organization and high sequence homology between many of the ebov and marv encoded proteins, marv vp unlike ebov vp , does not inhibit jak/stat signaling [ ] . this is consistent with the observation that regions important for karyopherin alpha binding are different between these two vp s [ ] . in contrast, tyrosine phosphorylation of jak and stat is inhibited by the marv matrix protein vp that also inhibits this ifn-ii signaling. interestingly, also jak -dependent and il- -induced tyrosine phosphorylation of stat and stat are targeted by marv vp suggesting that marv may globally inhibit jak dependent cytokine signaling with mechanisms different from that employed by ebov [ ] . among the escape mechanisms used by bacteria to evade immune responses, some have been recently reported to target ifn-i signaling molecules. having found that influenza viruses replicate to a higher efficiency in cells co-infected with staphylococcus aureus, warnking et al. [ ] demonstrated that an impaired stat /stat dimerization is responsible for a poor induction of isg transcription in spite of an abundant secretion of ifn-i driven by the flu virus infection. similarly, the inhibition of the response to ifn-i by mycobacterium tuberculosis was observed in human macrophages and correlated with mycobacterial pathogenicity [ ] . in primary cells and thp- cells, indeed, mycobacterium tuberculosis specifically inhibits ifn-i signal transduction pathway by impairing the activation of stat , while the avirulent mycobacterium bovis bcg fails to do so. alteration in isgf- complex formation was instead observed in human macrophages infected with nonpathogenic lactobacillus rhamnosus gg where only stat homodimers were found. in contrast, the pathogenic streptococcus pyogenes led to formation of not only stat homodimers but also of isgf- [ ] . based on these finding the authors speculated that the efficient induction of ifn-i production and related transcription factor activation by streptococci would lead to fast and effective immune responses that, however, could play a role in the pathogenesis. although the first antiviral isgs were discovered decades ago, until recently the mechanisms of action was defined for only a limited number of isg-encoded proteins. the renewed interest in the innate immune response to retroviruses with the identification of how several host restriction factors may limit retroviral infection [ ] [ ] [ ] , as well as large-scale functional screening of isgs, have identified genes that coordinately control the infection of a range of rna and dna viruses and have begun to dissect their mechanism of action [ , ] . interestingly, some of the most potent antiviral effectors reinforce the system by further inducing ifn or isgs. thus, by directly disarming and/or making the use of individual ifn-induced effector proteins, the antiviral effect of host cells may still be attenuated even though ifn-i is induced, and viruses can ensure protection from both autocrine and paracrine effects of secreted ifn-i. here, we will primarily focus on isgs for which viral countermeasures have been identified in the context of viruses covered in the present review. recent reviews report more extensively on isg viral antagonists and specifically on ifn-induced hiv restriction factors that are not covered here [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pkr is one of the major effectors of the ifn-i-induced antiviral state. upon activation from binding dsrna molecules via a dsrna binding domain, pkr phosphorylates the translation initiation factor, eif ␣, thus blocking cellular and viral protein synthesis in infected cells. due to its ability to bind dsrna molecules pkr is also a nucleic acid sensor, as mentioned above [ ] . viruses have evolved specific mechanisms to inhibit pkr activity or escape its action downstream. these include: the production of small and highly structured rna molecules that prevent the dsrnainduced dimerization and activation of pkr; expression of proteins that bind directly to and inhibit the activity of pkr or pkr activators; proteins that behave as pseudosubstrate and competitive inhibitors of pkr [ ] [ ] [ ] . amongst viruses covered in this review, both antiviral and proviral roles of pkr have been reported for hcv. the hcv ns a and e proteins directly interfere with the antiviral action of pkr. ns a binds directly to pkr, while the glyco-protein e acts as competitive substrate with eif ␣ for pkr binding, resulting in inhibition of pkr kinase activity and in increased hcv replication. the full length ires of hcv rna, which is recognized by pkr, may mediate either activation or inhibition of pkr [ ] . moreover, ns a, by binding to various domains of the ires, can alter the activation of pkr [ ] . another indirect inhibitory strategy mediated by the hcv ires has been recently reported. upon hcv rna sensing, pkr activates the mavs/traf /irf pathway that, however, does not induce ifn but a set of isgs including isg . as mentioned, isg deubiquitinates rig-i to negatively control the rig-i/mavs pathway and prevent uncontrolled detrimental ifn-i expression in physiological conditions. thus, hcv hijacks a protective cellular pathway to curtail host innate response [ ] . through a specific ires-mediated inhibition of eif ␣-dependent translation, the hcv ires also regulates the translational activity of pkr [ ] . eif ␣ is, indeed, essential to the translation of capped mrna, as those of isgs, while non-capped mrnas, as those of hcv, are translated independently from this factor. a general attenuation of isgs expression by hcv ires can be achieved also through a direct activation of pkr [ ] . interestingly, this attenuation of isg expression is observed in acute but not persistent infection, where, instead, a sustained isg expression occurs [ , ] . other proteins from flaviviruses have been shown to inhibit pkr including the ns a protein of jev that physically interacts with pkr and blocks its activation in response to several stimuli [ ] . ebov vp also interferes with the pathway regulated by pkr by blocking and also reversing pkr activation, thereby preventing translational arrest of viral mrnas. this pkr antagonism seems to be functionally different from dsrna binding and irf inhibition [ ] [ ] [ ] . ebov vp also associates with pact (pkr-associated activator). this complex abolishes pact interaction with the cterminal domain of rig-i, which is required for full activation of rig-i [ ] . as for retroviruses, hiv- infection does not activate pkr due to both viral and cellular controls (reviewed in [ ] ). in vitro, pkr is activated by low amounts of tar rna, whereas high concentrations inhibit the kinase function. the viral tat protein also counteracts pkr activation by several other mechanisms: it sequesters the activating dsrna; it can act as a substrate homologue of eif ␣ preventing the pkr mediated inhibition of protein synthesis; it prevents pkr auto-phosphorylation and exploits pkr activity to get phosphorylated and increase its binding to tar rna. moreover, hiv- replicates only in cells that have high levels of the tar rna binding protein (trbp), a strong inhibitor of pkr activation. interestingly, during hiv- infection of lymphocytes, when hiv- replicates at high levels, increased amounts of adar , an ifn-induced rna editing enzyme that binds to pkr to inhibit its activation, have been observed. moreover, pact contributes to pkr dephosphorylation during hiv- replication probably due to its binding to adar [ ] . thus, hiv- has evolved to replicate in cells with high levels of trbp, to induce the expression of adar and to change the function of pact for pkr inhibition [ ] . isg is an ubiquitin-like modifier that is induced rapidly by ifn-i and possesses antiviral activity against a number of viruses. isg antiviral functions include inhibition of virus release, isgylation of both viral and host proteins and immunomodulatory cytokine-like properties in its unconjugated and secreted form, as recently reviewed [ , ] . more than host proteins that are isgylated have been identified including irf , pkr and rig-i. isgylation preferentially targets newly translated proteins and, as a consequence of isgylation, degradation of the target protein is reduced by competition with ubiquitin conjugation [ , ] . to date, only few viral proteins have been shown to be isgylated and functional characterizations of isg conjugation has not been always verified under conditions of endogenous protein expression [ ] . moreover, often, isg -mediated protection might not be a result of direct antagonism of virus replication. as an example, isg has been shown to inhibit the release of hiv- and ebov. this effect is mediated by an ubiquitin antagonism. isg disrupts the ubiquitin-mediated regulation of ebov vp , necessary to produce budding and release of vp vlps [ ] , as well as the ubiquitination of the hiv- gag protein, which is required for the interaction with the cellular protein tsg to mediate hiv- budding and release [ ] . several viruses have, thus, developed countermeasures against isg and/or its conjugation. strategies, identified so far, include viral proteins that bind isg or that remove isg from target proteins (reviewed in [ ] ). sars-cov plp has both deubiquitinating and deisgylating activities [ , ] . recently, the structural basis of recognition and processing of deubiquitin and isg by plp has been reported [ ] . interestingly, despite mers-cov encodes a single plp similar to sars-cov, there is little to no sequence conservation among residues important for the deubiquitinating and deisgylating activity, suggesting that mers-cov plp is likely to recognize and process ubiquitin and isg substrates differently than sars-cov plp [ , ] . however, by affecting this post-translational modification, both viruses may modify cellular protein localization, protein activity and stability as well as signal transduction in order to increase viral replication and severity of infection. nevertheless, although these results stem from in vitro overexpression or mutant studies, the direct evidence for isg antagonism by these proteins remains to be demonstrated during viral infection. despite the well-characterized role in restricting replication of several viruses, isg may actually also promote the replication of specific viruses, as hcv. in hcv infections both anti-viral or proviral effects exerted by isg could be related to the net effect of isgylation on the various viral and host proteins targeted by isg . an antiviral effect has been reported when isg could conjugate to hcv ns a, thereby enhancing the inhibitory effect of ifn-i on hcv replication [ ] . in contrast, several groups have reported that isg and isgylation promote hcv production in a cell culture model independently of upstream ifn signaling [ , ] . although counter-intuitive, this finding may be explained by the observed inhibition of ifn-i induction by hcv upon isg overexpression that negatively controls the rig-i/mavs pathway at the level of rig-i ubiquitination [ ] . a negative regulation of ifn-i expression may also be mediated by usp , an isg displaying a potent inhibitory effect on the ifn-i pathway, to prevent autoinflammatory consequences of uncontrolled ifn-i production. similarly, usp may dampen the detrimental role that the hyperactivation of ifn-i signaling plays in the pathogenesis of some viral and bacterial infections, including hiv- , hcv and mycobacterium tuberculosis. usp is, indeed, stabilized by isg in an unconjugated free form [ ] . in this respect, the suggested potential role of the isg /usp pathway in hcv persistence is consistent with the observation that increased expression of hepatic isgs before ifn treatment is associated with an absent or poor response in patients chronically infected with hcv [ ] . thus, isg /usp pathway might explain the paradox that the preactivation of the endogenous ifn system, while fails to clear the infection, instead, may stimulate hcv production and blunt the effect of exogenous ifn-i. usp is similarly induced during some bacterial infections, including salmonella and mycobacterium tuberculosis. the decreased survival of mice that carry a point mutation in usp results from higher salmonella load in the spleen and liver, an increased inflammatory response and increased ifn-i signaling. similarly, these usp mutant mice are more susceptible to mycobacterium tuberculosis infection and have increased bacterial load in the lung and spleen, elevated inflammatory cytokine production and more severe lung pathology [ ] . in line with these findings, the results of dorhoi et al. [ ] have shown that ifnar deficient mice were protected from death upon aerogenic infection with mycobacterium tuberculosis. moreover, a rather detrimental effect of ifn-i was also found in whole blood of patients with tuberculosis, where a neutrophil-driven, ifn-inducible transcriptional signature was associated with clinical severity [ ] [ ] [ ] . these results thus reveal that some viruses and bacteria, to push replication and persistence, utilize an opposite, but as much as effectual strategy, consisting in enhancing/perpetuating an ifn-i response by targeting negative regulators of ifn-i expression. anti-microbial molecules, such as nitric oxide (no) radicals and reactive oxygen species (ros) mediate the antibacterial properties of ifn-i. the key producers of no is inducible nitric oxide synthase (inos), an enzyme that can be induced by both ifn-i and -ii, although the latter is the conventional inducer that plays a crucial role in fighting the infection of intracellular bacteria [ ] . similarly, ifn-i and -ii induce the subunits of the phagocyte nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which generates ros for killing organisms [ ] . thus, pathogens have evolved several ways of avoiding ros-and no-mediated killing. in spite of the fact that no data are available, so far, on the strategies exploited by bacteria to contrast the ifn-mediated transcriptional regulation of inos and nadph oxidase, a common theme for successful intracellular pathogens is the ability to avoid the colocalization with these harmful host enzymes. intracellular salmonella, which resides within a specialized membrane compartment called the salmonella-containing vacuole (scv) in macrophages, uses a t ss called salmonella pathogenicity island (spi ) to mediate protection from no and ros intermediates [ ] . intracellular organisms have also developed mechanisms to detoxify and repair no-mediated damage [ ] , as well as to avoid the induction of inos activity [ ] . given the crucial role played by these antimicrobial enzymes in contrasting bacterial infection, it is likely that strategies pinpointed by pathogens to inhibit the ifn-driven expression of these molecules will be discovered in the nearest future. to effectively resist the continual microbial threat from the environment, vertebrates possess several defense mechanisms including innate and adaptive immunity. as an essential component of the innate immunity, the ifn system constitutes the first line of defense against a number of pathogens to clear an incoming infection and instructing an ensuing adaptive response. successful pathogens have, thus, evolved sophisticated strategies to subvert and/or exploit the host immune system where blocking the ifn response, in the first place, is required to replicate and survive. the elucidation of some of these strategies has led to the identification of several, thus far, poorly recognized features of the innate immune response. in parallel, with the enormous recent advances in the comprehension of the molecular mechanisms of innate immune responses to pathogens, specific processes by which pathogenic microorganisms subvert these innate immune pathways, including the ifn system, is becoming progressively appreciated and it is reasonable to assume that many more will be discovered in the near future. by learning from the anti-immune strategies of pathogens we can, thus, not only identify key pathogen regulators as useful target to exploit to the host advantage, but we can also unveil weaknesses of host defenses and intervene to more precisely tune the immune response. the number and diversity of pathogen strategies for counteracting at each step the ifn system is stupefying. although beyond the scope of this review to discuss all antagonisms in detail, the ones that we have here reported, represent common and recurrent strategies used by a number of pathogens. this is illustrated by the existence of both viral and bacterial examples of pamp modifications as well as of viral and bacterial proteins that share cellular-like domain or cellular-like enzymatic properties that can compete with the host counterparts to dampen their physiological activities. in this respect, common hubs in the signaling pathways downstream pathogen sensors that trigger ifn-i, as few common adaptors or cofactors and transcription factors, are attractive targets of pathogen antagonism. the recognition of the mechanisms involved in microbial countermeasures may have several translation implications. the definition of microbial ability to elude the detection, as the methylation of their rna caps, can suggest strategies to utilize methyltransferase mutants as successful vaccine candidates against a number of different viruses as already suggested for denv [ ] . many of the pathogen proteins responsible for ifn-i antagonism are also determinants of virulence and pathogenesis and, as such, they are highly conserved and may, thus, constitute attractive targets for the development of promising therapeutics against various clinically relevant pathogens reducing the bias of resistance mutations. in turn, some of the cellular identified targets of pathogen proteins as well as protein interacting partners might turn out to be new drug targets for treating a range of different diseases that disarme common components of the ifn pathway. in this respect, the resolution of the crystallographic structures of viral antagonists in complex with their different viral and cellular ligands, is then crucial for the rational design of new drugs. the system biology approach and the ability to simultaneously investigate diverse pathways has led to appreciate the interconnection between these pathways also in terms of shared components and stimulation by different pathogens. thus, a single therapeutic strategy could modulate multiple pathways to the host benefit. nevertheless, each approach needs to be complemented with effective treatments that also overcome other concurrent strategies that often the same pathogen put in place. on the other side of the coin, recovery of a full innate response must be finely tuned. ifn-i is, indeed, not always protective but can instead play a pathogenic role as reported for some bacterial and viral infections, where an uncontrolled ifn production is a determinant of disease progression. even in these cases, however, insights in the mechanisms involved in turning an ifn protective response into a pathogenetic one, may be as well relevant for nonpathogen-induced diseases, as autoimmunity and inflammatory diseases. in this context, cell death and inflammasome activation have been described as crucial ifn-i-regulated events exploited by both pathogen and host to get their own advantage. despite inflammasome facilitates pathogen clearance and is beneficial to the host, in some instances, ifn-induced non-canonical nlrp inflammasome activation and pyroptosis appear to be detrimental due to excessive cell death, inflammation, and collateral tissue damage in vital organs [ ] . so pathogen proteins themselves or modified versions of them could be used as therapeutics working in suppressing inappropriate immune activation. this would be a bright way of hijacking molecules evolved during pathogen adaptation and associated fitness, to shift the balance to the host advantage. similarly, some identified targets of viral proteins in prr signaling pathways might be turned out to be new targets for treating a range of diseases. to find the way to generally induce an ifn response that is protective against a number of different infectious diseases, may be particularly relevant during an outbreak of unknown etiology or during the arising of newly emerging and re-emerging strains. likewise, finding the key to unlock the detrimental outcome of excessive ifn-i production during an infectious disease can open the way to cure autoimmune and inflammatory diseases. the authors declare no competing 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hepatitis c virus nonstructural protein - a: the swiss army knife of hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus dissociation of a mavs/ips- /visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns - a proteolytic cleavage cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system hepatitis c virus ns b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity immune evasion by hepatitis c virus ns / a protease-mediated cleavage of the tolllike receptor adaptor protein trif hcv-induced mir- contributes to evasion of host immune system by targeting myd and irak hepatitis c virus nonstructural protein a modulates the toll-like receptor-myd -dependent signaling pathway in macrophage cell lines expression of toll-like receptors in chronic hepatitis c virus infection distinct toll-like receptor and expression in peripheral blood mononuclear cells from patients with chronic hepatitis c infection hepatitis c virus infection decreases the expression of toll-like receptors and via upregulation of mir- plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome interferon-induced gene expression is a stronger predictor of treatment response than il b genotype in patients with hepatitis c hiv and hcv 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yopj targets traf proteins to inhibit tlr-mediated nf-kappab, mapk and irf signal transduction the shigella flexneri effector ospi deamidates ubc to dampen the inflammatory response inhibition of tlr signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs triggering the interferon antiviral response through an ikk-related pathway ikkepsilon and tbk are essential components of the irf signaling pathway regulation of immunity and oncogenesis by the irf transcription factor family interferon regulatory factors in immune cell development and host response to infection evidence for licensing of ifn-gamma-induced ifn regulatory factor transcription factor by myd in toll-like receptor-dependent gene induction program peroxisomes are signaling platforms for antiviral innate immunity a diverse range of gene products are effectors of the type i interferon antiviral response regulation of irf- -dependent innate immunity by the papain-like protease domain of the 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interferon regulatory factor- activation by hepatitis c virus core protein basic amino acid region dead/h box (ddx ) helicase binds the rig-i adaptor ips- to up-regulate ifn-beta-inducing potential hepatitis c virus core protein abrogates the ddx function that enhances ips- -mediated ifn-beta induction repression of interferon regulatory factor by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes hepatitis c virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor- and signal transducer and activator of transcription (stat) protein expression battistini, hiv- targeting of ifn regulatory factors human immunodeficiency virus type mediates global disruption of innate antiviral signaling and immune defenses within infected cells hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation vpu-deficient hiv strains stimulate innate immune signaling responses in target cells hiv- vpu induces caspase-mediated cleavage of irf apoptosis as an hiv strategy to escape immune attack hiv infection of dendritic cells subverts the ifn induction pathway via irf- and inhibits type ifn production irf- is required for full nf-kappab transcriptional activity at the human immunodeficiency virus type long terminal repeat enhancer modulation of human immunodeficiency virus replication by interferon regulatory factors intracellular hiv- tat protein represses constitutive lmp transcription increasing proteasome activity by interfering with the binding of irf- to stat ikappab kinase epsilon targets interferon regulatory factor in activated t lymphocytes the ebola virus vp protein inhibits activation of interferon regulatory factor ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- basic residues within the ebolavirus vp protein are required for its viral polymerase cofactor function ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery virus infection triggers sumoylation of irf and irf , leading to the negative regulation of type i interferon gene expression beneficial innate signaling interference for antibacterial responses by a toll-like receptor-mediated enhancement of the mkp-irf axis the sars coronavirus a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type interferon receptor severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferoninduced responses through downregulation of extracellular signal-regulated kinase -mediated signalling pathways severe acute respiratory syndrome coronavirus orf antagonizes stat function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type i interferon receptor a conserved peptide in west nile virus ns a protein contributes to proteolytic processing and is essential for replication inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns as an interferon antagonist identification of residues critical for the interferon antagonist function of langat virus ns reveals a role for the rna-dependent rna polymerase domain inhibition of alpha/beta interferon signaling by the ns b protein of flaviviruses dengue virus ns inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription phosphorylation ns of dengue virus mediates stat binding and degradation dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism tick-borne encephalitis virus ns associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling activation of the ras/raf/mek pathway facilitates hepatitis c virus replication via attenuation of the interferon-jak-stat pathway expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling- hcv structural proteins interfere with interferon-alpha jak/stat signalling pathway expression of hcv structural proteins impairs ifn-mediated antiviral response identification of the nonstructural protein b of hepatitis c virus as a factor that inhibits the antiviral activity of interferonalpha hepatitis c virus inhibits interferon signaling through up-regulation of protein phosphatase a intracellular innate immune cascades and interferon defenses that control hepatitis c virus hcv ns a inhibits interferon-alpha signaling through suppression of stat phosphorylation in hepatocyte-derived cell lines ebola virus vp proteins inhibit the interaction of npi- subfamily karyopherin alpha proteins with activated stat ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling ebola virus vp targets a unique nls binding site on karyopherin alpha to selectively compete with nuclear import of phosphorylated stat the ebola virus interferon antagonist vp directly binds stat and has a novel, pyramidal fold marburg virus evades interferon responses by a mechanism distinct from ebola virus super-infection with staphylococcus aureus inhibits influenza virusinduced type i ifn signaling through impaired stat -stat dimerization inhibition of response to alpha interferon by mycobacterium tuberculosis lactobacilli and streptococci activate nf-kappa b and stat signaling pathways in human macrophages interferon-stimulated genes: roles in viral pathogenesis host restriction factors in retroviral infection: promises in virus-host interaction 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double-stranded rna-dependent protein kinase pkr ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp protein and the rig-i activator pact determines infection outcome multiple levels of pkr inhibition during hiv- replication the pkr activator, pact, becomes a pkr inhibitor during hiv- replication hiv- translation and its regulation by cellular factors pkr and pact the antiviral activities of isg interferon-induced isg pathway: an ongoing virus-host battle positive regulation of interferon regulatory factor activation by herc via isg modification the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg isg inhibits ebola vp vlp budding in an l-domain-dependent manner by blocking nedd ligase activity innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns a the interferon stimulated gene functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment human intracellular isg prevents interferon-alpha/beta overamplification and auto-inflammation activation of endogenous type i ifn signaling contributes to persistent hcv infection contribution of increased isg , isgylation and deregulated type i ifn signaling in usp mutant mice during the course of bacterial infections type i ifn signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics genome-wide expression profiling identifies type interferon response pathways in active tuberculosis an interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis common patterns and disease-related signatures in tuberculosis and sarcoidosis inducible nitric oxide synthase and control of intracellular bacterial pathogens nadph oxidases: an overview from structure to innate immunity-associated pathologies salmonella pathogenicity island -dependent evasion of the phagocyte nadph oxidase modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice rational design of a live attenuated dengue vaccine: -o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques role of type i interferons in inflammasome activation, cell death, and disease during microbial infection we apologize to the many colleagues whose data and influence have been overlooked due to space or our knowledge limitations. a special thank to members of the e.m. coccia's and a. battistini's laboratory for helpful discussion and critical reading of the manuscript and eugenio morassi for preparing drawings. our work is supported in part by grant rf- from italian ministry of health (to emc) and from istituto superiore di sanità (to ab). key: cord- -bjqwwzam authors: zhang, lei; wang, hao; zhang, yi-qing title: against ebola: type i interferon guard risk and mesenchymal stromal cell combat sepsis date: - - journal: journal of zhejiang university-science b doi: . /jzus.b sha: doc_id: cord_uid: bjqwwzam the ebola outbreak in west africa triggered a global crisis. nine countries have reported more than infection cases in total and nearly lives have been lost. the actual death toll is likely much higher than this figure; the death rate is as high as %, considering confirmed cases. the ebola virus launches its destruction by shutting down the host’s innate and adaptive immune systems. the virus then replicates itself out of control and causes a cytokine storm in the host. consequently, the host’s overdriven immune system attacks its own endothelial cells and this leads to multiple organ hemorrhagic damage, the host dies of septic shock finally. under current circumstances where no specific interventions have shown effectiveness against the virus, our opinions are justified in applying a non-specific anti-viral approach during the incubation period of virus infection as an essential protection to put the host’s immune system into an alert state and henceforth to slow down the viral replication. when the viral infection proceeds to the terminal stage, the key factor would be applying a non-specific immune modulation approach to suppress the cytokine storm that causes multiple organ failure, in an attempt to open a time window for the host’s immune system to recover. the ebola outbreak in west africa triggered a global crisis. nine countries have reported more than infection cases in total and nearly lives have been lost. the actual death toll is likely much higher than this figure; the death rate is as high as %, considering confirmed cases. the ebola virus launches its destruction by shutting down the host's innate and adaptive immune systems. the virus then replicates itself out of control and causes a cytokine storm in the host. consequently, the host's overdriven immune system attacks its own endothelial cells and this leads to multiple organ hemorrhagic damage, the host dies of septic shock finally. under current circumstances where no specific interventions have shown effectiveness against the virus, our opinions are justified in applying a non-specific anti-viral approach during the incubation period of virus infection as an essential protection to put the host's immune system into an alert state and henceforth to slow down the viral replication. when the viral infection proceeds to the terminal stage, the key factor would be applying a non-specific immune modulation approach to suppress the cytokine storm that causes multiple organ failure, in an attempt to open a time window for the host's immune system to recover. the ebola virus invades antigen presenting cells (apcs) quietly and turns their alarm system off essentially, so that the immune system remains inactivated toward the virus. the virus then grows uncontrollably and invades many organs. eventually, many premature cells start dying and exploding, and these cells release all their contents, including signal molecules, into the blood. the signals eventually trigger the extreme immune attacks, which cause arteries, veins and capillaries to leak blood and plasma. the host's body temperature drops and blood pressure falls, causing the host to go into a severe septic shock. it has been shown that the ebola virus primarily targets macrophages and dendritic cells (dcs); later, the membrane-associated glycoprotein (gp) in the virus can bind to endothelial cells, causing cell death and vascular permeability. the latter allows the virus to spread to vital organs and execute severe and extensive damage to the body. the symptom of bleeding appears when the virus gains entry into hepatocytes and causes liver failure. although neutrophils, lymphocytes, and natural killer (nk) cells appear to remain uninfected, those cells undergo 'bystander apoptosis', presumably induced by inflammatory mediators and/or the loss of support signals from dcs. dissemination to regional lymph nodes results in further rounds of virus replication, followed by a spread through the bloodstream to dcs, fixed and mobile macrophages in the liver, spleen, thymus, and other lymphoid tissues, resulting in an extensive tissue necrosis (mohamadzadeh et al., ; ansari, ) (fig. ). the ebola virus applies multiple physical and biological mechanisms to evade host innate and acquired humoral and cellular immune responses. interferon (ifn) belongs to the cytokines that are used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. inhibition of the ifn alarm seems to be one of the most important aspects in the pathogenesis of ebola. the virus primarily antagonizes both the ifn-α and ifn-β responses in target cells, especially macrophages, monocytes, and dcs, by utilizing the viral protein vp to block the phosphorylation of the ifn regulatory factor (irf ), which acts as a transcription factor for the ifn production (cárdenas et al., ) , whereas vp to block the ifn-mediated antiviral response (xu et al., ) . macrophages play a critical role in nonspecific defense, and they also help to initiate specific defense mechanisms by recruiting other immune cells such as lymphocytes. massive infection of macrophages and related cells by the ebola virus indicates that it is able to block or evade the cells' innate antiviral mechanisms. the nk cells respond in an antigen-independent manner to viral infections and kill infected cells. nk cell numbers dramatically drop during the course of infection and almost all undergo apoptosis. dcs have a crucial role in both innate and adaptive immunity; however, infected dcs do not produce proinflammatory cytokines or costimulatory molecules, their ability to support t-cell proliferation is thus impaired and the infected dcs undergo anomalous maturation (hoenen et al., ) . impaired apcs function and lymphocyte apoptosis contribute to the failure of specific immune responsiveness. ebola triggers the systemic dysregulation of immunity that likely results in an uncontrolled virus replication. macrophage-produced pro-inflammatory cytokines are the key players of the host defense system against pathogens. in most patients, ebola viral burden elevates by time and triggers an extremely strong immune attack-a phenomenon called 'cytokine storm' (sullivan et al., ) , during which monocytes and/or macrophages produce a massive amount of pro-inflammatory cytokines, including tumor necrosis factor-α (tnf-α) and interleukins (ils), and this figure was created in reference to mohamadzadeh et al. ( ) in combination with our own considerations. nk: natural killer; tnf: tumor necrosis factor; il: interleukin; m-csf: macrophage colony-stimulating factor; mip: macrophage inflammatory protein; mcp: macrophage/monocyte chemotactic protein; no: nitric oxide; sgp: soluble virus glycoprotein chemokines, such as macrophage inflammatory protein α/β (mip- α/β), macrophage/monocyte chemotactic protein- (mcp- ), macrophage colonystimulating factor (m-csf), nitric oxide (no), and eotaxin (baize et al., ) . by now, the body response to ebola infection would be too severe and too late, and the cytokine storm leads to exaggerated inflammatory responses that contribute to lymphoid cell apoptosis and sepsis (ansari, ) . the virus eventually disables the vascular system and causes blood leakage combined with massive viremia and intravascular coagulopathy. the terminal stage of the virus infection usually includes diffusive bleeding and hypotensive shock, which would eventually kill the patients. ebola infection is generally composed of the incubation period, the symptomatic/acute stage, and the convalescent/terminal stage. the virus burden, inflammatory response, and specific antibodies are the main contributors to different outcomes: mortality, survival, or symptomless infection (fig. ) , suggesting that the appropriate intervention strategy in each stage would accordingly be able to control the ebola virus. whether an inflammatory response executes protective or damaging effects depends not only on the specific cytokine profile, but also on a delicate balance between the individual host immune response and the incoming virus (sullivan et al., ) . some patients have a delayed and prolonged inflammatory response that leads to a cytokine storm characterized by extremely high circulating levels of numerous pro-inflammatory cytokines; these patients generally cannot survive in the viral infection (misasi and sullivan, ) . the average levels of the proinflammatory cytokines in these patients' blood have been found to be - times higher than those in the patients who recovered from the viral infection (wauquier et al., ; ansari, ) . survivors tend to have a short-lived, balanced pro-and anti-inflammatory responses characterized by the presence of il- β, il- , and tnf-α in the early clinical course (bray and mahanty, ) , but no ifn-α was detected in either survivors or nonsurvivors (wauquier et al., ) . some viral infection cases are asymptomatic. an immediate, proper inflammatory response is critical to these cases, which is characterized by transient high levels of il- β, il- , tnf-α, mcp- , and mip- α/β in plasma within d of the first putative infectious contact (baxter, ; leroy et al., ; baize et al., ) . it is believed that in these cases, viral replications are suppressed by effective inflammatory responses. why some infected patients can develop a proper inflammatory response, whereas the others cannot, remains unclear. nonetheless, this phenomenon does give clues to the treatment against ebola virus disease (evd)-early activated innate immune responses may prevent the viral infection. prompt and appropriate adaptive immune responses to ebola seem to be important to the resolution of infection. both virus-specific humoral and cellular mechanisms are required for clearing the viral infection; the humoral immunity is more important at the acute stage for halting viral spread, whereas the cellular immunity is more important for eliminating virus-infected cells that could continuously serve as a source of virus (ansari, ) . fatal cases have been found to associate with a defective humoral response without specific igg production, in these cases only low levels of specific igm were detected in % of the patients (baize et al., ; ksiazek et al., ) . survivors were generally associated more significantly with the early emergence of specific humoral responses and regulated activation of cytotoxic cells that coincided with clearance of viral antigens from the blood. survivors produced specific igm as early as in the first d of symptoms onset and specific igg in - d (baize et al., ; ksiazek et al., ) . asymptomatic individuals produced specific igm and igg within - weeks after initial exposure to viral sources, these antibody productions reached only moderate levels one month later (leroy et al., ) . the outcome of an early viral load is unclear as the viral titers in the plasma were undetectable during the incubation period, whereas the virus load was similar between fatalities and survivors during the first days of the symptom stage. typically, million or more copies of the ebola virus per milliliter in plasma can be reached as early as two days after the onset of symptoms in fatal cases. the level of circulating antigens keeps rising until death. moreover, the virus load at or near the time of death can be -fold greater in fatal cases in comparison with nonfatal cases (schieffelin et al., ) . survivors' plasma often contained fewer than copies of the virus per milliliter of plasma. the level of circulating antigens began declining d after the onset of symptoms and dropped to an undetectable level when the patients recovered (baize et al., ; hoenen et al., ) . the viral load was the lowest in asymptomatic individuals, but the virus rna was detectable in these subjects for up to three weeks after initial exposure (leroy et al., ; baize et al., ) . ebola virus, but they undergo massive 'bystander apoptosis' (hoenen et al., ) . a strong depletion of both cd + and cd + lymphocytes and plasma cells was found in fatal cases (geisbert et al., ) , followed by extensive intravascular apoptosis, vascular dysfunction, and loss of endothelial barrier function, which kill the patients (baize et al., ) . in the symptomatic stage, the supportive care is mainly toward aggressive prevention of intravascular volume depletion, correction of profound electrolyte abnormalities, and prevention of the shock complications (fowler et al., ) . there is neither precaution for the incubation period nor effective medication for the terminal stage. we propose that a preventative antiviral intervention for the incubation period may lower the consequent virus burden. furthermore, we propose that an immunomodulatory strategy for the terminal stage may reduce the damages caused by the cytokine storm, thus prolonging the survival time of the patients, making it possible for the patients' adoptive immunity to recover and beat the infection. type i ifns are cytokines that are secreted by infected cells. they induce cell-intrinsic antiviral states in infected and neighbor cells, they also modulate innate immune responses in a balanced manner and activate the adaptive immune system (le bon and tough, ; ivashkiv and donlin, ) (fig. ) . this figure was created in reference to ivashkiv and donlin ( ) in combination with our own considerations ifn: interferon; dc: dendritic cell; isg: interferon-stimulated gene type i ifns have a broad spectrum of antiviral capability, which is able to fight most virus infections. to give an example, the recombinant ifn-α b has been shown to have a significant nonspecific inhibition of herpes simplex virus, influenza virus, and severe acute respiratory syndrome (sars) coronary virus replication (cao et al., ) . the ebola virus blocks the production of type i ifn by apcs including dcs and macrophages; it also blocks the ifn-mediated antiviral response by virus proteins. it is a crucial mechanism whereby the viruses evade attacks from the host immune system directly. however, recombinant ifn-α b ( iu/ml) can suppress ebola replication by -folds in vero cells in vitro, early treatment of ebola-infected cynomolgus with recombinant ifn-α b delayed onset of viremia and death by several days (jahrling et al., ) . in addition, ifn-β treatment was associated with reducing the plasma and tissue viral burden; it thus significantly increased survival time in macaques infected with the ebola virus in vivo (smith et al., ) . collectively, these results indicate that type i ifn may have therapeutic potential: it is reasonable to postulate direct effects on viral replication as well as adaptive immune response. the observation that the virus titers were not detectable in the incubation period indicates that the vast majority of cells were not infected by the ebola virus. based on this fact, we suggest that exogenous administration of type i ifn may induce uninfected cells into an antiviral state. type i ifn might limit the spread of the ebola virus and prolong survival if administered immediately after exposure to ebola viruses. some patients indeed recovered from the ebola infection without receiving specific interventions despite ebola infection's high mortality rate. this fact suggests that appropriate immune responses can help the body heal itself. the outcome highly depends on two cellular-level competing mechanisms: the apoptosis of endothelial cells executed by autoimmune attacks and vascular regeneration by stem cells. interestingly, the patients under the age of years had a lower fatality rate of %, whereas the fatality rate was up to % in those over the age of years, between these two age groups, the fatality rate was % (schieffelin et al., ) . stem cells are the essential for the regenerative processes of almost all tissues and organs. the significant differences indicate that patients' cellular-level healing capacity in different age groups may be relevant to the consumption of age-related changes in the stem cell reservoir. we suggest that treatment with mesenchymal stromal cells (mscs) during the terminal stage of evd should be considered. mscs are mesoderm-origin, multipotent cells that exist in many tissues and are capable of differentiating into several different cell types, and the numbers or potential of msc populations in adult organs decline during aging (stolzing et al., ; toledano et al., ) . after exogenous administration, mscs migrate to injured tissue sites where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. mscs operate through a variety of effector mechanisms on key cells of the innate and adaptive immune systems, mostly through manipulating the cell cycle or inducing maturation arrest without apoptosis (tyndall and pistoia, ) . the therapeutic effects of mscs may depend largely on the capacity of mscs to regulate inflammation and tissue homeostasis via an array of immunosuppressive factors, cytokines, growth factors, and differentiation factors. mscs reduce inflammation by shutting down the tnf-α pathway for immune cell activation and prevent a cytokine storm by inhibiting or disabling t-cell response. mscs reprogram macrophages, neutrophils, nk cells, dcs, t lymphocytes, and b lymphocytes, all of which counteract sepsis (plock et al., ) (fig. ) . mscs can specifically communicate with the inflammatory microenvironment and this immunoregulatory function of mscs is highly plastic . while stimulating tissue repair by mitogenic and angiogenic effects, mscs inhibit ongoing inflammation, apoptosis, and later fibrosis of injured tissue, and support endothelial cell growth and blood vessel repair; this strategy can help avoid the abuse of steroid hormones and various sequelae. it has been reported that graft-versus-host disease (gvhd), systemic lupus erythematosus (sle), and sepsis can be successfully treated by mscs (kebriaei et al., ; sun et al., ; wannemuehler et al., ; pedrazza et al., ) . it would be interesting to use the available nonhuman primate models of evd to test such a therapeutic hypothesis. it has been almost years since the first ebola outbreak in . to date, there are no effective therapeutic or prophylactic interventions available to prevent this infection. several experimental interventions are in early stages of development, and their availability is limited and intermittent (zhang and wang, ) . positive results were observed in several cases where zmapp and tkm-ebola were administrated, but it was unclear whether the positive outcome was due to the drugs or due to better supportive care in western countries than that of africa. clinical trial on an ebola vaccine developed by merck and newlink has been suspended due to unexpected side effects recently. meanwhile, médecins sans frontières (msf) has selected three existing interventions for clinical trials, they are favipiravir approved in japan for treating influenza, brincidofivir approved in the usa for virus treatment, and ebola convalescent serum, and they might all be present less of a supply challenge or are already approved for other purposes. however, preliminary data show that their potency against the ebola virus is limited. the outlook for the development of ebola therapeutics is not optimistic. there are many different kinds of viruses. these viruses mutate their dna/rna signatures and evolve rapidly. there has been no specific therapy for even the most common human viral infection such as hbv, hcv, hiv, hpv, and avian influenza. realistically, the non-specific treatment is an essential strategy against viral diseases in the foreseeable future. when our immune system is given sufficient time for intentional activation when we are exposed to deadly plock et al. ( ) in combination with our own considerations. nk: natural killer; il: interleukin; pge : prostaglandin e ; ido: indoleamine , -dioxygenase; shla-g : soluble human leukocyte antigen-g ; tnfr: tumor necrosis factor receptor; ifn: interferon; egf: epidermal growth factor viruses, there is a good chance that it can gear up and eliminate the viruses by itself. clinical features and pathobiology of ebolavirus infection defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virus-infected patients inflammatory responses in ebola virus-infected patients symptomless infection with ebola virus ebola hemorrhagic fever and septic shock antivirus evaluation and clinical evaluation of recombinant human interferon α b spray ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling caring for critically ill patients with ebola virus disease. perspectives from west africa apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses ebola virus: unravelling pathogenesis to combat a deadly disease regulation of type i interferon responses evaluation of immune globulin and recombinant interferon-α b for treatment of experimental ebola virus infections adult human mesenchymal stem cells added to corticosteroid therapy for the treatment of acute graft-versus-host disease clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kikwit, democratic republic of the congo links between innate and adaptive immunity via type i interferon human asymptomatic ebola infection and strong inflammatory response early immune responses accompanying human asymptomatic ebola infections camouflage and misdirection: the full-on assault of ebola virus disease how ebola and marburg viruses battle the immune system mesenchymal stem cells decrease splenocytes apoptosis in a sepsis experimental model perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation clinical illness and outcomes in patients with ebola in sierra leone interferon-β therapy prolongs survival in rhesus macaque models of ebola and marburg hemorrhagic fever age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies ebola virus pathogenesis: implications for vaccines and therapies mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans the let- -imp axis regulates ageing of the drosophila testis stem-cell niche mesenchymal stem cells combat sepsis plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications advances in mesenchymal stem cell research in sepsis human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis ebola virus vp targets a unique nls binding site on karyopherin alpha to selectively compete with nuclear import of phosphorylated stat forty years of the war against ebola we thank paul leufkens of neurophyxia b.v. (the netherlands) for critical reading of the manuscript. lei zhang, hao wang, and yi-qing zhang declare that they have no conflict of interest.this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord- -lrpfzsog authors: devos, elizabeth; jacobson, lisa title: approach to adult patients with acute dyspnea date: - - journal: emerg med clin north am doi: . /j.emc. . . sha: doc_id: cord_uid: lrpfzsog undifferentiated patients in respiratory distress require immediate attention in the emergency department. using a thorough history and clinical examination, clinicians can determine the most likely causes of dyspnea. understanding the pathophysiology of the most common diseases contributing to dyspnea guides rational testing and informed, expedited treatment decisions. respiratory distress is responsible for nearly million emergency department (ed) visits each year and is one of the most common presenting complaints in the elderly. when a patient presents with dyspnea, the primary task of the emergency physician is to assess for and ensure stability of the patient's airway, breathing, and circulation. in the case of dyspnea, presentations may range from minor symptoms to extremis. rapid assessment may necessitate the use of intubation, bipap, nebulizations, decompression, or other therapies in the immediate period following the patient's arrival, to treat dyspnea. the american thoracic society suggests that "dyspnea results from a . mismatch between central respiratory motor activity and incoming afferent information from receptors in the airways, lungs and chest wall structures." this dissociation can result from increased metabolic demand, decreased compliance, increased dead-space volume, or many other disorders that are discussed later. each patient presenting short of breath uses a different set of phrases to describe the symptoms and examination reveals a different combination of disorders. the clinician's ability to interpret these varying constellations is necessary to provide appropriate treatment to these patients, who are often in serious distress. acute dyspnea, or shortness of breath, is one of the most common chief complaints in the ed. the differential diagnosis includes many disorders that can be divided based on obstructive, parenchymal, cardiac, and compensatory features. a careful history can begin to narrow this wide differential. in addition to common symptoms, consider risk factors such as past medical and family history, trauma, travel, medications, and exposures. schwartzstein and lewis use the analogy of a machine to identify different causes of dyspnea based on pathophysiologic data. dysfunctions of the respiratory system may be caused by faulty controllers, ventilatory pumps, or gas exchangers ( table ) . this table makes it easier to understand the causes of shortness of breath related to respiratory causes. cardiovascular disease manifests as dyspnea by causing disruptions of the system that pumps oxygenated blood to tissues and then transports the carbon dioxide back to the lung. decreases in cardiac output or increases in resistance limit oxygen delivery. similarly, decreased oxygen carrying capacity in anemia plays a role in its presentation with dyspnea. a detailed physical examination also provides important guidance ( table ) . respiratory rate and oxygen saturation are obtained with vital signs. the clinician should assess the patient's work of breathing, looking for any tripoding or retractions. crepitance in the chest may indicate subcutaneous air and pneumothorax. lung sounds such as wheezing, rales, and rhonchi further guide the differential. decreased sounds, hyperresonance, or egophony may also provide additional clues. jugular venous distension, s gallop, and peripheral edema indicate that a patient has fluid overload. conjunctival pallor, capillary refill, and temperature of extremities can provide clues about blood volume and general circulation. pulses must also be assessed. multiple tests are available to narrow the differential diagnosis of acute dyspnea. when using tests to augment clinical decision making, be sure to weigh the information they may provide with any risks involved in performing the tests ( table ) . ultrasonography provides valuable information about the origin of symptoms, and, often, diagnosis in the initial assessment of an acutely dyspneic patient. these images may be obtained during or shortly after initial assessment, potentially other protocols include assessments to assess for other cardiac causes of dyspnea. [ ] [ ] [ ] focused evaluation of global left ventricular function, diastolic function, right ventricular size, and any pericardial effusion facilitates rapid assessment for massive myocardial infarction, cardiac tamponade, and massive pulmonary embolism at the bedside. in addition, inferior vena cava measurement can be used to assess for right-sided heart failure and to estimate central venous pressure. computed tomography (ct) use to evaluate acute dyspnea has increased in the last decade. risks include contrast reactions and nephropathy as well as radiationinduced cancers. recent american college of physicians recommendations advocate avoidance of ct as an initial test to evaluate patients at low risk for pulmonary embolism (pe). further, nearly one-fourth of patients undergoing ct for pe evaluation assess for lung sliding anterior absence of lung sliding occurs with a disruption of the normal sliding of viscera on parietal pleura or separation of the two. in m mode, absence of lung slide is seen as the stratosphere sign (also known as bar-code sign) absence of lung sliding in the presence of a lines necessitates search for pneumothorax. lung point is the ultrasonography finding in which lung slide is seen in the same view with the abolished lung slide and a lines in the same location, indicating the tip of the lung have clinically significant incidental findings. although ct may provide vital diagnostic information, clinicians must not only consider the scan's necessity but also plan appropriate follow-up for any clinically important incidental findings. always consider whether ct is necessary or whether less risky modalities, such as chest radiograph or ultrasonography, will answer pertinent questions. adult patients with acute dyspnea consider the -year-old woman discussed earlier. medics report tachypnea with very poor air movement during transport. as she rolls through the ambulance bay doors, you are already assessing her. adept clinicians can spot respiratory distress from across the room. she is diaphoretic, her shoulders are held adjacent to her ears, and she is breathing extremely rapidly with minimal air movement. you decide devos & jacobson to aggressively treat her for a severe asthma exacerbation, starting bipap ventilation with continuous nebulized albuterol and order adjunct therapies including intravenous steroids, intravenous magnesium, and intramuscular epinephrine. after minutes at her bedside, she begins to breathe more comfortably with the bipap machine and repeat auscultation reveals diffuse wheezing and improved air movement. as she begins to improve, ems returns with another patient. his breath sounds are audible to everyone in the resuscitation bay. he appears diaphoretic and panicked. examination reveals stridor, periorbital edema, tachycardia, and hypotension. immediate intervention for anaphylactic shock begins and, after rounds of epinephrine, fluid boluses, antihistamines, and steroids, he too begins to look better. wheezing, or musical respiratory sounds, typically result from partial airway obstruction. because this obstruction can result from inflammation, secretions, or even a foreign body, patients with noisy or whistling breathing need close evaluation to devos & jacobson determine whether the noise is inspiratory or expiratory, and whether it is from the lower airways or the upper airways. stridor from a swollen airway, foreign body, or other airway obstruction is imminently dangerous. although patients in anaphylaxis may benefit from the nebulized beta-agonist treatment used to treat an asthma exacerbation, it is not sufficient to save their lives. as opposed to wheezing, which is a lower airway expiratory sound, stridor is an upper airway sound transmitted when there is obstruction to the inflow of air during inspiration. the obstruction may be fixed (food bolus; fig. ) or inflammatory (anaphylaxis), but in any situation must be emergently managed. national and world organizations define asthma "by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation." the reversibility of airflow obstruction is the hallmark distinguishing asthma from other obstructive respiratory disorders. in contrast, chronic obstructive pulmonary disease (copd)/ emphysema is defined as "persistent airflow limitation that is usually progressive and associated with enhanced chronic inflammatory responses in the airways and the lungs." these patients also frequently wheeze, but may have a different course of acute and chronic disease. table highlights the differences between these similar, at times overlapping, diseases. asthma is an obstructive disease resulting from increased airway resistance. it is a reversible but recurrent chronic inflammatory disorder that characteristically causes severe dyspnea, wheezing, and coughing. there are main problems in asthma: chronically inflamed airways and hyperresponsive airways. intermittent airflow obstruction in symptomatic patients results in decreased ability to expire, leading to hyperinflation, stenting open the alveoli, and increasing the work of breathing. early in an exacerbation, symptoms are bronchospastic secondary to smooth muscle contraction. as an episode progresses, inflammatory changes in the airways can cause increased airway resistance and lead to vq mismatch (fig. ) . the severity of an exacerbation can be assessed clinically and should dictate how aggressively a patient is treated ( table ) . adult patients with acute dyspnea copd is defined by the global initiative for chronic obstructive lung disease (gold) as "persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases." the pathophysiology in each patient is typically a mix of lung parenchymal destruction, as seen in emphysema, and small airway inflammation with airway obstruction, or obstructive bronchiolitis. an exacerbation of copd presents as dyspnea, cough, and increased sputum production. in the emergent devos & jacobson setting, clinicians must treat the airflow limitation. as with asthma, monitoring of pulse oximetry, degree of respiratory distress, and hemodynamic stability can help clinicians anticipate the degree of severity of a particular exacerbation. more specific testing may also have a role, because radiographs and electrocardiograms may help differentiate other causes of shortness of breath from a copd exacerbation. in addition, an increase in sputum production or the presence of purulent sputum should be treated with antibiotics, regardless of other infectious symptoms. anaphylaxis is a sudden, potentially fatal, allergic reaction involving multiple organ systems. , the second symposium on the definition and management of anaphylaxis lists the following clinical criteria for diagnosis of anaphylaxis: . acute onset of an illness (minutes to several hours) with involvement of the skin, mucosal tissue, or both (eg, generalized hives; pruritus or flushing; swollen lips, tongue, uvula) and at least of the following: a. respiratory compromise (eg, dyspnea, wheeze-bronchospasm, stridor, reduced peak expiratory flow, hypoxemia) adult patients with acute dyspnea b. reduced blood pressure (bp) or associated symptoms of end-organ dysfunction (eg, hypotonia [collapse], syncope, incontinence) . two or more of the following that occur rapidly after exposure to a likely allergen for that patient: a. involvement of the skin-mucosal tissue b. respiratory compromise c. reduced bp or associated symptoms d. persistent gastrointestinal symptoms (eg, crampy abdominal pain, vomiting) . reduced bp after exposure to known allergen for that patient: a. infants and children: low systolic bp (age specific) or greater than % decrease in systolic bp b. adults: systolic bp of less than mm hg or greater than % decrease from that person's baseline it is the respiratory compromise that is relevant to this article, and it is important to recognize that treating the allergic component of these symptoms is necessary to save the patients. now ems is at the back door with a -year-old patient with a history of copd and congestive heart failure (chf). he is in respiratory distress with audible wheezing and tripoding. he is diaphoretic, hypertensive, and has pitting edema to his knees. they have given albuterol with no improvement of his symptoms. acute dyspnea is the most common symptom of patients presenting with heart failure. eighty percent of patients with acutely decompensated heart failure present through the ed with a chief complaint of dyspnea. this symptom is related to both pulmonary and systemic fluid overload and also low cardiac output. american college of emergency physicians clinical policy makes level b recommendations that standard clinical judgment can be improved with the use of a single b-type natriuretic peptide (bnp) or n-terminal pro-b-type natriuretic peptide measurement to rule in or out the diagnosis of chf. however, the true utility, may be in patients with dyspnea not expected to have a chf exacerbation, when finding a positive bnp would change management and allow a faster initiation of treatment. carpenter and colleagues found that the classic constellation of symptoms (jugular venous distension, peripheral edema, rales, and s ) were no more predictive of patients with both pulmonary edema on chest radiograph and an increased bnp level greater than pg/dl than any individual finding alone. although rales were the most sensitive finding tested for either outcome, they had specificity of only about % each. jugular venous distention and s gallop were the individual findings most predictive for pulmonary edema on radiograph or increased bnp level. ultrasonography measurements of the inferior vena cava also improve diagnostic accuracy versus bnp and chest radiograph alone. the medics are back in your department, this time with a -year-old man with cough and fever. his family is worried that he has been eating less and is sleepier than at hospital discharge last week. in pneumonia, the diffusion of oxygen is limited by alveolar infiltrates, leading to shortness of breath. common complaints and findings in community-acquired pneumonia include fever, cough, pleuritic chest pain, and sputum production, along with dyspnea. however, these clinical criteria may have a sensitivity as low as % compared with a chest radiograph. on examination, many patients have crackles or evidence of consolidation. guidelines from the infectious diseases society of america and the american thoracic society, recommend chest radiograph in patients with suspected pneumonia, which may show lobular consolidation, interstitial infiltrate, or cavitation. although infiltrate with suggestive symptoms makes the diagnosis, infiltrate may not be visible initially on patients with volume depletion. it is appropriate to treat empirically for to hours in these cases and to reimage when hydration is restored. the management of pneumonia requires history to allow classification based on the setting in which the illness was acquired. the infectious disease society of america and american thoracic society define the types of pneumonia as follows: hospitalacquired pneumonia (hap) is "pneumonia that occurs hours or more after admission which was not incubating at the time of admission." ventilator-associated pneumonia (vap) arises "more than - hours after endotracheal intubation." in addition, health care-associated pneumonia (hcap) is diagnosed in any patient who is "hospitalized in an acute care hospital for two or more days within days of the infection, resided in a nursing home or long-term care facility, received recent iv antibiotic therapy, chemotherapy or wound care within the past days of the current infection or attended a hospital or hemodialysis clinic." community-acquired pneumonia is not acquired in any of these situations. these classifications identify typical pathogens and guide appropriate initial management. important historical exposures and risk factors to refine treatment are summarized in table . the american thoracic society along with the infectious disease society of america's consensus statement offers important principles in the initial management and evaluation of adult patients with bacterial hap, vap, or hcap; the most important to be accomplished in the ed is to promptly treat with "appropriate and adequate therapy" to decrease mortality. after a brief delay, you see a -year-old woman with shortness of breath and chest pain. she smokes, uses hormonal birth control, and reports that her symptoms started when she came back from a business trip. pulmonary embolism (pe) interferes with both ventilation and perfusion. it ultimately causes circulatory collapse because of obstruction of right ventricular outflow eventually causing increased pulmonary artery pressure and failure of the right then left ventricles. before circulatory collapse, echocardiography can show signs of right ventricular (rv) strain, including dilatation of the right ventricle, rv hypokinesis, paradoxic septal wall motion, mcconnell sign (hypokinesis of the free rv wall with sparing of the apex), and tricuspid regurgitation. dresden and colleagues supported the use of ultrasonography in moderate-risk to high-risk patients to determine whether the patients were appropriate for anticoagulation while awaiting definitive imaging. early anticoagulation is recommended to improve mortality and there is evidence to support anticoagulation before diagnosis in patients with a wells score greater than who will have a delay to diagnosis of more than hour and minutes. , the assessment of patients with dyspnea and concern for pe requires a series of risk stratification. one common method is to use wells criteria (box ) in patients with suspicion for pe; although other stratification tools exist, none has been shown to be clearly superior. when there is low clinical suspicion for pe, perc (pulmonary embolism rule-out criteria) rules or d-dimer testing may be applied. if perc (box ) is negative, or there is intermediate pretest probability for pe with negative high-sensitivity d-dimer, no further testing for pe is required. when further testing is needed (positive d-dimer or high-sensitivity d-dimer not available), negative ct angiogram or low-probability vq scan may be used to rule out pe. in the next bed is a middle-aged woman with diabetes complaining of shortness of breath today. it was associated with some vague nausea and she says that she just does not feel good. angina pectoris is cardiac chest pain in which oxygen demand outweighs myocardial oxygen supply; in this case caused by occlusion of coronary arteries. although typically chest pain is a part of the presentation, dyspnea alone may be the initial complaint, termed an anginal equivalent. in one recent large series of patients undergoing stress testing, patients with dyspnea alone were at increased risk of death from cardiac causes. patients asked simply whether they experienced shortness of breath were considered dyspneic. the subset with no prior known coronary artery disease had more than times the risk of sudden cardiac death versus asymptomatic patients and more than twice the risk of those with typical angina. clinicians should consider past medical history and risk factors when assessing dyspnea for cardiac causes such as acute myocardial infarction and acute coronary syndrome. appropriate testing includes bedside electrocardiogram, troponin, and chest radiograph. the department eventually settles down and you are able to do some charting until a young man comes in with visible respiratory distress. he is tall and thin, smokes regularly, and reports sudden onset of severely painful breathing. pneumothorax occurs when air enters the plural space between the chest wall and the lung. typically only a thin serous layer exists between the visceral and parietal pleura. air enters this potential space only when there is damage to the lung or chest wall, or a gas-producing pleural space infection. the classic risk factors for bleb rupture causing spontaneous heart rate greater than beats/min: . immobilization > days or surgery in last weeks: . history of prior pe/deep venous thrombosis (dvt): . hemoptysis: malignancy with treatment within months or palliative: less than or equal to . pneumothorax are tall men, although smoking has been suggested to increase the risk of rupture by damaging the pleural layer. pneumothoraces may be identified by ultrasonography, chest radiograph, or ct. treatment may be guided by cause, severity, comorbidities, interventions such as positive pressure ventilation, size of the pneumothorax, and patient's preference. recent studies suggest that uncomplicated spontaneous pneumothorax in patients not undergoing positive pressure ventilation may be treated as successfully with needle aspiration as with other more invasive chest drains, regardless of size. tension pneumothorax is a serious event requiring immediate needle decompression to avert loss of cardiac output and arrest. however, recent review shows that the classic presentation of tension pneumothorax with hypotension, absent breath sounds, and deviated trachea may not be immediately seen in patients with spontaneous, unassisted respiration. because of the slower development of the accumulation of air and pressure variations, spontaneously breathing patients may compensate much longer and present atypically, as shown in table . thus, clinicians must remain vigilant. this article focuses on the cardiopulmonary system as the source of the problem in acutely dyspneic patients. it is important to also consider that the appearance of shortness of breath, tachypnea, or other typical symptoms of dyspnea may result from changing metabolic demands. these patients may appear, on the surface, to be in respiratory distress; they may be tachypneic, tachycardic, even pale or diaphoretic. in these cases, the clinician's responsibility is to identify and fix the true problem in order to improve the respiratory symptoms. severely anemic patients have limited oxygen carrying capacity. their bodies therefore experience oxygen hunger, which can manifest as shortness of breath. patients with dysfunctional hemoglobins secondary to irreversibly bound atoms or toxins may also be functionally anemic with the same symptoms. people's bodies attempt at all costs to maintain equilibrium. therefore, in metabolic acidoses (such as diabetic ketoacidosis), chemoreceptors detect acidosis and stimulate the respiratory center to hyperventilate. both the rate and the depth of ventilation often increase, leading to both tachypnea and hyperpnea, at times referred to as kussmaul respirations. this compensatory response is crucial for survival and should not be mistaken for dyspnea. it is equally important to realize that an increase in alveolar ventilation is not always a compensatory response (to acidosis or to primary pulmonary disorders) and hypocapnia may cause primary respiratory alkalosis, from central nervous system compromise, toxins (eg, salicylates), anxiety, or pain. in these patients, imaging rarely reveals a source of dyspnea, but clinical suspicion based on history and examination, including signs such as the fruity breath of ketonemia, the pallor of anemia, or the cyanosis of toxic hemoglobinopathies, directs providers toward appropriate laboratory testing and treatment. in addition, sometimes dyspnea is not dyspnea. acute anxiety and panic disorder can present as shortness of breath, tachypnea, or hyperventilation. patients with panic disorder often describe symptoms similar to those of patients with true airway obstruction despite their normal pulmonary function. it has been suggested that these patients have abnormal proprioception, experiencing dyspnea without abnormal stimuli. however, patients with a history of pulmonary disease can also have pure panic episodes. arterial blood gas may be useful in diagnosing anxiety-related hyperventilation. severe pain can also induce abnormal respiratory patterns. like compensatory problems, pain and anxiety can be managed by managing their causes. treat pain. reduce stress and anxiety with words, behaviors, or, if necessary, medications. however, air hunger and difficulty breathing also make individuals anxious. be sure to avoid premature diagnosis of a purely anxiety-based concern without first evaluating for more dangerous disorders. acute dyspnea presents commonly to the ed and it is imperative that emergency physicians be prepared to stabilize patients' oxygenation and ventilation, which requires careful and efficient consideration of the differential diagnosis. using cues from the history and physical examination, practitioners may guide the work-up and treatment to identify a parenchymal, obstructive, circulatory, or compensatory cause of dyspnea. early use of bedside testing, including ultrasonography, may limit unnecessary tests and save time in determining the best treatment course. thus ensuring both the best care for the patient and also the physician's ability to readily respond to the next case. the epidemiology and outcome of prehospital respiratory distress dyspnea. mechanisms, assessment, and management: a consensus statement chapter : dyspnea rapid evaluation by lung-cardiac inferior vena cava (lci) integrated ultrasound for differentiating heart failure from pulmonary disease as the cause of acute dyspnea in the emergency setting diagnosing heart failure among acutely dyspneic patients with cardiac, inferior vena cava and lung ultrasonography diagnosing acute heart failure in patients with undifferentiated dyspnea: a lung and 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thoracostomy? clinical presentation of patients with tension pneumothorax: a systematic review metabolic acidosis in the critically ill: part . classification and pathophysiology panic anxiety, dyspnea, and respiratory disease value of arterial blood gas analysis in patients with acute dyspnea: an observational study high resolution computed tomography for the diagnosis of community-acquired pneumonia key: cord- -pzjj wxc authors: smith, geof title: antimicrobial decision making for enteric diseases of cattle date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: pzjj wxc diarrhea in neonatal and adult cattle is common and can be caused by several etiologic agents. as diagnostic testing is not always readily available, practitioners must often decide on a course of treatment based on knowledge of the likely pathogen and their own clinical experience. antimicrobials have long been used to treat diarrhea in adults and neonates; however, there is increased pressure to prevent unnecessary use of antibiotics in food animal species. this article reviews existing data on the use of antibiotics given to cattle with enteric diseases to decide when they are necessary and which antimicrobials should be used. antibiotics to calves to prevent diarrhea cannot be recommended. however, the use of certain antimicrobials to treat select cases of calf diarrhea may be effective in reducing mortality and decreasing the severity and duration of diarrhea. unfortunately, it is unlikely that any of the antibiotics that are currently approved for the treatment of diarrhea in the united states would be effective. instead of mass medicating large numbers of animals, antimicrobial therapy should be targeted to specific animals that are likely to develop septicemia or have systemic signs of disease. there are many causes of diarrhea in adult cattle, and the vast majority of these do not warrant antimicrobial therapy. common enteric diseases of cattle include simple indigestion, rumen acidosis, parasites, coccidiosis, bovine viral diarrhea (bvd), winter dysentery, salmonella, paratuberculosis (johne disease), molybdenosis (copper deficiency), and malignant catarrhal fever (mcf), along with a wide range of toxicities including a host of poisonous plants. the only disease on this list that is likely to truly benefit from antimicrobial therapy is salmonella enteritis; however, an argument could be made for bvd and mcf. both these diseases suppress normal immune function and can lead to an increased occurrence of secondary bacterial infections. it is well understood that bvd is associated with the bovine respiratory disease complex and can lead to higher rates of bacterial pneumonia. however, cases of severe salmonella enteritis have also been reported after bvd infection in cattle causing significant mortality. therefore, it would not be inappropriate to administer a broad-spectrum antimicrobial to cattle suspected of having bvd or mcf, likely one that is labeled for metaphylactic use in cattle at high risk for developing respiratory disease. it is also important to note that many toxic cows with severe mastitis, metritis, or peritonitis often have diarrhea that is a direct result of endotoxemia. the mechanism of endotoxin-induced diarrhea is not completely understood; however, it seems to involve both prostaglandins and nitric oxide. the administration of endotoxin leads to abundant accumulation of fluid inside the small intestines of animals, which is thought to be prostaglandin mediated. endotoxin also increases the enzyme activities of nitric oxide synthase in intestinal smooth muscle, which changes the propagation of jejunal contractions resulting in rapid intestinal transit. the diarrhea observed during endotoxemia in cattle is not profuse but is generally described as low volume. in these cases, choosing to use an antimicrobial would likely not benefit the diarrhea or enteric disease present in the cows but would almost certainly be indicated from the standpoint of treating the primary disease condition. despite the limited number of enteric diseases in adult cattle that would benefit from antimicrobial therapy, surveys indicate that diarrhea is a relatively common reason for the use of antibiotics. in the national animal health monitoring survey (nahms) dairy study, mastitis was the most common reason for antimicrobial use on dairy farms followed by lameness, reproductive diseases (metritis), respiratory disease, and then diarrhea or other enteric disease. results of the survey showed that about % of cattle on dairy farms from the survey population had been treated with an antimicrobial for diarrhea in the preceding -month period and % of farms said they routinely had cows that received antimicrobial drugs because of diarrhea. data from the nahms feedlot study indicated that % of feedlots reported diarrhea or other enteric disease in calves after arrival with . % of calves showing evidence of diarrhea. further data from the study indicated that % of calves with diarrhea received treatment upon arrival. when the survey looked into what specific therapy was administered, % of calves received an injectable antimicrobial, while % of the cattle received an oral antibiotic. when reviewing the data of both the nahms dairy and feedlot studies, it becomes clear that enteric disease is not the primary reason for antimicrobial use in adult cattle. however, it is also apparent that diarrhea is one of the top or reasons cattle receive antimicrobials and that at least half of the cattle diagnosed with diarrhea receive either a parenteral and/or an oral antibiotic. most of the time dairy or beef cattle have diarrhea, it is not clear what the cause is, and therefore they are empirically treated with antibiotics. the assumption in many cases is that the animal has salmonellosis or some other bacterial enteritis. although this is certainly true in some cases, it is very likely that most cases of diarrhea are because of simple indigestion caused by an abrupt diet change, moldy feed, spoiled feed, or perhaps a mild grain overload (rumen acidosis). however, simple indigestion is often difficult or impossible to diagnose definitively and therefore cattle are treated empirically with antimicrobials. if salmonella are the main target of antimicrobial therapy in adult cattle with diarrhea, drug selection should ideally be based on the results of susceptibility testing using bacterial strains recovered from that particular dairy or feedlot. broad-spectrum antimicrobials are usually used pending the availability of susceptibility test results. salmonella show variable resistance patterns to ampicillin, amoxicillin, ceftiofur, florfenicol, neomycin, streptomycin, sulfonamides, tetracycline, and trimethoprim-sulfa and general resistance to penicillin, erythromycin, and tylosin. [ ] [ ] [ ] the most recently published data indicated that salmonella isolates from cattle were most commonly resistant to streptomycin, ampicillin, and sulfonamides, whereas resistance to ceftiofur was extremely low. as salmonella are facultative intracellular pathogens, selecting an antimicrobial with good tissue penetration and the ability to attain intracellular therapeutic drug concentrations within macrophages is desirable. in summary, antimicrobials for the treatment of diarrhea in adult cattle are likely being overused at present in the cattle industry. although diarrhea occurs fairly commonly, most causes are unlikely to respond to antimicrobials. treatment should be primarily supportive care, including fluid therapy, anthelmintics if needed, and provision of good-quality pasture or other forages. mortality rates in most cases of diarrhea in mature cattle are low, and the diarrhea generally resolves within a few days. diseases such as paratuberculosis would have a higher mortality but would still not be likely to respond to antimicrobial therapy. however, when cattle have signs of systemic infection such as pyrexia or bloody diarrhea, it may be rational to begin antimicrobial therapy, particularly on farms that have a history of salmonellosis. when examining an adult ruminant with enteric disease, the practitioner should consider the age of the animal; the onset, severity, and duration of diarrhea (acute vs chronic); the number of cattle affected (is this an individual animal or a herd problem); clinical signs in the animal other than diarrhea (does the animal show systemic signs of disease); nutritional history (especially recent changes in the diet), and whether there has been an introduction of new animals (bvd). all these help to determine a list of possible causes for the diarrhea and may help reduce the use of antimicrobial drugs in cattle that are unlikely to benefit from therapy. prudent use of antimicrobial drugs is recommended with an emphasis on establishing a herd diagnosis and conducting susceptibility testing for the specific salmonella serotype or other bacterial pathogen present and choosing an appropriate antibiotic. calf health should be a priority on both beef and dairy farms. despite this importance, the united states department of agriculture dairy study shows a preweaned antimicrobial decision making heifer calf mortality rate of . % and reports that only % of farms can supply an adequate number of replacements from their own herd. although mortality is slightly less in beef calves, % to % still die before weaning. in both beef and dairy calves, diarrhea represents the most common reason for loss due to death before weaning. therefore, practitioners and producers spend a significant amount of time trying to prevent diarrhea and also making sure good treatment programs are in place when diarrhea does occur. the main principles of diarrhea prevention in both beef and dairy cattle include ( ) using a vaccine in late gestation cattle containing enterotoxigenic escherichia coli, rotavirus, and coronavirus; ( ) making sure a good colostrum program is in place ensuring adequate intake of immunoglobulins by the calf; and ( ) decreasing the load of enteric pathogens in the environment through sanitation, hygiene, housing, and pasture management. historically, many producers (particularly in the dairy and veal industries) have used feeding of oral antibiotics to prevent diarrhea and hopefully decrease mortality in newborn calves. however, the practice of continually feeding antibiotics to calves is now prohibited in many countries, and the efficacy of feeding antibiotics to calves as a method of diarrhea prevention has not proven to be effective in recent studies. almost years ago, a thorough review was published on the efficacy of antibiotics for preventing diarrhea and improving weight gain in dairy calves. the investigator concluded that the addition of chlortetracycline and oxytetracycline to milk replacer in the first weeks of life decreased the incidence and severity of diarrhea. the minimum daily doses necessary for efficacy in this study were . to . mg/lb, which led to the routine inclusion of these antibiotics in milk replacers throughout the united states. unfortunately, this study did not look at critical factors such as mortality rate in calves or incidence of diarrhea. the primary benefits of oral antibiotics were found to be higher weight gain and decreased severity and duration of diarrhea. as discussed in a previous review article, there were several studies done in the s and the s using various antibiotics (including ampicillin, chlortetracycline, furazolidone, neomycin, oxytetracycline, and streptomycin) to prevent diarrhea in calves. although the results of these studies varied, only study documented a decrease in mortality rate from diarrhea due to prophylactic oral administration of chlortetracycline. a few studies did find a decrease in the total number of days of diarrhea associated with antibiotics ; however, other studies (particularly with neomycin) found increased rates of diarrhea in antibiotic-treated calves. , quite a few of these older studies found that oral administration of various antibiotics did not change the incidence of diarrhea in calves when compared with untreated controls. more recent studies have found that either oral antibiotics had no effect on decreasing calf diarrhea or in some cases diarrhea rates actually increased in calves fed antibiotics. for example, a study in california fed group of holstein heifers monensin in the starter ration, whereas another group was fed lasalocid and chlortetracycline (aureomycin) for the first weeks of life (in addition to nonmedicated milk replacer or whole milk). antibiotic-treated calves had no difference in average daily gain, feed efficiency, or the proportion of calves treated for diarrhea. in another study, holstein heifers were fed milk replacer medicated with oxytetracycline and neomycin or an unmedicated milk replacer that contained a probiotic (enteroguard-no longer commercially available). once again, body weight gain, feed efficiency, and the incidence and severity of diarrhea were similar between groups. in a third study, dairy calves were divided into groups: medicated milk replacer (neomycin and tetracycline for the first days of life) plus the administration of trimethoprim-sulfamethoxazole, spectinomycin, penicillin, and bismuth pectin for the treatment of diarrhea (referred to as conventional therapy); medicated milk replacer for the first days of life, bismuth pectin for diarrhea, and other antibiotics only in cases of fever or depressed attitude (targeted therapy); nonmedicated milk replacer with antimicrobial treatment of diarrhea (same treatments as the conventional therapy group above); and nonmedicated milk replacer with targeted therapy. calves fed a medicated milk replacer had % more days with diarrhea when compared with calves fed nonmedicated milk replacer. in a survey, about % of dairy farms in the united states fed medicated milk replacers to preweaned heifer calves, most commonly a combination of oxytetracycline and neomycin. however, a new federal regulation that began in restricts the feeding of medicated milk replacers to a period of to days. thus continuous feeding of antibiotics in the milk from birth to weaning is no longer permitted, and this is meant to transition the use of oral antibiotics in calves from prophylactic to therapeutic. medicated milk replacers should now be reserved for the treatment of bacterial enteritis (diarrhea) and bacterial pneumonia in dairy calves and not for prophylactic prevention. since the late s, the european union has prohibited the sale of milk replacers and other animal feeds containing antibiotics. all the feed and milk replacers for dairy cattle must be sold as nonmedicated, and then antibiotics can be added only for therapeutic use (for example, in calves with diarrhea). australia and new zealand also have strict laws regarding the importation of any animal feed, and these products are generally nonmedicated as well. overall, the conventional practice of adding antibiotics to milk or milk replacers for prophylactic use is being discouraged worldwide. most modern studies fail to find any benefit of using antibiotics as a prevention for diarrhea, and their use in this manner should be discouraged. the use of antibiotics as a treatment in calves with diarrhea is a controversial topic with strong opinions on both sides. several articles have been published indicating that antibiotics are contraindicated in calves with diarrhea or that they serve no beneficial purpose. , in contrast, other studies have indicated that antibiotics are effective in reducing mortality rate and speeding recovery in calves with diarrhea. , to begin the discussion, it is important to establish a reason to use antibiotics in calves with diarrhea. the primary treatment goals of an antibiotic in calves with diarrhea would be ( ) to prevent bacteremia and ( ) to decrease the number of coliform bacteria in the small intestine. several studies have reported that a significant number of calves with diarrhea subsequently develop bacteremia. an initial study in the early s reported that colostrum-deprived calves with diarrhea were frequently bacteremic ( / calves or %). in contrast, none of the diarrheic calves in this study that had received colostrum were bacteremic ( . or %). a study conducted on a large calf-rearing facility in california examined dairy calves with severe diarrhea ; of the calves ( %) had failure of passive transfer and ( %) calves were bacteremic (predominantly e coli). another study done in prince edward island, canada, looked at the prevalence of bacteremia in calves with diarrhea ; of the ( %) calves in this study were bacteremic (predominantly e coli). as noted previously, the percentage of calves with bacteremia was significantly higher in the failure of passive transfer group ( / or %) than in adequate passive transfer group ( / or %). taken together these studies indicate that it can be assumed that one-third of the calves with severe diarrhea are bacteremic and that the percentage is likely significantly higher in calves with failure of passive transfer. although some have argued that antibiotic use in calves with diarrhea is inappropriate and leads to the emergence antimicrobial decision making of resistant bacteria, a case can be made that the use of antibiotics to prevent and/or treat bacteremia in calves with diarrhea and systemic signs of disease is warranted. withholding effective treatment (antibiotics) for a life-threatening disease (such as bacteremia in calves with diarrhea) should not be condoned on animal welfare grounds. another potential reason for antibiotic therapy in calves with diarrhea is coliform overgrowth of the small intestine (fig. ) . research conducted in the s documented increased numbers of e coli in the abomasum, duodenum, and jejunum of calves with diarrhea. , more recent studies have consistently found increased numbers of intestinal e coli in calves with naturally acquired diarrhea regardless of the age of the calf or the cause of the diarrhea. , specifically, the numbers of e coli bacteria increase from -to , -fold in the duodenum, jejunum, and ileum of calves with scours, even when rotavirus or coronavirus is identified as the cause of diarrhea. this small intestinal overgrowth of the intestines with coliform bacteria can persist after the pathogen causing the diarrhea is gone. the increased numbers of coliform bacteria in the small intestine of calves with diarrhea is associated with altered small intestinal function, morphologic damage, and increased susceptibility to bacteremia. therefore there is some logic to the use of antimicrobials in scouring calves to decrease the number of intestinal coliform bacteria. this use could potentially prevent the development of bacteremia, decrease calf mortality, and decrease damage to the small intestine, facilitating digestion and absorption and increasing growth rate. several of which would be illegal to use in the united states (ie, chloramphenicol, furazolidone, or marbofloxacin). the results indicated that specific antibiotics were effective in reducing mortality and increasing growth rate when administered to calves with diarrhea. several studies provided evidence that even calves with simple diarrhea (without systemic signs of disease) seemed to recover faster with antibiotics as opposed to calves that did not receive antibiotics. some veterinarians feel that oral or parenteral administration of antibiotics to calves with diarrhea is contraindicated. the arguments most commonly used to support this approach include: ( ) oral antibiotics alter intestinal flora and thereby induce diarrhea or exacerbate existing diarrhea, ( ) antibiotics harm good intestinal bacteria more than bad bacteria, ( ) antimicrobial use in calves with diarrhea is not effective, and ( ) the use of antibiotics provides a selection pressure on the enteric bacterial population likely leading to increased antimicrobial resistance. there is solid evidence to indicate that the use of antimicrobial drugs can decrease mortality in calves and there is no evidence to support the argument that antimicrobials harm good bacteria more than the bad. however, the emergence of resistant bacteria is certainly serious and is something the veterinarian must take into account before treating calves with diarrhea. table contains a list of antimicrobials currently approved for the treatment or prevention of diarrhea in the united states. at present, oxytetracycline administered parenterally and chlortetracycline, neomycin, oxytetracycline, sulfamethazine, and tetracycline administered orally are the only antimicrobials labeled in the united states for the treatment of calf diarrhea. of these, none have been shown to be consistently efficacious in peer-reviewed studies. as discussed above, when treating calves with diarrhea the primary goals of therapy are to ( ) decrease the number of e coli bacteria in the small intestine and ( ) treat potential e coli bacteremia. with these goals in mind, the target of antimicrobial therapy in calves with diarrhea should be coliform bacteria both in the blood and in the small intestine. as none of the approved drugs for treating diarrhea in the united states are likely to be effective, extralabel use is likely justified. some efficacy has been described for oral amoxicillin in the treatment of calves with experimentally induced diarrhea, , but was not effective in the treatment of naturally acquired diarrhea in beef calves. amoxicillin trihydrate ( mg/kg administered orally every h) or amoxicillin trihydrate-clavulanate ( . mg combined drug/kg administered orally every h) for at least days is one antimicrobial approach that likely has some efficacy for calves with diarrhea. amoxicillin is partially absorbed from the calf small intestine with absorption being similar in both milk-fed and fasted calves. high amoxicillin concentrations are found in bile and intestinal contents after oral administration, with lower concentrations in serum. oral ampicillin could also be used, and its efficacy in one study was shown to be equivalent to that of amoxicillin. although very popular in the united states, oral sulfonamides cannot be recommended for treating calves with diarrhea because of the lack of efficacy studies. most antimicrobial susceptibility studies done in the past years indicate that sulfamethazine (and other sulfonamide drugs) would have poor sensitivity against coliform bacteria in the blood or small intestine. the most logical antimicrobial for parenteral treatment of calf diarrhea in the united states is ceftiofur ( . mg/kg given intramuscularly [im] every h) for at least days. ceftiofur is a broad-spectrum antibiotic that is resistant to b-lactamase. the labeled dose maintains plasma concentrations of ceftiofur above the minimum concentration required to inhibit the growth of % of e coli (mic ) in young calves ( . mg/ml). furthermore, % of the active metabolite (desfuroylceftiofur) is excreted into the intestinal tract of cattle providing activity in both the blood and the small intestine. parenteral ampicillin ( mg/kg im every h) is another antibiotic that would be likely to have efficacy in calves with diarrhea. in europe, parenteral enrofloxacin is labeled for the treatment of calf diarrhea, and several studies have documented efficacy with using fluoroquinolone antibiotics in calves with diarrhea. [ ] [ ] [ ] however, it must be emphasized that the extralabel use of fluoroquinolone antibiotics in the united states is illegal and obviously not recommended. historically, gentamicin was also considered an appropriate treatment for use in calves with diarrhea. however, parenteral administration of aminoglycosides cannot be recommended in calves with diarrhea because of the lack of published efficacy studies, prolonged slaughter withdrawal times ( months), potential for nephrotoxicity in dehydrated calves, and availability of other drugs likely to be equally successful (ceftiofur, amoxicillin, and ampicillin). the issue of whether or not to use antibiotics in a calf with simple diarrhea (without systemic signs of disease) is a little more controversial. although there have been studies to show that these calves gain more weight and recover faster than calves not given antibiotics, there are other studies that indicate no benefit to using antibiotics in these cases. , the clinician must weigh any potential benefit of antimicrobial therapy against the possibility of increasing the population of resistant bacteria on the farm. a fairly recent study demonstrated that individual treatment of sick calves with antibiotics increased the level of resistance to e coli isolates; however, the change in antimicrobial susceptibility was only transient. the next logical question is whether or not antimicrobial susceptibility testing should play a role in determining which drug is used to treat calves with diarrhea. historically, culture and susceptibility results from fecal culture have been routinely used to guide treatment decisions; however, it is not clear whether or not this has any clinical relevance. research validating susceptibility testing as being predictive of treatment outcome for calves with diarrhea is currently not available. part of the problem is that our target is coliform bacteria in the blood and small intestine, which are likely different from fecal bacterial flora. older studies have demonstrated that the predominant strain of e coli in the manure of calves with diarrhea usually changes several times during the course of disease. , these studies also show that about % of calves have different e coli strains isolated from the upper and lower parts of small intestine. so it is logical to conclude that fecal coliform isolates are not representative of what is happening in the intestine. another potential problem with using susceptibility testing to guide antimicrobial selection in cases of diarrhea is that most of the bacterial cultures submitted usually come from dead animals, which represent treatment failures and may have already received antibiotics. preferential growth of resistant bacterial strains can start within a few hours after antibiotic administration, and therefore culture results from dead calves may not be representative of the actual clinical problem. to the author's knowledge, the only study that has tried to assess the predictive ability of fecal antimicrobial susceptibility testing found that it was an inaccurate predictor of clinical outcome. in a large group of experiments evaluating the efficacy of amoxicillin for treating calf diarrhea, calves were divided into groups that either received amoxicillin or did not. diarrhea was experimentally induced using enterotoxigenic e coli and smith rectal swab culture, and susceptibility testing was done. most calves ( %) developed diarrhea after challenge; however, in only about % of cases did calves shed the actual challenge strain of e coli. recovery or treatment success in these studies was defined as normal feces within days after the start of treatment, while treatment failure was defined as death or scouring for more than days. among calves in which the e coli cultured from rectal swabs were susceptible to amoxicillin, % died and % recovered with . as the mean number of days scouring. outcomes were not different in calves that had amoxicillin-resistant strains of e coli cultured from rectal swabs with % death loss, % recovery rates, and . scouring days. in calves given a placebo instead of amoxicillin, mortality was significantly increased ( %), recovery rates were decreased ( %), and the number of scouring days was longer ( . ). the investigators concluded that amoxicillin had a significant effect on disease by decreasing mortality and number of scouring days; however, treatment success could not be predicted by whether the e coli cultured from rectal swabs was susceptible or resistant to the antimicrobial being used. two studies have concluded that there was a good correlation between in vitro antimicrobial susceptibility of fecal e coli isolates and clinical response to treatment; however, neither study had data to statistically analyze this association. , in contrast, other studies reported no correlation between in vitro susceptibility results for coliform isolates and response to antimicrobial treatment. , however, these studies did not differentiate enterotoxigenic and nonenterotoxigenic strains of e coli and also failed to do any statistical analysis of the data. there is a significant need for antimicrobial susceptibility data from e coli and salmonella isolates collected from the small intestine of untreated calves with diarrhea. minimum inhibitory concentrations (mic) could then be compared with free antimicrobial concentrations that are actually achievable in the intestinal tract of calves to determine the best drug to use along with the optimal dosing interval. however, it should be emphasized that antimicrobial concentrations can be altered by multiple variables, such as intestinal ph, which may be quite different between healthy calves and those with diarrhea. therefore even after establishing mic values and setting appropriate breakpoints, these need to be validated through clinical trials examining the use of specific antimicrobial drugs in calves with diarrhea as compared to the pathogen isolated and disease outcome. until then the use of fecal culture and susceptibility testing to guide antimicrobial selection for treating calf diarrhea is probably of little value. drug selection is based on knowledge of the likely pathogen (e coli in the blood and small intestine), pharmacokinetics of the drug (can it achieve therapeutic concentrations at the site of infection), and evaluation of the response to treatment (does the animal get better). on farms in which salmonella or e coli septicemia is a problem, looking at susceptibility results from blood cultures is likely much more appropriate than fecal culture. certainly the overuse of antibiotics is a concern, and the overall philosophy in veterinary medicine is to use antibiotics conservatively to preserve the efficacy of these drugs in both animals and humans. based on the need to minimize the use of antibiotics and because of the lack of any demonstrated recent efficacy, the feeding of antimicrobials to calves as a method of diarrhea prevention is not recommended. however, calves with diarrhea and systemic signs of illness should receive antibiotics targeted toward coliform bacteria in the blood (because of likelihood of bacteremia) and the small intestine (because of bacterial overgrowth). a clinical sepsis scoring system to predict bacteremia based on physical examination does not seem to be sufficiently accurate to guide antimicrobial decision making, and therefore the clinician should assume that calves are bacteremic when they exhibit inappetence, coinfection with bovine viral diarrhea virus and mycoplasma bovis in feedlot cattle with chronic pneumonia outbreak of salmonella enterica serotype newport in a beef cow-calf herd associated with exposure to bovine viral diarrhea virus supportive therapy of the toxic cow effect of lipopolysaccharide on diarrhea and gastrointestinal transit in mice: roles of nitric oxide and prostaglandin e effects of endotoxin on regulation of intestinal smooth muscle nitric oxide synthase and intestinal transit united states department of agriculture animal plant health inspection service (usda aphis) feedlot part iv: health and health management on u.s. feedlots with a capacity of , or more head salmonella in calves antimicrobial susceptibility of salmonella isolates recovered from calves with diarrhea in australia prevalence of antimicrobial resistance among salmonella on midwest and northeast usa dairy farms antibiotics as growth stimulants for dairy cattle: a review use of antibiotics to prevent calf diarrhea and septicemia antibiotics and calf diarrhea diarrhea and malabsorption in calves associated with therapeutic doses of antibiotics: absorptive and clinical changes adverse effect of oral antibacterial prophylaxis and therapy on incidence of neonatal calf diarrhea evaluation of an oral glucose-glycine-electrolyte formulation and amoxicillin for the treatment of diarrhea in calves a field trial comparing the effects of supplementation with aureomycin plus lasalocid or monensin on the health and production performance of dairy calves growth and health of holstein calves fed milk replacers supplemented with antibiotics or enteroguard targeting therapy to minimize antimicrobial use in preweaned calves: effects on health, growth, and treatment costs no effect of a homeopathic preparation on neonatal calf diarrhea in a randomized double-blind, placebo-controlled clinical trial a rational approach to treatment of calf diarrhea antimicrobial use in the treatment of calf diarrhea treatment of calf diarrhea: antimicrobial and ancillary treatments observations on the etiology of neonatal diarrhea (scours) in calves bacteriological culture of blood from critically ill neonatal calves model to predict septicemia in diarrheic calves the distribution of the colon-aerogenes group of bacteria in the alimentary tract of calves the bacteriology of the intestinal tract of young calves with special reference to early diarrhea distribution and virulence of escherichia coli in the small intestine of calves with and without diarrhea changes in small intestinal morphology and flora associated with decreased energy digestibility in calves with naturally occurring diarrhea pathogenesis and prevention of infectious diarrhea (scours) of newborn calves amoxycillin: distribution and clinical efficacy in calves a clinical evaluation of antimicrobial agents and temporary starvation in the treatment of acute undifferentiated diarrhea in newborn calves oral absorption and bioavailability of ampicillin derivatives in calves clinical efficacy of amoxicillin in calves with colibacillosis field evaluation of efficacy of marbofloxacin bolus in the treatment of naturally occurring diarrhea in the new born calf comparison of danofloxacin with baquiloprim/sulphadimidine for the treatment of experimentally induced escherichia coli diarrhea in calves efficacy of danofloxacin % injectable solution in the treatment of escherichia coli diarrhoea in young calves in europe field trial evaluating the influence of prophylactic and therapeutic antimicrobial administration on antimicrobial resistance of fecal escherichia coli in dairy calves the typing of e. coli by bacteriophage, its application to the study of e. coli populations of the intestinal tract of healthy calves and of calves suffering from white scours passage of antibiotics through the digestive tract of normal and scouring calves and their effect upon the bacterial flora discrepancy between antibiotic (amoxycillin) resistance in vitro and efficacy in calf diarrhea further observations on the effect of chemotherapy on the presence of drug-resistant bacterium coli in the intestinal tract of calves the sensitivity to chemotherapeutic agents of a further series of strains of bacterium coli from cases of white scours: the relationship between sensitivity tests and response to treatment neonatal diarrhea in calves escherichia coli and salmonella newport in calves: efficacy of prophylactic and therapeutic treatment dehydration, lethargy, or fever. in calves with diarrhea and no systemic signs of illness (normal appetite for milk, no fever), evidence suggests that the clinician continue to monitor the health of the calf and not administer antibiotics unless the calf's condition deteriorates. key: cord- -exmk gmu authors: chan, sophia s.c.; leung, doris y.p.; leung, angela y.m.; lam, cindy; hung, ivan; chu, daniel; chan, chi kuen; johnston, janice; liu, shao haei; liang, raymond; lam, tai hing; yuen, kwok yung title: a nurse-delivered brief health education intervention to improve pneumococcal vaccination rate among older patients with chronic diseases: a cluster randomized controlled trial date: - - journal: international journal of nursing studies doi: . /j.ijnurstu. . . sha: doc_id: cord_uid: exmk gmu abstract background the -valent pneumococcal polysaccharide vaccine is recommended for elders, especially those with chronic conditions. objective the aim of this study was to determine if an additional multi-component health education intervention increases the uptake rate of the pneumococcal vaccination among older patients with chronic diseases. methods a cluster randomized controlled trial was conducted from december to march . the clusters were the individual weeks within five hong kong outpatient clinics over a -week period. a sample of patients aged or above with chronic diseases was recruited. intervention group received a -min brief telephone education intervention before and a -min face-to-face intervention during scheduled medical appointments at the respective clinics. all subjects received standard care including health education leaflets and/or a video show at the clinics. pneumococcal vaccination rate and awareness of the vaccination at -month follow up were measured. results the vaccination rate was higher in the intervention group compared to the control group ( % vs %; relative risk= . , % ci= . – . ), but the two groups did not differ significantly in their awareness of the vaccination at -month follow up ( % vs %, relative risk= . , % ci= . – . ). discussion a nurse-delivered brief health education intervention was effective in increasing uptake of pneumococcal vaccination among older patients with chronic diseases. what is already known about this topic? pneumococcal vaccination has been recommended for elders by the hong kong sar government since october . pneumococcal vaccination is relatively new to people, especially in hong kong. no study on testing the use of health educational programmes to promote pneumococcal vaccination uptake in the asian countries. evaluation of a nurse-delivered health education intervention which aimed to improve pneumococcal vaccination rate among older patients with chronic diseases. the vaccination rate was higher in older patients who received a -min brief telephone education intervention and/or a -min face-to-face intervention than those only received standard care including health education leaflets and/or a video show at the clinics. the two groups did not differ significantly in their awareness of the vaccination at -month follow up. streptococcus pneumoniae causes invasive pneumococcal diseases (ipd) including septicaemia, meningitis and bacteraemic pneumonia in all age groups, especially in children, elders, and persons with chronic illnesses (centers for disease control and prevention, ) . the incidence rate of ipd in older people is triple of those aged - years, and they have the highest risk of death from ipd (robinson et al., ) . globally, ipd had caused . million deaths annually in (world health organization, . in developed countries, the annual incidence rates of ipd range from to per , with higher incidence rates in those aged years ( - per , ), whilst in hong kong the average annual incidence rate of ipd was . per , from to (center for health protection, ). the -valent pneumococcal polysaccharide vaccine (ppv) is recommended for elders, especially those with chronic conditions. ppv is efficacious in reducing the risk of systematic infection in institutionalized elders (hutchison et al., ) , and preventing mortality due to pneumonia (fisman et al., ; jackson et al., ; loeb, ) from observational studies, but its efficacy from randomized controlled trials in older patients with chronic diseases, is still unclear and subject of debate (moberley et al., ) . as pneumococcal infections become increasingly difficult to treat due to drug resistance, vaccination is an important and efficient way for preventing ipd due to s. pneumonia (spindler et al., ) . despite recommendations made by governments in many western countries including finland, sweden, uk, us and some regions in spain to deliver ppv to the elders and patients with chronic diseases, the vaccination rates vary substantially across countries. the uptake rates ranged from % in finland to % in the us (ruutu et al., ; united states department of health and human services, ) . several strategies involving health care professionals in improving the uptake rate of ppv have been proved effective in randomized trials. a computerized system reminding health professionals about the eligibility of patients for ppv increased the uptake rate from . % to . %, while an education program for patients through videotape-brochure was more effective than video-only and the control group (dexter et al., ; thomas et al., ) . a program with educational outreaching visits about the importance of vaccination to practicing physicians and a nurse-delivered education intervention to patients on discharge increased the vaccination rates (siriwardena et al., ; thomas et al., ) . however, to the best of our knowledge, no study on testing the use of a nurse-delivered brief telephone and face-to-face health education intervention to promote ppv uptake in asian countries has been reported. ppv has been recommended for elders by the hong kong sar government since october (hutchison et al., ) . prior to , vaccination for pneumococcal infection was not common and the estimated use of ppv was less than % for those aged over years (ho et al., ) . the uptake rate of influenza vaccination was also very low even after the outbreak of severe acute respiratory syndrome and substantial promotion from the government, as reflected by a study in hong kong which estimated that about % of patients visiting a public clinic did not have influenza vaccination within the past years of the clinic visit (mok et al., ) . consequently, additional efforts, other than mass media promotion, are needed to improve the uptake rate of vaccination. as ppv is relatively new to people in hong kong, it is important to implement and evaluate appropriate health education interventions to promote ppv and improve the vaccination rate, especially in vulnerable older patients with chronic diseases. nurses are the largest group of health care professionals who have the greatest frequency and duration of contact with patients, and thus have a strong potential to influence patients' behaviors. results: the vaccination rate was higher in the intervention group compared to the control group ( % vs %; relative risk = . , % ci = . - . ), but the two groups did not differ significantly in their awareness of the vaccination at -month follow up ( % vs %, relative risk = . , % ci = . - . ). discussion: a nurse-delivered brief health education intervention was effective in increasing uptake of pneumococcal vaccination among older patients with chronic diseases. ß elsevier ltd. all rights reserved. this large cluster randomized controlled trial, therefore, was conducted to test the effectiveness of a nursedelivered multiple component health education intervention on the uptake rate of ppv and awareness of ppv at month follow up among older patients with chronic diseases in hong kong. a single-blinded cluster randomized controlled trial with stratification by clinic was conducted from december to march for weeks (excluding the weeks of christmas and chinese new year) in the five hong kong hospital authority west cluster outpatient clinics. each week of the week study period was randomized to either intervention or control using random numbers generated by http://www.random.org. for each location, five weeks were allocated to intervention and five to the control. thus, there were clusters (i.e. study weeks) in the intervention and clusters in the control, making a total of clusters. two of the participating clinics are specialist outpatient clinics (sopcs) providing acute patient care and specialist services in cardiothoracic and pulmonary diseases, respectively. the remaining three clinics are general outpatient clinics (gopcs) which provide comprehensive primary medical care, serving an estimated population of . million in the central, western and southern districts of hong kong island, hong kong. subjects were eligible for inclusion if they were aged years or above, had chronic diseases (hypertension, cardiac diseases, diabetes, respiratory diseases, kidney diseases, liver diseases, and cancers), had no prior ppv and had scheduled medical appointments at the study sites during the study period. older patients who were cognitively impaired, not able to communicate effectively, had any febrile respiratory illnesses or other active infections were excluded. this study was approved by university of hong kong and the hong kong west cluster of hospital authority institutional review board on november , . lists of all patients with medical appointments from : am to : pm of the five study sites each of the study weeks were obtained from the respective clinics. one week before the medical appointments, two groups of trained research nurses (rns) of the project called the eligible subjects in the intervention and control groups separately to confirm whether their self-reported medical diagnosis matched the inclusion criteria, and if yes, they were invited to participate in the study while those could not be reached before their scheduled appointments were considered as missing the opportunity for enrollment into the study. after obtaining oral consent, the project rns responsible for the intervention group administered the baseline questionnaire and delivered the telephone health education intervention. the project rns responsible for the control group administered the baseline questionnaire only. the baseline questionnaire collected information on subjects' awareness of ppv, perceived stress, self-efficacy to manage disease in general, history of diseases and vaccinations, lifestyles and demographics. during the selected -week medical appointment sessions of the participating clinics, all patients, regardless of whether they had consented to participate in the study, received either the face-to-face health education intervention or standard care including promotional leaflets, poster displays, and health education video show, according to the cluster randomization. the -min face-to-face health education was delivered by the project rns or a group of two medical/nursing students (during their clinical practicum) supervised by one of the project rns in case there were students attending clinical practicum at the study clinics. however, only those who had consented and completed the baseline questionnaires before the medical appointments were included in the current study. ppv, if accepted, was administered by the nurses of the participating clinics to the patients according to the respective clinical procedures. all subjects were contacted via telephone at -week and -month follow-up after their medical consultations. trained research assistants who were blinded to subject group assignments conducted the telephone interviews using a structured follow-up questionnaire. the follow-up questionnaire collected information on subjects' selfreported ppv status, awareness and beliefs of ppv, barriers of not up taking ppv, perceived stress and self-efficacy to manage disease in general. a subject was considered as lost-to-follow-up after non-response to eight telephone calls made at different times of the day/night. the health education intervention comprised of parts, a -min brief health education telephone intervention before and a -min face-to-face health education intervention during subjects' medical appointments. the telephone briefing and face-to-face interventions, designed using the framework of pragmatism (baert, ) , included learning the facts of the health problem (pneumonia) and intervention (vaccination), and then the older patients were guided to interpret the given information from their own perspectives (to consider the pros and cons of receiving or rejecting the vaccination) so as to make decisions on whether to take the vaccination. the -min brief health education telephone intervention focused on the advantages and side effects of ppv, and highlighted the vaccine was free-of-charge at the selected clinics at the selected time. the face-to-face health education covered knowledge of ppv, including its nature and benefits, possible side-effects and care and support after receiving ppv. the control group received a reminder on their upcoming medical appointment after completing the baseline questionnaire. all study sites provided standard care including promotional leaflets, poster displays, and health education video show throughout the study period. fig. records the details of the health education intervention used in the current study. all the rns and medical and nursing students who were responsible to deliver the intervention had attended a oneday workshop delivered by the research team leaders on knowledge of ppv including its benefits and side effects, details of both parts of the health education interventions, study procedures and questionnaires, as well as communication skills with older patients. the primary outcome was the hospital authority clinical management system (cms)-recorded uptake rate of ppv by may . the secondary outcome was awareness of ppv at -month follow-up. in addition, reasons for not taking the ppv were examined at -week follow-up. the estimated % uptake rate of ppv reported previously in hong kong was not used for sample size calculation due to the changing situation in ppv provision in the community where ppv used to be only available in private clinics on a fee-for-service basis, and the hksar government now provides free ppv in selected public hospitals. thus, the required sample size calculated in the current study was on the basis of an increase in the vaccination rate from % to % (thomas et al., ) , a ratio of control to intervention participants of : , % power, and a significance level of %. we did not have prior information regarding the intra-class correlation coefficient (icc) on ppv in outpatient clinics in hong kong. previous studies reported icc was typically around . for primary care trials and usually less than . for community-based randomized trials (campbell et al., ) . as the current study included chronic patients receiving secondary care treatment in the clinics, we took the icc = . in calculating the sample size. this gave a target sample size of a total subjects, with subjects in each group. we enrolled subjects in each study week (one cluster) since there were study weeks ( weeks in clinics) available in the trial. proportions and means were used to summarize subject characteristics. generalized estimating equations (gee) at the individual level to allow adjustment for clustering by study week using an exchangeable correlation structure were performed to compare the primary outcome and the secondary outcomes between the intervention and control groups. in gee, models were fitted with adjustment for study week, clinic, three baseline variables of patient's educational level (primary level or below vs above primary level), whether had any type of vaccination in the past months, and whether was aware of ppv. a sensitivity analysis using gee was conducted by treating patients in the control group who also received the -min face-to-face health education at the clinic in the intervention group. relative risks (rrs) and their % confidence intervals (cis) were reported whenever applicable. fisher exact tests were used to compare the reasons for not receiving ppv at -week follow up. all analyses (except the sensitivity analysis) were performed using spssv . in and by intentionto-treat principle which is a strategy that compares study groups in terms of the treatment to which they were randomly allocated, rather than the treatment they actually received. in other words, analyses were performed according to the assigned treatment group, regardless of participant compliance, withdrawal or protocol deviation. also, for the secondary outcome of awareness of ppv at month, the patients who were lost to follow-up were treated as not aware of ppv. a total of , patients were screened for study eligibility in the participating clinics. of the eligible subjects, a total of ( %) agreed to participate with in the intervention group and in the control group. in the intervention group, ( %) of the subjects had received both the brief telephone intervention and the on-site face-to-face intervention (full compliance), while ( %) received the brief telephone intervention about ppv only (partial compliance). however, an on-site nurse reported that subjects in the control group also had also received the intervention but they remained in the control group by the intention-to-treat principle. a total of subjects ( %) in the intervention group and ( %) in the control group were successfully followed up at -week (follow up rate of %); and ( %) in the intervention group and ( %) in the control group at -month follow up (follow up rate of %) (fig. ) . the -month follow up was completed in june . the baseline variables between the intervention and control groups were similar (table ) . by may , , a total of ( %) subjects received the ppv injection during the study period as recorded in the cms, with ( %) subjects in the intervention group and ( %) in the control group. the icc of the cmsrecorded ppv uptake in the study by randomizing the study time in the participating clinics was . . when adjusted for clustering effects using gee, the adjusted relative risk (arr) for cms-recorded ppv uptake associated with the intervention was . ( % ci = . - . ). the intervention effect remained significant (arr = . , % ci = . - . ) after treating the patients in the control telephone briefing one week before the scheduled medical consultation . pneumococcal vaccine is now available free-of-charge at the selected sopcs/gopcs . you are encouraged to take this vaccination in the upcoming medical consultation at these sopcs/gopcs . the benefits of taking this vacc ination and possible side effects . we will meet you at the scheduled consultation session for further discussion . what is pneumococcal vaccine? . how does pneumococcal vaccine help to prevent pneumonia? . what are the possible side-effects? . how often should the vaccination take place? . what should be done in the post-vaccination period? . pneumococcal vaccine is now available free-of-charge at the sopcs/gopcs and you are encouraged to take this vaccination group who received the face-to-face health education as intervention subjects in the sensitivity analysis. the two groups, however, were similar in awareness of ppv at -month follow up (n = , % vs n = , %) and its corresponding icc was . . from the gee results, the adjusted relative risk for the awareness of ppv at month follow up was . ( % ci = . - . ), indicating no intervention effect. the intervention effect on the awareness of ppv remained insignificant (arr = . , % ci = . - . ) in the sensitivity analysis. at -month follow up, ( %) subjects of the intervention group and ( %) of the control group reported that the nurses had advised them to receive ppv, while only ( %) subjects of the intervention group and ( %) of the control group reported they received advice from the doctors. of the subjects completed -week follow up survey, ( %) subjects in intervention group and ( %) in control group did not receive the vaccination. exploratory analysis of these subjects revealed fewer subjects in the intervention group reported no need for the vaccination, had received all necessary vaccinations, and lack of knowledge about the availability and accessibility of the vaccination, while more subjects in the intervention group reported they did not want to receive ppv, as compared to the control group (table ) and these differences were statistically significant. it is worth noting that ( . %) subjects of the intervention group and ( . %) of the control group reported that they were suggested not to receive ppv by their physicians. a multiple-component brief health education intervention including a -min telephone briefing and a -min faceto-face education intervention was effective in motivating ( %) of the older patients to receive ppv vs ( %) in the control group, a % increase. although not directly comparable, the uptake rate in the intervention group was similar to a previous study using a nurse-delivered intervention with face-to-face patient contact. winston et al. ( ) reported a much lower uptake rate using a similar 'telephone outreach' intervention (< %) among a community older adult group (winston et al., ) ; and other studies reported a comparably higher uptake rate ( %) with a more intensive approach such as promotional intervention, educational brochures, and a reply card (krieger et al., ) . these results indicated that there are multiple strategies to improve vaccination uptake, and it seems that uptake rates may increase with intensity of the intervention. furthermore, both intervention and control groups showed a marked increase in the overall awareness of ppv (n = , %) at -month follow up comparing at baseline (n = , %), although there was no between group difference. the overall standard promotion of ppv at the clinics, and the telephone calls prior to the scheduled appointments for completing the baseline questionnaires might have exerted some effects on raising the awareness of ppv in both groups. providing health information such as promotional leaflet, poster, and educational video can raise the awareness of patients about the health problems, but adding a multiple component health education intervention, with direct communication with the older patients offering an opportunity for support can further motivate older patients to receive ppv. subjects in this study were older patients with chronic diseases and low education levels, and table baseline characteristics of subjects in the intervention and control groups, n (%) unless otherwise stated. intervention group (n = ) ppv, pneumococcal polysaccharide vaccine. a subjects in the intervention group and subjects in the control group did not complete the scale and were excluded from the analysis. b subjects in the intervention group and subjects in the control group did not complete the scale and were excluded from the analysis. hence these patients might need a more direct approach in communicating health messages such as face-to-face counseling, so that opportunities were given to ask questions, if any. about one-third of the patients who did not receive the vaccination reported that there was no need to receive the vaccine, suggesting more intensive intervention is needed to understand the reasons behind such thoughts and to motivate this group to consider the vaccination. although previous studies showed that advice from physicians on ppv was one of the crucial factors in promoting the uptake of the vaccination (united states department of health and human services, ) , family doctors at the clinics in hong kong have a very heavy workload that they can only spend a few minutes with every patient (hedley, ) . hence nurses can fill this important gap, and as indicated in this study, about two-third of the patients reported they had received advices from nurses about receiving ppv. it is worth noting that % (n = ) of the intervention group who did not receive ppv reported it was because of the expected side effects of ppv as compared to % (n = ) in the control group. this result suggested that the older patients might have negative perception on the health information provided by the nurses and developed some anxiety toward the vaccination leading to an adverse effect on the ppv uptake rates in the intervention group. further programmes thus should allow more time to discuss the side effects and adopt an appropriate balance of highlighting the benefits and possible side effects of ppv injection. finally, the study has provided estimate of icc on older patients with chronic diseases with regular visits to clinics for secondary care prevention which is very important in large vaccination studies as such an estimate of icc can enable accurate sample size estimation in further studies in a similar context. nevertheless, there were a few limitations in this study. first, the subjects were limited to older patients with chronic diseases and hence the results may not be entirely comparable to other studies which targeted all elders aged over years. furthermore, although all the five selected clinics were from the same region in hong kong, they had variations in their services provided and population served. for example, the three gopcs provided primary medical care services and their patients had less acute chronic illnesses such as hypertension, while the two sopcs provided follow up consultations, and their patients might have more acute illnesses and usually require more frequent follow ups. further studies with a wider coverage of all elders from different regions in hong kong would be desirable. second, only . % (n = ) of the patients in the intervention group had received both telephone briefing and on-site faceto-face intervention in the real clinical situation, hence the intervention effect could have been weakened. this situation was due to the busy day-to-day clinic routine, thus preventing the nurses from approaching all the randomized patients. in addition, some patients in both groups did not turn up at their scheduled appointments and hence did not receive the onsite intervention, leading to a . % (n = ) of the intervention group only received telephone briefing. although the sensitive analysis showed the intervention effect remained significant by treating the patients in the control group who had received the faceto-face health education at the clinics, future studies would require integrating health education into clinic procedures to improve efficiency. third, we had underestimated the icc in sample size calculation that the icc in this study was similar to clinical studies, not in between clinical and community studies (campbell et al., ) as expected. the underestimation of icc inevitably lowered the statistical power of the study. a retrospective calculation of the power of the study using an estimated icc of . indicating the study only has a power of . to detect a difference on % in the ppv uptake rates between the two groups at a % significance level. fourth, the physical space of some clinics was very small and crowded with patients with no designated space for delivering health education, hence limiting the quality of delivering the intervention. fifth, we had included medical and nursing students to deliver the second component of the intervention which might have induced variability in the quality of the intervention, although prior training was provided. because they had less experience in health promotion, the intervention effect might have been weakened. finally, only one-third of the eligible patients consented to the study which might also limited the generalizability of the current findings. the situation, however, was expected as ppv was very new to people in hong kong and a certain degree of reluctance to try a new type of vaccination is expected. furthermore, the study included multiple components and follow-up telephone calls and older patients might not want to be engaged into these activities. as a result, those participated in the study could be more cooperative and willing to take risks (from the patient's perspective) and hence willingness to receive ppv might be greater in both groups. a nurse-delivered brief multi-component health education intervention was effective in promoting and increasing uptake of ppv among older patients with chronic diseases. the traditional mode of uni-directional health education using pamphlets and videos can increase the awareness of health actions, but may not be able to change behaviors. physicians' advice has shown to be effective in increasing the uptake of ppv, but as nurses have more frequent contact with patients, they can fill this important gap and provide more direct communication with patients in motivating them to take appropriate health actions such as receiving ppv. pragmatism as a philosophy of the social sciences developments in cluster randomized trials and statistics in medicine prevention of pneumococcal disease: recommendations of the advisory committee on immunization practices (acip) a computerized reminder system to increase the use of preventive care for hospitalized patients prior pneumococcal vaccination is associated with reduced death, complications, and length of stay among hospitalized adults with community-acquired pneumonia problems in the delivery of primary health care in the public and private sections fluoroquinolone and other antimicrobial resistance in invasive pneumococci clinical effectiveness of pneumococcal vaccine. meta-analysis effectiveness of pneumococcal polysaccharide vaccine in older adults increasing influenza and pneumococcal immunization rates: a randomized controlled study of a senior center-based intervention pneumonia in older persons vaccines for preventing pneumococcal infection in adults prevalence of influenza vaccination and correlates of intention to be vaccinated among hong kong chinese epidemiology of invasive streptococcus pneumoniae infections in the united states, - : opportunities for prevention in the conjugate vaccine era communicable diseases cluster randomised controlled trial of an educational outreach visit to improve influenza and pneumococcal immunisation rates in primary care effects of a large-scale introduction of the pneumococcal polysaccharide vaccine among elderly persons in improving rates of pneumococcal vaccination on discharge from a tertiary center medical teaching unit: a prospective intervention patient education strategies to improve pneumococcal vaccination rates: randomized trial united states department of health and human services increasing pneumococcal vaccination in managed care through telephone outreach pneumococcal conjugate vaccines we would like to express gratitude to the five hong kong hospital authority west cluster outpatient clinics under hospital authority for their assistance during the study period.conflict of interest: none declared. funding: this study was supported by a donation from the li ka shing foundation, the university of hong kong and the hospital authority. key: cord- -gl i s authors: tang, qin; song, yulong; shi, mijuan; cheng, yingyin; zhang, wanting; xia, xiao-qin title: inferring the hosts of coronavirus using dual statistical models based on nucleotide composition date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: gl i s many coronaviruses are capable of interspecies transmission. some of them have caused worldwide panic as emerging human pathogens in recent years, e.g., severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). in order to assess their threat to humans, we explored to infer the potential hosts of coronaviruses using a dual-model approach based on nineteen parameters computed from spike genes of coronaviruses. both the support vector machine (svm) model and the mahalanobis distance (md) discriminant model achieved high accuracies in leave-one-out cross-validation of training data consisting of representative coronaviruses ( . % and . % respectively). predictions on additional coronaviruses precisely conformed to conclusions or speculations by other researchers. our approach is implemented as a web server that can be accessed at http://bioinfo.ihb.ac.cn/seq hosts. scientific reports | : | doi: . /srep invasion, its sequence must encode the information related to specific hosts; therefore, it is especially useful in identifying hosts of given coronaviruses. as the result of natural selection and evolution, different genomes are characterized with different preferences for nucleotides. according to probability principles, a shorter nucleotide fragment has a lower chance of variation due to evolution, and the copies of this fragment in a genome tend not to change significantly. this phenomenon is helpful for evolutionary analysis. dinucleotides are the most stable of these fragments because they are the shortest, and their bias values are usually diverse among species and they are highly invariant for a given individual genome . dinucleotide abundance has been proven to be reliable in the identification and classification of sequences from viral genomes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . support vector machines (svms) are a group of supervised machine learning methods that were originally introduced by vapnik as a linear classifier . their current standard incarnation (soft margin) comprises associated learning algorithms for classification and regression analysis . the basic principle of class separation for a svm is mapping vectors into a high-dimensional feature space and finding an optimal separating hyperplane between the two classes in this space by maximizing the margin between the classes' closest points. the points on the boundaries are referred to as support vectors, and the middle of the margin is the optimal separating hyperplane, which forms the largest gap between two sets of data . based on this gap, the points of different attributes fall into different classes. several types of algorithms exist for a svm to address classification problems for multiple classes and high-dimension data. svms perform well in multiple areas of biological analysis, including the evaluation of microarray expression data, the detection of remote protein homologies, and the recognition of translation initiation sites . instances in which the established classification is questionable or wrong can be identified if an svm is used for prediction of training samples. mahalanobis distance (md) discrimination is a classical and accurate method that is extensively applied in cluster analysis and classification techniques . md measures the distance between a point and a population and considers the variance of the population distribution; the points are sorted to the closest population in distance. another method-fisher's linear discriminant analysis-has been applied to infer hosts for three novel picorna-like viruses . as it requires data that have a normal distribution, which is not the case for our data, md is adopted in this study. previous studies of coronaviruses were primarily focused on the evolution of genomes or specific genes, serum-neutralization assays for identification of receptors, and crystal structure analysis of spike protein and receptor binding domains. in this study, we analysed the compositions of mononucleotides and dinucleotides in coronavirus spike genes. based on the data matrix of nucleotide composition, the md and svm were applied to predict hosts of coronaviruses. the results of this technique may provide hints regarding natural hosts or potential hosts of the virus and can be used to guide the selection of the cells for virus isolation or to explore the probability of interspecies transmission of coronaviruses. nucleotide composition analysis. nineteen parameters, including three mononucleotide frequencies (g, c and t) and dinucleotide biases, were computed from spike gene sequences (see supplementary table s ). all parameters show significant differences across the host groups (kruskal-wallis tests, p < . e- ); therefore, they were subsequently employed as factors in statistical models for discriminant analyses. empirically, a dinucleotide relative abundance or dinucleotide bias (e.g., ρ xy ) is significantly high if ρ ≥ . . among the dinucleotides in this study, the cpa and tpg show an average abundance that is significantly higher than the expected values (ρ ca = . , ρ tg = . ), whereas the average bias of cpg is extremely low (ρ cg = . ). this result indicates that the observed abundances of cpa and tpg are significantly higher than their expected values, and the observed abundance of cpg is significantly lower than the expected value. the g+ c content is minimal ( - %) . this finding indicates that coronaviruses exhibit a low density of nucleotide sequences and may be sensitive to heat or alkali. the low g+ c content also indicates a preference for codons ending with a or t and a higher mutability. training and validation of statistical models. the data matrix with factors as columns and samples as rows was fitted to svm and md models, all predictions in leave-one-out cross-validations were listed in supplementary table s and summarized in table according to host species. the validations indicate that both models achieved high accuracies on the training data set: . % for the svm and . % for the md. all incorrect cases in unsupervised predictions are listed in table . the only incorrect prediction by the svm is sample nc_ . , which is isolated from an avian species but was predicted to infect humans. among all incorrect predictions by md, bats are the common predicted hosts. no sample was incorrectly predicted by both models. the trained models were applied to additional samples, and the predictions unveiled clues regarding potential interspecies transmission (see table ). sequences - comprise spike genes of coronaviruses that were primarily isolated from palm civets from restaurants, animal markets, or farms in southern china when sars wreaked havoc in . the sequences of these coronaviruses (civet-covs) are similar not only to each other but also to sars-cov. cross-host evolution research of sars-cov in palm civet and humans indicated that the variations in spike genes seemed to be essential for the transition of coronavirus from animal-to-human transmission to human-to-human transmission . in addition to cross-neutralization with sars-cov, these sars-like civet-covs can use human ace as an entry receptor . bats are the reservoir hosts of a number of coronaviruses, and a recent study also suggests that bats are natural reservoirs of these sars-like coronaviruses, whereas palm civets and humans are intermediate hosts . all hosts predicted by the svm are humans, which supports the previously mentioned research. the md identified both bats and humans as hosts of these samples, but bats are the preferable hosts for samples - and the second choice for samples - . this finding is also expected as bats are considered to be natural hosts of these viruses. sequences - comprise spike genes of mers-covs from dromedaries after the outbreak in the middle east in . mers-covs are similar to the bat coronaviruses hku and hku in their amino acid sequences , and they can use human dpp as an entry receptor . mers-covs was assumed to originate from hku in pipistrelle, which is a type of japanese bat . in our study, these mers-covs isolated from camels were predicted to be capable of infecting humans; and bats are also likely hosts next to humans in predictions by md. this result is obviously consistent with above speculations, and also supports the who advices about avoiding close contact with camels (http://www.who.int/csr/ don/ _ _ _mers/en/). the st sample was a sars-associated coronavirus that was transmitted from human to pig , and both svm and md detected its threat to humans. bat and avian might be potential hosts since both models suggest that they are more vulnerable than porcine. samples - (rsshc , rs and sl-cov-wiv ) consist of three sars-like coronaviruses from bats . analyses based on the sequence similarities and cultures in the cell lines suggest that rs and sl-cov-wiv are capable of using a sars-cov receptor for cell entry and pose a threat to humans, whereas rsshc cannot . our study provides a precise support to these conclusions. the md correctly predicts bats as the natural hosts of the three viruses, and the svm indicates that rs and sl-cov-wiv are harmful to humans. the th sample was isolated from an alpaca by jin et al. in with a serotype of bovine; the phylogenetic analysis suggests that it shares the same ancestor with bovine-coronaviruses . our analysis supports the finding that this coronavirus is capable of infecting bovine. these analyses imply that this strain is capable of interspecies transmission between bovines and alpacas. samples and are enteric coronaviruses from bovines and humans; they have been identified as the same strain named "human enteric coronavirus " in the ncbi database due to the similarity between their spike protein sequences of . %. although they are similar to the human coronavirus oc and the bovine coronavirus, evidences from morphological, immunological, and genomic studies indicate that they are closer to bovine coronavirus than to human coronavirus (unpublished research, from personal communication). this finding is consistent with our analysis. in addition, avian and bat are worthy of attentions as potential hosts due to the small md values. two groups of two-dimensional data are plotted in n( , ) , and the red points represent a "tight" population with a smaller sd of n( . , . ). the red line separates the two groups classified by the md, and the groups predicted by the svm are delimited by the blue line. in this figure, two individuals (the red triangles between the two lines) from the "tight" population were classified into the "loose" group by the md, whereas the svm accidentally excluded four points (the blue reversed triangles between the two lines) from the "loose" population. this example shows that md and svm have inverse tendencies in some cases, i.e., when a "loose" population is close to a "tight" population, md intends to classify outliers of the "tight" population into the former. the opposite situation is valid for the svm. nucleotide composition analysis revealed the overrepresentation of cpa and tpg dinucleotides and the suppression of cpg dinucleotides (see supplementary table s ), which indicates that coronaviruses generally prefer motifs that contain cpas and tpgs and avoid cpgs in sequences. these dinucleotide biases are common characteristics of rna viruses in vertebrates , , , . as most vertebrates exhibit a very low cpg representation in genomes, rna viruses may gradually adapt to the accumulation of host mutations and mimic the host gene's dinucleotide patterns for survival . for dna viruses, the most-accepted mechanisms for the suppression of cpg dinucleotides are the methylation of cpg nucleotides and the subsequent deamination of -methylcytosine, which renders cpg a mutational hotspot . for rna viruses, a different hypothesis is that the rna viruses encounter different selection pressures when they switch to a new host, and viral rna genes mimic host mrnas to avoid immune detection . similar to other human ssrna viruses, coronaviruses show a strong correlation between cpg pressure and c+ g content (pearson's correlation coefficient, r = . , p < . e- , our data). a lower c+ g content usually indicates that the nucleotide sequence of the virus is unstable or is highly variable under evolutionary selection pressure. considering that the mutation rates for rna viruses are significantly higher than the mutation rates for dna viruses , mutational pressure may be the most important determinant of the bias in codon usage in human rna viruses, such as coronaviruses . the capabilities to bind with receptors and to replicate in host cells are essential for any virus to infect hosts. different genes contribute to these biological processes. variations on these genes may enable a table . the isolate sources and predicted hosts of coronaviruses. predictions consist of hosts with minimal md or p values, those with md < = or p < = . for svm, and those with md or p values no greater than corresponding values of isolate sources if the isolate sources are among the six categories of hosts. all predictions are listed in ascending of md or p values. * p < = . or md < = . ** p < = . or md < = . scientific reports | : | doi: . /srep virus to transmit cross-species. one famous example would be the polymerase (pb ) of influenza a virus, in which amino acid change from e to k at its th position would render the virus to replicate in mammalian cells [ ] [ ] [ ] . in coronaviruses, the spike protein is functionally associated with recognition of hosts and the rna-dependent rna polymerase (rdrp) is related to proliferation of virus. however, there are two obstacles limiting the use of rdrp gene: ( ) the similarities among nucleotide sequences is too high to train md model, i.e., the variation rate of rdrp sequence is slower and cannot provide enough resolution to discriminate different coronaviruses; ( ) even worse, available full-length cdss in public databases are very limited -only or so. on the contrary, the spike gene perfectly satisfied the requirements for variation rate and availability, therefore was adopted as markers in this study. md and svm show opposite tendencies in judging outliers (see fig. ) , which reflects the different principles of the two classification approaches. unlike the euclidian distance (ed), which measures the absolute distance between points or mass centres in space, the mahalanobis distance considers the variances within a population and the covariance between variables. in some cases, especially when a population with individuals who are scattered across a wide range is located close to a "tight" population with smaller internal variations, the md may classify marginal individuals from the latter into the "loose" population even if they are "close" to a "tight" population according to the ed. the md enables "loose" populations to have a greater number of points. the svm has a different philosophy. svm separates populations by finding a hyperplane that maximizes the distances between populations. when a "loose" population is close to the boundary of a "tight" population, svm is more likely to find this hyperplane within the former. this finding explains svm's tendency to exclude outliers from a "loose" population. bats are the reservoir hosts of a number of coronaviruses that can survive in bats and accumulate variations in the long evolutionary process , , . thus, coronaviruses in bats constitute a "loose" population with larger internal gaps. we assume that some strains of viruses in bats gain sufficient variation to enable them to infect other organisms; these viruses form a new "tight" population at the edge of the original group. in this case, the md emphasizes the connection of a virus with the original source, whereas the svm may be more sensitive to the possibility of infecting new hosts. therefore, the incorporation of analyses using the md and svm can be especially helpful for revealing the profile of interspecies transmission. according to the predictions by md, bats are not only the hosts in all incorrect cases from training data set (see table ), but also in the host list of each coronaviruses for testing (see table ). furthermore, bats were predicted to host of . % training samples isolated from other hosts (see table ). these facts convincingly support the notion that these viruses originated from bats and shifted to other hosts. next to bats, avians could be infected by . % samples from other hosts. if bats are the only reservoir hosts and coronaviruses spread from bats to avians and other animals, according to the stochastic event model, the probability of co-infectivity to both bat and avian can be the product of the infectivity probabilities to each of them, i.e., . ( . × . , see table ), then ( . × ) samples are expected to be of co-infectivity. however, only samples were predicted to be of co-infectivity to bats and avians. so avians might be the second independent source of coronavirus in parallel to bats. if this speculation is true, people will have to maintain vigilance to avian coronaviruses apart from avian influenza viruses. especially, due to the high accuracy of the svm in cross-validation, we should seriously consider its only "wrong" prediction: perhaps it is sensible to investigate whether the nc_ . virus from avian is capable of infecting humans. for the viruses that are capable of spreading across a host species barrier, the combination of the md and the svm is valuable for assessing their potential threat. the origin and interspecies transmission of coronaviruses have been extensively discussed in the past ten years, and the coronaviruses of most mammals are believed to originate from their ancestors in bats , , . our analysis with dual statistical models support the finding that sars-covs and mers-covs spread from bats to humans and other animals. in most cases, our approach provided convincing predictions. the dual-model approach can be expected to become a useful tool in future studies. typically, when a novel coronavirus is isolated, the combination of the md and the svm may provide meaningful hints regarding its origin and potential threat to humans or other animals. as soon as more virus genomes are sequenced, this approach can be applied to investigate the interspecies transmission route of other threatening viruses, including the recent ebola outbreak in west africa. data preparation. all genome sequences and complete coding sequences (cdss) of spike genes were downloaded from the national centre for biotechnology information (ncbi) database (http://www. ncbi.nlm.nih.gov/) on july , . sequences of spike genes were extracted from the coronavirus genomes and pooled with downloaded cdss. then, we removed replicate sequences and sequences that contained non-standard bases or were incapable of coding complete products. the length of each sequence is longer than , bases. among all valid nucleotide sequences that are listed in supplementary data s , sequences fall into six categories according to different hosts: for humans, for porcines, for bovines, for bats, for murines and for avians. the majority of the remaining viruses were isolated from the two epidemic diseases caused by the coronavirus in the past years. although we only listed the hosts from which they were isolated, these viruses have been verified or suspected to have the ability to infect different hosts; thus, all sequences were employed scientific reports | : | doi: . /srep to explore interspecies transmission of coronaviruses. viruses from other mammals, including canines, felines, rabbits, equines, alpacas and whales, were excluded from the data set as the number of spike sequences for each host is insufficient for establishing a separate group. spike sequences were computed using our original python scripts. dinucleotide bias is the ratio of the observed value to the expected frequency of each of the dinucleotides: where ρ xy is the dinucleotide bias, f xy is the frequency of dinucleotide xy, f x and f y are the frequencies of nucleotide x and nucleotide y , respectively. in this study, we considered factors, including three mononucleotide frequencies (g, c and t) and dinucleotide biases. as none of the frequencies has a normal distribution, the nonparametric "kruskal-wallis test" was employed to investigate the difference in each factor among six categories. as a result, significant differences across categories were detected for each factor; thus, all factors were employed for modelling. modelling, validation and prediction. as a classifier, the svm can efficiently perform a nonlinear classification using a kernel technique that is rooted in structural risk minimization. in this study, the r package e (version: . - ) was employed for the svm analysis. "c-classification" was adopted as the model type and "radial" was adopted as the svm kernel in our analysis. the md is a measure of the distance from a point to the centre of a distribution; the principle of this discriminant is that individuals belong to the closest group in the distance. the md is defined as , where x denotes the population, x denotes the individual, μ is the mean value of the population, t denotes the matrix transpose, and ∑ denotes the covariance matrix of population . the r program "distinguish.distance.r" was employed in the md analysis. leave-one-out cross-validation was employed for both svm and md analyses. when the trained models are applied to a sequence for testing, each of the six categories of hosts will obtain a p value from svm and a md value. based on p values and md values, three steps will be taken to determine candidate hosts. first, the host of minimal p value or md value is reasonably regarded as the preferable host. then, two adjustable empirical thresholds can be used for each model to pick out other potential hosts. in this study, we adopted . and . for p value, and for md value; i.e., likely hosts were determined if p < = . or md < = , and very likely hosts were defined by p < = . or md < = . the two steps are unsupervised prediction. in case that the isolate source is among the six host groups for modelling, a supervised prediction can be applied as the third step, i.e., all host species with p values or md values no more than those of the observed host will be listed as potential hosts, which can be practical references for researchers to evaluate a virus's threats to human or other animals. compare the tendencies of md and svm in predictions. two groups of two-dimensional vectors were generated in silico as two populations. the number of vectors in the first population are randomly generated from the normal distribution n( , ), and the number of vectors in the second population are randomly generated from n( . , . ). as the first population has a larger standard deviation (sd), we refer to it as the "loose" population and refer to the second population as the "tight" population. the two groups of data are employed for the leave-one-out cross-validations of md and svm. all python and r scripts employed in this study are available from the authors upon request. the prediction can be performed using the spike gene sequences of the coronaviruses on our web server, which is available to the public at no cost at http://bioinfo.ihb.ac.cn/seq hosts. interspecies transmission and emergence of novel viruses: lessons from bats and birds virus taxonomy, the ninth report of the international committee on taxonomy of viruses genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus a decade after sars: 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dnasequences mahalanobis distance. resonance statistical modeling and r software this work was supported by the -talent program grant from the chinese academy of sciences. q.t. designed the research, collected and analyzed data, and wrote the paper. y.s., m.s., y.c. and w.z. analyzed data, x.-q.x. designed the research, re-analyzed data, wrote the paper, and created the web server. all authors reviewed the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord- - fxvzj authors: chen, haidan; pang, tikki title: human genomics in asia date: - - journal: international encyclopedia of the social & behavioral sciences doi: . /b - - - - . - sha: doc_id: cord_uid: fxvzj in the past decade, asia has been actively engaged in human genomic studies and has made great contributions to the field. there is an increase in the number of genomics institutes, consortiums, and initiatives across the continent to study the association between genetic variation and disease. despite these laudable efforts, asia faces tremendous challenges in terms of funding, regulation, collaboration, and ethical, legal, and social issues related to genomics. these need to be addressed in the near future to promote the development of genomic medicine. the past decade has seen the advancement of human genetics and genomics worldwide. various countries have invested seriously in this field, with the expectation of gaining a better understanding of human health and disease. biobanks and genetic databases have been built to facilitate interdisciplinary clinical research. regional and international efforts have also been made to promote the success of genomic medicine. for example, in europe, the biobanking and biomolecular resources research infrastructure has been constructed to secure global competitiveness of research and industry of the european union, and benefit the health of european citizens. international genomic research collaborations, such as the human genome project (hgp), the international hapmap project, and the international cancer genome consortium, have produced open-access genetic databases for researchers around the globe (kaye, ; vaught et al., ) . the completion of the sequencing of the human genome, the mathematical analysis of dna variants called single nucleotide polymorphisms (snps), and the progress in genome-wide association studies have contributed dramatically to biomedical research. yet the majority of medical genomics research has mainly focused on people of european descent, the results of which will not be beneficial to the greatest segment of the world's population. given that the lack of diversity in these studies will bias our understanding of the association between genetic variants and diseases, researchers have recognized the importance of inclusion of populational diversity in pharmacogenomic and genome-wide disease studies (bustamante et al., ) . the geopolitical continent of asia contains almost twothirds of the world population, with rich cultural, ethnic, linguistic, and genetic diversity. asian nations, particularly those still developing, are often depicted as the epicenter of pandemics, e.g., severe acute respiratory syndrome (sars) and avian influenza outbreaks (tan et al., ) . at the same time, these countries also face growing chronic disease burdens, but the causes of diseases in asian populations remain unknown (jemal et al., ) . thus, genomic research on asian populations is essential to further enhance our knowledge of the genetic basis of complex chronic and infectious diseases, and to cope with asian and global public health challenges. in this article, we first give a brief introduction to the contributions of asian nations to human genomics, placing greater focus on the genomics consortiums and initiatives in the region: asian cohort consortium (acc), human genome organization (hugo) pan-asia snp consortium (pasnp . ) and hugo pan-asia population genomics initiative (pasnp . ). we then discuss the challenges that asia faces in bringing genomic science and technology to bear on applications of genomic medicine. finally, we outline several future perspectives for the advancement of genomics in asia. the world's largest geopolitical continent, asia, exhibits heterogeneous geographical, cultural, and socioeconomic characteristics. the investment and progress of genomic science and technology of each country across the continent are uneven. wealthy countries, such as singapore and japan, have made tremendous investments in genomics research and development (r&d) toward efficient diagnosis tools and personalized medicine. the genome institute of singapore (gis), located at singapore's biomedical hub, biopolis, is one of the centers of excellence in genomics in asia and indeed the world. gis gained world recognition through its rapid response to sars and identification of five strains of sars coronavirus in to effectively contain a pandemic. the strength of gis's research lies in three milestone arenas: "infectious diseases, cross-national science diplomacy, and regenerative medicine" (fischer, ) . japan has established several research institutions, e.g., riken center for genomic medicine, and research infrastructures, e.g., biobank japan, which are steering the nation ahead on the path toward genomic applications in medicine and health (yoshizawa et al., ) . emerging economies, such as china and india, are the rising powers in the global biomedicine field and are now challenging the developed economies (salter and faulkner, ) . china was the sole developing country to participate in the hgp, contributing toward sequencing of % of the human genome. the beijing genomics institute (bgi) has become a global sequencing powerhouse that sequenced the first asian human genome, and a number of plant and animal genomes, such as rice, silkworm, chicken, and panda (normile, ) . india is endeavoring to foster economic development and address local health needs through investment in genomics-based innovation. the indian genome variation consortium, a government-funded collaborative network of several local institutions, is one of the examples that reflect these efforts (hardy et al., ) . still other asian countries, such as indonesia and philippines, have emerged in recent years as active actors in the field of genomics, partly due to their ethnic and linguistic diversity (liu, ) . such efforts by nations across the continent are crucial for asia's genomic research collaboration networks, such as acc, apsnp . and . . we introduce these networks in detail below. first proposed in november , in seoul, the acc is a collaborative cancer cohort research project, involving more than one million healthy people across asia who will be followed over time until various disease endpoints are reached. the acc seeks to understand the relationship between genetics, environmental exposures, and the etiology of disease, and to discover early detection biomarkers. the acc has two missions: "(i) to serve as a platform for cross-cohort collaborative projects and combined analysis and (ii) to act as an incubator for new cohorts" (song et al., ) . the acc has approximately active members from bangladesh, china, india, iran, japan, south korea, malaysia, singapore, taiwan, and the united states, among others. it is cochaired by john potter of the fred hutchinson cancer research center, seattle, washington, usa, and daehee kang of seoul national university college of medicine, south korea. its coordinating center is located at the fred hutchinson cancer research center to provide support for scientific collaboration, coordination and communication, data operations, and statistical consultation (rolland et al., ) . as most studies on the associations between body mass index (bmi) and the risk of death have been conducted on north american and european populations, and less is known about these associations in asian populations, the focus of acc's projects is on bmi in asian populations. to date, the cross-cohort collaborative projects of acc have yielded about seven articles, showing the association between bmi and risk of death; bmi and diabetes; bmi, tobacco smoking, alcohol consumption, and risk of cancer of the small intestine; bmi and risk of death from pancreatic cancer; meat consumption and cause-specific mortality; and bmi and cardiovascular disease mortality in asian populations (https://www.asiacohort.org/ index.html (accessed . . .)). the snp consortium was established in early through a collaborative effort among major pharmaceutical companies, the wellcome trust, and five leading academic centers to provide a resource pool for clinical genomic research and for the discovery of novel diagnosis and personalized therapies (holden, ) . this consortium largely focused on caucasian populations, while genomic variation among asian peoples remained unexplored. in , hugo pan-asia snp consortium (pasnp . ) was set up with researchers from institutions in asian countries to map human genetic diversity in asia. coordinated from the gis, the pasnp took the first steps toward collaboration among asian scientists. based on strong bioinformatics teams from china, india, japan, singapore, etc., diverse ethnicities from indonesia, malaysia, philippines, and taiwan, and the scientists' common interests, the consortium used samples from more than individuals representing populations to conduct migration studies (normile, (normile, , . the pasnp consortium's first report, published in science in december , provided a physical map of human variation in southeast asian, east asian (ea), and central south asian populations, and showed strong evidence for the southern migration route to asia, though it did not completely rule out a two-wave model of migration (abdulla et al., ) . in order to advance the work of pasnp, the hugo pan-asia population genomics initiative was launched in as a version . of pasnp . . it established a larger network with hundreds of researchers, and constructed a gene pool with diverse people, data, and cultures, including those from mainland central asia and the pacific islands. the goal of pasnp . is to further explore asian migration patterns, asian genetic diversity, and local adaptation, and eventually to translate genomics knowledge to the practice of genomic medicine. the data collected by pasnp . will be open to the worldwide scientific community for further genomic studies (http://papgi.org/index.php/about_us (accessed . . .)). despite the achievements that asian countries and consortiums have made, they still face formidable challenges in terms of funding, regulation, collaboration, and the ethical, legal, and social issues (elsi) of genomics in asia. as we show in the next section, the development of human genomics in asia faces not only similar issues as do other national and transnational endeavors, such as funding, standardization of data and samples, harmonization of elsi practices and regulations, and gaining public trust, but also some asia-specific issues and local concerns in the practice of asian science and collaboration. funding funding for genomic science is becoming more competitive and more difficult to obtain, both from the government and from industry sources. for this reason, ensuring sustainable funding is one of the major challenges for genomic studies (vaught et al., ) . even in singapore, where the government had attracted many top scientists from the west with substantial funds, after two -year periods of multibillion dollar investments, with little or no warning the core budgets for bioscience institutes under the agency for science, technology and research (a*star) was cut in the third-year period beginning in . a*star bioscientists, including those from gis, had to compete for grants from the new industry alignment fund, and collaborate with clinicians on industrial and clinical applications (fischer, ; normile, ) . elsewhere, bgi took an entrepreneurial approach to fuel its growth of sequencing and bioinformatics capabilities. it borrowed . billion u.s. dollars from a government-owned bank and must begin to pay back the principal within years. since sequences do not make money, bgi established subsidiary companies to expand the application of genomic science and technology in order to pay off the debt and generate more income (normile, ) . for example, bgi diagnostics offers health services, such as noninvasive prenatal test and tests for various genetic disorders; bgi ark biotechnology co. ltd has set up a transgenic platform, a cloning platform, an experimental farm, and an animal model department (see bgi's website). in addition to the challenges of funding, some asian countries also find regulation a problem. when appointed as the first director of the hgp, james watson resolved that the u.s. national institutes of health should allocate part of its funds to study the elsi of genomics in the face of unprecedented challenges posed by genomics and its future application. research into the elsi of genomics was initiated first in the usa, thence spreading to canada and europe (zwart and nelis, ). awareness of the issues in the east happened much later, but such research was supposed to study similar issues as those in the west for the purpose of some outcomes leading to better regulation of genomics. more recently, yoshizawa et al. identified heterogeneous regulatory frameworks and elsi practice among ea countries. they found that ea countries' regulation of genomics had a relatively inconsistent and mixed character. consider the aspect of privacy as an example: japan, singapore, south korea, and taiwan have regulations and oversights to protect personal information, while china and indonesia exert little or no control over privacy issues. a similar situation is seen with ethics review. even though there are ethics review committees in nearly all of these countries, discrepancies in ethics review capacities and the implementation of oversight between different countries are noticeable (yoshizawa et al., ) . what is perhaps of greater concern is that, in overall terms, there are limited or even absent regulations in many developing countries. some of these countries may have no or limited capacity to regulate innovative genomic medicine. this could be a major barrier to the application of emerging genomic medicine products to improve global health. therefore, it is significant to include the developing countries in the international conference on harmonization, as well as other international consortia, so that developed countries can help improve the regulatory capacity of developing countries (hardy et al., ). yet international health research collaboration between developed and developing countries in the field of human genetics is often a sensitive issue. there are concerns about possible exploitation of the populations in the developing countries in the commercialization of human tissues, and unfairness in benefit sharing and ownership between developed and developing countries (schulz-baldes et al., ) . biodiversity-rich countries like china, india, and indonesia are concerned about the exploitation of genetic resources by more developed countries. they have been uneasy about equitable benefit sharing, ownership, and intellectual property rights in international research collaboration. as a consequence, china promulgated the interim measures for the administration of human genetic resources, issued by its ministry of science and technology and the ministry of health in . this directive stipulates that only through collaboration with chinese parties can foreign researchers get access to chinese genetic resources (chen, ) . fearing that indonesia may not benefit from vaccines developed by international scientists and multinational pharmaceutical companies, the indonesian government stopped sending h n virus specimens to the world health organization in . this stance was supported by malaysia, thailand, and other developing countries to communicate their desire for mutual trust, transparency, and equity between the developed and developing nations in the virus sharing mechanism. moreover, indonesia revised its health law and enacted the ministry of health regulation on material transfer agreement in in order to safeguard national sovereignty over its biological materials (sedyaningsih et al., ) . although india has ethical guidelines and regulations in place to prevent biopiracy, there is a lack of adherence to regulations and an absence of strict implementation of measures to monitor the misuse of genetic samples (kumar, ) . china faces similar problems. to protect the use of chinese genetic resources in research collaboration, the office of legislative affairs of the state council, people's republic of china, published the exposure draft of measures for the administration of human genetic resources online in to solicit advice from the public. as of this writing, the official measures have still not been issued (http://www.gov.cn/gzdt/ - / /content_ .htm (accessed . . .)). the biopiracy issue reflects complexities in cross-border collaboration as well as the importance of mutual trust and equity among scientists in using human genetics. although there are potential dangers of biopiracy and unfair benefit sharing in collaboration between developing countries and more developed countries (north-south collaborations), collaboration should nevertheless be promoted as a strategy to help build developing nations' research and translational capacities (schulz-baldes et al., ) . increasingly, there is also a trend toward collaboration among developing countries (south-south collaborations) in building human resource capacity (ivers et al., ) . in the case of the pasnp consortium, edison liu, one of the key organizers of the consortium, found it challenging to coordinate science collaboration among asian scientists. as he pointed out: scientists in asia have a tendency to look past each other and focus on collaborations with the united states or europe, partly because these collaborations get them more credit from their school administrations. also, in asia, most countries see each other as competitors. just getting people together is an accomplishment. liu, the science culture in asia is different from that among scientists in the west and tends to be more hierarchical and bureaucratic. another challenge rests with the disparity in research capacities and research infrastructures between asian countries (liu, ) . besides these challenges, asian genomic research collaborations face similar challenges as those of the international science communities more generally. as largescale genomic research collaborations require data comparisons and validations across different populations, the lack of harmonized elsi and lack of a regulatory infrastructure for genomic research and application are major hurdles. it is also difficult but crucial to ensure the quality of samples and the interoperability of data for collaboration (song et al., ; vaught et al., ) . finally, the translation of genomics knowledge to genomic medicine and innovative medicine products necessitates the interaction and collaboration of all stakeholders. they are not only scientists, clinicians, policy-makers, and industry people, but also patients, consumers, and healthy individuals. engaging the public in biobank and genomic studies is of great importance because researchers need access to patients' and to the public's biological samples. there are a good number of qualitative and quantitative studies about public perception of biobanks and genetic databases in western countries to explore the public's reasons for participating and not participating in biobank studies; their views of issues such as informed consent, benefit sharing, commercialization, and internationalization; and their concerns over privacy, discrimination, ownership, and the return of results. however, except for a few limited studies in asian countries, among them china, japan, and singapore, little is known about the asian public's perception of biobanks . investigation of public perceptions of biobanks reveals several factors that affect the public's willingness to donate their samples for research and to participate in the studies. these factors include the public's understanding of biobanks, its trust in research institutions and the scientists running those institutions, and its consideration of privacy, discrimination, and commercialization . in asia, there is tendency for the public to distrust science. for example, singapore's growing emphasis on commercialization of science discourages the public's participation in research. in japan, public trust in expertise was eroded following the earthquake, tsunami, and nuclear accident in (arimoto and sato, ) . there are cases reporting public distrust of experts, and public concerns over the misuse of genetic information and genetic discrimination after genetic testing in china and south korea. in taiwan, tensions and distrust arose between the public and authorities of the taiwan biobank due to a lack of open science communication (yoshizawa et al., ) . wellknown cases of scientific fraud in south korea (bonetta, ) , japan (tsurimoto et al., ) , and china (lin, ) have also contributed to the lack of trust between the public and scientists. all of these signal a call for an interface of science and society to actively interact with the public. as a result of rich and diverse genetic resources, together with political will of various countries, and the great efforts that scientists have made, asia is rising in the field of human genomics in the postgenomic era. building regional collaborative genomics networks, such as acc and pasnp . and . , is the first and vital step toward better science, medicine, and health in asia. the research conducted by these networks is competitive in the global arena and bridges the gaps between asia and western countries. however, compared with research collaboration in europe and north america, asian genomics networks face more challenges than their counterparts in the west. the emerging economies and the developing countries in asia have not established consistent and robust regulatory frameworks to govern the conduct of genomic research. furthermore, their elsi practice and regulation is often influenced by local sociopolitical and cultural concerns and tends to be heterogeneous. this has been a disincentive to regional collaboration on genomic science and technology, which requires the harmonization of elsi practice and regulation across asia. therefore, in the future, it will be helpful to place more emphasis on research into elsi of genomics and develop regulatory infrastructures, which could become a tool to secure the public's trust in science. it is also important to encourage both north-south collaborations and south-south collaborations. experience gained from pasnp could contribute to further collaboration on the basis of previous networks and research collaboration. editor's note this encyclopedia (see cross references) see also: anthropology, genomics, and human variation: national roots; biobanking: ethical issues ethical, legal, and social implications program at the national human genome research institute gene-environment interactions in health and well-being; genetics and indigenous communities: ethical issues mapping human genetic diversity in asia rebuilding public trust in science for policy-making the aftermath of scientific fraud genomics for the world governing international biobank collaboration: a case study of china kadoorie biobank biopolis: asian science in the global circuitry biobanks and the phantom public connecting the public with biobank research: reciprocity matters science and society -the next steps for genomic medicine: challenges and opportunities for the developing world genomic applications in medicine and health in india the snp consortium: summary of a private consortium effort to develop an applied map of the human genome south-south collaboration in scale-up of hiv care: building human capacity for care global cancer statistics from single biobanks to international networks: developing e-governance india's preparedness in tackling biopiracy and biobanking: still miles to go genomics and health in the developing world why serious academic fraud occurs in china genomics takes hold in asia. interview by david cyranoski genetic diversity -consortium hopes to map human history in asia snp study supports southern migration route to asia winds of change leave bioscientists scrambling china's sequencing powerhouse comes of age the iranian human mutation gene bank: a data and sample resource for worldwide collaborative genetics research natural forces: the regulation and discourse of genomics and advanced medical technologies in israel coordinating centers in cancer epidemiology research: the asia cohort consortium coordinating center state strategies of governance in biomedical innovation: aligning conceptual approaches for understanding 'rising powers' in the global context sharing benefits in international health research. research-capacity building as an example of an indirect collective benefit towards mutual trust, transparency and equity in virus sharing mechanism: the avian influenza case of indonesia asia cohort consortium: challenges for collaborative research global health governance and the rise of asia report from the working group of the molecular biology society of japan for the investigation of fraud in research papers a review of international biobanks and networks: success factors and key benchmarks elsi practices in genomic research in east asia: implications for research collaboration and public participation what is elsa genomics? science & society series on convergence research main_page -pan-asian population genomics initiative key: cord- -o rlqsq authors: ghosh, arun k.; brindisi, margherita title: organic carbamates in drug design and medicinal chemistry date: - - journal: j med chem doi: . /jm s sha: doc_id: cord_uid: o rlqsq [image: see text] the carbamate group is a key structural motif in many approved drugs and prodrugs. there is an increasing use of carbamates in medicinal chemistry and many derivatives are specifically designed to make drug–target interactions through their carbamate moiety. in this perspective, we present properties and stabilities of carbamates, reagents and chemical methodologies for the synthesis of carbamates, and recent applications of carbamates in drug design and medicinal chemistry. carbamate-bearing molecules play an important role in modern drug discovery and medicinal chemistry. organic carbamates (or urethanes) are structural elements of many approved therapeutic agents. structurally, the carbamate functionality is related to amide-ester hybrid features and, in general, displays very good chemical and proteolytic stabilities. carbamates are widely utilized as a peptide bond surrogate in medicinal chemistry. this is mainly due to their chemical stability and capability to permeate cell membranes. another unique feature of carbamates is their ability to modulate inter-and intramolecular interactions with the target enzymes or receptors. the carbamate functionality imposes a degree of conformational restriction due to the delocalization of nonbonded electrons on nitrogen into the carboxyl moiety. in addition, the carbamate functionality participates in hydrogen bonding through the carboxyl group and the backbone nh. therefore, substitution on the o-and n-termini of a carbamate offers opportunities for modulation of biological properties and improvement in stability and pharmacokinetic properties. carbamates have been manipulated for use in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamate derivatives are widely represented in agricultural chemicals, such as pesticides, fungicides, and herbicides. they play a major role in the chemical and paint industry as starting materials, intermediates, and solvents. furthermore, organic carbamates serve a very important role as optimum protecting groups for amines and amino acids in organic synthesis and peptide chemistry. in recent years, carbamate derivatives have received much attention due to their application in drug design and discovery. however, there are hardly any reviews on this subject in the literature. in the present perspective, we plan to provide an overview of the leading role of organic carbamates in medicinal chemistry, with particular focus on therapeutic carbamates and carbamate-based prodrugs. in this context, we will highlight the chemical methodologies adopted for the synthesis of these carbamate derivatives. also, we will outline successful designs of organic carbamates, including a variety of cyclic ether-derived carbamates, as suitable amide bond surrogates leading to a wide range of novel organic carbamates as potent hiv- protease, βsecretase, serine protease, and cysteine protease inhibitors. this information may be useful in further design of carbamate-based molecules as drugs or prodrugs. peptide-based molecules are an important starting point for drug discovery, especially in the design of enzyme inhibitors. because of their high affinity and specificity toward biological functions, peptide-based molecules also serve as valuable research tools. however, the poor in vivo stability, inadequate pharmacokinetic properties, and low bioavailability have generally limited their broader utility. hence, a variety of peptide mimics are being developed to improve drug-like character along with increased potency, target specificity, and longer duration of action. − to this end, several classes of peptidomimetics are tailored by replacing the native amide bond with unnatural linkages − such as retro-amide, urea, − carbamate, and heterocycles , as peptide bond surrogates. these functionalities confer metabolic stability toward aminopeptidases, the enzymes involved in the metabolism of peptidelike drugs. the carbamate's emerging role in medicinal chemistry is also due to its chemical stability and to its capability to increase permeability across cellular membranes. these attributes of organic carbamates have been exploited in drug design. as a result, the carbamate motif is becoming the choice for peptide bond surrogates. other uses of carbamates are well-known. particularly, the employment of carbamates in various industries as agrochemicals, in the polymer industry, and also in peptide syntheses. − in addition, among the various amineprotecting groups, carbamates are commonly used to enhance their chemical stability toward acids, bases, and hydrogenation. one important feature of organic carbamates is represented by the amide resonance. the amide resonance in carbamates has been studied in detail employing both experimental and theoretical methods by estimating the c−n bond rotational barriers. − the amide resonance in carbamates has been shown to be about − kcal mol − lower than those of amides, owing to the steric and electronic perturbations due to the additional oxygen. three possible resonance structures (a, b, and c, figure ) contribute to the stabilization of the carbamate moiety. carbamate motifs are characterized by a pseudo double bond. this implies the potential deconjugation of the heteroatom-(σ-bond)-carbon-(π-bond)-heteroatom system that restricts the free rotation about the formal single σ-bond. therefore, two isomers, syn and anti, may coexist in carbamates ( figure ) . , although carbamates display close similarity to amides, they show preference for the anti-isomer conformation. the anti rotamer is usually favored by . − . kcal mol − for steric and electrostatic reasons with respect to the syn counterpart. in many cases, the energy difference may be close to zero. as a result, those carbamates are found as an approximately : mixture of syn and anti isomers, as in the case of a number of boc-protected amino acid derivatives. this issue is of key importance since this balanced rotamer equilibria and the low activation energies render carbamates as optimal conformational switches in molecular devices. the influence of the r and r substituents on the free-energy difference between the two conformations has been investigated. beyond steric effects, electronegativity of r must be considered since it may affect the conformation in many ways, including changes in the dipole moment and bond angles. only the anti conformation would be expected in five-, six-, and seven-membered cyclic carbamates. calculations of the dipole moment for the carbamate group support this expectation. solvent, concentration, salts, and ph strongly influence the free energy difference of the syn and anti isomers of carbamates as well. intra-and intermolecular hydrogen bonding may also perturb the syn−anti isomer equilibrium of carbamates. , , a representative example of hydrogen bonding and concentration dependence was provided by gottlieb, nudelman, and collaborators. the authors took into consideration n-boc-amino acids and their corresponding methyl esters. an unusual abundance of syn-rotamer for n-boc-amino acids was detected. n-boc-amino acid esters give the expected spectra, consistent with previous reports of only a single species being observed at room temperature. concentration-dependent h nmr spectra indicate that the proportion of the syn-rotamers increases with concentration, supporting the existence of an aggregation process. since decreasing temperature is another method for stabilizing oligomerization, nmr experiments were also performed at different temperatures. as expected, when the temperature increases, the favored rotamer switches from syn to anti. overall, the collected data strongly supports the concept that the syn rotamers of n-carbamoylated amino acids form intermolecularly h-bonded species and the oh of the carboxylic acid must be involved in this process, as the corresponding esters do not behave similarly. to explain this phenomenon, the formation of a dimer was suggested ( figure ). support of this hypothesis was provided by adding increasing amounts of acetic acid to a solution of a carbamoylated amino acid ester. as expected, the syn rotamer appeared, and its concentration increased as a function of the amount of acid added. in contrast, addition of acetic acid to a solution of the corresponding carbamoylated amino acid did not affect the anti/syn ratio. in this context, moraczewski and co-workers designed a more effective hydrogen-bonding system that selectively perturbs the syn/anti rotamer equilibrium of a target carbamate group. the authors examined the abilities of acetic acid and , -bis(octylamido)pyridine ( ) to perturb the syn/ anti ratio of carbamates and ( figure ). in a cdcl solution, acetic acid moderately stabilizes double hydrogen bonding of the syn rotamer of phenyl carbamate ( figure a) , with no relevant effect on the syn/anti ratio for pyridyl carbamate ( figure b ). in the second case, the carboxylic acid favors donation of a hydrogen bond to the more basic pyridyl nitrogen and forms the complex shown in figure b . on the contrary, in the case of the donor−acceptor−donor triad , it strongly stabilizes the syn rotamer of ( figure d ) over the anti rotamer ( figure c ). there is no effect on the syn/anti ratio for , presumably because of a steric deterrent to the formation of a hydrogen-bonded complex ( figure e ). the carbamate moiety plays a noteworthy role in medicinal chemistry, not only because it is found in drugs but also for its presence in a number of prodrugs. the rate and level of their hydrolysis is a key issue for the duration and intensity of their pharmacological activity. fast hydrolysis of carbamate-bearing drugs may result in weak or shortened activity. on the contrary, carbamate-based prodrugs must undergo extensive hydrolysis at a suitable rate for releasing an active drug and obtaining the expected activity profile. vacondio et al. recently proposed an interesting study in which they compiled a large number of reliable literature data on the metabolic hydrolysis of therapeutic carbamates. the authors were able to exploit the collected data to gain a qualitative relationship between molecular structure and lability to metabolic hydrolysis. a trend was extrapolated, according to which the metabolic lability of carbamates decreased in the following series: aryl-oco-nhalkyl ≫ alkyl-oco-nhalkyl ∼ alkyl-oco-n(alkyl) ≥ alkyl-oco-n(endocyclic) ≥ aryl-oco-n(alkyl) ∼ aryl-oco-n(endocyclic) ≥ alkyl-oco-nharyl ∼ alkyl-oco-nhacyl ≫ alkyl-oco-nh > cyclic carbamates. therefore, carbamates derived from ammonia or aliphatic amines are sufficiently long-lived. an example is represented by cefoxitin ( ), a second-generation cephalosporin antibiotic ( figure ). cyclic fiveor six-membered carbamates are quite stable and do not usually undergo metabolic ring opening. the antibacterial agent linezolid ( ) is a representative example of this class ( figure ). for these drugs, carbamate hydrolysis is not necessarily the half-lifedetermining metabolic reaction. on the contrary, fatty acid amide hydrolase (faah) inhibitor (urb ) showed significant hydrolysis in buffer at physiological ph after h ( figure ). other representative therapeutic carbamate drugs and prodrugs will be discussed in sections and , respectively. organic carbamates play an important role in organic synthesis, especially as subunits of biologically active compounds. accordingly, simple and efficient methods for the synthesis of carbamates are of great interest. a number of methods have been developed for the synthesis of carbamates. . . carbamate synthesis via traditional methods. over the years, a variety of carabamates have been prepared by utilizing the hofmann rearrangement of amides, − the curtius rearrangement of acyl azides, , the reductive carbonylation of nitroaromatics, the carbonylation of amines, the reaction of alcohols with isocyanates, and carbon dioxide alkylation. − the hofmann rearrangement (method i, scheme ) is wellrecognized as a useful method to convert primary carboxamides to amines or carbamates, characterized by the reduction of one carbon in the structure. much effort has been devoted to the development of modified reagents to optimize the hofmann rearrangement since the classical method for this transformation, involving the use of an alkaline solution of bromine, is unsatisfactory and unreliable. a variety of oxidants and bases have been proposed as modified agents, e.g., iodine(iii) reagents such as phi(oac) , meobr, nbs-ch ona, nbs-koh, lead tetraacetate, and benzyltrimethylammonium tribromide. these modified methods, however, require more than equiv or an excess amount of the oxidizing reagent, which is not very convenient. the curtius rearrangement (method ii, scheme ) is the thermal decomposition of acyl azides into the isocyanate intermediate. this method is widely employed in the transformation of carboxylic acids into carbamates and ureas. acyl azides are usually prepared from carboxylic acid derivatives such as acyl chlorides, , mixed anhydrides, , and hydrazides. , subsequent isocyanate intermediates can be trapped by a variety of nucleophiles to provide the carbamate derivatives. the acid chloride method is not suitable for acidsensitive functionalities. one-pot transformations of carboxylic acids into carbamates avoids the isolation of unstable acyl azides. however, protocols involving the use of diphenylphosphoryl azide (dppa) for the one-pot curtius reaction are also characterized by issues related to toxicity and the high boiling point of dppa, which creates difficulties during workup and purification. − other general methods for carbamate preparation involve the use of the highly toxic phosgene, phosgene derivatives, , or isocyanates. significant efforts have been made to find an alternative to the phosgene process. a very attractive substitute for phosgene is carbon dioxide because it is a classic renewable resource (method iii, scheme ). in addition, its use is also very attractive due to its environmentally benign nature (nontoxic, noncorrosive, and nonflammable). carbon dioxide is wellknown to react rapidly with amines to form carbamic acid ammonium salts. the majority of the approaches in this context rely on the creation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles. nevertheless, since the nucleophilicity of the carbamate anion is lower than that of the amine formed in the equilibrium of the salt formation, the subsequent reaction of the carbamate salts with alkyl halides does not selectively provide urethanes. , the formation of carbamates from isocyanates (method iv, scheme ) is fundamentally important to polyurethane industries. synthetic limitations and toxicity issues, however, are associated with the use of phosgene, the most common route to obtain isocyanates. the readily available alkyl chloroformates are the most frequently used reagents for the preparation of carbamates (method v, scheme ). however, these reagents display major drawbacks, as a large excess of base and a long reaction time are required in order to gain acceptable reaction efficiency. moreover, excess reagents are not suitable for the synthesis of molecules bearing multiple functionalities in which the chemoselectivity is critical. . . carbamate synthesis via activated mixed carbonates. a number of organic carbonates have been developed as low-cost and benign alternatives to the phosgenebased routes for the synthesis of organic carbamates. in this context, several new alkoxycarbonylating agents ( − ) based on mixed carbonates have been developed ( figure ). these methods are often used for the synthesis of carbamates in drug design. − mixed carbonates with a p-nitrophenyl moiety are frequently used for the preparation of a large range of carbamates. − for this, p-nitrophenyl chloroformate ( , pnpcocl), when treated with the suitable alcohol in the presence of base, furnishes the corresponding activated carbonates, which have been shown to be useful and effective alkoxycarbonylating reagents for suitable amines (scheme ). examples of carbamate derivatives are shown in table . several alkoxycarbonylating reagents for amino groups having heterocyclic groups, such as n-hydroxyimide, have scheme . traditional synthetic methodologies adopted for the synthesis of carbamates been reported. moreover, the utility and versatility of carbonates and oxalates containing an electron-withdrawing group, such as n-hydroxyimide and benzotriazole derivatives as reagents for various tranformations, have been described. , , takeda et al. reported that -alkoxy[ -(trifluoromethyl)benzotriazolyl]carbonates easily derived from , -bis[ -(trifluoromethyl)benzotriazolyl]carbonate ( , btbc) showed high acylating reactivity toward alcohols as well as amino groups. btbc was prepared from -trifluoromethyl- hydroxybenzotriazole and trichloromethyl chloroformate and purified by washing with dry ether. moreover, it can be stored for several months in a freezer. btbc was allowed to react with primary alcohols in acetonitrile at room temperature to give stable activated carbonates. the carbonates were treated with amines in the presence of -dimethylaminopyridine (dmap), providing the corresponding carbamates (scheme and table ). in connection with our research work aimed at synthesizing biologically active polyfunctional molecules for probing enzyme active sites, we required a more general and synthetically reliable method for the synthesis of various carbamate derivatives. in , we described the utility of di( -pyridyl) carbonate ( , dpc) as an efficient, high-yielding, and convenient alkoxycarbonylation reagent for amines overcoming many of the limitations of existing methodologies. dpc was readily prepared from commercially available -hydroxypyridine and triphosgene in the presence of triethylamine and subsequently reacted with the suitable primary or secondary alcohol (e.g., (+)-menthol) to provide a mixed carbonate. alkoxycarbonylation of primary and secondary amines with the mixed carbonates was carried out in the presence of triethylamine and furnished the corresponding carbamates in good yields (scheme , method a, and table ). potassium hydride was used in the place of triethylamine in the preparation of the mixed carbonates containing tertiary alcohols (scheme and table ). subsequently, we investigated the scope of n,n′-disuccinimidyl carbonate ( , dsc) promoted alkoxycarbonylation of amines with a host of alcohols under mild conditions. rich and co-workers highlighted the convenience of succinimidylbased mixed carbonates for the high-yielding introduction of a -(trimethylsilyl)ethoxycarbonyl (teoc) protecting group to amino acids, without oligopeptide byproduct formation. dsc was found to be a highly effective alkoxycarbonylating reagent for a variety of primary and sterically hindered secondary alcohols. dsc is commercially available, or it can be conveniently prepared from n-hydroxysuccinimide following a procedure tracing out the synthesis of dpc. the ready availability of dsc, the stability of the mixed carbonates, and the mildness of the reaction procedure render this method a reliable route to organic carbamates (scheme and table ). since azides were extensively employed as incipient amines in the context of amino sugar and amino acid syntheses, their conversion into the corresponding carbamate derivatives could provide a novel, effective route for medicinal chemistry applications. in this context, a facile synthetic protocol to transform various azides into the corresponding functionalized journal of medicinal chemistry urethanes in high yields has been developed. in general, mixed carbonates of variously protected alcohols were prepared by reaction of excess dsc or dpc, as described previously. exposure of mixed carbonates to catalytic hydrogenation conditions with azides in the presence of % palladium on charcoal in tetrahydrofuran furnished the corresponding carbamates. interestingly, the use of triethylamine as a promoter has a notable effect on the yield and the rate of the alkoxycarbonylation process (scheme and table ). more recently, yoon and co-workers exploited -substitutedpyridazin- ( h)-ones as electrophilic transfer reagents. , in particular, the authors investigated the carbonylation potency of phenyl , -dichloro- -oxopyridazine- ( h)-carboxylate ( ) to amines for the preparation of phenylcarbamates (scheme and table ). compound is stable in air and in organic solvents at high temperature and is prepared easily from cheap and commercially available , -dichloropyridazin- ( h)-one ( ) in the presence of phenylchloroformate and triethylamine (scheme ). . . recent methodologies for carbamate synthesis. the application of carbon dioxide in organic synthesis has recently attracted much interest. most of the approaches rely on the generation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles, usually alkyl halides. − in this context, a mild and efficient preparation of alkyl carbamates on solid supports was described by jung et al. amines and anilines were coupled with merrifield's resin through a co linker in the presence of cesium carbonate and tetrabutylammonium iodide (tbai). carbon dioxide was supplied by bubbling it into the reaction suspension, where n,n-dimethylformamide (dmf) was the solvent of choice (scheme ). the reaction conditions are convenient for purification, and the reactions undergo complete conversions. the method is convenient for the generation of large combinatorial libraries for rapid screening of bioactive molecules. chiral substrates susceptible to racemization have survived the conditions (table ) . later, these authors reported a one-pot synthesis of n-alkyl carbamates starting from primary amines (scheme ). carbamates were generated via a three-component coupling of primary amines, co , and an alkyl halide in the presence of cesium carbonate and tbai in anhydrous dmf (scheme and table ). direct n-alkylation of the intermediate carbamate a in the presence of additional cesium carbonate by using a different alkyl halide gave rise to the desired n-alkyl carbamate b (scheme ). isolation of the intermediate a proved to be unnecessary, offering shortened synthetic sequences. it is interesting to note that tbai helps to minimize the overalkylation of the produced carbamate, presumably by enhancing the rate of co incorporation and/or stabilizing the incipient carbamate anion through conjugation with the tetrabutylammonium cation. sakakura and co-workers reported urethane synthesis by the reaction of dense carbon dioxide with amines and alcohols by a procedure that is not only phosgene-free but also completely halogen-free (scheme ). dialkyl carbonate synthesis from an alcohol and co is catalyzed by metal complexes such as dialkyl(oxo)tin and dialkyl(dichloro)tin. however, the alcohol conversion is very poor. similarly, the direct reaction of an amine, an alcohol, and carbon dioxide in the presence of dialkyltin compounds produced urethane only in a poor yield. the low conversion observed was attributed by the authors to thermodynamic limitations and catalyst deactivation by coproduced water. in order to overcome this issue, a new reaction system utilizing acetals as a chemical dehydrating agent, with subsequent alcohol regeneration (scheme ), was developed. in order to obtain urethane in good yields, dense-phase co under high pressure was necessary to lower the major side reactions, namely imine formation from acetone and alkylation of amines by alcohols. however, developing less toxic and more active catalysts based on metals other than tin was required. later, these authors reported novel nickel-based catalytic systems for dehydrative urethane formation from carbon dioxide, amines, and alcohols (scheme ). interestingly, adding nitrogenbased bidentate ligands efficiently improved the catalytic activity of ni(oac) -based catalysts (scheme and table ). bipyridines and phenanthrolines with strong coordinating abilities (low steric hindrance and high electron densities) were the better choice for obtaining urethanes in high yields. it is important to note that the ni-phenanthroline system is more active and less toxic than dialkyl(oxo)tin under the same reaction conditions. it is also noteworthy that the catalytic activity of the ni(oac) -( , ′-dimethylbipyridine) system is highly dependent on the ligand/metal ratio (table ). peterson and co-workers proposed a method for rapid sar development of compounds bearing urea or carbamate functionalities (scheme ). for carbamate formation, an journal of medicinal chemistry amine, in principle, could proceed through the carbamic acid− isocyanate reaction, and subsequent reaction with an alcohol may provide a carbamate product. while this is precedented by an intramolecular reaction variant to produce cyclic carbamates, the desired intermolecular coupling was not fruitful under the proposed reaction conditions. carbamic acids produced from secondary amines, however, did react with alcohols under mitsunobu conditions (dibenzyl azodicarboxylate, dbad, and tributylphosphine) in a dbu-catalyzed reaction with gaseous carbon dioxide, providing the corresponding carbamates (scheme and table ). this reaction did not proceed through the isocyanate intermediate but rather through an s n displacement of the activated alcohol. this hypothesis is supported by the observed inversion of stereochemistry upon conversion of a chiral secondary alcohol to the corresponding carbamate (table ) . very recently, jiao and co-workers reported a practical, pdcl -catalyzed efficient assembly of organic azides, carbon monoxide, and alcohols for the direct synthesis of carbamates via isocyanate formation and application in situ (scheme ). mild and neutral reaction conditions and generation of harmless n as the byproduct render this protocol very useful, particularly for the synthesis of bioactive compounds. moreover, the employment of co at atmospheric pressure and the use of a small amount of pdcl catalyst ( mol %) in the absence of any ligand represent a real alternative to customary carbamate synthetic methods (table ) . the synthesis of carbamates through the generation of carbamoyl chlorides is not convenient because of the requirement of the toxic phosgene. also, such carbamoyl chlorides are highly reactive, prone to hydrolysis, unstable, and not suitable for long-term storage. for these problems, batey and co-workers identified the use of carbamoylimidazolium salts as convenient n,n′-disubstituted carbamoyl transfer reagents, showing increased reactivity over carbamoylimidazoles as a result of the imidazolium effect (scheme ). − these salts are readily prepared by the sequential treatment of secondary amines with n,n′-carbonyldiimidazole (cdi) and iodomethane (scheme ). authors envisaged that the carbamoylimidazolium salts, while relatively unreactive with alcohols, would react with nucleophlic alkoxides to produce the corresponding carbamates (table ). in the case of phenols, tertiary amines are appropriate bases for the in situ generation of the reactive phenoxides. the lower acidity of aliphatic alcohols presumably prevents the formation of the alkoxide anion, which would serve as the reactive nucleophile. less acidic alcohols react with carbamoylimidazolium after their conversion into more nucleophilic sodium alkoxides (scheme ). the use of solid-supported reagents has become ubiquitous due to enhanced reactivity and selectivity, milder reaction conditions, convenient work-ups, and decreased solvent waste. the modified hofmann rearrangement, proposed by gogoi et al., is operationally simple, inexpensive and applicable to a variety of aliphatic and aromatic amides for the synthesis of methyl carbamates (scheme ). kf/al o represents a useful and interesting solid-supported strong base, which replaces organic bases in a variety of reactions. sodium hypochlorite is an inexpensive, convenient, and safe alternative to the currently employed oxidants. this prompted the authors to investigate kf/al o along with naocl as an efficient reagent system for hofmann rearrangement. kf/al o basicity stems from the formation of koh in the initial preparation of the solid-supported material by the reaction of kf with alumina supports. under these highly basic reaction conditions, hypochlorite ion is the predominant form of chlorine, reacting with the amide to form an n-chloroamide, which later undergoes rearrangement to the isocyanate. in the presence of methanol, the isocyanate is rapidly converted into the corresponding methyl carbamate (table ) . modifications of the curtius rearrangement have also been explored. lebel and co-workers have reported a useful protocol for the preparation of tert-butyl carbamates from the corresponding carboxylic acids. their reaction with di-tert- (scheme a and table ) . these authors extended the same methodology to the direct synthesis of carbamates of aromatic amines using aromatic carboxylic acids (scheme b and table ) . in particular, the reaction of a chloroformate or di-tert-butyl dicarbonate and sodium azide with an aromatic carboxylic acid produced the corresponding acyl azide, presumably through the formation of an azidoformate. in contrast to what was observed with aliphatic carboxylic acids, using similar reaction conditions, aromatic carboxylic acids led mainly to the formation of the corresponding tert-butyl ester, likely via the displacement of an azide leaving group with tert-butoxide. this may be ascribed to the higher stability of aromatic acyl azides with respect to their aliphatic counterparts. therefore, for these substrates, the curtius rearrangement can be promoted only at higher temperatures ( vs °c). , as mentioned, alkyl chloroformates are the most frequently used reagents for the preparation of carbamates, although the need of an excess amount limits their usefulness. a promising method for preparing carbamates involves the use of a catalytic promoter. − lately, indium-mediated reactions have gained significant consideration due to the high reactivity and unique properties of indium reagents, among them nontoxicity and inertness toward air and water. − moreover, pretreatment is not required for activating indium metal. in this context, jang and co-workers developed a simple, efficient, and selective method for synthesizing carbamates from amines, employing a catalytic amount of indium and only an equimolar amount of alkyl chloroformate (scheme ). the method shows the generality for a wide variety of sterically diverse amines and alcohols and can also be applicable for the selective protection of amino groups under mild conditions (table ) . arndtsen et al. proposed another application of indium-based reagents for the generation of n-protected amines in a single step (scheme and table ). since organoindium reagents readily transfer their organic groups to an imine carbon, only one-third of an equivalent is required, and the only byproduct is represented by indium trichloride. tetraorganoindium reagents can also be employed in a similar fashion for transferring all four organic groups. therefore, one-fourth of an equivalent of indium is necessary for their reaction with imines. copper(i) chloride ( %) was found to be the most efficient catalyst. sodeoka and colleagues reported the use of -alkoxycarbonyl- -nitro- , , -triazole reagents as useful intermediates for the preparation of carbamates (scheme ). to achieve a rapid and clean reaction, the features of the leaving group have a key role. an ideal leaving group should have a highly electronwithdrawing element in order to increase the electrophilicity of the carbonyl carbon, and the nucleophilicity should be low to avoid side reactions. it should also be easily separated from the reaction product. -nitro- , , -triazole (nt), although showing nucleophilicity, could be easily removed from the table . nickel-catalyzed urethane synthesis from co a conversion of amine. b urethane/consumed amide × . c ni/l = : . d ni/l = : . reaction due to its insolubility in dichloromethane or chloroform. nt-based reagents have a series of benefits such as high stability, since they can be stored for long periods without decomposition. reactions of these nt reagents with primary and secondary amines proceeded quickly to give the corresponding carbamates in > % yield (scheme a and table ). in contrast to aliphatic amines, aromatic amines were less reactive. however, the addition of triethylamine was found to be effective in promoting the reactions (scheme b and table ). the reductive carbonylation of aromatic nitro compounds to the corresponding carbamates has remained a subject of great interest both from mechanistic and application standpoints (scheme ). in this section, we will briefly mention the methodologies involving the use of an alcohol, although other procedures employing chloroformates have also been recently reported. , cheng and collaborators report the use of ru(co) − and ru (co) complexes for the catalysis of this reaction and highlighted the key effect of alcohol on the selectivity of carbamates (table ) . the results clearly indicate that low selectivity of carbamate is closely related to the ability of the alcohol to reduce nitroarenes to amino derivatives. therefore, the employment of an alcohol that cannot reduce nitroarene greatly increases the selectivity of carbamate. later, the binuclear rhodium complex [(ph p) rh (μ-oh) ]· c h was employed as an effective catalyst for the reductive carbonylation of nitrobenzenes to carbamate esters (table ) . palladiumbased catalysts have also been explored (table ) . − carbamate synthesis via transfunctionalization of substituted ureas and carbonates in the presence of di-n-butyltin oxide (dbto) as the catalyst was reported by chaudhari and colleagues (scheme a and table ). the carbonate reactivity pattern seems to be driven by the leaving group ability of the alkoxides and phenoxide to form the carbamate observed in aminolysis of carbonates. it has been shown that basicity of reacting urea plays a vital role in the catalytic activity of this reaction. indeed, aliphatic ureas show higher reactivity compared to aromatic ureas due to their higher basicity. the basic dbto is supposed to work as a nucleophile by attacking the carbonyl carbon of the carbonate, thus generating the catalytically active species dibutyl alkoxy carbonato tin [a]. use of dialkyl carbonates as environmentally friendly and nontoxic phosgene substitutes in alkoxycarbonylation reactions has also been exploited by porco et al. (scheme ). particularly, the authors examined the scope of zr(iv)catalyzed carbonate−carbamate exchange processes to prepare carbamates from dialkyl carbonates employing -hydroxypyridine (hyp) as a catalytic additive (table ) . recently, padiya and co-workers reported a useful method for preparing carbamates in an aqueous media (scheme ). interestingly, they found that , ′-carbonyldiimidazole (cdi), although unstable in water, rapidly reacts in aqueous media with amine to give good yields of the corresponding nsubstituted carbonylimidazolide. carbonylimidazolide derived from the primary amine reacts in situ with a nucleophile such as phenol, providing the corresponding carbamate. the product precipitates out from the reaction mixture and can be obtained in high purity by filtration, making the method simple and scalable (table ) . cdi was also found to mediate the lossen rearrangement, which occurs in the transformation of an activated hydroxamic acid into the corresponding isocyanate (scheme ). the proposed methodology is experimentally efficient and mild, being characterized by imidazole and co as the only stoichiometric byproducts. this method is a green and unconventional alternative to the curtius and hofmann rearrangements (table ) . another method based on the lossen rearrangement was recently proposed. the methodology envisaged the reaction of a hydroxamic acid with an alcohol, promoted by , , -trichloro- , , -triazine (cyanuric chloride; tct) in the presence of an excess of n-methyl morpholine (nmm) (scheme and table ). carbamates are inherent to many fda approved drugs. this structural motif is also a key functionality in numerous medicinal agents with clinical potential. in this section, a series of therapeutic carbamates with a variety of applications is outlined. . . miscellaneous carbamates with clinical relevance. . . . rivastigmine. rivastigmine ( , figure ) tartrate (exelon, novartis pharma) is a carbamate derivative that reversibly inhibits the metabolism of acetylcholinesterase (ache) and butyrylcholinesterase (buche) preferentially in the central nervous system (cns). it is used for the treatment of mild-to-moderate alzheimer's disease (ad) dementia and dementia due to parkinson's disease. , the drug can be administered orally or via a transdermal patch. the transdermal patch reduces side effects such as nausea and vomiting. rivastigmine undergoes extensive metabolism by che-mediated hydrolysis to the decarbamylated metabolite, without involvement of the major cytochrome p (cyp ) isozymes. the metabolite may undergo n-demethylation as well as conjugation. the pharmacokinetic half-life of rivastigmine in ad patients is around . h. when given orally, rivastigmine is well-absorbed, with a bioavailability of about % administered as a mg dose. , . . . muraglitazar. muraglitazar ( ) contains a carbamate functionality. it is a potent, novel nonthiazolidindione peroxisome proliferator-activated receptor dual agonist (pparα/γ) that demonstrated highly efficacious glucose and lipid lowering activities in vivo, along with an excellent adme profile. in a double-blind randomized clinical trial, muraglitazar resulted in a statistically significant improvement in plasma triglyceride, hdl cholesterol, apob, and non-hdl cholesterol concentrations at week . muraglitazar reduced triglyceride concentrations to a larger extent than did pioglitazone, regardless of baseline triglyceride levels. muraglitazar and pioglitazone treatment was associated with slight ( − %) increases in ldl cholesterol. however, muraglitazar development was discontinued due to major adverse cardiovascular side effects. . . . roxifiban. roxifiban ( ) is a carbamate derivative with a methyl ester prodrug. it is a potent, nonpeptide antagonist of the glycoprotein iib/iiia receptor. , the free acid resulting from roxifiban hydrolysis blocks the binding of fibrinogen to the receptor, thereby inhibiting platelet aggregation and providing a mechanism for antithrombotic mebendazole ( ) is a methyl carbamate derivative showing broad-spectrum anthelmintic properties. it demonstrated efficacy in the oral treatment of ascariasis, uncinariasis, oxyuriasis, and trichuriasis. like other benzimidazole anthelmintics, mebendazole's primary mechanism of action is consistent with tubulin binding. mebendazole was discontinued in . . . . flupirtine and retigabine. flupirtine ( ) and retigabine ( ) are ethyl carbamate derivatives. flupirtine is a centrally acting nonopioid analgesic that was identified within an antiepileptic drug discovery program by the u.s. national institutes of health. the doses used in a small clinical trial exceeded those established for analgesic activity. on the basis of this data, subsequent structural optimization resulted in retigabine. retigabine has anticonvulsant properties that appear to be mediated by opening or activating neuronal voltage-gated potassium channels. flupirtine showed n-methyl-d-aspartate (nmda) receptor antagonist properties. . . . felbamate. felbamate ( , felbatol, meda pharmaceuticals) is an alkyl carbamate derivative. it is an antiepileptic drug. the mechanism of action of felbamate involves a dual mechanism involving inhibition of n-methyl-d-aspartate (nmda) receptor response and positive modulation of γamino butyric acid subtype a (gaba a ) receptor, thus decreasing neuronal excitation. felbamate is rapidly absorbed (t max = − h) with an oral bioavailability > %. felbamate undergoes moderate metabolism via cyp a and cyp e isoenzymes, which are amenable to inhibition and induction effects. , the clinical use of scheme . synthesis of carbamates by modified curtius rearrangement it is a nonnucleoside reverse transcriptase inhibitor (nnrti). the drug is used as part of highly active antiretroviral therapy (haart). , however, its use is associated with variable treatment response and adverse effects, in most part because of the large differences in pharmacokinetics. cyp b is the main enzyme catalyzing the major clearance mechanism of efavirenz ( -hydroxylation to -hydroxyefavirenz) in vivo. , . . . zafirlukast. zafirlukast ( , accolate, astrazeneca) is a cyclopentyl n-aryl carbamate derivative. it is a selective and competitive receptor antagonist of the cysteinyl leukotrienes d- and e- , which is indicated for the prophylaxis and treatment of mild-to-moderate persistent and chronic asthma. both o → ch and o → nh bioisosteric analogues of zafirlukast were found to be potent. the carbamate moiety present in zafirlukast provided an excellent in vitro and in vivo profile and high oral bioavailability. zafirlukast undergoes hepatic metabolism, where hydroxylation by cytochrome cyp c is the major biotransformation pathway. the metabolites of zafirlukast do not significantly contribute to its overall activity. . . . mitomycin c. mitomycin c ( , mmc, mutamycin) is a complex carbamate derivative. it is an antitumor antibiotic that was identified in the s in fermentation cultures of the gram-negative bacteria streptomyces caespitosus. mmc is a site-specific, nondistorting dna cross-linking agent. , however, recent reports suggest that dna may not be the primary target of the drug. in particular, interaction of mmc with rrna and subsequent inhibition of protein translation has been proposed. mmc is customarily used as a chemotherapeutic agent in the treatment of several types of cancer, such as bladder, colon, and breast cancers. . . therapeutic carbamates as hiv protease inhibitors. hiv protease is an aspartic acid protease responsible for the cleavage of the gag−pol polyprotein into functional proteins essential for the production of infections progeny virus. inactivation of hiv- protease either by site-directed mutagenesis or by chemical inhibition results in the formation of immature, noninfections virus particles. as a consequence, hiv- protease is an attractive target in antiviral therapy. hiv protease is a c -symmetric, -amino acid homodimeric aspartyl protease in which each protein subunit contributes one asp-thr-gly motif to the single active site. the x-ray crystallographic analysis of the native protein and subsequent protein−ligand complexes and extensive research programs on other aspartyl proteases, including human renin, provided a path toward accelerated drug discovery programs targeting hiv protease. − a number of fda-approved hiv protease inhibitor drugs contain an important carbamate functionality. in this section, currently approved protease inhibitor drugs are discussed (figure ) . . . . ritonavir. ritonavir ( , norvir, abt- , a- , abbvie, inc.) structure possesses a thiazolyl methyl carbamate functionality. it is a peptidomimetic inhibitor of both the hiv- and hiv- proteases and was approved by the fda in march . this first-generation protease inhibitor was developed at abbott laboratories. the discovery of ritonavir was based on of . μm, bioavailability of %, and a plasma half-life of . h. ritonavir has a high molecular weight; however, it showed excellent pharmacokinetic properties. this is possibly due to the increased stability of the thiazole groups to oxidative metabolism and also due to its effect on cytochrome p oxidative enzymes. ritonavir is a type ii heme ligand that fits into the cyp a active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen. amprenavir was identified as a potent, orally bioavailable hiv- protease inhibitor with a low molecular weight and a mean ic of nm. it is marketed with a twice-a-day dosing format. amprenavir structure bears a stereochemically defined tetrahydrofuranylcarbamate engaging in a weak backbone interaction with the protease. , in vitro and in vivo studies have shown that amprenavir is primarily metabolized by cyp a , and the two major metabolites result from oxidation of the tetrahydrofuran and aniline moieties. properties. drv also maintains high potency against multidrugresistant hiv- strains. the design of drv originated from the backbone binding concept envisaging that an effective protease inhibitor maximizes rich networks of hydrogen-bonding interactions with the backbone atoms throughout the active site of the protease. the bis-thf moiety present in drv was designed based on the x-ray structure of inhibitor-hiv- protease complexes. the bis-thf carbamate moiety of drv was found to be essential for enzyme affinity (see figure for details). drv demonstrated exceptional potency against both wild-type hiv isolates and a wide range of resistant variants. hiv- variants. in , drv received full approval for the treatment of therapy-naive adults and children. drv is metabolized by the isoenzyme cyp a . , however, in the presence of a low dose of ritonavir, drv exhibits very good pharmacokinetic properties in patients. metabolism prodrugs are chemically modified forms of the actual pharmacologically active drug that undergo in vivo transformation to release the active drug molecule. this is a wellestablished strategy to improve drug disposition properties (physicochemical, biopharmaceutical, or pharmacokinetic properties) of pharmacologically relevant compounds and thereby increase their drug-like profile. , a prodrug strategy helps to overcome a variety of hurdles in drug formulation and delivery such as (i) poor oral absorption and aqueous solubility, (ii) poor lipid solubility, (iii) chemical instability, (iv) rapid presystemic metabolism, (v) toxicity and local irritation, and (vi) lack of site-selective delivery. a functional group on the parent drug may be used to form a chemical bond with the promoiety. generally, the linker should be self-removing or cleavable so that the parent drug can be released spontaneously or under a certain triggering condition, such as the presence of an enzyme or a change in ph. the promoiety coupled to the parent drug provides the ability to improve the drug-like properties or overcome the barriers in delivering the drug to its target cells. carbamates are the esters of carbamic acid, preferentially used in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamates are typically enzymatically more stable than the corresponding esters. they are, in general, more susceptible to hydrolysis than amides. thus, bioconversion of carbamate prodrugs requires esterases for the release of the parent drug. upon hydrolysis, carbamate esters release the parent phenol or alcohol drug and carbamic acid, which, due to its chemical instability, breaks down to the corresponding amine and carbon dioxide. carbamates of primary amines can also fragment into isocyanates and alcohols on treatment with bases, a further potential pathway for metabolic degradation. , the oh-catalyzed hydrolysis of these carbamate esters (r′-nhco-or) is strongly dependent on both the pk a of the proton on the leaving group (roh) and the degree of substitution on the nitrogen of the carbamate ester. since phenols have a lower pk a with respect to alcohols, carbamate esters of phenols are generally more chemically labile than those of alcohols. in the case of alcohols, both the n-monosubstituted and n,n-disubstituted carbamates are chemically stable toward hydrolysis. in phenols, n,ndisubstituted carbamates are chemically stable, whereas nmonosubstituted carbamates are the most labile toward chemical hydrolysis. short-lived carbamates have also been used as prodrugs of heteroaromatic amines (e.g., capecitabine, ) and amidines (lefradafiban ( ), dabigatran). . . alcohol and phenol carbamate prodrugs. most of the therapeutically relevant carbamate prodrugs have been designed as substrates of specific enzymes. antibody-directed enzyme prodrug therapy (adept) , and gene-directed enzyme prodrug therapy (gdept) are new strategies for targeting tumors. carboxypeptidase g (cpg ), an enzyme of bacterial origin, has been shown to catalyze the cleavage of an amide, carbamate, or urea linkage between glutamic acid and an on the basis of this specificity, a large number of prodrugs have been designed and synthesized for cpg . as shown in figure , the prodrug (zd p) is activated by hydrolysis at the carbamate bond by cpg to the corresponding potent di-iodophenol mustard ( ) . was found to possess the best profile in terms of enzymatic kinetics, cytotoxicity, and in vivo efficacy. it was selected for clinical development. the half-life (t / ) of the drug is approximately min, which is enough for diffusion into the tumor cell from the local release site and to minimize peripheral toxicity. , irinotecan was designed to deliver camptothecin as a predominant topoisomerase i inhibitor for anticancer therapy. irinotecan hydrochloride salt (cpt- , camptosar; pfizer) is a parenteral aqueous soluble carbamate prodrug of antineoplastic topoisomerase i inhibitor (sn- , -ethyl- -hydroxy-camptothecin). the potent antitumor activity of irinotecan is due to rapid formation of active metabolite in vivo (figure ). in this molecule, a dipiperidino ionizable promoiety is linked to the phenol functionality by a carbamate bond, thus improving the overall aqueous solubility. − the bioconversion back to occurs primarily by human liver microsomal carboxylesterases, ces a and ces , which release the ionizable piperidinopiperidine promoiety and , the active form of the drug. beyond minimizing the rate of enzymatic hydrolysis of its prodrug, sustained drug action can also be provided by decreasing the rate of drug metabolism. this is the case of bambuterol ( , bambec, astrazeneca), a bis-dimethyl carbamate prodrug of the β -agonist terbutaline ( ), which is used as a bronchodilator in the treatment of asthma. the phenolic moiety of terbutaline is subjected to rapid presystemic metabolism. in bambuterol, protection of this functionality also avoids first-pass intestinal and hepatic metabolism. this prodrug is inactive, however, after oral administration; it is slowly converted to terbutaline, mainly outside the lungs, by a series of hydrolysis and oxidation reactions (maily catalyzed by plasma cholinesterase, pche, and by cyp , figure ). , this allows a once-daily bambuterol treatment with respect to the three daily terbutaline administrations. an n,n′-dimethyl ethylenediamine spacer, used for the evaluation of cyclization-elimination-based prodrugs of phenols and alcohols, has been used for the development of prodrugs as a part of the adept activation strategy. when activated by a specific enzyme, the terminal amino group on the spacer activates and initiates an intramolecular cyclization reaction to eliminate a phenol or alcohol parent drug with parallel release of the cyclized spacer. in one such application, scherren et al. explored paclitaxel- ′-carbamates. this is particularly interesting because a free ′-hydroxyl group is important for biological activity. in general, carbamate linkages are more stable in vivo than esters and carbonates. since the proteolytic active form of plasmin is located in the tumor, linking a cytotoxic drug to a plasmin substrate may result in tumor-selective delivery. on the basis of this rationale, following plasmin hydrolysis, the spacer is expected to undergo spontaneous cyclization to yield a cyclic urea derivative (imidazolidinone), thereby releasing paclitaxel ( ) , as illustrated in scheme . , . . amine and amidine carbamate prodrugs. the amine group is one of the most common functional groups in many approved drugs. amines in drugs can cause physicochemical hurdles that have the potential to limit their safety and effective delivery to desired sites of action. therefore, a variety of prodrugs of amines have been designed to overcome formulation and delivery barriers. the carbamate functionality has been utilized in many prodrug strategies designed for amines. short-lived carbamates are also used as prodrugs of heteroaromatic amines and amidines. gabapentin ( , neurontin; pfizer, figure ) is a structural analogue of γ-aminobutyric acid (gaba). it is marketed as an anticonvulsant and an analgesic agent. gabapentin shows a number of limitations, including saturable absorption, high interpatient variability, lack of dose proportionality, and a short half-life. gabapentin enacarbil ( , horizant, previously known as xp ) is a carbamate prodrug of gabapentin. the prodrug is benefited by a monocarboxylate transporter type (mct ). mct is expressed in all segments of the colon and upper gastrointestinal tract. the prodrug also helps the sodium-dependent multivitamin transporter (smv t), responsible for absorption of multiple essential nutrients. , following absorption via these pathways, the prodrug is rapidly converted to gabapentin by nonspecific esterases, mainly in enterocytes and to a lesser extent in the liver. during conversion to gabapentin, each molecule of also generates carbon dioxide, acetaldehyde, and isobutyrate (figure ). the oral bioavailability of was improved from to % in monkeys. it showed doseproportional gabapentin exposure in humans. in , xenoport received fda approval (horizant) for the treatment of moderate-to-severe restless legs syndrome. in , horizant was also approved for the management of postherpetic neuralgia (phn) in adults. capecitabine ( , xeloda, roche) was designed to achieve greater selectivity than its active form, -fluorouracil ( , -fu). it is an orally administered carbamate prodrug of -fu, belonging to the fluoropyrimidine carbamate class. it requires a cascade of three enzymes for the bioconversion to the active drug. as shown in figure , the enzymatic bioconversion starts in the liver, where human carboxylesterases and (ces and ces ) cleave the carbamate ester bond. intact capecitabine is absorbed in the intestine, and its bioconversion in the liver releases the parent drug. to some extent, its bioconversion proceeds in tumors, thus avoiding any systemic toxicity. in particular, the remaining transformations to -fu are catalyzed by cytidine deaminase and thymidine phosphorylase. the latter enzyme is highly enriched in tumors, thus providing selective release of -fu in cancer cells. , the absorption of capecitabine is evident since % of an orally administered dose is recovered in urine and the t max of -fu is reached in approximately . − h. capecitabine is currently approved as a first line of therapy for colorectal and breast cancers and is also approved for use in combination with other anticancer drugs. , alkoxycarbonyl derivatives can serve as useful prodrugs for benzamidines. for example, the methoxycarbonyl methyl ester lefradafiban ( , bibu , boehringer ingelheim, germany) is effectively converted to the active platelet aggregation inhibitor fradafiban ( , bibu ) after oral administration. this was revealed by monitoring the plasma concentrations of and by ex vivo platelet aggregation studies. lefradafiban is the orally active prodrug of fradafiban, a glycoprotein iib/iiia receptor antagonist. esterases, but not cyp -dependent enzymes, are involved in the conversion of lefradafiban to fradafiban in vivo (figure ). over the years, we have developed a series of novel hiv- protease inhibitors incorporating cyclic ether-derived carbamates designed based on the x-ray structures of inhibitor-hiv- protease complexes. , in this endeavor, we have specifically developed stereochemically defined cyclic ether templates, where the cyclic ether oxygen could effectively replace a peptide carbonyl oxygen. the advantage of such replacement is to reduce peptidic features and improve metabolic stability of compounds. these cyclic ligands have been incorporated as carbamate derivatives. the evolution of the carbamate structural template is shown in figure . on the basis of the x-ray crystal structure of saquinavir ( )-bound hiv- protease, we first investigated -(r)-tetrahydrofuranylglycine so that the -(r)-thf ring oxygen would interact with the asp nh, similar to the asparagine side chain carbonyl oxygen of saquinavir (compound ). − in an effort to reduce molecular weight, the p quinoline was removed, and the amide bond was replaced with a carbamate to provide inhibitor with significant reduction of molecular weight ( da from da). the x-ray crystal structure of bound hiv- protease revealed that the ring oxygen of the -(s)-tetrahydrofuran ( -(s)-thf) is within proximity to form a hydrogen bond with the asp nh bond in the s subsite. the importance of the carbamate moiety is evident. the carbamate nh forms a hydrogen bond with the backbone carbonyl of gly , and the carbamate carbonyl functionality makes a tightly bound water-mediated hydrogen bond with the backbone nh's of the flap ile and ile ′ in the active site. our further investigation of the -(s)-thf in inhibitors containing a hydroxyethylene isostere led to a series of exceptionally potent inhibitors. as shown in table , -(s)-thf-containing carbamate drivatives (compounds − ) provided very potent inhibitors in antiviral assays. the potency enhancing effect of -(s)-thf carbamate was subsequently demonstrated in inhibitors containing the (r)-(hydroxyethyl)sulfonamide isostere. clinical development of inhibitor (vx ) led to fda approval of amprenavir for the treatment of hiv/aids patients. , further development of carbamate-derived novel hiv- protease inhibitors is shown in figure . we have designed a variety of inhibitors incorporating cyclic sulfones and bicyclic ligands (figure , compounds − ) . , these ligands were conceived in order to maximize hydrogen-bonding interactions with the protease backbone as well as to fill in the hydrophobic pocket in the s subsite. on the basis of the x-ray structure of saquinavir-bound hiv- protease, we then designed a fused bicyclic tetrahydrofuran (bis-thf) ligand to form hydrogen bonds with backbone aspartates in the s subsite as well as to fill in the hydrophobic site adjacent to the p -quinoline ring of saquinavir ( figure ) . , an x-ray structural analysis of -bound hiv- protease revealed that the bis-thf carbamate mimics the majority of p −p -amide bonds of saquinavir. a detailed structure−activity study also established that the stereochemistry of the bis-thf ring, and the position of the ring oxygens is critical to potency. with the development of a bis-thf carbamate that could form a network of hydrogen bonds in the s subsite of hiv- protease, we investigated transition state isosteres that can be functionalized to form hydrogen bonds in the s ′ subsite. our basic hypothesis was to design inhibitors that form a network of hydrogen bonds with the protease backbone atoms throughout the active site of hiv- protease, from s to s ′ subsites. this backbone binding strategy to combat drug resistance led to the development of a series of very potent carbamate-derived protease inhibitors. , , as shown in figure , we incorporated the bis-thf ligand in the (r)-hydroxyethylsulfonamide isostere bearing p-methoxysulfonamide as the p ′ ligand so that the methoxy oxygen can interact with aspartate backbone atoms in the s ′ subsite. the resulting inhibitors exhibited notable potency. , inhibitor with a ( r, as, ar)-bis-thf as the p ligand is significantly more potent in an antiviral assay than corresponding inhibitor with an enantiomeric bis-thf ligand. an x-ray structure of -bound hiv- protease revealed that the carbamate nh formed a hydrogen bond with the backbone gly carbonyl group and that carbamate carbonyl of is involved in an interesting tetra-coordinated hydrogen-bonding interaction with the structural water molecule, inhibitor sulfonamide oxygen, and the flap ile nh residues. also, the structure revealed interactions with the backbone atoms in both the s and s ′ subsites. , further replacement of the p-methoxy group at the s ′ to a p-amino group led to inhibitor ( figure ) . this inhibitor showed marked enzyme inhibitory activity as well as antiviral activity. an in-depth antiviral study revealed that maintained excellent antiviral activity against multidrug-resistant hiv- variants. − the x-ray structural studies of darunavir-bound hiv- protease showed extensive active site interactions ( figure ) . particularly, it formed a network of hydrogen bonds with the protein backbone throughout the active site. darunavir also exhibited favorable pharmacokinetic properties. subsequently, clinical development led to its fda approval as darunavir for the treatment of hiv/aids patients. , the carbamate functionality of darunavir ( ) was assembled as shown in scheme . ( r , a s , a r ) - -hydroxyhexahydrofuro[ , -b]furan (bis-thf) was treated with disuccinimidyl carbonate to provide activated mixed carbonate . reaction of this activated carbonate with hydroxyethylsulfonamide isostere provided darunavir. , the backbone binding inhibitor design strategies to combat drug resistance have been further utilized by us and others to advance a number of other preclinical and clinical inhibitors with carbamates. , figure shows selected bis-thfderived carbamates ( − ) with marked enzyme and antiviral activities. − like darunavir, inhibitor-bound x-ray structures of these inhibitors showed a network of hydrogen bonds in both s and s ′ subsites of hiv- protease. the inhibitor side chains as well as the bis-thf bicyclic framework also effectively filled the hydrophobic pockets in the active site. we have outlined a selected number of cyclic ether-derived carbamates that have been developed based on the backbone table . carbamates from cdi-and tct-mediated lossen rearrangement , scheme . tct-mediated lossen rearrangement for carbamate synthesis binding concept in figure . , particularly, incorporation of these stereochemically defined oxacyclic ligands such as cp-thf, tp-thf, tris-thf, and fluoro-bis-thf provided exceptionally potent inhibitors ( and − ) with clinical potential. − the importance of the carbamate functionality in these inhibitors is particularly worthy of note. x-ray crystal structures of these inhibitors in complex with hiv- protease provided the ligand-binding site interactions responsible for their respective antiviral potency against wild-type and multidrug-resistant viruses. in general, inhibitors are involved in hydrogen-bonding interactions with asp , asp , gly , asp , asp ′, and asp ′ in the hiv- protease active site. furthermore, the ring cycles adequately fill the hydrophobic pockets in the active site. inhibitors the search for an effective treatment for alzheimer's disease (ad) remains a major challenge in medicine. one of the pathological hallmarks of ad is the formation of β-amyloid (aβ) peptides in the cortex of ad patients. aβ-peptides are generated from β-amyloid precursor protein (app) by sequential cleavage by β-secretase (also known as bace or memapsin ) and γ-secretase. due to this central role of aβproduction, both β-secretase and γ-secretase have been implicated as important therapeutic targets for ad intervention. , as a result, design and synthesis of selective βsecretase and γ-secretase inhibitors have become an intense area of research over the years. . . development of β-secretase inhibitors. following the discovery of β-secretase, the first-generation β-secretase inhibitors were designed and synthesized by ghosh, tang, and co-workers. as shown in figure , utilizing a carbamate derivative of the leu−ala isostere , potent pseudopeptide inhibitors and were identified. the x-ray crystal structure of -bound β-secretase was determined to provide molecular insight into the ligand binding site interactions. the in-depth structural analysis thus provided critical drug design templates and led to the beginning of structure-based design approaches to peptidomimetic/nonpeptide β-secretase inhibitors. , the x-ray structure of -bound β-secretase revealed that the p asparagine side chain carboxamide nitrogen formed an intermolecular hydrogen bond with the p glutamic acid carbonyl group. on the basis of this molecular insight, a number of − membered cycloamide-carbamate-based macrocyclic inhibitors were designed and synthesized. as shown in figure , acyclic carbamate derivatives ( and ) were less potent than their corresponding cyclic inhibitors. inhibitor , with a -membered macrocycle containing a trans-olefin, amide and carbamate functionalities within the macrocycle, showed good β-secretase inhibitory activity. saturated inhibitor is less potent against bace , but it showed enhanced potency for bace . x-ray structural studies of inhibitor -bound secretase revealed that the carbamate carbonyl forms a hydrogen bond with the gln side chain carboxamide residue. interestingly, unsaturated inhibitor showed slight selectivity against memapsin (k i = nm). the design of a selective inhibitor is important for reducing toxicity through off-target effects. particularly, selectivity over other aspartic proteases, such as bace , pepsin, renin, cathepsin d (cat-d), and cathepsin e, may be important for the reduction of side effects and drug efficiency. on the basis of our detailed structure−activity studies and xray structural analysis, we have designed a variety of highly selective and potent bace inhibitors. in this perspective, we will highlight only the development of bace inhibitors bearing carbamate functionalities. as shown in figure , inhibitor is a potent bace inhibitor. however, it did not show selectivity against bace or cat-d. subsequent structure-based design led to the development of selective inhibitors and , which contain a pyrazolylmethyl and oxazolymethyl carbamate at the p position, respectively. inhibitor showed excellent bace potency and selectivity over bace and cathepsin d. the x-ray crystal structure of -bound β-secretase revealed that the carbamate carbonyl formed a hydrogen bond with the thr- backbone nh. also, figure . examples of phenol carbamate prodrugs and their metabolic activation. ethylenediamine spacer and release of paclitaxel the pyrazole nitrogen formed a strong hydrogen bond with the thr- side chain hydroxyl group. the p -sulfonyl functionality formed a number of hydrogen bonds in the s subsite as well. on the basis of this molecular insight, oxazole-derived was designed to provide a more stable and selective inhibitor. the synthesis of inhibitors and is outlined in scheme . urethanes and were prepared by treatment of , -dimethylpyrazolylmethanol ( ) or , dimethyl- -oxazolemethanol ( ) with triphosgene in the presence of triethylamine, followed by l-methionine methyl ester hydrochloride ( ) . saponification of the resulting methyl esters provided the corresponding acids. coupling of amine with acids and , as described previously, and subsequent oxidation of the sulfides with m-chloroperbenzoic acid furnished inhibitors and . freskos and co-workers have reported a series of β-secretase inhibitors that incorporated polar carbamate derivatives as the p ligand. , this strategy led to improve the cat-d selectivity. it was hypothesized that the s subsite of cat-d is more lipophilic and less tolerant of polar groups. as can be seen in figure , benzyl carbamate derivative displayed -fold selectivity over cat-d. however, polar -pyridylmethyl derivative improved selectivity nearly -fold. the corresponding -pyridyl methyl compound provided a reduction in selectivity (∼ -fold). -(s)-tetrahydrofuranyl carbamate showed a nearly -fold selectivity over cat-d. these inhibitors have also shown good to excellent ic values in hek cells. . . development of γ-secretase inhibitors. over the years, many structural classes of potent and selective γ-secretase inhibitors have been reported. a number of inhibitors displayed drug-like properties and also inhibited aβ production in animal models. in this section, we will review inhibitors with carbamate functionality. on the basis of γ-secretase inhibitor (ly- ), peters and co-workers designed a series of carbamate derivatives of dibenzazepinone as potent and metabolically stable γ-secretase inhibitors. as shown in figure nm. absolute stereochemistry of the active enantiomer was not determined. piperidine carbamate also showed good potency. carbamate derivative displayed a good membrane aβ ic value; however, it showed poor cyp properties. further modification led to compound with good inhibitory activity and improved cyp properties. bergstrom and co-workers reported a series of carbamateappended n-alkyl sulfonamides as γ-secretase inhibitors. , figure depicts selected examples that show potent aβ inhibitory activity. sulfonamide derivative was identified as a potent γ-secretase inhibitor. exploration of carbamateappended n-alkylsulfonamides resulted in potent inhibitors such as − . tertiary carbamate showed significant reduction of brain aβ in transgenic mice compared to that of its benzyl derivative. this compound also showed improved brainto-plasma ratio and good absolute brain concentration. hepatitis c virus (hcv) is a bloodborne virus that is found worldwide. there are multiple strains or genotypes of the hcv virus. hcv infections lead to progressive liver damage, cirrhosis, and liver cancer. in recent years, there have been a number of new and effective antiviral drugs developed for the treatment of hepatitis c. these include the development of hcv ns / a protease inhibitors and inhibitors hcv ns a. in this section, carbamate-derived therapeutics will be discussed. . . carbamate-derived serine protease inhibitors. serine proteases are a large family of proteolytic enzymes that play a variety of critical roles in many physiological processes. , deregulation of serine proteases has been related to the pathogenesis of diseases such as stroke, inflammation, alzheimer's disease, cancer, and arthritis. therefore, significant research efforts have been focused in the discovery of serine protease inhibitors. the active site of all serine proteases consists of a catalytic triad of ser, his, and asp. the nucleophilic attack by the hydroxyl group of serine at the carbonyl carbon of the scissile bond of the substrate, via general base catalysis by histidine, leads to the tetrahedral transition state. the tetrahedral intermediate ultimately collapses, leading to cleavage products. − these key active residues are conserved in all serine proteases. x-ray structural studies revealed that these residues are superimposable in the majority of serine proteases. , therefore, selectivity over other serine proteases represents a key issue to be taken into consideration during inhibitor design. most early inhibitors acted via a covalent mechanism in which an electrophilic group formed a covalent bond with the serine hydroxyl of the catalytic triad. the electrophilic groups are commonly referred to as serine traps or warheads. however, covalent inhibitors lack selectivity and specificity against other proteases in the same class or clan. the rational design of covalent serine protease inhibitors usually involves the selection of a good substrate to be linked to a serine trap/warhead. chloromethyl ketones, diphenyl phosphonate esters, trifluoromethyl ketones, peptidyl boronic acids, α-ketoheterocycles, and β-lactam derivatives are usually employed as warheads. on the basis of these warheads, a variety of irreversible and reversible covalent serine protease inhibitors were designed. in this section, representative serine protease inhibitors containing a carbamate functionality will be outlined. carbamate derivative (figure ), a diphenyl phosphonate ester containing a cbz group and bearing a single amino acid side chain, showed very good inhibitory activity against human plasma kallikrein, useful for the treatment of hereditary angioedema. , thrombin is an attractive therapeutic target for drug development against pulmonary embolism, thrombosis, and related diseases. , compound showed good potency and selectivity against human thrombin. it is stable and displayed no activity against acetylcholinesterase and no selectivity over cysteine proteases. peptidyl boronic acid-based thrombin inhibitors were developed by dupont-merck. in particular, n-boc derived inhibitor is a potent inhibitor (k i = . nm). , imperiali and co-workers introduced trifluoromethyl ketones as specific serine protease inhibitors, particularly for chymotrypsin and elastase. researchers at astrazeneca designed numerous peptidyl trifluoromethyl ketone derivatives as potent human elastase inhibitors. − further optimization of features resulted in the development of a number of orally active inhibitors. in particular, methyl carbamate derivative inhibitor was shown to be a very potent inhibitor (k i = nm) with excellent oral bioavailability in laboratory animals. , optically pure compound with an (s)-configuration at the p isopropyl side chain became a candidate for clinical development for potential treatment of elastase-implicated respiratory diseases. peptidomimetic boronic acid-based hepatitis c virus (hcv) ns / a protease inhibitors were designed and synthesized for the treatment of chronic hcv infections. hcv infections can lead to progressive liver damage, cirrhosis, and liver cancer. the ns / a serine protease plays a critical role in virus replication and has become an antiviral drug development target. , the first specific and potent hcv protease inhibitor with good oral bioavailability was ciluprevir, which contains a carbamate functionality ( , biln , figure ). this noncovalent macrocyclic peptidic inhibitor was the result of a substrate-based approach for the design of active site inhibitors. this inhibitor is very active in enzymatic (ic = nm) and cell-based replicon assays (ic = . nm) of hcv genotype . ciluprevir was later discontinued due to cardiac toxicity in animal models, but its development paved the way to boceprevir (victrelis, schering-plough, approved by fda in may ) and telaprevir (vx- , vertex pharmaceuticals and johnson & johnson). , in particular, for the development of boceprevir, the introduction of a ketoamide moiety, together with p and p optimization, led to inhibitor showing a k i of nm. its x-ray crystal structure in complex with the enzyme also provided insight for further optimization. indeed, a cyclopropylalanine residue was found to be optimal at p , and the resulting carbamate derivative showed a k i of nm. although inhibitors and displayed good enzyme inhibitory potency, they did not display cellular activity in a subgenomic hcv replicon assay, possibly because of their strong peptidic character. the discovery that an nmethylated leucine at p was critical for both enzymatic potency and cellular activity led to the potential of cyclopropylfused proline being envisaged as an optimum, conformationally constrained surrogate for this part of the inhibitor. combination of the p -optimized ligand with previously optimized p and p residues provided carbamate derivative with a k i = . nm and ic = nm. finally, truncation and p optimization, by the employment of a cyclobutyl moiety, led to compound (k i = nm), the direct boceprevir ancestor. subsequently, compounds and ( figure ) with a carbamate containing p proline core showed very potent inhibitory activity (ic = nm for and nm for ). similarly, macrocyclic inhibitor with an α-amino cyclic boronate showed good potency (ic = nm). the electron-withdrawing effect of the ester and amide functionalities was also utilized in the design of α-ketoesteror α-ketoamide-derived transition state inhibitors. a range of hcv ns / a protease inhibitors were designed and synthesized, incorporating α-ketoamide templates at the scissile site. structure-based design led to a variety of potent acyclic and cyclic inhibitors with ketoamide templates, as exemplified in compounds (ic tripeptidyl α-ketobenzoxazole inhibited human neutrophil elastase (hne) with an ic of nm. the ketooxazolinederived inhibitor displayed very potent activity against hne (ic = . nm). . . hcv ns a inhibitors. carbamate derivatives also play a key role as inhibitors of hcv ns a, which represents a new and promising target for hcv therapy. hcv ns a is a zincbinding phosphoprotein, and its role in the hcv virus life cycle is still not clear. however, it plays a critical role in hcv rna replication. also, it is involved in virion morphogenesis. due to the lack of enzymatic function, inhibitors of this viral-encoded protein have been pursued. researchers at bristol-myers squibb screened a library of compounds for their ability to inhibit hcv rna replication. this led to the identification of a lead compound specifically interfering with rna replication and later proving to inhibit the activity of ns a protein. subsequent optimization was focused on broadening the genotype specificity and improving pharmacokinetic properties of compounds. symmetry of the molecule played an important role in inhibitory potency. this finally led to the discovery of daclatasvir ( , bms- , figure ) a first-in-class inhibitor of the hcv ns a replication complex. daclatasvir was approved in europe in august, . ledipasvir ( , gs- , gilead sciences, figure ) is another carbamate-containing hcv ns a inhibitor with potent antiviral activity against hcv genotypes a and b. harvoni, a combination of ledipasvir and sofosbuvir (a nucleotide polymerase inhibitor), was approved by the fda in october for the treatment of chronic hcv genotype infection. this also represents the first approved regimen that does not require administration with interferon or ribavirin. , . carbamates as cysteine protease inhibitors cysteine proteases, also known as thiol proteases, are proteolytic enzymes responsible for the degradation of proteins. these enzymes are divided into three classes based on their sequence homology: the papain, caspase, and picornaviridae families. the papain family of proteases is the most known and studied. cysteine proteases have been identified in a variety of diverse organisms, such as bacteria, eukaryotic micro-organisms, plants, and animals and are divided into the clans ca, cd, ce, cf, and ch in the merops peptidase database. the largest subfamily among the class of cysteine proteases is the papain-like cysteine proteases, originating from papain as the archetype of the cysteine proteases. clan ca proteases utilize catalytic cys, his, and asn residues that are invariably in this order in the primary sequence of the protease. clan ca, family c (papain-family) cysteine proteases are wellcharacterized for many eukaryotic organisms. also, the best characterized plasmodium cysteine proteases, namely, the falcipains, belong to papain-family (clan ca) enzymes. clan cd presents two catalytic residues, his and cys, in sequence; clan ce has a triad formed by his, glu, or asp and cys at the c-terminus; in clan cf, the asparagine residue of the catalytic triad is replaced by a glutamate residue and the catalytic triad is ordered as glu, cys, and his; clan cg has a dyad of two cysteine residues, and clan ch presents a cys, thr, and his triad with the catalytic cysteine at the n-terminus. the proteolytic mechanism involves the formation of a thiolate−imidazolium ion pair, which provides a highly nucleophilic cysteine thiol. over the years, many cysteine protease inhibitors have been designed by appropriately linking electrophilic warheads to the specific recognition sequence of peptide substrates. reversible inhibitor warheads include aldehydes, α-ketoamides, α-ketoesters, and α-ketoacids. these inhibitors interact with the protease active site, forming the tetrahedral intermediate, but are eventually hydrolyzed, regenerating both the enzyme and the inhibitor in an equilibrium reaction. irreversible inhibitors of cysteine proteases include epoxides, aziridines, haloketones, vinyl sulfones, and acyloxymethylketones. these inhibitors inactivate the target through alkylation of the active site cysteine thiol, permanently disabling enzyme function. , , − the occurrence of severe acute respiratory syndrome (sars) in and the subsequent identification of a novel coronavirus as the etiological agent recognized cysteine proteases sars-cov clpro and sars-cov plpro (papainlike protease) as possible targets for drug design. , subsequent structure-based design based on a previous inhibitor's x-ray co-crystal structure with the enzyme provided carbamate derivative ( figure ) as a potent sars-cov clpro inhibitor (ic = μm). a wide variety of human rhinovirus c (hrv c) protease inhibitors were developed by the incorporation of α,βunsaturated carbonyl moieties as warheads. hanzlik et al. reported the first hrv c protease inhibitors containing a peptide portion and incorporating α,β-unsaturated esters. the peptide parts were selected based on the substrate cleavage site. the representative carbamate-containing inhibitor ( figure ) showed an ic value of nm. human cathepsin k plays a critical role in bone resorption. in an effort to block bone resorption, noncovalent cathepsin k inhibitors were developed. kim et al. provided carbamate derivative ( figure ) as a noncovalent and reversible cathepsin k (ic = . μm) and l inhibitor (ic = . μm). glaxowellcome scientists developed carbamate-containing ketoamide-based cathepsin k inhibitors such as ( figure ) (ic = . nm). starting from a potent ketone-based inhibitor with unsatisfactory drug-like properties, , incorporation of p −p elements from the ketoamide-based inhibitor led to a hybrid series of ketone-based cathepsin k inhibitors with improved bioavailability, as exemplified in inhibitor ( figure ) (ic = nm). cathepsin s has been suggested for the development of agents against a range of immune disorders. a new class of nonpeptidic and noncovalent cathepsin s inhibitors was reported in . subsequent structural optimization resulted in a very potent and competitive noncovalent carbamate-containing inhibitor ( figure ) (ic = nm). metabolizing enzyme inhibitors carbamates have been employed in the design of serine hydrolase inhibitiors. in this section, we will focus on inhibitors of endocannabinoid metabolizing enzymes, in which the carbamate functionality plays an important role. the endocannabinoid system is known to be a ubiquitous neuromodulatory system with a wide range of action that can be found in every primitive organism. it is composed of cannabinoid receptors (cbrs), endogenous cannabinoids (endocannabinoids, ecs), and the enzymes responsible for their production, transport, and degradation. , ecs are a class of signaling lipids, such as n-arachidonoyl ethanolamine (anandamide, aea), oleamide, and -arachidonoyl glycerol ( -ag), that exert their biological actions through the interaction with two g-protein coupled receptors, cb and cb . they modulate a range of responses and processes including pain, inflammation, appetite, motility, sleep, thermoregulation, and cognitive and emotional states. the actions of these signaling lipids are rapidly terminated by cellular reuptake and subsequent hydrolysis operated by a number of enzymes. an attractive approach involved the modulation of the ec system and aimed at eliciting the desirable effects of cbrs activation through the pharmacological inactivation of the main endocannabinoid metabolizing enzymes, namely, monoacylglycerol lipase (magl) and α/β-hydrolase domain containing and (abhd and abhd ). these three serine hydrolases account for approximately % of -ag hydrolysis in the cns, whereas fatty acid amide hydrolase (faah) is responsible for aea inactivation. inactivation of these enzymes would elevate the endogenous concentrations of all of its substrate and consequently prolong and potentiate their beneficial effects on pain and anxiety without evoking the classical cb r agonists side effects (hypomotility, hypothermia, and catalepsy). monoacylglycerol lipase (magl) is the primary enzyme responsible for the hydrolysis of -ag in the cns. about % of the total -ag hydrolysis in the brain is ascribed to magl. magl is a kda membrane enzyme belonging to the superfamily of the serine hydrolases with a catalytic triad represented by ser , his , and asp . it is ubiquitously present in the brain (cortex, hippocampus, cerebellum, thalamu, and striatum), where it localizes to presynaptic terminals, even if lower levels are found in the brainstem and hypothalamus. a concomitant distribution in membranes as well as in the cytosol has been reported. magl shares a common folding motif called the α/β-hydrolase fold. studies in recent years have shown that magl inhibitors elicit antinociceptive, anxiolytic, and antiemetic responses. magl inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. recently, the potential of magl inhibitors for the therapy of fragile x syndrome has been reported. the early discovered magl inhibitors were molecules able to target the cysteine residues present in the active site of the enzyme. later, the research has been focused on the synthesis of compounds covalently binding to ser of the catalytic triad. among them, carbamate (urb , figure ) was the first selective inhibitor of -ag degradation, although its potency remained limited (ic = μm on rat brain). selective magl inhibitors bearing a carbamate scaffold were developed by cravatt and coworkers. inhibitor (jzl , figure ) exhibited selectivity toward faah in vitro (ic = . nm and μm for human recombinant magl and faah, respectively). more recently, cravatt and co-workers reported a distinct class of o-hexafluoroisopropyl (hfip) carbamates bearing a reactive group that is bioisosteric with endocannabinoid substrates. the representative compound, (kml , figure , ic = . nm, human magl), displays excellent potency and in vivo. in comparison to previously described o-aryl carbamates, inhibitor showed enhanced selectivity over faah and other serine hydrolases. abhd gene encodes a ∼ kda protein containing an nterminal transmembrane region followed by a catalytic domain that includes the canonical gxsxg active-site motif of serine hydrolases. abhd is a unique and highly conserved enzyme in mammals and is mainly expressed in the brain, liver, kidney, and brown adipose tissue. as a member of the serine hydrolase class, abhd is predicted to hydrolyze esters, amides, or thioester bonds in substrates that could include small molecules, lipids, or peptides. although, the full range of substrates regulated by this enzyme in vivo is currently unknown. recent studies have also shown that abhd carbamate inhibitors produce anti-inflammatory and neuroprotective effects in a mouse model of traumatic brain injury. among them, optimized inhibitor ( figure ) displayed an ic value of nm and notable selectivity. faah is a membrane-bound enzyme belonging to the amidase family. the analysis of its crystal structure revealed a core composed of a characteristic ser-ser-lys catalytic triad. the catalytic residues of faah are buried deep within the enzyme and are accessible by two narrow channels. the importance of faah was demonstrated by the generation of faah knockout mice. faah −/− mice showed an elevated resting brain concentration of aea and manifested (i) an analgesic phenotype in both the carrageenan model of inflammatory pain and in the formalin model of spontaneous pain, (ii) a reduction in inflammatory responses, and (iii) improvements in slow wave sleep and memory acquisition. , the urb class of compounds was the first class of inhibitors identified for faah, and it is well-represented by (urb , figure , ic = . nm). the n-( phenyl)hexylcarbamate analogue (jp , figure , ic = nm) is another very potent compound representative of the biphenyl series of inhibitors. gattinoni and co-workers developed a series of oxime carbamate inhibitors. compound ( figure , ic = nm) displayed good affinity and selectivity toward faah. more recently, butini et al. developed a new class of potent and selective faah reversible carbamate inhibitors. among them, compound (nf , figure , k i = . nm on mouse brain faah) showed excellent activity. the compound showed impressive selectivity toward all the enzymes and receptors of the endocannabinoid system. in this perspective, the role of carbamates in drug design and medicinal chemistry has been highlighted. in particular, the perspective covers physical properties of carbamates and the development of novel chemical methodologies overcoming the historical safety and toxicity issues related to their preparation. furthermore, the importance of carbamate-derived compounds in medicinal chemistry and their widespread employment as drugs and prodrugs have been discussed. also showcased is the exploitation of organic carbamates in the development of numerous aspartic acid, serine, and cysteine protease inhibitors. we hope that this perspective will stimulate further use of organic carbamate as a structural motif in drug design and medicinal chemistry. the authors declare no competing financial interest. financial support of this research by the national institutes of health (gm ) and purdue university is gratefully acknowledged. we would like to thank our colleagues anthony tomaine, heather osswald, and anindya sarkar (purdue university) for helpful discussions. ad, alzheimer's disease; cat-d, cathespsin d; faah, fatty acid amide hydrolase; ppar, peroxisome proliferator-activated receptor; gaba, γ-amino butyric acid; haart, highly active antiretroviral therapy; bis-thf, bis-tetrahydrofuran; bace , beta-site amyloid precursor protein cleaving enzyme ; mdr, multidrug resistant; nnrti, non-nucleoside reverse transcriptase inhibitors; adept, antibody-directed enzyme prodrug therapy; gdept, gene-directed enzyme prodrug therapy; mct , monocarboxylate transporter type ; smvt, sodiumdependent multivitamin transporter; ces , carboxylesterases ; magl, monoacylglycerol lipase ■ references unusually low barrier to carbamate c−n rotation amide resonance in thioand seleno-carbamates: a theoretical study role of conjugation in the stabilities and rotational barriers of formamide and thioformamide. an ab initio valence-bond study on the stabilization of the syn-rotamer of amino acid carbamate derivatives by hydrogen bonding the dipole moment and structure of the carbamate group chemical aspects of the restricted rotation of esters, amides, and related compounds hydrolysis in drug and prodrug metabolismchemistry qualitative structuremetabolism relationships in the 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-amino]-carbonyloxycamptothecin, by human carboxylesterases ces a , ces , and a newly expressed carboxylesterase isoenzyme bioactivation of the anticancer agent cpt- to sn- by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions hydrolysis of h-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro oral bambuterol versus terbutaline in patients with asthma cyclization-activated prodrugs. basic carbamates of -hydroxyanisole cyclization-activated prodrugs. basic esters of -bromo- ′-deoxyuridine synthesis of self-immolative glucuronide-based prodrugs of a phenol mustard. anti-cancer drug des bioreduction activated prodrugs of camptothecin: molecular design, synthesis, activation mechanism and hypoxia selective cytotoxicity synthesis and biological evaluation of ′-carbamate-linked and ′-carbonate-linked prodrugs of paclitaxel: selective activation by the tumor-associated protease plasmin elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release alpha-isobutanoyloxyethoxy)-carbonyl] aminomethyl)- -cyclohexane acetic acid], a novel gabapentin prodrug: ii. improved oral bioavailability, dose proportionality, and colonic absorption compared with gabapentin in rats and monkeys ] aminomethyl)- -cyclohexane acetic acid], a novel gabapentin prodrug: i. design, synthesis, enzymatic conversion to gabapentin, and transport by intestinal solute transporters clinical pharmacokinetics of xp , a novel transported prodrug of gabapentin hydrolysis of capecitabine to ′-deoxy- -fluorocytidine by human carboxylesterases and inhibition by loperamide design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates -fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue rational development of capecitabine capecitabine: a review profound and sustained inhibition of platelet aggregation by fradafiban, a nonpeptide platelet glycoprotein iib/iiia antagonist, and its orally active prodrug, lefradafiban, in men bis-tetrahydrofuran: a privileged ligand for darunavir and a new generation of hiv protease inhibitors that combat drug resistance enhancing protein backbone bindinga fruitful concept for combating drug-resistant hiv crystal structure of an in vivo hiv- protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance potent hiv protease inhibitors: the development of tetrahydrofuranylglycines as novel p -ligands and pyrazine amides as p -ligands anti-hiv protease inhibitor darunavir inhibitors of hiv- protease containing the novel and potent (r)-(hydroxyethyl)sulfonamide isostere design and synthesis of amprenavir, a novel hiv protease inhibitor the development of cyclic sulfolanes as novel and high-affinity p ligands for hiv- protease inhibitors nonpeptidal p ligands for hiv protease inhibitors: structure-based design, synthesis, and biological evaluation structure-based design of non-peptide hiv protease inhibitors potent hiv protease inhibitors incorporating high-affinity p -ligands and (r)-(hydroxyethylamino)sulfonamide isostere high resolution crystal structures of hiv- protease with a potent non-peptide inhibitor (uic- ) active against multi-drug-resistant clinical strains ultra-high resolution crystal structure of hiv- protease mutant reveals two binding sites for clinical inhibitor tmc a potent human immunodeficiency virus type protease inhibitor, uic- (tmc- ), and selection of a novel (a s) mutation in the protease active site potent inhibition of hiv- replication by novel nonpeptidyl small molecule inhibitors of protease dimerization design of hiv- protease inhibitors active on multidrug-resistant virus darunavir, a conceptually new hiv- protease inhibitor for the treatment of drugresistant hiv darunavir, a new pi with dual mechanism: from a novel drug design concept to new hope against drug-resistant hiv stereoselective photochemical , -dioxolane addition to -alkoxymethyl- ( h)-furanone: synthesis of bis-tetrahydrofuranyl ligand for hiv protease inhibitor uic- (tmc- ) synthesis and optical resolution of high affinity p -ligands for hiv- protease inhibitors a novel bistetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (pi), grl- , is potent against multiple-pi-resistant human immunodeficiency virus in vitro ultra-potent p modified arylsulfonamide hiv protease inhibitors: the discovery of gw suppression of hiv- protease inhibitor resistance by phosphonate-mediated solvent anchoring tmc , a novel human immunodeficiency virus type protease inhibitor, shows in vitro an improved resistance profile and higher genetic barrier to resistance compared with current protease inhibitors flexible cyclic ethers/polyethers as novel p -ligands for hiv- protease inhibitors: design, synthesis, biological evaluation, and protein-ligand x-ray studies design and synthesis of potent hiv- protease inhibitors incorporating hexahydrofuropyranol-derived high affinity p( ) ligands: structure-activity studies and biological evaluation probing multidrug-resistance and protein-ligand interactions with oxatricyclic designed ligands in hiv- protease inhibitors grl- and grl- , difluoride-containing nonpeptidic hiv- protease inhibitors (pis) that inhibit the replication of multi-pi-resistant hiv- in vitro and possess favorable lipophilicity that may allow blood-brain barrier penetration the discovery of β-secretase and development toward a clinical inhibitor for ad: an exciting academic collaboration design of potent inhibitors for human brain memapsin (βsecretase) structure of the protease domain of memapsin (beta-secretase) complexed with inhibitor structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin (beta-secretase) cathepsin d is membrane-associated in macrophage endosomes design, synthesis and x-ray structure of protein-ligand complexes: important insight into selectivity of memapsin (beta-secretase) inhibitors design of potent inhibitors of human beta-secretase design of potent inhibitors of human beta-secretase. part chronic treatment with the gammasecretase inhibitor ly- , inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation studies of abeta pharmacodynamics in the brain, cerebrospinal fluid, and plasma in young (plaque-free) tg mice using the gammasecretase inhibitor n -[( s)- -( , -difluorophenyl)- -hydroxyethanoyl]-n -[( s)- -methyl- -oxo- , -dihydro- h-dibenzo quantitative measurement of changes in amyloid-beta( ) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor ly- novel orally active, dibenzazepinone-based gamma-secretase inhibitors design, synthesis, and evaluation of tetrahydroquinoline and pyrrolidine sulfonamide carbamates as gamma-secretase inhibitors discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors , -disubstituted n-arylsulfonyl piperidines as gamma-secretase inhibitors nitrogen-appended n-alkylsulfonamides as inhibitors of gamma-secretase carbamate-appended nalkylsulfonamides as inhibitors of gamma-secretase serine proteases of the human immune system in health and disease proteases: multifunctional enzymes in life and disease irreversible inhibitors of serine, cysteine, and threonine proteases strategies for the inhibition of serine proteases protease inhibitors: current status and future prospects structural basis of substrate specificity in the serine proteases novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases synthesis and kinetic studies of diphenyl -(npeptidylamino)alkanephosphonate esters and their biotinylated derivatives as inhibitors of serine proteases and probes for lymphocyte granzymes thrombin inhibitors: a new generation of antithrombotics the current status of coagulation the solution conformation of (d)phe-pro-containing peptides: implications on the activity of ac-(d)phe-pro-boroarg-oh, a potent thrombin inhibitor synthesis of conformationally-restricted boropeptide thrombin inhibitors inhibition of serine proteases by peptidyl fluoromethyl ketones nonpeptidic inhibitors of human leukocyte elastase. . design, synthesis, and x-ray crystallography of a series of orally active -aminopyrimidin- -one-containing trifluoromethyl ketones nonpeptidic inhibitors of human leukocyte elastase. . design of a potent, intratracheally active, pyridone-based trifluoromethyl ketone discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase orally active trifluoromethyl ketone inhibitors of human leukocyte elastase clinical consequences of hepatitis c virus infection challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection hepatitis c virus ns - a protease inhibitors: countering viral subversion in vitro and showing promise in the clinic an ns protease inhibitor with antiviral effects in humans infected with hepatitis c virus discovery of ( r, s)-n-[ -amino- -(cyclobutylmethyl)- , -dioxopropyl sch , a mechanism-based inhibitor of hepatitis c virus ns protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells hepatitis c virus ns - a serine protease inhibitors: sar of p′ moiety with improved potency depeptidization efforts on p -p ′ alpha-ketoamide inhibitors of hcv ns - a serine protease: effect on hcv replicon activity discovery of sch (sch ): a new ketoamide inhibitor of the hcv ns serine protease and hcv subgenomic rna replication discovery of boceprevir, a direct-acting ns / a protease inhibitor for treatment of chronic hepatitis c infections synthesis and antiviral activity of hcv ns / a peptidomimetic boronic acid inhibitors novel macrocyclic hcv ns protease inhibitors derived from alpha-amino cyclic boronates inhibition of cathepsin b and papain by peptidyl alpha-keto esters, alpha-keto amides, alpha-diketones, and alpha-keto acids design, synthesis, and biological activity of m-tyrosine-based -and -membered macrocyclic inhibitors of hepatitis c virus ns serine protease in vitro antiviral activity of sch (sch ), a novel inhibitor of the hepatitis c virus ns serine protease discovery and structure−activity relationship of p −p ketoamide derived macrocyclic inhibitors of hepatitis c virus ns protease synthetic inhibitors of elastase hcv-targeted antivirals: current status and future challenges small molecule inhibitors of the hepatitis c virus-encoded ns a protein chemical genetics strategy identifies an hcv ns a inhibitor with a potent clinical effect modeling shows that the ns a inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis c virus half-life a phase , randomized, placebocontrolled, -day, dose-ranging study of gs- , an ns a inhibitor, in patients with genotype hepatitis c ledipasvir and sofosbuvir for untreated hcv genotype infection fda approves first combination pill to treat hepatitis c evolutionary lines of cysteine peptidases thiol proteases: inhibitors and potential therapeutic targets review: novel cysteine proteases of the papain family cysteinyl proteinases and their selective inactivation michael acceptors as cysteine protease inhibitors use of cysteine-reactive small molecules in drug discovery for trypanosomal disease identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov clpro inhibitors synthesis and evaluation of peptidyl michael acceptors that inactivate human rhinovirus c protease and inhibit virus replication -piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin k inhibitors p −p conformationally constrained ketoamide-based inhibitors of cathepsin k azepanone-based inhibitors of human and rat cathepsin k an azepanone-based inhibitor of human cathepsin k with improved oral bioavailability in the rat and the monkey acyclic, orally bioavailable ketone-based cathepsin k inhibitors nonpeptidic, noncovalent inhibitors of the cysteine protease cathepsin s discovery and development of fatty acid amide hydrolase (faah) inhibitors the endocannabinoid system: drug targets, lead compounds, and potential therapeutic applications cannabinoid receptors: where they are and what they do a comprehensive profile of brain enzymes that hydrolyze the endocannabinoid -arachidonoylglycerol crystal structure of the human monoacylglycerol lipase, a key actor in endocannabinoid signaling therapeutic potential of monoacylglycerol lipase inhibitors uncoupling of the endocannabinoid signalling complex in a mouse model of fragile x syndrome urb inhibits monoacylglycerol lipase and selectively blocks -arachidonoylglycerol degradation in intact brain slices selective blockade of -arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects highly selective inhibitors of monoacylglycerol lipase bearing a reactive group that is bioisosteric with endocannabinoid substrates discovery and optimization of piperidyl- , , -triazole ureas as potent, selective, and in vivo-active inhibitors of alpha/beta-hydrolase domain containing (abhd ) selective inhibition of alpha/betahydrolase domain attenuates neurodegeneration, alleviates blood brain barrier breakdown, and improves functional recovery in a mouse model of traumatic brain injury a functional proteomic strategy to discover inhibitors for uncharacterized hydrolases structural adaptations in a membrane enzyme that terminates endocannabinoid signaling mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptormediated phenotypic hypoalgesia the endogenous cannabinoid system protects against colonic inflammation characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase inhibition of fatty-acid amide hydrolase accelerates acquisition and extinction rates in a spatial memory task. neuropsychopharmacology design, synthesis, and structure-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors mechanism of carbamate inactivation of faah: implications for the design of covalent inhibitors and in vivo functional probes for enzymes a new group of oxime carbamates as reversible inhibitors of fatty acid amide hydrolase discovery of potent inhibitors of human and mouse fatty acid amide hydrolases key: cord- - e fchy authors: boisguérin, prisca; deshayes, sébastien; gait, michael j.; o'donovan, liz; godfrey, caroline; betts, corinne a.; wood, matthew j.a.; lebleu, bernard title: delivery of therapeutic oligonucleotides with cell penetrating peptides() date: - - journal: adv drug deliv rev doi: . /j.addr. . . sha: doc_id: cord_uid: e fchy oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. clinical translation has been often hampered by poor biodistribution, however. cell-penetrating peptides (cpps) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids. this review focuses on cpp-delivery of several classes of oligonucleotides (ons), namely antisense oligonucleotides, splice switching oligonucleotides (ssos) and sirnas. two main strategies have been used to transport ons with cpps: covalent conjugation (which is more appropriate for charge-neutral on analogues) and non-covalent complexation (which has been used for sirna delivery essentially). chemical synthesis, mechanisms of cellular internalization and various applications will be reviewed. a comprehensive coverage of the enormous amount of published data was not possible. instead, emphasis has been put on strategies that have proven to be effective in animal models of important human diseases and on examples taken from the authors' own expertise. in recent years there has been a dramatic re-evaluation of how genes are regulated and of gene expression modalities. two major sets of discoveries centre on the key roles of non-coding rnas, and in particular those involved in the rna interference (rnai) pathway, as well as the mosaic structure of eukaryotic genes. the antisense on strategy was proposed more than two decades ago for the artificial regulation of eukaryotic gene expression in cultured cells via the hybridization of short ons to mrna targets through the pioneering work of stephenson and zamecnik [ , ] . the immense potential of this strategy, which in principle only requires knowledge of the target mrna sequence, was quickly realized within both academic and industrial laboratories. interestingly, "mother nature" has also exploited this potential, as convincingly demonstrated in bacteria [ ] and later on in eukaryotes [ ] by their ability to capitalize on such potent biochemical and genetic tools. the best demonstration that the antisense concept is exploited came from the discovery that antisense genes are able to control transcription in bacteria (and with less experimental evidence in eukaryotic cells) and finely tune the expression of target genes. however, these natural antisense rnas turned out to be long and highly structured, and attempts to use this knowledge in the design of synthetic antisense genes proved disappointing. most studies so far in the antisense field have instead focussed on short singlestranded dna mimics, the hybridization of which allows recruitment of the cellular enzyme rnase h and as a consequence leads to the destruction of the rna target [ ] . somewhat unexpectedly, eukaryotic coding genes are transcribed as immature mrna precursors, the splicing of which by the complex nuclear machinery leads to intron removal. it became rapidly realized that a major outcome is the possibility of exon re-assortment and that this is the case for the majority of human genes. importantly in terms of potential clinical translation, several human diseases are associated with dysfunction of the splicing machinery (as in β-thalassemia) or with the preferential use of one splicing event rather than another. intervention with the process of exon selection using low molecular weight drugs has turned out to be difficult and the most promising strategy was proposed instead by ryszard kole and colleagues as detailed in section of this issue. in contrast to the "classical" antisense on strategy, splice switching oligonucleotides (ssos) are designed to prevent (or promote) the insertion of exons through high affinity binding at obligatory splicing sequences in the nuclear pre-mrna (for example donor or acceptor splice sites) and therefore rnase hincompetent on analogues must be used [ ] . the discovery of rna interference by fire et al. has also revolutionized our concepts of gene expression regulation [ ] . the current detailed knowledge of rnai processing and recognition by the rna-induced silencing complex (risc) has led to the possibility of rational design and synthesis of artificial sirnas able to recognize any mrna on the sole basis of their sequences. as for rnase h-competent antisense on, sequence-specific recognition of the rna target leads to its degradation by the risc-associated nuclease. further, several hundred human genes have now been identified to code for short stem-loop structures, known as micro rnas (mirnas), which are processed to allow targeting of the ′-utrs of mrnas and many such mrna targets have been identified [ ] [ ] [ ] . astonishingly, mirnas do not need to hybridize over their entire sequence to promote the down-regulation of an mrna target [ ] . as a consequence, a single mirna is able to regulate the expression of a complex set of mrnas, which are often related in terms of cellular function. although still incomplete, ongoing studies show that specific sets of mirnas control most cellular functions and that dysregulation of their expression is associated with many human diseases. regulation of the levels of certain mirnas or interference with their binding to mrna targets are both now heavily explored strategies. synthetic antisense ons, ssos, sirnas and mirnas have become routine and invaluable tools to dissect cellular functions and they can be efficiently transfected into most laboratory cell lines using commercially available reagents such as cationic lipid formulations. however, their systemic in vivo administration has been plagued by toxicity and by low efficiency in the presence of serum proteins. therapeutic developments centred on the use of naked ons have thus met with only limited successes. indeed, more than two decades of therapeutic developments have only led to three fda approved drugs, with two used for easier-to-manage topical ocular applications (fomivirsen as an antisense treatment for ocular cytomegalovirus infections in immunocompromised patients [ ] and ranibizumab, an aptamer for the treatment of macular degeneration [ ] ). of more promise is the recent approval in the us (but not yet in europe) of mipomersen [ ] , an antisense on for the treatment of homozygous familial hypercholesterolemia, which capitalizes on the accumulation of phosphorothioate (ps) ons in the liver after systemic administration. encouraging small scale clinical data have also been reported in the treatment of duchenne muscular dystrophy with ′-o-methyl phosphorothioate ( ′-omeps) ons (drisapersen [ ] [ ] [ ] ) and phosphorodiamidate morpholino oligonucleotides (pmo, eteplirsen [ ] [ ] [ ] ), as will be extensively discussed later in section of this chapter. despite these successes, it is generally agreed that degradation in biological fluids, passage across cellular barriers and intracellular trafficking are all limiting factors in nucleic acids-based therapies [ ] . extensive searches for on chemical modifications have improved their metabolic stabilities significantly as well as their affinities for rna targets, and have to some extent reduced off-target effects. no on chemical modification has significantly improved cellular uptake or tissue targeting, however. the design of efficient and non-toxic delivery vectors for on-based drugs has therefore become a major concern in both academic and industrial laboratories. among the many proposed tools for delivery, cell penetrating peptides (cpps) have appeared as an easy to implement strategy and have led to encouraging data at least in murine models of human diseases, as will be reviewed later in this chapter. this review does not pretend to be exhaustive and will be restricted ( ) to the cpp delivery of nucleic acids-based drugs and mainly to ssos and sirnas and ( ) to delivery strategies which have turned efficient in animal models of human diseases. the harnessing of cationic peptides to deliver drugs across biological membranes was first attempted by ryser and his colleagues [ ] . they demonstrated that an anticancer drug could be delivered as a poly-llysine (pll) conjugate in drug resistant cells in vitro and in vivo in mouse models [ ] . likewise, the chemical conjugation of antisense ons to pll led to the generation of a potent antiviral activity in several in vitro models of viral infections [ ] . unfortunately, conjugates were poorly characterized in view of the polydispersed character of commercial pll preparations and, more importantly, they led to acute cytotoxicity upon systemic administration in mice. it was discovered serendipitously by virologists that much shorter stretches of cationic peptides could promote the cellular uptake of macromolecules. for example, it was found that the full size purified hiv- tat protein trans-activates the hiv- ltr promoter when incubated with cells [ ] . more astonishingly, this same tat protein was able as a conjugate to promote the cellular internalization of the non-permeable protein β-galactosidase and this paved the way for the use of tat as a delivery vector for biomolecules. along the same lines, the purified antennapedia protein from drosophila was able to exert its transcriptional activity when incubated with nerve cells and this property was ascribed to a short peptide named penetratin [ ] . dissection of the tat protein did also show that cell penetration was due to a small, basic amino acids-rich peptide known as the tat peptide [ ] . initial studies of the cellular trafficking of both penetratin and tat suggested an unusual non-receptor dependent mechanism of direct translocation across the plasma membrane. although challenged later on as detailed in section , this mechanism and the possibility to use such so called cell-penetrating peptides (cpps) as non-viral delivery vectors for biomolecules, fostered a very large interest. many cpps were rapidly discovered and proposed as vectors for the transport of various drugs across biological membranes, starting from low molecular mass drugs to very large molecular entities such as nanoparticles. most cpps were designed for the transport of a chemically conjugated cargo (see table ). since the chemical conjugation and purification of negatively charged ons with the most popular cationic cpps has turned out to be difficult, most applications have concerned chargeneutral on analogues such as peptide nucleic acids (pnas) and pmo (see section ). the most advanced studies have involved the use of these conjugates for rna splicing regulation. early work described the use of penetratin for the delivery of conjugated ps ons [ ] and of transportan (another popular cpp) for pna transport [ ] . unexpectedly, in a well-characterized hela cell assay with a positive read-out ( fig. ) , splicing redirection using pna or pmo oligomers conjugated to various standard cpps (tat, penetratin or oligo-arginines) was not achieved in our research groups [ ] . hela pluc cells were stably transfected with a construction in which the coding sequence of the luciferase gene is interrupted by a mutated intron of the human β-globin gene. this mutation creates a ′splice site and activates a ′splice site. masking of the ′splice site by a rnase h-incompetent antisense on ( ) restores the production of functional luciferase mrna and protein. this assay was provided by kole and colleagues [ ] and allows a reliable and easy to implement comparative evaluation of on analogues and on-delivery vectors. intronic point mutations in a β-thalassemia globin gene activate cryptic splice sites leading to the aberrant splicing of this intron and as a consequence to a non-functional protein. masking of the mutated site with a steric-block on re-orients the splicing machinery toward complete removal of the intron and leads to the production of a correctly spliced mrna. this mutated intron has been introduced into the coding region of a reporter luciferase gene and the construction has been stably transfected in hela cells, which are available from atcc (atcc® ccl- ™). luciferase expression can be easily monitored enzymatically or by pcr. this assay is advantageous in providing a positive read-out with a low background and a large dynamic range. it has been adopted by many laboratories in the field thus allowing easy comparisons. extensive studies of their cellular trafficking revealed that these cpp-on conjugates were efficiently taken up by cells but remained stuck in endocytic vesicles. in keeping with this hypothesis, further incubation with chloroquine (an endosomolytic drug) or with saponin (a membrane-permeabilizing agent) allowed by-pass of this restriction [ ] . similar conclusions were reached using the same well-characterized assay by the nielsen group [ ] . understanding the mechanisms of cell uptake using cpps via comparison of literature data has proved extremely difficult, since the behaviours of cpps seem to be strongly influenced by experimental conditions. among relevant factors are cpp sequence, type of cargo, concentration (which seems to be crucial to foster endocytosis or direct translocation) and cell type [ ] . the first data showing strong activity in the hela splicing redirection assay were obtained with a derivative of oligo-arginines in which the spatial distribution of guanidinium side chains was optimized by the use of a non-natural aminohexanoic acid spacer [ ] . similarly, the addition of arginine residues on the n-terminus of penetratin also resulted in strong activity in this hela assay for pna conjugates [ ] . this led to the development of several arginine-rich peptides as pmo conjugates for use in muscle cells and in vivo mouse models of dmd as outlined in section . in addition to covalent conjugation, some cpps have been designed for use as complexes, particularly for sirna delivery (table ) . this is because, as previously alluded to, chemical conjugation and purification of ons and sirnas with cationic cpps has been difficult to achieve. for example, in the case of ons, the heitz and divita group described a new class of such cpps, with mpg as the lead compound, which can be complexed with negatively charged ons essentially through electrostatic interactions. this and other complexation peptides are detailed in section . since practically all cpps have a high cationic charge (due to lys and arg residues), it has proved extremely hard technically for covalent conjugation of most cpps to standard negatively charged (phosphodiester or phosphorothioate) ons because of aggregation or precipitation that can occur. it was possible for thiol linker-functionalised ons (for example mixmers of ′-o-methyl/lna steric blocking ons) to be conjugated with cysteine-functionalised classic cpps such as tat and penetratin to give disulphide linkages, if the conjugations were carried out in the presence of a denaturing agent such as formamide [ ] . in this way, it was found that fluorescent versions of such conjugates were taken up much better into endosomal compartments of model hela cells than unconjugated versions, but release into the nucleus to generate steric block rna targeting activity proved not to be achievable [ ] . a disulphide linkage is also not thought to be compatible with sufficient conjugate stability using systemic delivery, although lung delivery by disulphide-linked penetratin and tat conjugated sirna has been attempted [ ] . thus the few rare examples of peptide-conjugated negatively charged ons used in vivo have generally utilised a thiol-maleimide linkage between on and peptide (fig. ). in one case the on was ′functionalised during synthesis with an aminohexyl linker and the resulting amino group reacted with a bifunctional n-(gammamaleimidobutyrloxy)-succinimide (gmbs) reagent to give a maleimide derivative. the maleimide group was subsequently reacted with a cyscontaining peptide, a cationic cpp known as f , to furnish the desired conjugate ( fig. a ) [ ] . surprisingly, chain aggregation was not reported in this conjugation reaction. alternatively and more conveniently, -mer non-cationic peptides discovered by a phage display technique were functionalised at the nterminus by reaction with maleimidopropionic acid at the final stage of peptide synthesis and after purification were conjugated to a ′-o-me phosphorothioate on synthesized with a ′-thiohexyl linker ( fig. b ). such conjugates were designed for enhanced uptake in muscle and heart due to use of homing peptides selected by a phage display method [ ] . non-cationic homing peptides are likely to be explored further for delivery of ons to specific tissue types. indeed there was a recent report of a phage-selected space peptide conjugated covalently to sirna showing enhanced skin penetration [ ] . one might also expect increasing use of conjugations using the modern "click" chemistry, such as via the copper-catalysed reaction between an azido functionality and an alkyne group, where each of the peptide and on contains a clickable functionality [ ] or where the on contains a ′-o-propyargyl functionality [ ] . despite the large body of literature on the use of peptide-conjugated pnas in cell culture [ ] there are surprisingly few examples of their use in vivo. in the case of short peptide attachments to pna, these can be assembled directly on the n or c-terminus of the pna chain during solid-phase synthesis without needing any specific conjugation step. the amide couplings of protected amino acids are identical to that of protected pna monomers as long as a compatible protecting group scheme is used. in the standard syntheses of pna, it has been common to add a few lys residues in any case, particularly to enhance the aqueous solubility of the pna. thus the first report of pna activity involved merely use of (lys) n-terminally functionalised pna (the lys residues effectively acting as a cpp) in an in vivo splicing assay using a green fluorescent protein reporter that was up-regulated through redirection of splicing by the pna [ ] . further, in vivo results were obtained using (lys) derivatives of pna, synthesized in the same continuous way on the n-terminus of the pna [ ] . the results showed that an (lys) -pna conjugate was rapidly cleared from the circulation but distributed relatively broadly in a mouse with highest concentrations reached in liver, kidney, and spleen. only very low amounts were detected in lungs, heart, skeletal muscle and testes, however. in vivo analysis was extended to an amphipathic lys and leu-containing d-peptide and another rich in arg and homo-arg as pna conjugates, again synthesized continuously on solid phase [ ] . neither showed splicing redirection in liver and only the lys/leu peptide-pna conjugate showed activity in kidney, but both showed significant activity in adipose tissue. however, the lack of potency compared to rnase-h activating ons and their perceived toxicity profile at high doses led to abandonment of peptide-pna conjugates as a drug modality for isis pharmaceuticals. a continuously synthesized nuclear localization signal (nls) peptide (pkkkrkv) attached to a pna was also reported to be active in a severe combined immunodeficiency (scid) mouse model in intronic targeting of c-myc in burkitt's lymphoma-aimed therapy [ ] . further, a penetratin peptide was synthesized continuously at the c-terminus of a triplex-forming pna to target chromosomal dna for genetic modification of haematopoietic progenitor cells in mice [ ] . promising microrna knockdown results were obtained also when an antisense lys-pna-(lys) derivative was tested in mouse spleen for targeting microrna- [ ] . very recently an anti-microrna pna disulphide-conjugated (fig. a) to a tumour-targeting phlip peptide has been shown to have potent activity in a mouse model of lymphoma [ ] . the only other example of a peptide-pna active in vivo that was not synthesized by continuous solid-phase assembly was an arg-rich cpp pip a (and related pip b) conjugated through a thioether bridge by reaction of a c-terminal cys residue on the peptide to an nterminal bromoacetyl group on the pna (fig. b ) [ ] . the conjugates were active by intramuscular injection into the mdx mouse, a model of duchenne muscular dystrophy (dmd), but later higher activity was found in vivo when using pmo rather than pna as the on material [ ] . despite results of mixed fortunes in vivo for cpp conjugates of pna, these materials deserve further investigation especially in the case of tissues that are harder to reach with other types of on. a new method of parallel synthesis of arrays of cpp-pna conjugates (known as selpepcon) could prove useful for pre-screening of suitable drug candidates in cell assays or for example by intramuscular delivery by far the majority of successfully in vivo used cpp conjugates have utilised pmo as on cargo. the application of cpp-pmo to muscular dystrophies is outlined in section . regarding peptide-pmo (p-pmo) synthesis, suffice it to say that it has not been possible to date to use continuous synthesis methods for p-pmo conjugates, since synthesis has been restricted to commercial pmo suppliers. methods of conjugation are therefore limited to what is available commercially regarding functionalised pmo or instead must employ un-functionalised pmo. practically all cpps used with pmo to date have been arg-rich starting from the initial observations of moulton and colleagues [ ] . early work involved use of a bi-functional cross-linker to couple a ′piperazino pmo through a maleimide linkage to the peptide (fig. a ). later the ′-piperazine was directly conjugated through an amide linkage using c-terminally activated peptides (fig. b ) [ ] . ′-conjugation can also be carried out with commercially available amino-link functionalised pmo by direct coupling to the c-terminal carboxylic acid of the peptide through an amide link [ , ] . more generally now, the secondary amine of the ′-morpholino group is coupled directly to the c-terminal carboxylic acid of the pmo via an amide link ( fig. c) [ , ] . for direct amide conjugation, it is not necessary for arg-containing peptides to be chemically protected. however, lyscontaining cpps require a protecting group on the epsilon amino group removable after conjugation. direct ′-amide conjugation of un-functionalised pmo also allows for peptides to be conjugated to the pmo containing for example alkyne functionalities to allow for azide click coupling of a fluorescent label to the p-pmo [ ] . in addition click chemistry conjugation features in an adaptation for p-pmo of the selpepcon method of parallel conjugate synthesis (fig. d ) [ ] . combinations of compatible conjugation techniques such as use of click chemistries are likely to dominate future p-pmo synthesis methods. the term muscular dystrophy describes a large group of hereditary diseases characterized by progressive weakness and degeneration of skeletal muscle [ ] . a variety of gene therapies are being developed for this clinically and genetically heterogeneous group of disorders, including antisense on mediated splice modulation (sso). recent advances have placed duchenne muscular dystrophy (dmd) at the forefront of developments in sso therapy. in order to overcome the remaining obstacles to the success of this approach, peptide-conjugated ssos have been extensively investigated in animal models of dmd. dmd is the most common subtype of muscular dystrophy in the uk [online mendelian inheritance in man (omim [ ] ) database reference: ] affecting one in new born boys. this severe, x-linked recessive disease results from mutations in the dmd gene [ ] . the disorder is characterized by progressive muscle degeneration and wasting, along with the emergence of respiratory and cardiac complications, ultimately leading to premature death [ ] . the majority of mutations underlying dmd are genomic deletions, encompassing multiple exons thereby producing a premature truncation of the open reading frame and resulting in the absence of the dystrophin protein. dystrophin is an integral component of the dystrophin associated protein complex (dapc), forming a crucial connection between the intracellular actin-cytoskeleton and the extracellular matrix. exon skipping therapy utilises ssos to target specific regions of the dmd transcript, inducing the exclusion of individual exons, leading to the restoration of aberrant reading frames and resulting in the production of an internally deleted, yet largely functional, dystrophin protein [ ] . following development in animal models, two ao chemistries have undergone proof-of-concept studies and repeat systemic doseescalation studies, which have now been completed in the clinical trial setting [ , [ ] [ ] [ ] ] . systemic administration of both pmo and ′ omeps ssos have yielded specific exon exclusion and partial restoration of dystrophin protein in peripheral muscle of dmd boys. despite the undoubted potential of exon skipping on therapy for dmd, the successful application of this approach is currently limited by the relatively inefficient targeting of skeletal muscle, as well as by the inadequate targeting of ssos to other affected tissues such as the heart [ ] . as such, cationic cpps, which may be co-administered by conjugation with ssos, have shown dramatic improvement in delivery and induce high levels of dystrophin correction in muscle at comparatively low doses. the majority of work has been performed in the mdx dmd mouse model, which is the most widely utilised model in the dmd field. the dystrophic murine phenotype arises as a result of a spontaneous mutation in exon which encodes for a premature termination site, thus preventing the production of dystrophin protein [ , ] . the targeted exclusion of exon in this mouse models the therapeutic strategy in patients by restoring the production of dystrophin protein. the majority of cpps utilised in the dmd field (tables and ) may be covalently linked to pmo [ , ] . however, another class of noncovalent peptides from the transportan family has been engineered to deliver anionic ssos such as ′omeps, as detailed in section [ ] . a number of modifications of stearylated tp [ ] gave rise to cpps capable of inducing high splice correction in vitro in murine h k mdx myotubes (pepfect ; [ ] and pepfect ; [ ] ) ( table ) . it should be noted that the in vivo efficacies of these cpps have yet to be assessed. the covalent class of cpps comprises subtypes, specifically oligoarginine derivatives, phage peptides, and penetratin derivatives. oligoarginines spaced by -aminohexanoic acid and/or β-alanine exhibit high splicing efficiency and serum stability in vitro [ ] [ ] [ ] . the (rxr) peptide was the first cpp conjugate to be administered in the mdx mouse at a range of doses, time-intervals and via different delivery routes. generally, a single intravenous administration induced high dystrophin exon skipping in skeletal muscle, diaphragm and, for the first time, in heart [ ] . another arginine-rich peptide, (rxrrbr) peptide (b-peptide), identified from a screen using the egfp- splicing reporter mouse model [ ] , also gave rise to impressive exon skipping notably in the heart, when delivered using higher doses and via the retro-orbital route [ ] . improvements in cardiac function such as resistance to dobutamine stress testing and improvements in end systolic volume and end diastolic volume were also observed. the identification of phage motifs from phage display-libraries allows the specific homing of a target tissue. as such, peptides with preferential binding to muscle and cardiac tissue were identified specifically for the treatment of dmd. these include muscle specific peptide (msp) which exhibits enhanced in vivo muscle binding capacity [ ] , a -mer peptide (m ) which revealed better splicing efficiency over msp [ ] , and a -mer peptide (p ) which exhibited a moderate improvement in splicing activity over naked sso [ ] . the beneficial tissue targeting attributes of phage peptides may be further enhanced when combined with the delivery efficiency of cpps, to develop a chimeric peptide. this was demonstrated when msp was coupled to the bpeptide to determine the combined efficacy in mdx mice [ , ] . the specific orientation of these peptides was crucial to dystrophin splicing activity, and when coupled in the configuration 'b-msp-pmo' revealed a - fold improvement in skeletal muscle restoration compared to b-pmo [ ] . however, no improvement in dystrophin restoration was observed in cardiac muscle. more recently, the pna/pmo internalization peptide (pip) series was derived from the parent penetratin peptide [ , ] . subsequent modifications including the addition of arginine residues to the nterminus (r -penetratin) [ ] , the addition of a c-terminal cysteine residue, and the utilisation of disulphide conjugation methods [ ] fashioned this group of peptides into the established 'pip' sequence conformation, consisting of a central hydrophobic core flanked on either side by arginine-rich sequences. additional in vivo screening of pip-pmo compounds was carried out to identify optimal cpps for dystrophin splicing and correction in mdx mice. in the pip -series (pip e-o), the core sequence, ilfqy, was retained and there was alteration in the composition and length of the flanking regions [ ] . the number of arginine residues ranged between and , and the number and placement of -aminohexanoic acid (x) and ß-alanine (b) spacer residues also varied in the flanking regions (table ). pip e-, pip j-, pip l-, and pip n-pmo resulted in the greatest number of dystrophin positive fibres following intramuscular administration (tibialis anterior muscle). of these highly efficient cpps, pip e-pmo induced the highest levels of exon skipping and dystrophin restoration body wide including in the heart, following a single mg/kg intravenous administration. when directly compared to b-pmo, pip e-pmo was shown to restore considerably greater dystrophin protein levels in the heart (intravenous administration comparison). as b-pmo and pip e-pmo comprised similar arginine sequence and content, it was deduced that the core region of pip e-(ilfqy) was responsible for the splicing activity in heart. therefore, the pip -series was designed with identical flanking regions to pip e and the core sequence was altered (table ) . pip a-, pip b-and pip f-pmo, which maintained a amino acid core, exhibited the greatest dystrophin splicing activity in heart over the previous lead candidate, pip e-pmo [ ] (fig. ) . peptides such as pip c-and pip d-pmo, with a shortened core, resulted in a substantial reduction in efficacy. as cardiac and respiratory complications are the leading causes of death amongst dmd patients, the ability of pip -pmo to restore cardiac function is vital. these compounds demonstrated their restorative ability in a long term, low dose administration study ( mg/kg over month time-course) in which % dystrophin protein was restored in heart and the onset of cardiomyopathy was prevented in an exercised mdx mouse model (unpublished data). liver and kidney toxicity has been assessed weeks after the administration of pip-pmo conjugates in mdx mice with no sign of toxicity [ , ] . further toxicology studies are currently being pursued however have not been published yet. of course, continuous optimisations to dose, pharmacokinetics and toxicity are underway which will facilitate the progression of this class of cpps to clinical trial. the development of cpp-on conjugates for the treatment of dmd has served as a foundation for the treatment of other diseases facing similar antisense on delivery challenges. antisense ons have been used to modulate rna processing in the triplet repeat disorder myotonic dystrophy [omim: , ]. this degenerative disease arises fig. . immunohistochemical staining of dystrophin following pip a-pmo treatment. dystrophin staining in the tibialis anterior and heart of c bl/ , untreated mdx and pip a-pmo treated mdx mice. the treated cohort received a single, intravenous . mg/kg dose, and tissues were harvested weeks later. from the expansion of pathogenic microsatellite repeats within noncoding regions of either the dystrophia myotonica-protein kinase (dmpk) or cchc-type zinc finger, nucleic acid binding protein (cnbp) gene loci [ , ] . a variety of antisense on strategies have been used to combat the effect of these toxic rna expansions, although currently the most promising is the use of steric block ons [ ] . using this approach, ons were designed to bind to the dmpk repeat regions, therefore preventing the detrimental sequestering of rna-binding proteins to within the expansion. due to the multisystemic nature of myotonic dystrophy, a cpp conjugated antisense on strategy has recently been tested in a mouse model to induce body wide distribution [ ] . spinal muscular atrophy [omim: , , , ] is a neuromuscular disorder that results from the loss of motor neurons and skeletal muscle atrophy. this autosomal recessive disease is caused by mutations in the survival of motor neuron (smn ) gene. the related smn gene undergoes alternative splicing due to a single nucleotide polymorphism, and therefore lacks exon in the majority of transcripts, resulting in low protein expression. in order to functionally compensate for the loss of smn protein in patients, ssos have been used to promote the inclusion of exon in smn transcripts, and therefore increase the production of smn protein [ ] . substantial headway has been made with naked ssos in animal models of sma [ ] and clinical trials are ongoing. however, the effective delivery of antisense ons to motor neurons in the cns constitutes a major challenge, which may be combatted by cpp conjugation. as described, multiple diseases may benefit from antisense on therapy and indeed some have reached clinical trial and demonstrated significant therapeutic potential. these successes may be further amassed with the utility of cpps, which is a rapidly evolving field of research. whilst studies pertaining to toxicity and bio-distribution are underway, it is anticipated that cpp-on therapies will reach clinical trial in the near future. amongst antiviral applications, cpp-pnas were designed several years ago for targeting the hiv- trans-activation responsive element tar [ , ] . reassuringly, a tat-pna was found to be non-toxic in mice at high ( mg/kg) doses [ ] . however, no further in vivo antiviral studies of cpp-pna have been published since. by contrast, cpp-pmo antiviral applications have been plentiful for a range of viruses, such as dengue, cocksackie, ebola and marburg viruses [ ] . only a few in vivo studies of cpp-directed antiviral delivery have been published, all of these involving arg-rich cpps, notably (rxr) xb or r f , as conjugates of pmo (table ) . for example, (rxr) xb-pmo blocked viral replication of human respiratory syncytial virus (rsv) expression in balb/c mice, where the pmo sequence was targeted to the start of the coding region [ ] . there was also a reduction in lung viral titres seen when mice were treated h after infection, but not h after infection. this suggested that the pmo must be present in the cell soon after infection if it is to have an impact on virus production. promising results were obtained in pre-and post-infection (rxr) xb-pmo treatment of -week old piglets infected with porcine reproductive and respiratory syndrome virus (prrsv) both in reducing viremia and interstitial pneumonia [ ] . an (rxr) xb-pmo conjugate, where the pmo was targeted to the ′-terminus of the genomic rna strand, was found to be substantially more active than naked pmo in reducing viral titers in livers of mice infected with murine hepatitis virus (mhv), a coronavirus, and protected mice from tissue-associated liver damage [ ] . cpp-pmo treatment also prolonged survival in two lethal challenge models. however in the case of high dose viral challenge and delayed treatment, the cpp-pmo was not protective and there was some evidence of toxicity in the diseased mice. ag mice treated by intra-peritoneal injection with (rxr) xb-pmo targeted to the ′-terminus or the ′-cyclization sequence (cs) regions of the dengue virus (denv- ) pre-infection (but not post-infection) were able to increase the average survival time to up to days, but viral rebound was seen eventually [ ] . similarly, dosing of (rxr) xb-pmo targeted either to the ′-terminus or the ′-cs region of west nile virus (wnv) rna partially protected mice from viral challenge but higher doses caused toxicity [ ] . unconjugated pmo had no efficacy. although the cpp-pmo approach is very promising in principle for targeting viral rna, an alternative antiviral approach involves use of pmo analogues with cationic piperazine groups within the pmo. such "second generation" pmos, known as pmoplus™, have now generally supplanted cpp-pmo in antiviral applications and these have been reviewed [ , ] . cpp-pna was suggested more than years ago for targeting specific essential bacterial genes [ ] and subsequently both cpp-pna and cpp-pmo have been studied extensively in bacterial cell culture [ ] , but surprisingly few experiments showing in vivo efficacy have been published. the first proof of principle in vivo was shown in where a single intravenous injection of a (kff) k-pna targeted to acpp (an essential gene) rna administered into escherichia coli k- infected balb/c mice min after bacterial challenge reduced bacterial blood titres and enhanced survival of the infected mice [ ] . similar results were obtained for the arg-rich (rff) rxb-pmo conjugate targeted to the same acpp gene when dosed in mice h after infection with e. coli w [ ] . the effect on potency of varying the peptide sequence was investigated in this model and (rxr) xb was found to be the most potent of various arg-rich cpps studied in mice [ ] . more recently, cpp-pna targeting rpod, a gene that encodes an rna polymerase primary σ subunit essential for bacterial growth, showed broad inhibition in multidrug-resistant escherichia coli, salmonella enterica, klebsiella pneumoniae, and shigella flexneri both in cells and in vivo, with (rxr) xb cpp as a pna conjugate having higher activity in general than (kff) k cpp [ ] . (rff) r-pmo targeted to acpp or gyra genes was found to be highly effective to increase mouse survival time when mice were challenged with the deadly ames strain of bacillus anthracis (the causative agent of anthrax) [ ] , suggesting that such treatment of anthrax-infected humans might one day become possible. however, just like in the case of pathogenic viruses, cationic backbone-containing pmo (pmoplus™) although not achieving the same efficiency as cpp-pmo [ ] , may prove to have a more favourable toxicity profile for therapeutic development. as briefly mentioned above, the non-covalent strategy is based on short peptides which are able to form complexes with cargoes without requiring any cross-linking or chemical modifications [ , ] . most of the cpps used in the non-covalent approach have an amphipathic character which enables a combination of electrostatic and hydrophobic interactions with the cargo. the amphipathicity may arise from either the primary structure or the secondary structure. primary amphipathic peptides can be defined as the sequential assembly of hydrophobic residues with hydrophilic residues, whereas secondary amphipathic peptides are generated by the conformational state that allows distribution of hydrophobic and hydrophilic residues on opposite sides of the molecule [ ] . though originally based on amphipathic peptides, the non-covalent approach has been extended to peptides and peptidic analogues that are able to self-assemble with ons to form stable cpp:on complexes [ ] and several of them have been reported to improve on delivery into mammalian cells [ , ] . the non-covalent approach was originally developed for the delivery of large molecules and several peptides able to bind and condense dna such as gala, kala, jts have been used to improve gene delivery [ , ] . in , the divita and heitz group designed the mpg peptide for the delivery of single and double stranded short ons [ ] and the strategy was then extended to proteins and peptides by the development of pep- [ ] . since mpg, numerous cpp:on complexes have been developed for the delivery of different types of ons including ao, sso and sirna. the main advantage of the non-covalent strategy over covalent conjugation lies in its simplicity and the protection that cpp:on complexes confer to the on from digestion by nucleases [ ] . compared to a chemical cpp-on conjugate, the non-covalent complexes usually involve a one-step process consisting of a simple mixing of both partners, cpp and on [ ] (fig. ) . cpp:on complexes do not require any chemical cleavage, prevent steric hindrance between peptide and on, favour a better release of the on inside the targeted cells and facilitate modifications to increase specificity for the on and/or the target [ ] . with regard to the mechanism of internalization, there is no consensus point of view for the cell entry of non-covalent complexes (see section ). however, as these complexes rely on electrostatic and hydrophobic interactions between positively charged cpps and negatively charged ons, their cellular uptake mechanism could be also controlled by structural polymorphism of peptides, particle stability and the nature of complexes/membrane interactions [ ] . interestingly, some cpp:on non-covalent complexes were shown to enter cells through a direct translocation process [ ] . most non-covalent cpps enable a wide range of chemical modifications and combinations and the modular nature of both peptides and resulting cpp:ons particles also allows new developments. the first developments of mpg were based on peptide/on interactions and investigations of cpp:on affinity by fluorescence spectrometry to measure the dissociation constant (kd) as well as the optimal molar ratio and/or charge ratio to obtain stable complexes [ , ] . a molar ratio (mr) of peptides per on and a charged-corrected (n/p) ratio of positively charged amino acids (n) for a phosphate group (p) were estimated [ ] . similar approaches were used for other non-covalent cpps such as pep- for hypna-ppna [ ] , cady, c and mpeg-pcl-ch r h c for sirna condensation [ ] [ ] [ ] [ ] . fluorescence assays were also combined to circular dichroism (cd) analyses to monitor conformational changes that might occur in the presence of the on and during cpp:on complex formation [ , [ ] [ ] [ ] . although both fluorescence and cd investigations suggest interactions and formation of cpp:on complexes, the use of gel shift assays has been generalized to demonstrate the formation of complexes [ , ] . however, none gives a clear characterization of cpp:on particles in terms of colloidal properties (size, charge and shape). studies on colloidal properties of cpp:on complexes have therefore been witnessed over recent years. methods used for other nanomedicines were applied in order to characterize the size, surface charge and morphology of these cpp:on complexes. in , law et al. were the first to report the hydrodynamic diameter and the zeta potential of r :sirna non-covalent complexes [ ] . after uv-visible absorbance and cd investigations suggesting r /sirna interactions, size and zeta potential were measured for several charge ratios (+/−) and revealed that r forms particles with sirna, with a hydrodynamic diameter of~ μm at sirna saturation [ ] . zeta potential increased with the hydrodynamic diameter of particles, supporting the positive contribution of the guanidinium group of r in the surface charge. size and zeta potential have to be estimated in parallel since, according to dvlo theory for colloidal systems, low zeta potential suggest low electrostatic repulsion which induce aggregation of particles whereas high absolute value of zeta potential are indicative of stable particles suspension [ ] . in this light, investigations of size and charge were generalized to other cpp:on complexes, for different types of ons (sirna, sso, and as-on). however, these parameters might be very different according to the cpp, the nature of the on or the formulation protocol [ ] . for example, kim et al. identified small r :sirna nanoparticles with a nm diameter and a + mv zeta potential for a charge-corrected n/p ratio of in pbs [ ] whilst cady:sirna nanoparticles have a size of nm and surface charge of + mv whatever the mr (mr to mr ) in mm nacl [ ] . cady:sirna nanoparticles have a polydispersity index (pdi) of . , suggesting homogeneous and unimodal nanoparticles [ ] . for other cpp:on complexes, the size and the biological activity clearly vary with the mr [ ] . size and charge can also depend on the environment which is important since nanoparticles stability and homogeneity might vary according to the nature of the buffer, as described for c m :sirna particles [ ] . in addition physiological conditions such as serum addition can influence colloidal properties. pepfect :sirna complexes form homogenous unimodal nanoparticles of - nm (pdi~ . - . ), which remained stable in water, whilst the presence of serum proteins induces larger particles of - nm with a wider distribution [ ] . cpp:on complexes can also be stabilized by specific excipients, such as lactose [ ] , % mannitol [ ] or albumin [ ] . since the shape of nanoparticles also influences the bloodstream circulation half-life [ ] , the morphology of pbn was studied by electronic microscopy and atomic force microscopy (afm) and most studies pointed to globular or spherical nano-objects [ , , ] . in fine colloidal characterization suggested a redefinition of the cpp:on complexes, and with regard to chemical modifications that were included in the design of non-covalent cpp as well as the discrepancy in the formulation of complexes, the more appropriate term of "peptide-based-nanoparticles" (pbns) was proposed [ , ] . in this light, formulation of pbn clearly became the key point to consider for further developments, especially with regard to in vivo administration. as pbns aim for in vivo perspectives, they have to be improved for membrane permeation and cell targeting abilities, whilst being biocompatible and biodegradable, with minimal cytotoxicity and inflammatory response (see section . ). few pbn have been successfully used for the in vivo delivery of antisense ons, ssos or sirna [ ] [ ] [ ] , , ] . with regard to sirna, the first reports on pbn-mediated delivery of sirna in vivo involved mpg-Δ nls peptide and a cholesterol oligo-d-arginine (chol-r ) [ , ] . mpg-Δ nls was used for the in vivo delivery of sirna targeting oct- into mouse blastocytes. oct- / (pou f ) is one of the earliest transcription factors expressed in the embryo and both the pluripotency and the fate of es cells depend upon a tight control of oct- / expression. mpg-Δ nls -based nanoparticles induced a sirna-mediated inhibition of up regulation of oct- / in es cells which prevents their specification toward the mesoderm and their differentiation into cardiomyocytes [ ] . similarly, injection of pbn in the inner cell mass of blastocysts impairs cardiogenesis in early embryos [ ] . chol-r was applied for the delivery of sirna targeting vegf (sivgef) in ct- cells xenografted tumour. intratumoural administration of chol-r :sivgef complexes induced a significant inhibition of tumour growth associated with a pronounced decrease in vegf level in the tumour, suggesting a synergistic effect between the d-amino acids of the cpp and the cholesterol moiety [ ] . in a similar approach, mpg-Δ nls was modified in a shortened (mpg- ) and cholesterol-functionalized (mpg- -chol) peptide analogue. mpg- -mediated delivery of sirna targeting cyclin b induces a significant down regulation of both protein and mrna levels and a systemic administration of mpg- -based nanoparticles targeting cyclin b prevented tumour growth in a xenografted tumour mouse model [ ] . intravenous injections of . mg/kg reduced tumour growth for % by day , and administration of . mg/kg pbn entirely abolished tumour growth [ ] . in addition formulation of a mix of mpg- and mpg- -chol ( %) in pbn revealed that cholesterol increases the biodistribution of sirna in the tumour by maintaining sirna in the plasma [ ] . cytokine levels in the plasma were quantified in order to assess the ability of mpg- :sirna and mpg- /mpg- -chol:sirna formulations to induce innate immune response in vivo. no increase in cytokine level was observed h after injection of any formulation, suggesting the lack of immune response induction [ ] . in a similar approach, pepfect was tested for in vivo rnai silencing. the hypoxanthine phosphoribosyltransferase (hprt ) gene was targeted due to the long cellular half-life of the protein (~ h) and the minimal impact on the viability of the transfected cell. systemic administration of pepfect :sirna nanoparticles ( mg/kg) induced a significant down regulation (n %) of the hprt mrna level in liver, kidney, and lung, without induction of immune response [ ] . in addition, pepfect -based nanoparticles were also used for rnaimediated silencing of luciferase (luc-sirna) in mice expressing luciferase in the liver and intravenous injection of pepfect :luc-sirna ( mg/kg) displayed decrease of bioluminescence for weeks, reaching % gene silencing whilst naked luc-sirna was unable to induce a rnai response [ ] . likewise, chol-r , r :sirna nanoparticles were formulated for sirna-mediated knockdown of human epidermal growth factor receptor (her- ) in human ovary adenocarcinoma cells (sk-ov- ). a reduction of her- expression was associated with the inhibition of sk-ov- xenograft tumour growth [ ] . intratumoural injection of r :sirna nanoparticles, every days at a dose of μg sirna per mouse, significantly reduced tumour growth without significant toxicity [ ] . furthermore, different strategies were investigated to stabilize the nanoparticles and to increase blood circulation such as exemplified by albumin-or polyethylene glycol (peg)-coated nanoparticles. for example, hou and co-workers have shown that albumin-coated formulation of p rhh exhibits remarkable transfection efficiency attributable to ph triggered nanoparticle disassembly [ ] . furthermore, tanaka and co-workers have demonstrated that intravenous injection of mpeg-pcl-ch r h c/sirna complexes had a significantly higher anti-tumour effect in sarcoma-bearing mice [ ] . most non-covalent cpps tested in vivo have been formulated for sirna delivery. however the non-covalent strategy is also intended to deliver other types of ons, such as-ons or ssos, in vivo. in this light, pep- was formulated with the antisense cyclin b hypna-ppna and the resulting pbn were evaluated on pc xenografted mice [ ] . pbn were administrated through intratumoural or intravenous injection every days, and the biological effect of the antisense hypna-ppna was evaluated by monitoring tumour growth during weeks after injection. intratumoural administration of pbn induced a % inhibition of tumour growth at a mg on dose and more than % at a mg dose, whereas intravenous injection only reduced tumour growth by % at a mg dose. consequently in order to improve the in vivo stability of pbn, small amounts of pegylated-pep- were included in formulation [ ] . intravenous administration of mg of on with pbn containing % of pegylated-pep- significantly improved pbn stability and inhibited tumour growth by more than %, which was - -fold more efficient than un-pegylated-pep- [ ] . these data emphasize the potential of non-covalent cpp for in vivo as-on delivery and the importance of pegylation on nanoparticle stability. in addition to the physicochemical properties of pbn, some specific characteristics of formulations have to be addressed. parameters such as affinity, molar or charge ratios, excipients, size and colloidal properties have a strong influence on biological interactions and minor changes can have a major impact on drug delivery efficacy [ ] . thus, as in the fda draft guidance for liposomal products, the physicochemical properties and specifications should include: morphology, net charge, particle size of pbn, spectroscopic data, light scattering index, in vitro release, content of peptide-engaged in pbn versus free peptide, biodegradation of the pbn. one of the most challenging questions concerning cpps is the mechanism by which these peptides enter cells. early studies using fluorescent dyes linked to cpps concluded that internalization was energy-and temperature-independent. however, this was later found to be an experimental artefact due to strong cpp adherence to the cell membrane leading to fluorescence overestimation and/or to fixation protocols using methanol allowing the cpp-dye complex to enter cells [ ] . recent studies have suggested that cpp uptake takes place by both endocytosis and energy-independent translocation, with the balance between these two pathways influenced by factors such as cpp sequence [ ] , temperature and cpp concentration [ ] [ ] [ ] [ ] . it is now admitted that both biophysical methods, cell biology assays and biological end-points to assess activity need to be used to dissect the cell import mechanism. molecules approaching a cell first encounter a layer of oligosaccharidesthe endothelial glycocalyx which is a network of membranebound proteoglycans and glycoproteins, covering the endothelium luminally [ ] . proteoglycans in particular carry large o-linked oligosaccharides consisting of highly negatively charged repeating disaccharide units, the glycosaminoglycans (gags), such as heparan sulphates (hs). gags are involved in the cellular binding and uptake of several viruses [ , ] as well as cationic cpps and polycationic nanoparticles [ , ] . oligo-arginines bind to hs with affinities in the upper nanomolar range suggesting that gags may act as cpp receptors (reviewed in [ ] ). for the interaction of cationic cpps with gags, the number of positive charges (number of arginines) was shown critical [ ] . as examples, tp -pna and penetratin-pna conjugates are mainly internalized via macropinocytosis after initial hs interaction on the cell surface [ , ] . beside the important role of the positive charges, sagan and co-workers also show a strong positive correlation between the number of tryptophan residues, gag binding and cell uptake [ ] . likewise, cady:sirna complexes interact with hspgs, which probably allow the binding and accumulation of the particles at the cell surface as demonstrated by electromobility gel shift assay and nanoparticle dissociation [ ] . several models to explain a direct membrane translocation of cpps have been proposed, such as the formation of inverted micelles, pore formation (toroidal or barrel slave), the carpet model and the sinkingraft model. these models have been initially proposed for the uptake of membrane-active peptides (reviewed in [ ] [ ] [ ] ) and then adapted for cpps, such as oligo-arginines [ , ] or transportan [ ] . likewise, the amphipathic mpg and cady peptides are internalized via direct membrane translocation. on the basis of physico-chemical investigations (e.g., circular dichroism, fourier transform infrared and electrophysiological measurements on model membranes), two very similar models have been proposed for mpg:sirna and cady:sirna pbns based on the formation of transient pore-like structures [ , ] . a partial conformational change takes place upon mpg complexation with nucleic acids, and an increase in β-sheet content upon association with the cell membrane [ ] . cady:sirna pbns were characterized in more details (reviewed in [ ] ). cady-mediated cellular uptake of sirna is extremely rapid [ ] . moreover, internalization and biological activity of cady:sirna occurred even at °c and under inhibition of mitochondrial oxidative phosphorylation revealing an energy-independent mechanism [ ] . fluorescence microscopy revealed that neither transferrin, nor rab co-localized with cady:sirna nanoparticles whereas co-localization with lysotracker was observed to some extent [ ] . these data further support an uptake mechanism largely independent of classical endocytosis with a degradation of the nanoparticles via the lysosomal pathway. apart for the few cases described in section . , the present consensus is that cell uptake occurs mainly by endocytosis for arginine-rich cpps at least at low concentrations [ ] . whether clathrin-coated pit endocytosis, macropinocytosis, or another endocytic route is used is still a matter of debate and the route might differ with cell type, nature of the payload, or concentration [ , ] . the internalization mechanism was mainly characterized using the hela splicing redirection assay (fig. ) by correlating cellular uptake and biological efficiency of cpp-pna or cpp-pmo conjugates. several conjugates have been analysed in detail such as (lys) -pna-lys [ ] , (rxr) -pmo [ ] or pip b-pna [ ] . cell uptake is energy-dependent and leads to sequestration of conjugates in cytoplasmic vesicles after an endocytic mechanism of internalization. the limitations provided by the endosome sequestration of the payload are well illustrated by the two following sets of data. pepfect , a stearylated version of transportan [ ] , is able to complex ssos and these pepfect :sso pbns are rather efficient in the hela splicing-redirection assay. however, a significant part of the complexes still remains entrapped in endosomes and can be released upon chloroquine treatment [ ] . based on these results, endosomolytic trifluoromethylquinoline (qn) moieties were grafted on the pepfect cpp leading to pepfect with largely improved cytoplasmic delivery of the transfected on and an increased splicing redirection activity. likewise, pepfect was efficient to transfect sirnas and to promote gene silencing at low concentrations at variance to the pepfect formulation [ ] . along the same line and more recently, we could relate the high efficiency of pip a-pmo in h k mdx skeletal muscles with an efficient release from endocytic vesicles after internalization via caveolaedependent endocytosis. at variance, this same conjugate is internalized by clathrin-dependent endocytosis in primary mdx cardiomyocytes, is less efficiently released from endocytic vesicles and has a lower exon skipping activity [ ] . several strategies can be used to study the cellular internalization of cpp-on conjugates or cpp:on complexes [ , ] . . biophysical characterization of cpp/on using circular dichroism, infra-red spectroscopy, dynamic light scattering or by insertion studies into phospholipid layers. . inhibition of a specific endocytic pathway and assessment of its effect on cpp/on internalization and biological activity. . co-localization microscopy studies of the association of cpp/on with fluorescently labelled endocytic markers the association of cpps with membranes induces the modification of several physical properties, such as the surface pressure of monolayer (langmuir blodgett) and the secondary structure of the peptide (ft-ir, cd, etc.). deformation of the lipid bilayer due to hydrophobic and hydrophilic interactions between the components as well as the peptide localization in the membrane can be also assessed using nmr, x-ray diffraction, coupled plasmon waveguide resonance, epr or fret [ ] . assessing endosomal escape directly has turned difficult and can only be monitored using artificial models such as liposomal leakage assays. different protocols were developed using large unilamellar vesicles (luvs) with entrapped dyes such as calcein [ ] , carboxyfluorescein [ ] or ants (fluorescent dye)/dpx (quencher) [ , ] . in cellulo, clathrin-mediated endocytosis can be predominantly characterized by the use of transferrin. its uptake in cells can indeed be inhibited by a clathrin-specific sirna or via cell transfection cells with a mutant form of dynamin [ ] . however, dynamin is now known to regulate other endocytic and membrane trafficking pathways. alternatively pharmacological inhibitors, such as chlorpromazine or dynasore (a dynamin inhibitor), can be used to inhibit this pathway even if these reagents cause significant cell toxicity and have to be used over short incubation times [ ] [ ] [ ] . potassium depletion or hypertonic medium incubation is also commonly used to investigate clathrin-mediated endocytosis [ , ] . caveolae are invaginations of the plasma membrane that are localized in lipid rafts. a number of ligands, such as cholera toxin b, sv virus and albumin have been shown to be internalized more or less specifically via caveolae. the glycosphingolipid analogue lactosylceramide (laccer), a fluorescent probe, was also shown to be internalized via this route and may represent a more selective marker for this pathway [ ] . a range of pharmacological inhibitors of this pathway have been described and, in the main, they are agents that deplete cholesterol synthesis (lovastatin), agents that rapidly extract cholesterol from lipid rafts (methyl-ß-cyclodextrin), and other cholesterol-interacting molecules such as the antibiotics nystatin and filipin. dowdy, futaki and their colleagues have shown that tat and other arginine-rich cpps induce a ubiquitous form of fluid-phase endocytosis termed micropinocytosis [ , ] . for example, the uptake of octaarginine (r ) peptide by hela cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (eipa) and the f-actin polymerization inhibitor cytochalasin d, suggesting a role for macropinocytosis in the uptake of the peptide. as mentioned above, cpps are taken up primarily by endocytic pathways, and in order to promote endosomal escape and increase the transfection efficiency of cpps, many different strategies have been used. we have mentioned the insertion of endosomolytic moieties in section . and additional strategies are described below. since endosomes have a low ph, researchers have incorporated histidine residues or appended oligo-histidine tails in the cpp sequence, aiming at capitalizing on a "proton sponge" effect (see review [ ] ). due to its protonation at ph . , the imidazole ring of histidine (which is a weak base) counterbalances the accumulation of protons generated by a specific atpase inside acidic vesicles, neutralizes the lumen of endocytic vesicles and increases their osmolarity. as a consequence, the endosomal vesicles swell and their content is delivered into the cytosol. this has been exploited with success for the tat-cpp [ ] , for microsphere coated with ornithine and histidine repeats (o h ) and for self-assembling nano-constructs of amphiphilic copolymers (see review [ ] ). mason and co-workers have focused on amphipathic α-helical peptides incorporating ph sensitive residues [ ] . the histidine residues in the lah peptide are uncharged at neutral ph but when the ph of the endosomal lumen drops, the side chains become protonated, large numbers of peptides are released from the complex and adopt a conformation and alignment in the membrane that induces membrane disorder [ , ] . other groups have focused on the addition of cholesterol or on fatty acid modifications. for example, chol-r improves sirna delivery in a mouse model bearing a subcutaneous tumour [ ] . tat-pna-mediated splice correction is increased by up to two orders of magnitude when conjugated with decanoic acid [ ] . stearylation of cpps represents another strategy to increase endosome escape and as a consequence to increase the transfection efficiency of sirna [ ] and phosphorothioate ′-ome rna [ ] as already detailed in section . for in vivo applications, the most frequently modification involves the grafting of a polyethylene glycol (peg) moiety to prevent rapid clearance. as reported for pep- -mediated delivery of antisense-cyclin b -charged-pna which blocks tumour growth in vivo upon intratumoural and intravenous injection, pegylation of pep- significantly improves complex stability and efficiency [ ] . in the context of clinical translation, it will be crucial to improve the tissue specificity of cpps through their functionalization. the screening by in vivo phage display has enabled the identification of numerous peptides that home specifically to various organs under normal or pathological conditions [ ] . for example the five residues homing peptides creka, which was identified by in vivo screening of phagedisplayed peptide libraries in tumour-bearing mmtv-pymt transgenic breast cancer mice [ ] , has been combined to the pvec peptide to yield a cpp with tumour homing specificity [ ] . despite their huge potential, the clinical use of nucleic acids-based drugs has been limited by their poor biodistribution. cell penetrating peptides have therefore been considered as a possible strategy to improve passage across biological barriers and intracellular delivery as reviewed extensively in this chapter. both covalent conjugates with neutral antisense on mimics and noncovalent complexes with either charged antisense ons or sirnas have been engineered. several such constructs have undergone extensive evaluation in animal models (mainly in mice) of both acquired (e.g., viral infections or cancers, in particular) and genetic (e.g., duchenne muscular dystrophy) human diseases. cpp-delivery has been convincingly shown to increase significantly the efficiency of these nucleic acids cargoes. clinical trials have however not yet been started to our knowledge. extensive studies of biodistribution and of possible toxic effects have in particular to be completed. despite many studies, mechanisms responsible for the extravasation and for the cellular trafficking of these cpp-drug conjugates or complexes are still poorly understood. direct translocation across cell membranes appears to be operational in some instances. in most cases however, cell internalization occurs through endocytosis and escape from endocytic vesicles limits biological efficiency. likewise, cpp delivery does not occur with a similar efficiency in all tissues after systemic administration. understanding better these limitations will obviously help in engineering of new cpp generations with a superior potential to target various tissues (and pathological conditions) and to deliver their payload within the appropriate cellular compartment. efforts in these directions have already been started as described in this chapter but much remains to be done. inhibition of rous sarcoma viral rna translation by a specific oligodeoxyribonucleotide inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide regulation by small rnas in bacteria: expanding frontiers the antisense transcriptomes of human cells antisense technology: a selective tool for gene expression regulation and gene targeting therapeutic potential of splice-switching oligonucleotides potent and specific genetic interference by double-stranded rna in caenorhabditis elegans targeting rna to treat neuromuscular disease use of 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cells in mice following systemic administration of a pna-peptide conjugate efficient inhibition of mir- function in vivo by peptide nucleic acids chemical structure requirements and cellular targeting of microrna- by peptide nucleic acids anti-mirs delivery of sirna and other macromolecules into skin and cells using a peptide enhancer induction of in vivo synthetic lethal rnai responses to treat glioblastoma inhibition of respiratory syncytial virus infections with morpholino oligomers in cell cultures and in mice inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer antiviral effects of antisense morpholino oligomers in murine coronavirus infection models cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of escherichia coli in vitro and in vivo variations in amino acid composition of antisense peptide-phosphorodiamidate morpholino oligomer affect potency against escherichia coli in 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vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro cell penetration by transportan efficient splicing correction by pna conjugation to an r -penetratin delivery peptide up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides cell-penetrating-peptide-based delivery of oligonucleotides: an overview vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents disulfide conjugation of peptides to oligonucleotides and their analogs lung delivery studies using sirna conjugated to tat( - ) and penetratin reveal peptide induced reduction in gene expression and induction of innate 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current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers an endosomolytic tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for dna transfection multifunctional polymeric micelles for delivery of drugs and sirna cationic amphipathic histidine-rich peptides for gene delivery effective endogenous gene silencing mediated by ph responsive peptides proceeds via multiple pathways design and evaluation of histidine-rich amphipathic peptides for sirna delivery improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (catlip) domain octaarginine-modified multifunctional envelope-type nano device for sirna delivery of nucleic acids with a stearylated (rxr) peptide using a non-covalent co-incubation strategy homing peptides as targeted delivery vehicles biomimetic amplification of nanoparticle homing to tumors design of a tumor homing cell-penetrating peptide for drug delivery acknowledgements lo'd was supported by a grant from the french muscular dystrophy association afm (programme number ). the work in the laboratory of mjg was supported by the medical research council (mrc programme number u ). bl and mjaw were supported by the afm grant "advances in oligonucleotide-mediated exon skipping for dmd and related disorders". work in the laboratory of pb and sd was partly funded by the feder (fonds européen de développement regional)/la région languedoc-roussillon, by the agence nationale de la recherche (anr) and by the centre national de la recherche scientifique (cnrs). key: cord- - q tk fd authors: zhu, qinchang; liu, ge; kai, masaaki title: dna aptamers in the diagnosis and treatment of human diseases date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: q tk fd aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. compared with rna aptamers, dna aptamers have inherent advantages in stability and facility of generation and synthesis. to better understand the specific potential of dna aptamers, an overview of the progress in the generation and application of dna aptamers in human disease diagnosis and therapy are presented in this review. special attention is given to researches that are relatively close to practical application. dna aptamers are expected to have great potential in the diagnosis and treatment of human diseases. aptamers can be broadly defined as short biomolecules like oligonucleotides and peptides that bind to specific targets with extremely high affinity based on their structural conformations. since the early s, systematic evolution of ligands by exponential enrichment (selex) and similar methods have been reported to efficiently select rna and dna aptamers [ ] [ ] [ ] [ ] . thereafter, nucleic acid aptamers have been extensively researched and applied. nucleic acid aptamers are rna and single-stranded (ss) dna oligonucleotides with lengths typically ranging from to mers, which have the same level of target-binding affinity as monoclonal antibodies (the dissociation constant (k d ) usually ranges from . to nm) [ , ] . compared with antibodies, nucleic acid aptamers have many advantages in their suitability for clinical application and industrialization, including almost no immunogenicity, efficient penetration, less batch variation, easy modification, cost-effectiveness and short production times [ , ] . in the past years, much progress has been made in the use of nucleic acid aptamers, particularly rna aptamers, as therapeutic agents, diagnostic and analytical tools, vehicles for targeted drug delivery, biosensors and even genetic control devices [ , [ ] [ ] [ ] [ ] . until now, although only one aptamer (macugen, pfizer/eyetech) has been approved for the therapeutic use in the clinic [ ] , ten other aptamers are being evaluated at different stages of clinical trials [ , ] . currently, the application in diagnostics, research and development is expected to account for the largest share of the aptamer market. the global aptamer market was estimated to be valued at $ . million in and to reach $ . million by [ ] . dna aptamers are expected to account for the largest share of the global market in [ ] , which indicates an increase in the application of dna aptamers. dna and rna aptamers are functionally similar but have some differences in their stability and accessibility. compared with dna aptamers, rna aptamers are chemically unstable because of the presence of a reactive hydroxyl group (-oh) at the position of the ribose sugar in rna nucleotides. this -oh group easily gets deprotonated in solution, especially in alkaline solutions. the resulting anionic -o´may nucleophilically attack the phosphorus atom of the phosphodiester linkage, leading to the hydrolysis of rna molecules [ ] . the nuclease resistance of rna aptamers was found to increase when the -hydroxyl group was removed from the sugars of rna [ , ] . dna aptamers are less reactive and relatively stable because of the c-h bonds at the position of the deoxyribose sugar of dna nucleotides. this chemical difference gives dna aptamers an inherent advantage in stability over rna aptamers. our previous study also confirmed that dna aptamers are much more stable than natural rna aptamers in % fetal bovine serum (fbs) and human serum [ ] . that is the reason why extra chemical modifications are usually added to rna aptamers to improve their chemical stability [ ] . however, it should be noted that the reactivity of rna nucleotides and non-watson-crick base pairing makes rna oligonucleotides prone to forming more diverse and complex three-dimensional ( d) structures [ , ] , which is helpful for selecting aptamers with high affinity and specificity from rna libraries [ ] . therefore, dna aptamers are usually selected from the libraries containing longer randomized regions for the purpose of obtaining more complicated structures. another potential advantage of dna aptamers over rna aptamers is their relatively simple selection process. the selection of rna aptamers requires reverse transcription and in vitro transcription in every round of selection, as well as an initial transcription for generating the rna library from a dna library in most cases [ , ] . but the selection process of dna aptamers does not require these extra steps [ , ] . moreover, once selected, the cost of producing dna aptamers is lower than that for rna aptamers. since the first ssdna aptamer was selected for human thrombin in [ ] , dna aptamers have been researched and applied in various fields, especially in diagnosis and treatment of human diseases. dna aptamers are usually explained to work in a similar way to rna aptamers. because of the potential advantages described above, dna aptamers have gained increasing attention in recent years. however, they have seldom been reviewed systematically and independently. to better understand the unique potential of dna aptamers, this article will review the recent progress of dna aptamers in regard to their preparation and application in the diagnosis and treatment of human diseases. the advantages and remaining challenges to develop and use dna-aptamer-based diagnostic tools and therapeutics will also be discussed. selex, an interactive in vitro selection procedure, is the basic method used to engineer aptamers, which was first reported for screening of rna aptamers independently by ellington's and tuerk's groups years ago [ , ] . selex was then adapted to generate dna aptamers [ , ] . in principle, aptamers are selected from an initial random ssdna pool based on the binding of the oligonucleotides to the target molecules under optimal conditions. the ssdna pool contains - random sequences of synthetic dna (the typical length is - nucleotides, flanked by two constant regions with primer sites for polymerase chain reaction [pcr] amplification). the unbound sequences are separated from the bound molecules, and the target-bound sequences are amplified by pcr. the amplified products (double stranded dna) are converted to ssdna through various ways [ ] and then used as a new aptamer pool for the next selection round. enriched aptamer sequences are finally cloned and identified by sequencing. usually after - rounds of selection, the specific aptamers with the strongest affinity for the target molecules are obtained. selection of aptamers using conventional in vitro selex requires purified and soluble target proteins. the processes used to obtain purified protein targets are time-consuming. sometimes it is difficult to purify target proteins. moreover, sometimes the aptamers selected using a non-native protein do not interact with the protein in a native conformation. to solve these problems, a strategy using whole live cells as targets for aptamer selection has been developed, which is known as cell-selex. this selex is able to generate dna aptamers that recognize the cell-surface or intracellular target protein in their native conformation, which shows great potential in cell-specific therapeutics and diagnostic applications [ ] [ ] [ ] . this method first selects the specific aptamers that bind to the target cells by a positive selection step, and then eliminates the non-target cell-specific aptamers by a negative selection step using non-target cells, followed by the selex process as summarized in figure a . recently, modified cell-selex methods have been developed to select aptamers targeting specific cells like disease state cells and metastatic cells. they are stimulus-response cell-selex [ ] and metastatic-cell-based selex [ ] . to shorten the time of conventional cell-selex, a two-step stimulus-response cell-selex method has been developed, which utilizes asymmetric pcr or streptavidin-biotin magnetic separation for the generation of single-stranded dna to reduce the selection cycle to two steps [ ] . competitive cell-selex is another approach to improve selection efficiency and affinity, which utilizes a nitrocellulose membrane that contains the target cells and negative control cells to select target aptamers [ ] . this method can reduce the selection time, since the negative selection step is not required. the conventional selex procedure is time-consuming and requires several steps to obtain a specific dna aptamer. to effectively generate highly specific aptamers, several novel methods using different selection approaches have been developed. combining selex with other method is one of the most effective ways to further improve the selection efficiency and binding affinity of dna aptamers. for example, cell-selex coupled with in silico maturation was developed to improve aptamer specificity [ ] . the method consists of cell-selex, a post-selex in silico process and in vitro screening. after the normal cell-selex, an extra in silico process was performed to further evolve improved aptamers from the selex-selected sequences through successive rounds of sequence shuffling and random mutation based on a genetic algorithm. this is followed by in vitro functional screening and selection of enhanced aptamers from the shuffling and mutated sequences. this method permits the evolution of functionally enhanced aptamer sequences recognizing targets of interest. in addition to selex-like methods, other methods have also been developed; magnetic-assisted rapid aptamer selection (maras) is a method that uses magnetic beads and an externally applied rotating magnetic field to provide the competitive mechanism for the rapid selection of aptamers with different affinity to the molecular target as summarized in figure b [ , ] . this method uses biofunctionalized magnetic nanoparticles to separate target-bound dna oligonucleotides from a library, selecting those interactions that survive a disruptive force generated by the movement of the particles in an externally applied rotating magnetic field. it abandons the multi-cycle evolutionary process used in conventional selex and thus can achieve rapid selection (completed in less than one hour). a one-step selection approach is another promising way to increase the rate of aptamer generation. recently, this rapid one-step selection method was developed to select specific dna aptamers using only one pcr step as summarized in figure c [ ] . in this method, a target immobilized on a glass coverslip was subjected to carboxyfluorescein (fam)-labeled nucleic acid pool binding, extensive washing and microscopy examination, followed by pcr enrichment of the selected aptamers. a control experiment used a labeled target and a labeled dna library was used to make sure the specificity of the selection. in the overlay image of the immobilized target labeled with alexa fluor (red color) and target bound fam-labeled dna aptamer (green fluorescence), an orange color is observed for target-specific selection. although the binding affinity of aptamers selected with the current version of the one-step method was in the low micromolar range, it was believed that it can be further improved by using larger targets, increasing the stringency of selection, and by combining it with a capillary electrophoresis separation. this method was described as a user-friendly, low-cost and easy way to select dna aptamers. in particular, this method allows the use of a chemically modified nucleic acid library directly as it requires only one pcr step. moreover, a dna microarray has been utilized to achieve one-step aptamer identification, in which the sequences of interest can be produced on the arrays for selection and pcr, cloning and sequencing are not required [ , ] . molecules , , page-page selected with the current version of the one-step method was in the low micromolar range, it was believed that it can be further improved by using larger targets, increasing the stringency of selection, and by combining it with a capillary electrophoresis separation. this method was described as a user-friendly, low-cost and easy way to select dna aptamers. in particular, this method allows the use of a chemically modified nucleic acid library directly as it requires only one pcr step. moreover, a dna microarray has been utilized to achieve one-step aptamer identification, in which the sequences of interest can be produced on the arrays for selection and pcr, cloning and sequencing are not required [ , ] . the target-coated magnetic beads are incubated with the ssdna pool. the beads with bound sequences are then separated from the unbound sequences with a u-shaped magnet or magnetic stand. after re-dispersion, the beads are put in an externally applied rotating or alternating magnetic field. during this process, the weak and non-specific binding sequences are released and separated. the strong-binding sequences are finally released from the beads by heating and then incubated with the beads without a target for the negative selection. the selected sequences are amplified, cloned and sequenced as usual; (c) one-step selection: a fam-labeled oligonucleotide library is incubated with a target immobilized on a glass coverslip that was coated with n-hydroxysuccinimide (nhs) functionalized polyethylene glycol (peg). unbound sequences are discarded by extensive washing followed by monitoring with fluorescence microscopy. the coverslip is later crushed and the bound sequences are eluted by heating in water. the selected aptamers are amplified, cloned and sequenced as usual. dna aptamers are well known to be more stable than rna aptamers, which allows them to be readily used in the primary stage of developing diagnostic tools. however, the degradation of dna aptamers by nuclease is still a serious problem that limits their clinical application, when they are subjected to complicated biological samples. to improve the nuclease resistance of dna aptamers, one those that do not bind to negative targets are retained and amplified by pcr. the pcr products are separated into ssdna for further rounds. after - cycles, the selected ssdnas are cloned and sequenced for aptamer identification; (b) magnetic-assisted rapid aptamer selection (maras): the target-coated magnetic beads are incubated with the ssdna pool. the beads with bound sequences are then separated from the unbound sequences with a u-shaped magnet or magnetic stand. after re-dispersion, the beads are put in an externally applied rotating or alternating magnetic field. during this process, the weak and non-specific binding sequences are released and separated. the strong-binding sequences are finally released from the beads by heating and then incubated with the beads without a target for the negative selection. the selected sequences are amplified, cloned and sequenced as usual; (c) one-step selection: a fam-labeled oligonucleotide library is incubated with a target immobilized on a glass coverslip that was coated with n-hydroxysuccinimide (nhs) functionalized polyethylene glycol (peg). unbound sequences are discarded by extensive washing followed by monitoring with fluorescence microscopy. the coverslip is later crushed and the bound sequences are eluted by heating in water. the selected aptamers are amplified, cloned and sequenced as usual. dna aptamers are well known to be more stable than rna aptamers, which allows them to be readily used in the primary stage of developing diagnostic tools. however, the degradation of dna aptamers by nuclease is still a serious problem that limits their clinical application, when they are subjected to complicated biological samples. to improve the nuclease resistance of dna aptamers, one of the effective solutions is to use a library with chemically modified dna sequences in the screening process. the modified dna library can be prepared by pcr amplification using specific polymerase and catalysis reactions using modified dna enzymes [ , ] . modification of the aptamers can also be performed after the in vitro selection from natural nucleic acid library. modifications of the sugar phosphate backbone or the pyrimidine are the strategies used to increase the stability and nuclease resistance of aptamers [ ] [ ] [ ] . capping the end of aptamers is also utilized to enhance aptamer stability against nucleases [ ] . incorporation of unnatural nucleotides is another approach to overcome aptamer instability. locked nucleic acid (lna) is one of the most prominent and successful nucleic acid analogues because of their pronounced stability [ ] [ ] [ ] . generation of "mirror aptamers", which are also known as spiegelmers, is another approach to improve aptamer stability against nucleases [ ] . generation of highly specific aptamers with a long lifetime is very important for their therapeutic application. because most dna aptamers have a small molecular weight (ranging from - kda), dna aptamers are readily removed via renal filtration and metabolic processes, limiting their therapeutic application. one of the effective ways to control the lifetime of dna aptamer in vivo is to conjugate the aptamers with bioavailable materials. conjugation of dna aptamers with polyethylene glycol (peg) is commonly used to prolong their circulation in the bloodstream [ , ] . coating dna aptamers with other nanomaterials such as nanoparticles [ , ] , liposomes [ ] and copolymers [ ] has been successfully used to improve the lifetime of dna aptamers. once selected to bind to disease-related biomarkers or pathogens, dna aptamers can be developed as biosensors through chemical modification with luminophores or linkage to nanoparticles in various formats [ ] . for example, in an earlier study, dna aptamers selected for bacillus anthracic spores were developed to detect anthrax spores in an aptamer-magnetic bead-electrochemiluminescence (am-ecl) sandwich mode [ ] . the dynal m- magnetic beads were covered with the aptamer and used as the capturer to capture the spores, while another biotinylated aptamer was used as the reporter and the signal was finally transduced through the streptavidin-ru(bpy) + ecl. since the first dna aptamer was selected, dozens of dna aptamers have been selected for the use of disease-related detection. although there are no aptamer-based diagnostic tools that are in clinical use at the moment, many preclinical studies indicate that dna aptamers have great potential to be used in this way. to profile the features of recent studies that use dna aptamers as a diagnostic tool for human diseases, we listed some dna aptamers selected for this purpose in table , with a special attention to parameters like the sensitivity and specificity, which have a great impact on their potential for clinical use. in theory, aptamers can be selected for developing diagnostic tools for various diseases, as long as definite targets, such as disease-specific biomarkers or pathogens, are available. to date, dna aptamers have been explored mainly for the diagnosis of infectious diseases, cancer and cardiovascular diseases. because of the convenience of cell-selex, which does not require preparation and purification of target molecules, many reported dna aptamers are selected with cancer cells, aiming to diagnose and image cancer tissue. these cancer cells include pancreatic [ ] , colon [ ] , liver [ , ] , cholangio [ ] , gastric [ , ] , prostate [ ] , breast [ ] and glioblastoma [ ] cancer cells. usually, the selection consists of positive selection with cancer cells and negative selection with normal cells from the same organs, as described in section . . . it is worth noting that, because the diagnosis and monitoring of metastasis of cancer cells is important for the treatment of cancer, several dna aptamers have been selected with metastatic cancer cells and negatively selected with non-metastatic cancer cells [ , , ] . target-induced dissociation (aunp) . µm % (n = amino acids) [ ] ns.: non-specified. for example, li et al. selected dna aptamers for metastatic colon cancer cells using sw cells derived from a metastatic site lymph node in the positive selection and sw cells from a primary colon adenocarcinoma of the same patient in the negative selection [ ] . the resulting aptamer (xl- ) was found to possess specific affinity to the metastatic colon cancer cells (k d = . nm). its truncated form (xl- - ) was used to image the cancer tissue after labeling with fluorescein amidite (fam), displaying an . % detection rate against colon cancer tissue with metastasis in regional lymph nodes and . % specificity against nonmetastatic colon cancer tissue. the biomarker molecules that specifically express or overexpress on the cancer cells were also used for the selection of diagnostic dna aptamers. in addition to the previously known prostate-specific membrane antigen (psma) [ ] and mucin (muc ) [ ] , other biomarkers like epidermal growth factor receptor variant iii (egfrviii) [ ] , epithelial cell adhesion molecule (epcam) [ ] and vascular endothelial growth factor (vegf ) [ ] have also been used to select dna aptamers for cancer detection. by directly using cancer biomarkers to select cancer-cell-recognized aptamers, you obtain highly specific and affinitive aptamers, but the potential risk is that the aptamers might not recognize the natural biomarkers on the surface of cancer cells because of the difference in the d structure of the purified biomarkers and the natural biomarkers. for the diagnosis of infectious agents, lots of dna aptamers have been selected predominantly with cell-selex and magnetic beads-based methods by targeting various viruses and bacteria or their antigen protein [ ] , including targets such as norovirus, influenza virus, severe acute respiratory syndrome coronavirus (sars-cov), hepatitis c virus (hcv), hepatitis b virus (hbv), human immunodeficiency virus (hiv), human papillomavirus (hpv), salmonella typhimurium, and pathogenic e. coli. because of the low cost and short production time of dna aptamers, the dna aptamer based diagnostic tools hold particular advantages for the diagnosis of infections that need point-of-care testing, such as infections caused by highly pathogenic pandemic influenza virus, hiv, sars-cov and malaria. avian influenza virus h n is a highly pathogenic subtype of the influenza virus, which can also infect humans and has a high mortality rate. wang et al. selected a dna aptamer targeting h n using both the virus antigen and the whole virus particle using the selex method [ ] . for the first four selection cycles, the purified virus antigen hemagglutinin (ha) was used as the target protein, while for the remaining eight cycles the entire h n virus particles were used as the targets. the selected aptamer showed high affinity (k d = . nm) and specificity to the h n virus (table ). using such a mixed selection mode might be a good strategy to obtain ideal aptamers, which can overcome the drawbacks of selection using only free biomarkers or cells. in the case of cardiovascular diseases, markers in the blood, such as myoglobin, c-reactive protein, l-homocysteine and thrombin have been used to select dna aptamer for the diagnosis of related diseases. myoglobin increases after acute myocardial infarction, which is an important early marker in urgent diagnosis of cardiovascular diseases. wang et al. selected dna aptamers against myoglobin using a fluidic chip method [ ] . the dna aptamer with the lowest k d value ( . nm) was subjected to the development of different biosensors for the detection of myoglobin, including the myoglobin-induced structural switching supersandwich biosensor [ ] and the antibody-myoglobin-aptamer sandwich biosensor [ ] . human c-reactive protein (crp) is a homopentameric oligoprotein, which has been validated as a powerful predictor and risk factor of inflammation and cardiovascular disease [ ] . yang et al. selected a dna aptamer (k d = . nm) targeting crp and used it to develop a sensor based on surface plasmon resonance technology [ ] . l-homocysteine is an amino acid intermediate, whose elevated level in the blood is associated with coronary heart disease [ ] . mckeague et al. selected dna aptamers targeting l-homocysteine and developed an aptamer-aunp sensor, in which the dna aptamers first coil around the surface of the gold particles and release the particles after binding to the homocysteine, which leads to salt-induced aggregation and colorization of the particles [ ] . thrombin is a serine protease that plays a critical role in the formation of obstructive blood clots, or thrombosis, which is involved in various diseases including chronic cardiovascular diseases [ ] . the first reported dna aptamer is the thrombin aptamer. since then, thrombin aptamers have been used as model aptamers to explore the mode of aptamer-based biosensors. it is the most frequently used dna aptamer for the demonstration of the proof-of-principle of various detection methods [ ] . the general modalities and assay formats for using aptamers in sensors have been well summarized in many comprehensive reviews [ ] [ ] [ ] [ ] [ ] . they can be roughly classified into four types based on the way the signal is generated: ( ) direct binding-based mode; ( ) target-induced structural switching mode; ( ) sandwich-like mode; and ( ) target-induced assembly or dissociation mode. these protocols are illustrated in figure . in the direct binding-based mode (figure a ), aptamers labeled with signal molecules directly bind to the target. the signal molecules on the aptamer-bound target can be detected directly. the aptamers for cancer cells imaging usually use this mode (table ) . it is a simple and direct mode, which is especially suitable for the situations like in vivo imaging that do not allow complex detection. but it does not contain signal amplification, which makes it difficult to increase the detection sensitivity. in target-induced structural switching mode, the binding of the target will cause a specific conformational change to the aptamer, which will "switch on or switch off" the signal generation [ , ] . for example, in a bio-chromophoric target-induced structural switching approach, a fluorophore-labeled dna aptamer forms a partial duplex with a small oligonucleotide modified with a quenching moiety (denoted qdna) in the absence of the target, bringing the fluorophore (f) and the quencher (q) into close proximity for maximum fluorescence quenching. when the target is introduced, the aptamer prefers to form the aptamer-target complex. the binding of the target to the aptamer will change the structure of the aptamers and release the partially complementary quencher, which will trigger the increase of the signal ( figure b ). this mode is suited for the development of aptamer-based reporters for real-time sensing applications. but the limitation of this mode is the decreased aptamer binding affinity because of competition from the qdna [ ] . in the sandwich-like mode, the target is "the meat" inserted within "two pieces of bread" consisting of an aptamer/aptamer or antibody/aptamer, which is very similar to the enzyme-linked immunosorbent assay (elisa) ( figure c ). aptamers in this mode function as reporter and/or capturer. this method permits double recognition of the target and signal amplification, which can greatly improve the specificity and sensitivity of aptamer-based detection. moreover, because of the small size of the aptamer, the aptamer sandwich mode can conquer the potential limitation of antibody elisa in detecting small molecules [ ] . but this mode requires extensive washing during the detection. in the target-induced assembly or dissociation mode, binding of the target to the aptamer will trigger the assembly of split aptamers or dissociation of bound aptamers, along with the change of detectable signals [ , ] (figure d ). in addition, it should be noted that various detection methods have been applied by dna aptamer based biosensors, including electrochemical, chemiluminescence, fluorescence, colorimetric, quantum dots-based and mass-sensitive detections [ ] . for imaging of cancer cells, most of the dna aptamer based methods use fluorophore-labeled aptamers to recognize the cancer cells directly (table ) . for the small targets like microorganisms and the marker protein, methods based on different modes have been explored extensively. the sandwich-like mode is frequently used ( figure c ) [ , ] . in the example of leishmania detection [ ] , the aptamer (lmwc- r) targeting the promastigote of leishmania was immobilized on the surface of the m magnetic beads. after the target leishmania promastigote were captured by lmwc- r, another -biotinylated reporter aptamer (lmhsp- b/ r) that targets the hydrophilic surface protein (hsp) of leishmania promastigote was added. then the beads with aptamer-captured leishmania promastigote and reporters were collected on the magnetic rack. streptavidin-horseradish peroxidase (sav-hrp) and the substrate ample red were added to transduce and display the signal. another frequently used mode is the target-induced dissociation mode, in which the system was in a non-signal status until the single-stranded aptamer dissociated from the initial state as a result of binding to the target [ , , ] . the detection of l-homocysteine mentioned above is a good example of the target-induced dissociation mode [ ] (figure d ). dna aptamers coil around the surface of the gold nanoparticles (aunps) and prevent the salt-induced aggregation of aunps. when the target molecules appear, the dna aptamers fold into a rigid structure and bind to the targets, and therefore release from the aunps. the aunps without the coverage of aptamers aggregate in the salt solution and change color from red to purple. the signal-molecule-labeled aptamers directly bind to the immobilized or free target. the signal molecules on the target are detected directly; (b) target-induced structural switching mode: a fluorophore-labeled dna aptamer forms a partial duplex with a small oligonucleotide modified with a quenching moiety (qdna) in the absence of the target, bringing the fluorophore (f) and the quencher (q) into close proximity for maximum fluorescence quenching. when the target is introduced, the aptamer prefers to form the aptamer-target complex. the binding of the target to the aptamer will change the structure of the aptamers and release the partially complementary quencher, which will trigger the increase of the signal; (c) sandwich-like mode: the aptamer or antibody is immobilized on the solid phase as the capturer. the captured target is reported by the biotinylated aptamer, which displays the signal through further binding to the streptavidin-conjugated hrp or aunp; (d) target-induced dissociation mode: dna aptamers coil around the surface of the gold nanoparticles (aunps) and stop the salt-induced aggregation of aunps. when the target appears, the dna aptamers bind to the target and release from the aunps. the aunps without the cover of aptamers aggregate in the salt solution and change color from red to purple. one of the biggest challenges for aptamer-based diagnostic tools and their clinical use is the possible invalidation in a real biological environment as a result of degradation from nucleases or interference from other matrix factors. using chemical modified dna aptamers is an effective way to prolong the lifetime of dna aptamer in the biological samples. for ultimate success, sufficient studies with clinical samples or in vivo studies are indispensable. currently, although most of the studies about dna aptamer based diagnostic tools for human diseases are still in the primary stage of laboratory studies, some of them have gone further by testing them with clinical samples and animal studies in vivo [ , , , ] . the dna aptamer xq- d ( table ) that targets the pancreatic ductal adenocarcinomas (pdac) cells has been tested with clinical pdac tissue sections and in vivo imaging of pdac tumor-bearing mice [ ] . out of pdac tissues and two out of eight normal pancreatic tissues were detected by the cy -labeled xq- d. in the in vivo imaging test, cy -labeled xq- d was injected into the balb/c-nude mice grafted with pdac through the tail vein. it was found to illuminate the tumor site up to h post-injection, while the cy -labeled library control did not give any signal. for this study, both phosphorothioate and ′-o-methyl modifications were tried for the dna aptamer. the four bases at the ′ and ′ termini of xq- d were replaced by ether phosphorothioate oligonucleotides or ′-o-methyl oligonucleotides. however, the phosphorothioate modification was the signal-molecule-labeled aptamers directly bind to the immobilized or free target. the signal molecules on the target are detected directly; (b) target-induced structural switching mode: a fluorophore-labeled dna aptamer forms a partial duplex with a small oligonucleotide modified with a quenching moiety (qdna) in the absence of the target, bringing the fluorophore (f) and the quencher (q) into close proximity for maximum fluorescence quenching. when the target is introduced, the aptamer prefers to form the aptamer-target complex. the binding of the target to the aptamer will change the structure of the aptamers and release the partially complementary quencher, which will trigger the increase of the signal; (c) sandwich-like mode: the aptamer or antibody is immobilized on the solid phase as the capturer. the captured target is reported by the biotinylated aptamer, which displays the signal through further binding to the streptavidin-conjugated hrp or aunp; (d) target-induced dissociation mode: dna aptamers coil around the surface of the gold nanoparticles (aunps) and stop the salt-induced aggregation of aunps. when the target appears, the dna aptamers bind to the target and release from the aunps. the aunps without the cover of aptamers aggregate in the salt solution and change color from red to purple. one of the biggest challenges for aptamer-based diagnostic tools and their clinical use is the possible invalidation in a real biological environment as a result of degradation from nucleases or interference from other matrix factors. using chemical modified dna aptamers is an effective way to prolong the lifetime of dna aptamer in the biological samples. for ultimate success, sufficient studies with clinical samples or in vivo studies are indispensable. currently, although most of the studies about dna aptamer based diagnostic tools for human diseases are still in the primary stage of laboratory studies, some of them have gone further by testing them with clinical samples and animal studies in vivo [ , , , ] . the dna aptamer xq- d ( table ) that targets the pancreatic ductal adenocarcinomas (pdac) cells has been tested with clinical pdac tissue sections and in vivo imaging of pdac tumor-bearing mice [ ] . out of pdac tissues and two out of eight normal pancreatic tissues were detected by the cy -labeled xq- d. in the in vivo imaging test, cy -labeled xq- d was injected into the balb/c-nude mice grafted with pdac through the tail vein. it was found to illuminate the tumor site up to h post-injection, while the cy -labeled library control did not give any signal. for this study, both phosphorothioate and -o-methyl modifications were tried for the dna aptamer. the four bases at the and termini of xq- d were replaced by ether phosphorothioate oligonucleotides or -o-methyl oligonucleotides. however, the phosphorothioate modification was found to weaken the binding ability of the aptamer, whereas the -o-methyl modification did not significantly affect the binding ability. moreover, the -o-methyl modified xq- d could last for h in % fbs medium, but the unmodified xq- d was completely degraded after -h incubation. an important application of aptamers is their use as small-molecule therapeutic agents. dna aptamers exhibit significant advantages in therapeutic application and have been developed as attractive therapeutic agents in competition with antibodies. generally, dna aptamers used for therapeutic applications function in two ways. first, dna aptamers can inhibit protein-protein interactions by specifically binding to the target protein and thereby functioning as antagonists. second, dna aptamers can function as agonists, which promotes the function of the target protein upon binding to the target protein. although dna aptamers act similarly to antibodies, the non-immunogenicity of dna aptamers make it more notable in therapeutics. compared with antibodies, dna aptamers are easier to uptake because of their small size. importantly, aptamers can specifically recognize a wide range of targets including small molecules, proteins and cells. moreover, given that dna aptamers can be designed and selected in vitro, they have lower production cost than antibodies. the properties and advantages of dna aptamers mentioned above facilitate the promising application of dna aptamers in the field of therapeutics. although there are currently no dna aptamer therapeutics in clinical use, four dna aptamers are being evaluated in clinical trials for their effect on hematological disease, macular degeneration disease and cancer ( table ) . as (antisoma) is a guanine-rich aptamer with a guanine quadruplex structure, targeting nucleolin. nucleolin is a eukaryotic nucleolar phosphoprotein that is involved in the synthesis and maturation of ribosomes and has been reported as a target for anti-cancer therapies. the guanine quadruplex structure can help to enhance the nuclease degradation resistance and cell uptake of as . as possesses anti-cancer activity against breast cancer cells [ ] , metastatic renal cell carcinoma [ ] and acute myeloid leukemia [ ] . the phase clinical trial for using as to treat renal cell carcinoma was completed in (nct ), while another phase clinical trial for treating acute myeloid leukemia was completed in (nct and nct ). arc (achemix), a pegylated dna aptamer, recognizes platelet ligand receptor von willebrand factor that mediates platelet recruitment. arc blocks the binding between von willebrand factor and the platelet, thereby inducing an antithrombotic effect. the efficacy of arc in platelet inhibition has been demonstrated and a phase clinical trial for treating von willebrand factor related platelet function disorders (nct ) has been completed. recent studies show that arc can effectively prevent thromboembolism [ ] . a phase clinical study shows that arc exhibits favorable pharmacokinetic, pharmacodynamic and safety properties in patients with congenital thrombotic thrombocytopenic purpura [ ] . nu (arca biopharma) is an unmodified dna aptamer targeting thrombin, which can prolong blood clotting. the phase clinical trial using nu as anticoagulation agent in patients undergoing off-pump coronary artery bypass graft (cabg) surgery (snap-cabg-off) has been completed (nct ). e (ophthotech), a pegylated dna aptamer, functions as an antagonist of platelet-derived growth factor. e in combination with anti-vegf agent can effectively prevent angiogenesis [ ] . currently, a phase clinical study of e in combination with ranibizumab (lucentis ) for wet age-related macular degeneration treatment is ongoing (nct ). besides the dna aptamers in clinical trials, many promising dna aptamers are in preclinical studies for treating various diseases, such as virus infections, tumors and central nervous system diseases. a dna aptamer recognizing the receptor-binding region of influenza a hemagglutinin was found to inhibit viral infection in an animal model against different influenza strains, as manifested by a %- % reduction of the virus burden in the lungs of treated mice [ ] . dna aptamer nas- was found to bind to vimentin and then cause apoptosis of mouse ascites adenocarcinoma cells in vitro and in vivo [ ] . recently, dna aptamers targeting human epidermal growth factor receptor (erbb- /her ) was demonstrated to retard the tumorigenic growth of gastric cancer in mice with more effective activity than anti-erbb- /her monoclonal antibody [ ] . moreover, remyelination was induced by a dna aptamer in a mouse model of multiple sclerosis (inflammatory disease of the central nervous system), which highlights the potential therapeutic application of dna aptamers in the treatment of multiple sclerosis [ ] . cell-specific drug delivery can help to increase the efficiency of a drug and reduce side effects. in addition to functioning as therapeutic agents, dna aptamers have also been explored as delivery vehicles in targeted delivery of drugs or small oligonucleotides such as small interference rna (sirna) and microrna (mirna). the ability of aptamers to specifically recognize the target and to be readily modified makes it a potential targeted delivery tool. dna aptamers are used in two ways for targeted delivery of drugs: ( ) directly linked to the drug molecules; ( ) in combination with nanoparticles to form the delivery platform. conjugating drug molecules directly to specific dna aptamers is a potential way to deliver the drugs specifically, and thus reduce the risk of off-target drugs. by linking dna aptamers to drugs or packing the drug into an aptamer-folded structure, dna aptamers-drug conjugates can efficiently deliver drugs to target cells with increased specificity. many dna aptamers have been selected to efficiently deliver chemotherapy drugs in vitro or in vivo, such as doxorubicin (dox) [ ] , fluorouracil [ ] and epirubicin [ ] . dimeric or dendrimer dna aptamers in conjugation with drugs have been developed to further enhance the efficiency of target delivery [ , ] . with regard to small oligonucleotides delivery, dna aptamers were directly linked to the small oligonucleotides to form a dna aptamer-oligonucleotide chimera, which could help to prevent non-specific internalization as well as decrease the cellular toxicity towards non-target cells. we have reported that a dna aptamer-sirna chimera could specifically enter into cd (+) t cells and efficiently decrease the expression of exogenous the hiv protease gene [ ] . an anti-mucin dna aptamer covalently linked to mirna- b was found to deliver mirna- b into ovarian cancer cells specifically and induce significant apoptosis of the cancer cells [ ] . dna aptamers in combination with nanoparticles as a delivery vehicle is another promising targeted delivery approach. a number of nanomaterials have been explored to conjugate with dna aptamers to form the delivery platform [ , ] . dna aptamer conjugated liposome likely has the highest potential as a delivery system, and liposome-based drug delivery systems have been evaluated in clinical trials. for example, as aptamer conjugated liposome was found be able to enhance the delivery specificity and uptake of dox in tumor cells, as well as to increase the accumulation of dox in the tumor tissues with reduced cardiotoxicity in vivo [ ] . micelles, aggregation of lipid molecules, are also used in dna aptamer-nanoparticles delivery systems. recently, as aptamer conjugated peg-poly(lactic-co-glycolic acid) (plga) nanoparticles have been developed to facilitate antiglioma delivery of paclitaxel in vivo [ ] . this aptamer-peg-plga delivery system can prolong circulation time and enhance target accumulation of paclitaxel, which facilitates tumor inhibition. gold nanoparticles are another attractive material used in drug delivery system, because of their high stability, low or no toxicity and facile conjugation. a dna aptamer (sgc c) conjugated gold nanoparticle system has been found to increase the uptake of dox into cancer cells [ ] . other nanomaterials such as silica, carbon nanotubes and quantum dots have also been used in dna aptamer-nanoparticles delivery systems to enhance the specificity and prolong the circulation of drug molecules [ ] . by summarizing the progress of the generation of dna aptamers and their application in human disease diagnosis and therapy, we have shown that dna aptamers have great potential to be used as an alternative to antibodies. since the first monoclonal antibody was produced in s, antibodies have been successfully and extensively used in the diagnosis and therapy of human diseases. aptamers are expected to achieve a similar success to antibodies. because of their stability, low cost and facile manipulation, dna aptamers will continue to be extensively studied and applied. although modified rna aptamers have enhanced stability, the high cost of chemically modified rna might limit their study and application. however, rna aptamers hold an advantage in providing more complex and diverse d structures, which is helpful for selecting aptamers with high affinity for complex targets needed for disease therapy. therefore, dna aptamers might have more promising application in diagnosis and in vivo imaging, while modified rna aptamers might have more promising application in therapy. in the era of personalized medicine, dna aptamer-based therapeutics and diagnostics are believed to have great potential for extensive application because of their flexibility to specifically bind to any molecule targets. before they can be widely applied, there are still many problems that remain to be addressed. problems like nuclease degradation, quick renal excretion and potential cross-reactivity of aptamers should be analyzed in future studies. systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t dna polymerase in vitro selection of rna molecules that bind specific ligands selection in vitro of single-stranded dna molecules that fold into specific ligand-binding structures selection of single-stranded dna molecules that bind and inhibit human thrombin aptamers as therapeutics single-stranded dna aptamers against pathogens and toxins: identification and biosensing applications a highlight of recent advances in aptamer technology and its application aptamers and their biological applications applications of aptamers for chemistry analysis, medicine and food security outlook for aptamers after twenty five years rna aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression pegaptanib (macugen): treating neovascular age-related macular degeneration and current role in clinical practice aptamer nanomedicine for cancer therapeutics: barriers and potential for translation aptamers market-global forecast to post-selex chemical optimization of a trypanosome-specific rna aptamer building oligonucleotide therapeutics using non-natural chemistries inhibition of hiv- protease expression in t cells owing to dna aptamer-mediated specific delivery of sirna improving the stability of aptamers by chemical modification influence of the -hydroxyl group conformation on the stability of a-form helices in rna oligonucleotide aptamers: new tools for targeted cancer therapy in vitro selection of functional nucleic acids aptamers overview: selection, features and applications single-stranded dna (ssdna) production in dna aptamer generation development of a novel dna aptamer ligand targeting to primary cultured tumor endothelial cells by a cell-based selex method dna aptamer evolved by cell-selex for recognition of prostate cancer development of an efficient targeted cell-selex procedure for dna aptamer reagents a two-step stimulus-response cell-selex method to generate a dna aptamer to recognize inflamed human aortic endothelial cells as a potential in vivo molecular probe for atherosclerosis plaque detection evolution of dna aptamers through in vitro metastatic-cell-based systematic evolution of ligands by exponential enrichment for metastatic cancer recognition and imaging in silico maturation of binding-specificity of dna aptamers against proteus mirabilis a novel protocol for generating high-affinity ssdna aptamers by using alternating magnetic fields magnetic-assisted rapid aptamer selection (maras) for generating high-affinity dna aptamer using rotating magnetic fields rapid one-step selection method for generating nucleic acid aptamers: development of a dna aptamer against alpha-bungarotoxin array-based evolution of dna aptamers allows modelling of an explicit sequence-fitness landscape single-round patterned dna library microarray aptamer lead identification the application of a modified nucleotide in aptamer selection: novel thrombin aptamers containing -( -pentynyl)- -deoxyuridine molecular evolution of functional nucleic acids with chemical modifications novel combinatorial selection of phosphorothioate oligonucleotide aptamers chemically modified nucleic acid aptamers for in vitro selections: evolving evolution effect of -end capping of aptamer with various , -bridged nucleotides: enzymatic post-modification toward a practical use of polyclonal aptamers application of locked nucleic acids to improve aptamer in vivo stability and targeting function locked nucleic acids: a promising molecular family for gene-function analysis and antisense drug development selection of lna-containing dna aptamers against recombinant human cd synthesis and properties of mirror-image dna simple peg modification of dna aptamer based on copper ion coordination for tumor targeting a multivalent dna aptamer specific for the b-cell receptor on human lymphoma and leukemia affinity analysis of dna aptamer-peptide interactions using gold nanoparticles aptamer-functionalized peg-plga nanoparticles for enhanced anti-glioma drug delivery selective delivery of an anticancer drug with aptamer-functionalized liposomes to breast cancer cells in vitro and in vivo dna aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells applications of aptasensors in clinical diagnostics in vitro selection of dna aptamers to anthrax spores with electrochemiluminescence detection dna aptamer selected against pancreatic ductal adenocarcinoma for in vivo imaging and clinical tissue recognition cell-selex based selection and characterization of dna aptamer recognizing human hepatocarcinoma molecular recognition of human liver cancer cells using dna aptamers generated via cell-selex cell-selex based selection and optimization of dna aptamers for specific recognition of human cholangiocarcinoma qbc- cells a cell-based single-stranded dna aptamer specifically targets gastric cancer a dna aptamer with high affinity and specificity for molecular recognition and targeting therapy of gastric cancer in vitro selection of dna aptamers for metastatic breast cancer cell recognition and tissue imaging selection of dna aptamers against glioblastoma cells with high affinity and specificity dna aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant iii in vitro selection of dna aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture probing high affinity sequences of dna aptamer against vegf selection of dna aptamers against vegf( ) using a protein competitor and the aptamer blotting method in vitro selection of dna aptamers to glioblastoma multiforme selection and characterization of dna aptamers for use in detection of avian influenza virus h n development of a fluorescent enzyme-linked dna aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies label-free detection of prion protein with its dna aptamer through the formation of t-hg +-t configuration application of a novel in vitro selection technique to isolate and characterise high affinity dna aptamers binding mammalian prion proteins structural basis for discriminatory recognition of plasmodium lactate dehydrogenase by a dna aptamer screening of dna aptamers against myoglobin using a positive and negative selection units integrated microfluidic chip and its biosensing application development of a dna aptamer for direct and selective homocysteine detection in human serum dna aptamer-based detection of prostate cancer dna aptamers against the muc tumour marker: design of aptamer-antibody sandwich elisa for the early diagnosis of epithelial tumours sensitive point-of-care monitoring of cardiac biomarker myoglobin using aptamer and ubiquitous personal glucose meter c-reactive protein, inflammation and coronary heart disease dna aptamer-based surface plasmon resonance sensing of human c-reactive protein homocysteine level and coronary heart disease incidence: a systematic review and meta-analysis thrombin, inflammation, and cardiovascular disease: an epidemiologic perspective aptamer binding assays for proteins: the thrombin example-a review design strategies for aptamer-based biosensors aptamer-based molecular recognition for biosensor development aptamer in bioanalytical applications aptamer-based biosensors for biomedical diagnostics structure-switching signaling aptamers structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling enzyme-linked small-molecule detection using split aptamer ligation target-induced conjunction of split aptamer fragments and assembly with a water-soluble conjugated polymer for improved protein detection an aptamer-based biosensor for sensitive thrombin detection the nucleolin targeting aptamer as destabilizes bcl- messenger rna in human breast cancer cells a phase ii trial of as (a novel nucleolin-targeted dna aptamer) in metastatic renal cell carcinoma randomized phase ii trial of the nucleolin targeting aptamer as combined with high-dose cytarabine in relapsed/refractory acute myeloid leukemia (aml) the von willebrand inhibitor arc reduces cerebral embolization after carotid endarterectomy: a randomized trial a dose ranging phase i/ii trial of the von willebrand factor inhibiting aptamer arc in patients with congenital thrombotic thrombocytopenic purpura emerging pharmacologic therapies for wet age-related macular degeneration as- , a guanosine-rich oligonucleotide aptamer targeting nucleolin for the potential treatment of cancer, including acute myeloid leukemia arc- , a pegylated aptamer antagonist of von willebrand factor for potential use as an anticoagulant or antithrombotic agent a dna aptamer prevents influenza infection by blocking the receptor binding region of the viral hemagglutinin dna-aptamer targeting vimentin for tumor therapy in vivo aptamer to erbb- /her enhances degradation of the target and inhibits tumorigenic growth remyelination induced by a dna aptamer in a mouse model of multiple sclerosis molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells automated modular synthesis of aptamer-drug conjugates for targeted drug delivery epirubicin loaded super paramagnetic iron oxide nanoparticle-aptamer bioconjugate for combined colon cancer therapy and imaging in vivo dimeric dna aptamer complexes for high-capacity-targeted drug delivery using ph-sensitive covalent linkages a controllable aptamer-based self-assembled dna dendrimer for high affinity targeting, bioimaging and drug delivery anticancer role of muc aptamer-mir- b chimera in epithelial ovarian carcinoma cells through regulation of pten methylation aptamer-conjugated nanomaterials and their applications aptamer-mediated targeted delivery of chemotherapeutic drugs and sirna for cancer therapy an as aptamer-conjugated liposomal system containing a bubble-generating agent for tumor-specific chemotherapy that overcomes multidrug resistance dna and aptamer stabilized gold nanoparticles for targeted delivery of anticancer therapeutics cell-type-specific, aptamer-functionalized agents for targeted disease therapy the authors would like to thank the nagasaki university for covering the cost to publish this paper in open access. the funder had no role in decision to publish and preparation of the manuscript.author contributions: mk, qz and gl conceived the review. qz and gl wrote the manuscript, and then mk revised the manuscript. the authors declare no conflict of interests. key: cord- -dfh b ln authors: mesch, gustavo s.; schwirian, kent p. title: social and political determinants of vaccine hesitancy: lessons learned from the h n pandemic of - date: - - journal: american journal of infection control doi: . /j.ajic. . . sha: doc_id: cord_uid: dfh b ln background public acceptance of vaccination programs is essential for vaccine preventable diseases. however, increasing sectors of the population have expressed hesitancy about participating in such programs, leading to the re-emergence of vaccine preventable diseases. in this study we rely on a recreancy hypothesis to test the association between confidence in the government and local hospitals and the willingness to take the vaccine. methods a secondary analysis of a survey that used a large sample of the u.s. population conducted in october was used (n = ). results the results indicate that . % of the respondents expressed willingness to be vaccinated. those with the greatest trust in the government were the most likely to be vaccinated ( . %), and those least confident were the least willing ( . %). from the ones reporting being confident in the local health system, . % were willing to be vaccinated, and from those not confident, only . % were willing to be vaccinated. conclusion the decision to get vaccinated in the midst of a contagious outbreak involves many considerations. trust in the government's technical and organization skill to deal with the infectious outbreak along with trust in medical organizations predict the adoption of recommended protection measures. the results indicate that public compliance with vaccination plans in health crisis requires the development of social and institutional trust. public acceptance of vaccination and collaboration with vaccination policymakers are essential for the prevention of contagious diseases. however, increasing sectors of the population have expressed hesitancy about participating in such programs. consequently, vaccination hesitancy has become a public health problem with social determinants. research has shown that in vaccination programs for preventable diseases, such as measles, mumps and rubella, varicella, and pertussis, a number of social factors have contributed to hesitancy. less is known about hesitancy during pandemic outbreaks. in contrast with programs for preventable diseases, pandemics provide a different context for vaccination hesitancy. during pandemics, the public usually lacks understandable, dependable, and timely information about the outbreak. social and institutional trust that provides legitimacy to policymakers' actions might be undermined because of lack of reliable and truthful information. often a new vaccine must be quickly manufactured and often may be accompanied by the misgivings of a portion of the population. furthermore, governments tend to become involved at all stages of the response to the pandemic, thereby opening the possibility that partisan politics may infringe on the scientific response to the outbreak. we examine social factors in relation to vaccine hesitancy: recreancy, perceived risk of infection, and political partisanship. recreancy is the concept used to describe the relationships among trust and risk. according to recreancy theory, the trust that individuals have in society's institutions is based on the perception of important considerations: the institution's competency to perform the tasks normally associated with it, and the institution's fiduciary responsibility in the sense that it is consciously working for the best interests of the population. in social risk situations, such as epidemics, trust in the government to respond effectively is important for the public to feel that their health interests will be maximally attended to. institutional recreancy is important at both the national and local levels and provides the basis for the legitimate exercise of state authority and the extent that is seen as entitled to be obeyed. the nature of public health, as a set of interventions requiring behavioral actions and lifestyle changes, requires legitimacy to achieve implementation of health policy. the local community may be viewed as a problem-solving system organized to improve the health of its residents and to be the main line of defense against contagions. the local health care system therefore comes under public scrutiny and is ultimately deemed to be positive or recreant. for this reason we distinguish between trust in the federal government's ability to fight outbreaks and trust in the local hospitals' ability. the health belief model is the most common model used to study health-related behaviors. a central component of the health belief model is the perceived threat of disease. , the basic model argues that people are likely to engage in disease prevention behaviors if they perceive that they are highly susceptible to the disease; that the disease has severe consequences; and that preventive behaviors such as vaccination are beneficial. , in recent years, an affective dimension (eg, fear, apprehension, worry, anxiety) has been added to the model and has proven to be a strong predictor of health behavior. h n studies have shown that perceived risk of infection is greater for older adults, women, and those that follow media reports about the outbreak. major health issues, problems, projects, and events, such as the outbreak of h n , are inherently political. cross-national comparative research has shown that the political agendas of governing political parties have not only direct effects on health through health care legislation but also indirect effects through policy and programs dealing with labor force and social welfare. in the united states the ideology of the democrats leans liberal and the ideology of the republicans leans conservative. the organization and delivery of health care are topics of serious disagreement between the parties. the republican party tends to favor less government involvement than the democratic party. although individual members of the parties do not necessarily agree with their party's position on all matters, their behavior and attitudes in many issues reflect their party's position. at issue here is if members of the major parties differ in terms of willingness to receive the h n vaccine. the purpose of the study is to investigate the relationships of recreancy, perceived risk of infection, and political partisanship in the public's vaccine hesitancy during the h n epidemic of - . we draw on the basic theoretical framework previously discussed. they are recreancy theory, which emphasizes the role of institutional trust at both the national and local level in health behavior decision-making; the health belief model, which brings together both the rational process of decision-making in health agency and the affective elements of things such as fear and worry; and the political partisanship perspective, which argues that all health care matters are inherently political, become differentially embedded in the philosophies of political parties, and form the responses of party members to specific matters. our first hypothesis is that those individuals who trust in the government's ability to deal with the h n outbreak were more likely to be willing to be vaccinated than those lacking such trust. our second hypothesis is that those individuals who trust in the ability of local hospitals and health agencies to deal with the h n outbreak were more likely to be willing to be vaccinated than those lacking such trust. our third hypothesis is that those individuals concerned with the possibility of h n infection were more willing to be vaccinated than those who were not concerned. our fourth hypothesis is that those individuals whose political partisanship supported the government's health care program (affordable care act) were more likely to be willing to be vaccinated than those not in support. in addition to these hypotheses we ask the following question: what factors are correlated with trust in the government's ability to deal with an epidemic outbreak? the data were gathered through a random representative survey sample of the u.s. population conducted in october as part of the abc news/washington post poll. the results from the full survey have a margin of sampling error of ae points. sampling and data were collected by tns of horsham, pennsylvania. the sample of participants included those contacted both by landlines and cell phones. the original data set is comparable with the u.s. population in terms of age and sex. there is a slight underrepresentation of ethnic minority groups and some over-representation of college graduates. to correct for these slight biases, a weighted procedure was used. the data were downloaded from the roper center for public opinion, and we conducted a secondary analysis of the responses of the participants (n ¼ ). the dependent variable was the willingness to take the flu vaccination. respondents were asked to indicate if they planned to get the swine flu vaccine this year. responses were coded as (yes) and (no). the independent variable recreancy was measured by questions. the first was confidence in the federal government's ability to respond effectively to an outbreak of swine flu in the united states. participants were asked to rank their degree of confidence on a -point likert scale with higher values expressing greater confidence. in the same manner, we measured confidence in local hospitals and health care systems on a -point scale. we measured the participants' perceived risk of infection with a question that asked them to indicate if they were concerned that they or someone in their immediate family might catch the h n virus known as swine flu. responses were coded dichotomously as (yes) and (no). political partisanship was measured by a question that asked the respondents to identify the political party with which they identified. there were categories: republican, democratic, and independent. the variables that served as controls included demographics that measured age ( categories), sex (measured dichotomously), presence of children in the home (children between months and years), level of education ( categories: less than high school, high school graduate, or college or graduate school), ethnicity ( categories: white, black, or hispanic), and social attitudesliberalism/conservatism ( categories: liberal, moderate, or conservative) ( table ) . the final sample included respondents. the average age of the respondents was . ae . years. women were slightly over-represented ( . %). children between months and years were in homes of . % of the sample members. in terms of education, . % reported having less than high school education, . % graduated from high school, and . % had college or graduate school education. in terms of race, . % reported being white, % were black, and . % were hispanics. regarding social attitudes, . % reported being liberals, . % reported being ideologically moderate, and . % described themselves as conservatives. a slight majority of the respondents ( . %) were concerned about the risk of flu infection. the democratic party had the largest percentage of respondent organized party identification ( . %), republicans had the smallest percentage ( . %), and political nonparty independents made up . %. overall, . % expressed trust in the government's ability to deal with the h n outbreak, but trust in the local hospitals was higher and reached . %. as table illustrates, for the entire sample, . % expressed willingness to be vaccinated. this willingness varies according to the independent variables. the level of significance for testing the null hypotheses is p < . , but we report the calculated value. perceived risk of infection was associated with the willingness to take the flu vaccine (p < . ). as concern about possible infection increased, so too did the willingness to be vaccinated. of those who were very concerned, . % were willing to become vaccinated, whereas only . % of those not concerned at all were willing to do so. political partisanship had a significant effect on the willingness to become vaccinated (p < . ). democrats ( . %) were more willing to be vaccinated than either republicans ( . %) or independents ( . %). the multiple logistic regressions that follow compare democrats with republicans. at the same time, although this willingness varied based on political partisanship, it did not vary based on social attitudes. the differences in the percentages of individuals holding liberal, moderate, and conservative attitudes in terms of vaccination willingness were statistically nonsignificant (p < . ). trust in government was statistically significant in willingness to be vaccinated (p < . ). those with the greatest trust in the government were the most willing to be vaccinated ( . %), and those least confident in the government were the least willing ( . %). similarly, trust in the local hospital and health care system made a statistically significant difference as well (p < . ). for those very confident in the local system, . % were willing to be vaccinated, whereas only . % of those who were not confident were willing to be vaccinated. of the demographic variables, only age was statistically significant (p < . ), with those > years of age ( . %) being the most willing to be vaccinated, and the least willing ( . %) was among the youngest, those through years of age. willing to be vaccinated: multivariate analysis table shows results for the multiple logistic regression analysis. the results show that of the net of other factors, several variables were significantly related to the willingness to be vaccinated. those who trust the government were more willing to get vaccinated than those lacking such trust (adjusted odds ratio [aor] ¼ . , p < . ). furthermore, those who trusted the local health care system were also more willing to get vaccinated than those who had little trust in the system (aor ¼ . , p < . ). the data also showed that those concerned about contracting the flu were more willing to become vaccinated than those who were not worried about contracting the flu (aor ¼ . , p < . ). similarly, the odds of republicans taking the vaccine were less than those of democrats (aor ¼ . , p < . ). with trust, fear, and partisanship taken into account, older respondents were more willing to be vaccinated (aor ¼ . , p < . ), and whites (aor ¼ . , p < . ) and hispanics (aor ¼ . , p < . ) were more willing to be vaccinated than blacks. table presents the results of the logistic regression conducted to determine the relationship between the variables and trust in the governments' ability to deal with the swine flu outbreak. people who trusted the local health care system were more likely to trust the government than were those who did not trust the local system (aor ¼ . , p < . ). republicans were less trusting than democrats (aor ¼ . , p < . ). those concerned about being infected with the flu were more trusting of the government's ability than those less concerned about infection (aor ¼ . , p < . ). in addition, the regression showed that older people were more likely than young people to trust the government (aor ¼ . , p < . ). hispanics were more likely than blacks (aor ¼ . , p < . ) or whites (aor ¼ . , p > . ) to trust the government's ability to deal with the swine flu crisis. in this study of vaccination resistance during the h n epidemic, the hypotheses based on theoretical models were supported by the data. the models are as follows: recreancy theory, health belief model, and political partisanship. several factors correlated with trust in government also were identified. as with all studies, our research suffers from several limitations, one of which is that it deals with the outbreak of a single disease. was the fear associated with h n generated by the rate of mortality and rapidity of the spread of the disease that made it seem more virulent than other outbreaks such as severe acute respiratory syndrome? would different outbreaks produce different results? given the fact that viral outbreaks appear somewhere in the world much of the time, we have additional opportunities to test the recreancy hypothesis in different cultural settings. investigations of these cases will help us determine whether vaccination is truly the most important public health development in recent years. at this point, the findings suggest several lessons that may be learned from the h n outbreak of : . trust: trust in the national government's ability to deal with an epidemic outbreak is important to a segment of the population in its willingness to be vaccinated. trust extends to local community health care organizations as well. . political partisanship: to a significant degree, trust in the government's ability to deal with the h n outbreak is based on political partisan attitudes about the proper role of government; members of political parties endorsing social health care programs are more willing to be vaccinated than others. . vulnerability: age, family composition, and ethnicity are also factors influencing willingness to be vaccinated. collectively, they point to a perceived vulnerability factor on the part of many that may be operating in the decision to seek vaccination. getting the flu is significant in willingness to be vaccinated, the fear, the more willing to be vaccinated. the decision to seek vaccination for a serious disease is clearly complex. we suggest that epidemics present situations that differ from the normal context of vaccination decision-making and are therefore worthy of additional investigation. future studies should build on this study and expand the study of the role of social trust in vaccination to the sources of such social and institutional trust. for example, an important topic for future study is the role of media content in trust in the ability of the government and local hospitals to deal with a health crisis and the role of media content on trust to vaccines. the most salient result of our study is that in , during the h n outbreak, only . % of the sample expressed willingness to get the vaccine that was developed to cope with the health crisis. a large percentage of the sample expressed hesitancy to be vaccinated. our study was directed at understanding the sources of this hesitancy and indicated that increasing the likelihood of the population to participate in vaccination programs requires interventions directed at increasing the trust of the population in the government public health capabilities and the role of the local hospitals. a development of a comprehensive policy to increase recreancy in situations of real social risk is needed. declining vaccination rates. contemporary pediatrics public health: an injection of trust what does the public know about ebola? the public's risk perception regarding the current ebola outbreak in a yet unaffected country the political ecology of plague in the global network of cities: the sars epidemic of risk and recreancy: weber, the division of labor, and the rationality of risk perceptions the perception of risk community ecology: a new theory and an illustrative test the health belief model the health belief model and personal health behavior health behavior and health education: theory, research and practice behavioral change in influenza vaccination; factors influencing increased uptake of the pandemic h n versus seasonal influenza vaccine in health care personnel emotions and preventive health behavior: worry, regret, and influenza vaccination the politics of public health politics and health outcomes political polarization in the american public we thank the roper institute for providing us with the data on which this study is based. key: cord- -xihpfidg authors: ford, julian d.; grasso, damion j.; elhai, jon d.; courtois, christine a. title: social, cultural, and other diversity issues in the traumatic stress field date: - - journal: posttraumatic stress disorder doi: . /b - - - - . -x sha: doc_id: cord_uid: xihpfidg this chapter describes how the impact of psychological trauma and posttraumatic stress disorder (ptsd) differ, depending on individual differences and the social and cultural context and culture-specific teachings and resources available to individuals, families, and communities. a social-ecological framework is used to differentiate the impact of exposure to traumatic stressors and the development of (or resistance to) ptsd, based on the individual’s or group’s (i) personal, unique physical characteristics, including skin color, racial background, gender, and sexual orientation; and (ii) family, ethnocultural, and community membership, including majority or minority group status, religious beliefs and practices, socioeconomic resources, and political and civic affiliations. while personal, familial, social, and cultural factors can be a positive resource contributing to safety and well-being, they also can be a basis for placing the person, group, or entire community or population in harm’s way or at heightened risk of developing ptsd. this is an adaptive response in one sense, providing an awareness and readiness to respond should the genocide or any associated forms of stigma, discrimination, or violence ever recur with impunity. however, it can also become a form of persistent hypervigilance similar to that seen in ptsd, placing a strain upon the individual's or group's daily life that may compromise their well-being. our discussion of the impact of exposure to traumatic stressors and ptsd on ethnoracial groups and individuals whose forebears have experienced historical trauma will bear this fact in mind. in addition, gender-based biases and beliefs, many of which are based on longstanding religious and cultural traditions, have caused women to be systematically discriminated against and subject to routine physical and sexual assault. genderbased discrimination and violence against females (whether intra-or extrafamilial) have been so widespread as to be implicated in what kristof and wudunn ( ) term "gendercide." in their recent book, they cite examples of selective abortions based on a fetus's gender and differential nutrition and care beginning in infancy also based on gender preference. they are then often followed by lifelong major disparities in education and restricted role and career opportunities for females as compared to males. unfortunately, even today, with all the advances that have occurred predominantly in western societies, these same issues remain in place around the world. the increased recognition of the underclass status of the majority of women and girls and the discrimination they face, along with the violence perpetrated against them (often seemingly with impunity), in countries around the world (whether industrialized and "advanced," or relatively primitive) has led to the recent development of major initiatives against global violence and discrimination against women. malala yousafzai, who was shot by the taliban for her espousal of universal education for girls, was awarded the nobel peace prize, the youngest recipient to date. the brutality of the attack against her was shocking, yet it served to highlight the traumatic threat to which many girls and women across the world are exposed when targeted for hateful acts by those who believe this is necessary to maintain the status quo and the subservience of females. discrimination and violence based on sexual orientation and transgender/intergender status are yet other sources of traumatic victimization that must be well recognized. sexual orientation is both a personal and social characteristic that is more complex than the gender that a person inherits based on inborn sexual characteristics. when socially ascribed gender and culturally promulgated expectations for gender-based activities, such as mating, are a mismatch to an individual's sense of his or her own true sexual preferences and identity, the conflict can be psychologically devastating. global initiatives therefore are underway to prevent or ameliorate the adverse impact of discrimination, stigma, and violence based on sexual orientation and identity providing an essential foundation for the basic liberties, freedom from assault, and the right to marry to gay, lesbian, bisexual, and transgendered (glbt) individuals. it should also be noted that boys and men are also subject to abuse and assault at rates that are not yet adequately researched. males may be more subject to violence when they are in a position of vulnerability of some sort due to being a member of a group that is targeted and/or of lesser status/lesser strength. depending on social, cultural, and other diversity issues in the traumatic stress field their cultural background and its traditions and beliefs, individuals may also have "multiple vulnerability status"-that is, to be members of more than one group or to have characteristic that cause them to be even more susceptible to discrimination or victimization (i.e., adolescent black male in the united states; a baby born with physical or developmental disabilities in a culture that endorses selective resources to the ablebodied; a gay man or lesbian woman of color in a highly homophobic and racist society). age is yet another vulnerability factor dimension that has not received adequate recognition, with individuals at either end of the life span as most vulnerable. research has substantiated that children and adolescents are the most at-risk segment of the population globally (finkelhor, ) . victimization of the elderly and the lessabled/disabled members of the population is now documented as widespread in many societies and is increasingly under investigation. like other forms of abuse, victimization of the elderly and less-abled is often based on the victim's relative degree of dependence and his powerlessness to defend himself. the extent and impact of exposure to traumatic stressors experienced by each of these vulnerable populations is discussed in this chapter, as are the efforts of international non-governmental organizations (ngos) to provide them with resources to reduce their exposure to traumatic stressors or to mitigate the adverse effects of traumatic exposure and ptsd (box . ). box . key points . culture, ethnicity, gender, sexual orientation, and disability are potential sources of resilience, but they also may lead to chronic stressors such as social stigma, discrimination, and oppression, which can increase psychological trauma and ptsd. . cumulative adversities are faced by many persons, communities, ethnocultural minority groups, and societies that may lead to-as well as worsen the impact of-ptsd: • persons of ethnoracial minority backgrounds; • persons discriminated against due to gender or sexual orientation; • persons with developmental or physical disabilities; • economically impoverished persons and groups, including the homeless; • victims of political repression, genocide, "ethnic cleansing," or torture; • persons chronically or permanently displaced from their homes and communities due to catastrophic armed conflicts or disasters. . members of ethnoracial minority groups have been found to be more likely in some cases to develop ptsd than other persons, but in other cases they are less likely to develop ptsd (e.g., persons of asian or african descent). . members of ethnoracial minority groups often encounter disparities in access to social, educational, economic, and health care resources; it is these disparities that are the most likely source of the increased vulnerability of these persons to psychological trauma and ptsd. (continued ) to psychological trauma in the immediate or most distant past, or both. psychological trauma and ptsd occur across the full spectrum of gender, racial, ethnic, and cultural groups in the united states (pole, gone, & kulkarni, ) . psychological trauma and ptsd are epidemic internationally as well, particularly for ethnoracial minority groups (which include a much broader range of ethnicities and cultures and manifestations of ptsd than typically recognized in studies of ptsd in the united states; de jong, komproe, spinazzola, van der kolk, & van ommeren, ; de jong, komproe, & van ommeren, ) . the scientific and clinical study of ptsd and its treatment among gender and ethnoracial majority and minority groups is of great importance, especially given the disparities, adversities, and traumas to which they have been subjected historically (miranda, mcguire, williams, & wang, )-and to which they are still exposed in health and health care, education and income, and adult criminal and juvenile justice (ford, ) . although latinos (and possibly african americans) persons are at greater risk than european americans for ptsd based on available research findings (pole, gone, & kulkarni, ) , it is possible that the elevated prevalence may be due to differences in the extent or types of exposure to psychological trauma (including prior traumas that often are not assessed in ptsd clinical or epidemiological studies; eisenman, gelberg, liu, & shapiro, ) or to differences in exposure to other risk or protective factors such as poverty, education, or gender-based violence (turner & lloyd, , . in addition, there is sufficient diversity (in norms, beliefs, values, roles, practices, language, and history) within categorical ethnocultural groups such as african americans or latinos to call into question any sweeping generalizations about their exposure and vulnerability or resilience to psychological trauma (pole et al., ) . race, ethnicity, gender and sexual identity, and culture tend to be described with shorthand labels that appear to distinguish homogeneous subgroups but that actually obscure the true heterogeneity within as well as between different groups (marsella, friedman, gerrity, & scurfield, ) . one partial antidote for this problem is for clinicians and researchers to be curious about these issues and to ask study participants or clinical patients to self-identify their own racial, ethnic, and cultural background and to essentially educate them about their unique characteristics and associated belief systems and traditions (brown, ; brown, hitlin, & elder, ) . it also is important to carefully assess factors that are associated with differential exposure to adverse experiences (such as racial-ethnic discrimination) or differential access to protective resources (such as income, health care, education, police protection), rather than assuming that each member of an ethnocultural group is identical on these crucial dimensions. however, when systematic disparities in exposure to stressors or deprivation of resources are identified for specific groups, such as persons from indigenous culturesthe original inhabitants of a geographic area who have been displaced or marginalized by colonizing national/cultural groups-are found to have a generally increased risk of discrimination, poverty, addiction, family violence, and poor health (harris et al., ; liberato, pomeroy, & fennell, ) , it is crucial not to mistakenly conclude that those persons are less resilient than others when they are confronted with traumatic stressors. commonly, the very opposite is true: persons and groups who are subjected to chronic stressors or deprivations tend to be more resilient than others, but they also are more exposed to and less protected from traumatic stressors (pole et al., ) . racism and associated discrimination and mistreatment are particularly chronic stressors faced by many members of ethnoracial and other minority groups. racism may constitute a form of psychological trauma in and of itself, increasing the risk of exposure to psychological trauma, and exacerbating its impact by increasing the risk of ptsd (ford, ) . as of yet, few systematic studies have directly examined racism as a risk factor for exposure to psychological trauma, although the connection is increasingly recognized (carter & forsyth, ; hunter & schmidt, ; miller, ) . perhaps, the holocaust and other forms of genocide have been the most investigated to date. studies of survivors of the holocaust and other types of ethnic annihilation provide particularly graphic and tragic evidence of the infliction of psychological trauma en masse in the name of racism (staub, ; yule, ) . studies are needed that systematically compare persons and groups who are exposed to different types and degrees of racism in order to test whether (and under what conditions) racism is a form of, or leads to exposure to other types of, traumatic stressors (ford, ) . when racism leads to the profiling and targeting of ethnoracial minority groups for violence, dispossession, dislocation, or annihilation, the risk of ptsd increases in proportion to type and degree of the traumatization involved (pole et al., ) . for example, studies based in the united states (pole et al., ) and internationally (macdonald, chamberlain, & long, ) suggest that racial discrimination may have played a role in placing military personnel from ethnoracial minority groups at risk for more extensive and severe combat trauma exposure. one study found that self-reported experiences of racial discrimination increased the risk of ptsd among latino and african american police officers (pole, best, metzler, & marmar, ) . another study with asian american military veterans from the vietnam war era showed that exposure to multiple race-related stressors that met ptsd criteria for psychological trauma was associated with more severe ptsd than when only one or no such race-related traumas were reported (loo, fairbank, & chemtob, ) . this study more precisely operationalized racism than any prior study, utilizing two psychometrically validated measures of race-related stressors and ptsd. however, as in the pole et al. ( ) study, the stressors/traumas and ptsd symptoms were assessed by contemporaneous self-report, so the actual extent of racism experienced by the participants cannot be definitely determined. the loo et al. ( ) study also did not control for traumatic stressor exposure other than that which was related to racism. in order to extend the valuable work these studies have begun, it will be important to utilize measures based on operationally specific criteria for categorizing and quantifying exposure to discrimination (e.g., wiking, johansson, & sundquist, ) as a distinct class of stressors that can be assessed separately as well as concurrently with exposure to psychological trauma. research also is needed to determine to what extent the adverse outcomes of racial disparities are the direct result of racism as a stressor (e.g., racially motivated stigmatization, mistreatment, subjugation, and deprivation resulting in personal and community depression and destabilization), as opposed to the indirect effects of racism (such as microaggressions that accrue over time). racism can also indirectly reduce access to protective factors (adequate nutrition and other socioeconomic and community resources) that protect against the adverse effects of stressors (such as poverty, pollution, disaster) and traumatic stressors (such as accidents, crime, or violence). hurricane katrina and its aftermath provided just such an example. it is important to determine whether ptsd is the product of either the direct or indirect effects of racism, or both, particularly given its demonstrated association with other psychiatric conditions (such as depression, anxiety, and addiction) and with increased risk of physical illness (such as cancer and cardiovascular disease) in ethnoracial minorities (e.g., among american indians; sawchuk et al., ) . education is a particularly relevant example of a socioeconomic resource to which ethnoracial minorities often have restricted access as that as a protective factor mitigating against the risk of ptsd (dirkzwager, bramsen, & van der ploeg, ) and overall health status (wiking et al., ) . racial disparities in access to education are due both to direct influences (such as lower funding for inner-city schools that disproportionately serve minority students) and indirect associations with other racial disparities (such as disproportionate juvenile and criminal justice confinement of ethnoracial minority persons). racial disparities in education are both the product of and a contributor to reduced access by minorities to other socioeconomic and health resources (such as income, health insurance, adequate nutrition) (harris et al., ) . when investigating risk and protective factors for ptsd, it is essential therefore to consider race and ethnicity in the context not only of ethnocultural identity and group membership but also of racism and other sources of racial disparities in access to socioeconomic resources. although all ethnoracial minority groups tend to be disproportionately disadvantaged with regard to the more privileged majority population, particularly severe disparities in access to vital resources often are complicated by exposure to pervasive (both intrafamilial and community) violence and by the loss of ties to family, home, and community. when family and community relationships are severed-as occurs with massive political upheaval, war, genocide, slavery, colonization, or catastrophic disasters-racial and ethnocultural groups may find themselves scattered and subject to further victimization and exploitation. for example, there continue to be massively displaced populations in central and south america, the balkans, central asia, and africa. when primary social ties are cut or diminished as a result of disaster, violence, or political repression, the challenge expands beyond survival of traumatic life-threatening danger to preserving a viable life, community, and culture in the face of lifealtering losses and suppression of those very factors needed to maintain (garbarino & kostelny, ; rabalais, ruggiero, & scotti, ) . ethnoracial groups that have been able to preserve or regenerate core elements of their original cultural norms, practices, and relationships within intact or reconstituted families may actually be particularly resilient to traumatic stressors and protected against the development of ptsd. for example, persons of asian or african descent have been found to be less likely than those of other ethnocultural backgrounds to develop ptsd. whether this is due to factors other than ethnicity per se, such as having cultural practices and beliefs that sustain family integrity and social ties, is a question that has not been scientifically studied and should be a focus for research (pole et al., ) . a recurring theme is that the psychological trauma inflicted in service of racial discrimination may lead not only to ptsd but also to a range of insidious psychosocial problems that result from adverse effects upon the psychobiological development of the affected persons. when families and entire communities are destroyed or displaced, the impact on the psychobiological development of children and young adults may lead to complex forms of ptsd that involve not only persistent fear and anxiety but also core problems with relatedness and self-regulation of emotion, consciousness, and bodily health that are described as "complex ptsd" (herman, ) or "disorders of extreme stress" (de jong et al., ) . a critical question not yet answered by studies of ptsd and racial discrimination (pole et al., ) and race-related stress (loo et al., ) , as well as by the robust literature that shows evidence of intergenerational transmission of risk for ptsd (kellerman, ) , is whether racism constitutes a "hidden" (crenshaw & hardy, ) or "invisible" (franklin, boyd-franklin, & kelly, ) form of traumatization that may be transmitted across generations. recent research findings demonstrating highly adverse effects of emotional abuse in childhood (teicher, samson, polcari, & mcgreenery, ) are consistent with a view that chronic denigration, shaming, demoralization, and coercion may constitute a risk factor for severe ptsd and associated psychobiological problems. research is needed to better describe how emotional violence or abuse related to racism may (along with physical violence) constitute a form of traumatic stress and how this may adversely affect not only current but also future generations. a fully articulated conceptual model for the scientific study and social/clinical prevention and treatment of the adverse impact of psychological trauma and ptsd requires principles and practices informed by this diversity of factors, rather than a "black and white" view of race, ethnicity, or culture that misrepresents the individual's and group's heritage, nature, and needs. treatment preferences, in terms of characteristics of the therapist as well as the therapy model, differ substantially not only across but also within ethnoracial groups (pole et al., ) . as a result, it is not possible as yet-and may never be possible-to precisely prescribe how best to select or train therapists and design or adapt therapies to fit different ethnocultural groups and the individuals within them. a culturally competent (brown, (brown, , a (brown, , b approach to treating ptsd (ford, ) begins with a collaborative discussion in which the therapist adopts the stance of a respectful visitor to the client's outer and inner world-clarifying the client's expectations and preferences, and the meaning of sensitive interpersonal communication modalities (such as spatial proximity, gaze, choice of names, private versus public topics, synchronizing of talk and listening, use of colloquialisms, providing advice or education). ptsd therapists thus must avoid stereotypic assumptions and become both a host and guest in the client's psychic world in order to ensure that assessment and treatment are genuinely collaborative and sensitive to each client's ethnocultural traditions, expectations, goals, and preferences (parson, ; stuart, ) . at times, it is helpful to involve other members of the family or culture in assisting with the treatment. religious beliefs and spirituality are other dimensions of culture that have not yet been given sufficient focus in most psychotherapy but must also be assessed and understood by the therapist (walker, courtois, & aten, ) . cultural competence means many things to many people, and unfortunately, it is often mistakenly equated with being of the same racial, ethnic, cultural, religious, or national background as the persons involved in a study or receiving services, or knowing in advance exactly what each person believes and expects, how they communicate with and are most receptive to learning from others, and what their experience has been in relation to sensitive matters such as psychological trauma or ptsd. this is likely to be a serious mistake for several reasons. sharing some general racial, ethnic, cultural, or national features (or an apparently identical language or religion) is not synonymous with shared identity, knowledge, or history. even persons from as virtually identical backgrounds as monozygotic twins raised in the same family have substantial differences in physical and temperamental characteristics as well as often quite distinct social learning histories, and thus rarely if ever can reliably read one another's minds or exactly know one another's vulnerabilities and strengths. therefore, cultural competence should not be defined in terms of stereotypic assumptions about identity or prescience but instead based upon a respectful interest in learning from each person and community what they have experienced and how they understand and are affected by psychological trauma and ptsd. we should also note that the idioms of distress can differ by culture and tradition. professionals from industrialized nations and anglo cultures must be cautious and respectful in working with individuals and communities from other cultures that are challenged by ptsd in the aftermath of exposure to violence or disasters. before offering or providing education or therapeutic assistance, it is essential to become aware of how the potential recipients understand and prefer to communicate about traumatic stress and the process of healing from traumatization. the implication for psychometric assessment of psychological trauma and ptsd with clients of ethnocultural minority groups (hall, ; marsella et al., ) is that it is essential to carefully select protocols that do not confront individuals with questions that are inadvertently disrespectful of their values or practices (e.g., including peyote as an example of an illicit drugs in a native american tribe that uses it for religious rituals), irrelevant (e.g., distinguishing blood family from close friends in a group that considers all community members as family), or incomplete (e.g., limiting health care to western medical or therapeutic services, to the exclusion of traditional forms of healing). a systematic assessment of trauma history and ptsd thus should include not only a recitation of events in a person's life and symptomatic or resilient responses in the aftermath but how the person interpreted these events and reactions based on their cultural framework, beliefs, and values (manson, ) . interventions for prevention or treatment of ptsd typically have been developed within the context of the western medical model (parson, ; but see andres-hyman, ortiz, anez, paris, & davidson, ; hinton et al., ; hwang, , for examples of culturally sensitive adaptations). evidence-based ptsd treatment models are not necessarily incompatible with culturally specific healing practices and have in common the goal of fostering not just symptom reduction but a bolstering of resilience and mastery (see chapters and ). the integration of culturally specific methods and rituals in prevention or treatment interventions for ptsd, however, requires careful ethnographic study (i.e., observing and learning about the values, norms, beliefs, and practices endorsed and enforced by different cultural subgroups and their particular idioms (ways of describing and explaining) traumatic stress and ptsd) so the ptsd clinician and researcher can truly work collaboratively withrather than imposing external assumptions and standards upon-the members of the wide range of ethnic and cultural communities. in most cultures, girls and women are subject to discrimination in the form of limitations on their access to crucial socioeconomic resources. women earn - % lower wages or salaries than men in most job classes in the united states (http://www.payequity.org/info.html) and europe (http://www.eurofound.europa.eu/ewco/ / / es i.htm). although girls and women are approaching parity with boys and men in access to education in most areas of the world (and exceed the enrollment of boys or men in secondary and college/university education), in sub-saharan africa and asia, women and girls are as much as % less likely to be able to enroll in education and to have achieved literacy as adults (http://www.uis.unesco.org/template/ pdf/educgeneral/uisfactsheet_ _no% _en.pdf). girls and women also may be systematically subjected to extreme forms of psychological and physical trauma as a result of their gender being equated with second-class citizenship and social norms that permit or even encourage exploitation. sexual exploitation of women and girls is an international epidemic, including abuse and molestation, harassment, rape and punishment of rape victims, forced marriage, genital mutilation, and sex trafficking or slavery (box . ). physical abuse or assault of women and girls is tolerated-and in some cases actually prescribed as a form of social control-in both mainstream cultures and subcultures that span the globe and include most religions and developed as well as developing or preindustrial societies. similar potentially traumatic forms of violence are directed at many glbt persons as a result of both formal and informal forms of social stigma and discrimination. epidemiological studies have been conducted with samples of glbt youth (d'augelli, grossman, & starks, ) and adults (herek, ) in the united states, suggesting that they are often subjected to potentially traumatic violence as a result of their nontraditional sexual orientation and behavior. instances of violence specifically related to sexual orientation include: • - % of gay men and % of lesbians who were physically assaulted in the past year; • - % of glb adults who were subjected to actual or threatened violence toward their person or a property crime at some point in their lives; • - % of glb adolescents reported past incidents of physical or sexual violence. in contrast to the general pattern of stigma-related violence being directed toward girls and women, gay and bisexual boys (d'augelli et al., ) and men (herek, ) were more likely to report violent victimization or threats than lesbian or bisexual women or girls. the findings from the survey of glb adolescents suggest that stigma and victimization begin early in life, with physical and sexual attacks occurring as early as ages - years old. one in eleven glb adolescents met criteria for ptsd, box . "making the harm visible": sexual exploitation of women and girls women from every world region report that the sexual exploitation of women and girls is increasing. all over the world, brothels and prostitution rings exist underground on a small scale, and on an increasingly larger scale, entire sections of cities are informally zoned into brothels, bars, and clubs that house, and often enslave, women for the purposes of prostitution. the magnitude and violence of these practices of sexual exploitation constitute an international human rights crisis of contemporary slavery. in prostitution: a form of modern slavery, dorchen leidholdt, the coexecutive director of the coalition against trafficking in women, examines the definitions of slavery and shows how prostitution, and related forms of sexual exploitation, fit into defined forms of slavery. in some parts of the world, such as the philippines, prostitution is illegal but well entrenched from providing "recreational services" to military personnel. in "blazing trails, confronting challenges: the sexual exploitation of women and girls in the philippines," aida f. santos describes the harmful conditions for women and girls in prostitution in the philippines, with problems related to health, violence, the legal system, and services. in other regions, such as northern norway, organized prostitution is a more recent problem, stemming from the economic crisis in russia. in "russian women in norway," asta beate håland describes how an entire community is being transformed by the trafficking of women for prostitution from russia to campgrounds and villages across the border in norway. political changes combined with economic crises have devastated entire world regions, increasing the supply of vulnerable women willing to risk their lives to earn money for themselves and their families. aurora javate de dios, president of the coalition against trafficking in women, discusses the impact of the southeast asian economic crisis on women's lives in "confronting trafficking, prostitution and sexual exploitation: the struggle for survival and dignity." economic globalization controlled by a handful of multinational corporations located in a few industrialized countries continues to shift wealth from poorer to richer countries. in her paper "globalization, human rights and sexual exploitation," aida f. santos shows us the connection between global economics and the commodification and sexual exploitation of women and girls, especially in the philippines. structural adjustment programs implemented by international financial institutions impose loan repayment plans on poor countries, which sacrifice social and educational programs in order to service their debt to rich nations and banks. fatoumata sire diakite points to structural adjustment programs as one of the factors contributing to poverty and sexual exploitation in her paper "prostitution in mali." zoraida ramirez rodriguez writes in "report on latin america" that the foreign debt and policies of the international monetary fund are primary factors in creating poverty for women and children. these forces leave women with few options, increasing the supply of women vulnerable to recruitment into bride trafficking and the prostitution industry. (continued ) social problems such as sexual and physical abuse within families force girls and women to leave in search of safety and a better life, but often they find more exploitation and violence. physical and sexual abuse of girls and women in their families and by intimate partners destroys girls' and women's sense of self and resiliency, making them easy targets for pimps and traffickers who prey on those who have few options left to them. these factors are evident in many of the papers from all world regions in this volume, such as jill leighton and katherine depasquale's, "a commitment to living," and martha daguno's, "support groups for survivors of the prostitution industry in manila." government policies and practices also fuel the demand for prostitution, as they legalize prostitution or refuse to enforce laws against pimps, traffickers, and male buyers. in making the harm visible, we see how countries with governmental structures and ideological foundations as different as the netherlands and iran, both promote and legalize sexual violence and exploitation of girls and women. in "legalizing pimping, dutch style," marie-victoire louis exposes the liberal laws and policies that legalize prostitution and tolerate brothels in the netherlands. at the other extreme, religious fundamentalists in iran have legalized the sexual exploitation of girls and women in child and temporary marriages and the sexual torture of women in prison. sarvnaz chitsaz and soona samsami document this harm and violation of human rights in "iranian women and girls: victims of exploitation and violence." global media and communication tools, such as the internet, make access to pornography, catalogs of mail-order brides, advertisements for prostitution tours, and information on where and how to buy women and girls in prostitution widely available. this open advertisement normalizes and increases the demand by men for women and girls to use in these different forms of exploitation. donna m. hughes describes her findings on how the internet is being used to promote the sexual exploitation of women and children in "the internet and the global prostitution industry." in this milieu, women and girls become commoditiesbought and sold locally and trafficked from one part of the world to another. how do we make the harm of sexual exploitation visible? in a world where sexual exploitation is increasingly normalized and industrialized, what is needed to make people see the harm and act to stop it? the women in making the harm visible recommend four ways to make the harm of sexual exploitation visible: listen to the experiences of survivors, expose the ideological constructions that hide the harm, expose the agents that profit from the sexual exploitation of women and children, and document harm and conduct research that reveals the harm and offers findings that can be used for policy initiatives. reprinted with permission from the introduction to making the harm visible, edited by d. hughes - times the prevalence of children (copeland, keeler, angold, & costello, ) and adolescents (kilpatrick et al., ) in national samples in the united states. although gender and sexual orientation may seem intuitively to be simpler phenomena than race or ethnicity, in reality they are quite complex in terms of referring to not just biological characteristics but many aspects of psychological identity and social affiliations. being a female or a male, let alone gay, lesbian, bisexual, or transgendered/ intergendered, means many different things to different people. although more stable than changeable, sexual orientation and even gender may be changed for the same person over time. it is inaccurate to assume that all or even most people of a given gender or sexual orientation are identical or even similar without careful and objective assessment of how they view themselves and how they actually act, think, and feel. in relationship to psychological trauma and ptsd, therefore, the broad generalizations that have been suggested by research concerning gender and sexual orientation relate more to the way in which people of a gender or sexual orientation are generally viewed and treated (which varies, depending on the society and culture) than to inherent qualities of a given gender or sexual orientation (which is highly individual across all societies and cultures). the finding that girls and women are more often subjected to sexual and intrafamilial traumatic stressors, while boys and men more often experience physical, accidental, combat, and assaultive traumatic stressors is consistent with stereotypic sex roles that are found in many (but not all) cultures that assign females to the role of subservient helper and caregiver, while males are assigned to the role of leader and warrior. there are biological foundations for these differences-such as due to distinct levels of the sex-linked hormones estrogen and testosterone, and brain chemicals that differentially affect females and males (oxytocin and vasopressin; see chapter ). however, biology need not dictate a person's or a group's destiny, so it is inaccurate to assume that males or females must always fill these sex role stereotypes, particularly when there are severe adverse consequences, such as the epidemics of abuse of girls and women and of boys and men killed as violent combatants or as the "spoils of war." stereotypes can be even more insidious and damaging in relation to sexual orientation. only in the past decades has homosexuality been rescued from the status of a psychiatric disorder (as it was in the first three editions of the diagnostic and statistical manual). stigma and harassment evidently are still experienced, potentially with traumatic results when violent acts are tolerated or even encouraged, by glbt adults and youth. it is not surprising that the prevalence of ptsd is greater among persons with other than heterosexual sexual identities, and the extent to which this is the result of the pernicious stigma directed at such individuals in most cultures or of outright traumatic violence, or both, remains to be tested. persons with physical or developmental disabilities are another group of persons who unfortunately may be subjected to stigma and discrimination. physical disabilities are more common in developing countries than in more industrialized and affluent nations in which medical technology and accident and illness prevention have reduced the risk of severe injury or genetically based physical disabilities (mueser, hiday, goodman, & valentini-hein, ) . persons with physical disabilities may be at risk for exposure to traumatic accidents or maltreatment as children and as adults due to limitations in their abilities to care for themselves and live independently, particularly if they have cognitive impairment due to conditions such as mental retardation or serious mental illness. only one study that examined the prevalence of exposure to potentially traumatic events among physically disabled persons could be located. that was a national survey of women with physical disabilities by the center for research on women with disabilities (nosek, howland, & young, ) . on the one hand, the study found that disabled women were no more likely to report exposure to physical or sexual abuse than women without physical disabilities. however, in more detailed interviews with a subsample, more than % reported instances of abuse, on average two incidents per woman (each often lasting for a lengthy time period). for example, the report provides verbatim quotations: more than half of all respondents ( % with disabilities, % with no disability) reported a history of either physical or sexual abuse, or both, which is a substantially higher prevalence than that reported in epidemiological surveys of nationally representative samples of women. notably, women with disabilities were more likely than women without disability to report emotional abuse from a caregiver or family member and to have experienced all forms of abuse for a longer time period than women without disability. although ptsd was not assessed, women younger than years old with spina bifida ( %), amputation, traumatic brain injury (tbi), or multiple sclerosis (> %) were highly likely to be diagnosed with depression than women with no disability. in light of the extensive histories of potentially traumatic abuse and of depression, it appears that women with physical disabilities-particularly those in early to midlife adulthood with disabilities that involve progressive deterioration or mental or psychological disfiguration-may be at risk for having experienced traumatic interpersonal violence and other forms of abuse and suffering from undetected ptsd. tbi is a special case of physical disability because it involves physical injury that specifically compromises mental functioning. tbi ranges from mild (no more than minutes of unconsciousness and hours of amnesia) to severe (coma of at least hours or amnesia for more than hours). studies with adults and children of both genders who have sustained tbi demonstrate that they are as likely to develop ptsd as persons in equally severe accidents or assaults who have not (mcmillan, williams, & bryant, ) . fewer studies have been conducted with persons with severe than mild tbi, but they have not been found to be less likely to develop ptsd, as was originally hypothesized-due to not being able to experience or later recall the psychologically traumatic aspects of the injury as vividly as a person who does not lose consciousness or have amnesia. a subsequent study confirmed that adults with tbi were less likely to report acute traumatic stress symptoms immediately after the injury and to recall having felt helpless when interviewed several weeks later but that months after the accident, they were equally likely to report ptsd symptoms as injury survivors with no tbi (jones, harvey, & brewin, ) . tbi definitely exacerbates, and indeed may cause, ptsd, as tragically is illustrated by the extremely high estimates of prevalence of ptsd among military veterans of the iraq and afghanistan wars with tbi. thus, ptsd warrants careful assessment when tbi has occurred. concerning developmental disabilities, similarly, only one published study of ptsd could be located (ryan, ) . in that study, adults receiving services for learning disability were more likely than other adults (kessler, sonnega, bromet, & hughes, ) to report exposure to traumatic stressors ( % prevalence, on average two past traumatic events). however, they had no greater risk than adults in the general population when exposed to traumatic stressors. the most frequently reported types of traumatic exposures were multiple experiences of sexual abuse by multiple perpetrators (commonly starting in childhood), physical abuse, or life-threatening neglect. traumatic losses involving a caregiver or close relative or friend (including witnessing the death in several instances (such as witnessing a sibling dying in a fire, a close friend die during a seizure or an accident, or a parent commit suicide by shooting himself in the head with a gun)) were also reported by at least % of the participants. most of the learning-disabled adults who met criteria for ptsd had been referred for treatment for violent or disruptive behavior, typically with no psychiatric diagnosis or a diagnosis of schizophrenia, autism, or intermittent explosive disorder. when ptsd was diagnosed, major depression was a frequent comorbid disorder; yet, neither ptsd nor depression typically had been identified prior to the clinical assessment study. the findings of this study suggest that adults with developmental disorders often have been targets for abuse or neglect in childhood or have sustained severe traumatic losses and that their ptsd and depression tend to go undiagnosed as clinicians make their behavioral difficulties the focus of treatment services (box . ). poverty is an adverse result of having low "social status." this does not mean that a person or group is objectively deficient but rather that he or she is identified socially and politically as either not deserving or not possessing the social mandate to have access to resources such as money, safety, housing, transportation, health care and nutrition, education, and gainful employment. kubiak's ( ) social location theory states that each individual possesses identities within their society that are defined ( ) show the adverse impact of undetected ptsd: "a -year-old girl with a learning disability has suffered early abuse of a physical and sexual nature, including neglect. she presented [for medical evaluation] in early childhood with behavioral problems of aggression. she settled in a residential school from age to before an act of arson. she later revealed that she had experienced inappropriate sexual behaviors with peers at school. she complained of intrusive thoughts and images, along with depressive symptoms. at times she shows sexually inappropriate behavior and self-harm." "a -year-old man with a moderate learning disability who had been sexually assaulted by a care[giver] presented [for medical evaluation] in [an] acute state with disturbance of appetite, sleep, loss of skills, and emotional numbness, but the abuse was revealed only months later. on being exposed to the perpetrator at a later date, he showed a deterioration in mental state with acute symptoms of anxiety, and later developed a depressive disorder requiring medication. his level of functioning never returned to that prior to the traumatic event." a third case illustrates the therapeutic gains that a ptsd perspective can provide: a -year-old woman with learning disabilities and pervasive developmental disorder had been diagnosed at age with schizophrenia and subsequently had been diagnosed with schizoaffective disorder, bipolar disorder, and borderline personality disorder. for years after the first psychiatric diagnosis and hospitalization, she had been psychiatrically hospitalized more than times due to episodes of acute suicidality complicated by auditory command hallucinations (i.e., she believed she was hearing voices telling her to kill herself) and compulsive self-harm behavior (she used sharp objects to cut virtually every area on both arms and legs). treatment included high doses of antipsychotic, antiseizure, antidepressant, and antianxiety medications and two courses of electroconvulsive therapy, with periods of relative stabilization sufficient for her to live in an assisted living residential home and on two occasions to live in an independent apartment with in-home daily case management and nursing care. each period of improvement was relatively brief, lasting no longer than - months, at which time she experienced severe worsening in the apparently psychotic, depressive, and anxiety symptoms, requiring multiple rehospitalizations and progressive loss of social and cognitive abilities. for several years, family therapy was conducted, and the patient's history of traumatic stressors was assessed gradually in order not to lead to further destabilization. in addition to potential episodes of sexual assault as an adolescent and young adult, her mother disclosed that her biological father had been severely domestically violent during the patient's first years of life, until the mother ended that relationship. when ptsd was confirmed and accepted by the treatment team and the patient and her family as the primary diagnosis, the patient felt that she finally understood why she was experiencing the cyclic surges in distress and was able to utilize affect by factors such as their race, socioeconomic class, gender, age, residential status, and legal status. the greater the number of oppressed identities that one possesses, the more likely one will be "poor," including not only low income but also living in neighborhoods plagued with high crime, gang violence, abandoned buildings, drugs, teen pregnancy, high unemployment rate, underfunded schools, housing shortage, food of limited nutritional value, and unresponsive police. thus, poverty fundamentally is a breakdown of the social order as well as a resultant deprivation of resources for some people. the relationship between low income and exposure to psychological trauma and ptsd has been studied primarily in relationship to women and families, including those who currently have stable housing and those who do not. morrell-bellai, goering, and boydell ( ) identify poverty as a core risk factor for homelessness, because the socioeconomic benefits provided by a diminishing societal safety net and the typically insufficient employment wages provided by marginal jobs force people to rely on an increasingly limited pool of subsidized housing or to become homeless. associated risk factors include a lack of education, lack of work skill, physical or mental disability, substance abuse problem, minority status, sole support parent status, or the absence of an economically viable support system (fischer & breakey, ; morrell-bellai et al., ) . snow and anderson ( ) found that the most common reasons for homelessness reported in a survey of men and women living "on the street" were family-related problems such as marital breakup; family caregivers becoming unwilling or unable to care for a mentally ill or substance-abusing family member; escape from a dysfunctional and/or abusive family; or not having a family to turn to for support. poverty and homelessness involve a vicious cycle in which socioeconomic adversities are compounded by the experience of homelessness, leading to psychological disaffiliation, hopelessness, and loss of self-efficacy, and often substance dependence (bentley, ; hopper & baumohl, ; morrell-bellai et al., ) -which thus tends to perpetuate poverty and homelessness. a recent study by frisman, ford, lin, mallon, and chang ( ) reported that % of a sample of very low-income homeless women caring for children had experienced at least one type (and on average, five different types) of psychologically traumatic events, usually repeatedly and over long periods of time, with one in three having experienced full or partial ptsd at regulation skills (taught using dialectic behavior therapy and trauma affect regulation: guide for education and therapy; see chapter ). over the next year, her medications were carefully reduced to the lower therapeutic range for attentional problems and anxiety, with a sustained improvement in mood and social and cognitive functioning such that she was able to successfully work as a skilled volunteer in an assisted living center for older adults. some time in their lives. in addition, ford and frisman ( ) found that one in three of these homeless women with children had experienced a complex variant of ptsd involving problems with dysregulated affect or impulses, dissociation, somatization, and alterations in fundamental beliefs about self, relationships, and the future (i.e., "complex ptsd" or "disorders of extreme stress"; ford, ) . more than half of the sample had a history of either or both ptsd and its complex variant. exposure to violence and other forms of victimization begins in childhood for many homeless individuals, in part due to their exposure and the vulnerability of their living conditions (north, smith, & spitznagel, ) . rates of childhood physical abuse as high as % among homeless adolescents have been reported (maclean, paradise, & cauce, ) , and this figure may be on the low end. extremely poor women, whether homeless or not, have elevated rates of lifetime ptsd or other mental illness, and a history of such disorders is associated with having grown up in family and community environments with violence, threat, and anger (bassuk, dawson, perloff, & weinrub, ; davies-netzley, hurlburt, & hough, ) . however, homelessness per se may confer additional risk: homeless mothers and their children have higher lifetime rates of violent abuse and assault than equally impoverished housed mothers (bassuk et al., ) . thus, poverty puts people at risk for traumatic violence, but not having a stable residence compounds this risk and the likelihood of developing ptsd. victims of political repression, genocide ("ethnic cleansing"), and torture when political power is used to repress free speech and citizens' self-determination, there is an increase in the risk to members of that nation or community and its neighbors and associates of psychological trauma. domestic violence (see box . ) is a microcosm that shares much in common with large-scale political repression, because physical, psychological, and economic power is used to entrap, systematically break down, and coercively control the thoughts as well as the actions and relationships of the victim. on a larger scale, political repression involves similar psychological (and often physical as well) assaults by the people and institutions in power on the people, families, communities, and organizations that are deprived of access to political power and socioeconomic resources-and therefore also on their fundamental freedoms and values. without access to self-determination and the resources necessary to sustain independence, people are vulnerable to not just traumatic exploitation and violence but also to the traumatic loss of their intimate relationships, their families, their way of life, and their values (box . ). genocide (also described as "ethnic cleansing") involves the planned and systematic elimination of an entire collectivity of people, based on discrimination against them. historically, genocide has occurred often when conquering nations not only dominated and subjugated other nations but sought to eradicate their core culture and its leaders and teachers and to kill off or enslave the entire population. examples in the twentieth century include the armenian genocide in turkey, the holocaust box . the lost boys of sudan: complex ptsd in the wake of societal breakdown in the book what is the what?, by dave eggers ( ), valentino achak deng (a fictional character based upon a real person) provides an autobiography that includes his trials and tribulations in his current home in atlanta, georgia, after a traumatic journey of many years as a "lost boy" fleeing from his family's home in a rural village in southern sudan to refugee camps in ethiopia and kenya. valentino graphically describes a relentless series of traumatic experiences that include his village becoming a war zone, the deaths of family and friends, starvation and continual threats of being killed while traveling by foot with thousands of other "lost" children to escape sudan, witnessing brutal acts of violence by children as well as adults (e.g., a boy beating another boy to death in a fight over food rations), and being robbed and beaten unconscious in his own home in atlanta by a predatory african american couple. valentino is a good example of a person who suffers from chronic and complex ptsd, yet is extremely articulate, intelligent, and resourceful. valentino struggles with both unwanted memories and the need to keep his memories so that he ultimately can make sense of what has happened to him: what is the what? by writing his autobiography, he did what the therapy for children or adults with ptsd is intended to do: making sense of, rather than attempting to avoid, memories and reminders of traumatic experiences as a part-albeit horrible or tragic-of one's complete life story (see chapters and ). for example, in trauma-focused cognitive behavior therapy, the therapist helps the child to write (or in other creative ways to depict, such as by drawing pictures; using puppets, dolls, or action figures; or using collage or music) a "story" of what happened to them before, during, and after traumatic experience(s) and to share this "story" with a parent who can help the child with feelings of guilt and fear so that the traumatic memory can be "over" in the child's mind. because valentino was not able to get that kind of help, his autobiography as an adult (the book) is a kind of second attempt to achieve a sense of resolution by telling his story. but we see how this is very difficult to do when current life involves new problems and dangers that interfere with achieving a sense of safety. whether valentino succeeds in achieving some degree of emotional resolution about what he and his loved ones have suffered is an open question. what is clear is that he never stops trying to do so. it also is apparent that valentino's ethnic identification and heritage as an african man from the dinka tribe is very important as a protective factor enabling him to retain a small but significant fragment of his sense of personal identity and his intimate ties to his family and community. he experiences an odyssey as a victim fleeing the scene of horrific trauma, an initially reluctant but eventually drug-induced savage combatant, and a refugee "stranger in a strange land" when he is able to escape to what seems like an entirely different planet in the cosmopolitan urban setting of atlanta and the southern united states. it is the psychological trauma that he experiences on this odyssey, and the chronic stressors and societal breakdown and oppression that led him-and millions of others of all ages and a multiplicity of ethnocultural groups-on this journey of crisis and survival, and not his ethnicity or cultural background that is responsible for the profound symptoms of ptsd that he develops. inflicted on jewish people in europe by the nazis, the "ethnic cleansing" in bosnia and serbo-croatia in the s, and the massacres and mass starvation and epidemics perpetrated in rwanda in , in sudan beginning in , and in somalia, kenya, and zimbabwe most recently. genocide was first used as a term in by raphael lemkin, combining the words genos, from the greek for "race" or "kind," and cidere, which is latin and can be translated as "kill" (brom & kleber, ) . in , the term was adopted by the united nations general assembly and defined by the united nations convention on the prevention and punishment of the crime of genocide (cppcg) as follows: gregory stanton, the president of genocide watch, described " stages of genocide"; http://www.genocidewatch.org/aboutgenocide/ stagesofgenocide.htm. accessed / / : . classification-earliest stage, dividing people into "us" and "them" (the victim group). . symbolization-assigning particular symbols to designate the victim group members. . dehumanization-equating certain people with subhuman animals, vermin, or insects. . organization-militias or special units created for the purpose of genocide. . polarization-broadcasting of propaganda aimed at marginalizing the out-group. . preparation-out-group members are physically separated or confined in a "ghetto." . extermination-murder, starvation, infection, or other forms of inflicting pain and death. . denial-refusal to accept responsibility or admit wrongdoing, maintaining the self-righteous position that the victim group deserved annihilation and were subhuman. these stages are approximate and vary in each separate incident, but they demonstrate how genocide differs from other forms of even very horrific violence (such as war) because the aim is not simply to subdue, harm, or exploit but to dehumanize, exterminate, and annihilate. genocide thus involves several traumatic features, including loss of self-worth and allegiance to core values and institutions; prolonged pain and suffering; bereavement; terror and horror of annihilation; injury; helplessness while witnessing demeaning, cruel, and violent events; and confinement. survival responses to genocide are described by brom and kleber ( ) as: … a narrowing of functioning and awareness in order to maximize the chances of survival [often involving] psychic closing off (also called robotization-that is, acting and feeling emotionally and mentally empty or on "automatic pilot" like a "robot"), [and] regression-that is, feeling, thinking, and acting like a child (or in the case of children, like a much younger age than actual chronological age). often victims also experience a strong dependence on perpetrators who decide on life and death. the "muselman effect" … manifested by complete physical decrepitude, apathy, slowing of movement, and gradual disintegration of personality (including loss of the capacity for rational reasoning) may result when individuals have been exposed to long-term and extreme circumstances. an additional phenomenon that is well documented is the so-called "death imprint" resulting when substantial witnessing of death continues to haunt the survivor. these reactions closely parallel the symptoms of both asd (such as dissociation and regression) and ptsd (such as intrusive reexperiencing and emotional numbing). the adverse long-term effects of experiencing genocide are severe and pervasive. more than one in three survivors become clinically depressed and develop ptsd. the social support of caring family members (and for children, parents, or other caregivers) and relationships and activities that individuals to retain their spiritual or religious beliefs and their sense of self-respect are crucial protective factors against ptsd and depression. however, even the most resilient and socially supported person is likely to experience distressing memories and survivor guilt years or even decades later. studies with elderly holocaust survivors who are physically and emotionally very hardy (often well into their s and s) have documented significant persisting emotional distress and ptsd symptoms or more years later (brom & kleber, ) . moreover, the offspring of holocaust survivors with ptsd are more likely than offspring whose parents do not have ptsd to themselves experience ptsd as adults (yehuda et al., (yehuda et al., , . genocide often involves physical hardships that compromise physical health and may lead to long-term illnesses and depletion of the body's immune system. for example, the physical exertion and pain involved in torture, untreated physical illnesses, insufficient sleep, starvation, exposure to extreme temperatures, and forced labor may accelerate the aging process (brom & kleber, ) . genocide also often includes separating individuals from their families and community groups. this not only deprives the survivor of crucial social support but engenders a sense of isolation, distrust, and shame and of being permanently psychologically damaged (herman, ) . survivors also are faced with a choice of holding to their allegiance to their family, nation, culture, and racial identity, despite the punishment inflicted by the perpetrators, or abandoning these basic commitments and rejecting themselves and people like them. faced with this impossible choice (as epitomized in william styron's classic novel, sophie's choice), survivors often believe that they failed utterly and let down not only themselves but their family and culture no matter how resiliently they coped and the integrity of their efforts. survivor guilt is an expression of a sense of grief, powerlessness, and failure, including questioning why they survived and others did not. torture. torture is a terrible special case of political repression that involves "malicious intent and a total disregard for the recipient's dignity and humanity. thus, torture is among the most egregious violations of a person's fundamental right to personal integrity and a pathological form of human interaction" (quiroga & jaranson, ) . the united nations (un) office of high commissioner for human rights established a "convention against torture and other cruel, inhuman or degrading treatment or punishment (cat)," which has been endorsed by nations and defines torture as follows: for the purpose of this convention, the term "torture" means any act by which severe pain or suffering, whether physical or mental, is intentionally inflicted on a person for such purpose as obtaining from him or a third person information or a confession, punishing him for an act he or a third person has committed, or is suspected of having committed, or intimidating or coercing him or a third person, or for any reason based on discrimination of any kind, when such pain or suffering is inflicted by, or at the instigation of, or with the consent or acquiescence of, a public official or other person acting in an official capacity. it does not include pain or suffering arising only from, inherent in, or incidental to lawful sanctions. (http://www.unhchr.ch/html/menu /b/h_cat .htm accessed / / ) an amnesty international worldwide survey found that % of countries practice torture systematically, despite the absolute prohibition of torture and cruel and inhuman treatment under international law. torture may be euphemistically referred to as "enhanced interrogation techniques" and condoned in order to obtain "intelligence" from designated enemies of the nation, although this is completely prohibited by the un resolution (quiroga & jaranson, ) . widespread controversy has attended the use of such techniques by the us central intelligence agency in response to the september , , terrorism incidents, controversy that peaked in with the presidential decision to close the guantanamo bay military prison, and the release of the us senate report revealing and questioning the legality and morality of torture tactics used in interrogation and incarceration. basoglu, livanou, and crnobaric ( ) , in a sample from the balkan war ( - ) studied from to , showed that the torture need not inflict physical pain in order to produce ptsd. psychological assessment of torture survivors was systematized by the istanbul protocol, a manual on the effective investigation and documentation of torture and other cruel, inhumane, or degrading treatment or punishment that includes modules for medical, psychological, and legal professionals united nations resolution / on december , (quiroga & jaranson, ) . the psychological problems most often reported by torture survivors are emotional symptoms (anxiety, depression, irritability/aggressiveness, emotional liability, self-isolation, alienation from others, withdrawal); cognitive symptoms (confusion/disorientation, memory and concentration impairments); and neuro-vegetative symptoms (lack of energy and stamina, insomnia, nightmares, sexual dysfunction) (quiroga & jaranson, ) . the most frequent psychiatric diagnoses are ptsd and major depression, other anxiety disorders such as panic disorder and generalized anxiety disorder, and substance use disorders. longer-term effects include changes in personality or worldview, consistent with complex ptsd (quiroga & jaranson, ) . the greater the degree of distress and loss of sense of control during torture, the greater the likelihood of ptsd and depression. resilience, through being able maintain a sense of personal control, efficacy, and hope while enduring torture, is associated with less distress during torture and lower risk of ptsd (quiroga & jaranson, ) . social, cultural, and other diversity issues in the traumatic stress field however, quiroga and jaranson ( ) cited a study by olsen showing that years after torture, physical pain was still prevalent even if torture was primarily psychological in nature. based on this finding and related studies, they conclude the following (p. ): the most important physical consequence of torture is chronic, long-lasting pain experienced in multiple areas of the body. all [physical] torture victims show some acute injuries, sometimes temporary, such as bruises, hematomas, lacerations, cuts, burns, and fractures of teeth or bones, if examined soon after the torture episode. permanent lesions, such as skin scars on different parts of the body, have been found in % to % of torture victims. … falanga, beating the sole of the feet with a wooden or metallic baton, has been studied extensively. survivors complain of chronic pain, a burning sensation. … acute renal failure secondary to rhabdomyolysis, or destruction of skeletal muscle, is a possible consequence of severe beating involving damage to muscle tissue. this condition can be fatal without hemodialysis. … a severe traumatic brain injury that is caused by a blow or jolt to the head or a penetrating head injury may disrupt the function of the brain by causing a fracture of the skull, brain hemorrhage, brain edema, seizures, and dementia. the effects of less severe brain injury have not been well studied. treatment for torture survivors must be multidisciplinary and involves a long-term approach. several treatment modalities have been developed, but little consensus exists concerning the standard of practice, and treatment effectiveness has not been scientifically validated by treatment outcome studies (quiroga & jaranson, ) . a key element that is widely agreed upon is to pay careful attention to not inadvertently replicating in benign ways aspects of torture in the treatment (such as by pressing a survivor to recount traumatic memories without the survivor's informed and voluntary consent; by encouraging or discouraging political, family, and social activities except as initiated by the survivor; or by behaving in authoritarian ways rather than seeking to be collaborative with the survivor). it also is best to use medical, psychiatric medication, and psychotherapy modalities to address the ptsd symptoms of impaired sleep, nightmares, hyperarousal, startle reactions, and irritability. quiroga and jaranson ( ) also recommend using groups for socializing and supportive activities to reestablish a sense of family and cultural values, and supporting the traditional religious and cultural beliefs of the survivor. currently, nearly torture survivor treatment centers exist worldwide, of them accredited by the international rehabilitation council of torture victims (quiroga & jaranson, ) . most of these centers also involve the survivors' families and communities in developing shared approaches to recovery and reparation of the harm done to all. the controversy concerning the use of torture on detainees in the so-called "war on terror" has led to deep concern on the part of not only the public at large but specifically by mental health professionals. the issue is that psychiatry and psychology professionals who are in the military or consult to the military have been involved in the detention and interrogation of suspected terrorists at high-security facilities such as the military base at guantanamo bay and the military prison in iraq, abu ghraib. as a result, guidelines for mental health professionals working in these or similar facilities in which prolonged detention and interrogation may involve practices that constitute torture have been developed by a special committee of the american psychological association's division ( ) on trauma psychology (box . ). the apa council of representatives … included in its "unequivocal condemnation" all techniques considered torture or cruel, inhuman or degrading treatment or punishment under the united nations convention against torture and other cruel, inhuman, or degrading treatment or punishment; the geneva conventions; the principles of medical ethics relevant to the role of health personnel, particularly physicians, in the protection of prisoners and detainees against torture and other cruel, inhuman, or degrading treatment or punishment; the basic principles for the treatment of prisoners, the mccain amendment, the united nations principles on the effective investigation and documentation of torture and other cruel, inhuman, or degrading treatment or punishment an "absolute prohibition against mock executions; waterboarding or any other form of simulated drowning or suffocation; sexual humiliation; rape; cultural or religious humiliation; exploitation of fears, phobias, or psychopathology; induced hypothermia; the use of psychotropic drugs or mind-altering substances; hooding; forced nakedness; stress positions; the use of dogs to threaten or intimidate; physical assault, including slapping or shaking; exposure to extreme heat or cold; threats of harm or death; isolation; sensory deprivation and overstimulation; sleep deprivation; or the threat [of these] to an individual or to members of an individual's family. psychologists are absolutely prohibited from knowingly planning, designing, participating in, or assisting in the use of all condemned techniques at any time and may not enlist others to employ these techniques. we have come to the conclusion that the united states' harsh interrogationdetention program is potentially trauma-inducing both in general (e.g., indefinite detention, little contact with lawyers, no contact with relatives or significant others, prolonged absence of due process, awareness that other prisoners have been tortured, lack of predictability or control regarding potential threats to survival or bodily integrity) and in terms of some of its specific components (e.g., prolonged isolation, waterboarding, humiliation, painful stress positions). in other words, these potentials for trauma extend beyond the narrow procedures that meet international definitions of torture. the evidence for risk of psychological trauma to detained enemy combatants is particularly compelling and well grounded in formal research, but there is also suggestive anecdotal and theoretical evidence of trauma induction in interrogators and the broader society. we were particularly struck by the fact that the potentially traumatic elements include not only activities designed to extract information from prisoners but also much of the detention process as it is currently conceived, beyond much oversight, or compliance with international law. given the pervasiveness of these traumatogenic elements, it is questionable whether psychologists can function in these settings without participating in, or being adversely affected by, heightened risk for trauma. nonetheless, as a group of psychologists with expertise in preventing traumatic stress and ameliorating debilitating posttraumatic sequelae, we believe that certain steps could … minimize the risk of psychological trauma. they are as follows: . we believe that the risk of traumatic stress and negative posttraumatic sequelae will be reduced if psychologists adhere to both the apa ethical standards and subsequent refinements of apa policies pertaining to interrogation, detention, and torture. such adherence would be more likely if the apa ethics code were revised to incorporate, as enforceable standards, the specific interrogation and torture-related policy refinements that have occurred since . psychologists should promote situations that maintain the risk of traumatic stress at acceptably low levels and avoid situations that heighten the risk for traumatic stress occurring. among other things, this means that psychologists should not provide professional services in secret prisons that appear to be beyond the reach of normal standards of international law or in settings in which torture and other human rights abuses have been credibly documented to be permitted on the basis of local laws. it also suggests that psychologists should not support or participate in any detention or interrogation procedure that constitutes cruel or inhumane treatment or that otherwise has been shown to elevate risk of traumatic stress (e.g., prolonged isolation). . if psychologists work in settings in which detention and interrogations are conducted, then they should conduct or seek an assessment of the potential traumatic features of the treatment of detainees before, during, and after interrogation. this assessment can be informal or formal, depending on whether other systems of oversight are in place. this assessment should include an examination of the social psychological factors that could elevate risk of trauma. because not all psychologists have expertise in assessing traumatic stress risk and/or social psychological (continued ) factors, the assessment should be conducted by psychologists who have this specific expertise. such assessments could inform decisions not only by psychologists but also by others working in facilities in which detention and interrogation occur. it is recommended therefore that apa advocate for appropriate governmental authorities to appoint an independent oversight committee for each facility of this type and that the oversight committees include psychologists identified by apa as having relevant expertise. . if psychologists work in settings in which risk of traumatic stress is found to be elevated then they should (i) formally recommend alterations that could reduce the traumatogenic potential of the detention and interrogation process (n.b. some recommendations may be aimed at policy makers rather than local authorities); (ii) conduct or seek an assessment of posttraumatic stress symptoms and associated features (e.g., depression, dissociation) in detainees, interrogators, and other directly or indirectly involved staff; (iii) recommend appropriate psychological interventions for any detainees or personnel found to be suffering from clinically significant psychological difficulties; and (iv) refuse to participate in any activities that significantly increase risk of traumatic stress. if a psychologist working in such settings does not have specific expertise required to meet some of the above recommendations, then she or he should consult with psychologist(s) who have this expertise to make the appropriate determination. . because some detainee abuses have been credibly linked to an absence of appropriate training and/or expertise, psychologists should advocate for, participate in designing, and/or assist with providing appropriate and comprehensive training to all personnel involved in interacting with detainees. this training should include (i) clear ethical guidelines emphasizing the prohibition of causing harm and the importance of protecting detainee rights, (ii) a research-based overview of the nature and consequences of traumatic stress and posttraumatic impairment as they relate to the interrogation and confinement process and all parties involved in layperson terms with practical implications, and (iii) detailed review of research on false confessions, in layperson terms, with practical implications for enhancing the validity and utility of information gathered in the course of interrogation and detention. because not all psychologists have expertise in these specific matters, apa should develop standardized training materials that cover the current state of psychological knowledge and practices on these important topics, and ensure that these materials are regularly updated by qualified psychologists in consultation with experts from other fields such as law enforcement, the military, and human rights. . because protecting human rights reduces the risk for traumatic stress and posttraumatic impairment, psychologists should collaborate with legal, military, and other colleagues to advocate for due process for all detainees, including providing clear guidelines about finite lengths of detention prior to formal hearing or trial and enforcing the recent supreme court decision to reinstate habeas corpus and other international standards of human rights. psychologists' support for these actions should not come from a blanket support for adherence to law but rather from an informed judgment that these … laws reduce the risk for harm. psychologists should be prepared to disagree with any future international laws or us supreme court decisions that heighten risk for traumatic stress. box . continued . psychologists should support increased transparency during the detention and interrogation process. such increased transparency could reduce the likelihood of traumatizing practices, increase the likelihood that traumatizing practices will be identified and stopped as early as possible, and protect ethical psychologists and other workers within the system from being falsely accused of acting unethically. we recognize that this recommendation raises an apparent conflict with the goal of secrecy commonly endorsed by national security organizations. we concur that full transparency is unreasonable and counterproductive. yet, we do believe that increased transparency is a safeguard against traumatizing practices. though the details of resolving this conflict are beyond the scope of this task force's expertise, we believe that reasonable, knowledgeable intelligence experts, in consultation with psychologists, can construct a system of oversight that will both retain credible independence from the military chain of command and guard classified information. one suggestion may be to establish a greater presence of psychological expertise within a framework of oversight protection. . if psychologists are going to continue to be involved in interrogations, then it will be important to continue to segregate the function of interrogation consultant from that of mental health provider to reduce risk of perceived or actual betrayal by the detainee. it is unknown whether betrayal of trust due to dual roles can constitute a direct form of traumatization under these circumstances, but it is likely that betrayal in this context could exacerbate traumatic stress that occurs of other aspects of detention and interrogation (especially in light of the ways that such detention appears to disrupt attachment as outlined in the body of our report). maintaining separate roles also may enable the psychologist to more effectively assist detainees with traumatic stress reactions by fostering a trusting therapeutic relationship. . psychologists should advocate for extra protections for detainees who are from vulnerable populations such as minors, ethnic minorities, or other groups that have limited access to socioeconomic or political resources or are potentially subject to societal discrimination or prejudice because such groups may be more likely to receive coercive interrogations and/or excessive force and less likely to be sympathetically viewed by the general public. for this purpose, psychologists may work within sponsor/authorizing organizations to institute developmental, gender, and culture sensitivity trainings for interrogators and should review evidence concerning the impact of different forms of traumatic stressors and differential sensitivity to the interrogation/detention setting/process on different (and particularly vulnerable) ages, genders, and cultural backgrounds. such psychologists should, to whatever extent possible, guard against such information being used to exploit vulnerable populations and instead emphasize ways to enhance safety and psychological wellbeing in the interrogation process. if psychologists lack relevant expertise to meet the recommendations, … they should seek or advocate for outside expert consultation. . psychologists should collaborate with colleagues from a variety of professions and organizations, including the military and intelligence organizations, to conduct ethical research on several aspects of the detention and interrogation process, especially its potential for inducing trauma. recent reviews suggest that most of the interrogation procedures used today have not received recent rigorous study (intelligence science board, ) . furthermore, very little of the recent study has been directed toward understanding the psychological effects of interrogation on not only the detainees but also the people working within and outside the interrogation and detention system. political violence not only leads to traumatic harm to people while they are living in their communities but also often when victims are forced to flee their homes either to another country or while remaining within their country. refugees are defined by the united nations high commissioner for refugees (unhcr) as persons who have left their nation of origin to escape violence. article one of the united nations convention relating to the status of refugees defines a refugee as someone who "owing to well-founded fear of being persecuted for reasons of race, religion, nationality, membership of a particular social group or political opinion, is outside the country of his nationality and is unable or, owing to such fear, is unwilling to avail himself of the protection of that country; or who, not having a nationality and being outside the country of his former habitual residence as a result of such events, is unable or, owing to such fear, is unwilling to return to it" (unhcr, p. ; http:// www.unhcr.org/home/publ/ b e ea .pdf; accessed . . ). therefore, refugees are distinct from both legal and illegal immigrants, economic migrants, environmental migrants, and labor migrants (weine, ) . refugees must involuntarily leave home, community, and family and friends, often with limited resources or preparation and usually without knowing whom they can trust and where they can find safe passage and a safe haven. thus, both prior to and during the displacement, refugees often suffer psychologically traumatic experiences, including having their community or homes attacked or destroyed due to war; racially, genderbased, or ethnically targeted genocide or terrorism; institutionally orchestrated deprivation and violence; along with torture, atrocities, rape, witnessing violence, fear for their lives, hunger, lack of adequate shelter, separation from loved ones, and destruction and loss of property (weine, ) . estimating the number of refugees is very difficult. a minimum estimate that is probably much lower than the actual number is calculated by the united nations based on the number of persons in resettlement camps or individually recognized by a host country. based on this definition, there were million refugees worldwide in (www.unhcr.org) (figure . ). in côte d'ivoire, and was quickly followed by others in libya, somalia, sudan, and elsewhere. in all, . million people were newly displaced, with a full , of these fleeing their countries and becoming refugees. " saw suffering on an epic scale. for so many lives to have been thrown into turmoil over so short a space of time means enormous personal cost for all who were affected," said the unhcr antónio guterres. "we can be grateful only that the international system for protecting such people held firm for the most part and that borders stayed open. these are testing times." worldwide, . million people ended either as refugees ( . million), internally displaced ( . million), or in the process of seeking asylum ( , ). despite the high number of new refugees, the overall figure was lower than the total of . million people, due mainly to the offsetting effect of large numbers of idps returning home: . million, the highest rate of returns of idps in more than a decade. among refugees, and notwithstanding an increase in voluntary repatriation over levels, was the third lowest year for returns ( , ) in a decade. viewed on a -year basis, the report shows several worrying trends. one is that forced displacement is affecting larger numbers of people globally, with the annual level exceeding million people for each of the last years. another is that a person who becomes a refugee is likely to remain one for many years-often stuck in a camp or living precariously in an urban location. of the . million refugees under unhcr's mandate, almost three-quarters ( . million) have been in exile for at least years awaiting a solution. overall, afghanistan remains the biggest producer of refugees ( . million), followed by iraq ( . million), somalia ( . million), sudan ( , ), and the democratic republic of the congo ( , ) . around four-fifths of the world's refugees flee to their neighboring countries, reflected in the large refugee populations seen, for example, in pakistan ( . million people), iran ( , ) , kenya ( , ), and chad ( , many people displaced from their communities by violence remain in their home country. they are not considered refugees, but "idps": "people or groups … who have been forced to leave their homes" due to "armed conflict, situations of generalized violence, violations of human rights, or natural-or human-made disasters, and who have not crossed an international border" (www.unhcr.org). as of , there were more than three times as many idps as refugees, an estimated million worldwide, million social, cultural, and other diversity issues in the traumatic stress field due to armed conflict and million due to mass natural disasters (www.unhcr.org). between one and more than million idps were known to be in several countries in , including colombia, congo, iraq, somalia, sudan, and uganda. azerbaijan, cote due n'orde, and sri lanka had more than , known idps each. at least another million persons are considered "stateless"-that is, to not be citizens of any nation, in . nepal and bangladesh have the majority of the stateless persons in the world, although nearly million persons in those two countries were made citizens in . palestine and iraq are the other countries with large numbers of stateless persons. there may be millions more stateless individuals, because only countries assisted the united nations in its census of stateless persons in (www.unhcr.org). the impact of forced displacement often is not just extremely stressful but traumatic. refugees, idps, and stateless persons have few protections and often must live in confined camps or crowded public shelters, where they are vulnerable to assaults (including rape), robbery, and illness. many have witnessed horrific violence associated with wars, genocide, or other forms of mass armed conflict that caused them to flee. loss of family and friends due to violence or illness is common, as well as due to being separated with no way to communicate. studies have documented high prevalence levels of ptsd and depression among refugees or idps from armed conflicts in central america, southeast asia, the middle east, and the balkans (fazel, wheeler, & danesh, ; marshall, schell, elliott, berthold, & chun, ) at least times higher than the - % prevalence estimates from epidemiological surveys (see chapter ). ptsd prevalence estimates that are more than three times higher than these very high levels, as high as - %, have been reported among disabled central american refugees (rivera, mari, andreoli, quintana, & ferraz, ) and among afghan mothers (seino, takano, mashal, hemat, & nakamura, ) . other studies have more specifically investigated physical displacement in the traumatic stress experienced by refugees. displacement may involve many stressors, and a research review found that "living in institutional accommodation, experiencing restricted economic opportunity, [being] displaced internally within their own country [or] repatriated to a country they had previously fled or whose initiating conflict was unresolved" were particularly problematic. this review of reports involving , participants ( , refugees and , persons who were not displaced) showed that displacement alone was associated with more severe mental health problems, including ptsd (porter & haslam, ) . in contrast to most research findings on the etiology (see chapter ) and epidemiology (see chapter ) of ptsd, "refugees who were older, more educated, and female and who had higher predisplacement socioeconomic status and rural residence also had worse outcomes" (porter & haslam, , p. ) . people become "internally displaced" as often due to mass natural disasters as to armed violence. in the united states, several hundred thousand people had to leave the new orleans area following hurricane katrina in august . almost , received medical care at american red cross shelters within the next month (mills, edmondson, & park, ) . many displaced persons already were severely disadvantaged due to living in poverty (roughly % of the population of new orleans), having limited access to quality health care, and exposure to community violence (mills et al., ) . in a study of adult evacuees from new orleans and surrounding parishes ( % men, average age years old, % black, % low income (annual income less than $ , ), % reporting a prehurricane psychiatric disorder ( % depression, % anxiety disorder, % bipolar disorder)), most ( %) waited several days to be evacuated, and a majority reported sustaining minor to severe injuries ( %) and mild to severe illness ( %) in the hurricane or evacuation process. one in seven lost a loved one due to death in the hurricane or its aftermath, and most ( %) were separated from a family member for a day or more. many ( %) lost their home, two-thirds of whom were without property insurance. almost two in three ( %), particularly women, people with a prior psychiatric disorder, and those who recalled feeling their lives were in danger during the hurricane or its aftermath, were injured physically, or felt they had limited control over their current life circumstances, reported symptoms sufficient to qualify for a diagnosis of asd. natural disasters of several magnitudes greater have occurred in less developed and affluent areas of the world. for example, the tsunami that struck on december , , in the wake of the sumatra-andaman earthquake killed an estimated , people along the coastlines of the indian ocean, including , indonesians. another half a million indonesians were displaced from their communities. studies of survivors of this tsunami from indonesian areas such as aceh and north sumatra (frankenberg et al., ) , as well as from thailand (van griensven et al., ) and sri lanka (hollifield et al., ) , have demonstrated that posttraumatic stress, anxiety, and depression are suffered by hundreds of thousands, and perhaps millions, of people who experienced psychological trauma due to the tsunami (box . ). box . refugee posttraumatic stress in the wake of mass natural disaster frankenberg et al. ( ) reported a unique study of the impact of a massive natural disaster: the indian ocean tsunami that struck the day after christmas . unlike most research on mental health after disasters, this study began with a survey of a representative sample of persons in the host country (indonesia) almost years before the disaster. this "national socioeconomic survey (susenas)" provided a registry of respondents and predisaster data on health and socioeconomic characteristics of people throughout indonesia. the "study of the tsunami aftermath and recovery (star)" attempted to recontact , persons interviewed in communities by the susenas. the study also was able to determine the extent of damage caused to each community by the tsunami. the researchers got data from the national aeronautics and space administration's moderate resolution imaging spectroradiometer sensor collected year prior to the tsunami, and again immediately after the tsunami, to assess the degree to which the pretsunami ground cover visible in the first image had been replaced by bare earth in the second image. communities with at least % loss of ground cover were classified as heavily damaged ( % of the surveyed communities). those with some loss of ground cover were categorized as moderately damaged ( % of all locales), and % with no loss of ground cover were classified as undamaged by the tsunami. community leaders' and field observers' estimates of damage strongly correlated (r= . and . ) with these satellite-based estimates. one in three of the survey respondents (average age years old) heard the tsunami wave or people screaming. fewer sustained injuries ( %), lost a spouse ( %), lost a parent or child ( %), or witnessed family or friends "struggle or disappear" ( %), but % lost a family member or friend, % lost their home, and % lost their farming land, livestock, or equipment. posttraumatic stress was assessed by asking every respondent years or older to answer seven of the items from the ptsd checklist (see chapter ), as follows: since the tsunami, have you ever experienced (never, rarely, sometimes, or exposure to probable traumatic stress due to hearing the wave or screams, being injured, or seeing friends or family members "struggle or disappear," doubled the severity of pcl-c scores. consistent with this finding, compared to the sleep difficulties reported before the tsunami, after the tsunami, there was a large increase in the likelihood of sleep difficulties only in the most heavily damaged areas. pcl-c scores increased the most in the worst damaged locales, followed by the moderately damaged ones, with little change in the nondamaged communities. pcl-c scores averaged . , . , and . for the heavily, moderately, and undamaged areas, respectively, at the time of the interview and had been % higher at their peak after the tsunami (based on respondents' recollections). this is consistent with other studies that have reported persistent ptsd symptoms among the worst exposed persons but a substantial decline in ptsd symptom severity over time even among heavily exposed persons (see chapter ). women reported higher pcl-c scores than men, but primarily only in the heavily damaged areas. age was a factor in all communities: persons younger than years reporting an increase after the tsunami and persons years and older reporting lower pcl-c scores after the tsunami. interestingly, respondents who had a parent alive before the tsunami had lower pcl-c scores after the tsunami, but marital status, education, and income were not related to posttsunami pcl-c scores. property damage also correlated with posttsunami pcl-c scores. (continued ) as frankenberg et al. ( ) noted, these findings probably understate the severity and widespread nature of the harm, including posttraumatic stress, caused by a massive disaster such as this tsunami. however, the study provides the strongest evidence to date that a disaster that is not only life threatening for many but that displaces tens or hundreds of thousands of persons from their homes, families, neighbors, and way of life has the strongest adverse impact on communities that are most directly affected. another study (van griensven et al., ) conducted weeks after the tsunami in six southwestern provinces of thailand (where more than persons died or were unaccounted for and another were injured) included random samples of displaced persons and nondisplaced persons from the most heavily hit area and persons from less damaged areas. even though the extent of death and destruction was less in the worst-hit areas of thailand than in indonesia, symptoms of ptsd were reported by % of displaced and % of nondisplaced persons in the most damaged area of thailand (and by % in the less damaged areas). anxiety or depression symptoms were reported by three times as many persons, with similar proportions depending on displacement and the severity of damage to the community. thus, this study adds to the findings of the study from indonesia by demonstrating that displacement from home and community was a factor in ptsd and related symptoms soon after a mass natural disaster. the thailand study also resurveyed participants from the worst-damaged area months later ( months after the tsunami) and confirmed that displaced persons continued to be more likely than nondisplaced persons to suffer from ptsd, anxiety, and depression symptoms. consistent with other studies (ford, adams, & dailey, ) of postdisaster recovery (including the indonesia study), as the first anniversary of the disaster approached, about % of each group had recovered sufficiently to no longer report severe symptoms. whereas the indonesia study examined the extent of damage to participants' homes and (for most) the source of their incomes (farmland, animals, and equipment), the thailand study inquired directly as to whether respondents had lost their source of income and found that loss of livelihood was the strongest correlate of ptsd, anxiety, and depression symptoms. thus, the thailand study showed that losing not only home or community but also one's ability to generate an independent income through gainful work may contribute to the development and persistence of ptsd and related anxiety and depression symptoms. the defining characteristics of becoming a refugee in the wake of disaster therefore include (i) exposure to life-threatening catastrophe; (ii) loss of or separation from family and friends, (iii) loss of home and community; and (iv) loss of one's personal or family livelihood. each of these factors may result in acute posttraumatic distress, and the combination of several places people at risk for persistent ptsd. as weine ( ) describes, resettlement of refugees is a substantial challenge not only for displaced persons themselves but also for the host country. relatively stable and affluent countries in asia (such as pakistan, due to afghan refugees), the middle east (such as lebanon and syria, due to palestinian refugees), and africa (such as kenya and south africa), as well as most european and british commonwealth nations and the united states, have had a large influx of refugees. the half of all refugees who are resettled in cities experience economic pressures due to poverty and low-wage work and must live in communities that are crowded, segregated economically and culturally, and often adversely affected by crime, gangs, drugs, aids, and troubled schools (weine, ) . another half of all refugees are resettled in suburban and rural areas, which are more isolated (www.unhcr.org). in either case, refugees often face prolonged separations from family, friends, and loved ones, as well as the burden of having to find a way to subsist while saving money to bring others to their new home and to provide support to those back home who have stayed behind. refugee children have additional needs and challenges, including having to survive life-threatening experiences without adult help or guidance and then, if they are fortunate enough to be permanently resettled, having to return to being a "child" with a new family, community, and culture (henderson, ) . refugee children often display not only the symptoms of ptsd but also behavior problems (such as control, aggression, or defiance of authority), profound bereavement, and developmental, learning, or educational delays or deficits that are understandable in light of their often chronic deprivations before and during displacement. however, children also can be particularly resilient in the face of the psychological losses and traumas of being a refugee, and often they are a key source of hope for their families in the resettlement process (weine, ) . many refugees have opportunities to receive mental health services, either in the context of a refugee camp or after resettlement, but many do not seek or utilize these services. survival; getting stable and predictable access to food, money, housing, transportation, and safety; renewing communication with friends and family; and sustaining or regaining connection to cultural and religious traditions, values, and practices may take precedence over mental health treatment (and may in fact be the best form of therapy for many, under the circumstances). in resettlement settings, clinical treatment for refugee trauma is typically organized through refugee mental health clinics or specialized torture victim treatment centers, with services including crisis intervention, psychopharmacology, individual psychotherapy, group psychotherapy, and self-help groups and activities (weine, ) . to deliver culturally appropriate services, many programs involve traditional healers, socialization or mutual support groups, multifamily groups, and culturally based activities (weine, ) . services also tend to be provided by staff who themselves are members of the refugees' ethnic community, in collaboration with traumatic stress specialists and mental health professionals. the traditional model of western professional "expert" doctor or consultant who unilaterally tells local staff or clients how best to do assessment, diagnosis, or treatment has been justly criticized as culturally insensitive and potentially harmful rather than helpful (weine, ) . instead, the joint experience and expertise of the refugee client, local professional and paraprofessional alike, traditional healers, and traumatic stress professionals are taken together in a team approach that validates the client's and local helpers' cultures and traditions. this approach enhances the providers' ability to make a true cross-cultural assessment of symptoms and diagnoses, to adapt interventions to reflect different cultural beliefs and practices, and to engage not just individual clients but families and communities in recovery from ptsd. such an approach is consistent with new theoretical views of refugee traumatic stress, which include "the concepts of cultural bereavement, cultural trauma, family consequences of refugee trauma, community trauma, and social suffering" (weine, ) . this more culturally grounded view of refugees' experiences of traumatic stress and recovery from ptsd has led to the development of innovative therapy approaches (such as incorporating personal testimony and reconciliation into treatment) that address refugees' psychological vulnerabilities but strongly acknowledge their (and their families' and communities') hopes and strengths (weine, ) . when mass catastrophes, whether human-made or "acts of god" in origin, including natural disasters such as tsunamis, tornadoes, hurricanes, floods, or earthquakes, or public health emergencies, such as aids, severe acute respiratory syndrome (sars), pandemic influenza, ebola, or human-made disasters such as terrorist attacks, airline crashes, ferry capsizes, and train derailments, cause tens or hundreds of thousands or even millions of people and families to experience psychological trauma, the resultant suffering and needs are generally beyond the capacities of traditional mental health services and other forms of government-sponsored services. ngos play a critical role supporting and assisting persons and communities affected by catastrophic disaster or violence, including providing psychological support through clinical and nonclinical behavioral health services (hamilton & dodgen, ) . ngo responses to the mental health needs of mass-disaster survivors are based on the core belief that "all disasters are local" (hamilton & dodgen, ) . this means that local responders such as law enforcement, police, emergency medical teams, and professionals from the health care facilities, schools, and government are invariably first on the scene and frequently remain involved for months or years afterward. when insufficient resources are available, a local community may request help from the country, state, or provincial governments, which in turn may request regional or national assistance from both government and private sectors. for that reason, ngos that provide assistance following disasters, such as the american red cross, the national voluntary organizations active in disaster (nvoad), the united way, and the salvation army do so through their local chapters, which organize the initial relief efforts to provide shelter, food, legal aid, health and mental health care, and humanitarian assistance. organized in , nvoad is the umbrella organization coordinating all disaster relief services provided by volunteer organizations such as the american red cross throughout the united states. ngos also work closely with faith-based organizations (fbos) in the united states (such as catholic charities united states, church world service, lutheran disaster response, national association of jewish chaplains) within the national response framework of the federal emergency management agency (fema), which guides the nation's "all-hazards incident response" (hamilton & dodgen, ) . for example, american red cross disaster mental health (dmh) volunteers provide mental health services to people in shelters, while the church of the brethren provides crisis intervention to young children through their disaster child care program (hamilton & dodgen, ) . the american red cross is the most widely recognized ngo providing dmh services in the united states. in , congress chartered the american red cross to "carry on a system of national and international relief in time of peace" to reduce and prevent the suffering caused by national calamities program (hamilton & dodgen, ) . in , the american red cross established a formal dmh services program and began training licensed and certified mental health professionals to volunteer and assist other red cross workers to cope with and recover from the traumatic stress (or "vicarious trauma") of their disaster relief work. initially only licensed psychologists and social workers were permitted to become red cross dmh volunteers, but recently professionals from other disciplines, such as psychiatry and masters-level marriage and family therapy or counseling professions, also have become eligible. the american red cross has set up formal agreements with the american psychiatric association, the american psychological association, the national association of social workers, the american counseling association, and the american association of marriage and family therapy. the agreements provide that in the event of a mass disaster, the red cross will notify each professional associations to put out a call to their memberships for professionals who have completed red cross preparatory training and who can take time out from their ordinary work to serve as dmh volunteers for weeks or more at red cross disaster services sites. the american red cross sets up and oversees family assistance centers for disaster-affected communities, provides crisis and grief counseling through its dmh volunteers, and coordinates with federal agencies such as fema and the national transportation safety board (for airline or mass transportation disasters) to provide child care services and interfaith memorial services. the red cross also works closely with disaster-focused ngos such as the national organization for victim assistance, disaster psychiatry outreach, and the international critical incident stress foundation, inc. (icisf). founded in , the icisf trains mental health professionals, emergency responders, clergy, and chaplains to conduct critical incident stress management (see chapter ) teams to support disaster services personnel. in , the american red cross broadened the scope of dmh services to include assisting disaster-affected persons who are seeking red cross assistance, as well as red cross volunteers. all dmh volunteers now are trained in psychological first aid (see chapter ) so that they will provide mental health services to disaster victims in an appropriately circumscribed manner that is therapeutic without attempting to conduct psychotherapy at a disaster relief site. two other freestanding programs participating in a dmh response are the green cross assistance program, which provides trained traumatology specialists and the association of traumatic stress specialists, an association of mental health professionals and paraprofessionals who assist survivors of psychological trauma (hamilton & dodgen, ) . a number of us ngos also work internationally to provide psychosocial support and traumatic stress counseling to survivors of disasters and mass conflicts. these include the international services of the american red cross, the united methodist committee on relief, church world services, green cross, action aid-the united states, the american refugee committee, the center for victims of torture, and doctors without borders (hamilton & dodgen, ) . the international federation of red cross and red crescent societies also assist many nations' red cross organizations in serving their own and neighboring countries. a analysis by the united states homeland security institute found that fbos and ngos had a significant beneficial impact during and after hurricanes katrina and rita, with mental health and spiritual support among types of services (hamilton & dodgen, ) . the study reported that while fbos and ngos faced significant limitations and challenges in providing services, mental health and spiritual support was one of the three best-applied special practices, particularly services designed to preserve family unity within disaster relief shelters. hamilton and dodgen ( , p. ) describe how ngos can work together to meet critical needs in times of mass crisis, using the september , , terrorist attacks in new york, washington, and pennsylvania, as a case in point: local mental health providers working in mental health settings mobilized quickly, but needs were expected to surpass local capability. the american red cross dispatched dmh providers from local and adjacent communities to provide mental health support and stress reduction assistance. national volunteers recruited from across the country arrived within a few days to augment that mission. concurrently, icisf-trained volunteers, some of whom were already part of military mental health systems, also arrived to provide assistance. other agencies, such as fbos, also organized support for victims. in washington, the military was the gatekeeper for volunteers and worked closely with the american red cross to coordinate mental health support. in new york, civilian authorities collaborated with the american red cross. as family assistance centers were set up to aid grieving families, national dmh volunteers continued providing mental health support. because the terrorist attacks created a crime scene, access was controlled and ngos needed official standing to provide assistance. incorporating lessons learned from / , a similar event today would be different in several ways: all ngos and government agencies would organize their response under the national incident management system (nims) and the nrf, thus creating a more centralized, coordinated response and reducing overlapping or competing activities on the part of nvoads. because of ongoing coordination and outreach efforts since / , a greater array of disciplines and specific types of expertise would be available through ngos. the benefits of these efforts were seen during the responses to hurricanes katrina and rita in . personal and community characteristics that reflect ethnocultural, national, gender, age, and disability factors are crucial in defining the identity of every human being. when traumatic stress occurs in a person's or community's life, its impact is influenced by these identity factors. when identity is used as a basis for stigma, discrimination, or socioeconomic disadvantage, those stressors compound the effect of traumatic stressors and can be traumatic in and of themselves. by addressing the vulnerability that this combination in a scientifically and clinically responsible manner (alcantara, casement, & lewis-fernandez, ; c'de baca, castillo, & qualls, ; ghafoori, barragan, tohidian, & palinkas, ) to assist rather than stigmatize persons and communities (ruglass et al., ) , the traumatic stress professional can play a crucial role in our society's quest for social justice. 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oriented psychotherapy for trauma refugees ethnicity, acculturation, and self reported health. a population based study among immigrants from poland, turkey, and iran in sweden low cortisol and risk for ptsd in adult offspring of holocaust survivors from pogroms to "ethnic cleansing": meeting the needs of war affected children key: cord- -fawcn em authors: liu, chunyan; xiao, yan; zhang, jing; ren, lili; li, jianguo; xie, zhengde; xu, baoping; yang, yan; qian, suyun; wang, jianwei; shen, kunling title: adenovirus infection in children with acute lower respiratory tract infections in beijing, china, to date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: fawcn em background: human adenoviruses (hadv) play a significant role in pediatric respiratory tract infections. to date, over types of hadv have been identified. here, hadv types are characterized in children in the beijing area with acute lower respiratory tract infections (alrtis) and the clinical features and laboratory findings of hospitalized hadv-infected cases are described. methods: respiratory specimens were collected from pediatric patients with alrtis in the emergency department or from those admitted to beijing children’s hospital between march and december . infections with common respiratory viruses were determined by pcr or rt-pcr. hadv positive samples were further typed by pcr and sequencing. results: among patients with alrtis, ( . %) were found to have hadv infection. hadv infection was primarily confined to children ( . %) less than years of age. a total of different types of hadv were detected throughout the study period, with hadv-b ( . %) and hadv-b ( . %) as the most prevalent types, followed by hadv-c ( . %) and hadvc ( . %). newly emerging and re-emergent types or variants, hadv-b (n = ), hadv-c (n = ), and hadv-b p (n = ), were identified. results also included the reported first case of co-infection with hadv-c and hadv-c . clinical entities of patients with single hadv infection (n = ) were similar to those with mixed hadv/respiratory syncytial virus (rsv) infections (n = ). patients with hadv-b infection had longer duration of fever and higher serum levels of muscle enzymes than hadv-b -infected patients. conclusions: during the study period, hadv-b and hadv-b were the predominant types identified in pediatric alrtis. hadv-b infection tends to have more severe clinical consequences. the presence of newly emerging types or variants and co-infection with different types of hadv highlights the need for constant and close surveillance of hadv infection. acute lower respiratory tract infections (alrtis) are the leading cause of pediatric morbidity and mortality worldwide, particularly in developing countries. in infants and young children, alrtis are most frequently caused by respiratory viruses. one such virus, human adenovirus (hadv), plays a significant role in pediatric respiratory tract infections, accounting for - % of the overall respiratory illnesses and - % of the pneumonias [ , ] . although most cases are mild and indistinguishable from other viral causes, alrtis caused by hadv can be severe, or even fatal, and are associated with the highest risk of long term respiratory sequelae [ ] . thus, hadvassociated alrtis are of particular interest to both clinicians and researchers. hadv are responsible for a wide spectrum of clinical diseases, including respiratory illness (both upper and lower respiratory tract), pharyngoconjunctival fever, conjunctivitis, cystitis, gastroenteritis, and neurologic and venereal disease [ ] . hadv were first isolated in as respiratory pathogens [ , ] . to date, over types of hadv have been identified and classified into seven species (a to g) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cases of severe infection, outbreaks in closed populations, and even epidemic outbreaks have been associated with the newly emerging or re-emergent types or variants [ ] [ ] [ ] . interestingly, different types of hadv display various tissue tropisms that correlate with different clinical manifestations of infection. hadv infections of the respiratory tract are predominantly caused by hadv-b (including subspecies b and b ), hadv-c, or hadv-e. the predominant types vary among different countries and regions and they change over time because transmission of novel strains between countries or across continents may occur [ ] . type identification is critical to epidemiological surveillance, detection of new strains, and understanding of hadv pathogenesis. however, because most clinical laboratories do not type the isolates, there is little published information about epidemiologic and clinical features of hadv infections by type in children with alrtis. to identify hadv types and species in children with alrtis in beijing area and to characterize clinical features and laboratory findings of hospitalized hadvinfected cases, respiratory specimens were collected from hospital-admitted pediatric patients with alrtis and typed hadv positive samples using pcr and sequencing. the study protocol was approved by the ethical review committee of beijing children's hospital. individual written informed consent was obtained from the parents or guardians of all participants. from march to december , pediatric patients with alrtis who presented in emergency department or were admitted to respiratory department or intensive care unit, beijing children's hospital, were recruited for the study. the study site hospital is a tertiary comprehensive pediatric hospital with over beds and more than twenty clinical departments. alrtis were defined as the presence of signs and symptoms of respiratory tract infection (i.e., fever, coughing, rhinorrhea, oropharyngeal hyperemia, swelling of tonsils), and lower respiratory signs (tachypnea, dyspnea, retractions, or wheezing/rales upon auscultation). the patients were diagnosed with bronchitis, bronchiolitis or pneumonia. chest x-rays were taken for all patients and the criteria for diagnosing pneumonia are the presence of lung infiltrates indicated by chest radiography. nasopharyngeal aspirate or throat swab specimens were collected in virus transport media from each patient. no repeated samples were collected from any patient. all samples were stored at − °c prior to use. total nucleic acids (dna and rna) were extracted from μl nasopharyngeal aspirate or throat swab specimens using the nuclisens easymag™ automated extraction system (biomérieux, marcy l'etoile, france) according to the manufacturer's instructions and eluted in μl elution buffer. the presence of common respiratory viral agents, including parainfluenza virus (piv) type - , influenza virus (ifv), respiratory syncytial virus (rsv), human rhinovirus (hrv), enterovirus (ev), human coronavirus (hcov e, nl , hku , and oc ), human metapneumovirus (hmpv), human bocavirus (hbov), and hadv was determined by multiplex rt-pcr, single rt-pcr, or pcr assays as previously described [ , ] . blank virus transport media here served as a negative control for nucleic acid extraction and pcr. hadv positive samples were further amplified using a nested pcr procedure that targeted hyper variable regions - of the hexon gene as described by lu and erdman [ ] . expected amplicons ranged from bp to bp (secondary amplification) in length. sequencing was performed in both directions using the amplification primers. sequences were proof read and assembled using seqman software v . . (dnastar inc., wi, u.s.). for assignment of molecular identity and identification of the closest match, sequence alignment was performed using the basic local alignment search tool (blast) against ncbi genbank database (http://www.ncbi.nlm.nih.gov). clinical data were retrospectively recorded by careful analysis of patient medical files in beijing children's hospital, using a predefined microsoft excel spreadsheet. patients' demographic, clinical, and radiologic findings were collected. continuous variables were summarized as means ± standard deviations (sd) or medians. for categorical variables, percentages of patients in each category were calculated. differences between groups were assessed using pearson's chi square test or fisher's exact test for categorical variables and the one way anova, independent-samples t test, mann-whitney u test, and kruskal-wallis test for continuous variables. all analyses were performed using spss software, version . (ibm corporation, ny, u.s.). all tests were calculated in a two-tailed manner and a p value of < . was considered statistically significant. from march through december , a total of patients with alrtis ( with pneumonia, with bronchitis and with bronchiolitis) were enrolled in this study. the mean age of study participants was . ± . years (median year; age range, . month to years and months). there were male participants with a male-to-female ratio of . : . at least one respiratory virus was detected in nasopharyngeal aspirate or throat swab specimens of ( . %) enrolled participants. rsv ( . %) was the most commonly detected viral pathogen, followed by hrv ( . %) and piv ( . %). one hundred and ninety-four patients ( . %, / ) were found to have hadv infection, representing . % ( / ) of patients with positive respiratory samples. male paticipants were more likely to be infected with hadv ( boys and girls, male to female ratio = . : ). the mean age of infection was . ± . years (median, year; age range, month to years). most of hadv-infected cases ( . %) were under years of age and the highest percentage of hadv infections ( . %) occurred in infants (age group -< year), followed by the age group -< years ( . %). additionally, one or more other respiratory viruses were detected in . % (n = ) of hadv-infected participants. dual viral infection was identified in cases, triple infection in cases, quadruple in and quintuple in . rsv (n = ) was the most frequently co-detected virus, followed by hrv (n = ) and piv (n = ). hbov (n = ), hcov (n = ), ifv (n = ), ev (n = ), and hmpv (n = ) were also found to be co-infected with hadv. one hundred and ninety-four hadv-positive specimens were all successfully typed by hexon gene amplifying and sequencing. throughout the study period, four species (a, b, c, e) of hadv, including different types were identified. additionally, hadv-b (n = ; . %) and hadv-b (n = ; . %), which belong to species b, were the most prevalent hadv types, accounting for . % of all hadv-associated infections. hadv-c ( . %), hadv-c ( . %), hadv-c ( . %), hadv-b ( . %), hadv-c ( . %), hadv-c ( . %), hadv-a ( . %), hadv-b ( . %), and hadv-e ( . %) were also detected. interestingly, sequencing results from one specimen showed superimposed peaks in the chromatograms. to confirm the possibility of multiple hadv strains in that sample, pcr products were cloned and sequenced further. distinct hexon genes of different types (hadv-c and hadv-c ) were verified. hadv detection rate varied through the years, ranging from . % in to . % in (fig. ) . additionally, although hadv was detected throughout the year, cases commonly peaked in winter and spring season (fig. ) . furthermore, different types of hadv did not remain constant across the whole study period (fig. ) . specifically, hadv-c , −c , −b , and -b were detected throughout the study; hadv-c in all years except ; hadv-c and hadv-c in years , , among the hadv-positive cases, hospitalized cases were included in the clinical analysis, and cases from the emergency department for which the details of the medical records were not available were excluded. pneumonia (n = , . %) was the most common clinical diagnosis, followed by bronchitis (n = ) and bronchiolitis (n = ). additionally, almost all hospitalized hadv-infected patients presented with fever ( / , . %) and coughing ( / , . %) ( table ). the mean peak body temperature was . ± . °c (n = , range . − . °c) and febrile seizures were noted in two febrile patients. in addition to respiratory symptoms, diarrhea, vomiting, skin rash, and conjunctivitis were noted in . %, . %, . % and . % of the patients respectively. twenty-two patients ( . %) had underlying diseases, which included congenital heart disease ( patients), airway anomaly (malacia, stenosis, patients), bronchopulmonary dysplasia ( patient), asthma ( patients), or primary immunodeficiency ( patient). seventeen patients ( . %) required admission to the intensive care unit and patients ( . %) received mechanical ventilation including both noninvasive (n = ) and invasive (n = ) modes. analysis revealed that the mean value of white blood cell (wbc) count was . ± . × /l ( table ) . leukocytosis (wbc > . × /l) was observed in ( . %) patients. eighty-one patients ( . %) had elevated serum c-reaction protein (crp). given rsv was the virus most frequently co-detected with hadv, differences among patients with single hadv infection (n = ) and those with hadv/rsv coinfections, including both dual infections (n = ) and multiple infections (hadv/rsv with one or more other respiratory viruses, n = ), were assessed ( table ). the mean age of patients with multiple infections ( . ± . ) and dual infections ( . ± . ) was significantly younger than those with single hadv infection ( . ± . ) (p = . ). however clinical characteristics and laboratory findings showed no significant differences among different groups. because hadv-b and hadv-b were the most predominant type among patients with hadv infection, the clinical entities of patients with single hadv-b infection (n = ) and those with single hadv-b infection (n = ) were also compared to exclude the possible effect of other respiratory virus infection (table ) . patients with single hadv-b infection showed longer duration of fever ( . ± . vs . ± . , p = . ) than patients with hadv-b alone. immunoglobulin was more frequently used in single hadv-b infected patients than in single hadv-b group (p = . ). patients with hadv-b alone also tend to require longer hospital stay ( . ± . vs . ± . , p = . ) than those with single hadv-b infection, although no significant difference was found. biochemical tests demonstrated aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh) and hydroxybutyrate dehydrogenase (hbdh) levels were significantly higher in the single hadv-b infected group. two patients died in-hospital. both of them required icu admission and died of multiple organ failure. one was a month-old boy with multiple underlying conditions of complex congenital heart disease and tracheobronchial malformation. the other was a previously healthy month-old boy. analysis indicated that both fatal patients were infected with hadv-b but no other respiratory viruses. hadv is a significant causative agent of respiratory tract illnesses in both children and adults. here, the molecular showed that the hadv infection rate in the current study population was . %, which was consistent with previous reports from china and other countries [ ] [ ] [ ] . results showed that most patients with hadv infection were younger than years ( . %), which is similar to numbers reported in previous studies [ , , , [ ] [ ] [ ] . this may because the immune systems of young children are not well developed, which leaves them prone to more severe hadv disease. this may also suggest that schoolage children are exposed to the most common endemic types of hadv early in life, thereby establishing a protective immunity resulting only in mild clinical symptoms, such that upper respiratory tract infection does not require care in an emergency department or hospital in this age group. over a period of years, different types of hadv belonging to species (hadv-a, b, c, e) were identified in respiratory specimens from children with alrtis. hadv- and hadv- of species b comprised the most prevalent types and presented throughout the duration of the study. although these results were consistent with previous reports from korea and argentina [ , , ] , investigations from croatia, peru, canada, france showed that species c predominated [ , , , ] . this difference in type prevalence may be attributed to difference in regions, year of study, and population recruited. notably, some newly emerging or re-emergent types or variants were here identified, although only in rare cases. five patients were found to have hadv-b (formerly named hadv- a), which is an uncommon re-emergent type that once caused an outbreak of respiratory tract infection in a senior high school in shanxi province, china in , including one fatal case [ ] . subsequently, hadv-b has been associated with several outbreaks of respiratory disease in other provinces in china [ ] . an emerging variant, hadv-b p (formerly known as a), was also found. recently, hadv-b p has been associated with several large outbreaks of acute respiratory infection, which included severe and even fatal cases in the united states and europe [ , ] . additionally, in , an outbreak of febrile respiratory illness that affected students in gansu province, china was reported to be caused by hadv-b p [ ] . one hadv-b infected patient who presented with bronchopneumonia and required hospitalization in april was identified. by further sequencing the fiber gene (data not shown), this strain was confirmed to be hadv-b p because it contained a unique characteristic -nuleotid deletion in fiber knob region as reported by kajon et al. [ ] . last, this is the first report of detection of hadv-c in respiratory samples collected from pediatric patients with alrtis and the first of co-detection of hadv-c with hadvc- . hadv-c (formerly designated strain ) was first isolated from the feces of a healthy child as part of an acute flaccid paralysis surveillance program. computational genomic and bioinformatic analysis showed hadv-c to be a recombinant virus with fiber gene nearly identical to hadv-c and a unique hexon distinct from all viruses in species hadv-c [ , ] . out of the three hadv-c -infected cases identified here, one was a previously healthy month-old male who presented with bronchopneumonia and conjunctivitis requiring hospitalization. because only a small number of hadv-c positive cases were found here and all were co-infected with other respiratory viruses, the pathogenic role of hadv-c in respiratory infections will require further investigation. hadv type is traditionally determined by virus isolation and subsequently serum neutralization tests, in which antibodies raised against specific type are used to suppress cytopathic effects in tissue culture assays. by nature of its design, this test can only reveal the dominant type. by applying pcr-based identification targeting hexon or fiber genes, co-infections with multiple hadv types (types from same or different species) have been reported in both immunocompromised and immunocompetent patients [ , , [ ] [ ] [ ] . in current study, results showed that one specimen contained both hadv-c and hadv-c by cloned sequencing the pcr products. these were amplified directly from respiratory samples using universal primers of hexon gene. this co-infected phenomenon was confirmed using the fiber gene sequencing results with type-specific primers (data not shown). the specimen was collected from a previously healthy . -year old boy, presenting with fever, coughing and seizure at emergency department on december , . co-infection of different hadv types has never been reported in any previous studies of mainland china. the clinical implications of such co-infection remain unclear, and its role in hadv pathogenesis and evolution will require further study. consistent with the report from guangzhou, southern china [ ] , results here showed that . % of hadvinfected participants were co-infected with one or more other respiratory tract viruses and that rsv was the most frequently co-detected virus. however, no significant differences in clinical characteristics and laboratory findings were found between patients with single hadv infection and those co-infected with rsv except that coinfections were more frequently observed in younger children. similarly, a study from peru also did found no higher prevalence of any clinical manifestations in coinfected patients than in those infected with hadv alone [ ] . the results of another report from chile showed the clinical severity to be the same in patients with single hadv infection and those with mixed rsv-hadv infections [ ] . these data demonstrate that, as more sensitive molecular methods become more frequently used to identify pathogens, co-detection of different viruses in the same specimen may also become more common. however, the clinical role of such coinfections will still require independent investigations. both hadv-b and hadv-b may cause severe or even fatal pneumonia in even immunocompetent children. several previous studies showed that patients infected with hadv-b tend to have higher case-fatality rates than those with hadv-b [ , ] . two fatal cases were recorded during the study period, and both of these patients were infected with hadv-b alone. analysis revealed that patients with hadv-b infection had longer duration of fever and higher serum levels of muscle enzymes than hadv-b -infected patients. patients with hadv-b infection also tended to require longer hospital stays although no significant difference was found. these differences have excluded the possible interference by any other co-infected respiratory viruses since this work only evaluated the patients with hadv infection alone. such results may suggest that hadv-b infection tended to cause more extrapulmonary tissue damage (such as liver and heart) and may have more severe clinical consequence. this is a cross-sectional study. only one respiratory sample was collected from each patient and no viral load analysis was performed. although hadv is a pathogen that for long has been known to cause respiratory tract infection, asymptomatic carriage of the virus may persist for weeks [ ] . the detection of hadv in nasopharyngeal aspirate or throat swab with the use of a pcr assay could represent convalescent-phase shedding, so detection may not suggest the current infection. in summary, a total of different types of hadv were identified in children with alrtis and hadv-b and hadv-b were the most predominant types. clinical entities of patients with single hadv infection were 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outbreaks of lower respiratory tract infections in children adenovirus serotype and infection with acute respiratory failure in children in taiwan we would like to thank all participating physicians and nurses of beijing children's hospital for their assistance and collaboration in the sample and clinical data collection. this study was supported by grants from the national major s & t research projects for the control and prevention of major infectious diseases in china ( zx - ) and national science and technology supported projects ( bai b ). the authors report no conflicts of interests.authors' contributions cl analyzed data, performed statistical analysis, drafted and reviewed manuscript. yx and jz carried out the molecular studies. lr participated in study design and coordination and helped to review the manuscript. jl designed the primers of sequencing typing. zx analyzed data and reviewed manuscript. bx, yy and sq collected samples and data. jw and ks conceived of the study. all authors read and approved of the final manuscript. availability of data and materials not applicable submit your next manuscript to biomed central and take full advantage of: key: cord- - xrdv m authors: nowland, megan h.; brammer, david w.; garcia, alexis; rush, howard g. title: biology and diseases of rabbits date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: xrdv m beginning in , an inbred rabbit colony was developed at the phipps institute for the study, treatment and prevention of tuberculosis at the university of pennsylvania. this colony was used to study natural resistance to infection with tuberculosis (robertson et al., ). other inbred colonies or well-defined breeding colonies were also developed at the university of illinois college of medicine center for genetics, the laboratories of the international health division of the rockefeller foundation, the university of utrecht in the netherlands, and jackson laboratories. these colonies were moved or closed in the years to follow. since , the u.s. department of agriculture has reported the total number of certain species of animals used by registered research facilities ( ). in , , rabbits were used in research. there has been an overall decrease in numbers of rabbits used. this decreasing trend started in the mid- s. in , , rabbits were used in research. despite the overall drop in the number used in research, the rabbit is still a valuable model and tool for many disciplines. the rabbit has been utilized in immunology research for many years especially in regard to the structure of immunoglobulins and the genetic control of their formation. in addition, the rabbit is commonly used for the production of polyclonal antibodies for use as immunologic reagents (mage, ; pinheiro et al., ) . the relatively large body size and blood volume, easy access to the vascular system, and an existent large body of information on the purification of rabbit immunoglobulins are a few reasons the rabbit is preferred over other common laboratory animal species for polyclonal antibody production (stills, ) . the organization of the lymphoid system of the rabbit is comparable to that of other mammals. however, the rabbit does possess two gut-associated lymphoid tissues (galt) with specialized functions in the maturation of igm + b cells. these are the vermiform appendix at the distal end of the cecum and the sacculus rotundus at the ileocecal junction (mage, ) . for many years, the lack of rabbit-specific immunological reagents has limited the study of inflammation and immunity in the rabbit. the use of real-time polymerase chain reaction (rt-pcr) techniques has overcome this limitation and permitted such studies in many species other than man and mice. a quantitative real-time rt-pcr assay for measuring mrna for rabbit cytokines ifn-γ, il- , il- , il- , and tnf-α has been described (godornes et al., ) . recently, schnupf and sansonetti ( ) reported on rt-pcr primer pairs for analysis of three chemokines and ccl ) and cytokines (il- β, il- , il- , il- , il- , il- p , il p , il- a, il- f, . the profile of cytokines in the rabbit appears similar to other mammals. in mice and humans, the primary antibody repertoire is created by combinatorial rearrangement of a large number of immunoglobulin gene segments. other species (chicken, sheep, cattle, and rabbit) that have a limited number of gene segments utilize somatic gene conversion and/or somatic hypermutation (pinheiro et al., ) . in the former, a portion of the immunoglobulin gene is replaced with a gene sequence from a nonfunctional pseudogene. in the latter, single-nucleotide changes are made in the immunoglobulin genes (jenne et al., ) . in the rabbit, gene diversification occurs initially in the fetus and the neonate in sites such as the bone marrow. subsequently, between and weeks of age, immature igm + b cells undergo further diversification in the galt (appendix, sacculus rotundus, and peyer's patches) (mage et al., ; pinheiro et al., ) . furthermore, certain species of intestinal bacteria (bacteroides fragilis and bacillus subtilis) are required for appendix follicle development and antibody diversification to occur (hanson and lanning, ; mage et al., ) . most mammals express five classes of immunoglobulins: igm, igd, igg, iga, and ige. however, the rabbit lacks igd (sun et al., ) . the area of cardiovascular research has used the rabbit in a variety of different models. numerous dietary modifications will induce or exacerbate cholesterolinduced atherosclerosis in the rabbit. a brief overview of some of these dietary modifications can be found elsewhere (jayo et al., ) . research efforts into cholesterol metabolism have used the watanabe heritable hyperlipidemic (whhl) (atkinson et al., ; kita et al., ) and the st. thomas hospital strain rabbits (laville et al., ) . the whhl rabbit has a marked deficiency of low-density lipoprotein (ldl) receptors in the liver and other tissues. selective breeding of the whhl rabbit will increase the incidence of coronary artery atherosclerosis without increasing the incidence of aortic atherosclerosis (watanabe et al., ) . in contrast, the st. thomas hospital strain has a normal functioning ldl receptor but still maintains a hypercholesterolemic state (laville et al., ) . genetically modified rabbits have been created via both intracytoplasmic injection (li et al., ) and retroviral vectors (hiripi et al., ) . this has resulted in a multitude of new strains to address interesting research questions. cardiovascular disease (lombardi et al., ; peng, ; sanbe et al., ; stanley et al., ) including models of long qt interval for exploration of treatments (biermann et al., ; jindal et al., ; liu et al., a; peng, ; sanbe et al., ; ziv et al., ) and atherosclerosis (araki et al., ; masson et al., ; tjwa et al., ) are the main focus of model development. strains have also been developed that express human recombinant proteins in rabbit milk (chrenek et al., ; dragin et al., ; hiripi et al., ; houser et al., ; lipinski et al., ; simon et al., ; soler et al., ) . this ability can be passed down for multiple generations (chrenek et al., ; dragin et al., ) . these human proteins have resulted in antigen production for rotavirus vaccine creation, human factor viii that could be used to treat hemophilia (chrenek et al., ; krylov et al., ; simon et al., ) and human growth hormone that could supplement a deficiency in that hormone (lipinski et al., ) . rabbits that express enhanced green fluorescent protein (egfp) in various tissues have been created for the purpose tracking cells, which is important for tissue engineering and regenerative medicine studies takahashi et al., ; yin et al., ) . the mouth of the rabbit is relatively small, and the oral cavity and pharynx are long and narrow. the dental formula is i / , c / , pm / , m - / × = or teeth. a small pair of incisors is present directly caudal to the primary maxillary incisors and is referred to as 'peg' teeth. the peg teeth are used along with the primary incisors to bite and shear food. the absence of second incisors has been noted in some rabbit colonies as a dominant trait (i /i or i /i ). the teeth of rabbits erupt continuously throughout life and therefore will continue to grow unless normal occlusion and use are sufficient to wear teeth to a normal length. molars do not have roots and are characterized by deep enamel folds. rabbits normally masticate with a chewing motion that facilitates grinding of food by movement of the premolars and molars from side to side and front to back. the rabbit has four pairs of salivary glands, including the parotid, submaxillary, sublingual, and zygomatic. the parotid is the largest and lies laterally just below the base of the ear. the zygomatic salivary gland does not have a counterpart in humans. the esophagus of the rabbit has three layers of striated muscle that extend the length of the esophagus down to, and including, the cardia of the stomach. this is in contrast to humans and many other species, which have separate portions of striated and smooth muscle along the length of the esophagus. there are no mucous glands in the esophagus of the rabbit. although the stomach of the rabbit holds approximately % of the volume of the gastrointestinal tract, it is never entirely empty in the healthy rabbit. the gastric contents often include a large amount of hair ingested as the result of normal grooming activity. the stomach is divided into the cardia, fundus, and pylorus. the liver has four lobes. the gallbladder is located on the right. from the liver, the common bile duct empties into the duodenum posterior to the pylorus. rabbits produce relatively large amounts of bile compared to other common species. the pancreas is diffuse within the mesentery of the small intestine and enters the duodenum - mm distal to the common bile duct. the small intestine of the rabbit is short relative to that of other species and comprises approximately % of the total length of the gastrointestinal (gi) tract. because the gi tract of the rabbit is relatively impermeable to large molecules, kits receive most of their passive immunity via the yolk sac prior to birth rather than by colostrum. peyer's patches are found along the ileum, particularly near the cecal junction. the sacculus rotundus is a large bulb of lymphoid tissue located at this junction. the large intestine includes the cecum, the ascending colon, the transverse colon, and the descending colon. the ileocecal valve regulates flow of chyme into the cecum and retards reverse flow back into the ileum. the cecum is very large with a capacity approximately -times that of the stomach. the cecum ends in a blind sac, the appendix. the colon is divided into proximal and distal portions by the fusus coli, which serves to regulate the elimination of hard versus soft fecal pellets. hard pellets comprise about two-thirds of the fecal output. soft pellets, or 'cecotrophs,' have a high moisture content and are rich in nitrogen-containing compounds (ferrando et al., ) and the Β vitamins niacin, riboflavin, pantothenate, and cyanocobalamin. rabbits consume cecotrophs directly from the anus to obtain significant nutritional benefit. soft pellets are sometimes termed 'night feces,' since they are generally produced at night in domestic rabbits. in contrast, the circadian rhythm of cecotrophy is reversed in wild rabbits, occurring during the day when the animals are in their burrows (hornicke, ) . nostrils of rabbits are well equipped with touch cells, and they have a well-developed sense of smell. nasal breathing in rabbits is characterized by twitching of the nostrils at rates varying from to times per minute, although twitching may be absent in the relaxed rabbit. it has been speculated that inspiration occurs as the nostril moves up and that this serves to direct the flow of air over the turbinate bones where the olfactory cells are most concentrated. the musculature of the thoracic wall contributes little to respiratory efforts. instead, rabbits rely mostly on the activity of the diaphragm. because of this, artificial respiration is easily performed by alternating the head of the rabbit between the up and down positions, - times per minute, while holding the animal. compression and release of the chest wall is an ineffective means of artificial respiration in the rabbit. the pharynx of the rabbit is long and narrow, and the tongue is relatively large. these features make endotracheal intubation difficult. the procedure is further laboratory animal medicine complicated by the propensity of the rabbit to laryngospasm during attempts to intubate the trachea. the rabbit lungs consist of six lobes. both right and left sides have cranial, middle, and caudal lobes, with the right caudal being further subdivided into lateral and medial portions. flow volume of air to the left lung is higher than that to the right due to the lower resistance of the proximal airways per unit volume (yokoyama, ) . in rabbits, lung volume increases with age, in contrast to that of humans and dogs, in which it decreases. bronchial-associated lymphoid tissue (balt) is present as distinct tissue. a unique feature of the cardiovascular system of the rabbit is that the tricuspid valve of the heart has only two cusps, rather than three as in many other mammals. a small group of pacemaker cells generate the impulse of the sinoatrial (sa) node in the rabbit, a feature that facilitates precise determination of the location of the pacemaker (bleeker et al., ; hoffman, ; west, ) . the sa and atrioventricular (av) nodes are slender and elongated, and the av node is separated from the annulus fibrosus by a layer of fat (truex and smythe, ) . additional unique anatomic features of the cardiovascular system of the rabbit have been utilized to advantage. the aortic nerve subserves no known chemoreceptors (kardon et al., ; stinnett and sepe, ) and responds to baroreceptors only. because the aortic nerve, which becomes the depressor nerve, runs alongside but separate from the vagosympathetic trunk, it lends itself readily to implantation of electrodes (karemaker et al., ) . the blood supply to the brain is restricted mainly to the internal carotid artery. blood supplied via the vertebral arteries is limited. the aorta of the rabbit demonstrates rhythmic contractions that arise from neurogenic stimulation in a pattern related to the pulse wave (mangel et al., ) . the kidneys of the rabbit are unipapillate in contrast to those of most other mammals, which are multipapillate. this feature increases the ease with which cannulization is performed. the right kidney lies more cranial than the left. glomeruli increase in number after birth in rabbits, whereas all of the glomeruli are present at birth in humans (smith, ) . ectopic glomeruli are normal in the rabbit (steinhausen et al., ) . blood vessels that perfuse the medulla remain open during many conditions under which vasoconstriction of the cortical tissue occurs; thus, the medullary tissue may be perfused, while the cortex is ischemic (trueta et al., ) . the urine of adult rabbits is typically cloudy due to a relatively high concentration of ammonium magnesium phosphate and calcium carbonate monohydrate precipitates (flatt and carpenter, ) . the urine may also take on hues ranging from yellow or reddish to brown. in contrast, the urine of young rabbits is typically clear, although healthy young rabbits may have albuminuria. the urine is normally yellow but can also take on reddish or brown hues once animals begin to eat green feed and cereal grains. normal rabbits have few cells, bacteria, or casts in their urine. the ph of the urine is typically alkaline at about . (williams, ) . a normal adult rabbit produces approximately - ml/kg of urine daily (gillett, ) , with does urinating more copiously than bucks. the urethral orifice of the buck is rounded, whereas that of the doe is slit-like. this feature is useful for distinguishing the sexes. the testes of the adult male usually lie within the scrotum; however, the inguinal canals that connect the abdominal cavity to the inguinal pouches do not close in the rabbit. for this reason, the testes can easily pass between the scrotum and the abdominal cavity. this feature necessitates closure of the superficial inguinal ring following orchiectomy by open technique to prevent herniation. the reproductive tract of the doe is characterized by two uterine horns that are connected to the vagina by separate cervices (bicornuate uterus). a common tube, the urogenital sinus or vestibulum, is present where the urethra enters the vagina. the placenta is hemochorial, and maternal blood flows into sinus-like spaces where the transfer of nutrients and other substances to the fetal circulation occurs (jones and hunt, ) . inguinal pouches are located lateral to the genitalia in both sexes. the pouches are blind and contain scent glands that produce white to brown secretions that may accumulate in the pouch. the metabolic rate of endotherms is generally related to the body surface area. including the ears, the rabbit has a relatively low metabolic rate (mr); however, if the surface area of the ears is discounted, the mr of the rabbit is similar to that of other endotherms. neonatal rabbits have an amount of body fat comparable to that of the human infant ( % of body weight) (cornblath and schwartz, ) . the neonatal rabbit is essentially an ectotherm until about day (gelineo, ) . the glucose reserves of the neonatal rabbit are quickly depleted, usually within about h after birth (shelley, ) . the fasting neonatal rabbit quickly becomes hypoglycemic and ketotic (callikan and girard, ) . the normal rectal temperature of the adult new zealand white rabbit at rest is approximately . - . °c (ruckebusch et al., ) . the ears serve an important thermoregulatory function. because they have a large surface area and are highly vascular with an extensive arteriovenous anastomotic system, the ears help the rabbit sense laboratory animal medicine and respond to cold versus warm temperatures (kluger et al., ) . in addition, the ears serve as a countercurrent heat-exchange system to help adjust body temperature. early studies found that the body of the adult rabbit ( kg body weight) consists of greater than % water ( %), with a half-time turnover of about . days and a loss of about ml daily (richmond et al., ) . the amount of water ingested varies with the amount and type of feed consumed and the environmental temperature. in general, rabbits will drink more water when consuming dry, pelleted feed than when consuming foodstuffs high in moisture, such as fresh greens. conversely, rabbits deprived of water will decrease food consumption. after days of complete water deprivation, food intake falls to less than % of normal (cizek, ) . normal values for various systems and parameters are provided as a general indication for these values in the rabbit. it is important to recognize, however, that most of these values have been obtained through the study of adult new zealand white rabbits. as with any experiment, values can vary significantly between breeds, laboratories, methods of sampling and measurement, and individual rabbits due to age, sex, breed, health, handling, and husbandry (hewitt et al., ; lidena and trautschold, ; mitruka and rawnsley, ; wolford et al., ; yu et al., ) . for this reason, individual laboratories should strive to establish their own normal values, whenever possible. values for hematologic parameters are shown in table . . these values represent those typical of adult new zealand white rabbits. in general, males have slightly greater hematocrit and hemoglobin values than females (mitruka and rawnsley, ) . anisocytosis is normal and accounts for variation in reported values for red blood cell diameter (sanderson and phillips, ) . reticulocyte values are usually between % and % in healthy rabbits (corash et al., ) . the neutrophil of the rabbit is sometimes referred to as a 'pseudoeosinophil' or 'heterophil,' due to the presence of red-staining granules in the cytoplasm. the heterophil ( - mm in diameter) is, however, smaller than the eosinophil ( - mm in diameter) (sanderson and phillips, ) . in addition, the red granules of the heterophil are smaller than the red granules of the eosinophil. the nucleus of the eosinophil may be either bilobed or horseshoe-shaped. as mentioned earlier, chemistry values can vary because of a number of factors. for this reason, each laboratory should establish its own normal values. aspartate aminotransferase (ast) is present in the liver, heart, skeletal muscle, kidney, and pancreas. collection of blood samples in rabbits by decapitation, cardiac puncture, or aortic incision, or the use of restraint that causes exertion will elevate ast levels due to muscle damage (lidena and trautschold, ) . similarly, levels of creatinine kinase are sensitive to muscle damage since that enzyme is present in the skeletal muscle, brain, and heart (lidena and trautschold, ; mitruka and rawnsley, ) . although most mammals have two isoenzymes (intestinal and a liver/kidney/bone form) of alkaline phosphatase (ap), rabbits are unique in having three forms of ap, including an intestinal form and two forms that are both present in the liver and the kidney (noguchi and yamashita, ) . values for blood and serum chemistry are shown in table . . cardiovascular and respiratory functions are often altered with experimental manipulation, anesthesia, or disease. normal values for these parameters and other miscellaneous biologic characteristics of the rabbit are listed in table . . values obtained from the following sources: burns and delannoy ( ) , gillett ( ) , kabata et al. ( ) , mitruka and rawnsley ( ), and woolford et al. ( ) . laboratory animal medicine rabbits are strictly herbivorous with a preferred diet of herbage that is low in fiber and high in protein and soluble carbohydrate (cheeke, ; cheeke, ) . rabbits will generally accept a pelleted feed more readily than one in meal form. when a meal diet is needed, a period of adjustment should be allowed for the rabbits to accommodate to the new diet. examples of adequate diets are shown in table . . the requirement for fiber in the diet of rabbits has been reviewed (gidenne, ) . fiber is especially important in the early postweaning period when low fiber intake is associated with an increase in digestive disorders (gidenne, ) . the exact nutrient requirements for individual rabbits vary with age, reproductive status, and health of the animal. on occasion, the need arises for use of highly purified diets. a suggested purified diet has been described elsewhere (subcommittee on rabbit nutrition, ) . it should be noted that overfeeding of values obtained from the following sources: burns and delannoy ( ) , fox ( ) , gillett ( ) , kraus et al. ( ), and loeb and quimby ( ) . values obtained from the following sources: barzago et al. ( ) , curiel et al. ( ) , gillett ( ) , kozma et al. ( ) , sanford and colby ( ) , suckow and douglas ( ), and zurovsky et al. ( ) . laboratory animal medicine laboratory rabbits resulting in obesity is common, but can be prevented by either reducing the amount of feed or by providing a low-energy, high-fiber maintenance diet (donnelly, ) . as mentioned earlier, rabbits engage in cecotrophy, and by doing so supplement their supply of protein and Β vitamins (carabaño et al., ; gidenne et al., ) . rabbits fed a diet high in fiber ingest a greater quantity of cecotropes than those on a lower fiber diet (fekete and bokori, ) . unlike most other species, both calcium absorption in the small intestine and serum calcium levels increase in proportion to the amount of calcium in the diet (cheeke, ) . prolonged feeding of diets high in calcium, such as those with a high level of alfalfa meal, can result in renal disease. consumption of diets containing excessive vitamin d can result in calcification of soft tissues, including the liver, kidney, vasculature, and muscles (besch-williford et al., ; lebas, ) . diets that are either too high or too low in vitamin a can result in reproductive dysfunction and congenital hydrocephalus (cheeke, ; digiacomo et al., ) . the exact requirement for vitamin a in the rabbit has not been determined; however, a level of - , iu/kg of diet is generally adequate (lebas, ) . vitamin e deficiency has been associated with infertility, muscular dystrophy, fetal death, neonatal death, and colobomatous microphthalmos in rabbits (lebas, ; nielsen and carlton, ; ringler and abrams, ; ringler and abrams, ) . mcdowell ( ) suggested that serum vitamin e levels of less than . μg/ml are indicative of hypovitaminosis e. relative to other species, rabbits have a high water intake. in general, daily water intake is approximately ml/kg of body weight. consumption of water is influenced by environmental temperature, disease states, and feed composition and intake (cizek, ; tschudin et al., ) . consumption of diets high in dry matter results in increased water intake (tschudin et al., ) . water consumption also increases with food deprivation. rabbits are social animals and attempts at group housing often meet with success, although mature males will fight and can inflict serious injury on one another (love, ; podberscek et al., ; whary et al., ) . group-penned female rabbits allowed to choose between single or paired housing prefer being in the same cage with other rabbits (huls et al., ) . in general, rabbits are timid and nonaggressive. some animals will display defensive behavior, typically characterized by thumping the cage floor with the rear feet, biting, and charging toward the front of the cage when opened. laboratory-housed rabbits demonstrate diurnal behavior, in contrast to the nocturnal pattern exhibited by wild rabbits (jilge, ) . the ethogram of the laboratory rabbit has been described (chu et al., ; gunn and morton, ) . the most common behaviors of individually housed rabbits included lie alert, doze, groom, sleep, and eat. individually housed rabbits were inactive the majority of the time (gunn and morton, ) . individually housed female rabbits showed an increase in abnormal behaviors compared to pair-housed rabbits (chu et al., ) . rabbits housed in pairs in double-wide cages locomoted more than individually housed rabbits (chu et al., ). the age of puberty varies with the breed of rabbit. puberty generally occurs at - months of age in small breeds, - months in medium breeds, and - months in large breeds (donnelly, ) . female new zealand white rabbits reach maturity at months of age and males at - months. the breeding life of a doe typically lasts approximately - years, although some remain productive for up to or years. in later years, litter sizes usually diminish. in comparison, most bucks will remain reproductively useful for an average of - years. because does often will engage in reproductive behavior before being able to ovulate, it is advisable not to breed does until they are fully grown. does do not have a distinct estrous cycle, but rather demonstrate a rhythm with respect to receptivity to the buck. receptivity is punctuated by periods ( - days every - days) of anestrus and seasonal variations in reproductive performance (hafez, ) . during periods of receptivity, the vulva of the doe usually becomes swollen, moist, and dark pink or red. receptivity of the doe is usually signaled by lordosis in response to the buck's attempt to mount, vulvar changes as described above, restlessness, and rubbing of the chin on the hutch or cage (donnelly, ) . vaginal cytology is generally not useful for determination of estrus or receptivity in the rabbit. typically, the doe is brought to the buck's cage for breeding, since the doe can be very territorial and may attack the male in her own quarters. a period of - min is usually sufficient to determine compatibility of the doe and buck. if receptive, the doe will lie in the mating position and raise her hindquarters to allow copulation. if fighting or lack of breeding is observed, the doe may be tried with another buck. a single buck is usually sufficient to service - does. ovulation is induced and occurs approximately - h after copulation (donnelly, ) . up to % of does fail to ovulate following copulation. ovulation can also be induced by administration of luteinizing hormone (kennelly and foote, ) , human chorionic gonadotropin (williams et al., ) , or gonadotropic releasing hormone (foote and simkin, ) . does may be bred immediately after kindling; however, most breeders delay until after the kits have been weaned. success at postpartum breeding varies, but one can produce a large number of kits in a relatively short time period by foster nursing the young and rebreeding the doe immediately. while conventional breeding, nursing, and weaning schedules allow for only litters per year, early postpartum breeding allows for up to litters per year. pregnancy can often be confirmed as early as day of gestation by palpation of the fetuses within the uterus. radiographic procedures permit pregnancy determination as early as day . conception rates have been observed to have an inverse relationship with ambient temperature but not light cycle. gestation in rabbits usually lasts for - days (donnelly, ) . does beyond - weeks of gestation will usually refuse a buck. does begin hair pulling and nest building during the last - days of gestation (donnelly, ) . a nesting box with shredded paper or other soft material such as straw should be provided to the doe several days prior to the expected kindling (parturition) date. the doe will usually line the box with her own hair. the nesting box should not be placed in the corner of the cage where the individual doe has been observed to urinate. pseudopregnancy is common in rabbits and can follow a variety of stimuli, including mounting by other does, sterile matings by bucks, administration of luteinizing hormone, or the presence of bucks nearby. in such circumstances, ovulation is followed by a persistent corpus luteum that lasts - days. the corpus luteum or corpora lutea secretes progesterone during this time, causing the uterus and mammae to enlarge. the doe may have the appearance of a normally pregnant rabbit. toward the end of pseudopregnancy, many does will begin to pull hair as part of ritual nest-building behavior. the process of parturition is referred to as 'kindling' when it relates to rabbits. kindling normally occurs during the early morning hours and takes approximately - min. impending kindling is often signaled by nest building and decreased food consumption during the preceding - days. both anterior and breech presentations are normal in the rabbit. fetuses retained beyond days generally die and may harm future reproductive ability of the doe if not expelled. the average number of kits born is seven to nine per litter, although smaller litters and litters of up to kits are not uncommon. breed, parity, nutritional status, and environmental factors influence litter size. polish rabbits usually have fewer than four kits per litter; dutch or flemish giant, four to five; and new zealand white, eight to ten. after the young have been cleaned following parturition, the doe typically consumes the placenta. cannibalism of the young by the doe sometimes occurs and may be related to environmental or hereditary factors or due to environmental stressors. does usually have either four or five pairs of nipples, whereas bucks have none. during the last week of pregnancy, marked development of the mammary gland occurs. the doe normally nurses the kits once daily for several minutes, usually in the early morning or in the evening, regardless of how many kits are present or laboratory animal medicine how many times they attempt to suckle. milk yield is normally between and g/day. during the first week of life, kits consume - g of milk per day. milk intake increases gradually to a maximum of g/day between and days of age (gidenne et al., ) . maximum output occurs at weeks following kindling and then declines during the fourth week. rabbit milk contains approximately . % protein, % fat, % lactose, and . % minerals. nursing may last - weeks. kits may begin consuming solid food by weeks of age, with weaning generally occurring by - weeks of age. the facilities present in most modern research animal facilities would be suitable for housing rabbits. general construction should include adequate heating, ventilation, and air conditioning to house rabbits at appropriate temperature and humidity. in addition, lighting should be adequate to allow easy visualization of the rabbits. surfaces, such as the floors, walls, and ceilings, should be easily sanitizable (national research council, ) . rabbit cages should provide a safe environment with easy access to food and water. adults can be caged individually or in compatible groups and should have sufficient floor space to lie down and stretch out. in the united states, minimum cage sizes are determined by the animal welfare act (awa) and the guide for the care and use of laboratory animals (guide). in both cases, sizes vary with the weight of the animal. currently, the awa regulations and the guide require . ft of floor space and in of cage height for rabbits weighing - kg (national research council, ) . cages should be constructed of durable materials that will resist corrosion and harsh detergents and disinfectants used in cleaning. consequently, in the research environment, rabbit cages are most often constructed of stainless steel or plastics. rabbits are usually housed in cages with mesh or slatted floors to permit urine and feces to drop through into a catch pan. mesh floors with catch pans do not prevent rabbits from engaging in the normal practice of coprophagy. information on environmental enrichment of laboratory rabbits has been published (baumans, ) . the behavior of rabbits in conventional cages was compared to that of rabbits provided with enriched cages that contained shelter, a shelf, and increased vertical space. rabbits in conventional cages were more restless, groomed excessively, exhibited more bar-gnawing, and were more timid than those housed in enriched cages (hansen and berthelsen, ) . indeed, fecal glucocorticoid levels in rabbits declined when they were provided with a wooden structure for resting and gnawing (buijs et al., ) . rabbits will play with objects placed in their cages. huls et al. ( ) noted that rabbits would use wooden sticks, wooden rings, and brass wire balls as toys. rabbits provided with objects (toys) spent significantly more time chewing than rabbits without toys (poggiagliolmi et al., ) . female rabbits can also be housed in compatible pairs or groups. singly housed female rabbits exhibited more abnormal behaviors compared to pair housed rabbits (chu et al., ) . group housing of unfamiliar males is not recommended because of the likelihood of fighting and injury. rabbits are optimally housed in cooler room temperatures than most other common species of laboratory animals. the guide recommends that temperatures in rabbit rooms be maintained between and °f. no specific illumination requirements for rabbits have been described. it is common practice to provide rabbits with - h of light in the light-dark cycle. in breeding colonies, females should be provided with - h of light. rabbits are easily startled by sudden, loud noises. for this reason, they should not be housed near noisy species such as dogs or monkeys, nor should they be housed near noise-generating operations such as the cage-wash area. catch pans should be cleaned as often as necessary to prevent the formation of ammonia. cages are generally sanitized on at least a weekly basis. rabbit urine contains large amounts of protein and minerals, and often forms deposits on cages and catch pans. it is common practice to soak equipment having urine deposits in acid washes to remove the scale before washing. ammonia production in rabbit rooms can be a significant problem; therefore, rabbit rooms should be ventilated at - air changes per hour (national research council, ) . it is also important to change excreta pans often to prevent the buildup of ammonia. etiology pasteurella multocida is a gram-negative nonmotile coccobacillus that causes pasteurellosis, also known as 'snuffles', the primary respiratory disease affecting domestic rabbits (deeb and digiacomo, ; guo et al., ) . historically, serogroup a isolates have laboratory animal medicine been associated with pneumonic and septicemic pasteurellosis in laboratory rabbits; however, capsular type a is also isolated from rabbits that appear clinically healthy (confer et al., ; el tayeb et al., ) . clinical signs pasteurella multocida infection is often subclinical, but pasteurellosis may cause fever, coughing, dyspnea, rhinitis (nasal discharge (serous to mucopurulent), sneezing, and upper airway stentor), pneumonia, otitis, septicemia, meningitis, abscesses (of viscera and subcutaneous sites), and death (al-lebban et al., ; confer et al., ; franco and cronin, ; guo et al., ; suckow et al., ; wilkie et al., ) . pasteurellosis may also be associated with pericarditis, pleuritis, sinusitis, dacryocystitis, conjunctivitis, iritis/ uveitis, phlegmon, mastitis, endometritis, pyometra, salpingitis, and orchitis (deeb and digiacomo, ; ferreira et al., ; stahel et al., ; williams, ) . epizootiology p. multocida can be endemic in rabbitries and is carried in the rabbit's nasal cavity (confer et al., ; deeb et al., ; digiacomo et al., ; suckow et al., ) . transmission is by direct contact between rabbits (wilkie et al., ) . coinfection with bordetella bronchiseptica may be observed in clinically affected rabbits (deeb et al., ) . stress-related factors associated with pasteurellosis include crowded or unsanitary conditions, transportation, and high ammonia concentrations in the air (confer et al., ) . previous studies reported a high prevalence of p. multocida infection (jaslow et al., ) . colonization in immature rabbits occurs more commonly in the sinuses followed by the trachea, middle ears, and lungs (glass and beasley, ) . similar to cats and dogs, rabbits may transmit p. multocida infection to humans (per et al., ; silberfein et al., ) . a study utilizing repetitive extragenic palindromic pcr (rep-pcr) and sequencing determined that % of the isolates were characterized as p. multocida subsp. multocida, % as p. multocida subsp. septica, % as atypical subspecies of p. multocida, % as p. canis, and % as an unknown species of the family pasteurellaceae (stahel et al., ) . the pathogenesis of p. multocida has been reviewed (wilkie et al., ) . the ptfa gene, encoding a type fimbrial subunit and involved in bacterial fixation on the surface of epithelial cells, may be highly prevalent in p. multocida isolates from rabbits (ferreira et al., ) . the p. multocida toxin is a major virulence factor in atrophic rhinitis of rabbits and acts by causing constitutive activation of g proteins frymus et al., ; orth et al., ; suckow et al., ) . pathology the specific pathologic findings will vary with the site of infection, but the underlying host response is characterized by acute or chronic suppurative inflammation with the infiltration of large numbers of neutrophils. rhinitis and sinusitis are accompanied by a mucopurulent nasal exudate. neutrophil infiltration of the tissues is extensive. the nasal passages are edematous, inflamed, and congested, and there may be mucosal ulcerations. the turbinate bones may atrophy digiacomo et al., ) . purulent conjunctivitis may be present. pneumonia is primarily cranioventral in distribution. the lungs can exhibit consolidation, atelectasis, and abscess formation. a purulent to fibrinopurulent exudate is evident, and there may be areas of hemorrhage and necrosis. in some rabbits, fibrinopurulent pleuritis and pericarditis are prominent features (glavits and magyar, ) . this is probably due to elaboration of a heat-labile toxin in some strains of the bacteria . acute hepatic necrosis and splenic lymphoid atrophy are also seen in association with the pleuritis and pneumonia induced by toxigenic strains. otitis media is characterized by a suppurative exudate with goblet cell proliferation and lymphocytic and plasma cell infiltration. in female rabbits with genital tract infections, the uterus may be enlarged and dilated. in the early stages of infection, the exudate is watery; later it thickens and is cream-colored. the exudate contains numerous neutrophils. focal endometrial ulceration can be found (johnson and wolf, ) . in the male, the testes are enlarged and may contain abscesses. systemic and visceral abscesses are characterized by a necrotic center, an infiltrate made up of polymorphonuclear neutrophils, and a fibrous capsule. septicemia may only present as congestion and petechial hemorrhages in many organs. severe pleuritis with accumulation of fibrinopurulent exudate in the thoracic cavity, serous rhinitis and tracheitis, acute hepatitis with necrotic foci in the parenchyma, and atrophy of lymphoid organs and tissues have been observed after experimental p. multocida infection in rabbits (glavits and magyar, ) . diagnosis sterile swabs can be used to collect samples from the nares or nasal cavity of rabbits for culture (ferreira et al., ; jaslow et al., ) . nasal lavage can also be used as a culture sample to isolate pasteurella (suckow et al., ) . p. multocida isolates can be classified into five serogroups based on capsular antigens (a, b, d, e, and f) and into serotypes based on somatic lps antigens (adler et al., ; liu et al., b; manning, ) . biochemical characterization of isolates may show high heterogeneity; however, rep-pcr and phylogenetic analysis using s ribosomal rna and rpob genes can be used for precise characterization of rabbit isolates (stahel et al., ) . classification of p. multocida into subspecies and/or by virulence profiles is useful for epidemiological investigations (ferreira et al., ; stahel et al., ) . random amplified polymorphic laboratory animal medicine dna pcr (rapd-pcr) has also been used to subtype rabbit p. multocida isolates (al-haddawi et al., ; dabo et al., ; williams et al., ) . pcr can detect capsule biosynthesis genes cap a, b, d, e, and f as well as virulence-related genes (ferreira et al., ) . serological tests can be used to detect antibodies against p. multocida (deeb et al., ; delong et al., ; digiacomo et al., ; glass and beasley, ; lukas et al., ) . differential diagnoses if radiographs reveal an internal mass associated with p. multocida infection, the differential diagnoses should include abscess, granuloma, neoplasia, and parasitic cyst (franco and cronin, ) . treatment, prevention, and control previous studies have investigated the use of vaccines to protect rabbits against p. multocida infection (confer et al., ) . immunization of rabbits with inactivated heat-labile p. multocida toxin or a commercial swine p. multocida bacterin-toxoid conferred protective immunity against challenge with the p. multocida heat-labile toxin (suckow, ; suckow et al., ) . a vaccine administered intranasally stimulated immunity against experimental pneumonic pasteurellosis and significantly reduced nasal bacterial counts (confer et al., ) . oral immunization of rabbits with a p. multocida thiocyanate extract (pte) in microparticles was immunogenic and significantly reduced the colony-forming units of homologous p. multocida recovered from the lungs and nasopharynx (suckow et al., ) . protective immunity to a heterologous strain of p. multocida can be achieved by vaccinating rabbits with pte via the subcutaneous route (suckow et al., ) . a p. multocida bacterin known as bunnyvac is currently licensed by the usda and is intended to be effective in preventing death and limiting disease due to pasteurella in rabbits. bunnyvac is manufactured by colorado serum company and distributed by pan american veterinary laboratories (http://pavlab. com/). control of pasteurellosis in rabbitries entails testing and culling animals that are positive for pasteurella spp. (ferreira et al., ) . furthermore, rabbits free of pasteurella and other infectious agents can be obtained by enrofloxacin treatment and through cesarean section or hysterectomy rederivation (pleasants, ; suckow et al., ; syukuda, ) . commercial suppliers of laboratory rabbits tend to exclude pasteurella from their colonies. treatment with antibiotics should be based on culture and sensitivity. antibiotic treatment of affected rabbits can alleviate clinical signs or delay disease progression but may not eradicate the disease (el tayeb et al., ; ferreira et al., ) . antibiotic treatment may suppress virulence gene expression without complete elimination of p. multocida . internal abscesses may not be treatable using antibiotics (franco and cronin, ) . penicillin therapy does not seem to be effective against pasteurella infection and may also lead to diarrhea and clostridium difficile colitis in rabbits (jaslow et al., ; rehg and lu, ) . one study from brazil determined that all tested strains were sensitive to ceftiofur, florfenicol, norfloxacin, enrofloxacin, ciprofloxacin, tetracycline, and doxycycline (ferreira et al., ) . other studies also indicate that fluoroquinolones are useful for the treatment of p. multocida infection in rabbits (abo-el-sooud and goudah, ; broome and brooks, ; franco and cronin, ; hanan et al., ; okewole and olubunmi, ) . oral ciprofloxacin ( mg/kg per day for days) has been used in rabbits (hanan et al., ) . research complications pasteurellosis can cause considerable economic losses (el tayeb et al., ; ferreira et al., ; stahel et al., ) and has the potential to affect different types of research studies using rabbits due to the multisystemic nature of the disease, and the possibility of high morbidity and mortality. therefore, pasteurella should be excluded from laboratory rabbit colonies. the class clostridia belongs to the phylum firmicutes (yutin and galperin, ) . recent genomic analyses suggest assigning some clostridium species that fall outside the family clostridiaceae into new genera. the genera tyzzerella, erysipelatoclostridium, and peptoclostridium have been proposed for c. piliforme, c. spiroforme, and c. difficile, respectively (yutin and galperin, ) . etiology c. piliforme is a pleomorphic, gramnegative, spore-forming, motile, obligate intracellular rod-shaped bacterium that causes tyzzer's disease and infects various animals including mice, nonhuman primates, gerbils, rats, rabbits, and others (allen et al., ; ganaway et al., ; pritt et al., ) . infection has also been reported in a human patient with human immunodeficiency virus- (smith et al., ) . phylogenetic analyses determined that microorganisms identified as c. piliforme form three clusters within a single clade and that the nearest related distinguishable species is c. colinum (feldman et al., ) . clinical signs the first reported outbreaks in laboratory rabbits described profuse watery to mucoid diarrhea usually followed by death in - h in -to -week old rabbits (allen et al., ) . rabbits in affected litters usually died within a week after the first fatality (allen et al., ) . the dams of affected litters occasionally died after a diarrheal disease that was more protracted than that of the offspring (allen et al., ) . these outbreaks lasted for - months and affected multiple rabbit rooms. c. piliforme infection may also be subclinical and transient as immunocompetent hosts may clear the infection (ganaway et al., ; pritt et al., ) . weanling rabbits with the acute form of tyzzer's disease exhibit diarrhea, listlessness, anorexia, and dehydration usually followed by death within h (cutlip et al., ) . the mortality rate in clinically affected rabbits was estimated to be - % (cutlip et al., ) . anorexia and stunting were observed in chronic cases associated with intestinal stenosis (cutlip et al., ) . acute and chronic tyzzer's disease types have been described in rabbits; however, large numbers of 'attaching' escherichia coli were recovered from the cecum of most rabbits (prescott, ) . epizootiology the vegetative cell is the active stage responsible for the disease and depends on the intracellular environment (ganaway, ) . therefore, the spore, a resistant stage, appears to be the essential element in the transmission of tyzzer's disease (ganaway, ; ganaway et al., ) . contact with soiled bedding or diseased rabbits have been used experimentally to transmit the disease to other rabbits (allen et al., ) . it is possible that subclinically infected rabbits (carriers) may introduce the organism into a colony (allen et al., ; pritt et al., ) . in mice, increased susceptibility to infection has been associated with stress (allen et al., ) . furthermore, treatment with cyclophosphamide, cortisone, and prednisolone has been used experimentally to reproduce the disease in animals, suggesting that immunosuppression plays a role in pathogenesis (allen et al., ; cutlip et al., ; pritt et al., ) . animals stressed by poor environmental conditions including overcrowding and extreme temperatures can develop the disease (cutlip et al., ; wobeser et al., ). significant modifications of the intestinal flora and an impaired immune system may play a role in pathogenesis (licois, ) . c. piliforme may be transported from the intestine to the liver through the portal circulation and to the heart through the lymphatics (allen et al., ) . some c. piliforme isolates can induce cytopathic effects on cell cultures, and in vivo, concomitant infection with other enteric pathogens such as e. coli may contribute to the severity of the disease (prescott, ; riley et al., ) . pathology lesions can be found in the distal ileum, cecum, proximal colon, liver, and heart (allen et al., ) . intestinal lesions are common, and histologically are characterized by necrosis of the mucosa and edema of the submucosa and serosa (allen et al., ) . bacilli appear as bundles of parallel rods or as criss crossed sticks in the cytoplasm of epithelial cells distributed from the surface of the mucosa to the base of the glands (allen et al., ) . the lesions in the liver are punctate areas of parenchymal necrosis that appear grossly as white spots, usually ≤ mm in diameter. large numbers of bacilli are found in the cytoplasm of cells in the zone of transition between the necrotic lesion and the healthy parenchyma (allen et al., ) . myocardial lesions appear as white streaks . - mm wide and - mm long extending from the region of the left interventricular groove laterally across the left ventricle (allen et al., ) . in the myocardium, bacilli may be noted in partially degenerated and normal looking cells at the sharply delineated borders of the lesions (allen et al., ) . diagnosis c. piliforme cannot be cultured in artificial (cell-free) media making its diagnosis difficult (allen et al., ; cutlip et al., ; ganaway et al., ; niepceron and licois, ) . other bacteria, including e. coli, have been isolated from the liver of diseased rabbits and are considered secondary invaders (allen et al., ; cutlip et al., ) . the isolation of the tyzzer's agent using liver extract agar has been described (kanazawa and imai, ) . c. piliforme can be grown in primary monolayer cultures of adult mouse hepatocytes, in mouse fibroblasts, in rat hepatocytes, and in embryonated eggs (craigie, ; duncan et al., ; ganaway et al., ; kawamura et al., ; pritt et al., ; riley et al., ) . serology for c. piliforme is commonly used for surveillance of laboratory animals because it is rapid and inexpensive (pritt et al., ) . immunofluorescence assay (ifa) and multiplexed fluorometric immunoassay (mfia) have been utilized (pritt et al., ) . in addition, c. piliforme pcr assays have been developed (feldman et al., ; gao et al., ; niepceron and licois, ; pritt et al., ) . clostridium piliforme seropositive rabbits may be negative for the organism by pcr and histopathological evaluation (pritt et al., ) . therefore, positive serological findings are not sufficient for a definitive diagnosis of c. piliforme infection and pcr testing and/or histopathology should be used for confirmation (pritt et al., ) . definitive diagnosis is based on identification of typical gross lesions and histological demonstration of intracellular c. piliforme at the periphery of the necrotic foci (niepceron and licois, ; pritt et al., ) . giemsa solution (ph ), warthin-starry silver method, levaditi silver method, and the periodic acid-schiff (pas) reaction have been used to demonstrate c. piliforme (allen et al., ; cutlip et al., ) . different morphologic forms of c. piliforme can be observed microscopically (allen et al., ; ganaway et al., ) . differential diagnoses clinically, other diarrheal diseases of rabbits can be included in the differential diagnoses. grossly, the multifocal white areas on the liver could be from eimeria stiedae infection (hepatic coccidiosis). treatment, prevention, and control for prevention, avoid introduction of rabbits of unknown c. piliforme status into a colony. minimize stress-related factors especially in young animals. good husbandry practices including regular bedding changes and disinfection laboratory animal medicine should decrease the likelihood of spreading c. piliforme in a colony. in one report, a tyzzer's disease outbreak was observed - days after rabbits were weaned and transferred to a facility in which the temperature fluctuated from to °c. the outbreak was controlled by transferring weanling rabbits to a building maintained at the same temperature as the breeder house ( - °c) (cutlip et al., ) . spores treated with heat ( or °c) or with either peracetic acid ( %) in a wetting agent (sodium alkylarylsulfonate) or sodium hypochlorite solution ( . %) for min lose infectivity (ganaway, ) . however, spores do not lose infectivity when treated with a phenolic germicidal agent, ethanol, or quaternary ammonium compounds (containing % or % benzalkonium chloride) (ganaway, ) . sodium hypochlorite solution ( . %) has been recommended as a surface disinfectant in animal facilities for prevention and control of tyzzer's disease (ganaway, ) . the sensitivity of c. piliforme to antibiotics has been investigated (kanazawa and imai, ) . in one study, none of the antibacterials tested were completely inhibitory (ganaway et al., ) . group treatment of rabbits with tetracyclines in the drinking water and food was effective in lowering the incidence of diarrhea and death (prescott, ) . research complications the high morbidity and mortality associated with tyzzer's disease can affect the overall population of rabbits in a colony thereby decreasing the number of rabbits suitable or available for experimentation. in addition, research studies involving experimental infection with enteric pathogens in rabbits may be confounded by c. piliforme-associated intestinal pathology. enterotoxemia refers to conditions of the bowel caused by toxigenic clostridia (carman and evans, ) . diagnosis of enterotoxemia should be based on culture of a toxigenic clostridium and demonstration of the toxin from the intestinal contents of the diseased animal (carman and evans, ; songer, ) . i. clostridium spiroforme etiology c. spiroforme is a gram-positive, sporebearing, helically coiled, semicircular, anaerobic bacterium that can produce iota toxin (borriello and carmen, ; carman and borriello, ; peeters et al., ) . the disease caused by c. spiroforme is known as 'iota enterotoxemia' (keel and songer, ; peeters et al., ) . clinical signs diarrhea, fecal soiling of the perineum, and cyanosis may be observed (carman and borriello, ) . diarrhea may be peracute and may lead to 'spontaneous' death or a moribund state (carman and borriello, ) . epizootiology c. spiroforme is thought to be acquired from the environment (carman and evans, ; songer, ) . weaning is associated with spontaneous disease and administration of antibiotics can also induce the disease (borriello and carmen, ; carman and borriello, ) . a study determined that disease results from exposure of weanling rabbits to c. spiroforme and also from exposure of adult rabbits with a disrupted gut flora (due to clindamycin treatment) to c. spiroforme suggesting that this bacterium is not a normal component of the rabbit gut flora (carman and borriello, ) . c. spiroforme-mediated diarrhea may be favored by maldigestion initiated by infectious agents and/or nutritional factors (peeters et al., ) . other clostridia, e. coli (epec), viruses, and parasites, may coinfect rabbits (peeters et al., ) . the iota toxin of c. spiroforme binds the same host cell receptor as the iota toxin of c. perfringens and the binary toxin of c. difficile (papatheodorou et al., ) . poor hygiene, stress, and diet can influence pathogenesis of the disease (bain et al., ; songer, ) . pathology grossly, ceca may be enlarged with gas, may exhibit serosal hemorrhages, and have liquid contents (carman and borriello, ) . cecal contents may be stained with blood (peeters et al., ) . c. spiroforme is mainly associated with lesions in the cecum, but may also involve lesions in the proximal colon and distal ileum (keel and songer, ) . mucosal necrosis can be observed microscopically (keel and songer, ) . the mucosa of the ileum, cecum, and colon may be denuded. cellular debris, red blood cells, and fibrin may be found in the intestinal lumen. polymorphonuclear cell infiltration and edema can be found in the lamina propria and submucosa. epithelial degeneration and dilation were found in the renal tubules of some rabbits (peeters et al., ) . diagnosis gram staining of smears prepared from intestinal contents can be used to detect c. spiroforme (bain et al., ) . clostridial culture and toxin detection assays have been described (agnoletti et al., ; bain et al., ; borriello and carmen, ; peeters et al., ) . c. spiroforme can be isolated from the intestinal contents of rabbits by high-speed centrifugation (holmes et al., ) . pcr assays for c. spiroforme and the iota toxin (binary toxin) have been developed (drigo et al., ) . differential diagnoses the differential diagnoses should include other clostridia, e. coli, viruses, and parasites (peeters et al., ) . treatment, prevention, and control the iota toxin from c. spiroforme is neutralized by serum prepared against the iota toxin of c. perfringens type e (borriello and carmen, ; carman and borriello, ; songer, ) . prevention, via reduction of risk factors and prudent use of antibiotics, is probably more important laboratory animal medicine than treatment (agnoletti et al., ) . cholestyramine has been used to prevent experimental enterotoxemia induced by clindamycin in rabbits (lipman et al., ) . fecal flora transplants using nonpathogenic c. spiroforme or c. difficile have been suggested for competitive inhibition of toxigenic strains (carman and evans, ) . no commercial vaccines are available for rabbits; however, vaccination of weanling rabbits with a toxoid imparted protection to intraperitoneal challenge with iota toxin (songer, ) . administration of antibiotics and change in diet are usually the treatment for c. spiroforme infections (songer, ) . the antibiotic susceptibility of c. spiroforme has been investigated (agnoletti et al., ; carman and wilkins, ) . c. spiroforme can have a wide range of resistance to antimicrobial classes used in rabbit therapy (agnoletti et al., ) . feeding fresh meadow hay has been suggested (bain et al., ) . research complications the mortality due to enterotoxemia caused by c. spiroforme would be disruptive to ongoing studies. no other complications have been reported. ii. clostridium difficile etiology c. difficile is a gram-positive, sporeforming, anaerobic bacillus commonly associated with diarrhea and colitis in humans and animals (keel and songer, ) . clinical signs c. difficile infection may be associated with anorexia, depression, diarrhea, fecal-staining of the perineum, decreased fecal output, abdominal distention, and death (perkins et al., ; rehg and lu, ) . peracute death, without clinical signs, is also a common presentation in rabbits (keel and songer, ; perkins et al., ) . epizootiology the spread of c. difficile involves carrier animals that do not show clinical signs of disease (keel and songer, ) . the carrier state may depend on the age of the individual (keel and songer, ) . c. difficile is thought to be acquired from the environment due to persistent contamination with spores (keel and songer, ) . disease is associated with antibiotic treatment but can also develop spontaneously (without antibiotic treatment) (perkins et al., ; rehg and lu, ) . the disease may also occur after stressful events such as weaning, sudden dietary changes, lactation, kindling, and illness (perkins et al., ) . rabbits that have been recently weaned are the most susceptible (perkins et al., ) . newborn rabbits are resistant to c. difficile disease possibly due to the lack of receptors for the toxins (keel and songer, ) . similar to c. spiroforme, the pathogenesis is associated with disruption of the gut flora and with colonization and proliferation of toxigenic clostridium. pathology grossly, a fluid-filled cecum and colon may be found on necropsy (rehg and lu, ) . spontaneous disease in rabbits is associated with lesions in the small intestine, most commonly in the ileum (keel and songer, ) . in one study, the small intestine was distended with fluid and the cecum was distended with chyme (perkins et al., ) . c. difficile is also associated with hemorrhagic typhlitis in hares (dabard et al., ) . c. difficile causes severe jejunal lesions in rabbits, but cecal lesions may occur (keel and songer, ; perkins et al., ) . mural hemorrhages, mucosal necrosis, and submucosal edema have been observed (perkins et al., ) . toxins a (enterotoxin) and b (cytotoxin) act synergistically and are essential virulence factors of c. difficile that enter the cells through receptor-mediated endocytosis (keel and songer, ) . toxins a and b disrupt the actin cytoskeleton by disrupting rho-subtype intracellular signaling molecules that affect cellular function (keel and songer, ) . inflammation and neurogenic stimuli also are involved in the pathogenesis of c. difficile disease (keel and songer, ) . in addition to toxins a and b, some c. difficile strains produce an actinspecific adp-ribosyltransferase or binary toxin (stubbs et al., ) . diagnosis c. difficile isolation and toxin assays have been described (keel and songer, ; perkins et al., ; rehg and lu, ) . c. difficile selective agar is commercially available. the tissue culture cytotoxin assay for c. difficile toxin b is considered the 'gold standard' (belanger et al., ) . c. difficile toxin b can be neutralized with c. sordelli antiserum, but not with c. spiroforme antiserum (perkins et al., ) . commercially available enzyme immunoassays to detect c. difficile toxin(s) have been used to diagnose rabbit cases (garcia et al., ; perkins et al., ) . pcr assays have been developed (belanger et al., ; goldenberg et al., ; houser et al., ; pallis et al., ) . differential diagnoses the differential diagnosis of peracute death in rabbits should include infection with clostridium spp. and/or ehec infection (garcia et al., ; perkins et al., ) . treatment, prevention, and control as with c. spiroforme, the reduction of risk factors and the prudent use of antibiotics are recommended (agnoletti et al., ) . cholestyramine may also be used for prevention (lipman et al., ) . fecal flora transplants have been suggested and commercial probiotic strains are able to inhibit c. difficile and c. perfringens in vitro (carman and evans, ; schoster et al., ) . research complications the sporadic nature of deaths due to c. difficile infection is unlikely to result in significant complications to research. iii. clostridium perfringens c. perfringens type e produces alpha and iota toxins and is an laboratory animal medicine uncommon cause of enterotoxemia in rabbits (redondo et al., ; songer, ) . because of the similarity between the iota toxins of c. spiroforme and c. perfringens type e, detection of toxin alone for diagnostic purposes will not differentiate between the two organisms (songer, ) . pcr can be used for typing c. perfringens based on amplification of toxin genes (daube et al., ) . historically, a disease process associated with e. coli infection was known as colibacillosis. currently, e. coli is classified based on the virulence factors that are genetically encoded and expressed in the bacteria. different virulence factors are associated with different e. coli 'pathotypes'. pathotypes may be associated with three general clinical syndromes: enteric/diarrheal disease, urinary tract infections, and sepsis/meningitis (kaper et al., ) . the centers for disease control and prevention currently recognizes six pathotypes associated with diarrhea in humans: enteropathogenic e. coli (epec), shiga toxin (stx)-producing e. coli (stec; also known as enterohemorrhagic e. coli (ehec) or verocytotoxin-producing e. coli (vtec)), enterotoxigenic e. coli (etec), enteroaggregative e. coli (eaec), enteroinvasive e. coli (eiec), and diffusely adherent e. coli (daec) (http://www.cdc.gov/ecoli/general/). comparative genomic analyses identified genes that were isolateand pathovar-specific and clustered strains according to pathotypes (lukjancenko et al., ; rasko et al., ) . two more emerging pathotypes have been suggested: adherent invasive e. coli (aiec; associated with crohn's disease in humans) and shiga toxin-producing enteroaggregative e. coli (steaec; associated with a large outbreak of hemolytic uremic syndrome (hus) in europe) (clements et al., ) . of these pathotypes, epec and stec are associated with natural disease in rabbits (cantey and blake, ; garcia et al., ) . in addition, necrotoxigenic e. coli (ntec) are associated with disease in rabbits (blanco et al., ) . pathogenic animal and human strains are very closely related and have virulence genes in common (clermont et al., ) . therefore, it is important to determine which e. coli pathotype(s) are associated with disease in rabbits in order to characterize new diseases and/or more accurately diagnose, prevent, control, and treat the condition as well as for epidemiological investigations. etiology epec carry the eae gene that encodes intimin, a protein involved in induction of attaching and effacing lesions in the intestine. e. coli serotype o , also known as rdec- , is the prototype epec strain which was isolated from rabbits with diarrhea and has been used experimentally as a model to study epec-induced disease (cantey and blake, ) . epec is an important cause of potentially fatal infant diarrhea in developing countries (kaper et al., ; swennes et al., ) . clinical signs ehec o was isolated from an outbreak of bloody diarrhea and sudden death in dutch belted rabbits (fig. . ) (garcia et al., ) . acute diarrhea following shipment was associated with epec o :h infection in laboratory rabbits (swennes et al., ) . laboratory rabbits can be reservoir hosts of pathogenic e. coli without exhibiting clinical signs (garcía and fox, ; swennes et al., ) . patent or occult blood may be detected in the feces of infected rabbits (camguilhem and milon, ; garcia et al., ) . epizootiology epec and ehec can be enzootic in rabbit colonies and these bacteria are transmitted by the fecal-oral route (garcía and fox, ; swennes et al., ; swennes et al., ) . epec and ehec coinfections are possible (garcía and fox, ; garcia et al., ) . ehec are a subset of stec that carry stx gene(s) that encode stx(s) and also carry the eae gene that encodes intimin (melton-celsa et al., ) . rabbits can harbor stec strains and are recognized as their vectors and reservoir hosts (bailey et al., ; blanco et al., ; garcía and fox, ; kim et al., ; leclercq and mahillon, ; pohl et al., ; pritchard et al., ; scaife et al., ) . pathology grossly, paintbrush hemorrhages of the cecal serosa may be observed after experimental infection with epec (camguilhem and milon, ) . also, experimentally, the serosal surface of the cecum and/or proximal colon can develop petechial or echymotic hemorrhages and may become edematous and thickened (garcía et al., ) . histologically and ultrastructurally, attaching and effacing lesions with pedestal formation can be observed with epec or ehec infections (kaper et al., ; peeters et al., ) . enterocolitis, nephropathy, and thrombotic microangiopathy can be observed in ehec-infected rabbits (garcía et al., ) . diagnosis feces or intestinal contents can be enriched in broth and then cultured using blood agar, macconkey agar, or ehec-selective media such as sorbitol macconkey agar or raibow® agar (garcía and fox, ; tarr, ; tarr et al., ) . after isolation of e. coli in pure culture, samples can be biotyped using commercial methods such as the api® e strips (bio-mérieux). serotyping and molecular characterization of isolates can be performed by the e. coli reference center (http://ecoli.cas.psu.edu/) at the pennsylvania state university. pcr assays can be utilized to detect virulence factors characteristic of epec, ehec, or other pathogenic e. coli as well as for high-resolution genotyping for epidemiological studies (blanco et al., ; garcía and fox, ) . molecular characterization of stec strains can be performed by the stec center (http://www. shigatox.net/new/) at michigan state university. differential diagnoses the differential diagnoses should include other causes of diarrhea in rabbits including the clostridial diseases and intestinal coccidiosis. treatment, prevention, and control for prevention, avoid introduction of rabbits of unknown pathogenic e. coli status into a colony. rabbits should be screened by culture and e. coli isolates characterized for virulence factors by pcr. also, since it is known that ehec can contaminate plants and vegetables, laboratory personnel should be aware that rabbit feeds such as hay, alfalfa, and other greens have the potential to introduce enteric pathogens such as ehec into laboratory rabbits (berger et al., ; garcía and fox, ) . ehec o can survive for days in grass hay feed (davis et al., ) . cesarean section rederivation and antibiotic treatment have been suggested for eradication of pathogenic e. coli in rabbits (swennes et al., ) . a 'one health' approach should be incorporated to control ehec infections because outbreaks such as with ehec o in humans was linked to consumption of spinach contaminated by feral swine and was additionally isolated from domestic cattle, surface water, sediment, and soil (garcia et al., ) -a good example of integrating human, animal, and environmental health (monath et al., ) . antibiotic treatment should be based on culture and sensitivity. importantly, in humans infected with ehec, treatment with antibiotics is controversial due to the possibility of induction of stx-encoding bacteriophages and worsening of the clinical condition due to hemolytic uremic syndrome (tarr et al., ) ; therefore, antibiotic treatment of rabbits infected with ehec may not be recommended. clinically affected rabbits can be treated with fluids as this intervention is nephroprotective in humans (hickey et al., ) . in addition, rabbit epec strains may carry extended-spectrum beta-lactamases making them resistant to antibiotics (poeta et al., ) . parenteral enrofloxacin administered prior to shipment decreased morbidity and mortality associated with endemic epec (swennes et al., ) . research complications epec infection can cause high morbidity and mortality in laboratory rabbit colonies and can affect studies involving intestinal physiology in rabbits. epec and ehec present a zoonotic risk (garcia et al., ; poeta et al., ; swennes et al., ) . etiology treponema paraluiscuniculi is a noncultivable species that infects rabbits and causes venereal spirochetosis or treponematosis (also known as rabbit syphilis, vent disease, or cuniculosis) (smajs et al., ) . although its genome structure is closely related to other pathogenic treponema species including t. pallidum subsp. pallidum, the etiological agent of human syphilis, t. paraluiscuniculi does not infect humans (smajs et al., ) . genome sequencing revealed that t. paraluiscuniculi evolved from a t. pallidum-like ancestor and adapted to rabbits during loss of infectivity to humans (smajs et al., ) . t. paraluiscuniculi can also infect hares, and causes seroconversion, but no clinical signs. in contrast, the related organism, t. paraluisleporis, can infect and induce disease in rabbits and hares. the close phylogenetic association between t. paraluiscuniculi and t. paraluisleporis suggests that these organisms could be given a subspecies or ecovar status rather than species status (lumeij et al., ) . clinical signs in naturally infected rabbits lesions commonly occur in the vulva or prepuce (cunliffe-beamer and fox, a) . other parts of the body that may be affected, in descending order, include the anal region, nose, eyelid, and lip (cunliffe-beamer and fox, a) . naturally infected rabbits develop lesions of the ear, face, prepuce, and anus (small and newman, ) . in a study involving intratesticular inoculation of t. paraluiscuniculi, single lesions were found in the prepuce or scrotum and multiple lesions were found in the nose, mouth, ear, prepuce, foot, and scrotum (small and newman, ) . all lesions had abundant treponemes by dark-field examination (small and newman, ) . epizootiology susceptibility to, and expression of venereal spirochetosis, may vary with the strain of rabbit (cunliffe-beamer and fox, b) . the prevalence of t. paraluiscuniculi infection increased with parity in adult females and most adult males seroconverted within months of entering the breeding program. these findings suggested that t. paraluiscuniculi spreads by horizontal transmission in adult rabbits (digiacomo et al., ) . in an enzootically infected conventional rabbit colony, the frequency of venereal spirochetosis was lower in rabbits less than months of age than in adult rabbits (cunliffe-beamer and fox, b) . experiments involving cross fostering of newborns indicated that infection occurred at birth (vertical transmission) and during the suckling period (cunliffe-beamer and fox, b) . in addition, horizontal transmission by coitus and skin contact occurs (small and newman, ) . experimental topical or intradermal-subcutaneous genital inoculation of adult rabbits confirmed these routes of transmission (cunliffe-beamer and fox, b) . the t. pallidum repeat (tpr) genes in t. pallidum subsp. pallidum are thought to code for potential virulence factors. tprk was the only tpr homolog found in t. paraluiscuniculi that induced antibody and t cell responses after experimental inoculation of rabbits indicating that tprk may be an important virulence factor in venereal spirochetosis (giacani et al., ) . virulence factors and pathogenesis have been recently reviewed (smajs et al., ) . pathology the lesions include erythemathous macules or papules to erosions, ulcers, and crusts (cunliffe-beamer and fox, a) . diagnosis serologic tests that have been used include the nontreponemal antigen tests (venereal disease research laboratory (vdrl) and rapid plasma reagin), microhemagglutination, and fluorescent treponemal antibody absorption tests (digiacomo et al., ) . although the nontreponemal antigen tests were not completely satisfactory, the vdrl test was more sensitive and the plasma reagin test was more specific in detecting t. paraluiscuniculi infection (digiacomo et al., ) . the sensitivity and specificity of the microhemagglutination test compared favorably with the fluorescent treponemal antibody absorption test and was recommended as the optimal assay to make a diagnosis (digiacomo et al., ) . detection of t. paraluiscuniculi in lesions can be achieved by dark-field microscopic examination of scrapings from lesions and by histological evaluation of silver-stained testicular sections (cunliffe-beamer and fox, a; faine, ) . pcr has been used for molecular characterization of treponemes including t. paraluiscuniculi (cejkova et al., ) . differential diagnoses the skin lesions may be confused with abrasions (trauma), mycotic infections, and lesions of ectoparasites (acariasis) (small and newman, ) . treatment, prevention, and control there are no vaccines available at this time to prevent treponematosis in rabbits; however, rabbits have been used as experimental models to test vaccines against t. pallidum in humans (ho and lukehart, ) . hysterectomy derivation can eliminate venereal spirochetosis (cunliffe-beamer and fox, b) . a study investigating two different doses ( , or , iu/kg body weight/ week) of benzathine penicillin g-procaine penicillin g to treat rabbits at -day intervals found that both dosages were effective. lesions healed within weeks of the first treatment and the plasma reagin titers declined markedly or disappeared by the sixth week after the first treatment (cunliffe-beamer and fox, c) . research complications t. paraluiscuniculi can affect studies of t. pallidum in rabbits (small and newman, ) . partial immunological cross-protection has been observed between t. paraluiscuniculi and t. pallidum (smajs et al., ; turner and hollander, ) . etiology lawsonia intracellularis is a gram-negative, curved to spiral-shaped, obligate intracellular bacterium that causes proliferative enteropathy in rabbits and other species of animals (sait et al., ; schauer et al., ) . clinical signs an intraepithelial 'vibrio' was associated with acute typhlitis in rabbits - weeks after weaning (moon et al., ) . diarrhea was reported in japanese white rabbits with presumptive l. intracellularis infection and histiocytic enteritis (umemura et al., ) . in another report, sucklings and weanlings were affected and the feces of most of the rabbits were characterized as semifluid and mucinous or pasty, and three rabbits had watery diarrhea (schoeb and fox, ) . these affected rabbits were afebrile and lethargic, refused food and water, and most died within a few days after the onset of diarrhea (schoeb and fox, ) . diarrhea, depression, and dehydration that resolved over the course of - weeks were reported in -to -week-old new zealand white (nzw) rabbits (hotchkiss et al., ) . diarrhea and weight loss were reported in a -month-old rabbit (horiuchi et al., ). an outbreak of diarrhea with high ( %) mortality was reported in -to -month-old nzw rabbits with proliferative enterocolitis associated with l. intracellularis and epec (schauer et al., ) . epizootiology proliferative enteropathy generally occurs as isolated cases or occasional minor outbreaks in species other than the pig, blue fox, and hamster (lawson and gebhart, ) . infected rabbits can serve as reservoir hosts for l. intracellularis infection in other species including foals (pusterla et al., a (pusterla et al., , . however, l. intracellularis appears to adapt to the specific animal species it infects (vannucci et al., ) . the pathogenesis of l. intracellularis infection has been reviewed (lawson and gebhart, ; smith and lawson, ) . studies using interferon (ifn)-gamma receptor knockout mice determined that interferon ifngamma plays a significant role in limiting intracellular infection and increased cellular proliferation associated with l. intracellularis infection (smith et al., ) . lawsonia surface antigen (lsaa) plays a role during l. intracellularis attachment to and entry into intestinal epithelial cells (mccluskey et al., ) . balb/ca mice are susceptible to rabbit l. intracellularis isolates but not to pig l. intracellularis isolates suggesting that there are biological differences between the proliferative enteropathy isolates from rabbits and pigs (murakata et al., ) . pathology distention and diffuse mucosal thickening of the jejunum and proximal ileum with enlarged cranial mesenteric lymph nodes was observed in -to -month-old rabbits (umemura et al., ) . thickening of the mucosa was associated with distention of the lamina propria with macrophages and the enlargement of the lymph nodes was also associated with infiltration of macrophages (umemura et al., ) . minute bacilli were observed in the apical cytoplasm of mucosal epithelial cells using toluidine blue (umemura et al., ) . thickening of the cecum and proximal colon has also been reported (hotchkiss et al., ) . in another study, no gross lesions were found in the small intestine, but two suckling rabbits had reddened ceca with congested vessels (schoeb and fox, ) . in this study two types of microscopic lesions were characterized: ( ) erosive and suppurative cecitis and colitis, and ( ) proliferative lesions in the cecum, sacculated colon, ileum, and distal jejunum, or a combination of these (schoeb and fox, ) . some animals had both erosive and proliferative lesions. narrow curved or spiral bacteria were detected in rabbits with erosive and proliferative lesions using warthin-starry stain and these bacteria were more abundant in cases with severe lesions (schoeb and fox, ) . proliferative intestinal lesions contained curved to spiral argyrophilic intracellular bacteria in the apical cytoplasm of crypt enterocytes (hotchkiss et al., ) . diagnosis the s ribosomal dna sequences from l. intracellularis isolates from different species of animals are highly similar (cooper et al., a) . however, antigenic differences have been found between pig and rabbit isolates . the complete genome sequence of a porcine strain has been recently reported (sait et al., ) . l. intracellularis can be detected in feces from healthy and diarrheic rabbits (lim et al., ) . pcr assays to detect l. intracellularis dna in feces have been evaluated (pedersen et al., ) . these assays can be used for ante mortem diagnosis of proliferative enteropathy in pigs (pedersen et al., ) . pcr primers used to diagnose lawsonia in other animals species have been used in rabbit cases (cooper et al., b; duhamel et al., ; fox et al., ; horiuchi et al., ; hotchkiss et al., ; jones et al., ) . other diagnostic methods include enzyme-linked immunosorbent assay (elisa) using synthetic peptides of lsaa and immunomagnetic separation using anti-lsaa antibody to capture l. intracellularis in fecal samples followed by detection with atp bioluminescence (watarai et al., (watarai et al., , . in tissue sections, l. intracellularis can be detected using silver stains such as warthin-starry stain (duhamel et al., ; horiuchi et al., ; hotchkiss et al., ; schauer et al., ; schoeb and fox, ) . indirect immunofluorescence has also been used in deparaffinized intestinal sections from infected rabbits (schoeb and fox, ) . immunohistochemistry using antiserum against synthetic peptides of lsaa has also been used to detect l. intracellularis in the ileum of a naturally infected rabbit (watarai et al., ) . electron microscopy reveals organisms that are ~ . - . μm wide and ≤ . μm long in the apical cytoplasm of villous and crypt epithelial cells (duhamel et al., ) . l. intracellularis can be cultured from homogenized intestinal tissue in cell lines including iec- (rat small intestinal cells) and mccoy cells (mouse fibroblasts) (lawson and gebhart, ; watarai et al., ) . a quantitative pcr (qpcr) assay that is able to assess the growth of l. intracellularis in cultured cells has also been used to detect the organisms in pig fecal samples and could be used in other animal species (drozd et al., ) . differential diagnoses clinically, the differential diagnosis should include other causes of diarrhea in rabbits. mycobacterium avium subsp. paratuberculosis can infect rabbits and induce thickening of the intestinal mucosa (beard et al., ; greig et al., ) . therefore, rabbit intestinal sections should be examined for acidfast organisms using stains such as ziehl-neelsen stain (duhamel et al., ; horiuchi et al., ; schoeb and fox, ; umemura et al., ) . furthermore, other intestinal organisms may colonize the intestine during l. intracellularis infection in rabbits (duhamel et al., ; hotchkiss et al., ; lim et al., ; schauer et al., ) . other bacterial diseases have been sporadically reported in rabbits. these are summarized in table . . treatment, prevention, and control vaccination strategies have been tested and developed for pigs and horses, but not for rabbits (nogueira et al., ; pusterla et al., b; weibel et al., ) . testing rabbits by pcr prior to introduction to a laboratory colony may be necessary for exclusion of this organism. oral neomycin ( mg/rabbit) was used to treat surviving rabbits during an outbreak of presumptive l. intracellularis and the diarrhea subsided (umemura et al., ) . because l. intracellularis infects the intestine and ifn-gamma appears to be involved in pathogenesis, research involving rabbit gastrointestinal pathology and immune responses may be confounded by infection with this organism. research complications the mortality associated with l. intracellularis infection would be disruptive to ongoing studies. etiology myxomatosis is caused by myxoma virus, a member of the family poxviridae, genus leporipoxvirus (kerr and donnelly, ; spiesschaert et al., ) . clinical signs the severity of disease varies with the strain of virus and the host species and breed. rabbits of the genus oryctolagus are particularly susceptible and often develop a fatal disease characterized by mucinous skin lesions and tumors. affected animals also exhibit edema around the mouth, nose, anus, and genitals as well as progressive conjunctivitis with serous and mucopurulent secretions from the eyes and nose (brabb and di giacomo, ; kerr and donnelly, ; spiesschaert et al., ) . bacterial pneumonia commonly develops and animals die - days after infection. the virus is spread by arthropod vectors and direct contact. epizootiology myxomatosis has a worldwide distribution. various species of sylvilagus and lepus are naturally susceptible (brabb and di giacomo, ) . the myxoma virus genome encodes for a number of immunomodulatory proteins which greatly affect the host immune response by inhibiting apoptosis, interfering with leukocyte chemotaxis, and suppressing leukocyte activation, thereby fostering viral replication and spread (spiesschaert et al., ) . pathology histopathology shows these 'myxomas' to be composed of undifferentiated stellate mesenchymal cells embedded in a matrix of mucinous material and interspersed with capillaries and inflammatory cells (brabb and di giacomo, ; kerr and donnelly, ) . diagnosis diagnosis can be made by pcr or elisa. definitive diagnosis depends on culture of the virus from infected tissues. differential diagnoses rabbits of the genus sylvilagus develop fibroma-like lesions that may be indistinguishable from those caused by rabbit fibroma virus. the two diseases have been distinguished by inoculation of fibroma material into oryctolagus rabbits. they develop fatal disease if the myxoma virus is the etiologic agent, or fibromas if rabbit fibroma virus is responsible. treatment, prevention, and control since the disease is spread by fleas and mosquitoes as well as by direct contact, control measures should include prevention of contact with arthropods and quarantine of infected rabbits. vaccines have been used in europe with some success. most recently, a live recombinant vaccine for both myxomatosis and rabbit hemorrhagic disease has been released in the united kingdom (spibey et al., ) . research complications none have been reported. rabbit (shope) fibroma virus is a leporipoxvirus that is antigenically related to myxoma virus. fibromatosis is endemic in wild rabbits; however, an outbreak in commercial rabbits caused extensive mortality (joiner et al., ) . usually, less virulent strains cause skin tumors in domestic rabbits (raflo et al., ) . the disease is probably spread by arthropods (brabb and di giacomo, ; kerr and donnelly, ) . fibromas are flat, subcutaneous, easily movable tumors, and most often found on the legs and face. tumors may persist for some time but eventually regress. metastasis does not occur. rabbit pox is a rare disease induced by an orthopoxvirus taxonomically similar to vaccina virus that has caused outbreaks of fatal disease in laboratory rabbits in the united states and holland (brabb and di giacomo, ) . rabbits with the disease may or may not present with 'pox' lesions in the skin. the animals have a fever and nasal discharge or days after infection. most rabbits have eye lesions including blepharitis, conjunctivitis, and keratitis with subsequent corneal ulceration. skin lesions, when present, are widespread. they begin as a macular rash and progress to papules up to cm in diameter by days postinfection. the lymph nodes are enlarged, the face is often edematous, and there may be lesions in the oral and nasal cavity. at gross necropsy, nodules can be found in the liver, gall bladder, spleen, lung, and reproductive organs. necrosis is widespread. characteristic cytoplasmic inclusions seen in many poxvirus infections are rare in this disease. mortality is high in affected animals. the virus is apparently spread by aerosols and is difficult to control. rabbit pox is used as a model of smallpox in humans in response to the potential use of smallpox as a bioterrorism agent. it is an effective model for the evaluation of potential therapies against smallpox (nalca and nichols, ; rice et al., ) . four herpesviruses (leporid herpesviruses , , , and ) have been isolated from rabbits and hares (davison, ) . leporid herpesvirus (lhv- ) was isolated from cottontail rabbits and is also known as cottontail rabbit herpesvirus. it is not pathogenic for domestic rabbits. lhv- (herpesvirus cuniculi) was isolated from domestic rabbits (o. cuniculus) and causes subclinical infections. lhv- (herpesvirus sylvilagus) was isolated from cottontail rabbits. cottontail rabbits infected with the virus develop a lymphoproliferative disease with lymphoid infiltration of many organs (hesselton et al., ) . lhv- does not infect domestic rabbits. lhv- - are tentatively classified in the genus radinovirus, subfamily gammaherpesvirinae. lhv- was isolated from domestic rabbits and caused severe disease in preweanlings (jin et al., a, b) . clinical signs included weakness, anorexia, conjunctivitis, keratitis, and periocular swelling. some animals also developed skin ulcers. lhv- is genus simplexvirus, subfamily alphaherpesvirinae. the cottontail rabbit is the natural host of the cottontail (shope) papillomavirus, a kappapapillomavirus, laboratory animal medicine which causes horny warts primarily on the neck, shoulders, and abdomen. the disease has a wide geographic distribution with the highest incidence occurring in rabbits in the midwest (brabb and di giacomo, ) . as many as % of infected sylvilagus rabbits develop squamous cell carcinomas. natural outbreaks in domestic rabbits have been reported (hagen, ) . in these natural outbreaks, papillomas were more common on the eyelids and ears. the virus is transmitted by arthropod vectors. this virus is used extensively as a model for the study of oncogenic virus biology and as a model for the treatment and prevention of papillomavirus infections in humans (christensen, ; salmon et al., ; sundarum et al., ) . oral papillomatosis in domestic rabbits is caused by a kappapapillomavirus that is related to but distinct from the cottontail rabbit papilloma virus. naturally occurring lesions have been seen in laboratory rabbits and appear as small, white, discrete growths on the ventral surface of the tongue (kerr and donnelly, ) . lesions may ulcerate. microscopic examination shows them to be typical papillomas. most lesions eventually regress spontaneously (brabb and di giacomo, ; kerr and donnelly, ) . etiology rabbit rotavirus is a member of the family reoviridae. all isolates of rabbit rotavirus have been classified as group a and have been serotype (brabb and di giacomo, ; kerr and donnelly, ) . clinical signs the severity of disease in naturally occurring outbreaks has been variable. in severe outbreaks, affected animals exhibit anorexia, dehydration, and watery to mucoid diarrhea and mortality can be quite high. in other reported outbreaks, mild, transient diarrhea has been reported (brabb and di giacomo, ; kerr and donnelly, ) . similarly, attempts to experimentally produce clinical disease have had variable results. mild diarrhea is usually seen, but in some studies there has been significant mortality. it is probable that other factors, such as maternal antibodies, diet, and the presence of pathogenic bacteria, affect the severity of clinical disease in outbreaks. for example, in combined experimental infections with both rotavirus and e. coli, the inoculation of both organisms led to more serious clinical signs than when given alone, indicating that rotavirus may have been a more significant determinant in the manifestation of this disease (thouless et al., ) . these investigators also showed that older rabbits were naturally more resistant to the combined infection with rotavirus and e. coli. epizootiology rotavirus infections of domestic rabbits are common (brabb and di giacomo, ) . many colonies of rabbits are serologically positive, and rotavirus can be isolated readily from rabbit feces. in endemically infected colonies, maternal antibodies to rotaviruses are passed transplacentally and decline at around the time of weaning (brabb and di giacomo, ) . rabbits of weaning age are most susceptible. very young rabbits appear to be protected from rotavirus infection by passive immunity, when present, but are quite susceptible when there is none (schoeb et al., ) . this is also the time when they are most likely to be subjected to diet changes that may contribute to a change in microbial flora. pathology in affected animals, there is villous atrophy and loss of epithelial cells in the small intestines. a lymphocytic infiltrate is present. diagnosis immunoassays (elisa and multiplex fluorescent immunoassay) are commercially available for rabbit rotavirus. a commercial immunochromatography kits for detecting human rotavirus infection was used successfully to diagnose rabbit rotavirus infection (fushuku and fukuda, ) . differential diagnoses c. piliforme, c. spiroforme, c. difficile, e. coli, lawsonia intracellularis, coronavirus, coccidiosis, and intestinal parasites should be considered. research complications colony mortality would be disruptive to ongoing studies. pleural effusion disease/infectious cardiomyopathy was diagnosed in rabbits inoculated with t. palliduminfected stocks of testicular tissue. because these treponemes could not be grown in vitro, the organism was propagated by passage in rabbits. the stocks were contaminated with a coronavirus, although it is not known whether this virus originated from rabbits or was a virus of human origin that had adapted to rabbits. with continued passage, the virus became more virulent, and significant mortality ensued. evidence indicated that it was not transmitted by direct contact. rabbits died due to congestive heart failure, and microscopic examination showed there was widespread necrosis of the heart muscle. it has been suggested that infection with this virus might be a model for the study of virus-induced cardiomyopathy (brabb and di giacomo, ; kerr and donnelly, ) . rabbit enteric coronavirus has been isolated from tissue cultures from rabbits (brabb and di giacomo, ; kerr and donnelly, ; lapierre et al., ) and has been associated with one naturally occurring outbreak of diarrhea in a barrier-maintained breeding colony (eaton, ) . these rabbits developed severe diarrhea, and most died within h of onset of clinical signs. attempts to reproduce the disease led to watery diarrhea, which lasted a short time; however, none of the rabbits died. it is quite probable that other microorganisms or laboratory animal medicine unknown environmental factors contributed to the severity of this outbreak. etiology rabbit hemorrhagic disease virus is a calicivirus of the genus lagovirus and is the causative agent of rabbit hemorrhagic disease (rhd) (abrantes et al., ; brabb and di giacomo, ; kerr and donnelly, ) . clinical signs three clinical syndromes are seen (abrantes et al., ) . the peracute form is characterized by sudden death without clinical signs. acutely affected animals demonstrate anorexia and depression. in addition, neurologic signs, respiratory signs, ocular hemorrhage, and epistaxis may be seen. morbidity and mortality are extremely high. lymphopenia and abnormalities in coagulation parameters are also seen. in the subacute form, similar signs may occur but are considerably milder and most of these rabbits survive (abrantes et al., ; kerr and donnelly, ) . epizootiology rabbit hemorrhagic disease was first reported in china in and is currently endemic in europe, asia, africa, australia, and new zealand. in addition, isolated outbreaks have been reported in numerous countries. the virus is transmitted by the fecal-oral route. the role of fomites and arthropod vectors is also suspected (brabb and di giacomo, ; kerr and donnelly, ) . the incubation period may be as short as or days, and sudden death with no previous signs is common. pathology periportal hepatic necrosis is the only consistent microscopic lesion, and the animals die due to disseminated intravascular coagulopathy and thrombosis (abrantes et al., ; kerr and donnelly, ) . diagnosis the virus has not been successfully grown in vitro; however, diagnosis can be confirmed with negative-contrast electron microscopy of liver tissue. specific antibodies can be detected by elisa or by hemagglutination inhibition. differential diagnoses a related calicivirus, european brown hare virus, has caused disease in hares in several countries in europe (brabb and di giacomo, ) . animals present with necrotic hepatitis, hemorrhages in the trachea and lungs, and pulmonary edema. a monoclonal antibody elisa is available for serodiagnosis, and control measures are similar to those for rhd. the agent resists drying, can be carried on fomites, and may be transmitted via respiratory and intestinal secretions (mitro and krauss, ) . any rabbit colonies with this disease should be quarantined and depopulated, and the environment thoroughly cleansed and disinfected. research complications colony mortality would be disruptive to ongoing studies. other viral infections several other viruses have been isolated from rabbit tissues, but have not been shown to produce disease. these include paramyxoviruses and bunyaviruses. serologic titers to togaviruses and flaviviruses have also been demonstrated in rabbits (brabb and di giacomo, ; kerr and donnelly, ) . etiology hepatic coccidiosis is caused by the parasite eimeria stiedae, which has also been referred to as monocystis stiedae, coccidium oviforme, and c. cuniculi (hofing and kraus, ) . clinical signs the clinical disease has a wide range of manifestations. mild infections often result in no apparent disease. most clinical signs are the result of interruption of normal hepatic function and blockage of the bile ducts. these signs are more common in juvenile rabbits and can include hepatomegaly, icterus, and anorexia (schoeb et al., ) . diarrhea can occur at the terminal stages of the disease (hofing and kraus, ) . decreased growth rates and weight loss are common. joyner et al. ( ) demonstrated that infected rabbits begin to lose weight within days. enlargement of the liver (hepatomegaly) is common. the liver normally is approximately . % of the body weight, but rabbits with severe hepatic coccidiosis may have livers that contribute to greater than % of the body weight (lund, b) . the age of the host strongly affects parasite development and oocyst production. four-month-old, coccidia-free rabbits experimentally infected with e. stiedae produced fewer oocysts than similarly infected -monthold rabbits (gomez-bautista et al., ) . epizootiology e. stiedae is found worldwide, although rabbits bred for use in research are commonly free of the parasite. transmission occurs by the fecaloral route, as for other coccidia. the organism has also been experimentally transmitted by intravenous, intraperitoneal, and intramuscular administration of oocysts (pellérdy, ) . smetana ( ) demonstrated that infection of the entire liver occurred following ligation of the right bile duct and inoculation of e. stiedae oocysts. the study also showed that infection occurred earliest within the small intrahepatic ducts, leading to the theory that infection occurred via blood or lymph. the precise life cycle is still undetermined, although a number of studies have examined it (horton, ; owen, ; rose, ) . sporozoites have been demonstrated in the lymph nodes following experimental inoculation (horton, ; rose, ) . pathology necropsy often shows the liver to be enlarged and discolored, with multifocal yellowish laboratory animal medicine white lesions of varying size (fig. . ) . exudate in the biliary tree is common, along with dilatation of bile ducts. microscopically, papillomatous hyperplasia of the ducts along with multiple life-cycle stages of the organism can be observed in the biliary epithelium ( fig. . ) . diagnosis infected rabbits may have decreased fibrinogen when compared to uninfected rabbits (cam et al., ) . serum bilirubin levels can rise to mg/ dl, increasing as soon as day of infection and increasing through days - before moderating (rose, ) . leukocytosis and anemia can be observed and acute phase proteins are notably increased by days post infection (freitas et al., ) . diagnosis can be made by examination of fecal material, by either flotation or concentration methods. oocysts can also be detected within the gallbladder exudate (hofing and kraus, ) . alternatively, oocysts can sometimes be observed by microscopic examination of impression smears of the cut surface of the liver. ultrasonography may be a useful tool for diagnosis, with dilated vessels and bile ducts and increased echogenicity of the liver parenchyma (cam et al., ) . differential diagnoses the hyperplastic biliary ducts can be mistaken grossly for neoplasia. other types of parasitic hepatitis should be considered as differential diagnoses. less frequently, hepatitis secondary to bacterial infections can occur. treatment, prevention, and control control of the infection until development of natural immunity is one strategy to minimize the severity of disease. davies et al. ( ) demonstrated that immunity occurs following a light infection with e. stiedae. in the rabbit, immunity to eimeria may be lifelong (niilo, ; pellérdy, ) . prevention of hepatic coccidiosis with sulfaquinozaline in the feed ( ppm) was shown to prevent infection when experimental challenged with , sporulated oocysts (joyner et al., ) . sulfonamides have been shown effective against eimeria spp. (hagen, ; horton-smith, ; jankiewicz, ; lund, a; tsunoda et al., ) . treatment with toltrazuril ( ppm in drinking water for one day) has been shown to effectively treat infected animals (cam et al., ) . thorough sanitation of potentially contaminated surfaces is critical to control of coccidiosis. research complications potential research complications arising from hepatic coccidiosis are considerable. the resulting liver damage and decreased weight gains can complicate both the supply of rabbits for research as well as adversely affect research protocols. etiology there are at least different pathogenic species of intestinal coccidia in rabbits, including eimeria coecicola, e. elongate, e exigua, e. intestinalis, e. flavescens, e. irresidua, e. magna, e. matsubayashii, e. media, e. nagnurensis, e. neoleporis, e. piriformis, e. vejdovskyi, and e. perforans (pakandl, ). all of these coccidia are presented here as a group rather than as individual species of intestinal coccidia. clinical signs although intestinal coccidiosis may be subclinical, clinical signs can range from mild to severe and can result in death of the animal. postweanling rabbits are the most likely to experience mortality related to intestinal coccidiosis. suckling rabbits (< days old) are generally considered to be resistant to infection (pakandl and hlaskova, ) . clinical signs also depend on the species of coccidia that are present. severe diarrhea, weight loss, or mild reduction in growth rate are all laboratory animal medicine possibilities. fecal occult blood may be detected with e. perforans infection (li and ooi, ) . death is usually associated with severe dehydration subsequent to diarrhea (frenkel, ) . epizootiology intestinal coccidiosis is a common rabbit disease worldwide (varga, ) . transmission is by the fecal-oral route through ingestion of sporocysts. unsporulated oocysts are passed in the feces and are not infective. such oocysts will, however, sporulate to an infective stage within days after shedding; thus, it is important that sanitation be frequent enough to remove infective stages from the environment. the oocyst burden of feces can be enormous. gallazzi (gallazzi, ) demonstrated that a subclinical carrier of intestinal coccidia had , oocysts/gram of feces and that a rabbit with diarrhea could shed in excess of , oocysts/gram of feces. environmental contamination with oocysts can be a problem when large numbers of oocysts are being excreted. the life cycles of eimeria spp. are similar to those of other coccidia. schizogony, gametogony, and sporogony are the three phases of this life cycle. other sources can be consulted for greater detail on the life cycle of these protozoans (davies et al., ; pakandl, ; pakandl and jelinkova, ; pellérdy, ; rutherford, ) . pathology lesions are apparent in the small and large intestines. necrotic areas of the intestinal wall appear as white foci (pakes, ; pakes and gerrity, ) . the location and extent of the lesions depend on the species of coccidia. diagnosis diagnosis of intestinal coccidiosis can be made through identification of the oocysts in the feces (pakes and gerrity, ) . a pcr has been developed (oliveira et al., ) that differentiates between of the different eimeria species that infect the domestic rabbit. this test has excellent sensitivity, with the ability to detect . - . sporulated oocysts per sample. smaller scale pcr for detection and differentiation between the more pathogenic species (e. intestinalis, e. flavicenens, and e. stiedae) has also been developed (yan et al., ) . differential diagnoses other causes of diarrhea in rabbits should be considered including tyzzer's disease, the clostridial diseases, colibacillosis, l. intracellularis, enteric coronovairus and rotavirus, protozoons, or intestinal parasites. treatment, prevention, and control because intestinal coccidiosis is most common in postweanling rabbits, prevention of the disease should focus on the preweaning period. an oral vaccination has been developed and consists of a nonpathogenic strain of e. magna. this vaccine is sprayed into the nest box when rabbits are days of age. the preweanling rabbits develop immunity subsequent to infection with the nonpathogenic strain and are then resistant to wild-type strains of e. magna at days of age (drouet-viard et al., ) . other oral vaccines developed from various eimeria strains are also in development (akpo et al., ) . prevention and control of infection can be accomplished by providing . % sulfamerazine or . % sulfaquinoxaline in the drinking water (kraus et al., ) . a combination of sulfaquinoxaline, strict sanitation, and elimination of infected animals has been shown to eliminate intestinal coccidiosis from a rabbit breeding colony (pakes and gerrity, ) . as for hepatic coccidiosis, sulfaquinoxaline provided in the feed ( ppm) is an effective treatment. research complications intestinal coccidiosis can impact studies of the gastrointestinal tract, or have an impact on survival of postweanling rabbits. etiology the protozoan organism cryptosporidium cuniculus has been found in the intestinal tract of the rabbit (hadfield and chalmers, ; inman and takeuchi, ; kaupke et al., ; rehg et al., ; robinson et al., ; shiibashi et al., ; zhang et al., ) . clinical signs clinical signs related to cryptosporidiosis seem to be quite variable in the rabbit. a large farm outbreak (kaupke et al., ) had rabbits that presented with lethargy, anorexia and diarrhea. animals showing clinical signs died within - days. the stress of weaning is thought to have exacerbated these signs. another report describes small intestinal dilatation observed during surgery in a rabbit without other clinical signs (inman and takeuchi, ) . epizootiology transmission is likely via ingestion of thick-walled sporulated oocysts. experimentally infected juvenile rabbits began shedding oocysts in their feces - days post infection and continued to shed until days post infection without clinical signs (robinson et al., ) . pathology histopathology of the small intestine of the reported rabbit was characterized by shortened, blunted villi and mild edema of the lamina propria. the lacteals of the ileum were also dilated, and an inflammatory response was observed (inman and takeuchi, ) . diagnosis c. cuniculus is emerging as a potential zoonotic pathogen with several reports in recent years (chalmers et al., (chalmers et al., , zhang et al., ) . in response to this, real-time pcr assays are in development (hadfield and chalmers, ) that detect and differentiate c. cuniculus from c. parvum and c. hominis. differential diagnoses c. cuniculus can only be differentiated from c. hominis and c. parvum via genetic analysis (robinson et al., ) . differential diagnoses would include infection with clostridium piliforme, c. spiroforme, c. difficile, e. coli, lawsonia intracellularis, coronavirus, rotavirus, protozoans, or intestinal parasites. treatment, prevention, and control minimizing stress can possibly prevent or reduce clinical signs laboratory animal medicine (kaupke et al., ) . antibiotics were ineffective in the large farm outbreak. presumably, supportive care (fluids) would be indicated in animals showing clinical signs (schoeb et al., ) . prevention requires husbandry and sanitation practices that prevent exposure. research complications this organism is emerging as a human pathogen, so appropriate precautions should be made to protect research personnel from rabbits positive for c. cuniculus. etiology the etiologic agent responsible for encephalitozoonosis is encephalitozoon cuniculi. this agent is historically known by the name nosema cuniculi (pakes and gerrity, ) and has been divided into three strains (i -rabbit strain, ii -mouse strain, iiidog strain) (didier et al., ) . the disease was first described in as an infectious encephalomyelitis causing motor paralysis in young rabbits (wright and craighead, ) . clinical signs encephalitozoonosis typically has a delayed onset (weeks to months post infection) prior to the exhibition of clinical signs. early infection affects the kidney, liver and lung, while alterations later in the infection are most severe in the kidneys and brain (kunzel and joachim, ) . the organism can be found in the tissues without an inflammatory response (pakes and gerrity, ) . although named for the motor paralysis in young rabbits, the disease is usually latent. if clinical signs are present, they can include convulsions, tremors, torticollis, paresis, and coma (pattison et al., ) as well as signs of kidney failure. intrauterine infection can result in phacoclastic uveitis leading to rupture of the lens capsule (kunzel and joachim, ) . epizootiology transmission is likely horizontal via direct contact or environmental contamination (kunzel and joachim, ) , primarily from ingestion of infected urine (schoeb et al., ; wasson and peper, ) . the pathogen can also be transmitted vertically, as evidenced by in utero pcr positivity reported by baneux and pognan ( ) . pathology the kidneys commonly have lesions at necropsy. typically, there are multiple white, pinpoint areas or gray, indented areas on the renal cortical surface (kraus et al., ) . microscopically, these areas are characterized by granulomatous inflammation. interstitial infiltration of lymphocytes and plasma cells and tubular degeneration may also be present (flatt and jackson, ) . granulomatous encephalitis is a characteristic lesion (fig. . ) (pakes and gerrity, ) . lesions of the spinal cord can also occur (koller, ) . the organisms are often not observed in histologic sections of the lesions. organisms may be seen floating free in the tubules of the kidney (pakes and gerrity, ) . diagnosis diagnosis of encephalitozoonosis can be made using several different methods. histologic examination of tissues and observation of the organism is definitive. brain and kidney samples yield the best detection rates for histopathological diagnosis (leipig et al., ) . the encephalitozoon organism does not stain well with hematoxylin and eosin, and is better demonstrated using giemsa stain, gram stain, or goodpasture's carbol fuchsin stain (pakes, ) . many different serologic tests exist for the organism. indirect fluorescence antibody technique and elisa are both available and reliable (kunzel and joachim, ) . advances in diagnostic techniques have been made in human medicine due to the susceptibility of immunosuppressed patients to this particular infection. several pcr tests for diagnosis and species differentiation of encephalitozoonosis have been developed (croppo et al., ; franzen et al., ; weiss and vossbrinck, ) . pcr can be performed on the intestine, brain, heart, liver, lung, or kidney tissue with a good ( %) overall detection rate reported (leipig et al., ) . differential diagnoses if the animals are demonstrating motor paralysis, conditions such as splay leg should be considered. for neurological signs, consider bacterial meningitis due to p. multocida infection or rabbit hemorrhagic disease. treatment, prevention, and control prevention and control of the organism in the colony are done by elimination of the organism from the colony of infected rabbits. because this is a latent disease in rabbits, serologic methods must be used to identify carriers of the organism. the indirect fluorescence antibody test has laboratory animal medicine been used successfully to identify infected rabbits (cox, ) . the elimination of infected rabbits must be accompanied by disinfection of the environment. several disinfectants have been effective against this organism. encephalitozoon was killed by % (v/v) lysol, % (v/v) formalin, and % (v/v) ethanol (shadduck and polley, ) % hydrogen peroxide, and % sodium hydroxide (kunzel and joachim, ) . successful treatment and prevention of e. cuniculi in the rabbit has been reported with use of fenbendazole (suter et al., ) . for cases of phacoclastic uveitis, removal of the lens is the treatment of choice (kunzel and joachim, ) . research complications encephalitozoonosis is most commonly subclinical disease, which makes it difficult to determine the effects it may have on research. granulomatous reactions would complicate renal physiology and neurologic research. depression of the igg response and an increase in the igm response to brucella abortus antigens has been demonstrated in rabbits infected with encephalitozoon organisms (cox, ) . encephalitozoonosis is also a recognized disease in immunodeficient humans. it is recommended that such individuals seek medical counsel prior to handling rabbits. isolates from humans have been shown to be infectious for rabbits (mathis et al., ) . etiology psoroptes cuniculi is a nonburrowing mite and the causative agent of psoroptic mange, also called ear mange, ear canker, or otoacariasis. the organism is distributed worldwide, but with modern husbandry practices, it is mostly historical in laboratory rabbit colonies (schoeb et al., ) . clinical signs lesions occur primarily in the inner surfaces of the external ear. the lesions are pruritic and can result in scratching, head shaking, pain, and even self-mutilation (hofing and kraus, ) . a tan, crusty exudate accumulates in the ears over the lesions and can become quite extensive and thick (fig. . ) . the skin under the crust is moist and reddened. the ears may become malodorous. epizootiology all stages of the mite (egg, larva, protonymph, and adult) occur on the host. early in the infestation, mites feed on sloughed skin cells and lipids. as local inflammation increases, they ingest serum, hemoglobin, and red blood cells (deloach and wright, ; hofing and kraus, ) . the entire life cycle is complete in days. mites are relatively resistant to drying and temperature and can survive off the host for - days in a temperature range of - °c and relative humidity of - %. pathology lesions are characterized histologically by chronic inflammation, hypertrophy of the malpighian layer, parakeratosis, and epithelial sloughing. a hypersensitivity response to the mites, mite feces, and saliva likely contributes to lesions (hofing and kraus, ) . diagnosis mites are large enough to be seen with the unaided eye or with an otoscope. material scraped from the inner surface of the ear can also be examined using a dissecting microscope. mites are oval-shaped with well-developed legs that project beyond the body margin. adult males measure - μm × - μm, and females measure - μm × - μm (hofing and kraus, ) . differential diagnoses rarely, infection with sarcoptes scabiei or cheyletiella parasitovorax should be considered as differential diagnoses. treatment, prevention, and control several successful treatments have been reported. prior to local treatment, the ears should be cleaned gently to remove accumulated exudate. one treatment involves the application of % rotenone in mineral oil ( : ) every days for days. ivermectin is an effective treatment at dosages of - μg/kg sc or im (curtis et al., ; mckellar et al., ; wright and riner, ) . one or two doses were utilized for effective treatment. treatment of moderate to severe infestations with ivermectin alone can fail. using adjunct vitamin therapy to minimize oxidative tissue damage has been shown to enhance treatment success (singh et al., ) . a single dose of topical selamectin at a minimum of mg/kg selamectin (kurtdede et al., ) and a single injection of laboratory animal medicine eprinomectin at or μg/kg (pan et al., ) were found to be effective treatments. regardless of treatment modality, it is generally recommended that the entire group of rabbits be treated at the same time. heat ( °c) and desiccation (< % humidity) will kill parasites that are not on the host (arlain et al., ) . vaccine targets have been investigated, with gut surface antigen being the primary focus (rossi et al., ) . research complications p. cuniculi has been associated with immune suppression and a systemic inflammatory reaction (shang et al., ) . ear trauma secondary to psoroptes infestation can limit access to the auricular artery and veins. . cheyletiella spp. (c. parasitovorax, c. takahasii, c. ochotonae, c. johnsoni) etiology cheyletiella mites are nonburrowing skin mites of rabbits. they are distributed worldwide. several closely related species have been reported to occur on rabbits, namely, c. parasitovorax, c. takahasii, c. ochotonae, and c. johnsoni (hofing and kraus, ) . clinical signs the anatomic site most commonly infested is the area over the scapulae. there may be mild hair loss in the area, and the skin may have a gray-white scale (cloyd and moorhead, ) . affected rabbits do not scratch, and there is no evidence of pruritus. skin lesions are mild or nonexistent. epizootiology all stages (egg, larva, pupa, and adult) in the life cycle occur on the host. mites remain in association with the keratin layer of the skin and feed on tissue fluid (myktowycz, ) . transmission is probably by direct contact (schoeb et al., ) . pathology when present, skin lesions are characterized by mild dermatitis, hyperkeratosis, and an inflammatory cell infiltrate (hofing and kraus, ) . diagnosis mites can be isolated by scraping or brushing fur in the affected areas onto a slide. clearing samples with - % potassium hydroxide will improve visibility of the mites, which can then be identified using a dissecting microscope. the female measures × μm, and the male is × μm. cheyletiella mites have a large, distinctive curved claw on the palpi (pegg, ) . differential diagnoses other skin mites (such as sarcoptes scabei) or fur mites (leporacarus gibbus) that can affect rabbits should be considered as well as the possibility of dermatophytosis. treatment, prevention, and control topical acaricides are often used and are effective at controlling infestation. ivermectin (subcutaneous or subcutaneous and oral) and selamectin (topical) treatments have been used successfully. eggs in the environment can reinfest the host, so posttreatment environmental sanitation is important (mellgren and bergvall, ) . research complications cheyletid mites can cause a transient dermatitis in humans who are in close contact with infested animals (cohen, ; lee, ) . for this reason, these mites can be considered a zoonotic pathogen. etiology sarcoptes scabiei is a burrowing mite and the causative agent of sarcoptic mange. mites of the genus sarcoptes are generally considered to be one species, s. scabiei, but are often further identified by a variety name corresponding to the host species (e.g., s. scabiei var. cuniculi). the organisms are commonly referred to as itch or scab mites. the disease has a worldwide distribution. clinical signs affected rabbits will exhibit intense pruritus with hair loss and abrasions as a resulting from scratching. serous encrustations on the skin and secondary bacterial infections are common. there has been one report of a secondary infection with the yeast malassezia (radi, ) . anemia and leukopenia can also be observed in affected rabbits (arlain et al., ) . epizootiology sarcoptic are similar to notoedric mites (notoedres cati) in morphology, life cycle, and public health significance. mites burrow and produce an intensely pruritic dermatitis. lesions are most common on the head (hofing and kraus, ) . all stages of sarcoptic mange mites occur on the host. the females burrow into the skin to lay eggs. young larvae can also be found in the skin, whereas older larvae, nymphs, and males reside on the skin surface. mites feed on lymph and epithelial cells (hofing and kraus, ) . pathology amyloidosis of the liver and glomerulus have been reported in rabbits with severe infestation (arlain et al., ) . the skin itself is hyperplastic and hyperkeratotic, with inflammatory response evident in the dermis (schoeb et al., ) . diagnosis because sarcoptes is a burrowing mite, skin scrapings are necessary to diagnose infestation. samples may be cleared with - % potassium hydroxide. female mites measure - μm × - μm. the body shape is round, and the legs are very short. differential diagnoses other causes of dermatitis in rabbits (such as cheyletiella spp., p. cuniculi or dermatophytosis) should be considered. ivermectin is effective at eliminating infestation at μg/kg administered subcutaneously. a single topical dose of selamectin at - mg/kg reduced the number of mites found on skin scrapings of angora rabbits (kurtdede et al., ) and eliminated clinical signs and parasitic infestation in a group of mixed-breed rabbits at a dose of mg/rabbit (farmaki et al., ) . as with psoroptes, more 'natural' treatments are being investigated with good preliminary results from eugenol (pasay et al., ) and eupatorium spp. (nong et al., ) . research complications no specific research complications have been reported. sarcoptes can cause a selflimiting dermatitis in humans. a wide variety of arthropod parasites has been reported in wild rabbits but they are extremely rare in laboratory rabbits. for an extensive listing the reader is referred to other sources (hofing and kraus, ) . etiology historically, the rabbit pinworm was identified as oxyuris ambigua, but is now known as passalurus ambiguus (hofing and kraus, ) . clinical signs even when rabbits have heavy oxyurid burdens, clinical signs are not usually apparent (erikson, ; soulsby, ). one case report described unsatisfactory breeding performance and poor condition in a rabbit colony infested with the parasite. epizootiology p. ambiguus has a direct life cycle. mature pinworms are found in the lumen of the cecum or colon of the rabbit. after ingestion, the eggs hatch in the small intestine, and the larvae molt with maturation in the cecum. the prepatent period is between and days (taffs, ) . transmission occurs easily via ingestion, given that individual rabbits have been found with over adult parasites (hofing and kraus, ) and that embryonated eggs pass out in the feces and are immediately infective (schoeb et al., ; taffs, ) . pathology minimal to no lesions are associated with this pinworm (schoeb et al., ) . diagnosis eggs can be found in feces, cecum, or colon. differential diagnoses this is the only reported pinworm in rabbits and it is not known to cause lesions or disease. treatment, prevention, and control several successful treatment strategies for rabbit oxyuriasis have been reported. piperazine citrate at mg/ ml of drinking water for day was successful in eliminating infestation (hofing and kraus, ) . at and ppm, fenbendazole mixed in the food for days eliminated all immature and adult pinworms (duwell and brech, ) . subcutaneous doses of ivermectin ( . mg/kg) were reported to be ineffective in reducing the burden of passalurus organisms in field populations of snowshoe hares (lepus americanus) (sovell and holmes, ) . due to the direct life cycle, strict husbandry and sanitation practices are required to prevent introduction and spread throughout a rabbit colony (schoeb et al., ) . research complications none have been described. etiology dermatophytosis, also known as ' ringworm' or 'tinea', refers to a skin infection caused by a dermatophyte, a keratinophilic and keratinolytic fungus (chermette et al., ; mendez-tovar, ; robert and pihet, ) . dermatophytes are a group of closely related filamentous fungi that are able to invade the stratum corneum of the epidermis and keratinized tissues including the skin, nail, and hair (kanbe, ) . dermatophytes can infect various animal species, including humans, and the disease is considered contagious and zoonotic (cafarchia et al., b; chermette et al., ; kramer et al., ) . the zoophilic dermatophytes trichophyton mentagrophytes and microsporum canis infect rabbits (cafarchia et al., (cafarchia et al., , a chermette et al., ; kramer et al., ) . clinical signs the general presentation of dermatophytosis in animals is an area of circular alopecia with erythematous margin and thin desquamation (chermette et al., ) . pruritus is generally absent and lesions can be single or multiple (chermette et al., ) . although lesions can be localized in any region, the anterior part of the body and the head seem to be more frequently involved (chermette et al., ) . in rabbits, lesions are often found on the ears and the face (around the eyes and on the nose) and these lesions show scaling and crusting (chermette et al., ; kramer et al., ) . infected rabbits may not exhibit clinical signs and may serve as carriers (balsari et al., ; cafarchia et al., cafarchia et al., , a chermette et al., ; lopez-martinez et al., ) . epizootiology although dermatophytosis is a common cutaneous disease of rabbits and other animals, its incidence is low in well-managed laboratory animal facilities (chermette et al., ; connole et al., ) . contact with infected animals or contaminated environments represent the major risk of infection (chermette et al., ) . young or immunocompromised rabbits are more susceptible (connole et al., ; kramer et al., ) . on rabbit farms, the occurrence of lesions, the age of the rabbits, and farm management practices were identified as the most significant risk factors for the occurrence of dermatophytosis (cafarchia et al., ) . clinically, disease expression varies depending on the host, fungal species, and enzyme production (cafarchia et al., a; vermout et al., ) . the pathogenesis involves contact, adherence, germination, invasion, and penetration (cafarchia et al., a; mendez-tovar, ; vermout et al., ) . these stages can be associated with the secretion of enzymes that degrade the infected tissue components (cafarchia et al., a) . t. mentagrophytes isolates from rabbits with skin lesions showed a significantly higher elastase and gelatinase activity compared laboratory animal medicine to isolates from clinically unaffected rabbits and from the environment (cafarchia et al., a) . furthermore, m. canis isolates from rabbits with skin lesions showed a significantly higher lipase activity compared to isolates from clinically unaffected rabbits and from the environment (cafarchia et al., a) . pathology histopathologic changes consist of mild to severe dermatitis. diagnosis the wood's lamp (ultraviolet light) method and direct examination of hairs and scales are fast and affordable tests (chermette et al., ; robert and pihet, ) . the wood's lamp can be used to screen for infections caused by m. canis (chermette et al., ) . m. canis-infected hairs fluoresce with an apple-green color and can be collected for microscopic examination and culture (chermette et al., ) . the results of the wood's lamp examination should be systematically confirmed by direct examination of hairs and/or fungal culture (chermette et al., ) . deep skin scraping should be performed to obtain hair and scales and confirm the absence of ectoparasites such as mites that can be associated with dermatophytosis (cafarchia et al., ; chermette et al., ) . clearing solutions such as chlorolactophenol or % potassium hydroxide (koh) can then be used to digest keratin prior to microscopic examination (chermette et al., ; robert and pihet, ) . the surface of the hair typically demonstrates clusters or chains of arthroconidia (chermette et al., ) . giemsa-stained skin scrapings allow observation of the arthroconidia along the hair (chermette et al., ) . fungal culture is the 'gold standard' for the diagnosis of dermatophytosis and the only method for the phenotypic identification of dermatophyte species (chermette et al., ) . the fungal culture must be complemented with direct examination of samples for optimal interpretation of the results (chermette et al., ; robert and pihet, ) . samples for fungal culture may include hairs, scales, crusts, skin scrapes, and tissue biopsies (chermette et al., ) . samples that are obtained from the margin of new skin lesions enhance fungal recovery by culture (chermette et al., ) . a brush can also be impressed on the surface of the culture medium after combing the fur and obtaining fungal spores with hair and debris (robert and pihet, ) . two media that can be used for fungal culture include sabouraud dextrose agar (supplemented with cycloheximide and antibiotics) and dermatophyte test media (dtm) (chermette et al., ; robert and pihet, ) . if histological examination is performed, periodic acid schiff (pas), or methylamine silver stain can be used to detect arthroconidia and hyphae (chermette et al., ) . molecular methods to identify dermatophytes have also been described and include pcr-rflp and sequencing of the internal transcribed spacer (its) region (chermette et al., ; kanbe, ; robert and pihet, ) . specific identification of the dermatophyte is essential for a better understanding of the epidemiology and prevention of the disease (chermette et al., ) . differential diagnoses the differential diagnoses can include other dermatoses caused by bacteria or ectoparasites (cafarchia et al., ; chermette et al., ) . treatment, prevention, and control dermatophytosis is considered a self-limiting disease in immunocompetent animals (chermette et al., ) . however, rabbits with dermatophytosis should be culled or separated from a laboratory animal colony due to the contagious and zoonotic nature of the disease (chermette et al., ) . the best method to prevent dermatophyte infection is to prevent contact with infected animals and contaminated environments including fomites (chermette et al., ) . an animal that contacts an infected animal or a contaminated environment can be washed with antifungal shampoo (chermette et al., ) . two vaccines incorporating live attenuated cells of t. mentagrophytes have been used to prevent disease in rabbits and other animals (lund and deboer, ) . the mentavak vaccine is from russia and the trichopelen vaccine (http://www. bioveta.cz/en/veterinary-division/home/) is from the czech republic (lund and deboer, ) . trichopelen is also indicated for treatment of dermatophytosis (lund and deboer, ) . enzootic dermatophytosis may be the result of the high resistance of the arthroconidia in the environment, the number of host species involved, and the close confinement of animals (chermette et al., ) . isolation or culling of infected animals plus environmental and equipment disinfection are required to control this disease (chermette et al., ) . a : dilution of household bleach or a . % enilconazole solution can be used to disinfect the environment (chermette et al., ) . infected animals should be handled with care to avoid zoonotic transmission (chermette et al., ) . if treatment is elected, antifungal treatment shortens the course of the infection and reduces dissemination of arthroconidia to other animals and into the environment (chermette et al., ) . systemic and topical antifungal treatment can be used in combination (chermette et al., ) . systemic drugs include griseofulvin (gold standard) or azole derivatives such as itraconazole (chermette et al., ; vella, ) . it is important to know that these drugs can have side effects and be contraindicated due to their teratogenic potential (chermette et al., ) . topical treatment may include . % enilconazole, a combination of % miconazole and % chlorhexidine, or lime sulfur (chermette et al., ; vella, ) . treatment can be discontinued after two negative fungal culture results (chermette et al., ) . research complications dematophyte lesions could confound histological studies involving the skin (connole et al., ) . etiology pneumocystosis in rabbits is caused by the fungus pneumocystis oryctolagi (dei-cas et al., ) . clinical signs infected rabbits may not develop clinical signs, but immunocompromised hosts can develop severe interstitial pneumonitis (dei-cas et al., ; sheldon, ) . epizootiology corticosteroid treatment can induce disease in infected rabbits; however, spontaneous disease (not associated with drug treatment) can also occur (dei-cas et al., ; sheldon, ; soulez et al., ) . p. oryctolagi is transmitted through the transplacental route (cere et al., a; sanchez et al., ) and through direct contact and aerosolization (cere and polack, ; cere et al., b; hughes, ; wakefield, wakefield, , . spontaneous pneumocystosis can occur at weaning, evolves during - days, and induces lung lesions and blood biochemical profile changes (dei-cas et al., ; soulez et al., ) . the organisms attach specifically to type epithelial alveolar cells and proliferate, filling up pulmonary alveoli cavities leading to respiratory failure (dei-cas et al., ) . changes in surfactant appear to be necessary for pneumocystis proliferation (prevost et al., ) . pneumocystis colonization decreases and becomes very low in -day-old rabbits (dei-cas et al., ) . most rabbits recover from pneumocystosis within - weeks (dei-cas et al., ) . the spontaneous resolution of pneumocystosis in rabbits may be associated with expression of interferon gamma (allaert et al., ) . immunosuppression may be suspected in cases of severe pulmonary disease associated with spontaneous pneumocystosis (sheldon, ) . pathology histologically, cystic forms of the organism can be detected in the lungs using toluidine blue o (tbo), gms, or pas stains (dei-cas et al., ) . interstitial thickening of alveolar septa and increased numbers of type epithelial alveolar cells are characteristic of this infection (creusy et al., ) . diagnosis for diagnosis, samples from nasal cavity wash, or post-mortem, from terminal bronchoalveolar lavage (bal) or lung homogenates can be used for pneumocystis detection by nested pcr (dei-cas et al., ; tamburrini et al., ; wakefield, ) . serological diagnosis can also be performed (tamburrini et al., ) . lung impression smears, lung-homogenate smears, and bal fluid samples can be stained for microscopic detection of pneumocystis (dei-cas et al., ) . useful stains include tbo, gomori-grocott's methenamine silver nitrate (gms), and methanol-giemsa or giemsalike stains (dei-cas et al., ) . other useful detection methods include phase-contrast microscopy and the use of pneumocystis-specific fluorescein-labeled antibodies (dei-cas et al., ) . differential diagnoses p. multocida can induce respiratory disease in rabbits and can be included in the differential diagnoses. treatment, prevention, and control for prevention, new rabbits should be negative for pneumocystis. cotrimoxazole treatment and nested pcr have been used as a screening mechanism to eliminate pneumocystis from colony-maintained rabbits (cere et al., c) . decontamination practices and air filtration were also important for eradication (cere et al., c) . confirmation of a pneumocystis-free status in a rabbit colony was demonstrated by negative pcr results and/ or failure to induce pneumocystosis after experimental corticosteroid challenge (dei-cas et al., ) . research complications research studies may be affected if rabbits of unknown pneumocystis status are experimentally treated with corticosteroids or other immunosuppressant drugs (sheldon, ) . pulmonary lesions may be found in infected rabbits and could potentially confound respiratory research studies (sheldon, ) . etiology unknown. clinical signs trichobezoar is often subclinical. if the trichobezoar causes partial or complete blockage, clinical signs of gastric or intestinal obstruction will result. death can occur due to prolonged anorexia and metabolic imbalances (gillett et al., ). it appears that obstruction of the pylorus, and not the volume of the gastric mass, is the critical factor in determining the clinical progress of the animal (leary et al., ) . epizootiology the condition occurs sporadically in rabbit colonies. pathology the discovery of a hairball in a rabbit is often an incidental finding during necropsy. up to % of rabbits have been found to have gastric trichobezoars during routine necropsy (leary et al., ) . gastric rupture can also result from an obstructive trichobezoar (gillett et al., ) . diagnosis diagnosis is often difficult because the clinical signs are nonspecific and the disease often progresses gradually. some cases involving acute pyloric obstruction result in sudden clinical disease and rapid clinical decline of the animal. manual palpation may indicate the presence of a firm mass in the cranial abdomen. gastric radiographs using contrast media may aid in the diagnosis, but definitive diagnosis is often made during exploratory surgery (gillett et al., ) . differential diagnoses constipation and intestinal foreign body should be considered in the differential list. treatment, prevention, and control treatment of trichobezoar is often unsuccessful. oral administration of mineral oil at ml/day has been reported (suckow and douglas, ) . alternatively, oral administration of - ml of fresh pineapple juice daily has been reported as a possible treatment modality (harkness and wagner, ) . if medical treatment does not resolve the condition, a gastrotomy should be performed. early surgical intervention is important in such cases, as other, subsequent metabolic abnormalities may quickly increase the surgical risk to the rabbit (bergdall and dysko, ) . research complications none have been reported. etiology subluxation or compression fractures of lumbar vertebrae are often secondary to struggling during restraint, particularly when the hindquarters of the rabbit are not supported (bergdall and dysko, ) . the seventh lumbar vertebra (l ) or its caudal articular processes are considered the most frequent sites of fractures, with fracture occurring more commonly than dislocation (flatt et al., ) . clinical signs clinical signs include posterior paresis or paralysis, loss of sensation in the hindlimbs, urinary and/or fecal incontinence, and perineal staining. pathology spinal cord hemorrhage and necrosis can be found. diagnosis diagnosis is based on clinical signs, history of recent restraint, struggling or other trauma, and palpation or radiographic analysis of the vertebral column. differential diagnoses spinal cord trauma. treatment, prevention, and control euthanasia of affected animals is usually warranted. moderate cases (subluxation with spinal edema) may resolve over time. the decision to euthanize should be based on severity of clinical signs. supportive care includes regular expression of the urinary bladder and prevention and treatment of decubital ulcers. corticosteroid and diuretic therapy may be effective for cases of subluxation with spinal edema (bergdall and dysko, ) . research complications loss of valuable research animals is the primary complication. although the condition is often referred to as 'sore hocks,' the correct name is ulcerative pododermatitis. despite the name, the condition rarely affects the hocks, but rather occurs most frequently on the plantar surface of the metatarsal and, to a lesser extent, the metacarpal regions. the condition is believed to be initiated by wire-floor housing, foot stomping, or having thin plantar fur pads. poor sanitation may worsen the condition. solid resting areas on the cage floors are associated with a decreased incidence of ulcerative pododermatitis, whereas a high-energy diet and increased body condition scores are associated with an increased incidence (sanchez et al., ) . the whole genome sequence from a single female rabbit of the partially inbred thorbecke rabbit strain was published in (orycun . ; accession aagw ). the annotated assembly is now available at the national center for biotechnology information (ncbi), the university of california santa cruz (ucsc), and ensembl. the rabbit chosen by the broad institute for sequencing was obtained from covance in . the assembly has . gbp in autosome and x chromosomes and mbp in unplaced scaffolds including mitochondria (gertz et al., ) . the nucleotide sequence of the complete mitochondrial dna (mtdna) molecule of the o. cuniculus has been determined (gissi et al., ) . the compositional differences between the two mtdna strands have also been detailed (gissi et al., ) . the sequencing of the rabbit genome, understanding of rabbit reproduction, and advances in genetic manipulation in the mouse production colonies have led to the ability to produce genetically engineered rabbits. the rabbit offers an alternative model when size or tissue characteristics of a genetically modified mouse are not appropriate. these genetic manipulation techniques were first described by robl (robl and burnside, ) . additional methods have been developed and include pronuclear injection of single cell embryos, injection of genetically modified embryonic stem cells into blastocysts, sperm-mediated gene transfer, and genetically modified somatic cell and nuclear transfer (christensen and peng, ) . commercial companies have been formed to provide genetic modification services with emphasis on production of a unique protein in the milk of rabbits. this section will outline spontaneous hereditary conditions of the rabbit that have been well characterized. some conditions represent conditions that have been identified in humans and other conditions offer insight into the mechanism(s) of particular organ or immune function. hydrocephalus refers to dilatation of the cerebral ventricles and is usually accompanied by accumulation of cerebrospinal fluid within the dilated spaces. some cases of hydrocephalus in rabbits have been presumed to be related to a single autosomal recessive gene (hy/hy); however, occurrence with other abnormalities suggests that inheritance may be more complicated (lindsey and fox, ) . in some cases, the condition appears to be inherited along with various ocular anomalies as an autosomal gene with incomplete dominance. hydrocephalus may also occur in rabbits as a congenital condition related to hypovitaminosis a in pregnant does (lindsey and fox, ) . etiology buphthalmia is inherited as an autosomal recessive trait, although penetrance is presumably incomplete since severity and the age of onset vary greatly and some bu/bu individuals do not develop buphthalmia (hanna et al., ) . clinical signs rabbits with hereditary glaucoma develop ocular changes that resemble human congenital glaucoma and buphthalmia. newborn bu/bu rabbits initially have normal intraocular pressure (iop; - mmhg) but increased pressures of - mmhg may develop after - months of age (burrows et al., ; knepper et al., ) . the eyes become progressively buphthalmic (either uni-or bilaterally) but the iop can return to normal or to sub-normal levels after - months. typical clinical changes include increased corneal diameter as the globe enlarges because the sclera is still immature. the cornea may develop a cloudy or bluish tint, corneal edema, increased corneal vascularity, and flattening of the cornea. structural changes may include widening of the angle, thickening of descmet's membrane, atrophy of the ciliary process, and excavation of the optic disk. impaired aqueous outflow may be due to incomplete cleavage of the drainage angle with abnormal insertion of uveal tissue into the cornea (tesluk et al., ) . in some cases, the cornea ulcerates and ruptures. there is also a marked reduction in semen concentration in buphthalmics, with a decrease in libido and decreased spermatogenesis in affected males (fox et al., ) . epizootiology the condition is common in new zealand white rabbits. pathology by weeks of age, the morphology of the congenital glaucoma trabecular network becomes abnormal with a smaller entrance to the trabecular network at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and by weeks, iris pillars with extensive lateral extensions in the angle recess can be observed. most intertrabecular spaces remain open; however, the inner intertrabecular spaces adjacent to the aqueous plexus become compressed. diagnosis diagnosis is based on clinical signs and measurement of intraocular pressure. treatment, prevention, and control specific treatment of buphthalmia has not been described for rabbits; however, affected individuals should not be used for breeding purposes. research complications loss of valuable research animals is the primary complication. etiology mandibular prognathism is the most common inherited disease of domestic rabbits. the condition is inherited as an autosomal recessive trait (mp/mp) with incomplete penetrance (fox and crary, ; huang et al., ; lindsey and fox, ) . clinical signs malocclusion related to mandibular prognathia may be clinically apparent as early as - weeks of age, but is more typically seen in older rabbits post weaning. clinical signs may include anorexia and weight loss. if severe enough and left untreated, affected animals will starve since they cannot properly prehend and masticate food. epizootiology normally, the lower incisors occlude with the large upper incisors, as well as with a pair of small secondary incisors that are immediately caudal to the primary maxillary incisors. the lower set of incisors typically wear against the upper set during normal biting activity, along an arc formed by biting movements of the lower incisors, whereas the maxillary secondary incisors wear at right angles to the mandibular incisors. the incisors wear more quickly at the posterior aspect in rabbits, partly because the enamel layer is thinner on that side. affected rabbits have a normal dental formula. the specific abnormality associated with mandibular prognathism is that the maxilla is short relative to a mandible of normal length. thus, although the mandible appears abnormally long, the primary defect involves the maxilla. in rabbits, the teeth (including the molars and premolars) grow continuously throughout life. the incisors, for example, grow at the rate of . - . mm/ week. when occlusion is normal, the teeth wear against one another and in this way remain a normal length. however, when occlusion is abnormal because of conditions that include mandibular prognathia, the teeth may become greatly elongated because typical attrition of the incisors does not occur. in affected animals the lower incisors often extend anterior to the upper incisors and protrude from the mouth, whereas the upper primary incisors grow past the lower incisors and curl within the mouth. in some instances, the upper incisors curl around dorsally and lacerate the mucosa of the hard palate. secondary infection and abscessation may occur in such cases. diagnosis diagnosis is based on clinical signs. differential diagnoses malocclusion secondary to mandibular or maxillary fracture should be considered. treatment, prevention, and control overgrown teeth should be trimmed every - weeks or more frequently if needed. trimming is preferably performed with a dental bur to avoid cracking the tooth, which laboratory animal medicine may happen more frequently if a bone or wire cutter is used. care should be taken to avoid exposing the pulp cavity as the result of excessive trimming. because the condition is hereditary, use of affected animals as breeding stock should be avoided. research complications no specific research complications have been reported. a number of disorders characterized clinically by complete abduction of one or more legs and the inability to assume a normal standing position are described by the term 'splay leg'. young kits of - weeks of age are most commonly affected. affected rabbits cannot adduct limbs and have difficulty in making normal locomotory movements. most commonly, animals are affected in the right rear limb, although the condition may be uni-or bilateral and may affect the anterior, posterior, or all four limbs. rabbits with splay leg may have difficulty in accessing food and water; thus, attention to adequate nutrition is required as part of proper clinical care. the clinical signs of splay leg may be due to an overall imbalance of development of the neural, muscular, and skeletal systems. possibly, some animals compensate with torsion and exorotation of the limb at the hip, whereas rabbits that are unable to compensate are clinically affected. although the precise pathogenesis of splay leg is not entirely understood, at least some cases are ascribed to inherited disorders. typical clinical signs are secondary to femoral endotorsion, with a shallow acetabulum but without luxation of the femur at the hip. the semitendinosus muscle of affected animals is abnormal, with smaller fibers and abnormal mitochondria. some reports suggest that the condition is associated with inherited achondroplasia of the hip and shoulder, whereas others indicate that a recessively inherited anteversion of the femoral head can be involved. self-mutilating behavior in a checkered cross (cross between english spot, german checkered giant, and checkered of rhineland rabbits) was reported to occur as an inherited trait (iglauer et al., ) . autotraumatization of the feet and pads was observed. the abnormal behavior could be interrupted by administration of haloperidol. although not manifested as a disease, the presence of serum atropine esterase allows rabbits to inactivate atropine when administered for therapeutic purposes (liebenberg and linn, ; stormont and suzuki, ) . the enzyme also permits rabbits to consume diets containing belladonna compounds. the enzyme is produced by a semidominant gene est- f. three phenotypes are recognized depending on the number of genes expressed. the enzyme first appears in the serum at month of age, and enzyme levels are greater in females than in males (lindsey and fox, ) . the est- f gene is linked to genes controlling the black pigment in the coat (forster and hannafin, ; fox and van zutphen, ; sawin and crary, ) . hereditary deficiency of the third component of complement (c ) was found in a strain of rabbits. this same strain also exhibited a hereditary c alpha-gamma deficiency. the serum c concentration, hemolytic c activity, and total complement hemolytic activity of these animals were significantly reduced. the low level of serum c in these rabbits was not due to c conversion, partial c antigenicity, and presence of a c inhibitor or hypercatabolism of normal c . the c deficiency was transmitted as a simple autosomal co-dominant trait. rabbits with this trait have a lower survival at months than normal rabbits (komatsu et al., ) . this complement deficiency syndrome in the rabbit has been well characterized. this syndrome was initially reported in in a strain of rabbits that lacked the sixth component of the hemolytic complement system (rother et al., ) . whole blood clotting time in glass or plastic was prolonged and prothrombin consumption was decreased in blood from the deficient animals. other parameters of blood coagulation were normal, including prothrombin time, partial thromboplastin time, specific clotting factor activities, platelet factor iii function, platelet count, and bleeding time (zimmerman et al., ) . abnormal platelet response is also characteristic of this syndrome in the rabbit (lee et al., ) . complement c -deficient rabbits are protected against diet-induced atherosclerosis despite having similar profiles in cholesterol levels and plasma lipoprotein. when compared to normal rabbits, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all normal rabbits while absent in all c -deficient animals (schmiedt et al., ) . the inheritance pattern for this defect is autosomal recessive (abe et al., ) . a progressive neurological syndrome has also been observed in the c -deficient rabbits. this syndrome is clinically characterized by subacute motor neuropathy. pathological studies of affected animals revealed ( ) severe axonal degeneration in the sciatic nerve involving mainly motor fibers; ( ) occasional peripheral axonal enlargement closely associated with axonal laboratory animal medicine degeneration; ( ) presence of structured abnormal material in normal-size myelinated fibers of the central and peripheral nervous systems; and ( ) widespread occurrence of dystrophic axons and axonal spheroids in the gray matter of the central nervous system. by ultrastructural examination, dystrophic axons were filled with tubulovesicular material, appearing as stalks of parallel membranes and dense bodies similar to what is described in human neuroaxonal dystrophies (nad). the disease manifested by c -deficient rabbits may represent an animal model of primary human nad (giannini et al., ) . genetic deficiency of the alpha-gamma-subunit of the eighth complement component (c alpha-gamma) was found in a substrain of the new zealand white rabbits. the serum of this deficient rabbit lacked the immunochemical and functional alpha-gamma-subunit of c (c d). this syndrome is transmitted as a simple autosomal recessive trait. the syndrome is characterized by smaller body weight compared to those of heterozygous and normal rabbits. in addition, survival rates for the first months of life of the deficient animals tended to be lower than those of heterozygous and normal littermates (komatsu et al., ) . all c d rabbits (more than animals obtained thus far) were consistently smaller than normal littermates from birth to adulthood, i.e., % of normal size at birth, % of normal size at days of age, and % of normal size at adulthood. the c α-γ deficiency in rabbits is always associated with dwarfism. furthermore, there appears to be a discrete recessive dwarf gene (dw- ), whose locus is not linked to c d. rabbits double-homozygous for c d and dw- (severe dwarf) were smaller than the c d or dwarf rabbits and almost all of the severe dwarf rabbits died within days after birth. the actual and relative weights of the thymus in the c d rabbits were consistently lower than those of normal rabbits, but histological examination of the c d thymus did not reveal any abnormalities. the c d and dwarf rabbits were fertile; however, crosses of c d females with c d or dwarf males led to a reduced delivery rate and small litter size. the c d locus is loosely linked to the c hypocomplementemic locus (c -hypo) (map distance cm) but not to the hemoglobin blood group locus (komatsu et al., ) . the inherited characteristics of the kurosawa and kusanagi hypercholesterolemic (khc) rabbit include persistent hypercholesterolemia. this strain of rabbits was produced by inbreeding mutants discovered in . these khc rabbits had serum cholesterol, triglyceride, and phospholipid levels - times greater than clinically normal o. cuniculus. the khc rabbits also had decreased serum high-density lipoprotein cholesterol concentration, about one-third the value in clinically normal rabbits. in addition, the serum lipoprotein electrophoretic patterns were characterized by a strong, broad beta-lipoprotein band and a diminished alphalipoprotein band. fractionation of lipoprotein lipids revealed increased cholesterol, phospholipid, and triglyceride in the ldl fraction; increased cholesterol and phospholipid in the very ldl fraction; and decreased cholesterol and triglyceride in the high-density lipoprotein fraction. the inheritance is thought to be a single autosomal recessive gene mutation, and analysis of the ldl receptor indicated that the khc rabbit has a -base pair deletion in the ldl receptor mrna. macroscopic analysis of the aorta revealed the atheromatous lesions at months of age, drastically increased lesional areas in the total aortic surface at months of age, and a high incidence of coronary atheromas and xanthomas (kurosawa et al., ) . a spontaneous phenotype in a rabbit was discovered with an elevation of serous lipid ingredients including cholesterol and beta-lipoprotein (beta-lp). atherosclerotic lesions were evident in the aorta and renal arteries. nodular xanthomas were also present on the front and rear feet. the hlr strain was inbred to accentuate these characteristics (watanabe et al., ) . the strain was eventually designated the watanabe-heritable hyperlipidemic rabbit (whhl-rabbit). an additional report of this strain of rabbits indicated that the whhl-rabbits spontaneous developed aortic atherosclerosis by months of age and xanthoma of digital joints in % of the rabbits aged to months (watanabe, ) . historically, spontaneous neoplasia in the laboratory rabbit has not been widely reported because neoplasia in the rabbit is very uncommon before years of age and many laboratory rabbits are not maintained beyond this age (weisbroth, ) . endometrial adenocarcinoma is the most common tumor in aged female rabbits, with an incidence of % reported in a colony of -year-old rabbits (baba and von haam, ) . tinkey et al. complied an extensive review of the literature dealing with spontaneous neoplasia in the domestic rabbit. this review contained data on case reports, descriptions of biologic aspects of naturally occurring tumors and reports of experimentally induced tumor models. neoplasia in sylvilagus and lepus were also discussed (tinkey et al., ) . uterine adenocarcinoma is by far the most common tumor in rabbits. typically, the disease is present as multiple tumors and is malignant, often metastasizing to the liver, lungs, and other organs. there is evidence that inheritance plays a role in susceptibility, but parity does not. uterine leiomyomas and leiomyosarcomas are much less common (weisbroth, ) . there are a few reports of vaginal squamous cell carcinomas (weisbroth, ) and an ovarian hemangioma has been described (greene and strauss, ) . mammary adenocarcinomas are fairly common in older female rabbits and may occur in animals with uterine adenocarcinoma (weisbroth, ) . papillomas have been described, but mammary adenocarcinomas are much more important. these malignant tumors may metastasize, but the cause of death in affected rabbits is often due to uterine adenocarcinoma. serial biopsy studies indicate that these tumors are preceded by cystic mastopathy as well as changes in the adrenal and pituitary glands (greene, ) . there may also be small prolactin-secreting pituitary adenomas in rabbits with mammary dysplasia (lipman et al., ) . testicular tumors in the rabbit appear to be relatively uncommon. interstitial tumors are the most common testicular tumor in the rabbit. seminomas and teratomas have also been reported (weisbroth, ) . embryonal nephromas are one of the most common tumors in laboratory rabbits. these tumors are often found incidentally, occur in younger animals, and seldom cause clinical signs (weisbroth, ) . there has been one report of a renal carcinoma in the rabbit (kaufman and quist, ) and one report of a leiomyoma arising in the urinary bladder (weisbroth, ) . malignant lymphomas (lymphosarcomas) are relatively common in rabbits. they may occur in rabbits that are less than years of age (weisbroth, ) , but older rabbits may also be affected. according to (weisbroth, ) , a tetrad of lesions is often seen. these lesions include enlarged kidneys, splenomegaly, hepatomegaly, and lymphadenopathy. older rabbits have presented with skin nodules and eye lesions; however, malignant lymphomas in the rabbit are seldom leukemic. most cases of malignant lymphoma appear to resemble the lymphoblastic subtype as seen in humans and mice. malignant lymphoma is more prevalent in some strains of rabbits than in others, and there is some evidence for a retroviral cause of lymphomas in rabbits (weisbroth, ) . true thymomas (containing both lymphoid and epithelial components) (vernau et al., ) and plasma cell myelomas (pascal, ) are rare in rabbits. one case of myeloid leukemia has been reported (meier et al., ) . basal cell tumors are reported to be rare (weisbroth, ) , but they may be underreported (li and schlafer, ) . squamous cell carcinomas are also uncommon, and there is no apparent predilection for any particular area of the body (weisbroth, ) . other cited skin-associated tumors include a trichoepithelioma (altman et al., ) , a sebaceous gland carcinoma (port and sidor, ) , and two malignant melanomas (hotchkiss et al., ) . osteosarcomas are extremely rare in rabbits, and most have arisen in the mandible or maxilla, with only one found in a long bone (weisbroth, ) . no primary tumors arising in cartilage have been described, although some of the reported osteosarcomas have had cartilaginous elements. one tumor of skeletal muscle, a rhabdomyosarcoma, has been reported. a few fibrosarcomas and one fibrosarcoma involving the foot have been reported (weisbroth, ) . a number of case reports of single tumors are found in the literature. these include a peritoneal mesothelioma (lichtensteiger and leathers, ) , an intracranial teratoma (bishop, ) , an ependymoma (kinkier and jepsen, ) , a neurofibrosarcoma, two hemangiosarcomas (pletcher and murphy, ) , and a malignant fibrous histiocytoma (yamamoto and fujishiro, ). there are a few very old reports of lung tumors dating to the first part of the th century (weisbroth, ) . there are several tumor models in which the cells used for inoculation were originally derived from rabbit tumors. these include the vx- carcinoma (kidd and rous, ) , the brown pearce carcinoma (brown and pearce, ) , and the greene melanoma (greene, ) . the vx- carcinoma originated from a squamous cell carcinoma in a rabbit carrying a shope papilloma. the most common modern use of this transplantable tumor is as a model for the study of various cancer treatment modalities for metastatic tumors (stetson et al., ) . the brown pearce carcinoma arose from a tumor in a rabbit testis, but the exact tissue of origin of the tumor was never determined. the tumor was readily transplantable and caused stable metastases. because some tumors regress, even after widespread metastases, this tumor has been used as a model for the study of tumor immunology (weisbroth, ) . the brown pearce laboratory animal medicine carcinoma, although extensively characterized and historically used, has been reported in the literature only five times from to (tinkey et al., ) . hydrometra has been described as a clinical condition of rabbits. all cases were in unmated rabbits that were used experimentally for the production of serum antibodies (bray et al., ; hobbs and parker, ; morrell, ) . clinical signs included abdominal distension and tachypnea. cases were characterized by distension of the uterine horns with a transudative fluid. one case was associated with uterine torsion (hobbs and parker, ) . one case had resolved with diuretic therapy, only to return later (bray et al., ) . most cases of liver lobe torsion in rabbits involve the caudate lobe (bergdall and dysko, ) , although one case report described torsion of the left hepatic lobe (wilson et al., ) . most reported cases have been incidental findings at necropsy. incidental hepatic lobe torsions have also been identified in three adult new zealand white rabbits that died from pasteurellosis (weisbroth, ) . three cases of hepatic torsion in pet rabbits were reported by wenger in (wenger et al., ) . all rabbits presented with an acute onset of lethargy, anorexia, abdominal pain, pale mucous membranes, and jaundice. one rabbit also had hematuria. another report of caudate liver lobe torsion also described a rabbit that was jaundiced, anemic, and anorexic, with elevated alanine aminotransferase. torsion of the caudate liver lobe was seen at necropsy (fitzgerald and fitzgerald, ) . in all reported clinical cases, rabbits were euthanized, or died during postoperative recovery. calcium carbonate and triple phosphate crystals are present in the urine of normal rabbits. these crystals contribute to the cloudy consistency of the urine (williams, ) . a -year retrospective study of hematuria in new zealand white rabbits was conducted by garibaldi (garibaldi et al., ) . physical examination, laboratory tests, radiography, and postmortem examination were utilized in most cases to verify the presence of hematuria and to determine its etiology. uterine adenocarcinoma was diagnosed in two rabbits. three rabbits had uterine polyps with hemorrhage. renal infarction with hemorrhage was diagnosed in three rabbits. urolithiasis with secondary urethral obstruction and hemorrhagic cystitis was identified as the cause of hematuria in four rabbits. other causes of hematuria included chronic cystitis, disseminated intravascular coagulation, bladder polyps and pyelonephritis. hematuria of undetermined origin was observed in one rabbit which emphasizes that hyperpigmented urine should be a rule out in all cases of suspected hematuria in rabbits (garibaldi et al., ) . one case of urolithiasis with hydronephrosis in a new zealand white rabbit was also reported (labranche and renegar, ) . this condition must be distinguished from hematuria caused by endometrial venous aneurysm in female rabbits (bray et al., ) . herniation of the kidney along with perinephric fat has been reported (suckow and grigdesby, ) . the affected rabbit was clinically normal except for a subcutaneous mass that had passed through the body wall. the precise etiology is not known, although it was speculated that herniation might have occurred as the result of unreported trauma. occlusion of the nasolacrimal duct, presumably due to accumulation of fat droplets, has been described as a putative cause of epiphora in some rabbits (marini et al., ) . although the obstruction occurred at the dorsal flexure, it is not clear if this was due to congenital rather than acquired stenosis. in a retrospective study of rabbits it was determined that the mean age of the rabbits presenting with ocular discharge from the nasolacrimal duct was . years. in rabbits ( %), dacryocystitis was a unilateral finding. no underlying cause could be determined in animals ( %). dental malocclusion was observed in rabbits ( %) and rhinitis in two animals ( %), with one animal showing both signs ( %). one rabbit ( %) presented with panophthalmitis. most animals ( %) received topical antibiotic treatment. regarding the clinical outcome, animals ( %) showed complete recovery, eight rabbits ( %) were euthanized, three ( %) died due to unrelated causes, and three ( %) were lost to follow-up. two rabbits ( %) continued to display signs of dacryocystitis (florin et al., ) . development and genetic differences of complement activity in rabbits influence of pasteurella multocida infection on 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(orycun . ) and localization of the igh locus to chromosome tpr homologs in treponema paraluiscuniculi cuniculi a strain inherited neuroaxonal dystrophy in c deficient rabbits fibres in rabbit feeding for digestive troubles prevention: respective role of low-digested and digestible fibre feeding behaviour of rabbits selected drug dosages and clinical reference data medical and surgical management of gastric obstruction from a hairball in the rabbit the complete mitochondrial dna sequence of the rabbit infection with and antibody response to pasteurella multocida and bordetella bronchiseptica in immature rabbits the pathology of experimental respiratory infection with pasteurella multocida and bordetella bronchiseptica in rabbits quantitation of rabbit cytokine mrna by real-time rt-pcr detection of toxigenic clostridium difficile in diarrheal stools by rapid real-time polymerase chain reaction the effect of host's age on the pathology of eimeria stiedae infection in rabbits a spontaneous melanoma in the hamster with a propensity for amelanotic alteration and sarcomatous transformation during transplantation diseases of the rabbit multiple primary tumors in the rabbit paratuberculosis in wild rabbits (oryctolagus cuniculus) inventory of the behaviour of new zealand white rabbits in laboratory cages identification of genes transcribed by pasteurella multocida in rabbit livers through the selective capture of transcribed sequences detection and characterization of cryptosporidium cuniculus by real-time pcr . rabbits. in: reproduction and breeding techniques for laboratory animals. lea & febiger the effects of continuous sulfaquinoxaline feeding on rabbit mortality spontaneous papillomatosis in domestic rabbits antibacterial efficacy and pharmacokinetic studies of ciprofloxacin on pasteurella multocida infected rabbits recessive buphthalmos in the rabbit the effect of environmental enrichment on the behaviour of caged rabbits microbial induction of b and t cell areas in rabbit appendix the biology and medicine of rabbits and rodents pathogenesis of herpesvirus sylvilagus infection in cottontail rabbits normal biochemical and hematological values in new zealand white rabbits early volume expansion during diarrhea and relative nephroprotection during subsequent hemolytic uremic syndrome transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector syphilis: using modern approaches to understand an old disease uterine torsion associated with either hydrometra or endometritis in two rabbits atrioventricular conduction in mammalian hearts arthropod and helminth parasites isolation of clostridium spiroforme from rabbits proliferative enteropathy involving lawsonia intracellularis infection in rabbits (oryctlagus cuniculus) coecotrophy in rabbits-a circadian function the route of migration of eimeria stiedae (lindemann, ) sporozoites between the duodenum and bile duct of the rabbit the treatment of hepatic coccidiosis in rabbits malignant melanomas in two rabbits proliferative enteropathy of rabbits: the intracellular campylobacter-like organism is closely related to lawsonia intracellularis real-time multiplex polymerase chain reaction assay for rapid detection of clostridium difficile toxin-encoding strains mandibular prognathism in the rabbit: discrimination between single-locus and multifactorial models of inheritance natural mode of acquisition for de novo infection with pneumocystis carinii response of adult new zealand white rabbits to enrichment objects and paired housing hereditary compulsive self-mutilating behaviour in laboratory rabbits spontaneous cryptosporidiosis in an adult female rabbit liver coccidiosis prevented by sulfasuxidine pasteurella associated rhinitis of rabbits: efficacy of penicillin therapy atherosclerotic research a new model of sheep ig diversification: shifting the emphasis toward combinatorial mechanisms and away from hypermutation the rabbit: a diurnal or nocturnal animal? characterization of a novel alphaherpesvirus associated with fatal infections of domestic rabbits an outbreak of fatal herpesvirus infection in domestic rabbits in alaska proteomic analyses of transgenic lqt and lqt rabbit hearts elucidate an increase in expression and activity of energy producing enzymes ovarian abscesses and pyometra in a domestic rabbit an epizootic of shope fibromatosis in a commercial rabbitry enhanced detection of intracellular organism of swine proliferative enteritis, ileal symbiont intracellularis, in feces by polymerase chain reaction eimeria stiedae in rabbits: the demonstration of responses to chemotherapy pure culture of the pathogenic agent of tyzzer's disease of mice molecular approaches in the diagnosis of dermatophytosis pathogenic escherichia coli beat-to-beat regulation of heart rate by afferent stimulation of the aortic nerve implantable stimulating electrode for baroreceptor afferent nerves in rabbits spontaneous renal carcinoma in a new zealand white rabbit an outbreak of massive mortality among farm rabbits associated with cryptosporidium infection growth of tyzzer's organism in primary monolayer cultures of adult mouse hepatocytes the comparative pathology of clostridium difficile-associated disease superovulatory response of pre-and post-pubertal rabbits to commercially available gonadotropins viral infections of rabbits a transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form cloning and sequence analysis of another shiga-like toxin iie variant gene (slt-iiera) from an escherichia coli r strain isolated from rabbit ependymoma in a rabbit deficiency of low density lipoprotein receptors in liver and adrenal gland of the whhl rabbit, an animal model of familial hypercholesterolemia peripheral thermal sensitivity in the rabbit ultrastructural studies of primary congenital glaucoma in rabbits spontaneous nosema cuniculi infection in laboratory rabbits genetic deficiency of the alpha-gamma-subunit of the eighth complement component in the rabbit hereditary c hypocomplementemia in the rabbit hereditary c α-γ deficiency associated with dwarfism in the rabbit environmental factors, clinical signs, therapy and zoonotic risk of rabbits with dermatophytosis biology and diseases of rabbits localization of human coagulation factor viii (hfviii) in transgenic rabbit by fish-tsa: identification of transgene copy number and transmission to the next generation encephalitozoonosis in rabbits new mutant rabbit strain with hypercholesterolemia and atherosclerotic lesions produced by serial inbreeding use of selamectin for the treatment of psoroptic and sarcoptic mite infestation in rabbits urinary calculus and hydronephrosis in a new zealand white rabbit preliminary report on the observation of a coronavirus in the intestine of the laboratory rabbit hereditary hyperlipidemia in the rabbit due to overproduction of lipoproteins, i. biochemical studies proliferative enteropathy experimental and naturally-occuring gastric foreign bodies in laboratory rabbits vitamins in rabbit nutrition: literature review and recommendations farmed rabbits and ducks as vectors for vtec o :h cheyletiella dermatitis: a report of fourteen cases abnormal platelet response to thromboplastin infusion in rabbits deficient in the sixth component of complement value of histopathology, immunohistochemistry, and real-time polymerase chain reaction in the confirmatory diagnosis of encephalitozoon cuniculi infection in rabbits fecal occult blood manifestation of intestinal eimeria spp. infection in rabbit production of transgenic rabbit embryos through intracytoplasmic sperm injection a spontaneous skin basal cell tumor in a black french minilop rabbit peritoneal mesothelioma in the rabbit tyzzer's disease catalytic enzyme activity concentration in plasma of man, sheep, dog, cat, rabbit, guinea pig, rat, and mouse seasonal and sexual influence on rabbit atropinesterase prevalence of lawsonia intracellularis, salmonella spp. and eimeria spp. in healthy and diarrheic pet rabbits inherited diseases and variations expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome q utilization of cholestyramine resin as a preventative treatment for antibiotic (clindamycin) induced enterotoxemia in the rabbit prolactin-secreting pituitary adenomas with mammary dysplasia in new zealand white rabbits differential conditions for early after-depolarizations and triggered activity in cardiomyocytes derived from transgenic lqt and lqt rabbits complete genome sequence of pasteurella multocida hn , a toxigenic strain of serogroup d resolution of established cardiac hypertrophy and fibrosis and prevention of systolic dysfunction in a transgenic rabbit model of human cardiomyopathy through thiol-sensitive mechanisms dermatophytes isolated from laboratory animals group housing: meeting the physical and social needs of the laboratory rabbit an enzymelinked immunosorbent assay to detect serum igg to pasteurella multocida in naturally and experimentally infected rabbits comparison of sequenced escherichia coli genomes is there a difference between hare syphilis and rabbit syphilis? cross infection experiments between rabbits and hares immunoprophylaxis of dermatophytosis in animals the effect of sulfaquinoxaline on the course of eimeria stiedae infection in the domestic rabbit estimating relative pollution of the environment with oocysts of eimeria stiedae immunology of lagomorphs b cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues rhythmic contractile activity of the in vivo rabbit aorta serology of pasteurella multocida in laboratory rabbits: a review microbiologic, radiographic, and anatomic study of the nasolacrimal duct apparatus in the rabbit (oryctolagus cuniculus) worsening of diet-induced atherosclerosis in a new model of transgenic rabbit expressing the human plasma phospholipid transfer protein two encephalitozoon cuniculi strains of human origin are infectious to rabbits lsaa, an antigen involved in cell attachment and invasion, is expressed by lawsonia intracellularis during infection in vitro and in vivo vitamins in animal nutrition clinical and pharmacological properties of ivermectin in rabbits and guinea pigs myeloid leukemia in the rabbit treatment of rabbit chyletiellosis with selamectin or ivermectin: a retrospective case study pathogenesis of shiga-toxin producing escherichia coli pathogenesis of dermatophytosis and tinea versicolor rabbit hemorrhagic disease: a review with special reference to its epizootiology clinical biochemical and hematological reference values in normal experimental animals and normal humans introduction: one health perspective intraepithelial vibrio associated with acute typhlitis of young rabbits infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy ectoparasites of the wild rabbit, oryctolagus cuniculus (l.) in australia rabbitpox: a model of airborne transmission of smallpox colobomatous microphthalmos in a new zealand white rabbit, arising from a colony with suspected vitamin e deficiency development of a high-sensitivity nested pcr assay for the detection of clostridium piliforme in clinical samples acquired resistance to reinfection of rabbits with eimeria magna the rabbit differs from other mammalian species in the tissue distribution of alkaline phosphatase isoenzymes immunological responses to vaccination following experimental lawsonia intracellularis virulent challenge in pigs clinical efficacy of botanical extracts from eupatorium adenophorum against the sarcoptes scabiei (sarcoptidae: sarcoptes) in rabbits walker's mammals of the world antibiograms of pathogenic bacteria isolated from laboratory rabbits in ibadan eimeria species of the domestic rabbit (oryctolagus cuniculus) pasteurella multocida toxin activation of heterotrimeric g proteins by deamidation life cycle of eimeria stiedae the reproduction of eimeria flavescens and eimeria intestinalis in suckling rabbits the rabbit coccidium eimeria piriformis: selection of a precocious line and life-cycle study protozoal diseases protozoal diseases rapid detection of clostridium difficile toxins from stool samples using real-time multiplex pcr efficacy of an injectable formulation of eprinomectin against psoroptes cuniculi, the ear mange mite in rabbits identification of the cellular receptor of clostridium spiroforme toxin acaricidal activity of eugenol based compounds against scabies mites plasma cell myeloma in the brain of a rabbit an outbreak of encephalomyelitis in broiler rabbits caused by nosema cuniculi diagnostic performance of different fecal lawsonia intracellularis-specific polymerase chain reaction assays as diagnostic tests for proliferative enteropathy in pigs: a review significance of clostridium spiroforme in the enteritis-complex of commercial rabbits biotype, serotype, and pathogenicity of attaching and effacing enteropathogenic escherichia coli strains isolated from diarrheic commercial rabbits three ectoparasites of veterinary interest communicable to man coccidia and coccidiosis parenteral infection experiments with eimeria stiedae transgenic rabbit models for studying human cardiovascular diseases meningitis and subgaleal, subdural, epidural empyema due to pasteurella multocida detection of clostridium difficile toxins from the small intestine and cecum of rabbits with naturally acquired enterotoxemia molecular bases of genetic diversity and evolution of the immunoglobulin heavy chain variable region (ighv) gene locus in leporids rearing germfree cesarean-born rats, mice, and rabbits through weaning spontaneous malignant hemangiosarcomas in two rabbits the behaviour of group penned and individually caged laboratory rabbits genetic characterization of antibiotic resistance in enteropathogenic escherichia coli carrying extendedspectrum beta-lactamases recovered from diarrhoeic rabbits environmental enrichment of new zealand white rabbits living in laboratory cages identification of eae sequences in enteropathogenic escherichia coli strains from rabbits a sebaceous gland carcinoma in a rabbit tyzzer's disease in rabbits in britain pneumocystosis in humans or in corticosteroid-untreated animal models: interactions between pulmonary surfactant changes and pneumocystis carinii in vivo or in vitro growth wild rabbits-a novel vector for verocytotoxigenic escherichia coli o evaluation of available diagnostic methods for clostridium piliforme in laboratory rabbits (oryctolagus cuniculus) further investigation of exposure to lawsonia intracellularis in wild and feral animals captured on horse properties with equine proliferative enteropathy efficacy of an avirulent live vaccine against lawsonia intracellularis in the prevention of proliferative enteropathy in experimentally infected weanling foals transmission of lawsonia intracellularis to weanling foals using feces from experimentally infected rabbits outbreak of sarcoptic mange and malasseziasis in rabbits (oryctolagus cuniculus) characterization of a fibroma virus isolated from naturally-occurring skin tumors in domestic rabbits the pangenome structure of escherichia coli: comparative genomic analysis of e. coli commensal and pathogenic isolates sudden death syndrome in adult cows associated with clostridium perfringens type e cryptosporidium cuniculus in the rabbit (oryctolagus cuniculus) clostridium difficile colitis in a rabbit following antibiotic therapy for pasteurellosis efficacy of cmx as a prophylactic and presymptomatic antiviral agent in new zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans comparative metabolism of tritiated water by mammals cytotoxicity of bacillus piliformis nutritional muscular dystrophy and neonatal mortality in a rabbit breeding colony laboratory diagnosis of vitamin e deficiency in rabbits fed a faulty commercial ration conventional methods for the diagnosis of dermatophytosis hydrocephalus and cleft palate in an inbred rabbit colony re-description of cryptosporidium cuniculus inman and takeuchi, (apicomplexa: cryptosporidiidae): morphology, biology and phylogeny production of transgenic rabbits a study of the life cycle of eimeria stiedae (lindemann, ) and the immunological response of the host immunocytochemistry of psoroptes cuniculi stained by sera from naive and infested rabbits: preliminary results deficiency of the sixth component of complement in rabbits with an inherited complement defect the life cycle of four intestinal coccidia of the domestic rabbit genome sequence of lawsonia intracellularis strain n , isolated from a sow with hemorrhagic proliferative enteropathy a cottontail rabbit papillomavirus strain (crpvb) with striking divergent e and e oncoproteins: an insight in the evolution of papillomaviruses transgenic rabbit model for human troponin i-based hypertrophic cardiomyopathy exploring transplacental transmission of pneumocystis oryctolagi in first-time pregnant and multiparous rabbit does health and body condition of lactating females on rabbit farms atropinesterase, a genetically determined enzyme in the rabbit wild rabbits (oryctolagus cuniculus) as potential carriers of verocytotoxin-producing escherichia coli proliferative enterocolitis associated with dual infection with enteropathogenic escherichia coli and lawsonia intracellularis in rabbits complement c deficiency protects against diet-induced atherosclerosis in rabbits quantitative rt-pcr profiling of the rabbit immune response: assessment of acute shigella flexneri infection enterocecocolitis associated with intraepithelial campylobacter-like bacteria in rabbits (oryctolagus cuniculus) rotavirus-associated diarrhea in a commercial rabbitry parasites of rabbits in vitro inhibition of clostridium difficile and clostridium perfringens by commercial probiotic strains some factors in the in vitro infectivity and replication of encephalitozoon cuniculi the oxidative status and inflammatory level of the peripheral blood of rabbits infested with psoroptes cuniculi glycogen reserves and their changes at birth cryptosporidium infection in juvenile pet rabbits aortic endograft infection due to pasteurella multocida following a rabbit bite analysis of the expression of platelet antigens cd and cd / in transgenic rabbits with the integrated human blood clotting factor viii gene construct psoroptes cuniculi induced oxidative imbalance in rabbits and its alleviation by using vitamins a, d , e, and h as adjunctive remedial complete genome sequence of treponema paraluiscuniculi, strain cuniculi a: the loss of infectivity to humans is associated with genome decay genetic diversity in treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws venereal spirochetosis of rabbits (rabbit syphilis) due to treponema cuniculi: a clinical, serological, and histopathological study coccidiosis of the live rabbit ii, experimental study on the mode of infection of the liver by sporozoites of eimeria stiedae lawsonia intracellularis: getting inside the pathogenesis of proliferative enteropathy gamma interferon influences intestinal epithelial hyperplasia caused by lawsonia intracellularis infection in mice bacillus piliformis infection (tyzzer's disease) in a patient infected with hiv- : confirmation with s ribosomal rna sequence analysis production of two vaccinating recombinant rotavirus proteins in the milk of transgenic rabbits clostridial enteric diseases of domestic animals the young rabbit: a nonimmunosuppressed model for pneumocystis carinii pneumonia helminth, arthropods, and protozoa of domestic animals efficacy of ivermectin against nematodes infecting field populations of snowshoe hares (lepus americanus) in yukon novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease the current status and future directions of myxoma virus, a master in immune evasion phenotypic and genetic characterization of pasteurella multocida and related isolates from rabbits in switzerland altered myofilament stoichiometry in response to heart failure in a cardioprotective alpha-myosin heavy chain transgenic rabbit model glomerular blood flow biochemical modulation of -bromo- '-deoxyuridine and -iodo- '-deoxyuridine incorporation into dna in vx tumor-bearing rabbits polyclonal antibody production rabbit cardiovascular responses during peep before and after vagotomy atropinesterase and cocainesterase of rabbit serum: localization of the enzyme activity in isozymes production of actin-specific adp-ribosyltransferase (binary toxin) by strains of clostridium difficile the laboratory rabbit spontaneous lateral abdominal (lumbar) hernia in a new zealand white rabbit immunization of rabbits against pasteurella multocida using a commercial swine vaccine heat-labile toxinproducing isolates of pasteurella multocida from rabbits protective immunity to pasteurella multocida heat-labile toxin by intranasal immunization in rabbits derivation of pasteurella multocida-free rabbit litters by enrofloxacin treatment immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine field trial of a pasteurella multocida extract vaccine in rabbits a comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (crpv) l gene protects against virus challenge prevention and treatment of encephalitozoon cuniculi infection in rabbits with fenbendazole enteropathogenic escherichia coli prevalence in laboratory rabbits enzootic enteropathogenic escherichia coli infection in laboratory rabbits rearing of germfree rabbits and establishment of an spf rabbit colony pinworm infection in laboratory rodents: a review establishment and characterization of cag/ egfp transgenic rabbit line pneumocystis carinii infection in young non-immunosuppressed rabbits. kinetics of infection and of the primary specific immune response shiga toxin-associated hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: distinct mechanisms of pathogenesis shiga-toxin-producing escherichia coli and haemolytic uraemic syndrome a clinical and pathological study of inherited glaucoma in new zealand white rabbits the effect of combined rotavirus and escherichia coli infection in rabbits rabbit neoplasia novel transgenic rabbit model sheds light on the puzzling role of matrix metalloproteinase- in atherosclerosis studies on the renal circulation comparative morphology of the cardiac conduction tissue in animals water intake in domestic rabbits (oryctolagus cuniculus) from open dishes and nipple drinkers under different water and feeding regimes intermittent medication of sulfadimethoxine and sulfamonomethoxine for the treatment of coccidiosis in domestic rabbits biology of the treponematoses based on studies carried out at the international treponematosis laboratory center of the johns hopkins university under the auspices of the world health organization histiocytic enteritis of rabbits evidence of host adaptation in lawsonia intracellularis infections large-scale management systems and parasite populations: coccidia in rabbits clinical veterinary advisor birds and exotic pets pathogenesis of dermatophytosis thymoma in a rabbit with hypercalcemia and periodic exophthalmus detection of dna sequences identical to pneumocystis carinii in samples of ambient air dna sequences identical to pneumocystis carinii f. sp. carinii and pneumocystis carinii f. sp. hominis in samples of air spora mammalian microsporidiosis serial inbreeding of rabbits with hereditary hyperlipidemia (whhl-rabbit) breeding of a rabbit strain of hyperlipidemia and characteristic of this strain (author's transl) the effect of selective breeding on the development of coronary atherosclerosis in whhl rabbits. an animal model for familial hypercholesterolemia enzyme-linked immunosorbent assay to detect lawsonia intracellularis in rabbits with proliferative enteropathy detection of lawsonia intracellularis using immunomagnetic beads and atp bioluminescence cultivation and characterization of lawsonia intracellularis isolated from rabbit and pig efficacy of simultaneous vaccination with enterisol(r) ileitis and ingelvac(r) circoflextm in a swiss breeding farm neoplastic diseases torsion of the caudate lobe of the liver in the domestic rabbit (oryctolagus) neoplastic diseases microsporidiosis: molecular and diagnostic aspects liver lobe torsion in three adult rabbits ultramicroelectric recording from cardiac pacemaker the effects of group housing on the research use of the laboratory rabbit pasteurella multocida: diseases and pathogenesis practical guide to laboratory animals ophthalmology of exotic pets the effects of ethylene dibromide on semen quality and fertility in the rabbit: evaluation of a model for human seminal characteristics dna polymorphisms amplified by arbitrary primers are useful as genetic markers liver lobe torsion in a rabbit tularemia, plague, yersiniosis, and tyzzer's disease in wild rodents and lagomorphs in canada: a review reference range data base for serum chemistry and hematology values in laboratory animals infectious motor paralysis in young rabbits comparative efficacy of injection routes and doses of ivermectin against psoroptes in rabbits pathology of spontaneous malignant fibrous histiocytoma in a japanese white rabbit (oryctolagus cuniculus) simultaneous identification of three highly pathogenic eimeria species in rabbits using a multiplex pcr diagnostic assay based on its - . s rrna-its fragments the oct promoter-egfp transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells flow-volume curves of excised right and left rabbit lungs biochemical parameters of normal rabbit serum a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia a blood coagulation abnormality in rabbits deficient in the sixth component of complement (c ) and its correction by purified c origin of complex behaviour of spatially discordant alternans in a transgenic rabbit model of type long qt syndrome key: cord- -gxmae rs authors: wang, jianzhong; cong, yanlong; yin, renfu; feng, na; yang, songtao; xia, xianzhu; xiao, yueqiang; wang, wenxiu; liu, xiufan; hu, shunlin; ding, chan; yu, shengqing; wang, chunfeng; ding, zhuang title: generation and evaluation of a recombinant genotype vii newcastle disease virus expressing vp protein of goose parvovirus as a bivalent vaccine in goslings date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: gxmae rs newcastle disease virus (ndv) and goose parvovirus (gpv) are considered to be two of the most important and widespread viruses infecting geese. in this study, we generated a recombinant rmna-vp , expressing gpv vp using a modified goose-origin ndv na- by changing the multi-basic cleavage site motif rrqkr↓f of the f protein to the dibasic motif grqgr↓l as that of the avirulent strain lasota as a vaccine vector. expression of the vp protein in rmna-vp infected cells was detected by immunofluorescence and western blot assay. the genetic stability was examined by serially passaging times in -day-old embryonated spf chicken eggs. goslings were inoculated with rmna-vp showed no apparent signs of disease and developed a strong gpv and ndv neutralizing antibodies response. this is the first study demonstrating that recombinant ndv has the potential to serve as bivalent live vaccine against goose parvovirus and newcastle disease virus infection in birds. goose parvovirus (gpv) infection also known as derzsy's disease, goose hepatitis, or gosling plague is an acute, contagious, and fatal disease to domestic goslings and muscovy ducklings (derzsy, ; gough et al., ; schettler, ). this is a gastrointestinal disease characterized mainly by diarrhea and fibrous hemorrhagic necrotic enteritis. with high mortality and morbidity, gpv has caused substantial economic losses to goose farming countries of europe and asia (jansson et al., ; takehara et al., ) . to control the disease caused by gpv, live attenuated vaccines are widely used in breeder geese or goslings in addition to flock management (gough and spackman, ; kisary, ) . usually, the attenuated viruses for vaccines were obtained by serial passages in cultured goose-embryo fibroblasts (gef) or embryonated goose eggs to reduce the pathogenicity . however, the potential risk of reversion to virulence of attenuated live vaccines is always a concern. additionally, the current available vaccines are prepared with chorioallantoic fluid collected from infected embryos, which is costly and inefficient due to the limited availability of spf (specific pathogen free) goose and duck embryos (ju et al., ; lee et al., ) . therefore, the development of alternative strategies for producing safe and effective vaccines may be of great significance. newcastle disease, caused by newcastle disease virus (ndv), is one of the most serious infectious diseases of many avian species and has caused substantial losses in the poultry industry worldwide (aldous and alexander, ; alexander, the basis of their pathogenicity for birds, ndv has been classified into three different pathotypes: velogenic (highly virulent), mesogenic (moderately virulent) or lentogenic (low virulence) viruses. the molecular basis for the pathogenicity of ndv is mainly determined by the amino acid sequence of the protease cleavage site located in f protein. mesogenic and velogenic viruses have a polybasic amino acid motif of r/kr-q-k/r-r-f , while viruses of low virulence have a monobasic amino acid sequence of g/e-k/r-q-g/e-r-l at the cleavage site (choi et al., ; panda et al., ; peeters et al., ) . lentogenic and, in some cases, mesogenic strains of ndv are widely used as live attenuated vaccines in poultry. in recent years, they also have been developed as a vector to express foreign protective antigens based on reverse genetic techniques. the use of such live vector vaccines for immunization not only protect birds against ndv and another poultry pathogens, such as highly pathogenic avian influenza virus (hpaiv) (ge et al., ) and infectious bursal disease virus (ibdv) (huang et al., b) , but also has the potential to serve as effective vaccines for human use against sars-cov or other emerging viruses (dinapoli et al., a (dinapoli et al., ,b, . in this study, we generated a recombinant ndv expressing the vp protein of gpv using a modified avirulent virus vector derived from ndv na- strain isolated from goose flocks. the safety, stability, and feasibility of this recombinant rmna-vp to serve as a bivalent live vaccine in goslings were evaluated. bhk- (atcc, ccl- ) and df- (atcc, crl- ) cell lines were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and maintained at • c with % co . the gpv vaccine strain, gd- , was obtained from the china veterinary culture collection. the viral stock was inoculated into the chorioallantoic cavity of -day-old embryonated goose eggs. ndv strain na- was a virulent strain isolated by our laboratory from geese stocks in nong'an county of jilin province, china in (genbank access no. dq ) and recovered from the full-length antigenomic cdna using a reverse genetics system as describe previously (wang et al., ) . to attenuate the ndv na- strain the sequence encoding the protease cleavage site of the f protein was modified by means of pcr mutagenesis. thus, a fragment between pmei and sacii restriction sites (nt - ) was amplified from the full-length cdna clone of na- , and subcloned into pbluescript ii sk+ (stratagene) at the ecorv site. then, the fragment pmei-sacii of pci-na- was pcr amplified using the primers fmut-pf ( -gcttgtttaaacaa-aacaacagccctctctcaccc- , with pmei site underlined) and fmut-pr ( -ccaagagctacactgccaataacggcacctataa-ggcgcccctgtctccctcctccagacgtggacacagac- , with modified sequence bolded). the pcr products were subjected to a second round pcr with the primes fmut-pf and fmut-pr ( -gggccgcggctgctgttatctgtgccgctgttgcaaccccaagagct-acactgccaataacggc- , with sacii site underlined) resulting in a new fragment encoding an avirulent protease cleavage site grqgr↓l of lasota mutated from rrqkr↓f of na- . finally, the new fragment was excised from pbluescript ii sk+ using the restriction enzymes pmei and sacii and was used to replace the corresponding fragment in the full-length cdna clone of the pci-na- . the resultant plasmid was designated as pci-mna- . the open reading frame (orf) of vp gene of gpv strain gd- was amplified from the genome dna by using the primer pair of -gcgcgtttaaacttaagaaaaaatacgggtagaagccgccaccat-ggcagagggaggaggcgg- and -gcgcgtttaaacttacagat-tttgagttagatatc- , in which the gene end and gene start sequences of ndv (bold), the optimal kozak sequence (italic), and the pmei restriction sites (underlined) were included. the amplified product was ligated into ecorv-cut pbluescript ii sk+ vector, sequenced, and inserted into the ndv full-length cdna clone contained in pci-mna- through the introduced pmei site in the p-m noncoding region at nucleotide position of the ndv genome, as described previously (wang et al., ) . the new full-length plasmid was designated as pci-na-vp . rescue of the infectious viruses were performed by transfecting the full-length cdna clone and supporting plasmids into bhk- cells as described previously (wang et al., ) .the rescued viruses were named rmna- and rmna-vp , respectively. a % solution of phosphotungstic acid (ph . ) was added to the virus samples for negative staining. then the mixtures were transferred to carbon-coated grids, and the specimens were photographed with an electron microscope h- (hitachi ltd., hitachi, japan). for immunofluorescence analysis, bhk- cells were plated on coverslips in -mm-diameter dishes and infected with ndv rmna- or rmna-vp . after h, the cells were fixed in ice-cold % paraformaldehyde in phosphate buffered saline (pbs) for min at room temperature and then washed with pbs times. cells were blocked in pbs containing % (w/v) bovine serum albumin (bsa) at • c for h. cells were then incubated with chicken serum anti-ndv or horse serum against gpv for h at room temperature and were washed times with pbs containing . % tween . then, the cells were stained with fitc (fluorescein isothiocyanate)conjugated goat anti-chicken antibody (sigma) or an alexa fluor -conjugated rabbit anti-horse igg (bioss) for min. finally, cells were washed times with pbs, and dapi ( , -diamidino- -phenylindole) was used to stain the cell nucleus. cells were analyzed with a confocal laser microscope (olympus corp, tokyo, japan). df cells were infected with rescued virus rmna- or rmna-vp at a multiplicity of infection (moi) of and incubated for h. the expression of cell-associated proteins was analyzed by western blotting. proteins from the lysates of infected cells were separated using sds-page under denaturing conditions and analyzed with chicken serum anti-ndv or rabbit anti-vp polyclonal antibody (bioss), or mono-clonal antibody against ␤-actin (cell signaling). immunostained proteins were visualized using a , diaminobenzidine (dab) reagent. mock-infected df cells were used as the negative control. to evaluate the genetic stability of the recombinant viruses, rmna- or rmna-vp was serially passaged by times in day-old embryonated eggs, respectively. the viral genome rna was isolated from allantoic fluids of each passage and subjected to rt-pcr to check the modified region in the f gene cleavage site of rmna- or rmna-vp and confirm the presence of the vp gene in the genome of rmna-vp by dna sequencing. the pathogenicity of the modified virus was evaluated by the mean death time (mdt) and the intracerebral pathogenicity index (icpi) according to the standard pathogenicity assay (alexander, ) . ten-day-old embryonated chicken eggs were inoculated with rmna- , rmna-vp and rna- , at tcid per egg. viral growth was analyzed at different time points after inoculation. a % tissue culture infective dose (tcid ) of each virus was determined by immunofluorescence assay. ninety-six well plates of %-confluent df cells were infected with serial -fold dilutions of virus (four wells per dilution). cells were incubated for days and fixed with % cold acetone. viral antigens were detected with chicken serum anti-ndv and fitc-conjugated goat anti-chicken antibody. goslings at days of age were randomized into three groups of birds each. group and group were injected with eid of the rescued rmna- or rmna-vp in a . -ml volume by subcutaneous administration and a booster injection was performed weeks later. the third group of goslings was injected with pbs used as negative controls. all animals were housed in a specific pathogen-free facility with free access to water and food. blood samples were collected at , , , and weeks post-immunization and the sera were separated by low speed centrifugation and stored at − • c. the neutralizing antibodies (nas) of individual goose serum samples against gpv and ndv were tested in the primary goose embryo fibroblasts (gefs) and df cells respectively. for the gpv nas assay, gefs were prepared from -day-old goose embryos and double dilutions of serum samples were pre-incubated with tcid gpv gd- at • c for h and then added to gef cells in -well plates. for the ndv nas assay, tcid ndv na- was incubated for h with serial dilutions of the test sera and then were added to df cells in -well plates. cells were observed microscopically for cytopathic effects (cpe) starting at h post-infection and the titers were calculated by using the method detailed by reed and muench ( ) . statistical analysis was performed using of the analysis of variance (anova) method and considered significant when p ≤ . . data are shown as means ± standard deviation (sd). to generate an attenuated ndv na- strain, modification were made to the full-length cdna clone of na- by changing the amino acid sequence of the f protein cleavage site from the naturally occurring rrqkr↓f to an avirulent motif grqgr↓l, resulting in a new cdna clone pci-mna- (fig. a) . the recovery of the modified virus rmna- was confirmed by rt-pcr and sequence analysis of the f gene from the ha-positive allantoic fluid (fig. b) . pathogenicity of the recombinant viruses was assessed by performing mdt and icpi tests. the results showed that the mdt of rmna- was h, while that of rna- was h. additionally, the icpi of rmna- was , while that of rna- was . . these results suggest that rmna- is highly attenuated. to investigate the capability of cpe and the trypsin-dependent infectivity in cell culture, df cells were infected with rna- , rmna- , or lasota viruses at a moi of . in the presence or absence of % normal allantoic fluid as exogenous protease supplementation (fig. c) . the results showed that syncytium formation could be observed in cells infected by parental virus rna- with or without exogenous protease. lasota only induced the formation of syncytia in the presence of added protease. in contrast, rmna- , which has exactly the same f protein cleavage site as lasota, did not cause any apparent syncytia even in the presence of exogenous protease. the genetic stability of the modified sites of rmna- was examined by serially passaging them times in -day-old embryonated chicken eggs and sequencing the f gene from each passage. the results indicate the cleavage sites were preserved and stably maintained after passages in chicken embryos (data not shown). to obtain the cdna clone pci-mna-vp , an artificial transcription cassette coding for the vp protein of gpv was inserted into pci-mna- between the p and m genes of the genome (fig. a) . the resultant recombinant virus, rmna-vp , was recovered entirely from this cdna by using our established reverse genetics procedures (wang et al., ) . the presence of the vp gene in the genome was confirmed by rt-pcr (data not show). the virions of rmna-vp were examined by observation with tem after negative staining, and compared with rna- and rmna- , the vp gene inserted into the genome did not affect viral morphology (fig. b) . expression of the vp protein by rmna-vp was examined by indirect confocal immunofluorescence staining infected bhk- cells. as expected, cells infected with rmna- were not stained by horse serum against gpv, but they were positive for immunostaining using chicken serum against ndv (fig. a ). cells infected with rmna-vp were stained by horse serum against gpv as well as chicken serum against ndv (fig. a) . the vp expression by the recombinant virus was further confirmed by western blotting analysis of infected df cell lysates with rabbit anti-vp polyclonal antibody (fig. b) , whereas no band was detected in purified rmna-vp virus (data not shown). these results suggested that the recombinant rmna-vp stably expressed thevp protein and that the vp protein was probably not incorporated into the virions. the biological properties of the recombinant viruses were compared to parental rna- by determination of growth characteristics in tissue culture and in embryonated chicken eggs. as the cleavage site of f protein was modified to a monobasic motif from the dibasic amino acid motif, both the rmna- and rmna-vp cannot be propagated as well as rna- in tissue culture (data not shown). the growth kinetics of the recombinant viruses were determined and compared in embryonated chicken eggs at different time points after inoculation. a % tissue culture infective dose (tcid ) of each virus was determined in df- cells by immunofluorescence assay. the results showed that both rmna-vp and rmna- have similar growth patterns compared to parental rna- . at h postinoculation, rna- and rmna- reached the maximum titers of . tcid /ml and tcid /ml respectively, whereas the maximal viral titer of rmna-vp achieved at h post-inoculation and was approximately -fold lower (fig. a) . the genetic stability of rmna-vp was also examined by serial passage times in -day-old embryonated spf chicken eggs as described previously. rt-pcr confirmed the presence of the vp gene in each passage, and sequence analysis showed that the vp gene remains unchanged during passages. also the mutant sequence at the cleavage sites in the f gene were proved to be preserved and stably maintained after serial passage through chicken embryos by dna sequencing (data not shown). the pathogenicity of recombinant ndv expressing the vp protein was evaluated by the mdt and icpi assays (fig. b) . the value of mdt exceeds h and the icpi was . these results show that rmna-vp continues to keep the characteristics of the vector virus rmna- . fig. . analysis of expression of the vp protein by rmna-vp . (a) immunofluorescence analysis of vp protein expression. bhk- cells were infected with rmna- or rmna-vp at an moi of . . the infected cells were fixed and probed with chicken serum against ndv and horse serum against gpv and then incubated with a fitcconjugated goat anti-chicken antibody and an alexa fluor -conjugated rabbit anti-horse igg. dapi (sigma) was used for cell nucleus staining. cells were analyzed by using a confocal laser microscope. (b) western blot analyses of vp expression. lysates of df cells infected with rmna-vp or rmna- were incubated with chicken serum anti-ndv, rabbit anti-vp polyclonal antibody or mono-clonal antibody against ␤-actin. binding was visualized with , -diaminobenzidine reagent after incubation with peroxidase-conjugated secondary antibodies. the locations of marker proteins are indicated on the left and the antiserum or antibody used is indicated on the right. to investigate the immunogenicity of the recombinant rmna-vp , groups of -day-old goslings were inoculated with rmna- , rmna-vp , or pbs. during the experimental period, the birds inoculated with the recombinant virus appeared healthy without any signs of vaccine side-effects. serum samples of the birds were collected before and after vaccination. the antibody titres against gpv were determined by virus neutralization test. at weeks after the first dose, the mean titer of gpv na in the blood of goslings inoculated with rmna-vp was . log (fig. a) . at weeks after the second dose, the mean titer reached up to log . then the mean titer of . log was detected at weeks after first immunization and still maintained a titer of . log until weeks. no gpv-specific antibodies were detected from goslings in the rmna- or pbs vaccine control group after either initial or boost vaccination. meanwhile, ndv nas were detected in all serum samples (fig. b) . the two groups of goslings inoculated with rmna- or rmna-vp displayed no significant differences in ndv vnas antibody titers (p > . ). the mean titers of . log in rmna-vp group and . log in rmna- group were detected at weeks after the first dose. three weeks after the second dose, the mean titer for the two groups reached . log and log , respectively, showing substantial boost responses. after weeks, the antibody titers are maintained at . log in rmna-vp group and at . log in rmna- group, and at weeks after vaccination the mean titers is still . log and . log respectively. fig. . immunogenicity evaluation in goslings. groups of goslings were inoculated subcutaneously with eid of rmna- or rmna-vp or with pbs as a control in a . -ml volume respectively. at weeks after initial vaccination, the goslings received a second dose. the serum samples from goslings were collected prior to vaccination and at different times after vaccination and the vna titers against gpv (a) and ndv (b) were detected. in this study, we generated a recombinant rmna-vp , expressing gpv vp using a modified goose-origin ndv na- as an avirulent, safe, and effective vaccine vector, and evaluated its potential as a bivalent vaccine against gosling plague and nd in goslings. the results of the gosling immunizing experiment show that rmna-vp could induce strong na responses to gpv and ndv simultaneously. this is the first study to demonstrate that a highly virulent ndv could serve as vaccine vector expressing exogenous protein after modification by changing the multi-basic cleavage sites of the f protein to the dibasic sequence of avirulent strain lasota. the recombinant rmna-vp has the potential to serve as a bivalent live vaccine against gosling plague and nd. gpv is a pathogen of major economic importance to the goose industry worldwide. currently, prophylactic vaccination is used as protection against gpv infection. two types of immunization strategies were adopted in field: vaccination of breeder geese before laying, by which the offspring was protected depending on maternally derived antibodies in the egg yolks, and vaccination of susceptible goslings, where the virulence of the vaccine strains needs to be much lower than that of strains used to immunize parent flocks. preparation of vaccines with varying levels of virulence makes the production very costly and labor-effective. therefore, the development of a safe, effective and low-cost vaccine is necessary. among the possible strategies, one of the most promising is the live viral vectored vaccines. the genomes of gpv encode non-structural and structural proteins, including three capsid proteins of vp , vp , and vp . the vp protein is the most abundant of the three core proteins in purified virions and can induce neutralizing antibodies with high immunogenicity (le gall-reculé et al., ; le gall-recule and jestin, ) . therefore, vp may be a suitable candidate antigen for developing a vaccine against gpv. as a viral vector, ndv has outstanding advantages. firstly, ndv grows to very high titers in many cell lines and embryonated eggs, which allows cost-effective and easy manufacture of the vaccine. secondly, ndv has a simple genome encoding only a few proteins than other viruses such as fowl pox virus and adenovirus. thirdly, the live ndv vaccines can induce not only humoral immunity and cellular immunity simultaneously, but also a strong mucosal immunity, which makes this expression vector more attractive (huang et al., ) . ndv lentogenic vaccine strains such as lasota can be developed in vectors directly; while mesogenic strains should be modified by changing the multi-basic cleavage site sequence of the f protein to the dibasic sequence of avirulent strains because these strains might cause disease in poultry and most strains are classified as select agents (kim et al., ) . recently, it has been demonstrated that the virulent isolates also can be attenuated by modification in the same way to serve as a live-attenuated vaccine candidate (hu et al., (hu et al., , xiao et al., ) . ndv has a single serotype, but many genotypes. to date, ndv had caused four major nd panzootics and different genotypes contributed to each panzootic. epidemiological studies revealed that genotype vii ndv strains were circulating predominantly worldwide after the fourth panzootic (huang et al., a; miller et al., miller et al., , and found to be responsible for the nd outbreaks in asia, africa, middle east, and south america (munir et al., ; perozo et al., ; wang et al., ) . these strains also caused many outbreaks of nd in domestic geese with high mortality and morbidity in southern and eastern china since the late s, which was referred as goose paramyxovirus (gpmv) in earlier reports (cai et al., ; jinding et al., ) . however, the genotype ii vaccine strains such as lasota or hitcher b remain in current and widespread use, and the differences in their antigenicity and genotypes between the presently circulating strains and commercial vaccine strains are considered the main reasons for the nd outbreaks in vaccinated poultry flocks (liu et al., ; miller et al., ) . recent work has demonstrated that genotype-matched vaccine strains provide better protection than conventional vaccines in terms of reducing virus shedding and transmission following challenge with ndv virulent virus (hu et al., (hu et al., , xiao et al., ) . in , a highly virulent genotype vii ndv strain na- was isolated from outbreaks in goose flocks in our lab (xu et al., ) . to evaluate whether this genotype vii isolate could be used as a vaccine vector for geese we generated a recombinant virus rmna-vp expressing vp protein of gpv after modifying the polybasic f cleavage site of na- to the dibasic motif of lasota. it is interesting that the modified na- which has exactly the same f protein cleavage site as avirulent lasota did not form syncytium in virus-infected cells as lasota did. even in the presence of exogenous protease no syncytium was observed (fig. c) . these results are repeatedly confirmed and consistent with previous research on the mutation of a highly virulent indonesian strain (xiao et al., ) . it is demonstrated that the syncytium formation is not determined by the motif of the f protein cleavage site alone; some other factors may also be involved. anyway, the inability to induce the formation of syncytia, may be indicative of a highly attenuated of the modified viruses comparing with lasota. as a feasibility study, one concern that requires investigation is that whether the mutated f protein cleavage site of vector virus might revert to a wt or wt-like sequence after serial passage. to evaluate its heredity stability, the recombinant virus rmna- and rmna-vp were passaged continuously in chicken embryos and the sequence of f protein cleavage site was determined every generation. the results demonstrated that the mutated f protein cleavage site always remained stable after passages in chicken embryos. the safety issue of the recombinant viruses should be further evaluated through its serial passage in geese prior to its use in field. immunogenicity of the recombinant virus rmna-vp was evaluated in goslings. a detectable immune response against gpv was evidenced after the first immunization dose, and after the second immunization administered weeks later, significant booster responses to vna of gpv were induced. moreover, the antibody level did not drop markedly during at least the first weeks. meanwhile, the neutralizing antibodies against ndv vaccine vector were also detected. the mean titer induced by rmna-vp was comparable to that induced by rmna- , indicating that this recombinant virus holds its characteristic in stimulating effective immune response against ndv infection. in summary, as a new type of engineered vaccine the recombinant virus rmna-vp has several advantages over the existing gpv vaccines. firstly, the recombinant rmna-vp will be highly economical for the poultry industry, because rmna-vp can be cultured in many cell lines and embryonated eggs. secondly, the recombinant virus can potentially overcome maternal antibody interference. therefore, it can be used for vaccination with or without maternal antibody. even in the presence of the maternal antibody, it can be used to enhance protection in the early danger period of gosling life. finally, this recombinant virus would be developed an in-ovo vaccine against gpv after further modified as describe in in-ovo vaccination against ibdv and ndv elsewhere (ge et al., ) . (i) all the authors have agreed to its submission and are responsible for its contents; (ii) all the authors have agreed that zhuang ding may act on their behalf regarding any subsequent processing of the paper; (iii) the authors have declared that no competing interests exist. detection and differentiation of newcastle disease virus (avian paramyxovirus type ). avian pathol newcastle disease and other avian paramyxoviruses newcastle disease, other avian paramyxoviruses, and pneumovirus infections genetic characterization and evolutionary analysis of newcastle disease virus isolate full genomes from waterbirds in south china during antigenic and immunogenic investigation of the virulence motif of the newcastle disease virus fusion protein a viral disease of goslings. i. epidemiological, clinical, pathological and aetiological studies newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens newcastle disease 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chicken eggs genomic sequence of an isolate of newcastle disease virus isolated from an outbreak in geese: a novel six nucleotide insertion in the non-coding region of the nucleoprotein gene recombinant newcastle disease virus as a vaccine vector a recombinant newcastle disease virus (ndv) expressing vp protein of infectious bursal disease virus (ibdv) protects against ndv and ibdv epidemiologic investigation of an outbreak of goose parvovirus infection in sweden a goose-sourced paramyxovirus isolated from southern china goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose modified newcastle disease virus vectors expressing the h hemagglutinin induce enhanced protection against highly pathogenic h n avian influenza virus in chickens immunological aspects of derzsy's disease in goslings expression of muscovy duck parvovirus capsid proteins (vp and vp ) in a baculovirus expression system 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protein is a major determinant for virulence biological and phylogenetic characterization of a genotype vii newcastle disease virus from venezuela: efficacy of field vaccination a simple method of estimating fifty per cent endpoints virus hepatitis of geese. ii. host range of goose hepatitis virus an outbreak of goose parvovirus infection in japan cloning of the genome of a goose parvovirus vaccine strain syg v and rescue of infectious virions from recombinant plasmid in embryonated goose eggs development of a reverse genetics system based on rna polymerase ii for newcastle disease virus genotype vii genotyping of newcastle disease viruses isolated from to in china generation by reverse genetics of an effective, stable, live-attenuated newcastle disease virus vaccine based on a currently circulating, highly virulent indonesian strain genomic analysis of newcastle disease virus strain na- isolated from geese in china the research was supported by the national natural scientific fund ( , , and ), and chinese special fund for agro-scientific research in the public interest ( ). key: cord- -heoj ji authors: hubbard, amelia; lewis, clare m; yoshida, kentaro; ramirez-gonzalez, ricardo h; de vallavieille-pope, claude; thomas, jane; kamoun, sophien; bayles, rosemary; uauy, cristobal; saunders, diane go title: field pathogenomics reveals the emergence of a diverse wheat yellow rust population date: - - journal: genome biol doi: . /s - - - sha: doc_id: cord_uid: heoj ji background: emerging and re-emerging pathogens imperil public health and global food security. responding to these threats requires improved surveillance and diagnostic systems. despite their potential, genomic tools have not been readily applied to emerging or re-emerging plant pathogens such as the wheat yellow (stripe) rust pathogen puccinia striiformis f. sp. tritici (pst). this is due largely to the obligate parasitic nature of pst, as culturing pst isolates for dna extraction remains slow and tedious. results: to counteract the limitations associated with culturing pst, we developed and applied a field pathogenomics approach by transcriptome sequencing infected wheat leaves collected from the field in . this enabled us to rapidly gain insights into this emerging pathogen population. we found that the pst population across the united kingdom (uk) underwent a major shift in recent years. population genetic structure analyses revealed four distinct lineages that correlated to the phenotypic groups determined through traditional pathology-based virulence assays. furthermore, the genetic diversity between members of a single population cluster for all pst field samples was much higher than that displayed by historical uk isolates, revealing a more diverse population of pst. conclusions: our field pathogenomics approach uncovered a dramatic shift in the pst population in the uk, likely due to a recent introduction of a diverse set of exotic pst lineages. the methodology described herein accelerates genetic analysis of pathogen populations and circumvents the difficulties associated with obligate plant pathogens. in principle, this strategy can be widely applied to a variety of plant pathogens. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. results: to counteract the limitations associated with culturing pst, we developed and applied a field pathogenomics approach by transcriptome sequencing infected wheat leaves collected from the field in . this enabled us to rapidly gain insights into this emerging pathogen population. we found that the pst population across the united kingdom (uk) underwent a major shift in recent years. population genetic structure analyses revealed four distinct lineages that correlated to the phenotypic groups determined through traditional pathology-based virulence assays. furthermore, the genetic diversity between members of a single population cluster for all pst field samples was much higher than that displayed by historical uk isolates, revealing a more diverse population of pst. conclusions: our field pathogenomics approach uncovered a dramatic shift in the pst population in the uk, likely due to a recent introduction of a diverse set of exotic pst lineages. the methodology described herein accelerates genetic analysis of pathogen populations and circumvents the difficulties associated with obligate plant pathogens. in principle, this strategy can be widely applied to a variety of plant pathogens. emerging and re-emerging diseases of humans, animals and plants pose a significant hazard to public health and food security. these threats can arise from newly discovered pathogens, such as the middle east respiratory syndrome (mers) coronavirus in humans [ ] , or novel host adaptation, as in zoonotic influenza [ ] . recent disease outbreaks in plants have been associated with expansions of pathogen geographic distribution and increased virulence of known pathogens, such as in the european outbreak of ash dieback [ ] and wheat stem rust across africa and the middle east [ ] . independent of the host organism, the scale and frequency of emerging diseases have increased with the globalization and industrialization of food production systems [ ] . improved surveillance mechanisms and diagnostic tools are needed to rapidly respond to these emerging threats. with recent advances in dna and rna sequencing, bacteriologists and virologists are capitalizing on these technological advances by integrating high-resolution genotypic data into pathogen surveillance activities [ ] . however, the application of genomics to emerging filamentous plant pathogens has lagged. filamentous plant pathogens tend to have large genomes and are often obligate parasites that cannot be axenically cultured in the laboratory. the time-consuming and tedious protocols required to maintain these pathogens on their hosts have impeded the translation of genomic technologies into surveillance and diagnostics methods. traditional diagnostic tools for pathogens have been based on targeted cultures, pcr-based approaches and/ or phenotypic evaluation of disease response in specific plant genotypes [ ] . these methods detect only known pathogenic agents, can introduce bias, and can fail to recognize novel variants or races due to their narrow scope [ ] . however, next-generation sequencing technologies can circumvent these limitations to provide a rich source of data for the development of surveillance and diagnostic tools. the high resolution of these approaches also enables exploration of the genetic determinants underpinning pathogenicity. whole-genome sequencing has emerged as a preferred technology, especially for viruses with relatively small genomes (approximately kb on average) [ ] , although this methodology is less tractable in pathogens with large genomes such as filamentous plant pathogens, which have genomes that range from to mb [ ] . alternatively, rna sequencing (rna-seq), which focuses solely on the expressed fraction of the genome, reduces the sequence space of the sample and provides relevant transcriptome data for both the pathogen and host in situ [ ] . despite modern agricultural practices, diseases of the major food crops cause up to % pre-harvest yield loss [ ] . among these crops, wheat is a critical staple providing % of the calories and over % of the protein consumed by humans [ ] . one of the major fungal diseases of wheat is yellow (stripe) rust caused by the obligate fungus puccinia striiformis westend. f. sp. tritici eriks (pst) [ ] . this disease is widespread across the major wheat-producing areas of the world and can cause significant reductions in both grain quality and yield in susceptible cultivars [ ] . in the past decade, new pst races have emerged that are capable of adapting to warmer temperatures, have expanded virulence profiles, and are more aggressive than previously characterized races [ ] . more recently, a series of pst races have arisen in europe and overcome many of the major resistance genes in european germplasm [ ] . for instance, in a race group collectively called 'warrior' (based on the virulence of one of the initial variants of this group to the uk wheat variety warrior) emerged as a serious threat to wheat production. however, the origin of this new race and its relationship with previously characterized races remain unclear. an important first step towards the development of more effective surveillance and diagnostic tools is the availability of a draft reference genome and annotation. cantu et al. [ ] published a first draft sequence of pst isolate (pst- ) with , annotated proteincoding sequences across the . mb assembly. more recently, zheng et al. [ ] published a mb draft sequence of chinese pst isolate cyr using a 'fosmidto-fosmid' approach and annotated , proteincoding sequences. these genomic resources can be used to identify pathogenicity determinants, such as secreted effector proteins [ ] that are recognized in certain host genotypes, where they induce an immune response that prevents disease progression. avirulence effector proteins are under strong selective pressure to adapt in order to evade detection by the host plant immune system [ ] . the signatures of adaptation and gene expression patterns of pathogen isolates with distinct virulence profiles can provide a powerful means of identifying specific avirulence/virulence proteins that can be used to track pathotypes at a national and international level. furthermore, publication of these draft reference genomes also provides an opportunity to characterize pathogen populations at a considerably higher resolution and on a much wider scale through re-sequencing of pst isolates. in this study, we developed a robust and rapid 'field pathogenomics' strategy, using transcriptome sequencing of pst-infected wheat leaves to gain insight into the population structure of an emerging pathogen. our analysis uncovered a dramatic shift in the pst population in the uk and supports the hypothesis that recent introduction of a diverse set of exotic pst lineages may have displaced the previous pst populations. our field pathogenomics approach circumvents the difficulties associated with less-tractable filamentous plant pathogens and can be applied to other emerging populations of pathogens. genotyping pathogens and their hosts using rna-seq of field-collected infected leaves to characterize the genotypic diversity of pst at the field level, we collected samples of wheat and triticale infected with pst from different counties across the uk in the spring and summer of ( figure a ; table s in additional file ). from these, we selected pst-infected wheat samples from wheat varieties that spanned the resistance spectrum, and pst-infected triticale samples (table s in additional file ). total rna was extracted from each sample and subjected to rna-seq analysis ( figure a ). after filtering, an average of % (standard deviation . %) reads aligned to the pst- reference genome [ ] , indicating that fungal transcripts account for a high percentage of the transcripts in pst-infected plant tissue (table s in additional file ). to address whether each sample comprised a single pst genotype without considerable bias in allelespecific expression, we calculated the distribution of read counts for biallelic single nucleotide polymorphisms (snps), determined from alignment to the pst- genome. as a dikaryon, the pst mean of read counts at heterokaryotic positions is expected to have a single mode at . , with two alternative alleles each representing one of the two haploid nuclei (additional file ) [ ] . based on the presence of only two alleles and the shape of the distribution being comparable to purified isolates when heterokaryotic snps were considered, we concluded that all samples likely represent a predominantly single genotype with little bias in allele expression (additional files and ). we next used our data to confirm the wheat variety in a particular pst-infected sample. to this end, we extracted the wheat sequences flanking a set of , genetically mapped wheat snps (table s in additional file ) [ ] . nine of the pst-infected wheat samples were collected from wheat varieties with identified snps (donal o'sullivan (university of reading) and james cockram (niab), personal communication) and for each of these samples, reads were independently aligned against the wheat sequences extracted above. each of the , snp positions with ≥ × coverage was then assessed for correlation against the available sequence data for seven wheat varieties. this analysis confirmed the wheat variety recorded at the point of sample collection as the most likely variety for all nine pst-infected wheat samples ( figure ). furthermore, for samples taken from the wheat variety oakley, the second highest matching variety was kws santiago, whose parents are sherbourne and oakley. oakley has been used widely in the parentage of various wheat varieties as reflected by the level of similarity between pst-infected oakley samples and all other varieties ( figure ). this analysis demonstrates that the transcriptomic data from pst-infected field samples can be used successfully to determine the host wheat variety. transcriptome sequencing was carried out on samples to generate transcript data from both the pathogen and host. for the pathogen, the data were used to assess the pathogen population diversity and differential gene expression. for the host, the data were used to confirm the host variety within a particular sample. snp, single nucleotide polymorphism. (b) field isolates (dark blue squares) are distinct and highly diverse when compared with the older uk population (light blue squares). phylogenetic analysis was undertaken using the third codon position of , pst- gene models ( , , sites) with ≥ % breadth of coverage for all pst isolates using a maximum likelihood model. stars indicate samples in which both the genome and transcriptome were sequenced from the same pst isolate. to determine the relationship between the pst field isolates and previously prevalent pst populations, the genomes of uk and french purified pst isolates collected between and were sequenced using an illumina whole-genome shotgun approach figure identification of wheat varieties using transcriptome data generated directly from pst-infected field samples. a total of , snp positions were used to differentiate wheat varieties. each of the , snp positions with ≥ × coverage was assessed for correlation against the available sequence data for seven wheat varieties. for each snp position, if the pst-infected field sample matched the sequence at a snp site for a particular variety (for example, variety = aa; field sample = aa) the position was scored , if the site only partially matched (for example, variety = aa; field sample = ac) then the position was scored . , and if the site had no match (for example, variety = aa; field sample = cc) then the position was given a score of . for each sample, the total score was determined and visualized for each of the seven wheat varieties. numbers in parentheses represent scores associated with differential markers for a particular wheat variety (blue shading). monomorphic markers across all varieties are represented in red. background colour and header relate to the reported variety for a given sample. warrior- , w ; warrior- , w . ( table s in additional file ). after filtering, reads were independently aligned to the pst- reference genome. phylogenetic analysis was undertaken using the third codon position of , pst- gene models ( , , sites) with ≥ % breadth of coverage for all pst isolates using a maximum likelihood model. this analysis illustrated that of the historical uk pst isolates and all french isolates clustered together in a single clade with little genetic variation (figure b) . by contrast, the pst field isolates collected in were distantly related to the older uk population, and included several diverse lineages. furthermore, a subset of of the pst field isolates were also genetically similar to a characterized 'warrior' type pst isolate from (pst- / ; figure b) . this indicates that a diverse pst population that contained the 'warrior' pathotype was prevalent across the uk in . with the first record of the 'warrior' pathotype occurring in the uk in , we decided to investigate the distribution of this lineage further by sequencing the genome of two purified pst isolates with known virulence profiles from and two from [ ] . after filtering, reads were aligned to the pst- reference genome. phylogenetic analysis revealed that two pst isolates from (pst- / and pst- / ) were more closely related to the older uk population, whereas the remaining isolates clustered within the 'warrior' type lineage ( figure b) . to further support the topology of the phylogenetic tree, we extracted rna from a susceptible wheat variety infected independently with six pst isolates (pst- / , pst- / , pst- / , pst- / , pst- / and pst- / ) that were also subjected to genome sequencing. the distribution of biallelic snps, from alignment to the pst- genome, confirmed that each sample comprised predominantly a single pst genotype without considerable bias in allelespecific expression (additional file ). when snp sites with sufficient depth of coverage in both the genomic and rna-seq samples were compared, an average of . % were identical between the genomic and rnaseq datasets (table s in additional file ). this indicates that allele-specific gene expression had a negligible effect on the topology of the phylogenetic tree. this analysis further supports the recent emergence of a diverse pst population that may have now displaced the previous pst population in the uk. to elucidate the population structure among the pst uk field isolates, we generated a list of , synonymous snp sites, of which , were biallelic. we used multivariate discriminant analysis of principal components (dapc) with the , biallelic snp sites to define the population structure and identify groups of genetically related pst isolates. the bayesian information criterion supported the division of pst isolates into four population clusters, which were clearly distinct in a scatterplot of the five principal components of the dapc (figure a ,b). in addition, bayesian-based clustering of the full set of , synonymous snp sites using the program structure classified the pst isolates into four population clusters (figure c ,d) that differed only in the partitioning of two isolates (pst- / and pst- / ) compared with the dapc assignment (additional file ). phylogenetic analyses were also undertaken using the third codon position of , genes ( , , sites) with ≥ % breadth of coverage for all pst isolates using a maximum likelihood model. this analysis supported the assignment of pst isolates to the four population clusters as reported by the bayesian-based clustering method ( figure d ). cluster-specific snps were converted into pcr-based assays and shown to differentiate the pst lineages (table s in additional file ; additional file ). furthermore, within the pst field samples collected in , all pst isolates sampled from triticale clustered within a single genetically distinct lineage ( figure d , cluster ii), potentially indicating a degree of host specificity within the pst population in the uk. next, to determine if the observed population structure was reflected in the phenotypic characteristics of the pst field isolates, we purified and cultured a subset of isolates for virulence profiling. four pst isolates from each of the three population clusters derived from pst-infected wheat samples were inoculated on a series of differential wheat varieties. disease severity was recorded to days post-inoculation (table s in additional file ). phylogenetic analysis of the pst isolates using a maximum likelihood model ( , genes; , , sites) was combined with their virulence profiles to assess correlations between the population substructure and pathology data. this analysis revealed distinct phenotypic characteristics for each population cluster that were different from members of other population clusters, but were largely conserved between isolates of a similar genetic background ( figure a ). furthermore, principal component analysis of the phenotypic characteristics supported the clear division of the isolates into three phenotypic groups that correlated directly with the genetic population clusters (figure b ). this reflects a clear association between the genotypic and phenotypic diversity displayed by the pst field isolates. cluster i isolates displayed the least phenotypic diversity between pst isolates. this correlated with much lower nucleotide diversity between members of this cluster compared with other clusters (figure c) . overall, however, the degree of genetic diversity between members of a single population cluster for all pst field samples was much higher than that displayed by the older uk and french isolates collected between and , excluding pst- / (figure c ). substantial genetic differentiation was also identified in all pair-wise comparisons of the four population clusters, with f st values ranging from . to . (figure c ). the variation in gene expression between members of a population cluster did not influence the calculation of genetic diversity (additional files and ). taken together, this supports the hypothesis that the new uk pst population is derived from a highly diverse founder population. polymorphic and differentially expressed effector candidates can be linked to the virulence profiles of the pst field isolates we also used our field pathogenomics approach to look for two signatures of adaptation, namely mutation and differential gene expression, by treating all isolates within a population cluster as replicates in the analysis. specifically, we sought to identify potential effector proteins and link these to the distinct virulence profiles within the pst population. first, to identify polymorphic effector candidates, we discriminated , homokaryotic and heterokaryotic snp sites that induced non-synonymous substitutions from alignment of all pst field isolates against the pst- reference genome. of these non-synonymous sites, we identified , snp sites where the amino acid residue was conserved among all members of a single population cluster with coverage in the region, but differed from the amino acid encoded by all members from at least one other population cluster (table s in additional file ). these , snp sites figure the pst field isolates are highly diverse and group genetically into four distinct population clusters. (a) scatterplot using the first two principal components (y-axis and x-axis, respectively) of the discriminant analysis of principal components (dapc) analysis of , synonymous single nucleotide polymorphism (snp) sites. each symbol represents a single pst isolate, coloured according to assignment to one of four population clusters. all four population clusters are clearly separated by dapc analysis. (b) the first three eigenvalue components from the dapc analysis, supporting the maintenance of three discriminant functions in the dapc analysis. (c) the optimal predicted number of population clusters k for the dataset is four. the y-axis corresponds to the bayesian information criterion (bic), a goodness-of-fit measurement calculated for each k. the elbow in the bic values (k = ) indicates the optimal number of populations. (d) phylogenetic analysis using a maximum likelihood model ( , , sites) and bayesian-based clustering of , synonymous snp sites classified pst field isolates into four population clusters. all pst isolates sampled from triticale clustered within a single genetically distinct lineage, cluster ii. bar charts represent structure analysis, with each bar representing estimated membership fractions for each individual. stars highlight isolates purified for virulence profiling. coloured circles represent uk counties in which samples were collected (table s in additional file ). were dispersed among , genes that displayed clusterspecific unique amino acid substitutions, of which had detectable secretion signals (figure a ). using the most highly ranked pst effector candidates from our previous study [ ] , we identified genes that encoded cluster-specific polymorphic proteins that displayed features typical of characterized effector proteins ( figure a ). next, to assess whether the gene expression profiles of the pst field isolates could be associated with cluster-specific disparity in virulence profiles, reads from each isolate were aligned independently to the pst- genome. differential expression analysis was conducted after normalization to identify genes that were significantly differentially regulated between the four population clusters (false discovery rate < . ; p-value < . ). all isolates within each population cluster were used as replicates in the analysis (table s in additional file ; additional file ). of the genes that were identified as significantly down-and up-regulated for all isolates within a particular population cluster, between . and . % could be annotated with potential structural or enzymatic functions (figure b ; additional file ). of those that were not annotated, an average of . % (standard deviation . %) were predicted to encode proteins with detectable secretion signals (figure b) . furthermore, we identified up-regulated and down-regulated genes that were among the most highly ranked pst effector candidates from our previous study (figure b) . one of these candidates, pst _ , was significantly down-regulated by isolates in cluster iii and had two amino acid substitutions that were specific and conserved among cluster i isolates (figure c ; additional file ). exploiting transcriptome sequencing for surveillance and population analysis of (re)-emerging pathogens human, animal and plant pathogens necessitate constant monitoring to preserve public health and food security. with the advent of next-generation sequencing technologies, it is now possible to integrate high-resolution dna and rna sequencing into pathogen surveillance programs. however, many pathogens cannot be axenically cultured, limiting access to pure dna and rna preparations. furthermore, large-scale population analysis of fungal pathogens by whole-genome sequencing remains limited by the lengthy processes associated with purification and multiplication of isolates for high molecular weight dna extraction and the cost of sequencing large genomes. we have developed an approach for pathogen population surveillance based on high-resolution transcriptome data acquired directly from field samples of pathogen-infected wheat and triticale. even though the analyzed samples consist of a mixture of pathogen and host rna, we recovered enough pathogen sequences for analysis. also, the rna-seq data were deep enough for reliable genotypic characterization. similar approaches using shotgun genome sequencing could have been problematic due to the large size of the genome of wheat (approximately gb) compared with that of pst (approximately mb) [ , ] . our approach also captures the pst population directly from the field and negates any biases that might be caused by purification and multiplication of the pathogen in the laboratory, a lengthy process that can impose artificial selection on the pathogen. using field pathogenomics, we could detect only a single pst genotype within each lesion. furthermore, using comparative analysis of rna-seq and genomic sequence data from six independent pst isolates (pst- / , pst- / , pst- / , pst- / , pst- / and pst- / ), we were able to confirm that allelicspecific expression between the two pst nuclei had minimal effect on genotypic analysis. together these results demonstrate that rna-seq analysis of pst-infected plant material is a useful approach for accurately genotyping isolates of pst directly from the field. however, our findings contrast with studies of mycosphaerella graminicola on wheat and rhynchosporium secalis on barley, where co-infection with multiple genotypes is common [ , ] . analyses of field pathogenomics data may be more complex in such pathosystems. whilst effectively capturing pathogen diversity, transcriptome sequencing of infected host tissue can also be leveraged to assess the genotype of the host. the (c) pst _ is a previously identified effector candidate that was significantly down-regulated by isolates in cluster iii and had two amino acid substitutions that were specific and conserved among cluster i isolates. the five carboxy-terminal amino acids were not defined due to poor coverage. availability of high-throughput snp chips for wheat [ ] and snp marker information for the majority of wheat varieties in the uk [ ] (and elsewhere) provides an unprecedented opportunity to exploit sequence data to confirm outbreaks on particular wheat varieties and look for associations between pathogen genotypes and host pedigrees. in this study, we developed an accurate system to associate samples from known wheat varieties with their corresponding snp markers. in the future, this will provide a rapid means of confirming whether previously resistant wheat varieties have indeed been broken by virulent races of the pathogen, using samples submitted directly to national pathology surveys. this would reduce delays associated with current protocols, which include pathogen propagation, subsequent virulence profiling and confirmation of a specific wheat variety using protein gels from harvested grains or similar distinctness, uniformity and stability assessments [ ] . traditionally, the surveillance of rust fungal pathogens in agroecosystems has hinged on field biology and race pathotype surveys to provide phenotypic information on pathogen diversity [ ] . however, assessments of genotypic diversity are not included routinely and when employed are restricted to just a handful of markers such as simple sequence repeats or amplified fragment length polymorphisms [ ] . our field pathogenomics approach enables the integration of high-resolution genotypic data into pathogen surveillance activities. for instance, more than million nucleotide positions were used to assess pst population diversity in this study. these highresolution genotypic data are vital to improve our understanding of the genetic substructure within a population, which provides essential information on the evolutionary forces that drive pathogen evolution within an agroecosystem. this study uncovered four genetically distinct lineages within the uk pst population, and each of these lineages had unique virulence profiles revealing a direct link between genotype and pathotype. although such a correlation has been reported for rust fungi [ , ] , our findings contrast to distantly related filamentous plant pathogens such as magnaporthe oryzae [ ] and colletotrichum lindemuthianum [ ] where a relationship between genotype and pathotype has not been detected. the time-consuming nature of traditional surveillance methods limits the number of pst isolates assessed each year. for instance, in the uk, a target number of pst-infected wheat samples are tested each year, specifically focusing on wheat varieties with a previous record of good resistance in the field. with new pst pathotypes/genotypes arising on susceptible varieties by mutation, recombination or through exotic incursions, it is unlikely that a new pathotype would be detected in a timely fashion by the current surveillance system. furthermore, an exotic isolate that displays similar phenotypic characteristics to a subset of the existing population would not be recognized as such. in this study, we uncovered a group of pst isolates (population cluster iii) that displayed identical phenotypic characteristics to a subset of the old uk population, but in fact belonged to a new emergent lineage that appears to be new to the uk. none of these isolates would have been identified as belonging to an emergent lineage based on phenotypic data alone. however, such population shifts may bear significance on disease incidence as the new population may carry important epidemiological traits other than pathogen virulence. rapid and systematic application of field pathogenomics should transform current disease surveillance systems by generating high-resolution genotypic information (additional file ) that inform disease incidence models, agronomic practices, and the selection of pst isolates for subsequent labor-intensive phenotypic characterization. the emergent pst population in the uk is now dominated by a number of newly selected, virulent clones that are adapted to an array of widely cultivated wheat varieties. by revealing genotype/pathotype-specific polymorphisms, the data we generated could prove useful in identifying candidate avirulence effectors that contribute to a pathogen's ability to evade recognition on particular host genotypes. herein, our analysis identified a small number of candidate effector genes with conserved mutations or expression profiles between members of the same population cluster that shared similar virulence profiles. ultimately, such information could be used to develop polymorphic markers to track the long-distance migration of pathotypes across wheat growing regions. we uncovered a dramatic shift in the pst population that could have serious implications for wheat production in the uk. whilst there have been widespread reports of recent changes in the pst population based on phenotypic characteristics [ ] , we report a comprehensive genetic analysis of this emergent pst population. plant-pathogenic fungi rely predominantly on recombination and mutation as the evolutionary forces that drive the emergence of new races and pathotypes [ ] . however, within a pathogen population, gene and genotype flow can shape the population substructure as propagules are exchanged between geographically separated epidemiological areas [ ] . given the clonal population structure of pst in northwestern europe, mutation and genotype flow are the primary inducers of diversity [ ] . the fact that none of the pst field isolates showed genetic similarity to the great majority of the older uk population (collected between and ; excluding pst- / ) indicates that the population is likely an exotic pst population that appears to have displaced the previous population. furthermore, the highest level of genetic diversity between the four emergent pst lineages (f st ranging from . to . ) was similar to that detected using simple sequence repeat markers and comparing pst isolates from different continents [ ] . this is indicative of distant ancestry or relatively low levels of gene flow between these emergent uk pst lineages. based on this evidence, we hypothesize that the change in pst population structure may have arisen from exotic incursions from multiple sources over recent years. future studies will focus on defining the origin(s) of this pst population. a subset of the emergent pst population we characterized displays the 'warrior' pathotype that was first detected in in the uk and is virulent on an array of previously resistant wheat varieties, including alchemy, warrior, and claire [ ] . our findings illustrate how pathogen genotype flow can trigger abrupt changes in the landscape of wheat genetic resistance to yellow rust. breeders are now at a crossroads in the uk, with few sources of yellow rust disease resistance available and the prospect of new varieties being rapidly taken off the official recommended list due to poor yellow rust resistance, as happened with torch ( year on the recommended list) and warrior ( years). with anthropogenic activities having a marked influence on the size of genetic neighborhoods [ ] , pathogen genotype flow is no longer dependent on life history traits and natural dispersal alone. the next step will be to define the boundaries of these ever-expanding genetic neighborhoods to inform surveillance strategies and breeding programs that need to take into account the full pathogen population within an isolated genetic neighborhood to breed for durable resistance. the pst isolates displayed a much higher degree of nucleotide diversity when compared with the older uk population. this reflects an increase in pst evolutionary potential in the uk pathogen population that could enhance their ability to overcome genetic resistance in the host. given that the highest levels of pst genotypic diversity have been reported in the himalayas and neighboring regions, it is possible that the emerging pst population is derived from one or more migration events from a geographic area with high sexual reproduction rates and a recombinant population structure [ ] . this is further supported by similarity in pathotypes between one lineage (cluster i) of the emergent uk population and those previously reported for exotic pst isolates [ ] . for instance, three chinese isolates that were collected in and a nepalese isolate from were shown to be virulent on the wheat variety spaldings prolific [ ] , which is a key determinant for the cluster i ('warrior') pathotype [ ] . furthermore, ali et al. [ ] previously classified two chinese isolates collected in as belonging to the northern french genotypic group (g ). future studies will focus on comparative sequence analysis between the pst isolates reported herein and global isolates of pst to determine the specific geographic origin(s) for this diverse pst population in the uk. the agronomic consequences of long-distance pathogen migration are currently unpredictable. although a pathogen population may not pose a significant threat to crop production in the country of origin, it can have devastating consequences in a new environment. for instance, in a severe stem rust epidemic in ethiopia was caused by a race similar to those detected in egypt, germany and turkey between and . however, despite the widespread devastation reported in ethiopia, other countries reported no negative effect of this race on wheat production. this episode illustrates the importance of global pathogen surveillance networks, to enable early warning systems that assess the threat of pathotypes to all crop genotypes planted within a single genetic neighborhood. field pathogenomics provides the means to generate enough markers to comprehensively genotype the pst population. high-resolution snp marker arrays would allow tracking pathogen dispersal on a global scale and clear definition of the pathogen population genetic structure. the approach reported herein uses attenuated pst-infected field samples, thereby negating the limitations associated with movement of live samples. whilst genotyping is undertaken in state-of-the-art molecular laboratories, the complementary virulence profiling can be carried out in national centers, thereby preventing any threat posed by transportation of live samples between countries. once genotypic information is generated, subsequent phenotypic characterization can focus on the most notable and representative samples ensuring the best possible use of limited national resources. in this study, we developed a robust and rapid method based on rna sequencing directly from infected host samples to gain insight into emerging pathogen populations. field pathogenomics should be applicable to surveillance of many pathogens besides wheat rust pathogens, and could contribute to addressing human, animal, and plant health issues. our approach enabled us to discover a dramatic shift in the uk pst population in essentially months after collecting the field samples. the emergent pst population has high levels of genetic diversity compared with historical uk isolates and appeared to be unrelated to the older population. this led us to conclude that the pst population was most probably derived from the recent introduction into the uk of diverse assemblage of exotic pst lineages, and that these introduced lineages may have rapidly displaced the previous pst population. such detailed knowledge of population shifts and dynamics is important for our understanding of emerging plant diseases and has consequences for the management of such diseases. a total of single lesion leaf samples of pst-infected wheat and triticale were collected directly from the field and stored in rna later solution at °c (life technologies, paisley, uk). the single lesion consisted of a to cm leaf section taken from a single infection site. total rna was extracted from of these samples using the qiagen rneasy mini kit according to the manufacturer's instructions (qiagen, manchester, uk). in addition, we extracted rna in a similar manner from infected leaves of susceptible wheat variety vuka inoculated independently with six pst isolates (pst- / , pst- / , pst- / , pst- / , pst- / and pst- / ). the quantity and quality of rna extracted were assessed using the agilent bioanalyzer (agilent technologies, edinburgh, uk). cdna libraries were prepared using the illumina truseq rna sample preparation kit (illumina, cambridge, uk). library quality was confirmed before sequencing using the agilent bioanalyzer (agilent technologies, edinburgh, uk). libraries were sequenced on the illumina gaiix at the sainsbury laboratory (for rb and rb ) or the illumina hiseq machine at the genome analysis centre, uk. adapter and barcode trimming and quality filtering were carried out using the fastx-toolkit. the -bp (gaiix) or -bp (hiseq) paired-end reads were aligned to the pst- assembly [ ] using the tophat package (version . . ) and bowtie alignment program (version . . ) with default parameters [ , ] . a similar approach was used for whole genome sequencing of pst isolates, except that gdna was extracted for each isolate from dried urediniospores using the ctab method as described by chen et al. [ ] and dna quantity was confirmed using the qubit . fluorometer. dna libraries were prepared using the illumina truseq dna sample preparation kit (illumina, cambridge, uk). sequencing of all gdna samples was carried out on an illumina hiseq machine at the genome analysis centre, uk, generating -bp paired-end reads which were aligned to the pst- assembly [ ] using bwa with default parameters [ ] . the illumina reads from all rna-seq and gdna runs were deposited in the short read archive (genbank; prjna and prjna ). first, from a set of , high-density wheat snps, , genetically mapped wheat snps were extracted [ ] . up to bp up-and down-stream of each snp site were extracted from the wheat chromosome arm survey sequence [ ] to create a reference for subsequent sequence alignments. nine pst-infected field samples were collected on wheat varieties with known varietal snp information (donal o'sullivan (university of reading) and james cockram (niab), personal communication). reads from each of these nine samples were independently aligned to the wheat genome sequences extracted above using the tophat package (version . . ) and bowtie alignment program (version . . ) with default parameters [ , ] . each of the , snp positions with ≥ × coverage was then assessed for correlation against the available sequence data for the seven wheat varieties. for each snp position, if the pst-infected field sample matched the sequence at a snp site for a particular variety (for example, variety = aa; field sample = aa) the position was scored , if the site only partially matched (for example, variety = aa; field sample = ac) then the position was scored . , and if the site had no match (for example, variety = aa; field sample = cc) then the position was given a score of . for each sample, the total score was determined and visualized for each of the seven wheat varieties. calling single nucleotide polymorphisms bam files were sorted and indexed, and snps determined using raw allele counts for each position that were obtained using pileup from samtools [ ] . heterokaryotic sites were identified as sites with allelic frequencies ranging from . to . . homokaryotic sites were those with allelic frequencies below . or above . . for both hetero-and homokaryotic sites to be reported, they had to satisfy a minimum depth of coverage of × for rna-seq data and × for genomic dna data. read frequencies were calculated for biallelic heterokaryotic snp sites and plotted using ggplot in r [ ] . homokaryotic and heterokaryotic snp sites that induced synonymous and non-synonymous substitutions were identified using snpeff, version . [ ] . all phylogenetic analysis of pst isolates was conducted using a maximum likelihood approach. first, for both genomic and rna-seq samples, nucleotide residues that differed from the pst- reference were identified and recorded if they satisfied a minimum of × or × depth of coverage, respectively. next, sites that were identical to the reference were recorded when they satisfied a minimum of × depth of coverage. finally, these sites were used to generate synthetic gene sets for each isolate and genes with a minimum of % breadth of coverage for all samples in a comparison were selected. the third codon position of these genes was then used to build maximum likelihood trees using raxml . . with replicates using the rapid bootstrap algorithm [ ] . phylogenetic trees were visualized in mega . [ ] . for the rna-seq samples, results from structure analysis were incorporated into the phylogenetic tree using itol [ ] . genetic differentiation of the pst field isolates was examined using the bayesian model-based approach implemented in the software structure, version . . [ ] via the python strauto program, version . [ ]. first, a list of , sites that introduced a synonymous change in at least one isolate was generated. then, the nucleotide at this position was extracted for all rnaseq samples. the 'admixture' model was used with three replicates of , markov chain monte carlo generations for k = to , where k is the number of populations. for each run the first , generations were discarded as burn-in before collecting data. to identify the k value the average log probability (lnp(d)) of each k value was calculated [ ] . the genetic differentiation of the field isolates was further assessed using the multivariate dapc within the adegenet package [ ] . first, , biallelic snp sites that introduced a synonymous change in at least one isolate were identified. using these data, principal component analysis was carried out to summarize genetic variation between and within potential population clusters. the optimum number of clusters was determined as the one showing the lowest bayesian information criterion. dapc analysis was then used to assign individuals to each of the population clusters. to assess the genetic diversity both within and between pst population clusters, all heterokaryotic and homokaryotic snps determined above from individual alignment of each isolate to the pst- reference were incorporated into a synthetic gene set for that isolate. the synthetic genes were combined for all pst field isolates within a population group, and genes with > % breadth of coverage for all isolates were selected. to calculate the degree of nucleotide diversity between isolates of a single population group, the degree of polymorphism between these gene sets was calculated using the dnasp software package, version . . [ ] . to determine the proportion of total genetic variance attributable to inter-population differences, the , sites that introduced a synonymous change in at least one isolate were used as input in the program genepop version . [ ] to calculate the wright's f st statistic. virulence phenotyping of pst isolates was based on the reactions of wheat cultivars possessing known resistances to pst, together with a number of cultivars possessing resistances which have not yet been fully described. tests were carried out on seedlings under controlled environment conditions [ ] , with infection types being assessed on the first seedling leaf using a to scale. infection types and were considered to represent a compatible interaction between host genotype and pathogen isolate, indicating the absence of avr alleles (that is, virulence) at the corresponding locus in the pathogen. the host resistance genes covered by the differential set were yr , yr , yr , yr , yr , yr , yr , yr , yr , yr , yr , yr , yr , yr , yr and the resistance in spaldings prolific. other discriminating differentials included the cultivars robigus, solstice, timber, warrior, ambition, and rendezvous. to distinguish the internal structure and variance within the pathology data, the scores associated with the reactions of each isolate on the differential wheat cultivars were used for principal component analysis in r [ ] . quantification of reads mapping to the pst- gene set from the pst field isolates was determined using the program htseq-count [ ] . next, the fisher's exact test, implemented as part of the edger package [ ] , was used to identify genes that were significantly differentially regulated between the four population clusters (false discovery rate < . ; p-value < . ). all isolates within each population cluster were used as replicates in the analysis to ( ) limit the influence of environmental factors on the expression profiles, as samples were collected at various sites throughout the season, and ( ) to link gene expression profiles to the virulence profiles that were unique to these genotypic groups. to identify potential effector proteins with signatures of adaptation such as mutation and variation in gene expression profiles, we focused on accessing those that were ranked the highest in our previous effector mining study [ ] . previously, we clustered protein sequences based on sequence similarity and ordered the resulting protein families based on the association of known effector features and pstspecific annotation [ ] . this resulted in overall scores for each family that reflected their likelihood of containing potential effector proteins [ ] . those within the top protein families were considered herein. primers were designed with primer version . . [ ] carrying standard fam or hex compatible tails (fam tail: ′ gaaggtgaccaagttcatgct ′; hex tail: ′ gaaggtcggagtcaacggatt ′) and with the target snp at the ′ end. oligonucleotides were ordered from sigma-aldrich (gillingham, uk) and primer mixes were as recommended by the manufacturer ( μl dh o, μl common primer ( μm), and μl each tailed primer ( μm); lgc genomics, teddington, uk). assays were carried out as described previously [ ] with the following modifications: μl reactions were used (composed of μl template ( to ng dna), . μl v × kaspar mix, and . μl primer mix)), pcr cycling was performed in an eppendorf mastercycler pro and well optically clear plates (catalogue number e , starlab, milton keynes, uk) were read on a tecan safire plate reader. data analysis was performed manually using klustercaller software (version . . . , lgc). additional file : contains supplementary tables s to s . microsoft excel workbook containing nine worksheets. table s : all pst-infected field samples collected in . table s : fungal transcripts account for a high percentage of the transcripts in pst-infected plant tissue. table s : wheat variety snps from iselect wheat snp chip (wang et al. [ ] ). table s : virulence profiles of pst isolates subjected to full genome sequencing. table s : comparison of snp sites between genomic and rna-seq datasets generated from the same pst isolate. table s : genotype data for nine pst field samples generated from kasp assays. table s : full seedling virulence tests of selected pst isolates. table s : non-synonymous snp sites where the amino acid residue was conserved among all members of a single population cluster with coverage in the region, but differed from the amino acid encoded by all members of at least one other population cluster. table s : differential expression analysis based on fisher's exact test of all pair-wise comparisons of population clusters. additional file : distribution of biallelic read counts. (a,c) read frequency at biallelic single nucleotide polymorphisms (snps) for a gdna sample known to consist of multiple mixed genotypes. the presence of several alleles with three copies and the presence of an uneven frequency distribution are indicators of more than a single genotype in the sample. (b,d) read frequency at biallelic snps for a purified gdna sample that consists of only a single pst genotype. the even frequency distribution and presence of only two alleles support the presence of a single genotype in the sample. the mode for each distribution is given in parentheses. isolation of a novel coronavirus from a man with pneumonia in saudi arabia influenza virus evolution, host adaptation, and pandemic formation hymenoscyphus pseudoalbidus, the causal agent of european ash dieback the emergence of ug races of the stem rust fungus is a threat to world wheat production the genomics of emerging pathogens transforming clinical microbiology with bacterial genome sequencing fungal molecular diagnostics: a mini review a cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples viral metagenomics genome evolution in filamentous plant pathogens: why bigger can be better dual rna-seq of pathogen and host the 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rust puccinia striiformis f. sp tritici isolation by distance evidence of genetic recombination in wheat yellow rust populations of a chinese oversummering area reduction in the sex ability of worldwide clonal populations of puccinia striiformis f.sp tritici differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks ultrafast and memory-efficient alignment of short dna sequences to the human genome relationship between virulence variation and dna polymorphism in puccinia striiformis fast and accurate short read alignment with burrows-wheeler transform international wheat genome sequencing c. a chromosome-based draft sequence of the hexaploid bread wheat (triticum aestivum) genome the sequence alignment/map format and samtools ggplot : elegant graphics for data analysis a program for annotating and predicting the effects of single nucleotide polymorphisms, snpeff: snps in the genome of drosophila melanogaster strain w maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models mega : molecular evolutionary genetics analysis version . interactive tree of life (itol): an online tool for phylogenetic tree display and annotation inference of population structure using multilocus genotype data adegenet: a r package for the multivariate analysis of genetic markers dnasp v : a software for comprehensive analysis of dna polymorphism data : a complete re-implementation of the genepop software for windows and linux identification of specific resistances against puccinia striiformis (yellow rust) in winter wheat varieties. . establishment of a set of type varieties for adult plant tests r: a language and environment for statistical computing. r foundation for statistical computing a python framework to work with high-throughput sequencing data edger: a bioconductor package for differential expression analysis of digital gene expression data primer -new capabilities and interfaces submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank all those who submitted pst-infected wheat and triticale samples to the ukcpvs in . we thank henk-jan schoonbeek, francesca stefanato, clare domoney, tina barsby, andrew dawson, jan bettgenhaeuser and matthew moscou for logistic support; albor dobon and laura reese for assistance with wet-lab experiments; luis enrique cabrera quio for bioinformatics assistance; and mark mcmullan for useful discussions regarding population genetic analyses. this project was funded by the sustainable crop production research for international development additional file : distribution of biallelic read counts for purified uk isolates subjected to full genome sequencing. the purified uk pst isolates that were selected for genome sequencing show varying patterns of read frequencies for biallelic single nucleotide polymorphisms. the mode for each distribution is given in parentheses.additional file : all pst-infected plant samples consist of a single pst genotype. distribution of biallelic single nucleotide polymorphism read frequencies for all pst field samples (rna-seq). the mode for each distribution is given in parentheses. additional file : differential expression analysis reveals population cluster-specific expression profiles. (a) there were to genes significantly down-regulated and to genes significantly up-regulated for all isolates within a particular population cluster. differential expression analysis was undertaken using fisher's exact test from the edger package to identify genes that were significantly differentially regulated between the four population clusters (false discovery rate < . ; p-value < . ). (b) of the significantly up-or down-regulated genes, between . and . % could be annotated with potential structural or enzymatic functions based on sequence similarity searches. (c) the effector candidate pst _ was significantly down-regulated by isolates of cluster iii.additional file : snp markers developed from the field pathogenomics data could differentiate the four emergent pst lineages. of the fifteen kasp assays developed, could be used to differentiate particular lineages within the emergent pst population in the uk. each closed circle represents the genotype of a single pst field sample. blue circles, x:x; green circles, x:y; red circles, y:y; open circles, h negative control; grey circles, not determined. where shown, background color reflects grouping of field samples within particular population clusters: cluster = pink; cluster = green; cluster = blue; cluster = red. numbers reflect population clusters within each genotype group.abbreviations bp: base pair; dapc: discriminant analysis of principal components; pst: puccinia striiformis westend. f. sp. tritici eriks; snp: single nucleotide polymorphism. the authors declare they have no competing interests.authors' contributions ky, jt, sk, rb, cu and dgos conceived and designed the project; dgos designed and performed bioinformatics analysis; ky advised on population genetic analysis; ah, cml, cv-p and dgos conducted wet-lab experiments;rhr-g assisted with wheat snp analysis and designed kasp assays; cu and dgos prepared the manuscript. all authors read and approved the final manuscript. key: cord- - nyew i authors: chen, yi-ning; loa, chien chang; ababneh, mustafa mohammed-khair; wu, ching ching; lin, tsang long title: genotyping of turkey coronavirus field isolates from various geographic locations in the unites states based on the spike gene date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: nyew i turkey flocks have experienced turkey coronaviral enteritis sporadically in the united states since the s. twenty-four field isolates of turkey coronavirus (tcov) from multiple states in the united states were recovered from to to determine the genetic relationships among them. the entire spike (s) gene of each tcov isolate was amplified and sequenced. pairwise comparisons were performed using the clustal w program, revealing . % to . % sequence identity in the full-length s protein, . % to . % in the amino terminus of the s subunit (containing one hypervariable region in s a), and . % to . % in the s subunit at the deduced amino acid sequence level. the conserved motifs, including two cleavage recognition sequences of the s protein, two heptad repeats, the transmembrane domain, and the golgi retention signal were identified in all tcov isolates. phylogenetic analysis based on the full-length s gene was used to distinguish north american tcov isolates from french tcov isolates. among the north american tcov isolates, three distinct genetic groups with % bootstrap support were observed. north carolina isolates formed group i, texas isolates formed group ii, and minnesota isolates formed group iii. the s genes of tcov isolates from the united states remained conserved because they contained predominantly synonymous substitutions. the findings of the present study suggest endemic circulation of distinct tcov genotypes in different geographic locations. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. turkey coronaviral enteritis of varying severity, caused by turkey coronavirus (tcov), has been reported in turkey flocks from multiple states in the united states since the s. the major clinical signs of tcov infection include depression, ruffled feathers, diarrhea, decreased body weight, and uneven flock growth. the most apparent gross lesions are markedly distended intestines with gaseous and watery content, particularly in the ileum and ceca. salient histopathologic findings include shortening of the intestinal villi, an increase in crypt depth, and widening of intervillous spaces [ ] . when turkeys are infected with tcov and other infectious agents such as astrovirus, small round virus, and escherichia coli (e. coli), they can develop poult enteritis-mortality syndrome (pems), which causes high mortality [ , ] . subsequent experimental studies of the tcov isolates vr- and tcov/on/mg / from canada have shown that tcov can cause symptoms electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. & tsang long lin tllin@purdue.edu similar to those caused by pems [ , ] . therefore, tcov has been suggested to be the major causative pathogen for turkey enteritis, and secondary infections caused by other opportunistic microorganisms enhance the severity of tcov enteritis and contribute to the development of pems. turkey enteritis associated with tcov infection has caused substantial economic losses in indiana, north carolina, arkansas, and other states in the united states [ , ] , as well as in canada [ ] , europe [ , ] , and brazil [ ] . currently, there are no vaccines to prevent the disease, and treatment of infected turkeys is often unsuccessful. a member of the species avian coronavirus (cov) in the genus gammacoronavirus and family coronaviridae, tcov has a positive single-stranded rna genome that is approximately kb in size. the major structural proteins of tcov include the spike (s), envelope (e), matrix (m), and nucleocapsid (n) proteins. comparisons of '-end coding regions [ , ] as well as the full genomes [ ] of tcov isolates and infectious bronchitis virus (ibv) have suggested that tcov arises through recombination in the s gene, because pairwise comparisons of s gene sequences have revealed only a % similarity between tcov isolates and ibv strains, whereas gene , m gene, gene , and n gene sequences have over % similarity [ , , ] . the s gene sequences of different tcov isolates ( %- . %) are more conserved than those of various ibv strains ( . %- . %), which could explain the close antigenic relationship of tcov isolates compared with the distant antigenicity of different ibv serotypes [ , , , ] . investigations during tcov outbreaks and genomic analyses of tcov isolates have revealed that distinct tcov isolates tend to circulate endemically, and their respective sequences group phylogenetically according to their state of origin [ , , ] . however, these observations may have been biased because of the small number of tcov sequences that were analyzed. in the present study, tcov isolates were recovered from clinical cases submitted to the indiana animal disease diagnostic laboratory at purdue university by turkey farms in minnesota, indiana, north carolina, missouri, arkansas, texas, south carolina, and pennsylvania between and . the objective of the present study was to elucidate the relationship between the genotypes and geographic distribution of tcov isolates from turkey farms in multiple states in the united states by using sequence analysis and comparing the full-length s gene. twenty-four field isolates of tcov were recovered from clinical cases submitted to the animal disease diagnostic laboratory at purdue university by turkey farms in minnesota, indiana, north carolina, missouri, arkansas, texas, south carolina, and pennsylvania between and (table ). field cases of tcov were confirmed by clinical signs, gross lesions, histopathologic findings, immunofluorescence antibody (ifa) assay with antiserum against tcov/in/ / , electron microscopy, and reverse transcription polymerase chain reaction (rt-pcr). all tcov isolates were propagated five times in embryonated turkey eggs as described previously [ ] . in brief, intestines from tcov-infected turkeys were homogenized as % suspensions in chilled sterile phosphate-buffered saline and clarified by centrifugation at rpm for minutes at °c. the supernatant was filtered through a . -lm membrane filter (millipore, bedford, ma, usa). the filtrate was inoculated into the amniotic cavity of -day-old embryonated turkey eggs. the embryo intestines were harvested after days of incubation for virus purification. the harvested intestines were homogenized and clarified at rpm for minutes at °c. the supernatant was layered on top of % and % sucrose and clarified using ultracentrifugation in an sw rotor at , rpm for hours at °c in an optima xl- k ultracentrifuge (beckman coulter, fullerton, ca, usa). the interface between % and % sucrose was collected and placed on top of a continuous %- % sucrose gradient and clarified by ultracentrifugation at , rpm for hours at °c. a band of buoyant density . - . g/ml (containing tcov) was collected and saved at - °c as the viral stock. the viral rna was extracted from the purified virus using rnapure tm reagent (genhunter, nashville, tn, usa) and chloroform, followed by precipitation using cold isopropyl alcohol and ethanol. the extracted rna was reverse transcribed to cdna using superscript tm iii reverse transcriptase (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the first reaction was minutes of incubation with the rna, random hexamer primer ( ng/ll), and mm dntps at °c, followed by minute on ice. the second reaction was minutes of incubation with a mixture of x firststrand buffer, . m of dithiothreitol (dtt), u of superscript tm iii reverse transcriptase, and u of rnaseout tm (invitrogen, carlsbad, ca, usa) at °c, followed by hour of incubation at °c and minutes of inactivation at °c. the full-length s gene (approximately . kb) was amplified by pcr using the cdna of each tcov isolate with the primers sup and sdown (online resource ). the mixture ( : , v:v) of taq (promega corp., madison, wi, usa) and pfu dna polymerases (stratagene, la jolla, ca, usa) with proofreading ability was used in a -well thermal cycler (geneamp, perkin-elmer cetus corp., norwalk, ct, usa) to maintain the fidelity of the pcr [ ] . the pcr products were electrophoresed on % agarose gels and purified using a zymoclean tm gel dna recovery kit twenty-four tcov isolates collected from eight states in the united states from to were purified and sequenced in our laboratory. tcov/in/ / was analyzed in a previous study [ ] , and the full-length s gene sequences of the other tcov isolates were published for the first time in the present study ( table ). the putative peptide cleavage site separating the amino-terminus of the s subunit from the carboxyl terminus of the s subunits as well as a second possible peptide cleavage site in the s subunit were detected using the prop server (http://www. cbs.dtu.uk/services/prop/). the s subunit of tcov was further designated as s a at the amino-terminus ( - in tcov/in/ / ) and s b at the carboxyl-terminus ( - in tcov/in/ / ) [ ] . figure shows a diagram of the s protein of tcov. the nucleotide and deduced amino acid sequence similarities of the s genes of all tcov isolates were analyzed using the clustal w alignment method in mega [ ] . a phylogenetic tree based on fulllength nucleotide sequences of the s gene was constructed using the maximum-likelihood method and the kimura -parameter model. a phylogenetic tree based on deduced amino acid sequences of the s a subunit, containing a hypervariable region (hvr), was constructed using the neighbor-joining method and the jones-taylor-thornton model. a codon-based z-test of positive selection for s gene sequences of various tcov isolates was conducted to analyze the differences in the number of nonsynonymous (dn) and synonymous (ds) substitutions per site by using the nei-gojobori method [ ] in mega . the variance of both trees and codon-based z-test were validated using bootstrap replicates. the s sequences of tcov isolates reported in the present study were submitted to the genbank databse, and their accession numbers ranged from kf to kf . the accession numbers of other covs used for phylogenetic analysis are also listed in table . the sizes of the s genes of tcov isolates reported in the present study ranged from to nucleotides. all tcov isolates exhibited similar s protein sequences (fig. ) . the consensus transcription-regulating sequence (trs), ctgaacaa, was identified nucleotides upstream of the start codon of the s protein. several conserved motifs and one hvr were found in all tcov isolates, and their sequences are listed for comparison in table . the consensus motif rxrr/x (x is any amino critical arginine was mutated to glycine and the second probable protein cleavage site was lost. rather than nqgr/s, french tcov isolates had the pqgr/s sequence as the conserved cleavage motif in the s subunit. only one hvr, spanning amino acid positions to (tcov/in/ / ) was found in the tcov isolates, rather than the three hvrs identified in ibv [ ] . two -aminoacid insertions in heptad repeats (hr and hr ) , the consensus motif (yikwpwyvwl) in the transmembrane domain, and the late golgi retention signal (yyttf) for s protein were also observed in all tcov isolates. among the amino acid residues of the neutralizing-epitopecontaining s fragment in the s subunit identified in a previous study [ ] , consensus residues were observed among the tcov isolates (online resource ). a pairwise comparison of the deduced amino acid sequences of the tcov isolates showed that the sequence identity ranged from . % to . % for the full-length s protein, . % to . % for the s a subunit containing hvr, and . % to . % for the s subunit phylogenetic trees based on the full-length s nucleotide sequences ( fig. a) and s a amino acid sequences containing hvrs (fig. b ) of different covs of the genus gammacoronavirus were generated. as shown in figures a and b, the ibv strains were separated from tcov isolates, and north american tcov isolates were separated from french tcov isolates. three genetic groups, referred to as groups i, ii, and iii, were observed in north american tcov isolates ( fig. a) because of the high degree of variation, most phylogenetic groupings based on the s a deduced amino acid sequences did not have a bootstrap value over % (fig. b) . nevertheless, the texas tcov isolates of group ii and all three tcov isolates of group iii shown in the phylogenetic tree based on the full-length s nucleotide sequences still clustered according to their s a amino acid sequences containing their hvr. turkey coronavirus isolates from different geographic areas in the united states have been shown to be antigenically related to one another [ ] . in the present study, an antiserum against the isolate tcov/in/ / reacted with all tcov isolates from tcov-infected turkeys and embryos by ifa assay (data not shown). the close antigenicity among tcov isolates was associated with the high similarity of the s gene sequences, which ranged from . % to . %. the s genes of tcov isolates were conserved compared with the diverse s genes among various ibv strains, which range from . % to %, resulting in the existence of many serotypes of ibv [ ] . the emergence of new ibv serotypes has been postulated to involve the recombination of s genes of vaccine strains of ibv in the field [ , ] . in a previous study, different serotypes of tcov/va/ / , tx/ / , and in/ / were identified by using a neutralization test in conjunction with real-time rt-pcr despite the high level of amino acid sequence identity ( % to %) among these tcov isolates [ ] . additional studies are necessary to clarify the antigenic relationships among the various tcov isolates and serotypes. in the present study, similar to previous findings with ibv strains, most of the variations in the s protein sequences among tcov isolates were observed in the amino-terminal half. along the alignment of these s protein sequences, the region of sequence with the most variation was between residues and of tcov/in/ / from the start codon of the s protein. various deletions occurred in this region in different tcov isolates. this region is in the vicinity of hvr ii (residues to ) of the ibv s protein. hvr i (residues to ), ii, and iii (residues to ) of the ibv s protein are associated with three neutralizing epitopes. the sequences of these regions could be used for differential diagnosis of ibv serotypes [ , ] . by contrast, similar regions of high variation corresponding to hvr i or iii of ibv were not detected among the tcov isolates examined. these differences illustrate why the s proteins of ibv strains are more diverse than those of tcov isolates. similar phylogenetic trees were constructed using the full-length s and s a proteins. thus, genotyping tcov field isolates based on the s a sequence containing hvr rather than the whole s gene or full-length s gene is more practical. the observation that tcov isolates originating from the same state were closely clustered together in the phylogenetic tree suggested endemic circulation of distinct tcov genotypes in various geographic locations. endemic circulation of distinct tcov genotypes in france and north american are recognized because french tcov isolates share only % amino acid sequence identity in the s protein with north american tcov isolates [ ] . distinct sources of recombination promoting the emergence of tcov in north america and europe has been suggested [ , ] . the groupings of north carolina, texas, and minnesota tcov isolates also support the theory of endemic tcov genotypes. the tcov isolates from the outbreaks in arkansas and north carolina in also clustered geographically [ ] and could be placed phylogenetically in group i in the present study. the . % amino acid sequence identity of the s proteins of two tcov isolates recovered years apart in minnesota (tcov/mn/atcc/ and tcov/mn/ / ) implied that the tcov isolate mn/atcc/ remained endemic and that no substantial genetic changes occurred over two decades. conservation of tcov isolates is also shown in the result that no positive selection of the s protein was found among the tcov isolates. because indiana isolates from and clustered in group i with most north carolina isolates and indiana isolates from and clustered in group ii with texas isolates, it is most likely that the turkey sources were the same for the turkey in conclusion, the relationship between tcov genotypes and the geographic distribution of tcov presented in the present study provides crucial information for the monitoring and control of diseases associated with tcov infection in the united states. characterization of turkey coronavirus from turkey poults with acute enteritis detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in canada antigenic relationship of turkey coronavirus 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of polyprotein protein and genome organization of turkey coronavirus mega : molecular evolutionary genetics analysis version . simple methods for estimating the numbers of synonymous nucleotide substitutions acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells identification and characterization of a neutralizing-epitope-containing spike protein fragment in turkey coronavirus genotypic and phenotypic characterization of the california (cal ) variant of infectious bronchitis virus comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different arkansas delmarva poultry industry -derived infectious bronchitis virus vaccines typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene reverse genetics system for the avian coronavirus infectious bronchitis virus changes in nonstructural protein are associated with attenuation in avian coronavirus infectious bronchitis virus novel avian coronavirus and fulminating disease in guinea fowl identification of a novel coronavirus from a beluga whale by using a panviral microarray key: cord- -l cdllh authors: saraste, j.; marie, m. title: intermediate compartment: a sorting station between the endoplasmic reticulum and the golgi apparatus date: - - journal: encyclopedia of cell biology doi: . /b - - - - . - sha: doc_id: cord_uid: l cdllh the intermediate compartment (ic) is a pleiomorphic membrane system that mediates two-way trafficing in the early biosynthetic-secretory pathway of mammalian cells. the ic associates with the cytoskeleton, binds copi (coat protein i) coats and generates vesicular, tubular and saccular transport carriers. it recieves newly made proteins and lipids from the endoplasmic reticulum (er) and sorts them for transport to the golgi apparatus or recycling back to the er. although the ic appears to be functionally complex and resides at the crossroads of multiple transport routes, it is still disputed whether it represents a transient or stable structure. newly synthesized proteins and lipids leave the endoplasmic reticulum (er) at specialized transitional regions called er exit sites (eres) (jamieson and palade, ; sesso et al., ; bannykh et al., ; hammond and glick, ; tang et al., ) and enter the intermediate compartment (ic) that has been shown to operate as an obligatory a post-er sorting station in the early biosynthetic-secretory trafficking of mammalian cells. from the ic they are typically transported to the cisternal stacks of the golgi apparatus, prior to their delivery to the different organelles of the endomembrane system or secretion to the extracellular space. bidirectional er-golgi trafficking involves the sequential operation of membrane-bound coat protein ii (copii) and copi coats (aridor and balch, ; scales et al., ; stephens et al., ) . er-derived copii vesicles mediate forward (anterograde) transport, while ic-and golgi-derived copi vesicles are thought to act in the opposite (retrograde) direction (lee et al., ; rabouille and klumperman, ) . retrograde transport also involves copiindependent routes (kano et al., ) . despite the conservation of the transport machineries (such as the cop coats), the organization of the er-golgi interface varies in different eukaryotic cells. for example, in plants, certain yeasts, and the fruit fly drosophila melanogaster, the individual golgi stacks lie next to the widespread eres, establishing units for short range er-golgi communication. by contrast, in animal cells the golgi stacks are linked together into a continuous ribbon around the microtubule-organizing center (mtoc)/centrosome, whereas the er extends throughout the cytoplasm. hence, a large proportion of the eres reside at the cell periphery and er-golgi trafficking depends on the long distance movements of the ic elements along mt tracks (saraste and svensson, ; presley et al., ; scales et al., ; brandizzi and barlowe, ; day et al., ) . thus, it has been proposed that the ic represents a late evolutionary invention, which developed to meet the special sorting, transport and recycling requirements of the large-sized animal cells, but lacks lower eukaryotes. however, results showing that the ic constitutes an extensive membrane system that can be compared with the endosomal network in complexity, are questioning this view. electron microscopic (em) studies using a temperature-sensitive mutant of semliki forest virus (sfv ts- ) to synchronize the transport of viral membrane glycoproteins from er to the plasma membrane (pm) showed that when the cells are shifted from c to c the proteins exit the er, but accumulate in vacuoles/saccules (up to . mm in diameter), tubules, and vesicles in the cis-golgi region and more peripheral locations (saraste and kuismanen, ) . by light microscopy (lm) the proteins were localized at c to scattered globular structures in the cytoplasm, a pattern distinct from that of er or golgi (kuismanen and saraste, ) . the transport block was readily reversible, and the proteins entered the golgi stacks and reached the pm following the transfer of cells from c to c, showing that these structures are normal transport intermediates. studies employing a similar mutant of vesicular stomatitis virus (vsv tso ) showed that the ' c compartment' also acts as a way station during the transport of the vsv g glycoprotein (balch et al., ; bonatti et al., ; schweizer et al., ; duden et al., ; lotti et al., ) . like the sfv proteins, the g-protein was found to maintain its er-type high-mannose glycans at c, indicating lack of processing by golgi enzymes. furthermore, cell fractionation experiments showed that newly synthesized secretory proteins are arrested in a post-er location when pancreatic exocrine cells are kept at c (saraste et al., ) . live cell imaging of green fluorescent protein (gfp) equipped with an er targeting signal (ssgfp) and em studies of procollagen and growth hormone constructs verified that the transport of membrane and secretory proteins is similarly affected at - c (blum et al., ; volchuk et al., ; trucco et al., ) . in addition, the transport of virus glycoproteins and cholesterol appears to be blocked at the same site at this temperature (urbani and simoni, ; heino et al. ) . em of mouse hepatitis virus (mhv)-infected cells showed that the budding of progeny virus initially takes place at tubulovesicular membranes located in the transitional region between the er and the golgi apparatus (tooze et al., ) . the first step of o-glycosylation, the attachment of nacetyl-galactosamine (galnac) to the viral membrane (m) glycoprotein, was suggested to occur in this compartment, causing its reactivity with the lectin helix pomatia, which specifically binds galnac (tooze et al., ; krijnse-locker et al., ) . subsequent studies showed that the intracellular maturation of various coronaviruses occurs at the same budding site, which corresponds to the ic (klumperman et al., ) . the discovery of the lys-asp-glu-leu tetra-peptide (kdel)motif in abundant, lumenal er proteins lead to the proposal that the c/budding compartment is the post-er site from which these proteins are retrieved to the er (munro and pelham, ; warren, ; pelham, ). the first mammalian kdel-receptor, a multispanning membrane protein, was identified and shown to predominantly localize to the ic and cis-golgi (lewis and pelham, ; tang et al., ; griffiths et al., ; orci et al., ) . also, kdel proteins, such as the immunoglobulin binding protein (bip/grp ), glucoseregulated protein of kda (grp ), protein disulphide isomerase (pdi) and calreticulin (cr), are present in the ic connolly et al., ; zuber et al., ; ying et al., ; breuza et al., ) . following binding to their receptor, the proteins are retrieved to the er in copi vesicles (orci et al., ; martínez-menárguez et al., ; majoul et al., ) . attachment of the kdel-motif to lysosomal enzymes and the use of the c block suggested that the enzyme that initiates the formation of their lysosomal targeting signal (mannose- -phosphate) resides in the ic (pelham, ; lazzarino and gabel, ; dittmer and von figura, ) . the cytoplasmic tails of certain type i integral membrane proteins were shown to contain dilysine (kkxx)motifs, which by directly interacting with copi coats result in their retrieval from the ic/cis-golgi to the er (nilsson et al., ; jackson et al., jackson et al., , cosson and letourneur, ) . the first endogenous ic markers rat p and human er-golgi intermediate compartment (ergic)- ( % homology) were identified by the generation of antibodies against the c post-er fraction isolated from pancreatic acinar cells (saraste et al., ) and a golgi fraction derived from epithelial caco- cells (schweizer et al., ) , respectively. the cytoplasmic c-terminal tails of these non-glycosylated, hexameric, type- integral membrane proteins (schindler et al., ; lahtinen et al., lahtinen et al., , neve et al., ) contain a kkff-motif, which interacts with copii and copi coats and gives rise to their continuous cycling between er, ic, and cis-golgi (kappeler et al., ; tisdale et al., ) . at c the recycling of p /ergic- to the er is inhibited and the proteins pile up in the same pre-golgi structures that contain the sfv or vsv membrane proteins saraste and svensson, ; plutner et al., ) , verifying that the p /ergic- compartment (figures and ) is equivalent to the site where cargo is arrested at low temperature. ergic- and the related vip were shown to share homology with leguminous plant lectins (fiedler and simons, ) and to be identical with a mannose-binding protein mr of human myelomonocytic (hl ) cells (arar et al., ) . the n-terminal domain of p /ergic- binds mannose in a calcium-dependent manner (itin et al., ; velloso et al., ; ; hence the name lectin mannose-binding protein (lman ). it is the best characterized mammalian cargo receptor, facilitating the copii vesicle-mediated export of a subset of soluble glycoproteins from the er (nichols et al., ; appenzeller et al., ; hauri et al., ) . most well-characterized ic proteins are components of the transport machinery. many of them cycle at the er-golgi boundary and are also found in cis-golgi, supporting the view of the ic as a transient structure (see below). in fact, em studies showing the specific metal (osmium) staining of the ic and cis-golgi provided the first indication of their compositional similarity (friend and murray, ; rambourg et al., ) . however, the fungal compound brefeldin a (bfa) helps to discriminate between ic and golgi components. it releases copi coats, disassembles the golgi stacks and redistributes golgi components to the er, whereas cycling proteins (such as p /ergic- /lman and the kdelreceptor) are arrested in the drug-resistant ic elements (lippincott-schwartz et al., ; saraste and svensson, ; tang et al., b; füllekrug et al., ; ward et al., ; marie et al., ) . the bfa resistance of the ic has been utilized for its proteomics analysis (breuza et al., ) , and indicates its stability. like p /ergic- , the p family proteins contain motifs for copii and copi binding, resulting in their cycling between the er, ic, and cis-golgi (rojo et al., ; dominguez et al., ; blum et al., ; gommel et al., ; strating and martens, ). as abundant type- transmembrane proteins they participate in the biogenesis of copi vesicles (stamnes et al., ; majoul et al., ; beck et al., ) , but have also been implicated in tubulation and the formation of membrane domains (lavoie et al., ; emery et al., ) . studies in yeast first suggested their function as receptors for the exit of glycosylphosphatidylinositol (gpi)-anchored proteins from the er (schimmöller et al., ) . the copi coats are mainly recruited to the cytoplasmic surface of ic and cis-golgi membranes, but also associate with the lateral rims of the golgi stacks (duden et al., ; oprins et al., ; griffiths et al., ; orci et al., ; klumperman et al., ) . copi vesicle budding depends on the activation of small gtpases of the adp-ribosylation factor (arf) family (popoff et al., ) . the guanine nucleotide exchange factor (gef) of these arfs, gbf , is the master regulator of copi recruitment and the target of bfa (niu et al., ) . it localizes to the ic and cis-golgi and plays a key role in er-to-golgi trafficking (kawamoto et al., ; garcia-mata et al., ; zhao et al., ; szul et al., ) . gbf knock down or specific inhibition by golgicide a releases copi coats and arrests the vsv g-protein in the ic, indicating its involvement in anterograde ic-to-golgi transport (manolea et al., ; sáenz et al., ) . however, the prevailing view is that the main function of copi vesicles is to recycle membrane and selected proteins to the er. in pancreatic exocrine cells approximately % of the coats associate with the vtcs, suggesting that the ic represents the main point for recycling (martínez-menárguez et al., ) . the roles of two gtpases of the large rab family, rab and rab , in er-golgi trafficking are relatively well understood (plutner et al., ; tisdale et al., ) . both interact with multiple effectors suggesting that they coordinate successive transport steps (barnekow et al., ) . the association of rab with the cytoplasmic surface of ic and cis-golgi membranes, and its absence from the er, has been demonstrated by em saraste et al., ; satoh et al., ; marie et al., ; figure ). the two rab isoforms, rab a and rab b ( % homology), are recruited to ic membranes at eres, show similar localizations by lm (mochizuki et al., ; figure (a)), but seem to play distinct roles in tubular and vesicular (long and short range) trafficking within the ic. accordingly, live cell imaging and cell fractionation have shown that rab a mainly associates with ic tubules (sannerud et al., ; marie et al., ) , while rab b interacts with gbf and evidently modulates copi recruitment to globular ic domains monetta et al., ; see below) . also, rab a is specifically nu * * * * figure (a) immunofluorescence lm localization of p /ergic- in baby hamster kidney, (b) mouse myeloma, (c) rat neuroendocrine pc , and (d) human hela cells. the protein marks the ic elements accumulating in the perinuclear golgi region (asterisks), scattered throughout the cytoplasm, and located close to the pm (arrows). the reticular staining indicates the er pool of p /ergic- , which varies in the different cell types. nu, nucleus. bar: mm. phosphorylated at mitosis (bailly et al., ) , correlating with the cessation of tubular ic dynamics, whereas copi-mediated vesicular transport continues (marie et al., ) . rab a can functionally replace ypt , which coordinates two-way er-golgi trafficking in the yeast saccharomyces cerevisiae (haubruck et al. ; segev, ; kamena et al., ) . rab has been localized to ic and cis-golgi membranes (chavrier et al., ; lotti et al., ) and proposed to regulate the formation of retrograde copi vesicles that contain p /ergic- (tisdale, ) . it has also been implicated in the recruitment of the motor protein dynein to ic membranes and the association of ic elements with mts (tisdale et al., ; see below) . most of the known rab and rab effectors are cytoplasmically oriented, long coiled-coil proteins, which function in the tethering of vesicle and organelle membranes preceding their soluble nsf attachment protein receptor (snare)-mediated fusion (barnekow et al., ; sztul and lupashin, ). besides the ic/cis-golgi tethers gm and gmap- (rios et al., ) , rab has been shown to interact with medialand trans-golgi tethers, suggesting a more widespread function (short et al., ; sinka et al., ) . the rab effector p is functional already at the peripheral eres (alvarez et al., ) , while gm (bound to its membrane anchor grasp ) cycles between central ic and cis-golgi (marra et al., ) and regulates membrane tethering at a later transport step (alvarez et al., ; marra et al., ) . rab also interacts with the transmembrane tethers golgin- and giantin, which appear to act in copi vesicle trafficking at the lateral rims of the golgi stacks (diao et al., ; malsam et al., ; barnekow et al., ; munro, ) . the membrane fusion machinery (snare proteins) operating in er-golgi trafficking in the yeast saccharomyces cerevisiae has been well characterized (cai et al., ; barlowe and miller, ) , whereas the fusion events that take place in mammalian cells remain enigmatic. the determination of the exact fusion steps is complicated by the continuous cycling of the snares in copi vesicles (hay et al., ; martinez-menárguez et al., ) . in analogy to yeast, rab (ypt ) has been suggested to recruit p (uso p) to copii vesicles at eres (allan et al., ) , followed by the formation of a snare complex (sec b, membrin, bet and syntaxin ), which mediates either homotypic fusion of copii vesicles or their heterotypic fusion with the stationary ic (zhang et al., ; xu et al., ; see below) . another snare complex (syntaxin , gs , bet and ykt ) is involved in a later cis-golgi transport step (zhang and hong, ) . by em the ic elements can be readily distinquished from the er and golgi, but share structural similarity with endosomes. they reside close to peripheral and central eres as clusters of vesicles and tubules (vtcs; figure (f)) that frequently contain copi coats (balch et al., ; bannykh et al., ; martínez-menárguez et al., ) . however, they display considerable size heterogeneity (ying et al., ) and also include large saccules that are found within the membrane clusters, free in the cytoplasm, or at the cis-face of the golgi stacks (saraste and svensson, ; lahtinen et al., , connolly et al., stinchcombe et al., ; ladinsky et al., ; fan et al., ; figures (a)- (e)). these pleiomorphic saccules can accommodate large-sized cargo, such as procollagen or lumenal protein aggregates (volchuk et al., ; trucco et al., ; zuber et al., ) , and based on correlative microscopy (lm/em) correspond to many of the mobile carriers that are visible in living cells (mironov et al., ) . like endosomes, they extend narrow tubules and also bind copi coats, indicating that they represent sites for vesicle budding (saraste and kuismanen, ; volchuk et al., ; horstman et al., ; figures (c) and (d)). the hypertrophy of the saccular domains most likely gives rise to the pre-golgi vacuoles seen in cells kept at c (saraste and kuismanen, ; trucco et al., ) . lm shows the division of the ic into globular and tubular domains (presley et al., ) . the former contain anterograde cargo, cargo receptors and copi coats, most likely corresponding to individual vtcs or free saccules, while the latter are highlighted by rab a (sannerud et al., ) . the tubules extend from the globular domains ( figure ) . some of them contain recycling proteins, but lack anterograde cargo, indicating that they function in retrograde transport (palokangas et al., ; simpson et al., ) . however, under synchronized conditions cargo is also detected in the tubular domain, due to overload or the existence of different types of ic tubules ( figure ). for example, incubation of cells at - c inhibits the formation of ic tubules, but causes the expansion of the globular domain, while the shift of cells to c generates tubular networks containing both antero-and retrograde markers (blum et al., ; ben-takaya et al., ; simpson et al., ) . proliferation of the tubules is induced when copi function is compromised (szul et al., ; marie et al., ; ben-takaya et al., ; tomás et al., ; hamlin et al., ) , but also occurs under physiological conditions. in differentiating neuroendocrine pc cells the rab a-positive tubular ic domain expands and the tubules move from the cell body to the forming neurites accumulating in their growth cones (sannerud et al., ; figure ). an analogous pathway connects the ic with the leading edge of fibroblasts ( figure ) . live imaging of various fluorescent ic markers indicates that the tubules are highly dynamic, while the globular domains are typically more stationary. due to their differential localization within these domains, the constructs highlight different aspects of ic dynamics (see below). long distance er-to-golgi transport involves the movement of ic elements from the cell periphery to the central cis-golgi region, resulting in the division of the ic into spatially distinct early (eresadjacent) and late (cis-golgi-adjacent) domains (saraste and svensson, ; presley et al., ; scales et al., ; marra et al., ) . two types of anterogradely moving ic elements can be resolved by lm, narrow tubules and large pleiomorphic structures. some of the latter appear to represent saccular elements that transform into elongated structures ('thick tubules') (presley et al., ; marie et al., ). in addition, narrow tubules establish dynamic connections between the globular ic domains (ben-tekaya et al., ; sannerud et al., ) . the long distance movements of the ic elements depend on mts (murshid and presley, ; palmer et al., (b) and ). the plus-and minus-end directed motor proteins kinesin and dynein associate with the ic elements (lippincott-schwartz et al., ; roghi and allan, ; stauber et al., ) , explaining their bidirectional movements even along the same mt tracks (sannerud et al., ) . when the mts in mammalian cells are depolymerized by nocodazole the ic elements become immobile and accumulate close to eres (saraste and svensson, ; hammond and glick, ; ben-tekaya et al., ; sannerud et al., ) . although the central golgi ribbon breaks down, the formation of golgi ministacks in the vicinity of eres re-establishes er-golgi communication as a short range process (as in plants), explaining the ongoing golgi modification and secretion of proteins (saraste and svensson, ; cole et al., ; storrie et al., ) . the rho family gtpase cdc , which regulates actin dynamics, interacts with copi coats and affects dynein function, suggesting functional coupling between the actin filament system and mt-dependent motility of ic/cis-golgi carriers (luna et al., ; chen et al., ) . similarly, whamm, which promotes actin nucleation and interacts with mts, has been localized to the ic and implicated in the formation and transport of pre-golgi tubules (campellone et al., ) . based on imaging of fluorescent cargo, such as the vsv g-protein and procollagen, in living cells (presley et al., ; scales et al., ; stephens and pepperkok, ) , it has been proposed that the ic represents a collection of large, pleiomorphic transport complexes (tcs) (bannykh and balch, ; stephens and pepperkok, ) , which form via homotypic fusion of copii vesicles, or protrude directly from the er (mironov et al., ; xu and hay, ; yu et al., ) . thereafter, they move in a mt-and dynein-dependent (burkhardt et al., ) 'stop-and-go' fashion to the golgi region, where they either fuse with or transform into cis-golgi cisternae (figure (a) ). in other words, the ic elements are transient structures which are first formed de novo at eres and then consumed at cis-golgi. these mobile structures (speed of about mm/sec) corresponding to saccules or more complex, pleiomorphic elements (mironov et al., ) appear to consist of subdomains enriched in antero-or retrograde cargo, or copi (shima et al., ; stephens et al., ) . they have also been shown to contain other machinery proteins, such as p / , p , vip , membrin and rab a (blum et al., ; golgi nu figure immunofluorescence lm localization of rab in differentiating pc cells, illustrating the interconnected globular (arrowheads) and tubular (arrows) domains of the ic. two confocal sections of the same cell are shown. the ic has expanded in response to a h treatment with the nerve growth factor (ngf) and the tubules are found both in the cell body and the neurite-like extensions (right panel). nu, nucleus; golgi, perinuclear golgi region. bar: mm. time projection figure bidirectional movements of gfp-rab a-positive tubules in a living nrk cell. selected confocal images from a movie obtained by timelapse microscopy are shown. tubules extending from the same relatively stationary globular ic element (black arrowhead) pinch off and move either in the direction of the cell's leading edge (upper panels; yellow arrowheads), or toward the golgi indicated by an asterisk (lower panels; red arrowheads). the paths taken by the tubules are highlighted in the time projection on the right. adapted from sannerud, r., marie, m., nizak, c., et al., chao et al., ; dahm et al., ; sannerud et al., ; monetta et al., ; tomás et al., ) . another model of the ic (figure (b) ) is largely based on the imaging of gfp-ergic- dynamics in living cells (ben-tekaya et al., ) . it proposes that the ic consists of stationary, long-lived membrane clusters, located close to the eres, which communicate with the er and golgi via distinct transport carriers (appenzeller-herzog and hauri, ) . thus, in a two-step transport process cargo is first transfered from er to the ic via heterotypic fusion of copii vesicles. the ic then generates novel (possibly copi-coated) anterograde carriers (acs) that move along mts to the cis-golgi. the recycling of gfp-ergic- to the er evidently occurs mainly from the eres-associated ic, since it was not detected in the acs containing soluble, golgi-destined cargo. the identification of a motif in the neuronal gaba transporter, that seems to be required for its exit from the ic, supports the stable nature of the ic (farhan et al., ) . visualization of ic dynamics using gfp-rab a showed that the pleiomorphic transport carriers arriving along mts from the cell periphery do not move directly to cis-golgi, but are targeted to the mtoc/centrosome that is normally positioned next to the golgi ribbon. in cells displaying centrosome motility, for example, cells that are migrating or entering mitosis, the centrosome-targeted membranes can be resolved as a separate compartment, called the pericentrosomal ic (pcic), which is distinct from the cis-golgi-adjacent ic domain (marie et al., , mochizuki et al., ; figure (a)). live imaging further showed that the separated pcic and the golgi communicate via tubular and globular carriers. the pcic contains its own pool of copi coats, and mediates the bfainduced, copi-independent backflow of golgi enzymes to the er, suggesting that forward pcic-to-golgi transport depends on copi coats, and thus is blocked by bfa . both the peripheral ic elements and the pcic persist upon golgi disassembly by bfa and maintain their communication with via dynamic tubules . accordingly, the ic has been proposed to constitute a dynamic, but stable membrane network due to its anchoring next to the eres and the centrosome figure (c) ). this model is supported by studies of mitotic cells, showing that the ic persists despite golgi breakdown and the rearrangement of the mts, and maintains its spatial organization due to its ongoing association with the spindle mts and the centrosomes at the spindle poles (marie et al., ; figure (a) ). in the endocytic pathway, soluble proteins and particles bound to lysosomes accumulate in the lumen of vacuolar endosomes, while many membrane proteins are sorted into narrow tubules for recycling to the pm. the observed major the ic consists of stationary membrane clusters located close to eres. in this case er-to-golgi trafficking involves two distinct transport steps, since the ic recieves cargo from the er via heterotypic fusion of copii vesicles and forms special anterograde carriers (ac) that move along mts to cis-golgi. (c) the ic represents a stable interconnected network that is anchored both next to eres and the centrosome. bidirectional transport between these sites and between the central ic elements and the golgi stacks involves vesicular, tubular or saccular carriers. eres-ic communication via copii vesicles occurs as in model b. copi, copii and clathrin coats are shown in gray, orange, and blue, respectively, and the centrosome in red. concentration of soluble secretory proteins within the ic (oprins et al., ) could be explained by similar geometrybased sorting, namely, their exclusion from vesicles and tubules, which recycle lipids and membrane-bound proteins back to the er (martínez-menárguez et al., ) . membrane recycling is a major function of both the ic and endosomes in mammalian cells. due to the similarity of the tubular domain of the ic with the endocytic recycling compartment (erc), its 'mirror compartment' next to the centrosome, it has been designated as the biosynthetic recycling compartment (brc; saraste and goud, ; marie et al., ) . lumenal conditions: receptor-mediated retrieval of kdel proteins from the ic requires that it is discontinuous with the er and maintains special lumenal conditions (pelham, ) . the finding of low ph-dependent binding of kdel-ligands to their receptor in vitro suggested the existence of a ph gradient between the er and cis-golgi (wilson et al., ) . the phsensitive interactions of the low density lipoprotein (ldl)receptor-related protein (lrp) and procollagen with the chaperones rap and hsp , respectively (bu et al., ; satoh et al., ) , and partial co-localization of damp (a marker for acidic compartments) and p /ergic- in central ic elements (palokangas et al., ) , are in accordance with this idea. the ph of the er and cis-golgi has been estimated to be about . and . - . , respectively (paroutis et al., ; vavassori et al., ) . although the effect ph on the binding of mannose-containing cargo to p /ergic- /lman remains unclear, it appears that cargo release is caused by a drop in free calcium concentration between the er and ic lumen (appenzeller-herzog et al., ; bentley et al., ; . on the other hand, the depletion of lumenal calcium stores affects the morphology of the ic and the recycling of cargo receptors at the er-golgi boundary, and the ic has been reported to contain the calcium-atpase serca, as well as the calciumbinding proteins bip, grp , cr, and calnuc (ying et al., ; breuza et al., ; howe et al., ; bentley et al., ) , suggesting its role in intracellular calcium storage. role of copi coats: there are at least three subtypes of copi coats (beck et al., ) , and four arf gtpases that regulate their membrane binding (popoff et al., ) . three arfs appear to act at the er-golgi boundary and two of these associate with membranes in a bfa-resistant manner (volpicelli-daley et al., ; chun et al., ; duijsings et al., ; ben-tekaya et al., ; hamlin et al., ) , suggesting that different types of copi vesicles mediate two-way trafficking at the level of the ic. the role of copi in anterograde transport has been considered for some time (hosobuchi et al., ; pepperkok et al., ; peter et al., ; orci et al., ; malsam et al., ) . in addition, although both p /ergic- and kdel-receptor employ copi vesicles in their recycling, their transport itineraries differ (tang et al., a; marie et al., marie et al., , . in addition to acting in vesicle budding the copi coats may form membrane domains (presley et al., ) . golgi bypass: besides mediating bidirectional er-golgi trafficking, the ic has been suggested to be involved in golgiindependent pathway(s) (marie et al., ; saraste et al., ) . the route between the ic and the leading edge or growth cone of motile fibroblasts or neurons, respectively ( figures and ) , could participate in cholesterol and integrin trafficking (urbani and simoni, ; sannerud et al., ; wang et al., ) or correspond to the bfa-resistant er-ic-pm route that supports phagocytosis (becker et al., ; saraste and goud, ; see below) . direct pericentrosomal communication between the ic and the endosomal system seems to be used by the cystic fibrosis transmembrane conductance regulator (cftr) during its golgi-independent transport to the cell surface (yoo et al., ; marie et al., ; gee et al., ) . further, the presence of the ic in neuronal dendrites may provide a golgi-independent satellite pathway for local dendritic trafficking (krijnse-locker et al., ; sannerud et al., ; ehlers, ) . questioning the studies on coronavirus maturation, which suggested that o-glycosylation is initiated in the ic (see above), subsequent em studies showed that the galnactransferases are predominantly found in the golgi. however, the activation of cells (by epidermal growth factor) causes their incorporation into copi vesicles and relocation to the ic and er, which consequently become positive for the lectin helix pomatia (gill et al., ) . the cycling of cis-golgi proteins to the ic could be a constitutive event (lin et al., ; marra et al., ; jarvela and linstedt, ) . moreover, the construction of the sugar chains on proteoglycans has been suggested to begin in the ic (jönsson et al., ) . the kdel-containing molecular chaperones present in the ic (bip, pdi, and grp ; see above) could be cycling while still bound to their unfolded client proteins, opening for a post-er level of protein folding and quality control. the presence of quality control machinery in the ic and the finding that the er-associated degradation of certain proteins requires their er export is in accordance with this idea (zuber et al., ; anelli and sitia, ) . the pdi family member erp and p / ergic- cooperate in the ic/cis-golgi in sequential assembly of igm polymers (anelli and sitia, ) . unassembled igm or t-cell antigen receptor subunits bound to erp or bip, respectively, can be retrieved to the er by the kdel-receptor in a ph-dependent manner (yamamoto et al., ; vavassori et al., ) , similarly as the overexpressed, misfolded vsv g-protein (hammond and helenius, ) and mutant v vasopressin receptors (hermosilla et al., ) . additional proof for a post-er checkpoint is provided by studies showing the accumulation of the deletion mutant df of cftr (gilbert et al., ) , misfolded mhc class i proteins (hsu et al., ; raposo et al., ) , and proinsulin (zuber et al., ) in expanded ic elements. the level of p /ergic- mrna is up-regulated by the unfolded protein response (upr), which also requires yeast ypt function, supporting a link between this signaling pathway and er-golgi trafficking (nyfeler et al., ; tsvetanova et al., ) . protein kinases src, apkc, and scyl have been implicated in copi function at the level of the ic (tisdale and artalejo, ; hamlin et al., ) and pkc and its downstream effectors appear to control ic morphology (ben-tekaya et al., ; sugawara et al., ) . the activation of neuronal trk receptor tyrosine kinases in the ic initiates signaling via the mek pathway leading to golgi fragmentation (schecterson et al., ) . membranes enriched in ic markers (p /ergic- /lman , kdel-receptor, and sec b) play a role in the biogenesis of autophagosomes by representing the primary membrane source for the lipidation of lc , which triggers this process (ge et al., ) . moreover, ypt /rab is a key regulator of autophagy in yeast and mammals (lynch- day et al. ; winslow et al., ; zoppino et al., ; huang et al., ; lipatova et al., ) . a phosphatidylinositol -phosphatase (mtmr ) acting in vesicle transport and autophagy is regulated by rab b and localizes predominantly to the pcic (mochizuki et al., ) , further supporting the role of the ic in autophagy. supporting the existence of an unconventional pathway that connects the er with the pm-derived phagosome (gagnon et al., ) , the ic-enriched snare sec b/ers- (zhang et al., ) was shown to influence phagocytosis independently of its role in er-golgi trafficking (becker et al., ; hatsuzawa et al., ) . in dendritic cells the delivery of certain proteinssuch as the peptide transporter (tap), cr and tapasinfrom er to the phagosome is required for antigen cross-presentation. this pathway depends on the interaction between sec b and the pm snare syntaxin and involves efficient recruitment of the integral ic components sec b and p /ergic- to the phagosomes (cebrian et al., ) , in accordance with the idea that the ic provides an important membrane source for their formation. the presence of cr, tapasin, and functional tap in the ic/cis-golgi support this possibility (kleijmeer et al., ; howe et al., ; ghanem et al., ) . the ic plays a role in the golgi-independent trafficking of cftr (see above). mutations in p /ergic- /lman , or its partner mcfd (multiple coagulation factor deficiency protein ) result in an autosomal recessive bleeding disorder called combined deficiency of coagulation factors v and viii. they disrupt the lectin mannose-binding protein (lman )-mcfd receptor complex, thereby inhibiting the secretion of these factors and reducing their serum levels (nichols et al., ; zheng and zhang, ) . parkinson's disease-related cytotoxic protein, α-synuclein, has been suggested to interfere with er-golgi trafficking and to arrest autophagy by inhibiting rab function (cooper et al., ; winslow et al., ) . many phagocytosed bacterial pathogens, such as legionella, hijack the er-to-ic-to-phagosome pathway during their intracellular replication (isberg et al., ; arasaki et al., ; see above) . in addition to coronaviruses, the ic has been implicated in the replication of bunya-, entero-, flavi-, picorna, and vacciniaviruses (jäntti et al., ; risco et al., ; miller and krijnse-locker, ; hsu et al., ) . surprisingly, p / ergic- interacts in a lectin-independent manner with fusion proteins of a number of membrane viruses and participates in different stages of their life cycle (klaus et al., ) . rab recruitment of p into a cis-snare complex: programming budding copii vesicles for fusion er to golgi transport: requirement for p at a pre-golgi vtc stage copi recruitment is modulated by a rab b-dependent mechanism the p -interactive proteins gm and giantin participate in endoplasmic reticulum-golgi traffic protein quality control in the early secretory pathway the lectin ergic- is a cargo transport receptor for glycoproteins the er-golgi intermediate compartment (ergic): in search of its identity and function ph-induced conversion of the transport lectin ergic- triggers glycoprotein release ergic- , a membrane protein of the endoplasmic reticulum-golgi intermediate compartment, is identical to mr , an intracellular mannose-specific lectin of myelomonocytic cells the legionella pneumophila effector drra is sufficient to stimulate snare-dependent membrane fusion principles of selective transport: coat complexes hold the key phosphorylation of two small gtp-binding proteins of the rab family by p cdc atp-coupled transport of vesicular stomatitis virus g protein between the endoplasmic reticulum and golgi vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum membrane dynamics at the endoplasmic reticulum-golgi interface the organization of endoplasmic reticulum export complexes secretory protein biogenesis and traffic in the early secretory pathway rab proteins and their interaction partners the copi system: molecular mechanisms and function differential use of endoplasmic reticulum membrane for phagocytosis in j macrophages adp ribosylation factors and and group via phospholipase a regulate morphology and intraorganellar traffic in the endoplasmic reticulum-golgi intermediate compartment live imaging of bidirectional traffic from the ergic vesicular calcium regulates coat retention, fusogenicity, and size of pre-golgi intermediates intracellular localization and in vivo trafficking of p a and p lumenal targeted gfp, used as a marker of soluble cargo, visualises rapid ergic to golgi traffic by a tubulo-vesicular network palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum organization of the er-golgi interface for membrane traffic control proteomics of endoplasmic reticulum-golgi intermediate compartment (ergic) membranes from brefeldin a-treated hepg cells identifies ergic- , a new cycling protein that interacts with human erv binding and endocytosis of kda protein by mdbk cells overexpression of the dynamitin (p ) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution coats, tethers, rabs, and snares work together to mediate the intracellular destination of a transport vesicle whamm is an arp / complex activator that binds microtubules and functions in er to golgi transport sec b regulates phagosomal maturation and antigen crosspresentation by dendritic cells snare membrane trafficking dynamics in vivo localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments coatomer-bound cdc regulates dynein recruitment to copi vesicles characterization of class i and class ii adp-ribosylation factors in live cells: gdp-bound class ii arfs associate with the er-golgi intermediate compartment independently of gbf golgi dispersal during microtubule disruption: regeneration of golgi stacks at peripheral endoplasmic reticulum exit sites transport into and out of the golgi complex studied by transfecting cells with cdnas encoding horseradish peroxidase alpha-synuclein blocks er-golgi traffic and rab rescues neuron loss in parkinson's models coatomer (copi)-coated vesicles: role in intracellular transport and protein sorting quantitative er golgi transport kinetics and protein separation upon golgi exit revealed by vesicular integral membrane protein dynamics in live cells a three-stage model of golgi structure and function the coiled-coil membrane protein golgin- is a novel rab effector required for golgi ribbon formation phosphorylation of arylsulphatase a occurs through multiple interactions with the udp-n-acetylglucosamine- -phosphotransferase proximal and distal to its retrieval site by the kdel receptor gp l/emp /p protein family members of the cis-golgi network bind both cop i and ii coatomer beta-cop, a kd protein associated with non-clathrin-coated vesicles and the golgi complex, shows homology to beta-adaptin differential membrane association properties and regulation of class i and class ii arfs dendritic trafficking for neuronal growth and plasticity the transmembrane protein p forms highly specialized domains that regulate membrane composition and dynamics ultrastructural analysis of transitional endoplasmic reticulum and pre-golgi intermediates: a highway for cars and trucks signal-dependent export of gaba transporter from the er-golgi intermediate compartment is specified by a c-terminal motif a putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins osmium impregnation of the golgi apparatus characterization of brefeldin a-induced vesicular structures containing cycling proteins of the intermediate compartment/cis-golgi network endoplasmic reticulummediated phagocytosis is a mechanism of entry into macrophages adp-ribosylation factor/copidependent events at the endoplasmic reticulum-golgi interface are regulated by the guanine nucleotide exchange factor gbf the er-golgi intermediate compartment is a key membrane source for the lc lipidation step of autophagosome biogenesis rescue of Δf -cftr trafficking via a grasp-dependent unconventional secretion pathway the transporter associated with antigen processing (tap) is active in a post-er compartment delta f cftr localizes in the endoplasmic reticulum-golgi intermediate compartment in cystic fibrosis cells regulation of o-glycosylation through golgi-to-er relocation of initiation enzymes p and p , the major transmembrane proteins of copi-coated transport vesicles, form heterooligomeric complexes and cycle between the organelles of the early secretory pathway localization of the lys, asp, glu, leu tetrapeptide receptor to the golgi complex and the intermediate compartment in mammalian cells immunocytochemical localization of beta-cop to the er-golgi boundary and the tgn scyl scaffolds class ii arfs to specific subcomplexes of coatomer through the γ-cop appendage domain dynamics of transitional endoplasmic reticulum sites in vertebrate cells quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the er, intermediate compartment, and golgi apparatus sec b is a negative regulator of phagocytosis in macrophages the ras-related mouse ypt protein can functionally replace the ypt gene product in yeast ergic- and traffic in the secretory pathway localization, dynamics, and protein interactions reveal distinct roles for er and golgi snares dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface disease-causing v( ) vasopressin receptors are retained in different compartments of the early secretory pathway ultrastructural characterization of endoplasmic reticulum-golgi transport containers (egtc) sec is a gene required for er to golgi protein transport that encodes a subunit of a yeast coatomer calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of mhc class i molecules viral reorganization of the secretory pathway generates distinct organelles for rna replication a recycling pathway between the endoplasmic reticulum and the golgi apparatus for retention of unassembled mhc class i molecules antibacterial autophagy occurs at pi( )p-enriched domains of the endoplasmic reticulum and requires rab gtpase the legionella pneumophila replication vacuole: making a cosy niche inside host cells ergic- is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum retrieval of transmembrane proteins to the endoplasmic reticulum intracellular transport of secretory proteins in the pancreatic exocrine cell. i. role of the peripheral elements of the golgi complex immunocytochemical analysis of uukuniemi virus budding compartments: role of the intermediate compartment and the golgi stack in virus maturation irradiation-induced protein inactivation reveals golgi enzyme cycling to cell periphery initiation of the decorin glycosaminoglycan chain in the endoplasmic reticulum-golgi intermediate compartment ypt p is essential for retrograde golgi-er transport and for golgi maintenance in s. cerevisiae yip a regulates the copiindependent retrograde transport from the golgi complex to the er the recycling of ergic- in the early secretory pathway. ergic- carries a cytosolic endoplasmic reticulum-exit determinant interacting with copii gbf , a guanine nucleotide exchange factor for adp-ribosylation factors, is localized to the cis-golgi and involved in membrane association of the copi coat the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles location of mhc-encoded transporters in the endoplasmic reticulum and cis-golgi coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding the recycling pathway of protein ergic- and dynamics of the er-golgi intermediate compartment characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step the organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons low temperature-induced transport blocks as tools to manipulate membrane traffic golgi structure in three dimensions: functional insights from the normal rat kidney cell characterization of a kda cis-golgi protein in pancreatic exocrine cells molecular cloning and expression of a -kda cis-golgi and intermediate compartment protein roles for alpha( )p and copi in endoplasmic reticulum cargo exit site formation biosynthesis of the mannose- -phosphate recognition marker in transport-impaired mouse lymphoma cells. demonstration of a two-step phosphorylation bi-directional protein transport between the er and golgi a human homologue of the yeast hdel receptor er/golgi intermediates acquire golgi enzymes by brefeldin a-sensitive retrograde transport in vitro regulation of selective autophagy onset by a ypt/rab gtpase module kinesin is the motor for microtubule-mediated golgi-to-er membrane traffic microtubuledependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway immunocytochemical analysis of the transfer of vesicular stomatitis virus g glycoprotein from the intermediate compartment to the golgi complex regulation of protein transport from the golgi complex to the endoplasmic reticulum by cdc and n-wasp trs directs a ypt gef, trappiii, to the phagophore to promote autophagy kdel-cargo regulates interactions between proteins involved in copi vesicle traffic: measurements in living cells using fret golgin tethers define subpopulations of copi vesicles distinct functions for arf nucleotide exchange factors at the golgi complex: gbf and bigs are required for assembly and maintenance of the golgi stack and tgn, respectively division of the intermediate compartment at the onset of mitosis provides a mechanism for golgi inheritance the function of the intermediate compartment in pre-golgi trafficking involves its stable connection with the centrosome take the 'a' train: on fast tracks to the cell surface the gm and grasp golgi proteins cycle through and define a subdomain of the intermediate compartment the biogenesis of the golgi ribbon: the roles of membrane input from the er and of gm vesicular tubular clusters between the er and golgi mediate concentration of soluble secretory proteins by exclusion from copi-coated vesicles peri-golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-golgi transport modification of intracellular membrane structures for virus replication er-to-golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized er exit domains phosphatidylinositol -phosphatase myotubularin-related protein (mtmr ) is regulated by small gtpase rab b in the early secretory and autophagic pathways rab b interacts with gbf and modulates both arf dynamics and copi association the golgin coiled-coil proteins of the golgi apparatus a c-terminal signal prevents secretion of luminal er proteins er-to-golgi transport and cytoskeletal interactions in animal cells oligomerization and interacellular localization of the glycoprotein receptor ergic- is independent of disulfide bonds mutations in the er-golgi intermediate compartment protein ergic- cause combined deficiency of coagulation factors v and viii short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum dynamics of gbf , a brefeldin a-sensitive arf exchange factor at the golgi the cargo receptor ergic- is a target of the unfolded protein response beta-cop localizes mainly to the cis-golgi side in exocrine pancreas the er-to-golgi interface is the major concentration site of secretory proteins in the exocrine pancreatic cell bidirectional transport by distinct populations of copi-coated vesicles the role of microtubules in transport between the endoplasmic reticulum and golgi apparatus in mammalian cells retrograde transport from the pre-golgi intermediate compartment and the golgi complex is affected by the vacuolar h þ -atpase inhibitor bafilomycin a the ph of the secretory pathway: measurement, determinants, and regulation evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment control of protein exit from the endoplasmic reticulum beta-cop is essential for biosynthetic membrane transport from the endoplasmic reticulum to the golgi complex in vivo β-cop is essential for transport of protein from the endoplasmic reticulum to the golgi in vitro rab b regulates vesicular transport between the endoplasmic reticulum and successive golgi compartments morphological analysis of protein transport from the er to golgi membranes in digitonin-permeabilized cells: role of the p -containing compartment several adp-ribosylation factor (arf) isoforms support copi vesicle formation dissection of copi and arf dynamics in vivo and role in golgi membrane transport er-to-golgi transport visualized in living cells golgi membrane dynamics the maturing role of copi vesicles in intra-golgi transport three-dimensional structure of the osmium-impregnated golgi apparatus as seen in the high voltage electron microscope misfolded major histocompatibility complex class i molecules accumulate in an expanded er-golgi intermediate compartment gmap- recruits γ-tubulin complexes to cis-golgi membranes and is required for golgi ribbon formation endoplasmic reticulum-golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly dynamic association of cytoplasmic dynein heavy chain a with the golgi apparatus and intermediate compartment involvement of the transmembrane protein p in biosynthetic protein transport golgicide a reveals essential roles for gbf in golgi assembly and function rab defines a novel pathway connecting the pre-golgi intermediate compartment with the cell periphery emerging new roles of the pre-golgi intermediate compartment in biosynthetic-secretory trafficking functional symmetry of endomembranes pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the golgi complex localization of the small gtp-binding protein rab to early compartments of the secretory pathway temperature-sensitive steps in the transport of secretory proteins through the golgi complex in exocrine pancreatic cells antibodies to rat pancreas golgi subfractions: identification of a kda cis-golgi protein distribution of the intermediate elements operating in er to golgi transport golgin- is a rab binding partner involved in golgi structure intracellular interaction of collagen-specific stress protein hsp with newly synthesized procollagen visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi trk activation in the secretory pathway promotes golgi fragmentation the absence of emp p, a component of er-derived copii-coated vesicles, causes a defect in transport of selected proteins to the golgi ergic- , a membrane protein of the er-golgi intermediate compartment, carries an er retention motif identification, by a monoclonal antibody, of a -kd protein associated with a tubulo-vesicular compartment at the cis-side of the golgi apparatus identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to golgi apparatus ypt and rab gtpases: insight into functions through novel interactions a three-dimensional reconstruction study of the rough er-golgi interface in serial thin sections of the pancreatic acinar cell of the rat segregation of copi-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-golgi transport complexes a grasp -rab effector complex linking golgi structure to membrane traffic biogenesis of tubular er-to-golgi transport intermediates golgi coiled-coil proteins contain multiple binding sites for rab family g proteins an integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding a role for kinesin- in copi-dependent recycling between the er and the golgi complex copi-coated er-to-golgi transport complexes segregate from copii in close proximity to er exit sites illuminating the secretory pathway: when do we need vesicles imaging of procollagen transport reveals copidependent cargo sorting during er-to-golgi transport in mammalian cells anterograde and retrograde traffic between the rough endoplasmic reticulum and the golgi complex recycling of golgi-resident glycosyltransferases through the er reveals a novel pathway and provides an explanation for nocodazole-induced golgi scattering the p family and selective transport processes at the er-golgi interface pkcδ and ε regulate the morphological integrity of the er-golgi intermediate compartment (ergic) but not the anterograde and retrograde transports via the golgi apparatus role of vesicle tethering factors in er-golgi membrane traffic dissecting the role of the arf guanine nucleotide exchange factor gbf in golgi biogenesis and protein trafficking segregation of ergic- and the mammalian kdel-receptor upon exit from the c compartment differential response of resident proteins and cycling proteins of the golgi to brefeldin a copii and exit from the endoplasmic reticulum molecular cloning, characterization, subcellular localization and dynamics of p , the mammalian kdel receptor a rab mutant with impaired gtpase activity stimulates vesicle formation from pre-golgi intermediates src-dependent atypical protein kinase c iota/lambda (apkciota/lambda) tyrosine phosphorylation is required for apkciota/lambda association with rab and glyceraldehyde- -phosphate dehydrogenase on pre-golgi intermediates rab utilizes glyceraldehyde- -phosphate dehydrogenase and protein kinase c{iota} to associate with microtubules and to recruit dynein gtpbinding mutants of rab and rab are potent inhibitors of vesicular transport from the endoplasmic reticulum to the golgi complex p / binds copi and is required for selective transport through the early secretory pathway regulation of er-golgi intermediate compartment tubulation and mobility by copi coats, motor proteins and microtubules replication of coronavirus mhv-a in saccells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-a secretory traffic triggers the formation of tubular continuities across golgi subcompartments the yeast rab gtpase ypt modulates unfolded protein response dynamics by regulating the stability of hac rna cholesterol and vesicular stomatitis virus g protein take separate routes from the endoplasmic reticulum to the plasma membrane a ph-regulated quality control cycle for surveillance of secretory protein assembly the crystal structure of the carbohydrate-recognition domain of the glycoprotein sorting receptor p /ergic- reveals an unpredicted metal-binding site and conformational changes associated with calcium ion binding megavesicles implicated in the rapid transport of intracisternal aggregates across the golgi stack isoform-selective effects of the depletion of adp-ribosylation factors - on membrane traffic regulation of integrin β recycling to lipid rafts by rab a to promote cell migration maintenance of golgi structure and function depends on the integrity of er export signals and salvage sequences ph-dependent binding of kdel to its receptor in vitro α-synuclein impairs macroautophagy: implications for parkinson's disease reconstitution of copii vesicle fusion to generate a pre-golgi intermediate compartment subunit structure of a mammalian er/golgi snare complex the kdel receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum the p -positive pre-golgi intermediates consist of distinct subpopulations of particles that show differential binding of copi and copii coats and contain vacuolar h þ -atpase colocalization of ca þ -atpase and grp with p and the effects of thapsigargin on protein recycling suggest the participation of the pre-golgi intermediate compartment in intracellular ca þ -storage non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway mbet p is required for homotypic copii vesicle tethering in mammalian cells ykt forms a snare complex with syntaxin , gs , and bet and participates in a late stage in endoplasmic reticulum-golgi transport the mammalian protein (rbet ) homologous to yeast bet p is primarily associated with the pre-golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the golgi apparatus morphological and functional association of sec b/ers- with the pre-golgi intermediate compartment gbf , a cis-golgi and vtcs-localized arf-gef, is implicated in er-to-golgi protein traffic structural characterization of carbohydrate binding by lman protein provides new insight into the endoplasmic reticulum export of factors v (fv) and viii (fviii) combined deficiency of coagulation factors v and viii: an update autophagosome formation depends on the small gtpase rab and functional er exit sites immunolocalization of udp-glucose: glycoprotein glucosyltransferase indicates involvement of pre-golgi intermediates in protein quality control misfolded proinsulin accumulates in expanded pre-golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of akita mice further reading getting into the golgi molecular motors and the golgi complex: staying put and moving through the kdel-receptor: new functions for an old protein protein sorting receptors in the early secretory pathway the golgi apparatus: years of progress and controversy architecture of the mammalian golgi secretory protein trafficking and organelle dynamics in living cells copii and the regulation of protein sorting in mammals key: cord- - snl jfx authors: elbadawi, lina i.; haupt, thomas; reisdorf, erik; danz, tonya; davis, jeffrey p. title: use and interpretation of a rapid respiratory syncytial virus antigen detection test among infants hospitalized in a neonatal intensive care unit — wisconsin, march date: - - journal: mmwr morb mortal wkly rep doi: nan sha: doc_id: cord_uid: snl jfx on march , , the wisconsin division of public health was notified of a possible respiratory syncytial virus (rsv) infection outbreak among infants hospitalized in a neonatal intensive care unit (nicu). on march , the index patient (neonate a), aged days, had feeding intolerance and apnea. a nasopharyngeal swab specimen collected from neonate a was tested using a single-manufacturer rapid rsv antigen detection test (rradt) at the hospital laboratory; the result was positive. the following day, because of concern about the possibility of more widespread rsv infection, rradt was used to test nasopharyngeal swab specimens from neonate b, aged month, who had resided in a different hospital room in the nicu and had developed an increased oxygen requirement, apnea, and poor feeding that day, as well as from two asymptomatic neonates who were hospitalized in the same room with neonate a; all three were positive. later that day, nasopharyngeal swab specimens from the remaining asymptomatic nicu patients were tested using the same rradt; seven tests were positive, making a total of positives. all rradts were performed at the hospital laboratory. on march , , the wisconsin division of public health was notified of a possible respiratory syncytial virus (rsv) infection outbreak among infants hospitalized in a neonatal intensive care unit (nicu). on march , the index patient (neonate a), aged days, had feeding intolerance and apnea. a nasopharyngeal swab specimen collected from neonate a was tested using a single-manufacturer rapid rsv antigen detection test (rradt) at the hospital laboratory; the result was positive. the following day, because of concern about the possibility of more widespread rsv infection, rradt was used to test nasopharyngeal swab specimens from neonate b, aged month, who had resided in a different hospital room in the nicu and had developed an increased oxygen requirement, apnea, and poor feeding that day, as well as from two asymptomatic neonates who were hospitalized in the same room with neonate a; all three were positive. later that day, nasopharyngeal swab specimens from the remaining asymptomatic nicu patients were tested using the same rradt; seven tests were positive, making a total of positives. all rradts were performed at the hospital laboratory. on march , the same nasopharyngeal specimens were sent to the wisconsin state laboratory of hygiene for confirmatory testing using a multiplex respiratory virus real-time polymerase chain reaction (pcr) panel (esensor, genmark diagnostics, inc.) that targets viruses, including rsv subgroups a and b. sixteen nasopharyngeal specimens were negative for all virus targets; three were positive for rsv-a, including the specimens from neonates a and b and from one asymptomatic neonate whose rradt result was positive. a nasopharyngeal swab specimen from one other asymptomatic neonate with a positive rradt tested positive for human coronavirus e by pcr. all nasopharyngeal specimen pcr results were confirmed at cdc. therefore, among specimens that were rsv-negative by pcr, eight were positive by rradt, for a false-positivity rate of %. the sensitivity (percentage of persons with the disease who have a positive test) and specificity (percentage of persons without the disease who have a negative test) of rradts for detecting rsv are characteristics of the test. however, test result interpretation depends on the positive predictive value (ppv) (i.e., the proportion of test-positive patients who have rsv infection), which is influenced by rsv infection prevalence. studies among infants and young children with symptoms consistent with respiratory illness during peak rsv season (late january through march) demonstrated a sensitivity, specificity, and ppv for rradt of %- %, %- %, and %- %, respectively ( - ) . however, the reported ppv of a test might not be applicable if the patient being tested is dissimilar to the population evaluated to determine the ppv; in this case, the ppv of a test used on symptomatic infants might not necessarily apply to asymptomatic infants, even if both are tested during peak rsv season. other possible contributors to the high rate of false positives include contaminated viral transport media or applied topical preparations, such as emollients to the neonates' nares. aliquots from all infant nasopharyngeal specimens were provided to the rradt manufacturer without personal identifying information for validation and verification; testing of these specimens was conducted by the manufacturer, and the hospital laboratory rradt results were replicated. at the conclusion of the investigation, wisconsin division of public health recommended to the facility that the rradt be used only for testing symptomatic neonates in accordance with manufacturer guidelines. in addition, the division recommended that any positive rradt results be confirmed by real-time pcr that would detect rsv a and b. diagnostic tests indicated for use in patients with a characteristic clinical illness might produce misleading results if used for another purpose, such as for screening of asymptomatic patients. wisconsin department of health services. respiratory virus surveillance report for the week ending head-to-head comparison of the diagnostic accuracies of bd veritor™ system rsv and quidel® sofia® rsv fia systems for respiratory syncytial virus (rsv) diagnosis evaluation of respiratory syncytial virus (rsv) direct antigen detection assays for use in point-ofcare testing key: cord- -s ypy hf authors: wang, dang; fan, jinxiu; fang, liurong; luo, rui; ouyang, haiping; ouyang, chao; zhang, huan; chen, huanchun; li, kui; xiao, shaobo title: the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-κb signaling by means of its deubiquitinating activity date: - - journal: mol immunol doi: . /j.molimm. . . sha: doc_id: cord_uid: s ypy hf since its emergence in the late s, porcine reproductive and respiratory syndrome (prrs) has been devastating the swine industry worldwide. the causative agent is an arterivirus, referred to as prrs virus (prrsv). the pathogenic mechanisms of prrs are poorly understood, but are believed to correlate with the ability of prrsv to inhibit immune responses of the host. however, precisely how the virus is capable of doing so remains obscure. in this study, we showed that prrsv infection led to reduced ubiquitination of cellular proteins. screening all of the nonstructural proteins (nsps) encoded by prrsv revealed that, apart from the nsp which contains the deubiqintinating (dub) ovarian tumor (otu) domain, nsp , which encodes a unique and conserved endoribonuclease (nendou) throughout the nidovirus order, also possesses dub activity. in vivo assay demonstrated that nsp specifically removed lysine (k )-linked polyubiquitin chains and the conserved sites c , h , d , k , and y were critical for its dub activity. remarkably, dub activity was responsible for the capacity of nsp to inhibit nuclear factor κb (nf-κb) activation. mutations abrogating the dub activity of nsp toward k -linked polyubiquitin chains of iκbα nullified the suppressive effect on nf-κb. our data add nsp to the list of dubs encoded by prrsv and uncover a novel mechanism by which prrsv cripples host innate immune responses. protein ubiquitination is a reversible process that plays a vital role in nearly every aspect of cellular physiology, including protein degradation, protein trafficking, transcription, cell-cycle control, and cell signaling; (liu et al., ; pickart, ) . not surprisingly, ubiquitination is targeted for manipulation by a wide range of microbial pathogens (randow and lehner, ). in particular, many viruses have evolved elaborate strategies to inhibit or redirect the ubiquitination machinery of the host for their survival (viswanathan et al., ) . for example, human immunodeficiency virus (hiv- ) prevents antiviral interferon response via vpr-and vif-directed, ubiquitin-mediated proteosomal degradation of interferon regulatory factor (irf- ) (okumura et al., ) ; the papain-like protease (plpro) domains of many coronaviruses, such as severe acute respiratory syndrome (sars) coronavirus (sars-cov), human coronavirus nl (hcov-nl ), and mouse hepatitis virus a (mhv-a ), have deubiquitinating (dub) activity that blocks type i interferons (ifns) induction (barretto et al., ; chen et al., b; clementz et al., ; devaraj et al., ; frieman et al., ; lindner et al., ; zheng et al., ) ; the leader proteinase (l pro ) of foot-and-mouth virus (fmdv) acts as a deubiquitinase that cleaves ubiquitin chains from retinoic acid-inducible gene i (rig-i), tank-binding kinase (tbk ), tnf receptor associated factor (traf ), and traf , thereby inhibiting the activation of type i ifn signaling ; the n-terminal protease (npro) of bovine viral diarrhea virus interacts with irf- and promotes its polyubiquitination and subsequent degradation through the proteasome (chen et al., a) ; the latency associated protein orf of murid herpesvirus- (muhv- ) associates with the host ubiquitin-ligase complex to promote poly-ubiquitination and subsequent proteasomal degradation of p /rela, which inhibits the activity of nuclear factor b (nf-b) to facilitate the establishment of muhv- latency (rodrigues et al., ) . collectively, these previous findings reveal that hijacking of the cellular ubiquitin system is an emerging, central theme around virus replication. in this regard, studying uniquitination events in virus-infected cells holds great promise to unravel important modulators of the intricate relationship between host and pathogen. not less important, understanding the mechanisms by which viral products interact with the ubiquitin system provides novel insights into viral pathogenesis and informs approaches to antiviral drug development. porcine reproductive and respiratory syndrome (prrs) is a relatively new viral infectious disease of the swine (rossow, ) . it is characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs. since it was first reported in the united states in and in europe in , prrs has devastated the swine industry worldwide, causing tremendous economic losses. as such, prrs is now considered to be one of the most important diseases in countries with intensive swine industries (meulenberg, ; murtaugh et al., ; neumann et al., ) . the causative agent, prrs virus (prrsv), is a single-stranded positive-sense rna virus classified within the order nidovirales, family arteriviridae, which also includes equine arteritis virus (eav), murine lactate dehydrogenase-elevating virus (ldv), and simian hemorrhagic fever virus (shfv) (cavanagh, ) . at least nine open reading frames (orfs) have been identified in the prrsv genome. orf a and orf b, which are situated in the -proximal three quarters of the genome, encode the viral non-structural proteins (nsps): nsp ␣, nsp ␤, and nsp to nsp . orf a, orf b, and orf to orf are located at the end of the genome and encode the viral structural proteins gp , e, gp , gp , gp , m, and n, respectively. because the nsps constitute ∼ % of the prrsv genome coding capacity, much attention has been garnered in studying the functions and immuno-modulatory roles of prrsv nsps (dokland, ) . to date, several nsps, including nsp ␣/␤, nsp , nsp , and nsp have been reported to have inhibitory effect on activation of the ifn-␤ promoter (beura et al., ; kim et al., ; li et al., ; shi et al., ; song et al., ) . more recently, the cysteine protease domain (cp) of prrsv nsp was identified as a member of the ovarian tumor domain (otu) family of deubiqintinating (dub) enzymes. it was shown that the otu domain of prrsv nsp antagonizes the induction of type i ifns by interfering with the nf-b pathway . however, it remains unclear whether other nsps encoded by prrsv have dub activity. in the present study, we screened the nsps of prrsv and found both nsp and nsp possess dub activity. interestingly, nsp specifically targeted lysine (k )-linked but not k -linked ubiquitination chains for cleavage. this attenuated ligand-induced degradation of inhibitor of nf-b alpha (ib␣), thereby inhibiting the activation of nf-b. our data identify nsp as a second dub encoded by prrsv and describe a novel mechanism by which prrsv antagonizes innate immunity of the host. marc- cells, pk- -cd cells and hek cells were maintained in dulbecco's modified eagle media (dmem, invitrogen) supplemented with % heated-inactivated fetal calf serum (fbs), u/ml penicillin, g/ml streptomycin sulfate at • c in a humidified % co incubator. pk- -cd cells, a porcine kidney cell line stably expressing the receptor cd of prrsv, were gifts of dr. en-min zhou (northwest a&f university, china) (wang et al., ) . the wuh strain of prrsv (li et al., ) , which was isolated from the brains of pigs that contracted the "high fever" syndrome in china at the end of , was used in this study. prrsv was propagated in marc- cells or pk- -cd cells, and the supernatants of infected cells were clarified and stored at − • c in aliquots. poly(i:c) (sigma-aldrich) and tumor necrosis factor ␣ (tnf-␣) (sigma-aldrich) were also used to stimulate cells. full-length ha-tagged ubiquitin (ub) plasmid (ha-ub) and ha-ub mutants in which all but one lys residue (ha-k -ub or ha-k -ub) were substituted with arg were gifts of dr. t. ohta (st. marianna university school of medicine, japan) (nishikawa et al., ) . pcdna . -flag-ub was previously described (clementz et al., ) . the luciferase report plasmid pnf-b-luc was purchased from stratagene. the hemagglutinin (ha) or v epitope tag was amplified by pcr and cloned into the pcaggs-mcs vector (niwa et al., ) to generate pcaggs-ha or pcaggs-v plasmid with n-terminally ha or v tag, respectively. for construction of the mammalian expression plasmids encoding various nonstructural proteins of prrsv, cdna fragments encoding full-length nsp ␣, nsp ␤, nsp - , nsp - of prrsv strain wuh (genbank accession no. hm ) were amplified by pcr and inserted into the pcaggs-ha or pcaggs-v plasmid. c a, c a, h a, h a, k a, d a, d a, and y a mutants of nsp were generated by overlap extension pcr in the pcaggs-v -nsp backbone. detailed sequences of the mutagenesis primers are available upon request. all mutants were validated by dna sequencing. the mammalian expression plasmid for ib␣ was constructed by pcr amplifying the cdna of ib␣ (genbank accession no. nm ) from hek cells, followed by cloning into the pcmv-tag b vector (stratagene). the monoclonal antibody (mab) a f used for detection of prrsv nsp was produced from hybridoma cells derived from sp / myeloma cells and spleen cells of balb/c mice immunized with recombinant nsp protein of prrsv strain wuh . the a f mab specifically recognized prrsv nsp in western blot and indirect immunofluorescence assays (unpublished data). the anti-beta-actin (biotechnology, china), anti-flag (macgene, china), anti-ha (mbl, japan), anti-v (mbl, japan), and anti-ubiquitin (santa cruz, ca) antibodies were used to detect the indicated proteins. horseradish peroxidase-conjugated anti-mouse or antirabbit igg antibodies were obtained from beyotime institute of biotechnology (jiangsu, china). marc- cells and pk- -cd cells were infected with prrsv strain wuh at different mois or sham-infected with dmem. at different time points post infection, cell lysates were collected and subjected to western blot analysis with an anti-ubiquitin antibody to measure the abundance of ubiquitinated proteins in the cell. hek cells grown in -well plates were co-transfected with . g/well of luciferase reporter plasmid pnf-b-luc along with . g/well of prl-tk plasmid (promega, for normalization of transfection efficiency) and various viral nsp-encoding plasmids or an empty control plasmid. in some experiments, cells were further transfected with poly(i:c) ( . g/well) at h after the initial co-transfection. cells were harvested h later and firefly luciferase and renilla luciferase activities were determined using the dual-luciferase reporter assay system (promega) according to the manufacturer's protocol. data represent relative firefly luciferase activity normalized to renilla luciferase activity and are representative of three independently conducted experiments. data are presented as means ± standard deviation (sd). a p-value of less than . was considered highly statistically significant. to determine the effect of nsp on expression of il- , il- , and ccl , hek cells in -well plates were transfected with g of empty vector or a plasmid encoding v -nsp . h later, cells were mock-transfected or transfected with g of poly(i:c) for h. total rna was extracted from the cells using trizol reagent (invitrogen, u.s.a.). one microgram of this total rna was reverse transcribed to cdna using amv reverse transcriptase (toyobo, japen), which ( l of l cdna) was subsequently used in a sybr green pcr assay (applied biosystems, u.s.a.). the abundance of individual mrna transcript in each sample was assayed three times and normalized to that of porcine glyceraldehyde- phosphate dehydrogenase (gapdh) mrna (as an internal control). the primers were designed by primer express software v. . (applied biosystems, u.s.a.). detailed sequences of the primers used are available upon request. hek cells cultured in -mm dishes were cotransfected with . g of ha-ub, ha-k -ub, or ha-k -ub plus appropriate amounts of vector encoding wild-type prrsv nsp or the indicated mutant using lipofectamine (invitrogen). where applicable, the empty vector was supplemented to keep the total amount of dna transfected constant. at h post transfection, cells were harvested by adding l lysis buffer a (lba) ( mm tris-hcl [ph . ], % sodium dodecyl sulfate, % dl-dithiothreitol, and % glycerol) containing mm n-ethylmaleimide (nem) (sigma) and mm iodoacetamine (sigma). cell lysates were then analyzed for expression of ubiquitin-conjugated proteins by western blot with an anti-ha antibody ( : , ) (mbl, japan). to verify the expression levels of nsp and the mutants, an anti-v antibody (mbl, japan) was used to detect the v -tagged proteins. beta-actin was immunoblotted with anti-beta-actin mab (beyotime, china) to demonstrate equal protein sample loading. transient transfection of hek cells with the indicated plasmids was performed routinely using lipofectamine as per the manufacturer's instructions (invitrogen). transfected hek cells from each -mm dish were lysed by adding l ml of lysis buffer ( mm tris-hcl [ph . ], mm nacl, % triton x- , nm phenylmethylsulfonyl fluoride [pmsf]), and the protein concentration was measured and adjusted. for immunoprecipitation, g of total cell lysates were incubated with . g of the indicated antibody and l of protein + g-agarose (beyotime, china) overnight at • c. the agarose beads were then washed three times with ml of lysis buffer. the immunoprecipitates were subjected to % sds-page and subsequent immunoblot analysis using the indicated antibodies. to investigate the levels of ubiquitinated cellular proteins during prrsv infection, we infected marc- cells (a clone of the african green monkey kidney cell line ma- ) with prrsv strain wuh at an multiplicity of infection (moi) of . . cell lysates were collected at different time points post infection and subjected to western blot analysis with anti-ubiquitin. as shown in fig. a , while the level of ubiquitinated cellular proteins was steady in mock-infected cells (lanes - ), it varied dynamically during the course of prrsv infection. a decrease in ubiquitination was first observed at h post infection (h.p.i.) (compare lanes vs. ), reaching a plateau phase between and h.p.i. (lanes - vs. ). although there was a slight rebound at h.p.i. (lane ), the level of ubiquitinated cellular proteins remained substantially lower than that of mock-infected cells (lane ). when marc- cells were infected with prrsv at increasing mois, the levels of ubiquitinated cellular proteins were reduced in a dose-dependent manner (fig. b) . importantly, infection with uv-inactivated prrsv, which is capable of receptor binding and internalization but not viral gene synthesis, did not alter the cellular level of ubiquitinated protein conjugates (fig. b) . similar results were observed in prrsvinfected pk- -cd cells (fig. c) . these data demonstrate that the level of ubiquitinated cellular proteins is reduced in prrsvinfected cells and that this is a result of active viral replication. of note, the latter notion was also supported by the western blot data that measured expression of prrsv nsp protein at different time points post infection (fig. a) . because the decreased cellular protein ubiquitination depended on prrsv replication and it has been shown that prrsv nsp has dub activity, we sought to determine whether other nsp(s) also contributes to protein deubiquitination. to this end, all of the nsps of prrsv strain wuh , except nsp (which encodes a very short peptide of amino acids), were cloned into a mammalian expression vector pcaggs-ha, such that they would be expressed as n-terminal ha-tagged fusion proteins. these nsp constructs were transiently transfected into cells and their expression were verified by western blot using an anti-ha antibody (data not shown). subsequently, each of these nsp constructs was co-transfected with a plasmid encoding flag-tagged ubiquitin (pcdna . -flag-ub) into hek cells (human embryonic kidney epithelial cells) and western blot was performed to detect the ubiquitin-conjugated proteins. as a negative control, the empty pcaggs-ha plasmid was used in place of the nsp-encoding vectors. as shown in fig. a , of the tested nsps, ectopic expression of nsp or nsp resulted in markedly reduced levels of ubiquitinconjugated proteins. notably, the effect of nsp appeared to be stronger than that of nsp (compare lanes vs. ). since, the dub activity of nsp had been characterized in previous studies, we focused on nsp in subsequent experiments. to further confirm the dub activity of nsp , hek cells were transfected with the empty vector or increasing amounts of v -tagged nsp expression plasmid along with ha-tagged ubiquitin vector (ha-ub) and the levels of ubiquitin-conjugated proteins were monitored at h post-transfection. as shown in fig. b , compared to the control vector transfected cells (lane ), the degree of deubiquitination directly correlated with the amount of nsp expressed (lanes - ). these data strongly suggest that nsp is directly responsible for the decrease in ubiquitinated cellular proteins. protein ubiquitination is an important posttranslational modification that has an essential role in the positive and negative regulation of nf-b signal transduction pathway, among different ubiquitination types k -and k -linked polyubiquitin chains are of great significance (wertz and dixit, ) . to further identify which ub linkage type is targeted by nsp , hek cells were transfected with ha-k -ub or ha-k -ub in place of ha-ub. these constructs allow solely the formation of k -or k -linked polyubiquitin chains, respectively. as shown in fig. c and d, while the accumulation of k -linked ubiquitinated proteins was reduced by nsp in a dose-dependent manner (fig. c) , k linked ub moieties were left intact upon nsp coexpression (fig. d) . these data indicate that prrsv nsp specifically targets k -linked polyubiquitin chains. . . the c residue is a potential catalytic site for nsp dub activity there are five families of dubs characterized by specific structural domains: ubiquitin c-terminal hydrolases (uchs), ubiquitin-specific proteases (usps), ovarian tumor proteases (otus), josephins and jab /mov metalloenzymes (jamms). uchs, usps, otus and josephins function as cysteine proteases, whereas jamms are zinc dependent metalloproteases. based on the structure of their catalytic domains, the human dubs are classified into five subfamilies, most of which are cysteine proteases characterized by a cysteine (cys, c) active site located within the amino terminus (nijman et al., ; wilkinson et al., ) . to identify the potential cysteine catalytic site for nsp dub activity, the amino acid sequences of nsp from various prrsv strains were aligned using the clustal w program. sequence alignment showed that cys of nsp is highly conserved across different genotypes of prrsv. however, cys is only identical among type genotype prrsv (fig. a) . to determine whether these two cysteines are critical residues involved in the dub activity of nsp of prrsv wuh strain, we constructed c a and c a nsp mutants and compared them with wt nsp for the ability to reduce protein ubiquitination. as shown in fig. b , overexpression of wt nsp or the c a mutant significantly inhibited k -linked ubiquitination of cellular proteins (compare lanes and vs. , respectively). in contrast, the c a mutant had no such effect (compare lanes vs. ). these data suggest that the c residue is pivotal for the dub activity of prrsv nsp . . . residues d , k , d and y are also associated with the dub activity of nsp prrsv nsp encodes an endoribonuclease (nendou), which is conserved throughout the nidovirales order but has not been identified in rna viruses of other families (ivanov et al., ; nedialkova et al., ) . nedialkova et al. ( ) compared the amino acid sequences of the nendou domain of arterivirus (in nsp ) and its counterpart in nsp of sars-cov and found that at least residues, corresponding to h , h , k , d , d , and y in prrsv nsp , are highly conserved among prrsv strain vr , sars-cov strain frankfurt , and eav strain bucyrus (nedialkova et al., ) . considering that prrsv is divided into distinct genotypes and even exhibits remarkable genetic diversity within each genotype, we compared the amino acid sequences of nsp of several representative prrsv strains isolated from different geographical regions in different years. as shown in fig. a , the six residues reported by nedialkova et al. ( ) are indeed highly conserved among different genotypes of prrsv and eav. to determine whether these residues contribute to the dub activity of nsp , we performed alanine substitution at each site. each of these mutants was co-transfected with ha-ub vector into hek cells and western blot was performed to detect the expression of ubiquitin-conjugated proteins and the nsp mutant. as shown in fig. c , while mutants bearing the h a or d a substitution (lanes and , respectively) acted as effective as wt nsp (lane ) in preventing the accumulation of ubiquitination of cellular proteins, mutants harboring h a, k a ory a substitution remarkably lost the dub activity (lane , and ), as compared to vector-transfected cells (lane ). intermediate dub activity was observed for the d a mutant (lane ). collectively, these data suggest that residues h , k , d and y are also associated with the dub activity of nsp . given that mutation at h or d destroys the nendou activity (nedialkova et al., ) , our data also indicate that the dub activity of nsp is uncoupled from its nendou activity. ␤, , , , , , , , , , ) or empty vector ( . g). cell lysates were prepared at h posttransfection and analyzed for ub-conjugated proteins by western blot with an anti-flag antibody. western blot with anti-beta-actin serves as a protein loading control. (b) hek cells grown in -mm dishes were transfected with ha-tagged ub expression plasmids ( . g), along with increasing quantities ( , . , . , . , or . g) of plasmid encoding v -nsp , using lipofectamine . cell lysates were prepared at h posttransfection and analyzed for ub-conjugated proteins by western blot with an anti-ha antibody. western blot with anti-v antibody shows expression of nsp , and western blot for beta-actin serves as a protein loading control. (c-d) the experiments were performed similarly to those in panel b, except that the ha-k -ub or ha-k -ub plasmid was used in lieu of ha-ub. ubiquitination is an important regulatory mechanism of nf-b signaling (wertz and dixit, ) . the discovery that nsp has dub activity would imply that nsp may affect nf-b activity. indeed, it has been shown that ectopic expression of prrsv nsp inhibits nf-b activation through unknown mechanism(s). in agreement with these previous reports (beura et al., ) , our luciferase reporter assays showed that nsp down-regulated poly(i:c)-induced activation of a synthetic nf-b-dependent promoter in a dose-dependent fashion (fig. a) . we next asked the question whether nsp also attenuates expression of poly(i:c)induced, endogenous nf-b-responsive genes. to this end, hek cells were transfected with a plasmid encoding v -nsp or the empty control vector. twenty-four hours post-transfection, cells were further transfected with poly(i:c) or mock-transfected. total rna was extracted from the cells and analyzed for the abundance of endogenous interleukin- (il- ), il- and chemokine (c-c motif) ligand (ccl , also known as rantes), mrnas by sybr green real-time rt-pcr. while poly(i:c) robustly induced the expression of il- ( fig. b) , il- (fig. c) , and ccl (fig. d) in cells transfected with control vector, it was substantially less effective in cells expressing nsp . these results show that nsp negatively regulates the induction of nf-b-target genes following stimulation by intracellular dsrna. generally, attachment of k- linked ub chains promotes the degradation of a protein by the ub-proteasome system (ups), one g) , along with the indicated nsp expression plasmids ( . g). cell lysates were prepared at h posttransfection and analyzed for ub-conjugated proteins by western blot with an anti-ha antibody. western blot with anti-v antibody shows expression of nsp , and western blot for beta-actin serves as a protein loading control. of the most important machineries for non-lysosomal degradation of cytoplasmic and nuclear proteins, while modification by k linked ubiquitination mediates largely non-proteolytic functions such as protein trafficking or kinase and phosphatase activation (ikeda and dikic, ) . a key step that leads to nf-b activation in response to many extracellular stimuli is the degradation of ib␣, which sequesters nf-b proteins in the cytoplasm in resting cells (wertz and dixit, ) . because prrsv nsp selectively removes k -linked ubiquitin moieties (fig. c) , we hypothesized that it may inhibit nf-b activation by interfering with the ubiquitination of ib␣ and subsequent proteosomal degradation. to test our hypothesis, hek cells were transfected with an nsp -encoding vector or the empty vector (as a control), followed by stimulation by tnf-␣. at and min post-stimulation, cell lysates were collected to detect ib␣ protein by western blot. as shown in fig. a and b, tnf-␣-induced degradation of ib␣ was significantly attenuated in cells expressing nsp , compared to cells transfected with the empty vector, indicating that nsp inhibits ligand-induced (a) hek cells grown in -well plates were transfected with . g/well of × nf-b-luc reporter plasmid, along with . g/well of prl-tk plasmid and increasing quantities ( , . , . , or . g) of plasmid encoding v -nsp , using lipofectamine . twenty-four hours after the initial transfection, the cells were further treated with poly(i:c) or mock treated. luciferase assays were performed at h after infection. (b-d) hek cells were transfected with g of plasmid encoding v -nsp or an empty vector, and, h later, the cells were transfected with g of poly(i:c). twenty-four hours after the second transfection, total rna was extracted and the expression of il- (b), il- (c) and ccl (d) and gapdh genes were evaluated by quantitative real-time rt-pcr. results are expressed as increases in mrna levels relative to those in cells transfected in the absence of poly(i:c) and were normalized by using gapdh housekeeping gene expression. results are representative of those from three independent experiments. ib␣ degradation. to further test whether nsp removes k -ub from ib␣, hek cells were transiently co-transfected with an nsp expression construct, ha-k -ub, and an ib␣-encoding vector. at h post transfection, mg (a proteasome inhibitor) was added for h to retain ubiquitinated ib␣. cell lysates were (a) hek cells in -mm dishes were cotransfected with the expression plasmids encoding ha-nsp or the pcaggs-ha (vector) empty plasmid ( g). transfected cells were treated with tnf-␣ ( ng/ml) for the amounts of time ( min, min) and subsequently immunoblotted. western blots were analyzed for total ib␣. the beta-actin was detected as a loading control. (b) relative levels of ib␣ were estimated by densitometric scanning after normalization against beta-actin and are shown as bar diagrams. data represent means of three replicates. (c) hek cells grown in -mm dishes were transfected with the expression plasmids encoding v -nsp ( . g), ha-k -ub ( g), pcmv-tag-ib␣ ( . g) using lipofectamine . mg ( nm) was treated at h after transfection. cell lysates were prepared at h after treatment and immunoprecipitated with anti-flag antibody and ubiquitin conjugation of protein was verified by immunoblotting with anti-ha antibody. the input tagged proteins were verified with indicated antibodies. then collected for co-ip assay. as shown in fig. c , ib␣ was conjugated with k -ub in cells without nsp coexpression (lane ). however, in cells expressing nsp , there were very few k -ub moieties attached to ib␣, despite the latter being expressed at comparable levels (lane ). in aggregate, these results suggest that nsp inhibits nf-b through removing k -linked polyubiquitin chains from ib␣ and thus preventing subsequent ib␣ degradation. g) , and the designated nsp expression plasmids ( . g), using lipofectamine . twenty-four hours after the initial transfection, the cells were further treated with poly(i:c) or mock treated. luciferase assays were performed at h after treatment. (b-c) hek cells in -mm dishes were transfected with the expression plasmids encoding v -nsp and mutants ( . g), ha-k -ub ( g), pcmv-tag-ib␣ ( . g), using lipofectamine . mg ( nm) was treated at h after transfection. cell lysates were prepared at h after treatment and immunoprecipitated with anti-flag antibody and ubiquitin conjugation of protein was verified by immunoblotting with anti-ha antibody. the input tagged proteins were verified with indicated antibodies. to delineate the molecular determinants of nsp involved in nf-b inhibition, various nsp mutants with differing dub activities ( fig. b and c) were analyzed for their abilities to affect poly(i:c)-induced nf-b activation in hek cells by lucifease reporter gene assay. as shown in fig. a , the results showed that mutants with impaired dub activity were attenuated for the ability to suppress nf-b activation. notably, the c a, h a and d a mutants with dub activities comparable to that of wt nsp were as effective as the latter in blocking activation of nf-b, while the c a, h a, k a and y a mutants with little or no dub activity were without inhibitory effect. the d a mutant possessing intermediate dub activity had a moderate inhibition on nf-b. taken together, these data show that dub activity is proportional to the suppressive effect on nf-b activation. next, we determined the abilities of these nsp mutants to remove k linked polyubiquitin chains from ib␣. as shown in fig. b and c, the dub activity toward polyubiquitinated ib␣ of the individual nsp mutant directly correlated with the capacity to inhibit nf-b activation. collectively, these results provide further support to the notion that prrsv nsp inhibits nf-b activation by means of its dub activity toward polyubiquitinated ib␣. in this study, we attempted to elucidate the mechanism(s) employed by prrsv to reduce cellular protein polyubiquitination and accidently found that prrsv nsp possesses dub activity. we also demonstrated that nsp specifically cleave k -linked, but not k -linked polyubiquitin chains. importantly, our data showed the dub activity is responsible for the ability of nsp to inhibit nf-b activation, revealing a novel mechanism evolved by prrsv to disarm host innate immune responses. at present, we favor a model in which nsp removes k -linked ubiquitin moieties from ib␣, thereby preventing the proteasomal degradation of ib␣ and subsequent liberation of nf-b. this is based on our data that the inhibitory effects of nsp mutants on nf-b correlated with their abilities to deubiquitinate ib␣ conjugated with k -linked ubiquitin chains. however, our study do not rule out the possibility nsp may also act on other cellular targets to regulate nf-b activity. previous studies have revealed that prrsv nsp contains endoribonuclease (nendou) activity (nedialkova et al., ) . although, nendou is highly conserved throughout the nidovirales order, thus far no nendou homologs have been identified in rna viruses of other families. in this regard, nendou is considered to be a genetic marker of nidoviruses (ivanov et al., ) . adopting a catalytic mechanism resembling that of rnase a, nendou acts independent of mn + and with a preference for uridylate, as has been demonstrated for nendous encoded by eav, sars-cov and prrsv (nedialkova et al., ) . through as-yet-unknown mechanisms, nendou facilitates viral rna synthesis (kang et al., ; posthuma et al., ) . in this study, we demonstrated that prrsv nsp also has dub activity, ascribing a novel function to this enigmatic protein. in addition, our mutational analyses suggested that the dub activity of nsp is separated from its nendou activity and that the former rather than the latter mediates the suppressive effect on nf-b. it will be interesting to investigate in future studies whether the nsp counterparts encoded by other nidoviruses also possess dub and nf-b-inhibitory activities and whether these relate to the nendou activity. lee et al. ( ) firstly demonstrated that prrsv infection activates nf-b signaling in marc- cells and pams through inducing ib degradation and p nuclear translocation. our group also showed that prrsv infection triggers activation of nf-b and that the nucleocapsid (n) protein of prrsv could elicit this process in marc- cells luo et al., ) . however, activated nf-b could only be detected after h post infection. given the recent reports that several prrsv nsps, including nsp ␣, nsp ␤, nsp , and nsp , could function as negative regulators of nf-b (beura et al., ; song et al., ; sun et al., ) , it is plausible that the regulation of nf-b is a dynamic process during the course of prrsv infection, and that prrsv has developed sophisticated strategies to either activate or inhibit nf-b at various stages of the viral life cycle to facilitate viral replication and/or disrupting host innate immune responses, offering the virus a maximal survival advantage. previous studies have shown that the nsp s of prrsv strains fl and bj- inhibited ifn-␤ production in hela and marc- cells (beura et al., ; shi et al., ) . we also found that the nsp encoded by the highly pathogenic wuh strain, which is prevalent in china and surrounding countries, could inhibit activation of irf- and ifn-␤ transcription in hek cells by luciferase reporter assays (data no shown). to our surprise, all of the nsp mutants characterized in the current study were impaired for the ability to block sev-induced ifn-␤ and irf- activation (data no shown). this result contrasted with the effects on nf-b, in that only the nsp mutants losing the dub activity were attenuated (fig. ) . thus, while the dub activity of nsp mediates the inhibition on nf-b activation, other mechanisms are involved in the nsp blockage of ifn-␤ production and irf- activation. apart from regulating innate immune signaling, ubiquitination has been demonstrated to orchestrate aspects of viral life cycle (isaacson and ploegh, ; randow and lehner, ) . for instance, the assembly and release of some viruses are subjected to ubiquitination-dependent regulation. a number of viral proteins, such as hiv gag (demirov et al., ; garrus et al., ) , ebola virus vp (yasuda et al., ) , epstein-barr virus (ebv) lmp a (ikeda et al., ) , and human papillomavirus (hpv) e (kamio et al., ; oh et al., ) , can be directly modified by ubiquitin or ubiquitin-like proteins, and these modifications play an important role in virion egress. while the precise role(s) of ubiquitination and/or deubiquitination in prrsv infection remains to be established, the findings that prrsv encodes at least two dubs, i.e., nsp and nsp , signify that these posttranslational modifications may play an important part in prrsv propagation and/or pathogenesis. for example, it is possible that nsp may cleave k -linked polyubiquitin chains attached to viral replicase proteins and/or structural components to control the half-lives of these viral products, providing a fine-tuned 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syndrome virus non-structural protein antagonizes interferon regulatory factor activation the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme immunity by ubiquitylation: a reversible process of modification activation of nf-kappab by nucleocapsid protein of the porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferon-beta production by interfering with the rig-i signaling pathway prrsv, the virus the ever-expanding diversity of porcine reproductive and respiratory syndrome virus biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states a genomic and functional inventory of deubiquitinating enzymes mass spectrometric and mutational analyses reveal lys- -linked polyubiquitin chains catalyzed by brca -bard ubiquitin ligase efficient selection for high-expression transfectants with a novel eukaryotic vector the papillomavirus e oncoprotein is ubiquitinated by ubch and cullin -and skp -containing e ligase hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation mechanisms underlying ubiquitination site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle viral avoidance and exploitation of the ubiquitin system termination of nf-kappab activity through a gammaherpesvirus protein that assembles an ec s ubiquitin-ligase porcine reproductive and respiratory syndrome endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction nonstructural protein alpha subunit-based inhibition of nf-kappab activation and suppression of interferon-beta production by porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions viral hijacking of the host ubiquitin system to evade interferon responses the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase pk- cells transfected with porcine cd by piggybac transposon system are susceptible to porcine reproductive and respiratory syndrome virus signaling to nf-kappab: regulation by ubiquitination metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase t nedd regulates egress of ebola virus-like particles from host cells plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production we thank dr. t. ohta for providing expression constructs. this work was supported by the national science fund for distinguished young scholars of china ( ), the national basic research program ( ) of china ( cb ), the national natural sciences foundation of china ( , ), and the key grant project of chinese ministry of education ( ). key: cord- -nx fg yn authors: mari, viviana; losurdo, michele; lucente, maria stella; lorusso, eleonora; elia, gabriella; martella, vito; patruno, giovanni; buonavoglia, domenico; decaro, nicola title: multiplex real-time rt-pcr assay for bovine viral diarrhea virus type , type and hobi-like pestivirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nx fg yn hobi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (bvdv) and . as a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. the aim of the present study was to develop a multiplex real-time rt-pcr assay, based on the taqman technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging hobi-like strains. the assay was found to be sensitive, specific and repeatable, ensuring detection of as few as ( )– ( ) viral rna copies. no cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. analysis of field samples tested positive for bvdv- , bvdv- or hobi-like virus by a nested pcr protocol revealed that the developed taqman assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time rt-pcr assay previously developed for hobi-like pestivirus detection showed cross-reactivity with few high-titre bvdv- samples. the genus pestivirus belongs to the flaviviridae family and includes recognised species: bovine viral diarrhea virus and (bvdv- and bvdv- ), classical swine fever virus (csfv), and border disease virus (bdv) (simmonds et al., ) . pestiviruses have a positive single-stranded rna genome, approximately . kb in length, composed by a single open reading frame (orf), preceded and followed by untranslated regions ( and utr). the single orf encodes for a polyprotein that gives origin to smaller proteins by viral cleavage: n pro , c, e rns , e , e , p , ns /ns , ns a, ns b, ns a, ns b (simmonds et al., ) . among all the genomic regions, the utr, n pro and e are widely used for comparison and phylogenetic analysis (bauermann et al., ) . on the basis of the capacity to cause a cytopathic effect (cpe) in cell cultures, two bvdv biotypes are known, cytopathogenic (cp) and non-cytopathogenic (ncp), both involved in the pathogenesis of mucosal disease (md), a fatal outcome of bvdv infection in persistently infected (pi) calves (brownlie et al., ; bolin et al., ) . pestivirus infection leads to significant economic losses worldwide showing a wide range of clinical signs, including mild upper respiratory signs, a transient decrease in circulating white blood cells, and a low-grade, short-term fever. however, there are virulent strains that cause severe respiratory disease, gastroenteric disorders, hemorrhagic syndrome, and pneumonia (baker, ; brownlie, ; corapi et al., ) . reproductive disorders represent one of the most important consequences of the bvdv infection (houe et al., ) . infection of pregnant cows during the first trimester of gestation with ncp bvdv strains, leads to failure of fertilization, return to estrus, abortion, congenital malformations, stillbirths, or the birth of pi animals which may appear normal or sometimes smaller and with congenital malformations. they are constantly viremic and bvdv seronegative, representing the main source of bvdv infection in the herd through their body fluids, so that eradication programs rely on the detection and slaughtering of these animals (bauermann et al., ) . because of their rna nature, pestivirus genomes accumulate several mutations during replication that may lead to the emergence of new strains, lineages or species (kirkland et al., ; schirrmeier et al., ; vilcek et al., ) . recently, additional four pestivirus species have been proposed within the genus pestivirus: pestivirus of giraffe, pronghorn virus, bungowannah virus, and hobi-like pestivirus (bauermann et al., ) . the prototype strain (hobi d / ) of this emerging group of pestiviruses, also known as bvdv- or atypical pestiviruses (larska et al., ; liu et al., ) , was detected as contaminant of a batch of fetal bovine serum (fbs) imported from brazil (schirrmeier et al., ) . natural infections by hobi-like pestiviruses have been reported in south america (cortez et al., ; weber et al., ) , asia (kampa et al., ; haider et al., ; mishra et al., ) and italy (decaro et al., (decaro et al., , a (decaro et al., , c (decaro et al., , a (decaro et al., , b . the virus has been associated to respiratory disease (decaro et al., (decaro et al., , c (decaro et al., , a and abortions (decaro et al., a) . several molecular methods are available for the detection and characterisation of pestiviruses. most of them either do not detect hobi-like pestiviruses at all or detect them with low efficiency (schirrmeier et al., ; ståhl et al., ståhl et al., , . a recently developed real-time rt-pcr assay is able to detect hobi-like pestiviruses without providing any simultaneous detection of bvdv- and bvdv- (liu et al., ) . however, the oligonucleotides used in this assay cross-react with high-titre bvdv- samples (decaro et al., b (decaro et al., , b . on the other hand, the nested pcr approach established by sullivan and akkina ( ) misclassifies hobi-like viruses as bvdv- (decaro et al., b) . in order to detect all pestiviruses infecting cattle using a single tool, a novel real-time rt-pcr assay has been recently developed (losurdo et al., ) . in addition, a nested pcr (npcr) protocol has been set up for pestivirus typing (decaro et al., b) . this assay has been proven to be specific as no cross-reactions between bvdv- , bvdv- and hobi-like pestiviruses were observed. however, the test is time consuming and labor intensive and can be exposed to a high risk of crosscontamination due to the post-pcr manipulations. to overcome these limitations, we have developed a multiplex real-time rt-pcr assay for simultaneous detection of the different species of bovine pestiviruses, including the emerging hobi-like group, allowing a rapid, sensitive and specific diagnosis of pestivirus infection and characterisation of the viral species. the full-length genome of reference strains of bvdv- , bvdv- , hobi-like pestivirus, bdv and csfv were obtained from the genbank database (http://www.ncbi.nlm.nih.gov/genbank/index. html) and aligned using the bioedit software package (hall, ) . forward and reverse primers were designed using primer software, version . (http://frodo.wi.mit.edu/primer /) to amplify a -nt fragment of the utr conserved region of all aligned pestiviruses. three specific probes to detect bvdv- , bvdv- and hobi-like pestivirus were designed using beacon designer software, version . (premier biosoft international, palo alto, ca, usa) (fig. ) . primers and probes were synthesised by eurofins genomics and are reported in table . pestivirus strains bvdv- nadl (courtesy of dr ferrari, istituto zooprofilattico sperimentale di lombardia ed emilia romagna, brescia, italy), bvdv- / (decaro et al., ) , hobi-like strain / - italy (decaro et al., ) , bdv bd (buonavoglia et al., ) , and an rna extract of the csfv lapinised chinese vaccine (courtesy of istituto zooprofilattico sperimentale di lombardia ed emilia romagna, brescia, italy) were used to evaluate the specificity of the test. to rule out any cross-reactivity between bovine pestiviruses and other bovine viral pathogens, isolates of the following viruses were also tested: bovine coronavirus (decaro et al., ) , bovine rotaviruses (pratelli et al., ) , bovine respiratory syncytial virus (vaccine strain brsv/ , cattlemaster , zoetis italia srl), bovine parainfluenza virus (vaccine strain ts rlb , cattlemaster , zoetis italia srl), and bovine herpesvirus types (thiry et al., ) and (tempesta et al., ) . a total of pestivirus positive field samples were analysed. ninety-eight samples were recruited from a previous study and had been already characterised by the npcr assay (decaro et al., b) , whereas additional specimens were detected more recently during an epidemiological survey for hobi-like pestivirus (unpublished data). the analysed samples included tissue samples from aborted fetuses, respiratory specimens from calves with respiratory disease, fecal samples from calves with enteritis and edta-blood samples from pi animals. rna was extracted from all samples using qiaamp ® cador ® pathogen mini kit (qiagen s.p.a., milan, italy), according to manufacturer's instructions. rna standards for bvdv- , bvdv- and hobi-like pestivirus were obtained amplifying a fragment of the utr region of reference strains bvdv- nadl, bvdv- / and hobi-like pestivirus italy- / - , using the common forward primer (vilcek et al., ) and three different pestivirus specific reverse primers (bvd - r: -tctatgcacacataaatgtggta- , bvd - r: -actaccggtcactctgccaactctccta- , bvd - r: -tcggtacacacatacatgtgata- ), designed using primer software, version . . . the rt-pcr products were cloned into topo ® xl pcr cloning vector (invitrogen srl, milan, italy) and transcribed with ribomax tm large scale rna production system-t (promega italia, milan, italy) from the t promoter, according to the manufacturer's guidelines. after dnase treatment, the transcripts were purified using qiaamp ® rna easy kit (qiagen s.p.a., milan, italy) and quantified by spectrophotometric analysis. ten-fold dilutions of the rna transcripts, representing to copies rna l − of template, were carried out in a mixed fecal/nasal swab suspension from a calf that tested pcr negative for pestivirus rna (sullivan and akkina, ; decaro et al., b) . aliquots of each dilution were frozen at − • c and used only once. reverse transcription of l of duplicates of the standard dilutions and rna extracts was carried out using geneamp ® rna pcr kit (life technologies italia applera italia, monza, italy) in a l reaction volume containing pcr buffer × (kcl mm, tris-hcl mm, ph . ), mgcl mm, mm of each deoxynucleotide (datp, dctp, dgtp, dttp), rnase inhibitor u, mulv reverse transcriptase . u, random hexamers . u. reverse-transcription was carried out at • c for min, followed by a denaturation step at • c for min. the triplex real-time pcr targeting the utr gene of bvdv- , bvdv- and hobi-like pestivirus was performed on a cfx tm real-time system (bio-rad laboratories srl, milan, italy) in a -l reaction mixture containing . l of itaq tm universal probes supermix (bio-rad laboratories srl, milan, italy), nm of primers pesti-qf and pesti-qr, nm of probes bvd -pb and bvd -pb and nm of probe bvd -pb, and l of c-dna. the thermal protocol consisted of activation of itaq dna polymerase at • c for min, followed by cycles of denaturation at • c for s and annealing/extension at • c for min. the detection of the table primers and taqman probes used in the multiplex real-time rt-pcr assay for discrimination of bovine pestiviruses and other oligonucleotides used in the study. increasing fluorescent signal was carried out during the extension step of the reaction and the data was analysed with the appropriate sequence detector software (bio-rad cfx manager v. . , bio-rad laboratories srl). in order to verify the absence of rna losses during the extraction step and the presence of rt-pcr inhibitors in the rna templates, an internal control (ic), consisting of an rna synthetic transcript containing the m gene of canine coronavirus (ccov) type ii (decaro et al., ) , was added to the lysis buffer (avl buffer, qiagen s.p.a.) at a concentration of , rna copies ml − of buffer prior to nucleic acid extraction. the fixed amount of the ic added to each sample had been calculated to give a mean c t value in a genotypespecific real-time rt-pcr assay (decaro et al., ) of . with a s.d. of . as calculated by separate runs. samples in which the c t value for the ic was > . (average plus s.d.) were excluded from the analysis. specificity of the assay was evaluated by testing pestivirus reference strains and other bovine viruses including bovine respiratory syncytial virus, bovine coronavirus, bovine rotavirus, bovine herpesvirus and and bovine parainfluenza virus. serial ten-fold dilutions of the bvdv- , bvdv- and hobi-like pestivirus standards containing from to copies of rna transcripts and the correlate c t values were used to set the standard curves for respective absolute quantifications. bovine nasal and faecal swabs and edta-blood samples that had tested negative for pestivirus and distilled water were used as negative controls and blank, respectively. the sensitivity of the multiplex real-time rt-pcr assay was evaluated using -fold dilutions of edta-blood samples containing about , and copies of bvdv- , bvdv- and hobi-like pestivirus rna, respectively, made in a edta-blood sample from a calf tested negative for bvdv. the same sample dilutions were submitted to npcr (decaro et al., b) and to the hobi-like taqman assay (liu et al., ) for a comparison. intra-assay repeatability was evaluated testing times the same samples in one experiment, and the inter-assay repeatability was verified repeating the experiment times. clinical samples containing virus amounts spanning the whole sensitivity limits of the multiplex real-time rt-pcr assay were selected for repeatability evaluation. coefficients of variation (cvs) were calculated by dividing the standard deviation of each tested sample by its mean and multiplying that result by . the detection of pestivirus rna in clinical samples and rna transcript dilutions was carried out using a npcr protocol previously developed for the characterisation of bovine pestiviruses (decaro et al., b) . first-and second-step amplifications were carried out using superscript tm one-step rt-pcr for long templates (life technologies italia) and amplitaq gold (life technologies italia), as previously described (decaro et al., b) . oligonucleotides are reported in table . to compare the performance of the developed triplex assay with the only existing real-time rt-pcr assay claimed to detect specifically this group of viruses, all clinical samples were tested by means of the liu's assay (liu et al., ) . reverse transcription and realtime pcr were carried out as described for the triplex assay using oligonucleotides listed in table . no fluorescence signal was detected from either negative controls or distilled water and all of the other bovine pathogens, including the related pestiviruses bdv and csfv, were not detected by the developed multiplex real-time rt-pcr. the assay was proven to be species specific since bvdv- , bvdv- and hobi-like pestivirus were correctly detected by the specific probes and no cross-reaction between the three pestiviruses was observed. the standard curves generated for each pestivirus using ten-fold dilutions of standard rna covered a linear range of at least nine orders of magnitude (from / to copies of standard rna) and linearity was observed over the entire quantification range (slopes of − . , − . and − . for bvdv- , bvdv- and hobi-like pestivirus, respectively). coefficients of regression (r ) were . , . and . for bvdv- , bvdv- and hobi-like pestivirus, respectively. the sensitivity of the assay was set at rna copies for bvdv- and at rna copies for bvdv- and hobi-like pestivirus. nested pcr had the same sensitivity in the case of bvdv- and hobi-like pestivirus, but it was -log less sensitive than the taqman assay when bvdv- was processed. in addition, the specific taqman assay by liu et al. ( ) was able to detect as few as hobi-like pestivirus rna copies. the repeatability was evaluated by calculating the intra-and interassay cvs. bvdv- cvs ranged from . % (samples containing × copies rna l − ) to . % ( . × copies rna l − ); bvdv- cvs varied from . % ( × copies rna l − ) to . % ( × copies rna l − ); hobi-like pestivirus cvs were between . % ( × copies rna l − ) and . % ( × copies rna l − ). interassay cvs ranges were comprised between . % ( × copies rna l − ) and . % ( × copies rna l − ) for bvdv- , between . % × copies rna l − ) and . % ( × copies rna l − ) for bvdv- ; between . % ( × copies rna l − ) and . % ( × copies rna l − ) for hobilike pestivirus. the ic was detected in all the examined samples, with c t values below the threshold value of . , thus confirming the absence of rna losses during nucleic acid extraction or dna polymerase inhibition during real-time pcr. in order to rule out any interference between the speciesspecific taqman probes contained in the same mix, a pestivirus negative edta-blood sample was also spiked with low ( copies) and high ( copies) concentrations of standard rna of bvdv- , bvdv- and hobi-like pestivirus and the viral loads in the spiked samples were quantified using the developed assay. the rna titres of the pestivirus species were calculated correctly, showing that no interference occurred during detection and quantitation of the different viruses contained in the same sample (data not shown). by analysis of field samples tested positive by npcr (decaro et al., b) , there was a perfect agreement between gel-based and real-time rt-pcr assays. bvdv- and bvdv- were detected in and samples, respectively, whereas specimens that had been collected from two different cattle herds in southern italy tested positive for hobi-like pestivirus. the amount of rna detected in the samples covered a wide range of copies per microlitre of template, ranging from . × to . × (bvdv- ), from . × to . × (bvdv- ) and from . × to . × (hobi-like pestivirus). the liu assay (liu et al., ) was able to type correctly all hobi-like strains and did not recognise any of the bvdv- positive samples, but out of bvdv- strains were mistyped as hobi-like viruses. the bvdv- titres of the mistyped samples were above rna copies l − of template. several pcr-based methods have been developed for sensitive and rapid detection of bvdv in clinical samples (bhudevi and weinstock, ; young et al., ; la rocca and sandvik, ; yan et al., ; fan et al., ; zhang et al., ; losurdo et al., ) , but only few methods are currently available for unambiguous discrimination between bvdv- and bvdv- (sullivan and akkina, ; letellier and kerkhofs, ; baxi et al., ; leblanc et al., ) . the emergence of hobi-like pestivirus that causes clinical pictures overlapping those induced by bvdv- and bvdv- (bauermann et al., (bauermann et al., , (bauermann et al., , , posed several concerns as for the ability of existing diagnostic methods to efficiently detect this emerging group of pestiviruses (schirrmeier et al., ) . the panpestivirus rt-pcr developed by vilcek et al. ( ) , which is commonly used for bvdv molecular screening, fails to detect or detects with low efficiency hobi-like strains due to the presence of a mismatch at the end of primer that prevents the correct primer annealing. other conventional and real-time rt-pcr protocols are able to detect the novel pestivirus but do not provide any virus characterisation, which is helpful to assess virus epidemiology (elvander et al., ; letellier et al., ; gaede et al., ; hoffmann et al., ; losurdo et al., ) . a taq-man assay that was claimed to be specific for hobi-like pestivirus was recently developed (liu et al., ) , but this assay could not discriminate simultaneously bvdv- and bvdv- and showed partial cross-reaction with high-titre bvdv- samples (decaro et al., b (decaro et al., , b . this cross-reaction was confirmed by the present study since out bvdv- positive samples reacted as hobi-like strains when tested by the liu's assay. recently, a npcr assay was established for simultaneous discrimination of all pestiviruses infecting cattle, including hobi-like pestivirus (decaro et al., b) . this method was specific and reliable, but also cumbersome as it requires two separate steps, an rt-pcr followed by the nested amplification; in addition, it presents a certain risk of cross-contamination between positive and negative samples. to overcome the limitations of existing methods, we have developed a triplex real-time rt-pcr assay, based on the taq-man chemistry, which was able to differentiate efficiently bvdv- , bvdv- and hobi-like pestivirus. the assay was proven to be repeatable and linear over a range of at least orders of magnitude, from / to rna copies, thus ensuring an accurate measurement of pestivirus rna loads in clinical samples. in comparison with npcr, the real-time rt-pcr assay was equally or slightly more sensitive and less time-consuming. labeling the speciesspecific probes with different fluorophores enables a correct characterisation of the pestiviral strains contained in clinical samples. in addition, the -well format of the real-time pcr plates ensures a high throughput, with the chance to test simultaneously several samples, which is useful for large-scale epidemiological surveys. the developed assay is a closed system in which the tube is never opened post-amplification, and this reduces the possibility of cross-contamination of new samples with previously amplified products. a certain carryover may occur due to the separation between rt and fluorogenic pcr, but we preferred a two-step assay, since one-tube methods are less sensitive than a two-step rt-pcr procedure (nakamura et al., ; bustin, ) and the risk of rna degradation is increased if analyses are performed over a long period of time (postollec et al., ) . in order to further reduce the risk of contamination, we have strictly separated the different working steps and carried out pipetting in different laminar flow hoods. in addition, the two-step assay requires more work, thus reducing the laboratory capacity. another limitation of the study is that the analysed pestiviral strains were geographically homogeneous, so that theoretically less common subtypes or divergent viruses circulating in different countries could not be correctly characterised. however, sequence alignment of the oligonucleotide binding regions showed that they are conserved within the same viral species for bvdv- and bvdv- . as for hobi-like viruses, even the more divergent strains recently identified in asia (mishra et al., ; haider et al., ) displayed only few mismatches, which should be tolerated by the oligonucleotides, including the taqman probe (fig. ) . anyway, the assay needs to be validated with those divergent strains that seem to be widespread in the asian continent. the developed assay does not recognise bdv, which was recently detected in cattle (strong et al., ; braun et al., ) . however, at the moment the epidemiological situation does not require including bdv 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characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species family flaviviridae natural infection of cattle with an atypical 'hobi'-like pestivirus-implications for bvd control and for the safety of biological products atypical 'hobi'-like pestiviruses-recent findings and implications antigenic and genetic characterisation of border disease viruses isolated from uk cattle a nested polymerase chain reaction assay to differentiate pestiviruses evaluation of the pathogenicity of a bovine herpesvirus- (bhv- ) strain in pregnant rabbits a live attenuated glycoprotein e negative bovine herpesvirus vaccine induces a partial cross-protection against caprine herpesvirus infection in goats pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis characterization of a novel pestivirus originating from a pronghorn antelope clinical presentation resembling mucosal disease associated with 'hobi'-like pestivirus in a field outbreak combination of reverse transcription real-time polymerase chain reaction and antigen capture enzymelinked immunosorbent assay for the detection of animals persistently infected with bovine viral diarrhea virus real-time rt-pcr detection of bovine viral diarrhoea virus in whole blood using an external rna reference comparison of conventional rt-pcr, reverse-transcription loop-mediated isothermal amplification, and sybr green i-based real-time rt-pcr in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches this work was supported by grants from italian ministry of university, project pon / "micromap -sviluppo di una piattaforma tecnologica multiplex per diagnostica molecolare, portatile ed automatizzata, basata sulla logica strumentale del labon-chip, in grado di consentire applicazioni multiparametriche in campo infettivologico". key: cord- - v exya authors: chua, amelia ze; lo, daryl yk; ho, wilbert hh; koh, yun qing; lim, daniel sy; tam, john kc; liaw, sok ying; koh, gerald ch title: the effectiveness of a shared conference experience in improving undergraduate medical and nursing students’ attitudes towards inter-professional education in an asian country: a before and after study date: - - journal: bmc med educ doi: . /s - - - sha: doc_id: cord_uid: v exya background: in recent years, increasing emphasis has been placed on the importance of collaboration within multi-disciplinary healthcare teams, so as to facilitate holistic patient care and thus allow improved treatment outcomes. there is hence an urgent need to educate healthcare undergraduates early in their professional careers on the importance of and complexities involved in cooperating with counterparts from other allied healthcare professions. in conjunction with this, a milestone student-led conference for undergraduate students, the th student medical-nursing education conference (smec), was organised in to provide a unique opportunity for shared learning among the entire cohort of undergraduate medical and nursing students in singapore matriculating in that year. methods: this study evaluated the effectiveness of the th smec as a shared conference experience in improving the attitudes of undergraduate medical and nursing students in singapore towards inter-professional education (ipe). a -point readiness for inter-professional learning scale (ripls) questionnaire comprising three subscales was administered to participants both before and after the conference. responses were collected, giving a response rate of . %. results were analysed using paired-samples t-tests with statistical significance set at p = . . results: improvements in overall scores for both medical and nursing students were reported for all three ripls subscales. examining the ripls items individually, significant improvement in scores for both medical and nursing students was obtained in all items. prior exposure to ipe activities was not a predictor of improvement in ipe attitudes. conclusion: the authors propose that student-led jointly-organised conference experiences are effective in improving healthcare students’ attitudes towards ipe. this study provides valuable insights to facilitate the development of further ipe programs to allow for the rapid and effective promotion of cooperation and collaboration between students across various healthcare disciplines. traditionally, doctors have been trained to be self-reliant and independent, with the profession relying more on expertise, autonomy and responsibility rather than interdependence, deliberation and dialogue [ ] . in recent years however, increasing focus has been placed on the importance of team-based care and collaboration between various healthcare professionals. critical to this shift is the advent of inter-professional education (ipe). ipe can be defined as an "educational process through which students and practitioners are provided with structured opportunities for 'shared learning'" [ ] , allowing healthcare students to understand the intricacies of working together with members of other healthcare professions. "working together" involves "acknowledging that all participants bring equally valid knowledge and expertise from their professional and personal experiences", and can result in novel methods of problem solving [ ] , improving the effectiveness of patient care in the process while also allowing for superior treatment outcomes. the literature suggests that ipe at the level of undergraduate learning could translate to improved working relations and understanding between the different healthcare professions. it is recommended that ipe be introduced early in the commencement of undergraduate healthcare courses, as this may help amend negative attitudes and avoid the formation of stereotypical views [ ] [ ] [ ] . medical and nursing students in singapore have in fact responded positively towards the concept of incorporating ipe into their professional education [ ] . it was hence decided that the th student medical education conference (smec), the only student-led healthcare-focused conference for undergraduate medical students in singapore, would be expanded to encompass both medical and nursing disciplines. the resultant th student medical-nursing education conference ( th smec ) was aptly accorded the theme of "under one roof", providing a milestone joint ipe event for first-year undergraduate medical and nursing students. in this paper, the authors present an assessment of the effectiveness of the th smec in improving attitudes of conference participants towards ipe. the th smec was held for first-year medical and nursing students at the very start of the - academic year in august. notably, with the opening of singapore's second undergraduate medical school that same year, the conference was able to transcend institutional boundaries as well, ultimately reaching out to all matriculating undergraduate medical and nursing students across the nation. participants encompassed undergraduate medical students from the yong loo lin school of medicine, national university of singapore and the lee kong chian school of medicine, nanyang technological university, as well as undergraduate nursing students from the alice lee center for nursing studies, yong loo lin school of medicine, national university of singapore. with this, the programme of the conference was especially tailored to ensure that all participants were able to gain insight from qualified professionals and educators in both the medical and nursing fields. table provides details on the ipe-focused plenary session and workshops that comprised the bulk of the conference. the conference was assessed via the administration of the readiness for inter-professional learning scale (ripls) to all conference participants. the ripls was originally formulated in by parsell and bligh [ ] as a -item questionnaire consisting of subscales (teamwork and collaboration; professional identity; and roles and responsibilities). as the first instrument designed to evaluate the "readiness" of healthcare students for shared activities, the ripls allows educators to quantify the impact of interventions on healthcare students [ , ] . ripls has subsequently been proven to be a valid and useful tool for measuring student attitudes towards ipe in the undergraduate context [ ] . for each item, participants were asked to provide their response using a likert scale with representing "strongly disagree" and representing "strongly agree". the questionnaire was administered twice to all participants -before and after the ipe components of the conference (prior to the plenary session and after the small-group workshops) to determine the effectiveness of the conference in improving students' attitudes towards ipe. participation in this study was voluntary, with consent taken after provision of a participation information sheet containing details of the study. the study was approved by the national university of singapore institutional review board. six of the items in the ripls were negatively worded in the survey form; however for the sake of presentation, the scores recorded in this paper are such that a higher score is always indicative of a more positive attitude towards ipe. cronbach alpha values were calculated to determine the internal consistency of the ripls instrument in our study population. paired-samples t-tests were employed for each of the items, as well as the subscale scores and overall total score in order to evaluate changes in the conference participants' attitudes towards ipe between before and after the conference. chi-squared (χ ) tests were used to compare ripls scores between those who had prior exposure to ipe versus those who did not, so as to evaluate the effect of prior exposure to ipe on changes in ripls scores before and after the conference. statistical significance was set at the conventional p < . . a total of responses were collected out of a total possible . . % of medical students from the yong loo lin school of medicine, . % of medical students from the lee kong chian school of medicine and . % of nursing students from the alice lee center for nursing studies responded, giving an overall response rate of . %. the demographics of the students who responded to the survey are illustrated in table . the internal reliability of the pre and post-conference questionnaires was assessed separately. cronbach's α coefficients of . and . respectively were obtained, indicating a high internal consistency of the ripls questionnaire used. the results obtained from the participants' responses are shown in table with respondents stratified according to their course of study (medicine or nursing). improvements in overall scores for both medical and nursing students were observed for all three ripls subscales. the scores for both medical and nursing students also improved significantly for all individual ripls items. prior exposure to ipe activities was not a predictor of improvement in ipe attitudes. thirty-seven conference participants were found to have had previous exposure to ipe activities. healthcare students who had undergone previous ipe experiences had a significantly higher baseline score (pre-conference) as compared to those without such experiences. however, there was no significant difference in the improvement in scores between those who had prior exposure to ipe and those who did not. results obtained for all ripls subscales showed overall significant improvements in scores, indicating that the th smec was effective in improving the attitudes of singaporean healthcare students towards ipe. notably, these improvements were obtained for both medical students as well as nursing students, implying that the th smec was not only able to improve attitudes towards ipe in both groups, but was also able to promote the importance of teamwork specifically between these two healthcare professions. looking individually at the ripls items; with the exception of a minority of questions, the baseline scores for both medical and nursing students were already high prior to the conference and additionally showed statistically significant improvements post-conference. this overall positive result is extremely encouraging as it not only small-group workshops participants were given the opportunity to select the workshop that interested them the most out of a choice of . each workshop was co-facilitated by at least one doctor and one nurse; with some facilitators inviting additional colleagues to share at the session. facilitators were given the freedom to conduct each workshop in any preferred format, but with the guideline that focus should be on the teamwork and cooperation between various healthcare professionals in daily practice. activities chosen by the facilitators ranged widely from video presentations to role-playing and group discussions. many also chose to use examples from key events in the history of healthcare in singapore, such as the epidemic of severe acute respiratory syndrome (sars). it was encouraging that all facilitators displayed a great keenness to share their thoughts on ipe with the conference participants. the workshop topics were as follows: (additional non-ipe events held during the conference included a symposium on "surviving medical and nursing school", as well as a scientific poster competition and a symposium on hospital residency for senior medical students.) indicates that ipe activities are highly effective in improving students' attitudes, but also suggests that singaporean healthcare students display a high readiness to participate in and learn from such activities. this reflects the findings of studies on ipe activities in other countries [ ] . with regards to question of subscale (see table ); the baseline scores for both medical and nursing students were high. however, while the cohort of medical students who participated showed improved attitudes towards ipe as a whole, it is noted that for this particular question, only the score for nursing students showed a significant improvement post-conference, whereas that for medical students showed only a slight increase that was not statistically significant. we hypothesize that this result is due to medical students' perception of doctors as being preeminent members of the healthcare team who work independently, such that medical students accordingly have a tendency to view teamwork and collaboration with less importance as compared to other healthcare students. this appears to be a global phenomenon, with studies from new zealand, the united arab emirates and sweden reporting similar results [ , , ] . notably, the literature indicates that such perceptions extend past graduation into working life as well, with doctors valuing teamwork less as compared to other healthcare professionals such as nurses and pharmacists [ ] . it is thus particularly important to correct this perception when attempting to improve medical students' attitudes towards ipe. the authors hence suggest that additional studies are necessary to further elicit the detailed reasons for the prevalence among medical students of these specific perceptions as well as to identify the best ways of correcting such beliefs within the context of ipe. healthcare students who had undergone previous ipe experiences had a significantly higher baseline score (pre-conference) as compared to those without such experiences. however, there was no significant difference in improvement in ripls scores between those who had prior exposure to ipe compared to those who did not. this suggests that the th smec was able to prove insightful by the same extent even to those who had prior exposures to ipe. it is hence worth considering whether participation in multiple ipe events would allow one to have a linear or even an exponential increase in the extent to which students are able to appreciate and work with members of other healthcare professions. a notable aspect of this study is that almost the entire cohort of singapore undergraduate medical and nursing students matriculating in was surveyed. with participants comprising the majority of first-year students from both undergraduate medical and nursing schools in singapore, and coupled with the high response rate of . %, this study provides a comprehensive indication of both the attitudes of local singaporean medical and nursing students towards ipe, as well as the potential effectiveness of ipe initiatives in the singaporean context. as the th smec was one of the few healthcare conferences that are organised for students, by students, the results of this study suggest that student-run initiatives can be highly effective in improving attitudes towards ipe. given that ultimately, healthcare students make up the target audience of these initiatives, it is sensible that such initiatives be organised by fellow students who are most equipped to identify the needs of their current generation; being the most able to organise the fulfilling, informative and enjoyable ipe experiences for their peers. this is supported by literature which has concluded that student-run initiatives should not just be considered to be nonessential electives, but as cherished events that will fuel the growth of ipe [ ] . perhaps another factor contributing to the effectiveness of the th smec was the timing at which it was conducted; specifically being approximately one week into the beginning of university education for the undergraduate student participants. holding ipe events at such an early stage of education makes it easier to avoid the phenomenon of students pigeonholing members of other healthcare professions, a phenomenon extremely common in post-graduate education should students not have previously participated in ipe [ ] . studies have in fact shown that holding ipe events in post-graduate education is much less effective, due to healthcare professionals having already developed their professional identity and hence holding more rigid views on inter-professional collaboration [ , , ] . within the period of undergraduate education itself, the literature in fact indicates that students who have just joined health profession courses are more receptive to ipe as compared to students who are nearing the end of their course [ ] [ ] [ ] . it is acknowledged that there are limitations to the study. it is noted that this paper is unable to evaluate the long-term impact of the conference on participants' ipe attitudes post-graduation. at the time of writing, it was not possible to elicit this as the cohort surveyed is yet to graduate for several more years. however, the authors suggest that given the long timeframe, any follow-up studies will be limited in significance, as the scope of further ipe and clinical exposures that will affect participants' attitudes are likely to vary significantly among participants by the time of graduation. hence it would very likely be inappropriate for any such studies to draw direct links between the effects of the conference in first year and ipe attitudes post-graduation. nevertheless, the inability to extrapolate the results presented here to predict changes in the long-run does not preclude the fact that the conference was able to have a significant impact on participants. rather, the authors suggest that the possibility of such impacts having an effect only in the shortterm means that further ipe exposures throughout the course of undergraduate education may be necessary to supplement the shared conference experience described in this study. additionally, the vast majority of conference participants were of the same age; hence it may not be appropriate to generalize our results to graduate medical and nursing students, or students from non-asian countries. the use of open-ended questions in addition to multiple-choice questions in the questionnaire could possibly be useful in allowing for a more comprehensive approach when studying this aspect in the future [ , ] . a further extension to this study could be to consider if a similar conference targeted at other combinations of healthcare professions such as dentistry, pharmacy and occupational therapy for example would prove to be as effective. the authors also suggest that while ipe is known to have an important impact on improving patient care, it would be interesting to further explore the ways in which an improvement in ipe attitudes affects healthcare professionals' specific working practices, possibly allowing for more targeted interventions to be implemented. our study found that participation in a student-led jointly-organised conference event was effective in improving medical and nursing students' improve attitudes towards ipe. for students barely into the first year of healthcare education, such an ipe event is extremely important and vital to developing a readiness to work with other members of the healthcare profession, a very important quality to possess especially in light of today's scientific advancements that herald interdependent healthcare cooperation. ipe is a relatively new pedagogical tool in medical and nursing education, thus further research should be undertaken to elicit more ways in which ipe can be incorporated into the curriculum. it is hoped that this paper will assist others in conveying the concepts of fostering teamwork, collaboration, an awareness of one's roles and responsibilities as well as uniqueness of one's discipline through their own ipe experiences. getting health professionals to work together interprofessional learning are first-year healthcare undergraduates at an asian university ready for interprofessional education? the need for multiprofessional health education in undergraduate studies the development of a questionnaire to assess the readiness of health care students for interprofessional learning (ripls) multiprofessional learning: the attitudes of medical, nursing and pharmacy students to shared learning a comparison of student attitudes and perceptions before and after an introductory interprofessional education experience validating the readiness for interprofessional learning scale (ripls) in the postgraduate context: are health care professionals ready for ipl? are senior uae medical and nursing students ready for interprofessional learning? validating the ripl scale in a middle eastern context are female students in general and nursing students more ready for teamwork and interprofessional collaboration in healthcare student leadership in interprofessional education: benefits, challenges and implications for educators, researchers and policymakers exploring the value of interprofessional shared learning the test-retest reliability of a revised version of the readiness for collaborative learning for collaborative working? initial findings from a longitudinal study of health and social care students from students to professionals: results of a longitudinal study of attitudes to pre-qualifying collaborative learning and working in health and social care in the united kingdom research design: qualitative, quantitative, and mixed methods approaches qualitative inquiry and research design: choosing among five approaches the authors would like to thank the following members of the smec organising committee who contributed in data collection and entry: douglas wc chee , eunice jh goh , lee hanyi , joshua sh lim , ooi tong li , chloe pawa , nur khairunnizzafiqah , tee shi yun . yong loo lin school of medicine, national university of singapore, national university health system, singapore alice lee center for nursing studies, yong loo lin school of medicine, national university of singapore, national university health system, singapore the authors would also like to thank the medical education unit, yong loo lin school of medicine, national university of singapore, national university health system, singapore, for helping to facilitate the running of the th smec . the authors declare that they have no competing interest. the authors alone are responsible for the content and writing of the article.authors' contributions gchk, jkct and lsy contributed to the concept and design of the study. azec, dykl, whhh, kyq and dsyl acquired the data. gchk, azec, dykl, whhh, kyq and dsyl analysed the data and all authors interpreted the data. azec, dykl, whhh, kyq and dsyl drafted the manuscript and gchk, jkct and lsy revised it critically for important intellectual content. all authors approved the final manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -qdehf rb authors: yun, heather c.; young, adam n.; caballero, manuel y.; lott, lisa; cropper, thomas l.; murray, clinton k. title: changes in clinical presentation and epidemiology of respiratory pathogens associated with acute respiratory illness in military trainees after reintroduction of adenovirus vaccine date: - - journal: open forum infect dis doi: . /ofid/ofv sha: doc_id: cord_uid: qdehf rb background. adenovirus (ad) has long been the predominant cause of acute respiratory illness (ari) in military trainees. in , live oral ad vaccines for serotypes and were reintroduced into us basic military training populations. this study evaluated the impact on clinical presentations and other respiratory pathogens. methods. the center for advanced molecular detection at joint base san antonio-lackland prospectively collects demographic, clinical, and polymerase chain reaction data from respiratory specimens (throat swab and nasal wash) among air force trainees presenting for care of ari. results. from june to august , trainees enrolled and were tested for selected respiratory pathogens. post-vaccine introduction (vi), reported systemic symptoms were less frequent, including fever ( % vs %) and myalgia ( % vs %; p < . ). median temperature and heart rate decreased ( . vs . °f, vs beats per minute; p < . ). ad detection decreased for all ad ( % vs %), ad ( % vs %), ( % vs %), ( % vs %), and ( . % vs %); p < . ). rhinovirus and cases with no pathogen identified increased in frequency ( % vs %, % vs %; p < . ). conclusions. acute respiratory illness in military trainees post-vi is associated with decreased severity of systemic symptoms and reduced fever and heart rate. marked reductions in frequency of ad serotypes are seen, including those in the vaccine, with no serotype shift. however, detection of several other respiratory pathogens, most notably rhinovirus, is observed in increasing proportions, and a majority are now undiagnosed clinical syndromes. reduction in fri due to ad [ ] . however, the military was the only purchaser of vaccine, which was produced by a sole manufacturer, and vaccine production ceased in when funding requirements for updating manufacturing facilities were not met. attempts by the department of defense to find an alternative solution were unsuccessful, and existing vaccine stocks were depleted in . ad quickly re-emerged as the major cause of morbidity, causing numerous outbreaks with both vaccine and nonvaccine serotypes, and directly resulting in approximately death per year [ ] [ ] [ ] [ ] [ ] . from to , ad ( primarily serotype ) caused % of all fri in the basic training environment [ ] . it is estimated that associated medical care and time lost from training resulted in costs of $ -$ million per year [ , ] . in , the us food and drug administration's (fda) newproduct approval process was initiated for resumption of production of ad and ad vaccines by a new manufacturer [ ] . phase vaccine trials demonstrated ( ) % efficacy for ad and ( ) ad (which was not circulating at the time) seroconversion rates of % [ ] . in , the vaccines were approved by the fda and reintroduced nearly simultaneously at all us military basic training locations in october/november of that year. since then, surveillance reports have consistently demonstrated great reductions in fri and ad-related illness. early data indicated a % decrease in fri, and proportions of collected specimens positive for ad decreased from % to % in the months surrounding vaccine introduction (vi) [ ] . nearly all of this was attributable to ad , with rare detections of serotypes , , , and , and cases involving vaccine-type p. follow-up data published in late , evaluating surveillance data from to , reported reductions in ad disease burden from . to . cases/person-week [ ] . the authors estimated that the current vaccines prevent cases of fri, - hospitalizations, and death per year. after vi, ad became the most prevalent circulating serotype, although actual number of cases detected decreased from approximately per year to in . this large study reported comprehensive surveillance data for overall ad and fri, but clinical data were not captured. whether the clinical presentation of those trainees who do present for care with a respiratory illness has changed post-vi is unclear. because respiratory illness remains a leading cause of presentation for care among military trainees, understanding of trends in clinical presentation and emerging, non-ad respiratory pathogens in the post-vi era requires evaluation. the purpose of this study was to evaluate ( ) changes in clinical presentations of ari pre-and post-vi, and ( ) reductions in proportions of disease due to ad. we also sought to further evaluate for evidence of nonvaccine type serotype shift and to determine whether the frequencies of common non-ad respiratory pathogens have changed after vi, in trainees presenting for care of ari, which have not previously been described in the published literature. joint base san antonio (jbsa)-lackland, texas, is the sole basic military training site for the us air force. training lasts . weeks, with - recruits present at any given time, and approximately training per year. units consisting of - individuals train together and live in open bay dormitories. the population is approximately % male. ill or injured trainees present for care at an outpatient medical clinic; those with respiratory illness and fever are then cohorted until well enough to return to training. those requiring hospitalization are admitted to the local military tertiary care hospital. vaccines, including ad vaccine beginning week of , and influenza vaccine seasonally, are administered during the first week. in , influenza vaccine against pdm h n influenza was available and administered after december , . oseltamivir was used for prophylaxis of close contacts of confirmed influenza cases throughout the study period. prophylaxis for streptococcus pyogenes is also administered to all trainees during the first week, consisting of benzathine penicillin, or azithromycin for penicillin allergic individuals. since , the center for advanced molecular detection ( th medical wing/science and technology, air education and training command) has prospectively evaluated epidemiology of respiratory pathogens and novel technologies for detection. for the purposes of this substudy, data were evaluated from june to august . ill recruits presenting for clinical care of ari at the outpatient clinic or hospital were approached by study personnel regarding participation. inclusion criteria were met if the trainee was ≥ years of age and endorsed any symptom of respiratory infection, including cough, coryza, sore throat, or nasal or sinus congestion. for those consenting to enroll in the study, demographic information was collected, along with a symptom questionnaire, including self-reported stress levels, and clinical signs, including vital signs and physical examination findings recorded during the medical encounter. provider diagnoses given at the time of the visit were also recorded when available. provider clinical diagnosis extracted from the note, if any, associated with the visit, was also explored with reference to diagnoses of upper respiratory tract infection (urti) vs lower respiratory tract infection (lrti). the diagnoses, "rhinitis, conjunctivitis, otitis, sinusitis, pharyngitis, sore throat" were considered to be representative of urti, and the terms "bronchitis, pneumonia" were representative of lrti. terms including "common cold", "viral syndrome", "fever", or "cough" were not included in the urti vs lrti analysis given their lack of anatomical description. nasal washes and throat swabs were collected for polymerase chain reaction (pcr) assays. duplicate presentations for multiple ari-related visits were excluded; each case represents an individual subject. specimen processing was performed as previously described [ ] . respiratory specimens were characterized daily by pcr on applied biosystems (abi) and htfast (applied biosystems, ca) and the viia real-time pcr systems. specimens were tested for ad ( panad and serotypes , , , , , , , and ); rhinovirus; influenza a, including h and h ; influenza b, enterovirus, human coronaviruses oc and e, bocavirus, human metapneumovirus (hmpv), parainfluenza virus type (hpiv), s pyogenes, streptococcus pneumoniae, mycoplasma pneumoniae, and chlamydophila pneumoniae. all primer and probe sequences used have been previously reported (association of public health laboratories guidelines for realtime reverse transcription-pcr assays of influenza respiratory viruses; non-influenza respiratory viruses from clinical respiratory specimens; respiratory bacterial pathogens) [ , ] . influenza a and b, enterovirus and rhinovirus thermocycling conditions were minutes at °c, minutes at °c, followed by seconds at °c and seconds at °c for ×. coronaviruses, hmpv, and hpiv thermocycling conditions were minutes at °c, minutes at °c, followed by seconds at °c and minute at °c for ×. streptococcus pneumoniae, s pyogenes, and bocavirus thermocyling conditions were minutes at °c followed by seconds at °c and seconds at °c for ×. thermocycling conditions for m pneumoniae and c pneumoniae were minutes at °c, minutes at °c, followed by seconds at °c and minute at °c for ×. analyses were performed using spss (spss, version . , spss). dichotomous variables were compared using χ or fisher's exact test as applicable. continuous variables were analyzed using mann-whitney u test for nonparametric data. all reported p values are -tailed with statistical significance set at <. . subjects provided voluntary, written informed consent in the presence of ombudsmen, and the study was approved by the jbsa-lackland institutional review board. this research was conducted in compliance with all applicable international and federal regulations regarding protection of human subjects. from june to august , trainees enrolled and had specimens tested for respiratory pathogens. ad vi took place week , ; % of this cohort enrolled pre-vi vs % post-vi. none of the pre-vi subjects received ad vaccine, vs % post-vi. overall, % were male, with a median age of years. enrollments ranged from to a peak of in for years with a complete calendar-year of data. trainees reported a perceived stress level of on a -point likert scale. post-vi, the male predominance of enrolled subjects decreased from % to % (p < . ) (see table ). clinical characteristics are presented in table . before vi, % of subjects had a recorded oral temperature > . °f, vs % afterward. one or more diagnoses representative or uri, lrti, or both, were present in . % pre-vi and . % post-vi. terms associated with lower respiratory tract infection (lrti) were more commonly included post-vi ( . % vs . %, p < . ) and terms associated with uri were less commonly included post-vi ( . % vs . %, p < . ). those with the diagnosis of "pneumonia" in the post-vi period were predominantly afebrile ( . %). supporting radiographs, if performed, were unavailable. the term "allergic" or "allergy" was included in none of the diagnoses pre-vi, but was included in . % post-vi (p < . ); % of these were listed in combination with some other diagnosis of uri or lrti. clinical signs and symptoms specifically associated with ad vs rhinovirus were also compared, with similar findings as those seen in general for pre-and post-vi (data not shown). the exceptions were the loss of statistical significance between those describing malaise and with abnormal tympanic membrane examinations. those with ad vs rhinovirus also more often described sore throat ( . %, . %, p < . ) and had more documented tonsillitis ( . % vs . %, p < . ). influenza a detections were less frequent post-vi; h accounted for the majority of the pre-vi detections, and these were all in . in subjects with documented fever post-vi (n = ), rhinovirus accounted for the largest proportion of abbreviations: iqr, interquartile range; vi, vaccine introduction. a abnormal examination: any of the following: "decreased breath sounds, rales, crackles, rhonchi, wheezes" for lungs, "abnormal, dull, erythematous, effusion" for tympanic membranes; "gallop, murmur, abnormal rhythm" for cardiac; "abnormal bowel sounds, distended, tender" for abdominal. these (n = ), with influenza detected in cases and any ad in ( serotype , serotype ). twenty-one of these had no pathogen detected the rate of detection of rhinovirus doubled in the post-vi period; an increase in raw numbers of rhinovirus positive enrollments per month was also seen despite lower enrollments in general (see table ). pre-vi, there were . rhinovirus positive cases per week, and . enrolled/week; post-vi, . / week were rhinovirus positive, among . enrolled/week. reintroduction of the ad vaccine in basic training populations has again been extraordinarily successful in reducing the burden of both ad-related respiratory illness and the burden of respiratory illness with fever. % reductions in fri have been demonstrated by others, including at joint base san antonio-lackland where this study was conducted, against the backdrop of a . % reduction in the weekly rate of ad-related illness [ , ] . sustaining the commitment to prevention of ad-related illness in uniquely susceptible trainees will be necessary if history is not to repeat itself. however, prevention of respiratory illness in this population is a complex task. risk factors for transmission of respiratory pathogens will continue to be present in conditions inherent to basic military training. adenovirus, despite its preeminence as a pathogen of interest in this group, has never been the entire story, and large outbreaks of non-vaccine serotype ads have occurred even while vaccine serotypes were circulating [ ] . influenza causes annual epidemics which, in the context of an effective vaccine program, are typically limited in this population, but which can have considerable impact when new strains emerge. in summer and fall of , influenza was responsible for % of fri in those who were tested [ , ] . large outbreaks of pharyngitis caused by s. pyogenes, complicated by acute rheumatic fever, pneumonia, necrotizing skin and soft tissue infections, and other suppurative and immunologic complications, have been reported throughout the past century, prompting widespread use of antimicrobial prophylaxis at training sites [ ] [ ] [ ] . pneumococcal outbreaks have also occurred despite such prophylaxis, including pneumonia and fatal meningitis [ , ] . others, including neisseria meningitidis, bordetella pertussis, m. pneumoniae, and c. pneumoniae, have been well-described in this population [ ] . horizontal efforts at respiratory infection prevention, such as promoting hand hygiene, environmental including gas mask disinfection, cohorting of ill trainees, and respiratory etiquette, will require continued emphasis, even with near-elimination of ad-related illness. however, vertical measures targeting specific organisms have also been demonstrated to have significant impact, with ad vaccine as the prime example, and ongoing exploration into post-vi causes of illness will be necessary to direct further interventions. furthermore, although widespread efforts exist to monitor fri rates and conduct surveillance for common respiratory viruses, not all acute respiratory illness is febrile. clearly, trainees are still presenting for illness, but those without fever, which now represent > % of those presenting for care, will not have respiratory pathogen analysis via dod-directed surveillance mechanisms. few prior data inform clinical differences between those presenting with ad vs other respiratory pathogens. recent comparisons of pdm( )h n influenza and ad, including an analysis from this cohort, corroborated a predominance of coryza and cough presentations for influenza, vs pharyngitis for ad [ , ] . this evaluation again emphasizes a classic presentation of ad-related illness: fever, systemic complaints, and pharyngitis, distinct from the afebrile, coryza/ cough presentations of those presenting post-vi and with rhinovirus. interestingly, documentation of abnormal lung examination findings increased post-vi, as did use of clinical diagnostic terms suggesting lrti, including both bronchitis and pneumonia. it is likely that most of those labelled "pneumonia" were never confirmed with radiographs, and absence of fever with these argues against that diagnosis in this young, otherwise healthy population. the predominant organism identified among these was rhinovirus, which, while not classically associated with lower respiratory disease in healthy adults, has been described including within military training populations [ , ] . nevertheless, the combination of increased lrti diagnoses, and the increase in cough as well as physical exam findings of the same, provide signal of an increase in lrti post-vi which should be explored with targeted research. it is also considerable that, despite a relatively broad panel of respiratory pathogens targeted with molecular methods, > % post-vi had no pathogen detected. some of these may have been noninfectious, as suggested by the increase in clinical diagnostic terms relating to allergies, but this represents a significant research gap. the nature of respiratory illness itself in basic military trainees has changed after reintroduction of ad vaccine, transitioning from a febrile pharyngitis marked by systemic signs and symptoms, to an afebrile, cough and coryza predominant illness. the ecologic niche occupied by vaccine-serotype ad in this population was remarkable, causing approximately % of all fri historically and with % of trainees infected by the end of training [ , ] . during ad vi in , molecular methods for pathogen surveillance were not available; despite this, serotype shift was observed. initially, ad was the only serotype included in the vaccine program, but ad was later added after this emerged and replaced ad as the predominant cause of fri [ ] . since that time, dozens of additional ad serotypes and other respiratory viral pathogens such as bocavirus and human metapneumovirus have been identified. respiratory pathogens cause outbreaks, which may come and go independently of a vaccine program's effect, so changes in frequency must be interpreted with caution. however, it is reassuring that ad has not yet reemerged in this population and, in fact, decreased in frequency since vi, a finding which has been corroborated by others, and potentially related to crossprotection with ad immunity [ , ] . the decrease in frequency of influenza a was driven by the unusually high number of influenza a cases in , which contributed to the total of during the entire study. the trend toward a decrease in s. pyogenes (with no changes in antibiotic prophylaxis during the study period) is potentially biologically plausible with viral coinfection increasing the likelihood of streptococcal illness, although specific associations between ad and s. pyogenes have not been established, and rates of s. pyogenes illness are not known to have changed during the first iteration of the ad vaccine program. the small increases seen in detection of m. pneumoniae, bocavirus and coronavirus oc may be due to chance alone or natural variation, but bear further observation. most significant was the increase seen in detections of rhinovirus, which increased as a proportion of detected pathogens, in rates of positive tests among those tested for rhinovirus, and in raw numbers despite fewer overall enrollments. rhinovirus may be associated with decreased probability of detecting other respiratory viral pathogens, including ad. an evaluation of virus pairs in a pcr-based analysis of co-detections demonstrated a negative association between ad and rhinovirus. while multiple additional respiratory viruses also demonstrated a negative association with rhinovirus, no others correlated either positively or negatively with ad [ ] . a negative interaction between these pathogens has previously been reported in military recruits with and without symptoms of respiratory illness, and with ad, but not rhinovirus, clearly associated with illness [ ] . the most significant strength of the study is the collection of a wide array of respiratory pathogen data alongside a detailed collection of clinical and demographic data, allowing thorough evaluations for ecologic niche replacement, as well as changes in clinical illness, throughout a major change in vaccine administration, in > trainees. others include the high uptake of ad vaccine, and the consistency in access to care, living and training conditions, and preventive medicine measures throughout the study period. the study also has a number of limitations. this is a single center which, although spanning several years, cannot account for natural variability of respiratory pathogens here or at other training sites, and some pathogens were tested for only from a subset of samples. these represent a convenience sample of the overall burden of trainees presenting to medical care for respiratory illness. trainees, for a number of reasons, may be reluctant to self-identify when ill and present for care. while we have no reason to believe this limitation changed over the course of the study, this limits the ability to extrapolate frequencies of detection to rates of disease. since asymptomatic subjects are not captured, it also limits the ability to determine colonization vs correlation with clinical illness. however, study procedures and approaches to enrolling trainees were unchanged over the course of the study period. there may have been increases in healthcare-seeking behavior in during the influenza pandemic, and this year saw the highest number of enrollments over the course of the study. finally, comorbidities in this group were not captured, although significant known comorbidities are typically disqualifying for military accession. in conclusion, reintroduction of ad vaccine in military trainees has markedly reduced the frequency of ad detection in trainees presenting for care of respiratory illness, and been associated with a -fold reduction in the proportion presenting with objective fever. acute respiratory illness has transitioned away from a febrile pharyngitis with systemic signs and symptoms to a predominantly afebrile coryza and cough illness. no evidence of ad serotype shift has been demonstrated, but rhinovirus is emerging as a potential pathogen of importance, and despite a broad array of tested viruses and bacteria, a majority now results with no pathogen identified. respiratory infections are no longer the leading cause of medical encounters among military recruits; now they are the second-leading cause [ ] . their ongoing importance epidemiologically requires continued surveillance and research into additional 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co-detection during acute respiratory tract infections? respiratory illness post ad vaccine • ofid • broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses surveillance snapshot: illness and injury burdens among u.s. military recruit trainees we express our gratitude to dr. sandra valtier and the research coordinators at the camd for recruiting the subjects and assisting in the study logistics. we are also grateful to the trainees who participated in the study.disclaimers. the opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the department of the army, department of the air force, department of defense or the us government. this work was prepared as part of their official duties and, as such, there is no copyright to be transferred. financial support. the work was supported by the united states air force, th medical wing/science and technology, air education and training command.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -jglk f d authors: zeng, sai‐zhen; xiao, ni‐guang; zhong, li‐li; yu, tian; zhang, bing; duan, zhao‐jun title: clinical features of human metapneumovirus genotypes in children with acute lower respiratory tract infection in changsha, china date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: jglk f d to explore the epidemiological and clinical features of different human metapneumovirus (hmpv) genotypes in hospitalized children. reverse transcription polymerase chain reaction (rt‐pcr) or pcr was employed to screen for both hmpv and other common respiratory viruses in nasopharyngeal aspirate specimens collected from children with lower respiratory tract infections from september to february (a period of . years). the demographics and clinical presentations of patients infected with different genotypes of hmpv were compared. a total of samples were positive for hmpv (positive detection rate: . %). co‐infection with other viruses was observed in . % ( / ) of cases, and human bocavirus was the most common additional respiratory virus. the most common symptoms included cough, fever, and wheezing. the m gene was sequenced for isolates; of these, genotype a was identified in . % ( / ) of patients, and genotype b was identified in . % ( / ) of patients. the predominant genotype of hmpv changed over the . ‐year study period from genotype a b to a b or b and then to predominantly b . most of clinical features were similar between patients infected with different hmpv genotypes. these results suggested that hmpv is an important viral pathogen in pediatric patients with acute lower respiratory tract infection in changsha. the hmpv subtypes a b and b were found to co‐circulate. the different hmpv genotypes exhibit similar clinical characteristics. j. med. virol. : – , . © wiley periodicals, inc. human metapneumovirus (hmpv) is a new respiratory viral pathogen identified in the netherlands in [van den hoogen et al., ] . hmpv is an rna virus with a negative-sense, single-stranded, nonsegmental genome. hmpv belongs to the metapneumovirus genus within the pneumovirinae subfamily of the paramyxoviridae family and is currently the only member of the genus metapneumovirus that is known to cause disease in humans. hmpv is widespread throughout the world [feuillet et al., ] and infects human populations of different age groups, especially children younger than years of age [mcadam et al., ; vicente et al., ; matsuzaki et al., ; zhu et al., ; zhang,,et al., ; lu et al., ; wei et al., ; xiao et al., ] . hmpv causes acute upper and lower respiratory tract infections. the clinical manifestations of hmpv infection are similar to those of respiratory syncytial virus infection [van den hoogen et al., ; van den hoogen et al., ] . based on the coding genes, hmpv has been classified into genotypes a and b, which are divided into sub-genotypes: a , a , b , and b . the sub-genotype a is divided further into sub-genotypes a a and a b. the new sub-genotype a b was discovered in children with respiratory tract infections by german researchers in [huck et al., ] . the previous studies have identified distinct predominant prevalences of hmpv genotypes in different regions and different years [matsuzaki et al., ; pitoiset et al., ; apostoli et al., ; kim et al., ; lu et al., ; wei et al., ] . however, the data on the epidemiological and clinical characteristics of various genotypes of hmpv are currently limited. xiao et al in our research group has reported that hmpv is an important pathogen causing acute lower respiratory tract infections in children of the hunan province [xiao et al., ] . to further explore the epidemiological and clinical features of various genotypes of hmpv, we summarized the data on the hmpv positive patients hospitalized for acute lower respiratory tract infections in hunan province over a period of . years (sep. -feb. ) nasopharyngeal aspirates samples were collected from children hospitalized for acute lower respiratory tract infection at hunan provincial people's hospital, china, during fixed days of each week between september and february . all enrolled hospitalized patients were years of age or younger and were admitted for acute lower respiratory tract infection (pneumonia, bronchitis, bronchiolitis, and asthma complicate pulmonary infection). acute lower respiratory tract infection was diagnosed on the basis of clinical and radiologic findings. the enrolled children were consulting with an illness where an acute or worsened cough was the main or dominant symptom, or had a clinical presentation that suggested a lower respiratory tract infection, with a duration of days. all nasopharyngeal aspirates were collected within - days of admission. demographic data and details of the clinical findings were recorded from case history. informed consent was obtained from the parents of all children who provided specimens. the study protocol was approved by the hospital ethics committee. all nasopharyngeal aspirates were collected and transported immediately to the laboratory at the national institute for viral disease control and prevention, china cdc, and stored at - ˚c until required for further analysis. viral dna and rna were extracted from ml of each nasopharyngeal aspirates specimen using the qiaamp viral dna and the qiaamp viral rna mini kits (qiagen, shanghai, china) according to the manufacturer's instructions. cdna was synthesized using random hexamer primers with the superscript ii rhreverse transcriptase (invitrogen, carlsbad, ca). screening for hmpv was conducted using traditional pcr methods. hmpv forward ( -ccc ttt gtt tca ggc caa- ) and reverse ( -gca gct tca aca gta gct g- ) primers, which target the m gene and generate a -bp product, were used as described previously [bellau-pujol et al., ] . all pcr products were purified using the qiaquick pcr purification kit (qiagen). the reaction mix contained pmol of each primer and . units of extaq dna polymerase (takara bio inc., tokyo, japan). reactions were incubated at ˚c for min, followed by cycles at ˚c for s, ˚c for s, and ˚c for s, followed by a final extension at ˚c for min. all pcr products were purified using the qiaquick pcr purification kit (qiagen) and cloned into the pgem-t easy vector (promega, madison, wi) and then sequenced. sequences were determined and analyzed using the dnastar software package. phylogeneticanalysis was performed with mega version . by using , bootstrapped replicates and the neighborjoining algorithm. the genbank accession numbers of the previously published sequences are as follows: can - (ay ), can - (ay ), can - (ay ), can - (ay ), can - (ay ), can - (ay ), can - (ay ), bj (dq ) and jps - (ay ). a standard reverse transcription-pcr technique was used to screen for respiratory syncytial virus (rsv), human rhinovirus (hrv), influenza viruses (ifva, ifvb), parainfluenza viruses (piv types - ), human coronaviruses hku (hcov-hku ), and nl (hcov-nl ), and pcr for adenovirus (adv) and human bocavirus (hbov) [hierholzer et al., ; bellau-pujol et al., ] . inferential statistics performed included non-parametric mann-whitney u test for continuous variables, with data reported as medians and th and th interquartile ranges (iqr). categorical variables were compared using the chi-square test or fisher's exact test. multivariate logistic regression analysis was used to identify independent risk factors. variables with a plausible relationship with p values less than . in the univariate analysis were entered into the multivariate analyses. results are summarized as odds ratios (or) and % confidence intervals (ci). all analyses were performed using spss version . software (spss inc., chicago, il). p < . was considered statistically significant. in total, patients were included, and about cases were excluded for samples missing when those were transporting from changsha to beijing (march-june ). the enrolled study samples represented . % of admissions for acute lower respiratory tract infection to hunan provincial people's hospital, china. the ages of enrolled children ranged from day to months, with a median age of (iqr - ) months. the majority of the specimens ( / , . %) were collected from patients under months old (figure ), and the male to female ratio was . : ( : ). of respiratory specimens tested in the study period, at least one respiratory virus was detected in ( . %). the most commonly detected virus was rsv ( , . %), followed by hrv ( , . %), piv- ( , . %), hbov ( , . %), adv ( , . %), hmpv ( , . %), ifvb ( , . %), ifva ( , . %), hcov-hku ( , . %), piv- ( , . %), piv- ( , . %), hcov-nl ( , . %). sixty-two of ( . %) hmpv-positive samples were found to be coinfected with other respiratory viruses, including twenty-two with hbov, seventeen with rsv, fifteen with hrv, thirteen with piv , six with adv, three with ifvb, two with piv , two with hcov-hku , and one with hcov-nl . hbov was the most common coinfecting virus, accounting for / ( . %) coinfections. among the patients with co-detections, . % ( / ) were positive for hmpv plus one additional viral agent and . % ( / ) were positive for hmpv plus two or three additional viruses; of these . %, seven patients were positive for three and six patients were positive for four viral agents. the epidemiological and clinical characteristics of patients infected with hmpv single infection and patients with hmpv codetections were similar. however, rhonchus were more common in patients with hmpv co-detections (x ¼ . , p ¼ . ) ( table i ). the univariates of cyanosis, crackles, and rhonchus were entered into the multivariate analyses, and rhonchus was associated with hmpv co-infection (or ¼ . ; %ci . - . ; p ¼ . ). approximately . % ( / ) of the males and . % ( / ) of the females tested positive for hmpv in this study. the male-to-female ratio in the hmpvinfected group was . : and differed significantly between the patients with and without hmpv infections (x ¼ . , p ¼ . ). the age of patients infected with hmpv varied from days to years of age (median (iqr), ( - ) months), and . % ( / ) were months of age. children - months of age exhibited the highest infection rate ( . %). the hmpv infection rates among four age groups differed significantly (x ¼ . , p ¼ . ; fig. ). hmpv infections occurred most frequently in spring. the positive rate of hmpv in spring reached . % ( / ). the seasonal distribution exhibited statistically significant differences in the positive rates of hmpv among four different seasons (x ¼ . , p ¼ . ; fig. ). among the pediatric patients with acute lower respiratory tract infections who tested positive for hmpv, the majority had developed a cough ( patients, . %), experienced a fever ( . %), suffered from wheezing ( . %), ( . %) developed moist rales, and rhonchi were audible in patients ( . %). in addition, patients experienced shortness of breath ( . %), developed cyanosis ( . %), had diarrhea ( . %), and required supplemental oxygen ( . %). thirty-five patients developed underlying disorders or complications ( . %), including patients had thrush, patients had granulocytopenia, children had congenital heart defects, children had cytomegaovirus infection, and some other diseases or complications. all hospitalized patients exhibited good prognoses and the median length of hospital stay was (iqr - ) days. the sequence of positive products and standard sequences from genbank exhibited high homology ( %- %). phylogenetic analyses indicated that the hmpv specimens were classified into the two main genetic lineages, a and b. ninety-eight ( / , . %) hmpv strains were subgroup a b, thirty-six ( . %) strains were subgroup b , one was subgroup b , and none were either subgroup a and a a. the nucleotide and deduced amino acid sequences of the m gene of hmpv specimens were compared with those of hmpv strains available at the genbank site (fig. ) . the epidemiological and clinical data of pediatric patients infected with type a ( cases) or type b hmpv ( cases) were summarized and analyzed. (fig. ) . comparison studies revealed that the epidemiological data and clinical characteristics were largely similar between the patients infected with genotype a hmpv and patients infected with genotype b hmpv. however, patients infected with genotype b hmpv developed diarrhea more frequently compared with pediatric patients infected with genotype a hmpv (x ¼ . , p ¼ . ). excluding the factor of mixed infection, the epidemiological and clinical characteristics of patients infected with type a hmpv alone and patients infected with type b hmpv alone were also basically similar. however, rhonchus were more common in patients with type a hmpv infection (x ¼ . , p ¼ . ). the univariates of fever, wheezing, cyanosis, . phylogenetic analysis of nucleotide sequences from the partial m gene of the hmpv strains from nasopharyngeal aspirates. phylogenetic trees were constructed by the neighbor-joining method using mega . . the viral sequences in black solid rhombus were generated from genbank (genbank accession no. can - (ay ), can - (ay ), can - (ay ), can - (ay ), can - (ay ), can - (ay ), can - (ay ), bj (dq ) and jps - (ay )); other reference sequences were obtained from the present study. genotypes of the previously published hmpv sequences: can - and can - were subgroup a a; bj and jps - were subgroup a b; can - and can - were subgroup a ; can - was subgroup b ; can - and can - were subgroup b . rhonchus were entered into the multivariate analyses, and rhonchus was associated with type a hmpv infection (or ¼ . ; %ci . - . ; p ¼ . ). the present study summarized the prevalence and clinical features of hmpv infections in hunan province between september and february , including the hmpv cases (sep. -aug. reported in the preliminary study [xiao et al., ] . the present study revealed that the hmpv positive rate was . %, in hospitalized children with acute lower respiratory tract infections in hunan province, that was similar to beijing [zhu et al., ; lu et al., ] , higher than southern china ( . %) [cai et al., ] , and lower than chongqing ( . %) [zhang et al., ] and taiwan ( %) [wei et al., ] . compared with studies in other countries, that was higher than the reported rate in japan ( . %) [matsuzaki et al., ] , lower than the reported rate in germany ( . %) [reiche et al., ] , and similar to the reported rates in the united states [van den hoogen et al., ; edwards et al., ] and south korea [kim et al., ] ( % and . %, respectively). the variation in hmpv detection rates between various regions may be related to the differences in climate, geographic locations, year and human populations. the present study found that the hmpv positive rate was higher in male pediatric patients, which is consistent with the most of the previous studies [peiris et al., ; van den hoogen et al., ; wei et al., ] , but different from a study in the united states which found hmpv infections were equally distributed between males and females [mcadam et al., ] . furthermore most previous studies have indicated that the majority of hmpv-positive patients are children of young ages [mcadam et al., ; vicente et al., ; lu et al., ; wei et al., ; reiche et al., ] , which is similar to the findings of the present study. the peak of hmpv infections was found in the spring in the present study, which is similar to that of beijing [zhu et al., ] , but different from that of chongqing where the peak of hmpv epidemics are observed in the spring/summer and the winter/spring seasons [zhang et al., ] . in the united states, hmpv infections have been reported to occur predominately in the winter and spring seasons [mcadam et al., ] . these unique characteristics of hmpv infections may be related to the geographic region and climate, and requires the further study for verification. in the present study the most common clinical symptoms of hmpv-positive patients included coughing, wheezing and fever, which in some pediatric patients, were accompanied by shortness of breath, cyanosis and moist rales heard in pulmonary auscultation, whcih were similar to those reported previously [zhang et al., ; lu et al., ; wei et al., ] . the present study revealed a rather high mixed infection of hmpv with other viruses ( . %) and hbov was the most frequently detected virus with hmpv. the rates of hmpv coinfection of previous studies range from approximately . % to . % [zhu et al., ; fathima et al., ; kim et al., ; zhang et al., ; lu et al., ; wei et al., ; cai et al., ] , and the commonly codetected viruses include rsv, hrv and piv- . these discrepancies are most likely due to the differences in the types of viruses examined and the test methods employed in the studies. however, the discrepancies may also be related to the selection of research objects, geographic regions and research periods. previous studies have indicated that mixed infection may aggravate the diseases and affect prognosis [greensill et al., ]. in the present study, lung auscultation did reveal that rhonchi were more common in patients with mixed hmpv infection compared with patients with single hmpv infection. however, no other significant differences were found between the patients, including the requirement of supplemental oxygen and duration of hospitalization. consistent with the findings reported by xiao et al [ ] , the present study revealed that both hmpv genotype a and genotype b were prevalent in the changsha area from september to february . hmpv genotypes a and b were the most predominant genotypes. the annual distribution indicated that genotype a and genotype b prevailed alternately. previous studies have reported the prevalence of certain predominant hmpv genotypes in a given year. hmpv sub-genotypes may vary in different years and alternative predominance of hmpv sub-genotypes has been observed [matsuzaki et al., ; pitoiset et al., ; kim et al., ; lu et al., ; wei et al., ] . the present study found that the epidemiological characteristics and clinical features were fundamentally similar among patients infected with different genotypes of hmpv, which is consistent with the results of some studies [bosis et al., ; wei et al., ] . diarrhea was a clinical manifestation that occurred more frequently in patients infected with type b hmpv compared with patients infected with type a hmpv in this study. however, there are different epidemiological and clinical findings in other two studies [matsuzaki et al., ; kim et al., ] . furthermore, a study indicated that patients with type a hmpv infection are often associated with more severe symptoms [vicente et al., ] . however, there is no apparent link between the genotype of hmpv and the severity factors of the disease in other studies [agapov et al., ; pitoiset et al., ] . certainly, more data are needed to elucidate this issue. in conclusion, the present study explores the epidemiological and clinical manifestations of infections with various genotypes of hmpv and especially the predominant prevalence of hmpv genotypes in different years. however, retrospective analysis was conducted in the present study without control samples, and the sample sizes of certain sub-genotypes of hmpv were small. these are the major limitations. and a long period of data accumulation with a larger sample size and control is required in the future study. genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness human metapneumovirus-associated hospital admissions over five consecutive epidemic seasons: evidence for alternating circulation of different genotypes development of three multiplex rt-pcr assays for the detection of respiratory rna viruses association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection respiratory virus infections among children in south china new vaccine surveillance network. burden of human metapneumovirus infection in young children use of an innovative 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epidemiological analysis in germany prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children clinical features of different genotypes/genogroups of human metapneumovirus in hospitalized children prevalence of human metapneumovirus in children with acute lower respiratory infection in changsha clinical impact of human metapneumovirus genotypes and genotype-specific seroprevalence in yamagata detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in southwest china characterization of human metapneumovirus from pediatric patients with acute respiratory infections in a -year period in beijing. china key: cord- -wrztpeb authors: zhang, xin; shi, hongyan; chen, jianfei; shi, da; dong, hui; feng, li title: identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: wrztpeb nucleocapsid (n) protein of transmissible gastroenteritis virus (tgev) packages viral rna genome to form a ribonucleoprotein complex. in addition to its function as a structural protein, n protein is involved in cell apoptosis or cell-cycle regulation. n protein possibly interacts with host factors to modulate cellular functions. to identify cellular proteins that interacted with n protein of tgev, methods of gst pull-down and co-ip were utilized to precipitate cellular proteins of swine testicular (st). bound cellular proteins were resolved by sds-page. analysis of interacting proteins by mass spectrometry allowed identification of cellular protein bands representative of cellular proteins including vimentin that bound to n protein. furthermore, the function of vimentin cytoskeleton in st cells during tgev infection was examined. vimentin cytoskeleton was required for virus replication. the present study thus provides protein-related information about interaction of tgev n protein with host cell that should be useful for understanding host cell response to coronavirus pathogenesis infection and the underlying mechanism of coronavirus replication. coronaviruses (covs), a genus in the coronaviridae family, are pleomorphic, enveloped viruses (perlman and netland, ) . covs have been clustered in the cornavirinae subfamily, which includes three approved genera, alpha-, beta-and gammacoronavirus, as well as a tentative new genus, the deltacoronavirus (de groot et al., ; reguera et al., ) . transmissible gastroenteritis virus (tgev) is a representative cov in the alphacoronavirus genus; severe acute respiratory syndrome-related coronavirus (sars-related cov) is a representative of the betacoronavirus genus; infectious bronchitis virus (ibv) is a representative of the gammacoronavirus genus; and bulbul-cov is a representative of the deltacoronavirus genus (de groot et al., ) . the coronaviridae are involved in respiratory, enteric, hepatic and neuronal infectious disease in animals and humans (perlman and netland, ) . tgev infection causes severe diarrhea in suckling piglets (about weeks old), which results in enormous economic loss in swine-producing areas in the world (kim and chae, ; sestak et al., ) . about two-thirds of the tgev genome ( . kb) encodes the replicase gene (rep) at the end, and one-third of the genome encodes other viral genes at the end in the order -s- a- b-e-m-n- - (penzes et al., ) . the genome of the tgev encodes four structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) . n proteins of covs are highly basic with a molecular mass ranging from to kda, depending on the species and strains. n protein binds to the rna genome, forming a helical nucleocapsid (escors et al., ; sturman et al., ) . recently, some reports showed that n protein of tgev play an important role in host cell for virus replication. n protein of tgev facilitates template switching and is required for efficient transcription (zuniga et al., ) . n protein underwent proteolysis in parallel with the activation of caspases within host cell and n protein of tgev is a substrate for caspases (eleouet et al., ) . tgev n protein nucleolus localization was found in transfection experiments and might induced a cell cycle delay or arrest to facilitate virus replication (wurm et al., ) . in contrast, tgev n protein was not accumulated in the nucleus in the infection context (calvo et al., ) . in addition, the role of tgev n protein in cell cycle arrest has been recently reported (ding et al., ) . n protein of tgev may function through direct or indirect interaction with cellular proteins. for better understanding of the mechanisms associated with pleiotropic functions of n protein, cellular proteins of swine testicular (st) cells were pulled down associated with n protein using glutathione (gst)-tagged full-length n proteins immobilized on gst agarose. and cellular proteins of st cells were precipitated using n protein mab. by sds-page coupled with mass spectrometry (ms), a total of cellular proteins interacting with n protein were successfully identified. information on the expanded repertoire of cellular proteins interacting with n protein will provide a framework for future biochemical analyses of these protein functions in tgev infection. st cells were grown in rpmi- medium supplemented with % fetal calf serum under standard culture conditions ( % co , • c). tgev infectious strain h (accession no. fj ) (wang et al., ) was propagated on an st cell monolayer. mouse mab to glyceraldehyde- -phosphate dehydrogenase (gapdh) (ab ) and rabbit poloclonal antibody vimentin (ab ) were purchased from abcam. fitc-labeled goat antimouse igg was purchased from kirkegaard and perry laboratories (kpl). tritc-labeled goat anti-rabbit igg was purchased from sigma. the mab to n protein of tgev were donated from mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china). st cells were plated in six-well plates day prior to infection with tgev infectious strain h at a multiplicity of infection (moi) of . tgev not-infected samples were exposed to culture medium alone. after adsorption for h, cells were washed twice and incubated in fresh rpmi- . the prokaryotic expression plasmid pgex-tgev-n was constructed previously . escherichia coli bl (de ) strain containing pgex-tgev-n plasmid was expressed under induction of mm isopropyl-␤-d-thiogalactopyranoside. gst pulldown assay was performed as previously described . expressed gst protein was used as a control. the lysate of tgev-infected st cells was prepared with ripa lysis buffer ( mm tris-hcl, ph . , mm nacl, % np- , . % deoxycholate) containing a protease inhibitor phenylmethanesulfonyl fluoride (pmsf) ( mm). after centrifugation at , × g for min, lysate supernatant was pretreated with l mouse igg control (beyotime) and protein a/g plus-agarose (santa cruz biotechnology) for min at • c to eliminate non-specific binding to agarose gel. the lysate supernatant ( g) was incubated with g of mab to n protein of tgev for overnight at • c. then, l resuspended protein a/g plus-agarose was added to this mixture and incubated at • c on a rocker platform for h. after washing four times with lysis buffer, isolated immunoprecipitated proteins (boiling min with page sample loading buffer) were then analyzed by % page analysis. the lysate of tgev not-infected st cells was used as a control. . . protein identification by matrix-assisted laser desorption/ionization time of flight (maldi-tof/tof) ms the gels described above stained with phastgel blue r (ge healthcare). protein bands of interest were manually excised from gels. maldi-tof/tof was performed as previously described (zhang et al., ). data were searched by gps explorer (ver. . ) with the search engine mascot (ver. . ). the search parameters were as follows: national center for biotechnology information non-redundant (ncbinr) database (release date, july ), and the database sus ( , sequences; , , residues); a trypsin digest was performed with one missing cleavage, ms tolerance was set at ppm and ms/ms tolerance at . da. known contaminant ions (tryptic autodigest peptides) were excluded. mascot protein scores (based on combined ms and ms/ms spectra) > were considered statistically significant (p ≤ . ). individual ms/ms spectrum, with a statistically significant (confidence interval ≥ %) ion score (based on ms/ms spectra), was accepted. to eliminate redundancy of proteins that appeared in database under different names and accession numbers, single protein member belonging to species sus or with the highest protein score (top rank) was separated from multi-protein family. equivalent amounts of cell lysates were subjected to % page and then transferred to . m nitrocellulose membranes (hybond-c extra, amersham biosciences). after blotting, membranes were incubated with mouse pab to vimentin or with mouse mab to n protein ( : ) at • c for h. after washing three times with pbst, membranes were inoculated with dylight tm -labeled antibody to mouse igg (h+l) ( : , , kpl, usa) or dylight tm -labeled antibody to rabbit igg (h+l) ( : , kpl, usa) at • c for min. images were visualized by odyssey infrared imaging system (li-cor). st cells inoculated with tgev were cultured for , , , , , and h. cells were washed twice with pbs and fixed with paraformaldehyde ( %) for min at • c, and then allowed to air dry. after blotting with % skimmed milk powder, the fixed cells were incubated with mab to tgev n protein ( : ) and rabbit pab to vimentin ( : , abcam) for h at • c in a humidified chamber. after washing three times with pbst, the fixed cells were incubated with fitc-labeled goat anti-mouse igg ( : , kpl) and tritc-labeled goat anti-rabbit igg ( : , sigma). additional nuclear staining with , -diamidino- -phenylindole (dapi, sigma) was performed as described previously (jungmann et al., ) . the triple-stained cells were washed three times with pbst and subsequently examined under a leica tcs sp laser confocal microscopy. . . transfection of sirna against vimentin sirna against vimentin (genepharma) was used for transfection. sequence of the sirna strands (two rounds of silencing) were as follows: -gcuaacuaccaagacacuatt- (sense) and -uagugucuugguaguuagctt- (antisense); -ccucugguu-gacacccauutt- (sense) and -aaugggugucaaccagaggtt- (antisense). negative control sirna strands were as follows: -uucuccgaacgugucacgutt- (sense), -acguga-cacguucggagaatt- (antisense). transfection with sirna was performed with lipofectamine reagent (invitrogen) by following the manufacturer's instructions. st cells were cultured overnight in six-well tissue culture plates. the sirna ( nm) was complexed with lipofectamine reagent by incubating together at room temperature for min. after removing the cell culture supernatant, the complex was added. after incubation for h, the cells were infected with tgev. total rna was extracted from the st cells transfected with sirna against vimentin and negative control sirna, using the rneasy mini kit (qiagen) according to the manufacturer's protocol. cdna synthesis was performed with total cellular rna using a reverse transcriptase m-mlv kit (takara), according to the manufacturer's protocol. real time rt-pcr was performed using a lightcycler ii (roche) in a total volume of l containing ng of cdna template, × sybr ® premix ex taq tm ii (perfect real time, takara), and a . m concentration of each primer. after initial denaturation at • c for min, the amplification was performed for cycles, each consisting of denaturation at • c for s and primer annealing at • c for s. melting curves were obtained, and quantitative analysis of the data was performed in a relative quantification ( − ct ) study model. parallel tgev notinfected st cells were used as control (relative expression = ) and gapdh as an internal reference gene. st cells were re-plated day before infection in well plates for the % infectious dose (tcid ) assays. treated samples and their paired controls were thawed as described and immediately serially diluted. cell cultures were then infected for h. after h of incubation, cpe was observed. tcid is calculated using the method of reed and munch. virus titer assay were performed three times for each condition and were performed using the student's t-test. the expressed gst-n protein immobilized on gst-agarose beads was used as a bait to pull down cellular proteins of tgevinfected st cells that form a complex with n protein. gst protein was used as control to eliminate non-specifically binding proteins. after extensive washing the resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. , cellular protein bands that were not seen in gst control gel. the protein bands of interest were manually excised from gels and plated into -well microplates for ms identification. the mab to n protein was used as a bait to precipitate cellular proteins of tgev-infected st cells that form a complex with n protein. after extensive washing resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. , cellular protein bands that were not seen in tgev not-infected st cells. the protein bands of interest were manually excised from the gels and plated into -well microplates for ms identification. tgev n protein-associated cellular proteins were identified using in-gel tryptic digestion, and maldi-tof/tof identification. protein bands binding to n protein were subjected to in-gel trypsin digestion, and peptides were analyzed by ms. database searches with the peptide masses resulted in positive identification for different cellular proteins (table , table s , and fig. s ). two atp-dependent rna helicase proteins were identified as interacting with n protein. protein band was identified as atpdependent rna helicase a (rha) and protein band was identified as atp-dependent rna helicase ddx (dbp-rb). four cytoskeleton proteins were identified as interacting with n protein. protein band and band were identified as ␣-actinin- (actn ). protein band and band were identified as vimentin (vim). protein band and band were identified as myosin. four ribosome-associated proteins were identified as interacting with n protein: heterogeneous nuclear ribonucleoprotein u (hnrnp u) (band and band ); s ribosomal protein s (band ); s ribosomal protein l (band ); and s ribosomal protein s (band ). one proteinbiosynthesis-associated protein (elongation factor -␣, band ) and one cell-cycle-arrest protein (vasohibin- , band ) were identified as interacting with n protein. maldi-tof ms spectra and tof/tof spectra of vimentin are shown in fig. . three cellular proteins, hnrnp u, actn , and vimentin, were identified both by gst-n pull down and co-ip in tgev-infected cells, which should have more biological importance in the context of infection. to verify the proteins identified by ms, western blotting was performed. many cytoskeleton associated proteins play important roles in virus infection (dohner and sodeik, ) . vimentin protein was identified not only in gst pull-down assay but also in co-ip assay. therefore, the identified protein vimentin was selected for western blot analysis. cellular proteins immobilized on gst-agarose beads in gst pull-down assay were examined with specific antibodies to vimentin (fig. ) . results validated the maldi-tof/tof identification of cellular proteins. to confirm the interaction between n protein of tgev and cellular vimentin, immunoprecipitation assay was utilized to identify whether tgev n protein interacted with cellular vimentin in tgevinfected st cells. from the immunoprecipitation results (fig. ) , we can see that the n protein of tgev was precipitated by the pab to cellular vimentin in tgev-infected st cells but not in tgev not-infected st cells. results demonstrate that cellular vimentin interacted with n protein of tgev. subcellular localization of vimentin was investigated in tgevinfected st cells using indirect immunofluorescence confocal microscopy. results indicated that subcellular localization of vimentin was distributed in cytoplasm with enrichment in the perinuclear region after tgev infection (fig. ) . cellular vimentin labeled with tritc was overlaid with n protein of tgev labeled with fitc, indicating that cellular vimentin was co-localized with n protein of tgev within the st cells during infection. to further investigate the role of vimentin in virus infection, vimentin protein of st cells was inhibited using sirna. the decrease level of vimentin mrna was validated by rt-pcr (fig. a) . st cells transfected with vimentin-specific sirna expressed lower level of vimentin by western blot analysis, compared to that transfected with the control sirna (fig. b ). after transfection with sirna, st cells were infected with tgev for another h after transfection with sirna at an moi of . virus titer assay were performed three times for each condition and were performed using the student's t-test. tcid of virus in control sirna group was . /ml and the vimentin sirna group was . /ml. fig. c shows that knock-down of vimentin resulted in significant reduction of cellassociated virus, which reflected viral replication. identifying host cell protein interacting with viral protein is a critical step for understanding protein function and viral replication. recently, affinity purification followed by ms has been widely used to find protein-protein interactions (dunham et al., ) . in the present study, using gst pull-down and co-ip coupled with ms identification, cellular protein bands representative of cellular proteins interacting with tgev n protein were identified. several cellular proteins found in this study were identical to those previously described interacting with cov n protein. ef a, ddx and hnrnp u were also observed in infectious bronchitis virus (ibv) n protein using quantitative proteomic approaches (emmott et al., ) . interaction between ef a and n protein of tgev and sars-cov were founded (zhou et al., ) . the hnrnp u protein was identified as binding preferentially tgev -end (galan et al., ) . a very recent work describes the key role of ibv n protein interaction with ddx in viral transcription (wu et al., ) . four cytoskeleton-associated proteins were identified that associated with n protein in this study. the cytoskeleton consists of a filament scaffold within cell. filaments are dynamic and divided into three types: microfilaments (actin filaments), microtubules, and intermediate filament (naghavi and goff, ) . during infection, viruses interact with components of the cytoskeleton to reach their appropriate intracellular sites of replication (radtke et al., ) . in previous study, differentially expressed cytoskeletal proteins were found in tgev infected st cells using two-dimensional difference gel electrophoresis (zhang et al., ) . cytoskeletal proteins found in this study provides new insights for understanding the mechanisms of tgev replication. cov replication uses complex mechanisms that involve viral and cellular proteins. similar to other positive-strand rna viruses, cov genome replication takes place in the cytoplasm in a membraneprotected microenvironment that contains all the protein required for viral rna synthesis (enjuanes et al., ) . electron microscopy studies of mouse-hepatitis-virus-infected cells have shown that replication complexes of virus consist of double-membrane vesicles (gosert et al., ) . cov replication complex formation possibly utilizes components of cellular autophagy (prentice et al., ) . although cov replication essentially takes place within the cytoplasm, the virus may control cell machinery by locating some of its proteins in host cell nucleus (enjuanes et al., ) . in the present study, vimentin was identified in pull-down assay and co-ip assay, suggesting that it plays an important role in tgev infection. costaining of cellular vimentin and n protein on immunofluorescence microscopy also supported an association between proteins. based on these findings, we speculate that vimentin plays a role in tgev replication. some studies demonstrate that vimentin plays important role in virus infection cycle. vimentin binding is critical for virus entry, such as human cytomegalovirus (cmv) (miller and hertel, ), japanese encephalitis virus (jev) (liang et al., ) , and cowpea mosaic virus (cpmv) (koudelka et al., ) . vimentin is required for virus replication, such as bluetongue virus (btv) (bhattacharya et al., ) , and dengue virus (denv) (kanlaya et al., ) . in this study, vimentin is required for tgev replication. therefore, these may potentially serve as novel therapeutic targets for controlling tgev infection. the efficiency of tgev infection depends on the presence and integrity of vimentin cytoskeleton, which may facilitate virus infection at different steps. vimentin may promote viral replication by interaction with n protein, which may help virions to transport through a functional golgi complex for viral maturation (risco et al., ) . additional work will be needed to pinpoint the exact steps during viral infection. overall, this study was the first to identified cellular proteins interaction with tgev n protein using proteomics method. a total of cellular proteins were identified, which provided new information to understand the mechanism of coronavirus replication. the interaction between the cellular vimentin and n protein of tgev was confirmed in tgev-infected st cells. knockdown of vimentin impairs tgev replication in st cells. the present study thus provides insights into interaction of tgev n protein with host cellular proteins that would be useful for further studying viral replication and pathogenesis. interaction between bluetongue virus outer capsid protein vp and vimentin is necessary for virus egress phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells coronaviridae tgev nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p signaling the role of the cytoskeleton during viral infection affinity-purification coupled to mass spectrometry: basic principles and strategies the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and - during tgev-induced apoptosis the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology biochemical aspects of coronavirus replication and virus-host interaction organization of 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contribute to attenuation nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division ef a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication identification of cellular proteome using two-dimensional difference gel electrophoresis in st cells infected with transmissible gastroenteritis coronavirus differential proteome analysis of host cells infected with porcine circovirus type the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor alpha coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was supported by the national natural science foundation of china (grant nos. , ), heilongjiang provincial natural science foundation (grant no. jc ) and heilongjiang provincial department of education ( td ). we thank mr. zhou xin-wen (fudan university, china) for help with maldi-tof/tof mass spectrometry and mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china) for the donation of mab to n protein of tgev. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.virusres. . . . key: cord- -sxyeuiw authors: mcintosh, kenneth; perlman, stanley title: coronaviruses, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) date: - - journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: . /b - - - - . - sha: doc_id: cord_uid: sxyeuiw nan the family coronaviridae, within the order nidovirales, contains two subfamilies, the coronavirinae and the torovirinae. coronaviruses (covs) are a large group of viruses infecting mammals and birds and producing a wide variety of diseases. they have been divided into four genera, two of which contain viruses infecting humans (see later). all human coronaviruses (hcovs) are primarily respiratory pathogens. during the winter of to , an alarming new disease appeared, severe acute respiratory syndrome (sars), which was quickly attributed to a new cov, the sars-cov. the outbreak originated in southern people's republic of china, with evidence that the virus was first derived from bats and was transmitted to man through an intermediate host, probably the palm civet (paguma larvata) or raccoon dog (nyctereutes procyonoides). [ ] [ ] [ ] the sars epidemic was controlled through a massive effort at case identification and containment, and the last known case occurred in mid- . in retrospect, the emergence of sars is consistent with what is known about covs as a group: they are important pathogens in animals causing a wide variety of diseases through a wide variety of pathogenic mechanisms, and they have been noted to mutate frequently and infect new species. , more recently, a related but different cov producing severe respiratory disease has emerged, the middle east respiratory syndrome coronavirus (mers-cov). mers-cov was grown in june , from a sputum sample obtained from a man in saudi arabia who died of overwhelming pneumonia. the virus was quickly identified as a new cov most closely related to several bat covs. this report was followed by a number of other reports identifying a total of infected individuals, all of whom had acute respiratory symptoms, severe in most, and fatal in (as of may , ) . human-to-human transmission has been documented, especially in hospital settings, a but appears to be inefficient. in , tyrrell and bynoe cultured a virus obtained from the respiratory tract of a boy with a common cold by passage in human embryonic tracheal organ cultures. the media from these cultures consistently produced colds in volunteers. the agent was ether sensitive but not related to any known human virus. subsequently, electron microscopy of fluids from infected organ cultures revealed particles that resembled infectious bronchitis virus of chickens. at about the same time, hamre and procknow recovered a cytopathic agent in tissue culture from medical students with colds. the prototype virus was named e and was found on electron microscopy to have a similar or identical morphology ( fig. - ) . using techniques similar to those used by tyrrell and bynoe, mcintosh and colleagues reported the recovery of several infectious bronchitis-like agents from the human respiratory tract, the prototype of which was named oc (oc for organ culture). at much the same time, mouse hepatitis virus and transmissible gastroenteritis virus of swine were shown to have the same morphology on electron microscopy. , shortly thereafter, the name coronavirus (the prefix corona denoting the crownlike appearance of the surface projections) was chosen to signify this new genus. the number of animal covs quickly grew, including viruses causing diseases in rats, mice, chickens, turkeys, various other bird species, cattle, several wild ruminants, beluga whales, dogs, cats, rabbits, and pigs, with manifestations in the respiratory and gastrointestinal tracts, central nervous system (cns), liver, reproductive tract, and others. through sequencing and antigenicity studies, the animal and human covs (hcovs) initially were divided into three groups: group , which • covs are members of the nidovirales order, single-stranded, positive-sense rna viruses with a large genome. they mutate and also recombine frequently. • laboratory diagnosis is best accomplished by finding viral rna through polymerase chain reaction. • there are no accepted effective antiviral drugs for covs. • prevention is through epidemiologic methods. the sars epidemic was halted through careful case finding, quarantine, and use of barrier precautions. a cov was independently and almost simultaneously isolated from sars patients by several laboratories and found by sequencing to be only distantly related to previously characterized covs. [ ] [ ] [ ] [ ] [ ] the sars outbreak stimulated a rapid and intense public health response coordinated by the world health organization, and by july , transmission had ceased throughout the world. despite this effort, however, probable cases had occurred in countries, with deaths. with the identification of the sars-cov, the hcov field became much more active. sensitive molecular methods were developed to detect rna from viruses identical or closely related to hcov- e and hcov-oc in the respiratory tract, and two new species were discovered: nl , an alphacoronavirus, and hku , a betacoronavirus. [ ] [ ] [ ] hcov-nl was found independently by three groups, two in the netherlands and, somewhat later, the third in new haven, figure - phylogenetic relationships among members of the subfamily coronavirinae. a rooted neighbor-joining tree was generated from amino-acid sequence alignments of coronaviridae-wide conserved domains in replicase polyprotein (adrp, nsp ; mpro, nsp ; rdrp, nsp ; hel, nsp ; exon, nsp ; nendou, nsp ; o-mt, nsp ) for coronaviruses, each a representative of a currently recognized coronavirus species. five of the six known human coronaviruses (hcov- e, hcov-nl , hcov-hku , sars-cov, and mers-cov) are indicated. hcov-oc is closely related to bovine coronavirus, which is shown in the figure. equine torovirus berne served as the outgroup. virus names are given with strain specifications; species and genus names are in italics as per convention. the tree shows the four main monophyletic clusters, corresponding to genera alpha-, beta-, gamma to be established numerous reports of cov-like particles (covlps) found by electron microscopy in human fecal matter, but these particles have been difficult to characterize further. more recent efforts to detect cov rna in feces using polymerase chain reaction (pcr) and primers for respiratory hcovs have had limited success and have failed to associate covs with gastrointestinal disease. , toroviruses were, like covs, first described in animals. they were first detected in the feces of cattle (breda virus) and horses (berne virus). , shortly thereafter, beards and colleagues examined human fecal material and reported finding particles with a similar appearance that aggregated in the presence of antiserum to the bovine and equine viruses. the human toroviruses, like the bovine toroviruses, do not grow in tissue culture, and thus almost all existing information about them is based on electron micrographic data. unlike animal toroviruses, , pcr-amplified torovirus rna sequences have not been found in human stool samples. a report of genome sequences amplified from particles purified from human stool was subsequently retracted and considered to reflect laboratory contamination from porcine strains. at this time, there are no reports definitively showing the existence of human toroviruses. the cov nucleic acid is rna, approximately kilobases in length, of positive sense, single stranded, polyadenylated, and infectious. the rna, the largest known viral rna ( fig. - ) , codes for (in order from the ′ end) a large polyprotein that is cleaved by virus-encoded connecticut. in all three cases, positive samples were from infants and children with respiratory disease. notably, hcov-nl and hcov- e were estimated to originate from a common precursor and diverge approximately years ago. hcov-hku was found in hong kong in an adult with respiratory disease. these two new hcov strains subsequently have been found worldwide and appear to have pathogenicity similar to that of hcov- e and hcov-oc , with the possible exception that nl is more frequently found in children with croup. , the mers-cov was found when a man was admitted in june to a hospital in jeddah, saudi arabia with overwhelming acute pneumonia with renal failure, and a sample of sputum grew a cytopathic virus that, on sequencing, proved to be a cov, classified as a betacoronavirus, and most closely related to two bat covs, hku- and hku- . over the next months additional cases ( fatal) were found, all but a few of them sporadic or hospital-based and in individuals living or traveling in the middle east. in the remainder of this chapter, the group of respiratory hcovs first discovered in the s and containing hcovs e, oc , nl , and hku- are referred to as community-acquired hcovs to distinguish them from the sars-cov and the mers-cov. in view of the prominence of covs in animal enteric diseases, there have been extensive efforts to identify enteric hcovs. there are figure - genome organization of representative human coronaviruses. all coronavirus genomes have the same basic structure and mechanism of replication. the ′ end of each genome encodes a leader sequence, which is attached to each virus-specific messenger rna transcript by a novel mechanism of discontinuous replication. the first two thirds of each genome encode replicase-associated genes. gene is translated as two large polyproteins, with the first expressed from orf a and the second from orf a/b following a − frameshift event. these polyproteins are then cleaved into individual proteins by two virus-encoded proteases. the major structural genes, the hemagglutinin-esterase (he), surface (s), envelope (e), transmembrane (m), and nucleocapsid (n) proteins, are indicated in green. the nonreplicase, accessory genes located at the ′ end of the genome are indicated with open boxes. the functions of these proteins are largely not known, and there is no sequence homology between accessory proteins of different coronaviruses. some of these proteins are virion associated, but none are required for virus replication. the open reading frames (orfs) encoding these proteins are numbered in order of appearance from the ′ end of the genome, with the exception that ns . of hcov-oc . i is an internal protein expressed from an alternative reading frame located within the n gene. it is equivalent to sars-cov-specific protein b and the mers-cov-specific protein b. (figure prepared by (dpp- ), which, like ace and apn, is an ectopeptidase that is abundantly expressed in the respiratory tract. all the community-acquired respiratory hcovs grow only with difficulty in tissue culture. despite this, both e and nl were discovered because they produced a detectable cytopathic effect, the first in human embryonic kidney and the second in llc-mk cells. both the sars-cov and the mers-cov were initially isolated and grew readily in vero cells. , hcovs oc and hku- have been grown in tissue culture after laboratory adaptation or in primary human airway epithelial cells. [ ] [ ] [ ] detection of all these viruses in clinical specimens is most conveniently and sensitively achieved using pcr. likewise, the enteric covs have been difficult to cultivate in vitro. all but a few strains have been detected only by electron microscopy of human fecal material. [ ] [ ] [ ] , some strains have been characterized by immune electron microscopy and found to be related to hcov-oc . two strains obtained from an outbreak of necrotizing enterocolitis in texas and passaged in intestinal organ cultures were reported to contain four or five proteins with apparent molecular weights similar to those of other covs but not related antigenically to known human strains or mouse hepatitis virus a . the evidence favors the view that these isolates, as well as particles antigenically related to hcov-oc , are members of the family coronaviridae, although their association with human disease is not yet proven. surveys of children with diarrhea using pcr imply that diarrhea associated with the four well-described hcov strains is unusual. , epidemiology evidence of community-acquired respiratory cov infections has been found wherever in the world it has been sought. in temperate climates, respiratory cov infections occur more often in the winter and spring than in the summer and fall. the contribution of cov infections to the total number of upper respiratory illnesses may be as high as % during times of peak viral activity. overall, the proportion of adult colds produced by covs may be reasonably estimated at %. early studies of hcov-oc and e in the united states demonstrated periodicity, with large epidemics occurring at -to -year intervals. strain hcov- e tended to be epidemic throughout the united states, whereas strain hcov-oc appeared in localized outbreaks. similar studies of nl and hku have not been done, but it seems from the available data that they also vary widely in incidence from year to year and place to place. reinfection is common and may be due to the rapid diminution of antibody levels after infection. infection occurs at all ages but is most common in children. the ratio of symptomatic to total infections varies between % and %, depending on the age of the population studied, the method of virus detection, and the definition of "infection. " , among adult volunteers % of those infected with hcov- e developed colds. the first report of a new cov causing severe pneumonia appeared in promed mail on september , . a man from jeddah, saudi arabia had developed pneumonia in june and died of respiratory and renal failure, and a virus was grown from a sputum sample that was subsequently sequenced and found to be a betacoronavirus thought at the time to be most closely related to bat covs hku and hku . between then and may , a total of cases occurred, all infected by this virus, now termed the middle east respiratory syndrome coronavirus (mers-cov). one hundred forty-five of the cases were fatal. more than of these have been acquired and diagnosed in the kingdom of saudi arabia, with most of the remainder in the united arab emirates, qatar, jordan, and kuwait. cases originating in the arabian peninsula have also occurred in travelers to egypt, tunisia, germany, italy, great britain, malaysia, the philippines, and the united states, with a few secondary cases sometimes occurring in those locations through close family or hospital spread. in the united states,these include two unrelated mers cases, and a third case related to one of these. a while infections with severe respiratory involvement have proteases to form several nonstructural proteins, including an rnadependent rna polymerase, methyltransferases, and a helicase, followed by either four or five structural proteins intermingled with a variable number of nonstructural and minor structural proteins. the first of the major structural proteins is a surface hemagglutinin-esterase protein, present on hcovs oc and hku and some animal betacoronaviruses, that may play some role in the attachment or release of the particle, or both, at the cell surface. this gene contains sequences similar to the hemagglutinin of influenza c virus, likely evidence of an interfamily recombinational event that occurred many years ago. the next gene encodes the surface glycoprotein that forms the petal-shaped surface projections and is responsible for attachment and the stimulation of neutralizing antibody. this is followed by a small envelope protein, a membrane glycoprotein, and a nucleocapsid protein that is complexed with the rna. there are several other open reading frames whose coding functions are not clear. the strategy of replication of covs is similar to that of other nidoviruses, in that all messenger rnas form a nested set with common polyadenylated ′ ends, with only the unique portion of the ′ end being translated. as in other rna viruses, mutations are common in nature, although the mutation rate is much lower, approximately × − per site per replication cycle. unlike other rna viruses, covs encode a ′- ′ exonuclease that has proofreading activities, playing a critical role in maintaining replication fidelity in cell cultures and in animals. covs are also capable of genetic recombination if two viruses infect the same cell at the same time. all covs develop exclusively in the cytoplasm of infected cells (fig. - ) . they bud into cytoplasmic vesicles from membranes of the pre-golgi endoplasmic reticulum. these virus-filled vesicles are then extruded by the exocytic secretory pathway. the resultant virus particles have a diameter of to nm on thin-section electron microscopy and to nm on negative staining. they are pleomorphic, with widely spaced, petal-shaped projections nm long (see fig. - ) . the cellular receptor for e and most other alphacoronaviruses is aminopeptidase n (apn). interestingly, nl , the other known human alphacoronavirus, uses as its cellular receptor angiotensinconverting enzyme ii (ace ), the same receptor as is used by the sars-cov. mouse hepatitis virus, a betacoronavirus related to strain oc , uses as its receptor a member of the carcinoembryonic antigen family. hcov-oc and bcov, which is closely related to hcov-oc , bind to -o-acetylated neuraminic acid as part of the entry process. the host cell receptor for mers-cov is dipeptidyl peptidase as %. there was no mortality in children or young adults younger than the age of years. in response to the global spread and associated severe disease, the world health organization coordinated a rapid and effective control program that included isolation of cases, careful attention to contact, droplet and airborne infection control procedures, quarantine of exposed persons in some settings, and efforts to control spread between countries through travel advisories and travel alerts. presumably as a result of these efforts, global transmission ceased by july . a few subsequent cases of sars were detected, but all were either a result of laboratory spread or individual cases related to presumed contact with civet cats or other intermediate hosts. the last known case occurred in mid- . spread of sars to humans is thought to have occurred primarily through droplet or contact transmission, with a possible role for fomites. in most instances, an individual case transmitted to very few others, although several well-documented instances of small-particle airborne transmission occurred, resulting in super-spreading events. spread in hospital settings appeared to be surprisingly efficient, but it could be effectively suppressed with the enforcement of droplet and contact precautions. containment measures were efficacious, in part, because patients were most contagious only after lower respiratory disease developed. the chain of spread was finally broken in the people's republic of china, the last country to experience endemic spread, in june . it now seems almost certain that the human epidemic began with the spread of a closely related bat virus first to palm civets or other animals sold in live wild game markets and then to humans in guangdong province in the people's republic of china, and that the virus adapted itself through mutation and possibly recombination, until it transmitted readily among humans. [ ] [ ] [ ] the virus that spread worldwide came largely from a single infected individual who traveled from occurred at all ages, the elderly and those with underlying conditions (diabetes, renal disease, immunosuppression) have been most often severely or fatally affected. the who and the cdc have published a case definition, as well as surveillance instructions to aid in epidemiologic control of the mers-cov. , the putative bat origin of this virus was strengthened by the finding that the virus grew readily in primary bat tissue culture. nevertheless, and while bats sampled in the middle east, africa, and europe were found to carry viruses closely related to the mers-cov, epidemiologic studies suggested there was likely to be at least one intermediate host. serologic and virologic studies indicated that camels in the middle east and africa were frequently infected by viruses very similar to some of those found in human mers cases. a acquisition of mers from camels appears likely, although the proportion of camel-acquired cases (versus those acquired from person-to-person contact or through another animal intermediate) is not clear. case clusters indicate that person-to-person hospital spread is more common than spread within families, and that casual-contact spread is unusual. a the sars epidemic began in guangdong province in the people's republic of china in mid-november . it came to worldwide attention in march when cases of severe, acute pneumonia were reported to the world health organization from hong kong, hanoi, and singapore. disease spread in hospitals to health care workers, visitors, and patients, among family members, and, on occasion, in hotels, apartment complexes, markets, and airplanes. worldwide spread was rapid but focal ( fig. - ) . the largest numbers of cases were reported from the people's republic of china, hong kong, taiwan, singapore, and toronto, canada. the overall case-fatality rates in these locations ranged from % to %, but persons with underlying medical conditions and those older than years of age had mortality rates as high involvement in other organ systems, including diarrhea, leukopenia, thrombocytopenia, and, most notably, pan-lymphopenia. virus has been detected in respiratory secretions, blood, stool, and urine specimens and tissue from the lung and kidney. on the basis of pcr testing, virus titer is highest during the second week of illness and can often be detected into the third week of illness and sometimes for as long as several months. , pulmonary symptoms may worsen late in the course of the illness, with the development of adult respiratory distress syndrome. there may also be late evidence of liver and kidney involvement. the pulmonary pathology of infection by the sars-cov has been described extensively, , , but little has been published about the pathology in other organ systems. [ ] [ ] [ ] the extrapulmonary pathologic changes found most consistently at autopsy are extensive necrosis of the white pulp of the spleen and a generalized small vessel arteritis. , in the lung, there is hyaline membrane formation, interstitial infiltration with lymphocytes and mononuclear cells, and desquamation of pneumocytes in the alveolar spaces. giant cells are a constant finding and usually have macrophage markers. in bronchoalveolar lavage, biopsy, and autopsy specimens viral particles have been noted in type i and ii pneumocytes. at this point, little is known about the pathogenesis of mers-cov because the infection was only recently described and no pathological specimens are yet available. it is anticipated that the pathologic changes in the lungs of patients with severe disease will be similar to those observed in patients with sars or other patients with acute respiratory distress syndrome (ards). almost all the antigenically distinct respiratory cov strains that were isolated in the s have been administered to volunteers, and all these produce illness with similar characteristics. , a summary of these characteristics is given in table - , in which a comparison is made with colds produced by rhinoviruses in similarly inoculated volunteers. the incubation period of cov colds was longer and their duration somewhat shorter, but the symptoms were similar. asymptomatic infection was sometimes seen and, indeed, has been a feature guangdong province to hong kong and infected a large number of individuals before himself succumbing to the disease. in contrast, the virus that was epidemic in the people's republic of china was more variable. although an etiologic role is not proven, enteric covs (or covlps) have been most frequently associated with gastrointestinal disease in neonates and infants younger than months. particles have been found in the stools of adults with the acquired immunodeficiency syndrome. , asymptomatic shedding is common, particularly in tropical climates and in populations living in poor hygienic conditions. the viruses can be detected for prolonged periods , , and without any apparent seasonal pattern. community-acquired respiratory covs (hcov- e, oc , nl , hku- ) generally replicate in ciliated and nonciliated (hcov- e) epithelial cells of the nasopharynx, probably producing both direct cell degeneration and an outpouring of chemokines and interleukins, with a resultant common-cold symptom complex similar to that produced by rhinovirus infection. the incubation period is, on average, days, and the peak of respiratory symptoms, as well as viral shedding, is reached at approximately or days after inoculation. the pattern of virus replication of covs must be at least in part determined by virus-receptor interaction. the two best-defined receptors for the respiratory covs are aminopeptidase n for strain hcov- e and angiotensin-converting enzyme ii for nl . the pathogenicity of sars is more complex and involves systemic spread. the route of infection of the sars-cov is probably through the respiratory tract. after an incubation period that is usually to days, but can be as long as to days, the disease begins, starting usually with fever and other systemic (influenza-like) symptoms, with cough and dyspnea developing a few days to a week later. although the lung is the focus of the disease process, there are often signs of levels decreased, suggesting that disease was partly immune mediated. clinical improvement was associated with the onset of a virusspecific antibody response. pediatric disease was, interestingly, significantly less severe than adult disease, although the features were similar. disease during pregnancy was severe, with high mortality in both the mother and fetus. congenital transmission was not described. the nature of the illness associated with enteric cov infection is much less clear. one study found a significant association of gastroenteritis in infants to months of age with the presence of covlps in the stool. another study, confined to infants in a neonatal intensive care unit, found highly significant associations between the presence of covlps in the stool and the presence of water-loss stools, bloody stools, abdominal distention, and bilious gastric aspirates. a further study of symptomatic infants shedding covlps pointed to possible differences between covlp-associated diarrhea and rotavirus diarrhea: although fever and vomiting were of similar incidence, stools were more often occult blood positive ( % in covlp-associated vs. % in rotavirus-associated disease), less often watery ( % vs. %), and more often mucoid ( % vs. %). finally, covs have been associated with at least three outbreaks of necrotizing enterocolitis in newborns, , , and the best characterized strains were isolated in infants with this illness. surveys seeking hcov rna by pcr using primers that would detect the known community-acquired respiratory hcovs in stool have been quite disappointing. in one study of fecal samples from children with gastrointestinal complaints tested over years, all four hcov species were found, but in all but of hcov-positive cases, either rotavirus or norovirus was also present. in addition, about half of the children in this survey had respiratory and gastrointestinal symptoms. in the same study, asymptomatic children were sampled and were positive. another study sampled symptomatic children, and were found to have hku rna. molecular studies using primers that would broadly detect new covs should be considered to resolve questions about the role of covs in human gastrointestinal disease. like many other viruses, covs have been sought as possible etiologic agents in multiple sclerosis. the search has been stimulated by the capacity of jhm, a well-studied strain of mouse hepatitis virus, to produce in mice and rats an immune-mediated chronic demyelinating encephalitis histologically similar to multiple sclerosis. hcov-oc , and hcov- e have been detected in brain tissue from multiple sclerosis patients using virus isolation, in situ hybridization, immunohistology, and pcr. moreover, t-cell lines established from patients with multiple sclerosis by stimulation with myelin basic protein or hcov- e were found to be cross-reactive with the opposite antigen, suggesting that molecular mimicry might be a possible pathogenic mechanism for the disease association. an adolescent boy with acute demyelinating encephalitis was found to have hcov-oc rna in both the respiratory tract and the cerebrospinal fluid. despite these intriguing reports, compelling evidence is lacking to establish an etiologic or pathogenetic association of covs with cns disease in humans. although some human respiratory covs grow in tissue culture directly from clinical samples and although antigen detection systems have been developed for both hcov-oc and hcov- e, , laboratory diagnosis of cov respiratory infections is best accomplished by molecular methods. reverse-transcriptase pcr (rt-pcr) systems have been developed using many different primers and detectors. from a clinical point of view, a single generic test for respiratory covs would be desirable, and such tests have been developed. however, when tested side by side with specific systems, the generic systems have a somewhat lower sensitivity. systems that combine primers and probes specific for several covs have also had considerable success. of both serologic surveys and pcr-based studies of natural infection of infants, children, and adults. , more serious respiratory tract illness is probably also caused by all four strains of community-acquired hcov. the evidence for this is not conclusive, but it seems likely that all strains can produce pneumonia and bronchiolitis in infants, , , , otitis and exacerbations of asthma in children and young adults, [ ] [ ] [ ] pneumonia in healthy adults, exacerbations of asthma and chronic bronchitis in adults, , both serious bronchitis and pneumonia in the elderly, , and pneumonia in the immunocompromised host. , hcovs are found in asymptomatic individuals of all ages, and, when accompanied by illness, are also sometimes accompanied by infections with other potential respiratory pathogens. these characteristics (infection without disease, coinfection during disease) are features of many respiratory pathogens, including particularly rhinoviruses, adenoviruses, human metapneumovirus, human bocavirus, and parainfluenza viruses, but also (although less frequently) respiratory syncytial virus and influenza virus. because infections with respiratory hcovs are so common, however, it is possible that they are responsible for a significant portion of these serious lower respiratory tract diseases, even though the basic pathogenicity of hcovs (judging from volunteer studies) is similar to that of rhinoviruses, and clearly less than that of respiratory syncytial virus, influenza viruses, and certain adenovirus types. there is some evidence that hcov-oc is more pathogenic in the elderly than hcov- e and also some evidence that infection with nl in children is different from the other respiratory hcovs in that several series have found an excess of children with croup. , information about the clinical presentation of patients infected with the mers-cov is limited. it is clear that there is a spectrum of illness with some infections consisting of mild upper respiratory symptoms only, and others characterized by cough and fever with progression to respiratory failure over about a week. , , , renal failure, as well as pericarditis and adult respiratory distress syndrome has been part of the reported clinical picture. the mers-cov host cell receptor, dpp- , is expressed at high levels in the kidney, raising the possibility that direct infection of this organ contributes to renal disease. a case definition that will lead to further epidemiologic studies has been published by the world health organization. severe acute respiratory syndrome coronavirus the first symptom in most cases of sars was fever, usually accompanied by headache, malaise, or myalgia. this was followed, usually in a few days, but as long as a week later, by a nonproductive cough and, in more severe cases, dyspnea. approximately % of patients had diarrhea. interestingly, upper respiratory symptoms such as rhinorrhea and sore throat usually did not occur. , , [ ] [ ] [ ] [ ] the chest radiograph was frequently abnormal, showing scattered air-space opacification, usually in the periphery and lower zones of the lung. spiral computed tomography demonstrated both ground-glass opacification and consolidation, often in a subpleural distribution. [ ] [ ] [ ] lymphopenia was common, , , with normal or somewhat depressed neutrophils. paradoxically, neutrophilia was associated with poor outcomes. the decrease in lymphocytes in the blood was most marked for cd cells but was seen in all t-cell phenotypes, including cd and cd , as well as natural killer cells. creatine kinase was often abnormal, as were lactic dehydrogenase and aspartate aminotransferase. levels of proinflammatory cytokines were elevated at early times during infection in patients with severe clinical disease and decreased in those patients who resolved the infection. approximately % of patients developed severe pulmonary disease that progressed to adult respiratory distress syndrome. adult respiratory distress syndrome with sars-cov infection was most likely to develop in patients older than years or with underlying disease such as diabetes, cardiac disease, and chronic hepatitis. , , , the overall mortality rate was between % and %, with the highest rates in the elderly and adults with underlying liver disease. in some patients, clinical deterioration occurred during the second week of illness, as virus ribavirin. it is now known that ribavirin has little activity against sars-cov in vitro, and there is no evidence that either intervention improved outcomes. , lopinavir/ritonavir and intravenous immune globulin were also used in some patients, again without conclusive evidence that they were helpful or harmful. there is anecdotal and at least partially controlled evidence of the benefit of either interferon-α or interferon-β treatment. further, treatment of sars-cov-infected cynomolgus monkeys with pegylated interferon-α resulted in improved outcomes, lending credence to the use of this therapy if sars recurred. treatment of mers-cov infection depends entirely on supportive measures. no antiviral drugs are recommended, although several studies have indicated that mers-cov is more sensitive to interferonα or interferon-β than sars-cov. , standard droplet precautions should be used, with aerosol precautions during certain high-risk procedures. rigorous application of hospital infection control procedures, particularly those directed at contact and droplet spread, was shown to have a major beneficial effect on the spread of the sars-cov. the containment of the global sars outbreak is a testament to the power of the cooperation and collaboration engendered by the world health organization to address a major public health threat. similar precautions are recommended for patients with suspected or confirmed mers-cov infections. vaccines for animal covs have been developed and widely used with variable efficacy. in one instance, a vaccine for feline infectious peritonitis appeared to lead to enhanced disease with subsequent natural infection. if sars does return or mers reaches epidemic proportions, an effective vaccine would be extremely helpful in control efforts, and a variety of vaccination strategies, including inactivated, subunit, and live-attenuated vaccines, are being pursued. , in addition, hospitals have been advised on improvement of infection control procedures so that in future epidemics of respiratory viruses, they will not be a major source of spread of infection, as occurred in the epidemic of sars. the mers-cov was originally isolated in vero and llc-mk cells, and there are several published methodologies for detection and identification by pcr. current recommendations from who are that definition of a possible case should be immediately reported to national authorities, and clinical, epidemiologic, and microbiologic investigations should be carried out. although sars-cov was grown from respiratory tract specimens in vero e and fetal rhesus monkey kidney cells, the more sensitive and rapid rt-pcr assays were most widely used to detect infection. virus was detected by rt-pcr in upper and lower respiratory tract, blood, stool, and urine specimens. early in the illness, specimens were found positive only in approximately one third of patients. use of samples from multiple sources increased the yield. virus was detected most frequently during the second week of illness. , , antibody tests have been developed using tissue culture-grown virus and indirect immunofluorescence or enzyme-linked immunosorbent assay. immunoglobulin m antibody can be detected in most patients for a limited period of time, and immunoglobulin g antibody appears first approximately days after onset of fever in patients with good outcomes and becomes essentially universal after weeks. laboratory diagnosis of the gastrointestinal covs depends now entirely on electron microscopy of stool specimens and detection of characteristic particles in negatively stained specimens. such testing is best performed in laboratories with extensive previous experience. given the severity of sars, clinicians throughout the world empirically treated most patients with corticosteroids and intravenous or oral the 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review of treatment effects pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus the spike protein of sars-cova target for vaccine and therapeutic development key: cord- -pcsuu authors: chan, kuan rong; ong, eugenia z; mok, darren zl; ooi, eng eong title: fc receptors and their influence on efficacy of therapeutic antibodies for treatment of viral diseases date: - - journal: expert rev anti infect ther doi: . / . . sha: doc_id: cord_uid: pcsuu the lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. in recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, ebola virus and hendra virus. the binding affinity of these antibodies can directly impact their therapeutic efficacy. however, we and others have also demonstrated that the subtype of fc-gamma receptors (fcγrs) engaged influences the stoichiometric requirement for virus neutralization. hence, the development of therapeutic antibodies against infectious diseases should consider the fcγrs engaged and fc-effector functions involved. this review highlights the current state of knowledge about fcγrs and fcγr effector functions involved in virus neutralization, with emphasis on factors that can affect fcγr engagement. a better understanding of fc-fcγr interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. the lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. in recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, ebola virus and hendra virus. the binding affinity of these antibodies can directly impact their therapeutic efficacy. however, we and others have also demonstrated that the subtype of fc-gamma receptors (fcgrs) engaged influences the stoichiometric requirement for virus neutralization. hence, the development of therapeutic antibodies against infectious diseases should consider the fcgrs engaged and fc-effector functions involved. this review highlights the current state of knowledge about fcgrs and fcgr effector functions involved in virus neutralization, with emphasis on factors that can affect fcgr engagement. a better understanding of fc-fcgr interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. while vaccination now protects against several important infectious diseases, many others still lack effective vaccines. the ability of viruses to rapidly mutate and alter their antigenic makeup (e.g., influenza and hiv), as well as the potential interference between different virus serotypes (e.g., dengue virus) has made vaccine development a challenging task. given that infection with such viruses can result in a significant number of cases and high mortality rates, rapid development of potent therapeutic measures can potentially save lives and limit the magnitude of outbreaks. with technological advances in the field of immunoglobulin g (igg) research and generally good safety profiles observed during antibody-based therapies, therapeutic antibodies have become attractive candidates for treating infectious diseases. the recent ebola outbreak in west africa is a good example of the potential of mab cocktails as an anti-infective strategy [ ] . other examples include the use of mab m . against hendra virus [ ] and palivizumab that targets respiratory syncytial virus (rsv) [ ] , resulting in significant reduction in the incidence of hospitalization. given the need for new antiviral therapies and considerable advances in igg research, there is a growing interest in developing therapeutic antibodies against infectious diseases. the binding affinity of antibodies to viruses can directly impact the efficacy of mabs [ ] , suggesting that target-specific mechanisms likely account for much of the efficacy of therapeutic mabs. however, many studies have also highlighted the contribution of fc-mediated immune effector functions in modulating the efficacy of these mabs [ ] . antigen-presenting cells such as dendritic cells, monocytes and macrophages can internalize antibody-opsonized viruses via fc-receptor-mediated phagocytosis. this results in antigen processing and presentation to effector cells of the adaptive immune system, eventually leading to virus clearance. on the other hand, neutrophils, monocytes, macrophages, mast cells and natural killer (nk) cells kill infected cells opsonized with antibodies in a process described as antibody-dependent cell-mediated cytotoxicity (adcc). while immune complexes are expected to be phagocytosed by fcreceptor bearing cells and cleared from the blood circulation, delivering viruses to monocytes or macrophages at subneutralizing antibody levels can result in antibody-enhanced infection due to their ability to serve as a host for viral replication. in support of this theory, subneutralizing levels of antibodies have been shown to result in increased infection of viruses such as dengue virus (denv), ross river virus, influenza, hiv- and epstein-barr virus (ebv) [ ] [ ] [ ] [ ] [ ] . hence, therapeutic antibodies ought to be delivered at doses high enough to prevent antibodyenhanced virus infection and avoid the cytokine storm that can result from excessive stimulation of fc-gamma receptors (fcgrs). given the delicate balance between antibody efficacy and adverse side effects, there is a need to develop methods that can better assess fc-mediated effector functions triggered by therapeutic antibodies. this could be beneficial in the future development of antibodies engineered to possess enhanced therapeutic activity [ , ] . here, we review the significance of the various aspects of fc-mediated effector functions involved in virus neutralization and factors that can potentially affect fcgr engagement. an improved understanding of these processes would allow identification of predictive factors to better assess the efficacy of therapeutic antibodies and facilitate development of engineered therapeutic antibodies with improved efficacy. among the different classes of immunoglobulins (iga, igd, ige, igg and igm), igg is the most commonly used and hence, will be the focus of this review. in general, antibodies consist of two distinct functional units: the fab and the fc regions (figure ). the fab portion of the antibody contains the variable region comprising three hypervariable complementarity-determining regions that bind specific antigenic epitopes. the binding affinity of the fab to viruses can be determined by binding assays such as elisa or by more specialized and precise methods such as biacore surface plasmon resonance. fab binding to viruses can block receptor interaction with the host, thereby hindering attachment and subsequent virus entry. in addition, several studies have suggested that antibodies that target regions other than the receptor-binding sites can also be neutralizing. for instance, antibodies that bind gp , a hiv- fusogenic transmembrane protein, did not inhibit virus-cell binding but was able to neutralize hiv- infection by recognizing fusionintermediate forms of gp at a postattachment step [ , ] . similarly, neutralizing antibodies that bind to west nile virus and denv have been shown to block infection at the postattachment step [ , ] . altogether, these investigations suggest that the fab portion of neutralizing antibodies can function by either blocking virus attachment or inhibiting virus fusion. by contrast, the fc portion of the antibody binds to fcgrbearing immune cells of the mononuclear phagocyte system such as monocytes, macrophages and dendritic cells. fcgrs have been shown to be important in modulating the efficacy of therapeutic mabs [ ] due to their involvement in fcgrmediated phagocytosis, cytokine production, adcc and complement-dependent cytotoxicity (cdc) that aids in virus neutralization (figure ). however, whether these fc effector functions are activated or inhibited depends on the subtypes of fcgrs engaged. among the human-activating fcgrs, fcgria has the highest affinity for both monomeric igg and immune complexes. fcgriia and fcgriiia, on the other hand, bind strongly to immune complexes but not to monomeric igg [ ] . both fcgria and fcgriiia require an accessory immunoreceptor tyrosine-based activation motif (itam) bearing gammachain for signal transduction. by contrast, the itam motif of fcgriia is located within its cytoplasmic tail (figure ). when activating fcgrs are clustered, src-related tyrosine kinases (e.g., src, fyn, fgr, hck and lyn) are activated and phosphorylated, resulting in itam phosphorylation that mediates recruitment and activation of protein kinases of the spleen tyrosine kinase (syk) and zap- family kinases [ ] . these activated kinases then catalyze the activation of downstream kinases, initiating a signaling cascade in several pathways to trigger immune effector functions. on the other hand, clustering of the inhibitory receptor fcgriib with activating fcgrs results in phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (itims) that recruit and activate phosphatases such as src homology (sh )-domain-containing inositol phosphatases ship- , ship- and sh -containing tyrosine phosphatases shp- [ , , ] . these phosphatases then dephosphorylate the kinases downstream of itam-mediated signal transduction to inhibit activating fcgr signaling (figure ). thus, coligation of inhibitory fcgrs with activating fcgrs results in abrogation of itam-mediated immune effector functions [ , ] . interaction of activating fcgrs with antibodies can directly affect efficacy of therapeutic antibodies. for instance, the efficacy of clinically approved anticancer mabs such as rituximab (anti-cd mab) and trastuzumab (humanized anti-her mab) was significantly reduced in mice deficient in activating fcgrs compared to wild-type mice [ , ] . this is in contrast to mice deficient in the inhibitory fcgrs, in which increased antibody-mediated cytolytic activity was observed [ ] . in addition, common single-nucleotide polymorphisms in fcgriia gene at position (rs ) and in fcgriiia gene at position (rs ) can lead to significant reduction in binding to igg [ ] and response to clinically approved mabs such as trastuzumab, rituximab and cetuximab (anti-egf mab) [ ] [ ] [ ] [ ] [ ] . furthermore, fc modifications and the use of f (ab) that alter the interaction between fc and fcgrs can directly affect fc-effector functions and hence therapeutic outcome [ ] . for example, hiatt et al. demonstrated that different glycan variants of palivizumab mediated different levels of adcc, affecting in vivo efficacy of palivizumab against rsv [ ] . overall, these studies suggest that therapeutic mabs directed against similar antigenic targets may differ in their clinical profile depending on fc-fcgr interaction. the neonatal fc receptor (fcrn) is a structurally distinct fc-receptor expressed in various cell types (e.g., vascular endothelium cells, monocytes and macrophages) [ , ] . fcrn binds to igg within the acidic environment of endocytic vacuoles but not at physiological ph. immune complexes that bind to fcrn are sorted to antigen-processing endosomes or recycling endosomes. however, monomeric iggs are sorted into recycling endosomes, which recycle iggs back to the cell surface following endocytosis, thus preventing intracellular degradation of iggs. this allows antibodies or immune complexes to persist in the blood circulation for up to several weeks after treatment. mutations at positions (thr gln) and (met leu) can significantly increase binding to fcrn and half-life of antibodies without affecting antigen-binding and fc-mediated effector functions [ , ] . while improved affinity to fcrn can extend the half-life of antibodies [ ] ; this has never been linked with increased clinical efficacy. however, in vivo studies with a hfcrn/rag knockout mouse model suggest that the antitumor activity of bevacizumab (anti-vegf) and cetuximab (anti-egf) can be significantly improved when binding to fcrn was increased [ ] . furthermore, recent studies have shown that administration of a broadly neutralizing antibody with enhanced fcrn affinity can improve protection against hiv- infection in nonhuman primates, not only by increasing serum half-life but also by enhancing transcytosis of antibodies to mucosal surfaces [ , ] . with slower clearance of antibodies and improved protection, less frequent dosing schedules would be expected, potentially reducing compliance issues. moreover, the extended half-life would ensure that the levels of circulating antibodies are kept sufficiently high for virus neutralization. this would also minimize the risk of antibody-dependent enhancement (ade) of virus infection, which would be particularly useful for viruses such as denv, where ade is hypothesized to result in increased viremia and increased risk of severe dengue [ , [ ] [ ] [ ] . however, the effects of improved half-life of these antibodies should be tested to avoid any potential toxic adverse effects. given the importance of fcgrs in mediating virus neutralization and fc effector functions, a better understanding of how therapeutic antibodies neutralize virus infections in fcgrbearing cells will impact implementation of dosing regiments and allow development of improved therapeutic antibodies against infectious diseases. here, we discuss the approaches that can be used to test for various fc-mediated effector functions and describe how these processes could contribute to efficient neutralization of viruses. virus neutralization in the presence of fcgr-mediated phagocytosis fcgr-mediated phagocytosis plays a critical role in clearing igg-opsonized pathogens from the systemic circulation and augmenting antigen presentation [ ] . cross-linking of fcgrs on phagocytes initiates downstream signaling that results in phosphorylation and activation of kinases such as phosphatidyl inositol -kinase, p s kinase and akt [ , ] , which are directly involved in reorganization of the actin cytoskeleton and membrane remodeling [ ] . antibody-opsonized viruses are then directed to lysosomes, where the antigens are processed into peptides. in human monocytes, fcgri has been shown to be involved in neutralization of virus immune complexes. for instance, reduction of antibody-opsonized hiv- infection in monocyte-derived macrophages was observed upon inhibition of fcgri [ ] . reducing fcgri but not fcgriia expression using small interfering rna also resulted in reduced antibody required for denv neutralization in monocytes, reinforcing the role of fcgri in denv neutralization [ ] . while the exact mechanism involved remains to be fully elucidated, activation of fcgri has been demonstrated to induce expression of proinflammatory mediators and is associated with trafficking of immune complexes that directly targets virus immune complexes to the late endosomes or lysosomes for enhanced antigen processing and antigen presentation to cd + t-cells [ ] . this can occur as early as min after fcgri cross-linking. in contrast, cross-linking of fcgriia resulted in slower trafficking of immune complexes, as trafficking to late endosomes or lysosomal compartments was not observed during the -min time interval [ ] . in the late endosomes or lysosomes, the viral proteins are degraded to peptide fragments that bind to mhc class ii molecules and presented to the plasma membrane for cd + t-cell activation. besides cd + responses, immune complexes have also been shown to increase cd + responses [ , ] as immune complexes can be shuttled to antigen-processing endosomes to stimulate the mhc class i cross-presentation machinery [ , ] . at present, the precise activating fcgrs involved in mhc class i cross-presentation still remain speculative. besides neutralization in antigen-processing endosomes, antibody-opsonized viruses internalized into the cell may also bind to tripartite motif-containing protein , a cytosolic igg receptor that targets antibody-opsonized viruses for proteasomal degradation via the e ubiquitin ligase activity [ , ] . of interest, this mode of neutralization has been shown to be particularly effective against nonenveloped viruses such as adenovirus [ , ] . the significance of antigen-presentation in the presence of antigen uptake has also been demonstrated in vivo, where protection mediated by antibodies was eliminated in mice with dendritic cells deprived of b- microglobulin, transporter associated with antigen processing (tap) or mhc-ii [ ] . however, igg-mediated uptake of viruses is not always favorable for the host. monocytes and macrophages are important sites of replication for viruses such as denv, ross river virus, influenza, hiv- and epstein-barr virus (ebv). increased internalization of antibody-opsonized viruses via fcgrs may, therefore, result in enhanced infection if these antibodies are not present at sufficient levels required for virus neutralization. this can occur via extrinsic or intrinsic ade [ ] . in extrinsic ade, immune complexes formed between viruses and subneutralizing levels of antibodies can lead to an increased number of infected cells due to increased uptake by fcgr-mediated phagocytosis. besides increased uptake, fcgr-mediated signaling can also increase production of anti-inflammatory cytokines such as il- that acts via the suppressor of cytokine signaling system to suppresses innate immunity, resulting in increased virus replication and output by infected cells [ ] . in addition, some viruses can also directly ligate other inhibitory receptors to suppress innate immunity. for instance, we have recently demonstrated that antibody-opsonized dengue can coligate the inhibitory receptor, leukocyte immunoglobulin-like receptor subfamily b member (lilrb ), to activate the phosphatase shp- that inhibits activation of signal transducer and activator of transcription (stat- ) and subsequent interferonstimulated gene (isg) expression [ ] . lilrb has also been shown to interact with other viral antigens such as human cytomegalovirus glycoprotein ul- [ , ] . although both fcgri and fcgriia can mediate ade of virus infection, fcgriia appears to do so more effectively [ , ] . hence, for pathogens that can replicate efficiently in monocytes and macrophages, it is important to ensure that therapeutic antibodies can neutralize these viruses even in the presence of fcgr-mediated phagocytosis to minimize the risk of ade. fcgr-mediated phagocytosis is affected by the size of immune aggregates, as immune complex size can affect the types of fc-receptors engaged [ ] . aggregation refers to the self-association of a number of molecules to form dimers, oligomers or even submicron to micron-sized complexes, which can be assessed by dynamic light scattering or nanoparticle tracking analysis [ ] . antibody concentration, affinity and stability can influence the size of immune complexes. our previous observations indicate that while small immune complexes coligate activating fcgrs, increased antibody concentrations can result in the formation of large immune aggregates that coligate the lowly expressed inhibitory receptor fcgriib to inhibit phagocytosis (figure ) [ ] . in line with our observations, morefield et al. observed that dendritic cells preferentially phagocytose smaller aggregates of~ mm compared with aggregates greater than mm [ ] . in another study by fifis et al. that used model polystyrene particles, smaller particles of < . um (especially - nm) were delivered more efficiently to dec + dendritic cells, triggered the production of higher antibody titers and promoted better type i cd and cd t cell responses when compared with larger particles [ ] . finally, antibody-mediated protection against cryptococcus neoformans was shown to be protective at optimal doses, but administration of large amounts of antibodies abrogates protection as phagocytic index was shown to decline at higher antibody concentrations [ ] . as such, determining whether the virus immune complexes can be neutralized intracellularly could potentially predict efficacy of the therapeutic antibody. these antibodies should preferably neutralize virus infections at levels that do not form viral aggregates, as this would ensure that virus neutralization occurs in the presence of fcgrmediated entry to result in enhanced antigen presentation and activation of adaptive immune responses. cytokine production triggered by activating fcgr signaling events promotes antiviral responses and activation of dcs, leading to increased antigen uptake and processing. in addition, these cytokines allow recruitment of monocytes and dcs to the site of infection so as to aid in virus clearance [ ] . activating fcgr signaling has been shown to phosphorylate spleen tyrosine kinase (syk) that results in up-regulation and activation of stat- , leading to up-regulation of cytokines and isgs critical for antiviral responses. while the intermediate signaling molecules have yet to be completely elucidated, we and others have shown that the activation of stat- can occur independently of interferon signaling [ , ] . the significance of stat- in immunity has been shown in stat knockout studies, where stat knockout results in significant defects in isgs production and dysregulation of tcell, macrophage and dc differentiation, resulting in t h -biased immune responses that delay viral clearance [ ] . besides stat- , protein kinase c expression and activation have been shown to be important in mhc class ii stimulation and presentation [ ] , a process pivotal for optimal cd + t-cell activation. the strength and nature of immune responses triggered by activating fcgrs, however, are determined by pairing of activating and inhibitory responses. activating fcgrs signal through itam to recruit and activate kinases to trigger immune effector functions. by contrast, inhibitory receptors signal through immunoreceptor tyrosine-based inhibitory motif (itim) to activate phosphatases that can inhibit itam function. for example, immune aggregates can coligate fcgriib, resulting in recruitment of phosphatases such as ship- and shp- that inhibit fcgrmediated phagocytosis [ , ] . as described previously, some viruses can also coligate inhibitory receptors such as lilrb to activate shp- to reduce cytokine and isg production. inhibitory receptors hence control a range of cellular responses mediated by activating fcgr-signaling, which include fc-effector functions such as fcgr-mediated phagocytosis, cytokine production, adcc and cdc. while inhibitory receptor coligation can potentially reduce the therapeutic efficacy of mabs, not all of these effects are undesirable. for instance, intravenous immunoglobulin can signal through fcgriib to induce antiinflammatory responses that can potentially reduce the risk of cytokine release syndrome in mab therapy [ ] [ ] [ ] . given the importance of kinases and phosphatases in mediating innate and adaptive immune responses, determining kinase and phosphatase profiles in fcgr-bearing cells mediated by therapeutic antibodies could hence predict the efficacy of these antibodies in controlling virus infection. although kinases triggered by igg and immune complexes are important for fc-mediated effector functions, overproduction of cytokines (e.g., il- b, tnfa, il- and il- ) due to immune complexes can result in immunogenicity-related adverse events [ ] . for instance, the phase i trial of the anti-cd mab tgn resulted in immune-mediated cytokine storm and multiorgan failure in healthy volunteers, highlighting the importance of assessing the likelihood of such adverse events [ ] . the need to assess the risk of such adverse events is even more crucial for engineered antibodies that have modified fc regions and increased binding to activating fcgrs. while improved fc-fcgr interactions can increase therapeutic efficacy of antibodies, this can also lead to increased risk of adverse events. hence, a cytokine signature encompassing the relevant proinflammatory and anti-inflammatory genes can potentially adcc is critical for clearance of antibody-opsonized cell surface receptor targets. when surface antigens are opsonized with antibodies, fc-receptor interaction with the host immune cells such as nk cells, monocytes, macrophages, neutrophils and mast cells can result in direct lysis of the infected cells, eventually leading to virus clearance. as many of these cell types express both activating and inhibitory fcgrs, fcgriib may hence affect activating fcgr-dependent adcc. among the activating fcgrs, fcgriiia is the most abundant fcgr expressed on nk cells, and adcc signaling is better characterized with fcgriiia relative to other activating fcgrs [ ] . the importance of fcgriiia in mediating adcc has been highlighted by studies showing a lack in clinical efficacy for several antibodies (e.g., infliximab and rituximab) in individuals with different fcgriiia polymorphisms [ ] . these observations were correlated with in vitro adcc analysis where peripheral blood mononuclear cells obtained from fcgriia h/h and/or fcgriiia v/v genotype showed higher adcc activity compared with those of other genotypes with lesser affinity to igg [ ] . previous research has also demonstrated the importance of adcc in viral defense and clearance. for instance, studies have shown both in vivo and in vitro the importance of adcc in controlling influenza, rsv, and hiv- infections in vivo and in human studies [ ] [ ] [ ] [ ] [ ] . complement-mediated activation by antibodies, on the other hand, can cause direct cell lysis by formation of the membrane attack complex (mac). this process, also termed as cdc, is initiated by binding of c q to antibody complexes. this results in the formation of membrane-bound c and c convertases that cleave and activate c and c , respectively. c cleavage by c convertase then forms c a and c b, where c b mediates the formation of membrane attack complex (mac) that can directly cause cdc by forming membrane pores on targeted cells. simultaneously, the cleavage of c results in the formation of c b that can be rapidly degraded into fragments ic b and c dg. deposition of these fragments on the target cells can then bind to cr expressed on leukocytes, leading to phagocytosis and adcc (figure ) [ ] , resulting in the neutralization of viruses. several clinically approved mabs have been shown to mediate their functions by activating complement. for instance, antibodies such as edrecolomab (antiepithelial cell adhesion molecule mouse mab) and alemtuzumab (anti-cd mab) can activate complement and mediate adcc [ , ] . several modifications to igg can directly affect binding to activating fcgrs and subsequent cellular activation. for instance, the igg triple mutant (s a/e a/l a) has been shown to increase binding to fcgriiia and adcc activity [ ] . fc variants (s d/i e and s d/i e/a l mutations) designed computationally possessed increased binding to fcgriiia and reduced binding to fcgriib, resulting in increased effector functions [ ] . the s d/i e/a l mutation, however, led to significant reduction in cdc [ ] . an anti-her antibody engineered to bear multiple substitutions (l v, f l, r p, y l and p l) also showed increased binding to fcgriiia and enhanced killing of her -expressing cancer cells [ ] . finally, given the critical role of fc-linked glycans in fcgr-binding, differences in glycan structure can affect binding and function of therapeutic antibodies. for example, afucosylated antibodies bind fcgriiia with better affinity and have enhanced ability to mediate adcc [ ] . while these modified antibodies can have improved efficacy, it is essential to ensure that they do not cause immunogenicity or adverse events when administered in patients. as such, assays to assess antidrug antibody responses are now a mainstay at most drug regulatory authorities for evaluating therapeutic antibodies during preclinical development. by improving binding to activating fcgrs and enhancing effector functions, fc modifications could benefit patients with fcgr polymorphisms that render them less responsive to therapeutic antibody treatment. enhanced adcc and cdc effector functions could also engage the immune system in a synergistic manner for more potent virus neutralization. the treatment of viral infections using therapeutic antibodies remains a challenging task. besides generating high-affinity antibodies against viruses, therapeutic antibodies should also trigger fc-mediated effector functions that facilitate virus neutralization. these effector functions include increased uptake via fcgr-mediated phagocytosis, increased cytokine production leading to enhanced antigen presentation and antiviral responses, increased adcc and cdc. combinatorial therapeutic strategies that trigger fc-mediated effector functions while occluding the interaction with immune inhibitory receptors could also enhance the clinical efficacy of therapeutic antibodies. finally, for viruses that infect and replicate within fcgr-bearing cells, therapeutic antibodies should mediate virus neutralization even in the presence of fcgr-mediated uptake, thus mitigating the risks of ade. with the increased focus on antibody-based therapeutics, there is a burgeoning need to develop robust methods that can rapidly and accurately assess antibody binding and fc-receptormediated effector functions. routine testing and measurement of fc-mediated effects should hence be considered for characterization of therapeutic antibodies, as this would allow selection and development of therapeutic antibodies with improved efficacy and reduced side effects. in addition, as the antiviral antibody responses are polyclonal and comprise different antibody isotypes, the effects of therapeutic antibodies in the presence of these antibodies should also be tested. given the importance of fc-fcgr interaction in antibodymediated effector functions, fc modification could lead to the development of therapeutic antibodies with improved interaction to activating fcgrs. this could enhance fcgr-mediated uptake, cytokine production, antigen presentation, adcc and cdc. future optimization to improve interaction with fcrn may also increase half-life of therapeutic antibodies, thereby reducing the risk of ade associated with the use of therapeutic antibodies against viruses that infect fcgr-bearing cells. with a growing number of methods to assess fc-mediated effector functions and technologies in antibody modification, we believe that more therapeutic antibodies against viral diseases will be developed in the near future. this will be useful for disease management, especially during epidemics. given the absence of licensed vaccines for many viruses, we believe that therapeutic antibodies will likely be important for treatment of viral infections, particularly during epidemics. we speculate that future advancement in antibody engineering resulting in improved fc-fcgr interaction will continue to aid the development of therapeutic antibodies that possess improved clinical efficacy. better and more robust methods that can predict fc functionality and fc-associated effects clinically will also continue to be essential to select for therapeutic antibodies with good efficacy but with minimal side effects. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. . the lack of effective vaccines against multiple infectious diseases necessitates the development of new therapeutics. . therapeutic antibodies are increasingly used for the treatment of infectious diseases as they are well-established and well-tolerated by humans. . therapeutic antibodies targeted against similar antigenic targets may differ in clinical profile, depending on the type of fcgrs that are engaged. . interaction between 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lack of fucose on human igg n-linked oligosaccharide improves binding to human fcgamma riii and antibody-dependent cellular toxicity papers of special note have been highlighted as: . of interest .. of considerable interest palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. key: cord- -n fpu s authors: Álvarez, e.; donado-campos, j.; morilla, f. title: new coronavirus outbreak. lessons learned from the severe acute respiratory syndrome epidemic date: - - journal: epidemiol infect doi: . /s x sha: doc_id: cord_uid: n fpu s system dynamics approach offers great potential for addressing how intervention policies can affect the spread of emerging infectious diseases in complex and highly networked systems. here, we develop a model that explains the severe acute respiratory syndrome coronavirus (sars-cov) epidemic that occurred in hong kong in . the dynamic model developed with system dynamics methodology included variables (five states, four flows, eight auxiliary variables, six parameters), five differential equations and algebraic equations. the parameters were optimized following an iterative process of simulation to fit the real data from the epidemics. univariate and multivariate sensitivity analyses were performed to determine the reliability of the model. in addition, we discuss how further testing using this model can inform community interventions to reduce the risk in current and future outbreaks, such as the recently middle east respiratory syndrome coronavirus (mers-cov) epidemic. middle east respiratory syndrome (mers), is a respiratory illness caused by a novel coronavirus (cov) [ ] . the disease was reported for the first time in saudi arabia in june and spread to several countries in africa, asia, americas and europe [ , ] . the capability of human-to-human transmission has been observed in at least four hospital settings [ ] [ ] [ ] [ ] . significantly, mers-cov shares certain similarities with the severe acute respiratory syndrome (sars)-cov that produced a global epidemic with more than human cases in - [ , ] . first, a number of patients infected with both viruses developed an acute respiratory disease that in some cases resulted in death [ , ] . in this sense, mers-cov appears to be highly pathogenic with an estimated case-fatality rate of around %, although this might be an overestimation as many infected patients may not have sought hospital assistance [ ] . second, both mers-cov and sars-cov, belong to the genus betacoronavirus and are closely related to coronaviruses isolated from bats [ ] [ ] [ ] . this strongly suggests that mers-cov and sars-cov may have been transmitted from bats to humans through intermediate species (e.g. camels for mers-cov and civet cats for sars-cov). third, since they are new emerging viruses, there are no effective vaccines or antiviral treatments. the sars epidemic is a clear example of how a networked health system can respond to a new threat to human health. one of the best-characterized outbreaks during the sars epidemic was in hong kong in where there were confirmed cases with deaths (who; http://www.who.int/csr/sars/country/en/index. html). the outbreak began in mid-february caused by an infected person who travelled from guangdong to hong kong [ ] . an important fact in the generation of a model of the outbreak is that the hong kong health authorities quickly implemented contagion control procedures [ ] . in general, two interventions were introduced to prevent the spread of sars-cov. the first was implementation of quarantine measures to isolate healthy people who had been in contact with infected people and therefore potentially in contact with the virus; isolating those that could be infected and asymptomatic during the incubation period and isolating and treating patients who had developed the disease. the other intervention was the application of protective measures by healthy people who were in contact with infected people to avoid becoming infected, such as respiratory protection for healthcare workers and daily disinfection of the environment of affected rooms [ ] . these control interventions were implemented progressively in the hong kong special administrative region from mid-march to late april [ ] . the application of these procedures allowed the rapid control of the outbreak in the subsequent months. in this sense, it is estimated that the epidemic in hong kong ended in late june. system dynamics has been proved to be a powerful instrument for analysing social, economic, ecological and biological systems [ ] . in addition, disease epidemiology has been studied using this approach, whereby system dynamics offers the practical application of concepts by computerized models that allow the systematic test of different scenarios and alternative policies [ ] [ ] [ ] . in this work, we performed a modelling of the hong kong sars-cov outbreak using system dynamics. the developed model contains five states, four flows, eight auxiliary variables and six parameters that interact through five differential and algebraic equations. the parameters of the model were optimized following an iterative process of simulation to obtain a model that largely fits the data available to the epidemic. moreover, the credibility of our model and its parameters are supported by both univariate and multivariate sensitivity analyses. the model reproduces how the implementation of control measures was effective in preventing the spread of infection to the rest of the population. basically, these measures result in a sustained reduction in the frequency of contacts. at present, the application of similar measures for infection containment can help to prevent the spread of new emerging epidemics, such as the outbreak caused by mers-cov. data on the total population of hong kong in was obtained from statistics of the census and statistic department, the government of the hong kong special administrative region (http://www.censtatd.gov.hk/home/). data on cumulative cases, deaths and recoveries during the sars epidemics in hong kong was obtained from global alert and response databases of the who (http://www.who. int/csr/sars/country/en/index.html). the model was developed following the four-step sequence proposed by system dynamics methodology [ ] . first, the real data from the hong kong sars epidemics ( fig. ) together with other evidences and our professional experience were used to create a mental modelling of the reality of the outbreak. second, the model structure that is able to explain the evolution of the epidemics was represented as a forrester diagram (fig. ) . third, the outbreak was mathematically modelled as a continuous dynamic process represented by a set of differential and algebraic equations (tables - ) . finally, the model was optimized to fit the real data from figure . a dynamic compartmental model provides a framework for the study of transport between different compartments of a system, i.e. well known epidemiological compartmental models [ ] . to explain how the protective measures taken by the government of hong kong allowed the rapid control of the epidemic we consider a dynamic model with five compartments (states) and four transitions (physical flows) between them. this model is based on two assumptions. first, the individuals are classified into five subgroups (susceptible, latent, infected, recovered, dead). although, the last subgroup is not strictly needed, it is used to keep an account of those dead. second, every day there is a different number of people: who are infected without symptoms of the disease (incidence); who develop signs and symptoms of the illness (sick per day); who recover from the disease (daily recovered), and who die (daily deaths) as consequence of the outbreak. the model structure built with vensim software (ventana systems inc., usa) ( fig. ) contains the five states and the four flows mentioned above and also eight auxiliary variables (inside circles) and six parameters (in bold). these elements are linked by the physical flows (double line with arrow) and by the information transmissions (single line with arrow) according to the mathematical model represented by the set of differential and algebraic equations (tables - ). the five differential equations in table establish the mass balance (inflows minus outflows) in the compartments, and as such, they describe the changes in the number of people (stocks) in the five subgroups. the four algebraic equations of table express that the physical transitions depend directly from the stock in the compartment of origin and indirectly from other stocks and parameters through the corresponding auxiliary variables. these equations involve three stocks (susceptible, latent, infected), three auxiliary variables (prevalence, contagion rate, recovery rate) and three parameters (incubation period, case fatality, disease duration). for instance, the incidence flow is proportional to the number of susceptible people, the prevalence and the contagion rate. the contagion rate or transmission coefficient ( ) prevalence recovery rate recovery rate(t) = ( − case fatality)/(disease duration) contagion rate contagion rate(t) = frequency of contacts(t) × infectivity cumulative cases cumulative cases(t) = dead(t) + recovered(t) + infected(t) people ( ) cumulative attack rate cumulative attack rate (t) = cumulative cases(t)/population(t) dimensionless ( ) frequency of contacts frequency of contacts(t) = daily contacts /( + (cumulative attack rate/ threshold of cumulative attack rate) ) basic reproductive number(t) = contagion rate(t)/recovery rate(t) dimensionless theoretically depends on the number of contacts per unit time and the probability of effective contact, i.e. the probability that a contact between an infectious source and susceptible host results in a successful transfer whereby the susceptible host becomes infected. the first three algebraic equations of table express the auxiliary variables (prevalence, contagion rate, recovery rate) used in the equations of table . in turn, these depend on other auxiliary variables, stocks and parameters. for instance, prevalence is defined as the ratio between number of infected people and the total population [ table , equation ( )]. the recovery rate depends on the illness duration and case fatality [ table , equation ( )]. equation ( ) in table expresses the contagion rate which is directly dependent on the auxiliary variable 'frequency of contacts' and the parameter 'infectivity'. one of the key variables in the model is frequency of contacts because it tries to reproduce the control measures carried out by the hong kong government to control the sars outbreak. we assumed that: ( ) the control measures gave, as consequence, a marked reduction of daily contacts in hong kong; ( ) the control measures were based on the cumulative attack rate, measured as the ratio between cumulative cases and total population [ table , equation ( )]. therefore, we decided to use the frequency of contacts as the daily contacts modulated by the repression hill function (fig. ), in accordance with the equation ( ) of table . despite the complexity level of biological systems several cases have been modelled using the hill function, in order to simulate repressor activities of enzymatic reactions and the regulation mechanisms of several transcription factors [ ] . in our model this repressor function allows the establishment of the relationship between frequency of contacts and the cumulative attack rate. note that the cumulative attack rate is used as an active repressor, so halfmaximal repression occurs when the cumulative attack rate is equal to the threshold, and almost total repression occurs when this cumulative attack rate is double the threshold. equation ( ) in table is used to report on the basic reproductive number (r ), which is defined as the expected number of secondary infectious cases generated by an average infectious case in an entirely susceptible population [ ] . in our model this number is calculated as the ratio between the contagion rate and the recovery rate. if r < , then the infected individuals in the total population fail to replace themselves, and the disease does not spread. however, if r > , the number of cases generally increases over time and the disease spreads. one of the most challenging issues in system dynamics modelling is to establish the value of the model parameters. the parameters can be estimated taking advantage of the known information available in the literature and can be optimized by means of an iterative process of simulation. using this approach, we set values for the six parameters of our model which are summarized in table . infectivity expresses the ability of the pathogen to penetrate, survive and multiply in the host and it is measured through the secondary attack rate which is defined as the probability that infection occurs in susceptible persons within a reasonable incubation period following known contact with an infectious person or an infectious source. epidemiological studies in ( ) of table ]. the dotted line corresponds to the threshold repression function (when n tends to infinity). the representation is normalized to the threshold of cumulative attack rate on the abscissa axis and the value of the discontinuity (daily contacts) on the ordinate axis. singapore showed that % of individuals infected with sars-cov did not cause secondary infections, suggesting low infectivity [ ] . after the optimization process, the final value set for infectivity was · , which is in agreement with a previous work examining the probability of transmission for sars-cov [ ] . the incubation period of the disease, during which time the individual is asymptomatic appears to be between and days with an average value of · days [ ] . similarly, although the duration of the disease is variable depending on the case, the average of the most severe and the mildest cases is days [ ] . during this time period the virus can be transmitted to other people. several studies have shown that the case fatality of sars-cov was also variable in the outbreak depending on different factors. it has been estimated that the mean case fatality for sars in hong kong was around · [ ] . social contact patterns have been shown to be highly relevant on the transmission dynamics of respiratory infections such as measles, rubella and influenza [ ] [ ] [ ] . quantifying this parameter is critical for estimating the impact of such infections, for designing and targeting preventive interventions, and for modelling their impact. in our model the daily contacts parameter, indicating the mean number of contacts of a person in day, was set to · after the optimization process, which is in agreement with previous studies quantifying these social mixing patterns [ , ] . the parameter 'threshold of cumulative attack rate' is critical in our model since it allows setting the value of the cumulative attack rate at which control measures were established by the hong kong authorities. this parameter was finally set at · × − . therefore, according to the hill function of figure , for any number of the cumulative attack rate below the cases by million, the frequency of contacts will be greater than · (half of the daily contacts), and in the opposite case the frequency of contacts will be markedly reduced. as explained before, our dynamic model was subjected to successive rounds of simulation and optimization in order to fine-tune the parameters of the model. in all these simulations we assumed that the hong kong epidemic originated from a traveller from guangdong in china [ ] , and at the beginning of the outbreak the entire population of hong kong was susceptible to sars-cov infection. these assumptions are represented by the initial values in the five stocks (table ) . moreover, based on the reported data, the simulation period was set to days with a time step of day. parameters of system dynamics models are subject to uncertainty. sensitivity analyses were conducted to provide insight into how uncertainty in the parameters affects the model outputs and which parameters tend to drive these variations. in this task it is essential to define the probabilistic distribution patterns of the model parameters, which are shown in table , together with the references that support these patterns. the most influential parameters were estimated by univariate analyses, in which changes in the model output were studied after disturbance in each parameter value independently. in complex models, univariate sensitivity analysis can be insufficient for a comprehensive study of the model. simultaneous fluctuations in the value of more than one parameter may create an unexpected output change due to nonlinear relationships in different model components. thus, to test the influence of simultaneous changes in the model parameters, the univariate analyses were followed by monte-carlo multivariate sensitivity analysis, in which the values of the six parameters were altered at the same time. modelling process, simulations and sensitivity analyses were performed using vensim dss software v. . a (ventana systems). figure shows a graphic comparison between the simulation results using the parameters of table and the real data of the hong kong outbreak. we compared only the variables of the model for which records were found in the public databases, which are the same six variables shown in figure . the simulation output for the variable 'sick per day' fit the data reported by the hong kong authorities (fig. a) , suggesting that the model was able to reproduce the epidemic curve. we observed that the number of new cases per day obtained in the simulation grew during the first days. from this time, the number of new infections gradually fell to values < at later stages of the outbreak. as a consequence, the auxiliary variable that stores the cumulative sars cases showed a characteristic sigmoidal growth (fig. b) , consistent with the real data. we observed that the number of sars cases grew from one at the beginning of the epidemic to around at the final stage of the outbreak, similar to the cases reported by the authorities. moreover, we observed a high fit between the model predictions and the real data. as expected, the number of deaths and recovered people in the simulation also grew with a sigmoidal shape to reach values similar to those found in the public databases (fig. c, d) . however, we observed a partial fit of these stock variables to the data reported during the outbreak. these slight mismatches were also observed in the flow variables for recovered and daily deaths (fig. e , f) that may be due to delays in reporting of cases by the authorities. in short, looking the simulation results of figure we can conclude that our model is able to reproduce largely the most important indicators of the sars epidemic that occurred in hong kong in . focusing on the evolution of the basic reproduction number (fig. ) , we note that during the early stages of the epidemic, r > , which is consistent with the observed disease spread. moreover, after day , r starts to drop to values < , probably due to the implementation of containment measures by the hong kong authorities after the issuance of the first global alert against sars on march . these results are consistent with a previous report showing the basic reproductive numbers for different sars epidemic curves, which supports the notion that our model is able to largely replicate the disease outbreak in hong kong [ ] . the results of the univariate sensitivity analysis are shown in figure . we focused our attention on the epidemic wave although the analysis is possible in other variables, as shown in the multivariate analysis of figure . variations in case fatality, threshold of cumulative attack rate and disease duration induced little changes in the epidemic curve (fig. -c) , while more extensive alterations in the epidemic wave were observed after changes in infectivity, daily contacts and incubation period (fig. d-f) . variations in the case-fatality parameter do not alter the output of the variable sick per day (fig. a) , although other variables from the model such as recovered and dead are highly impacted (data not shown). small changes in the shape and the maximum of the curve are observed after modification of the parameters 'threshold of cumulative attack rate' and 'disease duration' (fig. b, c) . by contrast, changes in infectivity, daily contacts and to a lesser extent in incubation period significantly alter both the position and height of the maximum of the epidemic curve (fig. d-f ) . the results of the multivariate sensitivity analysis are shown in figure , we analysed the output of four variables: sick per day, infected, recovered, and dead ( fig. a-d, respectively) . variations in the model parameters clearly change the shape of the epidemic curve, altering both the position and the height of the maximum of this variable (fig. a) . similarly, the output of the variables infected and recovered is highly impacted by the changes in parameters carried out in the multivariate sensitivity analysis (fig. b, c) . the alterations of variable outputs are clearly exemplified by the variable 'dead' (fig. d) . certain changes in the parameters of the model can explain an increase in the total number deaths during the epidemic rising from about to about . the observed variations in model output when the value of the parameters is changed support the idea that this model might be able to simulate different scenarios and epidemic conditions. system dynamics modelling has been successfully applied to study complex public health issues such as the design of optimal policies in healthcare [ ] , the impact of public health intervention in different situations [ , ] , and to study disease epidemiology [ ] [ ] [ ] . in the latter, system dynamics technology has become a powerful tool to understand and predict the impact of infectious diseases. epidemiological models can help health authorities to make recommendations regarding intervention to fight the spread of directly transmissible pathogens, especially when empirical data is limited. in this sense, mathematical models have been previously used to advise health policies against diseases such as pandemic influenza [ , ] and sars [ , , ] . several models studying sars transmission and interventions have been published. these are detailed hybrid stochastic and compartmental models that successfully explain the behaviour of the epidemic [ , , ] . here, trying to follow ockham's razor principle, we have built a simpler deterministic model, which is also able to reproduce the behaviour of the epidemic based on its natural history and the intervention measures taken in hong kong. the use of a less complicated model could be helpful in understanding the disease epidemic and also facilitating its reuse under other conditions. the epidemiological models depend on the consistency of the chosen parameters. therefore, the accurate quantification of these parameters is critical to estimate the path of a disease, to predict the impact of possible interventions, and to inform planning and decision making. here, we have combined reported information from the sars epidemic with an iterative optimization process to set the final values for the model parameters. under these conditions, the model output fits the epidemic curve observed in the hong kong sars-cov outbreak (fig. ) . of the factors that influence the dynamics of infectious diseases, the person-to-person contact pattern has been shown to be essential in disease spread [ ] . our model takes this essential factor into account through the auxiliary variable 'frequency of contacts', which is dependent on the auxiliary variable 'cumulative attack rate' and the parameters 'daily contacts' and 'threshold of cumulative attack rate' (fig. ) . a previous work showed that mixing patterns and contact characteristics were remarkably similar across the different european countries analysed in that table . the solid black line represents the simulation output and the grey area represents the % confidence bounds. study even though the average number of contacts recorded differed. interestingly, the authors suggests that the results may well be applicable to other countries with similar social structures, and that the initial epidemic phase of an emerging infection in susceptible populations, such as sars was in , is likely to be very similar [ ] . during the sars outbreak, health authorities, hospitals, and the overall population progressively implemented quarantine and protection policies to prevent the transmission of the disease [ ] . with this in mind, we made the assumption that the intervention of the health authorities caused a decrease in the frequency of contacts, which in turn led a decrease in the rate of contagion. to mimic this event in our model, the number of daily contacts is regulated by means of a repression hill function, which has been used to simulate repressor activities in complex biological systems [ ] . the basic reproductive number is a key epidemiological variable that characterizes the potential of a disease to spread. several works have estimated that prior to the first global alert the basic reproductive number for sars was > , correlating with an exponential increase in the number of cases. however, the implementation of effective control measures, such as quarantine, isolation, and strict hygiene practice in hospitals led a sudden decrease in r [ , ] . the fact that, in our model, r drops to values < around the date of the global alert reinforces the idea that a decrease in the frequency of contacts is able to effectively simulate the effects of the control measures established in the first stages of the epidemic. it is important to note that estimations of r in previous publications are considerably lower (around - ) than ours, which is almost at the beginning of the outbreak [ , , ] . nevertheless, the estimated value of r also differs in these works and the credible intervals surrounding these deterministic estimations were wide, reaching superior values of almost . this high variability can be explained in part by the superspreading events that occurred in sars epidemics. superspreading is an unusual situation, in which a single individual directly infects a large number of other people that has a large influence on the early course of the epidemic [ ] . interestingly, the fact that our model does not explicitly account these events could partially explain the very high estimated value of r at the beginning of the outbreak. the reliability of the model parameters is supported by univariate and multivariate sensitivity analyses (figs and ) . furthermore, sensitivity analysis is a powerful tool to analyse the influence of certain decision making in the evolution of the epidemic. taken together, these findings strongly suggest that table . the solid black line represents the simulation output and the grey area represents the % confidence bounds. our model, together with other system dynamics models can be used by epidemiologists to investigate the likely consequences of future re-emergences of sars-cov based on analysis of the previous known epidemics. in addition, by adapting the key parameters of these models or with a little change in the model structures, they can be used to face emerging outbreaks of infectious diseases, such as the recent mers-cov epidemic. in this regard, there are several similarities and differences which should be taken into account when using this model. both sars-cov and mers-cov may cause severe respiratory failure, extrapulmonary features such as diarrhoea and also mild or asymptomatic cases. in contrast with sars, mers has lower human-to-human transmission potential, affects predominantly older people with more comorbid illness and has a higher case-fatality rate [ ] . these factors would affect the output of the model variables (e.g. epidemic curve, cumulative cases, number of dead, etc.). middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) -worldwide isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence of person-to-person transmission within a family cluster of novel coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) investigations and implications for the prevention of 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social contacts and mixing patterns relevant to the spread of infectious diseases different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures using system dynamics to help develop and implement policies and programmes in health care in england work disability related to musculoskeletal pain: a system dynamics approach evaluating hmo policies with a computer simulation model prevention and rehabilitation as a means of cost containment: the example of myocardial infarction modelling the epidemiological consequences of hiv infection and aids: a contribution from operational research model based scenarios for the epidemiology of hiv/aids: the consequences of highly active antiretroviral therapy containing pandemic influenza at the source cost-effective strategies for mitigating a future influenza pandemic with h n characteristics real-time estimates in early detection of sars entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation transmission dynamics and control of severe acute respiratory syndrome mathematical models of infectious disease transmission severe acute respiratory syndrome vs. the middle east respiratory syndrome this research was supported by an intramural contract from universidad autónoma de madrid awarded to e. Álvarez. the institutional grant awarded to the centro de biología molecular 'severo ochoa' by the fundación ramón areces is also acknowledged. none. key: cord- -lk ao m authors: annamalai, thavamathi; saif, linda j.; lu, zhongyan; jung, kwonil title: age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: lk ao m porcine epidemic diarrhea (ped) is an enteric coronaviral infection that causes severe morbidity and mortality in suckling pigs, but less severe disease in older pigs. consequently, it causes significant economic losses to the pork industry. there are limited studies on the innate immune responses to ped virus (pedv) in pigs. the aims of our study were to investigate differences in innate immune responses to pedv infection in suckling and weaned pigs and to examine if disease severity coincides with reduced innate immune responses. weaned -day-old pigs (n = ) and -day-old nursing pigs (n = ) were assigned to pedv inoculated or uninoculated control groups. the pigs were observed daily for clinical signs, virus shedding and were euthanized at post-inoculation days (pids) and to assay immune responses. blood samples were collected at pids , and . the natural killer (nk) cell frequencies, nk cell activities (lysis of target k tumor cells in vitro), cd +cd + t cell and cd +cd + t cell frequencies were measured in blood and ileum at pids and . the pedv infected suckling pigs showed severe diarrhea and vomiting at pid , whereas the pedv infected weaned pigs showed milder clinical signs starting at pid . pedv infected suckling pigs had significantly higher diarrhea scores, earlier fecal pedv rna shedding and significantly higher viremia (viral rna in serum) compared to weaned pigs. there was no mortality in either infected suckling or infected weaned pigs. the control pigs not inoculated with pedv did not show any clinical signs and no detectable fecal or serum pedv rna. strikingly, pedv infected suckling pigs had significantly lower nk cell frequencies, undetectable nk cell activity and lower ifnγ producing nk cells in blood and ileum compared to pedv infected weaned pigs. pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from those of pedv infected weaned pigs and coincided with onset of fecal pedv rna shedding and serum pedv rna titers. the infected suckling pigs have higher and earlier increases in serum ifnα, but lower serum il- and tnfα levels compared to infected weaned pigs. cd +cd + t cell frequencies were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in cd +cd + t cell frequencies. in conclusion, the observations of impaired lytic activity and ifn-γ production by nk cells in suckling pigs coincided with the increased severity of pedv infection in the suckling pigs compared with the weaned pigs. porcine epidemic diarrhea virus (pedv) is an enteric coronavirus (genus alphacoronavirus, family coronaviridae, order nidovirales) causing significant morbidity and mortality in suckling pigs. pedv was first diagnosed in the usa in may, (stevenson et al., ) and has spread throughout the usa and was also reported in mexico and canada (vlasova et al., ) . the estimated annual economic losses in the us from pedv is $ million to $ . billion (paarlberg, ) . pedv causes severe enteric disease in suckling pigs (chen et al., ; stevenson et al., ) , but milder disease in older weaned pigs . this is similar to earlier observations for another enteric coronavirus infection of pigs, transmissible gastroenteritis (tge) (saif et al., ) . therefore, similar to tge virus (tgev) infection, biofeedback of intestinal contents of affected pigs to older pigs to build herd immunity is considered as an important method to reduce losses from pedv (jung and saif, ) . viral infections induce both innate and adaptive immunity. innate immunity involves production of cytokines and interferons as well as recruitment of innate immune cells such as http://dx.doi.org/ . /j.vetimm. . . - /© elsevier b.v. all rights reserved. dendritic cells, macrophages and natural killer (nk) cells (rouse and sehrawat, ) . the innate immune response plays a significant role in controlling primary viral infections and in development of adaptive immune responses (aoshi et al., ; janeway and medzhitov, ) . nk cells are innate immune cells that display cytotoxic action against virus infected host cells and tumor cells (campbell and hasegawa, ; herberman et al., ; trinchieri, ) and thus play an important initial role in containing the viral infection. nk cells are also a major source of certain cytokines such as ifn␥ and tnf␣ (fauriat et al., ; vivier et al., ) . they play a key role in initial clearance of infection in viral diseases (brandstadter and yang, ) . cytokines are important in viral infections in that they are necessary for cell to cell communication for inflammation and immune responses (akira and kishimoto, ) . the early cytokines secreted during a viral infection help to modulate immune responses. the cytokines examined in this study are early cytokines that have mainly proinflammatory and antiviral action. interferons are a group of cytokines whose major function is antiviral activity (isaacs and lindenmann, ; wheelock, ) . ifn␣ is a type i interferon produced by most cells in response to viral infection, with the major source being innate immune cells such as monocytes and dendritic cells (trinchieri et al., ) . ifn␥ is a type ii interferon produced initially by innate immune cells such as macrophages, dendritic cells and nk cells, and later on by activated t cells (sen, ) . ifn␥ is important in enhancing the activities of phagocytic cells such as macrophages and nk cells (carnaud et al., ) . il- is produced by various cell types and is a proinflammatory cytokine due to its chemoattractive properties for inflammatory cells (arndt et al., ; huber et al., ) . il- is a proinflammatory cytokine secreted by th cells, as well as ␥␦t cells (innate immune cells in mucosa) (jin and dong, ) . it stimulates the inflammatory response to viral infections (ryzhakov et al., ) . il- is a proinflammatory cytokine produced mainly by phagocytic cells and is involved in activation of nk cell activity including ifn␥ production by nk cells (trinchieri, ) . tnf␣ is a proinflammatory cytokine secreted mainly by macrophages that regulates cell death, differentiation and inflammation (bradley, ) . studies of rotavirus infection of children showed that the cytokine responses varied depending on severity of clinical signs in individuals (jiang et al., ) . studies of human rotavirus infected gnotobiotic pigs showed similarly that the proinflammatory cytokine responses were more marked with virulent virus compared with attenuated virus infection (azevedo et al., ) . the above cytokines were examined in the present study to understand if differences in proinflammatory cytokine responses between suckling and weaned pigs may be involved in susceptibility of suckling pigs to severe disease by pedv infection. there is a lack of information on innate immune responses of young and older pigs to pedv infection that might explain some of the differences in disease severity between young and older pigs. in the present study, we investigated the innate immune responses such as cytokine and nk cell activity as well as changes in frequencies of t cells to examine if differences coincide with the higher disease severity of suckling versus weaned pigs. the virus inoculum used in this study was the wild-type virulent us pedv strain pc a which was from the intestinal contents of a pedv positive field piglet, then serially passaged two times in gnotobiotic pigs (jung et al., ) . the original sample was negative by pcr for other enteric viruses such as tgev/porcine respiratory coronavirus (prcv), porcine deltacoronavirus, rotavirus groups a, b, and c, porcine enteric caliciviruses, st-valerien-like viruses, porcine astroviruses, enterovirus, kobuvirus, and bocavirus (amimo et al., a,b; chung et al., ; jung et al., b; kim et al., ; sisay et al., ; wang et al., ) . immune electron microscopy of the original sample using gnotobiotic pig hyperimmune serum to pedv showed only pedv particles. the gnotobiotic pig passaged pc a intestinal contents were diluted in minimum essential medium (mem) and used as inoculum in this study as noted below. seronegative pregnant sows and -day-old, pedvseronegative weaned, large white × duroc crossbred pigs were obtained from a pedv-free specific pathogen free (spf) (confirmed by history, lack of qrt-pcr-pedv positive fecal samples and pedv antibodies) swine herd of the ohio state university. the spf osu herd was also seronegative for antibodies to porcine respiratory and reproductive syndrome virus, prcv, tgev and porcine circovirus type . the sows farrowed naturally and nursed their piglets until the end of the study. the four experimental groups in the study were as follows. group : pedv inoculated -day-old suckling pigs (n = ); group : mem only inoculated -day-old suckling pigs (n = ); group : pedv inoculated day-old weaned pigs (n = ); group : mem only inoculated -day-old weaned pigs (n = ). all experimental procedures on animals were approved by the institutional animal care and use committee of the ohio state university. pigs in pedv groups were inoculated orally with pedv inoculum [ . log ge (genomic equivalents) (≈ . log plaque forming unit)/pig] and pigs in mem only inoculated groups received mem. the inoculation dose was based on a previous pathogenicity study in our lab (jung et al., ) . following pedv inoculation, pigs were monitored for clinical signs daily until necropsy. diarrhea was assessed by scoring fecal consistency. fecal consistency was scored as, = solid; = pasty; = semi-liquid; = liquid, with scores of or more considered diarrheic. inoculated and mock pigs (n = - /group at each time-point) were euthanized for immunological studies at an acute stage on post inoculation day (pid) and at a later stage (pid ) of infection. blood samples were taken at pid (n = - pigs per group), pid (n = - pigs per group) and pid (from euthanized pigs, n = - per group) and separate serum aliquots were prepared for cytokine analysis and viral rna quantification. rectal swabs were collected from all pigs on the designated pids to determine fecal ped viral shedding (pedv rna quantified by rt-qpcr). two rectal swabs were suspended in ml mem (jung et al., ) . the rna was extracted from l of serum or supernatants following centrifugation of the fecal suspensions ( × g for min at • c), using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. pedv rna titers in rectal swab supernatants and sera were determined by rt-qpcr as described previously (jung et al., ) . blood and ileum were collected on the day of euthanasia and processed for isolation of mnc as previously described (yuan et al., ) . the isolated cells were resuspended in rpmi medium (roswell park memorial institute medium) containing % fetal bovine serum, mm l-glutamine, mm sodium pyruvate, . mm suckling pigs had more severe clinical signs, earlier fecal pedv rna shedding and higher serum pedv rna compared to weaned piglets. following pedv inoculation, pigs were monitored every day and rectal swabs and blood for serum were collected at pid , and . fecal consistency scores of or more considered diarrheic. fecal shedding and serum pedv rna were determined by rtqpcr. the detection limit of rt-qpcr was genomic equivalents (ge) per reaction, corresponding to . log ge/ml of rectal swab fluid or . ge/ml of serum. therefore, viral rna titers more than . log ge/ml in rectal swab fluid or . ge/ml in serum are considered positive. statistical analysis was done to compare suckling and weaned pigs (across rows) for different parameters measured. values with different alphabetical superscript within a parameter and time point are considered different at p < . . the uninoculated pigs did not show any clinical signs. the rectal swab fluids and serum of uninoculated pigs were tested at similar time points and no pedv rna was detected. nonessential amino acids, mm hepes and antibiotics (e-rpmi) and used for assays. k (human erythroleukemia cell line) tumor cells were used as target cells and the assay was done as described previously with a few modifications (cao et al., ; park et al., ) . the k cells were initially stained with carboxy fluorescein succinimidyl ester (cfse) (ebioscience, usa), washed and used for the assay. mncs from blood and ileum were used as effector cells. effector: target cell ratios of : , . : and . : were used. the cells were mixed at the specified ratios and incubated overnight in e-rpmi at • c. the cells were then incubated with -aminoactinomycin d ( -aad) (life technologies, usa) for min at • c to stain dead cells. the cells were examined by flow cytometry and the percentage of cfse positive cells that were also stained with -aad were assessed as dead k cells. cfse labeled k cells incubated without mncs and stained similarly with -aad were used as controls for spontaneous death of k cells. the procedure was followed as described previously (chattha et al., ; yuan et al., ) with a few modifications. mononuclear cells from blood and ileum were cultured for h at • c in e-rpmi. the protein transport inhibitor, brefeldin a ( mg/ml; sigma-aldrich, usa), was added for the last h to prevent secretion of ifn␥ produced by the cells. the cells were stained with cd -fitc (fluorescein isothiocyanate) (clone ppt ; southern biotech, birmingham, al, usa), cd -sprd (spectral red) (clone - - ; bd biosciences, usa), and cd -biotin followed by streptavidin apc (allophycocyanin) (bd biosciences, usa) as secondary antibody. samples were stained intracellularly with anti-porcine ifn-␥-pe (phycoerythrin) (clone p g ; bd biosciences, usa). cd -cd -cd + ifn-␥+ cells were expressed as percentage of cd -cd -cd + nk cells. isotype antibody-labeled cells were used as controls. to determine the frequencies of t helper cells (cd +cd +), cytotoxic t cells (cd +cd +) and nk cells (cd -cd -cd +), cell samples were stained with anti-porcine cd -fitc, cd -pe (clone - - ; bd biosciences), and cd -sprd for min at • c. the frequencies of t cells or nk cells were expressed as percentage of lymphocytes expressing the respective markers. cells stained with isotype antibodies were used as controls. serum was separated by centrifuging blood at × g for min, and the collected serum was stored at − • c until tested. il- , il- , il- and ifn␣ were measured as previously described (azevedo et al., ; chattha et al., ) . for tnf␣, a porcine tnf␣ elisa kit was used per manufacturer's recommendations (kingfisher biotechnologies, st. paul, mn). all values are expressed as the means ± standard error of the means (sem). fecal consistency scores and viral rna titers in rectal fluids were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). nk cell activity, nk cell numbers, t cell numbers and cytokine amounts were analyzed by one-way anova using graphpad prism software. a value of p < . was considered statistically significant. . . suckling pigs had more severe clinical signs, earlier fecal pedv rna shedding and higher serum pedv rna titers compared to weaned pigs the suckling pigs showed severe diarrhea and vomiting at pid , whereas the weaned pigs showed milder clinical signs starting only at pid ( table ). the fecal consistency scores were significantly higher in suckling pigs compared to weaned pigs at all time points examined (p < . ) ( table ). the fecal shedding pedv rna titer was high in suckling pigs starting from pid , whereas the weaned pigs started shedding pedv rna at pid and shed at significantly higher titers at pid compared to the suckling pigs (p < . ) ( table ). the serum pedv rna titers were significantly higher in suckling pigs at all time-points sampled ( table ). the higher severity of disease in suckling pigs coincided with the higher serum pedv rna titers in suckling pigs compared to weaned pigs. the mem only inoculated control pigs had no detectable pedv rna in either serum or feces. the suckling pigs had no detectable nk cell activity in blood and ileal mncs regardless of pedv infection, whereas the pedv infected and uninfected weaned pigs had low, but detectable nk cell activity (percentage of target lysis: . - . ) at pids and (fig. a-d) . suckling piglets had lower nk cell activity (% lysis of k cells) compared to weaned piglets in blood and ileal mononuclear cells at pid (a and b) and pid (c and d). blood and ileal mononuclear cells were co-cultured with cfse stained k cells at indicated ratios overnight. the dead cells were stained by incubating with -aad. the cells were observed by flow cytometry to obtain % of dead k cells. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the bar graphs labeled with different alphabetical letters are significantly different (p < . ). the infected weaned pigs had significantly increased nk cell activity in ileum at pid compared to uninfected weaned pigs (p = . ) (fig. d ). the nk cell activity was similar for infected versus uninfected weaned pigs at pid ( fig. a and b ). this coincides with the delayed onset of virus shedding in the infected weaned pigs. the nk cell activity in ileum of uninfected weaned pigs was about -fold higher than in the blood of uninfected weaned pigs (p < . ). the nk cell activity in ileum of infected weaned pigs was about -fold higher than in the blood of infected weaned pigs at pid (p < . ) ( fig. a and b) and about -fold higher at pid (p < . ) ( fig. c and d). there was no significant difference in the nk cell activity of blood mncs of infected and uninfected weaned pigs at pids and ( fig. a and c). the increase in nk cell activity was not correlated to the increase in nk cell frequencies in the ileal mncs. . . weaned infected pigs had significantly higher nk cell frequencies in blood and ileum at pid compared to suckling infected pigs the uninfected weaned pigs had significantly higher blood nk cell frequencies compared to uninfected suckling pigs (p < . ) ( fig. a) . the infected weaned pigs also had significantly higher nk cell frequencies in blood and ileum at pid (p < . ) compared to infected suckling pigs ( fig. a and b) . at pid , the infected weaned pigs had similar nk cell frequencies in blood to uninfected weaned pigs. at pid , the infected suckling pigs showed a significant increase in nk cell frequencies in blood compared to uninfected suckling pigs (p < . ) (fig. a) . the ileal nk cell frequencies were similar across groups at pid (fig. b) . however, at pid , the infected weaned pigs had significantly higher nk cell frequencies in ileum compared to infected suckling pigs, although there was an increase in nk cell frequencies in ileum of infected suckling and weaned pigs compared to uninfected suckling and weaned pigs, respectively (p < . ) (fig. b ). the nk cell frequencies of suckling and weaned pigs were higher in blood mncs than in ileal mncs at all time-points. . . ifn producing cd -cd -cd + nk cell frequencies were significantly higher in blood of weaned pigs compared to suckling pigs at pid and , regardless of pedv infection status and in ileum at pid for pedv-infected pigs like for the nk cell activity ( fig. a and c) , the ifn␥ producing nk cells were undetectable in blood of suckling piglets (fig. a) . the infected weaned pigs had a significantly higher ifn␥ producing nk cell frequency in blood than uninfected weaned pigs at pid (p < . ), although there was no difference between the two groups at pid . the ifn␥ producing nk cell frequency in ileum was similar in uninfected/infected suckling (pid ) and weaned pigs (pid , ) (fig. b) . at pid , the infected weaned pigs had similar ileal ifn␥ producing nk cell frequencies compared to infected suckling pigs. the infected suckling pigs had significantly lower ileal ifn␥ producing nk cell frequencies than the infected weaned pigs at pid (p < . ). at pid , the ifn␥ producing nk cell frequency was significantly lower in the ileum of infected suckling pigs compared to uninfected suckling pigs (p < . ). however, in the weaned fig. . weaned infected piglets had higher nk cell frequencies compared to suckling infected piglets in blood (a) and ileum (b) at pid . blood and ileal mncs were stained with anti-porcine cd -fitc, cd -pe and cd -sprd. the nk cell (cd -cd -cd +) frequency was expressed as percentage of lymphocytes. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). infected pigs there was no significant reduction in the ileal ifn␥ producing nk cell frequency compared to uninfected weaned pigs. . . pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from pedv infected weaned pigs and coincided with fecal and serum pedv rna titers there was a marked induction of serum ifn␣ in infected suckling pigs at pid which declined significantly thereafter. for infected weaned pigs, the response was highest at pid (fig. a ). infected suckling pigs had significantly higher ifn␣ levels compared to infected weaned pigs at pid (p < . ). at pids and , there was no difference in the ifn␣ induction levels in infected weaned and suckling pigs. overall, the peak induction of serum ifn␣ in suckling pigs was at pid and was much higher than the peak induction of ifn-␣ in weaned pigs which was at pid (p < . ); both peaks coincided with the peaks of pedv rna shedding in feces and serum (suckling pigs) or serum (weaned pigs) and onset of diarrhea. the uninfected suckling and weaned pigs had similar low serum ifn␣ levels. serum il- levels followed a similar trend. il- levels of infected suckling pigs were highest at pid , whereas for infected weaned pigs, the induction was highest at pid (fig. b) . there was no statistical difference in the peak il- levels between the infected suckling pigs at pid and infected weaned pigs at pid . however, serum il- levels were significantly higher in infected suckling pigs compared to infected weaned pigs (p < . ) at pid . the infected weaned pigs had significantly higher serum il- fig. . ifn␥ producing cd -cd -cd + nk cell frequencies were higher in weaned pigs compared to suckling pigs at pid and in blood (a) and at pid in ileum (b). blood and ileal mncs were cultured in e-rpmi with protein transporter inhibitor, brefeldin a added. the cells were stained with cd -fitc, cd -sprd, and cd -biotin followed by streptavidin apc as secondary antibody. samples were stained intracellularly with anti-porcine ifn␥-pe. flow cytometric analysis was done and the percentage of cd -cd -cd + nk cells that were also ifn␥ positive was calculated. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). levels at pid compared to infected suckling pigs (p < . ). the uninfected suckling and weaned pigs had similar serum il- levels at all days. overall, serum il- levels were much lower than for all other cytokines measured. serum il- levels were significantly higher only in infected weaned pigs compared to infected suckling pigs at pid (p < . ) (data not shown). otherwise, there were no significant differences in the serum il- between weaned and suckling infected pigs at pids and . the uninfected suckling and weaned pigs had similar serum il- levels. serum il- levels were significantly higher only in infected weaned pigs compared to infected suckling pigs at pid (p < . ) (fig. c) . there was no difference in the serum il- between weaned and suckling infected pigs at pids and . the uninfected suckling and weaned pigs had similar serum il- levels. serum tnf␣ levels also followed similar trends to ifn␣ including the pids of peak levels in infected weaned and suckling pigs. infected suckling pigs had highest tnf␣ levels at pid , whereas for infected weaned pigs, the response was highest at pids and (fig. d) . serum tnf␣ was significantly higher in weaned infected pigs than in infected suckling pigs at pids and (p < . ), whereas there was no significant difference in the serum tnf␣ between infected suckling and infected weaned pigs at pid . the peak tnf␣ level in weaned pigs at pid was significantly higher than the peak tnf␣ level in suckling pigs at pid (p < . ). in suckling and weaned pigs at pids , and . blood samples were collected from the pigs at the specified time points and sera separated by centrifugation and frozen at − • c until use. the cytokine assays were done using standard assays. the groups that were compared were, infected suckling versus infected weaned at different time points, uninfected suckling or weaned versus infected suckling or weaned at different time points. the different time points within infected groups of suckling or weaned pigs were also compared. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). the serum ifn␥, il- and tgf␤ levels were measured and there were no differences in their levels between the treatment groups (data not shown). . . cd +cd + t cell frequencies were higher in blood and ileum of suckling pigs than in weaned pigs, whereas there was no difference in cd +cd + t cell frequencies in ileum of suckling and weaned pigs the cd +cd + t cell frequencies in blood and ileum of uninfected suckling pigs were significantly higher than those in uninfected weaned pigs at pids and (p < . ) (fig. a and b) . however, the infected suckling pigs had statistically similar cd +cd + t cell frequencies in blood at pid compared to those of infected weaned pigs, whereas blood (fig. a) and ileum (fig. b ) of infected suckling pigs had higher cd +cd + t cell frequencies compared to infected weaned pigs at pid (p < . ). the cd +cd + t cell frequency in blood did not differ between pids and within the infected or uninfected groups of suckling or weaned pigs. the cd +cd + t cell frequencies in blood and ileal mncs were lower in infected suckling pigs compared to uninfected suckling pigs at pid (p < . ) (fig. a and b) . the frequency fig. . cd +cd + cells were higher in ileum of suckling pigs than weaned pigs whereas there was no difference in cd +cd + cells in ileum of suckling and weaned pigs. the graphs show the percentage of cd +cd + cells in blood (a) and ileum (b) of suckling and infected pigs at pids and , and the cd +cd + cells in blood (c) and ileum (d) of suckling and infected pigs at pids and . blood and ileal mononuclear cells were stained with cd -fitc, cd -pe and cd -sprd for min at • c. the frequency of t cells or nk cells was expressed as percentage of lymphocytes. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). of ileal cd +cd + t cells was higher at pid compared to pid for suckling infected pigs (p < . ). the infected suckling pigs had transient relative leukopenia of cd +cd + and cd +cd + t cells at pid compared to uninfected suckling pigs. there was no difference in the cd +cd + t cell frequency in blood and ileal mncs in weaned infected and uninfected pigs at either time point. the cd +cd + t cell frequency in blood mncs was significantly lower in infected suckling pigs compared to uninfected suckling pigs at pid (p = . ). the infected weaned pigs had significantly higher frequency of cd +cd + t cells in blood at pid compared to pid . there was no difference in the cd +cd + t cell frequency in blood mncs in infected and uninfected weaned pigs at either time point. there was no difference between the suckling and weaned uninfected pigs in cd +cd + t cell frequencies in blood (fig. c ) and ileal (fig. d ) mncs. suckling infected pigs had significantly lower cd +cd + t cell frequency in blood mncs compared to weaned infected pigs at pid . the cd +cd + t cell frequency in ileal mncs was similar in uninfected suckling and weaned pigs. there was also no difference in the ileal cd +cd + t cell frequency between the infected weaned and suckling pigs. the cd +cd + t cell frequency in ileal mncs of infected weaned pigs was significantly higher compared to uninfected weaned pigs (p < . ) and the same was true for infected and uninfected suckling pigs (p < . ). the infected suckling pigs had significantly higher frequency of cd +cd + t cells in ileum at pid compared to pid . similarly, infected weaned pigs had significantly higher frequency of cd +cd + t cells in ileal mncs at pid compared to pid (p < . ). in the present study, we investigated the differences between suckling and weaned pigs in innate immune responses to pedv infection and attempted to assess if these differences coincide with the greater severity of ped in suckling pigs. the major findings of this study were: ( ) suckling pigs had earlier onset and more severe diarrhea and earlier fecal and higher serum pedv rna titers compared to weaned pigs; ( ) pedv infected and uninfected suckling pigs had lower nk cell frequencies and no activity and lower ifn␥ producing nk cell frequencies compared to weaned pigs; ( ) ifn␣ and pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from pedv infected weaned pigs. in infected suckling and weaned groups, peak ifn␣, il- and tnf␣ levels coincided with onset of diarrhea and fecal pedv rna shedding and peak serum pedv rna titers (pids and , respectively); and ( ) frequencies of cd +cd + t cells were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in frequency of cd +cd + t cells. there was a transient relative leukopenia in cd +cd + and cd +cd + t cells in blood of suckling, but not weaned pigs at pid . the nursing pigs showed severe clinical signs and fecal and serum pedv rna as early as pid as reported previously (jung et al., ; stevenson et al., ) , but the weaned pigs showed mild and delayed clinical signs and delayed shedding of pedv rna in feces, in agreement with previous findings . in general, neonates of various species are more susceptible to severe disease than adults (camacho-gonzalez et al., ; rose, ; rosenberg et al., ; tregoning and schwarze, ; wohlfender et al., ). one of the reasons for the high susceptibility of neonates to severe disease from infections is deficient innate and adaptive immune responses/memory (levy, ; levy et al., ) . in the absence of previous exposure to pathogens, and therefore no adaptive immune responses, the newborn depends on innate immune responses to clear or reduce viral infection. in instances when the virus infection overwhelms the already weak innate immunity of the neonate, severe disease results (firth et al., ) . the virus levels in blood were directly related to severity of certain enteroviral diseases in infants (dagan et al., ; yen et al., ) as well as in other viral infections (maggi et al., ; vaughn et al., ) . interestingly in the present study, the moderate titer of pedv rna in serum of suckling pigs and -fold lower titers in weaned pigs coincided with the greater severity of disease in suckling pigs compared to weaned pigs. the severity of disease in suckling pigs also coincided with the early high titer pedv rna fecal shedding. porcine nk cells are identified as cd -cd + cells (gerner et al., ) . suckling pigs had no detectable nk cell activity in blood or ileal mncs and the activity did not change with infection, although the suckling pigs had comparable nk cell frequencies in most instances (except pids of infected pigs) to weaned pigs. this is in confirmation of previous studies of nk cell frequency and cytotoxic activity of lymphocytes of healthy suckling pigs against k cells (onizuka et al., ; yang et al., ) . newborn pigs also did not have nk cell mediated cytotoxic activity against pk- cells persistently infected with tgev (cepica and derbyshire, ) . studies of tge showed that vaccination with a modified live tgev vaccine did not increase nk cell activity in newborn pigs (raymond and wilkie, ) , but in vitro stimulation of adult monocytes increased nk cell activity (charley et al., ) . in the present study, pedv infected weaned pigs showed increased frequencies as well as activity of nk cells in the ileum. although uninfected suckling pigs had comparable nk cell frequencies to weaned pigs, their frequencies were reduced in blood and slightly increased in ileum following infection, but nk cell activity remained undetectable. studies of human infants showed that neonates had significantly lower nk cell lytic activity and the activity was further reduced by infection or sepsis (georgeson et al., ; uksila et al., ) . nk cells of infants also had reduced antiviral activity such as defective killing of virus infected cells and reduced cytokine secretion (jacobson et al., ) . collectively, reduced nk cell activity and nk cell frequencies may have contributed to the early onset of virus shedding and the severe clinical signs in the suckling versus weaned pigs. coinciding with nk cell cytotoxic activity, the weaned pigs also had higher frequency of ifn␥ positive nk cells compared to suckling pigs. further, pedv infection reduced the frequencies of ifn␥ producing nk cells in ileum of suckling pigs. neonatal mncs are deficient in ifn␥ production in response to antigenic stimuli (wilson et al., ) . nk cells are the major source of ifn␥ and activation of nk cells by ifn␥ is essential for their lytic activity (wang et al., ) . studies have shown that the increase in frequency, activity of nk cells and ifn␥ production by nk cells is essential for reduction of viral load in mice infected with lymphocytic choriomeningitis virus or influenza virus (biron et al., ; mack et al., ; stein-streilein et al., ) . further, production of ifn␤ and ifn␥ in intestine of mice was essential and sufficient to induce innate immune cells to eliminate certain bacterial pathogens in mice (sotolongo et al., ) . since the suckling pigs showed reduced ifn␥ positive nk cell frequencies, this could result in reduced viral clearance contributing to the enhanced disease. similar to our observations, foot and mouth disease virus decreased the lytic activity of nk cells in swine (toka et al., ) . studies showed that il- production stimulates ifn␥ production and il- production is less in intestinal epithelium of neonatal pigs compared to older pigs (muneta et al., ) . thus, it appears that the failure to induce sufficient ifn␥ production by nk cells might be one reason for increased severity of disease and higher systemic load of pedv in suckling pigs. the weaned pigs had a delayed pro-inflammatory cytokine induction compared with suckling pigs which coincided with delayed infection, disease and shedding of pedv rna in feces of weaned pigs. ifn␣ is an antiviral cytokine produced in response to viral infection by monocytes and dendritic cells (hansmann et al., ; siegal et al., ) . consistent with fecal virus shedding and serum viral rna titers, the suckling pigs had the highest serum ifn␣ induction at pid . the higher systemic ifn␣ response in suckling pigs at the early stage of infection coincides with the serum pedv rna titer and very severe infection. collectively, lack of nk cell activity, reduced nk cell frequency and ifn␥ production might have contributed to the early onset of viral shedding, viremia (viral rna in serum) and severity of clinical signs. this is similar to the ifn␣ induction by tgev infection in suckling pigs (la bonnardiere and laude, ) . notably, treatment of newborn pigs with the interferon inducer, polyinosinic: polycytidylic acid complexed with poly-l-lysine, resulted in a delayed onset of clinical signs when pigs were infected with tgev (lesnick and derbyshire, ) and the ifn␣ treatment enhanced nk cell activity (charley et al., ) . also, treatment of tgev infected suckling pigs with oral human ifn␣ increased their survival rates (cummins et al., ) . in vitro studies of human dendritic cells stimulated with viruses show that neonatal cells are capable of producing ifn␣ responses similar to those of adults (renneson et al., ). the increased ifn␣ may be the result of infection in suckling pigs in the early stages and may have led to the reduced pedv rna shedding in feces at a later stage compared to the weaned pigs. the present studies showed that weaned infected pigs had higher il- at pid and higher tnf␣ induction at pids and compared to suckling pigs. tnf␣ is a proinflammatory cytokine secreted by activated macrophages, nk cells, t cells and other cells (bradley, ) . serum levels of tnf␣ are often associated with severity of disease (jiang et al., ; waage et al., ) . studies of rotavirus infected children show that higher tnf␣ and lower ifn␥ are associated with more severe symptoms of infection (jiang et al., ) . similarly, studies of human rotavirus infection in a gnotobiotic pig model show that levels of cytokines like ifn␥ and tnf␣ are higher when pigs are infected with virulent virus compared to attenuated virus (azevedo et al., ) . human neonatal monocytes/macrophages stimulated in vitro with bacterial lipopolysaccharides or virus showed reduced tnf␣ responses when compared to monocytes/macrophages from adults (levy et al., ; valero et al., ) . in the present study, although the suckling pigs had more severe disease, their tnf␣ levels were similar to weaned pigs at pid and less than weaned pigs at pids and . il- is a chemoattractant for neutrophils and is secreted by various cell types (bickel, ) . pigs infected with porcine respiratory and reproductive syndrome virus that subsequently cleared the infection had higher il- than pigs that did not clear the infection (kim and chae, ) . therefore, the higher il- in weaned pigs may have contributed to lower severity of disease. similar to the pattern of ifn␣, the serum levels of cytokines il- and il- were high in suckling pigs at pid and in weaned pigs at pids or and coincided with the delay in onset of clinical signs and fecal pedv rna shedding in weaned pigs. studies of human neonates show that they are capable of producing certain pro-inflammatory cytokines in comparable levels to adults (schnurr et al., ) . therefore, suckling piglets could have a similar ability to produce these cytokines as the weaned pigs. thus, pro-inflammatory cytokine profiles of suckling and weaned pigs mirrored the severity of infection and viremia. the suckling pigs lacked innate immune activity to delay onset of viral shedding and viremia that resulted in more cells infected and higher ifn␣ (jung et al., a) . in contrast, the robust innate immune activity in weaned pigs may have delayed the onset of viral shedding and viremia, resulting in a lower ifn␣ response. however, the innate immune response was not enough to prevent the higher fecal viral shedding at pid in weaned pigs although there was reduced diarrhea compared to suckling pigs. the results also indicated the ability of suckling pigs to produce proinflammatory cytokines comparable to weaned pigs. cd + t cells represent t helper cells that aid in the humoral immune response and are responsible for th cytokine production. cd + t cells represent cytotoxic t cells that are important in killing virus infected cells (germain, ; gerner et al., ) . the cd +cd + t cell frequency was higher in the uninfected suckling pigs compared to uninfected weaned pigs, whereas infected suckling pigs had transient relative leukopenia at pid and then had increased cd +cd + t cell frequencies at pid compared to uninfected suckling pigs. neonates have a polarity toward th responses dominated by cd +cd + t cells and this changes when they are given viral vaccines and it also changes with age (dowling and levy, ; kelly et al., ; kovarik and siegrist, ; siegrist et al., ) . the present study confirmed that the neonatal swine intestine has increased cd +cd + t cells that can be altered by viral infection. the cd +cd + t cell frequency was comparable in suckling and weaned uninfected pigs and infection increased the intestinal cd +cd + t cell frequency in both groups. thus, the observed differences in pedv infectivity between suckling and weaned pigs might not be influenced by early cd +cd + t cell frequency. since cd +cd + t cells are involved in early antiviral adaptive immune responses, these cells are expected to play important roles in pedv infection of the intestine. longer observations are required to determine if there are differences in adaptive immune responses between the suckling and weaned pigs. 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deficiencies diseases in neonatal foals. part : potential risk factors for a higher incidence of infectious diseases during the first days post partum isolation and characterization of porcine natural killer (nk) cells viral load in blood is correlated with disease severity of neonatal coxsackievirus b infection: early diagnosis and predicting disease severity is possible in severe neonatal enterovirus infection systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease virus-specific intestinal ifn-gamma producing t cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with care of experimental animals. we also thank xiaohong wang, kyle scheuer, dr sukumar kandasamy, bryan eyerly and john blakenship for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported by a grant from the oardc seeds, grant # oaoh (jung k, pi). key: cord- - bvym i authors: francoz, d.; buczinski, s.; bélanger, a.m.; forté, g.; labrecque, o.; tremblay, d.; wellemans, v.; dubuc, j. title: respiratory pathogens in québec dairy calves and their relationship with clinical status, lung consolidation, and average daily gain date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: bvym i background: bovine respiratory disease (brd) is of the most important causes of morbidity and mortality in dairy calves. surprisingly, field data are scant concerning the prevalence of respiratory pathogens involved in brd in preweaned dairy calves, especially in small herds. objectives: to identify the main respiratory pathogens isolated from calves in québec dairy herds with a high incidence of brd, and to determine if there is an association between the presence of these pathogens and clinical signs of pneumonia, lung consolidation, or average daily gain. animals: cross‐sectional study using a convenience sample of preweaned dairy calves from dairy herds. methods: at enrollment, calves were weighed, clinically examined, swabbed (nasal and nasopharyngeal), and lung ultrasonography was performed. one month later, all calves were reweighed. results: twenty‐two calves had clinical brd and had ultrasonographic evidence of lung consolidation. pasteurella multocida, mannheimia haemolytica, and histophilus somni were isolated in , , and calves, respectively. mycoplasma bovis was identified by pcr testing or culture in calves, and calves were found to be positive for mycoplasma spp. bovine coronavirus was detected in calves and bovine respiratory syncytial virus in . only the presence of m. bovis was associated with higher odds of clinical signs, lung consolidation, and lower average daily gain. conclusions and clinical importance: results suggested that nasopharyngeal carriage of m. bovis was detrimental to health and growth of dairy calves in small herds with a high incidence of brd. b ovine respiratory disease (brd) is of the most important causes of morbidity and mortality in dairy calves and affects both preweaned and weaned calves. brd is a syndrome of diverse etiology that is caused by or more of a wide range of organisms, including bacteria and viruses. among bacteria, pasteurella multocida, mannheimia haemolytica, histophilus somni, mycoplasma bovis, and trueperella pyogenes are more commonly reported. viruses involved in the development of brd are mainly bovine respiratory syncytial virus (brsv), parainfluenza virus type (pi- ), bovine herpesvirus type (bhv- ), bovine viral diarrhea virus (bvdv), and bovine coronavirus (bcv). most of these pathogens are considered omnipresent in cattle populations and the exact role of some of them, such as mycoplasma spp. or bcv, in the development of brd is still unclear. [ ] [ ] [ ] the relative importance of these organisms in brd pathogenesis also may be different among farms, depending on which pathogens are most prevalent and how they interact together. over the past - years, there has been emergence or re-emergence of respiratory pathogens such as bcv and mycoplasma bovis, which were shown to be involved in the development of brd in dairy calves. currently, data on the prevalence and relative importance of these different brd pathogens in preweaned dairy calves are scant, and available data are usually from large dairy farms. therefore, it is unclear if the prevalence of brd pathogens is the same in large herds as in small herds such as those located in eastern canada. prevalence information of the main brd pathogens could help to better understand how to control and prevent brd in eastern canada. therefore, the first objective of this study was to identify the main respiratory pathogens found in preweaned dairy calves from herds with a high prevalence of brd. the second objective of this study was to determine if there was an association between the presence of these pathogens and various clinical outcomes such as the presence of clinical signs of brd, lung consolidation, and average daily gain (adg). our first hypothesis was that brd pathogens found in small dairy herds with a high prevalence of calf respiratory disease problems would be similar to those found in larger dairy herds or beef cattle operations. secondly, we hypothesized that the presence of some of these pathogens would be associated with cases of clinical brd, cases of lung consolidation, or have a detrimental impact on adg. the study protocol was approved by the animal care committee of the universit e de montr eal. a cross-sectional study was conducted on dairy calves from herds that were regular clients of the bovine ambulatory clinic of the facult e de m edecine v et erinaire of the universit e de montr eal (saint-hyacinthe, qc, canada). herd selection was based on convenience of proximity (within km radius of the ambulatory clinic), history of brd diagnosed on the farm, and accessible accurate, detailed health records for the calves. herds were considered to have enzootic brd problems if they had at least cases of brd in calves confirmed by veterinary diagnosis over the last -month period, and if the veterinarian overseeing the herd agreed that there was an active problem of brd in calves. within each selected herd, the oldest preweaned female calves were selected for enrollment. if herds had < preweaned calves, all available preweaned calves were enrolled. sample size estimation for the study was based on finding a significant difference of . kg/d of adg between groups of animals (pathogen present: . kg/d; pathogen absent: . kg/d), considering alpha and beta errors of . and . , respectively, and after accounting for covariable and herd clustering effect. a similar sample size calculation was performed for a difference of % in abnormal clinical status (pathogen present: %; pathogen absent: %) or lung consolidation (pathogen present: %; pathogen absent: %). in all of these calculations, a sample size of animals per group (pathogen present or absent) was targeted for an estimated total count of calves to be enrolled. all participating herds were visited twice by veterinarians and a technician between november and january . during the first farm visit, enrolled calves were identified, clinically examined, weighed, and sampled for brd pathogen detection. weight was estimated by girth circumference using a tape measure specific for this purpose. a information on any previous treatments that had been administered to the calf also was collected, with particular attention paid to previous treatments for brd. participating producers were asked to collect on a specific sheet all disease and treatment events that occurred after the first farm visit. participating herds were visited for a second time month after the first visit by the same veterinarians and technician. during the second visit, data collection sheets were saved and enrolled calves were weighed. during the first visit, each calf was identified, clinically evaluated, and scored by the same veterinarian using the calf respiratory scoring criteria (crsc) from the university of wisconsin. briefly, this -point score is based on different criteria including rectal temperature, cough, nasal discharge, eye, and ear scores. each criterion is scored on a - scale, with associated with the lowest risk of being sick and with the highest risk of brd. the lung area of every calf was scanned by ultrasound from the th to the th intercostal space, as previously described. the ultrasonography was performed by the same person (gf) using an . mhz linear probe b directly applied on the thorax. isopropyl alcohol ( %) was applied on the area of interest as contact media for acceptable image quality without clipping body hair. c, the abnormality noted during lung ultrasonography was the presence of consolidated lung. depth of consolidation (depth) was considered relevant when it was ≥ cm. c during the first farm visit, midnasal swab (nasa) was taken from the left nostril of each calf and then placed in transport media d for viral and m. bovis detection by bacteriologic culture and pcr testing. a deep nasopharyngeal swab (naso) e was taken as previously reported from the right nostril for routine bacterial and mycoplasma cultures and then placed in transport media. f all laboratory analyses were performed in veterinary accredited diagnostic laboratories (see data s ). all conventional and mycoplasma bacteriologic cultures were performed at the laboratoire d' epid emiosurveillance animale du qu ebec of the minist ere de l'agriculture, des pêcheries et de l'alimentation du qu ebec. deep nasopharyngeal swabs were refrigerated and conventional culture was done within - hours of sampling. mycoplasma culture was performed on naso and nasa within - hours after sampling. midnasal swabs were refrigerated and submitted for pcr testing within hours of sampling. polymerase chain reaction testing included detection of m. bovis, brsv, bhv- , bvdv, pi- , and bcv. all pcr testing was performed at the molecular diagnostic laboratory of the facult e de m edecine v et erinaire de l'universit e de montr eal. the experimental unit of this study was the calf. a value of adg was determined for each calf by calculating the weight gain between first and second farm visits and dividing this result by the number of days between the visits. identification of the optimal clinical score (crsc) threshold to predict lung consolidation was performed. for this purpose, the sensitivity and specificity for each different clinical score from to was calculated using the freq procedures of sas. g a receiver operating characteristic (roc) curve also was obtained and the youden index calculated with the roc module of medcalc h to determine the threshold (diagnostic criterion) that maximized the total sum of sensitivity and specificity to predict lung consolidation. descriptive statistics were performed using the means and freq procedures of sas for continuous variables (adg and age at first visit) and binary variables (clinical status, lung consolidation, treatment before first visit, and bacteriologic results), respectively. univariable analyses were performed using the freq procedure of sas (pearson chi-square test), considering the dependent variables to be "clinical status" or "lung consolidation." univariable associations with a p ≤ . were retained for further modeling. final multivariable logistic regression models were built (glimmix procedure in sas) considering the dependent variables to be "clinical status" or "lung consolidation," adjusting for herd as a random effect, and forcing age at first visit into all models. model building was performed using a backward elimination strategy until p values of all remaining variables were ≤. . a univariable linear mixed model (mixed procedure in sas) considering adg as dependent variable and adjusting for herd as a random effect was used to screen for potential predictors. univariable associations with p values ≤. were retained for further modeling. a final multivariable mixed linear model was built for adg, considering herd as a random effect and forcing the variable age at first visit into the model. model building was performed using a backward elimination strategy as previously described. a total of preweaned female holstein calves from different herds were enrolled in the study. the size of participating herds ranged from to cows in lactation (median, cows). the weaning time was between and weeks of age in all herds, except in which it was - weeks. sensitivities and specificities of clinical scores between and to predict lung consolidation are presented in table . based on roc curve calculation (fig ) and the youden index, a score of ≥ on the crsc was determined to be the optimal threshold for predicting calves with clinical signs of brd (ie, sick calves). therefore, calves of ( %) were considered to have clinical pneumonia in this study. descriptive statistics for age at the first visit, clinical status, transthoracic ultrasonography, number of dead calves between visits, adg, and prevalence of treatment before the first visit are presented in table . overall, calves ( %) had ultrasonographic evidence of consolidation (depth ≥ cm). among these calves, ( %) had crsc scores < . on the other hand, among the calves with scores ≥ , only ( %) had no signs of lung consolidation on ultrasonography. pasteurella multocida was isolated in calves ( %) from herds. mannheimia haemolytica was isolated in calves ( %) from herds. histophilus somni was isolated in calves ( %) from different herds. more than of these bacteria were identified in herds and in calves. pasteurella multocida was cultured in association with m. haemolytica and h. somni in and calves, respectively. seventy-eight calves ( %) were positive for mycoplasma species other than m. bovis. mycoplasma spp. were cultured only from the nasa in calves, only from naso in calves, and from both swabs in calves. mycoplasma spp. was cultured at least once in all herds. nineteen calves ( %) were positive by culture, pcr, or both, of the nasa, naso, or both swabs for m. bovis (table ) . mycoplasma bovis was identified only by pcr testing in calves, by culture from the nasa and naso in calves, by pcr testing and culture of the naso in calf, and by pcr testing and culture of the nasa and naso in calves. mycoplasma bovis was identified with m. haemolytica in calves, p. multocida in calves, and p. multocida as well as h. somni in calves. bovine coronavirus and brsv were the only viruses detected in the study. brsv was detected in calf in the only herd in which no vaccination program for brd was carried out. bcv was detected in calves ( %) from different herds. the presence of p. multocida was negatively associated with treatment before first visit (p = . ). conversely, univariable analyses showed an association between the presence of m. bovis in naso (p = . ) and nasa cultures (p = . ) and calf clinical status. no other significant associations were found in the univariable association. the final multivariable model showed that, after adjusting for herd clustering and age at the first visit, m. bovis in nasa culture was associated with the clinical status of the animal at the first examination (negative nasa culture: % clinically affected; % ci, - ; positive nasa culture: % clinically affected; % ci, - ; p = . ). the final multivariable model to determine the effect of laboratory results on lung consolidation showed that after adjusting for herd clustering and age at the first visit, bacteriologic culture results for m. bovis from naso sampling were associated with lung consolidation (negative naso culture: % consolidated; % ci, - ; positive naso culture: % consolidated; % ci, - ; p = . ). two different models were built to determine the effect of laboratory results on adg; model was built for m. bovis results from nasa sampling and another model was built for m. bovis from naso sampling. after adjusting for herd clustering and age at the first visit, positive nasa results for m. bovis were associated with a lower subsequent adg (negative nasa culture: . kg/d; % ci, . - . ; positive nasa culture: . kg/d; % ci, . - . ; p < . ). a similar association was found for positive naso results for m. bovis (negative naso culture: . kg/d; % ci, . - . ; positive naso culture: . kg/d; % ci, . - . ; p < . ). the current study focuses on the prevalence of microbial pathogens in cases of brd in preweaned dairy calves in relatively small herds using an objective measure (lung consolidation) for the definition of cases of brd in living animals. among all the brd pathogens detected in this study, only m. bovis isolated from the nasopharynx or the nasal cavity had a negative impact on adg, and was associated with higher odds of having lung consolidation. pasteurella multocida, mycoplasma spp., and bcv were the main bacteria and virus identified in the study. however, none of them were associated with clinical score, lung consolidation on transthoracic ultrasonography, or adg. in addition, results of this study emphasize the importance of the adaptation of respiratory clinical score charts used in the particular population studied. mycoplasma bovis was the third most frequently found bacteria in the current study, but it was the only associated with clinical score, lung consolidation, and poor subsequent adg. mycoplasma bovis already has been shown to be associated with chronic debilitating diseases that respond poorly to treatment. , in a recent study, m. bovis identified by pcr in lung tissues was associated with pneumonic lesions in young dairy calves of < month of age. mycoplasma bovis also was reported to be associated with decreased adg in stocker calves. to our knowledge, ours is of the first reports to show the association between m. bovis culture results and decreased adg in dairy calves. interestingly, results of the present study showed that antimicrobial treatment before the first examination of participating calves was associated with higher odds of finding m. bovis at sampling. a possible explanation for this finding is that because m. bovis is well known to have multiple antimicrobial resistance, antimicrobial treatments could have decreased the presence of other bacterial inhabitants of the nasal cavity. such a situation potentially could promote the growth of m. bovis. definition of a clinical case is a critical point when studying brd in cattle. in the present study, the clinical status of calves was determined using previously published clinical score criteria. these criteria have been developed as a useful practical tool for calf ranchers when screening dairy calves for brd in the context of large dairy herds. the weight given to each parameter studied in the global score may be different according to different management practices such as those used in smaller herds. consequently, it seemed important to determine a threshold that would be suitable for the particular population studied. in the present study, the optimal clinical score threshold for predicting lung consolidation (an objective measure of lung lesions) was determined based on the highest sum of sensitivity and specificity. in other words, this optimal threshold would provide the lowest possible number of classification errors (diseased or not). determination of disease definitions in the absence of a gold standard by using an objective outcome for comparison has been commonly used for conditions such as hyperketonemia and postpartum endometritis in dairy cows. , such an approach represents an improvement to the current standard procedure for conditions that have substantial detrimental impacts on health, culling, or reproductive performance, and that are difficult to diagnose clinically (mostly subclinical diseases). one could argue that this could have biased the clinical status definition used in the study toward more severe and chronic cases compared with the definition commonly used on farms. in the case of brd, the use of an objective outcome is crucial considering that, in an experimental model, lung consolidation was seen by transthoracic ultrasonography as soon as hours after infection with m. haemolytica. i therefore, it is very likely that lung consolidation could appear much earlier than clinical signs of disease. this may be an explanation for the fact that % of calves with lung lesions had clinical scores < . on the other hand, other explanation could be that calves have chronic lesions not associated with clinical signs. the use of clinical disease status then should be targeted at predicting the lung consolidation status of animals with the greatest accuracy. as in previous reports in dairy calves, [ ] [ ] [ ] p. multocida and mycoplasma spp. were the most common bacteria isolated. pasteurella multocida is well accepted as a respiratory pathogen. , , it was not associated with the clinical status of calves, lung consolidation, or adg in this study, which is in opposition to previous studies in dairy calves or cattle. , , [ ] [ ] [ ] the presence of p. multocida was negatively associated with previous antimicrobial treatment, which could suggest that previous treatment decreased its prevalence. clinical status and lung consolidation also were only evaluated once. consequently, it was not possible to determine which stage of the diseases (ie, very acute, clinically active, or chronic) affected the calves. in other words, it was possible that calves were sick before or after the evaluation. therefore, the role and impact of p. multocida in brd could have been underestimated in the present study. the role of mycoplasma spp. (other than m. bovis) in the development of brd is not clear. numerous mycoplasma species have been isolated from the nasal flora of healthy calves, but some of them, such m. dispar, m. canis, and m. californicum, have been associated with bdr in calves. they are mainly associated with subclinical infections and are suspected to play a role in the induction of brd by negatively affecting the immune system and upper respiratory defense mechanism. in the present study, no association was found between the isolation of mycoplasma spp. and clinical status, lung consolidation, or adg. unfortunately, these mycoplasma spp. were not specifically identified in the present study. mycoplasma spp. identified by pcr in lung tissue have been reported to be associated with pneumonic lesions in young dairy calves of < month of age. in recent study on young dairy calves, culture of mycoplasma spp. in midnasal and deep pharyngeal swabs was associated with clinical cases of brd. however, no distinction between m. bovis and other mycoplasma species was made, which could be an important confounding factor for the association between mycoplasma spp. and brd. these discrepancies point out the importance of the identification of mycoplasma spp. in cases of brd and the necessity for additional studies for the determination of the pathogenic role of mycoplasma species other than m. bovis in young dairy calves experiencing brd. among all viruses investigated in the present study, bcv was by far the most commonly detected, but no association was found between the presence of bcv and clinical status, lung consolidation, or adg. this finding is consistent with the results of another study conducted in dairy calves from large herds in california. the exact role of this virus in the development of brd is still unknown and its pathogenic role in brd is still unclear. in young dairy calves, bcv has been associated with mild clinical signs of pneumonia, cough, rhinitis, fever, and anorexia, usually in association with enteritis. in norwegian study, dairy calves in herds with bcv seropositive calves were at a higher risk of developing pneumonia compared with calves in herds with only seronegative calves. however, frequent and intermittent nasal shedding has been reported in dairy calves with or without clinical signs of respiratory disease. , in opposition to a study in the united states in which brsv was isolated in . % of dairy calves, it was only detected once in this study ( %). this is similar to the finding of a scottish study in which . % of dairy calves were positive. the sample size, herd, and calf recruitment procedure in the present study potentially could explain the low prevalence of brsv in the study population. bovine viral diarrhea and ibr and pi- viruses were not found in the present study. this finding is in agreement with another study conducted in california in which there were no animals positive for bvd or bhv- . in another study conducted on dairy calves, pi- was detected in . % of animals. all herds included in the study except (in which brsv was detected) had vaccination protocols for the prevention of infection by these viruses, and these protocols may have controlled and precluded dissemination of these viruses within the herds. nonetheless, viral excretion in the nasal cavity is generally of a short duration (usually < week, depending on the virus), which makes viral isolation within a herd or an animal difficult if repetitive sampling is not performed. consequently, it is difficult to conclude based on the absence of these viruses that they are not important pathogens in brd in dairy calves in qu ebec. a total of calves were included in the present study. this sample size was based on a calculation of the sample size needed to provide % power to detect a difference of . g/d (pathogen present: . kg/d; pathogen absent: . kg/d) in adg between groups, and a difference of % in clinical status (pathogen present: %; pathogen absent: %) or lung consolidation (pathogen present: %; pathogen absent: %). unfortunately, the prevalence of some pathogens was lower than expected, leading to the number of infected animals being < per group. this was especially important for all viruses, m. haemolytica, h. somni, and mycoplasma. therefore, although no association between these pathogens and adg, clinical status, or lung consolidation was reported in this study, statistical power could have been insufficient in some cases (if the difference between groups was the same as expected in sample size calculation) to support this lack of association with confidence. also, results of the current study were obtained from herds recruited based on a high incidence brd problems. another limitation was the fact that it was a convenience sample of calves. however, in herds, the sample represented all or most of the calves in the herd. in addition, the median age of the selected calves was similar to the median age of the median age of first treatment for brd in a recent study. it is unclear if sampling herds with few or no respiratory problems or younger or older preweaned calves would have provided similar prevalence results for these pathogens. this situation deserves further assessment. the bronchopneumonias (respiratory disease complex of cattle, sheep and goats) bovine pasteurellosis and other bacterial infections of the respiratory tract pathogenesis and pathology of bovine pneumonia bovine respiratory coronavirus mycoplasma bovis pneumonia in cattle disease management of dairy calves and heifers short communication: ultrasonographic assessment of the thorax as a fast technique to assess pulmonary lesions in dairy calves with bovine respiratory disease use of deep nasopharyngeal swabs as a predictive diagnostic method for natural respiratory infections in calves mycoplasma bovis infections in cattle mycoplasma bovis infection in respiratory disease of dairy calves less than one month old associations between the prevalence of mollicutes and mycoplasma bovis and health and performance in stocker calves definitions and diagnosis of postpartum endometritis in dairy cows impact of hyperketonemia in early lactation dairy cows on health and production prevalence of viral and bacterial pathogens in nasopharyngeal recess regions of holstein calves with and without signs of clinical bovine respiratory disease. tulare calf project pasteurella multocida and bovine respiratory disease the microbial flora of the upper and lower respiratory tracts of feedlot calves with undifferentiated bovine respiratory disease respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response ter laak ea, noordergraaf jh, boomsluiter e. the nasal mycoplasma flora of healthy calves and cows outbreak of pneumonia and arthritis in beef calves associated with mycoplamsa bovis and mycoplasma californicum epidemiologic factors and isotype-specific antibody responses in serum and mucosal secretions of dairy calves with bovine coronavirus respiratory tract and enteric tract infections factors associated with morbidity, mortality, and growth of dairy heifer calves up to months of age the authors acknowledge the participating dairy producers as well as marie-eve r emy and jean-philippe pelletier for their technical assistance. this research project was funded by merck animal health (montr eal, qc, canada). the ultrasound unit was supplied by ei-medical imaging, loveland, co.conflict of interest declaration: the authors disclose no conflict of interest.off-label antimicrobial declaration: the authors declare no off-label use of antimicrobials. additional supporting information may be found online in supporting information:data s . complementory information on the bacteriological cultures and pcr testings perfomed during the study. key: cord- -xbwilp w authors: kin, nathalie; miszczak, fabien; lin, wei; ar gouilh, meriadeg; vabret, astrid title: genomic analysis of human coronaviruses oc (hcov-oc s) circulating in france from to reveals a high intra-specific diversity with new recombinant genotypes date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xbwilp w human coronavirus oc (hcov-oc ) is one of five currently circulating human coronaviruses responsible for respiratory infections. like all coronaviruses, it is characterized by its genome’s high plasticity. the objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in lower normandy between and . to this end, we sequenced complete nsp , s, and n genes of molecular isolates of hcov-oc from clinical samples and compared them to available data from the usa, belgium, and hong-kong. a new cluster e was invariably detected from nsp , s, and n data while the analysis of nsp and n genes revealed the existence of new f and g clusters respectively. the association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. identification of these recombinant viruses, together with temporal analysis and tmrca estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses. ay ), coding for the glomerular part of the s protein (sub-unit s ) of hcov-oc s from clinical samples collected in at the university hospital of caen. they observed four genetically distinct subgroups. one of the subgroups constitutes an outlier group located between hcov-oc and bovine coronavirus (bcov), containing a -nucleotide deletion in common with bcov but not with other hcov-oc s [ ] . more recently, four genetically distinct hcov-oc genotypes have been identified from respiratory specimens sampled at the public hospital of china over a -year period ( to ) [ ] . in this study, lau et al. based their genotype definition upon the complete sequence of nsp , s and n genes [ ] . genotype a contains the prototype vr strain isolated in . genotypes b and c are two naturally circulating hcov-oc s. genotype d is a recombinant virus of genotypes b and c, obtained from a specimen from [ ] . based on a bootscan analysis of the complete genome of the hcov-oc s belonging to the circulating genotypes b, c, and d, it was assumed that a hot spot was likely located between the nsp and s genes, more precisely at the nsp /nsp junction. our objective was to investigate the presence, the temporal distribution and the recombination patterns of hcov-oc in lower normandy over years ( to ), using the methodology and the hcov-oc genotype definition elaborated by lau et al. in [ ] . this study focuses on the sequences of the nsp , s, and n genes of hcov-oc s detected in respiratory specimens sampled from to . of the hcov-oc s and the prototype strain vr , six, eight, and three overlapping sequences were obtained for the nsp , s, and n genes, respectively. after assembly, these overlapping fragments encompassed the full nsp , s, and n genes. in this study, all hcov-oc s including the vr prototype strain are associated with three accession numbers in genbank, for nsp , s, and n genes ( table ). the sequences of hcov-oc s were named according to the following nomenclature: virus/fra-epi/location of sampling/year of sampling/specimen number. epi is an abbreviation for epicorem consortium. for the prototype strain, the specimen number is replaced by vr . the phylogenetic analysis was conducted by comparing the topology of the three phylogenetic trees obtained from the nsp , s, and n genes. figure shows the three trees obtained by the neighbor joining method [ ] . on each tree, five to six clusters were observed, including an outlier group compounded with the three bcovs sequences mebus, kakegawa, and quebec that root hcov-oc sequences [ ] [ ] [ ] . the clustering is supported with high bootstrap values. we used part of the nomenclature of genotypes proposed by lau et al. in [ ] . these authors described the genotypes a, b, c, and d from nsp , s and n genes. we used this nomenclature to name each genotype using three letters corresponding to clusters in which the different sequences are located in nsp , s, and n trees, respectively. following this nomenclature, genotype d is a recombinant genotype b/c/c. the three previously described clusters-a, b, and c-are observed in our three gene trees in addition to a newly described cluster e. the nsp tree depicts a new cluster we named "f" that roots all other hcov-oc s. according to the n gene, a sixth cluster we named "g" separates from cluster e and roots clusters a, b, and c. among the set of sequences obtained from the hcov-oc s of our study, three are distributed in two non-recombinant genotypes as follows: the vr sequence belongs to the genotype aaa; hcov-oc /fra-epi/caen/ / and hcov-oc /fra-epi/caen/ / belong to genotype bbb. the other sets of sequences are allocated among six recombinant genotypes as follows: five genotypes bcc, defined as genotype d by lau et al.; two genotypes cee, three genotypes cbe, one genotype bcg, one genotype cce, and one genotype ceb. among the american sets of sequences, seven are distributed into two non-recombinant genotypes as follows: one ccc and six eee. the seven other sets of sequences are distributed among four recombinant genotypes as follows: three genotypes cca, two genotypes fcb, one genotype ccb, and one genotype cee. among the two sets of sequences from hong-kong, one is a non-recombinant genotype ccc and the other is a recombinant genotype bcc. among the two hcov-oc s from belgium, one is a non-recombinant genotype bbb and the other is a recombinant genotype bcc. finally, the two last sequences of hcov-oc vr (accession number: ay and nc ) belong to the non-recombinant genotype aaa [ , ] . the results of the phylogenetic analysis are summarized in table . the two alignments constructed from the dated sequences of s and n genes have been used to set up a molecular clock calibration in order to estimate the date of emergence of mean clusters. figure shows bayesian trees inferred from the alignments of s and n genes. the dates of emergence of the clusters and the corresponding % highest posterior density ( % hpd) are indicated. based on the sequence data of the s gene, the tmrca of bcov and hcov-oc is estimated in , with a % hpd from to . for the hcov-oc , cluster e is predicted to have emerged in ( % hpd to ), and genotype a is estimated to have emerged in ( % hpd, to ). genotypes b and c are predicted to have emerged from their tmrca in ( % hpd, to ). the molecular clock conducted from sequence data of n gene allows us to estimate the trmca of bcov and hcov-oc in ( % hpd, to ). genotypes e and a are predicted to have emerged in ( % hpd, to ) and ( % hpd, to ), respectively. genotypes b and c are predicted to have emerged from their tmrca in ( % hpd, to ). phylogenetic analysis of the complete nsp , s, and n genes of the hcov-oc strains and bcov strains. the phylogenetic trees were constructed by the neighbor joining method [ ] . bootstrap values were calculated from replicates. bootstrap values over % are shown [ ] . the evolutionary distances were computed using the kimura -parameter method (kimura) and units are the number of base substitutions per site. evolutionary analyses were conducted in mega, version . . [ ] . blue triangle, isolates from caen; red circle, isolates from usa; green circle, isolates from hong-kong; black diamond, isolates from belgium; purple square, atcc-vr strains, empty black square, bcov strains. results of bayesian analysis based on s and n gene sequence data. inferences were calculated with the one parametric coalescent model with a constant size, under the tn +g substitution parameter, using the beast package (version . . ) [ ] . the length of mcmc was fixed at states for s and n genes. the dates of emergence of mean clusters are indicated, associated with the % hpd. the hcovs-oc , e, nl , and hku -belong to the viral molecular panel tested during the virological routine diagnostics of acute respiratory infections in humans. among these four circulating hcovs, oc and nl seem to show the greatest prevalence and incidence. these viruses also proved to be the viruses encountered at the earliest age of childhood [ ] . these hcovs circulate widely in epidemic form in the general population, causing infections that are most often benign. infants, immunosuppressed patients, and very elderly patients may however develop very severe pathologies. these four circulating hcovs must be distinguished from the emerging hcovs, sars-cov, and mers-cov, causes of much graver and potentially fatal respiratory pathologies. control of the latter hcovs requires the implementation of international health measures [ ] [ ] [ ] . the evolutionary potential of coronaviruses brings into play point mutations and recombination events. such genetic diversity generated in this way may have an impact on the performance of molecular detection techniques used in the virological diagnostic process. the study of intra-specific diversity is thus useful in the monitoring of means of detection. specifically, it allows for the detection of new circulating variants, and provides information about the evolutionary dynamics of the family of respiratory viruses being monitored, with special attention given to coronaviruses. our study focuses solely on the hcov-oc . the first analyses of the hcov-oc s gene were conducted in and revealed a potential spatial and temporal distribution of genetic clusters [ ] . in , s. lau and his colleagues were the first to propose a genotypic classification of hcov-oc from the complete sequencing of three genes: nsp (rdrp), s (spike), and n (nucleocapsid). four genotypes-a, b, c, and d-were identified: genotype a corresponds to the sequences of the hcov-oc prototype strain vr , adapted to culture cell line hrt- (human adenocarsinoma rectal) or rd (rhabdomyosarcoma); genotypes b and c correspond to contemporary circulating strains, and genotype d is described as a recombinant b/c virus. this study was performed on hcov-oc s detected in respiratory samples collected in hong kong over a period of years ( to ), in a non-homogenous temporal distribution. more precisely, the majority of these samples ( of ) were collected in , and the others ( of ) fall between and [ ] . our study defines the genotype of hcov-oc s detected from upper respiratory samples frozen at − °c, collected over a period of years from to at the rate of one to two samples per year. the hcov-oc s were detected in patients hospitalized in our university hospital. of these patients, were male and five were female, at ages from months to years. the symptomatology proved variable ( table ). the infections were essentially located in upper and lower respiratory airways. the presence of associated gastrointestinal symptoms in some patients should be noted. the prototype strain hcov-oc atcc vr , grown on cell line hrt- , was used as a control, allowing for the validation of the methodology used by lau et al. [ ] . this methodology had to be adapted since some of the primers published by the authors did not allow for amplification of all the hcov-oc s. of these sequences, we selected that reflect the genetic diversity and temporal distribution of the whole, and we introduced them into the alignment. in the end, three respective alignments of complete genes-nsp , s, and n-of hcov-oc s identified in three different regions of the world (hong kong, europe, and the usa) over a period of years (from to ) were generated and analyzed. they served as the basis for the construction of phylogenetic trees. within the three phylogenetic trees-nsp , s, and n-we identified six different clusters named a, b, c, e, f, and g. a comparative study of the topology of phylogenetic trees corresponding to several genes of the same virus is often used in phylogeny to define recombinant viruses [ , , ] . we thus compared the topology of nsp , s, and n trees by observing the contextual position of the corresponding sequences of the same hcov-oc molecular isolate. the complexity of the results of our analysis led us to establish a nomenclature allowing for the simplest expression of the genotypes: each genotype is expressed in the form of a three-letter code, linking each one to the member cluster of the different nsp , s, and n sequences. from here, we defined different genotypes, of which four were non-recombinant (aaa, bbb, ccc, and eee). the nine remaining genotypes correspond to different recombinant genotypes (table ) . among the hcov-oc sequences examined in this study, hcov-oc s of so-called non-recombinant genotypes were found: three hcov-oc s of genotype aaa corresponding to prototype strains vr ; three hcov-oc s of genotype bbb (origin, france and , and belgium ); two hcov-oc s of genotype ccc (origin, usa and hong kong ); and lastly, six hcov-oc s of genotype eee, all originating in the usa, and having circulated from to . the foremost important point to underline is that these results suggest a high level of recombination among circulating hcov-oc epidemic strains. the limited number of sequences studied in time and space does not allow for conclusive evidence of a possible temporal and spatial distribution of different genotypes hcov-oc . however, it should be noted that the e cluster has not yet been described. in the present study, it is identified only in hcov-oc sequences from the usa and france and data suggest that it has been circulating since the s after being the first known group to differentiate from bcov. the analysis of alignments shows that cluster e is characterized by a deletion of nucleotides in the s gene, more precisely in the s gene that codes the glomerular part of the s protein (position , - , , genbank accession number nc_ ). this deletion of nucleotides in the coding phase results in a deletion of amino acids, tqdg, in the corresponding protein sequence. this deletion was also present in the bovine coronavirus strains used in the alignment, as shown in figure . the analysis of s bcov sequences available in genbank shows that this deletion is expressed in all bcovs, and described in bovine coronaviruses associated with both enteric and respiratory tropisms. this deletion is therefore not a specific marker of viral tropism. it is located in the lectin domain of the s protein of bcov, and is involved in the attachment of the virus to the cellular receptor, identified as a derivative of neuraminic acid (n-acetyl- -o-acetylneuraminic acid or neu , ac ). no protein receptor has yet been described for the bcov or, more broadly, for betacoronavirus bovine-like clade a. we analyzed all the sequences of this s hcov-oc region available in genbank, for a total of sequences, and the deletion of nucleotides is found in only of them. of these sequences, correspond to hcov-oc s detected in respiratory samples from the usa and sequenced by town et al. four correspond to hcov-oc s detected in france (three in the current study, and one published by our team in ); two correspond to hcov-oc sequences deposited by browlin et al. under patent in the united kingdom (ep and wo ), and finally, the remaining two correspond to hcov-oc s adapted to vero cells and mdck i, published in by f. künkel and herrler g [ ] . the hcov-oc e cluster is phylogenetically close to the bcov cluster, and the presence of the genetic marker common to these two clusters provides an argument supporting the hypothesis put forth by vijgen in that would identify a crossing of the species barrier occurring from cattle to human [ ] . a molecular clock analysis was inferred using the beast software version . . (http://beast.bio.ed.ac.uk) based on sequence alignments of s and n genes. sequences of the nsp gene were not used because they were uninformative due to the low variability of this gene. results were compared with those previously obtained by vijgen from betacoronavirus clade a sequences (nine bcovs, three phevs, and eight hcov-oc s). it should be noted that eight sets of hcov-oc sequences used in the analysis of in , wertheim et al. demonstrated that a strong purifying selection could lead to the underestimation of the evolutionary rate of coronaviruses. in this study, the authors proposed an alternative theory of origin for all coronaviruses. they estimate the tmrca of coronaviruses infecting mammalian species (alpha-and betacoronavirus genus) and avian species (gamma-and deltacoronavirus genus) at about million years, as opposed to , years as previously estimated [ ] . these authors found a general agreement in the inferred branch lengths between evolutionary models for the shorter branches (inferior to . substitution per site). as expected in the phylogeny of two species of the same coronavirus genus involved in a relatively recent zoonotic transmission event, all the branches of neighbor joining and bayesian trees were shorter than . substitutions per site, as shown in figures and . it can be speculated that the phylogenetic analyses presented in this study did not underestimate the date of emergence of the different clusters nor the estimation of tmrca for hcov-oc and bcov. the molecular clock analysis indicated that hcov-oc and bcov strains have emerged from their tmrca around ( - ), which was consistent with the data published by vijgen et al. in [ ] . the emergence of cluster e, characterized by a tqgd amino acid deletion, is estimated at around , and would have occurred at the same time as that of cluster a, in the - time range. the cluster a includes the first hcov-oc strain identified in from a respiratory sample [ ] . the b and c clusters would have circulated later, since the s. the molecular clock results therefore suggest that, since their emergence in the late nineteenth century, the evolution of circulating hcov-oc epidemic strains would have been marked by the occurrence of an insertion of four amino acids, tqdg, in the n-terminal region of the s attachment protein, as opposed to the occurrence of the deletion of these four amino acids. the pursuit of this study should lead to a means of determining the functional impact of this insertion, especially with regard to tropism and the clinical expression of infection by hcov-oc . note that the frequent presence of gastrointestinal symptoms in the clinical landscape of hcov-oc respiratory infections has yet to be accounted for. in , hu et al. studied the genetic diversity of the hcov-oc circulating in beijing, china [ ] . the authors performed partial sequencing of the s and n genes ( , - , and , - , for s and n respectively, using the hcov-oc sequence associated with the accession number nc_ as reference) of hcov-oc s detected in the respiratory samples, all taken in . they included the sequences published by lau et al. in their analysis in , and used the nomenclature established by these same authors to identify various clusters obtained [ , ] . in addition to the a and b clusters, they identified other groups, designated unt , unt(b), unt(c/d), and (c/d), respectively. several clusters were found to correspond to those previously described by lau et al. in [ ] . the unt(b) cluster or "untyped b" is a group close to the b cluster. the c/d cluster corresponds to the c cluster, named "c/d", due to the presence of the hk _ strain defined by lau et al. as a genotype d, referring to a recombination virus belonging to the b cluster on the nsp sequence and to the c cluster on the s and n sequences [ ] . the unt(c/d) cluster or "untyped (c/d)" corresponds to a group close to the preceding cluster. finally, the unt cluster or "untyped ", which is close to the bcov sequences included in this analysis, might correspond to the e cluster describe in the current study. in the report by hu et al. ( ) , the unt cluster includes only five hcov-oc s (accession numbers z , hc , cq , dd , and z ) [ ] . we have verified that they all have the characteristic nucleotide deletion of nucleotides. this very recent report supports the results determined in the current study in that they both demonstrate a high genetic diversity of circulating hcov-oc s. the present study highlights the difficulty of investigating intraspecific genetic variability of hcov-oc s, though the analysis of partial viral genomes (complete genes) allows the description of clear recombination patterns. nevertheless, due to the high evolutionary potential of hcov-oc , analyzing complete genomes would reveal a more detailed and perhaps more complex recombination pattern. in addition, the data reported and analyzed here illustrate the dynamic diversification of hcov-oc following its emergence in human. this remarkable diversification contrasts with the relative stability of bcov in cattle (unpublished data). the present study therefore draws attention to the importance of increasing our knowledge of the evolution dynamism of coronavirus in combating the permanent emergence risk that they pose in the human population, evidenced by the sars pandemic of - and the recently identified mers-coronavirus in the middle-east. fifteen human respiratory specimens (upper and lower) positive for hcov-oc using molecular detection, sampled from to (one to two for each year) were chosen among the specimens available at the virology department of the university hospital of caen. these specimens were sampled from patients suffering from acute upper or lower respiratory diseases, and were sent to our laboratory for a viral diagnosis. the detection of hcov-oc was performed using an in-house real-time quantitative rt-pcr, for which the original primers may be found in table . clinical signs, age, and sex of patients were investigated and compiled in table . we also used a culture supernatant of the total nucleic acids of μl of clinical specimens and vr cultures supernatant were extracted using the qiasymphony automate and the qiasynphony virus/bacteria kit (qiagen, holden, germany), following instructions of manufacturer. to determine the genomic sequence of nsp , s, and n genes, a set of overlapping rt-pcr products, from to nucleotides, were generated. these rt-pcr products encompassed the entire three genes. twelve primers were used to amplify and sequence the nsp gene, composed of nucleotides and located from nucleotides , to , (reference to vr , nc ). eighteen primers were used to amplify and sequence the s gene composed of nucleotides. this gene is located in the last third of the hcov-oc genome, between bases , and , (reference to hcov-oc vr , nc ). six primers were used to amplify and sequence the n gene composed of nucleotides and located at the ' end of the hcov-oc genome, from nucleotide , to , (reference to hcov-oc vr , nc ). for both rt-pcr and sequencing reactions, primers were selected in conserved domains from an alignment of the previously published hcov-oc . some of the selected primers were published by lau in , but others were designed using primer-blast software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) [ ] . all of them were tested in silico and in vitro with the vr prototype strain to test their efficacy. in vitro experimentations have also been conducted using the vr strain to test the primer abilities to provide us with quality sequencing products. table , associated with the corresponding targeted gene and with the fragment size. rt-pcr reactions were performed using onestep rt-pcr system (qiagen, holden, germany), from . μl of nucleic acid in a final volume of μl. the same primers were used to perform the complete bidirectional sequencing of the rt-pcr products, with a sanger derived method. . μl of each rt-pcr product were first purified by adding μl of exosap-it (usb corporation, cleveland, oh, usa), and by heating it at °c for min, followed by min at °c to disable enzymatic activities. two sequencing reactions were performed for each rt-pcr product with either a forward or a reverse primer. to μl of purified rt-pcr product were used in a final volume of μl of reaction mixture, made up with the bigdye terminator cycle sequencing kit, version . (applied biosystems, foster city, ca, usa), in addition to μl of one of the corresponding primers. the cycling parameters for the sequencing rt-pcr were °c for min, followed by cycles of °c for s, °c for s, and °c for min. the analysis of labelled products was performed by the capillary electrophoresis abi prism genetic analyzer (applied biosystems, foster city, ca, usa), by the molecular genetics laboratory of the university hospital of caen. in this study, the term of molecular isolate was use to designate the sequence of hcov-oc detected directly from clinical sample. sequences were assembled in contigs corresponding to the entire nsp , s or n genes with codoncode aligner software, version . . (codoncode corporation, centerville, ma, usa). multiple sequence alignment and phylogenetic analysis were performed using mega software, version . (http://www.megasoftware.net). twenty-one sequences available in genbank were added to the alignment, including hcov-oc s hku _ and hku _ from hong-kong [ ] ; hcov-oc s be and be from belgium [ , , ] . the accession numbers corresponding to these sequences are given in table . phylogenetic trees were constructed using the method of neighbor joining, with the substitution model of kimura- , implemented in mega [ , ] . these trees were used to perform the comparative analysis of our results with those of lau et al in [ ] . in order to support neighbour joining trees with a probabilistic method and to estimate divergence time between groups and subgroups, trees of s and n genes were also inferred using the same sequences and a bayesian markov chain monte carlo method, implemented in the beast package, version . . [ , ] . inferences were calculated with the one parametric coalescent model with a constant size, under the tn +g substitution model according to bayesian information criterion (bic) and akaike information criterion (aic) values of a model test carried out on mega software. the tmrca was estimated using a relaxed molecular clock with an uncorrelated lognormal distribution. the length of mcmc was fixed at × − states for the both genes. the tracer software, version . , implemented in the beast package, was used to assess the fitness of the model used, focusing the effective sampling size (ess) data, after a burning of %. the target trees were obtained with the maximum credibility clades, with a posterior probability limit of . . the tmrca age for each genotype was compared for s and n genes. evolving the largest rna virus genome mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses ratification vote on taxonomic proposals to the international committee on taxonomy of viruses virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences coronavirus diversity, phylogeny and interspecies jumping two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia genomic characterization of equine coronavirus 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findings from a retrospective investigation severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia human respiratory coronavirus oc : genetic stability and neuroinvasion complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event implications of altered replication fidelity on the evolution and pathogenesis of coronaviruses lack of evidence for proofreading mechanisms associated with an rna virus polymerase high-frequency rna recombination of murine coronaviruses inter-and intra-variant genetic heterogeneity of human coronavirus oc strains in france molecular epidemiology of human coronavirus oc reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination circulation of genetically distinct contemporary human coronavirus oc strains the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular analysis of the s glycoprotein gene of bovine coronaviruses isolated in japan from to pathology of neonatal calf diarrhea induced by a coronavirus-like agent full-length genomic sequence of bovine coronavirus ( kb). completion of the open reading frame a/ b sequences evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc confidence limits on phylogenies: an approach using the bootstrap molecular evolutionary genetics analysis version . bayesian phylogenetics with beauti and the beast . the dominance of human coronavirus oc and nl infections in infants severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers progress in global surveillance and response capacity years after severe acute respiratory syndrome the first pandemic of the st century genomic analysis of colorado human nl coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene and evidence of recombination comparative analysis of coronavirus hku genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku structural and functional analysis of the s proteins of two human coronavirus oc strains adapted to growth in different cells prevalence and genetic diversity analysis of human coronavirus oc among adult patients with acute respiratory infections in beijing a cis-acting function for the coronavirus leader in defective interfering rna replication a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences prospects for inferring very large phylogenies by using the neighbor-joining method this work was supported by the french agence nationale de la recherche (anr) on the behalf of epicorem project (eco-epidemiology of coronaviruses, from wildlife to human: emergence prototype strains vr as reference and for validation of our method. this strain was obtained from the atcc and maintained in hrt- cells. threat assessment), grant anr- -bsv - . we thank paul brown for the english proofreading of the manuscript. a.v. and n.k. design the study. n.k. and w.l. performed the experiments. nk. and m.a.g. performed the analyses. n.k. conceived the manuscript with contributions from f.m., m.a.g. and a.v. all authors contributed toward the preparation, editing and proofreading of the manuscript. the authors declare no conflict of interest. key: cord- -pqn qhm authors: nan title: an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea, date: - - journal: infect chemother doi: . /ic. . . . sha: doc_id: cord_uid: pqn qhm this report includes a summary of a current outbreak of the middle east respiratory syndrome coronavirus infection in the republic of korea as of june , . epidemiologic, clinical, and laboratory investigations of this outbreak are ongoing. between may and june , there was an outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection with a considerable number of cases in the republic of korea. this report includes an overview of the epidemiologic investigations and public health responses in several affected hospitals as of june , . epidemiologic, clinical, and laboratory investigations of this outbreak are ongoing. the index patient (patient ) was a -year-old korean man. from april to may , , he had traveled to the middle east region (bahrain, united arab emirates, and saudi arabia). on may , while in asan-si, chungcheongnam-do, he experienced fever and myalgia. he visited a clinic on may , then moved to hospital a in pyeongtaek-si, gyeonggi-do, where inpatient care was advised. persistent fever, myalgia, cough, and dyspnea lead to a diagnosis of pneumonia. he decided to move from hospital a to receive better care, and then visited another clinic and emergency room of hospital b in seoul on may . on may , he was admitted to hospital b. a meticulous interview regarding his travel history by an infectious disease specialist resulted in the diagnosis of mers-cov infection after pcr confirmation by the korean centers for disease control and prevention (kcdc) on may . he was transferred to an isolation unit of hospital b. as of june , a cluster of persons including healthcare workers with confirmed mers-cov are known to have had direct or indirect contact with the index patient. among those, five patients (pa-http://dx.doi.org/ . /ic. . . . • infect chemother ; ( ): - www.icjournal.org tients , , , , and ), who were transferred from hospital a to other hospitals brought about subsequent clusters in five different hospitals. patient had pneumonia and stayed in hospital a between may and . he may have been exposed to the index patient on the same floor (eighth) between may and . he was transferred to another hospital on may , but as his pneumonia deteriorated further; he left the hospital and then came to seoul. he decided to visit the emergency room of hospital b on may , but he was intubated on may , remaining in the emergency room of hospital b before being transferred to an isolation unit. mers-cov infection was confirmed on may . by june , persons with confirmed mers-cov are known to have had direct or indirect contact with patient . other than clusters from patients and , there were several clusters in different hospitals. as of june , a total of confirmed mers cases have been reported to the kcdc. these reported mers cases include deaths. cases of mers continue to be reported throughout the republic of korea. the korean government launched a joint task force board called the "immediate response task force for mers (irt-fm)," which was composed of government officials and infectious disease experts and representatives of the korean society for infectious diseases (ksid) and korean society for healthcare-associated infection control and prevention (koshic) for all-out efforts against the epidemic. ) the irt-fm supported mers hospitals with updated and adapted scientific guidelines for patient care, infection control, and laboratory handling for medium-and small-sized hospitals. ) the board members voluntarily became involved in mers hospital intervention for infection control, contact tracing policy, and decisions to close hospitals. ) the ksid and koshic proposed several press releases regarding the mers-cov epidemic situation and the mode of transmission issue in the republic of korea. ) the ksid and koshic representatives aimed to support their members and interactively shared updates on epidemic data to stop further inter-hospital spreads using social network services (sns). as of june , many efforts for contact tracing have identified a total of , persons who had any close contact with confirmed cases, and these people have been quarantined for days. to control the outbreak, much stronger control measures with contact tracing, quarantine, and contact surveillance continue to be applied. the outbreak is now the second largest worldwide and the largest reported outside the middle east region owing to larg-er population density in the far east region, especially with large hospitals [ , ] . however, there still is no sound evidence of community transmission; the mers-cov infection in the republic of korea is healthcare-associated, accelerated by inter-hospital spread. on the basis of the reported cases, droplet and contact transmission appear to be the major modes of transmission, and airborne transmission is unlikely in the community [ ] . because of unexpected expansion of the epidemic even after proper contact tracing, infection-control measures currently applied in most hospitals focused not only on droplet and contact transmission prevention but also on preventing airborne transmission [ ] . the low barrier to healthcare access that leads to easy patient access to hospitals and the crowdedness of emergency rooms and wards in large hospitals, especially in highly populated metropolitan areas, has been suggested to be related with the unexpectedly larger outbreak than in saudi arabia. several additional weeks will be required to confirm whether the outbreak is being controlled [ ]. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) we thank jun yong choi, tae hyong kim, young hwa choi, hong bin kim, hee jung yoon, ji hyun yoon, jacob lee, joong sik eom, joon young song, sang-oh lee, won sup oh, kyung mi kim, sun young jeong, hee jin cheong, young goo song, jung-hyun choi, jin-hong yoo, and woo joo kim for their efforts in drafting this report. key: cord- -zhf jdcl authors: patil, satish; lis, lev g.; schumacher, robert j.; norris, beverly j.; morgan, monique l.; cuellar, rebecca a. d.; blazar, bruce r.; suryanarayanan, raj; gurvich, vadim j.; georg, gunda i. title: phosphonooxymethyl prodrug of triptolide: synthesis, physicochemical characterization, and efficacy in human colon adenocarcinoma and ovarian cancer xenografts date: - - journal: j med chem doi: . /acs.jmedchem. b sha: doc_id: cord_uid: zhf jdcl [image: see text] a disodium phosphonooxymethyl prodrug of the antitumor agent triptolide was prepared from the natural product in three steps ( % yield) and displayed excellent aqueous solubility at ph . ( mg/ml) compared to the natural product ( μg/ml). the estimated shelf life (t( )) for hydrolysis of the prodrug at °c and ph . was found to be two years. in a mouse model of human colon adenocarcinoma (ht- ), the prodrug administered intraperitoneally was effective in reducing or eliminating xenograft tumors at dose levels as low as . mg/kg when given daily and at . mg/kg when given less frequently. when given via intraperitoneal and oral routes at daily doses of . and . mg/kg, the prodrug was also effective and well tolerated in a mouse model of human ovarian cancer (a ). triptolide ( , figure ), a diterpene triepoxide, was first isolated from the medicinal plant tripterygium wilfordii hook f (twhf) and structurally characterized in . in the following years, it has been reported to be active in vitro by inhibiting proliferation and inducing apoptosis of various cancer cell lines and as preventing tumor growth and metastases in vivo. more recently, triptolide has been found to be effective against various cancers such as pancreatic cancer, neuroblastoma, breast cancer, and prostate cancer. the mechanisms of action of triptolide have been extensively investigated, , and evidence has been provided that triptolide can covalently modify proteins presumably by epoxide ring opening reactions. , , − it was recently shown that triptolide inhibits the atpase activity of human xpb (xeroderma pigmentosum b) by covalently binding cys of xpb to the , -epoxide. , because xpb is part of transcription factor thfiih, rna ii polymerase-mediated transcription and dna excision repair are inhibited. many of the observed anticancer effects can be explained by this mechanism, but other mechanisms have been observed such as epigenetic modifications, suppressing kinases, and hsp expression. , , triptolide was also reported to be the first dctpp (dctp pyrophosphatase) inhibitor. it is of interest to note that this is a noncovalent interaction of triptolide with the target protein. furthermore, the compound has multiple biological activities that could have value in other therapeutic areas. , triptolide was reported to stimulate polycystin- channel opening, thereby restoring calcium signaling and resulting in attenuation of cyst formation in a mouse model of polycystic kidney disease. it is also known for its reversible male antifertility effects. in addition, triptolide was shown to preserve cognitive function in transgenic mouse models of alzheimer's disease. , the anticancer and other activities of triptolide such as its immunosuppressive and anti-inflammatory properties are more thoroughly described in recent reviews. , , recent studies focus on the development of triptolide and its derivatives as potential antileukemic and antineoplastic agents. , despite its promising bioactivities, poor aqueous solubility, dose-dependent toxicity, narrow therapeutic window, and lack of patent protection of triptolide are impediments to its preclinical development and clinical success. two early stage clinical trials of triptolide as a potential drug for rheumatoid arthritis were conducted in the us over a decade ago. , the -hydroxytriptolide derivative -hydroxytriptolide (lldt- , , figure ) is currently in phase i clinical trial in china for rheumatoid arthritis. prodrug strategies, involving carboxylic and amino acid esters, have been utilized previously with the intent to achieve desirable water solubility of triptolide. , while several prodrugs of triptolide have been reported in the literature, triptolide succinate (omtriptolide, f , pg - , , figure ) is the only one reported to have entered clinical trials. unfortunately, the cleavage of the prodrug moiety was slow and incomplete, and significant interpatient variability was reported. this does not discount the prodrug strategy but suggests that other prodrugs that provide a reliable release of triptolide are needed. a nontoxic, water-soluble, chemically stable, and patentable prodrug approach would be a viable option to overcome some of the physicochemical limitations of triptolide for the clinical development of this natural product. historically, the use of a phosphate group as a promoiety has successfully overcome numerous delivery problems of potential drugs. − these prodrugs are formed by either direct linkage of a phosphate moiety onto a hydroxyl group of a parent drug in the form of a phosphomonoester or by attaching it to the parent drug via a chemical linker. the phosphate promoiety is ionized at physiological ph, resulting in a significant increase in aqueous solubility of poorly soluble phenol-and alcoholbearing parent drugs. − additionally, these phosphomonoester prodrugs are typically stable with long shelf-lives and undergo an alkaline phosphatase (ec . . . )-catalyzed bioconversion in vivo to release the parent alcohol or phenol drug and inorganic phosphate. numerous phosphomonoester prodrugs have shown good in vitro − and in vivo − conversion to the parent drug in the presence of alkaline phosphatases. the aforementioned omtriptolide was ineffective in clinical trials due to its incomplete and slow bioconversion in vivo. we hypothesized that the succinate promoiety directly attached to the -oh group of triptolide would not be easily accessible for enzymatic cleavage due to steric crowding. therefore, the prerequisites for a novel prodrug strategy of triptolide were three-fold: enhanced aqueous solubility, chemical stability, and fast, complete bioconversion in vivo. we aimed to achieve these objectives by incorporating the phosphonooxymethyl promoiety, as it possesses a favorable combination of high aqueous solubility and chemical stability. this strategy has been successful for solubility enhancement of paclitaxel and propofol, and, in both cases, the promoiety was attached to a sterically hindered hydroxyl group. previously, the synthesis and evaluation of the phosphonooxymethyl derivative of triptolide ( figure ) for pancreatic cancer , and several other preclinical cancer models has been reported. − we are now describing an improved synthesis for , its physicochemical characterization, and its pharmacodynamic evaluation in human colon adenocarcinoma and ovarian cancer xenografts via intraperitoneal and oral routes and using less frequent dosing schedules than employed in previous studies. synthesis. our initial attempts to prepare in two steps from triptolide by either o-alkylation with chloromethyl phosphate diesters or direct alkylation of the hydroxyl group with chloroiodomethane were not successful. , , therefore, an alternative strategy based on methylthiomethylation of the hydroxyl group was selected (schemes and ) to furnish key article intermediate . we observed complete conversion of triptolide during the pummerer rearrangement, but a mixture of products was obtained. the products were separated by column chromatography and identified by nmr. while the target methylthiomethyl ether was the main component ( − % yield), acetoxymethyl ether was identified as a major byproduct ( % yield), and triptonide ( ) formed in % (scheme ). this composition is similar to what was previously observed for this type of reaction using phenylsulfonyl derivatives. our attempts to carry out a basic hydrolysis of the acetoxymethyl ether resulted in decomposition of the product, but triptolide could be recovered by acidic hydrolysis of in − % yield. an alternative approach (scheme ) based on treating triptolide with dimethyl sulfide and benzoyl peroxide in acetonitrile resulted in a similar yield of compound ( %) with triptonide as the only side product in %. this methodology, however, allowed for significant reduction of the reaction time from days to h and also made purification of easier. in the next step (scheme ), the conversion of methylthiomethyl ether to dibenzyl phosphate was achieved using an n-iodosuccinimide-mediated (nis) nucleophilic displacement with dibenzyl phosphate in ch cl /thf in the presence of Å molecular sieves. while the yield was high ( %), chromatographic purification on silica gel was difficult because of pronounced decomposition due to instability of the benzylic esters of the phosphate. the − mg scale flash chromatography experiments revealed approximately % degradation. however, in larger scale preparations where contact between the compound and silica gel is required for a longer period of time, − % degradation was observed depending on the amount of substrate and the level of deactivation of the silica gel. attempts to use alternate sorbents such as florisil or alumina, as well as triethylamine deactivation of silica gel, resulted in similar levels of degradation. it was not possible to avoid purification prior to hydrogenation due to the various sulfur derivatives formed during the course of the synthesis. these compounds, even in minute quantities, can poison the pd/c catalyst used in the benzyl ether cleavage. this led to increased catalytic loading and longer reaction time, which in turn resulted in a larger number and quantity of impurities that contaminated the final product. additionally, during the formation of dibenzyl phosphate , − % of succinimide was produced from the reagent nis, which coeluted during column chromatography. an alternative purification methodology was developed utilizing a sequence of extractions that are described in experimental section, which removed most of the impurities while avoiding product decomposition. this methodology has proven reliable, efficient, and scalable. it results in the removal of most sulfur derivatives and succinimide without the use of chromatographic purification. in the penultimate step, dibenzyl phosphate was subjected to hydrogenation in the presence of % pd/c. the resultant phosphate was converted to by treatment with sodium carbonate. the final composition of the product included up to % of triptolide as a result of hydrolysis during the final chemical step. because the product decomposes during silica gel column chromatography, it was isolated in % purity by preparative hplc using a c column. the material is highly hygroscopic and was stored under nitrogen or argon gas. physicochemical characterization. physicochemical characterizations of were conducted to assess its solubility and chemical stability. aqueous solubility. adjusting solution ph can be a simple and effective method to increase the water solubility of a weakly acidic or basic injectable drug. however, ph adjustment is not a viable approach to increase the solubility of molecules such as triptolide that lack ionizable groups. for such compounds, various strategies including the use of cosolvents, surfactants, and complexing agents (for example, parenterally safe cyclodextrins) have been used to overcome aqueous solubility limitations. however, some of these techniques can contribute significantly to toxicity. ideally phosphonooxymethyl prodrugs will have good water solubility, particularly at physiological ph. avoiding ph extremes and/or cosolvent addition could allow for rapid parenteral infusion without the risk of drug precipitation. using hplc, the aqueous solubility (buffered to ph . with tris at room temperature) of triptolide was determined to be μg/ml while that of was mg/ml. thus, by prodrug formation, the solubility was enhanced times. we also estimated the second dissociation constant (pk a ) of . in solution, depending on the ph, the prodrug would exist in its diacidic, monobasic, or dibasic states as represented in scheme . the pk a has limited pharmaceutical relevance, because the ph of most physiologically acceptable solutions would ensure ionization of the diacidic species. however, the pk a is significant given its influence on aqueous solubility, chemical stability, and effectiveness as an enzyme substrate to ensure bioconversion. therefore, within the pharmaceutically relevant ph range, would exist in an equilibrium between its monobasic and dibasic species. thus, titration of an equilibrium mixture of these two species provides an acid ionization constant, pk a , whose value corresponds to the inflection point. estimation of this pk a using p nmr is advantageous, as the p isotope has high natural abundance ( %) and its chemical shift is indicative of the change in ionization state of the phosphorus moiety. the p nmr spectrum of was obtained as a function of ph. the experimental data shown for the change in observed phosphorus chemical shifts of in solutions at varying ph ( figure ) was used to determine pk a (eq , experimental part). the ph dependency of this observed chemical shift measurement resulted in a sigmoidal curve, which is typical of an acid−base titration. the pk a value of was found to be . , which compares favorably to estimates from the literature. − at ph . , would exist predominantly as a dianion, thus ensuring high solubility. article chemical stability determination. following oral dosing, prodrugs are exposed to a wide ph range in the gi tract, from highly acidic in the stomach to neutral in the colon. also, a wide array of hydrolytic enzymes, such as pepsin and pancreatin, are present in the gi tract. these enzymes have the natural function of digesting macromolecules for use as nutrients, but they can also bind and hydrolyze drug compounds. therefore, prodrugs must be stable in the acidic, basic, and enzymatic conditions of the gi tract for in vivo oral dosing. the excipients in a formulation may also promote prodrug decomposition. therefore, in vitro physiological stability of prodrugs in various buffers (ph − ) and in simulated gastrointestinal fluids should be assessed to gain insight into the stability of prodrugs. thus, was incubated with simulated gastric fluid (sgf), simulated intestinal fluid (sif), and various buffers (ph − ). oral dosing exposes compounds to ph − in the stomach, ph . at the beginning of the small intestine, ph . on average for the small intestine, and ph − in the colon. the gastric emptying time varies from . − h in the fasted state to several hours after a heavy meal. sgf simulates stomach fluid and incorporates acidic and enzymatic hydrolysis conditions. sif mimics the ph and hydrolytic enzymes in the intestine. incubation of in these solutions is a rapid way to determine if would be stable under conditions found in the gi tract. compound demonstrated ph-dependent stability and rapidly degraded with conversion to triptolide in simulated gastric fluid and at very low ph values ( − ) and in simulated gastric fluid whereas it was markedly stable at higher ph values ( − ) and in simulated intestinal fluid ( table ). the shorter half-life at low ph for could result in acid-mediated removal of the promoiety in the stomach. however, can be "protected" from the stomach acid by enteric coating. prodrugs, by their nature, tend to be less chemically stable than the parent drugs. to be practically useful, prodrugs must possess adequate chemical stability under the conditions of their use. prodrugs intended for intravenous administration should exhibit adequate solution state stability so that a readyto-use preparation with a reasonably long shelf life (i.e., or more years) can be formulated. if the solution state stability is inadequate, the drug can be formulated as a dry powder (usually by lyophilization) and reconstituted into solution right before administration. for example, safadi and co-workers described the development of phosphonooxymethyl carbonates as novel, water-soluble prodrugs of hindered alcohols. however, the lack of adequate chemical stability limited the commercial and clinical potential of this prodrug concept. the chemical hydrolysis of was followed at ph of . because the neutral ph range is perfectly suited for formulation delivery from a physiological perspective. we found that the hydrolysis of followed pseudo-first-order kinetics. using the pseudofirst-order rate constants, determined at several temperatures between and °c (table ) , the arrhenius plot was drawn (not shown). from this plot, the activation energy (e a ) was calculated to be . kj/mol. by extrapolating the plot to lower temperatures, the k obsd at and at °c were obtained. this enabled the calculation of the half-life (t / ) and shelf life (t ) of at and °c (table ) . thus, the chemical stability of at neutral ph was found to be quite satisfactory giving an estimated shelf life of approximately years (at °c). therefore, the formulation of in a freeze-dried form or as an aqueous solution at ph . is predicted to have an acceptable shelf life. enzymatic conversion of to triptolide. a prerequisite for a successful phosphate prodrug strategy is conversion catalyzed by alkaline phosphatase. in general, phosphomonoesters, such as phosphonooxymethyl prodrugs, are good substrates for both acid and alkaline phosphatases which are found in a wide variety of living organisms including bacteria, plants, and animals. alkaline phosphatase is found throughout the body and is mainly associated with plasma membranes of the intestine, placenta, bone, liver, and kidney at high concentrations. this enzyme participates in hydrolase/ article transferase reactions on a variety of phosphate-containing compounds under physiological conditions. for the in vitro evaluation of the dephosphorylation of , alkaline phosphatase from bovine intestinal mucosa was chosen. as implied by its name, this enzyme has maximum activity in the alkaline region (ph . ). the in vitro assay for phosphate monoester prodrugs is typically performed at ph . , an optimum ph for the activity of alkaline phosphatases. the evaluation of bioconversion kinetics is, however, more relevant if performed at physiological ph. thus, a change in ph of reaction media to . may, in principle, reduce the catalytic efficiency of the enzyme. however, for proof-of-concept, confirming that was indeed the substrate of alkaline phosphatase was paramount. clearly, if is administered parenterally in humans, the overwhelming presence of alkaline phosphate in vivo should guarantee bioconversion. the in vitro enzymatic lability of was recently reported by us. we carried out the bioconversion of into triptolide in the presence of alkaline phosphatase in glycine buffer at ph . (scheme ). exposure of to phosphatases cleaves the phosphate group and releases the hydroxymethyl derivative , which is chemically unstable and spontaneously releases triptolide and formaldehyde. the release of formaldehyde from prodrugs could raise a possible concern because of perceived toxicity. however, it is well-known that the turnover of formaldehyde in the human body from endogenous formaldehyde production by normal metabolism and from exogenous exposure (for example from food) is in the range of − g per day. because prodrugs only release milligram amounts of formaldehyde from phosphonooxymethyl prodrugs per day, the small amount of formaldehyde adds very little to the baseline exposure of gram quantities produced by normal metabolism. in addition, given the short half-life of . min for formaldehyde, which is converted to formic acid, exposure to formaldehyde produced by a prodrug would be limited to approximately min. both the disappearance of and the formation of triptolide were measured. compound degradation was a first-order process. the half-life (t / ) of was determined to be min in the presence of alkaline phosphatase, showing rapid conversion of the modified drug into its parent compound. additionally, was found to be stable for h in a similar assay conducted in the absence of alkaline phosphatase. the short half-life of in this assay indicates that the enzymatically catalyzed breakdown of occurred at a fast rate. a short half-life for is consistent with our hypothesis that a methylene-linked phosphate prodrug would be released rapidly and not be hampered by steric hindrance as seen with the succinate prodrug omtriptolide. steric hindrance has been noted in the bioconversion of a number of phosphate and phosphonooxymethyl prodrugs. , evaluation of for efficacy in mouse models of cancer. it was previously reported that compound can reduce tumor growth, prevent tumor progression, and improve survival in multiple mouse models of pancreatic cancer. , the efficacy of has also been demonstrated in preclinical models of osteosarcoma, nonsmall cell lung carcinoma, human papillomavirus-positive head and neck squamous cell carcinoma, and ovarian cancer. − these studies evaluated the efficacy of after daily intraperitoneal (ip) administration. here we used tumor xenograft models to evaluate the efficacy of when administered on a less frequent schedule and when administered by the oral route. efficacy study in a mouse model of human colon adenocarcinoma. this study was designed to evaluate the potential efficacy and toxicity of when administered ip daily (qd) over a range of doses and when administered on various schedules for days. the animal model used in the study was the ht- human colon adenocarcinoma cell line implanted in female athymic nude mice as a subcutaneous tumor. treatment groups with ten mice per group included four dose levels ( . , . , . , and . mg/kg) and five dose regimens over the week treatment period (daily [qd] for weeks; qd for weeks then no treatment for weeks; qd for weeks and with no treatment during weeks and ; ×/week for weeks; qd for weeks then ×/week). compound administered daily ip was found to be effective in reducing or eliminating xenograft tumors of the human colon adenocarcinoma ht- in this animal model at dose levels from . to . mg/kg ( figure ). dose regimens in which . mg/kg of was administered less frequently than daily or with a break from daily dosing were also found to be effective ( figure ). in the groups that received . mg/kg daily for weeks or . mg/kg daily for weeks followed by ×/week, all mice that survived until the end of the study ( / and / , respectively) were tumor free. compound was generally well tolerated in female athymic nude mice with the only test-article related deaths observed in those mice receiving . mg/kg doses for at least consecutive days ( in the "qd for weeks" group and each in the "qd for weeks" and the "qd for weeks then ×/ week" groups). clinical signs of toxicity were noted in some mice in higher dose groups after days of dosing. the frequency of mortality and severity of adverse clinical signs escalated with dose level and frequency. the mice receiving a daily dose of . mg/kg had the least mean group weight gain and the highest incidence of skin irritation and necrosis toward the end of the study. groups receiving a lower dose of or . mg/kg on an intermittent schedule had fewer clinical signs of toxicity and higher weight gain over the course of the study. efficacy study in a mouse model of human ovarian cancer. this study was designed to evaluate the efficacy and tolerability of in a mouse model of human ovarian cancer when administered via an intraperitoneal and oral route. the animal model used in this study was the a human ovarian cancer cell line implanted in female athymic nude mice as a subcutaneous tumor. treatments included daily doses of ranging from . mg/kg to . mg/kg (ten mice per group). article administration of by intraperitoneal injection was effective and well tolerated in female athymic nude mice with subcutaneous xenograft tumors of the human a ovarian carcinoma at daily doses of . or . mg/kg (data not shown). administration of by oral gavage was also effective at daily doses of . or . mg/kg but with slightly higher rates of morbidity and mortality ( figure ). frequency of mortality and severity of adverse clinical signs escalated with dose level and only two mice in the . mg/kg oral dose group survived beyond day (data not shown). clinical signs of acute toxicity included anorexia, dehydration, and moribund condition or death. in the vehicle control group, of mice were euthanized before the end of the study because tumor volume had surpassed the tumor volume end point. however, in the . mg/kg and . mg/kg groups, most of the mice survived until the end of the study ( of and of , respectively) and all but one in each group was tumor free. to overcome solubility problems associated with the natural products, the disodium phosphonooxymethyl prodrug of triptolide was prepared. the synthesis, physicochemical characterization, and in vivo efficacy in mouse models of human colon adenocarcinoma and human ovarian carcinoma demonstrated that has suitable properties to be a clinical candidate. because the synthesis could be accomplished in three steps from the natural product, scale-up of this method for the clinical use of the prodrug does not pose a problem. the chemical stability of , with a predicted shelf life of about years at °c, will allow storage of the prodrug over an extended time period. the in vivo mouse models of human colon adenocarcinoma and human ovarian carcinoma provide additional information about the efficacy and tolerability of , article suggesting that daily administration may not be required and that may be effective when administered orally. after additional preclinical safety and toxicity testing, compound entered phase i clinical trials in for evaluation in advanced gastrointestinal tumors. ■ experimental section chemistry. unless otherwise specified, all materials, reagents, and solvents were obtained from commercial suppliers and were used without further purification. the progress of a synthetic procedure was monitored, where possible, by thin layer chromatography (tlc) and the compounds of interest were visualized by short-wave uv lamp or ceric ammonium molybdate stain. tlc was conducted on silica gel μm, f plates. all solvents were removed using standard rotary evaporator techniques. flash column chromatography was performed on silica gel. h nmr spectra were recorded on a mhz and c nmr spectra were recorded on a mhz spectrometer. chemical shifts are reported in parts per million (ppm) using the solvent (cdcl ) residual peak as the internal standard (for h nmr: . ppm and for c nmr: . ppm). coupling constants (j) are reported in hertz. the multiplicities of the signals are assigned using the following abbreviations: s = singlet, d = doublet, t = triplet, br = broad, m = multiplet. high-resolution mass spectrometry (hrms) was performed by the university of minnesota mass spectrometry facility. the hplc system used consisted of a waters alliance hplc with waters photodiode array detector (milford, ma). the column used for analysis was a phenomenex c ( ) × . mm, μm particle size column (torrance, ca). for the analysis of triptolide and , the mobile phase consisted of acetonitrile ( % to % v/v) and a mm sodium phosphate monobasic buffer solution adjusted to ph with n naoh solution ( % to % v/v) for gradient elution over min. this was pumped at a flow rate of . ml/min. the injection volume was μl with detection at nm. the retention times were . and . min for and triptolide, respectively. the purity of was > %. ( bs, as, ar, r, as, as, bs, as, bs)- a-isopropyl- bmethyl- -((methylthio)methoxy)- , , b, , a, , a, a, b, bdecahydrotris(oxireno)[ ′, ′: b, ; ″, ″: , ; ‴, ‴: a, ]phenanthro-[ , -c]furan- ( h)-one ( ) . procedure a. a solution of triptolide ( ; . g, . mmol) in acetic acid ( ml, . mol) and a solution of acetic anhydride ( ml, mmol) in dmso ( ml, mmol) were mixed and stirred at room temperature for a period of days. the reaction mixture was then poured into water ( l) and neutralized with solid sodium bicarbonate added portionwise. the mixture was extracted with ethyl acetate ( × ml), and the combined organic extract was dried over anhydrous sodium sulfate. the solvent was removed under reduced pressure. the oily residue was purified by flash chromatography (ethyl acetate−hexanes : ) to produce compound ( . g; %) as a white foam. the nmr data matched those reported. procedure b. to a solution of triptolide ( . g, . mmol) in anhydrous acetonitrile ( ml) at °c dimethyl sulfide ( . ml, mmol; equiv) was added. then benzoyl peroxide ( . g, mmol, equiv) was added portionwise during the course of h. thereafter, the reaction mixture was stirred at °c for h, diluted with ethyl acetate ( ml), washed with diluted sodium carbonate (saturated na co : h o, : ) ( × ml) and brine ( ml), and dried over sodium sulfate overnight. the solvent was then removed under reduced pressure, and the resulting viscous mass containing crystals was filtered through a glass filter and washed with cold ethyl acetate ( ml). the collected solid was air-dried to yield triptonide ( ; . g). the filtrate's volume was removed under reduced pressure, and the residue was dried overnight under high vacuum to produce . g of crude product. the crude product was purified by column chromatography on silica gel using etoac−hexanes ( : ) and then etoac−ch cl ( : followed by : ) mixtures as eluents to produce the target compound ( . g; . %) and additional amounts of triptonide ( ; . g; the total yield of triptonide was . g, %). dibenzyl (((( bs, as, ar, r, as, as, bs, as, bs article phenanthro[ , -c]furan- -yl)oxy)methyl) phosphate ( ) . to a solution of compound ( . g; . mmol) in dry dichloromethane ( ml) were added powdered Å molecular sieves ( . g). the reaction mixture was placed under dry nitrogen, and then a solution of dibenzyl phosphate ( . g ( . mmol) and n-iodosuccinimide ( . g; . mmol) in anhydrous tetrahydrofuran ( ml) was added slowly at − °c. after the addition was completed, the reaction mixture was stirred at rt for a period of h, filtered, and diluted with dichloromethane ( ml). the solution was shaken with . m thiosulfate ( ml) until fully decolorized and then washed with saturated sodium bicarbonate ( ml) and brine ( ml). the organic solution was dried over sodium sulfate for . h and filtered, and then the solvent was removed under reduced pressure. the residue was dissolved in anhydrous acetonitrile ( ml), and the solution was extracted with pentane ( × ml). the acetonitrile solution was evaporated on a rotary evaporator followed by high vacuum overnight drying. yield: . g ( %) of dibenzyl ester , which was used directly in the next step without additional purification. the small sample was purified by silica gel flash chromatography ( % etoac/hexanes) to give compound as white foam. sodium ((( bs, as, ar, r, as, as, bs, as, bs )- a-isopropyl- b-methyl- -oxo- , , , , b, , a, , a, a, b, b-dodecahydrotris-(oxireno)[ ′, ′: b, ; ″, ″: , ; ‴, ‴: a, ]phenanthro[ , -c]furan- yl)oxy)methyl phosphate (minnelide, ). to a l round-bottom flask equipped with septa and stir bar were added pd/c ( . g) and anhydrous tetrahydrofuran ( ml). the flask was cooled in an ice bath and saturated with hydrogen gas using a bubbler under stirring up to full replacement of air. the bubbler was removed, and a solution of dibenzyl ester ( . g; . mmol) in dry thf ( ml) was added into the flask through a cannula while stirring and cooling the flask with an ice bath. then the reaction mixture was saturated with hydrogen again and left under stirring in the hydrogen atmosphere for h. the reaction mixture was monitored by tlc (etoac−hexanes, : ) until disappearance of dibenzyl ester . after completion, the reaction mixture was purged with nitrogen and filtered through a pad of celite. the thf solution was cooled to − °c and an ice cold solution of anhydrous sodium carbonate ( . g; . mmol) in deionized water ( ml) was added slowly with stirring to keep the temperature below °c. then the solvents were removed on a rotary evaporator followed by high vacuum evaporation to give a slightly cloudy aqueous solution. the solution was placed into equipment for continuous extraction with dichloromethane and underwent extraction for h. then the aqueous solution was separated from ch cl and extracted with ethyl acetate ( × ml). traces of organic solvent were removed by rotary evaporation, and the aqueous solution was passed through a . μm acrodisc syringe filter to give a clear aqueous solution. the solution was freeze-dried, producing ( . g; %). the product includes % of triptolide. purification by preparative hplc eluted with % methanol in water provides % pure as a colorless hygroscopic powder. aqueous solubility determination. approximately mg of each compound was weighed into ml glass vials (in triplicate); . ml of tris buffer (ph . ) was added for samples of and ml for triptolide samples. each buffered solution was saturated with or triptolide. the vials were then capped, sonicated, and vortexed prior to submersion in a constant temperature shaking bath ( shakes/min maintained at °c) for h after which time excess solid drug was removed from the saturated solution by centrifugation and filtration. the clear filtrates were then sampled and appropriately diluted for quantification by hplc. estimation of the second dissociation constant (pk a ) of using p nmr chemical shift. a mm solution of was prepared in % d o in an isotonic solution to prepare stock solutions of ml volume. samples of varying ph were prepared by adding small volumes (μl) of n hcl or n naoh solution and recording the resulting ph (denver instrument, bohemia, ny). aliquots ( . ml) were withdrawn from the stock solution after each alteration of ph and transferred to nmr tubes. twelve to fifteen such samples for analysis were prepared in the ph range of − . solutions were analyzed by p nmr spectroscopy using a mhz nmr instrument. the change in chemical shift was recorded as a function of ph. the p probe was calibrated using % h po as an external standard for the chemical shift. in solution, depending on the ph and the dissociation constant (k a ), exists in its diacidic, monobasic, or dibasic fractions as shown in scheme . the fraction of in its diacidic fraction may be ignored given the low and unlikely ph of such a solution within a physiological scenario. the fraction of in the monobasic form ( f mb ) and the fraction in the dibasic form (f db ) can, therefore, be expressed by eqs and , respectively: [h + ] represents the molar hydrogen ion concentration. the observed chemical shift (δ obsd ) of the p signal is a product of the mole fraction of the prodrug species multiplied by its chemical shift and is expressed by eq . δ mb and δ db represent the chemical shifts for the monobasic and the dibasic fraction of , respectively. substituting eqs and into , gives eq . the experimental data obtained indicate a shift in the p chemical shift with a variation in ph. this change in observed chemical shift as a function of ph is fit to eq using the graphpad prism graphing software (version . , graphpad software, inc., la jolla, ca) to estimate the second dissociation constant (pk a ) of . stability studies. a . mg amount of was dissolved in sodium chloride injection usp, and the volume was made up to ml. similarly, . mg of was dissolved in sodium chloride injection usp and the volume made up to ml. these two constituted the "high" and "low" concentration formulations, respectively. ampules were sterilized, to each ampule was added . ml of the solution of , and then they were flame-sealed. ampules were stored in ovens maintained at , in vitro stability study with . simulated gastric fluid (sgf): the fluid was generated by preparing a solution of sodium chloride ( . g), concentrated hcl ( . ml), and pepsin (sigma, p- , . g) in deionized water ( ml). the final volume was adjusted to ml by addition of deionized water. simulated intestinal fluid (sif): the fluid was generated by preparing a solution of monobasic potassium phosphate (kh po , . g), . n aqueous sodium hydroxide ( ml), and pancreatin (sigma, p- , . g) in deionized water ( ml). the ph of the solution was adjusted to . by addition of n aqueous sodium hydroxide, and then the final volume was adjusted to ml by addition of deionized water. efficacy studies in mouse xenograft models. the source of cell lines for tumor xenografts was a cryopreserved vial supplied by atcc. the ht- human colorectal adenocarcinoma cell line was established from the tumor tissue of a -year old adult female. cells were grown as a monolayer in cell culture-treated disposable flasks and cultured in hyclone mccoy's a culture media supplemented with % v/v fetal bovine serum. the original vial was thawed and cultured to create a master cell bank (mcb). the mcb vials were tested for contamination by mycoplasma organisms and for a panel of selected rodent pathogens and confirmed negative prior to culture of cells for implantation in research animals. mice were injected subcutaneously on the right flank with . × ht- tumor cells in a solution of % martigel/ % unsupplemented mccoy's a culture medium in a volume of . ml using a gauge needle. the a human ovarian cancer cell line was established from tumor tissue from an untreated patient. the source of the cell line for tumor xenografts in this study was a cryopreserved vial ordered from the ecacc (european collection of cell cultures) via the vendor sigma-aldrich. cells were grown as a monolayer in cell culture-treated disposable flasks and cultured in rpmi culture media supplemented with % v/v fetal bovine serum. the original vial was thawed and cultured to create an mcb. the mcb vials were tested for contamination by mycoplasma organisms and for a panel of selected rodent pathogens and confirmed negative prior to culture of cells for implantation in research animals. mice were each injected subcutaneously on the right or left flank with . × tumor cells in a solution of % matrigel/ % unsupplemented rpmi culture medium in a volume of . ml. female athymic nude mice, age to weeks, were received from taconic farms (albany, ny). the animals were examined and weighed on the day following receipt, and weights were measured and recorded twice weekly thereafter. all animals were allowed to acclimate to the laboratory environment for at least days prior to subcutaneous injection of cancer cells. the average tumor volume at randomization was mm for the ht- study and mm for the a study. there were mice in each group, and the duration of treatment was days for the ht- study and days for the a study. the dose concentration for each animal was based on the average of the body weight measurement for all of the animals in its respective dose group. the carboplatin dose in the a study was mg/kg administered twice each week. the test article was formulated in usp saline and delivered in a volume of . ml. the following parameters and end points were evaluated: tumor volume, tumor volume changes, tumor weight at study end, clinical signs, survival, body weights, and body weight changes. tumor measurements were performed at least twice weekly following the first appearance of tumors. a calibrated digital caliper was used to measure the length and width of each tumor. tumor volume was calculated by the formula l × w / , where w is the smallest dimension recorded. tumor volumes and tumor weights are expressed as mean ± sem. all authors participated in drafting and/or revising the manuscript. individual contributions: satish patil developed the synthesis of and designed and carried out the stability studies and was involved in the analysis and interpretation of data. lev lis developed the scale-up synthesis method for . robert schumacher designed and directed the in vivo studies and led the analysis and interpretation of in vivo study data. beverly norris conducted the in vivo studies and participated in the analysis and interpretation of in vivo study data. monique morgan conducted the in vivo studies and participated in the analysis and interpretation of in vivo study data. rebecca cuellar contributed to the analysis and interpretation of data. bruce blazar contributed to the development of the in vivo studies and to the analysis and interpretation of the in vivo study data. raj suryanarayanan participated in the stability studies and was involved in the analysis and interpretation of data. vadim j. gurvich was involved in development of the scale-up synthesis method for and in the analysis and interpretation of data. gunda georg was involved in the design of the prodrug, its synthesis, the stability studies, and the analysis and interpretation of data. notes e a , activation energy; k e , elimination rate constant; k obsd , pseudo-first-order rate constant; k r , reversion rate constant pk a , second dissociation constant sgf, simulated gastric fluid sif, simulated intestinal fluid united states pharmacopeia; xpb, xeroderma pigmentosum b; tfhii, transcription factor ii h ■ references tumor inhibitors. lxxiv. triptolide and tripdiolide, novel antileukemic diterpenoid triepoxides from tripterygium wilfordii medicinal chemistry and pharmacology of genus tripterygium (celastraceae) triptolide induces pancreatic cancer cell death via inhibition of heat shock protein triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo effect of triptolide on focal adhesion kinase and survival in mcf- breast cancer cells triptolide and its expanding multiple pharmacological functions mechanism of action of the anti-cancer agent triptolide triptolide is an inhibitor of rna polymerase i and ii-dependent transcription leading predominantly to down-regulation of short-lived mrna covalent modification of a cysteine residue in the xpb subunit of the general transcription factor tfiih through single epoxide cleavage of the transcription inhibitor triptolide triptolide binds covalently to a kda nuclear protein. role of epoxides in binding and activity the main anticancer bullets of the chinese medicinal herb, thunder god vine triptolide induces the expression of mir- − p: a negative regulator of heat shock protein and pancreatic cancer cell proliferation role of hsp- in triptolide-mediated cell death of neuroblastoma triptolide directly inhibits dctp pyrophosphatase triptolide: structural modifications, structure-activity relationships, bioactivities, clinical development and mechanisms triptolide is a traditional chinese medicine-derived inhibitor of polycystic kidney disease a potential male contraceptive triptolide preserves cognitive function and reduces neuropathology in a mouse model of alzheimer's disease triptolide treatment reduces alzheimer's disease (ad)-like pathology through inhibition of bace in a transgenic mouse model of ad preclinical antileukemic activity, toxicology, toxicokinetics and formulation development of triptolide derivative mrx design, synthesis and structure−activity relationships studies on the d ring of the natural product triptolide total synthesis of novel d-ring-modified triptolide analogues: structure-cytotoxic activity relationship studies on the d-ring of triptolide a phase i study of ethyl acetate extract of the chinese antirheumatic herb tripterygium wilfordii hook f in rheumatoid arthritis benefit of an extract of tripterygium wilfordii hook f in patients with rheumatoid arthritis -a double-blind, placebo-controlled study preparation of triptolide prodrugs having high aqueous solubility synthesis of triptolide prodrugs having high aqueous solubility for immunosuppressive and anti-inflammatory treatment pg − , a derivative of triptolide, causes tumor regression and sensitizes tumors to chemotherapy phase i dose-escalation study of f , a novel apoptosis inducer, in patients with advanced solid tumors rationale for design of biologically reversible drug derivatives: prodrugs fosamprenavir -a novel protease inhibitor and prodrug of amprenavir a case for prodrugs: fosphenytoin. adv pharmacokinetic evaluation of betamethasone and its water-soluble phosphate ester in humans synthesis, in vitro evaluation, and antileishmanial activity of water-soluble prodrugs of buparvaquone synthesis and pharmacological activity of a phosphate ester of delta- -tetrahydrocannabinol the in vitro enzymatic liabilities of chemically distinct phosphomonoester prodrugs phosphoryloxymethyl carbamates and carbonates -novel water-soluble prodrugs for amines and hindered alcohols reconversion of fosphenytoin in the presence of intestinal alkaline-phosphatase pharmacokinetics of estramustine phosphate (estracyt) in prostatic-cancer patients bioavailability of dexamethasone . dexamethasone phosphate pharmacokinetics of triamcinolone acetonide and its phosphate ester synthesis and antitumor evaluation of paclitaxel phosphonooxymethyl ethers: a novel class of water soluble paclitaxel pro-drugs a preclinical evaluation of minnelide as a therapeutic agent against pancreatic cancer cd + tumor initiating cells (tic) in a syngenic murine model of pancreatic cancer respond to minnelide minnelide: a novel therapeutic that promotes apoptosis in non-small cell lung carcinoma in vivo minnelide reduces tumor burden in preclinical models of osteosarcoma wild-type p reactivation by small-molecule minnelide tm in human papillomavirus (hpv)-positive head and neck squamous cell carcinoma inhibition of epithelial ovarian cancer by minnelide, a water-soluble pro-drug phosphonooxymethyl prodrugs of the broad spectrum antifungal azole, ravuconazole: synthesis and biological properties watersoluble prodrugs of hindered alcohols or phenols. us anandamide prodrugs. . water-soluble phosphate esters of arachidonylethanolamide and rmethanandamide hydrolysis of pharmaceutically relevant phosphate monoester monoanions: correlation to an established structure−reactivity relationship the acid strength of mono and diesters of phosphoric acid. the n-alkyl esters from me to bu, the esters of biological importance and the natural guanidinephosphoric acids formulationrelated problems associated with intravenous drug delivery human placental alkaline phosphatase in liver and intestine your prodrug releases formaldehyde: should you be concerned? no! water-soluble prodrugs of the human immunodeficiency virus protease inhibitors lopinavir and ritonavir identifier nct enantioselective total synthesis of (−)-triptolide, (−)-triptonide, (+)-triptophenolide, and (+)-triptoquinonide the authors declare no competing financial interest. the authors are grateful to the masonic cancer center (nih p ca ), the center for translational medicine, and the college of pharmacy for generous funding provided for this research. these studies were also supported by the vince and mcknight presidential chairs (to g.i.g.) and the william and mildred peters endowment fund (raj s.). we thank mary a. smart for the purification of by hplc, michael maher and vincent lanctot for assistance with the xenograft studies, and jing wang for his help with the stability studies. key: cord- -n tmn ph authors: cui, binglin; zhang, dangui; pan, hui; zhang, fan; farrar, jeremy; law, frieda; van doorn, h rogier; wu, beiyan; ba-thein, william title: viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: n tmn ph background: acute respiratory infections (aris) are common in children and mostly caused by viruses, but the significance of the detection of multiple viruses in aris is unclear. this study investigated respiratory viruses in aris among children and associated meteorological factors in shantou, southern china. methods: paired nasal/throat-flocked swabs collected from , children with aris, who visited outpatient walk-in clinics in a tertiary hospital between december and november , were examined for fourteen respiratory viruses - influenza viruses (flua, flub), respiratory syncytial viruses (rsv a and b), human coronaviruses (hcov: e, oc , hku , nl ), human metapneumoviruses (hmpv a and b), parainfluenza viruses (piv - ), human rhinoviruses (hrv a, b, c), enteroviruses (ev), adenoviruses (adv), human bocavirus (hbov), and human parechoviruses (hpev) - by multiplex real-time pcr. results: we identified at least one virus in . % ( / , ) and multiple viruses in . % ( / , ) of patients. ev and hrv were the most frequently detected single viruses ( . %, / and . %, / respectively) and co-detected pair ( . %, / ). overlapping seasonal trends of viruses were recorded over the year, with dual peaks for ev and single peaks for the others. by logistic regression analysis, ev was positively associated with the average temperature and humidity, hcov, and piv , but negatively with hrv, piv , and hbov. hrv was inversely associated with ev and piv . conclusions: this study reports high viral detection and co-detection rates in pediatric ari cases mainly due to ev and hrv. many viruses circulated throughout the year with similar seasonal trends in association with temperature, humidity, and wind velocity. statistically significant associations were present among the viruses. understanding the polyviral etiology and viral interactions in the cases with multiple viruses warrants further studies. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory infections (aris) are one of the illnesses of highest morbidity and mortality in children worldwide [ ] [ ] [ ] . the pathogens causing aris vary geographically and by season, but globally viruses play a major role. respiratory syncytial virus (rsv) is by far the most common pathogen associated with severe respiratory diseases as bronchiolitis, exacerbation of asthma, or pneumonia in early life, and is a leading cause of hospitalization in children under two [ ] . influenza viruses have the greatest potential to cause severe respiratory diseases in the very young, the elderly and those with underlying chronic conditions [ ] . enteroviruses including human rhinoviruses (hrv) and human enteroviruses (ev), previously identified in childhood upper respiratory tract infections, are commonly associated with milder aris and have been suspected as major etiological agents of lower respiratory tract infections leading to bronchiolitis and pneumonia in infants [ ] . it has also been reported that human metapneumovirus (hmpv) causes approximately - % of all aris in children and adults [ ] and adenoviruses (adv) account for - % of respiratory infections in children [ ] . respiratory illnesses can be attributable to other viruses such as parainfluenza viruses (piv) and human coronaviruses hcov- e, oc [ ] . with rapid progress in molecular diagnostics, newly discovered viruses including human bocavirus (hbov), human coronaviruses (hcov-nl , hcov-hku ), human parechoviruses (hpev), and polyomaviruses wu (wupyv) and ki (kipyv) have also been detected in children with respiratory infections, with varying levels of proof of causation [ ] . hospital-based studies in children published over the last decade worldwide have identified viruses in up to % of ari episodes, with a single virus found in - % and multiple viruses in - % of infected patients [ , , ] . coinfection is reportedly related to the time of year when circulations of multiple viruses occur [ ] . some studies have shown that the prevalence of co-infections is not related to the absolute prevalence of individual viruses [ ] . factors such as young age, male gender, and history of immunosuppression are associated with an increased chance of viral co-infections [ , , ] . there could be likely interactions between climatic, environmental, and behavioral factors, and complex interplay between circulating viruses and population-level immunity regarding viral coinfections. understanding these factors may help us prevent transmission of these infections. recent etiologic studies on pediatric respiratory infections mostly report the prevalence in hospitalized children and the seasonality of viruses without elaborating viral co-infection. therefore, the significance of the detection of multiple viral pathogens in aris is unclear. here, we investigated fourteen common respiratory viruses among pediatric outpatients in southern china during - and their associations with meteorological factors. this study was conducted at the pediatric outpatient walk-in clinics, the first affiliated hospital of shantou university medical college. the pediatric department provides both primary and tertiary care (common practice in china) for approximately , children per year in the chaoshan region of southern china. the chaoshan region is in the subtropical zone with an average annual temperature of . °c and excellent to lightly polluted air quality levels (air quality index, aqi: - , in - ) . based on modified who standard case definition of aris [ ] , eligible participants were defined as a child - years of age presenting within days of onset of illness with at least two of the following: fever, sore throat, cough, rhinorrhea, nasal congestion, and hoarseness of voice. patients with any condition preventing swab collection were excluded. we recruited eligible patients in the morning, during which approximately % of patient visits are made, on a daily basis except public holidays from december to november . participants' demographic details and clinical features are shown in table . paired nose and throat-flocked swabs (copan, brescia, italy, cat. no. cs and cs ) were collected from each participant, combined in one tube, and stored within h of collection at − °c until further processing. multiplex real-time pcr was performed using roche, lightcycler ii (roche diagnostics, penzberg, germany) to identify the following respiratory viruses: influenza a (flua), influenza b (flub), respiratory syncytial viruses a and b (rsv), human coronaviruses e, oc , hku and nl (hcov), human metapneumoviruses a and b (hmpv), human parainfluenza virus types , , , and (piv , piv , piv , and piv ), human rhinoviruses a, b, and c (hrv), human enteroviruses (ev), human adenoviruses (adv), human bocavirus (hbov), and human parechoviruses (hpev). nucleic acid extraction was performed using the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany, cat. no. ). reverse transcription and realtime pcr assays were performed as described previously [ ] , except for the primers and/or probes for hrv, hpev, and internal control equine arteritis virus (eav, see the sequences of viruses in additional file ). due to known cross-reactivity between enteroviruses [ ] [ ] [ ] , hrv was detected using two sets of primers and probes: hrv-v (version ) for screening and hrv-v (version ) for confirmation. real-time pcr results were interpreted as described previously [ ] . the pcr was considered positive or negative when the cp value was less than cycles or exceeded cycles, respectively, and the positive control showed the expected cp value, negative control was negative, and internal control showed the expected cp value. a negative internal control signal was accepted in case of a positive target sequence with correct positive and negative control signals. meteorological data, including the average daily temperature (°c), the average daily humidity (%), and the average daily wind velocity (km/h), were collected from the official website of shantou meteorology, tutiempo. net (http://www.tutiempo.net/en/climate/shantou/ / .htm). we used chi-square test to compare differences in the distribution of categorical variables, anova and kruskall wallis tests to compare medians, and the pearson correlation analysis to evaluate the associations between the meteorological factors and viruses and among viruses. the variables with significant associations were further analyzed in multivariate logistic regression models, in which symptoms and positivity of viruses were treated as dependent and independent variables to assess virussymptom associations; and individual viruses were treated as dependent variables with meteorological factors or other viruses as independent variables to investigate meteorological factor-virus and virus-virus associations. a two-tailed p-value of < . was considered significant. all these analyses were performed with spss statistics version . . the study was approved by the ethics committee of the first affiliated hospital of shantou university medical college and the oxford university tropical research ethical committee (oxtrec). written informed consent was obtained from parents or legal guardians of children enrolled in the study. of , children ( . % male) recruited, . % ( / , ) were > - years old (table ). at least one virus was identified in . % ( / , ) of the patients, with single virus in . % ( / , ) and multiple viruses in . % ( / , ). hpev was not detected. compared with virusnegative patients, virus-positive patients were less likely to have fever (or: . , % ci: . - . , p = . ). patients with multiple viruses were more likely to have rhinorrhea than those with single virus (or: . , % ci: . - . , p < . , table ). hcov (or: . , % ci: . - . ) and piv (or: . , % ci: . - . ) were more prevalent in the > year age group than in the ≤ year group (all p ≤ . ), while hbov (or: . , % ci: . - . ) and rsv (or: . , % ci: . - . ) were less frequently found in the > year group (all p < . ). chi-square test and multivariate logistic regression analysis showed that cough was positively associated with hrv and rsv, and negatively with ev; rhinorrhea was positively associated with hrv, piv , and hbov, and negatively with ev; fever was positively associated with ev, and negatively with hrv and piv ; and nasal congestion was positively associated with rsv, and negatively with ev and hcov (all p < . , table ). viruses detected alone or co-detected with other viruses are shown in table . the most frequently detected virus was ev ( . %, / ), followed by hrv ( . %, / ), and hcov ( . %, / ). ev and hrv were most commonly co-detected with other viruses (table ) and also the most commonly co-detected pair of viruses ( . %, / , see the distribution pattern of viruses in additional file ). screening with hrv-v identified cases co-positive for hrv and ev, and subsequent confirmation with hrv-v primers/ probes [ ] resulted in only positive cases ( . %, / ). the temporal circulation and co-circulation patterns of viruses are shown in figures and . there were overlapping seasonal trends of many viruses throughout the year, with dual peaks for ev in july and september and single peaks for the other viruses. both ev and hrv circulated throughout the year. hcov and piv circulated predominantly between april and may but sporadically throughout the year. piv , rsv, flua, and adv peaked in january, while hbov peaked in march. flub circulated mostly from february to july with a peak in april. codetection of - viruses occurred all in may (see additional file ). the optimal average daily temperature, humidity, and wind velocity for these viruses are shown in table . table shows the multivariate logistic regression models for independent associations between the viruses and meteorological factors and between the viruses. ev was positively associated with the average temperature and humidity and the presence of hcov and piv , but negatively with hrv, piv , and hbov. hrv was negatively associated with the presence of ev and piv . hcov was positively associated with the average temperature and humidity and the presence of ev and piv . piv was positively associated with the average humidity and the presence of rsv and flua, but negatively with the average temperature and wind velocity, and the presence of ev, hrv, and hbov. piv was positively associated with the average temperature and the presence of hcov and rsv, however, negatively with the wind velocity. hbov was positively associated with rsv and flua, but negatively with the average temperature and humidity and the presence of ev and piv . rsv was positively associated with the presence of piv - , hbov, and flua, but negatively with the average temperature and wind velocity. flua was positively associated with the presence of piv - , hbov, and rsv, but negatively with the average temperature. this is the first prospective study reporting the associations between meteorological parameters and co-circulation patterns of common respiratory viruses. the viral detection rate among pediatric outpatients with aris in this study ( . %, / , ) was higher than those reported from nanjing, china ( viruses, . %, / ) [ ] and other countries, including honduras ( viruses, . %, / ) [ ] and greece ( viruses, . %, / ) [ ] in the same study period. enteroviruses (ev, . % and hrv, . %) were most frequently detected in our outpatient children. influenza viruses and rsv, the leading pathogens in pediatric outpatients in only statistically significant results (p < . by chi-square test for individual comparisons of proportions within each group) are shown as "√" with reference(s) shown as "-". the optimal temperature for hcov and hbov was - °c. the optimal relative humidity was - % for piv and - % for hcov. similar studies [ , [ ] [ ] [ ] , were detected in . % and . % of our cases, with hcovs ( e, oc , hku , and nl ) in . %, and relatively recently discovered viral pathogens hbov and hmpv in . % and . % of cases, respectively ( table ) . the viral co-detection rate ( . %, / , ) was also high among our study population. reported rates of codetection vary widely, from . % among pediatric patients with influenza-like illness [ ] to % among infants with acute bronchiolitis [ ] . detection of dual viruses is common, and co-detection of five [ ] or even six viruses [ ] is not anecdotal. all the cases with - viruses in this study were in may, the end of the cold season in the chaoshan region. this may be in part due to past viral infections, as some viruses can still be detectable by pcr several weeks after infection [ , ] . most studies have shown that rsv is the predominant respiratory pathogen co-detected in hospitalized children, followed by hrv, piv, hmpv, hbov, and flua [ , ] . in this study, ev, hrv, hcov, and piv - were involved in the majority of co-detections, with ev-hrv as the most frequently co-detected pair ( % of codetections). ev and hrv were included in the panels in many studies globally [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and the ev-hrv pair was the most commonly detected pair among outpatient children with aris in finland ( . % of codetections) [ ] and infants with acute bronchiolitis in brazil [ ] . the co-detection rate of ev-hrv in this study is similar to that in finland [ ] . varying detection rates of multiple viruses in different studies may reflect the differences in the study period and location, study population, environmental factors, the number of respiratory pathogens tested, and/or the diagnostic methods/techniques used. likely reasons behind high detection rates of single and multiple viruses in this study could be due to improved recovery of viruses by using flocked swabs [ ] and/or combined nasal and throat swabs [ ] . there are advantages and disadvantages of multiplex pcr technique in diagnosing respiratory viral infections. while its high sensitivity and specificity facilitate simultaneous detection of a large spectrum of viruses, including those difficult to be identified by traditional methods [ ] , its capacity to detect low amounts of viral nucleic acids in some cases during viral incubation period, asymptomatic infection, or post-infectious shedding makes it difficult to interpret the results [ , ] . the development and validation of standardized quantitative pcr with clinically relevant cutoff values [ ] or combining qpcr with serology could be helpful for etiologic understanding of simultaneous presence of multiple viruses. certain host-specific risk factors may predispose a child to respiratory co-infection. younger age [ , , ] , male gender, and history of immunosuppression are associated with increased risk of viral co-detections [ ] . nonetheless, similar associations were not found in this study. viral co-detection is not random; clear associations for certain viral co-occurrence have been described [ ] . the viruses circulating at the same time of a year are more likely to accompany each other [ , , ] . this may be driven by meteorological factors which actually work behind seasonal variations, or by interactions of certain coexisting viruses. temperature, humidity, and wind velocity are the most commonly studied factors significantly associated with the overall number of ari hospitalizations and the prevalence of various respiratory viruses [ ] [ ] [ ] . the average temperature is the key climatic parameter associated with the prevalence of many respiratory viruses. some viruses survive and/or replicate better at low temperatures, having peak prevalence in the colder months. in our study, the detection rates of piv , rsv, flua, and adv were negatively associated with temperature and were highest at temperatures between °c and °c (tables and ), supporting the notion that low temperature is suitable for the survival of lipidenveloped air-borne viruses [ ] . low temperatures have been found to favor rsv in southeast china [ ] , malaysia [ ] , nepal [ ] , brazil [ ] and germany [ ] , influenza in japan [ ] and germany, and adv in germany [ ] ; however, high temperatures favored piv in southeast china [ , ] and nepal [ ] , rsv in singapore [ ] , hong kong [ ] , and indonesia [ ] , and adv in southeast china [ ] . no association between temperature and flua activity was found in nepal [ ] and brazil [ ] . in our study, other viruses such as ev, hcov, and piv were more often detected during months with higher temperatures, having peaks at temperatures between °c and °c (tables and ). in contrast to our findings, hcov was negatively associated with temperature, and no association between ev and temperature was found among children with aris in germany [ ] . association of humidity and viral detection rates has been reported from germany [ ] , singapore, hong kong, brisbane, and vancouver [ ] . in this study, three viruses (ev, hcov, and piv ) were positively associated with the average humidity ( table ). the optimal average humidity ranges for ev and hcov were - % and - % respectively, supporting a previous finding that high average humidity ( %) had a protective effect on the survival of hcov [ ] . our findings on piv are inconsistent with animal and laboratory observations that lipid-enveloped viruses such as piv survived better in cooler, less humid environment [ ] . hbov was negatively associated with the average humidity, and its optimal humidity was - % (tables and ). no climatic data is available for comparison regarding this virus. previously reported association between flua and the average humidity [ ] was not found in our study. wind velocity piv and piv have been reported to be negatively associated with wind velocity [ ] . in low wind speed environment, viruses can easily colonize in the epithelium of upper respiratory tract [ ] . an increased wind velocity is correlated with rsv activity in germany [ ] . in our study, piv , piv , and rsv were inversely associated with the wind velocity. although we observed higher rates of ev and flub but lower rate of hrv in low wind velocity, we could not confirm these associations by logistic regression analysis. the underlying reasons for the observed associations between virus circulations and meteorological factors are unclear. climate could have a direct or indirect effect on viral survival, transmission efficiency, host immunity, and social behavior change [ , ] . cold and dry conditions might favor the transmission of viruses, and cold or rainy days could decrease outdoor activities of children and increase the probabilities of close contact and transmission of infections [ ] . holidays (supported by our data with less cases in february as chinese new year and july-august summer holiday, figure and additional file ), could also play a role in an annual epidemic cycle [ ] . it is likely that several factors interact in complex ways in the development of observed epidemics under optimal climatic conditions and that the contributions of individual factors vary for different viruses. further investigations such as time series model over many years are needed to account for their inherent autocorrelations [ ] , and thus the observed associations between meteorological parameters and viruses in this exploratory analysis should be interpreted with caution. viral co-detection patterns may be the reflection of interactions between viruses. co-detection of viruses has been frequently reported [ , , , , , ] . here, we have assured their associations by mathematical models (table ) . we identified many pairs of viruses with positive associations, including ev-hcov, hcov-piv , piv -rsv, piv -flua, piv -rsv, hbov-rsv, hbov-flua, and rsv-flua. negative associations for ev-hrv, ev-piv , ev-hbov, hrv-piv , and piv -hbov were also found in this study. both belonging to the enteroviruses genus, hrv and ev have similarities in the highly conserved sequence of the ' noncoding region, which is the preferred site for molecular assay development [ , , , ] . cross-reactivity between the primers of hrv with evs has been reported and is among others attributable to ev-d , an emerging pathogen frequently undetected and misdiagnosed as hrv [ , , , ] . confirmation of cross-reacting ev types in this geographic region should be done in future studies. there is no consensus in the literature on the clinical implications of the viral detection and co-detection. some studies linked multiple viral detections with fever [ ] , or increased hospitalization and intensive care admission [ ] , while others described a very similar prognosis as in single infection [ , , ] , or even milder presentations [ ] . in this study, the virus-negative patients had fever more often, which may be caused by other pathogens such as bacteria. we also found that rhinorrhea was more frequently present in patients with multiple viruses than in those with a single virus, and some viruses were more (or less) likely to exist in certain age groups or were accompanied with certain symptoms. since we did not follow the cases, the associated clinical course and outcome (such as hospitalization) remain unknown. a better understanding on the clinical courses of single and multiple viral etiologies requires further studies. the current study has several limitations. the majority of outpatients enrolled in this study were mild and moderate cases. therefore, we could have missed pathogens responsible for severe aris. as healthy or asymptomatic controls were not included, their viral carriage burdens and the actual role of virus infections could not be elucidated. following up the cases for clinical burdens and serologic testing would be required in future studies. air quality indicators such as ozone and pm . , which might influence the host's susceptibility or virus circulation, should be included to investigate meteorological factors. in summary, this study reports a high viral carriage in pediatric ari cases with high viral co-detection rates mainly due to ev and hrv. there were overlapping seasonal trends of many viruses throughout the year. meteorological factors, including temperature, humidity, and wind velocity, were associated with the viral detection rates. statistically significant associations were present among the viruses. further studies are needed to address polyviral etiology and viral interaction in multiple virus positive cases. additional file : primers and probes of viruses. additional file : the distribution pattern of viruses in pediatric outpatients with acute respiratory infections, aris (n= ). the authors declare that they have no competing interests. authors' contributions blc and hp designed and performed the experiments, analyzed the data, and wrote the paper. dgz designed 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exacerbations in children with cystic fibrosis submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank the pediatricians from the pediatric department, the first affiliated hospital of shantou university medical college for their generous support, the children and their guardians for participation in this study, richard molenkamp at the university of amsterdam, academic medical center for technique and knowledge transfer to set up the multiplex real-time pcr, jieling chen at the shantou-oxford clinical research unit for technical assistance, and staff in the international institute of infection and immunity, shantou university medical college for their assistance with real-time pcr. this study was supported by the li ka shing foundation, shantou university medical college, and the university of oxford (grant no. b rsrt - ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord- -mqq fmsp authors: waumans, yannick; baerts, lesley; kehoe, kaat; lambeir, anne-marie; de meester, ingrid title: the dipeptidyl peptidase family, prolyl oligopeptidase, and prolyl carboxypeptidase in the immune system and inflammatory disease, including atherosclerosis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mqq fmsp research from over the past years has implicated dipeptidyl peptidase (dpp) iv and its family members in many processes and different pathologies of the immune system. most research has been focused on either dppiv or just a few of its family members. it is, however, essential to consider the entire dpp family when discussing any one of its members. there is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. in this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases dppiv, fap, dpp , dpp , dipeptidyl peptidase ii, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. we highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease. research from over the past years has implicated the dipeptidyl peptidase (dpp) family in various physiological processes and pathologies of the immune system. usually only four prolyl-specific peptidases are considered: dppiv (ec . . . ), fibroblast activation protein α (fap; ec . . .b ), and the more recently discovered dpp and dpp (ec . . ). however, due to similarities in substrate specificity and structural homology, it is more relevant to consider a broader family that also includes prolyl oligopeptidase (prep; ec . . . ), dipeptidyl peptidase ii (dppii) (ec . . . ), and prolyl carboxypeptidase (prcp; ec . . . ). first, dppii and prcp share the α/β hydrolase fold with the other dpps and the catalytic triad is completely conserved in both enzymes ( ) . moreover, dppii can cleave several dppiv substrates in vitro ( ) . conversely, due to its substrate preference for tripeptides ( ) , dppii could actually be considered as a prolyl carboxytripeptidase, emphasizing its similarities to prcp. another argument for considering a broader family stems from the fact that functional studies on the role of peptidases rely heavily on the use of enzyme inhibitors and many of the inhibitors used in earlier studies are now known to inhibit more than one family member. for example, early studies on dppiv used inhibitors which we now know also inhibit dppii, dpp , dpp , fap, and/or prep due to their sequential and/or structural similarity [e.g., ref. ( ) ( ) ( ) ( ) ( ) ]. prcp is known to be inhibited by kyp- and z-pro-prolinal at higher concentrations, which have often been used for the functional study of prep [e.g., ref. ( ) ( ) ( ) ]. table summarizes the most commonly used dpp inhibitors and their selectivity compared to dppiv. in view of the aforementioned reasons and for the sake of simplicity, we will use "dpp family" as a blanket term, which includes dppii, prcp, and prep even though strictly speaking they are not dpps. figure provides a general overview of this broadly defined dpp family. the roles of various family members in certain aspects of the immune system or immune dysfunction have been reviewed in the past [e.g., ref. ( ) ( ) ( ) ]. in this review, we provide a comprehensive discussion and update on the roles of dppiv, dppii, dpp , dpp , fap, prep, and prcp in the immune system and inflammatory disease. we highlight the role of these enzymes in atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease, as the dpp family encompasses possible therapeutic targets for the prevention and treatment of this disease. the prototypical dpp, dppiv (often dpp in medical jargon) cleaves off an n-terminal dipeptide from peptides with pro or ala on the penultimate position. its localization as a soluble enzyme in body fluids, or anchored in the plasma membrane of cells provides it with the necessary access to cleave a wide range of bioactive peptides. as such, it can modify their biological activity. glucagon-like peptide (glp)- and - , and glucosedependent insulinotropic peptide (gip) ( , ), substance p ( ), neuropeptide y (npy) ( ), stromal cell-derived factor- α/β (sdf- α/β or cxcl ) ( ), granulocyte macrophage colony-stimulating factor (gm-csf) ( ), cxcl ( - ), and high-mobility group box (hmgb ) ( ) have been identified as physiological substrates, while others, such as rantes, have been proposed based on in vitro experiments [e.g., ref. ( )]. dppiv also performs many of its physiological functions through interactions with other proteins, such as collagen, fibronectin, adenosine deaminase (ada), caveolin- , and the mannose- -phosphate/insulin-like growth factor ii receptor (m p/igfiir) ( - ). some of those will be discussed in more detail below. dipeptidyl peptidase iv is well known for its role in glucose homeostasis. it has become a validated therapeutic target for the treatment of type diabetes (t d) ( ). dppiv inhibitors reduce the rate of glp- inactivation (boxes and ). it has also been shown to be involved in cancer biology. the role of the dpp family in cancer has been addressed in several other reviews ( , - ). finally, dppiv has recently come back into the center of attention as the receptor for the mers coronavirus ( ). the incretins are a group of glucose-lowering molecules produced by the intestines. the best known incretin is glucagon-like peptide- (glp- ). this incretin is derived from proglucagon and secreted after a meal from l-cells in the distal ileum and colon. in the pancreas, it induces insulin secretion and biosynthesis while lowering glucagon secretion. in addition, glp- increases the β-cell mass, thereby restoring insulin production. it is clear that glp- also has functions outside glucose metabolism. its receptor, glp- -r, is not only found in the pancreas but also expressed in brain, lung, kidney, stomach, and heart ( , ). recently, it was shown that stimulation after myocardial infarction reduces the infarct size ( , ). currently, glp- agonists are approved for the treatment of type diabetes. these incretin mimetics seem to have a slightly better efficacy as dppiv inhibitors and lead more frequently to weight loss. unfortunately, an important drawback for their therapeutic use is that they can only be administered by subcutaneous injection ( ). fibroblast activation protein α, also known as seprase can present itself as a type ii transmembrane protein or as a shedded plasma protease ( ). in the latter case, it is also known as antiplasmincleaving enzyme, which converts α -antiplasmin into a more active form, suppressing fibrinolysis ( ). some of the known dppiv substrates were later found to be cleaved in vitro by fap as well ( ), though any physiological relevance remains unclear. unlike dppiv, fap also possesses a gelatinase activity. this enables fap to degrade proteins of the extracellular matrix ( ). this is of particular interest with regard to its involvement in a number of pathological processes ( ). fap is highly induced during inflammation, activation of hepatic stellate cells in liver cirrhosis and strongly expressed by mesenchymal cells of remodeling tissue ( , ). fap is also a key regulator during tumor growth and metastasis ( ). as all these processes require degradation of the extracellular matrix, fap's involvement in these pathologies is most likely associated with its gelatinase activity ( ). its role in cancer biology has been reviewed before ( , ). it is interesting to note that, so far, in clinical trials talabostat has shown minimal or no clinical benefit for the treatment of metastatic colorectal cancer, advanced non-small cell lung cancer, or stage iv melanoma ( - ). it should be mentioned, however, that talabostat is a broad-range inhibitor also targeting dppiv, dpp , and dpp . dipeptidyl peptidases and dpp show dppiv-like activity and share a very high-sequence similarity to each other ( % aa similarity, % aa identity) ( ). these cytoplasmic enzymes have several isoforms. it has been a matter of debate whether all are expressed as protein in cells and, if so, whether they are active ( - ). interestingly, the n-terminal extension of the longer dpp variant contains a nuclear localization signal and, indeed, this form localizes to the nucleus ( ). dpp has been shown to cleave a number of dppiv chemokine substrates in vitro ( ). another dppiv substrate, npy, has indirectly been shown to be box dppiv inhibitors. dipeptidyl peptidase iv inhibitors prolong the biological half-life of the incretins and are therefore used for the treatment of type diabetes. sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin are dppiv inhibitors currently available on the market for treatment of type diabetes. sitagliptin and alogliptin are highly selective toward dppiv in vitro, whereas vildagliptin and saxagliptin are less selective with regard to dpp and , and linagliptin with regard to fap ( ). their clinical efficacy and safety in the use of type diabetes seem comparable as far as can be judged from the data available. there is a growing interest toward a use outside type diabetes as it has become clear that dppiv inhibitors have pleiotropic effects. while negative effects have been found in heart failure ( ), some studies suggest them as a possible therapeutic strategy in cardiovascular pathologies ( , ). the sitagrami trial and follow-up studies revealed that the combination of a dppiv inhibitor with granulocyte-colony-stimulating factor or in monotherapy presents a therapeutic option after myocardial infarction ( , ). as stated above, the mechanism is not yet clear but may be explained by a longer biological half-life of dppiv substrates, glucagon-like peptide- , b-type natriuretic peptide, and stromal cell-derived factor- α/β. all three peptides have a cardioprotective effect that is abolished by dppiv-mediated cleavage. a dpp and dpp substrate as well ( ). efforts have been made to find intracellular dpp and substrates using a peptidomic approach ( ), but so far it has been hard to attribute physiological relevance to the possible substrates beyond the role of dpp and in intracellular peptide turnover ( ). the physiological functions of dpp and dpp are still not properly understood. mainly, a lack of available knockout animals, specific inhibitors, and substrates has hampered progress ( ). a mouse model has been established with a targeted inactivation of dpp enzymatic activity ( ), but homozygous dpp inactive neonates die within - h after birth. despite these limitations, some indications toward their role are surfacing. using immunohistochemistry, dpp and were found associated with spermatozoids and spermatids and the short mrna of dpp is predominantly expressed in testes ( , ), suggesting a role in spermatogenesis and male fertility. recent work has found sumo to be an allosteric activator of dpp ( ), whereas a small peptide corresponding to the interaction surface of sumo is a non-competitive inhibitor of dpp and dpp ( ). a genomewide association study has linked dpp to idiopathic pulmonary fibrosis ( ). finally, a number of studies have shown a role for dpp and dpp in apoptosis ( , - ). two studies showed that overexpression enhanced induced apoptosis and impaired cell adhesion and migration ( , ). conversely, dpp / inhibition in tumor cells decreased the number of viable cells because of a decreased cleavage of pro-apoptotic npy ( ). in macrophages, inhibition caused a marginal, yet significant increase in apoptosis, independent of npy cleavage ( ). interestingly, vildagliptin, a dppiv inhibitor already on the market to treat type diabetes, but with poorer selectivity toward dpp and , was shown to enhance parthenolide's anti-leukemic activity through its inhibition of dpp and , and not dppiv ( ). prolyl carboxypeptidase, also called angiotensinase c or lysosomal pro-x carboxypeptidase, is a lysosomal carboxypeptidase sharing strong sequence homology with the likewise lysosomal dppii ( , ) . prcp preferentially cleaves off the c-terminal amino acid when ala or pro is in the penultimate position, while dppii targets n-terminal x-pro or x-ala dipeptides ( , ). in addition to a structural similarity, prcp and dppii have partially overlapping substrate specificities due to dppii's preference for tripeptide substrates ( ) . perhaps surprisingly, gly-pro-pna and ala-pro-pna, two typical synthetic dpp substrates, have actually been used to perform prcp activity measurements ( ). prolyl carboxypeptidase is particularly known as one of the key enzymes of the renin-angiotensin system (ras). it inactivates the vasoactive peptides angiotensin ii and angiotensin iii by cleaving off the c-terminal phe ( ). α-melanocyt-stimulating hormone - , an anorexigenic neuromodulator, is inactivated by prcp, implying a role in body weight control ( ). based on the involvement of prcp in the conversion of these peptide hormones, the enzyme has also been associated with diseases, such as hypertension, diabetes mellitus, obesity, inflammation, and cardiovascular dysfunction ( , ). dipeptidyl peptidase ii has no known natural substrates. the dppiv substrate substance p has been shown to be cleaved by dppii in vitro ( ), but much less efficiently, casting doubt over any physiological relevance. it has been shown that inhibition or silencing of dppii causes apoptosis of quiescent g lymphocytes ( - ). on the other hand, a highly specific dppii inhibitor, uamc , did not induce apoptosis, autophagy, or necrosis in human leukocytes ( , ), but this study did not specifically look at quiescent cells or lymphocytes. finally, changes in dppii activity levels have been observed in a number of pathologies, such as neurodegenerative disorders, myopathies, cancer, and gastro-intestinal disorders ( ). prolyl oligopeptidase is an oligopeptidase with endopeptidase activity. it has been shown to be localized in the cytoplasm ( ) ( ) ( ) ( ) , but given its ability to inactivate several neuropeptides in vitro by limited proteolysis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , its involvement in the in vivo generation of immunoactive peptides n-acetyl-prolyl-glycylproline and n-acetyl-seryl-aspartyl-lysyl-proline ( , ) , and its presence in plasma ( , ) , it most likely also has an extracellular role. initial interest for prep derived from the positive effects of prep inhibitors on scopolamine-induced amnesia in rats ( ) ( ) ( ) ( ) . prep inhibition was also found to promote neuronal survival and neurite outgrowth of cerebellar granule cells ( ) . however, a recent study in mice shows that the lack of prep in vivo causes a reduction of synaptic spine density in the hippocampal region along with reduced long-term potentiation and memory functions ( ) . many of prep's functions are mediated through its interactions with other proteins. prep is known to interact with gap- ( , ) , α-tubulin ( ) , and gadph ( ) . its most studied interaction is with α-synuclein ( ), reviewed in ref. ( ) . prep and α-synuclein have been shown to co-localize in cell models of stress and in the substantia nigra of post-mortem parkinson's disease brain ( , ) . in vitro, the aggregation rate of α-synuclein increases in the presence of high concentrations of prep, which is abolished through active site inhibitors of prep and absent with a catalytically impaired prep mutant ( ) . in vivo, prep inhibition reduces α-synuclein aggregates in a cellular and animal model for parkinson's disease ( ) . the role of dppiv in monocytes and macrophages has been somewhat contested. whereas dppiv's presence on monocytes and macrophages has been shown repeatedly in mice and rats ( ) ( ) ( ) , its expression in human monocytes and macrophages is less obvious. figure shows an overview of the expression of dppiv throughout the immune system. in visceral obesity, dppiv expression is low on peripheral blood monocytes, macrophages, and dendritic cells, but it is upregulated in vitro frontiers in immunology | www.frontiersin.org august | volume | article after differentiation and activation of isolated monocytes into macrophages or dendritic cells, and in vivo locally in adipose tissue ( ) . interestingly, the authors showed that macrophage-or dendritic cell-associated dppiv most likely binds ada, promoting local degradation of adenosine, a t-cell proliferation suppressor, thereby inducing t-cell proliferation ( ) . three other studies also found no to low dppiv expression or activity associated with human monocytes and/or macrophages ( , [ ] [ ] [ ] . others have investigated dppiv in monocyte-or macrophage-like cell lines ( , , ( ) ( ) ( ) ( ) ( ) ( ) . in hl- cells, its expression has been found to be regulated by differentiation into macrophage-like cells ( ) . dppiv inhibitor alogliptin can affect erk activation, mmp and il- secretion in u cells ( , ) . however, these studies employed alogliptin at concentrations lower than its ic for dppiv. it is therefore questionable whether the observed effects were mediated by dppiv at all. on the other hand, proliferation is reduced in the presence of a dpp inhibitor in u cells expressing high levels of dppiv, but not in the same cell type expressing low levels of dppiv ( ) . moreover, the same inhibitor causes the former cells to secrete lower amounts of il- β, but higher amounts of tnfα ( ) . it could be that inhibition merely increases tnfα's half-life, as dppiv has been implicated in its degradation in u cells ( ) . in thp- cells, dppiv inhibitors alogliptin and sitagliptin both reduced these cells' chemotactic potential ( ) . dppiv inhibitors sitagliptin and nvpdpp also reduced nlrp , tlr , and il- β expression and increased glp- r expression in thp- cells and this effect was blocked through pma differentiation ( ) . importantly, such cell lines have been derived from different types of myeloid leukemia, and as it is known that dppiv expression is often dysregulated in cancer ( - ), the physiological relevance of these findings remains uncertain. fap has been shown on tumor-associated macrophages in human breast cancer ( ) . dipeptidyl peptidase / activity has been found in human monocytes and u cells ( ) . dpp was found associated with activated microglia/macrophages in a rat model of cerebral ischemia ( ) . dpp and are abundantly present in macrophage-rich regions of atherosclerotic plaques ( ). interestingly, dpp is upregulated after in vitro monocyte-to-macrophage differentiation. moreover, inhibition or rna silencing of dpp attenuates pro-inflammatory m , but not m , macrophage activation ( ). in rats, dppii is expressed in tissue-resident macrophages ( , ) . humans show dppii activity in monocytes as well as u cells ( , ) . human blood derived alveolar macrophages show high-prcp activity ( , ) . interestingly, in a mouse in vivo angiogenesis assay, macrophage infiltration into the wound was increased in mice with a prcp deletion ( ) . prolyl oligopeptidase activity has been shown in mouse and rat peritoneal macrophages and in rat pulmonary macrophages ( , , ) . its activity in mouse peritoneal macrophages is increased after thioglycollate ellicitation ( ) . in addition, prep has been identified as a neurotoxic component in the supernatant of activated thp- cells, which are monocyte-like cells ( ) . apparently, these cells secrete prep upon activation with ifnγ and lps and partly because of this, their supernatant is toxic to neuroblastoma sh-sy y cells, as shown through the use of prepspecific inhibitors ( ) . prep's mode of action in this remains unclear. recently, a study showed that dppiv acts as a chemorepellent for human and murine neutrophils ( ) . adding recombinant dppiv to purified human neutrophils in an insall chamber causes the neutrophils to migrate away from the higher concentration of dppiv. this effect is blocked by dppiv inhibitors, meaning that the effect is mediated through dppiv's enzymatic activity, although a candidate substrate is not obvious. moreover, in a mouse model of acute respiratory distress syndrome, oropharyngeal aspiration of dppiv prevented accumulation of neutrophils in the lung ( ) . by contrast, prep is involved in the generation of prolyl-glycyl-proline, a collagen fragment that is an efficient neutrophil chemoattractant ( ) . human peripheral blood neutrophils contain prep activity and are themselves capable of generating prolyl-glycyl-proline after lps-activation, alluding to a self-sustaining pathway of neutrophil inflammation ( ) . prcp is also abundantly expressed in human neutrophils ( ). the recruitment of eosinophils is affected by dppiv activity. ccl , also known as eotaxin, is a dppiv substrate and cleavage by dppiv prevents the activation of its receptor ccr ( ) . in rats, it was shown that administration of ccl results in eosinophil recruitment and this recruitment is significantly more effective in dppiv-deficient f mutants ( ) . finally, dppii activity has been reported in the granules of mast cells in several publications ( , , ) . it is released from peritoneal mast cells upon degranulation and is apparently inhibited by histamine and zn + at concentrations present in the granules of mast cells ( ) . dipeptidyl peptidase is present in low amounts on freshly isolated human nk cells and its expression is only upregulated in a small subpopulation after il- stimulation ( ) . in that study, it was also shown that dppiv inhibition suppresses dna synthesis and cell cycle progression of nk cells, but these effects may be dpp / mediated as the inhibitors used in that study are now known to also inhibit dpp / activity ( ) . another study shows that dppiv is actually only expressed by a small subpopulation of peripheral nk cells ( ) . the natural cytotoxicity of nk cells is not influenced by the presence or absence of dppiv on their cell surface ( , ) . however, dppiv-negative nk cells show significantly less cd -dependent lysis than dppiv-positive nk cells ( ) . interestingly, nk cytolytic function against tumor cells was diminished in dppiv-deficient rats in a model for lung metastasis ( ) . figure shows an overview of published data on the dpp family in the innate immune system. only about % of freshly isolated cd -positive b cells express dppiv, but this fraction grows significantly upon pokeweed mitogen (pwm) or s. aureus protein stimulation ( ) . similar to nk cells, dppiv inhibitors significantly suppress dna synthesis in b-lymphocytes ( ) , but again these inhibitors are now known to also inhibit dpp and ( ) . mouse spleen-derived b-lymphocytes only express low amounts of dppiv mrna ( ) . dpp and mrna, on the other hand, are expressed at much greater levels in these cells, and they are upregulated in raji cells, a b-lymphocyte-like cell line, after pwm, lps stimulation or mitomycin c treatment, and downregulated after dtt treatment ( ) . dpp and have also been shown immunohistochemically in human lymph follicular lymphocytes ( ) . dppii activity has also been shown in human b-lymphocytes ( ). dipeptidyl peptidase iv was originally described as a surface marker for t-lymphocytes, in which case it is better known as cd , and later more specifically for a subset of cd -positive memory cells, cd + cd ro + cd + cells, which respond maximally to recall antigen tetanus toxoid and induce b-cell igg synthesis ( , ) . indeed, cd surface expression is augmented along with the antigen sensitivity of a particular cd + t-cell clone ( ) . cd high cd + t-cells belong to the early effector memory t-cell subset ( ) . cd is also a marker for t-cell activation ( , ( ) ( ) ( ) . cd expression on cd + tcells correlates with t h responses. stimuli that typically induce a t h phenotype tend to induce cd expression ( ) . additionally, the cd + t cells capable of transendothelial migration in vitro are characterized by a bright expression of cd ( , ) , but cd does not seem to be actually involved in t-cell adhesion to endothelial cells or fibroblasts ( ) . recently, it was shown that up to % of all t h cells show very high cd expression, with mean fluorescent intensity on these cells almost twice as high as on t h or t h cells. therefore, the authors of this study suggest cd as a marker for t h cells ( ) . conversely, cd has been proposed as a negative marker for the selection treg cells due to its very low-surface expression on these cells ( ) ( ) ( ) . cd is also a costimulatory molecule for t-cell activation. crosslinking of cd , along with cd , stimulates t-cell activation and proliferation ( , , ) . cd can also directly activate t-cells in an alternative activation pathway, but this requires the presence of the tcr/cd complex ( ) ( ) ( ) . during costimulation, cd is mannose- phosphorylated and internalized, the latter of which is mediated in part by its interaction with m p/igfiir ( ) . it then localizes to lipid rafts where it might interact with cd , required for tcr signaling, facilitating colocalization of this molecule with tcr signaling molecules ( , ) . a number of candidate binding partners for costimulation have been proposed. ada and cd are known binding partners ( ) . even though ada binding to cd does not seem to be essential for immune functions in humans ( ) , the nanomolar affinity of this interaction probably reflects its importance ( ) . indeed, association with free ada or ada presented by ada-anchoring proteins on dendritic cells seems to costimulate t-cells through cd binding ( , ) . on the other hand, it has been shown that soluble dppiv enhances t-cell proliferation independent of its enzyme activity or ada-binding capability ( ) . interestingly, the ada-cd interaction can be inhibited by hiv- external envelope protein gp and this requires interaction of gp with cxcr ( ) . in fact, evidence suggests a physical association between cxcr and cd on peripheral blood lymphocytes ( ) . fibronectin is another known binding partner of cd involved in t-cell costimulation ( ) ( ) ( ) . finally, cd interacts with caveolin- on monocytes. this interaction causes an upregulation of cd on these cells, which potentiates antigen-specific t-cell activation ( ) . most studies seem to find no need for dppiv's enzymatic activity for succesful costimulation, as evidenced through the use of inhibitors and catalytically impaired dppiv mutants ( ) ( ) ( ) ( ) . dipeptidyl peptidase and are present in baboon spleen interfollicular t-lymphocytes and jurkat t cells ( ) . they are upregulated in the latter after pwm and lps, but not pha, stimulation ( , , ) . activation of pwm-stimulated t-cells is suppressed after dppiv/ / inhibition. moreover, dna synthesis and t-cell proliferation are reduced, as well as production of il- , - , - , and ifn-γ. this is due to an induction of tgf-β secretion ( , ( ) ( ) ( ) ( ) . inhibition also upregulates ctla- and downregulates dppiv expression ( , ) . these observations might be physiologically relevant as endogenous inhibitors of dpps are known which have similar effects in cell-based experiments as the synthetic inhibitors ( , ) . dipeptidyl peptidase ii activity is higher in t-lymphocytes than in b-lymphocytes ( ) and absence of dppii steers t-lymphocytes toward a t h phenotype. t-lymphocytes of dppii ko mice hyperproliferate and secrete il- after cd crosslinking or after in vivo priming and in vitro antigen-specific restimulation ( ) . prep activity has also been shown in mouse t-lymphocytes ( ) . its activity is significantly higher in immature, double-positive thymocytes compared to mature, single-positive thymocytes, or peripheral t-cells. t-cells stimulated with con a followed by il- show a time-dependent increase in prep activity and pretreatment of cells with a prep inhibitor renders them resistant to activation-induced cell death ( ) . figure shows an overview of in vitro data on dpp involvement in primary human t cell activation. the dpp family has been reported to be dysregulated or even involved in a number of inflammatory disorders. expression levels of a number of family members are modulated in rheumatoid arthritis. whereas the density of cd on peripheral t cells is increased in patients, it is low on synovial fluid t cells ( ) ( ) ( ) . dppiv activity in plasma, serum, or synovial fluid of patients has also been found to be decreased, similar to results in several rat models of arthritis ( ) ( ) ( ) ( ) ( ) ( ) ( ) . interestingly, rats resistant to induction of arthritis show higher plasma dppiv levels ( ) . by contrast, dppii and prep activity are increased in serum or synovial fluid of arthritis patients ( ) ( ) ( ) . likewise, fap immunoreactivity is much higher in fibroblast-like synoviocytes of rheumatoid arthritis patients compared to osteoarthritis controls ( ) . dppiv's involvement in rheumatoid arthritis has been studied, but remains unclear. on the one hand, inhibition can suppress development of arthritis in rats ( ) . note, however, that effects mediated through other dpps are hard to exclude as these inhibitors were developed before dpp and dpp were discovered. on the other hand, induced arthritis is more severe in dppiv-deficient mice ( ) . this may be due to increased levels of circulating cxcl ( ), a dppiv substrate shown to be involved in rheumatoid arthritis. several case reports in patients seem to suggest a link between the development of rheumatoid arthritis and the use of dppiv inhibitors ( ) ( ) ( ) . prcp has also been associated with rheumatoid arthritis as its activity was shown in synovial fluid isolated from arthritic joints ( ) . inflammatory bowel disease shows a distinct expression pattern of the dpp family. dppiv serum or plasma activity seems to be lower in patients, whereas there is an increase of circulating cd + cd + cells with a higher cd surface expression ( , ) . fap is heavily expressed by myofibroblasts in the submucosa strictures in crohn's disease, and is upregulated after stimulation with tnfα or tgfβ ( ) . in a mouse model, colonic dppii and dpp mrna and dppii activity are increased, while colonic dpp / activity only increases significantly in mice that are also dppiv knockouts ( ) . in mouse models, inhibition or abrogation of dppiv seems to at least partially ameliorate symptoms, possibly by increasing circulating glp- , impairing neutrophil recruitment, and maintaining treg populations ( ) ( ) ( ) ( ) ( ) ( ) . some of those beneficial effects may be mediated in part by the other dpps, as additive effects were found for dppiv ko and the dpp inhibitors ( , , ) . a recent study suggests that the ameliorative effects of dpp inhibitors are most likely not mediated through glp- protection ( ) . the dpp family has also been studied in neuroinflammation. ischemia-induced neuroinflammation in rats prompts a distinct expression and activity pattern of the dpps. in the days following ischemia, the brain of these rats undergoes a complex reorganization of dpp expression with changes in mrna, protein, and activity levels of dppii, , , and in cortical neurons, microglia, and macrophages ( ) . similarly, prep seems to be associated with astrocytes and microglia in lesioned inflamed brains of rats ( ) . dppiv and prep also may be involved in multiple sclerosis. cd + t cells were found to correlate with disease scores ( ) . soluble dppiv levels are elevated in cerebrospinal fluid of patients ( ) . plasma prep activity, on the other hand, is lower in patients with relapsing-remitting or primary progressive multiple sclerosis and in clinically isolated syndrome ( , ) . interestingly, prep inhibition seems to aggravate symptoms in a mouse model of multiple sclerosis ( ) . in systemic lupus erythematosus, dpps also seem to be dysregulated. in mouse models, dppii and prep activities are increased in plasma, spleen, kidney, and liver, whereas dppiv activity is decreased ( , ) . human patients also show elevated dppii and reduced dppiv activities in serum, along with reduced numbers of cd + t cells ( , ) . interestingly, serum dppiv levels are inversely correlated with disease score ( ) . fap immunoreactivity is decreased in the synovium of lupus patients ( ) . finally, dppiv has been studied in psoriasis, an immunemediated chronic inflammatory disorder with primary involvement of skin and joints. its mrna, protein levels and activity are higher in psoriatic skin samples ( , ) . by contrast, serum dppiv levels and activity seem to be lower in patients ( , ) , accompanied by a reduction of peripheral cd + cd + t cells ( , ) . two case reports suggest a link between the use of dppiv inhibitor sitagliptin and psoriasis. while one woman developed a psoriaform eruption days after starting sitagliptin treatment ( ), another patient's psoriatic lesions gradually diminished and were effectively gone months after the start of sitagliptin treatment ( ) . dipeptidyl peptidase iv has recently received much attention for its potential as a therapeutic target for the treatment of atherosclerosis (box ) ( ). this is not surprising considering the current use of dppiv inhibitors in the treatment of t d and the fact that t d is associated with a higher risk for atherosclerosis ( , ). in the apoe −/− mouse model of atherosclerosis, dppiv inhibition generally reduces plaque area and monocyte and macrophage plaque infiltration ( ) ( ) ( ) . a reduction in the number of plaque lesions or in smooth muscle cell content have also been observed ( , ) , as well as lower plaque mmp and higher plaque collagen levels, suggesting increased plaque stability ( ) . one study reported effects of dppiv inhibition on atherosclerotic plaques of only diabetic apoee −/− mice ( ), but more recently, terasaki et al. found similar effects in non-diabetic and diabetic apoe −/− mice ( ) . likely, such differences can be explained by the fact that different dppiv inhibitors were employed. effects of dppiv on atherogenesis similar to those observed in apoe −/− mice have been reproduced in ldlr −/− mice ( , ) . in human atherosclerotic plaques, atherosclerosis is the most common underlying cause of cardiovascular diseases and should be regarded as an inflammatory disease. it starts with dysfunction of the endothelium leading to the expression of leukocyte adhesion molecules, such as selectins and integrins. locally produced proinflammatory cytokines attract the immune cells into the inner layer of the endothelium. however, not only leukocytes are found in the plaque but also low-density lipoprotein particles (ldl) and their oxidized counterparts (oxldl). in the plaque, monocytes differentiate into macrophages, phagocytose the oxldl and turn into so-called pro-atherogenic foam cells. this process leads to a self-sustaining, local inflammation leading to plaque growth, and migration of smooth muscle cells into the core. a plaque is defined as stable as long as it is contained by a thick fibrous cap. however, the latter is slowly degraded by the proteolytic enzymes from the leukocytes. this eventually leads to rupture and the formation of arterial thrombi ( , dppiv immunoreactivity could only be found on endothelium of neovessels ( ). it was recently found that dppiv activity may be a predictor for the onset of atherosclerosis in otherwise healthy chinese individuals ( ) . another prospective study investigated the influence of vildagliptin or sitagliptin treatment on intima-media thickness, a surrogate marker for atherosclerosis. this study found that treatment with vildagliptin or sitagliptin reduced intima-media thickness, suggesting that dppiv inhibition might be beneficial in atherosclerosis in humans as well ( ) . moreover, treatment naïve t d patients treated with alogliptin for months saw a significant decrease in their circulating atherogenic lipids ( ) . it has been suggested that dppiv inhibitors' anti-atherogenic effects are mainly mediated through decreased monocyte infiltration, as dppiv inhibitors suppress monocyte activation and chemotaxis in vitro ( , ) . dppiv inhibition also reduces in vitro foam cell formation in exudate peritoneal macrophages from apoe −/− mice ( ) . moreover, soluble dppiv stimulates in vitro proliferation of smooth muscle cells and this can be reduced through the addition of a dppiv inhibitor ( , ) . finally, active circulating glp- levels are augmented and this improves endothelial dysfunction ( , ) . probably, dppiv inhibition improves atherosclerosis through a combination of all these mechanisms. indeed, incretin antagonists only partially attenuate the anti-atherogenic effects of dppiv inhibition, suggesting that other mechanisms beyond incretin preservation are in play ( ) . interestingly, monocyte-endothelial cell adhesion is abrogated by an anti-sdf- α antibody in vitro ( ) . ldl seems to induce sdf- α expression and leads to smooth muscle cell proliferation and inhibition of cell apoptosis ( , ) . sdf- α is a dppiv substrate, which loses its biological activity after cleavage ( ) . as dppiv inhibition seems to improve atherosclerosis, whereas intact sdf- α appears to be deletorious, it could be argued that sdf- α cleavage by dppiv does not play a major role in atherosclerosis. dipeptidyl peptidase and have been found to be abundantly present in the macrophage-rich regions of human atherosclerotic plaques and considering dpp 's role in macrophage activation, it might potentially be involved in atherogenesis ( ). fap expression is enhanced in some, but not all types of human atheromata. it is found on smooth muscle cells, and its expression correlates with macrophage burden, probably due to the fact that tnfα upregulates fap in smooth muscle cells in vitro. as it is mainly associated with collagen-poor regions and can digest type i collagen and gelatin in vitro, fap probably contributes to plaque instability ( ) . interestingly, many of the studies reviewed above show the potential of targeting dpp family members for the treatment of atherosclerosis (see figure ). fap inhibition might reduce plaque instability by decreasing collagen breakdown; dpp inhibition is likely to attenuate m macrophage activation, reducing the local inflammatory cascade; dppiv inhibition may decrease monocyte infiltration, foam cell formation, improve endothelial dysfunction, and reduce smooth muscle cell proliferation; and finally, prep inhibition might reduce neutrophil infiltration, preventing endothelial dysfunction, and monocyte infiltration. all of this shows the possibilities of repositioning dppiv inhibitors, currently being used to treat type diabetes, as well as the potential of targeting other members of the dpp family. caution should be taken when interpreting results from literature data based on dpp inhibitors, especially from older studies. it is now known that, under the experimental conditions used, many of these inhibitors are not specific for one particular family member. the reported findings, however, remain interesting. this review has shown extensive involvement of members of the dpp family in the immune system. it is clear that these enzymes hold great potential as targets for the treatment of certain inflammatory disorders. particularly, the possibility of targeting dpp family members for the prevention and treatment of atherosclerosis warrants further investigation. in vivo effects of a potent, selective dppii inhibitor: uamc is a possible tool for the elucidation of the physiological function of dppii. adv exp med dipeptidylpeptidase negatively regulates colony-stimulating factor activity and stress hematopoiesis structures of human dpp reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases purification of two dipeptidyl aminopeptidases ii from rat brain and their action on proline-containing neuropeptides dipeptidyl peptidase ii (dppii), a review thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase iv. . variation of the p position of xaa-boropro dipeptides structure-activity relationship of diaryl phosphonate esters as potent irreversible dipeptidyl peptidase iv inhibitors rapid parallel synthesis of dipeptide diphenyl phosphonate esters as inhibitors of dipeptidyl peptidases fluoro-olefins as peptidomimetic inhibitors of dipeptidyl peptidases prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner a prolyl oligopeptidase inhibitor, kyp- , reduces α-synuclein protein levels and aggregates in cellular and animal models of parkinson's disease subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion subcellular distribution of prolyl endopeptidase and cation-sensitive neutral endopeptidase in rabbit brain expression and traffic of cellular prolyl oligopeptidase are regulated during cerebellar granule cell differentiation, maturation, and aging distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues partial purification and characterization of post-proline cleaving enzyme: enzymatic inactivation of neurohypophyseal hormones by kidney preparations of various species changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of substance p by the purified enzyme porcine muscle prolyl endopeptidase and its endogenous substrates purification and characterization of prolyl endopeptidase from pig brain purification and characterization of prolyl endopeptidase from rat skin effect of s , a novel prolyl endopeptidase inhibitor, on substance p and alpha-melanocyte-stimulating hormone breakdown in the rat brain an evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro prolyl oligopeptidase: a potential target for the treatment of cognitive disorders on the role of prolyl oligopeptidase in health and disease degradation of neurotensin by rabbit brain endo-oligopeptidase a and endo-oligopeptidase b (prolineendopeptidase) proline specific peptidases brain endo-oligopeptidase b: a post-proline cleaving enzyme that inactivates angiotensin i and ii inactivation of thyrotropin-releasing hormone (trh) and ( me-his) trh by brain peptidases studied by highperformance liquid chromatography evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase i in the metabolism of lhrh and trh in brain proline-specific proteases in cultivated neuronal and glial cells effect of a novel prolyl endopeptidase inhibitor, jtp- , on prolyl endopeptidase activity and substance pand arginine-vasopressin-like immunoreactivity in the brains of aged rats neutrophils contain prolyl endopeptidase and generate the chemotactic peptide, pgp, from collagen prolyl oligopeptidase is involved in release of the antifibrotic peptide ac-sdkp alteration of prolyl oligopeptidase and activated α- -macroglobulin in multiple sclerosis subtypes and in the clinically isolated syndrome prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis specific inhibitors for prolyl endopeptidase and their anti-amnesic effect jtp- : a novel prolyl endopeptidase inhibitor with potential as a cognitive enhancer pharmacological studies of a novel prolyl endopeptidase inhibitor, jtp- , in rats with middle cerebral artery occlusion effects of prolyl endopeptidase inhibitors and neuropeptides on delayed neuronal death in rats ono- , a potential antidementia drug, delays age-induced apoptosis and suppresses overexpression of glyceraldehyde- -phosphate dehydrogenase in cultured central nervous system neurons prolyl endopeptidase-deficient mice have reduced synaptic spine density in the ca region of the hippocampus, impaired ltp, and spatial learning and memory prolyl oligopeptidase binds to gap- and functions without its peptidase activity gap shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase prolyl oligopeptidase is a glyceraldehyde- -phosphate dehydrogenase-binding protein that regulates genotoxic stress-induced cell death interaction of prolyl oligopeptidase with α-synuclein prolyl oligopeptidase colocalizes with α-synuclein, β-amyloid, tau protein and astroglia in the post-mortem brain samples with parkinson's and alzheimer's diseases prolyl oligopeptidase stimulates the aggregation of alpha-synuclein the anti-inflammatory effect of neuropeptide y (npy) in rats is dependent on dipeptidyl peptidase (dp ) activity and age soluble dpp originates in part from bone marrow cells and not from the kidney representative aminopeptidases and prolyl endopeptidase from murine macrophages: comparative activity levels in resident and elicited cells a potential role for dendritic cell/macrophage-expressing dpp in obesity-induced visceral inflammation dipeptidyl peptidase / -like activity in human leukocytes human u cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha plasma membrane-bound and lysosomal peptidases in human alveolar macrophages divergent regulation of cell surface protease expression in hl- cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid dpp- (cd ) inhibitor alogliptin inhibits tlr -mediated erk activation and erkdependent mmp- expression by u histiocytes dpp- (cd ) inhibitor alogliptin inhibits atherosclerosis in diabetic apolipoprotein edeficient mice longterm dipeptidyl-peptidase inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis dpp- inhibitors repress nlrp inflammasome and interleukin- beta via glp- receptor in macrophages through protein kinase c pathway inhibitors of dipeptidyl peptidase iv (dp iv, cd ) specifically suppress proliferation and modulate cytokine production of strongly cd expressing u cells fibroblast activation protein expression by stromal cells and tumor-associated macrophages in human breast cancer dipeptidyl peptidase iv, aminopeptidase n and dpiv/apn-like proteases in cerebral ischemia cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase ii in macrophages and mast cells cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase ii in the normal rat prolylcarboxypeptidase (angiotensinase c) in human lung and cultured cells prolylcarboxypeptidase promotes angiogenesis and vascular repair a prolyl endopeptidase from murine macrophages, its assay and specific inactivation cathepsin b and prolyl endopeptidase activity in rat peritoneal and alveolar macrophages. stimulation of peritoneal macrophages by saline lavage prolyl endopeptidase is revealed following silac analysis to be a novel mediator of human microglial and thp- cell neurotoxicity dipeptidyl peptidase iv is a human and murine neutrophil chemorepellent a novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation inhibition of cd /dipeptidyl peptidase iv enhances ccl /eotaxinmediated recruitment of eosinophils in vivo rat peritoneal mast cells release dipeptidyl peptidase ii expression and functional role of dipeptidyl peptidase iv (cd ) on human natural killer cells dipeptidyl peptidase iv inhibition for the treatment of type diabetes, potential importance of selectivity over dipeptidyl peptidases and the cd antigen is coupled to protein tyrosine phosphorylation and implicated in cd -mediated lysis in natural killer cells cd expression determines lung metastasis in mutant f rats: involvement of nk cell function and soluble cd functional role of cd on human b lymphocytes regulation of dipeptidyl peptidase and expression in activated lymphocytes and injured liver the in vivo expression of dipeptidyl peptidases and f , a novel cell surface molecule, involved in helper function of cd cells dipeptidyl peptidase iv of human lymphocytes -evidence for specific hydrolysis of glycylproline p-nitroanilide in t-lymphocytes influence of cd and integrins on the antigen sensitivity of human memory t cells cd -mediated co-stimulation in human cd (+) t cells provokes effector function via proinflammatory cytokine production dipeptidyl peptidase iv in human t lymphocytes. an approach to the role of a membrane peptidase in the immune system dipeptidyl peptidase iv in the immune system a novel pathway of human t cell activation via a kd t cell activation antigen cell surface characterization of t lymphocytes and allergen-specific t cell clones: correlation of cd expression with t(h ) subsets phenotypic characterization of cd + t cells that exhibit a transendothelial migratory capacity characterization of the c antigen involved in transendothelial migration of cd hi t cells after tight adhesion to human umbilical vein endothelial cell monolayers cd (dipeptidyl peptidase iv) on human t lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts human th cells express high levels of enzymatically active dipeptidylpeptidase iv (cd ) cd : a negative selection marker for human treg cells human treg cells are characterized by low/negative cd expression cloning and functional expression of the t cell activation antigen cd costimulation of cd + and cd + t cells through cd : the ada-binding epitope is not essential for complete signaling triggering of cytotoxic t lymphocytes and nk cells via the tp pathway is dependent on the expression of the t cell receptor/cd complex function of dipeptidyl peptidase iv (cd , tp ) in transfected human t cells fcr-mediated crosslinking of ta (cdw ) induces human t lymphocyte activation internalization of cd by mannose -phosphate/insulin-like growth factor ii receptor contributes to t cell activation cd -mediated signaling for t cell activation occurs in lipid rafts through its association with cd ro coassociation of cd (dipeptidyl peptidase iv) with cd on the surface of human t lymphocytes direct association of adenosine deaminase with a t cell activation antigen the binding site of human adenosine deaminase for cd /dipeptidyl peptidase iv: the arg gln mutation impairs binding to cd but does not cause immune deficiency the hiv- gp inhibits the binding of adenosine deaminase to cd by a mechanism modulated by cd and cxcr expression cd , adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse expression of ecto-adenosine deaminase and cd in human t cells triggered by the tcr-cd complex. possible role of adenosine deaminase as costimulatory molecule soluble cd /dipeptidyl peptidase iv enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding comodulation of cxcr and cd in human lymphocytes a novel consensus motif in fibronectin mediates dipeptidyl peptidase iv adhesion and metastasis fibronectin promotes proliferation of naive and memory t cells by signaling through both the vla- and vla- integrin molecules vla- mediates cd -dependent cd + t cell activation via the cs alternatively spliced domain of fibronectin cd up-regulates expression of cd on antigen-presenting cells by means of caveolin- the costimulatory activity of the cd antigen requires dipeptidyl peptidase iv enzymatic activity enzymatic activity of cd (dipeptidylpeptidase iv) is not required for its signalling function in t cells molecular analysis of cd -mediated signal transduction in t cells unchanged signaling capacity of mutant cd /dipeptidylpeptidase iv molecules devoid of enzymatic activity biochemical properties and expression profile of human prolyl dipeptidase dpp cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (dpp) iv homolog, dpp role of dipeptidyl peptidase iv (dp iv)-like enzymes in t lymphocyte activation: investigations in dp iv/cd -knockout mice inhibitors of dipeptidyl peptidase iv induce secretion of transforming growth factor-ß in pwm-stimulated pbmc and t cells dipeptidyl peptidase iv (dp iv/cd ) mrna expression in pwm-stimulated tcells is suppressed by specific dp iv inhibition, an effect mediated by tgfbeta( ) dipeptidyl peptidase iv on activated t cells as a target molecule for therapy of rheumatoid arthritis the expression of t-cell surface antigens ctla- , cd , and cd is modulated by inhibition of dipeptidylpeptidase iv (dpp iv, cd ) activity in murine stress-induced abortions downregulation of t cell activation following inhibition of dipeptidyl peptidase iv/cd by the n-terminal part of the thromboxane a receptor non-substrate peptides influencing dipeptidyl peptidase iv/cd activity and immune cell function th differentiation is the default program for dpp -deficient t cell differentiation murine t cells expressing high activity of prolyl endopeptidase are susceptible to activationinduced cell death expression and functional role of f (cd ) antigen on peripheral blood and synovial fluid t cells in rheumatoid arthritis patients cd surface molecule involvement in t cell activation and lymphokine synthesis in rheumatoid and other inflammatory synovitis in active chronic rheumatoid arthritis, dipeptidyl peptidase iv density is increased on monocytes and cd (+) t lymphocytes circulating cd is negatively associated with inflammation in human and experimental arthritis serum levels of soluble cd and cd and their clinical significance in patients with rheumatoid arthritis levels of dipeptidyl peptidase iv/cd substrates neuropeptide y and vasoactive intestinal peptide in rheumatoid arthritis patients activities of dipeptidyl peptidase ii and dipeptidyl peptidase iv in synovial fluid from patients with rheumatoid arthritis and osteoarthritis activities of dipeptidyl peptidase ii, dipeptidyl peptidase iv, prolyl endopeptidase, and collagenaselike peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis activities of dipeptidyl peptidase ii and dipeptidyl peptidase iv in mice with lupus erythematosus-like syndrome and in patients with lupus erythematosus and rheumatoid arthritis neutral aminopeptidase and dipeptidyl peptidase iv in the development of collagen ii-induced arthritis fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes antiarthritic effects of the novel dipeptidyl peptidase iv inhibitors tmc- a and tsl- polyarthropathy in type diabetes patients treated with dpp inhibitors sitagliptin (dpp- inhibitor)-induced rheumatoid arthritis in type diabetes mellitus: a case report acute onset of rheumatoid arthritis associated with administration of a dipeptidyl peptidase- (dpp- ) inhibitor to patients with diabetes mellitus dipeptidyl peptidase iv (dp iv, cd ) in patients with inflammatory bowel disease dipeptidyl peptidase- expression is reduced in crohn's disease fibroblast activation protein expression in crohn's disease strictures dipeptidyl peptidase expression during experimental colitis in mice the dpp-iv inhibitor er- has a proliferative effect on the colonic epithelium and a minimal effect in the amelioration of colitis dipeptidyl peptidase iv (dp iv, cd ) and aminopeptidase n (apn, cd ) as regulators of t cell function and targets of immunotherapy in cns inflammation inhibiting dipeptidyl peptidase activity partially ameliorates colitis in mice contribution of dipeptidyl peptidase iv to the severity of dextran sulfate sodium-induced colitis in the early phase dipeptidyl peptidase- inhibitor anagliptin facilitates restoration of dextran sulfate sodium-induced colitis biochemical and histological changes in the small intestine of mice with dextran sulfate sodium colitis the effects of a tgr agonist and a dipeptidyl peptidase iv inhibitor on dextran sulfate sodium-induced colitis in mice prolyl oligopeptidase: a rising star on the stage of neuroinflammation research a longitudinal study of the t cell activation marker cd in chronic progressive multiple sclerosis soluble cd and cd levels in csf and sera of patients with relapsing neuromyelitis optica activities of dipeptidyl peptidases in bxsb mice and mrl/lpr mice with lupus erythematosus-like syndrome reduction of serum soluble cd /dipeptidyl peptidase iv enzyme activity and its correlation with disease activity in systemic lupus erythematosus identification of distinct gene expression profiles in the synovium of patients with systemic lupus erythematosus cd /dipeptidyl-peptidase iv in psoriatic skin: upregulation and topographical changes distribution of dipeptidyl-peptidase iv on keratinocytes in the margin zone of a psoriatic lesion: a comparison with hyperproliferation and aberrant differentiation markers cd /dipeptidyl-peptidase iv and adenosine deaminase serum levels in psoriatic patients treated with cyclosporine, etanercept, and psoralen plus ultraviolet a phototherapy serum soluble cd levels: diagnostic efficiency for atopic dermatitis, cutaneous t-cell lymphoma and psoriasis in combination with serum thymus and activation-regulated chemokine levels expression of dipeptidyl-peptidase iv (cd ) on cd + t cells is significantly decreased in patients with psoriasis vulgaris and atopic dermatitis reduced cd bright expression of peripheral blood cd + tcell subsets in psoriatic patients psoriasiform eruption triggered by a dipeptidyl peptidase iv inhibitor sitagliptin, a dipeptidyl peptidase-iv inhibitor, improves psoriasis dpp- inhibitors and atherosclerosis: the promise primary prevention of cardiovascular diseases in people with diabetes mellitus: a scientific statement from the american heart association and the american diabetes association effects of pkf - , a dipeptidyl peptidase- inhibitor, on the development of atherosclerotic lesions in apolipoprotein e-null mice dpp- inhibitor, suppresses proliferation of vascular smooth muscles and monocyte inflammatory reaction and attenuates atherosclerosis in male apo e-deficient mice dipeptidyl peptidase- inhibitor, sitagliptin, improves endothelial dysfunction in association with its anti-inflammatory effects in patients with coronary artery disease and uncontrolled diabetes sitagliptin reduces plaque macrophage content and stabilises arteriosclerotic lesions in apoe (-/-) mice preventive effect of dipeptidyl peptidase- inhibitor on atherosclerosis is mainly attributable to incretin's actions in nondiabetic and diabetic apolipoprotein e-null mice dipeptidyl-peptidase- inhibitor, alogliptin, attenuates arterial inflammation and neointimal formation after injury in low-density lipoprotein (ldl) receptor-deficient mice increased plasma dpp activities predict new-onset atherosclerosis in association with its proinflammatory effects in chinese over a four year period: a prospective study decreased carotid atherosclerotic process by control of daily acute glucose fluctuations in diabetic patients treated by dpp-iv inhibitors alogliptin: a new dipeptidyl peptidase- inhibitor with potential anti-atherogenic properties inflammation and atherosclerosis the immune response in atherosclerosis: a doubleedged sword a dipeptidyl peptidase- inhibitor, des-fluoro-sitagliptin, improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein e-deficient mice upregulation of sdf- is associated with atherosclerosis lesions induced by ldl concentration polarization sdf- promotes ox-ldl induced vascular smooth muscle cell proliferation fibroblast activation protein is induced by inflammation and degrades type i collagen in thin-cap fibroatheromata key: cord- -glm dxhh authors: hwang, mihyun; phares, timothy w; hinton, david r; stohlman, stephen a; bergmann, cornelia c; min, booki title: distinct cd t-cell effects on primary versus recall cd t-cell responses during viral encephalomyelitis date: - - journal: immunology doi: . /imm. sha: doc_id: cord_uid: glm dxhh cd t-cell help is not a universal requirement for effective primary cd t cells but is essential to generate memory cd t cells capable of recall responses. this study examined how cd t cells affect primary and secondary anti-viral cd t-cell responses within the central nervous system (cns) during encephalomyelitis induced by sublethal gliatropic coronavirus. cd t-cell depletion before infection did not impair peripheral expansion, interferon-γ production, cns recruitment or initial cns effector capacity of virus-specific cd t cells ex vivo. nevertheless, impaired virus control in the absence of cd t cells was associated with gradually diminished cns cd t-cell interferon-γ production. furthermore, within the cd t-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher t-cell turnover. transfer of memory cd t cells to reduce viral load in cd -depleted mice reverted the recipient cns cd t-cell phenotype to that in wild-type control mice. however, memory cd t cells primed without cd t cells and transferred into infected cd -sufficient recipients expanded less efficiently and were not sustained in the cns, contrasting with their helped counterparts. these data suggest that cd t cells are dispensable for initial expansion, cns recruitment and differentiation of primary resident memory cd t cells as long as the duration of antigen exposure is limited. by contrast, cd t cells are essential to prolong primary cd t-cell function in the cns and imprint memory cd t cells for recall responses. a major role of cd t cells during infections is to support priming, programming and/or effector function of cd t cells. help can be provided by cytokine production or regulation of co-stimulatory factors, such as cd /cd ligand via antigen-presenting cells. , nevertheless, the requirement for cd t-cell help in primary cd t-cell responses is not universal and depends on the in vivo milieu during initial t-cell activation. primary cd t-cell responses against infectious agents are mostly cd t-cell independent, whereas responses to non-inflammatory stimulation or non-replicating vaccines are dependent on cd t-cell help. [ ] [ ] [ ] [ ] irrespective of the requirement for cd t-cell help for primary cd t-cell responses, it is accepted that cd t-cell help is necessary for the generation of memory cd t cells capable of efficient recall responses. , , cd t cells also play a key role in optimal cd t-cell expansion in the draining lymph node (ln), subsequent mobilization of activated cd t cells into inflamed tissues, as well as their maintenance and survival at effector sites. , [ ] [ ] [ ] [ ] while imprinting of cd t cells on cd t-cell function and survival has been extensively studied in peripheral and balb/c (h- d ) mice, which resolves into a persistent infection associated with chronic demyelination. initial activation of adaptive immunity occurs in the draining cervical ln (cln). activated cd and cd t cells subsequently cross the blood-brain barrier and enter the cns, where they are re-stimulated to secrete interferon-c (ifn-c), express granzyme b, and lyse virus-infected target cells. , cd t cells are the major effectors controlling viral load via both ifn-c and perforin-mediated mechanisms. [ ] [ ] [ ] nevertheless, sustained viral rna indicates persistence at low levels. the role of cd t cells is complex because they not only promote cd t-cell function and survival within the cns , and directly contribute to viral control, but also enhance pathology. [ ] [ ] [ ] [ ] [ ] a recent study to assess whether cd t cells influence cd t cells at the activation or effector stage during jhmv infection revealed that cd t cells not only enhance cd t-cell expansion in the cln during priming, but also exert helper function within the cns by locally promoting cd t-cell effector function and survival. cd t cells were incapable of controlling virus in the cns without cd t cells, even when primed in the presence of cd t cells. the latter results were obtained in h- b mice, in which the dominant cd t-cell response is directed to an epitope in a hypervariable region of the viral spike (s) protein restricted to h- d b . in the present report, we set out to assess the extent of cd t-cell imprinting not only on primary cd t-cell responses, but also on memory formation and recall cd t-cell responses in the cns. balb/c mice were chosen for these studies because they mount a prominent h- l d restricted cd t-cell response to an epitope in the highly conserved nucleocapsid (n) protein, which is expressed at much higher levels than the s protein, , potentially leading to distinct t-cell activation requirements. an accelerated cd t-cell response to the n relative to s epitope is indicated by earlier detection of n-specific relative to s-specific responses in cln of infected balb/c and c bl/ mice, respectively, as well as an early preponderance of n-specific over s-specific cd t cells in the cns of jhmv-infected (balb/c c bl/ ) f mice. moreover, adoptive transfers indicate that virusspecific cd t cells induced in the context of h- d have more potent antiviral activity than virus-specific cd t cells induced in the context of h- b . , surprisingly, herein we show that peripheral expansion of virus-specific cd t cells was not impaired in the absence of cd t cells in balb/c mice, as distinct from c bl/ mice. furthermore, cd t-cell help during priming was dispensable for cns accumulation and initial function of primary virus-specific cd effector t cells. however, uncontrolled cns virus replication in the absence of cd t cells ultimately resulted in loss of ifnc production, higher cd t-cell turnover, and inability to acquire an effector memory phenotype. nevertheless, the unhelped cd t-cell phenotype was rescued when virus replication was controlled by transfer of memory cd t cells, indicating that the unhelped cd t-cell phenotype during primary responses is regulated by antigen load, rather than lack of cd t-cell imprinting. by contrast, unhelped memory cd t cells mounted poor recall responses when transferred into cd t-cell-sufficient mice and could not be sustained in the cns, despite efficient virus control. mice, virus and cd t-cell depletion balb/c (h- d ) mice were obtained from the national cancer institute (frederick, md). all mice were used at - weeks of age and infected intracranially in the right hemisphere with plaque-forming units of the glia tropic monoclonal antibody (mab) -derived . v- variant of mouse hepatitis virus strain jhm (jhmv). virus titres were determined as previously described. briefly, clarified supernatants from individual brain homogenates were used to measure virus by plaque assay on a murine astrocytoma cell line, designated dbt. plaques were counted after hr incubation at °. clinical disease was scored daily as described elsewhere: , healthy; , ruffled fur and hunched back; , hind limb paralysis/ inability to turn to upright position; , complete hind limb paralysis and wasting; , moribund/dead. cd t cells were depleted by intraperitoneal injection with lg purified anti-mouse cd mab (gk . ; bioxcell, west lebanon, nh) at days À and of infection. cd t cells remained below Á % in mab-treated mice for at least days. controls received the same amount of isotype control anti-b-gal mab (gl ). memory cd t cells were generated by intraperitoneal injection of thy- . balb/c mice with plaqueforming units of jhmv. donor mice were treated with anti-mouse cd or control mab at day À and relative to intraperitoneal immunization for comparative analysis of 'unhelped' versus 'helped' cd t cells. after - weeks splenic donor cd t cells were isolated by negative selection using fitc-labelled anti-cd (clone rm - ), cd (clone d ), mhc class ii (clone m / . . ), fcc iii/ii receptor (clone . g ) and nk . (clone pk ), in combination with anti-fitc-coated magnetic beads (miltenyi biotec, inc., auburn, ca) and transferred into either naive balb/c mice at day before intracranial infection ( Á to Á /mouse) or at days after intracranial infection ( Á /mouse) as indicated. all procedures were carried out under animal protocols approved by the institutional animal care and use committees of cleveland clinic foundation. for lymphocyte isolation from the cns, brains and spinal cords were removed from mice perfused with cold pbs (ph Á ). tissues were homogenized in rpmi- containing collagenase ( mg/ml; roche, indianapolis, in) and dnase i ( mg/ml, roche) using gentlemacstm tubes, a gentlemacs dissociator (miltenyi biotec), and cell strainers (bd falcon, durham, nc). homogenates were centrifuged at g for min at °. the cells were resuspended in cold pbs, adjusted to % percoll (ge healthcare, uppsala, sweden), underlayed with % percoll and centrifuged at g for min at °as described previously. mononuclear cells were collected from the %/ % interface, washed with rpmi- and counted before analysis. peripheral lymphocytes were isolated from cln. rna from brains of naive and jhmv-infected mice was extracted using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions and cdna was subsequently generated by superscript iii rtase (invitrogen) with oligo-dt( - ) primers (invitrogen). taqman primers/probes specific for gapdh (mm _g ), ifn-c (mm _m ) and cxcl (mm _m ) were purchased from applied biosystems (foster city, ca) and rna levels were determined using steponeplus tm real-time pcr systems (applied biosystems) and stepone tm software v . (applied biosystems). gene expression was normalized to gapdh expression and converted to a linearized value using the formula: [ e(ct gapdh À ct gene )] . single-cell suspensions were blocked with rat anti-mouse cd / mab (clone . g : bd biosciences, san diego, ca) for min on ice before staining. for four-or fivecolour flow cytometry, cells were stained with fitc-, phycoerythrin (pe) -, pe-cy -, allophycocyanin-and pe-cy -conjugated mab specific for cd (clone -f ), cd (clone - . ), klrg (clone f ), cd (clone a r ) and mhc ii (clone m / . . ) (all from ebioscience, san diego ca) in pbs containing Á % bsa. for intracellular staining for ki (clone b ; bd biosciences), cells were stained with surface molecules before permeabilization with fix ( Á % pfa)/perm buffer (pbs containing Á % bsa and Á % saponin). virus-specific cd t cells were detected with pe-cy -conjugated anti-cd and pe-conjugated l d /pn class i tetramer at Á lg/ Á to Á cells as directed by the supplier (nih tetramer core facility, atlanta, ga). for virus-specific ifn-c production by cd t cells, cells were cultured with or without lm pn peptide for - hr with ll of golgi stop (bd bioscience)/ml. after stimulation, cells were stained for cd surface expression, fixed and permeabilized to detect intracellular ifn-c (clone s.b ; ebiosience). samples were analysed on a facs lsrii (bd biosciences). forward and side scatter signals obtained in linear mode were used to establish a gate containing live cells, while excluding dead cells and tissue debris. data were analysed using flowjo ( . . ) software (tree star inc., ashland, or). for cytotoxicity assays target cells were prepared by pulsing ammonium-chloride-potassium lysing buffer (invitrogen) -treated splenocytes with the immunodominant l d -restricted pn peptide ( lm) for hr at °. peptidepulsed and untreated targets were then labeled in nm cfse and Á nm cfse, respectively. equal numbers of peptide pulsed (cfse high ) and control (cfse low ) targets were mixed together. total target cells were plated into a v-bottom -well plate at per/well, and virus-specific cd t cells were added at effector : target ratios of : , : or : . target cells incubated alone were used as negative controls. the plate was centrifuged ( g for min) to optimize cell contact and incubated for hr at °. the proportion of cfse high and cfse low cells was determined by flow analysis. specific lysis was calculated with the formula: [ À (ratio of targets only/ratio of targets + t cells)] . brains and spinal cords from pbs-perfused mice were fixed with % neutral zinc-buffered formalin and embedded in paraffin. sections were stained with either haematoxylin & eosin or luxol fast blue (lfb) to determine inflammation and myelin integrity, respectively, as described previously. distribution of viral antigen was determined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlingame, ca) using the anti-jhmv mab j . specific for the c-terminus of the viral n protein as the primary antibody, and horse antimouse as the secondary antibody (vector laboratory) as described elsewhere. , , results are expressed as the mean ae sem for each group of mice. results are expressed as the mean ae sem for each group of mice. statistics were determined using unpaired two-tailed student's t-test and verified using two-way analysis of variance (anova) with bonferroni post-test. any variance between statistical evaluations resulting in a change from significant to non-significant or vice versa are indicated in respective figure legends (figs , , ). for the data sets indicated in legends, graphs were plotted using a graphpad prism . software (graphpad software, inc., la jolla, ca). to assess whether cd t-cell help for cd t cells during jhmv cns infection is dependent on the nature of the viral antigen and genetic background, balb/c mice were treated with depleting anti-cd or irrelevant anti-b-gal mab before infection. distinct from h- b mice, the absence of cd t cells did not affect expansion of virusspecific cd t cells in the cln (fig. a) . interferon-c production by virus-specific cd t cells was also not affected ( fig. b) , confirming a redundant role of cd t cells in priming effector cd t cells in h- d mice. these results prompted us to further examine the efficacy of primary anti-viral cd t cells within the cns, when primed in the absence (unhelped) or presence (helped) of cd t cells. disease onset and severity were initially similar in cd depleted and control infected balb/c mice (fig. a) . however, whereas controls recovered after day postinfection (p.i.) with a survival rate > %, cd -depleted mice exhibited progressively increased clinical symptoms, with mortality beginning at day p.i. and culminating at % before day p.i. (fig. a,b) . the sustained symptoms leading to mortality were associated with uncontrolled virus replication (fig. c ). whereas infectious virus in the brain progressively declined to undetectable levels by day p.i. in control mice, cd depleted mice failed to control infectious virus after day p.i. (fig. c) . these data demonstrated a critical role of cd t cells in controlling cns virus replication at least to day p.i., when viral clearance in both the h- b and h- d genetic backgrounds is independent of humoral immunity. , consistent with the inability to clear infectious virus from the brain, immunohistochemistry revealed an increased number of virus-infected cells in cd -depleted mice at day p.i. (fig. a) . interestingly, in the brain many of the virus-infected cells exhibited the morphology of neurons (fig. a, inset) . by contrast, only very rare virus-infected cells were detectable in the brains of con-trol mice at day p.i. virus-infected cells were also more abundant in spinal cords of cd -depleted mice compared with controls (fig. b) . in cd -depleted mice virus-infected cells were predominantly glia in appearance (fig. b , left inset) with occasional infected neurons (fig. b, right inset) . in control mice the spinal cords showed only rare virus-infected cells with the appearance of macrophages (fig. b, inset) . in contrast to the increased number of virus-infected cells and disease severity, overall inflammation in the spinal cord and the extent of demyelination were comparable between the groups (fig. c) . as t cells are the primary mediators of jhmv control in the cns during the first days p.i. , the inability of cd -depleted mice to reduce viral load suggested impaired cd t-cell recruitment or function. in balb/c mice infected with a lethal jhmv strain, activated cd t cells gained access to the cns independent of cd t cells; however, their numbers in the cns were reduced by~ %. following sublethal jhmv infection of balb/ c mice, cd t cells were more prevalent than cd t cells in the cns at day p.i. and accumulated to peak numbers by day p.i. (~ Á ), similar to cd t cells (~ ). to distinguish whether cd t cells affected cd t-cell recruitment and/or effector function following sublethal infection, cns accumulation of virusspecific cd t cells was monitored. cd t-cell depletion did not alter the proportion of virus-specific t cells within the cd t-cell population infiltrating the brain or spinal cord (fig. a , data not shown). while the frequencies of virus-specific cd t cells were equally low at day p.i., peak frequencies were reached by day p.i. in both groups. moreover, the absolute numbers of virus-specific cd t cells were also similar at all times p.i. (fig. b) , indicating that cd t cells were not required for virusspecific cd t-cell recruitment or retention. in the spinal cord virus-specific cd t cells were also virtually identical throughout infection, irrespective of the presence or absence of cd t cells during priming (fig. b) . the failure to clear infectious virus in the absence of cd t cells (fig. c ) therefore suggested either that the effector functions of cd t cells within the target tissue are impaired, or that cd t cells contribute directly to virus control. the primary anti-viral mediators reducing infectious jhmv within the cns are t-cell-derived ifn-c and perforin. [ ] [ ] [ ] the ifn-c controls virus directly in oligodendrocytes and is also critical to up-regulate mhc class i on oligodendrocytes as well as mhc class ii on microglia and macrophages. , , perforin-mediated cytolysis specifically controls virus in microglia/macrophages, but not oligodendrocytes. we therefore examined potential defects in effector functions of brain-derived unhelped virus-specific cd t cells by measurement of ex vivo cytolytic activity and ifn-c expression. cytolytic capacity of both unhelped and helped cd t cells showed no differences between the groups at day p.i. (data not shown). similarly, cd t-cell depletion did not impair the frequency of ifn-c-producing cells in brain-derived cd t cells at day p.i. (fig. a) . at day p.i. the frequency of ifn-c-producing unhelped cd t cells remained similar to day p.i., but was reduced relative to helped cd t cells. however, by day p.i. the frequency of ifn-c-producing virus-specific cd t cells in the unhelped group declined to < %, whereas it was sustained at~ % in controls. in addition to reduced frequencies, the extent of ifn-c production as assessed by mean fluorescence intensity (mfi) was also lower in the unhelped groups at days and p.i. (fig. a) . in spinal cords the frequencies of ifn-c-producing cd t cells were highest at day p.i. and declined in both groups out to day p.i., with no statistically significant differences between both groups (data not shown). although a trend towards reduced ifn-c secretion on a per cell basis was indicated by reduced mfi in the unhelped cd t-cell population, differences were not significant (data not shown). these results suggested that cd t cells support sustained ifn-c expression by virusspecific cd t cells under conditions of prolonged antigen exposure. no defects in initial generation and cns recruitment of ifn-c-producing virus-specific cd t cells suggested that the failure to control infectious virus was attributed to inefficient triggering of t-cell effector function in vivo. interferon-c mrna expression in the brain was indeed reduced in the cns of cd -depleted mice compared with controls (fig. b) . to assess functional ifn-c protein in vivo we further monitored ifn-c dependent up-regulation of cxcl mrna and mhc class ii on microglia. decreased levels of cxcl mrna supported reduced ifn-c activity (fig. b) . moreover, mhc class ii expression was barely detectable on microglia at day p.i. in cd -depleted mice, but was already up-regulated on % of microglia in controls (fig. c) . although > % of microglia in both groups expressed mhc class ii by day p.i., overall expression levels assessed by mfi were lower in cd -depleted mice. cd t cells therefore produced sufficient ifn-c in vivo to up-regulate mhc class ii on microglia by day p.i. in the absence of cd t cells. however, ifn-c levels were insufficient to achieve optimal mhc class ii induction, cxcl mrna up-regulation, or unhelped cd t cells do not acquire a memory precursor effector phenotype these data indicated that maintenance of ifn-c-producing cd t cells in the cns appears optimal when cd t cells are present during priming and/or the effector phase. upon activation, cd t cells undergo a complex differentiation programme determined by the nature of inflammatory signals. , however, two effector cell subsets whose differential expression of the interleukin- receptor a chain (cd ) and killer cell lectin-like receptor g (klrg ) is associated with fate determination and development of memory cells are common to many infections or immunizations. up-regulation of klrg on effector t cells directly coincides with the magnitude of t-bet expression and serves as a marker of terminally differentiated effector cells. , by contrast, cd is down-regulated upon activation, and activated cells with sustained cd expression survive the cd contraction phase to form the memory pool. hence, during differentiation cd t cells expressing a cd À klrg + phenotype are generally considered to be short-lived effector cells (slec), whereas a cd + klrg À phenotype is indicative of long-lived memory precursor effector cells (mpec). we therefore examined whether the absence of cd t cells at priming alters the differentiation phenotype of cns infiltrated cd t cells based on cd and klrg expression (fig. a) . although virus-specific cd t cells were low at day p.i. (fig. ) , the majority had downregulated cd ( % versus %, in cd -depleted versus control mice, respectively; data not shown). however, at this early time, only % of virus-specific cd t cells had a cd À klrg + slec phenotype in cd depleted mice, while~ % of helped cd t cells displayed this terminal effector phenotype (fig. b) . although this profile suggested a more activated virusspecific cd t-cell population in cd -sufficient mice, these early differences resolved by day p.i., when the relative populations of slec were similar at~ %, irrespective of cd t cells (fig. b) . by days and p.i. the proportion of klrg + cells and slec within virusspecific cd t cells had dropped significantly in both groups, although slec continued to be higher in cd depleted mice (fig b) . cd + klrg À mpec were similarly low in both groups at day p.i. and gradually increased throughout infection. however, whereas~ % of helped virus-specific cd t cells exhibited an mpec phenotype by day p.i., this population only reached % in cd -depleted mice (fig. b) . a minor fraction ranging from % to % of virus-specific cd t cells expressed both cd and klrg throughout infection in both groups. whereas this population increased with time p.i. in cd -depleted mice, it decreased in controls that had cleared virus (fig. b) . cd expression on klrg + cells has been observed under conditions of repetitive antigen re-stimulation and may mark long-lived effector memory t cells. , overall, the proportion of klrg -expressing jhmv-specific cd t cells was relatively low compared with differentiation of slec during infection with viruses replicating in non-cns tissues, e.g. following lymphocytic choriomeningitis virus infection or following malaria parasite immunization. , , weak klrg expression may be attributed to the unique cns environment and/or the redundant role of interleukin- , a strong inducer of klrg , during jhmv infection. overall, these results are consistent with the notion that early differences in virus-specific cd population arise from the absence of cd t cells during priming, whereas the significantly reduced efficacy to acquire an mpec phenotype, coincident with retention of a larger proportion of slec, is driven by ongoing viral antigen stimulation. the impaired ability of unhelped virus-specific cd t cells to acquire an mpec phenotype, yet the absence of a preferential decline of unhelped relative to helped cd t cells, is consistent with the notion that ongoing exposure to viral antigen drives continual renewal. , we therefore compared the homeostatic turnover of virus-specific cd t cells in the cns of cd -depleted and control mice during acute and persistent infection. consistent with similar numbers of virus-specific cd t cells within the cns, their proliferation was comparable at~ % at day p.i. (fig. c) . the proportions of proliferating virus-specific cd t cells within the cns gradually declined in both groups starting at day p.i. however, % of unhelped cd t cells still showed evidence of proliferation at days and p.i., whereas proliferation by helped cd t cells had declined to~ % (fig. c) . these data suggest that elevated virus load sustains proliferation, yet also promotes activation-induced cell death. indeed, while the frequency of apoptotic virus-specific cd t cells was similarly high in both groups at day p.i., it remained significantly higher in cd -depleted mice at day p.i. (fig. c) . therefore, in the absence of cd t cells continued proliferation by virus-specific cd t cells was balanced by activation-induced cell death, resulting in comparable total numbers of virus-specific cd t cells within the cns. similar phenotypic subsets, as well as activity of helped and unhelped virus-specific cd t cells in the cns at day p.i. suggested that cd t cells do not imprint peripheral cd t-cell activation or initial cns effector function. to eliminate the variable effect of viral load in altering cd t cells, cd -depleted infected mice received jhmv-specific memory cd t cells to reduce viral load. memory cd t cells were derived from thy- . mice immunized with jhmv - weeks post immunization and transferred into jhmv-infected cd -depleted or control thy- . recipients at day p.i. this strategy allowed activation of endogenous cd t cells and monitoring of both donor and recipient t cells based on the thy- congenic marker. both cd -depleted and control recipients developed mild signs of paralysis, and completely recovered from clinical symptoms by day p.i. without mortality (fig. a) . donor memory cd t cells prevented mortality consistent with the inability to recover infectious virus at day p.i. flow cytometric analysis at day p.i. confirmed essentially equivalent cns recruitment of total as well as virus-specific thy- . donor cd t cells in both cd -depleted and control recipients (fig. b,c) . the vast majority of donor cd t cells recruited to the cns were virus specific ( - %), despite constituting only - % of the donor population before transfer (data not shown). virus-specific recipient thy- . cd t cells were slightly higher in the brain of cd -depleted mice (fig d) , whereas those in spinal cord were similar (fig. d , data not shown) confirming that endogenous cd t-cell populations were not skewed by the transfer (fig. d) . importantly, > % of virus-specific endogenous cd t cells in the cns of cd -depleted mice expressed a cd + klrg À mpec phenotype at day p.i. (fig e) . the slec proportion was reduced to < %, showing a similar phenotype distribution as in control mice (fig. e) . the relative mpec and slec populations were also similar in spinal cords in both recipient groups (data not shown). the capacity of endogenous unhelped cd t cells to produce ifn-c was also restored to that of helped cd t cells (fig. f) . these findings suggest that the altered phenotype of primary cns cd t cells in cd -depleted mice is mainly driven by sustained viral load, rather than lack of cd t-cell help or imprinting. in peripheral infections, cd t-cell imprinting on cd t cells is most evident during memory recall responses. , we therefore determined if cd t-cell imprinting is critical for cd t-cell recall responses and survival within the cns. to generate unhelped or helped donor memory cd t cells, thy- . mice were either treated with anti-cd or control mab before jhmv immunization. unhelped or helped memory thy- . cd t cells were transferred into naive thy- . recipients day before infection. mice with no donor t cells served as controls. recipients of helped memory cd t cells displayed enhanced virus control at day p.i., but all groups controlled infectious virus by day p.i. (fig. a) . however, in contrast to their helped memory counterparts or primary cd t cells (figs and ) , unhelped virus-specific memory cd t cells were significantly impaired in expansion in the draining cln, as well as early accumulation in the cns, (fig. b) . the relative proportion and total numbers of thy- . + cd t cells was significantly lower in the unhelped, relative to helped, donor populations in both cln as well the cns at days and p.i. (fig b,c) . the difference was most apparent at day p.i. in the cns and was largely resolved by day p.i. moreover, the number of virusspecific donor cd t cells capable of ifn-c production was reduced in both cln and cns of recipients of unhelped versus helped memory cd t cells at days and p.i. (fig. d) . despite an apparent equilibration towards similar numbers of unhelped versus helped donor cd t cells in the cns by day p.i., survival of the unhelped memory cd t cells was profoundly reduced by day p.i. during the chronic phase. although helped donor thy- . + cd t cells comprised % of the total cd t-cell population, the proportion of unhelped cd t cells had declined > % (fig. e) . this vast reduction was also reflected by the total numbers of unhelped versus helped thy- . + donor cells within the cns (fig. e) . unhelped memory cells also failed to survive in the draining cln (data not shown). nonetheless, despite their low number, the relative proportion of ifn-c-producing cells was comparable between the groups (fig. f) . these results suggest that cd t-cell help at priming plays an essential role in generating long-lasting memory t cells following recall responses within the cns. numerous studies on the role of cd t cells in regulation of cd t-cell immunity reveal that help for functional primary cd t-cell responses is dependent on the pathogen and possibly the target tissue. for example, cd t-cell help is dispensable for activation and differentiation of naive cd t cells during lymphocytic choriomeningitis virus during herpes simplex virus infection. irrespective of the diverse effects of cd t-cell help on primary cd tcell responses, memory cd t cells generated in the absence of cd t cells are typically defective in recall responses. , , our studies demonstrate that cd t cells differentially influence cd t-cell immunity during primary and recall responses mounted to jhmv infection in h- d mice and that the necessity of cd t-cell help during priming is distinct from infected h- b mice. cd t cells primed in the absence of cd t cells in h- d mice had no defects in expansion or ifn-c expression in the periphery similar to vaccinia virus infection of h- b mice. accumulation and subsequent retention of virus-specific cd t cells within the cns also remained unaffected by the absence of cd t cells in h- d mice. moreover, cytolytic activity was similar and ifn-c-producing virus-specific cd effector cells were equally abundant in the cns regardless of the presence or absence of cd t cells during priming and acute infection. cns cd t cells were nevertheless incapable of reducing infectious virus when primed without cd t cells, potentially because of delayed and suboptimal mhc up-regulation in the absence of ifn-cproducing cd t cells. a role for cd help in maintaining effector function during prolonged antigen exposure was indicated by the decline in both the percentage and i. density plot shows representative staining of donor thy- . + cd t cells in the brain. bar graphs represent absolute numbers of helped or unhelped donor thy- . + cd t cells. (f) cns-derived cells were incubated with pn peptide to determine ifn-c-producing thy- . + cd t cells. data represent the mean ae sem. statistical significance was determined using an unpaired student's t-test. *p < Á ; **p < Á ; ***p < Á . evaluation by analysis of variance showed slightly increased statistical significance for values in (b) for sc and cln at day and in (d) for cln at day . by contrast, values in (b) for brain at day and cln at day , in (c) for brain and sc at day and cln at day , as well as (d) for brain at day and , and sc and cln at day did not reach statistically significant differences. the failure to clear infectious virus from the cns in cd -depleted mice also resulted in higher proliferation and apoptosis of virus-specific cd t cells within the cns, consistent with the inability to acquire an mpec phenotype. nevertheless, equilibration of viral load in cd -depleted mice and controls by transfer of memory cd t cells revealed that the unhelped cd t-cell phenotype was prominently driven by viral load rather than the absence of cd t-cell imprinting. the mechanisms underlying the differences in cd tcell imprinting during the priming phase in h- b versus h- d mice are unclear, but highlight critical epitope-specific and genetic influences. the inability to detect jhmv replication in cln suggests a minimal influence of virusdriven pro-inflammatory responses, especially as type i ifn induction by jhmv is very low. , differences may rather reside in the magnitude and kinetics of antigen processing or peptide presentation as l d restricted n protein-specific responses appear to arise earlier compared with d b -restricted s protein-specific responses. moreover, the breadth of cd t-cell responses appear to differ, as numerous h- b epitopes have been defined, while they appear more limited in h- d mice. , irrespectively, uncontrolled viral replication despite intact accumulation of virus-specific cd t cells was the consequence of cd t-cell depletion in both h- b and h- d mice; , while an early defect in cns cd t-cell responses in infected h- b mice suggested a local beneficial effect of cd t cells on cd t cells, a similar but more subtle effect in h- d mice cannot be excluded. however, we have been unable to distinguish a direct contribution of cd t cells to viral control, e.g. via early ifn-c secretion, from an indirect contribution to cd t-cell function involving antigenpresenting cells or soluble mediators. these findings imply that primary cd t-cell anti-viral immunity during encephalitis may vary significantly in an outbred population based on provision of cd t-cell help at multiple stages and anatomical locations. nevertheless, irrespective of potentially distinct mechanisms, cd t cells appear essential for effective local anti-viral cd t-cell function in the cns. a number of studies have also revealed that cd t cells facilitate the generation of functional memory cd t cells during recall responses. , , similar to peripheral infections, the role for cd t cells in cd t-cell recall responses was essential following jhmv cns infection. compared with their helped counterparts, jhmv-specific unhelped memory cd cells expanded less efficiently in the cln, were delayed in cns accumulation, and exhibited reduced survival. a potential mechanism underlying cd help is tumour necrosis factor-related apoptosisinducing ligand (trail). since helped cd t cells are capable of down-regulating trail expression, they are less vulnerable to trail-mediated apoptosis. however, we previously reported that purified cd t cells from the cns of cd -depleted h- b mice had only slightly increased transcript levels of trail mrna compared with controls. whether trail expression influences the limited expansion and survival of unhelped memory cd t cells in h- d mice remains to be examined. distinct from many peripheral infections, both cd and cd t-cell effector functions are associated with demyelination and clinical disease during jhmv encephalomyelitis. infection of oligodendrocytes, the primary targets of jhmv infection, is a prerequisite for demyelination but is insufficient to induce myelin stripping without t cells. , in this context it is of interest to note that uncontrolled virus replication during persistence did not exacerbate demyelination in cd -depleted mice. this contrasts with increased demyelination associated with similarly uncontrolled viral replication in mice deficient in humoral immunity and supports the notion that cd t cells promote pathology. , finally, it is interesting to note that uncontrolled viral replication in cd -depleted h- d mice resulted in viral spread to cells with neuronal morphology. while neuronal infection is sparse in immune competent mice, it is clearly evident under conditions of uncontrolled virus replication in infected ifn-a/b receptor-deficient, b-cell deficient, as well as scid mice, , , potentially contributing to the lethal phenotype. in summary, these data demonstrate that the roles for cd t cells in generating fully functional effector/memory cd t-cell responses following cns virus infection could be manifold depending on the stages of activation and differentiation, effector site of cd t-cell function, as well as genetic background. identifying the cellular mechanism by which cd t-cell immunity is regulated by cd t cells will provide a key insight into understanding cd -cd cooperation for the development of effective primary as well as memory responses within the cns. revealing the role of cd + t cells in viral immunity t-cell help for cytotoxic t lymphocytes is mediated by cd -cd l interactions a novel helper role for cd t cells cd + t cells are required to sustain cd + cytotoxic t-cell responses during chronic viral infection requirement for cd t cell help in generating functional cd t cell memory longitudinal requirement for cd + t cell help for adenovirus vector-elicited cd + t cell responses defective cd t cell memory following acute infection without cd t cell help cd + t cells are required for secondary expansion and memory in cd + t lymphocytes cd t cells promote cd t cell immunity at the priming and effector site during viral encephalitis ctl effector function within the central nervous system requires cd + t cells cd + t cells are required for the maintenance, not programming, of memory cd + t cells after acute infection cd + t lymphocyte mobilization to virusinfected tissue requires cd + t-cell help coronavirus infection of the central nervous system: host-virus stand-off kinetics of virus-specific cd + tcell expansion and trafficking following central nervous system infection perforin and c interferon-mediated control of coronavirus central nervous system infection by cd t cells in the absence of cd t cells mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis ifn-c is required for viral clearance from central nervous system oligodendroglia evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection cd and cd t cells have redundant but not identical roles in virus-induced demyelination effective clearance of mouse hepatitis virus from the central nervous system requires both cd + and cd + t cells cd t cells contribute to virus control and pathology following central nervous system infection with neurotropic mouse hepatitis virus a central role for cd + t cells and rantes in virus-induced central nervous system inflammation and demyelination memory cd + tcell-mediated protection from lethal coronavirus encephalomyelitis the jhm strain of mouse hepatitis virus induces a spike protein-specific db-restricted cytotoxic t cell response characterization of the ld-restricted cytotoxic t-lymphocyte epitope in the mouse hepatitis virus nucleocapsid protein inverted immunodominance and impaired cytolytic function of cd + t cells during viral persistence in the central nervous system bystander cd t cell-mediated demyelination after viral infection of the central nervous system pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies mechanisms of central nervous system viral persistence: the critical role of antibody and b cells antibody prevents virus reactivation within the central nervous system inhibition of interferon-c signaling in oligodendroglia delays coronavirus clearance without altering demyelination perforinmediated effector function within the central nervous system requires ifn-c-mediated mhc up-regulation heterogeneity and cell-fate decisions in effector and memory cd + t cell differentiation during viral infection inflammation directs memory precursor and short-lived effector cd + t cell fates via the graded expression of t-bet transcription factor selective expression of the interleukin receptor identifies effector cd t cells that give rise to long-lived memory cells stimulation history dictates memory cd t cell phenotype: implications for prime-boost vaccination cd stimulation promotes the frequency of il- receptor-expressing memory precursors and prevents il- -mediated loss of cd + t cell memory in the absence of cd + t cell help pathogen-induced inflammatory environment controls effector and memory cd + t cell differentiation short-lived effector cd t cells induced by genetically attenuated malaria parasite vaccination express cd c interleukin- (il- ), but not il- , deficiency ameliorates viral encephalitis without affecting viral control antigen-independent memory cd t cells do not develop during chronic viral infection cd t cells are required for cd t cell survival during both primary and memory recall responses cd + t cells are required for the priming of cd + t cells following infection with herpes simplex virus type oligodendroglia are limited in type i interferon induction and responsiveness in vivo type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells antigen specificity of cd t cell response in the central nervous system of mice infected with mouse hepatitis virus immunogenicity of jhm virus proteins: characterization of a cd + t cell epitope on nucleocapsid protein which induces different t-helper cell subsets enhancement of proliferation and downregulation of trail expression on cd + t cells by il- cd + t-cell help controls cd + t-cell memory via trail-mediated activation-induced cell death we sincerely thank wenqiang wei, eric barron, ernesto barron and jennifer powers for exceptional technical assistance. mh designed and performed the experiments, analysed the results, drafted the figures and manuscript. twp and drh participated in experiments, analysed the results and drafted the figures. sas, ccb and bm designed the study, interpreted the data and edited the manuscript. this work was supported by the national institutes of health grant po ns and cancer center support grant p ca . there are no financial conflicts of interest to declare. key: cord- -mz y am authors: yang, benjamin; yang, andrew; peng, shiwen; pang, xiaowu; roden, richard b.s.; wu, t.-c.; hung, chien-fu title: co-administration with dna encoding papillomavirus capsid proteins enhances the antitumor effects generated by therapeutic hpv dna vaccination date: - - journal: cell biosci doi: . /s - - -y sha: doc_id: cord_uid: mz y am background: dna vaccines have emerged as attractive candidates for the control of human papillomavirus (hpv)-associated malignancies. however, dna vaccines suffer from limited immunogenicity and thus strategies to enhance dna vaccine potency are needed. we have previously demonstrated that for dna vaccines encoding hpv- e antigen (crt/e ) linkage with calreticulin (crt) linked enhances both the e -specific cd (+) t cell immune responses and antitumor effects against e -expressing tumors. in the current study, we aim to introduce an approach to elicit potent cd (+) t cell help for the enhancement of antigen-specific cd (+) t cell immune responses generated by crt/e dna vaccination by using co-administration of a dna vector expressing papillomavirus major and minor capsid antigens, l and l . result: we showed that co-administration of vectors containing codon-optimized bovine papillomavirus type (bpv- ) l and l in combination with dna vaccines could elicit enhanced antigen-specific cd (+) in both crt/e and ovalbumin (ova) antigenic systems. we also demonstrated that co-administration of vectors expressing bpv- l and/or l dna with crt/e dna led to the generation of l /l -specific cd (+) t cell immune responses and l -specific neutralizing antibodies. furthermore, we showed that co-administration with dna encoding bpv l significantly enhances the therapeutic antitumor effects generated by crt/e dna vaccination. in addition, the observed enhancement of cd (+) t cell immune responses by dna encoding l and l was also found to extend to hpv- l /l system. conclusion: our strategy elicits both potent neutralizing antibody and therapeutic responses and may potentially be extended to other antigenic systems beyond papillomavirus for the control of infection and/or cancer. the knowledge that high risk human papillomavirus (hpv) is a necessary etiological factor for the development of cervical cancer provides the opportunity for control of cervical cancer and/or other hpv-associated malignancies through vaccination against hpv. currently, the two commercially available prophylactic vaccines, gardasil and cervarix, based upon virus-like particles (vlp) derived from the major capsid protein l effectively protect against infection by the two most common hpv types found in cervical cancer (for review see [ ] ). the minor capsid antigen l also shows promise for preventive vaccination in animal models, although it is less immunogenic than l vlp. however, neither l vlp nor l -based vaccines generate therapeutic effects against established hpv infection [ ] . therefore, given the significant burden of hpvassociated lesions worldwide, there is an urgent need to develop therapeutic hpv vaccines for the control of existing hpv infection and associated malignancies. quite different from preventive vaccines which target the viral capsid proteins l and/or l , therapeutic hpv vaccines focus on targeting the hpv e and e , since only these oncoproteins are consistently expressed in hpv-associated cancers and are responsible for the malignant transformation. among the various therapeutic hpv vaccines currently being tested, dna vaccines have emerged as attractive candidates for the treatment of cervical cancer and associated malignancies. naked dna is relatively safe, stable, and easy to produce and transport (for review see [ ] ). furthermore, dna vaccines are capable of sustained cellular gene expression, promote mhc class i antigen presentation, and have the capacity for repeated administration since they do not lead to the generation of neutralizing antibodies. however, an important limitation of dna vaccines is limited potency since they lack the intrinsic ability to amplify and spread in vivo. therefore, it is important to consider strategies to improve dna vaccine potency strategies (for review, see [ , ] ). one strategy to enhance dna vaccine potency is to improve antigen expression, processing, and presentation in antigen-presenting cells using intracellular targeting strategies (for review, see [ , ] ). several of our previous studies have employed dna vaccines encoding calreticulin (crt) linked to hpv- e antigen (crt/ e ) [ ] [ ] [ ] [ ] [ ] [ ] because this fusion greatly enhances the e specific cd + t cell immune responses in vaccinated mice. this phenomenon likely reflects the efficient recognition of calreticulin by dendritic cells and improved targeting to the mhci pathway by targeting to the endoplasmic reticulum [ ] . activation and proliferation of cd + t cells is crucial to the success of both humoral and cell-mediated responses to viral infection. although the fusion of e with crt enables such dna vaccines to elicit significant e -specific cd t cell immunity in the absence of cd t cell help [ ] , approaches to boost cd + t cell help are likely to enhance dna vaccine potency. indeed, cd + t cells play a significant role in priming effector cd + t cells, thus augmenting the cd + t cell responses, as well as generation of memory t cell populations (for review see [ ] ). cd + t cells help to differentiate naïve cd + t cells into effector cells by providing activation signals to dendritic cells (dcs), most notably il- , thus promoting cd + t cell proliferation. thus, strategies to induce cd + t helper cells at sites of cd + t cell priming can potentially enhance ctl immune responses. in the current study, we aim to combine intracellular targeting strategies using crt with a strategy to enhance cd + t help for the development of a therapeutic hpv dna vaccine. since papillomavirus l or l antigens likely contain cd + t cell epitopes [ ] [ ] [ ] [ ] [ ] , we reasoned that co-administration of vectors containing codon-optimized bovine papillomavirus (bpv) l and l may provide cd + t cell help and enhance the antigenspecific cd + t cell immune responses generated by dna vaccines. an additional potential benefit would be the induction of neutralizing antibody and protective immunity [ , ] . we showed that co-administration of vectors containing bpv l or l dna in combination with dna vaccines could elicit enhanced antigen-specific cd + in both crt/e and ovalbumin (ova) antigenic systems. we also demonstrated that co-administration of vectors containing bpv l ± l dna with crt/e dna led to the generation of l /l -specific cd + t cell immune responses as well as l -specific neutralizing antibodies. furthermore, we showed that co-administration with bpv l significantly enhances the therapeutic antitumor effects generated by crt/e dna vaccination. in addition, the observed enhancement of cd + t cell immune responses by dna encoding l and l was also found to extend to hpv- l /l system. overall, our data suggest that co-administration of dna encoding papillomavirus l or l can be used to enhance antigenspecific cd + t cell immune responses generated by therapeutic hpv dna vaccination for the control of hpv infection and hpv-associated tumors. furthermore, our approach can also generate neutralizing antibodies against papillomavirus for potential prevention against infection. this strategy also provides the opportunity to combine preventive and therapeutic approaches. our strategy may potentially be extended to other antigenic systems for the control of infection and/or cancer. co-administration with vectors encoding papillomavirus l or l significantly enhances the antigen-specific cd + t cell immune responses generated by crt/e or ova dna vaccination in order to characterize the antigen-specific cd + t cell immune responses generated by vaccination with crt/ e or ova dna in combination with vectors containing codon-optimized bpv l or l dna, c bl/ mice (five per group) were vaccinated intradermally via gene gun with crt/e or ova dna with or without bpv l or l dna twice at -week intervals. splenocytes from vaccinated mice were collected week after last immunization and the e or ova-specific t cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. as shown in fig. , mice vaccinated with crt/e or ova dna vaccine in combination with bpv -l or l dna generated significantly higher e -specific and ovaspecific cd + t cell immune responses compared to mice vaccinated with crt/e or ova dna alone. to evaluate whether the enhancement of antigen-specific cd + t cell immune responses by co-administration of bpv l or l dna is also observed in other papillomavirus systems, we co-administered hpv l or l dna with crt/e or ova dna vaccination. of note, co-administration of hpv l or l dna with crt/ e or ova dna vaccination also generated significantly higher e -specific cd + t cell responses compared to crt/e or ova dna vaccination alone in mice (fig. ) . thus, our data indicate that co-administration with papillomavirus l or l dna significantly enhances the antigen-specific cd + t cell immune responses generated by dna vaccination. co-administration of papillomavirus l or l dna with crt/e or ova dna led to the generation of l /l specific cd + t cell immune responses in order to determine whether the co-administration with bpv l or l dna with crt/e dna will lead to the generation of l or l -specific cd + t cell immune responses, c bl/ mice (five per group) were vaccinated intradermally via gene gun with crt/e dna with bpv l or l dna. mice vaccinated with crt/e dna alone were used as negative controls. splenocytes from vaccinated mice were collected week after last immunization and incubated with bpv l / l virus-like particles (vlps). the l or l -specific cd + t cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. as shown in fig. a and b, mice vaccinated with bpv l in combination with crt/e dna led to the generation of l -specific cd + t cell immune responses. similarly, vaccination with bpv l with crt/e dna led to significant level of l -specific cd + t cell immune responses compared to vaccination with crt/e alone. again, to observe whether the cd + t cell responses elicited by this vaccination strategy extend to other papillomavirus systems, we coadministered hpv l or l dna with crt/e or ova dna vaccination. as shown in fig. c and d, compared to vaccination with crt/e or ova dna alone, mice vaccinated with hpv- l or l dna in combination with crt/e or ova dna led to the generation of l or l -specific cd + t cell immune responses. these results suggest that co-administration with dna encoding papillomavirus l or l with fig. characterization of antigen-specific cd + t cell immune responses generated by antigen-specific dna vaccine mixed with vectors containing bpv l or l dna. c bl/ mice (five per group) were vaccinated intradermally via gene gun with μg/mouse of crt/e or ova dna with or without bpv l or l dna twice at -week intervals. splenocytes from vaccinated mice were collected week after last immunization and the e or ova-specific t cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. a representative flow cytometry data depicting the number of e (upper panel) or ova(lower panel)-specific cd + t cells. b bar graph representing the number of e (upper panel) or ova (lower panel) -specific cd + t cells/ x splenocytes (mean ± sd). data shown are representative of two experiments performed. * indicates p < . crt/e dna was able to generate appreciable l or l -specific cd + t cell immune responses respectively in vaccinated mice. co-administration with bpv l dna significantly enhances the therapeutic antitumor effects generated by crt/e dna vaccination in order to determine if the observed enhancement of antigen-specific cd + t cell immune responses by coadministration of bpv l dna can translate into potent therapeutic antitumor effects, we performed in vivo tumor treatment experiments using an hpv- e expressing murine tumor cell line, tc- . tc- also expresses hpv e , but does not contain either l or l . c bl/ mice (five per group) were first challenged with tc- tumor cells subcutaneously. one week after tumor challenge, mice were treated intradermally via gene gun with crt/e dna alone, bpv l dna alone or crt/ e dna in combination with bpv l dna. vaccinated mice were boosted twice at -week intervals with the same dose and regimen. tumor growth were monitored twice weekly by caliper measurements and palpations. as shown in fig. , tumor-bearing mice treated with crt/e dna vaccine in combination with bpv l dna generated significantly reduced tumor volume and prolonged survival compared to mice treated with crt/ e dna alone or bpv l dna alone. thus, our data indicate that co-administration with bpv l dna is capable of significantly enhancing the therapeutic antitumor effects generated by crt/e dna vaccination. the enhancement in e -specific cd + t cell immune responses are contributed by cd + t helper cells in order to determine the mechanism underlying the observed enhancement of antigen-specific cd + t cell immune responses generated by coadministration with l or l dna vectors, we have generated pcdna encoding the reverse sequences of l (bpv l (−)) or l dna (bpv l (−)). c bl/ mice (five per group) were vaccinated intradermally via gene gun with crt/e dna with or without the reverse sequence of bpv l or l dna twice at -week intervals. splenocytes from vaccinated mice were collected week after last immunization and the e -specific t cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. we found that mice vaccinated with crt/e dna vaccine in combination with the reverse sequence bpv -l or l dna did not lead to the increased frequency of e -specific cd + t cell immune responses observed in mice fig. characterization of antigen-specific cd + t cell immune responses generated by antigen-specific dna vaccine mixed with hpv- l or l dna. c bl/ mice (five per group) were vaccinated intradermally via gene gun with μg/mouse of crt/e or ova dna with or without hpv- l or l dna twice at -week intervals. splenocytes from vaccinated mice were collected week after last immunization and the e or ovaspecific t cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. a representative flow cytometry data depicting the number of e (upper panel) or ova (lower panel)-specific cd + t cells. b bar graph representing the number of e (upper panel) or ova (lower panel)-specific cd + t cells/ x splenocytes (mean ± sd). data shown are representative of two experiments performed. * indicates p < . fig. characterization of bpv and hpv- l or l -specific cd + t cell immune responses generated by crt/e or ova dna mixed with dna encoding bpv or hpv- l or l . c bl/ mice (five per group) were vaccinated intradermally via gene gun with μg/mouse of crt/ e with or without bpv or hpv- l or l dna twice at -week intervals. splenocytes from vaccinated mice were collected week after last immunization and pulsed with μg/ml of bpv or hpv- l /l vlps. the bpv l / l -specific cd + t cell immune responses were characterized using intracellular cytokine staining followed by flow cytometry analysis. a representative flow cytometry data depicting the number of bpv l /l -specific cd + t cells. b bar graph representing the number of bpv l /l -specific cd + t cells/ x splenocytes (mean ± sd). c representative flow cytometry data depicting the number of hpv- l /l -specific cd + t cells. d bar graph representing the number of hpv- l /l -specific cd + t cells/ x splenocytes (mean ± sd). data shown are representative of two experiments performed. * indicates p < . vaccinated with crt/e dna with bpv -l or l dna (data not shown). the insert sequences does not express into l and l that activate cd + t cell help, thereby cannot enhance the e -specific cd + t cell responses of the crt/e vaccine. the result suggests that cd + t cell help generated by co-expression of l or l protein promotes e -specific cd + t cell immune responses to crt/e dna vaccination. co-administration of bpv l dna with crt/e dna led to the generation of l -specific neutralizing antibodies in order to determine if co-administration of bpv l dna with crt/e dna will lead to the generation of bpv l -specific neutralizing antibodies, c bl/ mice (three per group) were immunized on days , , and intradermally via gene gun with crt/e and/or bpv l dna. in vitro neutralization assays were performed using bpv l pseudovirus on twofold dilutions of antisera collected from the mice weeks after the final immunization. mice vaccinated with crt/e in combination with bpv l dna were found to generate similar neutralizing antibody responses compared to mice vaccinated with bpv l dna alone (fig. ) . co-administration of bpv l or l dna with ova dna vaccination generated ova-specific cd + t cell response through intramuscular administration finally, we evaluate whether co-administration with papillomavirus l or l dna can elicit potent antigenspecific cd + t cell responses when applied through different route of administration. c bl/ mice (three per group) were vaccinated intramuscularly with ova dna with or without bpv l or l dna twice at one-week intervals. one week after the last immunization, pbmcs were collected and the ova-specific cd + t cell immune responses were characterized through flow cytometry analysis. as shown in fig. , mice vaccinated intramuscularly with ova dna in combination with bpv l or l dna generated significantly higher percentage of ovaspecific cd + t cells compared to mice vaccinated intramuscularly with ova dna alone. this data indicates that co-administration with papillomavirus l or l dna can lead to enhanced antigen-specific cd + t cell immune responses through the intramuscular route of administration. in the current study, we showed that co-administration of vectors containing codon-optimized bpv or hpv- l or l in combination with dna vaccines could elicit enhanced antigen-specific cd + in both crt/e and ovalbumin (ova) antigenic systems. we also demonstrated that co-administration of bpv or hpv- l or l dna with crt/e dna led to the generation of l /l -specific cd + t cell immune responses. in addition, the observed enhancement of e -specific cd + t cell immune responses by l dna also confers improved therapeutic antitumor effects against an e expressing tumor. moreover, co-administration of bpv l dna induced generation of l -specific neutralizing antibodies, which may serve to prevent further papillomavirus infections. of note, the enhanced antigen-specific cd + t cell responses are observed in intradermal as well as intramuscular routes of administration. indeed, we have already shown that vaccination with vector expressing l is protective against vaginal challenge with papillomavirus pseudovirions [ ] . taken together, our study suggests the promise of this approach for future clinical translation, and it can potentially be applied to other antigenic systems. here we show that cd + t cell help plays an important role in the enhancement of antigen-specific cd + t cell immune responses observed in our vaccination regimen as co-administration with the reverse sequence of bpv l or l did not enhance the antigen-specific cd + t cell responses. studies have shown that l and l are generally much more effective in eliciting cd + t cell responses [ ] [ ] [ ] [ ] [ ] . while it is possible that bpv l or l could elicit some cd + t cell responses, we characterization of bpv -specific neutralizing antibody responses generated by mice vaccinated with crt/e and/or bpv l dna. c bl/ mice (three per group) were immunized on days , , and intradermally using a gene gun with μg of dna per mouse of crt/e and/or bpv l dna. in vitro neutralization assays were performed using bpv l pseudovirus on twofold dilutions of antisera collected from the mice weeks after the final immunization. endpoint titers achieving % neutralization are plotted and the means shown as horizontal lines. pi = pre immune; l = bpv l dna vaccine; e = crt/e dna vaccine believe the cd + t cell help generated by l or l dna is the main contributor to the enhanced antigenspecific cd + t cell response observed. furthermore, it has been shown that cd + t cells can help generate memory t cells [ , ] . though the current study focuses on characterizing the antigen-specific cd + t cell therapeutic antitumor effect by our vaccination strategy, it will be of interest for future studies to further characterize the complete effect of cd + t cell and its ability to generate memory t cell responses for prolonged protection. our vaccination strategy was able to generate a potent therapeutic antitumor effect in tumor-bearing mice. interestingly, co-administration of bpv l dna with crt/e dna generated l -specific neutralizing antibodies, which confers prophylactic value. it has also been shown that vaccination with l dna induced l -specific neutralizing antibodies in balb/c mice [ ] . therefore, our vaccination strategy of co-administration of l dna can generate potent antibody responses in more than one genetic background. of note, studies have shown that papillomavirus l is generally not as effective in generating l -specific neutralizing antibodies [ , ] , thus co-administration with l dna should be prioritized in future translation. in the current study we show that co-administration of bpv l dna with crt/e led to potent therapeutic antitumor effects and prolonged survival due to the enhanced antigen-specific cd + t cell responses by cd + t cell help. since co-administration with hpv l dna with crt/e also significantly enhances antigenspecific cd + t cell responses by cd + t cell help, we believe that potent therapeutic antitumor effects should also be observed. to further promote clinical translation, subsequent investigations focusing on directly characterizing the antitumor effects of co-administrating hpv l with crt/e , and whether reversing the sequence of hpv l abolishes the antigen-specific cd + t cell response enhancement should be conducted. in addition, since our vaccination regimen achieved complete tumor suppression, the frequency of vaccination may be modified and further studied to determine the optimal vaccination regimen. importantly, our data show that the current vaccination strategy can generate enhanced antigen-specific cd + t cell responses by both intradermal and intramuscular vaccination. since dna vaccines are commonly applied intramuscularly in the clinic, future translation of the current vaccination technology may focus on the intramuscular route of administration. fig. comparison of ova-specific cd + t cell responses induced by pcdna -ova vaccination with or without co-administration of bpv l or l . a. schematic illustration of the experiment. briefly, ~ weeks old female c bl/ mice ( mice/group) were vaccinated with μg/mouse of pcdna -ova, with either μg/mouse of pcdna , or pcdna -bpvl , or pcdna -bpvl via intramuscular injection. the mice were boosted with the same regimen once after one week. days after the last vaccination, pbmcs were collected from peripheral blood, stained with fitcconjugated anti-mouse cd a antibody, pe-conjugated ova peptide (siinfekl) loaded h- k b tetramer. the data were acquired with facscalibur and analyzed with cellquest. b. representative flow cytometry image of pbmc staining. c. summary of the flow cytometry data several strategies have been employed to induce cd + t helper cells to enhance antigen-specific cd + t cellmediated immune responses generated by therapeutic hpv dna vaccines. we have previously demonstrated that a dna vaccine encoding invariant chain (ii) with the class ii-associated invariant peptide (clip) region replaced with the pan hla-dr binding epitope (padre) could elicit potent padre-specific cd + t cell responses in vaccinated mice [ ] . in addition, a coadministration of this dna construct (ii-padre) with dna encoding hpv- e generated significantly greater cd + t cell immune responses relative to a coadministration of dna encoding hpv- e with dna encoding unmodified ii [ ] . thus, the current study represents another promising approach to enhance cd + t help for the improvement of dna vaccine potency, but with the added benefit of inducing prophylactic immunity too. it will be of interest in the future to perform a headto-head comparison between vectors containing bpv l / l dna with ii-padre dna for their ability to enhance antigen-specific cd + t cell responses by therapeutic hpv dna vaccines. such information will facilitate the selection of the most effective or desirable dna construct for improving dna vaccine potency. another potential mechanism for enhancing the therapeutic hpv dna vaccine potency through the coadministration with l /l dna is the potential formation of vlps in transfected cells. it has been shown that the expression of l can lead to the formation of vlps in eukaryotic cells [ ] . furthermore, previous studies have demonstrated that papillomavirus vlps can directly activate dendritic cells and thereby increase expression of costimulatory markers and mhc class i and ii molecules [ ] [ ] [ ] . thus, the co-administration of l dna may lead to the local activation of dcs, resulting in further enhancement of antigen-specific cd + t cell immune responses generated by dna vaccination. however, since the l only expression construct had a similar effect to l dna, this suggests that the presence of vlp does not explain the enhanced cd t cell response upon coadministration with crt-e or ova constructs. strategies to enhance cd + t cell help may potentially be combined with other strategies to further enhance dna vaccine potency. we have previously demonstrated a significant enhancement of dna vaccine potency by combining a strategy to prolong dendritic cell life and intracellular targeting strategies with a strategy to boost cd + t cell help [ ] . since all these strategies function via different mechanisms, the combination may potentially result in significantly enhanced antigen-specific immune responses and improved antitumor effects. for clinical translation, it will be desirable to identify the best combination of the different strategies in order to achieve the best dna vaccine potency. in summary, our study demonstrates that the employment of dna encoding papillomavirus l or l can lead to generation of antigen-specific cd + t cells and neutralizing antibodies, resulting in the improvement of therapeutic and preventive hpv dna vaccine potency. our strategy may potentially be extended to other antigenic systems for the control of infection and/or cancer. mice c bl/ mice ( - weeks old) were purchased from the national cancer institute (frederick, md). all animals were maintained under specific pathogen-free conditions at the johns hopkins hospital (baltimore, md). all procedures were performed according to the johns hopkins institutional care and use committee approved protocols and in accordance with recommendations for the proper care of laboratory animals. tc- cells were obtained by co-transformation of primary c bl/ mouse lung epithelial cells with hpv- e and e and an activated ras oncogene as described previously [ ] . they were maintained in rpmi medium supplemented with mm glutamine, mm sodium pyruvate, mm -( -hydroxyethyl)- -piperazineethanesulfonic acid (hepes), x m β-mercaptoethanol, iu ml − penicillin, μg ml − streptomycin, % fetal bovine serum, and cultured at °c in a humidified incubator with % co . the pcdna -ova and pcdna -crt/e dna constructs were generated as described previously [ , ] . dna-coated gold particles were prepared as described previously [ ] . dna-coated gold particles were delivered to the shaved abdominal region of mice using a helium-driven gene gun (bio-rad laboratories inc., hercules, ca, usa) with a discharge pressure of p.s.i. c bl/ mice were immunized with μg of plasmid dna to each mouse encoding pcdna -crt/e mixed with pcdna -bpv -l or l or hpv-l or l delivered to the shaved abdomen. the mice received a homologous boost week later. c bl/ mice were vaccinated with μg of pcdna -ova dna with μg of pcdna , pcdna -bpvl , or pcdna -bpvl intramuscularly in the thigh muscle. the mice received a homologous boost week later intracellular cytokine staining and flow cytometry analysis splenocytes were harvested from mice week after the last vaccination. prior to intracellular cytokine staining, x splenocytes from each vaccination group were incubated for h with μg ml − hpv- e h- db epitope (rahynivtf) or ova peptide (isqavhaa-haeineagr) [ ] , or μg/ml bpv or hpv- l /l vlps in the presence of golgiplug (bd pharmingen) ( μl ml − ). the stimulated splenocytes were then washed once with facscan buffer and stained with phycoerythrin-conjugated monoclonal rat anti-mouse cd α or cd . cells were subjected to intracellular cytokine staining using the cytofix/cytoperm kit according to the manufacturer's instructions (bd pharmingen). intracellular ifn-γ was stained with fluorescein isothiocyanate-conjugated rat anti-mouse ifn-γ to identify the immune response and cytokine levels. pbmcs were collected from peripheral blood week after last intramuscular vaccination and stained with anti-mouse cd α and ova peptide (siinfekl) loaded h- k b tetramer. flow cytometry analysis was performed using facscalibur with cellquest software (bd biosciences, mountain view, ca, usa). in vivo tumor treatment experiments c bl/ mice (five per group) were inoculated subcutaneously with xl tc- tumor cells per mouse on the left flank. after week, when tumor progression is usually observed, mice were vaccinated with dna constructs pcdna -bpv l or l or pcdna -hpv l or l in conjunction with pcdna -crt/e or pcdna -ova or the control empty vector. a homologous boost was administered week after the first immunization. mice were monitored for tumor growth by measuring diameters with calipers twice a week. the bpv l , l and hpv l , l pseudovirions with encapsulated secreted alkaline phosphatase (seap) were generated by co-transfection of tt cells with plasmids encoding bpv l , l or hpv l and l and a seap reporter plasmid as described previously [ ] . cells collected after transfection were treated overnight with brij ( . %), benzonase ( . %) and purified by centrifugation on an optiprep step gradient ( , , and %) at , rpm for . h. pseudovirus neutralization assays were carried out as outlined previously [ ] . briefly, the pseudovirus and the pooled mouse immune sera were incubated for h and the mixture was used to infect tt cells. - h post-infection, the supernatants were collected and seap activity in the supernatants was measured by colorimetric assay. serum neutralization titers were defined as the highest dilution that caused at least a % reduction in seap activity, compared to control pre-immune serum samples. all data expressed as means ± s.d. are representative of at least two different experiments. data for intracellular cytokine staining with flow cytometry analysis and tumor treatment experiments were evaluated by analysis of variance. comparisons between individual data points were made using student's t-test. all p values < . were considered significant. how will hpv vaccines affect cervical cancer? an update of prophylactic human papillomavirus l virus-like particle vaccine clinical trial results dna vaccines: immunology, application, and optimization* modifying professional antigen-presenting cells to enhance dna vaccine potency enhancing dna vaccine potency by modifying the properties of antigen-presenting cells tumor-specific immunity and antiangiogenesis generated by a dna vaccine encoding calreticulin linked to a tumor antigen comparison of hpv dna vaccines employing intracellular targeting strategies development of a dna vaccine targeting human papillomavirus type oncoprotein e a combination of dna vaccines targeting human papillomavirus type e and e generates potent antitumor effects vaccination with dendritic cells transfected with bak and bax sirna enhances antigenspecific immune responses by prolonging dendritic cell life generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus control of hpv-associated tumors by innovative therapeutic hpv dna vaccine in the absence of cd + t cells cooperation between cd + and cd + t cells: when, where, and how t-cell responses to human papillomavirus type among women with different grades of cervical neoplasia listeria monocytogenes delivery of hpv- major capsid protein l induces systemic and mucosal cell-mediated cd + and cd + t-cell responses after oral immunization cellular immune responses to human papillomavirus (hpv)- l in healthy volunteers immunized with recombinant hpv- l virus-like particles effect of vaccine delivery system on the induction of hpv l -specific humoral and cell-mediated immune responses in immunized rhesus macaques analysis of cd (+) t-cell responses to human papillomavirus (hpv) type l in healthy adults reveals a high degree of responsiveness and cross-reactivity with other hpv types enhancement of capsid gene expression: preparing the human papillomavirus type major structural gene l for dna vaccination purposes multivalent human papillomavirus l dna vaccination utilizing electroporation requirement for cd t cell help in generating functional cd t cell memory cd + t cells modulate expansion and survival but not functional properties of effector and memory cd + t cells induced by malaria sporozoites mapping of linear b cell epitopes on capsid proteins of bovine papillomavirus: identification of three external type-restricted epitopes dna vaccines encoding ii-padre generates potent padre-specific cd (+) t-cell immune responses and enhances vaccine potency definition of linear antigenic regions of the hpv l capsid protein using synthetic virion-like particles activation of dendritic cells by human papillomavirus-like particles through tlr and nf-kappab-mediated signalling, moderated by tgf-beta papillomavirus-like particles stimulate murine bone marrow-derived dendritic cells to produce alpha interferon and th immune responses via myd human papillomavirus l l -e virus-like particles partially mature human dendritic cells and elicit e -specific t-helper responses from patients with cervical intraepithelial neoplasia or cervical cancer in vitro enhancing dna vaccine potency by combining a strategy to prolong dendritic cell life and intracellular targeting strategies with a strategy to boost cd + t cell treatment of established tumors with a novel vaccine that enhances major histocompatibility class ii presentation of tumor antigen enhancing dna vaccine potency by coadministration of dna encoding antiapoptotic proteins enhancement of dna vaccine potency by linkage of antigen gene to an hsp gene reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for hpv and hpv submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by nih/nci grants p ca , ro ca , r ai , ca and the cervical cancer spore program p ca . key: cord- - bkp mtj authors: choi, jun yong title: an outbreak of middle east respiratory syndrome coronavirus infection in south korea, date: - - journal: yonsei med j doi: . /ymj. . . . sha: doc_id: cord_uid: bkp mtj nan between may and july , there was an unexpected outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection in south korea. the outbreak has emerged as the largest one outside the middle east. as of july , there have been laboratory-confirmed mers cases, including deaths, recovered individuals discharged from the hospital, and patients who remain in hospitals (fig. ) . , the index patient was a -year-old korean man. the appearance of mers-cov was unexpected and unfamiliar to most physicians. infection prevention and control measures in hospitals were not optimal. extremely crowded emergency rooms and multi-bed rooms contributed significantly to nosocomial infection in some hospitals. the practice of seeking care at a number of medical facilities may have also been a contributing factor. additionally, the custom of having many friends and family members accompany or visit patients may have contributed to secondary spread of the infection. meanwhile, no evidence of community transmission has emerged. several super-spreading events, which happened within hospitals from patients , , , and , contributed to % of all subsequent cases. as well, whole genome sequencing of the mers-cov from this outbreak did not identify any major mutations different from global mers-cov. the factors that drove the super-spreading events of the outbreak have not yet been established. medical procedures that can generate aerosols from the lower respiratory tract of an undiagnosed patient with severe pneumonia could contribute as a super-spreading event. in addition, the crowdedness of the hospitals and environmental contamination could other reasons for the special event. strong infection control measures, including robust contact tracing, active surveillance, quarantine and isolation, have been applied to control the outbreak, since the initial recognition of the outbreak by the korean government. as of july , a total of persons have been quarantined for days, and persons have been discharged therefrom. the infection control measures are anticipated to control this outbreak successfully within several additional weeks. this large and complex outbreak, which arose in crowded hospitals within metropolitan cities, exposed several problems with the korean healthcare system, including emergency preparedness and response systems by the government, as well as infection prevention and control measures in hospitals. to prevent recurrence of a similar situation, we should not only seek to improve and strengthen such systems and measures, but also to develop trained experts and proper facilities. in addition, the outbreak raised several research questions on the epidemiology, virology, pathogenesis, infection control, and treatment of mers-cov infection that await answering. research into the korean outbreak will provide valuable lessons for better global public health. korea ministry of health and welfare and center for disease control and prevention. updates on mers: for press release korean society of infectious diseases; korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea korea ministry of health and welfare and center for disease control and prevention. identification of imported mers case: for press release middle east respiratory syndrome coronavirus (mers-cov)-republic of korea who recommends continuation of strong disease control measures to bring mers-cov outbreak in republic of korea to an end: for news release the author would like to thank dong-su jang, mfa (medical illustrator, medical research support section, yonsei university college of medicine, seoul, korea), for his help with the illustrations. key: cord- -haukpwtf authors: guo, jinlei; cao, yang; qin, kun; zhao, xiaopeng; wang, donghong; li, zi; xin, li; shu, yuelong; zhou, jianfang title: limited effect of recombinant human mannose-binding lectin on the infection of novel influenza a (h n ) virus in vitro date: - - journal: biochemical and biophysical research communications doi: . /j.bbrc. . . sha: doc_id: cord_uid: haukpwtf abstract mannose-binding lectin (mbl), a pattern-recognition molecule in serum, recognizes specific hexose sugars rich in mannose and n-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. it has been well-identified that mbl has antiviral effects via binding to seasonal influenza h and h subtype viruses. influenza a (h n ) virus, a novel reassortant virus to human population, possesses the surface hemagglutinin (ha) and neuraminidase (na) genes from duck and wild-bird influenza viruses and internal genes from poultry h n viruses. as of dec th, , a total of human infections and fatal cases have been identified. here, recombinant human (rh) mbl was tested for its binding and effects on hemagglutination inhibition (hi) and na activity inhibition (nai) of avian h n , h n and human h n viruses. we discovered that rhmbl exhibited a strong binding to h n virus as human h n did at high virus titers. however, it performed a significantly weaker hi activity effect on h n comparing to those of h n and h n , even at a much higher concentration ( . ± . vs. . ± . and . ± . μg/ml, respectively). similarly, minor nai effect of rhmbl, even at up to μg/ml, was found on h n virus while it displayed significant effects on both h n and h n at a lowest concentration of . ± . and . μg/ml, respectively. the hi and nai effects of rhmbl were calcium-dependent and mediated by lectin domain. our findings suggest that mbl, the host innate molecule, has differential interference effects with human and avian influenza virus and limited antiviral effect against h n virus. host innate immunity plays a critical role in the early phase of infection. this first-line defense against pathogens is mediated by a variety of pattern-recognition molecules including collectins, tolllike receptors and ficolins as well as inflammatory cytokines and type i interferon or macrophages and natural killer cells. mannosebinding lectin (mbl) is one of collectins circulating in the serum and synthesized by liver. it consists of collagenous domains and carbohydrate recognition domains (crd). the crds recognize sugars including d-mannose, n-acetylmannosamine, n-acetylglucosamine and l-fucose on the surface of many pathogens in a calcium-dependent manner [ ] . previous studies showed that mbl can bind to a range of clinically relevant microorganisms such as staphylococcus aureus, candida albicans [ ] , hiv, sars-cov, ebola virus, hsv, influenza virus [ e ] . the binding of mbl to microorganisms is presumed to induce mbl conformational changes that allow the molecule to initiate viral neutralization or kill virus via opsonization or complement activation [ ] . influenza a virus, a segmented single-stranded negative-sense rna virus, belongs to orthomyxoviridae and is subtyped according to the antigenic properties of their envelope glycoproteins, ha and na. currently, ha subtypes and na subtypes circulate in birds. among them, only seasonal h n and h n viruses circulate in human population [ ] . occasionally, some subtypes of avian influenza a virus can jump into human and cause diseases with a range of clinical symptoms and outcomes, such as conjunctivitis, mild upper respiratory tract disease, as well as severe pneumonia and death [ e ] . viral ha and na assist virus binding, entry and releasing during infection cycle. their potential n-linked glycosylation sites (ngs) can be glycosylated, which might allow their binding to host mbl. it has been found that the glycan at residue in h n ha was of high-mannose and mbl neutralized viral infectivity via it. many lines of evidences have shown that the mbl plays an important role in fighting against seasonal flu [ e ]. however, little is known about the interactions between avian influenza virus and the innate molecules. avian influenza h n virus is novel to human population [ , ] , which contains the surface ha and na genes from duck and wild-bird influenza viruses and internal genes from poultry h n viruses. unlike other h viruses that generally cause mild symptoms such as conjunctivitis or influenza-like illness (except one fatal case infected with h n in netherlands in ), h n virus usually results in severe pneumonia or respiratory failure in human. here, we examined the interactions of mbl with avian influenza virus h n , h n and human virus h n . furthermore, we studied the molecule mechanisms for them by structure modeling. the vaccine strain a/anhui/ / (h n ) (nibrg- ) was obtained from national institute for biological standards and control (uk), namely h n vac. the virus bears the ha and na of a/anhui/ / (h n ) and internal genes of a/puerto rico/ / (pr , h n ); a/brisbane/ / (h n ) was named as h n wt in the study; h n virus, a reassortant bearing the ha, na from a/hongkong/ / (h n ) and internal genes of pr , was named as h n rg. the reassortant h n ah haþpr na was with ha of a/ anhui/ / and seven genes of pr , which is rescued as previously reported [ ] . h n vac, h n wt and h n ah haþpr na were propagated in e -day-old embryonated chicken eggs, h n rg was grown in madin-darby canine kidney (mdck) cells (atcc, usa) with modified eagle's medium (invitrogen, usa)containing mg/ml n-tosyl-l-phenylalanine chloromethyl ketone (tpck)etreated trypsin (sigma, usa). virus stocks were purified by adsorption to and elution from turkey red blood cells (trbcs) and stored at À cuntil use [ ] . virus titer was determined by titration in mdck cells and the tissue culture infectious dose affecting % of the cultures (tcid ) is calculated by the reedemuench formula [ ] . recombinant human mbl (rhmbl) was purchased from sino biological inc (beijing, china). ninety-six-well plates were coated with  tcid influenza virus at a volume of ml/well for overnight at c, then were blocked for h with % bovine serum albumin (bsa, roche, switzerland) at c. different concentrations of rhmbl ( , , , , mg/ml) were added and incubated for h at c. the virus-dose dependent binding assay was conducted as that wells were precoated with  ,  ,  and  tcid influenza viruses per well. then mg/ml rhmbl was added and incubated for h at c. the binding was detected by the biotinylated human mbl pab ( . mg/ml) (r&d, usa), followed by streptavidin-horseradish peroxidase (hrp) ( : ) (r&d, usa) and tetramethylbenzidine substrate solution (bd, usa), the reaction was stopped by m h so and the optical density (od) at nm was measured by elisa reader (perkin-elmer, usa). the wells coated with mg/ml mannan from saccharomyces cerevisiae (sigma, usa) or coating buffer (kirkegaard & perry laboratories, usa) were used as positive control and negative control respectively. the test was performed in duplicates and in three independent experiments, absorbance from negative control was subtracted and results were normalized to positive control, data was expressed as a relative absorbance value using mean ± sem (%). hi assay was performed in v-bottom -well plates as previously described [ ] . briefly, ml influenza virus ( hau) was mixed with ml rhmbl of different concentrations diluted in hank's balanced salt solution (hbss) containing . mm ca þ for h at c, then ml % trbc was added to the mixture and incubate at room temperature for min. for hi reverse assay: rhmbl was diluted in hbss containing mm edta or mg/ml mannan, then incubated with hau of influenza virus. the results were expressed as the minimum inhibitory concentration (mic) of rhmbl that exhibited hi effect. influenza virus na activity was measured by elisa in which peanut agglutinin conjugated with hrp was used to detect b-dgalactose-n-acetylglucosamine exposed after removal of sialic acid from fetuin [ ] . appropriate amounts of virus in dulbecco's x pbs with cacl and mgcl (life technologies, usa) were used to perform the nai assays. different concentrations of rhmbl were diluted in hbss containing ca þ and mixed with influenza virus in a total volume of ml and preincubated at c for h, and then transferred to wells precoated with fetuin (sigma, usa) and incubated at c for h, after washing, ml of hrp-labeled peanut lectin ( mg/ml) was added and after h at room temperature, the wells were washed and o-phenylenediamine dihydrochloride in citrate buffer was added, reaction was stopped by m h so , and the od at nm was measured. the wells only with virus were used as the positive control, the od of wells with hbss used as a negative control was subtracted. results were expressed as relative na activity (%) calculated as the od of the tested wells with virus and rhmbl divided by the od of the wells with only virus. the ha and na d structures were predicted by using the homology modeling method of swiss-model [ ] . the modeling structures, corresponding templates and identities are shown in supplementary fig. s with pymol (delano scientific). the trimer structure of mbl was assembled using mbl crystal structure (pdb code: hup) with pisa [ ] which generate the oligomeric forms of protein according to the symmetry information. to quantify the distance between rbd/na activity region and each ngs, we employed the average euclidean distance of the center (c alpha atom) of every rbd/na activity region and the center (c alpha atom) of ngs. differences between groups were tested using non-parametric kruskalewallis analysis of variance (anova) for multiple comparisons using ibm spss statistics . (ibm, usa) and p < . was considered statistically significant. the elisa results showed that rhmbl bound both seasonal and avian influenza viruses in vitro. incubation with increasing concentrations of rhmbl resulted in elevated levels of mbl bound to immobilized influenza virus, reaching a plateau at mg/ml (fig. a) . then, mg/ml rhmbl was used to determine the affinity to increasing amounts of virus, revealing the virus-dose dependent binding feature (fig. b) . high binding of rhmbl to the human h n virus might be due to a relatively more ngs in its ha and na (table s ), in addition to the glycans attached at the site of as previously reported [ ] . beyond our expectation, h n virus exhibited stronger binding than h n virus, reaching a comparable level as human h n at higher titers of  and  , although fewer ngs was observed in avian h n than avian h n (table s ). the differential binding of rhmbl to avian virus might result from the types of oligosaccharide attached and further investigations are needed. the finding demonstrated here implied a possible antiviral effect initiated by rhmbl at early stage of novel avian virus infection since naïve host lacked the preexisted cross-reactive or specific anti-ha or anti-na antibodies. initially, we discovered that the lowest concentration for rhmbl (mg/ml, mean ± sem) to abolish virus-mediated trbc agglutination was . ± . , . ± . , . ± . for h n vac, h n rg and h n wt (table ) , respectively. the hi effect could be reversed in the presence of mm edta or mg/ml mannan, which indicates that hi was calcium-dependent and mannan-inhibitable. we then used a h n reassortant obtained ha from h n and other seven segments from pr to exclude a possible hemadsorption by n reported elsewhere [ ] . the mic against the h n was . ± . mg/ml (table ) , which was remarkably higher than those against h n or h n virus, indicating a weaker hi effect of rhmbl on h . as shown in fig. a , na activity of h n was inhibited by rhmbl in a dose-dependent manner, and the ic was . ± . mg/ ml. similarly, the rhmbl, at a concentration of . mg/ml, could inhibit the na activity of h n (fig. b) . nevertheless, the relative na activity of h n remained above % in the presence of increasing concentration of rhmbl even at up to mg/ml. by contrast, the positive control, oseltamivir (roche, switzerland) at the concentration of mm (equivalent to . mg/ml), could significantly inhibit the na activity of h n (fig. c) . furthermore, the inhibition of h n na activity by rhmbl could also be abolished in the presence of mm edta or mg/ml mannan, suggesting that the nai was calcium-dependent and mediated by lectin domain. rbd in ha, including the motifs of -helix, -and -loop, is one of important functional domains [ ] . na activity region is composed of functional residues (r , d , r , r , e , r , r and y ) and framework residues (e , r , w , s , d , i , e , h , e , n and e ) [ ] . to elucidate a possible interference between mbl and the ngs around the functional motifs: rbd in ha or the na activity domain, we compared ngs distribution in them by structure modeling (fig. s ). we also obtained the trimer form of mbl using the crystal structure (pdb code: hup) and measured the radius of mbl crd which is at least Å. although there are indications that trimers of mbl are not biologically active and at least a tetramer form is needed for activation of complement [ ] , Å can be regard as the minimum value. we subsequently calculated the average distances between the ngs located around rbd or na activity region ( table ) and speculated that their distance within the size of human mbl crd, Å, might affect the functions of ha or na. as listed in table s and fig. s , only one conserved ngs on ha head was detected at position in the avian h n while different pattern was found in h and h ( , , , , influenza a virus circulating in animal reservoirs is a continual cause for public health concern. in addition to the ever-present threat of seasonal influenza, we also face the threats from novel viruses such as h n and h n , pdm h n virus in recent years. since the viruses can escape from the protection of anti-ha or anti-na antibodies due to antigenic shift, the innate immunity in naïve host is crucial for battling against newly-emerging viruses. mbl, a pattern-recognition molecule in innate immunity, is known as a b inhibitor for seasonal influenza a virus. the average concentration of mbl in healthy human plasma (aged e years) is . ± . mg/ml [ ] , with about % of individuals present mbl levels below . mg/ml [ ] . the antiviral activity of mbl against seasonal influenza a virus is via its interactions with viral ha or na by blocking viral entry, fusion or releasing [ , ] . mbl could neutralize the virus either in a complement dependent or independent manner [ , ] , however, the effects of mbl may vary depending on specific strains. it also plays an important role in modulating inflammation and has been reported to contribute to deleterious inflammatory response to pdmh n and a h n avian isolate (a/quail/hong kong/g / ) [ ] . here, we found differential binding of rhmbl to human influenza h n and avian h n , h n viruses. specifically, rhmbl exhibited significant hi activity against h n and h n virus at a relatively low concentration ( . ± . , . ± . , respectively), while its hi activity on h n virus was around . ± . mg/ml, reaching the upper limit in plasma of healthy population. in contrast, for nai on h n virus, the rhmbl showed little effect even at a high concentration as mg/ml. as mbl is supposed to display a steric interference with ha or na when binding to the specific hexose sugars across or adjacent to rbd in ha or activity region in na, the limited impact of mbl on h n might result from its property of few ngs adjacent to functional region on the ha and na. of note, a serial of pathotypings including higher virus load, excess chemokine/cytokine response and (-) were incubated with increasing concentrations of rhmbl. na activity was measured by elisa, as described in materials and methods. c. the effects of mg/ml rhmbl and mm oseltamivir on na activity of h n vac. results were expressed as relative na activity (%) calculated as the od of the tested wells with virus and rhmbl divided by the od of the wells only with virus. the dashed line represents % of the original na activity. data are expressed as mean ± sem of three independent experiments. *p < . . functional impairments of b and t cells have been observed in h n infection cases, particularly in fatal cases [ , ] . the aberrant inflammatory response in h n -infected animals could be reversed partially by anti-c a, indicating a hyperactivated complement mediated [ ] . therefore, the strong mbl-h n virus interaction whereas limited effects on viral ha-receptor binding or na-mediated releasing, might amplify immune dysfunctions in vivo and confer clinical severity of h n infection via activating complement pathway and further investigates are needed. isolation and characterization of a mannan-binding protein from human serum mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition interaction of mannose-binding lectin with hiv type is sufficient for virus opsonization but not neutralization mannose-binding lectin in severe acute respiratory syndrome coronavirus infection mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type infection in mice mannose-binding lectin and innate immunity influenza a viruses: new research developments clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus human infection with influenza h n avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome clinical and epidemiological characteristics of a fatal case of avian influenza a h n virus infection: a descriptive study complement-dependent neutralization of influenza virus by a serum mannose-binding lectin human mannose-binding protein functions as an opsonin for influenza a viruses human mannan-binding lectin inhibits the infection of influenza a virus without complement human infection with a novel avian-origin influenza a (h n ) virus biological features of novel avian influenza a (h n ) virus mutations in polymerase genes enhanced the virulence of pandemic h n influenza virus in mice adsorption of influenza hemagglutinins and virus by red blood cells world health organization. manual for the laboratory diagnosis and virological surveillance of influenza measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates swiss-model: modelling protein tertiary and quaternary structure using evolutionary information inference of macromolecular assemblies from crystalline state neuraminidase hemadsorption activity, conserved in avian influenza a viruses, does not influence viral replication in ducks structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution mutations of neuraminidase implicated in neuraminidase inhibitors resistance loss of a single n-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice specific sites of n-linked glycosylation on the hemagglutinin of h n subtype influenza a virus determine sensitivity to inhibitors of the innate immune system and virulence in mice comparison of human blood concentrations of collectin kidney and mannan-binding lectin phase i safety, tolerability, and pharmacokinetic study of recombinant human mannan-binding lectin carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins mannose-binding lectin contributes to deleterious inflammatory response in pandemic h n and avian h n infection clinical findings in cases of influenza a (h n ) virus infection immune derangement occurs in patients with h n avian influenza treatment with anti-c a antibody improves the outcome of h n virus infection in african green monkeys we thank national institute for biological standards and control (uk) and centers for disease control and prevention (usa) for providing the viruses used in our study. we greatly appreciate yu lan for instructions on influenza bioinformatics. this work was supported by national mega-projects for infectious diseases ( zx - - ). none reported. supplementary data related to this article can be found at http:// dx.doi.org/ . /j.bbrc. . . . the transparency document associated with this article can be found in the online version at http://dx.doi.org/ . /j.bbrc. . . . key: cord- -rh zbehk authors: hutcheson, jessica m.; susta, leonardo; stice, steven l.; afonso, claudio l.; west, franklin d. title: delayed newcastle disease virus replication using rna interference to target the nucleoprotein date: - - journal: biologicals doi: . /j.biologicals. . . sha: doc_id: cord_uid: rh zbehk each year millions of chickens die from newcastle disease virus (ndv) worldwide leading to severe economic and food losses. current vaccination campaigns have limitations especially in developing countries, due to elevated costs, need of trained personnel for effective vaccine administration, and functional cold chain network to maintain vaccine viability. these problems have led to heightened interest in producing new antiviral strategies, such as rna interference (rnai). rnai methodology is capable of substantially decreasing viral replication at a cellular level, both in vitro and in vivo. in this study, we utilize microrna (mirna)-expressing constructs (a type of rna interference) in an attempt to target and knockdown five ndv structural rnas for nucleoprotein (np), phosphoprotein (p), matrix (m), fusion (f), and large (l) protein genes. immortalized chicken embryo fibroblast cells (df- ) that transiently expressed mirna targeting np mrna, showed increased resistance to ndv-induced cytopathic effects, as determined by cell count, relative to the same cells expressing mirna against alternative ndv proteins. upon infection with ndv, df- cells constitutively expressing the np mirna construct had improved cell survival up to h post infection (h.p.i) and decreased viral yield up to h.p.i. these results suggest that overexpression of the np mirna in cells and perhaps live animal may provide resistance to ndv. virulent strains of newcastle disease virus (ndv) are the causative agent of newcastle disease (nd), a devastating disease of poultry worldwide [ ] . since highly virulent ndv strains cause up to % mortality in infected flocks, adequate control of nd is vital to ensure healthy, productive poultry populations [ ] . while vaccine campaigns are routinely practiced, these are severely limited by elevated costs, need of trained personnel for adequate administration, and long term thermostability when transporting the vaccine [ ] . these problems are heightened in developing countries, where vaccine costs become excessive in subsistence farming settings [ e ] . such limitations have led researchers to explore new avenues to control problematic pathogens, such as ndv, to generate new, sustainable antiviral strategies [ , , ] . ribonucleic acid interference (rnai) is a naturally occurring intracellular process found in most organisms in which gene expression is controlled through silencing of specific messenger rnas (mrna) [ ] . rnai pathways are capable of being activated by several avenues including micro rna (mirna), small interfering rna duplexes (sirna) and short hairpin rna (shrna). these mechanisms of gene silencing are evolutionarily conserved and can silence mrna at multiple stages of expression including transcription, posttranscription, and translation [ , ] . in the mirna pathway, double stranded mirnas are processed by the protein drosha, further modified by dicer, and the product is then integrated into the rnainduced silencing complex (risc) [ ] . this multi-protein complex will then unravel the double stranded rna, and retain a single guide strand which will direct the knockdown of the target sequences bases on watson-crick base paring [ , ] . in the laboratory, synthetic vectors can be used to express mirna, sirna or shrna, and have been widely used in the scientific community as a means to decrease viral yields by lowering the amount of viable mrna that encodes for viral proteins, or other rna intermediates that are necessary for viral replication [ e ] . rnai approaches have been successfully used in studies of respiratory syncytial virus (rsv), avian influenza virus (aiv), parainfluenza virus, and coronavirus (including severe acute respiratory syndrome virus) [ e ]. alvarez et al. reported reduction in rsv viral concentrations induced by sirna without inducing off target pro-inflammatory effects, a potential problem [ ] . in similar research, suppression of aiv replication was achieved by using shrna to knockdown expression of the viral polymerase leading to reduced bird to bird transmission of the virus [ ] . these studies suggest rnai may be highly effective in disrupting viral replication and decreasing expression of virus genes [ ] . these approaches may now be applied to ndv. ndv is classified as avian paramyxovirus serotype (apmv- ), and is an enveloped virus with a negative sense, single stranded rna genome of approximately kb, which encodes six structural proteins, from to ': nucleoprotein (np), phosphoprotein (p), matrix protein (m), fusion protein (f), hemagglutinin-neuraminidase (hn), and the large polymerase protein (l) [ , ] . using pre-mirna to activate the cellular rnai pathway, a mirna can be used to target the messenger rna of ndv structural proteins, leading to the degradation of the transcripts and inhibiting viral replication [ ] . furthermore, by coupling mirna expression with a lentiviral (lv) delivery system, it is possible to create stable cell populations that constitutively express the mirna sequence [ ] . the use of lv vectors to incorporate exogenous genetic material into the host genome also lends to the possibility of creating transgenic animals capable of germline transmission of the transgene [ ] . an lv delivering a mirna that can induce resistance to ndv could be delivered to a donor bird at various stages resulting in an animal with an endogenous antiviral defense against ndv [ , ] . this approach could lead the basis for a functional, preventative antiviral strategy that does not require the use of additional prophylaxis in chickens. in this study, we attempted to determine if constitutive expression of mirna sequences targeting the mrna of five of the structural ndv proteins in chicken embryo fibroblast cells (df- ) would lead to decreased viral yield after infection, and/or resistance against ndv cytopathic effects. df- cells (chicken embryo fibroblast cell line; atcc crl- ) were cultured with fibroblast medium [dulbecco's modified eagle's medium (dmem) high glucose (hyclone) with % fetal bovine serum (fbs; hyclone), mm l-glutamine (gibco), and x pen/strep (gibco)] at c in a %co atmosphere. human embryonic kidney ft cells (invitrogen) were cultured at c in a %co atmosphere in complete medium [dmem high glucose (hyclone) with % fbs (hyclone), . mm mem non-essential amino acids (neaa; gibco), mm l-glutamine (gibco), mm mem sodium pyruvate (gibco), and x pen/strep (gibco)] supplemented with mg/ml geneticin (gibco cat# ). ndv strains lasota-virulent (ls-v) was obtained by the southeast poultry research laboratory (seprl) repository. ls-v is a virulent strain derived from the non-virulent lasota wild-type by site directed mutagenesis of the f gene cleavage site, and it has an intracerebral pathogenicity index (icpi) of . [ ] . ls-v stock was produced as follows [ ] . briefly, virus ( ul) was propagated in the chorioallantoic cavity of e day old embryonating specific pathogen free (spf) eggs (seprl white leghorn spf flock). dead eggs, or eggs surviving after days of incubation were chilled at c for h, and ha-positive allantoic fluid was extracted, pooled, clarified by centrifugation ( rpm for ), and divided into ml aliquots in cryovials, which were stored at À c. virus stock was tittered in df- cells in -well plates, and titer expressed as tissue culture infection dose % (tcid )/ml. for the purpose for this study, multiplicity of infection (moi) calculations were carried out using the viral titer expressed in tcid . sequences were designed based on block-it™ pol ii mir rnai expression vector kit (invitrogen (grand island, ca) cat# k - ) guidelines, and aimed against the transcribed sequences (mrna) of five ndv genes: one each for np, p, m, f, and three for the l gene (l , l , l ), for a total of seven mirna sequences. sequences were designed based on the consensus alignment of multiple ndv strains representing the most commonly circulating ndv genotype ii. a genotype ii strain representative (such as ls-v) was used since it had been characterized extensively by our group [ , ] . an additional sequence (scramble, scr) with no identity to known chicken or ndv genes was used as a control in all downstream experiments. the complete sequences of the mirnas are provided in table . no mirna was designed for the hn protein due to lack of highly conserved sequences for the protein. for each of the eight sequences, double-stranded oligonucleotides (ds oligo) containing the engineered pre-mirna cassettes were reconstituted by annealing two hplc-purified singlestranded oligos (custom made, integrated dna technologies). the oligonucleotides were designed according to the invitrogen manual guidelines (block-it™ pol ii mir rnai expression vector kit): from to , the top oligo contained a -nucleotide (nt) overhang for ligation in the vector, -nt reverse target sequence (table ) , a nt spacer (terminal loop), and a -nt sense target sequence, with an internal -nt deletion (inner loop). the bottom oligo consisted of the reverse complement to the top sequence, with overhang and no overhang. annealing of ds oligos was verified by ethidium bromide gel electrophoresis. each resulting ds oligo was ligated into pcdna™ . -gw/± emgfp-mir vector. ligated vectors were used to transform chemically competent e. coli cells (topo , invitrogen (grand island, ca) cat# c - ) following manufacturer's protocol. transformed cells were grown in lb agar plates with mg/ml blasticidin (gibco cat# a - ) for selection. dna was extracted from transformants using qiagen plasmid mini prep (qiagen (valencia, ca) cat# ) and insertion was verified by dna sequencing (ab- automated dna sequencer). mirna expression constructs were designated as mir-np, mir-p, mir-f, mir-l , mir-l , mir-l , mir-m, and mir-scr. previously verified expression constructs were subjected to rapid bp/lr recombination reaction per manufacturer's protocol (invitrogen's gateway ® technology; cat# k - ) in order to transfer the pre-mirna cassette to the plenti /v -dest destination vector (invitrogen (grand island, ca) cat# v - ), which is used for lentiviral packaging. plasmids were transformed into one shot ® stbl ™ chemically competent e. coli (invitrogen (grand island, ca) cat# c - ) according to manufacturer's protocol. transformed cells were selected in lb agar plates supplemented with mg/ml ampicillin (sigma cat# a ). transformant cells were grown in lb broth supplemented with ug/ml ampicillin for dna extraction using mini prep (qiagen (valencia, ca) cat# ), or midi prep (qiagen (valencia, ca) cat# ). ampicillin-resistant colonies were screened by electrophoretic banding upon double enzymatic restriction digestion with xhol and aflii nucleases, to assess correct insertion of the pre-mirna cassette (new england biolabs (ipswich, ma) cat# r ; cat# r s). dna sequencing was then used to confirm the restriction digestion results. confirmed lentiviral vectors were used to package lentiviruses, and to produce virus stocks. production of lentiviruses was conducted following virapower™ lentiviral expression system (invitrogen (grand island, ca) cat# ) protocol using t producer cell line (invitrogen (grand island, ca) cat# r - ). produced lentiviral populations were concentrated using peg-it™ virus precipitation solution ( x) following manufacturers protocol (system biosciences (mountain view, ca) cat# lv a- ). lentivirus stock was aliquoted in ml cryovials and stored at À c. in order to evaluate the efficacy of designed mirna cassettes to protect against ndv cytolytic effect, df- cells were transfected with lentiviral plasmids (able to express the mirna cassette) and subsequently infected with ls-v to assess the amount of cellular death. df -cells were plated at   df- cells were plated into well plates, h later cells were exposed to ml of x polybrene (sigma cat# h ) solution in fibroblast medium. previously frozen lentiviral stocks for the np and scr targets were thawed and ice and gently mixed. lentiviruses were diluted : in ml fibroblast medium, gently mixed by pipetting, then added to each well. plates were incubated for h at c in a humidified % co incubator. after h, medium was changed to fibroblast medium containing blasticidin ( mg/ml). since in the lentiviral constructs the emgfp gene is co-cistronic with the mirna cassette, transduced cells underwent two rounds of clonal sorting for gfp in order to produce cell populations expressing high level of mirnas. briefly, transduced df- cells were clonally sorted using beckman coulter moflo xdp based on the highest level of expression of the emgfp reporter system ( / bp filter) into well of a -well plate, and expanded in fibroblast medium. upon expansion, cells were sorted a second time using the same criteria and culture method. after passages, blasticidin ( mg/ml) was added to fibroblast medium for selection. in this way, two stably transduced, highly fluorescent df- expressing mirna for np, and scr were produced. stably transduced df- cells containing mirna for np and scr targets and naïve df- cells were plated at  cells/well into well plates, with three technical replicates for each group. h later, cells were infected with ls-v at moi of . (moi calculation was based on counting naïve df- in extra wells). cells were then infected with ls-vir ndv for h at c in modified fibroblast medium containing only % fbs, as previously described. post infections, cells were washed twice with pbs, and then returned to fibroblast medium. to assess viral growth in transduced cells, ul of supernatant were collected at , , and h.p.i., and replaced with ul fresh media each time. at h.p.i, phase and fluorescent images were collected, and viable cells counted by nexcelom bioscience cellometer auto t using trypan blue (sigma cat# t ) dye exclusion. the amount of virus in the collected supernatant was assessed by limiting dilution in df- cells in -well plates, and expressed as tcid /ml according to the spearman-karber method. table list of sequences in mirna design. sequence of mirna bold indicates directional overhangs for ligation into the expression construct. means from multiple groups in the experiment (both for cell count or virus titer per time point) were analyzed by anova with tukey post hoc test. when only two groups were compared, twosample t-test was performed. for all tests, significance was reported at the level of p . . to evaluate the ability of mirna constructs to targeting and knockdown np, p, f, l and m mrna, mirna constructs expressing mir-np, mir-p, mir-f, mir-l , mir-l , mir-l and mir-m were individually transfected into df- cells. to control for potential off target effects, a scramble mirna (mir-scr) was also transfected into df- cells. all constructs contained an emgfp reporter to determine if cells were successfully transfected. at h, phase images showed an intact monolayer consisting of healthy df- transfected cells (fig. aed, iel) . expression of emgfp in transfected cells showed df- cells are capable of being transfected and can successfully express constructs (fig. eeh, (fig. aed, iel) . however, df- cells transfected with mirna targeting the np mrna (mir-np) were able to maintain their monolayers up to h.p.i before displaying cpe ( fig. a) . cell counts also confirmed that targeting the np mrna could attenuate cell death triggered from ndv infection indicated by the significant increase (up to a fold increase) in cell survival at h.p.i (fig. ) . considering these observations, subsequent experiments were conducted exclusively using the mir-np construct as a potential viral knockdown target. to determine the potential of the mir-np construct to convey long term protection at the cellular level, df- cells were transduced with mir-np and mir-scr constructs that had been packaged in lv. to isolate a homogeneous population of cells that highly express plv-shnp and plv-shscr, transduced df- cells underwent two rounds of facs sorting to isolate cells that were highly gfp positive. due to the emgfp gene and mirna cassette being cocistronic, cells expressing the emgfp should also express the mirna product. after stable cultures of post-sorted cells were established, each of the df- transduced cell populations (plv-mir-np and plv-mir-scr) and a naïve control cell line (a nontransduced cell line) were challenged with ls-v at moi . . phase contrast images at and h.p.i showed similar results to transfection results (fig. aef) . plv-mir-np cultures retain an intact monolayer at h.p.i ( fig. a ; as indicated by arrow) while the scr control and naïve df- cultures show substantial syncytia formation and destruction of the monolayer (fig. bec ; as indicated by arrowheads). however, by h.p.i additional cpe were apparent in all of the cultures including plv-mir-np as demonstrated by the overwhelming presence of syncytia and little to no visibly healthy cells (fig. d) . further characterization of ndv resistance of plv-mir-np was evaluated by determining viral titers in supernatant collected after ls-v infection (moi . ) at , , and h.p.i. plv-mir-np resulted in significantly (p < . ) lower ndv viral titers at both and h time compared to plv-mir-scr (fig. g) . taken together, these results suggest that df- cells transduced with plv-mir-np are capably of decreasing the amount of ndv viral replication following in vitro viral challenge with a velogenic strain of ndv. in this study we demonstrated that knockdown of the np ndv viral mrna in df- cells could lead to decreased cell death and reduced titers (up to a -log decrease compared to plv-mir-scr control) during early stage infection with highly virulent lasota ndv. knockdown of the np protein resulted in delayed viral replication when compared to the scramble control. in unsegmented, negative strand rna viruses, such as ndv, the np protein plays a significant role in the replication and transcription of ndv [ ] . specifically, np, together with both the p and l proteins, interacts with the genomic rna to form the ribronucleoprotein (rnp) which is the template for rna synthesis. in this protein complex, np encapsulates the rna genome allowing proper function of the ndv polymerase [ , ] . np knockdown lowers the amount/ viability of rnp and, by disrupting this essential lifecycle step, it is reasonable to suspect that knocking down the np transcripts within infected cells resulted in the observed delay of viral replication, as shown by another group working with ndv [ ] . a similar study conducted with aiv (an orthomyxovirus) also used rnai to target several of aiv's proteins and reported knockdown of np was notably effective in limiting production of the virus compared to other targets [ ] . furthermore, other influenza studies have also targeted the np protein using rnai and observed a decrease in the amount of the reciprocal viral mrna, virion rna and its complementary rna [ ] . the np protein has able been indicated as a target for administering antiviral drugs [ ] . results such as these suggest that np may be a prime target for controlling and limiting viral replication ndv and similar viruses. plv-mir-np transduced df- cells were observably healthier than control cells types up to h after challenge with ls-v. however, these cells were unable to survive long term in culture with cells at h.p.i., showing significant cell death and syncytia formation by at this time point. while long-term survival was not observed in vitro, it is possible that delaying the rate of infection can ultimately lead to improved animal survival. commonly, a standard challenge dose between and eid is used experimentally to induce % infection and clinical signs in chickens [ ] . based on our observation that plv-mir-np led to a fold decrease in viral titers in vitro, in theory, a transgenic bird containing our construct may require an increased amount of virus to generate an infection. it is also reasonable to consider that a decreased amount of viral titers detected could translate to a reduction in viral shedding of infected birds. such is the case in a study completed by lyall et al. exploring the amount of viral shedding in transgenic chickens expressing an shrna against the polymerase of aiv [ ] . lyall describes that after viral challenge with a highly pathogenic strain of aiv, efficiency of transmission of the virus to other transgenic birds as well as non-transgenic animals was mitigated as assessed by histopathology and immunohistochemistry [ ] . while some birds died in the study, the decrease of viral transmission can translate into a reduction of viral propagation to birds in close contact, decreasing the spreading of the disease and substantially contributing to outbreak control. with the success of infectivity studies with aiv, it is conceivable that similar results could be obtained with ndv challenge studies in transgenic birds. other researchers have had success using the np protein as an anti-viral target utilizing rnai methodology to inhibit np expression and reduction of viral titers in culture, however this work was done in non-avian vero cells [ ] . it is more beneficial to conduct studies in an avian cell type as the ndv virus does not have to adapt to a non-native cell type, which may lead to mutations that are not naturally found in chickens. in addition, the study of rnai approaches in avian cultures is likely to be more representative with respect to the pathophysiology of ndv. yue et al. also showed that shrna targeting of np in chicken embryonic fibroblasts lead to knockdown of viral np mrna [ ] . however, this study failed to examine the effect of constitutive expression of the np shrna and only examine transient expression. understanding the effect of continued long term expression is a key component if this technology is ever to be translated to use in live animals in a production setting. in this study, the mirna sequences were designed based on conserved regions among genotype ii representatives. this genotype was selected because of the extensive characterization of ls-v (a representative of genotype ii), both in vivo and in vitro. in order to provide protection against the many ndv genotypes circulating worldwide (eighteen described so far [ ] ), other conserved genomic regions, in close proximity of the one used here, could be used to generate a broadly protective effect. for instance, this could be accomplished by deploying chaining pre-mirnas that could deliver multiple mirna species, therefore targeting multiple conserved regions at the same time. plv-mir-np constructs constitutively expressed in df- cells led to attenuated cpe and ls-v titers after ndv viral challenge. this study suggests that future transgenic animal studies are warranted. this would likely result in animals that are capable of having endogenous resistance to ndv infections and can have progressive benefits for small village farmers as well as large scale international poultry operations. with potential widespread adoption of resistant transgenic birds, one day the production of resistant animals maybe even more cost effective than the use of standard vaccine programs. traditional vaccine programs have associated costs of transportation, logistics and infrastructure to maintain a cold chain, labor costs to administer vaccines and the cost of lost birds due to ineffective vaccines. in addition, the use of mirna ndv resistant birds in economically impoverished countries and rural areas where vaccines are not readily available would be potentially paradigm changing by providing food and financial security. complete genome sequence of a subgenotype viid newcastle disease virus circulating predominantly in chickens in china virulent newcastle disease virus elicits a strong innate immune response in chickens newcastle disease: progress and gaps in the development of vaccines and diagnostic tools controlling animal disease in africa newcastle disease vaccines a future for transgenic livestock ribonucleic acid interference induced gene knockdown transfer and expression of small interfering rnas in mammalian cells using lentiviral vectors a direct comparison of strategies for 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replication by lentivirus-mediated rna interference high genetic diversity of newcastle disease virus in poultry in west and central africa: cocirculation of genotype xiv and newly defined genotypes xvii and xviii this project was supported by usda-ars cris number - - - d. the authors would like to acknowledge dawn williams-coplin and michele edenfield for dna sequencing. we would also like to thank mrs. julie nelson and the center for tropical and emerging global diseases flow cytometry facility, university of georgia. key: cord- -llnlto p authors: park, yong-shik; lee, changhwan; kim, kyung min; kim, seung woo; lee, keon-joo; ahn, jungmo; ki, moran title: the first case of the korean middle east respiratory syndrome outbreak date: - - journal: epidemiol health doi: . /epih/e sha: doc_id: cord_uid: llnlto p this study reviewed problems in the prevention of outbreak and spread of middle east respiratory syndrome (mers) and aimed to provide assistance in establishing policies to prevent and manage future outbreaks of novel infectious diseases of foreign origin via in-depth epidemiological investigation of the patient who initiated the mers outbreak in korea, . personal and phone interviews were conducted with the patient and his guardians, and his activities in saudi arabia were investigated with the help of the saudi arabian ministry of health. clinical courses and test results were confirmed from the medical records. the patient visited medical facilities and contacted people between may , , at symptom onset, and may , at admission to the national medical center; people were infected and diagnosed with mers thereafter. valuable lessons learned included: ( ) epidemiological knowledge on the mers transmission pattern and medical knowledge on its clinical course; ( ) improvement of epidemiological investigative methods via closed-circuit television, global positioning system tracking, and review of health insurance review and assessment service records; ( ) problems revealed in the existing preventive techniques, including early determination of the various people contacted; ( ) experiences with preventive methods used for the first time in korea, including cohort quarantine; ( ) reconsideration of the management systems for infectious disease outbreaks across the country, such as this case, at the levels of central government, local government, and the public; ( ) reconsideration of hospital infectious disease management systems, culture involving patient visitation, and emergency room environments. after landing in korea in may of , middle east respiratory syndrome (mers) resulted in a total of confirmed cases and deaths from may , when the symptoms occurred in the first patient, to july , when the last patient developed the symptoms [ ] . a novel coronavirus confirmed for the first time in is suspected to be the cause of mers and camels may be the host. human-to-human transmission is known to occur after transmission from a camel to a human, but the exact route has not been sufficiently investigated [ ] . the outbreak in korea started with a patient who arrived in may from saudi arabia, a country that had an outbreak of a large number of mers cases, and subsequently spread. a majority of infected patients were revealed to be cases of hospital-acquired infection [ , ] . this report is an epidemiological study of the first patient (patient # ) who initially brought mers into korea in . from may , when a diagnosis of mers was confirmed, the division of epidemic intelligence service, korea centers for disease control and prevention (kcdc), in cooperation with a group of civilian volunteers in epidemiology, traced the infection route and performed preventive measures for the spread of additional infections. through personal and phone interviews we contacted employees at business facility in saudi arabia who may have had contact with patient # during the incubation period; we investigated the places he visited, presence or absence of mers symptoms in the individuals he contacted, history of visiting medical facilities in the middle east, and history of consuming camel milk or meat, among other things. the patient's specific activities in saudi arabia were verified with the help of the saudi arabian ministry of health. additionally, the timing of symptom occurrence and the initial symptoms were reevaluated by confirming the history of visiting domestic medical facilities after arrival in korea via personal interviews with the patient and his guardians and examining his medical records. the kcdc performed a diagnostic serum antibody test for mers for individuals whose contact with patient # was confirmed by personal interviews or closed-circuit television (cctv) reviews. this study was conducted as an epidemiological investigation of the mers outbreak, and thus, additional processes for review and approval by institutional review board were not required on the basis of the life ethics and safety law enforcement rule item (human subject studies). patient # was a -year-old man and had underlying diseases including asthma, hypertension, dyslipidemia, and benign prostate hypertrophy. at the time of the study, he was a current smoker. he was in the greenhouse building business with a business facility in the middle east, specifically bahrain, as well as at a domestic business facility in asan, chungnam, korea. he visited the middle east region about once every months and stayed for approximately weeks during each visit. the most recent business trip was between april , and may , , for days. he had business visits to saudi arabia (may to ) and the united arab emirates (april to ) with bahrain as the base; both departure from and arrival at korea was via qatar. during his stay in the middle east, he had no history of direct contact with camels, consuming camel by products such as milk or meat, or visiting the medical facilities there. in saudi arabia, he stayed at his business facility and the hotel in the area of al muzahimiyah outside of the capital, riyadh. during his trips within the middle east, he had no contact with animals and did not eat or drink outside the hotels where he stayed. while staying in riyadh, he travelled with a driver, a guide, and the bahrain business facility manager, none of whom showed the symptoms suspected of mers. patient # returned to korea without any abnormal symptoms on may via flight oz and went to his domestic business place in asan on may . on may , fever broke for the first time, and he visited asan seoul clinic the next day (may ). at the time of the visit, his body temperature was . °c, and he complained of febrile sense and myalgia. on may , he visited the same place with a persistent high temperature ( . °c) and myalgia, and on may , he was transferred to pyeongtaek st. mary's hospital for inpatient treatment, because respiratory symptoms such as cough and sputum had developed, his body temperature was . °c, and the myalgia had worsened. after the transfer, he was in an outpatient exam room, a laboratory room, and a chest radiography room, and was put in a double-occupancy room at approximately pm (room , th floor). around : pm, a chest computed tomography (ct) scan was performed on the first underground floor. on the following day (may ) at approximately : am, he visited the radiography room on the first underground floor to undergo a chest radiogram. pneumonia in the right upper lobe was found on the chest ct scan, and infections with haemophilus influenzae and streptococcus pneu monia were confirmed on bacterial culture testing. from the evening of may , breathing difficulty and chest pain developed. symptoms did not improve, and the patient was discharged from the hospital at am on sunday, may ; thereafter, his wife drove him for treatment to the clinic, where he usually went for examination. the clinic referred him to a high-level medical center. he went to the samsung medical center in seoul emergency department, but returned home because a patient room was not available. on the following day (may ) at approximately am, his wife drove him again to the samsung medical center in seoul emergency department, and he was admitted. from pm, he wore a facial mask. on may , he was reported to the kcdc as a case suspected of mers in consideration of the clinical courses that deteriorated despite the antibiotic therapy and his history of travel to the middle east within the previous two weeks, which was revealed during a consultation with physician. on may , his sputum polymerase chain reaction (pcr) test was determined positive, following which he was transferred to the national medical center. patient # received antiviral, interferon, and antibiotic treatments, and me chanical ventilation therapy after an endo-tracheal intubation was performed due to worsening respiratory symptoms. on june , a sputum pcr test was confirmed negative for mers virus, and it was decided that he was cured. on september , he was discharged after completing treatment for a sacral sore (figure ). individuals who made contact with patient # and preventive efforts for them individuals who were in direct contact with patient # without appropriate personal protective equipment or who stayed with him in an enclosed space (patient room, office, etc.) were considered cases of close contact and were put under surveillance. the scope of close contact surveillance gradually expanded with the spread of the mers outbreak, and the final count of individuals under close contact surveillance was : employees at patient # 's domestic business facility in asan, medical staff members at the asan seoul clinic, medical staff members and patients (including patient # 's wife) at pyeongtaek st. mary's hospital, medical staff members and patients at the clinic, and medical staff members and patients at the samsung medical center in seoul. depending on the extent of contact, they were classified into either ) quarantine of a person or a place or ) active observational surveillance and were closely tracked and observed ( figure ). during surveillance, when symptoms suspected as mers occurred, respiratory samples were collected for testing, the suspected cases were transferred to a quarantined hospital, and epidemiological investigations were conducted. a total of people had contact with patient # from may , when the first symptoms occurred, to may , when he was transferred to the national medical center. of those, became infected, with an infection rate of . %. according to the characteristics of the contacted individuals, out of medical staff members ( . %); out of patients ( . %); and out of guardians, caregivers, and visitors ( . %) were infected. however, trying to correlate patient # 's transmission capacity or epidemiological characteristics to the infection rate had limitations because with the spread of mers, the scope of surveillance of contacted individuals gradually expanded, and the standards were not consistent across different institutions. nonetheless, based on a study analyzing epidemiological characteristics of the mers outbreak around pyeongtaek st. mary's hospital, the rate of infection was . % overall and was . % among medical staff members; . % among patients; and . % among guardians, caregivers, and visitors [ ] . aside from patient # , of individuals, in particular, with a confirmed diagnosis (# , # , and # ) individually infected more than individuals whose diagnosis was additionally confirmed, and they were thus labeled as "super spreaders." it is thought that, after contact with patient # , they visited different medical facilities and became epidemiologically associated with additional patients with a confirmed diagnosis [ ] . the mers outbreak in korea started with the infection of a businessman in his s who often traveled to the middle east for business. he did not show symptoms at his arrival in korea. a week later, he started experiencing symptoms and visited medical facilities. however, it took days for the mers infection to be confirmed at the th medical facility he visited, and, by then, the infection transmission had already begun at the previous medical facilities. thereafter, a "mers crisis" transpired for the first time wherein a total of patients were confirmed with mers infection; deaths occurred among these patients, and , individuals were quarantined for prevention. the clinical symptoms in the first patient deteriorated because of a delay in receiving appropriate treatment, and he was assisted with mechanical ventilation. on day after admission to the th hospital, tests for mers virus were negative, and the patient's condition recovered. from the epidemiological investigation, the course of transmission to this patient in the middle east was not clear. it can only be inferred that the first patient became infected while visiting saudi arabia, on the basis of a recent study showing that the mers virus that broke out in korea is genetically closest to the qatar strain [ , ] . however, to uncover the specific course of transmission, we must perform an epidemiological investigation in coordination with saudi arabia. various factors are considered as causes of the widespread outbreak. the first is health care workers' lack of knowledge regarding mers. since june , the kcdc had organized a national mers management team in preparation of a mers outbreak in korea, and mockup training had been performed to enhance the capacity to manage outbreaks of novel infectious diseases at quarantine stations and at the level of local government of cities and provinces. however, in cases such as the present case, wherein the symptoms do not manifest themselves when patients enter korea, preventing an outbreak of a novel infectious disease by imposing quarantine is not practical. in particular, health care workers did not receive sufficient education or communication against mers in anticipation of a case wherein a mers-infected individual, on whom quarantine was not imposed, would go to a regional medical facility. until the diagnosis was confirmed for patient # , the first medical facilities that he visited were unable to suspect a mers case. the doctor at the rd medical facility, who examined patient # and subsequently became infected, in an interview with the media after his discharge stated that he had not even heard of mers. the second factor communication about mers among travelers to the middle east was insufficient. since , when the likelihood of spread of mers would spread within the middle east region was indicated, the kcdc persistently promoted mers awareness among the travelers to that region. however, patient # , who frequently visited the region and his family members, had no idea about mers. thus, when he visited the general medical facilities with respiratory symptoms, he did not mention about his travel to the middle east, and only mentioned about his visit to bahrain when inquired about a travel history involving the middle east at the medical center where his diagnosis was confirmed. therefore, mers could not be diagnosed sooner. a third factor contributing to the rationale behind the outbreak was not kept in check earlier was that quarantine was not thoroughly imposed. after patient # 's diagnosis was confirmed, thorough quarantine was neither imposed on individuals who had close contact with him nor on those with casual contact (such as those who had a possibly contacted the doctors or the patients or those who were exposed to a space infected by him). lastly, in this outbreak, most patients were infected via hospital-acquired infection because korean medical institutions' patient rooms and emergency rooms were very crowded, and infected patients had to visit several hospitals because of the shortcomings of the medical care delivery system, which played a critical role in the spread of mers from hospital to hospital. through an investigation on the mers outbreak within korea, valuable lessons were learned. they include ( ) accumulation of epidemiological knowledge of the pattern of mers transmission as well as medical knowledge on its clinical courses; ( ) improvement of epidemiological investigative techniques using cctv, global positioning system tracking, and review of health insurance review and assessment service records, among other things; ( ) problems revealed in the existing preventive techniques, including the early determination of the scope of contacted people; ( ) accumulation of experiences with the preventive techniques used for the first time in korea, such as cohort quarantine; ( ) reconsideration of the management systems that deal with an outbreak of infectious diseases across the country, such as in the present case, at the levels of central government, local government, and the public; and ( ) reconsideration of infectious disease management system at hospitals, culture involving patient visitation, and emergency room environments. the mers outbreak in korea began with an infected businessman in his s who visited the middle east region. however, the spread of outbreak is attributed to insufficient preparation and management by the ministry of health and welfare. to prepare for an outbreak of novel infectious diseases in future, government organizations responsible for infectious diseases need to train personnel specialized in the area, and also promote awareness among medical staff at the primary and secondary medical facilities as well as among the public. the mers outbreak should serve as an opportunity to improve the level of managing hospital-acquired infection in korea. mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome current epidemiological situation of middle east respiratory syndrome coronavirus clusters and implications for public health response in south korea epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control the authors have no conflicts of interest to declare for this study. supplementary material (korean version) is available at http: //www.e-epih.org/. key: cord- - kzb ag authors: quinteros, josé a.; markham, philip f.; lee, sang-won; hewson, kylie a.; hartley, carol a.; legione, alistair r.; coppo, mauricio j. c.; vaz, paola k.; browning, glenn f. title: analysis of the complete genomic sequences of two virus subpopulations of the australian infectious bronchitis virus vaccine vics date: - - journal: avian pathol doi: . / . . sha: doc_id: cord_uid: kzb ag although sequencing of the ′ end of the genome of australian infectious bronchitis viruses (ibvs) has shown that their structural genes are distinct from those of ibvs found in other countries, their replicase genes have not been analysed. to examine this, the complete genomic sequences of the two subpopulations of the vics vaccine, vics-v and vics-del, were determined. compared with vics-v, the more attenuated vics-del strain had two non-synonymous changes in the non-structural protein (nsp ), a transmembrane (tm) domain that may participate in autocatalytic release of the -chymotrypsin-like protease, a polymorphic difference at the end of the s gene, which coincided with the body transcription-regulating sequence (b-trs) of mrna and a truncated open reading frame for a peptide encoded by gene ( b). these genetic differences could be responsible for the differences between these variants in pathogenicity in vivo, and replication in vitro. phylogenetic analysis of the whole genome showed that vics-v and vics-del did not cluster with strains from other countries, supporting the hypothesis that australian ibv strains have been evolving independently for some time, and analyses of individual polymerase peptide and s glycoprotein genes suggested a distant common ancestor with no recent recombination. this study suggests the potential role of the tm domain in nsp , the integrity of the s protein and the b-trs , and the putative accessory protein b, as well as the ′ untranslated region, in the virulence and replication of ibv and has provided a better understanding of relationships between the australian vaccine strain of ibv and those used elsewhere. infectious bronchitis is a highly contagious and widespread disease of chickens and is a major ongoing problem in all countries with an intensive poultry industry (sapats et al., ; casais et al., ) . the aetiological agent is infectious bronchitis virus (ibv), a member of the family coronaviridae (beaudette & hudson, ; tyrell, ) , subfamily coronavirinae and genus gammacoronavirus (group ; cavanagh et al., ) . while ibv has been detected in other avian species, including guinea fowl (numida meleagridis), partridge (alectoris sp.), peafowl (pavo cristato) and the blue-winged teal (anas sp.), clinically detectable disease has only been observed in chickens (liu et al., ; cavanagh, ; sun et al., ) . ibv has a positive sense, single-stranded rna genome approximately . kb in length (tannock, ; watkins et al., ; schochetman et al., ; jackwood & de witt, ) with the general organization ′ untranslated region (utr) - a, ab (or polymerase genes) -s - a, b, c (e) -m - b, c - a, b -n - b - ′ utr (cavanagh, ; ammayappan et al., ) . the genes encoding the structural proteins are s (spike glycoprotein), e (envelope glycoprotein), m (membrane glycoprotein) and n (nucleocapsid protein) (ignjatovic et al., ; cavanagh, ) , while the a, b, b, c, a, b and b genes encode accessory proteins (liu et al., ; liu & inglis, a, b; cao et al., ; hewson et al., ; bentley et al., ) , the functions of which are still unknown (liu et al., ) . the s glycoprotein is a virion surface, rod-shaped protein that is post-translationally cleaved into two subunits, s and s (cavanagh, ; stern & sefton, ; cavanagh, ) . during ibv infection, virions bind to the target cell receptors and release the viral genome into the cytoplasm of the host cell (ziebuhr, ; knoops et al., ) . the polyproteins a and ab (pp a and pp ab) are then translated, and the papain-like (plp) and -chymotrypsin-like ( cl) proteases are released from these polyproteins after an autocatalytic process. these proteases initiate the cleavage in trans of the peptides contained in pp a/pp ab. these peptides, probably together with some cellular proteins, form the replication-transcription complex. this initiates the transcription of a series of ′ nested subgenomic rnas that are translated into the viral structural and accessory proteins (gorbalenya et al., ; liu & brown, ; tibbles et al., ; liu et al., ; ziebuhr et al., ; ziebuhr, ) . in australia, three genotypic subgroups of ibv have been distinguished (hewson et al., ) . subgroup or "classical" strains were isolated between and and include the subtype b vaccines, which were classified using serological methods (wadey & faragher, ; ignjatovic & galli, ) and vaccine-related strains. subgroup or "novel" strains emerged between and , while subgroup strains, which emerged around , are recombinants derived from subgroup and strains (ignjatovic et al., ; hewson et al., ) . four different ibv strains are included in the commercially attenuated vaccines available for the control of infectious bronchitis in australia. three of these, vics, i (inghams) and s (steggles), belong to the same serotype (subtype b), while a (armidale) is serotypically distinct (subtype c; ignjatovic et al., ; hewson et al., ) . previous studies (hewson et al., ) have demonstrated that the vics vaccine contains two subpopulations, referred to as vics-v (the predominant subpopulation in the vaccine) and vics-del, which has a -nucleotide deletion in the ′ utr and multiple single-nucleotide differences in the structural protein genes compared with vics-v (hewson et al., ) . these viral subpopulations differ in their ability to replicate in vitro and in their pathogenicity in vivo. in vics-v inoculated groups, all the birds exhibit clinical signs, including head shaking and eye scratching/irritation, from day to day post-inoculation, whereas in the vicsdel inoculated group the clinical signs are less severe and only seen between days and post-inoculation. birds inoculated with vics-v also have significantly more severe tracheal lesions than those inoculated with vics-del. over the last few years, a number of complete genome sequences of many ibvs have been determined and the sequences of more than a hundred are now available in genbank (national center for biotechnology information, ncbi). however, the complete genome of an ibv isolated in australia has not been determined. as the pathogenicity and replication of the two ibv subpopulations, vics-v and vics-del, in the vics vaccine, differ in vivo and in vitro under laboratory conditions, the complete genome sequences of these two variants were compared in order to identify potential novel molecular determinants of pathogenicity and replication in ibv. virus. the vics-v and vics-del strains of ibv, purified previously in our laboratory, were propagated in specific pathogen-free embryonated hen eggs (australian spf services, woodend, victoria, australia), as described previously (hewson et al., ) . after - days of incubation, the allantoic cavities of the specific pathogen-free embryonated eggs were inoculated with one of the two strains. after h of incubation, the eggs were chilled for - h at °c and the allantoic fluid collected and stored at - °c. viral purification and nucleic acid extraction. the allantoic fluid was clarified as previously described (lougovskaia et al., ) , with slight modifications. the gradients were fractioned into ml volumes, and the fractions containing the highest concentration of virus were determined by pcr using primers and a protocol described previously (hewson et al., ) . after centrifugation at ,  g for h at °c the viral pellets were resuspended in - µl of tris-buffered saline (ph . ; . -m tris-hcl, . -m nacl) and stored at - °c. viral rna was extracted using rneasy™ extraction kits (qiagen pty. ltd., chadstone, victoria, australia), following the manufacturer's protocol, and then stored at - °c. complete genome sequencing. complete genome sequencing of the vics-v and vics-del strains of ibv was performed using the pgm ion torrent™ platform (life technologies australia pty. ltd., mulgrave, victoria, australia). rna was fragmented using rnase iii (life technologies) and purified using magnetic beads. to construct the libraries, the rna was reverse-transcribed using superscript™ iii (life technologies). the cdna was purified and amplified following the manufacturer's protocols and then sequenced using the chip, base sequencing ion onetouch™ kit v . all the reads that matched to chicken ribosomal rna and mitochondrial genome sequences were discarded. for vics-v, the remaining reads were mapped to the genomes of the beaudette strain [genbank accession number (gan) nc_ ], two vaccine strains from usa (conn and massachusetts, gan fj and gq , respectively) and two strains from china (saibk and sc , gan dq and eu , respectively) using geneious™ version . . (biomatters ltd., auckland, new zealand). because of the high level of sequence diversity in the s gene, the readings were also mapped to the previously determined sequence of structural protein genes from the vics vaccine (gan jn ). for vicsdel, the readings were mapped to the genome of vics-v and the previously determined sequence of the structural protein gene region of vics-del (gan jn ). all gaps and ambiguous sequences were corrected using sanger sequencing and the big dye™ terminator v . sequencing protocol (life technologies), following the manufacturer's instructions. as well as three turkey coronavirus (tcov) sequences and sequences of coronaviruses isolated from a duck and a peafowl in china (gan eu , tcov- ; eu , tcov-atcc; gq , tcov/tx-gl/ ; jf , duck cov; and ay , peafowl, respectively), were used for phylogenetic analyses. the sequences were aligned using clustal-omega (sievers et al., ; www.ebi.ac.uk) and the phylogenetic tree constructed using geneious™ . . , using the nearest neighbour interchange maximum likelihood heuristic method with bootstrap replications and the general time-reversible (gtr) substitution model, gamma distributed with invariant sites (g+i). the gtr+g+i substitution model was selected as the ideal model for these data using the "find best dna/protein model" tool, available in mega . (tamura et al., ) . phylogenetic trees were also constructed for individual peptides encoded by the polymerase genes, the main proteinase, also referred to as cl, the helicase (hel), the plp and the rna-dependent rna polymerase (rdrp), and also for the s glycoprotein. the sequences were aligned with clustalw (geneious™ . . ) and the phylogenetic trees constructed as described above. in these phylogenetic trees, only the vics-v sequence was included (except for the s glycoprotein tree, where both vics-v and vicsdel were included), as the vics-v and vics-del sequences were highly similar ( . %), and vics-v is the main component of the vics vaccine (hewson et al., ) . to assess the extent of potential recombination between viruses, a network tree was constructed using the same complete genome alignments as described above and splitstree . . (huson & bryant, ) . the distances were calculated with the uncorrected p-method and the network constructed with neighbor-net using bootstrap replicates. the sequences of the complete genomes of vics-v and vics-del have been submitted to genbank under accession numbers kf and kf , respectively. sequencing and coverage. for vics-v and vics-del, the ion-torrent chip generated . and . mb of data, comprising , and , reads, respectively, with mean read lengths of . and . nucleotides, respectively. after removal of the host-matched reads, a total of , and , reads were retained ( . % and . %), respectively. for vics-v, a total of , readings ( . %) were matched with the complete genome sequence of the beaudette strain. the mean depth of coverage was fold. a total of , reads could be mapped to the structural protein region, yielding a mean depth of coverage of fold and a minimum depth of fold. for vics-del, , reads ( . %) were matched with the complete genome sequence of the vics-v strain. the mean depth of coverage was fold. a total of , reads could be mapped to the structural protein region, yielding a mean depth of coverage of fold and a minimum depth of fold. the vics-v and vics-del genomes were , and , nucleotides in length, with a g + c content of . % and . %, respectively. vics-v had the typical ibv genome organization ( ′ utr - a/ ab -s - a, b, c (e)- a, b -m - a, b -n - ′ utr; stadler et al., ; cavanagh, ; thor et al., ) , while vics-del had a truncated b open reading frame (orf). comparisons of the complete genomes of vics-v and the more attenuated vics-del ( figure ) revealed several differences, which are summarized in table . the most significant of these differences found in vics-del were the absence of a glycine (gly) in two gly-gly motifs in vicsdel and two insertions-deletions (indels) in the nucleotide sequence of the non-structural protein (nsp ) gene, which would lead to a change in amino acids. this frameshift introduced two positively charged amino acids, changing the region from predominantly hydrophobic, and resulted in the loss of a third gly-gly motif. there was also a single-nucleotide polymorphism (snp) in the s gene at nucleotide , close to the ′ end of the gene, with approximately equal numbers of vics-v genomes having either a g or a u, while in vics-del most genomes had a u in this position. finally, a frameshift mutation resulting in the truncation of orf b and a -nucleotide deletion in the ′ utr region were recognized in the genome of vics-del. the snp in the s gene generated two non-synonymous codons, gaa (glutamate) or uaa (a premature stop codon), nine codons prior to the usual s gene stop codon. this snp also lies within the body transcription (b-trs) for mrna , which encodes the accessory proteins a and b, and the structural protein e. in vics-v, . % of the reads ( / ) contained the codon gaa and % contained the uaa stop codon. in vics-del, . % of the reads contained the gaa codon and . % the uaa codon (table ) . a second gly-gly motif, which has been described as a plp cleavage site (ziebuhr, ) , was identified in the vics-v sequence between nucleotides and . nucleotide alignment and phylogenetic analyses. phylogenetic analysis assigned most of the genomes into two main clusters (figure ). the first major cluster (a) included predominantly usa strains, except for the ck/ch/ ldl/ i (china) and peafowl (china) strains. the peafowl (china) isolate lay within the beaudette subcluster and has been described previously as very similar to a mass strain isolated in (liu et al., ) . the usa cluster can be subdivided into the mass vaccine, beaudette and holte subclusters. the second cluster (b) contained only strains of chinese origin, including a strain isolated from a duck, and these were subdivided in two subclusters (b and b in figure ). tcov strains lay in a separate cluster, closely related to usa ibv strains. there was a high level of similarity between the ita/ / (italy) and ck/swe/ / (sweden) strains, which have been described previously as european qx-like strains, a chinese origin genotype widely distributed in europe (ducatez et al., ; abro et al., ) . the strains ck/ch/lsd/ i (china) and tw / (taiwan) also shared a cluster. the vics-v and vics-del strains (australia) form their own cluster, while ibadan (nigeria) and snu (south korea) strains did not fall into any cluster. in the phylogenetic trees of the cl, hel, plp and rdrp genes (figure ) , the chinese and usa clusters were conserved. however, vics-v grouped within the chinese cluster in the cl and plp gene phylogenetic trees and within the usa cluster in the hel and rdrp gene phylogenetic trees. in the s glycoprotein gene trees ( figure ) the isolates fell into distinct chinese and usa clusters. in these trees, vics-v, vics-del and ibadan lay closer to the usa cluster, while the south korean, taiwanese and european strains were positioned closer to the chinese cluster. the georgia and delaware strains were distinct from all other strains, and were located between the ibv and tcov groups. a phylogenetic tree inferred using the amino acid sequences had the same cluster organization ( figure ). network tree analysis. in the analysis shown in figure , the boxes indicate the likelihood of recombination events between strains of ibv. the tree has the same cluster organization seen in the phylogenetic tree using whole genome sequences (figure ). the vics-v and vics-del branch splits from all clusters close to the centre of the tree, suggesting limited and historically distant recombination with other strains. although there has been considerable investigation of ibv and of coronaviruses in general, the determinants of their virulence and their capacity to replicate in vivo remain uncertain. as recent studies have suggested that the virulence determinants may not be located in the structural protein region of some ibvs (armesto et al., ) , it can be inferred that these determinants may be encoded within the a and ab polyproteins. therefore, studies of complete genome sequences could provide useful information about the position and characteristics of potential virulence determinants. in this study, the genome sequences of the strains vics-v and vics-del were completed. the a and ab polyproteins of ibv are post-translationally cleaved by virus-encoded proteinases into peptides (nsp to nsp ). nsp , which is found in all other coronaviruses, is absent in ibv, but nsp is considerably larger (liu et al., ; snijder et al., ; stadler et al., ; gorbalenya et al., ; ziebuhr, ; fang et al., ; sawicki et al., ; carbajo-lozoya et al., ) . in order to determine the location of the peptides encoded by the orfs in the a and ab polyproteins, we used the annotations of the beaudette strain (gan nc_ ). we complemented these annotations with those of ziebuhr ( ) . the locations of these peptides within orfs a and ab and their first and last amino acid residues are summarized in table . as discussed above, vics-v exhibits higher rates of replication in vitro and causes more severe clinical signs and tracheal lesions in vivo than vics-del. as the genomes of these two viruses have very high levels of nucleotide sequence identity, it can be inferred that any differences in their genomic sequences may influence these differences in virulence and replication. as described previously, gly-gly is the cleavage motif for plp (lim et al., ; ziebuhr, ) . vics-del lacks three gly-gly motifs seen in vics-v that are located in the middle of nsp , nsp and nsp . these motifs may result in additional proteolytic processing of these three nsps, and the absence of them in vics-del could alter their function. nsp and nsp are reported to contain transmembrane (tm) and exonuclease domains (ziebuhr, ) , while the function of nsp remains undetermined. highly conserved gly-gly motifs were also found between nucleotides and (within nsp ), and , and (within nsp ), and , and and (within nsp ) in both vics-v and vics-del and in other ibvs. these motifs may also be further processed by plp, although the previously characterized plp cleavage sites (between nsp and , and nsp and ) have an extended motif of lys-ala-gly-gly, which has been identified in all the complete genome sequences analysed in this study; this extended motif is not seen at these additional gly-gly sites. positions were based on ab from ibv beaudette strain (accession number nc_ ), and modified according to ziebuhr ( ) , with position the methionine at the beginning of orf a and ab. b based on phillips et al. ( ) . the mutations in nsp of vics-del (table ) may have implications for the replication of this virus, which would be consistent with data from previous in vivo studies (hewson et al., ) . during infection cl, the main protease, is auto-catalytically released (ziebuhr et al., ; ziebuhr, ) . there may be an interaction between the cl (nsp ), the tm (nsp ) and the tm (nsp ) hydrophobic domains (ziebuhr, ) , which are also referred to as mp and mp (tibbles et al., ) , during this proteolytic process. the importance of tm /mp , the transmembrane domain downstream of the cl proteinase, in the autocatalytic processing of cl has been suggested by in vitro assays (tibbles et al., ) . in this experiment, the further processing of a -kda immature protein product into smaller, post-translationally cleaved products of , , and kda (with the -kda peptide the size predicted for the fully processed form of cl protease) did not occur if these two hydrophobic domains were not associated with microsomal membranes. changes in tm / mp , such as those detected in vics-del, could affect the membrane association of this domain and thus the conformation of the protein, and the autocatalytic release of cl, which is essential for the initial stages of replication (ziebuhr, ) . the snp detected at the end of the s gene is also notable. the difference in the proportions of each of the nonsynonymous codons between the vics-v and vics-del populations, with a greater prevalence of the truncation phenotype in the vics-del population, suggests that truncation of the s protein may be related to the difference in virulence between these strains. the carboxyl terminal end of s anchors the s protein to the virion and also assists in membrane fusion with target cells (cavanagh, ) . alternatively, the differing b-trs sequences, cugaa-caa and cuuaacaa, which have both been identified previously in ibv (mondal & cardona, ; woo et al., ) , may differ in their efficiency in regulating replication and transcription and thus may influence production of mrna , which encodes the e protein. in the middle east respiratory syndrome coronavirus the deletion of the e protein results in a replication-competent, propagationdefective virus (almazán et al., ) . a nine amino acid truncation, resulting from the nucleotide change from u to g, has been described previously in an m strain variant, m h (kusters et al., ) . while these alternative codons have also been found in other ibv strains, the methods used for sequence determination would have precluded identification of differing subpopulations within these strains, as cloned, single cdna molecules were used for sequencing. the orfs b and c have been described in ibv (hewson et al., (hewson et al., , thor et al., ; phillips et al., ; bentley et al., ) , as well as in tcov (cao et al., ) , and have been synthesized in vitro (bentley et al., ) , but the products of these orfs are yet to be identified in infected cells, and their function remains unknown. both these orfs could be identified in the vics-v genome, but in vics-del, b was divided into two smaller orfs due to a singlenucleotide deletion. the vics vaccine is known to induce some clinical signs after vaccination. hewson et al. ( ) showed that vics-v caused more severe vaccine reactions than vics-del, had a higher replication rate in the trachea, kidneys and caecal tonsils, and induced more severe clinical signs. the putative b and c proteins have two interesting features: their high content of positively charged amino acids (lys and arg) and the presence of phosphorylation motifs, suggesting a possible role in the packaging of the genome, which may influence the rate of viral replication and therefore pathogenicity. phylogenetic analyses showed that the australian strains of ibv are distinct from strains isolated elsewhere in the world and that the most commonly used australian vaccine strain is very distinct from other ibv vaccine strains across the entire genome. this divergence has been shown previously in the structural genes (yu et al., ; bochkov et al., ) , but this is the first study to show that the divergence can be seen consistently throughout the genome. however, most of the fully sequenced ibv genomes are of strains with an origin in the usa or china, as only a few qx-type and qx-like strains (which now predominate in europe) have been fully sequenced, and this bias could have influenced the topology of the tree. these european strains were represented in this tree by strains from italy (ita/ / ) and sweden (ck/ swe/ / ). the phylogenetic tree constructed with the nucleotide sequences of the s protein ( figure ) includes more qx-type and qx-like strains, including ck/ uk/kg p/ and ck/uk/ / (uk), ck/ger/gb / (germany), ck/swe/ / and ck/swe/ / (sweden), ita/ / (italy) and ck/sp/ / and la/sp/ / (spain). phylogenetic trees of individual orfs (figures , and ) indicate that different regions of the vics-v genome varied in their similarity to the two major clusters, suggesting historical recombination events. however, this seems very unlikely to have happened recently, based on the results from the network tree analysis ( figure ). vics-v diverged and evolved as a distinct cluster, with the length of the vics-v branches, especially in the cl, hel and rdrp phylogenetic trees, suggesting more rapid evolution than most other strains. in conclusion, full genome analysis of vics-v and vicsdel has shown that the australian ibv vaccine vics has been evolving independently of strains elsewhere in the world for some time. the differences in the replication rate and pathogenicity of these two viral subpopulations may be attributable to the mutation in the tm domain in nsp and the snp found at the end of the s glycoprotein gene, described in this study for the first time. these newly described differences are additional to the previously described frameshift/truncation of the orf b and the nucleotide deletion in the ′ utr in vics-del (hewson et al., ) . further analyses of the complete genome sequences of other australian ibv strains may reveal additional information about relationships between these distinct vaccine strains and ibvs within the usa and chinese clusters. 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oligosaccharides of the avian infectious bronchitis virus glycoproteins a massachusetts prototype like coronavirus isolated from wild peafowls is pathogenic to chickens mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods the nucleic acid of infectious bronchitis virus recombination in avian gamma-coronavirus infectious bronchitis virus characterization in vitro of an autocatalytic processing activity associated with the predicted c-like proteinase domain of the coronavirus avian infectious bronchitis virus australian infectious bronchitis viruses: identification of nine subtypes by a neutralisation test the ribonucleic acid of infectious bronchitis virus coronavirus genomics and bioinformatics analysis. viruses characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens the coronavirus replicase the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond key: cord- -p a rboi authors: bibby, kyle; fischer, robert j.; casson, leonard w.; stachler, elyse; haas, charles n.; munster, vincent j. title: persistence of ebola virus in sterilized wastewater date: - - journal: environ sci technol lett doi: . /acs.estlett. b sha: doc_id: cord_uid: p a rboi [image: see text] in the wake of the ongoing / ebola virus outbreak, significant questions regarding the appropriate handling of ebola virus-contaminated liquid waste remain, including the persistence of ebola virus in wastewater. to address these uncertainties, we evaluated the persistence of ebola virus spiked in sterilized domestic sewage. the viral titer decreased approximately % within the first test day from an initial viral titer of ( ) tcid( ) ml(– ); however, it could not be determined if this initial rapid decrease was due to aggregation or inactivation of the viral particles. the subsequent viral titer decrease was less rapid, and infectious ebola virus particles persisted for all days of the test. the inactivation constant (k) was determined to be − . ( . days for a % viral titer decrease). due to experimental conditions, we believe these results to be an upper bound for ebola virus persistence in wastewater. wastewater composition is inherently heterogeneous; subsequently, we caution that interpretation of these results should be made within a holistic assessment, including the effects of wastewater composition, dilution, and potential exposure routes within wastewater infrastructure. while it remains unknown if ebola virus may be transmitted via wastewater, these data demonstrate a potential exposure route to infectious ebola virus via wastewater and emphasize the value of a precautionary approach to wastewater handling in an epidemic response. in march , an unprecedented outbreak of ebola virus disease (evd) began in western africa that as of july is ongoing and has claimed more than lives. the current outbreak is caused by viruses belonging to the species of the zaire ebolavirus, a member of the filoviridae family. , filoviridae are enveloped, filamentous viruses with lengths that may reach > nm. , the current outbreak has a reported case fatality rate of %. ebola virus can be excreted in bodily fluids, including vomit, stool, blood, saliva, semen, and breast milk. − ebola virus loads of up to genome copies ml − have been reported in blood, genome copies ml − in stool, and . genome copies ml − in urine; however, the conversion between genome copies and infectious units is unknown. the median infectious dose is believed to be < infectious viral particles. once infected, individuals may produce up to l of liquid waste per day, primarily watery diarrhea. ebola virus is considered a potential bioterrorism agent. , in response to the evd epidemic, both the world health organization (who) and the u.s. centers for disease control and prevention advised direct disposal of ebola-contaminated liquid waste into sewage systems (wastewater collection and treatment systems) and latrines without disinfection. initial recommendations were made on the basis of an expected limited persistence of ebola virus in the environment, as ebola virus is an enveloped virus, and a lack of strong evidence for a waterborne transmission route. as stated by a who guidance document, "ebola virus is likely to inactivate significantly faster in the environment than enteric viruses with known waterborne transmission (e.g., norovirus, hepatitis a virus)". however, as has been noted in a recent review, the persistence of enveloped viruses in the water environment varies by > orders of magnitude. recommendations for ebola virus-contaminated wastewater disposal were met with debate (e.g., refs − ) because of uncertainty about ebola virus persistence within wastewater matrices and the lack of a risk-based analysis for waste handling. wastewater handling recommendations have since been revised to recognize uncertainty in this area and to recommend disinfection of latrines and holding of wastewater prior to handling to allow ebola virus inactivation. − additionally, some facilities have opted to provide additional disinfection prior to disposal of liquid waste into sewer systems. recent research found both ethanol and hypochlorite to be effective disinfectants for ebola virus dried on surfaces; however, the disinfection kinetics of ebola virus within liquid matrices remains unknown. various wastewater disinfection approaches have been recently suggested for pathogen control in an outbreak setting. currently, no data on ebola virus persistence in wastewater exist, hindering risk estimation and examination of potential environmental exposure routes. the necessity of evaluating ebola virus persistence in wastewater matrices has previously been highlighted, as wastewater in ebola virus outbreak settings may be temporarily held in open containers or disposed of in open sewers. historically, the transmission of ebola virus via environmental routes (droplets, aerosols, or fomites) has been thought to be unlikely due to epidemiological evidence and environmental sampling. the primary ebola virus transmission route is via direct contact with bodily fluids. transmission has previously occurred without known direct contact with infected individuals, providing supporting evidence that ebola virus transmission may be possible via large droplets. the potential for transmission of ebola virus via wastewater is currently unknown. to address uncertainties regarding ebola virus persistence in wastewater, we have conducted an initial evaluation of ebola virus persistence within wastewater to address uncertainty and inform ongoing risk assessments. a current ebola virus outbreak strain from guinea (makona-wpgc ) was spiked to two end concentrations ( and tcid ml − ) into a domestic wastewater (untreated sewage) sample. the ebola virus-containing wastewater was sampled for days, and the viability of ebola virus was determined in these samples. study results are presented, and study limitations and implications are discussed, as well as recommendations for an ongoing research agenda. untreated wastewater was collected from an anonymous regional wastewater treatment facility in western pennsylvania that receives wastewater from seven communities (combined population of approximately ). the total raw wastewater flow to the treatment facility is < mgd (million gallons per day). approximately % of the raw wastewater originates from industrial sources. following collection, the wastewater was frozen at − °c to minimize compositional changes prior to analysis. wastewater characteristics (table ) were determined at an epa-certified analysis facility (microbac laboratories, marietta, oh). the region from which the sample was collected uses a combined sewer system that experiences significant infiltration, and the determined composition is typical for the region. ebola virus cultivation experiments were conducted at rocky mountain laboratories under bsl conditions. wastewater samples were shipped to rocky mountain laboratories overnight on ice. upon receipt, samples were sterilized with mrad of γ-irradiation. sterilization was performed to limit cell culture death due to wastewater microbial activity leading to a false positive. stock virus (ebola virus guinea makona-wpgc , . tcid ml − ) was handled as described previously. ebola virus was diluted in wastewater to achieve two separate virus titers ( and tcid ml − ), and both experiments were completed in triplicate. the ebola virus concentration in sewage has not been previously measured or estimated; thus, two separate concentrations were utilized to cover possible concentration scenarios. tcid is an end point dilution series that is used to determine at what dilution % of the infected wells produce cell death. the original infectious titer can be calculated utilizing the spearman−karber method. an approximation of focus-forming units (ffus) can be made from a poisson distribution utilizing the formula tcid × . , assuming each ffu is formed from a single virus. spiked wastewater was then distributed into three labeled vials for each concentration, and samples were taken daily for days. tests were conducted at °c and % relative humidity. next, μl from each well of the dilution plate was transferred to a -well cell culture plate seeded with vero cells. the cells were incubated with virus dilutions for h; then the medium was removed from the two highest concentrations and rinsed two times with pbs, and μl of fresh culture medium was added. fresh culture medium ( μl) was also added to the remaining wells in the plate. the plates were incubated at °c for days, inspected for the cytopathic effect (cpe), and scored. statistical analyses and graphing were completed with microsoft excel . the natural logarithm of c/c o tcid was plotted and fit with a linear trendline for estimation of the inactivation constant (k). a literature review was then performed to compare observed inactivation to other environmental matrices. ■ results and discussion ebola virus persistence in wastewater. ebola virus was spiked into wastewater at two concentrations and assayed for days to determine persistence in a wastewater matrix. the time zero time point was measured immediately following addition of ebola virus to the wastewater. no viable ebola virus was recovered from samples spiked with ebola virus tcid ml − after the initial time zero sampling. the limit of detection was . log tcid ml − . virus viabilities from the initial ebola virus tcid ml − concentration are shown in figure and detailed in with an inability to identify infectious ebola virus from the initial sample with ebola virus tcid ml − on day . there was a rapid decrease (approximately %) in ebola virus titer within the first day of the test. in addition to inactivation, viral particle aggregation or adsorption to wastewater particles may play a role in the apparent rapid viral decrease and enhanced viral persistence. in this case, aggregation would have two primary effects. first, aggregated particles would not be detected as multiple infectious units, resulting in an apparent rapid decrease. second, it has been previously recognized that aggregation increases viral persistence and resistance to inactivation stressors. , organic matter in wastewater has previously been suggested to enhance viral aggregation. similarly, viral association with particles has been recognized to provide protection from inactivation, including association of viral particles with wastewater solids. mechanistically, particle association is believed to protect viral particles from inactivation by shielding them from environmental stressors and is dependent on the organism and particle type. utilizing the current assay, it cannot be determined if the initial rapid decrease in viral titer was due to viral inactivation or aggregation. aggregation would result in an apparent viral titer decrease as each viral aggregate would function as an infectious unit in the cell culture assay. to address this uncertainty, we plotted two inactivation curves, both including ( figure a ) and excluding the measured time zero point ( figure b) . a linear trendline, as has been previously suggested for viral and ebola virus persistence, showed a lower fit (r = . including the time zero time point, and r = . excluding the time zero time point) than that previously observed for ebola virus in deionized water (r > . ), but a fit better than that previously observed for ebola virus in human blood (r < . ). the inactivation constant (k) was determined to be − . when including the time zero time point and − . when excluding the time zero time point. on the basis of the model fit, the t (time for % inactivation) would be . days including the time zero time point and . days excluding the time zero time point. the observed ebola virus inactivation in wastewater was slower than that observed for deionized water, which required . days for % inactivation at °c. the observed ebola virus inactivation in wastewater was more rapid than that reported for human blood, which required days for a % inactivation, and results are consistent with recent studies that identified viable ebola virus to persist in infected macaque blood for > days. in general, ebola virus was found to be less persistent in wastewater than model enteric viruses. while the t for ebola virus in wastewater was found to be < day, the t for hepatitis a is greater than days and the t for enteric adenovirus is days; however, the t for poliovirus is days, which is between the observed t values including or excluding the time zero time point. the results demonstrate a more rapid initial viral titer decrease but overall enhanced persistence of ebola virus in wastewater compared to the proposed enveloped surrogate bacteriophage phi . limitations. this study has two primary limitations that may alter the persistence of ebola virus compared to what may be observed in the field. first, the tested wastewater was more dilute than would be expected in typical latrine waste. , in general, interaction with constituents within the wastewater (e.g., ammonia) would be expected to contribute to more rapid inactivation of viruses; however, the true effect of these constituents on ebola virus persistence is unknown. second, the wastewater was frozen to minimize compositional changes and disinfected (γ-irradiation) prior to utilization to limit microbial activity resulting in false positive viral cell culture. microbial activity within wastewater matrices would be expected to contribute to more rapid inactivation of infectious viral particles; , however, the true effect of microbial activity on ebola virus persistence is unknown. microbial activity reduces viral persistence through both the production of metabolites detrimental to viral persistence and direct usage of the viral particles as a nutrient source. additionally, the influence of other environmental characteristics (e.g., temperature, ph, and mixing) on ebola virus persistence is unknown and may contribute to altered environmental behavior. as such, we believe these results to be an upper bound for ebola virus persistence in wastewater matrices. subsequently, we caution extrapolation of these results without a holistic assessment of all factors, including wastewater composition, dilution, and potential exposure routes. implications. the results of this study suggest a potential exposure route to infectious ebola virus via wastewater; however, any assessment of potential exposure routes must consider the effects of wastewater composition, dilution of contaminated wastewater, and inactivation of ebola virus during treatment and holding. additionally, the possibility for ebola virus transmission via wastewater and subsequent infection remains unknown. the who updated guidelines in january to recommend holding of latrine waste for week prior to further handling or transport. the objective of this holding period is to allow ebola virus die-off. on the basis of these results, it would be reasonable to approximate a three-log (i.e., . %) removal of ebola virus due to this holding period. the resulting risk would ultimately depend upon the initial ebola virus concentration in wastewater, potential for exposure, and susceptibility to infection via wastewater exposure. the wastewater travel times for wastewater via a sewer system to a centralized sewage treatment works would typically be < day, depending on system dynamics. further assessment is necessary to determine ebola inactivation and dilution within this period and potential human exposure routes, including workers within the sewer system and ebola virus persistence within wastewater sludges. the greatest exposure risk would be expected for persons in contact with contaminated wastewater prior to significant dilution, treatment, or holding. these results demonstrate a persistence of ebola virus in wastewater greater than what has previously been suggested and the potential of a wastewater exposure route to infectious ebola virus. while ebola virus was found to be generally less persistent than enteric viruses in wastewater, the identified survival period suggests value in a nuanced evaluation of wastewater exposure risks during an epidemic response. specifically, these findings highlight the value of a precautionary approach to wastewater handling within an outbreak scenario, in response to both ebola virus and other emerging viruses. the supporting information is available free of charge on the acs publications website at doi: . /acs.estlett. b . recorded tcid values and standard deviations of ebola virus spiked into sterilized domestic wastewater ( cdc ebola outbreak in west africa the natural history of ebola virus in africa. microbes infect ebola virus disease in west africa  the first months of the epidemic and forward projections ebola haemorrhagic fever characteristics of 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sana'a, yemen deployment of the microbial fuel cell latrine in ghana for decentralized sanitation virus survival in the environment with special attention to survival in sewage droplets and other environmental media of fecal or respiratory origin the authors declare no competing financial interest. this study was supported by the division of intramural research, national institute of allergy and infectious diseases, national institutes of health, and national science foundation grants (k.b. and l.w.c.) and (c.n.h.). we acknowledge the anonymous wastewater sampling site for assistance. key: cord- - bx ao authors: wu, andong; wang, yi; zeng, cong; huang, xingyu; xu, shan; su, ceyang; wang, min; chen, yu; guo, deyin title: prediction and biochemical analysis of putative cleavage sites of the c-like protease of middle east respiratory syndrome coronavirus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: bx ao coronavirus c-like protease ( clpro) is responsible for the cleavage of coronaviral polyprotein a/ ab (pp a/ ab) to produce the mature non-structural proteins (nsps) of nsp – . the nsp of the newly emerging middle east respiratory syndrome coronavirus (mers-cov) was identified as clpro and its canonical cleavage sites (between nsps) were predicted based on sequence alignment, but the cleavability of these cleavage sites remains to be experimentally confirmed and putative non-canonical cleavage sites (inside one nsp) within the pp a/ ab awaits further analysis. here, we proposed a method for predicting coronaviral clpro cleavage sites which balances the prediction accuracy and false positive outcomes. by applying this method to mers-cov, the canonical cleavage sites were readily identified and verified by the biochemical assays. the michaelis constant of the canonical cleavage sites of mers-cov showed that the substrate specificity of mers-cov clpro is relatively conserved. interestingly, nine putative non-canonical cleavage sites were predicted and three of them could be cleaved by mers-cov nsp . these results pave the way for identification and functional characterization of new nsp products of coronaviruses. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus carrying a genome of positive-sense rna (+ssrna). it was identified as the pathogen of a new viral respiratory disease outbreak in saudi arabia in june , named as middle east respiratory syndrome (mers). mers-cov emerged ten years after severe acute respiratory syndrome coronavirus (sars-cov) and quickly spread to several countries in middle east and europe (assiri et al., ; tashani et al., ) . soon after the first report, the mers-cov genome was sequenced and its genomic organization has been elucidated . this new coronavirus is classified in the lineage c of beta coronavirus, and is close to bat coronavirus hku and hku (de groot et al., ; lau et al., ) . like other coronaviruses (hussain et al., ; zuniga et al., ) , mers-cov contains a coterminal, nested set of seven subgenomic rnas (sgrnas), enabling translation of at least nine open reading frames (orfs). the -terminal two thirds of mers-cov genome contains a large open reading frame orf ab, which encodes polyprotein a (pp a, amino acids) and polyprotein ab (pp ab, amino acids), the latter being translated via a − ribosomal frameshifting at the end of orf a. these two polyproteins were predicted to be subsequently processed into non-structural proteins (nsps) by nsp , a papain-like protease (plpro), and nsp , a c-like protease ( clpro) (kilianski et al., ; van boheemen et al., ) . protease plays a key role during virus life cycle. it is essential for viral replication by mediating the maturation of viral replicases and thus becomes the target of potential antiviral drugs (thiel et al., ; ziebuhr et al., ) . investigating the cleavage sites of coronavirus proteases and the processing of polyproteins pp a/ ab will benefit to identify the viral proteins and their potential function for viral replication. some cleavage sites have been identified and confirmed by previous studies, including three cleavage sites of plpros of human coronavirus e (hcov e), mouse hepatitis virus (mhv), sars-cov, mers-cov and infectious bronchitis virus (ibv), whose cleavages release the first non-structural proteins (bonilla et al., ; kilianski et al., ; lim and liu, ; ziebuhr et al., ) . the canonical cleavage sites of clpros, the sites between the recognized nsps, have also been characterized, including all sites of mhv, ibv, sars-cov and a fraction of sites of hcov e which release the non-structural proteins from nsp to nsp (deming et al., ; grotzinger et al., ; liu et al., liu et al., , lu et al., ) . for clpro of mers-cov, two cleavage sites releasing nsp to nsp have been identified (kilianski et al., ) . however, other cleavage sites remain to be characterized. furthermore, efforts have been taken to predict these cleavages sites by sequence comparison. gorbalenya et al. ( ) made the first systematical prediction on ibv pp a/ ab according to the substrate specificity of c protease of picornaviruses. however, two of their predicted cleavage sites within nsp of ibv were proved uncleavable (liu et al., ; ng and liu, ) . gao et al. ( ) developed a software (zcurve cov) to predict the nsps as well as gene-encoded orfs of coronaviruses more accurately based on previous studies of clpros cleavage sites of ibv, mhv and hcov e. later on, non-orthogonal decision trees were used to mine the coronavirus protease cleavage data and to improve the sensitivity and accuracy of prediction (yang, ) . however, while these methods focus on the prediction of the canonical cleavage sites and target more and more on prediction accuracy to avoid false positives, potential non-canonical cleavage sites might be neglected. for example, a cleavage site between nsp and of mhv strain a is not predicted by above methods, but proved to be physiologically important since it produces a shorter nsp that can support the growth of mhv carrying a mutation on nsp - cleavage site (deming et al., ) . therefore, the substrate specificities of coronaviruses clpros are complicated. a clpro substrate library of four coronaviruses (hcov-nl , hcov-oc , sars-cov and ibv) containing amino acids × positions variants was constructed by making single amino acid (aa) substitution at each position from p to p , and their cleavage efficiencies were measured and analyzed to find out the most preferred residues at each position (chuck et al., ) . however, the non-canonical cleavage site with less preferred residues of clpro is adopted by coronaviruses (deming et al., ) . thus we speculate that other potential clpro cleavage sites may still exist in coronaviruses. in order to set up a more moderate and balanced criteria for protease cleavage site identification, we compared six scanning conditions with different stringency to systematically predict the clpro cleavage sites on pp a/ ab of five coronaviruses including mers-cov. as a representative, the cleavability of the predicted cleavage sites of mers-cov clpro was analyzed by the recombinant luciferase cleavage assay and the fluorescence resonance energy transfer (fret) assay. the results showed that all canonical cleavage sites of mers-cov pp a/ ab were cleavable in our experiments and three of nine predicted non-canonical cleavage sites appeared to be cleavable. our study points out a new direction regarding the prediction and identification of cleavage sites of proteases and contributes to understanding the mechanism of coronaviral polyprotein processing. the genome sequences of coronaviruses were downloaded from genebank database and the sequences of the clpro cleavage sites were collected from p to p (tables s -s ). the substrate profiles of each coronavirus group and the whole coronavirinae were summarized (table s ) . the coding sequence of mers-cov nsp (nc ) was synthesized chemically by genscript and cloned into vectors pet a and pgex- p- , respectively. the catalytic residue mutation c a was generated by over lapping pcr with mutagenic primers (table s ). all the clones and mutations were confirmed by dna sequencing. the expression vectors were transformed into escherichia coli strain bl (de ). the cells were grown at • c in lysogeny broth (lb) medium with antibiotics and induced with . mm isopropylbd-thiogalactopyranoside (iptg) at • c for h. the cells were harvested and resuspended in lysis buffer ( mm tris-hcl, ph . , mm nacl, mm edta, . % np , . mg/ml lysozyme and mm pmsf) at • c. after incubation for min on ice, mm mgcl and g/ml dnase i (sigma) were added to digest the genomic dna. the supernatant of cell lysate was applied to affinity chromatography column after centrifugation. the recombinant protein with his-tag was bound with nickel-nitrilotriacetic acid (ni-nta) resin (genscript) and washed with buffer a ( mm tris-hcl, ph . , mm nacl), buffer b ( mm tris-hcl, ph . , mm nacl, mm imidazole) and buffer c ( mm tris, ph . , mm nacl, mm imidazole). proteins were eluted with buffer d ( mm tris, ph . , mm nacl, mm imidazole). gst-tagged protein was bound with gst resin (genscript), washed with buffer a and eluted with buffer a supplemented with mm reduced glutathione (gsh). the purified proteins were desalted and concentrated by ultrafiltration using kda amicon ultra . -ml centrifugal filter (millipore). all the cleavage sites (eight residues, ranging from p to p ) were inserted into glo-sensor f linear vector. comparing to the wild type firefly luciferase ( aa), glo-sensor luciferase has short truncations at both termini with c-and n-part reversed, resulting in the new -aa n-and -aa c-terminal region respectively. the inserted sequence and the reversed arrangement of the nand c-terminal regions reduce the luciferase activity dramatically. after the recognition sequence was cut off by nsp , the luciferase recover its activity and luminescence in the presence of luciferase substrate. a back to front recombinant firefly luciferase inserted with different cleavage sites was expressed when the recombinant plasmids were co-incubated with a cell-free protein expression system extracted from wheat germ (promega). after incubation for h at • c, nsp was added into the system and the whole system was incubated at • c for h. then, the reaction system was diluted times and mixed thoroughly with equal volume of luciferase substrate. luciferase luminescence was measured by a luminometer (promega) after incubation for min at room temperature. all the conserved putative recognition sites were designed from p to p , synthesized and modified with a typical shorter wavelength fret pair, n-terminal dabcyl and c-terminal glu-edans by gl biochem (shanghai). the peptides were completely dissolved in dmso and the final concentration of dmso in the reaction system was %. m substrate peptide and . m tagged nsp were mixed in the solution of mm tris, ph . , mm edta, m dtt and incubated at • c for h. to calculate kcat/km, different amounts ( . - m) of substrate peptides were co-incubated with . m nsp . the reaction system was placed in giernor black plate and the fluorescence was detected by a microplate reader (molecular devices) with ex/em (nm/nm) = / . relative fluorescence unit (rfu) was collected every s for h. the initial slope (slope a = rfu/min) was generated from the linear interval of the rising stage. then, a linear equation was generated using the rfu at plateau (rfu max ) vs. the concentration of substrate. the slope (slope b = rfu/[s]) indicates the rfu change at per unit change of [s] . the initial reaction velocity (v = [s]/min) was calculated through dividing slope a by slope b. the michaelis-menten kinetic constants were generated by lineweaver-burk plot. the coronavirus clpros and their cleavage sites are evolutionarily conserved among different genera. to study the genetic diversity and evolution of clpro cleavage sites of coronaviruses pp a/ ab, primary sequences of clpro cleavage sites (ranging from p to p ) of species of coronaviruses were collected and listed in tables s -s , including the predicted and verified cleavage sites. canonical cleavage sites of each coronavirus were joined end to end to produce a spliced sequence which was then used to produce a phylogenetic tree (fig. a ). in addition, the sequences of all coronavirus clpro were used to generate another phylogenetic tree (fig. b) . the analyses showed that the phylogenetic distances and taxonomic positions of each virus, in both phylogenetic trees, were mostly consistent with that classified by the international committee on taxonomy of viruses (ictv) (http://www.ictvonline. org/virustaxonomy.asp). these results implied that the cleavage sites of coronaviral clpros might co-evolve with clpros, and the genetic diversity of both clpro and its cleavage sites are relatively conserved between different genera of coronaviruses. however, on the phylogenetic tree generated with clpro cleavage sites (fig. a) , the members of the genus gammacoronavirus, although clustered closely, is split into alphacoronaviruses and deltacoronaviruses, suggesting that the cleavage sites of gammacoronaviruses may have undergone recombination events during evolution. in order to develop an optimized method for cleavage site prediction that can cover all possible cleavage sites with fewer false positives, we have set three levels of criteria (stringent, moderate and mild) for cleavage site prediction. in the stringent rules, clpro cleavage sites only comprise the most preferred residues at each position based on previous description (chuck et al., ) . in moderate rules, clpro cleavage sites comprise residues which ever appeared in the cleavage sequences of congeneric coronaviruses at each particular position. as for mild rules, the cleavage sites could comprise any residues ever found in the cleavage sequences of all coronaviruses at each particular position. because the substrate preference at p and p is not strong, we decided to adopt two different lengths of cleavage sequences for prediction, one containing six residues from position p to p , and the other containing four residues from position p to p . these two lengths of cleavage sequences, combining with the three different criteria, made up a total of six search conditions for cleavage site predication with decreasing degree of stringency. the canonical cleavage sites of clpro for these seven groups of coronaviruses were summarized in tables s -s and used to set conditions iii to vi. possible residues at each particular position of clpro cleavage sites were predicted based on all six conditions to make the cleavage site profile of coronaviruses clpro (table s ). in principle, when condition i was employed, the least number of possible cleavage sites were identified in a scanned sequence, while condition vi predicted the largest number of possible cleavage sites in a scanned sequence. to the applicability, we applied all the six conditions on five representative coronaviruses, including hcov e from alphacoronavirus, mhv from betacoronavirus lineage a, sars-cov from beta coronavirus lineage b, mers-cov from betacoronavirus lineage c and ibv from gammacoronavirus. all possible cleavage sites predicted based on each condition were scanned on pp a/ ab of five representative coronaviruses and the results were summarized in table . as shown in table , increasing numbers of cleavages sites were found for each coronavirus when conditions from i to vi were applied. the results showed that condition i and ii were too strict to cover all canonical cleavages sites; condition v and vi were too loose so as to produce two to three times more than cleavages sites; condition iii could only cover the canonical cleavage sites for sars cov; only condition iv generates an appropriate number of cleavage sites for all five coronavirus. therefore, search condition iv was chosen for further analysis of the cleavage sites of mers-cov. by applying the search condition iv, putative cleavage sites (pss) as well as canonical cleavage sites (css) were predicted (table ) . although the canonical cleavage sites of mers-cov clpro have been predicted by sequence alignment with other coronavirus , our results suggested that the additional cleavage might occur in the process of mers-cov pp a/ ab processing. to verify the activity of mers-cov clpro and cleavability of the predicted cleavage sites, the biochemical assay systems of mers-cov clpro were established. as shown in fig. a and b, we first expressed and purified mers-cov clpro (nsp ) with different tags and mutation: n-terminally gst-tagged nsp (gnsp , . kda), n-terminally his-tagged ( extra amino acids with × his tag and linker provided by vector pet- a) nsp (hnsp , . kda), hnsp with catalytic residue mutation c a (hnsp m, . kda) (kilianski et al., ) and gst tag-gvlq-nsp with c a mutation and × his tag (gnsp mh, . kda), in which the sequence motif gvlq represents the last four residues of mers-cov nsp , mimicking the cleavage site of mers-cov nsp /nsp . in the biochemical assays, the gnsp mh with catalytic residue mutation c a could not undergo self-cleavage at the cleavage site to release gst in incubation for h (fig. c) , indicating that the clpro activity of mers-cov nsp in gnsp mh was inactivated by the mutation c a. thus, gnsp mh was used as protease substrate in the following biochemical assays. to verify the clpro activity of recombinant nsp s, gnsp and hnsp were incubated with substrate gnsp mh for min to h and analyzed by sds-page (fig. d) and western blotting, respectively (fig. e) . both gnsp and hnsp showed the proteolysis activity to cleave the substrate gnsp mh into two parts: gst ( . kda) and nsp mh ( . kda), which were confirmed by the correlation of their molecular weight (fig. d and e) . however, the clpro activity of gnsp was obviously weaker than that of hnsp , which could entirely cleave the substrate gnsp mh h post treatment ( fig. d and e) . these results could be explained by that the larger fusion tag at the n terminus of mers-cov clpro significantly reduced the proteolysis activity of clpro, which was consistent with the previous observation (xue et al., ) . in the biochemical assays, the the tree was generated by the sequence of nsp and the method is the same as described above. the number of cleavage sites in pp ab of representative coronaviruses predicted by using search conditions. condition iii condition iv condition v condition vi a canonical cleavage sites, which are located between recognized nsps. b putative cleavage sites, which are located inside various nsps. c six search conditions are designed: conditions i, iii and v cover six residues from p to p ; conditions ii, iv and vi cover four residues from p to p . conditions i and ii are set to comprise the most preferred residues at each position; conditions iii and iv comprise residues appeared in the cleavage sites of congeneric coronaviruses; conditions v and vi comprise residues appeared in the cleavage sequences of any coronaviruses. relatively lower proteolysis activity of clpro will benefit to observe the influence of different substrates. therefore, both recombinant gnsp and hnsp were used as mers-cov clpro in the following studies. to rapidly evaluate the proteolysis activity of mers-cov clpro toward the predicted cleavage sites of different substrates, a sensitive luciferase-based biosensor assay was adopted. as shown in fig. a , the canonical cleavage sites (cs) of mers-cov nsp /nsp (cs / ) and nsp /nsp (cs / ), which were experimentally confirmed in a previous study (kilianski et al., ) , were inserted into the inverted and circularly permuted luciferase construct pglo- f, in which the n-terminal and c-terminal halves of luciferase gene are separated. the resulting luciferase in translation system in vitro was inactive and could convert into an active luciferase when cleaved by recombinant viral protease at the engineered cleavage sites (such as cs / and cs / ). in this system, the luciferase signals were detected when incubated with both gnsp and hnsp , respectively (fig. b) . in contrast, the mutated nsp (hnsp m) could not convert the inactive luciferase into active form (fig. b ). this result indicated that the luciferase-based biosensor assay could be used to evaluate the proteolysis activity of mers-cov clpro. then, the other nine canonical cleavage sites and nine putative cleavage sites composed with aa from mers-cov pp a/ ab were inserted into the luciferase construct pglo- f, and the luciferase-based biosensor assays were performed using hnsp and hnsp m, respectively. as shown in fig. c , all the canonical cleavage sites of mers-cov clpro generated luciferase signal by hnsp at least . times higher than by the inactive hnsp m, indicating that all these canonical sites could be cleaved by mers-cov clpro. these results experimentally verified the existence of the predicted canonical cleavage sites. interestingly, among the nine putative cleavage sites, the luciferase signals of ps - , ps - and ps - remarkably increased more than folds when incubated with hnsp , indicating that the putative cleavage sites, located inside nsp and nsp of mers-cov respectively, might be cleavable (fig. d) . the other predicted putative sites (ps - , ps - , ps - , ps - , ps - , and ps - ) showed less than . folds increase of luciferase signal when they were treated by hnsp comparing with those treated by hnsp m (fig. c and d) . due to high sensitivity of the luciferase-based biosensor assay and the fact that the confirmed verification of the recombinant luciferase assays. inactive luciferase was synthesized in the cell-free translation system and the reaction mixture incubated at • c for h. after that, the protein mixture was divided into four parts and incubated with . m gnsp , hnsp , hnsp m or h o, respectively. after incubation for h at • c, the reaction product was diluted times and mixed with equal amount of luciferase substrate. after incubation at room temperature for min, the luciferase luminescence was measured. luciferase activation fold was calculated through dividing the signal value of the reaction system treated with active hnsp by the one treated with the inactive nsp mutant hnsp m. (c) the luciferase cleavage assay of predicted canonical cleavage sites and (d) putative cleavage sites. the luciferase expression vector inserted with cleavage sites were added to the wheat germ protein translation mix and incubated at • c for h, and the reaction mixture was divided and treated with hnsp and hnsp m, respectively. the dashed line indicates the lowest fold increase of luciferase signal by cleavage of previously confirmed clpro cleavage sites. the data presented here are the mean values ± sd derived from three independent experiments. canonical cleavage sites generated at least . times increase of luciferase signal, the cleavage signal of these six sites may represent the background level, indicating that they are likely uncleavable per se. these results suggest that previously unrecognized clpro cleavage sites may exist inside the nsps, which were regarded as non-canonical cleavage sites. the substrate specificity of coronaviruses clpro is determined by the residues from p to p positions of cleavage sites, especially depending on the p , p and p positions, which would benefit the prediction of cleavage site and design the broadspectrum inhibitors of coronaviruses clpro (chuck et al., ; hegyi and ziebuhr, ) . previous studies demonstrated that different canonical cleavage sites of some representative coronaviruses are not equally susceptible to proteolysis by recombinant clpro (fan et al., ; hegyi and ziebuhr, ) . to define the susceptibility of the canonical cleavage sites and substrate specificity of mers-cov clpro, -mer synthetic peptides representing corresponding canonical cleavage sites of mers-cov clpro were synthesized and modified with n-terminal dabcyl and c-terminal glu-edans (fig. a) . the fluorophore edans and quencher dabcyl are widely used in the biochemical assays based on the fluorescence resonance energy transfer (fret). as shown in fig. b , the peptides represented cleavage sites cs / and cs / were tested to optimize the fret assay, and the relative fluorescence unit (rfu) folds of both sites significantly increased when incubated with gnsp and hnsp . although the fret assay system is more costly and less sensitive than the luciferase-based biosensor assay (figs. b and b), it provides continuous read signals during the process of reaction, which could measure the kinetic characteristic of protease toward different substrates. the initial reaction rate (rfu/min) of all canonical cleavage sites of mers-cov were measured and shown in fig. c . the michaelis constants including kcat, km, kcat/km and relative kcat/km were then calculated (table ) . as shown in table , the substrate specificity of mers-cov clpro is relatively conserved with other coronaviruses as previously reported (fan et al., ; hegyi and ziebuhr, ; ziebuhr and siddell, ) . the relative kcat/km values of cs / and cs / indicated that the cleavage sites flanking mers-cov clpro are converted significantly faster than other sites. the efficient proteolysis at the sites flanking nsp implies that the nsp ( clpro) might be released from the polyprotein a/ ab at the very early stage of the maturation of viral nsps, which is similar with the hcov, tgev, sars-cov and mhv (fan et al., ; hegyi and ziebuhr, ) . however, the relative kcat/km value of cs / is lower than that of cs / (table ) , which is different from that of the coronaviruses (fan et al., ; hegyi and ziebuhr, ) . this could be explained by that the residue gly (g) at the p of cleavage site between nsp and nsp of mres-cov reduces the protease activity of clpro comparing with the residues ser (s), ala (a) and thr (t) of other coronaviruses (tables s -s ) as previous described (chuck et al., ) . whether such disparity plays any role in the replication and pathogenesis of mers-cov is unknown. the processing of viral polyprotein by clpro is essential for the replication of coronaviruses. besides the canonical cleavage sites of coronaviruses, some additional cleavage sites inside nsps, so-called non-canonical cleavage sites, have also been identified (deming et al., ) . therefore, more non-canonical clpro cleavage sites are to be identified in different coronaviruses. in this study, we designed six search conditions for predicting clpro cleavage sites, among which, the search condition iv provides a feasible way to reveal the potential cleavage sites of clpro within coronaviruses. based on the genetic diversity of different coronavirus genera (fig. ) , the scanning condition iv adopted the residues of clpro cleavage sites, which ever appeared in the cleavage sequences of congeneric coronaviruses at position p to p . in contrast, conditions i, ii, iii, v and vi were either too restrictive or generated too many false positive outcomes (table ). in the suggested condition iv, residues from position p to p were applied to the prediction of clpro cleavage site. by measuring the relative protease activities of clpro from different coronavirus genera against amino acids × positions of substrate variants, it is shown that the substrate specificity of position p , p and p are significantly lower than other positions (chuck et al., ) . therefore, the consideration of six or more residues is unnecessary, which could lead to leave-out of potential cleavage sites (table ) . comparing with the previous researches on the prediction and identification of clpro cleavage sites, the scanning condition iv showed its advantages. for example, the two nonexistent putative cleavage sites predicted within nsp of ibv (gorbalenya et al., ; liu et al., ; ng and liu, ) were avoided in our prediction method (data not shown). notably, the noncanonical cleavage site at the end of mhv nsp identified by deming et al. could be predicted using scanning condition iv. by using the search condition iv, putative cleavage sites were predicted in mers-cov pp ab in addition to the canonical cleavage sites. the luciferase signal of cs / increased . fold when treated with nsp in the recombinant luciferase cleavage assays, which is the lowest among the canonical cleavage sites (fig. c) . therefore, the . fold increase of luciferase signal was used arbitrarily as a threshold for judging positive and negative. among the nine predicted putative cleavage sites, three sites (ps - , ps - and ps - ) showed obviously increasing signals at least times above the background (fig. d ) and therefore were regarded as cleavable sites. the increase of signals of other six predicted putative cleavage sites was less than . times (fig. d) . therefore, they were regarded as non-cleavable sites and thus as false positives from the prediction. interestingly, the homologous sequence of ps - and ps - are conserved in lineage c of betacoronavirus including mers-cov, batcov hku and batcov hku (fig. a and b) . however, ps - is mers-cov unique sequence (fig. c) . moreover, the cleavability of a cleavage site in biochemical assays is a necessary but not sufficient condition for its physiological existence in the viral infection. a predicted cleavage site may or may not be accessible by a protease. the d structure model of mers-cov adpribose- -monophosphatase (adrp) domain built by comparative protein modeling and papain like protease (plpro) domain (bailey-elkin et al., ) showed that both ps - and ps - are located at the surface of adrp and plpro domain, opposite to the enzymatic active centers ( fig. d and e) , suggesting that these two sites are like approachable by the proteinase. most recently, the crystal structure of mers-cov clpro was determined (needle et al., ) . although ps - is also located at the surface of mers-cov clpro, the self-cleavage of mers-cov nsp was not observed in this study (fig. ) . therefore, the threshold we proposed in the luciferase-based biosensor system to exclude the false positive prediction results is reasonable (fig. d) . however, further studies are needed to identify the predicted cleavage products from the cells infected by mers-cov. currently, such work with live mers-cov is limited in our research facilities due to the biosafety rules, but it can be addressed in collaboration in the future. notably, the outcomes of the two cleavage assay systems were different. the signal fold change of highly sensitive luciferasebased biosensor assay is dependent on the accumulation of active luciferase cleaved by nsp during h (section ), while the outcome of the fret assay is instant relative fluorescence unit (rfu) signal. the rfu/min is the initial speed of the reaction, which reflects but not equals to the efficiency of the cleavage. these differences may be caused by the steric hindrance of the luciferase subunits, the distance between fluorophore and quencher of substrates for fret assay and substrate solubility. therefore, the activity observed in the two different systems cannot be compared directly. based on the characteristic of the two cleavage assay systems, the highly sensitive luciferase-based biosensor assay might be more suitable to high throughput screen the predicted putative cleavage site of protease while the fret assay better for cleavage kinetic analysis. according to the michaelis constants of mers-cov, the substrate specificity of mers-cov clpro is relatively conserved with other coronaviruses (fan et al., ; hegyi and ziebuhr, ) . notably, the pro (p) has been selected as result of evolution at position p of cleavage site between nsp and nsp (cs / ) of lineage c betacoronavirus, which is not preferred by the clpro based on the previous study (chuck et al., ) . however, the relative kcat/km value of mers-cov cs / is . , which is . fold higher than that of sars-cov (fan et al., ) . this indicated that the substrate preferences of some cleavage sites could still be varied among different genera of coronaviruses and the proposed scanning condition iv regarding the residues ever appearing in the cleavage sequences of congeneric coronaviruses is reasonable. in summary, we proposed an optimized search condition for predicting cleavage sites of coronavirus clpro. we verified the canonical cleavage sites of pp ab in biochemical assays. we further identified three non-canonical cleavage sites in the nsps of mers-cov. the results provide clues for possible identification of novel cleavage products of coronavirus nsps and will benefit the studies of the mechanisms of coronavirus replication. processing of polyprotein a/ ab by clpro is essential in coronavirus life cycle. the clpro cleavage site prediction methods established by previous studies are focus on the accuracy, while some noncanonical cleavage sites were missed. in this study, we built a moderate prediction method to balance the accuracy and false positive outcomes. using this method, putative cleavage sites, in addition to the canonical sites, were predicted in mers-cov pp ab and the cleavability of of them was experimentally confirmed. interestingly, all these non-canonical cleavage sites are located upstream to nsp , which is in contrast with previous understanding that the coronavirus cl protease only cleaves from nsp to nsp . this suggests a novel role of clpro in coronavirus pp a/ ab processing. however, the cleavability of these putative cleavage sites needs to be further verified in the viral proteins of mers-cov-infected cells. finally, the catalytic constants of the canonical cleavage sites of mers-cov clpro showed its conservation with the cousins in coronaviridae. hospital outbreak of middle east respiratory syndrome coronavirus crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression characterization of the leader papain-like proteinase of mhv-a : identification of a new in vitro cleavage site profiling of substrate specificities of c-like proteases from group , a, b, and coronaviruses middle 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specificities but distinct functions in viral replication processing of the human coronavirus e replicase polyproteins by the virus-encoded c-like proteinase: identification of proteolytic products and cleavage sites common to pp a and pp ab virus-encoded proteinases and proteolytic processing in the nidovirales sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.virusres. . . key: cord- -cpdpsg i authors: zheng, xiaotian; lee, stella; selvarangan, rangaraj; qin, xuan; tang, yi-wei; stiles, jeffrey; hong, tao; todd, kathleen; ratliff, amy e.; crabb, donna m.; xiao, li; atkinson, t. prescott; waites, ken b. title: macrolide-resistant mycoplasma pneumoniae, united states date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: cpdpsg i macrolide-resistant mycoplasma pneumoniae (mrmp) is highly prevalent in asia and is now being reported from europe. few data on mrmp are available in the united states. using genotypic and phenotypic methods, we detected high-level mrmp in . % of m. pneumoniae­–positive specimens from us locations. of alabama at birmingham used a laboratory-developed real-time pcr targeting repmp . testing for s rrna mutations conferring macrolide resistance was performed on original specimens by real-time pcr melt curve analysis ( ) and confirmed by dna sequencing at the lurie children's hospital (chicago). subculture and phenotypic antimicrobial susceptibility testing was performed by using a broth microdilution method approved by the clinical and laboratory standards institute at the university of alabama at birmingham. the study comprised m. pneumoniae-positive specimens. patients' ages ranged from months to years; ( . %) samples were from patients < years of age. specimen types were nasopharyngeal or nasal swabs ( samples), nasal aspirate/washes ( samples), bronchoalveolar lavage ( samples), and throat swab and tracheal aspirate samples ( sample each). we found a s rrna point mutation a g known to confer macrolide resistance in ( . %) of specimens by direct real-time pcr with melting curve analysis. all mutations were confirmed by dna sequencing. m. pneumoniae grew in subculture from ( . %) of these specimens. pcr and sequencing on those subcultures confirmed macrolide resistance in specimens that had been previously identified by direct real-time pcr and in additional specimens that had not previously been detected by pcr. the specimens most likely tested negative for mutations by direct pcr because of a low number of organisms in the original sample. macrolide resistance was not significantly correlated with patient age or specimen type (data not shown). mrmp was detected in a total of ( . %) samples (table) . we conducted phenotypic antimicrobial susceptibility testing on all m. pneumoniae isolates obtained by subculture. all macrolide-susceptible isolates showed very low erythromycin mics (< . µg/ml), whereas the isolates that had the a g mutation showed uniformly high erythromycin mics (> µg/ml). all isolates were susceptible to tetracycline (mic . - µg/ml) and levofloxacin (mic . - µg/ml). the filmarray respiratory pathogen panel detects multiple respiratory pathogens. of the respiratory specimens for which co-infection data were available, ( . %) had a viral pathogen detected along with united states mrmp prevalence has been reported to range from % to % in european countries ( , ) and is % in israel ( ). the prevalence is much higher in asia, where mrmp first emerged in , and now exceeds % in some areas of china and japan ( , ) . macrolide resistance has been documented in north america since ( , ) . the centers for disease control and prevention recently published results from specimens obtained from case-patients, small clusters, and outbreaks that occurred during - but did not specify geographic locations from which specimens were derived. an overall % rate of macrolide resistance was reported ( ) . our finding of high-level macrolide resistance in . % of specimens from all centers throughout a broad geographic area in the united states proves this problem has emerged in all regions of the nation and might increase over time, as it has in other countries. the design of our epidemiologic study provides the most accurate estimate of the point prevalence of mrmp available thus far. previous studies reported from the united states were limited primarily to individual case reports, clusters, outbreaks, or single geographic locations. the mechanism for macrolide resistance in m. pneumoniae is point mutations in a few positions of domain v of the peptidyl transferase loop of s rrna, the location of macrolide binding to the s bacterial ribosome subunit ( ) . the a g transition mutation has been the most common one detected in most studies and was the only mutation found in our study. although less common, other mutations conferring macrolide resistance, but not found in our study, include a t or c, a g, a g, and c a or g ( , , ) . unlike previous studies, our study used both molecular and phenotypic techniques to detect macrolide resistance. thus, we were able to correlate erythromycin mics with the presence of s rrna mutations. our results showed striking differences in erythromycin mics between the susceptible and resistant isolates and confirmed high-level resistance to erythromycin in every isolate. erythromycin is usually used for testing because mutations in a and a consistently have been shown to cause high resistance to both erythromycin and azithromycin ( , , ) . a limitation of the study is the nature of anonymous specimen collection. inclusion of patient information, especially antimicrobial therapy, will enhance data interpretation in prospectively conducted future studies. one notable observation from our study is the co-infection of m. pneumoniae with various viral pathogens. a viral co-infection rate > % supports the use of multiplex testing for viral and bacterial pathogens in children with respiratory infections of uncertain etiology. although our study has confirmed mrmp in geographically diverse us states, macrolides should remain the drugs of choice in children with m. pneumoniae respiratory infections. clinicians should be vigilant for macrolide treatment failures and consider using alternative drugs if necessary. countries such as japan and china that have a very high macrolide resistance rate for m. pneumoniae and other respiratory pathogens often have high consumption of macrolides. okada et al. ( ) reported that macrolides account for % of all oral antibacterial drugs in japan and concluded that the increase in macrolide-resistant bacteria during the past several years in that country was closely related to selective pressure resulting from widespread antimicrobial use. given the common use of macrolides in the united states to treat pediatric respiratory infections, judicious use of antimicrobial drugs should be emphasized. reevaluation of existing classes and investigation of new classes of antimicrobial agents may be prudent to have additional treatment alternatives for mrmp infections beyond tetracyclines and fluoroquinolones. surveillance for this resistance in the united states will help monitor the trend. society and the infectious diseases society of america antimicrobial susceptibilities and treatment options for pediatric mycoplasma pneumoniae infections-does macrolide resistance matter? macrolide-resistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of community-acquired pneumonia more complications occur in macrolide-resistant than in macrolidesensitive mycoplasma pneumoniae pneumonia rapid effectiveness of minocycline or doxycycline against macrolide-resistant mycoplasma pneumoniae infection in a outbreak among japanese children emerging macrolide resistance in mycoplasma pneumoniae in children: detection and characterization of resistant isolates clinical outcomes and macrolide resistance in mycoplasma pneumoniae infection in scotland, uk increased macrolide resistance of mycoplasma pneumoniae in france directly detected in clinical specimens by real-time pcr and melting curve analysis surveillance of macrolide-resistant mycoplasma pneumoniae in beijing nationwide surveillance of macrolide-resistant mycoplasma pneumoniae infection in pediatric patients detection of macrolide resistance in mycoplasma pneumoniae by real-time pcr and high-resolution melt analysis investigations of mycoplasma pneumoniae infections in the united states: trends in molecular typing and macrolide resistance from to rising rates of macrolide-resistant mycoplasma pneumoniae in the central united states macrolide-resistant mycoplasma pneumoniae in humans key: cord- - c ug authors: jackson, j. a. title: immunology in wild nonmodel rodents: an ecological context for studies of health and disease date: - - journal: parasite immunol doi: . /pim. sha: doc_id: cord_uid: c ug transcriptomic methods are set to revolutionize the study of the immune system in naturally occurring nonmodel organisms. with this in mind, the present article focuses on ways in which the use of ‘nonmodel’ rodents (not the familiar laboratory species) can advance studies into the classical, but ever relevant, epidemiologic triad of immune defence, infectious disease and environment. for example, naturally occurring rodents are an interesting system in which to study the environmental stimuli that drive the development and homeostasis of the immune system and, by extension, to identify where these stimuli are altered in anthropogenic environments leading to the formation of immunopathological phenotypes. measurement of immune expression may help define individual heterogeneity in infectious disease susceptibility and transmission and facilitate our understanding of infection dynamics and risk in the natural environment; furthermore, it may provide a means of surveillance that can filter individuals carrying previously unknown acute infections of potential ecological or zoonotic importance. finally, the study of immunology in wild animals may reveal interactions within the immune system and between immunity and other organismal traits that are not observable under restricted laboratory conditions. potentiating much of this is the possibility of combining gene expression profiles with analytical tools derived from ecology and systems biology to reverse engineer interaction networks between immune responses, other organismal traits and the environment (including symbiont exposures), revealing regulatory architecture. such holistic studies promise to link ecology, epidemiology and immunology in natural systems in a unified approach that can illuminate important problems relevant to human health and animal welfare and production. recent technological advances in the de novo sequencing and analysis of nucleic acids are revolutionizing the measurement of gene expression in nonmodel organisms, with promising applications in the study of the immune system ( ) . although other phenotypic measurements of immunity remain relevant and useful, albeit limited in scope or technically difficult to apply in nonmodel organisms ( ) , these advances mean that studying the immunology of such organisms in the natural environment has become easier and can take on a genomewide perspective embodied, for example in techniques such as rnaseq. this can, in turn, be accompanied by powerful analytical approaches derived from systems biology ( ) and statistical methodologies applied in ecology. when these elements are combined with the monitoring of natural fluctuation or experimental perturbation, it opens up the possibility of 'reverse engineering' the regulatory architecture of the immune system and its interaction with other organismal traits and with natural environmental pressures ( ) . such approaches, using natural systems, complement the strengths and weaknesses of modern immunology ( ) . here, the great strengths are derived from the very refined use of inbred and genetically manipulated mice under controlled conditions that negate environmental variation. this is very successful for unpicking the structure of molecular pathways and workings of cellular populations, but relevance for natural environmental variation disappears where genetically unrepresentative individuals are studied under homogenous laboratory conditions and in the absence of a natural flora and fauna of symbionts ( , ) . (here symbiont is defined as any organism involved in an intimate association with the host, including parasitic, commensal and mutualistic associations.) the present review will be concerned with how this 'blind spot' in modern immunology can be addressed by a focus on natural populations. it will scan the horizon for unique ways in which studies of nonmodel rodents can contribute to our wider understanding of the biology of the immune system and the way it interacts with the environment to determine health. additionally, it will consider how immunological measurement, interpreted in the light of paradigms from laboratory mouse immunology, can define individual variation relevant to ecological and epidemiological studies of infectious disease in the natural environment ( , ) . rather than produce an exhaustive list of possible interests, though, this review will concentrate on three broad reasons to study immunology in naturally occurring vertebrate hosts, reasons that seem particularly exciting because they could have major practical implications for human health and the welfare and productivity of domesticated animals. each of these themes will be considered in turn and then the reasons why nonmodel rodents (species excluding mus musculus, m. domesticus and rattus norvegicus) may be particularly useful models. finally, selected case studies will be discussed that have begun to approach some of the issues raised. whilst the focus in these case studies is on the measurement of expression in selected candidate genes, they also illustrate the potential for future work using broader transcriptomic approaches, such as rnaseq. much of the ill-health experienced in modern human populations is not caused directly by infectious agents but linked to aberrant inflammation ( ) . thus, conditions including cancer ( ) , diabetes ( ) , asthma and allergies ( ) and various neurological ( , ) and psychiatric disorders ( ) have, in part, immunological aetiologies. trends in these immune-based conditions have been, broadly, upward in 'westernized' human populations, the short time scale indicating the involvement of an environmental variable or variables ( ) associated with 'modern' anthropogenic environments. in addition, the effects of environmental factors on individuals are likely to be modulated by genetic variation inherited from wild ancestral populations ( , ) amplifying individual variability in propensity to disease. a major challenge facing biomedical science, then, is to pinpoint the environmental causes for this variance in health. in particular, given the observed epidemiological trends in immunologically based disease, the questions might be asked: what are the immunological changes that occur in the transition from natural to more anthropogenic environments, and what triggers these changes? several causal mechanisms have been considered, but to parasite immunologists one particularly influential and intuitive (but unproven) line of thought is that increased dysregulation of the immune system is ultimately caused by the host's co-adaptation to stimuli from co-evolved symbionts. these symbionts might include macroparasites and other agents of chronic infection that tend to be lost in anthropogenic environments ( ) . this idea is embodied in what has been termed the 'hygiene' ( ) or 'old friends' ( ) hypothesis. surprisingly, modern immunology is not well placed to take up the challenge of identifying real-world environmental drivers of the immune phenotype. this is because the remarkable laboratory models that have been established for revealing the molecular details of immunological pathways are unsuited to studying how these pathways interact with complex environments under natural conditions. it has often been noted that the genetics of laboratory mice does not reflect the natural situation ( ). typically, laboratory lineages are partly or completely inbred and often generated in a haphazard way that makes the range of allelic variation fixed in their genomes unrepresentative of natural variation ( ) . in the case of the fully inbred (isogenic) lines, their genomewide homozygosity is itself highly unnatural. whilst these disadvantages would perhaps be overcome ( ) by a range of carefully generated wild-derived inbred and outbred lines ( ) , a much less tractable limitation lies in the inability to recreate natural environmental influences in the laboratory ( ) . thus, wild animals experience a range of complex symbiont exposures and environmental stressors that cannot be sufficiently replicated in captivity ( ) . as such, wild mammals (and especially wild nonmodel rodents, due to some of the advantages discussed below) would seem a natural starting point to approach the problem of identifying environmental stimuli that drive immunological development and homeostasis in the wild. as previously considered in more detail by friberg et al. ( ) , progress could be made either through in situ studies in natural populations tracking the effects of environmental variables using manipulative experimental or observational approaches (see for example, the wood mouse case study below), or through transplantation of naturally occurring lineages to (and monitoring of the changes occurring in) experimentally manipulated anthropogenic environments. not all humans in modern environments develop immunologically based diseases (even though increasing numbers do), and those that succumb often have identifiable genetic predispositions. as noted above, causative environmental factors likely exert their effects upon a background of significant immunogenetic variability inherited from wild ancestral populations. the subject area of wild rodents as models for this immunogenetic variability was reviewed in detail by turner and paterson ( ) and will only be considered here sufficiently to provide a general overview relevant to the present article. briefly, a parallel challenge to the one of identifying environmental factors driving immunopathological phenotypes in anthropogenic environments described above, then, is the one of revealing genetic variation that places individuals at risk ( ) . in other words, finding genetic variation that natural selection has shaped in a way that, although adaptive in some natural settings, has the potential to be maladaptive in anthropogenic environments. such variation results from balancing selection, or from directional selection or genetic drift that has not proceeded to fixation, in ancestral populations. here, selective agents (for example, naturally occurring pathogens) that were present in the past, or neutral processes, may have driven into wild populations alleles that are broadly deleterious in novel anthropogenic environments. where potentially deleterious genes have been fixed, though, only variation due to environmental factors (see section above) or the wider genetic background is important. immunogenetic polymorphism driven by balancing or directional selection is thought to cause at least some of the variation underlying immunopathological conditions in modern humans ( ) ( ) ( ) ( ) ( ) ( ) and could, equally, be responsible for a great deal of natural variation in wild animals. although it is increasingly recognized that adaptive evolution has structured the polymorphism in genes controlling the vertebrate immune system ( ) ( ) ( ) , our knowledge of the dynamics of this selection is still rudimentary. crucially, the types of genes and pathways that tend to undergo balancing selection, or frequent intermittent directional selection, in the natural environment are poorly known. also, the nature of the selection involved and the phenotypic manifestations of the polymorphisms are not well understood. studies of wild populations ( ) ( ) ( ) ( ) ( ) are pivotal in this regard, as the wild is the only place in which natural selection, and the phenotypes upon which it operates, can be measured. furthermore, given the conservation of the mammalian immune system, it seems likely that similar genes and pathways may be the target of pathogen-mediated balancing selection across taxa. future studies into the causes of immunogenetic variation (and the characterization of the associated phenotypes) in natural populations are thus likely to feed insights into the biomedical and veterinary fields through focussing attention on the types of gene predisposed to drive immunopathology due to ancestral adaptive evolution. another goal of immunological studies in wildlife is to provide an improved understanding of the dynamics of infection in natural populations ( , ) . and rodents are of particular interest in this regard, given their ecological importance and role as reservoirs for many zoonotic infectious agents ( , ) . in general, the dynamics of zoonotic and other ecologically important infectious diseases are likely to be influenced by heterogeneities amongst individual hosts ( ) , which will affect disease severity and transmission. as the immune system is the host's defence against infectious agents, variation within it is likely to generate much of this heterogeneity ( , ) . the measurement of immunological variation in real ecological contexts may, then, allow us to better define individual heterogeneity, and moreover, to address its environmental causes and epidemiological consequences ( , ) . this could help anticipate infectious disease risks in the environment. for example, such information may help to identify variations in immune defence that occur in time [e.g. seasonal ( ) ( ) ( ) ] or space [e.g. habitat-specific or along invasion fronts ( , ) ] and that alter susceptibility to infectious agents; or it may help identify species ( , ) , or subsets within populations ( ) , whose immunological profiles indicate an increased likelihood to serve as permissive reservoir hosts for certain pathogens. for example, studies on variability in the expression of tnfa and mx genes in bank voles (myodes glareolus) have suggested the possibility of environmentally driven landscape-level patterns that may affect the epidemiology of zoonotic puumala hantavirus ( ). as the most numerous group of mammals (~ species) the rodents represent, through sheer weight of numbers and their wide distributions, very significant potential reservoirs for emerging zoonotic infections. much emphasis has recently been placed on emerging and re-emerging infectious diseases from wildlife reservoirs [for example, the one health initiative ( ) ] and the importance of undertaking pro-active monitoring programmes. in addition to infection risk for humans and domesticated animals, it is likely that epidemic and endemic infectious disease may also contribute significantly to the dynamics of wildlife themselves and indirectly to higher level ecosystem functioning. discovery of infectious agents of ecological importance, or that present a risk of zoonotic emergence is, however, limited by the fact that diagnostic techniques are specific to individual pathogens, or groups of pathogens. whilst next-generation sequencing (ngs) approaches are becoming available that allow very broad nonspecific surveys for pathogen sequences, these are costly and many individuals would have to be processed were a population to be sampled randomly. given this, the detection of potential emergent infectious agents in wildlife could, in some cases, be facilitated by the monitoring of immunological expression ( ) . this would narrowly focus attention on individuals with aberrant immunological profiles that might be indicative of infection states. for example, these individuals could then be selected for further ngs studies ( , ) in order to detect pathogen-specific sequences correlating with the aberrant immune expression profiles. whilst endemic reservoirs may be highly co-adapted with their pathogens and be relatively asymptomatic ( ) (perhaps without anomalous immune expression profiles), it is sometimes the case that infectious agents emerging from wildlife reservoirs may do so via an intermediate 'amplifier' host that does succumb to significant disease. such hosts may shed more infective particles into the environment than the endemic reservoir and also show signs of acute immune responses. for example, putative amplifier hosts may have been involved in the emergence of the sars coronavirus. here, the ultimate natural reservoir appears to include horseshoe bats (rhinolophus spp.), but the infection was likely transmitted to human hosts indirectly via other wild mammals, including the palm civet (paguma larvata) ( ), which is highly susceptible to the virus and sheds high titres of infective particles ( ) . in scenarios where the aim is to identify emergent disease risks before any infectious agent is specifically identified, immune expression studies may provide a way to filter potential diseased amplifier individuals from natural populations and focus attention on these for further study. it is also likely that the pattern of immune expression may be indicative of the type of pathogen involved [as is increasingly being exploited in medical diagnostics ( ) ] and that this may help target subsequent efforts at identification. studies in wild nonmodel rodents may also yield unexpected general insights into the fundamental biology of the mammalian immune system. thus, the laboratory model of mouse immunity cannot properly address many aspects of environmental variation seen in nature, and studies in humans are also limited in this way and by what tissues may be sampled or experimental approaches undertaken. on the other hand, immune responses in wild animals, once they can be interpreted using post-genomic and systems biology approaches, may reveal functional pathways and interaction networks that were not previously understood, precisely because they relate to stressful environmental conditions that cannot ethically be replicated in laboratory or domestic animals, or in humans. an example where a focus on natural populations may feed-back insights into general immunology is given in the section below dealing with immunodynamics in field vole populations ( ) . included in the fundamental biology of the immune system might be the costs of immunity embodied in classical ecological immunology ( , ) . this revolves around the concept of trade-offs: that immune responses impose a penalty in the form of competition for energy allocation to, or functional interference with, other life-history traits. understanding these costs, and the interaction of immunity with other organismal traits, is an active and exciting area of research ( ) that may generate insights relevant to human health and biomedical science. there is also relevance to agriculture, where production traits reflecting reproductive or growth parameters might interact with immunity in ways that are not yet fully appreciated. whilst many advances have been made in the field relating to costs of immunity ( ) , further studies in natural populations using the type of holistic, genomewide approach possible with transcriptomic methods hold the promise of further advances. why not just well-studied laboratory or farmed species? some studies may necessarily focus on particular species or local faunas because of specific concerns about infectious disease risks. where more general questions are to be answered, though, an obvious starting point for studies of immune function in natural systems would be to use the wild counterparts of standard laboratory models or other well-studied domesticated species. in particular, house mice (covered elsewhere in this special issue) would seem an obvious choice given their status as a central model in immunology. this would allow the use of robust measurements based on pre-existing antibody reagents (see next section) and would also allow interpretation of immunological patterns in the context of a very detailed organism-specific knowledge base. looking beyond the small number of laboratory and domesticated species, though, several considerations make it advisable to additionally consider other naturally occurring species. firstly, as they occur in the 'wild' today, rats and mice may have patchy population structures that are narrowly focussed on unnatural anthropogenic environments and are the result of a long history of anthropogenically linked dispersal. in the case of the mouse, for example, dispersal is believed to have occurred from the fertile crescent in the near east ( ) across most of the globe following the expansion of human civilizations ( ) . this history of co-habitation and dispersal means that house mice may harbour symbiont assemblages restricted by lineage sorting (extinction) during dispersal/colonization events and, as invasive organisms, they may have acquired new infections in their relatively recent evolutionary history. in response to selective forces acting during dispersal and gain/loss of infectious threats, such invasive species may also have undergone rapid genetically based changes in immune function ( , ) . these considerations (lack of a natural symbiont flora/fauna, an unusual evolutionary history of dispersal and frequent occupation of anthropogenic habitats) make rats and mice weaker natural models to assess 'hygiene hypothesis' or 'old friends' ideas. here, where the hypothesis is that immunopathological phenotypes in anthropogenic environments arise from a lack of stimuli from co-evolved symbionts, models are needed where the host co-exists in its natural setting with a complete assemblage of co-evolved symbionts. in contrast to the house mouse and norway rat, common naturally occurring murine and microtine rodent species (e.g. apodemus, myodes and microtus spp. in europe), whose ecology has been well studied, often occur in relatively extensive, evenly distributed and persistent populations. these are likelier to have been stably linked in the long term with natural or quasi-natural habitats and with a diverse, co-adapted symbiont assemblage. transcriptomic studies are facilitated by a previously annotated genome, and this may affect the choice of study species. increasing genomic information is becoming available for several well-studied taxa, including the european species apodemus sylvaticus, microtus agrestis and m. glareolus, and annotated genomes have been assembled for peromyscus maniculatus and microtus ochrogaster in north america. however, transcriptomic studies can be carried out through de novo assembly without a pre-existing species-specific genome ( ) and allow the exploitation of almost any species for ecological immunology studies. naturally occurring rodents recently used for ecological immunological studies in other parts of the world include, for example, organisms as diverse as the capybara (hydrochoerus hydrochaeris) ( , ) or the pallid atlantic forest rat (delomys sublineatus) ( ) in south america. in general, naturally occurring rodents represent unparalleled models for work in ecological immunology and immunogenetics. whilst their close relation to the laboratory mouse allows interpretation in terms of modern mechanistic immunology, their high population densities, amenability to longitudinal sampling and experimental manipulation in the field, short generation times, relatively spatially static populations and, ultimately, their adaptability to the laboratory environment, make these organisms uniquely tractable study systems. given the diversity and abundance of rodents, it is likely that most researchers will have, at close hand, naturally occurring rodent systems that may serve as useful ecological models or that are practically relevant as reservoirs of transmissible infectious disease. finally, it should not be forgotten that there is very likely to be value, for its own sake, in carrying out studies across a wide diversity of host systems ( ) . this will be useful in revealing the generalities in immune function across species and will also provide insights from the specialized adaptations that individual species use to meet specific sets of circumstances. there is a strong emphasis in modern immunology on the use of antibody reagents against immunological biomolecules for robust molecular phenotypic measurements. gene expression measurements at the mrna level, in comparison, are generally considered a more problematical approach that is 'resorted to' if necessary, particularly in the case of analyses of individual genes by real-time pcr (qpcr). this is based primarily on the fact that the expression of bioactive protein may not always track upstream mrna concentrations ( ) . complex kinetics in the pathway between mrna and protein, and the stability of the proteins themselves, can prevent a simple relationship. often there is poor concordance of matched transcriptomic and proteomic data sets across a range of organisms ( ) . such a lack of gene-by-gene correlation, though, is much less relevant than the information content that transcriptomic profiles carry in relation to the biological status of the individual. this information content is attested to by the increasing use of transcriptomic approaches in modern biology. more specifically, early indications in wild rodents ( , , , ) suggest that gene expression measurements, especially if interpreted with the complexity of post-transcriptional dynamics in mind, do contain much useful information. practical issues restrict the usefulness of antibody-based methods in nonmodel rodents. due to structural variability in many immune molecules (especially canonical cytokines and cell surface markers), the transferability of commercially available off-the-shelf reagents amongst rodents, even within the subfamily murinae, is limited. whilst some commercial assays and reagents may indeed be found to cross-react amongst rodent species (for example, many commercial antibodies targeting immunoglobulins), this approach may involve trialling dozens of others unsuccessfully. this has been the experience of researchers targeting cytokines in the cricetid, peromyscus maniculatus, in north america ( ), or even the murine, apodemus sylvaticus, in western europe ( , ) . thus, developing a broad panel of assays may require the de novo production of monoclonal and/or polyclonal antibodies, which is technically feasible but is costly and time-consuming ( - ), especially given that two separate antibodies may be required per target (if setting up a microplate elisa, for example). a promising and more economical strategy will be to explore further the potential of established and emerging nucleic acids measurement ( , , ) . this approach can focus on panels of candidate genes, selected on the basis of the existing knowledge base for mouse immunological pathways. even more powerfully, rnaseq ( , ) , because it encompasses the whole transcriptome ( s of thousands of genes, depending on sequencing coverage), largely removes concerns associated with measuring single genes as it allows a focus on whole pathways whose concerted variation is much more likely to reflect the phenotype. this is a technique that, although very expensive per sample, can be used in an unbiased way to identify informative and reliable marker genes that can then be measured by cheaper technologies in larger sample sizes. as an approach directed at the entire transcriptome, it is likely to supersede oligonucleotide microarray methods, due to general technical superiority ( ) and, particularly in the case of studies in nonmodel species, the requirement of microarrays for prior sequence information. targeting of single genes (or small panels of genes) may be carried out by qpcr (quantitative real-time pcr, often abbreviated as rt-pcr) or other emerging technologies that measure nucleic acids more directly, for example digital pcr ( ) or new developments of microarray-like systems ( ) . the latter technologies are likely to be technically more robust than qpcr, which although still invaluable, suffers from sensitivity to variable reaction kinetics amongst sam-ples; however, they are currently more expensive, and in the case of digital pcr less applicable to high-throughput applications. finally, the point should be made that, whatever molecular measurement approach taken (protein or rnabased), this should ultimately be cross-referenced to functional measures of immunity (such as susceptibility to pathogens). in natural populations, this cross-referencing can be achieved either by observational epidemiological studies, or, more powerfully (and with greater difficulty), by manipulative experimental infectious challenges. the advent of genomewide transcriptomic (and other high throughput) measurements arguably allows rapid progress in the study of immunity in natural systems through exploratory, data-led approaches that might loosely be covered by the term 'reverse engineering' ( ) . biologists often attempt to explain natural systems using a forwardengineering-like philosophy, where a high level model or concept is used to direct the interpretation of data. although this approach (in some form or other) is likely to remain indispensible and also corresponds to many biologists idea of the basic scientific method, it is vulnerable to arbitrary choice of starting model and is not necessarily the exclusive, or most direct, route to understand complex systems (especially when starting from a low knowledge base). in contrast, in reverse engineering, large sets of responses (as generated by, for example, transcriptomic or multiplexed protein measurements) can be correlated with environmental and organismal variables of interest across perturbations (either natural or experimental), allowing interaction networks to be inferred ( ). this can help establish how molecular pathways interact with each other and the environment to generate the observed phenotype. a reverse-engineering-like philosophy has, in part, featured in the examples dealt with below, and although these work with limited panels of measurements, they illustrate the potential for future studies using broader transcriptomic approaches. the immune system has co-evolved with commensal microbes to the extent that it requires cues from these organisms to programme its normal development ( , ) . moreover, as a result of this, immunopathological phenotypes are to be expected where microbial exposures occur that are outside the parameters within which natural selection has operated during evolutionary history ( , ) . this developmental interaction between microbiota and immune system is, in part, channelled by toll-like receptors (tlrs) of the innate immune system ( , , ) . the specificities of these receptors ( ) and the inflammatory programmes they recruit are essentially directed against microbes. however, recent studies in wild rodents, which will briefly be reviewed here, suggest that natural exposures to macroparasites modify systemic tlr-mediated responses to bacterial molecular patterns. macroparasites, given their widespread occurrence in natural vertebrate populations, may thus be part of an extended co-evolved interaction network, involving commensal microbes and innate antimicrobial responses, which can drive the development and homeostasis of the immune system. cotgrave forest, nottinghamshire a wood mouse population at cotgrave forest, nottinghamshire, was monitored ( , , ) over time, with a series of cross-sectional samples between and . a range of ex vivo tlr-mediated responses (to defined tlr agonists) were measured in cultured splenocytes from subsets of animals. due to a pattern of positive covariation amongst tlr-mediated tumour necrosis factor alpha (tnf-a) protein responses (tlrs , - , , ) measured in the early part of the sample series, and the especially strong associations of tlr -mediated tnf-a production with measures of macroparasite infection, this last response in particular was chosen to be measured in all samples. focussing on tlr -mediated response across the whole study period, this was found to be associated with certain macroparasites, especially the gastrointestinal heligmosomatid nematode, heligmosomoides polygyrus, and the blood-sucking ectoparasitic louse, polyplax serrata. the magnitude and direction of the associations, though, changed across the study period. the changing associations corresponded to different environmental conditions following a perturbation (population crash) in the mouse population during the middle part of the study. early in the study, the abundances of mice and macroparasites were high, but following the mouse population crash, the later part of the study was characterized by lower macroparasite abundance. corresponding changes occurred in the association between macroparasites (p. serrata and h. polygyrus) and tlr -mediated tnf-a responses, with a strong negative coefficient before the perturbation (at high infection levels) and a positive one subsequently (at lower infection levels). complementary laboratory experiments using the mouse-heligmosomoides bakeri model supported a causal effect of nematode infection upon tlr-mediated responses. this model is relevant because h. bakeri is a very close relative ( , ) of the h. polygyrus occurring in wood mice. previously na€ ıve inbred mice exposed to single (cba, balb/c, c bl/ , swr) and trickle h. bakeri infections (balb/c, c bl/ ), typically up-regulated tlr responses signalling through myeloid differentiation primary response gene (myd ) at some point during the infection course, usually coinciding with peak standing worm burdens. this is consistent with a permissive effect of myd signalling on gastrointestinal nematode infection demonstrated through experiments with myd À/À and interleukin one receptor one (il r ) À/À mice ( , ) . it is interesting, though, that in the trickle infection experiments, resistance developed in both balb/c and c bl/ . this contrasts with more permissive infection phenotypes previously achieved under similar experimental regimens, where the susceptible strain c bl/ supports chronic infection and can continue accumulating worms to the point of lethality ( ) . furthermore, in wild wood mice there is a linear accumulation of h. polygyrus with age and no indication of acquired immunity in the form of abundance as a decelerating function of age indicators ( ) . it may also be significant that the laboratory experiments, in initially na€ ıve animals, produced results consistent with the positive abundance -tlr response association seen in the wood mouse population during times of low parasite abundance, but were not consistent with the negative association seen at times of high parasite abundance. the latter negative association (and perhaps the permissive infection phenotypes noted above) could be accounted for by the well-known immunosuppressive effects of chronic heligmosomatid infections. these effects would likely involve adaptive regulatory t-cell responses that could feedback negatively onto innate immune responses ( ) . taken together, all of this information indicates that macroparasite infection exposures may have significant and context-dependent effects on the innate responses that mediate interactions of the immune system with microbes. the mechanism for this remains to be determined, but as discussed by friberg et al. ( ) , the possibilities include effects on tlr signalling by worms that are direct, via secreted immunomodulators, or indirect, via feedback from adaptive immune responses. other indirect effects could be mediated by altered exposure to microbes or innate damage signals resulting from the activities of macroparasites at their site of infection. there is some reason to believe that one or both of these last mechanisms might be important. thus, heligmosomatid excretory-secretory products ( ) , and the regulatory ( ) and th ( ) immune responses that these parasites typically trigger, generally reduce tlr-mediated signalling. in the laboratory experiments, though, heligmosomatid infections actually increased tlr responses (perhaps consistent with activation by tlr ligands, which are primarily microbialor damage-associated patterns). this re-emphasizes the possibility that co-infections with macroparasites contribute to the network of interactions between commensal microbes and the immune system. moreover, due to the epidemiological association of ectoparasitic lice with systemic responses in the cotgrave forest study, it seems that antiparasite responses at peripheral sites beyond the gut ( ) may also be involved. the existence of such an extended interaction network is highly relevant to our understanding of how microbiotal exposures programme immunity. because macroparasites are often absent in anthropogenic environments ( ) , this may contribute to the disruption of co-evolved interactions (cf. the hygiene hypothesis). thus, studies in a wild system have pointed towards the need for further work to establish the role of natural macroparasite communities in the formation of microbiotal assemblages and the contribution of this to health. background evolutionary fitness in the natural environment is not measureable in the laboratory, and so studies of hostpathogen community dynamics in the wild are essential to fully understand immune responses in their wider context as components of antipathogen strategies that maximize fitness. studies in wild field voles, briefly reviewed below, have aimed to identify distributional infection patterns associated with different antipathogen strategies in natural populations and to link these to expression signatures in immune-relevant genes. such gene expression markers can then be used to track the life-history correlates of different putative strategies and may also give insights into the immunological mechanisms involved. when considering adaptations to infection exposures, two strategies are available to a host and these may often be deployed together, although there may be some emphasis on one relative to the other. these strategies are resistance, where the host prevents infection (denies access to the pathogen), and tolerance, where the host allows access to the pathogen whilst actively mitigating the negative effects of infection. identifying patterns of tolerance and resistance in natural populations is problematical but can be approached using a general framework like that summarized in jackson et al. ( ) . in cross-sectional samples ('snapshot' destructive samples of individuals), tolerance in identifiable groups of animals may be measured as the regression slopes (reaction norms) of fitness measures (for example, body condition) against infection load. here there is the problem that, in individuals from natural populations, the infection dose and the time course of infection are not standardized. reaction norms, though, can also be supplemented by consideration of the phase curves (temporal trajectories of fitness in relation to infection load) of infection courses in longitudinally sampled individual animals. these allow a known infection load to be related to fitness measurements at a later time point. in the study briefly reviewed below, both approaches were used, with initial identification of a tolerance-like pattern in cross-sectional samples, which was then corroborated and extended by focussed analyses in longitudinal samples. immune gene expression profiles in field voles at kielder forest, northumberland gene expression measurements are increasingly being used in studies of infectious disease in nonmodel rodent systems ( , , , , ( ) ( ) ( ) . in work carried out in the well-studied kielder voles (m. agrestis) system ( ), measurements of a panel of candidate genes (representing different immunological pathways) were taken in sets of cross-sectional and longitudinal samples from individual voles at two sites in each of two seasons ( ) ( ) ( ) . this design aimed to capitalize on the respective strengths of the two sampling modes: the greater range and precision of measurement in destructively sampled 'snapshot' cross-sectional samples (where more tissues can be interrogated and manipulated in the laboratory), also the stronger inference of causality in time series data from repeat-sampled individually marked animals (longitudinal samples). in cross-sectional samples, expression profiles were measured in ex vivo stimulatory assays of cultured splenocytes (with stimulants including tlr and tlr agonists and mitogen), whilst in longitudinal samples, constitutive expression was recorded in peripheral blood. in addition, a range of infection and condition measures were recorded in the sample animals. early analyses in a partial cross-sectional data set for immune gene expression ( ) ( ) , and without considering pathogen data, suggested the value of the measurement approach through the existence of significant variation of expression in relation to season, life-history stage and individual condition ( ) . further analyses, on the full data set, searched for patterns of resistance or tolerance to pathogens. initially focussing on the more detailed cross-sectional data, and using body and organ condition (weight adjusted for standard length) as a fitness measure, it was possible to recognize a predominant pattern indicative of tolerance to macroparasite infection in mature males (where macroparasites accumulated with age indicators and were associated with increasing body condition) and a pattern indicative of resistance in immature males (where macroparasite abundances were decelerating functions of age indicators and not associated with body condition) ( ) . although unexpected, the positive association of body condition with macroparasite infection in mature males was in the context of negative changes in other life-history components and not inconsistent with a negative overall impact of infection exposure on fitness. high expression of the transcription factor gata-binding factor (gata ) in mitogen-stimulated splenocytes was found to mark both tolerant animals (amongst mature males) and resistant animals (amongst immature males) in the cross-sectional set. furthermore, analyses of timelagged associations in mature males in the longitudinal data suggested that macroparasite infections triggered gata responses [as might be expected in laboratory models ( ) ( ) ( ) ], which in turn gave rise to increases in body condition. this corroborated the cross-sectional analyses and supported a hypothesis that gata activity stimulated by macroparasites is part of a complex of (tolerance) responses leading to the readjustment of body condition in mature males. constitutive gata expression in peripheral blood also correlated with survival in longitudinally monitored animals, with high relative expression of gata predicting poorer survival in younger animals but having a progressively more positive effect on survivorship with increasing age. in this observational field study, the existence of confounding processes that might produce the cross-sectional patterns attributed to tolerance should be considered. indeed, the multifaceted nature of the kielder study, with cross-sectional and longitudinal components providing a range of measurements in different population strata, increases the opportunities for comparing predictions to data. two main confounding processes might be relevant in terms of their potential to generate tolerance-like patterns. one of these is differential mortality (dm): where, amongst animals heavily infected with macroparasites, those in poor condition die more quickly, biasing the average condition of the survivors upwards. another possibility is correlated risk (cr): where parasite load may be linked to good condition because hosts in good condition forage more or range more and encounter more parasite infective stages as a result. the dm and cr scenarios were poorer explanations for the observed patterns from a number of perspectives. under cr, increased condition would precede increased acquisition of parasites and the gata responses they trigger; but in temporal series for individual mature males, these two sets of events, in reality, occurred in the reversed sequence. this observed sequence and the direction of association also contradict the prediction of dm of a negative effect of macroparasite infection (and the gata responses triggered) on subsequent condition (which follows if infection is a major cause of mortality)in reality there was a positive association. perhaps most importantly, the dm and cr scenarios do not explain the age-specific changes in the relationship between gata expression and survival in longitudinal data, or in the relationship between gata , condition and macroparasite infection in the cross-sectional data. thus, if dm were true, gata expression in peripheral blood would be expected to show a consistent negative association with individual survival, especially in classes of animal with an apparent gata associated tolerance pattern. however, in reality gata expression decreases survival in smaller males (where a tolerance pattern is not seen) but tends to increase survival in larger animals (which do show the gata -associated tolerance pattern). furthermore, if cr were true, better conditioned animals would generally have higher parasite exposure and higher gata expression; but in reality, this is only seen in mature males and not in immature males. it might also be argued that a special case of cr, which could explain the latter age-specific pattern, is that better conditioned mature males range more, or have more social contacts, due to increased breeding activity. even in this case, though, the wide-ranging males would be expected to have better testis condition (if undergoing increased behaviours associated with breeding); but in our study, gata expression was negatively correlated with testis condition. thus, the details of the study were consistent with a hypothesis of elevated gata expression mediating resistance in immature males and tolerance in mature males; there were major inconsistencies with alternative dm and cr interpretations. gata is a master transcription factor involved in the differentiation and development of th (t-helper type cell) cells ( ) and would be expected to mark th activity in the splenocyte cultures studied. the seemingly dual aspect of gata (involved in tolerance and resistance) is not biologically implausible, given that th responses have long been associated with resistance to macroparasite infections in laboratory models but are also linked to wound-healing mechanisms that might be involved in tolerance ( ) . it would seem possible, however, that gata expressing th cells might drive different downstream effector responses in resistance and tolerance, and this is one aspect that is worthy of further study. whilst regulatory components of the immune system have previously been considered as possible mediators of tolerance ( ) , through their ability to dampen effector responses, this was not supported in the kielder study. thus, the antiinflammatory cytokines interleukin (il) and transforming growth factor beta one (tgf-b ) and the transcription factor forkhead box p (foxp ), whose expression characterizes many regulatory t-cell subsets, were measured but were not associated with tolerance patterns. gata expression in tolerant males was associated with a complex of life-history readjustments likely to impact fitness (represented by schematic phase curves in figure ). apart from the increase in body condition, there was a decrease in a fecundity indicator (testis condition) and increasing survival as animals aged. possibly, then, the tolerance strategy increases fitness via improvements in residual reproductive value (potential for future reproduction) during macroparasite infection. this is epidemiologically significant, as the transmission of infectious agents may become focussed through tolerant subsets of the population with consequences for the population dynamics, life histories and co-evolutionary dynamics of the interacting organisms. but there are also insights that can be fed back into laboratory immunology. the results raise the possibility that th cell activity may show ontogenetic changes, sometimes mediating resistance and at other times mediating tolerance. this dichotomy could be relevant to the lab-oratory as, typically, laboratory experiments are carried out in restricted life-history stages under restricted environmental conditions, and this may have skewed our view of what responses are likely to occur in systems outside of the laboratory. there is also a relevance to vaccination strategies in real-world situations, which may need to encompass the possibility that, under some conditions and in some subsets of a population, similar immunogenic stimuli may result in resistance or tolerance responses (that might not be protective in terms of preventing infection, but could have some benefit in terms of ameliorating disease). this may be especially significant, given that most vaccine adjuvants used in practical contexts are th -inducing aluminium salts (alums). although much interesting work has been done in nonmodel rodents using narrow focusses on individual immune responses, a renewed effort addressing the immune system (and its interaction with wider organismal traits and the environment) in a more holistic way seems likely to pay dividends. the pivotal technological means to do this are now accessible, in the form of nextgeneration sequencing analyses of the transcriptome. early indications from studies using qpcr panels of candidate genes suggest that gene expression measurements in natural populations do convey interpretable information about individual status, especially where combined with defined stimulatory treatments of cultured cell populations. associations occur with season, life-history stage and body condition. furthermore, there are strong indications that infection pressures are key drivers of many aspects of expression in the immune system in nature and that, as a result of these pressures, wild mammals can be confidently predicted to adopt phenotypes very different to those seen in laboratory rodents. all of this supports the utility of immune gene expression measurements for ecologically and epidemiologically motivated studies; it also confirms the interest for our basic understanding of the immune system: where the diverse combinations of environmental conditions seen in the wild may reveal interaction networks that remain hidden under controlled laboratory conditions. finally, studying the diversity of immune function in natural and anthropogenic environments (and, ultimately, further dissection of this variation under experimental conditions) will help us resolve the environmental stimuli (and genotype environment interactions) that affect the formation of immunopathological phenotypes such as those responsible for so much variation in human health and animal welfare. Δ body condition Δ survival probability Δ testis condition large mature male immature male gata blood gata mit-s m gata mit-s m figure life-history responses in male field voles (microtus agrestis) associated with gata expression triggered by macroparasite exposures, represented schematically as phase curves (temporal tracks through the plotted parameter space). gata mit-stim , gata expression in cultured mitogen-stimulated splenocytes; gata blood , constitutive gata expression in peripheral blood. the analysis of immunological profiles in wild animals: a case study on immunodynamics in the field vole, microtus agrestis beyond phytohaemagglutinin: assessing vertebrate immune function across ecological contexts towards a system level understanding of non-model organisms sampled from the environment: a network biology approach wild immunology macroparasites, innate immunity and immunoregulation: developing natural models measuring immune system variation to help understand host-pathogen community dynamics regulation of the immune system by biodiversity from the natural environment: an ecosystem service essential to health inflammation and cancer diagnosis and classification of autoimmune diabetes mellitus microbiome diversity and asthma and allergy risk the role of innate and adaptive immunity in parkinson's disease molecular mechanisms linking neuroinflammation and neurodegeneration in ms microbiota, immunoregulatory old friends and psychiatric disorders hygiene hypothesis and autoimmune diseases wild rodents as a model to discover genes and pathways underlying natural variation in infectious disease susceptibility the history of ecoimmunology and its integration with disease ecology review series on helminths, immune modulation and the hygiene hypothesis: immunity against helminths and immunological phenomena in modern human populations: coevolutionary legacies? 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expression profiling of lymph node cells from deer mice infected with andes virus saag- is a novel mosquito salivary protein that programmes host cd (+) t cells to express il- blood feeding by the rocky mountain spotted fever vector, dermacentor andersoni, induces interleukin- expression by cognate antigen responding cd (+) t cells the immune response to parasitic helminths: insights from murine models gata and the t-cell lineage: essential functions before and after t-helper- -cell differentiation evolution of th immunity: a rapid repair response to tissue destructive pathogens decomposing health: tolerance and resistance to parasites in animals acknowledgements i gratefully acknowledge the colleagues with whom i have worked on nonmodel rodent systems and whose ideas have influenced my own thinking, also acknowledged is funding from the leverhulme trust (rpg- ) and nerc (ne/l / ). key: cord- -vciposnk authors: ho, zheng jie marc; zhao, xiahong; cook, alex r; loh, jin phang; ng, sock hoon; tan, boon huan; lee, vernon j title: clinical differences between respiratory viral and bacterial mono- and dual pathogen detected among singapore military servicemen with febrile respiratory illness date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: vciposnk background: although it is known that febrile respiratory illnesses (fri) may be caused by multiple respiratory pathogens, there are no population-level studies describing its impact on clinical disease. methods: between may and october , fri patients and controls in the singapore military had clinical data and nasal wash samples collected prospectively and sent for pcr testing. patients with one pathogen detected (mono-pathogen) were compared with those with two pathogens (dual pathogen) for differences in basic demographics and clinical presentation. results: in total, . % had one pathogen detected, . % had two pathogens detected, . % had no pathogens detected, and . % had more than two pathogens. multiple pathogens were associated with recruits, those with asthma and non-smokers. influenza a ( . %), influenza b ( . %) and mycoplasma ( . %) were most commonly associated with mono-infections, while adenovirus was most commonly associated with dual infections ( . %). influenza a paired with s. pneumoniae had higher proportions of chills and rigors than their respective mono-pathogens (p = . , p = . ). h. influenzae paired with either enterovirus or parainfluenzae had higher proportions of cough with phlegm than their respective mono-pathogens. although there were observed differences in mean proportions of body temperature, nasal symptoms, sore throat, body aches and joint pains between viral and bacterial mono-pathogens, there were few differences between distinct dual-pathogen pairs and their respective mono-pathogen counterparts. conclusion: a substantial number of fri patients have multiple pathogens detected. observed clinical differences between patients of dual pathogen and mono-pathogen indicate the likely presence of complex microbial interactions between the various pathogens. febrile respiratory illnesses (fri) are caused by a wide range of pathogens, most commonly by viruses and bacteria, , some of which cause more serious clinical disease and morbidity. , it may also be due to multiple pathogens co-existing in a microenvironment of complex interactions, which is not unexpected as the respiratory mucosa has abundant resident flora to begin with. for instance, one study showed that . % of ambulatory patients with influenza-like illness had two viruses detected, and another found that in . % of children with community-acquired pneumonia, the illness was due to mixed viral-bacterial infections. others also previously described respiratory viral , and bacterial co-infections , in various settings, although most focus on specific pathogen combinations, especially of the synergism between influenza and streptococcus pneumoniae (s. pneumoniae). [ ] [ ] [ ] [ ] however, there are no population-level studies describing multiple pathogens among persons with upper respiratory tract infections and their impact on clinical disease. such information is of particular importance to countries within the tropical belt where there is a predilection towards multiple pathogens due to the year-round circulation of respiratory pathogens. , a previous study documented the clinical characteristics and epidemiology of viral mono-pathogens gleaned from the respiratory disease sentinel surveillance programme of the singapore military. here, we analyse additional data from the programme, compare patients with one (mono-pathogen) and two pathogens detected (dual pathogen), and describe observed differences in clinical characteristics. all singaporean males enter national service for years after high school or equivalent. during this period, the majority spend most of their time in communal living and training quarters in military camps and return home on weekends, resulting in a semi-closed environment with community interaction. sentinel surveillance for febrile respiratory patients were performed at five major sites. the period of study was from may to oct , and servicemen who sought primary health care at these camps during regular consultation hours were recruited. the fri inclusion criterion was having a body temperature of . °c and above with cough or sore throat. after obtaining informed consent, a standardised questionnaire was administered and nasal wash sampling performed by trained personnel followed by routine clinical assessment by an attending physician. repeat consultations were excluded if the patient was deemed to not have recovered from the first episode of illness. two weeks after the initial consultation, patients were reviewed (through case records and phone calls to patients, if necessary) to determine the number of patients who eventually required referral to hospitals for further evaluation, were diagnosed with pneumonia and/or were admitted for further treatment. randomly selected unmatched controls (at a rate of - persons per week) were also obtained across the year for comparative purposes of baseline commensal rates: these are soldiers from the same camps who were reporting sick at the medical centre for reasons other than respiratory symptoms or acute infections (e.g. those with muscle sprains were selected as controls). this is to prevent mild respiratory infections from being selected and confounding the baseline rates. informed consent was also sought from controls before recruitment. nasal wash samples were obtained from trained medical staff from each side of the nose and placed in universal transport media. these were stored in fridge at °c and transported to the laboratory using carriers with ice packs within h. an iso -accredited laboratory that regularly takes part in qcmd eqa programmes was used to perform molecular diagnostic testing. detailed laboratory methods have been described in the previous publication. briefly, this was done by the extraction of nucleic acids using the dna mini kit (qiagen, inc, valencia, ca, usa) and then tested using multiplex pcr assays coupled with bead array detection technology (resplex i and ii, version . , qiagen, inc, valencia, ca, usa) which can simultaneously detect and subtype different pathogens. first, pathogens of the same genus were grouped (e.g. 'influenza a' includes its various subtypes, and 'enterovirus' also includes coxsackievirus, echovirus and rhinovirus). demographic characteristics for controls, mono-pathogens, dual pathogens and patients with more than two pathogens were analysed and compared using descriptive statistics. analyses on the prevalence of co-existing pathogens were then performed. interval/ratio variables were compared using oneway analysis of variance (one-way anova). comparison of nominal variables with expected frequencies less than or equal to was done using fisher's exact test, while comparison of nominal variables with expected frequencies more than was done using pearson's chi-square test. pearson's chi-square test was conducted to identify trend in proportions. further analysis focussed on comparing patients with one and two pathogens. in this regard, i) controls, ii) patients with more than two pathogens as well as iii) mono-and dual pathogens with sample sizes of less than observations (considered too small for analysis) were excluded. as a result, a total of mono-pathogens and dual-pathogen pairs were available for comparison. permutation tests were conducted to compare the number of symptoms observed between mono-pathogen and dualpathogen patients for each pathogen as a proxy for severity of infection. to assess differences in symptom expression, dual pathogens were compared against mono-pathogens for mean proportions of symptoms (or signs). empirical proportions of symptoms with % confidence intervals (cis) for both mono-pathogens and dual pathogens were calculated and compared using pearson's chi-square test at a significance level of . . symptoms with onsets in at least % of patients for a minimum of one pathogen or combination were described in detail. in particular, dual infections with statistically different results from their respective viral monoinfections were highlighted. r statistical software (version . . ) was used to perform all statistical analyses. ethics approval was given by the singapore military joint medical committee for research and the national university of singapore's ethics review committee. of samples of patients tested, . % had monopathogens and . % had dual pathogens detected. no pathogens were picked up in . % samples, while . % samples had more than two pathogens. among dual pathogens, virus-bacterial pairs were most common at . %, followed by bacteria-bacteria ( . %) and virusvirus pairs ( . %). demographics for patients and controls are detailed in table . gender and the prevalence of heart disease were similar across all groups. mean age was slightly higher in controls, and the number of persons with asthma was higher among patients. multiple pathogens were also more commonly detected among recruits and in those not currently smoking. table shows the differences in detection of pathogens between patients and controls. there were no significant differences in rsv, m. pneumonia, s. pneumonia and n. meningitidis between the two groups. among dual pathogens, there were virus-bacteria, bacteria-bacteria and virus-virus combinations with more than observations each. the most common virus-virus pair was that of influenza a with enterovirus; and of bacteria-bacteria pairs, it was haemophilus influenzae (h. influenzae) with s. pneumoniae. the top three virus-bacteria observations were h. influenzae, paired with adenovirus, enterovirus and coronavirus, respectively. figure depicts the incidence of dual-pathogen pairs, with further details in table s . of the samples with more than pathogens detected, h. influenzae, s. pneumoniae, adenovirus and enterovirus were most commonly involved. the most common trio was adenovirus with s. pneumoniae and h. influenzae, which accounted for . % of samples with more than pathogens. number of symptoms mono-and dual-pathogen patients had similar symptom loads (with . symptoms on average). however, among dual-pathogen patients, those involving s. pneumoniae (p = . ), neisseria meningitidis (n. meningitidis) (p = . ) and h. influenzae (p = . ) displayed a higher number of symptoms than corresponding mono-pathogen patients. nine common symptoms, not ranked by severity, are presented in figure , with further details in table s and figure s . mean body temperature of viral mono-pathogen patients was slightly higher than that of bacterial mono-pathogen patients ( mean proportion of viral mono-pathogen patients with chills and rigors was lower than that of bacterial monopathogen patients ( . vs . ; p < . ). dual pathogens with both s. pneumoniae and influenza a were associated with high proportions of chills and rigors ( . , %ci . , . ). this was significantly more than s. pneumoniae ( . , %ci . , . ; p = . ) and cough with sputum mean proportions of viral and bacterial mono-pathogen patients having cough with sputum were similar, at . and . , respectively, although mean proportion of dual pathogens with the symptom was higher, at . (p < . ). specific dual pathogens with a higher proportion of cough with sputum than both respective bacterial and viral mono-pathogen patients were h. influenzae paired with enterovirus (p = . ; p = . ), or parainfluenzae (p = . ; p = . ). mean proportion of viral mono-pathogen patients having dry cough was higher than that of bacterial mono-pathogen patients ( . vs . ; p < . ). h. influenzae with enterovirus, with higher mean proportions of cough with phlegm as described above, showed a corresponding decrease in dry cough. the proportion among dualpathogen patients was also lower than the patients infected with the virus alone or the bacteria alone (p = . , p = . , respectively). mean proportion of viral mono-pathogen patients having nasal symptoms (sneezing, blocked nose and running nose) was higher than that of bacterial mono-pathogen patients ( . vs . , p < . ). mean proportion for dual infections with nasal symptoms lay in between at . , statistically different from both viral (p = . ) and bacterial (p < . ) mono-pathogen levels. however, no specific dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportion of viral mono-pathogen patients with sore throat was only slightly higher than that of bacterial monopathogen patients ( . vs . ; p = . ). the mean proportions for dual pathogens were similar to viral monopathogen levels ( . ) and likewise statistically higher than bacterial mono-pathogen levels (p = . ). interestingly, however, dual pathogens of coronavirus with s. pneumoniae (p = . ) or h. influenzae (p = . ) were instead found to be statistically lower than patients with coronavirus alone. mean proportions of viral mono-pathogen patients with headache were similar to that of bacterial mono-pathogen patients ( . vs . ). for dual pathogens ( . ), the mean proportion were only slightly higher than viral monopathogen patients (p = . ). however, no dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportions of viral mono-pathogen patients with body aches were similar to that of bacterial mono-pathogen patients ( . vs . ). mean proportion of dual pathogens with body aches ( . ) was slightly lower than viral monopathogen patients (p = . ). however, no dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportion of viral mono-pathogen patients with joint pains were higher than that of bacterial monopathogen patients ( . vs . ; p ≤ . ). mean proportions of dual infections with joint pains ( . ) were in between these two levels, being statistically different from both viral (p = . ) and bacterial (p = . ) monopathogen patients. however, no dual-pathogen pairs had statistically different levels than their respective viral monopathogens. patients were reviewed weeks after the first consultation to ascertain whether any complications had developed in the interim. the proportion of patients referred to hospitals (for further evaluation), as well as the proportion diagnosed with pneumonia, were found to increase significantly with the number of pathogens detected (p = . and p = . , respectively) (table ) . however, there were no clear trends in the number of patients eventually requiring inpatient treatment, possibly as a result of relatively small numbers. much emphasis in respiratory illness research that is based on clinical presentations has thus far centred on monoinfections, although in reality a substantial portion of patients may actually have two or more potential pathogens. our study shows that the prevalence of patients with two or more pathogens in a tropical setting was . %, most commonly due to virus-bacteria pairs. often, it seems that the role of 'less pathogenic' co-detected microbes are casually disregardedperhaps for ease of data interpretation. yet such assumptions are questionable especially because the impact of multiple pathogens on clinical characteristics has not been well studied. this formed the impetus for our analysis of the distribution of dual pathogens in ambulatory fri patients, and comparing associated clinical presentations between mono-and dual-pathogen patients. although we cannot conclude cause-effect relationships from the study, we noted a few interesting trends. the association between new recruits and multiple pathogens is likely due to the ease of transmission within the communal environment (of increased population density) on entry into military service, as described in clinical studies among similar cohorts. , these conditions also promote shifts in predominant circulating respiratory pathogens with time, as had been previously described, sometimes culminating in outbreaks of respiratory disease. , to prevent the occurrence of such incidents, mitigating measuressuch as appropriate education on hand and respiratory hygienehave been implemented. the higher prevalence of asthma in patients and the decreased number of pathogens among current smokers may also reflect the effects of the two on the upper respiratory tract. , for example, previous studies describe the effect of cigarette smoke in causing reduced competitive commensal organisms in the respiratory tract. , among dual infections, virus-virus pairs constitute only . % of the entire data set, within the lower end of range of viral co-infection studies in ambulatory settings ( . - . %). , , this may be due to local interactions between immune and microbial mechanisms preventing the occurrence of co-existing viral respiratory pathogens. such negative correlations have been previously described, including the replacement of one virus with another when the former is removed from the general population through vaccinations. the genus enterovirus was most prevalent ( . %) among viral-viral pairs, similar to two other viral coinfection studies reporting rhinovirus rates of . % and . %. , virus-bacterial pairs were most common, with a significant proportion involving adenovirus, particularly paired with h. influenzae ( . %). such a finding had also been previously observed among hospitalised children, where % of those with adenovirus were co-infected with various bacteria. previous chinchilla models on experimental otitis media also point towards possible synergisms between adenovirus and h. influenzae, although further studies are needed to conclusively determine whether such interactions exist in the upper respiratory tract. when it came to symptoms, the increased incidences of chills and rigors and elevated body temperatures in influenza a and influenza b, respectively, when paired with s. pneumoniae correspond to previous studies showing the disposition to superinfection caused by the influenza virus on respiratory epithelium, in both laboratory and hospital studies. [ ] [ ] [ ] [ ] [ ] our results show that these apply to ambulatory patients as well. however, we also noted that these systemictype symptoms appeared to be distinct from localised upper respiratory tract symptoms (such as running nose and cough), which were not found to be significantly different from patients with influenza alone. next, a higher prevalence of cough with phlegm was correlated with a number of dual-pathogen combinations, all of which involved the bacteria h. influenzae. although there are microbiological studies on the bacteria's interactions with rhinovirus, , there is insufficient information to conclusively explain the observations noted with parainfluenzae, warranting further studies. finally, diversity in the impact of dual pathogens on clinical manifestations, as seen through the results of other symptoms, is likely indicative of complex and diverse microbial interactions between respiratory pathogens in the upper respiratory tract. bosch et al. have detailed a number of known microbiologic mechanisms, including various modalities of synergisms and competition between species. these include pathogens that are usually associated with asymptomatic colonisation in healthy individuals (e.g. s. pneumoniae and h. influenzae), which are potentially pathogenic with shifts in the respiratory tract microenvironmentfor instance, the introduction of new microbes. [ ] [ ] [ ] many of these are not yet fully understood, and it is hoped that such epidemiological data may spur greater interest in co-pathogen microbiology research. our study does not explore patients infected with more than pathogens and co-pathogen pairs with < observations. although it identifies observed correlations between pairs and symptoms, it does not determine sequence of pathogens in relation to onset of symptoms or prove causality, which require further microbiological or case-control epidemiological research. severity of symptoms other than fever was not determined, actual diagnoses by doctors were not analysed, and further differences in the actual clinical impact could not be observed. although statistically significant differences have been described, the clinical significance of these findings have to be considered alongside as small differences may not be easily translatable to clinical practice and the large number of statistical comparisons increase the chances of type i (i.e. false-positive) errors. the study predominantly involved young adult males, limiting the generalizability to other populations. it is also conducted in a tropical setting with a fairly constant climate; thus, the effect of such changes on symptomology (e.g. in a temperate country) cannot be determined. by grouping pathogens of the same genus together in analysis, it is also not possible to determine whether specific subtypes are the cause for the observations made. we are unable to detect the presence of dual-pathogen patients involving two or more viruses from the same genus, especially within enteroviruses. although we compared differences in the detection of organisms between patients and controls, we are unable to conclude on whether certain organisms (such as n. meningitidis and adenovirus) are actually commensals, and pcr is not the optimal method for diagnosis of bacterial infections. we have described the aetiology of dual pathogens causing fri in the tropical setting and compared differences with monopathogens with regard to observed clinical manifestations. the presence of higher incidences of certain symptoms with specific pathogen pairs is indicative of underlying complex microbial interactions and affirms existing microbiological co-pathogen studies. however, many of these processes are still not well explored in existing literature, opening many opportunities for further research into this area. additional supporting information may be found in the online version of this article: figure s . proportions of each clinical symptom between dual pathogens and their mono-pathogen counterparts. table s . checkerboard of dual pathogens detected among cases. the dual-pathogen pairs for further analysis of symptoms (i.e. observations or more) are in bold. table s . mean proportions and comparisons between viral and bacterial mono-pathogens and dual pathogens. acute respiratory illness in the community. frequency of illness and the agents involved 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and viral infection increased h n infection rate in children with asthma cigarette smoking and mechanisms of susceptibility to infections of the respiratory tract and other organ systems recovery of potential pathogens and interfering bacteria in the nasopharynx of smokers and nonsmokers broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses single, dual and multiple respiratory virus infections and risk of hospitalization and mortality mixed infection is common in children with respiratory adenovirus infection synergistic effect of adenovirus type and nontypeable haemophilus influenzae in a chinchilla model of experimental otitis media immune dysfunction and bacterial coinfections following influenza insights into the interaction between influenza virus and pneumococcus influenza virus neuraminidase contributes to secondary bacterial pneumonia influenza a virus facilitates streptococcus pneumoniae transmission and disease interactions between streptococcus pneumoniae and influenza virus: a mutually beneficial relationship? influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam- and tlr expression human rhinovirus-induced isg selectively modulates epithelial antiviral immunity dynamics of nasopharyngeal colonization by potential respiratory pathogens human polymicrobial infections the ecology of nasal colonization of streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus: the role of competition and interactions with host's immune response the authors would like to acknowledge the staff from dso national laboratories for their tireless efforts in the collection of data and testing of laboratory samples, as well as staff from hq medical corps, singapore armed forces, and the centre for infectious disease epidemiology research (cider) at the saw swee hock school of public health, national university of singapore for their support. the programme was supported by a singapore ministry of defence-funded operational research programme and the centre for infectious diseases epidemiology and research in the saw swee hock school of public health of the national university of singapore and national university health system. the funders had no role in the study design, data collection, analysis, decision to publish or preparation of the manuscript. the authors declare that they have no competing interests. key: cord- -l q n ri authors: foss, stian; watkinson, ruth; sandlie, inger; james, leo c; andersen, jan terje title: trim : a cytosolic fc receptor with broad antibody isotype specificity date: - - journal: immunol rev doi: . /imr. sha: doc_id: cord_uid: l q n ri antibodies are key molecules in the fight against infections. although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. this postentry viral defense mechanism requires binding of the antibody to a cytosolic fc receptor named tripartite motif containing (trim ). in contrast to other fc receptors, trim shows remarkably broad isotype specificity as it does not only bind igg but also igm and iga. when viral pathogens coated with these antibody isotypes enter the cytosol, trim is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. in addition, inflammatory signaling is induced. as such, trim acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. here, we summarize our current understanding of how trim orchestrates humoral immunity in the cytosolic environment. antibodies are a crucial part of the immune response toward invading pathogens such as viruses, and the induction of so-called neutralizing antibodies is a primary goal of vaccination ( ) . in addition, there is great interest in the development of broadly neutralizing antibodies specific for major human pathogens such as human immunodeficiency virus (hiv) and influenza virus ( , ) . neutralization of viruses by antibodies is predicted to depend on high-affinity binding to specific epitopes of surface-exposed viral proteins that are required for binding to target cell receptors ( ) . such antibodies are thought to function according to the occupancy model that requires binding of a critical number of antibodies to a viral particle in such a way that most or all neutralizing epitopes are occupied ( ) . this may occur independently, or in concert with other antibody-mediated effector functions such as antibody-dependent cellular phagocytosis or antibody-dependent cellular cytotoxicity ( , ) . these effector functions are induced upon binding of antibody-virus immune complexes to classical fc c receptors (fccrs) expressed on the surface of hematopoietic cells such as natural killer (nk) cells, macrophages, and dendritic cells, which results in clearance and induction of t-cell responses ( ) . neutralizing antibodies also prevent infection in concert with non-classical fc receptors such as the neonatal fc receptor (fcrn) and the polymeric ig receptor (pigr) ( , ) . while fcrn mediates bidirectional transport of igg across mucosal epithelial surfaces ( ) ( ) ( ) ( ) ( ) , pigr mediates unidirectional transcytosis of iga and igm from the tissue into the luminal space ( ) . transcellular transport of neutralizing but also non-neutralizing antibody has been shown to facilitate protection against viral infection ( , ( ) ( ) ( ) ( ) ( ) ( ) . although neutralizing antibodies are produced during antiviral responses, the majority of the antibodies in the polyclonal response have no neutralizing activity by the classical definition ( , ) . this is due to the fact that they bind internal viral epitopes released from infected lysed cells, or viral surface proteins that are not involved in viral attachment and entry into host cells ( ) . in addition, viruses are known to display immune-dominant non-neutralizing epitopes that can bias the polyclonal response toward a nonneutralizing phenotype ( ) ( ) ( ) ( ) . as such, neutralization of viruses by antibodies has until recently been assumed to be a solely extracellular or intravesicular event. however, in recent years, it has become clear that the antiviral function of antibodies also extends into the cytosolic compartment of cells ( ) ( ) ( ) . this additional level of protection is orchestrated by the interferon (ifn)-inducible cytosolic fc receptor tripartite motif containing- (trim ). engagement of trim results in rapid postentry elimination of antibody-virus via recruitment of the proteasomal machinery ( , ) , in a mechanism termed antibody-dependent cellular neutralization (adin). concurrently, inflammatory signaling is also induced ( ) . therefore, antibodies that have failed to block entry of a virus particle into the cell and that is not intercepted by antibody-mediated effector functions operating in the extracellular environment may still be protective in the cytosolic compartment, even though they are, by the classical definition, non-neutralizing. instead, the cell takes advantage of trim to set up one last line of antiviral defense. trim can be distinguished from other fc receptors in two ways. firstly, trim shows remarkably broad antibody specificity as it can activate both of its functions upon binding to igg, igm as well as iga ( , ) , while other fc receptors display more restricted antibody isotype and subtype specificities ( ) ( ) ( ) . secondly, trim is broadly expressed by cells of most histogenic linages ( ) , while expression of classical fccrs is mainly restricted to hematopoietic cells ( ) . this suggests that a susceptible pathogen may be targeted by trim independently of the site of infection and local distribution of antibody isotypes. the trim -igg interaction was first described in a yeast two-hybrid screen in a study that investigated the role of trim as an autoantigen in immune disorders such as systemic lupus erythematosus and sj€ ogren's syndrome, in which it is referred to as ro or ss-a ( ) . in subsequent studies, trim was immunoprecipitated independently of antibody specificity, and the binding site for trim was postulated to be localized to the c h -c h interface of fc as it was found to compete with binding of staphylococcus protein a and streptococcus protein g ( , ) . furthermore, the corresponding binding site on trim was found localized to the c-terminal pryspry domain as truncation of this domain resulted in loss of binding. although the interaction between trim and igg was initially thought to be irrelevant due to the topologically distinct localization of the two proteins, a specific role in antiviral defense was more recently described ( ) . trim is an fc receptor that is structurally unrelated to all other classes of fc receptors ( , ) . it is part of the trim family which consists of over members in humans ( ) , with a diverse set of cellular roles including antiviral defense ( , ) . one of the most studied members is trim a, which mediates restriction of simian immunodeficiency virus via an antibody-independent mechanism ( ) . trim shares the same structural architecture as other trim proteins and consists of an n-terminal ring domain with e ubiquitin ligase activity, a b-box, and a central coiled-coil domain that is referred to as rbcc ( ) . it is, however, the c-terminal domain of trim proteins that determines ligand specificity and function, and in half of all known trim proteins this is a so-called pryspry domain. the pryspry domain of trim contains the antibody binding site, and is a globular fold comprising a b-sandwich of two antiparallel b-sheets connected by flexible loops ( ) , and is a fusion of pry and spry elements which are of distinct evolutionary origin ( ) . furthermore, trim proteins are known to form dimers or higher order structures via their coiled-coil domains and both heteromeric and homomeric trims have been described ( ) . crystallographic data of the trim coiled-coil have revealed that it has an antiparallel helical structure that places the n-terminal ring domains at opposite sides of the dimeric structure, while the c-terminal pryspry domains are positioned at the center ( ) . although a crystal structure of full-length trim has yet to be solved, the presence of the coiled-coil suggests that trim adopts a similar structural arrangement that would place its two pryspry domains in close proximity to each other. consistent with this, full-length trim has been shown to exist as a dimer in solution and form stable : complexes with human igg ( ) . thus, the two pryspry domains of a dimeric trim molecule may bind simultaneously to one igg fc ( ) . this symmetrical mode of binding will allow trim to rapidly intercept incoming antibodies ( , ) . a detailed understanding of the trim -igg interaction has been obtained from solving a co-crystal structure between the c-terminal pryspry domain of human trim and an fc fragment derived from human igg ( ) . the complex reveals a : stoichiometry where a pryspry domain binds to the interface between the c h and c h found on each side of the fc. as such, the binding site for trim is distinct from that of the classical fccrs and c q in the lower hinge and c h domain ( - ), but overlaps with the binding site for fcrn ( , ) as well as bacterial and viral fc receptors ( ) ( ) ( ) . in contrast to binding to fccrs, neither trim nor fcrn binding to igg is affected by removal of n -linked glycans of the c h domains ( ) . the core interaction site is formed between the spry element and the c h domain of igg fc ( , ) . here, the protruding and conserved fc loop encompassing residues - is inserted into a deep hydrophobic pocket on the spry surface where the apex fc residues h , n , and h (hnh motif) form a central hydrogen bond network surrounded by a hydrophobic shield of aromatic side chains that are engaged in aromatic stacking interactions. specifically, h fc and n fc interact with d spry located at the base of the spry binding pocket via hydrogen bonds, while h fc also interacts with w spry via aromatic stacking, which may also involve w spry . furthermore, h fc forms a hydrogen bond with d spry and stacks with f spry . in addition, i fc of the c h loop encompassing residues - is involved in a hydrophobic interaction with w pry . an overview of the co-crystal complex and the interaction network is presented in fig. . in contrast to the strict ph dependence of the fcrn-igg interaction, trim binds fc ph independently, but is sensitive to high salt concentrations ( ) . the interaction has been shown to be highly conserved in mammals, and displays wide cross-species reactivity as both human and mouse trim bind igg from a range of mammals ( ) . this is supported by inspection of the co-crystal structure between the mouse trim pryspry domain and an fc fragment derived from mouse igg a, which shows that the mouse and human mode of binding are highly similar ( ) . the binding affinity between human igg and the monomeric human pryspry domain has been measured to nm using isothermal titration calorimetry, while the murine interaction has an affinity of nm, where the human interaction occurs with more negative enthalpy ( , ) . however, the fact that full-length trim is a dimeric molecule has direct implications for its functional affinity to antibodies. the two pryspry domains of a dimeric trim molecule may bind simultaneously to one igg fc as suggested by the crystal structures ( ) . simultaneous engagement of both heavy chains likely explains the significantly increased interaction of dimeric trim , yielding an affinity of . nm for the igg-trim interaction as measured by fluorescence anisotropy ( ) . notably, this represents an increase of more than -fold relative to monomeric affinity, which makes trim the highest affinity fc receptor in humans. while fc receptors are highly selective in regard to isotype and subtype binding, trim not only binds igg but also igm and iga ( , ) . interestingly, the binding site for trim on iga and igm overlaps with that of fc a receptor i (fcari), fca/l receptor, pigr, as well as several pathogen-produced fc receptors ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the binding affinities of igm and iga toward monomeric human pryspry are considerably weaker, and have been measured to be and lm, respectively ( , ) . however, despite relatively weak monomeric binding, the dimeric nature of full-length trim suggests that the functional affinities for igm and iga are much stronger ( ) . assuming that the increase over monomeric affinity upon binding of full-length trim to igg is similar for igm and iga, the binding strength may be in the sub-micrometer range during physiological conditions ( ) . the weak monomeric binding of igm and iga is very likely due to the sequence variation in the stretch of amino acids corresponding to the c h -loop - of igg ( ) . specifically, the apex hnh motif of igg is pnr in igm and pla in iga. however, binding to trim is still accommodated, as there is structural conservation of the fc loop among the three isotypes. this has been addressed by molecular modeling showing that the structural loops in igg and igm superimpose closely at the secondary structure level allowing them to insert into the hydrophobic pryspry binding pocket ( ) . although the individual mutations h a, n a, n d, and h a in a murine igg antibody ( ) have been shown to abolish trim recognition, the specific replacements in igm and iga are predicted to form specific interactions with pryspry residues ( ) . in igm, conservation of the central n is thought to be crucial, as its hydrogen bonding potential with d spry is likely maintained, while replacement of h fc with proline will abolish its hydrogen bonding potential with d spry and its ability to interact with two fc residues simultane-ously. however, the proline residue may still interact with w spry and w spry . even though proline-tryptophan pairing is not a true aromatic stacking, it generates binding energy that is roughly double that of a hydrogen bond ( ) . furthermore, the replacement of h fc with arginine in igm abolishes its aromatic stacking potential with f spry , but the arginine may still bind d spry , given a favorable side chain conformation. in iga, p corresponding to h in igg is thought to have a similar role as in igm. the presence of l , the equivalent of n in igg, cannot interact with d spry , but is instead thought to form hydrophobic contacts with l s pry and l spry , while a fc is predicted not to contribute in binding ( ) . structural predictions of the interaction between igm and iga are shown in fig. . during infection with a non-enveloped virus, antibodies that are attached to the viral capsid are also carried into the cytosolic compartment of target cells where they are recognized by trim ( , ) (fig. ) . subsequently, trim engagement targets the virus for proteasomal degradation before it has had time to replicate and spread. this was first demonstrated using human adenovirus type (adv ) carrying the green fluorescent protein reporter gene as a model pathogen, incubated with anti-adv polyclonal serum from goat ( ) . upon infection of hela cells, protection from infection was found to correlate with the level of trim expression, as depletion of trim gave high infection levels while upregulation of trim by ifn-a stimulation increased adin ( ) . following endocytosis via the coxsackie and adenovirus receptor (car), adv in complex with non-neutralizing antibodies lyses the endosomal membrane and enters the cytosol where it is intercepted by trim . engagement of trim results in autoubiquitination in a manner that is dependent upon the e activity of its ring domain ( ) . ubiquitination leads to recruitment of the proteasomal machinery and rapid degradation of the viral complex, in a process that is perturbed by the proteasomal inhibitor, epoxomicin. the degradation process has been demonstrated to be strictly dependent on the aaa atpase v /vcp ( ) . vcp is a multi-domain protein complex that interacts directly with multi-ubiquitin chains via its n-terminal domain and is capable of extracting proteins from larger complexes as well as unfolding them ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . therefore, a general role for vcp in a preproteasomal degradation step has been suggested ( ) , and vcp was recently found to be recruited to stalled proteasomes ( ) . considering that the intact adv capsid measures nm in diameter ( ) , and that the diameter of the proteasomal barrel is only nm ( , ), such a predisassembly step is intuitively required ( ) . it is likely the challenging nature of the viral capsid rather than an intrinsic feature of adin that results in vcp dependence, as trim mediates the proteasomal turnover of ectopically expressed igg fc independently of vcp ( ) . the fast rate of adin is crucial as it must occur before adv reaches the nucleus for initiation of replication. this has been demonstrated using a fate-of-capsid assay, in which cellular levels of the major capsid protein, hexon, were degraded within h of infection ( ) . the adin mechanism is strictly dependent on ubiquitination, in which trim itself is the substrate for attachment of ubiquitin ( , ) . the ubiquitin machinery involves four enzymes: the ubiquitin activating (e ), the ubiquitin conjugating (e ), the substrate specifying ubiquitin ligase (e ), as well as the ubiquitin erasing deubiquitinase (dub), of which e is responsible for the ubiquitin chain linkage topology ( ) ( ) ( ) . the e enzyme ube w was recently shown to mediate attachment of k -linked monoubiquitin to trim ( ) . this makes trim accessible as a substrate for the e enzyme pair ube n/ube v , resulting in extension of k linked ubiquitin chain in vitro. furthermore, it was demonstrated that both ube w and ube n were required for attachment of both k and k -linked ubiquitin chains to trim in cells, in which the attachment of k -linked ubiquitin chains is thought to result in targeting to the proteasome ( ) . as trim undergo autoubiquitination, this could allow it to be effective against a diverse range of pathogens. this hypothesis is supported by the fact that antibody-coated beads transfected into cells become positive for ubiquitin, suggesting that any cytosolic particles bound by antibodies are potential targets for trim -mediated adin. during the initial stages of the antibody response, igm is the main antibody isotype produced. igm forms pentamers through association with the common joining chain (j-chain). iga is the main isotype at mucosal barriers and the second most prevalent isotype in serum at - mg/ml compared to mg/ml for igg. two subclasses of iga exist, iga and iga , and each is expressed as monomeric (miga), dimeric (diga), and secretory (s-iga) forms ( ) . in serum, miga is the prevalent form while s-iga dominates at mucosal sites ( , ) . immune complexes containing miga interact with fcari, which mediates phagocytosis, cellular cytotoxicity, antigen presentation, and cytokine release in professional cells, equivalent to the classical fccrs for igg ( ) . iga and igm bind the pigr, which mediates transport from the tissue into the lumen of body cavities, and upon release they are associated with the pigrderived secretory component (sc) to form s-iga and s-igm ( ) . interestingly, the sc-binding site on iga overlaps with the binding site for trim ( ), although sc is thought to associate with only one heavy chain in each iga monomer ( ) . this may explain the weak affinity between s-iga and the pryspry domain ( ) . during infection with adv , both igm-and iga-bound antibodies are recognized by trim and direct elimination of the virus via adin. this has been demonstrated with human igm and iga isolated from serum, and as observed for igg, ifn-a stimulation increases neutralization in a trim -dependent manner for both isotypes ( , ) . despite reduced affinity for s-iga, virus-specific s-iga from human serum was found to activate adin, as cellular depletion of trim partially restored viral infection. although the presence of the sc appears to inhibit trim function, data suggest that the disulfide bonds between iga and the sc may be broken upon exposure to the reducing environment of the cytosol, allowing attachment by trim ( ) . interestingly, in a recent study, it was demonstrated that s-iga forms trimers and tetramers in nasal secretions of healthy adults that contributed to enhanced neutralization potency ( ) . such higher forms of s-iga may likely reach the cytosol together with trim -susceptible pathogens. an intriguing feature of the adin mechanism is that only a few antibodies per virus are required for it to be effective as adin activity closely correlates with the expression level of trim rather than the extent of opsonization of the virus by a non-entry blocking antibody ( ) . using a murine monoclonal igg antibody ( c ) specific for the major capsid protein of adv, hexon, a non-entry neutralizing epitope, together with manipulation of cellular trim levels using ifn-a or trim shrna, allowed investigation of how antibody, trim and virus contribute to the efficiency of neutralization. in ifn-a-stimulated cells that expressed high levels of trim , as few as . antibody molecules per virus were found to be sufficient for neutralization, while five times more antibody was required in unstimulated cells ( ) . furthermore, adin was found to increase in a linearly incremental manner, demonstrating that each antibody associated with the virus contributes equally to its elimination upon binding to trim ( ) . this is in contrast to the occupancy model of entry neutralization, in which efficient prevention of infection can occur only after a critical number of antibodies occupy specific viral epitopes that are involved in cellular attachment and entry. consequently, this results in a lag phase in neutralization curves when infection levels are plotted against antibody concentration until the critical number of antibodies required to block entry has been reached ( ). this finding was supported by data demonstrating that polyclonal serumcontaining antibodies specific for adv required a higher antibody-virus stoichiometry for neutralization to occur when cellular trim was depleted, indicating a shift from cytosolic adin to entry blocking ( ) . thus, when the polyclonal response contains few entry neutralizing antibodies, adin may be responsible for the majority of the neutralization activity. low antibody-virus stoichiometry may also result in inefficient fccr-mediated effector functions by immune cells as efficient phagocytosis requires the formation of immune complexes and cross-binding to cell-surface fccrs. in addition, as trim also engages igm and iga, it is likely to contribute to early protection, and at the gate of entry of most viral pathogens, the mucosal barrier. this however, does not mean that trim -mediated protection becomes irrelevant in the later stages of infection when the amounts of entry neutralizing antibodies with high affinity are likely to increase. infection by several viruses has been shown to occur even at saturating levels of entry neutralizing antibodies ( ) . the virus particles that still infect their target cells under these conditions are known as the persistent fraction. in the case of adv , the magnitude of the persistent fraction has been shown to correlate with the expression level of trim , as a considerable increase in the persistent infection level was measured upon trim depletion ( ) . adin is also saturated at high viral loads, supporting the notion that trim levels are deterministic and suggesting that trim could be overcome by pathogens during high levels of viremia. as the efficacy of adin is also dependent on other cellular components such as vcp and the proteasome, the expression levels and rate of enzymatic turnover of these components are also likely to affect the ability of trim to induce adin ( ) . while extracellular detection of antibody-virus immune complexes by cell-surface-expressed fccrs results in phosphorylation of their intracellular immunoreceptor tyrosinebased activation motif, triggering phagocytosis and immune activation ( ) , known mechanisms for detection of pathogens in the intracellular environment occur independently of antibodies and are instead dependent upon recognition of pathogen-associated molecular patterns. this is achieved via pathogen recognition receptors (prrs) such as toll-like receptors ( ) , and cytosolic receptors for viral nucleic acid such as retinoic acid-inducible gene- (rig- ) and melanoma-differentiation associated protein (mda ) ( ). the immune system can also be activated by tissue damage as a result of pathogen replication via the mislocalization of selfmolecules. such host derived factors are known as damageassociated molecular patterns (damps) ( ) . even though the concentration of igg in human serum is as high as mg/ml, it is excluded from the cytosolic compartment of cells during normal physiological circumstances. however, during infection, when antibodies of the igg, igm, and iga isotypes enter the cytosol bound to a pathogen, they act like a damp upon trim recognition ( ) . importantly, in addition to adin, engagement of trim induces an antiviral state by activating inflammatory responses. unanchored k -linked ubiquitin is an immune second messenger that activates the transcription factor pathways nf-jb, ap- , irf , irf , and irf , via the enzyme complexes tak -tab -tab ( , ) and ikka-ikkb-nemo ( , ) , and inhibition of these complexes prevents trim -induced signaling ( ) . whenever a ubiquitin-modified substrate is targeted to the proteasome, there are three dubs in the s regulatory particle, usp /ubp , uch /uchl , and rpn /poh , that remove ubiquitin before substrate degradation ( ) . it was recently demonstrated that cells depleted of poh were unable to activate nf-jb signaling in response to adv -igg complexes, suggesting that poh cleaves off trim k -linked ubiquitin chains, which thereby activate an innate signaling response ( ) . this assure that trim only induces a signaling response if a virus antibody complex is targeted for proteasomal destruction, a mechanism that prevents adin from antagonizing signaling by rapid degradation of the virus, which thus synchronizes the activation of the two effector functions ( ) . activation of innate signaling pathways by trim induces rapid production of pro-inflammatory cytokines and chemokines, which has been shown to occur in both primary human lung fibroblasts and monocytes. specifically, the cytokines il- , il- , and ifn-c, as well as the chemokines cxcl and ccl were detected in response to igg-coated adv . furthermore, it was shown that inflammatory signaling was induced solely by trim upon detection of adv , and independently of known fccrs and prrs, as chemical inhibition of myd , trif, and the tyrosine kinase syk or knockdown of cytosolic rig- and mda did not impact signaling ( ) . activation of trim sensing places the cell in an antiviral state. this involves increased expression of major histocompatibility complex class i (mhc i), as well as the activating mhc i-like nk g d ligands in non-hematopoietic cells ( ) . as trim engagement results in rapid degradation of viral proteins, as well as inflammatory signaling that upregulates the expression of mhc i, it is possible that peptides derived from the virus could be transported via the transporter associated with antigen processing (tap) and loaded onto mhc i or nkg d molecules. this would then make the cell a target for the cytotoxic effects of nk cells and cd + t cells; however, this has not yet been tested experimentally. in principle, the cell could 'cure' itself from infection by adin rather than being killed by effector lymphocytes. induction of cell killing by lymphocytes requires clustering of mhc i molecules and t-cell receptors ( ) , and it may be speculated that the level of infection could be a factor that determines whether or not the cell would clear the virus solely via adin, or if t-cell help is required. an important question is how the induction of signaling via trim is regulated. the immune system has evolved specific mechanisms to prevent excessive inflammation and such regulation must also be active in the context of trim . for the classical fccrs, this is achieved by regulation of the expression levels of activating receptors and the inhibitory fccriib ( ) . as trim detection of cytosolic antibodies results in synchronized activation of both an effector and a signaling response, there must be strict regulation to avoid excessive inflammation at low pathogenic load. this may be a result of different requirements for activation of downstream ubiquitin-dependent processes, which must be related to how trim detects incoming antibodyvirus complexes, and is potentially also self-limiting because the signaling stimulus (i.e. intracellular antibody) is also rapidly degraded by the effector mechanism ( ). the full spectrum of pathogens that activates adin and trim signaling has yet to be determined. however, as both the effector and sensory functions of trim are strictly dependent on the presence of antibodies in the cytosol, the mechanism by which the pathogen enters the cell is crucial ( ) . regarding viral infections, the main targets for trim are non-enveloped viruses, as enveloped viruses shed their outer membrane coat upon fusion with the plasma or endosomal membrane and deliver their nucleocapsids into the cytosol. therefore, antibodies will be left behind at the cell membrane or inside endosomal vesicles. in line with this, the enveloped respiratory syncytial virus (rsv) is not detected by trim when added to cells in complex with anti-rsv antibodies ( ) . some non-enveloped viruses may also avoid trim detection due to their route of infection. for example, some rhinoviruses of the family picornaviridae are thought to inject their genome through a pore in the endosomal membrane, thereby leaving their nucleocapsid and any attached antibody behind inside the endosome ( ) . in the case of adv, its interaction with the cell-surface-expressed car triggers endocytosis ( ) . the virus then escapes endosomes and enters the cytosol by lysing the endosomal membrane, a process that relies on partial un-coating of the virus and exposure of an internal capsid protein that induces membrane damage ( ) . once inside the cytosol, the sub-viral particle uses microtubule-associated motor proteins to reach the nuclear membrane ( ) . subsequently, it docks to the nuclear pore complex through which the viral genome enters the nucleus ( ) . therefore, any attached antibody is readily accessible for trim recognition. viruses may also differ in their susceptibility to trim detection due to their behavior once they reach the cytosolic compartment ( , ) . although it is likely that some viruses have evolved to evade trim or even take advantage of trim as part of an un-coating mechanism, the rapid detection at a stage prior to synthesis of new viral proteins, as well as bridging by antibody, may make trim detection particularly difficult to antagonize ( ) . indeed, neutralization of several non-enveloped viruses by few antibody molecules has been described for adv, papillomaviruses, and poliovirus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . importantly, trim sensing is not restricted to virus infection as it can activate antibody-dependent nf-jb signaling in response to the intracellular bacterium salmonella enterica ( ) , which traffics between intracellular vesicles and the cytosol ( , ) . such trim -dependent immune signaling has been shown to be even more potent upon infection with a salmonella mutant (dsifa) that fails to maintain vesicle integrity, resulting in a greater proportion of bacteria in the cytosol ( ) . although bacteria are presumably too large to be degraded by the proteasome, and adin elimination of adv has been shown to happen independently of autophagy ( ), a possible link between trim and the formation of autophagosomes in the context of salmonella has been suggested ( ) . the fact that evolution has maintained strong binding between trim and antibodies across species points to its importance in host defense. humoral immunity during the course of an infection occurs by a variety of different mechanisms, and the relative contribution of each will depend on the nature of the pathogen as well as the polyclonal antibody response ( , , ) . therefore, to address whether or not trim makes a significant contribution to systemic protection in each case, this must be studied in the context of immune serum raised naturally against the pathogen. this has so far been investigated both in vitro and in vivo using the wildtype mouse adenovirus type- (mav- ) ( , ) which causes hemorrhagic encephalitis in mice. to recover from acute mav- infection, mice need a robust antibody response ( ) ( ) ( ) . the ability of anti-mav- serum to neutralize mav- was assessed in cells derived from wildtype or trim knockout mouse embryonic fibroblasts, and a significant proportion of neutralization was trim dependent ( ) . furthermore, upregulation of trim expression by ifn stimulation increased the adin potency of both pooled serum and immune serum collected from individual mice. the importance of trim for mav- protection in vivo was studied by comparing wildtype trim +/+ , trim heterozygote +/À , and trim knockout À/À c bl/ mice ( ) . upon challenge with mav- , it was shown that naive mice lacking trim had a higher viral load and increased mortality within the first week of infection. as these experiments were performed in naive animals, protection was presumably a result of interaction between anti-mav- igm and trim . in passive transfer experiments using antisera against mav- , it was demonstrated that survival closely correlated with trim expression, as transfer of serum was fully protective in wildtype, but not in trim -deficient, mice. heterozygous animals expressing intermediate levels of trim showed a haplo-insufficiency phenotype, with comparable mortality to mice homozygous null for trim , but an average brain viral load that was intermediate between wildtype and knockout animals at the end point of the experiment. moreover, in agreement with in vitro findings, ifn induction correlated with increased trim expression and reduced infection, highlighting the importance of trim for efficient adin activity. early anti-serum and diluted late anti-serum was more dependent on trim for protection compared to non-diluted late anti-serum ( ) . this indicates that when other antibodymediated mechanisms such as entry neutralization are incompletely protective, trim functions become increasingly important as they are able to operate under conditions of few antibodies per virus ( , ) . in addition, the use of a fully replicative wildtype virus in these studies demonstrates that trim is effective during a spreading infection ( , ) . as pathogens enter the body through mucosal surfaces, it raises an intriguing question as to whether trim is activated in polarized epithelial cells. iga and igm bind pigr, which mediates transport from the basolateral to the apical surface, and detection of viruses intracellularly may direct their transcytosis to the apical surface ( ) . in regard to igg, fcrn mediates bidirectional transport of monomeric igg or igg-immune complexes across polarized cellular layers ( ) ( ) ( ) ( ) ( ) . fcrn has even been shown to facilitate neutralization in a specific case where a ph-dependent igg antibody against influenza only neutralizes when the antibody-containing endosome undergoing fcrn transport fuses with a virus-containing acidic endosome ( ) . however, unpicking the antiviral contribution of immunoglobulin transport receptors like fcrn in vivo is complicated by their role in determining antibody serum half-life and biodistribution. an anti-hiv- igg engineered for improved fcrn binding was shown to have increased serum half-life, enhanced mucosal localization and superior protection against intrarectal infection with simian-hiv ( ) , but the mechanistic correlation between these properties was not determined. while there are many examples of viruses hijacking antibody transport receptors to facilitate infection, whether or not fcrn and pigr can directly prevent infection, or if there is crosstalk between these endosomal receptors and trim as they intercept viruses bound by antibody, remains to be addressed. during an infection, trim detects antibodies that are carried by pathogens into the cytosol. once antibodies are detected, trim mediates adin, a highly efficient viral elimination mechanism and concomitantly activates proinflammatory signaling resulting in a protective response. although the complete spectrum of pathogens that are susceptible has yet to be determined, the fast rate of detection and the adaptor function of antibodies are believed to make antagonism on the part of the virus particularly difficult. it is therefore likely that diverse non-enveloped viruses are targets for trim . trim has been shown to make a significant contribution to systemic protection, and thereby provide important immune defense alongside extracellular mechanisms. in addition, the ability of trim to interact with three antibody isotypes is a unique feature among known fc receptors. this, together with the broad expression profile of trim , allows it to function during all stages of an immune response at diverse sites. although the antibody binding properties and antiviral functions of trim have been well established, there are several questions regarding its biology that remain unanswered. of particular interest is how the effector and signaling responses are regulated in order to avoid excessive inflammation. as both arms of trim function depend on a complex sequential ubiquitination process, which results in synchronized activation, regulation may depend on different activation thresholds for downstream ubiquitindependent processes. it will also be important to address how trim synergizes with other immune responses. for instance, adin may contribute to cross-presentation of viral peptides on mhc class i molecules, resulting in the recruitment of cytotoxic lymphocytes to the infected cell. while trim is promiscuous in its antibody binding, the functional implications of this are largely unexplored as are the isotypespecific responses that might be elicited. for instance, while trim binds all four subtypes of human igg, it is not known whether their use during infection results in different responses. moreover, as the four igg subclasses differ significantly in their ability to interact with classical fc receptors, trim activity could be altered indirectly. the main gateway for viral pathogens into the body is via mucosal surfaces. this raises intriguing questions as to whether or not trim makes a significant contribution to protection at these body sites, and whether it functionally intersects with the transepithelial transport of antibodies by fcrn and the pigr. recently, it was demonstrated that complement factor c attached to different non-enveloped viruses can be carried inside the cytosol during infection ( ) . here, c detection resulted in proteasomal targeting of the virus and induction of inflammatory signaling in a process dependent upon mitochondrial antiviral-signaling protein (mavs) ( ) . as such, it could be speculated that other intracellular receptors specific for other immune serum proteins with antiviral functions have yet to be discovered. antibodies, viruses and vaccines broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses broadly neutralizing antiviral antibodies neutralization of animal viruses the antiviral activity of antibodies in vitro and in vivo broadly neutralizing hemagglutinin stalk-specific antibodies require fcgammar interactions for protection against influenza virus in vivo broadly neutralizing anti-hiv- antibodies require fc effector functions for in vivo activity fcgamma receptors as regulators of immune responses secretory iga: designed for antimicrobial defense immunoglobulin transport across polarized epithelial cells bidirectional fcrn-dependent igg transport in a polarized human intestinal epithelial cell line transcytosis and catabolism of antibody receptor-mediated immunoglobulin g transport across mucosal barriers in adult life: functional expression of fcrn in the mammalian lung fcrn-mediated antibody transport across epithelial cells revealed by electron tomography the recycling and transcytotic pathways for igg transport by fcrn are distinct and display an inherent polarity enhanced neonatal fc receptor function improves protection against primate shiv infection transfer of igg in the female genital tract by mhc class i-related neonatal fc receptor (fcrn) confers protective immunity to vaginal infection defense-in-depth by mucosally administered anti-hiv dimeric iga and systemic igg mabs: complete protection of rhesus monkeys from mucosal shiv challenge maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal fc receptor-mediated transcytosis intracellular neutralization of viral infection in polarized epithelial cells by neonatal fc receptor (fcrn)-mediated igg transport protective effect of rotavirus vp -specific iga monoclonal antibodies that lack neutralizing activity early high-affinity neutralizing anti-viral igg responses without further overall improvements of affinity the role of antibody concentration and avidity in antiviral protection antiviral antibody responses: the two extremes of a wide spectrum antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid neutralizing antibodies to adenovirus serotype vaccine vectors are directed primarily against the adenovirus hexon protein location, location, timing: analysis of cytomegalovirus epitopes for neutralizing antibodies hiv- evades antibodymediated neutralization through conformational masking of receptor-binding sites trim -dependent intracellular antibody neutralization of virus infection intracellular antibody immunity cellular self-defense: how cell-autonomous immunity protects against pathogens antibodies mediate intracellular immunity through tripartite motifcontaining (trim ) aaa atpase p /vcp is essential for trim -mediated virus neutralization intracellular antibodybound pathogens stimulate immune signaling via the fc receptor trim translocalized iga mediates neutralization and stimulates innate immunity inside infected cells fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond human antibody-fc receptor interactions illuminated by crystal structures specificity and affinity of human fcgamma receptors and their polymorphic variants for human igg subclasses the tripartite motif family identifies cell compartments protein-protein interactions between native ro and immunoglobulin g heavy chain the mw ro/ss-a autoantigen in sjogren's syndrome/systemic lupus erythematosus (ro ) is an interferongamma inducible tripartite motif protein associated with membrane proximal structures trim is a trimeric protein that binds igg fc via the b . domain structural basis for prysprymediated tripartite motif (trim) protein function trim is an igg receptor that is structurally, thermodynamically, and kinetically conserved trim proteins as ring finger e ubiquitin ligases trim family proteins: retroviral restriction and antiviral defence trimmunity: the roles of the trim e -ubiquitin ligase family in innate antiviral immunity anti-retroviral activity of trim alpha relationship between spry and b . protein domains. evolution of a component of immune defence? trim family: pleiotropy and diversification through homomultimer and heteromultimer formation the tripartite motif coiled-coil is an elongated antiparallel hairpin dimer the structure of a human type iii fcgamma receptor in complex with fc the . -a crystal structure of the human igg fc fragment-fc gammariii complex structure of fcgammari in complex with fc reveals the importance of glycan recognition for high-affinity igg binding mapping of the c q binding site on rituxan, a chimeric antibody with a human igg fc structural basis of phdependent antibody binding by the neonatal fc receptor structural insights into neonatal fc receptor-based recycling mechanisms crystallographic refinement and atomic models of a human fc fragment and its complex with fragment b of protein a from staphylococcus aureus at . -and . -a resolution model for the complex between protein g and an antibody fc fragment in solution crystal structure of the hsv- fc receptor bound to fc reveals a mechanism for antibody bipolar bridging structural requirements for the interaction of human iga with the human polymeric ig receptor the nonplanar secretory iga and near planar secretory iga solution structures rationalize their different mucosal immune responses identification of residues in the ch /ch domain interface of iga essential for interaction with the human fcalpha receptor (fcalphar) cd localization of the binding site for the monocyte immunoglobulin (ig) a-fc receptor (cd ) to the domain boundary between calpha and calpha in human iga the human iga-fc alpha receptor interaction and its blockade by streptococcal igabinding proteins streptococcal iga-binding proteins bind in the calpha -calpha interdomain region and inhibit binding of iga to human cd structural requirements for the interaction of human igm and iga with the human fcalpha/mu receptor sites in the ch domain of human iga that influence sensitivity to bacterial iga proteases regulation of virus neutralization and the persistent fraction by trim another role of proline: stabilization interactions in proteins and protein complexes concerning proline and tryptophane the aaa-atpase cdc /p regulates spindle disassembly at the end of mitosis cdc p is required for the cell cycle commitment point at start via degradation of the g -cdk inhibitor far p the ubiquitin-selective chaperone cdc- /p links myosin assembly to human myopathy emerging functions of the vcp/p aaa-atpase in the ubiquitin system distinct aaa-atpase p complexes function in discrete steps of nuclear assembly cdc /p promotes reformation of the nucleus by extracting the kinase aurora b from chromatin structure of the aaa atpase p valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation a conserved unfoldase activity for the p aaa-atpase in proteasomal degradation stalled proteasomes are directly relieved by p recruitment crystal structure of human adenovirus at . a resolution the s proteasome: assembly and function of a destructive machine structure, assembly and homeostatic regulation of the s proteasome sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim the e ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines noncanonical mms -encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for dna repair the mechanism of linkage-specific ubiquitin chain elongation by a single-subunit e the function of immunoglobulin a in immunity the immune geography of iga induction and function the structure and function of human iga iga fc receptors relationship of the quaternary structure of human secretory iga to neutralization of influenza virus occupancy and mechanism in antibody-mediated neutralization of animal viruses toll-like receptors and innate immunity viral evasion and subversion of pattern-recognition receptor signalling complexity of danger: the diverse nature of damage-associated molecular patterns trim is an innate immune sensor for the retrovirus capsid lattice direct activation of protein kinases by unanchored polyubiquitin chains activation of ikk by tnfalpha requires sitespecific ubiquitination of rip and polyubiquitin binding by nemo sensing of lys -linked polyubiquitination by nemo is a key event in nf-kappab activation trimming of ubiquitin chains by proteasomeassociated deubiquitinating enzymes mechanisms for t cell receptor triggering intracellular antibody-mediated immunity and the role of trim uncoating of human rhinoviruses drifting motions of the adenovirus receptor car and immobile integrins initiate virus uncoating and membrane lytic protein exposure adenovirus protein vi mediates membrane disruption following capsid disassembly dynein-and microtubulemediated translocation of adenovirus serotype occurs after endosomal lysis the role of the nuclear pore complex in adenovirus dna entry site of human rhinovirus rna uncoating revealed by fluorescent in situ hybridization opening of size-selective pores in endosomes during human rhinovirus serotype in vivo uncoating monitored by singleorganelle flow analysis two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at a resolution functional basis of poliovirus neutralization determined with monospecific neutralizing antibodies bivalent attachment of antibody onto poliovirus leads to conformational alteration and neutralization neutralizing antibody blocks adenovirus infection by arresting microtubuledependent cytoplasmic transport the interaction of neutralized poliovirus with hela cells. i. adsorption antipeptide antisera define neutralizing epitopes on the adenovirus hexon postentry neutralization of adenovirus type by an antihexon antibody neutralization of poliovirus by polyclonal antibodies requires binding of a single igg molecule per virion interaction between hela cells and adenovirus type virions neutralized by different antisera growth and killing of a salmonella enterica serovar typhimurium sifa mutant strain in the cytosol of different host cell lines autophagy recognizes intracellular salmonella enterica serovar typhimurium in damaged vacuoles antibody-and trim -dependent intracellular restriction of salmonella enterica simultaneous neutralization and innate immune detection of a replicating virus by trim intracellular antibody receptor trim prevents fatal viral infection fatal disseminated mouse adenovirus type infection in mice lacking b cells or bruton's tyrosine kinase mouse adenovirus type infection of natural killer cell-deficient mice mouse adenovirus type- replication is restricted to vascular endothelium in the cns of susceptible strains of mice intracellular sensing of complement c activates cell autonomous immunity this work was supported in part by the research council of norway through its center of excellence funding scheme (project number ). j. t. a. was supported by the research council of norway (grant no. / key: cord- -vzqof nj authors: mccallum, gabrielle b.; chatfield, mark d.; morris, peter s.; chang, anne b. title: risk factors for adverse outcomes of indigenous infants hospitalized with bronchiolitis date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: vzqof nj background: hospitalized bronchiolitis imposes a significant burden among infants, particularly among indigenous children. traditional or known risk factors for severe disease are well described, but there are limited data on risks for prolonged hospitalization and persistent symptoms. our aims were to determine factors (clinical and microbiological) associated with (i) prolonged length of stay (los); (ii) persistent respiratory symptoms at weeks; (iii) bronchiectasis up to ∼ months post‐hospitalisation; and (iv) risk of respiratory readmissions within months. methods: indigenous infants hospitalized with bronchiolitis were enrolled at royal darwin hospital between and . standardized forms were used to record clinical data. a nasopharyngeal swab was collected at enrolment to identify respiratory viruses and bacteria. results: the median age of infants was months (interquartile range – ); % male. on multivariate regression, our point severity score (including accessory muscle use) was the only factor associated with prolonged los but the effect was modest (+ . hr per point, %ci: . , . , p = . ). presence of cough at weeks increased the odds of bronchiectasis (or . , %ci: . , . , p = . ). factors associated with respiratory readmissions were: previous respiratory hospitalization (or . , %ci: . , . , p = . ) and household smoke (or . , %ci: . , . , p = . ). conclusion: increased severity score is associated with prolonged los in indigenous children hospitalized with bronchiolitis. as persistent symptoms at weeks post‐hospitalization are associated with future diagnosis of bronchiectasis, optimising clinical care beyond hospitalization is needed to improve long‐term respiratory outcomes for infants at risk of respiratory disease. pediatr pulmonol. ; : – . © wiley periodicals, inc. bronchiolitis is typically a self-limiting illness, but causes considerable morbidity and remains a leading cause for hospitalization among infants worldwide. the cost of hospitalized bronchiolitis has risen substantially in the usa over the last decade (increase of %). there are many studies that have reported on the risk factors for severe disease and length of stay (los) in hospital, for example, prematurity and cardio-respiratory disease. however, there is relatively little data on other factors (e.g. detection of bacteria with viruses) associated with los in hospital in an at-risk population (e.g., indigenous children who have more severe bronchiolitis) and future bronchiectasis. factors associated with severe illness and/or prolonged los include clinical severity (assessed by scoring systems), viruses, , , and secondary bacterial infection. respiratory syncytial virus (rsv) is implicated for - % of cases and is associated with more severe disease in some studies [ ] [ ] [ ] but not in others. whether single versus multiple viruses influence disease severity is also controversial. , further, a number of non-classical respiratory viruses (e.g. coronaviruses, bocaviruses) have been identified but the clinical relevance of these remains unclear, as these non-classical viruses are commonly present in asymptomatic children. in a hospital-based study, a virus (including non-classical viruses) was detected ( %) in the nasopharynx of children who did not have any respiratory symptoms. children with severe bronchiolitis requiring intensive care are likely to have a secondary bacterial infection. however, studies on bronchiolitis to date have not examined whether the presence of respiratory bacteria in the upper airways influences clinical outcomes in children hospitalized with bronchiolitis. examination for bacteria is not routine in bronchiolitis management. however, it is plausible that secondary bacterial infection that occur post viral infections are more likely when the nasopharynx is colonized with bacteria, as found in some populations such as in northern territory indigenous infants. in these children, early (at $ weeks of age), common (up to % of infants) and dense acquisition of respiratory bacteria (streptococcus pneumoniae, haemophilus influenzae and moraxella catarrhalis) have been documented. thus, in study settings like ours, the presence of bacteria in the nasopharynx may be particularly relevant. secondary bacterial infection may complicate bronchiolitis, and possibly contribute to poorer long-term outcomes such as chronic wet cough and bronchiectasis. it has been shown in children aged years hospitalized with first time wheezing, co-detection of viruses ( %) and bacteria ( %) in nasopharyngeal aspirates resulted in prolonged los. yet, it remains unknown whether co-detection of bacteria with viruses in the nasopharynx at the point of bronchiolitis hospitalization contributes to los. this data is lacking and is especially important for populations at high risk of poorer outcomes (e.g. secondary bacterial pneumonia, chronic wet cough etc). , further rationale for assessing viruses and bacteria in the infants involved in our studies was described in a previous paper. beyond hospitalization, persistence of respiratory symptoms (i.e., post-bronchiolitis syndrome) is increasingly appreciated. , cohort studies report symptoms in up to % of infants, - days post-hospitalization. , there are few data on what happens to these children in the medium term ( weeks to months). determining the clinical relevance of symptoms beyond hospitalization is particularly important in populations, for example, indigenous children who have a high risk of bronchiectasis. in the absence of any data from indigenous children, we combined data from three prospective studies that included indigenous infants hospitalized with a clinical diagnosis of bronchiolitis, to examine factors (clinical and microbiological) on admission that were associated with (i) prolonged los (aim- ); (ii) presence of persistent symptoms weeks after hospital discharge (aim- ); (iii) whether presence of cough at weeks was associated with bronchiectasis up to $ months posthospitalisation (aim- ); and (iv) re-hospitalization within months for a respiratory illness (aim- ). we hypothesized that co-detection of viruses and/or bacteria in the nasopharynx is associated with longer los, persistent respiratory symptoms and re-hospitalization with a respiratory illness within months of discharge. data from three prospective studies were combined ( fig. ) for the different aims; two studies were randomized controlled trials (rct) , and one a cohort study. here, we briefly describe these studies as the methods have been published. , , both rcts aimed to determine if different durations of azithromycin, compared to placebo, improved clinical outcomes for infants hospitalized with bronchiolitis (i.e., los, oxygen requirement and respiratory readmissions within months of hospital discharge). the cohort study aimed to determine the validity and reliability of a severity scoring system among infants presenting to royal darwin hospital (rdh) with bronchiolitis ( fig. ) . rdh is a bed referral center, servicing $ , people with geographical coverage of $ , km. in this study, only indigenous infants were included as our previous work showed being indigenous as an independent risk factor for more severe bronchiolitis. infants were eligible if they were from darwin, months (rct- ) or months old (rct- and cohort study), , and hospitalized with bronchiolitis. infants were excluded if they had very severe disease (admitted to intensive care), chronic lung disease, congenital heart disease, contraindications to macrolide use (rct- and rct- ), received macrolides (in last days), diarrhoea, or clinical and radiological features consistent with a primary diagnosis of pneumonia (as diagnosed by the attending medical team). infants were similar and contributed data only once for this study. standardized data collection forms were used to record demographic, medical history and clinical data ( table ) . other therapies and routine investigations were also documented. infants who received additional trial medication (e.g., azithromycin) were treated the same for this analysis as those who received placebo, as no clinically significant differences in either rct were observed. , severity was assessed using a score we validated in our setting. the score comprised of four components (respiratory rate, accessory muscle use, degree of wheezing and spo ). each component scored between and , providing a composite score between - . los was defined by the treating medical team as time from admission, to time for "ready for discharge" (sp consistently > % in air for > hr) and feeding adequately. infants were clinically reviewed by research nurses (urban-based infants) or at their local health clinic (remote-based infants) weeks after hospital discharge, to determine presence of persistent respiratory symptoms and signs. remoteness was described as more than km from a tertiary hospital. any respiratory readmission and investigations for bronchiectasis were monitored by a hospital based study (hrec / ). as rdh is the only hospital our target population accesses, all hospitalizations were accurately captured. a nasopharyngeal swab (nps) at enrolment was tested for common respiratory bacterial pathogens (streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and staphylococcus aureus) were identified on culture, data were entered on an access database and analyzed using stata version (statacorp college station, tx). as data were skewed, we log-transformed our main outcome measure los. exponentiating linear regression coefficients of log los gave us the multiplicative effect of a risk factor on geometric mean los, which we re-expressed (by assuming the median los for the reference group was hr) as the approximate increase in median los in hours. logistic regression was used to examine the association of independent variables on presence of cough at weeks, and respiratory readmissions within months. all factors with univariate p < . were included in multivariable regression models (except for viruses-rsv was entered and other variables relating to viruses were not). a two-tailed p . was considered significant. as this was a secondary analysis of available data, sample size calculation was not undertaken. demographic data are summarized in table for the indigenous infants included in this study ( % of the were from rct- , rct- % and cohort- %, fig. ). most children were aged months ( %), not born premature ( % born ! weeks) and required o supplementation ( %). no child required intensive care transfer. from the nps of indigenous infants (one refused, two did not provide consent for nps), ( %) had one or more viruses identified. rsv was the most common followed by hrv, wupyv and adenovirus (table ). at least one type of respiratory bacteria was detected in ( %) infants. the most common bacteria cultured was moraxella catarrhalis, followed by haemophilus influenzae, and streptococcus pneumoniae. the nps from ( %) infants was negative for both bacteria and virus. on univariate analysis, factors on admission significantly associated with prolonged los were age and severity score (table ) . notably, antibiotics prior to hospitalization, any virus and co-detection of virus/ bacteria were not associated with longer los. on multivariate regression, the only factor that remained significant was severity score. detection of rsv increased los by hr (p ¼ . ), but this did not reach statistical significance. we then examined which component of the severity score contributed the most to los (table ). in the univariate analysis, (considering each of the four components as single factors) all components on univariate analysis, factors at enrolment associated with prolonged los were age and severity score. on multivariate analysis, only severity score remained significant. à arising as a re-expression of the multiplicative effect on the geometric mean from a linear regression on log (los). except wheeze were significantly associated with prolonged los. on multivariate regression, only the "accessory muscle use" component remained significant. we repeated the main analyses, restricting the cohort to the n ¼ infants who had no previous respiratory hospitalizations. on multivariate analysis, the only factor which significantly prolonged los was the severity score (p ¼ . ). one hundred and fifty seven indigenous infants who had a clinical review at weeks contributed to this analysis (fig. ) . persistent respiratory symptoms were frequent beyond hospitalization (table ) . on univariate analysis, factors significantly associated with presence of cough weeks after discharge was age and presence of "any bacteria" cultured on nps. no factors remained significant on multivariate regression. as of th may , / ( %) infants had a chest ct scan requested by their treating pediatrician for clinical reasons when seen months (interquartile range (iqr) - ) post-hospitalization for bronchiolitis. all ( %) children had bronchiectasis documented in the ct scan, performed at median months (iqr - ) post the index bronchiolitis hospitalization. infants with persistent cough at weeks after hospitalization were significantly more likely to have bronchiectasis compared to those without a cough (or . , %ci: . - . , p ¼ . ). on univariate analysis, factors that significantly increased the odds of respiratory readmission -months after discharge were previous respiratory hospitalization in the univariate analysis, all four components except wheeze contributed to prolonged los. on multivariate regression, only accessory muscle use remained significantly associated with prolonged los. à arising as a re-expression of the multiplicative effect on the geometric mean from a linear regression on log (los). and exposure to household smoke. factors that significantly reduced the odds of readmission were detection of "any virus," "any bacteria," rsv and "rsv and hrv" on nps (table ) . on multivariate regression, previous respiratory hospitalization and exposure to household smoke were factors that significantly increased the odds of readmission but the presence of "any bacteria" and rsv reduced the odds of readmission ( table ). the presence of any respiratory abnormality (i.e., presence of cough, wheeze, or crackles) at weeks was not associated with the odds of respiratory readmissions (or . , % ci: . - . , p ¼ . ). in our study involving indigenous infants hospitalized with bronchiolitis, we found that a higher severity score on admission, particularly use of accessory muscles, was associated with prolonged hospital stay. the presence of a virus or viruses detected on hospitalization on multivariate regression, previous respiratory hospitalisation and exposure to household smoke significantly increasing odds of readmission but the presence of any bacteria and rsv reduced the odds of readmission. was not associated with poorer outcomes. beyond hospitalization, factors associated with presence of cough at weeks were previous respiratory hospitalization and presence of bacteria. by months, infants previously hospitalized with a respiratory illness and exposed to household smoke had an increased odds of re-hospitalization for a respiratory illness. in contrast however, infants with presence of rsv and culturable bacteria detected in nps had decreased odds of rehospitalization at months. further, infants who were coughing at weeks post-hospitalization were significantly more likely to be diagnosed with bronchiectasis a median of months later. our study involved only indigenous children as recurrent hospitalizations for respiratory infections and chronic suppurative lung disease is prevalent in our setting. indigenous children living in usa, canada, australia and new zealand share many similarities with respect to the burden of respiratory illness. thus, we were interested in finding factors associated with poorer clinical outcomes and possible future intervention points. studies on hospitalized bronchiolitis in canadian children found ethnicity, apnoea or respiratory arrest prior to hospitalization or pulmonary consolidation to have more complicated hospitalization. further, previous rsv bronchiolitis hospitalization was associated with chronic cough and recurrent respiratory infections in early childhood. well known or "traditional risk factors" associated with severe bronchiolitis in infants include prematurity, cardio-respiratory disease, and being indigenous. , in contrast, there are a few prospective studies which examined other factors that prolonged los. in these studies, factors significantly associated with prolonged los were young age, increased work of breathing, heart rate, respiratory rate, dehydration, hypoxia on admission, spo < %, respiratory distress assessment instrument (rdai) score > , apnoea, weight, rsv, ethnicity, and winter season. , [ ] [ ] [ ] retrospective studies have described that rsv, ethnicity, congenital syndromes, neuromuscular disorders and existing chronic respiratory diseases were associated with prolonged los. in our study, severity score on admission, particularly use of accessory muscles, was the only factor to prolong los, once other factors (n¼ ) were accounted for in the multivariate regression. none of the previous studies however had dissected which component of the scoring system contributed the most to los. further, despite the many studies undertaken for bronchiolitis, most scoring systems used were not validated. , , indeed the most commonly used system, the rdai score, has limited validity when systematically examined. within our study, we had validated the scoring system used and showed good inter-and intrarater reliability. the type and number of viruses (particularly rsv and hrv) did not significantly influence los in our study. our results are similar to some studies, , but dissimilar to others where rsv and/or hrv influenced disease severity. , , reasons for these variations are likely multi-factorial including sample size, different methodologies, sampling frame, age, number and types of viruses investigated. , we also found that although respiratory bacteria (h. influenzae, s. pneumoniae, and m. catarrhalis) and/or viruses were common ( %), their presence did not prolong los. while data relating to los and type of virus detection has been previously examined, none of these studies have examined the influence of the presence of bacteria in the upper airways. we found that the presence of bacteria detected in the nasopharynx with or without concurrent viruses did not influence los. our finding of high prevalence of concurrent bacteria with viruses (i.e., %) in the children's nasopharynx is similar to that reported by bisgaard and colleagues among young children of asthmatic mothers with acute wheeze in a non-hospitalised cohort. beyond the hospital phase, our study reported several novel outcomes. at weeks post-hospitalisation, the prevalence of respiratory symptoms in our study ( %) was similar to other studies ( - %). , , few studies however have prospectively recorded respiratory symptoms post-hospitalization, and no studies have examined for predictors of persistent respiratory symptoms otherwise known as "post-bronchiolitis syndrome." while presence of bacteria in the nps on admission was not significantly associated with persistence of cough at weeks, chronic cough is the most common symptom of an underlying lung disease and causes poorer quality of life. in this study, persistent cough was however a significant risk factor (or . , %ci . - . , p ¼ . ) for ct-confirmed bronchiectasis months later. bronchiectasis is particularly common in indigenous infants in the usa, australia and new zealand and hospitalization for respiratory disease is an independent risk factor. in the context that early treatment of bacterial infections in the lower airways prevents long-term lung damage, , our post-hospitalization data is both novel and important in high-risk population groups (e.g., indigenous children). post-bronchiolitis syndrome is likely highly important especially in high-risk populations. our data raises an important public health issue with respect to follow-up of children with bronchiolitis, to optimise clinical care beyond the immediate hospitalization phase to improve long-term respiratory outcomes for high-risk populations. we suggest that children should be clinically reviewed between weeks - after hospital discharge to assess for persistent symptoms and signs and managed accordingly. there is currently no high quality evidence on the most appropriate intervention in any groups of children with post-bronchiolitis syndrome. however, until further evidence is available, we suggest the use of antibiotics when the cough is wet and not improving in a group at high risk of chronic suppurative lung disease, in line with the treatment of protracted bacterial bronchitis. our post-hospitalization data also showed that children exposed to environmental tobacco smoke and those previously hospitalized with a respiratory illness were twice as likely to be readmitted with a respiratory illness within months of hospital discharge. surprisingly, we found that presence of rsvon admission and "any bacteria" significantly reduced the odds of re-hospitalization by months post index hospitalization. whether respiratory readmissions in this population were related to the initial bronchiolitis episode, a new infection or other causes can only be postulated. one possible reason for this could be that rsv detection is a marker (as opposed to a cause) for future asthma and as the majority of paediatric asthma is mild, having asthma is less likely to lead to re-hospitalisation compared to a respiratory infection in our setting. our study has several limitations. firstly, our sampling frame is restricted to indigenous children without the well-known, traditional risk factors (e.g. chronic lung disease, cardiac disease) and who did not require intensive care. reasons for our sampling frame were discussed above but it also means our data cannot be extrapolated to the general population or to non-hospitalized infants. secondly, we did not exclude infants that had been hospitalized previously; however, exclusion of these infants did not alter our main results. thirdly, % of children received antibiotics during hospitalization. this may have impacted on nasopharyngeal bacterial carriage, however removing these children did not alter our main results. fourthly, the children were systematically reviewed only at one time point, that is, at weeks posthospitalization. the decision by the treating paediatricians who reviewed these infants at a median of months post-hospitalization to subject the child to a chest ct scan was based on clinical assessments, not systemized and thus subject to external biases. thus, we could not systematically analyze the factors on admission that are associated with future development of bronchiectasis. also, while long-term, prospective, follow-up studies in high-risk populations are required, we cannot unethically subject all children to a chest ct (given the known risk of radiation in young children). in the first prospective study of indigenous infants hospitalized with bronchiolitis with post-hospitalization data at weeks and months, we found that the severity score on admission, particularly accessory muscle use, was the sole factor associated with prolonged los once other factors (clinical and microbiological) were accounted for. persistent respiratory symptoms at weeks increased the risk of detecting bronchiectasis and factors associated with persistent symptoms were prior hospitalisation for a respiratory illness and presence of bacteria on the nps on admission. thus, clinical review, with early treatment when necessary, should be undertaken in populations at high risk for chronic respiratory disease. follow-up studies beyond months are necessary to consolidate our findings and determine intervention points that can reduce the high burden of respiratory diseases in at-risk populations. anne chang conceptualized the study with modifications by peter morris and gabrielle mccallum. gabrielle mccallum designed the data collection instruments, collected data, coordinated the study, and carried out initial data analysis and manuscript. mark chatfield provided statistical support, carried out further data analysis, and edited the manuscript. all authors assisted in data interpretation, contributed to the manuscript and approved the manuscript as submitted. hernandez-cancio s. clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis temporal trends in emergency department visits for bronchiolitis in the united states sign guideline on bronchiolitis in infants risks of severity and readmission of indigenous and non-indigenous children hospitalised for bronchiolitis a single dose of azithromycin does not improve clinical outcomes of children hospitalised with bronchiolitis: a randomised, placebo-controlled trial clinical risk factors are more relevant than respiratory viruses in predicting bronchiolitis severity high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus 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fibrosis chronic suppurative lung disease/ bronchiectasis nasopharyngeal bacterial colonization during the first wheezing episode is associated with longer duration of hospitalization and higher risk of relapse in young children randomized placebo-controlled trial on azithromycin to reduce the morbidity of bronchiolitis in indigenous australian infants: rationale and protocol duration of illness in ambulatory children diagnosed with bronchiolitis duration of illness in infants with bronchiolitis evaluated in the emergency department three-weekly doses of azithromycin for indigenous infants hospitalised with bronchiolitis: a multicentre, randomised, placebo-controlled trial severity scoring systems: are they internally valid, reliable and predictive of oxygen use in children with acute bronchiolitis? streptococcus pneumoniae and noncapsular haemophilus influenzae nasal carriage and hand contamination in children-a comparison of two populations at risk of otitis media lung disease in indigenous children pediatric investigators collaborative network on infections in canada (picnic) prospective study of risk factors and outcomes in patients hospitalized with respiratory syncytial viral lower respiratory tract infection descriptive epidemiological features of bronchiolitis in a population-based cohort bronchiolitis study group for the pediatric emergency care applied research n. bronchiolitis: clinical characteristics associated with hospitalization and length of stay a validated clinical model to predict the need for admission and length of stay in children with acute bronchiolitis g (p) what predicts duration of hospital stay for bronchiolitis? factors predicting prolonged hospital stay for infants with bronchiolitis wheezing in infants: the response to epinephrine what is the clinical relevance of respiratory syncytial virus bronchiolitis?: findings from a multicenter, prospective study risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis association of bacteria and viruses with wheezy episodes in young children: prospective birth cohort study duration of symptoms of respiratory tract infections in children: systematic review a multicenter study on chronic cough in children: burden and etiologies based on a standardized management pathway state of the art-chronic wet cough: protracted bronchitis, chronic suppurative lung disease and bronchiectasis prospects for prevention of chronic bronchitis and bronchiectasis; rational management of bronchopulmonary infections by penicillin aerosol therapy antibiotics for persistent cough or wheeze following acute bronchiolitis in children chronic wet cough: protracted bronchitis, chronic suppurative lung disease and bronchiectasis we are grateful for all infants and families who participated in this study. we thank the remote health clinics for their support and attending the clinical review. we thank lesley versteegh and clare mckay for enrolling participants and collecting the clinical data. we thank vanya hampton, donna woltring, jemima beissbarth, jane gaydon and rebecca rockett for processing the viral and bacterial samples. key: cord- -mbwgi x authors: pang, junxiong; jin, jing; loh, jin phang; tan, boon huan; koh, wee hong victor; ng, sock hoon; ho, zheng jie marc; gao, qiuhan; cook, alex r; hsu, li yang; lee, vernon j; chen, mark i cheng title: risk factors for febrile respiratory illness and mono-viral infections in a semi-closed military environment: a case-control study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: mbwgi x background: febrile respiratory illness (fri) results in substantial burden in semi-closed environments. tackling risk factors may reduce transmission and infection. however, risk factors involved in one setting may not be generalizable in all settings due to differences in climate, residential environment, population genetic and cultural backgrounds. this study aims to identify risk factors of fri and mono-viral infections in a tropical military environment. methods: from year to , military personnel with temperature ≥ . °c, cough and/or sore throat, and personnel with no fever or no respiratory symptoms were recruited as cases and controls, respectively. subjects provided nasal wash specimens and answered a standardized questionnaire. resplex assays were used to determine the viral etiologies. descriptive, univariate and multivariate analyses of the variables were performed using appropriate descriptive tests and logistic regression modelling, respectively, with r program. results: a total of , fri cases and , non-fri study controls were recruited. increasing age [adjusted odds ratio (aor) = . ; % confidence interval (ci) = . - . ], recruit camp (aor = . ; % ci = . - . ) and smoker (aor = . ; % ci = . - . ) were independent risk factors of fri. malay ethnicity was positively associated with influenza a(h n )pdm (aor = . ; % ci = . - . ) and coxsackie/echovirus (aor = . ; % ci = . - . ) mono-infection. significant contact risk factors were stay-out personnel with ill household member (aor = . ; % ci = . - . ), and stay-in personnel with ill bunkmate and household member (aor = . ; % ci = . - . ). staying in camp with none ill in bunk and at home was a protective factor against fri (aor = . ; % ci = . - . ). these contact risk factors were similarly observed for the five most common viruses detected, namely adenovirus, rhinoviruses, influenza a and b, and coxsackie/echovirus. conclusion: increasing age, smoker, recruit-camp, stay-out personnel with ill household members and stay-in personnel with ill bunkmates were independent risk factors of fri in a semi-closed military environment. early identification and isolation of ill personnel from their bunk may be effective to prevent and reduce transmission and disease burden. febrile respiratory illness (fri) results in substantial disease burden in semi-closed environments such as in the households [ ] and militaries [ ] [ ] [ ] . fri is most commonly caused by viral infections, as observed in military respiratory surveillance programmes in finland [ ] , united kingdom [ , [ ] [ ] [ ] [ ] , netherlands [ ] , france [ , ] , south korea [ ] [ ] [ ] , west africa [ ] , taiwan [ ] , china [ ] , singapore [ ] [ ] [ ] [ ] [ ] [ ] , and the united states [ , [ ] [ ] [ ] [ ] [ ] . identifying risk factors of infection may provide guidance on policies and strategies for the prevention and control of fri. previous documented risk factors of fri in other countries included body mass index equal or greater than kg/m , previous respiratory tract infections [ ] , overcrowding and closed units [ , [ ] [ ] [ ] , presence of sand and dust storms, extreme temperature changes [ , ] , smoking [ ] , female, navy service, poor latrine facilities, increasing age and higher rank [ ] . however, these risk factors may not be generalizable to different environments, and may differ between specific predominant aetiological agents. the predominant viruses reported in the singapore armed forces (saf) comprised adenovirus, rhinoviruses, influenza a and b, and coxsackie/echovirus between and [ ] . adenovirus-associated respiratory disease, outbreaks and death have been reported in several countries amongst military recruits [ , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] . males and close contact with a person with respiratory symptoms within days before their own onset of illness were associated with adenovirus infection, but sleeping adjacent to someone ill with respiratory symptoms did not present higher risk to infection [ ] . influenza a and b viruses have also resulted in much morbidity in outbreaks, particularly influenza a(h n )pdm virus infection [ , , , , , , ] . some of the risk factors proposed were crowded living quarters defined as more than three personnel and age group less than year old [ ] , asthma and obesity [ ] , age group less than years old and the high proportion of military who had being seroconverted [ ] . human rhinoviruses are known to cause common cold as well as more complicated respiratory infections [ , [ ] [ ] [ ] [ ] [ ] . all known human rhinoviruses have been reported to be present in military recruits during respiratory infection [ ] . association of rhinovirus with lower respiratory tract infections is well documented [ ] . viral interference has also been proposed between rhinovirus and adenovirus infection [ ] . stress factor due to adaptation to new and different surroundings for military recruits was also proposed as risk factor for rhinovirus infection [ ] . in this study, we investigate the risk factors associated with fri and the predominant viral aetiologies of fri in a semi-closed military environment of the saf. the saf started a sentinel respiratory disease surveillance program in four major camps (including a recruit training camp) in may [ , ] to track febrile respiratory illness (fri) cases defined as patients with temperature ≥ . °c with cough or sore throat. patients visiting primary healthcare clinics in the camps between may and october during regular consultation hours who met the fri criteria were recruited. the sentinel respiratory disease surveillance program includes the written informed consent obtained by healthcare workers, a questionnaire, clinical specimens collection and a clinical examination of the participants. repeat consultations were excluded if the healthcare worker determined that the patient had not recovered from the first episode of illness. we also obtained samples from controls (those without respiratory symptoms or acute infections), who were recruited from the same medical center during the same week as the recruitment of cases with about to controls per week. this is to ensure that both cases and controls had similar health-seeking behaviour, and similar chance of exposure to a particular respiratory pathogen circulating in the same environment around the same period of the year to minimize potential misclassification bias. moreover, the controls were not matched or restricted by barrack, sex, age or symptom-onset. this is because of the fact that the aim of the study is to evaluate most of these variables as potential risk factors of fri. informed consent, the baseline questionnaire, and clinical specimens were also obtained from these controls. the questionnaire covers demographics, co-morbidities, vaccination status, stay-in camp status and contact details of ill member in bunk (for stay-in personnel) and at home (for both stay-in and stay-out personnel). stay-in personnel stay in camp on weekdays and stay outside camp only on weekends, and hence, have household members and bunkmates as their key contacts. stay-out personnel do not stay inside camp on weekdays and have to travel in and out of camp on weekdays to work. these stay-out personnel hence only have household members but no bunkmates as key contacts. before the influenza a(h n )pdm pandemic in , trivalent inactivated seasonal influenza vaccine (pre-pdm tiv) was in use. then, the pandemic monovalent influenza a(h n )pdm vaccine [pdm-a(h n )v] was first introduced to saf and administered to all recruits only from december . this was superseded by the new trivalent influenza vaccine (post-pdm tiv) which included the influenza a(h n )pdm strain, first introduced to saf in october , and routinely administered to all recruits in december , and then all other military personnel in november (fig. ). nasal washes from both side of the nose were taken by certified medical staff and sent to the laboratory for etiological testing within h. detailed laboratory methods have been described in a previous study [ , ] . briefly, we used a multiplex pcr panel which included different respiratory viruses. they are as following-adenovirus e, influenza a(h n ), rhinovirus, coxsackie/echovirus, influenza b, influenza a(h n )pdm , enterovirus (ev), human metapneumovirus (hmpv), parainfluenza (hpiv- ), hpiv- , hpiv- and hpiv- , coronavirus oc (cov-oc ), cov-nl , cov- e, cov-hku , respiratory syncytial virus a (rsv-a) and rsv-b and bocavirus (bv). additional singleplex pcr assays were then performed to determine the influenza subtype. total nucleic acids were extracted from each clinical specimen using the dna minikit (qiagen, inc, valencia, ca, usa) according to the manufacturer's instructions. a total of μl of dna extract were tested with resplex i and ii (version . , qiagen, inc., valencia, ca, usa) for the presence of respiratory micro-organisms on the liquichip workstation, according to the manufacturer's instructions. specimens that were resplex ii positive for flu-a were further subtyped with real-time pcr for h or h , or for ph n . briefly, μl of total genetic extracts were tested using an in-house developed assay based on the one-step superscriptiii/platinum taq kit (invitrogen, carlsbad, ca, usa) following the manufacturer's instructions on either the lightcycler machine from roche or the applied biosystems real-time pcr machine ( ). we compared variables of all fri subjects, and subsets of subjects with mono-viral infection (mvi) for the five most common viral pathogens (case groups) against the non-fri patients without viral infection detected from the panel (control group). specifically, the five most common viruses were influenza b, influenza a (h n ) pdm , coxsackie/echovirus, adenovirus e and rhinovirus. univariate logistic regression was conducted to identify statistically significant variables of interest. selected variables with high co-linearity were dropped, but all other variables significant at p < . were then included in a multivariable logistic regression model to determine the independent factors. the best model was determined using backward stepwise regression method. power calculation showed at least % power to detect a true positive association with effect size of . with % of the controls having the exposure of interest as cases. all tests were conducted at the % level of significance. we report odds ratio (or) and corresponding % confidence intervals (ci) where applicable. all statistical analyses were performed using an open source statistical software r . . (r core development team). written informed consent was obtained from the study participants. this study was reviewed and approved by the singapore military's joint medical committee for research, and the national university of singapore's ethics review committee. a total of , fri cases were recruited. of these, there were , fri cases ( . %) with mono-viral infection (mvi). of the , mvi cases, the five most common mvi were due to influenza b with cases ( . %), influenza a (h n )pdm with cases ( . %), coxsackie/echovirus with cases ( . %), adenovirus e with cases ( . %), and rhinovirus with cases ( %); the number of cases observed in each month from may to october is shown in fig. . of the , non-fri subjects recruited, , subjects ( . %) were confirmed to be negative for the whole panel of respiratory pathogens tested, and these served as the study controls in all subsequent analysis. the mean age of fri cases was . (± . ) as compared to . (± . ) years old for controls (p < . ; table ). a significantly higher proportion of cases ( . %) came from the recruit camp as compared to the controls ( . %; p < . ). the proportion of fri cases who had pre-pdm tiv and post-pdm tiv were significantly lower and higher than the study controls, respectively ( . % vs . %, p = . , and . % vs . %, p < . , respectively; table ). in addition, there were significant differences in the smoking status among the cases compared to the controls (p = . ), and there were significantly higher proportion of fri cases than the study controls ( . % vs . %, p = . ). in terms of movement history, there was a significantly lower proportion of cases who had travelled to other camp in the last days before clinical presentation than that of study controls ( . % vs . %; p < . ), and there were significantly higher proportion of fri cases who stayed in camp compared to the controls ( . % vs . %, p < . ; table ). increasing age was observed to be an independent risk factor for fri [adjusted odds ratio (aor) = . ; % confidence interval (ci) = . - . ; fig. (table ) . similarly, asthma (cor = . ; % = . - . ) was a potential risk factor of fri, but it was not independently associated with fri after adjusting for potential confounding factors (table ) . of the five most common mvi, increasing age was positively associated with coxsackie/echovirus(aor = . ; % ci = . - . ; fig. personnel who travelled to the community in the last days before clinical presentation had a significantly lower risk of adenovirus mono-infection (aor = . ; % ci = . - . ; fig. ) compared to personnel who did not. however, personnel travelling overseas in the last days before clinical presentation had . times higher risk of adenovirus mono-infection (aor = . ; % ci = . - . ) compared with personnel who did not travel overseas. compared to stay-out personnel with no ill household members in the last days before clinical presentation, stay-out personnel with ill household members had . times higher risk of fri (aor = . ; % ci = . - . ). moreover, compared to stay-out personnel with no ill household members, stay-in personnel who had neither ill bunkmates nor household members had . times lower risk of fri (aor = . ; % ci = . - . ). however, there was a higher risk of fri for stay-in personnel with an ill member in bunk regardless of whether they had any ill household members (aor = . ; % ci = . - . ) or not (aor = . ; % ci = . - . ) in the last days before clinical presentation. results for the analysis on each of the five most common mvi were very similar to those for all fri analysis (figs. and ). there was significantly higher risk of infection for all of the five most common mvi in stay-out personnel with ill household members compared with those who did not (fig. ) . regardless of whether they had ill household members or not, stay-in personnel with no ill bunkmates were not at significantly increased risk for any of the five most common mvi compared to stay-out personnel (with no ill household members). stayin personnel with ill bunkmates but without ill household members had significantly increased risk of all the mvi except adenovirus e (aor = . ; % ci = . - . ), where there was a non-significant increase in risk; having ill household members further increased the risk for all [ ] had simultaneously document the risk of fri due to a range of specific pathogens. in this study, we had shown that the five most common viral pathogens within our military environment was strongly associated with contact history, and had fairly similar trend of the fri risk factors identified. increasing age, recruit camp, and smokers were demographic risk factors for fri. increasing age was also reported as a risk factor for ari in us military personnel in overseas deployments [ ] . additional analyses showed that the risk was higher with increasing age for all the five mvi in this study, but only significantly so for coxsackie/ echovirus. in contrast, increasing age was previously reported to be a protective factor for seroconversion against influenza a(h n ) pdm in the local military during the initial wave of infections from june to october [ ] . these discrepant findings may be due to the changing age distribution of susceptible population towards influenza a(h n )pdm infections [ , ] , which might have shifted to involve more older individuals over the study period presented here (up to october ). in addition, this may be due to the increased in herd immunity effects among the new young cohorts of conscripts, where vaccine (initially as a monovalent formulation, and then later as part of the post-pandemic trivalent inactivated vaccine) was administered to all military recruits since november [ , ] . moreover, the lack or waning immunity against influenza a(h n )pdm in the older cohorts may have attributed to this trend, even though the individual level effects of vaccination against influenza a(h n )pdm (which was found to be significantly protective) was accounted for in the multivariate model. personnel in the recruit camp were at higher risk of fri as well as all the five most common mvi, particularly adenovirus e infection. this is likely due to the higher contact exposure rate in semi-closed environments, and increased stressors [ , , , , , , , , ] . alternatively, it could be due to the fact that personnel in non-recruit camps are already protected due to the adaptive immune response developed from the previous infections in recruit camp, where recruits usually only stay on a short term basis, before posted to non-recruit camp. smoking has been shown to increase risk of upper respiratory infection among recruits [ ] , hajj medical mission personnel [ ] , infants and children exposed to parental smoking [ ] . hence, it is not surprising to observe smoking as a risk factor of fri in our study. there are some studies that had shown that cigarette smoking impairs oral and respiratory tract immunity [ ] [ ] [ ] . this may have predispose smokers to a higher chance of viral infection resulting in fri. however, further study is warranted to investigate mechanism behind this observation. malay ethnicity was positively associated with both influenza-a(h n )pdm and coxsackie/ echovirus monoinfections. we had previously also found malays in the community to be at higher risk of influenza a(h n )pdm infection [ ] . however, a previous study in the saf found that malays conscripts actually had significantly higher score in hygiene practices and knowledge towards pandemic influenza as compared to chinese and indians [ ] . hence, there may be a potential genetic basis for the higher risk of infection in malays as compared to chinese and indians, given differences in genetic backgrounds of the hla class region which have been shown to result in weaker immune response against pathogen antigens [ ] . nevertheless, other unmeasured sociocultural and behavioural factors might explain these observations, and further studies are needed to confirm these observations and to understand the basis for the association. the protective effects of the influenza vaccine was largely in line with expectations, with the pre-pdm tiv protecting against influenza b but not against influenza a(h n )pdm , the pdm-a(h n )v protecting against influenza a(h n )pdm but not influenza b, and the post-pdm tiv protecting against both pdm-a(h n )v subtypes, as observed in our previous study [ ] . however, there were also some unexpected findings. these includes a potential protective effect (aor = . ; % ci = . - . ) of the pdm-a(h n )v against rhinovirus, and an increased risk (aor = . ; % ci = . - . ) of adenovirus e infection with the post-pdm tiv. these findings may have been due to non-specific interactions and interference between respiratory viruses which have been suggested by others [ ] , but could also have been due to the periodic nature of respiratory virus outbreaks. in particular, the post-pdm tiv period included a period of heightened adenovirus e activity (see fig. ) which might have been unrelated to changes in the vaccination policy, but which we could not adjust for due to co-linearity between the timing of these adenovirus fig. contact risk factors for fri and the five most common mono-viral infections e outbreaks and the phased roll-out of the influenza vaccine formulations. these unexpected findings would still require more scientific and epidemiological evidence for further conclusion. travelling overseas in the last days before clinical presentation was associated with a significantly increased risk for adenovirus e infection. we were not able to distinguish these as either military or personal overseas trips, but a previous outbreak of b human adenovirus e a strain in a military camp in singapore was also reported to be highly similar to other asian strains involved in outbreaks, suggesting a potential import of this strain from the neighbouring regions [ ] . as such, implementation of adenovirus vaccination may be useful to prevent sudden surge of cases with adenovirus e outbreak, given the high incidence of adenovirus infection in south-east asia [ , ] . one key finding was the relatively lower risk of fri and the five most common mvi for stay-in personnel as compared with stay-out personnel. at least for influenza b and a(h n )pdm , this could be due to the lower proportion of members in the households and the community who had the seasonal influenza vaccination [ ] , as compared to the camps where vaccination programme was implemented for all military personnel since the end of [ ] . as such, this may have resulted in a smaller pool of susceptible individuals and a larger herd immunity effects in camps as compared to within the community. the other explanation maybe that stay-in personnel have less exposure to younger household members, which was previously found to have a significant risk for seroconversion to influenza a(h n )pdm , and the risk was accentuated if the household member had fri [ ] . this also concurs with our findings on the effect of exposure to ill household members and bunkmates, and the effects are influenced by the domiciliary status of the soldier. for the five most common mvi, an ill household member was a major risk factor for stay-out personnel. moreover, the increase in risk for stay-in personnel from having ill household members was not as marked and mostly not significant. however, stay-in personnel with an ill bunkmate had a substantial increase in risk of infection. while our current study design does not allow us to attribute the cause of infection to contact with these ill household members or bunkmates, our findings do suggest that some of the transmission of these pathogens is mediated through close contacts, and support the use of preventive measures for fri aimed at reducing transmission from ill household members and bunkmates. this could be in the form of issuing advisories to emphasize hygiene during outbreaks, and identifying and isolating ill personnel early to break the transmission of fri. moreover, this finding also has potential applications in surveillance. we had previously reported on how it would be difficult for syndromic surveillance systems to detect outbreaks in larger military units given the high baseline rates of respiratory illness [ ] . given that the relevance of ill bunkmates is consistent for the predominant viral agents of fri, outbreak detection methods could instead focus on clusters of illness in those who share the same quarters, or are from the same military subunit as a reasonable proxy. we believe such an approach to syndromic surveillance deserves a prospective validation study where such clusters of illness are systematically sampled. there are several limitations to this study. first, there was the influenza a (h n )pdm pandemic in june to september during the early part of the study period, where the force of infection for influenza a (h n )pdm is likely to be higher than usual. however, the pandemic spread was well-contained with prompt protective and preventive measures such as vaccination (fig. ) , enhanced respiratory hygiene measures, isolation, quarantine, "ring prophylaxis" with oseltamivir during this period. as such, these measure are also likely to limit the risk of transmission of other circulating respiratory viruses during this specific period compared to other periods in the study. since different vaccines were used promptly and appropriately during the different study periods (fig. ) , vaccine type was used as a surrogate to account for the potential bias due to the enhanced protective and preventive measures applied during the influenza a (h n )pdm pandemic. nevertheless, this bias should be minimal because the controls were also recruited in the same period and camp as the cases. second, hand washing behaviour, allergy and military rank were not evaluated as potential risk factors of fri. this is because it was very challenging to accurately assess how frequent hand washing was performed by the soldiers. moreover, the soldiers may also tend to report the expected favourable hand washing behaviour. hence, the likelihood of recall bias and information bias are likely to be high and would make any form of interpretation challenging. allergy was not evaluated because the symptoms are very broad to specifically define as an allergy, and there would be significant potential information bias as it is less likely to clinically diagnosed allergy as compared with asthma, diabetes, hypertension and heart disease. furthermore, the aim of this study is not to study clinical signs and symptoms that are associated with fri. we did not consider military rank due to fact that there is a significant number of cases that were recruited from the recruit camp, where the population is mainly made up of recruits as compared to non-recruit camp, where the population is mainly made up of higher ranks (table ; p < . ). as such, it would be biased to include military rank as one of the variables. third, our data is limited to febrile presentations of viral respiratory infections and may not be applicable to milder acute respiratory infections. fourth, there is a lack of clinical and laboratory confirmation of the ill household members and bunkmates, and such data are hence subjected to recall bias. fifth, the prevalence for fri and mvi is about % and % respectively. as such, or values as proxies to rr would be similar for mvi, whereas the or of the risk factors for fri is likely an overestimation, to some extent, relative to rr. sixth, the resplex i assay (qiagen) was designed to also detect six bacterial respiratory pathogens. they were mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumomophila, streptococcus pneumoniae, neisseria meningitides and haemophilus influenza , , . however, fri subjects with bacterial causes were not excluded because one of the aims of the study is to determine the potential risk factors for fri, regardless of any detected or undetected respiratory virus and/or bacteria. lastly, this study involved predominantly young adult males in a military context, and hence, the results may not be generalizable to the overall population in the community, particularly for the contact risk factors. however, during the pandemic of influenza a(h n )pdm , clustering of febrile respiratory illness by classroom contact among school children [ ] and ill workplace contacts among healthcare workers were also observed [ ] . further studies in other settings such as nursing homes which collect contact history in a similar way should be attempted. increasing age, smokers, recruit camp, stay-out personnel with ill household members and stay-in personnel with ill bunkmates were independent risk factors of fri in a semi-closed military setting. early identification and isolation of ill bunkmates may be effective to prevent and to reduce further transmission in camp. public health campaigns and policy should take these risk factors into consideration to increase the effectiveness of interventions to reduce fri in the military environment. abbreviations fri: febrile respiratory illness; aor: adjusted odds ratio; ci: confidence interval; saf: singapore armed forces; mvi: mono-viral infection. frequency of acute respiratory illnesses and circulation of respiratory viruses in households with children over surveillance seasons epidemiology of community-acquired respiratory tract infections in adults. incidence, etiology, and impact recent trends of pneumonia morbidity in us naval personnel keep your hands clean influenza c virus infection in military recruits-symptoms and clinical manifestation respiratory infections in the military symptomatic respiratory syncytial virus infection in previously healthy young adults living in a crowded military environment respiratory syncytial virus: an important cause of acute respiratory illness among young adults undergoing military training viruses associated with acute 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in singapore epidemiology of the common cold in military recruits with emphasis on infections by rhinovirus types a, , and two unclassified rhinoviruses patterns of illness in rhinovirus infections of military personnel relationship of rhinovirus infection to mild upper respiratory disease. ii. epidemiologic observations in male military trainees relationship of rehinovirus infection to mild upper respiratory disease. . further epidemiologic observations in military personnel epidemiology of pathogen-specific respiratory infections among three us populations all known human rhinovirus species are present in sputum specimens of military recruits during respiratory infection rhinovirus and the lower respiratory tract a clinical diagnostic model for predicting influenza among young adult military personnel with febrile respiratory illness in singapore surveillance for febrile respiratory infections during cobra gold influenza disease burden in adults by subtypes following the initial epidemic of pandemic h n in singapore factors influencing infection by pandemic influenza a(h n )pdm over three epidemic waves in singapore acute respiratory tract infections among hajj medical mission personnel, saudi arabia the study protocol for a randomized controlled trial of a family-centred tobacco control program about environmental tobacco smoke (ets) to reduce respiratory illness in indigenous infants cigarette smoking impairs human pulmonary immunity to mycobacterium tuberculosis effect of smoking on immunity in human chronic periodontitis cigarette smoke effects on innate immune mechanisms in the nasal mucosa. potential effects on the microbiome risk factors for pandemic (h n ) seroconversion among adults knowledge, attitudes and practices towards pandemic influenza among cases, close contacts, and healthcare workers in tropical singapore: a cross-sectional survey ramifications of hla class i polymorphism and population genetics for vaccine development dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines an assessment of electronically captured data in the patient care enhancement system (paces) for syndromic surveillance teacher led school-based surveillance can allow accurate tracking of emerging infectious diseases -evidence from serial cross-sectional surveys of febrile respiratory illness during the h n influenza pandemic in singapore the work was supported by a singapore ministry of defence funded operational research program and the centre for infectious disease epidemiology and research in the saw swee hock school of public health of the national university of singapore and national university health system. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. biodefence centre, ministry of defence, singapore, singapore. yale-nus college, national university of singapore, singapore, singapore. program in health services and systems research, duke-nus graduate medical school, singapore, singapore. department of statistics and applied probability, national university of singapore, singapore, singapore. department of medicine, national university of singapore, singapore, singapore.received: january accepted: july the authors declare that they have no competing interests, except for vl who had previously received unrelated research grants from gsk.authors' contribution jp wrote the manuscript and analysed the data. jj analysed the data. lyh, arc and mic revised the manuscript and assisted with data analysis. vjl conceptualized the study and revised the manuscript. bht, jpl, whvk, shn were involved in the laboratory testing of the specimens. mh and qg were involved in subject recruitment and screening. all authors read and approved the final manuscript. key: cord- -w bpeqzg authors: wong, samson sai-yin; wong, sally cheuk-ying title: ebola virus disease in nonendemic countries date: - - journal: journal of the formosan medical association doi: . /j.jfma. . . sha: doc_id: cord_uid: w bpeqzg the west african outbreak of ebola virus disease was unprecedented in its scale and has resulted in transmissions outside endemic countries. clinicians in nonendemic countries will most likely face the disease in returning travelers, either among healthcare workers, expatriates, or visiting friends and relatives. clinical suspicion for the disease must be heightened for travelers or contacts presenting with compatible clinical syndromes, and strict infection control measures must be promptly implemented to minimize the risk of secondary transmission within healthcare settings or in the community. we present a concise review on human filoviral disease with an emphasis on issues that are pertinent to clinicians practicing in nonendemic countries. the largest outbreak of ebola virus disease (evd) in history has renewed interest in filoviruses and has provided an unprecedented impetus to the development of new therapeutics and vaccines for this highly lethal infection. hemorrhagic fevers caused by ebola and marburg virusesdalso collectively known as filoviral hemorrhagic fever (fhf)dpreviously caused dramatic, albeit limited, outbreaks in central africa. their impact on global health was rather small (except in the realm of biological warfare research) because of the high mortality rate, lack of effective antiviral therapies and vaccines, and potential for person-to-person transmission. the west african outbreak of evd proved that these filoviruses should no longer be considered as merely regional problems. a short review of evd and its clinical relevance to the nonendemic countries is presented. the current epidemic is caused by zaïre ebolavirus; however, references will also be made to the related marburgvirus, which shares many virological, clinical, and epidemiological characteristics. conflicts of interest: the authors have no conflicts of interest relevant to this article. the order mononegavirales consists of enveloped, nonsegmented, negative-sense, single-stranded rna viruses. the family filoviridae comprises three genera: cuevavirus, ebolavirus, and marburgvirus. in , the sole species of cuevavirus, lloviu cuevavirus, was described. it was discovered during an investigation of massive die-offs of miniopterus schreibersii bats in france, spain, and portugal in ; and the virus was detected in bat carcasses collected from northern spain. the genus ebolavirus the name "filovirus" describes a unique morphological characteristic of the viruses. the virions are generally filamentous (in latin, filum means "thread") with a diameter of approximately nm and a highly variable length of e , nm. they may also appear as branched filaments, short rods, u-shaped, circular, or hairpin-shaped. , the genomes of ebov and marv are approximately kb and consist of seven genes (from the to end): nucleoprotein (np), vp , vp , glycoprotein (gp), vp , vp , and rna-dependent rna polymerase (l). , ebolavirus expresses an additional protein through transcriptional editing of the gp gene. in addition to gp, a smaller secreted glycoprotein (sgp) is produced and excreted extracellularly. , at c, ebov and marv are stable and resist desiccation, which probably explains their stability in aerosol droplets. , they are however inactivated by heat and common disinfectants such as detergents, phenolics, and hypochlorites. the usual heat treatment of clinical samples at c for minutes may fail to render the specimen noninfectious. thermal inactivation at c for minutes or c for minutes is necessary. e gamma irradiation readily inactivates the filoviruses, although this method may not be readily available in routine clinical or laboratory settings. the genetics and molecular biology of the filoviral genome have been previously reviewed. , in addition to the essential functions for viral replication and assembly, many viral proteins exert their effects on the host immune system and may contribute to the pathogenesis of the infection (table ) . , , e , , , for example, vp and vp inhibit the normal antiviral activities of type i interferons at multiple steps of the pathway, whereas sgp may contribute to immune evasion by absorbing anti-gp antibodies (i.e., antigenic subversion). e because of the essential roles of many viral proteins in replication and assembly, some viral proteins (e.g., vp and vp ) are potential targets for antiviral agent development. , in recent years, the pathogenesis of fhf has been better elucidated. filoviruses are pantropic with the ability to infect different host cell types. the initial cells in which the viruses replicate are likely dendritic cells, macrophages, monocytes, and kupffer cells at the site of entry. a large number of lectins (e.g., dc-sign and l-sign) and immunorecognition receptors [i.e., triggering receptors expressed in myeloid cells (trem)] can serve as receptors for the viruses. after the initial multiplication in the aforementioned cell types, the viruses are transported to the reticuloendothelial system (e.g., lymph nodes, spleen, liver) and other organs where infection of other cell types occur. the resulting massive necrosis and end organ damage are reflected in the histopathology of human and primate infection models with necrosis in the liver, kidneys, lungs, lymphoid tissues, and other organs. , in addition, various mechanisms contribute to the development of coagulopathy and disseminated intravascular coagulation, which is a hallmark of viral hemorrhagic fevers. patients with fhf develop significant platelet dysfunction (which is not merely accountable for by thrombocytopenia), and this is contributed to by platelet activation and decreased vascular endothelium production of prostacyclin. another important target in the pathogenesis of fhf is the endothelium. human and primate endothelial cells are susceptible to infection by ebov, although direct cyopathic effects are not an important factor in the development of vasculopathy and coagulopathy. the release of vasoactive table filovirus genes and their functions. , , e , , , factors (e.g., nitric oxide) or cytokines and chemokines from monocytes and macrophages (e.g., tumor necrosis factor-alpha, interferons, monocyte chemoattactant protein- , interleukin- ) could be important in the genesis of vascular damage. the combined effects of increased secretions of proinflammatory cytokines, activation of the coagulation cascade, consumption and/or reduced production of protein c, thrombocytopenia and platelet dysfunction, hepatic damage with impaired production of coagulation factors, and vascular damage contribute to the development of coagulopathy in fhf. protective humoral and cellular immune responses can be demonstrated in patients who survive, and antibody levels persist for years. in addition to the various immune evasion mechanisms alluded to previously [e.g., binding of anti-gp antibodies by sgp (i.e., immune subversion), suppression of innate immune responses, and inhibition of interferon pathways], other potential virulence and pathogenic mechanisms of filoviruses such as the np and vp proteins have been identified. the generation of antibodies towards gp is critical for the protective efficacy of vaccines. ebolavirus and marv are geographically restricted to and cause outbreaks in sub-saharan africa; however, rebov is found in nonhuman primates and pigs in the philippines (table ) . , , , , , the first filovirus was discovered in after an outbreak of marburg hemorrhagic fever infection in germany. this infection originated from cercopithecus aethiops monkeys that were imported from uganda. in , the first natural outbreak of zebov occurred in northern zaïre (now called the democratic republic of the congo). in , sebov was discovered in an outbreak in nzara in sudan. in e , bebov caused an outbreak in uganda, followed by another outbreak in the democratic republic of the congo in . , in , tafv was discovered in a swiss biologist who acquired the infection in the republic of côte d'ivoire (i.e., the ivory coast). in , rebov was first discovered in monkeys (macaca fascicularis) that were exported from the philippines to reston, virginia, usa the infected monkeys were subsequently exported to italy ( e ) and to texas ( ) . cynomolgus macaques in the philippines are naturally infected, as are pigs. the rebov virus appears to be nonpathogenic to humans. e human filovirus infections are primary zoonotic diseases with a high propensity for interpersonal transmission. the primary animal reservoir of ebov and marv are bats (especially fruit bats). bats support long-term viral replication without developing the disease. virological and serological studies have confirmed filoviral infection in diverse bat species. , , in the philippines, china, and bangladesh, there is also evidence of rebov (and possibly other filoviral) infection in bats. e cases of fhf have been epidemiologically linked to contact with bat carcasses, spelunking among tourists, and outbreaks among gold miners who work in caves. e peak seasons of bat marv infection have been correlated with the incidence of human "spillover"infections. primates are also naturally infected with filoviruses, although they are likely to be infected by natural reservoir hosts (e.g., bats) and are not considered important reservoirs. , primates, nevertheless, remain important vectors for the introduction of the disease into humans in rural africa, and wild animal mortality sometimes precede human outbreaks of evd. other mammals that can be infected by evd include pigs (especially by rebov and zebov) and dogs; however, their role in causing human evd outbreaks is uncertain. e human infections typically occur with a few patterns of transmission. inhabitants of endemic areas, especially individuals dwelling in forests with occupational exposure to wild animals (e.g., hunters and people handling bushmeat), may develop symptomatic or subclinical infections, presumably because of the exposure to animal reservoirs of the virus. in some cases of human infection, a history of direct exposure to animal carcasses (such as primates, bats, duiker antelopes, and porcupines) or indirect exposure to bats in caves has occurred. e , , these single or multiple introductions to human populations may result in short chains of human transmission. when fhf patients are admitted to healthcare facilities where infection control measures are inadequate, a large outbreak may occur with transmission to other patients and healthcare workers. hospitals have been a major amplifying and disseminating factor in many previous outbreaks of fhf in africa. direct person-to-person transmission of ebov and marv occurs via blood or body fluids by percutaneous inoculation or by mucosal exposures. e during an outbreak of sebov, the virus was detected by viral culture or reverse transcription polymerase chain reaction (rt-pcr) in patients' saliva, skin swab, stool, tears, breast milk, and semen, but not in environmental samples. in view of the persistence of the viruses in semen and breast milk at days and days, respectively, after illness onset, , the use of condoms and withholding breastfeeding are recommended during convalescence. more than % of patients in the kitwit outbreak in the democratic republic of the congo had secondary cases in the household; the risk was highest for people with direct physical contact with the body fluids of symptomatic patients and exposure to patients in the late stages of the disease; this finding can be explained by the high viral load at this phase of disease. reusing needles or other medical instruments without adequate sterilization, needle stick injuries, lack of isolation facilities, and inadequate and inappropriate use of personal protective equipment all contribute to explosive hospital outbreaks. african burial customs of washing dead bodies and transporting bodies without barrier precautions further transmit the disease in the community. , , , infected pigs can transmit rebov to nonhuman primates via the airborne route and nonhuman primates can be infected experimentally by the inhalation of droplets (droplet size of . e . mm), although parenterally infected primates do not transmit the infection via the airborne route. e true airborne transmission of ebov between humans has likewise not been documented. up to % of patients in some ebov outbreaks may have no known contact or exposure history, although it remains unclear whether this is because of fomites, true airborne transmission, or inadequate investigation. , , , table outbreaks of human filovirus infection. , , , , , virus the ongoing evd outbreak at the time of this writing is unique in two aspects: ( ) its unprecedented scale and ( ) its origination in west africa (guinea) rather than in central africa. the first case occurred in guinea in february , and subsequently spread to the african countries of liberia, sierra leone, nigeria, senegal, and mali. in october , the world health organization declared the epidemic over in senegal and nigeria. , further epidemiological investigations suggested the index case was probably a year-old child who died on december , in southern guinea. the infection was subsequently transmitted from the child to family members, a village midwife, and a healthcare worker; some of these patients later caused outbreaks in different areas of guinea. ebola virus disease has caused infection in local healthcare workers of whom ( %) workers died (as of january , ); four of these infected healthcare workers were citizens of the united states of america, spain, and the united kingdom who returned to their respective countries for further management. on the other hand, asymptomatic infection was described in , as evidenced by seroconversion in % of close contacts of patients, in two zebov outbreaks in gabon. in % ( / ) of the seroconverted asymptomatic contacts, rt-pcr of the peripheral blood mononuclear cells was positive for the virus, and viremia persisted up to weeks in some contacts. another line of evidence for asymptomatic infections comes from seroprevalence studies in africa, in which . e . % of the surveyed population in central africa was seropositive for ebov with the seroprevalence consistently higher among forest-dwelling populations and hunters. e the incubation period of evd varies from days to days (commonly, e days), but a recent analysis of the cases in the zebov outbreak in kitwit suggested that the mean incubation period was . days, and the maximum incubation period was up to days. a biphasic illness has been described with an apparent remission of e days in between. , , e the disease usually begins abruptly with nonspecific symptoms such as fever ( e % of patients, ut infra), malaise ( e %), headache ( e %), sore throat, odynophagia or dysphagia ( e %), hiccoughs ( e %), and nonproductive cough ( e %). abdominal pain ( e %) or nausea and vomiting ( e %) often precede the onset of diarrhea ( e %), usually days after the onset of illness). the abdomen can be tender on palpation. in the absence of supportive therapy, diarrhea and vomiting may lead to fluid depletion and electrolyte disturbances such as hypokalemia. other symptoms include arthralgia or myalgia ( e %), chest pain ( e %), and conjunctival injection ( e %). a diffuse erythematous rash ( e %) appears towards the end of the st week which will later desquamate. the nonpruritic rash appears first on the trunk, and then spreads to the entire body with sparing of the face. three early symptoms of bilateral conjunctival injection, rash, and sore throat are suggestive of evd in the differential diagnosis. after the appearance of the rash, patients may either gradually recover or, in severe cases, progress over e days to the full-blown hemorrhagic fever syndrome with petechiae ( %), gum bleeding ( e %), melaena ( e %), hemoptysis ( e %), hematemesis ( e %), epistaxis ( e %), hematuria ( e %), menorrhagia, bleeding at venipuncture sites ( e %), and show features of disseminated intravascular coagulation. , the typical hemorrhagic fever picture, however, occurs only in approximately % of the patients (range, e %). , other manifestations in the late stage include evidence of multiorgan failure such as circulatory shock, obtundation, tachypnea, renal shutdown, convulsion, delirium, and coma. fever is often absent at this stage. death often occurs between days and , whereas patients who survive will show improvement around days to . intrauterine death is common in pregnant patients. mortality among pregnant women is substantial but may not be significantly higher than for nonpregnant patients; and pregnant women do not have an increased susceptibility to the infection. , survivors tend to improve from the nd week of illness. they make a slow recovery during which arthralgia (which could be asymmetric and migratory and often involves the large joints), uveitis, conjunctivitis, orchitis, parotitis, hearing loss, and tinnitus may occur. , chronic infection by filoviruses has not been documented, but male patients may shed the virus in the semen for e days after the onset of illness, and the transmission of marv has occurred via sexual intercourse. , , , common laboratory findings include lymphopenia, thrombocytopenia, elevated aminotransferases, hyperproteinemia, proteinuria, and prolonged prothrombin and activated partial thromboplastin time. with the progression of disease, evidence of disseminated intravascular coagulation and renal failure will appear. disease progression is also associated with worsening lymphopenia and rising antigenemia. compared to survivors, patients with fatal evd are more likely to have tachypnea. patients with disease also have a significantly higher level of viremia and a much weaker humoral immune response to the infection. significant differences in a number of cytokine and chemokine levels have also been detected between patients with nonfatal and fatal evd; in particular, the levels of many proinflammatory cytokines and nitric oxide are higher in nonsurvivors (with the possible exception of bebov), whereas the level of t cells and cd þ t cells were lower. , , e a high viral load is of prognostic significance. in sebov fhf, patients with fatal cases had an average of e (up to ) copies of rna/ml of serum, compared to the approximately copies of rna/mll of serum in survivors. patients with marv fhf likewise have high levels of viremia in blood with a median level of .  (range, .  e .  )/ml of serum serum. there are no pathognomonic signs or symptoms in the early stages of evd. the most useful history is epidemiological linkage to possible cases by travel history or by contact with known or suspected cases. laboratory investigations are necessary to confirm the diagnosis. other differential diagnoses (vide infra) should be excluded, as appropriate. all clinical specimens must be handled with great care from their collection to transport and testing in the laboratories. laboratory-acquired infection of ebov has occurred via percutaneous exposures. marburgvirus retains its infectivity in dried blood for at least days. filoviruses can be inactivated by heat [ c for minutes or c for minutes; or c for minutes in the presence of . % (final concentration) sodium dodecyl sulfate or . % (final concentration) tween ] or inactivated by % sodium deoxycholate solution, acetone, diethyl ether, % formalin, methanol, sodium hypochlorite, glutaraldehyde, % peracetic acid, phenolic disinfectants, and osmium tetroxide (used in fixation for electron microscopy). ultraviolet light is an effective means for surface disinfection. depending on the type of specimen and testing methodology, treatment of the sample with either triton x- , tween , sodium dodecyl sulfate, beta-propiolactone, chloramine b, or % acetic acid (ph . ) should be done before routine hematological, biochemical, and serological testing. heat inactivation is recommended for blood sodium, potassium, magnesium, urate, urea, creatinine, bilirubin, glucose, and c-reactive protein; however, other methods of inactivation would be necessary for calcium, phosphate, albumin, transaminases, gammaglutamyltransferase, and creatine kinase determination. standard virological techniques also apply to the diagnosis of fhf. these include viral culture, electron microscopy, serological tests using antigen or antibody detection, and nucleic acid amplification. , filoviruses can be cultivated from clinical specimens (especially blood and liver samples); however, because of the associated biohazards, a viral culture is notdand should not bedroutinely performed, except in facilities that can handle biosafety level agents. electron microscopy, which has excellent specificity because of the unique morphology of filoviruses, is also not routinely performed because of biosafety considerations, limited availability of electron microscope facilities in routine diagnostic settings, and the relatively high viral load necessary for visualization. the detection antibodies [e.g., immunoglobulin m (igm) or immunoglobulin g (igg)] is commonly achieved using immunofluorescent assays and enzyme-linked immunoassay (elisa) against recombinant np, gp, vp , vp , or vp antigens. e the appearance of igm and igg antibodies occurs at approximately days and e days, respectively, after the onset of illness. various antigen detection assays have been developed for the diagnosis of fhf and some have been used in field situations. the techniques include antigencapture elisa, immunofluorescent assay, dot-immunobinding assay, immunofiltration assay with different genus-specific or species-specific reactivity towards common targets such np, gp, and vp proteins. , , a practical limitation of these serological assays is the limited availability of these tests in laboratories in nonendemic countries. nucleic acid amplification is the diagnostic test of choice because of its high sensitivity (especially in the early phase of illness); its ability to differentiate between different agents of viral hemorrhagic fever; and its relatively lower biohazard, if the viruses are appropriately inactivated; and because antigen and antibody assays are often unavailable in laboratories in nonendemic countries. when the viral load is determined by quantitative assays, prognostic information can also be obtained. the most commonly used test is rt-pcr. a reverse transcriptioneloop-mediated isothermal amplification assay has also been developed for ebov and marv. , all diagnostic nucleic acid amplification tests must be adequately validated before being used clinically. if appropriately validated, the use of multiplex pcr/rt-pcr allows simultaneous detection of multiple pathogens that cause hemorrhagic fever. , the provision of nucleic acid amplification tests should preferably be centralized in national or regional reference laboratories to ensure adequate biosafety containment and quality of results. the rna of ebov can be detected in the sera by rt-pcr e hours earlier than by antigen capture; in some studies, it is detectable on day of the illness. , however, the viral load gradually reaches its peak at approximately e days after the onset of the disease. retesting is therefore necessary if rt-pcr is initially negative but clinical suspicion is high, especially when the first sample was obtained within days of the onset of disease. blood is the most commonly used sample for rt-pcr. oral fluid specimens can be a viable alternative to blood samples for rt-pcr in situations in which blood taking may be difficult or infeasible. common targets for nucleic acid amplification include the l, gp, and np genes. , , , , clinical management and vaccine development treatment of fhf is primarily supportive owing to the unavailability of approved, specific antiviral agents. concurrent infections such as malaria or bacterial sepsis should be treated, as appropriate. fluid and electrolyte replacement, blood product transfusion, renal replacement therapy, and ventilatory support such as extracorporeal membrane oxygenation should be administered, as necessary. , various experimental therapeutic approaches have been attempted in experimental animals or clinically; however, no randomized controlled trials prove their efficacy. examples include recombinant inhibitor of factor vii (rnapc ), recombinant human activated protein c, and interferon-beta. e as in cases of other severe viral infections, convalescent plasma from recovered patients has been used to treat fhf. this was deployed with apparent benefits in the evd outbreak in kitwit. the world health organization (who) published a guideline on the collection and preparation of convalescent plasma for use in the outbreak situation; however, the who recognizes the uncertainties in the efficacy of this treatment. cocktails of monoclonal antibodies have similarly been used recently with some success in reducing the mortality of ebov infection in nonhuman primates. these antibodies have been produced in plants and in mice. e based on these studies, an optimized cocktail of plant-derived monoclonal antibodies, called the zmapp, was produced; it protected % of rhesus macaques infected up to days with ebov. these antibodies have been used experimentally for treating human ebov patients in the west african outbreak, although the actual benefit to human evd remains to be confirmed. a second approach to the treatment of fhf lies in the development of specific antiviral agents. a current nucleotide analogue is favipiravir (t- ), which was developed and approved in japan for the treatment of influenza. favipiravir inhibits viral rna-dependent rna polymerase of the influenza virus. it was subsequently found to exhibit in vitro antiviral activities against certain other rna viruses such as bunyaviruses, arenaviruses, flaviviruses, alphaviruses, norovirus, and ebov. e animal studies also demonstrate the efficacy of favipiravir in the treatment of junín virus, arenavirus, and ebov hemorrhagic fevers, and the drug was used to treat human evd in the west african epidemic. e a dosing regimen of favipiravir for use in a clinical trial for the treatment of evd has been published. another nucleotide analogue, brincidofovir (a lipid conjugate of cidofovir), was previously developed to treat infections due to dna viruses such as adenoviruses, herpesviruses, and orthopoxviruses; the drug was granted emergency investigational new drug applications in october by the united states (us) food and drug administration for evaluation in evd treatment, and a clinical trial was started in january in monrovia, liberia. e the nucleoside analogue bcx was recently shown to inhibit rna polymerase of negative-and positive-sense rna viruses via chain termination effects. its in vivo activity against marv was demonstrated in guinea pigs and cynomolgus macaques. the development of bcx for human clinical trials will require a long time. another approach involves the screening of currently available nonantimicrobials for their activities on filoviruses. compounds such as selective estrogen receptor modulators (e.g., clomiphene, toremifene), amiodarone, dronedarone, and verapamil have antifilovirus activity in cell cultures and/or murine models. , in addition, rna interference using small interfering rnas provide postexposure protection of animals infected with ebov and marv. e a third approach to the specific management of filoviral infections explores the potential of postexposure prophylaxis. such regimens would benefit exposed healthcare workers and other social contacts, and laboratory staff experiencing accidental exposures. protective immunity towards filoviruses does exist, as demonstrated in the possible benefits of convalescent plasma and animal studies, in which humoral immunity (i.e., igg) protects against ebov infection. animals studies also confirm that passive immunization by neutralizing monoclonal antibodies is protective in primates. previously examined filovirus vaccine candidates that elicit protective humoral immunity experimentally include ebov-like particles containing gp, np, and vp ; replication-deficient ebov mutants that lack the vp gene; and ebov gp-containing fragment or fusion protein. e a phase clinical trial has tested dna vaccines encoding the glycoproteins of ebov and marv. one of the most promising vaccine candidates for filoviruses is the recombinant vesicular stomatitis virus-based vaccine system that expresses filoviral gp. this system elicits protective immunity against zebov, sebov, bebov, and marv; it also offers substantial postexposure prophylaxis in primate and murine models. e the vaccine was used for postexposure prophylaxis in a laboratory staff person who sustained a needle stick injury with zebov in . a similar vaccine system used replication-defective recombinant adenovirus that expressed ebov gp was protective in animal models. , phase clinical trials of the vesicular stomatitis virus-based and adenovirus-based vaccines began in late . ebola virus disease as a problem in nonendemic countries: issues on prevention and control the containment of fhf outbreaks in endemic countries requires substantial resources in coordination between the public health system and other authorities of the countries, surveillance of the disease, education and engagement of local citizens, isolation and treatment facilities, and laboratory support. , these requirements are often beyond the capability of endemic countries. significant international assistance is usually needed to contain major outbreaks. the discussion on these public health issues is beyond the scope of this article. for health authorities in nonendemic countries, a preparedness plan for emerging infections is an indispensable component of the public health system. the development and adoption of preparedness and response plans for emerging infectious diseases are first fostered in anticipation of pandemic influenza. the outbreaks of severe acute respiratory syndrome (sars), pandemic influenza a (i.e., h n ), middle east respiratory syndrome coronavirus (mers-cov), and, more recently, avian influenza a (i.e., h n ) and evd underscore the importance of such pre-emptive plans in preventing or mitigating the effects of disease transmission. , the details may vary, depending on the nature of the pathogens; however, key components in public health response include risk assessment; communication and education; establishing a rapid response team and management team to coordinate responses between different government ministries; monitoring and surveillance; providing adequate facilities and supplies for quarantine and infection control; developing laboratory diagnostics; and where appropriate, antimicrobial and vaccination policies, and/or stockpiling. , for clinicians in nonendemic countries, fhf will mostly be encountered in travelers returning from endemic areas. the sars epidemic and influenza a (h n ) pandemic demonstrated the efficiency of international traveldespecially air traveldin the global spread of infectious diseases. , as a continent, africa has a relatively small number of international travelers (only oceania has a smaller number of international tourist arrivals), although the exportation of fhf to nonendemic countries is well documented in the present epidemic. it is tempting to consider establishing travel restrictions to limit the spread of evd outside of africa; however, the actual benefit of such policies is limited. at the time of this writing, the who has issued no travel restrictions to the affected countries. however, it is prudent to issue health warnings to potential visitors to affected countries. pretravel education on transmission routes, precautionary measures, self-monitoring of signs and symptoms, and availability of medical care in destination countries should be provided. as in other subspecialties of travel medicine, the visiting friends and relatives (vfrs) are at particularly high risk of contracting travel-related infections. despite the fact that publicity and health alerts are often announced to the general public, vfrs do not always receive the necessary information on the risks because of cultural differences, language barriers, or different perceptions. hence, efforts must be targeted to vfrs (especially african communities in this context) to reduce the risk of disease importation. remote infrared thermal scanners have been used as a means of fever screening at airports in some countries. the practice first gained popularity during the sars epidemic, and was subsequently evaluated in the influenza a (h n ) pandemic. e to a lesser extent, this method has also been used for the screening of other febrile illness such as dengue fever. infrared thermal scanning is relatively popular in asian countries such as taiwan, japan, korea, and hong kong, and it is usually used in conjunction with health questionnaires for self-reported symptoms such as fever and travel history. the tympanic temperature would be measured for suspected cases. the sensitivity, specificity, and positive predictive value of thermal scanning are affected by a variety of factors such as the instruments used, the threshold temperature, the part of the body being measured, the distance of the instruments from the individual, and the previous use of antipyretic agents. patients in the incubation period of an infection obviously will not be detected by this screening method. despite the relatively low sensitivity, specificity, and positive predictive values of infrared thermal scanning, some investigators consider it a useful adjunctive measure for border screening, although it cannot be relied on as the sole method for screening. e contact tracing must be promptly initiated for any potential contacts of returned travelers diagnosed with a communicable infectious disease such as evd. detailed guidance on contact tracing for patients with evd and other forms of viral hemorrhagic fevers have been published. , all suspected contacts must undergo individual risk assessments, based on the travel history, the epidemic situation in the affected countries, and the nature of potential exposure before and during travel. detailed instructions and information must be provided to the contacts, who will then be monitored for fever and the development of symptoms. the monitoring may be performed in the community or within healthcare facilities, during which some limitations indor at least, advice againstdthe freedom to travel within the community or country may be necessary and interference of daily activities may be inevitable. close and empathetic liaison between the contacts and health departments is essential to ensure compliance with monitoring and to minimize psychological impacts. a preparedness plan for contact tracing, disease surveillance, clinical management, isolation precautions, and outbreak management must be in place to manage such incidents in nonendemic countries. hemorrhagic fever remains an uncommon cause of fever in returned travelers, although it is severe clinically and has substantial public health implications. the risk of fhf in returned travelers is low. ten cases of marburg hemorrhagic fever were reported from to , and all patients had a travel history to africa. cases of imported evd (excluding the nonpathogenic rebov) were described in south africa in (zebov) and in switzerland in (tafv). other causes of fever in such settings must be excluded by appropriate laboratory investigations. these include (depending on the itinerary and exposure history) other causes of fever with or without hemorrhagic presentations such as meningococcal infections, severe sepsis due to other bacterial infections (including rare infections such as anthrax and plague as guided by the clinical picture and exposure history), leptospirosis, typhoid and other causes of enteric fever, rickettsioses, malaria, trypanosomiasis, visceral leishmaniasis, dengue fever, and yellow fever. in particular, malaria (which is a very common treatable cause of fever in returned travelers and is endemic in sub-saharan africa) must be excluded by appropriate testing. depending on the travel destination, other causes of viral hemorrhagic fever have to be considered such as lassa fever, hantavirus infection, crimean-congo hemorrhagic fever, and rift valley fever. the travel history should also include human contacts with sick individuals in the community (e.g., attending local funerals) or in healthcare facilities (as in the case of volunteer workers). another important exposure history is interaction with bats and other wild animals (especially primates). for example, a history of spelunking has resulted in fhf among local citizens and foreign visitors. cases of marburg hemorrhagic fever have occurred after exposure of visitors to bats in caves in kenya and uganda. , when fhf is suspected based on travel and/or exposure histories, pre-emptive isolation is essential to minimize the risk of subsequent interpersonal spread, until the diagnosis is excluded. the routes of transmission of filoviruses are well documented. transmission can be interrupted, provided that proper infection control and public health measures are implemented. detailed infection control guidelines on caring for patients with fhf have been published. , e in essence, the principles of isolation and use of personal protective equipment are not significantly different from standard precautions and transmissionbased precautions (i.e., contact, droplet, and airborne) that are universally practiced in healthcare facilities. however, numerous studies and reviews have confirmed that the compliance with the choice and use of personal protective equipment among healthcare workers are almost always suboptimal. e essential factors that ensure the optimal implementation of infection control protocols are adequate training of healthcare workers on isolation procedures and on the appropriate use of personal protective equipment, preferably by interactive training with clear instructions; adequate manpower and organizational support; and the availability of timely and adequate guidance and support. e such training should not be limited to staff working in clinical areas; it must also include other healthcare workers such as laboratory workers and paramedical personnel. the key elements of infection control consist of patient placement (e.g., isolation facilities), strict adherence to hand hygiene (e.g., the who's "five moments for hand hygiene"), proper use of personal protective equipment such as gloves (e.g., double gloves in special circumstances), waterproof gowns, respiratory protection (e.g., surgical masks, respirators during aerosol-generating procedures), eye protection, rubber boots, and safe handling of sharp objects. support staff engaged in environmental disinfection, funerals, and burial services must also be adequately trained on the proper use of personal protective equipment and chemical disinfectants, and on the handling of human remains to ensure adequate disinfection and minimize the risk of accidental exposure to the virus. , , conclusion filoviral hemorrhagic fevers are uncommon, but they pose real clinical and public health threats to countries outside endemic areas. the routes of transmission, clinical manifestations, and infection control measures are well documented. prevention of secondary transmission is possible, provided that cases are promptly identified, and standard isolation and precautionary measures are strictly followed. as in cases of pandemic influenza or other epidemic-prone infections, a preparedness plan is essential to cope with the importation of the diseases and limit their subsequent spread. to minimize the risk of disease importation, it is essential to have a concerted program to engage vfrs and other at-risk travelers by community health education and advisories. the current evd outbreak provides an opportunity to evaluate new therapeutic and prophylactic strategies, whereas their actual value of these strategies in controlling the current and future epidemics require detailed clinical evaluations. ebola and marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for africa taxonomy of viruses. virus taxonomy: release discovery of an ebolavirus-like filovirus in europe ebolavirus and marburgvirus: insight the filoviridae family ebola and marburg hemorrhagic fever ebola haemorrhagic fever filoviridae: marburg and ebola viruses an introduction to ebola: the virus and the disease vervet monkey disease: studies on some physical and chemical properties of the 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in health-care settings, with focus on ebola personal protective equipment in the context of filovirus disease outbreak response. rapid advice guideline management of hazard group viral haemorrhagic fevers and similar human infectious diseases of high consequence european network for diagnostics of imported viral diseases. management and control of viral haemorrhagic fevers and other highly contagious viral pathogens canadian nosocomial infection surveillance program. are health care workers protected? an observational study of selection and removal of personal protective equipment in canadian acute care hospitals compliance with isolation precautions at a university hospital a review of the evidence for suboptimal compliance of healthcare practitioners to standard/universal infection control precautions sars hospital investigation team. factors associated with critical-care healthcare workers' adherence to recommended barrier precautions during the toronto severe acute respiratory syndrome outbreak behind the mask: determinants of nurse's adherence to facial protective equipment barriers to implementing infection prevention and control guidelines during crises: experiences of health care professionals guidance for safe handling of human remains of ebola patients in u.s. hospitals and mortuaries supplementary guidance for handling of dead bodies of suspected/confirmed ebola virus disease (evd) the ebola virus vp protein functions as a type i ifn antagonist ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling ebola virus disease centers for disease control and prevention. chronology of marburg hemorrhagic fever outbreaks key: cord- -oujdl authors: lung, o.; ohene‐adjei, s.; buchanan, c.; joseph, t.; king, r.; erickson, a.; detmer, s.; ambagala, a. title: multiplex pcr and microarray for detection of swine respiratory pathogens date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: oujdl porcine respiratory disease complex (prdc) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. no simple, single‐reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with prdc. furthermore, the detection of most of the bacterial pathogens implicated in prdc currently requires time‐consuming culture‐based methods that can take several days to obtain results. in this study, a novel prototype automated microarray that integrates and automates all steps of post‐pcr microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with prdc is described along with associated multiplex reverse transcriptase pcr. the user‐friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus (prrsv), influenza a virus, porcine circovirus type , porcine respiratory corona virus], four bacteria (mycoplasma hyopneumoniae, pasteurella multocida, salmonella enterica serovar choleraesuis, streptococcus suis), and further differentiated between type and type prrsv as well as toxigenic and non‐toxigenic p. multocida. the assay accurately identified and typed a panel of strains representing the eight targeted pathogens and was negative when tested with relevant and/or closely related non‐target bacterial and viral species. all targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material. the global pig industry produced approximately million pigs and million metric tons of pork in (http://faostat.fao.org). respiratory diseases are considered to be one of the main contributors to economic losses in the swine industry (opriessnig et al., ) . the united states department of agriculture (u.s.d.a.) national animal health monitoring system (nahms) study of swine production sites with or more pigs from major pork-producing states showed that respiratory problems are the main cause of nursery deaths ( . %) and grower/finisher pig mortality ( . %) (united states department of agriculture, ) . in canada, - % of pigs going for slaughter have cranioventral bronchopneumonia (hansen et al., a) . porcine respiratory disease complex (prdc) is multifactorial, with both infectious and non-infectious factors contributing to respiratory disease and predominantly seen in pigs between the ages of and months (opriessnig et al., ) . the interaction of viral and bacterial pathogens, environmental factors, pig-specific factors and management conditions all contribute to the development and impact the severity of prdc (opriessnig et al., ) . the type of pathogens involved in prdc is specific to the regions and countries where production occurs (opriessnig et al., ) . however, viruses most commonly associated with prdc include porcine reproductive and respiratory syndrome virus (prrsv) (rammohan et al., ) , porcine circovirus type (pcv ) (ellis et al., ; genzow et al., ) , influenza a virus (iav) and porcine respiratory corona virus (prcv) (pensaert et al., ; jung et al., ; renukaradhya et al., ) . bacteria such as mycoplasma hyopneumoniae (m. hyopneumoniae) (hansen et al., b) , pasteurella multocida (p. multocida) (davies, ) , salmonella enterica serovar choleraesuis (s.e choleraesuis) (reed et al., ; asai et al., ) and streptococcus suis (s. suis) (done and paton, ; silva et al., ; baums et al., ) are also commonly associated with prdc. porcine reproductive and respiratory syndrome virus is a major cause of swine production losses worldwide, and in the united states, reproductive and growing pig losses are an estimated $ million per year (neumann et al., ) . prrsv (genus arterivirus, family arteriviridae) is an enveloped virus with a single-stranded, positive-sense rna genome of approximately kb. prrsv is classified into two types with type predominating in europe and type predominating in north america and asia. pcv (genus circovirus, family ciroviridae) is a non-enveloped virus with a circular, covalently closed single-stranded dna genome of - nucleotides (meehan et al., ; hamel et al., ) . iav (genus influenzavirus a, family orthomyxoviridae) are enveloped viruses with a genome composed of eight single-stranded negative-sense rna segments. prcv (genus alphacoronavirus, family coronaviridae) are enveloped viruses with a single-stranded positivesense rna genome of approximately . kb. it was first isolated in belgium in , and it is a natural variant of transmissible gastroenteritis virus (tgev) (pensaert et al., ) that contains a deletion ( - nt in size) in the s gene which is used to differentiate prcv from tgev in pcr-based assays (kim et al., ; costantini et al., ) . prcv often causes subclinical infections. m. hyopneumoniae (hansen et al., b) causes mycoplasmal pneumonia of swine and is known to be one of the most common and economically important diseases found in pig farms worldwide, having low mortality, but high morbidity (otagiri et al., ) . p. multocida is a commensal found in the upper respiratory tract of pigs, but can also act as a primary or secondary pathogen responsible for pneumonia (davies, ) and atrophic rhinitis in pigs . s. e. choleraesuis is a host adapted salmonella serovar responsible for almost all types of salmonellosis in pigs in north america and europe (kingsley and baumler, ) . s. suis is a gram-positive, zoonotic bacterial pathogen important in polyserositis, septicaemia, arthritis, pneumonia and endocarditis in pigs (done and paton, ; silva et al., ; baums et al., ) . some of these bacteria are of low pathogenicity and exist as commensals in the upper respiratory tract of healthy pigs. they can cause severe respiratory disease by invading tissues already damaged due to a primary pathogen(s) (virus or bacteria), general immunosuppression and other factors such as poor environmental conditions and poor management practices. routine diagnostic methods for detection of viruses implicated in prdc include virus isolation in cell culture, antigen detection by direct fluorescent antibody staining, and enzyme immunoassay and culture-based methods for bacteria. such methods (grau-roma and segales, ) are time-consuming and require independent tests for each pathogen. furthermore, the detection of bacterial pathogens typically depends on time-consuming culture-based methods that can take several days to obtain results. due to their high sensitivity and ease of use, pcr and real-time pcr tests have been developed for several agents implicated in the prdc; however, these tests typically target single pathogens (lierz et al., ; lomonaco et al., ; tang et al., ) . a diagnostic test capable of simultaneous detection of multiple pathogens involved in prdc (atashpaz et al., ; wernike et al., ; xu et al., ) would save time, labour and cost by providing more information with each test performed. a multiplex pcr assay capable of detecting five porcine viruses including two porcine respiratory viruses was developed (giammarioli et al., ) . duplex and triplex real-time pcr for porcine respiratory viruses have also been recently described (chang et al., ; wu et al., ) . however, to date, there are no diagnostic tests capable of simultaneous detection of multiple major viral and bacterial porcine respiratory pathogens in a single reaction. microarray technology, with its capacity to incorporate a large number of capture probes, is a potentially useful tool for multiplexed detection and typing of pathogens. here, a microarray assay with associated multiplex rt-pcrs for detection and differentiation of four viruses and four bacteria involved in prdc using a novel user-friendly electronic microarray in which capture probe printing, hybridization, washing and reporting are fully integrated and automated is described. the electronic microarray contains test sites which can be activated independently via electrodes and utilizes electrophoretically driven hybridization that can be completed instantaneously. databases containing all available full and partial sequences of the genomic regions encoding the matrix proteins of iav (n = ) and prrsv (n = ), prcv spike protein (n = ), and pcv capsid protein (n = ) were compiled from the national centre for biotechnology information (ncbi). similarly, databases were compiled for the kmt (n = ) and toxa (n = ) genes of p. multocida, sly (n = ) and orfb (n = ) genes of s. suis, a bp portion of intergenic region ig- / (gardner and minion, ) of m. hyopneumoniae (n = ) and a bp region between open reading frames sc and sc previously identified as a metabolic island for s. e. choleraesuis (n = ). sequences were retrieved by searching ncbi's 'nucleotide' database using the gene names as keywords, as well as performing blast homology searches (altschul et al., ) with a representative sequence for each target, and downloading the aligned portion of all blast hits. redundant sequences were removed based on accession numbers. multiple sequence alignments for each genetic target were generated with clustalx v. . ( thompson et al., ; larkin et al., ) or mafft v. . (katoh and standley, ) using default settings. databases were maintained with either mega (tamura et al., ) or bioedit sequence alignment editor v. . . (hall, ) to ensure that all sequences were correctly oriented and aligned. similarly, representative whole-genome sequences, as well as full and partial sequences of homologous genes from related and unrelated non-targets such as tgev, porcine circovirus type (pcv ), as well as other salmonella enterica serovars, and mycoplasma species were downloaded for in silico analysis of probe specificity. several published pcr primers suitable for the assay were adopted from the literature (table ) . additional pcr primers (table ) and all target-specific capture probes (table ) were designed using either alleleid (premier biosoft international, palo alto, ca, usa) or bioedit sequence alignment editor v. . . (hall, ) based on the databases described above. primers and probes were designed to be - bp in length, with the melting temperatures between and °c and minimal secondary structures (dg ≥ À . kcal/mol). primers and probes identified by each software were compiled and examined in silico for specificity by blast (altschul et al., ) analysis using custom inhouse databases containing representative whole-genome sequences, as well as full or partial sequences of homologous genes from related and relevant non-targets. primers or probes that showed significant homology to closely related or unrelated non-target species were excluded from further investigation. a list of the viruses, bacteria and clinical samples used in this study is presented in table . the yield of the rneasy mini kit (qiagen, mississauga, on, canada), qiaamp viral rna mini kit (qiagen) and magmax total nucleic acid kit (ambion, austin, tx, usa) was evaluated with pcv (a non-enveloped dna virus) and prrsv (an enveloped rna virus) as per the manufacturers' recommendation. for these viruses, ll of a quantified stock was serially diluted ( À to À ) in swab material from -dayold piglets (prairie swine centre, saskatoon, sk, canada), previously tested to be negative for the targets. similarly, the ultraclean tissue and cells dna isolation kit (mobio laboratories, carlsbad, ca, usa), dneasy blood and tissue kit (qiagen) and magmax total nucleic acid kit were tested to evaluate the nucleic acid extraction efficiency of a gram-positive bacteria (s. suis) and a gram-negative bacteria (p. multocida). for these bacteria, ll of culture of known cfu/ml was serially diluted in the same swab material as above and extracted using each kit in parallel according to the manufacturers' instructions. the efficiency of the extraction kits was evaluated based on the limits of detection observed after rt-pcr amplification of the extracted material. the dneasy blood and tissue kit and the viral rna mini extraction kit were the most efficient for the tested bacteria and virus targets, respectively (data not shown). therefore, all subsequent nucleic acid extractions of laboratory amplified strains were performed using these kits. following preparation of nucleic acid extractions, the samples were subjected to pcr and microarray analysis. for the determination of the analytical sensitivity of the assay for viral targets, selected genes were amplified using the superscript tm iii one-step rt-pcr system with platinum â taq dna polymerase (life technologies, carlsbad, ca, usa) and cloned into the pjet . cloning vector using the clonejet pcr cloning kit (fisher scientific, ottawa, on, canada) according to the manufacturers' specifications. plasmids were extracted from successfully transformed bacteria using the qiaprep miniprep kit (qiagen) according to the manufacturer's specifications and were confirmed by sequencing (eurofins genomics, huntsville, al, usa). vectors containing the target genes for each virus, except for pcv , were linearized with the hind iii restriction enzyme (fisher scientific) and subjected to in vitro transcription using the megascript t transcription kit (life technologies) according to the manufacturer's specifications. template dna was eliminated using successive treatments with turbo dnase (life technologies,) before quantifying the rna using the rna br assay kit and the qubit . fluorometer (life technologies) according to the manufacturers' specifications. the rna was then serially diluted : in ultrapure distilled water (life technologies). the copy number was inferred using an online tool (http://endmemo.com/bio/dnacopynum. php) taking into account the nucleic acid concentration and nucleotide composition of the amplified region of each target. the copy number for the plasmid containing pcv capsid protein coding region was inferred based on the nucleic acid concentration and nucleotide composition over the entire plasmid. for the determination of the analytical sensitivity of the assay for bacterial targets (excluding m. hyopneumoniae), frozen cultures were streaked for single colonies onto % sheep blood agar plates (bbl tm blood agar base infusion agar; bd diagnostics, sparks, md, usa) and incubated at °c overnight. a single colony was inoculated into ml of miller's lb broth (life technologies) and grown overnight at °c on a shaking incubator ( rpm). the overnight lb broths were serially diluted : in pbs, and ll of material was spread onto blood agar plates in triplicate and grown overnight at °c for enumeration using the viable plate count method. the cultures were standardized to . cfu/ml (s. e choleraesuis and p. multocida) and . cfu/ml (s. suis), so a ll aliquot from each serial dilution in the series yielded cfus to the nearest power of base (i.e. , , etc.). for m. hyopneumoniae, an aliquot of genomic dna was quantified on the qubit . fluorometer, and a genomic copy number was inferred based on the nucleic acid continued) concentration and nucleotide composition over the entire genome. the analytical specificity of the viral and bacterial multiplex pcr assays was assessed by amplifying panels of non-target viruses and bacteria, respectively (table ) . the forward primers were modified with -phosphorylation (idt, coralville, ia, usa). all reverse primers were modified with either -tye â fluorophore using spc â attachment chemistry for the slide microarray or were synthesized with the reverse complementary sequence of the reporter probe at the end for the electronic microarray as described in lung et al. ( ) . rt-pcrs were performed using the superscript tm iii one-step rt-pcr system with platinum â taq dna polymerase. a multiplex rt-pcr with primers targeting the genomic regions encoding the iav and prrsv matrix proteins, prcv spike protein, pcv capsid protein, as well as an internal control, was developed (table ) . a multiplex pcr with primers targeting the kmt and toxa genes of p. multocida, sly and orfb genes of s. suis, intergenic space of m. hyopneumoniae, metabolic island of s. e. choleraesuis and an internal control was developed (table ) . a plasmid containing a fragment of the dengue virus genome was used as an internal pcr control for both the bacterial and viral rt-pcrs. both assays were optimized for buffer and magnesium concentration, annealing temperature, cycle number and internal control concentration. the finalized rt-pcr mixtures consisted of ll of nucleic acid, . pg internal control, ll of enzyme mix, lm of each primer in reaction buffer in a final volume of ll. reverse transcription was carried out for min at °c, followed by °c for min. pcr was carried out for cycles of °c for s, °c for laboratory samples and °c for clinical samples for s, °c for s, with a final extension step of °c for min. following pcr, unpurified material was assayed on the qiaxcel capillary gel electrophoresis system (qiagen) for visualization of amplicons. analytical sensitivity of each multiplex assay was determined using serial dilutions of quantified dna or reverse-transcribed rna as appropriate for each pathogen. the serial dilutions were amplified using the multiplex pcr assays as well as the singleplex pcr for each target. all pcr amplifications were carried out on the veriti thermocycler (life technologies) and visualized on the qiaxcel using the biocalculator v. . software (qiagen). the limit of detection was considered to be the last dilution where amplification was greater than the default threshold on the electropherogram and described in terms of the approximate total number rna or dna copies in the sample. a total of probes for the detection of four target viruses, probes for the detection of four bacterial targets and three control probes were initially screened by passive hybridization on low-cost conventional epoxy glass slide microarrays (corning, corning, ny, usa) that were printed and processed in-house according to protocols described previously (lung et al., ) . the probes were screened against a panel of five isolates representing the four target viruses, and three non-target viruses, and strains representing the four target bacteria, and non-target bacteria (table ) . microarray data were represented using the mean pixel intensity for each probe reaction. probe reactivity was calculated using the mean pixel fluorescent intensity (mfi) of all probes as a ratio of the non-template control. probe reactions above the ratio of the non-template control were considered positive. probes that showed good reactivity and specificity were selected for testing and validation on a novel automated electronic microarray in which capture probes are printed on streptavidin-containing acrylamide hydrogels and hybridization, washing and reporting are automated and computer controlled. capture probes used on the electronic microarray were modified with -biotin group to allow attachment to streptavidin-containing test sites (idt, coralville, ia, usa). a selected set of probes targeting the viruses, probes targeting the bacteria and three control probes (a negative probe and two probes targeting the internal control), which exhibited high reactivity and specificity on glass slide microarrays, were selected for validation on the electronic microarray. the viral probes were tested against an expanded panel of strains or isolates of the four target viruses and non-target viruses (table ) . similarly, the bacterial probes were tested against a panel of strains or isolates representing the four target bacteria and related or unrelated non-target bacteria ( table ). the electronic microarray assays were run using a protocol previously described (lung et al., ) with modifications. the modifications included the replacement of the 'touch down' washing protocol with a 'touch up' protocol in which washing steps were carried out using low salt buffer (nexogen, inc., san diego, ca, usa) with incremental increases rather than decreases in temperature. images were captured at each temperature increment. the red universal reporter probe was replaced with a -alexa fluor modified locked nucleic acid (lna) variant ( -/ alex n/tgt+ca+agc-g+at+at+act+gc- ) (idt, coralville, ia, usa) to increase its thermal stability over a more robust range of wash temperatures. all electronic microarray hybridizations were performed in duplicate, and a non-template pcr control (ntc) was included in all experiments. raw fluorescent intensity (fi) data from all utilized electrodes at each temperature increment were obtained and analysed using microsoft excel. for each probe, positive-to-non-template control (p : n) ratios were calculated by dividing the fi value from each analyte by the fi value produced by the ntc. for each assay, samples that produced p : n ratios of . or greater were considered positive. average p : n data derived from the microarrays were visualized with a heat map generated using treeview v. . (eisen et al., ) . oral and nasal swabs were obtained from specific pathogen free pigs at the cfia ottawa laboratory fallowfield, ontario, canada. oral and nasal swabs were screened for the target viral and bacterial pathogens, and pools of oral and nasal material that were negative for the target bacteria and viruses were used for spiking with target pathogens. bacterial strains were grown and quantified as described above, and supernatants containing virus from cell culture were used. for m. hyopneumoniae, culture was not performed and a freeze-dried cell pellet purchased from atcc was used after re-suspension in pbs and % glycerol. for inoculations with single agents, ll aliquots of oral and nasal samples were experimentally inoculated with ll of each live virus or bacteria. samples inoculated with multiple agents were prepared by pooling ll of each pathogen together, and adding ll of the pooled pathogens into ll of oral and nasal material (table ). nucleic acid from the full ll volume of the samples was extracted, pcr amplified and assayed on the electronic microarray as described in previous sections. approximately - mg of a panel of lung tissue submitted in to manitoba agriculture, food and rural initiatives (mafri) for diagnosis of porcine respiratory pathogens was ground in . ml of rlt buffer (qiagen), transferred to ll of magmax lysis buffer and processed for nucleic acid extraction in the kingfisher instrument (ambion). five to ll of extracted rna from ll of elution buffer was tested by real-time rt-pcr at mafri, and ll was tested at cfia by electronic microarray. multiplex pcr/rt-pcr two separate multiplex pcrs were developed for amplification of selected genes of four viruses and four bacteria involved in prdc, respectively. the multiplex pcr for bacteria consisted of a total of primers for the detection of the four target bacteria, typing of p. multocida and a pair of primers for an internal control (table ) . two genes from s. suis and p. multocida were each targeted for amplification by the multiplex pcr. amplification and detection of the toxa gene of p. multocida allowed for differentiation of virulent or pathogenic strains from non-pathogenic strains. primers for the orfb gene were added to a previously designed multiplex pcr with primers for the sly gene to allow detection of s. suis strains that lack the sly gene. the multiplex pcr generated products of the expected sizes ranging from approximately - bp (table ) when a panel of isolates representing the target species, including different serotypes of s. suis were tested (fig. ) . similarly, a multiplex rt-pcr with primers was developed and used to detect the four viruses and an internal control. the multiplex rt-pcr successfully amplified a panel of targeted viruses and generated amplicons of the expected size of approximately - bp ( table ) . the samples represented both genotypes of prrsv, as well as different subtypes of iav (fig. ) . the internal control variably amplified in both the bacterial and viral multiplex pcrs as a result of competitive pcr. in instances where targets were strongly amplified, amplification of the internal control was either weak or absent. conventional glass slide microarrays were processed manually as an initial low-cost screening tool to assess the specificity of the probe set (n = ) designed to detect the four target viruses, distinguish between genotypes and of prrsv, as well as differentiate prcv and tgev, and the probe set (n = ) designed to detect the four target bacteria and differentiate toxigenic and non-toxigenic strains of p. multocida. a selected set of probes targeting the viruses, probes targeting the bacteria and three control probes (a negative probe and two probes targeting the internal control), which exhibited the highest reactivity and specificity on slide microarrays, were selected for validation on the user-friendly automated electronic microarray. all target viruses and bacteria were accurately detected, and prrsv and p. multocida were accurately typed using the viral and bacterial probe set on the electronic microarray platform (fig. ) . the assay also successfully detected targeted pathogens in clinical lung specimens, as well as porcine oral and nasal swab material experimentally inoculated with single or multiple targets (table ). the results obtained were consistent with those obtained by singleplex assays with the exception of lt- which was positive for p. multocida based on the electronic microarray assay, but negative by bacterial culture. for both the rt-pcr and microarray assays, the analytical sensitivity varied with the different targets. for some targets, the multiplex assay had comparable sensitivity with the respective singleplex assay, while for other targets, the singleplex assays were more sensitive ( table ). the multiplex assay was most sensitive for detection of iav and s. suis for viral and bacterial targets, respectively (table ) . non-target bacteria samples did not react with the probes on either the conventional slide microarray or electronic microarray. other than tgev, a natural variant and highly related virus to prcv, no other non-target viruses showed amplification in the virus multiplex rt-pcr (data not shown). due to the strong amplification of tgev by the multiplex rt-pcr, the internal control failed to amplify (fig. b, sample ) . however, prcv and tgev were differentiated based on amplicon size, and in subsequent microarray characterization (fig. ). a user-friendly microarray for the simultaneous detection and differentiation of four viruses and four bacteria associated with prdc was developed as a test case for a novel automated 'amplicon-to-answer' electronic microarray technology. the electronic microarray integrates and automates all post-pcr steps required for microarray analysis (capture probe printing, hybridization, washing, reporting) and allows for simultaneous identification of eight pathogens, differentiation of the two prrsv genotypes and pathogenic versus non-pathogenic p. multocida strains. although the amplification of bacterial dna did not require a reverse transcriptase phase, an rt step was included in the pcr protocol as the amplicon yield was better than without the rt step. using the same protocol would also allow potential combination of the bacterial and viral multiplex pcr into a single multiplex pcr targeting all eight pathogens. a likely explanation for the increased amplification yield observed with rt-pcrs for bacterial targets is rt-pcr could utilize not just genomic dna, but also rna transcripts of target genes as template. in addition, the proprietary quantity of taq polymerase in the rt-pcr kit that was used in the rt-pcrs may be higher than that used in the pcr. initial screening of capture probes was performed using a conventional slide microarray platform due to the lower cost of screening large number of probes that were printed in conjunction with other projects. subsequently, the assay was adapted to a novel, rapid and user-friendly microarray platform that automates and integrates capture probe printing with all post-pcr steps of the assay, including electrophoretically driven hybridization, washing and reporting. the automated electronic microarray assay reduces the labour, time and number of instruments needed to acquire microarray results when compared with conventional microarrays which typically require substantial manual processing, slower passive hybridization of amplicons with capture probes and multiple pieces of equipment. the electronic microarray has an open platform format with test sites that can be individually activated and utilizes a single integrated instrument that automates capture probe printing and microarray processing using parameters set by the user. thus, the novel technology also reduces the skill level required to perform microarray assays and allows immediate, on-site modification of assays depending on the needs of the enduser. these unique features eliminate the need for anticipation of future needs, and the procurement and storage of manufactured arrays that are designed for a specific set of predetermined pathogens. for example, the system will allow immediate switching between assays that detects/ types all eight pathogens simultaneously to one that detects/types a subset of the pathogens, or to assays for detection and typing of other pathogens. as the user is able to control hybridization, wash and reporting temperature the assay can have excellent specificity and can be used for differentiating variants that are genetically very similar. however, the automated electronic microarray assay requires specialized arrays and investment in instru- probes p. multocida (toxigenic) fig. . summary of microarray results from the electronic microarray representing the four bacterial targets (a), the four viral targets (b) on the electronic microarray. the reactivity of specific reactions between targets and each pathogen-specific probes is outlined in yellow. nsbp = non-specific binding probe negative control. p : n ratios ≥ . are shown in red, and p : n ratios < . are in black. ntc = no template control. ic = internal control. amplification and detection of the internal control are not always observed when a target is present in high amounts. mentation. the cost of consumables for testing a sample with an electronic microarray is expected to be between one and a few real-time pcr assays and will depend on the assay design (i.e. reducing the number of probes will decrease the cost by allowing more samples to be tested on each array with test sites). in addition to glass and hydrogel (acrylamide)-based microarray matrices used in this study, the final probe sets have been tested successfully on reverse dot blots performed on nylon membranes (unpublished results). thus, the probe set described should have broad applications in hybridization-based assays that use a variety of different matrices. assays for simultaneous detection of multiple bacteria and virus implicated in prdc have not been described previously. the limit of detection of a lamp assay for pcv has been shown to be approximately copy of dna plasmid, much more sensitive than conventional pcr whose detection limit was copies (zhou et al., ) . however, lamp assays typically detect a single pathogen and are difficult to multiplex. in multiplex real-time pcr assays, a duplex pcr assay had a detection limit of tcid /ml for pcv and . tcid /ml for prrsv (chang et al., ) , while a triplex real-time pcr assay had a detection sensitivity of copies/ll for pcv and prsv and copies/ll for ppv (wu et al., ) . the multiplex pcr described here did not reduce the limits of detection for five targets although the detection limit of the pcr for three pathogens was an order of magnitude lower in the multiplex format. thus, further improvements in sensitivity are desirable and may be partly achieved by increasing the number of pcr cycles, reducing amplicon lengths or reducing the amount of internal control used in the assay. the limit of detection for pcv , prrsv, and m. hyoneumoniae was lower with the electronic microarray in comparison with the multiplex rt-pcr indicating the use of the electronic microarray platform reduces sensitivity, and unpublished results show that only amplicons that were visible after agarose gel electrophoresis can be detected by the electronic microarray. despite the reduced analytical sensitivity of the electronic microarray assay for some of the targets, the bacterial and viral targets were detected singly or in combination in clinical samples submitted for laboratory diagnosis. furthermore, the assay detected p. multocida in a clinical lung sample that was not detected by traditional culture methods. this discrepancy may be due to the higher sensitivity of the microarray assay, or the lack of active infection (e.g. the use of antibiotics). while primers and probes were evaluated using all sequences available on ncbi at the time of assay design and samples representative of both target and non-target bacteria and viruses were tested in this study, regular reevaluation of the coverage of the primers and probes and additional validation with clinical samples is desired. the four swine respiratory viruses targeted in this study have also been successfully detected with the virochip panviral array which consists of probes for detection of all known viruses at the time of design (nicholson et al., ) . the high-density virochip is a useful tool for virus discovery, but needs approximately h to obtain results and requires high viral titre for positive detection. in contrast, the electronic microarray assay described here can be completed in less than hours with little user handling plus approximately . h for the rt-pcr described. new instrumentation that further simplifies the workflow by integrating the pcr and array processes is now 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detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-pcr rapid multiplex pcr and real-time taqman pcr assays for detection of salmonella enterica and the highly virulent serovars choleraesuis and paratyphi c a sensitive multiplex real-time pcr panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders : development of multiplex pcr for simultaneous detection of six swine dna and rna viruses loop-mediated isothermal amplification for detection of porcine circovirus type . . . . prcv prrsv . . . . iav m. hyopneumoniae . p. multocida . . . . s. e. choleraesuis . . . . s. suis none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- - nrjpwh authors: wolff, cecilia; emanuelson, ulf; ohlson, anna; alenius, stefan; fall, nils title: bovine respiratory syncytial virus and bovine coronavirus in swedish organic and conventional dairy herds date: - - journal: acta vet scand doi: . /s - - -x sha: doc_id: cord_uid: nrjpwh background: infections with bovine respiratory syncytial virus (brsv) and bovine coronavirus (bocv) are endemic to the cattle populations in most countries, causing respiratory and/or enteric disease. it has been demonstrated that herds can remain free from these infections for several years also in high prevalence areas. organically managed (om) dairy herds have been shown to have lower seroprevalence of both viruses compared to conventionally managed (cm) herds. the objective of this study was to challenge the hypothesis of a lower occurrence of brsv and bocv in om compared to cm dairy herds. in november , may and may milk samples from four homebred primiparous cows were collected in to om and to cm herds. the antibody status regarding brsv and bocv was analysed with commercial indirect elisas. herds were classified as positive if at least one individual sample was positive. results: the prevalence of positive herds ranged from . % to . % for brsv and from . % to . % for bocv among om and cm herds, over the three sampling occasions. there was no statistically significant difference between om and cm herds at any sampling occasion. the incidence risk of newly infected herds did not differ statistically between om and cm herds at any sampling occasion, neither for brsv nor for bocv. the incidence of herds turning sero-negative between samplings corresponded to the incidence of newly infected. bulk tank milk (btm) samples were also sampled in the herds and analysed. several herds were negative on individual samples but positive in btm. herd-level data on production, health and reproduction were retrieved from vÄxa sweden and the study herds were representative of the source population. conclusion: there was no difference in prevalence of or incidence risk for brsv or bocv between swedish om and cm herds. because the incidence of herds becoming seropositive was balanced by herds becoming seronegative it should be possible to lower the prevalence of these two infections among swedish dairy cattle herds if biosecurity is improved. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. infections with bovine respiratory syncytial virus (brsv) [ ] [ ] [ ] [ ] and bovine coronavirus (bocv) [ , ] are endemic in the cattle populations in most countries. it has been demonstrated that a cattle herd can remain free from these infections for several years [ ] , even when located in high prevalence areas [ ] and in close proximity to herds experiencing an (brsv) outbreak [ ] . herds may become antibody negative to any of these infections within a few years provided the virus is not re-introduced into the herd [ , ] . brsv commonly cause respiratory disease, particularly in calves. disease can be caused by brsv only or in combination with other viruses (e.g. bocv) or secondary bacterial infection [ ] [ ] [ ] . bocv also causes enteric disease, in particular calf diarrhoea [ ] , and is the causative agent of winter dysentery, outbreak of diarrhoea, in adults [ ] . there have been reports of bocv as the single agent in outbreaks of respiratory disease as well [ , ] . in addition to impaired animal welfare due to illness, these infections may cause losses to production by reduced weight gain [ , ] , reduced milk yield [ , ] , increased bulk tank [ ] and individual [ ] milk somatic cell counts. after infection with brsv or bocv animals will remain seropositive for several years. this was demonstrated by bidokti et al. [ ] who found herd where the older cows were sero-positive while the younger cows were sero-negative, i.e. there had been no virus circulating for several years. maternal antibodies remain detectable for approximately months [ , ] , i.e. a never-infected heifer will be seronegative at the time of first calving. both milk and blood samples can be used to assess the serological status of cattle [ ] . when the herd's status is based on a bulk tank milk (btm) sample, which is convenient e. g. for screening a population, the result will reflect the long term, i.e. up to the life-span of the oldest cows, history of the herd. however, if primiparous homebred cows are sampled, the results will give a more accurate description of the recent, i.e. the life-span of the tested cows, history of the herd. although these viruses may spread during the warmer seasons, seroconversion with or without an outbreak of clinical disease is more frequent during the housing season (autumn and winter) [ , , ] . there is however, still a knowledge gap concerning what the most important routes for virus transmission between herds are. a few studies from the nordic countries have studied risk factors for herds to be and to become seropositive to brsv and bocv. risk factors at herd level have included a short distance to nearest herd, not providing boots to visitors, large herd size and a high density of cattle in the area [ , , , ] . a recent study found that organically managed (om) dairy herds had significantly lower seroprevalence of both bocv and brsv compared to conventionally managed (cm) herds. however, the study could not explain the reason for the differences in the two production systems [ ] . although the difference was statistically significant, the study was made with a relatively small sample of herds. it would be beneficial for the organic as well as the conventional dairy production if these results were validated and studied further. moreover, the swedish dairy industry has over the last decade undergone rapid structural changes with increasing herd sizes and decreasing herd numbers. simultaneously, the proportion of dairy cows under om management has increased from % in to % in [ , ] . this also calls for a new assessment of disease occurrence in the two management systems. the objective of this study was therefore to challenge the hypothesis of a lower occurrence of brsv and bocv in om compared to cm dairy herds and, specifically, to compare the herd prevalence and incidence. this was a prospective longitudinal observational study including om and cm dairy herds. the unit of interest was herd. the sampling frame was all dairy herds with a yearly average herd size of at least cows and enrolled in the swedish official milk recording scheme. the inclusion criterion of a yearly average herd-size of at least cows was chosen to exclude small herds which do not represent the "future" herd. geographically, all counties except for most southern skåne were included. skåne was excluded because it was known that there are very few brsv and bocv negative herds in this region. om herds were defined as herds with a dairy production certified according to the standards by the association krav (www.krav.se), the swedish member of the international federation of organic agriculture movements. the desired size of the study group was om and cm herds which was as many as the project would be able to manage. the number of invited herds was based on previous experiences of about % willingness among swedish dairy farmers to participate in similar observational studies. a simple random sample of cm herds from the in the sampling frame, and all eligible om herds (n = ) were sent a written invitation to the study in may . in total, ( %) and ( %) of farmers with om and cm herds, respectively, agreed to participate in the project. participants gave a written permission to access the herd's data from the milk recording scheme. study herds were sent instructions, material and protocol for sampling in november , may , and may . these occasions were chosen to include as many stall periods as possible during the project. at each sampling occasion, milk from four homebred primiparous cows and btm were sampled into test tubes with . mg of the preservative agent bronopol. samples were returned to the national veterinary institute by prepaid mail where they were stored at − °c until analysed. the antibody status regarding brsv and bocv was analysed with the commercial indirect elisas (svanovir brsv-ab and svanovir bcv-ab, boeringer ingelheim svanova, uppsala, sweden). the optical density (od) of samples was corrected by subtraction of negative control od, and the percent positivity (pp) value was calculated as the corrected od divided by the corrected od for positive controls and multiplied by . the samples from individuals were classified as negative if pp < , following the manufacturer's guidelines, and bulk tank samples if pp < . the sensitivity and specificity of the elisa test kit was % and %, respectively, for brsv and . % and %, respectively, for bocv, according to the manufacturer. after each completed sampling occasion, the farmers were sent information on their herd's serological status and information about basic biosecurity measures. a lottery ticket (value approx. euro) was enclosed to the letter. antibody status as measured in individual milk samples was used as an indicator of the herd's recent infection status regarding the two viruses. herds were defined as antibody positive if at least one of four samples from individual cows was positive. a stricter definition of positive herd where all four individual samples had to be positive was also applied. to assess any difference in antibody status between om and cm herds, the prevalences of positive herds at each sampling occasion among om and cm herds, respectively, were calculated with exact binomial confidence intervals using the package "binom" for r [ ] . data management and analysis was performed in r version . . [ ] . further, the incidence risk with exact binomial confidence intervals was calculated for the samplings in and , for om and cm herds, respectively. the period at risk was from the last sampling occasion, i.e. months and months, respectively. to explore if there were differences in antibody level in the bulk tank milk between the cm and om herds, the results from the btm samples (pp values) were categorised into six groups; pp < , ≤ pp < , ≤ pp < , ≤ pp < , ≤ pp < , and ≤ pp. the frequencies of om and cm herds in each group were tabulated for each year and virus. data on herd production, health and reproduction were retrieved from the swedish official milk recording scheme database managed by vÄxa sweden for all selected herds, i.e. including also the invited but nonparticipating herds. the information included averages of routinely collected parameters for the months before the start of the study. herd parameters were described and tested for difference between the om and cm herds that participated in i.e. entered the study, using a two-sided wilcoxon rank-sum test or fischer exact test. to study how the study group characteristics were affected from dropout herds leaving the study, herd parameters were described for the subsample of om and cm study herds that completed the last sampling in . to examine the representativeness of the study herds, herd parameters were also described for all invited om and cm that did not participate in the study. the number of study herds per year was , , and in , and , respectively (table ) based on the test results from sampled individuals the prevalence of brsv positive herds ranged from . % to . % over three sampling occasions among om and cm herds (table ). in , the prevalence of positive herds was lower in cm herds, and in and in om herds. for bocv the prevalence estimates ranged from . % to . % positive herds over three sampling occasions among om and cm herds (table ). in and the prevalence of positive herds was lower in cm herds and in in om herds. the confidence intervals of om and cm herds' prevalences were overlapping all years and it was therefore concluded that there was no statistical difference in the prevalence of brsv or bocv between om and cm herds. the prevalence of brsv-positive herds decreased to . - . % and for bocv to . - . % with the stricter the criteria of four out of four positive individual samples for a herd to be classified as positive at the sampling occasion ( table ) . the incidence risk of newly infected herds, i.e. herds that went from negative to having at least one of four primiparous cows with a positive test result, did not differ statistically between om and cm herds in any study year, neither for brsv nor for bocv (table ). there were in total herds that were antibody-negative to both viruses in ( . %), and herds in both ( . %) and ( . %) ( table ) . not more than , and herds were brsv negative and , , and herds were bocv negative in , and , respectively ( table ). all these herds negative in btm were also negative based on the individual samples. further, for both viruses, in a high proportion of all herds with btm pp < , all individual samples were negative. and, on the contrary, in a high proportion of herds with btm pp ≥ , all individual samples were positive ( table ) . at study start the om study herds were larger, had higher btm somatic cell counts, lower incidence of veterinary treated disease events but also a lower production compared to the cm study herds (see additional file : table s ). herd data for herds entering the study and herds completing the study were judged as comparable (see additional file : table s ). the characteristics of the herds entering the study were judged as comparable to the invited herds not entering the study (see additional file : table s ). in this study, there was no difference in incidence or prevalence of brsv or bocv antibody positivity between om and cm herds. this disagrees with previous results from sweden where the mean seroprevalence of antibodies in individual cows for brsv and bocv was found to be lower in om dairy herds compared to cm [ ] . ohlson et al. [ ] could not report any differences between om and cm herds with respect to brsv and bocv seropositivity, which agrees with the results in the present study. however, that study included only a small sample of om herds. the number of swedish om dairy cows increased from , in , when the herds in bidohkti et al. [ ] were recruited, compared to , in while during the same time period, the total number of dairy cows decreased from , to , [ , ] . a possible explanation to the different results in the current study compared to [ ] could be that the characteristics of the farmers with om and their herds as a group have changed from to . such a change of the "typical organic farmer" over time and in relation to market demand and political decisions, e.g. how incentives for organic farming might have developed from ideological for early converters to more financial for later, was discussed for denmark [ ] . interestingly, the total number of om herds going from positive to negative was higher compared to cm herds, but the opposite was true for herds going from negative to positive status (table ) . this could be hypothesised to be a result of better implementation of the biosecurity advice to participants by the farmers with om. although om herds were overrepresented in the study group, the overall prevalence estimates, as well as for om and cm separately, are in concordance with previous studies where herd prevalence of brsv in btm samples was - % in northern sweden and - % in the most southern parts [ ] , and % in a study with seven southern and northern counties [ ] . for bocv herd prevalence was reported as - % to - % in btm samples depending on region [ ] and % in a study with southern and northern counties [ ] . the national prevalence of these two infections seems not to have changed a lot from the late -ies. it would be interesting to further explore how changes of routines or characteristics of dairy herds during a bit more than a decade might be associated with the prevalence of the two infections. for example, if the greatly improved biosecurity in many farms might have been offset by poor routines in others during the last decade. the current sampling frame included all counties of sweden but the skåne county which from previous research was known to have a herd prevalence near % and therefore considered as of less interest in this study. in a screening of bulk tank milk samples in , including % of all swedish dairy herds, the herd prevalence in skåne was % (personal communication a ohlson) . every year several herds became antibody negative, and in other words gained brsv and/or bocv free status, but the incidence of number of herds becoming positive equalled out the number of herds clearing the infection. there were also a considerable number of herds with negative primiparous cows but positive btm, i.e. herds in which there had been no virus circulating for at least - years. apparently, there are good chances for a herd to free from these infections if good biosecurity is practiced and the virus is not reintroduced. this result agree with a norwegian survey where the incidence of herds becoming brsv positive (at least one antibody positive animal) and negative after six months was % and %, respectively [ ] . a high turnover of positive herds was also found in a swedish study over several years [ ] . if introduction of brsv and bocv to a herd can be eliminated or kept at lowest possible level, it would be possible to significantly reduce the prevalence of these infections in the swedish dairy population. many of the herds with positive btm samples, but with a low pp value, i.e. < had only negative individuals. this indicates that there had been no virus circulating in the herd the last to years (table ) but was rather due to antibodies from older cows who experienced the infections several years ago. this finding agrees with ohlson et al. [ ] where all sampled primiparous cows in herds with a btm sample pp value < were negative. we would therefore recommend sampling milk from young homebred cows over btm samples in prevalence studies/screenings, because of the advantage that herds where there has been no recent active infection can be identified. to save analysis expenditure individuals' samples, in addition, can be pooled for analysis without significant loss of sensitivity or specificity at herd level [ ] . also, the prevalence of positive herds decreased when "positive" was defined as four of four positive individuals ( ≤ pp) but the difference in prevalence using this stricter definition was not significant (table ). this supports that brsv and bocv virus have efficiently spread in the herd after introduction and results in a subsequent high within-herd seroprevalence. with a herd size of , the test characteristics of the two elisas used, and a cut-off point for positive herd of one positive out of four tested individuals, the hse for brsv is % and % at a within-herd prevalence of % and % respectively. for bocv the hse is % and % at a within-herd prevalence of % and %, respectively. hsp is % for both tests as calculated with the web based tool [ ] using the hypergeometric distribution. the herd sensitivity of the serological test can be increased by increasing the number of sampled individuals per herd; however, with a high within herd prevalence this becomes less influential. a limitation of the study is the less than perfect sensitivity of the two elisa tests used. because the aim was to compare om and cm herds, the true prevalence or incidence risk was not estimated. any misclassification bias due to test characteristics should be the same in the two groups. further, the different lengths of period at risk for the incidence estimates at the second and third sampling occasions was not optimal, because it made comparisons between sampling occasions impossible. this, however, does not influence the comparison of om and cm herds at the same sampling occasion. however, this comparison could have been improved with a larger number of study herds. for example, the number of herds going from positive to negative or the opposite was now low and the confidence intervals of estimates wide. it would also have been beneficial with a longer total study period, thus including more than two housing periods, and more frequent sampling to explore how herds' status change over time with better precision. we consider the om and cm study herds as representative of the om and cm dairy herds in sweden, because their production and health parameters did not differ substantially from the invited but non-participating herds (additional file : table s ). however, the study herds could still be biased towards more engaged and skilful farmers compared to non-participants and a higher response rate would have been desirable to minimize this risk. this is particularly valid for the cm herds, where the response rate was % compared to % for the om herds. this could mean that the farmers with om more accurately represent the om population, while a possibly stronger bias towards a stratum of "interested, progressive and better" farmers with cm could be envisioned. the invited cm herds were a random sample meaning the herds entering the study represented % of the eligible herds, while the invited om herds were a census, thus the participating herds represented % of the eligible herds. because the study included three sampling occasions the possible increase in response rate after reminders, e.g. by telephoning non-responders, was weighted against the risk of losing these additional participants to the next sampling occasion. the inclusion criterion of a yearly average herdsize of at least cows meant that , of the , dairy herds enrolled in milk recording in were not eligible for the study and the results should only be extrapolated to the larger herds. we judged that the study herds that participated to the last sampling occasion were comparable to study herds that participated at the start, i.e. herds leaving the study did not change the characteristics of the study group (additional file : table s ). there was no difference in prevalence of or incidence risk for brsv or bocv between organically and conventionally managed dairy herds in sweden. the incidence risk of herds becoming seropositive was balanced by herds becoming seronegative, but with improved biosecurity it should be possible to lower the prevalence of these two infections among swedish dairy cattle herds. additional file : table s . herd characteristics and production and health parameters at the time for the start of the study for organically managed (om) and conventionally managed (cm) herds entering the study, the study herds completing the full study period and invited but non-participating herds. bovine respiratory syncytial virus seroprevalence and risk factors in endemic dairy cattle 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immunoglobulins comparison of bovine coronavirus-specific and bovine respiratory syncytial virus-specific antibodies in serum versus milk samples detected by enzyme-linked immunosorbent assay dynamics of bovine respiratory syncytial virus infections: a longitudinal epidemiological study in dairy herds a nation-wide epidemiological study of acute bovine respiratory disease in france risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds risk factors for epidemic respiratory disease in norwegian cattle herds yearbook of agricultural statistics including food statistics dorai-raj s. binom: binomial confidence intervals for several parametrizations. r package version . - r: a language and environment for statistical computing. r foundation for statistical computing eleven years of organic dairy production in denmark: herd health and production related to time of conversion and compared to conventional production submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to express their gratitude to ms maj hjort for excellent technical assistance with the analyses of the milk samples. further, the authors would like to thank the swedish research council for environment, agricultural sciences and spatial planning, for the funding grant no: - - . the authors declare that they have no competing interests.authors' contributions nf and ue jointly conceived the study and designed it together with sa and ao. cw and nf planned and performed data collection. cw performed statistical analyses and drafted the manuscript. all authors revised and approved drafts and the final manuscript. key: cord- -w d fue authors: zuwała, kaja; golda, anna; kabala, wojciech; burmistrz, michał; zdzalik, michal; nowak, paulina; kedracka-krok, sylwia; zarebski, mirosław; dobrucki, jerzy; florek, dominik; zeglen, sławomir; wojarski, jacek; potempa, jan; dubin, grzegorz; pyrc, krzysztof title: the nucleocapsid protein of human coronavirus nl date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: w d fue human coronavirus (hcov) nl was first described in and is associated with respiratory tract disease of varying severity. at the genetic and structural level, hcov-nl is similar to other members of the coronavirinae subfamily, especially human coronavirus e (hcov- e). detailed analysis, however, reveals several unique features of the pathogen. the coronaviral nucleocapsid protein is abundantly present in infected cells. it is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. the aim of the present study was to characterize the hcov-nl nucleocapsid protein. biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the n-terminal domain responsible for nucleic acid binding and the c-terminal domain involved in protein oligomerization. surprisingly, analysis of the subcellular localization of the n protein of hcov-nl revealed that, differently than homologous proteins from other coronaviral species except for sars-cov, it is not present in the nucleus of infected or transfected cells. furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. this is in stark contrast with results obtained for other coronaviruses, except for the sars-cov. coronaviruses cause a variety of diseases in animals, whereas human infections are almost exclusively associated with respiratory tract infections (rti). contemporary taxonomy divides the coronavirinae subfamily into four genera (alpha, beta, gamma, and delta). only the alpha cell culture llc-mk cells (atcc: ccl- ; macaca mulatta kidney epithelial cell line) were maintained in minimal essential medium (mem), containing parts of hank's mem and part of earle's mem (paa laboratories, austria) supplemented with % heat-inactivated fetal bovine serum (fbs) (paa laboratories, austria), penicillin ( u/ml), and streptomycin ( μg/ml). cells were cultured on t flasks (tpp, switzerland) at °c with % co . t cells (ecacc: ; human embryonic kidney sv transformed, genetically modified) were maintained in dulbecco-modified eagle's medium (dmem; paa laboratories, austria) supplemented with % heat-inactivated fetal bovine serum (fbs; paa laboratories, austria), penicillin ( u/ml), and streptomycin ( μg/ml). cells were cultured on t flasks (tpp, switzerland) at °c with % co . t cells (atcc crl- ) were transfected with the plko. .-trc-ace plasmid using polyethylenimine (pei; sigma-aldrich, poland). the plasmid was based on the addgene plasmid [ ] . at h post-transfection, the cells were washed with sterile × pbs and cultured at °c for h in media supplemented with puromycin ( μg ml - ) at °c with % co . following selection, cells were passaged and the surviving clones were collected and analyzed. ace -expressing (ace + ) cells were maintained in dulbecco's mem (paa laboratories, austria) supplemented with % fbs, penicillin ( u ml - ), streptomycin ( μg ml - ), ciprofloxacin ( μg ml - ) and puromycin ( μg ml - ). t_ace + cells were maintained as wild type cells. human tracheobronchial epithelial cells were obtained from airway specimens resected from patients undergoing surgery under silesian center for heart diseases approved protocols. this study was approved by the bioethical committee of the medical university of silesia in katowice, poland (approval no: knw/ /kb / / dated on . . ) . a written informed consent was obtained from all patients ( adult patients). primary cells detached from human bronchi and trachea with pronase e were expanded on collagen-coated (collagen type iv, sigma-aldrich) plastic in bronchial epithelial growth media (begm) to generate passage cells and plated at density of × cells per well on permeable transwell supports ( . -mmdiameter; corning transwell-clear) in begm. cells were cultured at °c in presence of % co until confluence. human airway epithelium (hae) cultures were generated by changing the media to air liquid interface media (ali) and provision of an air-liquid interface for to weeks to form well-differentiated, polarized cultures that resemble the in vivo structure of pseudostratified mucociliary epithelium. all procedures were performed as previously described [ ] . all cell cultures were routinely screened for mycoplasma spp. contamination using hoechst staining. hcov-nl (amsterdam i strain) stock was generated by infecting llc-mk cells. infected cells were lysed days post-infection by two freeze-thaw cycles. the virus-containing fluid was cleared by centrifugation, aliquoted and stored at - °c. a control from mock infected cells was prepared in the same manner as the virus stocks. virus yield was assessed by titration on fully confluent llc-mk cells, according to reed and muench formula [ ] . cells on -well plates were incubated at °c for days and the cytopathic effect was scored using an inverted microscope. all experimental procedures were conducted as previously described [ ] . virus identity was confirmed by cdna sequencing. rna from viral and control cultures was extracted using genejet rna purification kit (thermo scientific, lithuania), according to manufacturer's protocol. isolated rna was stored at - °c. dna fragments were synthesized by a third party (genomed, poland). isolated viral rna was reverse transcribed using high capacity cdna kit (life technologies, poland) and used as a template for subsequent amplification. in order to obtain eukaryotic expression vector, nl -n gene was amplified using primers n_nl _ hindiii ( -gta caa gct tgc cac cat ggc tag tgt aaa ttg ggc c- ) and n_nl _ bamhi ( -gac tgg atc cgc atg caa aac ctc gtt gac aat- ) with marathon dna polymerase (a&a biotechnology, poland). resulting pcr product was subsequently gel purified using genejet gel extraction kit (thermo scientific, lithuania) and digested with hindiii and bamhi restriction enzymes. resulting fragment was cloned into the pmaxfp-green-n plasmid (lonza, switzerland) using corresponding restriction sites. plasmids (pmaxfp-green-n/nl -n) were recovered in dh α escherichia coli (life technologies, poland) and their identity and sequence were confirmed by dna sequencing (genomed, poland). expression plasmids for prokaryotic expression of the nl -n protein were prepared using pmaxfp-green-n/nl -n as a template. briefly, the fragments of n gene were amplified using primers given in parentheses: procnl -n ( -atg ccc atg ggc cat cac cat cat cac cac tct ggc gac gac gac gac aag gct agt gta aat tgg gcc gat g- and -atg cct cga gtt aat gca aaa cct cgt tga caa t- ), procnl - / -n ( -cat agg atc cag aaa acc tgt att ttc agg gat cat ttt aca tgc ctc ttt tg- and -cag caa gct ttt aag agc gat cct caa act caa c- ) and procnl - / -n ( -cat agg atc cag aaa acc tgt att ttc agg gat ctc aac cca ggg ctg ata ag- and -cag caa gct ttt atg act gca ttt ctt tga tag- ) and cloned into the pet duet plasmid (clonetech, usa). the element encoding × his tag was introduced at the n-terminus of the gene. three plasmids for prokaryotic expression were generated: procnl -n, procnl - / -n (for expression of the n terminal-domain), and procnl - / -n for expression of the cterminal domain. plasmid pmaxfp-green-n/nl -n or control plasmids were transfected to t cells using cationic carrier (polyethylenimine, pei; sigma-aldrich, poland). briefly, × cells were seeded onto collagen-coated (purecol; advanced biomatrix, usa) glass coverslips in a -well plate. next day media was removed, cells were washed with × pbs and overlaid with ml of dmem supplemented with μg of pei and μg of plasmid. h post-transfection coverslips were harvested for analysis. in order to test subcellular localization of the n protein in llc-mk cells, the maxfp-green-n/nl -n encoding rna was prepared based on the original plasmid. briefly, the plasmid was used as a template with primers sp _negfpmrna ( -act gac tga ttt agg tga cac tat aga agn gaa gct tgc cac cat ggc tag tg - ) and egfpmr-na_r ( -ttt ttt ttt ttt ttt ttt ttt cat taa tgc aaa acc tcg ttg ac - ), where the primer carries the sp promoter. in vitro transcription was carried out using the mmessage mmachine sp kit (life technologies, poland). further, rna was polyadenylated using poly(a) tailing kit (life technologies, poland) and transfected into cells using transit-mrna transfection kit (mirus, usa), as advised by the manufacturer. rna encoding maxfp-green protein (control) was prepared and transfected in the same manner using primers sp _gfpmrna (atg cat tta ggt gac act ata gat gga gag cga cga gag cgg cct gc) and gfpmrna_r (ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttc att att ctt cac cgg cat ctg cat c). in order to assess the influence of the n protein on cell cycle, the n-encoding rna was prepared based on the original plasmid and transfected in the same manner as described above. primers sp _nmrna ( -tcg gcc tcg tag gcc att tag gtg aca cta tag aag nct gag aga acc cac tgc tta c - ) and nmrna_r ( -ttt ttt ttt ttt ttt ttt ttt cat taa tgc aaa acc tcg ttg ac - ) were used, where the primer carries the sp promoter. cells transfected with mrna encoding the n protein or control cells were harvested h posttransfection by trypsinization and pelleted in sterile × pbs. after fixation in % etoh for h on ice, cells were incubated in staining solution ( μg/ml propidium iodide and μg/ml rnase a in sterile × pbs; sigma-aldrich, poland) for min at °c. the n protein was visualized with monoclonal antibody specific to nl -n (ingenasa, spain) and secondary alexa fluor goat anti-mouse antibody (life technologies, poland). cells expressing the n-nl protein were analyzed by flow cytometry (facscalibur, becton dickinson) as previously described [ ] . cells treated with nocodazole (sigma-aldrich, poland), a mitotic spindle poison, were sampled after h and evaluated as positive control for cell cycle arrest. obtained data were analyzed using modfit lt software (verity software house, usa). all experiments were conducted independently at least three times. cells were fixed using % formaldehyde solution in sterile × pbs for minutes. subsequently, cells were washed three times with × pbs and incubated with . % triton solution in × pbs to remove lipid fraction. further, cells were incubated in blocking buffer ( % of bsa; bio-shop, canada, . % tween ; bioshop, canada in × pbs) for minutes. for detection of hcov-nl n protein mouse monoclonal anti-hcov-nl -n antibody (diluted ×, ingenasa, spain) was incubated with the sample for hour at °c, followed by incubation with an anti-mouse alexa fluor (dilution ×, thermo fisher scientific, poland) for hour at °c. for visualization of nucleic acids, dapi dye ( μg/ml; sigma-aldrich, poland) was used. fluorescent images were acquired with leica tcs sp ii confocal microscope (leica microsystems gmbh, germany). images were pre-processed using leica application suite advanced fluorescence las af v. . . (leica microsystems gmbh) and further deconvolved with huygens essential package ver. . (scientific volume imaging b.v., the netherlands). all experiments were conducted independently at least three times. the nl -n ntd (amino acids - ) and ctd (amino acids - ) expression constructs were designed in silico by analysis of sequence alignments, comparative modeling and literature data. the sequences and structures of the homologous coronavirus nucleocapsid polypeptides used are listed in table . the strategy is described in the supporting information section. sequence sets were prepared using blast and spdbv. the comparative modeling was performed with spdbv, coot and pymol [ ] [ ] [ ] . procedure of gene amplification and plasmid preparation is described above. in order to express the n protein, ntd and ctd in e. coli, respective plasmids were transformed to bl cells and further cultured in lb media supplemented with ampicillin ( μg/ml) at °c, until the optical density (λ = nm) reached . - . . expression was induced by addition of iptg ( mm) and continued overnight at °c. subsequently cells were pelleted by centrifugation and suspended in lysis buffer ( mm tris, mm nacl, mm imidazol ph . ). for the full length protein, the buffer was supplemented with mm β-mercaptoethanol. bacterial cells were lyzed by sonication, and cellular debris was removed by centrifugation. proteins of interest were recovered by affinity chromatography (ni sepharose fast flow, ge healthcare, poland), ion-exchange chromatography (resource q, ge healthcare, poland) and size exclusion chromatography (superdex s , ge healthcare, poland). protein was detected using western-blotting technique and anti- × his antibodies (life technologies, poland). samples for mass spectrometry were prepared by dialysis into mm nh hco , ph . . measurements were performed using the microtof-qii mass spectrometer (bruker, germany) in positive ionization mode, using appollo source esi sprayer. prior to measurements the device was calibrated with tunemix solution. the obtained ms spectra were analyzed using data analysis . software (bruker, germany). molecular weight of proteins was confirmed using maximum entropy algorithm for ms spectra deconvolution (bruker, germany). protein preparations were overlaid on the poly-l-lysine coated glass slides (diameter of mm). samples were fixed with . % glutaraldehyde in . m sodium cacodylate buffer ph = . for minutes. subsequently, samples were washed with the abovementioned buffer and gently dehydrated by using solutions of ethanol in a graded series of concentrations. preparations were dried in a critical point dryer (quorum technologies, united kingdom). slides were mounted on holders using self-adhesive carbon discs (taab laboratories, united kingdom) and sputter coated with gold (ion sputter jfc- e; jeol, japan). electron micrographs were prepared using scanning electron microscope jsm- (jeol, japan). is heat capacity at the t m measured with respect to the chemical baseline. the ratio c exc;max p ∕ dhðt m Þis sensitive to the shape (width) of the transition. if two-state model holds true, the van't hoff and calorimetric enthalpies are equal within the experimental uncertainty, and so the ration = Δh(t m )/Δh vh should be equal to unity. protein electrophoresis in denaturing conditions was carried out in schagger & von jagow system. electrophoretic separation was carried out at v (stacking) / v (separation). proteins were visualized using coomassie brilliant blue g- (serva, germany). page ruler plus (thermo scientific, lithuania) was used as a prestained protein size marker. for emsa assay μg of rna or dna corresponding in sequence to the n-nl gene (prepared in the same manner as for the transfection of eukaryotic cells) was incubated in buffered solution ( mm tris, mm nacl, ph . ) with μg of the ntd or ctd for minutes at room temperature. subsequently, samples were separated on agarose gels and signal from nucleic acids was visualized with ethidium bromide staining. all experiments were conducted independently at least three times. in order to assess whether the ntd or ctd are able to form oligomers, μg of the protein was mixed with glutaraldehyde ( . %; serva). following minute incubation at room temperature . μl of m tris solution was added to the mixture, samples were mixed with protein sample buffer, denatured at °c and loaded onto the polyacrylamide gel. all experiments were conducted independently at least three times. the sequences of dna, rna and proteins used within the study correspond to those of hcov-nl isolate amsterdam (genbank accession number: nc_ ). accession numbers for n proteins of different coronaviruses are provided in s file. in silico analysis of nl -n nl -n is a basic protein (predicted pi, . ) comprising amino acids (aa). the predicted molecular weight is , . da. literature data indicate that the full length coronaviral nucleocapsid protein consists of two folded domains linked by an unstructured region. in more details the n protein includes following elements: n-tail, n-terminal domain (ntd), r.s.a.g. rich linker, c-terminal domain (ctd) and c-tail [ ] . the constructs of ntd and ctd used in this study were designed based on literature data, hcov-nl n protein amino acid sequence alignment with known homologs and on the comparative analysis of currently available crystal structures of these homologs. according to saikatendu et al. the ntd of hcov-nl n encompasses residues - [ ] . our sequence alignment and structural analysis suggests that nl -n - fragment better reflects the full n-terminal domain. nl -n fragment encompassing its ctd was chosen exclusively on the basis of sequence alignment and structural analysis which suggests that fragment - contains full, structurally stable ctd. the sequences and structures of n proteins used in above analysis are listed in table . the analysis strategy is summarized in s file and the amino acid sequences of the final constructs of ctd and ntd are presented in s file. in silico analysis conducted using psort ii revealed that two nuclear localization signals (nls) are buried within nl -n: pat (aa -kkpr- ) and pat (aa -prwkrvp- ). no bipartite nls were detected. the complete nl -n protein and its ctd and ntd were expressed in e. coli bl cells. ntd and ctd were purified to homogeneity whereas the full length n-protein was purified to about % homogeneity as demonstrated by sds-page (fig. ) . the identity of purified proteins was confirmed with mass spectrometry (data not shown). we next used differential scanning calorimetry (dsc) to examine thermal stability of the proteins. the dsc curves for the first heating scans obtained for nl -n, the ctd, and the ntd are shown in fig. . nl -n underwent irreversible denaturation and showed a broad transition curve. the denaturation temperature was estimated at . °c, with an enthalpy change of approximately kcal/mol ( table ). however, due to low signal to noise ratio the resulting baseline was variable and these values can only be treated as rough estimates. nevertheless, the low enthalpy value suggests that nl -n protein is relatively unstable. unfolding of the ntd was irreversible and accompanied by protein aggregation (indicated by the exotherm present in the high temperature region of the dsc curve). the transition temperature was °c and the Δh cal was . kcal/mol. surprisingly, thermal transition of the ctd was fully reversible, showing t t of . °c, a Δh cal of . kcal/mol, and a Δs cal of . kcal/ kmol. thermal transition of the ctd was cooperative, with a van't hoff enthalpy/calorimetric enthalpy ratio (Δh van'hoff / Δh cal ) of . . the coronaviral nucleocapsid forms a protective scaffold around the viral rna [ ] [ ] [ ] . the n protein forms oligomers via specific interactions between different regions within the protein [ ] [ ] [ ] . to confirm this assumption for hcov-nl , we performed mass spectrometry analyses. the results confirmed the presence of complete n protein dimers. furthermore, similar results were obtained for the ctd but not ntd, suggesting that ctd harbors the sites responsible for n protein dimerization ( table ) . the mass spectrometry results were confirmed by protein crosslinking studies. incubating the ctd in the presence of glutaraldehyde followed by sds-page analysis revealed the presence of protein dimers and higher molecular weight oligomers (fig. a) . similar results were obtained using size exclusion chromatography, showing that~ % of the protein is present as dimers (fig. b) . dimerization was not observed for ntd. we next performed an electrophoretic mobility shift assay to determine whether the n protein interacts with nucleic acids. briefly, samples containing nucleic acids were separated in agarose gel under native conditions in the presence/absence of the ctd or the ntd. nucleic acids were detected by ethidium bromide staining. as shown in fig. , the ntd binds nucleic acids (both dna and rna), as demonstrated by retarded rna and dna migration. the ctd did not bind nucleic acids. we also conducted similar analysis for the complete n protein (data not shown). obtained results suggested that the complete n protein has lower nucleic acid binding ability or is more specific compared to the ntd. however, due to rapid degradation of the complete n protein into separate domains and resulting presence of the free ntd in the solution these results were inconclusive. coronaviral n proteins localize to the cytoplasm, where they are involved in virus replication and assembly. however, the n proteins of almost all coronaviruses (except for sars-cov) also localize to the nucleus or to micronuclei [ ] [ ] [ ] [ ] . to examine the subcellular localization of the nl -n, cultures of t_ace + , llc-mk , and hae cultures were infected with the hcov-nl virus. subsequently, the cells were fixed and stained with antibodies specific for the n protein. in all cell types tested the protein localized exclusively in the cytoplasm (fig. , s , s , s files) . to test whether the observed lack of nuclear localization of nl -n does not result from insufficient nuclear staining, t cells were also transfected with pmaxfp-green-n/nl -n plasmid. llc-mk cells were transfected with maxfp-green/nl -n encoding mrna due to poor transfection efficiency using conventional dna delivery methods. maxfp-green protein was used as a control. we then examined the subcellular localization of nl -n using confocal microscopy ( fig. a and b ). maxfp-green-labeled nl -n localized exclusively to the cytoplasm in all tested cell types and no staining was observed in the nucleus. overexpression of the n protein resulted in formation of large deposits of the n protein in the cytoplasm, which may be attributed to vast overexpression of the protein, as infection of these cells did not result in formation of such structures. similar patterns were previously seen for other coronaviruses [ ] . previous reports show that expression of the coronaviral n protein results in delayed cellular growth. these observations were supported by biochemical studies showing that the n protein may actively participate in cell cycle regulation in infected cells by localizing to the nucleus and interacting with cyclins [ ] [ ] [ ] ] . to examine whether overexpression of the n protein affects cell cycle progression, we transfected llc-mk and t cells with rna encoding nl -n. in this particular case, we used rna transfection because it was more efficient than transfecting cells with dna vectors (unpublished observations). only cells expressing nl -n (as identified by staining with specific antibodies) were used for subsequent analyses. there was no significant difference in cell cycle progression in nl -n-expressing and non-expressing cells (fig. ) . since its discovery, hcov-nl was considered to be the closest relative of hcov- e. however, later identification of novel bat coronavirus species revealed several alphacoronaviruses closely clustering with the two human pathogens. such an observation raised speculations on zoonotic origin of hcov-nl [ , ] . considering that hcov-nl is rather an atypical alphacoronavirus (e.g., in terms of receptor usage and the predicted protease active site [ , ] , it has been suggested that, despite the high similarity between hcov- e and hcov-nl [ ] , these viruses may represent two distinct species that evolved from a common ancestor in bats, and were then introduced into human population via two independent zoonotic transmission events [ , ] . the nl -n is a basic protein comprising of aa. both the ntd and the complete n protein are unstable, as shown by the broad, irreversible dsc curves and obtained thermal parameters values: t m below °c and low Δh value for complete n protein. in general, Δh represents the energy amount of non-covalent bonds occurring within native protein. this is hcov-nl n protein consistent with our finding that the n protein rapidly degrades in aqueous solutions (data not shown). obtained results are consistent with previous reports [ , , ] . however, the ctd was surprisingly stable; no protein aggregation was observed upon heating and thermal denaturation was fully reversible, moreover judging from Δh(t m )/ Δh vh ratio it undergoes cooperative transition. thermodynamic protein stability is defined as the gibbs energy difference between the denatured and native states but, for some practical purposes, the denaturation temperature, t m (where Δg = for two-state reversible transition) may be more useful to measure the protein stability. irreversibility is a common feature of dsc measurements when the large, multidomain proteins are considered. irreversibility limits the use of a standard equilibrium thermodynamic analysis. for such a process, the denaturation temperature measured in dsc experiment is significantly lower than that corresponding to Δg = . in these cases, the t m (so called operational thermal stability) is kinetically controlled. the simplest model of irreversible unfolding can be described in the terms of kinetic analysis (rate equations) according to general lumry-erying scheme: where k is the equilibrium unfolding constant characterizing reversible unfolding step, k is the first order rate constant describing following irreversible step; n, u and d correspond to native, reversibly unfolded and irreversibly denatured monomer of the protein, respectively. therefore, d represents a modified denatured state existing in a quasi-two-state equilibrium with the native state [ , ] . in fact, for irreversible denaturation the enthalpy and t m become only apparent values since both parameters are dependent on the scan rate. the existence of the exotherm on the downhill part of endotherm dsc peak is the obvious evidence for the presence of association-aggregation processes-the most common reason of the protein unfolding irreversibility. lower stability of ntd and the complete n protein in comparison to ctd could be attributed to the distinct structural flexibility of these proteins. in turn, higher flexibility allows ntd to bind the nucleic acids which has obvious biological relevance for ntd and complete n protein function. one of the major functions of the n protein is to bind viral rna to form a nucleoprotein, which is then packed into new virions. therefore, we next examined the ability of nl -n to bind nucleic acids in an electrophoretic mobility shift assay. our results confirmed that the ntd efficiently bound rna and dna. however, previous studies show that coronaviral n protein has a higher affinity for the viral rna than non-viral nucleic acids and that only nucleoproteins with coronaviral genomic rna are efficiently incorporated into new virions [ , [ ] [ ] [ ] . observed limited binding of the nucleic acids by the complete n protein (data not shown) hypothetically resulted from interaction between the ntd and ctd. however, rapid n-protein degradation at the conditions of the experiment did not allow us to obtain conclusive answer. this phenomena requires further studies, as we were not able to distinguish at this stage hcov-nl n protein the mechanism of ntd-ctd interaction that limits nucleic acid binding and most likely increases the binding specificity. previous studies demonstrated that coronaviral n proteins form dimers, and to lesser extent higher order oligomers [ ] . the crystal structures of the sars-cov, infectious bronchitis virus (ibv), and mouse hepatitis virus (mhv)-n ctds revealed that all those domains are characterized by a similar polypeptide fold and are dimeric, strongly suggesting that the dimeric n protein is the unit that functions in vivo [ , ] . furthermore, tang et al. compared the n proteins from sars-cov and hcov- e [ ] demonstrating that they formed oligomers and bound to nucleic acids. moreover, lo et al. showed that oligomerization of the e-n protein is most likely also mediated by the ctd [ ] . here we show that the complete nl -n protein and the ctd can self-associate to form dimers and higher order oligomers. the role of aforementioned n-n interaction is, however, debatable, as nl -n strongly binds nucleic acids during formation of the ribonucleocapsid. it is therefore possible that this interaction may be important for stabilizing and shaping the ribonucleocapsid; however, it may also be important for other n-mediated processes [ ] . n protein of most coronaviruses is abundantly present in the nucleus of the infected cell [ ] [ ] [ ] [ ] . analysis of the nl -n protein using psort ii revealed the presence of two nls (pat and pat ), both of which are homologous to those observed in other coronaviral n proteins. for example, the ibv virus carries two very similar putative nls signals: pat rpkk [aa - ] and pat pkkekkl [aa - ]. one may, therefore, expect that the nl -n protein would also localize to the nucleus. surprisingly, no sign of nl -n localization to the nuclei of infected or transfected cells was detected, suggesting that these nls motifs are buried within the nl -n structure, as previously proposed for sars-cov [ , ] . to validate that the solely cytoplasmic localization of nl -n is found not only in established cell lines, we tested a fully differentiated human airway epithelium cultures, mimicking the natural environment of the human airway epithelium; identical results were obtained. it is, however, possible that the lack of nuclear staining observed after natural infection or transfection with the nl -n-encoding plasmid may be due to poor antibody staining within the nucleus [ ] . to address this problem, we used a vector encoding maxfp-green-n/nl -n fusion protein, but found no difference in the subcellular localization of the fusion protein and the native protein [ , , ] . also, we found that overexpression of maxfp-green-n/nl -n in the cells yielded a staining pattern identical to that shown by the native n protein. presented data show that the nl -n protein does not localize to the nucleus (or does so in a very limited fashion), similarly to the sars-cov n protein and differently than other coronaviral n proteins [ , ] . nuclear localization of the n protein may be important for several processes, including direct interference with the cell cycle. dysregulation of the cell cycle is a common strategy used by many dna and rna viruses, which enables them to hijack and exploit the host cell machinery for their own benefit. indeed, the n proteins of many coronaviruses (including sars-cov; although the n protein does not localize into the nucleus in this case) inhibit cell cycle progression [ ] [ ] [ ] ] . however, we found no difference in the proportion of cells in each phase of cell cycle for nl -n protein-expressing and non-expressing cells. in conclusion, we demonstrated here that although nl -n is largely similar to other coronaviral n proteins, it possesses some unique characteristics: it does not localize to the nucleus of the infected cell and its expression does not appear to affect the cell cycle progression. supporting information s file. design of the n-and c-terminal domains of hcov-nl n protein expression constructs was based on the sequence alignment with homologous n proteins together with the structural comparative analysis. upper panel: in order to predict polypeptide sequence encompassing the n-terminal domain of n protein from hcov-nl the following sequences of n proteins were aligned: q q r (uniprot id), hcov-nl ; p , avian infectious bronchitis virus (ibv, strain beaudette), sequence referring to the bxx crystal structure (protein data bank id); p , ibv (strain beaudette us), sequence referring to the c crystal structure; p , ibv (strain gray), sequence referring to the gec crystal structure; p , murine hepatitis virus (strain a ), sequence referring to the hd crystal structure; p , sars cov, sequence referring to the ofz crystal structure, p , sars cov, sequence referring to the og crystal structure. sequences referring to residues - of n protein from hcov-nl are presented as it is enough to reflect the full n-terminal domains of each sequence aligned. sequences reflecting residues defined by the electron density maps of the crystal structures ( bxx, c , gec, hd , ofz, og ) are colored green. the sequence of hcov-nl n protein predicted to constitute the structurally stable n-terminal domain is colored blue. to predict polypeptide sequence encompassing the c-terminal domain of n protein from hcov-nl the following sequences of n proteins were aligned: q q r , hcov hcov-nl n protein nl ; p , avian infectious bronchitis virus (ibv, strain beaudette), sequence referring to the ca crystal structure; p , ibv (strain gray), sequence referring to the ge crystal structure; p , ibv (strain gray), sequence referring to the ge crystal structure; p , human sars coronavirus, sequence referring to the cjr crystal structure; p , sars cov, sequence referring to the jw solution structure. sequences referring to residues - of n protein from hcov-nl are presented as it is enough to reflect the full c-terminal domains of each sequence aligned. sequences reflecting residues defined by the electron density maps of the n protein ctd crystal structures ( ca , ge , ge , cjr) and the residues of jw solution structure are colored violet. the sequence of hcov-nl n protein predicted to constitute the structurally stable c-terminal domain is colored red. lower panel: superposition of n protein ntd and ctd structures used in the comparative analysis. multiple molecules composing the asymmetric unit of given structure are colored the same. multialignment was performed in muscle [ ] . comparative structural analysis was done in spdbv, coot and pymol [ , ] . s file. subcellular localization of nl -n protein in fully differentiated human airway epithelium cultures cells infected with hcov-nl . virions were labelled with antibodies specific to the n-nl protein (green); dna was stained with dapi (blue). analysis was carried on with confocal microscopy 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nucleocapsid protein facilitates template switching and is required for efficient transcription m and n proteins of sars coronavirus induce apoptosis in hpf cells the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos- cells in the absence of growth factors the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor alpha characterisation of cyclin d downregulation in coronavirus infected cells the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism psort: a program for detecting sorting signals in proteins and predicting their subcellular localization a knowledge base for predicting protein localization sites in eukaryotic cells a lentiviral rnai library for human and mouse genes applied to an arrayed viral high-content screen well-differentiated human airway epithelial cell cultures a simple method of estimating fifty per cent endpoints infection with human coronavirus nl enhances streptococcal adherence to epithelial cells practical flow cytometry coot: model-building tools for molecular graphics automated protein modelling-the proteome in d basic local alignment search tool the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the n-terminal domain of n protein isolation and morphology of the internal component of human coronavirus, strain e ribonucleoprotein-like structures from coronavirus particles ribonucleoprotein of avian infectious bronchitis virus biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and e human coronaviruses x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation oligomerization of the carboxyl terminal domain of the human coronavirus e nucleocapsid protein trafficking motifs in the sars-coronavirus nucleocapsid protein characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization evidence supporting a zoonotic origin of human coronavirus strain nl distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus e in bats human coronaviruses e and nl : close yet still so far high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna low stability of nucleocapsid protein in sars virus differential scanning calorimetry of proteins a differential scanning calorimetry study of tetracycline repressor specific interaction between coronavirus leader rna and nucleocapsid protein mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding by using surface plasmon resonance coronavirus nucleocapsid protein is an rna chaperone characterization of n protein self-association in coronavirus ribonucleoprotein complexes nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus a higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus muscle: multiple sequence alignment with high accuracy and high throughput swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna solution structure of the c-terminal dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method key: cord- -kldu q x authors: oany, arafat rahman; sharmin, tahmina; chowdhury, afrin sultana; jyoti, tahmina pervin; hasan, md. anayet title: highly conserved regions in ebola virus rna dependent rna polymerase may be act as a universal novel peptide vaccine target: a computational approach date: - - journal: in silico pharmacol doi: . /s - - - sha: doc_id: cord_uid: kldu q x purpose: ebola virus (ebov) is such kind of virus which is responsible for , cases and deaths worldwide only in and with an average diseases fatality rate between % and %. although, medical technology has tried to handle the problems, there is no food and drug administration (fda)-approved therapeutics or vaccines available for the prevention, post exposure, or treatment of ebola virus disease (evd). methods: in the present study, we used the immunoinformatics approach to design a potential epitope-based vaccine against the rna-dependent rna polymerase-l of ebov. bioedit v . . sequence alignment editor, jalview v and clc sequence viewer v . . were used for the initial sequence analysis for securing the conservancy from the sequences. later the immune epitope database and analysis resource (iedb-ar) was used for the identification of t-cell and b-cellepitopes associated with type i and ii major histocompatibility complex molecules analysis. finally, the population coverage analysis was employed. results: the core epitope “fryeftapf” was found to be the most potential one, with % conservancy among all the strains of ebov. it also interacted with both type i and ii major histocompatibility complex molecules and is considered as nonallergenic in nature. finally, with impressive cumulative population coverage of . % for the both mhc-i and mhc-ii class throughout the world population was found for the proposed epitope. conclusion: to end, the projected peptide gave us a solid stand to propose for vaccine consideration and that might be experimented for its potency in eliciting immunity through humoral and cell mediated immune responses in vitro and in vivo. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. background evd, previously designated as ebola haemorrhagic fever, is a fatal disease in humans and other mammals (monkeys, chimpanzees and gorillas) (choi and croyle , leroy et al. , sullivan et al. . the fatality rate of edv is varied from to % with an average of about % (peters and peters ) (hoenen et al. ). the fifth one, reston virus (reston ebolavirus), is harmful to nonhuman primates, but not to humans (elisha and adegboro , geisbert et al. ). among the recognized species of ebolavirus, the notoriously deadly zaire ebolavirus is responsible for epidemics which have been taken place mainly in african countries including democratic republic of congo, uganda, sudan, the ivory coast, and gabon (baize et al. , chowell et al. , feldmann et al. , frieden et al. , hewlett and hewlett , kuhn et al. , li and chen , rouquet et al. . this virus is passed on people from wild animals and through human-to-human contact transmits in the human population. those are infected with this virus bear fearsome symptoms, including high fever, hemoptysis, impaired kidney and liver function and severe internal bleeding (gatherer , goeijenbier et al. , keiser et al. , peters and peters . in the fall of the ebola virus gained widespread attention when in west africa the largest outbreak has been reported in history. the ebov genome is a single-stranded, negative-sense, non-segmented rna approximately kb long. it codes for seven tandemly arranged viral genes which order is ′ leader-np (nucleoprotein) -vp (virion protein )-vp -gp (glycoprotein)-vp -vp -l (rna-dependent rna polymerase)-trailer − ′. transcription and translation of this viral genome result in the synthesis of seven structural proteins and a single non-structural, secreted glycoprotein (feldmann et al. ) . three of the structural proteins are membrane-associated proteins; gp is a type i transmembrane protein, while vp and vp are placed on the inner surface of the membrane. the remaining four, np, vp (transcription factor), vp (polymerase cofactor), and l (rna-dependent rna polymerase), are essential to viral genomic rna to form the ribonucleoprotein complex. these proteins have been shown to be necessary and sufficient for ebov transcription and replication (crary et al. , feldmann et al. , mühlberger et al. , takada et al. ). to date, information regarding the processing, structure and functions of ebola virus (ebov) protein l (ebol) demonstrates that it is an rna-dependent rna polymerase, with the assistance of vp . it also shows mrna (guanine-n ( )-)-methyltransferase, mrna guanylyltransferase and poly (a) synthetase activities which are essential for the replication and transcription of ebov (poch et al. ) . the viral mrna guanylyltransferase serves either as transcriptase or as replicase. the transcriptase synthesizes subgenomic rnas, assures their capping and polyadenylation. the transcriptase stutters on a specific sequence, leads to a co-transcriptional editing of the glycoprotein (gp) mrna. in replicase mode, the polymerase replicates the viral genome without recognizing the transcriptional signals. these reports suggest that ebol is an important cellular component for the transcription and replication of the ebov genome and, as such, plays a key role in the ebov life cycle. due to the emergence of ebola virus outbreak, there is an immediate need to determine novel therapeutic targets against this pathogen. the identification of specific epitopes derived from infectious pathogens has significantly advanced the development of epitope-based vaccines (evs). bettered understanding of the molecular basis of antigen recognition and hla binding motifs has resulted in the advancement of rationally designed vaccines depend on algorithms predicting the peptide's binding to human hla. in comparison to the conventional vaccines, peptide or epitope based vaccines are easy to develop, chemical stable, more specific, and free of any infectious or oncogenic potential hazard (holland and domingo , sette et al. ) . though evs have varied advantages, the wet lab based discovery of candidate epitopes is expensive and time consuming. furthermore, for the final selection of epitopes various immunological requirements are needed to be considered. as a result computational methods, an alternative in silico approaches (germain ) have recently been attracting growing interest of the researchers for predicting epitopes with reduced cost and time. the application of bioinformatics in immunology is termed as immunoinformatics. currently, numerous immunoinformatics tools are available for identifying b and t cell epitopes and human leukocyte antigen (hla) ligands (petrovsky and brusic , poland et al. , sette and fikes with high sensitivity and specificity. the 'immunoinformatics' approach has already proven its potency in the case of human immunodeficiency virus (wilson et al. ) , multiple sclerosis (bourdette et al. ) , tuberculosis (robinson and amara ) and malaria (lópez et al. ) with desired results. in the present study, we have followed immunoinformatics approaches for designing potential conserved epitope candidate for the utility of vaccine development against the deadly ebola virus, with an expectation of further wet lab validation. the protein sequences of the rna-dependent rna polymerase-l ) of the ebov were retrieved from the uniprotkb (apweiler et al. ) database in the fasta format. bioedit v . . sequence alignment editor (apweiler et al. ) was used for the identification of the conserved region among the sequences through multiple-sequence alignment (msa) with clustalw (hall ) . finally, jalview v tool (thompson et al. ) was used to retrieve the alignment and the clc sequence viewer v . . (http:// www.clcbio.com) was used for analysis of the divergence among the different strains of the ebov. vaxijen v . , a web-based server (waterhouse et al. , doytchinova and flower ) was used for the determination of the antigenicity of the conserved sequences. herein, we used the default parameters for the prediction, with a threshold value of . . for this study, two online servers were used. firstly, the netctl v . server (larsen et al. ) was used for predicting potential cytotoxic t lymphocyte (ctl) epitopes from the conserved peptides. here for predicting the epitopes, we used a combined algorithm including major histocompatibility complex class i (mhc-i) binding, transporter of antigenic peptide (tap) transport efficiency, and proteasomal c terminal cleavage prediction. depending on the score, the best candidates were picked for further investigation. the epitope prediction was confined to mhc-i supertypes. mhc-i binding and proteasomal cleavage were carried out through artificial neural networks and the weight matrix was used to estimate the tap transport efficiency. the threshold value for epitope identification was set at . for maintaining sensitivity and specificity of . and . , respectively during the analysis. this would support to assess the findings more decisively by developing more epitopes. finally, for confirming the prediction with default parameters, ctlpred (bhasin and raghava ) was employed additionally. furthermore, from the immune epitope database and analysis resource (iedb-ar), t cell epitope prediction tools was implied for the identification of mhc-i (hoof et al. , nielsen et al. ) and mhc-ii (wang et al. ; binding of the peptide. in order to calculate the half-maximal inhibitory concentration (ic ) values required for peptide binding to mhc-i molecules, stabilized matrix method (peters and sette ) was applied with a preset . -mer epitope. in case of mhc-ii binding analysis, the iedb-recommended method was used for the specific hla-dq, hla-dp, and hla-dr loci. herein, specific peptides were used to predict the mhc-ii interaction on the basis of mhc-i analysis and antigenic conservancy. population coverage for epitope was assessed by the iedb population coverage calculation tool (bui et al. ). here we used the allelic frequency of the interacting hla alleles for the prediction of the population coverage for the corresponding epitope. linear b cell epitopes are of different lengths of peptides from to in comparison to that of t cell epitopes. b-cell epitope produces immune response when it interacts with b lymphocytes. it then initiates the differentiation of b lymphocytes into plasma and memory cells (nair et al. ) . there are a number of web-based tools are available for the prediction of b-cell epitope which are hosted by iedb-ar. for the b-cell epitope prediction with high accuracy, multiple tools, including the emini surface accessibility prediction (emini et al. ) , kolaskar and tongaonkar antigenicity scale (kolaskar and tongaonkar ) , parker hydrophilicity prediction, (parker et al. ) and finally the chou and fasman beta turn prediction tool (chou and fasman ) were employed, because the antigenic parts of a protein belong to the beta turn regions (rini et al. ) . the structure of the conserved region was constructed by homology modelling using the modeller v (Šali et al. ) . modeller is a program that implements an automated approach to comparative protein structure modelling by satisfying spatial restraints (fiser et al. , sali and blundell ) . finally, the evaluation of the predicted model was verified by using two software tools, procheck (arnold et al. , laskowski et al. and qmean (benkert et al. ) . for predicting the disorder among the amino acid sequences, disopred v (ward et al. ) server was used. in order to calculate the protein variability index the protein variability server was implied where wu-kabat variability coefficient (garcia-boronat et al. ) has been used. the web-based allerhunter server (muh et al. ) was used to predict the allergenicity of our proposed epitope for vaccine development. this server predicts allergenicity through a combinational prediction, by using both integration of the food and agriculture organization (fao)/world health organization (who) allergenicity evaluation scheme and support vector machines (svm)-pairwise sequence similarity. allerhunter predicts allergens as well as nonallergens with high specificity. this makes allerhunter is a very useful program for allergen cross-reactivity prediction (liao and noble ) . epitope conservancy of the candidate epitopes was examined using a web-based epitope conservancy tool available in iedb analysis resource (bui et al. ). the conservancy level of each potential epitope was calculated by looking for identities in all rna-dependent rna polymerase-l protein sequences of different strains retrieved from database. a total of rna-dependent rna polymerase-l protein molecules from different variants of the ebov were retrieved from the uniprot database. the msa of the rnadependent polymerase-l proteins was retrieved from bioedit tool through clustalw with bootstrap replicates (additional file : figure s ). clc sequence viewer was used to construct phylograms from the msa obtained from bioedit, in order to analyze the divergence among the retrieved sequences. phylogram of rna-dependent rna polymerase-l is depicted in fig. . finally, the highly conserved region from the msa was retrieved for the further analysis. the selected conserved region is depicted in the fig. , from the msa number to . then the vaxijen v . server calculate the antigenicity of the conserved sequences with a score . . t-cell epitopes were selected firstly by using the netctl v . server where the epitope prediction was confined to mhc-i supertypes. based on the combined score, the top five epitopes (table ) were listed for further analysis. t-cell epitopes were again predicted by the ctlpred server (table ) . here a combined approach of artificial neural networks and support vector machines was applied. depending on the two analyses, the most common epitope-containing peptides, identified by both servers, was selected. the selected epitope was then used for the mhc-binding analysis. mhc-i-binding prediction, which was run through the stabilized matrix method, predicted a wide range of mhc-i allele interactions for the proposed t-cell epitopes. the mhc-i alleles for which the epitope showed higher affinity (ic < nm) are listed in table . the output of the mhc-ii interaction analysis is also shown in table . iedb population coverage tool analyzed the population coverage of the proposed epitope. the combined mhc-i and mhc-ii class were assessed against the whole world population with the selected mhc-i and mhc-ii interacted alleles (fig. ) . here, for predicting potential b-cell epitopes, we used amino acid-based methods. according to this procedure different analysis methods were applied for the identification of a continuous b cell epitope. the kolaskar and tongaonkar antigenicity scale was used for assessing the antigenic property of the peptides. the average antigenic propensity of the protein was . , with a maximum of . and a minimum of . . for the protein the antigenic determination threshold value was . , where all values equal or greater than . were potential antigenic determinants. the antigenic plot is depicted in the fig. . to be a potent b cell epitope, it must be surface accessible. hence, emini surface accessibility prediction was employed, with a maximum propensity score of . at threshold . (fig. ) . to strengthen our support for the prediction of the epitope to elicit b cell response the parker hydrophilicity and the chou and fasman beta turn prediction were employed. those are described in the figs. and . homology model of the conserved region was obtained by the modeller software, which is shown in fig. a and b. procheck server validated the stereochemical quality of the model through ramachandran plot (fig. c) , andqmean server also assessed the tertiary structure, with a qmean score of . . disopred v server predicted the disorder of the conserved peptide in order to get insight about the disorder among the conserved sequences, which is depicted in fig. . protein variability server predicted the variability of the conserved region of the rna-dependent rna polymerase-l ( fig. ) to ensure that the proposed epitope is within the invariable region. conservation analyses of the proposed epitopes were analyzed by the iedb conservancy analysis tool that is shown in table . allerhunter server predicted the our world is the habitation of more than seven billion people now. with the upgrade of medical science, new viruses along with their causing diseases are also emerging. ebola virus is such kinds of virus with a deadly outrage of their endemic nature especially in africa in recent time (evans and popova ) . till now there is no potential treatment for this virus to combat its deadly effects. recent time, the immunoinformatics approach give us some sort of hope for the design of an effective therapeutics, like vaccine, in association with the advancement of sequence based technology. similar approaches have been used successfully for identifying vaccine candidates in several pathogens viz. human corona virus (oany et al. ) , saint louis encephalitis virus (hasan et al. ) , crimean-congo hemorrhagic fever virus fig. parker hydrophilicity prediction of the epitope, with a minimum propensity score of − . and maximum score of . . notes: the x-and y-axes represent the sequence position and antigenic propensity score, respectively. the threshold value is . . the regions above the threshold are antigenic, shown in yellow fig. chou and fasman beta turn prediction of the epitope,with a minimum propensity score of . and maximum score of . . notes: the x-and y-axes represent the sequence position and antigenic propensity score, respectively. the threshold value is . . the regions above the threshold are antigenic, shown in yellow (oany et al. ) , chikungunya virus (hasan et al. ) and some others. the in vitro validation of this type of work has also been proven in recent time (khan et al. ) . though epitope-based vaccine designing has become a familiar approach, in the case of ebov no significant work yet has been done. ebov is an rna virus which has genetic blueprints made of rna instead of dna. creating vaccines is particularly difficult for rna viruses as they can quickly mutate their different exposed proteins (twiddy et al. ) . therefore the most potential way to create stable antiviral therapies against rna viruses including ebov is to target the transcription or replication machinery. scientists revealed that rnadependent rna polymerase-l (ebol) is an important cellular component for the transcription and replication of the ebov genome. when an ebov infects a cell, its rna genetic blueprint enters the cell along with rna-dependent rna polymerase-l. this polymerase normally "read" the rna genetic blueprint in order to synthesize mrna, which then leads to the formation of viral proteins as well as viral replication and more viral particles are produced. for these two vital involvements at the gateway, this protein was targeted to design most potential epitopes using in silico computational approaches. in the current study, firstly all the available sequences of rna-dependent rna polymerase-lwere retrieved from database. then antigenicity of the conserved peptides, generated by multiple sequence alignment was predicted by vaxijen, which suggested their ability to elicit potential immune response. sequence based bioinformatics approaches were applied to predict both b cell and t cell epitopes for conferring immunity in different ways. though at present, most of the vaccines are based on b cell immunity; vaccines based on t cell epitope have been encouraged recently. it is because, with time humoral response from memory b cells can be overcome easily by antigenic drift, while cell mediated immunity often provides long lasting immunity (bacchetta et al. , igietseme et al. ). cytotoxic cd + t lymphocytes (ctl) inhibit the spread of infectious agents by recognizing and killing infected cells or secreting specific antiviral cytokines (garcia et al. , shrestha and diamond ) . thus, vaccination based on t cell epitope is a unique approach to obtain strong immune response against infectious agents, such as, viruses (klein et al. ) . both netctl and ctlpred server were used to find epitopes for the activation of t-cell immunity with potential antigenicity. by examining the output it was predicted that fryeftapf would be the best epitope candidate and was further subjected for binding proficiency analysis. length is an important factor to consider for peptide antigen binding with mhc or tcr or both. t cell epitopes presented by mhc class i molecules are generally peptides between and amino acids in length. we therefore set peptide lengths at before making software based mhc class i t cell epitope identification using fig. protein variability index of the conserved peptides of all the sequences. the prediction suggests that our proposed epitope "fryeftapf" falls in the invariable region (blue line). notes: the conservancy threshold was . in this analysis. the x-axis indicates the amino acid positions in the sequences and the y-axis indicates the shannon variability score fig. disorder prediction of the conserved antigenic amino acid sequences. here, our proposed epitope lies outside ( - ) of the disordered region to secure its potentiality as an effective epitope. notes: amino acids in the input sequence are considered disordered when the blue line is above the gray dashed line, that is, when the confidence score is . . the orange line shows the confidence score of the disordered protein-binding residue predictions immune epitope database (iedb). analysis revealed that the core epitope "fryeftapf" would interact with ten different mhc class i alleles. on the other hand, the complete peptide "nlafryeftapfiey" interacts with the highest numbers of mhc class ii alleles (as many as alleles). along with the t-cell epitope, in our study, attention was also given to the b-cell epitope, which can induce both primary and secondary humoral immunity (trainor et al. ). multiple prediction methods were applied to determine the b-cell epitope considering several criteria of antigenicity, hydrophilicity, surface accessibility, and beta-turn. our proposed epitope has met all the criteria of the above b-cell prediction methods. the three-dimensional model of the conserved protein ensured the exact location of the epitope outside of the protein ( fig. a and b ) surface and the model validity was assessed by ramachandran plot (fig. c) , whereby . % amino acid residues were found within the favored region. the epitope was also treated as suitable candidate for vaccine through tenabled its position in the conserved sequence, by the discopred and protein variability server (figs. and ). conservancy is the most important criterion of an epitope to consider it for vaccine development. conservancy analysis of our proposed epitope showed % conservancy among all the available sequences. another important feature of the peptide vaccine is its allergenicity (mckeever et al. ) . in silico analysis revealed that the proposed epitope is nonallergenic in nature. wide range population coverage must be needed for a potential vaccine aspirant. at this point, our proposed epitope covers a remarkable population of . % for both types of mhc allele throughout the world population. that makes the epitope as a supreme candidate for vaccine consideration. finally, from the above in silico analysis, we are really optimistic that our proposed epitope would trigger an immune response in vitro and in vivo. a number of approaches exist for new vaccine development, such as recombinant vaccines, sub-unit protein and dna vaccines, auxotrophic organisms to deliver genes and so on. current study is an attempt to identify potential epitope targets against ebov using different computational tools. it is quite obvious that in order to minimize the deadly effects of ebov, highly potential drugs are immediately required and these in silico approaches will reduce the wet lab efforts with higher probability of success. therefore, it is concluded that the identified epitope may be exploited further for developing epitope-based vaccine against ebov. nevertheless, the initial hints we obtained will help to prioritize potential therapeutics for ebov. additional file : figure s . msa of the rna-dependent polymerase-l proteins of the different ebov. 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system for functional analysis of ebola virus glycoprotein clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mutation analysis of the fusion domain region of st. louis encephalitis virus envelope protein inferring the rate and time-scale of dengue virus evolution characterization of the l gene and ′trailer region of ebola virus a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach peptide binding predictions for hla dr, dp and dq molecules the disopred server for the prediction of protein disorder jalview version -a multiple sequence alignment editor and analysis workbench development of a dna vaccine designed to induce cytotoxic t lymphocyte responses to multiple conserved epitopes in hiv- the authors declare that they have no competing interests.authors' contributions aro has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. ts and asc carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. tpj worked for computational analysis. mah conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. convenient online submission rigorous peer review immediate publication on acceptance open access: articles freely available online high visibility within the fi eld retaining the copyright to your article submit your next manuscript at springeropen.com key: cord- - rukouk authors: zhong, jixin; gong, quan; goud, aditya; srinivasamaharaj, srividya; rajagopalan, sanjay title: recent advances in dipeptidyl-peptidase- inhibition therapy: lessons from the bench and clinical trials date: - - journal: j diabetes res doi: . / / sha: doc_id: cord_uid: rukouk dpp inhibitors (dpp i) are a class of newly developed antidiabetic drugs which preserve incretin hormones and promote postprandial insulin secretion. although the cardiovascular effect of dpp inhibition has been substantially studied, the exact role of dpp in cardiovascular disease especially in humans remains elusive. previous small studies and meta-analyses have suggested a benefit in both surrogate outcomes and cardiovascular events for these agents. however, there was growing evidence in recent years questioning the cardioprotective effect of dpp i. further, a signal of heart failure hospitalization in a recent large scale clinical trial savor-timi has called into question the safety of these agents and their utility in the treatment of cardiovascular disease. in this review, we will revisit the physiologic function of dpp and discuss its role in cardiometabolic disease based on recent experimental and clinical studies. dipeptidyl-peptidase- (dpp , also known as cd ) is a membrane glycoprotein that is well known for its role in the catalytic degradation of incretins. dpp inhibitors (dpp i), as a class of antidiabetic medications, have been accepted worldwide, owing to their ease of administration, modest effects on hba c, and lack of serious side effects. dpp inhibition in experimental models has uniformly demonstrated cardioprotective effects. indeed early meta-analyses of phase ii/iii data of dpp i used in the context of glycemia lowering have shown favorable protective effects of this class in terms of cardiovascular (cv) endpoints, leading to a widespread expectation that these drugs will show a benefit in appropriately designed efficacy trials from a cv standpoint [ ] [ ] [ ] . however, recently completed, appropriately designed, phase iii trials with the intent of demonstrating benefit from a cv perspective have not shown significant improvement in primary cv endpoints in patients treated with dpp i compared to placebo [ , ] . in this review, we will summarize the structure and function of dpp and its known roles in physiology. we will also review its importance in the pathophysiology of cardiometabolic disorders and provide recent clinical trial evidence that has tested its effects in cv disease. dpp is a transmembrane glycoprotein that forms a homodimer or tetramer on the plasma membrane and cleaves nterminal dipeptides from proteins with proline or alanine as the penultimate (p ) amino acids. dpp is highly conserved among species in terms of amino acid sequence. as shown in figure , dpp has a -amino-acid n-terminal cytoplasmic domain (aa - ), a -residue transmembrane domain (aa - ) , and a large c-terminal extracellular domain. the extracellular component contains a / -hydrolase domain and an eight-blade -propeller domain [ ] . this domain is responsible for its dipeptidyl-peptidase activity and its binding to proteins such as adenosine deaminase (ada) and fibronectin. residue and residues - within the cysteine-rich segment have been shown to be essential for ada binding [ , ] , while residues , , and are critical for the catalytic activity of dpp ( figure ). dpp is widely expressed in many organs, such as the kidney, spleen, lungs, pancreas, and prostate [ ] . it is expressed at high levels on endothelial cells, differentiated epithelial cells, and some immune cells such as t cells, dendritic cells, and macrophages. dpp is also present in plasma as a soluble form, which either comes from a shedding process driven by proteinases or is released into circulation by means of vesicles such as exosomes, ectosomes, and apoptotic bodies. altered expression of soluble dpp is commonly seen in many disorders such as solid tumors, autoimmune diseases, hepatitis c, type diabetes (t dm), and obesity [ ] [ ] [ ] . the regulation of dpp expression is not fully understood. studies have suggested that stat [ ] and hepatocyte nuclear factor- (hnf- ) [ ] mediate the transcription of dpp . in an in vitro experiment, cotransfection of hnf- and enhanced reporter gene expression under the control of dpp promoter [ ] . dpp promoter region also contains a gas (interferon gamma-activated sequence) motif, which is a binding site of stat . indeed, stat activation by administration of both interferons and retinoic acid leads to the binding of stat to the gas motif and a subsequent dpp transcription [ ] . in addition to transcriptional regulation, dpp is also regulated at posttranscriptional level. il- enhances the translation, but not transcription, of dpp in activated lymphocytes [ ] . many other cytokines are also involved in the regulation of dpp expression. il- has been shown to be responsible for the upregulation of dpp in fibroblast, epithelial cells, and stromal cells [ , ] . polarization to t h by tgf , il- , il- , il- , and il- also showed an increased expression of dpp [ ] . incretin peptides such as gastric inhibitory polypeptide (gip) and glucagonlike peptide (glp- ) are responsible for the modulation of postprandial blood glucose by promoting insulin secretion from pancreatic cells and via glucagonostatic effects. glp- and gip are rapidly inactivated by dpp , leading to a short half-life (minutes for both glp- and gip). mice lacking dpp (dpp −/− ) are protected from the development of dietinduced obesity and demonstrate improved postprandial glucose control [ , ] . pair-feeding and indirect calorimetry studies have shown that reduced food intake and increased energy expenditure accounted for the resistance to dietinduced obesity in dpp −/− mice. dpp −/− mice also demonstrate improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin-induced cell loss and hyperglycemia [ ] . pharmacological inhibition of dpp enzymatic activity improved glucose tolerance in wild-type but not in dpp −/− mice. dpp inhibition also improves glycemic control in glp r −/− mice, suggesting additional mechanisms for dpp inhibition-mediated antihyperglycemic effect [ ] . in addition to incretin peptides, dpp also cleaves a number of other proteins. the physiologic targets include glp , glp , brain natriuretic peptide (bnp), peptide yy, stromal-cell-derived factor- (sdf- ), erythropoietin, granulocyte colony-stimulating factor (g-csf), and substance p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . pharmacologic targets (evidence provided by in vitro cell culture and/or incubation experiments with substrate and dpp ) include gastrin-releasing peptide, growth-hormonereleasing factor, macrophage derived chemokine, eotaxin, ifn--induced protein- , granulocyte-macrophage colonystimulating factor, erythropoietin, il- , neuropeptide y, btype natriuretic peptide, and peptide yy. the catalytic activity of dpp has been extensively reviewed elsewhere and will not be discussed here in detail [ ] [ ] [ ] . in addition to its peptidase activity, dpp also possesses noncatalytic function via interactions with a range of ligands including ada, caveolin- , fibronectin, coronavirus spike protein, collagen, glypican- , insulin-like growth factor receptor, fibroblast activation protein, and cxcr . by interacting with these ligands, dpp plays a role in a variety of processes such as enhancing t cell activation and functional modulation of antigen presenting cells (apcs). the costimulatory function of dpp was first described in early s [ ] . ligation of dpp by ada or anti-dpp antibodies recognizing ada binding epitope enhanced t cell activation, proliferation, and cytokine production. crosslinking by anti-dpp antibody induced tyrosine phosphorylation of a subset of proteins [ ] . these phosphorylated molecules include signaling molecules downstream tcr/cd , such as p ck , p fyn , zap- , map kinase, c-cbl, and phospholipase c [ ] . since dpp has a very short intracellular domain ( aas), it relies on other proteins to transduce signaling in cells. torimoto et al. reported that activation of dpp by its ligand leads to coaggregation of cd ro into lipid rafts, suggesting that dpp may transduce costimulation via cd [ ] . this result is consistent with the observation that dpp high t cells are restricted to the cd ro + cells [ ] and cd + t cells lacking dpp cannot be triggered to elicit a memory t cell response [ ] . as we will discuss below, dpp -ada interaction may also promote t cell activation by degrading adenosine, an immunosuppressive metabolite. in addition, interaction of dpp with caveolin- may form a complex consisting of doo , carma , bcl , malt , and i b kinase in the lipid rafts on t cell membrane, leading to the activation of nf-b [ ] . respiratory syndrome (mers), a viral respiratory illness, was first reported in saudi arabia in . it was caused by the infection of a coronavirus, mers-cov. the mortality from mers is approximately % [ ] . dpp was subsequently identified as a functional receptor for the entry of mers-cov in human and bat cells [ ] . the engagement of the mers-cov spike protein s with dpp mediates viral attachment and internalization. the residues involved in the dpp virus binding are identical to the ada binding domain indicating a potential competition for dpp binding [ ] . human, macaque, horse, and rabbit dpp have been suggested to be able to bind mers-cov and therefore are susceptible to infection. however, small animals such as mice are more divergent with respect to the dpp virus binding region and are not susceptible to mers-cov infection [ ] . dpp has been previously reported as a cofactor for the entry of hiv in the cd + t cells [ ] . however, subsequent studies identified ccr and cxcr as the major coreceptors for hiv [ ] [ ] [ ] [ ] . the coexpression of dpp and ccr may partially explain the association between dpp expression and hiv infection [ ] . ohnuma et al. reported that dpp interacts with caveolin- present on apcs and initiates a signaling cascade in antigen loaded apcs, resulting in their activation [ , ] . upon binding to dpp , caveolin- is phosphorylated, resulting in the phosphorylation of irak- and its disassociation with tollip. phosphorylated irak- then activates nf-kappa b (nf b) [ , ] , which in turn upregulates cd [ ] . the interaction between dpp and caveolin- has been reported to be involved in the pathogenesis of arthritis [ ] . dpp has also been reported to bind multiple components of extracellular matrix such as collagen, fibronectin, and the hiv- tat protein [ , ] . interactions with these matrix components may play a role in sequestration of dpp and allow additional functions such as matrix remodeling, metastasis, and chemotaxis. circulating dpp activity has been reported to be increased in patients with obesity and t dm, positively correlating with hba c levels, degree of obesity, and measures of insulin resistance and inflammation [ , ] . lugari et al. reported that the increase of circulating dpp activity in diabetic patients results in a reduction of plasma glp- (fasting and in response to meals) [ ] . in addition to circulating dpp activity, the expression of dpp on t cells and dendritic cells is also increased in patients with t dm [ ] . however, there are also reports suggesting a decrease of circulating dpp activity in patients with t dm [ ] . this potential contradiction may relate to the fact that, in these studies, many patients were on concomitant medications. several widely used antidiabetic medications including thiazolidinedione, pioglitazone, and metformin have been reported to reduce circulating dpp [ ] [ ] [ ] and dpp expression on t cells [ ] . this may reflect improvement in glycemic control and other measures of inflammation resulting in the reciprocal decrease in dpp expression. an enhanced expression of hepatic dpp has also been reported in nonalcoholic fatty liver disease and its expression may adversely affect glucose metabolism in this condition [ ] . in vitro stimulation of hepg cells with high glucose increased the expression of dpp , whereas insulin, fatty acids, and cholesterol did not [ ] . incretin hormones, glp- and gip, both potentiate insulin secretion from pancreatic cells through g-protein-coupled receptors [ , ] . as mentioned above, both glp- and gip can be inactivated by dpp , resulting in a short half-life, less than min for glp- and less than min in rodents or min in human for gip [ ] [ ] [ ] . in patients with t dm, incretin response is attenuated with an increase in plasma dpp enzymatic activity as well as heightened tissue dpp expression and release in tissues such as visceral adipose. the increase in dpp levels and expression correlates with the degree of glycemia/insulin resistance, suggesting that dpp mediated incretin degradation is involved in the pathogenesis of t dm. there are several dpp i approved or being approved by fda or eu as antidiabetic drugs, such as sitagliptin, saxagliptin, linagliptin, vildagliptin, and alogliptin. most clinical trials with dpp i demonstrate approximately a . - . % lowering of hba c in patients with a baseline level around % [ ] . the placebo-subtracted hba c reductions with dpp i are generally greater in patients with higher baseline hba c. in general, most studies also corroborate improvements in homeostasis model assessment beta cell function index (homa-) and fasting proinsulin : insulin ratio, suggesting improvement in cell function [ ] . the incidence of side effects and hypoglycemia are very low with these agents [ , ] . in clinical trials, the most common reported side effects of dpp i include nasopharyngitis, upper respiratory tract infection, urinary tract infection, and headache [ ] . saxagliptin [ ] and linagliptin [ ] were recently approved by fda. in contrast to other members of the class, linagliptin has a primarily nonrenal route of excretion and therefore would not need dose adjustment, regardless of renal impairment [ ] . in addition to those approved drugs, more dpp i including dutogliptin and gemigliptin are under development and are awaiting fda approval. inflammation plays a key pathogenic role in the development of insulin resistance and t dm [ ] . innate immune mechanisms typified by macrophage infiltration and activation are widely believed to represent major mediators of adipose inflammation. however, recent findings suggest that t cells may also play an important role in this process. activated cd + effector t cells have been shown to promote adipose inflammation by enhancing macrophage recruitment and activation, while cd + t cells especially t h and regulatory t cells which are the t cell subpopulations expressing lowest levels of dpp [ ] were suggested to be protective in the development of adipose inflammation and insulin resistance [ ] . as mentioned above, dpp provides t cell costimulatory signals. therefore, dpp might play a role in the development of adipose inflammation and insulin resistance although there is a lack of direct evidence. in addition to mediating costimulatory signaling, interaction between dpp and adenosine deaminase (ada) may also facilitate t cell activation by providing a suitable microenvironment for t cell proliferation. inherited mutations in ada activity cause severe combined immunodeficiency (scid) in both human and mice [ ] [ ] [ ] . extracellular atp or adp is initially converted to amp by cd and cd to produce adenosine [ ] . adenosine is then processed by ada and converted to inosine [ ] . by anchoring ada onto the cell surface, dpp modulates pericellular adenosine levels and thus regulates t cell activation ( figure ). absence of ada activity results in the accumulation of adenosine, which dose-dependently inhibits t cell proliferation. jurkat cells expressing dpp mutant devoid of ada binding activity are sensitive to adenosine-mediated inhibition of t cell proliferation [ ] . in contrast, cells expressing ada and dpp on the surface are much more resistant to the inhibitory effect of adenosine [ , , ] . we have recently demonstrated that dpp expressed on adipose tissue macrophages is involved in inflammation and insulin resistance by interacting with ada [ ] . expression of dpp on adipose tissue macrophages was higher than that in circulation and was increased in obese and insulin resistant patients. furthermore, dpp level on adipose tissue macrophage positively correlated with degree of insulin resistance. dpp on antigen presenting cells, including macrophages and dendritic cells, facilitated t cell proliferation and activation through its noncatalytic activity as catalytic inhibition of dpp or addition of exogenous sdpp did not affect their capability to stimulate t cells. antigen presenting cell-expressing dpp was able to bind ada and promote t cell activation via removal of suppressive effect of adenosine [ ] . these results suggest that dpp on antigen presenting cells is capable of promoting inflammation and insulin resistance through its noncatalytic function. interestingly, murine and rat dpp do not bind ada [ ] . although adenovirus delivery of human dpp into mouse has been developed to study mers-cov infection [ ] , in vivo rodent models for the investigation of dpp -ada interaction of relevance to human diabetes are currently unavailable. biology. the signaling of glp- is mediated through glp- r. glp- r was originally identified in pancreatic cells but is widely expressed in many tissues and organs including the lungs, kidney, central/peripheral nervous system, and the cardiovascular system [ ] . the receptor for glp- (glp- r) is expressed in cardiovascular cells including endothelial cells, cardiomyocytes, and coronary smooth muscle cells [ ] . glp- r belongs to the family of g-protein-coupled receptors. the engagement of glp- r leads to the activation of adenylate cyclase through stimulatory gs subunit and subsequent accumulation of camp in classically responsive cells such as pancreatic cells [ , ] . for example, in cell, the activation of glp- activates pka, which subsequently reduces foxo and results in an increase of foxa . foxa then increases pdx- , a transcription factor for insulin [ ] . via a camp-dependent pathway, glp- r signaling may also induce the activation of pi k which further increases the expression of bcl- and bcl-xl, two antiapoptotic proteins [ ] . there is evidence indicating that glp- signaling is involved in the cardioprotective effects of dpp inhibition. for example, it has been shown that acute infusion of glp- improves endothelial dysfunction in patients with t dm [ , ] . exendin- , a glp- r agonist, was also shown to stimulate proliferation of human coronary artery endothelial cells through enos-, pka-, and pi k/akt-dependent pathways [ ] . both dpp i and glp- can increase endothelial progenitor cells (epcs), suggesting a role of a glp- dependent pathway in the development of epcs [ , ] . moreover, glp- can increase left ventricular developed pressure and coronary flow in isolated mouse hearts [ ] . further studies have confirmed that the protective effect of glp- on endothelial cells is mediated through increasing nitric oxide (no) production [ ] . activation of glp- r appears to have cardioprotective effect in humans and various animal models and its effects on contractility, blood pressure, and cardiac output appear to be independent of its antidiabetic effect [ ] . both enzymatic inhibition and genetic deletion of dpp reduced heart infarct size in ischemic models by preserving glp- [ , ] . in vitro treatment of cardiomyocyte with glp- r agonist reduced caspase- cleavage and apoptosis induced by various stimulations such as tnf , hypoxia, and lipidemia [ ] [ ] [ ] . zhao et al. reported that glp- increased p mapk activity, nitric oxide (no) production, and glut expression in isolated hearts and thus increased myocardial glucose uptake during aerobic perfusion [ ] . consistent with this, nikolaidis et al. reported that h continuous glp- infusion ( . pmol/kg/min) in a conscious dog increased myocardial glucose uptake and left ventricular hemodynamics [ ] . pretreatment of p mapk inhibitors could inhibit this effect [ ] . however, there have been conflicting observations of glp- effect on myocardial contractility. glp- was reported to be able to decrease contractility in primary cultured adult rat cardiomyocytes, despite increasing camp levels [ ] . the addition of . nm glp- to the perfusate reduced left ventricular developed pressure [ ] , while . nm glp- increased left ventricular developed pressure by % during langendorff aerobic perfusion of isolated mouse hearts [ ] . studies on glp r −/− mice suggest that the absence of glp- r increases baseline left ventricular developed pressure [ ] . although acute administration of glp- or exendin- increases heart rate and blood pressure at least in rodent models [ ] [ ] [ ] , chronic treatment of exendin- ( nmol/kg twice daily) for wk displayed a marked reduction in systolic blood pressure in both db/db mice and angiotensin-ii-infused c bl/ mice [ ] . in line with these animal studies, majority of clinical trials have also reported a reduction of blood pressure after chronic treatment of glp- r agonist [ ] [ ] [ ] . it has also been suggested that glp- exerts protective effect on atherosclerosis. continuous infusion of exendin- for weeks reduced neointimal formation [ ] , foam cell formation [ ] , and atherosclerotic lesion size [ , ] . some of these effects may relate to favorable effects of glp- on lipoprotein metabolism. infusion of glp- ( pmol/kg/min) via jugular vein reduces triacylglycerol absorption and intestinal apob- production in rats [ ] . hsieh et al. reported that exendin- ( nmol/kg, i.p.) and sitagliptin ( mg/kg, gavage) reduced postprandial triacylglycerol and apob- after oral fat load in c bl/ mice, whereas glp r −/− mice had an increased plasma level of triacylglycerol after oral fat load [ ] . human studies have shown that acute glp- infusion ( . pmol/kg/min) suppressed postprandial plasma triacylglycerol and free fatty acid levels [ ] . short-term treatment of glp- ( nmol sc injection before meals for days) decreased plasma vldltriacylglycerol by % [ ] . the lipid lowering effects of glp- and dpp have been reviewed in detail previously [ ] . glp- -dependent or -independent manner, although the functional significance of these effects has not been fully elucidated. by incubating isolated aorta rings with dpp enzymatic inhibitor, we provided direct evidence showing that dpp inhibition relaxes aorta through a glp- independent pathway. alogliptin, a dpp inhibitor, but not glp- , induces enos and akt phosphorylation (ser and ser , resp.) paralleled by a rapid increase in nitric oxide [ ] . inhibition of src kinase decreased enos and akt phosphorylation in response to alogliptin, in contrast to a lack of any effect on insulin mediated activation of the enos-akt, suggesting that alogliptin mediates vasodilation through src kinase mediated effects on enos-akt. dpp has also been shown to assist in angiogenesis by promoting epcs in a glp- -independent pathway. stromal derived factor- (sdf- ), a known substrate of dpp , is a regulator of epcs. by comparing four weeks of sitagliptin versus no additional treatment added to baseline metformin and/or sulfonylurea therapy in diabetic patients, fadini and coworkers demonstrated that patients with dpp inhibition had a -fold increase of epcs and a % increase of sdf- [ ] . dpp may also affect cardiovascular function through regulation of inflammation. dpp is highly expressed on many inflammatory cells such as t cells and regulates their biological function [ ] . both enzymatic and nonenzymatic functions of dpp play an important role in t cell activation. t cell activation can be blocked by enzymatic inhibition of dpp [ , ] . suppression of dipeptidyl-peptidase activity of dpp resulted in a reduced production of cytokines including il- , il- , il- , and ifn-by peripheral blood mononuclear cells and t cells [ , , ] . tgf- , an immunosuppressive cytokine, was shown to be upregulated by dpp inhibition [ , ] . t cells transfected with mutant dpp devoid of enzymatic activity displayed reduced activation compared to those cells transfected with wildtype dpp [ ] . consistent with this finding, the addition of soluble dpp promoted the recall antigen-induced proliferation of peripheral blood lymphocytes, while soluble dpp mutant without enzymatic activity did not, providing a direct evidence that enzymatic activity of dpp is involved in the t cell activation [ ] . as mentioned previously, dpp also promotes inflammation through catalytic independent mechanisms such as interactions with ada and caveolin- [ ] . however, there were also reports indicating that dpp may negatively regulate immune response. mice deficient for dpp showed enhanced severity of autoimmune encephalomyelitis and reduced tgf and increased production of ifn and tnf when immunized with myelin oligodendrocyte glycoprotein. inhibition of dpp activity has also been shown to enhance hematopoiesis by preserving colony-stimulating factor activity [ ] [ ] [ ] . those immunosuppressive functions may at least in part explain the lack of improvement in cardiovascular endpoints on t dm patients with dpp i. there is good evidence that dpp inhibition mediates protective effect on myocardial infarction, hypertension, and atherosclerosis. survival rate and infarct size significantly improved in dpp −/− mice after lad ligation, accompanied by enhanced prosurvival signal pathways, such as pakt, pgsk , and atrial natriuretic peptide in cardiac tissue [ ] . pharmacologic inhibition with sitagliptin also enhances expression of cardioprotective proteins and improved functional recovery after i/r injury in the murine heart [ ] . several studies in animal models support a favorable effect of dpp inhibition in improving endothelial function and blood pressure [ , , ] . saxagliptin treatment, for instance, has been shown to reduce blood pressure in the spontaneously hypertensive rat, an effect that was accompanied by an increase in aortic and glomerular no release with comparable reductions in peroxynitrite levels [ ] . in contrast to the positive effects in animal experiments, the effects of dpp i on endothelial function in humans have not always been positive. in a double blind study in t dm, treatment with vildagliptin for weeks improved forearm blood flow in response to intra-arterially delivered acetylcholine [ ] . in contrast, studies using flow mediated dilation (fmd) of the brachial artery have shown diametrically opposite results. in one study, dpp i (sitagliptin and alogliptin) actually appeared to worsen fmd when used to treat t dm [ ] . in another study, sitagliptin improved fmd in association with an increase in cd + cells [ ] . both glp- agonism and dpp inhibition reduce postprandial triacylglycerol and apob- in rodents [ , ] . human studies have shown that glp- infusion ( . pmol/kg/min) for hours suppresses postprandial plasma triacylglycerol and free fatty acid levels [ ] . a shortterm treatment of glp- ( nmol sc injection before meals for days) decreased plasma vldl-triacylglycerol by % [ ] . continuous infusion of exendin- for weeks reduces neointimal formation [ ] , foam cell formation [ ] , and atherosclerotic lesion size [ , ] in mice. by providing weeks of alogliptin treatment, shah et al. demonstrated that dpp inhibition reduced atherosclerotic plaque and vascular inflammation in atherosclerosis prone ldlr −/− and apoe −/− mice [ ] . however, human studies reveal contradictory results on the cardiovascular effects of dpp inhibitors. patil et al. reported in a recent meta-analysis including randomized clinical trials and , patients on dpp inhibition therapy and , patients on control treatment (other diabetic treatments or placebo) and demonstrated that dpp inhibitors are safe from a cardiovascular standpoint and have beneficial effects on cardiovascular events compared to other diabetic medications and placebo [ ] . another meta-analysis with trials and , patients performed by monami et al. also suggested that dpp inhibition reduced cardiovascular risk and all-cause mortality in diabetic patients [ ] . in contrast, a more recent meta-analysis of trials with , participants reported that dpp inhibitors have no difference in all-cause mortality, cv mortality, acute coronary syndrome, or stroke compared with placebo. furthermore, dpp inhibitors showed a statistically significant increase in heart failure outcomes (rr = . ; % ci . - . ; = . ) [ ] . the association between dpp inhibition and increased risk of heart failure is also supported by another meta-analysis by monami in [ ] . more importantly, the results from two large scale clinical trials completed recently also did not support the cardioprotective effect of dpp inhibition in humans. in the examine study, , diabetic patients with a recent (< days) myocardial infarction or unstable angina requiring hospitalization were randomized to the treatment of alogliptin or placebo. the patients were followed for a median period of months. although a safety trial was not designed to answer questions on efficacy, alogliptin treatment did not improve the combined outcome of primary endpoints including death from cardiovascular causes, nonfatal myocardial infarction, and nonfatal stroke [ ] . there was a trend towards reduction in cardiovascular death albeit nonstatistically significant. a total of , subjects with hba c . - % and one of the following cardiovascular risks: a history of mi, atherosclerosis, hypertension, smoking, or dyslipidemia, were recruited in savor-timi to evaluate the effect of saxagliptin on cardiovascular outcomes. the patients were randomized to saxagliptin or placebo group. additional antidiabetic agents were prescribed throughout the study. median follow-up time was . years and maximum was . years. in consistency with the results from examine, no improvements in cardiovascular outcomes were observed in saxagliptin treatment group compared to placebo-treated patients. it is noteworthy that the hospitalization rate for heart failure was higher in saxagliptin-treated subjects ( . % versus . %; hazard ratio, . ; % ci, . to . ; = . ) [ ] . no differences in multiple other safety endpoints (such as pancreatitis, cancer, and hypoglycemia) were observed across groups in both savor-timi and examine trials. these trials suggest that catalytic inhibition of dpp is basically safe from a cardiovascular standpoint but also does not improve cardiovascular endpoints at least in the short term. it must be noted that the median follow-up period for examine and savor-timi was . years and . years, respectively, and a longer follow-up period may be necessary to further confirm the results. at least from the results of current large scale trials, it is suggested that catalytic inhibition alone is insufficient to improve cardiovascular outcomes. the other ongoing trials assessing the cardiovascular safety of dpp i may provide further insights into the cardiovascular effect of dpp . failure. animal studies have suggested that dpp inhibition improved cardiac function and survival rate of heart failure. gomez et al. reported that dpp activity was positively associated with body weight in adult dog and was significantly higher in heart failure class compared with healthy heart and heart failure class , demonstrating that dpp might be involved in the early stage of heart failure [ ] . in a pressureoverload-induced heart failure animal model (transverse aortic constriction, tac), takahashi reported that vildagliptin ameliorated tac-induced left ventricular enlargement and dysfunction and improved survival rate on day (tac with vildagliptin, . %; tac without vildagliptin, . %; < . ). in accordance with this, shigeta et al. demonstrated that both pharmacological and genetic dpp inhibition reversed diabetic diastolic left ventricular dysfunction and pressure-overload-induced left ventricular dysfunction [ ] . improved left ventricular function assessed by ejection fraction, mitral annular systolic velocity, and peak systolic velocity was also observed in humans with dpp inhibition (sitagliptin, single dose or weeks of treatment) [ , ] . glp- agonists have also been shown to increase left ventricular ejection fraction [ ] [ ] [ ] (all in short term), although there have been inconsistencies [ , ] . despite the potential benefits in improving cardiac function from short-term use of dpp i and glp- analogs, reports on long-term cardiovascular effect of dpp i and glp- analogs are limited. surprisingly, scirica et al. reported an increased hospital admission rate for heart failure in saxagliptin-treated patients compared with placebo ( . % versus . %; hazard ratio, . ; % ci, . to . ; = . ) in savor-timi trial [ ] , raising the question whether dpp i increase the risk for heart failure [ ] . in a subsequent meta-analysis including trials and , patients, monami et al. reported that dpp i (including vildagliptin, sitagliptin, saxagliptin, alogliptin, linagliptin, and dutogliptin) increased the overall risk for heart failure with an or of . ( % ci: . to . ; = . ) although these results were heavily influenced by the savor-timi results [ ] . interestingly, another metaanalysis which pooled randomized controlled studies and , patients to assess the cardiovascular safety of saxagliptin reported that saxagliptin did not increase the incidence rates for cardiovascular events including heart failure, with an incidence rate ratio of . ( % ci: . to . ) for heart failure [ ] . in a study published july , weir et al. further evaluated the effect of sitagliptin (januvia) on cardiovascular endpoints (all-cause hospital admission/death as well as heart-failure-specific hospitalization/death in , diabetic patients recently diagnosed with heart failure) [ ] . although sitagliptin treatment did not increase the risk for primary endpoints or each component (hospital admission or death), it was noted to be associated with increased risk for heart failure hospitalization ( . % versus . %; or: . ; % ci: . - . ). the absolute risks associated with dpp i for heart failure are small. one limitation of this metaanalysis should not be overlooked with the principal criticism being that they include studies that were never designed for cardiovascular signals [ ] . are there risk factors that may identify patients at risk for this small risk seen with saxagliptin? in the savor-timi trial, scirica et al. reported that patients with increased risk for hospitalization for heart failure had either prior heart failure or elevated levels of natriuretic peptides or chronic kidney disease. however, the risks of the primary and secondary endpoints were similar between treatment groups even in patients at high risk for hospitalization for heart failure [ ] , suggesting that dpp inhibition by saxagliptin does not increase death, myocardial infarction, or stroke even in those with increased risk for heart failure [ ] . therefore, these recent findings may not limit the use of dpp i in t dm patients with cardiovascular disease. however, dpp i should be used with caution in t dm patients with increased vulnerability to dpp inhibition-associated heart failure, especially with saxagliptin such as history of heart failure or chronic kidney disease. the ongoing studies such as tecos, carmelina, and carolina should be informative of additional risks posed by these agents as a class. early studies on the safety suggested a well tolerability of dpp i with only some minor side effects such as gastrointestinal reaction (nausea, vomiting, diarrhea, etc.), flulike symptoms, and skin reactions [ , , ] . more importantly, they showed no weight gain and hypoglycemic risk compared to other oral antidiabetic medications [ ] . considering the diverse functions of dpp , the safety of long-term usage of dpp i on immune function such as antitumor and anti-infection was questioned [ ] . an altered expression level of dpp has been reported in many types of cancer [ ] . an increase in pancreatic cancer was noted in patients taking sitagliptin or exenatide compared with other therapies as evidenced by an evaluation of us fda adverse event database [ ] . but the incidence of all other cancers was similar between sitagliptin group and control drugs [ ] . another evaluation of the german adverse event database, in contrast, showed no significant association between pancreatic cancer and dpp i [ ] . several metaanalysis reports indicate an increased risk of infection such as nasopharyngitis and urinary tract infection in patients on dpp i [ , ] . however, there were also contradictory reports on infections. monami et al. reported in a metaanalysis that there was a significant increase in the risk of nasopharyngitis with sitagliptin (mantel-haenszel odds ratio (mh-or): . ; % ci: . - . ; = . ), but not with vildagliptin. no significant associations with upper respiratory tract infection and urinary tract infection were observed. the actual incidence of other infections was lower than in comparator groups [ ] . there were also some studies raising concerns that incretin-based therapy may potentially contribute to pancreatitis [ , ] . however, there are very limited clinical data examining the risk of dpp i on pancreatitis and the conclusion remains uncertain [ ] . a meta-analysis including trials on dpp i and pancreatitis risk showed no significant increase of pancreatitis with dpp i (mh-or: . ; % ci: . - . ; = . ) [ ] . it is noteworthy that the number of observed cases was small and further investigations are needed to confirm the result. elashoff et al. reported a -fold increase of pancreatitis in patients treated with sitagliptin compared with patients treated with control antidiabetic drugs [ ] . in contrast, a recent case-control study including , patients' first-time hospitalization for acute pancreatitis from to also reveals no associations between dpp i and acute pancreatitis [ ] . the development and application of specific dpp i in clinic reemphasized the importance of dpp in both physiological and pathological processes. recent studies suggest that dpp i are safe from cardiovascular perspective although no significant improvement was observed in cardiovascular endpoints on t dm patients with antidiabetic medications. however, it must be acknowledged that the durations of these two trials are less than years and all subjects were on medications such as statins which may interfere with the cardiovascular effects of dpp i. notably, at least one trial showed an increase of heart failure hospitalization in t dm patients with prior heart failure or chronic kidney disease, suggesting that dpp i should be used in caution in those patients. the absolute risks for heart failure agents are small with these agents and it must be acknowledged that not only diabetes but other therapies used for glycemia control such as sulfonylureas and thiazolidinediones also increase the risk for heart failure. further studies are required to investigate the exact role of dpp i, particularly their catalytic independent activity in cardiovascular disease. drs. jixin zhong, quan gong, aditya goud, and srividya srinivasamaharaj declare that there is no conflict of interests. dr. sanjay rajagopalan has received funding from takeda pharmaceuticals. genetic deletion or pharmacological inhibition of dipeptidyl peptidase- improves cardiovascular outcomes after myocardial infarction in mice 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hyperinsulinemic obesity and islet atrophy in spontaneously late-stage type diabetic rats similar increased serum dipeptidyl peptidase iv activity in chronic hepatitis c and other journal of diabetes research viral infections dipeptidyl peptidase-iv and related molecules: markers of malignancy? regulation of cd /dppiv gene expression by interferons and retinoic acid in tumor b cells role of hepatocyte nuclear factor and in the transcriptional regulation of human dipeptidyl peptidase iv during differentiation of caco- cells mechanisms of cd /dipeptidyl peptidase iv cytokine-dependent regulation on human activated lymphocytes increase of cd /dipeptidyl peptidase iv expression on human gingival fibroblasts upon stimulation with cytokines and bacterial components upregulation of cd expression in epithelial cells and stromal cells during wound-induced skin tumour formation human th cells express high levels of enzymatically active dipeptidylpeptidase iv (cd ) mice lacking dipeptidyl 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via cd in association with carma cdc, middle east respiratory syndrome (mers) adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase -mediated entry of the middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd mouse dipeptidyl peptidase is not a functional receptor for middle east respiratory syndrome coronavirus infection t cell activation antigen, cd , as a cofactor for entry of hiv in cd + cells cc ckr : a rantes, mip- , mip- receptor as a fusion cofactor for macrophage-tropic hiv- identification of a major co-receptor for primary isolates of hiv- hiv- entry into cd + cells is mediated by the chemokine receptor cc-ckr- the -chemokine receptors ccr and ccr facilitate infection by primary hiv- isolates ccr levels and expression pattern correlate with infectability by macrophagetropic hiv- , in vitro soluble cd / dipeptidyl peptidase iv induces t cell proliferation through cd up-regulation on apcs cd upregulates expression of cd on antigen-presenting cells by means of caveolin- cd mediates dissociation of tollip and irak- from caveolin- and induces upregulation of cd on antigen-presenting cells role of cd /dipeptidyl peptidase iv in human t cell activation and function t-cell activation via cd and caveolin- in rheumatoid synovium the cysteine-rich region of dipeptidyl, peptidase iv (cd ) is the collagen-binding site an emerging role of dipeptidyl peptidase (dpp ) beyond glucose control: potential implications in cardiovascular disease hyperglycaemia increases dipeptidyl peptidase iv activity in diabetes mellitus plasma dipeptidyl peptidase-iv activity in patients with type- diabetes mellitus correlates positively with hbalc levels, but is not acutely affected by food intake glucagon-like peptide (glp- ) secretion and plasma dipeptidyl peptidase iv (dpp-iv) activity in morbidly obese patients undergoing biliopancreatic diversion cd /dpp levels in peripheral blood and t cells in patients with type diabetes mellitus decreased dipeptidyl peptidase-iv activity and glucagon-like peptide- ( - )amide degradation in type diabetic subjects reduced serum dipeptidyl peptidase-iv after metformin and pioglitazone treatments inhibition of dipeptidyl peptidase iv activity by oral metformin in type diabetes inhibition of dipeptidyl peptidase-iv activity by metformin enhances the antidiabetic effects of glucagon-like peptide- increased hepatic expression of dipeptidyl peptidase- in non-alcoholic fatty liver disease and its association with insulin resistance and glucose metabolism cardiovascular biology of the incretin system the biology of incretin hormones degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide in vitro and in vivo by dipeptidyl peptidase iv glucagon-like peptide- ( - ) has a larger volume of distribution than glucagon-like peptide- ( - )amide in dogs and is degraded more quickly in vitro by dog plasma biology of incretins: glp- and gip new drugs for the treatment of diabetes part ii: incretin-based therapy and beyond efficacy and safety of the dipeptidyl peptidase- inhibitor sitagliptin as monotherapy in patients with type diabetes mellitus dpp inhibitors for diabetes-what next? cardiovascular effects of the dpp- inhibitors fda approves saxagliptin for type diabetes linagliptin approved for type diabetes linagliptin: a novel dipeptidyl peptidase inhibitor with a unique place in therapy inflammation and metabolic disorders adaptive immunity in obesity and insulin resistance new insights into adenosine-receptormediated immunosuppression and the role of adenosine in causing the immunodeficiency associated with adenosine deaminase deficiency ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ada)-deficient mice and corrects their immune and metabolic defects carrier frequency of a nonsense mutation in the adenosine deaminase (ada) gene implies a high incidence of ada-deficient severe combined immunodeficiency (scid) in somalia and a single, common haplotype indicates common ancestry adenosine generation catalyzed by cd and cd expressed on regulatory t cells mediates immune suppression ecto-enzyme and signaling functions of lymphocyte cd characterization of adenosine deaminase binding to human cd on t cells and its biologic role in immune response a potential role for dendritic cell/macrophage-expressing dpp in obesity-induced visceral inflammation the crystal structure of dipeptidyl peptidase iv (cd ) reveals its functional regulation and enzymatic mechanism biologists make first mouse model for mers tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide- receptor expression cloning of the pancreatic cell receptor for the gluco-incretin hormone glucagon-like peptide international union of pharmacology. xxxv. the glucagon receptor family transcription factor foxo mediates glucagon-like peptide- effects on pancreatic beta-cell mass glucagonlike peptide- inhibits apoptosis of insulin-secreting cells via a cyclic -adenosine monophosphate-dependent protein kinase a-and a phosphatidylinositol -kinase-dependent pathway effects of glucagonlike peptide- on endothelial function in type diabetes patients with stable coronary artery disease the possible protective role of glucagon-like peptide on endothelium during the meal and evidence for an 'endothelial resistance' to glucagon-like peptide in diabetes exendin- stimulates proliferation of human coronary artery endothelial cells through enos-, pka-and pi k/aktdependent pathways and requires glp- receptor glucagon-like peptide- improves proliferation and differentiation of endothelial progenitor cells via upregulating vegf generation dipeptidyl peptidase- inhibitor improves neovascularization by increasing circulating endothelial progenitor cells cardioprotective and vasodilatory actions of glucagon-like peptide receptor are mediated through both glucagon-like peptide receptor-dependent and -independent pathways a glucagon-like peptide- (glp- ) analogue, liraglutide, upregulates nitric oxide production and exerts anti-inflammatory action in endothelial cells renal and cardiac effects of dpp- inhibitors-from preclinical development to clinical research pretreatment with a dpp- inhibitor is infarct sparing in hearts from obese, pre-diabetic rats glp- r agonist liraglutide activates cytoprotective pathways and improves outcomes after experimental myocardial infarction in mice exenatide protects against hypoxia/reoxygenation-induced apoptosis by improving mitochondrial function in h c cells antiapoptotic effects of glp- in murine hl- cardiomyocytes direct effects of glucagon-like peptide- on myocardial contractility and glucose uptake in normal and postischemic isolated rat hearts recombinant glucagon-like peptide- increases myocardial glucose uptake and improves left ventricular performance in conscious dogs with pacing-induced dilated cardiomyopathy glucagon-like peptide- increases myocardial glucose uptake via p alpha map kinase-mediated, nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy glucagon-like peptide- increases camp but fails to augment contraction in adult rat cardiac myocytes subcutaneous glucagon-like peptide- ( - ) amide is insulinotropic and can cause hypoglycaemia in fasted healthy subjects changes in arterial blood pressure and heart rate induced by glucagon-like peptide- -( - ) amide in rats interactions of exendin-( - ) with the effects of glucagonlike peptide- -( - ) amide and of exendin- on arterial blood pressure and heart rate in rats exendin- has an antihypertensive effect in salt-sensitive mice model weight loss, glycemic control, and changes in cardiovascular biomarkers in patients with type diabetes receiving incretin therapies or insulin in a large cohort database interim analysis of the effects of exenatide treatment on a c, weight and cardiovascular risk factors over weeks in overweight patients with type diabetes duration- : exenatide once weekly produces sustained glycemic control and weight loss over weeks exendin- , a glucagonlike peptide- receptor agonist, reduces intimal thickening after vascular injury native incretins prevent the development of atherosclerotic lesions in apolipoprotein e knockout mice inhibition of monocyte adhesion to endothelial cells and attenuation of atherosclerotic lesion by a glucagon-like peptide- receptor agonist, exendin- glp- reduces intestinal lymph flow, triglyceride absorption, and apolipoprotein production in rats the glucagon-like peptide receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice glucagon-like peptide abolishes the postprandial rise in triglyceride concentrations and lowers levels of non-esterified fatty acids in humans the antidiabetogenic effect of glp- is maintained during a -day treatment period and improves diabetic dyslipoproteinemia in niddm patients lipoprotein effects of incretin analogs and dipeptidyl peptidase inhibitors acute dpp- inhibition modulates vascular tone through glp- independent pathways the oral dipeptidyl peptidase- inhibitor sitagliptin increases circulating endothelial progenitor cells in patients with type diabetes: possible role of stromal-derived factor- the role of dipeptidyl peptidase iv in human t lymphocyte activation. inhibitors and antibodies against dipeptidyl peptidase iv suppress lymphocyte proliferation and immunoglubolin synthesis in vitro inhibition of dipeptidyl aminopeptidase iv (dp-iv) by xaa-boropro dipeptides and use of these inhibitors to examine the role of dp-iv in t-cell function dipeptidyl peptidase iv (cd ) on human lymphocytes. synthetic inhibitors of and antibodies against dipeptidyl peptidase iv suppress the proliferation of pokeweed mitogen-stimulated peripheral blood mononuclear cells, and il- and il- production inhibitors of dipeptidyl peptidase iv (dp iv, cd ) induces secretion of transforming growth factor- (tgf- ) in stimulated mouse splenocytes and thymocytes inhibitors of dipeptidyl peptidase iv induce secretion of transforming growth factor- in pwm-stimulated pbmc and t cells the costimulatory activity of the cd antigen requires dipeptidyl peptidase iv enzymatic activity enhancement of antigen-induced t-cell proliferation by soluble cd / dipeptidyl peptidase iv dipeptidylpeptidase negatively regulates colony-stimulating factor activity and stress hematopoiesis sulphostin, a novel inhibitor of dipeptidyl peptidases iv (dppiv) that stimulates hematopoiesis in mice inhibition of cd enzyme activity with pro-boropro stimulates rat granulocyte/macrophage colony formation and thymocyte proliferation in vitro a dipeptidyl peptidase- inhibitor, des-fluoro-sitagliptin, improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein edeficient mice dipeptidylpeptidase inhibition is associated with improvement in blood pressure and diastolic function in insulin-resistant male zucker obese rats dipeptidyl peptidase- inhibition with saxagliptin enhanced nitric oxide release and reduced blood pressure and sicam- levels in hypertensive rats vildagliptin improves endothelium-dependent vasodilatation in type diabetes dipeptidyl peptidase- inhibitors attenuate endothelial function as evaluated by flow-mediated vasodilatation in type diabetic patients dpp- inhibitor and alpha-glucosidase inhibitor equally improve endothelial function in patients with type diabetes: edge study long-term dipeptidyl-peptidase inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis dipeptidyl peptidase- inhibitors and cardiovascular risk: ametaanalysis of randomized clinical trials dipeptidyl peptidase- inhibitors and cardiovascular outcomes: metaanalysis of randomized clinical trials with , participants dipeptidyl peptidase- inhibitors and heart failure: a meta-analysis of randomized clinical trials effect of heart failure on dipeptidyl peptidase iv activity in plasma of dogs cardioprotection against ischaemia induced by dobutamine stress using glucagon-like peptide- in patients with coronary artery disease glucagon-like peptide- infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure effects of glucagon-like peptide- in patients with acute myocardial infarction and left ventricular dysfunction after successful reperfusion effects of intravenous exenatide in type diabetic patients with congestive heart failure: a double-blind, randomised controlled clinical trial of efficacy and safety effect of glucagonlike peptide- (glp- ) on glycemic control and left ventricular function in patients undergoing coronary artery bypass grafting saxagliptin, alogliptin, and cardiovascular outcomes assessment of the cardiovascular safety of saxagliptin in patients with type diabetes mellitus: pooled analysis of clinical trials sitagliptin use in patients with diabetes and heart failure: a population-based retrospective cohort study do dipeptidyl peptidase- inhibitors increase the risk of heart failure? heart failure, saxagliptin and diabetes mellitus: observations from the savor-timi randomized trial dipeptidyl-peptidase- inhibitors and heart failure: class effect, substance-specific effect, or chance effect sitagliptin: a dipeptidyl peptidase iv inhibitor for the treatment of type diabetes saxagliptin: a new dipeptidyl peptidase- inhibitor for the treatment of type diabetes dipeptydil peptidase- inhibitors in type diabetes: a metaanalysis of randomized clinical trials inhibition of multifunctional dipeptidyl peptidase-iv: is there a risk of oncological and immunological adverse effects? pancreatitis, pancreatic, and thyroid cancer with glucagon-like peptide- based therapies glp and cancer: friend or foe? efficacy and safety of incretin therapy in type diabetes: systematic review and metaanalysis dipeptidyl peptidase- (dpp- ) inhibitors for type diabetes mellitus exenatide and rare adverse events glucagonlike peptide -based therapies and risk of hospitalization for acute pancreatitis in type diabetes mellitus: a population-based matched case-control study hope and fear for new classes of type diabetes drugs: is there preclinical evidence that journal of diabetes research incretin-based therapies alter pancreatic morphology? dipeptidyl peptidase- inhibitors and pancreatitis risk: a meta-analysis of randomized clinical trials incretin-based therapy and risk of acute pancreatitis: a nationwide population-based case-control study this work was supported by grants from aha ( post ) and nsfc ( ) to dr. zhong. key: cord- - vj oasa authors: song, xiangjun; zhao, xiaomin; huang, yong; xiang, hailing; zhang, wenlong; tong, dewen title: transmissible gastroenteritis virus (tgev) infection alters the expression of cellular microrna species that affect transcription of tgev gene date: - - journal: int j biol sci doi: . /ijbs. sha: doc_id: cord_uid: vj oasa transmissible gastroenteritis virus (tgev) is a member of coronaviridae family. tgev infection has emerged as a major cause of severe gastroenteritis and leads to alterations of many cellular processes. meanwhile, the pathogenic mechanism of tgev is still unclear. micrornas (mirnas) are a novel class of small non-coding rnas which are involved in the regulation of numerous biological processes such as viral infection and cell apoptosis. accumulating data show that mirnas are involved in the process of coronavirus infection such as replication of severe acute respiratory syndrome coronavirus (sars-cov). however, the link between mirnas and tgev infection is unknown. in this study, we performed microrna microarray assay and predicted targets of altered mirnas. the results showed tgev infection caused the change of mirnas profile. then we selected mir- for further analysis and subsequently identified cell division cycle-associated protein (cdca ) as the target of mir- . moreover, mir- showed the ability to inhibit transcription of tgev gene (a non-structure gene) via directly targeting cdca . in conclusion, differentially expressed mir- that is caused by tgev infection can suppress transcription of tgev gene via targeting cellular cdca . our key finding is that tgev selectively manipulates the expression of some cellular mirnas to regulate its subgenomic transcription. the mirnas are small rna species (containing about nucleotides) which are found in animals, plants, and some viruses. it has been shown that mirnas play a variety of roles in many cellular processes including development, differentiation, apoptosis, cell cycle (reviewed in reference [ ] ). generally, mirnas are derived from introns or intergenes of eukaryotic organisms or viruses and serve as regulators of gene expression [ ] . the mirna-mediated rna interference includes two patterns. one is perfectly base-paring with ' utr of target mrna leading to degradation of mrna. the other is imperfectly binding to target ' utr of mrna resulting in the inhibition of mrna translation. in mammalians, mirnas perform rnai through translation inhibition mediated through imperfect complementarity [ ] . as a result, a mirna can directly interact with multiple targets and a gene can be targeted by many mirnas [ ] . during the interactions between virus and host, mirnas are also playing important regulatory roles [ , ] . increasing evidence indicates that viral infection results in the changes of cellular mirnas species. in turn, the altered mirnas also affect viral infection [ , ] . for example, sars-cov infection up-regulates mir- *, - - p, and - , and down-regulates mir- and mir- . of these mirnas, mir- *, - - p, and - suppress sars-cov replication and ivyspring international publisher contribute to immune evasion in bronchoalveolar stem cells (basc) [ ] . tgev is an enveloped virus with a positive-sense single-stranded rna genome and causes a highly contagious, fatal gastroenteritis primarily in less than -week age piglets around the world. the mortality rate of seronegative suckling piglets infected with tgev can reach to as high as % [ , ] . the tgev genome contains a leader sequence at the ' end and a poly (a) tail at the ' end. the tgev genes are arranged in the order '-rep-s- a- b-e-m-n- - ' [ ] . the tgev gene locates at the 'end of the genome. during tgev infection, protein attenuates host antiviral response by interacting with protein phosphatase catalytic subunit (pp c), a key regulator of host antiviral defense [ ] , and reduces apoptosis, immune response, interferon response, and inflammation [ ] . the mammalian cellular mirnas are profoundly affected by viral infection and, in turn the mirnas also regulate viral gene expression via targeting viral genes or cellular genes. the mir- promotes hepatitis c virus (hcv) rna translation through hybridizing with the ' end of viral transcript [ , ] . the cellular mir- , mir- b, mir- , mir- , and mir- play important roles in the regulation of human immunodeficiency virus (hiv) gene expression through targeting the viral nef gene in cd + t-cells [ , ] . the mir- a binds to cellular ring finger (rnf) mrna to increase hendra virus replication [ ] . to date, complex regulatory networks associated with mirnas have not been comprehensively assessed in tgev infection. overall, we observed that tgev infection caused the change of mirna profile and mir- suppressed transcription of tgev gene via directly targeting cdca . cdca polyclonal antibody ( - -ap) was obtained from proteintech group (proteintech group, chicago, il, us). monoclonal β-actin (sc- ) was purchased from santacruz biotechnology (santa cruz, inc., ca, us). horseradish peroxidase (hrp)-conjugated secondary antibody was purchased from pierce (pierce, rockford, il, us). pk- cells were obtained from american type culture collection (atcc) (ccl- ) and grown in dulbecco's minimal essential medium (dmem) supplemented with % fetal bovine serum (gibco brl, gaithersburg, md, us), iu of penicillin and mg of streptomycin per ml, at ℃ in a % co atmosphere incubator. the tgev shaanxi strain was isolated from tgev-infected piglets by ding l et al [ ] . confluent pk- cells in -mm cell culture dish were infected with tgev for h at an moi of . . meanwhile, the mock infection was carried out. at hpi, total rna was extracted with trizol reagent (invitrogen, carlsbad, ca, us). microarray assay was performed as described previously [ ] using an updated version of the chip (swine mirna version , http://www.mirbase.org/). targets of differentially expressed mirnas were predicted by tar-getscan and miranda. the total rna was obtained using trizol reagent (invitrogen, carlsbad, ca, us) from pk- cells infected with tgev at . moi for h for the microarray analysis. reverse transcription reactions were performed as described previously [ ] . briefly, μg of total rna initially treated with dnase i (fermentas, st. leon-rot, germany), nm stem-loop rt primers, ×first strand buffer, . u/μl rnase inhibitor, u/μl m-mlv, and mm dtt (invitrogen, carlsbad, ca, us), were incubated at ℃ for min, ℃ for min, and ℃ for min. real-time pcr was carried out using the accupower ×greenstar qpcr master mix (bioneer, daejeon, korea) in a μl reaction volume including . μl ×greenstar master mix, . μl ×rox dye, . μl rt product, mm forward primer, and mm reverse primer. reactions were incubated at ℃ for min, followed by cycles of ℃ for sec, and ℃ for min on bio-rad iq real-time pcr system (bio-rad, usa). the relative quantification of mirnas was normalized to u using the ∆∆ct method [ ] . total rna was obtained using trizol reagent (invitrogen, carlsbad, ca, us) according to manufacturer's instructions. the primers for genomic rna (grna) and subgenomic mrnas (sgmrna) of tgev were described previously [ ] . a total of μg of rna was treated with dnase i (fermentas, st. leon-rot, germany) for min at ℃. the treated total rna was reversely transcribed with the first-strand cdna synthesis kit (invitrogen, carlsbad, ca, us). real-time pcr was performed using the accupower ×greenstar qpcr master mix (bioneer, daejeon, korea) in a μl reaction volume on bio-rad iq real-time pcr system (bio-rad, usa). fold variations of the sgmrnas were calculated (normalized to grna). ' utrs of candidate target genes containing the binding site of mir- were respectively amplified by pcr using primers containing sequences of xho i and not i cloning sites and were cloned into the vector psicheck- (promega, madison, wi, usa). to obtain mutation of mir- complementary sites within the ' utr of cdca , seed region was mutated following a mutagenesis protocol [ ] . the mir- mimics (sense strand '-uguggcug ugguguaggccagc- '; antisense strand '-gcug gccuacaccacagccac a- '), a negative control for mimics (an unrelated mimic, sense strand '-ucacaaccuccuagaaagaguaga- '; antisense strand '-ucuacucuuucuaggagguu guga- '), an inhibitor for mir- ( '-gcuggc cuacaccacagccaca- '), and a control rna inhibitor (a random sequence, '-ucuacucuuu cuaggagguuguga- ') were designed and synthesized by ribo biotech (ribobio, guangzhou, china). the mirna inhibitors were modified with '-o-methyl. for the luciferase reporter assay, pk- cells were seeded in -well plates and then co-transfected with ng plasmid and nm of mir- mimics, mir- inhibitors, or negative control, using lipofectamine (invitrogen, carlsbad, ca, us). at h post transfection (hpt), the luciferase activities were measured using dual-glo luciferase assay system (promega, madison, wi, usa) according to the manufacturer's manual. three sirnas (sicdca - , sicdca - , sicdca - ) silencing cdca gene and irrelevant sirna were synthesized by ribo biotech (ribobio, guangzhou, china). the most effective sirna (si-cdca - ), identified by western blot, was applied for the experiments. the sequence of si-cdca - is: sense sequence '-gaaguugauuuccaugg aadtdt- ' and antisense sequence '-dtdt cuucaacuaaagguaccuu- '. the negative control sirna is an irrelevant sirna. pk- cells were transfected with nm cdca -specific sicdca - or irrelevant sirna using lipofectamine (invitrogen, carlsbad, ca, us) according to the manufacturer's guidelines. cells were grown at ℃ for h and then infected with the tgev at moi of . . the total rna was isolated at and hpi. cell viability assay was carried out using cell counting kit- (cck- ) reagent (vazyme, piscataway, nj, usa). pk- cells were plated in -well dishes at a density of × cells/well. cells were transfected with nm sicdca - or irrelevant sirna using lipofectamine (invitrogen, carlsbad, ca, us) according to the manufacturer's guidelines. after h of incubation, the cells were treated with cck- reagent for h. absorbance was recorded with the microplate reader at nm. to construct the cdca expression plasmid, full-length cdca clone was produced by pcr amplification (forward '-ctagctagctagctgg ccgtcagcatggatg- '; reverse '-ctagtcta gactagcaagtggaaggcaggtagtgg- '). pcr products were digested and ligated into pci-neo (promega, madison, wi, usa) at nhe i and xho i sites. the correct constructed plasmid was named pci-neo-cdca . pk- cells were transfected with pci-neo vector (control) or pci-neo-cdca . at hours post-infection (hpt), cells were infected with tgev at moi of . . total rnas were extracted by trizol reagent (invitrogen, carlsbad, ca, us) at and hpi according to the manufacturer's protocol. real-time pcr was performed to detect the sgmrnas level. cells were treated with ripa lysis buffer with phenylmethyl sulfonylfluoride (pmsf). protein concentration was determined with bca protein assay reagent (pierce, rockford, il, us) and separated on a % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) gel and subsequently transferred to a polyvinylidene difluoride (pvdf) membranes (millipore corp, atlanta, ga, us). the pvdf membrane was blocked with % non-fat dry milk for h at room temperature, and then incubated with primary antibodies overnight at ℃ and subsequently with hrp-conjugated secondary antibody at room temperature for h. the signal was detected with enhanced chemiluminescence (ecl). pk- cells were seeded in -well plate ( × cells/well) in growth medium and transfected using lipofectamine with nm mir- mimics, mir- inhibitors, or negative control respectively. at hpt, the cells were infected with tgev at moi of . . the transcription of virus was determined by real-time pcr analysis after and hpi. to gain the overview of the impact of tgev in-fection on host cellular mirnas expression, pk- cells were infected with tgev shaanxi strain at a multiplicity of infection (moi) of . for microarray. the microarray was performed with the total rna extracted from pk- cells infected with tgev at hpi. the differential expression of multiple mirnas in tgev-infected cells in comparison with mock-infected cells was observed in fig. (a) . the expression levels of mirnas were changed remarkably (fold change > . , and p < . ). among them mirnas were up-regulated and mirnas were down-regulated. to validate the microarray results of differentially expressed mirnas, we tested the expression levels of them using real-time pcr. the fold changes of mirnas in tgev-infected cells were referred to that in mock-infected cells. the mirnas levels were normalized to u . the results were correlated with microarray (fig. b) . a total of differentially expressed mirnas were chosen for target prediction. the potential targets of differentially expressed mirnas were predicted with tar-getscan and miranda and , potential target genes were gained (supplementary table s ). to investigate the impact of differentially expressed mirnas on tgev transcription, we selected mir- as a representative based on p value (p < . ), fold change of mirna expression (fold change > . ), and mean fluorescence intensities (mfi) (mfi > ). pk- cells were transfected with mir- mimics, mimics control, inhibitors, or inhibitors control. subsequently all the samples and controls were infected with tgev. the mir- level of the samples was compared with the controls and normalized to u . the mir- level increased after transfection with the mirna mimics and decreased after transfection of the inhibitors in comparison with the control ( fig. a) . the transcription of tgev gene was decreased by overexpression of mir- and increased by inhibition of mir- at and hpi (fig. b) . except for gene , the replication of tgev genome and transcription of other subgenomes were not affected by mir- (data are not shown). to identify the targets that affect the transcription of tgev gene , targets of mir- were predicted by targetscan and miranda (supplementary table s ), and tgev gene does not contain mir- binding site. therefore, we hypothesized that mir- should target cellular gene to inhibit transcription of gene . in order to screen mir- target gene, recombinant dual-luciferase reporter plasmids that contain the binding site of mir- target gene at ' utr of the reporter were constructed for the dual-luciferase assay. the luciferase activity of plasmid containing the ' utr of cdca was markedly lower than that of the control (fig. a) . to generate ' utr mutant of cdca mrna containing mutations of conserved mir- binding site, the site-directed mutagenesis was performed using the dna sequence of wild-type ' utr as the template. two binding sites of mir- seed sequence at cdca ' utr were respectively mutated with the -bp substitution (fig. b) . to determine the direct interaction between mir- and ' utr of cdca mrna, the porcine cdca ' utr or its mutant was respectively subcloned into the ' utr of a renilla luciferase gene in a luciferase reporter psicheck- (fig. c) . the constructs were co-transfected into pk- cells with mir- mimics, mir- inhibitors, or negative control. compared with the control, introduction of exogenous mir- decreased the cdca -wt reporter activity by %, while overexpression and knockdown of mir- using mir- mimics and inhibitors did not affect cdca -mut reporter activity (fig. d) . to assess the specificity of mir- binding, mir- mimics, inhibitor, and cdca -wt reporter are co-transfected. the results showed that the mir- mimics decreased the cdca -wt reporter activity and the mir- mimics and inhibitors had specific binding activity (fig. e) . to determine whether mir- inhibits cdca expression, mir- mimics or mimics control was transfected into pk- cells, and cdca expression was assessed by western blot. introduction of exogenous mir- decreased expression of cdca in pk- cells compared with the control (fig. f) . knockdown of endogenous mir- using mir- inhibitors failed to increase the cdca expression (fig. g) . this is likely due to the lower level of endogenous mir- in pk- cells. moreover, we observed that tgev infection decreased cdca protein levels in pk- cells (fig. h) , while downregulation of endogenous mir- using mir- inhibitors reversed the effect of tgev infection on cdca expression (fig. i) . together, our data conclusively demonstrate that cdca is a direct target of mir- in pk- cells. the mirnas regulate target gene expression via binding to the ' utr of target mrna, and one mirna may target multiple genes. we confirmed that mir- suppressed transcription of gene and targeted cdca directly, but whether mir- inhibits transcription of gene by targeting cdca is unclear. to test the effect of artificial sirna of cdca , three sirnas with different sequences were respectively transfected into pk- cells and the effects of silencing were assessed by western blot analysis. the data indicated that sicdca - was the most effective sirna (fig. a) . the mrna level of cdca was significantly silenced by sicdca - (fig. b) , however the viability of cells was not affected by sicdca - in comparison with irrelevant sirna (fig. c) . to detect the overexpression effect of pci-neo-cdca , we measured the protein level of cdca at hpt using western blotting. as expected cdca expression level in transfected group increased compared with the control (fig. d) . to investigate the effect of cdca on the transcription of tgev gene , we detected transcription level of gene using real-time pcr. the results showed that silence of cdca using sicdca - decreased transcription of gene at and hpi and that overexpression of cdca facilitated transcription of gene at and hpi (fig. e) . to understand the relevance of cdca expression in viral infection, we determined the effect of cdca silencing and overexpression on tgev grna, other sgmrnas, and viral titers. the results showed that cdca had no effect on tgev grna level, other sgmrnas level, and viral titers (data are not shown). taken together, the data presented here demonstrated that tgev infection induced mirnas profiling changes and, among these altered mirnas the up-regulated mir- induced by tgev infection inhibited transcription of gene via directly targeting cdca . the major findings in this study are that tgev infection leads to the change of cellular mirnas expression profile, and altered mirnas regulate transcription of tgev gene through targeting cellular cdca . increasing evidence shows that mirnas play important roles in virus-host interactions including viral transcription, cell apoptosis, and antivi-ral effects of host [ ] . in this study, we examined the connection between mirnas and tgev infection and assessed global expression patterns of cellular mir-nas. based on the findings of this study, we proposed that mir- expression altered by tgev infection is involved in regulating transcription of tgev gene . the infection of coronavirus, such as sars-cov and infectious bronchitis virus (ibv), affects many cellular processes such as viral replication, host innate immunity or signaling pathways [ ] . in some cases, the altered processes are directed by the virus for its own advantage and other altered processes are cellu-lar defense responses to viral infection. in this study, we observed that the expression levels of cellular mirnas are regulated positively or negatively by tgev infection. the mirnas regulate viral infection through interacting with targets. therefore, the mirnas and targets form a complicated regulatory network during viral infection which suggests that this greatly expands the range of possible virus-host interactions. this is in good agreement with the notion that mirnas have evolved as a necessary and critical component to regulate host-virus interactions [ ] . in this study, we observed that tgev altered the mirnas profile and mir- was involved in regulating the interplay between tgev and pk- cells by modulating transcription of tgev gene . it was reported that sars-cov infection resulted in differentially expressed mirnas profile and some of altered mirnas regulated the interaction between sars-cov and host [ ] . these indicate coronaviruses may affect cellular mirnas expression and the altered mirnas are able to regulate viral infection. a key observation in this study was that mir- suppresses transcription of tgev gene . tgev gene , a non-structure gene, contributes to viral invasion and inhibits apoptosis in host cells [ ] . previously we reported that tgev induces apoptosis by multiple pathways [ ] [ ] [ ] . these observations suggest that mir- might be involved in regulating apoptosis through gene . therefore it will be important to detect effects of mir- on apoptosis. mir- was detected in porcine intestinal and milk by deep sequencing [ , ] , but the current studies related to mir- are very few. to identify what gene mir- will target to regulate the transcription of tgev gene , the potential targets of mir- were predicted using targetscan and mi-randa. more than potential targets were obtained (supplementary table s ). since tgev genes were not the potential targets of mir- , mir- must target the cellular genes. we identified the potential targets and obtained a new finding that exogenous mir- directly interacts with the ' utr of cdca mrna and suppresses cdca protein level. cdca is a transcriptional regulator that is directly targeted and activated by transcription factor e f [ ] . the e f plays a crucial role in controlling cell cycle and action of tumor suppressor proteins [ ] . in addition, cdca is a target gene of c-myc and the phosphorylated cdca regulates c-myc-dependent apoptosis and transformation [ ] . therefore it will be very important to discover the regulatory mechanism of cdca during tgev infection. in conclusion, the results of the present study provide evidence that tgev infection resulted in altered profiles of mirnas in pk- cells and the differentially expressed mir- was involved in regulation of tgev transcription by targeting cellular cdca . these findings will give insight into the regulatory mechanism of mirna to transcription of tgev gene . in addition, this opens a new avenue to target host cellular factors using sirna technologies to combat tgev transcription. there are limitations in this study that need to be addressed and resolved in future studies. the mir-nas play regulatory role at post-transcriptional level [ ] . an mirna may target multiple genes, and one gene may be targeted by many mirnas [ , ] . for mir- , it is supposed to take part in regulating the tgev infection process via targeting other genes. for the whole study, numerous differentially expressed mirnas and a quantity of targets of these mirnas form a complex regulatory network to modulate viral-host interactions at any stage of tgev infection. therefore, more functions of altered mirnas on tgev-host interactions need to uncover and more targets need to identify in the future studies. molecular mechanisms of rna interference microrna: a master regulator of cellular processes for bioengineering systems mechanisms of mirna-mediated gene regulation from common downregulation to mrna-specific 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protein modulates host innate immune response enhancement of hepatitis c viral rna abundance by precursor mir- molecules requirements for human dicer and trbp in microrna- regulation of hcv translation and rna abundance cellular micrornas contribute to hiv- latency in resting primary cd + t lymphocytes interplay between hiv- infection and host micrornas promotion of hendra virus replication by microrna a isolation and identification of porcine transmissible gastroenteritis virus shaanxi strain and sequence analysis of its n gene characterization of microrna expression profiles and the discovery of novel micrornas involved in cancer during human embryonic development real-time quantification of micrornas by stem-loop rt-pcr analysis of relative gene expression data using real-time quantitative pcr and the −ΔΔct method gene n proximal and distal rna motifs regulate coronavirus nucleocapsid mrna transcription gene splicing and mutagenesis by pcr-driven overlap extension rna viruses and 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mirna-mediated mechanisms in mammalian cells role of mir- in the regulation of lymphocyte immune function and disease runx : a microrna hub in normal and malignant hematopoiesis xiangjun song and xiaomin zhao designed the experiments, interpreted the data and wrote the article. xiangjun song and xiaomin zhao performed the experiments with assistance and advice from yong huang, hailing xiang, wenlong zhang. dewen tong revised the manuscript. all authors have read the manuscript and approved to submit it to this journal. supplementary the authors have declared that no competing interest exists. key: cord- -psog u authors: van asten, liselotte; bijkerk, paul; fanoy, ewout; van ginkel, annemarijn; suijkerbuijk, anita; van der hoek, wim; meijer, adam; vennema, harry title: early occurrence of influenza a epidemics coincided with changes in occurrence of other respiratory virus infections date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: psog u background: viral interaction in which outbreaks of influenza and other common respiratory viruses might affect each other has been postulated by several short studies. regarding longer time periods, influenza epidemics occasionally occur very early in the season, as during the pandemic. whether early occurrence of influenza epidemics impacts outbreaks of other common seasonal viruses is not clear. objectives: we investigated whether early occurrence of influenza outbreaks coincides with shifts in the occurrence of other common viruses, including both respiratory and non‐respiratory viruses. methods: we investigated time trends of and the correlation between positive laboratory diagnoses of eight common viruses in the netherlands over a ‐year time period ( – ): influenza viruses types a and b, respiratory syncytial virus (rsv), rhinovirus, coronavirus, norovirus, enterovirus, and rotavirus. we compared trends in viruses between early and late influenza seasons. results: between and , influenza b, rsv, and coronavirus showed shifts in their occurrence when influenza a epidemics occurred earlier than usual (before week ). although shifts were not always consistently of the same type, when influenza type a hit early, rsv outbreaks tended to be delayed, coronavirus outbreaks tended to be intensified, and influenza virus type b tended not to occur at all. occurrence of rhinovirus, norovirus, rotavirus, and enterovirus did not change. conclusion: when influenza a epidemics occured early, timing of the epidemics of several respiratory winter viruses usually occurring close in time to influenza a was affected, while trends in rhinoviruses (occurring in autumn) and trends in enteral viruses were not. it has been suggested that annual epidemics of different viral infections can interfere with each other, but clear trends over long time periods and underlying mechanisms are not known. [ ] [ ] [ ] [ ] a few population-level studies in europe were based on observations in one respiratory season only (the h n pandemic) in which the annually recurring influenza epidemic occurred relatively early. with the occurrence of several early influenza a seasons in recent years, an exploration of longer time trends of different viruses was considered useful to gain more insight in the suggested relationship between circulating viruses. understanding viral shifts and potential drivers thereof is relevant for further understanding whether certain viruses might promote or inhibit (pandemic) influenza spread and whether influenza vaccination could potentially affect trends in other respiratory viruses. in europe, influenza epidemics generally occur in winter, with the official start of the epidemic when influenza-like illness (ili) incidence in primary care sentinel surveillance exceeds an epidemic threshold (in combination with influenza a virus detection in clinical specimens collected from a subset of those ili patients). in the netherlands, the influenza epidemic threshold has been calculated at an ili incidence of Á / for minimally two consecutive weeks which is usually not exceeded before the turn of the year, [ ] [ ] [ ] [ ] but the timing of the first exceedance (i.e., start of the epidemic) can vary between november and march (based on data from to ). an extremely early influenza season was the / season when the influenza a (h n )pdm pandemic strain appeared and the ili epidemic threshold was reached by early october (week ). such early occurrence may affect the circulation of other seasonal pathogens, and theories on possible interference between outbreaks of different respiratory viruses have been postulated to be a possible cause of delays in expected seasonal outbreaks of other respiratory viruses. - while those earlier population-level studies focused mainly on the possible interaction between influenza a virus and rhinovirus circulation, there may also be a relationship between influenza a and other prevalent viruses. therefore, we investigated trends in several common viruses for which laboratory data were available from national surveillance in the netherlands for a longer time period of up to years including both respiratory and enteral viruses. we investigated trends in the reporting of common infectious respiratory and enteral viruses for which laboratory data were available for the - the data were available from the "weekly virological records system" of the dutch working group on clinical virology giving the number of positive laboratory diagnoses by year and week, but not providing data on the denominator nor on age or gender of the patient. submitting laboratories are associated with either hospitals or regional laboratories to which both gps and hospitals submit samples. the estimated proportion of all positive diagnostics captured by this national surveillance varies between the monitored pathogens and was estimated between % (for rotavirus) and % (for influenza virus) in a study of five pathogens. the types of tests used can differ between the submitting laboratories and over time. respiratory viruses were detected in throat swabs mainly by pcr-based methods in respiratory specimens from patients with respiratory disease symptoms. enteric viruses were detected in fecal samples by eia-based methods (rotavirus, norovirus) and by pcr-based methods (norovirus). enterovirus was detected mainly by pcr and in the early years also by culturing, in throat swabs, cnf, and in fecal specimens collected from patients suspected for an enterovirus infection. cross-reaction between rhinovirus and enterovirus pcrs for throat swab samples cannot be excluded completely. when collected from patients suspected for enterovirus infection, typing of enteroviruses indicated that this was a minority. when collected from patients with respiratory symptoms, enteroviruses might be misidentified as rhinovirus, as occurred during enterovirus d outbreaks. to determine whether an influenza a season occurred relatively early, we used both the influenza sentinel surveillance data and the laboratory data from the weekly virological records system. as general practitioner (gp) influenza sentinel surveillance is the current gold standard for influenza surveillance in the netherlands (with an epidemic threshold), early influenza a seasons were first identified using published dates of the influenza epidemics. we combined this information with visual inspection (as there is no threshold available) of trends in influenza a laboratory diagnoses reported in the weekly virological records system for confirmation of the early ili epidemics and for identification of any additional early influenza a seasons according to those laboratory data. a pre-defined cutoff point for the identification of early influenza seasons is not available. using the published ili epidemic periods, we identified two relatively early influenza a seasons wherein influenza epidemics started before the turn of the year (i.e., before week ): the / season (epidemic starting in week lasting to week ) and the / season (epidemic starting in week lasting to week ). , these two early epidemics were also confirmed by visible early increases in influenza laboratory data reported in the weekly virological records system ( figure ). after visual inspection of the laboratory data, the / season was additionally selected as an early season as influenza a diagnoses were clearly on the rise before week ( figure ). while the influenza epidemic in the / season did not start early (week ) according to the ili sentinel surveillance system, in the laboratory surveillance data this season seemed neither clearly early nor clearly late and was therefore excluded (influenza a laboratory diagnoses in this season are represented in appendix ). as most of the viruses under study peak in winter, we defined year-long seasons running from july to june rather than using calendar years (january to december). for comparability between the seasons, we presented the number of detected viruses per week as a percentage of the total number of detections during the study period per each respective virus. due to the intensified testing that occurred during the a (h n )pdm pandemic, the total number of influenza a virus laboratory diagnoses in the pandemic season was Á times higher than the average number of influenza a virus laboratory diagnoses in the - seasons, excluding the pandemic season. to facilitate the inclusion of such a high peak into the presented graphs, the number of influenza a virus laboratory diagnoses during the / season was scaled down (reduced by a factor Á , changing the height but not the shape of the epidemic in that year). as the number of influenza b virus laboratory diagnoses was very low during the pandemic, such downscaling was not performed for the influenza b virus laboratory diagnoses. smoothing of time series was performed by calculating -week moving averages (average of current, previous, and next week's value). correlation coefficients between different viruses were calculated using spearman's rank correlations for non-normally distributed data. we also shifted the time series forwards and backwards in time (À to + weeks) and compared whether the time shift with maximal correlation differed between early and late influenza seasons. the highest numbers of laboratory reports were available for rsv and the lowest for influenza b ( and reported diagnoses during the total study period, table ). time series of all considered pathogens are shown in figure . almost all of the included respiratory viruses (influenza a and b virus, rsv, coronavirus) except rhinovirus showed very clear seasonality in their reporting over time. the numbers increase, peak, and are elevated during winter season (december-february) or occasionally in very early spring (march-april) ( figure and appendix a-d). however, while influenza b virus diagnoses also displayed such winter seasonality, outbreaks did not occur each winter (five of the ten observed years showed clear influenza b epidemics, while the other years showed very low levels or even near-absence). in our data, rhinovirus levels showed less clear seasonality than the other respiratory viruses. they tended to be elevated over broad periods ( figure ) and seemed to be present year-round with less pronounced elevations during autumn/winter/spring. generally, rsv van asten et al. epidemics preceded influenza a virus epidemics, which in turn preceded influenza b epidemics (in the years that influenza b epidemics occurred). increases in coronavirus also usually preceded increases in influenza a (but not in the / season). the timing of rhinovirus levels relative to influenza a virus in laboratory diagnoses was less clear (due to seemingly year-round presence of rhinoviruses in specimens submitted for laboratory diagnoses). the two gastrointestinal viruses in the study (norovirus and rotavirus) both also presented with winter seasonality, often with norovirus preceding rotavirus epidemics ( figure and appendix c). norovirus and influenza a virus epidemics usually overlapped, but the norovirus epidemics had broader peaks, and numbers tended to start increasing earlier than influenza a virus diagnoses levels and decreased after the disappearance of influenza a virus. also, rotavirus outbreaks usually occurred before influenza a outbreaks started. enteroviruses, of which there are many types, can cause respiratory illness, but they also cause other illness. enterovirus data were sparse until , but in the - time period, enterovirus levels generally peak in summer (june-august). visually, we assessed whether seasons with early influenza a virus epidemics (as reported in the weekly virological records system) coincided with shifts in the reporting of other common pathogens or shifts in temperature and humidity trends. occurrence of seasons with relatively early influenza a virus circulation compared to seasons with later circulation of influenza a virus is shown in figure (early seasons in color, late seasons in gray). for the other viruses, the same grouping was made (based on early and late influenza seasons), with their trend during early influenza a seasons also given in color ( the correlation coefficients between influenza and other viruses were stratified by timing of the occurrence of the influenza epidemic (early versus late, table ). as the different viruses might differ in the timing of their occurrence relative to influenza a occurrence, the correlation coefficients were calculated at different time lags (shifted between weeks earlier up to weeks later) to determine the time shift with the maximum correlation with influenza a. when comparing seasons with early versus later occurrence of influenza epidemics, the maximum correlation coefficient was observed at different lags for all viruses. this difference in time lag relative to influenza a occurrence was smallest for influenza b, indicating that influenza b circulation relative to influenza a tends to occur at a more stable delay than for the other viruses. this suggests that changes in timing of influenza a circulation will be accompanied by changes in timing of influenza b circulation. however, such an overall correlation coefficient (calculated over multiple seasons combined) obscured the visual observation that in van asten et al. two of the three early influenza seasons, influenza b virus was almost absent (figure ). viruses that showed a shifted trend of reporting during years with early influenza a epidemics were of respiratory nature with clear winter seasonality and with epidemics occurring relatively close in time to influenza a virus epidemics. although per individual virus the shifts were not consistently of the same type or direction, generally said, in years when influenza a hit early, rsv tended to be delayed, coronavirus outbreaks tended to be intensified, and influenza b virus tended not to circulate at all. viruses that act on other organ systems (enteral) seemed not to be affected by the timing of influenza a outbreaks. in our data, rhinovirus showed little seasonality, but rather year-round presence, and therefore, no association with timing of influenza a could be observed. changes in seasonal patterns of respiratory pathogen laboratory reports have been observed in other, shorter, studies. in hong kong, after the early occurring a(h n ) pdm pandemic, increases were observed in adenovirus and parainfluenza virus detections, while the usual rsv summer peak disappeared in data from hospitals and clinics. rhinovirus and coronavirus were not included. in another study in beijing, all respiratory virus epidemics, except rhinovirus, were delayed after the pandemic influenza epidemic (data came from fever clinics which screen patients prior to being assigned to a specific hospital department). however, both regions have different climates from the netherlands (and from each other) complicating comparisons. other european observations have also been reported regarding the effect of rhinovirus on influenza virus circulation. while those reports hypothesize on viral interference between influenza virus and rhinovirus circulation (with rhinovirus delaying influenza spread), - , we (like the beijing study ) did not observe clear-cut trends in rhinovirus reports in our laboratory diagnoses data. the relationship previously reported between rhinovirus and influenza virus might perhaps be age specific because in one study, the reduced likelihood of a(h n )pdm detection in rhinovirus-positive samples was observed in a pediatric population, as was the reduced likelihood of detecting eight viruses in rhinovirus-positive samples in another study in children, while our data were not age specific and we did not have such information on concurrent infections. besides the reported possible interaction between rhinovirus and influenza a virus, much less is known about interaction between and with other (respiratory and nonrespiratory) viruses. our data suggest that respiratory viruses may impact each other's seasonality (in this study focused on the relationship between influenza a virus and other respiratory viruses), albeit through unknown mechanisms, as also hinted at by the beijing and hong kong data. , virtually all research suggesting interaction between respiratory viruses in human populations has focused on observational (ecological) studies such as ours, most including only one or a few seasons of data while our study included a -year period. only one prospective cohort study has been published (based on one season) which showed rhinovirus and coronavirus to interrupt the a(h n )pdm pandemic. our results could not confirm their finding of coronavirus inhibiting influenza a virus circulation, but we observed more coronavirus laboratory reports when influenza a virus circulated early. in contrast to most of the other studies, we could investigate whether patterns recurred due to our longer study period. this revealed that coronavirus laboratory reporting was more intense in / when it did not overlap with influenza a virus circulation. however, it was also more intense in the early influenza a year thereafter ( / ) when it actually completely overlapped with the influenza a epidemic hinting that direct biological inhibition of either virus by the other might be unlikely and thus apparent interaction reported by others may have depended on indirect factors or may be spurious. these variations by year in our data illustrate how results from shorter observational studies have to be interpreted with caution, especially when they focus only on year or one season. however, also in our longer study we have to remain cautious; to refute biological inhibition between coronavirus and influenza, it would be helpful to know whether the coronavirus and influenza virus specimens were from the same age group. unfortunately, as in other observational studies, surveillance artifacts could not be ruled out for any of the viruses in our study. these artifacts concern increases and decreases in laboratory testing over time that may not necessarily be related to changes in virus circulation in the population. like previous studies, we did not have information on issues potentially underlying such changes, such as shifts in laboratory testing policy, availability and use of new diagnostic tools, variations in testing intensity due to previous or new outbreaks, and possible outsourcing of certain tests. furthermore, information on age was not available in our data, possibly diluting trends that might have been visible were data investigated age specifically, or alternatively creating trends and shifts that might otherwise have been absent in certain age groups. if certain age groups are more likely to be hospitalized (such as infants with rsv infection) and/or to be sampled than other age groups and thus overrepresented in the laboratory data, our observed trends might not be representative for the total population if virus circulation trends actually differ between age groups. future ecological analyses should preferably include age-specific data as this may further clarify the true occurrence of shifted seasonality. comparison between the different studies (including ours) is also made difficult by unknowns other than age, such as illness severity and influenza vaccination status. as other ecological-type studies have suggested, also our observed shifts in the occurrence of influenza b virus, rsv, and coronavirus during early influenza a seasons suggest that viral interaction might play a role in the occurrence of virus epidemics. [ ] [ ] [ ] with our data, the precise direction of potential interactions is not clear, that is, which virus(es) impacts which. there are several types of virus-virus interactions, described in a review by da palma, ranging from (i) direct virus-virus interactions (e.g., when nucleic acids or proteins of one virus physically interact with the genes or gene products of a coinfecting virus); (ii) indirect interactions resulting from alterations in the host environment; to (iii) immunological interactions. however, with the current data we cannot determine whether the viruses impacted each other biologically. further, there are other issues that can impact the timing and magnitude of seasonal epidemics such as environmental, social, and behavioral phenomena that may also be the potential drivers of observed shifts in reporting patterns instead of viral inter-action (alone). to further attempt to disentangle these issues, future ecological studies should preferably include age-specific virological data over multiple seasons and better yet, cohort studies could be performed that (i) address the order of infection by different viruses (by serial sampling of subjects during the study period regardless of symptoms) as proposed by others; , (ii) include persons from the same household as was done by pascalis et al.; and (iii) include multiple years and thus multiple seasons per virus. there are several examples highlighting the importance of understanding drivers of viral shifts. as suggested by greer et al., further studies are relevant in light of the discussion whether rhinovirus promotes (pandemic) influenza spread through coughing and sneezing or whether it may paradoxically provide natural protection due to inhibition of influenza infection in those already infected with rhinovirus. further studies may also provide input to the question whether influenza vaccination can render persons more susceptible to other respiratory viruses due to the lack of temporary nonspecific immunity induced by actual influenza infection. canadian and australian researchers hypothesize that through this same mechanism of induced temporary non-specific table . correlation coefficients between number of weekly laboratory diagnoses of influenza a and weekly counts of other pathogens at different time lags ( weeks later to weeks earlier)* *bolded numbers indicate the largest correlation coefficient (with a negative correlation for enterovirus due to opposite seasonality with influenza a virus). immunity, the circulation of a seasonal influenza strain preceding a pandemic strain might decrease the susceptibility to that ensuing pandemic strain. not evaluated in our study but possibly also playing a role in virus shifts or virus-virus interactions is the variation in the circulation of influenza a subtypes from year to year. evaluation of such an effect might require evaluation of longer time series as the first general impression from our data was that there was no clear consistent association with subtype as the three early influenza years were not all dominated by the same influenza a type ( studying the association of climatic factors (an example of environmental phenomena potentially affecting virus circulation) with influenza [ ] [ ] [ ] was beyond the scope of this study, although covering the timespan of the study, a visual assessment of temperature and humidity trends showed no clear visual association between early influenza occurrence and early occurrence of low temperatures or low humidity. although two of the three early ili seasons ( / , / ) seem to have longer and colder cold spells (average weekly temperatures below °c) that also occur earlier in the winter season than in the other years, the first mentioned season (the / pandemic year) actually showed the cold spell occurring after the influenza season instead of at the beginning or during the influenza season (appendix e). unlike earlier studies, we included several non-respiratory pathogens, for which the results showed no shifts in trends, and therefore, interaction with influenza a virus seems unlikely. as norovirus, rotavirus, and enterovirus affect other organ systems, direct interaction may not be expected. however, sporadic examples do not rule out such unexpected interactions as others have suggested that live polio vaccine (against an enteral virus) might prevent otitis media (a respiratory infection), and decrease infantile diarrhea mortality (gastro-intestinal mortality). another issue is that, like others, we investigated shifts in virus circulation within the same seasons. whether early occurrence of influenza a may affect the circulation of seasonal pathogens in seasons thereafter is not known. in conclusion, when influenza hit early we observed shifts in patterns of several respiratory pathogens that occur close in time with influenza a. further research is needed to understand whether this was caused by (in)direct biological 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